Distinct Copy Number, Coding Sequence, and Locus Methylation Patterns Underlie Rhg1-Mediated Soybean Resistance to Soybean Cyst Nematode1[W][OPEN

PubMed Central

Cook, David E.; Bayless, Adam M.; Wang, Kai; Guo, Xiaoli; Song, Qijian; Jiang, Jiming; Bent, Andrew F.

2014-01-01

Copy number variation of kilobase-scale genomic DNA segments, beyond presence/absence polymorphisms, can be an important driver of adaptive traits. Resistance to Heterodera glycines (Rhg1) is a widely utilized quantitative trait locus that makes the strongest known contribution to resistance against soybean cyst nematode (SCN), Heterodera glycines, the most damaging pathogen of soybean (Glycine max). Rhg1 was recently discovered to be a complex locus at which resistance-conferring haplotypes carry up to 10 tandem repeat copies of a 31-kb DNA segment, and three disparate genes present on each repeat contribute to SCN resistance. Here, we use whole-genome sequencing, fiber-FISH (fluorescence in situ hybridization), and other methods to discover the genetic variation at Rhg1 across 41 diverse soybean accessions. Based on copy number variation, transcript abundance, nucleic acid polymorphisms, and differentially methylated DNA regions, we find that SCN resistance is associated with multicopy Rhg1 haplotypes that form two distinct groups. The tested high-copy-number Rhg1 accessions, including plant introduction (PI) 88788, contain a flexible number of copies (seven to 10) of the 31-kb Rhg1 repeat. The identified low-copy-number Rhg1 group, including PI 548402 (Peking) and PI 437654, contains three copies of the Rhg1 repeat and a newly identified allele of Glyma18g02590 (a predicted α-SNAP [α-soluble N-ethylmaleimide–sensitive factor attachment protein]). There is strong evidence for a shared origin of the two resistance-conferring multicopy Rhg1 groups and subsequent independent evolution. Differentially methylated DNA regions also were identified within Rhg1 that correlate with SCN resistance. These data provide insights into copy number variation of multigene segments, using as the example a disease resistance trait of high economic importance. PMID:24733883

The relationship of glutathione-S-transferases copy number variation and indoor air pollution to symptoms and markers of respiratory disease.

PubMed

Hersoug, Lars-Georg; Brasch-Andersen, Charlotte; Husemoen, Lise Lotte Nystrup; Sigsgaard, Torben; Linneberg, Allan

2012-07-01

Exposure to particulate matter (PM) may induce inflammation and oxidative stress in the airways. Carriers of null polymorphisms of glutathione S-transferases (GSTs), which detoxify reactive oxygen species, may be particularly susceptible to the effects of PM. To investigate whether deletions of GSTM1 and GSTT1 modify the potential effects of exposure to indoor sources of PM on symptoms and objective markers of respiratory disease. We conducted a population-based, cross-sectional study of 3471 persons aged 18-69 years. Information about exposure to indoor sources of PM and respiratory symptoms was obtained by a self-administered questionnaire. In addition, measurements of lung function (spirometry) and fractional exhaled nitric oxide were performed. Copy number variation of GSTM1 and GSTT1 was determined by polymerase chain reaction-based assays. We found that none of the symptoms and objective markers of respiratory disease were significantly associated with the GST null polymorphisms. An increasing number of positive alleles of the GSTM1 polymorphism tended to be associated lower prevalence of wheeze, cough, and high forced expiratory volume in 1 s (FEV(1) ), but these trends were not statistically significant. Furthermore, we did not observe any statistically significant interactions between GST copy number variation and exposure to indoor sources of PM in relation to respiratory symptoms and markers. In this adult population, GST copy number variations were not significantly associated with respiratory outcomes and did not modify the effects of self-reported exposure to indoor sources of PM on respiratory outcomes. © 2011 Blackwell Publishing Ltd.

Genome-wide copy number variant analysis in Holstein cattle reveals variants associated with 10 production traits including residual feed intake and dry matter intake

USDA-ARS?s Scientific Manuscript database

Copy number variation (CNV) is an important type of genetic variation contributing to phenotypic differences among mammals and may serve as an alternative molecular marker to single nucleotide polymorphism (SNP) for genome-wide association study (GWAS). Recently, GWAS analysis using CNV has been app...

High-resolution single-nucleotide polymorphism array-profiling in myeloproliferative neoplasms identifies novel genomic aberrations

PubMed Central

Stegelmann, Frank; Bullinger, Lars; Griesshammer, Martin; Holzmann, Karlheinz; Habdank, Marianne; Kuhn, Susanne; Maile, Carmen; Schauer, Stefanie; Döhner, Hartmut; Döhner, Konstanze

2010-01-01

Single-nucleotide polymorphism arrays allow for genome-wide profiling of copy-number alterations and copy-neutral runs of homozygosity at high resolution. To identify novel genetic lesions in myeloproliferative neoplasms, a large series of 151 clinically well characterized patients was analyzed in our study. Copy-number alterations were rare in essential thrombocythemia and polycythemia vera. In contrast, approximately one third of myelofibrosis patients exhibited small genomic losses (less than 5 Mb). In 2 secondary myelofibrosis cases the tumor suppressor gene NF1 in 17q11.2 was affected. Sequencing analyses revealed a mutation in the remaining NF1 allele of one patient. In terms of copy-neutral aberrations, no chromosomes other than 9p were recurrently affected. In conclusion, novel genomic aberrations were identified in our study, in particular in patients with myelofibrosis. Further analyses on single-gene level are necessary to uncover the mechanisms that are involved in the pathogenesis of myeloproliferative neoplasms. PMID:20015882

Variable-Number Tandem Repeats That Are Useful in Genotyping Isolates of Salmonella enterica subsp. enterica Serovars Typhimurium and Newport▿

PubMed Central

Witonski, D. ; Stefanova, R.; Ranganathan, A.; Schutze, G. E.; Eisenach, K. D.; Cave, M. D.

2006-01-01

The genome of Salmonella enterica subsp. enterica serovar Typhimurium strain LT2 was analyzed for direct repeats, and 54 sequences containing variable-number tandem repeat loci were identified. Ten primer pairs that anneal upstream and downstream of each selected locus were designed and used to amplify PCR targets in isolates of S. enterica serovars Typhimurium and Newport. Four of the 10 loci did not show polymorphism in the length of products. Six loci were selected for analysis. Isolates of S. enterica serovars Typhimurium and Newport that were related to specific outbreaks and showed identical pulsed-field gel electrophoresis patterns were indistinguishable by the length of the six variable-number tandem repeats. Isolates that differed in their pulsed-field gel electrophoresis patterns showed polymorphism in variable-number tandem repeat profiles. Length of the products was confirmed by DNA sequence analysis. Only 2 of the 10 loci contained exact integers of the direct repeat. Eight loci contained partial copies. The partial copies were maintained at the ends of the variable-number tandem repeat loci in all isolates. In spite of having partial copies that were maintained in all isolates, the number of direct repeats at a locus was polymorphic. Six variable-number tandem repeat loci were useful in distinguishing isolates of S. enterica serovars Typhimurium and Newport that had different pulsed-field gel electrophoresis patterns and in identifying outbreak-associated cases that shared a common pulsed-field gel pattern. PMID:16943354

Polymorphisms in α-Defensin–Encoding DEFA1A3 Associate with Urinary Tract Infection Risk in Children with Vesicoureteral Reflux

PubMed Central

Schwaderer, Andrew L.; Wang, Huanyu; Kim, SungHwan; Kline, Jennifer M.; Liang, Dong; Brophy, Pat D.; McHugh, Kirk M.; Tseng, George C.; Saxena, Vijay; Barr-Beare, Evan; Pierce, Keith R.; Shaikh, Nader; Manak, J. Robert; Cohen, Daniel M.; Becknell, Brian; Spencer, John D.; Baker, Peter B.; Yu, Chack-Yung

2016-01-01

The contribution of genetic variation to urinary tract infection (UTI) risk in children with vesicoureteral reflux is largely unknown. The innate immune system, which includes antimicrobial peptides, such as the α-defensins, encoded by DEFA1A3, is important in preventing UTIs but has not been investigated in the vesicoureteral reflux population. We used quantitative real–time PCR to determine DEFA1A3 DNA copy numbers in 298 individuals with confirmed UTIs and vesicoureteral reflux from the Randomized Intervention for Children with Vesicoureteral Reflux (RIVUR) Study and 295 controls, and we correlated copy numbers with outcomes. Outcomes studied included reflux grade, UTIs during the study on placebo or antibiotics, bowel and bladder dysfunction, and renal scarring. Overall, 29% of patients and 16% of controls had less than or equal to five copies of DEFA1A3 (odds ratio, 2.09; 95% confidence interval, 1.40 to 3.11; P<0.001). For each additional copy of DEFA1A3, the odds of recurrent UTI in patients receiving antibiotic prophylaxis decreased by 47% when adjusting for vesicoureteral reflux grade and bowel and bladder dysfunction. In patients receiving placebo, DEFA1A3 copy number did not associate with risk of recurrent UTI. Notably, we found that DEFA1A3 is expressed in renal epithelium and not restricted to myeloid-derived cells, such as neutrophils. In conclusion, low DEFA1A3 copy number associated with recurrent UTIs in subjects in the RIVUR Study randomized to prophylactic antibiotics, providing evidence that copy number polymorphisms in an antimicrobial peptide associate with UTI risk. PMID:26940096

Allele quantification using molecular inversion probes (MIP)

PubMed Central

Wang, Yuker; Moorhead, Martin; Karlin-Neumann, George; Falkowski, Matthew; Chen, Chunnuan; Siddiqui, Farooq; Davis, Ronald W.; Willis, Thomas D.; Faham, Malek

2005-01-01

Detection of genomic copy number changes has been an important research area, especially in cancer. Several high-throughput technologies have been developed to detect these changes. Features that are important for the utility of technologies assessing copy number changes include the ability to interrogate regions of interest at the desired density as well as the ability to differentiate the two homologs. In addition, assessing formaldehyde fixed and paraffin embedded (FFPE) samples allows the utilization of the vast majority of cancer samples. To address these points we demonstrate the use of molecular inversion probe (MIP) technology to the study of copy number. MIP is a high-throughput genotyping technology capable of interrogating >20 000 single nucleotide polymorphisms in the same tube. We have shown the ability of MIP at this multiplex level to provide copy number measurements while obtaining the allele information. In addition we have demonstrated a proof of principle for copy number analysis in FFPE samples. PMID:16314297

Altools: a user friendly NGS data analyser.

PubMed

Camiolo, Salvatore; Sablok, Gaurav; Porceddu, Andrea

2016-02-17

Genotyping by re-sequencing has become a standard approach to estimate single nucleotide polymorphism (SNP) diversity, haplotype structure and the biodiversity and has been defined as an efficient approach to address geographical population genomics of several model species. To access core SNPs and insertion/deletion polymorphisms (indels), and to infer the phyletic patterns of speciation, most such approaches map short reads to the reference genome. Variant calling is important to establish patterns of genome-wide association studies (GWAS) for quantitative trait loci (QTLs), and to determine the population and haplotype structure based on SNPs, thus allowing content-dependent trait and evolutionary analysis. Several tools have been developed to investigate such polymorphisms as well as more complex genomic rearrangements such as copy number variations, presence/absence variations and large deletions. The programs available for this purpose have different strengths (e.g. accuracy, sensitivity and specificity) and weaknesses (e.g. low computation speed, complex installation procedure and absence of a user-friendly interface). Here we introduce Altools, a software package that is easy to install and use, which allows the precise detection of polymorphisms and structural variations. Altools uses the BWA/SAMtools/VarScan pipeline to call SNPs and indels, and the dnaCopy algorithm to achieve genome segmentation according to local coverage differences in order to identify copy number variations. It also uses insert size information from the alignment of paired-end reads and detects potential large deletions. A double mapping approach (BWA/BLASTn) identifies precise breakpoints while ensuring rapid elaboration. Finally, Altools implements several processes that yield deeper insight into the genes affected by the detected polymorphisms. Altools was used to analyse both simulated and real next-generation sequencing (NGS) data and performed satisfactorily in terms of positive predictive values, sensitivity, the identification of large deletion breakpoints and copy number detection. Altools is fast, reliable and easy to use for the mining of NGS data. The software package also attempts to link identified polymorphisms and structural variants to their biological functions thus providing more valuable information than similar tools.

HvFT1 polymorphism and effect—survey of barley germplasm and expression analysis

PubMed Central

Loscos, Jorge; Igartua, Ernesto; Contreras-Moreira, Bruno; Gracia, M. Pilar; Casas, Ana M.

2014-01-01

Flowering time in plants is a tightly regulated process. In barley (Hordeum vulgare L.), HvFT1, ortholog of FLOWERING LOCUS T, is the main integrator of the photoperiod and vernalization signals leading to the transition from vegetative to reproductive state of the plant. This gene presents sequence polymorphisms affecting flowering time in the first intron and in the promoter. Recently, copy number variation (CNV) has been described for this gene. An allele with more than one copy was linked to higher gene expression, earlier flowering, and an overriding effect of the vernalization mechanism. This study aims at (1) surveying the distribution of HvFT1 polymorphisms across barley germplasm and (2) assessing gene expression and phenotypic effects of HvFT1 alleles. We analyzed HvFT1 CNV in 109 winter, spring, and facultative barley lines. There was more than one copy of the gene (2–5) only in spring or facultative barleys without a functional vernalization VrnH2 allele. CNV was investigated in several regions inside and around HvFT1. Two models of the gene were found: one with the same number of promoters and transcribed regions, and another with one promoter and variable number of transcribed regions. This last model was found in Nordic barleys only. Analysis of HvFT1 expression showed that association between known polymorphisms at the HvFT1 locus and the expression of the gene was highly dependent on the genetic background. Under long day conditions the earliest flowering lines carried a sensitive PpdH1 allele. Among spring cultivars with different number of copies, no clear relation was found between CNV, gene expression and flowering time. This was confirmed in a set of doubled haploid lines of a population segregating for HvFT1 CNV. Earlier flowering in the presence of several copies of HvFT1 was only seen in cultivar Tammi, which carries one promoter, suggesting a relation of gene structure with its regulation. HvCEN also affected to a large extent flowering time. PMID:24936204

LPA and PLG sequence variation and kringle IV-2 copy number in two populations.

PubMed

Crawford, Dana C; Peng, Ze; Cheng, Jan-Fang; Boffelli, Dario; Ahearn, Magdalena; Nguyen, Dan; Shaffer, Tristan; Yi, Qian; Livingston, Robert J; Rieder, Mark J; Nickerson, Deborah A

2008-01-01

Lp(a) levels have long been recognized as a potential risk factor for coronary heart disease that is almost completely under genetic control. Much of the genetics impacting Lp(a) levels has been attributed to the highly polymorphic LPA kringle IV-2 copy number variant, and most of the variance in Lp(a) levels in populations of European-descent is inversely correlated with kringle IV copy number. However, less of the variance is explained in African-descent populations for the same structural variation. African-descent populations have, on average, higher levels of Lp(a), suggesting other genetic factors contribute to Lp(a) level variability across populations. To identify potential cis-acting factors, we re-sequenced the gene LPA for single nucleotide polymorphism (SNP) discovery in 23 European-Americans and 24 African-Americans. We also re- sequenced the neighboring gene plasminogen (PLG) and genotyped the kringle IV copy number variant in the same reference samples. These data are the most comprehensive description of sequence variation in LPA and its relationship with the kringle IV copy number variant. With these data, we demonstrate that only a fraction of LPA sequence diversity has been previously documented. Also, we identify several high frequency SNPs present in the African-American sample but absent in the European-American sample. Finally, we show that SNPs within PLG are not in linkage disequilibrium with SNPs in LPA, and we show that kringle IV copy number variation is not in linkage disequilibrium with either LPA or PLG SNPs. Together, these data suggest that LPA SNPs could independently contribute to Lp(a) levels in the general population. Copyright (c) 2008 S. Karger AG, Basel.

Selection of Plasmodium falciparum Multidrug Resistance Gene 1 Alleles in Asexual Stages and Gametocytes by Artemether-Lumefantrine in Nigerian Children with Uncomplicated Falciparum Malaria ▿

PubMed Central

Happi, C. T.; Gbotosho, G. O.; Folarin, O. A.; Sowunmi, A.; Hudson, T.; O'Neil, M.; Milhous, W.; Wirth, D. F.; Oduola, A. M. J.

2009-01-01

We assessed Plasmodium falciparum mdr1 (Pfmdr1) gene polymorphisms and copy numbers as well as P. falciparum Ca2+ ATPase (PfATPase6) gene polymorphisms in 90 Nigerian children presenting with uncomplicated falciparum malaria and enrolled in a study of the efficacy of artemether-lumefantrine (AL). The nested PCR-restriction fragment length polymorphism and the quantitative real-time PCR methodologies were used to determine the alleles of the Pfmdr1 and PfATPase6 genes and the Pfmdr1 copy number variation, respectively, in patients samples collected prior to treatment and at the reoccurrence of parasites during a 42-day follow-up. The Pfmdr1 haplotype 86N-184F-1246D was significantly associated (P < 0.00001) with treatment failures and was selected for among posttreatment samples obtained from patients with newly acquired or recrudescing infections (P < 0.00001; χ2 = 36.5) and in gametocytes (log rank statistic = 5; P = 0.0253) after treatment with AL. All pre- and posttreatment samples as well as gametocytes harbored a single copy of the Pfmdr1 gene and the wild-type allele (L89) at codon 89 of the PfATPase6 gene. These findings suggest that polymorphisms in the Pfmdr1 gene are under AL selection pressure. Pfmdr1 polymorphisms may result in reduction in the therapeutic efficacy of this newly adopted combination treatment for uncomplicated falciparum malaria in Saharan countries of Africa. PMID:19075074

Selection of Plasmodium falciparum multidrug resistance gene 1 alleles in asexual stages and gametocytes by artemether-lumefantrine in Nigerian children with uncomplicated falciparum malaria.

PubMed

Happi, C T; Gbotosho, G O; Folarin, O A; Sowunmi, A; Hudson, T; O'Neil, M; Milhous, W; Wirth, D F; Oduola, A M J

2009-03-01

We assessed Plasmodium falciparum mdr1 (Pfmdr1) gene polymorphisms and copy numbers as well as P. falciparum Ca(2+) ATPase (PfATPase6) gene polymorphisms in 90 Nigerian children presenting with uncomplicated falciparum malaria and enrolled in a study of the efficacy of artemether-lumefantrine (AL). The nested PCR-restriction fragment length polymorphism and the quantitative real-time PCR methodologies were used to determine the alleles of the Pfmdr1 and PfATPase6 genes and the Pfmdr1 copy number variation, respectively, in patients samples collected prior to treatment and at the reoccurrence of parasites during a 42-day follow-up. The Pfmdr1 haplotype 86N-184F-1246D was significantly associated (P < 0.00001) with treatment failures and was selected for among posttreatment samples obtained from patients with newly acquired or recrudescing infections (P < 0.00001; chi(2) = 36.5) and in gametocytes (log rank statistic = 5; P = 0.0253) after treatment with AL. All pre- and posttreatment samples as well as gametocytes harbored a single copy of the Pfmdr1 gene and the wild-type allele (L89) at codon 89 of the PfATPase6 gene. These findings suggest that polymorphisms in the Pfmdr1 gene are under AL selection pressure. Pfmdr1 polymorphisms may result in reduction in the therapeutic efficacy of this newly adopted combination treatment for uncomplicated falciparum malaria in Saharan countries of Africa.

Plasmodium vivax multidrug resistance-1 gene polymorphism in French Guiana.

PubMed

Faway, Emilie; Musset, Lise; Pelleau, Stéphane; Volney, Béatrice; Casteras, Jessica; Caro, Valérie; Menard, Didier; Briolant, Sébastien; Legrand, Eric

2016-11-08

Plasmodium vivax malaria is a major public health problem in French Guiana. Some cases of resistance to chloroquine, the first-line treatment used against P. vivax malaria, have been described in the Brazilian Amazon region. The aim of this study is to investigate a possible dispersion of chloroquine-resistant P. vivax isolates in French Guiana. The genotype, polymorphism and copy number variation, of the P. vivax multidrug resistance gene-1 (pvmdr1) have been previously associated with modification of the susceptibility to chloroquine. The pvmdr1 gene polymorphism was evaluated by sequencing and copy number variation was assessed by real-time PCR, in P. vivax isolates obtained from 591 symptomatic patients from 1997 to 2013. The results reveal that 1.0% [95% CI 0.4-2.2] of French Guiana isolates carry the mutations Y976F and F1076L, and that the proportion of isolates with multiple copies of pvmdr1 has significantly decreased over time, from 71.3% (OR = 6.2 [95% CI 62.9-78.7], p < 0.0001) in 1997-2004 to 12.8% (OR = 0.03 [95% CI 9.4-16.9], p < 0.0001) in 2009-2013. A statistically significant relationship was found between Guf-A (harboring the single mutation T958M) and Sal-1 (wild type) alleles and pvmdr1 copy number. Few P. vivax isolates harboring chloroquine-resistant mutations in the pvmdr1 gene are circulating in French Guiana. However, the decrease in the prevalence of isolates carrying multiple copies of pvmdr1 might indicate that the P. vivax population in French Guiana is evolving towards a decreased susceptibility to chloroquine.

Parasites bearing a single copy of the multi-drug resistance gene (pfmdr-1) with wild-type SNPs predominate amongst Plasmodium falciparum isolates from Malawi.

PubMed

Nkhoma, Standwell; Nair, Shalini; Mukaka, Mavuto; Molyneux, Malcolm E; Ward, Stephen A; Anderson, Timothy J C

2009-07-01

We genotyped 160 P. falciparum infections from Malawi for pfmdr-1 copy number changes and SNPs associated with in vivo tolerance and poor in vitro sensitivity to the component drugs of Coartem. We also measured in vitro susceptibility of 49 of these isolates to a variety of drugs in clinical use or with a potential for use in Africa. All 160 infections carried a single copy of pfmdr-1 but 34% exhibited sequence variation at 4 of the 5 polymorphic sites in pfmdr-1. Isolates carrying 86-Asn and 184-Tyr pfmdr-1 alleles were significantly less sensitive (p<0.001) to mefloquine, lumefantrine, artemether and dihydroartemisinin compared with those bearing 86-Tyr and 184-Phe polymorphisms. This study provides baseline measures prior to policy change: continued surveillance for changes in baseline drug susceptibility, pfmdr-1 copy number and SNPs, and other putative Coartem resistance loci will be necessary to provide an early warning of emerging Coartem resistance in this setting.

Beneficial effect of a high number of copies of salivary amylase AMY1 gene on obesity risk in Mexican children.

PubMed

Mejía-Benítez, María A; Bonnefond, Amélie; Yengo, Loïc; Huyvaert, Marlène; Dechaume, Aurélie; Peralta-Romero, Jesús; Klünder-Klünder, Miguel; García Mena, Jaime; El-Sayed Moustafa, Julia S; Falchi, Mario; Cruz, Miguel; Froguel, Philippe

2015-02-01

Childhood obesity is a major public health problem in Mexico, affecting one in every three children. Genome-wide association studies identified genetic variants associated with childhood obesity, but a large missing heritability remains to be elucidated. We have recently shown a strong association between a highly polymorphic copy number variant encompassing the salivary amylase gene (AMY1 also known as AMY1A) and obesity in European and Asian adults. In the present study, we aimed to evaluate the association between AMY1 copy number and obesity in Mexican children. We evaluated the number of AMY1 copies in 597 Mexican children (293 obese children and 304 normal weight controls) through highly sensitive digital PCR. The effect of AMY1 copy number on obesity status was assessed using a logistic regression model adjusted for age and sex. We identified a marked effect of AMY1 copy number on reduced risk of obesity (OR per estimated copy 0.84, with the number of copies ranging from one to 16 in this population; p = 4.25 × 10(-6)). The global association between AMY1 copy number and reduced risk of obesity seemed to be mostly driven by the contribution of the highest AMY1 copy number. Strikingly, all children with >10 AMY1 copies were normal weight controls. Salivary amylase initiates the digestion of dietary starch, which is highly consumed in Mexico. Our current study suggests putative benefits of high number of AMY1 copies (and related production of salivary amylase) on energy metabolism in Mexican children.

Genetic variations in UGT2B28, UGT2B17, UGT2B15 genes and the risk of prostate cancer: A case-control study.

PubMed

Habibi, Mohsen; Mirfakhraie, Reza; Khani, Maryam; Rakhshan, Azadeh; Azargashb, Eznollah; Pouresmaeili, Farkhondeh

2017-11-15

Glucuronidation is a major pathway for elimination of exogenous and endogenous compounds such as environmental carcinogens and androgens from the body. This biochemical pathway is mediated by enzymes called uridine diphosphoglucuronosyltransferases (UGTs). Null (del/del) genes polymorphisms in UGT2B17, and UGT2B28 and D85Y single-nucleotide polymorphism (SNP) of UGT2B15 have been reported to increase the risk of prostate cancer. The goal of this study was to determine the association of mentioned genetic variants with the risk of prostate cancer. We investigated the copy number variations (CNVs) of UGT2B17 and UGT2B28 loci and the association between rs1902023 polymorphism of UGT2B15 gene in 360 subjects consisted of 120 healthy controls, 120 prostate cancer (PC) patients and 120 benign prostatic hyperplasia (BPH) patients. No association was detected for the mentioned polymorphisms and the risk of PC. However, a significant association was detected between UGT2B17 copy number variation and BPH risk (OR=2.189; 95% CI, 1.303-3.675; p=0.003). Furthermore, we observed that the D85Y polymorphism increases the risk of BPH when analyzed in combination with the copy number variation of UGT2B17 gene (OR=0.135; 95% CI, 0.036-0.512; p=0.003). Our findings suggest that the D85Y polymorphism of UGT2B15 and CNVs in UGT2B28 and UGT2B17 genes is not associated with prostate cancer risk in Iranian patients. To our knowledge, this is the first report that implicates the role of CNV of UGT2B17 gene in BPH. Copyright © 2017 Elsevier B.V. All rights reserved.

Association of Higher Defensin β-4 Genomic Copy Numbers with Behçet's Disease in Iraqi Patients.

PubMed

Hameed, Ammar F; Jaradat, Sameh; Al-Musawi, Bassam M; Sharquie, Khalifa; Ibrahim, Mazin J; Hayani, Raafa K; Norgauer, Johannes

2015-11-01

Behçet's disease (BD) is an immune-mediated small vessel systemic vasculitis. Human β-defensins are antimicrobial peptides associated with many inflammatory diseases and are encoded by the β-defensin family of multiple-copy genes. However, their role in BD necessitates further investigation. The aim of the present study was to investigate the possible association of BD in its various clinical forms with defensin β-4 (DEFB4) genomic copy numbers. This case-control study was conducted from January to September 2011 and included 50 control subjects and 27 unrelated Iraqi BD patients registered at Baghdad Teaching Hospital, Bagdad, Iraq. Copy numbers of the DEFB4 gene were determined using the comparative cycle threshold method by duplex real-time polymerase chain reaction technology at the Department of Dermatology of Jena University Hospital, Jena, Germany. DEFB4 genomic copy numbers were significantly higher in the BD group compared to the control group (P = 0.010). However, no statistically significant association was found between copy numbers and clinical variables within the BD group. The DEFB4 copy number polymorphism may be associated with BD; however, it is not associated with different clinical manifestations of the disease.

MERE1, a low-copy-number copia-type retroelement in Medicago truncatula active during tissue culture.

PubMed

Rakocevic, Alexandra; Mondy, Samuel; Tirichine, Leïla; Cosson, Viviane; Brocard, Lysiane; Iantcheva, Anelia; Cayrel, Anne; Devier, Benjamin; Abu El-Heba, Ghada Ahmed; Ratet, Pascal

2009-11-01

We have identified an active Medicago truncatula copia-like retroelement called Medicago RetroElement1-1 (MERE1-1) as an insertion in the symbiotic NSP2 gene. MERE1-1 belongs to a low-copy-number family in the sequenced Medicago genome. These copies are highly related, but only three of them have a complete coding region and polymorphism exists between the long terminal repeats of these different copies. This retroelement family is present in all M. truncatula ecotypes tested but also in other legume species like Lotus japonicus. It is active only during tissue culture in both R108 and Jemalong Medicago accessions and inserts preferentially in genes.

Application of Nexus copy number software for CNV detection and analysis.

PubMed

Darvishi, Katayoon

2010-04-01

Among human structural genomic variation, copy number variants (CNVs) are the most frequently known component, comprised of gains/losses of DNA segments that are generally 1 kb in length or longer. Array-based comparative genomic hybridization (aCGH) has emerged as a powerful tool for detecting genomic copy number variants (CNVs). With the rapid increase in the density of array technology and with the adaptation of new high-throughput technology, a reliable and computationally scalable method for accurate mapping of recurring DNA copy number aberrations has become a main focus in research. Here we introduce Nexus Copy Number software, a platform-independent tool, to analyze the output files of all types of commercial and custom-made comparative genomic hybridization (CGH) and single-nucleotide polymorphism (SNP) arrays, such as those manufactured by Affymetrix, Agilent Technologies, Illumina, and Roche NimbleGen. It also supports data generated by various array image-analysis software tools such as GenePix, ImaGene, and BlueFuse. (c) 2010 by John Wiley & Sons, Inc.

High throughput screening of human subtelomeric DNA for copy number changes using multiplex amplifiable probe hybridisation (MAPH).

PubMed

Hollox, E J; Atia, T; Cross, G; Parkin, T; Armour, J A L

2002-11-01

Subtelomeric regions of the human genome are gene rich, with a high level of sequence polymorphism. A number of clinical conditions, including learning disability, have been attributed to subtelomeric deletions or duplications, but screening for deletion in these regions using conventional cytogenetic methods and fluorescence in situ hybridisation (FISH) is laborious. Here we report that a new method, multiplex amplifiable probe hybridisation (MAPH), can be used to screen for copy number at subtelomeric regions. We have constructed a set of MAPH probes with each subtelomeric region represented at least once, so that one gel lane can assay copy number at all chromosome ends in one person. Each probe has been sequenced and, where possible, its position relative to the telomere determined by comparison with mapped clones. The sensitivity of the probes has been characterised on a series of cytogenetically verified positive controls and 83 normal controls were used to assess the frequency of polymorphic copy number with no apparent phenotypic effect. We have also used MAPH to test a cohort of 37 people selected from males referred for fragile X syndrome testing and found six changes that were confirmed by dosage PCR. MAPH can be used to screen subtelomeric regions of chromosomes for deletions and duplications before confirmation by FISH or dosage PCR. The high throughput nature of this technique allows it to be used for large scale screening of subtelomeric copy number, before confirmation by FISH. In practice, the availability of a rapid and efficient screen may allow subtelomeric analysis to be applied to a wider selection of patients than is currently possible using FISH alone.

High throughput screening of human subtelomeric DNA for copy number changes using multiplex amplifiable probe hybridisation (MAPH)

PubMed Central

Hollox, E; Atia, T; Cross, G; Parkin, T; Armour, J

2002-01-01

Background: Subtelomeric regions of the human genome are gene rich, with a high level of sequence polymorphism. A number of clinical conditions, including learning disability, have been attributed to subtelomeric deletions or duplications, but screening for deletion in these regions using conventional cytogenetic methods and fluorescence in situ hybridisation (FISH) is laborious. Here we report that a new method, multiplex amplifiable probe hybridisation (MAPH), can be used to screen for copy number at subtelomeric regions. Methods: We have constructed a set of MAPH probes with each subtelomeric region represented at least once, so that one gel lane can assay copy number at all chromosome ends in one person. Each probe has been sequenced and, where possible, its position relative to the telomere determined by comparison with mapped clones. Results: The sensitivity of the probes has been characterised on a series of cytogenetically verified positive controls and 83 normal controls were used to assess the frequency of polymorphic copy number with no apparent phenotypic effect. We have also used MAPH to test a cohort of 37 people selected from males referred for fragile X syndrome testing and found six changes that were confirmed by dosage PCR. Conclusions: MAPH can be used to screen subtelomeric regions of chromosomes for deletions and duplications before confirmation by FISH or dosage PCR. The high throughput nature of this technique allows it to be used for large scale screening of subtelomeric copy number, before confirmation by FISH. In practice, the availability of a rapid and efficient screen may allow subtelomeric analysis to be applied to a wider selection of patients than is currently possible using FISH alone. PMID:12414816

Genetic polymorphism in pvmdr1 and pvcrt-o genes in relation to in vitro drug susceptibility of Plasmodium vivax isolates from malaria-endemic countries.

PubMed

Lu, Feng; Lim, Chae Seung; Nam, Deok-Hwa; Kim, Kwonkee; Lin, Khin; Kim, Tong-Soo; Lee, Hyeong-Woo; Chen, Jun-Hu; Wang, Yue; Sattabongkot, Jetsumon; Han, Eun-Taek

2011-02-01

Treatment failure of chloroquine for Plasmodium vivax infection has increased in endemic countries. However, the molecular mechanisms for resistance and in vitro susceptibility of P. vivax to chloroquine remain elusive. We investigated the prevalence of mutations in the pvmdr1 and pvcrt-o genes, and the copy number of the pvmdr1 gene in isolates from the Republic of Korea (ROK), Thailand, the Union of Myanmar (Myanmar), and Papua New Guinea (PNG). We also measured in vitro susceptibility of Korean isolates to antimalarial drugs. The pvmdr1 analysis showed that mutations at amino acid position Y976F of pvmdr1 were found in isolates from Thailand (17.9%), Myanmar (13.3%), and PNG (100%), but none from the ROK, and mutation at position F1076L was present in isolates from the ROK (100%), Thailand (60.7%), and Myanmar (46.7%). One copy of the pvmdr1 gene was observed in most isolates and double copy numbers of the gene were observed in two Thai isolates. In the exons of the pvcrt-o gene that were sequenced, a K10 insertion was present in isolates from Thailand (56.0%) and Myanmar (46.2%), and the wild type was found in all Korean isolates. The results suggest that gene polymorphisms and copy number variation was observed in isolates of P. vivax from Southeast Asian countries. In Korean isolates polymorphism as limited to the F1076L variant, and no isolates with high level of resistance were found by in vitro susceptibility determinations. Moreover, our results provide a baseline for future prospective drug studies in malaria-endemic areas. 2010 Elsevier B.V. All rights reserved.

Multiply to conquer: Copy number variations at Ppd-B1 and Vrn-A1 facilitate global adaptation in wheat.

PubMed

Würschum, Tobias; Boeven, Philipp H G; Langer, Simon M; Longin, C Friedrich H; Leiser, Willmar L

2015-07-29

Copy number variation was found to be a frequent type of DNA polymorphism in the human genome often associated with diseases but its importance in crops and the effects on agronomic traits are still largely unknown. Here, we employed a large worldwide panel of 1110 winter wheat varieties to assess the frequency and the geographic distribution of copy number variants at the Photoperiod-B1 (Ppd-B1) and the Vernalization-A1 (Vrn-A1) loci as well as their effects on flowering time under field conditions. We identified a novel four copy variant of Vrn-A1 and based on the phylogenetic relationships among the lines show that the higher copy variants at both loci are likely to have arisen independently multiple times. In addition, we found that the frequency of the different copy number variants at both loci reflects the environmental conditions in the varieties' region of origin and based on multi-location field trials show that Ppd-B1 copy number has a substantial effect on the fine-tuning of flowering time. In conclusion, our results show the importance of copy number variation at Ppd-B1 and Vrn-A1 for the global adaptation of wheat making it a key factor for wheat success in a broad range of environments and in a wider context substantiate the significant role of copy number variation in crops.

ITALICS: an algorithm for normalization and DNA copy number calling for Affymetrix SNP arrays.

PubMed

Rigaill, Guillem; Hupé, Philippe; Almeida, Anna; La Rosa, Philippe; Meyniel, Jean-Philippe; Decraene, Charles; Barillot, Emmanuel

2008-03-15

Affymetrix SNP arrays can be used to determine the DNA copy number measurement of 11 000-500 000 SNPs along the genome. Their high density facilitates the precise localization of genomic alterations and makes them a powerful tool for studies of cancers and copy number polymorphism. Like other microarray technologies it is influenced by non-relevant sources of variation, requiring correction. Moreover, the amplitude of variation induced by non-relevant effects is similar or greater than the biologically relevant effect (i.e. true copy number), making it difficult to estimate non-relevant effects accurately without including the biologically relevant effect. We addressed this problem by developing ITALICS, a normalization method that estimates both biological and non-relevant effects in an alternate, iterative manner, accurately eliminating irrelevant effects. We compared our normalization method with other existing and available methods, and found that ITALICS outperformed these methods for several in-house datasets and one public dataset. These results were validated biologically by quantitative PCR. The R package ITALICS (ITerative and Alternative normaLIzation and Copy number calling for affymetrix Snp arrays) has been submitted to Bioconductor.

Copy number variants and genetic polymorphisms in TBX21, GATA3, Rorc, Foxp3 and susceptibility to Behcet's disease and Vogt-Koyanagi-Harada syndrome.

PubMed

Liao, Dan; Hou, Shengping; Zhang, Jun; Fang, Jing; Liu, Yunjia; Bai, Lin; Cao, Qingfeng; Kijlstra, Aize; Yang, Peizeng

2015-04-15

This study aimed to investigate the role of genetic variants including single nucleotide polymorphisms (SNPs) and copy number variants (CNVs) of TBX21, GATA3, Rorc and Foxp3 genes in Behcet's disease (BD) and Vogt-Koyanagi-Harada (VKH) syndrome in a Chinese Han population. Genotyping of 25 SNPs was performed by iPLEX system (Sequenom) or polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). TaqMan real time PCR was used to assess CNVs. The expression of Rorc and Foxp3 were examined by real-time PCR and cytokine production was measured by ELISA. High Rorc CNV was associated with the susceptibility to BD (P = 8.99 × 10(-8), OR = 3.0), and low Foxp3 CNV predisposed to BD in female patients (P = 1.92 × 10(-5), OR = 3.1). CNVs for the investigated genes were not altered in VKH syndrome. Further functional studies demonstrated that the relative mRNA expression levels of Rorc were increased in individuals with high Rorc copy number, but not for Foxp3. Increased production of IL-1β and IL-6 was found in individuals carrying a high CNV of Rorc. Our study showed that high CNVs of Rorc and low CNVs of Foxp3 confer risk for BD but not for VKH syndrome. The tested 25 SNPs in TBX21, GATA3, Rorc and Foxp3 did not associate with BD and VKH syndrome.

Differences in AMY1 Gene Copy Numbers Derived from Blood, Buccal Cells and Saliva Using Quantitative and Droplet Digital PCR Methods: Flagging the Pitfall.

PubMed

Ooi, Delicia Shu Qin; Tan, Verena Ming Hui; Ong, Siong Gim; Chan, Yiong Huak; Heng, Chew Kiat; Lee, Yung Seng

2017-01-01

The human salivary (AMY1) gene, encoding salivary α-amylase, has variable copy number variants (CNVs) in the human genome. We aimed to determine if real-time quantitative polymerase chain reaction (qPCR) and the more recently available Droplet Digital PCR (ddPCR) can provide a precise quantification of the AMY1 gene copy number in blood, buccal cells and saliva samples derived from the same individual. Seven participants were recruited and DNA was extracted from the blood, buccal cells and saliva samples provided by each participant. Taqman assay real-time qPCR and ddPCR were conducted to quantify AMY1 gene copy numbers. Statistical analysis was carried out to determine the difference in AMY1 gene copy number between the different biological specimens and different assay methods. We found significant within-individual difference (p<0.01) in AMY1 gene copy number between different biological samples as determined by qPCR. However, there was no significant within-individual difference in AMY1 gene copy number between different biological samples as determined by ddPCR. We also found that AMY1 gene copy number of blood samples were comparable between qPCR and ddPCR, while there is a significant difference (p<0.01) between AMY1 gene copy numbers measured by qPCR and ddPCR for both buccal swab and saliva samples. Despite buccal cells and saliva samples being possible sources of DNA, it is pertinent that ddPCR or a single biological sample, preferably blood sample, be used for determining highly polymorphic gene copy numbers like AMY1, due to the large within-individual variability between different biological samples if real time qPCR is employed.

Sequence polymorphisms at the growth hormone GH1/GH2-N and GH2-Z gene copies and their relationship with dairy traits in domestic sheep (Ovis aries).

PubMed

Vacca, G M; Dettori, M L; Balia, F; Luridiana, S; Mura, M C; Carcangiu, V; Pazzola, M

2013-09-01

The purpose was to analyze the growth hormone GH1/GH2-N and GH2-Z gene copies and to assess their possible association with milk traits in Sarda sheep. Two hundred multiparous lactating ewes were monitored. The two gene copies were amplified separately and each was used as template for a nested PCR, to investigate single strand conformation polymorphism (SSCP) of the 5'UTR, exon-1, exon-5 and 3'UTR DNA regions. SSCP analysis revealed marked differences in the number of polymorphic patterns between the two genes. Sequencing revealed five nucleotide changes at the GH1/GH2-N gene. Five nucleotide changes occurred at the GH2-Z gene: one was located in exon-5 (c.556G > A) and resulted in a putative amino acid substitution G186S. All the nucleotide changes were copy-specific, except c.*30delT, which was common to both GH1/GH2-N and GH2-Z. Variability in the promoter regions of each gene might have consequences on the expression level, due to the involvement in potential transcription factor binding sites. Both gene copies influenced milk yield. A correlation with milk protein and casein content was also evidenced. These results may have implications that make them useful for future breeding strategies in dairy sheep breeding.

Distinct Trajectories of Massive Recent Gene Gains and Losses in Populations of a Microbial Eukaryotic Pathogen

PubMed Central

Hartmann, Fanny E.; Croll, Daniel

2017-01-01

Abstract Differences in gene content are a significant source of variability within species and have an impact on phenotypic traits. However, little is known about the mechanisms responsible for the most recent gene gains and losses. We screened the genomes of 123 worldwide isolates of the major pathogen of wheat Zymoseptoria tritici for robust evidence of gene copy number variation. Based on orthology relationships in three closely related fungi, we identified 599 gene gains and 1,024 gene losses that have not yet reached fixation within the focal species. Our analyses of gene gains and losses segregating in populations showed that gene copy number variation arose preferentially in subtelomeres and in proximity to transposable elements. Recently lost genes were enriched in virulence factors and secondary metabolite gene clusters. In contrast, recently gained genes encoded mostly secreted protein lacking a conserved domain. We analyzed the frequency spectrum at loci segregating a gene presence–absence polymorphism in four worldwide populations. Recent gene losses showed a significant excess in low-frequency variants compared with genome-wide single nucleotide polymorphism, which is indicative of strong negative selection against gene losses. Recent gene gains were either under weak negative selection or neutral. We found evidence for strong divergent selection among populations at individual loci segregating a gene presence–absence polymorphism. Hence, gene gains and losses likely contributed to local adaptation. Our study shows that microbial eukaryotes harbor extensive copy number variation within populations and that functional differences among recently gained and lost genes led to distinct evolutionary trajectories. PMID:28981698

Novel applications of array comparative genomic hybridization in molecular diagnostics.

