Nonsyndromic cleft lip with or without cleft palate: Increased burden of rare variants within Gremlin-1, a component of the bone morphogenetic protein 4 pathway.

PubMed

Al Chawa, Taofik; Ludwig, Kerstin U; Fier, Heide; Pötzsch, Bernd; Reich, Rudolf H; Schmidt, Gül; Braumann, Bert; Daratsianos, Nikolaos; Böhmer, Anne C; Schuencke, Hannah; Alblas, Margrieta; Fricker, Nadine; Hoffmann, Per; Knapp, Michael; Lange, Christoph; Nöthen, Markus M; Mangold, Elisabeth

2014-06-01

The genes Gremlin-1 (GREM1) and Noggin (NOG) are components of the bone morphogenetic protein 4 pathway, which has been implicated in craniofacial development. Both genes map to recently identified susceptibility loci (chromosomal region 15q13, 17q22) for nonsyndromic cleft lip with or without cleft palate (nsCL/P). The aim of the present study was to determine whether rare variants in either gene are implicated in nsCL/P etiology. The complete coding regions, untranslated regions, and splice sites of GREM1 and NOG were sequenced in 96 nsCL/P patients and 96 controls of Central European ethnicity. Three burden and four nonburden tests were performed. Statistically significant results were followed up in a second case-control sample (n = 96, respectively). For rare variants observed in cases, segregation analyses were performed. In NOG, four rare sequence variants (minor allele frequency < 1%) were identified. Here, burden and nonburden analyses generated nonsignificant results. In GREM1, 33 variants were identified, 15 of which were rare. Of these, five were novel. Significant p-values were generated in three nonburden analyses. Segregation analyses revealed incomplete penetrance for all variants investigated. Our study did not provide support for NOG being the causal gene at 17q22. However, the observation of a significant excess of rare variants in GREM1 supports the hypothesis that this is the causal gene at chr. 15q13. Because no single causal variant was identified, future sequencing analyses of GREM1 should involve larger samples and the investigation of regulatory elements. © 2014 Wiley Periodicals, Inc.

Hepatitis C Virus Antigenic Convergence

PubMed Central

Campo, David S.; Dimitrova, Zoya; Yokosawa, Jonny; Hoang, Duc; Perez, Nestor O.; Ramachandran, Sumathi; Khudyakov, Yury

2012-01-01

Vaccine development against hepatitis C virus (HCV) is hindered by poor understanding of factors defining cross-immunoreactivity among heterogeneous epitopes. Using synthetic peptides and mouse immunization as a model, we conducted a quantitative analysis of cross-immunoreactivity among variants of the HCV hypervariable region 1 (HVR1). Analysis of 26,883 immunological reactions among pairs of peptides showed that the distribution of cross-immunoreactivity among HVR1 variants was skewed, with antibodies against a few variants reacting with all tested peptides. The HVR1 cross-immunoreactivity was accurately modeled based on amino acid sequence alone. The tested peptides were mapped in the HVR1 sequence space, which was visualized as a network of 11,319 sequences. The HVR1 variants with a greater network centrality showed a broader cross-immunoreactivity. The entire sequence space is explored by each HCV genotype and subtype. These findings indicate that HVR1 antigenic diversity is extensively convergent and effectively limited, suggesting significant implications for vaccine development. PMID:22355779

Integrated rare variant-based risk gene prioritization in disease case-control sequencing studies.

PubMed

Lin, Jhih-Rong; Zhang, Quanwei; Cai, Ying; Morrow, Bernice E; Zhang, Zhengdong D

2017-12-01

Rare variants of major effect play an important role in human complex diseases and can be discovered by sequencing-based genome-wide association studies. Here, we introduce an integrated approach that combines the rare variant association test with gene network and phenotype information to identify risk genes implicated by rare variants for human complex diseases. Our data integration method follows a 'discovery-driven' strategy without relying on prior knowledge about the disease and thus maintains the unbiased character of genome-wide association studies. Simulations reveal that our method can outperform a widely-used rare variant association test method by 2 to 3 times. In a case study of a small disease cohort, we uncovered putative risk genes and the corresponding rare variants that may act as genetic modifiers of congenital heart disease in 22q11.2 deletion syndrome patients. These variants were missed by a conventional approach that relied on the rare variant association test alone.

Variants of uncertain significance in newborn screening disorders: implications for large-scale genomic sequencing.

PubMed

Narravula, Alekhya; Garber, Kathryn B; Askree, S Hussain; Hegde, Madhuri; Hall, Patricia L

2017-01-01

As exome and genome sequencing using high-throughput sequencing technologies move rapidly into the diagnostic process, laboratories and clinicians need to develop a strategy for dealing with uncertain findings. A commitment must be made to minimize these findings, and all parties may need to make adjustments to their processes. The information required to reclassify these variants is often available but not communicated to all relevant parties. To illustrate these issues, we focused on three well-characterized monogenic, metabolic disorders included in newborn screens: classic galactosemia, caused by GALT variants; phenylketonuria, caused by PAH variants; and medium-chain acyl-CoA dehydrogenase (MCAD) deficiency, caused by ACADM variants. In 10 years of clinical molecular testing, we have observed 134 unique GALT variants, 46 of which were variants of uncertain significance (VUS). In PAH, we observed 132 variants, including 17 VUS, and for ACADM, we observed 64 unique variants, of which 33 were uncertain. After this review, 17 VUS (37%; 7 in ACADM, 9 in GALT, and 1 in PAH) were reclassified from uncertain (6 to benign or likely benign and 11 to pathogenic or likely pathogenic). We identified common types of missing information that would have helped make a definitive classification and categorized this information by ease and cost to obtain.Genet Med 19 1, 77-82.

Quick, sensitive and specific detection and evaluation of quantification of minor variants by high-throughput sequencing.

PubMed

Leung, Ross Ka-Kit; Dong, Zhi Qiang; Sa, Fei; Chong, Cheong Meng; Lei, Si Wan; Tsui, Stephen Kwok-Wing; Lee, Simon Ming-Yuen

2014-02-01

Minor variants have significant implications in quasispecies evolution, early cancer detection and non-invasive fetal genotyping but their accurate detection by next-generation sequencing (NGS) is hampered by sequencing errors. We generated sequencing data from mixtures at predetermined ratios in order to provide insight into sequencing errors and variations that can arise for which simulation cannot be performed. The information also enables better parameterization in depth of coverage, read quality and heterogeneity, library preparation techniques, technical repeatability for mathematical modeling, theory development and simulation experimental design. We devised minor variant authentication rules that achieved 100% accuracy in both testing and validation experiments. The rules are free from tedious inspection of alignment accuracy, sequencing read quality or errors introduced by homopolymers. The authentication processes only require minor variants to: (1) have minimum depth of coverage larger than 30; (2) be reported by (a) four or more variant callers, or (b) DiBayes or LoFreq, plus SNVer (or BWA when no results are returned by SNVer), and with the interassay coefficient of variation (CV) no larger than 0.1. Quantification accuracy undermined by sequencing errors could neither be overcome by ultra-deep sequencing, nor recruiting more variant callers to reach a consensus, such that consistent underestimation and overestimation (i.e. low CV) were observed. To accommodate stochastic error and adjust the observed ratio within a specified accuracy, we presented a proof of concept for the use of a double calibration curve for quantification, which provides an important reference towards potential industrial-scale fabrication of calibrants for NGS.

Next generation sequencing to identify novel genetic variants causative of autosomal dominant familial hypercholesterolemia associated with increased risk of coronary heart disease.

PubMed

Al-Allaf, Faisal A; Athar, Mohammad; Abduljaleel, Zainularifeen; Taher, Mohiuddin M; Khan, Wajahatullah; Ba-Hammam, Faisal A; Abalkhail, Hala; Alashwal, Abdullah

2015-07-01

Familial hypercholesterolemia (FH) is an autosomal dominant inherited disease characterized by elevated plasma low-density lipoprotein cholesterol (LDL-C). It is an autosomal dominant disease, caused by variants in Ldlr, ApoB or Pcsk9, which results in high levels of LDL-cholesterol (LDL-C) leading to early coronary heart disease. Sequencing whole genome for screening variants for FH are not suitable due to high cost. Hence, in this study we performed targeted customized sequencing of FH 12 genes (Ldlr, ApoB, Pcsk9, Abca1, Apoa2, Apoc3, Apon2, Arh, Ldlrap1, Apoc2, ApoE, and Lpl) that have been implicated in the homozygous phenotype of a proband pedigree to identify candidate variants by NGS Ion torrent PGM. Only three genes (Ldlr, ApoB, and Pcsk9) were found to be highly associated with FH based on the variant rate. The results showed that seven deleterious variants in Ldlr, ApoB, and Pcsk9 genes were pathological and were clinically significant based on predictions identified by SIFT and PolyPhen. Targeted customized sequencing is an efficient technique for screening variants among targeted FH genes. Final validation of seven deleterious variants conducted by capillary resulted to only one novel variant in Ldlr gene that was found in exon 14 (c.2026delG, p. Gly676fs). The variant found in Ldlr gene was a novel heterozygous variant derived from a male in the proband. Copyright © 2015 Elsevier B.V. All rights reserved.

Functional Assessment of Disease-Associated Regulatory Variants In Vivo Using a Versatile Dual Colour Transgenesis Strategy in Zebrafish

PubMed Central

Bhatia, Shipra; Gordon, Christopher T.; Foster, Robert G.; Melin, Lucie; Abadie, Véronique; Baujat, Geneviève; Vazquez, Marie-Paule; Amiel, Jeanne; Lyonnet, Stanislas; van Heyningen, Veronica; Kleinjan, Dirk A.

2015-01-01

Disruption of gene regulation by sequence variation in non-coding regions of the genome is now recognised as a significant cause of human disease and disease susceptibility. Sequence variants in cis-regulatory elements (CREs), the primary determinants of spatio-temporal gene regulation, can alter transcription factor binding sites. While technological advances have led to easy identification of disease-associated CRE variants, robust methods for discerning functional CRE variants from background variation are lacking. Here we describe an efficient dual-colour reporter transgenesis approach in zebrafish, simultaneously allowing detailed in vivo comparison of spatio-temporal differences in regulatory activity between putative CRE variants and assessment of altered transcription factor binding potential of the variant. We validate the method on known disease-associated elements regulating SHH, PAX6 and IRF6 and subsequently characterise novel, ultra-long-range SOX9 enhancers implicated in the craniofacial abnormality Pierre Robin Sequence. The method provides a highly cost-effective, fast and robust approach for simultaneously unravelling in a single assay whether, where and when in embryonic development a disease-associated CRE-variant is affecting its regulatory function. PMID:26030420

Whole exome sequencing identifies genetic variants in inherited thrombocytopenia with secondary qualitative function defects.

PubMed

Johnson, Ben; Lowe, Gillian C; Futterer, Jane; Lordkipanidzé, Marie; MacDonald, David; Simpson, Michael A; Sanchez-Guiú, Isabel; Drake, Sian; Bem, Danai; Leo, Vincenzo; Fletcher, Sarah J; Dawood, Ban; Rivera, José; Allsup, David; Biss, Tina; Bolton-Maggs, Paula Hb; Collins, Peter; Curry, Nicola; Grimley, Charlotte; James, Beki; Makris, Mike; Motwani, Jayashree; Pavord, Sue; Talks, Katherine; Thachil, Jecko; Wilde, Jonathan; Williams, Mike; Harrison, Paul; Gissen, Paul; Mundell, Stuart; Mumford, Andrew; Daly, Martina E; Watson, Steve P; Morgan, Neil V

2016-10-01

Inherited thrombocytopenias are a heterogeneous group of disorders characterized by abnormally low platelet counts which can be associated with abnormal bleeding. Next-generation sequencing has previously been employed in these disorders for the confirmation of suspected genetic abnormalities, and more recently in the discovery of novel disease-causing genes. However its full potential has not yet been exploited. Over the past 6 years we have sequenced the exomes from 55 patients, including 37 index cases and 18 additional family members, all of whom were recruited to the UK Genotyping and Phenotyping of Platelets study. All patients had inherited or sustained thrombocytopenia of unknown etiology with platelet counts varying from 11×10 9 /L to 186×10 9 /L. Of the 51 patients phenotypically tested, 37 (73%), had an additional secondary qualitative platelet defect. Using whole exome sequencing analysis we have identified "pathogenic" or "likely pathogenic" variants in 46% (17/37) of our index patients with thrombocytopenia. In addition, we report variants of uncertain significance in 12 index cases, including novel candidate genetic variants in previously unreported genes in four index cases. These results demonstrate that whole exome sequencing is an efficient method for elucidating potential pathogenic genetic variants in inherited thrombocytopenia. Whole exome sequencing also has the added benefit of discovering potentially pathogenic genetic variants for further study in novel genes not previously implicated in inherited thrombocytopenia. Copyright© Ferrata Storti Foundation.

Reanalysis of BRCA1/2 negative high risk ovarian cancer patients reveals novel germline risk loci and insights into missing heritability

PubMed Central

Dyson, Gregory; Levin, Nancy K.; Chaudhry, Sophia; Rosati, Rita; Kalpage, Hasini; Simon, Michael S.; Tainsky, Michael A.

2017-01-01

While up to 25% of ovarian cancer (OVCA) cases are thought to be due to inherited factors, the majority of genetic risk remains unexplained. To address this gap, we sought to identify previously undescribed OVCA risk variants through the whole exome sequencing (WES) and candidate gene analysis of 48 women with ovarian cancer and selected for high risk of genetic inheritance, yet negative for any known pathogenic variants in either BRCA1 or BRCA2. In silico SNP analysis was employed to identify suspect variants followed by validation using Sanger DNA sequencing. We identified five pathogenic variants in our sample, four of which are in two genes featured on current multi-gene panels; (RAD51D, ATM). In addition, we found a pathogenic FANCM variant (R1931*) which has been recently implicated in familial breast cancer risk. Numerous rare and predicted to be damaging variants of unknown significance were detected in genes on current commercial testing panels, most prominently in ATM (n = 6) and PALB2 (n = 5). The BRCA2 variant p.K3326*, resulting in a 93 amino acid truncation, was overrepresented in our sample (odds ratio = 4.95, p = 0.01) and coexisted in the germline of these women with other deleterious variants, suggesting a possible role as a modifier of genetic penetrance. Furthermore, we detected loss of function variants in non-panel genes involved in OVCA relevant pathways; DNA repair and cell cycle control, including CHEK1, TP53I3, REC8, HMMR, RAD52, RAD1, POLK, POLQ, and MCM4. In summary, our study implicates novel risk loci as well as highlights the clinical utility for retesting BRCA1/2 negative OVCA patients by genomic sequencing and analysis of genes in relevant pathways. PMID:28591191

Clinical Interpretation and Implications of Whole-Genome Sequencing

PubMed Central

Dewey, Frederick E.; Grove, Megan E.; Pan, Cuiping; Goldstein, Benjamin A.; Bernstein, Jonathan A.; Chaib, Hassan; Merker, Jason D.; Goldfeder, Rachel L.; Enns, Gregory M.; David, Sean P.; Pakdaman, Neda; Ormond, Kelly E.; Caleshu, Colleen; Kingham, Kerry; Klein, Teri E.; Whirl-Carrillo, Michelle; Sakamoto, Kenneth; Wheeler, Matthew T.; Butte, Atul J.; Ford, James M.; Boxer, Linda; Ioannidis, John P. A.; Yeung, Alan C.; Altman, Russ B.; Assimes, Themistocles L.; Snyder, Michael; Ashley, Euan A.; Quertermous, Thomas

2014-01-01

IMPORTANCE Whole-genome sequencing (WGS) is increasingly applied in clinical medicine and is expected to uncover clinically significant findings regardless of sequencing indication. OBJECTIVES To examine coverage and concordance of clinically relevant genetic variation provided by WGS technologies; to quantitate inherited disease risk and pharmacogenomic findings in WGS data and resources required for their discovery and interpretation; and to evaluate clinical action prompted by WGS findings. DESIGN, SETTING, AND PARTICIPANTS An exploratory study of 12 adult participants recruited at Stanford University Medical Center who underwent WGS between November 2011 and March 2012. A multidisciplinary team reviewed all potentially reportable genetic findings. Five physicians proposed initial clinical follow-up based on the genetic findings. MAIN OUTCOMES AND MEASURES Genome coverage and sequencing platform concordance in different categories of genetic disease risk, person-hours spent curating candidate disease-risk variants, interpretation agreement between trained curators and disease genetics databases, burden of inherited disease risk and pharmacogenomic findings, and burden and interrater agreement of proposed clinical follow-up. RESULTS Depending on sequencing platform, 10% to 19% of inherited disease genes were not covered to accepted standards for single nucleotide variant discovery. Genotype concordance was high for previously described single nucleotide genetic variants (99%-100%) but low for small insertion/deletion variants (53%-59%). Curation of 90 to 127 genetic variants in each participant required a median of 54 minutes (range, 5-223 minutes) per genetic variant, resulted in moderate classification agreement between professionals (Gross κ, 0.52; 95%CI, 0.40-0.64), and reclassified 69%of genetic variants cataloged as disease causing in mutation databases to variants of uncertain or lesser significance. Two to 6 personal disease-risk findings were discovered in each participant, including 1 frameshift deletion in the BRCA1 gene implicated in hereditary breast and ovarian cancer. Physician review of sequencing findings prompted consideration of a median of 1 to 3 initial diagnostic tests and referrals per participant, with fair interrater agreement about the suitability of WGS findings for clinical follow-up (Fleiss κ, 0.24; P < 001). CONCLUSIONS AND RELEVANCE In this exploratory study of 12 volunteer adults, the use of WGS was associated with incomplete coverage of inherited disease genes, low reproducibility of detection of genetic variation with the highest potential clinical effects, and uncertainty about clinically reportable findings. In certain cases, WGS will identify clinically actionable genetic variants warranting early medical intervention. These issues should be considered when determining the role of WGS in clinical medicine. PMID:24618965

A global reference for human genetic variation

PubMed Central

2016-01-01

The 1000 Genomes Project set out to provide a comprehensive description of common human genetic variation by applying whole-genome sequencing to a diverse set of individuals from multiple populations. Here we report completion of the project, having reconstructed the genomes of 2,504 individuals from 26 populations using a combination of low-coverage whole-genome sequencing, deep exome sequencing, and dense microarray genotyping. We characterized a broad spectrum of genetic variation, in total over 88 million variants (84.7 million single nucleotide polymorphisms (SNPs), 3.6 million short insertions/deletions (indels), and 60,000 structural variants), all phased onto high-quality haplotypes. This resource includes >99% of SNP variants with a frequency of >1% for a variety of ancestries. We describe the distribution of genetic variation across the global sample, and discuss the implications for common disease studies. PMID:26432245

Novel rare variations of the oxytocin receptor (OXTR) gene in autism spectrum disorder individuals.

PubMed

Liu, Xiaoxi; Kawashima, Minae; Miyagawa, Taku; Otowa, Takeshi; Latt, Khun Zaw; Thiri, Myo; Nishida, Hisami; Sugiyama, Toshiro; Tsurusaki, Yoshinori; Matsumoto, Naomichi; Mabuchi, Akihiko; Tokunaga, Katsushi; Sasaki, Tsukasa

2015-01-01

The oxytocin receptor (OXTR) gene has been implicated as a risk gene for autism spectrum disorder (ASD)-a neurodevelopmental disorder with essential features of impairments in social communication and reciprocal interaction. The genetic associations between common variations in OXTR and ASD have been reported in multiple ethnic populations. However, little is known about the distribution of rare variations within OXTR in ASD patients. In this study, we resequenced the full length of OXTR in 105 ASD individuals using an approach that combined the power of next-generation sequencing technology, long-range PCR and DNA pooling. We demonstrated that rare variants with minor allele frequency as low as 0.05% could be reliably detected by our method. We identified 28 novel variants including potential functional variants in the intron region and one rare missense variant (R150S). We subsequently performed Sanger sequencing and validated five novel variants located in previously suggested candidate regions in ASD individuals. Further sequencing of 312 healthy subjects showed that the burden of rare variants is significantly higher in ASDs compared with healthy individuals. Our results support that the rare variation in OXTR gene might be involved in ASD.

Utility of NIST Whole-Genome Reference Materials for the Technical Validation of a Multigene Next-Generation Sequencing Test.

PubMed

Shum, Bennett O V; Henner, Ilya; Belluoccio, Daniele; Hinchcliffe, Marcus J

2017-07-01

The sensitivity and specificity of next-generation sequencing laboratory developed tests (LDTs) are typically determined by an analyte-specific approach. Analyte-specific validations use disease-specific controls to assess an LDT's ability to detect known pathogenic variants. Alternatively, a methods-based approach can be used for LDT technical validations. Methods-focused validations do not use disease-specific controls but use benchmark reference DNA that contains known variants (benign, variants of unknown significance, and pathogenic) to assess variant calling accuracy of a next-generation sequencing workflow. Recently, four whole-genome reference materials (RMs) from the National Institute of Standards and Technology (NIST) were released to standardize methods-based validations of next-generation sequencing panels across laboratories. We provide a practical method for using NIST RMs to validate multigene panels. We analyzed the utility of RMs in validating a novel newborn screening test that targets 70 genes, called NEO1. Despite the NIST RM variant truth set originating from multiple sequencing platforms, replicates, and library types, we discovered a 5.2% false-negative variant detection rate in the RM truth set genes that were assessed in our validation. We developed a strategy using complementary non-RM controls to demonstrate 99.6% sensitivity of the NEO1 test in detecting variants. Our findings have implications for laboratories or proficiency testing organizations using whole-genome NIST RMs for testing. Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

No evidence that protein truncating variants in BRIP1 are associated with breast cancer risk: implications for gene panel testing

PubMed Central

Easton, Douglas F; Lesueur, Fabienne; Decker, Brennan; Michailidou, Kyriaki; Li, Jun; Allen, Jamie; Luccarini, Craig; Pooley, Karen A; Shah, Mitul; Bolla, Manjeet K; Wang, Qin; Dennis, Joe; Ahmad, Jamil; Thompson, Ella R; Damiola, Francesca; Pertesi, Maroulio; Voegele, Catherine; Mebirouk, Noura; Robinot, Nivonirina; Durand, Geoffroy; Forey, Nathalie; Luben, Robert N; Ahmed, Shahana; Aittomäki, Kristiina; Anton-Culver, Hoda; Arndt, Volker; Baynes, Caroline; Beckman, Matthias W; Benitez, Javier; Van Den Berg, David; Blot, William J; Bogdanova, Natalia V; Bojesen, Stig E; Brenner, Hermann; Chang-Claude, Jenny; Chia, Kee Seng; Choi, Ji-Yeob; Conroy, Don M; Cox, Angela; Cross, Simon S; Czene, Kamila; Darabi, Hatef; Devilee, Peter; Eriksson, Mikael; Fasching, Peter A; Figueroa, Jonine; Flyger, Henrik; Fostira, Florentia; García-Closas, Montserrat; Giles, Graham G; Glendon, Gord; González-Neira, Anna; Guénel, Pascal; Haiman, Christopher A; Hall, Per; Hart, Steven N; Hartman, Mikael; Hooning, Maartje J; Hsiung, Chia-Ni; Ito, Hidemi; Jakubowska, Anna; James, Paul A; John, Esther M; Johnson, Nichola; Jones, Michael; Kabisch, Maria; Kang, Daehee; Kosma, Veli-Matti; Kristensen, Vessela; Lambrechts, Diether; Li, Na; Lindblom, Annika; Long, Jirong; Lophatananon, Artitaya; Lubinski, Jan; Mannermaa, Arto; Manoukian, Siranoush; Margolin, Sara; Matsuo, Keitaro; Meindl, Alfons; Mitchell, Gillian; Muir, Kenneth; Nevelsteen, Ines; van den Ouweland, Ans; Peterlongo, Paolo; Phuah, Sze Yee; Pylkäs, Katri; Rowley, Simone M; Sangrajrang, Suleeporn; Schmutzler, Rita K; Shen, Chen-Yang; Shu, Xiao-Ou; Southey, Melissa C; Surowy, Harald; Swerdlow, Anthony; Teo, Soo H; Tollenaar, Rob A E M; Tomlinson, Ian; Torres, Diana; Truong, Thérèse; Vachon, Celine; Verhoef, Senno; Wong-Brown, Michelle; Zheng, Wei; Zheng, Ying; Nevanlinna, Heli; Scott, Rodney J; Andrulis, Irene L; Wu, Anna H; Hopper, John L; Couch, Fergus J; Winqvist, Robert; Burwinkel, Barbara; Sawyer, Elinor J; Schmidt, Marjanka K; Rudolph, Anja; Dörk, Thilo; Brauch, Hiltrud; Hamann, Ute; Neuhausen, Susan L; Milne, Roger L; Fletcher, Olivia; Pharoah, Paul D P; Campbell, Ian G; Dunning, Alison M; Le Calvez-Kelm, Florence; Goldgar, David E; Tavtigian, Sean V; Chenevix-Trench, Georgia

2016-01-01

Background BRCA1 interacting protein C-terminal helicase 1 (BRIP1) is one of the Fanconi Anaemia Complementation (FANC) group family of DNA repair proteins. Biallelic mutations in BRIP1 are responsible for FANC group J, and previous studies have also suggested that rare protein truncating variants in BRIP1 are associated with an increased risk of breast cancer. These studies have led to inclusion of BRIP1 on targeted sequencing panels for breast cancer risk prediction. Methods We evaluated a truncating variant, p.Arg798Ter (rs137852986), and 10 missense variants of BRIP1, in 48 144 cases and 43 607 controls of European origin, drawn from 41 studies participating in the Breast Cancer Association Consortium (BCAC). Additionally, we sequenced the coding regions of BRIP1 in 13 213 cases and 5242 controls from the UK, 1313 cases and 1123 controls from three population-based studies as part of the Breast Cancer Family Registry, and 1853 familial cases and 2001 controls from Australia. Results The rare truncating allele of rs137852986 was observed in 23 cases and 18 controls in Europeans in BCAC (OR 1.09, 95% CI 0.58 to 2.03, p=0.79). Truncating variants were found in the sequencing studies in 34 cases (0.21%) and 19 controls (0.23%) (combined OR 0.90, 95% CI 0.48 to 1.70, p=0.75). Conclusions These results suggest that truncating variants in BRIP1, and in particular p.Arg798Ter, are not associated with a substantial increase in breast cancer risk. Such observations have important implications for the reporting of results from breast cancer screening panels. PMID:26921362

Targeted exome sequencing of suspected mitochondrial disorders

PubMed Central

Lieber, Daniel S.; Calvo, Sarah E.; Shanahan, Kristy; Slate, Nancy G.; Liu, Shangtao; Hershman, Steven G.; Gold, Nina B.; Chapman, Brad A.; Thorburn, David R.; Berry, Gerard T.; Schmahmann, Jeremy D.; Borowsky, Mark L.; Mueller, David M.; Sims, Katherine B.

2013-01-01

Objective: To evaluate the utility of targeted exome sequencing for the molecular diagnosis of mitochondrial disorders, which exhibit marked phenotypic and genetic heterogeneity. Methods: We considered a diverse set of 102 patients with suspected mitochondrial disorders based on clinical, biochemical, and/or molecular findings, and whose disease ranged from mild to severe, with varying age at onset. We sequenced the mitochondrial genome (mtDNA) and the exons of 1,598 nuclear-encoded genes implicated in mitochondrial biology, mitochondrial disease, or monogenic disorders with phenotypic overlap. We prioritized variants likely to underlie disease and established molecular diagnoses in accordance with current clinical genetic guidelines. Results: Targeted exome sequencing yielded molecular diagnoses in established disease loci in 22% of cases, including 17 of 18 (94%) with prior molecular diagnoses and 5 of 84 (6%) without. The 5 new diagnoses implicated 2 genes associated with canonical mitochondrial disorders (NDUFV1, POLG2), and 3 genes known to underlie other neurologic disorders (DPYD, KARS, WFS1), underscoring the phenotypic and biochemical overlap with other inborn errors. We prioritized variants in an additional 26 patients, including recessive, X-linked, and mtDNA variants that were enriched 2-fold over background and await further support of pathogenicity. In one case, we modeled patient mutations in yeast to provide evidence that recessive mutations in ATP5A1 can underlie combined respiratory chain deficiency. Conclusion: The results demonstrate that targeted exome sequencing is an effective alternative to the sequential testing of mtDNA and individual nuclear genes as part of the investigation of mitochondrial disease. Our study underscores the ongoing challenge of variant interpretation in the clinical setting. PMID:23596069

Contributions of Function-Altering Variants in Genes Implicated in Pubertal Timing and Body Mass for Self-Limited Delayed Puberty.

PubMed

Howard, Sasha R; Guasti, Leonardo; Poliandri, Ariel; David, Alessia; Cabrera, Claudia P; Barnes, Michael R; Wehkalampi, Karoliina; O'Rahilly, Stephen; Aiken, Catherine E; Coll, Anthony P; Ma, Marcella; Rimmington, Debra; Yeo, Giles S H; Dunkel, Leo

2018-02-01

Self-limited delayed puberty (DP) is often associated with a delay in physical maturation, but although highly heritable the causal genetic factors remain elusive. Genome-wide association studies of the timing of puberty have identified multiple loci for age at menarche in females and voice break in males, particularly in pathways controlling energy balance. We sought to assess the contribution of rare variants in such genes to the phenotype of familial DP. We performed whole-exome sequencing in 67 pedigrees (125 individuals with DP and 35 unaffected controls) from our unique cohort of familial self-limited DP. Using a whole-exome sequencing filtering pipeline one candidate gene [fat mass and obesity-associated gene (FTO)] was identified. In silico, in vitro, and mouse model studies were performed to investigate the pathogenicity of FTO variants and timing of puberty in FTO+/- mice. We identified potentially pathogenic, rare variants in genes in linkage disequilibrium with genome-wide association studies of age at menarche loci in 283 genes. Of these, five genes were implicated in the control of body mass. After filtering for segregation with trait, one candidate, FTO, was retained. Two FTO variants, found in 14 affected individuals from three families, were also associated with leanness in these patients with DP. One variant (p.Leu44Val) demonstrated altered demethylation activity of the mutant protein in vitro. Fto+/- mice displayed a significantly delayed timing of pubertal onset (P < 0.05). Mutations in genes implicated in body mass and timing of puberty in the general population may contribute to the pathogenesis of self-limited DP. Copyright © 2017 Endocrine Society

Rare missense variants in CHRNB3 and CHRNA3 are associated with risk of alcohol and cocaine dependence.

PubMed

Haller, Gabe; Kapoor, Manav; Budde, John; Xuei, Xiaoling; Edenberg, Howard; Nurnberger, John; Kramer, John; Brooks, Andy; Tischfield, Jay; Almasy, Laura; Agrawal, Arpana; Bucholz, Kathleen; Rice, John; Saccone, Nancy; Bierut, Laura; Goate, Alison

2014-02-01

Previous findings have demonstrated that variants in nicotinic receptor genes are associated with nicotine, alcohol and cocaine dependence. Because of the substantial comorbidity, it has often been unclear whether a variant is associated with multiple substances or whether the association is actually with a single substance. To investigate the possible contribution of rare variants to the development of substance dependencies other than nicotine dependence, specifically alcohol and cocaine dependence, we undertook pooled sequencing of the coding regions and flanking sequence of CHRNA5, CHRNA3, CHRNB4, CHRNA6 and CHRNB3 in 287 African American and 1028 European American individuals from the Collaborative Study of the Genetics of Alcoholism (COGA). All members of families for whom any individual was sequenced (2504 African Americans and 7318 European Americans) were then genotyped for all variants identified by sequencing. For each gene, we then tested for association using FamSKAT. For European Americans, we find increased DSM-IV cocaine dependence symptoms (FamSKAT P = 2 × 10(-4)) and increased DSM-IV alcohol dependence symptoms (FamSKAT P = 5 × 10(-4)) among carriers of missense variants in CHRNB3. Additionally, one variant (rs149775276; H329Y) shows association with both cocaine dependence symptoms (P = 7.4 × 10(-5), β = 2.04) and alcohol dependence symptoms (P = 2.6 × 10(-4), β = 2.04). For African Americans, we find decreased cocaine dependence symptoms among carriers of missense variants in CHRNA3 (FamSKAT P = 0.005). Replication in an independent sample supports the role of rare variants in CHRNB3 and alcohol dependence (P = 0.006). These are the first results to implicate rare variants in CHRNB3 or CHRNA3 in risk for alcohol dependence or cocaine dependence.

Identification of Candidate Gene Variants in Korean MODY Families by Whole-Exome Sequencing.

PubMed

Shim, Ye Jee; Kim, Jung Eun; Hwang, Su-Kyeong; Choi, Bong Seok; Choi, Byung Ho; Cho, Eun-Mi; Jang, Kyoung Mi; Ko, Cheol Woo

2015-01-01

To date, 13 genes causing maturity-onset diabetes of the young (MODY) have been identified. However, there is a big discrepancy in the genetic locus between Asian and Caucasian patients with MODY. Thus, we conducted whole-exome sequencing in Korean MODY families to identify causative gene variants. Six MODY probands and their family members were included. Variants in the dbSNP135 and TIARA databases for Koreans and the variants with minor allele frequencies >0.5% of the 1000 Genomes database were excluded. We selected only the functional variants (gain of stop codon, frameshifts and nonsynonymous single-nucleotide variants) and conducted a case-control comparison in the family members. The selected variants were scanned for the previously introduced gene set implicated in glucose metabolism. Three variants c.620C>T:p.Thr207Ile in PTPRD, c.559C>G:p.Gln187Glu in SYT9, and c.1526T>G:p.Val509Gly in WFS1 were respectively identified in 3 families. We could not find any disease-causative alleles of known MODY 1-13 genes. Based on the predictive program, Thr207Ile in PTPRD was considered pathogenic. Whole-exome sequencing is a valuable method for the genetic diagnosis of MODY. Further evaluation is necessary about the role of PTPRD, SYT9 and WFS1 in normal insulin release from pancreatic beta cells. © 2015 S. Karger AG, Basel.

Functional Assays to Screen and Dissect Genomic Hits: Doubling Down on the National Investment in Genomic Research.

PubMed

Musunuru, Kiran; Bernstein, Daniel; Cole, F Sessions; Khokha, Mustafa K; Lee, Frank S; Lin, Shin; McDonald, Thomas V; Moskowitz, Ivan P; Quertermous, Thomas; Sankaran, Vijay G; Schwartz, David A; Silverman, Edwin K; Zhou, Xiaobo; Hasan, Ahmed A K; Luo, Xiao-Zhong James

2018-04-01

The National Institutes of Health have made substantial investments in genomic studies and technologies to identify DNA sequence variants associated with human disease phenotypes. The National Heart, Lung, and Blood Institute has been at the forefront of these commitments to ascertain genetic variation associated with heart, lung, blood, and sleep diseases and related clinical traits. Genome-wide association studies, exome- and genome-sequencing studies, and exome-genotyping studies of the National Heart, Lung, and Blood Institute-funded epidemiological and clinical case-control studies are identifying large numbers of genetic variants associated with heart, lung, blood, and sleep phenotypes. However, investigators face challenges in identification of genomic variants that are functionally disruptive among the myriad of computationally implicated variants. Studies to define mechanisms of genetic disruption encoded by computationally identified genomic variants require reproducible, adaptable, and inexpensive methods to screen candidate variant and gene function. High-throughput strategies will permit a tiered variant discovery and genetic mechanism approach that begins with rapid functional screening of a large number of computationally implicated variants and genes for discovery of those that merit mechanistic investigation. As such, improved variant-to-gene and gene-to-function screens-and adequate support for such studies-are critical to accelerating the translation of genomic findings. In this White Paper, we outline the variety of novel technologies, assays, and model systems that are making such screens faster, cheaper, and more accurate, referencing published work and ongoing work supported by the National Heart, Lung, and Blood Institute's R21/R33 Functional Assays to Screen Genomic Hits program. We discuss priorities that can accelerate the impressive but incomplete progress represented by big data genomic research. © 2018 American Heart Association, Inc.

Misregulation effect of a novel allelic variant in the Z promoter region found in cis with the CYP21A2 p.P482S mutation: implications for 21-hydroxylase deficiency.

PubMed

Fernández, Cecilia S; Bruque, Carlos D; Taboas, Melisa; Buzzalino, Noemí D; Espeche, Lucia D; Pasqualini, Titania; Charreau, Eduardo H; Alba, Liliana G; Ghiringhelli, Pablo D; Dain, Liliana

2015-09-01

The aim of the current study was to search for the presence of genetic variants in the CYP21A2 Z promoter regulatory region in patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Screening of the 10 most frequent pseudogene-derived mutations was followed by direct sequencing of the entire coding sequence, the proximal promoter, and a distal regulatory region in DNA samples from patients with at least one non-determined allele. We report three non-classical patients that presented a novel genetic variant-g.15626A>G-within the Z promoter regulatory region. In all the patients, the novel variant was found in cis with the mild, less frequent, p.P482S mutation located in the exon 10 of the CYP21A2 gene. The putative pathogenic implication of the novel variant was assessed by in silico analyses and in vitro assays. Topological analyses showed differences in the curvature and bendability of the DNA region bearing the novel variant. By performing functional studies, a significantly decreased activity of a reporter gene placed downstream from the regulatory region was found by the G transition. Our results may suggest that the activity of an allele bearing the p.P482S mutation may be influenced by the misregulated CYP21A2 transcriptional activity exerted by the Z promoter A>G variation.

αIIbβ3 variants defined by next-generation sequencing: Predicting variants likely to cause Glanzmann thrombasthenia

PubMed Central

Buitrago, Lorena; Rendon, Augusto; Liang, Yupu; Simeoni, Ilenia; Negri, Ana; Filizola, Marta; Ouwehand, Willem H.; Coller, Barry S.; Alessi, Marie-Christine; Ballmaier, Matthias; Bariana, Tadbir; Bellissimo, Daniel; Bertoli, Marta; Bray, Paul; Bury, Loredana; Carrell, Robin; Cattaneo, Marco; Collins, Peter; French, Deborah; Favier, Remi; Freson, Kathleen; Furie, Bruce; Germeshausen, Manuela; Ghevaert, Cedric; Gomez, Keith; Goodeve, Anne; Gresele, Paolo; Guerrero, Jose; Hampshire, Dan J.; Hadinnapola, Charaka; Heemskerk, Johan; Henskens, Yvonne; Hill, Marian; Hogg, Nancy; Johnsen, Jill; Kahr, Walter; Kerr, Ron; Kunishima, Shinji; Laffan, Michael; Natwani, Amit; Neerman-Arbez, Marguerite; Nurden, Paquita; Nurden, Alan; Ormiston, Mark; Othman, Maha; Ouwehand, Willem; Perry, David; Vilk, Shoshana Ravel; Reitsma, Pieter; Rondina, Matthew; Simeoni, Ilenia; Smethurst, Peter; Stephens, Jonathan; Stevenson, William; Szkotak, Artur; Turro, Ernest; Van Geet, Christel; Vries, Minka; Ward, June; Waye, John; Westbury, Sarah; Whiteheart, Sidney; Wilcox, David; Zhang, Bi

2015-01-01

Next-generation sequencing is transforming our understanding of human genetic variation but assessing the functional impact of novel variants presents challenges. We analyzed missense variants in the integrin αIIbβ3 receptor subunit genes ITGA2B and ITGB3 identified by whole-exome or -genome sequencing in the ThromboGenomics project, comprising ∼32,000 alleles from 16,108 individuals. We analyzed the results in comparison with 111 missense variants in these genes previously reported as being associated with Glanzmann thrombasthenia (GT), 20 associated with alloimmune thrombocytopenia, and 5 associated with aniso/macrothrombocytopenia. We identified 114 novel missense variants in ITGA2B (affecting ∼11% of the amino acids) and 68 novel missense variants in ITGB3 (affecting ∼9% of the amino acids). Of the variants, 96% had minor allele frequencies (MAF) < 0.1%, indicating their rarity. Based on sequence conservation, MAF, and location on a complete model of αIIbβ3, we selected three novel variants that affect amino acids previously associated with GT for expression in HEK293 cells. αIIb P176H and β3 C547G severely reduced αIIbβ3 expression, whereas αIIb P943A partially reduced αIIbβ3 expression and had no effect on fibrinogen binding. We used receiver operating characteristic curves of combined annotation-dependent depletion, Polyphen 2-HDIV, and sorting intolerant from tolerant to estimate the percentage of novel variants likely to be deleterious. At optimal cut-off values, which had 69–98% sensitivity in detecting GT mutations, between 27% and 71% of the novel αIIb or β3 missense variants were predicted to be deleterious. Our data have implications for understanding the evolutionary pressure on αIIbβ3 and highlight the challenges in predicting the clinical significance of novel missense variants. PMID:25827233

Burden of rare variants in ALS genes influences survival in familial and sporadic ALS.

PubMed

Pang, Shirley Yin-Yu; Hsu, Jacob Shujui; Teo, Kay-Cheong; Li, Yan; Kung, Michelle H W; Cheah, Kathryn S E; Chan, Danny; Cheung, Kenneth M C; Li, Miaoxin; Sham, Pak-Chung; Ho, Shu-Leong

2017-10-01

Genetic variants are implicated in the development of amyotrophic lateral sclerosis (ALS), but it is unclear whether the burden of rare variants in ALS genes has an effect on survival. We performed whole genome sequencing on 8 familial ALS (FALS) patients with superoxide dismutase 1 (SOD1) mutation and whole exome sequencing on 46 sporadic ALS (SALS) patients living in Hong Kong and found that 67% had at least 1 rare variant in the exons of 40 ALS genes; 22% had 2 or more. Patients with 2 or more rare variants had lower probability of survival than patients with 0 or 1 variant (p = 0.001). After adjusting for other factors, each additional rare variant increased the risk of respiratory failure or death by 60% (p = 0.0098). The presence of the rare variant was associated with the risk of ALS (Odds ratio 1.91, 95% confidence interval 1.03-3.61, p = 0.03), and ALS patients had higher rare variant burden than controls (MB, p = 0.004). Our findings support an oligogenic basis with the burden of rare variants affecting the development and survival of ALS. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Next-generation DNA sequencing identifies novel gene variants and pathways involved in specific language impairment.

PubMed

Chen, Xiaowei Sylvia; Reader, Rose H; Hoischen, Alexander; Veltman, Joris A; Simpson, Nuala H; Francks, Clyde; Newbury, Dianne F; Fisher, Simon E

2017-04-25

A significant proportion of children have unexplained problems acquiring proficient linguistic skills despite adequate intelligence and opportunity. Developmental language disorders are highly heritable with substantial societal impact. Molecular studies have begun to identify candidate loci, but much of the underlying genetic architecture remains undetermined. We performed whole-exome sequencing of 43 unrelated probands affected by severe specific language impairment, followed by independent validations with Sanger sequencing, and analyses of segregation patterns in parents and siblings, to shed new light on aetiology. By first focusing on a pre-defined set of known candidates from the literature, we identified potentially pathogenic variants in genes already implicated in diverse language-related syndromes, including ERC1, GRIN2A, and SRPX2. Complementary analyses suggested novel putative candidates carrying validated variants which were predicted to have functional effects, such as OXR1, SCN9A and KMT2D. We also searched for potential "multiple-hit" cases; one proband carried a rare AUTS2 variant in combination with a rare inherited haplotype affecting STARD9, while another carried a novel nonsynonymous variant in SEMA6D together with a rare stop-gain in SYNPR. On broadening scope to all rare and novel variants throughout the exomes, we identified biological themes that were enriched for such variants, including microtubule transport and cytoskeletal regulation.

Next-generation DNA sequencing identifies novel gene variants and pathways involved in specific language impairment

PubMed Central

Chen, Xiaowei Sylvia; Reader, Rose H.; Hoischen, Alexander; Veltman, Joris A.; Simpson, Nuala H.; Francks, Clyde; Newbury, Dianne F.; Fisher, Simon E.

2017-01-01

A significant proportion of children have unexplained problems acquiring proficient linguistic skills despite adequate intelligence and opportunity. Developmental language disorders are highly heritable with substantial societal impact. Molecular studies have begun to identify candidate loci, but much of the underlying genetic architecture remains undetermined. We performed whole-exome sequencing of 43 unrelated probands affected by severe specific language impairment, followed by independent validations with Sanger sequencing, and analyses of segregation patterns in parents and siblings, to shed new light on aetiology. By first focusing on a pre-defined set of known candidates from the literature, we identified potentially pathogenic variants in genes already implicated in diverse language-related syndromes, including ERC1, GRIN2A, and SRPX2. Complementary analyses suggested novel putative candidates carrying validated variants which were predicted to have functional effects, such as OXR1, SCN9A and KMT2D. We also searched for potential “multiple-hit” cases; one proband carried a rare AUTS2 variant in combination with a rare inherited haplotype affecting STARD9, while another carried a novel nonsynonymous variant in SEMA6D together with a rare stop-gain in SYNPR. On broadening scope to all rare and novel variants throughout the exomes, we identified biological themes that were enriched for such variants, including microtubule transport and cytoskeletal regulation. PMID:28440294

Experimental Assessment of Splicing Variants Using Expression Minigenes and Comparison with In Silico Predictions

PubMed Central

Sharma, Neeraj; Sosnay, Patrick R.; Ramalho, Anabela S.; Douville, Christopher; Franca, Arianna; Gottschalk, Laura B.; Park, Jeenah; Lee, Melissa; Vecchio-Pagan, Briana; Raraigh, Karen S.; Amaral, Margarida D.; Karchin, Rachel; Cutting, Garry R.

2015-01-01

Assessment of the functional consequences of variants near splice sites is a major challenge in the diagnostic laboratory. To address this issue, we created expression minigenes (EMGs) to determine the RNA and protein products generated by splice site variants (n = 10) implicated in cystic fibrosis (CF). Experimental results were compared with the splicing predictions of eight in silico tools. EMGs containing the full-length Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) coding sequence and flanking intron sequences generated wild-type transcript and fully processed protein in Human Embryonic Kidney (HEK293) and CF bronchial epithelial (CFBE41o-) cells. Quantification of variant induced aberrant mRNA isoforms was concordant using fragment analysis and pyrosequencing. The splicing patterns of c.1585−1G>A and c.2657+5G>A were comparable to those reported in primary cells from individuals bearing these variants. Bioinformatics predictions were consistent with experimental results for 9/10 variants (MES), 8/10 variants (NNSplice), and 7/10 variants (SSAT and Sroogle). Programs that estimate the consequences of mis-splicing predicted 11/16 (HSF and ASSEDA) and 10/16 (Fsplice and SplicePort) experimentally observed mRNA isoforms. EMGs provide a robust experimental approach for clinical interpretation of splice site variants and refinement of in silico tools. PMID:25066652

Rare missense variants in CHRNB3 and CHRNA3 are associated with risk of alcohol and cocaine dependence

PubMed Central

Haller, Gabe; Kapoor, Manav; Budde, John; Xuei, Xiaoling; Edenberg, Howard; Nurnberger, John; Kramer, John; Brooks, Andy; Tischfield, Jay; Almasy, Laura; Agrawal, Arpana; Bucholz, Kathleen; Rice, John; Saccone, Nancy; Bierut, Laura; Goate, Alison

2014-01-01

Previous findings have demonstrated that variants in nicotinic receptor genes are associated with nicotine, alcohol and cocaine dependence. Because of the substantial comorbidity, it has often been unclear whether a variant is associated with multiple substances or whether the association is actually with a single substance. To investigate the possible contribution of rare variants to the development of substance dependencies other than nicotine dependence, specifically alcohol and cocaine dependence, we undertook pooled sequencing of the coding regions and flanking sequence of CHRNA5, CHRNA3, CHRNB4, CHRNA6 and CHRNB3 in 287 African American and 1028 European American individuals from the Collaborative Study of the Genetics of Alcoholism (COGA). All members of families for whom any individual was sequenced (2504 African Americans and 7318 European Americans) were then genotyped for all variants identified by sequencing. For each gene, we then tested for association using FamSKAT. For European Americans, we find increased DSM-IV cocaine dependence symptoms (FamSKAT P = 2 × 10−4) and increased DSM-IV alcohol dependence symptoms (FamSKAT P = 5 × 10−4) among carriers of missense variants in CHRNB3. Additionally, one variant (rs149775276; H329Y) shows association with both cocaine dependence symptoms (P = 7.4 × 10−5, β = 2.04) and alcohol dependence symptoms (P = 2.6 × 10−4, β = 2.04). For African Americans, we find decreased cocaine dependence symptoms among carriers of missense variants in CHRNA3 (FamSKAT P = 0.005). Replication in an independent sample supports the role of rare variants in CHRNB3 and alcohol dependence (P = 0.006). These are the first results to implicate rare variants in CHRNB3 or CHRNA3 in risk for alcohol dependence or cocaine dependence. PMID:24057674

Mitochondrial DNA sequence data reveals association of haplogroup U with psychosis in bipolar disorder.

PubMed

Frye, Mark A; Ryu, Euijung; Nassan, Malik; Jenkins, Gregory D; Andreazza, Ana C; Evans, Jared M; McElroy, Susan L; Oglesbee, Devin; Highsmith, W Edward; Biernacka, Joanna M

2017-01-01

Converging genetic, postmortem gene-expression, cellular, and neuroimaging data implicate mitochondrial dysfunction in bipolar disorder. This study was conducted to investigate whether mitochondrial DNA (mtDNA) haplogroups and single nucleotide variants (SNVs) are associated with sub-phenotypes of bipolar disorder. MtDNA from 224 patients with Bipolar I disorder (BPI) was sequenced, and association of sequence variations with 3 sub-phenotypes (psychosis, rapid cycling, and adolescent illness onset) was evaluated. Gene-level tests were performed to evaluate overall burden of minor alleles for each phenotype. The haplogroup U was associated with a higher risk of psychosis. Secondary analyses of SNVs provided nominal evidence for association of psychosis with variants in the tRNA, ND4 and ND5 genes. The association of psychosis with ND4 (gene that encodes NADH dehydrogenase 4) was further supported by gene-level analysis. Preliminary analysis of mtDNA sequence data suggests a higher risk of psychosis with the U haplogroup and variation in the ND4 gene implicated in electron transport chain energy regulation. Further investigation of the functional consequences of this mtDNA variation is encouraged. Copyright © 2016. Published by Elsevier Ltd.

No evidence that protein truncating variants in BRIP1 are associated with breast cancer risk: implications for gene panel testing.

PubMed

Easton, Douglas F; Lesueur, Fabienne; Decker, Brennan; Michailidou, Kyriaki; Li, Jun; Allen, Jamie; Luccarini, Craig; Pooley, Karen A; Shah, Mitul; Bolla, Manjeet K; Wang, Qin; Dennis, Joe; Ahmad, Jamil; Thompson, Ella R; Damiola, Francesca; Pertesi, Maroulio; Voegele, Catherine; Mebirouk, Noura; Robinot, Nivonirina; Durand, Geoffroy; Forey, Nathalie; Luben, Robert N; Ahmed, Shahana; Aittomäki, Kristiina; Anton-Culver, Hoda; Arndt, Volker; Baynes, Caroline; Beckman, Matthias W; Benitez, Javier; Van Den Berg, David; Blot, William J; Bogdanova, Natalia V; Bojesen, Stig E; Brenner, Hermann; Chang-Claude, Jenny; Chia, Kee Seng; Choi, Ji-Yeob; Conroy, Don M; Cox, Angela; Cross, Simon S; Czene, Kamila; Darabi, Hatef; Devilee, Peter; Eriksson, Mikael; Fasching, Peter A; Figueroa, Jonine; Flyger, Henrik; Fostira, Florentia; García-Closas, Montserrat; Giles, Graham G; Glendon, Gord; González-Neira, Anna; Guénel, Pascal; Haiman, Christopher A; Hall, Per; Hart, Steven N; Hartman, Mikael; Hooning, Maartje J; Hsiung, Chia-Ni; Ito, Hidemi; Jakubowska, Anna; James, Paul A; John, Esther M; Johnson, Nichola; Jones, Michael; Kabisch, Maria; Kang, Daehee; Kosma, Veli-Matti; Kristensen, Vessela; Lambrechts, Diether; Li, Na; Lindblom, Annika; Long, Jirong; Lophatananon, Artitaya; Lubinski, Jan; Mannermaa, Arto; Manoukian, Siranoush; Margolin, Sara; Matsuo, Keitaro; Meindl, Alfons; Mitchell, Gillian; Muir, Kenneth; Nevelsteen, Ines; van den Ouweland, Ans; Peterlongo, Paolo; Phuah, Sze Yee; Pylkäs, Katri; Rowley, Simone M; Sangrajrang, Suleeporn; Schmutzler, Rita K; Shen, Chen-Yang; Shu, Xiao-Ou; Southey, Melissa C; Surowy, Harald; Swerdlow, Anthony; Teo, Soo H; Tollenaar, Rob A E M; Tomlinson, Ian; Torres, Diana; Truong, Thérèse; Vachon, Celine; Verhoef, Senno; Wong-Brown, Michelle; Zheng, Wei; Zheng, Ying; Nevanlinna, Heli; Scott, Rodney J; Andrulis, Irene L; Wu, Anna H; Hopper, John L; Couch, Fergus J; Winqvist, Robert; Burwinkel, Barbara; Sawyer, Elinor J; Schmidt, Marjanka K; Rudolph, Anja; Dörk, Thilo; Brauch, Hiltrud; Hamann, Ute; Neuhausen, Susan L; Milne, Roger L; Fletcher, Olivia; Pharoah, Paul D P; Campbell, Ian G; Dunning, Alison M; Le Calvez-Kelm, Florence; Goldgar, David E; Tavtigian, Sean V; Chenevix-Trench, Georgia

2016-05-01

BRCA1 interacting protein C-terminal helicase 1 (BRIP1) is one of the Fanconi Anaemia Complementation (FANC) group family of DNA repair proteins. Biallelic mutations in BRIP1 are responsible for FANC group J, and previous studies have also suggested that rare protein truncating variants in BRIP1 are associated with an increased risk of breast cancer. These studies have led to inclusion of BRIP1 on targeted sequencing panels for breast cancer risk prediction. We evaluated a truncating variant, p.Arg798Ter (rs137852986), and 10 missense variants of BRIP1, in 48 144 cases and 43 607 controls of European origin, drawn from 41 studies participating in the Breast Cancer Association Consortium (BCAC). Additionally, we sequenced the coding regions of BRIP1 in 13 213 cases and 5242 controls from the UK, 1313 cases and 1123 controls from three population-based studies as part of the Breast Cancer Family Registry, and 1853 familial cases and 2001 controls from Australia. The rare truncating allele of rs137852986 was observed in 23 cases and 18 controls in Europeans in BCAC (OR 1.09, 95% CI 0.58 to 2.03, p=0.79). Truncating variants were found in the sequencing studies in 34 cases (0.21%) and 19 controls (0.23%) (combined OR 0.90, 95% CI 0.48 to 1.70, p=0.75). These results suggest that truncating variants in BRIP1, and in particular p.Arg798Ter, are not associated with a substantial increase in breast cancer risk. Such observations have important implications for the reporting of results from breast cancer screening panels. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

Analysis of Toxic and Non-Toxic Alexandrium (Dinophyceae) Species Using Ribosomal RNA Gene Sequences

DTIC Science & Technology

1993-02-01

Therriault, J.-C. (1988). Cladistic analysis of electrophoretic variants within the toxic dinoflagellate genus Protogonyaulax. Botanica Marina 31: 39- 51. 8... Botanica Marina 34: 575-587. Halegraeff, G. M., and Bolch, C.J. (1992). Transport of toxic dinoflagellate cysts via ship’s ballast water: implications...analysis of electrophoretic variants within the toxic dinoflagellate genus Protogonv-u.!a,. Botanica Marina 31: 39-51. Curran, J., Baillie, D.L

Cumulative role of rare and common putative functional genetic variants at NPAS3 in schizophrenia susceptibility.

PubMed

González-Peñas, Javier; Arrojo, Manuel; Paz, Eduardo; Brenlla, Julio; Páramo, Mario; Costas, Javier

2015-10-01

Schizophrenia may be considered a human-specific disorder arisen as a maladaptive by-product of human-specific brain evolution. Therefore, genetic variants involved in susceptibility to schizophrenia may be identified among those genes related to acquisition of human-specific traits. NPAS3, a transcription factor involved in central nervous system development and neurogenesis, seems to be implicated in the evolution of human brain, as it is the human gene with most human-specific accelerated elements (HAEs), i.e., .mammalian conserved regulatory sequences with accelerated evolution in the lineage leading to humans after human-chimpanzee split. We hypothesize that any nucleotide variant at the NPAS3 HAEs may lead to altered susceptibility to schizophrenia. Twenty-one variants at these HAEs detected by the 1000 genomes Project, as well as five additional variants taken from psychiatric genome-wide association studies, were genotyped in 538 schizophrenic patients and 539 controls from Galicia. Analyses at the haplotype level or based on the cumulative role of the variants assuming different susceptibility models did not find any significant association in spite of enough power under several plausible scenarios regarding direction of effect and the specific role of rare and common variants. These results suggest that, contrary to our hypothesis, the special evolution of the NPAS3 HAEs in Homo relaxed the strong constraint on sequence that characterized these regions during mammalian evolution, allowing some sequence changes without any effect on schizophrenia risk. © 2015 Wiley Periodicals, Inc.

Protective Low-Frequency Variants for Preeclampsia in the Fms Related Tyrosine Kinase 1 Gene in the Finnish Population.

PubMed

Lokki, A Inkeri; Daly, Emma; Triebwasser, Michael; Kurki, Mitja I; Roberson, Elisha D O; Häppölä, Paavo; Auro, Kirsi; Perola, Markus; Heinonen, Seppo; Kajantie, Eero; Kere, Juha; Kivinen, Katja; Pouta, Anneli; Salmon, Jane E; Meri, Seppo; Daly, Mark; Atkinson, John P; Laivuori, Hannele

2017-08-01

Preeclampsia is a common pregnancy-specific vascular disorder characterized by new-onset hypertension and proteinuria during the second half of pregnancy. Predisposition to preeclampsia is in part heritable. It is associated with an increased risk of cardiovascular disease later in life. We have sequenced 124 candidate genes implicated in preeclampsia to pinpoint genetic variants contributing to predisposition to or protection from preeclampsia. First, targeted exomic sequencing was performed in 500 preeclamptic women and 190 controls from the FINNPEC cohort (Finnish Genetics of Preeclampsia Consortium). Then 122 women with a history of preeclampsia and 1905 parous women with no such history from the National FINRISK Study (a large Finnish population survey on risk factors of chronic, noncommunicable diseases) were included in the analyses. We tested 146 rare and low-frequency variants and found an excess (observed 13 versus expected 7.3) nominally associated with preeclampsia ( P <0.05). The most significantly associated sequence variants were protective variants rs35832528 (E982A; P =2.49E-4; odds ratio=0.387) and rs141440705 (R54S; P =0.003; odds ratio=0.442) in Fms related tyrosine kinase 1. These variants are enriched in the Finnish population with minor allele frequencies 0.026 and 0.017, respectively. They may also be associated with a lower risk of heart failure in 11 257 FINRISK women. This study provides the first evidence of maternal protective genetic variants in preeclampsia. © 2017 American Heart Association, Inc.

Simultaneous mutation and copy number variation (CNV) detection by multiplex PCR-based GS-FLX sequencing.

PubMed

Goossens, Dirk; Moens, Lotte N; Nelis, Eva; Lenaerts, An-Sofie; Glassee, Wim; Kalbe, Andreas; Frey, Bruno; Kopal, Guido; De Jonghe, Peter; De Rijk, Peter; Del-Favero, Jurgen

2009-03-01

We evaluated multiplex PCR amplification as a front-end for high-throughput sequencing, to widen the applicability of massive parallel sequencers for the detailed analysis of complex genomes. Using multiplex PCR reactions, we sequenced the complete coding regions of seven genes implicated in peripheral neuropathies in 40 individuals on a GS-FLX genome sequencer (Roche). The resulting dataset showed highly specific and uniform amplification. Comparison of the GS-FLX sequencing data with the dataset generated by Sanger sequencing confirmed the detection of all variants present and proved the sensitivity of the method for mutation detection. In addition, we showed that we could exploit the multiplexed PCR amplicons to determine individual copy number variation (CNV), increasing the spectrum of detected variations to both genetic and genomic variants. We conclude that our straightforward procedure substantially expands the applicability of the massive parallel sequencers for sequencing projects of a moderate number of amplicons (50-500) with typical applications in resequencing exons in positional or functional candidate regions and molecular genetic diagnostics. 2008 Wiley-Liss, Inc.

New genetic variants of LATS1 detected in urinary bladder and colon cancer.

PubMed

Saadeldin, Mona K; Shawer, Heba; Mostafa, Ahmed; Kassem, Neemat M; Amleh, Asma; Siam, Rania

2014-01-01

LATS1, the large tumor suppressor 1 gene, encodes for a serine/threonine kinase protein and is implicated in cell cycle progression. LATS1 is down-regulated in various human cancers, such as breast cancer, and astrocytoma. Point mutations in LATS1 were reported in human sarcomas. Additionally, loss of heterozygosity of LATS1 chromosomal region predisposes to breast, ovarian, and cervical tumors. In the current study, we investigated LATS1 genetic variations including single nucleotide polymorphisms (SNPs), in 28 Egyptian patients with either urinary bladder or colon cancers. The LATS1 gene was amplified and sequenced and the expression of LATS1 at the RNA level was assessed in 12 urinary bladder cancer samples. We report, the identification of a total of 29 variants including previously identified SNPs within LATS1 coding and non-coding sequences. A total of 18 variants were novel. Majority of the novel variants, 13, were mapped to intronic sequences and un-translated regions of the gene. Four of the five novel variants located in the coding region of the gene, represented missense mutations within the serine/threonine kinase catalytic domain. Interestingly, LATS1 RNA steady state levels was lost in urinary bladder cancerous tissue harboring four specific SNPs (16045 + 41736 + 34614 + 56177) positioned in the 5'UTR, intron 6, and two silent mutations within exon 4 and exon 8, respectively. This study identifies novel single-base-sequence alterations in the LATS1 gene. These newly identified variants could potentially be used as novel diagnostic or prognostic tools in cancer.

Implication of common and disease specific variants in CLU, CR1, and PICALM.

PubMed

Ferrari, Raffaele; Moreno, Jorge H; Minhajuddin, Abu T; O'Bryant, Sid E; Reisch, Joan S; Barber, Robert C; Momeni, Parastoo

2012-08-01

Two recent genome-wide association studies (GWAS) for late onset Alzheimer's disease (LOAD) revealed 3 new genes: clusterin (CLU), phosphatidylinositol binding clathrin assembly protein (PICALM), and complement receptor 1 (CR1). In order to evaluate association with these genome-wide association study-identified genes and to isolate the variants contributing to the pathogenesis of LOAD, we genotyped the top single nucleotide polymorphisms (SNPs), rs11136000 (CLU), rs3818361 (CR1), and rs3851179 (PICALM), and sequenced the entire coding regions of these genes in our cohort of 342 LOAD patients and 277 control subjects. We confirmed the association of rs3851179 (PICALM) (p = 7.4 × 10(-3)) with the disease status. Through sequencing we identified 18 variants in CLU, 3 of which were found exclusively in patients; 8 variants (out of 65) in CR1 gene were only found in patients and the 16 variants identified in PICALM gene were present in both patients and controls. In silico analysis of the variants in PICALM did not predict any damaging effect on the protein. The haplotype analysis of the variants in each gene predicted a common haplotype when the 3 single nucleotide polymorphisms rs11136000 (CLU), rs3818361 (CR1), and rs3851179 (PICALM), respectively, were included. For each gene the haplotype structure and size differed between patients and controls. In conclusion, we confirmed association of CLU, CR1, and PICALM genes with the disease status in our cohort through identification of a number of disease-specific variants among patients through the sequencing of the coding region of these genes. Published by Elsevier Inc.

Sequence variant classification and reporting: recommendations for improving the interpretation of cancer susceptibility genetic test results

PubMed Central

Plon, Sharon E.; Eccles, Diana M.; Easton, Douglas; Foulkes, William D.; Genuardi, Maurizio; Greenblatt, Marc S.; Hogervorst, Frans B.L.; Hoogerbrugge, Nicoline; Spurdle, Amanda B.; Tavtigian, Sean

2011-01-01

Genetic testing of cancer susceptibility genes is now widely applied in clinical practice to predict risk of developing cancer. In general, sequence-based testing of germline DNA is used to determine whether an individual carries a change that is clearly likely to disrupt normal gene function. Genetic testing may detect changes that are clearly pathogenic, clearly neutral or variants of unclear clinical significance. Such variants present a considerable challenge to the diagnostic laboratory and the receiving clinician in terms of interpretation and clear presentation of the implications of the result to the patient. There does not appear to be a consistent approach to interpreting and reporting the clinical significance of variants either among genes or among laboratories. The potential for confusion among clinicians and patients is considerable and misinterpretation may lead to inappropriate clinical consequences. In this article we review the current state of sequence-based genetic testing, describe other standardized reporting systems used in oncology and propose a standardized classification system for application to sequence based results for cancer predisposition genes. We suggest a system of five classes of variants based on the degree of likelihood of pathogenicity. Each class is associated with specific recommendations for clinical management of at-risk relatives that will depend on the syndrome. We propose that panels of experts on each cancer predisposition syndrome facilitate the classification scheme and designate appropriate surveillance and cancer management guidelines. The international adoption of a standardized reporting system should improve the clinical utility of sequence-based genetic tests to predict cancer risk. PMID:18951446

An Exome Sequencing Study to Assess the Role of Rare Genetic Variation in Pulmonary Fibrosis.

PubMed

Petrovski, Slavé; Todd, Jamie L; Durheim, Michael T; Wang, Quanli; Chien, Jason W; Kelly, Fran L; Frankel, Courtney; Mebane, Caroline M; Ren, Zhong; Bridgers, Joshua; Urban, Thomas J; Malone, Colin D; Finlen Copeland, Ashley; Brinkley, Christie; Allen, Andrew S; O'Riordan, Thomas; McHutchison, John G; Palmer, Scott M; Goldstein, David B

2017-07-01

Idiopathic pulmonary fibrosis (IPF) is an increasingly recognized, often fatal lung disease of unknown etiology. The aim of this study was to use whole-exome sequencing to improve understanding of the genetic architecture of pulmonary fibrosis. We performed a case-control exome-wide collapsing analysis including 262 unrelated individuals with pulmonary fibrosis clinically classified as IPF according to American Thoracic Society/European Respiratory Society/Japanese Respiratory Society/Latin American Thoracic Association guidelines (81.3%), usual interstitial pneumonia secondary to autoimmune conditions (11.5%), or fibrosing nonspecific interstitial pneumonia (7.2%). The majority (87%) of case subjects reported no family history of pulmonary fibrosis. We searched 18,668 protein-coding genes for an excess of rare deleterious genetic variation using whole-exome sequence data from 262 case subjects with pulmonary fibrosis and 4,141 control subjects drawn from among a set of individuals of European ancestry. Comparing genetic variation across 18,668 protein-coding genes, we found a study-wide significant (P < 4.5 × 10 -7 ) case enrichment of qualifying variants in TERT, RTEL1, and PARN. A model qualifying ultrarare, deleterious, nonsynonymous variants implicated TERT and RTEL1, and a model specifically qualifying loss-of-function variants implicated RTEL1 and PARN. A subanalysis of 186 case subjects with sporadic IPF confirmed TERT, RTEL1, and PARN as study-wide significant contributors to sporadic IPF. Collectively, 11.3% of case subjects with sporadic IPF carried a qualifying variant in one of these three genes compared with the 0.3% carrier rate observed among control subjects (odds ratio, 47.7; 95% confidence interval, 21.5-111.6; P = 5.5 × 10 -22 ). We identified TERT, RTEL1, and PARN-three telomere-related genes previously implicated in familial pulmonary fibrosis-as significant contributors to sporadic IPF. These results support the idea that telomere dysfunction is involved in IPF pathogenesis.

Pooled Sequencing of 531 Genes in Inflammatory Bowel Disease Identifies an Associated Rare Variant in BTNL2 and Implicates Other Immune Related Genes

PubMed Central

Prescott, Natalie J.; Lehne, Benjamin; Stone, Kristina; Lee, James C.; Taylor, Kirstin; Knight, Jo; Papouli, Efterpi; Mirza, Muddassar M.; Simpson, Michael A.; Spain, Sarah L.; Lu, Grace; Fraternali, Franca; Bumpstead, Suzannah J.; Gray, Emma; Amar, Ariella; Bye, Hannah; Green, Peter; Chung-Faye, Guy; Hayee, Bu’Hussain; Pollok, Richard; Satsangi, Jack; Parkes, Miles; Barrett, Jeffrey C.; Mansfield, John C.; Sanderson, Jeremy; Lewis, Cathryn M.; Weale, Michael E.; Schlitt, Thomas; Mathew, Christopher G.

2015-01-01

The contribution of rare coding sequence variants to genetic susceptibility in complex disorders is an important but unresolved question. Most studies thus far have investigated a limited number of genes from regions which contain common disease associated variants. Here we investigate this in inflammatory bowel disease by sequencing the exons and proximal promoters of 531 genes selected from both genome-wide association studies and pathway analysis in pooled DNA panels from 474 cases of Crohn’s disease and 480 controls. 80 variants with evidence of association in the sequencing experiment or with potential functional significance were selected for follow up genotyping in 6,507 IBD cases and 3,064 population controls. The top 5 disease associated variants were genotyped in an extension panel of 3,662 IBD cases and 3,639 controls, and tested for association in a combined analysis of 10,147 IBD cases and 7,008 controls. A rare coding variant p.G454C in the BTNL2 gene within the major histocompatibility complex was significantly associated with increased risk for IBD (p = 9.65x10−10, OR = 2.3[95% CI = 1.75–3.04]), but was independent of the known common associated CD and UC variants at this locus. Rare (<1%) and low frequency (1–5%) variants in 3 additional genes showed suggestive association (p<0.005) with either an increased risk (ARIH2 c.338-6C>T) or decreased risk (IL12B p.V298F, and NICN p.H191R) of IBD. These results provide additional insights into the involvement of the inhibition of T cell activation in the development of both sub-phenotypes of inflammatory bowel disease. We suggest that although rare coding variants may make a modest overall contribution to complex disease susceptibility, they can inform our understanding of the molecular pathways that contribute to pathogenesis. PMID:25671699

Fine-scale patterns of population stratification confound rare variant association tests.

PubMed

O'Connor, Timothy D; Kiezun, Adam; Bamshad, Michael; Rich, Stephen S; Smith, Joshua D; Turner, Emily; Leal, Suzanne M; Akey, Joshua M

2013-01-01

Advances in next-generation sequencing technology have enabled systematic exploration of the contribution of rare variation to Mendelian and complex diseases. Although it is well known that population stratification can generate spurious associations with common alleles, its impact on rare variant association methods remains poorly understood. Here, we performed exhaustive coalescent simulations with demographic parameters calibrated from exome sequence data to evaluate the performance of nine rare variant association methods in the presence of fine-scale population structure. We find that all methods have an inflated spurious association rate for parameter values that are consistent with levels of differentiation typical of European populations. For example, at a nominal significance level of 5%, some test statistics have a spurious association rate as high as 40%. Finally, we empirically assess the impact of population stratification in a large data set of 4,298 European American exomes. Our results have important implications for the design, analysis, and interpretation of rare variant genome-wide association studies.

BreaKmer: detection of structural variation in targeted massively parallel sequencing data using kmers.

PubMed

Abo, Ryan P; Ducar, Matthew; Garcia, Elizabeth P; Thorner, Aaron R; Rojas-Rudilla, Vanesa; Lin, Ling; Sholl, Lynette M; Hahn, William C; Meyerson, Matthew; Lindeman, Neal I; Van Hummelen, Paul; MacConaill, Laura E

2015-02-18

Genomic structural variation (SV), a common hallmark of cancer, has important predictive and therapeutic implications. However, accurately detecting SV using high-throughput sequencing data remains challenging, especially for 'targeted' resequencing efforts. This is critically important in the clinical setting where targeted resequencing is frequently being applied to rapidly assess clinically actionable mutations in tumor biopsies in a cost-effective manner. We present BreaKmer, a novel approach that uses a 'kmer' strategy to assemble misaligned sequence reads for predicting insertions, deletions, inversions, tandem duplications and translocations at base-pair resolution in targeted resequencing data. Variants are predicted by realigning an assembled consensus sequence created from sequence reads that were abnormally aligned to the reference genome. Using targeted resequencing data from tumor specimens with orthogonally validated SV, non-tumor samples and whole-genome sequencing data, BreaKmer had a 97.4% overall sensitivity for known events and predicted 17 positively validated, novel variants. Relative to four publically available algorithms, BreaKmer detected SV with increased sensitivity and limited calls in non-tumor samples, key features for variant analysis of tumor specimens in both the clinical and research settings. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

The Contribution of Mosaic Variants to Autism Spectrum Disorder.

PubMed

Freed, Donald; Pevsner, Jonathan

2016-09-01

De novo mutation is highly implicated in autism spectrum disorder (ASD). However, the contribution of post-zygotic mutation to ASD is poorly characterized. We performed both exome sequencing of paired samples and analysis of de novo variants from whole-exome sequencing of 2,388 families. While we find little evidence for tissue-specific mosaic mutation, multi-tissue post-zygotic mutation (i.e. mosaicism) is frequent, with detectable mosaic variation comprising 5.4% of all de novo mutations. We identify three mosaic missense and likely-gene disrupting mutations in genes previously implicated in ASD (KMT2C, NCKAP1, and MYH10) in probands but none in siblings. We find a strong ascertainment bias for mosaic mutations in probands relative to their unaffected siblings (p = 0.003). We build a model of de novo variation incorporating mosaic variants and errors in classification of mosaic status and from this model we estimate that 33% of mosaic mutations in probands contribute to 5.1% of simplex ASD diagnoses (95% credible interval 1.3% to 8.9%). Our results indicate a contributory role for multi-tissue mosaic mutation in some individuals with an ASD diagnosis.

De novo and inherited private variants in MAP1B in periventricular nodular heterotopia.

PubMed

Heinzen, Erin L; O'Neill, Adam C; Zhu, Xiaolin; Allen, Andrew S; Bahlo, Melanie; Chelly, Jamel; Chen, Ming Hui; Dobyns, William B; Freytag, Saskia; Guerrini, Renzo; Leventer, Richard J; Poduri, Annapurna; Robertson, Stephen P; Walsh, Christopher A; Zhang, Mengqi

2018-05-01

Periventricular nodular heterotopia (PVNH) is a malformation of cortical development commonly associated with epilepsy. We exome sequenced 202 individuals with sporadic PVNH to identify novel genetic risk loci. We first performed a trio-based analysis and identified 219 de novo variants. Although no novel genes were implicated in this initial analysis, PVNH cases were found overall to have a significant excess of nonsynonymous de novo variants in intolerant genes (p = 3.27x10-7), suggesting a role for rare new alleles in genes yet to be associated with the condition. Using a gene-level collapsing analysis comparing cases and controls, we identified a genome-wide significant signal driven by four ultra-rare loss-of-function heterozygous variants in MAP1B, including one de novo variant. In at least one instance, the MAP1B variant was inherited from a parent with previously undiagnosed PVNH. The PVNH was frontally predominant and associated with perisylvian polymicrogyria. These results implicate MAP1B in PVNH. More broadly, our findings suggest that detrimental mutations likely arising in immediately preceding generations with incomplete penetrance may also be responsible for some apparently sporadic diseases.

CDKL5 variants: Improving our understanding of a rare neurologic disorder.

PubMed

Hector, Ralph D; Kalscheuer, Vera M; Hennig, Friederike; Leonard, Helen; Downs, Jenny; Clarke, Angus; Benke, Tim A; Armstrong, Judith; Pineda, Mercedes; Bailey, Mark E S; Cobb, Stuart R

2017-12-01

To provide new insights into the interpretation of genetic variants in a rare neurologic disorder, CDKL5 deficiency, in the contexts of population sequencing data and an updated characterization of the CDKL5 gene. We analyzed all known potentially pathogenic CDKL5 variants by combining data from large-scale population sequencing studies with CDKL5 variants from new and all available clinical cohorts and combined this with computational methods to predict pathogenicity. The study has identified several variants that can be reclassified as benign or likely benign. With the addition of novel CDKL5 variants, we confirm that pathogenic missense variants cluster in the catalytic domain of CDKL5 and reclassify a purported missense variant as having a splicing consequence. We provide further evidence that missense variants in the final 3 exons are likely to be benign and not important to disease pathology. We also describe benign splicing and nonsense variants within these exons, suggesting that isoform hCDKL5_5 is likely to have little or no neurologic significance. We also use the available data to make a preliminary estimate of minimum incidence of CDKL5 deficiency. These findings have implications for genetic diagnosis, providing evidence for the reclassification of specific variants previously thought to result in CDKL5 deficiency. Together, these analyses support the view that the predominant brain isoform in humans (hCDKL5_1) is crucial for normal neurodevelopment and that the catalytic domain is the primary functional domain.

CDKL5 variants

PubMed Central

Kalscheuer, Vera M.; Hennig, Friederike; Leonard, Helen; Downs, Jenny; Clarke, Angus; Benke, Tim A.; Armstrong, Judith; Pineda, Mercedes; Bailey, Mark E.S.; Cobb, Stuart R.

2017-01-01

Objective: To provide new insights into the interpretation of genetic variants in a rare neurologic disorder, CDKL5 deficiency, in the contexts of population sequencing data and an updated characterization of the CDKL5 gene. Methods: We analyzed all known potentially pathogenic CDKL5 variants by combining data from large-scale population sequencing studies with CDKL5 variants from new and all available clinical cohorts and combined this with computational methods to predict pathogenicity. Results: The study has identified several variants that can be reclassified as benign or likely benign. With the addition of novel CDKL5 variants, we confirm that pathogenic missense variants cluster in the catalytic domain of CDKL5 and reclassify a purported missense variant as having a splicing consequence. We provide further evidence that missense variants in the final 3 exons are likely to be benign and not important to disease pathology. We also describe benign splicing and nonsense variants within these exons, suggesting that isoform hCDKL5_5 is likely to have little or no neurologic significance. We also use the available data to make a preliminary estimate of minimum incidence of CDKL5 deficiency. Conclusions: These findings have implications for genetic diagnosis, providing evidence for the reclassification of specific variants previously thought to result in CDKL5 deficiency. Together, these analyses support the view that the predominant brain isoform in humans (hCDKL5_1) is crucial for normal neurodevelopment and that the catalytic domain is the primary functional domain. PMID:29264392

A clinically driven variant prioritization framework outperforms purely computational approaches for the diagnostic analysis of singleton WES data.

PubMed

Stark, Zornitza; Dashnow, Harriet; Lunke, Sebastian; Tan, Tiong Y; Yeung, Alison; Sadedin, Simon; Thorne, Natalie; Macciocca, Ivan; Gaff, Clara; Oshlack, Alicia; White, Susan M; James, Paul A

2017-11-01

Rapid identification of clinically significant variants is key to the successful application of next generation sequencing technologies in clinical practice. The Melbourne Genomics Health Alliance (MGHA) variant prioritization framework employs a gene prioritization index based on clinician-generated a priori gene lists, and a variant prioritization index (VPI) based on rarity, conservation and protein effect. We used data from 80 patients who underwent singleton whole exome sequencing (WES) to test the ability of the framework to rank causative variants highly, and compared it against the performance of other gene and variant prioritization tools. Causative variants were identified in 59 of the patients. Using the MGHA prioritization framework the average rank of the causative variant was 2.24, with 76% ranked as the top priority variant, and 90% ranked within the top five. Using clinician-generated gene lists resulted in ranking causative variants an average of 8.2 positions higher than prioritization based on variant properties alone. This clinically driven prioritization approach significantly outperformed purely computational tools, placing a greater proportion of causative variants top or in the top 5 (permutation P-value=0.001). Clinicians included 40 of the 49 WES diagnoses in their a priori list of differential diagnoses (81%). The lists generated by PhenoTips and Phenomizer contained 14 (29%) and 18 (37%) of these diagnoses respectively. These results highlight the benefits of clinically led variant prioritization in increasing the efficiency of singleton WES data analysis and have important implications for developing models for the funding and delivery of genomic services.

Exome sequencing identifies a novel FOXP3 mutation in a 2-generation family with inflammatory bowel disease.

PubMed

Okou, David T; Mondal, Kajari; Faubion, William A; Kobrynski, Lisa J; Denson, Lee A; Mulle, Jennifer G; Ramachandran, Dhanya; Xiong, Yuning; Svingen, Phyllis; Patel, Viren; Bose, Promita; Waters, Jon P; Prahalad, Sampath; Cutler, David J; Zwick, Michael E; Kugathasan, Subra

2014-05-01

Inflammatory bowel disease (IBD) is heritable, but a total of 163 variants commonly implicated in IBD pathogenesis account for only 25% of the heritability. Rare, highly penetrant genetic variants may also explain mendelian forms of IBD and some of the missing heritability. To test the hypothesis that rare loss-of-function mutations can be causative, we performed whole exome sequencing (WES) on 5 members of a 2-generation family of European ancestry presenting with an early-onset and atypical form of IBD. WES was performed for all of the 5 family members; the mother and 3 male offspring were affected, whereas the father was unaffected. Mapping, annotation, and filtering criteria were used to reduce candidate variants. For functional testing we performed forkhead box P3 (FOXP3) staining and a T-cell suppression assay. We identified a novel missense variant in exon 6 of the X-linked FOXP3 gene. The c.694A>C substitution in FOXP3 results in a cysteine-to-glycine change at the protein position 232 that is completely conserved among all vertebrates. This variant (heterozygous in the mother and hemizygous in all 3 affected sons) did not impair FOXP3 protein expression, but significantly reduced the ability of the host's T regulatory cells to suppress an inappropriate autoimmune response. The variant results in a milder immune dysregulation, polyendocrinopathy, enteropathy, and X-linked phenotype with early-onset IBD. Our study illustrates the successful application of WES for making a definitive molecular diagnosis in a case of multiply affected families, with atypical IBD-like phenotype. Our results also have important implications for disease biology and disease-directed therapeutic development.

Functional phosphodiesterase 11A mutations may modify the risk of familial and bilateral testicular germ cell tumors

PubMed Central

Horvath, Anelia; Korde, Larissa; Greene, Mark H.; Libe, Rosella; Osorio, Paulo; Faucz, Fabio Rueda; Raffin-Sanson, Marie Laure; Tsang, Kit Man; Drori-Herishanu, Limor; Patronas, Yianna; Remmers, Elaine F; Nikita, Maria-Elena; Moran, Jason; Greene, Joseph; Nesterova, Maria; Merino, Maria; Bertherat, Jerome; Stratakis, Constantine A.

2009-01-01

Inactivating germline mutations in phosphodiesterase 11A (PDE11A) have been implicated in adrenal tumor susceptibility. PDE11A is highly-expressed in endocrine steroidogenic tissues, especially the testis, and mice with inactivated Pde11a exhibit male infertility, a known testicular germ cell tumor (TGCT) risk factor. We sequenced the PDE11A gene-coding region in 95 patients with TGCT from 64 unrelated kindreds. We identified 8 non-synonymous substitutions in 20 patients from 15 families: four (R52T; F258Y; G291R; V820M) were newly-recognized, three (R804H; R867G; M878V) were functional variants previously implicated in adrenal tumor predisposition, and one (Y727C) was a known polymorphism. We compared the frequency of these variants in our patients to unrelated controls that had been screened and found negative for any endocrine diseases: only the two previously-reported variants, R804H and R867G, known to be frequent in general population, were detected in these controls. The frequency of all PDE11A-gene variants (combined) was significantly higher among patients with TGCT (P=0.0002), present in 19% of the families of our cohort. Most variants were detected in the general population, but functional studies showed that all these mutations reduced PDE activity, and that PDE11A protein expression was decreased (or absent) in TGCT samples from carriers. This is the first demonstration of a PDE gene’s involvement in TGCT, although the cAMP signaling pathway has been investigated extensively in other reproductive organs and their diseases. In conclusion, we report that PDE11A-inactivating sequence variants may modify the risk of familial and bilateral TGCT. PMID:19549888

Diagnostics based on nucleic acid sequence variant profiling: PCR, hybridization, and NGS approaches.

PubMed

Khodakov, Dmitriy; Wang, Chunyan; Zhang, David Yu

2016-10-01

Nucleic acid sequence variations have been implicated in many diseases, and reliable detection and quantitation of DNA/RNA biomarkers can inform effective therapeutic action, enabling precision medicine. Nucleic acid analysis technologies being translated into the clinic can broadly be classified into hybridization, PCR, and sequencing, as well as their combinations. Here we review the molecular mechanisms of popular commercial assays, and their progress in translation into in vitro diagnostics. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Exome sequence analysis suggests genetic burden contributes to phenotypic variability and complex neuropathy

PubMed Central

Gonzaga-Jauregui, Claudia; Harel, Tamar; Gambin, Tomasz; Kousi, Maria; Griffin, Laurie B.; Francescatto, Ludmila; Ozes, Burcak; Karaca, Ender; Jhangiani, Shalini; Bainbridge, Matthew N.; Lawson, Kim S.; Pehlivan, Davut; Okamoto, Yuji; Withers, Marjorie; Mancias, Pedro; Slavotinek, Anne; Reitnauer, Pamela J; Goksungur, Meryem T.; Shy, Michael; Crawford, Thomas O.; Koenig, Michel; Willer, Jason; Flores, Brittany N.; Pediaditrakis, Igor; Us, Onder; Wiszniewski, Wojciech; Parman, Yesim; Antonellis, Anthony; Muzny, Donna M.; Katsanis, Nicholas; Battaloglu, Esra; Boerwinkle, Eric; Gibbs, Richard A.; Lupski, James R.

2015-01-01

Charcot-Marie-Tooth (CMT) disease is a clinically and genetically heterogeneous distal symmetric polyneuropathy. Whole-exome sequencing (WES) of 40 individuals from 37 unrelated families with CMT-like peripheral neuropathy refractory to molecular diagnosis identified apparent causal mutations in ~45% (17/37) of families. Three candidate disease genes are proposed, supported by a combination of genetic and in vivo studies. Aggregate analysis of mutation data revealed a significantly increased number of rare variants across 58 neuropathy associated genes in subjects versus controls; confirmed in a second ethnically discrete neuropathy cohort, suggesting mutation burden potentially contributes to phenotypic variability. Neuropathy genes shown to have highly penetrant Mendelizing variants (HMPVs) and implicated by burden in families were shown to interact genetically in a zebrafish assay exacerbating the phenotype established by the suppression of single genes. Our findings suggest that the combinatorial effect of rare variants contributes to disease burden and variable expressivity. PMID:26257172

A map of human genome variation from population-scale sequencing.

PubMed

Abecasis, Gonçalo R; Altshuler, David; Auton, Adam; Brooks, Lisa D; Durbin, Richard M; Gibbs, Richard A; Hurles, Matt E; McVean, Gil A

2010-10-28

The 1000 Genomes Project aims to provide a deep characterization of human genome sequence variation as a foundation for investigating the relationship between genotype and phenotype. Here we present results of the pilot phase of the project, designed to develop and compare different strategies for genome-wide sequencing with high-throughput platforms. We undertook three projects: low-coverage whole-genome sequencing of 179 individuals from four populations; high-coverage sequencing of two mother-father-child trios; and exon-targeted sequencing of 697 individuals from seven populations. We describe the location, allele frequency and local haplotype structure of approximately 15 million single nucleotide polymorphisms, 1 million short insertions and deletions, and 20,000 structural variants, most of which were previously undescribed. We show that, because we have catalogued the vast majority of common variation, over 95% of the currently accessible variants found in any individual are present in this data set. On average, each person is found to carry approximately 250 to 300 loss-of-function variants in annotated genes and 50 to 100 variants previously implicated in inherited disorders. We demonstrate how these results can be used to inform association and functional studies. From the two trios, we directly estimate the rate of de novo germline base substitution mutations to be approximately 10(-8) per base pair per generation. We explore the data with regard to signatures of natural selection, and identify a marked reduction of genetic variation in the neighbourhood of genes, due to selection at linked sites. These methods and public data will support the next phase of human genetic research.

Whole-genome sequencing in patients with ciliopathies uncovers a novel recurrent tandem duplication in IFT140.

PubMed

Geoffroy, Véronique; Stoetzel, Corinne; Scheidecker, Sophie; Schaefer, Elise; Perrault, Isabelle; Bär, Séverine; Kröll, Ariane; Delbarre, Marion; Antin, Manuela; Leuvrey, Anne-Sophie; Henry, Charline; Blanché, Hélène; Decker, Eva; Kloth, Katja; Klaus, Günter; Mache, Christoph; Martin-Coignard, Dominique; McGinn, Steven; Boland, Anne; Deleuze, Jean-François; Friant, Sylvie; Saunier, Sophie; Rozet, Jean-Michel; Bergmann, Carsten; Dollfus, Hélène; Muller, Jean

2018-04-24

Ciliopathies represent a wide spectrum of rare diseases with overlapping phenotypes and a high genetic heterogeneity. Among those, IFT140 is implicated in a variety of phenotypes ranging from isolated retinis pigmentosa to more syndromic cases. Using whole-genome sequencing in patients with uncharacterized ciliopathies, we identified a novel recurrent tandem duplication of exon 27-30 (6.7 kb) in IFT140, c.3454-488_4182+2588dup p.(Tyr1152_Thr1394dup), missed by whole-exome sequencing. Pathogenicity of the mutation was assessed on the patients' skin fibroblasts. Several hundreds of patients with a ciliopathy phenotype were screened and biallelic mutations were identified in 11 families representing 12 pathogenic variants of which seven are novel. Among those unrelated families especially with a Mainzer-Saldino syndrome, eight carried the same tandem duplication (two at the homozygous state and six at the heterozygous state). In conclusion, we demonstrated the implication of structural variations in IFT140-related diseases expanding its mutation spectrum. We also provide evidences for a unique genomic event mediated by an Alu-Alu recombination occurring on a shared haplotype. We confirm that whole-genome sequencing can be instrumental in the ability to detect structural variants for genomic disorders. © 2018 Wiley Periodicals, Inc.

Analysis of Genes Involved in Body Weight Regulation by Targeted Re-Sequencing.

PubMed

Volckmar, Anna-Lena; Han, Chung Ting; Pütter, Carolin; Haas, Stefan; Vogel, Carla I G; Knoll, Nadja; Struve, Christoph; Göbel, Maria; Haas, Katharina; Herrfurth, Nikolas; Jarick, Ivonne; Grallert, Harald; Schürmann, Annette; Al-Hasani, Hadi; Hebebrand, Johannes; Sauer, Sascha; Hinney, Anke

2016-01-01

Genes involved in body weight regulation that were previously investigated in genome-wide association studies (GWAS) and in animal models were target-enriched followed by massive parallel next generation sequencing. We enriched and re-sequenced continuous genomic regions comprising FTO, MC4R, TMEM18, SDCCAG8, TKNS, MSRA and TBC1D1 in a screening sample of 196 extremely obese children and adolescents with age and sex specific body mass index (BMI) ≥ 99th percentile and 176 lean adults (BMI ≤ 15th percentile). 22 variants were confirmed by Sanger sequencing. Genotyping was performed in up to 705 independent obesity trios (extremely obese child and both parents), 243 extremely obese cases and 261 lean adults. We detected 20 different non-synonymous variants, one frame shift and one nonsense mutation in the 7 continuous genomic regions in study groups of different weight extremes. For SNP Arg695Cys (rs58983546) in TBC1D1 we detected nominal association with obesity (pTDT = 0.03 in 705 trios). Eleven of the variants were rare, thus were only detected heterozygously in up to ten individual(s) of the complete screening sample of 372 individuals. Two of them (in FTO and MSRA) were found in lean individuals, nine in extremely obese. In silico analyses of the 11 variants did not reveal functional implications for the mutations. Concordant with our hypothesis we detected a rare variant that potentially leads to loss of FTO function in a lean individual. For TBC1D1, in contrary to our hypothesis, the loss of function variant (Arg443Stop) was found in an obese individual. Functional in vitro studies are warranted.

Clinical and molecular characterization of KCNT1-related severe early-onset epilepsy

PubMed Central

Nair, Umesh; Malhotra, Sony; Meyer, Esther; Trump, Natalie; Gazina, Elena V.; Papandreou, Apostolos; Ngoh, Adeline; Ackermann, Sally; Ambegaonkar, Gautam; Appleton, Richard; Desurkar, Archana; Eltze, Christin; Kneen, Rachel; Kumar, Ajith V.; Lascelles, Karine; Montgomery, Tara; Ramesh, Venkateswaran; Samanta, Rajib; Scott, Richard H.; Tan, Jeen; Whitehouse, William; Poduri, Annapurna; Scheffer, Ingrid E.; Chong, W.K. “Kling”; Cross, J. Helen; Topf, Maya; Petrou, Steven

2018-01-01

Objective To characterize the phenotypic spectrum, molecular genetic findings, and functional consequences of pathogenic variants in early-onset KCNT1 epilepsy. Methods We identified a cohort of 31 patients with epilepsy of infancy with migrating focal seizures (EIMFS) and screened for variants in KCNT1 using direct Sanger sequencing, a multiple-gene next-generation sequencing panel, and whole-exome sequencing. Additional patients with non-EIMFS early-onset epilepsy in whom we identified KCNT1 variants on local diagnostic multiple gene panel testing were also included. When possible, we performed homology modeling to predict the putative effects of variants on protein structure and function. We undertook electrophysiologic assessment of mutant KCNT1 channels in a xenopus oocyte model system. Results We identified pathogenic variants in KCNT1 in 12 patients, 4 of which are novel. Most variants occurred de novo. Ten patients had a clinical diagnosis of EIMFS, and the other 2 presented with early-onset severe nocturnal frontal lobe seizures. Three patients had a trial of quinidine with good clinical response in 1 patient. Computational modeling analysis implicates abnormal pore function (F346L) and impaired tetramer formation (F502V) as putative disease mechanisms. All evaluated KCNT1 variants resulted in marked gain of function with significantly increased channel amplitude and variable blockade by quinidine. Conclusions Gain-of-function KCNT1 pathogenic variants cause a spectrum of severe focal epilepsies with onset in early infancy. Currently, genotype-phenotype correlations are unclear, although clinical outcome is poor for the majority of cases. Further elucidation of disease mechanisms may facilitate the development of targeted treatments, much needed for this pharmacoresistant genetic epilepsy. PMID:29196579

Detection of a single nucleotide polymorphism in the human alpha-lactalbumin gene: implications for human milk proteins.

PubMed

Chowanadisai, Winyoo; Kelleher, Shannon L; Nemeth, Jennifer F; Yachetti, Stephen; Kuhlman, Charles F; Jackson, Joan G; Davis, Anne M; Lien, Eric L; Lönnerdal, Bo

2005-05-01

Variability in the protein composition of breast milk has been observed in many women and is believed to be due to natural variation of the human population. Single nucleotide polymorphisms (SNPs) are present throughout the entire human genome, but the impact of this variation on human milk composition and biological activity and infant nutrition and health is unclear. The goals of this study were to characterize a variant of human alpha-lactalbumin observed in milk from a Filipino population by determining the location of the polymorphism in the amino acid and genomic sequences of alpha-lactalbumin. Milk and blood samples were collected from 20 Filipino women, and milk samples were collected from an additional 450 women from nine different countries. alpha-Lactalbumin concentration was measured by high-performance liquid chromatography (HPLC), and milk samples containing the variant form of the protein were identified with both HPLC and mass spectrometry (MS). The molecular weight of the variant form was measured by MS, and the location of the polymorphism was narrowed down by protein reduction, alkylation and trypsin digestion. Genomic DNA was isolated from whole blood, and the polymorphism location and subject genotype were determined by amplifying the entire coding sequence of human alpha-lactalbumin by PCR, followed by DNA sequencing. A variant form of alpha-lactalbumin was observed in HPLC chromatograms, and the difference in molecular weight was determined by MS (wild type=14,070 Da, variant=14,056 Da). Protein reduction and digestion narrowed the polymorphism between the 33rd and 77th amino acid of the protein. The genetic polymorphism was identified as adenine to guanine, which translates to a substitution from isoleucine to valine at amino acid 46. The frequency of variation was higher in milk from China, Japan and Philippines, which suggests that this polymorphism is most prevalent in Asia. There are SNPs in the genome for human milk proteins and their implications for protein bioactivity and infant nutrition need to be considered.

Exploring the unknown: assumptions about allelic architecture and strategies for susceptibility variant discovery.

PubMed

McCarthy, Mark I

2009-07-03

Identification of common-variant associations for many common disorders has been highly effective, but the loci detected so far typically explain only a small proportion of the genetic predisposition to disease. Extending explained genetic variance is one of the major near-term goals of human genetic research. Next-generation sequencing technologies offer great promise, but optimal strategies for their deployment remain uncertain, not least because we lack a clear view of the characteristics of the variants being sought. Here, I discuss what can and cannot be inferred about complex trait disease architecture from the information currently available and review the implications for future research strategies.

An integrated map of structural variation in 2,504 human genomes.

PubMed

Sudmant, Peter H; Rausch, Tobias; Gardner, Eugene J; Handsaker, Robert E; Abyzov, Alexej; Huddleston, John; Zhang, Yan; Ye, Kai; Jun, Goo; Fritz, Markus Hsi-Yang; Konkel, Miriam K; Malhotra, Ankit; Stütz, Adrian M; Shi, Xinghua; Casale, Francesco Paolo; Chen, Jieming; Hormozdiari, Fereydoun; Dayama, Gargi; Chen, Ken; Malig, Maika; Chaisson, Mark J P; Walter, Klaudia; Meiers, Sascha; Kashin, Seva; Garrison, Erik; Auton, Adam; Lam, Hugo Y K; Mu, Xinmeng Jasmine; Alkan, Can; Antaki, Danny; Bae, Taejeong; Cerveira, Eliza; Chines, Peter; Chong, Zechen; Clarke, Laura; Dal, Elif; Ding, Li; Emery, Sarah; Fan, Xian; Gujral, Madhusudan; Kahveci, Fatma; Kidd, Jeffrey M; Kong, Yu; Lameijer, Eric-Wubbo; McCarthy, Shane; Flicek, Paul; Gibbs, Richard A; Marth, Gabor; Mason, Christopher E; Menelaou, Androniki; Muzny, Donna M; Nelson, Bradley J; Noor, Amina; Parrish, Nicholas F; Pendleton, Matthew; Quitadamo, Andrew; Raeder, Benjamin; Schadt, Eric E; Romanovitch, Mallory; Schlattl, Andreas; Sebra, Robert; Shabalin, Andrey A; Untergasser, Andreas; Walker, Jerilyn A; Wang, Min; Yu, Fuli; Zhang, Chengsheng; Zhang, Jing; Zheng-Bradley, Xiangqun; Zhou, Wanding; Zichner, Thomas; Sebat, Jonathan; Batzer, Mark A; McCarroll, Steven A; Mills, Ryan E; Gerstein, Mark B; Bashir, Ali; Stegle, Oliver; Devine, Scott E; Lee, Charles; Eichler, Evan E; Korbel, Jan O

2015-10-01

Structural variants are implicated in numerous diseases and make up the majority of varying nucleotides among human genomes. Here we describe an integrated set of eight structural variant classes comprising both balanced and unbalanced variants, which we constructed using short-read DNA sequencing data and statistically phased onto haplotype blocks in 26 human populations. Analysing this set, we identify numerous gene-intersecting structural variants exhibiting population stratification and describe naturally occurring homozygous gene knockouts that suggest the dispensability of a variety of human genes. We demonstrate that structural variants are enriched on haplotypes identified by genome-wide association studies and exhibit enrichment for expression quantitative trait loci. Additionally, we uncover appreciable levels of structural variant complexity at different scales, including genic loci subject to clusters of repeated rearrangement and complex structural variants with multiple breakpoints likely to have formed through individual mutational events. Our catalogue will enhance future studies into structural variant demography, functional impact and disease association.

Novel approach to genetic analysis and results in 3000 hemophilia patients enrolled in the My Life, Our Future initiative

PubMed Central

Johnsen, Jill M.; Fletcher, Shelley N.; Huston, Haley; Roberge, Sarah; Martin, Beth K.; Kircher, Martin; Josephson, Neil C.; Shendure, Jay; Ruuska, Sarah; Koerper, Marion A.; Morales, Jaime; Pierce, Glenn F.; Aschman, Diane J.

2017-01-01

Hemophilia A and B are rare, X-linked bleeding disorders. My Life, Our Future (MLOF) is a collaborative project established to genotype and study hemophilia. Patients were enrolled at US hemophilia treatment centers (HTCs). Genotyping was performed centrally using next-generation sequencing (NGS) with an approach that detected common F8 gene inversions simultaneously with F8 and F9 gene sequencing followed by confirmation using standard genotyping methods. Sixty-nine HTCs enrolled the first 3000 patients in under 3 years. Clinically reportable DNA variants were detected in 98.1% (2357/2401) of hemophilia A and 99.3% (595/599) of hemophilia B patients. Of the 924 unique variants found, 285 were novel. Predicted gene-disrupting variants were common in severe disease; missense variants predominated in mild–moderate disease. Novel DNA variants accounted for ∼30% of variants found and were detected continuously throughout the project, indicating that additional variation likely remains undiscovered. The NGS approach detected >1 reportable variants in 36 patients (10 females), a finding with potential clinical implications. NGS also detected incidental variants unlikely to cause disease, including 11 variants previously reported in hemophilia. Although these genes are thought to be conserved, our findings support caution in interpretation of new variants. In summary, MLOF has contributed significantly toward variant annotation in the F8 and F9 genes. In the near future, investigators will be able to access MLOF data and repository samples for research to advance our understanding of hemophilia. PMID:29296726

Development of a genotyping microarray for Usher syndrome.

PubMed

Cremers, Frans P M; Kimberling, William J; Külm, Maigi; de Brouwer, Arjan P; van Wijk, Erwin; te Brinke, Heleen; Cremers, Cor W R J; Hoefsloot, Lies H; Banfi, Sandro; Simonelli, Francesca; Fleischhauer, Johannes C; Berger, Wolfgang; Kelley, Phil M; Haralambous, Elene; Bitner-Glindzicz, Maria; Webster, Andrew R; Saihan, Zubin; De Baere, Elfride; Leroy, Bart P; Silvestri, Giuliana; McKay, Gareth J; Koenekoop, Robert K; Millan, Jose M; Rosenberg, Thomas; Joensuu, Tarja; Sankila, Eeva-Marja; Weil, Dominique; Weston, Mike D; Wissinger, Bernd; Kremer, Hannie

2007-02-01

Usher syndrome, a combination of retinitis pigmentosa (RP) and sensorineural hearing loss with or without vestibular dysfunction, displays a high degree of clinical and genetic heterogeneity. Three clinical subtypes can be distinguished, based on the age of onset and severity of the hearing impairment, and the presence or absence of vestibular abnormalities. Thus far, eight genes have been implicated in the syndrome, together comprising 347 protein-coding exons. To improve DNA diagnostics for patients with Usher syndrome, we developed a genotyping microarray based on the arrayed primer extension (APEX) method. Allele-specific oligonucleotides corresponding to all 298 Usher syndrome-associated sequence variants known to date, 76 of which are novel, were arrayed. Approximately half of these variants were validated using original patient DNAs, which yielded an accuracy of >98%. The efficiency of the Usher genotyping microarray was tested using DNAs from 370 unrelated European and American patients with Usher syndrome. Sequence variants were identified in 64/140 (46%) patients with Usher syndrome type I, 45/189 (24%) patients with Usher syndrome type II, 6/21 (29%) patients with Usher syndrome type III and 6/20 (30%) patients with atypical Usher syndrome. The chip also identified two novel sequence variants, c.400C>T (p.R134X) in PCDH15 and c.1606T>C (p.C536S) in USH2A. The Usher genotyping microarray is a versatile and affordable screening tool for Usher syndrome. Its efficiency will improve with the addition of novel sequence variants with minimal extra costs, making it a very useful first-pass screening tool.

Development of a genotyping microarray for Usher syndrome

PubMed Central

Cremers, Frans P M; Kimberling, William J; Külm, Maigi; de Brouwer, Arjan P; van Wijk, Erwin; te Brinke, Heleen; Cremers, Cor W R J; Hoefsloot, Lies H; Banfi, Sandro; Simonelli, Francesca; Fleischhauer, Johannes C; Berger, Wolfgang; Kelley, Phil M; Haralambous, Elene; Bitner‐Glindzicz, Maria; Webster, Andrew R; Saihan, Zubin; De Baere, Elfride; Leroy, Bart P; Silvestri, Giuliana; McKay, Gareth J; Koenekoop, Robert K; Millan, Jose M; Rosenberg, Thomas; Joensuu, Tarja; Sankila, Eeva‐Marja; Weil, Dominique; Weston, Mike D; Wissinger, Bernd; Kremer, Hannie

2007-01-01

Background Usher syndrome, a combination of retinitis pigmentosa (RP) and sensorineural hearing loss with or without vestibular dysfunction, displays a high degree of clinical and genetic heterogeneity. Three clinical subtypes can be distinguished, based on the age of onset and severity of the hearing impairment, and the presence or absence of vestibular abnormalities. Thus far, eight genes have been implicated in the syndrome, together comprising 347 protein‐coding exons. Methods: To improve DNA diagnostics for patients with Usher syndrome, we developed a genotyping microarray based on the arrayed primer extension (APEX) method. Allele‐specific oligonucleotides corresponding to all 298 Usher syndrome‐associated sequence variants known to date, 76 of which are novel, were arrayed. Results Approximately half of these variants were validated using original patient DNAs, which yielded an accuracy of >98%. The efficiency of the Usher genotyping microarray was tested using DNAs from 370 unrelated European and American patients with Usher syndrome. Sequence variants were identified in 64/140 (46%) patients with Usher syndrome type I, 45/189 (24%) patients with Usher syndrome type II, 6/21 (29%) patients with Usher syndrome type III and 6/20 (30%) patients with atypical Usher syndrome. The chip also identified two novel sequence variants, c.400C>T (p.R134X) in PCDH15 and c.1606T>C (p.C536S) in USH2A. Conclusion The Usher genotyping microarray is a versatile and affordable screening tool for Usher syndrome. Its efficiency will improve with the addition of novel sequence variants with minimal extra costs, making it a very useful first‐pass screening tool. PMID:16963483

Ovine Reference Materials and Assays for Prion Genetic Testing

USDA-ARS?s Scientific Manuscript database

Background: Genetic predisposition to scrapie in sheep is associated with variation in the peptide sequence of the ovine prion protein encoded by Prnp. Codon variants implicated in scrapie susceptibility or disease progression include those at amino acid positions 112, 136, 141, 154, and 171. Nin...

A structural variant in the 5’-flanking region of the TWIST2 gene affects melanocyte development in belted cattle

PubMed Central

Drögemüller, Cord; Jagannathan, Vidhya; Keller, Irene; Wüthrich, Daniel; Bruggmann, Rémy; Schütz, Ekkehard; Demmel, Steffi; Moser, Simon; Signer-Hasler, Heidi; Pieńkowska-Schelling, Aldona; Schelling, Claude; Sande, Marcos; Rongen, Ronald

2017-01-01

Belted cattle have a circular belt of unpigmented hair and skin around their midsection. The belt is inherited as a monogenic autosomal dominant trait. We mapped the causative variant to a 37 kb segment on bovine chromosome 3. Whole genome sequence data of 2 belted and 130 control cattle yielded only one private genetic variant in the critical interval in the two belted animals. The belt-associated variant was a copy number variant (CNV) involving the quadruplication of a 6 kb non-coding sequence located approximately 16 kb upstream of the TWIST2 gene. Increased copy numbers at this CNV were strongly associated with the belt phenotype in a cohort of 333 cases and 1322 controls. We hypothesized that the CNV causes aberrant expression of TWIST2 during neural crest development, which might negatively affect melanoblasts. Functional studies showed that ectopic expression of bovine TWIST2 in neural crest in transgenic zebrafish led to a decrease in melanocyte numbers. Our results thus implicate an unsuspected involvement of TWIST2 in regulating pigmentation and reveal a non-coding CNV underlying a captivating Mendelian character. PMID:28658273

Integrating evolutionary and regulatory information with a multispecies approach implicates genes and pathways in obsessive-compulsive disorder.

