Assessing copy number from exome sequencing and exome array CGH based on CNV spectrum in a large clinical cohort.

PubMed

Retterer, Kyle; Scuffins, Julie; Schmidt, Daniel; Lewis, Rachel; Pineda-Alvarez, Daniel; Stafford, Amanda; Schmidt, Lindsay; Warren, Stephanie; Gibellini, Federica; Kondakova, Anastasia; Blair, Amanda; Bale, Sherri; Matyakhina, Ludmila; Meck, Jeanne; Aradhya, Swaroop; Haverfield, Eden

2015-08-01

Detection of copy-number variation (CNV) is important for investigating many genetic disorders. Testing a large clinical cohort by array comparative genomic hybridization provides a deep perspective on the spectrum of pathogenic CNV. In this context, we describe a bioinformatics approach to extract CNV information from whole-exome sequencing and demonstrate its utility in clinical testing. Exon-focused arrays and whole-genome chromosomal microarray analysis were used to test 14,228 and 14,000 individuals, respectively. Based on these results, we developed an algorithm to detect deletions/duplications in whole-exome sequencing data and a novel whole-exome array. In the exon array cohort, we observed a positive detection rate of 2.4% (25 duplications, 318 deletions), of which 39% involved one or two exons. Chromosomal microarray analysis identified 3,345 CNVs affecting single genes (18%). We demonstrate that our whole-exome sequencing algorithm resolves CNVs of three or more exons. These results demonstrate the clinical utility of single-exon resolution in CNV assays. Our whole-exome sequencing algorithm approaches this resolution but is complemented by a whole-exome array to unambiguously identify intragenic CNVs and single-exon changes. These data illustrate the next advancements in CNV analysis through whole-exome sequencing and whole-exome array.Genet Med 17 8, 623-629.

Development of Genetic Markers in Eucalyptus Species by Target Enrichment and Exome Sequencing

PubMed Central

Dasgupta, Modhumita Ghosh; Dharanishanthi, Veeramuthu; Agarwal, Ishangi; Krutovsky, Konstantin V.

2015-01-01

The advent of next-generation sequencing has facilitated large-scale discovery, validation and assessment of genetic markers for high density genotyping. The present study was undertaken to identify markers in genes supposedly related to wood property traits in three Eucalyptus species. Ninety four genes involved in xylogenesis were selected for hybridization probe based nuclear genomic DNA target enrichment and exome sequencing. Genomic DNA was isolated from the leaf tissues and used for on-array probe hybridization followed by Illumina sequencing. The raw sequence reads were trimmed and high-quality reads were mapped to the E. grandis reference sequence and the presence of single nucleotide variants (SNVs) and insertions/ deletions (InDels) were identified across the three species. The average read coverage was 216X and a total of 2294 SNVs and 479 InDels were discovered in E. camaldulensis, 2383 SNVs and 518 InDels in E. tereticornis, and 1228 SNVs and 409 InDels in E. grandis. Additionally, SNV calling and InDel detection were conducted in pair-wise comparisons of E. tereticornis vs. E. grandis, E. camaldulensis vs. E. tereticornis and E. camaldulensis vs. E. grandis. This study presents an efficient and high throughput method on development of genetic markers for family– based QTL and association analysis in Eucalyptus. PMID:25602379

Probable Diagnosis of a Patient with Niemann-Pick Disease Type C: Managing Pitfalls of Exome Sequencing.

PubMed

Zeiger, William A; Jamal, Nasheed I; Scheuner, Maren T; Pittman, Patricia; Raymond, Kimiyo M; Morra, Massimo; Mishra, Shri K

2018-02-17

Here, we present a case of a 31-year-old man with progressive cognitive decline, ataxia, and dystonia. Extensive laboratory, radiographic, and targeted genetic studies over the course of several years failed to yield a diagnosis. Initial whole exome sequencing through a commercial laboratory identified several variants of uncertain significance; however, follow-up clinical examination and testing ruled each of these out. Eventually, repeat whole exome sequencing identified a known pathogenic intronic variant in the NPC1 gene (NM_000271.4, c.1554-1009G>A) and an additional heterozygous exonic variant of uncertain significance in the NPC1 gene (NM_000271.4, c.2524T>C). Follow-up biochemical testing was consistent with a diagnosis of probable Niemann-Pick disease Type C (NP-C). This case illustrates the potential of whole exome sequencing for diagnosing rare complex neurologic diseases. It also identifies several potential common pitfalls that must be navigated by clinicians when interpreting commercial whole exome sequencing results.

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.

Impact of exome sequencing in inflammatory bowel disease

PubMed Central

Cardinale, Christopher J; Kelsen, Judith R; Baldassano, Robert N; Hakonarson, Hakon

2013-01-01

Approaches to understanding the genetic contribution to inflammatory bowel disease (IBD) have continuously evolved from family- and population-based epidemiology, to linkage analysis, and most recently, to genome-wide association studies (GWAS). The next stage in this evolution seems to be the sequencing of the exome, that is, the regions of the human genome which encode proteins. The GWAS approach has been very fruitful in identifying at least 163 loci as being associated with IBD, and now, exome sequencing promises to take our genetic understanding to the next level. In this review we will discuss the possible contributions that can be made by an exome sequencing approach both at the individual patient level to aid with disease diagnosis and future therapies, as well as in advancing knowledge of the pathogenesis of IBD. PMID:24187447

Whole Exome Sequencing of Patients with Steroid-Resistant Nephrotic Syndrome.

PubMed

Warejko, Jillian K; Tan, Weizhen; Daga, Ankana; Schapiro, David; Lawson, Jennifer A; Shril, Shirlee; Lovric, Svjetlana; Ashraf, Shazia; Rao, Jia; Hermle, Tobias; Jobst-Schwan, Tilman; Widmeier, Eugen; Majmundar, Amar J; Schneider, Ronen; Gee, Heon Yung; Schmidt, J Magdalena; Vivante, Asaf; van der Ven, Amelie T; Ityel, Hadas; Chen, Jing; Sadowski, Carolin E; Kohl, Stefan; Pabst, Werner L; Nakayama, Makiko; Somers, Michael J G; Rodig, Nancy M; Daouk, Ghaleb; Baum, Michelle; Stein, Deborah R; Ferguson, Michael A; Traum, Avram Z; Soliman, Neveen A; Kari, Jameela A; El Desoky, Sherif; Fathy, Hanan; Zenker, Martin; Bakkaloglu, Sevcan A; Müller, Dominik; Noyan, Aytul; Ozaltin, Fatih; Cadnapaphornchai, Melissa A; Hashmi, Seema; Hopcian, Jeffrey; Kopp, Jeffrey B; Benador, Nadine; Bockenhauer, Detlef; Bogdanovic, Radovan; Stajić, Nataša; Chernin, Gil; Ettenger, Robert; Fehrenbach, Henry; Kemper, Markus; Munarriz, Reyner Loza; Podracka, Ludmila; Büscher, Rainer; Serdaroglu, Erkin; Tasic, Velibor; Mane, Shrikant; Lifton, Richard P; Braun, Daniela A; Hildebrandt, Friedhelm

2018-01-06

Steroid-resistant nephrotic syndrome overwhelmingly progresses to ESRD. More than 30 monogenic genes have been identified to cause steroid-resistant nephrotic syndrome. We previously detected causative mutations using targeted panel sequencing in 30% of patients with steroid-resistant nephrotic syndrome. Panel sequencing has a number of limitations when compared with whole exome sequencing. We employed whole exome sequencing to detect monogenic causes of steroid-resistant nephrotic syndrome in an international cohort of 300 families. Three hundred thirty-five individuals with steroid-resistant nephrotic syndrome from 300 families were recruited from April of 1998 to June of 2016. Age of onset was restricted to <25 years of age. Exome data were evaluated for 33 known monogenic steroid-resistant nephrotic syndrome genes. In 74 of 300 families (25%), we identified a causative mutation in one of 20 genes known to cause steroid-resistant nephrotic syndrome. In 11 families (3.7%), we detected a mutation in a gene that causes a phenocopy of steroid-resistant nephrotic syndrome. This is consistent with our previously published identification of mutations using a panel approach. We detected a causative mutation in a known steroid-resistant nephrotic syndrome gene in 38% of consanguineous families and in 13% of nonconsanguineous families, and 48% of children with congenital nephrotic syndrome. A total of 68 different mutations were detected in 20 of 33 steroid-resistant nephrotic syndrome genes. Fifteen of these mutations were novel. NPHS1 , PLCE1 , NPHS2 , and SMARCAL1 were the most common genes in which we detected a mutation. In another 28% of families, we detected mutations in one or more candidate genes for steroid-resistant nephrotic syndrome. Whole exome sequencing is a sensitive approach toward diagnosis of monogenic causes of steroid-resistant nephrotic syndrome. A molecular genetic diagnosis of steroid-resistant nephrotic syndrome may have important consequences

Exome sequencing for prenatal diagnosis of fetuses with sonographic abnormalities.

PubMed

Drury, Suzanne; Williams, Hywel; Trump, Natalie; Boustred, Christopher; Lench, Nicholas; Scott, Richard H; Chitty, Lyn S

2015-10-01

In the absence of aneuploidy or other pathogenic cytogenetic abnormality, fetuses with increased nuchal translucency (NT ≥ 3.5 mm) and/or other sonographic abnormalities have a greater incidence of genetic syndromes, but defining the underlying pathology can be challenging. Here, we investigate the value of whole exome sequencing in fetuses with sonographic abnormalities but normal microarray analysis. Whole exome sequencing was performed on DNA extracted from chorionic villi or amniocytes in 24 fetuses with unexplained ultrasound findings. In the first 14 cases sequencing was initially performed on fetal DNA only. For the remaining 10, the trio of fetus, mother and father was sequenced simultaneously. In 21% (5/24) cases, exome sequencing provided definitive diagnoses (Milroy disease, hypophosphatasia, achondrogenesis type 2, Freeman-Sheldon syndrome and Baraitser-Winter Syndrome). In a further case, a plausible diagnosis of orofaciodigital syndrome type 6 was made. In two others, a single mutation in an autosomal recessive gene was identified, but incomplete sequencing coverage precluded exclusion of the presence of a second mutation. Whole exome sequencing improves prenatal diagnosis in euploid fetuses with abnormal ultrasound scans. In order to expedite interpretation of results, trio sequencing should be employed, but interpretation can still be compromised by incomplete coverage of relevant genes. © 2015 John Wiley & Sons, Ltd.

Integrated sequencing of exome and mRNA of large-sized single cells.

PubMed

Wang, Lily Yan; Guo, Jiajie; Cao, Wei; Zhang, Meng; He, Jiankui; Li, Zhoufang

2018-01-10

Current approaches of single cell DNA-RNA integrated sequencing are difficult to call SNPs, because a large amount of DNA and RNA is lost during DNA-RNA separation. Here, we performed simultaneous single-cell exome and transcriptome sequencing on individual mouse oocytes. Using microinjection, we kept the nuclei intact to avoid DNA loss, while retaining the cytoplasm inside the cell membrane, to maximize the amount of DNA and RNA captured from the single cell. We then conducted exome-sequencing on the isolated nuclei and mRNA-sequencing on the enucleated cytoplasm. For single oocytes, exome-seq can cover up to 92% of exome region with an average sequencing depth of 10+, while mRNA-sequencing reveals more than 10,000 expressed genes in enucleated cytoplasm, with similar performance for intact oocytes. This approach provides unprecedented opportunities to study DNA-RNA regulation, such as RNA editing at single nucleotide level in oocytes. In future, this method can also be applied to other large cells, including neurons, large dendritic cells and large tumour cells for integrated exome and transcriptome sequencing.

Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets

DOE PAGES

Schulze, Kornelius; Imbeaud, Sandrine; Letouzé, Eric; ...

2015-03-30

Our genomic analyses promise to improve tumor characterization to optimize personalized treatment for patients with hepatocellular carcinoma (HCC). Exome sequencing analysis of 243 liver tumors identified mutational signatures associated with specific risk factors, mainly combined alcohol and tobacco consumption and exposure to aflatoxin B1. We identified 161 putative driver genes associated with 11 recurrently altered pathways. Associations of mutations defined 3 groups of genes related to risk factors and centered on CTNNB1 (alcohol), TP53 (hepatitis B virus, HBV) and AXIN1. These analyses according to tumor stage progression identified TERT promoter mutation as an early event, whereasFGF3, FGF4, FGF19 or CCND1more » amplification and TP53 and CDKN2A alterations appeared at more advanced stages in aggressive tumors. In 28% of the tumors, we identified genetic alterations potentially targetable by US Food and Drug Administration (FDA)–approved drugs. Finally, we identified risk factor–specific mutational signatures and defined the extensive landscape of altered genes and pathways in HCC, which will be useful to design clinical trials for targeted therapy.« less

Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets

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

Schulze, Kornelius; Imbeaud, Sandrine; Letouzé, Eric

Our genomic analyses promise to improve tumor characterization to optimize personalized treatment for patients with hepatocellular carcinoma (HCC). Exome sequencing analysis of 243 liver tumors identified mutational signatures associated with specific risk factors, mainly combined alcohol and tobacco consumption and exposure to aflatoxin B1. We identified 161 putative driver genes associated with 11 recurrently altered pathways. Associations of mutations defined 3 groups of genes related to risk factors and centered on CTNNB1 (alcohol), TP53 (hepatitis B virus, HBV) and AXIN1. These analyses according to tumor stage progression identified TERT promoter mutation as an early event, whereasFGF3, FGF4, FGF19 or CCND1more » amplification and TP53 and CDKN2A alterations appeared at more advanced stages in aggressive tumors. In 28% of the tumors, we identified genetic alterations potentially targetable by US Food and Drug Administration (FDA)–approved drugs. Finally, we identified risk factor–specific mutational signatures and defined the extensive landscape of altered genes and pathways in HCC, which will be useful to design clinical trials for targeted therapy.« less

SIMPLEX: Cloud-Enabled Pipeline for the Comprehensive Analysis of Exome Sequencing Data

PubMed Central

Fischer, Maria; Snajder, Rene; Pabinger, Stephan; Dander, Andreas; Schossig, Anna; Zschocke, Johannes; Trajanoski, Zlatko; Stocker, Gernot

2012-01-01

In recent studies, exome sequencing has proven to be a successful screening tool for the identification of candidate genes causing rare genetic diseases. Although underlying targeted sequencing methods are well established, necessary data handling and focused, structured analysis still remain demanding tasks. Here, we present a cloud-enabled autonomous analysis pipeline, which comprises the complete exome analysis workflow. The pipeline combines several in-house developed and published applications to perform the following steps: (a) initial quality control, (b) intelligent data filtering and pre-processing, (c) sequence alignment to a reference genome, (d) SNP and DIP detection, (e) functional annotation of variants using different approaches, and (f) detailed report generation during various stages of the workflow. The pipeline connects the selected analysis steps, exposes all available parameters for customized usage, performs required data handling, and distributes computationally expensive tasks either on a dedicated high-performance computing infrastructure or on the Amazon cloud environment (EC2). The presented application has already been used in several research projects including studies to elucidate the role of rare genetic diseases. The pipeline is continuously tested and is publicly available under the GPL as a VirtualBox or Cloud image at http://simplex.i-med.ac.at; additional supplementary data is provided at http://www.icbi.at/exome. PMID:22870267

Whole exome or genome sequencing: nurses need to prepare families for the possibilities.

PubMed

Prows, Cynthia A; Tran, Grace; Blosser, Beverly

2014-12-01

A discussion of whole exome sequencing and the type of possible results patients and families should be aware of before samples are obtained. To find the genetic cause of a rare disorder, whole exome sequencing analyses all known and suspected human genes from a single sample. Over 20,000 detected DNA variants in each individual exome must be considered as possibly causing disease or disregarded as not relevant to the person's disease. In the process, unexpected gene variants associated with known diseases unrelated to the primary purpose of the test may be incidentally discovered. Because family members' DNA samples are often needed, gene variants associated with known genetic diseases or predispositions for diseases can also be discovered in their samples. Discussion paper. PubMed 2009-2013, list of references in retrieved articles, Google Scholar. Nurses need a general understanding of the scope of potential genomic information that may be revealed with whole exome sequencing to provide support and guidance to individuals and families during their decision-making process, while waiting for results and after disclosure. Nurse scientists who want to use whole exome sequencing in their study design and methods must decide early in study development if they will return primary whole exome sequencing research results and if they will give research participants choices about learning incidental research results. It is critical that nurses translate their knowledge about whole exome sequencing into their patient education and patient advocacy roles and relevant programmes of research. © 2014 John Wiley & Sons Ltd.

Somatic mutation profiles of clear cell endometrial tumors revealed by whole exome and targeted gene sequencing.

PubMed

Le Gallo, Matthieu; Rudd, Meghan L; Urick, Mary Ellen; Hansen, Nancy F; Zhang, Suiyuan; Lozy, Fred; Sgroi, Dennis C; Vidal Bel, August; Matias-Guiu, Xavier; Broaddus, Russell R; Lu, Karen H; Levine, Douglas A; Mutch, David G; Goodfellow, Paul J; Salvesen, Helga B; Mullikin, James C; Bell, Daphne W

2017-09-01

The molecular pathogenesis of clear cell endometrial cancer (CCEC), a tumor type with a relatively unfavorable prognosis, is not well defined. We searched exome-wide for novel somatically mutated genes in CCEC and assessed the mutational spectrum of known and candidate driver genes in a large cohort of cases. We conducted whole exome sequencing of paired tumor-normal DNAs from 16 cases of CCEC (12 CCECs and the CCEC components of 4 mixed histology tumors). Twenty-two genes-of-interest were Sanger-sequenced from another 47 cases of CCEC. Microsatellite instability (MSI) and microsatellite stability (MSS) were determined by genotyping 5 mononucleotide repeats. Two tumor exomes had relatively high mutational loads and MSI. The other 14 tumor exomes were MSS and had 236 validated nonsynonymous or splice junction somatic mutations among 222 protein-encoding genes. Among the 63 cases of CCEC in this study, we identified frequent somatic mutations in TP53 (39.7%), PIK3CA (23.8%), PIK3R1 (15.9%), ARID1A (15.9%), PPP2R1A (15.9%), SPOP (14.3%), and TAF1 (9.5%), as well as MSI (11.3%). Five of 8 mutations in TAF1, a gene with no known role in CCEC, localized to the putative histone acetyltransferase domain and included 2 recurrently mutated residues. Based on patterns of MSI and mutations in 7 genes, CCEC subsets molecularly resembled serous endometrial cancer (SEC) or endometrioid endometrial cancer (EEC). Our findings demonstrate molecular similarities between CCEC and SEC and EEC and implicate TAF1 as a novel candidate CCEC driver gene. Cancer 2017;123:3261-8. © 2017 American Cancer Society. © 2017 American Cancer Society.

Efficacy of Exome-Targeted Capture Sequencing to Detect Mutations in Known Cerebellar Ataxia Genes.

PubMed

Coutelier, Marie; Hammer, Monia B; Stevanin, Giovanni; Monin, Marie-Lorraine; Davoine, Claire-Sophie; Mochel, Fanny; Labauge, Pierre; Ewenczyk, Claire; Ding, Jinhui; Gibbs, J Raphael; Hannequin, Didier; Melki, Judith; Toutain, Annick; Laugel, Vincent; Forlani, Sylvie; Charles, Perrine; Broussolle, Emmanuel; Thobois, Stéphane; Afenjar, Alexandra; Anheim, Mathieu; Calvas, Patrick; Castelnovo, Giovanni; de Broucker, Thomas; Vidailhet, Marie; Moulignier, Antoine; Ghnassia, Robert T; Tallaksen, Chantal; Mignot, Cyril; Goizet, Cyril; Le Ber, Isabelle; Ollagnon-Roman, Elisabeth; Pouget, Jean; Brice, Alexis; Singleton, Andrew; Durr, Alexandra

2018-05-01

Molecular diagnosis is difficult to achieve in disease groups with a highly heterogeneous genetic background, such as cerebellar ataxia (CA). In many patients, candidate gene sequencing or focused resequencing arrays do not allow investigators to reach a genetic conclusion. To assess the efficacy of exome-targeted capture sequencing to detect mutations in genes broadly linked to CA in a large cohort of undiagnosed patients and to investigate their prevalence. Three hundred nineteen index patients with CA and without a history of dominant transmission were included in the this cohort study by the Spastic Paraplegia and Ataxia Network. Centralized storage was in the DNA and cell bank of the Brain and Spine Institute, Salpetriere Hospital, Paris, France. Patients were classified into 6 clinical groups, with the largest being those with spastic ataxia (ie, CA with pyramidal signs [n = 100]). Sequencing was performed from January 1, 2014, through December 31, 2016. Detected variants were classified as very probably or definitely causative, possibly causative, or of unknown significance based on genetic evidence and genotype-phenotype considerations. Identification of variants in genes broadly linked to CA, classified in pathogenicity groups. The 319 included patients had equal sex distribution (160 female [50.2%] and 159 male patients [49.8%]; mean [SD] age at onset, 27.9 [18.6] years). The age at onset was younger than 25 years for 131 of 298 patients (44.0%) with complete clinical information. Consanguinity was present in 101 of 298 (33.9%). Very probable or definite diagnoses were achieved for 72 patients (22.6%), with an additional 19 (6.0%) harboring possibly pathogenic variants. The most frequently mutated genes were SPG7 (n = 14), SACS (n = 8), SETX (n = 7), SYNE1 (n = 6), and CACNA1A (n = 6). The highest diagnostic rate was obtained for patients with an autosomal recessive CA with oculomotor apraxia-like phenotype (6 of 17 [35.3%]) or

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.

Illustrative case studies in the return of exome and genome sequencing results

PubMed Central

Amendola, Laura M; Lautenbach, Denise; Scollon, Sarah; Bernhardt, Barbara; Biswas, Sawona; East, Kelly; Everett, Jessica; Gilmore, Marian J; Himes, Patricia; Raymond, Victoria M; Wynn, Julia; Hart, Ragan; Jarvik, Gail P

2015-01-01

Whole genome and exome sequencing tests are increasingly being ordered in clinical practice, creating a need for research exploring the return of results from these tests. A goal of the Clinical Sequencing and Exploratory Research (CSER) consortium is to gain experience with this process to develop best practice recommendations for offering exome and genome testing and returning results. Genetic counselors in the CSER consortium have an integral role in the return of results from these genomic sequencing tests and have gained valuable insight. We present seven emerging themes related to return of exome and genome sequencing results accompanied by case descriptions illustrating important lessons learned, counseling challenges specific to these tests and considerations for future research and practice. PMID:26478737

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...

Molecular Findings Among Patients Referred for Clinical Whole-Exome Sequencing

PubMed Central

Yang, Yaping; Muzny, Donna M.; Xia, Fan; Niu, Zhiyv; Person, Richard; Ding, Yan; Ward, Patricia; Braxton, Alicia; Wang, Min; Buhay, Christian; Veeraraghavan, Narayanan; Hawes, Alicia; Chiang, Theodore; Leduc, Magalie; Beuten, Joke; Zhang, Jing; He, Weimin; Scull, Jennifer; Willis, Alecia; Landsverk, Megan; Craigen, William J.; Bekheirnia, Mir Reza; Stray-Pedersen, Asbjorg; Liu, Pengfei; Wen, Shu; Alcaraz, Wendy; Cui, Hong; Walkiewicz, Magdalena; Reid, Jeffrey; Bainbridge, Matthew; Patel, Ankita; Boerwinkle, Eric; Beaudet, Arthur L.; Lupski, James R.; Plon, Sharon E.; Gibbs, Richard A.; Eng, Christine M.

2015-01-01

IMPORTANCE Clinical whole-exome sequencing is increasingly used for diagnostic evaluation of patients with suspected genetic disorders. OBJECTIVE To perform clinical whole-exome sequencing and report (1) the rate of molecular diagnosis among phenotypic groups, (2) the spectrum of genetic alterations contributing to disease, and (3) the prevalence of medically actionable incidental findings such as FBN1 mutations causing Marfan syndrome. DESIGN, SETTING, AND PATIENTS Observational study of 2000 consecutive patients with clinical whole-exome sequencing analyzed between June 2012 and August 2014. Whole-exome sequencing tests were performed at a clinical genetics laboratory in the United States. Results were reported by clinical molecular geneticists certified by the American Board of Medical Genetics and Genomics. Tests were ordered by the patient’s physician. The patients were primarily pediatric (1756 [88%]; mean age, 6 years; 888 females [44%], 1101 males [55%], and 11 fetuses [1% gender unknown]), demonstrating diverse clinical manifestations most often including nervous system dysfunction such as developmental delay. MAIN OUTCOMES AND MEASURES Whole-exome sequencing diagnosis rate overall and by phenotypic category, mode of inheritance, spectrum of genetic events, and reporting of incidental findings. RESULTS A molecular diagnosis was reported for 504 patients (25.2%) with 58% of the diagnostic mutations not previously reported. Molecular diagnosis rates for each phenotypic category were 143/526 (27.2%; 95% CI, 23.5%–31.2%) for the neurological group, 282/1147 (24.6%; 95% CI, 22.1%–27.2%) for the neurological plus other organ systems group, 30/83 (36.1%; 95% CI, 26.1%–47.5%) for the specific neurological group, and 49/244 (20.1%; 95% CI, 15.6%–25.8%) for the nonneurological group. The Mendelian disease patterns of the 527 molecular diagnoses included 280 (53.1%) autosomal dominant, 181 (34.3%) autosomal recessive (including 5 with uniparental disomy

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

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.

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.

Exome Sequence Analysis of 14 Families With High Myopia.

PubMed

Kloss, Bethany A; Tompson, Stuart W; Whisenhunt, Kristina N; Quow, Krystina L; Huang, Samuel J; Pavelec, Derek M; Rosenberg, Thomas; Young, Terri L

2017-04-01

To identify causal gene mutations in 14 families with autosomal dominant (AD) high myopia using exome sequencing. Select individuals from 14 large Caucasian families with high myopia were exome sequenced. Gene variants were filtered to identify potential pathogenic changes. Sanger sequencing was used to confirm variants in original DNA, and to test for disease cosegregation in additional family members. Candidate genes and chromosomal loci previously associated with myopic refractive error and its endophenotypes were comprehensively screened. In 14 high myopia families, we identified 73 rare and 31 novel gene variants as candidates for pathogenicity. In seven of these families, two of the novel and eight of the rare variants were within known myopia loci. A total of 104 heterozygous nonsynonymous rare variants in 104 genes were identified in 10 out of 14 probands. Each variant cosegregated with affection status. No rare variants were identified in genes known to cause myopia or in genes closest to published genome-wide association study association signals for refractive error or its endophenotypes. Whole exome sequencing was performed to determine gene variants implicated in the pathogenesis of AD high myopia. This study provides new genes for consideration in the pathogenesis of high myopia, and may aid in the development of genetic profiling of those at greatest risk for attendant ocular morbidities of this disorder.

Whole-Exome Sequencing Study of Thyrotropin-Secreting Pituitary Adenomas.

PubMed

Sapkota, Santosh; Horiguchi, Kazuhiko; Tosaka, Masahiko; Yamada, Syozo; Yamada, Masanobu

2017-02-01

Thyrotropin (TSH)-secreting pituitary adenomas (TSHomas) are a rare cause of hyperthyroidism, and the genetic aberrations responsible remain unknown. To identify somatic genetic abnormalities in TSHomas. A single-nucleotide polymorphism (SNP) array analysis was performed on 8 TSHomas. Four tumors with no allelic losses or limited loss of heterozygosity were selected, and whole-exome sequencing was performed, including their corresponding blood samples. Somatic variants were confirmed by Sanger sequencing. A set of 8 tumors was also assessed to validate candidate genes. Twelve patients with sporadic TSHomas were examined. The overall performance of whole-exome sequencing was good, with an average coverage of each base in the targeted region of 97.6%. Six DNA variants were confirmed as candidate driver mutations, with an average of 1.5 somatic mutations per tumor. No mutations were recurrent. Two of these mutations were found in genes with an established role in malignant tumorigenesis (SMOX and SYTL3), and 4 had unknown roles (ZSCAN23, ASTN2, R3HDM2, and CWH43). Similarly, an SNP array analysis revealed frequent chromosomal regions of copy number gains, including recurrent gains at loci harboring 4 of these 6 genes. Several candidate somatic mutations and changes in copy numbers for TSHomas were identified. The results showed no recurrence of mutations in the tumors studied but a low number of mutations, thereby highlighting their benign nature. Further studies on a larger cohort of TSHomas, along with the use of epigenetic and transcriptomic approaches, may reveal the underlying genetic lesions. Copyright © 2017 by the Endocrine Society

Whole exome sequencing to estimate alloreactivity potential between donors and recipients in stem cell transplantation

PubMed Central

Sampson, Juliana K.; Sheth, Nihar U.; Koparde, Vishal N.; Scalora, Allison F.; Serrano, Myrna G.; Lee, Vladimir; Roberts, Catherine H.; Jameson-Lee, Max; Ferreira-Gonzalez, Andrea; Manjili, Masoud H.; Buck, Gregory A.; Neale, Michael C.; Toor, Amir A.

2016-01-01

Summary Whole exome sequencing (WES) was performed on stem cell transplant donor-recipient (D-R) pairs to determine the extent of potential antigenic variation at a molecular level. In a small cohort of D-R pairs, a high frequency of sequence variation was observed between the donor and recipient exomes independent of human leucocyte antigen (HLA) matching. Nonsynonymous, nonconservative single nucleotide polymorphisms were approximately twice as frequent in HLA-matched unrelated, compared with related D-R pairs. When mapped to individual chromosomes, these polymorphic nucleotides were uniformly distributed across the entire exome. In conclusion, WES reveals extensive nucleotide sequence variation in the exomes of HLA-matched donors and recipients. PMID:24749631

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

Genetic Variants Identified from Epilepsy of Unknown Etiology in Chinese Children by Targeted Exome Sequencing

PubMed Central

Wang, Yimin; Du, Xiaonan; Bin, Rao; Yu, Shanshan; Xia, Zhezhi; Zheng, Guo; Zhong, Jianmin; Zhang, Yunjian; Jiang, Yong-hui; Wang, Yi

2017-01-01

Genetic factors play a major role in the etiology of epilepsy disorders. Recent genomics studies using next generation sequencing (NGS) technique have identified a large number of genetic variants including copy number (CNV) and single nucleotide variant (SNV) in a small set of genes from individuals with epilepsy. These discoveries have contributed significantly to evaluate the etiology of epilepsy in clinic and lay the foundation to develop molecular specific treatment. However, the molecular basis for a majority of epilepsy patients remains elusive, and furthermore, most of these studies have been conducted in Caucasian children. Here we conducted a targeted exome-sequencing of 63 trios of Chinese epilepsy families using a custom-designed NGS panel that covers 412 known and candidate genes for epilepsy. We identified pathogenic and likely pathogenic variants in 15 of 63 (23.8%) families in known epilepsy genes including SCN1A, CDKL5, STXBP1, CHD2, SCN3A, SCN9A, TSC2, MBD5, POLG and EFHC1. More importantly, we identified likely pathologic variants in several novel candidate genes such as GABRE, MYH1, and CLCN6. Our results provide the evidence supporting the application of custom-designed NGS panel in clinic and indicate a conserved genetic susceptibility for epilepsy between Chinese and Caucasian children. PMID:28074849

Whole exome sequencing to estimate alloreactivity potential between donors and recipients in stem cell transplantation.

PubMed

Sampson, Juliana K; Sheth, Nihar U; Koparde, Vishal N; Scalora, Allison F; Serrano, Myrna G; Lee, Vladimir; Roberts, Catherine H; Jameson-Lee, Max; Ferreira-Gonzalez, Andrea; Manjili, Masoud H; Buck, Gregory A; Neale, Michael C; Toor, Amir A

2014-08-01

Whole exome sequencing (WES) was performed on stem cell transplant donor-recipient (D-R) pairs to determine the extent of potential antigenic variation at a molecular level. In a small cohort of D-R pairs, a high frequency of sequence variation was observed between the donor and recipient exomes independent of human leucocyte antigen (HLA) matching. Nonsynonymous, nonconservative single nucleotide polymorphisms were approximately twice as frequent in HLA-matched unrelated, compared with related D-R pairs. When mapped to individual chromosomes, these polymorphic nucleotides were uniformly distributed across the entire exome. In conclusion, WES reveals extensive nucleotide sequence variation in the exomes of HLA-matched donors and recipients. © 2014 John Wiley & Sons Ltd.

Scalable whole-exome sequencing of cell-free DNA reveals high concordance with metastatic tumors.

PubMed

Adalsteinsson, Viktor A; Ha, Gavin; Freeman, Samuel S; Choudhury, Atish D; Stover, Daniel G; Parsons, Heather A; Gydush, Gregory; Reed, Sarah C; Rotem, Denisse; Rhoades, Justin; Loginov, Denis; Livitz, Dimitri; Rosebrock, Daniel; Leshchiner, Ignaty; Kim, Jaegil; Stewart, Chip; Rosenberg, Mara; Francis, Joshua M; Zhang, Cheng-Zhong; Cohen, Ofir; Oh, Coyin; Ding, Huiming; Polak, Paz; Lloyd, Max; Mahmud, Sairah; Helvie, Karla; Merrill, Margaret S; Santiago, Rebecca A; O'Connor, Edward P; Jeong, Seong H; Leeson, Rachel; Barry, Rachel M; Kramkowski, Joseph F; Zhang, Zhenwei; Polacek, Laura; Lohr, Jens G; Schleicher, Molly; Lipscomb, Emily; Saltzman, Andrea; Oliver, Nelly M; Marini, Lori; Waks, Adrienne G; Harshman, Lauren C; Tolaney, Sara M; Van Allen, Eliezer M; Winer, Eric P; Lin, Nancy U; Nakabayashi, Mari; Taplin, Mary-Ellen; Johannessen, Cory M; Garraway, Levi A; Golub, Todd R; Boehm, Jesse S; Wagle, Nikhil; Getz, Gad; Love, J Christopher; Meyerson, Matthew

2017-11-06

Whole-exome sequencing of cell-free DNA (cfDNA) could enable comprehensive profiling of tumors from blood but the genome-wide concordance between cfDNA and tumor biopsies is uncertain. Here we report ichorCNA, software that quantifies tumor content in cfDNA from 0.1× coverage whole-genome sequencing data without prior knowledge of tumor mutations. We apply ichorCNA to 1439 blood samples from 520 patients with metastatic prostate or breast cancers. In the earliest tested sample for each patient, 34% of patients have ≥10% tumor-derived cfDNA, sufficient for standard coverage whole-exome sequencing. Using whole-exome sequencing, we validate the concordance of clonal somatic mutations (88%), copy number alterations (80%), mutational signatures, and neoantigens between cfDNA and matched tumor biopsies from 41 patients with ≥10% cfDNA tumor content. In summary, we provide methods to identify patients eligible for comprehensive cfDNA profiling, revealing its applicability to many patients, and demonstrate high concordance of cfDNA and metastatic tumor whole-exome sequencing.

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.

Exome sequencing results in successful riboflavin treatment of a rapidly progressive neurological condition

PubMed Central

Petrovski, Slavé; Shashi, Vandana; Petrou, Steven; Schoch, Kelly; McSweeney, Keisha Melodi; Dhindsa, Ryan S.; Krueger, Brian; Crimian, Rebecca; Case, Laura E.; Khalid, Roha; El-Dairi, Maysantoine A.; Jiang, Yong-Hui; Mikati, Mohamad A.; Goldstein, David B.

2015-01-01

Genetically targeted therapies for rare Mendelian conditions are improving patient outcomes. Here, we present the case of a 20-mo-old female suffering from a rapidly progressing neurological disorder. Although diagnosed initially with a possible autoimmune condition, analysis of the child's exome resulted in a diagnosis of Brown–Vialetto–Van Laere syndrome 2 (BVVLS2). This new diagnosis led to a change in the therapy plan from steroids and precautionary chemotherapy to high-dose riboflavin. Improvements were reported quickly, including in motor strength after 1 mo. In this case, the correct diagnosis and appropriate treatment would have been unlikely in the absence of exome sequencing and careful interpretation. This experience adds to a growing list of examples that emphasize the importance of early genome-wide diagnostics. PMID:27148561

Outcomes of Diagnostic Exome Sequencing in Patients With Diagnosed or Suspected Autism Spectrum Disorders.

PubMed

Rossi, Mari; El-Khechen, Dima; Black, Mary Helen; Farwell Hagman, Kelly D; Tang, Sha; Powis, Zöe

2017-05-01

Exome sequencing has recently been proved to be a successful diagnostic method for complex neurodevelopmental disorders. However, the diagnostic yield of exome sequencing for autism spectrum disorders has not been extensively evaluated in large cohorts to date. We performed diagnostic exome sequencing in a cohort of 163 individuals with autism spectrum disorder (66.3%) or autistic features (33.7%). The diagnostic yield observed in patients in our cohort was 25.8% (42 of 163) for positive or likely positive findings in characterized disease genes, while a candidate genetic etiology was reported for an additional 3.3% (4 of 120) of patients. Among the positive findings in the patients with autism spectrum disorder or autistic features, 61.9% were the result of de novo mutations. Patients presenting with psychiatric conditions or ataxia or paraplegia in addition to autism spectrum disorder or autistic features were significantly more likely to receive positive results compared with patients without these clinical features (95.6% vs 27.1%, P < 0.0001; 83.3% vs 21.2%, P < 0.0001, respectively). The majority of the positive findings were in recently identified autism spectrum disorder genes, supporting the importance of diagnostic exome sequencing for patients with autism spectrum disorder or autistic features as the causative genes might evade traditional sequential or panel testing. These results suggest that diagnostic exome sequencing would be an efficient primary diagnostic method for patients with autism spectrum disorders or autistic features. Moreover, our data may aid clinicians to better determine which subset of patients with autism spectrum disorder with additional clinical features would benefit the most from diagnostic exome sequencing. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Whole-Exome Sequencing in Adults With Chronic Kidney Disease: A Pilot Study.

PubMed

Lata, Sneh; Marasa, Maddalena; Li, Yifu; Fasel, David A; Groopman, Emily; Jobanputra, Vaidehi; Rasouly, Hila; Mitrotti, Adele; Westland, Rik; Verbitsky, Miguel; Nestor, Jordan; Slater, Lindsey M; D'Agati, Vivette; Zaniew, Marcin; Materna-Kiryluk, Anna; Lugani, Francesca; Caridi, Gianluca; Rampoldi, Luca; Mattoo, Aditya; Newton, Chad A; Rao, Maya K; Radhakrishnan, Jai; Ahn, Wooin; Canetta, Pietro A; Bomback, Andrew S; Appel, Gerald B; Antignac, Corinne; Markowitz, Glen S; Garcia, Christine K; Kiryluk, Krzysztof; Sanna-Cherchi, Simone; Gharavi, Ali G

2018-01-16

The utility of whole-exome sequencing (WES) for the diagnosis and management of adult-onset constitutional disorders has not been adequately studied. Genetic diagnostics may be advantageous in adults with chronic kidney disease (CKD), in whom the cause of kidney failure often remains unknown. To study the diagnostic utility of WES in a selected referral population of adults with CKD. Observational cohort. A major academic medical center. 92 adults with CKD of unknown cause or familial nephropathy or hypertension. The diagnostic yield of WES and its potential effect on clinical management. Whole-exome sequencing provided a diagnosis in 22 of 92 patients (24%), including 9 probands with CKD of unknown cause and encompassing 13 distinct genetic disorders. Among these, loss-of-function mutations were identified in PARN in 2 probands with tubulointerstitial fibrosis. PARN mutations have been implicated in a short telomere syndrome characterized by lung, bone marrow, and liver fibrosis; these findings extend the phenotype of PARN mutations to renal fibrosis. In addition, review of the American College of Medical Genetics actionable genes identified a pathogenic BRCA2 mutation in a proband who was diagnosed with breast cancer on follow-up. The results affected clinical management in most identified cases, including initiation of targeted surveillance, familial screening to guide donor selection for transplantation, and changes in therapy. The small sample size and recruitment at a tertiary care academic center limit generalizability of findings among the broader CKD population. Whole-exome sequencing identified diagnostic mutations in a substantial number of adults with CKD of many causes. Further study of the utility of WES in the evaluation and care of patients with CKD in additional settings is warranted. New York State Empire Clinical Research Investigator Program, Renal Research Institute, and National Human Genome Research Institute of the National Institutes of

Identification of rare paired box 3 variant in strabismus by whole exome sequencing.

PubMed

Gong, Hui-Min; Wang, Jing; Xu, Jing; Zhou, Zhan-Yu; Li, Jing-Wen; Chen, Shu-Fang

2017-01-01

To identify the potentially pathogenic gene variants that contributes to the etiology of strabismus. A Chinese pedigree with strabismus was collected and the exomes of two affected individuals were sequenced using the next-generation sequencing technology. The resulting variants from exome sequencing were filtered by subsequent bioinformatics methods and the candidate mutation was verified as heterozygous in the affected proposita and her mother by sanger sequencing. Whole exome sequencing and filtering identified a nonsynonymous mutation c.434G-T transition in paired box 3 (PAX3) in the two affected individuals, which were predicted to be deleterious by more than 4 bioinformatics programs. This altered amino acid residue was located in the conserved PAX domain of PAX3. This gene encodes a member of the PAX family of transcription factors, which play critical roles during fetal development. Mutations in PAX3 were associated with Waardenburg syndrome with strabismus. Our results report that the c.434G-T mutation (p.R145L) in PAX3 may contribute to strabismus, expanding our understanding of the causally relevant genes for this disorder.

Targeted exome sequencing of Korean triple-negative breast cancer reveals homozygous deletions associated with poor prognosis of adjuvant chemotherapy-treated patients

PubMed Central

Jeong, Hae Min; Kim, Ryong Nam; Kwon, Mi Jeong; Oh, Ensel; Han, Jinil; Lee, Se Kyung; Choi, Jong-Sun; Park, Sara; Nam, Seok Jin; Gong, Gyung Yup; Nam, Jin Wu; Choi, Doo Ho; Lee, Hannah; Nam, Byung-Ho; Choi, Yoon-La; Shin, Young Kee

2017-01-01

Triple-negative breast cancer is characterized by the absence of estrogen and progesterone receptors and human epidermal growth factor receptor 2, and is associated with a poorer outcome than other subtypes of breast cancer. Moreover, there are no accurate prognostic genes or effective therapeutic targets, thereby necessitating continued intensive investigation. This study analyzed the genetic mutation landscape in 70 patients with triple-negative breast cancer by targeted exome sequencing of tumor and matched normal samples. Sequencing showed that more than 50% of these patients had deleterious mutations and homozygous deletions of DNA repair genes, such as ATM, BRCA1, BRCA2, WRN, and CHEK2. These findings suggested that a large number of patients with triple-negative breast cancer have impaired DNA repair function and that therefore a poly ADP-ribose polymerase inhibitor may be an effective drug in the treatment of this disease. Notably, homozygous deletion of three genes, EPHA5, MITF, and ACSL3, was significantly associated with an increased risk of recurrence or distant metastasis in adjuvant chemotherapy-treated patients. PMID:28977883

Whole-exome sequencing for mutation detection in pediatric disorders of insulin secretion: Maturity onset diabetes of the young and congenital hyperinsulinism.

PubMed

Johnson, S R; Leo, P J; McInerney-Leo, A M; Anderson, L K; Marshall, M; McGown, I; Newell, F; Brown, M A; Conwell, L S; Harris, M; Duncan, E L

2018-06-01

To assess the utility of whole-exome sequencing (WES) for mutation detection in maturity-onset diabetes of the young (MODY) and congenital hyperinsulinism (CHI). MODY and CHI are the two commonest monogenic disorders of glucose-regulated insulin secretion in childhood, with 13 causative genes known for MODY and 10 causative genes identified for CHI. The large number of potential genes makes comprehensive screening using traditional methods expensive and time-consuming. Ten subjects with MODY and five with CHI with known mutations underwent WES using two different exome capture kits (Nimblegen SeqCap EZ Human v3.0 Exome Enrichment Kit, Nextera Rapid Capture Exome Kit). Analysis was blinded to previously identified mutations, and included assessment for large deletions. The target capture of five exome capture technologies was also analyzed using sequencing data from >2800 unrelated samples. Four of five MODY mutations were identified using Nimblegen (including a large deletion in HNF1B). Although targeted, one mutation (in INS) had insufficient coverage for detection. Eleven of eleven mutations (six MODY, five CHI) were identified using Nextera Rapid (including the previously missed mutation). On reconciliation, all mutations concorded with previous data and no additional variants in MODY genes were detected. There were marked differences in the performance of the capture technologies. WES can be useful for screening for MODY/CHI mutations, detecting both point mutations and large deletions. However, capture technologies require careful selection. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Whole-exome sequencing and targeted gene sequencing provide insights into the role of PALB2 as a male breast cancer susceptibility gene.

PubMed

Silvestri, Valentina; Zelli, Veronica; Valentini, Virginia; Rizzolo, Piera; Navazio, Anna Sara; Coppa, Anna; Agata, Simona; Oliani, Cristina; Barana, Daniela; Castrignanò, Tiziana; Viel, Alessandra; Russo, Antonio; Tibiletti, Maria Grazia; Zanna, Ines; Masala, Giovanna; Cortesi, Laura; Manoukian, Siranoush; Azzollini, Jacopo; Peissel, Bernard; Bonanni, Bernardo; Peterlongo, Paolo; Radice, Paolo; Palli, Domenico; Giannini, Giuseppe; Chillemi, Giovanni; Montagna, Marco; Ottini, Laura

2017-01-01

Male breast cancer (MBC) is a rare disease whose etiology appears to be largely associated with genetic factors. BRCA1 and BRCA2 mutations account for about 10% of all MBC cases. Thus, a fraction of MBC cases are expected to be due to genetic factors not yet identified. To further explain the genetic susceptibility for MBC, whole-exome sequencing (WES) and targeted gene sequencing were applied to high-risk, BRCA1/2 mutation-negative MBC cases. Germ-line DNA of 1 male and 2 female BRCA1/2 mutation-negative breast cancer (BC) cases from a pedigree showing a first-degree family history of MBC was analyzed with WES. Targeted gene sequencing for the validation of WES results was performed for 48 high-risk, BRCA1/2 mutation-negative MBC cases from an Italian multicenter study of MBC. A case-control series of 433 BRCA1/2 mutation-negative MBC and female breast cancer (FBC) cases and 849 male and female controls was included in the study. WES in the family identified the partner and localizer of BRCA2 (PALB2) c.419delA truncating mutation carried by the proband, her father, and her paternal uncle (all affected with BC) and the N-acetyltransferase 1 (NAT1) c.97C>T nonsense mutation carried by the proband's maternal aunt. Targeted PALB2 sequencing detected the c.1984A>T nonsense mutation in 1 of the 48 BRCA1/2 mutation-negative MBC cases. NAT1 c.97C>T was not found in the case-control series. These results add strength to the evidence showing that PALB2 is involved in BC risk for both sexes and indicate that consideration should be given to clinical testing of PALB2 for BRCA1/2 mutation-negative families with multiple MBC and FBC cases. Cancer 2017;123:210-218. © 2016 American Cancer Society. © 2016 American Cancer Society.

Rapid prenatal diagnosis using targeted exome sequencing: a cohort study to assess feasibility and potential impact on prenatal counseling and pregnancy management.

PubMed

Chandler, Natalie; Best, Sunayna; Hayward, Jane; Faravelli, Francesca; Mansour, Sahar; Kivuva, Emma; Tapon, Dagmar; Male, Alison; DeVile, Catherine; Chitty, Lyn S

2018-03-29

PurposeUnexpected fetal abnormalities occur in 2-5% of pregnancies. While traditional cytogenetic and microarray approaches achieve diagnosis in around 40% of cases, lack of diagnosis in others impedes parental counseling, informed decision making, and pregnancy management. Postnatally exome sequencing yields high diagnostic rates, but relies on careful phenotyping to interpret genotype results. Here we used a multidisciplinary approach to explore the utility of rapid fetal exome sequencing for prenatal diagnosis using skeletal dysplasias as an exemplar.MethodsParents in pregnancies undergoing invasive testing because of sonographic fetal abnormalities, where multidisciplinary review considered skeletal dysplasia a likely etiology, were consented for exome trio sequencing (both parents and fetus). Variant interpretation focused on a virtual panel of 240 genes known to cause skeletal dysplasias.ResultsDefinitive molecular diagnosis was made in 13/16 (81%) cases. In some cases, fetal ultrasound findings alone were of sufficient severity for parents to opt for termination. In others, molecular diagnosis informed accurate prediction of outcome, improved parental counseling, and enabled parents to terminate or continue the pregnancy with certainty.ConclusionTrio sequencing with expert multidisciplinary review for case selection and data interpretation yields timely, high diagnostic rates in fetuses presenting with unexpected skeletal abnormalities. This improves parental counseling and pregnancy management.Genetics in Medicine advance online publication, 29 March 2018; doi:10.1038/gim.2018.30.

Whole-exome sequencing for diagnosis of hereditary ichthyosis.

PubMed

Sitek, J C; Kulseth, M A; Rypdal, K B; Skodje, T; Sheng, Y; Retterstøl, L

2018-02-14

Hereditary ichthyosis constitutes a diverse group of cornification disorders. Identification of the molecular cause facilitates optimal patient care. We wanted to estimate the diagnostic yield of applying whole-exome sequencing (WES) in the routine genetic workup of inherited ichthyosis. During a 3-year-period, all ichthyosis patients, except X-linked and mild vulgar ichthyosis, consecutively admitted to a university hospital clinic were offered WES with subsequent analysis of ichthyosis-related genes as a first-line genetic investigation. Clinical and molecular data have been collected retrospectively. Genetic variants causative for the ichthyosis were identified in 27 of 34 investigated patients (79.4%). In all, 31 causative mutations across 13 genes were disclosed, including 12 novel variants. TGM1 was the most frequently mutated gene, accounting for 43.7% of patients suffering from autosomal recessive congenital ichthyosis (ARCI). Whole-exome sequencing appears an effective tool in disclosing the molecular cause of patients with hereditary ichthyosis seen in clinical practice and should be considered a first-tier genetic test in these patients. © 2018 European Academy of Dermatology and Venereology.

Exome Sequencing Identifies Potentially Druggable Mutations in Nasopharyngeal Carcinoma.

PubMed

Chow, Yock Ping; Tan, Lu Ping; Chai, San Jiun; Abdul Aziz, Norazlin; Choo, Siew Woh; Lim, Paul Vey Hong; Pathmanathan, Rajadurai; Mohd Kornain, Noor Kaslina; Lum, Chee Lun; Pua, Kin Choo; Yap, Yoke Yeow; Tan, Tee Yong; Teo, Soo Hwang; Khoo, Alan Soo-Beng; Patel, Vyomesh

2017-03-03

In this study, we first performed whole exome sequencing of DNA from 10 untreated and clinically annotated fresh frozen nasopharyngeal carcinoma (NPC) biopsies and matched bloods to identify somatically mutated genes that may be amenable to targeted therapeutic strategies. We identified a total of 323 mutations which were either non-synonymous (n = 238) or synonymous (n = 85). Furthermore, our analysis revealed genes in key cancer pathways (DNA repair, cell cycle regulation, apoptosis, immune response, lipid signaling) were mutated, of which those in the lipid-signaling pathway were the most enriched. We next extended our analysis on a prioritized sub-set of 37 mutated genes plus top 5 mutated cancer genes listed in COSMIC using a custom designed HaloPlex target enrichment panel with an additional 88 NPC samples. Our analysis identified 160 additional non-synonymous mutations in 37/42 genes in 66/88 samples. Of these, 99/160 mutations within potentially druggable pathways were further selected for validation. Sanger sequencing revealed that 77/99 variants were true positives, giving an accuracy of 78%. Taken together, our study indicated that ~72% (n = 71/98) of NPC samples harbored mutations in one of the four cancer pathways (EGFR-PI3K-Akt-mTOR, NOTCH, NF-κB, DNA repair) which may be potentially useful as predictive biomarkers of response to matched targeted therapies.

Exome Sequencing Identifies Potentially Druggable Mutations in Nasopharyngeal Carcinoma

PubMed Central

Chow, Yock Ping; Tan, Lu Ping; Chai, San Jiun; Abdul Aziz, Norazlin; Choo, Siew Woh; Lim, Paul Vey Hong; Pathmanathan, Rajadurai; Mohd Kornain, Noor Kaslina; Lum, Chee Lun; Pua, Kin Choo; Yap, Yoke Yeow; Tan, Tee Yong; Teo, Soo Hwang; Khoo, Alan Soo-Beng; Patel, Vyomesh

2017-01-01

In this study, we first performed whole exome sequencing of DNA from 10 untreated and clinically annotated fresh frozen nasopharyngeal carcinoma (NPC) biopsies and matched bloods to identify somatically mutated genes that may be amenable to targeted therapeutic strategies. We identified a total of 323 mutations which were either non-synonymous (n = 238) or synonymous (n = 85). Furthermore, our analysis revealed genes in key cancer pathways (DNA repair, cell cycle regulation, apoptosis, immune response, lipid signaling) were mutated, of which those in the lipid-signaling pathway were the most enriched. We next extended our analysis on a prioritized sub-set of 37 mutated genes plus top 5 mutated cancer genes listed in COSMIC using a custom designed HaloPlex target enrichment panel with an additional 88 NPC samples. Our analysis identified 160 additional non-synonymous mutations in 37/42 genes in 66/88 samples. Of these, 99/160 mutations within potentially druggable pathways were further selected for validation. Sanger sequencing revealed that 77/99 variants were true positives, giving an accuracy of 78%. Taken together, our study indicated that ~72% (n = 71/98) of NPC samples harbored mutations in one of the four cancer pathways (EGFR-PI3K-Akt-mTOR, NOTCH, NF-κB, DNA repair) which may be potentially useful as predictive biomarkers of response to matched targeted therapies. PMID:28256603

CEQer: a graphical tool for copy number and allelic imbalance detection from whole-exome sequencing data.

PubMed

Piazza, Rocco; Magistroni, Vera; Pirola, Alessandra; Redaelli, Sara; Spinelli, Roberta; Redaelli, Serena; Galbiati, Marta; Valletta, Simona; Giudici, Giovanni; Cazzaniga, Giovanni; Gambacorti-Passerini, Carlo

2013-01-01

Copy number alterations (CNA) are common events occurring in leukaemias and solid tumors. Comparative Genome Hybridization (CGH) is actually the gold standard technique to analyze CNAs; however, CGH analysis requires dedicated instruments and is able to perform only low resolution Loss of Heterozygosity (LOH) analyses. Here we present CEQer (Comparative Exome Quantification analyzer), a new graphical, event-driven tool for CNA/allelic-imbalance (AI) coupled analysis of exome sequencing data. By using case-control matched exome data, CEQer performs a comparative digital exonic quantification to generate CNA data and couples this information with exome-wide LOH and allelic imbalance detection. This data is used to build mixed statistical/heuristic models allowing the identification of CNA/AI events. To test our tool, we initially used in silico generated data, then we performed whole-exome sequencing from 20 leukemic specimens and corresponding matched controls and we analyzed the results using CEQer. Taken globally, these analyses showed that the combined use of comparative digital exon quantification and LOH/AI allows generating very accurate CNA data. Therefore, we propose CEQer as an efficient, robust and user-friendly graphical tool for the identification of CNA/AI in the context of whole-exome sequencing data.

Molecular diagnosis of putative Stargardt disease probands by exome sequencing

PubMed Central

2012-01-01

Background The commonest genetic form of juvenile or early adult onset macular degeneration is Stargardt Disease (STGD) caused by recessive mutations in the gene ABCA4. However, high phenotypic and allelic heterogeneity and a small but non-trivial amount of locus heterogeneity currently impede conclusive molecular diagnosis in a significant proportion of cases. Methods We performed whole exome sequencing (WES) of nine putative Stargardt Disease probands and searched for potentially disease-causing genetic variants in previously identified retinal or macular dystrophy genes. Follow-up dideoxy sequencing was performed for confirmation and to screen for mutations in an additional set of affected individuals lacking a definitive molecular diagnosis. Results Whole exome sequencing revealed seven likely disease-causing variants across four genes, providing a confident genetic diagnosis in six previously uncharacterized participants. We identified four previously missed mutations in ABCA4 across three individuals. Likely disease-causing mutations in RDS/PRPH2, ELOVL, and CRB1 were also identified. Conclusions Our findings highlight the enormous potential of whole exome sequencing in Stargardt Disease molecular diagnosis and research. WES adequately assayed all coding sequences and canonical splice sites of ABCA4 in this study. Additionally, WES enables the identification of disease-related alleles in other genes. This work highlights the importance of collecting parental genetic material for WES testing as the current knowledge of human genome variation limits the determination of causality between identified variants and disease. While larger sample sizes are required to establish the precision and accuracy of this type of testing, this study supports WES for inherited early onset macular degeneration disorders as an alternative to standard mutation screening techniques. PMID:22863181

Identification of rare paired box 3 variant in strabismus by whole exome sequencing

PubMed Central

Gong, Hui-Min; Wang, Jing; Xu, Jing; Zhou, Zhan-Yu; Li, Jing-Wen; Chen, Shu-Fang

2017-01-01

AIM To identify the potentially pathogenic gene variants that contributes to the etiology of strabismus. METHODS A Chinese pedigree with strabismus was collected and the exomes of two affected individuals were sequenced using the next-generation sequencing technology. The resulting variants from exome sequencing were filtered by subsequent bioinformatics methods and the candidate mutation was verified as heterozygous in the affected proposita and her mother by sanger sequencing. RESULTS Whole exome sequencing and filtering identified a nonsynonymous mutation c.434G-T transition in paired box 3 (PAX3) in the two affected individuals, which were predicted to be deleterious by more than 4 bioinformatics programs. This altered amino acid residue was located in the conserved PAX domain of PAX3. This gene encodes a member of the PAX family of transcription factors, which play critical roles during fetal development. Mutations in PAX3 were associated with Waardenburg syndrome with strabismus. CONCLUSION Our results report that the c.434G-T mutation (p.R145L) in PAX3 may contribute to strabismus, expanding our understanding of the causally relevant genes for this disorder. PMID:28861346

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

PubMed Central

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.

2016-01-01

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 11×109/L to 186×109/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. PMID:27479822

Identification of a Heterozygous SPG11 Mutation by Clinical Exome Sequencing in a Patient With Hereditary Spastic Paraplegia: A Case Report.

PubMed

Oh, Ja-Young; Do, Hyun Jung; Lee, Seungok; Jang, Ja-Hyun; Cho, Eun-Hae; Jang, Dae-Hyun

2016-12-01

Next-generation sequencing, such as whole-genome sequencing, whole-exome sequencing, and targeted panel sequencing have been applied for diagnosis of many genetic diseases, and are in the process of replacing the traditional methods of genetic analysis. Clinical exome sequencing (CES), which provides not only sequence variation data but also clinical interpretation, aids in reaching a final conclusion with regards to genetic diagnosis. Sequencing of genes with clinical relevance rather than whole exome sequencing might be more suitable for the diagnosis of known hereditary disease with genetic heterogeneity. Here, we present the clinical usefulness of CES for the diagnosis of hereditary spastic paraplegia (HSP). We report a case of patient who was strongly suspected of having HSP based on her clinical manifestations. HSP is one of the diseases with high genetic heterogeneity, the 72 different loci and 59 discovered genes identified so far. Therefore, traditional approach for diagnosis of HSP with genetic analysis is very challenging and time-consuming. CES with TruSight One Sequencing Panel, which enriches about 4,800 genes with clinical relevance, revealed compound heterozygous mutations in SPG11 . One workflow and one procedure can provide the results of genetic analysis, and CES with enrichment of clinically relevant genes is a cost-effective and time-saving diagnostic tool for diseases with genetic heterogeneity, including HSP.

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

Whole-exome sequencing reveals GPIHBP1 mutations in infantile colitis with severe hypertriglyceridemia.

PubMed

Gonzaga-Jauregui, Claudia; Mir, Sabina; Penney, Samantha; Jhangiani, Shalini; Midgen, Craig; Finegold, Milton; Muzny, Donna M; Wang, Min; Bacino, Carlos A; Gibbs, Richard A; Lupski, James R; Kellermayer, Richard; Hanchard, Neil A

2014-07-01

Severe congenital hypertriglyceridemia (HTG) is a rare disorder caused by mutations in genes affecting lipoprotein lipase (LPL) activity. Here we report a 5-week-old Hispanic girl with severe HTG (12,031 mg/dL, normal limit 150 mg/dL) who presented with the unusual combination of lower gastrointestinal bleeding and milky plasma. Initial colonoscopy was consistent with colitis, which resolved with reduction of triglycerides. After negative sequencing of the LPL gene, whole-exome sequencing revealed novel compound heterozygous mutations in GPIHBP1. Our study broadens the phenotype of GPIHBP1-associated HTG, reinforces the effectiveness of whole-exome sequencing in Mendelian diagnoses, and implicates triglycerides in gastrointestinal mucosal injury.

Exome sequencing in amyotrophic lateral sclerosis identifies risk genes and pathways.

PubMed

Cirulli, Elizabeth T; Lasseigne, Brittany N; Petrovski, Slavé; Sapp, Peter C; Dion, Patrick A; Leblond, Claire S; Couthouis, Julien; Lu, Yi-Fan; Wang, Quanli; Krueger, Brian J; Ren, Zhong; Keebler, Jonathan; Han, Yujun; Levy, Shawn E; Boone, Braden E; Wimbish, Jack R; Waite, Lindsay L; Jones, Angela L; Carulli, John P; Day-Williams, Aaron G; Staropoli, John F; Xin, Winnie W; Chesi, Alessandra; Raphael, Alya R; McKenna-Yasek, Diane; Cady, Janet; Vianney de Jong, J M B; Kenna, Kevin P; Smith, Bradley N; Topp, Simon; Miller, Jack; Gkazi, Athina; Al-Chalabi, Ammar; van den Berg, Leonard H; Veldink, Jan; Silani, Vincenzo; Ticozzi, Nicola; Shaw, Christopher E; Baloh, Robert H; Appel, Stanley; Simpson, Ericka; Lagier-Tourenne, Clotilde; Pulst, Stefan M; Gibson, Summer; Trojanowski, John Q; Elman, Lauren; McCluskey, Leo; Grossman, Murray; Shneider, Neil A; Chung, Wendy K; Ravits, John M; Glass, Jonathan D; Sims, Katherine B; Van Deerlin, Vivianna M; Maniatis, Tom; Hayes, Sebastian D; Ordureau, Alban; Swarup, Sharan; Landers, John; Baas, Frank; Allen, Andrew S; Bedlack, Richard S; Harper, J Wade; Gitler, Aaron D; Rouleau, Guy A; Brown, Robert; Harms, Matthew B; Cooper, Gregory M; Harris, Tim; Myers, Richard M; Goldstein, David B

2015-03-27

Amyotrophic lateral sclerosis (ALS) is a devastating neurological disease with no effective treatment. We report the results of a moderate-scale sequencing study aimed at increasing the number of genes known to contribute to predisposition for ALS. We performed whole-exome sequencing of 2869 ALS patients and 6405 controls. Several known ALS genes were found to be associated, and TBK1 (the gene encoding TANK-binding kinase 1) was identified as an ALS gene. TBK1 is known to bind to and phosphorylate a number of proteins involved in innate immunity and autophagy, including optineurin (OPTN) and p62 (SQSTM1/sequestosome), both of which have also been implicated in ALS. These observations reveal a key role of the autophagic pathway in ALS and suggest specific targets for therapeutic intervention. Copyright © 2015, American Association for the Advancement of Science.

Exome Sequencing Reveals Primary Immunodeficiencies in Children with Community-Acquired Pseudomonas aeruginosa Sepsis.

PubMed

Asgari, Samira; McLaren, Paul J; Peake, Jane; Wong, Melanie; Wong, Richard; Bartha, Istvan; Francis, Joshua R; Abarca, Katia; Gelderman, Kyra A; Agyeman, Philipp; Aebi, Christoph; Berger, Christoph; Fellay, Jacques; Schlapbach, Luregn J

2016-01-01

One out of three pediatric sepsis deaths in high income countries occur in previously healthy children. Primary immunodeficiencies (PIDs) have been postulated to underlie fulminant sepsis, but this concept remains to be confirmed in clinical practice. Pseudomonas aeruginosa ( P. aeruginosa ) is a common bacterium mostly associated with health care-related infections in immunocompromised individuals. However, in rare cases, it can cause sepsis in previously healthy children. We used exome sequencing and bioinformatic analysis to systematically search for genetic factors underpinning severe P. aeruginosa infection in the pediatric population. We collected blood samples from 11 previously healthy children, with no family history of immunodeficiency, who presented with severe sepsis due to community-acquired P. aeruginosa bacteremia. Genomic DNA was extracted from blood or tissue samples obtained intravitam or postmortem. We obtained high-coverage exome sequencing data and searched for rare loss-of-function variants. After rigorous filtrations, 12 potentially causal variants were identified. Two out of eight (25%) fatal cases were found to carry novel pathogenic variants in PID genes, including BTK and DNMT3B . This study demonstrates that exome sequencing allows to identify rare, deleterious human genetic variants responsible for fulminant sepsis in apparently healthy children. Diagnosing PIDs in such patients is of high relevance to survivors and affected families. We propose that unusually severe and fatal sepsis cases in previously healthy children should be considered for exome/genome sequencing to search for underlying PIDs.

Exome Sequencing Reveals Primary Immunodeficiencies in Children with Community-Acquired Pseudomonas aeruginosa Sepsis

PubMed Central

Asgari, Samira; McLaren, Paul J.; Peake, Jane; Wong, Melanie; Wong, Richard; Bartha, Istvan; Francis, Joshua R.; Abarca, Katia; Gelderman, Kyra A.; Agyeman, Philipp; Aebi, Christoph; Berger, Christoph; Fellay, Jacques; Schlapbach, Luregn J.; Posfay-Barbe, Klara

2016-01-01

One out of three pediatric sepsis deaths in high income countries occur in previously healthy children. Primary immunodeficiencies (PIDs) have been postulated to underlie fulminant sepsis, but this concept remains to be confirmed in clinical practice. Pseudomonas aeruginosa (P. aeruginosa) is a common bacterium mostly associated with health care-related infections in immunocompromised individuals. However, in rare cases, it can cause sepsis in previously healthy children. We used exome sequencing and bioinformatic analysis to systematically search for genetic factors underpinning severe P. aeruginosa infection in the pediatric population. We collected blood samples from 11 previously healthy children, with no family history of immunodeficiency, who presented with severe sepsis due to community-acquired P. aeruginosa bacteremia. Genomic DNA was extracted from blood or tissue samples obtained intravitam or postmortem. We obtained high-coverage exome sequencing data and searched for rare loss-of-function variants. After rigorous filtrations, 12 potentially causal variants were identified. Two out of eight (25%) fatal cases were found to carry novel pathogenic variants in PID genes, including BTK and DNMT3B. This study demonstrates that exome sequencing allows to identify rare, deleterious human genetic variants responsible for fulminant sepsis in apparently healthy children. Diagnosing PIDs in such patients is of high relevance to survivors and affected families. We propose that unusually severe and fatal sepsis cases in previously healthy children should be considered for exome/genome sequencing to search for underlying PIDs. PMID:27703454

CEQer: A Graphical Tool for Copy Number and Allelic Imbalance Detection from Whole-Exome Sequencing Data

PubMed Central

Piazza, Rocco; Magistroni, Vera; Pirola, Alessandra; Redaelli, Sara; Spinelli, Roberta; Redaelli, Serena; Galbiati, Marta; Valletta, Simona; Giudici, Giovanni; Cazzaniga, Giovanni; Gambacorti-Passerini, Carlo

2013-01-01

Copy number alterations (CNA) are common events occurring in leukaemias and solid tumors. Comparative Genome Hybridization (CGH) is actually the gold standard technique to analyze CNAs; however, CGH analysis requires dedicated instruments and is able to perform only low resolution Loss of Heterozygosity (LOH) analyses. Here we present CEQer (Comparative Exome Quantification analyzer), a new graphical, event-driven tool for CNA/allelic-imbalance (AI) coupled analysis of exome sequencing data. By using case-control matched exome data, CEQer performs a comparative digital exonic quantification to generate CNA data and couples this information with exome-wide LOH and allelic imbalance detection. This data is used to build mixed statistical/heuristic models allowing the identification of CNA/AI events. To test our tool, we initially used in silico generated data, then we performed whole-exome sequencing from 20 leukemic specimens and corresponding matched controls and we analyzed the results using CEQer. Taken globally, these analyses showed that the combined use of comparative digital exon quantification and LOH/AI allows generating very accurate CNA data. Therefore, we propose CEQer as an efficient, robust and user-friendly graphical tool for the identification of CNA/AI in the context of whole-exome sequencing data. PMID:24124457

SEXCMD: Development and validation of sex marker sequences for whole-exome/genome and RNA sequencing.

PubMed

Jeong, Seongmun; Kim, Jiwoong; Park, Won; Jeon, Hongmin; Kim, Namshin

2017-01-01

Over the last decade, a large number of nucleotide sequences have been generated by next-generation sequencing technologies and deposited to public databases. However, most of these datasets do not specify the sex of individuals sampled because researchers typically ignore or hide this information. Male and female genomes in many species have distinctive sex chromosomes, XX/XY and ZW/ZZ, and expression levels of many sex-related genes differ between the sexes. Herein, we describe how to develop sex marker sequences from syntenic regions of sex chromosomes and use them to quickly identify the sex of individuals being analyzed. Array-based technologies routinely use either known sex markers or the B-allele frequency of X or Z chromosomes to deduce the sex of an individual. The same strategy has been used with whole-exome/genome sequence data; however, all reads must be aligned onto a reference genome to determine the B-allele frequency of the X or Z chromosomes. SEXCMD is a pipeline that can extract sex marker sequences from reference sex chromosomes and rapidly identify the sex of individuals from whole-exome/genome and RNA sequencing after training with a known dataset through a simple machine learning approach. The pipeline counts total numbers of hits from sex-specific marker sequences and identifies the sex of the individuals sampled based on the fact that XX/ZZ samples do not have Y or W chromosome hits. We have successfully validated our pipeline with mammalian (Homo sapiens; XY) and avian (Gallus gallus; ZW) genomes. Typical calculation time when applying SEXCMD to human whole-exome or RNA sequencing datasets is a few minutes, and analyzing human whole-genome datasets takes about 10 minutes. Another important application of SEXCMD is as a quality control measure to avoid mixing samples before bioinformatics analysis. SEXCMD comprises simple Python and R scripts and is freely available at https://github.com/lovemun/SEXCMD.

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.

Ion Torren Semiconductor Sequencing Allows Rapid, Low Cost Sequencing of the Human Exome (7th Annual SFAF Meeting, 2012)

ScienceCinema

Jenkins, David

2018-01-10

David Jenkins on "Ion Torrent semiconductor sequencing allows rapid, low-cost sequencing of the human exome" at the 2012 Sequencing, Finishing, Analysis in the Future Meeting held June 5-7, 2012 in Santa Fe, New Mexico.

Ion Torren Semiconductor Sequencing Allows Rapid, Low Cost Sequencing of the Human Exome (7th Annual SFAF Meeting, 2012)

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

Jenkins, David

David Jenkins on "Ion Torrent semiconductor sequencing allows rapid, low-cost sequencing of the human exome" at the 2012 Sequencing, Finishing, Analysis in the Future Meeting held June 5-7, 2012 in Santa Fe, New Mexico.

Whole-Exome Sequencing Reveals GPIHBP1 Mutations in Infantile Colitis With Severe Hypertriglyceridemia

PubMed Central

Gonzaga-Jauregui, Claudia; Mir, Sabina; Penney, Samantha; Jhangiani, Shalini; Midgen, Craig; Finegold, Milton; Muzny, Donna M.; Wang, Min; Bacino, Carlos A.; Gibbs, Richard A.; Lupski, James R.; Kellermayer, Richard; Hanchard, Neil A.

2014-01-01

Severe congenital hypertriglyceridemia (HTG) is a rare disorder caused by mutations in genes affecting lipoprotein lipase (LPL) activity. Here we report a 5-week-old Hispanic girl with severe HTG (12,031 mg/dL, normal limit 150 mg/dL) who presented with the unusual combination of lower gastrointestinal bleeding and milky plasma. Initial colonoscopy was consistent with colitis, which resolved with reduction of triglycerides. After negative sequencing of the LPL gene, whole-exome sequencing revealed novel compound heterozygous mutations in GPIHBP1. Our study broadens the phenotype of GPIHBP1-associated HTG, reinforces the effectiveness of whole-exome sequencing in Mendelian diagnoses, and implicates triglycer-ides in gastrointestinal mucosal injury. PMID:24614124

Targeted exome sequencing and chromosomal microarray for the molecular diagnosis of nevoid basal cell carcinoma syndrome.

PubMed

Matsudate, Yoshihiro; Naruto, Takuya; Hayashi, Yumiko; Minami, Mitsuyoshi; Tohyama, Mikiko; Yokota, Kenji; Yamada, Daisuke; Imoto, Issei; Kubo, Yoshiaki

2017-06-01

Nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant disorder mainly caused by heterozygous mutations of PTCH1. In addition to characteristic clinical features, detection of a mutation in causative genes is reliable for the diagnosis of NBCCS; however, no mutations have been identified in some patients using conventional methods. To improve the method for the molecular diagnosis of NBCCS. We performed targeted exome sequencing (TES) analysis using a multi-gene panel, including PTCH1, PTCH2, SUFU, and other sonic hedgehog signaling pathway-related genes, based on next-generation sequencing (NGS) technology in 8 cases in whom possible causative mutations were not detected by previously performed conventional analysis and 2 recent cases of NBCCS. Subsequent analysis of gross deletion within or around PTCH1 detected by TES was performed using chromosomal microarray (CMA). Through TES analysis, specific single nucleotide variants or small indels of PTCH1 causing inferred amino acid changes were identified in 2 novel cases and 2 undiagnosed cases, whereas gross deletions within or around PTCH1, which are validated by CMA, were found in 3 undiagnosed cases. However, no mutations were detected even by TES in 3 cases. Among 3 cases with gross deletions of PTCH1, deletions containing the entire PTCH1 and additional neighboring genes were detected in 2 cases, one of which exhibited atypical clinical features, such as severe mental retardation, likely associated with genes located within the 4.3Mb deleted region, especially. TES-based simultaneous evaluation of sequences and copy number status in all targeted coding exons by NGS is likely to be more useful for the molecular diagnosis of NBCCS than conventional methods. CMA is recommended as a subsequent analysis for validation and detailed mapping of deleted regions, which may explain the atypical clinical features of NBCCS cases. Copyright © 2017 Japanese Society for Investigative Dermatology. Published by

Exome-wide DNA capture and next generation sequencing in domestic and wild species.

PubMed

Cosart, Ted; Beja-Pereira, Albano; Chen, Shanyuan; Ng, Sarah B; Shendure, Jay; Luikart, Gordon

2011-07-05

Gene-targeted and genome-wide markers are crucial to advance evolutionary biology, agriculture, and biodiversity conservation by improving our understanding of genetic processes underlying adaptation and speciation. Unfortunately, for eukaryotic species with large genomes it remains costly to obtain genome sequences and to develop genome resources such as genome-wide SNPs. A method is needed to allow gene-targeted, next-generation sequencing that is flexible enough to include any gene or number of genes, unlike transcriptome sequencing. Such a method would allow sequencing of many individuals, avoiding ascertainment bias in subsequent population genetic analyses.We demonstrate the usefulness of a recent technology, exon capture, for genome-wide, gene-targeted marker discovery in species with no genome resources. We use coding gene sequences from the domestic cow genome sequence (Bos taurus) to capture (enrich for), and subsequently sequence, thousands of exons of B. taurus, B. indicus, and Bison bison (wild bison). Our capture array has probes for 16,131 exons in 2,570 genes, including 203 candidate genes with known function and of interest for their association with disease and other fitness traits. We successfully sequenced and mapped exon sequences from across the 29 autosomes and X chromosome in the B. taurus genome sequence. Exon capture and high-throughput sequencing identified thousands of putative SNPs spread evenly across all reference chromosomes, in all three individuals, including hundreds of SNPs in our targeted candidate genes. This study shows exon capture can be customized for SNP discovery in many individuals and for non-model species without genomic resources. Our captured exome subset was small enough for affordable next-generation sequencing, and successfully captured exons from a divergent wild species using the domestic cow genome as reference.