PubMed

Cheung, Sau W; Bi, Weimin

2018-05-31

In 2004, the implementation of array comparative genomic hybridization (array comparative genome hybridization [CGH]) into clinical practice marked a new milestone for genetic diagnosis. Array CGH and single-nucleotide polymorphism (SNP) arrays enable genome-wide detection of copy number changes in a high resolution, and therefore microarray has been recognized as the first-tier test for patients with intellectual disability or multiple congenital anomalies, and has also been applied prenatally for detection of clinically relevant copy number variations in the fetus. Area covered: In this review, the authors summarize the evolution of array CGH technology from their diagnostic laboratory, highlighting exonic SNP arrays developed in the past decade which detect small intragenic copy number changes as well as large DNA segments for the region of heterozygosity. The applications of array CGH to human diseases with different modes of inheritance with the emphasis on autosomal recessive disorders are discussed. Expert commentary: An exonic array is a powerful and most efficient clinical tool in detecting genome wide small copy number variants in both dominant and recessive disorders. However, whole-genome sequencing may become the single integrated platform for detection of copy number changes, single-nucleotide changes as well as balanced chromosomal rearrangements in the near future.

CCL3L1 copy number, CCR5 genotype and susceptibility to tuberculosis.

PubMed

Carpenter, Danielle; Taype, Carmen; Goulding, Jon; Levin, Mike; Eley, Brian; Anderson, Suzanne; Shaw, Marie-Anne; Armour, John A L

2014-01-09

Tuberculosis is a major infectious disease and functional studies have provided evidence that both the chemokine MIP-1α and its receptor CCR5 play a role in susceptibility to TB. Thus by measuring copy number variation of CCL3L1, one of the genes that encode MIP-1α, and genotyping a functional promoter polymorphism -2459A > G in CCR5 (rs1799987) we investigate the influence of MIP-1α and CCR5, independently and combined, in susceptibility to clinically active TB in three populations, a Peruvian population (n = 1132), a !Xhosa population (n = 605) and a South African Coloured population (n = 221). The three populations include patients with clinically diagnosed pulmonary TB, as well as other, less prevalent forms of extrapulmonary TB. Copy number of CCL3L1 was measured using the paralogue ratio test and exhibited ranges between 0-6 copies per diploid genome (pdg) in Peru, between 0-12 pdg in !Xhosa samples and between 0-10 pdg in South African Coloured samples. The CCR5 promoter polymorphism was observed to differ significantly in allele frequency between populations (*A; Peru f = 0.67, !Xhosa f = 0.38, Coloured f = 0.48). The case-control association studies performed however find, surprisingly, no evidence for an influence of variation in genes coding for MIP-1α or CCR5 individually or together in susceptibility to clinically active TB in these populations.

Screening for common copy-number variants in cancer genes.

PubMed

Tyson, Jess; Majerus, Tamsin M O; Walker, Susan; Armour, John A L

2010-12-01

For most cases of colorectal cancer that arise without a family history of the disease, it is proposed that an appreciable heritable component of predisposition is the result of contributions from many loci. Although progress has been made in identifying single nucleotide variants associated with colorectal cancer risk, the involvement of low-penetrance copy number variants is relatively unexplored. We have used multiplex amplifiable probe hybridization (MAPH) in a fourfold multiplex (QuadMAPH), positioned at an average resolution of one probe per 2 kb, to screen a total of 1.56 Mb of genomic DNA for copy number variants around the genes APC, AXIN1, BRCA1, BRCA2, CTNNB1, HRAS, MLH1, MSH2, and TP53. Two deletion events were detected, one upstream of MLH1 in a control individual and the other in APC in a colorectal cancer patient, but these do not seem to correspond to copy number polymorphisms with measurably high population frequencies. In summary, by means of our QuadMAPH assay, copy number measurement data were of sufficient resolution and accuracy to detect any copy number variants with high probability. However, this study has demonstrated a very low incidence of deletion and duplication variants within intronic and flanking regions of these nine genes, in both control individuals and colorectal cancer patients. Copyright © 2010 Elsevier Inc. All rights reserved.

Copy number polymorphism of the salivary amylase gene: implications in human nutrition research.

PubMed

Santos, J L; Saus, E; Smalley, S V; Cataldo, L R; Alberti, G; Parada, J; Gratacòs, M; Estivill, X

2012-01-01

The salivary α-amylase is a calcium-binding enzyme that initiates starch digestion in the oral cavity. The α-amylase genes are located in a cluster on the chromosome that includes salivary amylase genes (AMY1), two pancreatic α-amylase genes (AMY2A and AMY2B) and a related pseudogene. The AMY1 genes show extensive copy number variation which is directly proportional to the salivary α-amylase content in saliva. The α-amylase amount in saliva is also influenced by other factors, such as hydration status, psychosocial stress level, and short-term dietary habits. It has been shown that the average copy number of AMY1 gene is higher in populations that evolved under high-starch diets versus low-starch diets, reflecting an intense positive selection imposed by diet on amylase copy number during evolution. In this context, a number of different aspects can be considered in evaluating the possible impact of copy number variation of the AMY1 gene on nutrition research, such as issues related to human diet gene evolution, action on starch digestion, effect on glycemic response after starch consumption, modulation of the action of α-amylases inhibitors, effect on taste perception and satiety, influence on psychosocial stress and relation to oral health. Copyright © 2012 S. Karger AG, Basel.

DYZ1 copy number variation, Y chromosome polymorphism and early recurrent spontaneous abortion/early embryo growth arrest.

PubMed

Yan, Junhao; Fan, Lingling; Zhao, Yueran; You, Li; Wang, Laicheng; Zhao, Han; Li, Yuan; Chen, Zi-Jiang

2011-12-01

To find the association between recurrent spontaneous abortion (RSA)/early embryo growth arrest and Y chromosome polymorphism. Peripheral blood samples of the male patients of big Y chromosome, small Y chromosome and other male patients whose partners suffered from unexplained RSA/early embryo growth arrest were collected. PCR and real-time fluorescent quantitative PCR were used to test the deletion and the copy number variation of DYZ1 region in Y chromosome of the patients. A total of 79 big Y chromosome patients (48 of whose partners suffered from RSA or early embryo growth arrest), 7 small Y chromosome patients, 106 other male patients whose partners had suffered from unexplained RSA or early embryo growth arrest, and 100 normal male controls were enrolled. There was no fraction deletion of DYZ1 detected both in big Y patients and in normal men. Of RSA patients, 1 case showed deletion of 266bp from the gene locus 25-290bp, and 2 cases showed deletion of 773bp from 1347 to 2119bp. Of only 7 small Y chromosome patients, 2 cases showed deletion of 266bp from 25 to 290bp, and 4 cases showed deletion of 773bp from 1347 to 2119bp and 275bp from 3128 to 3420bp. The mean of DYZ1 copies was 3900 in normal control men; the mean in big Y patients was 5571, in RSA patients was 2655, and in small Y patients was 1059. All of the others were significantly different (P<0.01) compared with normal control men, which meant that DYZ1 copy number in normal control men was less than that of big Y chromosome patients, and was more than that of unexplained early RSA patients and small Y patients. The integrity and copy number variation of DYZ1 are closely related to the Y chromosome length under microscope. The cause of RSA/early embryo growth arrest in some couples may be the increase (big Y patients) or decrease of DYZ1 copy number in the husbands' Y chromosome. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Large scale variation in DNA copy number in chicken breeds

USDA-ARS?s Scientific Manuscript database

Background Detecting genetic variation is a critical step in elucidating the molecular mechanisms underlying phenotypic diversity. Until recently, such detection has mostly focused on single nucleotide polymorphisms (SNPs) because of the ease in screening complete genomes. Another type of variant, c...

Distinct Trajectories of Massive Recent Gene Gains and Losses in Populations of a Microbial Eukaryotic Pathogen.

PubMed

Hartmann, Fanny E; Croll, Daniel

2017-11-01

Differences in gene content are a significant source of variability within species and have an impact on phenotypic traits. However, little is known about the mechanisms responsible for the most recent gene gains and losses. We screened the genomes of 123 worldwide isolates of the major pathogen of wheat Zymoseptoria tritici for robust evidence of gene copy number variation. Based on orthology relationships in three closely related fungi, we identified 599 gene gains and 1,024 gene losses that have not yet reached fixation within the focal species. Our analyses of gene gains and losses segregating in populations showed that gene copy number variation arose preferentially in subtelomeres and in proximity to transposable elements. Recently lost genes were enriched in virulence factors and secondary metabolite gene clusters. In contrast, recently gained genes encoded mostly secreted protein lacking a conserved domain. We analyzed the frequency spectrum at loci segregating a gene presence-absence polymorphism in four worldwide populations. Recent gene losses showed a significant excess in low-frequency variants compared with genome-wide single nucleotide polymorphism, which is indicative of strong negative selection against gene losses. Recent gene gains were either under weak negative selection or neutral. We found evidence for strong divergent selection among populations at individual loci segregating a gene presence-absence polymorphism. Hence, gene gains and losses likely contributed to local adaptation. Our study shows that microbial eukaryotes harbor extensive copy number variation within populations and that functional differences among recently gained and lost genes led to distinct evolutionary trajectories. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Tailoring Nutritional Advice for Mexicans Based on Prevalence Profiles of Diet-Related Adaptive Gene Polymorphisms

PubMed Central

Ojeda-Granados, Claudia; Panduro, Arturo; Gonzalez-Aldaco, Karina; Sepulveda-Villegas, Maricruz; Rivera-Iñiguez, Ingrid

2017-01-01

Diet-related adaptive gene (DRAG) polymorphisms identified in specific populations are associated with chronic disorders in carriers of the adaptive alleles due to changes in dietary and lifestyle patterns in recent times. Mexico’s population is comprised of Amerindians (AM) and Mestizos who have variable AM, European (EUR) and African genetic ancestry and an increased risk of nutrition-related chronic diseases. Nutritional advice based on the Mexican genome and the traditional food culture is needed to develop preventive and therapeutic strategies. Therefore, we aimed to provide a prevalence profile of several DRAG polymorphisms in the Mexican population, including Central West (CW) Mexico subpopulations. Geographic heat maps were built using ArcGIS10 (Esri, Redlands, CA, USA) software, based on the published data of the MTHFR C677T (rs1801133), ABCA1 Arg230Cys (rs9282541), APOE T388C (rs429358)/C526T (rs7412), LCT C-13910T (rs4988235) polymorphisms and AMY1 copy number variation (CNV). Also, new data obtained by allelic discrimination-real-time polymerase chain reaction (RT-PCR) assays for the MTHFR, ABCA1, and APOE polymorphisms as well as the AMY1 CNV in the CW Mexico subpopulations with different proportions of AM and EUR ancestry were included. In the CW region, the highest frequency of the MTHFR 677T, ABCA1 230C and APOE ε4 adaptive alleles was observed in the AM groups, followed by Mestizos with intermediate AM ancestry. The LCT-13910T allele frequency was highest in Mestizos-EUR but extremely low in AM, while the AMY1 diploid copy number was 6.82 ± 3.3 copies. Overall, the heat maps showed a heterogeneous distribution of the DRAG polymorphisms, in which the AM groups revealed the highest frequencies of the adaptive alleles followed by Mestizos. Given these genetic differences, genome-based nutritional advice should be tailored in a regionalized and individualized manner according to the available foods and Mexican traditional food culture that may lead to a healthier dietary pattern. PMID:29125573

CCL3L1 copy number, CCR5 genotype and susceptibility to tuberculosis

PubMed Central

2014-01-01

Background Tuberculosis is a major infectious disease and functional studies have provided evidence that both the chemokine MIP-1α and its receptor CCR5 play a role in susceptibility to TB. Thus by measuring copy number variation of CCL3L1, one of the genes that encode MIP-1α, and genotyping a functional promoter polymorphism -2459A > G in CCR5 (rs1799987) we investigate the influence of MIP-1α and CCR5, independently and combined, in susceptibility to clinically active TB in three populations, a Peruvian population (n = 1132), a !Xhosa population (n = 605) and a South African Coloured population (n = 221). The three populations include patients with clinically diagnosed pulmonary TB, as well as other, less prevalent forms of extrapulmonary TB. Methods and results Copy number of CCL3L1 was measured using the paralogue ratio test and exhibited ranges between 0–6 copies per diploid genome (pdg) in Peru, between 0–12 pdg in !Xhosa samples and between 0–10 pdg in South African Coloured samples. The CCR5 promoter polymorphism was observed to differ significantly in allele frequency between populations (*A; Peru f = 0.67, !Xhosa f = 0.38, Coloured f = 0.48). Conclusions The case–control association studies performed however find, surprisingly, no evidence for an influence of variation in genes coding for MIP-1α or CCR5 individually or together in susceptibility to clinically active TB in these populations. PMID:24405814

Characterization of the variable-number tandem repeats in vrrA from different Bacillus anthracis isolates

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

Jackson, P.J.; Walthers, E.A.; Richmond, K.L.

1997-04-01

PCR analysis of 198 Bacillus anthracis isolates revealed a variable region of DNA sequence differing in length among the isolates. Five Polymorphisms differed by the presence Of two to six copies of the 12-bp tandem repeat 5{prime}-CAATATCAACAA-3{prime}. This variable-number tandem repeat (VNTR) region is located within a larger sequence containing one complete open reading frame that encodes a putative 30-kDa protein. Length variation did not change the reading frame of the encoded protein and only changed the copy number of a 4-amino-acid sequence (QYQQ) from 2 to 6. The structure of the VNTR region suggests that these multiple repeats aremore » generated by recombination or polymerase slippage. Protein structures predicted from the reverse-translated DNA sequence suggest that any structural changes in the encoded protein are confined to the region encoded by the VNTR sequence. Copy number differences in the VNTR region were used to define five different B. anthracis alleles. Characterization of 198 isolates revealed allele frequencies of 6.1, 17.7, 59.6, 5.6, and 11.1% sequentially from shorter to longer alleles. The high degree of polymorphism in the VNTR region provides a criterion for assigning isolates to five allelic categories. There is a correlation between categories and geographic distribution. Such molecular markers can be used to monitor the epidemiology of anthrax outbreaks in domestic and native herbivore populations. 22 refs., 4 figs., 3 tabs.« less

A survey of copy number variation in the porcine genome detected from whole-genome sequence

USDA-ARS?s Scientific Manuscript database

An important challenge to post-genomic biology is relating observed phenotypic variation to the underlying genotypic variation. Genome-wide association studies (GWAS) have made thousands of connections between single nucleotide polymorphisms (SNPs) and phenotypes, implicating regions of the genome t...

Genomic diversity of the human intestinal parasite Entamoeba histolytica

PubMed Central

2012-01-01

Background Entamoeba histolytica is a significant cause of disease worldwide. However, little is known about the genetic diversity of the parasite. We re-sequenced the genomes of ten laboratory cultured lines of the eukaryotic pathogen Entamoeba histolytica in order to develop a picture of genetic diversity across the genome. Results The extreme nucleotide composition bias and repetitiveness of the E. histolytica genome provide a challenge for short-read mapping, yet we were able to define putative single nucleotide polymorphisms in a large portion of the genome. The results suggest a rather low level of single nucleotide diversity, although genes and gene families with putative roles in virulence are among the more polymorphic genes. We did observe large differences in coverage depth among genes, indicating differences in gene copy number between genomes. We found evidence indicating that recombination has occurred in the history of the sequenced genomes, suggesting that E. histolytica may reproduce sexually. Conclusions E. histolytica displays a relatively low level of nucleotide diversity across its genome. However, large differences in gene family content and gene copy number are seen among the sequenced genomes. The pattern of polymorphism indicates that E. histolytica reproduces sexually, or has done so in the past, which has previously been suggested but not proven. PMID:22630046

High-Resolution SNP/CGH Microarrays Reveal the Accumulation of Loss of Heterozygosity in Commonly Used Candida albicans Strains

PubMed Central

Abbey, Darren; Hickman, Meleah; Gresham, David; Berman, Judith

2011-01-01

Phenotypic diversity can arise rapidly through loss of heterozygosity (LOH) or by the acquisition of copy number variations (CNV) spanning whole chromosomes or shorter contiguous chromosome segments. In Candida albicans, a heterozygous diploid yeast pathogen with no known meiotic cycle, homozygosis and aneuploidy alter clinical characteristics, including drug resistance. Here, we developed a high-resolution microarray that simultaneously detects ∼39,000 single nucleotide polymorphism (SNP) alleles and ∼20,000 copy number variation loci across the C. albicans genome. An important feature of the array analysis is a computational pipeline that determines SNP allele ratios based upon chromosome copy number. Using the array and analysis tools, we constructed a haplotype map (hapmap) of strain SC5314 to assign SNP alleles to specific homologs, and we used it to follow the acquisition of loss of heterozygosity (LOH) and copy number changes in a series of derived laboratory strains. This high-resolution SNP/CGH microarray and the associated hapmap facilitated the phasing of alleles in lab strains and revealed detrimental genome changes that arose frequently during molecular manipulations of laboratory strains. Furthermore, it provided a useful tool for rapid, high-resolution, and cost-effective characterization of changes in allele diversity as well as changes in chromosome copy number in new C. albicans isolates. PMID:22384363

A large-scale survey of genetic copy number variations among Han Chinese residing in Taiwan

PubMed Central

Lin, Chien-Hsing; Li, Ling-Hui; Ho, Sheng-Feng; Chuang, Tzu-Po; Wu, Jer-Yuarn; Chen, Yuan-Tsong; Fann, Cathy SJ

2008-01-01

Background Copy number variations (CNVs) have recently been recognized as important structural variations in the human genome. CNVs can affect gene expression and thus may contribute to phenotypic differences. The copy number inferring tool (CNIT) is an effective hidden Markov model-based algorithm for estimating allele-specific copy number and predicting chromosomal alterations from single nucleotide polymorphism microarrays. The CNIT algorithm, which was constructed using data from 270 HapMap multi-ethnic individuals, was applied to identify CNVs from 300 unrelated Han Chinese individuals in Taiwan. Results Using stringent selection criteria, 230 regions with variable copy numbers were identified in the Han Chinese population; 133 (57.83%) had been reported previously, 64 displayed greater than 1% CNV allele frequency. The average size of the CNV regions was 322 kb (ranging from 1.48 kb to 5.68 Mb) and covered a total of 2.47% of the human genome. A total of 196 of the CNV regions were simple deletions and 27 were simple amplifications. There were 449 genes and 5 microRNAs within these CNV regions; some of these genes are known to be associated with diseases. Conclusion The identified CNVs are characteristic of the Han Chinese population and should be considered when genetic studies are conducted. The CNV distribution in the human genome is still poorly characterized, and there is much diversity among different ethnic populations. PMID:19108714

Ty1-copia elements reveal diverse insertion sites linked to polymorphisms among flax (Linum usitatissimum L.) accessions.

PubMed

Galindo-González, Leonardo; Mhiri, Corinne; Grandbastien, Marie-Angèle; Deyholos, Michael K

2016-12-07

Initial characterization of the flax genome showed that Ty1-copia retrotransposons are abundant, with several members being recently inserted, and in close association with genes. Recent insertions indicate a potential for ongoing transpositional activity that can create genomic diversity among accessions, cultivars or varieties. The polymorphisms generated constitute a good source of molecular markers that may be associated with phenotype if the insertions alter gene activity. Flax, where accessions are bred mainly for seed nutritional properties or for fibers, constitutes a good model for studying the relationship of transpositional activity with diversification and breeding. In this study, we estimated copy number and used a type of transposon display known as Sequence-Specific Amplification Polymorphisms (SSAPs), to characterize six families of Ty1-copia elements across 14 flax accessions. Polymorphic insertion sites were sequenced to find insertions that could potentially alter gene expression, and a preliminary test was performed with selected genes bearing transposable element (TE) insertions. Quantification of six families of Ty1-copia elements indicated different abundances among TE families and between flax accessions, which suggested diverse transpositional histories. SSAPs showed a high level of polymorphism in most of the evaluated retrotransposon families, with a trend towards higher levels of polymorphism in low-copy number families. Ty1-copia insertion polymorphisms among cultivars allowed a general distinction between oil and fiber types, and between spring and winter types, demonstrating their utility in diversity studies. Characterization of polymorphic insertions revealed an overwhelming association with genes, with insertions disrupting exons, introns or within 1 kb of coding regions. A preliminary test on the potential transcriptional disruption by TEs of four selected genes evaluated in three different tissues, showed one case of significant impact of the insertion on gene expression. We demonstrated that specific Ty1-copia families have been active since breeding commenced in flax. The retrotransposon-derived polymorphism can be used to separate flax types, and the close association of many insertions with genes defines a good source of potential mutations that could be associated with phenotypic changes, resulting in diversification processes.

Copy Number Variation in Fungi and Its Implications for Wine Yeast Genetic Diversity and Adaptation

PubMed Central

Steenwyk, Jacob L.; Rokas, Antonis

2018-01-01

In recent years, copy number (CN) variation has emerged as a new and significant source of genetic polymorphisms contributing to the phenotypic diversity of populations. CN variants are defined as genetic loci that, due to duplication and deletion, vary in their number of copies across individuals in a population. CN variants range in size from 50 base pairs to whole chromosomes, can influence gene activity, and are associated with a wide range of phenotypes in diverse organisms, including the budding yeast Saccharomyces cerevisiae. In this review, we introduce CN variation, discuss the genetic and molecular mechanisms implicated in its generation, how they can contribute to genetic and phenotypic diversity in fungal populations, and consider how CN variants may influence wine yeast adaptation in fermentation-related processes. In particular, we focus on reviewing recent work investigating the contribution of changes in CN of fermentation-related genes in yeast wine strains and offer notable illustrations of such changes, including the high levels of CN variation among the CUP genes, which confer resistance to copper, a metal with fungicidal properties, and the preferential deletion and duplication of the MAL1 and MAL3 loci, respectively, which are responsible for metabolizing maltose and sucrose. Based on the available data, we propose that CN variation is a substantial dimension of yeast genetic diversity that occurs largely independent of single nucleotide polymorphisms. As such, CN variation harbors considerable potential for understanding and manipulating yeast strains in the wine fermentation environment and beyond. PMID:29520259

Genome-wide association study of preeclampsia detects novel maternal single nucleotide polymorphisms and copy-number variants in subsets of the Hyperglycemia and Adverse Pregnancy Outcome (HAPO) study cohort

PubMed Central

Zhao, Linlu; Bracken, Michael B.; DeWan, Andrew T.

2013-01-01

Summary A genome-wide association study was undertaken to identify maternal single nucleotide polymorphisms (SNPs) and copy-number variants (CNVs) associated with preeclampsia. Case-control analysis was performed on 1070 Afro-Caribbean (n=21 cases and 1049 controls) and 723 Hispanic (n=62 cases and 661 controls) mothers and 1257 mothers of European ancestry (n=50 cases and 1207 controls) from the Hyperglycemia and Adverse Pregnancy Outcome (HAPO) study. European ancestry subjects were genotyped on Illumina Human610-Quad and Afro-Caribbean and Hispanic subjects were genotyped on Illumina Human1M-Duo BeadChip microarrays. Genome-wide SNP data were analyzed using PLINK. CNVs were called using three detection algorithms (GNOSIS, PennCNV, and QuantiSNP), merged using CNVision, and then screened using stringent criteria. SNP and CNV findings were compared to those of the Study of Pregnancy Hypertension in Iowa (SOPHIA), an independent preeclampsia case-control dataset of Caucasian mothers (n=177 cases and 116 controls). A list of top SNPs were identified for each of the HAPO ethnic groups, but none reached Bonferroni-corrected significance. Novel candidate CNVs showing enrichment among preeclampsia cases were also identified in each of the three ethnic groups. Several variants were suggestively replicated in SOPHIA. The discovered SNPs and copy-number variable regions present interesting candidate genetic variants for preeclampsia that warrant further replication and investigation. PMID:23551011

A genome-wide survey reveals a deletion polymorphism associated with resistance to gastrointestinal nematodes in Angus cattle

USDA-ARS?s Scientific Manuscript database

Gastrointestinal (GI) nematode infections are a worldwide threat to animal health and production. In this study, we performed a genome-wide association study between copy number variations (CNV) and resistance to GI nematodes in an Angus cattle population. Using a linear regression analysis, we iden...

Polymorphisms in genes of interleukin 12 and its receptors and their association with protection against severe malarial anaemia in children in western Kenya.

PubMed

Zhang, Lyna; Prather, Donald; Vanden Eng, Jodi; Crawford, Sara; Kariuki, Simon; ter Kuile, Feiko; Terlouw, Dianne; Nahlen, Bernard; Lal, Altaf A; Slutsker, Laurence; Udhayakumar, Venkatachalam; Shi, Ya Ping

2010-03-29

Malarial anaemia is characterized by destruction of malaria infected red blood cells and suppression of erythropoiesis. Interleukin 12 (IL12) significantly boosts erythropoietic responses in murine models of malarial anaemia and decreased IL12 levels are associated with severe malarial anaemia (SMA) in children. Based on the biological relevance of IL12 in malaria anaemia, the relationship between genetic polymorphisms of IL12 and its receptors and SMA was examined. Fifty-five tagging single nucleotide polymorphisms covering genes encoding two IL12 subunits, IL12A and IL12B, and its receptors, IL12RB1 and IL12RB2, were examined in a cohort of 913 children residing in Asembo Bay region of western Kenya. An increasing copy number of minor variant (C) in IL12A (rs2243140) was significantly associated with a decreased risk of SMA (P = 0.006; risk ratio, 0.52 for carrying one copy of allele C and 0.28 for two copies). Individuals possessing two copies of a rare variant (C) in IL12RB1 (rs429774) also appeared to be strongly protective against SMA (P = 0.00005; risk ratio, 0.18). In addition, children homozygous for another rare allele (T) in IL12A (rs22431348) were associated with reduced risk of severe anaemia (SA) (P = 0.004; risk ratio, 0.69) and of severe anaemia with any parasitaemia (SAP) (P = 0.004; risk ratio, 0.66). In contrast, AG genotype for another variant in IL12RB1 (rs383483) was associated with susceptibility to high-density parasitaemia (HDP) (P = 0.003; risk ratio, 1.21). This study has shown strong associations between polymorphisms in the genes of IL12A and IL12RB1 and protection from SMA in Kenyan children, suggesting that human genetic variants of IL12 related genes may significantly contribute to the development of anaemia in malaria patients.

Copy number variation of CBF-A14 at the Fr-A2 locus determines frost tolerance in winter durum wheat.

PubMed

Sieber, Alisa-Naomi; Longin, C Friedrich H; Leiser, Willmar L; Würschum, Tobias

2016-06-01

Frost tolerance in durum wheat is mainly controlled by copy number variation of CBF - A14 at the Fr - A2 locus. Frost tolerance is a key trait for successful breeding of winter durum wheat (Triticum durum) which can increase the yield performance in regions favoring autumn-sown winter cereals. The aim of this study was to investigate the genetic architecture of frost tolerance in order to provide molecular support for the breeding of winter durum wheat. To this end, a diverse panel of 170 winter and 14 spring durum wheat genotypes of worldwide origin was evaluated for frost tolerance in the field, as well as in a semi-controlled test. A total of 30,611 polymorphic genome-wide markers obtained by a genotyping-by-sequencing approach and markers for candidate loci were used to assess marker-trait associations. One major QTL was detected on chromosome 5A, likely corresponding to Frost Resistance-A2 (Fr-A2). Further analyses strongly support the conclusion that copy number variation of CBF-A14 at the Fr-A2 locus is the causal polymorphism underlying this major QTL. It explains 91.6 % of the genotypic variance and a haploblock of two strongly associated markers in the QTL region also allowed to capture the variance of this QTL. In addition to this major QTL, a much smaller contribution of 4.2 % was observed for Fr-B2. We further investigated this major QTL and found that the copy number of CBF-A14 and the frequency of the frost tolerant haplotype mirrored the climatic conditions in the genotypes' country of origin, suggesting selection through breeding. Two functional KASP markers were developed which facilitate a high-throughput screening of the haploblock and thus a marker-based breeding of frost tolerance in winter durum wheat.

The Evolutionary Fates of a Large Segmental Duplication in Mouse

PubMed Central

Morgan, Andrew P.; Holt, J. Matthew; McMullan, Rachel C.; Bell, Timothy A.; Clayshulte, Amelia M.-F.; Didion, John P.; Yadgary, Liran; Thybert, David; Odom, Duncan T.; Flicek, Paul; McMillan, Leonard; de Villena, Fernando Pardo-Manuel

2016-01-01

Gene duplication and loss are major sources of genetic polymorphism in populations, and are important forces shaping the evolution of genome content and organization. We have reconstructed the origin and history of a 127-kbp segmental duplication, R2d, in the house mouse (Mus musculus). R2d contains a single protein-coding gene, Cwc22. De novo assembly of both the ancestral (R2d1) and the derived (R2d2) copies reveals that they have been subject to nonallelic gene conversion events spanning tens of kilobases. R2d2 is also a hotspot for structural variation: its diploid copy number ranges from zero in the mouse reference genome to >80 in wild mice sampled from around the globe. Hemizygosity for high copy-number alleles of R2d2 is associated in cis with meiotic drive; suppression of meiotic crossovers; and copy-number instability, with a mutation rate in excess of 1 per 100 transmissions in some laboratory populations. Our results provide a striking example of allelic diversity generated by duplication and demonstrate the value of de novo assembly in a phylogenetic context for understanding the mutational processes affecting duplicate genes. PMID:27371833

Genome-wide association study of CNVs in 16,000 cases of eight common diseases and 3,000 shared controls.

PubMed

Craddock, Nick; Hurles, Matthew E; Cardin, Niall; Pearson, Richard D; Plagnol, Vincent; Robson, Samuel; Vukcevic, Damjan; Barnes, Chris; Conrad, Donald F; Giannoulatou, Eleni; Holmes, Chris; Marchini, Jonathan L; Stirrups, Kathy; Tobin, Martin D; Wain, Louise V; Yau, Chris; Aerts, Jan; Ahmad, Tariq; Andrews, T Daniel; Arbury, Hazel; Attwood, Anthony; Auton, Adam; Ball, Stephen G; Balmforth, Anthony J; Barrett, Jeffrey C; Barroso, Inês; Barton, Anne; Bennett, Amanda J; Bhaskar, Sanjeev; Blaszczyk, Katarzyna; Bowes, John; Brand, Oliver J; Braund, Peter S; Bredin, Francesca; Breen, Gerome; Brown, Morris J; Bruce, Ian N; Bull, Jaswinder; Burren, Oliver S; Burton, John; Byrnes, Jake; Caesar, Sian; Clee, Chris M; Coffey, Alison J; Connell, John M C; Cooper, Jason D; Dominiczak, Anna F; Downes, Kate; Drummond, Hazel E; Dudakia, Darshna; Dunham, Andrew; Ebbs, Bernadette; Eccles, Diana; Edkins, Sarah; Edwards, Cathryn; Elliot, Anna; Emery, Paul; Evans, David M; Evans, Gareth; Eyre, Steve; Farmer, Anne; Ferrier, I Nicol; Feuk, Lars; Fitzgerald, Tomas; Flynn, Edward; Forbes, Alistair; Forty, Liz; Franklyn, Jayne A; Freathy, Rachel M; Gibbs, Polly; Gilbert, Paul; Gokumen, Omer; Gordon-Smith, Katherine; Gray, Emma; Green, Elaine; Groves, Chris J; Grozeva, Detelina; Gwilliam, Rhian; Hall, Anita; Hammond, Naomi; Hardy, Matt; Harrison, Pile; Hassanali, Neelam; Hebaishi, Husam; Hines, Sarah; Hinks, Anne; Hitman, Graham A; Hocking, Lynne; Howard, Eleanor; Howard, Philip; Howson, Joanna M M; Hughes, Debbie; Hunt, Sarah; Isaacs, John D; Jain, Mahim; Jewell, Derek P; Johnson, Toby; Jolley, Jennifer D; Jones, Ian R; Jones, Lisa A; Kirov, George; Langford, Cordelia F; Lango-Allen, Hana; Lathrop, G Mark; Lee, James; Lee, Kate L; Lees, Charlie; Lewis, Kevin; Lindgren, Cecilia M; Maisuria-Armer, Meeta; Maller, Julian; Mansfield, John; Martin, Paul; Massey, Dunecan C O; McArdle, Wendy L; McGuffin, Peter; McLay, Kirsten E; Mentzer, Alex; Mimmack, Michael L; Morgan, Ann E; Morris, Andrew P; Mowat, Craig; Myers, Simon; Newman, William; Nimmo, Elaine R; O'Donovan, Michael C; Onipinla, Abiodun; Onyiah, Ifejinelo; Ovington, Nigel R; Owen, Michael J; Palin, Kimmo; Parnell, Kirstie; Pernet, David; Perry, John R B; Phillips, Anne; Pinto, Dalila; Prescott, Natalie J; Prokopenko, Inga; Quail, Michael A; Rafelt, Suzanne; Rayner, Nigel W; Redon, Richard; Reid, David M; Renwick; Ring, Susan M; Robertson, Neil; Russell, Ellie; St Clair, David; Sambrook, Jennifer G; Sanderson, Jeremy D; Schuilenburg, Helen; Scott, Carol E; Scott, Richard; Seal, Sheila; Shaw-Hawkins, Sue; Shields, Beverley M; Simmonds, Matthew J; Smyth, Debbie J; Somaskantharajah, Elilan; Spanova, Katarina; Steer, Sophia; Stephens, Jonathan; Stevens, Helen E; Stone, Millicent A; Su, Zhan; Symmons, Deborah P M; Thompson, John R; Thomson, Wendy; Travers, Mary E; Turnbull, Clare; Valsesia, Armand; Walker, Mark; Walker, Neil M; Wallace, Chris; Warren-Perry, Margaret; Watkins, Nicholas A; Webster, John; Weedon, Michael N; Wilson, Anthony G; Woodburn, Matthew; Wordsworth, B Paul; Young, Allan H; Zeggini, Eleftheria; Carter, Nigel P; Frayling, Timothy M; Lee, Charles; McVean, Gil; Munroe, Patricia B; Palotie, Aarno; Sawcer, Stephen J; Scherer, Stephen W; Strachan, David P; Tyler-Smith, Chris; Brown, Matthew A; Burton, Paul R; Caulfield, Mark J; Compston, Alastair; Farrall, Martin; Gough, Stephen C L; Hall, Alistair S; Hattersley, Andrew T; Hill, Adrian V S; Mathew, Christopher G; Pembrey, Marcus; Satsangi, Jack; Stratton, Michael R; Worthington, Jane; Deloukas, Panos; Duncanson, Audrey; Kwiatkowski, Dominic P; McCarthy, Mark I; Ouwehand, Willem; Parkes, Miles; Rahman, Nazneen; Todd, John A; Samani, Nilesh J; Donnelly, Peter

2010-04-01

Copy number variants (CNVs) account for a major proportion of human genetic polymorphism and have been predicted to have an important role in genetic susceptibility to common disease. To address this we undertook a large, direct genome-wide study of association between CNVs and eight common human diseases. Using a purpose-designed array we typed approximately 19,000 individuals into distinct copy-number classes at 3,432 polymorphic CNVs, including an estimated approximately 50% of all common CNVs larger than 500 base pairs. We identified several biological artefacts that lead to false-positive associations, including systematic CNV differences between DNAs derived from blood and cell lines. Association testing and follow-up replication analyses confirmed three loci where CNVs were associated with disease-IRGM for Crohn's disease, HLA for Crohn's disease, rheumatoid arthritis and type 1 diabetes, and TSPAN8 for type 2 diabetes-although in each case the locus had previously been identified in single nucleotide polymorphism (SNP)-based studies, reflecting our observation that most common CNVs that are well-typed on our array are well tagged by SNPs and so have been indirectly explored through SNP studies. We conclude that common CNVs that can be typed on existing platforms are unlikely to contribute greatly to the genetic basis of common human diseases.

Single Nucleotide Polymorphism Array Analysis of Bone Marrow Failure Patients Reveals Characteristic Patterns of Genetic Changes

PubMed Central

Babushok, Daria V.; Xie, Hongbo M.; Roth, Jacquelyn J.; Perdigones, Nieves; Olson, Timothy S.; Cockroft, Joshua D.; Gai, Xiaowu; Perin, Juan C.; Li, Yimei; Paessler, Michele E.; Hakonarson, Hakon; Podsakoff, Gregory M.; Mason, Philip J.; Biegel, Jaclyn A.; Bessler, Monica

2013-01-01

Summary The bone marrow failure syndromes (BMFS) are a heterogeneous group of rare blood disorders characterized by inadequate haematopoiesis, clonal evolution, and increased risk of leukaemia. Single nucleotide polymorphism arrays (SNP-A) have been proposed as a tool for surveillance of clonal evolution in BMFS. To better understand the natural history of BMFS and to assess the clinical utility of SNP-A in these disorders, we analysed 124 SNP-A from a comprehensively characterized cohort of 91 patients at our BMFS centre. SNP-A were correlated with medical histories, haematopathology, cytogenetic and molecular data. To assess clonal evolution, longitudinal analysis of SNP-A was performed in 25 patients. We found that acquired copy number-neutral loss of heterozygosity (CN-LOH) was significantly more frequent in acquired aplastic anaemia (aAA) than in other BMFS (odds ratio 12.2, p<0.01). Homozygosity by descent was most common in congenital BMFS, frequently unmasking autosomal recessive mutations. Copy number variants (CNVs) were frequently polymorphic, and we identified CNVs enriched in neutropenia and aAA. Our results suggest that acquired CN-LOH is a general phenomenon in aAA that is probably mechanistically and prognostically distinct from typical CN-LOH of myeloid malignancies. Our analysis of clinical utility of SNP-A shows the highest yield of detecting new clonal haematopoiesis at diagnosis and at relapse. PMID:24116929

Single nucleotide polymorphism array analysis of bone marrow failure patients reveals characteristic patterns of genetic changes.

PubMed

Babushok, Daria V; Xie, Hongbo M; Roth, Jacquelyn J; Perdigones, Nieves; Olson, Timothy S; Cockroft, Joshua D; Gai, Xiaowu; Perin, Juan C; Li, Yimei; Paessler, Michele E; Hakonarson, Hakon; Podsakoff, Gregory M; Mason, Philip J; Biegel, Jaclyn A; Bessler, Monica

2014-01-01

The bone marrow failure syndromes (BMFS) are a heterogeneous group of rare blood disorders characterized by inadequate haematopoiesis, clonal evolution, and increased risk of leukaemia. Single nucleotide polymorphism arrays (SNP-A) have been proposed as a tool for surveillance of clonal evolution in BMFS. To better understand the natural history of BMFS and to assess the clinical utility of SNP-A in these disorders, we analysed 124 SNP-A from a comprehensively characterized cohort of 91 patients at our BMFS centre. SNP-A were correlated with medical histories, haematopathology, cytogenetic and molecular data. To assess clonal evolution, longitudinal analysis of SNP-A was performed in 25 patients. We found that acquired copy number-neutral loss of heterozygosity (CN-LOH) was significantly more frequent in acquired aplastic anaemia (aAA) than in other BMFS (odds ratio 12·2, P < 0·01). Homozygosity by descent was most common in congenital BMFS, frequently unmasking autosomal recessive mutations. Copy number variants (CNVs) were frequently polymorphic, and we identified CNVs enriched in neutropenia and aAA. Our results suggest that acquired CN-LOH is a general phenomenon in aAA that is probably mechanistically and prognostically distinct from typical CN-LOH of myeloid malignancies. Our analysis of clinical utility of SNP-A shows the highest yield of detecting new clonal haematopoiesis at diagnosis and at relapse. © 2013 John Wiley & Sons Ltd.

Transcriptionally active LTR retrotransposons in Eucalyptus genus are differentially expressed and insertionally polymorphic.

PubMed

Marcon, Helena Sanches; Domingues, Douglas Silva; Silva, Juliana Costa; Borges, Rafael Junqueira; Matioli, Fábio Filippi; Fontes, Marcos Roberto de Mattos; Marino, Celso Luis

2015-08-14

In Eucalyptus genus, studies on genome composition and transposable elements (TEs) are particularly scarce. Nearly half of the recently released Eucalyptus grandis genome is composed by retrotransposons and this data provides an important opportunity to understand TE dynamics in Eucalyptus genome and transcriptome. We characterized nine families of transcriptionally active LTR retrotransposons from Copia and Gypsy superfamilies in Eucalyptus grandis genome and we depicted genomic distribution and copy number in two Eucalyptus species. We also evaluated genomic polymorphism and transcriptional profile in three organs of five Eucalyptus species. We observed contrasting genomic and transcriptional behavior in the same family among different species. RLC_egMax_1 was the most prevalent family and RLC_egAngela_1 was the family with the lowest copy number. Most families of both superfamilies have their insertions occurring <3 million years, except one Copia family, RLC_egBianca_1. Protein theoretical models suggest different properties between Copia and Gypsy domains. IRAP and REMAP markers suggested genomic polymorphisms among Eucalyptus species. Using EST analysis and qRT-PCRs, we observed transcriptional activity in several tissues and in all evaluated species. In some families, osmotic stress increases transcript values. Our strategy was successful in isolating transcriptionally active retrotransposons in Eucalyptus, and each family has a particular genomic and transcriptional pattern. Overall, our results show that retrotransposon activity have differentially affected genome and transcriptome among Eucalyptus species.

New insights into mitogenomic phylogeny and copy number in eight indigenous sheep populations based on the ATP synthase and cytochrome c oxidase genes.

PubMed

Xiao, P; Niu, L L; Zhao, Q J; Chen, X Y; Wang, L J; Li, L; Zhang, H P; Guo, J Z; Xu, H Y; Zhong, T

2017-11-16

The origins and phylogeny of different sheep breeds has been widely studied using polymorphisms within the mitochondrial hypervariable region. However, little is known about the mitochondrial DNA (mtDNA) content and phylogeny based on mtDNA protein-coding genes. In this study, we assessed the phylogeny and copy number of the mtDNA in eight indigenous (population size, n=184) and three introduced (n=66) sheep breeds in China based on five mitochondrial coding genes (COX1, COX2, ATP8, ATP6 and COX3). The mean haplotype and nucleotide diversities were 0.944 and 0.00322, respectively. We identified a correlation between the lineages distribution and the genetic distance, whereby Valley-type Tibetan sheep had a closer genetic relationship with introduced breeds (Dorper, Poll Dorset and Suffolk) than with other indigenous breeds. Similarly, the Median-joining profile of haplotypes revealed the distribution of clusters according to genetic differences. Moreover, copy number analysis based on the five mitochondrial coding genes was affected by the genetic distance combining with genetic phylogeny; we also identified obvious non-synonymous mutations in ATP6 between the different levels of copy number expressions. These results imply that differences in mitogenomic compositions resulting from geographical separation lead to differences in mitochondrial function.

The effect of input DNA copy number on genotype call and characterising SNP markers in the humpback whale genome using a nanofluidic array.

PubMed

Bhat, Somanath; Polanowski, Andrea M; Double, Mike C; Jarman, Simon N; Emslie, Kerry R

2012-01-01

Recent advances in nanofluidic technologies have enabled the use of Integrated Fluidic Circuits (IFCs) for high-throughput Single Nucleotide Polymorphism (SNP) genotyping (GT). In this study, we implemented and validated a relatively low cost nanofluidic system for SNP-GT with and without Specific Target Amplification (STA). As proof of principle, we first validated the effect of input DNA copy number on genotype call rate using well characterised, digital PCR (dPCR) quantified human genomic DNA samples and then implemented the validated method to genotype 45 SNPs in the humpback whale, Megaptera novaeangliae, nuclear genome. When STA was not incorporated, for a homozygous human DNA sample, reaction chambers containing, on average 9 to 97 copies, showed 100% call rate and accuracy. Below 9 copies, the call rate decreased, and at one copy it was 40%. For a heterozygous human DNA sample, the call rate decreased from 100% to 21% when predicted copies per reaction chamber decreased from 38 copies to one copy. The tightness of genotype clusters on a scatter plot also decreased. In contrast, when the same samples were subjected to STA prior to genotyping a call rate and a call accuracy of 100% were achieved. Our results demonstrate that low input DNA copy number affects the quality of data generated, in particular for a heterozygous sample. Similar to human genomic DNA, a call rate and a call accuracy of 100% was achieved with whale genomic DNA samples following multiplex STA using either 15 or 45 SNP-GT assays. These calls were 100% concordant with their true genotypes determined by an independent method, suggesting that the nanofluidic system is a reliable platform for executing call rates with high accuracy and concordance in genomic sequences derived from biological tissue.