PubMed

Noh, Hyun Ji; Tang, Ruqi; Flannick, Jason; O'Dushlaine, Colm; Swofford, Ross; Howrigan, Daniel; Genereux, Diane P; Johnson, Jeremy; van Grootheest, Gerard; Grünblatt, Edna; Andersson, Erik; Djurfeldt, Diana R; Patel, Paresh D; Koltookian, Michele; M Hultman, Christina; Pato, Michele T; Pato, Carlos N; Rasmussen, Steven A; Jenike, Michael A; Hanna, Gregory L; Stewart, S Evelyn; Knowles, James A; Ruhrmann, Stephan; Grabe, Hans-Jörgen; Wagner, Michael; Rück, Christian; Mathews, Carol A; Walitza, Susanne; Cath, Daniëlle C; Feng, Guoping; Karlsson, Elinor K; Lindblad-Toh, Kerstin

2017-10-17

Obsessive-compulsive disorder is a severe psychiatric disorder linked to abnormalities in glutamate signaling and the cortico-striatal circuit. We sequenced coding and regulatory elements for 608 genes potentially involved in obsessive-compulsive disorder in human, dog, and mouse. Using a new method that prioritizes likely functional variants, we compared 592 cases to 560 controls and found four strongly associated genes, validated in a larger cohort. NRXN1 and HTR2A are enriched for coding variants altering postsynaptic protein-binding domains. CTTNBP2 (synapse maintenance) and REEP3 (vesicle trafficking) are enriched for regulatory variants, of which at least six (35%) alter transcription factor-DNA binding in neuroblastoma cells. NRXN1 achieves genome-wide significance (p = 6.37 × 10 -11 ) when we include 33,370 population-matched controls. Our findings suggest synaptic adhesion as a key component in compulsive behaviors, and show that targeted sequencing plus functional annotation can identify potentially causative variants, even when genomic data are limited.Obsessive-compulsive disorder (OCD) is a neuropsychiatric disorder with symptoms including intrusive thoughts and time-consuming repetitive behaviors. Here Noh and colleagues identify genes enriched for functional variants associated with increased risk of OCD.

Neuroligin 2 nonsense variant associated with anxiety, autism, intellectual disability, hyperphagia, and obesity.

PubMed

Parente, Daniel J; Garriga, Caryn; Baskin, Berivan; Douglas, Ganka; Cho, Megan T; Araujo, Gabriel C; Shinawi, Marwan

2017-01-01

Neuroligins are post-synaptic, cellular adhesion molecules implicated in synaptic formation and function. NLGN2 is strongly linked to inhibitory, GABAergic signaling and is crucial for maintaining the excitation-inhibition balance in the brain. Disruption of the excitation-inhibition balance is associated with neuropsychiatric disease. In animal models, altered NLGN2 expression causes anxiety, developmental delay, motor discoordination, social impairment, aggression, and sensory processing defects. In humans, mutations in NLGN3 and NLGN4 are linked to autism and schizophrenia; NLGN2 missense variants are implicated in schizophrenia. Copy number variants encompassing NLGN2 on 17p13.1 are associated with autism, intellectual disability, metabolic syndrome, diabetes, and dysmorphic features, but an isolated NLGN2 nonsense variant has not yet been described in humans. Here, we describe a 15-year-old male with severe anxiety, obsessive-compulsive behaviors, developmental delay, autism, obesity, macrocephaly, and some dysmorphic features. Exome sequencing identified a heterozygous, de novo, c.441C>A p.(Tyr147Ter) variant in NLGN2 that is predicted to cause loss of normal protein function. This is the first report of an NLGN2 nonsense variant in humans, adding to the accumulating evidence that links synaptic proteins with a spectrum of neurodevelopmental phenotypes. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Whole-exome Sequence Analysis Implicates Rare Il17REL Variants in Familial and Sporadic Inflammatory Bowel Disease.

PubMed

Sasaki, Mark M; Skol, Andrew D; Hungate, Eric A; Bao, Riyue; Huang, Lei; Kahn, Stacy A; Allan, James M; Brant, Steven R; McGovern, Dermot P B; Peter, Inga; Silverberg, Mark S; Cho, Judy H; Kirschner, Barbara S; Onel, Kenan

2016-01-01

Rare variants (<1%) likely contribute significantly to risk for common diseases such as inflammatory bowel disease (IBD) in specific patient subsets, such as those with high familiality. They are, however, extraordinarily challenging to identify. To discover candidate rare variants associated with IBD, we performed whole-exome sequencing on 6 members of a pediatric-onset IBD family with multiple affected individuals. To determine whether the variants discovered in this family are also associated with nonfamilial IBD, we investigated their influence on disease in 2 large case-control (CC) series. We identified 2 rare variants, rs142430606 and rs200958270, both in the established IBD-susceptibility gene IL17REL, carried by all 4 affected family members and their obligate carrier parents. We then demonstrated that both variants are associated with sporadic ulcerative colitis (UC) in 2 independent data sets. For UC in CC 1: rs142430606 (odds ratio [OR] = 2.99, Padj = 0.028; minor allele frequency [MAF]cases = 0.0063, MAFcontrols = 0.0021); rs200958270 (OR = 2.61, Padj = 0.082; MAFcases = 0.0045, MAFcontrols = 0.0017). For UC in CC 2: rs142430606 (OR = 1.94, P = 0.0056; MAFcases = 0.0071, MAFcontrols = 0.0045); rs200958270 (OR = 2.08, P = 0.0028; MAFcases = 0.0071, MAFcontrols = 0.0042). We discover in a family and replicate in 2 CC data sets 2 rare susceptibility variants for IBD, both in IL17REL. Our results illustrate that whole-exome sequencing performed on disease-enriched families to guide association testing can be an efficient strategy for the discovery of rare disease-associated variants. We speculate that rare variants identified in families and confirmed in the general population may be important modifiers of disease risk for patients with a family history, and that genetic testing of these variants may be warranted in this patient subset.

Functional annotation of HOT regions in the human genome: implications for human disease and cancer

PubMed Central

Li, Hao; Chen, Hebing; Liu, Feng; Ren, Chao; Wang, Shengqi; Bo, Xiaochen; Shu, Wenjie

2015-01-01

Advances in genome-wide association studies (GWAS) and large-scale sequencing studies have resulted in an impressive and growing list of disease- and trait-associated genetic variants. Most studies have emphasised the discovery of genetic variation in coding sequences, however, the noncoding regulatory effects responsible for human disease and cancer biology have been substantially understudied. To better characterise the cis-regulatory effects of noncoding variation, we performed a comprehensive analysis of the genetic variants in HOT (high-occupancy target) regions, which are considered to be one of the most intriguing findings of recent large-scale sequencing studies. We observed that GWAS variants that map to HOT regions undergo a substantial net decrease and illustrate development-specific localisation during haematopoiesis. Additionally, genetic risk variants are disproportionally enriched in HOT regions compared with LOT (low-occupancy target) regions in both disease-relevant and cancer cells. Importantly, this enrichment is biased toward disease- or cancer-specific cell types. Furthermore, we observed that cancer cells generally acquire cancer-specific HOT regions at oncogenes through diverse mechanisms of cancer pathogenesis. Collectively, our findings demonstrate the key roles of HOT regions in human disease and cancer and represent a critical step toward further understanding disease biology, diagnosis, and therapy. PMID:26113264

Functional annotation of HOT regions in the human genome: implications for human disease and cancer.

PubMed

Li, Hao; Chen, Hebing; Liu, Feng; Ren, Chao; Wang, Shengqi; Bo, Xiaochen; Shu, Wenjie

2015-06-26

Advances in genome-wide association studies (GWAS) and large-scale sequencing studies have resulted in an impressive and growing list of disease- and trait-associated genetic variants. Most studies have emphasised the discovery of genetic variation in coding sequences, however, the noncoding regulatory effects responsible for human disease and cancer biology have been substantially understudied. To better characterise the cis-regulatory effects of noncoding variation, we performed a comprehensive analysis of the genetic variants in HOT (high-occupancy target) regions, which are considered to be one of the most intriguing findings of recent large-scale sequencing studies. We observed that GWAS variants that map to HOT regions undergo a substantial net decrease and illustrate development-specific localisation during haematopoiesis. Additionally, genetic risk variants are disproportionally enriched in HOT regions compared with LOT (low-occupancy target) regions in both disease-relevant and cancer cells. Importantly, this enrichment is biased toward disease- or cancer-specific cell types. Furthermore, we observed that cancer cells generally acquire cancer-specific HOT regions at oncogenes through diverse mechanisms of cancer pathogenesis. Collectively, our findings demonstrate the key roles of HOT regions in human disease and cancer and represent a critical step toward further understanding disease biology, diagnosis, and therapy.

Germline recessive mutations in PI4KA are associated with perisylvian polymicrogyria, cerebellar hypoplasia and arthrogryposis

PubMed Central

Pagnamenta, Alistair T.; Howard, Malcolm F.; Wisniewski, Eva; Popitsch, Niko; Knight, Samantha J.L.; Keays, David A.; Quaghebeur, Gerardine; Cox, Helen; Cox, Phillip; Balla, Tamas; Taylor, Jenny C.; Kini, Usha

2015-01-01

Polymicrogyria (PMG) is a structural brain abnormality involving the cerebral cortex that results from impaired neuronal migration and although several genes have been implicated, many cases remain unsolved. In this study, exome sequencing in a family where three fetuses had all been diagnosed with PMG and cerebellar hypoplasia allowed us to identify regions of the genome for which both chromosomes were shared identical-by-descent, reducing the search space for causative variants to 8.6% of the genome. In these regions, the only plausibly pathogenic mutations were compound heterozygous variants in PI4KA, which Sanger sequencing confirmed segregated consistent with autosomal recessive inheritance. The paternally transmitted variant predicted a premature stop mutation (c.2386C>T; p.R796X), whereas the maternally transmitted variant predicted a missense substitution (c.5560G>A; p.D1854N) at a conserved residue within the catalytic domain. Functional studies using expressed wild-type or mutant PI4KA enzyme confirmed the importance of p.D1854 for kinase activity. Our results emphasize the importance of phosphoinositide signalling in early brain development. PMID:25855803

Effect of S267F variant of NTCP on the patients with chronic hepatitis B.

PubMed

Lee, Hye Won; Park, Hye Jung; Jin, Bora; Dezhbord, Mehrangiz; Kim, Do Young; Han, Kwang-Hyub; Ryu, Wang-Shick; Kim, Seungtaek; Ahn, Sang Hoon

2017-12-15

Sodium taurocholate cotransporting polypeptide (NTCP) was identified as an entry receptor for hepatitis B virus (HBV) infection. The substitution of serine at position 267 of NTCP with phenylalanine (S267F) is an Asian-specific variation that hampers HBV entry in vitro. In this study, we aimed to evaluate the prevalence of S267F polymorphism in Korean patients with chronic hepatitis B (CHB) and its association with disease progression and potential viral evolution in the preS1 domain of HBV. We found that the frequency of the S267F variant of NTCP in CHB patients and controls was 2.7% and 5.7% (P = 0.031), respectively, and that those who had S267F variant were less susceptible to chronic HBV infection. The frequency of the S267F variant in CHB, cirrhosis and hepatocellular carcinoma (HCC) patients was 3.3%, 0.9%, and 3.5%, respectively. Thus, the S267F variant correlated significantly with a lower risk for cirrhosis (P = 0.036). Sequencing preS1 domain of HBV from the patients who had S267F variant revealed no significant sequence change compared to the wild type. In conclusion, the S267F variant of NTCP is clinically associated with a lower risk of chronic HBV infection and cirrhosis development, which implicates suppressing HBV entry could reduce the disease burden.

Mutation analysis of genes within the dynactin complex in a cohort of hereditary peripheral neuropathies.

PubMed

Tey, S; Ahmad-Annuar, A; Drew, A P; Shahrizaila, N; Nicholson, G A; Kennerson, M L

2016-08-01

The cytoplasmic dynein-dynactin genes are attractive candidates for neurodegenerative disorders given their functional role in retrograde transport along neurons. The cytoplasmic dynein heavy chain (DYNC1H1) gene has been implicated in various neurodegenerative disorders, and dynactin 1 (DCTN1) genes have been implicated in a wide spectrum of disorders including motor neuron disease, Parkinson's disease, spinobulbar muscular atrophy and hereditary spastic paraplegia. However, the involvement of other dynactin genes with inherited peripheral neuropathies (IPN) namely, hereditary sensory neuropathy, hereditary motor neuropathy and Charcot-Marie-Tooth disease is under reported. We screened eight genes; DCTN1-6 and ACTR1A and ACTR1B in 136 IPN patients using whole-exome sequencing and high-resolution melt (HRM) analysis. Eight non-synonymous variants (including one novel variant) and three synonymous variants were identified. Four variants have been reported previously in other studies, however segregation analysis within family members excluded them from causing IPN in these families. No variants of disease significance were identified in this study suggesting the dynactin genes are unlikely to be a common cause of IPNs. However, with the ease of querying gene variants from exome data, these genes remain worthwhile candidates to assess unsolved IPN families for variants that may affect the function of the proteins. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Human papillomavirus type-16 variants in Quechua aboriginals from Argentina.

PubMed

Picconi, María Alejandra; Alonio, Lidia Virginia; Sichero, Laura; Mbayed, Viviana; Villa, Luisa Lina; Gronda, Jorge; Campos, Rodolfo; Teyssié, Angélica

2003-04-01

Cervical carcinoma is the leading cause of cancer death in Quechua indians from Jujuy (northwestern Argentina). To determine the prevalence of HPV-16 variants, 106 HPV-16 positive cervical samples were studied, including 33 low-grade squamous intraepithelial lesions (LSIL), 28 high-grade squamous intraepithelial lesions (HSIL), 9 invasive cervical cancer (ICC), and 36 samples from women with normal colposcopy and cytology. HPV genome variability was examined in the L1 and E6 genes by PCR-hybridization. In a subset of 20 samples, a LCR fragment was also analyzed by PCR-sequencing. Most variants belonged to the European branch with subtle differences that depended on the viral gene fragment studied. Only about 10% of the specimens had non-European variants, including eight Asian-American, two Asian, and one North-American-1. E6 gene analysis revealed that 43% of the samples were identical to HPV-16 prototype, while 57% corresponded to variants. Interestingly, the majority (87%) of normal smears had HPV-16 prototype, whereas variants were detected mainly in SIL and ICC. LCR sequencing yielded 80% of variants, including 69% of European, 19% Asian-American, and 12% Asian. We identified a new variant, the Argentine Quechua-51 (AQ-51), similar to B-14 plus two additional changes: G7842-->A and A7837-->C; phylogenetic inference allocated it in the Asian-American branch. The high proportion of European variants may reflect Spanish colonial influence on these native Inca descendants. The predominance of HPV-16 variants in pathologic samples when compared to normal controls could have implications for the natural history of cervical lesions. Copyright 2003 Wiley-Liss, Inc.

High-throughput sequencing of mGluR signaling pathway genes reveals enrichment of rare variants in autism.

PubMed

Kelleher, Raymond J; Geigenmüller, Ute; Hovhannisyan, Hayk; Trautman, Edwin; Pinard, Robert; Rathmell, Barbara; Carpenter, Randall; Margulies, David

2012-01-01

Identification of common molecular pathways affected by genetic variation in autism is important for understanding disease pathogenesis and devising effective therapies. Here, we test the hypothesis that rare genetic variation in the metabotropic glutamate-receptor (mGluR) signaling pathway contributes to autism susceptibility. Single-nucleotide variants in genes encoding components of the mGluR signaling pathway were identified by high-throughput multiplex sequencing of pooled samples from 290 non-syndromic autism cases and 300 ethnically matched controls on two independent next-generation platforms. This analysis revealed significant enrichment of rare functional variants in the mGluR pathway in autism cases. Higher burdens of rare, potentially deleterious variants were identified in autism cases for three pathway genes previously implicated in syndromic autism spectrum disorder, TSC1, TSC2, and SHANK3, suggesting that genetic variation in these genes also contributes to risk for non-syndromic autism. In addition, our analysis identified HOMER1, which encodes a postsynaptic density-localized scaffolding protein that interacts with Shank3 to regulate mGluR activity, as a novel autism-risk gene. Rare, potentially deleterious HOMER1 variants identified uniquely in the autism population affected functionally important protein regions or regulatory sequences and co-segregated closely with autism among children of affected families. We also identified rare ASD-associated coding variants predicted to have damaging effects on components of the Ras/MAPK cascade. Collectively, these findings suggest that altered signaling downstream of mGluRs contributes to the pathogenesis of non-syndromic autism.

High-Throughput Sequencing of mGluR Signaling Pathway Genes Reveals Enrichment of Rare Variants in Autism

PubMed Central

Hovhannisyan, Hayk; Trautman, Edwin; Pinard, Robert; Rathmell, Barbara; Carpenter, Randall; Margulies, David

2012-01-01

Identification of common molecular pathways affected by genetic variation in autism is important for understanding disease pathogenesis and devising effective therapies. Here, we test the hypothesis that rare genetic variation in the metabotropic glutamate-receptor (mGluR) signaling pathway contributes to autism susceptibility. Single-nucleotide variants in genes encoding components of the mGluR signaling pathway were identified by high-throughput multiplex sequencing of pooled samples from 290 non-syndromic autism cases and 300 ethnically matched controls on two independent next-generation platforms. This analysis revealed significant enrichment of rare functional variants in the mGluR pathway in autism cases. Higher burdens of rare, potentially deleterious variants were identified in autism cases for three pathway genes previously implicated in syndromic autism spectrum disorder, TSC1, TSC2, and SHANK3, suggesting that genetic variation in these genes also contributes to risk for non-syndromic autism. In addition, our analysis identified HOMER1, which encodes a postsynaptic density-localized scaffolding protein that interacts with Shank3 to regulate mGluR activity, as a novel autism-risk gene. Rare, potentially deleterious HOMER1 variants identified uniquely in the autism population affected functionally important protein regions or regulatory sequences and co-segregated closely with autism among children of affected families. We also identified rare ASD-associated coding variants predicted to have damaging effects on components of the Ras/MAPK cascade. Collectively, these findings suggest that altered signaling downstream of mGluRs contributes to the pathogenesis of non-syndromic autism. PMID:22558107

G23D: Online tool for mapping and visualization of genomic variants on 3D protein structures.

PubMed

Solomon, Oz; Kunik, Vered; Simon, Amos; Kol, Nitzan; Barel, Ortal; Lev, Atar; Amariglio, Ninette; Somech, Raz; Rechavi, Gidi; Eyal, Eran

2016-08-26

Evaluation of the possible implications of genomic variants is an increasingly important task in the current high throughput sequencing era. Structural information however is still not routinely exploited during this evaluation process. The main reasons can be attributed to the partial structural coverage of the human proteome and the lack of tools which conveniently convert genomic positions, which are the frequent output of genomic pipelines, to proteins and structure coordinates. We present G23D, a tool for conversion of human genomic coordinates to protein coordinates and protein structures. G23D allows mapping of genomic positions/variants on evolutionary related (and not only identical) protein three dimensional (3D) structures as well as on theoretical models. By doing so it significantly extends the space of variants for which structural insight is feasible. To facilitate interpretation of the variant consequence, pathogenic variants, functional sites and polymorphism sites are displayed on protein sequence and structure diagrams alongside the input variants. G23D also provides modeling of the mutant structure, analysis of intra-protein contacts and instant access to functional predictions and predictions of thermo-stability changes. G23D is available at http://www.sheba-cancer.org.il/G23D . G23D extends the fraction of variants for which structural analysis is applicable and provides better and faster accessibility for structural data to biologists and geneticists who routinely work with genomic information.

Information Topics of Greatest Interest for Return of Genome Sequencing Results among Women Diagnosed with Breast Cancer at a Young Age.

PubMed

Seo, Joann; Ivanovich, Jennifer; Goodman, Melody S; Biesecker, Barbara B; Kaphingst, Kimberly A

2017-06-01

We investigated what information women diagnosed with breast cancer at a young age would want to learn when genome sequencing results are returned. We conducted 60 semi-structured interviews with women diagnosed with breast cancer at age 40 or younger. We examined what specific information participants would want to learn across result types and for each type of result, as well as how much information they would want. Genome sequencing was not offered to participants as part of the study. Two coders independently coded interview transcripts; analysis was conducted using NVivo10. Across result types, participants wanted to learn about health implications, risk and prevalence in quantitative terms, causes of variants, and causes of diseases. Participants wanted to learn actionable information for variants affecting risk of preventable or treatable disease, medication response, and carrier status. The amount of desired information differed for variants affecting risk of unpreventable or untreatable disease, with uncertain significance, and not health-related. Women diagnosed with breast cancer at a young age recognize the value of genome sequencing results in identifying potential causes and effective treatments and expressed interest in using the information to help relatives and to further understand their other health risks. Our findings can inform the development of effective feedback strategies for genome sequencing that meet patients' information needs and preferences.

Walking the interactome for candidate prioritization in exome sequencing studies of Mendelian diseases

DOE PAGES

Smedley, Damian; Kohler, Sebastian; Czeschik, Johanna Christina; ...

2014-07-30

Here, whole-exome sequencing (WES) has opened up previously unheard of possibilities for identifying novel disease genes in Mendelian disorders, only about half of which have been elucidated to date. However, interpretation of WES data remains challenging. As a result, we analyze protein–protein association (PPA) networks to identify candidate genes in the vicinity of genes previously implicated in a disease. The analysis, using a random-walk with restart (RWR) method, is adapted to the setting of WES by developing a composite variant-gene relevance score based on the rarity, location and predicted pathogenicity of variants and the RWR evaluation of genes harboring themore » variants. Benchmarking using known disease variants from 88 disease-gene families reveals that the correct gene is ranked among the top 10 candidates in ≥50% of cases, a figure which we confirmed using a prospective study of disease genes identified in 2012 and PPA data produced before that date. In conclusion, we implement our method in a freely available Web server, ExomeWalker, that displays a ranked list of candidates together with information on PPAs, frequency and predicted pathogenicity of the variants to allow quick and effective searches for candidates that are likely to reward closer investigation.« less

Walking the interactome for candidate prioritization in exome sequencing studies of Mendelian diseases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smedley, Damian; Kohler, Sebastian; Czeschik, Johanna Christina

Here, whole-exome sequencing (WES) has opened up previously unheard of possibilities for identifying novel disease genes in Mendelian disorders, only about half of which have been elucidated to date. However, interpretation of WES data remains challenging. As a result, we analyze protein–protein association (PPA) networks to identify candidate genes in the vicinity of genes previously implicated in a disease. The analysis, using a random-walk with restart (RWR) method, is adapted to the setting of WES by developing a composite variant-gene relevance score based on the rarity, location and predicted pathogenicity of variants and the RWR evaluation of genes harboring themore » variants. Benchmarking using known disease variants from 88 disease-gene families reveals that the correct gene is ranked among the top 10 candidates in ≥50% of cases, a figure which we confirmed using a prospective study of disease genes identified in 2012 and PPA data produced before that date. In conclusion, we implement our method in a freely available Web server, ExomeWalker, that displays a ranked list of candidates together with information on PPAs, frequency and predicted pathogenicity of the variants to allow quick and effective searches for candidates that are likely to reward closer investigation.« less

mirVAFC: A Web Server for Prioritizations of Pathogenic Sequence Variants from Exome Sequencing Data via Classifications.

PubMed

Li, Zhongshan; Liu, Zhenwei; Jiang, Yi; Chen, Denghui; Ran, Xia; Sun, Zhong Sheng; Wu, Jinyu

2017-01-01

Exome sequencing has been widely used to identify the genetic variants underlying human genetic disorders for clinical diagnoses, but the identification of pathogenic sequence variants among the huge amounts of benign ones is complicated and challenging. Here, we describe a new Web server named mirVAFC for pathogenic sequence variants prioritizations from clinical exome sequencing (CES) variant data of single individual or family. The mirVAFC is able to comprehensively annotate sequence variants, filter out most irrelevant variants using custom criteria, classify variants into different categories as for estimated pathogenicity, and lastly provide pathogenic variants prioritizations based on classifications and mutation effects. Case studies using different types of datasets for different diseases from publication and our in-house data have revealed that mirVAFC can efficiently identify the right pathogenic candidates as in original work in each case. Overall, the Web server mirVAFC is specifically developed for pathogenic sequence variant identifications from family-based CES variants using classification-based prioritizations. The mirVAFC Web server is freely accessible at https://www.wzgenomics.cn/mirVAFC/. © 2016 WILEY PERIODICALS, INC.

Novel and ultra-rare damaging variants in neuropeptide signaling are associated with disordered eating behaviors

PubMed Central

Bahl, Ethan; Hannah, Claire; Hofammann, Dabney; Acevedo, Summer; Cui, Huxing; McAdams, Carrie J.

2017-01-01

Objective Eating disorders develop through a combination of genetic vulnerability and environmental stress, however the genetic basis of this risk is unknown. Methods To understand the genetic basis of this risk, we performed whole exome sequencing on 93 unrelated individuals with eating disorders (38 restricted-eating and 55 binge-eating) to identify novel damaging variants. Candidate genes with an excessive burden of predicted damaging variants were then prioritized based upon an unbiased, data-driven bioinformatic analysis. One top candidate pathway was empirically tested for therapeutic potential in a mouse model of binge-like eating. Results An excessive burden of novel damaging variants was identified in 186 genes in the restricted-eating group and 245 genes in the binge-eating group. This list is significantly enriched (OR = 4.6, p<0.0001) for genes involved in neuropeptide/neurotrophic pathways implicated in appetite regulation, including neurotensin-, glucagon-like peptide 1- and BDNF-signaling. Administration of the glucagon-like peptide 1 receptor agonist exendin-4 significantly reduced food intake in a mouse model of ‘binge-like’ eating. Conclusions These findings implicate ultra-rare and novel damaging variants in neuropeptide/neurotropic factor signaling pathways in the development of eating disorder behaviors and identify glucagon-like peptide 1-receptor agonists as a potential treatment for binge eating. PMID:28846695

Missense-depleted regions in population exomes implicate ras superfamily nucleotide-binding protein alteration in patients with brain malformation

PubMed Central

Ge, Xiaoyan; Gong, Henry; Dumas, Kevin; Litwin, Jessica; Phillips, Joanna J; Waisfisz, Quinten; Weiss, Marjan M; Hendriks, Yvonne; Stuurman, Kyra E; Nelson, Stanley F; Grody, Wayne W; Lee, Hane; Kwok, Pui-Yan; Shieh, Joseph T C

2016-01-01

Genomic sequence interpretation can miss clinically relevant missense variants for several reasons. Rare missense variants are numerous in the exome and difficult to prioritise. Affected genes may also not have existing disease association. To improve variant prioritisation, we leverage population exome data to identify intragenic missense-depleted regions (MDRs) genome-wide that may be important in disease. We then use missense depletion analyses to help prioritise undiagnosed disease exome variants. We demonstrate application of this strategy to identify a novel gene association for human brain malformation. We identified de novo missense variants that affect the GDP/GTP-binding site of ARF1 in three unrelated patients. Corresponding functional analysis suggests ARF1 GDP/GTP-activation is affected by the specific missense mutations associated with heterotopia. These findings expand the genetic pathway underpinning neurologic disease that classically includes FLNA. ARF1 along with ARFGEF2 add further evidence implicating ARF/GEFs in the brain. Using functional ontology, top MDR-containing genes were highly enriched for nucleotide-binding function, suggesting these may be candidates for human disease. Routine consideration of MDR in the interpretation of exome data for rare diseases may help identify strong genetic factors for many severe conditions, infertility/reduction in reproductive capability, and embryonic conditions contributing to preterm loss. PMID:28868155

Isolation and characterization of alternatively spliced variants of the mouse sigma1 receptor gene, Sigmar1.

PubMed

Pan, Ling; Pasternak, David A; Xu, Jin; Xu, Mingming; Lu, Zhigang; Pasternak, Gavril W; Pan, Ying-Xian

2017-01-01

The sigma1 receptor acts as a chaperone at the endoplasmic reticulum, associates with multiple proteins in various cellular systems, and involves in a number of diseases, such as addiction, pain, cancer and psychiatric disorders. The sigma1 receptor is encoded by the single copy SIGMAR1 gene. The current study identifies five alternatively spliced variants of the mouse sigma1 receptor gene using a polymerase chain reaction cloning approach. All the splice variants are generated by exon skipping or alternative 3' or 5' splicing, producing the truncated sigma1 receptor. Similar alternative splicing has been observed in the human SIGMAR1 gene based on the molecular cloning or genome sequence prediction, suggesting conservation of alternative splicing of SIGMAR1 gene. Using quantitative polymerase chain reactions, we demonstrate differential expression of several splice variants in mouse tissues and brain regions. When expressed in HEK293 cells, all the splice variants fail to bind sigma ligands, implicating that each truncated region in these splice variants is important for ligand binding. However, co-immunoprecipitation (Co-IP) study in HEK293 cells co-transfected with tagged constructs reveals that all the splice variants maintain their ability to physically associate with a mu opioid receptor (mMOR-1), providing useful information to correlate the motifs/sequences necessary for their physical association. Furthermore, a competition Co-IP study showed that all the variants can disrupt in a dose-dependent manner the dimerization of the original sigma1 receptor with mMOR-1, suggesting a potential dominant negative function and providing significant insights into their function.

Whole exome sequencing implicates eye development, the unfolded protein response and plasma membrane homeostasis in primary open-angle glaucoma

PubMed Central

Souzeau, Emmanuelle; Sharma, Shiwani; Landers, John; Mills, Richard; Goldberg, Ivan; Healey, Paul R.; Graham, Stuart; Hewitt, Alex W.; Mackey, David A.; Galanopoulos, Anna; Casson, Robert J.; Ruddle, Jonathan B.; Ellis, Jonathan; Leo, Paul; Brown, Matthew A.; MacGregor, Stuart; Lynn, David J.; Burdon, Kathryn P.; Craig, Jamie E.

2017-01-01

Purpose To identify biological processes associated with POAG and its subtypes, high-tension (HTG) and normal-tension glaucoma (NTG), by analyzing rare potentially damaging genetic variants. Methods A total of 122 and 65 unrelated HTG and NTG participants, respectively, with early onset advanced POAG, 103 non-glaucoma controls and 993 unscreened ethnicity-matched controls were included in this study. Study participants without myocilin disease-causing variants and non-glaucoma controls were subjected to whole exome sequencing on an Illumina HiSeq2000. Exomes of participants were sequenced on an Illumina HiSeq2000. Qualifying variants were rare in the general population (MAF < 0.001) and potentially functionally damaging (nonsense, frameshift, splice or predicted pathogenic using SIFT or Polyphen2 software). Genes showing enrichment of qualifying variants in cases were selected for pathway and network analysis using InnateDB. Results POAG cases showed enrichment of rare variants in camera-type eye development genes (p = 1.40×10–7, corrected p = 3.28×10–4). Implicated eye development genes were related to neuronal or retinal development. HTG cases were significantly enriched for key regulators in the unfolded protein response (UPR) (p = 7.72×10–5, corrected p = 0.013). The UPR is known to be involved in myocilin-related glaucoma; our results suggest the UPR has a role in non-myocilin causes of HTG. NTG cases showed enrichment in ion channel transport processes (p = 1.05×10–4, corrected p = 0.027) including calcium, chloride and phospholipid transporters involved in plasma membrane homeostasis. Network analysis also revealed enrichment of the MHC Class I antigen presentation pathway in HTG, and the EGFR1 and cell-cycle pathways in both HTG and NTG. Conclusion This study suggests that mutations in eye development genes are enriched in POAG. HTG can result from aberrant responses to protein misfolding which may be amenable to molecular chaperone therapy. NTG is associated with impaired plasma membrane homeostasis increasing susceptibility to apoptosis. PMID:28264060

Next-Generation Sequencing in Oncology: Genetic Diagnosis, Risk Prediction and Cancer Classification

PubMed Central

Kamps, Rick; Brandão, Rita D.; van den Bosch, Bianca J.; Paulussen, Aimee D. C.; Xanthoulea, Sofia; Blok, Marinus J.; Romano, Andrea

2017-01-01

Next-generation sequencing (NGS) technology has expanded in the last decades with significant improvements in the reliability, sequencing chemistry, pipeline analyses, data interpretation and costs. Such advances make the use of NGS feasible in clinical practice today. This review describes the recent technological developments in NGS applied to the field of oncology. A number of clinical applications are reviewed, i.e., mutation detection in inherited cancer syndromes based on DNA-sequencing, detection of spliceogenic variants based on RNA-sequencing, DNA-sequencing to identify risk modifiers and application for pre-implantation genetic diagnosis, cancer somatic mutation analysis, pharmacogenetics and liquid biopsy. Conclusive remarks, clinical limitations, implications and ethical considerations that relate to the different applications are provided. PMID:28146134

Clinical implications of SCN1A missense and truncation variants in a large Japanese cohort with Dravet syndrome.

PubMed

Ishii, Atsushi; Watkins, Joseph C; Chen, Debbie; Hirose, Shinichi; Hammer, Michael F

2017-02-01

Two major classes of SCN1A variants are associated with Dravet syndrome (DS): those that result in haploinsufficiency (truncating) and those that result in an amino acid substitution (missense). The aim of this retrospective study was to describe the first large cohort of Japanese patients with SCN1A mutation-positive DS (n = 285), and investigate the relationship between variant (type and position) and clinical expression and response to treatment. We sequenced all exons and intron-exon boundaries of SCN1A in our cohort, investigated differences in the distribution of truncating and missense variants, tested for associations between variant type and phenotype, and compared these patterns with those of cohorts with milder epilepsy and healthy individuals. Unlike truncation variants, missense variants are found at higher density in the S4 voltage sensor and pore loops and at lower density in the domain I-II and II-III linkers and the first three segments of domain II. Relative to healthy individuals, there is an increased frequency of truncating (but not missense) variants in the noncoding C-terminus. The rate of cognitive decline is more rapid for patients with truncation variants regardless of age at seizure onset, whereas age at onset is a predictor of the rate of cognitive decline for patients with missense variants. We found significant differences in the distribution of truncating and missense variants across the SCN1A sequence among healthy individuals, patients with DS, and those with milder forms of SCN1A-variant positive epilepsy. Testing for associations with phenotype revealed that variant type can be predictive of rate of cognitive decline. Analysis of descriptive medication data suggests that in addition to conventional drug therapy in DS, bromide, clonazepam and topiramate may reduce seizure frequency. Wiley Periodicals, Inc. © 2016 International League Against Epilepsy.

Excessive burden of lysosomal storage disorder gene variants in Parkinson's disease.

PubMed

Robak, Laurie A; Jansen, Iris E; van Rooij, Jeroen; Uitterlinden, André G; Kraaij, Robert; Jankovic, Joseph; Heutink, Peter; Shulman, Joshua M

2017-12-01

Mutations in the glucocerebrosidase gene (GBA), which cause Gaucher disease, are also potent risk factors for Parkinson's disease. We examined whether a genetic burden of variants in other lysosomal storage disorder genes is more broadly associated with Parkinson's disease susceptibility. The sequence kernel association test was used to interrogate variant burden among 54 lysosomal storage disorder genes, leveraging whole exome sequencing data from 1156 Parkinson's disease cases and 1679 control subjects. We discovered a significant burden of rare, likely damaging lysosomal storage disorder gene variants in association with Parkinson's disease risk. The association signal was robust to the exclusion of GBA, and consistent results were obtained in two independent replication cohorts, including 436 cases and 169 controls with whole exome sequencing and an additional 6713 cases and 5964 controls with exome-wide genotyping. In secondary analyses designed to highlight the specific genes driving the aggregate signal, we confirmed associations at the GBA and SMPD1 loci and newly implicate CTSD, SLC17A5, and ASAH1 as candidate Parkinson's disease susceptibility genes. In our discovery cohort, the majority of Parkinson's disease cases (56%) have at least one putative damaging variant in a lysosomal storage disorder gene, and 21% carry multiple alleles. Our results highlight several promising new susceptibility loci and reinforce the importance of lysosomal mechanisms in Parkinson's disease pathogenesis. We suggest that multiple genetic hits may act in combination to degrade lysosomal function, enhancing Parkinson's disease susceptibility. © The Author (2017). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Identification of point mutations in clinical Staphylococcus aureus strains that produce small-colony variants auxotrophic for menadione.

PubMed

Dean, Melissa A; Olsen, Randall J; Long, S Wesley; Rosato, Adriana E; Musser, James M

2014-04-01

Staphylococcus aureus small-colony variants (SCVs) are implicated in chronic and relapsing infections that are difficult to diagnose and treat. Despite many years of study, the underlying molecular mechanisms and virulence effect of the small-colony phenotype remain incompletely understood. We sequenced the genomes of five S. aureus SCV strains recovered from human patients and discovered previously unidentified nonsynonymous point mutations in three genes encoding proteins in the menadione biosynthesis pathway. Analysis of genetic revertants and complementation with wild-type alleles confirmed that these mutations caused the SCV phenotype and decreased virulence for mice.

Genetic diversity of Grapevine virus A in Washington and California vineyards.

PubMed

Alabi, Olufemi J; Al Rwahnih, Maher; Mekuria, Tefera A; Naidu, Rayapati A

2014-05-01

Grapevine virus A (GVA; genus Vitivirus, family Betaflexiviridae) has been implicated with the Kober stem grooving disorder of the rugose wood disease complex. In this study, 26 isolates of GVA recovered from wine grape (Vitis vinifera) cultivars from California and Washington were analyzed for their genetic diversity. An analysis of a portion of the RNA-dependent RNA polymerase (RdRp) and complete coat protein (CP) sequences revealed intra- and inter-isolate sequence diversity. Our results indicated that both RdRp and CP are under strong negative selection based on the normalized values for the ratio of nonsynonymous substitutions per nonsynonymous site to synonymous substitutions per synonymous site. A global phylogenetic analysis of CP sequences revealed segregation of virus isolates into four major clades with no geographic clustering. In contrast, the RdRp-based phylogenetic tree indicated segregation of GVA isolates from California and Washington into six clades, independent of geographic origin or cultivar. Phylogenetic network coupled with recombination analyses showed putative recombination events in both RdRp and CP sequence data sets, with more of these events located in the CP sequence. The preponderance of divergent variants of GVA co-replicating within individual grapevines could increase viral genotypic complexity with implications for phylogenetic analysis and evolutionary history of the virus. The knowledge of genetic diversity of GVA generated in this study will provide a foundation for elucidating the epidemiological characteristics of virus populations at different scales and implementing appropriate management strategies for minimizing the spread of genetic variants of the virus by vectors and via planting materials supplied to nurseries and grape growers.

The protein cofactor allows the sequence of an RNase P ribozyme to diversify by maintaining the catalytically active structure of the enzyme.

PubMed Central

Kim, J J; Kilani, A F; Zhan, X; Altman, S; Liu, F

1997-01-01

To study the effect proteins have on the catalysis and evolution of RNA enzymes, we simulated evolution of RNase P catalytic M1 RNA in vitro, in the presence and absence of its C5 protein cofactor. In the presence of C5, functional M1 sequence variants (not catalytically active in the absence of C5) were selected in addition to those identical to M1. C5 maintains the catalytically active structure of the variants and allows for an enhanced spectrum of M1 molecules to function in the context of a ribonucleoprotein (RNP) complex. The generation of an RNP enzyme, requiring both RNA and protein components, from a catalytically active RNA molecule has implications for how modern RNP complexes evolved from ancestral RNAs. PMID:9174096

Fast single-pass alignment and variant calling using sequencing data

USDA-ARS?s Scientific Manuscript database

Sequencing research requires efficient computation. Few programs use already known information about DNA variants when aligning sequence data to the reference map. New program findmap.f90 reads the previous variant list before aligning sequence, calling variant alleles, and summing the allele counts...

Clinical Sequencing Exploratory Research Consortium: Accelerating Evidence-Based Practice of Genomic Medicine.

PubMed

Green, Robert C; Goddard, Katrina A B; Jarvik, Gail P; Amendola, Laura M; Appelbaum, Paul S; Berg, Jonathan S; Bernhardt, Barbara A; Biesecker, Leslie G; Biswas, Sawona; Blout, Carrie L; Bowling, Kevin M; Brothers, Kyle B; Burke, Wylie; Caga-Anan, Charlisse F; Chinnaiyan, Arul M; Chung, Wendy K; Clayton, Ellen W; Cooper, Gregory M; East, Kelly; Evans, James P; Fullerton, Stephanie M; Garraway, Levi A; Garrett, Jeremy R; Gray, Stacy W; Henderson, Gail E; Hindorff, Lucia A; Holm, Ingrid A; Lewis, Michelle Huckaby; Hutter, Carolyn M; Janne, Pasi A; Joffe, Steven; Kaufman, David; Knoppers, Bartha M; Koenig, Barbara A; Krantz, Ian D; Manolio, Teri A; McCullough, Laurence; McEwen, Jean; McGuire, Amy; Muzny, Donna; Myers, Richard M; Nickerson, Deborah A; Ou, Jeffrey; Parsons, Donald W; Petersen, Gloria M; Plon, Sharon E; Rehm, Heidi L; Roberts, J Scott; Robinson, Dan; Salama, Joseph S; Scollon, Sarah; Sharp, Richard R; Shirts, Brian; Spinner, Nancy B; Tabor, Holly K; Tarczy-Hornoch, Peter; Veenstra, David L; Wagle, Nikhil; Weck, Karen; Wilfond, Benjamin S; Wilhelmsen, Kirk; Wolf, Susan M; Wynn, Julia; Yu, Joon-Ho

2016-06-02

Despite rapid technical progress and demonstrable effectiveness for some types of diagnosis and therapy, much remains to be learned about clinical genome and exome sequencing (CGES) and its role within the practice of medicine. The Clinical Sequencing Exploratory Research (CSER) consortium includes 18 extramural research projects, one National Human Genome Research Institute (NHGRI) intramural project, and a coordinating center funded by the NHGRI and National Cancer Institute. The consortium is exploring analytic and clinical validity and utility, as well as the ethical, legal, and social implications of sequencing via multidisciplinary approaches; it has thus far recruited 5,577 participants across a spectrum of symptomatic and healthy children and adults by utilizing both germline and cancer sequencing. The CSER consortium is analyzing data and creating publically available procedures and tools related to participant preferences and consent, variant classification, disclosure and management of primary and secondary findings, health outcomes, and integration with electronic health records. Future research directions will refine measures of clinical utility of CGES in both germline and somatic testing, evaluate the use of CGES for screening in healthy individuals, explore the penetrance of pathogenic variants through extensive phenotyping, reduce discordances in public databases of genes and variants, examine social and ethnic disparities in the provision of genomics services, explore regulatory issues, and estimate the value and downstream costs of sequencing. The CSER consortium has established a shared community of research sites by using diverse approaches to pursue the evidence-based development of best practices in genomic medicine. Copyright © 2016 American Society of Human Genetics. All rights reserved.

Identification of novel mutations and sequence variants in the SOX2 and CHX10 genes in patients with anophthalmia/microphthalmia

PubMed Central

Zhou, Jie; Kherani, Femida; Bardakjian, Tanya M.; Katowitz, James; Hughes, Nkecha; Schimmenti, Lisa A.; Schneider, Adele

2008-01-01

Purpose Mutations in the SOX2 and CHX10 genes have been reported in patients with anophthalmia and/or microphthalmia. In this study, we evaluated 34 anophthalmic/microphthalmic patient DNA samples (two sets of siblings included) for mutations and sequence variants in SOX2 and CHX10. Methods Conformational sensitive gel electrophoresis (CSGE) was used for the initial SOX2 and CHX10 screening of 34 affected individuals (two sets of siblings), five unaffected family members, and 80 healthy controls. Patient samples containing heteroduplexes were selected for sequence analysis. Base pair changes in SOX2 and CHX10 were confirmed by sequencing bidirectionally in patient samples. Results Two novel heterozygous mutations and two sequence variants (one known) in SOX2 were identified in this cohort. Mutation c.310 G>T (p. Glu104X), found in one patient, was in the region encoding the high mobility group (HMG) DNA-binding domain and resulted in a change from glutamic acid to a stop codon. The second mutation, noted in two affected siblings, was a single nucleotide deletion c.549delC (p. Pro184ArgfsX19) in the region encoding the activation domain, resulting in a frameshift and premature termination of the coding sequence. The shortened protein products may result in the loss of function. In addition, a novel nucleotide substitution c.*557G>A was identified in the 3′-untranslated region in one patient. The relationship between the nucleotide change and the protein function is indeterminate. A known single nucleotide polymorphism (c. *469 C>A, SNP rs11915160) was also detected in 2 of the 34 patients. Screening of CHX10 identified two synonymous sequence variants, c.471 C>T (p.Ser157Ser, rs35435463) and c.579 G>A (p. Gln193Gln, novel SNP), and one non-synonymous sequence variant, c.871 G>A (p. Asp291Asn, novel SNP). The non-synonymous polymorphism was also present in healthy controls, suggesting non-causality. Conclusions These results support the role of SOX2 in ocular development. Loss of SOX2 function results in severe eye malformation. CHX10 was not implicated with microphthalmia/anophthalmia in our patient cohort. PMID:18385794

Population-based rare variant detection via pooled exome or custom hybridization capture with or without individual indexing.

PubMed

Ramos, Enrique; Levinson, Benjamin T; Chasnoff, Sara; Hughes, Andrew; Young, Andrew L; Thornton, Katherine; Li, Allie; Vallania, Francesco L M; Province, Michael; Druley, Todd E

2012-12-06

Rare genetic variation in the human population is a major source of pathophysiological variability and has been implicated in a host of complex phenotypes and diseases. Finding disease-related genes harboring disparate functional rare variants requires sequencing of many individuals across many genomic regions and comparing against unaffected cohorts. However, despite persistent declines in sequencing costs, population-based rare variant detection across large genomic target regions remains cost prohibitive for most investigators. In addition, DNA samples are often precious and hybridization methods typically require large amounts of input DNA. Pooled sample DNA sequencing is a cost and time-efficient strategy for surveying populations of individuals for rare variants. We set out to 1) create a scalable, multiplexing method for custom capture with or without individual DNA indexing that was amenable to low amounts of input DNA and 2) expand the functionality of the SPLINTER algorithm for calling substitutions, insertions and deletions across either candidate genes or the entire exome by integrating the variant calling algorithm with the dynamic programming aligner, Novoalign. We report methodology for pooled hybridization capture with pre-enrichment, indexed multiplexing of up to 48 individuals or non-indexed pooled sequencing of up to 92 individuals with as little as 70 ng of DNA per person. Modified solid phase reversible immobilization bead purification strategies enable no sample transfers from sonication in 96-well plates through adapter ligation, resulting in 50% less library preparation reagent consumption. Custom Y-shaped adapters containing novel 7 base pair index sequences with a Hamming distance of ≥2 were directly ligated onto fragmented source DNA eliminating the need for PCR to incorporate indexes, and was followed by a custom blocking strategy using a single oligonucleotide regardless of index sequence. These results were obtained aligning raw reads against the entire genome using Novoalign followed by variant calling of non-indexed pools using SPLINTER or SAMtools for indexed samples. With these pipelines, we find sensitivity and specificity of 99.4% and 99.7% for pooled exome sequencing. Sensitivity, and to a lesser degree specificity, proved to be a function of coverage. For rare variants (≤2% minor allele frequency), we achieved sensitivity and specificity of ≥94.9% and ≥99.99% for custom capture of 2.5 Mb in multiplexed libraries of 22-48 individuals with only ≥5-fold coverage/chromosome, but these parameters improved to ≥98.7 and 100% with 20-fold coverage/chromosome. This highly scalable methodology enables accurate rare variant detection, with or without individual DNA sample indexing, while reducing the amount of required source DNA and total costs through less hybridization reagent consumption, multi-sample sonication in a standard PCR plate, multiplexed pre-enrichment pooling with a single hybridization and lesser sequencing coverage required to obtain high sensitivity.

Targeted deep sequencing identifies rare loss-of-function variants in IFNGR1 for risk of atopic dermatitis complicated by eczema herpeticum.

PubMed

Gao, Li; Bin, Lianghua; Rafaels, Nicholas M; Huang, Lili; Potee, Joseph; Ruczinski, Ingo; Beaty, Terri H; Paller, Amy S; Schneider, Lynda C; Gallo, Rich; Hanifin, Jon M; Beck, Lisa A; Geha, Raif S; Mathias, Rasika A; Barnes, Kathleen C; Leung, Donald Y M

2015-12-01

A subset of atopic dermatitis is associated with increased susceptibility to eczema herpeticum (ADEH+). We previously reported that common single nucleotide polymorphisms (SNPs) in the IFN-γ (IFNG) and IFN-γ receptor 1 (IFNGR1) genes were associated with the ADEH+ phenotype. We sought to interrogate the role of rare variants in interferon pathway genes for the risk of ADEH+. We performed targeted sequencing of interferon pathway genes (IFNG, IFNGR1, IFNAR1, and IL12RB1) in 228 European American patients with AD selected according to their eczema herpeticum status, and severity was measured by using the Eczema Area and Severity Index. Replication genotyping was performed in independent samples of 219 European American and 333 African American subjects. Functional investigation of loss-of-function variants was conducted by using site-directed mutagenesis. We identified 494 single nucleotide variants encompassing 105 kb of sequence, including 145 common, 349 (70.6%) rare (minor allele frequency <5%), and 86 (17.4%) novel variants, of which 2.8% were coding synonymous, 93.3% were noncoding (64.6% intronic), and 3.8% were missense. We identified 6 rare IFNGR1 missense variants, including 3 damaging variants (Val14Met [V14M], Val61Ile, and Tyr397Cys [Y397C]) conferring a higher risk for ADEH+ (P = .031). Variants V14M and Y397C were confirmed to be deleterious, leading to partial IFNGR1 deficiency. Seven common IFNGR1 SNPs, along with common protective haplotypes (2-7 SNPs), conferred a reduced risk of ADEH+ (P = .015-.002 and P = .0015-.0004, respectively), and both SNP and haplotype associations were replicated in an independent African American sample (P = .004-.0001 and P = .001-.0001, respectively). Our results provide evidence that both genetic variants in the gene encoding IFNGR1 are implicated in susceptibility to the ADEH+ phenotype. Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Leveraging network analytics to infer patient syndrome and identify causal genes in rare disease cases.

PubMed

Krämer, Andreas; Shah, Sohela; Rebres, Robert Anthony; Tang, Susan; Richards, Daniel Rene

2017-08-11

Next-generation sequencing is widely used to identify disease-causing variants in patients with rare genetic disorders. Identifying those variants from whole-genome or exome data can be both scientifically challenging and time consuming. A significant amount of time is spent on variant annotation, and interpretation. Fully or partly automated solutions are therefore needed to streamline and scale this process. We describe Phenotype Driven Ranking (PDR), an algorithm integrated into Ingenuity Variant Analysis, that uses observed patient phenotypes to prioritize diseases and genes in order to expedite causal-variant discovery. Our method is based on a network of phenotype-disease-gene relationships derived from the QIAGEN Knowledge Base, which allows for efficient computational association of phenotypes to implicated diseases, and also enables scoring and ranking. We have demonstrated the utility and performance of PDR by applying it to a number of clinical rare-disease cases, where the true causal gene was known beforehand. It is also shown that PDR compares favorably to a representative alternative tool.

Rapid-Onset Obesity with Hypothalamic Dysfunction, Hypoventilation, and Autonomic Dysregulation (ROHHAD): exome sequencing of trios, monozygotic twins and tumours.

PubMed

Barclay, Sarah F; Rand, Casey M; Borch, Lauren A; Nguyen, Lisa; Gray, Paul A; Gibson, William T; Wilson, Richard J A; Gordon, Paul M K; Aung, Zaw; Berry-Kravis, Elizabeth M; Ize-Ludlow, Diego; Weese-Mayer, Debra E; Bech-Hansen, N Torben

2015-08-25

Rapid-onset Obesity with Hypothalamic Dysfunction, Hypoventilation, and Autonomic Dysregulation (ROHHAD) is thought to be a genetic disease caused by de novo mutations, though causative mutations have yet to be identified. We searched for de novo coding mutations among a carefully-diagnosed and clinically homogeneous cohort of 35 ROHHAD patients. We sequenced the exomes of seven ROHHAD trios, plus tumours from four of these patients and the unaffected monozygotic (MZ) twin of one (discovery cohort), to identify constitutional and somatic de novo sequence variants. We further analyzed this exome data to search for candidate genes under autosomal dominant and recessive models, and to identify structural variations. Candidate genes were tested by exome or Sanger sequencing in a replication cohort of 28 ROHHAD singletons. The analysis of the trio-based exomes found 13 de novo variants. However, no two patients had de novo variants in the same gene, and additional patient exomes and mutation analysis in the replication cohort did not provide strong genetic evidence to implicate any of these sequence variants in ROHHAD. Somatic comparisons revealed no coding differences between any blood and tumour samples, or between the two discordant MZ twins. Neither autosomal dominant nor recessive analysis yielded candidate genes for ROHHAD, and we did not identify any potentially causative structural variations. Clinical exome sequencing is highly unlikely to be a useful diagnostic test in patients with true ROHHAD. As ROHHAD has a high risk for fatality if not properly managed, it remains imperative to expand the search for non-exomic genetic risk factors, as well as to investigate other possible mechanisms of disease. In so doing, we will be able to confirm objectively the ROHHAD diagnosis and to contribute to our understanding of obesity, respiratory control, hypothalamic function, and autonomic regulation.

Population genomic analysis of strain variation in Leptospirillum group II bacteria involved in acid mine drainage formation.

PubMed

Simmons, Sheri L; Dibartolo, Genevieve; Denef, Vincent J; Goltsman, Daniela S Aliaga; Thelen, Michael P; Banfield, Jillian F

2008-07-22

Deeply sampled community genomic (metagenomic) datasets enable comprehensive analysis of heterogeneity in natural microbial populations. In this study, we used sequence data obtained from the dominant member of a low-diversity natural chemoautotrophic microbial community to determine how coexisting closely related individuals differ from each other in terms of gene sequence and gene content, and to uncover evidence of evolutionary processes that occur over short timescales. DNA sequence obtained from an acid mine drainage biofilm was reconstructed, taking into account the effects of strain variation, to generate a nearly complete genome tiling path for a Leptospirillum group II species closely related to L. ferriphilum (sampling depth approximately 20x). The population is dominated by one sequence type, yet we detected evidence for relatively abundant variants (>99.5% sequence identity to the dominant type) at multiple loci, and a few rare variants. Blocks of other Leptospirillum group II types ( approximately 94% sequence identity) have recombined into one or more variants. Variant blocks of both types are more numerous near the origin of replication. Heterogeneity in genetic potential within the population arises from localized variation in gene content, typically focused in integrated plasmid/phage-like regions. Some laterally transferred gene blocks encode physiologically important genes, including quorum-sensing genes of the LuxIR system. Overall, results suggest inter- and intrapopulation genetic exchange involving distinct parental genome types and implicate gain and loss of phage and plasmid genes in recent evolution of this Leptospirillum group II population. Population genetic analyses of single nucleotide polymorphisms indicate variation between closely related strains is not maintained by positive selection, suggesting that these regions do not represent adaptive differences between strains. Thus, the most likely explanation for the observed patterns of polymorphism is divergence of ancestral strains due to geographic isolation, followed by mixing and subsequent recombination.

Chromatin accessibility prediction via a hybrid deep convolutional neural network.

PubMed

Liu, Qiao; Xia, Fei; Yin, Qijin; Jiang, Rui

2018-03-01

A majority of known genetic variants associated with human-inherited diseases lie in non-coding regions that lack adequate interpretation, making it indispensable to systematically discover functional sites at the whole genome level and precisely decipher their implications in a comprehensive manner. Although computational approaches have been complementing high-throughput biological experiments towards the annotation of the human genome, it still remains a big challenge to accurately annotate regulatory elements in the context of a specific cell type via automatic learning of the DNA sequence code from large-scale sequencing data. Indeed, the development of an accurate and interpretable model to learn the DNA sequence signature and further enable the identification of causative genetic variants has become essential in both genomic and genetic studies. We proposed Deopen, a hybrid framework mainly based on a deep convolutional neural network, to automatically learn the regulatory code of DNA sequences and predict chromatin accessibility. In a series of comparison with existing methods, we show the superior performance of our model in not only the classification of accessible regions against background sequences sampled at random, but also the regression of DNase-seq signals. Besides, we further visualize the convolutional kernels and show the match of identified sequence signatures and known motifs. We finally demonstrate the sensitivity of our model in finding causative noncoding variants in the analysis of a breast cancer dataset. We expect to see wide applications of Deopen with either public or in-house chromatin accessibility data in the annotation of the human genome and the identification of non-coding variants associated with diseases. Deopen is freely available at https://github.com/kimmo1019/Deopen. ruijiang@tsinghua.edu.cn. Supplementary data are available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Population Genomic Analysis of Strain Variation in Leptospirillum Group II Bacteria Involved in Acid Mine Drainage Formation

PubMed Central

Denef, Vincent J; Goltsman, Daniela S. Aliaga; Thelen, Michael P; Banfield, Jillian F

2008-01-01

Deeply sampled community genomic (metagenomic) datasets enable comprehensive analysis of heterogeneity in natural microbial populations. In this study, we used sequence data obtained from the dominant member of a low-diversity natural chemoautotrophic microbial community to determine how coexisting closely related individuals differ from each other in terms of gene sequence and gene content, and to uncover evidence of evolutionary processes that occur over short timescales. DNA sequence obtained from an acid mine drainage biofilm was reconstructed, taking into account the effects of strain variation, to generate a nearly complete genome tiling path for a Leptospirillum group II species closely related to L. ferriphilum (sampling depth ∼20×). The population is dominated by one sequence type, yet we detected evidence for relatively abundant variants (>99.5% sequence identity to the dominant type) at multiple loci, and a few rare variants. Blocks of other Leptospirillum group II types (∼94% sequence identity) have recombined into one or more variants. Variant blocks of both types are more numerous near the origin of replication. Heterogeneity in genetic potential within the population arises from localized variation in gene content, typically focused in integrated plasmid/phage-like regions. Some laterally transferred gene blocks encode physiologically important genes, including quorum-sensing genes of the LuxIR system. Overall, results suggest inter- and intrapopulation genetic exchange involving distinct parental genome types and implicate gain and loss of phage and plasmid genes in recent evolution of this Leptospirillum group II population. Population genetic analyses of single nucleotide polymorphisms indicate variation between closely related strains is not maintained by positive selection, suggesting that these regions do not represent adaptive differences between strains. Thus, the most likely explanation for the observed patterns of polymorphism is divergence of ancestral strains due to geographic isolation, followed by mixing and subsequent recombination. PMID:18651792

Mosaic PPM1D mutations are associated with predisposition to breast and ovarian cancer.

PubMed

Ruark, Elise; Snape, Katie; Humburg, Peter; Loveday, Chey; Bajrami, Ilirjana; Brough, Rachel; Rodrigues, Daniel Nava; Renwick, Anthony; Seal, Sheila; Ramsay, Emma; Duarte, Silvana Del Vecchio; Rivas, Manuel A; Warren-Perry, Margaret; Zachariou, Anna; Campion-Flora, Adriana; Hanks, Sandra; Murray, Anne; Ansari Pour, Naser; Douglas, Jenny; Gregory, Lorna; Rimmer, Andrew; Walker, Neil M; Yang, Tsun-Po; Adlard, Julian W; Barwell, Julian; Berg, Jonathan; Brady, Angela F; Brewer, Carole; Brice, Glen; Chapman, Cyril; Cook, Jackie; Davidson, Rosemarie; Donaldson, Alan; Douglas, Fiona; Eccles, Diana; Evans, D Gareth; Greenhalgh, Lynn; Henderson, Alex; Izatt, Louise; Kumar, Ajith; Lalloo, Fiona; Miedzybrodzka, Zosia; Morrison, Patrick J; Paterson, Joan; Porteous, Mary; Rogers, Mark T; Shanley, Susan; Walker, Lisa; Gore, Martin; Houlston, Richard; Brown, Matthew A; Caufield, Mark J; Deloukas, Panagiotis; McCarthy, Mark I; Todd, John A; Turnbull, Clare; Reis-Filho, Jorge S; Ashworth, Alan; Antoniou, Antonis C; Lord, Christopher J; Donnelly, Peter; Rahman, Nazneen

2013-01-17

Improved sequencing technologies offer unprecedented opportunities for investigating the role of rare genetic variation in common disease. However, there are considerable challenges with respect to study design, data analysis and replication. Using pooled next-generation sequencing of 507 genes implicated in the repair of DNA in 1,150 samples, an analytical strategy focused on protein-truncating variants (PTVs) and a large-scale sequencing case-control replication experiment in 13,642 individuals, here we show that rare PTVs in the p53-inducible protein phosphatase PPM1D are associated with predisposition to breast cancer and ovarian cancer. PPM1D PTV mutations were present in 25 out of 7,781 cases versus 1 out of 5,861 controls (P = 1.12 × 10(-5)), including 18 mutations in 6,912 individuals with breast cancer (P = 2.42 × 10(-4)) and 12 mutations in 1,121 individuals with ovarian cancer (P = 3.10 × 10(-9)). Notably, all of the identified PPM1D PTVs were mosaic in lymphocyte DNA and clustered within a 370-base-pair region in the final exon of the gene, carboxy-terminal to the phosphatase catalytic domain. Functional studies demonstrate that the mutations result in enhanced suppression of p53 in response to ionizing radiation exposure, suggesting that the mutant alleles encode hyperactive PPM1D isoforms. Thus, although the mutations cause premature protein truncation, they do not result in the simple loss-of-function effect typically associated with this class of variant, but instead probably have a gain-of-function effect. Our results have implications for the detection and management of breast and ovarian cancer risk. More generally, these data provide new insights into the role of rare and of mosaic genetic variants in common conditions, and the use of sequencing in their identification.

Mosaic PPM1D mutations are associated with predisposition to breast and ovarian cancer

PubMed Central

Ruark, Elise; Snape, Katie; Humburg, Peter; Loveday, Chey; Bajrami, Ilirjana; Brough, Rachel; Rodrigues, Daniel Nava; Renwick, Anthony; Seal, Sheila; Ramsay, Emma; Duarte, Silvana Del Vecchio; Rivas, Manuel A.; Warren-Perry, Margaret; Zachariou, Anna; Campion-Flora, Adriana; Hanks, Sandra; Murray, Anne; Pour, Naser Ansari; Douglas, Jenny; Gregory, Lorna; Rimmer, Andrew; Walker, Neil M.; Yang, Tsun-Po; Adlard, Julian W.; Barwell, Julian; Berg, Jonathan; Brady, Angela F.; Brewer, Carole; Brice, Glen; Chapman, Cyril; Cook, Jackie; Davidson, Rosemarie; Donaldson, Alan; Douglas, Fiona; Eccles, Diana; Evans, D. Gareth; Greenhalgh, Lynn; Henderson, Alex; Izatt, Louise; Kumar, Ajith; Lalloo, Fiona; Miedzybrodzka, Zosia; Morrison, Patrick J.; Paterson, Joan; Porteous, Mary; Rogers, Mark T.; Shanley, Susan; Walker, Lisa; Gore, Martin; Houlston, Richard; Brown, Matthew A.; Caufield, Mark J.; Deloukas, Panagiotis; McCarthy, Mark I.; Todd, John A.; Turnbull, Clare; Reis-Filho, Jorge S.; Ashworth, Alan; Antoniou, Antonis C.; Lord, Christopher J.; Donnelly, Peter; Rahman, Nazneen

2013-01-01

Improved sequencing technologies offer unprecedented opportunities for investigating the role of rare genetic variation in common disease. However, there are considerable challenges with respect to study design, data analysis and replication1. Here, using pooled next-generation sequencing of 507 genes implicated in the repair of DNA in 1,150 samples, an analytical strategy focussed on protein truncating variants (PTVs) and a large-scale sequencing case-control replication experiment in 13,642 individuals, we show that rare PTVs in the p53 inducible protein phosphatase PPM1D are associated with predisposition to breast cancer and to ovarian cancer. PPM1D PTV mutations were present in 25/7781 cases vs 1/5861 controls; P=1.12×10−5, which included 18 mutations in 6,912 individuals with breast cancer; P = 2.42×10−4 and 12 mutations in 1,121 individuals with ovarian cancer; P = 3.10×10−9. Notably, all the identified PPM1D PTVs were mosaic in lymphocyte DNA and clustered within a 370 bp region in the final exon of the gene, C-terminal to the phosphatase catalytic domain. Functional studies demonstrated that the mutations result in enhanced suppression of p53 in response to ionising radiation exposure, suggesting the mutant alleles encode hyperactive PPM1D isoforms. Thus, although the mutations cause premature protein truncation, they do not result in the simple loss-of-function typically associated with this class of variant, but instead likely have a gain-of-function effect. Our results have implications for the detection and management of breast and ovarian cancer risk. More generally, these data provide new insights into the role of rare and of mosaic genetic variants in common conditions, and the utility of sequencing in their identification. PMID:23242139

Recurrent variants in OTOF are significant contributors to prelingual nonsydromic hearing loss in Saudi patients

PubMed Central

Almontashiri, Naif A M; Alswaid, Abdulrahman; Oza, Andrea; Al-Mazrou, Khalid A; Elrehim, Omnia; Tayoun, Ahmad Abou; Rehm, Heidi L; Amr, Sami S

2018-01-01

Purpose Hearing loss is more prevalent in the Saudi Arabian population than in other populations; however, the full range of genetic etiologies in this population is unknown. We report the genetic findings from 33 Saudi hearing-loss probands of tribal ancestry, with predominantly prelingual severe to profound hearing loss. Methods Testing was performed over the course of 2012–2016, and involved initial GJB2 sequence and GJB6-D13S1830 deletion screening, with negative cases being reflexed to a next-generation sequencing panel with 70, 71, or 87 hearing-loss genes. Results A “positive” result was reached in 63% of probands, with two recurrent OTOF variants (p.Glu57* and p.Arg1792His) accountable for a third of all “positive” cases. The next most common cause was pathogenic variants in MYO7A and SLC26A4, each responsible for three “positive” cases. Interestingly, only one “positive” diagnosis had a DFNB1-related cause, due to a homozygous GJB6-D13S1830 deletion, and no sequence variants in GJB2 were detected. Conclusion Our findings implicate OTOF as a potential major contributor to hearing loss in the Saudi population, while highlighting the low contribution of GJB2, thus offering important considerations for clinical testing strategies for Saudi patients. Further screening of Saudi patients is needed to characterize the genetic spectrum in this population. PMID:29048421

GenProBiS: web server for mapping of sequence variants to protein binding sites.

PubMed

Konc, Janez; Skrlj, Blaz; Erzen, Nika; Kunej, Tanja; Janezic, Dusanka

2017-07-03

Discovery of potentially deleterious sequence variants is important and has wide implications for research and generation of new hypotheses in human and veterinary medicine, and drug discovery. The GenProBiS web server maps sequence variants to protein structures from the Protein Data Bank (PDB), and further to protein-protein, protein-nucleic acid, protein-compound, and protein-metal ion binding sites. The concept of a protein-compound binding site is understood in the broadest sense, which includes glycosylation and other post-translational modification sites. Binding sites were defined by local structural comparisons of whole protein structures using the Protein Binding Sites (ProBiS) algorithm and transposition of ligands from the similar binding sites found to the query protein using the ProBiS-ligands approach with new improvements introduced in GenProBiS. Binding site surfaces were generated as three-dimensional grids encompassing the space occupied by predicted ligands. The server allows intuitive visual exploration of comprehensively mapped variants, such as human somatic mis-sense mutations related to cancer and non-synonymous single nucleotide polymorphisms from 21 species, within the predicted binding sites regions for about 80 000 PDB protein structures using fast WebGL graphics. The GenProBiS web server is open and free to all users at http://genprobis.insilab.org. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Comparison of Constitutional and Replication Stress-Induced Genome Structural Variation by SNP Array and Mate-Pair Sequencing

PubMed Central

Arlt, Martin F.; Ozdemir, Alev Cagla; Birkeland, Shanda R.; Lyons, Robert H.; Glover, Thomas W.; Wilson, Thomas E.

2011-01-01

Copy-number variants (CNVs) are a major source of genetic variation in human health and disease. Previous studies have implicated replication stress as a causative factor in CNV formation. However, existing data are technically limited in the quality of comparisons that can be made between human CNVs and experimentally induced variants. Here, we used two high-resolution strategies—single nucleotide polymorphism (SNP) arrays and mate-pair sequencing—to compare CNVs that occur constitutionally to those that arise following aphidicolin-induced DNA replication stress in the same human cells. Although the optimized methods provided complementary information, sequencing was more sensitive to small variants and provided superior structural descriptions. The majority of constitutional and all aphidicolin-induced CNVs appear to be formed via homology-independent mechanisms, while aphidicolin-induced CNVs were of a larger median size than constitutional events even when mate-pair data were considered. Aphidicolin thus appears to stimulate formation of CNVs that closely resemble human pathogenic CNVs and the subset of larger nonhomologous constitutional CNVs. PMID:21212237

Rare ADAR and RNASEH2B variants and a type I interferon signature in glioma and prostate carcinoma risk and tumorigenesis.

PubMed

Beyer, Ulrike; Brand, Frank; Martens, Helge; Weder, Julia; Christians, Arne; Elyan, Natalie; Hentschel, Bettina; Westphal, Manfred; Schackert, Gabriele; Pietsch, Torsten; Hong, Bujung; Krauss, Joachim K; Samii, Amir; Raab, Peter; Das, Anibh; Dumitru, Claudia A; Sandalcioglu, I Erol; Hakenberg, Oliver W; Erbersdobler, Andreas; Lehmann, Ulrich; Reifenberger, Guido; Weller, Michael; Reijns, Martin A M; Preller, Matthias; Wiese, Bettina; Hartmann, Christian; Weber, Ruthild G

2017-12-01

In search of novel germline alterations predisposing to tumors, in particular to gliomas, we studied a family with two brothers affected by anaplastic gliomas, and their father and paternal great-uncle diagnosed with prostate carcinoma. In this family, whole-exome sequencing yielded rare, simultaneously heterozygous variants in the Aicardi-Goutières syndrome (AGS) genes ADAR and RNASEH2B co-segregating with the tumor phenotype. AGS is a genetically induced inflammatory disease particularly of the brain, which has not been associated with a consistently increased cancer risk to date. By targeted sequencing, we identified novel ADAR and RNASEH2B variants, and a 3- to 17-fold frequency increase of the AGS mutations ADAR,c.577C>G;p.(P193A) and RNASEH2B,c.529G>A;p.(A177T) in the germline of familial glioma patients as well as in test and validation cohorts of glioblastomas and prostate carcinomas versus ethnicity-matched controls, whereby rare RNASEH2B variants were significantly more frequent in familial glioma patients. Tumors with ADAR or RNASEH2B variants recapitulated features of AGS, such as calcification and increased type I interferon expression. Patients carrying ADAR or RNASEH2B variants showed upregulation of interferon-stimulated gene (ISG) transcripts in peripheral blood as seen in AGS. An increased ISG expression was also induced by ADAR and RNASEH2B variants in tumor cells and was blocked by the JAK inhibitor Ruxolitinib. Our data implicate rare variants in the AGS genes ADAR and RNASEH2B and a type I interferon signature in glioma and prostate carcinoma risk and tumorigenesis, consistent with a genetic basis underlying inflammation-driven malignant transformation in glioma and prostate carcinoma development.

Isolation and characterization of alternatively spliced variants of the mouse sigma1 receptor gene, Sigmar1

PubMed Central

Pan, Ling; Pasternak, David A.; Xu, Jin; Xu, Mingming; Lu, Zhigang; Pasternak, Gavril W.

2017-01-01

The sigma1 receptor acts as a chaperone at the endoplasmic reticulum, associates with multiple proteins in various cellular systems, and involves in a number of diseases, such as addiction, pain, cancer and psychiatric disorders. The sigma1 receptor is encoded by the single copy SIGMAR1 gene. The current study identifies five alternatively spliced variants of the mouse sigma1 receptor gene using a polymerase chain reaction cloning approach. All the splice variants are generated by exon skipping or alternative 3’ or 5’ splicing, producing the truncated sigma1 receptor. Similar alternative splicing has been observed in the human SIGMAR1 gene based on the molecular cloning or genome sequence prediction, suggesting conservation of alternative splicing of SIGMAR1 gene. Using quantitative polymerase chain reactions, we demonstrate differential expression of several splice variants in mouse tissues and brain regions. When expressed in HEK293 cells, all the splice variants fail to bind sigma ligands, implicating that each truncated region in these splice variants is important for ligand binding. However, co-immunoprecipitation (Co-IP) study in HEK293 cells co-transfected with tagged constructs reveals that all the splice variants maintain their ability to physically associate with a mu opioid receptor (mMOR-1), providing useful information to correlate the motifs/sequences necessary for their physical association. Furthermore, a competition Co-IP study showed that all the variants can disrupt in a dose-dependent manner the dimerization of the original sigma1 receptor with mMOR-1, suggesting a potential dominant negative function and providing significant insights into their function. PMID:28350844

Hundreds of variants clustered in genomic loci and biological pathways affect human height

PubMed Central

Lango Allen, Hana; Estrada, Karol; Lettre, Guillaume; Berndt, Sonja I.; Weedon, Michael N.; Rivadeneira, Fernando; Willer, Cristen J.; Jackson, Anne U.; Vedantam, Sailaja; Raychaudhuri, Soumya; Ferreira, Teresa; Wood, Andrew R.; Weyant, Robert J.; Segrè, Ayellet V.; Speliotes, Elizabeth K.; Wheeler, Eleanor; Soranzo, Nicole; Park, Ju-Hyun; Yang, Jian; Gudbjartsson, Daniel; Heard-Costa, Nancy L.; Randall, Joshua C.; Qi, Lu; Smith, Albert Vernon; Mägi, Reedik; Pastinen, Tomi; Liang, Liming; Heid, Iris M.; Luan, Jian'an; Thorleifsson, Gudmar; Winkler, Thomas W.; Goddard, Michael E.; Lo, Ken Sin; Palmer, Cameron; Workalemahu, Tsegaselassie; Aulchenko, Yurii S.; Johansson, Åsa; Zillikens, M.Carola; Feitosa, Mary F.; Esko, Tõnu; Johnson, Toby; Ketkar, Shamika; Kraft, Peter; Mangino, Massimo; Prokopenko, Inga; Absher, Devin; Albrecht, Eva; Ernst, Florian; Glazer, Nicole L.; Hayward, Caroline; Hottenga, Jouke-Jan; Jacobs, Kevin B.; Knowles, Joshua W.; Kutalik, Zoltán; Monda, Keri L.; Polasek, Ozren; Preuss, Michael; Rayner, Nigel W.; Robertson, Neil R.; Steinthorsdottir, Valgerdur; Tyrer, Jonathan P.; Voight, Benjamin F.; Wiklund, Fredrik; Xu, Jianfeng; Zhao, Jing Hua; Nyholt, Dale R.; Pellikka, Niina; Perola, Markus; Perry, John R.B.; Surakka, Ida; Tammesoo, Mari-Liis; Altmaier, Elizabeth L.; Amin, Najaf; Aspelund, Thor; Bhangale, Tushar; Boucher, Gabrielle; Chasman, Daniel I.; Chen, Constance; Coin, Lachlan; Cooper, Matthew N.; Dixon, Anna L.; Gibson, Quince; Grundberg, Elin; Hao, Ke; Junttila, M. Juhani; Kaplan, Lee M.; Kettunen, Johannes; König, Inke R.; Kwan, Tony; Lawrence, Robert W.; Levinson, Douglas F.; Lorentzon, Mattias; McKnight, Barbara; Morris, Andrew P.; Müller, Martina; Ngwa, Julius Suh; Purcell, Shaun; Rafelt, Suzanne; Salem, Rany M.; Salvi, Erika; Sanna, Serena; Shi, Jianxin; Sovio, Ulla; Thompson, John R.; Turchin, Michael C.; Vandenput, Liesbeth; Verlaan, Dominique J.; Vitart, Veronique; White, Charles C.; Ziegler, Andreas; Almgren, Peter; Balmforth, Anthony J.; Campbell, Harry; Citterio, Lorena; De Grandi, Alessandro; Dominiczak, Anna; Duan, Jubao; Elliott, Paul; Elosua, Roberto; Eriksson, Johan G.; Freimer, Nelson B.; Geus, Eco J.C.; Glorioso, Nicola; Haiqing, Shen; Hartikainen, Anna-Liisa; Havulinna, Aki S.; Hicks, Andrew A.; Hui, Jennie; Igl, Wilmar; Illig, Thomas; Jula, Antti; Kajantie, Eero; Kilpeläinen, Tuomas O.; Koiranen, Markku; Kolcic, Ivana; Koskinen, Seppo; Kovacs, Peter; Laitinen, Jaana; Liu, Jianjun; Lokki, Marja-Liisa; Marusic, Ana; Maschio, Andrea; Meitinger, Thomas; Mulas, Antonella; Paré, Guillaume; Parker, Alex N.; Peden, John F.; Petersmann, Astrid; Pichler, Irene; Pietiläinen, Kirsi H.; Pouta, Anneli; Ridderstråle, Martin; Rotter, Jerome I.; Sambrook, Jennifer G.; Sanders, Alan R.; Schmidt, Carsten Oliver; Sinisalo, Juha; Smit, Jan H.; Stringham, Heather M.; Walters, G.Bragi; Widen, Elisabeth; Wild, Sarah H.; Willemsen, Gonneke; Zagato, Laura; Zgaga, Lina; Zitting, Paavo; Alavere, Helene; Farrall, Martin; McArdle, Wendy L.; Nelis, Mari; Peters, Marjolein J.; Ripatti, Samuli; van Meurs, Joyce B.J.; Aben, Katja K.; Ardlie, Kristin G; Beckmann, Jacques S.; Beilby, John P.; Bergman, Richard N.; Bergmann, Sven; Collins, Francis S.; Cusi, Daniele; den Heijer, Martin; Eiriksdottir, Gudny; Gejman, Pablo V.; Hall, Alistair S.; Hamsten, Anders; Huikuri, Heikki V.; Iribarren, Carlos; Kähönen, Mika; Kaprio, Jaakko; Kathiresan, Sekar; Kiemeney, Lambertus; Kocher, Thomas; Launer, Lenore J.; Lehtimäki, Terho; Melander, Olle; Mosley, Tom H.; Musk, Arthur W.; Nieminen, Markku S.; O'Donnell, Christopher J.; Ohlsson, Claes; Oostra, Ben; Palmer, Lyle J.; Raitakari, Olli; Ridker, Paul M.; Rioux, John D.; Rissanen, Aila; Rivolta, Carlo; Schunkert, Heribert; Shuldiner, Alan R.; Siscovick, David S.; Stumvoll, Michael; Tönjes, Anke; Tuomilehto, Jaakko; van Ommen, Gert-Jan; Viikari, Jorma; Heath, Andrew C.; Martin, Nicholas G.; Montgomery, Grant W.; Province, Michael A.; Kayser, Manfred; Arnold, Alice M.; Atwood, Larry D.; Boerwinkle, Eric; Chanock, Stephen J.; Deloukas, Panos; Gieger, Christian; Grönberg, Henrik; Hall, Per; Hattersley, Andrew T.; Hengstenberg, Christian; Hoffman, Wolfgang; Lathrop, G.Mark; Salomaa, Veikko; Schreiber, Stefan; Uda, Manuela; Waterworth, Dawn; Wright, Alan F.; Assimes, Themistocles L.; Barroso, Inês; Hofman, Albert; Mohlke, Karen L.; Boomsma, Dorret I.; Caulfield, Mark J.; Cupples, L.Adrienne; Erdmann, Jeanette; Fox, Caroline S.; Gudnason, Vilmundur; Gyllensten, Ulf; Harris, Tamara B.; Hayes, Richard B.; Jarvelin, Marjo-Riitta; Mooser, Vincent; Munroe, Patricia B.; Ouwehand, Willem H.; Penninx, Brenda W.; Pramstaller, Peter P.; Quertermous, Thomas; Rudan, Igor; Samani, Nilesh J.; Spector, Timothy D.; Völzke, Henry; Watkins, Hugh; Wilson, James F.; Groop, Leif C.; Haritunians, Talin; Hu, Frank B.; Kaplan, Robert C.; Metspalu, Andres; North, Kari E.; Schlessinger, David; Wareham, Nicholas J.; Hunter, David J.; O'Connell, Jeffrey R.; Strachan, David P.; Wichmann, H.-Erich; Borecki, Ingrid B.; van Duijn, Cornelia M.; Schadt, Eric E.; Thorsteinsdottir, Unnur; Peltonen, Leena; Uitterlinden, André; Visscher, Peter M.; Chatterjee, Nilanjan; Loos, Ruth J.F.; Boehnke, Michael; McCarthy, Mark I.; Ingelsson, Erik; Lindgren, Cecilia M.; Abecasis, Gonçalo R.; Stefansson, Kari; Frayling, Timothy M.; Hirschhorn, Joel N

2010-01-01

Most common human traits and diseases have a polygenic pattern of inheritance: DNA sequence variants at many genetic loci influence phenotype. Genome-wide association (GWA) studies have identified >600 variants associated with human traits1, but these typically explain small fractions of phenotypic variation, raising questions about the utility of further studies. Here, using 183,727 individuals, we show that hundreds of genetic variants, in at least 180 loci, influence adult height, a highly heritable and classic polygenic trait2,3. The large number of loci reveals patterns with important implications for genetic studies of common human diseases and traits. First, the 180 loci are not random, but instead are enriched for genes that are connected in biological pathways (P=0.016), and that underlie skeletal growth defects (P<0.001). Second, the likely causal gene is often located near the most strongly associated variant: in 13 of 21 loci containing a known skeletal growth gene, that gene was closest to the associated variant. Third, at least 19 loci have multiple independently associated variants, suggesting that allelic heterogeneity is a frequent feature of polygenic traits, that comprehensive explorations of already-discovered loci should discover additional variants, and that an appreciable fraction of associated loci may have been identified. Fourth, associated variants are enriched for likely functional effects on genes, being over-represented amongst variants that alter amino acid structure of proteins and expression levels of nearby genes. Our data explain ∼10% of the phenotypic variation in height, and we estimate that unidentified common variants of similar effect sizes would increase this figure to ∼16% of phenotypic variation (∼20% of heritable variation). Although additional approaches are needed to fully dissect the genetic architecture of polygenic human traits, our findings indicate that GWA studies can identify large numbers of loci that implicate biologically relevant genes and pathways. PMID:20881960

Chondrodysplasia with multiple dislocations: comprehensive study of a series of 30 cases.

PubMed

Ranza, E; Huber, C; Levin, N; Baujat, G; Bole-Feysot, C; Nitschke, P; Masson, C; Alanay, Y; Al-Gazali, L; Bitoun, P; Boute, O; Campeau, P; Coubes, C; McEntagart, M; Elcioglu, N; Faivre, L; Gezdirici, A; Johnson, D; Mihci, E; Nur, B G; Perrin, L; Quelin, C; Terhal, P; Tuysuz, B; Cormier-Daire, V

2017-06-01

The group of chondrodysplasia with multiple dislocations includes several entities, characterized by short stature, dislocation of large joints, hand and/or vertebral anomalies. Other features, such as epiphyseal or metaphyseal changes, cleft palate, intellectual disability are also often part of the phenotype. In addition, several conditions with overlapping features are related to this group and broaden the spectrum. The majority of these disorders have been linked to pathogenic variants in genes encoding proteins implicated in the synthesis or sulfation of proteoglycans (PG). In a series of 30 patients with multiple dislocations, we have performed exome sequencing and subsequent targeted analysis of 15 genes, implicated in chondrodysplasia with multiple dislocations, and related conditions. We have identified causative pathogenic variants in 60% of patients (18/30); when a clinical diagnosis was suspected, this was molecularly confirmed in 53% of cases. Forty percent of patients remain without molecular etiology. Pathogenic variants in genes implicated in PG synthesis are of major importance in chondrodysplasia with multiple dislocations and related conditions. The combination of hand features, growth failure severity, radiological aspects of long bones and of vertebrae allowed discrimination among the different conditions. We propose key diagnostic clues to the clinician. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Comparison and evaluation of two exome capture kits and sequencing platforms for variant calling.

PubMed

Zhang, Guoqiang; Wang, Jianfeng; Yang, Jin; Li, Wenjie; Deng, Yutian; Li, Jing; Huang, Jun; Hu, Songnian; Zhang, Bing

2015-08-05

To promote the clinical application of next-generation sequencing, it is important to obtain accurate and consistent variants of target genomic regions at low cost. Ion Proton, the latest updated semiconductor-based sequencing instrument from Life Technologies, is designed to provide investigators with an inexpensive platform for human whole exome sequencing that achieves a rapid turnaround time. However, few studies have comprehensively compared and evaluated the accuracy of variant calling between Ion Proton and Illumina sequencing platforms such as HiSeq 2000, which is the most popular sequencing platform for the human genome. The Ion Proton sequencer combined with the Ion TargetSeq Exome Enrichment Kit together make up TargetSeq-Proton, whereas SureSelect-Hiseq is based on the Agilent SureSelect Human All Exon v4 Kit and the HiSeq 2000 sequencer. Here, we sequenced exonic DNA from four human blood samples using both TargetSeq-Proton and SureSelect-HiSeq. We then called variants in the exonic regions that overlapped between the two exome capture kits (33.6 Mb). The rates of shared variant loci called by two sequencing platforms were from 68.0 to 75.3% in four samples, whereas the concordance of co-detected variant loci reached 99%. Sanger sequencing validation revealed that the validated rate of concordant single nucleotide polymorphisms (SNPs) (91.5%) was higher than the SNPs specific to TargetSeq-Proton (60.0%) or specific to SureSelect-HiSeq (88.3%). With regard to 1-bp small insertions and deletions (InDels), the Sanger sequencing validated rates of concordant variants (100.0%) and SureSelect-HiSeq-specific (89.6%) were higher than those of TargetSeq-Proton-specific (15.8%). In the sequencing of exonic regions, a combination of using of two sequencing strategies (SureSelect-HiSeq and TargetSeq-Proton) increased the variant calling specificity for concordant variant loci and the sensitivity for variant loci called by any one platform. However, for the sequencing of platform-specific variants, the accuracy of variant calling by HiSeq 2000 was higher than that of Ion Proton, specifically for the InDel detection. Moreover, the variant calling software also influences the detection of SNPs and, specifically, InDels in Ion Proton exome sequencing.

A novel homozygous truncating GNAT1 mutation implicated in retinal degeneration.

PubMed

Carrigan, Matthew; Duignan, Emma; Humphries, Pete; Palfi, Arpad; Kenna, Paul F; Farrar, G Jane

2016-04-01

The GNAT1 gene encodes the α subunit of the rod transducin protein, a key element in the rod phototransduction cascade. Variants in GNAT1 have been implicated in stationary night-blindness in the past, but unlike other proteins in the same pathway, it has not previously been implicated in retinitis pigmentosa. A panel of 182 retinopathy-associated genes was sequenced to locate disease-causing mutations in patients with inherited retinopathies. Sequencing revealed a novel homozygous truncating mutation in the GNAT1 gene in a patient with significant pigmentary disturbance and constriction of visual fields, a presentation consistent with retinitis pigmentosa. This is the first report of a patient homozygous for a complete loss-of-function GNAT1 mutation. The clinical data from this patient provide definitive evidence of retinitis pigmentosa with late onset in addition to the lifelong night-blindness that would be expected from a lack of transducin function. These data suggest that some truncating GNAT1 variants can indeed cause a recessive, mild, late-onset retinal degeneration in human beings rather than just stationary night-blindness as reported previously, with notable similarities to the phenotype of the Gnat1 knockout mouse. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

aes, the gene encoding the esterase B in Escherichia coli, is a powerful phylogenetic marker of the species.

PubMed

Lescat, Mathilde; Hoede, Claire; Clermont, Olivier; Garry, Louis; Darlu, Pierre; Tuffery, Pierre; Denamur, Erick; Picard, Bertrand

2009-12-29

Previous studies have established a correlation between electrophoretic polymorphism of esterase B, and virulence and phylogeny of Escherichia coli. Strains belonging to the phylogenetic group B2 are more frequently implicated in extraintestinal infections and include esterase B2 variants, whereas phylogenetic groups A, B1 and D contain less virulent strains and include esterase B1 variants. We investigated esterase B as a marker of phylogeny and/or virulence, in a thorough analysis of the esterase B-encoding gene. We identified the gene encoding esterase B as the acetyl-esterase gene (aes) using gene disruption. The analysis of aes nucleotide sequences in a panel of 78 reference strains, including the E. coli reference (ECOR) strains, demonstrated that the gene is under purifying selection. The phylogenetic tree reconstructed from aes sequences showed a strong correlation with the species phylogenetic history, based on multi-locus sequence typing using six housekeeping genes. The unambiguous distinction between variants B1 and B2 by electrophoresis was consistent with Aes amino-acid sequence analysis and protein modelling, which showed that substituted amino acids in the two esterase B variants occurred mostly at different sites on the protein surface. Studies in an experimental mouse model of septicaemia using mutant strains did not reveal a direct link between aes and extraintestinal virulence. Moreover, we did not find any genes in the chromosomal region of aes to be associated with virulence. Our findings suggest that aes does not play a direct role in the virulence of E. coli extraintestinal infection. However, this gene acts as a powerful marker of phylogeny, illustrating the extensive divergence of B2 phylogenetic group strains from the rest of the species.

A Common Mutation in DEFB126 Causes Impaired Sperm Function and Subfertility

PubMed Central

Tollner, Theodore L.; Venners, Scott A.; Hollox, Edward J.; Yudin, Ashley I.; Liu, Xue; Tang, Genfu; Xing, Houxun; Kays, Robert J.; Lau, Tsang; Overstreet, James W.; Xu, Xiping; Bevins, Charles L.; Cherr, Gary N.

2013-01-01

A glycosylated polypeptide, β-defensin 126 (DEFB126), derived from the epididymis and adsorbed onto the sperm surface, has been implicated in immunoprotection and efficient movement of sperm in mucosal fluids of the female reproductive tract. Here, we report a sequence variant in DEFB126 that has a 2-nucleotide deletion in the open reading frame, which generates a non-stop mRNA. The allele frequency of this variant sequence is high in both a European (0.47) and a Chinese (0.45) population cohort. Binding of the Agaricus bisporus lectin to the sperm surface glycocalyx was significantly lower in men with the homozygous variant (del/del) genotype than in those with either a del/wt or wt/wt genotype, suggesting an altered sperm glycocalyx with fewer O-linked oligosaccharides in del/del men. Moreover, sperm from the del/del donors exhibited an 84% reduction in the rate of penetration of a hyaluronic acid (HA) gel, a surrogate for cervical mucus, compared to the other genotypes. This reduction in sperm performance in HA gels was not a result of decreased progressive motility (average curvilinear velocity) or morphological deficits. However, DEFB126 genotype and lectin binding were highly correlated with performance in the penetration assays. In a prospective cohort study of newly married couples who were trying to conceive by natural means, couples were less likely to become pregnant and took longer to achieve a live birth if the male partner was homozygous for the variant sequence. This common sequence variation in DEFB126, and its apparent cause of impaired reproductive function, provides an opportunity to better understand, clinically evaluate, and possibly treat human infertility. PMID:21775668

Comprehensive Rare Variant Analysis via Whole-Genome Sequencing to Determine the Molecular Pathology of Inherited Retinal Disease.

PubMed

Carss, Keren J; Arno, Gavin; Erwood, Marie; Stephens, Jonathan; Sanchis-Juan, Alba; Hull, Sarah; Megy, Karyn; Grozeva, Detelina; Dewhurst, Eleanor; Malka, Samantha; Plagnol, Vincent; Penkett, Christopher; Stirrups, Kathleen; Rizzo, Roberta; Wright, Genevieve; Josifova, Dragana; Bitner-Glindzicz, Maria; Scott, Richard H; Clement, Emma; Allen, Louise; Armstrong, Ruth; Brady, Angela F; Carmichael, Jenny; Chitre, Manali; Henderson, Robert H H; Hurst, Jane; MacLaren, Robert E; Murphy, Elaine; Paterson, Joan; Rosser, Elisabeth; Thompson, Dorothy A; Wakeling, Emma; Ouwehand, Willem H; Michaelides, Michel; Moore, Anthony T; Webster, Andrew R; Raymond, F Lucy

2017-01-05

Inherited retinal disease is a common cause of visual impairment and represents a highly heterogeneous group of conditions. Here, we present findings from a cohort of 722 individuals with inherited retinal disease, who have had whole-genome sequencing (n = 605), whole-exome sequencing (n = 72), or both (n = 45) performed, as part of the NIHR-BioResource Rare Diseases research study. We identified pathogenic variants (single-nucleotide variants, indels, or structural variants) for 404/722 (56%) individuals. Whole-genome sequencing gives unprecedented power to detect three categories of pathogenic variants in particular: structural variants, variants in GC-rich regions, which have significantly improved coverage compared to whole-exome sequencing, and variants in non-coding regulatory regions. In addition to previously reported pathogenic regulatory variants, we have identified a previously unreported pathogenic intronic variant in CHM in two males with choroideremia. We have also identified 19 genes not previously known to be associated with inherited retinal disease, which harbor biallelic predicted protein-truncating variants in unsolved cases. Whole-genome sequencing is an increasingly important comprehensive method with which to investigate the genetic causes of inherited retinal disease. Copyright © 2017. Published by Elsevier Inc.

Macrophage-tropic variants initiate human immunodeficiency virus type 1 infection after sexual, parenteral, and vertical transmission.

PubMed Central

van't Wout, A B; Kootstra, N A; Mulder-Kampinga, G A; Albrecht-van Lent, N; Scherpbier, H J; Veenstra, J; Boer, K; Coutinho, R A; Miedema, F; Schuitemaker, H

1994-01-01

Macrophage-tropic, non-syncytium-inducing, HIV-1 variants predominate in the asymptomatic phase of infection and may be responsible for establishing infection in an individual exposed to the mixture of HIV-1 variants. Here, genotypical and phenotypical characteristics of virus populations, present in sexual, parenteral, or vertical donor-recipient pairs, were studied. Sequence analysis of the V3 domain confirmed the presence of a homogeneous virus population in recently infected individuals. Biological HIV-1 clones were further characterized for syncytium inducing capacity on the MT2 cell line and for macrophage tropism as defined by the appearance of proviral DNA upon inoculation of monocyte-derived macrophages. Both sexual and parenteral transmission cases revealed a selective outgrowth in the recipient of the most macrophage-tropic variant(s) present in the donor. In three out of five vertical transmission cases, more than one highly macrophage-tropic virus variant was present in the child shortly after birth, suggestive of transmission of multiple variants. In three primary infection cases, homogeneous virus populations of macrophage-tropic, non-syncytium-inducing variants were present prior to seroconversion, thus excluding humoral immunity as the selective pressure in favour of macrophage-tropic variants. These observations may have important implications for vaccine development. PMID:7962552

Identification of Point Mutations in Clinical Staphylococcus aureus Strains That Produce Small-Colony Variants Auxotrophic for Menadione

PubMed Central

Dean, Melissa A.; Olsen, Randall J.; Long, S. Wesley; Rosato, Adriana E.

2014-01-01

Staphylococcus aureus small-colony variants (SCVs) are implicated in chronic and relapsing infections that are difficult to diagnose and treat. Despite many years of study, the underlying molecular mechanisms and virulence effect of the small-colony phenotype remain incompletely understood. We sequenced the genomes of five S. aureus SCV strains recovered from human patients and discovered previously unidentified nonsynonymous point mutations in three genes encoding proteins in the menadione biosynthesis pathway. Analysis of genetic revertants and complementation with wild-type alleles confirmed that these mutations caused the SCV phenotype and decreased virulence for mice. PMID:24452687

Targeted Deep Sequencing Identifies Rare ‘loss-of-function’ Variants in IFNGR1 for Risk of Atopic Dermatitis Complicated by Eczema Herpeticum

PubMed Central

Gao, Li; Rafaels, Nicholas M; Huang, Lili; Potee, Joseph; Ruczinski, Ingo; Beaty, Terri H.; Paller, Amy S.; Schneider, Lynda C.; Gallo, Rich; Hanifin, Jon M.; Beck, Lisa A.; Geha, Raif S.; Mathias, Rasika A.; Leung, Donald Y. M.

2015-01-01

Background A subset of atopic dermatitis (AD) is associated with increased susceptibility to eczema herpeticum (ADEH+). We previously reported that common single nucleotide polymorphisms (SNPs) in interferon-gamma (IFNG) and receptor 1 (IFNGR1) were associated with ADEH+ phenotype. Objective To interrogate the role of rare variants in IFN-pathway genes for risk of ADEH+. Methods We performed targeted sequencing of interferon-pathway genes (IFNG, IFNGR1, IFNAR1 and IL12RB1) in 228 European American (EA) AD patients selected according to their EH status and severity measured by Eczema Area and Severity Index (EASI). Replication genotyping was performed in independent samples of 219 EA and 333 African Americans (AA). Functional investigation of ‘loss-of-function’ variants was conducted using site-directed mutagenesis. Results We identified 494 single nucleotide variants (SNVs) encompassing 105kb of sequence, including 145 common, 349 (70.6%) rare (minor allele frequency (MAF) <5%) and 86 (17.4%) novel variants, of which 2.8% were coding-synonymous, 93.3% were non-coding (64.6% intronic), and 3.8% were missense. We identified six rare IFNGR1 missense including three damaging variants (Val14Met (V14M), Val61Ile and Tyr397Cys (Y397C)) conferring a higher risk for ADEH+ (P=0.031). Variants V14M and Y397C were confirmed to be deleterious leading to partial IFNGR1 deficiency. Seven common IFNGR1 SNPs, along with common protective haplotypes (2 to 7-SNPs) conferred a reduced risk of ADEH+ (P=0.015-0.002, P=0.0015-0.0004, respectively), and both SNP and haplotype associations were replicated in an independent AA sample (P=0.004-0.0001 and P=0.001-0.0001, respectively). Conclusion Our results provide evidence that both genetic variants in the gene encoding IFNGR1 are implicated in susceptibility to the ADEH+ phenotype. CAPSULE SUMMARY We provided the first evidence that rare functional IFNGR1 mutations contribute to a defective systemic IFN-γ immune response that accounts for the propensity of AD patients to disseminated viral skin infections. PMID:26343451

XLID-Causing Mutations and Associated Genes Challenged in Light of Data From Large-Scale Human Exome Sequencing

PubMed Central

Piton, Amélie; Redin, Claire; Mandel, Jean-Louis

2013-01-01

Because of the unbalanced sex ratio (1.3–1.4 to 1) observed in intellectual disability (ID) and the identification of large ID-affected families showing X-linked segregation, much attention has been focused on the genetics of X-linked ID (XLID). Mutations causing monogenic XLID have now been reported in over 100 genes, most of which are commonly included in XLID diagnostic gene panels. Nonetheless, the boundary between true mutations and rare non-disease-causing variants often remains elusive. The sequencing of a large number of control X chromosomes, required for avoiding false-positive results, was not systematically possible in the past. Such information is now available thanks to large-scale sequencing projects such as the National Heart, Lung, and Blood (NHLBI) Exome Sequencing Project, which provides variation information on 10,563 X chromosomes from the general population. We used this NHLBI cohort to systematically reassess the implication of 106 genes proposed to be involved in monogenic forms of XLID. We particularly question the implication in XLID of ten of them (AGTR2, MAGT1, ZNF674, SRPX2, ATP6AP2, ARHGEF6, NXF5, ZCCHC12, ZNF41, and ZNF81), in which truncating variants or previously published mutations are observed at a relatively high frequency within this cohort. We also highlight 15 other genes (CCDC22, CLIC2, CNKSR2, FRMPD4, HCFC1, IGBP1, KIAA2022, KLF8, MAOA, NAA10, NLGN3, RPL10, SHROOM4, ZDHHC15, and ZNF261) for which replication studies are warranted. We propose that similar reassessment of reported mutations (and genes) with the use of data from large-scale human exome sequencing would be relevant for a wide range of other genetic diseases. PMID:23871722

Exome Sequencing and the Management of Neurometabolic Disorders.

PubMed

Tarailo-Graovac, Maja; Shyr, Casper; Ross, Colin J; Horvath, Gabriella A; Salvarinova, Ramona; Ye, Xin C; Zhang, Lin-Hua; Bhavsar, Amit P; Lee, Jessica J Y; Drögemöller, Britt I; Abdelsayed, Mena; Alfadhel, Majid; Armstrong, Linlea; Baumgartner, Matthias R; Burda, Patricie; Connolly, Mary B; Cameron, Jessie; Demos, Michelle; Dewan, Tammie; Dionne, Janis; Evans, A Mark; Friedman, Jan M; Garber, Ian; Lewis, Suzanne; Ling, Jiqiang; Mandal, Rupasri; Mattman, Andre; McKinnon, Margaret; Michoulas, Aspasia; Metzger, Daniel; Ogunbayo, Oluseye A; Rakic, Bojana; Rozmus, Jacob; Ruben, Peter; Sayson, Bryan; Santra, Saikat; Schultz, Kirk R; Selby, Kathryn; Shekel, Paul; Sirrs, Sandra; Skrypnyk, Cristina; Superti-Furga, Andrea; Turvey, Stuart E; Van Allen, Margot I; Wishart, David; Wu, Jiang; Wu, John; Zafeiriou, Dimitrios; Kluijtmans, Leo; Wevers, Ron A; Eydoux, Patrice; Lehman, Anna M; Vallance, Hilary; Stockler-Ipsiroglu, Sylvia; Sinclair, Graham; Wasserman, Wyeth W; van Karnebeek, Clara D

2016-06-09

Whole-exome sequencing has transformed gene discovery and diagnosis in rare diseases. Translation into disease-modifying treatments is challenging, particularly for intellectual developmental disorder. However, the exception is inborn errors of metabolism, since many of these disorders are responsive to therapy that targets pathophysiological features at the molecular or cellular level. To uncover the genetic basis of potentially treatable inborn errors of metabolism, we combined deep clinical phenotyping (the comprehensive characterization of the discrete components of a patient's clinical and biochemical phenotype) with whole-exome sequencing analysis through a semiautomated bioinformatics pipeline in consecutively enrolled patients with intellectual developmental disorder and unexplained metabolic phenotypes. We performed whole-exome sequencing on samples obtained from 47 probands. Of these patients, 6 were excluded, including 1 who withdrew from the study. The remaining 41 probands had been born to predominantly nonconsanguineous parents of European descent. In 37 probands, we identified variants in 2 genes newly implicated in disease, 9 candidate genes, 22 known genes with newly identified phenotypes, and 9 genes with expected phenotypes; in most of the genes, the variants were classified as either pathogenic or probably pathogenic. Complex phenotypes of patients in five families were explained by coexisting monogenic conditions. We obtained a diagnosis in 28 of 41 probands (68%) who were evaluated. A test of a targeted intervention was performed in 18 patients (44%). Deep phenotyping and whole-exome sequencing in 41 probands with intellectual developmental disorder and unexplained metabolic abnormalities led to a diagnosis in 68%, the identification of 11 candidate genes newly implicated in neurometabolic disease, and a change in treatment beyond genetic counseling in 44%. (Funded by BC Children's Hospital Foundation and others.).

Exome Sequencing and the Management of Neurometabolic Disorders

PubMed Central

Tarailo-Graovac, M.; Shyr, C.; Ross, C.J.; Horvath, G.A.; Salvarinova, R.; Ye, X.C.; Zhang, L.-H.; Bhavsar, A.P.; Lee, J.J.Y.; Drögemöller, B.I.; Abdelsayed, M.; Alfadhel, M.; Armstrong, L.; Baumgartner, M.R.; Burda, P.; Connolly, M.B.; Cameron, J.; Demos, M.; Dewan, T.; Dionne, J.; Evans, A.M.; Friedman, J.M.; Garber, I.; Lewis, S.; Ling, J.; Mandal, R.; Mattman, A.; McKinnon, M.; Michoulas, A.; Metzger, D.; Ogunbayo, O.A.; Rakic, B.; Rozmus, J.; Ruben, P.; Sayson, B.; Santra, S.; Schultz, K.R.; Selby, K.; Shekel, P.; Sirrs, S.; Skrypnyk, C.; Superti-Furga, A.; Turvey, S.E.; Van Allen, M.I.; Wishart, D.; Wu, J.; Wu, J.; Zafeiriou, D.; Kluijtmans, L.; Wevers, R.A.; Eydoux, P.; Lehman, A.M.; Vallance, H.; Stockler-Ipsiroglu, S.; Sinclair, G.; Wasserman, W.W.; van Karnebeek, C.D.

2016-01-01

BACKGROUND Whole-exome sequencing has transformed gene discovery and diagnosis in rare diseases. Translation into disease-modifying treatments is challenging, particularly for intellectual developmental disorder. However, the exception is inborn errors of metabolism, since many of these disorders are responsive to therapy that targets pathophysiological features at the molecular or cellular level. METHODS To uncover the genetic basis of potentially treatable inborn errors of metabolism, we combined deep clinical phenotyping (the comprehensive characterization of the discrete components of a patient’s clinical and biochemical phenotype) with whole-exome sequencing analysis through a semiautomated bioinformatics pipeline in consecutively enrolled patients with intellectual developmental disorder and unexplained metabolic phenotypes. RESULTS We performed whole-exome sequencing on samples obtained from 47 probands. Of these patients, 6 were excluded, including 1 who withdrew from the study. The remaining 41 probands had been born to predominantly nonconsanguineous parents of European descent. In 37 probands, we identified variants in 2 genes newly implicated in disease, 9 candidate genes, 22 known genes with newly identified phenotypes, and 9 genes with expected phenotypes; in most of the genes, the variants were classified as either pathogenic or probably pathogenic. Complex phenotypes of patients in five families were explained by coexisting monogenic conditions. We obtained a diagnosis in 28 of 41 probands (68%) who were evaluated. A test of a targeted intervention was performed in 18 patients (44%). CONCLUSIONS Deep phenotyping and whole-exome sequencing in 41 probands with intellectual developmental disorder and unexplained metabolic abnormalities led to a diagnosis in 68%, the identification of 11 candidate genes newly implicated in neurometabolic disease, and a change in treatment beyond genetic counseling in 44%. (Funded by BC Children’s Hospital Foundation and others.) PMID:27276562

VariantBam: filtering and profiling of next-generational sequencing data using region-specific rules.

PubMed

Wala, Jeremiah; Zhang, Cheng-Zhong; Meyerson, Matthew; Beroukhim, Rameen

2016-07-01

We developed VariantBam, a C ++ read filtering and profiling tool for use with BAM, CRAM and SAM sequencing files. VariantBam provides a flexible framework for extracting sequencing reads or read-pairs that satisfy combinations of rules, defined by any number of genomic intervals or variant sites. We have implemented filters based on alignment data, sequence motifs, regional coverage and base quality. For example, VariantBam achieved a median size reduction ratio of 3.1:1 when applied to 10 lung cancer whole genome BAMs by removing large tags and selecting for only high-quality variant-supporting reads and reads matching a large dictionary of sequence motifs. Thus VariantBam enables efficient storage of sequencing data while preserving the most relevant information for downstream analysis. VariantBam and full documentation are available at github.com/jwalabroad/VariantBam rameen@broadinstitute.org Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

BlackOPs: increasing confidence in variant detection through mappability filtering.

PubMed

Cabanski, Christopher R; Wilkerson, Matthew D; Soloway, Matthew; Parker, Joel S; Liu, Jinze; Prins, Jan F; Marron, J S; Perou, Charles M; Hayes, D Neil

2013-10-01

Identifying variants using high-throughput sequencing data is currently a challenge because true biological variants can be indistinguishable from technical artifacts. One source of technical artifact results from incorrectly aligning experimentally observed sequences to their true genomic origin ('mismapping') and inferring differences in mismapped sequences to be true variants. We developed BlackOPs, an open-source tool that simulates experimental RNA-seq and DNA whole exome sequences derived from the reference genome, aligns these sequences by custom parameters, detects variants and outputs a blacklist of positions and alleles caused by mismapping. Blacklists contain thousands of artifact variants that are indistinguishable from true variants and, for a given sample, are expected to be almost completely false positives. We show that these blacklist positions are specific to the alignment algorithm and read length used, and BlackOPs allows users to generate a blacklist specific to their experimental setup. We queried the dbSNP and COSMIC variant databases and found numerous variants indistinguishable from mapping errors. We demonstrate how filtering against blacklist positions reduces the number of potential false variants using an RNA-seq glioblastoma cell line data set. In summary, accounting for mapping-caused variants tuned to experimental setups reduces false positives and, therefore, improves genome characterization by high-throughput sequencing.