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.

Exome sequencing-driven discovery of coding polymorphisms associated with common metabolic phenotypes.

PubMed

Albrechtsen, A; Grarup, N; Li, Y; Sparsø, T; Tian, G; Cao, H; Jiang, T; Kim, S Y; Korneliussen, T; Li, Q; Nie, C; Wu, R; Skotte, L; Morris, A P; Ladenvall, C; Cauchi, S; Stančáková, A; Andersen, G; Astrup, A; Banasik, K; Bennett, A J; Bolund, L; Charpentier, G; Chen, Y; Dekker, J M; Doney, A S F; Dorkhan, M; Forsen, T; Frayling, T M; Groves, C J; Gui, Y; Hallmans, G; Hattersley, A T; He, K; Hitman, G A; Holmkvist, J; Huang, S; Jiang, H; Jin, X; Justesen, J M; Kristiansen, K; Kuusisto, J; Lajer, M; Lantieri, O; Li, W; Liang, H; Liao, Q; Liu, X; Ma, T; Ma, X; Manijak, M P; Marre, M; Mokrosiński, J; Morris, A D; Mu, B; Nielsen, A A; Nijpels, G; Nilsson, P; Palmer, C N A; Rayner, N W; Renström, F; Ribel-Madsen, R; Robertson, N; Rolandsson, O; Rossing, P; Schwartz, T W; Slagboom, P E; Sterner, M; Tang, M; Tarnow, L; Tuomi, T; van't Riet, E; van Leeuwen, N; Varga, T V; Vestmar, M A; Walker, M; Wang, B; Wang, Y; Wu, H; Xi, F; Yengo, L; Yu, C; Zhang, X; Zhang, J; Zhang, Q; Zhang, W; Zheng, H; Zhou, Y; Altshuler, D; 't Hart, L M; Franks, P W; Balkau, B; Froguel, P; McCarthy, M I; Laakso, M; Groop, L; Christensen, C; Brandslund, I; Lauritzen, T; Witte, D R; Linneberg, A; Jørgensen, T; Hansen, T; Wang, J; Nielsen, R; Pedersen, O

2013-02-01

Human complex metabolic traits are in part regulated by genetic determinants. Here we applied exome sequencing to identify novel associations of coding polymorphisms at minor allele frequencies (MAFs) >1% with common metabolic phenotypes. The study comprised three stages. We performed medium-depth (8×) whole exome sequencing in 1,000 cases with type 2 diabetes, BMI >27.5 kg/m(2) and hypertension and in 1,000 controls (stage 1). We selected 16,192 polymorphisms nominally associated (p < 0.05) with case-control status, from four selected annotation categories or from loci reported to associate with metabolic traits. These variants were genotyped in 15,989 Danes to search for association with 12 metabolic phenotypes (stage 2). In stage 3, polymorphisms showing potential associations were genotyped in a further 63,896 Europeans. Exome sequencing identified 70,182 polymorphisms with MAF >1%. In stage 2 we identified 51 potential associations with one or more of eight metabolic phenotypes covered by 45 unique polymorphisms. In meta-analyses of stage 2 and stage 3 results, we demonstrated robust associations for coding polymorphisms in CD300LG (fasting HDL-cholesterol: MAF 3.5%, p = 8.5 × 10(-14)), COBLL1 (type 2 diabetes: MAF 12.5%, OR 0.88, p = 1.2 × 10(-11)) and MACF1 (type 2 diabetes: MAF 23.4%, OR 1.10, p = 8.2 × 10(-10)). We applied exome sequencing as a basis for finding genetic determinants of metabolic traits and show the existence of low-frequency and common coding polymorphisms with impact on common metabolic traits. Based on our study, coding polymorphisms with MAF above 1% do not seem to have particularly high effect sizes on the measured metabolic traits.

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

Single-cell whole exome and targeted sequencing in NPM1/FLT3 positive pediatric acute myeloid leukemia.

PubMed

Walter, Christiane; Pozzorini, Christian; Reinhardt, Katarina; Geffers, Robert; Xu, Zhenyu; Reinhardt, Dirk; von Neuhoff, Nils; Hanenberg, Helmut

2018-02-01

The small portion of leukemic stem cells (LSCs) in acute myeloid leukemia (AML) present in children and adolescents is often masked by the high background of AML blasts and normal hematopoietic cells. The aim of the current study was to establish a simple workflow for reliable genetic analysis of single LSC-enriched blasts from pediatric patients. For three AMLs with mutations in nucleophosmin 1 and/or fms-like tyrosine kinase 3, we performed whole genome amplification on sorted single-cell DNA followed by whole exome sequencing (WES). The corresponding bulk bone marrow DNAs were also analyzed by WES and by targeted sequencing (TS) that included 54 genes associated with myeloid malignancies. Analysis revealed that read coverage statistics were comparable between single-cell and bulk WES data, indicating high-quality whole genome amplification. From 102 single-cell variants, 72 single nucleotide variants and insertions or deletions (70%) were consistently found in the two bulk DNA analyses. Variants reliably detected in single cells were also present in TS. However, initial screening by WES with read counts between 50-72× failed to detect rare AML subclones in the bulk DNAs. In summary, our study demonstrated that single-cell WES combined with bulk DNA TS is a promising tool set for detecting AML subclones and possibly LSCs. © 2017 Wiley Periodicals, Inc.

Self-guided management of exome and whole-genome sequencing results: changing the results return model.

PubMed

Yu, Joon-Ho; Jamal, Seema M; Tabor, Holly K; Bamshad, Michael J

2013-09-01

Researchers and clinicians face the practical and ethical challenge of if and how to offer for return the wide and varied scope of results available from individual exome sequencing and whole-genome sequencing. We argue that rather than viewing individual exome sequencing and whole-genome sequencing as a test for which results need to be "returned," that the technology should instead be framed as a dynamic resource of information from which results should be "managed" over the lifetime of an individual. We further suggest that individual exome sequencing and whole-genome sequencing results management is optimized using a self-guided approach that enables individuals to self-select among results offered for return in a convenient, confidential, personalized context that is responsive to their value system. This approach respects autonomy, allows individuals to maximize potential benefits of genomic information (beneficence) and minimize potential harms (nonmaleficence), and also preserves their right to an open future to the extent they desire or think is appropriate. We describe key challenges and advantages of such a self-guided management system and offer guidance on implementation using an information systems approach.

Whole-exome sequencing of 228 patients with sporadic Parkinson's disease.

PubMed

Sandor, Cynthia; Honti, Frantisek; Haerty, Wilfried; Szewczyk-Krolikowski, Konrad; Tomlinson, Paul; Evetts, Sam; Millin, Stephanie; Keane, Thomas; McCarthy, Shane A; Durbin, Richard; Talbot, Kevin; Hu, Michele; Webber, Caleb; Ponting, Chris P; Wade-Martins, Richard

2017-01-24

Parkinson's disease (PD) is the most common neurodegenerative movement disorder, affecting 1% of the population over 65 years characterized clinically by both motor and non-motor symptoms accompanied by the preferential loss of dopamine neurons in the substantia nigra pars compacta. Here, we sequenced the exomes of 244 Parkinson's patients selected from the Oxford Parkinson's Disease Centre Discovery Cohort and, after quality control, 228 exomes were available for analyses. The PD patient exomes were compared to 884 control exomes selected from the UK10K datasets. No single non-synonymous (NS) single nucleotide variant (SNV) nor any gene carrying a higher burden of NS SNVs was significantly associated with PD status after multiple-testing correction. However, significant enrichments of genes whose proteins have roles in the extracellular matrix were amongst the top 300 genes with the most significantly associated NS SNVs, while regions associated with PD by a recent Genome Wide Association (GWA) study were enriched in genes containing PD-associated NS SNVs. By examining genes within GWA regions possessing rare PD-associated SNVs, we identified RAD51B. The protein-product of RAD51B interacts with that of its paralogue RAD51, which is associated with congenital mirror movements phenotypes, a phenotype also comorbid with PD.

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.

Exome sequencing of bilateral testicular germ cell tumors suggests independent development lineages.

PubMed

Brabrand, Sigmund; Johannessen, Bjarne; Axcrona, Ulrika; Kraggerud, Sigrid M; Berg, Kaja G; Bakken, Anne C; Bruun, Jarle; Fosså, Sophie D; Lothe, Ragnhild A; Lehne, Gustav; Skotheim, Rolf I

2015-02-01

Intratubular germ cell neoplasia, the precursor of testicular germ cell tumors (TGCTs), is hypothesized to arise during embryogenesis from developmentally arrested primordial germ cells (PGCs) or gonocytes. In early embryonal life, the PGCs migrate from the yolk sac to the dorsal body wall where the cell population separates before colonizing the genital ridges. However, whether the malignant transformation takes place before or after this separation is controversial. We have explored the somatic exome-wide mutational spectra of bilateral TGCT to provide novel insight into the in utero critical time frame of malignant transformation and TGCT pathogenesis. Exome sequencing was performed in five patients with bilateral TGCT (eight tumors), of these three patients in whom both tumors were available (six tumors) and two patients each with only one available tumor (two tumors). Selected loci were explored by Sanger sequencing in 71 patients with bilateral TGCT. From the exome-wide mutational spectra, no identical mutations in any of the three bilateral tumor pairs were identified. Exome sequencing of all eight tumors revealed 87 somatic non-synonymous mutations (median 10 per tumor; range 5-21), some in already known cancer genes such as CIITA, NEB, platelet-derived growth factor receptor α (PDGFRA), and WHSC1. SUPT6H was found recurrently mutated in two tumors. We suggest independent development lineages of bilateral TGCT. Thus, malignant transformation into intratubular germ cell neoplasia is likely to occur after the migration of PGCs. We reveal possible drivers of TGCT pathogenesis, such as mutated PDGFRA, potentially with therapeutic implications for TGCT patients. Copyright © 2014 Neoplasia Press, Inc. Published by Elsevier Inc. All rights reserved.

Exome sequencing and arrayCGH detection of gene sequence and copy number variation between ILS and ISS mouse strains.

PubMed

Dumas, Laura; Dickens, C Michael; Anderson, Nathan; Davis, Jonathan; Bennett, Beth; Radcliffe, Richard A; Sikela, James M

2014-06-01

It has been well documented that genetic factors can influence predisposition to develop alcoholism. While the underlying genomic changes may be of several types, two of the most common and disease associated are copy number variations (CNVs) and sequence alterations of protein coding regions. The goal of this study was to identify CNVs and single-nucleotide polymorphisms that occur in gene coding regions that may play a role in influencing the risk of an individual developing alcoholism. Toward this end, two mouse strains were used that have been selectively bred based on their differential sensitivity to alcohol: the Inbred long sleep (ILS) and Inbred short sleep (ISS) mouse strains. Differences in initial response to alcohol have been linked to risk for alcoholism, and the ILS/ISS strains are used to investigate the genetics of initial sensitivity to alcohol. Array comparative genomic hybridization (arrayCGH) and exome sequencing were conducted to identify CNVs and gene coding sequence differences, respectively, between ILS and ISS mice. Mouse arrayCGH was performed using catalog Agilent 1 × 244 k mouse arrays. Subsequently, exome sequencing was carried out using an Illumina HiSeq 2000 instrument. ArrayCGH detected 74 CNVs that were strain-specific (38 ILS/36 ISS), including several ISS-specific deletions that contained genes implicated in brain function and neurotransmitter release. Among several interesting coding variations detected by exome sequencing was the gain of a premature stop codon in the alpha-amylase 2B (AMY2B) gene specifically in the ILS strain. In total, exome sequencing detected 2,597 and 1,768 strain-specific exonic gene variants in the ILS and ISS mice, respectively. This study represents the most comprehensive and detailed genomic comparison of ILS and ISS mouse strains to date. The two complementary genome-wide approaches identified strain-specific CNVs and gene coding sequence variations that should provide strong candidates to

Whole exome sequencing identifies a homozygous nonsense variation in ALMS1 gene in a patient with syndromic obesity.

PubMed

Das Bhowmik, Aneek; Gupta, Neerja; Dalal, Ashwin; Kabra, Madhulika

In the present study we report on genetic analysis in a patient with developmental delay, truncal obesity and vision problem, to find the causative mutation. Whole exome sequencing was performed on genomic DNA extracted from whole blood of the patient which revealed a homozygous nonsense variant (c.2816T>A) in exon 8 of ALMS1 gene that results in a stop codon and premature truncation at codon 939 (p.L939Ter) of the protein. The mutation was confirmed by Sanger sequencing. Exome sequencing was helpful in establishing diagnosis of Alstrom syndrome in this patient. This case highlights the utility of exome sequencing in clinical practice. Copyright © 2016 Asia Oceania Association for the Study of Obesity. Published by Elsevier Ltd. All rights reserved.

Unexpected allelic heterogeneity and spectrum of mutations in Fowler syndrome revealed by next-generation exome sequencing.

PubMed

Lalonde, Emilie; Albrecht, Steffen; Ha, Kevin C H; Jacob, Karine; Bolduc, Nathalie; Polychronakos, Constantin; Dechelotte, Pierre; Majewski, Jacek; Jabado, Nada

2010-08-01

Protein coding genes constitute approximately 1% of the human genome but harbor 85% of the mutations with large effects on disease-related traits. Therefore, efficient strategies for selectively sequencing complete coding regions (i.e., "whole exome") have the potential to contribute our understanding of human diseases. We used a method for whole-exome sequencing coupling Agilent whole-exome capture to the Illumina DNA-sequencing platform, and investigated two unrelated fetuses from nonconsanguineous families with Fowler Syndrome (FS), a stereotyped phenotype lethal disease. We report novel germline mutations in feline leukemia virus subgroup C cellular-receptor-family member 2, FLVCR2, which has recently been shown to cause FS. Using this technology, we identified three types of genetic abnormalities: point-mutations, insertions-deletions, and intronic splice-site changes (first pathogenic report using this technology), in the fetuses who both were compound heterozygotes for the disease. Although revealing a high level of allelic heterogeneity and mutational spectrum in FS, this study further illustrates the successful application of whole-exome sequencing to uncover genetic defects in rare Mendelian disorders. Of importance, we show that we can identify genes underlying rare, monogenic and recessive diseases using a limited number of patients (n=2), in the absence of shared genetic heritage and in the presence of allelic heterogeneity.

Promises, pitfalls and practicalities of prenatal whole exome sequencing.

PubMed

Best, Sunayna; Wou, Karen; Vora, Neeta; Van der Veyver, Ignatia B; Wapner, Ronald; Chitty, Lyn S

2018-01-01

Prenatal genetic diagnosis provides information for pregnancy and perinatal decision-making and management. In several small series, prenatal whole exome sequencing (WES) approaches have identified genetic diagnoses when conventional tests (karyotype and microarray) were not diagnostic. Here, we review published prenatal WES studies and recent conference abstracts. Thirty-one studies were identified, with diagnostic rates in series of five or more fetuses varying between 6.2% and 80%. Differences in inclusion criteria and trio versus singleton approaches to sequencing largely account for the wide range of diagnostic rates. The data suggest that diagnostic yields will be greater in fetuses with multiple anomalies or in cases preselected following genetic review. Beyond its ability to improve diagnostic rates, we explore the potential of WES to improve understanding of prenatal presentations of genetic disorders and lethal fetal syndromes. We discuss prenatal phenotyping limitations, counselling challenges regarding variants of uncertain significance, incidental and secondary findings, and technical problems in WES. We review the practical, ethical, social and economic issues that must be considered before prenatal WES could become part of routine testing. Finally, we reflect upon the potential future of prenatal genetic diagnosis, including a move towards whole genome sequencing and non-invasive whole exome and whole genome testing. © 2017 John Wiley & Sons, Ltd. © 2017 John Wiley & Sons, Ltd.

Exome sequencing in 32 patients with anophthalmia/microphthalmia and developmental eye defects.

PubMed

Slavotinek, A M; Garcia, S T; Chandratillake, G; Bardakjian, T; Ullah, E; Wu, D; Umeda, K; Lao, R; Tang, P L-F; Wan, E; Madireddy, L; Lyalina, S; Mendelsohn, B A; Dugan, S; Tirch, J; Tischler, R; Harris, J; Clark, M J; Chervitz, S; Patwardhan, A; West, J M; Ursell, P; de Alba Campomanes, A; Schneider, A; Kwok, P-Y; Baranzini, S; Chen, R O

2015-11-01

Anophthalmia/microphthalmia (A/M) is a genetically heterogeneous birth defect for which the etiology is unknown in more than 50% of patients. We used exome sequencing with the ACE Exome(TM) (Personalis, Inc; 18 cases) and UCSF Genomics Core (21 cases) to sequence 28 patients with A/M and four patients with varied developmental eye defects. In the 28 patients with A/M, we identified de novo mutations in three patients (OTX2, p.(Gln91His), RARB, p.Arg387Cys and GDF6, p.Ala249Glu) and inherited mutations in STRA6 in two patients. In patients with developmental eye defects, a female with cataracts and cardiomyopathy had a de novo COL4A1 mutation, p.(Gly773Arg), expanding the phenotype associated with COL4A1 to include cardiomyopathy. A male with a chorioretinal defect, microcephaly, seizures and sensorineural deafness had two PNPT1 mutations, p.(Ala507Ser) and c.401-1G>A, and we describe eye defects associated with this gene for the first time. Exome sequencing was efficient for identifying mutations in pathogenic genes for which there is no clinical testing available and for identifying cases that expand phenotypic spectra, such as the PNPT1 and COL4A1-associated disorders described here. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Application of exome sequencing in the search for genetic causes of rare disorders of copper metabolism.

PubMed

Fuchs, Sabine A; Harakalova, Magdalena; van Haaften, Gijs; van Hasselt, Peter M; Cuppen, Edwin; Houwen, Roderick H J

2012-07-01

The genetic defect in a number of rare disorders of metal metabolism remains elusive. The limited number of patients with these disorders impedes the identification of the causative gene through positional cloning, which requires numerous families with multiple affected individuals. However, with next-generation sequencing all coding DNA (exomes) or whole genomes of patients can be sequenced to identify genes that are consistently mutated in patients. With this strategy only a limited number of patients and/or pedigrees is needed, bringing the elucidation of the genetic cause of even very rare diseases within reach. The main challenge associated with whole exome sequencing is the identification of the disease-causing mutation(s) among abundant genetic candidate variants. We describe several strategies to manage this data wealth, including comparison with control databases, increasing the number of patients and controls, and reducing the genomic region under investigation through homozygosity mapping. In this review we introduce a number of rare disorders of copper metabolism, with a suspected but yet unknown monogenetic cause, as an attractive target for this strategy. We anticipate that use of these novel techniques will identify the basic defect in the disorders described in this review, as well as in other genetic disorders of metal metabolism, in the next few years.

Whole Exome Sequencing of Pediatric Gastric Adenocarcinoma Reveals an Atypical Presentation of Li-Fraumeni Syndrome

PubMed Central

Chang, Vivian Y.; Federman, Noah; Martinez-Agosto, Julian; Tatishchev, Sergei F.; Nelson, Stanley F.

2014-01-01

Background Gastric adenocarcinoma is a rare diagnosis in childhood. A 14-year old male patient presented with metastatic gastric adenocarcinoma, and a strong family history of colon cancer. Clinical sequencing of CDH1 and APC were negative. Whole exome sequencing was therefore applied to capture the majority of protein-coding regions for the identification of single-nucleotide variants, small insertion/deletions, and copy number abnormalities in the patient’s germline as well as primary tumor. Materials and Methods DNA was extracted from the patient’s blood, primary tumor, and the unaffected mother’s blood. DNA libraries were constructed and sequenced on Illumina HiSeq2000. Data were post-processed using Picard and Samtools, then analyzed with the Genome Analysis Toolkit. Variants were annotated using an in-house Ensembl-based program. Copy number was assessed using ExomeCNV. Results Each sample was sequenced to a mean depth of coverage of greater than 120×. A rare non-synonymous coding SNV in TP53 was identified in the germline. There were 10 somatic cancer protein-damaging variants that were not observed in the unaffected mother genome. ExomeCNV comparing tumor to the patient’s germline, identified abnormal copy number, spanning 6,946 genes. Conclusion We present an unusual case of Li-Fraumeni detected by whole exome sequencing. There were also likely driver somatic mutations in the gastric adenocarcinoma. These results highlight the need for more thorough and broad scale germline and cancer analyses to accurately inform patients of inherited risk to cancer and to identify somatic mutations. PMID:23015295

An Observational Study of Children's Involvement in Informed Consent for Exome Sequencing Research.

PubMed

Miller, Victoria A; Werner-Lin, Allison; Walser, Sarah A; Biswas, Sawona; Bernhardt, Barbara A

2017-02-01

The goal of this study was to examine children's involvement in consent sessions for exome sequencing research and associations of involvement with provider and parent communication. Participants included 44 children (8-17 years) from five cohorts who were offered participation in an exome sequencing study. The consent sessions were audiotaped, transcribed, and coded. Providers attempted to facilitate the child's involvement in the majority (73%) of sessions, and most (75%) children also verbally participated. Provider facilitation was strongly associated with likelihood of child participation. These findings underscore that strategies such as asking for children's opinions and soliciting their questions show respect for children and may increase the likelihood that they are engaged and involved in decisions about research participation.

Exome-wide Sequencing Shows Low Mutation Rates and Identifies Novel Mutated Genes in Seminomas.

PubMed

Cutcutache, Ioana; Suzuki, Yuka; Tan, Iain Beehuat; Ramgopal, Subhashini; Zhang, Shenli; Ramnarayanan, Kalpana; Gan, Anna; Lee, Heng Hong; Tay, Su Ting; Ooi, Aikseng; Ong, Choon Kiat; Bolthouse, Jonathan T; Lane, Brian R; Anema, John G; Kahnoski, Richard J; Tan, Patrick; Teh, Bin Tean; Rozen, Steven G

2015-07-01

Testicular germ cell tumors are the most common cancer diagnosed in young men, and seminomas are the most common type of these cancers. There have been no exome-wide examinations of genes mutated in seminomas or of overall rates of nonsilent somatic mutations in these tumors. The objective was to analyze somatic mutations in seminomas to determine which genes are affected and to determine rates of nonsilent mutations. Eight seminomas and matched normal samples were surgically obtained from eight patients. DNA was extracted from tissue samples and exome sequenced on massively parallel Illumina DNA sequencers. Single-nucleotide polymorphism chip-based copy number analysis was also performed to assess copy number alterations. The DNA sequencing read data were analyzed to detect somatic mutations including single-nucleotide substitutions and short insertions and deletions. The detected mutations were validated by independent sequencing and further checked for subclonality. The rate of nonsynonymous somatic mutations averaged 0.31 mutations/Mb. We detected nonsilent somatic mutations in 96 genes that were not previously known to be mutated in seminomas, of which some may be driver mutations. Many of the mutations appear to have been present in subclonal populations. In addition, two genes, KIT and KRAS, were affected in two tumors each with mutations that were previously observed in other cancers and are presumably oncogenic. Our study, the first report on exome sequencing of seminomas, detected somatic mutations in 96 new genes, several of which may be targetable drivers. Furthermore, our results show that seminoma mutation rates are five times higher than previously thought, but are nevertheless low compared to other common cancers. Similar low rates are seen in other cancers that also have excellent rates of remission achieved with chemotherapy. We examined the DNA sequences of seminomas, the most common type of testicular germ cell cancer. Our study identified 96

Experience of targeted Usher exome sequencing as a clinical test

PubMed Central

Besnard, Thomas; García-García, Gema; Baux, David; Vaché, Christel; Faugère, Valérie; Larrieu, Lise; Léonard, Susana; Millan, Jose M; Malcolm, Sue; Claustres, Mireille; Roux, Anne-Françoise

2014-01-01

We show that massively parallel targeted sequencing of 19 genes provides a new and reliable strategy for molecular diagnosis of Usher syndrome (USH) and nonsyndromic deafness, particularly appropriate for these disorders characterized by a high clinical and genetic heterogeneity and a complex structure of several of the genes involved. A series of 71 patients including Usher patients previously screened by Sanger sequencing plus newly referred patients was studied. Ninety-eight percent of the variants previously identified by Sanger sequencing were found by next-generation sequencing (NGS). NGS proved to be efficient as it offers analysis of all relevant genes which is laborious to reach with Sanger sequencing. Among the 13 newly referred Usher patients, both mutations in the same gene were identified in 77% of cases (10 patients) and one candidate pathogenic variant in two additional patients. This work can be considered as pilot for implementing NGS for genetically heterogeneous diseases in clinical service. PMID:24498627

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.

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 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

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.

Comprehensive comparison of three commercial human whole-exome capture platforms.

PubMed

Asan; Xu, Yu; Jiang, Hui; Tyler-Smith, Chris; Xue, Yali; Jiang, Tao; Wang, Jiawei; Wu, Mingzhi; Liu, Xiao; Tian, Geng; Wang, Jun; Wang, Jian; Yang, Huangming; Zhang, Xiuqing

2011-09-28

Exome sequencing, which allows the global analysis of protein coding sequences in the human genome, has become an effective and affordable approach to detecting causative genetic mutations in diseases. Currently, there are several commercial human exome capture platforms; however, the relative performances of these have not been characterized sufficiently to know which is best for a particular study. We comprehensively compared three platforms: NimbleGen's Sequence Capture Array and SeqCap EZ, and Agilent's SureSelect. We assessed their performance in a variety of ways, including number of genes covered and capture efficacy. Differences that may impact on the choice of platform were that Agilent SureSelect covered approximately 1,100 more genes, while NimbleGen provided better flanking sequence capture. Although all three platforms achieved similar capture specificity of targeted regions, the NimbleGen platforms showed better uniformity of coverage and greater genotype sensitivity at 30- to 100-fold sequencing depth. All three platforms showed similar power in exome SNP calling, including medically relevant SNPs. Compared with genotyping and whole-genome sequencing data, the three platforms achieved a similar accuracy of genotype assignment and SNP detection. Importantly, all three platforms showed similar levels of reproducibility, GC bias and reference allele bias. We demonstrate key differences between the three platforms, particularly advantages of solutions over array capture and the importance of a large gene target set.

Clinical exome sequencing reports: current informatics practice and future opportunities.

PubMed

Swaminathan, Rajeswari; Huang, Yungui; Astbury, Caroline; Fitzgerald-Butt, Sara; Miller, Katherine; Cole, Justin; Bartlett, Christopher; Lin, Simon

2017-11-01

The increased adoption of clinical whole exome sequencing (WES) has improved the diagnostic yield for patients with complex genetic conditions. However, the informatics practice for handling information contained in whole exome reports is still in its infancy, as evidenced by the lack of a common vocabulary within clinical sequencing reports generated across genetic laboratories. Genetic testing results are mostly transmitted using portable document format, which can make secondary analysis and data extraction challenging. This paper reviews a sample of clinical exome reports generated by Clinical Laboratory Improvement Amendments-certified genetic testing laboratories at tertiary-care facilities to assess and identify common data elements. Like structured radiology reports, which enable faster information retrieval and reuse, structuring genetic information within clinical WES reports would help facilitate integration of genetic information into electronic health records and enable retrospective research on the clinical utility of WES. We identify elements listed as mandatory according to practice guidelines but are currently missing from some of the clinical reports, which might help to organize the data when stored within structured databases. We also highlight elements, such as patient consent, that, although they do not appear within any of the current reports, may help in interpreting some of the information within the reports. Integrating genetic and clinical information would assist the adoption of personalized medicine for improved patient care and outcomes. © The Author 2017. Published by Oxford University Press on behalf of the American Medical Informatics Association. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Whole exome sequencing is an efficient, sensitive and specific method of mutation detection in osteogenesis imperfecta and Marfan syndrome

PubMed Central

McInerney-Leo, Aideen M; Marshall, Mhairi S; Gardiner, Brooke; Coucke, Paul J; Van Laer, Lut; Loeys, Bart L; Summers, Kim M; Symoens, Sofie; West, Jennifer A; West, Malcolm J; Paul Wordsworth, B; Zankl, Andreas; Leo, Paul J; Brown, Matthew A; Duncan, Emma L

2013-01-01

Osteogenesis imperfecta (OI) and Marfan syndrome (MFS) are common Mendelian disorders. Both conditions are usually diagnosed clinically, as genetic testing is expensive due to the size and number of potentially causative genes and mutations. However, genetic testing may benefit patients, at-risk family members and individuals with borderline phenotypes, as well as improving genetic counseling and allowing critical differential diagnoses. We assessed whether whole exome sequencing (WES) is a sensitive method for mutation detection in OI and MFS. WES was performed on genomic DNA from 13 participants with OI and 10 participants with MFS who had known mutations, with exome capture followed by massive parallel sequencing of multiplexed samples. Single nucleotide polymorphisms (SNPs) and small indels were called using Genome Analysis Toolkit (GATK) and annotated with ANNOVAR. CREST, exomeCopy and exomeDepth were used for large deletion detection. Results were compared with the previous data. Specificity was calculated by screening WES data from a control population of 487 individuals for mutations in COL1A1, COL1A2 and FBN1. The target capture of five exome capture platforms was compared. All 13 mutations in the OI cohort and 9/10 in the MFS cohort were detected (sensitivity=95.6%) including non-synonymous SNPs, small indels (<10 bp), and a large UTR5/exon 1 deletion. One mutation was not detected by GATK due to strand bias. Specificity was 99.5%. Capture platforms and analysis programs differed considerably in their ability to detect mutations. Consumable costs for WES were low. WES is an efficient, sensitive, specific and cost-effective method for mutation detection in patients with OI and MFS. Careful selection of platform and analysis programs is necessary to maximize success. PMID:24501682

Whole exome sequence analysis of Peters anomaly

PubMed Central

Weh, Eric; Reis, Linda M.; Happ, Hannah C.; Levin, Alex V.; Wheeler, Patricia G.; David, Karen L.; Carney, Erin; Angle, Brad; Hauser, Natalie

2015-01-01

Peters anomaly is a rare form of anterior segment ocular dysgenesis, which can also be associated with additional systemic defects. At this time, the majority of cases of Peters anomaly lack a genetic diagnosis. We performed whole exome sequencing of 27 patients with syndromic or isolated Peters anomaly to search for pathogenic mutations in currently known ocular genes. Among the eight previously recognized Peters anomaly genes, we identified a de novo missense mutation in PAX6, c.155G>A, p.(Cys52Tyr), in one patient. Analysis of 691 additional genes currently associated with a different ocular phenotype identified a heterozygous splicing mutation c.1025+2T>A in TFAP2A, a de novo heterozygous nonsense mutation c.715C>T, p.(Gln239*) in HCCS, a hemizygous mutation c.385G>A, p.(Glu129Lys) in NDP, a hemizygous mutation c.3446C>T, p.(Pro1149Leu) in FLNA, and compound heterozygous mutations c.1422T>A, p.(Tyr474*) and c.2544G>A, p.(Met848Ile) in SLC4A11; all mutations, except for the FLNA and SLC4A11 c.2544G>A alleles, are novel. This is the frst study to use whole exome sequencing to discern the genetic etiology of a large cohort of patients with syndromic or isolated Peters anomaly. We report five new genes associated with this condition and suggest screening of TFAP2A and FLNA in patients with Peters anomaly and relevant syndromic features and HCCS, NDP and SLC4A11 in patients with isolated Peters anomaly. PMID:25182519

Exome Sequencing Fails to Identify the Genetic Cause of Aicardi Syndrome.

PubMed

Lund, Caroline; Striano, Pasquale; Sorte, Hanne Sørmo; Parisi, Pasquale; Iacomino, Michele; Sheng, Ying; Vigeland, Magnus D; Øye, Anne-Marte; Møller, Rikke Steensbjerre; Selmer, Kaja K; Zara, Federico

2016-09-01

Aicardi syndrome (AS) is a well-characterized neurodevelopmental disorder with an unknown etiology. In this study, we performed whole-exome sequencing in 11 female patients with the diagnosis of AS, in order to identify the disease-causing gene. In particular, we focused on detecting variants in the X chromosome, including the analysis of variants with a low number of sequencing reads, in case of somatic mosaicism. For 2 of the patients, we also sequenced the exome of the parents to search for de novo mutations. We did not identify any genetic variants likely to be damaging. Only one single missense variant was identified by the de novo analyses of the 2 trios, and this was considered benign. The failure to identify a disease gene in this study may be due to technical limitations of our study design, including the possibility that the genetic aberration leading to AS is situated in a non-exonic region or that the mutation is somatic and not detectable by our approach. Alternatively, it is possible that AS is genetically heterogeneous and that 11 patients are not sufficient to reveal the causative genes. Future studies of AS should consider designs where also non-exonic regions are explored and apply a sequencing depth so that also low-grade somatic mosaicism can be detected.

Exome sequencing supports a de novo mutational paradigm for schizophrenia

PubMed Central

Xu, Bin; Roos, J. Louw; Dexheimer, Phillip; Boone, Braden; Plummer, Brooks; Levy, Shawn; Gogos, Joseph A.; Karayiorgou, Maria

2011-01-01

Despite high heritability, a large fraction of cases with schizophrenia do not have a family history of the disease (sporadic cases). Here, we examine the possibility that rare de novo protein-altering mutations contribute to the genetic component of schizophrenia by sequencing the exome of 53 sporadic cases, 22 unaffected controls and their parents. We identified 40 de novo mutations in 27 patients affecting 40 genes including a potentially disruptive mutation in DGCR2, a gene removed by the recurrent schizophrenia-predisposing 22q11.2 microdeletion. Comparison to rare inherited variants revealed that the identified de novo mutations show a large excess of nonsynonymous changes in cases, as well as a greater potential to affect protein structure and function. Our analysis reveals a major role of de novo mutations in schizophrenia and also a large mutational target, which together provide a plausible explanation for the high global incidence and persistence of the disease. PMID:21822266

Targeted exome sequencing identifies novel compound heterozygous mutations in P3H1 in a fetus with osteogenesis imperfecta type VIII.

PubMed

Huang, Yanru; Mei, Libin; Lv, Weigang; Li, Haoxian; Zhang, Rui; Pan, Qian; Tan, Hu; Guo, Jing; Luo, Xiaomei; Chen, Chen; Liang, Desheng; Wu, Lingqian

2017-01-01

Osteogenesis imperfecta (OI) is a highly clinically and genetically heterogeneous group of disorders. It is difficult to identify severe OI in the perinatal period. Here, a Chinese woman with a suspected history of fetal OI was referred to our institution at 19weeks of gestation, due to ultrasound inspection during antenatal screening, which revealed bulbous metaphyses, short humeri, and short thick bent femora in the fetus. Using targeted exome sequencing of 248 genes known to be involved in skeletal system diseases, we identified novel compound heterozygous mutation in the P3H1 gene in the fetus with OI type VIII: c.105_120del (p.D36Rfs*16) and c.2164C>T (p.Q722*). These two mutations were inherited from the father and mother, respectively. The mRNA level of P3H1 wasn't changed suggested that mRNA with this mutation escaped from nonsense-mediated RNA decay. Besides, the level of P3H1 was absence while the CRTAP was mildly decreased. In conclusion, our findings imply this novel compound heterozygous mutation as the molecular pathogenetic in a Chinese fetus with OI type VIII, and demonstrate that targeted next-generation sequencing (NGS) is an accurate, rapid, and cost-effective method in the genetic diagnosis of fetal skeletal dysplasia with genetic and clinical heterogeneity, especially for autosomal recessive skeletal disorders. Copyright © 2016 Elsevier B.V. All rights reserved.

Evaluation of somatic copy number estimation tools for whole-exome sequencing data.

PubMed

Nam, Jae-Yong; Kim, Nayoung K D; Kim, Sang Cheol; Joung, Je-Gun; Xi, Ruibin; Lee, Semin; Park, Peter J; Park, Woong-Yang

2016-03-01

Whole-exome sequencing (WES) has become a standard method for detecting genetic variants in human diseases. Although the primary use of WES data has been the identification of single nucleotide variations and indels, these data also offer a possibility of detecting copy number variations (CNVs) at high resolution. However, WES data have uneven read coverage along the genome owing to the target capture step, and the development of a robust WES-based CNV tool is challenging. Here, we evaluate six WES somatic CNV detection tools: ADTEx, CONTRA, Control-FREEC, EXCAVATOR, ExomeCNV and Varscan2. Using WES data from 50 kidney chromophobe, 50 bladder urothelial carcinoma, and 50 stomach adenocarcinoma patients from The Cancer Genome Atlas, we compared the CNV calls from the six tools with a reference CNV set that was identified by both single nucleotide polymorphism array 6.0 and whole-genome sequencing data. We found that these algorithms gave highly variable results: visual inspection reveals significant differences between the WES-based segmentation profiles and the reference profile, as well as among the WES-based profiles. Using a 50% overlap criterion, 13-77% of WES CNV calls were covered by CNVs from the reference set, up to 21% of the copy gains were called as losses or vice versa, and dramatic differences in CNV sizes and CNV numbers were observed. Overall, ADTEx and EXCAVATOR had the best performance with relatively high precision and sensitivity. We suggest that the current algorithms for somatic CNV detection from WES data are limited in their performance and that more robust algorithms are needed. © The Author 2015. Published by Oxford University Press. For Permissions, please email: journals.permissions@oup.com.

Identification of Small Exonic CNV from Whole-Exome Sequence Data and Application to Autism Spectrum Disorder

PubMed Central

Poultney, Christopher S.; Goldberg, Arthur P.; Drapeau, Elodie; Kou, Yan; Harony-Nicolas, Hala; Kajiwara, Yuji; De Rubeis, Silvia; Durand, Simon; Stevens, Christine; Rehnström, Karola; Palotie, Aarno; Daly, Mark J.; Ma’ayan, Avi; Fromer, Menachem; Buxbaum, Joseph D.

2013-01-01

Copy number variation (CNV) is an important determinant of human diversity and plays important roles in susceptibility to disease. Most studies of CNV carried out to date have made use of chromosome microarray and have had a lower size limit for detection of about 30 kilobases (kb). With the emergence of whole-exome sequencing studies, we asked whether such data could be used to reliably call rare exonic CNV in the size range of 1–30 kilobases (kb), making use of the eXome Hidden Markov Model (XHMM) program. By using both transmission information and validation by molecular methods, we confirmed that small CNV encompassing as few as three exons can be reliably called from whole-exome data. We applied this approach to an autism case-control sample (n = 811, mean per-target read depth = 161) and observed a significant increase in the burden of rare (MAF ≤1%) 1–30 kb CNV, 1–30 kb deletions, and 1–10 kb deletions in ASD. CNV in the 1–30 kb range frequently hit just a single gene, and we were therefore able to carry out enrichment and pathway analyses, where we observed enrichment for disruption of genes in cytoskeletal and autophagy pathways in ASD. In summary, our results showed that XHMM provided an effective means to assess small exonic CNV from whole-exome data, indicated that rare 1–30 kb exonic deletions could contribute to risk in up to 7% of individuals with ASD, and implicated a candidate pathway in developmental delay syndromes. PMID:24094742

Exome and deep sequencing of clinically aggressive neuroblastoma reveal somatic mutations that affect key pathways involved in cancer progression

PubMed Central

Lasorsa, Vito Alessandro; Formicola, Daniela; Pignataro, Piero; Cimmino, Flora; Calabrese, Francesco Maria; Mora, Jaume; Esposito, Maria Rosaria; Pantile, Marcella; Zanon, Carlo; De Mariano, Marilena; Longo, Luca; Hogarty, Michael D.; de Torres, Carmen; Tonini, Gian Paolo; Iolascon, Achille; Capasso, Mario

2016-01-01

The spectrum of somatic mutation of the most aggressive forms of neuroblastoma is not completely determined. We sought to identify potential cancer drivers in clinically aggressive neuroblastoma. Whole exome sequencing was conducted on 17 germline and tumor DNA samples from high-risk patients with adverse events within 36 months from diagnosis (HR-Event3) to identify somatic mutations and deep targeted sequencing of 134 genes selected from the initial screening in additional 48 germline and tumor pairs (62.5% HR-Event3 and high-risk patients), 17 HR-Event3 tumors and 17 human-derived neuroblastoma cell lines. We revealed 22 significantly mutated genes, many of which implicated in cancer progression. Fifteen genes (68.2%) were highly expressed in neuroblastoma supporting their involvement in the disease. CHD9, a cancer driver gene, was the most significantly altered (4.0% of cases) after ALK. Other genes (PTK2, NAV3, NAV1, FZD1 and ATRX), expressed in neuroblastoma and involved in cell invasion and migration were mutated at frequency ranged from 4% to 2%. Focal adhesion and regulation of actin cytoskeleton pathways, were frequently disrupted (14.1% of cases) thus suggesting potential novel therapeutic strategies to prevent disease progression. Notably BARD1, CHEK2 and AXIN2 were enriched in rare, potentially pathogenic, germline variants. In summary, whole exome and deep targeted sequencing identified novel cancer genes of clinically aggressive neuroblastoma. Our analyses show pathway-level implications of infrequently mutated genes in leading neuroblastoma progression. PMID:27009842

Exome and deep sequencing of clinically aggressive neuroblastoma reveal somatic mutations that affect key pathways involved in cancer progression.

PubMed

Lasorsa, Vito Alessandro; Formicola, Daniela; Pignataro, Piero; Cimmino, Flora; Calabrese, Francesco Maria; Mora, Jaume; Esposito, Maria Rosaria; Pantile, Marcella; Zanon, Carlo; De Mariano, Marilena; Longo, Luca; Hogarty, Michael D; de Torres, Carmen; Tonini, Gian Paolo; Iolascon, Achille; Capasso, Mario

2016-04-19

The spectrum of somatic mutation of the most aggressive forms of neuroblastoma is not completely determined. We sought to identify potential cancer drivers in clinically aggressive neuroblastoma.Whole exome sequencing was conducted on 17 germline and tumor DNA samples from high-risk patients with adverse events within 36 months from diagnosis (HR-Event3) to identify somatic mutations and deep targeted sequencing of 134 genes selected from the initial screening in additional 48 germline and tumor pairs (62.5% HR-Event3 and high-risk patients), 17 HR-Event3 tumors and 17 human-derived neuroblastoma cell lines.We revealed 22 significantly mutated genes, many of which implicated in cancer progression. Fifteen genes (68.2%) were highly expressed in neuroblastoma supporting their involvement in the disease. CHD9, a cancer driver gene, was the most significantly altered (4.0% of cases) after ALK.Other genes (PTK2, NAV3, NAV1, FZD1 and ATRX), expressed in neuroblastoma and involved in cell invasion and migration were mutated at frequency ranged from 4% to 2%.Focal adhesion and regulation of actin cytoskeleton pathways, were frequently disrupted (14.1% of cases) thus suggesting potential novel therapeutic strategies to prevent disease progression.Notably BARD1, CHEK2 and AXIN2 were enriched in rare, potentially pathogenic, germline variants.In summary, whole exome and deep targeted sequencing identified novel cancer genes of clinically aggressive neuroblastoma. Our analyses show pathway-level implications of infrequently mutated genes in leading neuroblastoma progression.

Use of whole exome sequencing for the identification of Ito-based arrhythmia mechanism and therapy.

PubMed

Sturm, Amy C; Kline, Crystal F; Glynn, Patric; Johnson, Benjamin L; Curran, Jerry; Kilic, Ahmet; Higgins, Robert S D; Binkley, Philip F; Janssen, Paul M L; Weiss, Raul; Raman, Subha V; Fowler, Steven J; Priori, Silvia G; Hund, Thomas J; Carnes, Cynthia A; Mohler, Peter J

2015-05-26

Identified genetic variants are insufficient to explain all cases of inherited arrhythmia. We tested whether the integration of whole exome sequencing with well-established clinical, translational, and basic science platforms could provide rapid and novel insight into human arrhythmia pathophysiology and disease treatment. We report a proband with recurrent ventricular fibrillation, resistant to standard therapeutic interventions. Using whole-exome sequencing, we identified a variant in a previously unidentified exon of the dipeptidyl aminopeptidase-like protein-6 (DPP6) gene. This variant is the first identified coding mutation in DPP6 and augments cardiac repolarizing current (Ito) causing pathological changes in Ito and action potential morphology. We designed a therapeutic regimen incorporating dalfampridine to target Ito. Dalfampridine, approved for multiple sclerosis, normalized the ECG and reduced arrhythmia burden in the proband by >90-fold. This was combined with cilostazol to accelerate the heart rate to minimize the reverse-rate dependence of augmented Ito. We describe a novel arrhythmia mechanism and therapeutic approach to ameliorate the disease. Specifically, we identify the first coding variant of DPP6 in human ventricular fibrillation. These findings illustrate the power of genetic approaches for the elucidation and treatment of disease when carefully integrated with clinical and basic/translational research teams. © 2015 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

Paired Exome Analysis Reveals Clonal Evolution and Potential Therapeutic Targets in Urothelial Carcinoma.

PubMed

Lamy, Philippe; Nordentoft, Iver; Birkenkamp-Demtröder, Karin; Thomsen, Mathilde Borg Houlberg; Villesen, Palle; Vang, Søren; Hedegaard, Jakob; Borre, Michael; Jensen, Jørgen Bjerggaard; Høyer, Søren; Pedersen, Jakob Skou; Ørntoft, Torben F; Dyrskjøt, Lars

2016-10-01

Greater knowledge concerning tumor heterogeneity and clonality is needed to determine the impact of targeted treatment in the setting of bladder cancer. In this study, we performed whole-exome, transcriptome, and deep-focused sequencing of metachronous tumors from 29 patients initially diagnosed with early-stage bladder tumors (14 with nonprogressive disease and 15 with progressive disease). Tumors from patients with progressive disease showed a higher variance of the intrapatient mutational spectrum and a higher frequency of APOBEC-related mutations. Allele-specific expression was also higher in these patients, particularly in tumor suppressor genes. Phylogenetic analysis revealed a common origin of the metachronous tumors, with a higher proportion of clonal mutations in the ancestral branch; however, 19 potential therapeutic targets were identified as both ancestral and tumor-specific alterations. Few subclones were present based on PyClone analysis. Our results illuminate tumor evolution and identify candidate therapeutic targets in bladder cancer. Cancer Res; 76(19); 5894-906. ©2016 AACR. ©2016 American Association for Cancer Research.

Amplicon-based semiconductor sequencing of human exomes: performance evaluation and optimization strategies.

PubMed

Damiati, E; Borsani, G; Giacopuzzi, Edoardo

2016-05-01

The Ion Proton platform allows to perform whole exome sequencing (WES) at low cost, providing rapid turnaround time and great flexibility. Products for WES on Ion Proton system include the AmpliSeq Exome kit and the recently introduced HiQ sequencing chemistry. Here, we used gold standard variants from GIAB consortium to assess the performances in variants identification, characterize the erroneous calls and develop a filtering strategy to reduce false positives. The AmpliSeq Exome kit captures a large fraction of bases (>94 %) in human CDS, ClinVar genes and ACMG genes, but with 2,041 (7 %), 449 (13 %) and 11 (19 %) genes not fully represented, respectively. Overall, 515 protein coding genes contain hard-to-sequence regions, including 90 genes from ClinVar. Performance in variants detection was maximum at mean coverage >120×, while at 90× and 70× we measured a loss of variants of 3.2 and 4.5 %, respectively. WES using HiQ chemistry showed ~71/97.5 % sensitivity, ~37/2 % FDR and ~0.66/0.98 F1 score for indels and SNPs, respectively. The proposed low, medium or high-stringency filters reduced the amount of false positives by 10.2, 21.2 and 40.4 % for indels and 21.2, 41.9 and 68.2 % for SNP, respectively. Amplicon-based WES on Ion Proton platform using HiQ chemistry emerged as a competitive approach, with improved accuracy in variants identification. False-positive variants remain an issue for the Ion Torrent technology, but our filtering strategy can be applied to reduce erroneous variants.

Molecular Diagnostic Experience of Whole-Exome Sequencing in Adult Patients

PubMed Central

Posey, Jennifer E.; Rosenfeld, Jill A.; James, Regis A.; Bainbridge, Matthew; Niu, Zhiyv; Wang, Xia; Dhar, Shweta; Wiszniewski, Wojciech; Akdemir, Zeynep H.C.; Gambin, Tomasz; Xia, Fan; Person, Richard E.; Walkiewicz, Magdalena; Shaw, Chad A.; Sutton, V. Reid; Beaudet, Arthur L.; Muzny, Donna; Eng, Christine M.; Yang, Yaping; Gibbs, Richard A.; Lupski, James R.; Boerwinkle, Eric; Plon, Sharon E.

2015-01-01

Purpose Whole exome sequencing (WES) is increasingly used as a diagnostic tool in medicine, but prior reports focus on predominantly pediatric cohorts with neurologic or developmental disorders. We describe the diagnostic yield and characteristics of whole exome sequencing in adults. Methods We performed a retrospective analysis of consecutive WES reports for adults from a diagnostic laboratory. Phenotype composition was determined using Human Phenotype Ontology terms. Results Molecular diagnoses were reported for 17.5% (85/486) of adults, lower than a primarily pediatric population (25.2%; p=0.0003); the diagnostic rate was higher (23.9%) in those 18–30 years of age compared to patients over 30 years (10.4%; p=0.0001). Dual Mendelian diagnoses contributed to 7% of diagnoses, revealing blended phenotypes. Diagnoses were more frequent among individuals with abnormalities of the nervous system, skeletal system, head/neck, and growth. Diagnostic rate was independent of family history information, and de novo mutations contributed to 61.4% of autosomal dominant diagnoses. Conclusion Early WES experience in adults demonstrates molecular diagnoses in a substantial proportion of patients, informing clinical management, recurrence risk and recommendations for relatives. A positive family history was not predictive, consistent with molecular diagnoses often revealed by de novo events, informing the Mendelian basis of genetic disease in adults. PMID:26633545

Data Interoperability of Whole Exome Sequencing (WES) Based Mutational Burden Estimates from Different Laboratories

PubMed Central

Qiu, Ping; Pang, Ling; Arreaza, Gladys; Maguire, Maureen; Chang, Ken C. N.; Marton, Matthew J.; Levitan, Diane

2016-01-01

Immune checkpoint inhibitors, which unleash a patient’s own T cells to kill tumors, are revolutionizing cancer treatment. Several independent studies suggest that higher non-synonymous mutational burden assessed by whole exome sequencing (WES) in tumors is associated with improved objective response, durable clinical benefit, and progression-free survival in immune checkpoint inhibitors treatment. Next-generation sequencing (NGS) is a promising technology being used in the clinic to direct patient treatment. Cancer genome WES poses a unique challenge due to tumor heterogeneity and sequencing artifacts introduced by formalin-fixed, paraffin-embedded (FFPE) tissue. In order to evaluate the data interoperability of WES data from different sources to survey tumor mutational landscape, we compared WES data of several tumor/normal matched samples from five commercial vendors. A large data discrepancy was observed from vendors’ self-reported data. Independent data analysis from vendors’ raw NGS data shows that whole exome sequencing data from qualified vendors can be combined and analyzed uniformly to derive comparable quantitative estimates of tumor mutational burden. PMID:27136543

Exome sequencing of extreme phenotypes identifies DCTN4 as a modifier of chronic Pseudomonas aeruginosa infection in cystic fibrosis.

PubMed

Emond, Mary J; Louie, Tin; Emerson, Julia; Zhao, Wei; Mathias, Rasika A; Knowles, Michael R; Wright, Fred A; Rieder, Mark J; Tabor, Holly K; Nickerson, Deborah A; Barnes, Kathleen C; Gibson, Ronald L; Bamshad, Michael J

2012-07-08

Exome sequencing has become a powerful and effective strategy for the discovery of genes underlying Mendelian disorders. However, use of exome sequencing to identify variants associated with complex traits has been more challenging, partly because the sample sizes needed for adequate power may be very large. One strategy to increase efficiency is to sequence individuals who are at both ends of a phenotype distribution (those with extreme phenotypes). Because the frequency of alleles that contribute to the trait are enriched in one or both phenotype extremes, a modest sample size can potentially be used to identify novel candidate genes and/or alleles. As part of the National Heart, Lung, and Blood Institute (NHLBI) Exome Sequencing Project (ESP), we used an extreme phenotype study design to discover that variants in DCTN4, encoding a dynactin protein, are associated with time to first P. aeruginosa airway infection, chronic P. aeruginosa infection and mucoid P. aeruginosa in individuals with cystic fibrosis.

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

Molecular defects identified by whole exome sequencing in a child with Fanconi anemia.

PubMed

Zheng, Zhaojing; Geng, Juan; Yao, Ru-En; Li, Caihua; Ying, Daming; Shen, Yongnian; Ying, Lei; Yu, Yongguo; Fu, Qihua

2013-11-10

Fanconi anemia is a rare genetic disease characterized by bone marrow failure, multiple congenital malformations, and an increased susceptibility to malignancy. At least 15 genes have been identified that are involved in the pathogenesis of Fanconi anemia. However, it is still a challenge to assign the complementation group and to characterize the molecular defects in patients with Fanconi anemia. In the current study, whole exome sequencing was used to identify the affected gene(s) in a boy with Fanconi anemia. A recurring, non-synonymous mutation was found (c.3971C>T, p.P1324L) as well as a novel frameshift mutation (c.989_995del, p.H330LfsX2) in FANCA gene. Our results indicate that whole exome sequencing may be useful in clinical settings for rapid identification of disease-causing mutations in rare genetic disorders such as Fanconi anemia. © 2013 Elsevier B.V. All rights reserved.

Identification of small exonic CNV from whole-exome sequence data and application to autism spectrum disorder.

PubMed

Poultney, Christopher S; Goldberg, Arthur P; Drapeau, Elodie; Kou, Yan; Harony-Nicolas, Hala; Kajiwara, Yuji; De Rubeis, Silvia; Durand, Simon; Stevens, Christine; Rehnström, Karola; Palotie, Aarno; Daly, Mark J; Ma'ayan, Avi; Fromer, Menachem; Buxbaum, Joseph D

2013-10-03

Copy number variation (CNV) is an important determinant of human diversity and plays important roles in susceptibility to disease. Most studies of CNV carried out to date have made use of chromosome microarray and have had a lower size limit for detection of about 30 kilobases (kb). With the emergence of whole-exome sequencing studies, we asked whether such data could be used to reliably call rare exonic CNV in the size range of 1-30 kilobases (kb), making use of the eXome Hidden Markov Model (XHMM) program. By using both transmission information and validation by molecular methods, we confirmed that small CNV encompassing as few as three exons can be reliably called from whole-exome data. We applied this approach to an autism case-control sample (n = 811, mean per-target read depth = 161) and observed a significant increase in the burden of rare (MAF ≤1%) 1-30 kb CNV, 1-30 kb deletions, and 1-10 kb deletions in ASD. CNV in the 1-30 kb range frequently hit just a single gene, and we were therefore able to carry out enrichment and pathway analyses, where we observed enrichment for disruption of genes in cytoskeletal and autophagy pathways in ASD. In summary, our results showed that XHMM provided an effective means to assess small exonic CNV from whole-exome data, indicated that rare 1-30 kb exonic deletions could contribute to risk in up to 7% of individuals with ASD, and implicated a candidate pathway in developmental delay syndromes. Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

AUDIOME: a tiered exome sequencing-based comprehensive gene panel for the diagnosis of heterogeneous nonsyndromic sensorineural hearing loss.

PubMed

Guan, Qiaoning; Balciuniene, Jorune; Cao, Kajia; Fan, Zhiqian; Biswas, Sawona; Wilkens, Alisha; Gallo, Daniel J; Bedoukian, Emma; Tarpinian, Jennifer; Jayaraman, Pushkala; Sarmady, Mahdi; Dulik, Matthew; Santani, Avni; Spinner, Nancy; Abou Tayoun, Ahmad N; Krantz, Ian D; Conlin, Laura K; Luo, Minjie

2018-03-29

PurposeHereditary hearing loss is highly heterogeneous. To keep up with rapidly emerging disease-causing genes, we developed the AUDIOME test for nonsyndromic hearing loss (NSHL) using an exome sequencing (ES) platform and targeted analysis for the curated genes.MethodsA tiered strategy was implemented for this test. Tier 1 includes combined Sanger and targeted deletion analyses of the two most common NSHL genes and two mitochondrial genes. Nondiagnostic tier 1 cases are subjected to ES and array followed by targeted analysis of the remaining AUDIOME genes.ResultsES resulted in good coverage of the selected genes with 98.24% of targeted bases at >15 ×. A fill-in strategy was developed for the poorly covered regions, which generally fell within GC-rich or highly homologous regions. Prospective testing of 33 patients with NSHL revealed a diagnosis in 11 (33%) and a possible diagnosis in 8 cases (24.2%). Among those, 10 individuals had variants in tier 1 genes. The ES data in the remaining nondiagnostic cases are readily available for further analysis.ConclusionThe tiered and ES-based test provides an efficient and cost-effective diagnostic strategy for NSHL, with the potential to reflex to full exome to identify causal changes outside of the AUDIOME test.Genetics in Medicine advance online publication, 29 March 2018; doi:10.1038/gim.2018.48.

Whole exome sequencing in recurrent early pregnancy loss.

PubMed

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

2016-05-01

Exome sequencing can identify genetic causes of idiopathic recurrent pregnancy loss (RPL). 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. 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. 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. 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

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

Exome Sequencing in the Clinical Diagnosis of Sporadic or Familial Cerebellar Ataxia

PubMed Central

Fogel, Brent L.; Lee, Hane; Deignan, Joshua L.; Strom, Samuel P.; Kantarci, Sibel; Wang, Xizhe; Quintero-Rivera, Fabiola; Vilain, Eric; Grody, Wayne W.; Perlman, Susan; Geschwind, Daniel H.; Nelson, Stanley F.

2015-01-01

IMPORTANCE Cerebellar ataxias are a diverse collection of neurologic disorders with causes ranging from common acquired etiologies to rare genetic conditions. Numerous genetic disorders have been associated with chronic progressive ataxia and this consequently presents a diagnostic challenge for the clinician regarding how to approach and prioritize genetic testing in patients with such clinically heterogeneous phenotypes. Additionally, while the value of genetic testing in early-onset and/or familial cases seems clear, many patients with ataxia present sporadically with adult onset of symptoms and the contribution of genetic variation to the phenotype of these patients has not yet been established. OBJECTIVE To investigate the contribution of genetic disease in a population of patients with predominantly adult- and sporadic-onset cerebellar ataxia. DESIGN, SETTING, AND PARTICIPANTS We examined a consecutive series of 76 patients presenting to a tertiary referral center for evaluation of chronic progressive cerebellar ataxia. MAIN OUTCOMES AND MEASURES Next-generation exome sequencing coupled with comprehensive bioinformatic analysis, phenotypic analysis, and clinical correlation. RESULTS We identified clinically relevant genetic information in more than 60% of patients studied (n = 46), including diagnostic pathogenic gene variants in 21% (n = 16), a notable yield given the diverse genetics and clinical heterogeneity of the cerebellar ataxias. CONCLUSIONS AND RELEVANCE This study demonstrated that clinical exome sequencing in patients with adult-onset and sporadic presentations of ataxia is a high-yield test, providing a definitive diagnosis in more than one-fifth of patients and suggesting a potential diagnosis in more than one-third to guide additional phenotyping and diagnostic evaluation. Therefore, clinical exome sequencing is an appropriate consideration in the routine genetic evaluation of all patients presenting with chronic progressive cerebellar ataxia

Panel-based Genetic Diagnostic Testing for Inherited Eye Diseases is Highly Accurate and Reproducible and More Sensitive for Variant Detection Than Exome Sequencing

PubMed Central

Bujakowska, Kinga M.; Sousa, Maria E.; Fonseca-Kelly, Zoë D.; Taub, Daniel G.; Janessian, Maria; Wang, Dan Yi; Au, Elizabeth D.; Sims, Katherine B.; Sweetser, David A.; Fulton, Anne B.; Liu, Qin; Wiggs, Janey L.; Gai, Xiaowu; Pierce, Eric A.

2015-01-01

Purpose Next-generation sequencing (NGS) based methods are being adopted broadly for genetic diagnostic testing, but the performance characteristics of these techniques have not been fully defined with regard to test accuracy and reproducibility. Methods We developed a targeted enrichment and NGS approach for genetic diagnostic testing of patients with inherited eye disorders, including inherited retinal degenerations, optic atrophy and glaucoma. In preparation for providing this Genetic Eye Disease (GEDi) test on a CLIA-certified basis, we performed experiments to measure the sensitivity, specificity, reproducibility as well as the clinical sensitivity of the test. Results The GEDi test is highly reproducible and accurate, with sensitivity and specificity for single nucleotide variant detection of 97.9% and 100%, respectively. The sensitivity for variant detection was notably better than the 88.3% achieved by whole exome sequencing (WES) using the same metrics, due to better coverage of targeted genes in the GEDi test compared to commercially available exome capture sets. Prospective testing of 192 patients with IRDs indicated that the clinical sensitivity of the GEDi test is high, with a diagnostic rate of 51%. Conclusion The data suggest that based on quantified performance metrics, selective targeted enrichment is preferable to WES for genetic diagnostic testing. PMID:25412400

Exome Sequencing in 32 Patients with Anophthalmia/Microphthalmia and Developmental Eye Defects

PubMed Central

Slavotinek, Anne M.; Garcia, Sarah T.; Chandratillake, Gemma; Bardakjian, Tanya; Ullah, Ehsan; Wu, Di; Umeda, Kyle; Lao, Richard; Tang, Paul Ling-Fung; Wan, Eunice; Madireddy, Lohith; Lyalina, Svetlana; Mendelsohn, Bryce A.; Dugan, Sarah; Tirch, Jean; Tischler, Reana; Harris, Jason; Clark, Michael J.; Chervitz, Stephen; Patwardhan, Anil; West, John M.; Ursell, Phillip; de Alba Campomanes, Alejandra; Schneider, Adele; Kwok, Pui-yan; Baranzini, Sergio; Chen, Richard O.

2014-01-01

Anophthalmia/microphthalmia (A/M) is a genetically heterogeneous birth defect for which the etiology is unknown in more than 50% of patients. We used exome sequencing with the ACE Exome™ (Personalis, Inc; 18 cases) and UCSF Genomics Core (21 cases) to sequence 28 patients with A/M and four patients with varied developmental eye defects. In the 28 patients with A/M, we identified de novo mutations in three patients (OTX2, p.(Gln91His), RARB, p.Arg387Cys and GDF6, p.Ala249Glu) and inherited mutations in STRA6 in two patients. In patients with developmental eye defects, a female with cataracts and cardiomyopathy had a de novo COL4A1 mutation, p.(Gly773Arg), expanding the phenotype associated with COL4A1 to include cardiomyopathy. A male with a chorioretinal defect, microcephaly, seizures and sensorineural deafness had two PNPT1 mutations, p.(Ala507Ser) and c.401-1G>A, and we describe eye defects associated with this gene for the first time. Exome sequencing was efficient for identifying mutations in pathogenic genes for which there is no clinical testing available and for identifying cases that expand phenotypic spectra, such as the PNPT1 and COL4A1-associated disorders described here. PMID:25457163

Utility of whole exome sequencing in the diagnosis of Usher syndrome: Report of novel compound heterozygous MYO7A mutations.

PubMed

Ramzan, Khushnooda; Al-Owain, Mohammed; Huma, Rozeena; Al-Hazzaa, Selwa A F; Al-Ageel, Sarah; Imtiaz, Faiqa; Al-Sayed, Moeenaldeen

2018-05-01

Next generation sequencing (NGS), such as targeted panel sequencing, whole-exome sequencing and whole-genome sequencing has led to an exponential increase of elucidated genetic causes in both rare diseases, and common but heterogeneous disorders. NGS is applied in both research and clinical settings, and the clinical exome sequencing (CES), which provides not only the sequence variation data but also clinical interpretation, aids in reaching a final conclusion with regards to a genetic diagnosis. Usher syndrome is a group of disorders, characterized by bilateral sensorineural hearing loss, with or without vestibular dysfunction and retinitis pigmentosa. The index patient, a 2-year-old child was initially diagnosed with nonsyndromic hearing impairment. Homozygosity mapping followed by CES was utilized as a diagnostic tool to identify the genetic basis of his hearing loss. A paternally inherited novel insertion, c.198_199insA (p.Val67Serfs*73) and a maternally inherited novel deletion, c.1219_1226del (p.Phe407Aspfs*33) in gene MYO7A were found in compound heterozygous state in the index patient. The result expands the mutational spectrum of MYO7A. In addition it helped in early diagnosis of the syndrome, for planning and adjustments for the patient, and as well as for future family planning. This study highlights the clinical effectiveness of CES for Usher syndrome diagnosis in a child presented with congenital hearing loss. Copyright © 2018. Published by Elsevier B.V.

Whole-exome sequencing identifies novel homozygous mutation in NPAS2 in family with nonobstructive azoospermia.

PubMed

Ramasamy, Ranjith; Bakırcıoğlu, M Emre; Cengiz, Cenk; Karaca, Ender; Scovell, Jason; Jhangiani, Shalini N; Akdemir, Zeynep C; Bainbridge, Matthew; Yu, Yao; Huff, Chad; Gibbs, Richard A; Lupski, James R; Lamb, Dolores J

2015-08-01

To investigate the genetic cause of nonobstructive azoospermia (NOA) in a consanguineous Turkish family through homozygosity mapping followed by targeted exon/whole-exome sequencing to identify genetic variations. Whole-exome sequencing (WES). Research laboratory. Two siblings in a consanguineous family with NOA. Validating all variants passing filter criteria with Sanger sequencing to confirm familial segregation and absence in the control population. Discovery of a mutation that could potentially cause NOA. A novel nonsynonymous mutation in the neuronal PAS-2 domain (NPAS2) was identified in a consanguineous family from Turkey. This mutation in exon 14 (chr2: 101592000 C>G) of NPAS2 is likely a disease-causing mutation as it is predicted to be damaging, it is a novel variant, and it segregates with the disease. Family segregation of the variants showed the presence of the homozygous mutation in the three brothers with NOA and a heterozygous mutation in the mother as well as one brother and one sister who were both fertile. The mutation is not found in the single-nucleotide polymorphism database, the 1000 Genomes Project, the Baylor College of Medicine cohort of 500 Turkish patients (not a population-specific polymorphism), or the matching 50 fertile controls. With the use of WES we identified a novel homozygous mutation in NPAS2 as a likely disease-causing variant in a Turkish family diagnosed with NOA. Our data reinforce the clinical role of WES in the molecular diagnosis of highly heterogeneous genetic diseases for which conventional genetic approaches have previously failed to find a molecular diagnosis. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Exome capture sequencing identifies a novel mutation in BBS4

PubMed Central

Wang, Hui; Chen, Xianfeng; Dudinsky, Lynn; Patenia, Claire; Chen, Yiyun; Li, Yumei; Wei, Yue; Abboud, Emad B.; Al-Rajhi, Ali A.; Lewis, Richard Alan; Lupski, James R.; Mardon, Graeme; Gibbs, Richard A.; Perkins, Brian D.

2011-01-01

Purpose Leber congenital amaurosis (LCA) is one of the most severe eye dystrophies characterized by severe vision loss at an early stage and accounts for approximately 5% of all retinal dystrophies. The purpose of this study was to identify a novel LCA disease allele or gene and to develop an approach combining genetic mapping with whole exome sequencing. Methods Three patients from King Khaled Eye Specialist Hospital (KKESH205) underwent whole genome single nucleotide polymorphism genotyping, and a single candidate region was identified. Taking advantage of next-generation high-throughput DNA sequencing technologies, whole exome capture sequencing was performed on patient KKESH205#7. Sanger direct sequencing was used during the validation step. The zebrafish model was used to examine the function of the mutant allele. Results A novel missense mutation in Bardet-Biedl syndrome 4 protein (BBS4) was identified in a consanguineous family from Saudi Arabia. This missense mutation in the fifth exon (c.253G>C;p.E85Q) of BBS4 is likely a disease-causing mutation as it segregates with the disease. The mutation is not found in the single nucleotide polymorphism (SNP) database, the 1000 Genomes Project, or matching normal controls. Functional analysis of this mutation in zebrafish indicates that the G253C allele is pathogenic. Coinjection of the G253C allele cannot rescue the mislocalization of rhodopsin in the retina when BBS4 is knocked down by morpholino injection. Immunofluorescence analysis in cell culture shows that this missense mutation in BBS4 does not cause obvious defects in protein expression or pericentriolar localization. Conclusions This mutation likely mainly reduces or abolishes BBS4 function in the retina. Further studies of this allele will provide important insights concerning the pleiotropic nature of BBS4 function. PMID:22219648

Clinical germline diagnostic exome sequencing for hereditary cancer: Findings within novel candidate genes are prevalent.

PubMed

Powis, Zöe; Espenschied, Carin R; LaDuca, Holly; Hagman, Kelly D; Paudyal, Tripti; Li, Shuwei; Inaba, Hiroto; Mauer, Ann; Nathanson, Katherine L; Knost, James; Chao, Elizabeth C; Tang, Sha

2018-08-01

Clinical diagnostic exome sequencing (DES) has been effective in diagnosing individuals with suspected genetic conditions; nevertheless little has been described regarding its clinical utility in individuals with a personal and family history of cancer. This study aimed to assess diagnostic yield and clinical characteristics of pediatric and adult patients undergoing germline DES for hereditary cancer. We retrospectively reviewed 2171 patients referred for DES; cases with a personal and/or family history of cancer were further studied. Of 39 cancer patients, relevant alterations were found in eight individuals (21%), including one (3%) positive pathogenic alteration within a characterized gene, two (5%) uncertain findings in characterized genes, and five (13%) alterations in novel candidate genes. Two of the 5 pediatric patients, undergoing testing, (40%) had findings in novel candidate genes, with the remainder being negative. We include brief case studies to illustrate the variety of challenging issues related to these patients. Our observations demonstrate utility of family-based exome sequencing in patients for suspected hereditary cancer, including familial co-segregation analysis, and comprehensive medical review. DES may be particularly useful when traditional approaches do not result in a diagnosis or in families with unique phenotypes. This work also highlights the importance and complexity of analysis of uncharacterized genes in exome sequencing for hereditary cancer. Copyright © 2018 Elsevier Inc. All rights reserved.

Targeted cancer exome sequencing reveals recurrent mutations in myeloproliferative neoplasms

PubMed Central

Tenedini, E; Bernardis, I; Artusi, V; Artuso, L; Roncaglia, E; Guglielmelli, P; Pieri, L; Bogani, C; Biamonte, F; Rotunno, G; Mannarelli, C; Bianchi, E; Pancrazzi, A; Fanelli, T; Malagoli Tagliazucchi, G; Ferrari, S; Manfredini, R; Vannucchi, A M; Tagliafico, E

2014-01-01

With the intent of dissecting the molecular complexity of Philadelphia-negative myeloproliferative neoplasms (MPN), we designed a target enrichment panel to explore, using next-generation sequencing (NGS), the mutational status of an extensive list of 2000 cancer-associated genes and microRNAs. The genomic DNA of granulocytes and in vitro-expanded CD3+T-lymphocytes, as a germline control, was target-enriched and sequenced in a learning cohort of 20 MPN patients using Roche 454 technology. We identified 141 genuine somatic mutations, most of which were not previously described. To test the frequency of the identified variants, a larger validation cohort of 189 MPN patients was additionally screened for these mutations using Ion Torrent AmpliSeq NGS. Excluding the genes already described in MPN, for 8 genes (SCRIB, MIR662, BARD1, TCF12, FAT4, DAP3, POLG and NRAS), we demonstrated a mutation frequency between 3 and 8%. We also found that mutations at codon 12 of NRAS (NRASG12V and NRASG12D) were significantly associated, for primary myelofibrosis (PMF), with highest dynamic international prognostic scoring system (DIPSS)-plus score categories. This association was then confirmed in 66 additional PMF patients composing a final dataset of 168 PMF showing a NRAS mutation frequency of 4.7%, which was associated with a worse outcome, as defined by the DIPSS plus score. PMID:24150215

Distribution and clinical impact of functional variants in 50,726 whole-exome sequences from the DiscovEHR study.

PubMed

Dewey, Frederick E; Murray, Michael F; Overton, John D; Habegger, Lukas; Leader, Joseph B; Fetterolf, Samantha N; O'Dushlaine, Colm; Van Hout, Cristopher V; Staples, Jeffrey; Gonzaga-Jauregui, Claudia; Metpally, Raghu; Pendergrass, Sarah A; Giovanni, Monica A; Kirchner, H Lester; Balasubramanian, Suganthi; Abul-Husn, Noura S; Hartzel, Dustin N; Lavage, Daniel R; Kost, Korey A; Packer, Jonathan S; Lopez, Alexander E; Penn, John; Mukherjee, Semanti; Gosalia, Nehal; Kanagaraj, Manoj; Li, Alexander H; Mitnaul, Lyndon J; Adams, Lance J; Person, Thomas N; Praveen, Kavita; Marcketta, Anthony; Lebo, Matthew S; Austin-Tse, Christina A; Mason-Suares, Heather M; Bruse, Shannon; Mellis, Scott; Phillips, Robert; Stahl, Neil; Murphy, Andrew; Economides, Aris; Skelding, Kimberly A; Still, Christopher D; Elmore, James R; Borecki, Ingrid B; Yancopoulos, George D; Davis, F Daniel; Faucett, William A; Gottesman, Omri; Ritchie, Marylyn D; Shuldiner, Alan R; Reid, Jeffrey G; Ledbetter, David H; Baras, Aris; Carey, David J

2016-12-23

The DiscovEHR collaboration between the Regeneron Genetics Center and Geisinger Health System couples high-throughput sequencing to an integrated health care system using longitudinal electronic health records (EHRs). We sequenced the exomes of 50,726 adult participants in the DiscovEHR study to identify ~4.2 million rare single-nucleotide variants and insertion/deletion events, of which ~176,000 are predicted to result in a loss of gene function. Linking these data to EHR-derived clinical phenotypes, we find clinical associations supporting therapeutic targets, including genes encoding drug targets for lipid lowering, and identify previously unidentified rare alleles associated with lipid levels and other blood level traits. About 3.5% of individuals harbor deleterious variants in 76 clinically actionable genes. The DiscovEHR data set provides a blueprint for large-scale precision medicine initiatives and genomics-guided therapeutic discovery. Copyright © 2016, American Association for the Advancement of Science.

Whole-exome sequencing identifies USH2A mutations in a pseudo-dominant Usher syndrome family.

PubMed

Zheng, Sui-Lian; Zhang, Hong-Liang; Lin, Zhen-Lang; Kang, Qian-Yan

2015-10-01

Usher syndrome (USH) is an autosomal recessive (AR) multi-sensory degenerative disorder leading to deaf-blindness. USH is clinically subdivided into three subclasses, and 10 genes have been identified thus far. Clinical and genetic heterogeneities in USH make a precise diagnosis difficult. A dominant‑like USH family in successive generations was identified, and the present study aimed to determine the genetic predisposition of this family. Whole‑exome sequencing was performed in two affected patients and an unaffected relative. Systematic data were analyzed by bioinformatic analysis to remove the candidate mutations via step‑wise filtering. Direct Sanger sequencing and co‑segregation analysis were performed in the pedigree. One novel and two known mutations in the USH2A gene were identified, and were further confirmed by direct sequencing and co‑segregation analysis. The affected mother carried compound mutations in the USH2A gene, while the unaffected father carried a heterozygous mutation. The present study demonstrates that whole‑exome sequencing is a robust approach for the molecular diagnosis of disorders with high levels of genetic heterogeneity.

Analyzing Somatic Genome Rearrangements in Human Cancers by Using Whole-Exome Sequencing

PubMed Central

Yang, Lixing; Lee, Mi-Sook; Lu, Hengyu; Oh, Doo-Yi; Kim, Yeon Jeong; Park, Donghyun; Park, Gahee; Ren, Xiaojia; Bristow, Christopher A.; Haseley, Psalm S.; Lee, Soohyun; Pantazi, Angeliki; Kucherlapati, Raju; Park, Woong-Yang; Scott, Kenneth L.; Choi, Yoon-La; Park, Peter J.