Sequenza: allele-specific copy number and mutation profiles from tumor sequencing data.

PubMed

Favero, F; Joshi, T; Marquard, A M; Birkbak, N J; Krzystanek, M; Li, Q; Szallasi, Z; Eklund, A C

2015-01-01

Exome or whole-genome deep sequencing of tumor DNA along with paired normal DNA can potentially provide a detailed picture of the somatic mutations that characterize the tumor. However, analysis of such sequence data can be complicated by the presence of normal cells in the tumor specimen, by intratumor heterogeneity, and by the sheer size of the raw data. In particular, determination of copy number variations from exome sequencing data alone has proven difficult; thus, single nucleotide polymorphism (SNP) arrays have often been used for this task. Recently, algorithms to estimate absolute, but not allele-specific, copy number profiles from tumor sequencing data have been described. We developed Sequenza, a software package that uses paired tumor-normal DNA sequencing data to estimate tumor cellularity and ploidy, and to calculate allele-specific copy number profiles and mutation profiles. We applied Sequenza, as well as two previously published algorithms, to exome sequence data from 30 tumors from The Cancer Genome Atlas. We assessed the performance of these algorithms by comparing their results with those generated using matched SNP arrays and processed by the allele-specific copy number analysis of tumors (ASCAT) algorithm. Comparison between Sequenza/exome and SNP/ASCAT revealed strong correlation in cellularity (Pearson's r = 0.90) and ploidy estimates (r = 0.42, or r = 0.94 after manual inspecting alternative solutions). This performance was noticeably superior to previously published algorithms. In addition, in artificial data simulating normal-tumor admixtures, Sequenza detected the correct ploidy in samples with tumor content as low as 30%. The agreement between Sequenza and SNP array-based copy number profiles suggests that exome sequencing alone is sufficient not only for identifying small scale mutations but also for estimating cellularity and inferring DNA copy number aberrations. © The Author 2014. Published by Oxford University Press on behalf of the European Society for Medical Oncology.

"SLC2A3" Single-Nucleotide Polymorphism and Duplication Influence Cognitive Processing and Population-Specific Risk for Attention-Deficit/Hyperactivity Disorder

ERIC Educational Resources Information Center

Merker, Sören; Reif, Andreas; Ziegler, Georg C.; Weber, Heike; Mayer, Ute; Ehlis, Ann-Christine; Conzelmann, Annette; Johansson, Stefan; Müller-Reible, Clemens; Nanda, Indrajit; Haaf, Thomas; Ullmann, Reinhard; Romanos, Marcel; Fallgatter, Andreas J.; Pauli, Paul; Strekalova, Tatyana; Jansch, Charline; Vasquez, Alejandro Arias; Haavik, Jan; Ribasés, Marta; Ramos-Quiroga, Josep Antoni; Buitelaar, Jan K.; Franke, Barbara; Lesch, Klaus-Peter

2017-01-01

Background: Attention-deficit/hyperactivity disorder (ADHD) is a common, highly heritable neurodevelopmental disorder with profound cognitive, behavioral, and psychosocial impairments with persistence across the life cycle. Our initial genome-wide screening approach for copy number variants (CNVs) in ADHD implicated a duplication of…

Detection and assessment of copy number variation using PacBio long-read and Illumina sequencing in New Zealand dairy cattle.

PubMed

Couldrey, C; Keehan, M; Johnson, T; Tiplady, K; Winkelman, A; Littlejohn, M D; Scott, A; Kemper, K E; Hayes, B; Davis, S R; Spelman, R J

2017-07-01

Single nucleotide polymorphisms have been the DNA variant of choice for genomic prediction, largely because of the ease of single nucleotide polymorphism genotype collection. In contrast, structural variants (SV), which include copy number variants (CNV), translocations, insertions, and inversions, have eluded easy detection and characterization, particularly in nonhuman species. However, evidence increasingly shows that SV not only contribute a substantial proportion of genetic variation but also have significant influence on phenotypes. Here we present the discovery of CNV in a prominent New Zealand dairy bull using long-read PacBio (Pacific Biosciences, Menlo Park, CA) sequencing technology and the Sniffles SV discovery tool (version 0.0.1; https://github.com/fritzsedlazeck/Sniffles). The CNV identified from long reads were compared with CNV discovered in the same bull from Illumina sequencing using CNVnator (read depth-based tool; Illumina Inc., San Diego, CA) as a means of validation. Subsequently, further validation was undertaken using whole-genome Illumina sequencing of 556 cattle representing the wider New Zealand dairy cattle population. Very limited overlap was observed in CNV discovered from the 2 sequencing platforms, in part because of the differences in size of CNV detected. Only a few CNV were therefore able to be validated using this approach. However, the ability to use CNVnator to genotype the 557 cattle for copy number across all regions identified as putative CNV allowed a genome-wide assessment of transmission level of copy number based on pedigree. The more highly transmissible a putative CNV region was observed to be, the more likely the distribution of copy number was multimodal across the 557 sequenced animals. Furthermore, visual assessment of highly transmissible CNV regions provided evidence supporting the presence of CNV across the sequenced animals. This transmission-based approach was able to confirm a subset of CNV that segregates in the New Zealand dairy cattle population. Genome-wide identification and validation of CNV is an important step toward their inclusion in genomic selection strategies. The Authors. Published by the Federation of Animal Science Societies and Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

Synthetic and Evolutionary Construction of a Chlorate-Reducing Shewanella oneidensis MR-1.

PubMed

Clark, Iain C; Melnyk, Ryan A; Youngblut, Matthew D; Carlson, Hans K; Iavarone, Anthony T; Coates, John D

2015-05-19

Despite evidence for the prevalence of horizontal gene transfer of respiratory genes, little is known about how pathways functionally integrate within new hosts. One example of a mobile respiratory metabolism is bacterial chlorate reduction, which is frequently encoded on composite transposons. This implies that the essential components of the metabolism are encoded on these mobile elements. To test this, we heterologously expressed genes for chlorate reduction from Shewanella algae ACDC in the non-chlorate-reducing Shewanella oneidensis MR-1. The construct that ultimately endowed robust growth on chlorate included cld, a cytochrome c gene, clrABDC, and two genes of unknown function. Although strain MR-1 was unable to grow on chlorate after initial insertion of these genes into the chromosome, 11 derived strains capable of chlorate respiration were obtained through adaptive evolution. Genome resequencing indicated that all of the evolved chlorate-reducing strains replicated a large genomic region containing chlorate reduction genes. Contraction in copy number and loss of the ability to reduce chlorate were also observed, indicating that this phenomenon was extremely dynamic. Although most strains contained more than six copies of the replicated region, a single strain with less duplication also grew rapidly. This strain contained three additional mutations that we hypothesized compensated for the low copy number. We remade the mutations combinatorially in the unevolved strain and determined that a single nucleotide polymorphism (SNP) upstream of cld enabled growth on chlorate and was epistatic to a second base pair change in the NarP binding sequence between narQP and nrfA that enhanced growth. The ability of chlorate reduction composite transposons to form functional metabolisms after transfer to a new host is an important part of their propagation. To study this phenomenon, we engineered Shewanella oneidensis MR-1 into a chlorate reducer. We defined a set of genes sufficient to endow growth on chlorate from a plasmid, but found that chromosomal insertion of these genes was nonfunctional. Evolution of this inoperative strain into a chlorate reducer showed that tandem duplication was a dominant mechanism of activation. While copy number changes are a relatively rapid way of increasing gene dosage, replicating almost 1 megabase of extra DNA is costly. Mutations that alleviate the need for high copy number are expected to arise and eventually predominate, and we identified a single nucleotide polymorphism (SNP) that relieved the copy number requirement. This study uses both rational and evolutionary approaches to gain insight into the evolution of a fascinating respiratory metabolism. Copyright © 2015 Clark et al.

MixHMM: Inferring Copy Number Variation and Allelic Imbalance Using SNP Arrays and Tumor Samples Mixed with Stromal Cells

PubMed Central

Schulz, Vincent; Chen, Min; Tuck, David

2010-01-01

Background Genotyping platforms such as single nucleotide polymorphism (SNP) arrays are powerful tools to study genomic aberrations in cancer samples. Allele specific information from SNP arrays provides valuable information for interpreting copy number variation (CNV) and allelic imbalance including loss-of-heterozygosity (LOH) beyond that obtained from the total DNA signal available from array comparative genomic hybridization (aCGH) platforms. Several algorithms based on hidden Markov models (HMMs) have been designed to detect copy number changes and copy-neutral LOH making use of the allele information on SNP arrays. However heterogeneity in clinical samples, due to stromal contamination and somatic alterations, complicates analysis and interpretation of these data. Methods We have developed MixHMM, a novel hidden Markov model using hidden states based on chromosomal structural aberrations. MixHMM allows CNV detection for copy numbers up to 7 and allows more complete and accurate description of other forms of allelic imbalance, such as increased copy number LOH or imbalanced amplifications. MixHMM also incorporates a novel sample mixing model that allows detection of tumor CNV events in heterogeneous tumor samples, where cancer cells are mixed with a proportion of stromal cells. Conclusions We validate MixHMM and demonstrate its advantages with simulated samples, clinical tumor samples and a dilution series of mixed samples. We have shown that the CNVs of cancer cells in a tumor sample contaminated with up to 80% of stromal cells can be detected accurately using Illumina BeadChip and MixHMM. Availability The MixHMM is available as a Python package provided with some other useful tools at http://genecube.med.yale.edu:8080/MixHMM. PMID:20532221

DcSto: carrot Stowaway-like elements are abundant, diverse, and polymorphic

USDA-ARS?s Scientific Manuscript database

We investigated nine families of Stowaway-like MITEs in the carrot genome, named DcSto1 to DcSto9. All of them were AT-rich and shared a highly conserved 6 bp-long TIR typical for Stowaways. The copy number of DcSto1 elements was estimated as ca. 5,000 per diploid genome. We observed preference for ...

Pfmdr1 copy number and arteminisin derivatives combination therapy failure in falciparum malaria in Cambodia.

PubMed

Lim, Pharath; Alker, Alisa P; Khim, Nimol; Shah, Naman K; Incardona, Sandra; Doung, Socheat; Yi, Poravuth; Bouth, Denis Mey; Bouchier, Christiane; Puijalon, Odile Mercereau; Meshnick, Steven R; Wongsrichanalai, Chansuda; Fandeur, Thierry; Le Bras, Jacques; Ringwald, Pascal; Ariey, Frédéric

2009-01-12

The combination of artesunate and mefloquine was introduced as the national first-line treatment for Plasmodium falciparum malaria in Cambodia in 2000. However, recent clinical trials performed at the Thai-Cambodian border have pointed to the declining efficacy of both artesunate-mefloquine and artemether-lumefantrine. Since pfmdr1 modulates susceptibility to mefloquine and artemisinin derivatives, the aim of this study was to assess the link between pfmdr1 copy number, in vitro susceptibility to individual drugs and treatment failure to combination therapy. Blood samples were collected from P. falciparum-infected patients enrolled in two in vivo efficacy studies in north-western Cambodia: 135 patients were treated with artemether-lumefantrine (AL group) in Sampovloun in 2002 and 2003, and 140 patients with artesunate-mefloquine (AM group) in Sampovloun and Veal Veng in 2003 and 2004. At enrollment, the in vitro IC50 was tested and the strains were genotyped for pfmdr1 copy number by real-time PCR. The pfmdr1 copy number was analysed for 115 isolates in the AM group, and for 109 isolates in the AL group. Parasites with increased pfmdr1 copy number had significantly reduced in vitro susceptibility to mefloquine, lumefantrine and artesunate. There was no association between pfmdr1 polymorphisms and in vitro susceptibilities. In the patients treated with AM, the mean pfmdr1copy number was lower in subjects with adequate clinical and parasitological response compared to those who experienced late treatment failure (n = 112, p < 0.001). This was not observed in the patients treated with AL (n = 96, p = 0.364). The presence of three or more copies of pfmdr1 were associated with recrudescence in artesunate-mefloquine treated patients (hazard ratio (HR) = 7.80 [95%CI: 2.09-29.10], N = 115), p = 0.002) but not with recrudescence in artemether-lumefantrine treated patients (HR = 1.03 [95%CI: 0.24-4.44], N = 109, p = 0.969). This study shows that pfmdr1 copy number is a molecular marker of AM treatment failure in falciparum malaria on the Thai-Cambodian border. However, while it is associated with increased IC50 for lumefantrine, pfmdr1 copy number is not associated with AL treatment failure in the area, suggesting involvement of other molecular mechanisms in AL treatment failures in Cambodia.

Copy number variations in "classical" obesity candidate genes are not frequently associated with severe early-onset obesity in children.

PubMed

Windholz, Jan; Kovacs, Peter; Schlicke, Marina; Franke, Christin; Mahajan, Anubha; Morris, Andrew P; Lemke, Johannes R; Klammt, Jürgen; Kiess, Wieland; Schöneberg, Torsten; Pfäffle, Roland; Körner, Antje

2017-05-01

Obesity is genetically heterogeneous and highly heritable, although polymorphisms explain the phenotype in only a small proportion of obese children. We investigated the presence of copy number variations (CNVs) in "classical" genes known to be associated with (monogenic) early-onset obesity in children. In 194 obese Caucasian children selected for early-onset and severe obesity from our obesity cohort we screened for deletions and/or duplications by multiplex ligation-dependent probe amplification reaction (MLPA). As we found one MLPA probe to interfere with a polymorphism in SIM1 we investigated its association with obesity and other phenotypic traits in our extended cohort of 2305 children. In the selected subset of most severely obese children, we did not find CNV with MLPA in POMC, LEP, LEPR, MC4R, MC3R or MC2R genes. However, one SIM1 probe located at exon 9 gave signals suggestive for SIM1 insufficiency in 52 patients. Polymerase chain reaction (PCR) analysis identified this as a false positive result due to interference with single nucleotide polymorphism (SNP) rs3734354/rs3734355. We, therefore, investigated for associations of this polymorphism with obesity and metabolic traits in our extended cohort. We found rs3734354/rs3734355 to be associated with body mass index-standard deviation score (BMI-SDS) (p = 0.003), but not with parameters of insulin metabolism, blood pressure or food intake. In our modest sample of severely obese children, we were unable to find CNVs in well-established monogenic obesity genes. Nevertheless, we found an association of rs3734354 in SIM1 with obesity of early-onset type in children, although not with obesity-related traits.

SNP genotypes of Mycobacterium leprae isolates in Thailand and their combination with rpoT and TTC genotyping for analysis of leprosy distribution and transmission.

PubMed

Phetsuksiri, Benjawan; Srisungngam, Sopa; Rudeeaneksin, Janisara; Bunchoo, Supranee; Lukebua, Atchariya; Wongtrungkapun, Ruch; Paitoon, Soontara; Sakamuri, Rama Murthy; Brennan, Patrick J; Vissa, Varalakshmi

2012-01-01

Based on the discovery of three single nucleotide polymorphisms (SNPs) in Mycobacterium leprae, it has been previously reported that there are four major SNP types associated with different geographic regions around the world. Another typing system for global differentiation of M. leprae is the analysis of the variable number of short tandem repeats within the rpoT gene. To expand the analysis of geographic distribution of M. leprae, classified by SNP and rpoT gene polymorphisms, we studied 85 clinical isolates from Thai patients and compared the findings with those reported from Asian isolates. SNP genotyping by PCR amplification and sequencing revealed that all strains like those in Myanmar were SNP type 1 and 3, with the former being predominant, while in Japan, Korea, and Indonesia, the SNP type 3 was found to be more frequent. The pattern of M. leprae distribution in Thailand and Myanmar is quite similar, except that SNP type 2 was not found in Thailand. In addition, the 3-copy hexamer genotype in the rpoT gene is shared among the isolates from these two neighboring countries. On the basis of these two markers, we postulate that M. leprae in leprosy patients from Myanmar and Thailand has a common historical origin. Further differentiation among Thai isolates was possible by assessing copy numbers of the TTC sequence, a more polymorphic microsatellite locus.

A Likelihood-Based Framework for Association Analysis of Allele-Specific Copy Numbers.

PubMed

Hu, Y J; Lin, D Y; Sun, W; Zeng, D

2014-10-01

Copy number variants (CNVs) and single nucleotide polymorphisms (SNPs) co-exist throughout the human genome and jointly contribute to phenotypic variations. Thus, it is desirable to consider both types of variants, as characterized by allele-specific copy numbers (ASCNs), in association studies of complex human diseases. Current SNP genotyping technologies capture the CNV and SNP information simultaneously via fluorescent intensity measurements. The common practice of calling ASCNs from the intensity measurements and then using the ASCN calls in downstream association analysis has important limitations. First, the association tests are prone to false-positive findings when differential measurement errors between cases and controls arise from differences in DNA quality or handling. Second, the uncertainties in the ASCN calls are ignored. We present a general framework for the integrated analysis of CNVs and SNPs, including the analysis of total copy numbers as a special case. Our approach combines the ASCN calling and the association analysis into a single step while allowing for differential measurement errors. We construct likelihood functions that properly account for case-control sampling and measurement errors. We establish the asymptotic properties of the maximum likelihood estimators and develop EM algorithms to implement the corresponding inference procedures. The advantages of the proposed methods over the existing ones are demonstrated through realistic simulation studies and an application to a genome-wide association study of schizophrenia. Extensions to next-generation sequencing data are discussed.

Variegated clonality and rapid emergence of new molecular lesions in xenografts of acute lymphoblastic leukemia are associated with drug resistance.

PubMed

Nowak, Daniel; Liem, Natalia L M; Mossner, Maximilian; Klaumünzer, Marion; Papa, Rachael A; Nowak, Verena; Jann, Johann C; Akagi, Tadayuki; Kawamata, Norihiko; Okamoto, Ryoko; Thoennissen, Nils H; Kato, Motohiro; Sanada, Masashi; Hofmann, Wolf-Karsten; Ogawa, Seishi; Marshall, Glenn M; Lock, Richard B; Koeffler, H Phillip

2015-01-01

The use of genome-wide copy-number analysis and massive parallel sequencing has revolutionized the understanding of the clonal architecture of pediatric acute lymphoblastic leukemia (ALL) by demonstrating that this disease is composed of highly variable clonal ancestries following the rules of Darwinian selection. The current study aimed to analyze the molecular composition of childhood ALL biopsies and patient-derived xenografts with particular emphasis on mechanisms associated with acquired chemoresistance. Genomic DNA from seven primary pediatric ALL patient samples, 29 serially passaged xenografts, and six in vivo selected chemoresistant xenografts were analyzed with 250K single-nucleotide polymorphism arrays. Copy-number analysis of non-drug-selected xenografts confirmed a highly variable molecular pattern of variegated subclones. Whereas primary patient samples from initial diagnosis displayed a mean of 5.7 copy-number alterations per sample, serially passaged xenografts contained a mean of 8.2 and chemoresistant xenografts a mean of 10.5 copy-number alterations per sample, respectively. Resistance to cytarabine was explained by a new homozygous deletion of the DCK gene, whereas methotrexate resistance was associated with monoallelic deletion of FPGS and mutation of the remaining allele. This study demonstrates that selecting for chemoresistance in xenografted human ALL cells can reveal novel mechanisms associated with drug resistance. Copyright © 2015 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

Genetic Structures of Copy Number Variants Revealed by Genotyping Single Sperm

PubMed Central

Luo, Minjie; Cui, Xiangfeng; Fredman, David; Brookes, Anthony J.; Azaro, Marco A.; Greenawalt, Danielle M.; Hu, Guohong; Wang, Hui-Yun; Tereshchenko, Irina V.; Lin, Yong; Shentu, Yue; Gao, Richeng; Shen, Li; Li, Honghua

2009-01-01

Background Copy number variants (CNVs) occupy a significant portion of the human genome and may have important roles in meiotic recombination, human genome evolution and gene expression. Many genetic diseases may be underlain by CNVs. However, because of the presence of their multiple copies, variability in copy numbers and the diploidy of the human genome, detailed genetic structure of CNVs cannot be readily studied by available techniques. Methodology/Principal Findings Single sperm samples were used as the primary subjects for the study so that CNV haplotypes in the sperm donors could be studied individually. Forty-eight CNVs characterized in a previous study were analyzed using a microarray-based high-throughput genotyping method after multiplex amplification. Seventeen single nucleotide polymorphisms (SNPs) were also included as controls. Two single-base variants, either allelic or paralogous, could be discriminated for all markers. Microarray data were used to resolve SNP alleles and CNV haplotypes, to quantitatively assess the numbers and compositions of the paralogous segments in each CNV haplotype. Conclusions/Significance This is the first study of the genetic structure of CNVs on a large scale. Resulting information may help understand evolution of the human genome, gain insight into many genetic processes, and discriminate between CNVs and SNPs. The highly sensitive high-throughput experimental system with haploid sperm samples as subjects may be used to facilitate detailed large-scale CNV analysis. PMID:19384415

Pathogenesis and Consequences of Uniparental Disomy in Cancer

PubMed Central

Makishima, Hideki; Maciejewski, Jaroslaw P.

2012-01-01

Systematic application of new genome-wide single nucleotide polymorphism arrays has demonstrated that somatically acquired regions of loss of heterozygosity (LOH) without changes in copy number frequently occur in many types of cancer. Until recently, the ubiquity of this type of chromosomal defect had remained unrecognized as it cannot be detected using routine cytogenetic technologies. Random and recurrent patterns of copy-neutral LOH, also referred to as uniparental disomy (UPD), can be found in specific cancer types and probably contribute to clonal outgrowth owing to various mechanisms. In this review we explore the types, topography, genesis, pathophysiological consequences and clinical implications of UPD. PMID:21518781

VCS: Tool for Visualizing Copy Number Variation and Single Nucleotide Polymorphism.

PubMed

Kim, HyoYoung; Sung, Samsun; Cho, Seoae; Kim, Tae-Hun; Seo, Kangseok; Kim, Heebal

2014-12-01

Copy number variation (CNV) or single nucleotide phlyorphism (SNP) is useful genetic resource to aid in understanding complex phenotypes or deseases susceptibility. Although thousands of CNVs and SNPs are currently avaliable in the public databases, they are somewhat difficult to use for analyses without visualization tools. We developed a web-based tool called the VCS (visualization of CNV or SNP) to visualize the CNV or SNP detected. The VCS tool can assist to easily interpret a biological meaning from the numerical value of CNV and SNP. The VCS provides six visualization tools: i) the enrichment of genome contents in CNV; ii) the physical distribution of CNV or SNP on chromosomes; iii) the distribution of log2 ratio of CNVs with criteria of interested; iv) the number of CNV or SNP per binning unit; v) the distribution of homozygosity of SNP genotype; and vi) cytomap of genes within CNV or SNP region.

Diabetic Nephropathy Induced by Increased Ace Gene Dosage Is Associated with High Renal Levels of Angiotensin (1–7) and Bradykinin

PubMed Central

Bertoncello, Nádia; Moreira, Roseli Peres; Arita, Danielle Yuri; Aragão, Danielle S.; Watanabe, Ingrid Kazue Mizuno; Dantas, Patricia S.; Santos, Ralmony; Mattar-Rosa, Rodolfo; Yokota, Rodrigo; Cunha, Tatiana Sousa; Casarini, Dulce Elena

2015-01-01

Population studies have shown an association between diabetic nephropathy (DN) and insertion/deletion (I/D) polymorphism of the angiotensin-converting enzyme (ACE) gene (ACE in humans, Ace in mice). The aim was to evaluate the modulation of Ace copies number and diabetes mellitus (DM) on renal RAS and correlate it with indicators of kidney function. Increased number of copies of the Ace gene, associated with DM, induces renal dysfunction. The susceptibility to the development of DN in 3 copies of animals is associated with an imbalance in activity of RAS enzymes leading to increased synthesis of Ang II and Ang-(1–7). Increased concentration of renal Ang-(1–7) appears to potentiate the deleterious effects triggered by Ang II on kidney structure and function. Results also show increased bradykinin concentration in 3 copies diabetic group. Taken together, results indicate that the deleterious effects described in 3 copies diabetic group are, at least in part, due to a combination of factors not usually described in the literature. Thus, the data presented here show up innovative and contribute to understanding the complex mechanisms involved in the development of DN, in order to optimize the treatment of patients with this complication. PMID:26442284

Segmental Duplications and Copy-Number Variation in the Human Genome

PubMed Central

Sharp, Andrew J. ; Locke, Devin P. ; McGrath, Sean D. ; Cheng, Ze ; Bailey, Jeffrey A. ; Vallente, Rhea U. ; Pertz, Lisa M. ; Clark, Royden A. ; Schwartz, Stuart ; Segraves, Rick ; Oseroff, Vanessa V. ; Albertson, Donna G. ; Pinkel, Daniel ; Eichler, Evan E. 

2005-01-01

The human genome contains numerous blocks of highly homologous duplicated sequence. This higher-order architecture provides a substrate for recombination and recurrent chromosomal rearrangement associated with genomic disease. However, an assessment of the role of segmental duplications in normal variation has not yet been made. On the basis of the duplication architecture of the human genome, we defined a set of 130 potential rearrangement hotspots and constructed a targeted bacterial artificial chromosome (BAC) microarray (with 2,194 BACs) to assess copy-number variation in these regions by array comparative genomic hybridization. Using our segmental duplication BAC microarray, we screened a panel of 47 normal individuals, who represented populations from four continents, and we identified 119 regions of copy-number polymorphism (CNP), 73 of which were previously unreported. We observed an equal frequency of duplications and deletions, as well as a 4-fold enrichment of CNPs within hotspot regions, compared with control BACs (P < .000001), which suggests that segmental duplications are a major catalyst of large-scale variation in the human genome. Importantly, segmental duplications themselves were also significantly enriched >4-fold within regions of CNP. Almost without exception, CNPs were not confined to a single population, suggesting that these either are recurrent events, having occurred independently in multiple founders, or were present in early human populations. Our study demonstrates that segmental duplications define hotspots of chromosomal rearrangement, likely acting as mediators of normal variation as well as genomic disease, and it suggests that the consideration of genomic architecture can significantly improve the ascertainment of large-scale rearrangements. Our specialized segmental duplication BAC microarray and associated database of structural polymorphisms will provide an important resource for the future characterization of human genomic disorders. PMID:15918152

Expression and phylogenetic analyses reveal paralogous lineages of putatively classical and non-classical MHC-I genes in three sparrow species (Passer).

PubMed

Drews, Anna; Strandh, Maria; Råberg, Lars; Westerdahl, Helena

2017-06-26

The Major Histocompatibility Complex (MHC) plays a central role in immunity and has been given considerable attention by evolutionary ecologists due to its associations with fitness-related traits. Songbirds have unusually high numbers of MHC class I (MHC-I) genes, but it is not known whether all are expressed and equally important for immune function. Classical MHC-I genes are highly expressed, polymorphic and present peptides to T-cells whereas non-classical MHC-I genes have lower expression, are more monomorphic and do not present peptides to T-cells. To get a better understanding of the highly duplicated MHC genes in songbirds, we studied gene expression in a phylogenetic framework in three species of sparrows (house sparrow, tree sparrow and Spanish sparrow), using high-throughput sequencing. We hypothesize that sparrows could have classical and non-classical genes, as previously indicated though never tested using gene expression. The phylogenetic analyses reveal two distinct types of MHC-I alleles among the three sparrow species, one with high and one with low level of polymorphism, thus resembling classical and non-classical genes, respectively. All individuals had both types of alleles, but there was copy number variation both within and among the sparrow species. However, the number of highly polymorphic alleles that were expressed did not vary between species, suggesting that the structural genomic variation is counterbalanced by conserved gene expression. Overall, 50% of the MHC-I alleles were expressed in sparrows. Expression of the highly polymorphic alleles was very variable, whereas the alleles with low polymorphism had uniformly low expression. Interestingly, within an individual only one or two alleles from the polymorphic genes were highly expressed, indicating that only a single copy of these is highly expressed. Taken together, the phylogenetic reconstruction and the analyses of expression suggest that sparrows have both classical and non-classical MHC-I genes, and that the evolutionary origin of these genes predate the split of the three investigated sparrow species 7 million years ago. Because only the classical MHC-I genes are involved in antigen presentation, the function of different MHC-I genes should be considered in future ecological and evolutionary studies of MHC-I in sparrows and other songbirds.

Genetic and intermediate phenotypic susceptibility markers of gastric cancer in Hispanic Americans: a case-control study.

PubMed

Sun, Yuhui; Gu, Jian; Ajani, Jaffer A; Chang, David W; Wu, Xifeng; Stroehlein, John R

2014-10-01

Hispanics are the largest nonwhite ethnic group in the US population, and they have higher incidence and mortality rates for gastric cancer (GC) than whites and Asians. Studies have identified several genetic susceptibility loci and intermediate phenotypic biomarkers for GC in whites and Asians. No studies have evaluated genetic susceptibility and intermediate phenotypic biomarkers in Hispanics. In a case-control study of 132 Hispanic patients with GC (cases) and a control group of 125 Hispanics (controls), the authors evaluated the association of 5 single nucleotide polymorphisms (SNPs) that predispose whites and/or Asians to GC and of 2 intermediate phenotypic markers in peripheral blood leukocytes, ie, telomere length and mitochondrial DNA (mtDNA) copy number, with the GC risk. The variant C allele of the reference SNP rs2294008 in the PSCA gene was associated with a significantly reduced risk of GC (per allele-adjusted odds ratio [aOR], 0.51; 95% confidence interval [CI], 0.33-0.77; P = .002). Leukocyte mtDNA copy numbers were significantly lower in GC cases (mean ± standard deviation, 0.91 ± 0.28) than in controls (1.29 ± 0.42; P < .001). When individuals were dichotomized into high and low mtDNA copy number groups based on the median mtDNA copy number value in the controls, those who had a low mtDNA copy number had a significantly increased risk of GC (aOR, 11.00; 95% CI, 4.79-25.23; P < .001) compared with those who had a high mtDNA copy number. Telomere length was not associated significantly with the risk of GC (aOR, 1.21; 95% CI, 0.65-2.27; P = .551). Hispanics share certain genetic susceptibility loci and intermediate phenotypic GC biomarkers with whites and Asians and may also have distinct genetic susceptibility factors. © 2014 American Cancer Society.

Genome-Wide Copy Number Variation Association Analyses for Age at Menarche

PubMed Central

Li, Jian; Pan, Rong; Shen, Hui; Tian, Qing; Zhou, Yu; Liu, Yong-Jun

2012-01-01

Context: Menarche is a significant physiological event for women. Age at menarche (AAM) is a heritable trait associated with many common female diseases. The genetic basis and the mechanism for AAM are largely unknown. Copy number variation (CNV) is a common type of genetic variation underlying human complex traits. The importance of CNV to AAM variation is unclear. Objective: The objective of the study was to identify CNV important to AAM variation. Design: We performed the first genome-wide CNV study of AAM in 1654 Caucasian females using Affymetrix human single-nucleotide polymorphism 6.0 array. We also replicated our findings in another Chinese cohort containing 752 women. Results: We identified a CNV, variation_38399, in the 2q14.2 region, for association with AAM (P = 1.03 × 10−3). The CNV has two variants (one copy and two copy), with a mean AAM of 14.00 yr and 12.90 yr, respectively. Interestingly, in a Chinese sample containing 752 women, this CNV has been replicated both with a marginally significant P = 0.090 and with a same direction of effect (a lower copy number for a later AAM). The CNV is located approximately 75 kb upstream of the diazepam binding inhibitor (DBI), a gene known to regulate estrogen levels, a key factor for menarche. Conclusion: Our findings for the first time identified a novel CNV and suggested the DBI-mediated endocrinological pathway as a potential mechanism for AAM regulation. PMID:22904172

[Advances in congenital vertebral malformation caused by genomic copy number variation].

PubMed

Liu, Zhenlei; Wu, Nan; Wu, Zhihong; Zuo, Yuzhi; Qiu, Guixing

2016-04-01

Congenital vertebral malformation (CVM) is a congenital vertebral structural deformity caused by abnormal somitogenesis during embryonic development, of which the reason lies in gene mutation or abnormal regulation of the genes that coordinate somitogenesis during embryonic period. ICVAS had proposed a new classification algorithm for CVM, which facilitated exploration for its genetic etiology. Genomic Copy Number Variation (CNV) is a kind of DNA mutation, which is important for human evolution, phenotype polymorphism and diseases. Series of advances have been made on genetic causes of CVM, especially on CVM caused by CNV. CNVs of chromosome 16p11.2, 10q24.31, 17p11.2, 20p11, 22q11.2 and a few other regions are associated with CVM, indicating that gene dosage may play important roles in the development of the spinal cord.

Human beta-globin gene polymorphisms characterized in DNA extracted from ancient bones 12,000 years old.

PubMed

Béraud-Colomb, E; Roubin, R; Martin, J; Maroc, N; Gardeisen, A; Trabuchet, G; Goosséns, M

1995-12-01

Analyzing the nuclear DNA from ancient human bones is an essential step to the understanding of genetic diversity in current populations, provided that such systematic studies are experimentally feasible. This article reports the successful extraction and amplification of nuclear DNA from the beta-globin region from 5 of 10 bone specimens up to 12,000 years old. These have been typed for beta-globin frameworks by sequencing through two variable positions and for a polymorphic (AT) chi (T) gamma microsatellite 500 bp upstream of the beta-globin gene. These specimens of human remains are somewhat older than those analyzed in previous nuclear gene sequencing reports and considerably older than those used to study high-copy-number human mtDNA. These results show that the systematic study of nuclear DNA polymorphisms of ancient populations is feasible.

Validation of rearrangement break points identified by paired-end sequencing in natural populations of Drosophila melanogaster.

PubMed

Cridland, Julie M; Thornton, Kevin R

2010-01-13

Several recent studies have focused on the evolution of recently duplicated genes in Drosophila. Currently, however, little is known about the evolutionary forces acting upon duplications that are segregating in natural populations. We used a high-throughput, paired-end sequencing platform (Illumina) to identify structural variants in a population sample of African D. melanogaster. Polymerase chain reaction and sequencing confirmation of duplications detected by multiple, independent paired-ends showed that paired-end sequencing reliably uncovered the break points of structural rearrangements and allowed us to identify a number of tandem duplications segregating within a natural population. Our confirmation experiments show that rates of confirmation are very high, even at modest coverage. Our results also compare well with previous studies using microarrays (Emerson J, Cardoso-Moreira M, Borevitz JO, Long M. 2008. Natural selection shapes genome wide patterns of copy-number polymorphism in Drosophila melanogaster. Science. 320:1629-1631. and Dopman EB, Hartl DL. 2007. A portrait of copy-number polymorphism in Drosophila melanogaster. Proc Natl Acad Sci U S A. 104:19920-19925.), which both gives us confidence in the results of this study as well as confirms previous microarray results.We were also able to identify whole-gene duplications, such as a novel duplication of Or22a, an olfactory receptor, and identify copy-number differences in genes previously known to be under positive selection, like Cyp6g1, which confers resistance to dichlorodiphenyltrichloroethane. Several "hot spots" of duplications were detected in this study, which indicate that particular regions of the genome may be more prone to generating duplications. Finally, population frequency analysis of confirmed events also showed an excess of rare variants in our population, which indicates that duplications segregating in the population may be deleterious and ultimately destined to be lost from the population.

Evolution and selection of Rhg1, a copy-number variant nematode-resistance locus

PubMed Central

Lee, Tong Geon; Kumar, Indrajit; Diers, Brian W; Hudson, Matthew E

2015-01-01

The soybean cyst nematode (SCN) resistance locus Rhg1 is a tandem repeat of a 31.2 kb unit of the soybean genome. Each 31.2-kb unit contains four genes. One allele of Rhg1, Rhg1-b, is responsible for protecting most US soybean production from SCN. Whole-genome sequencing was performed, and PCR assays were developed to investigate allelic variation in sequence and copy number of the Rhg1 locus across a population of soybean germplasm accessions. Four distinct sequences of the 31.2-kb repeat unit were identified, and some Rhg1 alleles carry up to three different types of repeat unit. The total number of copies of the repeat varies from 1 to 10 per haploid genome. Both copy number and sequence of the repeat correlate with the resistance phenotype, and the Rhg1 locus shows strong signatures of selection. Significant linkage disequilibrium in the genome outside the boundaries of the repeat allowed the Rhg1 genotype to be inferred using high-density single nucleotide polymorphism genotyping of 15 996 accessions. Over 860 germplasm accessions were found likely to possess Rhg1 alleles. The regions surrounding the repeat show indications of non-neutral evolution and high genetic variability in populations from different geographic locations, but without evidence of fixation of the resistant genotype. A compelling explanation of these results is that balancing selection is in operation at Rhg1. PMID:25735447

Associations of GBP2 gene copy number variations with growth traits and transcriptional expression in Chinese cattle.

PubMed

Zhang, Gui-Min; Zheng, Li; He, Hua; Song, Cheng-Chuang; Zhang, Zi-Jing; Cao, Xiu-Kai; Lei, Chu-Zhao; Lan, Xian-Yong; Qi, Xing-Lei; Chen, Hong; Huang, Yong-Zhen

2018-03-20

Copy number variations (CNVs) recently have been recognized as another important genetic variability followed single nucleotide polymorphisms (SNPs). The guanylate binding protein 2 (GBP2) gene plays an important role in cell proliferation. This study was performed to determine the presence of GBP2 CNV (relative to Angus cattle) in 466 individuals representing six main cattle breeds from China, identify its relationship with growth, and explore the biological effects of gene expression. There were two CNV regions in the GBP2 gene, for three types, CNV1 loss type (relative to Angus cattle) was more frequent in XN than other breeds, and CNV2 loss type (relative to Angus cattle) was more frequent in XN and CDM than other breeds. Though the GBP2 gene copy number presented no correlation with the transcriptional expression of JX (P > .05), but the transcriptional expression in heart is higher than other tissues, and the copy number in muscles and fat of JX is higher than others breeds. Statistical analysis revealed that the GBP2 gene CNV1 and CNV2 were significantly associated with growth traits (P < .05). In conclusion, this research established the correlations between CNVs of GBP2 gene and growth traits in different cattle breeds, and our results suggested that the CNVs in GBP2 gene may be considered markers for the molecular breeding of Chinese beef cattle. Copyright © 2018. Published by Elsevier B.V.

The genomic and transcriptomic architecture of 2,000 breast tumours reveals novel subgroups.

PubMed

Curtis, Christina; Shah, Sohrab P; Chin, Suet-Feung; Turashvili, Gulisa; Rueda, Oscar M; Dunning, Mark J; Speed, Doug; Lynch, Andy G; Samarajiwa, Shamith; Yuan, Yinyin; Gräf, Stefan; Ha, Gavin; Haffari, Gholamreza; Bashashati, Ali; Russell, Roslin; McKinney, Steven; Langerød, Anita; Green, Andrew; Provenzano, Elena; Wishart, Gordon; Pinder, Sarah; Watson, Peter; Markowetz, Florian; Murphy, Leigh; Ellis, Ian; Purushotham, Arnie; Børresen-Dale, Anne-Lise; Brenton, James D; Tavaré, Simon; Caldas, Carlos; Aparicio, Samuel

2012-04-18

The elucidation of breast cancer subgroups and their molecular drivers requires integrated views of the genome and transcriptome from representative numbers of patients. We present an integrated analysis of copy number and gene expression in a discovery and validation set of 997 and 995 primary breast tumours, respectively, with long-term clinical follow-up. Inherited variants (copy number variants and single nucleotide polymorphisms) and acquired somatic copy number aberrations (CNAs) were associated with expression in ~40% of genes, with the landscape dominated by cis- and trans-acting CNAs. By delineating expression outlier genes driven in cis by CNAs, we identified putative cancer genes, including deletions in PPP2R2A, MTAP and MAP2K4. Unsupervised analysis of paired DNA–RNA profiles revealed novel subgroups with distinct clinical outcomes, which reproduced in the validation cohort. These include a high-risk, oestrogen-receptor-positive 11q13/14 cis-acting subgroup and a favourable prognosis subgroup devoid of CNAs. Trans-acting aberration hotspots were found to modulate subgroup-specific gene networks, including a TCR deletion-mediated adaptive immune response in the ‘CNA-devoid’ subgroup and a basal-specific chromosome 5 deletion-associated mitotic network. Our results provide a novel molecular stratification of the breast cancer population, derived from the impact of somatic CNAs on the transcriptome.

Copy number variation of lipocalin family genes for male-specific proteins in tilapia and its association with gender.

PubMed

Shirak, A; Golik, M; Lee, B-Y; Howe, A E; Kocher, T D; Hulata, G; Ron, M; Seroussi, E

2008-11-01

Lipocalins are involved in the binding of small molecules like sex steroids. We show here that the previously reported tilapia male-specific protein (MSP) is a lipocalin encoded by a variety of paralogous and homologous genes in different tilapia species. Exon-intron boundaries of MSP genes were typical of the six-exon genomic structure of lipocalins, and the transcripts were capable of encoding 200 amino-acid polypeptides that consisted of a putative signal peptide and a lipocalin domain. Cysteine residues are conserved in positions analogous to those forming the three disulfide bonds characteristic of the ligand pocket. The calculated molecular mass of the secreted MSP (20.4 kDa) was less than half of that observed, suggesting that it is highly glycosylated like its homologue tributyltin-binding protein. Analysis of sequence variations revealed three types of paralogs MSPA, MSPB and MSPC. Expression of both MSPA and MSPB was detected in testis. In haploid Oreochromis niloticus embryos, each of these types consisted of two closely related paralogs, and asymmetry between MSP copy numbers on the maternal (six copies) and the paternal (three copies) chromosomes was observed. Using this polymorphism we mapped MSPA and MSPC to linkage group 12 of an F(2) mapping family derived from a cross between O. niloticus and Oreochromis aureus. Females with high MSP copy number were more frequent by more than twofold than males. Gender-MSPC combinations showed significant deviation from expected Mendelian segregation (P=0.009) suggesting elimination of males with MSPC copies. We discuss different hypotheses to explain this elimination, including possibility for allelic conflict resulted by the hybridization.

Analysis of Copy Number Variation in the Abp Gene Regions of Two House Mouse Subspecies Suggests Divergence during the Gene Family Expansions

PubMed Central

Pezer, Željka; Chung, Amanda G.; Karn, Robert C.