Identification of Rare Variants in TNNI3 with Atrial Fibrillation in a Chinese GeneID Population

PubMed Central

Wang, Chuchu; Wu, Manman; Qian, Jin; Li, Bin; Tu, Xin; Xu, Chengqi; Li, Sisi; Chen, Shanshan; Zhao, Yuanyuan; Huang, Yufeng; Shi, Lisong; Cheng, Xiang; Liao, Yuhua; Chen, Qiuyun; Xia, Yunlong; Yao, Wei; Wu, Gang; Cheng, Mian; Wang, Qing K.

2015-01-01

Despite advances by genome-wide association studies (GWAS), much of heritability of common human diseases remains missing, a phenomenon referred to as ‘missing heritability’. One potential cause for ‘missing heritability’ is the rare susceptibility variants overlooked by GWAS. Atrial fibrillation (AF) is the most common arrhythmia seen at hospitals and increases risk of stroke by 5-fold and doubles risk of heart failure and sudden death. Here we studied one large Chinese family with AF and hypertrophic cardiomyopathy (HCM). Whole-exome sequencing analysis identified a mutation in TNNI3, R186Q, that co-segregated with the disease in the family, but did not exist in >1,583 controls, suggesting that R186Q causes AF and HCM. High-resolution melting curve analysis and direct DNA sequence analysis were then used to screen mutations in all exons and exon-intron boundaries of TNNI3 in a panel of 1,127 unrelated AF patients and 1,583 non-AF subjects. Four novel missense variants were identified in TNNI3, including E64G, M154L, E187G and D196G in four independent AF patients, but no variant was found in 1,583 non-AF subjects. All variants were not found in public databases, including the ExAC Browser database with 60,706 exomes. These data suggests that rare TNNI3 variants are associated with AF (P=0.03). TNNI3 encodes troponin I, a key regulator of the contraction-relaxation function of cardiac muscle and was not previously implicated in AF. Thus, this study may identify a new biological pathway for the pathogenesis of AF and provides evidence to support the rare variant hypothesis for missing heritability. PMID:26169204

Variant of TREM2 Associated with the Risk of Alzheimer’s Disease

PubMed Central

Jonsson, Thorlakur; Stefansson, Hreinn; Steinberg, Stacy; Jonsdottir, Ingileif; Jonsson, Palmi V.; Snaedal, Jon; Bjornsson, Sigurbjorn; Huttenlocher, Johanna; Levey, Allan I.; Lah, James J.; Rujescu, Dan; Hampel, Harald; Giegling, Ina; Andreassen, Ole A.; Engedal, Knut; Ulstein, Ingun; Djurovic, Srdjan; Ibrahim-Verbaas, Carla; Hofman, Albert; Ikram, M. Arfan; van Duijn, Cornelia M; Thorsteinsdottir, Unnur; Kong, Augustine; Stefansson, Kari

2013-01-01

BACKGROUND Sequence variants, including the ε4 allele of apolipoprotein E, have been associated with the risk of the common late-onset form of Alzheimer’s disease. Few rare variants affecting the risk of late-onset Alzheimer’s disease have been found. METHODS We obtained the genome sequences of 2261 Icelanders and identified sequence variants that were likely to affect protein function. We imputed these variants into the genomes of patients with Alzheimer’s disease and control participants and then tested for an association with Alzheimer’s disease. We performed replication tests using case–control series from the United States, Norway, the Netherlands, and Germany. We also tested for a genetic association with cognitive function in a population of unaffected elderly persons. RESULTS A rare missense mutation (rs75932628-T) in the gene encoding the triggering receptor expressed on myeloid cells 2 (TREM2), which was predicted to result in an R47H substitution, was found to confer a significant risk of Alzheimer’s disease in Iceland (odds ratio, 2.92; 95% confidence interval [CI], 2.09 to 4.09; P = 3.42×10−10). The mutation had a frequency of 0.46% in controls 85 years of age or older. We observed the association in additional sample sets (odds ratio, 2.90; 95% CI, 2.16 to 3.91; P = 2.1×10−12 in combined discovery and replication samples). We also found that carriers of rs75932628-T between the ages of 80 and 100 years without Alzheimer’s disease had poorer cognitive function than noncarriers (P = 0.003). CONCLUSIONS Our findings strongly implicate variant TREM2 in the pathogenesis of Alzheimer’s disease. Given the reported antiinflammatory role of TREM2 in the brain, the R47H substitution may lead to an increased predisposition to Alzheimer’s disease through impaired containment of inflammatory processes. (Funded by the National Institute on Aging and others.) PMID:23150908

Selecting sequence variants to improve genomic predictions for dairy cattle

USDA-ARS?s Scientific Manuscript database

Millions of genetic variants have been identified by population-scale sequencing projects, but subsets are needed for routine genomic predictions or to include on genotyping arrays. Methods of selecting sequence variants were compared using both simulated sequence genotypes and actual data from run ...

Sequence variants in four genes underlying Bardet-Biedl syndrome in consanguineous families

PubMed Central

Ullah, Asmat; Umair, Muhammad; Yousaf, Maryam; Khan, Sher Alam; Nazim-ud-din, Muhammad; Shah, Khadim; Ahmad, Farooq; Azeem, Zahid; Ali, Ghazanfar; Alhaddad, Bader; Rafique, Afzal; Jan, Abid; Haack, Tobias B.; Strom, Tim M.; Meitinger, Thomas; Ghous, Tahseen

2017-01-01

Purpose To investigate the molecular basis of Bardet-Biedl syndrome (BBS) in five consanguineous families of Pakistani origin. Methods Linkage in two families (A and B) was established to BBS7 on chromosome 4q27, in family C to BBS8 on chromosome 14q32.1, and in family D to BBS10 on chromosome 12q21.2. Family E was investigated directly with exome sequence analysis. Results Sanger sequencing revealed two novel mutations and three previously reported mutations in the BBS genes. These mutations include two deletions (c.580_582delGCA, c.1592_1597delTTCCAG) in the BBS7 gene, a missense mutation (p.Gln449His) in the BBS8 gene, a frameshift mutation (c.271_272insT) in the BBS10 gene, and a nonsense mutation (p.Ser40*) in the MKKS (BBS6) gene. Conclusions Two novel mutations and three previously reported variants, identified in the present study, further extend the body of evidence implicating BBS6, BBS7, BBS8, and BBS10 in causing BBS. PMID:28761321

Novel pedigree analysis implicates DNA repair and chromatin remodeling in multiple myeloma risk

PubMed Central

Curtin, Karen; Rajamanickam, Venkatesh; Jayabalan, David; Atanackovic, Djordje; Rajkumar, S. Vincent; Kumar, Shaji; Slager, Susan; Galia, Perrine; Demangel, Delphine; Salama, Mohamed; Joseph, Vijai; Lipkin, Steven M.; Dumontet, Charles; Vachon, Celine M.

2018-01-01

The high-risk pedigree (HRP) design is an established strategy to discover rare, highly-penetrant, Mendelian-like causal variants. Its success, however, in complex traits has been modest, largely due to challenges of genetic heterogeneity and complex inheritance models. We describe a HRP strategy that addresses intra-familial heterogeneity, and identifies inherited segments important for mapping regulatory risk. We apply this new Shared Genomic Segment (SGS) method in 11 extended, Utah, multiple myeloma (MM) HRPs, and subsequent exome sequencing in SGS regions of interest in 1063 MM / MGUS (monoclonal gammopathy of undetermined significance–a precursor to MM) cases and 964 controls from a jointly-called collaborative resource, including cases from the initial 11 HRPs. One genome-wide significant 1.8 Mb shared segment was found at 6q16. Exome sequencing in this region revealed predicted deleterious variants in USP45 (p.Gln691* and p.Gln621Glu), a gene known to influence DNA repair through endonuclease regulation. Additionally, a 1.2 Mb segment at 1p36.11 is inherited in two Utah HRPs, with coding variants identified in ARID1A (p.Ser90Gly and p.Met890Val), a key gene in the SWI/SNF chromatin remodeling complex. Our results provide compelling statistical and genetic evidence for segregating risk variants for MM. In addition, we demonstrate a novel strategy to use large HRPs for risk-variant discovery more generally in complex traits. PMID:29389935

Novel pedigree analysis implicates DNA repair and chromatin remodeling in multiple myeloma risk.

PubMed

Waller, Rosalie G; Darlington, Todd M; Wei, Xiaomu; Madsen, Michael J; Thomas, Alun; Curtin, Karen; Coon, Hilary; Rajamanickam, Venkatesh; Musinsky, Justin; Jayabalan, David; Atanackovic, Djordje; Rajkumar, S Vincent; Kumar, Shaji; Slager, Susan; Middha, Mridu; Galia, Perrine; Demangel, Delphine; Salama, Mohamed; Joseph, Vijai; McKay, James; Offit, Kenneth; Klein, Robert J; Lipkin, Steven M; Dumontet, Charles; Vachon, Celine M; Camp, Nicola J

2018-02-01

The high-risk pedigree (HRP) design is an established strategy to discover rare, highly-penetrant, Mendelian-like causal variants. Its success, however, in complex traits has been modest, largely due to challenges of genetic heterogeneity and complex inheritance models. We describe a HRP strategy that addresses intra-familial heterogeneity, and identifies inherited segments important for mapping regulatory risk. We apply this new Shared Genomic Segment (SGS) method in 11 extended, Utah, multiple myeloma (MM) HRPs, and subsequent exome sequencing in SGS regions of interest in 1063 MM / MGUS (monoclonal gammopathy of undetermined significance-a precursor to MM) cases and 964 controls from a jointly-called collaborative resource, including cases from the initial 11 HRPs. One genome-wide significant 1.8 Mb shared segment was found at 6q16. Exome sequencing in this region revealed predicted deleterious variants in USP45 (p.Gln691* and p.Gln621Glu), a gene known to influence DNA repair through endonuclease regulation. Additionally, a 1.2 Mb segment at 1p36.11 is inherited in two Utah HRPs, with coding variants identified in ARID1A (p.Ser90Gly and p.Met890Val), a key gene in the SWI/SNF chromatin remodeling complex. Our results provide compelling statistical and genetic evidence for segregating risk variants for MM. In addition, we demonstrate a novel strategy to use large HRPs for risk-variant discovery more generally in complex traits.

Precise detection of de novo single nucleotide variants in human genomes.

PubMed

Gómez-Romero, Laura; Palacios-Flores, Kim; Reyes, José; García, Delfino; Boege, Margareta; Dávila, Guillermo; Flores, Margarita; Schatz, Michael C; Palacios, Rafael

2018-05-22

The precise determination of de novo genetic variants has enormous implications across different fields of biology and medicine, particularly personalized medicine. Currently, de novo variations are identified by mapping sample reads from a parent-offspring trio to a reference genome, allowing for a certain degree of differences. While widely used, this approach often introduces false-positive (FP) results due to misaligned reads and mischaracterized sequencing errors. In a previous study, we developed an alternative approach to accurately identify single nucleotide variants (SNVs) using only perfect matches. However, this approach could be applied only to haploid regions of the genome and was computationally intensive. In this study, we present a unique approach, coverage-based single nucleotide variant identification (COBASI), which allows the exploration of the entire genome using second-generation short sequence reads without extensive computing requirements. COBASI identifies SNVs using changes in coverage of exactly matching unique substrings, and is particularly suited for pinpointing de novo SNVs. Unlike other approaches that require population frequencies across hundreds of samples to filter out any methodological biases, COBASI can be applied to detect de novo SNVs within isolated families. We demonstrate this capability through extensive simulation studies and by studying a parent-offspring trio we sequenced using short reads. Experimental validation of all 58 candidate de novo SNVs and a selection of non-de novo SNVs found in the trio confirmed zero FP calls. COBASI is available as open source at https://github.com/Laura-Gomez/COBASI for any researcher to use. Copyright © 2018 the Author(s). Published by PNAS.

Whole-genome sequencing and genetic variant analysis of a Quarter Horse mare.

PubMed

Doan, Ryan; Cohen, Noah D; Sawyer, Jason; Ghaffari, Noushin; Johnson, Charlie D; Dindot, Scott V

2012-02-17

The catalog of genetic variants in the horse genome originates from a few select animals, the majority originating from the Thoroughbred mare used for the equine genome sequencing project. The purpose of this study was to identify genetic variants, including single nucleotide polymorphisms (SNPs), insertion/deletion polymorphisms (INDELs), and copy number variants (CNVs) in the genome of an individual Quarter Horse mare sequenced by next-generation sequencing. Using massively parallel paired-end sequencing, we generated 59.6 Gb of DNA sequence from a Quarter Horse mare resulting in an average of 24.7X sequence coverage. Reads were mapped to approximately 97% of the reference Thoroughbred genome. Unmapped reads were de novo assembled resulting in 19.1 Mb of new genomic sequence in the horse. Using a stringent filtering method, we identified 3.1 million SNPs, 193 thousand INDELs, and 282 CNVs. Genetic variants were annotated to determine their impact on gene structure and function. Additionally, we genotyped this Quarter Horse for mutations of known diseases and for variants associated with particular traits. Functional clustering analysis of genetic variants revealed that most of the genetic variation in the horse's genome was enriched in sensory perception, signal transduction, and immunity and defense pathways. This is the first sequencing of a horse genome by next-generation sequencing and the first genomic sequence of an individual Quarter Horse mare. We have increased the catalog of genetic variants for use in equine genomics by the addition of novel SNPs, INDELs, and CNVs. The genetic variants described here will be a useful resource for future studies of genetic variation regulating performance traits and diseases in equids.

Sequence analysis of the mitochondrial DNA control region of ciscoes (genus Coregonus): taxonomic implications for the Great Lakes species flock.

PubMed

Reed, K M; Dorschner, M O; Todd, T N; Phillips, R B

1998-09-01

Sequence variation in the control region (D-loop) of the mitochondrial DNA (mtDNA) was examined to assess the genetic distinctiveness of the shortjaw cisco (Coregonus zenithicus). Individuals from within the Great Lakes Basin as well as inland lakes outside the basin were sampled. DNA fragments containing the entire D-loop were amplified by PCR from specimens of C. zenithicus and the related species C. artedi, C. hoyi, C. kiyi, and C. clupeaformis. DNA sequence analysis revealed high similarity within and among species and shared polymorphism for length variants. Based on this analysis, the shortjaw cisco is not genetically distinct from other cisco species.

Analysis of PAC1 receptor gene variants in Caucasian and African American infants dying of sudden infant death syndrome.

PubMed

Barrett, Karlene T; Rodikova, Ekaterina; Weese-Mayer, Debra E; Rand, Casey M; Marazita, Mary L; Cooper, Margaret E; Berry-Kravis, Elizabeth M; Bech-Hansen, N Torben; Wilson, Richard J A

2013-12-01

Stress peptide, pituitary adenylate cyclase-activating polypeptide (PACAP), has been implicated in sudden infant death syndrome (SIDS). The aim of this exploratory study was to determine whether variants in the gene encoding the PACAP-specific receptor, PAC1, are associated with SIDS in Caucasian and African American infants. Polymerase chain reaction and Sanger DNA sequencing was used to compare variants in the 5'-untranslated region, exons and intron-exon boundaries of the PAC1 gene in 96 SIDS cases and 96 race- and gender-matched controls. The intron 3 variant, A/G: rs758995 (variant 'h'), and the intron 6 variant, C/T: rs10081254 (variant 'n'), were significantly associated with SIDS in Caucasians and African Americans, respectively (p < 0.05). Also associated with SIDS were interactions between the variants rs2302475 (variant 'i') in PAC1 and rs8192597 and rs2856966 in PACAP among Caucasians (p < 0.02) and rs2267734 (variant 'q') in PAC1 and rs1893154 in PACAP among African Americans (p < 0.01). However, none of these differences survived post hoc analysis. Overall, this study does not support a strong association between variants in the PAC1 gene and SIDS; however, a number of potential associations between race-specific variants and SIDS were identified that warrant targeted investigations in future studies. ©2013 Foundation Acta Paediatrica. Published by John Wiley & Sons Ltd.

TIAM1 variants improve clinical outcome in neuroblastoma.

PubMed

Sanmartín, Elena; Yáñez, Yania; Fornés-Ferrer, Victoria; Zugaza, José L; Cañete, Adela; Castel, Victoria; Font de Mora, Jaime

2017-07-11

Identification of tumor driver mutations is crucial for improving clinical outcome using a personalized approach to the treatment of cancer. Neuroblastoma is a tumor of the peripheral sympathetic nervous system for which only a few driver alterations have been described including MYCN amplification and ALK mutations. We assessed 106 primary neuroblastoma tumors by next generation sequencing using a customized amplicon-based gene panel. Our results reveal that genetic variants in TIAM1 gene associate with better clinical outcome, suggesting a role for these TIAM1 variants in preventing progression of this disease. The detected variants are located within the different domains of TIAM1 that signal to the upstream regulator RAS and downstream effector molecules MYC and RAC, which are all implicated in neuroblastoma etiology and progression. Clinical outcome was improved in tumors where a TIAM1 variant was present concomitantly with either ALK mutation or MYCN amplification. Given the function of these signaling molecules in cell survival, proliferation, differentiation and neurite outgrowth, our data suggest that the TIAM1-mediated network is essential to neuroblastoma and thus, inhibiting TIAM1 reflects a rational strategy for improving therapy efficacy in neuroblastoma.

Germline EMSY sequence alterations in hereditary breast cancer and ovarian cancer families.

PubMed

Määttä, Kirsi M; Nurminen, Riikka; Kankuri-Tammilehto, Minna; Kallioniemi, Anne; Laasanen, Satu-Leena; Schleutker, Johanna

2017-07-24

BRCA1 and BRCA2 mutations explain approximately one-fifth of the inherited susceptibility in high-risk Finnish hereditary breast and ovarian cancer (HBOC) families. EMSY is located in the breast cancer-associated chromosomal region 11q13. The EMSY gene encodes a BRCA2-interacting protein that has been implicated in DNA damage repair and genomic instability. We analysed the role of germline EMSY variation in breast/ovarian cancer predisposition. The present study describes the first EMSY screening in patients with high familial risk for this disease. Index individuals from 71 high-risk, BRCA1/2-negative HBOC families were screened for germline EMSY sequence alterations in protein coding regions and exon-intron boundaries using Sanger sequencing and TaqMan assays. The identified variants were further screened in 36 Finnish HBOC patients and 904 controls. Moreover, one novel intronic deletion was screened in a cohort of 404 breast cancer patients unselected for family history. Haplotype block structure and the association of haplotypes with breast/ovarian cancer were analysed using Haploview. The functionality of the identified variants was predicted using Haploreg, RegulomeDB, Human Splicing Finder, and Pathogenic-or-Not-Pipeline 2. Altogether, 12 germline EMSY variants were observed. Two alterations were located in the coding region, five alterations were intronic, and five alterations were located in the 3'untranslated region (UTR). Variant frequencies did not significantly differ between cases and controls. The novel variant, c.2709 + 122delT, was detected in 1 out of 107 (0.9%) breast cancer patients, and the carrier showed a bilateral form of the disease. The deletion was absent in 897 controls (OR = 25.28; P = 0.1) and in 404 breast cancer patients unselected for family history. No haplotype was identified to increase the risk of breast/ovarian cancer. Functional analyses suggested that variants, particularly in the 3'UTR, were located within regulatory elements. The novel deletion was predicted to affect splicing regulatory elements. These results suggest that the identified EMSY variants are likely neutral at the population level. However, these variants may contribute to breast/ovarian cancer risk in single families. Additional analyses are warranted for rare novel intronic deletions and the 3'UTR variants predicted to have functional roles.

Global variation in CYP2C8–CYP2C9 functional haplotypes

PubMed Central

Speed, William C; Kang, Soonmo Peter; Tuck, David P; Harris, Lyndsay N; Kidd, Kenneth K

2009-01-01

We have studied the global frequency distributions of 10 single nucleotide polymorphisms (SNPs) across 132 kb of CYP2C8 and CYP2C9 in ∼2500 individuals representing 45 populations. Five of the SNPs were in noncoding sequences; the other five involved the more common missense variants (four in CYP2C8, one in CYP2C9) that change amino acids in the gene products. One haplotype containing two CYP2C8 coding variants and one CYP2C9 coding variant reaches an average frequency of 10% in Europe; a set of haplotypes with a different CYP2C8 coding variant reaches 17% in Africa. In both cases these haplotypes are found in other regions of the world at <1%. This considerable geographic variation in haplotype frequencies impacts the interpretation of CYP2C8/CYP2C9 association studies, and has pharmacogenomic implications for drug interactions. PMID:19381162

Evaluation of exome variants using the Ion Proton Platform to sequence error-prone regions.

PubMed

Seo, Heewon; Park, Yoomi; Min, Byung Joo; Seo, Myung Eui; Kim, Ju Han

2017-01-01

The Ion Proton sequencer from Thermo Fisher accurately determines sequence variants from target regions with a rapid turnaround time at a low cost. However, misleading variant-calling errors can occur. We performed a systematic evaluation and manual curation of read-level alignments for the 675 ultrarare variants reported by the Ion Proton sequencer from 27 whole-exome sequencing data but that are not present in either the 1000 Genomes Project and the Exome Aggregation Consortium. We classified positive variant calls into 393 highly likely false positives, 126 likely false positives, and 156 likely true positives, which comprised 58.2%, 18.7%, and 23.1% of the variants, respectively. We identified four distinct error patterns of variant calling that may be bioinformatically corrected when using different strategies: simplicity region, SNV cluster, peripheral sequence read, and base inversion. Local de novo assembly successfully corrected 201 (38.7%) of the 519 highly likely or likely false positives. We also demonstrate that the two sequencing kits from Thermo Fisher (the Ion PI Sequencing 200 kit V3 and the Ion PI Hi-Q kit) exhibit different error profiles across different error types. A refined calling algorithm with better polymerase may improve the performance of the Ion Proton sequencing platform.

A novel ATTR L32V mutation causes familial amyloid polyneuropathy in a Bolivian family.

PubMed

Martínez-Ulloa, Pedro L; Vallejo, Manuela; Corral, Iñigo; García-Barragán, Nuria; Alcazar, Alberto; Martínez-Alonso, Emma; Martínez-Poles, Javier; Pian, Hector; Jiménez-Escrig, Adriano

2017-09-01

We report a new transthyretin (ATTR) gene c.272C>G mutation and variant protein, p.Leu32Val, in a kindred of Bolivian origin with a rapid progressive peripheral neuropathy and cardiomyopathy. Three individuals from a kindred with peripheral nerve and cardiac amyloidosis were examined. Analysis of the TTR gene was performed by Sanger direct sequencing. Neuropathologic examination was obtained on the index patient with mass spectrometry study of the ATTR deposition. Direct DNA sequence analysis of exons 2, 3, and 4 of the TTR gene demonstrated a c.272 C>G mutation in exon 2 (p.L32V). Sural nerve biopsy revealed massive amyloid deposition in the perineurium, endoneurium and vasa nervorum. Mass spectrometric analyses of ATTR immunoprecipitated from nerve biopsy showed the presence of both wild-type and variant proteins. The observed mass results for the wild-type and variant proteins were consistent with the predicted values calculated from the genetic analysis data. The ATTR L32V is associated with a severe course. This has implications for treatment of affected individuals and counseling of family members. © 2017 Peripheral Nerve Society.

Identification of pathogenic gene variants in small families with intellectually disabled siblings by exome sequencing.

PubMed

Schuurs-Hoeijmakers, Janneke H M; Vulto-van Silfhout, Anneke T; Vissers, Lisenka E L M; van de Vondervoort, Ilse I G M; van Bon, Bregje W M; de Ligt, Joep; Gilissen, Christian; Hehir-Kwa, Jayne Y; Neveling, Kornelia; del Rosario, Marisol; Hira, Gausiya; Reitano, Santina; Vitello, Aurelio; Failla, Pinella; Greco, Donatella; Fichera, Marco; Galesi, Ornella; Kleefstra, Tjitske; Greally, Marie T; Ockeloen, Charlotte W; Willemsen, Marjolein H; Bongers, Ernie M H F; Janssen, Irene M; Pfundt, Rolph; Veltman, Joris A; Romano, Corrado; Willemsen, Michèl A; van Bokhoven, Hans; Brunner, Han G; de Vries, Bert B A; de Brouwer, Arjan P M

2013-12-01

Intellectual disability (ID) is a common neurodevelopmental disorder affecting 1-3% of the general population. Mutations in more than 10% of all human genes are considered to be involved in this disorder, although the majority of these genes are still unknown. We investigated 19 small non-consanguineous families with two to five affected siblings in order to identify pathogenic gene variants in known, novel and potential ID candidate genes. Non-consanguineous families have been largely ignored in gene identification studies as small family size precludes prior mapping of the genetic defect. Using exome sequencing, we identified pathogenic mutations in three genes, DDHD2, SLC6A8, and SLC9A6, of which the latter two have previously been implicated in X-linked ID phenotypes. In addition, we identified potentially pathogenic mutations in BCORL1 on the X-chromosome and in MCM3AP, PTPRT, SYNE1, and ZNF528 on autosomes. We show that potentially pathogenic gene variants can be identified in small, non-consanguineous families with as few as two affected siblings, thus emphasising their value in the identification of syndromic and non-syndromic ID genes.

Pathogenetics of alveolar capillary dysplasia with misalignment of pulmonary veins.

PubMed

Szafranski, Przemyslaw; Gambin, Tomasz; Dharmadhikari, Avinash V; Akdemir, Kadir Caner; Jhangiani, Shalini N; Schuette, Jennifer; Godiwala, Nihal; Yatsenko, Svetlana A; Sebastian, Jessica; Madan-Khetarpal, Suneeta; Surti, Urvashi; Abellar, Rosanna G; Bateman, David A; Wilson, Ashley L; Markham, Melinda H; Slamon, Jill; Santos-Simarro, Fernando; Palomares, María; Nevado, Julián; Lapunzina, Pablo; Chung, Brian Hon-Yin; Wong, Wai-Lap; Chu, Yoyo Wing Yiu; Mok, Gary Tsz Kin; Kerem, Eitan; Reiter, Joel; Ambalavanan, Namasivayam; Anderson, Scott A; Kelly, David R; Shieh, Joseph; Rosenthal, Taryn C; Scheible, Kristin; Steiner, Laurie; Iqbal, M Anwar; McKinnon, Margaret L; Hamilton, Sara Jane; Schlade-Bartusiak, Kamilla; English, Dawn; Hendson, Glenda; Roeder, Elizabeth R; DeNapoli, Thomas S; Littlejohn, Rebecca Okashah; Wolff, Daynna J; Wagner, Carol L; Yeung, Alison; Francis, David; Fiorino, Elizabeth K; Edelman, Morris; Fox, Joyce; Hayes, Denise A; Janssens, Sandra; De Baere, Elfride; Menten, Björn; Loccufier, Anne; Vanwalleghem, Lieve; Moerman, Philippe; Sznajer, Yves; Lay, Amy S; Kussmann, Jennifer L; Chawla, Jasneek; Payton, Diane J; Phillips, Gael E; Brosens, Erwin; Tibboel, Dick; de Klein, Annelies; Maystadt, Isabelle; Fisher, Richard; Sebire, Neil; Male, Alison; Chopra, Maya; Pinner, Jason; Malcolm, Girvan; Peters, Gregory; Arbuckle, Susan; Lees, Melissa; Mead, Zoe; Quarrell, Oliver; Sayers, Richard; Owens, Martina; Shaw-Smith, Charles; Lioy, Janet; McKay, Eileen; de Leeuw, Nicole; Feenstra, Ilse; Spruijt, Liesbeth; Elmslie, Frances; Thiruchelvam, Timothy; Bacino, Carlos A; Langston, Claire; Lupski, James R; Sen, Partha; Popek, Edwina; Stankiewicz, Paweł

2016-05-01

Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a lethal lung developmental disorder caused by heterozygous point mutations or genomic deletion copy-number variants (CNVs) of FOXF1 or its upstream enhancer involving fetal lung-expressed long noncoding RNA genes LINC01081 and LINC01082. Using custom-designed array comparative genomic hybridization, Sanger sequencing, whole exome sequencing (WES), and bioinformatic analyses, we studied 22 new unrelated families (20 postnatal and two prenatal) with clinically diagnosed ACDMPV. We describe novel deletion CNVs at the FOXF1 locus in 13 unrelated ACDMPV patients. Together with the previously reported cases, all 31 genomic deletions in 16q24.1, pathogenic for ACDMPV, for which parental origin was determined, arose de novo with 30 of them occurring on the maternally inherited chromosome 16, strongly implicating genomic imprinting of the FOXF1 locus in human lungs. Surprisingly, we have also identified four ACDMPV families with the pathogenic variants in the FOXF1 locus that arose on paternal chromosome 16. Interestingly, a combination of the severe cardiac defects, including hypoplastic left heart, and single umbilical artery were observed only in children with deletion CNVs involving FOXF1 and its upstream enhancer. Our data demonstrate that genomic imprinting at 16q24.1 plays an important role in variable ACDMPV manifestation likely through long-range regulation of FOXF1 expression, and may be also responsible for key phenotypic features of maternal uniparental disomy 16. Moreover, in one family, WES revealed a de novo missense variant in ESRP1, potentially implicating FGF signaling in the etiology of ACDMPV.

Pathogenetics of Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins

PubMed Central

Szafranski, Przemyslaw; Gambin, Tomasz; Dharmadhikari, Avinash V.; Akdemir, Kadir Caner; Jhangiani, Shalini N.; Schuette, Jennifer; Godiwala, Nihal; Yatsenko, Svetlana A.; Sebastian, Jessica; Madan-Khetarpal, Suneeta; Surti, Urvashi; Abellar, Rosanna G.; Bateman, David A.; Wilson, Ashley L.; Markham, Melinda H.; Slamon, Jill; Santos-Simarro, Fernando; Palomares, María; Nevado, Julián; Lapunzina, Pablo; Hon-Yin, Brian Chung; Wai-Lap, Wong; Chu, Yoyo Wing Yiu; Mok, Gary Tsz Kin; Eitan, Kerem; Reiter, Joel; Ambalavanan, Namasivayam; Anderson, Scott A.; Kelly, David R.; Shieh, Joseph; Rosenthal, Taryn C.; Scheible, Kristin; Steiner, Laurie; Iqbal, M. Anwar; McKinnon, Margaret; Hamilton, Sara Jane; Schlade-Bartusiak, Kamilla; English, Dawn; Hendson, Glenda; Roeder, Elizabeth R.; DeNapoli, Thomas S.; Littlejohn, Rebecca Okashah; Wolff, Daynna J.; Wagner, Carol L.; Yeung, Alison; Francis, David; Fiorino, Elizabeth K.; Edelman, Morris; Fox, Joyce; Hayes, Denise A.; Janssens, Sandra; De Baere, Elfride; Menten, Bjorn; Loccufier, Anne; Van Walleghem, Lieve; Moerman, Philippe; Sznajer, Yves; Lay, Amy S.; Kussmann, Jennifer L.; Chawla, Jasneek; Payton, Diane J.; Phillips, Gael E.; Brosens, Erwin; Tibboel, Dick; de Klein, Annelies; Maystadt, Isabelle; Fisher, Richard; Sebire, Neil; Male, Alison; Chopra, Maya; Pinner, Jason; Malcolm, Girvan; Peters, Gregory; Arbuckle, Susan; Lees, Melissa; Mead, Zoe; Quarrell, Oliver; Sayers, Richard; Owens, Martina; Shaw-Smith, Charles; Lioy, Janet; McKay, Eileen; de Leeuw, Nicole; Feenstra, Ilse; Spruijt, Liesbeth; Elmslie, Frances; Thiruchelvam, Timothy; Bacino, Carlos A.; Langston, Claire; Lupski, James R.; Sen, Partha; Popek, Edwina; Stankiewicz, Paweł

2017-01-01

Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a lethal lung developmental disorder caused by heterozygous point mutations or genomic deletion copy-number variants (CNVs) of FOXF1 or its upstream enhancer involving fetal lung-expressed long noncoding RNA genes LINC01081 and LINC01082. Using custom-designed array comparative genomic hybridization, Sanger sequencing, whole exome sequencing (WES), and bioinformatic analyses, we studied 22 new unrelated families (20 postnatal and two prenatal) with clinically diagnosed ACDMPV. We describe novel deletion CNVs at the FOXF1 locus in 13 unrelated ACDMPV patients. Together with the previously reported cases, all 31 genomic deletions in 16q24.1, pathogenic for ACDMPV, for which parental origin was determined, arose de novo with 30 of them occurring on the maternally inherited chromosome 16, strongly implicating genomic imprinting of the FOXF1 locus in human lungs. Surprisingly, we have also identified four ACDMPV families with the pathogenic variants in the FOXF1 locus that arose on paternal chromosome 16. Interestingly, a combination of the severe cardiac defects, including hypoplastic left heart, and single umbilical artery were observed only in children with deletion CNVs involving FOXF1 and its upstream enhancer. Our data demonstrate that genomic imprinting at 16q24.1 plays an important role in variable ACDMPV manifestation likely through long-range regulation of FOXF1 expression, and may be also responsible for key phenotypic features of maternal uniparental disomy 16. Moreover, in one family, WES revealed a de novo missense variant in ESRP1, potentially implicating FGF signaling in etiology of ACDMPV. PMID:27071622

Implicity Defined Neural Networks for Sequence Labeling

DTIC Science & Technology

2017-02-13

popularity of the Long Short - Term Memory (LSTM) (Hochreiter and Schmidhuber, 1997) and variants such as the Gated Recurrent Unit (GRU) (Cho et al., 2014...bidirectional lstm and other neural network architectures. Neural Net- works 18(5):602–610. Sepp Hochreiter and Jürgen Schmidhuber. 1997. Long short - term ...hid- den states of the network to coupled together, allowing potential improvement on problems with complex, long -distance dependencies. Initial

An efficient and scalable analysis framework for variant extraction and refinement from population-scale DNA sequence data.

PubMed

Jun, Goo; Wing, Mary Kate; Abecasis, Gonçalo R; Kang, Hyun Min

2015-06-01

The analysis of next-generation sequencing data is computationally and statistically challenging because of the massive volume of data and imperfect data quality. We present GotCloud, a pipeline for efficiently detecting and genotyping high-quality variants from large-scale sequencing data. GotCloud automates sequence alignment, sample-level quality control, variant calling, filtering of likely artifacts using machine-learning techniques, and genotype refinement using haplotype information. The pipeline can process thousands of samples in parallel and requires less computational resources than current alternatives. Experiments with whole-genome and exome-targeted sequence data generated by the 1000 Genomes Project show that the pipeline provides effective filtering against false positive variants and high power to detect true variants. Our pipeline has already contributed to variant detection and genotyping in several large-scale sequencing projects, including the 1000 Genomes Project and the NHLBI Exome Sequencing Project. We hope it will now prove useful to many medical sequencing studies. © 2015 Jun et al.; Published by Cold Spring Harbor Laboratory Press.

Principles and Recommendations for Standardizing the Use of the Next-Generation Sequencing Variant File in Clinical Settings.

PubMed

Lubin, Ira M; Aziz, Nazneen; Babb, Lawrence J; Ballinger, Dennis; Bisht, Himani; Church, Deanna M; Cordes, Shaun; Eilbeck, Karen; Hyland, Fiona; Kalman, Lisa; Landrum, Melissa; Lockhart, Edward R; Maglott, Donna; Marth, Gabor; Pfeifer, John D; Rehm, Heidi L; Roy, Somak; Tezak, Zivana; Truty, Rebecca; Ullman-Cullere, Mollie; Voelkerding, Karl V; Worthey, Elizabeth A; Zaranek, Alexander W; Zook, Justin M

2017-05-01

A national workgroup convened by the Centers for Disease Control and Prevention identified principles and made recommendations for standardizing the description of sequence data contained within the variant file generated during the course of clinical next-generation sequence analysis for diagnosing human heritable conditions. The specifications for variant files were initially developed to be flexible with regard to content representation to support a variety of research applications. This flexibility permits variation with regard to how sequence findings are described and this depends, in part, on the conventions used. For clinical laboratory testing, this poses a problem because these differences can compromise the capability to compare sequence findings among laboratories to confirm results and to query databases to identify clinically relevant variants. To provide for a more consistent representation of sequence findings described within variant files, the workgroup made several recommendations that considered alignment to a common reference sequence, variant caller settings, use of genomic coordinates, and gene and variant naming conventions. These recommendations were considered with regard to the existing variant file specifications presently used in the clinical setting. Adoption of these recommendations is anticipated to reduce the potential for ambiguity in describing sequence findings and facilitate the sharing of genomic data among clinical laboratories and other entities. Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Sequence data and association statistics from 12,940 type 2 diabetes cases and controls.

PubMed

Flannick, Jason; Fuchsberger, Christian; Mahajan, Anubha; Teslovich, Tanya M; Agarwala, Vineeta; Gaulton, Kyle J; Caulkins, Lizz; Koesterer, Ryan; Ma, Clement; Moutsianas, Loukas; McCarthy, Davis J; Rivas, Manuel A; Perry, John R B; Sim, Xueling; Blackwell, Thomas W; Robertson, Neil R; Rayner, N William; Cingolani, Pablo; Locke, Adam E; Tajes, Juan Fernandez; Highland, Heather M; Dupuis, Josee; Chines, Peter S; Lindgren, Cecilia M; Hartl, Christopher; Jackson, Anne U; Chen, Han; Huyghe, Jeroen R; van de Bunt, Martijn; Pearson, Richard D; Kumar, Ashish; Müller-Nurasyid, Martina; Grarup, Niels; Stringham, Heather M; Gamazon, Eric R; Lee, Jaehoon; Chen, Yuhui; Scott, Robert A; Below, Jennifer E; Chen, Peng; Huang, Jinyan; Go, Min Jin; Stitzel, Michael L; Pasko, Dorota; Parker, Stephen C J; Varga, Tibor V; Green, Todd; Beer, Nicola L; Day-Williams, Aaron G; Ferreira, Teresa; Fingerlin, Tasha; Horikoshi, Momoko; Hu, Cheng; Huh, Iksoo; Ikram, Mohammad Kamran; Kim, Bong-Jo; Kim, Yongkang; Kim, Young Jin; Kwon, Min-Seok; Lee, Juyoung; Lee, Selyeong; Lin, Keng-Han; Maxwell, Taylor J; Nagai, Yoshihiko; Wang, Xu; Welch, Ryan P; Yoon, Joon; Zhang, Weihua; Barzilai, Nir; Voight, Benjamin F; Han, Bok-Ghee; Jenkinson, Christopher P; Kuulasmaa, Teemu; Kuusisto, Johanna; Manning, Alisa; Ng, Maggie C Y; Palmer, Nicholette D; Balkau, Beverley; Stančáková, Alena; Abboud, Hanna E; Boeing, Heiner; Giedraitis, Vilmantas; Prabhakaran, Dorairaj; Gottesman, Omri; Scott, James; Carey, Jason; Kwan, Phoenix; Grant, George; Smith, Joshua D; Neale, Benjamin M; Purcell, Shaun; Butterworth, Adam S; Howson, Joanna M M; Lee, Heung Man; Lu, Yingchang; Kwak, Soo-Heon; Zhao, Wei; Danesh, John; Lam, Vincent K L; Park, Kyong Soo; Saleheen, Danish; So, Wing Yee; Tam, Claudia H T; Afzal, Uzma; Aguilar, David; Arya, Rector; Aung, Tin; Chan, Edmund; Navarro, Carmen; Cheng, Ching-Yu; Palli, Domenico; Correa, Adolfo; Curran, Joanne E; Rybin, Dennis; Farook, Vidya S; Fowler, Sharon P; Freedman, Barry I; Griswold, Michael; Hale, Daniel Esten; Hicks, Pamela J; Khor, Chiea-Chuen; Kumar, Satish; Lehne, Benjamin; Thuillier, Dorothée; Lim, Wei Yen; Liu, Jianjun; Loh, Marie; Musani, Solomon K; Puppala, Sobha; Scott, William R; Yengo, Loïc; Tan, Sian-Tsung; Taylor, Herman A; Thameem, Farook; Wilson, Gregory; Wong, Tien Yin; Njølstad, Pål Rasmus; Levy, Jonathan C; Mangino, Massimo; Bonnycastle, Lori L; Schwarzmayr, Thomas; Fadista, João; Surdulescu, Gabriela L; Herder, Christian; Groves, Christopher J; Wieland, Thomas; Bork-Jensen, Jette; Brandslund, Ivan; Christensen, Cramer; Koistinen, Heikki A; Doney, Alex S F; Kinnunen, Leena; Esko, Tõnu; Farmer, Andrew J; Hakaste, Liisa; Hodgkiss, Dylan; Kravic, Jasmina; Lyssenko, Valeri; Hollensted, Mette; Jørgensen, Marit E; Jørgensen, Torben; Ladenvall, Claes; Justesen, Johanne Marie; Käräjämäki, Annemari; Kriebel, Jennifer; Rathmann, Wolfgang; Lannfelt, Lars; Lauritzen, Torsten; Narisu, Narisu; Linneberg, Allan; Melander, Olle; Milani, Lili; Neville, Matt; Orho-Melander, Marju; Qi, Lu; Qi, Qibin; Roden, Michael; Rolandsson, Olov; Swift, Amy; Rosengren, Anders H; Stirrups, Kathleen; Wood, Andrew R; Mihailov, Evelin; Blancher, Christine; Carneiro, Mauricio O; Maguire, Jared; Poplin, Ryan; Shakir, Khalid; Fennell, Timothy; DePristo, Mark; de Angelis, Martin Hrabé; Deloukas, Panos; Gjesing, Anette P; Jun, Goo; Nilsson, Peter; Murphy, Jacquelyn; Onofrio, Robert; Thorand, Barbara; Hansen, Torben; Meisinger, Christa; Hu, Frank B; Isomaa, Bo; Karpe, Fredrik; Liang, Liming; Peters, Annette; Huth, Cornelia; O'Rahilly, Stephen P; Palmer, Colin N A; Pedersen, Oluf; Rauramaa, Rainer; Tuomilehto, Jaakko; Salomaa, Veikko; Watanabe, Richard M; Syvänen, Ann-Christine; Bergman, Richard N; Bharadwaj, Dwaipayan; Bottinger, Erwin P; Cho, Yoon Shin; Chandak, Giriraj R; Chan, Juliana Cn; Chia, Kee Seng; Daly, Mark J; Ebrahim, Shah B; Langenberg, Claudia; Elliott, Paul; Jablonski, Kathleen A; Lehman, Donna M; Jia, Weiping; Ma, Ronald C W; Pollin, Toni I; Sandhu, Manjinder; Tandon, Nikhil; Froguel, Philippe; Barroso, Inês; Teo, Yik Ying; Zeggini, Eleftheria; Loos, Ruth J F; Small, Kerrin S; Ried, Janina S; DeFronzo, Ralph A; Grallert, Harald; Glaser, Benjamin; Metspalu, Andres; Wareham, Nicholas J; Walker, Mark; Banks, Eric; Gieger, Christian; Ingelsson, Erik; Im, Hae Kyung; Illig, Thomas; Franks, Paul W; Buck, Gemma; Trakalo, Joseph; Buck, David; Prokopenko, Inga; Mägi, Reedik; Lind, Lars; Farjoun, Yossi; Owen, Katharine R; Gloyn, Anna L; Strauch, Konstantin; Tuomi, Tiinamaija; Kooner, Jaspal Singh; Lee, Jong-Young; Park, Taesung; Donnelly, Peter; Morris, Andrew D; Hattersley, Andrew T; Bowden, Donald W; Collins, Francis S; Atzmon, Gil; Chambers, John C; Spector, Timothy D; Laakso, Markku; Strom, Tim M; Bell, Graeme I; Blangero, John; Duggirala, Ravindranath; Tai, E Shyong; McVean, Gilean; Hanis, Craig L; Wilson, James G; Seielstad, Mark; Frayling, Timothy M; Meigs, James B; Cox, Nancy J; Sladek, Rob; Lander, Eric S; Gabriel, Stacey; Mohlke, Karen L; Meitinger, Thomas; Groop, Leif; Abecasis, Goncalo; Scott, Laura J; Morris, Andrew P; Kang, Hyun Min; Altshuler, David; Burtt, Noël P; Florez, Jose C; Boehnke, Michael; McCarthy, Mark I

2017-12-19

To investigate the genetic basis of type 2 diabetes (T2D) to high resolution, the GoT2D and T2D-GENES consortia catalogued variation from whole-genome sequencing of 2,657 European individuals and exome sequencing of 12,940 individuals of multiple ancestries. Over 27M SNPs, indels, and structural variants were identified, including 99% of low-frequency (minor allele frequency [MAF] 0.1-5%) non-coding variants in the whole-genome sequenced individuals and 99.7% of low-frequency coding variants in the whole-exome sequenced individuals. Each variant was tested for association with T2D in the sequenced individuals, and, to increase power, most were tested in larger numbers of individuals (>80% of low-frequency coding variants in ~82 K Europeans via the exome chip, and ~90% of low-frequency non-coding variants in ~44 K Europeans via genotype imputation). The variants, genotypes, and association statistics from these analyses provide the largest reference to date of human genetic information relevant to T2D, for use in activities such as T2D-focused genotype imputation, functional characterization of variants or genes, and other novel analyses to detect associations between sequence variation and T2D.

Sequence data and association statistics from 12,940 type 2 diabetes cases and controls

PubMed Central

Jason, Flannick; Fuchsberger, Christian; Mahajan, Anubha; Teslovich, Tanya M.; Agarwala, Vineeta; Gaulton, Kyle J.; Caulkins, Lizz; Koesterer, Ryan; Ma, Clement; Moutsianas, Loukas; McCarthy, Davis J.; Rivas, Manuel A.; Perry, John R. B.; Sim, Xueling; Blackwell, Thomas W.; Robertson, Neil R.; Rayner, N William; Cingolani, Pablo; Locke, Adam E.; Tajes, Juan Fernandez; Highland, Heather M.; Dupuis, Josee; Chines, Peter S.; Lindgren, Cecilia M.; Hartl, Christopher; Jackson, Anne U.; Chen, Han; Huyghe, Jeroen R.; van de Bunt, Martijn; Pearson, Richard D.; Kumar, Ashish; Müller-Nurasyid, Martina; Grarup, Niels; Stringham, Heather M.; Gamazon, Eric R.; Lee, Jaehoon; Chen, Yuhui; Scott, Robert A.; Below, Jennifer E.; Chen, Peng; Huang, Jinyan; Go, Min Jin; Stitzel, Michael L.; Pasko, Dorota; Parker, Stephen C. J.; Varga, Tibor V.; Green, Todd; Beer, Nicola L.; Day-Williams, Aaron G.; Ferreira, Teresa; Fingerlin, Tasha; Horikoshi, Momoko; Hu, Cheng; Huh, Iksoo; Ikram, Mohammad Kamran; Kim, Bong-Jo; Kim, Yongkang; Kim, Young Jin; Kwon, Min-Seok; Lee, Juyoung; Lee, Selyeong; Lin, Keng-Han; Maxwell, Taylor J.; Nagai, Yoshihiko; Wang, Xu; Welch, Ryan P.; Yoon, Joon; Zhang, Weihua; Barzilai, Nir; Voight, Benjamin F.; Han, Bok-Ghee; Jenkinson, Christopher P.; Kuulasmaa, Teemu; Kuusisto, Johanna; Manning, Alisa; Ng, Maggie C. Y.; Palmer, Nicholette D.; Balkau, Beverley; Stančáková, Alena; Abboud, Hanna E.; Boeing, Heiner; Giedraitis, Vilmantas; Prabhakaran, Dorairaj; Gottesman, Omri; Scott, James; Carey, Jason; Kwan, Phoenix; Grant, George; Smith, Joshua D.; Neale, Benjamin M.; Purcell, Shaun; Butterworth, Adam S.; Howson, Joanna M. M.; Lee, Heung Man; Lu, Yingchang; Kwak, Soo-Heon; Zhao, Wei; Danesh, John; Lam, Vincent K. L.; Park, Kyong Soo; Saleheen, Danish; So, Wing Yee; Tam, Claudia H. T.; Afzal, Uzma; Aguilar, David; Arya, Rector; Aung, Tin; Chan, Edmund; Navarro, Carmen; Cheng, Ching-Yu; Palli, Domenico; Correa, Adolfo; Curran, Joanne E.; Rybin, Dennis; Farook, Vidya S.; Fowler, Sharon P.; Freedman, Barry I.; Griswold, Michael; Hale, Daniel Esten; Hicks, Pamela J.; Khor, Chiea-Chuen; Kumar, Satish; Lehne, Benjamin; Thuillier, Dorothée; Lim, Wei Yen; Liu, Jianjun; Loh, Marie; Musani, Solomon K.; Puppala, Sobha; Scott, William R.; Yengo, Loïc; Tan, Sian-Tsung; Taylor, Herman A.; Thameem, Farook; Wilson, Gregory; Wong, Tien Yin; Njølstad, Pål Rasmus; Levy, Jonathan C.; Mangino, Massimo; Bonnycastle, Lori L.; Schwarzmayr, Thomas; Fadista, João; Surdulescu, Gabriela L.; Herder, Christian; Groves, Christopher J.; Wieland, Thomas; Bork-Jensen, Jette; Brandslund, Ivan; Christensen, Cramer; Koistinen, Heikki A.; Doney, Alex S. F.; Kinnunen, Leena; Esko, Tõnu; Farmer, Andrew J.; Hakaste, Liisa; Hodgkiss, Dylan; Kravic, Jasmina; Lyssenko, Valeri; Hollensted, Mette; Jørgensen, Marit E.; Jørgensen, Torben; Ladenvall, Claes; Justesen, Johanne Marie; Käräjämäki, Annemari; Kriebel, Jennifer; Rathmann, Wolfgang; Lannfelt, Lars; Lauritzen, Torsten; Narisu, Narisu; Linneberg, Allan; Melander, Olle; Milani, Lili; Neville, Matt; Orho-Melander, Marju; Qi, Lu; Qi, Qibin; Roden, Michael; Rolandsson, Olov; Swift, Amy; Rosengren, Anders H.; Stirrups, Kathleen; Wood, Andrew R.; Mihailov, Evelin; Blancher, Christine; Carneiro, Mauricio O.; Maguire, Jared; Poplin, Ryan; Shakir, Khalid; Fennell, Timothy; DePristo, Mark; de Angelis, Martin Hrabé; Deloukas, Panos; Gjesing, Anette P.; Jun, Goo; Nilsson, Peter; Murphy, Jacquelyn; Onofrio, Robert; Thorand, Barbara; Hansen, Torben; Meisinger, Christa; Hu, Frank B.; Isomaa, Bo; Karpe, Fredrik; Liang, Liming; Peters, Annette; Huth, Cornelia; O'Rahilly, Stephen P; Palmer, Colin N. A.; Pedersen, Oluf; Rauramaa, Rainer; Tuomilehto, Jaakko; Salomaa, Veikko; Watanabe, Richard M.; Syvänen, Ann-Christine; Bergman, Richard N.; Bharadwaj, Dwaipayan; Bottinger, Erwin P.; Cho, Yoon Shin; Chandak, Giriraj R.; Chan, Juliana CN; Chia, Kee Seng; Daly, Mark J.; Ebrahim, Shah B.; Langenberg, Claudia; Elliott, Paul; Jablonski, Kathleen A.; Lehman, Donna M.; Jia, Weiping; Ma, Ronald C. W.; Pollin, Toni I.; Sandhu, Manjinder; Tandon, Nikhil; Froguel, Philippe; Barroso, Inês; Teo, Yik Ying; Zeggini, Eleftheria; Loos, Ruth J. F.; Small, Kerrin S.; Ried, Janina S.; DeFronzo, Ralph A.; Grallert, Harald; Glaser, Benjamin; Metspalu, Andres; Wareham, Nicholas J.; Walker, Mark; Banks, Eric; Gieger, Christian; Ingelsson, Erik; Im, Hae Kyung; Illig, Thomas; Franks, Paul W.; Buck, Gemma; Trakalo, Joseph; Buck, David; Prokopenko, Inga; Mägi, Reedik; Lind, Lars; Farjoun, Yossi; Owen, Katharine R.; Gloyn, Anna L.; Strauch, Konstantin; Tuomi, Tiinamaija; Kooner, Jaspal Singh; Lee, Jong-Young; Park, Taesung; Donnelly, Peter; Morris, Andrew D.; Hattersley, Andrew T.; Bowden, Donald W.; Collins, Francis S.; Atzmon, Gil; Chambers, John C.; Spector, Timothy D.; Laakso, Markku; Strom, Tim M.; Bell, Graeme I.; Blangero, John; Duggirala, Ravindranath; Tai, E. Shyong; McVean, Gilean; Hanis, Craig L.; Wilson, James G.; Seielstad, Mark; Frayling, Timothy M.; Meigs, James B.; Cox, Nancy J.; Sladek, Rob; Lander, Eric S.; Gabriel, Stacey; Mohlke, Karen L.; Meitinger, Thomas; Groop, Leif; Abecasis, Goncalo; Scott, Laura J.; Morris, Andrew P.; Kang, Hyun Min; Altshuler, David; Burtt, Noël P.; Florez, Jose C.; Boehnke, Michael; McCarthy, Mark I.

2017-01-01

To investigate the genetic basis of type 2 diabetes (T2D) to high resolution, the GoT2D and T2D-GENES consortia catalogued variation from whole-genome sequencing of 2,657 European individuals and exome sequencing of 12,940 individuals of multiple ancestries. Over 27M SNPs, indels, and structural variants were identified, including 99% of low-frequency (minor allele frequency [MAF] 0.1–5%) non-coding variants in the whole-genome sequenced individuals and 99.7% of low-frequency coding variants in the whole-exome sequenced individuals. Each variant was tested for association with T2D in the sequenced individuals, and, to increase power, most were tested in larger numbers of individuals (>80% of low-frequency coding variants in ~82 K Europeans via the exome chip, and ~90% of low-frequency non-coding variants in ~44 K Europeans via genotype imputation). The variants, genotypes, and association statistics from these analyses provide the largest reference to date of human genetic information relevant to T2D, for use in activities such as T2D-focused genotype imputation, functional characterization of variants or genes, and other novel analyses to detect associations between sequence variation and T2D. PMID:29257133

A statistical method for the detection of variants from next-generation resequencing of DNA pools.

PubMed

Bansal, Vikas

2010-06-15

Next-generation sequencing technologies have enabled the sequencing of several human genomes in their entirety. However, the routine resequencing of complete genomes remains infeasible. The massive capacity of next-generation sequencers can be harnessed for sequencing specific genomic regions in hundreds to thousands of individuals. Sequencing-based association studies are currently limited by the low level of multiplexing offered by sequencing platforms. Pooled sequencing represents a cost-effective approach for studying rare variants in large populations. To utilize the power of DNA pooling, it is important to accurately identify sequence variants from pooled sequencing data. Detection of rare variants from pooled sequencing represents a different challenge than detection of variants from individual sequencing. We describe a novel statistical approach, CRISP [Comprehensive Read analysis for Identification of Single Nucleotide Polymorphisms (SNPs) from Pooled sequencing] that is able to identify both rare and common variants by using two approaches: (i) comparing the distribution of allele counts across multiple pools using contingency tables and (ii) evaluating the probability of observing multiple non-reference base calls due to sequencing errors alone. Information about the distribution of reads between the forward and reverse strands and the size of the pools is also incorporated within this framework to filter out false variants. Validation of CRISP on two separate pooled sequencing datasets generated using the Illumina Genome Analyzer demonstrates that it can detect 80-85% of SNPs identified using individual sequencing while achieving a low false discovery rate (3-5%). Comparison with previous methods for pooled SNP detection demonstrates the significantly lower false positive and false negative rates for CRISP. Implementation of this method is available at http://polymorphism.scripps.edu/~vbansal/software/CRISP/.

Characterization of the two intra-individual sequence variants in the 18S rRNA gene in the plant parasitic nematode, Rotylenchulus reniformis.

PubMed

Nyaku, Seloame T; Sripathi, Venkateswara R; Kantety, Ramesh V; Gu, Yong Q; Lawrence, Kathy; Sharma, Govind C

2013-01-01

The 18S rRNA gene is fundamental to cellular and organismal protein synthesis and because of its stable persistence through generations it is also used in phylogenetic analysis among taxa. Sequence variation in this gene within a single species is rare, but it has been observed in few metazoan organisms. More frequently it has mostly been reported in the non-transcribed spacer region. Here, we have identified two sequence variants within the near full coding region of 18S rRNA gene from a single reniform nematode (RN) Rotylenchulus reniformis labeled as reniform nematode variant 1 (RN_VAR1) and variant 2 (RN_VAR2). All sequences from three of the four isolates had both RN variants in their sequences; however, isolate 13B had only RN variant 2 sequence. Specific variable base sites (96 or 5.5%) were found within the 18S rRNA gene that can clearly distinguish the two 18S rDNA variants of RN, in 11 (25.0%) and 33 (75.0%) of the 44 RN clones, for RN_VAR1 and RN_VAR2, respectively. Neighbor-joining trees show that the RN_VAR1 is very similar to the previously existing R. reniformis sequence in GenBank, while the RN_VAR2 sequence is more divergent. This is the first report of the identification of two major variants of the 18S rRNA gene in the same single RN, and documents the specific base variation between the two variants, and hypothesizes on simultaneous co-existence of these two variants for this gene.

Characterization of the Two Intra-Individual Sequence Variants in the 18S rRNA Gene in the Plant Parasitic Nematode, Rotylenchulus reniformis

PubMed Central

Nyaku, Seloame T.; Sripathi, Venkateswara R.; Kantety, Ramesh V.; Gu, Yong Q.; Lawrence, Kathy; Sharma, Govind C.

2013-01-01

The 18S rRNA gene is fundamental to cellular and organismal protein synthesis and because of its stable persistence through generations it is also used in phylogenetic analysis among taxa. Sequence variation in this gene within a single species is rare, but it has been observed in few metazoan organisms. More frequently it has mostly been reported in the non-transcribed spacer region. Here, we have identified two sequence variants within the near full coding region of 18S rRNA gene from a single reniform nematode (RN) Rotylenchulus reniformis labeled as reniform nematode variant 1 (RN_VAR1) and variant 2 (RN_VAR2). All sequences from three of the four isolates had both RN variants in their sequences; however, isolate 13B had only RN variant 2 sequence. Specific variable base sites (96 or 5.5%) were found within the 18S rRNA gene that can clearly distinguish the two 18S rDNA variants of RN, in 11 (25.0%) and 33 (75.0%) of the 44 RN clones, for RN_VAR1 and RN_VAR2, respectively. Neighbor-joining trees show that the RN_VAR1 is very similar to the previously existing R. reniformis sequence in GenBank, while the RN_VAR2 sequence is more divergent. This is the first report of the identification of two major variants of the 18S rRNA gene in the same single RN, and documents the specific base variation between the two variants, and hypothesizes on simultaneous co-existence of these two variants for this gene. PMID:23593343

Whole exome sequencing for familial bicuspid aortic valve identifies putative variants.

PubMed

Martin, Lisa J; Pilipenko, Valentina; Kaufman, Kenneth M; Cripe, Linda; Kottyan, Leah C; Keddache, Mehdi; Dexheimer, Phillip; Weirauch, Matthew T; Benson, D Woodrow

2014-10-01

Bicuspid aortic valve (BAV) is the most common congenital cardiovascular malformation. Although highly heritable, few causal variants have been identified. The purpose of this study was to identify genetic variants underlying BAV by whole exome sequencing a multiplex BAV kindred. Whole exome sequencing was performed on 17 individuals from a single family (BAV=3; other cardiovascular malformation, 3). Postvariant calling error control metrics were established after examining the relationship between Mendelian inheritance error rate and coverage, quality score, and call rate. To determine the most effective approach to identifying susceptibility variants from among 54 674 variants passing error control metrics, we evaluated 3 variant selection strategies frequently used in whole exome sequencing studies plus extended family linkage. No putative rare, high-effect variants were identified in all affected but no unaffected individuals. Eight high-effect variants were identified by ≥2 of the commonly used selection strategies; however, these were either common in the general population (>10%) or present in the majority of the unaffected family members. However, using extended family linkage, 3 synonymous variants were identified; all 3 variants were identified by at least one other strategy. These results suggest that traditional whole exome sequencing approaches, which assume causal variants alter coding sense, may be insufficient for BAV and other complex traits. Identification of disease-associated variants is facilitated by the use of segregation within families. © 2014 American Heart Association, Inc.

Guidelines for investigating causality of sequence variants in human disease

PubMed Central

MacArthur, D. G.; Manolio, T. A.; Dimmock, D. P.; Rehm, H. L.; Shendure, J.; Abecasis, G. R.; Adams, D. R.; Altman, R. B.; Antonarakis, S. E.; Ashley, E. A.; Barrett, J. C.; Biesecker, L. G.; Conrad, D. F.; Cooper, G. M.; Cox, N. J.; Daly, M. J.; Gerstein, M. B.; Goldstein, D. B.; Hirschhorn, J. N.; Leal, S. M.; Pennacchio, L. A.; Stamatoyannopoulos, J. A.; Sunyaev, S. R.; Valle, D.; Voight, B. F.; Winckler, W.; Gunter, C.

2014-01-01

The discovery of rare genetic variants is accelerating, and clear guidelines for distinguishing disease-causing sequence variants from the many potentially functional variants present in any human genome are urgently needed. Without rigorous standards we risk an acceleration of false-positive reports of causality, which would impede the translation of genomic research findings into the clinical diagnostic setting and hinder biological understanding of disease. Here we discuss the key challenges of assessing sequence variants in human disease, integrating both gene-level and variant-level support for causality. We propose guidelines for summarizing confidence in variant pathogenicity and highlight several areas that require further resource development. PMID:24759409

Guidelines for investigating causality of sequence variants in human disease.

PubMed

MacArthur, D G; Manolio, T A; Dimmock, D P; Rehm, H L; Shendure, J; Abecasis, G R; Adams, D R; Altman, R B; Antonarakis, S E; Ashley, E A; Barrett, J C; Biesecker, L G; Conrad, D F; Cooper, G M; Cox, N J; Daly, M J; Gerstein, M B; Goldstein, D B; Hirschhorn, J N; Leal, S M; Pennacchio, L A; Stamatoyannopoulos, J A; Sunyaev, S R; Valle, D; Voight, B F; Winckler, W; Gunter, C

2014-04-24

The discovery of rare genetic variants is accelerating, and clear guidelines for distinguishing disease-causing sequence variants from the many potentially functional variants present in any human genome are urgently needed. Without rigorous standards we risk an acceleration of false-positive reports of causality, which would impede the translation of genomic research findings into the clinical diagnostic setting and hinder biological understanding of disease. Here we discuss the key challenges of assessing sequence variants in human disease, integrating both gene-level and variant-level support for causality. We propose guidelines for summarizing confidence in variant pathogenicity and highlight several areas that require further resource development.

Reliable Detection of Herpes Simplex Virus Sequence Variation by High-Throughput Resequencing.

PubMed

Morse, Alison M; Calabro, Kaitlyn R; Fear, Justin M; Bloom, David C; McIntyre, Lauren M

2017-08-16

High-throughput sequencing (HTS) has resulted in data for a number of herpes simplex virus (HSV) laboratory strains and clinical isolates. The knowledge of these sequences has been critical for investigating viral pathogenicity. However, the assembly of complete herpesviral genomes, including HSV, is complicated due to the existence of large repeat regions and arrays of smaller reiterated sequences that are commonly found in these genomes. In addition, the inherent genetic variation in populations of isolates for viruses and other microorganisms presents an additional challenge to many existing HTS sequence assembly pipelines. Here, we evaluate two approaches for the identification of genetic variants in HSV1 strains using Illumina short read sequencing data. The first, a reference-based approach, identifies variants from reads aligned to a reference sequence and the second, a de novo assembly approach, identifies variants from reads aligned to de novo assembled consensus sequences. Of critical importance for both approaches is the reduction in the number of low complexity regions through the construction of a non-redundant reference genome. We compared variants identified in the two methods. Our results indicate that approximately 85% of variants are identified regardless of the approach. The reference-based approach to variant discovery captures an additional 15% representing variants divergent from the HSV1 reference possibly due to viral passage. Reference-based approaches are significantly less labor-intensive and identify variants across the genome where de novo assembly-based approaches are limited to regions where contigs have been successfully assembled. In addition, regions of poor quality assembly can lead to false variant identification in de novo consensus sequences. For viruses with a well-assembled reference genome, a reference-based approach is recommended.

Ionotropic GABA and Glutamate Receptor Mutations and Human Neurologic Diseases.

PubMed

Yuan, Hongjie; Low, Chian-Ming; Moody, Olivia A; Jenkins, Andrew; Traynelis, Stephen F

2015-07-01

The advent of whole exome/genome sequencing and the technology-driven reduction in the cost of next-generation sequencing as well as the introduction of diagnostic-targeted sequencing chips have resulted in an unprecedented volume of data directly linking patient genomic variability to disorders of the brain. This information has the potential to transform our understanding of neurologic disorders by improving diagnoses, illuminating the molecular heterogeneity underlying diseases, and identifying new targets for therapeutic treatment. There is a strong history of mutations in GABA receptor genes being involved in neurologic diseases, particularly the epilepsies. In addition, a substantial number of variants and mutations have been found in GABA receptor genes in patients with autism, schizophrenia, and addiction, suggesting potential links between the GABA receptors and these conditions. A new and unexpected outcome from sequencing efforts has been the surprising number of mutations found in glutamate receptor subunits, with the GRIN2A gene encoding the GluN2A N-methyl-d-aspartate receptor subunit being most often affected. These mutations are associated with multiple neurologic conditions, for which seizure disorders comprise the largest group. The GluN2A subunit appears to be a locus for epilepsy, which holds important therapeutic implications. Virtually all α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor mutations, most of which occur within GRIA3, are from patients with intellectual disabilities, suggesting a link to this condition. Similarly, the most common phenotype for kainate receptor variants is intellectual disability. Herein, we summarize the current understanding of disease-associated mutations in ionotropic GABA and glutamate receptor families, and discuss implications regarding the identification of human mutations and treatment of neurologic diseases. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Ionotropic GABA and Glutamate Receptor Mutations and Human Neurologic Diseases

PubMed Central

Yuan, Hongjie; Low, Chian-Ming; Moody, Olivia A.; Jenkins, Andrew

2015-01-01

The advent of whole exome/genome sequencing and the technology-driven reduction in the cost of next-generation sequencing as well as the introduction of diagnostic-targeted sequencing chips have resulted in an unprecedented volume of data directly linking patient genomic variability to disorders of the brain. This information has the potential to transform our understanding of neurologic disorders by improving diagnoses, illuminating the molecular heterogeneity underlying diseases, and identifying new targets for therapeutic treatment. There is a strong history of mutations in GABA receptor genes being involved in neurologic diseases, particularly the epilepsies. In addition, a substantial number of variants and mutations have been found in GABA receptor genes in patients with autism, schizophrenia, and addiction, suggesting potential links between the GABA receptors and these conditions. A new and unexpected outcome from sequencing efforts has been the surprising number of mutations found in glutamate receptor subunits, with the GRIN2A gene encoding the GluN2A N-methyl-d-aspartate receptor subunit being most often affected. These mutations are associated with multiple neurologic conditions, for which seizure disorders comprise the largest group. The GluN2A subunit appears to be a locus for epilepsy, which holds important therapeutic implications. Virtually all α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor mutations, most of which occur within GRIA3, are from patients with intellectual disabilities, suggesting a link to this condition. Similarly, the most common phenotype for kainate receptor variants is intellectual disability. Herein, we summarize the current understanding of disease-associated mutations in ionotropic GABA and glutamate receptor families, and discuss implications regarding the identification of human mutations and treatment of neurologic diseases. PMID:25904555

Variant calling in low-coverage whole genome sequencing of a Native American population sample.

PubMed

Bizon, Chris; Spiegel, Michael; Chasse, Scott A; Gizer, Ian R; Li, Yun; Malc, Ewa P; Mieczkowski, Piotr A; Sailsbery, Josh K; Wang, Xiaoshu; Ehlers, Cindy L; Wilhelmsen, Kirk C

2014-01-30

The reduction in the cost of sequencing a human genome has led to the use of genotype sampling strategies in order to impute and infer the presence of sequence variants that can then be tested for associations with traits of interest. Low-coverage Whole Genome Sequencing (WGS) is a sampling strategy that overcomes some of the deficiencies seen in fixed content SNP array studies. Linkage-disequilibrium (LD) aware variant callers, such as the program Thunder, may provide a calling rate and accuracy that makes a low-coverage sequencing strategy viable. We examined the performance of an LD-aware variant calling strategy in a population of 708 low-coverage whole genome sequences from a community sample of Native Americans. We assessed variant calling through a comparison of the sequencing results to genotypes measured in 641 of the same subjects using a fixed content first generation exome array. The comparison was made using the variant calling routines GATK Unified Genotyper program and the LD-aware variant caller Thunder. Thunder was found to improve concordance in a coverage dependent fashion, while correctly calling nearly all of the common variants as well as a high percentage of the rare variants present in the sample. Low-coverage WGS is a strategy that appears to collect genetic information intermediate in scope between fixed content genotyping arrays and deep-coverage WGS. Our data suggests that low-coverage WGS is a viable strategy with a greater chance of discovering novel variants and associations than fixed content arrays for large sample association analyses.

A machine learning model to determine the accuracy of variant calls in capture-based next generation sequencing.

PubMed

van den Akker, Jeroen; Mishne, Gilad; Zimmer, Anjali D; Zhou, Alicia Y

2018-04-17

Next generation sequencing (NGS) has become a common technology for clinical genetic tests. The quality of NGS calls varies widely and is influenced by features like reference sequence characteristics, read depth, and mapping accuracy. With recent advances in NGS technology and software tools, the majority of variants called using NGS alone are in fact accurate and reliable. However, a small subset of difficult-to-call variants that still do require orthogonal confirmation exist. For this reason, many clinical laboratories confirm NGS results using orthogonal technologies such as Sanger sequencing. Here, we report the development of a deterministic machine-learning-based model to differentiate between these two types of variant calls: those that do not require confirmation using an orthogonal technology (high confidence), and those that require additional quality testing (low confidence). This approach allows reliable NGS-based calling in a clinical setting by identifying the few important variant calls that require orthogonal confirmation. We developed and tested the model using a set of 7179 variants identified by a targeted NGS panel and re-tested by Sanger sequencing. The model incorporated several signals of sequence characteristics and call quality to determine if a variant was identified at high or low confidence. The model was tuned to eliminate false positives, defined as variants that were called by NGS but not confirmed by Sanger sequencing. The model achieved very high accuracy: 99.4% (95% confidence interval: +/- 0.03%). It categorized 92.2% (6622/7179) of the variants as high confidence, and 100% of these were confirmed to be present by Sanger sequencing. Among the variants that were categorized as low confidence, defined as NGS calls of low quality that are likely to be artifacts, 92.1% (513/557) were found to be not present by Sanger sequencing. This work shows that NGS data contains sufficient characteristics for a machine-learning-based model to differentiate low from high confidence variants. Additionally, it reveals the importance of incorporating site-specific features as well as variant call features in such a model.

Overlap between Parkinson disease and Alzheimer disease in ABCA7 functional variants

PubMed Central

Nuytemans, Karen; Maldonado, Lizmarie; Ali, Aleena; John-Williams, Krista; Beecham, Gary W.; Martin, Eden; Scott, William K.

2016-01-01

Objective: Given their reported function in phagocytosis and clearance of protein aggregates in Alzheimer disease (AD), we hypothesized that variants in ATP-binding cassette transporter A7 (ABCA7) might be involved in Parkinson disease (PD). Methods: ABCA7 variants were identified using whole-exome sequencing (WES) on 396 unrelated patients with PD and 222 healthy controls. In addition, we used the publicly available WES data from the Parkinson's Progression Markers Initiative (444 patients and 153 healthy controls) as a second, independent data set. Results: We observed a higher frequency of loss-of-function (LOF) variants and rare putative highly functional variants (Combined Annotation Dependent Depletion [CADD] >20) in clinically diagnosed patients with PD than in healthy controls in both data sets. Overall, we identified LOF variants in 11 patients and 1 healthy control (odds ratio [OR] 4.94, Fisher exact p = 0.07). Four of these variants have been previously implicated in AD risk (p.E709AfsX86, p.W1214X, p.L1403RfsX7, and rs113809142). In addition, rare variants with CADD >20 were observed in 19 patients vs 3 healthy controls (OR 2.85, Fisher exact p = 0.06). Conclusion: The presence of ABCA7 LOF variants in clinically defined PD suggests that they might be risk factors for neurodegeneration in general, especially those variants hallmarked by protein aggregation. More studies will be needed to evaluate the overall impact of this transporter in neurodegenerative disease. PMID:27066581

Functional alterations due to amino acid changes and evolutionary comparative analysis of ARPKD and ADPKD genes.

PubMed

Edrees, Burhan M; Athar, Mohammad; Abduljaleel, Zainularifeen; Al-Allaf, Faisal A; Taher, Mohiuddin M; Khan, Wajahatullah; Bouazzaoui, Abdellatif; Al-Harbi, Naffaa; Safar, Ramzia; Al-Edressi, Howaida; Alansary, Khawala; Anazi, Abulkareem; Altayeb, Naji; Ahmed, Muawia A

2016-12-01

A targeted customized sequencing of genes implicated in autosomal recessive polycystic kidney disease (ARPKD) phenotype was performed to identify candidate variants using the Ion torrent PGM next-generation sequencing. The results identified four potential pathogenic variants in PKHD1 gene [c.4870C > T, p.(Arg1624Trp), c.5725C > T, p.(Arg1909Trp), c.1736C > T, p.(Thr579Met) and c.10628T > G, p.(Leu3543Trp)] among 12 out of 18 samples. However, one variant c.4870C > T, p.(Arg1624Trp) was common among eight patients. Some patient samples also showed few variants in autosomal dominant polycystic kidney disease (ADPKD) disease causing genes PKD1 and PKD2 such as c.12433G > A, p.(Val4145Ile) and c.1445T > G, p.(Phe482Cys), respectively. All causative variants were validated by capillary sequencing and confirmed the presence of a novel homozygous variant c.10628T > G, p.(Leu3543Trp) in a male proband. We have recently published the results of these studies (Edrees et al., 2016). Here we report for the first time the effect of the common mutation p.(Arg1624Trp) found in eight samples on the protein structure and function due to the specific amino acid changes of PKHD1 protein using molecular dynamics simulations. The computational approaches provide tool predict the phenotypic effect of variant on the structure and function of the altered protein. The structural analysis with the common mutation p.(Arg1624Trp) in the native and mutant modeled protein were also studied for solvent accessibility, secondary structure and stabilizing residues to find out the stability of the protein between wild type and mutant forms. Furthermore, comparative genomics and evolutionary analyses of variants observed in PKHD1 , PKD1 , and PKD2 genes were also performed in some mammalian species including human to understand the complexity of genomes among closely related mammalian species. Taken together, the results revealed that the evolutionary comparative analyses and characterization of PKHD1 , PKD1 , and PKD2 genes among various related and unrelated mammalian species will provide important insights into their evolutionary process and understanding for further disease characterization and management.

Genome sequencing reveals loci under artificial selection that underlie disease phenotypes in the laboratory rat.

PubMed

Atanur, Santosh S; Diaz, Ana Garcia; Maratou, Klio; Sarkis, Allison; Rotival, Maxime; Game, Laurence; Tschannen, Michael R; Kaisaki, Pamela J; Otto, Georg W; Ma, Man Chun John; Keane, Thomas M; Hummel, Oliver; Saar, Kathrin; Chen, Wei; Guryev, Victor; Gopalakrishnan, Kathirvel; Garrett, Michael R; Joe, Bina; Citterio, Lorena; Bianchi, Giuseppe; McBride, Martin; Dominiczak, Anna; Adams, David J; Serikawa, Tadao; Flicek, Paul; Cuppen, Edwin; Hubner, Norbert; Petretto, Enrico; Gauguier, Dominique; Kwitek, Anne; Jacob, Howard; Aitman, Timothy J

2013-08-01

Large numbers of inbred laboratory rat strains have been developed for a range of complex disease phenotypes. To gain insights into the evolutionary pressures underlying selection for these phenotypes, we sequenced the genomes of 27 rat strains, including 11 models of hypertension, diabetes, and insulin resistance, along with their respective control strains. Altogether, we identified more than 13 million single-nucleotide variants, indels, and structural variants across these rat strains. Analysis of strain-specific selective sweeps and gene clusters implicated genes and pathways involved in cation transport, angiotensin production, and regulators of oxidative stress in the development of cardiovascular disease phenotypes in rats. Many of the rat loci that we identified overlap with previously mapped loci for related traits in humans, indicating the presence of shared pathways underlying these phenotypes in rats and humans. These data represent a step change in resources available for evolutionary analysis of complex traits in disease models. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Genome Sequencing Reveals Loci under Artificial Selection that Underlie Disease Phenotypes in the Laboratory Rat

PubMed Central

Atanur, Santosh S.; Diaz, Ana Garcia; Maratou, Klio; Sarkis, Allison; Rotival, Maxime; Game, Laurence; Tschannen, Michael R.; Kaisaki, Pamela J.; Otto, Georg W.; Ma, Man Chun John; Keane, Thomas M.; Hummel, Oliver; Saar, Kathrin; Chen, Wei; Guryev, Victor; Gopalakrishnan, Kathirvel; Garrett, Michael R.; Joe, Bina; Citterio, Lorena; Bianchi, Giuseppe; McBride, Martin; Dominiczak, Anna; Adams, David J.; Serikawa, Tadao; Flicek, Paul; Cuppen, Edwin; Hubner, Norbert; Petretto, Enrico; Gauguier, Dominique; Kwitek, Anne; Jacob, Howard; Aitman, Timothy J.

2013-01-01

Summary Large numbers of inbred laboratory rat strains have been developed for a range of complex disease phenotypes. To gain insights into the evolutionary pressures underlying selection for these phenotypes, we sequenced the genomes of 27 rat strains, including 11 models of hypertension, diabetes, and insulin resistance, along with their respective control strains. Altogether, we identified more than 13 million single-nucleotide variants, indels, and structural variants across these rat strains. Analysis of strain-specific selective sweeps and gene clusters implicated genes and pathways involved in cation transport, angiotensin production, and regulators of oxidative stress in the development of cardiovascular disease phenotypes in rats. Many of the rat loci that we identified overlap with previously mapped loci for related traits in humans, indicating the presence of shared pathways underlying these phenotypes in rats and humans. These data represent a step change in resources available for evolutionary analysis of complex traits in disease models. PaperClip PMID:23890820

An analytical framework for whole-genome sequence association studies and its implications for autism spectrum disorder.

PubMed

Werling, Donna M; Brand, Harrison; An, Joon-Yong; Stone, Matthew R; Zhu, Lingxue; Glessner, Joseph T; Collins, Ryan L; Dong, Shan; Layer, Ryan M; Markenscoff-Papadimitriou, Eirene; Farrell, Andrew; Schwartz, Grace B; Wang, Harold Z; Currall, Benjamin B; Zhao, Xuefang; Dea, Jeanselle; Duhn, Clif; Erdman, Carolyn A; Gilson, Michael C; Yadav, Rachita; Handsaker, Robert E; Kashin, Seva; Klei, Lambertus; Mandell, Jeffrey D; Nowakowski, Tomasz J; Liu, Yuwen; Pochareddy, Sirisha; Smith, Louw; Walker, Michael F; Waterman, Matthew J; He, Xin; Kriegstein, Arnold R; Rubenstein, John L; Sestan, Nenad; McCarroll, Steven A; Neale, Benjamin M; Coon, Hilary; Willsey, A Jeremy; Buxbaum, Joseph D; Daly, Mark J; State, Matthew W; Quinlan, Aaron R; Marth, Gabor T; Roeder, Kathryn; Devlin, Bernie; Talkowski, Michael E; Sanders, Stephan J

2018-05-01

Genomic association studies of common or rare protein-coding variation have established robust statistical approaches to account for multiple testing. Here we present a comparable framework to evaluate rare and de novo noncoding single-nucleotide variants, insertion/deletions, and all classes of structural variation from whole-genome sequencing (WGS). Integrating genomic annotations at the level of nucleotides, genes, and regulatory regions, we define 51,801 annotation categories. Analyses of 519 autism spectrum disorder families did not identify association with any categories after correction for 4,123 effective tests. Without appropriate correction, biologically plausible associations are observed in both cases and controls. Despite excluding previously identified gene-disrupting mutations, coding regions still exhibited the strongest associations. Thus, in autism, the contribution of de novo noncoding variation is probably modest in comparison to that of de novo coding variants. Robust results from future WGS studies will require large cohorts and comprehensive analytical strategies that consider the substantial multiple-testing burden.

LIPT1 deficiency presenting as early infantile epileptic encephalopathy, Leigh disease, and secondary pyruvate dehydrogenase complex deficiency.

PubMed

Stowe, Robert C; Sun, Qin; Elsea, Sarah H; Scaglia, Fernando

2018-05-01

Lipoic acid is an essential cofactor for the mitochondrial 2-ketoacid dehydrogenase complexes and the glycine cleavage system. Lipoyltransferase 1 catalyzes the covalent attachment of lipoate to these enzyme systems. Pathogenic variants in LIPT1 gene have recently been described in four patients from three families, commonly presenting with severe lactic acidosis resulting in neonatal death and/or poor neurocognitive outcomes. We report a 2-month-old male with severe lactic acidosis, refractory status epilepticus, and brain imaging suggestive of Leigh disease. Exome sequencing implicated compound heterozygous LIPT1 pathogenic variants. We describe the fifth case of LIPT1 deficiency, whose phenotype progressed to that of an early infantile epileptic encephalopathy, which is novel compared to previously described patients whom we will review. Due to the significant biochemical and phenotypic overlap that LIPT1 deficiency and mitochondrial energy cofactor disorders have with pyruvate dehydrogenase deficiency and/or nonketotic hyperglycinemia, they are and have been presumptively under-diagnosed without exome sequencing. © 2018 Wiley Periodicals, Inc.

Exome sequencing reveals novel genetic loci influencing obesity-related traits in Hispanic children

USDA-ARS?s Scientific Manuscript database

To perform whole exome sequencing in 928 Hispanic children and identify variants and genes associated with childhood obesity.Single-nucleotide variants (SNVs) were identified from Illumina whole exome sequencing data using integrated read mapping, variant calling, and an annotation pipeline (Mercury...

Joint linkage and association analysis with exome sequence data implicates SLC25A40 in hypertriglyceridemia.

PubMed

Rosenthal, Elisabeth A; Ranchalis, Jane; Crosslin, David R; Burt, Amber; Brunzell, John D; Motulsky, Arno G; Nickerson, Deborah A; Wijsman, Ellen M; Jarvik, Gail P

2013-12-05

Hypertriglyceridemia (HTG) is a heritable risk factor for cardiovascular disease. Investigating the genetics of HTG may identify new drug targets. There are ~35 known single-nucleotide variants (SNVs) that explain only ~10% of variation in triglyceride (TG) level. Because of the genetic heterogeneity of HTG, a family study design is optimal for identification of rare genetic variants with large effect size because the same mutation can be observed in many relatives and cosegregation with TG can be tested. We considered HTG in a five-generation family of European American descent (n = 121), ascertained for familial combined hyperlipidemia. By using Bayesian Markov chain Monte Carlo joint oligogenic linkage and association analysis, we detected linkage to chromosomes 7 and 17. Whole-exome sequence data revealed shared, highly conserved, private missense SNVs in both SLC25A40 on chr7 and PLD2 on chr17. Jointly, these SNVs explained 49% of the genetic variance in TG; however, only the SLC25A40 SNV was significantly associated with TG (p = 0.0001). This SNV, c.374A>G, causes a highly disruptive p.Tyr125Cys substitution just outside the second helical transmembrane region of the SLC25A40 inner mitochondrial membrane transport protein. Whole-gene testing in subjects from the Exome Sequencing Project confirmed the association between TG and SLC25A40 rare, highly conserved, coding variants (p = 0.03). These results suggest a previously undescribed pathway for HTG and illustrate the power of large pedigrees in the search for rare, causal variants. Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Sequence analysis of the mitochondrial DNA control region of ciscoes (genus Coregonus): Taxonomic implications for the Great Lakes species flock

USGS Publications Warehouse

Reed, Kent M.; Dorschner, Michael O.; Todd, Thomas N.; Phillips, Ruth B.

1998-01-01

Sequence variation in the control region (D-loop) of the mitochondrial DNA (mtDNA) was examined to assess the genetic distinctiveness of the shortjaw cisco (Coregonus zenithicus). Individuals from within the Great Lakes Basin as well as inland lakes outside the basin were sampled. DNA fragments containing the entire D-loop were amplified by PCR from specimens ofC. zenithicus and the related species C. artedi, C. hoyi, C. kiyi, and C. clupeaformis. DNA sequence analysis revealed high similarity within and among species and shared polymorphism for length variants. Based on this analysis, the shortjaw cisco is not genetically distinct from other cisco species.

Novel sequence variants in the TMIE gene in families with autosomal recessive nonsyndromic hearing impairment

PubMed Central

Santos, Regie Lyn P.; El-Shanti, Hatem; Sikandar, Shaheen; Lee, Kwanghyuk; Bhatti, Attya; Yan, Kai; Chahrour, Maria H.; McArthur, Nathan; Pham, Thanh L.; Mahasneh, Amjad Abdullah; Ahmad, Wasim

2010-01-01

To date, 37 genes have been identified for nonsyndromic hearing impairment (NSHI). Identifying the functional sequence variants within these genes and knowing their population-specific frequencies is of public health value, in particular for genetic screening for NSHI. To determine putatively functional sequence variants in the transmembrane inner ear (TMIE) gene in Pakistani and Jordanian families with autosomal recessive (AR) NSHI, four Jordanian and 168 Pakistani families with ARNSHI that is not due to GJB2 (CX26) were submitted to a genome scan. Two-point and multipoint parametric linkage analyses were performed, and families with logarithmic odds (LOD) scores of 1.0 or greater within the TMIE region underwent further DNA sequencing. The evolutionary conservation and location in predicted protein domains of amino acid residues where sequence variants occurred were studied to elucidate the possible effects of these sequence variants on function. Of seven families that were screened for TMIE, putatively functional sequence variants were found to segregate with hearing impairment in four families but were not seen in not less than 110 ethnically matched control chromosomes. The previously reported c.241C>T (p.R81C) variant was observed in two Pakistani families. Two novel variants, c.92A>G (p.E31G) and the splice site mutation c.212–2A>C, were identified in one Pakistani and one Jordanian family, respectively. The c.92A>G (p.E31G) variant occurred at a residue that is conserved in the mouse and is predicted to be extracellular. Conservation and potential functionality of previously published mutations were also examined. The prevalence of functional TMIE variants in Pakistani families is 1.7% [95% confidence interval (CI) 0.3–4.8]. Further studies on the spectrum, prevalence rates, and functional effect of sequence variants in the TMIE gene in other populations should demonstrate the true importance of this gene as a cause of hearing impairment. PMID:16389551

Rare disruptive variants in the DISC1 Interactome and Regulome: association with cognitive ability and schizophrenia.

PubMed

Teng, S; Thomson, P A; McCarthy, S; Kramer, M; Muller, S; Lihm, J; Morris, S; Soares, D C; Hennah, W; Harris, S; Camargo, L M; Malkov, V; McIntosh, A M; Millar, J K; Blackwood, D H; Evans, K L; Deary, I J; Porteous, D J; McCombie, W R

2018-05-01

Schizophrenia (SCZ), bipolar disorder (BD) and recurrent major depressive disorder (rMDD) are common psychiatric illnesses. All have been associated with lower cognitive ability, and show evidence of genetic overlap and substantial evidence of pleiotropy with cognitive function and neuroticism. Disrupted in schizophrenia 1 (DISC1) protein directly interacts with a large set of proteins (DISC1 Interactome) that are involved in brain development and signaling. Modulation of DISC1 expression alters the expression of a circumscribed set of genes (DISC1 Regulome) that are also implicated in brain biology and disorder. Here we report targeted sequencing of 59 DISC1 Interactome genes and 154 Regulome genes in 654 psychiatric patients and 889 cognitively-phenotyped control subjects, on whom we previously reported evidence for trait association from complete sequencing of the DISC1 locus. Burden analyses of rare and singleton variants predicted to be damaging were performed for psychiatric disorders, cognitive variables and personality traits. The DISC1 Interactome and Regulome showed differential association across the phenotypes tested. After family-wise error correction across all traits (FWER across ), an increased burden of singleton disruptive variants in the Regulome was associated with SCZ (FWER across P=0.0339). The burden of singleton disruptive variants in the DISC1 Interactome was associated with low cognitive ability at age 11 (FWER across P=0.0043). These results identify altered regulation of schizophrenia candidate genes by DISC1 and its core Interactome as an alternate pathway for schizophrenia risk, consistent with the emerging effects of rare copy number variants associated with intellectual disability.

Whole-genome sequencing of monozygotic twins discordant for schizophrenia indicates multiple genetic risk factors for schizophrenia.

PubMed

Tang, Jinsong; Fan, Yu; Li, Hong; Xiang, Qun; Zhang, Deng-Feng; Li, Zongchang; He, Ying; Liao, Yanhui; Wang, Ya; He, Fan; Zhang, Fengyu; Shugart, Yin Yao; Liu, Chunyu; Tang, Yanqing; Chan, Raymond C K; Wang, Chuan-Yue; Yao, Yong-Gang; Chen, Xiaogang

2017-06-20

Schizophrenia is a common disorder with a high heritability, but its genetic architecture is still elusive. We implemented whole-genome sequencing (WGS) analysis of 8 families with monozygotic (MZ) twin pairs discordant for schizophrenia to assess potential association of de novo mutations (DNMs) or inherited variants with susceptibility to schizophrenia. Eight non-synonymous DNMs (including one splicing site) were identified and shared by twins, which were either located in previously reported schizophrenia risk genes (p.V24689I mutation in TTN, p.S2506T mutation in GCN1L1, IVS3+1G > T in DOCK1) or had a benign to damaging effect according to in silico prediction analysis. By searching the inherited rare damaging or loss-of-function (LOF) variants and common susceptible alleles from three classes of schizophrenia candidate genes, we were able to distill genetic alterations in several schizophrenia risk genes, including GAD1, PLXNA2, RELN and FEZ1. Four inherited copy number variations (CNVs; including a large deletion at 16p13.11) implicated for schizophrenia were identified in four families, respectively. Most of families carried both missense DNMs and inherited risk variants, which might suggest that DNMs, inherited rare damaging variants and common risk alleles together conferred to schizophrenia susceptibility. Our results support that schizophrenia is caused by a combination of multiple genetic factors, with each DNM/variant showing a relatively small effect size. Copyright © 2017 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. All rights reserved.

Polypeptide having or assisting in carbohydrate material degrading activity and uses thereof

DOEpatents

Schooneveld-Bergmans, Margot Elisabeth Francoise; Heijne, Wilbert Herman Marie; Los, Alrik Pieter

2016-02-16

The invention relates to a polypeptide which comprises the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 76% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 76% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional polypeptide and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.

Polypeptide having beta-glucosidase activity and uses thereof

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schoonneveld-Bergmans, Margot Elisabeth Francoise; Heijne, Wilbert Herman Marie; De Jong, Rene Marcel

The invention relates to a polypeptide comprising the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 96% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 96% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well asmore » the amino acid sequence of the full-length functional polypeptide and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.« less

Polypeptide having swollenin activity and uses thereof

DOEpatents

Schoonneveld-Bergmans, Margot Elizabeth Francoise; Heijne, Wilbert Herman Marie; Vlasie, Monica D; Damveld, Robbertus Antonius

2015-11-04

The invention relates to a polypeptide comprising the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 73% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 73% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional polypeptide and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.

Polypeptide having beta-glucosidase activity and uses thereof

DOEpatents

Schooneveld-Bergmans, Margot Elisabeth Francoise; Heijne, Wilbert Herman Marie; De Jong, Rene Marcel; Damveld, Robbertus Antonius

2015-09-01

The invention relates to a polypeptide comprising the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 70% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 70% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional polypeptide and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.

Polypeptide having cellobiohydrolase activity and uses thereof

DOEpatents

Sagt, Cornelis Maria Jacobus; Schooneveld-Bergmans, Margot Elisabeth Francoise; Roubos, Johannes Andries; Los, Alrik Pieter

2015-09-15

The invention relates to a polypeptide comprising the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 93% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 93% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional polypeptide and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.

Polypeptide having acetyl xylan esterase activity and uses thereof

DOEpatents

Schoonneveld-Bergmans, Margot Elisabeth Francoise; Heijne, Wilbert Herman Marie; Los, Alrik Pieter

2015-10-20

The invention relates to a polypeptide comprising the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 82% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 82% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional polypeptide and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.

Polypeptide having carbohydrate degrading activity and uses thereof

DOEpatents

Schooneveld-Bergmans, Margot Elisabeth Francoise; Heijne, Wilbert Herman Marie; Vlasie, Monica Diana; Damveld, Robbertus Antonius

2015-08-18

The invention relates to a polypeptide comprising the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 73% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 73% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional polypeptide and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.

Sensitivity of BRCA1/2 testing in high-risk breast/ovarian/male breast cancer families: little contribution of comprehensive RNA/NGS panel testing.

PubMed

Byers, Helen; Wallis, Yvonne; van Veen, Elke M; Lalloo, Fiona; Reay, Kim; Smith, Philip; Wallace, Andrew J; Bowers, Naomi; Newman, William G; Evans, D Gareth

2016-11-01

The sensitivity of testing BRCA1 and BRCA2 remains unresolved as the frequency of deep intronic splicing variants has not been defined in high-risk familial breast/ovarian cancer families. This variant category is reported at significant frequency in other tumour predisposition genes, including NF1 and MSH2. We carried out comprehensive whole gene RNA analysis on 45 high-risk breast/ovary and male breast cancer families with no identified pathogenic variant on exonic sequencing and copy number analysis of BRCA1/2. In addition, we undertook variant screening of a 10-gene high/moderate risk breast/ovarian cancer panel by next-generation sequencing. DNA testing identified the causative variant in 50/56 (89%) breast/ovarian/male breast cancer families with Manchester scores of ≥50 with two variants being confirmed to affect splicing on RNA analysis. RNA sequencing of BRCA1/BRCA2 on 45 individuals from high-risk families identified no deep intronic variants and did not suggest loss of RNA expression as a cause of lost sensitivity. Panel testing in 42 samples identified a known RAD51D variant, a high-risk ATM variant in another breast ovary family and a truncating CHEK2 mutation. Current exonic sequencing and copy number analysis variant detection methods of BRCA1/2 have high sensitivity in high-risk breast/ovarian cancer families. Sequence analysis of RNA does not identify any variants undetected by current analysis of BRCA1/2. However, RNA analysis clarified the pathogenicity of variants of unknown significance detected by current methods. The low diagnostic uplift achieved through sequence analysis of the other known breast/ovarian cancer susceptibility genes indicates that further high-risk genes remain to be identified.

Multiplexed resequencing analysis to identify rare variants in pooled DNA with barcode indexing using next-generation sequencer.

PubMed

Mitsui, Jun; Fukuda, Yoko; Azuma, Kyo; Tozaki, Hirokazu; Ishiura, Hiroyuki; Takahashi, Yuji; Goto, Jun; Tsuji, Shoji

2010-07-01

We have recently found that multiple rare variants of the glucocerebrosidase gene (GBA) confer a robust risk for Parkinson disease, supporting the 'common disease-multiple rare variants' hypothesis. To develop an efficient method of identifying rare variants in a large number of samples, we applied multiplexed resequencing using a next-generation sequencer to identification of rare variants of GBA. Sixteen sets of pooled DNAs from six pooled DNA samples were prepared. Each set of pooled DNAs was subjected to polymerase chain reaction to amplify the target gene (GBA) covering 6.5 kb, pooled into one tube with barcode indexing, and then subjected to extensive sequence analysis using the SOLiD System. Individual samples were also subjected to direct nucleotide sequence analysis. With the optimization of data processing, we were able to extract all the variants from 96 samples with acceptable rates of false-positive single-nucleotide variants.

An update on the genetic architecture of hyperuricemia and gout.

PubMed

Merriman, Tony R

2015-04-10

Genome-wide association studies that scan the genome for common genetic variants associated with phenotype have greatly advanced medical knowledge. Hyperuricemia is no exception, with 28 loci identified. However, genetic control of pathways determining gout in the presence of hyperuricemia is still poorly understood. Two important pathways determining hyperuricemia have been confirmed (renal and gut excretion of uric acid with glycolysis now firmly implicated). Major urate loci are SLC2A9 and ABCG2. Recent studies show that SLC2A9 is involved in renal and gut excretion of uric acid and is implicated in antioxidant defense. Although etiological variants at SLC2A9 are yet to be identified, it is clear that considerable genetic complexity exists at the SLC2A9 locus, with multiple statistically independent genetic variants and local epistatic interactions. The positions of implicated genetic variants within or near chromatin regions involved in transcriptional control suggest that this mechanism (rather than structural changes in SLC2A9) is important in regulating the activity of SLC2A9. ABCG2 is involved primarily in extra-renal uric acid under-excretion with the etiological variant influencing expression. At the other 26 loci, probable causal genes can be identified at three (PDZK1, SLC22A11, and INHBB) with strong candidates at a further 10 loci. Confirmation of the causal gene will require a combination of re-sequencing, trans-ancestral mapping, and correlation of genetic association data with expression data. As expected, the urate loci associate with gout, although inconsistent effect sizes for gout require investigation. Finally, there has been no genome-wide association study using clinically ascertained cases to investigate the causes of gout in the presence of hyperuricemia. In such a study, use of asymptomatic hyperurcemic controls would be expected to increase the ability to detect genetic associations with gout.

VarDict: a novel and versatile variant caller for next-generation sequencing in cancer research

PubMed Central

Lai, Zhongwu; Markovets, Aleksandra; Ahdesmaki, Miika; Chapman, Brad; Hofmann, Oliver; McEwen, Robert; Johnson, Justin; Dougherty, Brian; Barrett, J. Carl; Dry, Jonathan R.

2016-01-01

Abstract Accurate variant calling in next generation sequencing (NGS) is critical to understand cancer genomes better. Here we present VarDict, a novel and versatile variant caller for both DNA- and RNA-sequencing data. VarDict simultaneously calls SNV, MNV, InDels, complex and structural variants, expanding the detected genetic driver landscape of tumors. It performs local realignments on the fly for more accurate allele frequency estimation. VarDict performance scales linearly to sequencing depth, enabling ultra-deep sequencing used to explore tumor evolution or detect tumor DNA circulating in blood. In addition, VarDict performs amplicon aware variant calling for polymerase chain reaction (PCR)-based targeted sequencing often used in diagnostic settings, and is able to detect PCR artifacts. Finally, VarDict also detects differences in somatic and loss of heterozygosity variants between paired samples. VarDict reprocessing of The Cancer Genome Atlas (TCGA) Lung Adenocarcinoma dataset called known driver mutations in KRAS, EGFR, BRAF, PIK3CA and MET in 16% more patients than previously published variant calls. We believe VarDict will greatly facilitate application of NGS in clinical cancer research. PMID:27060149

FAVR (Filtering and Annotation of Variants that are Rare): methods to facilitate the analysis of rare germline genetic variants from massively parallel sequencing datasets

PubMed Central

2013-01-01

Background Characterising genetic diversity through the analysis of massively parallel sequencing (MPS) data offers enormous potential to significantly improve our understanding of the genetic basis for observed phenotypes, including predisposition to and progression of complex human disease. Great challenges remain in resolving genetic variants that are genuine from the millions of artefactual signals. Results FAVR is a suite of new methods designed to work with commonly used MPS analysis pipelines to assist in the resolution of some of the issues related to the analysis of the vast amount of resulting data, with a focus on relatively rare genetic variants. To the best of our knowledge, no equivalent method has previously been described. The most important and novel aspect of FAVR is the use of signatures in comparator sequence alignment files during variant filtering, and annotation of variants potentially shared between individuals. The FAVR methods use these signatures to facilitate filtering of (i) platform and/or mapping-specific artefacts, (ii) common genetic variants, and, where relevant, (iii) artefacts derived from imbalanced paired-end sequencing, as well as annotation of genetic variants based on evidence of co-occurrence in individuals. We applied conventional variant calling applied to whole-exome sequencing datasets, produced using both SOLiD and TruSeq chemistries, with or without downstream processing by FAVR methods. We demonstrate a 3-fold smaller rare single nucleotide variant shortlist with no detected reduction in sensitivity. This analysis included Sanger sequencing of rare variant signals not evident in dbSNP131, assessment of known variant signal preservation, and comparison of observed and expected rare variant numbers across a range of first cousin pairs. The principles described herein were applied in our recent publication identifying XRCC2 as a new breast cancer risk gene and have been made publically available as a suite of software tools. Conclusions FAVR is a platform-agnostic suite of methods that significantly enhances the analysis of large volumes of sequencing data for the study of rare genetic variants and their influence on phenotypes. PMID:23441864

Clinical Validation and Implementation of a Targeted Next-Generation Sequencing Assay to Detect Somatic Variants in Non-Small Cell Lung, Melanoma, and Gastrointestinal Malignancies

PubMed Central

Fisher, Kevin E.; Zhang, Linsheng; Wang, Jason; Smith, Geoffrey H.; Newman, Scott; Schneider, Thomas M.; Pillai, Rathi N.; Kudchadkar, Ragini R.; Owonikoko, Taofeek K.; Ramalingam, Suresh S.; Lawson, David H.; Delman, Keith A.; El-Rayes, Bassel F.; Wilson, Malania M.; Sullivan, H. Clifford; Morrison, Annie S.; Balci, Serdar; Adsay, N. Volkan; Gal, Anthony A.; Sica, Gabriel L.; Saxe, Debra F.; Mann, Karen P.; Hill, Charles E.; Khuri, Fadlo R.; Rossi, Michael R.

2017-01-01

We tested and clinically validated a targeted next-generation sequencing (NGS) mutation panel using 80 formalin-fixed, paraffin-embedded (FFPE) tumor samples. Forty non-small cell lung carcinoma (NSCLC), 30 melanoma, and 30 gastrointestinal (12 colonic, 10 gastric, and 8 pancreatic adenocarcinoma) FFPE samples were selected from laboratory archives. After appropriate specimen and nucleic acid quality control, 80 NGS libraries were prepared using the Illumina TruSight tumor (TST) kit and sequenced on the Illumina MiSeq. Sequence alignment, variant calling, and sequencing quality control were performed using vendor software and laboratory-developed analysis workflows. TST generated ≥500× coverage for 98.4% of the 13,952 targeted bases. Reproducible and accurate variant calling was achieved at ≥5% variant allele frequency with 8 to 12 multiplexed samples per MiSeq flow cell. TST detected 112 variants overall, and confirmed all known single-nucleotide variants (n = 27), deletions (n = 5), insertions (n = 3), and multinucleotide variants (n = 3). TST detected at least one variant in 85.0% (68/80), and two or more variants in 36.2% (29/80), of samples. TP53 was the most frequently mutated gene in NSCLC (13 variants; 13/32 samples), gastrointestinal malignancies (15 variants; 13/25 samples), and overall (30 variants; 28/80 samples). BRAF mutations were most common in melanoma (nine variants; 9/23 samples). Clinically relevant NGS data can be obtained from routine clinical FFPE solid tumor specimens using TST, benchtop instruments, and vendor-supplied bioinformatics pipelines. PMID:26801070

Geographic Population Structure in Epstein-Barr Virus Revealed by Comparative Genomics

PubMed Central

Chiara, Matteo; Manzari, Caterina; Lionetti, Claudia; Mechelli, Rosella; Anastasiadou, Eleni; Chiara Buscarinu, Maria; Ristori, Giovanni; Salvetti, Marco; Picardi, Ernesto; D’Erchia, Anna Maria; Pesole, Graziano; Horner, David S.

2016-01-01

Epstein-Barr virus (EBV) latently infects the majority of the human population and is implicated as a causal or contributory factor in numerous diseases. We sequenced 27 complete EBV genomes from a cohort of Multiple Sclerosis (MS) patients and healthy controls from Italy, although no variants showed a statistically significant association with MS. Taking advantage of the availability of ∼130 EBV genomes with known geographical origins, we reveal a striking geographic distribution of EBV sub-populations with distinct allele frequency distributions. We discuss mechanisms that potentially explain these observations, and their implications for understanding the association of EBV with human disease. PMID:27635051

High Resolution Melt analysis for mutation screening in PKD1 and PKD2

PubMed Central

2011-01-01

Background Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disorder. It is characterized by focal development and progressive enlargement of renal cysts leading to end-stage renal disease. PKD1 and PKD2 have been implicated in ADPKD pathogenesis but genetic features and the size of PKD1 make genetic diagnosis tedious. Methods We aim to prove that high resolution melt analysis (HRM), a recent technique in molecular biology, can facilitate molecular diagnosis of ADPKD. We screened for mutations in PKD1 and PKD2 with HRM in 37 unrelated patients with ADPKD. Results We identified 440 sequence variants in the 37 patients. One hundred and thirty eight were different. We found 28 pathogenic mutations (25 in PKD1 and 3 in PKD2 ) within 28 different patients, which is a diagnosis rate of 75% consistent with literature mean direct sequencing diagnosis rate. We describe 52 new sequence variants in PKD1 and two in PKD2. Conclusion HRM analysis is a sensitive and specific method for molecular diagnosis of ADPKD. HRM analysis is also costless and time sparing. Thus, this method is efficient and might be used for mutation pre-screening in ADPKD genes. PMID:22008521

Whole-genome sequence-based analysis of thyroid function.

PubMed

Taylor, Peter N; Porcu, Eleonora; Chew, Shelby; Campbell, Purdey J; Traglia, Michela; Brown, Suzanne J; Mullin, Benjamin H; Shihab, Hashem A; Min, Josine; Walter, Klaudia; Memari, Yasin; Huang, Jie; Barnes, Michael R; Beilby, John P; Charoen, Pimphen; Danecek, Petr; Dudbridge, Frank; Forgetta, Vincenzo; Greenwood, Celia; Grundberg, Elin; Johnson, Andrew D; Hui, Jennie; Lim, Ee M; McCarthy, Shane; Muddyman, Dawn; Panicker, Vijay; Perry, John R B; Bell, Jordana T; Yuan, Wei; Relton, Caroline; Gaunt, Tom; Schlessinger, David; Abecasis, Goncalo; Cucca, Francesco; Surdulescu, Gabriela L; Woltersdorf, Wolfram; Zeggini, Eleftheria; Zheng, Hou-Feng; Toniolo, Daniela; Dayan, Colin M; Naitza, Silvia; Walsh, John P; Spector, Tim; Davey Smith, George; Durbin, Richard; Richards, J Brent; Sanna, Serena; Soranzo, Nicole; Timpson, Nicholas J; Wilson, Scott G

2015-03-06

Normal thyroid function is essential for health, but its genetic architecture remains poorly understood. Here, for the heritable thyroid traits thyrotropin (TSH) and free thyroxine (FT4), we analyse whole-genome sequence data from the UK10K project (N=2,287). Using additional whole-genome sequence and deeply imputed data sets, we report meta-analysis results for common variants (MAF≥1%) associated with TSH and FT4 (N=16,335). For TSH, we identify a novel variant in SYN2 (MAF=23.5%, P=6.15 × 10(-9)) and a new independent variant in PDE8B (MAF=10.4%, P=5.94 × 10(-14)). For FT4, we report a low-frequency variant near B4GALT6/SLC25A52 (MAF=3.2%, P=1.27 × 10(-9)) tagging a rare TTR variant (MAF=0.4%, P=2.14 × 10(-11)). All common variants explain ≥20% of the variance in TSH and FT4. Analysis of rare variants (MAF<1%) using sequence kernel association testing reveals a novel association with FT4 in NRG1. Our results demonstrate that increased coverage in whole-genome sequence association studies identifies novel variants associated with thyroid function.