2016-01-01

Although exome sequencing data are generated primarily to detect single-nucleotide variants and indels, they can also be used to identify a subset of genomic rearrangements whose breakpoints are located in or near exons. Using >4,600 tumor and normal pairs across 15 cancer types, we identified over 9,000 high confidence somatic rearrangements, including a large number of gene fusions. We find that the 5′ fusion partners of functional fusions are often housekeeping genes, whereas the 3′ fusion partners are enriched in tyrosine kinases. We establish the oncogenic potential of ROR1-DNAJC6 and CEP85L-ROS1 fusions by showing that they can promote cell proliferation in vitro and tumor formation in vivo. Furthermore, we found that ∼4% of the samples have massively rearranged chromosomes, many of which are associated with upregulation of oncogenes such as ERBB2 and TERT. Although the sensitivity of detecting structural alterations from exomes is considerably lower than that from whole genomes, this approach will be fruitful for the multitude of exomes that have been and will be generated, both in cancer and in other diseases. PMID:27153396

Detection of clinically relevant copy-number variants by exome sequencing in a large cohort of genetic disorders

PubMed Central

Pfundt, Rolph; del Rosario, Marisol; Vissers, Lisenka E.L.M.; Kwint, Michael P.; Janssen, Irene M.; de Leeuw, Nicole; Yntema, Helger G.; Nelen, Marcel R.; Lugtenberg, Dorien; Kamsteeg, Erik-Jan; Wieskamp, Nienke; Stegmann, Alexander P.A.; Stevens, Servi J.C.; Rodenburg, Richard J.T.; Simons, Annet; Mensenkamp, Arjen R.; Rinne, Tuula; Gilissen, Christian; Scheffer, Hans; Veltman, Joris A.; Hehir-Kwa, Jayne Y.

2017-01-01

Purpose: Copy-number variation is a common source of genomic variation and an important genetic cause of disease. Microarray-based analysis of copy-number variants (CNVs) has become a first-tier diagnostic test for patients with neurodevelopmental disorders, with a diagnostic yield of 10–20%. However, for most other genetic disorders, the role of CNVs is less clear and most diagnostic genetic studies are generally limited to the study of single-nucleotide variants (SNVs) and other small variants. With the introduction of exome and genome sequencing, it is now possible to detect both SNVs and CNVs using an exome- or genome-wide approach with a single test. Methods: We performed exome-based read-depth CNV screening on data from 2,603 patients affected by a range of genetic disorders for which exome sequencing was performed in a diagnostic setting. Results: In total, 123 clinically relevant CNVs ranging in size from 727 bp to 15.3 Mb were detected, which resulted in 51 conclusive diagnoses and an overall increase in diagnostic yield of ~2% (ranging from 0 to –5.8% per disorder). Conclusions: This study shows that CNVs play an important role in a broad range of genetic disorders and that detection via exome-based CNV profiling results in an increase in the diagnostic yield without additional testing, bringing us closer to single-test genomics. Genet Med advance online publication 27 October 2016 PMID:28574513

Exome sequencing of a colorectal cancer family reveals shared mutation pattern and predisposition circuitry along tumor pathways.

PubMed

Suleiman, Suleiman H; Koko, Mahmoud E; Nasir, Wafaa H; Elfateh, Ommnyiah; Elgizouli, Ubai K; Abdallah, Mohammed O E; Alfarouk, Khalid O; Hussain, Ayman; Faisal, Shima; Ibrahim, Fathelrahamn M A; Romano, Maurizio; Sultan, Ali; Banks, Lawrence; Newport, Melanie; Baralle, Francesco; Elhassan, Ahmed M; Mohamed, Hiba S; Ibrahim, Muntaser E

2015-01-01

The molecular basis of cancer and cancer multiple phenotypes are not yet fully understood. Next Generation Sequencing promises new insight into the role of genetic interactions in shaping the complexity of cancer. Aiming to outline the differences in mutation patterns between familial colorectal cancer cases and controls we analyzed whole exomes of cancer tissues and control samples from an extended colorectal cancer pedigree, providing one of the first data sets of exome sequencing of cancer in an African population against a background of large effective size typically with excess of variants. Tumors showed hMSH2 loss of function SNV consistent with Lynch syndrome. Sets of genes harboring insertions-deletions in tumor tissues revealed, however, significant GO enrichment, a feature that was not seen in control samples, suggesting that ordered insertions-deletions are central to tumorigenesis in this type of cancer. Network analysis identified multiple hub genes of centrality. ELAVL1/HuR showed remarkable centrality, interacting specially with genes harboring non-synonymous SNVs thus reinforcing the proposition of targeted mutagenesis in cancer pathways. A likely explanation to such mutation pattern is DNA/RNA editing, suggested here by nucleotide transition-to-transversion ratio that significantly departed from expected values (p-value 5e-6). NFKB1 also showed significant centrality along with ELAVL1, raising the suspicion of viral etiology given the known interaction between oncogenic viruses and these proteins.

Bovine exome sequence analysis and targeted SNP genotyping of recessive fertility defects BH1, HH2, and HH3 reveal a putative causative mutation in SMC2 for HH3.

PubMed

McClure, Matthew C; Bickhart, Derek; Null, Dan; Vanraden, Paul; Xu, Lingyang; Wiggans, George; Liu, George; Schroeder, Steve; Glasscock, Jarret; Armstrong, Jon; Cole, John B; Van Tassell, Curtis P; Sonstegard, Tad S

2014-01-01

The recent discovery of bovine haplotypes with negative effects on fertility in the Brown Swiss, Holstein, and Jersey breeds has allowed producers to identify carrier animals using commercial single nucleotide polymorphism (SNP) genotyping assays. This study was devised to identify the causative mutations underlying defective bovine embryo development contained within three of these haplotypes (Brown Swiss haplotype 1 and Holstein haplotypes 2 and 3) by combining exome capture with next generation sequencing. Of the 68,476,640 sequence variations (SV) identified, only 1,311 genome-wide SNP were concordant with the haplotype status of 21 sequenced carriers. Validation genotyping of 36 candidate SNP identified only 1 variant that was concordant to Holstein haplotype 3 (HH3), while no variants located within the refined intervals for HH2 or BH1 were concordant. The variant strictly associated with HH3 is a non-synonymous SNP (T/C) within exon 24 of the Structural Maintenance of Chromosomes 2 (SMC2) on Chromosome 8 at position 95,410,507 (UMD3.1). This polymorphism changes amino acid 1135 from phenylalanine to serine and causes a non-neutral, non-tolerated, and evolutionarily unlikely substitution within the NTPase domain of the encoded protein. Because only exome capture sequencing was used, we could not rule out the possibility that the true causative mutation for HH3 might lie in a non-exonic genomic location. Given the essential role of SMC2 in DNA repair, chromosome condensation and segregation during cell division, our findings strongly support the non-synonymous SNP (T/C) in SMC2 as the likely causative mutation. The absence of concordant variations for HH2 or BH1 suggests either the underlying causative mutations lie within a non-exomic region or in exome regions not covered by the capture array.

Bovine Exome Sequence Analysis and Targeted SNP Genotyping of Recessive Fertility Defects BH1, HH2, and HH3 Reveal a Putative Causative Mutation in SMC2 for HH3

PubMed Central

McClure, Matthew C.; Bickhart, Derek; Null, Dan; VanRaden, Paul; Xu, Lingyang; Wiggans, George; Liu, George; Schroeder, Steve; Glasscock, Jarret; Armstrong, Jon; Cole, John B.; Van Tassell, Curtis P.; Sonstegard, Tad S.

2014-01-01

The recent discovery of bovine haplotypes with negative effects on fertility in the Brown Swiss, Holstein, and Jersey breeds has allowed producers to identify carrier animals using commercial single nucleotide polymorphism (SNP) genotyping assays. This study was devised to identify the causative mutations underlying defective bovine embryo development contained within three of these haplotypes (Brown Swiss haplotype 1 and Holstein haplotypes 2 and 3) by combining exome capture with next generation sequencing. Of the 68,476,640 sequence variations (SV) identified, only 1,311 genome-wide SNP were concordant with the haplotype status of 21 sequenced carriers. Validation genotyping of 36 candidate SNP identified only 1 variant that was concordant to Holstein haplotype 3 (HH3), while no variants located within the refined intervals for HH2 or BH1 were concordant. The variant strictly associated with HH3 is a non-synonymous SNP (T/C) within exon 24 of the Structural Maintenance of Chromosomes 2 (SMC2) on Chromosome 8 at position 95,410,507 (UMD3.1). This polymorphism changes amino acid 1135 from phenylalanine to serine and causes a non-neutral, non-tolerated, and evolutionarily unlikely substitution within the NTPase domain of the encoded protein. Because only exome capture sequencing was used, we could not rule out the possibility that the true causative mutation for HH3 might lie in a non-exonic genomic location. Given the essential role of SMC2 in DNA repair, chromosome condensation and segregation during cell division, our findings strongly support the non-synonymous SNP (T/C) in SMC2 as the likely causative mutation. The absence of concordant variations for HH2 or BH1 suggests either the underlying causative mutations lie within a non-exomic region or in exome regions not covered by the capture array. PMID:24667746

Whole exome sequencing frequently detects a monogenic cause in early onset nephrolithiasis and nephrocalcinosis.

PubMed

Daga, Ankana; Majmundar, Amar J; Braun, Daniela A; Gee, Heon Yung; Lawson, Jennifer A; Shril, Shirlee; Jobst-Schwan, Tilman; Vivante, Asaf; Schapiro, David; Tan, Weizhen; Warejko, Jillian K; Widmeier, Eugen; Nelson, Caleb P; Fathy, Hanan M; Gucev, Zoran; Soliman, Neveen A; Hashmi, Seema; Halbritter, Jan; Halty, Margarita; Kari, Jameela A; El-Desoky, Sherif; Ferguson, Michael A; Somers, Michael J G; Traum, Avram Z; Stein, Deborah R; Daouk, Ghaleb H; Rodig, Nancy M; Katz, Avi; Hanna, Christian; Schwaderer, Andrew L; Sayer, John A; Wassner, Ari J; Mane, Shrikant; Lifton, Richard P; Milosevic, Danko; Tasic, Velibor; Baum, Michelle A; Hildebrandt, Friedhelm

2018-01-01

The incidence of nephrolithiasis continues to rise. Previously, we showed that a monogenic cause could be detected in 11.4% of individuals with adult-onset nephrolithiasis or nephrocalcinosis and in 16.7-20.8% of individuals with onset before 18 years of age, using gene panel sequencing of 30 genes known to cause nephrolithiasis/nephrocalcinosis. To overcome the limitations of panel sequencing, we utilized whole exome sequencing in 51 families, who presented before age 25 years with at least one renal stone or with a renal ultrasound finding of nephrocalcinosis to identify the underlying molecular genetic cause of disease. In 15 of 51 families, we detected a monogenic causative mutation by whole exome sequencing. A mutation in seven recessive genes (AGXT, ATP6V1B1, CLDN16, CLDN19, GRHPR, SLC3A1, SLC12A1), in one dominant gene (SLC9A3R1), and in one gene (SLC34A1) with both recessive and dominant inheritance was detected. Seven of the 19 different mutations were not previously described as disease-causing. In one family, a causative mutation in one of 117 genes that may represent phenocopies of nephrolithiasis-causing genes was detected. In nine of 15 families, the genetic diagnosis may have specific implications for stone management and prevention. Several factors that correlated with the higher detection rate in our cohort were younger age at onset of nephrolithiasis/nephrocalcinosis, presence of multiple affected members in a family, and presence of consanguinity. Thus, we established whole exome sequencing as an efficient approach toward a molecular genetic diagnosis in individuals with nephrolithiasis/nephrocalcinosis who manifest before age 25 years. Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

Exome sequencing and genome-wide linkage analysis in 17 families illustrate the complex contribution of TTN truncating variants to dilated cardiomyopathy.

PubMed

Norton, Nadine; Li, Duanxiang; Rampersaud, Evadnie; Morales, Ana; Martin, Eden R; Zuchner, Stephan; Guo, Shengru; Gonzalez, Michael; Hedges, Dale J; Robertson, Peggy D; Krumm, Niklas; Nickerson, Deborah A; Hershberger, Ray E

2013-04-01

BACKGROUND- Familial dilated cardiomyopathy (DCM) is a genetically heterogeneous disease with >30 known genes. TTN truncating variants were recently implicated in a candidate gene study to cause 25% of familial and 18% of sporadic DCM cases. METHODS AND RESULTS- We used an unbiased genome-wide approach using both linkage analysis and variant filtering across the exome sequences of 48 individuals affected with DCM from 17 families to identify genetic cause. Linkage analysis ranked the TTN region as falling under the second highest genome-wide multipoint linkage peak, multipoint logarithm of odds, 1.59. We identified 6 TTN truncating variants carried by individuals affected with DCM in 7 of 17 DCM families (logarithm of odds, 2.99); 2 of these 7 families also had novel missense variants that segregated with disease. Two additional novel truncating TTN variants did not segregate with DCM. Nucleotide diversity at the TTN locus, including missense variants, was comparable with 5 other known DCM genes. The average number of missense variants in the exome sequences from the DCM cases or the ≈5400 cases from the Exome Sequencing Project was ≈23 per individual. The average number of TTN truncating variants in the Exome Sequencing Project was 0.014 per individual. We also identified a region (chr9q21.11-q22.31) with no known DCM genes with a maximum heterogeneity logarithm of odds score of 1.74. CONCLUSIONS- These data suggest that TTN truncating variants contribute to DCM cause. However, the lack of segregation of all identified TTN truncating variants illustrates the challenge of determining variant pathogenicity even with full exome sequencing.

IMPACT: a whole-exome sequencing analysis pipeline for integrating molecular profiles with actionable therapeutics in clinical samples

PubMed Central

Hintzsche, Jennifer; Kim, Jihye; Yadav, Vinod; Amato, Carol; Robinson, Steven E; Seelenfreund, Eric; Shellman, Yiqun; Wisell, Joshua; Applegate, Allison; McCarter, Martin; Box, Neil; Tentler, John; De, Subhajyoti

2016-01-01

Objective Currently, there is a disconnect between finding a patient’s relevant molecular profile and predicting actionable therapeutics. Here we develop and implement the Integrating Molecular Profiles with Actionable Therapeutics (IMPACT) analysis pipeline, linking variants detected from whole-exome sequencing (WES) to actionable therapeutics. Methods and materials The IMPACT pipeline contains 4 analytical modules: detecting somatic variants, calling copy number alterations, predicting drugs against deleterious variants, and analyzing tumor heterogeneity. We tested the IMPACT pipeline on whole-exome sequencing data in The Cancer Genome Atlas (TCGA) lung adenocarcinoma samples with known EGFR mutations. We also used IMPACT to analyze melanoma patient tumor samples before treatment, after BRAF-inhibitor treatment, and after BRAF- and MEK-inhibitor treatment. Results IMPACT Food and Drug Administration (FDA) correctly identified known EGFR mutations in the TCGA lung adenocarcinoma samples. IMPACT linked these EGFR mutations to the appropriate FDA-approved EGFR inhibitors. For the melanoma patient samples, we identified NRAS p.Q61K as an acquired resistance mutation to BRAF-inhibitor treatment. We also identified CDKN2A deletion as a novel acquired resistance mutation to BRAFi/MEKi inhibition. The IMPACT analysis pipeline predicts these somatic variants to actionable therapeutics. We observed the clonal dynamic in the tumor samples after various treatments. We showed that IMPACT not only helped in successful prioritization of clinically relevant variants but also linked these variations to possible targeted therapies. Conclusion IMPACT provides a new bioinformatics strategy to delineate candidate somatic variants and actionable therapies. This approach can be applied to other patient tumor samples to discover effective drug targets for personalized medicine. IMPACT is publicly available at http://tanlab.ucdenver.edu/IMPACT. PMID:27026619

Exome sequencing establishes a gelsolin mutation as the cause of inherited bulbar-onset neuropathy.

PubMed

Caress, James B; Johnson, Janel O; Abramzon, Yevgeniya A; Hawkins, Gregory A; Gibbs, J Raphael; Sullivan, Elizabeth A; Chahal, Chamanpreet S; Traynor, Bryan J

2017-11-01

Progressive bulbar motor neuropathy is primarily caused by bulbar-onset ALS. Hereditary amyloidosis type IV also presents with a bulbar neuropathy that mimics motor neuron disease. The disease is prevalent in Finland only and is not commonly included in the differential diagnosis of ALS. We studied 18 members of a family in which some had bulbar motor neuropathy, and we performed exome sequencing. Five affected family members were found to have a D187Y substitution in the GSN gene known to cause hereditary amyloidosis type IV. This American family presented with progressive bulbar neuropathy due to a gelsolin mutation not found in Finland. Hereditary amyloidosis type IV presents with bulbar motor neuropathy and not with peripheral neuropathy as occurs with common forms of amyloidosis. This report demonstrates the power of exome sequencing to determine the cause of rare hereditary diseases with incomplete or atypical phenotypes. Muscle Nerve 56: 1001-1005, 2017. © 2016 Wiley Periodicals, Inc.

Whole-exome sequencing identifies common and rare variant metabolic QTLs in a Middle Eastern population.

PubMed

Yousri, Noha A; Fakhro, Khalid A; Robay, Amal; Rodriguez-Flores, Juan L; Mohney, Robert P; Zeriri, Hassina; Odeh, Tala; Kader, Sara Abdul; Aldous, Eman K; Thareja, Gaurav; Kumar, Manish; Al-Shakaki, Alya; Chidiac, Omar M; Mohamoud, Yasmin A; Mezey, Jason G; Malek, Joel A; Crystal, Ronald G; Suhre, Karsten

2018-01-23

Metabolomics-genome-wide association studies (mGWAS) have uncovered many metabolic quantitative trait loci (mQTLs) influencing human metabolic individuality, though predominantly in European cohorts. By combining whole-exome sequencing with a high-resolution metabolomics profiling for a highly consanguineous Middle Eastern population, we discover 21 common variant and 12 functional rare variant mQTLs, of which 45% are novel altogether. We fine-map 10 common variant mQTLs to new metabolite ratio associations, and 11 common variant mQTLs to putative protein-altering variants. This is the first work to report common and rare variant mQTLs linked to diseases and/or pharmacological targets in a consanguineous Arab cohort, with wide implications for precision medicine in the Middle East.

cnvScan: a CNV screening and annotation tool to improve the clinical utility of computational CNV prediction from exome sequencing data.

PubMed

Samarakoon, Pubudu Saneth; Sorte, Hanne Sørmo; Stray-Pedersen, Asbjørg; Rødningen, Olaug Kristin; Rognes, Torbjørn; Lyle, Robert

2016-01-14

With advances in next generation sequencing technology and analysis methods, single nucleotide variants (SNVs) and indels can be detected with high sensitivity and specificity in exome sequencing data. Recent studies have demonstrated the ability to detect disease-causing copy number variants (CNVs) in exome sequencing data. However, exonic CNV prediction programs have shown high false positive CNV counts, which is the major limiting factor for the applicability of these programs in clinical studies. We have developed a tool (cnvScan) to improve the clinical utility of computational CNV prediction in exome data. cnvScan can accept input from any CNV prediction program. cnvScan consists of two steps: CNV screening and CNV annotation. CNV screening evaluates CNV prediction using quality scores and refines this using an in-house CNV database, which greatly reduces the false positive rate. The annotation step provides functionally and clinically relevant information using multiple source datasets. We assessed the performance of cnvScan on CNV predictions from five different prediction programs using 64 exomes from Primary Immunodeficiency (PIDD) patients, and identified PIDD-causing CNVs in three individuals from two different families. In summary, cnvScan reduces the time and effort required to detect disease-causing CNVs by reducing the false positive count and providing annotation. This improves the clinical utility of CNV detection in exome data.

Whole Exome Sequencing Reveals a Monogenic Cause of Disease in ≈43% of 35 Families With Midaortic Syndrome.

PubMed

Warejko, Jillian K; Schueler, Markus; Vivante, Asaf; Tan, Weizhen; Daga, Ankana; Lawson, Jennifer A; Braun, Daniela A; Shril, Shirlee; Amann, Kassaundra; Somers, Michael J G; Rodig, Nancy M; Baum, Michelle A; Daouk, Ghaleb; Traum, Avram Z; Kim, Heung Bae; Vakili, Khashayar; Porras, Diego; Lock, James; Rivkin, Michael J; Chaudry, Gulraiz; Smoot, Leslie B; Singh, Michael N; Smith, Edward R; Mane, Shrikant M; Lifton, Richard P; Stein, Deborah R; Ferguson, Michael A; Hildebrandt, Friedhelm

2018-04-01

Midaortic syndrome (MAS) is a rare cause of severe childhood hypertension characterized by narrowing of the abdominal aorta in children and is associated with extensive vascular disease. It may occur as part of a genetic syndrome, such as neurofibromatosis, or as consequence of a pathological inflammatory disease. However, most cases are considered idiopathic. We hypothesized that in a high percentage of these patients, a monogenic cause of disease may be detected by evaluating whole exome sequencing data for mutations in 1 of 38 candidate genes previously described to cause vasculopathy. We studied a cohort of 36 individuals from 35 different families with MAS by exome sequencing. In 15 of 35 families (42.9%), we detected likely causal dominant mutations. In 15 of 35 (42.9%) families with MAS, whole exome sequencing revealed a mutation in one of the genes previously associated with vascular disease ( NF1 , JAG1 , ELN , GATA6 , and RNF213 ). Ten of the 15 mutations have not previously been reported. This is the first report of ELN , RNF213 , or GATA6 mutations in individuals with MAS. Mutations were detected in NF1 (6/15 families), JAG1 (4/15 families), ELN (3/15 families), and one family each for GATA6 and RNF213 Eight individuals had syndromic disease and 7 individuals had isolated MAS. Whole exome sequencing can provide conclusive molecular genetic diagnosis in a high fraction of individuals with syndromic or isolated MAS. Establishing an etiologic diagnosis may reveal genotype/phenotype correlations for MAS in the future and should, therefore, be performed routinely in MAS. © 2018 American Heart Association, Inc.

Whole exome sequencing reveals recurrent mutations in BRCA2 and FAT genes in acinar cell carcinomas of the pancreas.

PubMed

Furukawa, Toru; Sakamoto, Hitomi; Takeuchi, Shoko; Ameri, Mitra; Kuboki, Yuko; Yamamoto, Toshiyuki; Hatori, Takashi; Yamamoto, Masakazu; Sugiyama, Masanori; Ohike, Nobuyuki; Yamaguchi, Hiroshi; Shimizu, Michio; Shibata, Noriyuki; Shimizu, Kyoko; Shiratori, Keiko

2015-03-06

Acinar cell carcinoma of the pancreas is a rare tumor with a poor prognosis. Compared to pancreatic ductal adenocarcinoma, its molecular features are poorly known. We studied a total of 11 acinar cell carcinomas, including 3 by exome and 4 by target sequencing. Exome sequencing revealed 65 nonsynonymous mutations and 22 indels with a mutation rate of 3.4 mutations/Mb per tumor, on average. By accounting for not only somatic but also germline mutations with loss of the wild-type allele, we identified recurrent mutations of BRCA2 and FAT genes. BRCA2 showed somatic or germline premature termination mutations, with loss of the wild-type allele in 3 of 7 tumors. FAT1, FAT3, and FAT4 showed somatic or germline missense mutations in 4 of 7 tumors. The germline FAT mutations were with loss of the wild-type allele. Loss of BRCA2 expression was observed in 5 of 11 tumors. One patient with a BRCA2-mutated tumor experienced complete remission of liver metastasis following cisplatinum chemotherapy. In conclusion, acinar cell carcinomas show a distinct mutation pattern and often harbor somatic or germline mutations of BRCA2 and FAT genes. This result may warrant assessment of BRCA2 abrogation in patients with the carcinoma to determine their sensitivity to chemotherapy.

Whole-Exome Sequencing in Two Extreme Phenotypes of Response to VEGF-Targeted Therapies in Patients With Metastatic Clear Cell Renal Cell Carcinoma.

PubMed

Fay, Andre P; de Velasco, Guillermo; Ho, Thai H; Van Allen, Eliezer M; Murray, Bradley; Albiges, Laurence; Signoretti, Sabina; Hakimi, A Ari; Stanton, Melissa L; Bellmunt, Joaquim; McDermott, David F; Atkins, Michael B; Garraway, Levi A; Kwiatkowski, David J; Choueiri, Toni K

2016-07-01

Advances in next-generation sequencing have provided a unique opportunity to understand the biology of disease and mechanisms of sensitivity or resistance to specific agents. Renal cell carcinoma (RCC) is a heterogeneous disease and highly variable clinical responses have been observed with vascular endothelial growth factor (VEGF)-targeted therapy (VEGF-TT). We hypothesized that whole-exome sequencing analysis might identify genotypes associated with extreme response or resistance to VEGF-TT in metastatic (mRCC). Patients with mRCC who had received first-line sunitinib or pazopanib and were in 2 extreme phenotypes of response were identified. Extreme responders (ERs) were defined as those with partial response or complete response for 3 or more years (n=13) and primary refractory patients (PRPs) were defined as those with progressive disease within the first 3 months of therapy (n=14). International Metastatic RCC Database Consortium prognostic scores were not significantly different between the groups (P=.67). Considering the genes known to be mutated in RCC at significant frequency, PBRM1 mutations were identified in 7 ERs (54%) versus 1 PRP (7%) (P=.01). In addition, mutations in TP53 (n=4) were found only in PRPs (P=.09). Our data suggest that mutations in some genes in RCC may impact response to VEGF-TT. Copyright © 2016 by the National Comprehensive Cancer Network.

Whole-exome sequencing analysis of Waardenburg syndrome in a Chinese family.

PubMed

Chen, Dezhong; Zhao, Na; Wang, Jing; Li, Zhuoyu; Wu, Changxin; Fu, Jie; Xiao, Han

2017-01-01

Waardenburg syndrome (WS) is a dominantly inherited, genetically heterogeneous auditory-pigmentary syndrome characterized by non-progressive sensorineural hearing loss and iris discoloration. By whole-exome sequencing (WES), we identified a nonsense mutation (c.598C>T) in PAX3 gene, predicted to be disease causing by in silico analysis. This is the first report of genetically diagnosed case of WS PAX3 c.598C>T nonsense mutation in Chinese ethnic origin by WES and in silico functional prediction methods.

Whole-exome sequencing analysis of Waardenburg syndrome in a Chinese family

PubMed Central

Chen, Dezhong; Zhao, Na; Wang, Jing; Li, Zhuoyu; Wu, Changxin; Fu, Jie; Xiao, Han

2017-01-01

Waardenburg syndrome (WS) is a dominantly inherited, genetically heterogeneous auditory-pigmentary syndrome characterized by non-progressive sensorineural hearing loss and iris discoloration. By whole-exome sequencing (WES), we identified a nonsense mutation (c.598C>T) in PAX3 gene, predicted to be disease causing by in silico analysis. This is the first report of genetically diagnosed case of WS PAX3 c.598C>T nonsense mutation in Chinese ethnic origin by WES and in silico functional prediction methods. PMID:28690861

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.

Exome sequencing identifies a DNAJB6 mutation in a family with dominantly-inherited limb-girdle muscular dystrophy.

PubMed

Couthouis, Julien; Raphael, Alya R; Siskind, Carly; Findlay, Andrew R; Buenrostro, Jason D; Greenleaf, William J; Vogel, Hannes; Day, John W; Flanigan, Kevin M; Gitler, Aaron D

2014-05-01

Limb-girdle muscular dystrophy primarily affects the muscles of the hips and shoulders (the "limb-girdle" muscles), although it is a heterogeneous disorder that can present with varying symptoms. There is currently no cure. We sought to identify the genetic basis of limb-girdle muscular dystrophy type 1 in an American family of Northern European descent using exome sequencing. Exome sequencing was performed on DNA samples from two affected siblings and one unaffected sibling and resulted in the identification of eleven candidate mutations that co-segregated with the disease. Notably, this list included a previously reported mutation in DNAJB6, p.Phe89Ile, which was recently identified as a cause of limb-girdle muscular dystrophy type 1D. Additional family members were Sanger sequenced and the mutation in DNAJB6 was only found in affected individuals. Subsequent haplotype analysis indicated that this DNAJB6 p.Phe89Ile mutation likely arose independently of the previously reported mutation. Since other published mutations are located close by in the G/F domain of DNAJB6, this suggests that the area may represent a mutational hotspot. Exome sequencing provided an unbiased and effective method for identifying the genetic etiology of limb-girdle muscular dystrophy type 1 in a previously genetically uncharacterized family. This work further confirms the causative role of DNAJB6 mutations in limb-girdle muscular dystrophy type 1D. Copyright © 2014 Elsevier B.V. All rights reserved.

Whole Exome Sequencing of a Patient with Metastatic Hidradenocarcinoma and Review of the Literature

PubMed Central

Gupta, Eva; Guthrie, Kimberly J.; Krishna, Murli; Asmann, Yan; Parker, Alexander S.; Joseph, Richard W.

2015-01-01

Hidradenocarcinoma is a rare malignancy of the sweat glands with only a few cases reported in literature. The management of these tumors is based on the extent of disease with local disease managed with surgical resection. These can tumors carry a high potential of lymphatic and vascular spread and local and distant metastases are not uncommon. Given the rarity of the tumor and lack of genetic and clinical data about these tumors, there is no consensus on the proper management of metastatic disease. Here in we report the first case of metastatic hidradenocarcinoma with detailed molecular profiling including whole exome sequencing. We identified mutations in multiple genes including two that are potentially targetable: PTCH1 and TCF7L1. Further work is necessary to not only confirm the presence of these mutations but also to confirm the clinical significance. PMID:25918615

Whole exome sequencing of a patient with metastatic hidradenocarcinoma and review of the literature.

PubMed

Gupta, Eva; Guthrie, Kimberly J; Krishna, Murli; Asmann, Yan; Parker, Alexander S; Joseph, Richard W

2015-02-11

Hidradenocarcinoma is a rare malignancy of the sweat glands with only a few cases reported in literature. The management of these tumors is based on the extent of disease with local disease managed with surgical resection. These can tumors carry a high potential of lymphatic and vascular spread and local and distant metastases are not uncommon. Given the rarity of the tumor and lack of genetic and clinical data about these tumors, there is no consensus on the proper management of metastatic disease. Here in we report the first case of metastatic hidradenocarcinoma with detailed molecular profiling including whole exome sequencing. We identified mutations in multiple genes including two that are potentially targetable: PTCH1 and TCF7L1. Further work is necessary to not only confirm the presence of these mutations but also to confirm the clinical significance.

Exome sequencing of a colorectal cancer family reveals shared mutation pattern and predisposition circuitry along tumor pathways

PubMed Central

Suleiman, Suleiman H.; Koko, Mahmoud E.; Nasir, Wafaa H.; Elfateh, Ommnyiah; Elgizouli, Ubai K.; Abdallah, Mohammed O. E.; Alfarouk, Khalid O.; Hussain, Ayman; Faisal, Shima; Ibrahim, Fathelrahamn M. A.; Romano, Maurizio; Sultan, Ali; Banks, Lawrence; Newport, Melanie; Baralle, Francesco; Elhassan, Ahmed M.; Mohamed, Hiba S.; Ibrahim, Muntaser E.

2015-01-01

The molecular basis of cancer and cancer multiple phenotypes are not yet fully understood. Next Generation Sequencing promises new insight into the role of genetic interactions in shaping the complexity of cancer. Aiming to outline the differences in mutation patterns between familial colorectal cancer cases and controls we analyzed whole exomes of cancer tissues and control samples from an extended colorectal cancer pedigree, providing one of the first data sets of exome sequencing of cancer in an African population against a background of large effective size typically with excess of variants. Tumors showed hMSH2 loss of function SNV consistent with Lynch syndrome. Sets of genes harboring insertions–deletions in tumor tissues revealed, however, significant GO enrichment, a feature that was not seen in control samples, suggesting that ordered insertions–deletions are central to tumorigenesis in this type of cancer. Network analysis identified multiple hub genes of centrality. ELAVL1/HuR showed remarkable centrality, interacting specially with genes harboring non-synonymous SNVs thus reinforcing the proposition of targeted mutagenesis in cancer pathways. A likely explanation to such mutation pattern is DNA/RNA editing, suggested here by nucleotide transition-to-transversion ratio that significantly departed from expected values (p-value 5e-6). NFKB1 also showed significant centrality along with ELAVL1, raising the suspicion of viral etiology given the known interaction between oncogenic viruses and these proteins. PMID:26442106

[Exome sequencing revealed Allan-Herndon-Dudley syndrome underlying multiple disabilities].

PubMed

Arvio, Maria; Philips, Anju K; Ahvenainen, Minna; Somer, Mirja; Kalscheuer, Vera; Järvelä, Irma

2014-01-01

Normal function of the thyroid gland is the cornerstone of a child's mental development and physical growth. We describe a Finnish family, in which the diagnosis of three brothers became clear after investigations that lasted for more than 30 years. Two of the sons have already died. DNA analysis of the third one, a 16-year-old boy, revealed in exome sequencing of the complete X chromosome a mutation in the SLC16A2 gene, i.e. MCT8, coding for a thyroid hormone transport protein. Allan-Herndon-Dudley syndrome was thus shown to be the cause of multiple disabilities.

Whole-Exome Sequencing Identifies Rare and Low-Frequency Coding Variants Associated with LDL Cholesterol

PubMed Central

Lange, Leslie A.; Hu, Youna; Zhang, He; Xue, Chenyi; Schmidt, Ellen M.; Tang, Zheng-Zheng; Bizon, Chris; Lange, Ethan M.; Smith, Joshua D.; Turner, Emily H.; Jun, Goo; Kang, Hyun Min; Peloso, Gina; Auer, Paul; Li, Kuo-ping; Flannick, Jason; Zhang, Ji; Fuchsberger, Christian; Gaulton, Kyle; Lindgren, Cecilia; Locke, Adam; Manning, Alisa; Sim, Xueling; Rivas, Manuel A.; Holmen, Oddgeir L.; Gottesman, Omri; Lu, Yingchang; Ruderfer, Douglas; Stahl, Eli A.; Duan, Qing; Li, Yun; Durda, Peter; Jiao, Shuo; Isaacs, Aaron; Hofman, Albert; Bis, Joshua C.; Correa, Adolfo; Griswold, Michael E.; Jakobsdottir, Johanna; Smith, Albert V.; Schreiner, Pamela J.; Feitosa, Mary F.; Zhang, Qunyuan; Huffman, Jennifer E.; Crosby, Jacy; Wassel, Christina L.; Do, Ron; Franceschini, Nora; Martin, Lisa W.; Robinson, Jennifer G.; Assimes, Themistocles L.; Crosslin, David R.; Rosenthal, Elisabeth A.; Tsai, Michael; Rieder, Mark J.; Farlow, Deborah N.; Folsom, Aaron R.; Lumley, Thomas; Fox, Ervin R.; Carlson, Christopher S.; Peters, Ulrike; Jackson, Rebecca D.; van Duijn, Cornelia M.; Uitterlinden, André G.; Levy, Daniel; Rotter, Jerome I.; Taylor, Herman A.; Gudnason, Vilmundur; Siscovick, David S.; Fornage, Myriam; Borecki, Ingrid B.; Hayward, Caroline; Rudan, Igor; Chen, Y. Eugene; Bottinger, Erwin P.; Loos, Ruth J.F.; Sætrom, Pål; Hveem, Kristian; Boehnke, Michael; Groop, Leif; McCarthy, Mark; Meitinger, Thomas; Ballantyne, Christie M.; Gabriel, Stacey B.; O’Donnell, Christopher J.; Post, Wendy S.; North, Kari E.; Reiner, Alexander P.; Boerwinkle, Eric; Psaty, Bruce M.; Altshuler, David; Kathiresan, Sekar; Lin, Dan-Yu; Jarvik, Gail P.; Cupples, L. Adrienne; Kooperberg, Charles; Wilson, James G.; Nickerson, Deborah A.; Abecasis, Goncalo R.; Rich, Stephen S.; Tracy, Russell P.; Willer, Cristen J.; Gabriel, Stacey B.; Altshuler, David M.; Abecasis, Gonçalo R.; Allayee, Hooman; Cresci, Sharon; Daly, Mark J.; de Bakker, Paul I.W.; DePristo, Mark A.; Do, Ron; Donnelly, Peter; Farlow, Deborah N.; Fennell, Tim; Garimella, Kiran; Hazen, Stanley L.; Hu, Youna; Jordan, Daniel M.; Jun, Goo; Kathiresan, Sekar; Kang, Hyun Min; Kiezun, Adam; Lettre, Guillaume; Li, Bingshan; Li, Mingyao; Newton-Cheh, Christopher H.; Padmanabhan, Sandosh; Peloso, Gina; Pulit, Sara; Rader, Daniel J.; Reich, David; Reilly, Muredach P.; Rivas, Manuel A.; Schwartz, Steve; Scott, Laura; Siscovick, David S.; Spertus, John A.; Stitziel, Nathaniel O.; Stoletzki, Nina; Sunyaev, Shamil R.; Voight, Benjamin F.; Willer, Cristen J.; Rich, Stephen S.; Akylbekova, Ermeg; Atwood, Larry D.; Ballantyne, Christie M.; Barbalic, Maja; Barr, R. Graham; Benjamin, Emelia J.; Bis, Joshua; Boerwinkle, Eric; Bowden, Donald W.; Brody, Jennifer; Budoff, Matthew; Burke, Greg; Buxbaum, Sarah; Carr, Jeff; Chen, Donna T.; Chen, Ida Y.; Chen, Wei-Min; Concannon, Pat; Crosby, Jacy; Cupples, L. Adrienne; D’Agostino, Ralph; DeStefano, Anita L.; Dreisbach, Albert; Dupuis, Josée; Durda, J. Peter; Ellis, Jaclyn; Folsom, Aaron R.; Fornage, Myriam; Fox, Caroline S.; Fox, Ervin; Funari, Vincent; Ganesh, Santhi K.; Gardin, Julius; Goff, David; Gordon, Ora; Grody, Wayne; Gross, Myron; Guo, Xiuqing; Hall, Ira M.; Heard-Costa, Nancy L.; Heckbert, Susan R.; Heintz, Nicholas; Herrington, David M.; Hickson, DeMarc; Huang, Jie; Hwang, Shih-Jen; Jacobs, David R.; Jenny, Nancy S.; Johnson, Andrew D.; Johnson, Craig W.; Kawut, Steven; Kronmal, Richard; Kurz, Raluca; Lange, Ethan M.; Lange, Leslie A.; Larson, Martin G.; Lawson, Mark; Lewis, Cora E.; Levy, Daniel; Li, Dalin; Lin, Honghuang; Liu, Chunyu; Liu, Jiankang; Liu, Kiang; Liu, Xiaoming; Liu, Yongmei; Longstreth, William T.; Loria, Cay; Lumley, Thomas; Lunetta, Kathryn; Mackey, Aaron J.; Mackey, Rachel; Manichaikul, Ani; Maxwell, Taylor; McKnight, Barbara; Meigs, James B.; Morrison, Alanna C.; Musani, Solomon K.; Mychaleckyj, Josyf C.; Nettleton, Jennifer A.; North, Kari; O’Donnell, Christopher J.; O’Leary, Daniel; Ong, Frank; Palmas, Walter; Pankow, James S.; Pankratz, Nathan D.; Paul, Shom; Perez, Marco; Person, Sharina D.; Polak, Joseph; Post, Wendy S.; Psaty, Bruce M.; Quinlan, Aaron R.; Raffel, Leslie J.; Ramachandran, Vasan S.; Reiner, Alexander P.; Rice, Kenneth; Rotter, Jerome I.; Sanders, Jill P.; Schreiner, Pamela; Seshadri, Sudha; Shea, Steve; Sidney, Stephen; Silverstein, Kevin; Smith, Nicholas L.; Sotoodehnia, Nona; Srinivasan, Asoke; Taylor, Herman A.; Taylor, Kent; Thomas, Fridtjof; Tracy, Russell P.; Tsai, Michael Y.; Volcik, Kelly A.; Wassel, Chrstina L.; Watson, Karol; Wei, Gina; White, Wendy; Wiggins, Kerri L.; Wilk, Jemma B.; Williams, O. Dale; Wilson, Gregory; Wilson, James G.; Wolf, Phillip; Zakai, Neil A.; Hardy, John; Meschia, James F.; Nalls, Michael; Singleton, Andrew; Worrall, Brad; Bamshad, Michael J.; Barnes, Kathleen C.; Abdulhamid, Ibrahim; Accurso, Frank; Anbar, Ran; Beaty, Terri; Bigham, Abigail; Black, Phillip; Bleecker, Eugene; Buckingham, Kati; Cairns, Anne Marie; Caplan, Daniel; Chatfield, Barbara; Chidekel, Aaron; Cho, Michael; Christiani, David C.; Crapo, James D.; Crouch, Julia; Daley, Denise; Dang, Anthony; Dang, Hong; De Paula, Alicia; DeCelie-Germana, Joan; Drumm, Allen DozorMitch; Dyson, Maynard; Emerson, Julia; Emond, Mary J.; Ferkol, Thomas; Fink, Robert; Foster, Cassandra; Froh, Deborah; Gao, Li; Gershan, William; Gibson, Ronald L.; Godwin, Elizabeth; Gondor, Magdalen; Gutierrez, Hector; Hansel, Nadia N.; Hassoun, Paul M.; Hiatt, Peter; Hokanson, John E.; Howenstine, Michelle; Hummer, Laura K.; Kanga, Jamshed; Kim, Yoonhee; Knowles, Michael R.; Konstan, Michael; Lahiri, Thomas; Laird, Nan; Lange, Christoph; Lin, Lin; Lin, Xihong; Louie, Tin L.; Lynch, David; Make, Barry; Martin, Thomas R.; Mathai, Steve C.; Mathias, Rasika A.; McNamara, John; McNamara, Sharon; Meyers, Deborah; Millard, Susan; Mogayzel, Peter; Moss, Richard; Murray, Tanda; Nielson, Dennis; Noyes, Blakeslee; O’Neal, Wanda; Orenstein, David; O’Sullivan, Brian; Pace, Rhonda; Pare, Peter; Parker, H. Worth; Passero, Mary Ann; Perkett, Elizabeth; Prestridge, Adrienne; Rafaels, Nicholas M.; Ramsey, Bonnie; Regan, Elizabeth; Ren, Clement; Retsch-Bogart, George; Rock, Michael; Rosen, Antony; Rosenfeld, Margaret; Ruczinski, Ingo; Sanford, Andrew; Schaeffer, David; Sell, Cindy; Sheehan, Daniel; Silverman, Edwin K.; Sin, Don; Spencer, Terry; Stonebraker, Jackie; Tabor, Holly K.; Varlotta, Laurie; Vergara, Candelaria I.; Weiss, Robert; Wigley, Fred; Wise, Robert A.; Wright, Fred A.; Wurfel, Mark M.; Zanni, Robert; Zou, Fei; Nickerson, Deborah A.; Rieder, Mark J.; Green, Phil; Shendure, Jay; Akey, Joshua M.; Bustamante, Carlos D.; Crosslin, David R.; Eichler, Evan E.; Fox, P. Keolu; Fu, Wenqing; Gordon, Adam; Gravel, Simon; Jarvik, Gail P.; Johnsen, Jill M.; Kan, Mengyuan; Kenny, Eimear E.; Kidd, Jeffrey M.; Lara-Garduno, Fremiet; Leal, Suzanne M.; Liu, Dajiang J.; McGee, Sean; O’Connor, Timothy D.; Paeper, Bryan; Robertson, Peggy D.; Smith, Joshua D.; Staples, Jeffrey C.; Tennessen, Jacob A.; Turner, Emily H.; Wang, Gao; Yi, Qian; Jackson, Rebecca; Peters, Ulrike; Carlson, Christopher S.; Anderson, Garnet; Anton-Culver, Hoda; Assimes, Themistocles L.; Auer, Paul L.; Beresford, Shirley; Bizon, Chris; Black, Henry; Brunner, Robert; Brzyski, Robert; Burwen, Dale; Caan, Bette; Carty, Cara L.; Chlebowski, Rowan; Cummings, Steven; Curb, J. David; Eaton, Charles B.; Ford, Leslie; Franceschini, Nora; Fullerton, Stephanie M.; Gass, Margery; Geller, Nancy; Heiss, Gerardo; Howard, Barbara V.; Hsu, Li; Hutter, Carolyn M.; Ioannidis, John; Jiao, Shuo; Johnson, Karen C.; Kooperberg, Charles; Kuller, Lewis; LaCroix, Andrea; Lakshminarayan, Kamakshi; Lane, Dorothy; Lasser, Norman; LeBlanc, Erin; Li, Kuo-Ping; Limacher, Marian; Lin, Dan-Yu; Logsdon, Benjamin A.; Ludlam, Shari; Manson, JoAnn E.; Margolis, Karen; Martin, Lisa; McGowan, Joan; Monda, Keri L.; Kotchen, Jane Morley; Nathan, Lauren; Ockene, Judith; O’Sullivan, Mary Jo; Phillips, Lawrence S.; Prentice, Ross L.; Robbins, John; Robinson, Jennifer G.; Rossouw, Jacques E.; Sangi-Haghpeykar, Haleh; Sarto, Gloria E.; Shumaker, Sally; Simon, Michael S.; Stefanick, Marcia L.; Stein, Evan; Tang, Hua; Taylor, Kira C.; Thomson, Cynthia A.; Thornton, Timothy A.; Van Horn, Linda; Vitolins, Mara; Wactawski-Wende, Jean; Wallace, Robert; Wassertheil-Smoller, Sylvia; Zeng, Donglin; Applebaum-Bowden, Deborah; Feolo, Michael; Gan, Weiniu; Paltoo, Dina N.; Sholinsky, Phyliss; Sturcke, Anne

2014-01-01

Elevated low-density lipoprotein cholesterol (LDL-C) is a treatable, heritable risk factor for cardiovascular disease. Genome-wide association studies (GWASs) have identified 157 variants associated with lipid levels but are not well suited to assess the impact of rare and low-frequency variants. To determine whether rare or low-frequency coding variants are associated with LDL-C, we exome sequenced 2,005 individuals, including 554 individuals selected for extreme LDL-C (>98th or <2nd percentile). Follow-up analyses included sequencing of 1,302 additional individuals and genotype-based analysis of 52,221 individuals. We observed significant evidence of association between LDL-C and the burden of rare or low-frequency variants in PNPLA5, encoding a phospholipase-domain-containing protein, and both known and previously unidentified variants in PCSK9, LDLR and APOB, three known lipid-related genes. The effect sizes for the burden of rare variants for each associated gene were substantially higher than those observed for individual SNPs identified from GWASs. We replicated the PNPLA5 signal in an independent large-scale sequencing study of 2,084 individuals. In conclusion, this large whole-exome-sequencing study for LDL-C identified a gene not known to be implicated in LDL-C and provides unique insight into the design and analysis of similar experiments. PMID:24507775

IMPACT: a whole-exome sequencing analysis pipeline for integrating molecular profiles with actionable therapeutics in clinical samples.

PubMed

Hintzsche, Jennifer; Kim, Jihye; Yadav, Vinod; Amato, Carol; Robinson, Steven E; Seelenfreund, Eric; Shellman, Yiqun; Wisell, Joshua; Applegate, Allison; McCarter, Martin; Box, Neil; Tentler, John; De, Subhajyoti; Robinson, William A; Tan, Aik Choon

2016-07-01

Currently, there is a disconnect between finding a patient's relevant molecular profile and predicting actionable therapeutics. Here we develop and implement the Integrating Molecular Profiles with Actionable Therapeutics (IMPACT) analysis pipeline, linking variants detected from whole-exome sequencing (WES) to actionable therapeutics. The IMPACT pipeline contains 4 analytical modules: detecting somatic variants, calling copy number alterations, predicting drugs against deleterious variants, and analyzing tumor heterogeneity. We tested the IMPACT pipeline on whole-exome sequencing data in The Cancer Genome Atlas (TCGA) lung adenocarcinoma samples with known EGFR mutations. We also used IMPACT to analyze melanoma patient tumor samples before treatment, after BRAF-inhibitor treatment, and after BRAF- and MEK-inhibitor treatment. IMPACT Food and Drug Administration (FDA) correctly identified known EGFR mutations in the TCGA lung adenocarcinoma samples. IMPACT linked these EGFR mutations to the appropriate FDA-approved EGFR inhibitors. For the melanoma patient samples, we identified NRAS p.Q61K as an acquired resistance mutation to BRAF-inhibitor treatment. We also identified CDKN2A deletion as a novel acquired resistance mutation to BRAFi/MEKi inhibition. The IMPACT analysis pipeline predicts these somatic variants to actionable therapeutics. We observed the clonal dynamic in the tumor samples after various treatments. We showed that IMPACT not only helped in successful prioritization of clinically relevant variants but also linked these variations to possible targeted therapies. IMPACT provides a new bioinformatics strategy to delineate candidate somatic variants and actionable therapies. This approach can be applied to other patient tumor samples to discover effective drug targets for personalized medicine.IMPACT is publicly available at http://tanlab.ucdenver.edu/IMPACT. © The Author 2016. Published by Oxford University Press on behalf of the American Medical

Identification of co-occurrence in a patient with Dent's disease and ADA2-deficiency by exome sequencing.

PubMed

Günthner, Roman; Wagner, Matias; Thurm, Tobias; Ponsel, Sabine; Höfele, Julia; Lange-Sperandio, Bärbel

2018-04-05

Patients with co-occurrence of two independent pathologies pose a challenge for clinicians as the phenotype often presents as an unclear syndrome. In these cases, exome sequencing serves as a powerful instrument to determine the underlying genetic causes. Here, we present the case of a 4-year old boy with proteinuria, microhematuria, hypercalciuria, nephrocalcinosis, livedo-like rash, recurrent abdominal pain, anemia and continuously elevated CRP. Single exome sequencing revealed the pathogenic nonsense mutation p.(Arg98*) in the CLCN5 gene causing the X-linked inherited, renal tubular disorder Dent's disease. Furthermore, the two pathogenic and compound heterozygous missense variants p.(Gly47Ala) and p.(Pro251Leu) in the CECR1 gene could be identified. Mutations in the CECR1 gene are associated with a hereditary form of polyarteritis nodosa, called ADA2-deficiency. Both parents were carriers of a single heterozygous variant in CECR1 and the mother was carrier of the CLCN5 variant. This case evidently demonstrates the advantage of whole exome sequencing compared to single gene testing as the pathology in the CECR1 gene might have only been diagnosed after the occurrence of signs of systemic vasculitis like strokes or hemorrhages. Therefore, treatment and prevention can now start early to improve the outcome of these patients. Copyright © 2018 Elsevier B.V. All rights reserved.

Targeted Exome Sequencing of Krebs Cycle Genes Reveals Candidate Cancer-Predisposing Mutations in Pheochromocytomas and Paragangliomas.

PubMed

Remacha, Laura; Comino-Méndez, Iñaki; Richter, Susan; Contreras, Laura; Currás-Freixes, María; Pita, Guillermo; Letón, Rocío; Galarreta, Antonio; Torres-Pérez, Rafael; Honrado, Emiliano; Jiménez, Scherezade; Maestre, Lorena; Moran, Sebastian; Esteller, Manel; Satrústegui, Jorgina; Eisenhofer, Graeme; Robledo, Mercedes; Cascón, Alberto

2017-10-15

Purpose: Mutations in Krebs cycle genes are frequently found in patients with pheochromocytomas/paragangliomas. Disruption of SDH, FH or MDH2 enzymatic activities lead to accumulation of specific metabolites, which give rise to epigenetic changes in the genome that cause a characteristic hypermethylated phenotype. Tumors showing this phenotype, but no alterations in the known predisposing genes, could harbor mutations in other Krebs cycle genes. Experimental Design: We used downregulation and methylation of RBP1, as a marker of a hypermethylation phenotype, to select eleven pheochromocytomas and paragangliomas for targeted exome sequencing of a panel of Krebs cycle-related genes. Methylation profiling, metabolite assessment and additional analyses were also performed in selected cases. Results: One of the 11 tumors was found to carry a known cancer-predisposing somatic mutation in IDH1 A variant in GOT2 , c.357A>T, found in a patient with multiple tumors, was associated with higher tumor mRNA and protein expression levels, increased GOT2 enzymatic activity in lymphoblastic cells, and altered metabolite ratios both in tumors and in GOT2 knockdown HeLa cells transfected with the variant. Array methylation-based analysis uncovered a somatic epigenetic mutation in SDHC in a patient with multiple pheochromocytomas and a gastrointestinal stromal tumor. Finally, a truncating germline IDH3B mutation was found in a patient with a single paraganglioma showing an altered α-ketoglutarate/isocitrate ratio. Conclusions: This study further attests to the relevance of the Krebs cycle in the development of PCC and PGL, and points to a potential role of other metabolic enzymes involved in metabolite exchange between mitochondria and cytosol. Clin Cancer Res; 23(20); 6315-24. ©2017 AACR . ©2017 American Association for Cancer Research.

Diagnosis of mitochondrial disorders by concomitant next-generation sequencing of the exome and mitochondrial genome

PubMed Central

Dinwiddie, Darrell L.; Smith, Laurie D.; Miller, Neil A.; Atherton, Andrea M.; Farrow, Emily G.; Strenk, Meghan E.; Soden, Sarah E.; Saunders, Carol J.; Kingsmore, Stephen F.

2015-01-01

Mitochondrial diseases are notoriously difficult to diagnose due to extreme locus and allelic heterogeneity, with both nuclear and mitochondrial genomes potentially liable. Using exome sequencing we demonstrate the ability to rapidly and cost effectively evaluate both the nuclear and mitochondrial genomes to obtain a molecular diagnosis for four patients with three distinct mitochondrial disorders. One patient was found to have Leigh syndrome due to a mutation in MT-ATP6, two affected siblings were discovered to be compound heterozygous for mutations in the NDUFV1 gene, which causes mitochondrial complex I deficiency, and one patient was found to have coenzyme Q10 deficiency due to compound heterozygous mutations in COQ2. In all cases conventional diagnostic testing failed to identify a molecular diagnosis. We suggest that additional studies should be conducted to evaluate exome sequencing as a primary diagnostic test for mitochondrial diseases, including those due to mtDNA mutations. PMID:23631824

Whole-exome sequencing identifies recurrent SF3B1 R625 mutation and comutation of NF1 and KIT in mucosal melanoma.

PubMed

Hintzsche, Jennifer D; Gorden, Nicholas T; Amato, Carol M; Kim, Jihye; Wuensch, Kelsey E; Robinson, Steven E; Applegate, Allison J; Couts, Kasey L; Medina, Theresa M; Wells, Keith R; Wisell, Joshua A; McCarter, Martin D; Box, Neil F; Shellman, Yiqun G; Gonzalez, Rene C; Lewis, Karl D; Tentler, John J; Tan, Aik Choon; Robinson, William A

2017-06-01

Mucosal melanomas are a rare subtype of melanoma, arising in mucosal tissues, which have a very poor prognosis due to the lack of effective targeted therapies. This study aimed to better understand the molecular landscape of these cancers and find potential new therapeutic targets. Whole-exome sequencing was performed on mucosal melanomas from 19 patients and 135 sun-exposed cutaneous melanomas, with matched peripheral blood samples when available. Mutational profiles were compared between mucosal subgroups and sun-exposed cutaneous melanomas. Comparisons of molecular profiles identified 161 genes enriched in mucosal melanoma (P<0.05). KIT and NF1 were frequently comutated (32%) in the mucosal subgroup, with a significantly higher incidence than that in cutaneous melanoma (4%). Recurrent SF3B1 R625H/S/C mutations were identified and validated in 7 of 19 (37%) mucosal melanoma patients. Mutations in the spliceosome pathway were found to be enriched in mucosal melanomas when compared with cutaneous melanomas. Alternative splicing in four genes were observed in SF3B1-mutant samples compared with the wild-type samples. This study identified potential new therapeutic targets for mucosal melanoma, including comutation of NF1 and KIT, and recurrent R625 mutations in SF3B1. This is the first report of SF3B1 R625 mutations in vulvovaginal mucosal melanoma, with the largest whole-exome sequencing project of mucosal melanomas to date. The results here also indicated that the mutations in SF3B1 lead to alternative splicing in multiple genes. These findings expand our knowledge of this rare disease.

Whole-exome sequencing of primary plasma cell leukemia discloses heterogeneous mutational patterns.

PubMed

Cifola, Ingrid; Lionetti, Marta; Pinatel, Eva; Todoerti, Katia; Mangano, Eleonora; Pietrelli, Alessandro; Fabris, Sonia; Mosca, Laura; Simeon, Vittorio; Petrucci, Maria Teresa; Morabito, Fortunato; Offidani, Massimo; Di Raimondo, Francesco; Falcone, Antonietta; Caravita, Tommaso; Battaglia, Cristina; De Bellis, Gianluca; Palumbo, Antonio; Musto, Pellegrino; Neri, Antonino

2015-07-10

Primary plasma cell leukemia (pPCL) is a rare and aggressive form of plasma cell dyscrasia and may represent a valid model for high-risk multiple myeloma (MM). To provide novel information concerning the mutational profile of this disease, we performed the whole-exome sequencing of a prospective series of 12 pPCL cases included in a Phase II multicenter clinical trial and previously characterized at clinical and molecular levels. We identified 1, 928 coding somatic non-silent variants on 1, 643 genes, with a mean of 166 variants per sample, and only few variants and genes recurrent in two or more samples. An excess of C > T transitions and the presence of two main mutational signatures (related to APOBEC over-activity and aging) occurring in different translocation groups were observed. We identified 14 candidate cancer driver genes, mainly involved in cell-matrix adhesion, cell cycle, genome stability, RNA metabolism and protein folding. Furthermore, integration of mutation data with copy number alteration profiles evidenced biallelically disrupted genes with potential tumor suppressor functions. Globally, cadherin/Wnt signaling, extracellular matrix and cell cycle checkpoint resulted the most affected functional pathways. Sequencing results were finally combined with gene expression data to better elucidate the biological relevance of mutated genes. This study represents the first whole-exome sequencing screen of pPCL and evidenced a remarkable genetic heterogeneity of mutational patterns. This may provide a contribution to the comprehension of the pathogenetic mechanisms associated with this aggressive form of PC dyscrasia and potentially with high-risk MM.

Whole exome sequencing: a state-of-the-art approach for defining (and exploring!) genetic landscapes in pediatric nephrology.

PubMed

Gulati, Ashima; Somlo, Stefan

2018-05-01

The genesis of whole exome sequencing as a powerful tool for detailing the protein coding sequence of the human genome was conceptualized based on the availability of next-generation sequencing technology and knowledge of the human reference genome. The field of pediatric nephrology enriched with molecularly unsolved phenotypes is allowing the clinical and research application of whole exome sequencing to enable novel gene discovery and provide amendment of phenotypic misclassification. Recent studies in the field have informed us that newer high-throughput sequencing techniques are likely to be of high yield when applied in conjunction with conventional genomic approaches such as linkage analysis and other strategies used to focus subsequent analysis. They have also emphasized the need for the validation of novel genetic findings in large collaborative cohorts and the production of robust corroborative biological data. The well-structured application of comprehensive genomic testing in clinical and research arenas will hopefully continue to advance patient care and precision medicine, but does call for attention to be paid to its integrated challenges.

Exome sequencing coupled with mRNA analysis identifies NDUFAF6 as a Leigh gene.

PubMed

Bianciardi, Laura; Imperatore, Valentina; Fernandez-Vizarra, Erika; Lopomo, Angela; Falabella, Micol; Furini, Simone; Galluzzi, Paolo; Grosso, Salvatore; Zeviani, Massimo; Renieri, Alessandra; Mari, Francesca; Frullanti, Elisa

2016-11-01

We report here the case of a young male who started to show verbal fluency disturbance, clumsiness and gait anomalies at the age of 3.5years and presented bilateral striatal necrosis. Clinically, the diagnosis was compatible with Leigh syndrome but the underlying molecular defect remained elusive even after exome analysis using autosomal/X-linked recessive or de novo models. Dosage of respiratory chain activity on fibroblasts, but not in muscle, underlined a deficit in complex I. Re-analysis of heterozygous probably pathogenic variants, inherited from one healthy parent, identified the p.Ala178Pro in NDUFAF6, a complex I assembly factor. RNA analysis showed an almost mono-allelic expression of the mutated allele in blood and fibroblasts and puromycin treatment on cultured fibroblasts did not lead to the rescue of the maternal allele expression, not supporting the involvement of nonsense-mediated RNA decay mechanism. Complementation assay underlined a recovery of complex I activity after transduction of the wild-type gene. Since the second mutation was not detected and promoter methylation analysis resulted normal, we hypothesized a non-exonic event in the maternal allele affecting a regulatory element that, in conjunction with the paternal mutation, leads to the autosomal recessive disorder and the different allele expression in various tissues. This paper confirms NDUFAF6 as a genuine morbid gene and proposes the coupling of exome sequencing with mRNA analysis as a method useful for enhancing the exome sequencing detection rate when the simple application of classical inheritance models fails. Copyright © 2016 Elsevier Inc. All rights reserved.

Further delineation of COG8-CDG: A case with novel compound heterozygous mutations diagnosed by targeted exome sequencing.

PubMed

Yang, Aram; Cho, Sung Yoon; Jang, Ja-Hyun; Kim, Jinsup; Kim, Sook Za; Lee, Beom Hee; Yoo, Han-Wook; Jin, Dong-Kyu

2017-08-01

Congenital disorders of glycosylation (CDG) are a rapidly expanding group of inherited metabolic disorders with highly variable clinical presentations caused by deficient glycosylation of proteins and/or lipids. CDG-IIh is a very rare subgroup of CDG caused by mutations in the conserved oligomeric Golgi (COG) complex gene, COG8, and so far, only two cases have been reported in the medical literature. Here, we describe an 8-year-old Korean boy with psychomotor retardation, hypotonia, failure to thrive, elevated serum liver enzymes, microcephaly, and talipes equinovarus. A liver biopsy of the patient showed only interface hepatitis with mild lobular activity, and brain magnetic resonance imaging revealed cerebellar atrophy. Compared with the previous two reported cases, our patient showed relatively mild psychomotor retardation without a seizure history. The transferrin isoelectric focusing profiles in the patient showed a CDG type II pattern with increased disialo- and trisialo-transferrin. Targeted exome sequencing was performed to screen all CDG type II-related genes, and two novel frameshift mutations were found: c.171dupG (p.Leu58Alafs*29) and c.1656dupC (p.Ala553Argfs*15) in COG8. The parents were heterozygous carriers of each variant. CDG should be included in the initial differential diagnosis for children with a suspected unknown syndrome or unclassified inherited metabolic disorder or children with diverse clinical presentations, such as psychomotor retardation, hypotonia, skeletal deformity, microcephaly, cerebellar atrophy, and unexplained transient elevated liver enzyme. Copyright © 2017 Elsevier B.V. All rights reserved.

Whole-exome sequencing identifies rare and low-frequency coding variants associated with LDL cholesterol.

PubMed

Lange, Leslie A; Hu, Youna; Zhang, He; Xue, Chenyi; Schmidt, Ellen M; Tang, Zheng-Zheng; Bizon, Chris; Lange, Ethan M; Smith, Joshua D; Turner, Emily H; Jun, Goo; Kang, Hyun Min; Peloso, Gina; Auer, Paul; Li, Kuo-Ping; Flannick, Jason; Zhang, Ji; Fuchsberger, Christian; Gaulton, Kyle; Lindgren, Cecilia; Locke, Adam; Manning, Alisa; Sim, Xueling; Rivas, Manuel A; Holmen, Oddgeir L; Gottesman, Omri; Lu, Yingchang; Ruderfer, Douglas; Stahl, Eli A; Duan, Qing; Li, Yun; Durda, Peter; Jiao, Shuo; Isaacs, Aaron; Hofman, Albert; Bis, Joshua C; Correa, Adolfo; Griswold, Michael E; Jakobsdottir, Johanna; Smith, Albert V; Schreiner, Pamela J; Feitosa, Mary F; Zhang, Qunyuan; Huffman, Jennifer E; Crosby, Jacy; Wassel, Christina L; Do, Ron; Franceschini, Nora; Martin, Lisa W; Robinson, Jennifer G; Assimes, Themistocles L; Crosslin, David R; Rosenthal, Elisabeth A; Tsai, Michael; Rieder, Mark J; Farlow, Deborah N; Folsom, Aaron R; Lumley, Thomas; Fox, Ervin R; Carlson, Christopher S; Peters, Ulrike; Jackson, Rebecca D; van Duijn, Cornelia M; Uitterlinden, André G; Levy, Daniel; Rotter, Jerome I; Taylor, Herman A; Gudnason, Vilmundur; Siscovick, David S; Fornage, Myriam; Borecki, Ingrid B; Hayward, Caroline; Rudan, Igor; Chen, Y Eugene; Bottinger, Erwin P; Loos, Ruth J F; Sætrom, Pål; Hveem, Kristian; Boehnke, Michael; Groop, Leif; McCarthy, Mark; Meitinger, Thomas; Ballantyne, Christie M; Gabriel, Stacey B; O'Donnell, Christopher J; Post, Wendy S; North, Kari E; Reiner, Alexander P; Boerwinkle, Eric; Psaty, Bruce M; Altshuler, David; Kathiresan, Sekar; Lin, Dan-Yu; Jarvik, Gail P; Cupples, L Adrienne; Kooperberg, Charles; Wilson, James G; Nickerson, Deborah A; Abecasis, Goncalo R; Rich, Stephen S; Tracy, Russell P; Willer, Cristen J

2014-02-06

Elevated low-density lipoprotein cholesterol (LDL-C) is a treatable, heritable risk factor for cardiovascular disease. Genome-wide association studies (GWASs) have identified 157 variants associated with lipid levels but are not well suited to assess the impact of rare and low-frequency variants. To determine whether rare or low-frequency coding variants are associated with LDL-C, we exome sequenced 2,005 individuals, including 554 individuals selected for extreme LDL-C (>98(th) or <2(nd) percentile). Follow-up analyses included sequencing of 1,302 additional individuals and genotype-based analysis of 52,221 individuals. We observed significant evidence of association between LDL-C and the burden of rare or low-frequency variants in PNPLA5, encoding a phospholipase-domain-containing protein, and both known and previously unidentified variants in PCSK9, LDLR and APOB, three known lipid-related genes. The effect sizes for the burden of rare variants for each associated gene were substantially higher than those observed for individual SNPs identified from GWASs. We replicated the PNPLA5 signal in an independent large-scale sequencing study of 2,084 individuals. In conclusion, this large whole-exome-sequencing study for LDL-C identified a gene not known to be implicated in LDL-C and provides unique insight into the design and analysis of similar experiments. Copyright © 2014 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

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.

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

Whole-Exome Sequencing to Identify Novel Biological Pathways Associated With Infertility After Pelvic Inflammatory Disease.

PubMed

Taylor, Brandie D; Zheng, Xiaojing; Darville, Toni; Zhong, Wujuan; Konganti, Kranti; Abiodun-Ojo, Olayinka; Ness, Roberta B; O'Connell, Catherine M; Haggerty, Catherine L

2017-01-01

Ideal management of sexually transmitted infections (STI) may require risk markers for pathology or vaccine development. Previously, we identified common genetic variants associated with chlamydial pelvic inflammatory disease (PID) and reduced fecundity. As this explains only a proportion of the long-term morbidity risk, we used whole-exome sequencing to identify biological pathways that may be associated with STI-related infertility. We obtained stored DNA from 43 non-Hispanic black women with PID from the PID Evaluation and Clinical Health Study. Infertility was assessed at a mean of 84 months. Principal component analysis revealed no population stratification. Potential covariates did not significantly differ between groups. Sequencing kernel association test was used to examine associations between aggregates of variants on a single gene and infertility. The results from the sequencing kernel association test were used to choose "focus genes" (P < 0.01; n = 150) for subsequent Ingenuity Pathway Analysis to identify "gene sets" that are enriched in biologically relevant pathways. Pathway analysis revealed that focus genes were enriched in canonical pathways including, IL-1 signaling, P2Y purinergic receptor signaling, and bone morphogenic protein signaling. Focus genes were enriched in pathways that impact innate and adaptive immunity, protein kinase A activity, cellular growth, and DNA repair. These may alter host resistance or immunopathology after infection. Targeted sequencing of biological pathways identified in this study may provide insight into STI-related infertility.

Whole-exome sequencing identifies novel candidate predisposition genes for familial polycythemia vera.

PubMed

Hirvonen, Elina A M; Pitkänen, Esa; Hemminki, Kari; Aaltonen, Lauri A; Kilpivaara, Outi

2017-04-20

Polycythemia vera (PV), characterized by massive production of erythrocytes, is one of the myeloproliferative neoplasms. Most patients carry a somatic gain-of-function mutation in JAK2, c.1849G > T (p.Val617Phe), leading to constitutive activation of JAK-STAT signaling pathway. Familial clustering is also observed occasionally, but high-penetrance predisposition genes to PV have remained unidentified. We studied the predisposition to PV by exome sequencing (three cases) in a Finnish PV family with four patients. The 12 shared variants (maximum allowed minor allele frequency <0.001 in Finnish population in ExAC database) predicted damaging in silico and absent in an additional control set of over 500 Finns were further validated by Sanger sequencing in a fourth affected family member. Three novel predisposition candidate variants were identified: c.1254C > G (p.Phe418Leu) in ZXDC, c.1931C > G (p.Pro644Arg) in ATN1, and c.701G > A (p.Arg234Gln) in LRRC3. We also observed a rare, predicted benign germline variant c.2912C > G (p.Ala971Gly) in BCORL1 in all four patients. Somatic mutations in BCORL1 have been reported in myeloid malignancies. We further screened the variants in eight PV patients in six other Finnish families, but no other carriers were found. Exome sequencing provides a powerful tool for the identification of novel variants, and understanding the familial predisposition of diseases. This is the first report on Finnish familial PV cases, and we identified three novel candidate variants that may predispose to the disease.

Identification of a novel PHEX mutation in a Chinese family with X-linked hypophosphatemic rickets using exome sequencing.

PubMed

Yuan, Lamei; Wu, Song; Xu, Hongbo; Xiao, Jingjing; Yang, Zhijian; Xia, Hong; Liu, An; Hu, Pengzhi; Lu, Anjie; Chen, Yulan; Xu, Fengping; Deng, Hao

2015-01-01

Familial hypophosphatemic rickets (HR), the most common inherited form of rickets, is a group of inherited renal phosphate wasting disorders characterized by growth retardation, rickets with bone deformities, osteomalacia, poor dental development, and hypophosphatemia. The purpose of this study was to identify the genetic defect responsible for familial HR in a four-generation Chinese Han pedigree by exome sequencing and Sanger sequencing. Clinical features include skeletal deformities, teeth abnormalities, hearing impairments and variable serum phosphate level in patients of this family. A novel deletion mutation, c.1553delT (p.F518Sfs*4), was identified in the X-linked phosphate regulating endopeptidase homolog gene (PHEX). The mutation is predicted to result in prematurely truncated and loss-of-function PHEX protein. Our data suggest that exome sequencing is a powerful tool to discover mutation(s) in HR, a disorder with genetic and clinical heterogeneity. The findings may also provide new insights into the cause and diagnosis of HR, and have implications for genetic counseling and clinical management.

Exome sequencing identifies SUCO mutations in mesial temporal lobe epilepsy.

PubMed

Sha, Zhiqiang; Sha, Longze; Li, Wenting; Dou, Wanchen; Shen, Yan; Wu, Liwen; Xu, Qi

2015-03-30

Mesial temporal lobe epilepsy (mTLE) is the main type and most common medically intractable form of epilepsy. Severity of disease-based stratified samples may help identify new disease-associated mutant genes. We analyzed mRNA expression profiles from patient hippocampal tissue. Three of the seven patients had severe mTLE with generalized-onset convulsions and consciousness loss that occurred over many years. We found that compared with other groups, patients with severe mTLE were classified into a distinct group. Whole-exome sequencing and Sanger sequencing validation in all seven patients identified three novel SUN domain-containing ossification factor (SUCO) mutations in severely affected patients. Furthermore, SUCO knock down significantly reduced dendritic length in vitro. Our results indicate that mTLE defects may affect neuronal development, and suggest that neurons have abnormal development due to lack of SUCO, which may be a generalized-onset epilepsy-related gene. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Whole-exome sequencing identifies recurrent SF3B1 R625 mutation and comutation of NF1 and KIT in mucosal melanoma

PubMed Central

Hintzsche, Jennifer D.; Gorden, Nicholas T.; Amato, Carol M.; Kim, Jihye; Wuensch, Kelsey E.; Robinson, Steven E.; Applegate, Allison J.; Couts, Kasey L.; Medina, Theresa M.; Wells, Keith R.; Wisell, Joshua A.; McCarter, Martin D.; Box, Neil F.; Shellman, Yiqun G.; Gonzalez, Rene C.; Lewis, Karl D.; Tentler, John J.

2017-01-01

Mucosal melanomas are a rare subtype of melanoma, arising in mucosal tissues, which have a very poor prognosis due to the lack of effective targeted therapies. This study aimed to better understand the molecular landscape of these cancers and find potential new therapeutic targets. Whole-exome sequencing was performed on mucosal melanomas from 19 patients and 135 sun-exposed cutaneous melanomas, with matched peripheral blood samples when available. Mutational profiles were compared between mucosal subgroups and sun-exposed cutaneous melanomas. Comparisons of molecular profiles identified 161 genes enriched in mucosal melanoma (P<0.05). KIT and NF1 were frequently comutated (32%) in the mucosal subgroup, with a significantly higher incidence than that in cutaneous melanoma (4%). Recurrent SF3B1 R625H/S/C mutations were identified and validated in 7 of 19 (37%) mucosal melanoma patients. Mutations in the spliceosome pathway were found to be enriched in mucosal melanomas when compared with cutaneous melanomas. Alternative splicing in four genes were observed in SF3B1-mutant samples compared with the wild-type samples. This study identified potential new therapeutic targets for mucosal melanoma, including comutation of NF1 and KIT, and recurrent R625 mutations in SF3B1. This is the first report of SF3B1 R625 mutations in vulvovaginal mucosal melanoma, with the largest whole-exome sequencing project of mucosal melanomas to date. The results here also indicated that the mutations in SF3B1 lead to alternative splicing in multiple genes. These findings expand our knowledge of this rare disease. PMID:28296713

Whole-Exome Sequencing to Decipher the Genetic Heterogeneity of Hearing Loss in a Chinese Family with Deaf by Deaf Mating

PubMed Central

Qing, Jie; Yan, Denise; Zhou, Yuan; Liu, Qiong; Wu, Weijing; Xiao, Zian; Liu, Yuyuan; Liu, Jia; Du, Lilin; Xie, Dinghua; Liu, Xue Zhong

2014-01-01

Inherited deafness has been shown to have high genetic heterogeneity. For many decades, linkage analysis and candidate gene approaches have been the main tools to elucidate the genetics of hearing loss. However, this associated study design is costly, time-consuming, and unsuitable for small families. This is mainly due to the inadequate numbers of available affected individuals, locus heterogeneity, and assortative mating. Exome sequencing has now become technically feasible and a cost-effective method for detection of disease variants underlying Mendelian disorders due to the recent advances in next-generation sequencing (NGS) technologies. In the present study, we have combined both the Deafness Gene Mutation Detection Array and exome sequencing to identify deafness causative variants in a large Chinese composite family with deaf by deaf mating. The simultaneous screening of the 9 common deafness mutations using the allele-specific PCR based universal array, resulted in the identification of the 1555A>G in the mitochondrial DNA (mtDNA) 12S rRNA in affected individuals in one branch of the family. We then subjected the mutation-negative cases to exome sequencing and identified novel causative variants in the MYH14 and WFS1 genes. This report confirms the effective use of a NGS technique to detect pathogenic mutations in affected individuals who were not candidates for classical genetic studies. PMID:25289672

Whole exome sequencing for determination of tumor mutation load in liquid biopsy from advanced cancer patients.