2017-01-01

Abstract The Androgen-binding protein (Abp) gene region of the mouse genome contains 64 genes, some encoding pheromones that influence assortative mating between mice from different subspecies. Using CNVnator and quantitative PCR, we explored copy number variation in this gene family in natural populations of Mus musculus domesticus (Mmd) and Mus musculus musculus (Mmm), two subspecies of house mice that form a narrow hybrid zone in Central Europe. We found that copy number variation in the center of the Abp gene region is very common in wild Mmd, primarily representing the presence/absence of the final duplications described for the mouse genome. Clustering of Mmd individuals based on this variation did not reflect their geographical origin, suggesting no population divergence in the Abp gene cluster. However, copy number variation patterns differ substantially between Mmd and other mouse taxa. Large blocks of Abp genes are absent in Mmm, Mus musculus castaneus and an outgroup, Mus spretus, although with differences in variation and breakpoint locations. Our analysis calls into question the reliance on a reference genome for interpreting the detailed organization of genes in taxa more distant from the Mmd reference genome. The polymorphic nature of the gene family expansion in all four taxa suggests that the number of Abp genes, especially in the central gene region, is not critical to the survival and reproduction of the mouse. However, Abp haplotypes of variable length may serve as a source of raw genetic material for new signals influencing reproductive communication and thus speciation of mice. PMID:28575204

Copy-Number Gains of HUWE1 Due to Replication- and Recombination-Based Rearrangements

PubMed Central

Froyen, Guy; Belet, Stefanie; Martinez, Francisco; Santos-Rebouças, Cíntia Barros; Declercq, Matthias; Verbeeck, Jelle; Donckers, Lene; Berland, Siren; Mayo, Sonia; Rosello, Monica; Pimentel, Márcia Mattos Gonçalves; Fintelman-Rodrigues, Natalia; Hovland, Randi; Rodrigues dos Santos, Suely; Raymond, F. Lucy; Bose, Tulika; Corbett, Mark A.; Sheffield, Leslie; van Ravenswaaij-Arts, Conny M.A.; Dijkhuizen, Trijnie; Coutton, Charles; Satre, Veronique; Siu, Victoria; Marynen, Peter

2012-01-01

We previously reported on nonrecurrent overlapping duplications at Xp11.22 in individuals with nonsyndromic intellectual disability (ID) harboring HSD17B10, HUWE1, and the microRNAs miR-98 and let-7f-2 in the smallest region of overlap. Here, we describe six additional individuals with nonsyndromic ID and overlapping microduplications that segregate in the families. High-resolution mapping of the 12 copy-number gains reduced the minimal duplicated region to the HUWE1 locus only. Consequently, increased mRNA levels were detected for HUWE1, but not HSD17B10. Marker and SNP analysis, together with identification of two de novo events, suggested a paternally derived intrachromosomal duplication event. In four independent families, we report on a polymorphic 70 kb recurrent copy-number gain, which harbors part of HUWE1 (exon 28 to 3′ untranslated region), including miR-98 and let-7f-2. Our findings thus demonstrate that HUWE1 is the only remaining dosage-sensitive gene associated with the ID phenotype. Junction and in silico analysis of breakpoint regions demonstrated simple microhomology-mediated rearrangements suggestive of replication-based duplication events. Intriguingly, in a single family, the duplication was generated through nonallelic homologous recombination (NAHR) with the use of HUWE1-flanking imperfect low-copy repeats, which drive this infrequent NAHR event. The recurrent partial HUWE1 copy-number gain was also generated through NAHR, but here, the homologous sequences used were identified as TcMAR-Tigger DNA elements, a template that has not yet been reported for NAHR. In summary, we showed that an increased dosage of HUWE1 causes nonsyndromic ID and demonstrated that the Xp11.22 region is prone to recombination- and replication-based rearrangements. PMID:22840365

Rapid evolution and copy number variation of primate RHOXF2, an X-linked homeobox gene involved in male reproduction and possibly brain function.

PubMed

Niu, Ao-lei; Wang, Yin-qiu; Zhang, Hui; Liao, Cheng-hong; Wang, Jin-kai; Zhang, Rui; Che, Jun; Su, Bing

2011-10-12

Homeobox genes are the key regulators during development, and they are in general highly conserved with only a few reported cases of rapid evolution. RHOXF2 is an X-linked homeobox gene in primates. It is highly expressed in the testicle and may play an important role in spermatogenesis. As male reproductive system is often the target of natural and/or sexual selection during evolution, in this study, we aim to dissect the pattern of molecular evolution of RHOXF2 in primates and its potential functional consequence. We studied sequences and copy number variation of RHOXF2 in humans and 16 nonhuman primate species as well as the expression patterns in human, chimpanzee, white-browed gibbon and rhesus macaque. The gene copy number analysis showed that there had been parallel gene duplications/losses in multiple primate lineages. Our evidence suggests that 11 nonhuman primate species have one RHOXF2 copy, and two copies are present in humans and four Old World monkey species, and at least 6 copies in chimpanzees. Further analysis indicated that the gene duplications in primates had likely been mediated by endogenous retrovirus (ERV) sequences flanking the gene regions. In striking contrast to non-human primates, humans appear to have homogenized their two RHOXF2 copies by the ERV-mediated non-allelic recombination mechanism. Coding sequence and phylogenetic analysis suggested multi-lineage strong positive selection on RHOXF2 during primate evolution, especially during the origins of humans and chimpanzees. All the 8 coding region polymorphic sites in human populations are non-synonymous, implying on-going selection. Gene expression analysis demonstrated that besides the preferential expression in the reproductive system, RHOXF2 is also expressed in the brain. The quantitative data suggests expression pattern divergence among primate species. RHOXF2 is a fast-evolving homeobox gene in primates. The rapid evolution and copy number changes of RHOXF2 had been driven by Darwinian positive selection acting on the male reproductive system and possibly also on the central nervous system, which sheds light on understanding the role of homeobox genes in adaptive evolution.

The utility of copy number variation (CNV) in studies of hypertension-related left ventricular hypertrophy (LVH): rationale, potential and challenges.

PubMed

Boonpeng, Hoh; Yusoff, Khalid

2013-03-01

The ultimate goal of human genetics is to understand the role of genome variation in elucidating human traits and diseases. Besides single nucleotide polymorphism (SNP), copy number variation (CNV), defined as gains or losses of a DNA segment larger than 1 kb, has recently emerged as an important tool in understanding heritable source of human genomic differences. It has been shown to contribute to genetic susceptibility of various common and complex diseases. Despite a handful of publications, its role in cardiovascular diseases remains largely unknown. Here, we deliberate on the currently available technologies for CNV detection. The possible utility and the potential roles of CNV in exploring the mechanisms of cardiac remodeling in hypertension will also be addressed. Finally, we discuss the challenges for investigations of CNV in cardiovascular diseases and its possible implications in diagnosis of hypertension-related left ventricular hypertrophy (LVH).

A large-scale dataset of single and mixed-source short tandem repeat profiles to inform human identification strategies: PROVEDIt.

PubMed

Alfonse, Lauren E; Garrett, Amanda D; Lun, Desmond S; Duffy, Ken R; Grgicak, Catherine M

2018-01-01

DNA-based human identity testing is conducted by comparison of PCR-amplified polymorphic Short Tandem Repeat (STR) motifs from a known source with the STR profiles obtained from uncertain sources. Samples such as those found at crime scenes often result in signal that is a composite of incomplete STR profiles from an unknown number of unknown contributors, making interpretation an arduous task. To facilitate advancement in STR interpretation challenges we provide over 25,000 multiplex STR profiles produced from one to five known individuals at target levels ranging from one to 160 copies of DNA. The data, generated under 144 laboratory conditions, are classified by total copy number and contributor proportions. For the 70% of samples that were synthetically compromised, we report the level of DNA damage using quantitative and end-point PCR. In addition, we characterize the complexity of the signal by exploring the number of detected alleles in each profile. Copyright © 2017 Elsevier B.V. All rights reserved.

Comparative Analysis of CNV Calling Algorithms: Literature Survey and a Case Study Using Bovine High-Density SNP Data.

PubMed

Xu, Lingyang; Hou, Yali; Bickhart, Derek M; Song, Jiuzhou; Liu, George E

2013-06-25

Copy number variations (CNVs) are gains and losses of genomic sequence between two individuals of a species when compared to a reference genome. The data from single nucleotide polymorphism (SNP) microarrays are now routinely used for genotyping, but they also can be utilized for copy number detection. Substantial progress has been made in array design and CNV calling algorithms and at least 10 comparison studies in humans have been published to assess them. In this review, we first survey the literature on existing microarray platforms and CNV calling algorithms. We then examine a number of CNV calling tools to evaluate their impacts using bovine high-density SNP data. Large incongruities in the results from different CNV calling tools highlight the need for standardizing array data collection, quality assessment and experimental validation. Only after careful experimental design and rigorous data filtering can the impacts of CNVs on both normal phenotypic variability and disease susceptibility be fully revealed.

Effects of Transgenic Bt+CpTI cotton on the abundance and diversity of rhizosphere ammonia oxidizing bacteria and archaea.

PubMed

Dong, Lianhua; Meng, Ying; Wang, Jing; Sun, Guoqing

2016-09-01

Genetically modified crops (GMCs) hold great promise for improving agricultural output, but at the same time present challenges in terms of environmental safety assessment. Ammonia oxidizers, including ammonia oxidizing bacteria (AOB) and archaea (AOA), are very important functional microbial groups in nitrogen cycle. The abundance and diversity of AOA and AOB in the rhizosphere of genetically modified cotton (SGK321) and non-GM cotton (SY321) across growth stages were investigated using real time quantitative PCR (qPCR) and terminal restriction fragment length polymorphism (T-RFLP). Results showed that cotton genotype had a significant effect on the change in abundance of AOA and AOB, as indicated by amoA copy number. Variations in AOB abundance in rhizosphere of SY321 differed from those in SGK321. The number of AOB in the rhizosphere of SY321 fluctuated considerably: It dramatically decreased from 1.2?106 copies g-1 dry soil to 3?105 copies g-1 dry soil during the flowering stage and then increased to 1.1?106 copies g-1 and 1.5?106 copies g-1 at the belling and boll opening stages, respectively. However, abundance of AOB in the rhizosphere of SGK321 was relatively stable during all the stages of growth. The effect of SGK321 and SY321 on AOA number was quite similar to that of AOB: AOA abundance in SGK321 increased smoothly from 1.0 ?105 copies g-1 dry soil to 1.4?106 copies g-1 dry soil during growth, but that in SY321 fluctuated. Correspondence analysis (CA), canonical CA (CCA), and partial CCA (pCCA) of T-RFLP profiles of AOA and AOB showed that AOB community changed across growth stages in both cotton genotypes, and cotton genotype was the most important factor affecting the AOA community. In conclusion, the current findings indicated no adverse effect of GM cotton on functional microorganisms.

Genome-wide high-resolution screening in Dupuytren's disease reveals common regions of DNA copy number alterations.

PubMed

Shih, Barbara B; Tassabehji, May; Watson, James S; McGrouther, Angus D; Bayat, Ardeshir

2010-07-01

Dupuytren's disease (DD) is a familial disorder with a high genetic susceptibility in white people; however, its etiopathogenesis remains unknown. Previous comparative genomic hybridization studies using lower-resolution, 44-k oligonucleotide-based arrays revealed no copy number variation (CNV) changes in DD. In this study, we used a higher-resolution genome-wide screening (next-generation microarrays) comprising 963,331 human sequences (3 kb spacing between probes) for whole genome DNA variation analysis. The objective was to detect cryptic chromosomal imbalances in DD. Agilent SurePrint G3 microarrays, one million format (Agilent Technologies, Santa Clara, CA), were used to detect CNV regions (CNVRs) in DNA extracted from nodules of 4 white men with DD (age, 69 +/- 4 y). Reference samples were from the DNA of 10 men who served as control patients. Copy number variations that were common to greater than 3 assessed DD individuals (p < .05) were selected as candidate loci for DD etiology. In addition, quantitative polymerase chain reactions (qPCR) assays were designed for selected CNVRs on DNA from 13 DD patients and 11 control patients. Independent t-tests and Fisher's exact tests were carried out for statistical analysis. Three novel CNVs previously unreported in the phenotypically normal population were detected in 3 DD cases, located at 10q22, 16p12.1, and 17p12. Nine polymorphic CNVRs potentially associated with DD were determined using our strategic selection criteria, locating to chromosomes 1q31, 6p21, 7p14, 8p11, 12p13, 14q11, 17q21 and 20p13. More than 3 of the DD cases tested had a CNVR located to a small region on 6p21 and 4 CNVRs within 6p21-22 of the human leukocyte antigen (HLA) genes. Three novel copy number alterations were observed in 3 unrelated patients with sporadic (no known family history) DD. Nine polymorphic CNVRs were found to be common among the DD cases. These variants might contain genes involved in DD formation, indicating that important gene networks expressed within the palmar fascia might contribute to genetic susceptibility of DD. Copyright 2010. Published by Elsevier Inc.

Molecular surveillance for drug-resistant Plasmodium falciparum in clinical and subclinical populations from three border regions of Burma/Myanmar: cross-sectional data and a systematic review of resistance studies.

PubMed

Brown, Tyler; Smith, Linda S; Oo, Eh Kalu Shwe; Shawng, Kum; Lee, Thomas J; Sullivan, David; Beyrer, Chris; Richards, Adam K

2012-09-19

Confirmation of artemisinin-delayed parasite clearance in Plasmodium falciparum along the Thai-Myanmar border has inspired a global response to contain and monitor drug resistance to avert the disastrous consequences of a potential spread to Africa. However, resistance data from Myanmar are sparse, particularly from high-risk areas where limited health services and decades of displacement create conditions for resistance to spread. Subclinical infections may represent an important reservoir for resistance genes that confer a fitness disadvantage relative to wild-type alleles. This study estimates the prevalence of resistance genotypes in three previously unstudied remote populations in Myanmar and tests the a priori hypothesis that resistance gene prevalence would be higher among isolates collected from subclinical infections than isolates collected from febrile clinical patients. A systematic review of resistance studies is provided for context. Community health workers in Karen and Kachin States and an area spanning the Indo-Myanmar border collected dried blood spots from 988 febrile clinical patients and 4,591 villagers with subclinical infection participating in routine prevalence surveys. Samples positive for P. falciparum 18 s ribosomal RNA by real-time PCR were genotyped for P. falciparum multidrug resistance protein (pfmdr1) copy number and the pfcrt K76T polymorphism using multiplex real-time PCR. Pfmdr1 copy number increase and the pfcrt K76 polymorphism were determined for 173 and 269 isolates, respectively. Mean pfmdr1 copy number was 1.2 (range: 0.7 to 3.7). Pfmdr1 copy number increase was present in 17.5%, 9.6% and 11.1% of isolates from Karen and Kachin States and the Indo-Myanmar border, respectively. Pfmdr1 amplification was more prevalent in subclinical isolates (20.3%) than clinical isolates (6.4%, odds ratio 3.7, 95% confidence interval 1.1 - 12.5). Pfcrt K76T prevalence ranged from 90-100%. Community health workers can contribute to molecular surveillance of drug resistance in remote areas of Myanmar. Marginal and displaced populations under-represented among previous resistance investigations can and should be included in resistance surveillance efforts, particularly once genetic markers of artemisinin-delayed parasite clearance are identified. Subclinical infections may contribute to the epidemiology of drug resistance, but determination of gene amplification from desiccated filter samples requires further validation when DNA concentration is low.

Complement Factor D in Age-Related Macular Degeneration

PubMed Central

Stanton, Chloe M.; Yates, John R.W.; den Hollander, Anneke I.; Seddon, Johanna M.; Swaroop, Anand; Stambolian, Dwight; Fauser, Sascha; Hoyng, Carel; Yu, Yi; Atsuhiro, Kanda; Branham, Kari; Othman, Mohammad; Chen, Wei; Kortvely, Elod; Chalmers, Kevin; Hayward, Caroline; Moore, Anthony T.; Dhillon, Baljean; Ueffing, Marius

2011-01-01

Purpose. To examine the role of complement factor D (CFD) in age-related macular degeneration (AMD) by analysis of genetic association, copy number variation, and plasma CFD concentrations. Methods. Single nucleotide polymorphisms (SNPs) in the CFD gene were genotyped and the results analyzed by binary logistic regression. CFD gene copy number was analyzed by gene copy number assay. Plasma CFD was measured by an enzyme-linked immunosorbent assay. Results. Genetic association was found between CFD gene SNP rs3826945 and AMD (odds ratio 1.44; P = 0.028) in a small discovery case-control series (462 cases and 325 controls) and replicated in a combined cohorts meta-analysis of 4765 cases and 2693 controls, with an odds ratio of 1.11 (P = 0.032), with the association almost confined to females. Copy number variation in the CFD gene was identified in 13 out of 640 samples examined but there was no difference in frequency between AMD cases (1.3%) and controls (2.7%). Plasma CFD concentration was measured in 751 AMD cases and 474 controls and found to be elevated in AMD cases (P = 0.00025). The odds ratio for those in the highest versus lowest quartile for plasma CFD was 1.81. The difference in plasma CFD was again almost confined to females. Conclusions. CFD regulates activation of the alternative complement pathway, which is implicated in AMD pathogenesis. The authors found evidence for genetic association between a CFD gene SNP and AMD and a significant increase in plasma CFD concentration in AMD cases compared with controls, consistent with a role for CFD in AMD pathogenesis. PMID:22003108

Modified screening and ranking algorithm for copy number variation detection.

PubMed

Xiao, Feifei; Min, Xiaoyi; Zhang, Heping

2015-05-01

Copy number variation (CNV) is a type of structural variation, usually defined as genomic segments that are 1 kb or larger, which present variable copy numbers when compared with a reference genome. The screening and ranking algorithm (SaRa) was recently proposed as an efficient approach for multiple change-points detection, which can be applied to CNV detection. However, some practical issues arise from application of SaRa to single nucleotide polymorphism data. In this study, we propose a modified SaRa on CNV detection to address these issues. First, we use the quantile normalization on the original intensities to guarantee that the normal mean model-based SaRa is a robust method. Second, a novel normal mixture model coupled with a modified Bayesian information criterion is proposed for candidate change-point selection and further clustering the potential CNV segments to copy number states. Simulations revealed that the modified SaRa became a robust method for identifying change-points and achieved better performance than the circular binary segmentation (CBS) method. By applying the modified SaRa to real data from the HapMap project, we illustrated its performance on detecting CNV segments. In conclusion, our modified SaRa method improves SaRa theoretically and numerically, for identifying CNVs with high-throughput genotyping data. The modSaRa package is implemented in R program and freely available at http://c2s2.yale.edu/software/modSaRa. Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Effects of genetic polymorphisms of glutathione S-transferase genes (GSTM1, GSTT1, GSTP1) on the risk of diabetic nephropathy: a meta-analysis.

PubMed

Orlewski, Jan; Orlewska, Ewa

2015-01-01

Glutathione S-transferases (GSTs) belong to a family of ubiquitous and multifunctional enzymes that protect the cells against oxidative stress. The aim of the study was to evaluate the association between the polymorphisms of glutathione-S-transferase (GST) genes and diabetic nephropathy (DN). PubMed, EMBASE, and Google Scholar databases were systematically searched to identify relevant studies. The odds ratio (OR) for the association was determined using a fixed or random effects model. Tests for heterogeneity of the results and sensitivity analyses were performed. A total of 9 publications (874 patients in the study group, 966 controls) were included. With the exception of 1 study, GSTT1 and GSTM1 genotypes were not assessed by methods that measure a gene copy number. A significantly increased risk of DN was found for the GSTM1(-) genotype (OR, 1.27; 95% CI, 1.02-1.58) and the combination of GSTT1(-)/GSTM1(-) (OR,2.02; 95% CI, 1.22-3.36). We did not observe a correlation between DN and the GSTT1(-) genotype or the presence of Val alleles. In a subgroup analysis, an association between DN and the GSTM1(-) genotype was significant in Asians but not in Caucasians. Our results indicate that the GSTM1(-) genotype and the combination of GSTT1(-)/GSTM1(-) increase the risk of DN. The combination of the GST polymorphisms rather than individual polymorphismshould be investigated. Genotyping allowing a trimodular determination of the GST copy number variations may better describe an association between the risk of disease and a given genotype.

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.

Detection of clinically relevant copy number alterations in oral cancer progression using multiplexed droplet digital PCR.

PubMed

Hughesman, Curtis B; Lu, X J David; Liu, Kelly Y P; Zhu, Yuqi; Towle, Rebecca M; Haynes, Charles; Poh, Catherine F

2017-09-19

Copy number alterations (CNAs), a common genomic event during carcinogenesis, are known to affect a large fraction of the genome. Common recurrent gains or losses of specific chromosomal regions occur at frequencies that they may be considered distinctive features of tumoral cells. Here we introduce a novel multiplexed droplet digital PCR (ddPCR) assay capable of detecting recurrent CNAs that drive tumorigenesis of oral squamous cell carcinoma. Applied to DNA extracted from oral cell lines and clinical samples of various disease stages, we found good agreement between CNAs detected by our ddPCR assay with those previously reported using comparative genomic hybridization or single nucleotide polymorphism arrays. Furthermore, we demonstrate that the ability to target specific locations of the genome permits detection of clinically relevant oncogenic events such as small, submicroscopic homozygous deletions. Additional capabilities of the multiplexed ddPCR assay include the ability to infer ploidy level, quantify the change in copy number of target loci with high-level gains, and simultaneously assess the status and viral load for high-risk human papillomavirus types 16 and 18. This novel multiplexed ddPCR assay therefore may have clinical value in differentiating between benign oral lesions from those that are at risk of progressing to oral cancer.

[Diagnostic value of MYB protein expression in adenoid cystic carcinoma and status of MYB gene copy number].

PubMed

Huo, Zhen; Zeng, Xuan; Wu, Shafei; Wu, Huanwen; Meng, Yunxiao; Liu, Yuanyuan; Luo, Yufeng; Cao, Jinling; Liang, Zhiyong

2015-08-01

To explore the diagnostic value of MYB protein expression for adenoid cystic carcinoma and its differential diagnosis from other salivary gland tumors, and to further investigate the status of MYB gene copy number. MYB expression was studied by immunohistochemistry in 34 adenoid cystic carcinomas, 55 non-adenoid cystic carcinomas (other salivary gland tumors) including 10 pleomorphic adenomas, 10 basal cell adenomas, 10 epithelial-myoepithelial carcinomas, 9 basal cell adenocarcinomas, 8 mucoepidermoid carcinomas, 4 carcinoma in pleomorphic adenomas, and 4 polymorphous low-grade adenocarcinoma. MYB gene copy number status was detected by FISH in MYB protein-positive cases. 82.4% (28/34) of adenoid cystic carcinomas were MYB protein-positive, compared with 9.1% (5/55) of non-adenoid cystic carcinomas, and the difference between the two groups was statistically significant (P < 0.01). 2/18 of adenoid cystic carcinomas had duplication of MYB gene by FISH, and all non-adenoid cystic carcinomas were negative although the difference was not statistically significant (P = 0.435). MYB protein expression is a useful diagnostic marker for adenoid cystic carcinomas in its separation from other salivary gland tumors. In addition, duplication of MYB gene is no a major mechanism for the MYB protein overexpression.

Impact of constitutional copy number variants on biological pathway evolution.

PubMed

Poptsova, Maria; Banerjee, Samprit; Gokcumen, Omer; Rubin, Mark A; Demichelis, Francesca

2013-01-23

Inherited Copy Number Variants (CNVs) can modulate the expression levels of individual genes. However, little is known about how CNVs alter biological pathways and how this varies across different populations. To trace potential evolutionary changes of well-described biological pathways, we jointly queried the genomes and the transcriptomes of a collection of individuals with Caucasian, Asian or Yoruban descent combining high-resolution array and sequencing data. We implemented an enrichment analysis of pathways accounting for CNVs and genes sizes and detected significant enrichment not only in signal transduction and extracellular biological processes, but also in metabolism pathways. Upon the estimation of CNV population differentiation (CNVs with different polymorphism frequencies across populations), we evaluated that 22% of the pathways contain at least one gene that is proximal to a CNV (CNV-gene pair) that shows significant population differentiation. The majority of these CNV-gene pairs belong to signal transduction pathways and 6% of the CNV-gene pairs show statistical association between the copy number states and the transcript levels. The analysis suggested possible examples of positive selection within individual populations including NF-kB, MAPK signaling pathways, and Alu/L1 retrotransposition factors. Altogether, our results suggest that constitutional CNVs may modulate subtle pathway changes through specific pathway enzymes, which may become fixed in some populations.

Impact of constitutional copy number variants on biological pathway evolution

PubMed Central

2013-01-01

Background Inherited Copy Number Variants (CNVs) can modulate the expression levels of individual genes. However, little is known about how CNVs alter biological pathways and how this varies across different populations. To trace potential evolutionary changes of well-described biological pathways, we jointly queried the genomes and the transcriptomes of a collection of individuals with Caucasian, Asian or Yoruban descent combining high-resolution array and sequencing data. Results We implemented an enrichment analysis of pathways accounting for CNVs and genes sizes and detected significant enrichment not only in signal transduction and extracellular biological processes, but also in metabolism pathways. Upon the estimation of CNV population differentiation (CNVs with different polymorphism frequencies across populations), we evaluated that 22% of the pathways contain at least one gene that is proximal to a CNV (CNV-gene pair) that shows significant population differentiation. The majority of these CNV-gene pairs belong to signal transduction pathways and 6% of the CNV-gene pairs show statistical association between the copy number states and the transcript levels. Conclusions The analysis suggested possible examples of positive selection within individual populations including NF-kB, MAPK signaling pathways, and Alu/L1 retrotransposition factors. Altogether, our results suggest that constitutional CNVs may modulate subtle pathway changes through specific pathway enzymes, which may become fixed in some populations. PMID:23342974

Genome-wide analysis of macrosatellite repeat copy number variation in worldwide populations: evidence for differences and commonalities in size distributions and size restrictions

PubMed Central

2013-01-01

Background Macrosatellite repeats (MSRs), usually spanning hundreds of kilobases of genomic DNA, comprise a significant proportion of the human genome. Because of their highly polymorphic nature, MSRs represent an extreme example of copy number variation, but their structure and function is largely understudied. Here, we describe a detailed study of six autosomal and two X chromosomal MSRs among 270 HapMap individuals from Central Europe, Asia and Africa. Copy number variation, stability and genetic heterogeneity of the autosomal macrosatellite repeats RS447 (chromosome 4p), MSR5p (5p), FLJ40296 (13q), RNU2 (17q) and D4Z4 (4q and 10q) and X chromosomal DXZ4 and CT47 were investigated. Results Repeat array size distribution analysis shows that all of these MSRs are highly polymorphic with the most genetic variation among Africans and the least among Asians. A mitotic mutation rate of 0.4-2.2% was observed, exceeding meiotic mutation rates and possibly explaining the large size variability found for these MSRs. By means of a novel Bayesian approach, statistical support for a distinct multimodal rather than a uniform allele size distribution was detected in seven out of eight MSRs, with evidence for equidistant intervals between the modes. Conclusions The multimodal distributions with evidence for equidistant intervals, in combination with the observation of MSR-specific constraints on minimum array size, suggest that MSRs are limited in their configurations and that deviations thereof may cause disease, as is the case for facioscapulohumeral muscular dystrophy. However, at present we cannot exclude that there are mechanistic constraints for MSRs that are not directly disease-related. This study represents the first comprehensive study of MSRs in different human populations by applying novel statistical methods and identifies commonalities and differences in their organization and function in the human genome. PMID:23496858

Prenatal Diagnosis of DNA Copy Number Variations by Genomic Single-Nucleotide Polymorphism Array in Fetuses with Congenital Heart Defects.

PubMed

Tang, Shaohua; Lv, Jiaojiao; Chen, Xiangnan; Bai, Lili; Li, Huanzheng; Chen, Chong; Wang, Ping; Xu, Xueqin; Lu, Jianxin

2016-01-01

To evaluate the usefulness of single-nucleotide polymorphism (SNP) array for prenatal genetic diagnosis of congenital heart defect (CHD), we used this approach to detect clinically significant copy number variants (CNVs) in fetuses with CHDs. A HumanCytoSNP-12 array was used to detect genomic samples obtained from 39 fetuses that exhibited cardiovascular abnormalities on ultrasound and had a normal karyotype. The relationship between CNVs and CHDs was identified by using genotype-phenotype comparisons and searching of chromosomal databases. All clinically significant CNVs were confirmed by real-time PCR. CNVs were detected in 38/39 (97.4%) fetuses: variants of unknown significance were detected in 2/39 (5.1%), and clinically significant CNVs were identified in 7/39 (17.9%). In 3 of the 7 fetuses with clinically significant CNVs, 3 rare and previously undescribed CNVs were detected, and these CNVs encompassed the CHD candidate genes FLNA (Xq28 dup), BCOR (Xp11.4 dup), and RBL2 (16q12.2 del). Compared with conventional cytogenetic genomics, SNP array analysis provides significantly improved detection of submicroscopic genomic aberrations in pregnancies with CHDs. Based on these results, we propose that genomic SNP array is an effective method which could be used in the prenatal diagnostic test to assist genetic counseling for pregnancies with CHDs. © 2015 S. Karger AG, Basel.

Analysis of Copy Number Variation in the Abp Gene Regions of Two House Mouse Subspecies Suggests Divergence during the Gene Family Expansions.

PubMed

Pezer, Željka; Chung, Amanda G; Karn, Robert C; Laukaitis, Christina M

2017-06-01

The Androgen-binding protein ( Abp ) gene region of the mouse genome contains 64 genes, some encoding pheromones that influence assortative mating between mice from different subspecies. Using CNVnator and quantitative PCR, we explored copy number variation in this gene family in natural populations of Mus musculus domesticus ( Mmd ) and Mus musculus musculus ( Mmm ), two subspecies of house mice that form a narrow hybrid zone in Central Europe. We found that copy number variation in the center of the Abp gene region is very common in wild Mmd , primarily representing the presence/absence of the final duplications described for the mouse genome. Clustering of Mmd individuals based on this variation did not reflect their geographical origin, suggesting no population divergence in the Abp gene cluster. However, copy number variation patterns differ substantially between Mmd and other mouse taxa. Large blocks of Abp genes are absent in Mmm , Mus musculus castaneus and an outgroup, Mus spretus , although with differences in variation and breakpoint locations. Our analysis calls into question the reliance on a reference genome for interpreting the detailed organization of genes in taxa more distant from the Mmd reference genome. The polymorphic nature of the gene family expansion in all four taxa suggests that the number of Abp genes, especially in the central gene region, is not critical to the survival and reproduction of the mouse. However, Abp haplotypes of variable length may serve as a source of raw genetic material for new signals influencing reproductive communication and thus speciation of mice. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

The genomic and transcriptomic architecture of 2,000 breast tumours reveals novel subgroups

PubMed Central

Curtis, Christina; Shah, Sohrab P.; Chin, Suet-Feung; Turashvili, Gulisa; Rueda, Oscar M.; Dunning, Mark J.; Speed, Doug; Lynch, Andy G.; Samarajiwa, Shamith; Yuan, Yinyin; Gräf, Stefan; Ha, Gavin; Haffari, Gholamreza; Bashashati, Ali; Russell, Roslin; McKinney, Steven; Langerød, Anita; Green, Andrew; Provenzano, Elena; Wishart, Gordon; Pinder, Sarah; Watson, Peter; Markowetz, Florian; Murphy, Leigh; Ellis, Ian; Purushotham, Arnie; Børresen-Dale, Anne-Lise; Brenton, James D.; Tavaré, Simon; Caldas, Carlos; Aparicio, Samuel

2012-01-01

The elucidation of breast cancer subgroups and their molecular drivers requires integrated views of the genome and transcriptome from representative numbers of patients. We present an integrated analysis of copy number and gene expression in a discovery and validation set of 997 and 995 primary breast tumours, respectively, with long-term clinical follow-up. Inherited variants (copy number variants and single nucleotide polymorphisms) and acquired somatic copy number aberrations (CNAs) were associated with expression in ~40% of genes, with the landscape dominated by cis- and trans-acting CNAs. By delineating expression outlier genes driven in cis by CNAs, we identified putative cancer genes, including deletions in PPP2R2A, MTAP and MAP2K4. Unsupervised analysis of paired DNA–RNA profiles revealed novel subgroups with distinct clinical outcomes, which reproduced in the validation cohort. These include a high-risk, oestrogen-receptor-positive 11q13/14 cis-acting subgroup and a favourable prognosis subgroup devoid of CNAs. Trans-acting aberration hotspots were found to modulate subgroup-specific gene networks, including a TCR deletion-mediated adaptive immune response in the ‘CNA-devoid’ subgroup and a basal-specific chromosome 5 deletion-associated mitotic network. Our results provide a novel molecular stratification of the breast cancer population, derived from the impact of somatic CNAs on the transcriptome. PMID:22522925

Mitochondria DNA change and oxidative damage in clinically stable patients with major depressive disorder.

PubMed

Chang, Cheng-Chen; Jou, Shaw-Hwa; Lin, Ta-Tsung; Lai, Te-Jen; Liu, Chin-San

2015-01-01

To compare alterations of mitochondria DNA (mtDNA) copy number, single nucleotide polymorphisms (SNPs), and oxidative damage of mtDNA in clinically stable patients with major depressive disorder (MDD). Patients met DSM-IV diagnostic criteria for MDD were recruited from the psychiatric outpatient clinic at Changhua Christian Hospital, Taiwan. They were clinically stable and their medications had not changed for at least the preceding two months. Exclusion criteria were substance-induced psychotic disorder, eating disorder, anxiety disorder or illicit substance abuse. Comparison subjects did not have any major psychiatric disorder and they were medically healthy. Peripheral blood leukocytes were analyzed to compare copy number, SNPs and oxidative damage of mtDNA between the two groups. 40 MDD patients and 70 comparison subjects were collected. The median age of the subjects was 42 years and 38 years in MDD and comparison groups, respectively. Leukocyte mtDNA copy number of MDD patients was significantly lower than that of the comparison group (p = 0.037). MDD patients had significantly higher mitochondrial oxidative damage than the comparison group (6.44 vs. 3.90, p<0.001). After generalized linear model adjusted for age, sex, smoking, family history, and psychotropic use, mtDNA copy number was still significantly lower in the MDD group (p<0.001). MtDNA oxidative damage was positively correlated with age (p<0.001) and MDD (p<0.001). Antipsychotic use was negatively associated with mtDNA copy number (p = 0.036). The study is cross-sectional with no longitudinal follow up. The cohort is clinically stable and generalizability of our result to other cohort should be considered. Our study suggests that oxidative stress and mitochondria may play a role in the pathophysiology of MDD. More large-scale studies are warranted to assess the interplay between oxidative stress, mitochondria dysfunction and MDD.

Transposable elements generate population-specific insertional patterns and allelic variation in genes of wild emmer wheat (Triticum turgidum ssp. dicoccoides).

PubMed

Domb, Katherine; Keidar, Danielle; Yaakov, Beery; Khasdan, Vadim; Kashkush, Khalil

2017-10-27

Natural populations of the tetraploid wild emmer wheat (genome AABB) were previously shown to demonstrate eco-geographically structured genetic and epigenetic diversity. Transposable elements (TEs) might make up a significant part of the genetic and epigenetic variation between individuals and populations because they comprise over 80% of the wild emmer wheat genome. In this study, we performed detailed analyses to assess the dynamics of transposable elements in 50 accessions of wild emmer wheat collected from 5 geographically isolated sites. The analyses included: the copy number variation of TEs among accessions in the five populations, population-unique insertional patterns, and the impact of population-unique/specific TE insertions on structure and expression of genes. We assessed the copy numbers of 12 TE families using real-time quantitative PCR, and found significant copy number variation (CNV) in the 50 wild emmer wheat accessions, in a population-specific manner. In some cases, the CNV difference reached up to 6-fold. However, the CNV was TE-specific, namely some TE families showed higher copy numbers in one or more populations, and other TE families showed lower copy numbers in the same population(s). Furthermore, we assessed the insertional patterns of 6 TE families using transposon display (TD), and observed significant population-specific insertional patterns. The polymorphism levels of TE-insertional patterns reached 92% among all wild emmer wheat accessions, in some cases. In addition, we observed population-specific/unique TE insertions, some of which were located within or close to protein-coding genes, creating allelic variations in a population-specific manner. We also showed that those genes are differentially expressed in wild emmer wheat. For the first time, this study shows that TEs proliferate in wild emmer wheat in a population-specific manner, creating new alleles of genes, which contribute to the divergent evolution of homeologous genes from the A and B subgenomes.

A Novel Center Star Multiple Sequence Alignment Algorithm Based on Affine Gap Penalty and K-Band

NASA Astrophysics Data System (ADS)

Zou, Quan; Shan, Xiao; Jiang, Yi

Multiple sequence alignment is one of the most important topics in computational biology, but it cannot deal with the large data so far. As the development of copy-number variant(CNV) and Single Nucleotide Polymorphisms(SNP) research, many researchers want to align numbers of similar sequences for detecting CNV and SNP. In this paper, we propose a novel multiple sequence alignment algorithm based on affine gap penalty and k-band. It can align more quickly and accurately, that will be helpful for mining CNV and SNP. Experiments prove the performance of our algorithm.

Low copy numbers of complement C4 and homozygous deficiency of C4A may predispose to severe disease and earlier disease onset in patients with systemic lupus erythematosus.

PubMed

Jüptner, M; Flachsbart, F; Caliebe, A; Lieb, W; Schreiber, S; Zeuner, R; Franke, A; Schröder, J O

2018-04-01

Objectives Low copy numbers and deletion of complement C4 genes are potent risk factors for systemic lupus erythematosus (SLE). However, it is not known whether this genetic association affects the clinical outcome. We investigated C4 copy number variation and its relationship to clinical and serological features in a Northern European lupus cohort. Methods We genotyped the C4 gene locus using polymerase chain reaction (PCR)-based TaqMan assays in 169 patients with SLE classified according to the 1997 revised American College of Rheumatology (ACR) criteria and in 520 matched controls. In the patient group the mean C4 serum protein concentrations nephelometrically measured during a 12-month period prior to genetic analysis were compared to C4 gene copy numbers. Severity of disease was classified according to the intensity of the immunosuppressive regimens applied and compared to C4 gene copy numbers, too. In addition, we performed a TaqMan based analysis of three lupus-associated single-nucleotide polymorphisms (SNPs) located inside the major histocompatibility complex (MHC) to investigate the independence of complement C4 in association with SLE. Results Homozygous deficiency of the C4A isotype was identified as the strongest risk factor for SLE (odds ratio (OR) = 5.329; p = 7.7 × 10 -3 ) in the case-control comparison. Moreover, two copies of total C4 were associated with SLE (OR = 3.699; p = 6.8 × 10 -3 ). C4 serum levels were strongly related to C4 gene copy numbers in patients, the mean concentration ranging from 0.110 g/l (two copies) to 0.256 g/l (five to six copies; p = 4.9 × 10 -6 ). Two copies of total C4 and homozygous deletion of C4A were associated with a disease course requiring cyclophosphamide therapy (OR = 4.044; p = 0.040 and OR = 5.798; p = 0.034, respectively). Homozygous deletion of C4A was associated with earlier onset of SLE (median 24 vs. 34 years; p = 0.019) but not significant after correction for multiple testing. SNP analysis revealed a significant association of HLA-DRB1*0301 with SLE (OR = 2.231; p = 1.33 × 10 -5 ). Conclusions Our findings confirm the important role of complement C4 genes in the development of SLE. Beyond the impact on the susceptibility for lupus, C4 copy numbers may be related to earlier onset and a more severe course of the disease. The association of homozygous deletion of C4A and SLE is accompanied by the presence of HLA-DRB1*0301 without a proven pathophysiological mechanism.

Impact of CYP2D6 polymorphisms on clinical efficacy & tolerability of metoprolol tartrate

PubMed Central

Hamadeh, Issam S.; Langaee, Taimour Y.; Dwivedi, Ruti; Garcia, Sofia; Burkley, Ben M.; Chapman, Arlene B.; Gums, John G.; Turner, Stephen T.; Gong, Yan; Cooper-DeHoff, Rhonda M.; Johnson, Julie A.

2014-01-01

Metoprolol is a selective β-1 adrenergic receptor blocker that undergoes extensive metabolism by the polymorphic enzyme, CYP2D6. Our objective was to investigate the influence of CYP2D6 polymorphisms on efficacy and tolerability of metoprolol tartrate. 281 study participants with uncomplicated hypertension received 50 mg of metoprolol twice daily followed by response guided titration to 100 mg twice daily. Phenotypes were assigned based on results of CYP2D6 genotyping and copy number variation assays. Clinical response to metoprolol and adverse effect rates were analyzed in relation to CYP2D6 phenotypes by using appropriate statistical tests. Heart rate response differed significantly by CYP2D6 phenotype (p-value <0.0001) with poor metabolizers & intermediate metabolizers showing greater HR reduction. However, blood pressure response and adverse effect rates were not significantly different by CYP2D6 phenotype. Other than a significant difference in heart rate response, CYP2D6 polymorphisms were not a determinant of the variability in response or tolerability to metoprolol. PMID:24637943

Sequence Polymorphisms and Structural Variations among Four Grapevine (Vitis vinifera L.) Cultivars Representing Sardinian Agriculture

PubMed Central

Mercenaro, Luca; Nieddu, Giovanni; Porceddu, Andrea; Pezzotti, Mario; Camiolo, Salvatore

2017-01-01

The genetic diversity among grapevine (Vitis vinifera L.) cultivars that underlies differences in agronomic performance and wine quality reflects the accumulation of single nucleotide polymorphisms (SNPs) and small indels as well as larger genomic variations. A combination of high throughput sequencing and mapping against the grapevine reference genome allows the creation of comprehensive sequence variation maps. We used next generation sequencing and bioinformatics to generate an inventory of SNPs and small indels in four widely cultivated Sardinian grape cultivars (Bovale sardo, Cannonau, Carignano and Vermentino). More than 3,200,000 SNPs were identified with high statistical confidence. Some of the SNPs caused the appearance of premature stop codons and thus identified putative pseudogenes. The analysis of SNP distribution along chromosomes led to the identification of large genomic regions with uninterrupted series of homozygous SNPs. We used a digital comparative genomic hybridization approach to identify 6526 genomic regions with significant differences in copy number among the four cultivars compared to the reference sequence, including 81 regions shared between all four cultivars and 4953 specific to single cultivars (representing 1.2 and 75.9% of total copy number variation, respectively). Reads mapping at a distance that was not compatible with the insert size were used to identify a dataset of putative large deletions with cultivar Cannonau revealing the highest number. The analysis of genes mapping to these regions provided a list of candidates that may explain some of the phenotypic differences among the Bovale sardo, Cannonau, Carignano and Vermentino cultivars. PMID:28775732

Rare Copy Number Variants Are a Common Cause of Short Stature

PubMed Central

Zahnleiter, Diana; Uebe, Steffen; Ekici, Arif B.; Hoyer, Juliane; Wiesener, Antje; Wieczorek, Dagmar; Kunstmann, Erdmute; Reis, André; Doerr, Helmuth-Guenther; Rauch, Anita; Thiel, Christian T.

2013-01-01

Human growth has an estimated heritability of about 80%–90%. Nevertheless, the underlying cause of shortness of stature remains unknown in the majority of individuals. Genome-wide association studies (GWAS) showed that both common single nucleotide polymorphisms and copy number variants (CNVs) contribute to height variation under a polygenic model, although explaining only a small fraction of overall genetic variability in the general population. Under the hypothesis that severe forms of growth retardation might also be caused by major gene effects, we searched for rare CNVs in 200 families, 92 sporadic and 108 familial, with idiopathic short stature compared to 820 control individuals. Although similar in number, patients had overall significantly larger CNVs (p-value<1×10−7). In a gene-based analysis of all non-polymorphic CNVs>50 kb for gene function, tissue expression, and murine knock-out phenotypes, we identified 10 duplications and 10 deletions ranging in size from 109 kb to 14 Mb, of which 7 were de novo (p<0.03) and 13 inherited from the likewise affected parent but absent in controls. Patients with these likely disease causing 20 CNVs were smaller than the remaining group (p<0.01). Eleven (55%) of these CNVs either overlapped with known microaberration syndromes associated with short stature or contained GWAS loci for height. Haploinsufficiency (HI) score and further expression profiling suggested dosage sensitivity of major growth-related genes at these loci. Overall 10% of patients carried a disease-causing CNV indicating that, like in neurodevelopmental disorders, rare CNVs are a frequent cause of severe growth retardation. PMID:23516380

CNVinspector: a web-based tool for the interactive evaluation of copy number variations in single patients and in cohorts.