Mitochondrial targeting sequence variants of the CHCHD2 gene are a risk for Lewy body disorders

PubMed Central

Ogaki, Kotaro; Koga, Shunsuke; Heckman, Michael G.; Fiesel, Fabienne C.; Ando, Maya; Labbé, Catherine; Lorenzo-Betancor, Oswaldo; Moussaud-Lamodière, Elisabeth L.; Soto-Ortolaza, Alexandra I.; Walton, Ronald L.; Strongosky, Audrey J.; Uitti, Ryan J.; McCarthy, Allan; Lynch, Timothy; Siuda, Joanna; Opala, Grzegorz; Rudzinska, Monika; Krygowska-Wajs, Anna; Barcikowska, Maria; Czyzewski, Krzysztof; Puschmann, Andreas; Nishioka, Kenya; Funayama, Manabu; Hattori, Nobutaka; Parisi, Joseph E.; Petersen, Ronald C.; Graff-Radford, Neill R.; Boeve, Bradley F.; Springer, Wolfdieter; Wszolek, Zbigniew K.; Dickson, Dennis W.

2015-01-01

Objective: To assess the role of CHCHD2 variants in patients with Parkinson disease (PD) and Lewy body disease (LBD) in Caucasian populations. Methods: All exons of the CHCHD2 gene were sequenced in a US Caucasian patient-control series (878 PD, 610 LBD, and 717 controls). Subsequently, exons 1 and 2 were sequenced in an Irish series (355 PD and 365 controls) and a Polish series (394 PD and 350 controls). Immunohistochemistry and immunofluorescence studies were performed on pathologic LBD cases with rare CHCHD2 variants. Results: We identified 9 rare exonic variants of unknown significance. These variants were more frequent in the combined group of PD and LBD patients compared to controls (0.6% vs 0.1%, p = 0.013). In addition, the presence of any rare variant was more common in patients with LBD (2.5% vs 1.0%, p = 0.050) compared to controls. Eight of these 9 variants were located within the gene's mitochondrial targeting sequence. Conclusions: Although the role of variants of the CHCHD2 gene in PD and LBD remains to be further elucidated, the rare variants in the mitochondrial targeting sequence may be a risk factor for Lewy body disorders, which may link CHCHD2 to other genetic forms of parkinsonism with mitochondrial dysfunction. PMID:26561290

Pooled-DNA Sequencing for Elucidating New Genomic Risk Factors, Rare Variants Underlying Alzheimer's Disease.

PubMed

Jin, Sheng Chih; Benitez, Bruno A; Deming, Yuetiva; Cruchaga, Carlos

2016-01-01

Analyses of genome-wide association studies (GWAS) for complex disorders usually identify common variants with a relatively small effect size that only explain a small proportion of phenotypic heritability. Several studies have suggested that a significant fraction of heritability may be explained by low-frequency (minor allele frequency (MAF) of 1-5 %) and rare-variants that are not contained in the commercial GWAS genotyping arrays (Schork et al., Curr Opin Genet Dev 19:212, 2009). Rare variants can also have relatively large effects on risk for developing human diseases or disease phenotype (Cruchaga et al., PLoS One 7:e31039, 2012). However, it is necessary to perform next-generation sequencing (NGS) studies in a large population (>4,000 samples) to detect a significant rare-variant association. Several NGS methods, such as custom capture sequencing and amplicon-based sequencing, are designed to screen a small proportion of the genome, but most of these methods are limited in the number of samples that can be multiplexed (i.e. most sequencing kits only provide 96 distinct index). Additionally, the sequencing library preparation for 4,000 samples remains expensive and thus conducting NGS studies with the aforementioned methods are not feasible for most research laboratories.The need for low-cost large scale rare-variant detection makes pooled-DNA sequencing an ideally efficient and cost-effective technique to identify rare variants in target regions by sequencing hundreds to thousands of samples. Our recent work has demonstrated that pooled-DNA sequencing can accurately detect rare variants in targeted regions in multiple DNA samples with high sensitivity and specificity (Jin et al., Alzheimers Res Ther 4:34, 2012). In these studies we used a well-established pooled-DNA sequencing approach and a computational package, SPLINTER (short indel prediction by large deviation inference and nonlinear true frequency estimation by recursion) (Vallania et al., Genome Res 20:1711, 2010), for accurate identification of rare variants in large DNA pools. Given an average sequencing coverage of 30× per haploid genome, SPLINTER can detect rare variants and short indels up to 4 base pairs (bp) with high sensitivity and specificity (up to 1 haploid allele in a pool as large as 500 individuals). Step-by-step instructions on how to conduct pooled-DNA sequencing experiments and data analyses are described in this chapter.

ABC Assay: Method Development and Application to Quantify the Role of Three DWV Master Variants in Overwinter Colony Losses of European Honey Bees.

PubMed

Kevill, Jessica L; Highfield, Andrea; Mordecai, Gideon J; Martin, Stephen J; Schroeder, Declan C

2017-10-27

Deformed wing virus (DWV) is one of the most prevalent honey bee viral pathogens in the world. Typical of many RNA viruses, DWV is a quasi-species, which is comprised of a large number of different variants, currently consisting of three master variants: Type A, B, and C. Little is known about the impact of each variant or combinations of variants upon the biology of individual hosts. Therefore, we have developed a new set of master variant-specific DWV primers and a set of standards that allow for the quantification of each of the master variants. Competitive reverse transcriptase polymerase chain reaction (RT-PCR) experimental design confirms that each new DWV primer set is specific to the retrospective master variant. The sensitivity of the ABC assay is dependent on whether DNA or RNA is used as the template and whether other master variants are present in the sample. Comparison of the overall proportions of each master variant within a sample of known diversity, as confirmed by next-generation sequence (NGS) data, validates the efficiency of the ABC assay. The ABC assay was used on archived material from a Devon overwintering colony loss (OCL) 2006-2007 study; further implicating DWV type A and, for the first time, possibly C in the untimely collapse of honey bee colonies. Moreover, in this study DWV type B was not associated with OCL. The use of the ABC assay will allow researchers to quickly and cost effectively pre-screen for the presence of DWV master variants in honey bees.

Comparative polymorphism of BAT-26 between healthy individuals and cancer patients and its cancer risk implication for local Chinese.

PubMed

Zheng, Yanying; Liu, Li; Sun, Yi; Chen, Jie; Wang, Jianrong; Zhu, Changle; Lai, Rensheng; Xie, Ling

2016-07-30

BAT-26 is one of the representative markers for microsatellite instability evaluation and presents different polymorphisms in different ethnic populations. The current knowledge of its comparative polymorphism between healthy individuals and cancer patients in the Chinese population is insufficient. This study aims to analyze germline polymorphic variations of BAT-26 between healthy individuals and cancer patients in Chinese from Jiangsu province and the associated cancer risk implications. The various BAT-26 alleles and their percentages in cervical cells from 500 healthy women were assessed by direct sequencing. Twenty of these samples were also analyzed by fragment analysis. BAT-26 of blood DNA from 24 healthy individuals and 247 cancer patients was analyzed by fragment analysis. Compared with the sequencing results, 122.6-122.9 bp, 123.4-123.8 bp and 124.1-124.8 bp corresponded to the A25, A26 and A27 alleles, respectively. The 524 healthy individuals showed 4.58%, 92.18% and 3.24% of A25, A26 and A27, respectively. The variant alleles A18, A24, A28, A29 and A32 were only found in cancer patients, accounting for 0.81%, 0.40%, 0.40%, 0.40% and 0.40%, respectively; the A25, A26 and A27 alleles in cancer patients accounted for 6.48%, 77.33% and 13.77%. Healthy individuals had a stable BAT-26 profile within the quasimonomorphic variation range (QMVR), but cancer patients harbored variant alleles outside QMVR and showed a trend from quasimonomorph to polymonomorph, suggesting that variant alleles of BAT-26 in germline cells may be regarded as a potential marker of higher cancer risk in the Chinese population from Jiangsu province.

A PYY Q62P variant linked to human obesity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ahituv, Nadav; Kavaslar, Nihan; Schackwitz, Wendy

2005-06-27

Members of the pancreatic polypeptide family and the irreceptors have been implicated in the control of food intake in rodents and humans. To investigate whether nucleotide changes in these candidate genes result in abnormal weight in humans, we sequenced the coding exons and splice sites of seven family members (NPY, PYY, PPY, NPY1R, NPY2R, NPY4R, and NPY5R) in a large cohort of extremely obese (n=379) and lean (n=378) individuals. In total we found eleven rare non-synonymous variants, four of which exhibited familial segregation, NPY1R L53P and PPY P63L with leanness and NPY2R D42G and PYY Q62P with obesity. Functional analysismore » of the obese variants revealed NPY2R D42G to have reduced cell surface expression, while previous cell culture based studies indicated variant PYY Q62P to have altered receptor binding selectivity and we show that it fails to reduce food intake through mouse peptide injection experiments. These results support that rare non-synonymous variants within these genes can alter susceptibility to human body mass index extremes.« less

Gain-of-function glutamate receptor interacting protein 1 variants alter GluA2 recycling and surface distribution in patients with autism

PubMed Central

Mejias, Rebeca; Adamczyk, Abby; Anggono, Victor; Niranjan, Tejasvi; Thomas, Gareth M.; Sharma, Kamal; Skinner, Cindy; Schwartz, Charles E.; Stevenson, Roger E.; Fallin, M. Daniele; Kaufmann, Walter; Pletnikov, Mikhail; Valle, David; Huganir, Richard L.; Wang, Tao

2011-01-01

Glutamate receptor interacting protein 1 (GRIP1) is a neuronal scaffolding protein that interacts directly with the C termini of glutamate receptors 2/3 (GluA2/3) via its PDZ domains 4 to 6 (PDZ4–6). We found an association (P < 0.05) of a SNP within the PDZ4-6 genomic region with autism by genotyping autistic patients (n = 480) and matched controls (n = 480). Parallel sequencing identified five rare missense variants within or near PDZ4–6 only in the autism cohort, resulting in a higher cumulative mutation load (P = 0.032). Two variants correlated with a more severe deficit in reciprocal social interaction in affected sibling pairs from proband families. These variants were associated with altered interactions with GluA2/3 and faster recycling and increased surface distribution of GluA2 in neurons, suggesting gain-of-function because GRIP1/2 deficiency showed opposite phenotypes. Grip1/2 knockout mice exhibited increased sociability and impaired prepulse inhibition. These results support a role for GRIP in social behavior and implicate GRIP1 variants in modulating autistic phenotype. PMID:21383172

High depth, whole-genome sequencing of cholera isolates from Haiti and the Dominican Republic.

PubMed

Sealfon, Rachel; Gire, Stephen; Ellis, Crystal; Calderwood, Stephen; Qadri, Firdausi; Hensley, Lisa; Kellis, Manolis; Ryan, Edward T; LaRocque, Regina C; Harris, Jason B; Sabeti, Pardis C

2012-09-11

Whole-genome sequencing is an important tool for understanding microbial evolution and identifying the emergence of functionally important variants over the course of epidemics. In October 2010, a severe cholera epidemic began in Haiti, with additional cases identified in the neighboring Dominican Republic. We used whole-genome approaches to sequence four Vibrio cholerae isolates from Haiti and the Dominican Republic and three additional V. cholerae isolates to a high depth of coverage (>2000x); four of the seven isolates were previously sequenced. Using these sequence data, we examined the effect of depth of coverage and sequencing platform on genome assembly and identification of sequence variants. We found that 50x coverage is sufficient to construct a whole-genome assembly and to accurately call most variants from 100 base pair paired-end sequencing reads. Phylogenetic analysis between the newly sequenced and thirty-three previously sequenced V. cholerae isolates indicates that the Haitian and Dominican Republic isolates are closest to strains from South Asia. The Haitian and Dominican Republic isolates form a tight cluster, with only four variants unique to individual isolates. These variants are located in the CTX region, the SXT region, and the core genome. Of the 126 mutations identified that separate the Haiti-Dominican Republic cluster from the V. cholerae reference strain (N16961), 73 are non-synonymous changes, and a number of these changes cluster in specific genes and pathways. Sequence variant analyses of V. cholerae isolates, including multiple isolates from the Haitian outbreak, identify coverage-specific and technology-specific effects on variant detection, and provide insight into genomic change and functional evolution during an epidemic.

AB053. NRG1 rare variant effects in Hirschsprung disease patients

PubMed Central

Gunadi; Budi, Nova; Iskandar, Kristy; Adrianto, Indra

2017-01-01

Background Hirschsprung disease (HSCR) is a heterogeneous genetic disorder characterized by absence of ganglion cells along intestines resulting in functional bowel obstruction. NRG1 gene has been implicated in the intestinal ganglionosis. This study aimed to investigate the contribution of NRG1 gene into the HSCR development in Indonesian population. Methods We performed Sanger sequencing to find NRG1 variants in 54 HSCR patients. Results All patients were sporadic non-syndromic HSCR with 53/54 (98%) and 1/54 (2%) were short-segment and long-segment patients, respectively. NRG1 analysis showed one rare variant, c.397G > C (p.V133L), and three common variants, rs7834206, rs3735774, and rs75155858. The p.V133L was predicted to reside within in a region of high mammalian conservation, overlap with the promoter and enhancer histone marks of relevant tissues such as digestive and smooth muscle tissues and alter AP-4_2, BDP1_disc3, Egr-1_known1, Egr-1_known4, HEN1_2 transcription factor binding motifs. Furthermore, this variant was absent in 92 controls. Conclusions This study is the first report of NRG1 rare variant associated with HSCR patients in South-East Asian ancestry and adds insights into the NRG1 effect in the molecular pathogenesis of HSCR.

CYP21A2 mutation update: Comprehensive analysis of databases and published genetic variants.

PubMed

Simonetti, Leandro; Bruque, Carlos D; Fernández, Cecilia S; Benavides-Mori, Belén; Delea, Marisol; Kolomenski, Jorge E; Espeche, Lucía D; Buzzalino, Noemí D; Nadra, Alejandro D; Dain, Liliana

2018-01-01

Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders of adrenal steroidogenesis. Disorders in steroid 21-hydroxylation account for over 95% of patients with CAH. Clinically, the 21-hydroxylase deficiency has been classified in a broad spectrum of clinical forms, ranging from severe or classical, to mild late onset or non-classical. Known allelic variants in the disease causing CYP21A2 gene are spread among different sources. Until recently, most variants reported have been identified in the clinical setting, which presumably bias described variants to pathogenic ones, as those found in the CYPAlleles database. Nevertheless, a large number of variants are being described in massive genome projects, many of which are found in dbSNP, but lack functional implications and/or their phenotypic effect. In this work, we gathered a total of 1,340 GVs in the CYP21A2 gene, from which 899 variants were unique and 230 have an effect on human health, and compiled all this information in an integrated database. We also connected CYP21A2 sequence information to phenotypic effects for all available mutations, including double mutants in cis. Data compiled in the present work could help physicians in the genetic counseling of families affected with 21-hydroxylase deficiency. © 2017 Wiley Periodicals, Inc.

XLID-causing mutations and associated genes challenged in light of data from large-scale human exome sequencing.

PubMed

Piton, Amélie; Redin, Claire; Mandel, Jean-Louis

2013-08-08

Because of the unbalanced sex ratio (1.3-1.4 to 1) observed in intellectual disability (ID) and the identification of large ID-affected families showing X-linked segregation, much attention has been focused on the genetics of X-linked ID (XLID). Mutations causing monogenic XLID have now been reported in over 100 genes, most of which are commonly included in XLID diagnostic gene panels. Nonetheless, the boundary between true mutations and rare non-disease-causing variants often remains elusive. The sequencing of a large number of control X chromosomes, required for avoiding false-positive results, was not systematically possible in the past. Such information is now available thanks to large-scale sequencing projects such as the National Heart, Lung, and Blood (NHLBI) Exome Sequencing Project, which provides variation information on 10,563 X chromosomes from the general population. We used this NHLBI cohort to systematically reassess the implication of 106 genes proposed to be involved in monogenic forms of XLID. We particularly question the implication in XLID of ten of them (AGTR2, MAGT1, ZNF674, SRPX2, ATP6AP2, ARHGEF6, NXF5, ZCCHC12, ZNF41, and ZNF81), in which truncating variants or previously published mutations are observed at a relatively high frequency within this cohort. We also highlight 15 other genes (CCDC22, CLIC2, CNKSR2, FRMPD4, HCFC1, IGBP1, KIAA2022, KLF8, MAOA, NAA10, NLGN3, RPL10, SHROOM4, ZDHHC15, and ZNF261) for which replication studies are warranted. We propose that similar reassessment of reported mutations (and genes) with the use of data from large-scale human exome sequencing would be relevant for a wide range of other genetic diseases. Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Evaluation of Nine Somatic Variant Callers for Detection of Somatic Mutations in Exome and Targeted Deep Sequencing Data.

PubMed

Krøigård, Anne Bruun; Thomassen, Mads; Lænkholm, Anne-Vibeke; Kruse, Torben A; Larsen, Martin Jakob

2016-01-01

Next generation sequencing is extensively applied to catalogue somatic mutations in cancer, in research settings and increasingly in clinical settings for molecular diagnostics, guiding therapy decisions. Somatic variant callers perform paired comparisons of sequencing data from cancer tissue and matched normal tissue in order to detect somatic mutations. The advent of many new somatic variant callers creates a need for comparison and validation of the tools, as no de facto standard for detection of somatic mutations exists and only limited comparisons have been reported. We have performed a comprehensive evaluation using exome sequencing and targeted deep sequencing data of paired tumor-normal samples from five breast cancer patients to evaluate the performance of nine publicly available somatic variant callers: EBCall, Mutect, Seurat, Shimmer, Indelocator, Somatic Sniper, Strelka, VarScan 2 and Virmid for the detection of single nucleotide mutations and small deletions and insertions. We report a large variation in the number of calls from the nine somatic variant callers on the same sequencing data and highly variable agreement. Sequencing depth had markedly diverse impact on individual callers, as for some callers, increased sequencing depth highly improved sensitivity. For SNV calling, we report EBCall, Mutect, Virmid and Strelka to be the most reliable somatic variant callers for both exome sequencing and targeted deep sequencing. For indel calling, EBCall is superior due to high sensitivity and robustness to changes in sequencing depths.

Evaluation of Nine Somatic Variant Callers for Detection of Somatic Mutations in Exome and Targeted Deep Sequencing Data

PubMed Central

Krøigård, Anne Bruun; Thomassen, Mads; Lænkholm, Anne-Vibeke; Kruse, Torben A.; Larsen, Martin Jakob

2016-01-01

Next generation sequencing is extensively applied to catalogue somatic mutations in cancer, in research settings and increasingly in clinical settings for molecular diagnostics, guiding therapy decisions. Somatic variant callers perform paired comparisons of sequencing data from cancer tissue and matched normal tissue in order to detect somatic mutations. The advent of many new somatic variant callers creates a need for comparison and validation of the tools, as no de facto standard for detection of somatic mutations exists and only limited comparisons have been reported. We have performed a comprehensive evaluation using exome sequencing and targeted deep sequencing data of paired tumor-normal samples from five breast cancer patients to evaluate the performance of nine publicly available somatic variant callers: EBCall, Mutect, Seurat, Shimmer, Indelocator, Somatic Sniper, Strelka, VarScan 2 and Virmid for the detection of single nucleotide mutations and small deletions and insertions. We report a large variation in the number of calls from the nine somatic variant callers on the same sequencing data and highly variable agreement. Sequencing depth had markedly diverse impact on individual callers, as for some callers, increased sequencing depth highly improved sensitivity. For SNV calling, we report EBCall, Mutect, Virmid and Strelka to be the most reliable somatic variant callers for both exome sequencing and targeted deep sequencing. For indel calling, EBCall is superior due to high sensitivity and robustness to changes in sequencing depths. PMID:27002637

International interlaboratory study comparing single organism 16S rRNA gene sequencing data: Beyond consensus sequence comparisons

PubMed Central

Olson, Nathan D.; Lund, Steven P.; Zook, Justin M.; Rojas-Cornejo, Fabiola; Beck, Brian; Foy, Carole; Huggett, Jim; Whale, Alexandra S.; Sui, Zhiwei; Baoutina, Anna; Dobeson, Michael; Partis, Lina; Morrow, Jayne B.

2015-01-01

This study presents the results from an interlaboratory sequencing study for which we developed a novel high-resolution method for comparing data from different sequencing platforms for a multi-copy, paralogous gene. The combination of PCR amplification and 16S ribosomal RNA gene (16S rRNA) sequencing has revolutionized bacteriology by enabling rapid identification, frequently without the need for culture. To assess variability between laboratories in sequencing 16S rRNA, six laboratories sequenced the gene encoding the 16S rRNA from Escherichia coli O157:H7 strain EDL933 and Listeria monocytogenes serovar 4b strain NCTC11994. Participants performed sequencing methods and protocols available in their laboratories: Sanger sequencing, Roche 454 pyrosequencing®, or Ion Torrent PGM®. The sequencing data were evaluated on three levels: (1) identity of biologically conserved position, (2) ratio of 16S rRNA gene copies featuring identified variants, and (3) the collection of variant combinations in a set of 16S rRNA gene copies. The same set of biologically conserved positions was identified for each sequencing method. Analytical methods using Bayesian and maximum likelihood statistics were developed to estimate variant copy ratios, which describe the ratio of nucleotides at each identified biologically variable position, as well as the likely set of variant combinations present in 16S rRNA gene copies. Our results indicate that estimated variant copy ratios at biologically variable positions were only reproducible for high throughput sequencing methods. Furthermore, the likely variant combination set was only reproducible with increased sequencing depth and longer read lengths. We also demonstrate novel methods for evaluating variable positions when comparing multi-copy gene sequence data from multiple laboratories generated using multiple sequencing technologies. PMID:27077030

Copy Number Variants in Obesity-Related Syndromes: Review and Perspectives on Novel Molecular Approaches

PubMed Central

Koiffmann, Celia Priszkulnik

2012-01-01

In recent decades, obesity has reached epidemic proportions worldwide and became a major concern in public health. Despite heritability estimates of 40 to 70% and the long-recognized genetic basis of obesity in a number of rare cases, the list of common obesity susceptibility variants by the currently published genome-wide association studies (GWASs) only explain a small proportion of the individual variation in risk of obesity. It was not until very recently that GWASs of copy number variants (CNVs) in individuals with extreme phenotypes reported a number of large and rare CNVs conferring high risk to obesity, and specifically deletions on chromosome 16p11.2. In this paper, we comment on the recent advances in the field of genetics of obesity with an emphasis on the genes and genomic regions implicated in highly penetrant forms of obesity associated with developmental disorders. Array genomic hybridization in this patient population has afforded discovery opportunities for CNVs that have not previously been detectable. This information can be used to generate new diagnostic arrays and sequencing platforms, which will likely enhance detection of known genetic conditions with the potential to elucidate new disease genes and ultimately help in developing a next-generation sequencing protocol relevant to clinical practice. PMID:23316347

Targeted DNA sequencing of non-small cell lung cancer identifies mutations associated with brain metastases.

PubMed

Wilson, George D; Johnson, Matthew D; Ahmed, Samreen; Cardenas, Paola Yumpo; Grills, Inga S; Thibodeau, Bryan J

2018-05-25

This study explores the hypothesis that dominant molecular oncogenes in non-small cell lung cancer (NSCLC) are associated with metastatic spread to the brain. NSCLC patient groups with no evidence of metastasis, with metastatic disease to a non-CNS site, who developed brain metastasis after diagnosis, and patients with simultaneous diagnosis of NSCLC and metastatic brain lesions were studied using targeted sequencing. In patients with brain metastasis versus those without, only 2 variants (one each in BCL6 and NOTHC2) were identified that occurred in ≥ 4 NSCLC of patients with brain metastases but ≤ 1 of the NSCLC samples without brain metastases. At the gene level, 20 genes were found to have unique variants in more than 33% of the patients with brain metastases. When analyzed at the patient level, these 20 genes formed the basis of a predictive test to discriminate those with brain metastasis. Further analysis showed that PI3K/AKT signaling is altered in both the primary and metastases of NSCLC patients with brain lesions. While no single variant was associated with brain metastasis, this study describes a potential gene panel for the identification of patients at risk and implicates PI3K/AKT signaling as a therapeutic target.

Contactin 4 as an Autism Susceptibility Locus

PubMed Central

Cottrell, Catherine E.; Bir, Natalie; Varga, Elizabeth; Alvarez, Carlos E.; Bouyain, Samuel; Zernzach, Randall; LambThrush, Devon; Evans, Johnna; Trimarchi, Michael; Butter, Eric M.; Cunningham, David; Gastier-Foster, Julie M.; McBride, Kim; Herman, Gail E.

2011-01-01

Scientific Abstract Structural and sequence variation have been described in several members of the contactin (CNTN) and contactin associated protein (CNTNAP) gene families in association with neurodevelopmental disorders, including autism. Using array comparative genome hybridization (CGH), we identified a maternally inherited ~535 kb deletion at 3p26.3 encompassing the 5′ end of the contactin 4 gene (CNTN4) in a patient with autism. Based on this finding and previous reports implicating genomic rearrangements of CNTN4 in autism spectrum disorders (ASDs) and 3p− microdeletion syndrome, we undertook sequencing of the coding regions of the gene in a local ASD cohort in comparison with a set of controls. Unique missense variants were identified in 4/75 unrelated individuals with an ASD, as well as in 1/107 controls. All of the amino acid substitutions were nonsynonomous, occurred at evolutionarily conserved positions, and were, thus, felt likely to be deleterious. However, these data did not reach statistical significance, nor did the variants segregate with disease within all of the ASD families. Finally, there was no detectable difference in binding of two of the variants to the interacting protein PTPRG in vitro. Thusadditional, larger studies will be necessary to determine whether CNTN4 functions as an autism susceptibility locus in combination with other genetic and/or environmental factors. PMID:21308999

Targeted DNA sequencing of non-small cell lung cancer identifies mutations associated with brain metastases

PubMed Central

Wilson, George D.; Johnson, Matthew D.; Ahmed, Samreen; Cardenas, Paola Yumpo; Grills, Inga S.; Thibodeau, Bryan J.

2018-01-01

Introduction This study explores the hypothesis that dominant molecular oncogenes in non-small cell lung cancer (NSCLC) are associated with metastatic spread to the brain. Methods NSCLC patient groups with no evidence of metastasis, with metastatic disease to a non-CNS site, who developed brain metastasis after diagnosis, and patients with simultaneous diagnosis of NSCLC and metastatic brain lesions were studied using targeted sequencing. Results In patients with brain metastasis versus those without, only 2 variants (one each in BCL6 and NOTHC2) were identified that occurred in ≥ 4 NSCLC of patients with brain metastases but ≤ 1 of the NSCLC samples without brain metastases. At the gene level, 20 genes were found to have unique variants in more than 33% of the patients with brain metastases. When analyzed at the patient level, these 20 genes formed the basis of a predictive test to discriminate those with brain metastasis. Further analysis showed that PI3K/AKT signaling is altered in both the primary and metastases of NSCLC patients with brain lesions. Conclusion While no single variant was associated with brain metastasis, this study describes a potential gene panel for the identification of patients at risk and implicates PI3K/AKT signaling as a therapeutic target. PMID:29899834

Targeted resequencing identifies defective variants of decoy receptor 3 in pediatric-onset inflammatory bowel disease.

PubMed

Cardinale, C J; Wei, Z; Panossian, S; Wang, F; Kim, C E; Mentch, F D; Chiavacci, R M; Kachelries, K E; Pandey, R; Grant, S F A; Baldassano, R N; Hakonarson, H

2013-10-01

Genome-wide association studies have implicated common variation at the 20q13 locus in inflammatory bowel disease, particularly for the pediatric Crohn's form. This locus harbors tumor necrosis factor receptor superfamily (TNFRSF6B), encoding a secreted protein, decoy receptor 3 (DcR3), which binds to and neutralizes pro-inflammatory cytokines of the tumor necrosis factor superfamily. We sought to further the evidence of DcR3's role in pediatric IBD by identifying missense mutations with functional significance within TNFRSF6B. We sequenced the exons of the gene in 528 Caucasian pediatric IBD cases and 549 Caucasian healthy controls to establish the frequency of such events in each population. Sequencing revealed that our IBD cohort harbored a greater number of missense variants, yielding an odds ratio of 3.9 (P-value=0.005). Using functional assays, we established that the frequency of mutants defective in secretion from cultured cells was greater in the Crohn's category than in the controls, yielding an odds ratio of 7.1 (P-value=0.004). These results suggest that rare defective variants in TNFRSF6B have a role in the pathogenesis of some cases of IBD and that interventions targeting this group of tumor necrosis factor-family members may benefit patients with IBD.

Missense variants in AIMP1 gene are implicated in autosomal recessive intellectual disability without neurodegeneration

PubMed Central

Iqbal, Zafar; Püttmann, Lucia; Musante, Luciana; Razzaq, Attia; Zahoor, Muhammad Yasir; Hu, Hao; Wienker, Thomas F; Garshasbi, Masoud; Fattahi, Zohreh; Gilissen, Christian; Vissers, Lisenka ELM; de Brouwer, Arjan PM; Veltman, Joris A; Pfundt, Rolph; Najmabadi, Hossein; Ropers, Hans-Hilger; Riazuddin, Sheikh; Kahrizi, Kimia; van Bokhoven, Hans

2016-01-01

AIMP1/p43 is a multifunctional non-catalytic component of the multisynthetase complex. The complex consists of nine catalytic and three non-catalytic proteins, which catalyze the ligation of amino acids to their cognate tRNA isoacceptors for use in protein translation. To date, two allelic variants in the AIMP1 gene have been reported as the underlying cause of autosomal recessive primary neurodegenerative disorder. Here, we present two consanguineous families from Pakistan and Iran, presenting with moderate to severe intellectual disability, global developmental delay, and speech impairment without neurodegeneration. By the combination of homozygosity mapping and next generation sequencing, we identified two homozygous missense variants, p.(Gly299Arg) and p.(Val176Gly), in the gene AIMP1 that co-segregated with the phenotype in the respective families. Molecular modeling of the variants revealed deleterious effects on the protein structure that are predicted to result in reduced AIMP1 function. Our findings indicate that the clinical spectrum for AIMP1 defects is broader than witnessed so far. PMID:26173967

Missense variants in AIMP1 gene are implicated in autosomal recessive intellectual disability without neurodegeneration.

PubMed

Iqbal, Zafar; Püttmann, Lucia; Musante, Luciana; Razzaq, Attia; Zahoor, Muhammad Yasir; Hu, Hao; Wienker, Thomas F; Garshasbi, Masoud; Fattahi, Zohreh; Gilissen, Christian; Vissers, Lisenka E L M; de Brouwer, Arjan P M; Veltman, Joris A; Pfundt, Rolph; Najmabadi, Hossein; Ropers, Hans-Hilger; Riazuddin, Sheikh; Kahrizi, Kimia; van Bokhoven, Hans

2016-03-01

AIMP1/p43 is a multifunctional non-catalytic component of the multisynthetase complex. The complex consists of nine catalytic and three non-catalytic proteins, which catalyze the ligation of amino acids to their cognate tRNA isoacceptors for use in protein translation. To date, two allelic variants in the AIMP1 gene have been reported as the underlying cause of autosomal recessive primary neurodegenerative disorder. Here, we present two consanguineous families from Pakistan and Iran, presenting with moderate to severe intellectual disability, global developmental delay, and speech impairment without neurodegeneration. By the combination of homozygosity mapping and next generation sequencing, we identified two homozygous missense variants, p.(Gly299Arg) and p.(Val176Gly), in the gene AIMP1 that co-segregated with the phenotype in the respective families. Molecular modeling of the variants revealed deleterious effects on the protein structure that are predicted to result in reduced AIMP1 function. Our findings indicate that the clinical spectrum for AIMP1 defects is broader than witnessed so far.

Rett-like phenotypes: expanding the genetic heterogeneity to the KCNA2 gene and first familial case of CDKL5-related disease.

PubMed

Allou, L; Julia, S; Amsallem, D; El Chehadeh, S; Lambert, L; Thevenon, J; Duffourd, Y; Saunier, A; Bouquet, P; Pere, S; Moustaïne, A; Ruaud, L; Roth, V; Jonveaux, P; Philippe, C

2017-03-01

Several genes have been implicated in Rett syndrome (RTT) in its typical and variant forms. We applied next-generation sequencing (NGS) to evaluate for mutations in known or new candidate genes in patients with variant forms of Rett or Rett-like phenotypes of unknown molecular aetiology. In the first step, we used NGS with a custom panel including MECP2, CDKL5, FOXG1, MEF2C and IQSEC2. In addition to a FOXG1 mutation in a patient with all core features of the congenital variant of RTT, we identified a missense (p.Ser240Thr) in CDKL5 in a patient who appeared to be seizure free. This missense was maternally inherited with opposite allele expression ratios in the proband and her mother. In the asymptomatic mother, the mutated copy of the CDKL5 gene was inactivated in 90% of blood cells. We also identified a premature stop codon (p.Arg926*) in IQSEC2 in a patient with a Rett-like phenotype. Finally, exome sequencing enabled us to characterize a heterozygous de novo missense (p.Val408Ala) in KCNA2 encoding the potassium channel Kv 1.2 in a girl with infantile-onset seizures variant of RTT. Our study expands the genetic heterogeneity of RTT and RTT-like phenotypes. Moreover, we report the first familial case of CDKL5-related disease. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Genomic prediction using preselected DNA variants from a GWAS with whole-genome sequence data in Holstein-Friesian cattle.

PubMed

Veerkamp, Roel F; Bouwman, Aniek C; Schrooten, Chris; Calus, Mario P L

2016-12-01

Whole-genome sequence data is expected to capture genetic variation more completely than common genotyping panels. Our objective was to compare the proportion of variance explained and the accuracy of genomic prediction by using imputed sequence data or preselected SNPs from a genome-wide association study (GWAS) with imputed whole-genome sequence data. Phenotypes were available for 5503 Holstein-Friesian bulls. Genotypes were imputed up to whole-genome sequence (13,789,029 segregating DNA variants) by using run 4 of the 1000 bull genomes project. The program GCTA was used to perform GWAS for protein yield (PY), somatic cell score (SCS) and interval from first to last insemination (IFL). From the GWAS, subsets of variants were selected and genomic relationship matrices (GRM) were used to estimate the variance explained in 2087 validation animals and to evaluate the genomic prediction ability. Finally, two GRM were fitted together in several models to evaluate the effect of selected variants that were in competition with all the other variants. The GRM based on full sequence data explained only marginally more genetic variation than that based on common SNP panels: for PY, SCS and IFL, genomic heritability improved from 0.81 to 0.83, 0.83 to 0.87 and 0.69 to 0.72, respectively. Sequence data also helped to identify more variants linked to quantitative trait loci and resulted in clearer GWAS peaks across the genome. The proportion of total variance explained by the selected variants combined in a GRM was considerably smaller than that explained by all variants (less than 0.31 for all traits). When selected variants were used, accuracy of genomic predictions decreased and bias increased. Although 35 to 42 variants were detected that together explained 13 to 19% of the total variance (18 to 23% of the genetic variance) when fitted alone, there was no advantage in using dense sequence information for genomic prediction in the Holstein data used in our study. Detection and selection of variants within a single breed are difficult due to long-range linkage disequilibrium. Stringent selection of variants resulted in more biased genomic predictions, although this might be due to the training population being the same dataset from which the selected variants were identified.

Effect of Next-Generation Exome Sequencing Depth for Discovery of Diagnostic Variants.

PubMed

Kim, Kyung; Seong, Moon-Woo; Chung, Won-Hyong; Park, Sung Sup; Leem, Sangseob; Park, Won; Kim, Jihyun; Lee, KiYoung; Park, Rae Woong; Kim, Namshin

2015-06-01

Sequencing depth, which is directly related to the cost and time required for the generation, processing, and maintenance of next-generation sequencing data, is an important factor in the practical utilization of such data in clinical fields. Unfortunately, identifying an exome sequencing depth adequate for clinical use is a challenge that has not been addressed extensively. Here, we investigate the effect of exome sequencing depth on the discovery of sequence variants for clinical use. Toward this, we sequenced ten germ-line blood samples from breast cancer patients on the Illumina platform GAII(x) at a high depth of ~200×. We observed that most function-related diverse variants in the human exonic regions could be detected at a sequencing depth of 120×. Furthermore, investigation using a diagnostic gene set showed that the number of clinical variants identified using exome sequencing reached a plateau at an average sequencing depth of about 120×. Moreover, the phenomena were consistent across the breast cancer samples.

Carbohydrate degrading polypeptide and uses thereof

DOEpatents

Sagt, Cornelis Maria Jacobus; Schooneveld-Bergmans, Margot Elisabeth Francoise; Roubos, Johannes Andries; Los, Alrik Pieter

2015-10-20

The invention relates to a polypeptide having carbohydrate material degrading activity which comprises the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 4, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 96% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 96% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional protein and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.

A Recurrent De Novo Variant in NACC1 Causes a Syndrome Characterized by Infantile Epilepsy, Cataracts, and Profound Developmental Delay.

PubMed

Schoch, Kelly; Meng, Linyan; Szelinger, Szabolcs; Bearden, David R; Stray-Pedersen, Asbjorg; Busk, Oyvind L; Stong, Nicholas; Liston, Eriskay; Cohn, Ronald D; Scaglia, Fernando; Rosenfeld, Jill A; Tarpinian, Jennifer; Skraban, Cara M; Deardorff, Matthew A; Friedman, Jeremy N; Akdemir, Zeynep Coban; Walley, Nicole; Mikati, Mohamad A; Kranz, Peter G; Jasien, Joan; McConkie-Rosell, Allyn; McDonald, Marie; Wechsler, Stephanie Burns; Freemark, Michael; Kansagra, Sujay; Freedman, Sharon; Bali, Deeksha; Millan, Francisca; Bale, Sherri; Nelson, Stanley F; Lee, Hane; Dorrani, Naghmeh; Goldstein, David B; Xiao, Rui; Yang, Yaping; Posey, Jennifer E; Martinez-Agosto, Julian A; Lupski, James R; Wangler, Michael F; Shashi, Vandana

2017-02-02

Whole-exome sequencing (WES) has increasingly enabled new pathogenic gene variant identification for undiagnosed neurodevelopmental disorders and provided insights into both gene function and disease biology. Here, we describe seven children with a neurodevelopmental disorder characterized by microcephaly, profound developmental delays and/or intellectual disability, cataracts, severe epilepsy including infantile spasms, irritability, failure to thrive, and stereotypic hand movements. Brain imaging in these individuals reveals delay in myelination and cerebral atrophy. We observe an identical recurrent de novo heterozygous c.892C>T (p.Arg298Trp) variant in the nucleus accumbens associated 1 (NACC1) gene in seven affected individuals. One of the seven individuals is mosaic for this variant. NACC1 encodes a transcriptional repressor implicated in gene expression and has not previously been associated with germline disorders. The probability of finding the same missense NACC1 variant by chance in 7 out of 17,228 individuals who underwent WES for diagnoses of neurodevelopmental phenotypes is extremely small and achieves genome-wide significance (p = 1.25 × 10 -14 ). Selective constraint against missense variants in NACC1 makes this excess of an identical missense variant in all seven individuals more remarkable. Our findings are consistent with a germline recurrent mutational hotspot associated with an allele-specific neurodevelopmental phenotype in NACC1. Copyright © 2017 American Society of Human Genetics. All rights reserved.

Implementation and utilization of genetic testing in personalized medicine

PubMed Central

Abul-Husn, Noura S; Owusu Obeng, Aniwaa; Sanderson, Saskia C; Gottesman, Omri; Scott, Stuart A

2014-01-01

Clinical genetic testing began over 30 years ago with the availability of mutation detection for sickle cell disease diagnosis. Since then, the field has dramatically transformed to include gene sequencing, high-throughput targeted genotyping, prenatal mutation detection, preimplantation genetic diagnosis, population-based carrier screening, and now genome-wide analyses using microarrays and next-generation sequencing. Despite these significant advances in molecular technologies and testing capabilities, clinical genetics laboratories historically have been centered on mutation detection for Mendelian disorders. However, the ongoing identification of deoxyribonucleic acid (DNA) sequence variants associated with common diseases prompted the availability of testing for personal disease risk estimation, and created commercial opportunities for direct-to-consumer genetic testing companies that assay these variants. This germline genetic risk, in conjunction with other clinical, family, and demographic variables, are the key components of the personalized medicine paradigm, which aims to apply personal genomic and other relevant data into a patient’s clinical assessment to more precisely guide medical management. However, genetic testing for disease risk estimation is an ongoing topic of debate, largely due to inconsistencies in the results, concerns over clinical validity and utility, and the variable mode of delivery when returning genetic results to patients in the absence of traditional counseling. A related class of genetic testing with analogous issues of clinical utility and acceptance is pharmacogenetic testing, which interrogates sequence variants implicated in interindividual drug response variability. Although clinical pharmacogenetic testing has not previously been widely adopted, advances in rapid turnaround time genetic testing technology and the recent implementation of preemptive genotyping programs at selected medical centers suggest that personalized medicine through pharmacogenetics is now a reality. This review aims to summarize the current state of implementing genetic testing for personalized medicine, with an emphasis on clinical pharmacogenetic testing. PMID:25206309

Complete cDNA sequence of SAP-like pentraxin from Limulus polyphemus: implications for pentraxin evolution.

PubMed

Tharia, Hazel A; Shrive, Annette K; Mills, John D; Arme, Chris; Williams, Gwyn T; Greenhough, Trevor J

2002-02-22

The serum amyloid P component (SAP)-like pentraxin Limulus polyphemus SAP is a recently discovered, distinct pentraxin species, of known structure, which does not bind phosphocholine and whose N-terminal sequence has been shown to differ markedly from the highly conserved N terminus of all other known horseshoe crab pentraxins. The complete cDNA sequence of Limulus SAP, and the derived amino acid sequence, the first invertebrate SAP-like pentraxin sequence, have been determined. Two sequences were identified that differed only in the length of the 3' untranslated region. Limulus SAP is synthesised as a precursor protein of 234 amino acid residues, the first 17 residues encoding a signal peptide that is absent from the mature protein. Phylogenetic analysis clusters Limulus SAP pentraxin with the horseshoe crab C-reactive proteins (CRPs) rather than the mammalian SAPs, which are clustered with mammalian CRPs. The deduced amino acid sequence shares 22% identity with both human SAP and CRP, which are 51% identical, and 31-35% with horseshoe crab CRPs. These analyses indicate that gene duplication of CRP (or SAP), followed by sequence divergence and the evolution of CRP and/or SAP function, occurred independently along the chordate and arthropod evolutionary lines rather than in a common ancestor. They further indicate that the CRP/SAP gene duplication event in Limulus occurred before both the emergence of the Limulus CRP variants and the mammalian CRP/SAP gene duplication. Limulus SAP, which does not exhibit the CRP characteristic of calcium-dependent binding to phosphocholine, is established as a pentraxin species distinct from all other known horseshoe crab pentraxins that exist in many variant forms sharing a high level of sequence homology. Copyright 2002 Elsevier Science Ltd.

Validation and optimization of the Ion Torrent S5 XL sequencer and Oncomine workflow for BRCA1 and BRCA2 genetic testing.

PubMed

Shin, Saeam; Kim, Yoonjung; Chul Oh, Seoung; Yu, Nae; Lee, Seung-Tae; Rak Choi, Jong; Lee, Kyung-A

2017-05-23

In this study, we validated the analytical performance of BRCA1/2 sequencing using Ion Torrent's new bench-top sequencer with amplicon panel with optimized bioinformatics pipelines. Using 43 samples that were previously validated by Illumina's MiSeq platform and/or by Sanger sequencing/multiplex ligation-dependent probe amplification, we amplified the target with the Oncomine™ BRCA Research Assay and sequenced on Ion Torrent S5 XL (Thermo Fisher Scientific, Waltham, MA, USA). We compared two bioinformatics pipelines for optimal processing of S5 XL sequence data: the Torrent Suite with a plug-in Torrent Variant Caller (Thermo Fisher Scientific), and commercial NextGENe software (Softgenetics, State College, PA, USA). All expected 681 single nucleotide variants, 15 small indels, and three copy number variants were correctly called, except one common variant adjacent to a rare variant on the primer-binding site. The sensitivity, specificity, false positive rate, and accuracy for detection of single nucleotide variant and small indels of S5 XL sequencing were 99.85%, 100%, 0%, and 99.99% for the Torrent Variant Caller and 99.85%, 99.99%, 0.14%, and 99.99% for NextGENe, respectively. The reproducibility of variant calling was 100%, and the precision of variant frequency also showed good performance with coefficients of variation between 0.32 and 5.29%. We obtained highly accurate data through uniform and sufficient coverage depth over all target regions and through optimization of the bioinformatics pipeline. We confirmed that our platform is accurate and practical for diagnostic BRCA1/2 testing in a clinical laboratory.

Genetic Architecture of Vitamin B12 and Folate Levels Uncovered Applying Deeply Sequenced Large Datasets

PubMed Central

Thorleifsson, Gudmar; Ahluwalia, Tarunveer S.; Steinthorsdottir, Valgerdur; Bjarnason, Helgi; Gudbjartsson, Daniel F.; Magnusson, Olafur T.; Sparsø, Thomas; Albrechtsen, Anders; Kong, Augustine; Masson, Gisli; Tian, Geng; Cao, Hongzhi; Nie, Chao; Kristiansen, Karsten; Husemoen, Lise Lotte; Thuesen, Betina; Li, Yingrui; Nielsen, Rasmus; Linneberg, Allan; Olafsson, Isleifur; Eyjolfsson, Gudmundur I.; Jørgensen, Torben; Wang, Jun; Hansen, Torben; Thorsteinsdottir, Unnur; Stefánsson, Kari; Pedersen, Oluf

2013-01-01

Genome-wide association studies have mainly relied on common HapMap sequence variations. Recently, sequencing approaches have allowed analysis of low frequency and rare variants in conjunction with common variants, thereby improving the search for functional variants and thus the understanding of the underlying biology of human traits and diseases. Here, we used a large Icelandic whole genome sequence dataset combined with Danish exome sequence data to gain insight into the genetic architecture of serum levels of vitamin B12 (B12) and folate. Up to 22.9 million sequence variants were analyzed in combined samples of 45,576 and 37,341 individuals with serum B12 and folate measurements, respectively. We found six novel loci associating with serum B12 (CD320, TCN2, ABCD4, MMAA, MMACHC) or folate levels (FOLR3) and confirmed seven loci for these traits (TCN1, FUT6, FUT2, CUBN, CLYBL, MUT, MTHFR). Conditional analyses established that four loci contain additional independent signals. Interestingly, 13 of the 18 identified variants were coding and 11 of the 13 target genes have known functions related to B12 and folate pathways. Contrary to epidemiological studies we did not find consistent association of the variants with cardiovascular diseases, cancers or Alzheimer's disease although some variants demonstrated pleiotropic effects. Although to some degree impeded by low statistical power for some of these conditions, these data suggest that sequence variants that contribute to the population diversity in serum B12 or folate levels do not modify the risk of developing these conditions. Yet, the study demonstrates the value of combining whole genome and exome sequencing approaches to ascertain the genetic and molecular architectures underlying quantitative trait associations. PMID:23754956

Factors influencing success of clinical genome sequencing across a broad spectrum of disorders

PubMed Central

Lise, Stefano; Broxholme, John; Cazier, Jean-Baptiste; Rimmer, Andy; Kanapin, Alexander; Lunter, Gerton; Fiddy, Simon; Allan, Chris; Aricescu, A. Radu; Attar, Moustafa; Babbs, Christian; Becq, Jennifer; Beeson, David; Bento, Celeste; Bignell, Patricia; Blair, Edward; Buckle, Veronica J; Bull, Katherine; Cais, Ondrej; Cario, Holger; Chapel, Helen; Copley, Richard R; Cornall, Richard; Craft, Jude; Dahan, Karin; Davenport, Emma E; Dendrou, Calliope; Devuyst, Olivier; Fenwick, Aimée L; Flint, Jonathan; Fugger, Lars; Gilbert, Rodney D; Goriely, Anne; Green, Angie; Greger, Ingo H.; Grocock, Russell; Gruszczyk, Anja V; Hastings, Robert; Hatton, Edouard; Higgs, Doug; Hill, Adrian; Holmes, Chris; Howard, Malcolm; Hughes, Linda; Humburg, Peter; Johnson, David; Karpe, Fredrik; Kingsbury, Zoya; Kini, Usha; Knight, Julian C; Krohn, Jonathan; Lamble, Sarah; Langman, Craig; Lonie, Lorne; Luck, Joshua; McCarthy, Davis; McGowan, Simon J; McMullin, Mary Frances; Miller, Kerry A; Murray, Lisa; Németh, Andrea H; Nesbit, M Andrew; Nutt, David; Ormondroyd, Elizabeth; Oturai, Annette Bang; Pagnamenta, Alistair; Patel, Smita Y; Percy, Melanie; Petousi, Nayia; Piazza, Paolo; Piret, Sian E; Polanco-Echeverry, Guadalupe; Popitsch, Niko; Powrie, Fiona; Pugh, Chris; Quek, Lynn; Robbins, Peter A; Robson, Kathryn; Russo, Alexandra; Sahgal, Natasha; van Schouwenburg, Pauline A; Schuh, Anna; Silverman, Earl; Simmons, Alison; Sørensen, Per Soelberg; Sweeney, Elizabeth; Taylor, John; Thakker, Rajesh V; Tomlinson, Ian; Trebes, Amy; Twigg, Stephen RF; Uhlig, Holm H; Vyas, Paresh; Vyse, Tim; Wall, Steven A; Watkins, Hugh; Whyte, Michael P; Witty, Lorna; Wright, Ben; Yau, Chris; Buck, David; Humphray, Sean; Ratcliffe, Peter J; Bell, John I; Wilkie, Andrew OM; Bentley, David; Donnelly, Peter; McVean, Gilean

2015-01-01

To assess factors influencing the success of whole genome sequencing for mainstream clinical diagnosis, we sequenced 217 individuals from 156 independent cases across a broad spectrum of disorders in whom prior screening had identified no pathogenic variants. We quantified the number of candidate variants identified using different strategies for variant calling, filtering, annotation and prioritisation. We found that jointly calling variants across samples, filtering against both local and external databases, deploying multiple annotation tools and using familial transmission above biological plausibility contributed to accuracy. Overall, we identified disease causing variants in 21% of cases, rising to 34% (23/68) for Mendelian disorders and 57% (8/14) in trios. We also discovered 32 potentially clinically actionable variants in 18 genes unrelated to the referral disorder, though only four were ultimately considered reportable. Our results demonstrate the value of genome sequencing for routine clinical diagnosis, but also highlight many outstanding challenges. PMID:25985138

Whole genome sequences of a male and female supercentenarian, ages greater than 114 years.

PubMed

Sebastiani, Paola; Riva, Alberto; Montano, Monty; Pham, Phillip; Torkamani, Ali; Scherba, Eugene; Benson, Gary; Milton, Jacqueline N; Baldwin, Clinton T; Andersen, Stacy; Schork, Nicholas J; Steinberg, Martin H; Perls, Thomas T

2011-01-01

Supercentenarians (age 110+ years old) generally delay or escape age-related diseases and disability well beyond the age of 100 and this exceptional survival is likely to be influenced by a genetic predisposition that includes both common and rare genetic variants. In this report, we describe the complete genomic sequences of male and female supercentenarians, both age >114 years old. We show that: (1) the sequence variant spectrum of these two individuals' DNA sequences is largely comparable to existing non-supercentenarian genomes; (2) the two individuals do not appear to carry most of the well-established human longevity enabling variants already reported in the literature; (3) they have a comparable number of known disease-associated variants relative to most human genomes sequenced to-date; (4) approximately 1% of the variants these individuals possess are novel and may point to new genes involved in exceptional longevity; and (5) both individuals are enriched for coding variants near longevity-associated variants that we discovered through a large genome-wide association study. These analyses suggest that there are both common and rare longevity-associated variants that may counter the effects of disease-predisposing variants and extend lifespan. The continued analysis of the genomes of these and other rare individuals who have survived to extremely old ages should provide insight into the processes that contribute to the maintenance of health during extreme aging.

Whole Genome Sequences of a Male and Female Supercentenarian, Ages Greater than 114 Years

PubMed Central

Sebastiani, Paola; Riva, Alberto; Montano, Monty; Pham, Phillip; Torkamani, Ali; Scherba, Eugene; Benson, Gary; Milton, Jacqueline N.; Baldwin, Clinton T.; Andersen, Stacy; Schork, Nicholas J.; Steinberg, Martin H.; Perls, Thomas T.

2012-01-01

Supercentenarians (age 110+ years old) generally delay or escape age-related diseases and disability well beyond the age of 100 and this exceptional survival is likely to be influenced by a genetic predisposition that includes both common and rare genetic variants. In this report, we describe the complete genomic sequences of male and female supercentenarians, both age >114 years old. We show that: (1) the sequence variant spectrum of these two individuals’ DNA sequences is largely comparable to existing non-supercentenarian genomes; (2) the two individuals do not appear to carry most of the well-established human longevity enabling variants already reported in the literature; (3) they have a comparable number of known disease-associated variants relative to most human genomes sequenced to-date; (4) approximately 1% of the variants these individuals possess are novel and may point to new genes involved in exceptional longevity; and (5) both individuals are enriched for coding variants near longevity-associated variants that we discovered through a large genome-wide association study. These analyses suggest that there are both common and rare longevity-associated variants that may counter the effects of disease-predisposing variants and extend lifespan. The continued analysis of the genomes of these and other rare individuals who have survived to extremely old ages should provide insight into the processes that contribute to the maintenance of health during extreme aging. PMID:22303384

BETASEQ: a powerful novel method to control type-I error inflation in partially sequenced data for rare variant association testing.

PubMed

Yan, Song; Li, Yun

2014-02-15

Despite its great capability to detect rare variant associations, next-generation sequencing is still prohibitively expensive when applied to large samples. In case-control studies, it is thus appealing to sequence only a subset of cases to discover variants and genotype the identified variants in controls and the remaining cases under the reasonable assumption that causal variants are usually enriched among cases. However, this approach leads to inflated type-I error if analyzed naively for rare variant association. Several methods have been proposed in recent literature to control type-I error at the cost of either excluding some sequenced cases or correcting the genotypes of discovered rare variants. All of these approaches thus suffer from certain extent of information loss and thus are underpowered. We propose a novel method (BETASEQ), which corrects inflation of type-I error by supplementing pseudo-variants while keeps the original sequence and genotype data intact. Extensive simulations and real data analysis demonstrate that, in most practical situations, BETASEQ leads to higher testing powers than existing approaches with guaranteed (controlled or conservative) type-I error. BETASEQ and associated R files, including documentation, examples, are available at http://www.unc.edu/~yunmli/betaseq

Using whole-exome sequencing to identify variants inherited from mosaic parents

PubMed Central

Rios, Jonathan J; Delgado, Mauricio R

2015-01-01

Whole-exome sequencing (WES) has allowed the discovery of genes and variants causing rare human disease. This is often achieved by comparing nonsynonymous variants between unrelated patients, and particularly for sporadic or recessive disease, often identifies a single or few candidate genes for further consideration. However, despite the potential for this approach to elucidate the genetic cause of rare human disease, a majority of patients fail to realize a genetic diagnosis using standard exome analysis methods. Although genetic heterogeneity contributes to the difficulty of exome sequence analysis between patients, it remains plausible that rare human disease is not caused by de novo or recessive variants. Multiple human disorders have been described for which the variant was inherited from a phenotypically normal mosaic parent. Here we highlight the potential for exome sequencing to identify a reasonable number of candidate genes when dominant disease variants are inherited from a mosaic parent. We show the power of WES to identify a limited number of candidate genes using this disease model and how sequence coverage affects identification of mosaic variants by WES. We propose this analysis as an alternative to discover genetic causes of rare human disorders for which typical WES approaches fail to identify likely pathogenic variants. PMID:24986828

Telomere extension by telomerase and ALT generates variant repeats by mechanistically distinct processes

PubMed Central

Lee, Michael; Hills, Mark; Conomos, Dimitri; Stutz, Michael D.; Dagg, Rebecca A.; Lau, Loretta M.S.; Reddel, Roger R.; Pickett, Hilda A.

2014-01-01

Telomeres are terminal repetitive DNA sequences on chromosomes, and are considered to comprise almost exclusively hexameric TTAGGG repeats. We have evaluated telomere sequence content in human cells using whole-genome sequencing followed by telomere read extraction in a panel of mortal cell strains and immortal cell lines. We identified a wide range of telomere variant repeats in human cells, and found evidence that variant repeats are generated by mechanistically distinct processes during telomerase- and ALT-mediated telomere lengthening. Telomerase-mediated telomere extension resulted in biased repeat synthesis of variant repeats that differed from the canonical sequence at positions 1 and 3, but not at positions 2, 4, 5 or 6. This indicates that telomerase is most likely an error-prone reverse transcriptase that misincorporates nucleotides at specific positions on the telomerase RNA template. In contrast, cell lines that use the ALT pathway contained a large range of variant repeats that varied greatly between lines. This is consistent with variant repeats spreading from proximal telomeric regions throughout telomeres in a stochastic manner by recombination-mediated templating of DNA synthesis. The presence of unexpectedly large numbers of variant repeats in cells utilizing either telomere maintenance mechanism suggests a conserved role for variant sequences at human telomeres. PMID:24225324

Detecting very low allele fraction variants using targeted DNA sequencing and a novel molecular barcode-aware variant caller.

PubMed

Xu, Chang; Nezami Ranjbar, Mohammad R; Wu, Zhong; DiCarlo, John; Wang, Yexun

2017-01-03

Detection of DNA mutations at very low allele fractions with high accuracy will significantly improve the effectiveness of precision medicine for cancer patients. To achieve this goal through next generation sequencing, researchers need a detection method that 1) captures rare mutation-containing DNA fragments efficiently in the mix of abundant wild-type DNA; 2) sequences the DNA library extensively to deep coverage; and 3) distinguishes low level true variants from amplification and sequencing errors with high accuracy. Targeted enrichment using PCR primers provides researchers with a convenient way to achieve deep sequencing for a small, yet most relevant region using benchtop sequencers. Molecular barcoding (or indexing) provides a unique solution for reducing sequencing artifacts analytically. Although different molecular barcoding schemes have been reported in recent literature, most variant calling has been done on limited targets, using simple custom scripts. The analytical performance of barcode-aware variant calling can be significantly improved by incorporating advanced statistical models. We present here a highly efficient, simple and scalable enrichment protocol that integrates molecular barcodes in multiplex PCR amplification. In addition, we developed smCounter, an open source, generic, barcode-aware variant caller based on a Bayesian probabilistic model. smCounter was optimized and benchmarked on two independent read sets with SNVs and indels at 5 and 1% allele fractions. Variants were called with very good sensitivity and specificity within coding regions. We demonstrated that we can accurately detect somatic mutations with allele fractions as low as 1% in coding regions using our enrichment protocol and variant caller.

Next-generation sequencing using a pre-designed gene panel for the molecular diagnosis of congenital disorders in pediatric patients.

PubMed

Lim, Eileen C P; Brett, Maggie; Lai, Angeline H M; Lee, Siew-Peng; Tan, Ee-Shien; Jamuar, Saumya S; Ng, Ivy S L; Tan, Ene-Choo

2015-12-14

Next-generation sequencing (NGS) has revolutionized genetic research and offers enormous potential for clinical application. Sequencing the exome has the advantage of casting the net wide for all known coding regions while targeted gene panel sequencing provides enhanced sequencing depths and can be designed to avoid incidental findings in adult-onset conditions. A HaloPlex panel consisting of 180 genes within commonly altered chromosomal regions is available for use on both the Ion Personal Genome Machine (PGM) and MiSeq platforms to screen for causative mutations in these genes. We used this Haloplex ICCG panel for targeted sequencing of 15 patients with clinical presentations indicative of an abnormality in one of the 180 genes. Sequencing runs were done using the Ion 318 Chips on the Ion Torrent PGM. Variants were filtered for known polymorphisms and analysis was done to identify possible disease-causing variants before validation by Sanger sequencing. When possible, segregation of variants with phenotype in family members was performed to ascertain the pathogenicity of the variant. More than 97% of the target bases were covered at >20×. There was an average of 9.6 novel variants per patient. Pathogenic mutations were identified in five genes for six patients, with two novel variants. There were another five likely pathogenic variants, some of which were unreported novel variants. In a cohort of 15 patients, we were able to identify a likely genetic etiology in six patients (40%). Another five patients had candidate variants for which further evaluation and segregation analysis are ongoing. Our results indicate that the HaloPlex ICCG panel is useful as a rapid, high-throughput and cost-effective screening tool for 170 of the 180 genes. There is low coverage for some regions in several genes which might have to be supplemented by Sanger sequencing. However, comparing the cost, ease of analysis, and shorter turnaround time, it is a good alternative to exome sequencing for patients whose features are suggestive of a genetic etiology involving one of the genes in the panel.

Human papillomavirus type 16 variants in cervical intraepithelial neoplasia and invasive carcinoma in San Luis Potosí City, Mexico

PubMed Central

López-Revilla, Rubén; Pineda, Marco A; Ortiz-Valdez, Julio; Sánchez-Garza, Mireya; Riego, Lina

2009-01-01

Background In San Luis Potosí City cervical infection by human papillomavirus type 16 (HPV16) associated to dysplastic lesions is more prevalent in younger women. In this work HPV16 subtypes and variants associated to low-grade intraepithelial lesions (LSIL), high-grade intraepithelial lesions (HSIL) and invasive cervical cancer (ICC) of 38 women residing in San Luis Potosí City were identified by comparing their E6 open reading frame sequences. Results Three European (E) variants (E-P, n = 27; E-T350G, n = 7; E-C188G, n = 2) and one AA-a variant (n = 2) were identified among the 38 HPV16 sequences analyzed. E-P variant sequences contained 23 single nucleotide changes, two of which (A334G, A404T) had not been described before and allowed the phylogenetic separation from the other variants. E-P A334G sequences were the most prevalent (22 cases, 57.9%), followed by the E-P Ref prototype (8 cases, 21.1%) and E-P A404T (1 case, 2.6%) sequences. The HSIL + ICC fraction was 0.21 for the E-P A334G variants and 0.00 for the E-P Ref variants. Conclusion We conclude that in the women included in this study the HPV16 E subtype is 19 times more frequent than the AA subtype; that the circulating E variants are E-P (71.1%) > E-T350G (18.4%) > E-C188G (5.3%); that 71.0% of the E-P sequences carry the A334G single nucleotide change and appear to correspond to a HPV16 variant characteristic of San Luis Potosi City more oncogenic than the E-P Ref prototype. PMID:19216802

Imputation of Exome Sequence Variants into Population- Based Samples and Blood-Cell-Trait-Associated Loci in African Americans: NHLBI GO Exome Sequencing Project

PubMed Central

Auer, Paul L.; Johnsen, Jill M.; Johnson, Andrew D.; Logsdon, Benjamin A.; Lange, Leslie A.; Nalls, Michael A.; Zhang, Guosheng; Franceschini, Nora; Fox, Keolu; Lange, Ethan M.; Rich, Stephen S.; O’Donnell, Christopher J.; Jackson, Rebecca D.; Wallace, Robert B.; Chen, Zhao; Graubert, Timothy A.; Wilson, James G.; Tang, Hua; Lettre, Guillaume; Reiner, Alex P.; Ganesh, Santhi K.; Li, Yun

2012-01-01

Researchers have successfully applied exome sequencing to discover causal variants in selected individuals with familial, highly penetrant disorders. We demonstrate the utility of exome sequencing followed by imputation for discovering low-frequency variants associated with complex quantitative traits. We performed exome sequencing in a reference panel of 761 African Americans and then imputed newly discovered variants into a larger sample of more than 13,000 African Americans for association testing with the blood cell traits hemoglobin, hematocrit, white blood count, and platelet count. First, we illustrate the feasibility of our approach by demonstrating genome-wide-significant associations for variants that are not covered by conventional genotyping arrays; for example, one such association is that between higher platelet count and an MPL c.117G>T (p.Lys39Asn) variant encoding a p.Lys39Asn amino acid substitution of the thrombpoietin receptor gene (p = 1.5 × 10−11). Second, we identified an association between missense variants of LCT and higher white blood count (p = 4 × 10−13). Third, we identified low-frequency coding variants that might account for allelic heterogeneity at several known blood cell-associated loci: MPL c.754T>C (p.Tyr252His) was associated with higher platelet count; CD36 c.975T>G (p.Tyr325∗) was associated with lower platelet count; and several missense variants at the α-globin gene locus were associated with lower hemoglobin. By identifying low-frequency missense variants associated with blood cell traits not previously reported by genome-wide association studies, we establish that exome sequencing followed by imputation is a powerful approach to dissecting complex, genetically heterogeneous traits in large population-based studies. PMID:23103231

Identification of genetic risk factors in the Chinese population implicates a role of immune system in Alzheimer's disease pathogenesis.

PubMed

Zhou, Xiaopu; Chen, Yu; Mok, Kin Y; Zhao, Qianhua; Chen, Keliang; Chen, Yuewen; Hardy, John; Li, Yun; Fu, Amy K Y; Guo, Qihao; Ip, Nancy Y

2018-02-20

Alzheimer's disease (AD) is a leading cause of mortality among the elderly. We performed a whole-genome sequencing study of AD in the Chinese population. In addition to the variants identified in or around the APOE locus (sentinel variant rs73052335, P = 1.44 × 10 -14 ), two common variants, GCH1 (rs72713460, P = 4.36 × 10 -5 ) and KCNJ15 (rs928771, P = 3.60 × 10 -6 ), were identified and further verified for their possible risk effects for AD in three small non-Asian AD cohorts. Genotype-phenotype analysis showed that KCNJ15 variant rs928771 affects the onset age of AD, with earlier disease onset in minor allele carriers. In addition, altered expression level of the KCNJ15 transcript can be observed in the blood of AD subjects. Moreover, the risk variants of GCH1 and KCNJ15 are associated with changes in their transcript levels in specific tissues, as well as changes of plasma biomarkers levels in AD subjects. Importantly, network analysis of hippocampus and blood transcriptome datasets suggests that the risk variants in the APOE , GCH1 , and KCNJ15 loci might exert their functions through their regulatory effects on immune-related pathways. Taking these data together, we identified common variants of GCH1 and KCNJ15 in the Chinese population that contribute to AD risk. These variants may exert their functional effects through the immune system. Copyright © 2018 the Author(s). Published by PNAS.

Investigation of rare and low-frequency variants using high-throughput sequencing with pooled DNA samples

PubMed Central

Wang, Jingwen; Skoog, Tiina; Einarsdottir, Elisabet; Kaartokallio, Tea; Laivuori, Hannele; Grauers, Anna; Gerdhem, Paul; Hytönen, Marjo; Lohi, Hannes; Kere, Juha; Jiao, Hong

2016-01-01

High-throughput sequencing using pooled DNA samples can facilitate genome-wide studies on rare and low-frequency variants in a large population. Some major questions concerning the pooling sequencing strategy are whether rare and low-frequency variants can be detected reliably, and whether estimated minor allele frequencies (MAFs) can represent the actual values obtained from individually genotyped samples. In this study, we evaluated MAF estimates using three variant detection tools with two sets of pooled whole exome sequencing (WES) and one set of pooled whole genome sequencing (WGS) data. Both GATK and Freebayes displayed high sensitivity, specificity and accuracy when detecting rare or low-frequency variants. For the WGS study, 56% of the low-frequency variants in Illumina array have identical MAFs and 26% have one allele difference between sequencing and individual genotyping data. The MAF estimates from WGS correlated well (r = 0.94) with those from Illumina arrays. The MAFs from the pooled WES data also showed high concordance (r = 0.88) with those from the individual genotyping data. In conclusion, the MAFs estimated from pooled DNA sequencing data reflect the MAFs in individually genotyped samples well. The pooling strategy can thus be a rapid and cost-effective approach for the initial screening in large-scale association studies. PMID:27633116

Targeted Deep Resequencing Identifies Coding Variants in the PEAR1 Gene That Play a Role in Platelet Aggregation

PubMed Central

Kim, Yoonhee; Suktitipat, Bhoom; Yanek, Lisa R.; Faraday, Nauder; Wilson, Alexander F.; Becker, Diane M.; Becker, Lewis C.; Mathias, Rasika A.

2013-01-01

Platelet aggregation is heritable, and genome-wide association studies have detected strong associations with a common intronic variant of the platelet endothelial aggregation receptor1 (PEAR1) gene both in African American and European American individuals. In this study, we used a sequencing approach to identify additional exonic variants in PEAR1 that may also determine variability in platelet aggregation in the GeneSTAR Study. A 0.3 Mb targeted region on chromosome 1q23.1 including the entire PEAR1 gene was Sanger sequenced in 104 subjects (45% male, 49% African American, age = 52±13) selected on the basis of hyper- and hypo- aggregation across three different agonists (collagen, epinephrine, and adenosine diphosphate). Single-variant and multi-variant burden tests for association were performed. Of the 235 variants identified through sequencing, 61 were novel, and three of these were missense variants. More rare variants (MAF<5%) were noted in African Americans compared to European Americans (108 vs. 45). The common intronic GWAS-identified variant (rs12041331) demonstrated the most significant association signal in African Americans (p = 4.020×10−4); no association was seen for additional exonic variants in this group. In contrast, multi-variant burden tests indicated that exonic variants play a more significant role in European Americans (p = 0.0099 for the collective coding variants compared to p = 0.0565 for intronic variant rs12041331). Imputation of the individual exonic variants in the rest of the GeneSTAR European American cohort (N = 1,965) supports the results noted in the sequenced discovery sample: p = 3.56×10−4, 2.27×10−7, 5.20×10−5 for coding synonymous variant rs56260937 and collagen, epinephrine and adenosine diphosphate induced platelet aggregation, respectively. Sequencing approaches confirm that a common intronic variant has the strongest association with platelet aggregation in African Americans, and show that exonic variants play an additional role in platelet aggregation in European Americans. PMID:23704978

Exonic Splicing Mutations Are More Prevalent than Currently Estimated and Can Be Predicted by Using In Silico Tools

PubMed Central

Soukarieh, Omar; Gaildrat, Pascaline; Hamieh, Mohamad; Drouet, Aurélie; Baert-Desurmont, Stéphanie; Frébourg, Thierry; Tosi, Mario; Martins, Alexandra

2016-01-01

The identification of a causal mutation is essential for molecular diagnosis and clinical management of many genetic disorders. However, even if next-generation exome sequencing has greatly improved the detection of nucleotide changes, the biological interpretation of most exonic variants remains challenging. Moreover, particular attention is typically given to protein-coding changes often neglecting the potential impact of exonic variants on RNA splicing. Here, we used the exon 10 of MLH1, a gene implicated in hereditary cancer, as a model system to assess the prevalence of RNA splicing mutations among all single-nucleotide variants identified in a given exon. We performed comprehensive minigene assays and analyzed patient’s RNA when available. Our study revealed a staggering number of splicing mutations in MLH1 exon 10 (77% of the 22 analyzed variants), including mutations directly affecting splice sites and, particularly, mutations altering potential splicing regulatory elements (ESRs). We then used this thoroughly characterized dataset, together with experimental data derived from previous studies on BRCA1, BRCA2, CFTR and NF1, to evaluate the predictive power of 3 in silico approaches recently described as promising tools for pinpointing ESR-mutations. Our results indicate that ΔtESRseq and ΔHZEI-based approaches not only discriminate which variants affect splicing, but also predict the direction and severity of the induced splicing defects. In contrast, the ΔΨ-based approach did not show a compelling predictive power. Our data indicates that exonic splicing mutations are more prevalent than currently appreciated and that they can now be predicted by using bioinformatics methods. These findings have implications for all genetically-caused diseases. PMID:26761715

Uptake, Results, and Outcomes of Germline Multiple-Gene Sequencing After Diagnosis of Breast Cancer.

PubMed

Kurian, Allison W; Ward, Kevin C; Hamilton, Ann S; Deapen, Dennis M; Abrahamse, Paul; Bondarenko, Irina; Li, Yun; Hawley, Sarah T; Morrow, Monica; Jagsi, Reshma; Katz, Steven J

2018-05-10

Low-cost sequencing of multiple genes is increasingly available for cancer risk assessment. Little is known about uptake or outcomes of multiple-gene sequencing after breast cancer diagnosis in community practice. To examine the effect of multiple-gene sequencing on the experience and treatment outcomes for patients with breast cancer. For this population-based retrospective cohort study, patients with breast cancer diagnosed from January 2013 to December 2015 and accrued from SEER registries across Georgia and in Los Angeles, California, were surveyed (n = 5080, response rate = 70%). Responses were merged with SEER data and results of clinical genetic tests, either BRCA1 and BRCA2 (BRCA1/2) sequencing only or including additional other genes (multiple-gene sequencing), provided by 4 laboratories. Type of testing (multiple-gene sequencing vs BRCA1/2-only sequencing), test results (negative, variant of unknown significance, or pathogenic variant), patient experiences with testing (timing of testing, who discussed results), and treatment (strength of patient consideration of, and surgeon recommendation for, prophylactic mastectomy), and prophylactic mastectomy receipt. We defined a patient subgroup with higher pretest risk of carrying a pathogenic variant according to practice guidelines. Among 5026 patients (mean [SD] age, 59.9 [10.7]), 1316 (26.2%) were linked to genetic results from any laboratory. Multiple-gene sequencing increasingly replaced BRCA1/2-only testing over time: in 2013, the rate of multiple-gene sequencing was 25.6% and BRCA1/2-only testing, 74.4%;in 2015 the rate of multiple-gene sequencing was 66.5% and BRCA1/2-only testing, 33.5%. Multiple-gene sequencing was more often ordered by genetic counselors (multiple-gene sequencing, 25.5% and BRCA1/2-only testing, 15.3%) and delayed until after surgery (multiple-gene sequencing, 32.5% and BRCA1/2-only testing, 19.9%). Multiple-gene sequencing substantially increased rate of detection of any pathogenic variant (multiple-gene sequencing: higher-risk patients, 12%; average-risk patients, 4.2% and BRCA1/2-only testing: higher-risk patients, 7.8%; average-risk patients, 2.2%) and variants of uncertain significance, especially in minorities (multiple-gene sequencing: white patients, 23.7%; black patients, 44.5%; and Asian patients, 50.9% and BRCA1/2-only testing: white patients, 2.2%; black patients, 5.6%; and Asian patients, 0%). Multiple-gene sequencing was not associated with an increase in the rate of prophylactic mastectomy use, which was highest with pathogenic variants in BRCA1/2 (BRCA1/2, 79.0%; other pathogenic variant, 37.6%; variant of uncertain significance, 30.2%; negative, 35.3%). Multiple-gene sequencing rapidly replaced BRCA1/2-only testing for patients with breast cancer in the community and enabled 2-fold higher detection of clinically relevant pathogenic variants without an associated increase in prophylactic mastectomy. However, important targets for improvement in the clinical utility of multiple-gene sequencing include postsurgical delay and racial/ethnic disparity in variants of uncertain significance.

Systematic comparison of variant calling pipelines using gold standard personal exome variants

PubMed Central

Hwang, Sohyun; Kim, Eiru; Lee, Insuk; Marcotte, Edward M.

2015-01-01

The success of clinical genomics using next generation sequencing (NGS) requires the accurate and consistent identification of personal genome variants. Assorted variant calling methods have been developed, which show low concordance between their calls. Hence, a systematic comparison of the variant callers could give important guidance to NGS-based clinical genomics. Recently, a set of high-confident variant calls for one individual (NA12878) has been published by the Genome in a Bottle (GIAB) consortium, enabling performance benchmarking of different variant calling pipelines. Based on the gold standard reference variant calls from GIAB, we compared the performance of thirteen variant calling pipelines, testing combinations of three read aligners—BWA-MEM, Bowtie2, and Novoalign—and four variant callers—Genome Analysis Tool Kit HaplotypeCaller (GATK-HC), Samtools mpileup, Freebayes and Ion Proton Variant Caller (TVC), for twelve data sets for the NA12878 genome sequenced by different platforms including Illumina2000, Illumina2500, and Ion Proton, with various exome capture systems and exome coverage. We observed different biases toward specific types of SNP genotyping errors by the different variant callers. The results of our study provide useful guidelines for reliable variant identification from deep sequencing of personal genomes. PMID:26639839

Genetic Relatedness among Hepatitis A Virus Strains Associated with Food-Borne Outbreaks

PubMed Central

Vaughan, Gilberto; Xia, Guoliang; Forbi, Joseph C.; Purdy, Michael A.; Rossi, Lívia Maria Gonçalves; Spradling, Philip R.; Khudyakov, Yury E.

2013-01-01

The genetic characterization of hepatitis A virus (HAV) strains is commonly accomplished by sequencing subgenomic regions, such as the VP1/P2B junction. HAV genome is not extensively variable, thus presenting opportunity for sharing sequences of subgenomic regions among genetically unrelated isolates. The degree of misrepresentation of phylogenetic relationships by subgenomic regions is especially important for tracking transmissions. Here, we analyzed whole-genome (WG) sequences of 101 HAV strains identified from 4 major multi-state, food-borne outbreaks of hepatitis A in the Unites States and from 14 non-outbreak-related HAV strains that shared identical VP1/P2B sequences with the outbreak strains. Although HAV strains with an identical VP1/P2B sequence were specific to each outbreak, WG were different, with genetic diversity reaching 0.31% (mean 0.09%). Evaluation of different subgenomic regions did not identify any other section of the HAV genome that could accurately represent phylogenetic relationships observed using WG sequences. The identification of 2–3 dominant HAV strains in 3 out of 4 outbreaks indicates contamination of the implicated food items with a heterogeneous HAV population. However, analysis of intra-host HAV variants from eight patients involved in one outbreak showed that only a single sequence variant established infection in each patient. Four non-outbreak strains were found closely related to strains from 2 outbreaks, whereas ten were genetically different from the outbreak strains. Thus, accurate tracking of HAV strains can be accomplished using HAV WG sequences, while short subgenomic regions are useful for identification of transmissions only among cases with known epidemiological association. PMID:24223112

Preconception Carrier Screening by Genome Sequencing: Results from the Clinical Laboratory.

PubMed

Punj, Sumit; Akkari, Yassmine; Huang, Jennifer; Yang, Fei; Creason, Allison; Pak, Christine; Potter, Amiee; Dorschner, Michael O; Nickerson, Deborah A; Robertson, Peggy D; Jarvik, Gail P; Amendola, Laura M; Schleit, Jennifer; Simpson, Dana Kostiner; Rope, Alan F; Reiss, Jacob; Kauffman, Tia; Gilmore, Marian J; Himes, Patricia; Wilfond, Benjamin; Goddard, Katrina A B; Richards, C Sue

2018-06-07

Advances in sequencing technologies permit the analysis of a larger selection of genes for preconception carrier screening. The study was designed as a sequential carrier screen using genome sequencing to analyze 728 gene-disorder pairs for carrier and medically actionable conditions in 131 women and their partners (n = 71) who were planning a pregnancy. We report here on the clinical laboratory results from this expanded carrier screening program. Variants were filtered and classified using the latest American College of Medical Genetics and Genomics (ACMG) guideline; only pathogenic and likely pathogenic variants were confirmed by orthologous methods before being reported. Novel missense variants were classified as variants of uncertain significance. We reported 304 variants in 202 participants. Twelve carrier couples (12/71 couples tested) were identified for common conditions; eight were carriers for hereditary hemochromatosis. Although both known and novel variants were reported, 48% of all reported variants were missense. For novel splice-site variants, RNA-splicing assays were performed to aid in classification. We reported ten copy-number variants and five variants in non-coding regions. One novel variant was reported in F8, associated with hemophilia A; prenatal testing showed that the male fetus harbored this variant and the neonate suffered a life-threatening hemorrhage which was anticipated and appropriately managed. Moreover, 3% of participants had variants that were medically actionable. Compared with targeted mutation screening, genome sequencing improves the sensitivity of detecting clinically significant variants. While certain novel variant interpretation remains challenging, the ACMG guidelines are useful to classify variants in a healthy population. Copyright © 2018 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

India Allele Finder: a web-based annotation tool for identifying common alleles in next-generation sequencing data of Indian origin.

PubMed

Zhang, Jimmy F; James, Francis; Shukla, Anju; Girisha, Katta M; Paciorkowski, Alex R

2017-06-27

We built India Allele Finder, an online searchable database and command line tool, that gives researchers access to variant frequencies of Indian Telugu individuals, using publicly available fastq data from the 1000 Genomes Project. Access to appropriate population-based genomic variant annotation can accelerate the interpretation of genomic sequencing data. In particular, exome analysis of individuals of Indian descent will identify population variants not reflected in European exomes, complicating genomic analysis for such individuals. India Allele Finder offers improved ease-of-use to investigators seeking to identify and annotate sequencing data from Indian populations. We describe the use of India Allele Finder to identify common population variants in a disease quartet whole exome dataset, reducing the number of candidate single nucleotide variants from 84 to 7. India Allele Finder is freely available to investigators to annotate genomic sequencing data from Indian populations. Use of India Allele Finder allows efficient identification of population variants in genomic sequencing data, and is an example of a population-specific annotation tool that simplifies analysis and encourages international collaboration in genomics research.

Population genetic implications from sequence variation in four Y chromosome genes.

PubMed

Shen, P; Wang, F; Underhill, P A; Franco, C; Yang, W H; Roxas, A; Sung, R; Lin, A A; Hyman, R W; Vollrath, D; Davis, R W; Cavalli-Sforza, L L; Oefner, P J

2000-06-20

Some insight into human evolution has been gained from the sequencing of four Y chromosome genes. Primary genomic sequencing determined gene SMCY to be composed of 27 exons that comprise 4,620 bp of coding sequence. The unfinished sequencing of the 5' portion of gene UTY1 was completed by primer walking, and a total of 20 exons were found. By using denaturing HPLC, these two genes, as well as DBY and DFFRY, were screened for polymorphic sites in 53-72 representatives of the five continents. A total of 98 variants were found, yielding nucleotide diversity estimates of 2.45 x 10(-5), 5. 07 x 10(-5), and 8.54 x 10(-5) for the coding regions of SMCY, DFFRY, and UTY1, respectively, with no variant having been observed in DBY. In agreement with most autosomal genes, diversity estimates for the noncoding regions were about 2- to 3-fold higher and ranged from 9. 16 x 10(-5) to 14.2 x 10(-5) for the four genes. Analysis of the frequencies of derived alleles for all four genes showed that they more closely fit the expectation of a Luria-Delbrück distribution than a distribution expected under a constant population size model, providing evidence for exponential population growth. Pairwise nucleotide mismatch distributions date the occurrence of population expansion to approximately 28,000 years ago. This estimate is in accord with the spread of Aurignacian technology and the disappearance of the Neanderthals.

ABC Assay: Method Development and Application to Quantify the Role of Three DWV Master Variants in Overwinter Colony Losses of European Honey Bees

PubMed Central

Kevill, Jessica L.; Highfield, Andrea; Mordecai, Gideon J.; Schroeder, Declan C.

2017-01-01

Deformed wing virus (DWV) is one of the most prevalent honey bee viral pathogens in the world. Typical of many RNA viruses, DWV is a quasi-species, which is comprised of a large number of different variants, currently consisting of three master variants: Type A, B, and C. Little is known about the impact of each variant or combinations of variants upon the biology of individual hosts. Therefore, we have developed a new set of master variant-specific DWV primers and a set of standards that allow for the quantification of each of the master variants. Competitive reverse transcriptase polymerase chain reaction (RT-PCR) experimental design confirms that each new DWV primer set is specific to the retrospective master variant. The sensitivity of the ABC assay is dependent on whether DNA or RNA is used as the template and whether other master variants are present in the sample. Comparison of the overall proportions of each master variant within a sample of known diversity, as confirmed by next-generation sequence (NGS) data, validates the efficiency of the ABC assay. The ABC assay was used on archived material from a Devon overwintering colony loss (OCL) 2006–2007 study; further implicating DWV type A and, for the first time, possibly C in the untimely collapse of honey bee colonies. Moreover, in this study DWV type B was not associated with OCL. The use of the ABC assay will allow researchers to quickly and cost effectively pre-screen for the presence of DWV master variants in honey bees. PMID:29077069

Simple and efficient identification of rare recessive pathologically important sequence variants from next generation exome sequence data.

PubMed

Carr, Ian M; Morgan, Joanne; Watson, Christopher; Melnik, Svitlana; Diggle, Christine P; Logan, Clare V; Harrison, Sally M; Taylor, Graham R; Pena, Sergio D J; Markham, Alexander F; Alkuraya, Fowzan S; Black, Graeme C M; Ali, Manir; Bonthron, David T

2013-07-01

Massively parallel ("next generation") DNA sequencing (NGS) has quickly become the method of choice for seeking pathogenic mutations in rare uncharacterized monogenic diseases. Typically, before DNA sequencing, protein-coding regions are enriched from patient genomic DNA, representing either the entire genome ("exome sequencing") or selected mapped candidate loci. Sequence variants, identified as differences between the patient's and the human genome reference sequences, are then filtered according to various quality parameters. Changes are screened against datasets of known polymorphisms, such as dbSNP and the 1000 Genomes Project, in the effort to narrow the list of candidate causative variants. An increasing number of commercial services now offer to both generate and align NGS data to a reference genome. This potentially allows small groups with limited computing infrastructure and informatics skills to utilize this technology. However, the capability to effectively filter and assess sequence variants is still an important bottleneck in the identification of deleterious sequence variants in both research and diagnostic settings. We have developed an approach to this problem comprising a user-friendly suite of programs that can interactively analyze, filter and screen data from enrichment-capture NGS data. These programs ("Agile Suite") are particularly suitable for small-scale gene discovery or for diagnostic analysis. © 2013 WILEY PERIODICALS, INC.

Regularized rare variant enrichment analysis for case-control exome sequencing data.

PubMed

Larson, Nicholas B; Schaid, Daniel J

2014-02-01

Rare variants have recently garnered an immense amount of attention in genetic association analysis. However, unlike methods traditionally used for single marker analysis in GWAS, rare variant analysis often requires some method of aggregation, since single marker approaches are poorly powered for typical sequencing study sample sizes. Advancements in sequencing technologies have rendered next-generation sequencing platforms a realistic alternative to traditional genotyping arrays. Exome sequencing in particular not only provides base-level resolution of genetic coding regions, but also a natural paradigm for aggregation via genes and exons. Here, we propose the use of penalized regression in combination with variant aggregation measures to identify rare variant enrichment in exome sequencing data. In contrast to marginal gene-level testing, we simultaneously evaluate the effects of rare variants in multiple genes, focusing on gene-based least absolute shrinkage and selection operator (LASSO) and exon-based sparse group LASSO models. By using gene membership as a grouping variable, the sparse group LASSO can be used as a gene-centric analysis of rare variants while also providing a penalized approach toward identifying specific regions of interest. We apply extensive simulations to evaluate the performance of these approaches with respect to specificity and sensitivity, comparing these results to multiple competing marginal testing methods. Finally, we discuss our findings and outline future research. © 2013 WILEY PERIODICALS, INC.

Inner retinal dystrophy in a patient with biallelic sequence variants in BRAT1.

PubMed

Oatts, Julius T; Duncan, Jacque L; Hoyt, Creig S; Slavotinek, Anne M; Moore, Anthony T

2017-12-01

Mutations in the BRCA1-associated protein required for the ataxia telangiectasia mutated (ATM) activation-1 (BRAT1) gene cause lethal neonatal rigidity and multifocal seizure syndrome characterized by rigidity and intractable seizures and a milder phenotype with intellectual disability, seizures, nonprogressive cerebellar ataxia or dyspraxia, and cerebellar atrophy. To date, nystagmus, cortical visual impairment, impairment of central vision, optic nerve hypoplasia, and optic atrophy have been described in this condition. This article describes the retinal findings in a patient with biallelic deleterious sequence variants in BRAT1. Case report of a child with biallelic sequence variants in the BRAT1 gene. This patient had developmental delay, microcephaly, nystagmus, and esotropia, and full-field electroretinography (ERG) revealed an inner retinal dystrophy. She was found on exome sequencing to have compound heterozygous sequence variants in the BRAT1 gene: one maternally inherited frameshift variant (c.294dupA, predicting p.Leu99Thrfs*92), which has previously been reported, and one paternally inherited novel missense variant (c.803G>A, p.Arg268His), which is likely to affect protein function. Biallelic sequence variants in BRAT1 have been reported to cause a variety of ocular and systemic manifestations, but to our knowledge, this is the first report of inner retinal dysfunction manifest as selective loss of full-field ERG scotopic and photopic b-wave amplitudes.

Amyotrophic lateral sclerosis onset is influenced by the burden of rare variants in known amyotrophic lateral sclerosis genes.

PubMed

Cady, Janet; Allred, Peggy; Bali, Taha; Pestronk, Alan; Goate, Alison; Miller, Timothy M; Mitra, Robi D; Ravits, John; Harms, Matthew B; Baloh, Robert H

2015-01-01

To define the genetic landscape of amyotrophic lateral sclerosis (ALS) and assess the contribution of possible oligogenic inheritance, we aimed to comprehensively sequence 17 known ALS genes in 391 ALS patients from the United States. Targeted pooled-sample sequencing was used to identify variants in 17 ALS genes. Fragment size analysis was used to define ATXN2 and C9ORF72 expansion sizes. Genotype-phenotype correlations were made with individual variants and total burden of variants. Rare variant associations for risk of ALS were investigated at both the single variant and gene level. A total of 64.3% of familial and 27.8% of sporadic subjects carried potentially pathogenic novel or rare coding variants identified by sequencing or an expanded repeat in C9ORF72 or ATXN2; 3.8% of subjects had variants in >1 ALS gene, and these individuals had disease onset 10 years earlier (p = 0.0046) than subjects with variants in a single gene. The number of potentially pathogenic coding variants did not influence disease duration or site of onset. Rare and potentially pathogenic variants in known ALS genes are present in >25% of apparently sporadic and 64% of familial patients, significantly higher than previous reports using less comprehensive sequencing approaches. A significant number of subjects carried variants in >1 gene, which influenced the age of symptom onset and supports oligogenic inheritance as relevant to disease pathogenesis. © 2014 American Neurological Association.

Gene Expression Elucidates Functional Impact of Polygenic Risk for Schizophrenia

PubMed Central

Fromer, Menachem; Roussos, Panos; Sieberts, Solveig K; Johnson, Jessica S; Kavanagh, David H; Perumal, Thanneer M; Ruderfer, Douglas M; Oh, Edwin C; Topol, Aaron; Shah, Hardik R; Klei, Lambertus L; Kramer, Robin; Pinto, Dalila; Gümüş, Zeynep H; Cicek, A. Ercument; Dang, Kristen K; Browne, Andrew; Lu, Cong; Xie, Lu; Readhead, Ben; Stahl, Eli A; Parvizi, Mahsa; Hamamsy, Tymor; Fullard, John F; Wang, Ying-Chih; Mahajan, Milind C; Derry, Jonathan M J; Dudley, Joel; Hemby, Scott E; Logsdon, Benjamin A; Talbot, Konrad; Raj, Towfique; Bennett, David A; De Jager, Philip L; Zhu, Jun; Zhang, Bin; Sullivan, Patrick F; Chess, Andrew; Purcell, Shaun M; Shinobu, Leslie A; Mangravite, Lara M; Toyoshiba, Hiroyoshi; Gur, Raquel E; Hahn, Chang-Gyu; Lewis, David A; Haroutunian, Vahram; Peters, Mette A; Lipska, Barbara K; Buxbaum, Joseph D; Schadt, Eric E; Hirai, Keisuke; Roeder, Kathryn; Brennand, Kristen J; Katsanis, Nicholas; Domenici, Enrico; Devlin, Bernie; Sklar, Pamela

2016-01-01

Over 100 genetic loci harbor schizophrenia associated variants, yet how these variants confer liability is uncertain. The CommonMind Consortium sequenced RNA from dorsolateral prefrontal cortex of schizophrenia cases (N = 258) and control subjects (N = 279), creating a resource of gene expression and its genetic regulation. Using this resource, ~20% of schizophrenia loci have variants that could contribute to altered gene expression and liability. In five loci, only a single gene was involved: FURIN, TSNARE1, CNTN4, CLCN3, or SNAP91. Altering expression of FURIN, TSNARE1, or CNTN4 changes neurodevelopment in zebrafish; knockdown of FURIN in human neural progenitor cells yields abnormal migration. Of 693 genes showing significant case/control differential expression, their fold changes are ≤ 1.33, and an independent cohort yields similar results. Gene co-expression implicates a network relevant for schizophrenia. Our findings show schizophrenia is polygenic and highlight the utility of this resource for mechanistic interpretations of genetic liability for brain diseases. PMID:27668389

Gene expression elucidates functional impact of polygenic risk for schizophrenia.

PubMed

Fromer, Menachem; Roussos, Panos; Sieberts, Solveig K; Johnson, Jessica S; Kavanagh, David H; Perumal, Thanneer M; Ruderfer, Douglas M; Oh, Edwin C; Topol, Aaron; Shah, Hardik R; Klei, Lambertus L; Kramer, Robin; Pinto, Dalila; Gümüş, Zeynep H; Cicek, A Ercument; Dang, Kristen K; Browne, Andrew; Lu, Cong; Xie, Lu; Readhead, Ben; Stahl, Eli A; Xiao, Jianqiu; Parvizi, Mahsa; Hamamsy, Tymor; Fullard, John F; Wang, Ying-Chih; Mahajan, Milind C; Derry, Jonathan M J; Dudley, Joel T; Hemby, Scott E; Logsdon, Benjamin A; Talbot, Konrad; Raj, Towfique; Bennett, David A; De Jager, Philip L; Zhu, Jun; Zhang, Bin; Sullivan, Patrick F; Chess, Andrew; Purcell, Shaun M; Shinobu, Leslie A; Mangravite, Lara M; Toyoshiba, Hiroyoshi; Gur, Raquel E; Hahn, Chang-Gyu; Lewis, David A; Haroutunian, Vahram; Peters, Mette A; Lipska, Barbara K; Buxbaum, Joseph D; Schadt, Eric E; Hirai, Keisuke; Roeder, Kathryn; Brennand, Kristen J; Katsanis, Nicholas; Domenici, Enrico; Devlin, Bernie; Sklar, Pamela

2016-11-01

Over 100 genetic loci harbor schizophrenia-associated variants, yet how these variants confer liability is uncertain. The CommonMind Consortium sequenced RNA from dorsolateral prefrontal cortex of people with schizophrenia (N = 258) and control subjects (N = 279), creating a resource of gene expression and its genetic regulation. Using this resource, ∼20% of schizophrenia loci have variants that could contribute to altered gene expression and liability. In five loci, only a single gene was involved: FURIN, TSNARE1, CNTN4, CLCN3 or SNAP91. Altering expression of FURIN, TSNARE1 or CNTN4 changed neurodevelopment in zebrafish; knockdown of FURIN in human neural progenitor cells yielded abnormal migration. Of 693 genes showing significant case-versus-control differential expression, their fold changes were ≤ 1.33, and an independent cohort yielded similar results. Gene co-expression implicates a network relevant for schizophrenia. Our findings show that schizophrenia is polygenic and highlight the utility of this resource for mechanistic interpretations of genetic liability for brain diseases.

Comparison of Ion Personal Genome Machine Platforms for the Detection of Variants in BRCA1 and BRCA2.

PubMed

Hwang, Sang Mee; Lee, Ki Chan; Lee, Min Seob; Park, Kyoung Un

2018-01-01

Transition to next generation sequencing (NGS) for BRCA1 / BRCA2 analysis in clinical laboratories is ongoing but different platforms and/or data analysis pipelines give different results resulting in difficulties in implementation. We have evaluated the Ion Personal Genome Machine (PGM) Platforms (Ion PGM, Ion PGM Dx, Thermo Fisher Scientific) for the analysis of BRCA1 /2. The results of Ion PGM with OTG-snpcaller, a pipeline based on Torrent mapping alignment program and Genome Analysis Toolkit, from 75 clinical samples and 14 reference DNA samples were compared with Sanger sequencing for BRCA1 / BRCA2 . Ten clinical samples and 14 reference DNA samples were additionally sequenced by Ion PGM Dx with Torrent Suite. Fifty types of variants including 18 pathogenic or variants of unknown significance were identified from 75 clinical samples and known variants of the reference samples were confirmed by Sanger sequencing and/or NGS. One false-negative results were present for Ion PGM/OTG-snpcaller for an indel variant misidentified as a single nucleotide variant. However, eight discordant results were present for Ion PGM Dx/Torrent Suite with both false-positive and -negative results. A 40-bp deletion, a 4-bp deletion and a 1-bp deletion variant was not called and a false-positive deletion was identified. Four other variants were misidentified as another variant. Ion PGM/OTG-snpcaller showed acceptable performance with good concordance with Sanger sequencing. However, Ion PGM Dx/Torrent Suite showed many discrepant results not suitable for use in a clinical laboratory, requiring further optimization of the data analysis for calling variants.

Human papillomavirus type 18 variant lineages in United States populations characterized by sequence analysis of LCR-E6, E2, and L1 regions.

PubMed

Arias-Pulido, Hugo; Peyton, Cheri L; Torrez-Martínez, Norah; Anderson, D Nelson; Wheeler, Cosette M

2005-07-20

While HPV 16 variant lineages have been well characterized, the knowledge about HPV 18 variants is limited. In this study, HPV 18 nucleotide variations in the E2 hinge region were characterized by sequence analysis in 47 control and 51 tumor specimens. Fifty of these specimens were randomly selected for sequencing of an LCR-E6 segment and 20 samples representative of LCR-E6 and E2 sequence variants were examined across the L1 region. A total of 2770 nucleotides per HPV 18 variant genome were considered in this study. HPV 18 variant nucleotides were linked among all gene segments analyzed and grouped into three main branches: Asian-American (AA), European (E), and African (Af). These three branches were equally distributed among controls and cases and when stratified by Hispanic and non-Hispanic ethnicities. Among invasive cervical cancer cases, no significant differences in the three HPV variant branches were observed among ethnic groups or when stratified by histopathology (squamous vs. adenocarcinoma). The Af branch showed the greatest nucleotide variability when compared to the HPV 18 reference sequence and was more closely related to HPV 45 than either AA or E branches. Our data also characterize nucleotide and amino acid variations in the L1 capsid gene among HPV 18 variants, which may be relevant to vaccine strategies and subsequent studies of naturally occurring HPV 18 variants. Several novel HPV 18 nucleotide variations were identified in this study.

VarBin, a novel method for classifying true and false positive variants in NGS data

PubMed Central

2013-01-01

Background Variant discovery for rare genetic diseases using Illumina genome or exome sequencing involves screening of up to millions of variants to find only the one or few causative variant(s). Sequencing or alignment errors create "false positive" variants, which are often retained in the variant screening process. Methods to remove false positive variants often retain many false positive variants. This report presents VarBin, a method to prioritize variants based on a false positive variant likelihood prediction. Methods VarBin uses the Genome Analysis Toolkit variant calling software to calculate the variant-to-wild type genotype likelihood ratio at each variant change and position divided by read depth. The resulting Phred-scaled, likelihood-ratio by depth (PLRD) was used to segregate variants into 4 Bins with Bin 1 variants most likely true and Bin 4 most likely false positive. PLRD values were calculated for a proband of interest and 41 additional Illumina HiSeq, exome and whole genome samples (proband's family or unrelated samples). At variant sites without apparent sequencing or alignment error, wild type/non-variant calls cluster near -3 PLRD and variant calls typically cluster above 10 PLRD. Sites with systematic variant calling problems (evident by variant quality scores and biases as well as displayed on the iGV viewer) tend to have higher and more variable wild type/non-variant PLRD values. Depending on the separation of a proband's variant PLRD value from the cluster of wild type/non-variant PLRD values for background samples at the same variant change and position, the VarBin method's classification is assigned to each proband variant (Bin 1 to Bin 4). Results To assess VarBin performance, Sanger sequencing was performed on 98 variants in the proband and background samples. True variants were confirmed in 97% of Bin 1 variants, 30% of Bin 2, and 0% of Bin 3/Bin 4. Conclusions These data indicate that VarBin correctly classifies the majority of true variants as Bin 1 and Bin 3/4 contained only false positive variants. The "uncertain" Bin 2 contained both true and false positive variants. Future work will further differentiate the variants in Bin 2. PMID:24266885

Unlocking hidden genomic sequence

PubMed Central

Keith, Jonathan M.; Cochran, Duncan A. E.; Lala, Gita H.; Adams, Peter; Bryant, Darryn; Mitchelson, Keith R.

2004-01-01

Despite the success of conventional Sanger sequencing, significant regions of many genomes still present major obstacles to sequencing. Here we propose a novel approach with the potential to alleviate a wide range of sequencing difficulties. The technique involves extracting target DNA sequence from variants generated by introduction of random mutations. The introduction of mutations does not destroy original sequence information, but distributes it amongst multiple variants. Some of these variants lack problematic features of the target and are more amenable to conventional sequencing. The technique has been successfully demonstrated with mutation levels up to an average 18% base substitution and has been used to read previously intractable poly(A), AT-rich and GC-rich motifs. PMID:14973330

Identification of rare X-linked neuroligin variants by massively parallel sequencing in males with autism spectrum disorder.

PubMed

Steinberg, Karyn Meltz; Ramachandran, Dhanya; Patel, Viren C; Shetty, Amol C; Cutler, David J; Zwick, Michael E

2012-09-28

Autism spectrum disorder (ASD) is highly heritable, but the genetic risk factors for it remain largely unknown. Although structural variants with large effect sizes may explain up to 15% ASD, genome-wide association studies have failed to uncover common single nucleotide variants with large effects on phenotype. The focus within ASD genetics is now shifting to the examination of rare sequence variants of modest effect, which is most often achieved via exome selection and sequencing. This strategy has indeed identified some rare candidate variants; however, the approach does not capture the full spectrum of genetic variation that might contribute to the phenotype. We surveyed two loci with known rare variants that contribute to ASD, the X-linked neuroligin genes by performing massively parallel Illumina sequencing of the coding and noncoding regions from these genes in males from families with multiplex autism. We annotated all variant sites and functionally tested a subset to identify other rare mutations contributing to ASD susceptibility. We found seven rare variants at evolutionary conserved sites in our study population. Functional analyses of the three 3' UTR variants did not show statistically significant effects on the expression of NLGN3 and NLGN4X. In addition, we identified two NLGN3 intronic variants located within conserved transcription factor binding sites that could potentially affect gene regulation. These data demonstrate the power of massively parallel, targeted sequencing studies of affected individuals for identifying rare, potentially disease-contributing variation. However, they also point out the challenges and limitations of current methods of direct functional testing of rare variants and the difficulties of identifying alleles with modest effects.

Identification of rare X-linked neuroligin variants by massively parallel sequencing in males with autism spectrum disorder

PubMed Central

2012-01-01

Background Autism spectrum disorder (ASD) is highly heritable, but the genetic risk factors for it remain largely unknown. Although structural variants with large effect sizes may explain up to 15% ASD, genome-wide association studies have failed to uncover common single nucleotide variants with large effects on phenotype. The focus within ASD genetics is now shifting to the examination of rare sequence variants of modest effect, which is most often achieved via exome selection and sequencing. This strategy has indeed identified some rare candidate variants; however, the approach does not capture the full spectrum of genetic variation that might contribute to the phenotype. Methods We surveyed two loci with known rare variants that contribute to ASD, the X-linked neuroligin genes by performing massively parallel Illumina sequencing of the coding and noncoding regions from these genes in males from families with multiplex autism. We annotated all variant sites and functionally tested a subset to identify other rare mutations contributing to ASD susceptibility. Results We found seven rare variants at evolutionary conserved sites in our study population. Functional analyses of the three 3’ UTR variants did not show statistically significant effects on the expression of NLGN3 and NLGN4X. In addition, we identified two NLGN3 intronic variants located within conserved transcription factor binding sites that could potentially affect gene regulation. Conclusions These data demonstrate the power of massively parallel, targeted sequencing studies of affected individuals for identifying rare, potentially disease-contributing variation. However, they also point out the challenges and limitations of current methods of direct functional testing of rare variants and the difficulties of identifying alleles with modest effects. PMID:23020841

Exome Pool-Seq in neurodevelopmental disorders.

PubMed

Popp, Bernt; Ekici, Arif B; Thiel, Christian T; Hoyer, Juliane; Wiesener, Antje; Kraus, Cornelia; Reis, André; Zweier, Christiane

2017-12-01

High throughput sequencing has greatly advanced disease gene identification, especially in heterogeneous entities. Despite falling costs this is still an expensive and laborious technique, particularly when studying large cohorts. To address this problem we applied Exome Pool-Seq as an economic and fast screening technology in neurodevelopmental disorders (NDDs). Sequencing of 96 individuals can be performed in eight pools of 12 samples on less than one Illumina sequencer lane. In a pilot study with 96 cases we identified 27 variants, likely or possibly affecting function. Twenty five of these were identified in 923 established NDD genes (based on SysID database, status November 2016) (ACTB, AHDC1, ANKRD11, ATP6V1B2, ATRX, CASK, CHD8, GNAS, IFIH1, KCNQ2, KMT2A, KRAS, MAOA, MED12, MED13L, RIT1, SETD5, SIN3A, TCF4, TRAPPC11, TUBA1A, WAC, ZBTB18, ZMYND11), two in 543 (SysID) candidate genes (ZNF292, BPTF), and additionally a de novo loss-of-function variant in LRRC7, not previously implicated in NDDs. Most of them were confirmed to be de novo, but we also identified X-linked or autosomal-dominantly or autosomal-recessively inherited variants. With a detection rate of 28%, Exome Pool-Seq achieves comparable results to individual exome analyses but reduces costs by >85%. Compared with other large scale approaches using Molecular Inversion Probes (MIP) or gene panels, it allows flexible re-analysis of data. Exome Pool-Seq is thus well suited for large-scale, cost-efficient and flexible screening in characterized but heterogeneous entities like NDDs.

Extensive Within-Host Diversity in Fecally Carried Extended-Spectrum-Beta-Lactamase-Producing Escherichia coli Isolates: Implications for Transmission Analyses.

PubMed

Stoesser, N; Sheppard, A E; Moore, C E; Golubchik, T; Parry, C M; Nget, P; Saroeun, M; Day, N P J; Giess, A; Johnson, J R; Peto, T E A; Crook, D W; Walker, A S

2015-07-01

Studies of the transmission epidemiology of antimicrobial-resistant Escherichia coli, such as strains harboring extended-spectrum beta-lactamase (ESBL) genes, frequently use selective culture of rectal surveillance swabs to identify isolates for molecular epidemiological investigation. Typically, only single colonies are evaluated, which risks underestimating species diversity and transmission events. We sequenced the genomes of 16 E. coli colonies from each of eight fecal samples (n = 127 genomes; one failure), taken from different individuals in Cambodia, a region of high ESBL-producing E. coli prevalence. Sequence data were used to characterize both the core chromosomal diversity of E. coli isolates and their resistance/virulence gene content as a proxy measure of accessory genome diversity. The 127 E. coli genomes represented 31 distinct sequence types (STs). Seven (88%) of eight subjects carried ESBL-positive isolates, all containing blaCTX-M variants. Diversity was substantial, with a median of four STs/individual (range, 1 to 10) and wide genetic divergence at the nucleotide level within some STs. In 2/8 (25%) individuals, the same blaCTX-M variant occurred in different clones, and/or different blaCTX-M variants occurred in the same clone. Patterns of other resistance genes and common virulence factors, representing differences in the accessory genome, were also diverse within and between clones. The substantial diversity among intestinally carried ESBL-positive E. coli bacteria suggests that fecal surveillance, particularly if based on single-colony subcultures, will likely underestimate transmission events, especially in high-prevalence settings. Copyright © 2015, Stoesser et al.

Genotype-specific signal generation based on digestion of 3-way DNA junctions: application to KRAS variation detection.

PubMed

Amicarelli, Giulia; Adlerstein, Daniel; Shehi, Erlet; Wang, Fengfei; Makrigiorgos, G Mike

2006-10-01

Genotyping methods that reveal single-nucleotide differences are useful for a wide range of applications. We used digestion of 3-way DNA junctions in a novel technology, OneCutEventAmplificatioN (OCEAN) that allows sequence-specific signal generation and amplification. We combined OCEAN with peptide-nucleic-acid (PNA)-based variant enrichment to detect and simultaneously genotype v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) codon 12 sequence variants in human tissue specimens. We analyzed KRAS codon 12 sequence variants in 106 lung cancer surgical specimens. We conducted a PNA-PCR reaction that suppresses wild-type KRAS amplification and genotyped the product with a set of OCEAN reactions carried out in fluorescence microplate format. The isothermal OCEAN assay enabled a 3-way DNA junction to form between the specific target nucleic acid, a fluorescently labeled "amplifier", and an "anchor". The amplifier-anchor contact contains the recognition site for a restriction enzyme. Digestion produces a cleaved amplifier and generation of a fluorescent signal. The cleaved amplifier dissociates from the 3-way DNA junction, allowing a new amplifier to bind and propagate the reaction. The system detected and genotyped KRAS sequence variants down to approximately 0.3% variant-to-wild-type alleles. PNA-PCR/OCEAN had a concordance rate with PNA-PCR/sequencing of 93% to 98%, depending on the exact implementation. Concordance rate with restriction endonuclease-mediated selective-PCR/sequencing was 89%. OCEAN is a practical and low-cost novel technology for sequence-specific signal generation. Reliable analysis of KRAS sequence alterations in human specimens circumvents the requirement for sequencing. Application is expected in genotyping KRAS codon 12 sequence variants in surgical specimens or in bodily fluids, as well as single-base variations and sequence alterations in other genes.