PubMed

Koeppel, Florence; Blanchard, Steven; Jovelet, Cécile; Genin, Bérengère; Marcaillou, Charles; Martin, Emmanuel; Rouleau, Etienne; Solary, Eric; Soria, Jean-Charles; André, Fabrice; Lacroix, Ludovic

2017-01-01

Tumor mutation load (TML) has been proposed as a biomarker of patient response to immunotherapy in several studies. TML is usually determined by tumor biopsy DNA (tDNA) whole exome sequencing (WES), therefore TML evaluation is limited by informative biopsy availability. Circulating cell free DNA (cfDNA) provided by liquid biopsy is a surrogate specimen to biopsy for molecular profiling. Nevertheless performing WES on DNA from plasma is technically challenging and the ability to determine tumor mutation load from liquid biopsies remains to be demonstrated. In the current study, WES was performed on cfDNA from 32 metastatic patients of various cancer types included into MOSCATO 01 (NCT01566019) and/or MATCHR (NCT02517892) molecular triage trials. Results from targeted gene sequencing (TGS) and WES performed on cfDNA were compared to results from tumor tissue biopsy. In cfDNA samples, WES mutation detection sensitivity was 92% compared to targeted sequencing (TGS). When comparing cfDNA-WES to tDNA-WES, mutation detection sensitivity was 53%, consistent with previously published prospective study comparing cfDNA-TGS to tDNA-TGS. For samples in which presence of tumor DNA was confirmed in cfDNA, tumor mutation load from liquid biopsy was correlated with tumor biopsy. Taken together, this study demonstrated that liquid biopsy may be applied to determine tumor mutation load. Qualification of liquid biopsy for interpretation is a crucial point to use cfDNA for mutational load estimation.

Whole exome sequencing for determination of tumor mutation load in liquid biopsy from advanced cancer patients

PubMed Central

Blanchard, Steven; Jovelet, Cécile; Genin, Bérengère; Marcaillou, Charles; Martin, Emmanuel; Rouleau, Etienne; Solary, Eric; Soria, Jean-Charles; André, Fabrice; Lacroix, Ludovic

2017-01-01

Tumor mutation load (TML) has been proposed as a biomarker of patient response to immunotherapy in several studies. TML is usually determined by tumor biopsy DNA (tDNA) whole exome sequencing (WES), therefore TML evaluation is limited by informative biopsy availability. Circulating cell free DNA (cfDNA) provided by liquid biopsy is a surrogate specimen to biopsy for molecular profiling. Nevertheless performing WES on DNA from plasma is technically challenging and the ability to determine tumor mutation load from liquid biopsies remains to be demonstrated. In the current study, WES was performed on cfDNA from 32 metastatic patients of various cancer types included into MOSCATO 01 (NCT01566019) and/or MATCHR (NCT02517892) molecular triage trials. Results from targeted gene sequencing (TGS) and WES performed on cfDNA were compared to results from tumor tissue biopsy. In cfDNA samples, WES mutation detection sensitivity was 92% compared to targeted sequencing (TGS). When comparing cfDNA-WES to tDNA-WES, mutation detection sensitivity was 53%, consistent with previously published prospective study comparing cfDNA-TGS to tDNA-TGS. For samples in which presence of tumor DNA was confirmed in cfDNA, tumor mutation load from liquid biopsy was correlated with tumor biopsy. Taken together, this study demonstrated that liquid biopsy may be applied to determine tumor mutation load. Qualification of liquid biopsy for interpretation is a crucial point to use cfDNA for mutational load estimation. PMID:29161279

Gratitude, protective buffering, and cognitive dissonance: How families respond to pediatric whole exome sequencing in the absence of actionable results.

PubMed

Werner-Lin, Allison; Zaspel, Lori; Carlson, Mae; Mueller, Rebecca; Walser, Sarah A; Desai, Ria; Bernhardt, Barbara A

2018-03-01

Clinical genome and exome sequencing (CGES) may identify variants leading to targeted management of existing conditions. Yet, CGES often fails to identify pathogenic diagnostic variants and introduces uncertainties by detecting variants of uncertain significance (VUS) and secondary findings. This study investigated how families understand findings and adjust their perspectives on CGES. As part of NIH's Clinical Sequencing Exploratory Research Consortium, children were recruited from clinics at the Children's Hospital of Pennsylvania (CHOP) and offered exome sequencing. Primary pathogenic and possibly pathogenic, and some secondary findings were returned. Investigators digitally recorded results disclosure sessions and conducted 3-month follow up interviews with 10 adolescents and a parent. An interdisciplinary team coded all transcripts. Participants were initially disappointed with findings, yet reactions evolved within disclosure sessions and at 3-month interviews toward acceptance and satisfaction. Families erroneously expected, and prepared extensively, to learn about risk for common conditions. During disclosure sessions, parents and adolescents varied in how they monitored and responded to each others reactions. Several misinterpreted, or overestimated, the utility of findings to attribute meaning and achieve closure for the CGES experience. Participants perceived testing as an opportunity to improve disease management despite results that did not introduce new treatments or diagnoses. Future research may examine whether families experience cognitive dissonance regarding discrepancies between expectations and findings, and how protective buffering minimizes the burden of disappointment on loved ones. As CGES is increasingly integrated into clinical care providers must contend with tempering family expectations and interpretations of findings while managing complex medical care. © 2018 Wiley Periodicals, Inc.

Exome sequencing in Thai patients with familial obesity.

PubMed

Kaewsutthi, S; Santiprabhob, J; Phonrat, B; Tungtrongchitr, A; Lertrit, P; Tungtrongchitr, R

2016-07-14

Obesity is a major worldwide health issue, with increasing prevalence in adults and children from developed and developing countries. Obesity causes several chronic diseases, including cardiovascular and respiratory diseases, osteoarthritis, hypertension, stroke, type II diabetes, obstructive sleep apnea, and several types of cancer. Previous genome-wide association studies have identified several genes associated with obesity, including LEP, LEPR, POMC, PCSK1, FTO, MC3R, MC4R, GNPDA2, TMEM18, QPCTL/GIPR, BDNF, ETV5, MAP2K5/SKOR1, SEC16B, SIM1, and TNKS/MSRA. However, most of these variants are found in the intronic or intergenic regions, making it difficult to elucidate the underlying mechanisms. Therefore, in this study, we performed a whole exome sequencing of the protein-coding regions in the total genome (exome) of two obese and one normal subject belonging to the same Thai family to identify the genes responsible for obesity. We identified 709 functional variants that were differentially expressed between obese and normal subjects; of these, 65 were predicted to be deleterious to protein structure or function. The minor allele frequency of 14 of these genes (ALOX5AP, COL9A2, DEFB126, GDPD4, HCRTR1, MLL3, OPLAH, OR4C45, PRIM2, RXFP2, TIGD6, TRPM8, USP49, and ZNF596) was low, indicating causal variants that could be associated with complex traits or diseases. Genotyping revealed HCRTR1, COL9A2, and TRPM8 to be associated with the regulation of feeding behavior and energy expenditure. These genes constituted a network of pathways, including lipid metabolism, signaling transduction, immune, membrane transport, and gene regulation pathways, and seemed to play important roles in obesity.

Rapid identification of kidney cyst mutations by whole exome sequencing in zebrafish

PubMed Central

Ryan, Sean; Willer, Jason; Marjoram, Lindsay; Bagwell, Jennifer; Mankiewicz, Jamie; Leshchiner, Ignaty; Goessling, Wolfram; Bagnat, Michel; Katsanis, Nicholas

2013-01-01

Forward genetic approaches in zebrafish have provided invaluable information about developmental processes. However, the relative difficulty of mapping and isolating mutations has limited the number of new genetic screens. Recent improvements in the annotation of the zebrafish genome coupled to a reduction in sequencing costs prompted the development of whole genome and RNA sequencing approaches for gene discovery. Here we describe a whole exome sequencing (WES) approach that allows rapid and cost-effective identification of mutations. We used our WES methodology to isolate four mutations that cause kidney cysts; we identified novel alleles in two ciliary genes as well as two novel mutants. The WES approach described here does not require specialized infrastructure or training and is therefore widely accessible. This methodology should thus help facilitate genetic screens and expedite the identification of mutants that can inform basic biological processes and the causality of genetic disorders in humans. PMID:24130329

Barley whole exome capture: a tool for genomic research in the genus Hordeum and beyond

PubMed Central

Mascher, Martin; Richmond, Todd A; Gerhardt, Daniel J; Himmelbach, Axel; Clissold, Leah; Sampath, Dharanya; Ayling, Sarah; Steuernagel, Burkhard; Pfeifer, Matthias; D'Ascenzo, Mark; Akhunov, Eduard D; Hedley, Pete E; Gonzales, Ana M; Morrell, Peter L; Kilian, Benjamin; Blattner, Frank R; Scholz, Uwe; Mayer, Klaus FX; Flavell, Andrew J; Muehlbauer, Gary J; Waugh, Robbie; Jeddeloh, Jeffrey A; Stein, Nils

2013-01-01

Advanced resources for genome-assisted research in barley (Hordeum vulgare) including a whole-genome shotgun assembly and an integrated physical map have recently become available. These have made possible studies that aim to assess genetic diversity or to isolate single genes by whole-genome resequencing and in silico variant detection. However such an approach remains expensive given the 5 Gb size of the barley genome. Targeted sequencing of the mRNA-coding exome reduces barley genomic complexity more than 50-fold, thus dramatically reducing this heavy sequencing and analysis load. We have developed and employed an in-solution hybridization-based sequence capture platform to selectively enrich for a 61.6 megabase coding sequence target that includes predicted genes from the genome assembly of the cultivar Morex as well as publicly available full-length cDNAs and de novo assembled RNA-Seq consensus sequence contigs. The platform provides a highly specific capture with substantial and reproducible enrichment of targeted exons, both for cultivated barley and related species. We show that this exome capture platform provides a clear path towards a broader and deeper understanding of the natural variation residing in the mRNA-coding part of the barley genome and will thus constitute a valuable resource for applications such as mapping-by-sequencing and genetic diversity analyzes. PMID:23889683

Thiamine pyrophosphokinase deficiency causes a Leigh Disease like phenotype in a sibling pair: identification through whole exome sequencing and management strategies.

PubMed

Fraser, Jamie L; Vanderver, Adeline; Yang, Sandra; Chang, Taeun; Cramp, Laura; Vezina, Gilbert; Lichter-Konecki, Uta; Cusmano-Ozog, Kristina P; Smpokou, Patroula; Chapman, Kimberly A; Zand, Dina J

2014-01-01

We present a sibling pair with Leigh-like disease, progressive hypotonia, regression, and chronic encephalopathy. Whole exome sequencing in the younger sibling demonstrated a homozygous thiamine pyrophosphokinase (TPK) mutation. Initiation of high dose thiamine, niacin, biotin, α-lipoic acid and ketogenic diet in this child demonstrated improvement in neurologic function and re-attainment of previously lost milestones. The diagnosis of TPK deficiency was difficult due to inconsistent biochemical and diagnostic parameters, rapidity of clinical demise and would not have been made in a timely manner without the use of whole exome sequencing. Molecular diagnosis allowed for attempt at dietary modification with cofactor supplementation which resulted in an improved clinical course.

Development and validation of a whole-exome sequencing test for simultaneous detection of point mutations, indels and copy-number alterations for precision cancer care

PubMed Central

Rennert, Hanna; Eng, Kenneth; Zhang, Tuo; Tan, Adrian; Xiang, Jenny; Romanel, Alessandro; Kim, Robert; Tam, Wayne; Liu, Yen-Chun; Bhinder, Bhavneet; Cyrta, Joanna; Beltran, Himisha; Robinson, Brian; Mosquera, Juan Miguel; Fernandes, Helen; Demichelis, Francesca; Sboner, Andrea; Kluk, Michael; Rubin, Mark A; Elemento, Olivier

2016-01-01

We describe Exome Cancer Test v1.0 (EXaCT-1), the first New York State-Department of Health-approved whole-exome sequencing (WES)-based test for precision cancer care. EXaCT-1 uses HaloPlex (Agilent) target enrichment followed by next-generation sequencing (Illumina) of tumour and matched constitutional control DNA. We present a detailed clinical development and validation pipeline suitable for simultaneous detection of somatic point/indel mutations and copy-number alterations (CNAs). A computational framework for data analysis, reporting and sign-out is also presented. For the validation, we tested EXaCT-1 on 57 tumours covering five distinct clinically relevant mutations. Results demonstrated elevated and uniform coverage compatible with clinical testing as well as complete concordance in variant quality metrics between formalin-fixed paraffin embedded and fresh-frozen tumours. Extensive sensitivity studies identified limits of detection threshold for point/indel mutations and CNAs. Prospective analysis of 337 cancer cases revealed mutations in clinically relevant genes in 82% of tumours, demonstrating that EXaCT-1 is an accurate and sensitive method for identifying actionable mutations, with reasonable costs and time, greatly expanding its utility for advanced cancer care. PMID:28781886

Usability study of clinical exome analysis software: top lessons learned and recommendations.

PubMed

Shyr, Casper; Kushniruk, Andre; Wasserman, Wyeth W

2014-10-01

New DNA sequencing technologies have revolutionized the search for genetic disruptions. Targeted sequencing of all protein coding regions of the genome, called exome analysis, is actively used in research-oriented genetics clinics, with the transition to exomes as a standard procedure underway. This transition is challenging; identification of potentially causal mutation(s) amongst ∼10(6) variants requires specialized computation in combination with expert assessment. This study analyzes the usability of user interfaces for clinical exome analysis software. There are two study objectives: (1) To ascertain the key features of successful user interfaces for clinical exome analysis software based on the perspective of expert clinical geneticists, (2) To assess user-system interactions in order to reveal strengths and weaknesses of existing software, inform future design, and accelerate the clinical uptake of exome analysis. Surveys, interviews, and cognitive task analysis were performed for the assessment of two next-generation exome sequence analysis software packages. The subjects included ten clinical geneticists who interacted with the software packages using the "think aloud" method. Subjects' interactions with the software were recorded in their clinical office within an urban research and teaching hospital. All major user interface events (from the user interactions with the packages) were time-stamped and annotated with coding categories to identify usability issues in order to characterize desired features and deficiencies in the user experience. We detected 193 usability issues, the majority of which concern interface layout and navigation, and the resolution of reports. Our study highlights gaps in specific software features typical within exome analysis. The clinicians perform best when the flow of the system is structured into well-defined yet customizable layers for incorporation within the clinical workflow. The results highlight opportunities to

Prenatal whole exome sequencing: the views of clinicians, scientists, genetic counsellors and patient representatives.

PubMed

Quinlan-Jones, Elizabeth; Kilby, Mark D; Greenfield, Sheila; Parker, Michael; McMullan, Dominic; Hurles, Matthew E; Hillman, Sarah C

2016-10-01

Focus groups were conducted with individuals involved in prenatal diagnosis to determine their opinions relating to whole exome sequencing in fetuses with structural anomalies. Five representatives of patient groups/charities (PRGs) and eight clinical professionals (CPs) participated. Three focus groups occurred (the two groups separately and then combined). Framework analysis was performed to elicit themes. A thematic coding frame was identified based on emerging themes. Seven main themes (consent, analysis, interpretation/reinterpretation of results, prenatal issues, uncertainty, incidental findings and information access) with subthemes emerged. The main themes were raised by both groups, apart from 'analysis', which was raised by CPs only. Some subthemes were raised by PRGs and CPs (with different perspectives). Others were raised either by PRGs or CPs, showing differences in patient/clinician agendas. Prenatal consent for whole exome sequencing is not a 'perfect' process, but consent takers should be fully educated regarding the test. PRGs highlighted issues involving access to results, feeling that women want to know all information. PRGs also felt that patients want reinterpretation of results over time, whilst CPs felt that interpretation should be performed at the point of testing only. © 2016 John Wiley & Sons, Ltd. © 2016 John Wiley & Sons, Ltd.

Somatic Genetic Variation in Solid Pseudopapillary Tumor of the Pancreas by Whole Exome Sequencing

PubMed Central

Guo, Meng; Luo, Guopei; Jin, Kaizhou; Long, Jiang; Cheng, He; Lu, Yu; Wang, Zhengshi; Yang, Chao; Xu, Jin; Ni, Quanxing; Yu, Xianjun; Liu, Chen

2017-01-01

Solid pseudopapillary tumor of the pancreas (SPT) is a rare pancreatic disease with a unique clinical manifestation. Although CTNNB1 gene mutations had been universally reported, genetic variation profiles of SPT are largely unidentified. We conducted whole exome sequencing in nine SPT patients to probe the SPT-specific insertions and deletions (indels) and single nucleotide polymorphisms (SNPs). In total, 54 SNPs and 41 indels of prominent variations were demonstrated through parallel exome sequencing. We detected that CTNNB1 mutations presented throughout all patients studied (100%), and a higher count of SNPs was particularly detected in patients with older age, larger tumor, and metastatic disease. By aggregating 95 detected variation events and viewing the interconnections among each of the genes with variations, CTNNB1 was identified as the core portion in the network, which might collaborate with other events such as variations of USP9X, EP400, HTT, MED12, and PKD1 to regulate tumorigenesis. Pathway analysis showed that the events involved in other cancers had the potential to influence the progression of the SNPs count. Our study revealed an insight into the variation of the gene encoding region underlying solid-pseudopapillary neoplasm tumorigenesis. The detection of these variations might partly reflect the potential molecular mechanism. PMID:28054945

Identification of a novel mutation in a Chinese family with Nance-Horan syndrome by whole exome sequencing*

PubMed Central

Hong, Nan; Chen, Yan-hua; Xie, Chen; Xu, Bai-sheng; Huang, Hui; Li, Xin; Yang, Yue-qing; Huang, Ying-ping; Deng, Jian-lian; Qi, Ming; Gu, Yang-shun

2014-01-01

Objective: Nance-Horan syndrome (NHS) is a rare X-linked disorder characterized by congenital nuclear cataracts, dental anomalies, and craniofacial dysmorphisms. Mental retardation was present in about 30% of the reported cases. The purpose of this study was to investigate the genetic and clinical features of NHS in a Chinese family. Methods: Whole exome sequencing analysis was performed on DNA from an affected male to scan for candidate mutations on the X-chromosome. Sanger sequencing was used to verify these candidate mutations in the whole family. Clinical and ophthalmological examinations were performed on all members of the family. Results: A combination of exome sequencing and Sanger sequencing revealed a nonsense mutation c.322G>T (E108X) in exon 1 of NHS gene, co-segregating with the disease in the family. The nonsense mutation led to the conversion of glutamic acid to a stop codon (E108X), resulting in truncation of the NHS protein. Multiple sequence alignments showed that codon 108, where the mutation (c.322G>T) occurred, was located within a phylogenetically conserved region. The clinical features in all affected males and female carriers are described in detail. Conclusions: We report a nonsense mutation c.322G>T (E108X) in a Chinese family with NHS. Our findings broaden the spectrum of NHS mutations and provide molecular insight into future NHS clinical genetic diagnosis. PMID:25091991

Identification of a novel mutation in a Chinese family with Nance-Horan syndrome by whole exome sequencing.

PubMed

Hong, Nan; Chen, Yan-hua; Xie, Chen; Xu, Bai-sheng; Huang, Hui; Li, Xin; Yang, Yue-qing; Huang, Ying-ping; Deng, Jian-lian; Qi, Ming; Gu, Yang-shun

2014-08-01

Nance-Horan syndrome (NHS) is a rare X-linked disorder characterized by congenital nuclear cataracts, dental anomalies, and craniofacial dysmorphisms. Mental retardation was present in about 30% of the reported cases. The purpose of this study was to investigate the genetic and clinical features of NHS in a Chinese family. Whole exome sequencing analysis was performed on DNA from an affected male to scan for candidate mutations on the X-chromosome. Sanger sequencing was used to verify these candidate mutations in the whole family. Clinical and ophthalmological examinations were performed on all members of the family. A combination of exome sequencing and Sanger sequencing revealed a nonsense mutation c.322G>T (E108X) in exon 1 of NHS gene, co-segregating with the disease in the family. The nonsense mutation led to the conversion of glutamic acid to a stop codon (E108X), resulting in truncation of the NHS protein. Multiple sequence alignments showed that codon 108, where the mutation (c.322G>T) occurred, was located within a phylogenetically conserved region. The clinical features in all affected males and female carriers are described in detail. We report a nonsense mutation c.322G>T (E108X) in a Chinese family with NHS. Our findings broaden the spectrum of NHS mutations and provide molecular insight into future NHS clinical genetic diagnosis.

Exome Sequencing Identifies Truncating Mutations in Human SERPINF1 in Autosomal-Recessive Osteogenesis Imperfecta

PubMed Central

Becker, Jutta; Semler, Oliver; Gilissen, Christian; Li, Yun; Bolz, Hanno Jörn; Giunta, Cecilia; Bergmann, Carsten; Rohrbach, Marianne; Koerber, Friederike; Zimmermann, Katharina; de Vries, Petra; Wirth, Brunhilde; Schoenau, Eckhard; Wollnik, Bernd; Veltman, Joris A.; Hoischen, Alexander; Netzer, Christian

2011-01-01

Osteogenesis imperfecta (OI) is a heterogeneous genetic disorder characterized by bone fragility and susceptibility to fractures after minimal trauma. After mutations in all known OI genes had been excluded by Sanger sequencing, we applied next-generation sequencing to analyze the exome of a single individual who has a severe form of the disease and whose parents are second cousins. A total of 26,922 variations from the human reference genome sequence were subjected to several filtering steps. In addition, we extracted the genotypes of all dbSNP130-annotated SNPs from the exome sequencing data and used these 299,494 genotypes as markers for the genome-wide identification of homozygous regions. A single homozygous truncating mutation, affecting SERPINF1 on chromosome 17p13.3, that was embedded into a homozygous stretch of 2.99 Mb remained. The mutation was also homozygous in the affected brother of the index patient. Subsequently, we identified homozygosity for two different truncating SERPINF1 mutations in two unrelated patients with OI and parental consanguinity. All four individuals with SERPINF1 mutations have severe OI. Fractures of long bones and severe vertebral compression fractures with resulting deformities were observed as early as the first year of life in these individuals. Collagen analyses with cultured dermal fibroblasts displayed no evidence for impaired collagen folding, posttranslational modification, or secretion. SERPINF1 encodes pigment epithelium-derived factor (PEDF), a secreted glycoprotein of the serpin superfamily. PEDF is a multifunctional protein and one of the strongest inhibitors of angiogenesis currently known in humans. Our data provide genetic evidence for PEDF involvement in human bone homeostasis. PMID:21353196

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.

Discovery of somatic mutations in the progression of chronic myeloid leukemia by whole-exome sequencing.

PubMed

Huang, Y; Zheng, J; Hu, J D; Wu, Y A; Zheng, X Y; Liu, T B; Chen, F L

2014-02-19

We performed whole-exome sequencing in samples representing accelerated phase (AP) and blastic crisis (BC) in a subject with chronic myeloid leukemia (CML). A total of 12.74 Gb clean data were generated, achieving a mean depth coverage of 64.45 and 69.53 for AP and BC samples, respectively, of the target region. A total of 148 somatic variants were detected, including 76 insertions and deletions (indels), 64 single-nucleotide variations (SNV), and 8 structural variations (SV). On the basis of annotation and functional prediction analysis, we identified 3 SNVs and 6 SVs that showed a potential association with CML progression. Among the genes that harbor the identified variants, GATA2 has previously been reported to play important roles in the progression from AP to BC in CML. Identification of these genes will allow us to gain a better understanding of the pathological mechanism of CML and represents a critical advance toward new molecular diagnostic tests for the development of potential therapies for CML.

Exome sequencing analysis reveals variants in primary immunodeficiency genes in patients with very early onset inflammatory bowel disease.

PubMed

Kelsen, Judith R; Dawany, Noor; Moran, Christopher J; Petersen, Britt-Sabina; Sarmady, Mahdi; Sasson, Ariella; Pauly-Hubbard, Helen; Martinez, Alejandro; Maurer, Kelly; Soong, Joanne; Rappaport, Eric; Franke, Andre; Keller, Andreas; Winter, Harland S; Mamula, Petar; Piccoli, David; Artis, David; Sonnenberg, Gregory F; Daly, Mark; Sullivan, Kathleen E; Baldassano, Robert N; Devoto, Marcella

2015-11-01

Very early onset inflammatory bowel disease (VEO-IBD), IBD diagnosed at 5 years of age or younger, frequently presents with a different and more severe phenotype than older-onset IBD. We investigated whether patients with VEO-IBD carry rare or novel variants in genes associated with immunodeficiencies that might contribute to disease development. Patients with VEO-IBD and parents (when available) were recruited from the Children's Hospital of Philadelphia from March 2013 through July 2014. We analyzed DNA from 125 patients with VEO-IBD (age, 3 wk to 4 y) and 19 parents, 4 of whom also had IBD. Exome capture was performed by Agilent SureSelect V4, and sequencing was performed using the Illumina HiSeq platform. Alignment to human genome GRCh37 was achieved followed by postprocessing and variant calling. After functional annotation, candidate variants were analyzed for change in protein function, minor allele frequency less than 0.1%, and scaled combined annotation-dependent depletion scores of 10 or less. We focused on genes associated with primary immunodeficiencies and related pathways. An additional 210 exome samples from patients with pediatric IBD (n = 45) or adult-onset Crohn's disease (n = 20) and healthy individuals (controls, n = 145) were obtained from the University of Kiel, Germany, and used as control groups. Four hundred genes and regions associated with primary immunodeficiency, covering approximately 6500 coding exons totaling more than 1 Mbp of coding sequence, were selected from the whole-exome data. Our analysis showed novel and rare variants within these genes that could contribute to the development of VEO-IBD, including rare heterozygous missense variants in IL10RA and previously unidentified variants in MSH5 and CD19. In an exome sequence analysis of patients with VEO-IBD and their parents, we identified variants in genes that regulate B- and T-cell functions and could contribute to pathogenesis. Our analysis could lead to the

Exome Sequencing Analysis Reveals Variants in Primary Immunodeficiency Genes in Patients With Very Early Onset Inflammatory Bowel Disease

PubMed Central

Kelsen, Judith R.; Dawany, Noor; Moran, Christopher J.; Petersen, Britt-Sabina; Sarmady, Mahdi; Sasson, Ariella; Pauly-Hubbard, Helen; Martinez, Alejandro; Maurer, Kelly; Soong, Joanne; Rappaport, Eric; Franke, Andre; Keller, Andreas; Winter, Harland S.; Mamula, Petar; Piccoli, David; Artis, David; Sonnenberg, Gregory F.; Daly, Mark; Sullivan, Kathleen E.; Baldassano, Robert N.; Devoto, Marcella

2016-01-01

Background & Aims Very early onset inflammatory bowel disease (VEO-IBD), IBD diagnosed ≤5 y of age, frequently presents with a different and more severe phenotype than older-onset IBD. We investigated whether patients with VEO-IBD carry rare or novel variants in genes associated with immunodeficiencies that might contribute to disease development. Methods Patients with VEO-IBD and parents (when available) were recruited from the Children's Hospital of Philadelphia from March 2013 through July 2014. We analyzed DNA from 125 patients with VEO-IBD (ages 3 weeks to 4 y) and 19 parents, 4 of whom also had IBD. Exome capture was performed by Agilent SureSelect V4, and sequencing was performed using the Illumina HiSeq platform. Alignment to human genome GRCh37 was achieved followed by post-processing and variant calling. Following functional annotation, candidate variants were analyzed for change in protein function, minor allele frequency <0.1%, and scaled combined annotation dependent depletion scores ≤10. We focused on genes associated with primary immunodeficiencies and related pathways. An additional 210 exome samples from patients with pediatric IBD (n=45) or adult-onset Crohn's disease (n=20) and healthy individuals (controls, n=145) were obtained from the University of Kiel, Germany and used as control groups. Results Four-hundred genes and regions associated with primary immunodeficiency, covering approximately 6500 coding exons totaling > 1 Mbp of coding sequence, were selected from the whole exome data. Our analysis revealed novel and rare variants within these genes that could contribute to the development of VEO-IBD, including rare heterozygous missense variants in IL10RA and previously unidentified variants in MSH5 and CD19. Conclusions In an exome sequence analysis of patients with VEO-IBD and their parents, we identified variants in genes that regulate B- and T-cell functions and could contribute to pathogenesis. Our analysis could lead to the

Whole-exome sequencing revealed two novel mutations in Usher syndrome.

PubMed

Koparir, Asuman; Karatas, Omer Faruk; Atayoglu, Ali Timucin; Yuksel, Bayram; Sagiroglu, Mahmut Samil; Seven, Mehmet; Ulucan, Hakan; Yuksel, Adnan; Ozen, Mustafa

2015-06-01

Usher syndrome is a clinically and genetically heterogeneous autosomal recessive inherited disorder accompanied by hearing loss and retinitis pigmentosa (RP). Since the associated genes are various and quite large, we utilized whole-exome sequencing (WES) as a diagnostic tool to identify the molecular basis of Usher syndrome. DNA from a 12-year-old male diagnosed with Usher syndrome was analyzed by WES. Mutations detected were confirmed by Sanger sequencing. The pathogenicity of these mutations was determined by in silico analysis. A maternally inherited deleterious frameshift mutation, c.14439_14454del in exon 66 and a paternally inherited non-sense c.10830G>A stop-gain SNV in exon 55 of USH2A were found as two novel compound heterozygous mutations. Both of these mutations disrupt the C terminal of USH2A protein. As a result, WES revealed two novel compound heterozygous mutations in a Turkish USH2A patient. This approach gave us an opportunity to have an appropriate diagnosis and provide genetic counseling to the family within a reasonable time. Copyright © 2015 Elsevier B.V. All rights reserved.

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.

Exome Sequencing of 18 Chinese Families with Congenital Cataracts: A New Sight of the NHS Gene

PubMed Central

Sun, Wenmin; Xiao, Xueshan; Li, Shiqiang; Guo, Xiangming; Zhang, Qingjiong

2014-01-01

Purpose The aim of this study was to investigate the mutation spectrum and frequency of 34 known genes in 18 Chinese families with congenital cataracts. Methods Genomic DNA and clinical data was collected from 18 families with congenital cataracts. Variations in 34 cataract-associated genes were screened by whole exome sequencing and then validated by Sanger sequencing. Results Eleven candidate variants in seven of the 34 genes were detected by exome sequencing and then confirmed by Sanger sequencing, including two variants predicted to be benign and the other pathogenic mutations. The nine mutations were present in 9 of the 18 (50%) families with congenital cataracts. Of the four families with mutations in the X-linked NHS gene, no other abnormalities were recorded except for cataract, in which a pseudo-dominant inheritance form was suggested, as female carriers also had different forms of cataracts. Conclusion This study expands the mutation spectrum and frequency of genes responsible for congenital cataract. Mutation in NHS is a common cause of nonsyndromic congenital cataract with pseudo-autosomal dominant inheritance. Combined with our previous studies, a genetic basis could be identified in 67.6% of families with congenital cataracts in our case series, in which mutations in genes encoding crystallins, genes encoding connexins, and NHS are responsible for 29.4%, 14.7%, and 11.8% of families, respectively. Our results suggest that mutations in NHS are the common cause of congenital cataract, both syndromic and nonsyndromic. PMID:24968223

Exome sequences of multiplex, multigenerational families reveal schizophrenia risk loci with potential implications for neurocognitive performance.

PubMed

Kos, Mark Z; Carless, Melanie A; Peralta, Juan; Curran, Joanne E; Quillen, Ellen E; Almeida, Marcio; Blackburn, August; Blondell, Lucy; Roalf, David R; Pogue-Geile, Michael F; Gur, Ruben C; Göring, Harald H H; Nimgaonkar, Vishwajit L; Gur, Raquel E; Almasy, Laura

2017-12-01

Schizophrenia is a serious mental illness, involving disruptions in thought and behavior, with a worldwide prevalence of about one percent. Although highly heritable, much of the genetic liability of schizophrenia is yet to be explained. We searched for susceptibility loci in multiplex, multigenerational families affected by schizophrenia, targeting protein-altering variation with in silico predicted functional effects. Exome sequencing was performed on 136 samples from eight European-American families, including 23 individuals diagnosed with schizophrenia or schizoaffective disorder. In total, 11,878 non-synonymous variants from 6,396 genes were tested for their association with schizophrenia spectrum disorders. Pathway enrichment analyses were conducted on gene-based test results, protein-protein interaction (PPI) networks, and epistatic effects. Using a significance threshold of FDR < 0.1, association was detected for rs10941112 (p = 2.1 × 10 -5 ; q-value = 0.073) in AMACR, a gene involved in fatty acid metabolism and previously implicated in schizophrenia, with significant cis effects on gene expression (p = 5.5 × 10 -4 ), including brain tissue data from the Genotype-Tissue Expression project (minimum p = 6.0 × 10 -5 ). A second SNP, rs10378 located in TMEM176A, also shows risk effects in the exome data (p = 2.8 × 10 -5 ; q-value = 0.073). PPIs among our top gene-based association results (p < 0.05; n = 359 genes) reveal significant enrichment of genes involved in NCAM-mediated neurite outgrowth (p = 3.0 × 10 -5 ), while exome-wide SNP-SNP interaction effects for rs10941112 and rs10378 indicate a potential role for kinase-mediated signaling involved in memory and learning. In conclusion, these association results implicate AMACR and TMEM176A in schizophrenia risk, whose effects may be modulated by genes involved in synaptic plasticity and neurocognitive performance. © 2017 Wiley Periodicals, Inc.

Analyzing Somatic Genome Rearrangements in Human Cancers by Using Whole-Exome Sequencing | Office of Cancer Genomics

Cancer.gov

Although exome sequencing data are generated primarily to detect single-nucleotide variants and indels, they can also be used to identify a subset of genomic rearrangements whose breakpoints are located in or near exons. Using >4,600 tumor and normal pairs across 15 cancer types, we identified over 9,000 high confidence somatic rearrangements, including a large number of gene fusions.

Clinical multiplexed exome sequencing distinguishes adult oligodendroglial neoplasms from astrocytic and mixed lineage gliomas.

PubMed

Cryan, Jane B; Haidar, Sam; Ramkissoon, Lori A; Bi, Wenya Linda; Knoff, David S; Schultz, Nikolaus; Abedalthagafi, Malak; Brown, Loreal; Wen, Patrick Y; Reardon, David A; Dunn, Ian F; Folkerth, Rebecca D; Santagata, Sandro; Lindeman, Neal I; Ligon, Azra H; Beroukhim, Rameen; Hornick, Jason L; Alexander, Brian M; Ligon, Keith L; Ramkissoon, Shakti H

2014-09-30

Classifying adult gliomas remains largely a histologic diagnosis based on morphology; however astrocytic, oligodendroglial and mixed lineage tumors can display overlapping histologic features. We used multiplexed exome sequencing (OncoPanel) on 108 primary or recurrent adult gliomas, comprising 65 oligodendrogliomas, 28 astrocytomas and 15 mixed oligoastrocytomas to identify lesions that could enhance lineage classification. Mutations in TP53 (20/28, 71%) and ATRX (15/28, 54%) were enriched in astrocytic tumors compared to oligodendroglial tumors of which 4/65 (6%) had mutations in TP53 and 2/65 (3%) had ATRX mutations. We found that oligoastrocytomas harbored mutations in TP53 (80%, 12/15) and ATRX (60%, 9/15) at frequencies similar to pure astrocytic tumors, suggesting that oligoastrocytomas and astrocytomas may represent a single genetic or biological entity. p53 protein expression correlated with mutation status and showed significant increases in astrocytomas and oligoastrocytomas compared to oligodendrogliomas, a finding that also may facilitate accurate classification. Furthermore our OncoPanel analysis revealed that 15% of IDH1/2 mutant gliomas would not be detected by traditional IDH1 (p.R132H) antibody testing, supporting the use of genomic technologies in providing clinically relevant data. In all, our results demonstrate that multiplexed exome sequencing can support evaluation and classification of adult low-grade gliomas with a single clinical test.

Whole-exome sequencing reveals the spectrum of gene mutations and the clonal evolution patterns in paediatric acute myeloid leukaemia.

PubMed

Shiba, Norio; Yoshida, Kenichi; Shiraishi, Yuichi; Okuno, Yusuke; Yamato, Genki; Hara, Yusuke; Nagata, Yasunobu; Chiba, Kenichi; Tanaka, Hiroko; Terui, Kiminori; Kato, Motohiro; Park, Myoung-Ja; Ohki, Kentaro; Shimada, Akira; Takita, Junko; Tomizawa, Daisuke; Kudo, Kazuko; Arakawa, Hirokazu; Adachi, Souichi; Taga, Takashi; Tawa, Akio; Ito, Etsuro; Horibe, Keizo; Sanada, Masashi; Miyano, Satoru; Ogawa, Seishi; Hayashi, Yasuhide

2016-11-01

Acute myeloid leukaemia (AML) is a molecularly and clinically heterogeneous disease. Targeted sequencing efforts have identified several mutations with diagnostic and prognostic values in KIT, NPM1, CEBPA and FLT3 in both adult and paediatric AML. In addition, massively parallel sequencing enabled the discovery of recurrent mutations (i.e. IDH1/2 and DNMT3A) in adult AML. In this study, whole-exome sequencing (WES) of 22 paediatric AML patients revealed mutations in components of the cohesin complex (RAD21 and SMC3), BCORL1 and ASXL2 in addition to previously known gene mutations. We also revealed intratumoural heterogeneities in many patients, implicating multiple clonal evolution events in the development of AML. Furthermore, targeted deep sequencing in 182 paediatric AML patients identified three major categories of recurrently mutated genes: cohesion complex genes [STAG2, RAD21 and SMC3 in 17 patients (8·3%)], epigenetic regulators [ASXL1/ASXL2 in 17 patients (8·3%), BCOR/BCORL1 in 7 patients (3·4%)] and signalling molecules. We also performed WES in four patients with relapsed AML. Relapsed AML evolved from one of the subclones at the initial phase and was accompanied by many additional mutations, including common driver mutations that were absent or existed only with lower allele frequency in the diagnostic samples, indicating a multistep process causing leukaemia recurrence. © 2016 John Wiley & Sons Ltd.

Whole exome sequencing of rare variants in EIF4G1 and VPS35 in Parkinson disease

PubMed Central

Nuytemans, Karen; Bademci, Guney; Inchausti, Vanessa; Dressen, Amy; Kinnamon, Daniel D.; Mehta, Arpit; Wang, Liyong; Züchner, Stephan; Beecham, Gary W.; Martin, Eden R.; Scott, William K.

2013-01-01

Objective: Recently, vacuolar protein sorting 35 (VPS35) and eukaryotic translation initiation factor 4 gamma 1 (EIF4G1) have been identified as 2 causal Parkinson disease (PD) genes. We used whole exome sequencing for rapid, parallel analysis of variations in these 2 genes. Methods: We performed whole exome sequencing in 213 patients with PD and 272 control individuals. Those rare variants (RVs) with <5% frequency in the exome variant server database and our own control data were considered for analysis. We performed joint gene-based tests for association using RVASSOC and SKAT (Sequence Kernel Association Test) as well as single-variant test statistics. Results: We identified 3 novel VPS35 variations that changed the coded amino acid (nonsynonymous) in 3 cases. Two variations were in multiplex families and neither segregated with PD. In EIF4G1, we identified 11 (9 nonsynonymous and 2 small indels) RVs including the reported pathogenic mutation p.R1205H, which segregated in all affected members of a large family, but also in 1 unaffected 86-year-old family member. Two additional RVs were found in isolated patients only. Whereas initial association studies suggested an association (p = 0.04) with all RVs in EIF4G1, subsequent testing in a second dataset for the driving variant (p.F1461) suggested no association between RVs in the gene and PD. Conclusions: We confirm that the specific EIF4G1 variation p.R1205H seems to be a strong PD risk factor, but is nonpenetrant in at least one 86-year-old. A few other select RVs in both genes could not be ruled out as causal. However, there was no evidence for an overall contribution of genetic variability in VPS35 or EIF4G1 to PD development in our dataset. PMID:23408866

Whole exome sequencing using Ion Proton system enables reliable genetic diagnosis of inherited retinal dystrophies

PubMed Central

Riera, Marina; Navarro, Rafael; Ruiz-Nogales, Sheila; Méndez, Pilar; Burés-Jelstrup, Anniken; Corcóstegui, Borja; Pomares, Esther

2017-01-01

Inherited retinal dystrophies (IRD) comprise a wide group of clinically and genetically complex diseases that progressively affect the retina. Over recent years, the development of next-generation sequencing (NGS) methods has transformed our ability to diagnose heterogeneous diseases. In this work, we have evaluated the implementation of whole exome sequencing (WES) for the molecular diagnosis of IRD. Using Ion ProtonTM system, we simultaneously analyzed 212 genes that are responsible for more than 25 syndromic and non-syndromic IRD. This approach was used to evaluate 59 unrelated families, with the pathogenic variant(s) successfully identified in 71.18% of cases. Interestingly, the mutation detection rate varied substantially depending on the IRD subtype. Overall, we found 63 different mutations (21 novel) in 29 distinct genes, and performed in vivo functional studies to determine the deleterious impact of variants identified in MERTK, CDH23, and RPGRIP1. In addition, we provide evidences that support CDHR1 as a gene responsible for autosomal recessive retinitis pigmentosa with early macular affectation, and present data regarding the disease mechanism of this gene. Altogether, these results demonstrate that targeted WES of all IRD genes is a reliable, hypothesis-free approach, and a cost- and time-effective strategy for the routine genetic diagnosis of retinal dystrophies. PMID:28181551

Whole exome sequencing reveals concomitant mutations of multiple FA genes in individual Fanconi anemia patients

PubMed Central

2014-01-01

Background Fanconi anemia (FA) is a rare inherited genetic syndrome with highly variable clinical manifestations. Fifteen genetic subtypes of FA have been identified. Traditional complementation tests for grouping studies have been used generally in FA patients and in stepwise methods to identify the FA type, which can result in incomplete genetic information from FA patients. Methods We diagnosed five pediatric patients with FA based on clinical manifestations, and we performed exome sequencing of peripheral blood specimens from these patients and their family members. The related sequencing data were then analyzed by bioinformatics, and the FANC gene mutations identified by exome sequencing were confirmed by PCR re-sequencing. Results Homozygous and compound heterozygous mutations of FANC genes were identified in all of the patients. The FA subtypes of the patients included FANCA, FANCM and FANCD2. Interestingly, four FA patients harbored multiple mutations in at least two FA genes, and some of these mutations have not been previously reported. These patients’ clinical manifestations were vastly different from each other, as were their treatment responses to androstanazol and prednisone. This finding suggests that heterozygous mutation(s) in FA genes could also have diverse biological and/or pathophysiological effects on FA patients or FA gene carriers. Interestingly, we were not able to identify de novo mutations in the genes implicated in DNA repair pathways when the sequencing data of patients were compared with those of their parents. Conclusions Our results indicate that Chinese FA patients and carriers might have higher and more complex mutation rates in FANC genes than have been conventionally recognized. Testing of the fifteen FANC genes in FA patients and their family members should be a regular clinical practice to determine the optimal care for the individual patient, to counsel the family and to obtain a better understanding of FA pathophysiology

Whole exome sequencing reveals concomitant mutations of multiple FA genes in individual Fanconi anemia patients.

PubMed

Chang, Lixian; Yuan, Weiping; Zeng, Huimin; Zhou, Quanquan; Wei, Wei; Zhou, Jianfeng; Li, Miaomiao; Wang, Xiaomin; Xu, Mingjiang; Yang, Fengchun; Yang, Yungui; Cheng, Tao; Zhu, Xiaofan

2014-05-15

Fanconi anemia (FA) is a rare inherited genetic syndrome with highly variable clinical manifestations. Fifteen genetic subtypes of FA have been identified. Traditional complementation tests for grouping studies have been used generally in FA patients and in stepwise methods to identify the FA type, which can result in incomplete genetic information from FA patients. We diagnosed five pediatric patients with FA based on clinical manifestations, and we performed exome sequencing of peripheral blood specimens from these patients and their family members. The related sequencing data were then analyzed by bioinformatics, and the FANC gene mutations identified by exome sequencing were confirmed by PCR re-sequencing. Homozygous and compound heterozygous mutations of FANC genes were identified in all of the patients. The FA subtypes of the patients included FANCA, FANCM and FANCD2. Interestingly, four FA patients harbored multiple mutations in at least two FA genes, and some of these mutations have not been previously reported. These patients' clinical manifestations were vastly different from each other, as were their treatment responses to androstanazol and prednisone. This finding suggests that heterozygous mutation(s) in FA genes could also have diverse biological and/or pathophysiological effects on FA patients or FA gene carriers. Interestingly, we were not able to identify de novo mutations in the genes implicated in DNA repair pathways when the sequencing data of patients were compared with those of their parents. Our results indicate that Chinese FA patients and carriers might have higher and more complex mutation rates in FANC genes than have been conventionally recognized. Testing of the fifteen FANC genes in FA patients and their family members should be a regular clinical practice to determine the optimal care for the individual patient, to counsel the family and to obtain a better understanding of FA pathophysiology.

Exome Sequencing and Linkage Analysis Identified Novel Candidate Genes in Recessive Intellectual Disability Associated with Ataxia.

PubMed

Jazayeri, Roshanak; Hu, Hao; Fattahi, Zohreh; Musante, Luciana; Abedini, Seyedeh Sedigheh; Hosseini, Masoumeh; Wienker, Thomas F; Ropers, Hans Hilger; Najmabadi, Hossein; Kahrizi, Kimia

2015-10-01

Intellectual disability (ID) is a neuro-developmental disorder which causes considerable socio-economic problems. Some ID individuals are also affected by ataxia, and the condition includes different mutations affecting several genes. We used whole exome sequencing (WES) in combination with homozygosity mapping (HM) to identify the genetic defects in five consanguineous families among our cohort study, with two affected children with ID and ataxia as major clinical symptoms. We identified three novel candidate genes, RIPPLY1, MRPL10, SNX14, and a new mutation in known gene SURF1. All are autosomal genes, except RIPPLY1, which is located on the X chromosome. Two are housekeeping genes, implicated in transcription and translation regulation and intracellular trafficking, and two encode mitochondrial proteins. The pathogenesis of these variants was evaluated by mutation classification, bioinformatic methods, review of medical and biological relevance, co-segregation studies in the particular family, and a normal population study. Linkage analysis and exome sequencing of a small number of affected family members is a powerful new technique which can be used to decrease the number of candidate genes in heterogenic disorders such as ID, and may even identify the responsible gene(s).

A scalable, fully automated process for construction of sequence-ready human exome targeted capture libraries

PubMed Central

2011-01-01

Genome targeting methods enable cost-effective capture of specific subsets of the genome for sequencing. We present here an automated, highly scalable method for carrying out the Solution Hybrid Selection capture approach that provides a dramatic increase in scale and throughput of sequence-ready libraries produced. Significant process improvements and a series of in-process quality control checkpoints are also added. These process improvements can also be used in a manual version of the protocol. PMID:21205303

Genomic complexity and dynamics of clonal evolution in childhood acute myeloid leukemia studied with whole-exome sequencing.

PubMed

Masetti, Riccardo; Castelli, Ilaria; Astolfi, Annalisa; Bertuccio, Salvatore Nicola; Indio, Valentina; Togni, Marco; Belotti, Tamara; Serravalle, Salvatore; Tarantino, Giuseppe; Zecca, Marco; Pigazzi, Martina; Basso, Giuseppe; Pession, Andrea; Locatelli, Franco

2016-08-30

Despite significant improvement in treatment of childhood acute myeloid leukemia (AML), 30% of patients experience disease recurrence, which is still the major cause of treatment failure and death in these patients. To investigate molecular mechanisms underlying relapse, we performed whole-exome sequencing of diagnosis-relapse pairs and matched remission samples from 4 pediatric AML patients without recurrent cytogenetic alterations. Candidate driver mutations were selected for targeted deep sequencing at high coverage, suitable to detect small subclones (0.12%). BiCEBPα mutation was found to be stable and highly penetrant, representing a separate biological and clinical entity, unlike WT1 mutations, which were extremely unstable. Among the mutational patterns underlying relapse, we detected the acquisition of proliferative advantage by signaling activation (PTPN11 and FLT3-TKD mutations) and the increased resistance to apoptosis (hyperactivation of TYK2). We also found a previously undescribed feature of AML, consisting of a hypermutator phenotype caused by SETD2 inactivation. The consequent accumulation of new mutations promotes the adaptability of the leukemia, contributing to clonal selection. We report a novel ASXL3 mutation characterizing a very small subclone (<1%) present at diagnosis and undergoing expansion (60%) at relapse. Taken together, these findings provide molecular clues for designing optimal therapeutic strategies, in terms of target selection, adequate schedule design and reliable response-monitoring techniques.

Rare Compound Heterozygous Frameshift Mutations in ALMS1 Gene Identified Through Exome Sequencing in a Taiwanese Patient With Alström Syndrome.

PubMed

Tsai, Meng-Che; Yu, Hui-Wen; Liu, Tsunglin; Chou, Yen-Yin; Chiou, Yuan-Yow; Chen, Peng-Chieh

2018-01-01

Alström syndrome (AS) is a rare autosomal recessive disorder that shares clinical features with other ciliopathy-related diseases. Genetic mutation analysis is often required in making differential diagnosis but usually costly in time and effort using conventional Sanger sequencing. Herein we describe a Taiwanese patient presenting cone-rod dystrophy and early-onset obesity that progressed to diabetes mellitus with marked insulin resistance during adolescence. Whole exome sequencing of the patient's genomic DNA identified a novel frameshift mutation in exons 15 (c.10290_10291delTA, p.Lys3431Serfs * 10) and a rare mutation in 16 (c.10823_10824delAG, p.Arg3609Alafs * 6) of ALMS1 gene. The compound heterozygous mutations were predicted to render truncated proteins. This report highlighted the clinical utility of exome sequencing and extended the knowledge of mutation spectrum in AS patients.

Genetic Mapping and Exome Sequencing Identify Variants Associated with Five Novel Diseases

PubMed Central

Puffenberger, Erik G.; Jinks, Robert N.; Sougnez, Carrie; Cibulskis, Kristian; Willert, Rebecca A.; Achilly, Nathan P.; Cassidy, Ryan P.; Fiorentini, Christopher J.; Heiken, Kory F.; Lawrence, Johnny J.; Mahoney, Molly H.; Miller, Christopher J.; Nair, Devika T.; Politi, Kristin A.; Worcester, Kimberly N.; Setton, Roni A.; DiPiazza, Rosa; Sherman, Eric A.; Eastman, James T.; Francklyn, Christopher; Robey-Bond, Susan; Rider, Nicholas L.; Gabriel, Stacey; Morton, D. Holmes; Strauss, Kevin A.

2012-01-01

The Clinic for Special Children (CSC) has integrated biochemical and molecular methods into a rural pediatric practice serving Old Order Amish and Mennonite (Plain) children. Among the Plain people, we have used single nucleotide polymorphism (SNP) microarrays to genetically map recessive disorders to large autozygous haplotype blocks (mean = 4.4 Mb) that contain many genes (mean = 79). For some, uninformative mapping or large gene lists preclude disease-gene identification by Sanger sequencing. Seven such conditions were selected for exome sequencing at the Broad Institute; all had been previously mapped at the CSC using low density SNP microarrays coupled with autozygosity and linkage analyses. Using between 1 and 5 patient samples per disorder, we identified sequence variants in the known disease-causing genes SLC6A3 and FLVCR1, and present evidence to strongly support the pathogenicity of variants identified in TUBGCP6, BRAT1, SNIP1, CRADD, and HARS. Our results reveal the power of coupling new genotyping technologies to population-specific genetic knowledge and robust clinical data. PMID:22279524

Genetic Diagnosis in Consanguineous Families With Kidney Disease by Homozygosity Mapping Coupled With Whole-Exome Sequencing

PubMed Central

Al-Romaih, Khaldoun I.; Genovese, Giulio; Al-Mojalli, Hamad; Al-Othman, Saleh; Al-Manea, Hadeel; Al-Suleiman, Mohammed; Al-Jondubi, Mohammed; Atallah, Nourah; Al-Rodhyan, Maha; Weins, Astrid; Pollak, Martin R.; Adra, Chaker N.

2011-01-01

Background Accurate diagnosis of the primary cause of an individual’s kidney disease can be essential for proper management. Some kidney diseases have overlapping histopathological features despite being caused by defects in different genes. In this report we describe two consanguineous Saudi Arabian families in which individuals presented with kidney failure and mixed clinical and histological features initially thought consistent with focal segmental glomerulosclerosis. Study Design Case series. Setting and participants We studied members of two apparently unrelated families from Saudi Arabia with kidney disease. Measurements Whole-genome single-nucleotide polymorphism analysis followed by targeted isolation and sequencing of exons using genomic DNA samples from affected members of these families, followed by additional focused genotyping and sequence analysis. Results The two apparently unrelated families shared a region of homozygosity on chromosome 2q13. Exome sequence from the affected individuals lacked any sequence reads from the NPHP1 gene, which is located within this homozygous region. Additional PCR based genotyping confirmed that affected individuals had NPHP1 deletions, rather than defects in a known FSGS-associated gene. Limitations The methods used here may not result in a clear genetic diagnosis in many cases of apparent familial kidney disease. Conclusions This analysis demonstrates the power of new high-throughput genotyping and sequencing technologies to aid in the rapid genetic diagnosis of individuals with an inherited form of kidney disease. We believe it is likely that such tools may become useful clinical genetic tools and alter the manner in which diagnoses are made in nephrology. PMID:21658830

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.

Germline whole exome sequencing and large-scale replication identifies FANCM as a likely high grade serous ovarian cancer susceptibility gene.

PubMed

Dicks, Ed; Song, Honglin; Ramus, Susan J; Oudenhove, Elke Van; Tyrer, Jonathan P; Intermaggio, Maria P; Kar, Siddhartha; Harrington, Patricia; Bowtell, David D; Group, Aocs Study; Cicek, Mine S; Cunningham, Julie M; Fridley, Brooke L; Alsop, Jennifer; Jimenez-Linan, Mercedes; Piskorz, Anna; Goranova, Teodora; Kent, Emma; Siddiqui, Nadeem; Paul, James; Crawford, Robin; Poblete, Samantha; Lele, Shashi; Sucheston-Campbell, Lara; Moysich, Kirsten B; Sieh, Weiva; McGuire, Valerie; Lester, Jenny; Odunsi, Kunle; Whittemore, Alice S; Bogdanova, Natalia; Dürst, Matthias; Hillemanns, Peter; Karlan, Beth Y; Gentry-Maharaj, Aleksandra; Menon, Usha; Tischkowitz, Marc; Levine, Douglas; Brenton, James D; Dörk, Thilo; Goode, Ellen L; Gayther, Simon A; Pharoah, D P Paul

2017-08-01

We analyzed whole exome sequencing data in germline DNA from 412 high grade serous ovarian cancer (HGSOC) cases from The Cancer Genome Atlas Project and identified 5,517 genes harboring a predicted deleterious germline coding mutation in at least one HGSOC case. Gene-set enrichment analysis showed enrichment for genes involved in DNA repair (p = 1.8×10 -3 ). Twelve DNA repair genes - APEX1, APLF, ATX, EME1, FANCL, FANCM, MAD2L2, PARP2, PARP3, POLN, RAD54L and SMUG1 - were prioritized for targeted sequencing in up to 3,107 HGSOC cases, 1,491 cases of other epithelial ovarian cancer (EOC) subtypes and 3,368 unaffected controls of European origin. We estimated mutation prevalence for each gene and tested for associations with disease risk. Mutations were identified in both cases and controls in all genes except MAD2L2 , where we found no evidence of mutations in controls. In FANCM we observed a higher mutation frequency in HGSOC cases compared to controls (29/3,107 cases, 0.96 percent; 13/3,368 controls, 0.38 percent; P=0.008) with little evidence for association with other subtypes (6/1,491, 0.40 percent; P=0.82). The relative risk of HGSOC associated with deleterious FANCM mutations was estimated to be 2.5 (95% CI 1.3 - 5.0; P=0.006). In summary, whole exome sequencing of EOC cases with large-scale replication in case-control studies has identified FANCM as a likely novel susceptibility gene for HGSOC, with mutations associated with a moderate increase in risk. These data may have clinical implications for risk prediction and prevention approaches for high-grade serous ovarian cancer in the future and a significant impact on reducing disease mortality.

Exome sequencing links corticospinal motor neuron disease to common neurodegenerative disorders.

PubMed

Novarino, Gaia; Fenstermaker, Ali G; Zaki, Maha S; Hofree, Matan; Silhavy, Jennifer L; Heiberg, Andrew D; Abdellateef, Mostafa; Rosti, Basak; Scott, Eric; Mansour, Lobna; Masri, Amira; Kayserili, Hulya; Al-Aama, Jumana Y; Abdel-Salam, Ghada M H; Karminejad, Ariana; Kara, Majdi; Kara, Bulent; Bozorgmehri, Bita; Ben-Omran, Tawfeg; Mojahedi, Faezeh; El Din Mahmoud, Iman Gamal; Bouslam, Naima; Bouhouche, Ahmed; Benomar, Ali; Hanein, Sylvain; Raymond, Laure; Forlani, Sylvie; Mascaro, Massimo; Selim, Laila; Shehata, Nabil; Al-Allawi, Nasir; Bindu, P S; Azam, Matloob; Gunel, Murat; Caglayan, Ahmet; Bilguvar, Kaya; Tolun, Aslihan; Issa, Mahmoud Y; Schroth, Jana; Spencer, Emily G; Rosti, Rasim O; Akizu, Naiara; Vaux, Keith K; Johansen, Anide; Koh, Alice A; Megahed, Hisham; Durr, Alexandra; Brice, Alexis; Stevanin, Giovanni; Gabriel, Stacy B; Ideker, Trey; Gleeson, Joseph G

2014-01-31

Hereditary spastic paraplegias (HSPs) are neurodegenerative motor neuron diseases characterized by progressive age-dependent loss of corticospinal motor tract function. Although the genetic basis is partly understood, only a fraction of cases can receive a genetic diagnosis, and a global view of HSP is lacking. By using whole-exome sequencing in combination with network analysis, we identified 18 previously unknown putative HSP genes and validated nearly all of these genes functionally or genetically. The pathways highlighted by these mutations link HSP to cellular transport, nucleotide metabolism, and synapse and axon development. Network analysis revealed a host of further candidate genes, of which three were mutated in our cohort. Our analysis links HSP to other neurodegenerative disorders and can facilitate gene discovery and mechanistic understanding of disease.

Genomic landscape of ovarian clear cell carcinoma via whole exome sequencing.

PubMed

Kim, Se Ik; Lee, Ji Won; Lee, Maria; Kim, Hee Seung; Chung, Hyun Hoon; Kim, Jae-Weon; Park, Noh Hyun; Song, Yong-Sang; Seo, Jeong-Sun

2018-02-01

To analyze whole exome sequencing (WES) data on ovarian clear cell carcinoma (OCCC) in Korean patients via the technique of next generation sequencing (NGS). Genomic profiles were compared between endometriosis-associated OCCC (EMS-OCCC) and Non-EMS-OCCC. We used serum samples and cancer tissues, stored at the Seoul National University Hospital Human Biobank, that were initially collected from women diagnosed with OCCC between 2012 and 2016. In total, 15 patients were enrolled: 5 with pathologically confirmed EMS-OCCC and 10 with Non-EMS-OCCC. We performed NGS WES on 15 fresh frozen OCCC tissues and matched serum samples, enabling comprehensive genomic characterization of OCCC. OCCC was characterized by complex genomic alterations, with a median of 178 exonic mutations (range, 111-25,798) and a median of 343 somatic copy number variations (range, 43-1,820) per tumor sample. In all, 54 somatic mutations were discovered across 14 genes, including PIK3CA (40%), ARID1A (40%), and KRAS (20%) in the 15 Korean OCCCs. Copy number gains in NTRK1 (33%), MYC (40%), and GNAS (47%) and copy number losses in TET2 (73%), TSC1 (67%), BRCA2 (60%), and SMAD4 (47%) were frequent. The significantly altered pathways were associated with proliferation and survival (including the PI3K/AKT, TP53, and ERBB2 pathways) in 87% of OCCCs and with chromatin remodeling in 47% of OCCCs. No significant differences in frequencies of genetic alterations were detected between EMS-OCCC and Non-EMS-OCCC groups. We successfully characterized the genomic landscape of 15 Korean patients with OCCC. We identified potential therapeutic targets for the treatment of this malignancy. Copyright © 2017. Published by Elsevier Inc.

A prospective evaluation of whole-exome sequencing as a first-tier molecular test in infants with suspected monogenic disorders.

PubMed

Stark, Zornitza; Tan, Tiong Y; Chong, Belinda; Brett, Gemma R; Yap, Patrick; Walsh, Maie; Yeung, Alison; Peters, Heidi; Mordaunt, Dylan; Cowie, Shannon; Amor, David J; Savarirayan, Ravi; McGillivray, George; Downie, Lilian; Ekert, Paul G; Theda, Christiane; James, Paul A; Yaplito-Lee, Joy; Ryan, Monique M; Leventer, Richard J; Creed, Emma; Macciocca, Ivan; Bell, Katrina M; Oshlack, Alicia; Sadedin, Simon; Georgeson, Peter; Anderson, Charlotte; Thorne, Natalie; Melbourne Genomics Health Alliance; Gaff, Clara; White, Susan M

2016-11-01

To prospectively evaluate the diagnostic and clinical utility of singleton whole-exome sequencing (WES) as a first-tier test in infants with suspected monogenic disease. Singleton WES was performed as a first-tier sequencing test in infants recruited from a single pediatric tertiary center. This occurred in parallel with standard investigations, including single- or multigene panel sequencing when clinically indicated. The diagnosis rate, clinical utility, and impact on management of singleton WES were evaluated. Of 80 enrolled infants, 46 received a molecular genetic diagnosis through singleton WES (57.5%) compared with 11 (13.75%) who underwent standard investigations in the same patient group. Clinical management changed following exome diagnosis in 15 of 46 diagnosed participants (32.6%). Twelve relatives received a genetic diagnosis following cascade testing, and 28 couples were identified as being at high risk of recurrence in future pregnancies. This prospective study provides strong evidence for increased diagnostic and clinical utility of singleton WES as a first-tier sequencing test for infants with a suspected monogenic disorder. Singleton WES outperformed standard care in terms of diagnosis rate and the benefits of a diagnosis, namely, impact on management of the child and clarification of reproductive risks for the extended family in a timely manner.Genet Med 18 11, 1090-1096.

Molecular Genetics of the Usher Syndrome in Lebanon: Identification of 11 Novel Protein Truncating Mutations by Whole Exome Sequencing

PubMed Central

Reddy, Ramesh; Fahiminiya, Somayyeh; El Zir, Elie; Mansour, Ahmad; Megarbane, Andre; Majewski, Jacek; Slim, Rima

2014-01-01

Background Usher syndrome (USH) is a genetically heterogeneous condition with ten disease-causing genes. The spectrum of genes and mutations causing USH in the Lebanese and Middle Eastern populations has not been described. Consequently, diagnostic approaches designed to screen for previously reported mutations were unlikely to identify the mutations in 11 unrelated families, eight of Lebanese and three of Middle Eastern origins. In addition, six of the ten USH genes consist of more than 20 exons, each, which made mutational analysis by Sanger sequencing of PCR-amplified exons from genomic DNA tedious and costly. The study was aimed at the identification of USH causing genes and mutations in 11 unrelated families with USH type I or II. Methods Whole exome sequencing followed by expanded familial validation by Sanger sequencing. Results We identified disease-causing mutations in all the analyzed patients in four USH genes, MYO7A, USH2A, GPR98 and CDH23. Eleven of the mutations were novel and protein truncating, including a complex rearrangement in GPR98. Conclusion Our data highlight the genetic diversity of Usher syndrome in the Lebanese population and the time and cost-effectiveness of whole exome sequencing approach for mutation analysis of genetically heterogeneous conditions caused by large genes. PMID:25211151

Molecular genetics of the Usher syndrome in Lebanon: identification of 11 novel protein truncating mutations by whole exome sequencing.

PubMed

Reddy, Ramesh; Fahiminiya, Somayyeh; El Zir, Elie; Mansour, Ahmad; Megarbane, Andre; Majewski, Jacek; Slim, Rima

2014-01-01

Usher syndrome (USH) is a genetically heterogeneous condition with ten disease-causing genes. The spectrum of genes and mutations causing USH in the Lebanese and Middle Eastern populations has not been described. Consequently, diagnostic approaches designed to screen for previously reported mutations were unlikely to identify the mutations in 11 unrelated families, eight of Lebanese and three of Middle Eastern origins. In addition, six of the ten USH genes consist of more than 20 exons, each, which made mutational analysis by Sanger sequencing of PCR-amplified exons from genomic DNA tedious and costly. The study was aimed at the identification of USH causing genes and mutations in 11 unrelated families with USH type I or II. Whole exome sequencing followed by expanded familial validation by Sanger sequencing. We identified disease-causing mutations in all the analyzed patients in four USH genes, MYO7A, USH2A, GPR98 and CDH23. Eleven of the mutations were novel and protein truncating, including a complex rearrangement in GPR98. Our data highlight the genetic diversity of Usher syndrome in the Lebanese population and the time and cost-effectiveness of whole exome sequencing approach for mutation analysis of genetically heterogeneous conditions caused by large genes.

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.

Ethical and legal implications of whole genome and whole exome sequencing in African populations.

PubMed

Wright, Galen E B; Koornhof, Pieter G J; Adeyemo, Adebowale A; Tiffin, Nicki

2013-05-28

Rapid advances in high throughput genomic technologies and next generation sequencing are making medical genomic research more readily accessible and affordable, including the sequencing of patient and control whole genomes and exomes in order to elucidate genetic factors underlying disease. Over the next five years, the Human Heredity and Health in Africa (H3Africa) Initiative, funded by the Wellcome Trust (United Kingdom) and the National Institutes of Health (United States of America), will contribute greatly towards sequencing of numerous African samples for biomedical research. Funding agencies and journals often require submission of genomic data from research participants to databases that allow open or controlled data access for all investigators. Access to such genotype-phenotype and pedigree data, however, needs careful control in order to prevent identification of individuals or families. This is particularly the case in Africa, where many researchers and their patients are inexperienced in the ethical issues accompanying whole genome and exome research; and where an historical unidirectional flow of samples and data out of Africa has created a sense of exploitation and distrust. In the current study, we analysed the implications of the anticipated surge of next generation sequencing data in Africa and the subsequent data sharing concepts on the protection of privacy of research subjects. We performed a retrospective analysis of the informed consent process for the continent and the rest-of-the-world and examined relevant legislation, both current and proposed. We investigated the following issues: (i) informed consent, including guidelines for performing culturally-sensitive next generation sequencing research in Africa and availability of suitable informed consent documents; (ii) data security and subject privacy whilst practicing data sharing; (iii) conveying the implications of such concepts to research participants in resource limited settings. We

Ethical and legal implications of whole genome and whole exome sequencing in African populations

PubMed Central

2013-01-01

Background Rapid advances in high throughput genomic technologies and next generation sequencing are making medical genomic research more readily accessible and affordable, including the sequencing of patient and control whole genomes and exomes in order to elucidate genetic factors underlying disease. Over the next five years, the Human Heredity and Health in Africa (H3Africa) Initiative, funded by the Wellcome Trust (United Kingdom) and the National Institutes of Health (United States of America), will contribute greatly towards sequencing of numerous African samples for biomedical research. Discussion Funding agencies and journals often require submission of genomic data from research participants to databases that allow open or controlled data access for all investigators. Access to such genotype-phenotype and pedigree data, however, needs careful control in order to prevent identification of individuals or families. This is particularly the case in Africa, where many researchers and their patients are inexperienced in the ethical issues accompanying whole genome and exome research; and where an historical unidirectional flow of samples and data out of Africa has created a sense of exploitation and distrust. In the current study, we analysed the implications of the anticipated surge of next generation sequencing data in Africa and the subsequent data sharing concepts on the protection of privacy of research subjects. We performed a retrospective analysis of the informed consent process for the continent and the rest-of-the-world and examined relevant legislation, both current and proposed. We investigated the following issues: (i) informed consent, including guidelines for performing culturally-sensitive next generation sequencing research in Africa and availability of suitable informed consent documents; (ii) data security and subject privacy whilst practicing data sharing; (iii) conveying the implications of such concepts to research participants in resource

Whole-exome sequencing identifies novel compound heterozygous mutations in USH2A in Spanish patients with autosomal recessive retinitis pigmentosa.

PubMed

Méndez-Vidal, Cristina; González-Del Pozo, María; Vela-Boza, Alicia; Santoyo-López, Javier; López-Domingo, Francisco J; Vázquez-Marouschek, Carmen; Dopazo, Joaquin; Borrego, Salud; Antiñolo, Guillermo

2013-01-01

Retinitis pigmentosa (RP) is an inherited retinal dystrophy characterized by extreme genetic and clinical heterogeneity. Thus, the diagnosis is not always easily performed due to phenotypic and genetic overlap. Current clinical practices have focused on the systematic evaluation of a set of known genes for each phenotype, but this approach may fail in patients with inaccurate diagnosis or infrequent genetic cause. In the present study, we investigated the genetic cause of autosomal recessive RP (arRP) in a Spanish family in which the causal mutation has not yet been identified with primer extension technology and resequencing. We designed a whole-exome sequencing (WES)-based approach using NimbleGen SeqCap EZ Exome V3 sample preparation kit and the SOLiD 5500×l next-generation sequencing platform. We sequenced the exomes of both unaffected parents and two affected siblings. Exome analysis resulted in the identification of 43,204 variants in the index patient. All variants passing filter criteria were validated with Sanger sequencing to confirm familial segregation and absence in the control population. In silico prediction tools were used to determine mutational impact on protein function and the structure of the identified variants. Novel Usher syndrome type 2A (USH2A) compound heterozygous mutations, c.4325T>C (p.F1442S) and c.15188T>G (p.L5063R), located in exons 20 and 70, respectively, were identified as probable causative mutations for RP in this family. Family segregation of the variants showed the presence of both mutations in all affected members and in two siblings who were apparently asymptomatic at the time of family ascertainment. Clinical reassessment confirmed the diagnosis of RP in these patients. Using WES, we identified two heterozygous novel mutations in USH2A as the most likely disease-causing variants in a Spanish family diagnosed with arRP in which the cause of the disease had not yet been identified with commonly used techniques. Our data

Whole-exome sequencing identifies novel compound heterozygous mutations in USH2A in Spanish patients with autosomal recessive retinitis pigmentosa

PubMed Central

Méndez-Vidal, Cristina; González-del Pozo, María; Vela-Boza, Alicia; Santoyo-López, Javier; López-Domingo, Francisco J.; Vázquez-Marouschek, Carmen; Dopazo, Joaquin; Borrego, Salud

2013-01-01

Purpose Retinitis pigmentosa (RP) is an inherited retinal dystrophy characterized by extreme genetic and clinical heterogeneity. Thus, the diagnosis is not always easily performed due to phenotypic and genetic overlap. Current clinical practices have focused on the systematic evaluation of a set of known genes for each phenotype, but this approach may fail in patients with inaccurate diagnosis or infrequent genetic cause. In the present study, we investigated the genetic cause of autosomal recessive RP (arRP) in a Spanish family in which the causal mutation has not yet been identified with primer extension technology and resequencing. Methods We designed a whole-exome sequencing (WES)-based approach using NimbleGen SeqCap EZ Exome V3 sample preparation kit and the SOLiD 5500×l next-generation sequencing platform. We sequenced the exomes of both unaffected parents and two affected siblings. Exome analysis resulted in the identification of 43,204 variants in the index patient. All variants passing filter criteria were validated with Sanger sequencing to confirm familial segregation and absence in the control population. In silico prediction tools were used to determine mutational impact on protein function and the structure of the identified variants. Results Novel Usher syndrome type 2A (USH2A) compound heterozygous mutations, c.4325T>C (p.F1442S) and c.15188T>G (p.L5063R), located in exons 20 and 70, respectively, were identified as probable causative mutations for RP in this family. Family segregation of the variants showed the presence of both mutations in all affected members and in two siblings who were apparently asymptomatic at the time of family ascertainment. Clinical reassessment confirmed the diagnosis of RP in these patients. Conclusions Using WES, we identified two heterozygous novel mutations in USH2A as the most likely disease-causing variants in a Spanish family diagnosed with arRP in which the cause of the disease had not yet been identified with

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

Novel DDR2 mutation identified by whole exome sequencing in a Moroccan patient with spondylo-meta-epiphyseal dysplasia, short limb-abnormal calcification type.

PubMed

Mansouri, Maria; Kayserili, Hülya; Elalaoui, Siham Chafai; Nishimura, Gen; Iida, Aritoshi; Lyahyai, Jaber; Miyake, Noriko; Matsumoto, Naomichi; Sefiani, Abdelaziz; Ikegawa, Shiro

2016-02-01

Spondylo-meta-epiphyseal dysplasia (SMED), short limb-abnormal calcification type (SMED, SL-AC), is a very rare autosomal recessive disorder with various skeletal changes characterized by premature calcification leading to severe disproportionate short stature. Twenty-two patients have been reported until now, but only five mutations (four missense and one splice-site) in the conserved sequence encoding the tyrosine kinase domain of the DDR2 gene has been identified. We report here a novel DDR2 missense mutation, c.370C > T (p.Arg124Trp) in a Moroccan girl with SMED, SL-AC, identified by whole exome sequencing. Our study has expanded the mutational spectrum of this rare disease and it has shown that exome sequencing is a powerful and cost-effective tool for the diagnosis of clinically heterogeneous disorders such as SMED. © 2015 Wiley Periodicals, Inc.

Exome capture sequencing reveals new insights into hepatitis B virus-induced hepatocellular carcinoma at the early stage of tumorigenesis.

PubMed

Chen, Yong; Wang, Lijuan; Xu, Hexiang; Liu, Xingxiang; Zhao, Yingren

2013-10-01

Hepatocellular carcinoma (HCC), the most common type of liver cancer, is the third primary cause of cancer-related mortality worldwide. The molecular mechanisms underlying the initiation and formation of HCC remain obscure. In the present study, we performed exome sequencing using tumor and normal tissues from 3 hepatitis B virus (HBV)-positive BCLC stage A HCC patients. Bioinformatic analysis was performed to find candidate protein-altering somatic mutations. Eighty damaging mutations were validated and 59 genes were reported to be mutated in HBV-related HCCs for the first time here. Further analysis using whole genome sequencing (WGS) data of 88 HBV-related HCC patients from the European Genome-phenome Archive database showed that mutations in 33 of the 59 genes were also detected in other samples. Variants of two newly found genes, ZNF717 and PARP4, were detected in more than 10% of the WGS samples. Several other genes, such as FLNA and CNTN2, are also noteworthy. Thus, the exome sequencing analysis of three BCLC stage A patients provides new insights into the molecular events governing the early steps of HBV-induced HCC tumorigenesis.

Using whole-exome sequencing to investigate the genetic bases of lysosomal storage diseases of unknown etiology.

PubMed

Wang, Nan; Zhang, Yeting; Gedvilaite, Erika; Loh, Jui Wan; Lin, Timothy; Liu, Xiuping; Liu, Chang-Gong; Kumar, Dibyendu; Donnelly, Robert; Raymond, Kimiyo; Schuchman, Edward H; Sleat, David E; Lobel, Peter; Xing, Jinchuan

2017-11-01

Lysosomes are membrane-bound, acidic eukaryotic cellular organelles that play important roles in the degradation of macromolecules. Mutations that cause the loss of lysosomal protein function can lead to a group of disorders categorized as the lysosomal storage diseases (LSDs). Suspicion of LSD is frequently based on clinical and pathologic findings, but in some cases, the underlying genetic and biochemical defects remain unknown. Here, we performed whole-exome sequencing (WES) on 14 suspected LSD cases to evaluate the feasibility of using WES for identifying causal mutations. By examining 2,157 candidate genes potentially associated with lysosomal function, we identified eight variants in five genes as candidate disease-causing variants in four individuals. These included both known and novel mutations. Variants were corroborated by targeted sequencing and, when possible, functional assays. In addition, we identified nonsense mutations in two individuals in genes that are not known to have lysosomal function. However, mutations in these genes could have resulted in phenotypes that were diagnosed as LSDs. This study demonstrates that WES can be used to identify causal mutations in suspected LSD cases. We also demonstrate cases where a confounding clinical phenotype may potentially reflect more than one lysosomal protein defect. © 2017 Wiley Periodicals, Inc.