PubMed

Knierim, Ellen; Schwarz, Jana Marie; Schuelke, Markus; Seelow, Dominik

2013-08-01

Many genetic disorders are caused by copy number variations (CNVs) in the human genome. However, the large number of benign CNV polymorphisms makes it difficult to delineate causative variants for a certain disease phenotype. Hence, we set out to create software that accumulates and visualises locus-specific knowledge and enables clinicians to study their own CNVs in the context of known polymorphisms and disease variants. CNV data from healthy cohorts (Database of Genomic Variants) and from disease-related databases (DECIPHER) were integrated into a joint resource. Data are presented in an interactive web-based application that allows inspection, evaluation and filtering of CNVs in single individuals or in entire cohorts. CNVinspector provides simple interfaces to upload CNV data, compare them with own or published control data and visualise the results in graphical interfaces. Beyond choosing control data from different public studies, platforms and methods, dedicated filter options allow the detection of CNVs that are either enriched in patients or depleted in controls. Alternatively, a search can be restricted to those CNVs that appear in individuals of similar clinical phenotype. For each gene of interest within a CNV, we provide a link to NCBI, ENSEMBL and the GeneDistiller search engine to browse for potential disease-associated genes. With its user-friendly handling, the integration of control data and the filtering options, CNVinspector will facilitate the daily work of clinical geneticists and accelerate the delineation of new syndromes and gene functions. CNVinspector is freely accessible under http://www.cnvinspector.org.

Activation of individual L1 retrotransposon instances is restricted to cell-type dependent permissive loci

PubMed Central

Philippe, Claude; Vargas-Landin, Dulce B; Doucet, Aurélien J; van Essen, Dominic; Vera-Otarola, Jorge; Kuciak, Monika; Corbin, Antoine; Nigumann, Pilvi; Cristofari, Gaël

2016-01-01

LINE-1 (L1) retrotransposons represent approximately one sixth of the human genome, but only the human-specific L1HS-Ta subfamily acts as an endogenous mutagen in modern humans, reshaping both somatic and germline genomes. Due to their high levels of sequence identity and the existence of many polymorphic insertions absent from the reference genome, the transcriptional activation of individual genomic L1HS-Ta copies remains poorly understood. Here we comprehensively mapped fixed and polymorphic L1HS-Ta copies in 12 commonly-used somatic cell lines, and identified transcriptional and epigenetic signatures allowing the unambiguous identification of active L1HS-Ta copies in their genomic context. Strikingly, only a very restricted subset of L1HS-Ta loci - some being polymorphic among individuals - significantly contributes to the bulk of L1 expression, and these loci are differentially regulated among distinct cell lines. Thus, our data support a local model of L1 transcriptional activation in somatic cells, governed by individual-, locus-, and cell-type-specific determinants. DOI: http://dx.doi.org/10.7554/eLife.13926.001 PMID:27016617

COPS: A Sensitive and Accurate Tool for Detecting Somatic Copy Number Alterations Using Short-Read Sequence Data from Paired Samples

PubMed Central

Krishnan, Neeraja M.; Gaur, Prakhar; Chaudhary, Rakshit; Rao, Arjun A.; Panda, Binay

2012-01-01

Copy Number Alterations (CNAs) such as deletions and duplications; compose a larger percentage of genetic variations than single nucleotide polymorphisms or other structural variations in cancer genomes that undergo major chromosomal re-arrangements. It is, therefore, imperative to identify cancer-specific somatic copy number alterations (SCNAs), with respect to matched normal tissue, in order to understand their association with the disease. We have devised an accurate, sensitive, and easy-to-use tool, COPS, COpy number using Paired Samples, for detecting SCNAs. We rigorously tested the performance of COPS using short sequence simulated reads at various sizes and coverage of SCNAs, read depths, read lengths and also with real tumor:normal paired samples. We found COPS to perform better in comparison to other known SCNA detection tools for all evaluated parameters, namely, sensitivity (detection of true positives), specificity (detection of false positives) and size accuracy. COPS performed well for sequencing reads of all lengths when used with most upstream read alignment tools. Additionally, by incorporating a downstream boundary segmentation detection tool, the accuracy of SCNA boundaries was further improved. Here, we report an accurate, sensitive and easy to use tool in detecting cancer-specific SCNAs using short-read sequence data. In addition to cancer, COPS can be used for any disease as long as sequence reads from both disease and normal samples from the same individual are available. An added boundary segmentation detection module makes COPS detected SCNA boundaries more specific for the samples studied. COPS is available at ftp://115.119.160.213 with username “cops” and password “cops”. PMID:23110103

Exploiting sequence similarity to validate the sensitivity of SNP arrays in detecting fine-scaled copy number variations.

PubMed

Wong, Gerard; Leckie, Christopher; Gorringe, Kylie L; Haviv, Izhak; Campbell, Ian G; Kowalczyk, Adam

2010-04-15

High-density single nucleotide polymorphism (SNP) genotyping arrays are efficient and cost effective platforms for the detection of copy number variation (CNV). To ensure accuracy in probe synthesis and to minimize production costs, short oligonucleotide probe sequences are used. The use of short probe sequences limits the specificity of binding targets in the human genome. The specificity of these short probeset sequences has yet to be fully analysed against a normal reference human genome. Sequence similarity can artificially elevate or suppress copy number measurements, and hence reduce the reliability of affected probe readings. For the purpose of detecting narrow CNVs reliably down to the width of a single probeset, sequence similarity is an important issue that needs to be addressed. We surveyed the Affymetrix Human Mapping SNP arrays for probeset sequence similarity against the reference human genome. Utilizing sequence similarity results, we identified a collection of fine-scaled putative CNVs between gender from autosomal probesets whose sequence matches various loci on the sex chromosomes. To detect these variations, we utilized our statistical approach, Detecting REcurrent Copy number change using rank-order Statistics (DRECS), and showed that its performance was superior and more stable than the t-test in detecting CNVs. Through the application of DRECS on the HapMap population datasets with multi-matching probesets filtered, we identified biologically relevant SNPs in aberrant regions across populations with known association to physical traits, such as height, covered by the span of a single probe. This provided empirical confirmation of the existence of naturally occurring narrow CNVs as well as the sensitivity of the Affymetrix SNP array technology in detecting them. The MATLAB implementation of DRECS is available at http://ww2.cs.mu.oz.au/ approximately gwong/DRECS/index.html.

Lactase persistence and augmented salivary alpha-amylase gene copy numbers might have been selected by the combined toxic effects of gluten and (food born) pathogens.

PubMed

Pruimboom, Leo; Fox, Tom; Muskiet, Frits A J

2014-03-01

Various positively selected adaptations to new nutrients have been identified. Lactase persistence is among the best known, conferring the ability for drinking milk at post weaning age. An augmented number of amylase gene (AMY1) copies, giving rise to higher salivary amylase activity, has been implicated in the consumption of starch-rich foods. Higher AMY1 copy numbers have been demonstrated in populations with recent histories of starchy-rich diets. It is however questionable whether the resulting polymorphisms have exerted positive selection only by providing easily available sources of macro and micronutrients. Humans have explored new environments more than any other animal. Novel environments challenge the host, but especially its immune system with new climatic conditions, food and especially pathogens. With the advent of the agricultural revolution and the concurrent domestication of cattle came new pathogens. We contend that specific new food ingredients (e.g., gluten) and novel pathogens drove selection for lactase persistence and higher AMY gene copy numbers. Both adaptations provide ample glucose for activating the sodium glucose-dependent co-transporter 1 (SGLT1), which is the principal glucose, sodium and water transporter in the gastro-intestinal tract. Their rapid uptake confers protection against potentially lethal dehydration, hyponatremia and ultimately multiple organ failure. Oral rehydration therapy aims at SGLT1 activity and is the current treatment of choice for chronic diarrhoea and vomiting. We hypothesize that lifelong lactase activity and rapid starch digestion should be looked at as the evolutionary covalent of oral rehydration therapy. Copyright © 2014 Elsevier Ltd. All rights reserved.

Copy number and haplotype variation at the VRN-A1 and central FR-A2 loci are associated with frost tolerance in hexaploid wheat.

PubMed

Zhu, Jie; Pearce, Stephen; Burke, Adrienne; See, Deven Robert; Skinner, Daniel Z; Dubcovsky, Jorge; Garland-Campbell, Kimberly

2014-05-01

The interaction between VRN - A1 and FR - A2 largely affect the frost tolerance of hexaploid wheat. Frost tolerance is critical for wheat survival during cold winters. Natural variation for this trait is mainly associated with allelic differences at the VERNALIZATION 1 (VRN1) and FROST RESISTANCE 2 (FR2) loci. VRN1 regulates the transition between vegetative and reproductive stages and FR2, a locus including several tandemly duplicated C-REPEAT BINDING FACTOR (CBF) transcription factors, regulates the expression of Cold-regulated genes. We identified sequence and copy number variation at these two loci among winter and spring wheat varieties and characterized their association with frost tolerance. We identified two FR-A2 haplotypes-'FR-A2-S' and 'FR-A2-T'-distinguished by two insertion/deletions and ten single nucleotide polymorphisms within the CBF-A12 and CBF-A15 genes. Increased copy number of CBF-A14 was frequently associated with the FR-A2-T haplotype and with higher CBF14 transcript levels in response to cold. Factorial ANOVAs revealed significant interactions between VRN1 and FR-A2 for frost tolerance in both winter and spring panels suggesting a crosstalk between vernalization and cold acclimation pathways. The model including these two loci and their interaction explained 32.0 and 20.7 % of the variation in frost tolerance in the winter and spring panels, respectively. The interaction was validated in a winter wheat F 4:5 population segregating for both genes. Increased VRN-A1 copy number was associated with improved frost tolerance among varieties carrying the FR-A2-T allele but not among those carrying the FR-A2-S allele. These results suggest that selection of varieties carrying the FR-A2-T allele and three copies of the recessive vrn-A1 allele would be a good strategy to improve frost tolerance in wheat.

Evolution of the human-specific microRNA miR-941

PubMed Central

Hu, Hai Yang; He, Liu; Fominykh, Kseniya; Yan, Zheng; Guo, Song; Zhang, Xiaoyu; Taylor, Martin S.; Tang, Lin; Li, Jie; Liu, Jianmei; Wang, Wen; Yu, Haijing; Khaitovich, Philipp

2012-01-01

MicroRNA-mediated gene regulation is important in many physiological processes. Here we explore the roles of a microRNA, miR-941, in human evolution. We find that miR-941 emerged de novo in the human lineage, between six and one million years ago, from an evolutionarily volatile tandem repeat sequence. Its copy-number remains polymorphic in humans and shows a trend for decreasing copy-number with migration out of Africa. Emergence of miR-941 was accompanied by accelerated loss of miR-941-binding sites, presumably to escape regulation. We further show that miR-941 is highly expressed in pluripotent cells, repressed upon differentiation and preferentially targets genes in hedgehog- and insulin-signalling pathways, thus suggesting roles in cellular differentiation. Human-specific effects of miR-941 regulation are detectable in the brain and affect genes involved in neurotransmitter signalling. Taken together, these results implicate miR-941 in human evolution, and provide an example of rapid regulatory evolution in the human linage. PMID:23093182

Heme Oxygenase 1 and 2 Common Genetic Variants and Risk for Essential Tremor

PubMed Central

Ayuso, Pedro; Agúndez, José A.G.; Alonso-Navarro, Hortensia; Martínez, Carmen; Benito-León, Julián; Ortega-Cubero, Sara; Lorenzo-Betancor, Oswaldo; Pastor, Pau; López-Alburquerque, Tomás; García-Martín, Elena; Jiménez-Jiménez, Félix J.

2015-01-01

Abstract Several reports suggested a role of heme oxygenase genes 1 and 2 (HMOX1 and HMOX2) in modifying the risk to develop Parkinson disease (PD). Because essential tremor (ET) and PD share phenotypical and, probably, etiologic factors of the similarities, we analyzed whether such genes are related with the risk to develop ET. We analyzed the distribution of allelic and genotype frequencies of the HMOX1 rs2071746, HMOX1 rs2071747, HMOX2 rs2270363, and HMOX2 rs1051308 single nucleotide polymorphisms, as well as the presence of copy number variations of these genes in 202 subjects with familial ET and 747 healthy controls. Allelic frequencies of rs2071746T and rs1051308G were significantly lower in ET patients than in controls. None of the studied polymorphisms influenced the disease onset. The present study suggests a weak association between HMOX1 rs2071746 and HMOX2 rs1051308 polymorphisms and the risk to develop ET in the Spanish population. PMID:26091465

Heme Oxygenase 1 and 2 Common Genetic Variants and Risk for Essential Tremor.

PubMed

Ayuso, Pedro; Agúndez, José A G; Alonso-Navarro, Hortensia; Martínez, Carmen; Benito-León, Julián; Ortega-Cubero, Sara; Lorenzo-Betancor, Oswaldo; Pastor, Pau; López-Alburquerque, Tomás; García-Martín, Elena; Jiménez-Jiménez, Félix J

2015-06-01

Several reports suggested a role of heme oxygenase genes 1 and 2 (HMOX1 and HMOX2) in modifying the risk to develop Parkinson disease (PD). Because essential tremor (ET) and PD share phenotypical and, probably, etiologic factors of the similarities, we analyzed whether such genes are related with the risk to develop ET. We analyzed the distribution of allelic and genotype frequencies of the HMOX1 rs2071746, HMOX1 rs2071747, HMOX2 rs2270363, and HMOX2 rs1051308 single nucleotide polymorphisms, as well as the presence of copy number variations of these genes in 202 subjects with familial ET and 747 healthy controls. Allelic frequencies of rs2071746T and rs1051308G were significantly lower in ET patients than in controls. None of the studied polymorphisms influenced the disease onset. The present study suggests a weak association between HMOX1 rs2071746 and HMOX2 rs1051308 polymorphisms and the risk to develop ET in the Spanish population.

Simple Sequence Repeats in Escherichia coli: Abundance, Distribution, Composition, and Polymorphism

PubMed Central

Gur-Arie, Riva; Cohen, Cyril J.; Eitan, Yuval; Shelef, Leora; Hallerman, Eric M.; Kashi, Yechezkel

2000-01-01

Computer-based genome-wide screening of the DNA sequence of Escherichia coli strain K12 revealed tens of thousands of tandem simple sequence repeat (SSR) tracts, with motifs ranging from 1 to 6 nucleotides. SSRs were well distributed throughout the genome. Mononucleotide SSRs were over-represented in noncoding regions and under-represented in open reading frames (ORFs). Nucleotide composition of mono- and dinucleotide SSRs, both in ORFs and in noncoding regions, differed from that of the genomic region in which they occurred, with 93% of all mononucleotide SSRs proving to be of A or T. Computer-based analysis of the fine position of every SSR locus in the noncoding portion of the genome relative to downstream ORFs showed SSRs located in areas that could affect gene regulation. DNA sequences at 14 arbitrarily chosen SSR tracts were compared among E. coli strains. Polymorphisms of SSR copy number were observed at four of seven mononucleotide SSR tracts screened, with all polymorphisms occurring in noncoding regions. SSR polymorphism could prove important as a genome-wide source of variation, both for practical applications (including rapid detection, strain identification, and detection of loci affecting key phenotypes) and for evolutionary adaptation of microbes.[The sequence data described in this paper have been submitted to the GenBank data library under accession numbers AF209020–209030 and AF209508–209518.] PMID:10645951

Identification of candidate genes involved in neuroblastoma progression by combining genomic and expression microarrays with survival data.

PubMed

Łastowska, M; Viprey, V; Santibanez-Koref, M; Wappler, I; Peters, H; Cullinane, C; Roberts, P; Hall, A G; Tweddle, D A; Pearson, A D J; Lewis, I; Burchill, S A; Jackson, M S

2007-11-22

Identifying genes, whose expression is consistently altered by chromosomal gains or losses, is an important step in defining genes of biological relevance in a wide variety of tumour types. However, additional criteria are needed to discriminate further among the large number of candidate genes identified. This is particularly true for neuroblastoma, where multiple genomic copy number changes of proven prognostic value exist. We have used Affymetrix microarrays and a combination of fluorescent in situ hybridization and single nucleotide polymorphism (SNP) microarrays to establish expression profiles and delineate copy number alterations in 30 primary neuroblastomas. Correlation of microarray data with patient survival and analysis of expression within rodent neuroblastoma cell lines were then used to define further genes likely to be involved in the disease process. Using this approach, we identify >1000 genes within eight recurrent genomic alterations (loss of 1p, 3p, 4p, 10q and 11q, 2p gain, 17q gain, and the MYCN amplicon) whose expression is consistently altered by copy number change. Of these, 84 correlate with patient survival, with the minimal regions of 17q gain and 4p loss being enriched significantly for such genes. These include genes involved in RNA and DNA metabolism, and apoptosis. Orthologues of all but one of these genes on 17q are overexpressed in rodent neuroblastoma cell lines. A significant excess of SNPs whose copy number correlates with survival is also observed on proximal 4p in stage 4 tumours, and we find that deletion of 4p is associated with improved outcome in an extended cohort of tumours. These results define the major impact of genomic copy number alterations upon transcription within neuroblastoma, and highlight genes on distal 17q and proximal 4p for downstream analyses. They also suggest that integration of discriminators, such as survival and comparative gene expression, with microarray data may be useful in the identification of critical genes within regions of loss or gain in many human cancers.

Targeted next-generation sequencing at copy-number breakpoints for personalized analysis of rearranged ends in solid tumors.

PubMed

Kim, Hyun-Kyoung; Park, Won Cheol; Lee, Kwang Man; Hwang, Hai-Li; Park, Seong-Yeol; Sorn, Sungbin; Chandra, Vishal; Kim, Kwang Gi; Yoon, Woong-Bae; Bae, Joon Seol; Shin, Hyoung Doo; Shin, Jong-Yeon; Seoh, Ju-Young; Kim, Jong-Il; Hong, Kyeong-Man

2014-01-01

The concept of the utilization of rearranged ends for development of personalized biomarkers has attracted much attention owing to its clinical applicability. Although targeted next-generation sequencing (NGS) for recurrent rearrangements has been successful in hematologic malignancies, its application to solid tumors is problematic due to the paucity of recurrent translocations. However, copy-number breakpoints (CNBs), which are abundant in solid tumors, can be utilized for identification of rearranged ends. As a proof of concept, we performed targeted next-generation sequencing at copy-number breakpoints (TNGS-CNB) in nine colon cancer cases including seven primary cancers and two cell lines, COLO205 and SW620. For deduction of CNBs, we developed a novel competitive single-nucleotide polymorphism (cSNP) microarray method entailing CNB-region refinement by competitor DNA. Using TNGS-CNB, 19 specific rearrangements out of 91 CNBs (20.9%) were identified, and two polymerase chain reaction (PCR)-amplifiable rearrangements were obtained in six cases (66.7%). And significantly, TNGS-CNB, with its high positive identification rate (82.6%) of PCR-amplifiable rearrangements at candidate sites (19/23), just from filtering of aligned sequences, requires little effort for validation. Our results indicate that TNGS-CNB, with its utility for identification of rearrangements in solid tumors, can be successfully applied in the clinical laboratory for cancer-relapse and therapy-response monitoring.

Parallel Evolution of Copy-Number Variation across Continents in Drosophila melanogaster

PubMed Central

Schrider, Daniel R.; Hahn, Matthew W.; Begun, David J.

2016-01-01

Genetic differentiation across populations that is maintained in the presence of gene flow is a hallmark of spatially varying selection. In Drosophila melanogaster, the latitudinal clines across the eastern coasts of Australia and North America appear to be examples of this type of selection, with recent studies showing that a substantial portion of the D. melanogaster genome exhibits allele frequency differentiation with respect to latitude on both continents. As of yet there has been no genome-wide examination of differentiated copy-number variants (CNVs) in these geographic regions, despite their potential importance for phenotypic variation in Drosophila and other taxa. Here, we present an analysis of geographic variation in CNVs in D. melanogaster. We also present the first genomic analysis of geographic variation for copy-number variation in the sister species, D. simulans, in order to investigate patterns of parallel evolution in these close relatives. In D. melanogaster we find hundreds of CNVs, many of which show parallel patterns of geographic variation on both continents, lending support to the idea that they are influenced by spatially varying selection. These findings support the idea that polymorphic CNVs contribute to local adaptation in D. melanogaster. In contrast, we find very few CNVs in D. simulans that are geographically differentiated in parallel on both continents, consistent with earlier work suggesting that clinal patterns are weaker in this species. PMID:26809315

The hOGG1 Ser326Cys polymorphism and male subfertility in Taiwanese patients with varicocele.

PubMed

Chen, S S-S; Chiu, L-P

2018-03-26

To investigate the association between the human 8-oxoguanine DNA glycosylase 1 (hOGG1) gene Ser326Cys polymorphism and male subfertility in Taiwanese patients with varicocele, we made a prospective study. Ninety young male patients with varicocele (group 1), 50 young male patients with subclinical varicocele (group 2) and 30 normal young male patients without varicocele (group 3) were recruited in this study. The hOGG1 null homozygous genotype (Cys/Cys) and the occurrence of a 4,977-bp deletion in mitochondrial DNA and mitochondrial copy number in spermatozoa were determined by polymerase chain reaction. The 8-hydroxy-2'-deoxyguanosine (8-OHdG) content of DNA in the spermatozoa was measured using high-performance liquid chromatography, and total antioxidant capacity (TAC) of seminal plasma was detected electrochemically. The rates of male subfertility were 31.1% (28/90) in group 1 and 22% (11/50) in group 2. Of 39 subfertile men, 74.4% (29/39) had the hOGG1 Cys/Cys genotype. Patients in groups 1 and 2 with hOGG1 Cys/Cys genotype had significantly higher 8-OHdG content in sperm DNA, lower mitochondrial copy number in spermatozoa and lower TAC in seminal plasma than those with Ser/Ser or Ser/Cys genotype. Clinicians should pay more attention to patients with varicocele with the hOGG1 Cys/Cys genotype. © 2018 Blackwell Verlag GmbH.

Molecular surveillance for drug-resistant Plasmodium falciparum in clinical and subclinical populations from three border regions of Burma/Myanmar: cross-sectional data and a systematic review of resistance studies

PubMed Central

2012-01-01

Background Confirmation of artemisinin-delayed parasite clearance in Plasmodium falciparum along the Thai-Myanmar border has inspired a global response to contain and monitor drug resistance to avert the disastrous consequences of a potential spread to Africa. However, resistance data from Myanmar are sparse, particularly from high-risk areas where limited health services and decades of displacement create conditions for resistance to spread. Subclinical infections may represent an important reservoir for resistance genes that confer a fitness disadvantage relative to wild-type alleles. This study estimates the prevalence of resistance genotypes in three previously unstudied remote populations in Myanmar and tests the a priori hypothesis that resistance gene prevalence would be higher among isolates collected from subclinical infections than isolates collected from febrile clinical patients. A systematic review of resistance studies is provided for context. Methods Community health workers in Karen and Kachin States and an area spanning the Indo-Myanmar border collected dried blood spots from 988 febrile clinical patients and 4,591 villagers with subclinical infection participating in routine prevalence surveys. Samples positive for P. falciparum 18 s ribosomal RNA by real-time PCR were genotyped for P. falciparum multidrug resistance protein (pfmdr1) copy number and the pfcrt K76T polymorphism using multiplex real-time PCR. Results Pfmdr1 copy number increase and the pfcrt K76 polymorphism were determined for 173 and 269 isolates, respectively. Mean pfmdr1 copy number was 1.2 (range: 0.7 to 3.7). Pfmdr1 copy number increase was present in 17.5%, 9.6% and 11.1% of isolates from Karen and Kachin States and the Indo-Myanmar border, respectively. Pfmdr1 amplification was more prevalent in subclinical isolates (20.3%) than clinical isolates (6.4%, odds ratio 3.7, 95% confidence interval 1.1 - 12.5). Pfcrt K76T prevalence ranged from 90-100%. Conclusions Community health workers can contribute to molecular surveillance of drug resistance in remote areas of Myanmar. Marginal and displaced populations under-represented among previous resistance investigations can and should be included in resistance surveillance efforts, particularly once genetic markers of artemisinin-delayed parasite clearance are identified. Subclinical infections may contribute to the epidemiology of drug resistance, but determination of gene amplification from desiccated filter samples requires further validation when DNA concentration is low. PMID:22992214

Simple sequence repeats in Escherichia coli: abundance, distribution, composition, and polymorphism.

PubMed

Gur-Arie, R; Cohen, C J; Eitan, Y; Shelef, L; Hallerman, E M; Kashi, Y

2000-01-01

Computer-based genome-wide screening of the DNA sequence of Escherichia coli strain K12 revealed tens of thousands of tandem simple sequence repeat (SSR) tracts, with motifs ranging from 1 to 6 nucleotides. SSRs were well distributed throughout the genome. Mononucleotide SSRs were over-represented in noncoding regions and under-represented in open reading frames (ORFs). Nucleotide composition of mono- and dinucleotide SSRs, both in ORFs and in noncoding regions, differed from that of the genomic region in which they occurred, with 93% of all mononucleotide SSRs proving to be of A or T. Computer-based analysis of the fine position of every SSR locus in the noncoding portion of the genome relative to downstream ORFs showed SSRs located in areas that could affect gene regulation. DNA sequences at 14 arbitrarily chosen SSR tracts were compared among E. coli strains. Polymorphisms of SSR copy number were observed at four of seven mononucleotide SSR tracts screened, with all polymorphisms occurring in noncoding regions. SSR polymorphism could prove important as a genome-wide source of variation, both for practical applications (including rapid detection, strain identification, and detection of loci affecting key phenotypes) and for evolutionary adaptation of microbes.

Loss of function of 1-FEH IIb has more impact on post-harvest inulin degradation in Cichorium intybus than copy number variation of its close paralog 1-FEH IIa

PubMed Central

Dauchot, Nicolas; Raulier, Pierre; Maudoux, Olivier; Notté, Christine; Draye, Xavier; Van Cutsem, Pierre

2015-01-01

Key Message: The loss of mini-exon 2 in the 1-FEH IIb glycosyl-hydrolase results in a putative non-functional allele. This loss of function has a strong impact on the susceptibility to post-harvest inulin depolymerization. Significant variation of copy number was identified in its close paralog 1-FEH IIa, but no quantitative effect of copy number on carbohydrates-related phenotypes was detected. Inulin polyfructan is the second most abundant storage carbohydrate in flowering plants. After harvest, it is depolymerized by fructan exohydrolases (FEHs) as an adaptive response to end-season cold temperatures. In chicory, the intensity of this depolymerization differs between cultivars but also between individuals within a cultivar. Regarding this phenotypic variability, we recently identified statistically significant associations between inulin degradation and genetic polymorphisms located in three FEHs. We present here new results of a systematic analysis of copy number variation (CNV) in five key members of the chicory (Cichorium intybus) GH32 multigenic family, including three FEH genes and the two inulin biosynthesis genes: 1-SST and 1-FFT. qPCR analysis identified a significant variability of relative copy number only in the 1-FEH IIa gene. However, this CNV had no quantitative effect. Instead, cloning of the full length gDNA of a close paralogous sequence (1-FEH IIb) identified a 1028 bp deletion in lines less susceptible to post-harvest inulin depolymerization. This region comprises a 9 bp mini-exon containing one of the three conserved residues of the active site. This results in a putative non-functional 1-FEH IIb allele and an observed lower inulin depolymerization. Extensive genotyping confirmed that the loss of mini-exon 2 in 1-FEH IIb and the previously identified 47 bp duplication located in the 3′UTR of 1-FEH IIa belong to a single haplotype, both being statistically associated with reduced susceptibility to post-harvest inulin depolymerization. Emergence of these haplotypes is discussed. PMID:26157446

Technical note: a novel method for routine genotyping of horse coat color gene polymorphisms.

PubMed

Royo, L J; Fernández, I; Azor, P J; Alvarez, I; Pérez-Pardal, L; Goyache, F

2008-06-01

The aim of this note is to describe a reliable, fast, and cost-effective real-time PCR method for routine genotyping of mutations responsible for most coat color variation in horses. The melanocortin-1 receptor, Agouti-signaling peptide, and membrane-associated transporter protein alleles were simultaneously determined using 2 PCR protocols. The assay described here is an alternative method for routine genotyping of a defined number of polymorphisms. Allelic variants are detected in real time and no post-PCR manipulations are required, therefore limiting costs and possible carryover contamination. Data can be copied to a Microsoft Excel spreadsheet for semiautomatic determination of the genotype using a macro freely available at http://www.igijon.com/personales/fgoyache/software_i.htm (last accessed February 26, 2007). The performance of the method is demonstrated on 156 Spanish Purebred horses.

A common copy number variation polymorphism in the CNTNAP2 gene: sexual dimorphism in association with healthy aging and disease.

PubMed

Iakoubov, Leonid; Mossakowska, Malgorzata; Szwed, Malgorzata; Puzianowska-Kuznicka, Monika

2015-01-01

New therapeutic targets are needed to fight aging-related diseases and increase life span. A new female-specific association with diseases and limited survival past 80 years was recently reported for a copy number variation (CNV) in the CNTNAP4 gene from the neurexin superfamily. We asked whether there are CNVs that are associated with aging phenotypes within other genes from the neurexin superfamily and whether this association is sex specific. Select CNV polymorphisms were genotyped with proprietary TaqMan qPCR assays. A case/control study, in which a group of 81- to 90-year-old community-dwelling Caucasians with no chronic diseases (case) was compared to a similar control group of 65- to 75-year-olds, revealed a negative association with healthy aging for the ins allele of common esv11910 CNV in the CNTNAP2 gene (n = 388; OR = 0.29, 95% CI: 0.14-0.59, p = 0.0004 for males, and OR = 0.82, 95% CI: 0.42-1.57, p = 0.625 for females). This male-specific association was validated in a study of an independent group of 76- to 80-year-olds. To look for a corresponding positive association of the allele with aging-related diseases, two case subgroups of 81- to 90-year-olds, one composed of individuals with cognitive impairment and the other with various diseases not directly related to the nervous system, such as cardiovascular diseases, etc., were compared to a healthy control subgroup of the same age. A positive male-specific association was found for both cases (OR = 2.75, p = 0.008 for association with cognitive impairment, and OR = 3.18, p = 0.002 for other diseases combined). A new male-specific association with aging is reported for a CNV in the CNTNAP2 gene. The polymorphism might be useful for diagnosing individual genetic predispositions to healthy aging versus aging complicated by chronic diseases. © 2014 S. Karger AG, Basel.

An association analysis between mitochondrial DNA content, G10398A polymorphism, HPV infection, and the prognosis of cervical cancer in the Chinese Han population.

PubMed

Feng, Dali; Xu, Hui; Li, Xin; Wei, Yuehua; Jiang, Huangang; Xu, Hong; Luo, Aihua; Zhou, Fuxiang

2016-04-01

The aim was to analyze quantitative (mitochondrial DNA (mtDNA) content) and qualitative (G10398A polymorphism) mtDNA alterations as well as human papillomavirus (HPV) infection in cervical cancer prognosis. One hundred and twenty-two cases of formalin-fixed paraffin-embedded cervical carcinoma specimens were collected from the Yichang Tumor Hospital and Zhongnan Hospital of Wuhan University in the recent 10 years together with medical records. A quantitative real-time PCR (RT-PCR) was used to determine the copy number of the mitochondrial DNA and HPV expression levels. G10398A polymorphism was determined by PCR-RFLP assay. The overall survival of patients with higher mtDNA content was significantly reduced compared with lower mtDNA content patients (P = 0.029). But there was no difference of prognosis between the mtDNA 10398 A allele and G allele. However, the Kaplan-Meier survival curve illustrated a significantly reduced overall survival in the patients with 10398A plus high mtDNA copy number compared with the other groups (P < 0.05). Although no association between HPV expression level and cervical cancer prognosis was observed, 10398A got increased mtDNA content compared with 10398G (P < 0.05) and 10398G displayed an increased HPV-positive rate compared with 10398A. Furthermore, HPV-18 and mtDNA content were positively related in the younger subgroup (≤45 years) (correlation coefficient = 0.456, P = 0.022). This study indicated that mtDNA content and HPV infection status are associated with cervical cancer prognosis. High mitochondrial DNA content plus 10398 A may be a marker of poor prognosis in cervical cancer. And mtDNA variation may potentially influence the predisposition to HPV infection and cervical carcinogenesis.

A Complex 6p25 Rearrangement in a Child With Multiple Epiphyseal Dysplasia

PubMed Central

Bedoyan, Jirair K.; Lesperance, Marci M.; Ackley, Todd; Iyer, Ramaswamy K.; Innis, Jeffrey W.; Misra, Vinod K.

2015-01-01

Genomic rearrangements are increasingly recognized as important contributors to human disease. Here we report on an 11½-year-old child with myopia, Duane retraction syndrome, bilateral mixed hearing loss, skeletal anomalies including multiple epiphyseal dysplasia, and global developmental delay, and a complex 6p25 genomic rearrangement. We have employed oligonucleotide-based comparative genomic hybridization arrays (aCGH) of different resolutions (44 and 244K) as well as a 1 M single nucleotide polymorphism (SNP) array to analyze this complex rearrangement. Our analyses reveal a complex rearrangement involving a ~2.21 Mb interstitial deletion, a ~240 kb terminal deletion, and a 70–80 kb region in between these two deletions that shows maintenance of genomic copy number. The interstitial deletion contains eight known genes, including three Forkhead box containing (FOX) transcription factors (FOXQ1, FOXF2, and FOXC1). The region maintaining genomic copy number partly overlaps the dual specificity protein phosphatase 22 (DUSP22) gene. Array analyses suggest a homozygous loss of genomic material at the 5′ end of DUSP22, which was corroborated using TaqMan® copy number analysis. It is possible that this homozygous genomic loss may render both copies of DUSP22 or its products non-functional. Our analysis suggests a rearrangement mechanism distinct from a previously reported replication-based error-prone mechanism without template switching for a specific 6p25 rearrangement with a 1.22 Mb interstitial deletion. Our study demonstrates the utility and limitations of using oligonucleotide-based aCGH and SNP array technologies of increasing resolutions in order to identify complex DNA rearrangements and gene disruptions. PMID:21204225

Characterization of the COL2A1 VNTR polymorphism

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

Berg, E.S.; Olaisen, B.

1993-05-01

The variable number of tandem repeat (VNTR) region 3{prime} to the collagen type II gene (COL2A1) was amplified in vitro by the polymerase chain reaction. Subsequent high-resolution gel electrophoresis showed that the five earlier reported alleles could be further subtyped. A total of 17 allelic variants with a heterozygosity of 73.0% were found in 202 unrelated Norwegians. DNA sequencing of 19 COL2A1 alleles has been performed. The internal organization of the VNTR was common for all alleles, as previously shown for a few alleles. Moreover, the polymorphism in the COL2A1 locus is mainly due to variation in the numbers ofmore » copies of two repeat units, containing 34 and 31 bp, respectively, and/or to small deletions in either of the two units. DNA sequencing of alleles with the same electrophoretic size revealed no heterogeneity such as an alternating order of the different units, a feature that might have been expected to be the result of unequal crossing-over events. The observed ordered structure of the VNTR and the possibility of single-stranded DNA from the cores in the VNTR forming hairpins and loops suggest that the COL2A1 polymorphism may have evolved mainly by replication slippage mechanisms. 23 refs., 2 figs., 3 tabs.« less

Copy number variability in Parkinson's disease: assembling the puzzle through a systems biology approach.

PubMed

La Cognata, Valentina; Morello, Giovanna; D'Agata, Velia; Cavallaro, Sebastiano

2017-01-01

Parkinson's disease (PD), the second most common progressive neurodegenerative disorder of aging, was long believed to be a non-genetic sporadic origin syndrome. The proof that several genetic loci are responsible for rare Mendelian forms has represented a revolutionary breakthrough, enabling to reveal molecular mechanisms underlying this debilitating still incurable condition. While single nucleotide polymorphisms (SNPs) and small indels constitute the most commonly investigated DNA variations accounting for only a limited number of PD cases, larger genomic molecular rearrangements have emerged as significant PD-causing mutations, including submicroscopic Copy Number Variations (CNVs). CNVs constitute a prevalent source of genomic variations and substantially participate in each individual's genomic makeup and phenotypic outcome. However, the majority of genetic studies have focused their attention on single candidate-gene mutations or on common variants reaching a significant statistical level of acceptance. This gene-centric approach is insufficient to uncover the genetic background of polygenic multifactorial disorders like PD, and potentially masks rare individual CNVs that all together might contribute to disease development or progression. In this review, we will discuss literature and bioinformatic data describing the involvement of CNVs on PD pathobiology. We will analyze the most frequent copy number changes in familiar PD genes and provide a "systems biology" overview of rare individual rearrangements that could functionally act on commonly deregulated molecular pathways. Assessing the global genome-wide burden of CNVs in PD patients may reveal new disease-related molecular mechanisms, and open the window to a new possible genetic scenario in the unsolved PD puzzle.

Cytokine signaling pathway polymorphisms and AIDS-related non-Hodgkin lymphoma risk in the Multicenter AIDS Cohort Study

PubMed Central

Wong, Hui-Lee; Breen, Elizabeth C.; Pfeiffer, Ruth M.; Aissani, Brahim; Martinson, Jeremy J.; Margolick, Joseph B.; Kaslow, Richard A.; Jacobson, Lisa P.; Ambinder, Richard F.; Chanock, Stephen; Martínez-Maza, Otoniel; Rabkin, Charles S.

2014-01-01

Cytokine stimulation of B-cell proliferation may be an important etiologic mechanism for acquired immunodeficiency syndrome (AIDS)-related non-Hodgkin lymphoma (NHL). The Epstein-Barr virus may be a co-factor, particularly for primary central nervous system (CNS) tumors, which are uniformly EBV-positive in the setting of AIDS. Thus, we examined associations of genetic variation in IL10 and related cytokine signaling molecules (IL10RA, CXCL12, IL13, IL4, IL4R, CCL5 and BCL6) with AIDS-related NHL risk and evaluated differences between primary CNS and systemic tumors. We compared 160 Multicenter AIDS Cohort Study (MACS) participants with incident lymphomas, of which 90 followed another AIDS diagnosis, to HIV-1-seropositive controls matched on duration of lymphoma-free survival post-HIV-1 infection (N=160) or post-AIDS diagnosis (N=90). We fit conditional logistic regression models to estimate odds ratios (ORs) and 95 percent confidence intervals (95%CIs). Carriage of at least one copy of the T allele for the IL10 rs1800871 (as compared to no copies) was associated with decreased AIDS-NHL risk specific to lymphomas arising from the CNS (CC vs. CT/TT: OR=0.3; 95%CI: 0.1, 0.7) but not systemically (CC vs. CT/TT: OR=1.0; 95%CI: 0.5, 1.9) (Pheterogeneity=0.03). Carriage of two copies of the “low IL10” haplotype rs1800896_A/rs1800871_T/rs1800872_A was associated with decreased lymphoma risk that varied by number of copies (Ptrend=0.02). None of the ORs for the other studied polymorphisms was significantly different from 1.0. Excessive IL10 response to HIV-1 infection may be associated with increased risk of NHL, particularly in the CNS. IL10 dysregulation may be an important etiologic pathway for EBV-related lymphomagenesis. PMID:20299965

The Diversity of REcent and Ancient huMan (DREAM): A New Microarray for Genetic Anthropology and Genealogy, Forensics, and Personalized Medicine

PubMed Central

Yusuf, Leeban; Anderson, Ainan I J; Pirooznia, Mehdi; Arnellos, Dimitrios; Vilshansky, Gregory; Ercal, Gunes; Lu, Yontao; Webster, Teresa; Baird, Michael L; Esposito, Umberto

2017-01-01

Abstract The human population displays wide variety in demographic history, ancestry, content of DNA derived from hominins or ancient populations, adaptation, traits, copy number variation, drug response, and more. These polymorphisms are of broad interest to population geneticists, forensics investigators, and medical professionals. Historically, much of that knowledge was gained from population survey projects. Although many commercial arrays exist for genome-wide single-nucleotide polymorphism genotyping, their design specifications are limited and they do not allow a full exploration of biodiversity. We thereby aimed to design the Diversity of REcent and Ancient huMan (DREAM)—an all-inclusive microarray that would allow both identification of known associations and exploration of standing questions in genetic anthropology, forensics, and personalized medicine. DREAM includes probes to interrogate ancestry informative markers obtained from over 450 human populations, over 200 ancient genomes, and 10 archaic hominins. DREAM can identify 94% and 61% of all known Y and mitochondrial haplogroups, respectively, and was vetted to avoid interrogation of clinically relevant markers. To demonstrate its capabilities, we compared its FST distributions with those of the 1000 Genomes Project and commercial arrays. Although all arrays yielded similarly shaped (inverse J) FST distributions, DREAM’s autosomal and X-chromosomal distributions had the highest mean FST, attesting to its ability to discern subpopulations. DREAM performances are further illustrated in biogeographical, identical by descent, and copy number variation analyses. In summary, with approximately 800,000 markers spanning nearly 2,000 genes, DREAM is a useful tool for genetic anthropology, forensic, and personalized medicine studies. PMID:29165562

Allele-Specific HLA Loss and Immune Escape in Lung Cancer Evolution.

PubMed

McGranahan, Nicholas; Rosenthal, Rachel; Hiley, Crispin T; Rowan, Andrew J; Watkins, Thomas B K; Wilson, Gareth A; Birkbak, Nicolai J; Veeriah, Selvaraju; Van Loo, Peter; Herrero, Javier; Swanton, Charles

2017-11-30

Immune evasion is a hallmark of cancer. Losing the ability to present neoantigens through human leukocyte antigen (HLA) loss may facilitate immune evasion. However, the polymorphic nature of the locus has precluded accurate HLA copy-number analysis. Here, we present loss of heterozygosity in human leukocyte antigen (LOHHLA), a computational tool to determine HLA allele-specific copy number from sequencing data. Using LOHHLA, we find that HLA LOH occurs in 40% of non-small-cell lung cancers (NSCLCs) and is associated with a high subclonal neoantigen burden, APOBEC-mediated mutagenesis, upregulation of cytolytic activity, and PD-L1 positivity. The focal nature of HLA LOH alterations, their subclonal frequencies, enrichment in metastatic sites, and occurrence as parallel events suggests that HLA LOH is an immune escape mechanism that is subject to strong microenvironmental selection pressures later in tumor evolution. Characterizing HLA LOH with LOHHLA refines neoantigen prediction and may have implications for our understanding of resistance mechanisms and immunotherapeutic approaches targeting neoantigens. VIDEO ABSTRACT. Copyright © 2017 The Francis Crick Institute. Published by Elsevier Inc. All rights reserved.

Differences in aggressive behavior and DNA copy number variants between BALB/cJ and BALB/cByJ substrains.

PubMed

Velez, Lady; Sokoloff, Greta; Miczek, Klaus A; Palmer, Abraham A; Dulawa, Stephanie C

2010-03-01

Some BALB/c substrains exhibit different levels of aggression. We compared aggression levels between male BALB/cJ and BALB/cByJ substrains using the resident intruder paradigm. These substrains were also assessed in other tests of emotionality and information processing including the open field, forced swim, fear conditioning, and prepulse inhibition tests. We also evaluated single nucleotide polymorphisms (SNPs) previously reported between these BALB/c substrains. Finally, we compared BALB/cJ and BALB/cByJ mice for genomic deletions or duplications, collectively termed copy number variants (CNVs), to identify candidate genes that might underlie the observed behavioral differences. BALB/cJ mice showed substantially higher aggression levels than BALB/cByJ mice; however, only minor differences in other behaviors were observed. None of the previously reported SNPs were verified. Eleven CNV regions were identified between the two BALB/c substrains. Our findings identify a robust difference in aggressive behavior between BALB/cJ and BALB/cByJ substrains, which could be the result of the identified CNVs.