Network Analysis of Sequence-Function Relationships and Exploration of Sequence Space of TEM β-Lactamases.

PubMed

Zeil, Catharina; Widmann, Michael; Fademrecht, Silvia; Vogel, Constantin; Pleiss, Jürgen

2016-05-01

The Lactamase Engineering Database (www.LacED.uni-stuttgart.de) was developed to facilitate the classification and analysis of TEM β-lactamases. The current version contains 474 TEM variants. Two hundred fifty-nine variants form a large scale-free network of highly connected point mutants. The network was divided into three subnetworks which were enriched by single phenotypes: one network with predominantly 2be and two networks with 2br phenotypes. Fifteen positions were found to be highly variable, contributing to the majority of the observed variants. Since it is expected that a considerable fraction of the theoretical sequence space is functional, the currently sequenced 474 variants represent only the tip of the iceberg of functional TEM β-lactamase variants which form a huge natural reservoir of highly interconnected variants. Almost 50% of the variants are part of a quartet. Thus, two single mutations that result in functional enzymes can be combined into a functional protein. Most of these quartets consist of the same phenotype, or the mutations are additive with respect to the phenotype. By predicting quartets from triplets, 3,916 unknown variants were constructed. Eighty-seven variants complement multiple quartets and therefore have a high probability of being functional. The construction of a TEM β-lactamase network and subsequent analyses by clustering and quartet prediction are valuable tools to gain new insights into the viable sequence space of TEM β-lactamases and to predict their phenotype. The highly connected sequence space of TEM β-lactamases is ideally suited to network analysis and demonstrates the strengths of network analysis over tree reconstruction methods. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

A novel homozygous variant in the SMOC1 gene underlying Waardenburg anophthalmia syndrome.

PubMed

Ullah, Asmat; Umair, Muhammad; Ahmad, Farooq; Muhammad, Dost; Basit, Sulman; Ahmad, Wasim

2017-01-01

Waardenburg anophthalmia syndrome (WAS), also known as ophthalmo-acromelic syndrome or anophthalmia-syndactyly, is a rare congenital disorder that segregates in an autosomal recessive pattern. Clinical features of the syndrome include malformation of the eyes and the skeleton. Mostly, WAS is caused by mutations in the SMOC-1 gene. The present report describes a large consanguineous family of Pakistani origin segregating Waardenburg anophthalmia syndrome in an autosomal recessive pattern. Genotyping followed by Sanger sequencing was performed to search for a candidate gene. SNP genotyping using AffymetrixGeneChip Human Mapping 250K Nsp array established a single homozygous region among affected members on chromosome 14q23.1-q24.3 harboring the SMOC1 gene. Sequencing of the gene revealed a novel homozygous missense mutation (c.812G>A; p.Cys271Tyr) in the family. This is the first report of Waardenburg anophthalmia syndrome caused by a SMOC1 variant in a Pakistani population. The mutation identified in the present investigation extends the body of evidence implicating the gene SMOC-1 in causing WAS.

Multiplexed enrichment of rare DNA variants via sequence-selective and temperature-robust amplification

PubMed Central

Wu, Lucia R.; Chen, Sherry X.; Wu, Yalei; Patel, Abhijit A.; Zhang, David Yu

2018-01-01

Rare DNA-sequence variants hold important clinical and biological information, but existing detection techniques are expensive, complex, allele-specific, or don’t allow for significant multiplexing. Here, we report a temperature-robust polymerase-chain-reaction method, which we term blocker displacement amplification (BDA), that selectively amplifies all sequence variants, including single-nucleotide variants (SNVs), within a roughly 20-nucleotide window by 1,000-fold over wild-type sequences. This allows for easy detection and quantitation of hundreds of potential variants originally at ≤0.1% in allele frequency. BDA is compatible with inexpensive thermocycler instrumentation and employs a rationally designed competitive hybridization reaction to achieve comparable enrichment performance across annealing temperatures ranging from 56 °C to 64 °C. To show the sequence generality of BDA, we demonstrate enrichment of 156 SNVs and the reliable detection of single-digit copies. We also show that the BDA detection of rare driver mutations in cell-free DNA samples extracted from the blood plasma of lung-cancer patients is highly consistent with deep sequencing using molecular lineage tags, with a receiver operator characteristic accuracy of 95%. PMID:29805844

Mitochondrial DNA sequence context in the penetrance of mitochondrial t-RNA mutations: A study across multiple lineages with diagnostic implications

PubMed Central

Queen, Rachel A.; Steyn, Jannetta S.; Lord, Phillip

2017-01-01

Mitochondrial DNA (mtDNA) mutations are well recognized as an important cause of inherited disease. Diseases caused by mtDNA mutations exhibit a high degree of clinical heterogeneity with a complex genotype-phenotype relationship, with many such mutations exhibiting incomplete penetrance. There is evidence that the spectrum of mutations causing mitochondrial disease might differ between different mitochondrial lineages (haplogroups) seen in different global populations. This would point to the importance of sequence context in the expression of mutations. To explore this possibility, we looked for mutations which are known to cause disease in humans, in animals of other species unaffected by mtDNA disease. The mt-tRNA genes are the location of many pathogenic mutations, with the m.3243A>G mutation on the mt-tRNA-Leu(UUR) being the most frequently seen mutation in humans. This study looked for the presence of m.3243A>G in 2784 sequences from 33 species, as well as any of the other mutations reported in association with disease located on mt-tRNA-Leu(UUR). We report a number of disease associated variations found on mt-tRNA-Leu(UUR) in other chordates, as the major population variant, with m.3243A>G being seen in 6 species. In these, we also found a number of mutations which appear compensatory and which could prevent the pathogenicity associated with this change in humans. This work has important implications for the discovery and diagnosis of mtDNA mutations in non-European populations. In addition, it might provide a partial explanation for the conflicting results in the literature that examines the role of mtDNA variants in complex traits. PMID:29161289

Linkage disequilibrium among commonly genotyped SNP and variants detected from bull sequence

USDA-ARS?s Scientific Manuscript database

Genomic prediction utilizing causal variants could increase selection accuracy above that achieved with SNP genotyped by commercial assays. A number of variants detected from sequencing influential sires are likely to be causal, but noticable improvements in prediction accuracy using imputed sequen...

Core sequence of PAPf39 amyloid fibrils and mechanism of pH-dependent fibril formation: the role of monomer conformation.

PubMed

French, Kinsley C; Makhatadze, George I

2012-12-21

PAPf39, a 39-residue peptide fragment from human prostatic acidic phosphatase, has been shown to form amyloid fibrils in semen (SEVI), which increase HIV infectivity by up to 5 orders of magnitude. The sequence of the PAPf39 fibrillar core was identified using hydrogen-deuterium exchange (HDX) mass spectrometry and protease protection assays. The central and C-terminal regions are highly protected from HDX and proteolytic cleavage and, thus, are part of the fibrillar core. Conversely, the N-terminal region is unprotected from HDX and proteolytic cleavage, suggesting that it is exposed and not part of the fibrillar core. This finding was tested using two N-terminal truncated variants, PAPf39Δ1-8 and PAPf39Δ1-13. Both variants formed amyloid fibrils at neutral pH. However, these variants showed a markedly different pH dependence of fibril formation versus that of PAPf39. PAPf39 fibrils can form at pH 7.7, but not at pH 5.5 or 2.5, while both N-terminally truncated variants can form fibrils at these pH values. Thus, the N-terminal region is not necessary for fibril formation but modulates the pH dependence of PAPf39 fibril formation. PAPf39Δ1-8 and PAPf39Δ1-13 are capable of seeding PAPf39 fibril formation at neutral pH, suggesting that these variants are structurally compatible with PAPf39, yet no mixed fibril formation occurs between the truncated variants and PAPf39 at low pH. This suggests that pH affects the PAPf39 monomer conformational ensemble, which is supported by far-UV circular dichroism spectroscopy. A conceptual model describing the pH dependence of PAPf39 aggregation is proposed and provides potential biological implications.

Detection of genetic variants between different Polish Landrace and Puławska pigs by means of RNA-seq analysis.

PubMed

Piórkowska, K; Żukowski, K; Ropka-Molik, K; Tyra, M

2018-06-01

Variant calling analysis based on RNA sequencing data provides information about gene variants. RNA-seq is cheaper and faster than is DNA sequencing. However, it requires individual hard filters during data processing due to post-transcriptional modifications such as splicing and RNA editing. In the present study, RNA-seq transcriptome data on two Polish pig breeds (Puławska, PUL, n = 8, and Polish Landrace, PL, n = 8) were included. The pig breeds are significantly different with regard to meat qualities such as texture, water exudation, growth traits and fat content in carcasses. A total of 2451 significant mutations were identified by a chi square tests, and functional analysis was carried out using Panther, KEGG and Kobas. Interesting missense gene variants and mutations located in regulatory regions were found in a few genes related to fatty acid metabolism and lipid storage such as ACSL5, ALDH3A2, FADS1, SCD, PLA2G12A and ATGL. A validation of mutational influences on pig traits was performed for ALDH3A2, ATGL, PLA2G12A and MYOM1 variants using association analysis including 215 pigs of the PL and PUL breeds. The ALDH3A2ENSSSCT00000019636.2:c.470T>C polymorphism was found to affect the weight of the ham and loin eye area. In turn, an ENSSSCT00000004091.2:c.2836G>A MYOM1 mutation, which could be implicated in myofibrillar network organisation, had an effect on meatiness and loin texture parameters. The study aimed to estimate the usefulness of RNA-seq results for a purpose other than differentially expressed gene analysis. The analysis performed indicated interesting gene variants that could be used in the future as markers during selection. © 2018 Stichting International Foundation for Animal Genetics.

Study designs for identification of rare disease variants in complex diseases: the utility of family-based designs.

PubMed

Ionita-Laza, Iuliana; Ottman, Ruth

2011-11-01

The recent progress in sequencing technologies makes possible large-scale medical sequencing efforts to assess the importance of rare variants in complex diseases. The results of such efforts depend heavily on the use of efficient study designs and analytical methods. We introduce here a unified framework for association testing of rare variants in family-based designs or designs based on unselected affected individuals. This framework allows us to quantify the enrichment in rare disease variants in families containing multiple affected individuals and to investigate the optimal design of studies aiming to identify rare disease variants in complex traits. We show that for many complex diseases with small values for the overall sibling recurrence risk ratio, such as Alzheimer's disease and most cancers, sequencing affected individuals with a positive family history of the disease can be extremely advantageous for identifying rare disease variants. In contrast, for complex diseases with large values of the sibling recurrence risk ratio, sequencing unselected affected individuals may be preferable.

High prevalence of DUOX2 mutations in Japanese patients with permanent congenital hypothyroidism or transient hypothyroidism.

PubMed

Matsuo, Kumihiro; Tanahashi, Yusuke; Mukai, Tokuo; Suzuki, Shigeru; Tajima, Toshihiro; Azuma, Hiroshi; Fujieda, Kenji

2016-07-01

Dual oxidase 2 (DUOX2) mutations are a cause of dyshormonogenesis (DH) and have been identified in patients with permanent congenital hypothyroidism (PH) and with transient hypothyroidism (TH). We aimed to elucidate the prevalence and phenotypical variations of DUOX2 mutations. Forty-eight Japanese DH patients were enroled and analysed for sequence variants of DUOX2, DUOXA2, and TPO using polymerase chain reaction-amplified direct sequencing. Fourteen sequence variants of DUOX2, including 10 novel variants, were identified in 11 patients. DUOX2 variants were more prevalent (11/48, 22.9%) than TPO (3/48, 6.3%) (p=0.020). The prevalence of DUOX2 variants in TH was slightly, but not significantly, higher than in PH. Furthermore, one patient had digenic heterozygous sequence variants of both DUOX2 and TPO. Our results suggest that DUOX2 mutations might be the most common cause of both PH and TH, and that phenotypes of these mutations might be milder than those of other causes.

Allele-Specific Methylation Occurs at Genetic Variants Associated with Complex Disease

PubMed Central

Hutchinson, John N.; Raj, Towfique; Fagerness, Jes; Stahl, Eli; Viloria, Fernando T.; Gimelbrant, Alexander; Seddon, Johanna; Daly, Mark; Chess, Andrew; Plenge, Robert

2014-01-01

We hypothesize that the phenomenon of allele-specific methylation (ASM) may underlie the phenotypic effects of multiple variants identified by Genome-Wide Association studies (GWAS). We evaluate ASM in a human population and document its genome-wide patterns in an initial screen at up to 380,678 sites within the genome, or up to 5% of the total genomic CpGs. We show that while substantial inter-individual variation exists, 5% of assessed sites show evidence of ASM in at least six samples; the majority of these events (81%) are under genetic influence. Many of these cis-regulated ASM variants are also eQTLs in peripheral blood mononuclear cells and monocytes and/or in high linkage-disequilibrium with variants linked to complex disease. Finally, focusing on autoimmune phenotypes, we extend this initial screen to confirm the association of cis-regulated ASM with multiple complex disease-associated variants in an independent population using next-generation bisulfite sequencing. These four variants are implicated in complex phenotypes such as ulcerative colitis and AIDS progression disease (rs10491434), Celiac disease (rs2762051), Crohn's disease, IgA nephropathy and early-onset inflammatory bowel disease (rs713875) and height (rs6569648). Our results suggest cis-regulated ASM may provide a mechanistic link between the non-coding genetic changes and phenotypic variation observed in these diseases and further suggests a route to integrating DNA methylation status with GWAS results. PMID:24911414

Functional variants in the LRRK2 gene confer shared effects on risk for Crohn's disease and Parkinson's disease.

PubMed

Hui, Ken Y; Fernandez-Hernandez, Heriberto; Hu, Jianzhong; Schaffner, Adam; Pankratz, Nathan; Hsu, Nai-Yun; Chuang, Ling-Shiang; Carmi, Shai; Villaverde, Nicole; Li, Xianting; Rivas, Manual; Levine, Adam P; Bao, Xiuliang; Labrias, Philippe R; Haritunians, Talin; Ruane, Darren; Gettler, Kyle; Chen, Ernie; Li, Dalin; Schiff, Elena R; Pontikos, Nikolas; Barzilai, Nir; Brant, Steven R; Bressman, Susan; Cheifetz, Adam S; Clark, Lorraine N; Daly, Mark J; Desnick, Robert J; Duerr, Richard H; Katz, Seymour; Lencz, Todd; Myers, Richard H; Ostrer, Harry; Ozelius, Laurie; Payami, Haydeh; Peter, Yakov; Rioux, John D; Segal, Anthony W; Scott, William K; Silverberg, Mark S; Vance, Jeffery M; Ubarretxena-Belandia, Iban; Foroud, Tatiana; Atzmon, Gil; Pe'er, Itsik; Ioannou, Yiannis; McGovern, Dermot P B; Yue, Zhenyu; Schadt, Eric E; Cho, Judy H; Peter, Inga

2018-01-10

Crohn's disease (CD), a form of inflammatory bowel disease, has a higher prevalence in Ashkenazi Jewish than in non-Jewish European populations. To define the role of nonsynonymous mutations, we performed exome sequencing of Ashkenazi Jewish patients with CD, followed by array-based genotyping and association analysis in 2066 CD cases and 3633 healthy controls. We detected association signals in the LRRK2 gene that conferred risk for CD (N2081D variant, P = 9.5 × 10 -10 ) or protection from CD (N551K variant, tagging R1398H-associated haplotype, P = 3.3 × 10 -8 ). These variants affected CD age of onset, disease location, LRRK2 activity, and autophagy. Bayesian network analysis of CD patient intestinal tissue further implicated LRRK2 in CD pathogenesis. Analysis of the extended LRRK2 locus in 24,570 CD cases, patients with Parkinson's disease (PD), and healthy controls revealed extensive pleiotropy, with shared genetic effects between CD and PD in both Ashkenazi Jewish and non-Jewish cohorts. The LRRK2 N2081D CD risk allele is located in the same kinase domain as G2019S, a mutation that is the major genetic cause of familial and sporadic PD. Like the G2019S mutation, the N2081D variant was associated with increased kinase activity, whereas neither N551K nor R1398H variants on the protective haplotype altered kinase activity. We also confirmed that R1398H, but not N551K, increased guanosine triphosphate binding and hydrolyzing enzyme (GTPase) activity, thereby deactivating LRRK2. The presence of shared LRRK2 alleles in CD and PD provides refined insight into disease mechanisms and may have major implications for the treatment of these two seemingly unrelated diseases. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Deep whole-genome sequencing of 90 Han Chinese genomes.

PubMed

Lan, Tianming; Lin, Haoxiang; Zhu, Wenjuan; Laurent, Tellier Christian Asker Melchior; Yang, Mengcheng; Liu, Xin; Wang, Jun; Wang, Jian; Yang, Huanming; Xu, Xun; Guo, Xiaosen

2017-09-01

Next-generation sequencing provides a high-resolution insight into human genetic information. However, the focus of previous studies has primarily been on low-coverage data due to the high cost of sequencing. Although the 1000 Genomes Project and the Haplotype Reference Consortium have both provided powerful reference panels for imputation, low-frequency and novel variants remain difficult to discover and call with accuracy on the basis of low-coverage data. Deep sequencing provides an optimal solution for the problem of these low-frequency and novel variants. Although whole-exome sequencing is also a viable choice for exome regions, it cannot account for noncoding regions, sometimes resulting in the absence of important, causal variants. For Han Chinese populations, the majority of variants have been discovered based upon low-coverage data from the 1000 Genomes Project. However, high-coverage, whole-genome sequencing data are limited for any population, and a large amount of low-frequency, population-specific variants remain uncharacterized. We have performed whole-genome sequencing at a high depth (∼×80) of 90 unrelated individuals of Chinese ancestry, collected from the 1000 Genomes Project samples, including 45 Northern Han Chinese and 45 Southern Han Chinese samples. Eighty-three of these 90 have been sequenced by the 1000 Genomes Project. We have identified 12 568 804 single nucleotide polymorphisms, 2 074 210 short InDels, and 26 142 structural variations from these 90 samples. Compared to the Han Chinese data from the 1000 Genomes Project, we have found 7 000 629 novel variants with low frequency (defined as minor allele frequency < 5%), including 5 813 503 single nucleotide polymorphisms, 1 169 199 InDels, and 17 927 structural variants. Using deep sequencing data, we have built a greatly expanded spectrum of genetic variation for the Han Chinese genome. Compared to the 1000 Genomes Project, these Han Chinese deep sequencing data enhance the characterization of a large number of low-frequency, novel variants. This will be a valuable resource for promoting Chinese genetics research and medical development. Additionally, it will provide a valuable supplement to the 1000 Genomes Project, as well as to other human genome projects. © The Authors 2017. Published by Oxford University Press.

Analysis of CHRNA7 rare variants in autism spectrum disorder susceptibility.

PubMed

Bacchelli, Elena; Battaglia, Agatino; Cameli, Cinzia; Lomartire, Silvia; Tancredi, Raffaella; Thomson, Susanne; Sutcliffe, James S; Maestrini, Elena

2015-04-01

Chromosome 15q13.3 recurrent microdeletions are causally associated with a wide range of phenotypes, including autism spectrum disorder (ASD), seizures, intellectual disability, and other psychiatric conditions. Whether the reciprocal microduplication is pathogenic is less certain. CHRNA7, encoding for the alpha7 subunit of the neuronal nicotinic acetylcholine receptor, is considered the likely culprit gene in mediating neurological phenotypes in 15q13.3 deletion cases. To assess if CHRNA7 rare variants confer risk to ASD, we performed copy number variant analysis and Sanger sequencing of the CHRNA7 coding sequence in a sample of 135 ASD cases. Sequence variation in this gene remains largely unexplored, given the existence of a fusion gene, CHRFAM7A, which includes a nearly identical partial duplication of CHRNA7. Hence, attempts to sequence coding exons must distinguish between CHRNA7 and CHRFAM7A, making next-generation sequencing approaches unreliable for this purpose. A CHRNA7 microduplication was detected in a patient with autism and moderate cognitive impairment; while no rare damaging variants were identified in the coding region, we detected rare variants in the promoter region, previously described to functionally reduce transcription. This study represents the first sequence variant analysis of CHRNA7 in a sample of idiopathic autism. © 2015 Wiley Periodicals, Inc.

Single nucleotide polymorphisms associated with nonsyndromic cryptorchidism in Mexican patients.

PubMed

Chávez-Saldaña, M; Vigueras-Villaseñor, R M; Yokoyama-Rebollar, E; Landero-Huerta, D A; Rojas-Castañeda, J C; Taja-Chayeb, L; Cuevas-Alpuche, J O; Zambrano, E

2018-02-01

Cryptorchidism is a frequent genitourinary malformation considered as an important risk factor for infertility and testicular malignancy. The aetiology of cryptorchidism is multifactorial in which certain SNPs, capable of inhibiting the development of the gubernaculum, are implicated. We analysed 16 SNPs by allelic discrimination and automated sequencing in 85 patients and 99 healthy people, with the objective to identify the association between these variants and isolated cryptorchidism. In two different patients with unilateral cryptorchidism, we found the variants rs121912556 and p.R105R of INSL3 gene in a heterozygous form associated with cryptorchidism, so we could considered them as risk factors for cryptorchidism. On the other hand, SNPs rs10421916 of INSL3 gene, as well as the variants rs1555633 and rs7325513 in the RXFP2 gene, and rs3779456 variant of the HOXA10 gene were statistically significant, when the patients and controls were compared and could be considered as protective factors since are predominantly present in controls. The genotype-phenotype correlation did not show statistical significance. With these results, we could conclude that these polymorphisms can be considered as important variants in our population and would contribute in the future knowledge of the aetiology and physiopathology of cryptorchidism. © 2017 Blackwell Verlag GmbH.

Identification of rare genetic variation of NLRP1 gene in familial multiple sclerosis.

PubMed

Maver, Ales; Lavtar, Polona; Ristić, Smiljana; Stopinšek, Sanja; Simčič, Saša; Hočevar, Keli; Sepčić, Juraj; Drulović, Jelena; Pekmezović, Tatjana; Novaković, Ivana; Alenka, Hodžić; Rudolf, Gorazd; Šega, Saša; Starčević-Čizmarević, Nada; Palandačić, Anja; Zamolo, Gordana; Kapović, Miljenko; Likar, Tina; Peterlin, Borut

2017-06-16

The genetic etiology and the contribution of rare genetic variation in multiple sclerosis (MS) has not yet been elucidated. Although familial forms of MS have been described, no convincing rare and penetrant variants have been reported to date. We aimed to characterize the contribution of rare genetic variation in familial and sporadic MS and have identified a family with two sibs affected by concomitant MS and malignant melanoma (MM). We performed whole exome sequencing in this primary family and 38 multiplex MS families and 44 sporadic MS cases and performed transcriptional and immunologic assessment of the identified variants. We identified a potentially causative homozygous missense variant in NLRP1 gene (Gly587Ser) in the primary family. Further possibly pathogenic NLRP1 variants were identified in the expanded cohort of patients. Stimulation of peripheral blood mononuclear cells from MS patients with putatively pathogenic NLRP1 variants showed an increase in IL-1B gene expression and active cytokine IL-1β production, as well as global activation of NLRP1-driven immunologic pathways. We report a novel familial association of MS and MM, and propose a possible underlying genetic basis in NLRP1 gene. Furthermore, we provide initial evidence of the broader implications of NLRP1-related pathway dysfunction in MS.

Exome Sequencing Identifies Three Novel Candidate Genes Implicated in Intellectual Disability

PubMed Central

Azam, Maleeha; Ayub, Humaira; Vissers, Lisenka E. L. M.; Gilissen, Christian; Ali, Syeda Hafiza Benish; Riaz, Moeen; Veltman, Joris A.; Pfundt, Rolph; van Bokhoven, Hans; Qamar, Raheel

2014-01-01

Intellectual disability (ID) is a major health problem mostly with an unknown etiology. Recently exome sequencing of individuals with ID identified novel genes implicated in the disease. Therefore the purpose of the present study was to identify the genetic cause of ID in one syndromic and two non-syndromic Pakistani families. Whole exome of three ID probands was sequenced. Missense variations in two plausible novel genes implicated in autosomal recessive ID were identified: lysine (K)-specific methyltransferase 2B (KMT2B), zinc finger protein 589 (ZNF589), as well as hedgehog acyltransferase (HHAT) with a de novo mutation with autosomal dominant mode of inheritance. The KMT2B recessive variant is the first report of recessive Kleefstra syndrome-like phenotype. Identification of plausible causative mutations for two recessive and a dominant type of ID, in genes not previously implicated in disease, underscores the large genetic heterogeneity of ID. These results also support the viewpoint that large number of ID genes converge on limited number of common networks i.e. ZNF589 belongs to KRAB-domain zinc-finger proteins previously implicated in ID, HHAT is predicted to affect sonic hedgehog, which is involved in several disorders with ID, KMT2B associated with syndromic ID fits the epigenetic module underlying the Kleefstra syndromic spectrum. The association of these novel genes in three different Pakistani ID families highlights the importance of screening these genes in more families with similar phenotypes from different populations to confirm the involvement of these genes in pathogenesis of ID. PMID:25405613

Efficient analysis of mouse genome sequences reveal many nonsense variants

PubMed Central

Steeland, Sophie; Timmermans, Steven; Van Ryckeghem, Sara; Hulpiau, Paco; Saeys, Yvan; Van Montagu, Marc; Vandenbroucke, Roosmarijn E.; Libert, Claude

2016-01-01

Genetic polymorphisms in coding genes play an important role when using mouse inbred strains as research models. They have been shown to influence research results, explain phenotypical differences between inbred strains, and increase the amount of interesting gene variants present in the many available inbred lines. SPRET/Ei is an inbred strain derived from Mus spretus that has ∼1% sequence difference with the C57BL/6J reference genome. We obtained a listing of all SNPs and insertions/deletions (indels) present in SPRET/Ei from the Mouse Genomes Project (Wellcome Trust Sanger Institute) and processed these data to obtain an overview of all transcripts having nonsynonymous coding sequence variants. We identified 8,883 unique variants affecting 10,096 different transcripts from 6,328 protein-coding genes, which is about 28% of all coding genes. Because only a subset of these variants results in drastic changes in proteins, we focused on variations that are nonsense mutations that ultimately resulted in a gain of a stop codon. These genes were identified by in silico changing the C57BL/6J coding sequences to the SPRET/Ei sequences, converting them to amino acid (AA) sequences, and comparing the AA sequences. All variants and transcripts affected were also stored in a database, which can be browsed using a SPRET/Ei M. spretus variants web tool (www.spretus.org), including a manual. We validated the tool by demonstrating the loss of function of three proteins predicted to be severely truncated, namely Fas, IRAK2, and IFNγR1. PMID:27147605

Identification of novel genetic causes of Rett syndrome-like phenotypes.

PubMed

Lopes, Fátima; Barbosa, Mafalda; Ameur, Adam; Soares, Gabriela; de Sá, Joaquim; Dias, Ana Isabel; Oliveira, Guiomar; Cabral, Pedro; Temudo, Teresa; Calado, Eulália; Cruz, Isabel Fineza; Vieira, José Pedro; Oliveira, Renata; Esteves, Sofia; Sauer, Sascha; Jonasson, Inger; Syvänen, Ann-Christine; Gyllensten, Ulf; Pinto, Dalila; Maciel, Patrícia

2016-03-01

The aim of this work was to identify new genetic causes of Rett-like phenotypes using array comparative genomic hybridisation and a whole exome sequencing approach. We studied a cohort of 19 Portuguese patients (16 girls, 3 boys) with a clinical presentation significantly overlapping Rett syndrome (RTT). Genetic analysis included filtering of the single nucleotide variants and indels with preference for de novo, homozygous/compound heterozygous, or maternally inherited X linked variants. Examination by MRI and muscle biopsies was also performed. Pathogenic genomic imbalances were found in two patients (10.5%): an 18q21.2 deletion encompassing four exons of the TCF4 gene and a mosaic UPD of chromosome 3. Variants in genes previously implicated in neurodevelopmental disorders (NDD) were identified in six patients (32%): de novo variants in EEF1A2, STXBP1 and ZNF238 were found in three patients, maternally inherited X linked variants in SLC35A2, ZFX and SHROOM4 were detected in two male patients and one homozygous variant in EIF2B2 was detected in one patient. Variants were also detected in five novel NDD candidate genes (26%): we identified de novo variants in the RHOBTB2, SMARCA1 and GABBR2 genes; a homozygous variant in EIF4G1; compound heterozygous variant in HTT. Network analysis reveals that these genes interact by means of protein interactions with each other and with the known RTT genes. These findings expand the phenotypical spectrum of previously known NDD genes to encompass RTT-like clinical presentations and identify new candidate genes for RTT-like phenotypes. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

RareVariantVis: new tool for visualization of causative variants in rare monogenic disorders using whole genome sequencing data.

PubMed

Stokowy, Tomasz; Garbulowski, Mateusz; Fiskerstrand, Torunn; Holdhus, Rita; Labun, Kornel; Sztromwasser, Pawel; Gilissen, Christian; Hoischen, Alexander; Houge, Gunnar; Petersen, Kjell; Jonassen, Inge; Steen, Vidar M

2016-10-01

The search for causative genetic variants in rare diseases of presumed monogenic inheritance has been boosted by the implementation of whole exome (WES) and whole genome (WGS) sequencing. In many cases, WGS seems to be superior to WES, but the analysis and visualization of the vast amounts of data is demanding. To aid this challenge, we have developed a new tool-RareVariantVis-for analysis of genome sequence data (including non-coding regions) for both germ line and somatic variants. It visualizes variants along their respective chromosomes, providing information about exact chromosomal position, zygosity and frequency, with point-and-click information regarding dbSNP IDs, gene association and variant inheritance. Rare variants as well as de novo variants can be flagged in different colors. We show the performance of the RareVariantVis tool in the Genome in a Bottle WGS data set. https://www.bioconductor.org/packages/3.3/bioc/html/RareVariantVis.html tomasz.stokowy@k2.uib.no Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Herfort, Lydie; Peterson, Tawnya D.; McCue, Lee Ann

For at least a decade, annually recurring blooms of the photosynthetic ciliate, Myrionecta rubra have been observed in the Columbia River estuary in late summer. In an effort to understand the dynamics of these blooms, we investigated the genetic variability of M. rubra and its cryptophyte plastids within three large estuarine blooms formed in consecutive years (2007-2009), and conducted a broader spatial survey along the coasts of Oregon/Washington. Analysis of the ‘18S-28S’ sequences specific for Mesodiniidae uncovered at least 7 variants of M. rubra within the Columbia River coastal margin in spring and summer, but only one of these M.more » rubra variants was implicated in estuary bloom formation. Using a multigene approach, we show that the bloom-forming variant of M. rubra appears to harbor the same cryptophyte chloroplast in recurring blooms. Analyses of chloroplast 16S rRNA, cryptophyte RuBisCO and Photosystem II D2 genes together suggest that the plastid is derived from Teleaulax amphioxeia. Free-living cells of this species and of other cryptophytes were practically absent from the bloom patches in the estuary main channels based on 18S rDNA sequence analyses. The respectively low and high proportions of T. amphioxeia nuclei and chloroplasts signals found in the M. rubra bloom of the Columbia River estuary in successive years supports the notion of a transient association between T. amphioxeia and the bloom-forming M. rubra variant, with loss of cryptophyte nuclei. The genetic variability of M. rubra uncovered here is relevant to the controversy in the literature regarding the cryptophyte /M. rubra association.« less

Host genetic variation in mucosal immunity pathways influences the upper airway microbiome.

PubMed

Igartua, Catherine; Davenport, Emily R; Gilad, Yoav; Nicolae, Dan L; Pinto, Jayant; Ober, Carole

2017-02-01

The degree to which host genetic variation can modulate microbial communities in humans remains an open question. Here, we performed a genetic mapping study of the microbiome in two accessible upper airway sites, the nasopharynx and the nasal vestibule, during two seasons in 144 adult members of a founder population of European decent. We estimated the relative abundances (RAs) of genus level bacteria from 16S rRNA gene sequences and examined associations with 148,653 genetic variants (linkage disequilibrium [LD] r 2  < 0.5) selected from among all common variants discovered in genome sequences in this population. We identified 37 microbiome quantitative trait loci (mbQTLs) that showed evidence of association with the RAs of 22 genera (q < 0.05) and were enriched for genes in mucosal immunity pathways. The most significant association was between the RA of Dermacoccus (phylum Actinobacteria) and a variant 8 kb upstream of TINCR (rs117042385; p = 1.61 × 10 -8 ; q = 0.002), a long non-coding RNA that binds to peptidoglycan recognition protein 3 (PGLYRP3) mRNA, a gene encoding a known antimicrobial protein. A second association was between a missense variant in PGLYRP4 (rs3006458) and the RA of an unclassified genus of family Micrococcaceae (phylum Actinobacteria) (p = 5.10 × 10 -7 ; q = 0.032). Our findings provide evidence of host genetic influences on upper airway microbial composition in humans and implicate mucosal immunity genes in this relationship.

Single-Exome sequencing identified a novel RP2 mutation in a child with X-linked retinitis pigmentosa.

PubMed

Lim, Hassol; Park, Young-Mi; Lee, Jong-Keuk; Taek Lim, Hyun

2016-10-01

To present an efficient and successful application of a single-exome sequencing study in a family clinically diagnosed with X-linked retinitis pigmentosa. Exome sequencing study based on clinical examination data. An 8-year-old proband and his family. The proband and his family members underwent comprehensive ophthalmologic examinations. Exome sequencing was undertaken in the proband using Agilent SureSelect Human All Exon Kit and Illumina HiSeq 2000 platform. Bioinformatic analysis used Illumina pipeline with Burrows-Wheeler Aligner-Genome Analysis Toolkit (BWA-GATK), followed by ANNOVAR to perform variant functional annotation. All variants passing filter criteria were validated by Sanger sequencing to confirm familial segregation. Analysis of exome sequence data identified a novel frameshift mutation in RP2 gene resulting in a premature stop codon (c.665delC, p.Pro222fsTer237). Sanger sequencing revealed this mutation co-segregated with the disease phenotype in the child's family. We identified a novel causative mutation in RP2 from a single proband's exome sequence data analysis. This study highlights the effectiveness of the whole-exome sequencing in the genetic diagnosis of X-linked retinitis pigmentosa, over the conventional sequencing methods. Even using a single exome, exome sequencing technology would be able to pinpoint pathogenic variant(s) for X-linked retinitis pigmentosa, when properly applied with aid of adequate variant filtering strategy. Copyright © 2016 Canadian Ophthalmological Society. Published by Elsevier Inc. All rights reserved.

The surface glycoprotein of a natural feline leukemia virus subgroup A variant, FeLV-945, as a determinant of disease outcome.

PubMed

Bolin, Lisa L; Ahmad, Shamim; Levy, Laura S

2011-10-15

Feline leukemia virus (FeLV) is a natural retrovirus of domestic cats associated with degenerative, proliferative and malignant diseases. Studies of FeLV infection in a cohort of naturally infected cats were undertaken to examine FeLV variation, the selective pressures operative in FeLV infection that lead to predominance of natural variants, and the consequences for infection and disease progression. A unique variant, designated FeLV-945, was identified as the predominant isolate in the cohort and was associated with non-T-cell diseases including multicentric lymphoma. FeLV-945 was assigned to the FeLV-A subgroup based on sequence analysis and receptor utilization, but was shown to differ in sequence from a prototype member of FeLV-A, designated FeLV-A/61E, in the long terminal repeat (LTR) and the surface glycoprotein gene (SU). A unique sequence motif in the FeLV-945 LTR was shown to function as a transcriptional enhancer and to confer a replicative advantage. The FeLV-945 SU protein was observed to differ in sequence as compared to FeLV-A/61E within functional domains known to determine receptor selection and binding. Experimental infection of newborn cats was performed using wild type FeLV-A/61E or recombinant FeLV-A/61E in which the LTR (61E/945L) or LTR and SU (61E/945SL) were exchanged for that of FeLV-945. Infection with either FeLV-A/61E or 61E/945L resulted in T-cell lymphoma of the thymus, although 61E/945L caused disease significantly more rapidly. In contrast, infection with 61E/945SL resulted in the rapid induction of a multicentric lymphoma of B-cell origin, thus recapitulating the outcome of natural infection and implicating FeLV-945 SU as a determinant of disease outcome. Recombinant FeLV-B was detected infrequently and at low levels in multicentric lymphomas, and was thereby not implicated in disease induction. Preliminary studies of receptor interaction indicated that virus particles bearing FeLV-945 SU bind to the FeLV-A receptor more efficiently than do particles bearing FeLV-A/61E SU, and that soluble SU proteins expressed from the viruses demonstrate the same differential binding phenotype. Preliminary mutational analysis of FeLV-945 was performed by exchanging regions containing either the primary receptor binding determinant, VRA, the secondary determinant, VRB, or a proline-rich region, PRR, with that of FeLV-A/61E. Results implicated a region containing VRA as a minor contributor, while a region containing VRB largely conferred increased binding efficiency. Copyright © 2011 Elsevier B.V. All rights reserved.

Mutation screening of SEMA3A and SEMA7A in patients with congenital hypogonadotropic hypogonadism.

PubMed

Känsäkoski, Johanna; Fagerholm, Rainer; Laitinen, Eeva-Maria; Vaaralahti, Kirsi; Hackman, Peter; Pitteloud, Nelly; Raivio, Taneli; Tommiska, Johanna

2014-05-01

Congenital hypogonadotropic hypogonadism (HH), a rare disorder characterized by absent, partial, or delayed puberty, can be caused by the lack or deficient number of hypothalamic gonadotropin-releasing hormone (GnRH) neurons. SEMA3A was recently implicated in the etiology of the disorder, and Sema7A-deficient mice have a reduced number of GnRH neurons in their brains. SEMA3A and SEMA7A were screened by Sanger sequencing in altogether 50 Finnish HH patients (34 with Kallmann syndrome (KS; HH with hyposmia/anosmia) and 16 with normosmic HH (nHH)). In 20 patients, mutation(s) had already been found in genes known to be implicated in congenital HH. Three heterozygous variants (c.458A>G (p.Asn153Ser), c.1253A>G (p.Asn418Ser), and c.1303G>A (p.Val435Ile)) were found in SEMA3A in three KS patients, two of which also had a mutation in FGFR1. Two rare heterozygous variants (c.442C>T (p.Arg148Trp) and c.1421G>A (p.Arg474Gln)) in SEMA7A were found in one male nHH patient with a previously identified KISS1R nonsense variant and one male KS patient with a previously identified mutation in KAL1, respectively. Our results suggest that heterozygous missense variants in SEMA3A and SEMA7A may modify the phenotype of KS but most likely are not alone sufficient to cause the disorder.

Whole exome sequencing in recurrent early pregnancy loss

PubMed Central

Qiao, Ying; Wen, Jiadi; Tang, Flamingo; Martell, Sally; Shomer, Naomi; Leung, Peter C.K.; Stephenson, Mary D.; Rajcan-Separovic, Evica

2016-01-01

STUDY HYPOTHESIS Exome sequencing can identify genetic causes of idiopathic recurrent pregnancy loss (RPL). STUDY FINDING We identified compound heterozygous deleterious mutations affecting DYNC2H1 and ALOX15 in two out of four families with RPL. Both genes have a role in early development. Bioinformatics analysis of all genes with rare and putatively pathogenic mutations in miscarriages and couples showed enrichment in pathways relevant to pregnancy loss, including the complement and coagulation cascades pathways. WHAT IS KNOWN ALREADY Next generation sequencing (NGS) is increasingly being used to identify known and novel gene mutations in children with developmental delay and in fetuses with ultrasound-detected anomalies. In contrast, NGS is rarely used to study pregnancy loss. Chromosome microarray analysis detects putatively causative DNA copy number variants (CNVs) in ∼2% of miscarriages and CNVs of unknown significance (predominantly parental in origin) in up to 40% of miscarriages. Therefore, a large number of miscarriages still have an unknown cause. STUDY DESIGN, SAMPLES/MATERIALS, METHODS Whole exome sequencing (WES) was performed using Illumina HiSeq 2000 platform on seven euploid miscarriages from four families with RPL. Golden Helix SVS v8.1.5 was used for data assessment and inheritance analysis for deleterious DNA variants predicted to severely disrupt protein-coding genes by introducing a frameshift, loss of the stop codon, gain of the stop codon, changes in splicing or the initial codon. Webgestalt (http://bioinfo.vanderbilt.edu/webgestalt/) was used for pathway and disease association enrichment analysis of a gene pool containing putatively pathogenic variants in miscarriages and couples in comparison to control gene pools. MAIN RESULTS AND THE ROLE OF CHANCE Compound heterozygous mutations in DYNC2H1 and ALOX15 were identified in miscarriages from two families with RPL. DYNC2H1 is involved in cilia biogenesis and has been associated with fetal lethality in humans. ALOX15 is expressed in placenta and its dysregulation has been associated with inflammation, placental, dysfunction, abnormal oxidative stress response and angiogenesis. The pool of putatively pathogenic single nucleotide variants (SNVs) and small insertions and deletions (indels) detected in the miscarriages showed enrichment in ‘complement and coagulation cascades pathway’, and ‘ciliary motility disorders’. We conclude that CNVs, individual SNVs and pool of deleterious gene mutations identified by exome sequencing could contribute to RPL. LIMITATIONS, REASONS FOR CAUTION The size of our sample cohort is small. The functional effect of candidate mutations should be evaluated to determine whether the mutations are causative. WIDER IMPLICATIONS OF THE FINDINGS This is the first study to assess whether SNVs may contribute to the pathogenesis of miscarriage. Furthermore, our findings suggest that collective effect of mutations in relevant biological pathways could be implicated in RPL. STUDY FUNDING AND COMPETING INTEREST(S) The study was funded by Canadian Institutes of Health Research (grant MOP 106467) and Michael Smith Foundation of Health Research Career Scholar salary award to ERS. PMID:26826164

Novel Genetic Variants of Sporadic Atrial Septal Defect (ASD) in a Chinese Population Identified by Whole-Exome Sequencing (WES)

PubMed Central

Liu, Yong; Cao, Yu; Li, Yaxiong; Lei, Dongyun; Li, Lin; Hou, Zong Liu; Han, Shen; Meng, Mingyao; Shi, Jianlin; Zhang, Yayong; Wang, Yi; Niu, Zhaoyi; Xie, Yanhua; Xiao, Benshan; Wang, Yuanfei; Li, Xiao; Yang, Lirong

2018-01-01

Background Recently, mutations in several genes have been described to be associated with sporadic ASD, but some genetic variants remain to be identified. The aim of this study was to use whole-exome sequencing (WES) combined with bioinformatics analysis to identify novel genetic variants in cases of sporadic congenital ASD, followed by validation by Sanger sequencing. Material/Methods Five Han patients with secundum ASD were recruited, and their tissue samples were analyzed by WES, followed by verification by Sanger sequencing of tissue and blood samples. Further evaluation using blood samples included 452 additional patients with sporadic secundum ASD (212 male and 240 female patients) and 519 healthy subjects (252 male and 267 female subjects) for further verification by a multiplexed MassARRAY system. Bioinformatic analyses were performed to identify novel genetic variants associated with sporadic ASD. Results From five patients with sporadic ASD, a total of 181,762 genomic variants in 33 exon loci, validated by Sanger sequencing, were selected and underwent MassARRAY analysis in 452 patients with ASD and 519 healthy subjects. Three loci with high mutation frequencies, the 138665410 FOXL2 gene variant, the 23862952 MYH6 gene variant, and the 71098693 HYDIN gene variant were found to be significantly associated with sporadic ASD (P<0.05); variants in FOXL2 and MYH6 were found in patients with isolated, sporadic ASD (P<5×10−4). Conclusions This was the first study that demonstrated variants in FOXL2 and HYDIN associated with sporadic ASD, and supported the use of WES and bioinformatics analysis to identify disease-associated mutations. PMID:29505555

Novel Genetic Variants of Sporadic Atrial Septal Defect (ASD) in a Chinese Population Identified by Whole-Exome Sequencing (WES).

PubMed

Liu, Yong; Cao, Yu; Li, Yaxiong; Lei, Dongyun; Li, Lin; Hou, Zong Liu; Han, Shen; Meng, Mingyao; Shi, Jianlin; Zhang, Yayong; Wang, Yi; Niu, Zhaoyi; Xie, Yanhua; Xiao, Benshan; Wang, Yuanfei; Li, Xiao; Yang, Lirong; Wang, Wenju; Jiang, Lihong

2018-03-05

BACKGROUND Recently, mutations in several genes have been described to be associated with sporadic ASD, but some genetic variants remain to be identified. The aim of this study was to use whole-exome sequencing (WES) combined with bioinformatics analysis to identify novel genetic variants in cases of sporadic congenital ASD, followed by validation by Sanger sequencing. MATERIAL AND METHODS Five Han patients with secundum ASD were recruited, and their tissue samples were analyzed by WES, followed by verification by Sanger sequencing of tissue and blood samples. Further evaluation using blood samples included 452 additional patients with sporadic secundum ASD (212 male and 240 female patients) and 519 healthy subjects (252 male and 267 female subjects) for further verification by a multiplexed MassARRAY system. Bioinformatic analyses were performed to identify novel genetic variants associated with sporadic ASD. RESULTS From five patients with sporadic ASD, a total of 181,762 genomic variants in 33 exon loci, validated by Sanger sequencing, were selected and underwent MassARRAY analysis in 452 patients with ASD and 519 healthy subjects. Three loci with high mutation frequencies, the 138665410 FOXL2 gene variant, the 23862952 MYH6 gene variant, and the 71098693 HYDIN gene variant were found to be significantly associated with sporadic ASD (P<0.05); variants in FOXL2 and MYH6 were found in patients with isolated, sporadic ASD (P<5×10^-4). CONCLUSIONS This was the first study that demonstrated variants in FOXL2 and HYDIN associated with sporadic ASD, and supported the use of WES and bioinformatics analysis to identify disease-associated mutations.

Short communication: Validation of 4 candidate causative trait variants in 2 cattle breeds using targeted sequence imputation.

PubMed

Pausch, Hubert; Wurmser, Christine; Reinhardt, Friedrich; Emmerling, Reiner; Fries, Ruedi

2015-06-01

Most association studies for pinpointing trait-associated variants are performed within breed. The availability of sequence data from key ancestors of several cattle breeds now enables immediate assessment of the frequency of trait-associated variants in populations different from the mapping population and their imputation into large validation populations. The objective of this study was to validate the effects of 4 putatively causative variants on milk production traits, male fertility, and stature in German Fleckvieh and Holstein-Friesian animals using targeted sequence imputation. We used whole-genome sequence data of 456 animals to impute 4 missense mutations in DGAT1, GHR, PRLR, and PROP1 into 10,363 Fleckvieh and 8,812 Holstein animals. The accuracy of the imputed genotypes exceeded 95% for all variants. Association testing with imputed variants revealed consistent antagonistic effects of the DGAT1 p.A232K and GHR p.F279Y variants on milk yield and protein and fat contents, respectively, in both breeds. The allele frequency of both polymorphisms has changed considerably in the past 20 yr, indicating that they were targets of recent selection for milk production traits. The PRLR p.S18N variant was associated with yield traits in Fleckvieh but not in Holstein, suggesting that it may be in linkage disequilibrium with a mutation affecting yield traits rather than being causal. The reported effects of the PROP1 p.H173R variant on milk production, male fertility, and stature could not be confirmed. Our results demonstrate that population-wide imputation of candidate causal variants from sequence data is feasible, enabling their rapid validation in large independent populations. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

A novel LPL intronic variant: g.18704C>A identified by re-sequencing Kuwaiti Arab samples is associated with high-density lipoprotein, very low-density lipoprotein and triglyceride lipid levels.

PubMed

Al-Bustan, Suzanne A; Al-Serri, Ahmad; Annice, Babitha G; Alnaqeeb, Majed A; Al-Kandari, Wafa Y; Dashti, Mohammed

2018-01-01

The role interethnic genetic differences play in plasma lipid level variation across populations is a global health concern. Several genes involved in lipid metabolism and transport are strong candidates for the genetic association with lipid level variation especially lipoprotein lipase (LPL). The objective of this study was to re-sequence the full LPL gene in Kuwaiti Arabs, analyse the sequence variation and identify variants that could attribute to variation in plasma lipid levels for further genetic association. Samples (n = 100) of an Arab ethnic group from Kuwait were analysed for sequence variation by Sanger sequencing across the 30 Kb LPL gene and its flanking sequences. A total of 293 variants including 252 single nucleotide polymorphisms (SNPs) and 39 insertions/deletions (InDels) were identified among which 47 variants (32 SNPs and 15 InDels) were novel to Kuwaiti Arabs. This study is the first to report sequence data and analysis of frequencies of variants at the LPL gene locus in an Arab ethnic group with a novel "rare" variant (LPL:g.18704C>A) significantly associated to HDL (B = -0.181; 95% CI (-0.357, -0.006); p = 0.043), TG (B = 0.134; 95% CI (0.004-0.263); p = 0.044) and VLDL (B = 0.131; 95% CI (-0.001-0.263); p = 0.043) levels. Sequence variation in Kuwaiti Arabs was compared to other populations and was found to be similar with regards to the number of SNPs, InDels and distribution of the number of variants across the LPL gene locus and minor allele frequency (MAF). Moreover, comparison of the identified variants and their MAF with other reports provided a list of 46 potential variants across the LPL gene to be considered for future genetic association studies. The findings warrant further investigation into the association of g.18704C>A with lipid levels in other ethnic groups and with clinical manifestations of dyslipidemia.

A novel LPL intronic variant: g.18704C>A identified by re-sequencing Kuwaiti Arab samples is associated with high-density lipoprotein, very low-density lipoprotein and triglyceride lipid levels

PubMed Central

Al-Serri, Ahmad; Annice, Babitha G.; Alnaqeeb, Majed A.; Al-Kandari, Wafa Y.; Dashti, Mohammed

2018-01-01

The role interethnic genetic differences play in plasma lipid level variation across populations is a global health concern. Several genes involved in lipid metabolism and transport are strong candidates for the genetic association with lipid level variation especially lipoprotein lipase (LPL). The objective of this study was to re-sequence the full LPL gene in Kuwaiti Arabs, analyse the sequence variation and identify variants that could attribute to variation in plasma lipid levels for further genetic association. Samples (n = 100) of an Arab ethnic group from Kuwait were analysed for sequence variation by Sanger sequencing across the 30 Kb LPL gene and its flanking sequences. A total of 293 variants including 252 single nucleotide polymorphisms (SNPs) and 39 insertions/deletions (InDels) were identified among which 47 variants (32 SNPs and 15 InDels) were novel to Kuwaiti Arabs. This study is the first to report sequence data and analysis of frequencies of variants at the LPL gene locus in an Arab ethnic group with a novel “rare” variant (LPL:g.18704C>A) significantly associated to HDL (B = -0.181; 95% CI (-0.357, -0.006); p = 0.043), TG (B = 0.134; 95% CI (0.004–0.263); p = 0.044) and VLDL (B = 0.131; 95% CI (-0.001–0.263); p = 0.043) levels. Sequence variation in Kuwaiti Arabs was compared to other populations and was found to be similar with regards to the number of SNPs, InDels and distribution of the number of variants across the LPL gene locus and minor allele frequency (MAF). Moreover, comparison of the identified variants and their MAF with other reports provided a list of 46 potential variants across the LPL gene to be considered for future genetic association studies. The findings warrant further investigation into the association of g.18704C>A with lipid levels in other ethnic groups and with clinical manifestations of dyslipidemia. PMID:29438437

Posttranscriptional regulation of the immediate-early gene EGR1 by light in the mouse retina.

PubMed

Simon, Perikles; Schott, Klaus; Williams, Robert W; Schaeffel, Frank

2004-12-01

Synaptic plasticity is modulated by differential regulation of transcription factors such as EGR1 which binds to DNA via a zinc finger binding domain. Inactivation of EGR1 has implicated this gene as a key regulator of memory formation and learning. However, it remains puzzling how synaptic input can lead to an up-regulation of the EGR-1 protein within only a few minutes. Here, we show by immunohistochemical staining that the EGR-1 protein is localized in synapses throughout the mouse retina. We demonstrate for the first time that two variants of Egr-1 mRNA are produced in the retina by alternative polyadenylation, with the longer version having an additional 293 base pairs at the end of the 3'UTR. Remarkably, the use of the alternative polyadenylation site is controlled by light. The additional 3'UTR sequence of the longer variant displays an even higher level of phylogenetic conservation than the coding region of this highly conserved gene. Additionally, it harbours a cytoplasmic polyadenylation element which is known to respond to NMDA receptor activation. The longer version of the Egr-1 mRNA could therefore rapidly respond to excitatory stimuli such as light or glutamate release whereas the short variant, which is predominantly expressed and contains the full coding sequence, lacks the regulatory elements for cytoplasmic polyadenylation in its 3'UTR.

Implication of Genes for the N-Methyl-D-Aspartate (NMDA) Receptor in Substance Addictions.

PubMed

Chen, Jiali; Ma, Yunlong; Fan, Rongli; Yang, Zhongli; Li, Ming D

2018-02-10

Drug dependence is a chronic brain disease with harmful consequences for both individual users and society. Glutamate is a primary excitatory neurotransmitter in the brain, and both in vivo and in vitro experiments have implicated N-methyl-D-aspartate (NMDA) receptor, a glutamate receptor, as an element in various types of addiction. Recent findings from genetics-based approaches such as genome-wide linkage, candidate gene association, genome-wide association (GWA), and next-generation sequencing have demonstrated the significant association of NMDA receptor subunit genes such as GluN3A, GluN2B, and GluN2A with various addiction-related phenotypes. Of these genes, GluN3A has been the most studied, and it has been revealed to play crucial roles in the etiology of addictions. In this communication, we provide an updated view of the genetic effects of NMDA receptor subunit genes and their functions in the etiology of addictions based on the findings from investigation of both common and rare variants as well as SNP-SNP interactions. To better understand the molecular mechanisms underlying addiction-related behaviors and to promote the development of specific medicines for the prevention and treatment of addictions, current efforts aim not only to identify more causal variants in NMDA receptor subunits by using large independent samples but also to reveal the molecular functions of these variants in addictions.

Identifying Common Genetic Risk Factors of Diabetic Neuropathies

PubMed Central

Witzel, Ini-Isabée; Jelinek, Herbert F.; Khalaf, Kinda; Lee, Sungmun; Khandoker, Ahsan H.; Alsafar, Habiba

2015-01-01

Type 2 diabetes mellitus (T2DM) is a global public health problem of epidemic proportions, with 60–70% of affected individuals suffering from associated neurovascular complications that act on multiple organ systems. The most common and clinically significant neuropathies of T2DM include uremic neuropathy, peripheral neuropathy, and cardiac autonomic neuropathy. These conditions seriously impact an individual’s quality of life and significantly increase the risk of morbidity and mortality. Although advances in gene sequencing technologies have identified several genetic variants that may regulate the development and progression of T2DM, little is known about whether or not the variants are involved in disease progression and how these genetic variants are associated with diabetic neuropathy specifically. Significant missing heritability data and complex disease etiologies remain to be explained. This article is the first to provide a review of the genetic risk variants implicated in the diabetic neuropathies and to highlight potential commonalities. We thereby aim to contribute to the creation of a genetic-metabolic model that will help to elucidate the cause of diabetic neuropathies, evaluate a patient’s risk profile, and ultimately facilitate preventative and targeted treatment for the individual. PMID:26074879

GWAS and fine-mapping of 35 production, reproduction and conformation traits with imputed sequences of 27K Holstein bulls

USDA-ARS?s Scientific Manuscript database

Fine-mapping of causal variants is becoming feasible for complex traits in livestock GWAS, as an increasing number of animals are sequenced. Imputation has been routinely applied to ascertain sequence variants in large genotyped populations based on small reference populations of sequenced animals. ...

Animal selection for whole genome sequencing by quantifying the unique contribution of homozygous haplotypes sequenced

USDA-ARS?s Scientific Manuscript database

Major whole genome sequencing projects promise to identify rare and causal variants within livestock species; however, the efficient selection of animals for sequencing remains a major problem within these surveys. The goal of this project was to develop a library of high accuracy genetic variants f...

GWAS and fine-mapping of 35 production, reproduction, and conformation traits with imputed sequences of 27K Holstein bulls

USDA-ARS?s Scientific Manuscript database

Imputation has been routinely applied to ascertain sequence variants in large genotyped populations based on reference populations of sequenced animals. With the implementation of the 1000 Bull Genomes Project and increasing numbers of animals sequenced, fine-mapping of causal variants is becoming f...

Germline genetic variants with implications for disease risk and therapeutic outcomes.

PubMed

Pasternak, Amy L; Ward, Kristen M; Luzum, Jasmine A; Ellingrod, Vicki L; Hertz, Daniel L

2017-10-01

Genetic testing has multiple clinical applications including disease risk assessment, diagnosis, and pharmacogenomics. Pharmacogenomics can be utilized to predict whether a pharmacologic therapy will be effective or to identify patients at risk for treatment-related toxicity. Although genetic tests are typically ordered for a distinct clinical purpose, the genetic variants that are found may have additional implications for either disease or pharmacology. This review will address multiple examples of germline genetic variants that are informative for both disease and pharmacogenomics. The discussed relationships are diverse. Some of the agents are targeted for the disease-causing genetic variant, while others, although not targeted therapies, have implications for the disease they are used to treat. It is also possible that the disease implications of a genetic variant are unrelated to the pharmacogenomic implications. Some of these examples are considered clinically actionable pharmacogenes, with evidence-based, pharmacologic treatment recommendations, while others are still investigative as areas for additional research. It is important that clinicians are aware of both the disease and pharmacogenomic associations of these germline genetic variants to ensure patients are receiving comprehensive personalized care. Copyright © 2017 the American Physiological Society.

Analysis of intra-host genetic diversity of Prunus necrotic ringspot virus (PNRSV) using amplicon next generation sequencing.

PubMed

Kinoti, Wycliff M; Constable, Fiona E; Nancarrow, Narelle; Plummer, Kim M; Rodoni, Brendan

2017-01-01

PCR amplicon next generation sequencing (NGS) analysis offers a broadly applicable and targeted approach to detect populations of both high- or low-frequency virus variants in one or more plant samples. In this study, amplicon NGS was used to explore the diversity of the tripartite genome virus, Prunus necrotic ringspot virus (PNRSV) from 53 PNRSV-infected trees using amplicons from conserved gene regions of each of PNRSV RNA1, RNA2 and RNA3. Sequencing of the amplicons from 53 PNRSV-infected trees revealed differing levels of polymorphism across the three different components of the PNRSV genome with a total number of 5040, 2083 and 5486 sequence variants observed for RNA1, RNA2 and RNA3 respectively. The RNA2 had the lowest diversity of sequences compared to RNA1 and RNA3, reflecting the lack of flexibility tolerated by the replicase gene that is encoded by this RNA component. Distinct PNRSV phylo-groups, consisting of closely related clusters of sequence variants, were observed in each of PNRSV RNA1, RNA2 and RNA3. Most plant samples had a single phylo-group for each RNA component. Haplotype network analysis showed that smaller clusters of PNRSV sequence variants were genetically connected to the largest sequence variant cluster within a phylo-group of each RNA component. Some plant samples had sequence variants occurring in multiple PNRSV phylo-groups in at least one of each RNA and these phylo-groups formed distinct clades that represent PNRSV genetic strains. Variants within the same phylo-group of each Prunus plant sample had ≥97% similarity and phylo-groups within a Prunus plant sample and between samples had less ≤97% similarity. Based on the analysis of diversity, a definition of a PNRSV genetic strain was proposed. The proposed definition was applied to determine the number of PNRSV genetic strains in each of the plant samples and the complexity in defining genetic strains in multipartite genome viruses was explored.

Whole exome sequencing in an Italian family with isolated maxillary canine agenesis and canine eruption anomalies.

PubMed

Barbato, Ersilia; Traversa, Alice; Guarnieri, Rosanna; Giovannetti, Agnese; Genovesi, Maria Luce; Magliozzi, Maria Rosa; Paolacci, Stefano; Ciolfi, Andrea; Pizzi, Simone; Di Giorgio, Roberto; Tartaglia, Marco; Pizzuti, Antonio; Caputo, Viviana

2018-07-01

The aim of this study was the clinical and molecular characterization of a family segregating a trait consisting of a phenotype specifically involving the maxillary canines, including agenesis, impaction and ectopic eruption, characterized by incomplete penetrance and variable expressivity. Clinical standardized assessment of 14 family members and a whole-exome sequencing (WES) of three affected subjects were performed. WES data analyses (sequence alignment, variant calling, annotation and prioritization) were carried out using an in-house implemented pipeline. Variant filtering retained coding and splice-site high quality private and rare variants. Variant prioritization was performed taking into account both the disruptive impact and the biological relevance of individual variants and genes. Sanger sequencing was performed to validate the variants of interest and to carry out segregation analysis. Prioritization of variants "by function" allowed the identification of multiple variants contributing to the trait, including two concomitant heterozygous variants in EDARADD (c.308C>T, p.Ser103Phe) and COL5A1 (c.1588G>A, p.Gly530Ser), specifically associated with a more severe phenotype (i.e. canine agenesis). Differently, heterozygous variants in genes encoding proteins with a role in the WNT pathway were shared by subjects showing a phenotype of impacted/ectopic erupted canines. This study characterized the genetic contribution underlying a complex trait consisting of isolated canine anomalies in a medium-sized family, highlighting the role of WNT and EDA cell signaling pathways in tooth development. Copyright © 2018 Elsevier Ltd. All rights reserved.

Pooled Resequencing of 122 Ulcerative Colitis Genes in a Large Dutch Cohort Suggests Population-Specific Associations of Rare Variants in MUC2.

PubMed

Visschedijk, Marijn C; Alberts, Rudi; Mucha, Soren; Deelen, Patrick; de Jong, Dirk J; Pierik, Marieke; Spekhorst, Lieke M; Imhann, Floris; van der Meulen-de Jong, Andrea E; van der Woude, C Janneke; van Bodegraven, Adriaan A; Oldenburg, Bas; Löwenberg, Mark; Dijkstra, Gerard; Ellinghaus, David; Schreiber, Stefan; Wijmenga, Cisca; Rivas, Manuel A; Franke, Andre; van Diemen, Cleo C; Weersma, Rinse K

2016-01-01

Genome-wide association studies have revealed several common genetic risk variants for ulcerative colitis (UC). However, little is known about the contribution of rare, large effect genetic variants to UC susceptibility. In this study, we performed a deep targeted re-sequencing of 122 genes in Dutch UC patients in order to investigate the contribution of rare variants to the genetic susceptibility to UC. The selection of genes consists of 111 established human UC susceptibility genes and 11 genes that lead to spontaneous colitis when knocked-out in mice. In addition, we sequenced the promoter regions of 45 genes where known variants exert cis-eQTL-effects. Targeted pooled re-sequencing was performed on DNA of 790 Dutch UC cases. The Genome of the Netherlands project provided sequence data of 500 healthy controls. After quality control and prioritization based on allele frequency and pathogenicity probability, follow-up genotyping of 171 rare variants was performed on 1021 Dutch UC cases and 1166 Dutch controls. Single-variant association and gene-based analyses identified an association of rare variants in the MUC2 gene with UC. The associated variants in the Dutch population could not be replicated in a German replication cohort (1026 UC cases, 3532 controls). In conclusion, this study has identified a putative role for MUC2 on UC susceptibility in the Dutch population and suggests a population-specific contribution of rare variants to UC.

Analysis of human papillomavirus 16 E6, E7 genes and Long Control Region in cervical samples from Uruguayan women.

PubMed

Ramas, Viviana; Mirazo, Santiago; Bonilla, Sylvia; Ruchansky, Dora; Arbiza, Juan

2018-05-15

This study aims to investigate the HPV16 variant distribution by sequence analyses of E6, E7 oncogenes and the Long Control Region (LCR), from cervical cells collected from Uruguayan women, and to reconstruct the phylogenetic relationships among variants. Forty-seven HPV16 variants, obtained from women with HSIL, LSIL, ASCUS and NILM cytological classes were analyzed for LCR and 12 were further studied for E6 and E7. Detailed sequence comparison, genetic heterogeneity analyses and phylogenetic reconstruction were performed. A high variability was observed among LCR sequences, which were distributed in 18 different variants. E6 and E7 sequences exhibited novel non-synonymous substitutions. Uruguayan sequences mainly belonged to the European lineage, and only 5 sequences clustered in non-European branches; 3 of them in the Asian-American and North-American linage and 2 in an African branch. Additionally, 6 new variants from European and African clusters were identified. HPV16 isolates mainly belonged to the European lineage, though strains from African and Asian-American lineages were also identified. Herein is reported for the first time the distribution and molecular characterization of HPV16 variants from Uruguay, providing novel insights on the molecular epidemiology of this infectious disease in the South America. A high variability among HPV 16 isolates mainly belonged to European lineage, provides an extensive sequence dataset from a country with high burden of cervical cancer. Copyright © 2018 Elsevier B.V. All rights reserved.

Whole Exome Sequencing Identifies Rare Protein-Coding Variants in Behçet's Disease.

PubMed

Ognenovski, Mikhail; Renauer, Paul; Gensterblum, Elizabeth; Kötter, Ina; Xenitidis, Theodoros; Henes, Jörg C; Casali, Bruno; Salvarani, Carlo; Direskeneli, Haner; Kaufman, Kenneth M; Sawalha, Amr H

2016-05-01

Behçet's disease (BD) is a systemic inflammatory disease with an incompletely understood etiology. Despite the identification of multiple common genetic variants associated with BD, rare genetic variants have been less explored. We undertook this study to investigate the role of rare variants in BD by performing whole exome sequencing in BD patients of European descent. Whole exome sequencing was performed in a discovery set comprising 14 German BD patients of European descent. For replication and validation, Sanger sequencing and Sequenom genotyping were performed in the discovery set and in 2 additional independent sets of 49 German BD patients and 129 Italian BD patients of European descent. Genetic association analysis was then performed in BD patients and 503 controls of European descent. Functional effects of associated genetic variants were assessed using bioinformatic approaches. Using whole exome sequencing, we identified 77 rare variants (in 74 genes) with predicted protein-damaging effects in BD. These variants were genotyped in 2 additional patient sets and then analyzed to reveal significant associations with BD at 2 genetic variants detected in all 3 patient sets that remained significant after Bonferroni correction. We detected genetic association between BD and LIMK2 (rs149034313), involved in regulating cytoskeletal reorganization, and between BD and NEIL1 (rs5745908), involved in base excision DNA repair (P = 3.22 × 10(-4) and P = 5.16 × 10(-4) , respectively). The LIMK2 association is a missense variant with predicted protein damage that may influence functional interactions with proteins involved in cytoskeletal regulation by Rho GTPase, inflammation mediated by chemokine and cytokine signaling pathways, T cell activation, and angiogenesis (Bonferroni-corrected P = 5.63 × 10(-14) , P = 7.29 × 10(-6) , P = 1.15 × 10(-5) , and P = 6.40 × 10(-3) , respectively). The genetic association in NEIL1 is a predicted splice donor variant that may introduce a deleterious intron retention and result in a noncoding transcript variant. We used whole exome sequencing in BD for the first time and identified 2 rare putative protein-damaging genetic variants associated with this disease. These genetic variants might influence cytoskeletal regulation and DNA repair mechanisms in BD and might provide further insight into increased leukocyte tissue infiltration and the role of oxidative stress in BD. © 2016, American College of Rheumatology.

Low-abundance HIV drug-resistant viral variants in treatment-experienced persons correlate with historical antiretroviral use.

PubMed

Le, Thuy; Chiarella, Jennifer; Simen, Birgitte B; Hanczaruk, Bozena; Egholm, Michael; Landry, Marie L; Dieckhaus, Kevin; Rosen, Marc I; Kozal, Michael J

2009-06-29

It is largely unknown how frequently low-abundance HIV drug-resistant variants at levels under limit of detection of conventional genotyping (<20% of quasi-species) are present in antiretroviral-experienced persons experiencing virologic failure. Further, the clinical implications of low-abundance drug-resistant variants at time of virologic failure are unknown. Plasma samples from 22 antiretroviral-experienced subjects collected at time of virologic failure (viral load 1380 to 304,000 copies/mL) were obtained from a specimen bank (from 2004-2007). The prevalence and profile of drug-resistant mutations were determined using Sanger sequencing and ultra-deep pyrosequencing. Genotypes were interpreted using Stanford HIV database algorithm. Antiretroviral treatment histories were obtained by chart review and correlated with drug-resistant mutations. Low-abundance drug-resistant mutations were detected in all 22 subjects by deep sequencing and only in 3 subjects by Sanger sequencing. In total they accounted for 90 of 247 mutations (36%) detected by deep sequencing; the majority of these (95%) were not detected by standard genotyping. A mean of 4 additional mutations per subject were detected by deep sequencing (p<0.0001, 95%CI: 2.85-5.53). The additional low-abundance drug-resistant mutations increased a subject's genotypic resistance to one or more antiretrovirals in 17 of 22 subjects (77%). When correlated with subjects' antiretroviral treatment histories, the additional low-abundance drug-resistant mutations correlated with the failing antiretroviral drugs in 21% subjects and correlated with historical antiretroviral use in 79% subjects (OR, 13.73; 95% CI, 2.5-74.3, p = 0.0016). Low-abundance HIV drug-resistant mutations in antiretroviral-experienced subjects at time of virologic failure can increase a subject's overall burden of resistance, yet commonly go unrecognized by conventional genotyping. The majority of unrecognized resistant mutations correlate with historical antiretroviral use. Ultra-deep sequencing can provide important historical resistance information for clinicians when planning subsequent antiretroviral regimens for highly treatment-experienced patients, particularly when their prior treatment histories and longitudinal genotypes are not available.

Low-Abundance HIV Drug-Resistant Viral Variants in Treatment-Experienced Persons Correlate with Historical Antiretroviral Use

PubMed Central

Le, Thuy; Chiarella, Jennifer; Simen, Birgitte B.; Hanczaruk, Bozena; Egholm, Michael; Landry, Marie L.; Dieckhaus, Kevin; Rosen, Marc I.; Kozal, Michael J.

2009-01-01

Background It is largely unknown how frequently low-abundance HIV drug-resistant variants at levels under limit of detection of conventional genotyping (<20% of quasi-species) are present in antiretroviral-experienced persons experiencing virologic failure. Further, the clinical implications of low-abundance drug-resistant variants at time of virologic failure are unknown. Methodology/Principal Findings Plasma samples from 22 antiretroviral-experienced subjects collected at time of virologic failure (viral load 1380 to 304,000 copies/mL) were obtained from a specimen bank (from 2004–2007). The prevalence and profile of drug-resistant mutations were determined using Sanger sequencing and ultra-deep pyrosequencing. Genotypes were interpreted using Stanford HIV database algorithm. Antiretroviral treatment histories were obtained by chart review and correlated with drug-resistant mutations. Low-abundance drug-resistant mutations were detected in all 22 subjects by deep sequencing and only in 3 subjects by Sanger sequencing. In total they accounted for 90 of 247 mutations (36%) detected by deep sequencing; the majority of these (95%) were not detected by standard genotyping. A mean of 4 additional mutations per subject were detected by deep sequencing (p<0.0001, 95%CI: 2.85–5.53). The additional low-abundance drug-resistant mutations increased a subject's genotypic resistance to one or more antiretrovirals in 17 of 22 subjects (77%). When correlated with subjects' antiretroviral treatment histories, the additional low-abundance drug-resistant mutations correlated with the failing antiretroviral drugs in 21% subjects and correlated with historical antiretroviral use in 79% subjects (OR, 13.73; 95% CI, 2.5–74.3, p = 0.0016). Conclusions/Significance Low-abundance HIV drug-resistant mutations in antiretroviral-experienced subjects at time of virologic failure can increase a subject's overall burden of resistance, yet commonly go unrecognized by conventional genotyping. The majority of unrecognized resistant mutations correlate with historical antiretroviral use. Ultra-deep sequencing can provide important historical resistance information for clinicians when planning subsequent antiretroviral regimens for highly treatment-experienced patients, particularly when their prior treatment histories and longitudinal genotypes are not available. PMID:19562031

Reducing false-positive incidental findings with ensemble genotyping and logistic regression based variant filtering methods.

PubMed

Hwang, Kyu-Baek; Lee, In-Hee; Park, Jin-Ho; Hambuch, Tina; Choe, Yongjoon; Kim, MinHyeok; Lee, Kyungjoon; Song, Taemin; Neu, Matthew B; Gupta, Neha; Kohane, Isaac S; Green, Robert C; Kong, Sek Won

2014-08-01

As whole genome sequencing (WGS) uncovers variants associated with rare and common diseases, an immediate challenge is to minimize false-positive findings due to sequencing and variant calling errors. False positives can be reduced by combining results from orthogonal sequencing methods, but costly. Here, we present variant filtering approaches using logistic regression (LR) and ensemble genotyping to minimize false positives without sacrificing sensitivity. We evaluated the methods using paired WGS datasets of an extended family prepared using two sequencing platforms and a validated set of variants in NA12878. Using LR or ensemble genotyping based filtering, false-negative rates were significantly reduced by 1.1- to 17.8-fold at the same levels of false discovery rates (5.4% for heterozygous and 4.5% for homozygous single nucleotide variants (SNVs); 30.0% for heterozygous and 18.7% for homozygous insertions; 25.2% for heterozygous and 16.6% for homozygous deletions) compared to the filtering based on genotype quality scores. Moreover, ensemble genotyping excluded > 98% (105,080 of 107,167) of false positives while retaining > 95% (897 of 937) of true positives in de novo mutation (DNM) discovery in NA12878, and performed better than a consensus method using two sequencing platforms. Our proposed methods were effective in prioritizing phenotype-associated variants, and an ensemble genotyping would be essential to minimize false-positive DNM candidates. © 2014 WILEY PERIODICALS, INC.

Targeted next-generation sequencing makes new molecular diagnoses and expands genotype-phenotype relationship in Ehlers-Danlos syndrome.

PubMed

Weerakkody, Ruwan A; Vandrovcova, Jana; Kanonidou, Christina; Mueller, Michael; Gampawar, Piyush; Ibrahim, Yousef; Norsworthy, Penny; Biggs, Jennifer; Abdullah, Abdulshakur; Ross, David; Black, Holly A; Ferguson, David; Cheshire, Nicholas J; Kazkaz, Hanadi; Grahame, Rodney; Ghali, Neeti; Vandersteen, Anthony; Pope, F Michael; Aitman, Timothy J

2016-11-01

Ehlers-Danlos syndrome (EDS) comprises a group of overlapping hereditary disorders of connective tissue with significant morbidity and mortality, including major vascular complications. We sought to identify the diagnostic utility of a next-generation sequencing (NGS) panel in a mixed EDS cohort. We developed and applied PCR-based NGS assays for targeted, unbiased sequencing of 12 collagen and aortopathy genes to a cohort of 177 unrelated EDS patients. Variants were scored blind to previous genetic testing and then compared with results of previous Sanger sequencing. Twenty-eight pathogenic variants in COL5A1/2, COL3A1, FBN1, and COL1A1 and four likely pathogenic variants in COL1A1, TGFBR1/2, and SMAD3 were identified by the NGS assays. These included all previously detected single-nucleotide and other short pathogenic variants in these genes, and seven newly detected pathogenic or likely pathogenic variants leading to clinically significant diagnostic revisions. Twenty-two variants of uncertain significance were identified, seven of which were in aortopathy genes and required clinical follow-up. Unbiased NGS-based sequencing made new molecular diagnoses outside the expected EDS genotype-phenotype relationship and identified previously undetected clinically actionable variants in aortopathy susceptibility genes. These data may be of value in guiding future clinical pathways for genetic diagnosis in EDS.Genet Med 18 11, 1119-1127.

Rare variants in RTEL1 are associated with familial interstitial pneumonia.

PubMed

Cogan, Joy D; Kropski, Jonathan A; Zhao, Min; Mitchell, Daphne B; Rives, Lynette; Markin, Cheryl; Garnett, Errine T; Montgomery, Keri H; Mason, Wendi R; McKean, David F; Powers, Julia; Murphy, Elissa; Olson, Lana M; Choi, Leena; Cheng, Dong-Sheng; Blue, Elizabeth Marchani; Young, Lisa R; Lancaster, Lisa H; Steele, Mark P; Brown, Kevin K; Schwarz, Marvin I; Fingerlin, Tasha E; Schwartz, David A; Lawson, William E; Loyd, James E; Zhao, Zhongming; Phillips, John A; Blackwell, Timothy S

2015-03-15

Up to 20% of cases of idiopathic interstitial pneumonia cluster in families, comprising the syndrome of familial interstitial pneumonia (FIP); however, the genetic basis of FIP remains uncertain in most families. To determine if new disease-causing rare genetic variants could be identified using whole-exome sequencing of affected members from FIP families, providing additional insights into disease pathogenesis. Affected subjects from 25 kindreds were selected from an ongoing FIP registry for whole-exome sequencing from genomic DNA. Candidate rare variants were confirmed by Sanger sequencing, and cosegregation analysis was performed in families, followed by additional sequencing of affected individuals from another 163 kindreds. We identified a potentially damaging rare variant in the gene encoding for regulator of telomere elongation helicase 1 (RTEL1) that segregated with disease and was associated with very short telomeres in peripheral blood mononuclear cells in 1 of 25 families in our original whole-exome sequencing cohort. Evaluation of affected individuals in 163 additional kindreds revealed another eight families (4.7%) with heterozygous rare variants in RTEL1 that segregated with clinical FIP. Probands and unaffected carriers of these rare variants had short telomeres (<10% for age) in peripheral blood mononuclear cells and increased T-circle formation, suggesting impaired RTEL1 function. Rare loss-of-function variants in RTEL1 represent a newly defined genetic predisposition for FIP, supporting the importance of telomere-related pathways in pulmonary fibrosis.

Silent genetic alterations identified by targeted next-generation sequencing in pheochromocytoma/paraganglioma: A clinicopathological correlations.

PubMed

Pillai, Suja; Gopalan, Vinod; Lo, Chung Y; Liew, Victor; Smith, Robert A; Lam, Alfred King Y

2017-02-01

The goal of this pilot study was to develop a customized, cost-effective amplicon panel (Ampliseq) for target sequencing in a cohort of patients with sporadic phaeochromocytoma/paraganglioma. Phaeochromocytoma/paragangliomas from 25 patients were analysed by targeted next-generation sequencing approach using an Ion Torrent PGM instrument. Primers for 15 target genes (NF1, RET, VHL, SDHA, SDHB, SDHC, SDHD, SDHAF2, TMEM127, MAX, MEN1, KIF1Bβ, EPAS1, CDKN2 & PHD2) were designed using ion ampliseq designer. Ion Reporter software and Ingenuity® Variant Analysis™ software (www.ingenuity.com/variants) from Ingenuity Systems were used to analysis these results. Overall, 713 variants were identified. The variants identified from the Ion Reporter ranged from 64 to 161 per patient. Single nucleotide variants (SNV) were the most common. Further annotation with the help of Ingenuity variant analysis revealed 29 of these 713variants were deletions. Of these, six variants were non-pathogenic and four were likely to be pathogenic. The remaining 19 variants were of uncertain significance. The most frequently altered gene in the cohort was KIF1B followed by NF1. Novel KIF1B pathogenic variant c.3375+1G>A was identified. The mutation was noted in a patient with clinically confirmed neurofibromatosis. Chromosome 1 showed the presence of maximum number of variants. Use of targeted next-generation sequencing is a sensitive method for the detecting genetic changes in patients with phaeochromocytoma/paraganglioma. The precise detection of these genetic changes helps in understanding the pathogenesis of these tumours. Copyright © 2016 Elsevier Inc. All rights reserved.

Genomic Rearrangements in Arabidopsis Considered as Quantitative Traits.

PubMed

Imprialou, Martha; Kahles, André; Steffen, Joshua G; Osborne, Edward J; Gan, Xiangchao; Lempe, Janne; Bhomra, Amarjit; Belfield, Eric; Visscher, Anne; Greenhalgh, Robert; Harberd, Nicholas P; Goram, Richard; Hein, Jotun; Robert-Seilaniantz, Alexandre; Jones, Jonathan; Stegle, Oliver; Kover, Paula; Tsiantis, Miltos; Nordborg, Magnus; Rätsch, Gunnar; Clark, Richard M; Mott, Richard

2017-04-01

To understand the population genetics of structural variants and their effects on phenotypes, we developed an approach to mapping structural variants that segregate in a population sequenced at low coverage. We avoid calling structural variants directly. Instead, the evidence for a potential structural variant at a locus is indicated by variation in the counts of short-reads that map anomalously to that locus. These structural variant traits are treated as quantitative traits and mapped genetically, analogously to a gene expression study. Association between a structural variant trait at one locus, and genotypes at a distant locus indicate the origin and target of a transposition. Using ultra-low-coverage (0.3×) population sequence data from 488 recombinant inbred Arabidopsis thaliana genomes, we identified 6502 segregating structural variants. Remarkably, 25% of these were transpositions. While many structural variants cannot be delineated precisely, we validated 83% of 44 predicted transposition breakpoints by polymerase chain reaction. We show that specific structural variants may be causative for quantitative trait loci for germination and resistance to infection by the fungus Albugo laibachii , isolate Nc14. Further we show that the phenotypic heritability attributable to read-mapping anomalies differs from, and, in the case of time to germination and bolting, exceeds that due to standard genetic variation. Genes within structural variants are also more likely to be silenced or dysregulated. This approach complements the prevalent strategy of structural variant discovery in fewer individuals sequenced at high coverage. It is generally applicable to large populations sequenced at low-coverage, and is particularly suited to mapping transpositions. Copyright © 2017 by the Genetics Society of America.

Process of labeling specific chromosomes using recombinant repetitive DNA

DOEpatents

Moyzis, R.K.; Meyne, J.

1988-02-12

Chromosome preferential nucleotide sequences are first determined from a library of recombinant DNA clones having families of repetitive sequences. Library clones are identified with a low homology with a sequence of repetitive DNA families to which the first clones respectively belong and variant sequences are then identified by selecting clones having a pattern of hybridization with genomic DNA dissimilar to the hybridization pattern shown by the respective families. In another embodiment, variant sequences are selected from a sequence of a known repetitive DNA family. The selected variant sequence is classified as chromosome specific, chromosome preferential, or chromosome nonspecific. Sequences which are classified as chromosome preferential are further sequenced and regions are identified having a low homology with other regions of the chromosome preferential sequence or with known sequences of other family members and consensus sequences of the repetitive DNA families for the chromosome preferential sequences. The selected low homology regions are then hybridized with chromosomes to determine those low homology regions hybridized with a specific chromosome under normal stringency conditions.

A survey of tools for variant analysis of next-generation genome sequencing data

PubMed Central

Pabinger, Stephan; Dander, Andreas; Fischer, Maria; Snajder, Rene; Sperk, Michael; Efremova, Mirjana; Krabichler, Birgit; Speicher, Michael R.; Zschocke, Johannes

2014-01-01

Recent advances in genome sequencing technologies provide unprecedented opportunities to characterize individual genomic landscapes and identify mutations relevant for diagnosis and therapy. Specifically, whole-exome sequencing using next-generation sequencing (NGS) technologies is gaining popularity in the human genetics community due to the moderate costs, manageable data amounts and straightforward interpretation of analysis results. While whole-exome and, in the near future, whole-genome sequencing are becoming commodities, data analysis still poses significant challenges and led to the development of a plethora of tools supporting specific parts of the analysis workflow or providing a complete solution. Here, we surveyed 205 tools for whole-genome/whole-exome sequencing data analysis supporting five distinct analytical steps: quality assessment, alignment, variant identification, variant annotation and visualization. We report an overview of the functionality, features and specific requirements of the individual tools. We then selected 32 programs for variant identification, variant annotation and visualization, which were subjected to hands-on evaluation using four data sets: one set of exome data from two patients with a rare disease for testing identification of germline mutations, two cancer data sets for testing variant callers for somatic mutations, copy number variations and structural variations, and one semi-synthetic data set for testing identification of copy number variations. Our comprehensive survey and evaluation of NGS tools provides a valuable guideline for human geneticists working on Mendelian disorders, complex diseases and cancers. PMID:23341494

Screening for rare variants in the PNPLA3 gene in obese liver biopsy patients.

PubMed

Zegers, Doreen; Verrijken, An; Francque, Sven; de Freitas, Fenna; Beckers, Sigri; Aerts, Evi; Ruppert, Martin; Hubens, Guy; Michielsen, Peter; Van Hul, Wim; Van Gaal, Luc F

2016-12-01

Previous research has clearly implicated the PNPLA3 gene in the etiology of nonalcoholic fatty liver disease as a polymorphism in the gene was found to be robustly associated to the disease. However, data on the involvement of rare PNPLA3 variants in the development of nonalcoholic fatty liver disease (NAFLD) is currently limited. Therefore, we performed an extensive mutation analysis study on a cohort of obese liver biopsy patients to determine PNPLA3 variation and its correlation with fatty liver disease. We screened the entire coding region of the PNPLA3 gene in DNA samples of 393 obese liver biopsy patients with varying degrees of fatty liver disease. Mutation analysis was performed by high-resolution melting curve analysis in combination with direct sequencing. We identified several common polymorphisms as well as one rare synonymous variant (c.867G>A rs139896256), one rare intronic variant (c.979+13C>T) and 3 nonsynonymous coding variants (p.A76T, p.A104V and p.T200M) in the PNPLA3 gene. In silico analysis indicated that the p.A104V variant will probably have no functional effect, whereas for the p.A76T and p.T200M variant a possible pathogenic effect is suggested. Overall, we showed that novel variants in PNPLA3 are very rare in our liver biopsy cohort, thereby indicating that their impact on the etiology of NAFLD is probably limited. Nevertheless, for the three rare coding variants that were identified in patients with advanced liver disease, further functional characterization will be essential to verify their potential disease causality. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Inferring Short-Range Linkage Information from Sequencing Chromatograms

PubMed Central

Beggel, Bastian; Neumann-Fraune, Maria; Kaiser, Rolf; Verheyen, Jens; Lengauer, Thomas

2013-01-01

Direct Sanger sequencing of viral genome populations yields multiple ambiguous sequence positions. It is not straightforward to derive linkage information from sequencing chromatograms, which in turn hampers the correct interpretation of the sequence data. We present a method for determining the variants existing in a viral quasispecies in the case of two nearby ambiguous sequence positions by exploiting the effect of sequence context-dependent incorporation of dideoxynucleotides. The computational model was trained on data from sequencing chromatograms of clonal variants and was evaluated on two test sets of in vitro mixtures. The approach achieved high accuracies in identifying the mixture components of 97.4% on a test set in which the positions to be analyzed are only one base apart from each other, and of 84.5% on a test set in which the ambiguous positions are separated by three bases. In silico experiments suggest two major limitations of our approach in terms of accuracy. First, due to a basic limitation of Sanger sequencing, it is not possible to reliably detect minor variants with a relative frequency of no more than 10%. Second, the model cannot distinguish between mixtures of two or four clonal variants, if one of two sets of linear constraints is fulfilled. Furthermore, the approach requires repetitive sequencing of all variants that might be present in the mixture to be analyzed. Nevertheless, the effectiveness of our method on the two in vitro test sets shows that short-range linkage information of two ambiguous sequence positions can be inferred from Sanger sequencing chromatograms without any further assumptions on the mixture composition. Additionally, our model provides new insights into the established and widely used Sanger sequencing technology. The source code of our method is made available at http://bioinf.mpi-inf.mpg.de/publications/beggel/linkageinformation.zip. PMID:24376502

Common and rare variants associated with kidney stones and biochemical traits

PubMed Central

Oddsson, Asmundur; Sulem, Patrick; Helgason, Hannes; Edvardsson, Vidar O.; Thorleifsson, Gudmar; Sveinbjörnsson, Gardar; Haraldsdottir, Eik; Eyjolfsson, Gudmundur I.; Sigurdardottir, Olof; Olafsson, Isleifur; Masson, Gisli; Holm, Hilma; Gudbjartsson, Daniel F.; Thorsteinsdottir, Unnur; Indridason, Olafur S.; Palsson, Runolfur; Stefansson, Kari

2015-01-01

Kidney stone disease is a complex disorder with a strong genetic component. We conducted a genome-wide association study of 28.3 million sequence variants detected through whole-genome sequencing of 2,636 Icelanders that were imputed into 5,419 kidney stone cases, including 2,172 cases with a history of recurrent kidney stones, and 279,870 controls. We identify sequence variants associating with kidney stones at ALPL (rs1256328[T], odds ratio (OR)=1.21, P=5.8 × 10−10) and a suggestive association at CASR (rs7627468[A], OR=1.16, P=2.0 × 10−8). Focusing our analysis on coding sequence variants in 63 genes with preferential kidney expression we identify two rare missense variants SLC34A1 p.Tyr489Cys (OR=2.38, P=2.8 × 10−5) and TRPV5 p.Leu530Arg (OR=3.62, P=4.1 × 10−5) associating with recurrent kidney stones. We also observe associations of the identified kidney stone variants with biochemical traits in a large population set, indicating potential biological mechanism. PMID:26272126

Common and rare variants associated with kidney stones and biochemical traits.

PubMed

Oddsson, Asmundur; Sulem, Patrick; Helgason, Hannes; Edvardsson, Vidar O; Thorleifsson, Gudmar; Sveinbjörnsson, Gardar; Haraldsdottir, Eik; Eyjolfsson, Gudmundur I; Sigurdardottir, Olof; Olafsson, Isleifur; Masson, Gisli; Holm, Hilma; Gudbjartsson, Daniel F; Thorsteinsdottir, Unnur; Indridason, Olafur S; Palsson, Runolfur; Stefansson, Kari

2015-08-14

Kidney stone disease is a complex disorder with a strong genetic component. We conducted a genome-wide association study of 28.3 million sequence variants detected through whole-genome sequencing of 2,636 Icelanders that were imputed into 5,419 kidney stone cases, including 2,172 cases with a history of recurrent kidney stones, and 279,870 controls. We identify sequence variants associating with kidney stones at ALPL (rs1256328[T], odds ratio (OR)=1.21, P=5.8 × 10(-10)) and a suggestive association at CASR (rs7627468[A], OR=1.16, P=2.0 × 10(-8)). Focusing our analysis on coding sequence variants in 63 genes with preferential kidney expression we identify two rare missense variants SLC34A1 p.Tyr489Cys (OR=2.38, P=2.8 × 10(-5)) and TRPV5 p.Leu530Arg (OR=3.62, P=4.1 × 10(-5)) associating with recurrent kidney stones. We also observe associations of the identified kidney stone variants with biochemical traits in a large population set, indicating potential biological mechanism.

From days to hours: reporting clinically actionable variants from whole genome sequencing.

PubMed

Middha, Sumit; Baheti, Saurabh; Hart, Steven N; Kocher, Jean-Pierre A

2014-01-01

As the cost of whole genome sequencing (WGS) decreases, clinical laboratories will be looking at broadly adopting this technology to screen for variants of clinical significance. To fully leverage this technology in a clinical setting, results need to be reported quickly, as the turnaround rate could potentially impact patient care. The latest sequencers can sequence a whole human genome in about 24 hours. However, depending on the computing infrastructure available, the processing of data can take several days, with the majority of computing time devoted to aligning reads to genomics regions that are to date not clinically interpretable. In an attempt to accelerate the reporting of clinically actionable variants, we have investigated the utility of a multi-step alignment algorithm focused on aligning reads and calling variants in genomic regions of clinical relevance prior to processing the remaining reads on the whole genome. This iterative workflow significantly accelerates the reporting of clinically actionable variants with no loss of accuracy when compared to genotypes obtained with the OMNI SNP platform or to variants detected with a standard workflow that combines Novoalign and GATK.

The genetic architecture of type 2 diabetes.

PubMed

Fuchsberger, Christian; Flannick, Jason; Teslovich, Tanya M; Mahajan, Anubha; Agarwala, Vineeta; Gaulton, Kyle J; Ma, Clement; Fontanillas, Pierre; Moutsianas, Loukas; McCarthy, Davis J; Rivas, Manuel A; Perry, John R B; Sim, Xueling; Blackwell, Thomas W; Robertson, Neil R; Rayner, N William; Cingolani, Pablo; Locke, Adam E; Tajes, Juan Fernandez; Highland, Heather M; Dupuis, Josee; Chines, Peter S; Lindgren, Cecilia M; Hartl, Christopher; Jackson, Anne U; Chen, Han; Huyghe, Jeroen R; van de Bunt, Martijn; Pearson, Richard D; Kumar, Ashish; Müller-Nurasyid, Martina; Grarup, Niels; Stringham, Heather M; Gamazon, Eric R; Lee, Jaehoon; Chen, Yuhui; Scott, Robert A; Below, Jennifer E; Chen, Peng; Huang, Jinyan; Go, Min Jin; Stitzel, Michael L; Pasko, Dorota; Parker, Stephen C J; Varga, Tibor V; Green, Todd; Beer, Nicola L; Day-Williams, Aaron G; Ferreira, Teresa; Fingerlin, Tasha; Horikoshi, Momoko; Hu, Cheng; Huh, Iksoo; Ikram, Mohammad Kamran; Kim, Bong-Jo; Kim, Yongkang; Kim, Young Jin; Kwon, Min-Seok; Lee, Juyoung; Lee, Selyeong; Lin, Keng-Han; Maxwell, Taylor J; Nagai, Yoshihiko; Wang, Xu; Welch, Ryan P; Yoon, Joon; Zhang, Weihua; Barzilai, Nir; Voight, Benjamin F; Han, Bok-Ghee; Jenkinson, Christopher P; Kuulasmaa, Teemu; Kuusisto, Johanna; Manning, Alisa; Ng, Maggie C Y; Palmer, Nicholette D; Balkau, Beverley; Stančáková, Alena; Abboud, Hanna E; Boeing, Heiner; Giedraitis, Vilmantas; Prabhakaran, Dorairaj; Gottesman, Omri; Scott, James; Carey, Jason; Kwan, Phoenix; Grant, George; Smith, Joshua D; Neale, Benjamin M; Purcell, Shaun; Butterworth, Adam S; Howson, Joanna M M; Lee, Heung Man; Lu, Yingchang; Kwak, Soo-Heon; Zhao, Wei; Danesh, John; Lam, Vincent K L; Park, Kyong Soo; Saleheen, Danish; So, Wing Yee; Tam, Claudia H T; Afzal, Uzma; Aguilar, David; Arya, Rector; Aung, Tin; Chan, Edmund; Navarro, Carmen; Cheng, Ching-Yu; Palli, Domenico; Correa, Adolfo; Curran, Joanne E; Rybin, Denis; Farook, Vidya S; Fowler, Sharon P; Freedman, Barry I; Griswold, Michael; Hale, Daniel Esten; Hicks, Pamela J; Khor, Chiea-Chuen; Kumar, Satish; Lehne, Benjamin; Thuillier, Dorothée; Lim, Wei Yen; Liu, Jianjun; van der Schouw, Yvonne T; Loh, Marie; Musani, Solomon K; Puppala, Sobha; Scott, William R; Yengo, Loïc; Tan, Sian-Tsung; Taylor, Herman A; Thameem, Farook; Wilson, Gregory; Wong, Tien Yin; Njølstad, Pål Rasmus; Levy, Jonathan C; Mangino, Massimo; Bonnycastle, Lori L; Schwarzmayr, Thomas; Fadista, João; Surdulescu, Gabriela L; Herder, Christian; Groves, Christopher J; Wieland, Thomas; Bork-Jensen, Jette; Brandslund, Ivan; Christensen, Cramer; Koistinen, Heikki A; Doney, Alex S F; Kinnunen, Leena; Esko, Tõnu; Farmer, Andrew J; Hakaste, Liisa; Hodgkiss, Dylan; Kravic, Jasmina; Lyssenko, Valeriya; Hollensted, Mette; Jørgensen, Marit E; Jørgensen, Torben; Ladenvall, Claes; Justesen, Johanne Marie; Käräjämäki, Annemari; Kriebel, Jennifer; Rathmann, Wolfgang; Lannfelt, Lars; Lauritzen, Torsten; Narisu, Narisu; Linneberg, Allan; Melander, Olle; Milani, Lili; Neville, Matt; Orho-Melander, Marju; Qi, Lu; Qi, Qibin; Roden, Michael; Rolandsson, Olov; Swift, Amy; Rosengren, Anders H; Stirrups, Kathleen; Wood, Andrew R; Mihailov, Evelin; Blancher, Christine; Carneiro, Mauricio O; Maguire, Jared; Poplin, Ryan; Shakir, Khalid; Fennell, Timothy; DePristo, Mark; de Angelis, Martin Hrabé; Deloukas, Panos; Gjesing, Anette P; Jun, Goo; Nilsson, Peter; Murphy, Jacquelyn; Onofrio, Robert; Thorand, Barbara; Hansen, Torben; Meisinger, Christa; Hu, Frank B; Isomaa, Bo; Karpe, Fredrik; Liang, Liming; Peters, Annette; Huth, Cornelia; O'Rahilly, Stephen P; Palmer, Colin N A; Pedersen, Oluf; Rauramaa, Rainer; Tuomilehto, Jaakko; Salomaa, Veikko; Watanabe, Richard M; Syvänen, Ann-Christine; Bergman, Richard N; Bharadwaj, Dwaipayan; Bottinger, Erwin P; Cho, Yoon Shin; Chandak, Giriraj R; Chan, Juliana C N; Chia, Kee Seng; Daly, Mark J; Ebrahim, Shah B; Langenberg, Claudia; Elliott, Paul; Jablonski, Kathleen A; Lehman, Donna M; Jia, Weiping; Ma, Ronald C W; Pollin, Toni I; Sandhu, Manjinder; Tandon, Nikhil; Froguel, Philippe; Barroso, Inês; Teo, Yik Ying; Zeggini, Eleftheria; Loos, Ruth J F; Small, Kerrin S; Ried, Janina S; DeFronzo, Ralph A; Grallert, Harald; Glaser, Benjamin; Metspalu, Andres; Wareham, Nicholas J; Walker, Mark; Banks, Eric; Gieger, Christian; Ingelsson, Erik; Im, Hae Kyung; Illig, Thomas; Franks, Paul W; Buck, Gemma; Trakalo, Joseph; Buck, David; Prokopenko, Inga; Mägi, Reedik; Lind, Lars; Farjoun, Yossi; Owen, Katharine R; Gloyn, Anna L; Strauch, Konstantin; Tuomi, Tiinamaija; Kooner, Jaspal Singh; Lee, Jong-Young; Park, Taesung; Donnelly, Peter; Morris, Andrew D; Hattersley, Andrew T; Bowden, Donald W; Collins, Francis S; Atzmon, Gil; Chambers, John C; Spector, Timothy D; Laakso, Markku; Strom, Tim M; Bell, Graeme I; Blangero, John; Duggirala, Ravindranath; Tai, E Shyong; McVean, Gilean; Hanis, Craig L; Wilson, James G; Seielstad, Mark; Frayling, Timothy M; Meigs, James B; Cox, Nancy J; Sladek, Rob; Lander, Eric S; Gabriel, Stacey; Burtt, Noël P; Mohlke, Karen L; Meitinger, Thomas; Groop, Leif; Abecasis, Goncalo; Florez, Jose C; Scott, Laura J; Morris, Andrew P; Kang, Hyun Min; Boehnke, Michael; Altshuler, David; McCarthy, Mark I

2016-08-04

The genetic architecture of common traits, including the number, frequency, and effect sizes of inherited variants that contribute to individual risk, has been long debated. Genome-wide association studies have identified scores of common variants associated with type 2 diabetes, but in aggregate, these explain only a fraction of the heritability of this disease. Here, to test the hypothesis that lower-frequency variants explain much of the remainder, the GoT2D and T2D-GENES consortia performed whole-genome sequencing in 2,657 European individuals with and without diabetes, and exome sequencing in 12,940 individuals from five ancestry groups. To increase statistical power, we expanded the sample size via genotyping and imputation in a further 111,548 subjects. Variants associated with type 2 diabetes after sequencing were overwhelmingly common and most fell within regions previously identified by genome-wide association studies. Comprehensive enumeration of sequence variation is necessary to identify functional alleles that provide important clues to disease pathophysiology, but large-scale sequencing does not support the idea that lower-frequency variants have a major role in predisposition to type 2 diabetes.

The genetic architecture of type 2 diabetes

PubMed Central

Ma, Clement; Fontanillas, Pierre; Moutsianas, Loukas; McCarthy, Davis J; Rivas, Manuel A; Perry, John R B; Sim, Xueling; Blackwell, Thomas W; Robertson, Neil R; Rayner, N William; Cingolani, Pablo; Locke, Adam E; Tajes, Juan Fernandez; Highland, Heather M; Dupuis, Josee; Chines, Peter S; Lindgren, Cecilia M; Hartl, Christopher; Jackson, Anne U; Chen, Han; Huyghe, Jeroen R; van de Bunt, Martijn; Pearson, Richard D; Kumar, Ashish; Müller-Nurasyid, Martina; Grarup, Niels; Stringham, Heather M; Gamazon, Eric R; Lee, Jaehoon; Chen, Yuhui; Scott, Robert A; Below, Jennifer E; Chen, Peng; Huang, Jinyan; Go, Min Jin; Stitzel, Michael L; Pasko, Dorota; Parker, Stephen C J; Varga, Tibor V; Green, Todd; Beer, Nicola L; Day-Williams, Aaron G; Ferreira, Teresa; Fingerlin, Tasha; Horikoshi, Momoko; Hu, Cheng; Huh, Iksoo; Ikram, Mohammad Kamran; Kim, Bong-Jo; Kim, Yongkang; Kim, Young Jin; Kwon, Min-Seok; Lee, Juyoung; Lee, Selyeong; Lin, Keng-Han; Maxwell, Taylor J; Nagai, Yoshihiko; Wang, Xu; Welch, Ryan P; Yoon, Joon; Zhang, Weihua; Barzilai, Nir; Voight, Benjamin F; Han, Bok-Ghee; Jenkinson, Christopher P; Kuulasmaa, Teemu; Kuusisto, Johanna; Manning, Alisa; Ng, Maggie C Y; Palmer, Nicholette D; Balkau, Beverley; Stančáková, Alena; Abboud, Hanna E; Boeing, Heiner; Giedraitis, Vilmantas; Prabhakaran, Dorairaj; Gottesman, Omri; Scott, James; Carey, Jason; Kwan, Phoenix; Grant, George; Smith, Joshua D; Neale, Benjamin M; Purcell, Shaun; Butterworth, Adam S; Howson, Joanna M M; Lee, Heung Man; Lu, Yingchang; Kwak, Soo-Heon; Zhao, Wei; Danesh, John; Lam, Vincent K L; Park, Kyong Soo; Saleheen, Danish; So, Wing Yee; Tam, Claudia H T; Afzal, Uzma; Aguilar, David; Arya, Rector; Aung, Tin; Chan, Edmund; Navarro, Carmen; Cheng, Ching-Yu; Palli, Domenico; Correa, Adolfo; Curran, Joanne E; Rybin, Denis; Farook, Vidya S; Fowler, Sharon P; Freedman, Barry I; Griswold, Michael; Hale, Daniel Esten; Hicks, Pamela J; Khor, Chiea-Chuen; Kumar, Satish; Lehne, Benjamin; Thuillier, Dorothée; Lim, Wei Yen; Liu, Jianjun; van der Schouw, Yvonne T; Loh, Marie; Musani, Solomon K; Puppala, Sobha; Scott, William R; Yengo, Loïc; Tan, Sian-Tsung; Taylor, Herman A; Thameem, Farook; Wilson, Gregory; Wong, Tien Yin; Njølstad, Pål Rasmus; Levy, Jonathan C; Mangino, Massimo; Bonnycastle, Lori L; Schwarzmayr, Thomas; Fadista, João; Surdulescu, Gabriela L; Herder, Christian; Groves, Christopher J; Wieland, Thomas; Bork-Jensen, Jette; Brandslund, Ivan; Christensen, Cramer; Koistinen, Heikki A; Doney, Alex S F; Kinnunen, Leena; Esko, Tõnu; Farmer, Andrew J; Hakaste, Liisa; Hodgkiss, Dylan; Kravic, Jasmina; Lyssenko, Valeriya; Hollensted, Mette; Jørgensen, Marit E; Jørgensen, Torben; Ladenvall, Claes; Justesen, Johanne Marie; Käräjämäki, Annemari; Kriebel, Jennifer; Rathmann, Wolfgang; Lannfelt, Lars; Lauritzen, Torsten; Narisu, Narisu; Linneberg, Allan; Melander, Olle; Milani, Lili; Neville, Matt; Orho-Melander, Marju; Qi, Lu; Qi, Qibin; Roden, Michael; Rolandsson, Olov; Swift, Amy; Rosengren, Anders H; Stirrups, Kathleen; Wood, Andrew R; Mihailov, Evelin; Blancher, Christine; Carneiro, Mauricio O; Maguire, Jared; Poplin, Ryan; Shakir, Khalid; Fennell, Timothy; DePristo, Mark; de Angelis, Martin Hrabé; Deloukas, Panos; Gjesing, Anette P; Jun, Goo; Nilsson, Peter; Murphy, Jacquelyn; Onofrio, Robert; Thorand, Barbara; Hansen, Torben; Meisinger, Christa; Hu, Frank B; Isomaa, Bo; Karpe, Fredrik; Liang, Liming; Peters, Annette; Huth, Cornelia; O'Rahilly, Stephen P; Palmer, Colin N A; Pedersen, Oluf; Rauramaa, Rainer; Tuomilehto, Jaakko; Salomaa, Veikko; Watanabe, Richard M; Syvänen, Ann-Christine; Bergman, Richard N; Bharadwaj, Dwaipayan; Bottinger, Erwin P; Cho, Yoon Shin; Chandak, Giriraj R; Chan, Juliana C N; Chia, Kee Seng; Daly, Mark J; Ebrahim, Shah B; Langenberg, Claudia; Elliott, Paul; Jablonski, Kathleen A; Lehman, Donna M; Jia, Weiping; Ma, Ronald C W; Pollin, Toni I; Sandhu, Manjinder; Tandon, Nikhil; Froguel, Philippe; Barroso, Inês; Teo, Yik Ying; Zeggini, Eleftheria; Loos, Ruth J F; Small, Kerrin S; Ried, Janina S; DeFronzo, Ralph A; Grallert, Harald; Glaser, Benjamin; Metspalu, Andres; Wareham, Nicholas J; Walker, Mark; Banks, Eric; Gieger, Christian; Ingelsson, Erik; Im, Hae Kyung; Illig, Thomas; Franks, Paul W; Buck, Gemma; Trakalo, Joseph; Buck, David; Prokopenko, Inga; Mägi, Reedik; Lind, Lars; Farjoun, Yossi; Owen, Katharine R; Gloyn, Anna L; Strauch, Konstantin; Tuomi, Tiinamaija; Kooner, Jaspal Singh; Lee, Jong-Young; Park, Taesung; Donnelly, Peter; Morris, Andrew D; Hattersley, Andrew T; Bowden, Donald W; Collins, Francis S; Atzmon, Gil; Chambers, John C; Spector, Timothy D; Laakso, Markku; Strom, Tim M; Bell, Graeme I; Blangero, John; Duggirala, Ravindranath; Tai, E Shyong; McVean, Gilean; Hanis, Craig L; Wilson, James G; Seielstad, Mark; Frayling, Timothy M; Meigs, James B; Cox, Nancy J; Sladek, Rob; Lander, Eric S; Gabriel, Stacey; Burtt, Noël P; Mohlke, Karen L; Meitinger, Thomas; Groop, Leif; Abecasis, Goncalo; Florez, Jose C; Scott, Laura J; Morris, Andrew P; Kang, Hyun Min; Boehnke, Michael; Altshuler, David; McCarthy, Mark I

2016-01-01

The genetic architecture of common traits, including the number, frequency, and effect sizes of inherited variants that contribute to individual risk, has been long debated. Genome-wide association studies have identified scores of common variants associated with type 2 diabetes, but in aggregate, these explain only a fraction of heritability. To test the hypothesis that lower-frequency variants explain much of the remainder, the GoT2D and T2D-GENES consortia performed whole genome sequencing in 2,657 Europeans with and without diabetes, and exome sequencing in a total of 12,940 subjects from five ancestral groups. To increase statistical power, we expanded sample size via genotyping and imputation in a further 111,548 subjects. Variants associated with type 2 diabetes after sequencing were overwhelmingly common and most fell within regions previously identified by genome-wide association studies. Comprehensive enumeration of sequence variation is necessary to identify functional alleles that provide important clues to disease pathophysiology, but large-scale sequencing does not support a major role for lower-frequency variants in predisposition to type 2 diabetes. PMID:27398621

Sequence Variation in the Small-Subunit rRNA Gene of Plasmodium malariae and Prevalence of Isolates with the Variant Sequence in Sichuan, China

PubMed Central

Liu, Qing; Zhu, Shenghua; Mizuno, Sahoko; Kimura, Masatsugu; Liu, Peina; Isomura, Shin; Wang, Xingzhen; Kawamoto, Fumihiko

1998-01-01

By two PCR-based diagnostic methods, Plasmodium malariae infections have been rediscovered at two foci in the Sichuan province of China, a region where no cases of P. malariae have been officially reported for the last 2 decades. In addition, a variant form of P. malariae which has a deletion of 19 bp and seven substitutions of base pairs in the target sequence of the small-subunit (SSU) rRNA gene was detected with high frequency. Alignment analysis of Plasmodium sp. SSU rRNA gene sequences revealed that the 5′ region of the variant sequence is identical to that of P. vivax or P. knowlesi and its 3′ region is identical to that of P. malariae. The same sequence variations were also found in P. malariae isolates collected along the Thai-Myanmar border, suggesting a wide distribution of this variant form from southern China to Southeast Asia. PMID:9774600

MYO7A and USH2A gene sequence variants in Italian patients with Usher syndrome.