Performance comparison of two commercial human whole-exome capture systems on formalin-fixed paraffin-embedded lung adenocarcinoma samples.

PubMed

Bonfiglio, Silvia; Vanni, Irene; Rossella, Valeria; Truini, Anna; Lazarevic, Dejan; Dal Bello, Maria Giovanna; Alama, Angela; Mora, Marco; Rijavec, Erika; Genova, Carlo; Cittaro, Davide; Grossi, Francesco; Coco, Simona

2016-08-30

Next Generation Sequencing (NGS) has become a valuable tool for molecular landscape characterization of cancer genomes, leading to a better understanding of tumor onset and progression, and opening new avenues in translational oncology. Formalin-fixed paraffin-embedded (FFPE) tissue is the method of choice for storage of clinical samples, however low quality of FFPE genomic DNA (gDNA) can limit its use for downstream applications. To investigate the FFPE specimen suitability for NGS analysis and to establish the performance of two solution-based exome capture technologies, we compared the whole-exome sequencing (WES) data of gDNA extracted from 5 fresh frozen (FF) and 5 matched FFPE lung adenocarcinoma tissues using: SeqCap EZ Human Exome v.3.0 (Roche NimbleGen) and SureSelect XT Human All Exon v.5 (Agilent Technologies). Sequencing metrics on Illumina HiSeq were optimal for both exome systems and comparable among FFPE and FF samples, with a slight increase of PCR duplicates in FFPE, mainly in Roche NimbleGen libraries. Comparison of single nucleotide variants (SNVs) between FFPE-FF pairs reached overlapping values >90 % in both systems. Both WES showed high concordance with target re-sequencing data by Ion PGM™ in 22 lung-cancer genes, regardless the source of samples. Exon coverage of 623 cancer-related genes revealed high coverage efficiency of both kits, proposing WES as a valid alternative to target re-sequencing. High-quality and reliable data can be successfully obtained from WES of FFPE samples starting from a relatively low amount of input gDNA, suggesting the inclusion of NGS-based tests into clinical contest. In conclusion, our analysis suggests that the WES approach could be extended to a translational research context as well as to the clinic (e.g. to study rare malignancies), where the simultaneous analysis of the whole coding region of the genome may help in the detection of cancer-linked variants.

Whole-exome sequencing for RH genotyping and alloimmunization risk in children with sickle cell anemia

PubMed Central

Flanagan, Jonathan M.; Vege, Sunitha; Luban, Naomi L. C.; Brown, R. Clark; Ware, Russell E.; Westhoff, Connie M.

2017-01-01

RH genes are highly polymorphic and encode the most complex of the 35 human blood group systems. This genetic diversity contributes to Rh alloimmunization in patients with sickle cell anemia (SCA) and is not avoided by serologic Rh-matched red cell transfusions. Standard serologic testing does not distinguish variant Rh antigens. Single nucleotide polymorphism (SNP)–based DNA arrays detect many RHD and RHCE variants, but the number of alleles tested is limited. We explored a next-generation sequencing (NGS) approach using whole-exome sequencing (WES) in 27 Rh alloimmunized and 27 matched non-alloimmunized patients with SCA who received chronic red cell transfusions and were enrolled in a multicenter study. We demonstrate that WES provides a comprehensive RH genotype, identifies SNPs not interrogated by DNA array, and accurately determines RHD zygosity. Among this multicenter cohort, we demonstrate an association between an altered RH genotype and Rh alloimmunization: 52% of Rh immunized vs 19% of non-immunized patients expressed variant Rh without co-expression of the conventional protein. Our findings suggest that RH allele variation in patients with SCA is clinically relevant, and NGS technology can offer a comprehensive alternative to targeted SNP-based testing. This is particularly relevant as NGS data becomes more widely available and could provide the means for reducing Rh alloimmunization in children with SCA. PMID:29296782

Exome Sequencing Identifies Mitochondrial Alanyl-tRNA Synthetase Mutations in Infantile Mitochondrial Cardiomyopathy

PubMed Central

Götz, Alexandra; Tyynismaa, Henna; Euro, Liliya; Ellonen, Pekka; Hyötyläinen, Tuulia; Ojala, Tiina; Hämäläinen, Riikka H.; Tommiska, Johanna; Raivio, Taneli; Oresic, Matej; Karikoski, Riitta; Tammela, Outi; Simola, Kalle O.J.; Paetau, Anders; Tyni, Tiina; Suomalainen, Anu

2011-01-01

Infantile cardiomyopathies are devastating fatal disorders of the neonatal period or the first year of life. Mitochondrial dysfunction is a common cause of this group of diseases, but the underlying gene defects have been characterized in only a minority of cases, because tissue specificity of the manifestation hampers functional cloning and the heterogeneity of causative factors hinders collection of informative family materials. We sequenced the exome of a patient who died at the age of 10 months of hypertrophic mitochondrial cardiomyopathy with combined cardiac respiratory chain complex I and IV deficiency. Rigorous data analysis allowed us to identify a homozygous missense mutation in AARS2, which we showed to encode the mitochondrial alanyl-tRNA synthetase (mtAlaRS). Two siblings from another family, both of whom died perinatally of hypertrophic cardiomyopathy, had the same mutation, compound heterozygous with another missense mutation. Protein structure modeling of mtAlaRS suggested that one of the mutations affected a unique tRNA recognition site in the editing domain, leading to incorrect tRNA aminoacylation, whereas the second mutation severely disturbed the catalytic function, preventing tRNA aminoacylation. We show here that mutations in AARS2 cause perinatal or infantile cardiomyopathy with near-total combined mitochondrial respiratory chain deficiency in the heart. Our results indicate that exome sequencing is a powerful tool for identifying mutations in single patients and allows recognition of the genetic background in single-gene disorders of variable clinical manifestation and tissue-specific disease. Furthermore, we show that mitochondrial disorders extend to prenatal life and are an important cause of early infantile cardiac failure. PMID:21549344

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

Use of whole-exome sequencing to determine the genetic basis of multiple mitochondrial respiratory chain complex deficiencies.

PubMed

Taylor, Robert W; Pyle, Angela; Griffin, Helen; Blakely, Emma L; Duff, Jennifer; He, Langping; Smertenko, Tania; Alston, Charlotte L; Neeve, Vivienne C; Best, Andrew; Yarham, John W; Kirschner, Janbernd; Schara, Ulrike; Talim, Beril; Topaloglu, Haluk; Baric, Ivo; Holinski-Feder, Elke; Abicht, Angela; Czermin, Birgit; Kleinle, Stephanie; Morris, Andrew A M; Vassallo, Grace; Gorman, Grainne S; Ramesh, Venkateswaran; Turnbull, Douglass M; Santibanez-Koref, Mauro; McFarland, Robert; Horvath, Rita; Chinnery, Patrick F

2014-07-02

Mitochondrial disorders have emerged as a common cause of inherited disease, but their diagnosis remains challenging. Multiple respiratory chain complex defects are particularly difficult to diagnose at the molecular level because of the massive number of nuclear genes potentially involved in intramitochondrial protein synthesis, with many not yet linked to human disease. To determine the molecular basis of multiple respiratory chain complex deficiencies. We studied 53 patients referred to 2 national centers in the United Kingdom and Germany between 2005 and 2012. All had biochemical evidence of multiple respiratory chain complex defects but no primary pathogenic mitochondrial DNA mutation. Whole-exome sequencing was performed using 62-Mb exome enrichment, followed by variant prioritization using bioinformatic prediction tools, variant validation by Sanger sequencing, and segregation of the variant with the disease phenotype in the family. Presumptive causal variants were identified in 28 patients (53%; 95% CI, 39%-67%) and possible causal variants were identified in 4 (8%; 95% CI, 2%-18%). Together these accounted for 32 patients (60% 95% CI, 46%-74%) and involved 18 different genes. These included recurrent mutations in RMND1, AARS2, and MTO1, each on a haplotype background consistent with a shared founder allele, and potential novel mutations in 4 possible mitochondrial disease genes (VARS2, GARS, FLAD1, and PTCD1). Distinguishing clinical features included deafness and renal involvement associated with RMND1 and cardiomyopathy with AARS2 and MTO1. However, atypical clinical features were present in some patients, including normal liver function and Leigh syndrome (subacute necrotizing encephalomyelopathy) seen in association with TRMU mutations and no cardiomyopathy with founder SCO2 mutations. It was not possible to confidently identify the underlying genetic basis in 21 patients (40%; 95% CI, 26%-54%). Exome sequencing enhances the ability to identify

Guidance to rational use of pharmaceuticals in gallbladder sarcomatoid carcinoma using patient-derived cancer cells and whole exome sequencing.

PubMed

Feng, Feiling; Cheng, Qingbao; Yang, Liang; Zhang, Dadong; Ji, Shunlong; Zhang, Qiangzu; Lin, Yihui; Li, Fugen; Xiong, Lei; Liu, Chen; Jiang, Xiaoqing

2017-01-17

Gallbladder sarcomatoid carcinoma is a rare cancer with no clinical standard treatment. With the rapid development of next generation sequencing, it has been able to provide reasonable treatment options for patients based on genetic variations. However, most cancer drugs are not approval for gallbladder sarcomatoid carcinoma indications. The correlation between drug response and a genetic variation needs to be further elucidated. Three patient-derived cells-JXQ-3D-001, JXQ-3D-002, and JXQ-3D-003, were derived from biopsy samples of one gallbladder sarcomatoid carcinoma patient with progression and have been characterized. In order to study the relationship between drug sensitivity and gene alteration, genetic mutations of three patient-derived cells were discovered by whole exome sequencing, and drug screening has been performed based on the gene alterations and related signaling pathways that are associated with drug targets. It has been found that there are differences in biological characteristics such as morphology, cell proliferation, cell migration and colony formation activity among these three patient-derived cells although they are derived from the same patient. Their sensitivities to the chemotherapy drugs-Fluorouracil, Doxorubicin, and Cisplatin are distinct. Moreover, none of common chemotherapy drugs could inhibit the proliferations of all three patient-derived cells. Comprehensive analysis of their whole exome sequencing demonstrated that tumor-associated genes TP53, AKT2, FGFR3, FGF10, SDHA, and PI3KCA were mutated or amplified. Part of these alterations are actionable. By screening a set of compounds that are associated with the genetic alteration, it has been found that GDC-0941 and PF-04691502 for PI3K-AKT-mTOR pathway inhibitors could dramatically decrease the proliferation of three patient-derived cells. Importantly, expression of phosphorylated AKT and phosphorylated S6 were markedly decreased after treatments with PI3K-AKT-mTOR pathway

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

Whole exome sequencing is an efficient, sensitive and specific method for determining the genetic cause of short-rib thoracic dystrophies.

PubMed

McInerney-Leo, A M; Harris, J E; Leo, P J; Marshall, M S; Gardiner, B; Kinning, E; Leong, H Y; McKenzie, F; Ong, W P; Vodopiutz, J; Wicking, C; Brown, M A; Zankl, A; Duncan, E L

2015-12-01

Short-rib thoracic dystrophies (SRTDs) are congenital disorders due to defects in primary cilium function. SRTDs are recessively inherited with mutations identified in 14 genes to date (comprising 398 exons). Conventional mutation detection (usually by iterative Sanger sequencing) is inefficient and expensive, and often not undertaken. Whole exome massive parallel sequencing has been used to identify new genes for SRTD (WDR34, WDR60 and IFT172); however, the clinical utility of whole exome sequencing (WES) has not been established. WES was performed in 11 individuals with SRTDs. Compound heterozygous or homozygous mutations were identified in six confirmed SRTD genes in 10 individuals (IFT172, DYNC2H1, TTC21B, WDR60, WDR34 and NEK1), giving overall sensitivity of 90.9%. WES data from 993 unaffected individuals sequenced using similar technology showed two individuals with rare (minor allele frequency <0.005) compound heterozygous variants of unknown significance in SRTD genes (specificity >99%). Costs for consumables, laboratory processing and bioinformatic analysis were

Jannovar: a java library for exome annotation.

PubMed

Jäger, Marten; Wang, Kai; Bauer, Sebastian; Smedley, Damian; Krawitz, Peter; Robinson, Peter N

2014-05-01

Transcript-based annotation and pedigree analysis are two basic steps in the computational analysis of whole-exome sequencing experiments in genetic diagnostics and disease-gene discovery projects. Here, we present Jannovar, a stand-alone Java application as well as a Java library designed to be used in larger software frameworks for exome and genome analysis. Jannovar uses an interval tree to identify all transcripts affected by a given variant, and provides Human Genome Variation Society-compliant annotations both for variants affecting coding sequences and splice junctions as well as untranslated regions and noncoding RNA transcripts. Jannovar can also perform family-based pedigree analysis with Variant Call Format (VCF) files with data from members of a family segregating a Mendelian disorder. Using a desktop computer, Jannovar requires a few seconds to annotate a typical VCF file with exome data. Jannovar is freely available under the BSD2 license. Source code as well as the Java application and library file can be downloaded from http://compbio.charite.de (with tutorial) and https://github.com/charite/jannovar. © 2014 WILEY PERIODICALS, INC.

Whole exome sequencing in neurogenetic odysseys: An effective, cost- and time-saving diagnostic approach.

PubMed

Córdoba, Marta; Rodriguez-Quiroga, Sergio Alejandro; Vega, Patricia Analía; Salinas, Valeria; Perez-Maturo, Josefina; Amartino, Hernán; Vásquez-Dusefante, Cecilia; Medina, Nancy; González-Morón, Dolores; Kauffman, Marcelo Andrés

2018-01-01

Diagnostic trajectories for neurogenetic disorders frequently require the use of considerable time and resources, exposing patients and families to so-called "diagnostic odysseys". Previous studies have provided strong evidence for increased diagnostic and clinical utility of whole-exome sequencing in medical genetics. However, specific reports assessing its utility in a setting such as ours- a neurogeneticist led academic group serving in a low-income country-are rare. To assess the diagnostic yield of WES in patients suspected of having a neurogenetic condition and explore the cost-effectiveness of its implementation in a research group located in an Argentinean public hospital. This is a prospective study of the clinical utility of WES in a series of 40 consecutive patients selected from a Neurogenetic Clinic of a tertiary Hospital in Argentina. We evaluated patients retrospectively for previous diagnostic trajectories. Diagnostic yield, clinical impact on management and economic diagnostic burden were evaluated. We demonstrated the clinical utility of Whole Exome Sequencing in our patient cohort, obtaining a diagnostic yield of 40% (95% CI, 24.8%-55.2%) among a diverse group of neurological disorders. The average age at the time of WES was 23 (range 3-70). The mean time elapsed from symptom onset to WES was 11 years (range 3-42). The mean cost of the diagnostic workup prior to WES was USD 1646 (USD 1439 to 1853), which is 60% higher than WES cost in our center. WES for neurogenetics proved to be an effective, cost- and time-saving approach for the molecular diagnosis of this heterogeneous and complex group of patients.

Sporadic autism exomes reveal a highly interconnected protein network of de novo mutations.

PubMed

O'Roak, Brian J; Vives, Laura; Girirajan, Santhosh; Karakoc, Emre; Krumm, Niklas; Coe, Bradley P; Levy, Roie; Ko, Arthur; Lee, Choli; Smith, Joshua D; Turner, Emily H; Stanaway, Ian B; Vernot, Benjamin; Malig, Maika; Baker, Carl; Reilly, Beau; Akey, Joshua M; Borenstein, Elhanan; Rieder, Mark J; Nickerson, Deborah A; Bernier, Raphael; Shendure, Jay; Eichler, Evan E

2012-04-04

It is well established that autism spectrum disorders (ASD) have a strong genetic component; however, for at least 70% of cases, the underlying genetic cause is unknown. Under the hypothesis that de novo mutations underlie a substantial fraction of the risk for developing ASD in families with no previous history of ASD or related phenotypes--so-called sporadic or simplex families--we sequenced all coding regions of the genome (the exome) for parent-child trios exhibiting sporadic ASD, including 189 new trios and 20 that were previously reported. Additionally, we also sequenced the exomes of 50 unaffected siblings corresponding to these new (n = 31) and previously reported trios (n = 19), for a total of 677 individual exomes from 209 families. Here we show that de novo point mutations are overwhelmingly paternal in origin (4:1 bias) and positively correlated with paternal age, consistent with the modest increased risk for children of older fathers to develop ASD. Moreover, 39% (49 of 126) of the most severe or disruptive de novo mutations map to a highly interconnected β-catenin/chromatin remodelling protein network ranked significantly for autism candidate genes. In proband exomes, recurrent protein-altering mutations were observed in two genes: CHD8 and NTNG1. Mutation screening of six candidate genes in 1,703 ASD probands identified additional de novo, protein-altering mutations in GRIN2B, LAMC3 and SCN1A. Combined with copy number variant (CNV) data, these results indicate extreme locus heterogeneity but also provide a target for future discovery, diagnostics and therapeutics.

Novel candidate genes may be possible predisposing factors revealed by whole exome sequencing in familial esophageal squamous cell carcinoma.

PubMed

Forouzanfar, Narjes; Baranova, Ancha; Milanizadeh, Saman; Heravi-Moussavi, Alireza; Jebelli, Amir; Abbaszadegan, Mohammad Reza

2017-05-01

Esophageal squamous cell carcinoma is one of the deadliest of all the cancers. Its metastatic properties portend poor prognosis and high rate of recurrence. A more advanced method to identify new molecular biomarkers predicting disease prognosis can be whole exome sequencing. Here, we report the most effective genetic variants of the Notch signaling pathway in esophageal squamous cell carcinoma susceptibility by whole exome sequencing. We analyzed nine probands in unrelated familial esophageal squamous cell carcinoma pedigrees to identify candidate genes. Genomic DNA was extracted and whole exome sequencing performed to generate information about genetic variants in the coding regions. Bioinformatics software applications were utilized to exploit statistical algorithms to demonstrate protein structure and variants conservation. Polymorphic regions were excluded by false-positive investigations. Gene-gene interactions were analyzed for Notch signaling pathway candidates. We identified novel and damaging variants of the Notch signaling pathway through extensive pathway-oriented filtering and functional predictions, which led to the study of 27 candidate novel mutations in all nine patients. Detection of the trinucleotide repeat containing 6B gene mutation (a slice site alteration) in five of the nine probands, but not in any of the healthy samples, suggested that it may be a susceptibility factor for familial esophageal squamous cell carcinoma. Noticeably, 8 of 27 novel candidate gene mutations (e.g. epidermal growth factor, signal transducer and activator of transcription 3, MET) act in a cascade leading to cell survival and proliferation. Our results suggest that the trinucleotide repeat containing 6B mutation may be a candidate predisposing gene in esophageal squamous cell carcinoma. In addition, some of the Notch signaling pathway genetic mutations may act as key contributors to esophageal squamous cell carcinoma.

Microcephaly-capillary malformation syndrome: Brothers with a homozygous STAMBP mutation, uncovered by exome sequencing.

PubMed

Naseer, Muhammad Imran; Sogaty, Sameera; Rasool, Mahmood; Chaudhary, Adeel G; Abutalib, Yousif Ahmed; Walker, Susan; Marshall, Christian R; Merico, Daniele; Carter, Melissa T; Scherer, Stephen W; Al-Qahtani, Mohammad H; Zarrei, Mehdi

2016-11-01

We describe two brothers from a consanguineous family of Egyptian ancestry, presenting with microcephaly, apparent global developmental delay, seizures, spasticity, congenital blindness, and multiple cutaneous capillary malformations. Through exome sequencing, we uncovered a homozygous missense variant in STAMBP (p.K303R) in the two siblings, inherited from heterozygous carrier parents. Mutations in STAMBP are known to cause microcephaly-capillary malformation syndrome (MIC-CAP) and the phenotype in this family is consistent with this diagnosis. We compared the findings in the present brothers with those of earlier reported patients. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Growth hormone deficiency with advanced bone age: phenotypic interaction between GHRH receptor and CYP21A2 mutations diagnosed by sanger and whole exome sequencing.

PubMed

Correa, Fernanda A; França, Marcela M; Fang, Qing; Ma, Qianyi; Bachega, Tania A; Rodrigues, Andresa; Ozel, Bilge A; Li, Jun Z; Mendonca, Berenice B; Jorge, Alexander A L; Carvalho, Luciani R; Camper, Sally A; Arnhold, Ivo J P

2017-12-01

Isolated growth hormone deficiency (IGHD) is the most common pituitary hormone deficiency and, clinically, patients have delayed bone age. High sequence similarity between CYP21A2 gene and CYP21A1P pseudogene poses difficulties for exome sequencing interpretation. A 7.5 year-old boy born to second-degree cousins presented with severe short stature (height SDS -3.7) and bone age of 6 years. Clonidine and combined pituitary stimulation tests revealed GH deficiency. Pituitary MRI was normal. The patient was successfully treated with rGH. Surprisingly, at 10.8 years, his bone age had advanced to 13 years, but physical exam, LH and testosterone levels remained prepubertal. An ACTH stimulation test disclosed a non-classic congenital adrenal hyperplasia due to 21-hydroxylase deficiency explaining the bone age advancement and, therefore, treatment with cortisone acetate was added. The genetic diagnosis of a homozygous mutation in GHRHR (p.Leu144His), a homozygous CYP21A2 mutation (p.Val282Leu) and CYP21A1P pseudogene duplication was established by Sanger sequencing, MLPA and whole-exome sequencing. We report the unusual clinical presentation of a patient born to consanguineous parents with two recessive endocrine diseases: non-classic congenital adrenal hyperplasia modifying the classical GH deficiency phenotype. We used a method of paired read mapping aided by neighbouring mis-matches to overcome the challenges of exome-sequencing in the presence of a pseudogene.

Rare coding variation in paraoxonase-1 is associated with ischemic stroke in the NHLBI Exome Sequencing Project.

PubMed

Kim, Daniel Seung; Crosslin, David R; Auer, Paul L; Suzuki, Stephanie M; Marsillach, Judit; Burt, Amber A; Gordon, Adam S; Meschia, James F; Nalls, Mike A; Worrall, Bradford B; Longstreth, W T; Gottesman, Rebecca F; Furlong, Clement E; Peters, Ulrike; Rich, Stephen S; Nickerson, Deborah A; Jarvik, Gail P

2014-06-01

HDL-associated paraoxonase-1 (PON1) is an enzyme whose activity is associated with cerebrovascular disease. Common PON1 genetic variants have not been consistently associated with cerebrovascular disease. Rare coding variation that likely alters PON1 enzyme function may be more strongly associated with stroke. The National Heart, Lung, and Blood Institute Exome Sequencing Project sequenced the coding regions (exomes) of the genome for heart, lung, and blood-related phenotypes (including ischemic stroke). In this sample of 4,204 unrelated participants, 496 had verified, noncardioembolic ischemic stroke. After filtering, 28 nonsynonymous PON1 variants were identified. Analysis with the sequence kernel association test, adjusted for covariates, identified significant associations between PON1 variants and ischemic stroke (P = 3.01 × 10(-3)). Stratified analyses demonstrated a stronger association of PON1 variants with ischemic stroke in African ancestry (AA) participants (P = 5.03 × 10(-3)). Ethnic differences in the association between PON1 variants with stroke could be due to the effects of PON1Val109Ile (overall P = 7.88 × 10(-3); AA P = 6.52 × 10(-4)), found at higher frequency in AA participants (1.16% vs. 0.02%) and whose protein is less stable than the common allele. In summary, rare genetic variation in PON1 was associated with ischemic stroke, with stronger associations identified in those of AA. Increased focus on PON1 enzyme function and its role in cerebrovascular disease is warranted.

Whole-exome sequencing for variant discovery in blepharospasm.

PubMed

Tian, Jun; Vemula, Satya R; Xiao, Jianfeng; Valente, Enza Maria; Defazio, Giovanni; Petrucci, Simona; Gigante, Angelo Fabio; Rudzińska-Bar, Monika; Wszolek, Zbigniew K; Kennelly, Kathleen D; Uitti, Ryan J; van Gerpen, Jay A; Hedera, Peter; Trimble, Elizabeth J; LeDoux, Mark S

2018-05-16

Blepharospasm (BSP) is a type of focal dystonia characterized by involuntary orbicularis oculi spasms that are usually bilateral, synchronous, and symmetrical. Despite strong evidence for genetic contributions to BSP, progress in the field has been constrained by small cohorts, incomplete penetrance, and late age of onset. Although several genetic etiologies for dystonia have been identified through whole-exome sequencing (WES), none of these are characteristically associated with BSP as a singular or predominant manifestation. We performed WES on 31 subjects from 21 independent pedigrees with BSP. The strongest candidate sequence variants derived from in silico analyses were confirmed with bidirectional Sanger sequencing and subjected to cosegregation analysis. Cosegregating deleterious variants (GRCH37/hg19) in CACNA1A (NM_001127222.1: c.7261_7262delinsGT, p.Pro2421Val), REEP4 (NM_025232.3: c.109C>T, p.Arg37Trp), TOR2A (NM_130459.3: c.568C>T, p.Arg190Cys), and ATP2A3 (NM_005173.3: c.1966C>T, p.Arg656Cys) were identified in four independent multigenerational pedigrees. Deleterious variants in HS1BP3 (NM_022460.3: c.94C>A, p.Gly32Cys) and GNA14 (NM_004297.3: c.989_990del, p.Thr330ArgfsTer67) were identified in a father and son with segmental cranio-cervical dystonia first manifest as BSP. Deleterious variants in DNAH17, TRPV4, CAPN11, VPS13C, UNC13B, SPTBN4, MYOD1, and MRPL15 were found in two or more independent pedigrees. To our knowledge, none of these genes have previously been associated with isolated BSP, although other CACNA1A mutations have been associated with both positive and negative motor disorders including ataxia, episodic ataxia, hemiplegic migraine, and dystonia. Our WES datasets provide a platform for future studies of BSP genetics which will demand careful consideration of incomplete penetrance, pleiotropy, population stratification, and oligogenic inheritance patterns. © 2018 The Authors. Molecular Genetics & Genomic Medicine published by Wiley

Allele-specific copy-number discovery from whole-genome and whole-exome sequencing

PubMed Central

Wang, WeiBo; Wang, Wei; Sun, Wei; Crowley, James J.; Szatkiewicz, Jin P.

2015-01-01

Copy-number variants (CNVs) are a major form of genetic variation and a risk factor for various human diseases, so it is crucial to accurately detect and characterize them. It is conceivable that allele-specific reads from high-throughput sequencing data could be leveraged to both enhance CNV detection and produce allele-specific copy number (ASCN) calls. Although statistical methods have been developed to detect CNVs using whole-genome sequence (WGS) and/or whole-exome sequence (WES) data, information from allele-specific read counts has not yet been adequately exploited. In this paper, we develop an integrated method, called AS-GENSENG, which incorporates allele-specific read counts in CNV detection and estimates ASCN using either WGS or WES data. To evaluate the performance of AS-GENSENG, we conducted extensive simulations, generated empirical data using existing WGS and WES data sets and validated predicted CNVs using an independent methodology. We conclude that AS-GENSENG not only predicts accurate ASCN calls but also improves the accuracy of total copy number calls, owing to its unique ability to exploit information from both total and allele-specific read counts while accounting for various experimental biases in sequence data. Our novel, user-friendly and computationally efficient method and a complete analytic protocol is freely available at https://sourceforge.net/projects/asgenseng/. PMID:25883151

Asparagine synthetase deficiency detected by whole exome sequencing causes congenital microcephaly, epileptic encephalopathy and psychomotor delay.

PubMed

Ben-Salem, Salma; Gleeson, Joseph G; Al-Shamsi, Aisha M; Islam, Barira; Hertecant, Jozef; Ali, Bassam R; Al-Gazali, Lihadh

2015-06-01

Deficiency of Asparagine Synthetase (ASNSD, MIM 615574) is a very rare autosomal recessive disorder presenting with some brain abnormalities. Affected individuals have congenital microcephaly and progressive encephalopathy associated with severe intellectual disability and intractable seizures. The loss of function of the asparagine synthetase (ASNS, EC 6.3.5.4), particularly in the brain, is the major cause of this particular congenital microcephaly. In this study, we clinically evaluated an affected child from a consanguineous Emirati family presenting with congenital microcephaly and epileptic encephalopathy. In addition, whole-exome sequencing revealed a novel homozygous substitution mutation (c.1193A > C) in the ASNS gene. This mutation resulted in the substitution of highly conserved tyrosine residue by cysteine (p.Y398C). Molecular modeling analysis predicts hypomorphic and damaging effects of this mutation on the protein structure and altering its enzymatic activity. Therefore, we conclude that the loss of ASNS function is most likely the cause of this condition in the studied family. This report brings the number of reported families with this very rare disorder to five and the number of pathogenic mutations in the ASNS gene to four. This finding extends the ASNS pathogenic mutations spectrum and highlights the utility of whole-exome sequencing in elucidation the causes of rare recessive disorders that are heterogeneous and/or overlap with other conditions.

Exome-first approach identified a novel gloss deletion associated with Lowe syndrome.

PubMed

Watanabe, Miki; Nakagawa, Ryuji; Kohmoto, Tomohiro; Naruto, Takuya; Suga, Ken-Ichi; Goji, Aya; Horikawa, Hideaki; Masuda, Kiyoshi; Kagami, Shoji; Imoto, Issei

2016-01-01

Lowe syndrome (LS) is an X-linked disorder affecting the eyes, nervous system and kidneys, typically caused by missense or nonsense/frameshift OCRL mutations. We report a 6-month-old male clinically suspected to have LS, but without the Fanconi-type renal dysfunction. Using a targeted-exome sequencing-first approach, LS was diagnosed by the identification of a deletion involving 1.7 Mb at Xq25-q26.1, encompassing the entire OCRL gene and neighboring loci.

Detection of 1p36 deletion by clinical exome-first diagnostic approach.

PubMed

Watanabe, Miki; Hayabuchi, Yasunobu; Ono, Akemi; Naruto, Takuya; Horikawa, Hideaki; Kohmoto, Tomohiro; Masuda, Kiyoshi; Nakagawa, Ryuji; Ito, Hiromichi; Kagami, Shoji; Imoto, Issei

2016-01-01

Although chromosome 1p36 deletion syndrome is considered clinically recognizable based on characteristic features, the clinical manifestations of patients during infancy are often not consistent with those observed later in life. We report a 4-month-old girl who showed multiple congenital anomalies and developmental delay, but no clinical signs of syndromic disease caused by a terminal deletion in 1p36.32-p36.33 that was first identified by targeted-exome sequencing for molecular diagnosis.

Assessing the Power of Exome Chips.

PubMed

Page, Christian Magnus; Baranzini, Sergio E; Mevik, Bjørn-Helge; Bos, Steffan Daniel; Harbo, Hanne F; Andreassen, Bettina Kulle

2015-01-01

Genotyping chips for rare and low-frequent variants have recently gained popularity with the introduction of exome chips, but the utility of these chips remains unclear. These chips were designed using exome sequencing data from mainly American-European individuals, enriched for a narrow set of common diseases. In addition, it is well-known that the statistical power of detecting associations with rare and low-frequent variants is much lower compared to studies exclusively involving common variants. We developed a simulation program adaptable to any exome chip design to empirically evaluate the power of the exome chips. We implemented the main properties of the Illumina HumanExome BeadChip array. The simulated data sets were used to assess the power of exome chip based studies for varying effect sizes and causal variant scenarios. We applied two widely-used statistical approaches for rare and low-frequency variants, which collapse the variants into genetic regions or genes. Under optimal conditions, we found that a sample size between 20,000 to 30,000 individuals were needed in order to detect modest effect sizes (0.5% < PAR > 1%) with 80% power. For small effect sizes (PAR <0.5%), 60,000-100,000 individuals were needed in the presence of non-causal variants. In conclusion, we found that at least tens of thousands of individuals are necessary to detect modest effects under optimal conditions. In addition, when using rare variant chips on cohorts or diseases they were not originally designed for, the identification of associated variants or genes will be even more challenging.

Identification of somatic mutations in non-small cell lung carcinomas using whole-exome sequencing

PubMed Central

Liu, Pengyuan; Morrison, Carl; Wang, Liang; Xiong, Donghai; Vedell, Peter; Cui, Peng; Hua, Xing; Ding, Feng; Lu, Yan; James, Michael; Ebben, John D.; Xu, Haiming; Adjei, Alex A.; Head, Karen; Andrae, Jaime W.; Tschannen, Michael R.; Jacob, Howard; Pan, Jing; Zhang, Qi; Van den Bergh, Francoise; Xiao, Haijie; Lo, Ken C.; Patel, Jigar; Richmond, Todd; Watt, Mary-Anne; Albert, Thomas; Selzer, Rebecca; Anderson, Marshall; Wang, Jiang; Wang, Yian; Starnes, Sandra; Yang, Ping; You, Ming

2012-01-01

Lung cancer is the leading cause of cancer-related death, with non-small cell lung cancer (NSCLC) being the predominant form of the disease. Most lung cancer is caused by the accumulation of genomic alterations due to tobacco exposure. To uncover its mutational landscape, we performed whole-exome sequencing in 31 NSCLCs and their matched normal tissue samples. We identified both common and unique mutation spectra and pathway activation in lung adenocarcinomas and squamous cell carcinomas, two major histologies in NSCLC. In addition to identifying previously known lung cancer genes (TP53, KRAS, EGFR, CDKN2A and RB1), the analysis revealed many genes not previously implicated in this malignancy. Notably, a novel gene CSMD3 was identified as the second most frequently mutated gene (next to TP53) in lung cancer. We further demonstrated that loss of CSMD3 results in increased proliferation of airway epithelial cells. The study provides unprecedented insights into mutational processes, cellular pathways and gene networks associated with lung cancer. Of potential immediate clinical relevance, several highly mutated genes identified in our study are promising druggable targets in cancer therapy including ALK, CTNNA3, DCC, MLL3, PCDHIIX, PIK3C2B, PIK3CG and ROCK2. PMID:22510280

Whole exome sequencing identifies novel candidate genes that modify chronic obstructive pulmonary disease susceptibility.

PubMed

Bruse, Shannon; Moreau, Michael; Bromberg, Yana; Jang, Jun-Ho; Wang, Nan; Ha, Hongseok; Picchi, Maria; Lin, Yong; Langley, Raymond J; Qualls, Clifford; Klensney-Tait, Julia; Zabner, Joseph; Leng, Shuguang; Mao, Jenny; Belinsky, Steven A; Xing, Jinchuan; Nyunoya, Toru

2016-01-07

Chronic obstructive pulmonary disease (COPD) is characterized by an irreversible airflow limitation in response to inhalation of noxious stimuli, such as cigarette smoke. However, only 15-20 % smokers manifest COPD, suggesting a role for genetic predisposition. Although genome-wide association studies have identified common genetic variants that are associated with susceptibility to COPD, effect sizes of the identified variants are modest, as is the total heritability accounted for by these variants. In this study, an extreme phenotype exome sequencing study was combined with in vitro modeling to identify COPD candidate genes. We performed whole exome sequencing of 62 highly susceptible smokers and 30 exceptionally resistant smokers to identify rare variants that may contribute to disease risk or resistance to COPD. This was a cross-sectional case-control study without therapeutic intervention or longitudinal follow-up information. We identified candidate genes based on rare variant analyses and evaluated exonic variants to pinpoint individual genes whose function was computationally established to be significantly different between susceptible and resistant smokers. Top scoring candidate genes from these analyses were further filtered by requiring that each gene be expressed in human bronchial epithelial cells (HBECs). A total of 81 candidate genes were thus selected for in vitro functional testing in cigarette smoke extract (CSE)-exposed HBECs. Using small interfering RNA (siRNA)-mediated gene silencing experiments, we showed that silencing of several candidate genes augmented CSE-induced cytotoxicity in vitro. Our integrative analysis through both genetic and functional approaches identified two candidate genes (TACC2 and MYO1E) that augment cigarette smoke (CS)-induced cytotoxicity and, potentially, COPD susceptibility.

Whole exome sequencing in neurogenetic odysseys: An effective, cost- and time-saving diagnostic approach

PubMed Central

Córdoba, Marta; Rodriguez-Quiroga, Sergio Alejandro; Vega, Patricia Analía; Salinas, Valeria; Perez-Maturo, Josefina; Amartino, Hernán; Vásquez-Dusefante, Cecilia; Medina, Nancy; González-Morón, Dolores; Kauffman, Marcelo Andrés

2018-01-01

Background Diagnostic trajectories for neurogenetic disorders frequently require the use of considerable time and resources, exposing patients and families to so-called “diagnostic odysseys”. Previous studies have provided strong evidence for increased diagnostic and clinical utility of whole-exome sequencing in medical genetics. However, specific reports assessing its utility in a setting such as ours- a neurogeneticist led academic group serving in a low-income country—are rare. Objectives To assess the diagnostic yield of WES in patients suspected of having a neurogenetic condition and explore the cost-effectiveness of its implementation in a research group located in an Argentinean public hospital. Methods This is a prospective study of the clinical utility of WES in a series of 40 consecutive patients selected from a Neurogenetic Clinic of a tertiary Hospital in Argentina. We evaluated patients retrospectively for previous diagnostic trajectories. Diagnostic yield, clinical impact on management and economic diagnostic burden were evaluated. Results We demonstrated the clinical utility of Whole Exome Sequencing in our patient cohort, obtaining a diagnostic yield of 40% (95% CI, 24.8%-55.2%) among a diverse group of neurological disorders. The average age at the time of WES was 23 (range 3–70). The mean time elapsed from symptom onset to WES was 11 years (range 3–42). The mean cost of the diagnostic workup prior to WES was USD 1646 (USD 1439 to 1853), which is 60% higher than WES cost in our center. Conclusions WES for neurogenetics proved to be an effective, cost- and time-saving approach for the molecular diagnosis of this heterogeneous and complex group of patients. PMID:29389947

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.

Identification of autism-related MECP2 mutations by whole-exome sequencing and functional validation.

PubMed

Wen, Zhu; Cheng, Tian-Lin; Li, Gai-Zhi; Sun, Shi-Bang; Yu, Shun-Ying; Zhang, Yi; Du, Ya-Song; Qiu, Zilong

2017-01-01

Methyl-CpG-binding protein-2 (MeCP2) is a critical regulator for neural development. Either loss- or gain-of-function leads to severe neurodevelopmental disorders, such as Rett syndrome (RTT) and autism spectrum disorder (ASD). We set out to screen for MECP2 mutations in patients of ASD and determine whether these autism-related mutations may compromise the proper function of MeCP2. Whole-exome sequencing was performed to screen MECP2 and other ASD candidate genes for 120 patients diagnosed with ASD. The parents of patients who were identified with MECP2 mutation were selected for further Sanger sequencing. Each patient accomplished the case report form including general information and clinical scales applied to assess their clinical features. Mouse cortical neurons and HEK-293 cells were cultured and transfected with MeCP2 wild-type (WT) or mutant to examine the function of autism-associated MeCP2 mutants. HEK-293 cells were used to examine the expression of MeCP2 mutant constructs with Western blot. Mouse cortical neurons were used to analyze neurites and axon outgrowth by immunofluorescence experiments. We identified three missense mutations of MECP2 from three autism patients by whole-exome sequencing: p.P152L (c.455C>T), p.P376S (c.1162C>T), and p.R294X (c.880C>T). Among these mutations, p.P152L and p.R294X were de novo mutations, whereas p.P376S was inherited maternally. The diagnosis of RTT was excluded in all three autism patients. Abnormalities of dendritic and axonal growth were found after autism-related MeCP2 mutants were expressed in mouse cortical neurons; suggesting that autism-related MECP2 mutations impair the proper development of neurons. Our study identified genetic mutations of the MECP2 gene in autism patients, which were previously considered to be associated primarily with RTT. This finding suggests that loss-of-function mutations of MECP2 may also lead to autism spectrum disorders.

Allele-specific copy-number discovery from whole-genome and whole-exome sequencing.

PubMed

Wang, WeiBo; Wang, Wei; Sun, Wei; Crowley, James J; Szatkiewicz, Jin P

2015-08-18

Copy-number variants (CNVs) are a major form of genetic variation and a risk factor for various human diseases, so it is crucial to accurately detect and characterize them. It is conceivable that allele-specific reads from high-throughput sequencing data could be leveraged to both enhance CNV detection and produce allele-specific copy number (ASCN) calls. Although statistical methods have been developed to detect CNVs using whole-genome sequence (WGS) and/or whole-exome sequence (WES) data, information from allele-specific read counts has not yet been adequately exploited. In this paper, we develop an integrated method, called AS-GENSENG, which incorporates allele-specific read counts in CNV detection and estimates ASCN using either WGS or WES data. To evaluate the performance of AS-GENSENG, we conducted extensive simulations, generated empirical data using existing WGS and WES data sets and validated predicted CNVs using an independent methodology. We conclude that AS-GENSENG not only predicts accurate ASCN calls but also improves the accuracy of total copy number calls, owing to its unique ability to exploit information from both total and allele-specific read counts while accounting for various experimental biases in sequence data. Our novel, user-friendly and computationally efficient method and a complete analytic protocol is freely available at https://sourceforge.net/projects/asgenseng/. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Exome Sequencing Finds a Novel PCSK1 Mutation in a Child With Generalized Malabsorptive Diarrhea and Diabetes Insipidus

PubMed Central

Yourshaw, Michael; Solorzano-Vargas, R. Sergio; Pickett, Lindsay A.; Lindberg, Iris; Wang, Jiafang; Cortina, Galen; Pawlikowska-Haddal, Anna; Baron, Howard; Venick, Robert S.; Nelson, Stanley F.; Martín, Martín G.

2014-01-01

Objectives Congenital diarrhea disorders are a group of genetically diverse and typically autosomal recessive disorders that have yet to be well characterized phenotypically or molecularly. Diagnostic assessments are generally limited to nutritional challenges and histologic evaluation, and many subjects eventually require a prolonged course of intravenous nutrition. Here we describe next-generation sequencing techniques to investigate a child with perplexing congenital malabsorptive diarrhea and other presumably unrelated clinical problems; this method provides an alternative approach to molecular diagnosis. Methods We screened the diploid genome of an affected individual, using exome sequencing, for uncommon variants that have observed protein-coding consequences. We assessed the functional activity of the mutant protein, as well as its lack of expression using immunohistochemistry. Results Among several rare variants detected was a homozygous nonsense mutation in the catalytic domain of the proprotein convertase subtilisin/kexin type 1 gene. The mutation abolishes prohormone convertase 1/3 endoprotease activity as well as expression in the intestine. These primary genetic findings prompted a careful endocrine reevaluation of the child at 4.5 years of age, and multiple significant problems were subsequently identified consistent with the known phenotypic consequences of proprotein convertase subtilisin/kexin type 1 (PCSK1) gene mutations. Based on the molecular diagnosis, alternate medical and dietary management was implemented for diabetes insipidus, polyphagia, and micropenis. Conclusions Whole-exome sequencing provides a powerful diagnostic tool to clinicians managing rare genetic disorders with multiple perplexing clinical manifestations. PMID:24280991

Exome sequencing finds a novel PCSK1 mutation in a child with generalized malabsorptive diarrhea and diabetes insipidus.

PubMed

Yourshaw, Michael; Solorzano-Vargas, R Sergio; Pickett, Lindsay A; Lindberg, Iris; Wang, Jiafang; Cortina, Galen; Pawlikowska-Haddal, Anna; Baron, Howard; Venick, Robert S; Nelson, Stanley F; Martín, Martín G

2013-12-01

Congenital diarrhea disorders are a group of genetically diverse and typically autosomal recessive disorders that have yet to be well characterized phenotypically or molecularly. Diagnostic assessments are generally limited to nutritional challenges and histologic evaluation, and many subjects eventually require a prolonged course of intravenous nutrition. Here we describe next-generation sequencing techniques to investigate a child with perplexing congenital malabsorptive diarrhea and other presumably unrelated clinical problems; this method provides an alternative approach to molecular diagnosis. We screened the diploid genome of an affected individual, using exome sequencing, for uncommon variants that have observed protein-coding consequences. We assessed the functional activity of the mutant protein, as well as its lack of expression using immunohistochemistry. Among several rare variants detected was a homozygous nonsense mutation in the catalytic domain of the proprotein convertase subtilisin/kexin type 1 gene. The mutation abolishes prohormone convertase 1/3 endoprotease activity as well as expression in the intestine. These primary genetic findings prompted a careful endocrine reevaluation of the child at 4.5 years of age, and multiple significant problems were subsequently identified consistent with the known phenotypic consequences of proprotein convertase subtilisin/kexin type 1 (PCSK1) gene mutations. Based on the molecular diagnosis, alternate medical and dietary management was implemented for diabetes insipidus, polyphagia, and micropenis. Whole-exome sequencing provides a powerful diagnostic tool to clinicians managing rare genetic disorders with multiple perplexing clinical manifestations.

Identification of a Novel Missense FBN2 Mutation in a Chinese Family with Congenital Contractural Arachnodactyly Using Exome Sequencing

PubMed Central

Deng, Hao; Lu, Qian; Xu, Hongbo; Deng, Xiong; Yuan, Lamei; Yang, Zhijian; Guo, Yi; Lin, Qiongfen; Xiao, Jingjing; Guan, Liping; Song, Zhi

2016-01-01

Congenital contractural arachnodactyly (CCA, OMIM 121050), also known as Beals-Hecht syndrome, is an autosomal dominant disorder of connective tissue. CCA is characterized by arachnodactyly, dolichostenomelia, pectus deformities, kyphoscoliosis, congenital contractures and a crumpled appearance of the helix of the ear. The aim of this study is to identify the genetic cause of a 4-generation Chinese family of Tujia ethnicity with congenital contractural arachnodactyly by exome sequencing. The clinical features of patients in this family are consistent with CCA. A novel missense mutation, c.3769T>C (p.C1257R), in the fibrillin 2 gene (FBN2) was identified responsible for the genetic cause of our family with CCA. The p.C1257R mutation occurs in the 19th calcium-binding epidermal growth factor-like (cbEGF) domain. The amino acid residue cysteine in this domain is conserved among different species. Our findings suggest that exome sequencing is a powerful tool to discover mutation(s) in CCA. Our results may also provide new insights into the cause and diagnosis of CCA, and may have implications for genetic counseling and clinical management. PMID:27196565

Detection of 1p36 deletion by clinical exome-first diagnostic approach

PubMed Central

Watanabe, Miki; Hayabuchi, Yasunobu; Ono, Akemi; Naruto, Takuya; Horikawa, Hideaki; Kohmoto, Tomohiro; Masuda, Kiyoshi; Nakagawa, Ryuji; Ito, Hiromichi; Kagami, Shoji; Imoto, Issei

2016-01-01

Although chromosome 1p36 deletion syndrome is considered clinically recognizable based on characteristic features, the clinical manifestations of patients during infancy are often not consistent with those observed later in life. We report a 4-month-old girl who showed multiple congenital anomalies and developmental delay, but no clinical signs of syndromic disease caused by a terminal deletion in 1p36.32-p36.33 that was first identified by targeted-exome sequencing for molecular diagnosis. PMID:28428889

Exome-first approach identified a novel gloss deletion associated with Lowe syndrome

PubMed Central

Watanabe, Miki; Nakagawa, Ryuji; Kohmoto, Tomohiro; Naruto, Takuya; Suga, Ken-ichi; Goji, Aya; Horikawa, Hideaki; Masuda, Kiyoshi; Kagami, Shoji; Imoto, Issei

2016-01-01

Lowe syndrome (LS) is an X-linked disorder affecting the eyes, nervous system and kidneys, typically caused by missense or nonsense/frameshift OCRL mutations. We report a 6-month-old male clinically suspected to have LS, but without the Fanconi-type renal dysfunction. Using a targeted-exome sequencing-first approach, LS was diagnosed by the identification of a deletion involving 1.7 Mb at Xq25-q26.1, encompassing the entire OCRL gene and neighboring loci. PMID:27867521

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

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.

Case Report: Application of whole exome sequencing for accurate diagnosis of rare syndromes of mineralocorticoid excess

PubMed Central

Narayanan, Ranjit; Karuthedath Vellarikkal, Shamsudheen; Jayarajan, Rijith; Verma, Ankit; Dixit, Vishal; Scaria, Vinod; Sivasubbu, Sridhar

2017-01-01

Syndromes of mineralocorticoid excess (SME) are closely related clinical manifestations occurring within a specific set of diseases. Overlapping clinical manifestations of such syndromes often create a dilemma in accurate diagnosis, which is crucial for disease surveillance and management especially in rare genetic disorders. Here we demonstrate the use of whole exome sequencing (WES) for accurate diagnosis of rare SME and report that p.R337C variation in the HSD11B2 gene causes progressive apparent mineralocorticoid excess (AME) syndrome in a South Indian family of Mappila origin. PMID:29067160

Practices and Policies of Clinical Exome Sequencing Providers: Analysis and Implications

PubMed Central

Jamal, Seema M.; Yu, Joon-Ho; Chong, Jessica X.; Dent, Karin M.; Conta, Jessie H.; Tabor, Holly K.; Bamshad, Michael J.

2013-01-01

Exome and whole genome sequencing (ES/WGS) offer potential advantages over traditional approaches to diagnostic genetic testing. Consequently, use of ES/WGS in clinical settings is rapidly becoming commonplace. Yet there are myriad moral, ethical, and perhaps legal implications attached to the use of ES and health care professionals and institutions will need to consider these implications in the context of the varied practices and policies of ES service providers. We developed “core elements” of content and procedures for informed consent, data sharing, and results management and a quantitative scale to assess the extent to which research protocols met the standards established by these core elements. We then used these tools to evaluate the practices and policies of each of the 6 U.S. CLIA-certified labs offering clinical ES. Approaches toward informed consent, data sharing, and results return vary widely among ES providers as do the overall potential merits and disadvantages of each, and more importantly, the balance between the two. PMID:23610049

Exome sequencing identifies MAX mutations as a cause of hereditary pheochromocytoma.

PubMed

Comino-Méndez, Iñaki; Gracia-Aznárez, Francisco J; Schiavi, Francesca; Landa, Iñigo; Leandro-García, Luis J; Letón, Rocío; Honrado, Emiliano; Ramos-Medina, Rocío; Caronia, Daniela; Pita, Guillermo; Gómez-Graña, Alvaro; de Cubas, Aguirre A; Inglada-Pérez, Lucía; Maliszewska, Agnieszka; Taschin, Elisa; Bobisse, Sara; Pica, Giuseppe; Loli, Paola; Hernández-Lavado, Rafael; Díaz, José A; Gómez-Morales, Mercedes; González-Neira, Anna; Roncador, Giovanna; Rodríguez-Antona, Cristina; Benítez, Javier; Mannelli, Massimo; Opocher, Giuseppe; Robledo, Mercedes; Cascón, Alberto

2011-06-19

Hereditary pheochromocytoma (PCC) is often caused by germline mutations in one of nine susceptibility genes described to date, but there are familial cases without mutations in these known genes. We sequenced the exomes of three unrelated individuals with hereditary PCC (cases) and identified mutations in MAX, the MYC associated factor X gene. Absence of MAX protein in the tumors and loss of heterozygosity caused by uniparental disomy supported the involvement of MAX alterations in the disease. A follow-up study of a selected series of 59 cases with PCC identified five additional MAX mutations and suggested an association with malignant outcome and preferential paternal transmission of MAX mutations. The involvement of the MYC-MAX-MXD1 network in the development and progression of neural crest cell tumors is further supported by the lack of functional MAX in rat PCC (PC12) cells and by the amplification of MYCN in neuroblastoma and suggests that loss of MAX function is correlated with metastatic potential.

Exome sequencing identifies CTSK mutations in patients originally diagnosed as intermediate osteopetrosis☆

PubMed Central

Pangrazio, Alessandra; Puddu, Alessandro; Oppo, Manuela; Valentini, Maria; Zammataro, Luca; Vellodi, Ashok; Gener, Blanca; Llano-Rivas, Isabel; Raza, Jamal; Atta, Irum; Vezzoni, Paolo; Superti-Furga, Andrea; Villa, Anna; Sobacchi, Cristina

2014-01-01

Autosomal Recessive Osteopetrosis is a genetic disorder characterized by increased bone density due to lack of resorption by the osteoclasts. Genetic studies have widely unraveled the molecular basis of the most severe forms, while cases of intermediate severity are more difficult to characterize, probably because of a large heterogeneity. Here, we describe the use of exome sequencing in the molecular diagnosis of 2 siblings initially thought to be affected by “intermediate osteopetrosis”, which identified a homozygous mutation in the CTSK gene. Prompted by this finding, we tested by Sanger sequencing 25 additional patients addressed to us for recessive osteopetrosis and found CTSK mutations in 4 of them. In retrospect, their clinical and radiographic features were found to be compatible with, but not typical for, Pycnodysostosis. We sought to identify modifier genes that might have played a role in the clinical manifestation of the disease in these patients, but our results were not informative. In conclusion, we underline the difficulties of differential diagnosis in some patients whose clinical appearance does not fit the classical malignant or benign picture and recommend that CTSK gene be included in the molecular diagnosis of high bone density conditions. PMID:24269275

Disease Variant Landscape of a Large Multiethnic Population of Moyamoya Patients by Exome Sequencing

PubMed Central

Shoemaker, Lorelei D.; Clark, Michael J.; Patwardhan, Anil; Chandratillake, Gemma; Garcia, Sarah; Chen, Rong; Morgan, Alexander A.; Leng, Nan; Kirk, Scott; Chen, Richard; Cook, Douglas J.; Snyder, Michael; Steinberg, Gary K.

2015-01-01

Moyamoya disease (MMD) is a rare disorder characterized by cerebrovascular occlusion and development of hemorrhage-prone collateral vessels. Approximately 10–12% of cases are familial, with a presumed low penetrance autosomal dominant pattern of inheritance. Diagnosis commonly occurs only after clinical presentation. The recent identification of the RNF213 founder mutation (p.R4810K) in the Asian population has made a significant contribution, but the etiology of this disease remains unclear. To further develop the variant landscape of MMD, we performed high-depth whole exome sequencing of 125 unrelated, predominantly nonfamilial, ethnically diverse MMD patients in parallel with 125 internally sequenced, matched controls using the same exome and analysis platform. Three subpopulations were established: Asian, Caucasian, and non-RNF213 founder mutation cases. We provided additional support for the previously observed RNF213 founder mutation (p.R4810K) in Asian cases (P = 6.01×10−5) that was enriched among East Asians compared to Southeast Asian and Pacific Islander cases (P = 9.52×10−4) and was absent in all Caucasian cases. The most enriched variant in Caucasian (P = 7.93×10−4) and non-RNF213 founder mutation (P = 1.51×10−3) cases was ZXDC (p.P562L), a gene involved in MHC Class II activation. Collapsing variant methodology ranked OBSCN, a gene involved in myofibrillogenesis, as most enriched in Caucasian (P = 1.07×10−4) and non-RNF213 founder mutation cases (P = 5.31×10−5). These findings further support the East Asian origins of the RNF213 (p.R4810K) variant and more fully describe the genetic landscape of multiethnic MMD, revealing novel, alternative candidate variants and genes that may be important in MMD etiology and diagnosis. PMID:26530418

Portero versus portador: Spanish interpretation of genomic terminology during whole exome sequencing results disclosure.

PubMed

Gutierrez, Amanda M; Robinson, Jill O; Statham, Emily E; Scollon, Sarah; Bergstrom, Katie L; Slashinski, Melody J; Parsons, Donald W; Plon, Sharon E; McGuire, Amy L; Street, Richard L

2017-11-01

Describe modifications to technical genomic terminology made by interpreters during disclosure of whole exome sequencing (WES) results. Using discourse analysis, we identified and categorized interpretations of genomic terminology in 42 disclosure sessions where Spanish-speaking parents received their child's WES results either from a clinician using a medical interpreter, or directly from a bilingual physician. Overall, 76% of genomic terms were interpreted accordantly, 11% were misinterpreted and 13% were omitted. Misinterpretations made by interpreters and bilingual physicians included using literal and nonmedical terminology to interpret genomic concepts. Modifications to genomic terminology made during interpretation highlight the need to standardize bilingual genomic lexicons. We recommend Spanish terms that can be used to refer to genomic concepts.

Extended exome sequencing identifies BACH2 as a novel major risk locus for Addison's disease.

PubMed

Eriksson, D; Bianchi, M; Landegren, N; Nordin, J; Dalin, F; Mathioudaki, A; Eriksson, G N; Hultin-Rosenberg, L; Dahlqvist, J; Zetterqvist, H; Karlsson, Å; Hallgren, Å; Farias, F H G; Murén, E; Ahlgren, K M; Lobell, A; Andersson, G; Tandre, K; Dahlqvist, S R; Söderkvist, P; Rönnblom, L; Hulting, A-L; Wahlberg, J; Ekwall, O; Dahlqvist, P; Meadows, J R S; Bensing, S; Lindblad-Toh, K; Kämpe, O; Pielberg, G R

2016-12-01

Autoimmune disease is one of the leading causes of morbidity and mortality worldwide. In Addison's disease, the adrenal glands are targeted by destructive autoimmunity. Despite being the most common cause of primary adrenal failure, little is known about its aetiology. To understand the genetic background of Addison's disease, we utilized the extensively characterized patients of the Swedish Addison Registry. We developed an extended exome capture array comprising a selected set of 1853 genes and their potential regulatory elements, for the purpose of sequencing 479 patients with Addison's disease and 1394 controls. We identified BACH2 (rs62408233-A, OR = 2.01 (1.71-2.37), P = 1.66 × 10 -15 , MAF 0.46/0.29 in cases/controls) as a novel gene associated with Addison's disease development. We also confirmed the previously known associations with the HLA complex. Whilst BACH2 has been previously reported to associate with organ-specific autoimmune diseases co-inherited with Addison's disease, we have identified BACH2 as a major risk locus in Addison's disease, independent of concomitant autoimmune diseases. Our results may enable future research towards preventive disease treatment. © 2016 The Authors. Journal of Internal Medicine published by John Wiley & Sons Ltd on behalf of Association for Publication of The Journal of Internal Medicine.

Characterization of X Chromosome Inactivation Using Integrated Analysis of Whole-Exome and mRNA Sequencing

PubMed Central

Szelinger, Szabolcs; Malenica, Ivana; Corneveaux, Jason J.; Siniard, Ashley L.; Kurdoglu, Ahmet A.; Ramsey, Keri M.; Schrauwen, Isabelle; Trent, Jeffrey M.; Narayanan, Vinodh; Huentelman, Matthew J.; Craig, David W.

2014-01-01

In females, X chromosome inactivation (XCI) is an epigenetic, gene dosage compensatory mechanism by inactivation of one copy of X in cells. Random XCI of one of the parental chromosomes results in an approximately equal proportion of cells expressing alleles from either the maternally or paternally inherited active X, and is defined by the XCI ratio. Skewed XCI ratio is suggestive of non-random inactivation, which can play an important role in X-linked genetic conditions. Current methods rely on indirect, semi-quantitative DNA methylation-based assay to estimate XCI ratio. Here we report a direct approach to estimate XCI ratio by integrated, family-trio based whole-exome and mRNA sequencing using phase-by-transmission of alleles coupled with allele-specific expression analysis. We applied this method to in silico data and to a clinical patient with mild cognitive impairment but no clear diagnosis or understanding molecular mechanism underlying the phenotype. Simulation showed that phased and unphased heterozygous allele expression can be used to estimate XCI ratio. Segregation analysis of the patient's exome uncovered a de novo, interstitial, 1.7 Mb deletion on Xp22.31 that originated on the paternally inherited X and previously been associated with heterogeneous, neurological phenotype. Phased, allelic expression data suggested an 83∶20 moderately skewed XCI that favored the expression of the maternally inherited, cytogenetically normal X and suggested that the deleterious affect of the de novo event on the paternal copy may be offset by skewed XCI that favors expression of the wild-type X. This study shows the utility of integrated sequencing approach in XCI ratio estimation. PMID:25503791

Rare coding variation in paraoxonase-1 is associated with ischemic stroke in the NHLBI Exome Sequencing Project[S

PubMed Central

Kim, Daniel Seung; Crosslin, David R.; Auer, Paul L.; Suzuki, Stephanie M.; Marsillach, Judit; Burt, Amber A.; Gordon, Adam S.; Meschia, James F.; Nalls, Mike A.; Worrall, Bradford B.; Longstreth, W. T.; Gottesman, Rebecca F.; Furlong, Clement E.; Peters, Ulrike; Rich, Stephen S.; Nickerson, Deborah A.; Jarvik, Gail P.

2014-01-01

HDL-associated paraoxonase-1 (PON1) is an enzyme whose activity is associated with cerebrovascular disease. Common PON1 genetic variants have not been consistently associated with cerebrovascular disease. Rare coding variation that likely alters PON1 enzyme function may be more strongly associated with stroke. The National Heart, Lung, and Blood Institute Exome Sequencing Project sequenced the coding regions (exomes) of the genome for heart, lung, and blood-related phenotypes (including ischemic stroke). In this sample of 4,204 unrelated participants, 496 had verified, noncardioembolic ischemic stroke. After filtering, 28 nonsynonymous PON1 variants were identified. Analysis with the sequence kernel association test, adjusted for covariates, identified significant associations between PON1 variants and ischemic stroke (P = 3.01 × 10−3). Stratified analyses demonstrated a stronger association of PON1 variants with ischemic stroke in African ancestry (AA) participants (P = 5.03 × 10−3). Ethnic differences in the association between PON1 variants with stroke could be due to the effects of PON1Val109Ile (overall P = 7.88 × 10−3; AA P = 6.52 × 10−4), found at higher frequency in AA participants (1.16% vs. 0.02%) and whose protein is less stable than the common allele. In summary, rare genetic variation in PON1 was associated with ischemic stroke, with stronger associations identified in those of AA. Increased focus on PON1 enzyme function and its role in cerebrovascular disease is warranted. PMID:24711634

Sustained therapeutic response to riboflavin in a child with a progressive neurological condition, diagnosed by whole-exome sequencing

PubMed Central

Shashi, Vandana; Petrovski, Slavé; Schoch, Kelly; Crimian, Rebecca; Case, Laura E.; Khalid, Roha; El-Dairi, Maysantoine A.; Jiang, Yong-Hui; Mikati, Mohamad A.; Goldstein, David B.

2015-01-01

One of the most promising outcomes of whole-exome sequencing (WES) is the alteration of medical management following an accurate diagnosis in patients with previously unresolved disorders. Although case reports of targeted therapies resulting from WES have been published, there are few reports with long-term follow-up that confirm a sustained therapeutic response. Following a diagnosis by WES of Brown–Vialetto–Van Laere Syndrome 2 (BVVLS2), high-dose riboflavin therapy was instituted in a 20-mo-old child. An immediate clinical response with stabilization of signs and symptoms was noted over the first 2–4 wk. Subsequent clinical follow-up over the following 8 mo demonstrates not just stabilization, but continuing and sustained improvements in all manifestations of this usually fatal condition, which generally includes worsening motor weakness, sensory ataxia, hearing, and vision impairments. This case emphasizes that early application of WES can transform patient care, enabling therapy that in addition to being lifesaving can sometimes reverse the disabling disease processes in a progressive condition. PMID:27148562

Exome capture from the spruce and pine giga-genomes.

PubMed

Suren, H; Hodgins, K A; Yeaman, S; Nurkowski, K A; Smets, P; Rieseberg, L H; Aitken, S N; Holliday, J A

2016-09-01

Sequence capture is a flexible tool for generating reduced representation libraries, particularly in species with massive genomes. We used an exome capture approach to sequence the gene space of two of the dominant species in Canadian boreal and montane forests - interior spruce (Picea glauca x engelmanii) and lodgepole pine (Pinus contorta). Transcriptome data generated with RNA-seq were coupled with draft genome sequences to design baits corresponding to 26 824 genes from pine and 28 649 genes from spruce. A total of 579 samples for spruce and 631 samples for pine were included, as well as two pine congeners and six spruce congeners. More than 50% of targeted regions were sequenced at >10× depth in each species, while ~12% captured near-target regions within 500 bp of a bait position were sequenced to a depth >10×. Much of our read data arose from off-target regions, which was likely due to the fragmented and incomplete nature of the draft genome assemblies. Capture in general was successful for the related species, suggesting that baits designed for a single species are likely to successfully capture sequences from congeners. From these data, we called approximately 10 million SNPs and INDELs in each species from coding regions, introns, untranslated and flanking regions, as well as from the intergenic space. Our study demonstrates the utility of sequence capture for resequencing in complex conifer genomes, suggests guidelines for improving capture efficiency and provides a rich resource of genetic variants for studies of selection and local adaptation in these species. © 2016 John Wiley & Sons Ltd.

Exome sequence analysis and follow up genotyping implicates rare ULK1 variants to be involved in susceptibility to schizophrenia

PubMed Central

Al Eissa, Mariam M.; Fiorentino, Alessia; Sharp, Sally I.; O'Brien, Niamh L.; Wolfe, Kate; Giaroli, Giovanni; Curtis, David; Bass, Nicholas J.

2017-01-01

Summary Schizophrenia (SCZ) is a severe, highly heritable psychiatric disorder. Elucidation of the genetic architecture of the disorder will facilitate greater understanding of the altered underlying neurobiological mechanisms. The aim of this study was to identify likely aetiological variants in subjects affected with SCZ. Exome sequence data from a SCZ cas–control sample from Sweden was analysed for likely aetiological variants using a weighted burden test. Suggestive evidence implicated the UNC‐51‐like kinase (ULK1) gene, and it was observed that four rare variants that were more common in the Swedish SCZ cases were also more common in UK10K SCZ cases, as compared to obesity cases. These three missense variants and one intronic variant were genotyped in the University College London cohort of 1304 SCZ cases and 1348 ethnically matched controls. All four variants were more common in the SCZ cases than controls and combining them produced a result significant at P = 0.02. The results presented here demonstrate the importance of following up exome sequencing studies using additional datasets. The roles of ULK1 in autophagy and mTOR signalling strengthen the case that these pathways may be important in the pathophysiology of SCZ. The findings reported here await independent replication. PMID:29148569

Molecular Diagnosis of Usher Syndrome: Application of Two Different Next Generation Sequencing-Based Procedures

PubMed Central

Licastro, Danilo; Mutarelli, Margherita; Peluso, Ivana; Neveling, Kornelia; Wieskamp, Nienke; Rispoli, Rossella; Vozzi, Diego; Athanasakis, Emmanouil; D'Eustacchio, Angela; Pizzo, Mariateresa; D'Amico, Francesca; Ziviello, Carmela; Simonelli, Francesca; Fabretto, Antonella; Scheffer, Hans; Gasparini, Paolo; Banfi, Sandro; Nigro, Vincenzo

2012-01-01

Usher syndrome (USH) is a clinically and genetically heterogeneous disorder characterized by visual and hearing impairments. Clinically, it is subdivided into three subclasses with nine genes identified so far. In the present study, we investigated whether the currently available Next Generation Sequencing (NGS) technologies are already suitable for molecular diagnostics of USH. We analyzed a total of 12 patients, most of which were negative for previously described mutations in known USH genes upon primer extension-based microarray genotyping. We enriched the NGS template either by whole exome capture or by Long-PCR of the known USH genes. The main NGS sequencing platforms were used: SOLiD for whole exome sequencing, Illumina (Genome Analyzer II) and Roche 454 (GS FLX) for the Long-PCR sequencing. Long-PCR targeting was more efficient with up to 94% of USH gene regions displaying an overall coverage higher than 25×, whereas whole exome sequencing yielded a similar coverage for only 50% of those regions. Overall this integrated analysis led to the identification of 11 novel sequence variations in USH genes (2 homozygous and 9 heterozygous) out of 18 detected. However, at least two cases were not genetically solved. Our result highlights the current limitations in the diagnostic use of NGS for USH patients. The limit for whole exome sequencing is linked to the need of a strong coverage and to the correct interpretation of sequence variations with a non obvious, pathogenic role, whereas the targeted approach suffers from the high genetic heterogeneity of USH that may be also caused by the presence of additional causative genes yet to be identified. PMID:22952768

Exome copy number variation detection: Use of a pool of unrelated healthy tissue as reference sample.

PubMed

Wenric, Stephane; Sticca, Tiberio; Caberg, Jean-Hubert; Josse, Claire; Fasquelle, Corinne; Herens, Christian; Jamar, Mauricette; Max, Stéphanie; Gothot, André; Caers, Jo; Bours, Vincent

2017-01-01

An increasing number of bioinformatic tools designed to detect CNVs (copy number variants) in tumor samples based on paired exome data where a matched healthy tissue constitutes the reference have been published in the recent years. The idea of using a pool of unrelated healthy DNA as reference has previously been formulated but not thoroughly validated. As of today, the gold standard for CNV calling is still aCGH but there is an increasing interest in detecting CNVs by exome sequencing. We propose to design a metric allowing the comparison of two CNV profiles, independently of the technique used and assessed the validity of using a pool of unrelated healthy DNA instead of a matched healthy tissue as reference in exome-based CNV detection. We compared the CNV profiles obtained with three different approaches (aCGH, exome sequencing with a matched healthy tissue as reference, exome sequencing with a pool of eight unrelated healthy tissue as reference) on three multiple myeloma samples. We show that the usual analyses performed to compare CNV profiles (deletion/amplification ratios and CNV size distribution) lack in precision when confronted with low LRR values, as they only consider the binary status of each CNV. We show that the metric-based distance constitutes a more accurate comparison of two CNV profiles. Based on these analyses, we conclude that a reliable picture of CNV alterations in multiple myeloma samples can be obtained from whole-exome sequencing in the absence of a matched healthy sample. © 2016 WILEY PERIODICALS, INC.

Improving mapping and SNP-calling performance in multiplexed targeted next-generation sequencing

PubMed Central

2012-01-01

Background Compared to classical genotyping, targeted next-generation sequencing (tNGS) can be custom-designed to interrogate entire genomic regions of interest, in order to detect novel as well as known variants. To bring down the per-sample cost, one approach is to pool barcoded NGS libraries before sample enrichment. Still, we lack a complete understanding of how this multiplexed tNGS approach and the varying performance of the ever-evolving analytical tools can affect the quality of variant discovery. Therefore, we evaluated the impact of different software tools and analytical approaches on the discovery of single nucleotide polymorphisms (SNPs) in multiplexed tNGS data. To generate our own test model, we combined a sequence capture method with NGS in three experimental stages of increasing complexity (E. coli genes, multiplexed E. coli, and multiplexed HapMap BRCA1/2 regions). Results We successfully enriched barcoded NGS libraries instead of genomic DNA, achieving reproducible coverage profiles (Pearson correlation coefficients of up to 0.99) across multiplexed samples, with <10% strand bias. However, the SNP calling quality was substantially affected by the choice of tools and mapping strategy. With the aim of reducing computational requirements, we compared conventional whole-genome mapping and SNP-calling with a new faster approach: target-region mapping with subsequent ‘read-backmapping’ to the whole genome to reduce the false detection rate. Consequently, we developed a combined mapping pipeline, which includes standard tools (BWA, SAMtools, etc.), and tested it on public HiSeq2000 exome data from the 1000 Genomes Project. Our pipeline saved 12 hours of run time per Hiseq2000 exome sample and detected ~5% more SNPs than the conventional whole genome approach. This suggests that more potential novel SNPs may be discovered using both approaches than with just the conventional approach. Conclusions We recommend applying our general �

Exome sequencing identifies CTSK mutations in patients originally diagnosed as intermediate osteopetrosis.

PubMed

Pangrazio, Alessandra; Puddu, Alessandro; Oppo, Manuela; Valentini, Maria; Zammataro, Luca; Vellodi, Ashok; Gener, Blanca; Llano-Rivas, Isabel; Raza, Jamal; Atta, Irum; Vezzoni, Paolo; Superti-Furga, Andrea; Villa, Anna; Sobacchi, Cristina

2014-02-01

Autosomal Recessive Osteopetrosis is a genetic disorder characterized by increased bone density due to lack of resorption by the osteoclasts. Genetic studies have widely unraveled the molecular basis of the most severe forms, while cases of intermediate severity are more difficult to characterize, probably because of a large heterogeneity. Here, we describe the use of exome sequencing in the molecular diagnosis of 2 siblings initially thought to be affected by "intermediate osteopetrosis", which identified a homozygous mutation in the CTSK gene. Prompted by this finding, we tested by Sanger sequencing 25 additional patients addressed to us for recessive osteopetrosis and found CTSK mutations in 4 of them. In retrospect, their clinical and radiographic features were found to be compatible with, but not typical for, Pycnodysostosis. We sought to identify modifier genes that might have played a role in the clinical manifestation of the disease in these patients, but our results were not informative. In conclusion, we underline the difficulties of differential diagnosis in some patients whose clinical appearance does not fit the classical malignant or benign picture and recommend that CTSK gene be included in the molecular diagnosis of high bone density conditions. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Extraction of Molecular Features through Exome to Transcriptome Alignment

PubMed Central

Mudvari, Prakriti; Kowsari, Kamran; Cole, Charles; Mazumder, Raja; Horvath, Anelia

2014-01-01

Integrative Next Generation Sequencing (NGS) DNA and RNA analyses have very recently become feasible, and the published to date studies have discovered critical disease implicated pathways, and diagnostic and therapeutic targets. A growing number of exomes, genomes and transcriptomes from the same individual are quickly accumulating, providing unique venues for mechanistic and regulatory features analysis, and, at the same time, requiring new exploration strategies. In this study, we have integrated variation and expression information of four NGS datasets from the same individual: normal and tumor breast exomes and transcriptomes. Focusing on SNPcentered variant allelic prevalence, we illustrate analytical algorithms that can be applied to extract or validate potential regulatory elements, such as expression or growth advantage, imprinting, loss of heterozygosity (LOH), somatic changes, and RNA editing. In addition, we point to some critical elements that might bias the output and recommend alternative measures to maximize the confidence of findings. The need for such strategies is especially recognized within the growing appreciation of the concept of systems biology: integrative exploration of genome and transcriptome features reveal mechanistic and regulatory insights that reach far beyond linear addition of the individual datasets. PMID:24791251

Analysis of exome sequence in 604 trios for recessive genotypes in schizophrenia

PubMed Central

Rees, E; Kirov, G; Walters, J T; Richards, A L; Howrigan, D; Kavanagh, D H; Pocklington, A J; Fromer, M; Ruderfer, D M; Georgieva, L; Carrera, N; Gormley, P; Palta, P; Williams, H; Dwyer, S; Johnson, J S; Roussos, P; Barker, D D; Banks, E; Milanova, V; Rose, S A; Chambert, K; Mahajan, M; Scolnick, E M; Moran, J L; Tsuang, M T; Glatt, S J; Chen, W J; Hwu, H-G; Faraone, Stephen V; Roe, Cheri A; Chandler, Sharon D; Liu, Chih-Min; Liu, Chen-Chung; Yeh, Ling-Ling; Ouyang, Wen-Chen; Chan, Hung-Yu; Chen, Chun-Ying; Neale, B M; Palotie, A; Sklar, P; Purcell, S M; McCarroll, S A; Holmans, P; Owen, M J; O'Donovan, M C

2015-01-01

Genetic associations involving both rare and common alleles have been reported for schizophrenia but there have been no systematic scans for rare recessive genotypes using fully phased trio data. Here, we use exome sequencing in 604 schizophrenia proband–parent trios to investigate the role of recessive (homozygous or compound heterozygous) nonsynonymous genotypes in the disorder. The burden of recessive genotypes was not significantly increased in probands at either a genome-wide level or in any individual gene after adjustment for multiple testing. At a system level, probands had an excess of nonsynonymous compound heterozygous genotypes (minor allele frequency, MAF ⩽1%) in voltage-gated sodium channels (VGSCs; eight in probands and none in parents, P=1.5 × 10−4). Previous findings of multiple de novo loss-of-function mutations in this gene family, particularly SCN2A, in autism and intellectual disability provide biological and genetic plausibility for this finding. Pointing further to the involvement of VGSCs in schizophrenia, we found that these genes were enriched for nonsynonymous mutations (MAF ⩽0.1%) in cases genotyped using an exome array, (5585 schizophrenia cases and 8103 controls), and that in the trios data, synaptic proteins interacting with VGSCs were also enriched for both compound heterozygosity (P=0.018) and de novo mutations (P=0.04). However, we were unable to replicate the specific association with compound heterozygosity at VGSCs in an independent sample of Taiwanese schizophrenia trios (N=614). We conclude that recessive genotypes do not appear to make a substantial contribution to schizophrenia at a genome-wide level. Although multiple lines of evidence, including several from this study, suggest that rare mutations in VGSCs contribute to the disorder, in the absence of replication of the original findings regarding compound heterozygosity, this conclusion requires evaluation in a larger sample of trios. PMID:26196440

RefCNV: Identification of Gene-Based Copy Number Variants Using Whole Exome Sequencing.