Genome-wide screening identifies a KCNIP1 copy number variant as a genetic predictor for atrial fibrillation

PubMed Central

Tsai, Chia-Ti; Hsieh, Chia-Shan; Chang, Sheng-Nan; Chuang, Eric Y.; Ueng, Kwo-Chang; Tsai, Chin-Feng; Lin, Tsung-Hsien; Wu, Cho-Kai; Lee, Jen-Kuang; Lin, Lian-Yu; Wang, Yi-Chih; Yu, Chih-Chieh; Lai, Ling-Ping; Tseng, Chuen-Den; Hwang, Juey-Jen; Chiang, Fu-Tien; Lin, Jiunn-Lee

2016-01-01

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia. Previous genome-wide association studies had identified single-nucleotide polymorphisms in several genomic regions to be associated with AF. In human genome, copy number variations (CNVs) are known to contribute to disease susceptibility. Using a genome-wide multistage approach to identify AF susceptibility CNVs, we here show a common 4,470-bp diallelic CNV in the first intron of potassium interacting channel 1 gene (KCNIP1) is strongly associated with AF in Taiwanese populations (odds ratio=2.27 for insertion allele; P=6.23 × 10−24). KCNIP1 insertion is associated with higher KCNIP1 mRNA expression. KCNIP1-encoded protein potassium interacting channel 1 (KCHIP1) is physically associated with potassium Kv channels and modulates atrial transient outward current in cardiac myocytes. Overexpression of KCNIP1 results in inducible AF in zebrafish. In conclusions, a common CNV in KCNIP1 gene is a genetic predictor of AF risk possibly pointing to a functional pathway. PMID:26831368

The Role of Constitutional Copy Number Variants in Breast Cancer

PubMed Central

Walker, Logan C.; Wiggins, George A.R.; Pearson, John F.

2015-01-01

Constitutional copy number variants (CNVs) include inherited and de novo deviations from a diploid state at a defined genomic region. These variants contribute significantly to genetic variation and disease in humans, including breast cancer susceptibility. Identification of genetic risk factors for breast cancer in recent years has been dominated by the use of genome-wide technologies, such as single nucleotide polymorphism (SNP)-arrays, with a significant focus on single nucleotide variants. To date, these large datasets have been underutilised for generating genome-wide CNV profiles despite offering a massive resource for assessing the contribution of these structural variants to breast cancer risk. Technical challenges remain in determining the location and distribution of CNVs across the human genome due to the accuracy of computational prediction algorithms and resolution of the array data. Moreover, better methods are required for interpreting the functional effect of newly discovered CNVs. In this review, we explore current and future application of SNP array technology to assess rare and common CNVs in association with breast cancer risk in humans. PMID:27600231

Copy Number Variations in Tilapia Genomes.

PubMed

Li, Bi Jun; Li, Hong Lian; Meng, Zining; Zhang, Yong; Lin, Haoran; Yue, Gen Hua; Xia, Jun Hong

2017-02-01

Discovering the nature and pattern of genome variation is fundamental in understanding phenotypic diversity among populations. Although several millions of single nucleotide polymorphisms (SNPs) have been discovered in tilapia, the genome-wide characterization of larger structural variants, such as copy number variation (CNV) regions has not been carried out yet. We conducted a genome-wide scan for CNVs in 47 individuals from three tilapia populations. Based on 254 Gb of high-quality paired-end sequencing reads, we identified 4642 distinct high-confidence CNVs. These CNVs account for 1.9% (12.411 Mb) of the used Nile tilapia reference genome. A total of 1100 predicted CNVs were found overlapping with exon regions of protein genes. Further association analysis based on linear model regression found 85 CNVs ranging between 300 and 27,000 base pairs significantly associated to population types (R 2  > 0.9 and P > 0.001). Our study sheds first insights on genome-wide CNVs in tilapia. These CNVs among and within tilapia populations may have functional effects on phenotypes and specific adaptation to particular environments.

Copy Number Variants and Exome Sequencing Analysis in Six Pairs of Chinese Monozygotic Twins Discordant for Congenital Heart Disease.

PubMed

Xu, Yuejuan; Li, Tingting; Pu, Tian; Cao, Ruixue; Long, Fei; Chen, Sun; Sun, Kun; Xu, Rang

2017-12-01

Congenital heart disease (CHD) is one of the most common birth defects. More than 200 susceptibility loci have been identified for CHDs, yet a large part of the genetic risk factors remain unexplained. Monozygotic (MZ) twins are thought to be completely genetically identical; however, discordant phenotypes have been found in MZ twins. Recent studies have demonstrated genetic differences between MZ twins. We aimed to test whether copy number variants (CNVs) and/or genetic mutation differences play a role in the etiology of CHDs by using single nucleotide polymorphism (SNP) genotyping arrays and whole exome sequencing of twin pairs discordant for CHDs. Our goal was to identify mutations present only in the affected twins, which could identify novel candidates for CHD susceptibility loci. We present a comprehensive analysis for the CNVs and genetic mutation results of the selected individuals but detected no consistent differences within the twin pairs. Our study confirms that chromosomal structure or genetic mutation differences do not seem to play a role in the MZ twins discordant for CHD.

Single-Nucleotide Polymorphism and Copy Number Variation of the Multidrug Resistance-1 Locus of Plasmodium vivax: Local and Global Patterns

PubMed Central

Vargas-Rodríguez, Rosa del Carmen Miluska; da Silva Bastos, Melissa; Menezes, Maria José; Orjuela-Sánchez, Pamela; Ferreira, Marcelo U.

2012-01-01

Emerging resistance to chloroquine (CQ) poses a major challenge for Plasmodium vivax malaria control, and nucleotide substitutions and copy number variation in the P. vivax multidrug resistance 1 (pvmdr-1) locus, which encodes a digestive vacuole membrane transporter, may modulate this phenotype. We describe patterns of genetic variation in pvmdr-1 alleles from Acre and Amazonas in northwestern Brazil, and compare then with those reported in other malaria-endemic regions. The pvmdr-1 mutation Y976F, which is associated with CQ resistance in Southeast Asia and Oceania, remains rare in northwestern Brazil (1.8%) and its prevalence mirrors that of CQ resistance worldwide. Gene amplification of pvmdr-1, which is associated with mefloquine resistance but increased susceptibility to CQ, remains relatively rare in northwestern Brazil (0.9%) and globally (< 4%), but became common (> 10%) in Tak Province, Thailand, possibly because of drug-mediated selection. The global database we have assembled provides a baseline for further studies of genetic variation in pvmdr-1 and drug resistance in P. vivax malaria. PMID:22949516

Single-nucleotide polymorphism and copy number variation of the multidrug resistance-1 locus of Plasmodium vivax: local and global patterns.

PubMed

Vargas-Rodríguez, Rosa del Carmen Miluska; da Silva Bastos, Melissa; Menezes, Maria José; Orjuela-Sánchez, Pamela; Ferreira, Marcelo U

2012-11-01

Emerging resistance to chloroquine (CQ) poses a major challenge for Plasmodium vivax malaria control, and nucleotide substitutions and copy number variation in the P. vivax multidrug resistance 1 (pvmdr-1) locus, which encodes a digestive vacuole membrane transporter, may modulate this phenotype. We describe patterns of genetic variation in pvmdr-1 alleles from Acre and Amazonas in northwestern Brazil, and compare then with those reported in other malaria-endemic regions. The pvmdr-1 mutation Y976F, which is associated with CQ resistance in Southeast Asia and Oceania, remains rare in northwestern Brazil (1.8%) and its prevalence mirrors that of CQ resistance worldwide. Gene amplification of pvmdr-1, which is associated with mefloquine resistance but increased susceptibility to CQ, remains relatively rare in northwestern Brazil (0.9%) and globally (< 4%), but became common (> 10%) in Tak Province, Thailand, possibly because of drug-mediated selection. The global database we have assembled provides a baseline for further studies of genetic variation in pvmdr-1 and drug resistance in P. vivax malaria.

Increased pfmdr1 gene copy number and the decline in pfcrt and pfmdr1 resistance alleles in Ghanaian Plasmodium falciparum isolates after the change of anti-malarial drug treatment policy.

PubMed

Duah, Nancy O; Matrevi, Sena A; de Souza, Dziedzom K; Binnah, Daniel D; Tamakloe, Mary M; Opoku, Vera S; Onwona, Christiana O; Narh, Charles A; Quashie, Neils B; Abuaku, Benjamin; Duplessis, Christopher; Kronmann, Karl C; Koram, Kwadwo A

2013-10-30

With the introduction of artemisinin-based combination therapy (ACT) in 2005, monitoring of anti-malarial drug efficacy, which includes the use of molecular tools to detect known genetic markers of parasite resistance, is important for first-hand information on the changes in parasite susceptibility to drugs in Ghana. This study investigated the Plasmodium falciparum multidrug resistance gene (pfmdr1) copy number, mutations and the chloroquine resistance transporter gene (pfcrt) mutations in Ghanaian isolates collected in seven years to detect the trends in prevalence of mutations. Archived filter paper blood blots collected from children aged below five years with uncomplicated malaria in 2003-2010 at sentinel sites were used. Using quantitative real-time polymerase chain reaction (qRT-PCR), 756 samples were assessed for pfmdr1 gene copy number. PCR and restriction fragment length polymorphism (RFLP) were used to detect alleles of pfmdr1 86 in 1,102 samples, pfmdr1 184, 1034, 1042 and 1246 in 832 samples and pfcrt 76 in 1,063 samples. Merozoite surface protein 2 (msp2) genotyping was done to select monoclonal infections for copy number analysis. The percentage of isolates with increased pfmdr1 copy number were 4, 27, 9, and 18% for 2003-04, 2005-06, 2007-08 and 2010, respectively. Significant increasing trends for prevalence of pfmdr1 N86 (×(2) = 96.31, p <0.001) and pfcrt K76 (×(2) = 64.50, p <0.001) and decreasing trends in pfmdr1 Y86 (x(2) = 38.52, p <0.001) and pfcrt T76 (x(2) = 43.49, p <0.001) were observed from 2003-2010. The pfmdr1 F184 and Y184 prevalence showed an increasing and decreasing trends respectively but were not significant (×(2) = 7.39,p=0.060; ×(2) = 7.49, p = 0.057 respectively). The pfmdr1 N86-F184-D1246 haplotype, which is alleged to be selected by artemether-lumefantrine showed a significant increasing trend (×(2) = 20.75, p < 0.001). Increased pfmdr1 gene copy number was observed in the isolates analysed and this finding has implications for the use of ACT in the country although no resistance has been reported. The decreasing trend in the prevalence of chloroquine resistance markers after change of treatment policy presents the possibility for future introduction of chloroquine as prophylaxis for malaria risk groups such as children and pregnant women in Ghana.

Somatic Mutation Allelic Ratio Test Using ddPCR (SMART-ddPCR): An Accurate Method for Assessment of Preferential Allelic Imbalance in Tumor DNA.

PubMed

de Smith, Adam J; Walsh, Kyle M; Hansen, Helen M; Endicott, Alyson A; Wiencke, John K; Metayer, Catherine; Wiemels, Joseph L

2015-01-01

The extent to which heritable genetic variants can affect tumor development has yet to be fully elucidated. Tumor selection of single nucleotide polymorphism (SNP) risk alleles, a phenomenon called preferential allelic imbalance (PAI), has been demonstrated in some cancer types. We developed a novel application of digital PCR termed Somatic Mutation Allelic Ratio Test using Droplet Digital PCR (SMART-ddPCR) for accurate assessment of tumor PAI, and have applied this method to test the hypothesis that heritable SNPs associated with childhood acute lymphoblastic leukemia (ALL) may demonstrate tumor PAI. These SNPs are located at CDKN2A (rs3731217) and IKZF1 (rs4132601), genes frequently lost in ALL, and at CEBPE (rs2239633), ARID5B (rs7089424), PIP4K2A (rs10764338), and GATA3 (rs3824662), genes located on chromosomes gained in high-hyperdiploid ALL. We established thresholds of AI using constitutional DNA from SNP heterozygotes, and subsequently measured allelic copy number in tumor DNA from 19-142 heterozygote samples per SNP locus. We did not find significant tumor PAI at these loci, though CDKN2A and IKZF1 SNPs showed a trend towards preferential selection of the risk allele (p = 0.17 and p = 0.23, respectively). Using a genomic copy number control ddPCR assay, we investigated somatic copy number alterations (SCNA) underlying AI at CDKN2A and IKZF1, revealing a complex range of alterations including homozygous and hemizygous deletions and copy-neutral loss of heterozygosity, with varying degrees of clonality. Copy number estimates from ddPCR showed high agreement with those from multiplex ligation-dependent probe amplification (MLPA) assays. We demonstrate that SMART-ddPCR is a highly accurate method for investigation of tumor PAI and for assessment of the somatic alterations underlying AI. Furthermore, analysis of publicly available data from The Cancer Genome Atlas identified 16 recurrent SCNA loci that contain heritable cancer risk SNPs associated with a matching tumor type, and which represent candidate PAI regions warranting further investigation.

Sex bias in copy number variation of olfactory receptor gene family depends on ethnicity.

PubMed

Shadravan, Farideh

2013-01-01

Gender plays a pivotal role in the human genetic identity and is also manifested in many genetic disorders particularly mental retardation. In this study its effect on copy number variation (CNV), known to cause genetic disorders was explored. As the olfactory receptor (OR) repertoire comprises the largest human gene family, it was selected for this study, which was carried out within and between three populations, derived from 150 individuals from the 1000 Genome Project. Analysis of 3872 CNVs detected among 791 OR loci, in which 307 loci showed CNV, revealed the following novel findings: Sex bias in CNV was significantly more prevalent in uncommon than common CNV variants of OR pseudogenes, in which the male genome showed more CNVs; and in one-copy number loss compared to complete deletion of OR pseudogenes; both findings implying a more recent evolutionary role for gender. Sex bias in copy number gain was also detected. Another novel finding was that the observed sex bias was largely dependent on ethnicity and was in general absent in East Asians. Using a CNV public database for sick children (International Standard Cytogenomic Array Consortium) the application of these findings for improving clinical molecular diagnostics is discussed by showing an example of sex bias in CNV among kids with autism. Additional clinical relevance is discussed, as the most polymorphic CNV-enriched OR cluster in the human genome, located on chr 15q11.2, is found near the Prader-Willi syndrome/Angelman syndrome bi-directionally imprinted region associated with two well-known mental retardation syndromes. As olfaction represents the primitive cognition in most mammals, arguably in competition with the development of a larger brain, the extensive retention of OR pseudogenes in females of this study, might point to a parent-of-origin indirect regulatory role for OR pseudogenes in the embryonic development of human brain. Thus any perturbation in the temporal regulation of olfactory system could lead to developmental delay disorders including mental retardation.

Molecular basis of non-syndromic hypospadias: systematic mutation screening and genome-wide copy-number analysis of 62 patients.

PubMed

Kon, M; Suzuki, E; Dung, V C; Hasegawa, Y; Mitsui, T; Muroya, K; Ueoka, K; Igarashi, N; Nagasaki, K; Oto, Y; Hamajima, T; Yoshino, K; Igarashi, M; Kato-Fukui, Y; Nakabayashi, K; Hayashi, K; Hata, K; Matsubara, Y; Moriya, K; Ogata, T; Nonomura, K; Fukami, M

2015-03-01

What percentage of cases with non-syndromic hypospadias can be ascribed to mutations in known causative/candidate/susceptibility genes or submicroscopic copy-number variations (CNVs) in the genome? Monogenic and digenic mutations in known causative genes and cryptic CNVs account for >10% of cases with non-syndromic hypospadias. While known susceptibility polymorphisms appear to play a minor role in the development of this condition, further studies are required to validate this observation. Fifteen causative, three candidate, and 14 susceptible genes, and a few submicroscopic CNVs have been implicated in non-syndromic hypospadias. Systematic mutation screening and genome-wide copy-number analysis of 62 patients. The study group consisted of 57 Japanese and five Vietnamese patients with non-syndromic hypospadias. Systematic mutation screening was performed for 25 known causative/candidate/susceptibility genes using a next-generation sequencer. Functional consequences of nucleotide alterations were assessed by in silico assays. The frequencies of polymorphisms in the patient group were compared with those in the male general population. CNVs were analyzed by array-based comparative genomic hybridization and characterized by fluorescence in situ hybridization. Seven of 62 patients with anterior or posterior hypospadias carried putative pathogenic mutations, such as hemizygous mutations in AR, a heterozygous mutation in BNC2, and homozygous mutations in SRD5A2 and HSD3B2. Two of the seven patients had mutations in multiple genes. We did not find any rare polymorphisms that were abundant specifically in the patient group. One patient carried mosaic dicentric Y chromosome. The patient group consisted solely of Japanese and Vietnamese individuals and clinical and hormonal information of the patients remained rather fragmentary. In addition, mutation analysis focused on protein-altering substitutions. Our data provide evidence that pathogenic mutations can underlie both mild and severe hypospadias and that HSD3B2 mutations cause non-syndromic hypospadias as a sole clinical manifestation. Most importantly, this is the first report documenting possible oligogenicity of non-syndromic hypospadias. This study was funded by the Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology; by the Grant-in-Aid from the Japan Society for the Promotion of Science; by the Grants from the Ministry of Health, Labour and Welfare, from the National Center for Child Health and Development and from the Takeda Foundation. The authors have no competing interests to disclose. Not applicable. © The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Cortisol responses to chronic stress in adult macaques: moderation by a polymorphism in the serotonin transporter gene.

PubMed

Qin, Dongdong; Rizak, Joshua; Feng, Xiaoli; Yang, Shangchuan; Yang, Lichuan; Fan, Xiaona; Lü, Longbao; Chen, Lin; Hu, Xintian

2015-02-01

Accumulating evidence has shown that a polymorphism in the promoter region of the serotonin transporter gene (5-HTTLPR) moderates the association between stress and depressive symptoms. However, the exact etiologies underlying this moderation are not well understood. Here it is reported that among adult female rhesus macaques, an orthologous polymorphism (rh5-HTTLPR) exerted an influence on cortisol responses to chronic stress. It was found that females with two copies of the short allele were associated with increased cortisol responses to chronic stress in comparison to their counterparts who have one or two copies of the long allele. In the absence of stress, no differences related to genotype were observed in these females. This genetic moderation was found without a genetic influence on exposure to stressful situations. Rather it was found to be a genetic modulation of cortisol responses to chronic stress. These findings indicate that the rh5-HTTLPR polymorphism is closely related to hypothalamus-pituitary-adrenal (HPA) axis reactivity, which may increase susceptibility to depression in females with low serotonin transporter efficiency and a history of stress. Copyright © 2014 Elsevier B.V. All rights reserved.

Genomic Hypomethylation in the Human Germline Associates with Selective Structural Mutability in the Human Genome

PubMed Central

Li, Jian; Harris, R. Alan; Cheung, Sau Wai; Coarfa, Cristian; Jeong, Mira; Goodell, Margaret A.; White, Lisa D.; Patel, Ankita; Kang, Sung-Hae; Shaw, Chad; Chinault, A. Craig; Gambin, Tomasz; Gambin, Anna; Lupski, James R.; Milosavljevic, Aleksandar

2012-01-01

The hotspots of structural polymorphisms and structural mutability in the human genome remain to be explained mechanistically. We examine associations of structural mutability with germline DNA methylation and with non-allelic homologous recombination (NAHR) mediated by low-copy repeats (LCRs). Combined evidence from four human sperm methylome maps, human genome evolution, structural polymorphisms in the human population, and previous genomic and disease studies consistently points to a strong association of germline hypomethylation and genomic instability. Specifically, methylation deserts, the ∼1% fraction of the human genome with the lowest methylation in the germline, show a tenfold enrichment for structural rearrangements that occurred in the human genome since the branching of chimpanzee and are highly enriched for fast-evolving loci that regulate tissue-specific gene expression. Analysis of copy number variants (CNVs) from 400 human samples identified using a custom-designed array comparative genomic hybridization (aCGH) chip, combined with publicly available structural variation data, indicates that association of structural mutability with germline hypomethylation is comparable in magnitude to the association of structural mutability with LCR–mediated NAHR. Moreover, rare CNVs occurring in the genomes of individuals diagnosed with schizophrenia, bipolar disorder, and developmental delay and de novo CNVs occurring in those diagnosed with autism are significantly more concentrated within hypomethylated regions. These findings suggest a new connection between the epigenome, selective mutability, evolution, and human disease. PMID:22615578

Prenatal detection of fetal triploidy from cell-free DNA testing in maternal blood.

PubMed

Nicolaides, Kypros H; Syngelaki, Argyro; del Mar Gil, Maria; Quezada, Maria Soledad; Zinevich, Yana

2014-01-01

To investigate potential performance of cell-free DNA (cfDNA) testing in maternal blood in detecting fetal triploidy. Plasma and buffy coat samples obtained at 11-13 weeks' gestation from singleton pregnancies with diandric triploidy (n=4), digynic triploidy (n=4), euploid fetuses (n=48) were sent to Natera, Inc. (San Carlos, Calif., USA) for cfDNA testing. Multiplex polymerase chain reaction amplification of cfDNA followed by sequencing of single nucleotide polymorphic loci covering chromosomes 13, 18, 21, X, and Y was performed. Sequencing data were analyzed using the NATUS algorithm which identifies copy number for each of the five chromosomes. cfDNA testing provided a result in 44 (91.7%) of the 48 euploid cases and correctly predicted the fetal sex and the presence of two copies each of chromosome 21, 18 and 13. In diandric triploidy, cfDNA testing identified multiple paternal haplotypes (indicating fetal trisomy 21, trisomy 18 and trisomy 13) suggesting the presence of either triploidy or dizygotic twins. In digynic triploidy the fetal fraction corrected for maternal weight and gestational age was below the 0.5th percentile. cfDNA testing by targeted sequencing and allelic ratio analysis of single nucleotide polymorphisms covering chromosomes 21, 18, 13, X, and Y can detect diandric triploidy and raise the suspicion of digynic triploidy. © 2013 S. Karger AG, Basel.

Mitochondrial haplotypes are not associated with mice selectively bred for high voluntary wheel running.

PubMed

Wone, Bernard W M; Yim, Won C; Schutz, Heidi; Meek, Thomas H; Garland, Theodore

2018-04-04

Mitochondrial haplotypes have been associated with human and rodent phenotypes, including nonshivering thermogenesis capacity, learning capability, and disease risk. Although the mammalian mitochondrial D-loop is highly polymorphic, D-loops in laboratory mice are identical, and variation occurs elsewhere mainly between nucleotides 9820 and 9830. Part of this region codes for the tRNA Arg gene and is associated with mitochondrial densities and number of mtDNA copies. We hypothesized that the capacity for high levels of voluntary wheel-running behavior would be associated with mitochondrial haplotype. Here, we analyzed the mtDNA polymorphic region in mice from each of four replicate lines selectively bred for 54 generations for high voluntary wheel running (HR) and from four control lines (Control) randomly bred for 54 generations. Sequencing the polymorphic region revealed a variable number of adenine repeats. Single nucleotide polymorphisms (SNPs) varied from 2 to 3 adenine insertions, resulting in three haplotypes. We found significant genetic differentiations between the HR and Control groups (F st  = 0.779, p ≤ 0.0001), as well as among the replicate lines of mice within groups (F sc  = 0.757, p ≤ 0.0001). Haplotypes, however, were not strongly associated with voluntary wheel running (revolutions run per day), nor with either body mass or litter size. This system provides a useful experimental model to dissect the physiological processes linking mitochondrial, genomic SNPs, epigenetics, or nuclear-mitochondrial cross-talk to exercise activity. Copyright © 2018. Published by Elsevier B.V.

Mismatch and G-Stack Modulated Probe Signals on SNP Microarrays

PubMed Central

Binder, Hans; Fasold, Mario; Glomb, Torsten

2009-01-01

Background Single nucleotide polymorphism (SNP) arrays are important tools widely used for genotyping and copy number estimation. This technology utilizes the specific affinity of fragmented DNA for binding to surface-attached oligonucleotide DNA probes. We analyze the variability of the probe signals of Affymetrix GeneChip SNP arrays as a function of the probe sequence to identify relevant sequence motifs which potentially cause systematic biases of genotyping and copy number estimates. Methodology/Principal Findings The probe design of GeneChip SNP arrays enables us to disentangle different sources of intensity modulations such as the number of mismatches per duplex, matched and mismatched base pairings including nearest and next-nearest neighbors and their position along the probe sequence. The effect of probe sequence was estimated in terms of triple-motifs with central matches and mismatches which include all 256 combinations of possible base pairings. The probe/target interactions on the chip can be decomposed into nearest neighbor contributions which correlate well with free energy terms of DNA/DNA-interactions in solution. The effect of mismatches is about twice as large as that of canonical pairings. Runs of guanines (G) and the particular type of mismatched pairings formed in cross-allelic probe/target duplexes constitute sources of systematic biases of the probe signals with consequences for genotyping and copy number estimates. The poly-G effect seems to be related to the crowded arrangement of probes which facilitates complex formation of neighboring probes with at minimum three adjacent G's in their sequence. Conclusions The applied method of “triple-averaging” represents a model-free approach to estimate the mean intensity contributions of different sequence motifs which can be applied in calibration algorithms to correct signal values for sequence effects. Rules for appropriate sequence corrections are suggested. PMID:19924253

Genome-wide copy number variation (CNV) detection in Nelore cattle reveals highly frequent variants in genome regions harboring QTLs affecting production traits.

PubMed

da Silva, Joaquim Manoel; Giachetto, Poliana Fernanda; da Silva, Luiz Otávio; Cintra, Leandro Carrijo; Paiva, Samuel Rezende; Yamagishi, Michel Eduardo Beleza; Caetano, Alexandre Rodrigues

2016-06-13

Copy number variations (CNVs) have been shown to account for substantial portions of observed genomic variation and have been associated with qualitative and quantitative traits and the onset of disease in a number of species. Information from high-resolution studies to detect, characterize and estimate population-specific variant frequencies will facilitate the incorporation of CNVs in genomic studies to identify genes affecting traits of importance. Genome-wide CNVs were detected in high-density single nucleotide polymorphism (SNP) genotyping data from 1,717 Nelore (Bos indicus) cattle, and in NGS data from eight key ancestral bulls. A total of 68,007 and 12,786 distinct CNVs were observed, respectively. Cross-comparisons of results obtained for the eight resequenced animals revealed that 92 % of the CNVs were observed in both datasets, while 62 % of all detected CNVs were observed to overlap with previously validated cattle copy number variant regions (CNVRs). Observed CNVs were used for obtaining breed-specific CNV frequencies and identification of CNVRs, which were subsequently used for gene annotation. A total of 688 of the detected CNVRs were observed to overlap with 286 non-redundant QTLs associated with important production traits in cattle. All of 34 CNVs previously reported to be associated with milk production traits in Holsteins were also observed in Nelore cattle. Comparisons of estimated frequencies of these CNVs in the two breeds revealed 14, 13, 6 and 14 regions in high (>20 %), low (<20 %) and divergent (NEL > HOL, NEL < HOL) frequencies, respectively. Obtained results significantly enriched the bovine CNV map and enabled the identification of variants that are potentially associated with traits under selection in Nelore cattle, particularly in genome regions harboring QTLs affecting production traits.

SNPitty: An Intuitive Web Application for Interactive B-Allele Frequency and Copy Number Visualization of Next-Generation Sequencing Data.

PubMed

van Riet, Job; Krol, Niels M G; Atmodimedjo, Peggy N; Brosens, Erwin; van IJcken, Wilfred F J; Jansen, Maurice P H M; Martens, John W M; Looijenga, Leendert H; Jenster, Guido; Dubbink, Hendrikus J; Dinjens, Winand N M; van de Werken, Harmen J G

2018-03-01

Exploration and visualization of next-generation sequencing data are crucial for clinical diagnostics. Software allowing simultaneous visualization of multiple regions of interest coupled with dynamic heuristic filtering of genetic aberrations is, however, lacking. Therefore, the authors developed the web application SNPitty that allows interactive visualization and interrogation of variant call format files by using B-allele frequencies of single-nucleotide polymorphisms and single-nucleotide variants, coverage metrics, and copy numbers analysis results. SNPitty displays variant alleles and allelic imbalances with a focus on loss of heterozygosity and copy number variation using genome-wide heterozygous markers and somatic mutations. In addition, SNPitty is capable of generating predefined reports that summarize and highlight disease-specific targets of interest. SNPitty was validated for diagnostic interpretation of somatic events by showcasing a serial dilution series of glioma tissue. Additionally, SNPitty is demonstrated in four cancer-related scenarios encountered in daily clinical practice and on whole-exome sequencing data of peripheral blood from a Down syndrome patient. SNPitty allows detection of loss of heterozygosity, chromosomal and gene amplifications, homozygous or heterozygous deletions, somatic mutations, or any combination thereof in regions or genes of interest. Furthermore, SNPitty can be used to distinguish molecular relationships between multiple tumors from a single patient. On the basis of these data, the authors demonstrate that SNPitty is robust and user friendly in a wide range of diagnostic scenarios. Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Dynamics of Copy Number Variation in Host Races of the Pea Aphid

PubMed Central

Duvaux, Ludovic; Geissmann, Quentin; Gharbi, Karim; Zhou, Jing-Jiang; Ferrari, Julia; Smadja, Carole M.; Butlin, Roger K.

2015-01-01

Copy number variation (CNV) makes a major contribution to overall genetic variation and is suspected to play an important role in adaptation. However, aside from a few model species, the extent of CNV in natural populations has seldom been investigated. Here, we report on CNV in the pea aphid Acyrthosiphon pisum, a powerful system for studying the genetic architecture of host-plant adaptation and speciation thanks to multiple host races forming a continuum of genetic divergence. Recent studies have highlighted the potential importance of chemosensory genes, including the gustatory and olfactory receptor gene families (Gr and Or, respectively), in the process of host race formation. We used targeted resequencing to achieve a very high depth of coverage, and thereby revealed the extent of CNV of 434 genes, including 150 chemosensory genes, in 104 individuals distributed across eight host races of the pea aphid. We found that CNV was widespread in our global sample, with a significantly higher occurrence in multigene families, especially in Ors. We also observed a decrease in the gene probability of being completely duplicated or deleted (CDD) with increase in coding sequence length. Genes with CDD variants were usually more polymorphic for copy number, especially in the P450 gene family where toxin resistance may be related to gene dosage. We found that Gr were overrepresented among genes discriminating host races, as were CDD genes and pseudogenes. Our observations shed new light on CNV dynamics and are consistent with CNV playing a role in both local adaptation and speciation. PMID:25234705

Parallel Evolution of Copy-Number Variation across Continents in Drosophila melanogaster.

PubMed

Schrider, Daniel R; Hahn, Matthew W; Begun, David J

2016-05-01

Genetic differentiation across populations that is maintained in the presence of gene flow is a hallmark of spatially varying selection. In Drosophila melanogaster, the latitudinal clines across the eastern coasts of Australia and North America appear to be examples of this type of selection, with recent studies showing that a substantial portion of the D. melanogaster genome exhibits allele frequency differentiation with respect to latitude on both continents. As of yet there has been no genome-wide examination of differentiated copy-number variants (CNVs) in these geographic regions, despite their potential importance for phenotypic variation in Drosophila and other taxa. Here, we present an analysis of geographic variation in CNVs in D. melanogaster. We also present the first genomic analysis of geographic variation for copy-number variation in the sister species, D. simulans, in order to investigate patterns of parallel evolution in these close relatives. In D. melanogaster we find hundreds of CNVs, many of which show parallel patterns of geographic variation on both continents, lending support to the idea that they are influenced by spatially varying selection. These findings support the idea that polymorphic CNVs contribute to local adaptation in D. melanogaster In contrast, we find very few CNVs in D. simulans that are geographically differentiated in parallel on both continents, consistent with earlier work suggesting that clinal patterns are weaker in this species. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Copy number variations at the Prader-Willi syndrome region on chromosome 15 and associations with obesity in whites.

PubMed

Chen, Yuan; Liu, Yong-Jun; Pei, Yu-Fang; Yang, Tie-Lin; Deng, Fei-Yan; Liu, Xiao-Gang; Li, Ding-You; Deng, Hong-Wen

2011-06-01

Obesity is a serious health problem with strong genetic determination. Copy number variation (CNV) is a common type of genomic variant associated with some complex human diseases. However, it is not clear how CNVs contribute to the etiology of obesity. In this study, we examined 1,000 unrelated US whites to search for CNVs that may predispose to obesity. We focused our analyses on the Prader-Willi syndrome (PWS) critical region (chromosome 15q11-q13), because the PWS region is a hotspot for CNV generation and obesity is one of the major clinical manifestations for chromosome abnormalities at this region. We constructed a map containing 39 CNVs at the PWS critical region with CNV occurrence rates higher than 1%. Among them, three CNVs were significantly associated with body fat mass (P < 0.05), with a higher copy number (CN) associated with an increase of 5.08-9.77 kg in body fat mass. These three CNVs are close to two known PWS genes, NDN (necdin homolog) and C15orf2 (chromosome 15 open reading frame 2), and partially overlap with another obesity gene PWRN1 (Prader-Willi region nonprotein-coding RNA 1). Interestingly, our recently published whole genome association scan study using the same sample by examining single-nucleotide polymorphisms (SNPs) did not find any significant associations at these CNV regions, suggesting the importance of examining both CNVs and SNPs for better understanding of genetic basis of obesity. Further studies are warranted to validate these CNVs and their importance to obesity.

Detection of genome-wide copy number variants in myeloid malignancies using next-generation sequencing.

PubMed

Shen, Wei; Paxton, Christian N; Szankasi, Philippe; Longhurst, Maria; Schumacher, Jonathan A; Frizzell, Kimberly A; Sorrells, Shelly M; Clayton, Adam L; Jattani, Rakhi P; Patel, Jay L; Toydemir, Reha; Kelley, Todd W; Xu, Xinjie

2018-04-01

Genetic abnormalities, including copy number variants (CNV), copy number neutral loss of heterozygosity (CN-LOH) and gene mutations, underlie the pathogenesis of myeloid malignancies and serve as important diagnostic, prognostic and/or therapeutic markers. Currently, multiple testing strategies are required for comprehensive genetic testing in myeloid malignancies. The aim of this proof-of-principle study was to investigate the feasibility of combining detection of genome-wide large CNVs, CN-LOH and targeted gene mutations into a single assay using next-generation sequencing (NGS). For genome-wide CNV detection, we designed a single nucleotide polymorphism (SNP) sequencing backbone with 22 762 SNP regions evenly distributed across the entire genome. For targeted mutation detection, 62 frequently mutated genes in myeloid malignancies were targeted. We combined this SNP sequencing backbone with a targeted mutation panel, and sequenced 9 healthy individuals and 16 patients with myeloid malignancies using NGS. We detected 52 somatic CNVs, 11 instances of CN-LOH and 39 oncogenic mutations in the 16 patients with myeloid malignancies, and none in the 9 healthy individuals. All CNVs and CN-LOH were confirmed by SNP microarray analysis. We describe a genome-wide SNP sequencing backbone which allows for sensitive detection of genome-wide CNVs and CN-LOH using NGS. This proof-of-principle study has demonstrated that this strategy can provide more comprehensive genetic profiling for patients with myeloid malignancies using a single assay. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Chromosomal microarray analysis as the first-tier test for the identification of pathogenic copy number variants in chromosome 9 pericentric regions and its challenge.

PubMed

Wang, Jia-Chi; Boyar, Fatih Z

2016-01-01

Chromosomal microarray analysis (CMA) has been recommended and practiced routinely in the large reference laboratories of U.S.A. as the first-tier test for the postnatal evaluation of individuals with intellectual disability, autism spectrum disorders, and/or multiple congenital anomalies. Using CMA as a diagnostic tool and without a routine setting of fluorescence in situ hybridization with labeled bacterial artificial chromosome probes (BAC-FISH) in the large reference laboratories becomes a challenge in the characterization of chromosome 9 pericentric region. This region has a very complex genomic structure and contains a variety of heterochromatic and euchromatic polymorphic variants. These variants were usually studied by G-banding, C-banding and BAC-FISH analysis. Chromosomal microarray analysis (CMA) was not recommended since it may lead to false positive results. Here, we presented a cohort of four cases, in which high-resolution CMA was used as the first-tier test or simultaneously with G-banding analysis on the proband to identify pathogenic copy number variants (CNVs) in the whole genome. CMA revealed large pathogenic CNVs from chromosome 9 in 3 cases which also revealed different G-banding patterns between the two chromosome 9 homologues. Although we demonstrated that high-resolution CMA played an important role in the identification of pathogenic copy number variants in chromosome 9 pericentric regions, the lack of BAC-FISH analysis or other useful tools renders significant challenges in the characterization of chromosome 9 pericentric regions. None; it is not a clinical trial, and the cases were retrospectively collected and analyzed.

Two sequence-ready contigs spanning the two copies of a 200-kb duplication on human 21q: partial sequence and polymorphisms.

PubMed

Potier, M; Dutriaux, A; Orti, R; Groet, J; Gibelin, N; Karadima, G; Lutfalla, G; Lynn, A; Van Broeckhoven, C; Chakravarti, A; Petersen, M; Nizetic, D; Delabar, J; Rossier, J

1998-08-01

Physical mapping across a duplication can be a tour de force if the region is larger than the size of a bacterial clone. This was the case of the 170- to 275-kb duplication present on the long arm of chromosome 21 in normal human at 21q11.1 (proximal region) and at 21q22.1 (distal region), which we described previously. We have constructed sequence-ready contigs of the two copies of the duplication of which all the clones are genuine representatives of one copy or the other. This required the identification of four duplicon polymorphisms that are copy-specific and nonallelic variations in the sequence of the STSs. Thirteen STSs were mapped inside the duplicated region and 5 outside but close to the boundaries. Among these STSs 10 were end clones from YACs, PACs, or cosmids, and the average interval between two markers in the duplicated region was 16 kb. Eight PACs and cosmids showing minimal overlaps were selected in both copies of the duplication. Comparative sequence analysis along the duplication showed three single-basepair changes between the two copies over 659 bp sequenced (4 STSs), suggesting that the duplication is recent (less than 4 mya). Two CpG islands were located in the duplication, but no genes were identified after a 36-kb cosmid from the proximal copy of the duplication was sequenced. The homology of this chromosome 21 duplicated region with the pericentromeric regions of chromosomes 13, 2, and 18 suggests that the mechanism involved is probably similar to pericentromeric-directed mechanisms described in interchromosomal duplications. Copyright 1998 Academic Press.

The Diversity of REcent and Ancient huMan (DREAM): A New Microarray for Genetic Anthropology and Genealogy, Forensics, and Personalized Medicine.

PubMed

Elhaik, Eran; Yusuf, Leeban; Anderson, Ainan I J; Pirooznia, Mehdi; Arnellos, Dimitrios; Vilshansky, Gregory; Ercal, Gunes; Lu, Yontao; Webster, Teresa; Baird, Michael L; Esposito, Umberto

2017-12-01

The human population displays wide variety in demographic history, ancestry, content of DNA derived from hominins or ancient populations, adaptation, traits, copy number variation, drug response, and more. These polymorphisms are of broad interest to population geneticists, forensics investigators, and medical professionals. Historically, much of that knowledge was gained from population survey projects. Although many commercial arrays exist for genome-wide single-nucleotide polymorphism genotyping, their design specifications are limited and they do not allow a full exploration of biodiversity. We thereby aimed to design the Diversity of REcent and Ancient huMan (DREAM)-an all-inclusive microarray that would allow both identification of known associations and exploration of standing questions in genetic anthropology, forensics, and personalized medicine. DREAM includes probes to interrogate ancestry informative markers obtained from over 450 human populations, over 200 ancient genomes, and 10 archaic hominins. DREAM can identify 94% and 61% of all known Y and mitochondrial haplogroups, respectively, and was vetted to avoid interrogation of clinically relevant markers. To demonstrate its capabilities, we compared its FST distributions with those of the 1000 Genomes Project and commercial arrays. Although all arrays yielded similarly shaped (inverse J) FST distributions, DREAM's autosomal and X-chromosomal distributions had the highest mean FST, attesting to its ability to discern subpopulations. DREAM performances are further illustrated in biogeographical, identical by descent, and copy number variation analyses. In summary, with approximately 800,000 markers spanning nearly 2,000 genes, DREAM is a useful tool for genetic anthropology, forensic, and personalized medicine studies. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

A single nucleotide polymorphism in the FADS1/FADS2 gene is associated with plasma lipid profiles in two genetically similar Asian ethnic groups with distinctive differences in lifestyle.

PubMed

Nakayama, Kazuhiro; Bayasgalan, Tumenbayer; Tazoe, Fumiko; Yanagisawa, Yoshiko; Gotoh, Takaya; Yamanaka, Kazuhiro; Ogawa, Ayumi; Munkhtulga, Lkhagvasuren; Chimedregze, Ulziiburen; Kagawa, Yasuo; Ishibashi, Shun; Iwamoto, Sadahiko

2010-06-01

Recent genome-wide association studies (GWASs) showed that single nucleotide polymorphisms (SNPs) in FADS1/FADS2 were associated with plasma lipid concentrations in populations with European ancestry. We investigated the associations between the SNPs in FADS1/FADS2 and plasma concentrations of triglycerides, high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) in two Asian groups, i.e., Japanese and Mongolians. The genotype of rs174547 (T/C), found to be associated with triglyceride and HDL-C concentrations in the GWAS, was determined in 21,004 Japanese and 1,203 Mongolian individuals. Genotype-phenotype association was assessed by using multiple linear regression models, assuming an additive model of inheritance. The copy number of the rs174547 C allele was significantly associated with increased triglyceride levels (P = 1.5 x 10(-6)) and decreased HDL-C levels (P = 0.03) in the Japanese population. On the other hand, in the Mongolian population, the rs174547 C allele copy number was strongly associated with decreased LDL-C levels (P = 2.6 x 10(-6)), but was not associated with triglyceride and HDL-C levels. The linkage disequilibrium pattern and haplotype structures of SNPs around the FADS1/FADS2 locus showed no marked dissimilarity between Japanese and Mongolian individuals. The present data indicate that the FADS1/FADS2 locus can be added to the growing list of loci involved in polygenic dyslipidemia in Asians. Furthermore, the variable effects of FADS1/FADS2 on plasma lipid profiles in Asians may result from differences in the dietary intake of polyunsaturated fatty acids, which serve as substrates for enzymes encoded by FADS1/FADS2.

Identifying allelic loss and homozygous deletions in pancreatic cancer without matched normals using high-density single-nucleotide polymorphism arrays.

PubMed

Calhoun, Eric S; Hucl, Tomas; Gallmeier, Eike; West, Kristen M; Arking, Dan E; Maitra, Anirban; Iacobuzio-Donahue, Christine A; Chakravarti, Aravinda; Hruban, Ralph H; Kern, Scott E

2006-08-15

Recent advances in oligonucleotide arrays and whole-genome complexity reduction data analysis now permit the evaluation of tens of thousands of single-nucleotide polymorphisms simultaneously for a genome-wide analysis of allelic status. Using these arrays, we created high-resolution allelotype maps of 26 pancreatic cancer cell lines. The areas of heterozygosity implicitly served to reveal regions of allelic loss. The array-derived maps were verified by a panel of 317 microsatellite markers used in a subset of seven samples, showing a 97.1% concordance between heterozygous calls. Three matched tumor/normal pairs were used to estimate the false-negative and potential false-positive rates for identifying loss of heterozygosity: 3.6 regions (average minimal region of loss, 720,228 bp) and 2.3 regions (average heterozygous gap distance, 4,434,994 bp) per genome, respectively. Genomic fractional allelic loss calculations showed that cumulative levels of allelic loss ranged widely from 17.1% to 79.9% of the haploid genome length. Regional increases in "NoCall" frequencies combined with copy number loss estimates were used to identify 41 homozygous deletions (19 first reports), implicating an additional 13 regions disrupted in pancreatic cancer. Unexpectedly, 23 of these occurred in just two lines (BxPc3 and MiaPaCa2), suggesting the existence of at least two subclasses of chromosomal instability (CIN) patterns, distinguished here by allelic loss and copy number changes (original CIN) and those also highly enriched in the genomic "holes" of homozygous deletions (holey CIN). This study provides previously unavailable high-resolution allelotype and deletion breakpoint maps in widely shared pancreatic cancer cell lines and effectively eliminates the need for matched normal tissue to define informative loci.