PubMed

Sodi, Andrea; Mariottini, Alessandro; Passerini, Ilaria; Murro, Vittoria; Tachyla, Iryna; Bianchi, Benedetta; Menchini, Ugo; Torricelli, Francesca

2014-01-01

To analyze the spectrum of sequence variants in the MYO7A and USH2A genes in a group of Italian patients affected by Usher syndrome (USH). Thirty-six Italian patients with a diagnosis of USH were recruited. They received a standard ophthalmologic examination, visual field testing, optical coherence tomography (OCT) scan, and electrophysiological tests. Fluorescein angiography and fundus autofluorescence imaging were performed in selected cases. All the patients underwent an audiologic examination for the 0.25-8,000 Hz frequencies. Vestibular function was evaluated with specific tests. DNA samples were analyzed for sequence variants of the MYO7A gene (for USH1) and the USH2A gene (for USH2) with direct sequencing techniques. A few patients were analyzed for both genes. In the MYO7A gene, ten missense variants were found; three patients were compound heterozygous, and two were homozygous. Thirty-four USH2A gene variants were detected, including eight missense variants, nine nonsense variants, six splicing variants, and 11 duplications/deletions; 19 patients were compound heterozygous, and three were homozygous. Four MYO7A and 17 USH2A variants have already been described in the literature. Among the novel mutations there are four USH2A large deletions, detected with multiplex ligation dependent probe amplification (MLPA) technology. Two potentially pathogenic variants were found in 27 patients (75%). Affected patients showed variable clinical pictures without a clear genotype-phenotype correlation. Ten variants in the MYO7A gene and 34 variants in the USH2A gene were detected in Italian patients with USH at a high detection rate. A selective analysis of these genes may be valuable for molecular analysis, combining diagnostic efficiency with little time wastage and less resource consumption.

Higher criticism approach to detect rare variants using whole genome sequencing data

PubMed Central

2014-01-01

Because of low statistical power of single-variant tests for whole genome sequencing (WGS) data, the association test for variant groups is a key approach for genetic mapping. To address the features of sparse and weak genetic effects to be detected, the higher criticism (HC) approach has been proposed and theoretically has proven optimal for detecting sparse and weak genetic effects. Here we develop a strategy to apply the HC approach to WGS data that contains rare variants as the majority. By using Genetic Analysis Workshop 18 "dose" genetic data with simulated phenotypes, we assess the performance of HC under a variety of strategies for grouping variants and collapsing rare variants. The HC approach is compared with the minimal p-value method and the sequence kernel association test. The results show that the HC approach is preferred for detecting weak genetic effects. PMID:25519367

ANGPTL8/Betatrophin R59W variant is associated with higher glucose level in non-diabetic Arabs living in Kuwaits.

PubMed

Abu-Farha, Mohamed; Melhem, Motasem; Abubaker, Jehad; Behbehani, Kazem; Alsmadi, Osama; Elkum, Naser

2016-02-11

ANGPTL8 (betatrophin) has been recently identified as a regulator of lipid metabolism through its interaction with ANGPTL3. A sequence variant in ANGPTL8 has been shown to associate with lower level of Low Density Lipoprotein (LDL) and High Density Lipoprotein (HDL). The objective of this study is to identify sequence variants in ANGPTL8 gene in Arabs and investigate their association with ANGPTL8 plasma level and clinical parameters. A cross sectional study was designed to examine the level of ANGPTL8 in 283 non-diabetic Arabs, and to identify its sequence variants using Sanger sequencing and their association with various clinical parameters. Using Sanger sequencing, we sequenced the full ANGPTL8 gene in 283 Arabs identifying two single nucleotide polymorphisms (SNPs) Rs.892066 and Rs.2278426 in the coding region. Our data shows for the first time that Arabs with the heterozygote form of (c.194C > T Rs.2278426) had higher level of Fasting Blood Glucose (FBG) compared to the CC homozygotes. LDL and HDL level in these subjects did not show significant difference between the two subgroups. Circulation level of ANGPTL8 did not vary between the two forms. No significant changes were observed between the various forms of Rs.892066 variant and FBG, LDL or HDL. Our data shows for the first time that heterozygote form of ANGPTL8 Rs.2278426 variant was associated with higher FBG level in Arabs highlighting the importance of these variants in controlling the function of betatrophin.

Identification of missing variants by combining multiple analytic pipelines.

PubMed

Ren, Yingxue; Reddy, Joseph S; Pottier, Cyril; Sarangi, Vivekananda; Tian, Shulan; Sinnwell, Jason P; McDonnell, Shannon K; Biernacka, Joanna M; Carrasquillo, Minerva M; Ross, Owen A; Ertekin-Taner, Nilüfer; Rademakers, Rosa; Hudson, Matthew; Mainzer, Liudmila Sergeevna; Asmann, Yan W

2018-04-16

After decades of identifying risk factors using array-based genome-wide association studies (GWAS), genetic research of complex diseases has shifted to sequencing-based rare variants discovery. This requires large sample sizes for statistical power and has brought up questions about whether the current variant calling practices are adequate for large cohorts. It is well-known that there are discrepancies between variants called by different pipelines, and that using a single pipeline always misses true variants exclusively identifiable by other pipelines. Nonetheless, it is common practice today to call variants by one pipeline due to computational cost and assume that false negative calls are a small percent of total. We analyzed 10,000 exomes from the Alzheimer's Disease Sequencing Project (ADSP) using multiple analytic pipelines consisting of different read aligners and variant calling strategies. We compared variants identified by using two aligners in 50,100, 200, 500, 1000, and 1952 samples; and compared variants identified by adding single-sample genotyping to the default multi-sample joint genotyping in 50,100, 500, 2000, 5000 and 10,000 samples. We found that using a single pipeline missed increasing numbers of high-quality variants correlated with sample sizes. By combining two read aligners and two variant calling strategies, we rescued 30% of pass-QC variants at sample size of 2000, and 56% at 10,000 samples. The rescued variants had higher proportions of low frequency (minor allele frequency [MAF] 1-5%) and rare (MAF < 1%) variants, which are the very type of variants of interest. In 660 Alzheimer's disease cases with earlier onset ages of ≤65, 4 out of 13 (31%) previously-published rare pathogenic and protective mutations in APP, PSEN1, and PSEN2 genes were undetected by the default one-pipeline approach but recovered by the multi-pipeline approach. Identification of the complete variant set from sequencing data is the prerequisite of genetic association analyses. The current analytic practice of calling genetic variants from sequencing data using a single bioinformatics pipeline is no longer adequate with the increasingly large projects. The number and percentage of quality variants that passed quality filters but are missed by the one-pipeline approach rapidly increased with sample size.

Characterization and mapping of the human rhodopsin kinase gene and screening of the gene for mutations in patients with retinitis pigmentosa

DOE Office of Scientific and Technical Information (OSTI.GOV)

Khani, S.C.; Lin, D.; Magovcevic, I.

1994-09-01

Rhodopsin kinase (RK) is a cytosolic enzyme in rod photoreceptors that initiates the deactivation of the phototransductions cascade by phosphorylating photoactivated rhodopsin. Although the cDNA sequence of bovine RK has been determined previously, no human cDNA or genomic sequence has thus far been available for genetic studies. In order to investigate the possible role of this candidate gene in retinitis pigmentosa (RP) and allied diseases, we have isolated and characterized human cDNA and genomic clones derived from the RK locus. The coding sequence of the human gene is 1692 nucleotides in length and is split into seven exons. The humanmore » and the bovine sequence show 84% identity at the nucleotide level and 92% identity at the amino acid level. Thus far, the intronic sequences flanking each exon except for one have been determined. We have also mapped the human RK gene to chromosome 13q34 using fluorescence in situ hybridization. To our knowledge, no RP gene has as yet been linked to this region. However, since the substrate for RK (rhodopsin) and other members of the phototransduction cascade have been implicated in the pathogenesis of RP, it is conceivable that defects in RK can also cause some forms of this disease. We are evaluating this possibility by screening DNA from 173 patients with autosomal recessive RP and 190 patients with autosomal dominant RP. So far, we have found 11 patients with variant bands. In one patient with autosomal dominant RP we discovered the missense change Ser536Leu. Cosegregation studies and further sequencing of the variant bands are currently underway.« less

Leveraging long read sequencing from a single individual to provide a comprehensive resource for benchmarking variant calling methods

PubMed Central

Mu, John C.; Tootoonchi Afshar, Pegah; Mohiyuddin, Marghoob; Chen, Xi; Li, Jian; Bani Asadi, Narges; Gerstein, Mark B.; Wong, Wing H.; Lam, Hugo Y. K.

2015-01-01

A high-confidence, comprehensive human variant set is critical in assessing accuracy of sequencing algorithms, which are crucial in precision medicine based on high-throughput sequencing. Although recent works have attempted to provide such a resource, they still do not encompass all major types of variants including structural variants (SVs). Thus, we leveraged the massive high-quality Sanger sequences from the HuRef genome to construct by far the most comprehensive gold set of a single individual, which was cross validated with deep Illumina sequencing, population datasets, and well-established algorithms. It was a necessary effort to completely reanalyze the HuRef genome as its previously published variants were mostly reported five years ago, suffering from compatibility, organization, and accuracy issues that prevent their direct use in benchmarking. Our extensive analysis and validation resulted in a gold set with high specificity and sensitivity. In contrast to the current gold sets of the NA12878 or HS1011 genomes, our gold set is the first that includes small variants, deletion SVs and insertion SVs up to a hundred thousand base-pairs. We demonstrate the utility of our HuRef gold set to benchmark several published SV detection tools. PMID:26412485

Structural analysis of two length variants of the rDNA intergenic spacer from Eruca sativa.

PubMed

Lakshmikumaran, M; Negi, M S

1994-03-01

Restriction enzyme analysis of the rRNA genes of Eruca sativa indicated the presence of many length variants within a single plant and also between different cultivars which is unusual for most crucifers studied so far. Two length variants of the rDNA intergenic spacer (IGS) from a single individual E. sativa (cv. Itsa) plant were cloned and characterized. The complete nucleotide sequences of both the variants (3 kb and 4 kb) were determined. The intergenic spacer contains three families of tandemly repeated DNA sequences denoted as A, B and C. However, the long (4 kb) variant shows the presence of an additional repeat, denoted as D, which is a duplication of a 224 bp sequence just upstream of the putative transcription initiation site. Repeat units belonging to the three different families (A, B and C) were in the size range of 22 to 30 bp. Such short repeat elements are present in the IGS of most of the crucifers analysed so far. Sequence analysis of the variants (3 kb and 4 kb) revealed that the length heterogeneity of the spacer is located at three different regions and is due to the varying copy numbers of repeat units belonging to families A and B. Length variation of the spacer is also due to the presence of a large duplication (D repeats) in the 4 kb variant which is absent in the 3 kb variant. The putative transcription initiation site was identified by comparisons with the rDNA sequences from other plant species.

Genetic Characterization of Circulating African Swine Fever Viruses in Nigeria (2007-2015).

PubMed

Luka, P D; Achenbach, J E; Mwiine, F N; Lamien, C E; Shamaki, D; Unger, H; Erume, J

2017-10-01

Sequencing and analysis of three discrete genome regions of African swine fever viruses (ASFV) from archival samples collected in 2007-2011 and active and passive surveillance between 2012 and 2015 in Nigeria were carried out. Analysis was conducted by genotyping of three single-copy African swine fever (ASF) genes. The E183L and B646L genes that encode structural proteins p54 and p72, respectively, were utilized to delineate genotypes before intragenotypic resolution by characterization of the tetrameric amino acid repeat region within the hypervariable central variable region of the B602L gene. The results showed no variation in the p72 and p54 gene regions sequenced. Phylogeny of p72 sequences revealed that all the Nigerian isolates belonged to genotype I, while that of the p54 recovered the Ia genotype. Analysis of B602L gene revealed the differences in the number of tetrameric repeats. Four new variants (Tet-15, Tet-17a, Tet-17b and Tet-48) were recovered, while a fifth variant (Tet-20) was the most widely distributed in the country displacing Tet-36 reported previously in 2003-2006. The viruses responsible for ASF outbreaks in Nigeria are from very closely related but mutated variants of the virus that have been circulating since 1997. A practical implication of the genetic variability of the Nigerian viral isolates in this study is the need for continuous sampling and analysis of circulating viruses, which will provide epidemiological information on the evolution of ASFV in the field versus new incursion for informed strategic control of the disease in the country. © 2016 Blackwell Verlag GmbH.

Ecological dynamics and co-occurrence among marine phytoplankton, bacteria and myoviruses shows microdiversity matters.

PubMed

Needham, David M; Sachdeva, Rohan; Fuhrman, Jed A

2017-07-01

Numerous ecological processes, such as bacteriophage infection and phytoplankton-bacterial interactions, often occur via strain-specific mechanisms. Therefore, studying the causes of microbial dynamics should benefit from highly resolving taxonomic characterizations. We sampled daily to weekly over 5 months following a phytoplankton bloom off Southern California and examined the extent of microdiversity, that is, significant variation within 99% sequence similarity clusters, operational taxonomic units (OTUs), of bacteria, archaea, phytoplankton chloroplasts (all via 16S or intergenic spacer (ITS) sequences) and T4-like-myoviruses (via g23 major capsid protein gene sequence). The extent of microdiversity varied between genes (ITS most, g23 least) and only temporally common taxa were highly microdiverse. Overall, 60% of taxa exhibited microdiversity; 59% of these had subtypes that changed significantly as a proportion of the parent taxon, indicating ecologically distinct taxa. Pairwise correlations between prokaryotes and myoviruses or phytoplankton (for example, highly microdiverse Chrysochromulina sp.) improved when using single-base variants. Correlations between myoviruses and SAR11 increased in number (172 vs 9, Spearman>0.65) and became stronger (0.61 vs 0.58, t-test: P<0.001) when using SAR11 ITS single-base variants vs OTUs. Whole-community correlation between SAR11 and myoviruses was much improved when using ITS single-base variants vs OTUs, with Mantel rho=0.49 vs 0.27; these results are consistent with strain-specific interactions. Mantel correlations suggested >1 μm (attached/large) prokaryotes are a major myovirus source. Consideration of microdiversity improved observation of apparent host and virus networks, and provided insights into the ecological and evolutionary factors influencing the success of lineages, with important implications to ecosystem resilience and microbial function.

Next-generation sequencing for genetic testing of familial colorectal cancer syndromes.

PubMed

Simbolo, Michele; Mafficini, Andrea; Agostini, Marco; Pedrazzani, Corrado; Bedin, Chiara; Urso, Emanuele D; Nitti, Donato; Turri, Giona; Scardoni, Maria; Fassan, Matteo; Scarpa, Aldo

2015-01-01

Genetic screening in families with high risk to develop colorectal cancer (CRC) prevents incurable disease and permits personalized therapeutic and follow-up strategies. The advancement of next-generation sequencing (NGS) technologies has revolutionized the throughput of DNA sequencing. A series of 16 probands for either familial adenomatous polyposis (FAP; 8 cases) or hereditary nonpolyposis colorectal cancer (HNPCC; 8 cases) were investigated for intragenic mutations in five CRC familial syndromes-associated genes (APC, MUTYH, MLH1, MSH2, MSH6) applying both a custom multigene Ion AmpliSeq NGS panel and conventional Sanger sequencing. Fourteen pathogenic variants were detected in 13/16 FAP/HNPCC probands (81.3 %); one FAP proband presented two co-existing pathogenic variants, one in APC and one in MUTYH. Thirteen of these 14 pathogenic variants were detected by both NGS and Sanger, while one MSH2 mutation (L280FfsX3) was identified only by Sanger sequencing. This is due to a limitation of the NGS approach in resolving sequences close or within homopolymeric stretches of DNA. To evaluate the performance of our NGS custom panel we assessed its capability to resolve the DNA sequences corresponding to 2225 pathogenic variants reported in the COSMIC database for APC, MUTYH, MLH1, MSH2, MSH6. Our NGS custom panel resolves the sequences where 2108 (94.7 %) of these variants occur. The remaining 117 mutations reside inside or in close proximity to homopolymer stretches; of these 27 (1.2 %) are imprecisely identified by the software but can be resolved by visual inspection of the region, while the remaining 90 variants (4.0 %) are blind spots. In summary, our custom panel would miss 4 % (90/2225) of pathogenic variants that would need a small set of Sanger sequencing reactions to be solved. The multiplex NGS approach has the advantage of analyzing multiple genes in multiple samples simultaneously, requiring only a reduced number of Sanger sequences to resolve homopolymeric DNA regions not adequately assessed by NGS. The implementation of NGS approaches in routine diagnostics of familial CRC is cost-effective and significantly reduces diagnostic turnaround times.

Increasing the Yield in Targeted Next-Generation Sequencing by Implicating CNV Analysis, Non-Coding Exons and the Overall Variant Load: The Example of Retinal Dystrophies

PubMed Central

Eisenberger, Tobias; Neuhaus, Christine; Khan, Arif O.; Decker, Christian; Preising, Markus N.; Friedburg, Christoph; Bieg, Anika; Gliem, Martin; Issa, Peter Charbel; Holz, Frank G.; Baig, Shahid M.; Hellenbroich, Yorck; Galvez, Alberto; Platzer, Konrad; Wollnik, Bernd; Laddach, Nadja; Ghaffari, Saeed Reza; Rafati, Maryam; Botzenhart, Elke; Tinschert, Sigrid; Börger, Doris; Bohring, Axel; Schreml, Julia; Körtge-Jung, Stefani; Schell-Apacik, Chayim; Bakur, Khadijah; Al-Aama, Jumana Y.; Neuhann, Teresa; Herkenrath, Peter; Nürnberg, Gudrun; Nürnberg, Peter; Davis, John S.; Gal, Andreas; Bergmann, Carsten; Lorenz, Birgit; Bolz, Hanno J.

2013-01-01

Retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) are major causes of blindness. They result from mutations in many genes which has long hampered comprehensive genetic analysis. Recently, targeted next-generation sequencing (NGS) has proven useful to overcome this limitation. To uncover “hidden mutations” such as copy number variations (CNVs) and mutations in non-coding regions, we extended the use of NGS data by quantitative readout for the exons of 55 RP and LCA genes in 126 patients, and by including non-coding 5′ exons. We detected several causative CNVs which were key to the diagnosis in hitherto unsolved constellations, e.g. hemizygous point mutations in consanguineous families, and CNVs complemented apparently monoallelic recessive alleles. Mutations of non-coding exon 1 of EYS revealed its contribution to disease. In view of the high carrier frequency for retinal disease gene mutations in the general population, we considered the overall variant load in each patient to assess if a mutation was causative or reflected accidental carriership in patients with mutations in several genes or with single recessive alleles. For example, truncating mutations in RP1, a gene implicated in both recessive and dominant RP, were causative in biallelic constellations, unrelated to disease when heterozygous on a biallelic mutation background of another gene, or even non-pathogenic if close to the C-terminus. Patients with mutations in several loci were common, but without evidence for di- or oligogenic inheritance. Although the number of targeted genes was low compared to previous studies, the mutation detection rate was highest (70%) which likely results from completeness and depth of coverage, and quantitative data analysis. CNV analysis should routinely be applied in targeted NGS, and mutations in non-coding exons give reason to systematically include 5′-UTRs in disease gene or exome panels. Consideration of all variants is indispensable because even truncating mutations may be misleading. PMID:24265693

Increasing the yield in targeted next-generation sequencing by implicating CNV analysis, non-coding exons and the overall variant load: the example of retinal dystrophies.

PubMed

Eisenberger, Tobias; Neuhaus, Christine; Khan, Arif O; Decker, Christian; Preising, Markus N; Friedburg, Christoph; Bieg, Anika; Gliem, Martin; Charbel Issa, Peter; Holz, Frank G; Baig, Shahid M; Hellenbroich, Yorck; Galvez, Alberto; Platzer, Konrad; Wollnik, Bernd; Laddach, Nadja; Ghaffari, Saeed Reza; Rafati, Maryam; Botzenhart, Elke; Tinschert, Sigrid; Börger, Doris; Bohring, Axel; Schreml, Julia; Körtge-Jung, Stefani; Schell-Apacik, Chayim; Bakur, Khadijah; Al-Aama, Jumana Y; Neuhann, Teresa; Herkenrath, Peter; Nürnberg, Gudrun; Nürnberg, Peter; Davis, John S; Gal, Andreas; Bergmann, Carsten; Lorenz, Birgit; Bolz, Hanno J

2013-01-01

Retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) are major causes of blindness. They result from mutations in many genes which has long hampered comprehensive genetic analysis. Recently, targeted next-generation sequencing (NGS) has proven useful to overcome this limitation. To uncover "hidden mutations" such as copy number variations (CNVs) and mutations in non-coding regions, we extended the use of NGS data by quantitative readout for the exons of 55 RP and LCA genes in 126 patients, and by including non-coding 5' exons. We detected several causative CNVs which were key to the diagnosis in hitherto unsolved constellations, e.g. hemizygous point mutations in consanguineous families, and CNVs complemented apparently monoallelic recessive alleles. Mutations of non-coding exon 1 of EYS revealed its contribution to disease. In view of the high carrier frequency for retinal disease gene mutations in the general population, we considered the overall variant load in each patient to assess if a mutation was causative or reflected accidental carriership in patients with mutations in several genes or with single recessive alleles. For example, truncating mutations in RP1, a gene implicated in both recessive and dominant RP, were causative in biallelic constellations, unrelated to disease when heterozygous on a biallelic mutation background of another gene, or even non-pathogenic if close to the C-terminus. Patients with mutations in several loci were common, but without evidence for di- or oligogenic inheritance. Although the number of targeted genes was low compared to previous studies, the mutation detection rate was highest (70%) which likely results from completeness and depth of coverage, and quantitative data analysis. CNV analysis should routinely be applied in targeted NGS, and mutations in non-coding exons give reason to systematically include 5'-UTRs in disease gene or exome panels. Consideration of all variants is indispensable because even truncating mutations may be misleading.

VaDiR: an integrated approach to Variant Detection in RNA.

PubMed

Neums, Lisa; Suenaga, Seiji; Beyerlein, Peter; Anders, Sara; Koestler, Devin; Mariani, Andrea; Chien, Jeremy

2018-02-01

Advances in next-generation DNA sequencing technologies are now enabling detailed characterization of sequence variations in cancer genomes. With whole-genome sequencing, variations in coding and non-coding sequences can be discovered. But the cost associated with it is currently limiting its general use in research. Whole-exome sequencing is used to characterize sequence variations in coding regions, but the cost associated with capture reagents and biases in capture rate limit its full use in research. Additional limitations include uncertainty in assigning the functional significance of the mutations when these mutations are observed in the non-coding region or in genes that are not expressed in cancer tissue. We investigated the feasibility of uncovering mutations from expressed genes using RNA sequencing datasets with a method called Variant Detection in RNA(VaDiR) that integrates 3 variant callers, namely: SNPiR, RVBoost, and MuTect2. The combination of all 3 methods, which we called Tier 1 variants, produced the highest precision with true positive mutations from RNA-seq that could be validated at the DNA level. We also found that the integration of Tier 1 variants with those called by MuTect2 and SNPiR produced the highest recall with acceptable precision. Finally, we observed a higher rate of mutation discovery in genes that are expressed at higher levels. Our method, VaDiR, provides a possibility of uncovering mutations from RNA sequencing datasets that could be useful in further functional analysis. In addition, our approach allows orthogonal validation of DNA-based mutation discovery by providing complementary sequence variation analysis from paired RNA/DNA sequencing datasets.

Rare variants and autoimmune disease.

PubMed

Massey, Jonathan; Eyre, Steve

2014-09-01

The study of rare variants in monogenic forms of autoimmune disease has offered insight into the aetiology of more complex pathologies. Research in complex autoimmune disease initially focused on sequencing candidate genes, with some early successes, notably in uncovering low-frequency variation associated with Type 1 diabetes mellitus. However, other early examples have proved difficult to replicate, and a recent study across six autoimmune diseases, re-sequencing 25 autoimmune disease-associated genes in large sample sizes, failed to find any associated rare variants. The study of rare and low-frequency variation in autoimmune diseases has been made accessible by the inclusion of such variants on custom genotyping arrays (e.g. Immunochip and Exome arrays). Whole-exome sequencing approaches are now also being utilised to uncover the contribution of rare coding variants to disease susceptibility, severity and treatment response. Other sequencing strategies are starting to uncover the role of regulatory rare variation. © The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

MToolBox: a highly automated pipeline for heteroplasmy annotation and prioritization analysis of human mitochondrial variants in high-throughput sequencing

PubMed Central

Diroma, Maria Angela; Santorsola, Mariangela; Guttà, Cristiano; Gasparre, Giuseppe; Picardi, Ernesto; Pesole, Graziano; Attimonelli, Marcella

2014-01-01

Motivation: The increasing availability of mitochondria-targeted and off-target sequencing data in whole-exome and whole-genome sequencing studies (WXS and WGS) has risen the demand of effective pipelines to accurately measure heteroplasmy and to easily recognize the most functionally important mitochondrial variants among a huge number of candidates. To this purpose, we developed MToolBox, a highly automated pipeline to reconstruct and analyze human mitochondrial DNA from high-throughput sequencing data. Results: MToolBox implements an effective computational strategy for mitochondrial genomes assembling and haplogroup assignment also including a prioritization analysis of detected variants. MToolBox provides a Variant Call Format file featuring, for the first time, allele-specific heteroplasmy and annotation files with prioritized variants. MToolBox was tested on simulated samples and applied on 1000 Genomes WXS datasets. Availability and implementation: MToolBox package is available at https://sourceforge.net/projects/mtoolbox/. Contact: marcella.attimonelli@uniba.it Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25028726

De novo assembly and next-generation sequencing to analyse full-length gene variants from codon-barcoded libraries.

PubMed

Cho, Namjin; Hwang, Byungjin; Yoon, Jung-ki; Park, Sangun; Lee, Joongoo; Seo, Han Na; Lee, Jeewon; Huh, Sunghoon; Chung, Jinsoo; Bang, Duhee

2015-09-21

Interpreting epistatic interactions is crucial for understanding evolutionary dynamics of complex genetic systems and unveiling structure and function of genetic pathways. Although high resolution mapping of en masse variant libraries renders molecular biologists to address genotype-phenotype relationships, long-read sequencing technology remains indispensable to assess functional relationship between mutations that lie far apart. Here, we introduce JigsawSeq for multiplexed sequence identification of pooled gene variant libraries by combining a codon-based molecular barcoding strategy and de novo assembly of short-read data. We first validate JigsawSeq on small sub-pools and observed high precision and recall at various experimental settings. With extensive simulations, we then apply JigsawSeq to large-scale gene variant libraries to show that our method can be reliably scaled using next-generation sequencing. JigsawSeq may serve as a rapid screening tool for functional genomics and offer the opportunity to explore evolutionary trajectories of protein variants.

A survey of single nucleotide polymorphisms identified from whole-genome sequencing and their functional effect in the porcine genome

USDA-ARS?s Scientific Manuscript database

Genetic variants detected from sequence have been used to successfully identify causal variants and map complex traits in several organisms. High and moderate impact variants, those expected to alter or disrupt the protein coded by a gene and those that regulate protein production, likely have a mor...

Characterization of alanine to valine sequence variants in the Fc region of nivolumab biosimilar produced in Chinese hamster ovary cells.

PubMed

Li, Yantao; Fu, Tuo; Liu, Tao; Guo, Huaizu; Guo, Qingcheng; Xu, Jin; Zhang, Dapeng; Qian, Weizhu; Dai, Jianxin; Li, Bohua; Guo, Yajun; Hou, Sheng; Wang, Hao

2016-07-01

Nivolumab is a therapeutic fully human IgG4 antibody to programmed death 1 (PD-1). In this study, a nivolumab biosimilar, which was produced in our laboratory, was analyzed and characterized. Sequence variants that contain undesired amino acid sequences may cause concern during biosimilar bioprocess development. We found that low levels of sequence variants were detected in the heavy chain of the nivolumab biosimilar by ultra performance liquid chromatography (UPLC) and tandem mass spectrometry. It was further identified with UPLC-MS/MS by IdeS or trypsin digestion. The sequence variant was confirmed through addition of synthetic mutant peptide. Subsequently, the mixing base signal of normal and mutant sequence was detected through DNA sequencing. The relative levels of mutant A424V in the Fc region of the heavy chain have been detected and demonstrated to be 12.25% and 13.54%, via base peak intensity (BPI) and UV chromatography of the tryptic peptide mapping, respectively. A424V variant was also quantified by real-time PCR (RT-PCR) at the DNA and RNA level, which was 19.2% and 16.8%, respectively. The relative content of the mutant was consistent at the DNA, RNA and protein level, indicating that the A424V mutation may have little influence at transcriptional or translational levels. These results demonstrate that orthogonal state-of-the-art techniques such as LC- UV- MS and RT-PCR should be implemented to characterize recombinant proteins and cell lines for development of biosimilars. Our study suggests that it is important to establish an integrated and effective analytical method to monitor and characterize sequence variants during antibody drug development, especially for antibody biosimilar products.

Polymorphisms and variants in the prion protein sequence of European moose (Alces alces), reindeer (Rangifer tarandus), roe deer (Capreolus capreolus) and fallow deer (Dama dama) in Scandinavia

PubMed Central

Wik, Lotta; Mikko, Sofia; Klingeborn, Mikael; Stéen, Margareta; Simonsson, Magnus; Linné, Tommy

2012-01-01

The prion protein (PrP) sequence of European moose, reindeer, roe deer and fallow deer in Scandinavia has high homology to the PrP sequence of North American cervids. Variants in the European moose PrP sequence were found at amino acid position 109 as K or Q. The 109Q variant is unique in the PrP sequence of vertebrates. During the 1980s a wasting syndrome in Swedish moose, Moose Wasting Syndrome (MWS), was described. SNP analysis demonstrated a difference in the observed genotype proportions of the heterozygous Q/K and homozygous Q/Q variants in the MWS animals compared with the healthy animals. In MWS moose the allele frequencies for 109K and 109Q were 0.73 and 0.27, respectively, and for healthy animals 0.69 and 0.31. Both alleles were seen as heterozygotes and homozygotes. In reindeer, PrP sequence variation was demonstrated at codon 176 as D or N and codon 225 as S or Y. The PrP sequences in roe deer and fallow deer were identical with published GenBank sequences. PMID:22441661

Reconstructing metastatic seeding patterns of human cancers

PubMed Central

Reiter, Johannes G.; Makohon-Moore, Alvin P.; Gerold, Jeffrey M.; Bozic, Ivana; Chatterjee, Krishnendu; Iacobuzio-Donahue, Christine A.; Vogelstein, Bert; Nowak, Martin A.

2017-01-01

Reconstructing the evolutionary history of metastases is critical for understanding their basic biological principles and has profound clinical implications. Genome-wide sequencing data has enabled modern phylogenomic methods to accurately dissect subclones and their phylogenies from noisy and impure bulk tumour samples at unprecedented depth. However, existing methods are not designed to infer metastatic seeding patterns. Here we develop a tool, called Treeomics, to reconstruct the phylogeny of metastases and map subclones to their anatomic locations. Treeomics infers comprehensive seeding patterns for pancreatic, ovarian, and prostate cancers. Moreover, Treeomics correctly disambiguates true seeding patterns from sequencing artifacts; 7% of variants were misclassified by conventional statistical methods. These artifacts can skew phylogenies by creating illusory tumour heterogeneity among distinct samples. In silico benchmarking on simulated tumour phylogenies across a wide range of sample purities (15–95%) and sequencing depths (25-800 × ) demonstrates the accuracy of Treeomics compared with existing methods. PMID:28139641

Whole-exome sequencing identified a variant in EFTUD2 gene in establishing a genetic diagnosis.

PubMed

Rengasamy Venugopalan, S; Farrow, E G; Lypka, M

2017-06-01

Craniofacial anomalies are complex and have an overlapping phenotype. Mandibulofacial Dysostosis and Oculo-Auriculo-Vertebral Spectrum are conditions that share common craniofacial phenotype and present a challenge in arriving at a diagnosis. In this report, we present a case of female proband who was given a differential diagnosis of Treacher Collins syndrome or Hemifacial Microsomia without certainty. Prior genetic testing reported negative for 22q deletion and FGFR screenings. The objective of this study was to demonstrate the critical role of whole-exome sequencing in establishing a genetic diagnosis of the proband. The participants were 14½-year-old affected female proband/parent trio. Proband/parent trio were enrolled in the study. Surgical tissue sample from the proband and parental blood samples were collected and prepared for whole-exome sequencing. Illumina HiSeq 2500 instrument was used for sequencing (125 nucleotide reads/84X coverage). Analyses of variants were performed using custom-developed software, RUNES and VIKING. Variant analyses following whole-exome sequencing identified a heterozygous de novo pathogenic variant, c.259C>T (p.Gln87*), in EFTUD2 (NM_004247.3) gene in the proband. Previous studies have reported that the variants in EFTUD2 gene were associated with Mandibulofacial Dysostosis with Microcephaly. Patients with facial asymmetry, micrognathia, choanal atresia and microcephaly should be analyzed for variants in EFTUD2 gene. Next-generation sequencing techniques, such as whole-exome sequencing offer great promise to improve the understanding of etiologies of sporadic genetic diseases. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

A power set-based statistical selection procedure to locate susceptible rare variants associated with complex traits with sequencing data.

PubMed

Sun, Hokeun; Wang, Shuang

2014-08-15

Existing association methods for rare variants from sequencing data have focused on aggregating variants in a gene or a genetic region because of the fact that analysing individual rare variants is underpowered. However, these existing rare variant detection methods are not able to identify which rare variants in a gene or a genetic region of all variants are associated with the complex diseases or traits. Once phenotypic associations of a gene or a genetic region are identified, the natural next step in the association study with sequencing data is to locate the susceptible rare variants within the gene or the genetic region. In this article, we propose a power set-based statistical selection procedure that is able to identify the locations of the potentially susceptible rare variants within a disease-related gene or a genetic region. The selection performance of the proposed selection procedure was evaluated through simulation studies, where we demonstrated the feasibility and superior power over several comparable existing methods. In particular, the proposed method is able to handle the mixed effects when both risk and protective variants are present in a gene or a genetic region. The proposed selection procedure was also applied to the sequence data on the ANGPTL gene family from the Dallas Heart Study to identify potentially susceptible rare variants within the trait-related genes. An R package 'rvsel' can be downloaded from http://www.columbia.edu/∼sw2206/ and http://statsun.pusan.ac.kr. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

The Spectrum of CFTR Variants in Nonwhite Cystic Fibrosis Patients: Implications for Molecular Diagnostic Testing.

PubMed

Schrijver, Iris; Pique, Lynn; Graham, Steve; Pearl, Michelle; Cherry, Athena; Kharrazi, Martin

2016-01-01

Despite the implementation of cystic fibrosis (CF) newborn screening programs across the United States, the identification of CFTR gene variants in nonwhite populations compared with whites remains suboptimal. Our objective was to establish the spectrum of CFTR variants and their frequencies in CF patients in the United States with African, Native American, Asian, East Indian, or Middle Eastern backgrounds. By using direct DNA sequencing, we identified two CFTR variants in 89 of 140 affected nonwhite individuals with uncharacterized genotypes. Seven variants were novel. Multiplex ligation-dependent probe amplification detected 14 rearrangements in the remaining 51 patients, 6 of which were novel. Deletions and duplications accounted for 17% of unidentified alleles. A cross-sectional analysis of genotyping data from the CF Foundation Patient Registry was performed, comparing 3496 nonwhite patients with 22,206 white CF patients. Patients of Hispanic, black, or Asian ancestry were less likely to have two identified CFTR variants (P < 0.0001 for Hispanics and blacks, P = 0.003 for Asians), and more likely to carry no mutations on the commonly used 23 mutation carrier screening panel (P < 0.0001). We analyzed the mutations recorded for each ancestry and summarized the most frequent ones. This research could facilitate equity in mutation detection between white and nonwhite or mixed-ethnicity CF patients, enabling an earlier diagnosis improving their quality of life. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Systematic RH genotyping and variant identification in French donors of African origin

PubMed Central

Kappler-Gratias, Sandrine; Auxerre, Carine; Dubeaux, Isabelle; Beolet, Marylise; Ripaux, Maryline; Le Pennec, Pierre-Yves; Pham, Bach-Nga

2014-01-01

Background RH molecular analysis has enabled the documentation of numerous variants of RHD and RHCE alleles, especially in individuals of African origin. The aim of the present study was to determine the type and frequency of D and/or RhCE variants among blood donors of African origin in France, by performing a systematic RH molecular analysis, in order to evaluate the implications for blood transfusion of patients of African origin. Materials and methods Samples from 316 African blood donors, whose origin was established by their Fy(a−b−) phenotype, were first analysed using the RHD and RHCE BeadChips Kit (BioArray Solutions, Immucor, Warren, NJ, USA). Sequencing was performed when necessary. Results RHD molecular analysis showed that 26.2% of donors had a variant RHD allele. It allowed the prediction of a partial D in 11% of cases. RHCE molecular analysis showed that 14.2% of donors had a variant RHCE allele or RH [RN or (C)ces] haplotype. A rare Rh phenotype associated with the loss of a high-prevalence antigen or partial RhCE antigens were predicted from RHCE molecular analysis in 1 (0.3%) and 17 (5%) cases, respectively. Discussion Systematic RHD and RHCE molecular analysis performed in blood donors of African origin provides transfusion-relevant information for individuals of African origin because of the frequency of variant RH alleles. RH molecular analysis may improve transfusion therapy of patients by allowing better donor and recipient matching, based not only on phenotypically matched red blood cell units, but also on units that are genetically matched with regards to RhCE variants. PMID:23867180

Association between Rare Variants in AP4E1, a Component of Intracellular Trafficking, and Persistent Stuttering

PubMed Central

Raza, M. Hashim; Mattera, Rafael; Morell, Robert; Sainz, Eduardo; Rahn, Rachel; Gutierrez, Joanne; Paris, Emily; Root, Jessica; Solomon, Beth; Brewer, Carmen; Basra, M. Asim Raza; Khan, Shaheen; Riazuddin, Sheikh; Braun, Allen; Bonifacino, Juan S.; Drayna, Dennis

2015-01-01

Stuttering is a common, highly heritable neurodevelopmental disorder characterized by deficits in the volitional control of speech. Whole-exome sequencing identified two heterozygous AP4E1 coding variants, c.1549G>A (p.Val517Ile) and c.2401G>A (p.Glu801Lys), that co-segregate with persistent developmental stuttering in a large Cameroonian family, and we observed the same two variants in unrelated Cameroonians with persistent stuttering. We found 23 other rare variants, including predicted loss-of-function variants, in AP4E1 in unrelated stuttering individuals in Cameroon, Pakistan, and North America. The rate of rare variants in AP4E1 was significantly higher in unrelated Pakistani and Cameroonian stuttering individuals than in population-matched control individuals, and coding variants in this gene are exceptionally rare in the general sub-Saharan West African, South Asian, and North American populations. Clinical examination of the Cameroonian family members failed to identify any symptoms previously reported in rare individuals carrying homozygous loss-of-function mutations in this gene. AP4E1 encodes the ε subunit of the heterotetrameric (ε-β4-μ4-σ4) AP-4 complex, involved in protein sorting at the trans-Golgi network. We found that the μ4 subunit of AP-4 interacts with NAGPA, an enzyme involved in the synthesis of the mannose 6-phosphate signal that targets acid hydrolases to the lysosome and the product of a gene previously associated with stuttering. These findings implicate deficits in intracellular trafficking in persistent stuttering. PMID:26544806

Group-based variant calling leveraging next-generation supercomputing for large-scale whole-genome sequencing studies.

PubMed

Standish, Kristopher A; Carland, Tristan M; Lockwood, Glenn K; Pfeiffer, Wayne; Tatineni, Mahidhar; Huang, C Chris; Lamberth, Sarah; Cherkas, Yauheniya; Brodmerkel, Carrie; Jaeger, Ed; Smith, Lance; Rajagopal, Gunaretnam; Curran, Mark E; Schork, Nicholas J

2015-09-22

Next-generation sequencing (NGS) technologies have become much more efficient, allowing whole human genomes to be sequenced faster and cheaper than ever before. However, processing the raw sequence reads associated with NGS technologies requires care and sophistication in order to draw compelling inferences about phenotypic consequences of variation in human genomes. It has been shown that different approaches to variant calling from NGS data can lead to different conclusions. Ensuring appropriate accuracy and quality in variant calling can come at a computational cost. We describe our experience implementing and evaluating a group-based approach to calling variants on large numbers of whole human genomes. We explore the influence of many factors that may impact the accuracy and efficiency of group-based variant calling, including group size, the biogeographical backgrounds of the individuals who have been sequenced, and the computing environment used. We make efficient use of the Gordon supercomputer cluster at the San Diego Supercomputer Center by incorporating job-packing and parallelization considerations into our workflow while calling variants on 437 whole human genomes generated as part of large association study. We ultimately find that our workflow resulted in high-quality variant calls in a computationally efficient manner. We argue that studies like ours should motivate further investigations combining hardware-oriented advances in computing systems with algorithmic developments to tackle emerging 'big data' problems in biomedical research brought on by the expansion of NGS technologies.

Rare Variants in RTEL1 Are Associated with Familial Interstitial Pneumonia

PubMed Central

Cogan, Joy D.; Zhao, Min; Mitchell, Daphne B.; Rives, Lynette; Markin, Cheryl; Garnett, Errine T.; Montgomery, Keri H.; Mason, Wendi R.; McKean, David F.; Powers, Julia; Murphy, Elissa; Olson, Lana M.; Choi, Leena; Cheng, Dong-Sheng; Blue, Elizabeth Marchani; Young, Lisa R.; Lancaster, Lisa H.; Steele, Mark P.; Brown, Kevin K.; Schwarz, Marvin I.; Fingerlin, Tasha E.; Schwartz, David A.; Lawson, William E.; Loyd, James E.; Zhao, Zhongming; Phillips, John A.; Blackwell, Timothy S.

2015-01-01

Rationale: Up to 20% of cases of idiopathic interstitial pneumonia cluster in families, comprising the syndrome of familial interstitial pneumonia (FIP); however, the genetic basis of FIP remains uncertain in most families. Objectives: To determine if new disease-causing rare genetic variants could be identified using whole-exome sequencing of affected members from FIP families, providing additional insights into disease pathogenesis. Methods: Affected subjects from 25 kindreds were selected from an ongoing FIP registry for whole-exome sequencing from genomic DNA. Candidate rare variants were confirmed by Sanger sequencing, and cosegregation analysis was performed in families, followed by additional sequencing of affected individuals from another 163 kindreds. Measurements and Main Results: We identified a potentially damaging rare variant in the gene encoding for regulator of telomere elongation helicase 1 (RTEL1) that segregated with disease and was associated with very short telomeres in peripheral blood mononuclear cells in 1 of 25 families in our original whole-exome sequencing cohort. Evaluation of affected individuals in 163 additional kindreds revealed another eight families (4.7%) with heterozygous rare variants in RTEL1 that segregated with clinical FIP. Probands and unaffected carriers of these rare variants had short telomeres (<10% for age) in peripheral blood mononuclear cells and increased T-circle formation, suggesting impaired RTEL1 function. Conclusions: Rare loss-of-function variants in RTEL1 represent a newly defined genetic predisposition for FIP, supporting the importance of telomere-related pathways in pulmonary fibrosis. PMID:25607374

Mitochondrial genomic variation associated with higher mitochondrial copy number: the Cache County Study on Memory Health and Aging.

PubMed

Ridge, Perry G; Maxwell, Taylor J; Foutz, Spencer J; Bailey, Matthew H; Corcoran, Christopher D; Tschanz, JoAnn T; Norton, Maria C; Munger, Ronald G; O'Brien, Elizabeth; Kerber, Richard A; Cawthon, Richard M; Kauwe, John S K

2014-01-01

The mitochondria are essential organelles and are the location of cellular respiration, which is responsible for the majority of ATP production. Each cell contains multiple mitochondria, and each mitochondrion contains multiple copies of its own circular genome. The ratio of mitochondrial genomes to nuclear genomes is referred to as mitochondrial copy number. Decreases in mitochondrial copy number are known to occur in many tissues as people age, and in certain diseases. The regulation of mitochondrial copy number by nuclear genes has been studied extensively. While mitochondrial variation has been associated with longevity and some of the diseases known to have reduced mitochondrial copy number, the role that the mitochondrial genome itself has in regulating mitochondrial copy number remains poorly understood. We analyzed the complete mitochondrial genomes from 1007 individuals randomly selected from the Cache County Study on Memory Health and Aging utilizing the inferred evolutionary history of the mitochondrial haplotypes present in our dataset to identify sequence variation and mitochondrial haplotypes associated with changes in mitochondrial copy number. Three variants belonging to mitochondrial haplogroups U5A1 and T2 were significantly associated with higher mitochondrial copy number in our dataset. We identified three variants associated with higher mitochondrial copy number and suggest several hypotheses for how these variants influence mitochondrial copy number by interacting with known regulators of mitochondrial copy number. Our results are the first to report sequence variation in the mitochondrial genome that causes changes in mitochondrial copy number. The identification of these variants that increase mtDNA copy number has important implications in understanding the pathological processes that underlie these phenotypes.

Common low-density lipoprotein receptor p.G116S variant has a large effect on plasma low-density lipoprotein cholesterol in circumpolar inuit populations.

PubMed

Dubé, Joseph B; Wang, Jian; Cao, Henian; McIntyre, Adam D; Johansen, Christopher T; Hopkins, Scarlett E; Stringer, Randa; Hosseinzadeh, Siyavash; Kennedy, Brooke A; Ban, Matthew R; Young, T Kue; Connelly, Philip W; Dewailly, Eric; Bjerregaard, Peter; Boyer, Bert B; Hegele, Robert A

2015-02-01

Inuit are considered to be vulnerable to cardiovascular disease because their lifestyles are becoming more Westernized. During sequence analysis of Inuit individuals at extremes of lipid traits, we identified 2 nonsynonymous variants in low-density lipoprotein receptor (LDLR), namely p.G116S and p.R730W. Genotyping these variants in 3324 Inuit from Alaska, Canada, and Greenland showed they were common, with allele frequencies 10% to 15%. Only p.G116S was associated with dyslipidemia: the increase in LDL cholesterol was 0.54 mmol/L (20.9 mg/dL) per allele (P=5.6×10(-49)), which was >3× larger than the largest effect sizes seen with other common variants in other populations. Carriers of p.G116S had a 3.02-fold increased risk of hypercholesterolemia (95% confidence interval, 2.34-3.90; P=1.7×10(-17)), but did not have classical familial hypercholesterolemia. In vitro, p.G116S showed 60% reduced ligand-binding activity compared with wild-type receptor. In contrast, p.R730W was associated with neither LDL cholesterol level nor altered in vitro activity. LDLR p.G116S is thus unique: a common dysfunctional variant in Inuit whose large effect on LDL cholesterol may have public health implications. © 2014 American Heart Association, Inc.

Epilepsy with auditory features

PubMed Central

Licchetta, Laura; Baldassari, Sara; Palombo, Flavia; Menghi, Veronica; D'Aurizio, Romina; Leta, Chiara; Stipa, Carlotta; Boero, Giovanni; d'Orsi, Giuseppe; Magi, Alberto; Scheffer, Ingrid; Seri, Marco; Tinuper, Paolo; Bisulli, Francesca

2015-01-01

Objective: To identify novel genes implicated in epilepsy with auditory features (EAF) in phenotypically heterogeneous families with unknown molecular basis. Methods: We identified 15 probands with EAF in whom an LGI1 mutation had been excluded. We performed electroclinical phenotyping on all probands and available affected relatives. We used whole-exome sequencing (WES) in 20 individuals with EAF (including all the probands and 5 relatives) to identify single nucleotide variants, small insertions/deletions, and copy number variants. Results: WES revealed likely pathogenic variants in genes that had not been previously associated with EAF: a CNTNAP2 intragenic deletion, 2 truncating mutations of DEPDC5, and a missense SCN1A change. Conclusions: EAF is a clinically and molecularly heterogeneous disease. The association of EAF with CNTNAP2, DEPDC5, and SCN1A mutations widens the phenotypic spectrum related to these genes. CNTNAP2 encodes CASPR2, a member of the voltage-gated potassium channel complex in which LGI1 plays a role. The finding of a CNTNAP2 deletion emphasizes the importance of this complex in EAF and shows biological convergence. PMID:27066544

Genetic fine-mapping and genomic annotation defines causal mechanisms at type 2 diabetes susceptibility loci

PubMed Central

Mahajan, Anubha; Locke, Adam; Rayner, N William; Robertson, Neil; Scott, Robert A; Prokopenko, Inga; Scott, Laura J; Green, Todd; Sparso, Thomas; Thuillier, Dorothee; Yengo, Loic; Grallert, Harald; Wahl, Simone; Frånberg, Mattias; Strawbridge, Rona J; Kestler, Hans; Chheda, Himanshu; Eisele, Lewin; Gustafsson, Stefan; Steinthorsdottir, Valgerdur; Thorleifsson, Gudmar; Qi, Lu; Karssen, Lennart C; van Leeuwen, Elisabeth M; Willems, Sara M; Li, Man; Chen, Han; Fuchsberger, Christian; Kwan, Phoenix; Ma, Clement; Linderman, Michael; Lu, Yingchang; Thomsen, Soren K; Rundle, Jana K; Beer, Nicola L; van de Bunt, Martijn; Chalisey, Anil; Kang, Hyun Min; Voight, Benjamin F; Abecasis, Goncalo R; Almgren, Peter; Baldassarre, Damiano; Balkau, Beverley; Benediktsson, Rafn; Blüher, Matthias; Boeing, Heiner; Bonnycastle, Lori L; Borringer, Erwin P; Burtt, Noël P; Carey, Jason; Charpentier, Guillaume; Chines, Peter S; Cornelis, Marilyn C; Couper, David J; Crenshaw, Andrew T; van Dam, Rob M; Doney, Alex SF; Dorkhan, Mozhgan; Edkins, Sarah; Eriksson, Johan G; Esko, Tonu; Eury, Elodie; Fadista, João; Flannick, Jason; Fontanillas, Pierre; Fox, Caroline; Franks, Paul W; Gertow, Karl; Gieger, Christian; Gigante, Bruna; Gottesman, Omri; Grant, George B; Grarup, Niels; Groves, Christopher J; Hassinen, Maija; Have, Christian T; Herder, Christian; Holmen, Oddgeir L; Hreidarsson, Astradur B; Humphries, Steve E; Hunter, David J; Jackson, Anne U; Jonsson, Anna; Jørgensen, Marit E; Jørgensen, Torben; Kerrison, Nicola D; Kinnunen, Leena; Klopp, Norman; Kong, Augustine; Kovacs, Peter; Kraft, Peter; Kravic, Jasmina; Langford, Cordelia; Leander, Karin; Liang, Liming; Lichtner, Peter; Lindgren, Cecilia M; Lindholm, Eero; Linneberg, Allan; Liu, Ching-Ti; Lobbens, Stéphane; Luan, Jian’an; Lyssenko, Valeriya; Männistö, Satu; McLeod, Olga; Meyer, Julia; Mihailov, Evelin; Mirza, Ghazala; Mühleisen, Thomas W; Müller-Nurasyid, Martina; Navarro, Carmen; Nöthen, Markus M; Oskolkov, Nikolay N; Owen, Katharine R; Palli, Domenico; Pechlivanis, Sonali; Perry, John RB; Platou, Carl GP; Roden, Michael; Ruderfer, Douglas; Rybin, Denis; van der Schouw, Yvonne T; Sennblad, Bengt; Sigurðsson, Gunnar; Stančáková, Alena; Steinbach, Gerald; Storm, Petter; Strauch, Konstantin; Stringham, Heather M; Sun, Qi; Thorand, Barbara; Tikkanen, Emmi; Tonjes, Anke; Trakalo, Joseph; Tremoli, Elena; Tuomi, Tiinamaija; Wennauer, Roman; Wood, Andrew R; Zeggini, Eleftheria; Dunham, Ian; Birney, Ewan; Pasquali, Lorenzo; Ferrer, Jorge; Loos, Ruth JF; Dupuis, Josée; Florez, Jose C; Boerwinkle, Eric; Pankow, James S; van Duijn, Cornelia; Sijbrands, Eric; Meigs, James B; Hu, Frank B; Thorsteinsdottir, Unnur; Stefansson, Kari; Lakka, Timo A; Rauramaa, Rainer; Stumvoll, Michael; Pedersen, Nancy L; Lind, Lars; Keinanen-Kiukaanniemi, Sirkka M; Korpi-Hyövälti, Eeva; Saaristo, Timo E; Saltevo, Juha; Kuusisto, Johanna; Laakso, Markku; Metspalu, Andres; Erbel, Raimund; Jöckel, Karl-Heinz; Moebus, Susanne; Ripatti, Samuli; Salomaa, Veikko; Ingelsson, Erik; Boehm, Bernhard O; Bergman, Richard N; Collins, Francis S; Mohlke, Karen L; Koistinen, Heikki; Tuomilehto, Jaakko; Hveem, Kristian; Njølstad, Inger; Deloukas, Panagiotis; Donnelly, Peter J; Frayling, Timothy M; Hattersley, Andrew T; de Faire, Ulf; Hamsten, Anders; Illig, Thomas; Peters, Annette; Cauchi, Stephane; Sladek, Rob; Froguel, Philippe; Hansen, Torben; Pedersen, Oluf; Morris, Andrew D; Palmer, Collin NA; Kathiresan, Sekar; Melander, Olle; Nilsson, Peter M; Groop, Leif C; Barroso, Inês; Langenberg, Claudia; Wareham, Nicholas J; O’Callaghan, Christopher A; Gloyn, Anna L; Altshuler, David; Boehnke, Michael; Teslovich, Tanya M; McCarthy, Mark I; Morris, Andrew P

2015-01-01

We performed fine-mapping of 39 established type 2 diabetes (T2D) loci in 27,206 cases and 57,574 controls of European ancestry. We identified 49 distinct association signals at these loci, including five mapping in/near KCNQ1. “Credible sets” of variants most likely to drive each distinct signal mapped predominantly to non-coding sequence, implying that T2D association is mediated through gene regulation. Credible set variants were enriched for overlap with FOXA2 chromatin immunoprecipitation binding sites in human islet and liver cells, including at MTNR1B, where fine-mapping implicated rs10830963 as driving T2D association. We confirmed that this T2D-risk allele increases FOXA2-bound enhancer activity in islet- and liver-derived cells. We observed allele-specific differences in NEUROD1 binding in islet-derived cells, consistent with evidence that the T2D-risk allele increases islet MTNR1B expression. Our study demonstrates how integration of genetic and genomic information can define molecular mechanisms through which variants underlying association signals exert their effects on disease. PMID:26551672

A novel de novo activating mutation in STAT3 identified in a patient with common variable immunodeficiency (CVID).

PubMed

Russell, Mark A; Pigors, Manuela; Houssen, Maha E; Manson, Ania; Kelsell, David; Longhurst, Hilary; Morgan, Noel G

2018-02-01

Common variable immunodeficiency (CVID) is characterised by repeated infection associated with primary acquired hypogammaglobulinemia. CVID frequently has a complex aetiology but, in certain cases, it has a monogenic cause. Recently, variants within the gene encoding the transcription factor STAT3 were implicated in monogenic CVID. Here, we describe a patient presenting with symptoms synonymous with CVID, who displayed reduced levels of IgG and IgA, repeated viral infections and multiple additional co-morbidities. Whole-exome sequencing revealed a de novo novel missense mutation in the coiled-coil domain of STAT3 (c.870A>T; p.K290N). Accordingly, the K290N variant of STAT3 was generated, and a STAT3 responsive dual-luciferase reporter assay revealed that the variant strongly enhances STAT3 transcriptional activity both under basal and stimulated (with IL-6) conditions. Overall, these data complement earlier studies in which CVID-associated STAT3 mutations are predicted to enhance transcriptional activity, suggesting that such patients may respond favourably to IL-6 receptor antagonists (e.g. tocilizumab). Copyright © 2017 Elsevier Inc. All rights reserved.

An Evaluation of Different Target Enrichment Methods in Pooled Sequencing Designs for Complex Disease Association Studies

PubMed Central

Day-Williams, Aaron G.; McLay, Kirsten; Drury, Eleanor; Edkins, Sarah; Coffey, Alison J.; Palotie, Aarno; Zeggini, Eleftheria

2011-01-01

Pooled sequencing can be a cost-effective approach to disease variant discovery, but its applicability in association studies remains unclear. We compare sequence enrichment methods coupled to next-generation sequencing in non-indexed pools of 1, 2, 10, 20 and 50 individuals and assess their ability to discover variants and to estimate their allele frequencies. We find that pooled resequencing is most usefully applied as a variant discovery tool due to limitations in estimating allele frequency with high enough accuracy for association studies, and that in-solution hybrid-capture performs best among the enrichment methods examined regardless of pool size. PMID:22069447

Protein-altering variants associated with body mass index implicate pathways that control energy intake and expenditure in obesity.

PubMed

Turcot, Valérie; Lu, Yingchang; Highland, Heather M; Schurmann, Claudia; Justice, Anne E; Fine, Rebecca S; Bradfield, Jonathan P; Esko, Tõnu; Giri, Ayush; Graff, Mariaelisa; Guo, Xiuqing; Hendricks, Audrey E; Karaderi, Tugce; Lempradl, Adelheid; Locke, Adam E; Mahajan, Anubha; Marouli, Eirini; Sivapalaratnam, Suthesh; Young, Kristin L; Alfred, Tamuno; Feitosa, Mary F; Masca, Nicholas G D; Manning, Alisa K; Medina-Gomez, Carolina; Mudgal, Poorva; Ng, Maggie C Y; Reiner, Alex P; Vedantam, Sailaja; Willems, Sara M; Winkler, Thomas W; Abecasis, Gonçalo; Aben, Katja K; Alam, Dewan S; Alharthi, Sameer E; Allison, Matthew; Amouyel, Philippe; Asselbergs, Folkert W; Auer, Paul L; Balkau, Beverley; Bang, Lia E; Barroso, Inês; Bastarache, Lisa; Benn, Marianne; Bergmann, Sven; Bielak, Lawrence F; Blüher, Matthias; Boehnke, Michael; Boeing, Heiner; Boerwinkle, Eric; Böger, Carsten A; Bork-Jensen, Jette; Bots, Michiel L; Bottinger, Erwin P; Bowden, Donald W; Brandslund, Ivan; Breen, Gerome; Brilliant, Murray H; Broer, Linda; Brumat, Marco; Burt, Amber A; Butterworth, Adam S; Campbell, Peter T; Cappellani, Stefania; Carey, David J; Catamo, Eulalia; Caulfield, Mark J; Chambers, John C; Chasman, Daniel I; Chen, Yii-Der I; Chowdhury, Rajiv; Christensen, Cramer; Chu, Audrey Y; Cocca, Massimiliano; Collins, Francis S; Cook, James P; Corley, Janie; Corominas Galbany, Jordi; Cox, Amanda J; Crosslin, David S; Cuellar-Partida, Gabriel; D'Eustacchio, Angela; Danesh, John; Davies, Gail; Bakker, Paul I W; Groot, Mark C H; Mutsert, Renée; Deary, Ian J; Dedoussis, George; Demerath, Ellen W; Heijer, Martin; Hollander, Anneke I; Ruijter, Hester M; Dennis, Joe G; Denny, Josh C; Di Angelantonio, Emanuele; Drenos, Fotios; Du, Mengmeng; Dubé, Marie-Pierre; Dunning, Alison M; Easton, Douglas F; Edwards, Todd L; Ellinghaus, David; Ellinor, Patrick T; Elliott, Paul; Evangelou, Evangelos; Farmaki, Aliki-Eleni; Farooqi, I Sadaf; Faul, Jessica D; Fauser, Sascha; Feng, Shuang; Ferrannini, Ele; Ferrieres, Jean; Florez, Jose C; Ford, Ian; Fornage, Myriam; Franco, Oscar H; Franke, Andre; Franks, Paul W; Friedrich, Nele; Frikke-Schmidt, Ruth; Galesloot, Tessel E; Gan, Wei; Gandin, Ilaria; Gasparini, Paolo; Gibson, Jane; Giedraitis, Vilmantas; Gjesing, Anette P; Gordon-Larsen, Penny; Gorski, Mathias; Grabe, Hans-Jörgen; Grant, Struan F A; Grarup, Niels; Griffiths, Helen L; Grove, Megan L; Gudnason, Vilmundur; Gustafsson, Stefan; Haessler, Jeff; Hakonarson, Hakon; Hammerschlag, Anke R; Hansen, Torben; Harris, Kathleen Mullan; Harris, Tamara B; Hattersley, Andrew T; Have, Christian T; Hayward, Caroline; He, Liang; Heard-Costa, Nancy L; Heath, Andrew C; Heid, Iris M; Helgeland, Øyvind; Hernesniemi, Jussi; Hewitt, Alex W; Holmen, Oddgeir L; Hovingh, G Kees; Howson, Joanna M M; Hu, Yao; Huang, Paul L; Huffman, Jennifer E; Ikram, M Arfan; Ingelsson, Erik; Jackson, Anne U; Jansson, Jan-Håkan; Jarvik, Gail P; Jensen, Gorm B; Jia, Yucheng; Johansson, Stefan; Jørgensen, Marit E; Jørgensen, Torben; Jukema, J Wouter; Kahali, Bratati; Kahn, René S; Kähönen, Mika; Kamstrup, Pia R; Kanoni, Stavroula; Kaprio, Jaakko; Karaleftheri, Maria; Kardia, Sharon L R; Karpe, Fredrik; Kathiresan, Sekar; Kee, Frank; Kiemeney, Lambertus A; Kim, Eric; Kitajima, Hidetoshi; Komulainen, Pirjo; Kooner, Jaspal S; Kooperberg, Charles; Korhonen, Tellervo; Kovacs, Peter; Kuivaniemi, Helena; Kutalik, Zoltán; Kuulasmaa, Kari; Kuusisto, Johanna; Laakso, Markku; Lakka, Timo A; Lamparter, David; Lange, Ethan M; Lange, Leslie A; Langenberg, Claudia; Larson, Eric B; Lee, Nanette R; Lehtimäki, Terho; Lewis, Cora E; Li, Huaixing; Li, Jin; Li-Gao, Ruifang; Lin, Honghuang; Lin, Keng-Hung; Lin, Li-An; Lin, Xu; Lind, Lars; Lindström, Jaana; Linneberg, Allan; Liu, Ching-Ti; Liu, Dajiang J; Liu, Yongmei; Lo, Ken S; Lophatananon, Artitaya; Lotery, Andrew J; Loukola, Anu; Luan, Jian'an; Lubitz, Steven A; Lyytikäinen, Leo-Pekka; Männistö, Satu; Marenne, Gaëlle; Mazul, Angela L; McCarthy, Mark I; McKean-Cowdin, Roberta; Medland, Sarah E; Meidtner, Karina; Milani, Lili; Mistry, Vanisha; Mitchell, Paul; Mohlke, Karen L; Moilanen, Leena; Moitry, Marie; Montgomery, Grant W; Mook-Kanamori, Dennis O; Moore, Carmel; Mori, Trevor A; Morris, Andrew D; Morris, Andrew P; Müller-Nurasyid, Martina; Munroe, Patricia B; Nalls, Mike A; Narisu, Narisu; Nelson, Christopher P; Neville, Matt; Nielsen, Sune F; Nikus, Kjell; Njølstad, Pål R; Nordestgaard, Børge G; Nyholt, Dale R; O'Connel, Jeffrey R; O'Donoghue, Michelle L; Olde Loohuis, Loes M; Ophoff, Roel A; Owen, Katharine R; Packard, Chris J; Padmanabhan, Sandosh; Palmer, Colin N A; Palmer, Nicholette D; Pasterkamp, Gerard; Patel, Aniruddh P; Pattie, Alison; Pedersen, Oluf; Peissig, Peggy L; Peloso, Gina M; Pennell, Craig E; Perola, Markus; Perry, James A; Perry, John R B; Pers, Tune H; Person, Thomas N; Peters, Annette; Petersen, Eva R B; Peyser, Patricia A; Pirie, Ailith; Polasek, Ozren; Polderman, Tinca J; Puolijoki, Hannu; Raitakari, Olli T; Rasheed, Asif; Rauramaa, Rainer; Reilly, Dermot F; Renström, Frida; Rheinberger, Myriam; Ridker, Paul M; Rioux, John D; Rivas, Manuel A; Roberts, David J; Robertson, Neil R; Robino, Antonietta; Rolandsson, Olov; Rudan, Igor; Ruth, Katherine S; Saleheen, Danish; Salomaa, Veikko; Samani, Nilesh J; Sapkota, Yadav; Sattar, Naveed; Schoen, Robert E; Schreiner, Pamela J; Schulze, Matthias B; Scott, Robert A; Segura-Lepe, Marcelo P; Shah, Svati H; Sheu, Wayne H-H; Sim, Xueling; Slater, Andrew J; Small, Kerrin S; Smith, Albert V; Southam, Lorraine; Spector, Timothy D; Speliotes, Elizabeth K; Starr, John M; Stefansson, Kari; Steinthorsdottir, Valgerdur; Stirrups, Kathleen E; Strauch, Konstantin; Stringham, Heather M; Stumvoll, Michael; Sun, Liang; Surendran, Praveen; Swift, Amy J; Tada, Hayato; Tansey, Katherine E; Tardif, Jean-Claude; Taylor, Kent D; Teumer, Alexander; Thompson, Deborah J; Thorleifsson, Gudmar; Thorsteinsdottir, Unnur; Thuesen, Betina H; Tönjes, Anke; Tromp, Gerard; Trompet, Stella; Tsafantakis, Emmanouil; Tuomilehto, Jaakko; Tybjaerg-Hansen, Anne; Tyrer, Jonathan P; Uher, Rudolf; Uitterlinden, André G; Uusitupa, Matti; Laan, Sander W; Duijn, Cornelia M; Leeuwen, Nienke; van Setten, Jessica; Vanhala, Mauno; Varbo, Anette; Varga, Tibor V; Varma, Rohit; Velez Edwards, Digna R; Vermeulen, Sita H; Veronesi, Giovanni; Vestergaard, Henrik; Vitart, Veronique; Vogt, Thomas F; Völker, Uwe; Vuckovic, Dragana; Wagenknecht, Lynne E; Walker, Mark; Wallentin, Lars; Wang, Feijie; Wang, Carol A; Wang, Shuai; Wang, Yiqin; Ware, Erin B; Wareham, Nicholas J; Warren, Helen R; Waterworth, Dawn M; Wessel, Jennifer; White, Harvey D; Willer, Cristen J; Wilson, James G; Witte, Daniel R; Wood, Andrew R; Wu, Ying; Yaghootkar, Hanieh; Yao, Jie; Yao, Pang; Yerges-Armstrong, Laura M; Young, Robin; Zeggini, Eleftheria; Zhan, Xiaowei; Zhang, Weihua; Zhao, Jing Hua; Zhao, Wei; Zhao, Wei; Zhou, Wei; Zondervan, Krina T; Rotter, Jerome I; Pospisilik, John A; Rivadeneira, Fernando; Borecki, Ingrid B; Deloukas, Panos; Frayling, Timothy M; Lettre, Guillaume; North, Kari E; Lindgren, Cecilia M; Hirschhorn, Joel N; Loos, Ruth J F

2018-01-01

Genome-wide association studies (GWAS) have identified >250 loci for body mass index (BMI), implicating pathways related to neuronal biology. Most GWAS loci represent clusters of common, noncoding variants from which pinpointing causal genes remains challenging. Here we combined data from 718,734 individuals to discover rare and low-frequency (minor allele frequency (MAF) < 5%) coding variants associated with BMI. We identified 14 coding variants in 13 genes, of which 8 variants were in genes (ZBTB7B, ACHE, RAPGEF3, RAB21, ZFHX3, ENTPD6, ZFR2 and ZNF169) newly implicated in human obesity, 2 variants were in genes (MC4R and KSR2) previously observed to be mutated in extreme obesity and 2 variants were in GIPR. The effect sizes of rare variants are ~10 times larger than those of common variants, with the largest effect observed in carriers of an MC4R mutation introducing a stop codon (p.Tyr35Ter, MAF = 0.01%), who weighed ~7 kg more than non-carriers. Pathway analyses based on the variants associated with BMI confirm enrichment of neuronal genes and provide new evidence for adipocyte and energy expenditure biology, widening the potential of genetically supported therapeutic targets in obesity.

Chromosome specific repetitive DNA sequences

DOEpatents

Moyzis, Robert K.; Meyne, Julianne

1991-01-01

A method is provided for determining specific nucleotide sequences useful in forming a probe which can identify specific chromosomes, preferably through in situ hybridization within the cell itself. In one embodiment, chromosome preferential nucleotide sequences are first determined from a library of recombinant DNA clones having families of repetitive sequences. Library clones are identified with a low homology with a sequence of repetitive DNA families to which the first clones respectively belong and variant sequences are then identified by selecting clones having a pattern of hybridization with genomic DNA dissimilar to the hybridization pattern shown by the respective families. In another embodiment, variant sequences are selected from a sequence of a known repetitive DNA family. The selected variant sequence is classified as chromosome specific, chromosome preferential, or chromosome nonspecific. Sequences which are classified as chromosome preferential are further sequenced and regions are identified having a low homology with other regions of the chromosome preferential sequence or with known sequences of other family me This invention is the result of a contract with the Department of Energy (Contract No. W-7405-ENG-36).

Loss-of-function DNA sequence variant in the CLCNKA chloride channel implicates the cardio-renal axis in interindividual heart failure risk variation.

PubMed

Cappola, Thomas P; Matkovich, Scot J; Wang, Wei; van Booven, Derek; Li, Mingyao; Wang, Xuexia; Qu, Liming; Sweitzer, Nancy K; Fang, James C; Reilly, Muredach P; Hakonarson, Hakon; Nerbonne, Jeanne M; Dorn, Gerald W

2011-02-08

Common heart failure has a strong undefined heritable component. Two recent independent cardiovascular SNP array studies identified a common SNP at 1p36 in intron 2 of the HSPB7 gene as being associated with heart failure. HSPB7 resequencing identified other risk alleles but no functional gene variants. Here, we further show no effect of the HSPB7 SNP on cardiac HSPB7 mRNA levels or splicing, suggesting that the SNP marks the position of a functional variant in another gene. Accordingly, we used massively parallel platforms to resequence all coding exons of the adjacent CLCNKA gene, which encodes the K(a) renal chloride channel (ClC-K(a)). Of 51 exonic CLCNKA variants identified, one SNP (rs10927887, encoding Arg83Gly) was common, in linkage disequilibrium with the heart failure risk SNP in HSPB7, and associated with heart failure in two independent Caucasian referral populations (n = 2,606 and 1,168; combined P = 2.25 × 10(-6)). Individual genotyping of rs10927887 in the two study populations and a third independent heart failure cohort (combined n = 5,489) revealed an additive allele effect on heart failure risk that is independent of age, sex, and prior hypertension (odds ratio = 1.27 per allele copy; P = 8.3 × 10(-7)). Functional characterization of recombinant wild-type Arg83 and variant Gly83 ClC-K(a) chloride channel currents revealed ≈ 50% loss-of-function of the variant channel. These findings identify a common, functionally significant genetic risk factor for Caucasian heart failure. The variant CLCNKA risk allele, telegraphed by linked variants in the adjacent HSPB7 gene, uncovers a previously overlooked genetic mechanism affecting the cardio-renal axis.

Analysis of selected genes associated with cardiomyopathy by next-generation sequencing.

PubMed

Szabadosova, Viktoria; Boronova, Iveta; Ferenc, Peter; Tothova, Iveta; Bernasovska, Jarmila; Zigova, Michaela; Kmec, Jan; Bernasovsky, Ivan

2018-02-01

As the leading cause of congestive heart failure, cardiomyopathy represents a heterogenous group of heart muscle disorders. Despite considerable progress being made in the genetic diagnosis of cardiomyopathy by detection of the mutations in the most prevalent cardiomyopathy genes, the cause remains unsolved in many patients. High-throughput mutation screening in the disease genes for cardiomyopathy is now possible because of using target enrichment followed by next-generation sequencing. The aim of the study was to analyze a panel of genes associated with dilated or hypertrophic cardiomyopathy based on previously published results in order to identify the subjects at risk. The method of next-generation sequencing by IlluminaHiSeq 2500 platform was used to detect sequence variants in 16 individuals diagnosed with dilated or hypertrophic cardiomyopathy. Detected variants were filtered and the functional impact of amino acid changes was predicted by computational programs. DNA samples of the 16 patients were analyzed by whole exome sequencing. We identified six nonsynonymous variants that were shown to be pathogenic in all used prediction softwares: rs3744998 (EPG5), rs11551768 (MGME1), rs148374985 (MURC), rs78461695 (PLEC), rs17158558 (RET) and rs2295190 (SYNE1). Two of the analyzed sequence variants had minor allele frequency (MAF)<0.01: rs148374985 (MURC), rs34580776 (MYBPC3). Our data support the potential role of the detected variants in pathogenesis of dilated or hypertrophic cardiomyopathy; however, the possibility that these variants might not be true disease-causing variants but are susceptibility alleles that require additional mutations or injury to cause the clinical phenotype of disease must be considered. © 2017 Wiley Periodicals, Inc.

Variants of beta-glucosidase

DOEpatents

Fidantsef, Ana; Lamsa, Michael; Gorre-Clancy, Brian

2015-07-14

The present invention relates to variants of a parent beta-glucosidase, comprising a substitution at one or more positions corresponding to positions 142, 183, 266, and 703 of amino acids 1 to 842 of SEQ ID NO: 2 or corresponding to positions 142, 183, 266, and 705 of amino acids 1 to 844 of SEQ ID NO: 70, wherein the variant has beta-glucosidase activity. The present invention also relates to nucleotide sequences encoding the variant beta-glucosidases and to nucleic acid constructs, vectors, and host cells comprising the nucleotide sequences.

Variants of beta-glucosidases

DOEpatents

Fidantsef, Ana; Lamsa, Michael; Gorre-Clancy, Brian

2014-10-07

The present invention relates to variants of a parent beta-glucosidase, comprising a substitution at one or more positions corresponding to positions 142, 183, 266, and 703 of amino acids 1 to 842 of SEQ ID NO: 2 or corresponding to positions 142, 183, 266, and 705 of amino acids 1 to 844 of SEQ ID NO: 70, wherein the variant has beta-glucosidase activity. The present invention also relates to nucleotide sequences encoding the variant beta-glucosidases and to nucleic acid constructs, vectors, and host cells comprising the nucleotide sequences.

Variants of beta-glucosidase

DOEpatents

Fidantsef, Ana [Davis, CA; Lamsa, Michael [Davis, CA; Gorre-Clancy, Brian [Elk Grove, CA

2009-12-29

The present invention relates to variants of a parent beta-glucosidase, comprising a substitution at one or more positions corresponding to positions 142, 183, 266, and 703 of amino acids 1 to 842 of SEQ ID NO: 2 or corresponding to positions 142, 183, 266, and 705 of amino acids 1 to 844 of SEQ ID NO: 70, wherein the variant has beta-glucosidase activity. The present invention also relates to nucleotide sequences encoding the variant beta-glucosidases and to nucleic acid constructs, vectors, and host cells comprising the nucleotide sequences.

Whole-exome sequencing reveals genetic variants associated with chronic kidney disease characterized by tubulointerstitial damages in North Central Region, Sri Lanka.

PubMed

Nanayakkara, Shanika; Senevirathna, S T M L D; Parahitiyawa, Nipuna B; Abeysekera, Tilak; Chandrajith, Rohana; Ratnatunga, Neelakanthi; Hitomi, Toshiaki; Kobayashi, Hatasu; Harada, Kouji H; Koizumi, Akio

2015-09-01

The familial clustering observed in chronic kidney disease of uncertain etiology (CKDu) characterized by tubulointerstitial damages in the North Central Region of Sri Lanka strongly suggests the involvement of genetic factors in its pathogenesis. The objective of the present study is to use whole-exome sequencing to identify the genetic variants associated with CKDu. Whole-exome sequencing of eight CKDu cases and eight controls was performed, followed by direct sequencing of candidate loci in 301 CKDu cases and 276 controls. Association study revealed rs34970857 (c.658G > A/p.V220M) located in the KCNA10 gene encoding a voltage-gated K channel as the most promising SNP with the highest odds ratio of 1.74. Four rare variants were identified in gene encoding Laminin beta2 (LAMB2) which is known to cause congenital nephrotic syndrome. Three out of four variants in LAMB2 were novel variants found exclusively in cases. Genetic investigations provide strong evidence on the presence of genetic susceptibility for CKDu. Possibility of presence of several rare variants associated with CKDu in this population is also suggested.

HGVS Recommendations for the Description of Sequence Variants: 2016 Update.

PubMed

den Dunnen, Johan T; Dalgleish, Raymond; Maglott, Donna R; Hart, Reece K; Greenblatt, Marc S; McGowan-Jordan, Jean; Roux, Anne-Francoise; Smith, Timothy; Antonarakis, Stylianos E; Taschner, Peter E M

2016-06-01

The consistent and unambiguous description of sequence variants is essential to report and exchange information on the analysis of a genome. In particular, DNA diagnostics critically depends on accurate and standardized description and sharing of the variants detected. The sequence variant nomenclature system proposed in 2000 by the Human Genome Variation Society has been widely adopted and has developed into an internationally accepted standard. The recommendations are currently commissioned through a Sequence Variant Description Working Group (SVD-WG) operating under the auspices of three international organizations: the Human Genome Variation Society (HGVS), the Human Variome Project (HVP), and the Human Genome Organization (HUGO). Requests for modifications and extensions go through the SVD-WG following a standard procedure including a community consultation step. Version numbers are assigned to the nomenclature system to allow users to specify the version used in their variant descriptions. Here, we present the current recommendations, HGVS version 15.11, and briefly summarize the changes that were made since the 2000 publication. Most focus has been on removing inconsistencies and tightening definitions allowing automatic data processing. An extensive version of the recommendations is available online, at http://www.HGVS.org/varnomen. © 2016 WILEY PERIODICALS, INC.

Exome Sequencing Identifies Potential Risk Variants for Mendelian Disorders at High Prevalence in Qatar

PubMed Central

Rodriguez-Flores, Juan L.; Fakhro, Khalid; Hackett, Neil R.; Salit, Jacqueline; Fuller, Jennifer; Agosto-Perez, Francisco; Gharbiah, Maey; Malek, Joel A.; Zirie, Mahmoud; Jayyousi, Amin; Badii, Ramin; Al-Marri, Ajayeb Al-Nabet; Chouchane, Lotfi; Stadler, Dora J.; Hunter-Zinck, Haley; Mezey, Jason G.; Crystal, Ronald G.

2013-01-01

Exome sequencing of families of related individuals has been highly successful in identifying genetic polymorphisms responsible for Mendelian disorders. Here, we demonstrate the value of the reverse approach, where we use exome sequencing of a sample of unrelated individuals to analyze allele frequencies of known causal mutations for Mendelian diseases. We sequenced the exomes of 100 individuals representing the three major genetic subgroups of the Qatari population (Q1 Bedouin, Q2 Persian-South Asian, Q3 African) and identified 37 variants in 33 genes with effects on 36 clinically significant Mendelian diseases. These include variants not present in 1000 Genomes and variants at high frequency when compared to 1000 Genomes populations. Several of these Mendelian variants were only segregating in one Qatari subpopulation, where the observed subpopulation specificity trends were confirmed in an independent population of 386 Qataris. Pre-marital genetic screening in Qatar tests for only 4 out of the 37, such that this study provides a set of Mendelian disease variants with potential impact on the epidemiological profile of the population that could be incorporated into the testing program if further experimental and clinical characterization confirms high penetrance. PMID:24123366

Diversity and Divergence of Dinoflagellate Histone Proteins

PubMed Central

Marinov, Georgi K.; Lynch, Michael

2015-01-01

Histone proteins and the nucleosomal organization of chromatin are near-universal eukaroytic features, with the exception of dinoflagellates. Previous studies have suggested that histones do not play a major role in the packaging of dinoflagellate genomes, although several genomic and transcriptomic surveys have detected a full set of core histone genes. Here, transcriptomic and genomic sequence data from multiple dinoflagellate lineages are analyzed, and the diversity of histone proteins and their variants characterized, with particular focus on their potential post-translational modifications and the conservation of the histone code. In addition, the set of putative epigenetic mark readers and writers, chromatin remodelers and histone chaperones are examined. Dinoflagellates clearly express the most derived set of histones among all autonomous eukaryote nuclei, consistent with a combination of relaxation of sequence constraints imposed by the histone code and the presence of numerous specialized histone variants. The histone code itself appears to have diverged significantly in some of its components, yet others are conserved, implying conservation of the associated biochemical processes. Specifically, and with major implications for the function of histones in dinoflagellates, the results presented here strongly suggest that transcription through nucleosomal arrays happens in dinoflagellates. Finally, the plausible roles of histones in dinoflagellate nuclei are discussed. PMID:26646152

Analysis of intra-host genetic diversity of Prunus necrotic ringspot virus (PNRSV) using amplicon next generation sequencing

PubMed Central

Constable, Fiona E.; Nancarrow, Narelle; Plummer, Kim M.; Rodoni, Brendan

2017-01-01

PCR amplicon next generation sequencing (NGS) analysis offers a broadly applicable and targeted approach to detect populations of both high- or low-frequency virus variants in one or more plant samples. In this study, amplicon NGS was used to explore the diversity of the tripartite genome virus, Prunus necrotic ringspot virus (PNRSV) from 53 PNRSV-infected trees using amplicons from conserved gene regions of each of PNRSV RNA1, RNA2 and RNA3. Sequencing of the amplicons from 53 PNRSV-infected trees revealed differing levels of polymorphism across the three different components of the PNRSV genome with a total number of 5040, 2083 and 5486 sequence variants observed for RNA1, RNA2 and RNA3 respectively. The RNA2 had the lowest diversity of sequences compared to RNA1 and RNA3, reflecting the lack of flexibility tolerated by the replicase gene that is encoded by this RNA component. Distinct PNRSV phylo-groups, consisting of closely related clusters of sequence variants, were observed in each of PNRSV RNA1, RNA2 and RNA3. Most plant samples had a single phylo-group for each RNA component. Haplotype network analysis showed that smaller clusters of PNRSV sequence variants were genetically connected to the largest sequence variant cluster within a phylo-group of each RNA component. Some plant samples had sequence variants occurring in multiple PNRSV phylo-groups in at least one of each RNA and these phylo-groups formed distinct clades that represent PNRSV genetic strains. Variants within the same phylo-group of each Prunus plant sample had ≥97% similarity and phylo-groups within a Prunus plant sample and between samples had less ≤97% similarity. Based on the analysis of diversity, a definition of a PNRSV genetic strain was proposed. The proposed definition was applied to determine the number of PNRSV genetic strains in each of the plant samples and the complexity in defining genetic strains in multipartite genome viruses was explored. PMID:28632759

A Multiple-Sequence Variant of the Multiple-Baseline Design: A Strategy for Analysis of Sequence Effects and Treatment Comparison.

ERIC Educational Resources Information Center

Noell, George H.; Gresham, Frank M.

2001-01-01

Describes design logic and potential uses of a variant of the multiple-baseline design. The multiple-baseline multiple-sequence (MBL-MS) consists of multiple-baseline designs that are interlaced with one another and include all possible sequences of treatments. The MBL-MS design appears to be primarily useful for comparison of treatments taking…

Targeted exome sequencing for the identification of a protective variant against Internet gaming disorder at rs2229910 of neurotrophic tyrosine kinase receptor, type 3 (NTRK3): A pilot study

PubMed Central

Kim, Jeong-Yu; Jeong, Jo-Eun; Rhee, Je-Keun; Cho, Hyun; Chun, Ji-Won; Kim, Tae-Min; Choi, Sam-Wook; Choi, Jung-Seok; Kim, Dai-Jin

2016-01-01

Background and aims Internet gaming disorder (IGD) has gained recognition as a potential new diagnosis in the fifth revision of the Diagnostic and Statistical Manual of Mental Disorders, but genetic evidence supporting this disorder remains scarce. Methods In this study, targeted exome sequencing was conducted in 30 IGD patients and 30 control subjects with a focus on genes linked to various neurotransmitters associated with substance and non-substance addictions, depression, and attention deficit hyperactivity disorder. Results rs2229910 of neurotrophic tyrosine kinase receptor, type 3 (NTRK3) was the only single nucleotide polymorphism (SNP) that exhibited a significantly different minor allele frequency in IGD subjects compared to controls (p = .01932), suggesting that this SNP has a protective effect against IGD (odds ratio = 0.1541). The presence of this potentially protective allele was also associated with less time spent on Internet gaming and lower scores on the Young’s Internet Addiction Test and Korean Internet Addiction Proneness Scale for Adults. Conclusions The results of this first targeted exome sequencing study of IGD subjects indicate that rs2229910 of NTRK3 is a genetic variant that is significantly related to IGD. These findings may have significant implications for future research investigating the genetics of IGD and other behavioral addictions. PMID:27826991

A founder large deletion mutation in Xeroderma pigmentosum-Variant form in Tunisia: implication for molecular diagnosis and therapy.

PubMed

Ben Rekaya, Mariem; Laroussi, Nadia; Messaoud, Olfa; Jones, Mariem; Jerbi, Manel; Naouali, Chokri; Bouyacoub, Yosra; Chargui, Mariem; Kefi, Rym; Fazaa, Becima; Boubaker, Mohamed Samir; Boussen, Hamouda; Mokni, Mourad; Abdelhak, Sonia; Zghal, Mohamed; Khaled, Aida; Yacoub-Youssef, Houda

2014-01-01

Xeroderma pigmentosum Variant (XP-V) form is characterized by a late onset of skin symptoms. Our aim is the clinical and genetic investigations of XP-V Tunisian patients in order to develop a simple tool for early diagnosis. We investigated 16 suspected XP patients belonging to ten consanguineous families. Analysis of the POLH gene was performed by linkage analysis, long range PCR, and sequencing. Genetic analysis showed linkage to the POLH gene with a founder haplotype in all affected patients. Long range PCR of exon 9 to exon 11 showed a 3926 bp deletion compared to control individuals. Sequence analysis demonstrates that this deletion has occurred between two Alu-Sq2 repetitive sequences in the same orientation, respectively, in introns 9 and 10. We suggest that this mutation POLH NG_009252.1: g.36847_40771del3925 is caused by an equal crossover event that occurred between two homologous chromosomes at meiosis. These results allowed us to develop a simple test based on a simple PCR in order to screen suspected XP-V patients. In Tunisia, the prevalence of XP-V group seems to be underestimated and clinical diagnosis is usually later. Cascade screening of this founder mutation by PCR in regions with high frequency of XP provides a rapid and cost-effective tool for early diagnosis of XP-V in Tunisia and North Africa.

A Founder Large Deletion Mutation in Xeroderma Pigmentosum-Variant Form in Tunisia: Implication for Molecular Diagnosis and Therapy

PubMed Central

Ben Rekaya, Mariem; Laroussi, Nadia; Messaoud, Olfa; Jones, Mariem; Jerbi, Manel; Bouyacoub, Yosra; Chargui, Mariem; Kefi, Rym; Fazaa, Becima; Boubaker, Mohamed Samir; Boussen, Hamouda; Mokni, Mourad; Abdelhak, Sonia; Zghal, Mohamed; Khaled, Aida; Yacoub-Youssef, Houda

2014-01-01

Xeroderma pigmentosum Variant (XP-V) form is characterized by a late onset of skin symptoms. Our aim is the clinical and genetic investigations of XP-V Tunisian patients in order to develop a simple tool for early diagnosis. We investigated 16 suspected XP patients belonging to ten consanguineous families. Analysis of the POLH gene was performed by linkage analysis, long range PCR, and sequencing. Genetic analysis showed linkage to the POLH gene with a founder haplotype in all affected patients. Long range PCR of exon 9 to exon 11 showed a 3926 bp deletion compared to control individuals. Sequence analysis demonstrates that this deletion has occurred between two Alu-Sq2 repetitive sequences in the same orientation, respectively, in introns 9 and 10. We suggest that this mutation POLH NG_009252.1: g.36847_40771del3925 is caused by an equal crossover event that occurred between two homologous chromosomes at meiosis. These results allowed us to develop a simple test based on a simple PCR in order to screen suspected XP-V patients. In Tunisia, the prevalence of XP-V group seems to be underestimated and clinical diagnosis is usually later. Cascade screening of this founder mutation by PCR in regions with high frequency of XP provides a rapid and cost-effective tool for early diagnosis of XP-V in Tunisia and North Africa. PMID:24877075

Complete Sequence of Four Multidrug-Resistant MOBQ1 Plasmids Harboring blaGES-5 Isolated from Escherichia coli and Serratia marcescens Persisting in a Hospital in Canada.

PubMed

Boyd, David; Taylor, Geoffrey; Fuller, Jeff; Bryce, Elizabeth; Embree, Joanne; Gravel, Denise; Katz, Kevin; Kibsey, Pamela; Kuhn, Magdalena; Langley, Joanne; Mataseje, Laura; Mitchell, Robyn; Roscoe, Diane; Simor, Andrew; Thomas, Eva; Turgeon, Nathalie; Mulvey, Michael

2015-06-01

The usefulness of carbapenems for gram-negative infections is becoming compromised by organisms harboring carbapenemases, enzymes which can hydrolyze the drug. Currently KPC (class A), NDM (class B), and OXA-48 types (class D) are the most globally widespread carbapenemases. However, among the GES-type class A extended-spectrum β-lactamases (ESBLs) there are variants that hydrolyze carbapenems, with blaGES-5 being the most common. Two Escherichia coli and two Serratia marcescens harboring blaGES-5 on plasmids were isolated by the Canadian Nosocomial Infection Surveillance Program (CNISP) from four different patients in a single hospital over a 2-year period. Complete sequencing of the blaGES-5 plasmids indicated that all four had nearly identical backbones consisting of genes for replication, partitioning, and stability, but contained variant accessory regions consisting of mobile elements and antimicrobial resistance genes. The plasmids were of a novel replicon type, but belonged to the MOBQ1 group based on relaxase sequences, and appeared to be mobilizable, but not self-transmissible. Considering the time periods of bacterial isolation, it would appear the blaGES-5 plasmid has persisted in an environmental niche for at least 2 years in the hospital. This has implications for infection control and clinical care when it is transferred to clinically relevant gram-negative organisms.

Genomic variation in macrophage-cultured European porcine reproductive and respiratory syndrome virus Olot/91 revealed using ultra-deep next generation sequencing.

PubMed

Lu, Zen H; Brown, Alexander; Wilson, Alison D; Calvert, Jay G; Balasch, Monica; Fuentes-Utrilla, Pablo; Loecherbach, Julia; Turner, Frances; Talbot, Richard; Archibald, Alan L; Ait-Ali, Tahar

2014-03-04

Porcine Reproductive and Respiratory Syndrome (PRRS) is a disease of major economic impact worldwide. The etiologic agent of this disease is the PRRS virus (PRRSV). Increasing evidence suggest that microevolution within a coexisting quasispecies population can give rise to high sequence heterogeneity in PRRSV. We developed a pipeline based on the ultra-deep next generation sequencing approach to first construct the complete genome of a European PRRSV, strain Olot/9, cultured on macrophages and then capture the rare variants representative of the mixed quasispecies population. Olot/91 differs from the reference Lelystad strain by about 5% and a total of 88 variants, with frequencies as low as 1%, were detected in the mixed population. These variants included 16 non-synonymous variants concentrated in the genes encoding structural and nonstructural proteins; including Glycoprotein 2a and 5. Using an ultra-deep sequencing methodology, the complete genome of Olot/91 was constructed without any prior knowledge of the sequence. Rare variants that constitute minor fractions of the heterogeneous PRRSV population could successfully be detected to allow further exploration of microevolutionary events.

A rare variant of the mtDNA HVS1 sequence in the hairs of Napoléon's family.

PubMed

Lucotte, Gérard

2010-10-04

This paper describes the finding of a rare variant in the sequence of the hypervariable segment (HVS1) of mitochondrial (mtDNA) extracted from two preserved hairs, authenticated as belonging to the French Emperor Napoléon I (Napoléon Bonaparte). This rare variant is a mutation that changes the base C to T at position 16,184 (16184C→T), and it constitutes the only mutation found in this HVS1 sequence. This mutation is rare, because it was not found in a reference database (P < 0.05). In a personal database (M. Pala) comprising 37,000 different sequences, the 16184C→T mutation was found in only three samples, thus in this database the mutation frequency was 0.00008%. This mutation 16184C→T was also the only variant found subsequently in the HVS1 sequences of mtDNAs extracted from Napoléon's mother (Letizia) and from his youngest sister (Caroline), confirming that this mutation is maternally inherited. This 16184C→T variant could be used for genetic verification to authenticate any doubtful material and determine whether it should indeed be attributed to Napoléon.

A rare variant of the mtDNA HVS1 sequence in the hairs of Napoléon's family

PubMed Central

2010-01-01

This paper describes the finding of a rare variant in the sequence of the hypervariable segment (HVS1) of mitochondrial (mtDNA) extracted from two preserved hairs, authenticated as belonging to the French Emperor Napoléon I (Napoléon Bonaparte). This rare variant is a mutation that changes the base C to T at position 16,184 (16184C→T), and it constitutes the only mutation found in this HVS1 sequence. This mutation is rare, because it was not found in a reference database (P < 0.05). In a personal database (M. Pala) comprising 37,000 different sequences, the 16184C→T mutation was found in only three samples, thus in this database the mutation frequency was 0.00008%. This mutation 16184C→T was also the only variant found subsequently in the HVS1 sequences of mtDNAs extracted from Napoléon's mother (Letizia) and from his youngest sister (Caroline), confirming that this mutation is maternally inherited. This 16184C→T variant could be used for genetic verification to authenticate any doubtful material and determine whether it should indeed be attributed to Napoléon. PMID:21092341

Novel pathogenic variant (c.3178G>A) in the SMC1A gene in a family with Cornelia de Lange syndrome identified by exome sequencing.

PubMed

Jang, Mi Ae; Lee, Chang Woo; Kim, Jin Kyung; Ki, Chang Seok

2015-11-01

Cornelia de Lange syndrome (CdLS) is a clinically and genetically heterogeneous congenital anomaly. Mutations in the NIPBL gene account for a half of the affected individuals. We describe a family with CdLS carrying a novel pathogenic variant of the SMC1A gene identified by exome sequencing. The proband was a 3-yr-old boy presenting with a developmental delay. He had distinctive facial features without major structural anomalies and tested negative for the NIPBL gene. His younger sister, mother, and maternal grandmother presented with mild mental retardation. By exome sequencing of the proband, a novel SMC1A variant, c.3178G>A, was identified, which was expected to cause an amino acid substitution (p.Glu1060Lys) in the highly conserved coiled-coil domain of the SMC1A protein. Sanger sequencing confirmed that the three female relatives with mental retardation also carry this variant. Our results reveal that SMC1A gene defects are associated with milder phenotypes of CdLS. Furthermore, we showed that exome sequencing could be a useful tool to identify pathogenic variants in patients with CdLS.

Whole genome sequencing and imputation in isolated populations identify genetic associations with medically-relevant complex traits

PubMed Central

Southam, Lorraine; Gilly, Arthur; Süveges, Dániel; Farmaki, Aliki-Eleni; Schwartzentruber, Jeremy; Tachmazidou, Ioanna; Matchan, Angela; Rayner, Nigel W.; Tsafantakis, Emmanouil; Karaleftheri, Maria; Xue, Yali; Dedoussis, George; Zeggini, Eleftheria

2017-01-01

Next-generation association studies can be empowered by sequence-based imputation and by studying founder populations. Here we report ∼9.5 million variants from whole-genome sequencing (WGS) of a Cretan-isolated population, and show enrichment of rare and low-frequency variants with predicted functional consequences. We use a WGS-based imputation approach utilizing 10,422 reference haplotypes to perform genome-wide association analyses and observe 17 genome-wide significant, independent signals, including replicating evidence for association at eight novel low-frequency variant signals. Two novel cardiometabolic associations are at lead variants unique to the founder population sequences: chr16:70790626 (high-density lipoprotein levels beta −1.71 (SE 0.25), P=1.57 × 10−11, effect allele frequency (EAF) 0.006); and rs145556679 (triglycerides levels beta −1.13 (SE 0.17), P=2.53 × 10−11, EAF 0.013). Our findings add empirical support to the contribution of low-frequency variants in complex traits, demonstrate the advantage of including population-specific sequences in imputation panels and exemplify the power gains afforded by population isolates. PMID:28548082

Electron holes appear to trigger cancer-implicated mutations

NASA Astrophysics Data System (ADS)

Miller, John; Villagran, Martha

Malignant tumors are caused by mutations, which also affect their subsequent growth and evolution. We use a novel approach, computational DNA hole spectroscopy [M.Y. Suarez-Villagran & J.H. Miller, Sci. Rep. 5, 13571 (2015)], to compute spectra of enhanced hole probability based on actual sequence data. A hole is a mobile site of positive charge created when an electron is removed, for example by radiation or contact with a mutagenic agent. Peaks in the hole spectrum depict sites where holes tend to localize and potentially trigger a base pair mismatch during replication. Our studies of reveal a correlation between hole spectrum peaks and spikes in human mutation frequencies. Importantly, we also find that hole peak positions that do not coincide with large variant frequencies often coincide with cancer-implicated mutations and/or (for coding DNA) encoded conserved amino acids. This enables combining hole spectra with variant data to identify critical base pairs and potential cancer `driver' mutations. Such integration of DNA hole and variance spectra could also prove invaluable for pinpointing critical regions, and sites of driver mutations, in the vast non-protein-coding genome. Supported by the State of Texas through the Texas Ctr. for Superconductivity.

Genetic identification of brain cell types underlying schizophrenia.

PubMed

Skene, Nathan G; Bryois, Julien; Bakken, Trygve E; Breen, Gerome; Crowley, James J; Gaspar, Héléna A; Giusti-Rodriguez, Paola; Hodge, Rebecca D; Miller, Jeremy A; Muñoz-Manchado, Ana B; O'Donovan, Michael C; Owen, Michael J; Pardiñas, Antonio F; Ryge, Jesper; Walters, James T R; Linnarsson, Sten; Lein, Ed S; Sullivan, Patrick F; Hjerling-Leffler, Jens

2018-06-01

With few exceptions, the marked advances in knowledge about the genetic basis of schizophrenia have not converged on findings that can be confidently used for precise experimental modeling. By applying knowledge of the cellular taxonomy of the brain from single-cell RNA sequencing, we evaluated whether the genomic loci implicated in schizophrenia map onto specific brain cell types. We found that the common-variant genomic results consistently mapped to pyramidal cells, medium spiny neurons (MSNs) and certain interneurons, but far less consistently to embryonic, progenitor or glial cells. These enrichments were due to sets of genes that were specifically expressed in each of these cell types. We also found that many of the diverse gene sets previously associated with schizophrenia (genes involved in synaptic function, those encoding mRNAs that interact with FMRP, antipsychotic targets, etc.) generally implicated the same brain cell types. Our results suggest a parsimonious explanation: the common-variant genetic results for schizophrenia point at a limited set of neurons, and the gene sets point to the same cells. The genetic risk associated with MSNs did not overlap with that of glutamatergic pyramidal cells and interneurons, suggesting that different cell types have biologically distinct roles in schizophrenia.

Cotransin induces accumulation of a cytotoxic clusterin variant that cotranslationally rerouted to the cytosol

DOE Office of Scientific and Technical Information (OSTI.GOV)

Choi, Ilho; Kim, Jiyeon; Park, Joong-Yeol

2013-05-01

Although clusterin (CLU) was originally identified as a secreted glycoprotein that plays cytoprotective role, several intracellular CLU variants have been recently identified in the diverse pathological conditions. The mechanistic basis of these variants is now believed to be alternative splicing and retrotranslocation. Here, we uncovered, an unglycosylated and signal sequence-unprocessed, CLU variant in the cytosol. This variant proved to be a product that cotranslationally rerouted to the cytosol during translocation. Cytosolic CLU was prone to aggregation at peri-nuclear region of cells and induced cell death. Signal sequence is shown to be an important determinant for cytosolic CLU generation and aggregation.more » These results provide not only a new mechanistic insight into the cytosolic CLU generation but also an idea for therapeutic mislocalization of CLU as a strategy for cancer treatment. - Highlights: ► Intracellular CLU variants have been recently identified in the diverse pathological conditions. ► Translocation of clusterin is less efficient than that of Prl. ► We identified a new cytotoxic clusterin variant whose signal sequence was unprocessed. ► This variant proved to be a product that cotranslationally rerouted to cytosol.« less

AmpliVar: mutation detection in high-throughput sequence from amplicon-based libraries.

PubMed

Hsu, Arthur L; Kondrashova, Olga; Lunke, Sebastian; Love, Clare J; Meldrum, Cliff; Marquis-Nicholson, Renate; Corboy, Greg; Pham, Kym; Wakefield, Matthew; Waring, Paul M; Taylor, Graham R

2015-04-01

Conventional means of identifying variants in high-throughput sequencing align each read against a reference sequence, and then call variants at each position. Here, we demonstrate an orthogonal means of identifying sequence variation by grouping the reads as amplicons prior to any alignment. We used AmpliVar to make key-value hashes of sequence reads and group reads as individual amplicons using a table of flanking sequences. Low-abundance reads were removed according to a selectable threshold, and reads above this threshold were aligned as groups, rather than as individual reads, permitting the use of sensitive alignment tools. We show that this approach is more sensitive, more specific, and more computationally efficient than comparable methods for the analysis of amplicon-based high-throughput sequencing data. The method can be extended to enable alignment-free confirmation of variants seen in hybridization capture target-enrichment data. © 2015 WILEY PERIODICALS, INC.

A survey of single nucleotide polymorphisms identified from whole-genome sequencing and their functional effect in the porcine genome.

PubMed

Keel, B N; Nonneman, D J; Rohrer, G A

2017-08-01

Genetic variants detected from sequence have been used to successfully identify causal variants and map complex traits in several organisms. High and moderate impact variants, those expected to alter or disrupt the protein coded by a gene and those that regulate protein production, likely have a more significant effect on phenotypic variation than do other types of genetic variants. Hence, a comprehensive list of these functional variants would be of considerable interest in swine genomic studies, particularly those targeting fertility and production traits. Whole-genome sequence was obtained from 72 of the founders of an intensely phenotyped experimental swine herd at the U.S. Meat Animal Research Center (USMARC). These animals included all 24 of the founding boars (12 Duroc and 12 Landrace) and 48 Yorkshire-Landrace composite sows. Sequence reads were mapped to the Sscrofa10.2 genome build, resulting in a mean of 6.1 fold (×) coverage per genome. A total of 22 342 915 high confidence SNPs were identified from the sequenced genomes. These included 21 million previously reported SNPs and 79% of the 62 163 SNPs on the PorcineSNP60 BeadChip assay. Variation was detected in the coding sequence or untranslated regions (UTRs) of 87.8% of the genes in the porcine genome: loss-of-function variants were predicted in 504 genes, 10 202 genes contained nonsynonymous variants, 10 773 had variation in UTRs and 13 010 genes contained synonymous variants. Approximately 139 000 SNPs were classified as loss-of-function, nonsynonymous or regulatory, which suggests that over 99% of the variation detected in our pigs could potentially be ignored, allowing us to focus on a much smaller number of functional SNPs during future analyses. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

The PBII gene of the human salivary proline-rich protein P-B produces another protein, Q504X8, with an opiorphin homolog, QRGPR.

PubMed

Saitoh, Eiichi; Sega, Takuya; Imai, Akane; Isemura, Satoko; Kato, Tetsuo; Ochiai, Akihito; Taniguchi, Masayuki

2018-04-01

The NCBI gene database and human-transcriptome database for alternative splicing were used to determine the expression of mRNAs for P-B (SMR3B) and variant form of P-B. The translational product from the former mRNA was identified as the protein named P-B, whereas that from the latter has not yet been elucidated. In the present study, we investigated the expression of P-B and its variant form at the protein level. To identify the variant protein of P-B, (1) cationic proteins with a higher isoelectric point in human pooled whole saliva were purified by a two dimensional liquid chromatography; (2) the peptide fragments generated from the in-solution of all proteins digested with trypsin separated and analyzed by MALDI-TOF-MS; and (3) the presence or absence of P-B in individual saliva was examined by 15% SDS-PAGE. The peptide sequences (I 37 PPPYSCTPNMNNCSR 52 , C 53 HHHHKRHHYPCNYCFCYPK 72 , R 59 HHYPCNYCFCYPK 72 and H 60 HYPCNYCFCYPK 72 ) present in the variant protein of P-B were identified. The peptide sequence (G 6 PYPPGPLAPPQPFGPGFVPPPPPPPYGPGR 36 ) in P-B (or the variant) and sequence (I 37 PPPPPAPYGPGIFPPPPPQP 57 ) in P-B were identified. The sum of the sequences identified indicated a 91.23% sequence identity for P-B and 79.76% for the variant. There were cases in which P-B existed in individual saliva, but there were cases in which it did not exist in individual saliva. The variant protein is produced by excising a non-canonical intron (CC-AC pair) from the 3'-noncoding sequence of the PBII gene. Both P-B and the variant are subject to proteolysis in the oral cavity. Copyright © 2018 Elsevier Ltd. All rights reserved.