PubMed

Chang, Lun-Ching; Das, Biswajit; Lih, Chih-Jian; Si, Han; Camalier, Corinne E; McGregor, Paul M; Polley, Eric

2016-01-01

With rapid advances in DNA sequencing technologies, whole exome sequencing (WES) has become a popular approach for detecting somatic mutations in oncology studies. The initial intent of WES was to characterize single nucleotide variants, but it was observed that the number of sequencing reads that mapped to a genomic region correlated with the DNA copy number variants (CNVs). We propose a method RefCNV that uses a reference set to estimate the distribution of the coverage for each exon. The construction of the reference set includes an evaluation of the sources of variability in the coverage distribution. We observed that the processing steps had an impact on the coverage distribution. For each exon, we compared the observed coverage with the expected normal coverage. Thresholds for determining CNVs were selected to control the false-positive error rate. RefCNV prediction correlated significantly (r = 0.96-0.86) with CNV measured by digital polymerase chain reaction for MET (7q31), EGFR (7p12), or ERBB2 (17q12) in 13 tumor cell lines. The genome-wide CNV analysis showed a good overall correlation (Spearman's coefficient = 0.82) between RefCNV estimation and publicly available CNV data in Cancer Cell Line Encyclopedia. RefCNV also showed better performance than three other CNV estimation methods in genome-wide CNV analysis.

Whole-exome sequencing reveals novel mutations and epigenetic regulation in hypopharyngeal carcinoma

PubMed Central

Wu, Ping; Wu, Honglong; Tang, Yaoyun; Luo, Shi; Fang, Xing; Xie, Chubo; He, Jian; Zhao, Suping; Wang, Xiaofeng; Xu, Jiajia; Chen, Xi; Li, Dongfang; Yang, Huanming; Wang, Jian

2017-01-01

Hypopharyngeal cancer (HPC) frequently presents at an advanced stage, resulting in poor prognosis. Although combined surgical therapy and chemoradiotherapy have improved the survival for patients with HPC over the past 3 decades, the mortality rate in late-stage diagnosis of HPC is unsatisfactory. In this study, we performed whole-exome sequencing (WES) of 23 hypopharyngeal tumor and paired adjacent normal tissue to identify novel candidate driver genes associated with hypopharyngeal carcinoma. We identified several copy number variants (CNVs) and 15 somatic mutation genes that were associated with hypopharyngeal carcinoma. Mutations in nine new genes (PRB4, NSD1, REC8, ZNF772, ZNF69, EI24, CYFIP2, NEFH, KRTAP4-5) were also indentified. PRB4 and NSD1 expression were significantly upregulated in hypopharyngeal carcinoma, which was confirmed in an independent cohort using IHC. There was a positive relationship between PRB4 and NSD1. Downregulation of PRB4 by siRNA could inhibit cell growth, colony formation and cell invasion. Notably, we here demonstrate that NSD1 could bind to the promoter regions of PRB4 and activate promoter activity by reducing the binding of H3K27me2 and increasing the binding of H3K36me2 on PRB4 promoter. In summary, we pinpoint the predominant mutations in hypopharyngeal carcinoma by WES, highlighting the substantial genetic alterations contributing to hypopharyngeal carcinoma tumorigenesis. We also indentify a novel epigenetically regulatory between PRB4 and NSD1 that contribute to hypopharyngeal carcinoma tumorigenesis. They may become potential prognostic biomarkers and therapeutic target for hypopharyngeal carcinoma treatment. PMID:29156722

Whole-Exome Sequencing Identifies Novel Variants for Tooth Agenesis.

PubMed

Dinckan, N; Du, R; Petty, L E; Coban-Akdemir, Z; Jhangiani, S N; Paine, I; Baugh, E H; Erdem, A P; Kayserili, H; Doddapaneni, H; Hu, J; Muzny, D M; Boerwinkle, E; Gibbs, R A; Lupski, J R; Uyguner, Z O; Below, J E; Letra, A

2018-01-01

Tooth agenesis is a common craniofacial abnormality in humans and represents failure to develop 1 or more permanent teeth. Tooth agenesis is complex, and variations in about a dozen genes have been reported as contributing to the etiology. Here, we combined whole-exome sequencing, array-based genotyping, and linkage analysis to identify putative pathogenic variants in candidate disease genes for tooth agenesis in 10 multiplex Turkish families. Novel homozygous and heterozygous variants in LRP6, DKK1, LAMA3, and COL17A1 genes, as well as known variants in WNT10A, were identified as likely pathogenic in isolated tooth agenesis. Novel variants in KREMEN1 were identified as likely pathogenic in 2 families with suspected syndromic tooth agenesis. Variants in more than 1 gene were identified segregating with tooth agenesis in 2 families, suggesting oligogenic inheritance. Structural modeling of missense variants suggests deleterious effects to the encoded proteins. Functional analysis of an indel variant (c.3607+3_6del) in LRP6 suggested that the predicted resulting mRNA is subject to nonsense-mediated decay. Our results support a major role for WNT pathways genes in the etiology of tooth agenesis while revealing new candidate genes. Moreover, oligogenic cosegregation was suggestive for complex inheritance and potentially complex gene product interactions during development, contributing to improved understanding of the genetic etiology of familial tooth agenesis.

Exome sequencing for the differential diagnosis of ciliary chondrodysplasias: Example of a WDR35 mutation case and review of the literature.

PubMed

Antony, Dinu; Nampoory, Narayanan; Bacchelli, Chiara; Melhem, Motasem; Wu, Kaman; James, Chela T; Beales, Philip L; Hubank, Mike; Thomas, Daisy; Mashankar, Anant; Behbehani, Kazem; Schmidts, Miriam; Alsmadi, Osama

2017-12-01

Exome sequencing is becoming widely popular and affordable, making it one of the most desirable methods for the identification of rare genetic variants for clinical diagnosis. Here, we report the clinical application of whole exome sequencing for the ultimate diagnosis of a ciliary chondrodysplasia case presented with an initial clinical diagnosis of Asphyxiating Thoracic Dystrophy (ATD, Jeune Syndrome). We have identified a novel homozygous missense mutation in WDR35 (c.206G > A), a gene previously associated with Sensenbrenner Syndrome, Ellis-van Creveld syndrome and Short-rib polydactyly syndrome type V. The genetic findings in this family led to the re-evaluation of the initial diagnosis and a differential diagnosis of Sensenbrenner Syndrome was made after cautious re-examination of the patient. Cell culture studies revealed normal subcellular localization of the mutant WDR35 protein in comparison to wildtype protein, pointing towards impaired protein-protein interaction and/or altered cell signaling pathways as a consequence of the mutated allele. This research study highlights the importance of including pathogenic variant identification in the diagnosis pipeline of ciliary chondrodysplasias, especially for clinically not fully defined phenotypes. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Whole-Exome Sequencing in Familial Parkinson Disease

PubMed Central

Farlow, Janice L.; Robak, Laurie A.; Hetrick, Kurt; Bowling, Kevin; Boerwinkle, Eric; Coban-Akdemir, Zeynep H.; Gambin, Tomasz; Gibbs, Richard A.; Gu, Shen; Jain, Preti; Jankovic, Joseph; Jhangiani, Shalini; Kaw, Kaveeta; Lai, Dongbing; Lin, Hai; Ling, Hua; Liu, Yunlong; Lupski, James R.; Muzny, Donna; Porter, Paula; Pugh, Elizabeth; White, Janson; Doheny, Kimberly; Myers, Richard M.; Shulman, Joshua M.; Foroud, Tatiana

2016-01-01

IMPORTANCE Parkinson disease (PD) is a progressive neurodegenerative disease for which susceptibility is linked to genetic and environmental risk factors. OBJECTIVE To identify genetic variants contributing to disease risk in familial PD. DESIGN, SETTING, AND PARTICIPANTS A 2-stage study design that included a discovery cohort of families with PD and a replication cohort of familial probands was used. In the discovery cohort, rare exonic variants that segregated in multiple affected individuals in a family and were predicted to be conserved or damaging were retained. Genes with retained variants were prioritized if expressed in the brain and located within PD-relevant pathways. Genes in which prioritized variants were observed in at least 4 families were selected as candidate genes for replication in the replication cohort. The setting was among individuals with familial PD enrolled from academic movement disorder specialty clinics across the United States. All participants had a family history of PD. MAIN OUTCOMES AND MEASURES Identification of genes containing rare, likely deleterious, genetic variants in individuals with familial PD using a 2-stage exome sequencing study design. RESULTS The 93 individuals from 32 families in the discovery cohort (49.5% [46 of 93] female) had a mean (SD) age at onset of 61.8 (10.0) years. The 49 individuals with familial PD in the replication cohort (32.6% [16 of 49] female) had a mean (SD) age at onset of 50.1 (15.7) years. Discovery cohort recruitment dates were 1999 to 2009, and replication cohort recruitment dates were 2003 to 2014. Data analysis dates were 2011 to 2015. Three genes containing a total of 13 rare and potentially damaging variants were prioritized in the discovery cohort. Two of these genes (TNK2 and TNR) also had rare variants that were predicted to be damaging in the replication cohort. All 9 variants identified in the 2 replicated genes in 12 families across the discovery and replication cohorts were

A de novo whole gene deletion of XIAP detected by exome sequencing analysis in very early onset inflammatory bowel disease: a case report.

PubMed

Kelsen, Judith R; Dawany, Noor; Martinez, Alejandro; Martinez, Alejuandro; Grochowski, Christopher M; Maurer, Kelly; Rappaport, Eric; Piccoli, David A; Baldassano, Robert N; Mamula, Petar; Sullivan, Kathleen E; Devoto, Marcella

2015-11-18

Children with very early-onset inflammatory bowel disease (VEO-IBD), those diagnosed at less than 5 years of age, are a unique population. A subset of these patients present with a distinct phenotype and more severe disease than older children and adults. Host genetics is thought to play a more prominent role in this young population, and monogenic defects in genes related to primary immunodeficiencies are responsible for the disease in a small subset of patients with VEO-IBD. We report a child who presented at 3 weeks of life with very early-onset inflammatory bowel disease (VEO-IBD). He had a complicated disease course and remained unresponsive to medical and surgical therapy. The refractory nature of his disease, together with his young age of presentation, prompted utilization of whole exome sequencing (WES) to detect an underlying monogenic primary immunodeficiency and potentially target therapy to the identified defect. Copy number variation analysis (CNV) was performed using the eXome-Hidden Markov Model. Whole exome sequencing revealed 1,380 nonsense and missense variants in the patient. Plausible candidate variants were not detected following analysis of filtered variants, therefore, we performed CNV analysis of the WES data, which led us to identify a de novo whole gene deletion in XIAP. This is the first reported whole gene deletion in XIAP, the causal gene responsible for XLP2 (X-linked lymphoproliferative Disease 2). XLP2 is a syndrome resulting in VEO-IBD and can increase susceptibility to hemophagocytic lymphohistocytosis (HLH). This identification allowed the patient to be referred for bone marrow transplantation, potentially curative for his disease and critical to prevent the catastrophic sequela of HLH. This illustrates the unique etiology of VEO-IBD, and the subsequent effects on therapeutic options. This cohort requires careful and thorough evaluation for monogenic defects and primary immunodeficiencies.

Identification of two novel pathogenic compound heterozygous MYO7A mutations in Usher syndrome by whole exome sequencing.

PubMed

Jia, Ying; Li, Xiaoge; Yang, Dong; Xu, Yi; Guo, Ying; Li, Xin

2018-01-01

The current study aims to identify the pathogenic sites in a core pedigree of Usher syndrome (USH). A core pedigree of USH was analyzed by whole exome sequencing (WES). Mutations were verified by polymerase chain reaction (PCR) amplification and Sanger sequencing. Two pathogenic variations (c.849+2T>C and c.5994G>A) in MYO7A were successfully identified and individually separated from parents. One variant (c.849+2T>C) was nonsense mutation, causing the protein terminated in advance, and the other one (c.5994G>A) located near the boundary of exon could cause aberrant splicing. This study provides a meaningful exploration for identification of clinical core genetic pedigrees. Copyright © 2017 Elsevier B.V. All rights reserved.

Mapping autosomal recessive intellectual disability: combined microarray and exome sequencing identifies 26 novel candidate genes in 192 consanguineous families.

PubMed

Harripaul, R; Vasli, N; Mikhailov, A; Rafiq, M A; Mittal, K; Windpassinger, C; Sheikh, T I; Noor, A; Mahmood, H; Downey, S; Johnson, M; Vleuten, K; Bell, L; Ilyas, M; Khan, F S; Khan, V; Moradi, M; Ayaz, M; Naeem, F; Heidari, A; Ahmed, I; Ghadami, S; Agha, Z; Zeinali, S; Qamar, R; Mozhdehipanah, H; John, P; Mir, A; Ansar, M; French, L; Ayub, M; Vincent, J B

2018-04-01

Approximately 1% of the global population is affected by intellectual disability (ID), and the majority receive no molecular diagnosis. Previous studies have indicated high levels of genetic heterogeneity, with estimates of more than 2500 autosomal ID genes, the majority of which are autosomal recessive (AR). Here, we combined microarray genotyping, homozygosity-by-descent (HBD) mapping, copy number variation (CNV) analysis, and whole exome sequencing (WES) to identify disease genes/mutations in 192 multiplex Pakistani and Iranian consanguineous families with non-syndromic ID. We identified definite or candidate mutations (or CNVs) in 51% of families in 72 different genes, including 26 not previously reported for ARID. The new ARID genes include nine with loss-of-function mutations (ABI2, MAPK8, MPDZ, PIDD1, SLAIN1, TBC1D23, TRAPPC6B, UBA7 and USP44), and missense mutations include the first reports of variants in BDNF or TET1 associated with ID. The genes identified also showed overlap with de novo gene sets for other neuropsychiatric disorders. Transcriptional studies showed prominent expression in the prenatal brain. The high yield of AR mutations for ID indicated that this approach has excellent clinical potential and should inform clinical diagnostics, including clinical whole exome and genome sequencing, for populations in which consanguinity is common. As with other AR disorders, the relevance will also apply to outbred populations.

Genome-Wide Linkage, Exome Sequencing and Functional Analyses Identify ABCB6 as the Pathogenic Gene of Dyschromatosis Universalis Hereditaria

PubMed Central

Wang, Na; Wang, Chuan; Chen, Xuechao; Sheng, Donglai; Fu, Xi’an; See, Kelvin; Foo, Jia Nee; Low, Huiqi; Liany, Herty; Irwan, Ishak Darryl; Liu, Jian; Yang, Baoqi; Chen, Mingfei; Yu, Yongxiang; Yu, Gongqi; Niu, Guiye; You, Jiabao; Zhou, Yan; Ma, Shanshan; Wang, Ting; Yan, Xiaoxiao; Goh, Boon Kee; Common, John E. A.; Lane, Birgitte E.; Sun, Yonghu; Zhou, Guizhi; Lu, Xianmei; Wang, Zhenhua; Tian, Hongqing; Cao, Yuanhua; Chen, Shumin; Liu, Qiji; Liu, Jianjun; Zhang, Furen

2014-01-01

Background As a genetic disorder of abnormal pigmentation, the molecular basis of dyschromatosis universalis hereditaria (DUH) had remained unclear until recently when ABCB6 was reported as a causative gene of DUH. Methodology We performed genome-wide linkage scan using Illumina Human 660W-Quad BeadChip and exome sequencing analyses using Agilent SureSelect Human All Exon Kits in a multiplex Chinese DUH family to identify the pathogenic mutations and verified the candidate mutations using Sanger sequencing. Quantitative RT-PCR and Immunohistochemistry was performed to verify the expression of the pathogenic gene, Zebrafish was also used to confirm the functional role of ABCB6 in melanocytes and pigmentation. Results Genome-wide linkage (assuming autosomal dominant inheritance mode) and exome sequencing analyses identified ABCB6 as the disease candidate gene by discovering a coding mutation (c.1358C>T; p.Ala453Val) that co-segregates with the disease phenotype. Further mutation analysis of ABCB6 in four other DUH families and two sporadic cases by Sanger sequencing confirmed the mutation (c.1358C>T; p.Ala453Val) and discovered a second, co-segregating coding mutation (c.964A>C; p.Ser322Lys) in one of the four families. Both mutations were heterozygous in DUH patients and not present in the 1000 Genome Project and dbSNP database as well as 1,516 unrelated Chinese healthy controls. Expression analysis in human skin and mutagenesis interrogation in zebrafish confirmed the functional role of ABCB6 in melanocytes and pigmentation. Given the involvement of ABCB6 mutations in coloboma, we performed ophthalmological examination of the DUH carriers of ABCB6 mutations and found ocular abnormalities in them. Conclusion Our study has advanced our understanding of DUH pathogenesis and revealed the shared pathological mechanism between pigmentary DUH and ocular coloboma. PMID:24498303

Exome sequencing identifies putative drivers of progression of transient myeloproliferative disorder to AMKL in infants with Down syndrome.

PubMed

Nikolaev, Sergey I; Santoni, Federico; Vannier, Anne; Falconnet, Emilie; Giarin, Emanuela; Basso, Giuseppe; Hoischen, Alexander; Veltman, Joris A; Groet, Jurgen; Nizetic, Dean; Antonarakis, Stylianos E

2013-07-25

Some neonates with Down syndrome (DS) are diagnosed with self-regressing transient myeloproliferative disorder (TMD), and 20% to 30% of those progress to acute megakaryoblastic leukemia (AMKL). We performed exome sequencing in 7 TMD/AMKL cases and copy-number analysis in these and 10 additional cases. All TMD/AMKL samples contained GATA1 mutations. No exome-sequenced TMD/AMKL sample had other recurrently mutated genes. However, 2 of 5 TMD cases, and all AMKL cases, showed mutations/deletions other than GATA1, in genes proven as transformation drivers in non-DS leukemia (EZH2, APC, FLT3, JAK1, PARK2-PACRG, EXT1, DLEC1, and SMC3). One patient at the TMD stage revealed 2 clonal expansions with different GATA1 mutations, of which 1 clone had an additional driver mutation. Interestingly, it was the other clone that gave rise to AMKL after accumulating mutations in 7 other genes. Data suggest that GATA1 mutations alone are sufficient for clonal expansions, and additional driver mutations at the TMD stage do not necessarily predict AMKL progression. Later in infancy, leukemic progression requires "third-hit driver" mutations/somatic copy-number alterations found in non-DS leukemias. Putative driver mutations affecting WNT (wingless-related integration site), JAK-STAT (Janus kinase/signal transducer and activator of transcription), or MAPK/PI3K (mitogen-activated kinase/phosphatidylinositol-3 kinase) pathways were found in all cases, aberrant activation of which converges on overexpression of MYC.

Reference genotype and exome data from an Australian Aboriginal population for health-based research

PubMed Central

Tang, Dave; Anderson, Denise; Francis, Richard W.; Syn, Genevieve; Jamieson, Sarra E.; Lassmann, Timo; Blackwell, Jenefer M.

2016-01-01

Genetic analyses, including genome-wide association studies and whole exome sequencing (WES), provide powerful tools for the analysis of complex and rare genetic diseases. To date there are no reference data for Aboriginal Australians to underpin the translation of health-based genomic research. Here we provide a catalogue of variants called after sequencing the exomes of 72 Aboriginal individuals to a depth of 20X coverage in ∼80% of the sequenced nucleotides. We determined 320,976 single nucleotide variants (SNVs) and 47,313 insertions/deletions using the Genome Analysis Toolkit. We had previously genotyped a subset of the Aboriginal individuals (70/72) using the Illumina Omni2.5 BeadChip platform and found ~99% concordance at overlapping sites, which suggests high quality genotyping. Finally, we compared our SNVs to six publicly available variant databases, such as dbSNP and the Exome Sequencing Project, and 70,115 of our SNVs did not overlap any of the single nucleotide polymorphic sites in all the databases. Our data set provides a useful reference point for genomic studies on Aboriginal Australians. PMID:27070114

Reference genotype and exome data from an Australian Aboriginal population for health-based research.

PubMed

Tang, Dave; Anderson, Denise; Francis, Richard W; Syn, Genevieve; Jamieson, Sarra E; Lassmann, Timo; Blackwell, Jenefer M

2016-04-12

Genetic analyses, including genome-wide association studies and whole exome sequencing (WES), provide powerful tools for the analysis of complex and rare genetic diseases. To date there are no reference data for Aboriginal Australians to underpin the translation of health-based genomic research. Here we provide a catalogue of variants called after sequencing the exomes of 72 Aboriginal individuals to a depth of 20X coverage in ∼80% of the sequenced nucleotides. We determined 320,976 single nucleotide variants (SNVs) and 47,313 insertions/deletions using the Genome Analysis Toolkit. We had previously genotyped a subset of the Aboriginal individuals (70/72) using the Illumina Omni2.5 BeadChip platform and found ~99% concordance at overlapping sites, which suggests high quality genotyping. Finally, we compared our SNVs to six publicly available variant databases, such as dbSNP and the Exome Sequencing Project, and 70,115 of our SNVs did not overlap any of the single nucleotide polymorphic sites in all the databases. Our data set provides a useful reference point for genomic studies on Aboriginal Australians.

New insights into the genetics of glioblastoma multiforme by familial exome sequencing

PubMed Central

Backes, Christina; Harz, Christian; Fischer, Ulrike; Schmitt, Jana; Ludwig, Nicole; Petersen, Britt-Sabina; Mueller, Sabine C.; Kim, Yoo-Jin; Wolf, Nadine M.; Katus, Hugo A.; Meder, Benjamin; Furtwängler, Rhoikos; Franke, Andre; Bohle, Rainer; Henn, Wolfram; Graf, Norbert; Keller, Andreas; Meese, Eckart

2015-01-01

Glioblastoma multiforme (GBM) is the most aggressive and malignant subtype of human brain tumors. While a family clustering of GBM has long been acknowledged, relevant hereditary factors still remained elusive. Exome sequencing of families offers the option to discover respective genetic factors. We sequenced blood samples of one of the rare affected families: while both parents were healthy, both children were diagnosed with GBM. We report 85 homozygous non-synonymous single nucleotide variations (SNVs) in both siblings that were heterozygous in the parents. Beyond known key players for GBM such as ERBB2, PMS2, or CHI3L1, we identified over 50 genes that have not been associated to GBM so far. We also discovered three accumulative effects potentially adding to the tumorigenesis in the siblings: a clustering of multiple variants in single genes (e.g. PTPRB, CROCC), the aggregation of affected genes on specific molecular pathways (e.g. Focal adhesion or ECM receptor interaction) and genomic proximity (e.g. chr22.q12.2, chr1.p36.33). We found a striking accumulation of SNVs in specific genes for the daughter, who developed not only a GBM at the age of 12 years but was subsequently diagnosed with a pilocytic astrocytoma, a common acute lymphatic leukemia and a diffuse pontine glioma. The reported variants underline the relevance of genetic predisposition and cancer development in this family and demonstrate that GBM has a complex and heterogeneous genetic background. Sequencing of other affected families will help to further narrow down the driving genetic causes for this disease. PMID:25537509

Targeted or whole genome sequencing of formalin fixed tissue samples: potential applications in cancer genomics.

PubMed

Munchel, Sarah; Hoang, Yen; Zhao, Yue; Cottrell, Joseph; Klotzle, Brandy; Godwin, Andrew K; Koestler, Devin; Beyerlein, Peter; Fan, Jian-Bing; Bibikova, Marina; Chien, Jeremy

2015-09-22

Current genomic studies are limited by the poor availability of fresh-frozen tissue samples. Although formalin-fixed diagnostic samples are in abundance, they are seldom used in current genomic studies because of the concern of formalin-fixation artifacts. Better characterization of these artifacts will allow the use of archived clinical specimens in translational and clinical research studies. To provide a systematic analysis of formalin-fixation artifacts on Illumina sequencing, we generated 26 DNA sequencing data sets from 13 pairs of matched formalin-fixed paraffin-embedded (FFPE) and fresh-frozen (FF) tissue samples. The results indicate high rate of concordant calls between matched FF/FFPE pairs at reference and variant positions in three commonly used sequencing approaches (whole genome, whole exome, and targeted exon sequencing). Global mismatch rates and C · G > T · A substitutions were comparable between matched FF/FFPE samples, and discordant rates were low (<0.26%) in all samples. Finally, low-pass whole genome sequencing produces similar pattern of copy number alterations between FF/FFPE pairs. The results from our studies suggest the potential use of diagnostic FFPE samples for cancer genomic studies to characterize and catalog variations in cancer genomes.

Whole-exome sequencing links TMCO1 defect syndrome with cerebro-facio-thoracic dysplasia.

PubMed

Pehlivan, Davut; Karaca, Ender; Aydin, Hatip; Beck, Christine R; Gambin, Tomasz; Muzny, Donna M; Bilge Geckinli, B; Karaman, Ali; Jhangiani, Shalini N; Gibbs, Richard A; Lupski, James R

2014-09-01

Whole-exome sequencing (WES) is a type of disruptive technology that has tremendous influence on human and clinical genetics research. An efficient and cost-effective method, WES is now widely used as a diagnostic tool for identifying the molecular basis of genetic syndromes that are often challenging to diagnose. Here we report a patient with a clinical diagnosis of cerebro-facio-thoracic dysplasia (CFTD; MIM#213980) in whom we identified a homozygous splice-site mutation in the transmembrane and coiled-coil domains 1 (TMCO1) gene using WES. TMCO1 mutations cause craniofacial dysmorphism, skeletal anomalies characterized by multiple malformations of the vertebrae and ribs, and intellectual disability (MIM#614132). A retrospective review revealed that clinical manifestations of both syndromes are very similar and overlap remarkably. We propose that mutations of TMCO1 are not only responsible for craniofacial dysmorphism, skeletal anomalies and mental retardation syndrome but also for CFTD.

Exome sequencing identifies recurrent somatic RAC1 mutations in melanoma

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

Krauthammer, Michael; Kong, Yong; Ha, Byung Hak

We characterized the mutational landscape of melanoma, the form of skin cancer with the highest mortality rate, by sequencing the exomes of 147 melanomas. Sun-exposed melanomas had markedly more ultraviolet (UV)-like C>T somatic mutations compared to sun-shielded acral, mucosal and uveal melanomas. Among the newly identified cancer genes was PPP6C, encoding a serine/threonine phosphatase, which harbored mutations that clustered in the active site in 12% of sun-exposed melanomas, exclusively in tumors with mutations in BRAF or NRAS. Notably, we identified a recurrent UV-signature, an activating mutation in RAC1 in 9.2% of sun-exposed melanomas. This activating mutation, the third most frequentmore » in our cohort of sun-exposed melanoma after those of BRAF and NRAS, changes Pro29 to serine (RAC1{sup P29S}) in the highly conserved switch I domain. Crystal structures, and biochemical and functional studies of RAC1{sup P29S} showed that the alteration releases the conformational restraint conferred by the conserved proline, causes an increased binding of the protein to downstream effectors, and promotes melanocyte proliferation and migration. These findings raise the possibility that pharmacological inhibition of downstream effectors of RAC1 signaling could be of therapeutic benefit.« less

SeqMule: automated pipeline for analysis of human exome/genome sequencing data.

PubMed

Guo, Yunfei; Ding, Xiaolei; Shen, Yufeng; Lyon, Gholson J; Wang, Kai

2015-09-18

Next-generation sequencing (NGS) technology has greatly helped us identify disease-contributory variants for Mendelian diseases. However, users are often faced with issues such as software compatibility, complicated configuration, and no access to high-performance computing facility. Discrepancies exist among aligners and variant callers. We developed a computational pipeline, SeqMule, to perform automated variant calling from NGS data on human genomes and exomes. SeqMule integrates computational-cluster-free parallelization capability built on top of the variant callers, and facilitates normalization/intersection of variant calls to generate consensus set with high confidence. SeqMule integrates 5 alignment tools, 5 variant calling algorithms and accepts various combinations all by one-line command, therefore allowing highly flexible yet fully automated variant calling. In a modern machine (2 Intel Xeon X5650 CPUs, 48 GB memory), when fast turn-around is needed, SeqMule generates annotated VCF files in a day from a 30X whole-genome sequencing data set; when more accurate calling is needed, SeqMule generates consensus call set that improves over single callers, as measured by both Mendelian error rate and consistency. SeqMule supports Sun Grid Engine for parallel processing, offers turn-key solution for deployment on Amazon Web Services, allows quality check, Mendelian error check, consistency evaluation, HTML-based reports. SeqMule is available at http://seqmule.openbioinformatics.org.

Gene identification in the congenital disorders of glycosylation type I by whole-exome sequencing.

PubMed

Timal, Sharita; Hoischen, Alexander; Lehle, Ludwig; Adamowicz, Maciej; Huijben, Karin; Sykut-Cegielska, Jolanta; Paprocka, Justyna; Jamroz, Ewa; van Spronsen, Francjan J; Körner, Christian; Gilissen, Christian; Rodenburg, Richard J; Eidhof, Ilse; Van den Heuvel, Lambert; Thiel, Christian; Wevers, Ron A; Morava, Eva; Veltman, Joris; Lefeber, Dirk J

2012-10-01

Congenital disorders of glycosylation type I (CDG-I) form a growing group of recessive neurometabolic diseases. Identification of disease genes is compromised by the enormous heterogeneity in clinical symptoms and the large number of potential genes involved. Until now, gene identification included the sequential application of biochemical methods in blood samples and fibroblasts. In genetically unsolved cases, homozygosity mapping has been applied in consanguineous families. Altogether, this time-consuming diagnostic strategy led to the identification of defects in 17 different CDG-I genes. Here, we applied whole-exome sequencing (WES) in combination with the knowledge of the protein N-glycosylation pathway for gene identification in our remaining group of six unsolved CDG-I patients from unrelated non-consanguineous families. Exome variants were prioritized based on a list of 76 potential CDG-I candidate genes, leading to the rapid identification of one known and two novel CDG-I gene defects. These included the first X-linked CDG-I due to a de novo mutation in ALG13, and compound heterozygous mutations in DPAGT1, together the first two steps in dolichol-PP-glycan assembly, and mutations in PGM1 in two cases, involved in nucleotide sugar biosynthesis. The pathogenicity of the mutations was confirmed by showing the deficient activity of the corresponding enzymes in patient fibroblasts. Combined with these results, the gene defect has been identified in 98% of our CDG-I patients. Our results implicate the potential of WES to unravel disease genes in the CDG-I in newly diagnosed singleton families.

Leveraging the Power of High Performance Computing for Next Generation Sequencing Data Analysis: Tricks and Twists from a High Throughput Exome Workflow

PubMed Central

Wonczak, Stephan; Thiele, Holger; Nieroda, Lech; Jabbari, Kamel; Borowski, Stefan; Sinha, Vishal; Gunia, Wilfried; Lang, Ulrich; Achter, Viktor; Nürnberg, Peter

2015-01-01

Next generation sequencing (NGS) has been a great success and is now a standard method of research in the life sciences. With this technology, dozens of whole genomes or hundreds of exomes can be sequenced in rather short time, producing huge amounts of data. Complex bioinformatics analyses are required to turn these data into scientific findings. In order to run these analyses fast, automated workflows implemented on high performance computers are state of the art. While providing sufficient compute power and storage to meet the NGS data challenge, high performance computing (HPC) systems require special care when utilized for high throughput processing. This is especially true if the HPC system is shared by different users. Here, stability, robustness and maintainability are as important for automated workflows as speed and throughput. To achieve all of these aims, dedicated solutions have to be developed. In this paper, we present the tricks and twists that we utilized in the implementation of our exome data processing workflow. It may serve as a guideline for other high throughput data analysis projects using a similar infrastructure. The code implementing our solutions is provided in the supporting information files. PMID:25942438

Exome-wide association analysis reveals novel coding sequence variants associated with lipid traits in Chinese.

PubMed

Tang, Clara S; Zhang, He; Cheung, Chloe Y Y; Xu, Ming; Ho, Jenny C Y; Zhou, Wei; Cherny, Stacey S; Zhang, Yan; Holmen, Oddgeir; Au, Ka-Wing; Yu, Haiyi; Xu, Lin; Jia, Jia; Porsch, Robert M; Sun, Lijie; Xu, Weixian; Zheng, Huiping; Wong, Lai-Yung; Mu, Yiming; Dou, Jingtao; Fong, Carol H Y; Wang, Shuyu; Hong, Xueyu; Dong, Liguang; Liao, Yanhua; Wang, Jiansong; Lam, Levina S M; Su, Xi; Yan, Hua; Yang, Min-Lee; Chen, Jin; Siu, Chung-Wah; Xie, Gaoqiang; Woo, Yu-Cho; Wu, Yangfeng; Tan, Kathryn C B; Hveem, Kristian; Cheung, Bernard M Y; Zöllner, Sebastian; Xu, Aimin; Eugene Chen, Y; Jiang, Chao Qiang; Zhang, Youyi; Lam, Tai-Hing; Ganesh, Santhi K; Huo, Yong; Sham, Pak C; Lam, Karen S L; Willer, Cristen J; Tse, Hung-Fat; Gao, Wei

2015-12-22

Blood lipids are important risk factors for coronary artery disease (CAD). Here we perform an exome-wide association study by genotyping 12,685 Chinese, using a custom Illumina HumanExome BeadChip, to identify additional loci influencing lipid levels. Single-variant association analysis on 65,671 single nucleotide polymorphisms reveals 19 loci associated with lipids at exome-wide significance (P<2.69 × 10(-7)), including three Asian-specific coding variants in known genes (CETP p.Asp459Gly, PCSK9 p.Arg93Cys and LDLR p.Arg257Trp). Furthermore, missense variants at two novel loci-PNPLA3 p.Ile148Met and PKD1L3 p.Thr429Ser-also influence levels of triglycerides and low-density lipoprotein cholesterol, respectively. Another novel gene, TEAD2, is found to be associated with high-density lipoprotein cholesterol through gene-based association analysis. Most of these newly identified coding variants show suggestive association (P<0.05) with CAD. These findings demonstrate that exome-wide genotyping on samples of non-European ancestry can identify additional population-specific possible causal variants, shedding light on novel lipid biology and CAD.

Whole-Exome Sequencing of Metastatic Cancer and Biomarkers of Treatment Response.

PubMed

Beltran, Himisha; Eng, Kenneth; Mosquera, Juan Miguel; Sigaras, Alexandros; Romanel, Alessandro; Rennert, Hanna; Kossai, Myriam; Pauli, Chantal; Faltas, Bishoy; Fontugne, Jacqueline; Park, Kyung; Banfelder, Jason; Prandi, Davide; Madhukar, Neel; Zhang, Tuo; Padilla, Jessica; Greco, Noah; McNary, Terra J; Herrscher, Erick; Wilkes, David; MacDonald, Theresa Y; Xue, Hui; Vacic, Vladimir; Emde, Anne-Katrin; Oschwald, Dayna; Tan, Adrian Y; Chen, Zhengming; Collins, Colin; Gleave, Martin E; Wang, Yuzhuo; Chakravarty, Dimple; Schiffman, Marc; Kim, Robert; Campagne, Fabien; Robinson, Brian D; Nanus, David M; Tagawa, Scott T; Xiang, Jenny Z; Smogorzewska, Agata; Demichelis, Francesca; Rickman, David S; Sboner, Andrea; Elemento, Olivier; Rubin, Mark A

2015-07-01

Understanding molecular mechanisms of response and resistance to anticancer therapies requires prospective patient follow-up and clinical and functional validation of both common and low-frequency mutations. We describe a whole-exome sequencing (WES) precision medicine trial focused on patients with advanced cancer. To understand how WES data affect therapeutic decision making in patients with advanced cancer and to identify novel biomarkers of response. Patients with metastatic and treatment-resistant cancer were prospectively enrolled at a single academic center for paired metastatic tumor and normal tissue WES during a 19-month period (February 2013 through September 2014). A comprehensive computational pipeline was used to detect point mutations, indels, and copy number alterations. Mutations were categorized as category 1, 2, or 3 on the basis of actionability; clinical reports were generated and discussed in precision tumor board. Patients were observed for 7 to 25 months for correlation of molecular information with clinical response. Feasibility, use of WES for decision making, and identification of novel biomarkers. A total of 154 tumor-normal pairs from 97 patients with a range of metastatic cancers were sequenced, with a mean coverage of 95X and 16 somatic alterations detected per patient. In total, 16 mutations were category 1 (targeted therapy available), 98 were category 2 (biologically relevant), and 1474 were category 3 (unknown significance). Overall, WES provided informative results in 91 cases (94%), including alterations for which there is an approved drug, there are therapies in clinical or preclinical development, or they are considered drivers and potentially actionable (category 1-2); however, treatment was guided in only 5 patients (5%) on the basis of these recommendations because of access to clinical trials and/or off-label use of drugs. Among unexpected findings, a patient with prostate cancer with exceptional response to treatment was

Novel compound heterozygous mutations identified by whole exome sequencing in a Japanese patient with geroderma osteodysplastica.

PubMed

Takeda, Ryojun; Takagi, Masaki; Shinohara, Hiroyuki; Futagawa, Hiroshi; Narumi, Satoshi; Hasegawa, Tomonobu; Nishimura, Gen; Yoshihashi, Hiroshi

2017-12-01

Geroderma osteodysplastica (GO) is a subtype of cutis laxa syndrome characterized by congenital wrinkly skin, a prematurely aged face, extremely short stature, and osteoporosis leading to recurrent fractures. GO exhibits an autosomal recessive inheritance pattern and is caused by loss-of-function mutations in GORAB, which encodes a protein important for Golgi-related transport. Using whole exome sequencing, we identified novel compound heterozygous nonsense mutations in the GORAB in a GO patient. The patient was a 14-year-old Japanese boy. Wrinkled skin and joint laxity were present at birth. At 1 year of age, he was clinically diagnosed with cutis laxa syndrome based on recurrent long bone fractures and clinical features, including wrinkled skin, joint laxity, and a distinctive face. He did not show retarded gross motor and cognitive development. At 11 years of age, he was treated with oral bisphosphonate and vitamin D owing to recurrent multiple spontaneous fractures of the vertebral and extremity bones associated with a low bone mineral density (BMD). Bisphosphonate treatment improved his BMD and fracture rate. Whole exome sequencing revealed two novel compound heterozygous nonsense mutations in the GORAB gene (p.Arg60* and p.Gln124*), and the diagnosis of GO was established. GO is a rare connective tissue disorder. Approximately 60 cases have been described to date, and this is the first report of a patient from Japan. Few studies have reported the effects of bisphosphonate treatment in GO patients with recurrent spontaneous fractures. Based on this case study, we hypothesize that oral bisphosphonate and vitamin D are effective and safe treatment options for the management of recurrent fractures in GO patients. It is important to establish a precise diagnosis of GO to prevent recurrent fractures and optimize treatment plans. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Comparison and integration of deleteriousness prediction methods for nonsynonymous SNVs in whole exome sequencing studies

PubMed Central

Dong, Chengliang; Wei, Peng; Jian, Xueqiu; Gibbs, Richard; Boerwinkle, Eric; Wang, Kai; Liu, Xiaoming

2015-01-01

Accurate deleteriousness prediction for nonsynonymous variants is crucial for distinguishing pathogenic mutations from background polymorphisms in whole exome sequencing (WES) studies. Although many deleteriousness prediction methods have been developed, their prediction results are sometimes inconsistent with each other and their relative merits are still unclear in practical applications. To address these issues, we comprehensively evaluated the predictive performance of 18 current deleteriousness-scoring methods, including 11 function prediction scores (PolyPhen-2, SIFT, MutationTaster, Mutation Assessor, FATHMM, LRT, PANTHER, PhD-SNP, SNAP, SNPs&GO and MutPred), 3 conservation scores (GERP++, SiPhy and PhyloP) and 4 ensemble scores (CADD, PON-P, KGGSeq and CONDEL). We found that FATHMM and KGGSeq had the highest discriminative power among independent scores and ensemble scores, respectively. Moreover, to ensure unbiased performance evaluation of these prediction scores, we manually collected three distinct testing datasets, on which no current prediction scores were tuned. In addition, we developed two new ensemble scores that integrate nine independent scores and allele frequency. Our scores achieved the highest discriminative power compared with all the deleteriousness prediction scores tested and showed low false-positive prediction rate for benign yet rare nonsynonymous variants, which demonstrated the value of combining information from multiple orthologous approaches. Finally, to facilitate variant prioritization in WES studies, we have pre-computed our ensemble scores for 87 347 044 possible variants in the whole-exome and made them publicly available through the ANNOVAR software and the dbNSFP database. PMID:25552646

Whole exome sequencing identifies driver mutations in asymptomatic computed tomography-detected lung cancers with normal karyotype.

PubMed

Belloni, Elena; Veronesi, Giulia; Rotta, Luca; Volorio, Sara; Sardella, Domenico; Bernard, Loris; Pece, Salvatore; Di Fiore, Pier Paolo; Fumagalli, Caterina; Barberis, Massimo; Spaggiari, Lorenzo; Pelicci, Pier Giuseppe; Riva, Laura

2015-04-01

The efficacy of curative surgery for lung cancer could be largely improved by non-invasive screening programs, which can detect the disease at early stages. We previously showed that 18% of screening-identified lung cancers demonstrate a normal karyotype and, following high-density genome scanning, can be subdivided into samples with 1) numerous; 2) none; and 3) few copy number alterations. Whole exome sequencing was applied to the two normal karyotype, screening-detected lung cancers, constituting group 2, as well as normal controls. We identified mutations in both tumors, including KEAP1 (commonly mutated in lung cancers) in one, and TP53, PMS1, and MSH3 (well-characterized DNA-repair genes) in the other. The two normal karyotype screening-detected lung tumors displayed a typical lung cancer mutational profile that only next generation sequencing could reveal, which offered an additional contribution to the over-diagnosis bias concept hypothesized within lung cancer screening programs. Copyright © 2015 Elsevier Inc. All rights reserved.

Best Practices and Joint Calling of the HumanExome BeadChip: The CHARGE Consortium

PubMed Central

Grove, Megan L.; Yu, Bing; Cochran, Barbara J.; Haritunians, Talin; Bis, Joshua C.; Taylor, Kent D.; Hansen, Mark; Borecki, Ingrid B.; Cupples, L. Adrienne; Fornage, Myriam; Gudnason, Vilmundur; Harris, Tamara B.; Kathiresan, Sekar; Kraaij, Robert; Launer, Lenore J.; Levy, Daniel; Liu, Yongmei; Mosley, Thomas; Peloso, Gina M.; Psaty, Bruce M.; Rich, Stephen S.; Rivadeneira, Fernando; Siscovick, David S.; Smith, Albert V.; Uitterlinden, Andre; van Duijn, Cornelia M.; Wilson, James G.; O’Donnell, Christopher J.; Rotter, Jerome I.; Boerwinkle, Eric

2013-01-01

Genotyping arrays are a cost effective approach when typing previously-identified genetic polymorphisms in large numbers of samples. One limitation of genotyping arrays with rare variants (e.g., minor allele frequency [MAF] <0.01) is the difficulty that automated clustering algorithms have to accurately detect and assign genotype calls. Combining intensity data from large numbers of samples may increase the ability to accurately call the genotypes of rare variants. Approximately 62,000 ethnically diverse samples from eleven Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) Consortium cohorts were genotyped with the Illumina HumanExome BeadChip across seven genotyping centers. The raw data files for the samples were assembled into a single project for joint calling. To assess the quality of the joint calling, concordance of genotypes in a subset of individuals having both exome chip and exome sequence data was analyzed. After exclusion of low performing SNPs on the exome chip and non-overlap of SNPs derived from sequence data, genotypes of 185,119 variants (11,356 were monomorphic) were compared in 530 individuals that had whole exome sequence data. A total of 98,113,070 pairs of genotypes were tested and 99.77% were concordant, 0.14% had missing data, and 0.09% were discordant. We report that joint calling allows the ability to accurately genotype rare variation using array technology when large sample sizes are available and best practices are followed. The cluster file from this experiment is available at www.chargeconsortium.com/main/exomechip. PMID:23874508

Increased frequency of de novo copy number variants in congenital heart disease by integrative analysis of single nucleotide polymorphism array and exome sequence data.

PubMed

Glessner, Joseph T; Bick, Alexander G; Ito, Kaoru; Homsy, Jason; Rodriguez-Murillo, Laura; Fromer, Menachem; Mazaika, Erica; Vardarajan, Badri; Italia, Michael; Leipzig, Jeremy; DePalma, Steven R; Golhar, Ryan; Sanders, Stephan J; Yamrom, Boris; Ronemus, Michael; Iossifov, Ivan; Willsey, A Jeremy; State, Matthew W; Kaltman, Jonathan R; White, Peter S; Shen, Yufeng; Warburton, Dorothy; Brueckner, Martina; Seidman, Christine; Goldmuntz, Elizabeth; Gelb, Bruce D; Lifton, Richard; Seidman, Jonathan; Hakonarson, Hakon; Chung, Wendy K

2014-10-24

Congenital heart disease (CHD) is among the most common birth defects. Most cases are of unknown pathogenesis. To determine the contribution of de novo copy number variants (CNVs) in the pathogenesis of sporadic CHD. We studied 538 CHD trios using genome-wide dense single nucleotide polymorphism arrays and whole exome sequencing. Results were experimentally validated using digital droplet polymerase chain reaction. We compared validated CNVs in CHD cases with CNVs in 1301 healthy control trios. The 2 complementary high-resolution technologies identified 63 validated de novo CNVs in 51 CHD cases. A significant increase in CNV burden was observed when comparing CHD trios with healthy trios, using either single nucleotide polymorphism array (P=7×10(-5); odds ratio, 4.6) or whole exome sequencing data (P=6×10(-4); odds ratio, 3.5) and remained after removing 16% of de novo CNV loci previously reported as pathogenic (P=0.02; odds ratio, 2.7). We observed recurrent de novo CNVs on 15q11.2 encompassing CYFIP1, NIPA1, and NIPA2 and single de novo CNVs encompassing DUSP1, JUN, JUP, MED15, MED9, PTPRE SREBF1, TOP2A, and ZEB2, genes that interact with established CHD proteins NKX2-5 and GATA4. Integrating de novo variants in whole exome sequencing and CNV data suggests that ETS1 is the pathogenic gene altered by 11q24.2-q25 deletions in Jacobsen syndrome and that CTBP2 is the pathogenic gene in 10q subtelomeric deletions. We demonstrate a significantly increased frequency of rare de novo CNVs in CHD patients compared with healthy controls and suggest several novel genetic loci for CHD. © 2014 American Heart Association, Inc.

Whole-exome sequencing reveals a rare interferon gamma receptor 1 mutation associated with myasthenia gravis.

PubMed

Qi, Guoyan; Liu, Peng; Gu, Shanshan; Yang, Hongxia; Dong, Huimin; Xue, Yinping

2018-04-01

Our study is aimed to explore the underlying genetic basis of myasthenia gravis. We collected a Chinese pedigree with myasthenia gravis, and whole-exome sequencing was performed on the two affected siblings and their parents. The candidate pathogenic gene was identified by bioinformatics filtering, which was further verified by Sanger sequencing. The homozygous mutation c.G40A (p.V14M) in interferon gamma receptor 1was identified. Moreover, the mutation was also detected in 3 cases of 44 sporadic myasthenia gravis patients. The p.V14M substitution in interferon gamma receptor 1 may affect the signal peptide function and the translocation on cell membrane, which could disrupt the binding of the ligand of interferon gamma and antibody production, contributing to myasthenia gravis susceptibility. We discovered that a rare variant c.G40A in interferon gamma receptor 1 potentially contributes to the myasthenia gravis pathogenesis. Further functional studies are needed to confirm the effect of the interferon gamma receptor 1 on the myasthenia gravis phenotype.

BALSA: integrated secondary analysis for whole-genome and whole-exome sequencing, accelerated by GPU.

PubMed

Luo, Ruibang; Wong, Yiu-Lun; Law, Wai-Chun; Lee, Lap-Kei; Cheung, Jeanno; Liu, Chi-Man; Lam, Tak-Wah

2014-01-01

This paper reports an integrated solution, called BALSA, for the secondary analysis of next generation sequencing data; it exploits the computational power of GPU and an intricate memory management to give a fast and accurate analysis. From raw reads to variants (including SNPs and Indels), BALSA, using just a single computing node with a commodity GPU board, takes 5.5 h to process 50-fold whole genome sequencing (∼750 million 100 bp paired-end reads), or just 25 min for 210-fold whole exome sequencing. BALSA's speed is rooted at its parallel algorithms to effectively exploit a GPU to speed up processes like alignment, realignment and statistical testing. BALSA incorporates a 16-genotype model to support the calling of SNPs and Indels and achieves competitive variant calling accuracy and sensitivity when compared to the ensemble of six popular variant callers. BALSA also supports efficient identification of somatic SNVs and CNVs; experiments showed that BALSA recovers all the previously validated somatic SNVs and CNVs, and it is more sensitive for somatic Indel detection. BALSA outputs variants in VCF format. A pileup-like SNAPSHOT format, while maintaining the same fidelity as BAM in variant calling, enables efficient storage and indexing, and facilitates the App development of downstream analyses. BALSA is available at: http://sourceforge.net/p/balsa.

Molecular Characterization of Colorectal Signet-Ring Cell Carcinoma Using Whole-Exome and RNA Sequencing.

PubMed

Nam, Jae-Yong; Oh, Bo Young; Hong, Hye Kyung; Bae, Joon Seol; Kim, Tae Won; Ha, Sang Yun; Park, Donghyun; Lee, Woo Yong; Kim, Hee Cheol; Yun, Seong Hyeon; Park, Yoon Ah; Joung, Je-Gun; Park, Woong-Yang; Cho, Yong Beom

2018-05-07

Signet-ring cell carcinoma (SRCC) is a very rare subtype of colorectal adenocarcinoma (COAD) with a poor clinical prognosis. Although understanding key mechanisms of tumor progression in SRCCs is critical for precise treatment, a comprehensive view of genomic alterations is lacking. We performed whole-exome sequencing of tumors and matched normal blood as well as RNA sequencing of tumors and matched normal colonic tissues from five patients with SRCC. We identified major somatic alterations and characterized transcriptional changes at the gene and pathway level. Based on high-throughput sequencing, the pattern of mutations and copy number variations was overall similar to that of COAD. Transcriptome analysis revealed that major transcription factors, such as SRF, HNF4A, ZEB1, and RUNX1, with potential regulatory roles in key pathways, including focal adhesion, the PI3K-Akt signaling pathway, and the MAPK signaling pathway, may play a role in the tumorigenesis of SRCC. Furthermore, significantly upregulated genes in SRCCs were enriched for epithelial-mesenchymal transition genes, and accumulation of mucin in intracytoplasm was associated with the overexpression of MUC2. The results indicate that the molecular basis of colorectal SRCC exhibits key differences from that of consensus COAD. Our findings clarify important genetic features of particular abnormalities in SRCCs. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Exome sequencing reveals a de novo POLD1 mutation causing phenotypic variability in mandibular hypoplasia, deafness, progeroid features, and lipodystrophy syndrome (MDPL).

PubMed

Elouej, Sahar; Beleza-Meireles, Ana; Caswell, Richard; Colclough, Kevin; Ellard, Sian; Desvignes, Jean Pierre; Béroud, Christophe; Lévy, Nicolas; Mohammed, Shehla; De Sandre-Giovannoli, Annachiara

2017-06-01

Mandibular hypoplasia, deafness, progeroid features, and lipodystrophy syndrome (MDPL) is an autosomal dominant systemic disorder characterized by prominent loss of subcutaneous fat, a characteristic facial appearance and metabolic abnormalities. This syndrome is caused by heterozygous de novo mutations in the POLD1 gene. To date, 19 patients with MDPL have been reported in the literature and among them 14 patients have been characterized at the molecular level. Twelve unrelated patients carried a recurrent in-frame deletion of a single codon (p.Ser605del) and two other patients carried a novel heterozygous mutation in exon 13 (p.Arg507Cys). Additionally and interestingly, germline mutations of the same gene have been involved in familial polyposis and colorectal cancer (CRC) predisposition. We describe a male and a female patient with MDPL respectively affected with mild and severe phenotypes. Both of them showed mandibular hypoplasia, a beaked nose with bird-like facies, prominent eyes, a small mouth, growth retardation, muscle and skin atrophy, but the female patient showed such a severe and early phenotype that a first working diagnosis of Hutchinson-Gilford Progeria was made. The exploration was performed by direct sequencing of POLD1 gene exon 15 in the male patient with a classical MDPL phenotype and by whole exome sequencing in the female patient and her unaffected parents. Exome sequencing identified in the latter patient a de novo heterozygous undescribed mutation in the POLD1 gene (NM_002691.3: c.3209T>A), predicted to cause the missense change p.Ile1070Asn in the ZnF2 (Zinc Finger 2) domain of the protein. This mutation was not reported in the 1000 Genome Project, dbSNP and Exome sequencing databases. Furthermore, the Isoleucine1070 residue of POLD1 is highly conserved among various species, suggesting that this substitution may cause a major impairment of POLD1 activity. For the second patient, affected with a typical MDPL phenotype, direct sequencing

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

Whole-exome sequencing links TMCO1 defect syndrome with cerebro-facio-thoracic dysplasia

PubMed Central

Pehlivan, Davut; Karaca, Ender; Aydin, Hatip; Beck, Christine R; Gambin, Tomasz; Muzny, Donna M; Bilge Geckinli, B; Karaman, Ali; Jhangiani, Shalini N; Gibbs, Richard A; Lupski, James R

2014-01-01

Whole-exome sequencing (WES) is a type of disruptive technology that has tremendous influence on human and clinical genetics research. An efficient and cost-effective method, WES is now widely used as a diagnostic tool for identifying the molecular basis of genetic syndromes that are often challenging to diagnose. Here we report a patient with a clinical diagnosis of cerebro-facio-thoracic dysplasia (CFTD; MIM#213980) in whom we identified a homozygous splice-site mutation in the transmembrane and coiled-coil domains 1 (TMCO1) gene using WES. TMCO1 mutations cause craniofacial dysmorphism, skeletal anomalies characterized by multiple malformations of the vertebrae and ribs, and intellectual disability (MIM#614132). A retrospective review revealed that clinical manifestations of both syndromes are very similar and overlap remarkably. We propose that mutations of TMCO1 are not only responsible for craniofacial dysmorphism, skeletal anomalies and mental retardation syndrome but also for CFTD. PMID:24424126

Exome Sequencing Establishes Diagnosis of Alström Syndrome in an Infant Presenting with Non-Syndromic Dilated Cardiomyopathy

PubMed Central

Long, Pamela A.; Evans, Jared M.; Olson, Timothy M.

2015-01-01

Idiopathic dilated cardiomyopathy is a heritable, genetically heterogeneous disorder characterized by progressive heart failure. Dilated cardiomyopathy typically exhibits autosomal dominant inheritance, yet frequently remains clinically silent until adulthood. We sought to discover the molecular basis of idiopathic, non-syndromic dilated cardiomyopathy in a one-month-old male presenting with severe heart failure. Previous comprehensive testing of blood, urine, and skin biopsy specimen was negative for metabolic, mitochondrial, storage, and infectious etiologies. Ophthalmologic examination was normal. Chromosomal microarray and commercial dilated cardiomyopathy gene panel testing failed to identify a causative mutation. Parental screening echocardiograms revealed no evidence of clinically silent dilated cardiomyopathy. Whole exome sequencing was carried out on the family trio on a research basis, filtering for rare, deleterious, recessive and de novo genetic variants. Pathogenic compound heterozygous truncating mutations were identified in ALMS1, diagnostic of Alström syndrome and prompting disclosure of genetic findings. Alström syndrome is a known cause for dilated cardiomyopathy in children yet delayed and mis-diagnosis are common owing to its rarity and age-dependent emergence of multisystem clinical manifestations. At six months of age the patient ultimately developed bilateral nystagmus and hyperopia, features characteristic of the syndrome. Early diagnosis is guiding clinical monitoring of other organ systems and allowing for presymptomatic intervention. Furthermore, recognition of recessive inheritance as the mechanism for sporadic disease has informed family planning. This case highlights a limitation of standard gene testing panels for pediatric dilated cardiomyopathy and exemplifies the potential for whole exome sequencing to solve a diagnostic dilemma and enable personalized care. PMID:25706677

Exome Sequence Reveals Mutations in CoA Synthase as a Cause of Neurodegeneration with Brain Iron Accumulation

PubMed Central

Dusi, Sabrina; Valletta, Lorella; Haack, Tobias B.; Tsuchiya, Yugo; Venco, Paola; Pasqualato, Sebastiano; Goffrini, Paola; Tigano, Marco; Demchenko, Nikita; Wieland, Thomas; Schwarzmayr, Thomas; Strom, Tim M.; Invernizzi, Federica; Garavaglia, Barbara; Gregory, Allison; Sanford, Lynn; Hamada, Jeffrey; Bettencourt, Conceição; Houlden, Henry; Chiapparini, Luisa; Zorzi, Giovanna; Kurian, Manju A.; Nardocci, Nardo; Prokisch, Holger; Hayflick, Susan; Gout, Ivan; Tiranti, Valeria

2014-01-01

Neurodegeneration with brain iron accumulation (NBIA) comprises a clinically and genetically heterogeneous group of disorders with progressive extrapyramidal signs and neurological deterioration, characterized by iron accumulation in the basal ganglia. Exome sequencing revealed the presence of recessive missense mutations in COASY, encoding coenzyme A (CoA) synthase in one NBIA-affected subject. A second unrelated individual carrying mutations in COASY was identified by Sanger sequence analysis. CoA synthase is a bifunctional enzyme catalyzing the final steps of CoA biosynthesis by coupling phosphopantetheine with ATP to form dephospho-CoA and its subsequent phosphorylation to generate CoA. We demonstrate alterations in RNA and protein expression levels of CoA synthase, as well as CoA amount, in fibroblasts derived from the two clinical cases and in yeast. This is the second inborn error of coenzyme A biosynthesis to be implicated in NBIA. PMID:24360804

Effectiveness of exome and genome sequencing guided by acuity of illness for diagnosis of neurodevelopmental disorders

PubMed Central

Soden, Sarah E.; Saunders, Carol J.; Willig, Laurel K.; Farrow, Emily G.; Smith, Laurie D.; Petrikin, Josh E.; LePichon, Jean-Baptiste; Miller, Neil A.; Thiffault, Isabelle; Dinwiddie, Darrell L.; Twist, Greyson; Noll, Aaron; Heese, Bryce A.; Zellmer, Lee; Atherton, Andrea M.; Abdelmoity, Ahmed T.; Safina, Nicole; Nyp, Sarah S.; Zuccarelli, Britton; Larson, Ingrid A.; Modrcin, Ann; Herd, Suzanne; Creed, Mitchell; Ye, Zhaohui; Yuan, Xuan; Brodsky, Robert A.; Kingsmore, Stephen F.

2014-01-01

Neurodevelopmental disorders (NDDs) affect more than 3% of children and are attributable to single-gene mutations at more than 1000 loci. Traditional methods yield molecular diagnoses in less than one-half of children with NDD. Whole-genome sequencing (WGS) and whole-exome sequencing (WES) can enable diagnosis of NDD, but their clinical and cost-effectiveness are unknown. One hundred families with 119 children affected by NDD received diagnostic WGS and/or WES of parent-child trios, wherein the sequencing approach was guided by acuity of illness. Forty-five percent received molecular diagnoses. An accelerated sequencing modality, rapid WGS, yielded diagnoses in 73% of families with acutely ill children (11 of 15). Forty percent of families with children with nonacute NDD, followed in ambulatory care clinics (34 of 85), received diagnoses: 33 by WES and 1 by staged WES then WGS. The cost of prior negative tests in the nonacute patients was $19,100 per family, suggesting sequencing to be cost-effective at up to $7640 per family. A change in clinical care or impression of the pathophysiology was reported in 49% of newly diagnosed families. If WES or WGS had been performed at symptom onset, genomic diagnoses may have been made 77 months earlier than occurred in this study. It is suggested that initial diagnostic evaluation of children with NDD should include trio WGS or WES, with extension of accelerated sequencing modalities to high-acuity patients. PMID:25473036

Molecular diagnostic experience of whole-exome sequencing in adult patients.

PubMed

Posey, Jennifer E; Rosenfeld, Jill A; James, Regis A; Bainbridge, Matthew; Niu, Zhiyv; Wang, Xia; Dhar, Shweta; Wiszniewski, Wojciech; Akdemir, Zeynep H C; Gambin, Tomasz; Xia, Fan; Person, Richard E; Walkiewicz, Magdalena; Shaw, Chad A; Sutton, V Reid; Beaudet, Arthur L; Muzny, Donna; Eng, Christine M; Yang, Yaping; Gibbs, Richard A; Lupski, James R; Boerwinkle, Eric; Plon, Sharon E

2016-07-01

Whole-exome sequencing (WES) is increasingly used as a diagnostic tool in medicine, but prior reports focus on predominantly pediatric cohorts with neurologic or developmental disorders. We describe the diagnostic yield and characteristics of WES in adults. We performed a retrospective analysis of consecutive WES reports for adults from a diagnostic laboratory. Phenotype composition was determined using Human Phenotype Ontology terms. Molecular diagnoses were reported for 17.5% (85/486) of adults, which is lower than that for a primarily pediatric population (25.2%; P = 0.0003); the diagnostic rate was higher (23.9%) for those 18-30 years of age compared to patients older than 30 years (10.4%; P = 0.0001). Dual Mendelian diagnoses contributed to 7% of diagnoses, revealing blended phenotypes. Diagnoses were more frequent among individuals with abnormalities of the nervous system, skeletal system, head/neck, and growth. Diagnostic rate was independent of family history information, and de novo mutations contributed to 61.4% of autosomal dominant diagnoses. Early WES experience in adults demonstrates molecular diagnoses in a substantial proportion of patients, informing clinical management, recurrence risk, and recommendations for relatives. A positive family history was not predictive, consistent with molecular diagnoses often revealed by de novo events, informing the Mendelian basis of genetic disease in adults.Genet Med 18 7, 678-685.

Construction of a combinatorial pipeline using two somatic variant  calling  methods  for whole exome sequence data of gastric cancer.

PubMed

Kohmoto, Tomohiro; Masuda, Kiyoshi; Naruto, Takuya; Tange, Shoichiro; Shoda, Katsutoshi; Hamada, Junichi; Saito, Masako; Ichikawa, Daisuke; Tajima, Atsushi; Otsuji, Eigo; Imoto, Issei

2017-01-01

High-throughput next-generation sequencing is a powerful tool to identify the genotypic landscapes of somatic variants and therapeutic targets in various cancers including gastric cancer, forming the basis for personalized medicine in the clinical setting. Although the advent of many computational algorithms leads to higher accuracy in somatic variant calling, no standard method exists due to the limitations of each method. Here, we constructed a new pipeline. We combined two different somatic variant callers with different algorithms, Strelka and VarScan 2, and evaluated performance using whole exome sequencing data obtained from 19 Japanese cases with gastric cancer (GC); then, we characterized these tumors based on identified driver molecular alterations. More single nucleotide variants (SNVs) and small insertions/deletions were detected by Strelka and VarScan 2, respectively. SNVs detected by both tools showed higher accuracy for estimating somatic variants compared with those detected by only one of the two tools and accurately showed the mutation signature and mutations of driver genes reported for GC. Our combinatorial pipeline may have an advantage in detection of somatic mutations in GC and may be useful for further genomic characterization of Japanese patients with GC to improve the efficacy of GC treatments. J. Med. Invest. 64: 233-240, August, 2017.

Effective Immunological Guidance of Genetic Analyses Including Exome Sequencing in Patients Evaluated for Hemophagocytic Lymphohistiocytosis.

PubMed

Ammann, Sandra; Lehmberg, Kai; Zur Stadt, Udo; Klemann, Christian; Bode, Sebastian F N; Speckmann, Carsten; Janka, Gritta; Wustrau, Katharina; Rakhmanov, Mirzokhid; Fuchs, Ilka; Hennies, Hans C; Ehl, Stephan

2017-11-01

We report our experience in using flow cytometry-based immunological screening prospectively as a decision tool for the use of genetic studies in the diagnostic approach to patients with hemophagocytic lymphohistiocytosis (HLH). We restricted genetic analysis largely to patients with abnormal immunological screening, but included whole exome sequencing (WES) for those with normal findings upon Sanger sequencing. Among 290 children with suspected HLH analyzed between 2010 and 2014 (including 17 affected, but asymptomatic siblings), 87/162 patients with "full" HLH and 79/111 patients with "incomplete/atypical" HLH had normal immunological screening results. In 10 patients, degranulation could not be tested. Among the 166 patients with normal screening, genetic analysis was not performed in 107 (all with uneventful follow-up), while 154 single gene tests by Sanger sequencing in the remaining 59 patients only identified a single atypical CHS patient. Flow cytometry correctly predicted all 29 patients with FHL-2, XLP1 or 2. Among 85 patients with defective NK degranulation (including 13 asymptomatic siblings), 70 were Sanger sequenced resulting in a genetic diagnosis in 55 (79%). Eight patients underwent WES, revealing mutations in two known and one unknown cytotoxicity genes and one metabolic disease. FHL3 was the most frequent genetic diagnosis. Immunological screening provided an excellent decision tool for the need and depth of genetic analysis of HLH patients and provided functionally relevant information for rapid patient classification, contributing to a significant reduction in the time from diagnosis to transplantation in recent years.

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.

Improved diagnostic yield compared with targeted gene sequencing panels suggests a role for whole-genome sequencing as a first-tier genetic test

PubMed Central

Lionel, Anath C; Costain, Gregory; Monfared, Nasim; Walker, Susan; Reuter, Miriam S; Hosseini, S Mohsen; Thiruvahindrapuram, Bhooma; Merico, Daniele; Jobling, Rebekah; Nalpathamkalam, Thomas; Pellecchia, Giovanna; Sung, Wilson W L; Wang, Zhuozhi; Bikangaga, Peter; Boelman, Cyrus; Carter, Melissa T; Cordeiro, Dawn; Cytrynbaum, Cheryl; Dell, Sharon D; Dhir, Priya; Dowling, James J; Heon, Elise; Hewson, Stacy; Hiraki, Linda; Inbar-Feigenberg, Michal; Klatt, Regan; Kronick, Jonathan; Laxer, Ronald M; Licht, Christoph; MacDonald, Heather; Mercimek-Andrews, Saadet; Mendoza-Londono, Roberto; Piscione, Tino; Schneider, Rayfel; Schulze, Andreas; Silverman, Earl; Siriwardena, Komudi; Snead, O Carter; Sondheimer, Neal; Sutherland, Joanne; Vincent, Ajoy; Wasserman, Jonathan D; Weksberg, Rosanna; Shuman, Cheryl; Carew, Chris; Szego, Michael J; Hayeems, Robin Z; Basran, Raveen; Stavropoulos, Dimitri J; Ray, Peter N; Bowdin, Sarah; Meyn, M Stephen; Cohn, Ronald D; Scherer, Stephen W; Marshall, Christian R

2018-01-01

Purpose Genetic testing is an integral diagnostic component of pediatric medicine. Standard of care is often a time-consuming stepwise approach involving chromosomal microarray analysis and targeted gene sequencing panels, which can be costly and inconclusive. Whole-genome sequencing (WGS) provides a comprehensive testing platform that has the potential to streamline genetic assessments, but there are limited comparative data to guide its clinical use. Methods We prospectively recruited 103 patients from pediatric non-genetic subspecialty clinics, each with a clinical phenotype suggestive of an underlying genetic disorder, and compared the diagnostic yield and coverage of WGS with those of conventional genetic testing. Results WGS identified diagnostic variants in 41% of individuals, representing a significant increase over conventional testing results (24% P = 0.01). Genes clinically sequenced in the cohort (n = 1,226) were well covered by WGS, with a median exonic coverage of 40 × ±8 × (mean ±SD). All the molecular diagnoses made by conventional methods were captured by WGS. The 18 new diagnoses made with WGS included structural and non-exonic sequence variants not detectable with whole-exome sequencing, and confirmed recent disease associations with the genes PIGG, RNU4ATAC, TRIO, and UNC13A. Conclusion WGS as a primary clinical test provided a higher diagnostic yield than conventional genetic testing in a clinically heterogeneous cohort. PMID:28771251

Improved diagnostic yield compared with targeted gene sequencing panels suggests a role for whole-genome sequencing as a first-tier genetic test.

PubMed

Lionel, Anath C; Costain, Gregory; Monfared, Nasim; Walker, Susan; Reuter, Miriam S; Hosseini, S Mohsen; Thiruvahindrapuram, Bhooma; Merico, Daniele; Jobling, Rebekah; Nalpathamkalam, Thomas; Pellecchia, Giovanna; Sung, Wilson W L; Wang, Zhuozhi; Bikangaga, Peter; Boelman, Cyrus; Carter, Melissa T; Cordeiro, Dawn; Cytrynbaum, Cheryl; Dell, Sharon D; Dhir, Priya; Dowling, James J; Heon, Elise; Hewson, Stacy; Hiraki, Linda; Inbar-Feigenberg, Michal; Klatt, Regan; Kronick, Jonathan; Laxer, Ronald M; Licht, Christoph; MacDonald, Heather; Mercimek-Andrews, Saadet; Mendoza-Londono, Roberto; Piscione, Tino; Schneider, Rayfel; Schulze, Andreas; Silverman, Earl; Siriwardena, Komudi; Snead, O Carter; Sondheimer, Neal; Sutherland, Joanne; Vincent, Ajoy; Wasserman, Jonathan D; Weksberg, Rosanna; Shuman, Cheryl; Carew, Chris; Szego, Michael J; Hayeems, Robin Z; Basran, Raveen; Stavropoulos, Dimitri J; Ray, Peter N; Bowdin, Sarah; Meyn, M Stephen; Cohn, Ronald D; Scherer, Stephen W; Marshall, Christian R

2018-04-01

PurposeGenetic testing is an integral diagnostic component of pediatric medicine. Standard of care is often a time-consuming stepwise approach involving chromosomal microarray analysis and targeted gene sequencing panels, which can be costly and inconclusive. Whole-genome sequencing (WGS) provides a comprehensive testing platform that has the potential to streamline genetic assessments, but there are limited comparative data to guide its clinical use.MethodsWe prospectively recruited 103 patients from pediatric non-genetic subspecialty clinics, each with a clinical phenotype suggestive of an underlying genetic disorder, and compared the diagnostic yield and coverage of WGS with those of conventional genetic testing.ResultsWGS identified diagnostic variants in 41% of individuals, representing a significant increase over conventional testing results (24%; P = 0.01). Genes clinically sequenced in the cohort (n = 1,226) were well covered by WGS, with a median exonic coverage of 40 × ±8 × (mean ±SD). All the molecular diagnoses made by conventional methods were captured by WGS. The 18 new diagnoses made with WGS included structural and non-exonic sequence variants not detectable with whole-exome sequencing, and confirmed recent disease associations with the genes PIGG, RNU4ATAC, TRIO, and UNC13A.ConclusionWGS as a primary clinical test provided a higher diagnostic yield than conventional genetic testing in a clinically heterogeneous cohort.

Exome sequencing identifies a novel mutation of the GDI1 gene in a Chinese non-syndromic X-linked intellectual disability family

PubMed Central

Duan, Yongheng; Lin, Sheng; Xie, Lichun; Zheng, Kaifeng; Chen, Shiguo; Song, Hui; Zeng, Xuchun; Gu, Xueying; Wang, Heyun; Zhang, Linghua; Shao, Hao; Hong, Wenxu; Zhang, Lijie; Duan, Shan

2017-01-01

Abstract X-linked intellectual disability (XLID) has been associated with various genes. Diagnosis of XLID, especially for non-syndromic ones (NS-XLID), is often hampered by the heterogeneity of this disease. Here we report the case of a Chinese family in which three males suffer from intellectual disability (ID). The three patients shared the same phenotype: no typical clinical manifestation other than IQ score ≤ 70. For a genetic diagnosis for this family we carried out whole exome sequencing on the proband, and validated 16 variants of interest in the genomic DNA of all the family members. A missense mutation (c.710G > T), which mapped to exon 6 of the Rab GDP-Dissociation Inhibitor 1 (GDI1) gene, was found segregating with the ID phenotype, and this mutation changes the 237th position in the guanosine diphosphate dissociation inhibitor (GDI) protein from glycine to valine (p. Gly237Val). Through molecular dynamics simulations we found that this substitution results in a conformational change of GDI, possibly affecting the Rab-binding capacity of this protein. In conclusion, our study identified a novel GDI1 mutation that is possibly NS-XLID causative, and showed that whole exome sequencing provides advantages for detecting novel ID-associated variants and can greatly facilitate the genetic diagnosis of the disease. PMID:28863211

Whole exome sequencing and array-based molecular karyotyping as aids to prenatal diagnosis in fetuses with suspected Simpson-Golabi-Behmel syndrome.

PubMed

Kehrer, Christina; Hoischen, Alexander; Menkhaus, Ralf; Schwab, Eva; Müller, Andreas; Kim, Sarah; Kreiß, Martina; Weitensteiner, Valerie; Hilger, Alina; Berg, Christoph; Geipel, Anne; Reutter, Heiko; Gembruch, Ulrich

2016-10-01

Simpson-Golabi-Behmel (SGBS) syndrome type 1 and type 2 represent rare X-linked prenatal overgrowth disorders. The aim of our study is to describe the prenatal sonographic features as well as the genetic work-up. Retrospective analysis of four cases with a pre- or postnatal diagnosis of SGBS in a single tertiary referral center within a period of 4 years. In the study period, four male fetuses with SGBS were detected. The final diagnosis was made prenatally in three cases. In all cases the second trimester anomaly scan revealed left sided congenital diaphragmatic hernia (CDH) with additional anomalies; three fetuses with SGBS type 1 showed fetal overgrowth. In two of these, whole exome sequencing showed a possible frameshift mutation and a point mutation in the gene GPC3, respectively. In the third case, multiplex ligation-dependent probe amplification (MLPA) revealed a hemizygous duplication of exon 3-7 in the gene GPC3. In the fourth case, SGBS type 2 was confirmed by array comparative genomic hybridization (CGH) of amniotic fluid cells showing a deletion of the gene OFD1. We could demonstrate, that in the presence of a CDH, syndromes of the fetus can be increasingly differentiated by detailed sonography followed by a selective and graded molecular diagnostic using microarray techniques and whole exome sequencing. © 2016 John Wiley & Sons, Ltd. © 2016 John Wiley & Sons, Ltd.

Exome sequence reveals mutations in CoA synthase as a cause of neurodegeneration with brain iron accumulation.