Genome wide analysis reveals single nucleotide polymorphisms associated with fatness and putative novel copy number variants in three pig breeds

PubMed Central

2013-01-01

Background Obesity, excess fat tissue in the body, can underlie a variety of medical complaints including heart disease, stroke and cancer. The pig is an excellent model organism for the study of various human disorders, including obesity, as well as being the foremost agricultural species. In order to identify genetic variants associated with fatness, we used a selective genomic approach sampling DNA from animals at the extreme ends of the fat and lean spectrum using estimated breeding values derived from a total population size of over 70,000 animals. DNA from 3 breeds (Sire Line Large White, Duroc and a white Pietrain composite line (Titan)) was used to interrogate the Illumina Porcine SNP60 Genotyping Beadchip in order to identify significant associations in terms of single nucleotide polymorphisms (SNPs) and copy number variants (CNVs). Results By sampling animals at each end of the fat/lean EBV (estimate breeding value) spectrum the whole population could be assessed using less than 300 animals, without losing statistical power. Indeed, several significant SNPs (at the 5% genome wide significance level) were discovered, 4 of these linked to genes with ontologies that had previously been correlated with fatness (NTS, FABP6, SST and NR3C2). Quantitative analysis of the data identified putative CNV regions containing genes whose ontology suggested fatness related functions (MCHR1, PPARα, SLC5A1 and SLC5A4). Conclusions Selective genotyping of EBVs at either end of the phenotypic spectrum proved to be a cost effective means of identifying SNPs and CNVs associated with fatness and with estimated major effects in a large population of animals. PMID:24225222

Population Structure Shapes Copy Number Variation in Malaria Parasites.

PubMed

Cheeseman, Ian H; Miller, Becky; Tan, John C; Tan, Asako; Nair, Shalini; Nkhoma, Standwell C; De Donato, Marcos; Rodulfo, Hectorina; Dondorp, Arjen; Branch, Oralee H; Mesia, Lastenia Ruiz; Newton, Paul; Mayxay, Mayfong; Amambua-Ngwa, Alfred; Conway, David J; Nosten, François; Ferdig, Michael T; Anderson, Tim J C

2016-03-01

If copy number variants (CNVs) are predominantly deleterious, we would expect them to be more efficiently purged from populations with a large effective population size (Ne) than from populations with a small Ne. Malaria parasites (Plasmodium falciparum) provide an excellent organism to examine this prediction, because this protozoan shows a broad spectrum of population structures within a single species, with large, stable, outbred populations in Africa, small unstable inbred populations in South America and with intermediate population characteristics in South East Asia. We characterized 122 single-clone parasites, without prior laboratory culture, from malaria-infected patients in seven countries in Africa, South East Asia and South America using a high-density single-nucleotide polymorphism/CNV microarray. We scored 134 high-confidence CNVs across the parasite exome, including 33 deletions and 102 amplifications, which ranged in size from <500 bp to 59 kb, as well as 10,107 flanking, biallelic single-nucleotide polymorphisms. Overall, CNVs were rare, small, and skewed toward low frequency variants, consistent with the deleterious model. Relative to African and South East Asian populations, CNVs were significantly more common in South America, showed significantly less skew in allele frequencies, and were significantly larger. On this background of low frequency CNV, we also identified several high-frequency CNVs under putative positive selection using an FST outlier analysis. These included known adaptive CNVs containing rh2b and pfmdr1, and several other CNVs (e.g., DNA helicase and three conserved proteins) that require further investigation. Our data are consistent with a significant impact of genetic structure on CNV burden in an important human pathogen. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Analysis and visualization of chromosomal abnormalities in SNP data with SNPscan

PubMed Central

Ting, Jason C; Ye, Ying; Thomas, George H; Ruczinski, Ingo; Pevsner, Jonathan

2006-01-01

Background A variety of diseases are caused by chromosomal abnormalities such as aneuploidies (having an abnormal number of chromosomes), microdeletions, microduplications, and uniparental disomy. High density single nucleotide polymorphism (SNP) microarrays provide information on chromosomal copy number changes, as well as genotype (heterozygosity and homozygosity). SNP array studies generate multiple types of data for each SNP site, some with more than 100,000 SNPs represented on each array. The identification of different classes of anomalies within SNP data has been challenging. Results We have developed SNPscan, a web-accessible tool to analyze and visualize high density SNP data. It enables researchers (1) to visually and quantitatively assess the quality of user-generated SNP data relative to a benchmark data set derived from a control population, (2) to display SNP intensity and allelic call data in order to detect chromosomal copy number anomalies (duplications and deletions), (3) to display uniparental isodisomy based on loss of heterozygosity (LOH) across genomic regions, (4) to compare paired samples (e.g. tumor and normal), and (5) to generate a file type for viewing SNP data in the University of California, Santa Cruz (UCSC) Human Genome Browser. SNPscan accepts data exported from Affymetrix Copy Number Analysis Tool as its input. We validated SNPscan using data generated from patients with known deletions, duplications, and uniparental disomy. We also inspected previously generated SNP data from 90 apparently normal individuals from the Centre d'Étude du Polymorphisme Humain (CEPH) collection, and identified three cases of uniparental isodisomy, four females having an apparently mosaic X chromosome, two mislabelled SNP data sets, and one microdeletion on chromosome 2 with mosaicism from an apparently normal female. These previously unrecognized abnormalities were all detected using SNPscan. The microdeletion was independently confirmed by fluorescence in situ hybridization, and a region of homozygosity in a UPD case was confirmed by sequencing of genomic DNA. Conclusion SNPscan is useful to identify chromosomal abnormalities based on SNP intensity (such as chromosomal copy number changes) and heterozygosity data (including regions of LOH and some cases of UPD). The program and source code are available at the SNPscan website . PMID:16420694

Studying Genome Heterogeneity within the Arbuscular Mycorrhizal Fungal Cytoplasm

PubMed Central

Halary, Sébastien; Bapteste, Eric; Hijri, Mohamed

2015-01-01

Although heterokaryons have been reported in nature, multicellular organisms are generally assumed genetically homogeneous. Here, we investigate the case of arbuscular mycorrhizal fungi (AMF) that form symbiosis with plant roots. The growth advantages they confer to their hosts are of great potential benefit to sustainable agricultural practices. However, measuring genetic diversity for these coenocytes is a major challenge: Within the same cytoplasm, AMF contain thousands of nuclei and show extremely high levels of genetic variation for some loci. The extent and physical location of polymorphism within and between AMF genomes is unclear. We used two complementary strategies to estimate genetic diversity in AMF, investigating polymorphism both on a genome scale and in putative single copy loci. First, we used data from whole-genome pyrosequencing of four AMF isolates to describe genetic diversity, based on a conservative network-based clustering approach. AMF isolates showed marked differences in genome-wide diversity patterns in comparison to a panel of control fungal genomes. This clustering approach further allowed us to provide conservative estimates of Rhizophagus spp. genomes sizes. Second, we designed new putative single copy genomic markers, which we investigated by massive parallel amplicon sequencing for two Rhizophagus irregularis and one Rhizophagus sp. isolates. Most loci showed high polymorphism, with up to 103 alleles per marker. This polymorphism could be distributed within or between nuclei. However, we argue that the Rhizophagus isolates under study might be heterokaryotic, at least for the putative single copy markers we studied. Considering that genetic information is the main resource for identification of AMF, we suggest that special attention is warranted for the study of these ecologically important organisms. PMID:25573960

Studying genome heterogeneity within the arbuscular mycorrhizal fungal cytoplasm.

PubMed

Boon, Eva; Halary, Sébastien; Bapteste, Eric; Hijri, Mohamed

2015-01-07

Although heterokaryons have been reported in nature, multicellular organisms are generally assumed genetically homogeneous. Here, we investigate the case of arbuscular mycorrhizal fungi (AMF) that form symbiosis with plant roots. The growth advantages they confer to their hosts are of great potential benefit to sustainable agricultural practices. However, measuring genetic diversity for these coenocytes is a major challenge: Within the same cytoplasm, AMF contain thousands of nuclei and show extremely high levels of genetic variation for some loci. The extent and physical location of polymorphism within and between AMF genomes is unclear. We used two complementary strategies to estimate genetic diversity in AMF, investigating polymorphism both on a genome scale and in putative single copy loci. First, we used data from whole-genome pyrosequencing of four AMF isolates to describe genetic diversity, based on a conservative network-based clustering approach. AMF isolates showed marked differences in genome-wide diversity patterns in comparison to a panel of control fungal genomes. This clustering approach further allowed us to provide conservative estimates of Rhizophagus spp. genomes sizes. Second, we designed new putative single copy genomic markers, which we investigated by massive parallel amplicon sequencing for two Rhizophagus irregularis and one Rhizophagus sp. isolates. Most loci showed high polymorphism, with up to 103 alleles per marker. This polymorphism could be distributed within or between nuclei. However, we argue that the Rhizophagus isolates under study might be heterokaryotic, at least for the putative single copy markers we studied. Considering that genetic information is the main resource for identification of AMF, we suggest that special attention is warranted for the study of these ecologically important organisms. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Increased pfmdr1 gene copy number and the decline in pfcrt and pfmdr1 resistance alleles in Ghanaian Plasmodium falciparum isolates after the change of anti-malarial drug treatment policy

PubMed Central

2013-01-01

Background With the introduction of artemisinin-based combination therapy (ACT) in 2005, monitoring of anti-malarial drug efficacy, which includes the use of molecular tools to detect known genetic markers of parasite resistance, is important for first-hand information on the changes in parasite susceptibility to drugs in Ghana. This study investigated the Plasmodium falciparum multidrug resistance gene (pfmdr1) copy number, mutations and the chloroquine resistance transporter gene (pfcrt) mutations in Ghanaian isolates collected in seven years to detect the trends in prevalence of mutations. Methods Archived filter paper blood blots collected from children aged below five years with uncomplicated malaria in 2003–2010 at sentinel sites were used. Using quantitative real-time polymerase chain reaction (qRT-PCR), 756 samples were assessed for pfmdr1 gene copy number. PCR and restriction fragment length polymorphism (RFLP) were used to detect alleles of pfmdr1 86 in 1,102 samples, pfmdr1 184, 1034, 1042 and 1246 in 832 samples and pfcrt 76 in 1,063 samples. Merozoite surface protein 2 (msp2) genotyping was done to select monoclonal infections for copy number analysis. Results The percentage of isolates with increased pfmdr1 copy number were 4, 27, 9, and 18% for 2003–04, 2005–06, 2007–08 and 2010, respectively. Significant increasing trends for prevalence of pfmdr1 N86 (×2 = 96.31, p <0.001) and pfcrt K76 (×2 = 64.50, p <0.001) and decreasing trends in pfmdr1 Y86 (×2 = 38.52, p <0.001) and pfcrt T76 (×2 = 43.49, p <0.001) were observed from 2003–2010. The pfmdr1 F184 and Y184 prevalence showed an increasing and decreasing trends respectively but were not significant (×2 = 7.39,p=0.060; ×2 = 7.49, p = 0.057 respectively). The pfmdr1 N86-F184-D1246 haplotype, which is alleged to be selected by artemether-lumefantrine showed a significant increasing trend (×2 = 20.75, p < 0.001). Conclusion Increased pfmdr1 gene copy number was observed in the isolates analysed and this finding has implications for the use of ACT in the country although no resistance has been reported. The decreasing trend in the prevalence of chloroquine resistance markers after change of treatment policy presents the possibility for future introduction of chloroquine as prophylaxis for malaria risk groups such as children and pregnant women in Ghana. PMID:24172030

Population-genetic properties of differentiated copy number variations in cattle.

PubMed

Xu, Lingyang; Hou, Yali; Bickhart, Derek M; Zhou, Yang; Hay, El Hamidi Abdel; Song, Jiuzhou; Sonstegard, Tad S; Van Tassell, Curtis P; Liu, George E

2016-03-23

While single nucleotide polymorphism (SNP) is typically the variant of choice for population genetics, copy number variation (CNV) which comprises insertion, deletion and duplication of genomic sequence, is an informative type of genetic variation. CNVs have been shown to be both common in mammals and important for understanding the relationship between genotype and phenotype. However, CNV differentiation, selection and its population genetic properties are not well understood across diverse populations. We performed a population genetics survey based on CNVs derived from the BovineHD SNP array data of eight distinct cattle breeds. We generated high resolution results that show geographical patterns of variations and genome-wide admixture proportions within and among breeds. Similar to the previous SNP-based studies, our CNV-based results displayed a strong correlation of population structure and geographical location. By conducting three pairwise comparisons among European taurine, African taurine, and indicine groups, we further identified 78 unique CNV regions that were highly differentiated, some of which might be due to selection. These CNV regions overlapped with genes involved in traits related to parasite resistance, immunity response, body size, fertility, and milk production. Our results characterize CNV diversity among cattle populations and provide a list of lineage-differentiated CNVs.

Molecular characterization of Mycobacterium tuberculosis isolated in the State of Parana in southern Brazil.

PubMed

Malaghini, Marcelo; Brockelt, Sonia Regina; Burger, Marion; Kritski, Afrânio; Thomaz-Soccol, Vanete

2009-01-01

Sequence IS6110 has been successfully used throughout the world for characterizing the Mycobacterium tuberculosis lineages. The aim of this study was to obtain data about circulating strains of M. tuberculosis in patients from the State of Parana in southern Brazil. Sixty-two clinical specimens obtained from sputum, bronchial aspirate, biopsy and urine from 62 patients clinically diagnosed with tuberculosis and admitted to the SUS-Brazil - The Brazilian Centralized Health Service System - were genotyped by the mixed-linker PCR DNA fingerprinting technique. The analysis demonstrated that the number of copies of the IS6110 sequence per isolates varied from four to 13 bands, with an average number of 8.5. From this, 93% of the isolates presented multiple copies. Isolates with no copies of the IS6110 element were not observed. The genetic analysis by UPGMA grouped the 62 isolates by similarity into three different groups: the first group contained two strains, the second was composed of 23, and the third, a more heterogeneous group, contained 37 isolates. Only two isolates (3.2%) formed a cluster; in other words, they presented a pattern of polymorphism with similarity above 95%. Such findings suggest that in the State of Parana, illness predominantly develops through reactivation of the latent infection as opposed to exogenous transmission. The methodology used (mixed-linker PCR DNA fingerprinting) allowed for 93.5% differentiation of the isolates tested, and proved to be a powerful tool for differentiation in the molecular genotyping of M. tuberculosis.

Mapping autism risk loci using genetic linkage and chromosomal rearrangements

PubMed Central

Szatmari, Peter; Paterson, Andrew; Zwaigenbaum, Lonnie; Roberts, Wendy; Brian, Jessica; Liu, Xiao-Qing; Vincent, John; Skaug, Jennifer; Thompson, Ann; Senman, Lili; Feuk, Lars; Qian, Cheng; Bryson, Susan; Jones, Marshall; Marshall, Christian; Scherer, Stephen; Vieland, Veronica; Bartlett, Christopher; Mangin, La Vonne; Goedken, Rhinda; Segre, Alberto; Pericak-Vance, Margaret; Cuccaro, Michael; Gilbert, John; Wright, Harry; Abramson, Ruth; Betancur, Catalina; Bourgeron, Thomas; Gillberg, Christopher; Leboyer, Marion; Buxbaum, Joseph; Davis, Kenneth; Hollander, Eric; Silverman, Jeremy; Hallmayer, Joachim; Lotspeich, Linda; Sutcliffe, James; Haines, Jonathan; Folstein, Susan; Piven, Joseph; Wassink, Thomas; Sheffield, Val; Geschwind, Daniel; Bucan, Maja; Brown, Ted; Cantor, Rita; Constantino, John; Gilliam, Conrad; Herbert, Martha; Lajonchere, Clara; Ledbetter, David; Lese-Martin, Christa; Miller, Janet; Nelson, Stan; Samango-Sprouse, Carol; Spence, Sarah; State, Matthew; Tanzi, Rudolph; Coon, Hilary; Dawson, Geraldine; Devlin, Bernie; Estes, Annette; Flodman, Pamela; Klei, Lambertus; Mcmahon, William; Minshew, Nancy; Munson, Jeff; Korvatska, Elena; Rodier, Patricia; Schellenberg, Gerard; Smith, Moyra; Spence, Anne; Stodgell, Chris; Tepper, Ping Guo; Wijsman, Ellen; Yu, Chang-En; Rogé, Bernadette; Mantoulan, Carine; Wittemeyer, Kerstin; Poustka, Annemarie; Felder, Bärbel; Klauck, Sabine; Schuster, Claudia; Poustka, Fritz; Bölte, Sven; Feineis-Matthews, Sabine; Herbrecht, Evelyn; Schmötzer, Gabi; Tsiantis, John; Papanikolaou, Katerina; Maestrini, Elena; Bacchelli, Elena; Blasi, Francesca; Carone, Simona; Toma, Claudio; Van Engeland, Herman; De Jonge, Maretha; Kemner, Chantal; Koop, Frederieke; Langemeijer, Marjolein; Hijmans, Channa; Staal, Wouter; Baird, Gillian; Bolton, Patrick; Rutter, Michael; Weisblatt, Emma; Green, Jonathan; Aldred, Catherine; Wilkinson, Julie-Anne; Pickles, Andrew; Le Couteur, Ann; Berney, Tom; Mcconachie, Helen; Bailey, Anthony; Francis, Kostas; Honeyman, Gemma; Hutchinson, Aislinn; Parr, Jeremy; Wallace, Simon; Monaco, Anthony; Barnby, Gabrielle; Kobayashi, Kazuhiro; Lamb, Janine; Sousa, Ines; Sykes, Nuala; Cook, Edwin; Guter, Stephen; Leventhal, Bennett; Salt, Jeff; Lord, Catherine; Corsello, Christina; Hus, Vanessa; Weeks, Daniel; Volkmar, Fred; Tauber, Maïté; Fombonne, Eric; Shih, Andy; Meyer, Kacie

2007-01-01

Autism spectrum disorders (ASD) are common, heritable neurodevelopmental conditions. The genetic architecture of ASD is complex, requiring large samples to overcome heterogeneity. Here we broaden coverage and sample size relative to other studies of ASD by using Affymetrix 10K single nucleotide polymorphism (SNP) arrays and 1168 families with ≥ 2 affected individuals to perform the largest linkage scan to date, while also analyzing copy number variation (CNV) in these families. Linkage and CNV analyses implicate chromosome 11p12-p13 and neurexins, respectively, amongst other candidate loci. Neurexins team with previously-implicated neuroligins for glutamatergic synaptogenesis, highlighting glutamate-related genes as promising candidates for ASD. PMID:17322880

CCL3L1 copy number and susceptibility to malaria

PubMed Central

Carpenter, Danielle; Färnert, Anna; Rooth, Ingegerd; Armour, John A.L.; Shaw, Marie-Anne

2012-01-01

Copy number variation can contribute to the variation observed in susceptibility to complex diseases. Here we present the first study to investigate copy number variation of the chemokine gene CCL3L1 with susceptibility to malaria. We present a family-based genetic analysis of a Tanzanian population (n = 922), using parasite load, mean number of clinical infections of malaria and haemoglobin levels as phenotypes. Copy number of CCL3L1 was measured using the paralogue ratio test (PRT) and the dataset exhibited copy numbers ranging between 1 and 10 copies per diploid genome (pdg). Association between copy number and phenotypes was assessed. Furthermore, we were able to identify copy number haplotypes in some families, using microsatellites within the copy variable region, for transmission disequilibrium testing. We identified a high level of copy number haplotype diversity and find some evidence for an association of low CCL3L1 copy number with protection from anaemia. PMID:22484763

CCL3L1 copy number and susceptibility to malaria.

PubMed

Carpenter, Danielle; Färnert, Anna; Rooth, Ingegerd; Armour, John A L; Shaw, Marie-Anne

2012-07-01

Copy number variation can contribute to the variation observed in susceptibility to complex diseases. Here we present the first study to investigate copy number variation of the chemokine gene CCL3L1 with susceptibility to malaria. We present a family-based genetic analysis of a Tanzanian population (n=922), using parasite load, mean number of clinical infections of malaria and haemoglobin levels as phenotypes. Copy number of CCL3L1 was measured using the paralogue ratio test (PRT) and the dataset exhibited copy numbers ranging between 1 and 10 copies per diploid genome (pdg). Association between copy number and phenotypes was assessed. Furthermore, we were able to identify copy number haplotypes in some families, using microsatellites within the copy variable region, for transmission disequilibrium testing. We identified a high level of copy number haplotype diversity and find some evidence for an association of low CCL3L1 copy number with protection from anaemia. Copyright © 2012 Elsevier B.V. All rights reserved.

Perceived discrimination, serotonin transporter linked polymorphic region status, and the development of conduct problems.

PubMed

Brody, Gene H; Beach, Steven R H; Chen, Yi-Fu; Obasi, Ezemenari; Philibert, Robert A; Kogan, Steven M; Simons, Ronald L

2011-05-01

This study examined the prospective relations of adolescents' perceptions of discrimination and their genetic status with increases in conduct problems. Participants were 461 African American youths residing in rural Georgia (Wave 1 mean age = 15.5 years) who provided three waves of data and a saliva sample from which a polymorphism in the SCL6A4 (serotonin transporter [5-HTT]) gene polymorphism known as the 5-HTT linked promoter region (5-HTTLPR) was genotyped. Data analyses using growth curve modeling indicated that perceived discrimination was significantly related to the slope of conduct problems. As hypothesized, interactions between perceived discrimination and genetic status emerged for male but not female youths. Compared with those carrying two copies of the long allele variant of 5-HTTLPR, male youths carrying one or two copies of its short allele variant evinced higher rates of conduct problems over time when they perceived high levels of racial discrimination. These findings are consistent with resilience and differential susceptibility propositions stating that genes can both foster sensitivity to adverse events and confer protection from those events.

Genotyping of Mycobacterium tuberculosis with additional markers enhances accuracy in epidemiological studies.

PubMed Central

Warren, R; Richardson, M; Sampson, S; Hauman, J H; Beyers, N; Donald, P R; van Helden, P D

1996-01-01

Two highly polymorphic Mycobacterium tuberculosis genomic domains, characterized by hybridization to the oligonucleotide (GTG)5, were identified as potential DNA fingerprinting probes. These domains were cloned [pMTB484(1) and pMTB484(2K4), respectively] and shown to be useful for genotype analysis by Southern blotting. These probes were used to genotype geographically linked strains of M. tuberculosis previously shown to have identical IS6110 fingerprints. Subsequent DNA fingerprints generated with MTB484(1) and MTB484(2K4) showed a high degree of polymorphism, allowing subclassification of IS6110-defined clusters into composites of smaller clusters and unique strains. Correlation of the molecular data with patient interviews and clinical records confirmed the sensitivity of these probes, as contacts were established only within subclusters. These findings demonstrate the requirement for multiple probes to accurately classify M. tuberculosis strains, even those with high copy numbers of IS6110. The enhanced accuracy of strain typing should, in turn, further our understanding of the epidemiology of tuberculosis. PMID:8862588

Germline Mutations and Polymorphisms in the Origins of Cancers in Women

PubMed Central

Hirshfield, Kim M.; Rebbeck, Timothy R.; Levine, Arnold J.

2010-01-01

Several female malignancies including breast, ovarian, and endometrial cancers can be characterized based on known somatic and germline mutations. Initiation and propagation of tumors reflect underlying genomic alterations such as mutations, polymorphisms, and copy number variations found in genes of multiple cellular pathways. The contributions of any single genetic variation or mutation in a population depend on its frequency and penetrance as well as tissue-specific functionality. Genome wide association studies, fluorescence in situ hybridization, comparative genomic hybridization, and candidate gene studies have enumerated genetic contributors to cancers in women. These include p53, BRCA1, BRCA2, STK11, PTEN, CHEK2, ATM, BRIP1, PALB2, FGFR2, TGFB1, MDM2, MDM4 as well as several other chromosomal loci. Based on the heterogeneity within a specific tumor type, a combination of genomic alterations defines the cancer subtype, biologic behavior, and in some cases, response to therapeutics. Consideration of tumor heterogeneity is therefore important in the critical analysis of gene associations in cancer. PMID:20111735

Chromosome 17: association of a large inversion polymorphism with corticosteroid response in asthma.

PubMed

Tantisira, Kelan G; Lazarus, Ross; Litonjua, Augusto A; Klanderman, Barbara; Weiss, Scott T

2008-08-01

A 900-kb inversion exists within a large region of conserved linkage disequilibrium (LD) on chromosome 17. CRHR1 is located within the inversion region and associated with inhaled corticosteroid response in asthma. We hypothesized that CRHR1 variants are in LD with the inversion, supporting a potential role for natural selection in the genetic response to corticosteroids. We genotyped six single nucleotide polymorphisms (SNPs) spanning chromosome 17: 40,410,565-42,372,240, including four SNPs defining inversion status. Similar allele frequencies and strong LD were noted between the inversion and a CRHR1 SNP previously associated with lung function response to inhaled corticosteroids. Each inversion-defining SNP was strongly associated with inhaled corticosteroid response in adult asthma (P values 0.002-0.005). The CRHR1 response to inhaled corticosteroids may thus be explained by natural selection resulting from inversion status or by long-range LD with another gene. Additional pharmacogenetic investigations into regions of chromosomal diversity, including copy number variation and inversions, are warranted.

Undermethylated DNA as a source of microsatellites from a conifer genome.

PubMed

Zhou, Y; Bui, T; Auckland, L D; Williams, C G

2002-02-01

Developing microsatellites from the large, highly duplicated conifer genome requires special tools. To improve the efficiency of developing Pinus taeda L. microsatellites, undermethylated (UM) DNA fragments were used to construct a microsatellite-enriched copy library. A methylation-sensitive restriction enzyme, McrBC, was used to enrich for UM DNA before library construction. Digested DNA fragments larger than 9 kb were then excised and digested with RsaI and used to construct nine dinucleotide and trinucleotide libraries. A total of 1016 microsatellite-positive clones were detected among 11 904 clones and 620 of these were unique. Of 245 primer sets that produced a PCR product, 113 could be developed as UM microsatellite markers and 70 were polymorphic. Inheritance and marker informativeness were tested for a random sample of 36 polymorphic markers using a three-generation outbred pedigree. Thirty-one microsatellites (86%) had single-locus inheritance despite the highly duplicated nature of the P. taeda genome. Nineteen UM microsatellites had highly informative intercross mating type configurations. Allele number and frequency were estimated for eleven UM microsatellites using a population survey. Allele numbers for these UM microsatellites ranged from 3 to 12 with an average of 5.7 alleles/locus. Frequencies for the 63 alleles were mostly in the low-common range; only 14 of the 63 were in the rare allele (q < 0.05) class. Enriching for UM DNA was an efficient method for developing polymorphic microsatellites from a large plant genome.

Detection of cryptic pathogenic copy number variations and constitutional loss of heterozygosity using high resolution SNP microarray analysis in 117 patients referred for cytogenetic analysis and impact on clinical practice.

PubMed

Bruno, D L; Ganesamoorthy, D; Schoumans, J; Bankier, A; Coman, D; Delatycki, M; Gardner, R J M; Hunter, M; James, P A; Kannu, P; McGillivray, G; Pachter, N; Peters, H; Rieubland, C; Savarirayan, R; Scheffer, I E; Sheffield, L; Tan, T; White, S M; Yeung, A; Bowman, Z; Ngo, C; Choy, K W; Cacheux, V; Wong, L; Amor, D J; Slater, H R

2009-02-01

Microarray genome analysis is realising its promise for improving detection of genetic abnormalities in individuals with mental retardation and congenital abnormality. Copy number variations (CNVs) are now readily detectable using a variety of platforms and a major challenge is the distinction of pathogenic from ubiquitous, benign polymorphic CNVs. The aim of this study was to investigate replacement of time consuming, locus specific testing for specific microdeletion and microduplication syndromes with microarray analysis, which theoretically should detect all known syndromes with CNV aetiologies as well as new ones. Genome wide copy number analysis was performed on 117 patients using Affymetrix 250K microarrays. 434 CNVs (195 losses and 239 gains) were found, including 18 pathogenic CNVs and 9 identified as "potentially pathogenic". Almost all pathogenic CNVs were larger than 500 kb, significantly larger than the median size of all CNVs detected. Segmental regions of loss of heterozygosity larger than 5 Mb were found in 5 patients. Genome microarray analysis has improved diagnostic success in this group of patients. Several examples of recently discovered "new syndromes" were found suggesting they are more common than previously suspected and collectively are likely to be a major cause of mental retardation. The findings have several implications for clinical practice. The study revealed the potential to make genetic diagnoses that were not evident in the clinical presentation, with implications for pretest counselling and the consent process. The importance of contributing novel CNVs to high quality databases for genotype-phenotype analysis and review of guidelines for selection of individuals for microarray analysis is emphasised.

Karyotype versus microarray testing for genetic abnormalities after stillbirth.

PubMed

Reddy, Uma M; Page, Grier P; Saade, George R; Silver, Robert M; Thorsten, Vanessa R; Parker, Corette B; Pinar, Halit; Willinger, Marian; Stoll, Barbara J; Heim-Hall, Josefine; Varner, Michael W; Goldenberg, Robert L; Bukowski, Radek; Wapner, Ronald J; Drews-Botsch, Carolyn D; O'Brien, Barbara M; Dudley, Donald J; Levy, Brynn

2012-12-06

Genetic abnormalities have been associated with 6 to 13% of stillbirths, but the true prevalence may be higher. Unlike karyotype analysis, microarray analysis does not require live cells, and it detects small deletions and duplications called copy-number variants. The Stillbirth Collaborative Research Network conducted a population-based study of stillbirth in five geographic catchment areas. Standardized postmortem examinations and karyotype analyses were performed. A single-nucleotide polymorphism array was used to detect copy-number variants of at least 500 kb in placental or fetal tissue. Variants that were not identified in any of three databases of apparently unaffected persons were then classified into three groups: probably benign, clinical significance unknown, or pathogenic. We compared the results of karyotype and microarray analyses of samples obtained after delivery. In our analysis of samples from 532 stillbirths, microarray analysis yielded results more often than did karyotype analysis (87.4% vs. 70.5%, P<0.001) and provided better detection of genetic abnormalities (aneuploidy or pathogenic copy-number variants, 8.3% vs. 5.8%; P=0.007). Microarray analysis also identified more genetic abnormalities among 443 antepartum stillbirths (8.8% vs. 6.5%, P=0.02) and 67 stillbirths with congenital anomalies (29.9% vs. 19.4%, P=0.008). As compared with karyotype analysis, microarray analysis provided a relative increase in the diagnosis of genetic abnormalities of 41.9% in all stillbirths, 34.5% in antepartum stillbirths, and 53.8% in stillbirths with anomalies. Microarray analysis is more likely than karyotype analysis to provide a genetic diagnosis, primarily because of its success with nonviable tissue, and is especially valuable in analyses of stillbirths with congenital anomalies or in cases in which karyotype results cannot be obtained. (Funded by the Eunice Kennedy Shriver National Institute of Child Health and Human Development.).

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 genetic aberrations on chromosome 22 outside the NF2 locus in schwannomatosis and neurofibromatosis type 2.

PubMed

Buckley, Patrick G; Mantripragada, Kiran K; Díaz de Ståhl, Teresita; Piotrowski, Arkadiusz; Hansson, Caisa M; Kiss, Hajnalka; Vetrie, David; Ernberg, Ingemar T; Nordenskjöld, Magnus; Bolund, Lars; Sainio, Markku; Rouleau, Guy A; Niimura, Michihito; Wallace, Andrew J; Evans, D Gareth R; Grigelionis, Gintautas; Menzel, Uwe; Dumanski, Jan P

2005-12-01

Schwannomatosis is characterized by multiple peripheral and cranial nerve schwannomas that occur in the absence of bilateral 8th cranial nerve schwannomas. The latter is the main diagnostic criterion of neurofibromatosis type 2 (NF2), which is a related but distinct disorder. The genetic factors underlying the differences between schwannomatosis and NF2 are poorly understood, although available evidence implicates chromosome 22 as the primary location of the gene(s) of interest. To investigate this, we comprehensively profiled the DNA copy number in samples from sporadic and familial schwannomatosis, NF2, and a large cohort of normal controls. Using a tiling-path chromosome 22 genomic array, we identified two candidate regions of copy number variation, which were further characterized by a PCR-based array with higher resolution. The latter approach allows the detection of minute alterations in total genomic DNA, with as little as 1.5 kb per measurement point of nonredundant sequence on the array. In DNA derived from peripheral blood from a schwannomatosis patient and a sporadic schwannoma sample, we detected rearrangements of the immunoglobulin lambda (IGL) locus, which is unlikely to be due to a B-cell specific somatic recombination of IGL. Analysis of normal controls indicated that these IGL rearrangements were restricted to schwannomatosis/schwannoma samples. In the second candidate region spanning GSTT1 and CABIN1 genes, we observed a frequent copy number polymorphism at the GSTT1 locus. We further describe missense mutations in the CABIN1 gene that are specific to samples from schwannomatosis and NF2 and make this gene a plausible candidate for contributing to the pathogenesis of these disorders. Copyright 2005 Wiley-Liss, Inc.

Common variants of the BRCA1 wild-type allele modify the risk of breast cancer in BRCA1 mutation carriers

PubMed Central

Cox, David G.; Simard, Jacques; Sinnett, Daniel; Hamdi, Yosr; Soucy, Penny; Ouimet, Manon; Barjhoux, Laure; Verny-Pierre, Carole; McGuffog, Lesley; Healey, Sue; Szabo, Csilla; Greene, Mark H.; Mai, Phuong L.; Andrulis, Irene L.; Thomassen, Mads; Gerdes, Anne-Marie; Caligo, Maria A.; Friedman, Eitan; Laitman, Yael; Kaufman, Bella; Paluch, Shani S.; Borg, Åke; Karlsson, Per; Stenmark Askmalm, Marie; Barbany Bustinza, Gisela; Nathanson, Katherine L.; Domchek, Susan M.; Rebbeck, Timothy R.; Benítez, Javier; Hamann, Ute; Rookus, Matti A.; van den Ouweland, Ans M.W.; Ausems, Margreet G.E.M.; Aalfs, Cora M.; van Asperen, Christi J.; Devilee, Peter; Gille, Hans J.J.P.; Peock, Susan; Frost, Debra; Evans, D. Gareth; Eeles, Ros; Izatt, Louise; Adlard, Julian; Paterson, Joan; Eason, Jacqueline; Godwin, Andrew K.; Remon, Marie-Alice; Moncoutier, Virginie; Gauthier-Villars, Marion; Lasset, Christine; Giraud, Sophie; Hardouin, Agnès; Berthet, Pascaline; Sobol, Hagay; Eisinger, François; Bressac de Paillerets, Brigitte; Caron, Olivier; Delnatte, Capucine; Goldgar, David; Miron, Alex; Ozcelik, Hilmi; Buys, Saundra; Southey, Melissa C.; Terry, Mary Beth; Singer, Christian F.; Dressler, Anne-Catharina; Tea, Muy-Kheng; Hansen, Thomas V.O.; Johannsson, Oskar; Piedmonte, Marion; Rodriguez, Gustavo C.; Basil, Jack B.; Blank, Stephanie; Toland, Amanda E.; Montagna, Marco; Isaacs, Claudine; Blanco, Ignacio; Gayther, Simon A.; Moysich, Kirsten B.; Schmutzler, Rita K.; Wappenschmidt, Barbara; Engel, Christoph; Meindl, Alfons; Ditsch, Nina; Arnold, Norbert; Niederacher, Dieter; Sutter, Christian; Gadzicki, Dorothea; Fiebig, Britta; Caldes, Trinidad; Laframboise, Rachel; Nevanlinna, Heli; Chen, Xiaoqing; Beesley, Jonathan; Spurdle, Amanda B.; Neuhausen, Susan L.; Ding, Yuan C.; Couch, Fergus J.; Wang, Xianshu; Peterlongo, Paolo; Manoukian, Siranoush; Bernard, Loris; Radice, Paolo; Easton, Douglas F.; Chenevix-Trench, Georgia; Antoniou, Antonis C.; Stoppa-Lyonnet, Dominique; Mazoyer, Sylvie; Sinilnikova, Olga M.

2011-01-01

Mutations in the BRCA1 gene substantially increase a woman's lifetime risk of breast cancer. However, there is great variation in this increase in risk with several genetic and non-genetic modifiers identified. The BRCA1 protein plays a central role in DNA repair, a mechanism that is particularly instrumental in safeguarding cells against tumorigenesis. We hypothesized that polymorphisms that alter the expression and/or function of BRCA1 carried on the wild-type (non-mutated) copy of the BRCA1 gene would modify the risk of breast cancer in carriers of BRCA1 mutations. A total of 9874 BRCA1 mutation carriers were available in the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA) for haplotype analyses of BRCA1. Women carrying the rare allele of single nucleotide polymorphism rs16942 on the wild-type copy of BRCA1 were at decreased risk of breast cancer (hazard ratio 0.86, 95% confidence interval 0.77–0.95, P = 0.003). Promoter in vitro assays of the major BRCA1 haplotypes showed that common polymorphisms in the regulatory region alter its activity and that this effect may be attributed to the differential binding affinity of nuclear proteins. In conclusion, variants on the wild-type copy of BRCA1 modify risk of breast cancer among carriers of BRCA1 mutations, possibly by altering the efficiency of BRCA1 transcription. PMID:21890493

A genetic polymorphism in the sex-linked ATP5A1 gene is associated with individual fitness in Ovenbirds (Seiurus aurocapilla)

Treesearch

Judith D. Toms; Lori S. Eggert; Wayne J. Arendt; John Faaborg

2012-01-01

While testing genetic sexing techniques in Ovenbirds (Seiurus aurocapilla),we found a genetic polymorphism in the ATP5A1 gene in 38% of individuals. The Z ' allele included changes in both intronic and exonic portions of the sequenced region, but there was no evidence that this changed the resulting ATP synthase product. Males that had one or more copies of...

Detection and correction of false segmental duplications caused by genome mis-assembly

PubMed Central

2010-01-01

Diploid genomes with divergent chromosomes present special problems for assembly software as two copies of especially polymorphic regions may be mistakenly constructed, creating the appearance of a recent segmental duplication. We developed a method for identifying such false duplications and applied it to four vertebrate genomes. For each genome, we corrected mis-assemblies, improved estimates of the amount of duplicated sequence, and recovered polymorphisms between the sequenced chromosomes. PMID:20219098

Characterization of a new high copy Stowaway family MITE, BRAMI-1 in Brassica genome

PubMed Central

2013-01-01

Background Miniature inverted-repeat transposable elements (MITEs) are expected to play important roles in evolution of genes and genome in plants, especially in the highly duplicated plant genomes. Various MITE families and their roles in plants have been characterized. However, there have been fewer studies of MITE families and their potential roles in evolution of the recently triplicated Brassica genome. Results We identified a new MITE family, BRAMI-1, belonging to the Stowaway super-family in the Brassica genome. In silico mapping revealed that 697 members are dispersed throughout the euchromatic regions of the B. rapa pseudo-chromosomes. Among them, 548 members (78.6%) are located in gene-rich regions, less than 3 kb from genes. In addition, we identified 516 and 15 members in the 470 Mb and 15 Mb genomic shotgun sequences currently available for B. oleracea and B. napus, respectively. The resulting estimated copy numbers for the entire genomes were 1440, 1464 and 2490 in B. rapa, B. oleracea and B. napus, respectively. Concurrently, only 70 members of the related Arabidopsis ATTIRTA-1 MITE family were identified in the Arabidopsis genome. Phylogenetic analysis revealed that BRAMI-1 elements proliferated in the Brassica genus after divergence from the Arabidopsis lineage. MITE insertion polymorphism (MIP) was inspected for 50 BRAMI-1 members, revealing high levels of insertion polymorphism between and within species of Brassica that clarify BRAMI-1 activation periods up to the present. Comparative analysis of the 71 genes harbouring the BRAMI-1 elements with their non-insertion paralogs (NIPs) showed that the BRAMI-1 insertions mainly reside in non-coding sequences and that the expression levels of genes with the elements differ from those of their NIPs. Conclusion A Stowaway family MITE, named as BRAMI-1, was gradually amplified and remained present in over than 1400 copies in each of three Brassica species. Overall, 78% of the members were identified in gene-rich regions, and it is assumed that they may contribute to the evolution of duplicated genes in the highly duplicated Brassica genome. The resulting MIPs can serve as a good source of DNA markers for Brassica crops because the insertions are highly dispersed in the gene-rich euchromatin region and are polymorphic between or within species. PMID:23547712

Pooled Enrichment Sequencing Identifies Diversity and Evolutionary Pressures at NLR Resistance Genes within a Wild Tomato Population

PubMed Central

Stam, Remco; Scheikl, Daniela; Tellier, Aurélien

2016-01-01

Nod-like receptors (NLRs) are nucleotide-binding domain and leucine-rich repeats containing proteins that are important in plant resistance signaling. Many of the known pathogen resistance (R) genes in plants are NLRs and they can recognize pathogen molecules directly or indirectly. As such, divergence and copy number variants at these genes are found to be high between species. Within populations, positive and balancing selection are to be expected if plants coevolve with their pathogens. In order to understand the complexity of R-gene coevolution in wild nonmodel species, it is necessary to identify the full range of NLRs and infer their evolutionary history. Here we investigate and reveal polymorphism occurring at 220 NLR genes within one population of the partially selfing wild tomato species Solanum pennellii. We use a combination of enrichment sequencing and pooling ten individuals, to specifically sequence NLR genes in a resource and cost-effective manner. We focus on the effects which different mapping and single nucleotide polymorphism calling software and settings have on calling polymorphisms in customized pooled samples. Our results are accurately verified using Sanger sequencing of polymorphic gene fragments. Our results indicate that some NLRs, namely 13 out of 220, have maintained polymorphism within our S. pennellii population. These genes show a wide range of πN/πS ratios and differing site frequency spectra. We compare our observed rate of heterozygosity with expectations for this selfing and bottlenecked population. We conclude that our method enables us to pinpoint NLR genes which have experienced natural selection in their habitat. PMID:27189991

Polymorphisms in Plasmodium falciparum Chloroquine Resistance Transporter and Multidrug Resistance 1 Genes: Parasite Risk Factors that Affect Treatment Outcomes for P. falciparum Malaria after Artemether-Lumefantrine and Artesunate-Amodiaquine

PubMed Central

Venkatesan, Meera; Gadalla, Nahla B.; Stepniewska, Kasia; Dahal, Prabin; Nsanzabana, Christian; Moriera, Clarissa; Price, Ric N.; Mårtensson, Andreas; Rosenthal, Philip J.; Dorsey, Grant; Sutherland, Colin J.; Guérin, Philippe; Davis, Timothy M. E.; Ménard, Didier; Adam, Ishag; Ademowo, George; Arze, Cesar; Baliraine, Frederick N.; Berens-Riha, Nicole; Björkman, Anders; Borrmann, Steffen; Checchi, Francesco; Desai, Meghna; Dhorda, Mehul; Djimdé, Abdoulaye A.; El-Sayed, Badria B.; Eshetu, Teferi; Eyase, Frederick; Falade, Catherine; Faucher, Jean-François; Fröberg, Gabrielle; Grivoyannis, Anastasia; Hamour, Sally; Houzé, Sandrine; Johnson, Jacob; Kamugisha, Erasmus; Kariuki, Simon; Kiechel, Jean-René; Kironde, Fred; Kofoed, Poul-Erik; LeBras, Jacques; Malmberg, Maja; Mwai, Leah; Ngasala, Billy; Nosten, Francois; Nsobya, Samuel L.; Nzila, Alexis; Oguike, Mary; Otienoburu, Sabina Dahlström; Ogutu, Bernhards; Ouédraogo, Jean-Bosco; Piola, Patrice; Rombo, Lars; Schramm, Birgit; Somé, A. Fabrice; Thwing, Julie; Ursing, Johan; Wong, Rina P. M.; Zeynudin, Ahmed; Zongo, Issaka; Plowe, Christopher V.; Sibley, Carol Hopkins

2014-01-01

Adequate clinical and parasitologic cure by artemisinin combination therapies relies on the artemisinin component and the partner drug. Polymorphisms in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and P. falciparum multidrug resistance 1 (pfmdr1) genes are associated with decreased sensitivity to amodiaquine and lumefantrine, but effects of these polymorphisms on therapeutic responses to artesunate-amodiaquine (ASAQ) and artemether-lumefantrine (AL) have not been clearly defined. Individual patient data from 31 clinical trials were harmonized and pooled by using standardized methods from the WorldWide Antimalarial Resistance Network. Data for more than 7,000 patients were analyzed to assess relationships between parasite polymorphisms in pfcrt and pfmdr1 and clinically relevant outcomes after treatment with AL or ASAQ. Presence of the pfmdr1 gene N86 (adjusted hazards ratio = 4.74, 95% confidence interval = 2.29 – 9.78, P < 0.001) and increased pfmdr1 copy number (adjusted hazards ratio = 6.52, 95% confidence interval = 2.36–17.97, P < 0.001) were significant independent risk factors for recrudescence in patients treated with AL. AL and ASAQ exerted opposing selective effects on single-nucleotide polymorphisms in pfcrt and pfmdr1. Monitoring selection and responding to emerging signs of drug resistance are critical tools for preserving efficacy of artemisinin combination therapies; determination of the prevalence of at least pfcrt K76T and pfmdr1 N86Y should now be routine. PMID:25048375

Folate and One-Carbon Metabolism Gene Polymorphisms and Their Associations With Oral Facial Clefts

PubMed Central

Boyles, Abee L.; Wilcox, Allen J.; Taylor, Jack A.; Meyer, Klaus; Fredriksen, Åse; Ueland, Per Magne; Drevon, Christian A.; Vollset, Stein Emil; Lie, Rolv Terje

2008-01-01

Folate metabolism plays a critical role in embryonic development. Prenatal folate supplementation reduces the risk of neural tube defects and probably oral facial clefts. Previous studies of related metabolic genes have associated polymorphisms in cystathionine-beta-synthase (CBS) and 5,10-methylenetetrahydrofolate reductase (MTHFR) with cleft risk. We explored associations between genes related to one-carbon metabolism and clefts in a Norwegian population-based study that included 362 families with cleft lip with or without cleft palate (CL/P) and 191 families with cleft palate only (CPO). We previously showed a 39% reduction in risk of CL/P with folic acid supplementation in this population. In the present study we genotyped 12 polymorphisms in nine genes related to one-carbon metabolism and looked for associations of clefting risk with fetal polymorphisms, maternal polymorphisms, as well as parent-of-origin effects, using combined likelihood-ratio tests (LRT). We also stratified by maternal periconceptional intake of folic acid (>400 μg) to explore gene-exposure interactions. We found a reduced risk of CL/P with mothers who carried the CBS C699T variant (rs234706); relative risk was 0.94 with one copy of the T allele (95% CI 0.63-1.4) and 0.50 (95% CI 0.26-0.96) with two copies (P = 0.008). We found no evidence of interaction of this variant with folate status. We saw no evidence of risk from the MTHFR C677T variant (rs1801133) either overall or after stratifying by maternal folate intake. No associations were found between any of the polymorphisms and CPO. Genetic variations in the nine metabolic genes examined here do not confer a substantial degree of risk for clefts. Published 2008 Wiley-Liss, Inc.† PMID:18203168

Sequencing and annotation of the chloroplast DNAs and identification of polymorphisms distinguishing normal male-fertile and male-sterile cytoplasms of onion.