Reducing false positive incidental findings with ensemble genotyping and logistic regression-based variant filtering methods

PubMed Central

Hwang, Kyu-Baek; Lee, In-Hee; Park, Jin-Ho; Hambuch, Tina; Choi, Yongjoon; Kim, MinHyeok; Lee, Kyungjoon; Song, Taemin; Neu, Matthew B.; Gupta, Neha; Kohane, Isaac S.; Green, Robert C.; Kong, Sek Won

2014-01-01

As whole genome sequencing (WGS) uncovers variants associated with rare and common diseases, an immediate challenge is to minimize false positive findings due to sequencing and variant calling errors. False positives can be reduced by combining results from orthogonal sequencing methods, but costly. Here we present variant filtering approaches using logistic regression (LR) and ensemble genotyping to minimize false positives without sacrificing sensitivity. We evaluated the methods using paired WGS datasets of an extended family prepared using two sequencing platforms and a validated set of variants in NA12878. Using LR or ensemble genotyping based filtering, false negative rates were significantly reduced by 1.1- to 17.8-fold at the same levels of false discovery rates (5.4% for heterozygous and 4.5% for homozygous SNVs; 30.0% for heterozygous and 18.7% for homozygous insertions; 25.2% for heterozygous and 16.6% for homozygous deletions) compared to the filtering based on genotype quality scores. Moreover, ensemble genotyping excluded > 98% (105,080 of 107,167) of false positives while retaining > 95% (897 of 937) of true positives in de novo mutation (DNM) discovery, and performed better than a consensus method using two sequencing platforms. Our proposed methods were effective in prioritizing phenotype-associated variants, and ensemble genotyping would be essential to minimize false positive DNM candidates. PMID:24829188

dbWGFP: a database and web server of human whole-genome single nucleotide variants and their functional predictions.

PubMed

Wu, Jiaxin; Wu, Mengmeng; Li, Lianshuo; Liu, Zhuo; Zeng, Wanwen; Jiang, Rui

2016-01-01

The recent advancement of the next generation sequencing technology has enabled the fast and low-cost detection of all genetic variants spreading across the entire human genome, making the application of whole-genome sequencing a tendency in the study of disease-causing genetic variants. Nevertheless, there still lacks a repository that collects predictions of functionally damaging effects of human genetic variants, though it has been well recognized that such predictions play a central role in the analysis of whole-genome sequencing data. To fill this gap, we developed a database named dbWGFP (a database and web server of human whole-genome single nucleotide variants and their functional predictions) that contains functional predictions and annotations of nearly 8.58 billion possible human whole-genome single nucleotide variants. Specifically, this database integrates 48 functional predictions calculated by 17 popular computational methods and 44 valuable annotations obtained from various data sources. Standalone software, user-friendly query services and free downloads of this database are available at http://bioinfo.au.tsinghua.edu.cn/dbwgfp. dbWGFP provides a valuable resource for the analysis of whole-genome sequencing, exome sequencing and SNP array data, thereby complementing existing data sources and computational resources in deciphering genetic bases of human inherited diseases. © The Author(s) 2016. Published by Oxford University Press.

Vascular Ehlers–Danlos Syndrome in siblings with biallelic COL3A1 sequence variants and marked clinical variability in the extended family

PubMed Central

Jørgensen, Agnete; Fagerheim, Toril; Rand-Hendriksen, Svend; Lunde, Per I; Vorren, Torgrim O; Pepin, Melanie G; Leistritz, Dru F; Byers, Peter H

2015-01-01

Vascular Ehlers–Danlos Syndrome (vEDS), also known as EDS type IV, is considered to be an autosomal dominant disorder caused by sequence variants in COL3A1, which encodes the chains of type III procollagen. We identified a family in which there was marked clinical variation with the earliest death due to extensive aortic dissection at age 15 years and other family members in their eighties with no complications. The proband was born with right-sided clubfoot but was otherwise healthy until he died unexpectedly at 15 years. His sister, in addition to signs consistent with vascular EDS, had bilateral frontal and parietal polymicrogyria. The proband and his sister each had two COL3A1 sequence variants, c.1786C>T, p.(Arg596*) in exon 26 and c.3851G>A, p.(Gly1284Glu) in exon 50 on different alleles. Cells from the compound heterozygote produced a reduced amount of type III procollagen, all the chains of which had abnormal electrophoretic mobility. Biallelic sequence variants have a significantly worse outcome than heterozygous variants for either null mutations or missense mutations, and frontoparietal polymicrogyria may be an added phenotype feature. This genetic constellation provides a very rare explanation for marked intrafamilial clinical variation due to sequence variants in COL3A1. PMID:25205403

VIPER: a web application for rapid expert review of variant calls.

PubMed

Wöste, Marius; Dugas, Martin

2018-06-01

With the rapid development in next-generation sequencing, cost and time requirements for genomic sequencing are decreasing, enabling applications in many areas such as cancer research. Many tools have been developed to analyze genomic variation ranging from single nucleotide variants to whole chromosomal aberrations. As sequencing throughput increases, the number of variants called by such tools also grows. Often employed manual inspection of such calls is thus becoming a time-consuming procedure. We developed the Variant InsPector and Expert Rating tool (VIPER) to speed up this process by integrating the Integrative Genomics Viewer into a web application. Analysts can then quickly iterate through variants, apply filters and make decisions based on the generated images and variant metadata. VIPER was successfully employed in analyses with manual inspection of more than 10 000 calls. VIPER is implemented in Java and Javascript and is freely available at https://github.com/MarWoes/viper. marius.woeste@uni-muenster.de. Supplementary data are available at Bioinformatics online.

A Bioinformatics Workflow for Variant Peptide Detection in Shotgun Proteomics*

PubMed Central

Li, Jing; Su, Zengliu; Ma, Ze-Qiang; Slebos, Robbert J. C.; Halvey, Patrick; Tabb, David L.; Liebler, Daniel C.; Pao, William; Zhang, Bing

2011-01-01

Shotgun proteomics data analysis usually relies on database search. However, commonly used protein sequence databases do not contain information on protein variants and thus prevent variant peptides and proteins from been identified. Including known coding variations into protein sequence databases could help alleviate this problem. Based on our recently published human Cancer Proteome Variation Database, we have created a protein sequence database that comprehensively annotates thousands of cancer-related coding variants collected in the Cancer Proteome Variation Database as well as noncancer-specific ones from the Single Nucleotide Polymorphism Database (dbSNP). Using this database, we then developed a data analysis workflow for variant peptide identification in shotgun proteomics. The high risk of false positive variant identifications was addressed by a modified false discovery rate estimation method. Analysis of colorectal cancer cell lines SW480, RKO, and HCT-116 revealed a total of 81 peptides that contain either noncancer-specific or cancer-related variations. Twenty-three out of 26 variants randomly selected from the 81 were confirmed by genomic sequencing. We further applied the workflow on data sets from three individual colorectal tumor specimens. A total of 204 distinct variant peptides were detected, and five carried known cancer-related mutations. Each individual showed a specific pattern of cancer-related mutations, suggesting potential use of this type of information for personalized medicine. Compatibility of the workflow has been tested with four popular database search engines including Sequest, Mascot, X!Tandem, and MyriMatch. In summary, we have developed a workflow that effectively uses existing genomic data to enable variant peptide detection in proteomics. PMID:21389108

Expansion of phenotype and genotypic data in CRB2-related syndrome.

PubMed

Lamont, Ryan E; Tan, Wen-Hann; Innes, A Micheil; Parboosingh, Jillian S; Schneidman-Duhovny, Dina; Rajkovic, Aleksandar; Pappas, John; Altschwager, Pablo; DeWard, Stephanie; Fulton, Anne; Gray, Kathryn J; Krall, Max; Mehta, Lakshmi; Rodan, Lance H; Saller, Devereux N; Steele, Deanna; Stein, Deborah; Yatsenko, Svetlana A; Bernier, François P; Slavotinek, Anne M

2016-10-01

Sequence variants in CRB2 cause a syndrome with greatly elevated maternal serum alpha-fetoprotein and amniotic fluid alpha-fetoprotein levels, cerebral ventriculomegaly and renal findings similar to Finnish congenital nephrosis. All reported patients have been homozygotes or compound heterozygotes for sequence variants in the Crumbs, Drosophila, Homolog of, 2 (CRB2) genes. Variants affecting CRB2 function have also been identified in four families with steroid resistant nephrotic syndrome, but without any other known systemic findings. We ascertained five, previously unreported individuals with biallelic variants in CRB2 that were predicted to affect function. We compiled the clinical features of reported cases and reviewed available literature for cases with features suggestive of CRB2-related syndrome in order to better understand the phenotypic and genotypic manifestations. Phenotypic analyses showed that ventriculomegaly was a common clinical manifestation (9/11 confirmed cases), in contrast to the original reports, in which patients were ascertained due to renal disease. Two children had minor eye findings and one was diagnosed with a B-cell lymphoma. Further genetic analysis identified one family with two affected siblings who were both heterozygous for a variant in NPHS2 predicted to affect function and separate families with sequence variants in NPHS4 and BBS7 in addition to the CRB2 variants. Our report expands the clinical phenotype of CRB2-related syndrome and establishes ventriculomegaly and hydrocephalus as frequent manifestations. We found additional sequence variants in genes involved in kidney development and ciliopathies in patients with CRB2-related syndrome, suggesting that these variants may modify the phenotype.

Comparative oncology DNA sequencing of canine T cell lymphoma via human hotspot panel

PubMed Central

Beheshti, Afshin; Pilichowska, Monika; Burgess, Kristine; Ricks-Santi, Luisel; McNiel, Elizabeth; London, Cheryl B.; Ravi, Dashnamoorthy; Evens, Andrew M.

2018-01-01

T-cell lymphoma (TCL) is an uncommon and aggressive form of human cancer. Lymphoma is the most common hematopoietic tumor in canines (companion animals), with TCL representing approximately 30% of diagnoses. Collectively, the canine is an appealing model for cancer research given the spontaneous occurrence of cancer, intact immune system, and phytogenetic proximity to humans. We sought to establish mutational congruence of the canine with known human TCL mutations in order to identify potential actionable oncogenic pathways. Following pathologic confirmation, DNA was sequenced in 16 canine TCL (cTCL) cases using a custom Human Cancer Hotspot Panel of 68 genes commonly mutated in human TCL. Sequencing identified 4,527,638 total reads with average length of 229 bases containing 346 unique variants and 1,474 total variants; each sample had an average of 92 variants. Among these, there were 258 germline and 32 somatic variants. Among the 32 somatic variants there were 8 missense variants, 1 splice junction variant and the remaining were intron or synonymous variants. A frequency of 4 somatic mutations per sample were noted with >7 mutations detected in MET, KDR, STK11 and BRAF. Expression of these associated proteins were also detected via Western blot analyses. In addition, Sanger sequencing confirmed three variants of high quality (MYC, MET, and TP53 missense mutation). Taken together, the mutational spectrum and protein analyses showed mutations in signaling pathways similar to human TCL and also identified novel mutations that may serve as drug targets as well as potential biomarkers. PMID:29854308

Designing two-in-one antibodies.

PubMed

Valladares, Ignacio Garcia; Espinoza, Luis R

2009-09-01

Evaluation of: Bostrom J, Shang-Fan Y, Kan D et al.: Variants of the antibody Herceptin that interact with HER2 and VEGF at the antigen binding site. Science 323, 1610-1614 (2009). The longstanding held notion that one antibody equals one antigen and, hence, one function has been challenged in recent years. Improved technology in antibody production, especially the accumulation of sequence data of immunoglobulin genes and the advent of PCR have made it possible to clone antibody gene repertoires. The current paper provides further challenge to the notion of one antibody = one antigen by developing 'two-in-one' antibodies with an antigen-binding site that binds two distinct proteins with high affinity. A therapeutic variant antibody of Herceptin (Genentech, CA, USA) was isolated that binds the human EGF receptor (HER)2 and also to VEGF. This development may represent a breakthrough discovery and may have significant implications in the therapy of malignant, infectious, allergic and autoimmune disorders.

Androgen Receptor Gene Polymorphisms and Alterations in Prostate Cancer: Of Humanized Mice and Men

PubMed Central

Robins, Diane M.

2011-01-01

Germline polymorphisms and somatic mutations of the androgen receptor (AR) have been intensely investigated in prostate cancer but even with genomic approaches their impact remains controversial. To assess the functional significance of AR genetic variation, we converted the mouse gene to the human sequence by germline recombination and engineered alleles to query the role of a polymorphic glutamine (Q) tract implicated in cancer risk. In a prostate cancer model, AR Q tract length influences progression and castration response. Mutation profiling in mice provides direct evidence that somatic AR variants are selected by therapy, a finding validated in human metastases from distinct treatment groups. Mutant ARs exploit multiple mechanisms to resist hormone ablation, including alterations in ligand specificity, target gene selectivity, chaperone interaction and nuclear localization. Regardless of their frequency, these variants permute normal function to reveal novel means to target wild type AR and its key interacting partners. PMID:21689727

Personalized Approaches to Clopidogrel Therapy: Are We There Yet?

PubMed Central

Anderson, Christopher D.; Biffi, Alessandro; Greenberg, Steven M.; Rosand, Jonathan

2010-01-01

Clopidogrel is one of the most commonly prescribed medications world-wide. Recent advisories from the US Food and Drug Administration (FDA) have drawn attention to the possibility of personalized decision-making for individuals who are candidates for clopidogrel. As is the case with antihypertensives, statins and warfarin, common genetic sequence variants can influence clopidogrel metabolism and its effect on platelet activity. These genetic variants have, in multiple studies, been associated with adverse clinical outcomes. Concurrent medication use also influences the body's handling of clopidogrel. Proton pump inhibitors, widely prescribed in conjunction with clopidogrel, may blunt its effectiveness. We address implications for bedside decision-making in light of accumulated data and current FDA advisories, and conclude that genetic testing for CYP2C19 genotype and limitation of PPI interactions do not yet appear to offer an opportunity to optimize treatment given the current state of knowledge. PMID:21030701

Filovirus RefSeq Entries: Evaluation and Selection of Filovirus Type Variants, Type Sequences, and Names

PubMed Central

Kuhn, Jens H.; Andersen, Kristian G.; Bào, Yīmíng; Bavari, Sina; Becker, Stephan; Bennett, Richard S.; Bergman, Nicholas H.; Blinkova, Olga; Bradfute, Steven; Brister, J. Rodney; Bukreyev, Alexander; Chandran, Kartik; Chepurnov, Alexander A.; Davey, Robert A.; Dietzgen, Ralf G.; Doggett, Norman A.; Dolnik, Olga; Dye, John M.; Enterlein, Sven; Fenimore, Paul W.; Formenty, Pierre; Freiberg, Alexander N.; Garry, Robert F.; Garza, Nicole L.; Gire, Stephen K.; Gonzalez, Jean-Paul; Griffiths, Anthony; Happi, Christian T.; Hensley, Lisa E.; Herbert, Andrew S.; Hevey, Michael C.; Hoenen, Thomas; Honko, Anna N.; Ignatyev, Georgy M.; Jahrling, Peter B.; Johnson, Joshua C.; Johnson, Karl M.; Kindrachuk, Jason; Klenk, Hans-Dieter; Kobinger, Gary; Kochel, Tadeusz J.; Lackemeyer, Matthew G.; Lackner, Daniel F.; Leroy, Eric M.; Lever, Mark S.; Mühlberger, Elke; Netesov, Sergey V.; Olinger, Gene G.; Omilabu, Sunday A.; Palacios, Gustavo; Panchal, Rekha G.; Park, Daniel J.; Patterson, Jean L.; Paweska, Janusz T.; Peters, Clarence J.; Pettitt, James; Pitt, Louise; Radoshitzky, Sheli R.; Ryabchikova, Elena I.; Saphire, Erica Ollmann; Sabeti, Pardis C.; Sealfon, Rachel; Shestopalov, Aleksandr M.; Smither, Sophie J.; Sullivan, Nancy J.; Swanepoel, Robert; Takada, Ayato; Towner, Jonathan S.; van der Groen, Guido; Volchkov, Viktor E.; Volchkova, Valentina A.; Wahl-Jensen, Victoria; Warren, Travis K.; Warfield, Kelly L.; Weidmann, Manfred; Nichol, Stuart T.

2014-01-01

Sequence determination of complete or coding-complete genomes of viruses is becoming common practice for supporting the work of epidemiologists, ecologists, virologists, and taxonomists. Sequencing duration and costs are rapidly decreasing, sequencing hardware is under modification for use by non-experts, and software is constantly being improved to simplify sequence data management and analysis. Thus, analysis of virus disease outbreaks on the molecular level is now feasible, including characterization of the evolution of individual virus populations in single patients over time. The increasing accumulation of sequencing data creates a management problem for the curators of commonly used sequence databases and an entry retrieval problem for end users. Therefore, utilizing the data to their fullest potential will require setting nomenclature and annotation standards for virus isolates and associated genomic sequences. The National Center for Biotechnology Information’s (NCBI’s) RefSeq is a non-redundant, curated database for reference (or type) nucleotide sequence records that supplies source data to numerous other databases. Building on recently proposed templates for filovirus variant naming [ ()////-], we report consensus decisions from a majority of past and currently active filovirus experts on the eight filovirus type variants and isolates to be represented in RefSeq, their final designations, and their associated sequences. PMID:25256396

Machine-learned analysis of the association of next-generation sequencing-based human TRPV1 and TRPA1 genotypes with the sensitivity to heat stimuli and topically applied capsaicin.

PubMed

Kringel, Dario; Geisslinger, Gerd; Resch, Eduard; Oertel, Bruno G; Thrun, Michael C; Heinemann, Sarah; Lötsch, Jörn

2018-03-27

Heat pain and its modulation by capsaicin varies among subjects in experimental and clinical settings. A plausible cause is a genetic component, of which TRPV1 ion channels, by their response to both heat and capsaicin, are primary candidates. However, TRPA1 channels can heterodimerize with TRPV1 channels and carry genetic variants reported to modulate heat pain sensitivity. To address the role of these candidate genes in capsaicin-induced hypersensitization to heat, pain thresholds acquired before and after topical application of capsaicin and TRPA1/TRPV1 exomic sequences derived by next-generation sequencing were assessed in n = 75 healthy volunteers and the genetic information comprised 278 loci. Gaussian mixture modeling indicated 2 phenotype groups with high or low capsaicin-induced hypersensitization to heat. Unsupervised machine learning implemented as swarm-based clustering hinted at differences in the genetic pattern between these phenotype groups. Several methods of supervised machine learning implemented as random forests, adaptive boosting, k-nearest neighbors, naive Bayes, support vector machines, and for comparison, binary logistic regression predicted the phenotype group association consistently better when based on the observed genotypes than when using a random permutation of the exomic sequences. Of note, TRPA1 variants were more important for correct phenotype group association than TRPV1 variants. This indicates a role of the TRPA1 and TRPV1 next-generation sequencing-based genetic pattern in the modulation of the individual response to heat-related pain phenotypes. When considering earlier evidence that topical capsaicin can induce neuropathy-like quantitative sensory testing patterns in healthy subjects, implications for future analgesic treatments with transient receptor potential inhibitors arise.This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal.

Annotation of Sequence Variants in Cancer Samples: Processes and Pitfalls for Routine Assays in the Clinical Laboratory.

PubMed

Lee, Lobin A; Arvai, Kevin J; Jones, Dan

2015-07-01

As DNA sequencing of multigene panels becomes routine for cancer samples in the clinical laboratory, an efficient process for classifying variants has become more critical. Determining which germline variants are significant for cancer disposition and which somatic mutations are integral to cancer development or therapy response remains difficult, even for well-studied genes such as BRCA1 and TP53. We compare and contrast the general principles and lines of evidence commonly used to distinguish the significance of cancer-associated germline and somatic genetic variants. The factors important in each step of the analysis pipeline are reviewed, as are some of the publicly available annotation tools. Given the range of indications and uses of cancer sequencing assays, including diagnosis, staging, prognostication, theranostics, and residual disease detection, the need for flexible methods for scoring of variants is discussed. The usefulness of protein prediction tools and multimodal risk-based or Bayesian approaches are highlighted. Using TET2 variants encountered in hematologic neoplasms, several examples of this multifactorial approach to classifying sequence variants of unknown significance are presented. Although there are still significant gaps in the publicly available data for many cancer genes that limit the broad application of explicit algorithms for variant scoring, the elements of a more rigorous model are outlined. Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Whole-Exome Sequencing of 10 Scientists: Evaluation of the Process and Outcomes.

PubMed

Lindor, Noralane M; Schahl, Kimberly A; Johnson, Kiley J; Hunt, Katherine S; Mensink, Kara A; Wieben, Eric D; Klee, Eric; Black, John L; Highsmith, W Edward; Thibodeau, Stephen N; Ferber, Matthew J; Aypar, Umut; Ji, Yuan; Graham, Rondell P; Fiksdal, Alexander S; Sarangi, Vivek; Ormond, Kelly E; Riegert-Johnson, Douglas L; McAllister, Tammy M; Farrugia, Gianrico; McCormick, Jennifer B

2015-10-01

To understand motivations, educational needs, and concerns of individuals contemplating whole-exome sequencing (WES) and determine what amount of genetic information might be obtained by sequencing a generally healthy cohort so as to more effectively counsel future patients. From 2012 to 2014, 40 medically educated, generally healthy scientists at Mayo Clinic were invited to have WES conducted on a research basis; 26 agreed to be in a drawing from which 10 participants were selected. The study involved pre- and posttest genetic counseling and completion of 4 surveys related to the experience and outcomes. Whole-exome sequencing was conducted on DNA from blood from each person. Most variants (76,305 per person; range, 74,505-77,387) were known benign allelic variants, variants in genes of unknown function, or variants of uncertain significance in genes of known function. The results of suspected pathogenic/pathogenic variants in Mendelian disorders and pharmacogenomic variants were disclosed. The mean number of suspected pathogenic/pathogenic variants was 2.2 per person (range, 1-4). Four pharmacogenomic genes were included for reporting; variants were found in 9 of 10 participants. This study provides data that may be useful in establishing reality-based patient expectations, outlines specific points to cover during counseling, and increases confidence in the feasibility of providing adequate preparation and counseling for WES in generally healthy individuals. Copyright © 2015 Mayo Foundation for Medical Education and Research. Published by Elsevier Inc. All rights reserved.

Macular xanthophylls, lipoprotein-related genes, and age-related macular degeneration1234

PubMed Central

Koo, Euna; Neuringer, Martha; SanGiovanni, John Paul

2014-01-01

Plant-based macular xanthophylls (MXs; lutein and zeaxanthin) and the lutein metabolite meso-zeaxanthin are the major constituents of macular pigment, a compound concentrated in retinal areas that are responsible for fine-feature visual sensation. There is an unmet need to examine the genetics of factors influencing regulatory mechanisms and metabolic fates of these 3 MXs because they are linked to processes implicated in the pathogenesis of age-related macular degeneration (AMD). In this work we provide an overview of evidence supporting a molecular basis for AMD-MX associations as they may relate to DNA sequence variation in AMD- and lipoprotein-related genes. We recognize a number of emerging research opportunities, barriers, knowledge gaps, and tools offering promise for meaningful investigation and inference in the field. Overviews on AMD- and high-density lipoprotein (HDL)–related genes encoding receptors, transporters, and enzymes affecting or affected by MXs are followed with information on localization of products from these genes to retinal cell types manifesting AMD-related pathophysiology. Evidence on the relation of each gene or gene product with retinal MX response to nutrient intake is discussed. This information is followed by a review of results from mechanistic studies testing gene-disease relations. We then present findings on relations of AMD with DNA sequence variants in MX-associated genes. Our conclusion is that AMD-associated DNA variants that influence the actions and metabolic fates of HDL system constituents should be examined further for concomitant influence on MX absorption, retinal tissue responses to MX intake, and the capacity to modify MX-associated factors and processes implicated in AMD pathogenesis. PMID:24829491

Association of levels of fasting glucose and insulin with rare variants at the chromosome 11p11.2-MADD locus: Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) Consortium Targeted Sequencing Study.

PubMed

Cornes, Belinda K; Brody, Jennifer A; Nikpoor, Naghmeh; Morrison, Alanna C; Chu, Huan; Ahn, Byung Soo; Wang, Shuai; Dauriz, Marco; Barzilay, Joshua I; Dupuis, Josée; Florez, Jose C; Coresh, Josef; Gibbs, Richard A; Kao, W H Linda; Liu, Ching-Ti; McKnight, Barbara; Muzny, Donna; Pankow, James S; Reid, Jeffrey G; White, Charles C; Johnson, Andrew D; Wong, Tien Y; Psaty, Bruce M; Boerwinkle, Eric; Rotter, Jerome I; Siscovick, David S; Sladek, Robert; Meigs, James B

2014-06-01

Common variation at the 11p11.2 locus, encompassing MADD, ACP2, NR1H3, MYBPC3, and SPI1, has been associated in genome-wide association studies with fasting glucose and insulin (FI). In the Cohorts for Heart and Aging Research in Genomic Epidemiology Targeted Sequencing Study, we sequenced 5 gene regions at 11p11.2 to identify rare, potentially functional variants influencing fasting glucose or FI levels. Sequencing (mean depth, 38×) across 16.1 kb in 3566 individuals without diabetes mellitus identified 653 variants, 79.9% of which were rare (minor allele frequency <1%) and novel. We analyzed rare variants in 5 gene regions with FI or fasting glucose using the sequence kernel association test. At NR1H3, 53 rare variants were jointly associated with FI (P=2.73×10(-3)); of these, 7 were predicted to have regulatory function and showed association with FI (P=1.28×10(-3)). Conditioning on 2 previously associated variants at MADD (rs7944584, rs10838687) did not attenuate this association, suggesting that there are >2 independent signals at 11p11.2. One predicted regulatory variant, chr11:47227430 (hg18; minor allele frequency=0.00068), contributed 20.6% to the overall sequence kernel association test score at NR1H3, lies in intron 2 of NR1H3, and is a predicted binding site for forkhead box A1 (FOXA1), a transcription factor associated with insulin regulation. In human HepG2 hepatoma cells, the rare chr11:47227430 A allele disrupted FOXA1 binding and reduced FOXA1-dependent transcriptional activity. Sequencing at 11p11.2-NR1H3 identified rare variation associated with FI. One variant, chr11:47227430, seems to be functional, with the rare A allele reducing transcription factor FOXA1 binding and FOXA1-dependent transcriptional activity. © 2014 American Heart Association, Inc.

Large-scale whole-genome sequencing of the Icelandic population.

PubMed

Gudbjartsson, Daniel F; Helgason, Hannes; Gudjonsson, Sigurjon A; Zink, Florian; Oddson, Asmundur; Gylfason, Arnaldur; Besenbacher, Soren; Magnusson, Gisli; Halldorsson, Bjarni V; Hjartarson, Eirikur; Sigurdsson, Gunnar Th; Stacey, Simon N; Frigge, Michael L; Holm, Hilma; Saemundsdottir, Jona; Helgadottir, Hafdis Th; Johannsdottir, Hrefna; Sigfusson, Gunnlaugur; Thorgeirsson, Gudmundur; Sverrisson, Jon Th; Gretarsdottir, Solveig; Walters, G Bragi; Rafnar, Thorunn; Thjodleifsson, Bjarni; Bjornsson, Einar S; Olafsson, Sigurdur; Thorarinsdottir, Hildur; Steingrimsdottir, Thora; Gudmundsdottir, Thora S; Theodors, Asgeir; Jonasson, Jon G; Sigurdsson, Asgeir; Bjornsdottir, Gyda; Jonsson, Jon J; Thorarensen, Olafur; Ludvigsson, Petur; Gudbjartsson, Hakon; Eyjolfsson, Gudmundur I; Sigurdardottir, Olof; Olafsson, Isleifur; Arnar, David O; Magnusson, Olafur Th; Kong, Augustine; Masson, Gisli; Thorsteinsdottir, Unnur; Helgason, Agnar; Sulem, Patrick; Stefansson, Kari

2015-05-01

Here we describe the insights gained from sequencing the whole genomes of 2,636 Icelanders to a median depth of 20×. We found 20 million SNPs and 1.5 million insertions-deletions (indels). We describe the density and frequency spectra of sequence variants in relation to their functional annotation, gene position, pathway and conservation score. We demonstrate an excess of homozygosity and rare protein-coding variants in Iceland. We imputed these variants into 104,220 individuals down to a minor allele frequency of 0.1% and found a recessive frameshift mutation in MYL4 that causes early-onset atrial fibrillation, several mutations in ABCB4 that increase risk of liver diseases and an intronic variant in GNAS associating with increased thyroid-stimulating hormone levels when maternally inherited. These data provide a study design that can be used to determine how variation in the sequence of the human genome gives rise to human diversity.

REVEL: An Ensemble Method for Predicting the Pathogenicity of Rare Missense Variants.

PubMed

Ioannidis, Nilah M; Rothstein, Joseph H; Pejaver, Vikas; Middha, Sumit; McDonnell, Shannon K; Baheti, Saurabh; Musolf, Anthony; Li, Qing; Holzinger, Emily; Karyadi, Danielle; Cannon-Albright, Lisa A; Teerlink, Craig C; Stanford, Janet L; Isaacs, William B; Xu, Jianfeng; Cooney, Kathleen A; Lange, Ethan M; Schleutker, Johanna; Carpten, John D; Powell, Isaac J; Cussenot, Olivier; Cancel-Tassin, Geraldine; Giles, Graham G; MacInnis, Robert J; Maier, Christiane; Hsieh, Chih-Lin; Wiklund, Fredrik; Catalona, William J; Foulkes, William D; Mandal, Diptasri; Eeles, Rosalind A; Kote-Jarai, Zsofia; Bustamante, Carlos D; Schaid, Daniel J; Hastie, Trevor; Ostrander, Elaine A; Bailey-Wilson, Joan E; Radivojac, Predrag; Thibodeau, Stephen N; Whittemore, Alice S; Sieh, Weiva

2016-10-06

The vast majority of coding variants are rare, and assessment of the contribution of rare variants to complex traits is hampered by low statistical power and limited functional data. Improved methods for predicting the pathogenicity of rare coding variants are needed to facilitate the discovery of disease variants from exome sequencing studies. We developed REVEL (rare exome variant ensemble learner), an ensemble method for predicting the pathogenicity of missense variants on the basis of individual tools: MutPred, FATHMM, VEST, PolyPhen, SIFT, PROVEAN, MutationAssessor, MutationTaster, LRT, GERP, SiPhy, phyloP, and phastCons. REVEL was trained with recently discovered pathogenic and rare neutral missense variants, excluding those previously used to train its constituent tools. When applied to two independent test sets, REVEL had the best overall performance (p < 10 -12 ) as compared to any individual tool and seven ensemble methods: MetaSVM, MetaLR, KGGSeq, Condel, CADD, DANN, and Eigen. Importantly, REVEL also had the best performance for distinguishing pathogenic from rare neutral variants with allele frequencies <0.5%. The area under the receiver operating characteristic curve (AUC) for REVEL was 0.046-0.182 higher in an independent test set of 935 recent SwissVar disease variants and 123,935 putatively neutral exome sequencing variants and 0.027-0.143 higher in an independent test set of 1,953 pathogenic and 2,406 benign variants recently reported in ClinVar than the AUCs for other ensemble methods. We provide pre-computed REVEL scores for all possible human missense variants to facilitate the identification of pathogenic variants in the sea of rare variants discovered as sequencing studies expand in scale. Copyright © 2016 American Society of Human Genetics. All rights reserved.

Protein-altering variants associated with body mass index implicate pathways that control energy intake and expenditure underpinning obesity

PubMed Central

Turcot, Valérie; Lu, Yingchang; Highland, Heather M; Schurmann, Claudia; Justice, Anne E; Fine, Rebecca S; Bradfield, Jonathan P; Esko, Tõnu; Giri, Ayush; Graff, Mariaelisa; Guo, Xiuqing; Hendricks, Audrey E; Karaderi, Tugce; Lempradl, Adelheid; Locke, Adam E; Mahajan, Anubha; Marouli, Eirini; Sivapalaratnam, Suthesh; Young, Kristin L; Alfred, Tamuno; Feitosa, Mary F; Masca, Nicholas GD; Manning, Alisa K; Medina-Gomez, Carolina; Mudgal, Poorva; Ng, Maggie CY; Reiner, Alex P; Vedantam, Sailaja; Willems, Sara M; Winkler, Thomas W; Abecasis, Goncalo; Aben, Katja K; Alam, Dewan S; Alharthi, Sameer E; Allison, Matthew; Amouyel, Philippe; Asselbergs, Folkert W; Auer, Paul L; Balkau, Beverley; Bang, Lia E; Barroso, Inês; Bastarache, Lisa; Benn, Marianne; Bergmann, Sven; Bielak, Lawrence F; Blüher, Matthias; Boehnke, Michael; Boeing, Heiner; Boerwinkle, Eric; Böger, Carsten A; Bork-Jensen, Jette; Bots, Michiel L; Bottinger, Erwin P; Bowden, Donald W; Brandslund, Ivan; Breen, Gerome; Brilliant, Murray H; Broer, Linda; Brumat, Marco; Burt, Amber A; Butterworth, Adam S; Campbell, Peter T; Cappellani, Stefania; Carey, David J; Catamo, Eulalia; Caulfield, Mark J; Chambers, John C; Chasman, Daniel I; Chen, Yii-Der Ida; Chowdhury, Rajiv; Christensen, Cramer; Chu, Audrey Y; Cocca, Massimiliano; Collins, Francis S; Cook, James P; Corley, Janie; Galbany, Jordi Corominas; Cox, Amanda J; Crosslin, David S; Cuellar-Partida, Gabriel; D'Eustacchio, Angela; Danesh, John; Davies, Gail; de Bakker, Paul IW; de Groot, Mark CH; de Mutsert, Renée; Deary, Ian J; Dedoussis, George; Demerath, Ellen W; den Heijer, Martin; den Hollander, Anneke I; den Ruijter, Hester M; Dennis, Joe G; Denny, Josh C; Di Angelantonio, Emanuele; Drenos, Fotios; Du, Mengmeng; Dubé, Marie-Pierre; Dunning, Alison M; Easton, Douglas F; Edwards, Todd L; Ellinghaus, David; Ellinor, Patrick T; Elliott, Paul; Evangelou, Evangelos; Farmaki, Aliki-Eleni; Farooqi, I. Sadaf; Faul, Jessica D; Fauser, Sascha; Feng, Shuang; Ferrannini, Ele; Ferrieres, Jean; Florez, Jose C; Ford, Ian; Fornage, Myriam; Franco, Oscar H; Franke, Andre; Franks, Paul W; Friedrich, Nele; Frikke-Schmidt, Ruth; Galesloot, Tessel E.; Gan, Wei; Gandin, Ilaria; Gasparini, Paolo; Gibson, Jane; Giedraitis, Vilmantas; Gjesing, Anette P; Gordon-Larsen, Penny; Gorski, Mathias; Grabe, Hans-Jörgen; Grant, Struan FA; Grarup, Niels; Griffiths, Helen L; Grove, Megan L; Gudnason, Vilmundur; Gustafsson, Stefan; Haessler, Jeff; Hakonarson, Hakon; Hammerschlag, Anke R; Hansen, Torben; Harris, Kathleen Mullan; Harris, Tamara B; Hattersley, Andrew T; Have, Christian T; Hayward, Caroline; He, Liang; Heard-Costa, Nancy L; Heath, Andrew C; Heid, Iris M; Helgeland, Øyvind; Hernesniemi, Jussi; Hewitt, Alex W; Holmen, Oddgeir L; Hovingh, G Kees; Howson, Joanna MM; Hu, Yao; Huang, Paul L; Huffman, Jennifer E; Ikram, M Arfan; Ingelsson, Erik; Jackson, Anne U; Jansson, Jan-Håkan; Jarvik, Gail P; Jensen, Gorm B; Jia, Yucheng; Johansson, Stefan; Jørgensen, Marit E; Jørgensen, Torben; Jukema, J Wouter; Kahali, Bratati; Kahn, René S; Kähönen, Mika; Kamstrup, Pia R; Kanoni, Stavroula; Kaprio, Jaakko; Karaleftheri, Maria; Kardia, Sharon LR; Karpe, Fredrik; Kathiresan, Sekar; Kee, Frank; Kiemeney, Lambertus A; Kim, Eric; Kitajima, Hidetoshi; Komulainen, Pirjo; Kooner, Jaspal S; Kooperberg, Charles; Korhonen, Tellervo; Kovacs, Peter; Kuivaniemi, Helena; Kutalik, Zoltán; Kuulasmaa, Kari; Kuusisto, Johanna; Laakso, Markku; Lakka, Timo A; Lamparter, David; Lange, Ethan M; Lange, Leslie A; Langenberg, Claudia; Larson, Eric B; Lee, Nanette R; Lehtimäki, Terho; Lewis, Cora E; Li, Huaixing; Li, Jin; Li-Gao, Ruifang; Lin, Honghuang; Lin, Keng-Hung; Lin, Li-An; Lin, Xu; Lind, Lars; Lindström, Jaana; Linneberg, Allan; Liu, Ching-Ti; Liu, Dajiang J; Liu, Yongmei; Lo, Ken Sin; Lophatananon, Artitaya; Lotery, Andrew J; Loukola, Anu; Luan, Jian'an; Lubitz, Steven A; Lyytikäinen, Leo-Pekka; Männistö, Satu; Marenne, Gaëlle; Mazul, Angela L; McCarthy, Mark I; McKean-Cowdin, Roberta; Medland, Sarah E; Meidtner, Karina; Milani, Lili; Mistry, Vanisha; Mitchell, Paul; Mohlke, Karen L; Moilanen, Leena; Moitry, Marie; Montgomery, Grant W; Mook-Kanamori, Dennis O; Moore, Carmel; Mori, Trevor A; Morris, Andrew D; Morris, Andrew P; Müller-Nurasyid, Martina; Munroe, Patricia B; Nalls, Mike A; Narisu, Narisu; Nelson, Christopher P; Neville, Matt; Nielsen, Sune F; Nikus, Kjell; Njølstad, Pål R; Nordestgaard, Børge G; Nyholt, Dale R; O'Connel, Jeffrey R; O’Donoghue, Michelle L.; Olde Loohuis, Loes M; Ophoff, Roel A; Owen, Katharine R; Packard, Chris J; Padmanabhan, Sandosh; Palmer, Colin NA; Palmer, Nicholette D; Pasterkamp, Gerard; Patel, Aniruddh P; Pattie, Alison; Pedersen, Oluf; Peissig, Peggy L; Peloso, Gina M; Pennell, Craig E; Perola, Markus; Perry, James A; Perry, John RB; Pers, Tune H; Person, Thomas N; Peters, Annette; Petersen, Eva RB; Peyser, Patricia A; Pirie, Ailith; Polasek, Ozren; Polderman, Tinca J; Puolijoki, Hannu; Raitakari, Olli T; Rasheed, Asif; Rauramaa, Rainer; Reilly, Dermot F; Renström, Frida; Rheinberger, Myriam; Ridker, Paul M; Rioux, John D; Rivas, Manuel A; Roberts, David J; Robertson, Neil R; Robino, Antonietta; Rolandsson, Olov; Rudan, Igor; Ruth, Katherine S; Saleheen, Danish; Salomaa, Veikko; Samani, Nilesh J; Sapkota, Yadav; Sattar, Naveed; Schoen, Robert E; Schreiner, Pamela J; Schulze, Matthias B; Scott, Robert A; Segura-Lepe, Marcelo P; Shah, Svati H; Sheu, Wayne H-H; Sim, Xueling; Slater, Andrew J; Small, Kerrin S; Smith, Albert Vernon; Southam, Lorraine; Spector, Timothy D; Speliotes, Elizabeth K; Starr, John M; Stefansson, Kari; Steinthorsdottir, Valgerdur; Stirrups, Kathleen E; Strauch, Konstantin; Stringham, Heather M; Stumvoll, Michael; Sun, Liang; Surendran, Praveen; Swift, Amy J; Tada, Hayato; Tansey, Katherine E; Tardif, Jean-Claude; Taylor, Kent D; Teumer, Alexander; Thompson, Deborah J; Thorleifsson, Gudmar; Thorsteinsdottir, Unnur; Thuesen, Betina H; Tönjes, Anke; Tromp, Gerard; Trompet, Stella; Tsafantakis, Emmanouil; Tuomilehto, Jaakko; Tybjaerg-Hansen, Anne; Tyrer, Jonathan P; Uher, Rudolf; Uitterlinden, André G; Uusitupa, Matti; van der Laan, Sander W; van Duijn, Cornelia M; van Leeuwen, Nienke; van Setten, Jessica; Vanhala, Mauno; Varbo, Anette; Varga, Tibor V; Varma, Rohit; Velez Edwards, Digna R; Vermeulen, Sita H; Veronesi, Giovanni; Vestergaard, Henrik; Vitart, Veronique; Vogt, Thomas F; Völker, Uwe; Vuckovic, Dragana; Wagenknecht, Lynne E; Walker, Mark; Wallentin, Lars; Wang, Feijie; Wang, Carol A; Wang, Shuai; Wang, Yiqin; Ware, Erin B; Wareham, Nicholas J; Warren, Helen R; Waterworth, Dawn M; Wessel, Jennifer; White, Harvey D; Willer, Cristen J; Wilson, James G; Witte, Daniel R; Wood, Andrew R; Wu, Ying; Yaghootkar, Hanieh; Yao, Jie; Yao, Pang; Yerges-Armstrong, Laura M; Young, Robin; Zeggini, Eleftheria; Zhan, Xiaowei; Zhang, Weihua; Zhao, Jing Hua; Zhao, Wei; Zhao, Wei; Zhou, Wei; Zondervan, Krina T; Rotter, Jerome I; Pospisilik, John A; Rivadeneira, Fernando; Borecki, Ingrid B; Deloukas, Panos; Frayling, Timothy M; Lettre, Guillaume; North, Kari E; Lindgren, Cecilia M; Hirschhorn, Joel N; Loos, Ruth JF

2018-01-01

Genome-wide association studies (GWAS) have identified >250 loci for body mass index (BMI), implicating pathways related to neuronal biology. Most GWAS loci represent clusters of common, non-coding variants from which pinpointing causal genes remains challenging. Here, we combined data from 718,734 individuals to discover rare and low-frequency (MAF<5%) coding variants associated with BMI. We identified 14 coding variants in 13 genes, of which eight in genes (ZBTB7B, ACHE, RAPGEF3, RAB21, ZFHX3, ENTPD6, ZFR2, ZNF169) newly implicated in human obesity, two (MC4R, KSR2) previously observed in extreme obesity, and two variants in GIPR. Effect sizes of rare variants are ~10 times larger than of common variants, with the largest effect observed in carriers of an MC4R stop-codon (p.Tyr35Ter, MAF=0.01%), weighing ~7kg more than non-carriers. Pathway analyses confirmed enrichment of neuronal genes and provide new evidence for adipocyte and energy expenditure biology, widening the potential of genetically-supported therapeutic targets to treat obesity. PMID:29273807

Investigating DNA-, RNA-, and protein-based features as a means to discriminate pathogenic synonymous variants.

PubMed

Livingstone, Mark; Folkman, Lukas; Yang, Yuedong; Zhang, Ping; Mort, Matthew; Cooper, David N; Liu, Yunlong; Stantic, Bela; Zhou, Yaoqi

2017-10-01

Synonymous single-nucleotide variants (SNVs), although they do not alter the encoded protein sequences, have been implicated in many genetic diseases. Experimental studies indicate that synonymous SNVs can lead to changes in the secondary and tertiary structures of DNA and RNA, thereby affecting translational efficiency, cotranslational protein folding as well as the binding of DNA-/RNA-binding proteins. However, the importance of these various features in disease phenotypes is not clearly understood. Here, we have built a support vector machine (SVM) model (termed DDIG-SN) as a means to discriminate disease-causing synonymous variants. The model was trained and evaluated on nearly 900 disease-causing variants. The method achieves robust performance with the area under the receiver operating characteristic curve of 0.84 and 0.85 for protein-stratified 10-fold cross-validation and independent testing, respectively. We were able to show that the disease-causing effects in the immediate proximity to exon-intron junctions (1-3 bp) are driven by the loss of splicing motif strength, whereas the gain of splicing motif strength is the primary cause in regions further away from the splice site (4-69 bp). The method is available as a part of the DDIG server at http://sparks-lab.org/ddig. © 2017 Wiley Periodicals, Inc.

Consequences of splitting whole-genome sequencing effort over multiple breeds on imputation accuracy.

PubMed

Bouwman, Aniek C; Veerkamp, Roel F

2014-10-03

The aim of this study was to determine the consequences of splitting sequencing effort over multiple breeds for imputation accuracy from a high-density SNP chip towards whole-genome sequence. Such information would assist for instance numerical smaller cattle breeds, but also pig and chicken breeders, who have to choose wisely how to spend their sequencing efforts over all the breeds or lines they evaluate. Sequence data from cattle breeds was used, because there are currently relatively many individuals from several breeds sequenced within the 1,000 Bull Genomes project. The advantage of whole-genome sequence data is that it carries the causal mutations, but the question is whether it is possible to impute the causal variants accurately. This study therefore focussed on imputation accuracy of variants with low minor allele frequency and breed specific variants. Imputation accuracy was assessed for chromosome 1 and 29 as the correlation between observed and imputed genotypes. For chromosome 1, the average imputation accuracy was 0.70 with a reference population of 20 Holstein, and increased to 0.83 when the reference population was increased by including 3 other dairy breeds with 20 animals each. When the same amount of animals from the Holstein breed were added the accuracy improved to 0.88, while adding the 3 other breeds to the reference population of 80 Holstein improved the average imputation accuracy marginally to 0.89. For chromosome 29, the average imputation accuracy was lower. Some variants benefitted from the inclusion of other breeds in the reference population, initially determined by the MAF of the variant in each breed, but even Holstein specific variants did gain imputation accuracy from the multi-breed reference population. This study shows that splitting sequencing effort over multiple breeds and combining the reference populations is a good strategy for imputation from high-density SNP panels towards whole-genome sequence when reference populations are small and sequencing effort is limiting. When sequencing effort is limiting and interest lays in multiple breeds or lines this provides imputation of each breed.

Validation of a next-generation sequencing assay for clinical molecular oncology.

PubMed

Cottrell, Catherine E; Al-Kateb, Hussam; Bredemeyer, Andrew J; Duncavage, Eric J; Spencer, David H; Abel, Haley J; Lockwood, Christina M; Hagemann, Ian S; O'Guin, Stephanie M; Burcea, Lauren C; Sawyer, Christopher S; Oschwald, Dayna M; Stratman, Jennifer L; Sher, Dorie A; Johnson, Mark R; Brown, Justin T; Cliften, Paul F; George, Bijoy; McIntosh, Leslie D; Shrivastava, Savita; Nguyen, Tudung T; Payton, Jacqueline E; Watson, Mark A; Crosby, Seth D; Head, Richard D; Mitra, Robi D; Nagarajan, Rakesh; Kulkarni, Shashikant; Seibert, Karen; Virgin, Herbert W; Milbrandt, Jeffrey; Pfeifer, John D

2014-01-01

Currently, oncology testing includes molecular studies and cytogenetic analysis to detect genetic aberrations of clinical significance. Next-generation sequencing (NGS) allows rapid analysis of multiple genes for clinically actionable somatic variants. The WUCaMP assay uses targeted capture for NGS analysis of 25 cancer-associated genes to detect mutations at actionable loci. We present clinical validation of the assay and a detailed framework for design and validation of similar clinical assays. Deep sequencing of 78 tumor specimens (≥ 1000× average unique coverage across the capture region) achieved high sensitivity for detecting somatic variants at low allele fraction (AF). Validation revealed sensitivities and specificities of 100% for detection of single-nucleotide variants (SNVs) within coding regions, compared with SNP array sequence data (95% CI = 83.4-100.0 for sensitivity and 94.2-100.0 for specificity) or whole-genome sequencing (95% CI = 89.1-100.0 for sensitivity and 99.9-100.0 for specificity) of HapMap samples. Sensitivity for detecting variants at an observed 10% AF was 100% (95% CI = 93.2-100.0) in HapMap mixes. Analysis of 15 masked specimens harboring clinically reported variants yielded concordant calls for 13/13 variants at AF of ≥ 15%. The WUCaMP assay is a robust and sensitive method to detect somatic variants of clinical significance in molecular oncology laboratories, with reduced time and cost of genetic analysis allowing for strategic patient management. Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Sequencing Structural Variants in Cancer for Precision Therapeutics.

PubMed

Macintyre, Geoff; Ylstra, Bauke; Brenton, James D

2016-09-01

The identification of mutations that guide therapy selection for patients with cancer is now routine in many clinical centres. The majority of assays used for solid tumour profiling use DNA sequencing to interrogate somatic point mutations because they are relatively easy to identify and interpret. Many cancers, however, including high-grade serous ovarian, oesophageal, and small-cell lung cancer, are driven by somatic structural variants that are not measured by these assays. Therefore, there is currently an unmet need for clinical assays that can cheaply and rapidly profile structural variants in solid tumours. In this review we survey the landscape of 'actionable' structural variants in cancer and identify promising detection strategies based on massively-parallel sequencing. Copyright © 2016 Elsevier Ltd. All rights reserved.

Rare Variant, Gene-Based Association Study of Hereditary Melanoma Using Whole-Exome Sequencing.

PubMed

Artomov, Mykyta; Stratigos, Alexander J; Kim, Ivana; Kumar, Raj; Lauss, Martin; Reddy, Bobby Y; Miao, Benchun; Daniela Robles-Espinoza, Carla; Sankar, Aravind; Njauw, Ching-Ni; Shannon, Kristen; Gragoudas, Evangelos S; Marie Lane, Anne; Iyer, Vivek; Newton-Bishop, Julia A; Timothy Bishop, D; Holland, Elizabeth A; Mann, Graham J; Singh, Tarjinder; Daly, Mark J; Tsao, Hensin

2017-12-01

Extraordinary progress has been made in our understanding of common variants in many diseases, including melanoma. Because the contribution of rare coding variants is not as well characterized, we performed an exome-wide, gene-based association study of familial cutaneous melanoma (CM) and ocular melanoma (OM). Using 11 990 jointly processed individual DNA samples, whole-exome sequencing was performed, followed by large-scale joint variant calling using GATK (Genome Analysis ToolKit). PLINK/SEQ was used for statistical analysis of genetic variation. Four models were used to estimate the association among different types of variants. In vitro functional validation was performed using three human melanoma cell lines in 2D and 3D proliferation assays. In vivo tumor growth was assessed using xenografts of human melanoma A375 melanoma cells in nude mice (eight mice per group). All statistical tests were two-sided. Strong signals were detected for CDKN2A (Pmin = 6.16 × 10-8) in the CM cohort (n = 273) and BAP1 (Pmin = 3.83 × 10-6) in the OM (n = 99) cohort. Eleven genes that exhibited borderline association (P < 10-4) were independently validated using The Cancer Genome Atlas melanoma cohort (379 CM, 47 OM) and a matched set of 3563 European controls with CDKN2A (P = .009), BAP1 (P = .03), and EBF3 (P = 4.75 × 10-4), a candidate risk locus, all showing evidence of replication. EBF3 was then evaluated using germline data from a set of 132 familial melanoma cases and 4769 controls of UK origin (joint P = 1.37 × 10-5). Somatically, loss of EBF3 expression correlated with progression, poorer outcome, and high MITF tumors. Functionally, induction of EBF3 in melanoma cells reduced cell growth in vitro, retarded tumor formation in vivo, and reduced MITF levels. The results of this large rare variant germline association study further define the mutational landscape of hereditary melanoma and implicate EBF3 as a possible CM predisposition gene.

Rare Variant, Gene-Based Association Study of Hereditary Melanoma Using Whole-Exome Sequencing

PubMed Central

Artomov, Mykyta; Stratigos, Alexander J; Kim, Ivana; Kumar, Raj; Lauss, Martin; Reddy, Bobby Y; Miao, Benchun; Daniela Robles-Espinoza, Carla; Sankar, Aravind; Njauw, Ching-Ni; Shannon, Kristen; Gragoudas, Evangelos S; Marie Lane, Anne; Iyer, Vivek; Newton-Bishop, Julia A; Timothy Bishop, D; Holland, Elizabeth A; Mann, Graham J; Singh, Tarjinder; Daly, Mark J; Tsao, Hensin

2017-01-01

Abstract Background Extraordinary progress has been made in our understanding of common variants in many diseases, including melanoma. Because the contribution of rare coding variants is not as well characterized, we performed an exome-wide, gene-based association study of familial cutaneous melanoma (CM) and ocular melanoma (OM). Methods Using 11 990 jointly processed individual DNA samples, whole-exome sequencing was performed, followed by large-scale joint variant calling using GATK (Genome Analysis ToolKit). PLINK/SEQ was used for statistical analysis of genetic variation. Four models were used to estimate the association among different types of variants. In vitro functional validation was performed using three human melanoma cell lines in 2D and 3D proliferation assays. In vivo tumor growth was assessed using xenografts of human melanoma A375 melanoma cells in nude mice (eight mice per group). All statistical tests were two-sided. Results Strong signals were detected for CDKN2A (Pmin = 6.16 × 10-8) in the CM cohort (n = 273) and BAP1 (Pmin = 3.83 × 10‐6) in the OM (n = 99) cohort. Eleven genes that exhibited borderline association (P < 10‐4) were independently validated using The Cancer Genome Atlas melanoma cohort (379 CM, 47 OM) and a matched set of 3563 European controls with CDKN2A (P = .009), BAP1 (P = .03), and EBF3 (P = 4.75 × 10‐4), a candidate risk locus, all showing evidence of replication. EBF3 was then evaluated using germline data from a set of 132 familial melanoma cases and 4769 controls of UK origin (joint P = 1.37 × 10‐5). Somatically, loss of EBF3 expression correlated with progression, poorer outcome, and high MITF tumors. Functionally, induction of EBF3 in melanoma cells reduced cell growth in vitro, retarded tumor formation in vivo, and reduced MITF levels. Conclusions The results of this large rare variant germline association study further define the mutational landscape of hereditary melanoma and implicate EBF3 as a possible CM predisposition gene. PMID:29522175

The UK10K project identifies rare variants in health and disease.

PubMed

Walter, Klaudia; Min, Josine L; Huang, Jie; Crooks, Lucy; Memari, Yasin; McCarthy, Shane; Perry, John R B; Xu, ChangJiang; Futema, Marta; Lawson, Daniel; Iotchkova, Valentina; Schiffels, Stephan; Hendricks, Audrey E; Danecek, Petr; Li, Rui; Floyd, James; Wain, Louise V; Barroso, Inês; Humphries, Steve E; Hurles, Matthew E; Zeggini, Eleftheria; Barrett, Jeffrey C; Plagnol, Vincent; Richards, J Brent; Greenwood, Celia M T; Timpson, Nicholas J; Durbin, Richard; Soranzo, Nicole

2015-10-01

The contribution of rare and low-frequency variants to human traits is largely unexplored. Here we describe insights from sequencing whole genomes (low read depth, 7×) or exomes (high read depth, 80×) of nearly 10,000 individuals from population-based and disease collections. In extensively phenotyped cohorts we characterize over 24 million novel sequence variants, generate a highly accurate imputation reference panel and identify novel alleles associated with levels of triglycerides (APOB), adiponectin (ADIPOQ) and low-density lipoprotein cholesterol (LDLR and RGAG1) from single-marker and rare variant aggregation tests. We describe population structure and functional annotation of rare and low-frequency variants, use the data to estimate the benefits of sequencing for association studies, and summarize lessons from disease-specific collections. Finally, we make available an extensive resource, including individual-level genetic and phenotypic data and web-based tools to facilitate the exploration of association results.

Frequency of EBV LMP-1 Promoter and Coding Variations in Burkitt Lymphoma Samples in Africa and South America and Peripheral Blood in Uganda.

PubMed

Liao, Hsiao-Mei; Liu, Hebing; Lei, Heiyan; Li, Bingjie; Chin, Pei-Ju; Tsai, Shien; Bhatia, Kishor; Gutierrez, Marina; Epelman, Sidnei; Biggar, Robert J; Nkrumah, Francis; Neequaye, Janet; Ogwang, Martin D; Reynolds, Steven J; Lo, Shyh-Ching; Mbulaiteye, Sam M

2018-06-02

Epstein-Barr virus (EBV) is linked to several cancers, including endemic Burkitt lymphoma (eBL), but causal variants are unknown. We recently reported novel sequence variants in the LMP-1 gene and promoter in EBV genomes sequenced from 13 of 14 BL biopsies. Alignments of the novel sequence variants for 114 published EBV genomes, including 27 from BL cases, revealed four LMP-1 variant patterns, designated A to D. Pattern A variant was found in 48% of BL EBV genomes. Here, we used PCR-Sanger sequencing to evaluate 50 additional BL biopsies from Ghana, Brazil, and Argentina, and peripheral blood samples from 113 eBL cases and 115 controls in Uganda. Pattern A was found in 60.9% of 64 BL biopsies evaluated. Compared to PCR-negative subjects in Uganda, detection of Pattern A in peripheral blood was associated with eBL case status (odds ratio [OR] 31.7, 95% confidence interval: 6.8⁻149), controlling for relevant confounders. Variant Pattern A and Pattern D were associated with eBL case status, but with lower ORs (9.7 and 13.6, respectively). Our results support the hypothesis that EBV LMP-1 Pattern A may be associated with eBL, but it is not the sole associated variant. Further research is needed to replicate and elucidate our findings.

Targeted Analysis of Whole Genome Sequence Data to Diagnose Genetic Cardiomyopathy

DOE PAGES

Golbus, Jessica R.; Puckelwartz, Megan J.; Dellefave-Castillo, Lisa; ...

2014-09-01

Background—Cardiomyopathy is highly heritable but genetically diverse. At present, genetic testing for cardiomyopathy uses targeted sequencing to simultaneously assess the coding regions of more than 50 genes. New genes are routinely added to panels to improve the diagnostic yield. With the anticipated $1000 genome, it is expected that genetic testing will shift towards comprehensive genome sequencing accompanied by targeted gene analysis. Therefore, we assessed the reliability of whole genome sequencing and targeted analysis to identify cardiomyopathy variants in 11 subjects with cardiomyopathy. Methods and Results—Whole genome sequencing with an average of 37× coverage was combined with targeted analysis focused onmore » 204 genes linked to cardiomyopathy. Genetic variants were scored using multiple prediction algorithms combined with frequency data from public databases. This pipeline yielded 1-14 potentially pathogenic variants per individual. Variants were further analyzed using clinical criteria and/or segregation analysis. Three of three previously identified primary mutations were detected by this analysis. In six subjects for whom the primary mutation was previously unknown, we identified mutations that segregated with disease, had clinical correlates, and/or had additional pathological correlation to provide evidence for causality. For two subjects with previously known primary mutations, we identified additional variants that may act as modifiers of disease severity. In total, we identified the likely pathological mutation in 9 of 11 (82%) subjects. We conclude that these pilot data demonstrate that ~30-40× coverage whole genome sequencing combined with targeted analysis is feasible and sensitive to identify rare variants in cardiomyopathy-associated genes.« less

Screening for single nucleotide variants, small indels and exon deletions with a next-generation sequencing based gene panel approach for Usher syndrome

PubMed Central

Krawitz, Peter M; Schiska, Daniela; Krüger, Ulrike; Appelt, Sandra; Heinrich, Verena; Parkhomchuk, Dmitri; Timmermann, Bernd; Millan, Jose M; Robinson, Peter N; Mundlos, Stefan; Hecht, Jochen; Gross, Manfred

2014-01-01

Usher syndrome is an autosomal recessive disorder characterized both by deafness and blindness. For the three clinical subtypes of Usher syndrome causal mutations in altogether 12 genes and a modifier gene have been identified. Due to the genetic heterogeneity of Usher syndrome, the molecular analysis is predestined for a comprehensive and parallelized analysis of all known genes by next-generation sequencing (NGS) approaches. We describe here the targeted enrichment and deep sequencing for exons of Usher genes and compare the costs and workload of this approach compared to Sanger sequencing. We also present a bioinformatics analysis pipeline that allows us to detect single-nucleotide variants, short insertions and deletions, as well as copy number variations of one or more exons on the same sequence data. Additionally, we present a flexible in silico gene panel for the analysis of sequence variants, in which newly identified genes can easily be included. We applied this approach to a cohort of 44 Usher patients and detected biallelic pathogenic mutations in 35 individuals and monoallelic mutations in eight individuals of our cohort. Thirty-nine of the sequence variants, including two heterozygous deletions comprising several exons of USH2A, have not been reported so far. Our NGS-based approach allowed us to assess single-nucleotide variants, small indels, and whole exon deletions in a single test. The described diagnostic approach is fast and cost-effective with a high molecular diagnostic yield. PMID:25333064

Screening for single nucleotide variants, small indels and exon deletions with a next-generation sequencing based gene panel approach for Usher syndrome.

PubMed

Krawitz, Peter M; Schiska, Daniela; Krüger, Ulrike; Appelt, Sandra; Heinrich, Verena; Parkhomchuk, Dmitri; Timmermann, Bernd; Millan, Jose M; Robinson, Peter N; Mundlos, Stefan; Hecht, Jochen; Gross, Manfred

2014-09-01

Usher syndrome is an autosomal recessive disorder characterized both by deafness and blindness. For the three clinical subtypes of Usher syndrome causal mutations in altogether 12 genes and a modifier gene have been identified. Due to the genetic heterogeneity of Usher syndrome, the molecular analysis is predestined for a comprehensive and parallelized analysis of all known genes by next-generation sequencing (NGS) approaches. We describe here the targeted enrichment and deep sequencing for exons of Usher genes and compare the costs and workload of this approach compared to Sanger sequencing. We also present a bioinformatics analysis pipeline that allows us to detect single-nucleotide variants, short insertions and deletions, as well as copy number variations of one or more exons on the same sequence data. Additionally, we present a flexible in silico gene panel for the analysis of sequence variants, in which newly identified genes can easily be included. We applied this approach to a cohort of 44 Usher patients and detected biallelic pathogenic mutations in 35 individuals and monoallelic mutations in eight individuals of our cohort. Thirty-nine of the sequence variants, including two heterozygous deletions comprising several exons of USH2A, have not been reported so far. Our NGS-based approach allowed us to assess single-nucleotide variants, small indels, and whole exon deletions in a single test. The described diagnostic approach is fast and cost-effective with a high molecular diagnostic yield.

Genome sequencing elucidates Sardinian genetic architecture and augments association analyses for lipid and blood inflammatory markers

PubMed Central

Zoledziewska, Magdalena; Mulas, Antonella; Pistis, Giorgio; Steri, Maristella; Danjou, Fabrice; Kwong, Alan; Ortega del Vecchyo, Vicente Diego; Chiang, Charleston W. K.; Bragg-Gresham, Jennifer; Pitzalis, Maristella; Nagaraja, Ramaiah; Tarrier, Brendan; Brennan, Christine; Uzzau, Sergio; Fuchsberger, Christian; Atzeni, Rossano; Reinier, Frederic; Berutti, Riccardo; Huang, Jie; Timpson, Nicholas J; Toniolo, Daniela; Gasparini, Paolo; Malerba, Giovanni; Dedoussis, George; Zeggini, Eleftheria; Soranzo, Nicole; Jones, Chris; Lyons, Robert; Angius, Andrea; Kang, Hyun M.; Novembre, John; Sanna, Serena; Schlessinger, David; Cucca, Francesco; Abecasis, Gonçalo R

2015-01-01

We report ~17.6M genetic variants from whole-genome sequencing of 2,120 Sardinians; 22% are absent from prior sequencing-based compilations and enriched for predicted functional consequence. Furthermore, ~76K variants common in our sample (frequency >5%) are rare elsewhere (<0.5% in the 1000 Genomes Project). We assessed the impact of these variants on circulating lipid levels and five inflammatory biomarkers. Fourteen signals, including two major new loci, were observed for lipid levels, and 19, including two novel loci, for inflammatory markers. New associations would be missed in analyses based on 1000 Genomes data, underlining the advantages of large-scale sequencing in this founder population. PMID:26366554

DaMold: A data-mining platform for variant annotation and visualization in molecular diagnostics research.

PubMed

Pandey, Ram Vinay; Pabinger, Stephan; Kriegner, Albert; Weinhäusel, Andreas

2017-07-01

Next-generation sequencing (NGS) has become a powerful and efficient tool for routine mutation screening in clinical research. As each NGS test yields hundreds of variants, the current challenge is to meaningfully interpret the data and select potential candidates. Analyzing each variant while manually investigating several relevant databases to collect specific information is a cumbersome and time-consuming process, and it requires expertise and familiarity with these databases. Thus, a tool that can seamlessly annotate variants with clinically relevant databases under one common interface would be of great help for variant annotation, cross-referencing, and visualization. This tool would allow variants to be processed in an automated and high-throughput manner and facilitate the investigation of variants in several genome browsers. Several analysis tools are available for raw sequencing-read processing and variant identification, but an automated variant filtering, annotation, cross-referencing, and visualization tool is still lacking. To fulfill these requirements, we developed DaMold, a Web-based, user-friendly tool that can filter and annotate variants and can access and compile information from 37 resources. It is easy to use, provides flexible input options, and accepts variants from NGS and Sanger sequencing as well as hotspots in VCF and BED formats. DaMold is available as an online application at http://damold.platomics.com/index.html, and as a Docker container and virtual machine at https://sourceforge.net/projects/damold/. © 2017 Wiley Periodicals, Inc.

Mutations in GBA are associated with familial Parkinson disease susceptibility and age at onset.

PubMed

Nichols, W C; Pankratz, N; Marek, D K; Pauciulo, M W; Elsaesser, V E; Halter, C A; Rudolph, A; Wojcieszek, J; Pfeiffer, R F; Foroud, T

2009-01-27

To characterize sequence variation within the glucocerebrosidase (GBA) gene in a select subset of our sample of patients with familial Parkinson disease (PD) and then to test in our full sample whether these sequence variants increased the risk for PD and were associated with an earlier onset of disease. We performed a comprehensive study of all GBA exons in one patient with PD from each of 96 PD families, selected based on the family-specific lod scores at the GBA locus. Identified GBA variants were subsequently screened in all 1325 PD cases from 566 multiplex PD families and in 359 controls. Nine different GBA variants, five previously reported, were identified in 21 of the 96 PD cases sequenced. Screening for these variants in the full sample identified 161 variant carriers (12.2%) in 99 different PD families. An unbiased estimate of the frequency of the five previously reported GBA variants in the familial PD sample was 12.6% and in the control sample was 5.3% (odds ratio 2.6; 95% confidence interval 1.5-4.4). Presence of a GBA variant was associated with an earlier age at onset (p = 0.0001). On average, those patients carrying a GBA variant had onset with PD 6.04 years earlier than those without a GBA variant. This study suggests that GBA is a susceptibility gene for familial Parkinson disease (PD) and patients with GBA variants have an earlier age at onset than patients with PD without GBA variants.

Genetic Analyses in Small-for-Gestational-Age Newborns.

PubMed

Stalman, Susanne E; Solanky, Nita; Ishida, Miho; Alemán-Charlet, Cristina; Abu-Amero, Sayeda; Alders, Marielle; Alvizi, Lucas; Baird, William; Demetriou, Charalambos; Henneman, Peter; James, Chela; Knegt, Lia C; Leon, Lydia J; Mannens, Marcel M A M; Mul, Adi N; Nibbering, Nicole A; Peskett, Emma; Rezwan, Faisal I; Ris-Stalpers, Carrie; van der Post, Joris A M; Kamp, Gerdine A; Plötz, Frans B; Wit, Jan M; Stanier, Philip; Moore, Gudrun E; Hennekam, Raoul C

2018-03-01

Small for gestational age (SGA) can be the result of fetal growth restriction, which is associated with perinatal morbidity and mortality. Mechanisms that control prenatal growth are poorly understood. The aim of the current study was to gain more insight into prenatal growth failure and determine an effective diagnostic approach in SGA newborns. We hypothesized that one or more copy number variations (CNVs) and disturbed methylation and sequence variants may be present in genes associated with fetal growth. A prospective cohort study of subjects with a low birth weight for gestational age. The study was conducted at an academic pediatric research institute. A total of 21 SGA newborns with a mean birth weight below the first centile and a control cohort of 24 appropriate-for-gestational-age newborns were studied. Array comparative genomic hybridization, genome-wide methylation studies, and exome sequencing were performed. The numbers of CNVs, methylation disturbances, and sequence variants. The genetic analyses demonstrated three CNVs, one systematically disturbed methylation pattern, and one sequence variant explaining SGA. Additional methylation disturbances and sequence variants were present in 20 patients. In 19 patients, multiple abnormalities were found. Our results confirm the influence of a large number of mechanisms explaining dysregulation of fetal growth. We concluded that CNVs, methylation disturbances, and sequence variants all contribute to prenatal growth failure. These genetic workups can be an effective diagnostic approach in SGA newborns.

Characterization of genetic sequence variation of 58 STR loci in four major population groups.

PubMed

Novroski, Nicole M M; King, Jonathan L; Churchill, Jennifer D; Seah, Lay Hong; Budowle, Bruce

2016-11-01

Massively parallel sequencing (MPS) can identify sequence variation within short tandem repeat (STR) alleles as well as their nominal allele lengths that traditionally have been obtained by capillary electrophoresis. Using the MiSeq FGx Forensic Genomics System (Illumina), STRait Razor, and in-house excel workbooks, genetic variation was characterized within STR repeat and flanking regions of 27 autosomal, 7 X-chromosome and 24 Y-chromosome STR markers in 777 unrelated individuals from four population groups. Seven hundred and forty six autosomal, 227 X-chromosome, and 324 Y-chromosome STR alleles were identified by sequence compared with 357 autosomal, 107 X-chromosome, and 189 Y-chromosome STR alleles that were identified by length. Within the observed sequence variation, 227 autosomal, 156 X-chromosome, and 112 Y-chromosome novel alleles were identified and described. One hundred and seventy six autosomal, 123 X-chromosome, and 93 Y-chromosome sequence variants resided within STR repeat regions, and 86 autosomal, 39 X-chromosome, and 20 Y-chromosome variants were located in STR flanking regions. Three markers, D18S51, DXS10135, and DYS385a-b had 1, 4, and 1 alleles, respectively, which contained both a novel repeat region variant and a flanking sequence variant in the same nucleotide sequence. There were 50 markers that demonstrated a relative increase in diversity with the variant sequence alleles compared with those of traditional nominal length alleles. These population data illustrate the genetic variation that exists in the commonly used STR markers in the selected population samples and provide allele frequencies for statistical calculations related to STR profiling with MPS data. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Rare and Coding Region Genetic Variants Associated With Risk of Ischemic Stroke: The NHLBI Exome Sequence Project.

PubMed

Auer, Paul L; Nalls, Mike; Meschia, James F; Worrall, Bradford B; Longstreth, W T; Seshadri, Sudha; Kooperberg, Charles; Burger, Kathleen M; Carlson, Christopher S; Carty, Cara L; Chen, Wei-Min; Cupples, L Adrienne; DeStefano, Anita L; Fornage, Myriam; Hardy, John; Hsu, Li; Jackson, Rebecca D; Jarvik, Gail P; Kim, Daniel S; Lakshminarayan, Kamakshi; Lange, Leslie A; Manichaikul, Ani; Quinlan, Aaron R; Singleton, Andrew B; Thornton, Timothy A; Nickerson, Deborah A; Peters, Ulrike; Rich, Stephen S

2015-07-01

Stroke is the second leading cause of death and the third leading cause of years of life lost. Genetic factors contribute to stroke prevalence, and candidate gene and genome-wide association studies (GWAS) have identified variants associated with ischemic stroke risk. These variants often have small effects without obvious biological significance. Exome sequencing may discover predicted protein-altering variants with a potentially large effect on ischemic stroke risk. To investigate the contribution of rare and common genetic variants to ischemic stroke risk by targeting the protein-coding regions of the human genome. The National Heart, Lung, and Blood Institute (NHLBI) Exome Sequencing Project (ESP) analyzed approximately 6000 participants from numerous cohorts of European and African ancestry. For discovery, 365 cases of ischemic stroke (small-vessel and large-vessel subtypes) and 809 European ancestry controls were sequenced; for replication, 47 affected sibpairs concordant for stroke subtype and an African American case-control series were sequenced, with 1672 cases and 4509 European ancestry controls genotyped. The ESP's exome sequencing and genotyping started on January 1, 2010, and continued through June 30, 2012. Analyses were conducted on the full data set between July 12, 2012, and July 13, 2013. Discovery of new variants or genes contributing to ischemic stroke risk and subtype (primary analysis) and determination of support for protein-coding variants contributing to risk in previously published candidate genes (secondary analysis). We identified 2 novel genes associated with an increased risk of ischemic stroke: a protein-coding variant in PDE4DIP (rs1778155; odds ratio, 2.15; P = 2.63 × 10(-8)) with an intracellular signal transduction mechanism and in ACOT4 (rs35724886; odds ratio, 2.04; P = 1.24 × 10(-7)) with a fatty acid metabolism; confirmation of PDE4DIP was observed in affected sibpair families with large-vessel stroke subtype and in African Americans. Replication of protein-coding variants in candidate genes was observed for 2 previously reported GWAS associations: ZFHX3 (cardioembolic stroke) and ABCA1 (large-vessel stroke). Exome sequencing discovered 2 novel genes and mechanisms, PDE4DIP and ACOT4, associated with increased risk for ischemic stroke. In addition, ZFHX3 and ABCA1 were discovered to have protein-coding variants associated with ischemic stroke. These results suggest that genetic variation in novel pathways contributes to ischemic stroke risk and serves as a target for prediction, prevention, and therapy.

Worldwide Distribution of Cytochrome P450 Alleles: A Meta-analysis of Population-scale Sequencing Projects.

PubMed

Zhou, Y; Ingelman-Sundberg, M; Lauschke, V M

2017-10-01

Genetic polymorphisms in cytochrome P450 (CYP) genes can result in altered metabolic activity toward a plethora of clinically important medications. Thus, single nucleotide variants and copy number variations in CYP genes are major determinants of drug pharmacokinetics and toxicity and constitute pharmacogenetic biomarkers for drug dosing, efficacy, and safety. Strikingly, the distribution of CYP alleles differs considerably between populations with important implications for personalized drug therapy and healthcare programs. To provide a global distribution map of CYP alleles with clinical importance, we integrated whole-genome and exome sequencing data from 56,945 unrelated individuals of five major human populations. By combining this dataset with population-specific linkage information, we derive the frequencies of 176 CYP haplotypes, providing an extensive resource for major genetic determinants of drug metabolism. Furthermore, we aggregated this dataset into spectra of predicted functional variability in the respective populations and discuss the implications for population-adjusted pharmacological treatment strategies. © 2017 The Authors Clinical Pharmacology & Therapeutics published by Wiley Periodicals, Inc. on behalf of American Society for Clinical Pharmacology and Therapeutics.

Common 5S rRNA variants are likely to be accepted in many sequence contexts

NASA Technical Reports Server (NTRS)

Zhang, Zhengdong; D'Souza, Lisa M.; Lee, Youn-Hyung; Fox, George E.

2003-01-01

Over evolutionary time RNA sequences which are successfully fixed in a population are selected from among those that satisfy the structural and chemical requirements imposed by the function of the RNA. These sequences together comprise the structure space of the RNA. In principle, a comprehensive understanding of RNA structure and function would make it possible to enumerate which specific RNA sequences belong to a particular structure space and which do not. We are using bacterial 5S rRNA as a model system to attempt to identify principles that can be used to predict which sequences do or do not belong to the 5S rRNA structure space. One promising idea is the very intuitive notion that frequently seen sequence changes in an aligned data set of naturally occurring 5S rRNAs would be widely accepted in many other 5S rRNA sequence contexts. To test this hypothesis, we first developed well-defined operational definitions for a Vibrio region of the 5S rRNA structure space and what is meant by a highly variable position. Fourteen sequence variants (10 point changes and 4 base-pair changes) were identified in this way, which, by the hypothesis, would be expected to incorporate successfully in any of the known sequences in the Vibrio region. All 14 of these changes were constructed and separately introduced into the Vibrio proteolyticus 5S rRNA sequence where they are not normally found. Each variant was evaluated for its ability to function as a valid 5S rRNA in an E. coli cellular context. It was found that 93% (13/14) of the variants tested are likely valid 5S rRNAs in this context. In addition, seven variants were constructed that, although present in the Vibrio region, did not meet the stringent criteria for a highly variable position. In this case, 86% (6/7) are likely valid. As a control we also examined seven variants that are seldom or never seen in the Vibrio region of 5S rRNA sequence space. In this case only two of seven were found to be potentially valid. The results demonstrate that changes that occur multiple times in a local region of RNA sequence space in fact usually will be accepted in any sequence context in that same local region.

Transcriptome Sequencing Revealed Significant Alteration of Cortical Promoter Usage and Splicing in Schizophrenia

PubMed Central

Wu, Jing Qin; Wang, Xi; Beveridge, Natalie J.; Tooney, Paul A.; Scott, Rodney J.; Carr, Vaughan J.; Cairns, Murray J.

2012-01-01

Background While hybridization based analysis of the cortical transcriptome has provided important insight into the neuropathology of schizophrenia, it represents a restricted view of disease-associated gene activity based on predetermined probes. By contrast, sequencing technology can provide un-biased analysis of transcription at nucleotide resolution. Here we use this approach to investigate schizophrenia-associated cortical gene expression. Methodology/Principal Findings The data was generated from 76 bp reads of RNA-Seq, aligned to the reference genome and assembled into transcripts for quantification of exons, splice variants and alternative promoters in postmortem superior temporal gyrus (STG/BA22) from 9 male subjects with schizophrenia and 9 matched non-psychiatric controls. Differentially expressed genes were then subjected to further sequence and functional group analysis. The output, amounting to more than 38 Gb of sequence, revealed significant alteration of gene expression including many previously shown to be associated with schizophrenia. Gene ontology enrichment analysis followed by functional map construction identified three functional clusters highly relevant to schizophrenia including neurotransmission related functions, synaptic vesicle trafficking, and neural development. Significantly, more than 2000 genes displayed schizophrenia-associated alternative promoter usage and more than 1000 genes showed differential splicing (FDR<0.05). Both types of transcriptional isoforms were exemplified by reads aligned to the neurodevelopmentally significant doublecortin-like kinase 1 (DCLK1) gene. Conclusions This study provided the first deep and un-biased analysis of schizophrenia-associated transcriptional diversity within the STG, and revealed variants with important implications for the complex pathophysiology of schizophrenia. PMID:22558445

Lessons learned from whole exome sequencing in multiplex families affected by a complex genetic disorder, intracranial aneurysm.

PubMed

Farlow, Janice L; Lin, Hai; Sauerbeck, Laura; Lai, Dongbing; Koller, Daniel L; Pugh, Elizabeth; Hetrick, Kurt; Ling, Hua; Kleinloog, Rachel; van der Vlies, Pieter; Deelen, Patrick; Swertz, Morris A; Verweij, Bon H; Regli, Luca; Rinkel, Gabriel J E; Ruigrok, Ynte M; Doheny, Kimberly; Liu, Yunlong; Broderick, Joseph; Foroud, Tatiana

2015-01-01

Genetic risk factors for intracranial aneurysm (IA) are not yet fully understood. Genomewide association studies have been successful at identifying common variants; however, the role of rare variation in IA susceptibility has not been fully explored. In this study, we report the use of whole exome sequencing (WES) in seven densely-affected families (45 individuals) recruited as part of the Familial Intracranial Aneurysm study. WES variants were prioritized by functional prediction, frequency, predicted pathogenicity, and segregation within families. Using these criteria, 68 variants in 68 genes were prioritized across the seven families. Of the genes that were expressed in IA tissue, one gene (TMEM132B) was differentially expressed in aneurysmal samples (n=44) as compared to control samples (n=16) (false discovery rate adjusted p-value=0.023). We demonstrate that sequencing of densely affected families permits exploration of the role of rare variants in a relatively common disease such as IA, although there are important study design considerations for applying sequencing to complex disorders. In this study, we explore methods of WES variant prioritization, including the incorporation of unaffected individuals, multipoint linkage analysis, biological pathway information, and transcriptome profiling. Further studies are needed to validate and characterize the set of variants and genes identified in this study.

An abundance of rare functional variants in 202 drug target genes sequenced in 14,002 people

PubMed Central

Nelson, Matthew R.; Wegmann, Daniel; Ehm, Margaret G.; Kessner, Darren; St. Jean, Pamela; Verzilli, Claudio; Shen, Judong; Tang, Zhengzheng; Bacanu, Silviu-Alin; Fraser, Dana; Warren, Liling; Aponte, Jennifer; Zawistowski, Matthew; Liu, Xiao; Zhang, Hao; Zhang, Yong; Li, Jun; Li, Yun; Li, Li; Woollard, Peter; Topp, Simon; Hall, Matthew D.; Nangle, Keith; Wang, Jun; Abecasis, Gonçalo; Cardon, Lon R.; Zöllner, Sebastian; Whittaker, John C.; Chissoe, Stephanie L.; Novembre, John; Mooser, Vincent

2015-01-01

Rare genetic variants contribute to complex disease risk; however, the abundance of rare variants in human populations remains unknown. We explored this spectrum of variation by sequencing 202 genes encoding drug targets in 14,002 individuals. We find rare variants are abundant (one every 17 bases) and geographically localized, such that even with large sample sizes, rare variant catalogs will be largely incomplete. We used the observed patterns of variation to estimate population growth parameters, the proportion of variants in a given frequency class that are putatively deleterious, and mutation rates for each gene. Overall we conclude that, due to rapid population growth and weak purifying selection, human populations harbor an abundance of rare variants, many of which are deleterious and have relevance to understanding disease risk. PMID:22604722

BEST1 sequence variants in Italian patients with vitelliform macular dystrophy

PubMed Central

Sodi, Andrea; Passerini, Ilaria; Caputo, Roberto; Bacci, Giacomo Maria; Bodoj, Mirela; Torricelli, Francesca; Menchini, Ugo

2012-01-01

Purpose To analyze the spectrum of sequence variants in the BEST1 gene in a group of Italian patients affected by Best vitelliform macular dystrophy (VMD). Methods Thirty Italian patients with a diagnosis of VMD and 20 clinically healthy relatives were recruited. They belonged to 19 Italian families predominantly originating from central Italy. They received a standard ophthalmologic examination, OCT scan, and electrophysiological tests (ERG and EOG). Fluorescein and ICG angiographies and fundus autofluorescence imaging were performed in selected cases. DNA samples were analyzed for sequence variants of the BEST1 gene by direct sequencing techniques. Results Nine missense variants and one deletion were found in the affected patients; each patient carried one mutation. Five variants [c.73C>T (p.Arg25Trp), c.652C>T (p.Arg218Cys), c.652C>G (p.Arg218Gly), c.728C>T (p.Ala243Val), c.893T>C (p.Phe298Ser)] have already been described in literature while another five variants [c.217A>C (p.Ile73Leu), c.239T>G (p.Phe80Cys), c.883_885del (p.Ile295del), c.907G>A (p.Asp303Asn), c.911A>G (p.Asp304Gly)] had not previously been reported. Affected patients, sometimes even from the same family, occasionally showed variable phenotypes. One heterozygous variant was also found in five clinically healthy relatives with normal fundus, visual acuity and ERG but with abnormal EOG. Conclusions Ten variants in the BEST1 gene were detected in a group of individuals with clinically apparent VMD, and in some clinically normal individuals with an abnormal EOG. The high prevalence of novel variants and the frequent report of a specific variant (p.Arg25Trp) that has rarely been described in other ethnic groups suggests a distribution of BEST1 variants peculiar to Italian VMD patients. PMID:23213274

A novel recurrent mutation in MITF predisposes to familial and sporadic melanoma

PubMed Central

Yokoyama, Satoru; Woods, Susan L.; Boyle, Glen M.; Aoude, Lauren G.; MacGregor, Stuart; Zismann, Victoria; Gartside, Michael; Cust, Anne E.; Haq, Rizwan; Harland, Mark; Taylor, John C.; Duffy, David L.; Holohan, Kelly; Dutton-Regester, Ken; Palmer, Jane M.; Bonazzi, Vanessa; Stark, Mitchell S.; Symmons, Judith; Law, Matthew H.; Schmidt, Christopher; Lanagan, Cathy; O’Connor, Linda; Holland, Elizabeth A.; Schmid, Helen; Maskiell, Judith A.; Jetann, Jodie; Ferguson, Megan; Jenkins, Mark A.; Kefford, Richard F.; Giles, Graham G.; Armstrong, Bruce K.; Aitken, Joanne F.; Hopper, John L.; Whiteman, David C.; Pharoah, Paul D.; Easton, Douglas F.; Dunning, Alison M.; Newton-Bishop, Julia A.; Montgomery, Grant W.; Martin, Nicholas G.; Mann, Graham J.; Bishop, D. Timothy; Tsao, Hensin; Trent, Jeffrey M.; Fisher, David E.; Hayward, Nicholas K.; Brown, Kevin M.

2012-01-01

So far, two familial melanoma genes have been identified, accounting for a minority of genetic risk in families. Mutations in CDKN2A account for approximately 40% of familial cases1, and predisposing mutations in CDK4 have been reported in a very small number of melanoma kindreds2. To identify other familial melanoma genes, here we conducted whole-genome sequencing of probands from several melanoma families, identifying one individual carrying a novel germline variant (coding DNA sequence c.G1075A; protein sequence p.E318K; rs149617956) in the melanoma-lineage-specific oncogene microphthalmia-associated transcription factor (MITF). Although the variant co-segregated with melanoma in some but not all cases in the family, linkage analysis of 31 families subsequently identified to carry the variant generated a log odds ratio (lod) score of 2.7 under a dominant model, indicating E318K as a possible intermediate risk variant. Consistent with this, the E318K variant was significantly associated with melanoma in a large Australian case–control sample. Likewise, it was similarly associated in an independent case–control sample from the United Kingdom. In the Australian sample, the variant allele was significantly over-represented in cases with a family history of melanoma, multiple primary melanomas, or both. The variant allele was also associated with increased naevus count and non-blue eye colour. Functional analysis of E318K showed that MITF encoded by the variant allele had impaired sumoylation and differentially regulated several MITF targets. These data indicate that MITF is a melanoma-predisposition gene and highlight the utility of whole-genome sequencing to identify novel rare variants associated with disease susceptibility. PMID:22080950

Germline Missense Variants in the BTNL2 Gene Are Associated with Prostate Cancer Susceptibility

PubMed Central

FitzGerald, Liesel M.; Kumar, Akash; Boyle, Evan A.; Zhang, Yuzheng; McIntosh, Laura M.; Kolb, Suzanne; Stott-Miller, Marni; Smith, Tiffany; Karyadi, Danielle M.; Ostrander, Elaine A.; Hsu, Li; Shendure, Jay; Stanford, Janet L.

2013-01-01

Background Rare, inherited mutations account for 5%–10% of all prostate cancer (PCa) cases. However, to date, few causative mutations have been identified. Methods To identify rare mutations for PCa, we performed whole-exome sequencing (WES) in multiple kindreds (n = 91) from 19 hereditary prostate cancer (HPC) families characterized by aggressive or early onset phenotypes. Candidate variants (n = 130) identified through family- and bioinformatics-based filtering of WES data were then genotyped in an independent set of 270 HPC families (n = 819 PCa cases; n = 496 unaffected relatives) for replication. Two variants with supportive evidence were subsequently genotyped in a population-based case-control study (n = 1,155 incident PCa cases; n = 1,060 age-matched controls) for further confirmation. All participants were men of European ancestry. Results The strongest evidence was for two germline missense variants in the butyrophilin-like 2 (BTNL2) gene (rs41441651, p.Asp336Asn and rs28362675, p.Gly454Cys) that segregated with affection status in two of the WES families. In the independent set of 270 HPC families, 1.5% (rs41441651; P = 0.0032) and 1.2% (rs28362675; P = 0.0070) of affected men, but no unaffected men, carried a variant. Both variants were associated with elevated PCa risk in the population-based study (rs41441651: OR = 2.7; 95% CI, 1.27–5.87; P = 0.010; rs28362675: OR = 2.5; 95% CI, 1.16–5.46; P = 0.019). Conclusions Results indicate that rare BTNL2 variants play a role in susceptibility to both familial and sporadic prostate cancer. Impact Results implicate BTNL2 as a novel PCa susceptibility gene. PMID:23833122

Association between Rare Variants in AP4E1, a Component of Intracellular Trafficking, and Persistent Stuttering.

PubMed

Raza, M Hashim; Mattera, Rafael; Morell, Robert; Sainz, Eduardo; Rahn, Rachel; Gutierrez, Joanne; Paris, Emily; Root, Jessica; Solomon, Beth; Brewer, Carmen; Basra, M Asim Raza; Khan, Shaheen; Riazuddin, Sheikh; Braun, Allen; Bonifacino, Juan S; Drayna, Dennis

2015-11-05

Stuttering is a common, highly heritable neurodevelopmental disorder characterized by deficits in the volitional control of speech. Whole-exome sequencing identified two heterozygous AP4E1 coding variants, c.1549G>A (p.Val517Ile) and c.2401G>A (p.Glu801Lys), that co-segregate with persistent developmental stuttering in a large Cameroonian family, and we observed the same two variants in unrelated Cameroonians with persistent stuttering. We found 23 other rare variants, including predicted loss-of-function variants, in AP4E1 in unrelated stuttering individuals in Cameroon, Pakistan, and North America. The rate of rare variants in AP4E1 was significantly higher in unrelated Pakistani and Cameroonian stuttering individuals than in population-matched control individuals, and coding variants in this gene are exceptionally rare in the general sub-Saharan West African, South Asian, and North American populations. Clinical examination of the Cameroonian family members failed to identify any symptoms previously reported in rare individuals carrying homozygous loss-of-function mutations in this gene. AP4E1 encodes the ε subunit of the heterotetrameric (ε-β4-μ4-σ4) AP-4 complex, involved in protein sorting at the trans-Golgi network. We found that the μ4 subunit of AP-4 interacts with NAGPA, an enzyme involved in the synthesis of the mannose 6-phosphate signal that targets acid hydrolases to the lysosome and the product of a gene previously associated with stuttering. These findings implicate deficits in intracellular trafficking in persistent stuttering. Copyright © 2015 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Golbus, Jessica R.; Puckelwartz, Megan J.; Dellefave-Castillo, Lisa

Background—Cardiomyopathy is highly heritable but genetically diverse. At present, genetic testing for cardiomyopathy uses targeted sequencing to simultaneously assess the coding regions of more than 50 genes. New genes are routinely added to panels to improve the diagnostic yield. With the anticipated $1000 genome, it is expected that genetic testing will shift towards comprehensive genome sequencing accompanied by targeted gene analysis. Therefore, we assessed the reliability of whole genome sequencing and targeted analysis to identify cardiomyopathy variants in 11 subjects with cardiomyopathy. Methods and Results—Whole genome sequencing with an average of 37× coverage was combined with targeted analysis focused onmore » 204 genes linked to cardiomyopathy. Genetic variants were scored using multiple prediction algorithms combined with frequency data from public databases. This pipeline yielded 1-14 potentially pathogenic variants per individual. Variants were further analyzed using clinical criteria and/or segregation analysis. Three of three previously identified primary mutations were detected by this analysis. In six subjects for whom the primary mutation was previously unknown, we identified mutations that segregated with disease, had clinical correlates, and/or had additional pathological correlation to provide evidence for causality. For two subjects with previously known primary mutations, we identified additional variants that may act as modifiers of disease severity. In total, we identified the likely pathological mutation in 9 of 11 (82%) subjects. We conclude that these pilot data demonstrate that ~30-40× coverage whole genome sequencing combined with targeted analysis is feasible and sensitive to identify rare variants in cardiomyopathy-associated genes.« less

Customisation of the exome data analysis pipeline using a combinatorial approach.

PubMed

Pattnaik, Swetansu; Vaidyanathan, Srividya; Pooja, Durgad G; Deepak, Sa; Panda, Binay

2012-01-01

The advent of next generation sequencing (NGS) technologies have revolutionised the way biologists produce, analyse and interpret data. Although NGS platforms provide a cost-effective way to discover genome-wide variants from a single experiment, variants discovered by NGS need follow up validation due to the high error rates associated with various sequencing chemistries. Recently, whole exome sequencing has been proposed as an affordable option compared to whole genome runs but it still requires follow up validation of all the novel exomic variants. Customarily, a consensus approach is used to overcome the systematic errors inherent to the sequencing technology, alignment and post alignment variant detection algorithms. However, the aforementioned approach warrants the use of multiple sequencing chemistry, multiple alignment tools, multiple variant callers which may not be viable in terms of time and money for individual investigators with limited informatics know-how. Biologists often lack the requisite training to deal with the huge amount of data produced by NGS runs and face difficulty in choosing from the list of freely available analytical tools for NGS data analysis. Hence, there is a need to customise the NGS data analysis pipeline to preferentially retain true variants by minimising the incidence of false positives and make the choice of right analytical tools easier. To this end, we have sampled different freely available tools used at the alignment and post alignment stage suggesting the use of the most suitable combination determined by a simple framework of pre-existing metrics to create significant datasets.

Challenges imposed by minor reference alleles on the identification and reporting of clinical variants from exome data.

PubMed

Koko, Mahmoud; Abdallah, Mohammed O E; Amin, Mutaz; Ibrahim, Muntaser

2018-01-15

The conventional variant calling of pathogenic alleles in exome and genome sequencing requires the presence of the non-pathogenic alleles as genome references. This hinders the correct identification of variants with minor and/or pathogenic reference alleles warranting additional approaches for variant calling. More than 26,000 Exome Aggregation Consortium (ExAC) variants have a minor reference allele including variants with known ClinVar disease alleles. For instance, in a number of variants related to clotting disorders, the phenotype-associated allele is a human genome reference allele (rs6025, rs6003, rs1799983, and rs2227564 using the assembly hg19). We highlighted how the current variant calling standards miss homozygous reference disease variants in these sites and provided a bioinformatic panel that can be used to screen these variants using commonly available variant callers. We present exome sequencing results from an individual with venous thrombosis to emphasize how pathogenic alleles in clinically relevant variants escape variant calling while non-pathogenic alleles are detected. This article highlights the importance of specialized variant calling strategies in clinical variants with minor reference alleles especially in the context of personal genomes and exomes. We provide here a simple strategy to screen potential disease-causing variants when present in homozygous reference state.

Isolation and Molecular Characterization of Novel Infectious Bronchitis Virus Variants from Vaccinated Broiler Flocks in Egypt.

PubMed

Abdel-Sabour, Mohammed A; Al-Ebshahy, Emad M; Khaliel, Samy A; Abdel-Wanis, Nabil A; Yanai, Tokuma

2017-09-01

The present study aimed to determine the molecular characteristics of circulating infectious bronchitis virus (IBV) strains in vaccinated broiler flocks in the Giza and Fayoum governorates. Thirty-four isolates were collected, and egg propagation revealed their ability to induce typical IBV lesions after three to five successive passages. Three selected isolates were identified as IBV using a real-time reverse transcriptase-PCR assay targeted the nucleocapsid (N) gene and further characterized by partial spike (S) gene sequence analysis. Phylogenetic analysis revealed their clustering into two variant groups. Group I consisted of one variant (VSVRI_F3), which had 99.1% nucleotide sequence identity to the Q1 reference strain. Group II consisted of variants VSVRI_G4 and VSVRI_G9, which showed 92.8%-94.3% nucleotide identity with the Egyptian variants Eg/12120S/2012, Eg/12197B/2012, and Eg/1265B/2012. Regarding the deduced amino acid sequence, the three variants had 77.1%-85.2% similarity with the vaccine strains currently used in Egypt. These findings highlight the importance of monitoring the prevalence of IBV variants in vaccinated broiler flocks as well as adopting an appropriate vaccination strategy.

[Fine mapping of complex disease susceptibility loci].

PubMed

Song, Qingfeng; Zhang, Hongxing; Ma, Yilong; Zhou, Gangqiao

2014-01-01

Genome-wide association studies (GWAS) using single nucleotide polymorphism (SNP) markers have identified more than 3800 susceptibility loci for more than 660 diseases or traits. However, the most significantly associated variants or causative variants in these loci and their biological functions have remained to be clarified. These causative variants can help to elucidate the pathogenesis and discover new biomarkers of complex diseases. One of the main goals in the post-GWAS era is to identify the causative variants and susceptibility genes, and clarify their functional aspects by fine mapping. For common variants, imputation or re-sequencing based strategies were implemented to increase the number of analyzed variants and help to identify the most significantly associated variants. In addition, functional element, expression quantitative trait locus (eQTL) and haplotype analyses were performed to identify functional common variants and susceptibility genes. For rare variants, fine mapping was carried out by re-sequencing, rare haplotype analysis, family-based analysis, burden test, etc.This review summarizes the strategies and problems for fine mapping.

Polymorphic variations in the FANCA gene in high-risk non-BRCA1/2 breast cancer individuals from the French Canadian population.

PubMed

Litim, Nadhir; Labrie, Yvan; Desjardins, Sylvie; Ouellette, Geneviève; Plourde, Karine; Belleau, Pascal; Durocher, Francine

2013-02-01

The majority of genes associated with breast cancer susceptibility, including BRCA1 and BRCA2 genes, are involved in DNA repair mechanisms. Moreover, among the genes recently associated with an increased susceptibility to breast cancer, four are Fanconi Anemia (FA) genes: FANCD1/BRCA2, FANCJ/BACH1/BRIP1, FANCN/PALB2 and FANCO/RAD51C. FANCA is implicated in DNA repair and has been shown to interact directly with BRCA1. It has been proposed that the formation of FANCA/G (dependent upon the phosphorylation of FANCA) and FANCB/L sub-complexes altogether with FANCM, represent the initial step for DNA repair activation and subsequent formation of other sub-complexes leading to ubiquitination of FANCD2 and FANCI. As only approximately 25% of inherited breast cancers are attributable to BRCA1/2 mutations, FANCA therefore becomes an attractive candidate for breast cancer susceptibility. We thus analyzed FANCA gene in 97 high-risk French Canadian non-BRCA1/2 breast cancer individuals by direct sequencing as well as in 95 healthy control individuals from the same population. Among a total of 85 sequence variants found in either or both series, 28 are coding variants and 19 of them are missense variations leading to amino acid change. Three of the amino acid changes, namely Thr561Met, Cys625Ser and particularly Ser1088Phe, which has been previously reported to be associated with FA, are predicted to be damaging by the SIFT and PolyPhen softwares. cDNA amplification revealed significant expression of 4 alternative splicing events (insertion of an intronic portion of intron 10, and the skipping of exons 11, 30 and 31). In silico analyzes of relevant genomic variants have been performed in order to identify potential variations involved in the expression of these spliced transcripts. Sequence variants in FANCA could therefore be potential spoilers of the Fanconi-BRCA pathway and as a result, they could in turn have an impact in non-BRCA1/2 breast cancer families. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Evaluation of targeted exome sequencing for 28 protein-based blood group systems, including the homologous gene systems, for blood group genotyping.

PubMed

Schoeman, Elizna M; Lopez, Genghis H; McGowan, Eunike C; Millard, Glenda M; O'Brien, Helen; Roulis, Eileen V; Liew, Yew-Wah; Martin, Jacqueline R; McGrath, Kelli A; Powley, Tanya; Flower, Robert L; Hyland, Catherine A

2017-04-01

Blood group single nucleotide polymorphism genotyping probes for a limited range of polymorphisms. This study investigated whether massively parallel sequencing (also known as next-generation sequencing), with a targeted exome strategy, provides an extended blood group genotype and the extent to which massively parallel sequencing correctly genotypes in homologous gene systems, such as RH and MNS. Donor samples (n = 28) that were extensively phenotyped and genotyped using single nucleotide polymorphism typing, were analyzed using the TruSight One Sequencing Panel and MiSeq platform. Genes for 28 protein-based blood group systems, GATA1, and KLF1 were analyzed. Copy number variation analysis was used to characterize complex structural variants in the GYPC and RH systems. The average sequencing depth per target region was 66.2 ± 39.8. Each sample harbored on average 43 ± 9 variants, of which 10 ± 3 were used for genotyping. For the 28 samples, massively parallel sequencing variant sequences correctly matched expected sequences based on single nucleotide polymorphism genotyping data. Copy number variation analysis defined the Rh C/c alleles and complex RHD hybrids. Hybrid RHD*D-CE-D variants were correctly identified, but copy number variation analysis did not confidently distinguish between D and CE exon deletion versus rearrangement. The targeted exome sequencing strategy employed extended the range of blood group genotypes detected compared with single nucleotide polymorphism typing. This single-test format included detection of complex MNS hybrid cases and, with copy number variation analysis, defined RH hybrid genes along with the RHCE*C allele hitherto difficult to resolve by variant detection. The approach is economical compared with whole-genome sequencing and is suitable for a red blood cell reference laboratory setting. © 2017 AABB.

Functional genetic selection of Helix 66 in Escherichia coli 23S rRNA identified the eukaryotic-binding sequence for ribosomal protein L2

PubMed Central

Kitahara, Kei; Kajiura, Akimasa; Sato, Neuza Satomi; Suzuki, Tsutomu

2007-01-01

Ribosomal protein L2 is a highly conserved primary 23S rRNA-binding protein. L2 specifically recognizes the internal bulge sequence in Helix 66 (H66) of 23S rRNA and is localized to the intersubunit space through formation of bridge B7b with 16S rRNA. The L2-binding site in H66 is highly conserved in prokaryotic ribosomes, whereas the corresponding site in eukaryotic ribosomes has evolved into distinct classes of sequences. We performed a systematic genetic selection of randomized rRNA sequences in Escherichia coli, and isolated 20 functional variants of the L2-binding site. The isolated variants consisted of eukaryotic sequences, in addition to prokaryotic sequences. These results suggest that L2/L8e does not recognize a specific base sequence of H66, but rather a characteristic architecture of H66. The growth phenotype of the isolated variants correlated well with their ability of subunit association. Upon continuous cultivation of a deleterious variant, we isolated two spontaneous mutations within domain IV of 23S rRNA that compensated for its weak subunit association, and alleviated its growth defect, implying that functional interactions between intersubunit bridges compensate ribosomal function. PMID:17553838

Position-specific automated processing of V3 env ultra-deep pyrosequencing data for predicting HIV-1 tropism

PubMed Central

Jeanne, Nicolas; Saliou, Adrien; Carcenac, Romain; Lefebvre, Caroline; Dubois, Martine; Cazabat, Michelle; Nicot, Florence; Loiseau, Claire; Raymond, Stéphanie; Izopet, Jacques; Delobel, Pierre

2015-01-01

HIV-1 coreceptor usage must be accurately determined before starting CCR5 antagonist-based treatment as the presence of undetected minor CXCR4-using variants can cause subsequent virological failure. Ultra-deep pyrosequencing of HIV-1 V3 env allows to detect low levels of CXCR4-using variants that current genotypic approaches miss. However, the computation of the mass of sequence data and the need to identify true minor variants while excluding artifactual sequences generated during amplification and ultra-deep pyrosequencing is rate-limiting. Arbitrary fixed cut-offs below which minor variants are discarded are currently used but the errors generated during ultra-deep pyrosequencing are sequence-dependant rather than random. We have developed an automated processing of HIV-1 V3 env ultra-deep pyrosequencing data that uses biological filters to discard artifactual or non-functional V3 sequences followed by statistical filters to determine position-specific sensitivity thresholds, rather than arbitrary fixed cut-offs. It allows to retain authentic sequences with point mutations at V3 positions of interest and discard artifactual ones with accurate sensitivity thresholds. PMID:26585833

Position-specific automated processing of V3 env ultra-deep pyrosequencing data for predicting HIV-1 tropism.

PubMed

Jeanne, Nicolas; Saliou, Adrien; Carcenac, Romain; Lefebvre, Caroline; Dubois, Martine; Cazabat, Michelle; Nicot, Florence; Loiseau, Claire; Raymond, Stéphanie; Izopet, Jacques; Delobel, Pierre

2015-11-20

HIV-1 coreceptor usage must be accurately determined before starting CCR5 antagonist-based treatment as the presence of undetected minor CXCR4-using variants can cause subsequent virological failure. Ultra-deep pyrosequencing of HIV-1 V3 env allows to detect low levels of CXCR4-using variants that current genotypic approaches miss. However, the computation of the mass of sequence data and the need to identify true minor variants while excluding artifactual sequences generated during amplification and ultra-deep pyrosequencing is rate-limiting. Arbitrary fixed cut-offs below which minor variants are discarded are currently used but the errors generated during ultra-deep pyrosequencing are sequence-dependant rather than random. We have developed an automated processing of HIV-1 V3 env ultra-deep pyrosequencing data that uses biological filters to discard artifactual or non-functional V3 sequences followed by statistical filters to determine position-specific sensitivity thresholds, rather than arbitrary fixed cut-offs. It allows to retain authentic sequences with point mutations at V3 positions of interest and discard artifactual ones with accurate sensitivity thresholds.

VirVarSeq: a low-frequency virus variant detection pipeline for Illumina sequencing using adaptive base-calling accuracy filtering.

PubMed

Verbist, Bie M P; Thys, Kim; Reumers, Joke; Wetzels, Yves; Van der Borght, Koen; Talloen, Willem; Aerssens, Jeroen; Clement, Lieven; Thas, Olivier

2015-01-01

In virology, massively parallel sequencing (MPS) opens many opportunities for studying viral quasi-species, e.g. in HIV-1- and HCV-infected patients. This is essential for understanding pathways to resistance, which can substantially improve treatment. Although MPS platforms allow in-depth characterization of sequence variation, their measurements still involve substantial technical noise. For Illumina sequencing, single base substitutions are the main error source and impede powerful assessment of low-frequency mutations. Fortunately, base calls are complemented with quality scores (Qs) that are useful for differentiating errors from the real low-frequency mutations. A variant calling tool, Q-cpileup, is proposed, which exploits the Qs of nucleotides in a filtering strategy to increase specificity. The tool is imbedded in an open-source pipeline, VirVarSeq, which allows variant calling starting from fastq files. Using both plasmid mixtures and clinical samples, we show that Q-cpileup is able to reduce the number of false-positive findings. The filtering strategy is adaptive and provides an optimized threshold for individual samples in each sequencing run. Additionally, linkage information is kept between single-nucleotide polymorphisms as variants are called at the codon level. This enables virologists to have an immediate biological interpretation of the reported variants with respect to their antiviral drug responses. A comparison with existing SNP caller tools reveals that calling variants at the codon level with Q-cpileup results in an outstanding sensitivity while maintaining a good specificity for variants with frequencies down to 0.5%. The VirVarSeq is available, together with a user's guide and test data, at sourceforge: http://sourceforge.net/projects/virtools/?source=directory. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Semiconductor Whole Exome Sequencing for the Identification of Genetic Variants in Colombian Patients Clinically Diagnosed with Long QT Syndrome.

PubMed

Burgos, Mariana; Arenas, Alvaro; Cabrera, Rodrigo

2016-08-01

Inherited long QT syndrome (LQTS) is a cardiac channelopathy characterized by a prolongation of QT interval and the risk of syncope, cardiac arrest, and sudden cardiac death. Genetic diagnosis of LQTS is critical in medical practice as results can guide adequate management of patients and distinguish phenocopies such as catecholaminergic polymorphic ventricular tachycardia (CPVT). However, extensive screening of large genomic regions is required in order to reliably identify genetic causes. Semiconductor whole exome sequencing (WES) is a promising approach for the identification of variants in the coding regions of most human genes. DNA samples from 21 Colombian patients clinically diagnosed with LQTS were enriched for coding regions using multiplex polymerase chain reaction (PCR) and subjected to WES using a semiconductor sequencer. Semiconductor WES showed mean coverage of 93.6 % for all coding regions relevant to LQTS at >10× depth with high intra- and inter-assay depth heterogeneity. Fifteen variants were detected in 12 patients in genes associated with LQTS. Three variants were identified in three patients in genes associated with CPVT. Co-segregation analysis was performed when possible. All variants were analyzed with two pathogenicity prediction algorithms. The overall prevalence of LQTS and CPVT variants in our cohort was 71.4 %. All LQTS variants previously identified through commercial genetic testing were identified. Standardized WES assays can be easily implemented, often at a lower cost than sequencing panels. Our results show that WES can identify LQTS-causing mutations and permits differential diagnosis of related conditions in a real-world clinical setting. However, high heterogeneity in sequencing depth and low coverage in the most relevant genes is expected to be associated with reduced analytical sensitivity.

Mutation analysis in 129 genes associated with other forms of retinal dystrophy in 157 families with retinitis pigmentosa based on exome sequencing.