PubMed

Dusi, Sabrina; Valletta, Lorella; Haack, Tobias B; Tsuchiya, Yugo; Venco, Paola; Pasqualato, Sebastiano; Goffrini, Paola; Tigano, Marco; Demchenko, Nikita; Wieland, Thomas; Schwarzmayr, Thomas; Strom, Tim M; Invernizzi, Federica; Garavaglia, Barbara; Gregory, Allison; Sanford, Lynn; Hamada, Jeffrey; Bettencourt, Conceição; Houlden, Henry; Chiapparini, Luisa; Zorzi, Giovanna; Kurian, Manju A; Nardocci, Nardo; Prokisch, Holger; Hayflick, Susan; Gout, Ivan; Tiranti, Valeria

2014-01-02

Neurodegeneration with brain iron accumulation (NBIA) comprises a clinically and genetically heterogeneous group of disorders with progressive extrapyramidal signs and neurological deterioration, characterized by iron accumulation in the basal ganglia. Exome sequencing revealed the presence of recessive missense mutations in COASY, encoding coenzyme A (CoA) synthase in one NBIA-affected subject. A second unrelated individual carrying mutations in COASY was identified by Sanger sequence analysis. CoA synthase is a bifunctional enzyme catalyzing the final steps of CoA biosynthesis by coupling phosphopantetheine with ATP to form dephospho-CoA and its subsequent phosphorylation to generate CoA. We demonstrate alterations in RNA and protein expression levels of CoA synthase, as well as CoA amount, in fibroblasts derived from the two clinical cases and in yeast. This is the second inborn error of coenzyme A biosynthesis to be implicated in NBIA. Copyright © 2014 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

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.

Genetic evolution of pancreatic cancer: lessons learnt from the pancreatic cancer genome sequencing project

PubMed Central

Iacobuzio-Donahue, Christine A

2012-01-01

Pancreatic cancer is a disease caused by the accumulation of genetic alterations in specific genes. Elucidation of the human genome sequence, in conjunction with technical advances in the ability to perform whole exome sequencing, have provided new insight into the mutational spectra characteristic of this lethal tumour type. Most recently, exomic sequencing has been used to clarify the clonal evolution of pancreatic cancer as well as provide time estimates of pancreatic carcinogenesis, indicating that a long window of opportunity may exist for early detection of this disease while in the curative stage. Moving forward, these mutational analyses indicate potential targets for personalised diagnostic and therapeutic intervention as well as the optimal timing for intervention based on the natural history of pancreatic carcinogenesis and progression. PMID:21749982

[Identification of novel pathogenic gene mutations in pediatric acute myeloid leukemia by whole-exome resequencing].

PubMed

Shiba, Norio

2015-12-01

A new class of gene mutations, identified in the pathogenesis of adult acute myeloid leukemia (AML), includes DNMT3A, IDH1/2, TET2 and EZH2. However, these mutations are rare in pediatric AML cases, indicating that pathogeneses differ between adult and pediatric forms of AML. Meanwhile, the recent development of massively parallel sequencing technologies has provided a new opportunity to discover genetic changes across entire genomes or proteincoding sequences. In order to reveal a complete registry of gene mutations, we performed whole exome resequencing of paired tumor-normal specimens from 19 pediatric AML cases using Illumina HiSeq 2000. In total, 80 somatic mutations or 4.2 mutations per sample were identified. Many of the recurrent mutations identified in this study involved previously reported targets in AML, such as FLT3, CEBPA, KIT, CBL, NRAS, WT1 and EZH2. On the other hand, several genes were newly identified in the current study, including BCORL1 and major cohesin components such as SMC3 and RAD21. Whole exome resequencing revealed a complex array of gene mutations in pediatric AML genomes. Our results indicate that a subset of pediatric AML represents a discrete entity that could be discriminated from its adult counterpart, in terms of the spectrum of gene mutations.

Identification of novel mutations in endometrial cancer patients by whole-exome sequencing.

PubMed

Chang, Ya-Sian; Huang, Hsien-Da; Yeh, Kun-Tu; Chang, Jan-Gowth

2017-05-01

The aim of the present study was to identify genomic alterations in Taiwanese endometrial cancer patients. This information is vitally important in Taiwan, where endometrial cancer is the second most common gynecological cancer. We performed whole-exome sequencing on DNA from 14 tumor tissue samples from Taiwanese endometrial cancer patients. We used the Genome Analysis Tool kit software package for data analysis, and the dbSNP, Catalogue of Somatic Mutations in Cancer (COSMIC) and The Cancer Genome Atlas (TCGA) databases for comparisons. Variants were validated via Sanger sequencing. We identified 143 non-synonymous mutations in 756 canonical cancer-related genes and 1,271 non-synonymous mutations in non-canonical cancer-related genes in 14 endometrial samples. PTEN, KRAS and PIK3R1 were the most frequently mutated canonical cancer-related genes. Our results revealed nine potential driver genes (MAPT, IL24, MCM6, TSC1, BIRC2, CIITA, DST, CASP8 and NOTCH2) and 21 potential passenger genes (ARMCX4, IGSF10, VPS13C, DCT, DNAH14, TLN1, ZNF605, ZSCAN29, MOCOS, CMYA5, PCDH17, UGT1A8, CYFIP2, MACF1, NUDT5, JAKMIP1, PCDHGB4, FAM178A, SNX6, IMP4 and PCMTD1). The detected molecular aberrations led to putative activation of the mTOR, Wnt, MAPK, VEGF and ErbB pathways, as well as aberrant DNA repair, cell cycle control and apoptosis pathways. We characterized the mutational landscape and genetic alterations in multiple cellular pathways of endometrial cancer in the Taiwanese population.

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.

Exome Sequencing Is an Efficient Tool for Variant Late-Infantile Neuronal Ceroid Lipofuscinosis Molecular Diagnosis

PubMed Central

Ortega-Recalde, Oscar; Nallathambi, Jeyabalan; Anandula, Venkata Ramana; Renukaradhya, Umashankar; Laissue, Paul

2014-01-01

The neuronal ceroid-lipofuscinoses (NCL) is a group of neurodegenerative disorders characterized by epilepsy, visual failure, progressive mental and motor deterioration, myoclonus, dementia and reduced life expectancy. Classically, NCL-affected individuals have been classified into six categories, which have been mainly defined regarding the clinical onset of symptoms. However, some patients cannot be easily included in a specific group because of significant variation in the age of onset and disease progression. Molecular genetics has emerged in recent years as a useful tool for enhancing NCL subtype classification. Fourteen NCL genetic forms (CLN1 to CLN14) have been described to date. The variant late-infantile form of the disease has been linked to CLN5, CLN6, CLN7 (MFSD8) and CLN8 mutations. Despite advances in the diagnosis of neurodegenerative disorders mutations in these genes may cause similar phenotypes, which rends difficult accurate candidate gene selection for direct sequencing. Three siblings who were affected by variant late-infantile NCL are reported in the present study. We used whole-exome sequencing, direct sequencing and in silico approaches to identify the molecular basis of the disease. We identified the novel c.1219T>C (p.Trp407Arg) and c.1361T>C (p.Met454Thr) MFSD8 pathogenic mutations. Our results highlighted next generation sequencing as a novel and powerful methodological approach for the rapid determination of the molecular diagnosis of NCL. They also provide information regarding the phenotypic and molecular spectrum of CLN7 disease. PMID:25333361

Exome sequencing is an efficient tool for variant late-infantile neuronal ceroid lipofuscinosis molecular diagnosis.

PubMed

Patiño, Liliana Catherine; Battu, Rajani; Ortega-Recalde, Oscar; Nallathambi, Jeyabalan; Anandula, Venkata Ramana; Renukaradhya, Umashankar; Laissue, Paul

2014-01-01

The neuronal ceroid-lipofuscinoses (NCL) is a group of neurodegenerative disorders characterized by epilepsy, visual failure, progressive mental and motor deterioration, myoclonus, dementia and reduced life expectancy. Classically, NCL-affected individuals have been classified into six categories, which have been mainly defined regarding the clinical onset of symptoms. However, some patients cannot be easily included in a specific group because of significant variation in the age of onset and disease progression. Molecular genetics has emerged in recent years as a useful tool for enhancing NCL subtype classification. Fourteen NCL genetic forms (CLN1 to CLN14) have been described to date. The variant late-infantile form of the disease has been linked to CLN5, CLN6, CLN7 (MFSD8) and CLN8 mutations. Despite advances in the diagnosis of neurodegenerative disorders mutations in these genes may cause similar phenotypes, which rends difficult accurate candidate gene selection for direct sequencing. Three siblings who were affected by variant late-infantile NCL are reported in the present study. We used whole-exome sequencing, direct sequencing and in silico approaches to identify the molecular basis of the disease. We identified the novel c.1219T>C (p.Trp407Arg) and c.1361T>C (p.Met454Thr) MFSD8 pathogenic mutations. Our results highlighted next generation sequencing as a novel and powerful methodological approach for the rapid determination of the molecular diagnosis of NCL. They also provide information regarding the phenotypic and molecular spectrum of CLN7 disease.

Application of Whole Exome Sequencing in Six Families with an Initial Diagnosis of Autosomal Dominant Retinitis Pigmentosa: Lessons Learned

PubMed Central

Fernandez-San Jose, Patricia; Liu, Yichuan; March, Michael; Pellegrino, Renata; Golhar, Ryan; Corton, Marta; Blanco-Kelly, Fiona; López-Molina, Maria Isabel; García-Sandoval, Blanca; Guo, Yiran; Tian, Lifeng; Liu, Xuanzhu; Guan, Liping; Zhang, Jianguo; Keating, Brendan; Xu, Xun

2015-01-01

This study aimed to identify the genetics underlying dominant forms of inherited retinal dystrophies using whole exome sequencing (WES) in six families extensively screened for known mutations or genes. Thirty-eight individuals were subjected to WES. Causative variants were searched among single nucleotide variants (SNVs) and insertion/deletion variants (indels) and whenever no potential candidate emerged, copy number variant (CNV) analysis was performed. Variants or regions harboring a candidate variant were prioritized and segregation of the variant with the disease was further assessed using Sanger sequencing in case of SNVs and indels, and quantitative PCR (qPCR) for CNVs. SNV and indel analysis led to the identification of a previously reported mutation in PRPH2. Two additional mutations linked to different forms of retinal dystrophies were identified in two families: a known frameshift deletion in RPGR, a gene responsible for X-linked retinitis pigmentosa and p.Ser163Arg in C1QTNF5 associated with Late-Onset Retinal Degeneration. A novel heterozygous deletion spanning the entire region of PRPF31 was also identified in the affected members of a fourth family, which was confirmed with qPCR. This study allowed the identification of the genetic cause of the retinal dystrophy and the establishment of a correct diagnosis in four families, including a large heterozygous deletion in PRPF31, typically considered one of the pitfalls of this method. Since all findings in this study are restricted to known genes, we propose that targeted sequencing using gene-panel is an optimal first approach for the genetic screening and that once known genetic causes are ruled out, WES might be used to uncover new genes involved in inherited retinal dystrophies. PMID:26197217

Whole Genome SNP Genotyping and Exome Sequencing Reveal Novel Genetic Variants and Putative Causative Genes in Congenital Hyperinsulinism

PubMed Central

Proverbio, Maria Carla; Mangano, Eleonora; Gessi, Alessandra; Bordoni, Roberta; Spinelli, Roberta; Asselta, Rosanna; Valin, Paola Sogno; Di Candia, Stefania; Zamproni, Ilaria; Diceglie, Cecilia; Mora, Stefano; Caruso-Nicoletti, Manuela; Salvatoni, Alessandro; De Bellis, Gianluca; Battaglia, Cristina

2013-01-01

Congenital hyperinsulinism of infancy (CHI) is a rare disorder characterized by severe hypoglycemia due to inappropriate insulin secretion. The genetic causes of CHI have been found in genes regulating insulin secretion from pancreatic β-cells; recessive inactivating mutations in the ABCC8 and KCNJ11 genes represent the most common events. Despite the advances in understanding the molecular pathogenesis of CHI, specific genetic determinants in about 50 % of the CHI patients remain unknown, suggesting additional locus heterogeneity. In order to search for novel loci contributing to the pathogenesis of CHI, we combined a family-based association study, using the transmission disequilibrium test on 17 CHI patients lacking mutations in ABCC8/KCNJ11, with a whole-exome sequencing analysis performed on 10 probands. This strategy allowed the identification of the potential causative mutations in genes implicated in the regulation of insulin secretion such as transmembrane proteins (CACNA1A, KCNH6, KCNJ10, NOTCH2, RYR3, SCN8A, TRPV3, TRPC5), cytosolic (ACACB, CAMK2D, CDKAL1, GNAS, NOS2, PDE4C, PIK3R3) and mitochondrial enzymes (PC, SLC24A6), and in four genes (CSMD1, SLC37A3, SULF1, TLL1) suggested by TDT family-based association study. Moreover, the exome-sequencing approach resulted to be an efficient diagnostic tool for CHI, allowing the identification of mutations in three causative CHI genes (ABCC8, GLUD1, and HNF1A) in four out of 10 patients. Overall, the present study should be considered as a starting point to design further investigations: our results might indeed contribute to meta-analysis studies, aimed at the identification/confirmation of novel causative or modifier genes. PMID:23869231

Application of whole-exome sequencing to unravel the molecular basis of undiagnosed syndromic congenital neutropenia with intellectual disability.

PubMed

Gauthier-Vasserot, Alexandra; Thauvin-Robinet, Christel; Bruel, Ange-Line; Duffourd, Yannis; St-Onge, Judith; Jouan, Thibaud; Rivière, Jean-Baptiste; Heron, Delphine; Donadieu, Jean; Bellanné-Chantelot, Christine; Briandet, Claire; Huet, Frédéric; Kuentz, Paul; Lehalle, Daphné; Duplomb-Jego, Laurence; Gautier, Elodie; Maystadt, Isabelle; Pinson, Lucile; Amram, Daniel; El Chehadeh, Salima; Melki, Judith; Julia, Sophia; Faivre, Laurence; Thevenon, Julien

2017-01-01

Neutropenia can be qualified as congenital when of neonatal onset or when associated with extra-hematopoietic manifestations. Overall, 30% of patients with congenital neutropenia (CN) remain without a molecular diagnosis after a multidisciplinary consultation and tedious diagnostic strategy. In the rare situations when neutropenia is identified and associated with intellectual disability (ID), there are few diagnostic hypotheses to test. This retrospective multicenter study reports on a clinically heterogeneous cohort of 10 unrelated patients with CN associated with ID and no molecular diagnosis prior to whole-exome sequencing (WES). WES provided a diagnostic yield of 40% (4/10). The results suggested that in many cases neutropenia and syndromic manifestations could not be assigned to the same molecular alteration. Three sub-groups of patients were highlighted: (i) severe, symptomatic chronic neutropenia, detected early in life, and related to a known mutation in the CN spectrum (ELANE); (ii) mild to moderate benign intermittent neutropenia, detected later, and associated with mutations in genes implicated in neurodevelopmental disorders (CHD2, HUWE1); and (iii) moderate to severe intermittent neutropenia as a probably undiagnosed feature of a newly reported syndrome (KAT6A). Unlike KAT6A, which seems to be associated with a syndromic form of CN, the other reported mutations may not explain the entire clinical picture. Although targeted gene sequencing can be discussed for the primary diagnosis of severe CN, we suggest that performing WES for the diagnosis of disorders associating CN with ID will not only provide the etiological diagnosis but will also pave the way towards personalized care and follow-up. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

SETD2 is recurrently mutated in whole-exome sequenced canine osteosarcoma.

PubMed

Sakthikumar, Sharadha; Elvers, Ingegerd; Kim, Jaegil; Arendt, Maja L; Thomas, Rachael; Turner-Maier, Jason; Swofford, Ross; Johnson, Jeremy; Schumacher, Steven E; Alföldi, Jessica; Axelsson, Erik; Couto, Guillermo; Kisseberth, William; Pettersson, Mats E; Getz, Gad; Meadows, Jennifer R S; Modiano, Jaime F; Breen, Matthew; Kierczak, Marcin; Forsberg-Nilsson, Karin; Marinescu, Voichita D; Lindblad-Toh, Kerstin

2018-05-03

Osteosarcoma (OSA) is a debilitating bone cancer that affects humans, especially children and adolescents. A homologous form of OSA spontaneously occurs in dogs, and its differential incidence observed across breeds allows for the investigation of tumor mutations in the context of multiple genetic backgrounds. Using whole-exome sequencing and dogs from three susceptible breeds (22 golden retrievers, 21 Rottweilers, and 23 greyhounds), we found that OSA tumors show a high frequency of somatic copy number alterations (SCNA) affecting key oncogenes and tumor suppressor genes. The across-breed results are similar to what has been observed for human OSA, but the disease frequency and somatic mutation counts vary in the three breeds. For all breeds, three mutational signatures (one of which has not been previously reported), and eleven significantly mutated genes were identified. TP53 was the most frequently altered gene (83% of dogs have either mutations or SCNA in TP53), recapitulating observations in human OSA. The second most frequently mutated gene, histone methyltransferase SETD2, has known roles in multiple cancers, but has not previously been strongly implicated in OSA. This study points to the likely importance of histone modifications in OSA and highlights the strong genetic similarities between human and dog OSA, suggesting that canine OSA may serve as an excellent model for developing treatment strategies in both species. Copyright ©2018, American Association for Cancer Research.

Next-generation sequencing in schizophrenia and other neuropsychiatric disorders.

PubMed

Schreiber, Matthew; Dorschner, Michael; Tsuang, Debby

2013-10-01

Schizophrenia is a debilitating lifelong illness that lacks a cure and poses a worldwide public health burden. The disease is characterized by a heterogeneous clinical and genetic presentation that complicates research efforts to identify causative genetic variations. This review examines the potential of current findings in schizophrenia and in other related neuropsychiatric disorders for application in next-generation technologies, particularly whole-exome sequencing (WES) and whole-genome sequencing (WGS). These approaches may lead to the discovery of underlying genetic factors for schizophrenia and may thereby identify and target novel therapeutic targets for this devastating disorder. © 2013 Wiley Periodicals, Inc.

Microfluidic droplet enrichment for targeted sequencing

PubMed Central

Eastburn, Dennis J.; Huang, Yong; Pellegrino, Maurizio; Sciambi, Adam; Ptáček, Louis J.; Abate, Adam R.

2015-01-01

Targeted sequence enrichment enables better identification of genetic variation by providing increased sequencing coverage for genomic regions of interest. Here, we report the development of a new target enrichment technology that is highly differentiated from other approaches currently in use. Our method, MESA (Microfluidic droplet Enrichment for Sequence Analysis), isolates genomic DNA fragments in microfluidic droplets and performs TaqMan PCR reactions to identify droplets containing a desired target sequence. The TaqMan positive droplets are subsequently recovered via dielectrophoretic sorting, and the TaqMan amplicons are removed enzymatically prior to sequencing. We demonstrated the utility of this approach by generating an average 31.6-fold sequence enrichment across 250 kb of targeted genomic DNA from five unique genomic loci. Significantly, this enrichment enabled a more comprehensive identification of genetic polymorphisms within the targeted loci. MESA requires low amounts of input DNA, minimal prior locus sequence information and enriches the target region without PCR bias or artifacts. These features make it well suited for the study of genetic variation in a number of research and diagnostic applications. PMID:25873629

Targeted gene panel sequencing in children with very early onset inflammatory bowel disease--evaluation and prospective analysis.

PubMed

Kammermeier, Jochen; Drury, Suzanne; James, Chela T; Dziubak, Robert; Ocaka, Louise; Elawad, Mamoun; Beales, Philip; Lench, Nicholas; Uhlig, Holm H; Bacchelli, Chiara; Shah, Neil

2014-11-01

Multiple monogenetic conditions with partially overlapping phenotypes can present with inflammatory bowel disease (IBD)-like intestinal inflammation. With novel genotype-specific therapies emerging, establishing a molecular diagnosis is becoming increasingly important. We have introduced targeted next-generation sequencing (NGS) technology as a prospective screening tool in children with very early onset IBD (VEOIBD). We evaluated the coverage of 40 VEOIBD genes in two separate cohorts undergoing targeted gene panel sequencing (TGPS) (n=25) and whole exome sequencing (WES) (n=20). TGPS revealed causative mutations in four genes (IL10RA, EPCAM, TTC37 and SKIV2L) discovered unexpected phenotypes and directly influenced clinical decision making by supporting as well as avoiding haematopoietic stem cell transplantation. TGPS resulted in significantly higher median coverage when compared with WES, fewer coverage deficiencies and improved variant detection across established VEOIBD genes. Excluding or confirming known VEOIBD genotypes should be considered early in the disease course in all cases of therapy-refractory VEOIBD, as it can have a direct impact on patient management. To combine both described NGS technologies would compensate for the limitations of WES for disease-specific application while offering the opportunity for novel gene discovery in the research setting. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

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.

Exome Sequencing Identified a Recessive RDH12 Mutation in a Family with Severe Early-Onset Retinitis Pigmentosa

PubMed Central

Gong, Bo; Wei, Bo; Huang, Lulin; Hao, Jilong; Li, Xiulan; Yang, Yin; Zhou, Yu; Hao, Fang; Cui, Zhihua; Zhang, Dingding; Wang, Le

2015-01-01

Retinitis pigmentosa (RP) is the most important hereditary retinal disease caused by progressive degeneration of the photoreceptor cells. This study is to identify gene mutations responsible for autosomal recessive retinitis pigmentosa (arRP) in a Chinese family using next-generation sequencing technology. A Chinese family with 7 members including two individuals affected with severe early-onset RP was studied. All patients underwent a complete ophthalmic examination. Exome sequencing was performed on a single RP patient (the proband of this family) and direct Sanger sequencing on other family members and normal controls was followed to confirm the causal mutations. A homozygous mutation c.437T

Exome sequencing reveals FAM20c mutations associated with fibroblast growth factor 23-related hypophosphatemia, dental anomalies, and ectopic calcification.

PubMed

Rafaelsen, Silje Hjorth; Raeder, Helge; Fagerheim, Anne Kristine; Knappskog, Per; Carpenter, Thomas O; Johansson, Stefan; Bjerknes, Robert

2013-06-01

Fibroblast growth factor 23 (FGF23) plays a crucial role in renal phosphate regulation, exemplified by the causal role of PHEX and DMP1 mutations in X-linked hypophosphatemic rickets and autosomal recessive rickets type 1, respectively. Using whole exome sequencing we identified compound heterozygous mutations in family with sequence similarity 20, member C (FAM20C) in two siblings referred for hypophosphatemia and severe dental demineralization disease. FAM20C mutations were not found in other undiagnosed probands of a national Norwegian population of familial hypophosphatemia. Our results demonstrate that mutations in FAM20C provide a putative new mechanism in human subjects leading to dysregulated FGF23 levels, hypophosphatemia, hyperphosphaturia, dental anomalies, intracerebral calcifications and osteosclerosis of the long bones in the absence of rickets. Copyright © 2013 American Society for Bone and Mineral Research.

New splicing mutation in the choline kinase beta (CHKB) gene causing a muscular dystrophy detected by whole-exome sequencing.

PubMed

Oliveira, Jorge; Negrão, Luís; Fineza, Isabel; Taipa, Ricardo; Melo-Pires, Manuel; Fortuna, Ana Maria; Gonçalves, Ana Rita; Froufe, Hugo; Egas, Conceição; Santos, Rosário; Sousa, Mário

2015-06-01

Muscular dystrophies (MDs) are a group of hereditary muscle disorders that include two particularly heterogeneous subgroups: limb-girdle MD and congenital MD, linked to 52 different genes (seven common to both subgroups). Massive parallel sequencing technology may avoid the usual stepwise gene-by-gene analysis. We report the whole-exome sequencing (WES) analysis of a patient with childhood-onset progressive MD, also presenting mental retardation and dilated cardiomyopathy. Conventional sequencing had excluded eight candidate genes. WES of the trio (patient and parents) was performed using the ion proton sequencing system. Data analysis resorted to filtering steps using the GEMINI software revealed a novel silent variant in the choline kinase beta (CHKB) gene. Inspection of sequence alignments ultimately identified the causal variant (CHKB:c.1031+3G>C). This splice site mutation was confirmed using Sanger sequencing and its effect was further evaluated with gene expression analysis. On reassessment of the muscle biopsy, typical abnormal mitochondrial oxidative changes were observed. Mutations in CHKB have been shown to cause phosphatidylcholine deficiency in myofibers, causing a rare form of CMD (only 21 patients reported). Notwithstanding interpretative difficulties that need to be overcome before the integration of WES in the diagnostic workflow, this work corroborates its utility in solving cases from highly heterogeneous groups of diseases, in which conventional diagnostic approaches fail to provide a definitive diagnosis.

Novel inborn error of folate metabolism: identification by exome capture and sequencing of mutations in the MTHFD1 gene in a single proband.

PubMed

Watkins, David; Schwartzentruber, Jeremy A; Ganesh, Jaya; Orange, Jordan S; Kaplan, Bernard S; Nunez, Laura Dempsey; Majewski, Jacek; Rosenblatt, David S

2011-09-01

An infant was investigated because of megaloblastic anaemia, atypical hemolytic uraemic syndrome, severe combined immune deficiency, elevated blood levels of homocysteine and methylmalonic acid, and a selective decreased synthesis of methylcobalamin in cultured fibroblasts. Exome sequencing was performed on patient genomic DNA. Two mutations were identified in the MTHFD1 gene, which encodes a protein that catalyses three reactions involved in cellular folate metabolism. This protein is essential for the generation of formyltetrahydrofolate and methylenetetrahydrofolate and important for nucleotide and homocysteine metabolism. One mutation (c.727+1G>A) affects the splice acceptor site of intron 8. The second mutation, c.517C>T (p.R173C), changes a critical arginine residue in the NADP-binding site of the protein. Mutations affecting this arginine have previously been shown to affect enzyme activity. Both parents carry a single mutation and an unaffected sibling carries neither mutation. The combination of two mutations in the MTHFRD1 gene, predicted to have severe consequences, in the patient and their absence in the unaffected sibling, supports causality. This patient represents the first case of an inborn error of folate metabolism affecting the trifunctional MTHFD1 protein. This report reinforces the power of exome capture and sequencing for the discovery of novel genes, even when only a single proband is available for study.

Debunking Occam's razor: Diagnosing multiple genetic diseases in families by whole-exome sequencing.

PubMed

Balci, T B; Hartley, T; Xi, Y; Dyment, D A; Beaulieu, C L; Bernier, F P; Dupuis, L; Horvath, G A; Mendoza-Londono, R; Prasad, C; Richer, J; Yang, X-R; Armour, C M; Bareke, E; Fernandez, B A; McMillan, H J; Lamont, R E; Majewski, J; Parboosingh, J S; Prasad, A N; Rupar, C A; Schwartzentruber, J; Smith, A C; Tétreault, M; Innes, A M; Boycott, K M

2017-09-01

Recent clinical whole exome sequencing (WES) cohorts have identified unanticipated multiple genetic diagnoses in single patients. However, the frequency of multiple genetic diagnoses in families is largely unknown. We set out to identify the rate of multiple genetic diagnoses in probands and their families referred for analysis in two national research programs in Canada. We retrospectively analyzed WES results for 802 undiagnosed probands referred over the past 5 years in either the FORGE or Care4Rare Canada WES initiatives. Of the 802 probands, 226 (28.2%) were diagnosed based on mutations in known disease genes. Eight (3.5%) had two or more genetic diagnoses explaining their clinical phenotype, a rate in keeping with the large published studies (average 4.3%; 1.4 - 7.2%). Seven of the 8 probands had family members with one or more of the molecularly diagnosed diseases. Consanguinity and multisystem disease appeared to increase the likelihood of multiple genetic diagnoses in a family. Our findings highlight the importance of comprehensive clinical phenotyping of family members to ultimately provide accurate genetic counseling. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Novel compound heterozygous mutations in the OTOF Gene identified by whole-exome sequencing in auditory neuropathy spectrum disorder.

PubMed

Tang, Fengzhu; Ma, Dengke; Wang, Yulan; Qiu, Yuecai; Liu, Fei; Wang, Qingqing; Lu, Qiutian; Shi, Min; Xu, Liang; Liu, Min; Liang, Jianping

2017-03-23

Many hearing-loss diseases are demonstrated to have Mendelian inheritance caused by mutations in single gene. However, many deaf individuals have diseases that remain genetically unexplained. Auditory neuropathy is a sensorineural deafness in which sounds are able to be transferred into the inner ear normally but the transmission of the signals from inner ear to auditory nerve and brain is injured, also known as auditory neuropathy spectrum disorder (ANSD). The pathogenic mutations of the genes responsible for the Chinese ANSD population remain poorly understood. A total of 127 patients with non-syndromic hearing loss (NSHL) were enrolled in Guangxi Zhuang Autonomous Region. A hereditary deafness gene mutation screening was performed to identify the mutation sites in four deafness-related genes (GJB2, GJB3, 12S rRNA, and SLC26A4). In addition, whole-exome sequencing (WES) was applied to explore unappreciated mutation sites in the cases with the singularity of its phenotype. Well-characterized mutations were found in only 8.7% (11/127) of the patients. Interestingly, two mutations in the OTOF gene were identified in two affected siblings with ANSD from a Chinese family, including one nonsense mutation c.1273C > T (p.R425X) and one missense mutation c.4994 T > C (p.L1665P). Furthermore, we employed Sanger sequencing to confirm the mutations in each subject. Two compound heterozygous mutations in the OTOF gene were observed in the two affected siblings, whereas the two parents and unaffected sister were heterozygous carriers of c.1273C > T (father and sister) and c.4994 T > C (mother). The nonsense mutation p.R425X, contributes to a premature stop codon, may result in a truncated polypeptide, which strongly suggests its pathogenicity for ANSD. The missense mutation p.L1665P results in a single amino acid substitution in a highly conserved region. Two mutations in the OTOF gene in the Chinese deaf population were recognized for the first time. These

Whole exome sequencing identifies a mutation for a novel form of corneal intraepithelial dyskeratosis

PubMed Central

Soler, Vincent José; Tran-Viet, Khanh-Nhat; Galiacy, Stéphane D; Limviphuvadh, Vachiranee; Klemm, Thomas Patrick; St Germain, Elizabeth; Fournié, Pierre R; Guillaud, Céline; Maurer-Stroh, Sebastian; Hawthorne, Felicia; Suarez, Cyrielle; Kantelip, Bernadette; Afshari, Natalie A; Creveaux, Isabelle; Luo, Xiaoyan; Meng, Weihua; Calvas, Patrick; Cassagne, Myriam; Arné, Jean-Louis; Rozen, Steven G; Malecaze, François; Young, Terri L

2014-01-01

Background Corneal intraepithelial dyskeratosis is an extremely rare condition. The classical form, affecting Native American Haliwa-Saponi tribe members, is called hereditary benign intraepithelial dyskeratosis (HBID). Herein, we present a new form of corneal intraepithelial dyskeratosis for which we identified the causative gene by using deep sequencing technology. Methods and results A seven member Caucasian French family with two corneal intraepithelial dyskeratosis affected individuals (6-year-old proband and his mother) was ascertained. The proband presented with bilateral complete corneal opacification and dyskeratosis. Palmoplantar hyperkeratosis and laryngeal dyskeratosis were associated with the phenotype. Histopathology studies of cornea and vocal cord biopsies showed dyskeratotic keratinisation. Quantitative PCR ruled out 4q35 duplication, classically described in HBID cases. Next generation sequencing with mean coverage of 50× using the Illumina Hi Seq and whole exome capture processing was performed. Sequence reads were aligned, and screened for single nucleotide variants and insertion/deletion calls. In-house pipeline filtering analyses and comparisons with available databases were performed. A novel missense mutation M77T was discovered for the gene NLRP1 which maps to chromosome 17p13.2. This was a de novo mutation in the proband’s mother, following segregation in the family, and not found in 738 control DNA samples. NLRP1 expression was determined in adult corneal epithelium. The amino acid change was found to destabilise significantly the protein structure. Conclusions We describe a new corneal intraepithelial dyskeratosis and how we identified its causative gene. The NLRP1 gene product is implicated in inflammation, autoimmune disorders, and caspase mediated apoptosis. NLRP1 polymorphisms are associated with various diseases. PMID:23349227

Dynamical System Modeling to Simulate Donor T Cell Response to Whole Exome Sequencing-Derived Recipient Peptides Demonstrates Different Alloreactivity Potential in HLA-Matched and -Mismatched Donor-Recipient Pairs.

PubMed

Abdul Razzaq, Badar; Scalora, Allison; Koparde, Vishal N; Meier, Jeremy; Mahmood, Musa; Salman, Salman; Jameson-Lee, Max; Serrano, Myrna G; Sheth, Nihar; Voelkner, Mark; Kobulnicky, David J; Roberts, Catherine H; Ferreira-Gonzalez, Andrea; Manjili, Masoud H; Buck, Gregory A; Neale, Michael C; Toor, Amir A

2016-05-01

Immune reconstitution kinetics and subsequent clinical outcomes in HLA-matched recipients of allogeneic stem cell transplantation (SCT) are variable and difficult to predict. Considering SCT as a dynamical system may allow sequence differences across the exomes of the transplant donors and recipients to be used to simulate an alloreactive T cell response, which may allow better clinical outcome prediction. To accomplish this, whole exome sequencing was performed on 34 HLA-matched SCT donor-recipient pairs (DRPs) and the nucleotide sequence differences translated to peptides. The binding affinity of the peptides to the relevant HLA in each DRP was determined. The resulting array of peptide-HLA binding affinity values in each patient was considered as an operator modifying a hypothetical T cell repertoire vector, in which each T cell clone proliferates in accordance with the logistic equation of growth. Using an iterating system of matrices, each simulated T cell clone's growth was calculated with the steady-state population being proportional to the magnitude of the binding affinity of the driving HLA-peptide complex. Incorporating competition between T cell clones responding to different HLA-peptide complexes reproduces a number of features of clinically observed T cell clonal repertoire in the simulated repertoire, including sigmoidal growth kinetics of individual T cell clones and overall repertoire, Power Law clonal frequency distribution, increase in repertoire complexity over time with increasing clonal diversity, and alteration of clonal dominance when a different antigen array is encountered, such as in SCT. The simulated, alloreactive T cell repertoire was markedly different in HLA-matched DRPs. The patterns were differentiated by rate of growth and steady-state magnitude of the simulated T cell repertoire and demonstrate a possible correlation with survival. In conclusion, exome wide sequence differences in DRPs may allow simulation of donor alloreactive T

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

Clinical characterization and diagnosis of cystic fibrosis through exome sequencing in Chinese infants with Bartter-syndrome-like hypokalemia alkalosis.

PubMed

Qiu, Liru; Yang, Fengjie; He, Yonghua; Yuan, Huiqing; Zhou, Jianhua

2018-03-09

Cystic fibrosis (CF) is a fatal autosomal-recessive disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. CF is characterized by recurrent pulmonary infection with obstructive pulmonary disease. CF is common in the Caucasian population but is rare in the Chinese population. The symptoms of early-stage CF are often untypical and may sometimes manifest as Bartter syndrome (BS)-like hypokalemic alkalosis. Therefore, the ability of doctors to differentiate CF from BS-like hypokalemic alkalosis in Chinese infants is a great challenge in the timely and accurate diagnosis of CF. In China, sporadic CF has not been diagnosed in children younger than three years of age to date. Three infants, who were initially admitted to our hospital over the period of June 2013 to September 2014 with BS-like hypokalemic alkalosis, were diagnosed with CF through exome sequencing and sweat chloride measurement. The compound heterozygous mutations of the CFTR gene were detected in two infants, and a homozygous missense mutation was found in one infant. Among the six identified mutations, two are novel point mutations (c.1526G > C and c.3062C > T) that are possibly pathogenic. The three infants are the youngest Chinese patients to have been diagnosed with sporadic CF at a very early stage. Follow-up examination showed that all of the cases remained symptom-free after early intervention, indicating the potential benefit of very early diagnosis and timely intervention in children with CF. Our results demonstrate the necessity of distinguishing CF from BS in Chinese infants with hypokalemic alkalosis and the significant diagnostic value of powerful exome sequencing for rare genetic diseases. Furthermore, our findings expand the CFTR mutation spectrum associated with CF.

Whole Exome Sequencing identifies a splicing mutation in NSUN2 as a cause of a Dubowitz-like syndrome

PubMed Central

Martinez, Fernando; Lee, Jeong Ho; Lee, Ji Eun; Blanco, Sandra; Nickerson, Elizabeth; Gabriel, Stacey; Frye, Michaela; Al-Gazali, Lihadh; Gleeson, Joseph G.

2016-01-01

Dubowitz Syndrome is an autosomal recessive disorder characterized by the constellation of mild microcephaly, growth and mental retardation, eczema and peculiar facies, but causes are still unknown. We studied a multiplex consanguineous family with many features of Dubowitz syndrome using whole exome sequencing and identified a splice mutation in NSUN2, encoding a conserved RNA methyltransferase. NSUN2 has been implicated in Myc-induced cell proliferation and mitotic spindle stability, which might help explain the varied clinical presentations that can include chromosomal instability and immunological defects. Patient cells displayed loss of NSUN2-specific methylation at two residues of the aspartate tRNA. Our findings establish NSUN2 as the first causal gene with relationship to the Dubowitz syndrome spectrum phenotype. PMID:22577224

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

Homozygosity Mapping and Whole Exome Sequencing to Detect SLC45A2 and G6PC3 Mutations in a Single Patient with Oculocutaneous Albinism and Neutropenia

PubMed Central

Cullinane, Andrew R.; Vilboux, Thierry; O’Brien, Kevin; Curry, James A.; Maynard, Dawn M.; Carlson-Donohoe, Hannah; Ciccone, Carla; Markello, Thomas C.; Gunay-Aygun, Meral; Huizing, Marjan; Gahl, William A.

2011-01-01

We evaluated a 32 year-old woman whose oculocutaneous albinism, bleeding diathesis, neutropenia, and history of recurrent infections prompted consideration of the diagnosis of Hermansky-Pudlak syndrome type 2 (HPS-2). This was ruled out due to the presence of platelet delta granules and absence of AP3B1 mutations. Since parental consanguinity suggested an autosomal recessive mode of inheritance, we employed homozygosity mapping, followed by whole exome sequencing, to identify two candidate disease-causing genes, SLC45A2 and G6PC3. Conventional di-deoxy sequencing confirmed pathogenic mutations in SLC45A2, associated with oculocutaneous albinism type 4 (OCA-4), and G6PC3, associated with neutropenia. The substantial reduction of SLC45A2 protein in the patient’s melanocytes caused the mis-localization of tyrosinase from melanosomes to the plasma membrane and also led to the incorporation of tyrosinase into exosomes and secretion into the culture medium, explaining the hypopigmentation in OCA-4. Our patient’s G6PC3 mRNA expression level was also reduced, leading to increased apoptosis of her fibroblasts under ER stress. This report describes the first North American patient with OCA-4, the first culture of human OCA-4 melanocytes, and the use of homozygosity mapping followed by whole exome sequencing to identify disease-causing mutations in multiple genes in a single affected individual. PMID:21677667

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.

Whole-genome and multisector exome sequencing of primary and post-treatment glioblastoma reveals patterns of tumor evolution

PubMed Central

Kim, Hoon; Zheng, Siyuan; Amini, Seyed S.; Virk, Selene M.; Mikkelsen, Tom; Brat, Daniel J.; Grimsby, Jonna; Sougnez, Carrie; Muller, Florian; Hu, Jian; Sloan, Andrew E.; Cohen, Mark L.; Van Meir, Erwin G.; Scarpace, Lisa; Laird, Peter W.; Weinstein, John N.; Lander, Eric S.; Gabriel, Stacey; Getz, Gad; Meyerson, Matthew; Chin, Lynda; Barnholtz-Sloan, Jill S.

2015-01-01

Glioblastoma (GBM) is a prototypical heterogeneous brain tumor refractory to conventional therapy. A small residual population of cells escapes surgery and chemoradiation, resulting in a typically fatal tumor recurrence ∼7 mo after diagnosis. Understanding the molecular architecture of this residual population is critical for the development of successful therapies. We used whole-genome sequencing and whole-exome sequencing of multiple sectors from primary and paired recurrent GBM tumors to reconstruct the genomic profile of residual, therapy resistant tumor initiating cells. We found that genetic alteration of the p53 pathway is a primary molecular event predictive of a high number of subclonal mutations in glioblastoma. The genomic road leading to recurrence is highly idiosyncratic but can be broadly classified into linear recurrences that share extensive genetic similarity with the primary tumor and can be directly traced to one of its specific sectors, and divergent recurrences that share few genetic alterations with the primary tumor and originate from cells that branched off early during tumorigenesis. Our study provides mechanistic insights into how genetic alterations in primary tumors impact the ensuing evolution of tumor cells and the emergence of subclonal heterogeneity. PMID:25650244

Molecular diagnosis of glycogen storage disease and disorders with overlapping clinical symptoms by massive parallel sequencing.

PubMed

Vega, Ana I; Medrano, Celia; Navarrete, Rosa; Desviat, Lourdes R; Merinero, Begoña; Rodríguez-Pombo, Pilar; Vitoria, Isidro; Ugarte, Magdalena; Pérez-Cerdá, Celia; Pérez, Belen

2016-10-01

Glycogen storage disease (GSD) is an umbrella term for a group of genetic disorders that involve the abnormal metabolism of glycogen; to date, 23 types of GSD have been identified. The nonspecific clinical presentation of GSD and the lack of specific biomarkers mean that Sanger sequencing is now widely relied on for making a diagnosis. However, this gene-by-gene sequencing technique is both laborious and costly, which is a consequence of the number of genes to be sequenced and the large size of some genes. This work reports the use of massive parallel sequencing to diagnose patients at our laboratory in Spain using either a customized gene panel (targeted exome sequencing) or the Illumina Clinical-Exome TruSight One Gene Panel (clinical exome sequencing (CES)). Sequence variants were matched against biochemical and clinical hallmarks. Pathogenic mutations were detected in 23 patients. Twenty-two mutations were recognized (mostly loss-of-function mutations), including 11 that were novel in GSD-associated genes. In addition, CES detected five patients with mutations in ALDOB, LIPA, NKX2-5, CPT2, or ANO5. Although these genes are not involved in GSD, they are associated with overlapping phenotypic characteristics such as hepatic, muscular, and cardiac dysfunction. These results show that next-generation sequencing, in combination with the detection of biochemical and clinical hallmarks, provides an accurate, high-throughput means of making genetic diagnoses of GSD and related diseases.Genet Med 18 10, 1037-1043.

Exome sequencing identifies rare LDLR and APOA5 alleles conferring risk for myocardial infarction.

PubMed

Do, Ron; Stitziel, Nathan O; Won, Hong-Hee; Jørgensen, Anders Berg; Duga, Stefano; Angelica Merlini, Pier; Kiezun, Adam; Farrall, Martin; Goel, Anuj; Zuk, Or; Guella, Illaria; Asselta, Rosanna; Lange, Leslie A; Peloso, Gina M; Auer, Paul L; Girelli, Domenico; Martinelli, Nicola; Farlow, Deborah N; DePristo, Mark A; Roberts, Robert; Stewart, Alexander F R; Saleheen, Danish; Danesh, John; Epstein, Stephen E; Sivapalaratnam, Suthesh; Hovingh, G Kees; Kastelein, John J; Samani, Nilesh J; Schunkert, Heribert; Erdmann, Jeanette; Shah, Svati H; Kraus, William E; Davies, Robert; Nikpay, Majid; Johansen, Christopher T; Wang, Jian; Hegele, Robert A; Hechter, Eliana; Marz, Winfried; Kleber, Marcus E; Huang, Jie; Johnson, Andrew D; Li, Mingyao; Burke, Greg L; Gross, Myron; Liu, Yongmei; Assimes, Themistocles L; Heiss, Gerardo; Lange, Ethan M; Folsom, Aaron R; Taylor, Herman A; Olivieri, Oliviero; Hamsten, Anders; Clarke, Robert; Reilly, Dermot F; Yin, Wu; Rivas, Manuel A; Donnelly, Peter; Rossouw, Jacques E; Psaty, Bruce M; Herrington, David M; Wilson, James G; Rich, Stephen S; Bamshad, Michael J; Tracy, Russell P; Cupples, L Adrienne; Rader, Daniel J; Reilly, Muredach P; Spertus, John A; Cresci, Sharon; Hartiala, Jaana; Tang, W H Wilson; Hazen, Stanley L; Allayee, Hooman; Reiner, Alex P; Carlson, Christopher S; Kooperberg, Charles; Jackson, Rebecca D; Boerwinkle, Eric; Lander, Eric S; Schwartz, Stephen M; Siscovick, David S; McPherson, Ruth; Tybjaerg-Hansen, Anne; Abecasis, Goncalo R; Watkins, Hugh; Nickerson, Deborah A; Ardissino, Diego; Sunyaev, Shamil R; O'Donnell, Christopher J; Altshuler, David; Gabriel, Stacey; Kathiresan, Sekar

2015-02-05

Myocardial infarction (MI), a leading cause of death around the world, displays a complex pattern of inheritance. When MI occurs early in life, genetic inheritance is a major component to risk. Previously, rare mutations in low-density lipoprotein (LDL) genes have been shown to contribute to MI risk in individual families, whereas common variants at more than 45 loci have been associated with MI risk in the population. Here we evaluate how rare mutations contribute to early-onset MI risk in the population. We sequenced the protein-coding regions of 9,793 genomes from patients with MI at an early age (≤50 years in males and ≤60 years in females) along with MI-free controls. We identified two genes in which rare coding-sequence mutations were more frequent in MI cases versus controls at exome-wide significance. At low-density lipoprotein receptor (LDLR), carriers of rare non-synonymous mutations were at 4.2-fold increased risk for MI; carriers of null alleles at LDLR were at even higher risk (13-fold difference). Approximately 2% of early MI cases harbour a rare, damaging mutation in LDLR; this estimate is similar to one made more than 40 years ago using an analysis of total cholesterol. Among controls, about 1 in 217 carried an LDLR coding-sequence mutation and had plasma LDL cholesterol > 190 mg dl(-1). At apolipoprotein A-V (APOA5), carriers of rare non-synonymous mutations were at 2.2-fold increased risk for MI. When compared with non-carriers, LDLR mutation carriers had higher plasma LDL cholesterol, whereas APOA5 mutation carriers had higher plasma triglycerides. Recent evidence has connected MI risk with coding-sequence mutations at two genes functionally related to APOA5, namely lipoprotein lipase and apolipoprotein C-III (refs 18, 19). Combined, these observations suggest that, as well as LDL cholesterol, disordered metabolism of triglyceride-rich lipoproteins contributes to MI risk.

New mutations in non-syndromic primary ovarian insufficiency patients identified via whole-exome sequencing.

PubMed

Patiño, Liliana Catherine; Beau, Isabelle; Carlosama, Carolina; Buitrago, July Constanza; González, Ronald; Suárez, Carlos Fernando; Patarroyo, Manuel Alfonso; Delemer, Brigitte; Young, Jacques; Binart, Nadine; Laissue, Paul

2017-07-01

Is it possible to identify new mutations potentially associated with non-syndromic primary ovarian insufficiency (POI) via whole-exome sequencing (WES)? WES is an efficient tool to study genetic causes of POI as we have identified new mutations, some of which lead to protein destablization potentially contributing to the disease etiology. POI is a frequently occurring complex pathology leading to infertility. Mutations in only few candidate genes, mainly identified by Sanger sequencing, have been definitively related to the pathogenesis of the disease. This is a retrospective cohort study performed on 69 women affected by POI. WES and an innovative bioinformatics analysis were used on non-synonymous sequence variants in a subset of 420 selected POI candidate genes. Mutations in BMPR1B and GREM1 were modeled by using fragment molecular orbital analysis. Fifty-five coding variants in 49 genes potentially related to POI were identified in 33 out of 69 patients (48%). These genes participate in key biological processes in the ovary, such as meiosis, follicular development, granulosa cell differentiation/proliferation and ovulation. The presence of at least two mutations in distinct genes in 42% of the patients argued in favor of a polygenic nature of POI. It is possible that regulatory regions, not analyzed in the present study, carry further variants related to POI. WES and the in silico analyses presented here represent an efficient approach for mapping variants associated with POI etiology. Sequence variants presented here represents potential future genetic biomarkers. This study was supported by the Universidad del Rosario and Colciencias (Grants CS/CIGGUR-ABN062-2016 and 672-2014). Colciencias supported Liliana Catherine Patiño´s work (Fellowship: 617, 2013). The authors declare no conflict of interest. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For

Exome Sequencing Links Mutations in PARN and RTEL1 with Familial Pulmonary Fibrosis and Telomere Shortening

PubMed Central

Stuart, Bridget D.; Choi, Jungmin; Zaidi, Samir; Xing, Chao; Holohan, Brody; Chen, Rui; Choi, Mihwa; Dharwadkar, Pooja; Torres, Fernando; Girod, Carlos E.; Weissler, Jonathan; Fitzgerald, John; Kershaw, Corey; Klesney-Tait, Julia; Mageto, Yolanda; Shay, Jerry W.; Ji, Weizhen; Bilguvar, Kaya; Mane, Shrikant; Lifton, Richard P.; Garcia, Christine Kim

2015-01-01

Idiopathic pulmonary fibrosis (IPF) is an age-related disease featuring progressive lung scarring. To elucidate the molecular basis of IPF, we performed exome sequencing of familial pulmonary fibrosis kindreds. Gene burden analysis comparing 78 European cases and 2,816 controls implicated PARN, an exoribonuclease with no prior connection to telomere biology or disease, with five novel heterozygous damaging mutations in unrelated cases and none in controls (P-value = 1.3 × 10−8); mutations were shared by all affected relatives (odds in favor of linkage = 4,096:1). RTEL1, an established locus for dyskeratosis congenita, harbored significantly more novel damaging and missense variants at conserved residues in cases than controls (P = 1.6 × 10−6). PARN and RTEL1 mutation carriers had shortened leukocyte telomere lengths and epigenetic inheritance of short telomeres was seen in family members. Together these genes explain ~7% of familial pulmonary fibrosis and strengthen the link between lung fibrosis and telomere dysfunction. PMID:25848748

Exome sequencing links mutations in PARN and RTEL1 with familial pulmonary fibrosis and telomere shortening.

PubMed

Stuart, Bridget D; Choi, Jungmin; Zaidi, Samir; Xing, Chao; Holohan, Brody; Chen, Rui; Choi, Mihwa; Dharwadkar, Pooja; Torres, Fernando; Girod, Carlos E; Weissler, Jonathan; Fitzgerald, John; Kershaw, Corey; Klesney-Tait, Julia; Mageto, Yolanda; Shay, Jerry W; Ji, Weizhen; Bilguvar, Kaya; Mane, Shrikant; Lifton, Richard P; Garcia, Christine Kim

2015-05-01

Idiopathic pulmonary fibrosis (IPF) is an age-related disease featuring progressive lung scarring. To elucidate the molecular basis of IPF, we performed exome sequencing of familial kindreds with pulmonary fibrosis. Gene burden analysis comparing 78 European cases and 2,816 controls implicated PARN, an exoribonuclease with no previous connection to telomere biology or disease, with five new heterozygous damaging mutations in unrelated cases and none in controls (P = 1.3 × 10(-8)); mutations were shared by all affected relatives (odds in favor of linkage = 4,096:1). RTEL1, an established locus for dyskeratosis congenita, harbored significantly more new damaging and missense variants at conserved residues in cases than in controls (P = 1.6 × 10(-6)). PARN and RTEL1 mutation carriers had shortened leukocyte telomere lengths, and we observed epigenetic inheritance of short telomeres in family members. Together, these genes explain ~7% of familial pulmonary fibrosis and strengthen the link between lung fibrosis and telomere dysfunction.

Discovery and prioritization of somatic mutations in diffuse large B-cell lymphoma (DLBCL) by whole-exome sequencing

PubMed Central

Lohr, Jens G.; Stojanov, Petar; Lawrence, Michael S.; Auclair, Daniel; Chapuy, Bjoern; Sougnez, Carrie; Cruz-Gordillo, Peter; Knoechel, Birgit; Asmann, Yan W.; Slager, Susan L.; Novak, Anne J.; Dogan, Ahmet; Ansell, Stephen M.; Zou, Lihua; Gould, Joshua; Saksena, Gordon; Stransky, Nicolas; Rangel-Escareño, Claudia; Fernandez-Lopez, Juan Carlos; Hidalgo-Miranda, Alfredo; Melendez-Zajgla, Jorge; Hernández-Lemus, Enrique; Schwarz-Cruz y Celis, Angela; Imaz-Rosshandler, Ivan; Ojesina, Akinyemi I.; Jung, Joonil; Pedamallu, Chandra S.; Lander, Eric S.; Habermann, Thomas M.; Cerhan, James R.; Shipp, Margaret A.; Getz, Gad; Golub, Todd R.

2012-01-01

To gain insight into the genomic basis of diffuse large B-cell lymphoma (DLBCL), we performed massively parallel whole-exome sequencing of 55 primary tumor samples from patients with DLBCL and matched normal tissue. We identified recurrent mutations in genes that are well known to be functionally relevant in DLBCL, including MYD88, CARD11, EZH2, and CREBBP. We also identified somatic mutations in genes for which a functional role in DLBCL has not been previously suspected. These genes include MEF2B, MLL2, BTG1, GNA13, ACTB, P2RY8, PCLO, and TNFRSF14. Further, we show that BCL2 mutations commonly occur in patients with BCL2/IgH rearrangements as a result of somatic hypermutation normally occurring at the IgH locus. The BCL2 point mutations are primarily synonymous, and likely caused by activation-induced cytidine deaminase–mediated somatic hypermutation, as shown by comprehensive analysis of enrichment of mutations in WRCY target motifs. Those nonsynonymous mutations that are observed tend to be found outside of the functionally important BH domains of the protein, suggesting that strong negative selection against BCL2 loss-of-function mutations is at play. Last, by using an algorithm designed to identify likely functionally relevant but infrequent mutations, we identify KRAS, BRAF, and NOTCH1 as likely drivers of DLBCL pathogenesis in some patients. Our data provide an unbiased view of the landscape of mutations in DLBCL, and this in turn may point toward new therapeutic strategies for the disease. PMID:22343534

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.

Targeted therapy according to next generation sequencing-based panel sequencing.

PubMed

Saito, Motonobu; Momma, Tomoyuki; Kono, Koji

2018-04-17

Targeted therapy against actionable gene mutations shows a significantly higher response rate as well as longer survival compared to conventional chemotherapy, and has become a standard therapy for many cancers. Recent progress in next-generation sequencing (NGS) has enabled to identify huge number of genetic aberrations. Based on sequencing results, patients recommend to undergo targeted therapy or immunotherapy. In cases where there are no available approved drugs for the genetic mutations detected in the patients, it is recommended to be facilitate the registration for the clinical trials. For that purpose, a NGS-based sequencing panel that can simultaneously target multiple genes in a single investigation has been used in daily clinical practice. To date, various types of sequencing panels have been developed to investigate genetic aberrations with tumor somatic genome variants (gain-of-function or loss-of-function mutations, high-level copy number alterations, and gene fusions) through comprehensive bioinformatics. Because sequencing panels are efficient and cost-effective, they are quickly being adopted outside the lab, in hospitals and clinics, in order to identify personal targeted therapy for individual cancer patients.

Exome sequencing of a large family identifies potential candidate genes contributing risk to bipolar disorder.

PubMed

Zhang, Tianxiao; Hou, Liping; Chen, David T; McMahon, Francis J; Wang, Jen-Chyong; Rice, John P

2018-03-01

Bipolar disorder is a mental illness with lifetime prevalence of about 1%. Previous genetic studies have identified multiple chromosomal linkage regions and candidate genes that might be associated with bipolar disorder. The present study aimed to identify potential susceptibility variants for bipolar disorder using 6 related case samples from a four-generation family. A combination of exome sequencing and linkage analysis was performed to identify potential susceptibility variants for bipolar disorder. Our study identified a list of five potential candidate genes for bipolar disorder. Among these five genes, GRID1(Glutamate Receptor Delta-1 Subunit), which was previously reported to be associated with several psychiatric disorders and brain related traits, is particularly interesting. Variants with functional significance in this gene were identified from two cousins in our bipolar disorder pedigree. Our findings suggest a potential role for these genes and the related rare variants in the onset and development of bipolar disorder in this one family. Additional research is needed to replicate these findings and evaluate their patho-biological significance. Copyright © 2017 Elsevier B.V. All rights reserved.

Diagnosis and molecular basis of mitochondrial respiratory chain disorders: exome sequencing for disease gene identification.

PubMed

Ohtake, A; Murayama, K; Mori, M; Harashima, H; Yamazaki, T; Tamaru, S; Yamashita, Y; Kishita, Y; Nakachi, Y; Kohda, M; Tokuzawa, Y; Mizuno, Y; Moriyama, Y; Kato, H; Okazaki, Y

2014-04-01

Mitochondrial disorders have the highest incidence among congenital metabolic diseases, and are thought to occur at a rate of 1 in 5000 births. About 25% of the diseases diagnosed as mitochondrial disorders in the field of pediatrics have mitochondrial DNA abnormalities, while the rest occur due to defects in genes encoded in the nucleus. The most important function of the mitochondria is biosynthesis of ATP. Mitochondrial disorders are nearly synonymous with mitochondrial respiratory chain disorder, as respiratory chain complexes serve a central role in ATP biosynthesis. By next-generation sequencing of the exome, we analyzed 104 patients with mitochondrial respiratory chain disorders. The results of analysis to date were 18 patients with novel variants in genes previously reported to be disease-causing, and 27 patients with mutations in genes suggested to be associated in some way with mitochondria, and it is likely that they are new disease-causing genes in mitochondrial disorders. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

Whole Exome Sequencing Identifies de Novo Mutations in GATA6 Associated with Congenital Diaphragmatic Hernia

PubMed Central

Yu, Lan; Bennett, James T.; Wynn, Julia; Carvill, Gemma L.; Cheung, Yee Him; Shen, Yufeng; Mychaliska, George B.; Azarow, Kenneth S.; Crombleholme, Timothy M.; Chung, Dai H.; Potoka, Douglas; Warner, Brad W.; Bucher, Brian; Lim, Foong-Yen; Pietsch, John; Stolar, Charles; Aspelund, Gudrun; Arkovitz, Marc S.; Mefford, Heather; Chung, Wendy K.

2014-01-01

Background Congenital diaphragmatic hernia (CDH) is a common birth defect affecting 1 in 3,000 births. It is characterized by herniation of abdominal viscera through an incompletely formed diaphragm. Although chromosomal anomalies and mutations in several genes have been implicated, the cause for most patients is unknown. Methods We used whole exome sequencing in two families with CDH and congenital heart disease, and identified mutations in GATA6 in both. Results In the first family, we identified a de novo missense mutation (c.1366C>T, p.R456C) in a sporadic CDH patient with tetralogy of Fallot. In the second, a nonsense mutation (c.712G>T, p.G238*) was identified in two siblings with CDH and a large ventricular septal defect. The G238* mutation was inherited from their mother, who was clinically affected with congenital absence of the pericardium, patent ductus arteriosus, and intestinal malrotation. Deep sequencing of blood and saliva derived DNA from the mother suggested somatic mosaicism as an explanation for her milder phenotype, with only approximately 15% mutant alleles. To determine the frequency of GATA6 mutations in CDH, we sequenced the gene in 378 patients with CDH. We identified one additional de novo mutation (c.1071delG, p.V358Cfs34*). Conclusions Mutations in GATA6 have been previously associated with pancreatic agenesis and congenital heart disease. We conclude that, in addition to the heart and the pancreas, GATA6 is involved in development of two additional organs, the diaphragm and the pericardium. In addition we have shown that de novo mutations can contribute to the development of CDH, a common birth defect. PMID:24385578

A novel NOTCH3 mutation identified in patients with oral cancer by whole exome sequencing.

PubMed

Yi, Yanjun; Tian, Zhuowei; Ju, Houyu; Ren, Guoxin; Hu, Jingzhou

2017-06-01

Oral cancer is a serious disease caused by environmental factors and/or susceptible genes. In the present study, in order to identify useful genetic biomarkers for cancer prediction and prevention, and for personalized treatment, we detected somatic mutations in 5 pairs of oral cancer tissues and blood samples using whole exome sequencing (WES). Finally, we confirmed a novel nonsense single-nucleotide polymorphism (SNP; chr19:15288426A>C) in the NOTCH3 gene with sanger sequencing, which resulted in a N1438T mutation in the protein sequence. Using multiple in silico analyses, this variant was found to mildly damaging effects on the NOTCH3 gene, which was supported by the results from analyses using PANTHER, SNAP and SNPs&GO. However, further analysis using Mutation Taster revealed that this SNP had a probability of 0.9997 to be 'disease causing'. In addition, we performed 3D structure simulation analysis and the results suggested that this variant had little effect on the solubility and hydrophobicity of the protein and thus on its function; however, it decreased the stability of the protein by increasing the total energy following minimization (-1,051.39 kcal/mol for the mutant and -1,229.84 kcal/mol for the native) and decreasing one stabilizing residue of the protein. Less stability of the N1438T mutant was also supported by analysis using I-Mutant with a DDG value of -1.67. Overall, the present study identified and confirmed a novel mutation in the NOTCH3 gene, which may decrease the stability of NOTCH3, and may thus prove to be helpful in cancer prognosis.

Outcome of Whole Exome Sequencing for Diagnostic Odyssey Cases of an Individualized Medicine Clinic: The Mayo Clinic Experience.

PubMed

Lazaridis, Konstantinos N; Schahl, Kimberly A; Cousin, Margot A; Babovic-Vuksanovic, Dusica; Riegert-Johnson, Douglas L; Gavrilova, Ralitza H; McAllister, Tammy M; Lindor, Noralane M; Abraham, Roshini S; Ackerman, Michael J; Pichurin, Pavel N; Deyle, David R; Gavrilov, Dimitar K; Hand, Jennifer L; Klee, Eric W; Stephens, Michael C; Wick, Myra J; Atkinson, Elizabeth J; Linden, David R; Ferber, Matthew J; Wieben, Eric D; Farrugia, Gianrico

2016-03-01

To describe the experience and outcome of performing whole-exome sequencing (WES) for resolution of patients on a diagnostic odyssey in the first 18 months of an individualized medicine clinic (IMC). The IMC offered WES to physicians of Mayo Clinic practice for patients with suspected genetic disease. DNA specimens of the proband and relatives were submitted to WES laboratories. We developed the Genomic Odyssey Board with multidisciplinary expertise to determine the appropriateness for IMC services, review WES reports, and make the final decision about whether the exome findings explain the disease. This study took place from September 30, 2012, to March 30, 2014. In the first 18 consecutive months, the IMC received 82 consultation requests for patients on a diagnostic odyssey. The Genomic Odyssey Board deferred 7 cases and approved 75 cases to proceed with WES. Seventy-one patients met with an IMC genomic counselor. Fifty-one patients submitted specimens for WES testing, and the results have been received for all. There were 15 cases in which a diagnosis was made on the basis of WES findings; thus, the positive diagnostic yield of this practice was 29%. The mean cost per patient for this service was approximately $8000. Medicaid supported 27% of the patients, and 38% of patients received complete or partial insurance coverage. The significant diagnostic yield, moderate cost, and notable health marketplace acceptance for WES compared with conventional genetic testing make the former method a rational diagnostic approach for patients on a diagnostic odyssey. Copyright © 2016 Mayo Foundation for Medical Education and Research. Published by Elsevier Inc. All rights reserved.

Analysis of whole exome sequencing with cardiometabolic traits using family-based linkage and association in the IRAS Family Study

PubMed Central

Tabb, Keri L.; Hellwege, Jacklyn N.; Palmer, Nicholette D.; Dimitrov, Latchezar; Sajuthi, Satria; Taylor, Kent D.; NG, Maggie C.Y.; Hawkins, Gregory A.; Chen, Yii-Der Ida; Brown, W. Mark; McWilliams, David; Williams, Adrienne; Lorenzo, Carlos; Norris, Jill M.; Long, Jirong; Rotter, Jerome I.; Curran, Joanne E.; Blangero, John; Wagenknecht, Lynne E.; Langefeld, Carl D.; Bowden, Donald W.

2017-01-01

Summary Family-based methods are a potentially powerful tool to identify trait-defining genetic variants in extended families, particularly when used to complement conventional association analysis. We utilized two-point linkage analysis and single variant association analysis to evaluate whole exome sequencing (WES) data from 1,205 Hispanic Americans (78 families) from the Insulin Resistance Atherosclerosis Family Study. WES identified 211,612 variants above the minor allele frequency threshold of ≥0.005. These variants were tested for linkage and/or association with 50 cardiometabolic traits after quality control checks. Two-point linkage analysis yielded 10,580,600 LOD scores with 1,148 LOD scores ≥3, 183 LOD scores ≥4, and 29 LOD scores ≥5. The maximal novel LOD score was 5.50 for rs2289043:T>C, in UNC5C with subcutaneous adipose tissue volume. Association analysis identified 13 variants attaining genome-wide significance (p<5×10-08), with the strongest association between rs651821:C>T in APOA5, and triglyceride levels (p=3.67×10-10). Overall, there was a 5.2-fold increase in the number of informative variants detected by WES compared to exome chip analysis in this population, nearly 30% of which were novel variants relative to dbSNP build 138. Thus, integration of results from two-point linkage and single-variant association analysis from WES data enabled identification of novel signals potentially contributing to cardiometabolic traits. PMID:28067407

A targeted sequencing panel identifies rare damaging variants in multiple genes in the cranial neural tube defect, anencephaly.

PubMed

Ishida, M; Cullup, T; Boustred, C; James, C; Docker, J; English, C; Lench, N; Copp, A J; Moore, G E; Greene, N D E; Stanier, P

2018-04-01

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. © 2017 The Authors. Clinical Genetics published by John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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.

Whole-exome sequencing of a rare case of familial childhood acute lymphoblastic leukemia reveals putative predisposing mutations in Fanconi anemia genes.

PubMed

Spinella, Jean-François; Healy, Jasmine; Saillour, Virginie; Richer, Chantal; Cassart, Pauline; Ouimet, Manon; Sinnett, Daniel

2015-07-23

Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer. While the multi-step model of pediatric leukemogenesis suggests interplay between constitutional and somatic genomes, the role of inherited genetic variability remains largely undescribed. Nonsyndromic familial ALL, although extremely rare, provides the ideal setting to study inherited contributions to ALL. Toward this goal, we sequenced the exomes of a childhood ALL family consisting of mother, father and two non-twinned siblings diagnosed with concordant pre-B hyperdiploid ALL and previously shown to have inherited a rare form of PRDM9, a histone H3 methyltransferase involved in crossing-over at recombination hotspots and Holliday junctions. We postulated that inheritance of additional rare disadvantaging variants in predisposing cancer genes could affect genomic stability and lead to increased risk of hyperdiploid ALL within this family. Whole exomes were captured using Agilent's SureSelect kit and sequenced on the Life Technologies SOLiD System. We applied a data reduction strategy to identify candidate variants shared by both affected siblings. Under a recessive disease model, we focused on rare non-synonymous or frame-shift variants in leukemia predisposing pathways. Though the family was nonsyndromic, we identified a combination of rare variants in Fanconi anemia (FA) genes FANCP/SLX4 (compound heterozygote - rs137976282/rs79842542) and FANCA (rs61753269) and a rare homozygous variant in the Holliday junction resolvase GEN1 (rs16981869). These variants, predicted to affect protein function, were previously identified in familial breast cancer cases. Based on our in-house database of 369 childhood ALL exomes, the sibs were the only patients to carry this particularly rare combination and only a single hyperdiploid patient was heterozygote at both FANCP/SLX4 positions, while no FANCA variant allele carriers were identified. FANCA is the most commonly mutated gene in FA and is essential for

MACARON: A python framework to identify and re-annotate multi-base affected codons in whole genome/exome sequence data.

PubMed

Khan, Waqasuddin; Saripella, Ganapathi Varma-; Ludwig, Thomas; Cuppens, Tania; Thibord, Florian; Génin, Emmanuelle; Deleuze, Jean-Francois; Trégouët, David-Alexandre

2018-05-03

Predicted deleteriousness of coding variants is a frequently used criterion to filter out variants detected in next-generation sequencing projects and to select candidates impacting on the risk of human diseases. Most available dedicated tools implement a base-to-base annotation approach that could be biased in presence of several variants in the same genetic codon. We here proposed the MACARON program that, from a standard VCF file, identifies, re-annotates and predicts the amino acid change resulting from multiple single nucleotide variants (SNVs) within the same genetic codon. Applied to the whole exome dataset of 573 individuals, MACARON identifies 114 situations where multiple SNVs within a genetic codon induce an amino acid change that is different from those predicted by standard single SNV annotation tool. Such events are not uncommon and deserve to be studied in sequencing projects with inconclusive findings. MACARON is written in python with codes available on the GENMED website (www.genmed.fr). david-alexandre.tregouet@inserm.fr. Supplementary data are available at Bioinformatics online.

Lynch Syndrome Patients' Views of and Preferences for Return of Results Following Whole Exome Sequencing

PubMed Central

Joseph, Galen; Guiltinan, Jenna; Kianmahd, Jessica; Youngblom, Janey; Blanco, Amie

2015-01-01

Whole exome sequencing (WES) uses next generation sequencing technology to provide information on nearly all functional, protein-coding regions in an individual's genome. Due to the vast amount of information and incidental findings that can be generated from this technology, patient preferences must be investigated to help clinicians consent and return results to patients. Patients (n=19) who were previously clinically diagnosed with Lynch syndrome, but received uninformative negative Lynch syndrome genetic results through traditional molecular testing methods participated in semi-structured interviews after WES testing but before return of results to explore their views of WES and preferences for return of results. Analyses of interview results found that nearly all participants believed that the benefits of receiving all possible results generated from WES outweighed the undesirable effects. The majority of participants conveyed that relative to coping with a cancer diagnosis, information generated from WES would be manageable. Importantly, participants' experience with Lynch syndrome influenced their notions of genetic determinism, tolerance for uncertain results, and family communication plans. Participants would prefer to receive WES results in person from a genetic counselor or medical geneticist so that an expert could help explain the meaning and implications of the potentially large quantity and range of complicated results. These results underscore the need to study various populations with regard to the clinical use of WES in order to effectively and empathetically communicate the possible implications of this new technology and return results. PMID:24449059

Exome Sequencing in an Admixed Isolated Population Indicates NFXL1 Variants Confer a Risk for Specific Language Impairment

PubMed Central

Villanueva, Pía; Nudel, Ron; Hoischen, Alexander; Fernández, María Angélica; Simpson, Nuala H.; Gilissen, Christian; Reader, Rose H.; Jara, Lillian; Echeverry, Maria Magdalena; Francks, Clyde; Baird, Gillian; Conti-Ramsden, Gina; O’Hare, Anne; Bolton, Patrick F.; Hennessy, Elizabeth R.; Palomino, Hernán; Carvajal-Carmona, Luis; Veltman, Joris A.; Cazier, Jean-Baptiste; De Barbieri, Zulema

2015-01-01

Children affected by Specific Language Impairment (SLI) fail to acquire age appropriate language skills despite adequate intelligence and opportunity. SLI is highly heritable, but the understanding of underlying genetic mechanisms has proved challenging. In this study, we use molecular genetic techniques to investigate an admixed isolated founder population from the Robinson Crusoe Island (Chile), who are affected by a high incidence of SLI, increasing the power to discover contributory genetic factors. We utilize exome sequencing in selected individuals from this population to identify eight coding variants that are of putative significance. We then apply association analyses across the wider population to highlight a single rare coding variant (rs144169475, Minor Allele Frequency of 4.1% in admixed South American populations) in the NFXL1 gene that confers a nonsynonymous change (N150K) and is significantly associated with language impairment in the Robinson Crusoe population (p = 2.04 × 10–4, 8 variants tested). Subsequent sequencing of NFXL1 in 117 UK SLI cases identified four individuals with heterozygous variants predicted to be of functional consequence. We conclude that coding variants within NFXL1 confer an increased risk of SLI within a complex genetic model. PMID:25781923

Exome sequencing in an admixed isolated population indicates NFXL1 variants confer a risk for specific language impairment.

PubMed

Villanueva, Pía; Nudel, Ron; Hoischen, Alexander; Fernández, María Angélica; Simpson, Nuala H; Gilissen, Christian; Reader, Rose H; Jara, Lillian; Echeverry, María Magdalena; Echeverry, Maria Magdalena; Francks, Clyde; Baird, Gillian; Conti-Ramsden, Gina; O'Hare, Anne; Bolton, Patrick F; Hennessy, Elizabeth R; Palomino, Hernán; Carvajal-Carmona, Luis; Veltman, Joris A; Cazier, Jean-Baptiste; De Barbieri, Zulema; Fisher, Simon E; Newbury, Dianne F

2015-03-01

Children affected by Specific Language Impairment (SLI) fail to acquire age appropriate language skills despite adequate intelligence and opportunity. SLI is highly heritable, but the understanding of underlying genetic mechanisms has proved challenging. In this study, we use molecular genetic techniques to investigate an admixed isolated founder population from the Robinson Crusoe Island (Chile), who are affected by a high incidence of SLI, increasing the power to discover contributory genetic factors. We utilize exome sequencing in selected individuals from this population to identify eight coding variants that are of putative significance. We then apply association analyses across the wider population to highlight a single rare coding variant (rs144169475, Minor Allele Frequency of 4.1% in admixed South American populations) in the NFXL1 gene that confers a nonsynonymous change (N150K) and is significantly associated with language impairment in the Robinson Crusoe population (p = 2.04 × 10-4, 8 variants tested). Subsequent sequencing of NFXL1 in 117 UK SLI cases identified four individuals with heterozygous variants predicted to be of functional consequence. We conclude that coding variants within NFXL1 confer an increased risk of SLI within a complex genetic model.

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

Targeted sequencing-based analyses of candidate gene variants in ulcerative colitis-associated colorectal neoplasia.

PubMed

Chakrabarty, Sanjiban; Varghese, Vinay Koshy; Sahu, Pranoy; Jayaram, Pradyumna; Shivakumar, Bhadravathi M; Pai, Cannanore Ganesh; Satyamoorthy, Kapaettu

2017-06-27

Long-standing ulcerative colitis (UC) leading to colorectal cancer (CRC) is one of the most serious and life-threatening consequences acknowledged globally. Ulcerative colitis-associated colorectal carcinogenesis showed distinct molecular alterations when compared with sporadic colorectal carcinoma. Targeted sequencing of 409 genes in tissue samples of 18 long-standing UC subjects at high risk of colorectal carcinoma (UCHR) was performed to identify somatic driver mutations, which may be involved in the molecular changes during the transformation of non-dysplastic mucosa to high-grade dysplasia. Findings from the study are also compared with previously published genome wide and exome sequencing data in inflammatory bowel disease-associated and sporadic colorectal carcinoma. Next-generation sequencing analysis identified 1107 mutations in 275 genes in UCHR subjects. In addition to TP53 (17%) and KRAS (22%) mutations, recurrent mutations in APC (33%), ACVR2A (61%), ARID1A (44%), RAF1 (39%) and MTOR (61%) were observed in UCHR subjects. In addition, APC, FGFR3, FGFR2 and PIK3CA driver mutations were identified in UCHR subjects. Recurrent mutations in ARID1A (44%), SMARCA4 (17%), MLL2 (44%), MLL3 (67%), SETD2 (17%) and TET2 (50%) genes involved in histone modification and chromatin remodelling were identified in UCHR subjects. Our study identifies new oncogenic driver mutations which may be involved in the transition of non-dysplastic cells to dysplastic phenotype in the subjects with long-standing UC with high risk of progression into colorectal neoplasia.

Bioinformatics Pipelines for Targeted Resequencing and Whole-Exome Sequencing of Human and Mouse Genomes: A Virtual Appliance Approach for Instant Deployment

PubMed Central

Saeed, Isaam; Wong, Stephen Q.; Mar, Victoria; Goode, David L.; Caramia, Franco; Doig, Ken; Ryland, Georgina L.; Thompson, Ella R.; Hunter, Sally M.; Halgamuge, Saman K.; Ellul, Jason; Dobrovic, Alexander; Campbell, Ian G.; Papenfuss, Anthony T.; McArthur, Grant A.; Tothill, Richard W.