PubMed

Xu, Yan; Guan, Liping; Xiao, Xueshan; Zhang, Jianguo; Li, Shiqiang; Jiang, Hui; Jia, Xiaoyun; Yang, Jianhua; Guo, Xiangming; Yin, Ye; Wang, Jun; Zhang, Qingjiong

2015-01-01

Mutations in 60 known genes were previously identified by exome sequencing in 79 of 157 families with retinitis pigmentosa (RP). This study analyzed variants in 129 genes associated with other forms of hereditary retinal dystrophy in the same cohort. Apart from the 73 genes previously analyzed, a further 129 genes responsible for other forms of hereditary retinal dystrophy were selected based on RetNet. Variants in the 129 genes determined by whole exome sequencing were selected and filtered by bioinformatics analysis. Candidate variants were confirmed by Sanger sequencing and validated by analysis of available family members and controls. A total of 90 candidate variants were present in the 129 genes. Sanger sequencing confirmed 83 of the 90 variants. Analysis of family members and controls excluded 76 of these 83 variants. The remaining seven variants were considered to be potential pathogenic mutations; these were c.899A>G, c.1814C>G, and c.2107C>T in BBS2; c.1073C>T and c.1669C>T in INPP5E; and c.3582C>G and c.5704-5C>G in CACNA1F. Six of these seven mutations were novel. The mutations were detected in five unrelated patients without a family history, including three patients with homozygous or compound heterozygous mutations in BBS2 and INPP5E, and two patients with hemizygous mutations in CACNA1F. None of the patients had mutations in the genes associated with autosome dominant retinal dystrophy. Only a small portion of patients with RP, about 3% (5/157), had causative mutations in the 129 genes associated with other forms of hereditary retinal dystrophy.

Analysis of Multiallelic CNVs by Emulsion Haplotype Fusion PCR.

PubMed

Tyson, Jess; Armour, John A L

2017-01-01

Emulsion-fusion PCR recovers long-range sequence information by combining products in cis from individual genomic DNA molecules. Emulsion droplets act as very numerous small reaction chambers in which different PCR products from a single genomic DNA molecule are condensed into short joint products, to unite sequences in cis from widely separated genomic sites. These products can therefore provide information about the arrangement of sequences and variants at a larger scale than established long-read sequencing methods. The method has been useful in defining the phase of variants in haplotypes, the typing of inversions, and determining the configuration of sequence variants in multiallelic CNVs. In this description we outline the rationale for the application of emulsion-fusion PCR methods to the analysis of multiallelic CNVs, and give practical details for our own implementation of the method in that context.

Screening of whole genome sequences identified high-impact variants for stallion fertility.

PubMed

Schrimpf, Rahel; Gottschalk, Maren; Metzger, Julia; Martinsson, Gunilla; Sieme, Harald; Distl, Ottmar

2016-04-14

Stallion fertility is an economically important trait due to the increase of artificial insemination in horses. The availability of whole genome sequence data facilitates identification of rare high-impact variants contributing to stallion fertility. The aim of our study was to genotype rare high-impact variants retrieved from next-generation sequencing (NGS)-data of 11 horses in order to unravel harmful genetic variants in large samples of stallions. Gene ontology (GO) terms and search results from public databases were used to obtain a comprehensive list of human und mice genes predicted to participate in the regulation of male reproduction. The corresponding equine orthologous genes were searched in whole genome sequence data of seven stallions and four mares and filtered for high-impact genetic variants using SnpEFF, SIFT and Polyphen 2 software. All genetic variants with the missing homozygous mutant genotype were genotyped on 337 fertile stallions of 19 breeds using KASP genotyping assays or PCR-RFLP. Mixed linear model analysis was employed for an association analysis with de-regressed estimated breeding values of the paternal component of the pregnancy rate per estrus (EBV-PAT). We screened next generation sequenced data of whole genomes from 11 horses for equine genetic variants in 1194 human and mice genes involved in male fertility and linked through common gene ontology (GO) with male reproductive processes. Variants were filtered for high-impact on protein structure and validated through SIFT and Polyphen 2. Only those genetic variants were followed up when the homozygote mutant genotype was missing in the detection sample comprising 11 horses. After this filtering process, 17 single nucleotide polymorphism (SNPs) were left. These SNPs were genotyped in 337 fertile stallions of 19 breeds using KASP genotyping assays or PCR-RFLP. An association analysis in 216 Hanoverian stallions revealed a significant association of the splice-site disruption variant g.37455302G>A in NOTCH1 with the de-regressed estimated breeding values of the paternal component of the pregnancy rate per estrus (EBV-PAT). For 9 high-impact variants within the genes CFTR, OVGP1, FBXO43, TSSK6, PKD1, FOXP1, TCP11, SPATA31E1 and NOTCH1 (g.37453246G>C) absence of the homozygous mutant genotype in the validation sample of all 337 fertile stallions was obvious. Therefore, these variants were considered as potentially deleterious factors for stallion fertility. In conclusion, this study revealed 17 genetic variants with a predicted high damaging effect on protein structure and missing homozygous mutant genotype. The g.37455302G>A NOTCH1 variant was identified as a significant stallion fertility locus in Hanoverian stallions and further 9 candidate fertility loci with missing homozygous mutant genotypes were validated in a panel including 19 horse breeds. To our knowledge this is the first study in horses using next generation sequencing data to uncover strong candidate factors for stallion fertility.

Mitochondrial Disease Sequence Data Resource (MSeqDR): a global grass-roots consortium to facilitate deposition, curation, annotation, and integrated analysis of genomic data for the mitochondrial disease clinical and research communities.

PubMed

Falk, Marni J; Shen, Lishuang; Gonzalez, Michael; Leipzig, Jeremy; Lott, Marie T; Stassen, Alphons P M; Diroma, Maria Angela; Navarro-Gomez, Daniel; Yeske, Philip; Bai, Renkui; Boles, Richard G; Brilhante, Virginia; Ralph, David; DaRe, Jeana T; Shelton, Robert; Terry, Sharon F; Zhang, Zhe; Copeland, William C; van Oven, Mannis; Prokisch, Holger; Wallace, Douglas C; Attimonelli, Marcella; Krotoski, Danuta; Zuchner, Stephan; Gai, Xiaowu

2015-03-01

Success rates for genomic analyses of highly heterogeneous disorders can be greatly improved if a large cohort of patient data is assembled to enhance collective capabilities for accurate sequence variant annotation, analysis, and interpretation. Indeed, molecular diagnostics requires the establishment of robust data resources to enable data sharing that informs accurate understanding of genes, variants, and phenotypes. The "Mitochondrial Disease Sequence Data Resource (MSeqDR) Consortium" is a grass-roots effort facilitated by the United Mitochondrial Disease Foundation to identify and prioritize specific genomic data analysis needs of the global mitochondrial disease clinical and research community. A central Web portal (https://mseqdr.org) facilitates the coherent compilation, organization, annotation, and analysis of sequence data from both nuclear and mitochondrial genomes of individuals and families with suspected mitochondrial disease. This Web portal provides users with a flexible and expandable suite of resources to enable variant-, gene-, and exome-level sequence analysis in a secure, Web-based, and user-friendly fashion. Users can also elect to share data with other MSeqDR Consortium members, or even the general public, either by custom annotation tracks or through the use of a convenient distributed annotation system (DAS) mechanism. A range of data visualization and analysis tools are provided to facilitate user interrogation and understanding of genomic, and ultimately phenotypic, data of relevance to mitochondrial biology and disease. Currently available tools for nuclear and mitochondrial gene analyses include an MSeqDR GBrowse instance that hosts optimized mitochondrial disease and mitochondrial DNA (mtDNA) specific annotation tracks, as well as an MSeqDR locus-specific database (LSDB) that curates variant data on more than 1300 genes that have been implicated in mitochondrial disease and/or encode mitochondria-localized proteins. MSeqDR is integrated with a diverse array of mtDNA data analysis tools that are both freestanding and incorporated into an online exome-level dataset curation and analysis resource (GEM.app) that is being optimized to support needs of the MSeqDR community. In addition, MSeqDR supports mitochondrial disease phenotyping and ontology tools, and provides variant pathogenicity assessment features that enable community review, feedback, and integration with the public ClinVar variant annotation resource. A centralized Web-based informed consent process is being developed, with implementation of a Global Unique Identifier (GUID) system to integrate data deposited on a given individual from different sources. Community-based data deposition into MSeqDR has already begun. Future efforts will enhance capabilities to incorporate phenotypic data that enhance genomic data analyses. MSeqDR will fill the existing void in bioinformatics tools and centralized knowledge that are necessary to enable efficient nuclear and mtDNA genomic data interpretation by a range of shareholders across both clinical diagnostic and research settings. Ultimately, MSeqDR is focused on empowering the global mitochondrial disease community to better define and explore mitochondrial diseases. Copyright © 2014 Elsevier Inc. All rights reserved.

Mitochondrial Disease Sequence Data Resource (MSeqDR): A global grass-roots consortium to facilitate deposition, curation, annotation, and integrated analysis of genomic data for the mitochondrial disease clinical and research communities

PubMed Central

Falk, Marni J.; Shen, Lishuang; Gonzalez, Michael; Leipzig, Jeremy; Lott, Marie T.; Stassen, Alphons P.M.; Diroma, Maria Angela; Navarro-Gomez, Daniel; Yeske, Philip; Bai, Renkui; Boles, Richard G.; Brilhante, Virginia; Ralph, David; DaRe, Jeana T.; Shelton, Robert; Terry, Sharon; Zhang, Zhe; Copeland, William C.; van Oven, Mannis; Prokisch, Holger; Wallace, Douglas C.; Attimonelli, Marcella; Krotoski, Danuta; Zuchner, Stephan; Gai, Xiaowu

2014-01-01

Success rates for genomic analyses of highly heterogeneous disorders can be greatly improved if a large cohort of patient data is assembled to enhance collective capabilities for accurate sequence variant annotation, analysis, and interpretation. Indeed, molecular diagnostics requires the establishment of robust data resources to enable data sharing that informs accurate understanding of genes, variants, and phenotypes. The “Mitochondrial Disease Sequence Data Resource (MSeqDR) Consortium” is a grass-roots effort facilitated by the United Mitochondrial Disease Foundation to identify and prioritize specific genomic data analysis needs of the global mitochondrial disease clinical and research community. A central Web portal (https://mseqdr.org) facilitates the coherent compilation, organization, annotation, and analysis of sequence data from both nuclear and mitochondrial genomes of individuals and families with suspected mitochondrial disease. This Web portal provides users with a flexible and expandable suite of resources to enable variant-, gene-, and exome-level sequence analysis in a secure, Web-based, and user-friendly fashion. Users can also elect to share data with other MSeqDR Consortium members, or even the general public, either by custom annotation tracks or through use of a convenient distributed annotation system (DAS) mechanism. A range of data visualization and analysis tools are provided to facilitate user interrogation and understanding of genomic, and ultimately phenotypic, data of relevance to mitochondrial biology and disease. Currently available tools for nuclear and mitochondrial gene analyses include an MSeqDR GBrowse instance that hosts optimized mitochondrial disease and mitochondrial DNA (mtDNA) specific annotation tracks, as well as an MSeqDR locus-specific database (LSDB) that curates variant data on more than 1,300 genes that have been implicated in mitochondrial disease and/or encode mitochondria-localized proteins. MSeqDR is integrated with a diverse array of mtDNA data analysis tools that are both freestanding and incorporated into an online exome-level dataset curation and analysis resource (GEM.app) that is being optimized to support needs of the MSeqDR community. In addition, MSeqDR supports mitochondrial disease phenotyping and ontology tools, and provides variant pathogenicity assessment features that enable community review, feedback, and integration with the public ClinVar variant annotation resource. A centralized Web-based informed consent process is being developed, with implementation of a Global Unique Identifier (GUID) system to integrate data deposited on a given individual from different sources. Community-based data deposition into MSeqDR has already begun. Future efforts will enhance capabilities to incorporate phenotypic data that enhance genomic data analyses. MSeqDR will fill the existing void in bioinformatics tools and centralized knowledge that are necessary to enable efficient nuclear and mtDNA genomic data interpretation by a range of shareholders across both clinical diagnostic and research settings. Ultimately, MSeqDR is focused on empowering the global mitochondrial disease community to better define and explore mitochondrial disease. PMID:25542617

Genome sequencing of idiopathic pulmonary fibrosis in conjunction with a medical school human anatomy course.

PubMed

Kumar, Akash; Dougherty, Max; Findlay, Gregory M; Geisheker, Madeleine; Klein, Jason; Lazar, John; Machkovech, Heather; Resnick, Jesse; Resnick, Rebecca; Salter, Alexander I; Talebi-Liasi, Faezeh; Arakawa, Christopher; Baudin, Jacob; Bogaard, Andrew; Salesky, Rebecca; Zhou, Qian; Smith, Kelly; Clark, John I; Shendure, Jay; Horwitz, Marshall S

2014-01-01

Even in cases where there is no obvious family history of disease, genome sequencing may contribute to clinical diagnosis and management. Clinical application of the genome has not yet become routine, however, in part because physicians are still learning how best to utilize such information. As an educational research exercise performed in conjunction with our medical school human anatomy course, we explored the potential utility of determining the whole genome sequence of a patient who had died following a clinical diagnosis of idiopathic pulmonary fibrosis (IPF). Medical students performed dissection and whole genome sequencing of the cadaver. Gross and microscopic findings were more consistent with the fibrosing variant of nonspecific interstitial pneumonia (NSIP), as opposed to IPF per se. Variants in genes causing Mendelian disorders predisposing to IPF were not detected. However, whole genome sequencing identified several common variants associated with IPF, including a single nucleotide polymorphism (SNP), rs35705950, located in the promoter region of the gene encoding mucin glycoprotein MUC5B. The MUC5B promoter polymorphism was recently found to markedly elevate risk for IPF, though a particular association with NSIP has not been previously reported, nor has its contribution to disease risk previously been evaluated in the genome-wide context of all genetic variants. We did not identify additional predicted functional variants in a region of linkage disequilibrium (LD) adjacent to MUC5B, nor did we discover other likely risk-contributing variants elsewhere in the genome. Whole genome sequencing thus corroborates the association of rs35705950 with MUC5B dysregulation and interstitial lung disease. This novel exercise additionally served a unique mission in bridging clinical and basic science education.

Sequence Variants and Haplotype Analysis of Cat ERBB2 Gene: A Survey on Spontaneous Cat Mammary Neoplastic and Non-Neoplastic Lesions

PubMed Central

Santos, Sara; Bastos, Estela; Baptista, Cláudia S.; Sá, Daniela; Caloustian, Christophe; Guedes-Pinto, Henrique; Gärtner, Fátima; Gut, Ivo G.; Chaves, Raquel

2012-01-01

The human ERBB2 proto-oncogene is widely considered a key gene involved in human breast cancer onset and progression. Among spontaneous tumors, mammary tumors are the most frequent cause of cancer death in cats and second most frequent in humans. In fact, naturally occurring tumors in domestic animals, more particularly cat mammary tumors, have been proposed as a good model for human breast cancer, but critical genetic and molecular information is still scarce. The aims of this study include the analysis of the cat ERBB2 gene partial sequences (between exon 17 and 20) in order to characterize a normal and a mammary lesion heterogeneous populations. Cat genomic DNA was extracted from normal frozen samples (n = 16) and from frozen and formalin-fixed paraffin-embedded mammary lesion samples (n = 41). We amplified and sequenced two cat ERBB2 DNA fragments comprising exons 17 to 20. It was possible to identify five sequence variants and six haplotypes in the total population. Two sequence variants and two haplotypes show to be specific for cat mammary tumor samples. Bioinformatics analysis predicts that four of the sequence variants can produce alternative transcripts or activate cryptic splicing sites. Also, a possible association was identified between clinicopathological traits and the variant haplotypes. As far as we know, this is the first attempt to examine ERBB2 genetic variations in cat mammary genome and its possible association with the onset and progression of cat mammary tumors. The demonstration of a possible association between primary tumor size (one of the two most important prognostic factors) and the number of masses with the cat ERBB2 variant haplotypes reveal the importance of the analysis of this gene in veterinary medicine. PMID:22489125

GTRAC: fast retrieval from compressed collections of genomic variants

PubMed Central

Tatwawadi, Kedar; Hernaez, Mikel; Ochoa, Idoia; Weissman, Tsachy

2016-01-01

Motivation: The dramatic decrease in the cost of sequencing has resulted in the generation of huge amounts of genomic data, as evidenced by projects such as the UK10K and the Million Veteran Project, with the number of sequenced genomes ranging in the order of 10 K to 1 M. Due to the large redundancies among genomic sequences of individuals from the same species, most of the medical research deals with the variants in the sequences as compared with a reference sequence, rather than with the complete genomic sequences. Consequently, millions of genomes represented as variants are stored in databases. These databases are constantly updated and queried to extract information such as the common variants among individuals or groups of individuals. Previous algorithms for compression of this type of databases lack efficient random access capabilities, rendering querying the database for particular variants and/or individuals extremely inefficient, to the point where compression is often relinquished altogether. Results: We present a new algorithm for this task, called GTRAC, that achieves significant compression ratios while allowing fast random access over the compressed database. For example, GTRAC is able to compress a Homo sapiens dataset containing 1092 samples in 1.1 GB (compression ratio of 160), while allowing for decompression of specific samples in less than a second and decompression of specific variants in 17 ms. GTRAC uses and adapts techniques from information theory, such as a specialized Lempel-Ziv compressor, and tailored succinct data structures. Availability and Implementation: The GTRAC algorithm is available for download at: https://github.com/kedartatwawadi/GTRAC Contact: kedart@stanford.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27587665

GTRAC: fast retrieval from compressed collections of genomic variants.

PubMed

Tatwawadi, Kedar; Hernaez, Mikel; Ochoa, Idoia; Weissman, Tsachy

2016-09-01

The dramatic decrease in the cost of sequencing has resulted in the generation of huge amounts of genomic data, as evidenced by projects such as the UK10K and the Million Veteran Project, with the number of sequenced genomes ranging in the order of 10 K to 1 M. Due to the large redundancies among genomic sequences of individuals from the same species, most of the medical research deals with the variants in the sequences as compared with a reference sequence, rather than with the complete genomic sequences. Consequently, millions of genomes represented as variants are stored in databases. These databases are constantly updated and queried to extract information such as the common variants among individuals or groups of individuals. Previous algorithms for compression of this type of databases lack efficient random access capabilities, rendering querying the database for particular variants and/or individuals extremely inefficient, to the point where compression is often relinquished altogether. We present a new algorithm for this task, called GTRAC, that achieves significant compression ratios while allowing fast random access over the compressed database. For example, GTRAC is able to compress a Homo sapiens dataset containing 1092 samples in 1.1 GB (compression ratio of 160), while allowing for decompression of specific samples in less than a second and decompression of specific variants in 17 ms. GTRAC uses and adapts techniques from information theory, such as a specialized Lempel-Ziv compressor, and tailored succinct data structures. The GTRAC algorithm is available for download at: https://github.com/kedartatwawadi/GTRAC CONTACT: : kedart@stanford.edu Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Integrating multiple genomic data to predict disease-causing nonsynonymous single nucleotide variants in exome sequencing studies.

PubMed

Wu, Jiaxin; Li, Yanda; Jiang, Rui

2014-03-01

Exome sequencing has been widely used in detecting pathogenic nonsynonymous single nucleotide variants (SNVs) for human inherited diseases. However, traditional statistical genetics methods are ineffective in analyzing exome sequencing data, due to such facts as the large number of sequenced variants, the presence of non-negligible fraction of pathogenic rare variants or de novo mutations, and the limited size of affected and normal populations. Indeed, prevalent applications of exome sequencing have been appealing for an effective computational method for identifying causative nonsynonymous SNVs from a large number of sequenced variants. Here, we propose a bioinformatics approach called SPRING (Snv PRioritization via the INtegration of Genomic data) for identifying pathogenic nonsynonymous SNVs for a given query disease. Based on six functional effect scores calculated by existing methods (SIFT, PolyPhen2, LRT, MutationTaster, GERP and PhyloP) and five association scores derived from a variety of genomic data sources (gene ontology, protein-protein interactions, protein sequences, protein domain annotations and gene pathway annotations), SPRING calculates the statistical significance that an SNV is causative for a query disease and hence provides a means of prioritizing candidate SNVs. With a series of comprehensive validation experiments, we demonstrate that SPRING is valid for diseases whose genetic bases are either partly known or completely unknown and effective for diseases with a variety of inheritance styles. In applications of our method to real exome sequencing data sets, we show the capability of SPRING in detecting causative de novo mutations for autism, epileptic encephalopathies and intellectual disability. We further provide an online service, the standalone software and genome-wide predictions of causative SNVs for 5,080 diseases at http://bioinfo.au.tsinghua.edu.cn/spring.

Exome Sequencing in Suspected Monogenic Dyslipidemias

PubMed Central

Stitziel, Nathan O.; Peloso, Gina M.; Abifadel, Marianne; Cefalu, Angelo B.; Fouchier, Sigrid; Motazacker, M. Mahdi; Tada, Hayato; Larach, Daniel B.; Awan, Zuhier; Haller, Jorge F.; Pullinger, Clive R.; Varret, Mathilde; Rabès, Jean-Pierre; Noto, Davide; Tarugi, Patrizia; Kawashiri, Masa-aki; Nohara, Atsushi; Yamagishi, Masakazu; Risman, Marjorie; Deo, Rahul; Ruel, Isabelle; Shendure, Jay; Nickerson, Deborah A.; Wilson, James G.; Rich, Stephen S.; Gupta, Namrata; Farlow, Deborah N.; Neale, Benjamin M.; Daly, Mark J.; Kane, John P.; Freeman, Mason W.; Genest, Jacques; Rader, Daniel J.; Mabuchi, Hiroshi; Kastelein, John J.P.; Hovingh, G. Kees; Averna, Maurizio R.; Gabriel, Stacey; Boileau, Catherine; Kathiresan, Sekar

2015-01-01

Background Exome sequencing is a promising tool for gene mapping in Mendelian disorders. We utilized this technique in an attempt to identify novel genes underlying monogenic dyslipidemias. Methods and Results We performed exome sequencing on 213 selected family members from 41 kindreds with suspected Mendelian inheritance of extreme levels of low-density lipoprotein (LDL) cholesterol (after candidate gene sequencing excluded known genetic causes for high LDL cholesterol families) or high-density lipoprotein (HDL) cholesterol. We used standard analytic approaches to identify candidate variants and also assigned a polygenic score to each individual in order to account for their burden of common genetic variants known to influence lipid levels. In nine families, we identified likely pathogenic variants in known lipid genes (ABCA1, APOB, APOE, LDLR, LIPA, and PCSK9); however, we were unable to identify obvious genetic etiologies in the remaining 32 families despite follow-up analyses. We identified three factors that limited novel gene discovery: (1) imperfect sequencing coverage across the exome hid potentially causal variants; (2) large numbers of shared rare alleles within families obfuscated causal variant identification; and (3) individuals from 15% of families carried a significant burden of common lipid-related alleles, suggesting complex inheritance can masquerade as monogenic disease. Conclusions We identified the genetic basis of disease in nine of 41 families; however, none of these represented novel gene discoveries. Our results highlight the promise and limitations of exome sequencing as a discovery technique in suspected monogenic dyslipidemias. Considering the confounders identified may inform the design of future exome sequencing studies. PMID:25632026

Exome sequencing for simultaneous mutation screening in children with hemophagocytic lymphohistiocytosis.

PubMed

Mukda, Ekchol; Trachoo, Objoon; Pasomsub, Ekawat; Tiyasirichokchai, Rawiphorn; Iemwimangsa, Nareenart; Sosothikul, Darintr; Chantratita, Wasun; Pakakasama, Samart

2017-08-01

In the present study, we used exome sequencing to analyze PRF1, UNC13D, STX11, and STXBP2, as well as genes associated with primary immunodeficiency disease (RAB27A, LYST, AP3B1, SH2D1A, ITK, CD27, XIAP, and MAGT1) in Thai children with hemophagocytic lymphohistiocytosis (HLH). We performed mutation analysis of HLH-associated genes in 25 Thai children using an exome sequencing method. Genetic variations found within these target genes were compared to exome sequencing data from 133 healthy individuals. Variants identified with minor allele frequencies <5% and novel mutations were confirmed using Sanger sequencing. Exome sequencing data revealed 101 non-synonymous single nucleotide polymorphisms (SNPs) in all subjects. These SNPs were classified as pathogenic (n = 1), likely pathogenic (n = 16), variant of unknown significance (n = 12), or benign variant (n = 72). Homozygous, compound heterozygous, and double-gene heterozygous variants, involving mutations in PRF1 (n = 3), UNC13D (n = 2), STXBP2 (n = 3), LYST (n = 3), XIAP (n = 2), AP3B1 (n = 1), RAB27A (n = 1), and MAGT1 (n = 1), were demonstrated in 12 patients. Novel mutations were found in most patients in this study. In conclusion, exome sequencing demonstrated the ability to identify rare genetic variants in HLH patients. This method is useful in the detection of mutations in multi-gene associated diseases.

Statistical method to compare massive parallel sequencing pipelines.

PubMed

Elsensohn, M H; Leblay, N; Dimassi, S; Campan-Fournier, A; Labalme, A; Roucher-Boulez, F; Sanlaville, D; Lesca, G; Bardel, C; Roy, P

2017-03-01

Today, sequencing is frequently carried out by Massive Parallel Sequencing (MPS) that cuts drastically sequencing time and expenses. Nevertheless, Sanger sequencing remains the main validation method to confirm the presence of variants. The analysis of MPS data involves the development of several bioinformatic tools, academic or commercial. We present here a statistical method to compare MPS pipelines and test it in a comparison between an academic (BWA-GATK) and a commercial pipeline (TMAP-NextGENe®), with and without reference to a gold standard (here, Sanger sequencing), on a panel of 41 genes in 43 epileptic patients. This method used the number of variants to fit log-linear models for pairwise agreements between pipelines. To assess the heterogeneity of the margins and the odds ratios of agreement, four log-linear models were used: a full model, a homogeneous-margin model, a model with single odds ratio for all patients, and a model with single intercept. Then a log-linear mixed model was fitted considering the biological variability as a random effect. Among the 390,339 base-pairs sequenced, TMAP-NextGENe® and BWA-GATK found, on average, 2253.49 and 1857.14 variants (single nucleotide variants and indels), respectively. Against the gold standard, the pipelines had similar sensitivities (63.47% vs. 63.42%) and close but significantly different specificities (99.57% vs. 99.65%; p < 0.001). Same-trend results were obtained when only single nucleotide variants were considered (99.98% specificity and 76.81% sensitivity for both pipelines). The method allows thus pipeline comparison and selection. It is generalizable to all types of MPS data and all pipelines.

Screening of the Filamin C Gene in a Large Cohort of Hypertrophic Cardiomyopathy Patients.

PubMed

Gómez, Juan; Lorca, Rebeca; Reguero, Julian R; Morís, César; Martín, María; Tranche, Salvador; Alonso, Belén; Iglesias, Sara; Alvarez, Victoria; Díaz-Molina, Beatriz; Avanzas, Pablo; Coto, Eliecer

2017-04-01

Recent exome sequencing studies identified filamin C ( FLNC ) as a candidate gene for hypertrophic cardiomyopathy (HCM). Our aim was to determine the rate of FLNC candidate variants in a large cohort of HCM patients who were also sequenced for the main sarcomere genes. A total of 448 HCM patients were next generation-sequenced (semiconductor chip technology) for the MYH7, MYBPC3 , TNNT2 , TNNI3 , ACTC1 , TNNC1 , MYL2 , MYL3 , TPM1 , and FLNC genes. We also sequenced 450 healthy controls from the same population. Based on the reported population frequencies, bioinformatic criteria, and familial segregation, we identified 20 FLNC candidate variants (13 new; 1 nonsense; and 19 missense) in 22 patients. Compared with the patients, only 1 of the control's missense variants was nonreported ( P =0.007; Fisher exact probability test). Based on the familial segregation and the reported functional studies, 6 of the candidate variants (in 7 patients) were finally classified as likely pathogenic, 10 as variants of uncertain significance, and 4 as likely benign. We provide a compelling evidence of the involvement of FLNC in the development of HCM. Most of the FLNC variants were associated with mild forms of HCM and a reduced penetrance, with few affected in the families to confirm the segregation. Our work, together with others who found FLNC variants among patients with dilated and restrictive cardiomyopathies, pointed to this gene as an important cause of structural cardiomyopathies. © 2017 American Heart Association, Inc.

Mass Spectrometric Determination of ILPR G-quadruplex Binding Sites in Insulin and IGF-2

PubMed Central

Xiao, JunFeng

2009-01-01

The insulin-linked polymorphic region (ILPR) of the human insulin gene promoter region forms G-quadruplex structures in vitro. Previous studies show that insulin and insulin-like growth factor-2 (IGF-2) exhibit high affinity binding in vitro to 2-repeat sequences of ILPR variants a and h, but negligible binding to variant i. Two-repeat sequences of variants a and h form intramolecular G-quadruplex structures that are not evidenced for variant i. Here we report on the use of protein digestion combined with affinity capture and MALDI-MS detection to pinpoint ILPR binding sites in insulin and IGF-2. Peptides captured by ILPR variants a and h were sequenced by MALDI-MS/MS, LC-MS and in silico digestion. On-bead digestion of insulin-ILPR variant a complexes supported the conclusions. The results indicate that the sequence VCG(N)RGF is generally present in the captured peptides and is likely involved in the affinity binding interactions of the proteins with the ILPR G-quadruplexes. The significance of arginine in the interactions was studied by comparing the affinities of synthesized peptides VCGERGF and VCGEAGF with ILPR variant a. Peptides from other regions of the proteins that are connected through disulfide linkages were also detected in some capture experiments. Identification of binding sites could facilitate design of DNA binding ligands for capture and detection of insulin and IGF-2. The interactions may have biological significance as well. PMID:19747845

Analysis of protein-altering variants in telomerase genes and their association with MUC5B common variant status in patients with idiopathic pulmonary fibrosis: a candidate gene sequencing study.

PubMed

Dressen, Amy; Abbas, Alexander R; Cabanski, Christopher; Reeder, Janina; Ramalingam, Thirumalai R; Neighbors, Margaret; Bhangale, Tushar R; Brauer, Matthew J; Hunkapiller, Julie; Reeder, Jens; Mukhyala, Kiran; Cuenco, Karen; Tom, Jennifer; Cowgill, Amy; Vogel, Jan; Forrest, William F; Collard, Harold R; Wolters, Paul J; Kropski, Jonathan A; Lancaster, Lisa H; Blackwell, Timothy S; Arron, Joseph R; Yaspan, Brian L

2018-06-08

Idiopathic pulmonary fibrosis (IPF) risk has a strong genetic component. Studies have implicated variations at several loci, including TERT, surfactant genes, and a single nucleotide polymorphism at chr11p15 (rs35705950) in the intergenic region between TOLLIP and MUC5B. Patients with IPF who have risk alleles at rs35705950 have longer survival from the time of IPF diagnosis than do patients homozygous for the non-risk allele, whereas patients with shorter telomeres have shorter survival times. We aimed to assess whether rare protein-altering variants in genes regulating telomere length are enriched in patients with IPF homozygous for the non-risk alleles at rs35705950. Between Nov 1, 2014, and Nov 1, 2016, we assessed blood samples from patients aged 40 years or older and of European ancestry with sporadic IPF from three international phase 3 clinical trials (INSPIRE, CAPACITY, ASCEND), one phase 2 study (RIFF), and US-based observational studies (Vanderbilt Clinical Interstitial Lung Disease Registry and the UCSF Interstitial Lung Disease Clinic registry cohorts) at the Broad Institute (Cambridge, MA, USA) and Human Longevity (San Diego, CA, USA). We also assessed blood samples from non-IPF controls in several clinical trials. We did whole-genome sequencing to assess telomere length and identify rare protein-altering variants, stratified by rs35705950 genotype. We also assessed rare functional variation in TERT exons and compared telomere length and disease progression across genotypes. We assessed samples from 1510 patients with IPF and 1874 non-IPF controls. 30 (3%) of 1046 patients with an rs35705950 risk allele had a rare protein-altering variant in TERT compared with 34 (7%) of 464 non-risk allele carriers (odds ratio 0·40 [95% CI 0·24-0·66], p=0·00039). Subsequent analyses identified enrichment of rare protein-altering variants in PARN and RTEL1, and rare variation in TERC in patients with IPF compared with controls. We expanded our study population to provide a more accurate estimation of rare variant frequency in these four loci, and to calculate telomere length. The proportion of patients with at least one rare variant in TERT, PARN, TERC, or RTEL1 was higher in patients with IPF than in controls (149 [9%] of 1739 patients vs 205 [2%] of 8645 controls, p=2·44 × 10 -8 ). Patients with IPF who had a variant in any of the four identified telomerase component genes had telomeres that were 3·69-16·10% shorter than patients without a variant in any of the four genes and had an earlier mean age of disease onset than patients without one or more variants (65·1 years [SD 7·8] vs 67·1 years [7·9], p=0·004). In the placebo arms of clinical trials, shorter telomeres were significantly associated with faster disease progression (1·7% predicted forced vital capacity per kb per year, p=0·002). Pirfenidone had treatment benefit regardless of telomere length (p=4·24 × 10 -8 for telomere length lower than the median, p=0·0044 for telomere length greater than the median). Rare protein-altering variants in TERT, PARN, TERC, and RTEL1 are enriched in patients with IPF compared with controls, and, in the case of TERT, particularly in individuals without a risk allele at the rs35705950 locus. This suggests that multiple genetic factors contribute to sporadic IPF, which might implicate distinct mechanisms of pathogenesis and disease progression. Genentech, National Institutes of Health, Francis Family Foundation, Pulmonary Fibrosis Foundation, Nina Ireland Program for Lung Health, US Department of Veterans Affairs. Copyright © 2018 Elsevier Ltd. All rights reserved.

Whole-Genome Sequences of Variants of Bacillus anthracis Sterne and Their Toxin Gene Deletion Mutants

PubMed Central

Staab, A.; Plaut, R. D.; Pratt, C.; Lovett, S. P.; Wiley, M. R.; Biggs, T. D.; Bernhards, R. C.; Beck, L. C.; Palacios, G. F.; Stibitz, S.; Jones, K. L.; Goodwin, B. G.; Smith, M. A.

2017-01-01

ABSTRACT Here, we report the draft genome sequences of three laboratory variants of Bacillus anthracis Sterne and their double (Δlef Δcya) and triple (Δpag Δlef Δcya) toxin gene deletion derivatives. PMID:29122874

The N-terminal sequence of albumin Redhill, a variant of human serum albumin.

PubMed

Hutchinson, D W; Matejtschuk, P

1985-12-02

Albumin Redhill, a variant human albumin, has been isolated by fast protein liquid chromatofocusing. The N-terminal sequence of this protein corresponded to that of albumin A except that one additional arginine residue was attached to the N-terminus.

Sanger Confirmation Is Required to Achieve Optimal Sensitivity and Specificity in Next-Generation Sequencing Panel Testing.

PubMed

Mu, Wenbo; Lu, Hsiao-Mei; Chen, Jefferey; Li, Shuwei; Elliott, Aaron M

2016-11-01

Next-generation sequencing (NGS) has rapidly replaced Sanger sequencing as the method of choice for diagnostic gene-panel testing. For hereditary-cancer testing, the technical sensitivity and specificity of the assay are paramount as clinicians use results to make important clinical management and treatment decisions. There is significant debate within the diagnostics community regarding the necessity of confirming NGS variant calls by Sanger sequencing, considering that numerous laboratories report having 100% specificity from the NGS data alone. Here we report our results from 20,000 hereditary-cancer NGS panels spanning 47 genes, in which all 7845 nonpolymorphic variants were Sanger- sequenced. Of these, 98.7% were concordant between NGS and Sanger sequencing and 1.3% were identified as NGS false-positives, located mainly in complex genomic regions (A/T-rich regions, G/C-rich regions, homopolymer stretches, and pseudogene regions). Simulating a false-positive rate of zero by adjusting the variant-calling quality-score thresholds decreased the sensitivity of the assay from 100% to 97.8%, resulting in the missed detection of 176 Sanger-confirmed variants, the majority in complex genomic regions (n = 114) and mosaic mutations (n = 7). The data illustrate the importance of setting quality thresholds for panel testing only after thousands of samples have been processed and the necessity of Sanger confirmation of NGS variants to maintain the highest possible sensitivity. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

OVAS: an open-source variant analysis suite with inheritance modelling.

PubMed

Mozere, Monika; Tekman, Mehmet; Kari, Jameela; Bockenhauer, Detlef; Kleta, Robert; Stanescu, Horia

2018-02-08

The advent of modern high-throughput genetics continually broadens the gap between the rising volume of sequencing data, and the tools required to process them. The need to pinpoint a small subset of functionally important variants has now shifted towards identifying the critical differences between normal variants and disease-causing ones. The ever-increasing reliance on cloud-based services for sequence analysis and the non-transparent methods they utilize has prompted the need for more in-situ services that can provide a safer and more accessible environment to process patient data, especially in circumstances where continuous internet usage is limited. To address these issues, we herein propose our standalone Open-source Variant Analysis Sequencing (OVAS) pipeline; consisting of three key stages of processing that pertain to the separate modes of annotation, filtering, and interpretation. Core annotation performs variant-mapping to gene-isoforms at the exon/intron level, append functional data pertaining the type of variant mutation, and determine hetero/homozygosity. An extensive inheritance-modelling module in conjunction with 11 other filtering components can be used in sequence ranging from single quality control to multi-file penetrance model specifics such as X-linked recessive or mosaicism. Depending on the type of interpretation required, additional annotation is performed to identify organ specificity through gene expression and protein domains. In the course of this paper we analysed an autosomal recessive case study. OVAS made effective use of the filtering modules to recapitulate the results of the study by identifying the prescribed compound-heterozygous disease pattern from exome-capture sequence input samples. OVAS is an offline open-source modular-driven analysis environment designed to annotate and extract useful variants from Variant Call Format (VCF) files, and process them under an inheritance context through a top-down filtering schema of swappable modules, run entirely off a live bootable medium and accessed locally through a web-browser.

Simultaneous detection of human mitochondrial DNA and nuclear-inserted mitochondrial-origin sequences (NumtS) using forensic mtDNA amplification strategies and pyrosequencing technology.

PubMed

Bintz, Brittania J; Dixon, Groves B; Wilson, Mark R

2014-07-01

Next-generation sequencing technologies enable the identification of minor mitochondrial DNA variants with higher sensitivity than Sanger methods, allowing for enhanced identification of minor variants. In this study, mixtures of human mtDNA control region amplicons were subjected to pyrosequencing to determine the detection threshold of the Roche GS Junior(®) instrument (Roche Applied Science, Indianapolis, IN). In addition to expected variants, a set of reproducible variants was consistently found in reads from one particular amplicon. A BLASTn search of the variant sequence revealed identity to a segment of a 611-bp nuclear insertion of the mitochondrial control region (NumtS) spanning the primer-binding sites of this amplicon (Nature 1995;378:489). Primers (Hum Genet 2012;131:757; Hum Biol 1996;68:847) flanking the insertion were used to confirm the presence or absence of the NumtS in buccal DNA extracts from twenty donors. These results further our understanding of human mtDNA variation and are expected to have a positive impact on the interpretation of mtDNA profiles using deep-sequencing methods in casework. © 2014 American Academy of Forensic Sciences.

Evolution of simeprevir-resistant variants over time by ultra-deep sequencing in HCV genotype 1b.

PubMed

Akuta, Norio; Suzuki, Fumitaka; Sezaki, Hitomi; Suzuki, Yoshiyuki; Hosaka, Tetsuya; Kobayashi, Masahiro; Kobayashi, Mariko; Saitoh, Satoshi; Ikeda, Kenji; Kumada, Hiromitsu

2014-08-01

Using ultra-deep sequencing technology, the present study was designed to investigate the evolution of simeprevir-resistant variants (amino acid substitutions of aa80, aa155, aa156, and aa168 positions in HCV NS3 region) over time. In Toranomon Hospital, 18 Japanese patients infected with HCV genotype 1b, received triple therapy of simeprevir/PEG-IFN/ribavirin (DRAGON or CONCERT study). Sustained virological response rate was 67%, and that was significantly higher in patients with IL28B rs8099917 TT than in those with non-TT. Six patients, who did not achieve sustained virological response, were tested for resistant variants by ultra-deep sequencing, at the baseline, at the time of re-elevation of viral loads, and at 96 weeks after the completion of treatment. Twelve of 18 resistant variants, detected at re-elevation of viral load, were de novo resistant variants. Ten of 12 de novo resistant variants become undetectable over time, and that five of seven resistant variants, detected at baseline, persisted over time. In one patient, variants of Q80R at baseline (0.3%) increased at 96-week after the cessation of treatment (10.2%), and de novo resistant variants of D168E (0.3%) also increased at 96-week after the cessation of treatment (9.7%). In conclusion, the present study indicates that the emergence of simeprevir-resistant variants after the start of treatment could not be predicted at baseline, and the majority of de novo resistant variants become undetectable over time. Further large-scale prospective studies should be performed to investigate the clinical utility in detecting simeprevir-resistant variants. © 2014 Wiley Periodicals, Inc.

Truncating variants in the majority of the cytoplasmic domain of PCDH15 are unlikely to cause Usher syndrome 1F.

PubMed

Perreault-Micale, Cynthia; Frieden, Alexander; Kennedy, Caleb J; Neitzel, Dana; Sullivan, Jessica; Faulkner, Nicole; Hallam, Stephanie; Greger, Valerie

2014-11-01

Loss of function variants in the PCDH15 gene can cause Usher syndrome type 1F, an autosomal recessive disease associated with profound congenital hearing loss, vestibular dysfunction, and retinitis pigmentosa. The Ashkenazi Jewish population has an increased incidence of Usher syndrome type 1F (founder variant p.Arg245X accounts for 75% of alleles), yet the variant spectrum in a panethnic population remains undetermined. We sequenced the coding region and intron-exon borders of PCDH15 using next-generation DNA sequencing technology in approximately 14,000 patients from fertility clinics. More than 600 unique PCDH15 variants (single nucleotide changes and small indels) were identified, including previously described pathogenic variants p.Arg3X, p.Arg245X (five patients), p.Arg643X, p.Arg929X, and p.Arg1106X. Novel truncating variants were also found, including one in the N-terminal extracellular domain (p.Leu877X), but all other novel truncating variants clustered in the exon 33 encoded C-terminal cytoplasmic domain (52 patients, 14 variants). One variant was observed predominantly in African Americans (carrier frequency of 2.3%). The high incidence of truncating exon 33 variants indicates that they are unlikely to cause Usher syndrome type 1F even though many remove a large portion of the gene. They may be tolerated because PCDH15 has several alternate cytoplasmic domain exons and differentially spliced isoforms may function redundantly. Effects of some PCDH15 truncating variants were addressed by deep sequencing of a panethnic population. Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Positional bias in variant calls against draft reference assemblies.

PubMed

Briskine, Roman V; Shimizu, Kentaro K

2017-03-28

Whole genome resequencing projects may implement variant calling using draft reference genomes assembled de novo from short-read libraries. Despite lower quality of such assemblies, they allowed researchers to extend a wide range of population genetic and genome-wide association analyses to non-model species. As the variant calling pipelines are complex and involve many software packages, it is important to understand inherent biases and limitations at each step of the analysis. In this article, we report a positional bias present in variant calling performed against draft reference assemblies constructed from de Bruijn or string overlap graphs. We assessed how frequently variants appeared at each position counted from ends of a contig or scaffold sequence, and discovered unexpectedly high number of variants at the positions related to the length of either k-mers or reads used for the assembly. We detected the bias in both publicly available draft assemblies from Assemblathon 2 competition as well as in the assemblies we generated from our simulated short-read data. Simulations confirmed that the bias causing variants are predominantly false positives induced by reads from spatially distant repeated sequences. The bias is particularly strong in contig assemblies. Scaffolding does not eliminate the bias but tends to mitigate it because of the changes in variants' relative positions and alterations in read alignments. The bias can be effectively reduced by filtering out the variants that reside in repetitive elements. Draft genome sequences generated by several popular assemblers appear to be susceptible to the positional bias potentially affecting many resequencing projects in non-model species. The bias is inherent to the assembly algorithms and arises from their particular handling of repeated sequences. It is recommended to reduce the bias by filtering especially if higher-quality genome assembly cannot be achieved. Our findings can help other researchers to improve the quality of their variant data sets and reduce artefactual findings in downstream analyses.

Genetic epidemiology of pharmacogenetic variants in South East Asian Malays using whole-genome sequences.

PubMed

Sivadas, A; Salleh, M Z; Teh, L K; Scaria, V

2017-10-01

Expanding the scope of pharmacogenomic research by including multiple global populations is integral to building robust evidence for its clinical translation. Deep whole-genome sequencing of diverse ethnic populations provides a unique opportunity to study rare and common pharmacogenomic markers that often vary in frequency across populations. In this study, we aim to build a diverse map of pharmacogenetic variants in South East Asian (SEA) Malay population using deep whole-genome sequences of 100 healthy SEA Malay individuals. We investigated the allelic diversity of potentially deleterious pharmacogenomic variants in SEA Malay population. Our analysis revealed 227 common and 466 rare potentially functional single nucleotide variants (SNVs) in 437 pharmacogenomic genes involved in drug metabolism, transport and target genes, including 74 novel variants. This study has created one of the most comprehensive maps of pharmacogenetic markers in any population from whole genomes and will hugely benefit pharmacogenomic investigations and drug dosage recommendations in SEA Malays.

Analysis of protein-coding genetic variation in 60,706 humans.

PubMed

Lek, Monkol; Karczewski, Konrad J; Minikel, Eric V; Samocha, Kaitlin E; Banks, Eric; Fennell, Timothy; O'Donnell-Luria, Anne H; Ware, James S; Hill, Andrew J; Cummings, Beryl B; Tukiainen, Taru; Birnbaum, Daniel P; Kosmicki, Jack A; Duncan, Laramie E; Estrada, Karol; Zhao, Fengmei; Zou, James; Pierce-Hoffman, Emma; Berghout, Joanne; Cooper, David N; Deflaux, Nicole; DePristo, Mark; Do, Ron; Flannick, Jason; Fromer, Menachem; Gauthier, Laura; Goldstein, Jackie; Gupta, Namrata; Howrigan, Daniel; Kiezun, Adam; Kurki, Mitja I; Moonshine, Ami Levy; Natarajan, Pradeep; Orozco, Lorena; Peloso, Gina M; Poplin, Ryan; Rivas, Manuel A; Ruano-Rubio, Valentin; Rose, Samuel A; Ruderfer, Douglas M; Shakir, Khalid; Stenson, Peter D; Stevens, Christine; Thomas, Brett P; Tiao, Grace; Tusie-Luna, Maria T; Weisburd, Ben; Won, Hong-Hee; Yu, Dongmei; Altshuler, David M; Ardissino, Diego; Boehnke, Michael; Danesh, John; Donnelly, Stacey; Elosua, Roberto; Florez, Jose C; Gabriel, Stacey B; Getz, Gad; Glatt, Stephen J; Hultman, Christina M; Kathiresan, Sekar; Laakso, Markku; McCarroll, Steven; McCarthy, Mark I; McGovern, Dermot; McPherson, Ruth; Neale, Benjamin M; Palotie, Aarno; Purcell, Shaun M; Saleheen, Danish; Scharf, Jeremiah M; Sklar, Pamela; Sullivan, Patrick F; Tuomilehto, Jaakko; Tsuang, Ming T; Watkins, Hugh C; Wilson, James G; Daly, Mark J; MacArthur, Daniel G

2016-08-18

Large-scale reference data sets of human genetic variation are critical for the medical and functional interpretation of DNA sequence changes. Here we describe the aggregation and analysis of high-quality exome (protein-coding region) DNA sequence data for 60,706 individuals of diverse ancestries generated as part of the Exome Aggregation Consortium (ExAC). This catalogue of human genetic diversity contains an average of one variant every eight bases of the exome, and provides direct evidence for the presence of widespread mutational recurrence. We have used this catalogue to calculate objective metrics of pathogenicity for sequence variants, and to identify genes subject to strong selection against various classes of mutation; identifying 3,230 genes with near-complete depletion of predicted protein-truncating variants, with 72% of these genes having no currently established human disease phenotype. Finally, we demonstrate that these data can be used for the efficient filtering of candidate disease-causing variants, and for the discovery of human 'knockout' variants in protein-coding genes.

Whole-Genome Sequencing and Variant Analysis of Human Papillomavirus 16 Infections.

PubMed

van der Weele, Pascal; Meijer, Chris J L M; King, Audrey J

2017-10-01

Human papillomavirus (HPV) is a strongly conserved DNA virus, high-risk types of which can cause cervical cancer in persistent infections. The most common type found in HPV-attributable cancer is HPV16, which can be subdivided into four lineages (A to D) with different carcinogenic properties. Studies have shown HPV16 sequence diversity in different geographical areas, but only limited information is available regarding HPV16 diversity within a population, especially at the whole-genome level. We analyzed HPV16 major variant diversity and conservation in persistent infections and performed a single nucleotide polymorphism (SNP) comparison between persistent and clearing infections. Materials were obtained in the Netherlands from a cohort study with longitudinal follow-up for up to 3 years. Our analysis shows a remarkably large variant diversity in the population. Whole-genome sequences were obtained for 57 persistent and 59 clearing HPV16 infections, resulting in 109 unique variants. Interestingly, persistent infections were completely conserved through time. One reinfection event was identified where the initial and follow-up samples clustered differently. Non-A1/A2 variants seemed to clear preferentially ( P = 0.02). Our analysis shows that population-wide HPV16 sequence diversity is very large. In persistent infections, the HPV16 sequence was fully conserved. Sequencing can identify HPV16 reinfections, although occurrence is rare. SNP comparison identified no strongly acting effect of the viral genome affecting HPV16 infection clearance or persistence in up to 3 years of follow-up. These findings suggest the progression of an early HPV16 infection could be host related. IMPORTANCE Human papillomavirus 16 (HPV16) is the predominant type found in cervical cancer. Progression of initial infection to cervical cancer has been linked to sequence properties; however, knowledge of variants circulating in European populations, especially with longitudinal follow-up, is limited. By sequencing a number of infections with known follow-up for up to 3 years, we gained initial insights into the genetic diversity of HPV16 and the effects of the viral genome on the persistence of infections. A SNP comparison between sequences obtained from clearing and persistent infections did not identify strongly acting DNA variations responsible for these infection outcomes. In addition, we identified an HPV16 reinfection event where sequencing of initial and follow-up samples showed different HPV16 variants. Based on conventional genotyping, this infection would incorrectly be considered a persistent HPV16 infection. In the context of vaccine efficacy and monitoring studies, such infections could potentially cause reduced reported efficacy or efficiency. Copyright © 2017 van der Weele et al.

Association analysis of bovine Foxa2 gene single sequence variant and haplotype combinations with growth traits in Chinese cattle.

PubMed

Liu, Mei; Li, Mijie; Wang, Shaoqiang; Xu, Yao; Lan, Xianyong; Li, Zhuanjian; Lei, Chuzhao; Yang, Dongying; Jia, Yutang; Chen, Hong

2014-02-25

Forkhead box A2 (Foxa2) has been recognized as one of the most potent transcriptional activators that is implicated in the control of feeding behavior and energy homeostasis. However, similar researches about the effects of genetic variations of Foxa2 gene on growth traits are lacking. Therefore, this study detected Foxa2 gene polymorphisms by DNA pool sequencing, PCR-RFLP and PCR-ACRS methods in 822 individuals from three Chinese cattle breeds. The results showed that four sequence variants (SVs) were screened, including two mutations (SV1, g. 7005 C>T and SV2, g. 7044 C>G) in intron 4, one mutation (SV3, g. 8449 A>G) in exon 5 and one mutation (SV4, g. 8537 T>C) in the 3'UTR. Notably, association analysis of the single mutations with growth traits in total individuals (at 24months) revealed that significant statistical difference was found in four SVs, and SV4 locus was highly significantly associated with growth traits throughout all three breeds (P<0.05 or P<0.01). Meanwhile, haplotype combination CCCCAGTC also indicated remarkably associated to better chest girth and body weight in Jiaxian Red cattle (P<0.05). We herein described a comprehensive study on the variability of bovine Foxa2 gene that was predictive of molecular markers in cattle breeding for the first time. Copyright © 2013 Elsevier B.V. All rights reserved.

The population genomics of rhesus macaques (Macaca mulatta) based on whole-genome sequences

PubMed Central

Xue, Cheng; Raveendran, Muthuswamy; Harris, R. Alan; Fawcett, Gloria L.; Liu, Xiaoming; White, Simon; Dahdouli, Mahmoud; Rio Deiros, David; Below, Jennifer E.; Salerno, William; Cox, Laura; Fan, Guoping; Ferguson, Betsy; Horvath, Julie; Johnson, Zach; Kanthaswamy, Sree; Kubisch, H. Michael; Liu, Dahai; Platt, Michael; Smith, David G.; Sun, Binghua; Vallender, Eric J.; Wang, Feng; Wiseman, Roger W.; Chen, Rui; Muzny, Donna M.; Gibbs, Richard A.; Yu, Fuli; Rogers, Jeffrey

2016-01-01

Rhesus macaques (Macaca mulatta) are the most widely used nonhuman primate in biomedical research, have the largest natural geographic distribution of any nonhuman primate, and have been the focus of much evolutionary and behavioral investigation. Consequently, rhesus macaques are one of the most thoroughly studied nonhuman primate species. However, little is known about genome-wide genetic variation in this species. A detailed understanding of extant genomic variation among rhesus macaques has implications for the use of this species as a model for studies of human health and disease, as well as for evolutionary population genomics. Whole-genome sequencing analysis of 133 rhesus macaques revealed more than 43.7 million single-nucleotide variants, including thousands predicted to alter protein sequences, transcript splicing, and transcription factor binding sites. Rhesus macaques exhibit 2.5-fold higher overall nucleotide diversity and slightly elevated putative functional variation compared with humans. This functional variation in macaques provides opportunities for analyses of coding and noncoding variation, and its cellular consequences. Despite modestly higher levels of nonsynonymous variation in the macaques, the estimated distribution of fitness effects and the ratio of nonsynonymous to synonymous variants suggest that purifying selection has had stronger effects in rhesus macaques than in humans. Demographic reconstructions indicate this species has experienced a consistently large but fluctuating population size. Overall, the results presented here provide new insights into the population genomics of nonhuman primates and expand genomic information directly relevant to primate models of human disease. PMID:27934697

Rare missense variants in POT1 predispose to familial cutaneous malignant melanoma

PubMed Central

Shi, Jianxin; Yang, Xiaohong R.; Ballew, Bari; Rotunno, Melissa; Calista, Donato; Fargnoli, Maria Concetta; Ghiorzo, Paola; Paillerets, Brigitte Bressac-de; Nagore, Eduardo; Avril, Marie Francoise; Caporaso, Neil E.; McMaster, Mary L.; Cullen, Michael; Wang, Zhaoming; Zhang, Xijun; Bruno, William; Pastorino, Lorenza; Queirolo, Paola; Banuls-Roca, Jose; Garcia-Casado, Zaida; Vaysse, Amaury; Mohamdi, Hamida; Riazalhosseini, Yasser; Foglio, Mario; Jouenne, Fanélie; Hua, Xing; Hyland, Paula L.; Yin, Jinhu; Vallabhaneni, Haritha; Chai, Weihang; Minghetti, Paola; Pellegrini, Cristina; Ravichandran, Sarangan; Eggermont, Alexander; Lathrop, Mark; Peris, Ketty; Scarra, Giovanna Bianchi; Landi, Giorgio; Savage, Sharon A.; Sampson, Joshua N.; He, Ji; Yeager, Meredith; Goldin, Lynn R.; Demenais, Florence; Chanock, Stephen J.; Tucker, Margaret A.; Goldstein, Alisa M.; Liu, Yie; Landi, Maria Teresa

2014-01-01

Although CDKN2A is the most frequent high-risk melanoma susceptibility gene, the underlying genetic factors for most melanoma-prone families remain unknown. Using whole exome sequencing, we identified a rare variant that arose as a founder mutation in the telomere shelterin POT1 gene (g.7:124493086 C>T, Ser270Asn) in five unrelated melanoma-prone families from Romagna, Italy. Carriers of this variant had increased telomere length and elevated fragile telomeres suggesting that this variant perturbs telomere maintenance. Two additional rare POT1 variants were identified in all cases sequenced in two other Italian families, yielding a frequency of POT1 variants comparable to that of CDKN2A mutations in this population. These variants were not found in public databases or in 2,038 genotyped Italian controls. We also identified two rare recurrent POT1 variants in American and French familial melanoma cases. Our findings suggest that POT1 is a major susceptibility gene for familial melanoma in several populations. PMID:24686846

Intersubtype Differences in the Effect of a Rare p24 Gag Mutation on HIV-1 Replicative Fitness

PubMed Central

Chopera, Denis R.; Cotton, Laura A.; Zawaira, Alexander; Mann, Jaclyn K.; Ngandu, Nobubelo K.; Ntale, Roman; Carlson, Jonathan M.; Mlisana, Koleka; Woodman, Zenda; de Assis Rosa, Debra; Martin, Eric; Miura, Toshiyuki; Pereyra, Florencia; Walker, Bruce D.; Gray, Clive M.; Martin, Darren P.; Ndung'u, Thumbi; Brockman, Mark A.; Karim, Salim Abdool

2012-01-01

Certain immune-driven mutations in HIV-1, such as those arising in p24Gag, decrease viral replicative capacity. However, the intersubtype differences in the replicative consequences of such mutations have not been explored. In HIV-1 subtype B, the p24Gag M250I mutation is a rare variant (0.6%) that is enriched among elite controllers (7.2%) (P = 0.0005) and appears to be a rare escape variant selected by HLA-B58 supertype alleles (P < 0.01). In contrast, in subtype C, it is a relatively common minor polymorphic variant (10 to 15%) whose appearance is not associated with a particular HLA allele. Using site-directed mutant viruses, we demonstrate that M250I reduces in vitro viral replicative capacity in both subtype B and subtype C sequences. However, whereas in subtype C downstream compensatory mutations at p24Gag codons 252 and 260 reduce the adverse effects of M250I, fitness costs in subtype B appear difficult to restore. Indeed, patient-derived subtype B sequences harboring M250I exhibited in vitro replicative defects, while those from subtype C did not. The structural implications of M250I were predicted by protein modeling to be greater in subtype B versus C, providing a potential explanation for its lower frequency and enhanced replicative defects in subtype B. In addition to accounting for genetic differences between HIV-1 subtypes, the design of cytotoxic-T-lymphocyte-based vaccines may need to account for differential effects of host-driven viral evolution on viral fitness. PMID:23015721

Genetic variants in post myocardial infarction patients presenting with electrical storm of unstable ventricular tachycardia.

PubMed

Rangaraju, Advithi; Krishnan, Shuba; Aparna, G; Sankaran, Satish; Mannan, Ashraf U; Rao, B Hygriv

2018-01-30

Electrical storm (ES) is a life threatening clinical situation. Though a few clinical pointers exist, the occurrence of ES in a patient with remote myocardial infarction (MI) is generally unpredictable. Genetic markers for this entity have not been studied. In the present study, we carried out genetic screening in patients with remote myocardial infarction presenting with ES by next generation sequencing and identified 25 rare variants in 19 genes predominantly in RYR2, SCN5A, KCNJ11, KCNE1 and KCNH2, CACNA1B, CACNA1C, CACNA1D and desmosomal genes - DSP and DSG2 that could potentially be implicated in electrical storm. These genes have been previously reported to be associated with inherited syndromes of Sudden Cardiac Death. The present study suggests that the genetic architecture in patients with remote MI and ES of unstable ventricular tachycardia may be similar to that of Ion channelopathies. Identification of these variants may identify post MI patients who are predisposed to develop electrical storm and help in risk stratification. Copyright © 2018 Indian Heart Rhythm Society. Production and hosting by Elsevier B.V. All rights reserved.

Genetic fine mapping and genomic annotation defines causal mechanisms at type 2 diabetes susceptibility loci.

PubMed

Gaulton, Kyle J; Ferreira, Teresa; Lee, Yeji; Raimondo, Anne; Mägi, Reedik; Reschen, Michael E; Mahajan, Anubha; Locke, Adam; Rayner, N William; Robertson, Neil; Scott, Robert A; Prokopenko, Inga; Scott, Laura J; Green, Todd; Sparso, Thomas; Thuillier, Dorothee; Yengo, Loic; Grallert, Harald; Wahl, Simone; Frånberg, Mattias; Strawbridge, Rona J; Kestler, Hans; Chheda, Himanshu; Eisele, Lewin; Gustafsson, Stefan; Steinthorsdottir, Valgerdur; Thorleifsson, Gudmar; Qi, Lu; Karssen, Lennart C; van Leeuwen, Elisabeth M; Willems, Sara M; Li, Man; Chen, Han; Fuchsberger, Christian; Kwan, Phoenix; Ma, Clement; Linderman, Michael; Lu, Yingchang; Thomsen, Soren K; Rundle, Jana K; Beer, Nicola L; van de Bunt, Martijn; Chalisey, Anil; Kang, Hyun Min; Voight, Benjamin F; Abecasis, Gonçalo R; Almgren, Peter; Baldassarre, Damiano; Balkau, Beverley; Benediktsson, Rafn; Blüher, Matthias; Boeing, Heiner; Bonnycastle, Lori L; Bottinger, Erwin P; Burtt, Noël P; Carey, Jason; Charpentier, Guillaume; Chines, Peter S; Cornelis, Marilyn C; Couper, David J; Crenshaw, Andrew T; van Dam, Rob M; Doney, Alex S F; Dorkhan, Mozhgan; Edkins, Sarah; Eriksson, Johan G; Esko, Tonu; Eury, Elodie; Fadista, João; Flannick, Jason; Fontanillas, Pierre; Fox, Caroline; Franks, Paul W; Gertow, Karl; Gieger, Christian; Gigante, Bruna; Gottesman, Omri; Grant, George B; Grarup, Niels; Groves, Christopher J; Hassinen, Maija; Have, Christian T; Herder, Christian; Holmen, Oddgeir L; Hreidarsson, Astradur B; Humphries, Steve E; Hunter, David J; Jackson, Anne U; Jonsson, Anna; Jørgensen, Marit E; Jørgensen, Torben; Kao, Wen-Hong L; Kerrison, Nicola D; Kinnunen, Leena; Klopp, Norman; Kong, Augustine; Kovacs, Peter; Kraft, Peter; Kravic, Jasmina; Langford, Cordelia; Leander, Karin; Liang, Liming; Lichtner, Peter; Lindgren, Cecilia M; Lindholm, Eero; Linneberg, Allan; Liu, Ching-Ti; Lobbens, Stéphane; Luan, Jian'an; Lyssenko, Valeriya; Männistö, Satu; McLeod, Olga; Meyer, Julia; Mihailov, Evelin; Mirza, Ghazala; Mühleisen, Thomas W; Müller-Nurasyid, Martina; Navarro, Carmen; Nöthen, Markus M; Oskolkov, Nikolay N; Owen, Katharine R; Palli, Domenico; Pechlivanis, Sonali; Peltonen, Leena; Perry, John R B; Platou, Carl G P; Roden, Michael; Ruderfer, Douglas; Rybin, Denis; van der Schouw, Yvonne T; Sennblad, Bengt; Sigurðsson, Gunnar; Stančáková, Alena; Steinbach, Gerald; Storm, Petter; Strauch, Konstantin; Stringham, Heather M; Sun, Qi; Thorand, Barbara; Tikkanen, Emmi; Tonjes, Anke; Trakalo, Joseph; Tremoli, Elena; Tuomi, Tiinamaija; Wennauer, Roman; Wiltshire, Steven; Wood, Andrew R; Zeggini, Eleftheria; Dunham, Ian; Birney, Ewan; Pasquali, Lorenzo; Ferrer, Jorge; Loos, Ruth J F; Dupuis, Josée; Florez, Jose C; Boerwinkle, Eric; Pankow, James S; van Duijn, Cornelia; Sijbrands, Eric; Meigs, James B; Hu, Frank B; Thorsteinsdottir, Unnur; Stefansson, Kari; Lakka, Timo A; Rauramaa, Rainer; Stumvoll, Michael; Pedersen, Nancy L; Lind, Lars; Keinanen-Kiukaanniemi, Sirkka M; Korpi-Hyövälti, Eeva; Saaristo, Timo E; Saltevo, Juha; Kuusisto, Johanna; Laakso, Markku; Metspalu, Andres; Erbel, Raimund; Jöcke, Karl-Heinz; Moebus, Susanne; Ripatti, Samuli; Salomaa, Veikko; Ingelsson, Erik; Boehm, Bernhard O; Bergman, Richard N; Collins, Francis S; Mohlke, Karen L; Koistinen, Heikki; Tuomilehto, Jaakko; Hveem, Kristian; Njølstad, Inger; Deloukas, Panagiotis; Donnelly, Peter J; Frayling, Timothy M; Hattersley, Andrew T; de Faire, Ulf; Hamsten, Anders; Illig, Thomas; Peters, Annette; Cauchi, Stephane; Sladek, Rob; Froguel, Philippe; Hansen, Torben; Pedersen, Oluf; Morris, Andrew D; Palmer, Collin N A; Kathiresan, Sekar; Melander, Olle; Nilsson, Peter M; Groop, Leif C; Barroso, Inês; Langenberg, Claudia; Wareham, Nicholas J; O'Callaghan, Christopher A; Gloyn, Anna L; Altshuler, David; Boehnke, Michael; Teslovich, Tanya M; McCarthy, Mark I; Morris, Andrew P

2015-12-01

We performed fine mapping of 39 established type 2 diabetes (T2D) loci in 27,206 cases and 57,574 controls of European ancestry. We identified 49 distinct association signals at these loci, including five mapping in or near KCNQ1. 'Credible sets' of the variants most likely to drive each distinct signal mapped predominantly to noncoding sequence, implying that association with T2D is mediated through gene regulation. Credible set variants were enriched for overlap with FOXA2 chromatin immunoprecipitation binding sites in human islet and liver cells, including at MTNR1B, where fine mapping implicated rs10830963 as driving T2D association. We confirmed that the T2D risk allele for this SNP increases FOXA2-bound enhancer activity in islet- and liver-derived cells. We observed allele-specific differences in NEUROD1 binding in islet-derived cells, consistent with evidence that the T2D risk allele increases islet MTNR1B expression. Our study demonstrates how integration of genetic and genomic information can define molecular mechanisms through which variants underlying association signals exert their effects on disease.

Hereditary spastic paraplegia type 43 (SPG43) is caused by mutation in C19orf12

PubMed Central

Landouré, Guida; Zhu, Peng-Peng; Lourenço, Charles M.; Johnson, Janel O.; Toro, Camilo; Bricceno, Katherine V.; Rinaldi, Carlo; Meilleur, Katherine G.; Sangaré, Modibo; Diallo, Oumarou; Pierson, Tyler M.; Ishiura, Hiroyuki; Tsuji, Shoji; Hein, Nichole; Fink, John K.; Stoll, Marion; Nicholson, Garth; Gonzalez, Michael; Speziani, Fiorella; Dürr, Alexandra; Stevanin, Giovanni; Biesecker, Leslie G.; Accardi, John; Landis, Dennis M. D.; Gahl, William A.; Traynor, Bryan J.; Marques, Wilson; Züchner, Stephan; Blackstone, Craig; Fischbeck, Kenneth H.; Burnett, Barrington G.

2013-01-01

We report here the genetic basis for a form of progressive hereditary spastic paraplegia (SPG43) previously described in two Malian sisters. Exome sequencing revealed a homozygous missense variant (c.187G>C; p.Ala63Pro) in C19orf12, a gene recently implicated in neurodegeneration with brain iron accumulation (NBIA). The same mutation was subsequently also found in a Brazilian family with features of NBIA, and we identified another NBIA patient with a three-nucleotide deletion (c.197_199del; p.Gly66del). Haplotype analysis revealed that the p.Ala63Pro mutations have a common origin, but MRI scans showed no brain iron deposition in the Malian SPG43 subjects. Heterologous expression of these SPG43 and NBIA variants resulted in similar alterations in the subcellular distribution of C19orf12. The SPG43 and NBIA variants reported here as well as the most common C19orf12 missense mutation reported in NBIA patients are found within a highly-conserved, extended hydrophobic domain in C19orf12, underscoring the functional importance of this domain. PMID:23857908

Frequency of genetic polymorphisms of PXR gene in the Brazilian population.

PubMed

Moreira, Ricardo P P; Jorge, Alexander A L; Mendonca, Berenice B; Bachega, Tânia A S S

2011-01-01

PXR polymorphisms have been implicated in modulating CYP3A4 and PXR expression, potentially accounting for interindividual differences in drug metabolism. The prevalence of PXR polymorphisms varies among ethnic groups and data on the allelic distribution in the highly mixed Brazilian population is lacking. The aim of this study was to analyze genetic variations in the PXR gene in Brazilians and to compare the results to other ethnic groups. DNA samples from 117 healthy Brazilians underwent PCR amplification and sequencing. Eleven polymorphisms were identified, 3 of which are highly associated with differences in CYP3A4 expression. We also identified 1 new synonymous variant in 1.3% of the alleles. Among the functional polymorphisms, -25913 C>T and -6994T>C occurred at a higher frequency comparedtothe Africanalleles (p < 0.05) but at a lower frequency compared to Caucasian alleles. The 8055 C>T allele was found at a similar frequency to those described in Caucasians and Africans (p > 0.05). We observed that functional variants of the PXR were frequent in our sample of the Brazilian population. Our results suggest that PXR gene variants may be of interest in pharmacogenetic studies involving Brazilians.

Determination of disease phenotypes and pathogenic variants from exome sequence data in the CAGI 4 gene panel challenge.

PubMed

Kundu, Kunal; Pal, Lipika R; Yin, Yizhou; Moult, John

2017-09-01

The use of gene panel sequence for diagnostic and prognostic testing is now widespread, but there are so far few objective tests of methods to interpret these data. We describe the design and implementation of a gene panel sequencing data analysis pipeline (VarP) and its assessment in a CAGI4 community experiment. The method was applied to clinical gene panel sequencing data of 106 patients, with the goal of determining which of 14 disease classes each patient has and the corresponding causative variant(s). The disease class was correctly identified for 36 cases, including 10 where the original clinical pipeline did not find causative variants. For a further seven cases, we found strong evidence of an alternative disease to that tested. Many of the potentially causative variants are missense, with no previous association with disease, and these proved the hardest to correctly assign pathogenicity or otherwise. Post analysis showed that three-dimensional structure data could have helped for up to half of these cases. Over-reliance on HGMD annotation led to a number of incorrect disease assignments. We used a largely ad hoc method to assign probabilities of pathogenicity for each variant, and there is much work still to be done in this area. © 2017 The Authors. **Human Mutation published by Wiley Periodicals, Inc.

Genomic diagnosis for children with intellectual disability and/or developmental delay.

PubMed

Bowling, Kevin M; Thompson, Michelle L; Amaral, Michelle D; Finnila, Candice R; Hiatt, Susan M; Engel, Krysta L; Cochran, J Nicholas; Brothers, Kyle B; East, Kelly M; Gray, David E; Kelley, Whitley V; Lamb, Neil E; Lose, Edward J; Rich, Carla A; Simmons, Shirley; Whittle, Jana S; Weaver, Benjamin T; Nesmith, Amy S; Myers, Richard M; Barsh, Gregory S; Bebin, E Martina; Cooper, Gregory M

2017-05-30

Developmental disabilities have diverse genetic causes that must be identified to facilitate precise diagnoses. We describe genomic data from 371 affected individuals, 309 of which were sequenced as proband-parent trios. Whole-exome sequences (WES) were generated for 365 individuals (127 affected) and whole-genome sequences (WGS) were generated for 612 individuals (244 affected). Pathogenic or likely pathogenic variants were found in 100 individuals (27%), with variants of uncertain significance in an additional 42 (11.3%). We found that a family history of neurological disease, especially the presence of an affected first-degree relative, reduces the pathogenic/likely pathogenic variant identification rate, reflecting both the disease relevance and ease of interpretation of de novo variants. We also found that improvements to genetic knowledge facilitated interpretation changes in many cases. Through systematic reanalyses, we have thus far reclassified 15 variants, with 11.3% of families who initially were found to harbor a VUS and 4.7% of families with a negative result eventually found to harbor a pathogenic or likely pathogenic variant. To further such progress, the data described here are being shared through ClinVar, GeneMatcher, and dbGaP. Our data strongly support the value of large-scale sequencing, especially WGS within proband-parent trios, as both an effective first-choice diagnostic tool and means to advance clinical and research progress related to pediatric neurological disease.

Gene and pathway level analyses of germline DNA-repair gene variants and prostate cancer susceptibility using the iCOGS-genotyping array.

PubMed

Saunders, Edward J; Dadaev, Tokhir; Leongamornlert, Daniel A; Al Olama, Ali Amin; Benlloch, Sara; Giles, Graham G; Wiklund, Fredrik; Gronberg, Henrik; Haiman, Christopher A; Schleutker, Johanna; Nordestgaard, Borge G; Travis, Ruth C; Neal, David; Pasayan, Nora; Khaw, Kay-Tee; Stanford, Janet L; Blot, William J; Thibodeau, Stephen N; Maier, Christiane; Kibel, Adam S; Cybulski, Cezary; Cannon-Albright, Lisa; Brenner, Hermann; Park, Jong Y; Kaneva, Radka; Batra, Jyotsna; Teixeira, Manuel R; Pandha, Hardev; Govindasami, Koveela; Muir, Ken; Easton, Douglas F; Eeles, Rosalind A; Kote-Jarai, Zsofia

2016-04-12

Germline mutations within DNA-repair genes are implicated in susceptibility to multiple forms of cancer. For prostate cancer (PrCa), rare mutations in BRCA2 and BRCA1 give rise to moderately elevated risk, whereas two of B100 common, low-penetrance PrCa susceptibility variants identified so far by genome-wide association studies implicate RAD51B and RAD23B. Genotype data from the iCOGS array were imputed to the 1000 genomes phase 3 reference panel for 21 780 PrCa cases and 21 727 controls from the Prostate Cancer Association Group to Investigate Cancer Associated Alterations in the Genome (PRACTICAL) consortium. We subsequently performed single variant, gene and pathway-level analyses using 81 303 SNPs within 20 Kb of a panel of 179 DNA-repair genes. Single SNP analyses identified only the previously reported association with RAD51B. Gene-level analyses using the SKAT-C test from the SNP-set (Sequence) Kernel Association Test (SKAT) identified a significant association with PrCa for MSH5. Pathway-level analyses suggested a possible role for the translesion synthesis pathway in PrCa risk and Homologous recombination/Fanconi Anaemia pathway for PrCa aggressiveness, even though after adjustment for multiple testing these did not remain significant. MSH5 is a novel candidate gene warranting additional follow-up as a prospective PrCa-risk locus. MSH5 has previously been reported as a pleiotropic susceptibility locus for lung, colorectal and serous ovarian cancers.

Exome-wide Association Study Identifies GREB1L Mutations in Congenital Kidney Malformations.

PubMed

Sanna-Cherchi, Simone; Khan, Kamal; Westland, Rik; Krithivasan, Priya; Fievet, Lorraine; Rasouly, Hila Milo; Ionita-Laza, Iuliana; Capone, Valentina P; Fasel, David A; Kiryluk, Krzysztof; Kamalakaran, Sitharthan; Bodria, Monica; Otto, Edgar A; Sampson, Matthew G; Gillies, Christopher E; Vega-Warner, Virginia; Vukojevic, Katarina; Pediaditakis, Igor; Makar, Gabriel S; Mitrotti, Adele; Verbitsky, Miguel; Martino, Jeremiah; Liu, Qingxue; Na, Young-Ji; Goj, Vinicio; Ardissino, Gianluigi; Gigante, Maddalena; Gesualdo, Loreto; Janezcko, Magdalena; Zaniew, Marcin; Mendelsohn, Cathy Lee; Shril, Shirlee; Hildebrandt, Friedhelm; van Wijk, Joanna A E; Arapovic, Adela; Saraga, Marijan; Allegri, Landino; Izzi, Claudia; Scolari, Francesco; Tasic, Velibor; Ghiggeri, Gian Marco; Latos-Bielenska, Anna; Materna-Kiryluk, Anna; Mane, Shrikant; Goldstein, David B; Lifton, Richard P; Katsanis, Nicholas; Davis, Erica E; Gharavi, Ali G

2017-11-02

Renal agenesis and hypodysplasia (RHD) are major causes of pediatric chronic kidney disease and are highly genetically heterogeneous. We conducted whole-exome sequencing in 202 case subjects with RHD and identified diagnostic mutations in genes known to be associated with RHD in 7/202 case subjects. In an additional affected individual with RHD and a congenital heart defect, we found a homozygous loss-of-function (LOF) variant in SLIT3, recapitulating phenotypes reported with Slit3 inactivation in the mouse. To identify genes associated with RHD, we performed an exome-wide association study with 195 unresolved case subjects and 6,905 control subjects. The top signal resided in GREB1L, a gene implicated previously in Hoxb1 and Shha signaling in zebrafish. The significance of the association, which was p = 2.0 × 10 -5 for novel LOF, increased to p = 4.1 × 10 -6 for LOF and deleterious missense variants combined, and augmented further after accounting for segregation and de novo inheritance of rare variants (joint p = 2.3 × 10 -7 ). Finally, CRISPR/Cas9 disruption or knockdown of greb1l in zebrafish caused specific pronephric defects, which were rescued by wild-type human GREB1L mRNA, but not mRNA containing alleles identified in case subjects. Together, our study provides insight into the genetic landscape of kidney malformations in humans, presents multiple candidates, and identifies SLIT3 and GREB1L as genes implicated in the pathogenesis of RHD. Copyright © 2017 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Bottlenecks and selective sweeps during domestication have increased deleterious genetic variation in dogs.

PubMed

Marsden, Clare D; Ortega-Del Vecchyo, Diego; O'Brien, Dennis P; Taylor, Jeremy F; Ramirez, Oscar; Vilà, Carles; Marques-Bonet, Tomas; Schnabel, Robert D; Wayne, Robert K; Lohmueller, Kirk E

2016-01-05

Population bottlenecks, inbreeding, and artificial selection can all, in principle, influence levels of deleterious genetic variation. However, the relative importance of each of these effects on genome-wide patterns of deleterious variation remains controversial. Domestic and wild canids offer a powerful system to address the role of these factors in influencing deleterious variation because their history is dominated by known bottlenecks and intense artificial selection. Here, we assess genome-wide patterns of deleterious variation in 90 whole-genome sequences from breed dogs, village dogs, and gray wolves. We find that the ratio of amino acid changing heterozygosity to silent heterozygosity is higher in dogs than in wolves and, on average, dogs have 2-3% higher genetic load than gray wolves. Multiple lines of evidence indicate this pattern is driven by less efficient natural selection due to bottlenecks associated with domestication and breed formation, rather than recent inbreeding. Further, we find regions of the genome implicated in selective sweeps are enriched for amino acid changing variants and Mendelian disease genes. To our knowledge, these results provide the first quantitative estimates of the increased burden of deleterious variants directly associated with domestication and have important implications for selective breeding programs and the conservation of rare and endangered species. Specifically, they highlight the costs associated with selective breeding and question the practice favoring the breeding of individuals that best fit breed standards. Our results also suggest that maintaining a large population size, rather than just avoiding inbreeding, is a critical factor for preventing the accumulation of deleterious variants.

Bottlenecks and selective sweeps during domestication have increased deleterious genetic variation in dogs

PubMed Central

Marsden, Clare D.; Ortega-Del Vecchyo, Diego; O’Brien, Dennis P.; Taylor, Jeremy F.; Ramirez, Oscar; Vilà, Carles; Marques-Bonet, Tomas; Schnabel, Robert D.; Wayne, Robert K.; Lohmueller, Kirk E.

2016-01-01

Population bottlenecks, inbreeding, and artificial selection can all, in principle, influence levels of deleterious genetic variation. However, the relative importance of each of these effects on genome-wide patterns of deleterious variation remains controversial. Domestic and wild canids offer a powerful system to address the role of these factors in influencing deleterious variation because their history is dominated by known bottlenecks and intense artificial selection. Here, we assess genome-wide patterns of deleterious variation in 90 whole-genome sequences from breed dogs, village dogs, and gray wolves. We find that the ratio of amino acid changing heterozygosity to silent heterozygosity is higher in dogs than in wolves and, on average, dogs have 2–3% higher genetic load than gray wolves. Multiple lines of evidence indicate this pattern is driven by less efficient natural selection due to bottlenecks associated with domestication and breed formation, rather than recent inbreeding. Further, we find regions of the genome implicated in selective sweeps are enriched for amino acid changing variants and Mendelian disease genes. To our knowledge, these results provide the first quantitative estimates of the increased burden of deleterious variants directly associated with domestication and have important implications for selective breeding programs and the conservation of rare and endangered species. Specifically, they highlight the costs associated with selective breeding and question the practice favoring the breeding of individuals that best fit breed standards. Our results also suggest that maintaining a large population size, rather than just avoiding inbreeding, is a critical factor for preventing the accumulation of deleterious variants. PMID:26699508

Single-Molecule Sequencing Reveals Complex Genome Variation of Hepatitis B Virus during 15 Years of Chronic Infection following Liver Transplantation

PubMed Central

Betz-Stablein, B. D.; Töpfer, A.; Littlejohn, M.; Yuen, L.; Colledge, D.; Sozzi, V.; Angus, P.; Thompson, A.; Revill, P.; Beerenwinkel, N.; Warner, N.

2016-01-01

ABSTRACT Chronic hepatitis B (CHB) is prevalent worldwide. The infectious agent, hepatitis B virus (HBV), replicates via an RNA intermediate and is error prone, leading to the rapid generation of closely related but not identical viral variants, including those that can escape host immune responses and antiviral treatments. The complexity of CHB can be further enhanced by the presence of HBV variants with large deletions in the genome generated via splicing (spHBV variants). Although spHBV variants are incapable of autonomous replication, their replication is rescued by wild-type HBV. spHBV variants have been shown to enhance wild-type virus replication, and their prevalence increases with liver disease progression. Single-molecule deep sequencing was performed on whole HBV genomes extracted from samples, including the liver explant, longitudinally collected from a subject with CHB over a 15-year period after liver transplantation. By employing novel bioinformatics methods, this analysis showed that the dynamics of the viral population across a period of changing treatment regimens was complex. The spHBV variants detected in the liver explant remained present posttransplantation, and a highly diverse novel spHBV population as well as variants with multiple deletions in the pre-S genes emerged. The identification of novel mutations outside the HBV reverse transcriptase gene that co-occurred with known drug resistance-associated mutations highlights the relevance of using full-genome deep sequencing and supports the hypothesis that drug resistance involves interactions across the full length of the HBV genome. IMPORTANCE Single-molecule sequencing allowed the characterization, in unprecedented detail, of the evolution of HBV populations and offered unique insights into the dynamics of defective and spHBV variants following liver transplantation and complex treatment regimens. This analysis also showed the rapid adaptation of HBV populations to treatment regimens with evolving drug resistance phenotypes and evidence of purifying selection across the whole genome. Finally, the new open-source bioinformatics tools with the capacity to easily identify potential spliced variants from deep sequencing data are freely available. PMID:27252524

Ranking viruses: measures of positional importance within networks define core viruses for rational polyvalent vaccine development.

PubMed

Anderson, Tavis K; Laegreid, William W; Cerutti, Francesco; Osorio, Fernando A; Nelson, Eric A; Christopher-Hennings, Jane; Goldberg, Tony L

2012-06-15

The extraordinary genetic and antigenic variability of RNA viruses is arguably the greatest challenge to the development of broadly effective vaccines. No single viral variant can induce sufficiently broad immunity, and incorporating all known naturally circulating variants into one multivalent vaccine is not feasible. Furthermore, no objective strategies currently exist to select actual viral variants that should be included or excluded in polyvalent vaccines. To address this problem, we demonstrate a method based on graph theory that quantifies the relative importance of viral variants. We demonstrate our method through application to the envelope glycoprotein gene of a particularly diverse RNA virus of pigs: porcine reproductive and respiratory syndrome virus (PRRSV). Using distance matrices derived from sequence nucleotide difference, amino acid difference and evolutionary distance, we constructed viral networks and used common network statistics to assign each sequence an objective ranking of relative 'importance'. To validate our approach, we use an independent published algorithm to score our top-ranked wild-type variants for coverage of putative T-cell epitopes across the 9383 sequences in our dataset. Top-ranked viruses achieve significantly higher coverage than low-ranked viruses, and top-ranked viruses achieve nearly equal coverage as a synthetic mosaic protein constructed in silico from the same set of 9383 sequences. Our approach relies on the network structure of PRRSV but applies to any diverse RNA virus because it identifies subsets of viral variants that are most important to overall viral diversity. We suggest that this method, through the objective quantification of variant importance, provides criteria for choosing viral variants for further characterization, diagnostics, surveillance and ultimately polyvalent vaccine development.

Whole Gene Capture Analysis of 15 CRC Susceptibility Genes in Suspected Lynch Syndrome Patients.

PubMed

Jansen, Anne M L; Geilenkirchen, Marije A; van Wezel, Tom; Jagmohan-Changur, Shantie C; Ruano, Dina; van der Klift, Heleen M; van den Akker, Brendy E W M; Laros, Jeroen F J; van Galen, Michiel; Wagner, Anja; Letteboer, Tom G W; Gómez-García, Encarna B; Tops, Carli M J; Vasen, Hans F; Devilee, Peter; Hes, Frederik J; Morreau, Hans; Wijnen, Juul T

2016-01-01

Lynch Syndrome (LS) is caused by pathogenic germline variants in one of the mismatch repair (MMR) genes. However, up to 60% of MMR-deficient colorectal cancer cases are categorized as suspected Lynch Syndrome (sLS) because no pathogenic MMR germline variant can be identified, which leads to difficulties in clinical management. We therefore analyzed the genomic regions of 15 CRC susceptibility genes in leukocyte DNA of 34 unrelated sLS patients and 11 patients with MLH1 hypermethylated tumors with a clear family history. Using targeted next-generation sequencing, we analyzed the entire non-repetitive genomic sequence, including intronic and regulatory sequences, of 15 CRC susceptibility genes. In addition, tumor DNA from 28 sLS patients was analyzed for somatic MMR variants. Of 1979 germline variants found in the leukocyte DNA of 34 sLS patients, one was a pathogenic variant (MLH1 c.1667+1delG). Leukocyte DNA of 11 patients with MLH1 hypermethylated tumors was negative for pathogenic germline variants in the tested CRC susceptibility genes and for germline MLH1 hypermethylation. Somatic DNA analysis of 28 sLS tumors identified eight (29%) cases with two pathogenic somatic variants, one with a VUS predicted to pathogenic and LOH, and nine cases (32%) with one pathogenic somatic variant (n = 8) or one VUS predicted to be pathogenic (n = 1). This is the first study in sLS patients to include the entire genomic sequence of CRC susceptibility genes. An underlying somatic or germline MMR gene defect was identified in ten of 34 sLS patients (29%). In the remaining sLS patients, the underlying genetic defect explaining the MMRdeficiency in their tumors might be found outside the genomic regions harboring the MMR and other known CRC susceptibility genes.

Computational evaluation of exome sequence data using human and model organism phenotypes improves diagnostic efficiency

PubMed Central

Bone, William P.; Washington, Nicole L.; Buske, Orion J.; Adams, David R.; Davis, Joie; Draper, David; Flynn, Elise D.; Girdea, Marta; Godfrey, Rena; Golas, Gretchen; Groden, Catherine; Jacobsen, Julius; Köhler, Sebastian; Lee, Elizabeth M. J.; Links, Amanda E.; Markello, Thomas C.; Mungall, Christopher J.; Nehrebecky, Michele; Robinson, Peter N.; Sincan, Murat; Soldatos, Ariane G.; Tifft, Cynthia J.; Toro, Camilo; Trang, Heather; Valkanas, Elise; Vasilevsky, Nicole; Wahl, Colleen; Wolfe, Lynne A.; Boerkoel, Cornelius F.; Brudno, Michael; Haendel, Melissa A.; Gahl, William A.; Smedley, Damian

2016-01-01

Purpose: Medical diagnosis and molecular or biochemical confirmation typically rely on the knowledge of the clinician. Although this is very difficult in extremely rare diseases, we hypothesized that the recording of patient phenotypes in Human Phenotype Ontology (HPO) terms and computationally ranking putative disease-associated sequence variants improves diagnosis, particularly for patients with atypical clinical profiles. Genet Med 18 6, 608–617. Methods: Using simulated exomes and the National Institutes of Health Undiagnosed Diseases Program (UDP) patient cohort and associated exome sequence, we tested our hypothesis using Exomiser. Exomiser ranks candidate variants based on patient phenotype similarity to (i) known disease–gene phenotypes, (ii) model organism phenotypes of candidate orthologs, and (iii) phenotypes of protein–protein association neighbors. Genet Med 18 6, 608–617. Results: Benchmarking showed Exomiser ranked the causal variant as the top hit in 97% of known disease–gene associations and ranked the correct seeded variant in up to 87% when detectable disease–gene associations were unavailable. Using UDP data, Exomiser ranked the causative variant(s) within the top 10 variants for 11 previously diagnosed variants and achieved a diagnosis for 4 of 23 cases undiagnosed by clinical evaluation. Genet Med 18 6, 608–617. Conclusion: Structured phenotyping of patients and computational analysis are effective adjuncts for diagnosing patients with genetic disorders. Genet Med 18 6, 608–617. PMID:26562225

Multi-species sequence comparison reveals conservation of ghrelin gene-derived splice variants encoding a truncated ghrelin peptide.

PubMed

Seim, Inge; Jeffery, Penny L; Thomas, Patrick B; Walpole, Carina M; Maugham, Michelle; Fung, Jenny N T; Yap, Pei-Yi; O'Keeffe, Angela J; Lai, John; Whiteside, Eliza J; Herington, Adrian C; Chopin, Lisa K

2016-06-01

The peptide hormone ghrelin is a potent orexigen produced predominantly in the stomach. It has a number of other biological actions, including roles in appetite stimulation, energy balance, the stimulation of growth hormone release and the regulation of cell proliferation. Recently, several ghrelin gene splice variants have been described. Here, we attempted to identify conserved alternative splicing of the ghrelin gene by cross-species sequence comparisons. We identified a novel human exon 2-deleted variant and provide preliminary evidence that this splice variant and in1-ghrelin encode a C-terminally truncated form of the ghrelin peptide, termed minighrelin. These variants are expressed in humans and mice, demonstrating conservation of alternative splicing spanning 90 million years. Minighrelin appears to have similar actions to full-length ghrelin, as treatment with exogenous minighrelin peptide stimulates appetite and feeding in mice. Forced expression of the exon 2-deleted preproghrelin variant mirrors the effect of the canonical preproghrelin, stimulating cell proliferation and migration in the PC3 prostate cancer cell line. This is the first study to characterise an exon 2-deleted preproghrelin variant and to demonstrate sequence conservation of ghrelin gene-derived splice variants that encode a truncated ghrelin peptide. This adds further impetus for studies into the alternative splicing of the ghrelin gene and the function of novel ghrelin peptides in vertebrates.

The Saccharomyces Genome Database Variant Viewer

PubMed Central

Sheppard, Travis K.; Hitz, Benjamin C.; Engel, Stacia R.; Song, Giltae; Balakrishnan, Rama; Binkley, Gail; Costanzo, Maria C.; Dalusag, Kyla S.; Demeter, Janos; Hellerstedt, Sage T.; Karra, Kalpana; Nash, Robert S.; Paskov, Kelley M.; Skrzypek, Marek S.; Weng, Shuai; Wong, Edith D.; Cherry, J. Michael

2016-01-01

The Saccharomyces Genome Database (SGD; http://www.yeastgenome.org) is the authoritative community resource for the Saccharomyces cerevisiae reference genome sequence and its annotation. In recent years, we have moved toward increased representation of sequence variation and allelic differences within S. cerevisiae. The publication of numerous additional genomes has motivated the creation of new tools for their annotation and analysis. Here we present the Variant Viewer: a dynamic open-source web application for the visualization of genomic and proteomic differences. Multiple sequence alignments have been constructed across high quality genome sequences from 11 different S. cerevisiae strains and stored in the SGD. The alignments and summaries are encoded in JSON and used to create a two-tiered dynamic view of the budding yeast pan-genome, available at http://www.yeastgenome.org/variant-viewer. PMID:26578556

Genetics of Combined Pituitary Hormone Deficiency: Roadmap into the Genome Era.

PubMed

Fang, Qing; George, Akima S; Brinkmeier, Michelle L; Mortensen, Amanda H; Gergics, Peter; Cheung, Leonard Y M; Daly, Alexandre Z; Ajmal, Adnan; Pérez Millán, María Ines; Ozel, A Bilge; Kitzman, Jacob O; Mills, Ryan E; Li, Jun Z; Camper, Sally A

2016-12-01

The genetic basis for combined pituitary hormone deficiency (CPHD) is complex, involving 30 genes in a variety of syndromic and nonsyndromic presentations. Molecular diagnosis of this disorder is valuable for predicting disease progression, avoiding unnecessary surgery, and family planning. We expect that the application of high throughput sequencing will uncover additional contributing genes and eventually become a valuable tool for molecular diagnosis. For example, in the last 3 years, six new genes have been implicated in CPHD using whole-exome sequencing. In this review, we present a historical perspective on gene discovery for CPHD and predict approaches that may facilitate future gene identification projects conducted by clinicians and basic scientists. Guidelines for systematic reporting of genetic variants and assigning causality are emerging. We apply these guidelines retrospectively to reports of the genetic basis of CPHD and summarize modes of inheritance and penetrance for each of the known genes. In recent years, there have been great improvements in databases of genetic information for diverse populations. Some issues remain that make molecular diagnosis challenging in some cases. These include the inherent genetic complexity of this disorder, technical challenges like uneven coverage, differing results from variant calling and interpretation pipelines, the number of tolerated genetic alterations, and imperfect methods for predicting pathogenicity. We discuss approaches for future research in the genetics of CPHD.

A targeted sequencing panel identifies rare damaging variants in multiple genes in the cranial neural tube defect, anencephaly

PubMed Central

Cullup, T.; Boustred, C.; James, C.; Docker, J.; English, C.; Lench, N.; Copp, A.J.; Moore, G.E.; Greene, N.D.E.; Stanier, P.

2018-01-01

Neural tube defects (NTDs) affecting the brain (anencephaly) are lethal before or at birth, whereas lower spinal defects (spina bifida) may lead to lifelong neurological handicap. Collectively, NTDs rank among the most common birth defects worldwide. This study focuses on anencephaly, which despite having a similar frequency to spina bifida and being the most common type of NTD observed in mouse models, has had more limited inclusion in genetic studies. A genetic influence is strongly implicated in determining risk of NTDs and a molecular diagnosis is of fundamental importance to families both in terms of understanding the origin of the condition and for managing future pregnancies. Here we used a custom panel of 191 NTD candidate genes to screen 90 patients with cranial NTDs (n = 85 anencephaly and n = 5 craniorachischisis) with a targeted exome sequencing platform. After filtering and comparing to our in‐house control exome database (N = 509), we identified 397 rare variants (minor allele frequency, MAF < 1%), 21 of which were previously unreported and predicted damaging. This included 1 frameshift (PDGFRA), 2 stop‐gained (MAT1A; NOS2) and 18 missense variations. Together with evidence for oligogenic inheritance, this study provides new information on the possible genetic causation of anencephaly. PMID:29205322

A novel FY*A allele with the 265T and 298A SNPs formerly associated exclusively with the FY*B allele and weak Fy(b) antigen expression: implication for genotyping interpretative algorithms.

PubMed

Lopez, G H; Condon, J A; Wilson, B; Martin, J R; Liew, Y-W; Flower, R L; Hyland, C A

2015-01-01

An Australian Caucasian blood donor consistently presented a serology profile for the Duffy blood group as Fy(a+b+) with Fy(a) antigen expression weaker than other examples of Fy(a+b+) red cells. Molecular typing studies were performed to investigate the reason for the observed serology profile. Blood group genotyping was performed using a commercial SNP microarray platform. Sanger sequencing was performed using primer sets to amplify across exons 1 and 2 of the FY gene and using allele-specific primers. The propositus was genotyped as FY*A/B, FY*X heterozygote that predicted the Fy(a+b+(w) ) phenotype. Sequencing identified the 265T and 298A variants on the FY*A allele. This link between FY*A allele and 265T was confirmed by allele-specific PCR. The reduced Fy(a) antigen reactivity is attributed to a FY*A allele-carrying 265T and 298A variants previously defined in combination only with the FY*B allele and associated with weak Fy(b) antigen expression. This novel allele should be considered in genotyping interpretative algorithms for generating a predicted phenotype. © 2014 International Society of Blood Transfusion.

Mutational screening of the USH2A gene in Spanish USH patients reveals 23 novel pathogenic mutations

PubMed Central

2011-01-01

Background Usher Syndrome type II (USH2) is an autosomal recessive disorder, characterized by moderate to severe hearing impairment and retinitis pigmentosa (RP). Among the three genes implicated, mutations in the USH2A gene account for 74-90% of the USH2 cases. Methods To identify the genetic cause of the disease and determine the frequency of USH2A mutations in a cohort of 88 unrelated USH Spanish patients, we carried out a mutation screening of the 72 coding exons of this gene by direct sequencing. Moreover, we performed functional minigene studies for those changes that were predicted to affect splicing. Results As a result, a total of 144 DNA sequence variants were identified. Based upon previous studies, allele frequencies, segregation analysis, bioinformatics' predictions and in vitro experiments, 37 variants (23 of them novel) were classified as pathogenic mutations. Conclusions This report provide a wide spectrum of USH2A mutations and clinical features, including atypical Usher syndrome phenotypes resembling Usher syndrome type I. Considering only the patients clearly diagnosed with Usher syndrome type II, and results obtained in this and previous studies, we can state that mutations in USH2A are responsible for 76.1% of USH2 disease in patients of Spanish origin. PMID:22004887

HapFABIA: Identification of very short segments of identity by descent characterized by rare variants in large sequencing data

PubMed Central

Hochreiter, Sepp

2013-01-01

Identity by descent (IBD) can be reliably detected for long shared DNA segments, which are found in related individuals. However, many studies contain cohorts of unrelated individuals that share only short IBD segments. New sequencing technologies facilitate identification of short IBD segments through rare variants, which convey more information on IBD than common variants. Current IBD detection methods, however, are not designed to use rare variants for the detection of short IBD segments. Short IBD segments reveal genetic structures at high resolution. Therefore, they can help to improve imputation and phasing, to increase genotyping accuracy for low-coverage sequencing and to increase the power of association studies. Since short IBD segments are further assumed to be old, they can shed light on the evolutionary history of humans. We propose HapFABIA, a computational method that applies biclustering to identify very short IBD segments characterized by rare variants. HapFABIA is designed to detect short IBD segments in genotype data that were obtained from next-generation sequencing, but can also be applied to DNA microarray data. Especially in next-generation sequencing data, HapFABIA exploits rare variants for IBD detection. HapFABIA significantly outperformed competing algorithms at detecting short IBD segments on artificial and simulated data with rare variants. HapFABIA identified 160 588 different short IBD segments characterized by rare variants with a median length of 23 kb (mean 24 kb) in data for chromosome 1 of the 1000 Genomes Project. These short IBD segments contain 752 000 single nucleotide variants (SNVs), which account for 39% of the rare variants and 23.5% of all variants. The vast majority—152 000 IBD segments—are shared by Africans, while only 19 000 and 11 000 are shared by Europeans and Asians, respectively. IBD segments that match the Denisova or the Neandertal genome are found significantly more often in Asians and Europeans but also, in some cases exclusively, in Africans. The lengths of IBD segments and their sharing between continental populations indicate that many short IBD segments from chromosome 1 existed before humans migrated out of Africa. Thus, rare variants that tag these short IBD segments predate human migration from Africa. The software package HapFABIA is available from Bioconductor. All data sets, result files and programs for data simulation, preprocessing and evaluation are supplied at http://www.bioinf.jku.at/research/short-IBD. PMID:24174545

Two Novel Variants Affecting CDKL5 Transcript Associated with Epileptic Encephalopathy.

PubMed

Neupauerová, Jana; Štěrbová, Katalin; Vlčková, Markéta; Sebroňová, Věra; Maříková, Tat'ána; Krůtová, Marcela; David, Staněk; Kršek, Pavel; Žaliová, Markéta; Seeman, Pavel; Laššuthová, Petra

2017-10-01

Variants in the human X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been reported as being etiologically associated with early infantile epileptic encephalopathy type 2 (EIEE2). We report on two patients, a boy and a girl, with EIEE2 that present with early onset epilepsy, hypotonia, severe intellectual disability, and poor eye contact. Massively parallel sequencing (MPS) of a custom-designed gene panel for epilepsy and epileptic encephalopathy containing 112 epilepsy-related genes was performed. Sanger sequencing was used to confirm the novel variants. For confirmation of the functional consequence of an intronic CDKL5 variant in patient 2, an RNA study was done. DNA sequencing revealed de novo variants in CDKL5, a c.2578C>T (p. Gln860*) present in a hemizygous state in a 3-year-old boy, and a potential splice site variant c.463+5G>A in heterozygous state in a 5-year-old girl. Multiple in silico splicing algorithms predicted a highly reduced splice site score for c.463+5G>A. A subsequent mRNA study confirmed an aberrant shorter transcript lacking exon 7. Our data confirmed that variants in the CDKL5 are associated with EIEE2. There is credible evidence that the novel identified variants are pathogenic and, therefore, are likely the cause of the disease in the presented patients. In one of the patients a stop codon variant is predicted to produce a truncated protein, and in the other patient an intronic variant results in aberrant splicing.

Rare Variant Association Test with Multiple Phenotypes

PubMed Central

Lee, Selyeong; Won, Sungho; Kim, Young Jin; Kim, Yongkang; Kim, Bong-Jo; Park, Taesung

2016-01-01

Although genome-wide association studies (GWAS) have now discovered thousands of genetic variants associated with common traits, such variants cannot explain the large degree of “missing heritability,” likely due to rare variants. The advent of next generation sequencing technology has allowed rare variant detection and association with common traits, often by investigating specific genomic regions for rare variant effects on a trait. Although multiply correlated phenotypes are often concurrently observed in GWAS, most studies analyze only single phenotypes, which may lessen statistical power. To increase power, multivariate analyses, which consider correlations between multiple phenotypes, can be used. However, few existing multi-variant analyses can identify rare variants for assessing multiple phenotypes. Here, we propose Multivariate Association Analysis using Score Statistics (MAAUSS), to identify rare variants associated with multiple phenotypes, based on the widely used Sequence Kernel Association Test (SKAT) for a single phenotype. We applied MAAUSS to Whole Exome Sequencing (WES) data from a Korean population of 1,058 subjects, to discover genes associated with multiple traits of liver function. We then assessed validation of those genes by a replication study, using an independent dataset of 3,445 individuals. Notably, we detected the gene ZNF620 among five significant genes. We then performed a simulation study to compare MAAUSS's performance with existing methods. Overall, MAAUSS successfully conserved type 1 error rates and in many cases, had a higher power than the existing methods. This study illustrates a feasible and straightforward approach for identifying rare variants correlated with multiple phenotypes, with likely relevance to missing heritability. PMID:28039885

Next-generation sequencing of the monogenic obesity genes LEP, LEPR, MC4R, PCSK1 and POMC in a Norwegian cohort of patients with morbid obesity and normal weight controls.

PubMed

Nordang, Gry B N; Busk, Øyvind L; Tveten, Kristian; Hanevik, Hans Ivar; Fell, Anne Kristin M; Hjelmesæth, Jøran; Holla, Øystein L; Hertel, Jens K

2017-05-01

Rare sequence variants in at least five genes are known to cause monogenic obesity. In this study we aimed to investigate the prevalence of, and characterize, rare coding and splice site variants in LEP, LEPR, MC4R, PCSK1 and POMC in patients with morbid obesity and normal weight controls. Targeted next-generation sequencing of all exons in LEP, LEPR, MC4R, PCSK1 and POMC was performed in 485 patients with morbid obesity and 327 normal weight population-based controls from Norway. In total 151 variants were detected. Twenty-eight (18.5%) of these were rare, coding or splice variants and five (3.3%) were novel. All individuals, except one control, were heterozygous for the 28 variants, and the distribution of the rare variants showed a significantly higher carrier frequency among cases than controls (9.9% vs. 4.9%, p=0.011). Four variants in MC4R were classified as pathogenic or likely pathogenic. Four cases (0.8%) of monogenic obesity were detected, all due to MC4R variants previously linked to monogenic obesity. Significant differences in carrier frequencies among patients with morbid obesity and normal weight controls suggest an association between heterozygous rare coding variants in these five genes and morbid obesity. However, additional studies in larger cohorts and functional testing of the novel variants identified are required to confirm the findings. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Inexpensive and Highly Reproducible Cloud-Based Variant Calling of 2,535 Human Genomes

PubMed Central

Shringarpure, Suyash S.; Carroll, Andrew; De La Vega, Francisco M.; Bustamante, Carlos D.

2015-01-01

Population scale sequencing of whole human genomes is becoming economically feasible; however, data management and analysis remains a formidable challenge for many research groups. Large sequencing studies, like the 1000 Genomes Project, have improved our understanding of human demography and the effect of rare genetic variation in disease. Variant calling on datasets of hundreds or thousands of genomes is time-consuming, expensive, and not easily reproducible given the myriad components of a variant calling pipeline. Here, we describe a cloud-based pipeline for joint variant calling in large samples using the Real Time Genomics population caller. We deployed the population caller on the Amazon cloud with the DNAnexus platform in order to achieve low-cost variant calling. Using our pipeline, we were able to identify 68.3 million variants in 2,535 samples from Phase 3 of the 1000 Genomes Project. By performing the variant calling in a parallel manner, the data was processed within 5 days at a compute cost of $7.33 per sample (a total cost of $18,590 for completed jobs and $21,805 for all jobs). Analysis of cost dependence and running time on the data size suggests that, given near linear scalability, cloud computing can be a cheap and efficient platform for analyzing even larger sequencing studies in the future. PMID:26110529

A massive parallel sequencing workflow for diagnostic genetic testing of mismatch repair genes

PubMed Central

Hansen, Maren F; Neckmann, Ulrike; Lavik, Liss A S; Vold, Trine; Gilde, Bodil; Toft, Ragnhild K; Sjursen, Wenche

2014-01-01

The purpose of this study was to develop a massive parallel sequencing (MPS) workflow for diagnostic analysis of mismatch repair (MMR) genes using the GS Junior system (Roche). A pathogenic variant in one of four MMR genes, (MLH1, PMS2, MSH6, and MSH2), is the cause of Lynch Syndrome (LS), which mainly predispose to colorectal cancer. We used an amplicon-based sequencing method allowing specific and preferential amplification of the MMR genes including PMS2, of which several pseudogenes exist. The amplicons were pooled at different ratios to obtain coverage uniformity and maximize the throughput of a single-GS Junior run. In total, 60 previously identified and distinct variants (substitutions and indels), were sequenced by MPS and successfully detected. The heterozygote detection range was from 19% to 63% and dependent on sequence context and coverage. We were able to distinguish between false-positive and true-positive calls in homopolymeric regions by cross-sample comparison and evaluation of flow signal distributions. In addition, we filtered variants according to a predefined status, which facilitated variant annotation. Our study shows that implementation of MPS in routine diagnostics of LS can accelerate sample throughput and reduce costs without compromising sensitivity, compared to Sanger sequencing. PMID:24689082

Diff-seq: A high throughput sequencing-based mismatch detection assay for DNA variant enrichment and discovery

PubMed Central

Karas, Vlad O; Sinnott-Armstrong, Nicholas A; Varghese, Vici; Shafer, Robert W; Greenleaf, William J; Sherlock, Gavin

2018-01-01

Abstract Much of the within species genetic variation is in the form of single nucleotide polymorphisms (SNPs), typically detected by whole genome sequencing (WGS) or microarray-based technologies. However, WGS produces mostly uninformative reads that perfectly match the reference, while microarrays require genome-specific reagents. We have developed Diff-seq, a sequencing-based mismatch detection assay for SNP discovery without the requirement for specialized nucleic-acid reagents. Diff-seq leverages the Surveyor endonuclease to cleave mismatched DNA molecules that are generated after cross-annealing of a complex pool of DNA fragments. Sequencing libraries enriched for Surveyor-cleaved molecules result in increased coverage at the variant sites. Diff-seq detected all mismatches present in an initial test substrate, with specific enrichment dependent on the identity and context of the variation. Application to viral sequences resulted in increased observation of variant alleles in a biologically relevant context. Diff-Seq has the potential to increase the sensitivity and efficiency of high-throughput sequencing in the detection of variation. PMID:29361139