2014-01-01

Targeted resequencing by massively parallel sequencing has become an effective and affordable way to survey small to large portions of the genome for genetic variation. Despite the rapid development in open source software for analysis of such data, the practical implementation of these tools through construction of sequencing analysis pipelines still remains a challenging and laborious activity, and a major hurdle for many small research and clinical laboratories. We developed TREVA (Targeted REsequencing Virtual Appliance), making pre-built pipelines immediately available as a virtual appliance. Based on virtual machine technologies, TREVA is a solution for rapid and efficient deployment of complex bioinformatics pipelines to laboratories of all sizes, enabling reproducible results. The analyses that are supported in TREVA include: somatic and germline single-nucleotide and insertion/deletion variant calling, copy number analysis, and cohort-based analyses such as pathway and significantly mutated genes analyses. TREVA is flexible and easy to use, and can be customised by Linux-based extensions if required. TREVA can also be deployed on the cloud (cloud computing), enabling instant access without investment overheads for additional hardware. TREVA is available at http://bioinformatics.petermac.org/treva/. PMID:24752294

Exome sequencing reveals a thrombopoietin ligand mutation in a Micronesian family with autosomal recessive aplastic anemia.

PubMed

Dasouki, Majed J; Rafi, Syed K; Olm-Shipman, Adam J; Wilson, Nathan R; Abhyankar, Sunil; Ganter, Brigitte; Furness, L Mike; Fang, Jianwen; Calado, Rodrigo T; Saadi, Irfan

2013-11-14

We recently identified 2 siblings afflicted with idiopathic, autosomal recessive aplastic anemia. Whole-exome sequencing identified a novel homozygous missense mutation in thrombopoietin (THPO, c.112C>T) in both affected siblings. This mutation encodes an arginine to cysteine substitution at residue 38 or residue 17 excluding the 21-amino acid signal peptide of THPO receptor binding domain (RBD). THPO has 4 conserved cysteines in its RBD that form 2 disulfide bonds. Our in silico modeling predicts that introduction of a fifth cysteine may disrupt normal disulfide bonding to cause poor receptor binding. In functional assays, the mutant-THPO-containing media shows two- to threefold reduced ability to sustain UT7-TPO cells, which require THPO for proliferation. Both parents and a sibling with heterozygous R17C change have reduced platelet counts, whereas a sibling with wild-type sequence has normal platelet count. Thus, the R17C partial loss-of-function allele results in aplastic anemia in the homozygous state and mild thrombocytopenia in the heterozygous state in our family. Together with the recent identification of THPO receptor (MPL) mutations and the effects of THPO agonists in aplastic anemia, our results have clinical implications in the diagnosis and treatment of patients with aplastic anemia and highlight a role for the THPO-MPL pathway in hematopoiesis in vivo.

Whole-genome and multisector exome sequencing of primary and post-treatment glioblastoma reveals patterns of tumor evolution.

PubMed

Kim, Hoon; Zheng, Siyuan; Amini, Seyed S; Virk, Selene M; Mikkelsen, Tom; Brat, Daniel J; Grimsby, Jonna; Sougnez, Carrie; Muller, Florian; Hu, Jian; Sloan, Andrew E; Cohen, Mark L; Van Meir, Erwin G; Scarpace, Lisa; Laird, Peter W; Weinstein, John N; Lander, Eric S; Gabriel, Stacey; Getz, Gad; Meyerson, Matthew; Chin, Lynda; Barnholtz-Sloan, Jill S; Verhaak, Roel G W

2015-03-01

Glioblastoma (GBM) is a prototypical heterogeneous brain tumor refractory to conventional therapy. A small residual population of cells escapes surgery and chemoradiation, resulting in a typically fatal tumor recurrence ∼ 7 mo after diagnosis. Understanding the molecular architecture of this residual population is critical for the development of successful therapies. We used whole-genome sequencing and whole-exome sequencing of multiple sectors from primary and paired recurrent GBM tumors to reconstruct the genomic profile of residual, therapy resistant tumor initiating cells. We found that genetic alteration of the p53 pathway is a primary molecular event predictive of a high number of subclonal mutations in glioblastoma. The genomic road leading to recurrence is highly idiosyncratic but can be broadly classified into linear recurrences that share extensive genetic similarity with the primary tumor and can be directly traced to one of its specific sectors, and divergent recurrences that share few genetic alterations with the primary tumor and originate from cells that branched off early during tumorigenesis. Our study provides mechanistic insights into how genetic alterations in primary tumors impact the ensuing evolution of tumor cells and the emergence of subclonal heterogeneity. © 2015 Kim et al.; Published by Cold Spring Harbor Laboratory Press.

Whole-exome sequencing supports genetic heterogeneity in childhood apraxia of speech.

PubMed

Worthey, Elizabeth A; Raca, Gordana; Laffin, Jennifer J; Wilk, Brandon M; Harris, Jeremy M; Jakielski, Kathy J; Dimmock, David P; Strand, Edythe A; Shriberg, Lawrence D

2013-10-02

Childhood apraxia of speech (CAS) is a rare, severe, persistent pediatric motor speech disorder with associated deficits in sensorimotor, cognitive, language, learning and affective processes. Among other neurogenetic origins, CAS is the disorder segregating with a mutation in FOXP2 in a widely studied, multigenerational London family. We report the first whole-exome sequencing (WES) findings from a cohort of 10 unrelated participants, ages 3 to 19 years, with well-characterized CAS. As part of a larger study of children and youth with motor speech sound disorders, 32 participants were classified as positive for CAS on the basis of a behavioral classification marker using auditory-perceptual and acoustic methods that quantify the competence, precision and stability of a speaker's speech, prosody and voice. WES of 10 randomly selected participants was completed using the Illumina Genome Analyzer IIx Sequencing System. Image analysis, base calling, demultiplexing, read mapping, and variant calling were performed using Illumina software. Software developed in-house was used for variant annotation, prioritization and interpretation to identify those variants likely to be deleterious to neurodevelopmental substrates of speech-language development. Among potentially deleterious variants, clinically reportable findings of interest occurred on a total of five chromosomes (Chr3, Chr6, Chr7, Chr9 and Chr17), which included six genes either strongly associated with CAS (FOXP1 and CNTNAP2) or associated with disorders with phenotypes overlapping CAS (ATP13A4, CNTNAP1, KIAA0319 and SETX). A total of 8 (80%) of the 10 participants had clinically reportable variants in one or two of the six genes, with variants in ATP13A4, KIAA0319 and CNTNAP2 being the most prevalent. Similar to the results reported in emerging WES studies of other complex neurodevelopmental disorders, our findings from this first WES study of CAS are interpreted as support for heterogeneous genetic origins of

What are Whole Exome Sequencing and Whole Genome Sequencing?

MedlinePlus

... the future. For more information about DNA sequencing technologies and their use: Genetics Home Reference discusses whether ... University in St. Louis describes the different sequencing technologies and what the new technologies have meant for ...

The Genome Sequencer FLX System--longer reads, more applications, straight forward bioinformatics and more complete data sets.

PubMed

Droege, Marcus; Hill, Brendon

2008-08-31

The Genome Sequencer FLX System (GS FLX), powered by 454 Sequencing, is a next-generation DNA sequencing technology featuring a unique mix of long reads, exceptional accuracy, and ultra-high throughput. It has been proven to be the most versatile of all currently available next-generation sequencing technologies, supporting many high-profile studies in over seven applications categories. GS FLX users have pursued innovative research in de novo sequencing, re-sequencing of whole genomes and target DNA regions, metagenomics, and RNA analysis. 454 Sequencing is a powerful tool for human genetics research, having recently re-sequenced the genome of an individual human, currently re-sequencing the complete human exome and targeted genomic regions using the NimbleGen sequence capture process, and detected low-frequency somatic mutations linked to cancer.

Whole-exome sequencing identifies novel MPL and JAK2 mutations in triple-negative myeloproliferative neoplasms

PubMed Central

Milosevic Feenstra, Jelena D.; Nivarthi, Harini; Gisslinger, Heinz; Leroy, Emilie; Rumi, Elisa; Chachoua, Ilyas; Bagienski, Klaudia; Kubesova, Blanka; Pietra, Daniela; Gisslinger, Bettina; Milanesi, Chiara; Jäger, Roland; Chen, Doris; Berg, Tiina; Schalling, Martin; Schuster, Michael; Bock, Christoph; Constantinescu, Stefan N.; Cazzola, Mario

2016-01-01

Essential thrombocythemia (ET) and primary myelofibrosis (PMF) are chronic diseases characterized by clonal hematopoiesis and hyperproliferation of terminally differentiated myeloid cells. The disease is driven by somatic mutations in exon 9 of CALR or exon 10 of MPL or JAK2-V617F in >90% of the cases, whereas the remaining cases are termed “triple negative.” We aimed to identify the disease-causing mutations in the triple-negative cases of ET and PMF by applying whole-exome sequencing (WES) on paired tumor and control samples from 8 patients. We found evidence of clonal hematopoiesis in 5 of 8 studied cases based on clonality analysis and presence of somatic genetic aberrations. WES identified somatic mutations in 3 of 8 cases. We did not detect any novel recurrent somatic mutations. In 3 patients with clonal hematopoiesis analyzed by WES, we identified a somatic MPL-S204P, a germline MPL-V285E mutation, and a germline JAK2-G571S variant. We performed Sanger sequencing of the entire coding region of MPL in 62, and of JAK2 in 49 additional triple-negative cases of ET or PMF. New somatic (T119I, S204F, E230G, Y591D) and 1 germline (R321W) MPL mutation were detected. All of the identified MPL mutations were gain-of-function when analyzed in functional assays. JAK2 variants were identified in 5 of 57 triple-negative cases analyzed by WES and Sanger sequencing combined. We could demonstrate that JAK2-V625F and JAK2-F556V are gain-of-function mutations. Our results suggest that triple-negative cases of ET and PMF do not represent a homogenous disease entity. Cases with polyclonal hematopoiesis might represent hereditary disorders. PMID:26423830

Whole-exome sequencing identifies novel MPL and JAK2 mutations in triple-negative myeloproliferative neoplasms.

PubMed

Milosevic Feenstra, Jelena D; Nivarthi, Harini; Gisslinger, Heinz; Leroy, Emilie; Rumi, Elisa; Chachoua, Ilyas; Bagienski, Klaudia; Kubesova, Blanka; Pietra, Daniela; Gisslinger, Bettina; Milanesi, Chiara; Jäger, Roland; Chen, Doris; Berg, Tiina; Schalling, Martin; Schuster, Michael; Bock, Christoph; Constantinescu, Stefan N; Cazzola, Mario; Kralovics, Robert

2016-01-21

Essential thrombocythemia (ET) and primary myelofibrosis (PMF) are chronic diseases characterized by clonal hematopoiesis and hyperproliferation of terminally differentiated myeloid cells. The disease is driven by somatic mutations in exon 9 of CALR or exon 10 of MPL or JAK2-V617F in >90% of the cases, whereas the remaining cases are termed "triple negative." We aimed to identify the disease-causing mutations in the triple-negative cases of ET and PMF by applying whole-exome sequencing (WES) on paired tumor and control samples from 8 patients. We found evidence of clonal hematopoiesis in 5 of 8 studied cases based on clonality analysis and presence of somatic genetic aberrations. WES identified somatic mutations in 3 of 8 cases. We did not detect any novel recurrent somatic mutations. In 3 patients with clonal hematopoiesis analyzed by WES, we identified a somatic MPL-S204P, a germline MPL-V285E mutation, and a germline JAK2-G571S variant. We performed Sanger sequencing of the entire coding region of MPL in 62, and of JAK2 in 49 additional triple-negative cases of ET or PMF. New somatic (T119I, S204F, E230G, Y591D) and 1 germline (R321W) MPL mutation were detected. All of the identified MPL mutations were gain-of-function when analyzed in functional assays. JAK2 variants were identified in 5 of 57 triple-negative cases analyzed by WES and Sanger sequencing combined. We could demonstrate that JAK2-V625F and JAK2-F556V are gain-of-function mutations. Our results suggest that triple-negative cases of ET and PMF do not represent a homogenous disease entity. Cases with polyclonal hematopoiesis might represent hereditary disorders. © 2016 by The American Society of Hematology.

Whole-exome sequencing identifies recurrent AKT1 mutations in sclerosing hemangioma of lung

PubMed Central

Jung, Seung-Hyun; Kim, Min Sung; Lee, Sung-Hak; Park, Hyun-Chun; Choi, Hyun Joo; Maeng, Leeso; Min, Ki Ouk; Kim, Jeana; Park, Tae In; Shin, Ok Ran; Kim, Tae-Jung; Xu, Haidong; Lee, Kyo Young; Kim, Tae-Min; Song, Sang Yong; Lee, Charles; Chung, Yeun-Jun; Lee, Sug Hyung

2016-01-01

Pulmonary sclerosing hemangioma (PSH) is a benign tumor with two cell populations (epithelial and stromal cells), for which genomic profiles remain unknown. We conducted exome sequencing of 44 PSHs and identified recurrent somatic mutations of AKT1 (43.2%) and β-catenin (4.5%). We used a second subset of 24 PSHs to confirm the high frequency of AKT1 mutations (overall 31/68, 45.6%; p.E17K, 33.8%) and recurrent β-catenin mutations (overall 3 of 68, 4.4%). Of the PSHs without AKT1 mutations, two exhibited AKT1 copy gain. AKT1 mutations existed in both epithelial and stromal cells. In two separate PSHs from one patient, we observed two different AKT1 mutations, indicating they were not disseminated but independent arising tumors. Because the AKT1 mutations were not found to co-occur with β-catenin mutations (or any other known driver alterations) in any of the PSHs studied, we speculate that this may be the single-most common driver alteration to develop PSHs. Our study revealed genomic differences between PSHs and lung adenocarcinomas, including a high rate of AKT1 mutation in PSHs. These genomic features of PSH identified in the present study provide clues to understanding the biology of PSH and for differential genomic diagnosis of lung tumors. PMID:27601661

Novel compound heterozygous mutations in the GPR98 (USH2C) gene identified by whole exome sequencing in a Moroccan deaf family.

PubMed

Bousfiha, Amale; Bakhchane, Amina; Charoute, Hicham; Detsouli, Mustapha; Rouba, Hassan; Charif, Majida; Lenaers, Guy; Barakat, Abdelhamid

2017-10-01

In the present work, we identified two novel compound heterozygote mutations in the GPR98 (G protein-coupled receptor 98) gene causing Usher syndrome. Whole-exome sequencing was performed to study the genetic causes of Usher syndrome in a Moroccan family with three affected siblings. We identify two novel compound heterozygote mutations (c.1054C > A, c.16544delT) in the GPR98 gene in the three affected siblings carrying post-linguale bilateral moderate hearing loss with normal vestibular functions and before installing visual disturbances. This is the first time that mutations in the GPR98 gene are described in the Moroccan deaf patients.

Whole-exome sequencing identified a homozygous FNBP4 mutation in a family with a condition similar to microphthalmia with limb anomalies.

PubMed

Kondo, Yukiko; Koshimizu, Eriko; Megarbane, Andre; Hamanoue, Haruka; Okada, Ippei; Nishiyama, Kiyomi; Kodera, Hirofumi; Miyatake, Satoko; Tsurusaki, Yoshinori; Nakashima, Mitsuko; Doi, Hiroshi; Miyake, Noriko; Saitsu, Hirotomo; Matsumoto, Naomichi

2013-07-01

Microphthalmia with limb anomalies (MLA), also known as Waardenburg anophthalmia syndrome or ophthalmoacromelic syndrome, is a rare autosomal recessive disorder. Recently, we and others successfully identified SMOC1 as the causative gene for MLA. However, there are several MLA families without SMOC1 abnormality, suggesting locus heterogeneity in MLA. We aimed to identify a pathogenic mutation in one Lebanese family having an MLA-like condition without SMOC1 mutation by whole-exome sequencing (WES) combined with homozygosity mapping. A c.683C>T (p.Thr228Met) in FNBP4 was found as a primary candidate, drawing the attention that FNBP4 and SMOC1 may potentially modulate BMP signaling. Copyright © 2013 Wiley Periodicals, Inc.

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.

Whole-Exome Sequencing in a South American Cohort Links ALDH1A3, FOXN1 and Retinoic Acid Regulation Pathways to Autism Spectrum Disorders.

PubMed

Moreno-Ramos, Oscar A; Olivares, Ana María; Haider, Neena B; de Autismo, Liga Colombiana; Lattig, María Claudia

2015-01-01

Autism spectrum disorders (ASDs) are a range of complex neurodevelopmental conditions principally characterized by dysfunctions linked to mental development. Previous studies have shown that there are more than 1000 genes likely involved in ASD, expressed mainly in brain and highly interconnected among them. We applied whole exome sequencing in Colombian-South American trios. Two missense novel SNVs were found in the same child: ALDH1A3 (RefSeq NM_000693: c.1514T>C (p.I505T)) and FOXN1 (RefSeq NM_003593: c.146C>T (p.S49L)). Gene expression studies reveal that Aldh1a3 and Foxn1 are expressed in ~E13.5 mouse embryonic brain, as well as in adult piriform cortex (PC; ~P30). Conserved Retinoic Acid Response Elements (RAREs) upstream of human ALDH1A3 and FOXN1 and in mouse Aldh1a3 and Foxn1 genes were revealed using bioinformatic approximation. Chromatin immunoprecipitation (ChIP) assay using Retinoid Acid Receptor B (Rarb) as the immunoprecipitation target suggests RA regulation of Aldh1a3 and Foxn1 in mice. Our results frame a possible link of RA regulation in brain to ASD etiology, and a feasible non-additive effect of two apparently unrelated variants in ALDH1A3 and FOXN1 recognizing that every result given by next generation sequencing should be cautiously analyzed, as it might be an incidental finding.

Whole-Exome Sequencing in a South American Cohort Links ALDH1A3, FOXN1 and Retinoic Acid Regulation Pathways to Autism Spectrum Disorders

PubMed Central

Moreno-Ramos, Oscar A.; Olivares, Ana María; Haider, Neena B.; de Autismo, Liga Colombiana; Lattig, María Claudia

2015-01-01

Autism spectrum disorders (ASDs) are a range of complex neurodevelopmental conditions principally characterized by dysfunctions linked to mental development. Previous studies have shown that there are more than 1000 genes likely involved in ASD, expressed mainly in brain and highly interconnected among them. We applied whole exome sequencing in Colombian—South American trios. Two missense novel SNVs were found in the same child: ALDH1A3 (RefSeq NM_000693: c.1514T>C (p.I505T)) and FOXN1 (RefSeq NM_003593: c.146C>T (p.S49L)). Gene expression studies reveal that Aldh1a3 and Foxn1 are expressed in ~E13.5 mouse embryonic brain, as well as in adult piriform cortex (PC; ~P30). Conserved Retinoic Acid Response Elements (RAREs) upstream of human ALDH1A3 and FOXN1 and in mouse Aldh1a3 and Foxn1 genes were revealed using bioinformatic approximation. Chromatin immunoprecipitation (ChIP) assay using Retinoid Acid Receptor B (Rarb) as the immunoprecipitation target suggests RA regulation of Aldh1a3 and Foxn1 in mice. Our results frame a possible link of RA regulation in brain to ASD etiology, and a feasible non-additive effect of two apparently unrelated variants in ALDH1A3 and FOXN1 recognizing that every result given by next generation sequencing should be cautiously analyzed, as it might be an incidental finding. PMID:26352270

Panel-based whole exome sequencing identifies novel mutations in microphthalmia and anophthalmia patients showing complex Mendelian inheritance patterns.

PubMed

Riera, Marina; Wert, Ana; Nieto, Isabel; Pomares, Esther

2017-11-01

Microphthalmia and anophthalmia (MA) are congenital eye abnormalities that show an extremely high clinical and genetic complexity. In this study, we evaluated the implementation of whole exome sequencing (WES) for the genetic analysis of MA patients. This approach was used to investigate three unrelated families in which previous single-gene analyses failed to identify the molecular cause. A total of 47 genes previously associated with nonsyndromic MA were included in our panel. WES was performed in one affected patient from each family using the AmpliSeq TM Exome technology and the Ion Proton TM platform. A novel heterozygous OTX2 missense mutation was identified in a patient showing bilateral anophthalmia who inherited the variant from a parent who was a carrier, but showed no sign of the condition. We also describe a new PAX6 missense variant in an autosomal-dominant pedigree affected by mild bilateral microphthalmia showing high intrafamiliar variability, with germline mosaicism determined to be the most plausible molecular cause of the disease. Finally, a heterozygous missense mutation in RBP4 was found to be responsible in an isolated case of bilateral complex microphthalmia. This study highlights that panel-based WES is a reliable and effective strategy for the genetic diagnosis of MA. Furthermore, using this technique, the mutational spectrum of these diseases was broadened, with novel variants identified in each of the OTX2, PAX6, and RBP4 genes. Moreover, we report new cases of reduced penetrance, mosaicism, and variable phenotypic expressivity associated with MA, further demonstrating the heterogeneity of such disorders. © 2017 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.

Evaluation of three read-depth based CNV detection tools using whole-exome sequencing data.

PubMed

Yao, Ruen; Zhang, Cheng; Yu, Tingting; Li, Niu; Hu, Xuyun; Wang, Xiumin; Wang, Jian; Shen, Yiping

2017-01-01

Whole exome sequencing (WES) has been widely accepted as a robust and cost-effective approach for clinical genetic testing of small sequence variants. Detection of copy number variants (CNV) within WES data have become possible through the development of various algorithms and software programs that utilize read-depth as the main information. The aim of this study was to evaluate three commonly used, WES read-depth based CNV detection programs using high-resolution chromosomal microarray analysis (CMA) as a standard. Paired CMA and WES data were acquired for 45 samples. A total of 219 CNVs (size ranged from 2.3 kb - 35 mb) identified on three CMA platforms (Affymetrix, Agilent and Illumina) were used as standards. CNVs were called from WES data using XHMM, CoNIFER, and CNVnator with modified settings. All three software packages detected an elevated proportion of small variants (< 20 kb) compared to CMA. XHMM and CoNIFER had poor detection sensitivity (22.2 and 14.6%), which correlated with the number of capturing probes involved. CNVnator detected most variants and had better sensitivity (87.7%); however, suffered from an overwhelming detection of small CNVs below 20 kb, which required further confirmation. Size estimation of variants was exaggerated by CNVnator and understated by XHMM and CoNIFER. Low concordances of CNV, detected by three different read-depth based programs, indicate the immature status of WES-based CNV detection. Low sensitivity and uncertain specificity of WES-based CNV detection in comparison with CMA based CNV detection suggests that CMA will continue to play an important role in detecting clinical grade CNV in the NGS era, which is largely based on WES.

A clinical utility study of exome sequencing versus conventional genetic testing in pediatric neurology.

PubMed

Vissers, Lisenka E L M; van Nimwegen, Kirsten J M; Schieving, Jolanda H; Kamsteeg, Erik-Jan; Kleefstra, Tjitske; Yntema, Helger G; Pfundt, Rolph; van der Wilt, Gert Jan; Krabbenborg, Lotte; Brunner, Han G; van der Burg, Simone; Grutters, Janneke; Veltman, Joris A; Willemsen, Michèl A A P

2017-09-01

Implementation of novel genetic diagnostic tests is generally driven by technological advances because they promise shorter turnaround times and/or higher diagnostic yields. Other aspects, including impact on clinical management or cost-effectiveness, are often not assessed in detail prior to implementation. We studied the clinical utility of whole-exome sequencing (WES) in complex pediatric neurology in terms of diagnostic yield and costs. We analyzed 150 patients (and their parents) presenting with complex neurological disorders of suspected genetic origin. In a parallel study, all patients received both the standard diagnostic workup (e.g., cerebral imaging, muscle biopsies or lumbar punctures, and sequential gene-by-gene-based testing) and WES simultaneously. Our unique study design allowed direct comparison of diagnostic yield of both trajectories and provided insight into the economic implications of implementing WES in this diagnostic trajectory. We showed that WES identified significantly more conclusive diagnoses (29.3%) than the standard care pathway (7.3%) without incurring higher costs. Exploratory analysis of WES as a first-tier diagnostic test indicates that WES may even be cost-saving, depending on the extent of other tests being omitted. Our data support such a use of WES in pediatric neurology for disorders of presumed genetic origin.Genet Med advance online publication 23 March 2017.

High diagnostic yield of clinical exome sequencing in Middle Eastern patients with Mendelian disorders.

PubMed

Yavarna, Tarunashree; Al-Dewik, Nader; Al-Mureikhi, Mariam; Ali, Rehab; Al-Mesaifri, Fatma; Mahmoud, Laila; Shahbeck, Noora; Lakhani, Shenela; AlMulla, Mariam; Nawaz, Zafar; Vitazka, Patrik; Alkuraya, Fowzan S; Ben-Omran, Tawfeg

2015-09-01

Clinical exome sequencing (CES) has become an increasingly popular diagnostic tool in patients with heterogeneous genetic disorders, especially in those with neurocognitive phenotypes. Utility of CES in consanguineous populations has not yet been determined on a large scale. A clinical cohort of 149 probands from Qatar with suspected Mendelian, mainly neurocognitive phenotypes, underwent CES from July 2012 to June 2014. Intellectual disability and global developmental delay were the most common clinical presentations but our cohort displayed other phenotypes, such as epilepsy, dysmorphism, microcephaly and other structural brain anomalies and autism. A pathogenic or likely pathogenic mutation, including pathogenic CNVs, was identified in 89 probands for a diagnostic yield of 60%. Consanguinity and positive family history predicted a higher diagnostic yield. In 5% (7/149) of cases, CES implicated novel candidate disease genes (MANF, GJA9, GLG1, COL15A1, SLC35F5, MAGE4, NEUROG1). CES uncovered two coexisting genetic disorders in 4% (6/149) and actionable incidental findings in 2% (3/149) of cases. Average time to diagnosis was reduced from 27 to 5 months. CES, which already has the highest diagnostic yield among all available diagnostic tools in the setting of Mendelian disorders, appears to be particularly helpful diagnostically in the highly consanguineous Middle Eastern population.

Whole exome sequencing identifies mutations in Usher syndrome genes in profoundly deaf Tunisian patients.

PubMed

Riahi, Zied; Bonnet, Crystel; Zainine, Rim; Lahbib, Saida; Bouyacoub, Yosra; Bechraoui, Rym; Marrakchi, Jihène; Hardelin, Jean-Pierre; Louha, Malek; Largueche, Leila; Ben Yahia, Salim; Kheirallah, Moncef; Elmatri, Leila; Besbes, Ghazi; Abdelhak, Sonia; Petit, Christine

2015-01-01

Usher syndrome (USH) is an autosomal recessive disorder characterized by combined deafness-blindness. It accounts for about 50% of all hereditary deafness blindness cases. Three clinical subtypes (USH1, USH2, and USH3) are described, of which USH1 is the most severe form, characterized by congenital profound deafness, constant vestibular dysfunction, and a prepubertal onset of retinitis pigmentosa. We performed whole exome sequencing in four unrelated Tunisian patients affected by apparently isolated, congenital profound deafness, with reportedly normal ocular fundus examination. Four biallelic mutations were identified in two USH1 genes: a splice acceptor site mutation, c.2283-1G>T, and a novel missense mutation, c.5434G>A (p.Glu1812Lys), in MYO7A, and two previously unreported mutations in USH1G, i.e. a frameshift mutation, c.1195_1196delAG (p.Leu399Alafs*24), and a nonsense mutation, c.52A>T (p.Lys18*). Another ophthalmological examination including optical coherence tomography actually showed the presence of retinitis pigmentosa in all the patients. Our findings provide evidence that USH is under-diagnosed in Tunisian deaf patients. Yet, early diagnosis of USH is of utmost importance because these patients should undergo cochlear implant surgery in early childhood, in anticipation of the visual loss.

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.

Nicotine and oxidative stress induced exomic variations are concordant and overrepresented in cancer-associated genes

PubMed Central

Bavarva, Jasmin H.; Tae, Hongseok; McIver, Lauren; Garner, Harold R.

2014-01-01

Although the connection between cancer and cigarette smoke is well established, nicotine is not characterized as a carcinogen. Here, we used exome sequencing to identify nicotine and oxidative stress-induced somatic mutations in normal human epithelial cells and its correlation with cancer. We identified over 6,400 SNVs, indels and microsatellites in each of the stress exposed cells relative to the control, of which, 2,159 were consistently observed at all nicotine doses. These included 429 nsSNVs including 158 novel and 79 cancer-associated. Over 80% of consistently nicotine induced variants overlap with variations detected in oxidative stressed cells, indicating that nicotine induced genomic alterations could be mediated through oxidative stress. Nicotine induced mutations were distributed across 1,585 genes, of which 49% were associated with cancer. MUC family genes were among the top mutated genes. Analysis of 591 lung carcinoma tumor exomes from The Cancer Genome Atlas (TCGA) revealed that 20% of non-small-cell lung cancer tumors in smokers have mutations in at least one of the MUC4, MUC6 or MUC12 genes in contrast to only 6% in non-smokers. These results indicate that nicotine induces genomic variations, promotes instability potentially mediated by oxidative stress, implicating nicotine in carcinogenesis, and establishes MUC genes as potential targets. PMID:24947164

Whole-exome sequencing, without prior linkage, identifies a mutation in LAMB3 as a cause of dominant hypoplastic amelogenesis imperfecta.

PubMed

Poulter, James A; El-Sayed, Walid; Shore, Roger C; Kirkham, Jennifer; Inglehearn, Chris F; Mighell, Alan J

2014-01-01

The conventional approach to identifying the defective gene in a family with an inherited disease is to find the disease locus through family studies. However, the rapid development and decreasing cost of next generation sequencing facilitates a more direct approach. Here, we report the identification of a frameshift mutation in LAMB3 as a cause of dominant hypoplastic amelogenesis imperfecta (AI). Whole-exome sequencing of three affected family members and subsequent filtering of shared variants, without prior genetic linkage, sufficed to identify the pathogenic variant. Simultaneous analysis of multiple family members confirms segregation, enhancing the power to filter the genetic variation found and leading to rapid identification of the pathogenic variant. LAMB3 encodes a subunit of Laminin-5, one of a family of basement membrane proteins with essential functions in cell growth, movement and adhesion. Homozygous LAMB3 mutations cause junctional epidermolysis bullosa (JEB) and enamel defects are seen in JEB cases. However, to our knowledge, this is the first report of dominant AI due to a LAMB3 mutation in the absence of JEB.

Whole Exome Analysis of Early Onset Alzheimer’s Disease

DTIC Science & Technology

2013-04-01

FTD), FTD with Parkinsonism , and early-onset Alzheimer Disease (EOAD)-like presentations. Using whole exome capture with subsequent sequencing, we...dementia. The MAPT R406W mutation is associated with EOAD-like symptoms and Parkinsonism without FTD, as well as distinct cognitive courses. KEY...OUTCOMES: Carney RM, Kohli MA, Kunkle BW, Naj AC, Gilbert JR, Züchner S, PERICAK-VANCE MA, Parkinsonism and distinct dementia patterns in a

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

A programmable method for massively parallel targeted sequencing

PubMed Central

Hopmans, Erik S.; Natsoulis, Georges; Bell, John M.; Grimes, Susan M.; Sieh, Weiva; Ji, Hanlee P.

2014-01-01

We have developed a targeted resequencing approach referred to as Oligonucleotide-Selective Sequencing. In this study, we report a series of significant improvements and novel applications of this method whereby the surface of a sequencing flow cell is modified in situ to capture specific genomic regions of interest from a sample and then sequenced. These improvements include a fully automated targeted sequencing platform through the use of a standard Illumina cBot fluidics station. Targeting optimization increased the yield of total on-target sequencing data 2-fold compared to the previous iteration, while simultaneously increasing the percentage of reads that could be mapped to the human genome. The described assays cover up to 1421 genes with a total coverage of 5.5 Megabases (Mb). We demonstrate a 10-fold abundance uniformity of greater than 90% in 1 log distance from the median and a targeting rate of up to 95%. We also sequenced continuous genomic loci up to 1.5 Mb while simultaneously genotyping SNPs and genes. Variants with low minor allele fraction were sensitively detected at levels of 5%. Finally, we determined the exact breakpoint sequence of cancer rearrangements. Overall, this approach has high performance for selective sequencing of genome targets, configuration flexibility and variant calling accuracy. PMID:24782526

Identification of Novel Single Nucleotide Polymorphisms Associated with Acute Respiratory Distress Syndrome by Exome-Seq

PubMed Central

Shortt, Katherine; Chaudhary, Suman; Grigoryev, Dmitry; Heruth, Daniel P.; Venkitachalam, Lakshmi; Zhang, Li Q.; Ye, Shui Q.

2014-01-01

Acute respiratory distress syndrome (ARDS) is a lung condition characterized by impaired gas exchange with systemic release of inflammatory mediators, causing pulmonary inflammation, vascular leak and hypoxemia. Existing biomarkers have limited effectiveness as diagnostic and therapeutic targets. To identify disease-associating variants in ARDS patients, whole-exome sequencing was performed on 96 ARDS patients, detecting 1,382,399 SNPs. By comparing these exome data to those of the 1000 Genomes Project, we identified a number of single nucleotide polymorphisms (SNP) which are potentially associated with ARDS. 50,190SNPs were found in all case subgroups and controls, of which89 SNPs were associated with susceptibility. We validated three SNPs (rs78142040, rs9605146 and rs3848719) in additional ARDS patients to substantiate their associations with susceptibility, severity and outcome of ARDS. rs78142040 (C>T) occurs within a histone mark (intron 6) of the Arylsulfatase D gene. rs9605146 (G>A) causes a deleterious coding change (proline to leucine) in the XK, Kell blood group complex subunit-related family, member 3 gene. rs3848719 (G>A) is a synonymous SNP in the Zinc-Finger/Leucine-Zipper Co-Transducer NIF1 gene. rs78142040, rs9605146, and rs3848719 are associated significantly with susceptibility to ARDS. rs3848719 is associated with APACHE II score quartile. rs78142040 is associated with 60-day mortality in the overall ARDS patient population. Exome-seq is a powerful tool to identify potential new biomarkers for ARDS. We selectively validated three SNPs which have not been previously associated with ARDS and represent potential new genetic biomarkers for ARDS. Additional validation in larger patient populations and further exploration of underlying molecular mechanisms are warranted. PMID:25372662

Exome sequencing identifies complex I NDUFV2 mutations as a novel cause of Leigh syndrome.

PubMed

Cameron, Jessie M; MacKay, Nevena; Feigenbaum, Annette; Tarnopolsky, Mark; Blaser, Susan; Robinson, Brian H; Schulze, Andreas

2015-09-01

Two siblings with hypertrophic cardiomyopathy and brain atrophy were diagnosed with Complex I deficiency based on low enzyme activity in muscle and high lactate/pyruvate ratio in fibroblasts. Whole exome sequencing results of fibroblast gDNA from one sibling was narrowed down to 190 SNPs or In/Dels in 185 candidate genes by selecting non-synonymous coding sequence base pair changes that were not present in the SNP database. Two compound heterozygous mutations were identified in both siblings in NDUFV2, encoding the 24 kDa subunit of Complex I. The intronic mutation (c.IVS2 + 1delGTAA) is disease causing and has been reported before. The other mutation is novel (c.669_670insG, p.Ser224Valfs*3) and predicted to cause a pathogenic frameshift in the protein. Subsequent investigation of 10 probands with complex I deficiency from different families revealed homozygosity for the intronic c.IVS2 + 1delGTAA mutation in a second, consanguineous family. In this family three of five siblings were affected. Interestingly, they presented with Leigh syndrome but no cardiac involvement. The same genotype had been reported previously in a two families but presenting with hypertrophic cardiomyopathy, trunk hypotonia and encephalopathy. We have identified NDUFV2 mutations in two families with Complex I deficiency, including a novel mutation. The diagnosis of Leigh syndrome expands the clinical phenotypes associated with the c.IVS2 + 1delGTAA mutation in this gene. Copyright © 2015 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.

Mutation Clusters from Cancer Exome.

PubMed

Kakushadze, Zura; Yu, Willie

2017-08-15

We apply our statistically deterministic machine learning/clustering algorithm *K-means (recently developed in https://ssrn.com/abstract=2908286) to 10,656 published exome samples for 32 cancer types. A majority of cancer types exhibit a mutation clustering structure. Our results are in-sample stable. They are also out-of-sample stable when applied to 1389 published genome samples across 14 cancer types. In contrast, we find in- and out-of-sample instabilities in cancer signatures extracted from exome samples via nonnegative matrix factorization (NMF), a computationally-costly and non-deterministic method. Extracting stable mutation structures from exome data could have important implications for speed and cost, which are critical for early-stage cancer diagnostics, such as novel blood-test methods currently in development.

Mutation Clusters from Cancer Exome

PubMed Central

Kakushadze, Zura; Yu, Willie

2017-01-01

We apply our statistically deterministic machine learning/clustering algorithm *K-means (recently developed in https://ssrn.com/abstract=2908286) to 10,656 published exome samples for 32 cancer types. A majority of cancer types exhibit a mutation clustering structure. Our results are in-sample stable. They are also out-of-sample stable when applied to 1389 published genome samples across 14 cancer types. In contrast, we find in- and out-of-sample instabilities in cancer signatures extracted from exome samples via nonnegative matrix factorization (NMF), a computationally-costly and non-deterministic method. Extracting stable mutation structures from exome data could have important implications for speed and cost, which are critical for early-stage cancer diagnostics, such as novel blood-test methods currently in development. PMID:28809811

Analysis and Visualization Tool for Targeted Amplicon Bisulfite Sequencing on Ion Torrent Sequencers

PubMed Central

Pabinger, Stephan; Ernst, Karina; Pulverer, Walter; Kallmeyer, Rainer; Valdes, Ana M.; Metrustry, Sarah; Katic, Denis; Nuzzo, Angelo; Kriegner, Albert; Vierlinger, Klemens; Weinhaeusel, Andreas

2016-01-01

Targeted sequencing of PCR amplicons generated from bisulfite deaminated DNA is a flexible, cost-effective way to study methylation of a sample at single CpG resolution and perform subsequent multi-target, multi-sample comparisons. Currently, no platform specific protocol, support, or analysis solution is provided to perform targeted bisulfite sequencing on a Personal Genome Machine (PGM). Here, we present a novel tool, called TABSAT, for analyzing targeted bisulfite sequencing data generated on Ion Torrent sequencers. The workflow starts with raw sequencing data, performs quality assessment, and uses a tailored version of Bismark to map the reads to a reference genome. The pipeline visualizes results as lollipop plots and is able to deduce specific methylation-patterns present in a sample. The obtained profiles are then summarized and compared between samples. In order to assess the performance of the targeted bisulfite sequencing workflow, 48 samples were used to generate 53 different Bisulfite-Sequencing PCR amplicons from each sample, resulting in 2,544 amplicon targets. We obtained a mean coverage of 282X using 1,196,822 aligned reads. Next, we compared the sequencing results of these targets to the methylation level of the corresponding sites on an Illumina 450k methylation chip. The calculated average Pearson correlation coefficient of 0.91 confirms the sequencing results with one of the industry-leading CpG methylation platforms and shows that targeted amplicon bisulfite sequencing provides an accurate and cost-efficient method for DNA methylation studies, e.g., to provide platform-independent confirmation of Illumina Infinium 450k methylation data. TABSAT offers a novel way to analyze data generated by Ion Torrent instruments and can also be used with data from the Illumina MiSeq platform. It can be easily accessed via the Platomics platform, which offers a web-based graphical user interface along with sample and parameter storage. TABSAT is freely

NCI-60 Whole Exome Sequencing and Pharmacological CellMiner Analyses

PubMed Central

Reinhold, William C.; Varma, Sudhir; Sousa, Fabricio; Sunshine, Margot; Abaan, Ogan D.; Davis, Sean R.; Reinhold, Spencer W.; Kohn, Kurt W.; Morris, Joel; Meltzer, Paul S.; Doroshow, James H.; Pommier, Yves

2014-01-01

Exome sequencing provides unprecedented insights into cancer biology and pharmacological response. Here we assess these two parameters for the NCI-60, which is among the richest genomic and pharmacological publicly available cancer cell line databases. Homozygous genetic variants that putatively affect protein function were identified in 1,199 genes (approximately 6% of all genes). Variants that are either enriched or depleted compared to non-cancerous genomes, and thus may be influential in cancer progression and differential drug response were identified for 2,546 genes. Potential gene knockouts are made available. Assessment of cell line response to 19,940 compounds, including 110 FDA-approved drugs, reveals ≈80-fold range in resistance versus sensitivity response across cell lines. 103,422 gene variants were significantly correlated with at least one compound (at p<0.0002). These include genes of known pharmacological importance such as IGF1R, BRAF, RAD52, MTOR, STAT2 and TSC2 as well as a large number of candidate genes such as NOM1, TLL2, and XDH. We introduce two new web-based CellMiner applications that enable exploration of variant-to-compound relationships for a broad range of researchers, especially those without bioinformatics support. The first tool, “Genetic variant versus drug visualization”, provides a visualization of significant correlations between drug activity-gene variant combinations. Examples are given for the known vemurafenib-BRAF, and novel ifosfamide-RAD52 pairings. The second, “Genetic variant summation” allows an assessment of cumulative genetic variations for up to 150 combined genes together; and is designed to identify the variant burden for molecular pathways or functional grouping of genes. An example of its use is provided for the EGFR-ERBB2 pathway gene variant data and the identification of correlated EGFR, ERBB2, MTOR, BRAF, MEK and ERK inhibitors. The new tools are implemented as an updated web-based Cell

A comprehensive iterative approach is highly effective in diagnosing individuals who are exome negative.

PubMed

Shashi, Vandana; Schoch, Kelly; Spillmann, Rebecca; Cope, Heidi; Tan, Queenie K-G; Walley, Nicole; Pena, Loren; McConkie-Rosell, Allyn; Jiang, Yong-Hui; Stong, Nicholas; Need, Anna C; Goldstein, David B

2018-06-15

Sixty to seventy-five percent of individuals with rare and undiagnosed phenotypes remain undiagnosed after exome sequencing (ES). With standard ES reanalysis resolving 10-15% of the ES negatives, further approaches are necessary to maximize diagnoses in these individuals. In 38 ES negative patients an individualized genomic-phenotypic approach was employed utilizing (1) phenotyping; (2) reanalyses of FASTQ files, with innovative bioinformatics; (3) targeted molecular testing; (4) genome sequencing (GS); and (5) conferring of clinical diagnoses when pathognomonic clinical findings occurred. Certain and highly likely diagnoses were made in 18/38 (47%) individuals, including identifying two new developmental disorders. The majority of diagnoses (>70%) were due to our bioinformatics, phenotyping, and targeted testing identifying variants that were undetected or not prioritized on prior ES. GS diagnosed 3/18 individuals with structural variants not amenable to ES. Additionally, tentative diagnoses were made in 3 (8%), and in 5 individuals (13%) candidate genes were identified. Overall, diagnoses/potential leads were identified in 26/38 (68%). Our comprehensive approach to ES negatives maximizes the ES and clinical data for both diagnoses and candidate gene identification, without GS in the majority. This iterative approach is cost-effective and is pertinent to the current conundrum of ES negatives.

Exome analysis in clinical practice: expanding the phenotype of Bartsocas-Papas syndrome.

PubMed

Gripp, Karen W; Ennis, Sara; Napoli, Joseph

2013-05-01

Exome analysis has had a dramatic impact on genetic research. We present the application of such newly generated information to patient care. The patient was a female, born with normal growth parameters to nonconsanguineous parents after an uneventful pregnancy. She had bilateral cleft lip/palate and ankyloblepharon. Sparse hair, dysplastic nails and hypohidrosis were subsequently noted. With exception of speech related issues, her development was normal. A clinical diagnosis of ankyloblepharon-ectodermal defects-cleft lip/palate or Hay-Wells syndrome resulted in TP63 sequence analysis. TP63 sequence and deletion/duplication analysis of all coding exons had a normal result, as did chromosome and SNP array analysis. Diagnostic exome analysis revealed a heterozygous nonsense mutation in KRT83 categorized as deleterious and associated with monilethrix. In addition, a homozygous missense variant of unknown clinical significance was reported in RIPK4. Using research based exome analysis, RIPK4 had just a few months prior been identified as pathogenic for Bartsocas-Papas syndrome. While the clinical diagnostic report implied the KRT83 mutation as a more likely cause for the patient's phenotype, clinical correlation, literature review and use of computerized mutation analysis programs allowed us to identify the homozygous RIPK4 (c.488G > A; p.Gly163Asp) mutation as the underlying pathogenic change. Consequently, we expand the phenotype of Bartsocas-Papas syndrome to an attenuated presentation resembling Hay-Wells syndrome, lacking lethality and pterygia. In contrast to the autosomal dominant Hay-Wells syndrome, Bartsocas-Papas syndrome is autosomal recessive, implying a 25% recurrence risk. Copyright © 2013 Wiley Periodicals, Inc.

Diagnostic Exome Sequencing Identifies a Novel Gene, EMILIN1, Associated with Autosomal-Dominant Hereditary Connective Tissue Disease.

PubMed

Capuano, Alessandra; Bucciotti, Francesco; Farwell, Kelly D; Tippin Davis, Brigette; Mroske, Cameron; Hulick, Peter J; Weissman, Scott M; Gao, Qingshen; Spessotto, Paola; Colombatti, Alfonso; Doliana, Roberto

2016-01-01

Heritable connective tissue diseases are a highly heterogeneous family of over 200 disorders that affect the extracellular matrix. While the genetic basis of several disorders is established, the etiology has not been discovered for a large portion of patients, likely due to rare yet undiscovered disease genes. By performing trio-exome sequencing of a 55-year-old male proband presenting with multiple symptoms indicative of a connective disorder, we identified a heterozygous missense alteration in exon 1 of the Elastin Microfibril Interfacer 1 (EMILIN1) gene, c.64G>A (p.A22T). The proband presented with ascending and descending aortic aneurysms, bilateral lower leg and foot sensorimotor peripheral neuropathy, arthropathy, and increased skin elasticity. Sanger sequencing confirmed that the EMILIN1 alteration, which maps around the signal peptide cleavage site, segregated with disease in the affected proband, mother, and son. The impaired secretion of EMILIN-1 in cells transfected with the mutant p.A22T coincided with abnormal protein accumulation within the endoplasmic reticulum. In skin biopsy of the proband, we detected less EMILIN-1 with disorganized and abnormal coarse fibrils, aggregated deposits underneath the epidermis basal lamina, and dermal cells apoptosis. These findings collectively suggest that EMILIN1 may represent a new disease gene associated with an autosomal-dominant connective tissue disorder. © 2015 The Authors. **Human Mutation published by Wiley Periodicals, Inc.

Whole Exome Sequencing and Heterologous Cellular Electrophysiology Studies Elucidate a Novel Loss-of-Function Mutation in the CACNA1A-Encoded Neuronal P/Q-Type Calcium Channel in a Child With Congenital Hypotonia and Developmental Delay.

PubMed

Weyhrauch, Derek L; Ye, Dan; Boczek, Nicole J; Tester, David J; Gavrilova, Ralitza H; Patterson, Marc C; Wieben, Eric D; Ackerman, Michael J

2016-02-01

A 4-year-old boy born at 37 weeks' gestation with intrauterine growth retardation presented with developmental delay with pronounced language and gross motor delay, axial hypotonia, and dynamic hypertonia of the extremities. Investigations including the Minnesota Newborn Screen, thyroid stimulating hormone/thyroxin, and inborn errors of metabolism screening were negative. Cerebral magnetic resonance imaging and spectroscopy were normal. Genetic testing was negative for coagulopathy, Smith-Lemli-Opitz, fragile X, and Prader-Willi/Angelman syndromes. Whole genome array analysis was unremarkable. Whole exome sequencing was performed through a commercial testing laboratory to elucidate the underlying etiology for the child's presentation. A de novo mutation was hypothesized. In attempt to establish pathogenicity of our candidate variant, cellular electrophysiologic functional analysis of the putative de novo mutation was performed using patch-clamp technology. Whole exome sequencing revealed a p.P1353L variant in the CACNA1A gene, which encodes for the α1-subunit of the brain-specific P/Q-type calcium channel (CaV2.1). This presynaptic high-voltage-gated channel couples neuronal excitation to the vesicular release of neurotransmitter and is implicated in several neurologic disorders. DNA Sanger sequencing confirmed that the de novo mutation was absent in both parents and present in the child only. Electrophysiologic analysis of P1353L-CACNA1A demonstrated near complete loss of function, with a 95% reduction in peak current density. Whole exome sequencing coupled with cellular electrophysiologic functional analysis of a de novoCACNA1A missense mutation has elucidated the probable underlying pathophysiologic mechanism responsible for the child's phenotype. Genetic testing of CACNA1A in patients with congenital hypotonia and developmental delay may be warranted. Copyright © 2016. Published by Elsevier Inc.

Exome sequencing reveals a novel Moroccan founder mutation in SLC19A3 as a new cause of early-childhood fatal Leigh syndrome.

PubMed

Gerards, Mike; Kamps, Rick; van Oevelen, Jo; Boesten, Iris; Jongen, Eveline; de Koning, Bart; Scholte, Hans R; de Angst, Isabel; Schoonderwoerd, Kees; Sefiani, Abdelaziz; Ratbi, Ilham; Coppieters, Wouter; Karim, Latifa; de Coo, René; van den Bosch, Bianca; Smeets, Hubert

2013-03-01

Leigh syndrome is an early onset, often fatal progressive neurodegenerative disorder caused by mutations in the mitochondrial or nuclear DNA. Until now, mutations in more than 35 genes have been reported to cause Leigh syndrome, indicating an extreme genetic heterogeneity for this disorder, but still only explaining part of the cases. The possibility of whole exome sequencing enables not only mutation detection in known candidate genes, but also the identification of new genes associated with Leigh syndrome in small families and isolated cases. Exome sequencing was combined with homozygosity mapping to identify the genetic defect in a Moroccan family with fatal Leigh syndrome in early childhood and specific magnetic resonance imaging abnormalities in the brain. We detected a homozygous nonsense mutation (c.20C>A; p.Ser7Ter) in the thiamine transporter SLC19A3. In vivo overexpression of wild-type SLC19A3 showed an increased thiamine uptake, whereas overexpression of mutant SLC19A3 did not, confirming that the mutation results in an absent or non-functional protein. Seventeen additional patients with Leigh syndrome were screened for mutations in SLC19A3 using conventional Sanger sequencing. Two unrelated patients, both from Moroccan origin and one from consanguineous parents, were homozygous for the same p.Ser7Ter mutation. One of these patients showed the same MRI abnormalities as the patients from the first family. Strikingly, patients receiving thiamine had an improved life-expectancy. One patient in the third family deteriorated upon interruption of the thiamine treatment and recovered after reinitiating. Although unrelated, all patients came from the province Al Hoceima in Northern Morocco. Based on the recombination events the mutation was estimated to have occurred 1250-1750 years ago. Our data shows that SLC19A3 is a new candidate for mutation screening in patients with Leigh syndrome, who might benefit from high doses of thiamine and/or biotin. Especially

Whole-exome Sequencing Identifies Rare Variants in ATP8B4 as a Risk Factor for Systemic Sclerosis

PubMed Central

Gao, Li; Emond, Mary J; Louie, Tin; Cheadle, Chris; Berger, Alan E.; Rafaels, Nicholas; Vergara, Candelaria; Kim, Yoonhee; Taub, Margaret A.; Ruczinski, Ingo; Mathai, Stephen C.; Rich, Stephen S; Nickerson, Deborah A; Hummers, Laura K.; Bamshad, Michael J; Hassoun, Paul M.; Mathias, Rasika A; Barnes, Kathleen C.

2015-01-01

Objective To determine the contribution of rare variants as genetic modifiers of the expressivity, penetrance, and severity of systemic sclerosis (SSc). Methods We performed whole-exome sequencing of 78 European American systemic sclerosis patients, including 35 patients without pulmonary arterial hypertension (SSc-PAH−) and 43 patients with PAH (SSc-PAH+). Association testing of case-control probability for rare variants was performed using the aSKAT-O method with small sample adjustment by comparing all SSc patients with a reference population of 3,179 controls from the ESP 5,500 exome dataset. Replication genotyping was performed in an independent sample of 3,263 patients (415 SSc and 2,848 controls). We conducted expression profiling of mRNA from 61 SSc patients (19 SSc-PAH− and 42 SSc-PAH+) and 41 corresponding controls. Results The ATP8B4 gene was associated with a significant increase in the risk of SSc (P = 3.18 × 10−7). Among the 64 ATP8B4 variants tested, a single missense variant, c.1308C>G (F436L, rs55687265), provided the most compelling evidence for association (P = 9.35 × 10−10; OR = 6.11), which was confirmed in the replication cohort (P = 0.012; OR = 1.86) and meta-analysis (P = 1.92 x 10−7; OR = 2.5). Genes involved in E3 ubiquitin-protein ligase complex (ASB10) and cyclic nucleotide gated channelopathies (CNGB3) as well as HLA-DRB5 and HSPB2 (aka heat shock protein 27) provided additional evidence for association (P < 10−5). Differential ATP8B4 expression was observed among the SSc patients compared to the controls (P = 0.0005). Conclusion ATP8B4 may represent a putative genetic risk factor for SSc and pulmonary vascular complications. PMID:26473621

Exome sequencing identifies a novel SMCHD1 mutation in facioscapulohumeral muscular dystrophy 2.

PubMed

Mitsuhashi, Satomi; Boyden, Steven E; Estrella, Elicia A; Jones, Takako I; Rahimov, Fedik; Yu, Timothy W; Darras, Basil T; Amato, Anthony A; Folkerth, Rebecca D; Jones, Peter L; Kunkel, Louis M; Kang, Peter B

2013-12-01

FSHD2 is a rare form of facioscapulohumeral muscular dystrophy (FSHD) characterized by the absence of a contraction in the D4Z4 macrosatellite repeat region on chromosome 4q35 that is the hallmark of FSHD1. However, hypomethylation of this region is common to both subtypes. Recently, mutations in SMCHD1 combined with a permissive 4q35 allele were reported to cause FSHD2. We identified a novel p.Lys275del SMCHD1 mutation in a family affected with FSHD2 using whole-exome sequencing and linkage analysis. This mutation alters a highly conserved amino acid in the ATPase domain of SMCHD1. Subject III-11 is a male who developed asymmetrical muscle weakness characteristic of FSHD at 13 years. Physical examination revealed marked bilateral atrophy at biceps brachii, bilateral scapular winging, some asymmetrical weakness at tibialis anterior and peroneal muscles, and mild lower facial weakness. Biopsy of biceps brachii in subject II-5, the father of III-11, demonstrated lobulated fibers and dystrophic changes. Endomysial and perivascular inflammation was found, which has been reported in FSHD1 but not FSHD2. Given the previous report of SMCHD1 mutations in FSHD2 and the clinical presentations consistent with the FSHD phenotype, we conclude that the SMCHD1 mutation is the likely cause of the disease in this family. Copyright © 2013 Elsevier B.V. All rights reserved.

Mutational and structural analysis of diffuse large B-cell lymphoma using whole genome sequencing | Office of Cancer Genomics

Cancer.gov

Abstract: Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous cancer comprising at least two molecular subtypes that differ in gene expression and distribution of mutations. Recently, application of genome/exome sequencing and RNA-seq to DLBCL has revealed numerous genes that are recurrent targets of somatic point mutation in this disease.

Uveal melanoma hepatic metastases mutation spectrum analysis using targeted next-generation sequencing of 400 cancer genes.

PubMed

Luscan, A; Just, P A; Briand, A; Burin des Roziers, C; Goussard, P; Nitschké, P; Vidaud, M; Avril, M F; Terris, B; Pasmant, E

2015-04-01

Uveal melanoma (UM) is the most common malignant tumour of the eye. Diagnosis often occurs late in the course of disease, and prognosis is generally poor. Recently, recurrent somatic mutations were described, unravelling additional specific altered pathways in UM. Targeted next-generation sequencing (NGS) can now be applied to an accurate and fast identification of somatic mutations in cancer. The aim of the present study was to characterise the mutation pattern of five UM hepatic metastases with well-defined clinical and pathological features. We analysed the UM mutation spectrum using targeted NGS on 409 cancer genes. Four previous reported genes were found to be recurrently mutated. All tumours presented mutually exclusive GNA11 or GNAQ missense mutations. BAP1 loss-of-function mutations were found in three UMs. SF3B1 missense mutations were found in the two UMs with no BAP1 mutations. We then searched for additional mutation targets. We identified the Arg505Cys mutation in the tumour suppressor FBXW7. The same mutation was previously described in different cancer types, and FBXW7 was recently reported to be mutated in UM exomes. Further studies are required to confirm FBXW7 implication in UM tumorigenesis. Elucidating the molecular mechanisms underlying UM tumorigenesis holds the promise for novel and effective targeted UM therapies. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

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.

Whole exome sequencing of an asbestos-induced wild-type murine model of malignant mesothelioma.

PubMed

Sneddon, Sophie; Patch, Ann-Marie; Dick, Ian M; Kazakoff, Stephen; Pearson, John V; Waddell, Nicola; Allcock, Richard J N; Holt, Robert A; Robinson, Bruce W S; Creaney, Jenette

2017-06-02

Malignant mesothelioma (MM) is an aggressive cancer of the pleural and peritoneal cavities caused by exposure to asbestos. Asbestos-induced mesotheliomas in wild-type mice have been used extensively as a preclinical model because they are phenotypically identical to their human counterpart. However, it is not known if the genetic lesions in these mice tumours are similar to in the human disease, a prerequisite for any new preclinical studies that target genetic abnormalities. We performed whole exome sequencing of fifteen asbestos-induced murine MM tumour cell lines from BALB/c, CBA and C57BL/6 mouse strains and compared the somatic mutations and copy number variations with those recurrently reported in human MM. We then catalogued and characterised the mutational landscape of the wild-type murine MM tumours. Quantitative RT-PCR was used to interrogate the expression of key MM genes of interest in the mRNA. Consistent with human MM tumours, we identified homozygous loss of the tumour suppressor Cdkn2a in 14/15 tumours. One tumour retained the first exon of both of the p16INK4a and p19ARF isoforms though this tumour also contained genetic amplification of Myc resulting in increased expression of the c-Myc proto-oncogene in the mRNA. There were no chromosomal losses in either the Bap1 or Nf2 regions. One tumour harbored homozygous loss of Trp53 in the DNA. Mutation rates were similar in tumours generated in the CBA and C57BL/6 strains when compared to human MM. Interestingly, all BALB/c tumour lines displayed high mutational loads, consistent with the known mutator phenotype of the host strain. The Wnt, MAPK and Jak-STAT signaling pathways were found to be the most commonly affected biological pathways. Mutations and copy number deletions also occurred in the Hedgehog and Hippo pathways. These data suggest that in the wild-type murine model asbestos causes mesotheliomas in a similar way to in human MM. This further supports the notion that the murine model of MM

Homozygous and hemizygous CNV detection from exome sequencing data in a Mendelian disease cohort

PubMed Central

Gambin, Tomasz; Akdemir, Zeynep C.; Yuan, Bo; Gu, Shen; Chiang, Theodore; Carvalho, Claudia M.B.; Shaw, Chad; Jhangiani, Shalini; Boone, Philip M.; Eldomery, Mohammad K.; Karaca, Ender; Bayram, Yavuz; Stray-Pedersen, Asbjørg; Muzny, Donna; Charng, Wu-Lin; Bahrambeigi, Vahid; Belmont, John W.; Boerwinkle, Eric; Beaudet, Arthur L.; Gibbs, Richard A.

2017-01-01

Abstract We developed an algorithm, HMZDelFinder, that uses whole exome sequencing (WES) data to identify rare and intragenic homozygous and hemizygous (HMZ) deletions that may represent complete loss-of-function of the indicated gene. HMZDelFinder was applied to 4866 samples in the Baylor–Hopkins Center for Mendelian Genomics (BHCMG) cohort and detected 773 HMZ deletion calls (567 homozygous or 206 hemizygous) with an estimated sensitivity of 86.5% (82% for single-exonic and 88% for multi-exonic calls) and precision of 78% (53% single-exonic and 96% for multi-exonic calls). Out of 773 HMZDelFinder-detected deletion calls, 82 were subjected to array comparative genomic hybridization (aCGH) and/or breakpoint PCR and 64 were confirmed. These include 18 single-exon deletions out of which 8 were exclusively detected by HMZDelFinder and not by any of seven other CNV detection tools examined. Further investigation of the 64 validated deletion calls revealed at least 15 pathogenic HMZ deletions. Of those, 7 accounted for 17–50% of pathogenic CNVs in different disease cohorts where 7.1–11% of the molecular diagnosis solved rate was attributed to CNVs. In summary, we present an algorithm to detect rare, intragenic, single-exon deletion CNVs using WES data; this tool can be useful for disease gene discovery efforts and clinical WES analyses. PMID:27980096

Military Health Care Dilemmas and Genetic Discrimination: A Family's Experience with Whole Exome Sequencing.

PubMed

Helm, Benjamin M; Langley, Katherine; Spangler, Brooke B; Schrier Vergano, Samantha A

2015-01-01

Whole-exome sequencing (WES) has increased our ability to analyze large parts of the human genome, bringing with it a plethora of ethical, legal, and social implications. A topic dominating discussion of WES is identification of "secondary findings" (SFs), defined as the identification of risk in an asymptomatic individual unrelated to the indication for the test. SFs can have considerable psychosocial impact on patients and families, and patients with an SF may have concerns regarding genomic privacy and genetic discrimination. The Genetic Information Nondiscrimination Act of 2008 (GINA) currently excludes protections for members of the military. This may cause concern in military members and families regarding genetic discrimination when considering genetic testing. In this report, we discuss a case involving a patient and family in which a secondary finding was discovered by WES. The family members have careers in the U.S. military, and a risk-predisposing condition could negatively affect employment. While beneficial medical management changes were made, the information placed exceptional stress on the family, who were forced to navigate career-sensitive "extra-medical" issues, to consider the impacts of uncovering risk-predisposition, and to manage the privacy of their genetic information. We highlight how information obtained from WES may collide with these issues and emphasize the importance of genetic counseling for anyone undergoing WES.

A clinical utility study of exome sequencing versus conventional genetic testing in pediatric neurology

PubMed Central

Vissers, Lisenka E.L.M.; van Nimwegen, Kirsten J.M.; Schieving, Jolanda H.; Kamsteeg, Erik-Jan; Kleefstra, Tjitske; Yntema, Helger G.; Pfundt, Rolph; van der Wilt, Gert Jan; Krabbenborg, Lotte; Brunner, Han G.; van der Burg, Simone; Grutters, Janneke; Veltman, Joris A.; Willemsen, Michèl A.A.P.

2017-01-01

Purpose: Implementation of novel genetic diagnostic tests is generally driven by technological advances because they promise shorter turnaround times and/or higher diagnostic yields. Other aspects, including impact on clinical management or cost-effectiveness, are often not assessed in detail prior to implementation. Methods: We studied the clinical utility of whole-exome sequencing (WES) in complex pediatric neurology in terms of diagnostic yield and costs. We analyzed 150 patients (and their parents) presenting with complex neurological disorders of suspected genetic origin. In a parallel study, all patients received both the standard diagnostic workup (e.g., cerebral imaging, muscle biopsies or lumbar punctures, and sequential gene-by-gene–based testing) and WES simultaneously. Results: Our unique study design allowed direct comparison of diagnostic yield of both trajectories and provided insight into the economic implications of implementing WES in this diagnostic trajectory. We showed that WES identified significantly more conclusive diagnoses (29.3%) than the standard care pathway (7.3%) without incurring higher costs. Exploratory analysis of WES as a first-tier diagnostic test indicates that WES may even be cost-saving, depending on the extent of other tests being omitted. Conclusion: Our data support such a use of WES in pediatric neurology for disorders of presumed genetic origin. Genet Med advance online publication 23 March 2017 PMID:28333917

An evaluation of copy number variation detection tools for cancer using whole exome sequencing data.

PubMed

Zare, Fatima; Dow, Michelle; Monteleone, Nicholas; Hosny, Abdelrahman; Nabavi, Sheida

2017-05-31

Recently copy number variation (CNV) has gained considerable interest as a type of genomic/genetic variation that plays an important role in disease susceptibility. Advances in sequencing technology have created an opportunity for detecting CNVs more accurately. Recently whole exome sequencing (WES) has become primary strategy for sequencing patient samples and study their genomics aberrations. However, compared to whole genome sequencing, WES introduces more biases and noise that make CNV detection very challenging. Additionally, tumors' complexity makes the detection of cancer specific CNVs even more difficult. Although many CNV detection tools have been developed since introducing NGS data, there are few tools for somatic CNV detection for WES data in cancer. In this study, we evaluated the performance of the most recent and commonly used CNV detection tools for WES data in cancer to address their limitations and provide guidelines for developing new ones. We focused on the tools that have been designed or have the ability to detect cancer somatic aberrations. We compared the performance of the tools in terms of sensitivity and false discovery rate (FDR) using real data and simulated data. Comparative analysis of the results of the tools showed that there is a low consensus among the tools in calling CNVs. Using real data, tools show moderate sensitivity (~50% - ~80%), fair specificity (~70% - ~94%) and poor FDRs (~27% - ~60%). Also, using simulated data we observed that increasing the coverage more than 10× in exonic regions does not improve the detection power of the tools significantly. The limited performance of the current CNV detection tools for WES data in cancer indicates the need for developing more efficient and precise CNV detection methods. Due to the complexity of tumors and high level of noise and biases in WES data, employing advanced novel segmentation, normalization and de-noising techniques that are designed specifically for cancer data is

Exome Sequencing Identifies a REEP1 Mutation Involved in Distal Hereditary Motor Neuropathy Type V

PubMed Central

Beetz, Christian; Pieber, Thomas R.; Hertel, Nicole; Schabhüttl, Maria; Fischer, Carina; Trajanoski, Slave; Graf, Elisabeth; Keiner, Silke; Kurth, Ingo; Wieland, Thomas; Varga, Rita-Eva; Timmerman, Vincent; Reilly, Mary M.; Strom, Tim M.; Auer-Grumbach, Michaela

2012-01-01

The distal hereditary motor neuropathies (dHMNs) are a heterogeneous group of neurodegenerative disorders affecting the lower motoneuron. In a family with both autosomal-dominant dHMN and dHMN type V (dHMN/dHMN-V) present in three generations, we excluded mutations in all genes known to be associated with a dHMN phenotype through Sanger sequencing and defined three potential loci through linkage analysis. Whole-exome sequencing of two affected individuals revealed a single candidate variant within the linking regions, i.e., a splice-site alteration in REEP1 (c.304-2A>G). A minigene assay confirmed complete loss of splice-acceptor functionality and skipping of the in-frame exon 5. The resulting mRNA is predicted to be expressed at normal levels and to encode an internally shortened protein (p.102_139del). Loss-of-function REEP1 mutations have previously been identified in dominant hereditary spastic paraplegia (HSP), a disease associated with upper-motoneuron pathology. Consistent with our clinical-genetic data, we show that REEP1 is strongly expressed in the lower motoneurons as well. Upon exogeneous overexpression in cell lines we observe a subcellular localization defect for p.102_139del that differs from that observed for the known HSP-associated missense mutation c.59C>A (p.Ala20Glu). Moreover, we show that p.102_139del, but not p.Ala20Glu, recruits atlastin-1, i.e., one of the REEP1 binding partners, to the altered sites of localization. These data corroborate the loss-of-function nature of REEP1 mutations in HSP and suggest that a different mechanism applies in REEP1-associated dHMN. PMID:22703882

Whole-exome sequencing reveals LRP5 mutations and canonical Wnt signaling associated with hepatic cystogenesis

PubMed Central

Cnossen, Wybrich R.; te Morsche, René H. M.; Hoischen, Alexander; Gilissen, Christian; Chrispijn, Melissa; Venselaar, Hanka; Mehdi, Soufi; Bergmann, Carsten; Veltman, Joris A.; Drenth, Joost P. H.

2014-01-01

Polycystic livers are seen in the rare inherited disorder isolated polycystic liver disease (PCLD) and are recognized as the most common extrarenal manifestation in autosomal dominant polycystic kidney disease. Hepatic cystogenesis is characterized by progressive proliferation of cholangiocytes, ultimately causing hepatomegaly. Genetically, polycystic liver disease is a heterogeneous disorder with incomplete penetrance and caused by mutations in PRKCSH, SEC63, PKD1, or PKD2. Genome-wide SNP typing and Sanger sequencing revealed no pathogenic variants in hitherto genes in an extended PCLD family. We performed whole-exome sequencing of DNA samples from two members. A heterozygous variant c.3562C > T located at a highly conserved amino acid position (p.R1188W) in the low density lipoprotein receptor-related protein 5 (LRP5) gene segregated with the disease (logarithm of odds score, 4.62) but was not observed in more than 1,000 unaffected individuals. Screening of LRP5 in a PCLD cohort identified three additional mutations in three unrelated families with polycystic livers (p.V454M, p.R1529S, and p.D1551N), again all undetected in controls. All variants were predicted to be damaging with profound structural effects on LRP5 protein domains. Liver cyst tissue and normal hepatic tissue samples from patients and controls showed abundant LRP5 expression by immunohistochemistry. Functional activity analyses indicated that mutant LRP5 led to reduced wingless signal activation. In conclusion, we demonstrate that germ-line LRP5 missense mutations are associated with hepatic cystogenesis. The findings presented in this study link the pathophysiology of PCLD to deregulation of the canonical wingless signaling pathway. PMID:24706814

X-exome sequencing identifies a HDAC8 variant in a large pedigree with X-linked intellectual disability, truncal obesity, gynaecomastia, hypogonadism and unusual face.

PubMed

Harakalova, Magdalena; van den Boogaard, Marie-Jose; Sinke, Richard; van Lieshout, Stef; van Tuil, Marc C; Duran, Karen; Renkens, Ivo; Terhal, Paulien A; de Kovel, Carolien; Nijman, Ies J; van Haelst, Mieke; Knoers, Nine V A M; van Haaften, Gijs; Kloosterman, Wigard; Hennekam, Raoul C M; Cuppen, Edwin; Ploos van Amstel, Hans Kristian

2012-08-01

We present a large Dutch family with seven males affected by a novel syndrome of X-linked intellectual disability, hypogonadism, gynaecomastia, truncal obesity, short stature and recognisable craniofacial manifestations resembling but not identical to Wilson-Turner syndrome. Seven female relatives show a much milder expression of the phenotype. We performed X chromosome exome (X-exome) sequencing in five individuals from this family and identified a novel intronic variant in the histone deacetylase 8 gene (HDAC8), c.164+5G>A, which disturbs the normal splicing of exon 2 resulting in exon skipping, and introduces a premature stop at the beginning of the histone deacetylase catalytic domain. The identified variant completely segregates in this family and was absent in 96 Dutch controls and available databases. Affected female carriers showed a notably skewed X-inactivation pattern in lymphocytes in which the mutated X-chromosome was completely inactivated. HDAC8 is a member of the protein family of histone deacetylases that play a major role in epigenetic gene silencing during development. HDAC8 specifically controls the patterning of the skull with the mouse HDAC8 knock-out showing craniofacial deformities of the skull. The present family provides the first evidence for involvement of HDAC8 in a syndromic form of intellectual disability.

Whole exome sequencing confirms the clinical diagnosis of Marfan syndrome combined with X-linked hypophosphatemia.

PubMed

Sheng, Xunlun; Chen, Xue; Lei, Bo; Chen, Rui; Wang, Hui; Zhang, Fangxia; Rong, Weining; Ha, Ruoshui; Liu, Yani; Zhao, Feng; Yang, Peizeng; Zhao, Chen

2015-06-04

To determine the genetic lesions and to modify the clinical diagnosis for a Chinese family with significant intrafamilial phenotypic diversities and unusual presentations. Three affected patients and the asymptomatic father were included and received comprehensive systemic examinations. Whole exome sequencing (WES) was performed for mutation detection. Structural modeling test was applied to analyze the potential structural changes caused by the missense substitution. The proband showed a wide spectrum of systemic anomalies, including bilateral ectopia lentis, atrial septal defect, ventricular septal defect, widening of tibial metaphysis with medial bowing, and dolichostenomelia in digits, while her mother and elder brother only demonstrated similar skeletal changes. A recurrent mutation, PHEX p.R291*, was found in all patients, while a de novo mutation, FBN1 p.C792F, was only detected in the proband. The FBN1 substitution was also predicted to cause significant conformational change in fibrillin-1 protein, thus changing its physical and biological properties. Taken together, we finalized the diagnosis for this family as X-linked hypophosphatemia (XLH), and diagnosed this girl as Marfan syndrome combined with XLH, and congenital heart disease. Our study also emphasizes the importance of WES in assisting the clinical diagnosis for complicated cases when the original diagnoses are challenged.

Whole-exome sequencing in obsessive-compulsive disorder identifies rare mutations in immunological and neurodevelopmental pathways

PubMed Central

Cappi, C; Brentani, H; Lima, L; Sanders, S J; Zai, G; Diniz, B J; Reis, V N S; Hounie, A G; Conceição do Rosário, M; Mariani, D; Requena, G L; Puga, R; Souza-Duran, F L; Shavitt, R G; Pauls, D L; Miguel, E C; Fernandez, T V

2016-01-01

Studies of rare genetic variation have identified molecular pathways conferring risk for developmental neuropsychiatric disorders. To date, no published whole-exome sequencing studies have been reported in obsessive-compulsive disorder (OCD). We sequenced all the genome coding regions in 20 sporadic OCD cases and their unaffected parents to identify rare de novo (DN) single-nucleotide variants (SNVs). The primary aim of this pilot study was to determine whether DN variation contributes to OCD risk. To this aim, we evaluated whether there is an elevated rate of DN mutations in OCD, which would justify this approach toward gene discovery in larger studies of the disorder. Furthermore, to explore functional molecular correlations among genes with nonsynonymous DN SNVs in OCD probands, a protein–protein interaction (PPI) network was generated based on databases of direct molecular interactions. We applied Degree-Aware Disease Gene Prioritization (DADA) to rank the PPI network genes based on their relatedness to a set of OCD candidate genes from two OCD genome-wide association studies (Stewart et al., 2013; Mattheisen et al., 2014). In addition, we performed a pathway analysis with genes from the PPI network. The rate of DN SNVs in OCD was 2.51 × 10−8 per base per generation, significantly higher than a previous estimated rate in unaffected subjects using the same sequencing platform and analytic pipeline. Several genes harboring DN SNVs in OCD were highly interconnected in the PPI network and ranked high in the DADA analysis. Nearly all the DN SNVs in this study are in genes expressed in the human brain, and a pathway analysis revealed enrichment in immunological and central nervous system functioning and development. The results of this pilot study indicate that further investigation of DN variation in larger OCD cohorts is warranted to identify specific risk genes and to confirm our preliminary finding with regard to PPI network enrichment for particular

Single molecule targeted sequencing for cancer gene mutation detection.

PubMed

Gao, Yan; Deng, Liwei; Yan, Qin; Gao, Yongqian; Wu, Zengding; Cai, Jinsen; Ji, Daorui; Li, Gailing; Wu, Ping; Jin, Huan; Zhao, Luyang; Liu, Song; Ge, Liangjin; Deem, Michael W; He, Jiankui

2016-05-19

With the rapid decline in cost of sequencing, it is now affordable to examine multiple genes in a single disease-targeted clinical test using next generation sequencing. Current targeted sequencing methods require a separate step of targeted capture enrichment during sample preparation before sequencing. Although there are fast sample preparation methods available in market, the library preparation process is still relatively complicated for physicians to use routinely. Here, we introduced an amplification-free Single Molecule Targeted Sequencing (SMTS) technology, which combined targeted capture and sequencing in one step. We demonstrated that this technology can detect low-frequency mutations using artificially synthesized DNA sample. SMTS has several potential advantages, including simple sample preparation thus no biases and errors are introduced by PCR reaction. SMTS has the potential to be an easy and quick sequencing technology for clinical diagnosis such as cancer gene mutation detection, infectious disease detection, inherited condition screening and noninvasive prenatal diagnosis.