PubMed

von Kohn, Christopher; Kiełkowska, Agnieszka; Havey, Michael J

2013-12-01

Male-sterile (S) cytoplasm of onion is an alien cytoplasm introgressed into onion in antiquity and is widely used for hybrid seed production. Owing to the biennial generation time of onion, classical crossing takes at least 4 years to classify cytoplasms as S or normal (N) male-fertile. Molecular markers in the organellar DNAs that distinguish N and S cytoplasms are useful to reduce the time required to classify onion cytoplasms. In this research, we completed next-generation sequencing of the chloroplast DNAs of N- and S-cytoplasmic onions; we assembled and annotated the genomes in addition to identifying polymorphisms that distinguish these cytoplasms. The sizes (153 538 and 153 355 base pairs) and GC contents (36.8%) were very similar for the chloroplast DNAs of N and S cytoplasms, respectively, as expected given their close phylogenetic relationship. The size difference was primarily due to small indels in intergenic regions and a deletion in the accD gene of N-cytoplasmic onion. The structures of the onion chloroplast DNAs were similar to those of most land plants with large and small single copy regions separated by inverted repeats. Twenty-eight single nucleotide polymorphisms, two polymorphic restriction-enzyme sites, and one indel distributed across 20 chloroplast genes in the large and small single copy regions were selected and validated using diverse onion populations previously classified as N or S cytoplasmic using restriction fragment length polymorphisms. Although cytoplasmic male sterility is likely associated with the mitochondrial DNA, maternal transmission of the mitochondrial and chloroplast DNAs allows for polymorphisms in either genome to be useful for classifying onion cytoplasms to aid the development of hybrid onion cultivars.

CONAN: copy number variation analysis software for genome-wide association studies

PubMed Central

2010-01-01

Background Genome-wide association studies (GWAS) based on single nucleotide polymorphisms (SNPs) revolutionized our perception of the genetic regulation of complex traits and diseases. Copy number variations (CNVs) promise to shed additional light on the genetic basis of monogenic as well as complex diseases and phenotypes. Indeed, the number of detected associations between CNVs and certain phenotypes are constantly increasing. However, while several software packages support the determination of CNVs from SNP chip data, the downstream statistical inference of CNV-phenotype associations is still subject to complicated and inefficient in-house solutions, thus strongly limiting the performance of GWAS based on CNVs. Results CONAN is a freely available client-server software solution which provides an intuitive graphical user interface for categorizing, analyzing and associating CNVs with phenotypes. Moreover, CONAN assists the evaluation process by visualizing detected associations via Manhattan plots in order to enable a rapid identification of genome-wide significant CNV regions. Various file formats including the information on CNVs in population samples are supported as input data. Conclusions CONAN facilitates the performance of GWAS based on CNVs and the visual analysis of calculated results. CONAN provides a rapid, valid and straightforward software solution to identify genetic variation underlying the 'missing' heritability for complex traits that remains unexplained by recent GWAS. The freely available software can be downloaded at http://genepi-conan.i-med.ac.at. PMID:20546565

Genome-Wide Mapping of Copy Number Variation in Humans: Comparative Analysis of High Resolution Array Platforms

PubMed Central

Haraksingh, Rajini R.; Abyzov, Alexej; Gerstein, Mark; Urban, Alexander E.; Snyder, Michael

2011-01-01

Accurate and efficient genome-wide detection of copy number variants (CNVs) is essential for understanding human genomic variation, genome-wide CNV association type studies, cytogenetics research and diagnostics, and independent validation of CNVs identified from sequencing based technologies. Numerous, array-based platforms for CNV detection exist utilizing array Comparative Genome Hybridization (aCGH), Single Nucleotide Polymorphism (SNP) genotyping or both. We have quantitatively assessed the abilities of twelve leading genome-wide CNV detection platforms to accurately detect Gold Standard sets of CNVs in the genome of HapMap CEU sample NA12878, and found significant differences in performance. The technologies analyzed were the NimbleGen 4.2 M, 2.1 M and 3×720 K Whole Genome and CNV focused arrays, the Agilent 1×1 M CGH and High Resolution and 2×400 K CNV and SNP+CGH arrays, the Illumina Human Omni1Quad array and the Affymetrix SNP 6.0 array. The Gold Standards used were a 1000 Genomes Project sequencing-based set of 3997 validated CNVs and an ultra high-resolution aCGH-based set of 756 validated CNVs. We found that sensitivity, total number, size range and breakpoint resolution of CNV calls were highest for CNV focused arrays. Our results are important for cost effective CNV detection and validation for both basic and clinical applications. PMID:22140474

Mitochondrial genomic variation associated with higher mitochondrial copy number: the Cache County Study on Memory Health and Aging.

PubMed

Ridge, Perry G; Maxwell, Taylor J; Foutz, Spencer J; Bailey, Matthew H; Corcoran, Christopher D; Tschanz, JoAnn T; Norton, Maria C; Munger, Ronald G; O'Brien, Elizabeth; Kerber, Richard A; Cawthon, Richard M; Kauwe, John S K

2014-01-01

The mitochondria are essential organelles and are the location of cellular respiration, which is responsible for the majority of ATP production. Each cell contains multiple mitochondria, and each mitochondrion contains multiple copies of its own circular genome. The ratio of mitochondrial genomes to nuclear genomes is referred to as mitochondrial copy number. Decreases in mitochondrial copy number are known to occur in many tissues as people age, and in certain diseases. The regulation of mitochondrial copy number by nuclear genes has been studied extensively. While mitochondrial variation has been associated with longevity and some of the diseases known to have reduced mitochondrial copy number, the role that the mitochondrial genome itself has in regulating mitochondrial copy number remains poorly understood. We analyzed the complete mitochondrial genomes from 1007 individuals randomly selected from the Cache County Study on Memory Health and Aging utilizing the inferred evolutionary history of the mitochondrial haplotypes present in our dataset to identify sequence variation and mitochondrial haplotypes associated with changes in mitochondrial copy number. Three variants belonging to mitochondrial haplogroups U5A1 and T2 were significantly associated with higher mitochondrial copy number in our dataset. We identified three variants associated with higher mitochondrial copy number and suggest several hypotheses for how these variants influence mitochondrial copy number by interacting with known regulators of mitochondrial copy number. Our results are the first to report sequence variation in the mitochondrial genome that causes changes in mitochondrial copy number. The identification of these variants that increase mtDNA copy number has important implications in understanding the pathological processes that underlie these phenotypes.

Copy-number and gene dependency analysis reveals partial copy loss of wild-type SF3B1 as a novel cancer vulnerability. | Office of Cancer Genomics

Cancer.gov

Genomic instability is a hallmark of human cancer, and results in widespread somatic copy number alterations. We used a genome-scale shRNA viability screen in human cancer cell lines to systematically identify genes that are essential in the context of particular copy-number alterations (copy-number associated gene dependencies). The most enriched class of copy-number associated gene dependencies was CYCLOPS (Copy-number alterations Yielding Cancer Liabilities Owing to Partial losS) genes, and spliceosome components were the most prevalent.

SLC22A1-ABCB1 haplotype profiles predict imatinib pharmacokinetics in Asian patients with chronic myeloid leukemia.

PubMed

Singh, Onkar; Chan, Jason Yongsheng; Lin, Keegan; Heng, Charles Chuah Thuan; Chowbay, Balram

2012-01-01

This study aimed to explore the influence of SLC22A1, PXR, ABCG2, ABCB1 and CYP3A5 3 genetic polymorphisms on imatinib mesylate (IM) pharmacokinetics in Asian patients with chronic myeloid leukemia (CML). Healthy subjects belonging to three Asian populations (Chinese, Malay, Indian; n = 70 each) and CML patients (n = 38) were enrolled in a prospective pharmacogenetics study. Imatinib trough (C(0h)) and clearance (CL) were determined in the patients at steady state. Haplowalk method was applied to infer the haplotypes and generalized linear model (GLM) to estimate haplotypic effects on IM pharmacokinetics. Association of haplotype copy numbers with IM pharmacokinetics was defined by Mann-Whitney U test. Global haplotype score statistics revealed a SLC22A1 sub-haplotypic region encompassing three polymorphisms (rs3798168, rs628031 and IVS7+850C>T), to be significantly associated with IM clearance (p = 0.013). Haplotype-specific GLM estimated that the haplotypes AGT and CGC were both associated with 22% decrease in clearance compared to CAC [CL (10(-2) L/hr/mg): CAC vs AGT: 4.03 vs 3.16, p = 0.017; CAC vs CGC: 4.03 vs 3.15, p = 0.017]. Patients harboring 2 copies of AGT or CGC haplotypes had 33.4% lower clearance and 50% higher C(0h) than patients carrying 0 or 1 copy [CL (10(-2) L/hr/mg): 2.19 vs 3.29, p = 0.026; C(0h) (10(-6) 1/ml): 4.76 vs 3.17, p = 0.013, respectively]. Further subgroup analysis revealed SLC22A1 and ABCB1 haplotypic combinations to be significantly associated with clearance and C(0h) (p = 0.002 and 0.009, respectively). This exploratory study suggests that SLC22A1-ABCB1 haplotypes may influence IM pharmacokinetics in Asian CML patients.

DangerTrack: A scoring system to detect difficult-to-assess regions.

PubMed

Dolgalev, Igor; Sedlazeck, Fritz; Busby, Ben

2017-01-01

Over recent years, multiple groups have shown that a large number of structural variants, repeats, or problems with the underlying genome assembly have dramatic effects on the mapping, calling, and overall reliability of single nucleotide polymorphism calls. This project endeavored to develop an easy-to-use track for looking at structural variant and repeat regions. This track, DangerTrack, can be displayed alongside the existing Genome Reference Consortium assembly tracks to warn clinicians and biologists when variants of interest may be incorrectly called, of dubious quality, or on an insertion or copy number expansion. While mapping and variant calling can be automated, it is our opinion that when these regions are of interest to a particular clinical or research group, they warrant a careful examination, potentially involving localized reassembly. DangerTrack is available at https://github.com/DCGenomics/DangerTrack.

Development of universal genetic markers based on single-copy orthologous (COSII) genes in Poaceae.

PubMed

Liu, Hailan; Guo, Xiaoqin; Wu, Jiasheng; Chen, Guo-Bo; Ying, Yeqing

2013-03-01

KEY MESSAGE : We develop a set of universal genetic markers based on single-copy orthologous (COSII) genes in Poaceae. Being evolutionary conserved, single-copy orthologous (COSII) genes are particularly useful in comparative mapping and phylogenetic investigation among species. In this study, we identified 2,684 COSII genes based on five sequenced Poaceae genomes including rice, maize, sorghum, foxtail millet, and brachypodium, and then developed 1,072 COSII markers whose transferability and polymorphism among five bamboo species were further evaluated with 46 pairs of randomly selected primers. 91.3 % of the 46 primers obtained clear amplification in at least one bamboo species, and 65.2 % of them produced polymorphism in more than one species. We also used 42 of them to construct the phylogeny for the five bamboo species, and it might reflect more precise evolutionary relationship than the one based on the vegetative morphology. The results indicated a promising prospect of applying these markers to the investigation of genetic diversity and the classification of Poaceae. To ease and facilitate access of the information of common interest to readers, a web-based database of the COSII markers is provided ( http://www.sicau.edu.cn/web/yms/PCOSWeb/PCOS.html ).

Evaluation of the class II region of the major histocompatibility complex of the greyhound with the genomic matching technique and sequence-based typing.

PubMed

Fliegner, R A; Holloway, S A; Lester, S; McLure, C A; Dawkins, R L

2008-08-01

The class II region of the major histocompatibility complex was evaluated in 25 greyhounds by sequence-based typing and the genomic matching technique (GMT). Two new DLA-DRB1 alleles were identified. Twenty-four dogs carried the DLA-DRB1*01201/DQA1*00401/DQB1*01303/DQB1*01701 haplotype, which carries two DQB1 alleles. One haplotype was identified from which DQB1 and DQA1 appeared to be deleted. The GMT enabled detection of DQB1 copy number, discrimination of the different class II haplotypes and the identification of new, possibly biologically relevant polymorphisms.

Combined array CGH plus SNP genome analyses in a single assay for optimized clinical testing

PubMed Central

Wiszniewska, Joanna; Bi, Weimin; Shaw, Chad; Stankiewicz, Pawel; Kang, Sung-Hae L; Pursley, Amber N; Lalani, Seema; Hixson, Patricia; Gambin, Tomasz; Tsai, Chun-hui; Bock, Hans-Georg; Descartes, Maria; Probst, Frank J; Scaglia, Fernando; Beaudet, Arthur L; Lupski, James R; Eng, Christine; Wai Cheung, Sau; Bacino, Carlos; Patel, Ankita

2014-01-01

In clinical diagnostics, both array comparative genomic hybridization (array CGH) and single nucleotide polymorphism (SNP) genotyping have proven to be powerful genomic technologies utilized for the evaluation of developmental delay, multiple congenital anomalies, and neuropsychiatric disorders. Differences in the ability to resolve genomic changes between these arrays may constitute an implementation challenge for clinicians: which platform (SNP vs array CGH) might best detect the underlying genetic cause for the disease in the patient? While only SNP arrays enable the detection of copy number neutral regions of absence of heterozygosity (AOH), they have limited ability to detect single-exon copy number variants (CNVs) due to the distribution of SNPs across the genome. To provide comprehensive clinical testing for both CNVs and copy-neutral AOH, we enhanced our custom-designed high-resolution oligonucleotide array that has exon-targeted coverage of 1860 genes with 60 000 SNP probes, referred to as Chromosomal Microarray Analysis – Comprehensive (CMA-COMP). Of the 3240 cases evaluated by this array, clinically significant CNVs were detected in 445 cases including 21 cases with exonic events. In addition, 162 cases (5.0%) showed at least one AOH region >10 Mb. We demonstrate that even though this array has a lower density of SNP probes than other commercially available SNP arrays, it reliably detected AOH events >10 Mb as well as exonic CNVs beyond the detection limitations of SNP genotyping. Thus, combining SNP probes and exon-targeted array CGH into one platform provides clinically useful genetic screening in an efficient manner. PMID:23695279

Genome-wide analysis of esophageal adenocarcinoma yields specific copy number aberrations that correlate with prognosis.

PubMed

Frankel, Adam; Armour, Nicola; Nancarrow, Derek; Krause, Lutz; Hayward, Nicholas; Lampe, Guy; Smithers, B Mark; Barbour, Andrew

2014-04-01

The incidence of esophageal adenocarcinoma (EAC) has been increasing rapidly for the past 3 decades in Western (Caucasian) populations. Curative treatment is based around esophagectomy, which has a major impact on quality of life. For those suitable for treatment with curative intent, 5-year survival is ∼30%. More accurate prognostic tools are therefore needed, and copy number aberrations (CNAs) may offer the ability to act as prospective biomarkers in this regard. We performed a genome-wide examination of CNAs in 54 samples of EAC using single-nucleotide polymorphism (SNP) arrays. Our aims were to describe frequent regions of CNA, to define driver CNAs, and to identify CNAs that correlated with survival. Regions of frequent amplification included oncogenes such as EGFR, MYC, KLF12, and ERBB2, while frequently deleted regions included tumor suppressor genes such as CDKN2A/B, PTPRD, FHIT, and SMAD4. The genomic identification of significant targets in cancer (GISTIC) algorithm identified 24 regions of gain and 28 regions of loss that were likely to contain driver changes. We discovered 61 genes in five regions that, when stratified by CNA type (gain or loss), correlated with a statistically significant difference in survival. Pathway analysis of the genes residing in both the GISTIC and prognostic regions showed they were significantly enriched for cancer-related networks. Finally, we discovered that copy-neutral loss of heterozygosity is a frequent mechanism of CNA in genes currently targetable by chemotherapy, potentially leading to under-reporting of cases suitable for such treatment. Copyright © 2014 Wiley Periodicals, Inc.

Synthetic and Evolutionary Construction of a Chlorate-Reducing Shewanella oneidensis MR-1

PubMed Central

Clark, Iain C.; Melnyk, Ryan A.; Youngblut, Matthew D.; Carlson, Hans K.; Iavarone, Anthony T.

2015-01-01

ABSTRACT Despite evidence for the prevalence of horizontal gene transfer of respiratory genes, little is known about how pathways functionally integrate within new hosts. One example of a mobile respiratory metabolism is bacterial chlorate reduction, which is frequently encoded on composite transposons. This implies that the essential components of the metabolism are encoded on these mobile elements. To test this, we heterologously expressed genes for chlorate reduction from Shewanella algae ACDC in the non-chlorate-reducing Shewanella oneidensis MR-1. The construct that ultimately endowed robust growth on chlorate included cld, a cytochrome c gene, clrABDC, and two genes of unknown function. Although strain MR-1 was unable to grow on chlorate after initial insertion of these genes into the chromosome, 11 derived strains capable of chlorate respiration were obtained through adaptive evolution. Genome resequencing indicated that all of the evolved chlorate-reducing strains replicated a large genomic region containing chlorate reduction genes. Contraction in copy number and loss of the ability to reduce chlorate were also observed, indicating that this phenomenon was extremely dynamic. Although most strains contained more than six copies of the replicated region, a single strain with less duplication also grew rapidly. This strain contained three additional mutations that we hypothesized compensated for the low copy number. We remade the mutations combinatorially in the unevolved strain and determined that a single nucleotide polymorphism (SNP) upstream of cld enabled growth on chlorate and was epistatic to a second base pair change in the NarP binding sequence between narQP and nrfA that enhanced growth. PMID:25991681

Comprehensive Survey of Genetic Diversity in Chloroplast Genomes and 45S nrDNAs within Panax ginseng Species

PubMed Central

Kim, Kyunghee; Lee, Sang-Choon; Lee, Junki; Lee, Hyun Oh; Joh, Ho Jun; Kim, Nam-Hoon; Park, Hyun-Seung; Yang, Tae-Jin

2015-01-01

We report complete sequences of chloroplast (cp) genome and 45S nuclear ribosomal DNA (45S nrDNA) for 11 Panax ginseng cultivars. We have obtained complete sequences of cp and 45S nrDNA, the representative barcoding target sequences for cytoplasm and nuclear genome, respectively, based on low coverage NGS sequence of each cultivar. The cp genomes sizes ranged from 156,241 to 156,425 bp and the major size variation was derived from differences in copy number of tandem repeats in the ycf1 gene and in the intergenic regions of rps16-trnUUG and rpl32-trnUAG. The complete 45S nrDNA unit sequences were 11,091 bp, representing a consensus single transcriptional unit with an intergenic spacer region. Comparative analysis of these sequences as well as those previously reported for three Chinese accessions identified very rare but unique polymorphism in the cp genome within P. ginseng cultivars. There were 12 intra-species polymorphisms (six SNPs and six InDels) among 14 cultivars. We also identified five SNPs from 45S nrDNA of 11 Korean ginseng cultivars. From the 17 unique informative polymorphic sites, we developed six reliable markers for analysis of ginseng diversity and cultivar authentication. PMID:26061692

Copy number variation and missense mutations of the agouti signaling protein (ASIP) gene in goat breeds with different coat colors.

PubMed

Fontanesi, L; Beretti, F; Riggio, V; Gómez González, E; Dall'Olio, S; Davoli, R; Russo, V; Portolano, B

2009-01-01

In goats, classical genetic studies reported a large number of alleles at the Agouti locus with effects on coat color and pattern distribution. From these early studies, the dominant A(Wt) (white/tan) allele was suggested to cause the white color of the Saanen breed. Here, we sequenced the coding region of the goat ASIP gene in 6 goat breeds (Girgentana, Maltese, Derivata di Siria, Murciano-Granadina, Camosciata delle Alpi, and Saanen), with different coat colors and patterns. Five single nucleotide polymorphisms (SNPs) were identified, 3 of which caused missense mutations in conserved positions of the cysteine-rich carboxy-terminal domain of the protein (p.Ala96Gly, p.Cys126Gly, and p.Val128Gly). Allele and genotype frequencies suggested that these mutations are not associated or not completely associated with coat color in the investigated goat breeds. Moreover, genotyping and sequencing results, deviation from Hardy-Weinberg equilibrium, as well as allele copy number evaluation from semiquantitative fluorescent multiplex PCR, indicated the presence of copy number variation (CNV) in all investigated breeds. To confirm the presence of CNV and evaluate its extension, we applied a bovine-goat cross-species array comparative genome hybridization (aCGH) experiment using a custom tiling array based on bovine chromosome 13. aCGH results obtained for 8 goat DNA samples confirmed the presence of CNV affecting a region of less that 100 kb including the ASIP and AHCY genes. In Girgentana and Saanen breeds, this CNV might cause the A(Wt) allele, as already suggested for a similar structural mutation in sheep affecting the ASIP and AHCY genes, providing evidence for a recurrent interspecies CNV. However, other mechanisms may also be involved in determining coat color in these 2 breeds. Copyright 2009 S. Karger AG, Basel.

Impact of Parental Bos taurus and Bos indicus Origins on Copy Number Variation in Traditional Chinese Cattle Breeds

PubMed Central

Zhang, Liangzhi; Jia, Shangang; Plath, Martin; Huang, Yongzhen; Li, Congjun; Lei, Chuzhao; Zhao, Xin; Chen, Hong

2015-01-01

Copy number variation (CNV) is an important component of genomic structural variation and plays a role not only in evolutionary diversification but also in domestication. Chinese cattle were derived from Bos taurus and Bos indicus, and several breeds presumably are of hybrid origin, but the evolution of CNV regions (CNVRs) has not yet been examined in this context. Here, we of CNVRs, mtDNA D-loop sequence variation, and Y-chromosomal single nucleotide polymorphisms to assess the impact of maternal and paternal B. taurus and B. indicus origins on the distribution of CNVRs in 24 Chinese domesticated bulls. We discovered 470 genome-wide CNVRs, only 72 of which were shared by all three Y-lineages (B. taurus: Y1, Y2; B. indicus: Y3), whereas 265 were shared by inferred taurine or indicine paternal lineages, and 228 when considering their maternal taurine or indicine origins. Phylogenetic analysis uncovered eight taurine/indicine hybrids, and principal component analysis on CNVs corroborated genomic exchange during hybridization. The distribution patterns of CNVRs tended to be lineage-specific, and correlation analysis revealed significant positive or negative co-occurrences of CNVRs across lineages. Our study suggests that CNVs in Chinese cattle partly result from selective breeding during domestication, but also from hybridization and introgression. PMID:26260653

A genome-wide assessment of rare copy number variants in colorectal cancer.

PubMed

Li, Zhenli; Yu, Dan; Gan, Meifu; Shan, Qiaonan; Yin, Xiaoyang; Tang, Shunli; Zhang, Shuai; Shi, Yongyong; Zhu, Yimin; Lai, Maode; Zhang, Dandan

2015-09-22

Colorectal cancer (CRC) is a complex disease with an estimated heritability of approximately 35%. However, known CRC-related common single nucleotide polymorphisms (SNPs) can only explain ~0.65% of the heritability. This "missing heritability" may be explained partially by rare copy number variants (CNVs). In this study, we performed a genome-wide scan using Illumina Human-Omni Express BeadChip, 694 sporadic CRC cases and 1641 controls were eventually included in our analysis after quality control. The global burden analysis revealed a 1.53-fold excess of rare CNVs in CRC cases compared with controls (P < 1 × 10(-6)), and the difference being more pronounced for genic rare CNVs and CNVs overlapped with coding regions (1.65-fold and 1.84-fold, respectively, both P < 1 × 10(-6)). Interestingly, both the cases in the lowest and middle tertile of age carried a higher burden of rare CNVs comparing to the highest tertile. Furthermore, 639 CNV-disrupted genes exclusive to CRC cases were found to be significantly enriched in gene ontology (GO) terms concerning nucleosome assembly and olfactory receptor activity. Our study was the first to evaluate the burden of rare CNVs in sporadic CRC and suggested that rare CNVs contributed to the missing heritability of CRC.

Improved Multiplex Ligation-dependent Probe Amplification (i-MLPA) for rapid copy number variant (CNV) detection.

PubMed

Saxena, Sonal; Gowdhaman, Kavitha; Kkani, Poornima; Vennapusa, Bhavyasri; Rama Subramanian, Chellamuthu; Ganesh Kumar, S; Mohan, Kommu Naga

2015-10-23

In Multiplex Ligation-dependent Probe Amplification (MLPA), copy number variants (CNVs) for specific genes are identified after normalization of the amounts of PCR products from ligated reference probes hybridized to genomic regions that are ideally free from normal variation. However, we observed ambiguous calls for two reference probes in an investigation of the human 15q11.2 region by MLPA among 20 controls, due to the presence of single nucleotide polymorphisms (SNPs) in the probe-binding regions. Further in silico analysis revealed that 18 out of 19 reference probes hybridize to regions subject to variation, underlining the requirement for designing new reference probes against variation-free regions. An improved MLPA (i-MLPA) method was developed by generating a new set of reference probes to reduce the chances of ambiguous calls and new reagents that reduce hybridization times to 30 min from 16h to obtain MLPA ratio data within 6h. Using i-MLPA, we screened 240 schizophrenia patients for CNVs in 15q11.2 region. Three deletions and two duplications were identified among the 240 schizophrenia patients. No variation was observed for the new reference probes. Taken together, i-MLPA procedure helps obtaining non-ambiguous CNV calls within 6h without compromising accuracy. Copyright © 2015 Elsevier B.V. All rights reserved.

Mixed Sequence Reader: A Program for Analyzing DNA Sequences with Heterozygous Base Calling

PubMed Central

Chang, Chun-Tien; Tsai, Chi-Neu; Tang, Chuan Yi; Chen, Chun-Houh; Lian, Jang-Hau; Hu, Chi-Yu; Tsai, Chia-Lung; Chao, Angel; Lai, Chyong-Huey; Wang, Tzu-Hao; Lee, Yun-Shien

2012-01-01

The direct sequencing of PCR products generates heterozygous base-calling fluorescence chromatograms that are useful for identifying single-nucleotide polymorphisms (SNPs), insertion-deletions (indels), short tandem repeats (STRs), and paralogous genes. Indels and STRs can be easily detected using the currently available Indelligent or ShiftDetector programs, which do not search reference sequences. However, the detection of other genomic variants remains a challenge due to the lack of appropriate tools for heterozygous base-calling fluorescence chromatogram data analysis. In this study, we developed a free web-based program, Mixed Sequence Reader (MSR), which can directly analyze heterozygous base-calling fluorescence chromatogram data in .abi file format using comparisons with reference sequences. The heterozygous sequences are identified as two distinct sequences and aligned with reference sequences. Our results showed that MSR may be used to (i) physically locate indel and STR sequences and determine STR copy number by searching NCBI reference sequences; (ii) predict combinations of microsatellite patterns using the Federal Bureau of Investigation Combined DNA Index System (CODIS); (iii) determine human papilloma virus (HPV) genotypes by searching current viral databases in cases of double infections; (iv) estimate the copy number of paralogous genes, such as β-defensin 4 (DEFB4) and its paralog HSPDP3. PMID:22778697

Obesity with associated developmental delay and/or learning disability in patients exhibiting additional features: report of novel pathogenic copy number variants.

PubMed

D'Angelo, Carla Sustek; Kohl, Ilana; Varela, Monica Castro; de Castro, Cláudia Irene Emílio; Kim, Chong Ae; Bertola, Débora Romeo; Lourenço, Charles Marques; Perez, Ana Beatriz Alvarez; Koiffmann, Celia Priszkulnik

2013-03-01

Obesity is a major threat to public health worldwide, and there is now mounting evidence favoring a role for the central nervous system (CNS) in weight control. A causal relationship has been recognized in both monogenic (e.g., BDNF, TRKB, and SIM1 deficiencies) and syndromic forms of obesity [e.g., Prader-Willi syndrome (PWS)]. Syndromic obesity arising from chromosomal abnormalities, that typically also affect learning and development, are often associated with congenital malformations and behavioral characteristics. We report on nine unrelated patients with a diagnosis of learning disability and/or developmental delay (DD) in addition to obesity that were found to have copy number variants (CNVs) by single nucleotide polymorphism array-based analysis. Each patient also had a distinct and complex phenotype, and most had hypotonia and other neuroendocrine issues, such as hyperphagia and hypogonadism. Molecular and clinical characterization of these patients enabled us to determine with confidence that the CNVs we observed were pathogenic or likely to be pathogenic. Overall, the CNVs reported here encompassed a candidate gene or region (e.g., SIM1) that has been reported in patients associating obesity and DD and/or intellectual disability (ID) and novel candidate genes and regions. Copyright © 2013 Wiley Periodicals, Inc.

Length Variation, Heteroplasmy and Sequence Divergence in the Mitochondrial DNA of Four Species of Sturgeon (Acipenser)

PubMed Central

Brown, J. R.; Beckenbach, K.; Beckenbach, A. T.; Smith, M. J.

1996-01-01

The extent of mtDNA length variation and heteroplasmy as well as DNA sequences of the control region and two tRNA genes were determined for four North American sturgeon species: Acipenser transmontanus, A. medirostris, A. fulvescens and A. oxyrhnychus. Across the Continental Divide, a division in the occurrence of length variation and heteroplasmy was observed that was concordant with species biogeography as well as with phylogenies inferred from restriction fragment length polymorphisms (RFLP) of whole mtDNA and pairwise comparisons of unique sequences of the control region. In all species, mtDNA length variation was due to repeated arrays of 78-82-bp sequences each containing a D-loop strand synthesis termination associated sequence (TAS). Individual repeats showed greater sequence conservation within individuals and species rather than between species, which is suggestive of concerted evolution. Differences in the frequencies of multiple copy genomes and heteroplasmy among the four species may be ascribed to differences in the rates of recurrent mutation. A mechanism that may offset the high rate of mutation for increased copy number is suggested on the basis that an increase in the number of functional TAS motifs might reduce the frequency of successfully initiated H-strand replications. PMID:8852850

GPHMM: an integrated hidden Markov model for identification of copy number alteration and loss of heterozygosity in complex tumor samples using whole genome SNP arrays

PubMed Central

Li, Ao; Liu, Zongzhi; Lezon-Geyda, Kimberly; Sarkar, Sudipa; Lannin, Donald; Schulz, Vincent; Krop, Ian; Winer, Eric; Harris, Lyndsay; Tuck, David

2011-01-01

There is an increasing interest in using single nucleotide polymorphism (SNP) genotyping arrays for profiling chromosomal rearrangements in tumors, as they allow simultaneous detection of copy number and loss of heterozygosity with high resolution. Critical issues such as signal baseline shift due to aneuploidy, normal cell contamination, and the presence of GC content bias have been reported to dramatically alter SNP array signals and complicate accurate identification of aberrations in cancer genomes. To address these issues, we propose a novel Global Parameter Hidden Markov Model (GPHMM) to unravel tangled genotyping data generated from tumor samples. In contrast to other HMM methods, a distinct feature of GPHMM is that the issues mentioned above are quantitatively modeled by global parameters and integrated within the statistical framework. We developed an efficient EM algorithm for parameter estimation. We evaluated performance on three data sets and show that GPHMM can correctly identify chromosomal aberrations in tumor samples containing as few as 10% cancer cells. Furthermore, we demonstrated that the estimation of global parameters in GPHMM provides information about the biological characteristics of tumor samples and the quality of genotyping signal from SNP array experiments, which is helpful for data quality control and outlier detection in cohort studies. PMID:21398628

Candida species biofilm and Candida albicans ALS3 polymorphisms in clinical isolates

PubMed Central

Bruder-Nascimento, Ariane; Camargo, Carlos Henrique; Mondelli, Alessandro Lia; Sugizaki, Maria Fátima; Sadatsune, Terue; Bagagli, Eduardo

2014-01-01

Over the last decades, there have been important changes in the epidemiology of Candida infections. In recent years, Candida species have emerged as important causes of invasive infections mainly among immunocompromised patients. This study analyzed Candida spp. isolates and compared the frequency and biofilm production of different species among the different sources of isolation: blood, urine, vulvovaginal secretions and peritoneal dialysis fluid. Biofilm production was quantified in 327 Candida isolates obtained from patients attended at a Brazilian tertiary public hospital (Botucatu, Sao Paulo). C. albicans ALS3 gene polymorphism was also evaluated by determining the number of repeated motifs in the central domain. Of the 198 total biofilm-positive isolates, 72 and 126 were considered as low and high biofilm producers, respectively. Biofilm production by C. albicans was significantly lower than that by non-albicans isolates and was most frequently observed in C. tropicalis. Biofilm production was more frequent among bloodstream isolates than other clinical sources, in urine, the isolates displayed a peculiar distribution by presenting two distinct peaks, one containing biofilm-negative isolates and the other containing isolates with intense biofilm production. The numbers of tandem-repeat copies per allele were not associated with biofilm production, suggesting the evolvement of other genetic determinants. PMID:25763043

Interactions between Early Parenting and a Polymorphism of the Child’s Dopamine Transporter Gene in Predicting Future Child Conduct Disorder Symptoms

PubMed Central

Lahey, Benjamin B.; Rathouz, Paul J.; Lee, Steve S.; Chronis-Tuscano, Andrea; Pelham, William E.; Waldman, Irwin D.; Cook, Edwin H.

2010-01-01

Mounting evidence suggests that genetic risks for mental disorders often interact with the social environment, but most studies still ignore environmental moderation of genetic influences. We tested interactions between maternal parenting and the variable number tandem repeat (VNTR) polymorphism in the 3′ untranslated region (UTR) of the dopamine transporter gene in the child to increase understanding of gene-environment interactions involving early parenting. Participants were part of a 9-year longitudinal study of 4–6-year-old children who met criteria for attention-deficit/hyperactivity disorder (ADHD) and demographically matched controls. Maternal parenting was observed during standard mother-child interactions in wave 1. The child’s conduct disorder (CD) symptoms 5–8 years later were measured using separate structured diagnostic interviews of the mother and youth. Controlling for ADHD symptoms and child disruptive behavior during the mother-child interaction, there was a significant inverse relation between levels of both positive and negative parenting at 4–6 years and the number of later CD symptoms, but primarily among children with two copies of the 9-repeat allele of the VNTR. The significant interaction with negative parenting was replicated in parent and youth reports of CD symptoms separately. PMID:21171728

Restriction Fragment Length Polymorphism Analysis Reveals High Levels of Genetic Divergence Among the Light Organ Symbionts of Flashlight Fish.

PubMed

Wolfe, C J; Haygood, M G

1991-08-01

Restriction fragment length polymorphisms within the lux and 16S ribosomal RNA gene regions were used to compare unculturable bacterial light organ symbionts of several anomalopid fish species. The method of Nei and Li (1979) was used to calculate phylogenetic distance from the patterns of restriction fragment lengths of the luxA and 16S rRNA regions. Phylogenetic trees constructed from each distance matrix (luxA and 16S rDNA data) have similar branching orders. The levels of divergence among the symbionts, relative to other culturable luminous bacteria, suggests that the symbionts differ at the level of species among host fish genera. Symbiont relatedness and host geographic location do not seem to be correlated, and the symbionts do not appear to be strains of common, free-living, luminous bacteria. In addition, the small number of hybridizing fragments within the 16S rRNA region of the symbionts, compared with that of the free-living species, suggests a decrease in copy number of rRNA operons relative to free-living species. At this level of investigation, the symbiont phylogeny is consistent with the proposed phylogeny of the host fish family and suggests that each symbiont strain coevolved with its host fish species.

Proximal 15q familial euchromatic variant and PWS/AS critical region duplication in the same patient: a cytogenetic pitfall.

PubMed

Carelle-Calmels, Nadège; Girard-Lemaire, Françoise; Guérin, Eric; Bieth, Eric; Rudolf, Gabrielle; Biancalana, Valérie; Pecheur, Hélène; Demil, Houria; Schneider, Thierry; de Saint-Martin, Anne; Caron, Olivier; Legrain, Michèle; Gaston, Valérie; Flori, Elisabeth

2008-01-01

Cytogenetically detectable elongation of the 15q proximal region can be associated with Prader-Willi/Angelman critical region interstitial duplications or with inherited juxtacentromeric euchromatic variants. The first category has been reported in association with developmental delay and autistic disorders. These pathogenic recurrent duplications are more frequently of maternal origin and originate from unequal meiotic crossovers between chromosome 15 low-copy repeats. 15q juxtacentromeric euchromatic variants reflect polymorphic copy number variations of segments containing pseudogenes and usually segregate without apparent phenotypic consequence. Pathogenic relevant 15q11-q13 duplications are not distinguishable from the innocuous euchromatic variants with conventional cytogenetic methods. We report cytogenetic and molecular studies of a patient with hypotonia, developmental delay and epilepsy, carrying, on the same chromosome 15, both a de novo 15q11-q13 interstitial duplication and an inherited 15q juxtacentromeric amplification from maternal origin. The duplication, initially suspected by fluorescent in situ hybridization (FISH), has been confirmed by molecular studies. The 15q juxtacentromeric region amplification, which segregates in the family for at least three generations, has been confirmed by FISH using BAC probes overlapping the NF1 and GABRA5 pseudogenes. This report emphasizes the importance to distinguish proximal 15q polymorphic variants from clinically significant duplications. In any patient with inherited 15q proximal variant but unexplained developmental delay suggesting 15q11-q13 pathology, a pathogenic rearrangement has to be searched with adapted strategies, in order to detect deletions as well as duplications of this region.

Application of droplet digital PCR to determine copy number of endogenous genes and transgenes in sugarcane.

PubMed

Sun, Yue; Joyce, Priya Aiyar

2017-11-01

Droplet digital PCR combined with the low copy ACT allele as endogenous reference gene, makes accurate and rapid estimation of gene copy number in Q208 A and Q240 A attainable. Sugarcane is an important cultivated crop with both high polyploidy and aneuploidy in its 10 Gb genome. Without a known copy number reference gene, it is difficult to accurately estimate the copy number of any gene of interest by PCR-based methods in sugarcane. Recently, a new technology, known as droplet digital PCR (ddPCR) has been developed which can measure the absolute amount of the target DNA in a given sample. In this study, we deduced the true copy number of three endogenous genes, actin depolymerizing factor (ADF), adenine phosphoribosyltransferase (APRT) and actin (ACT) in three Australian sugarcane varieties, using ddPCR by comparing the absolute amounts of the above genes with a transgene of known copy number. A single copy of the ACT allele was detected in Q208 A , two copies in Q240 A , but was absent in Q117. Copy number variation was also observed for both APRT and ADF, and ranged from 9 to 11 in the three tested varieties. Using this newly developed ddPCR method, transgene copy number was successfully determined in 19 transgenic Q208 A and Q240 A events using ACT as the reference endogenous gene. Our study demonstrates that ddPCR can be used for high-throughput genetic analysis and is a quick, accurate and reliable alternative method for gene copy number determination in sugarcane. This discovered ACT allele would be a suitable endogenous reference gene for future gene copy number variation and dosage studies of functional genes in Q208 A and Q240 A .

Developing a Risk-scoring Model for Ankylosing Spondylitis Based on a Combination of HLA-B27, Single-nucleotide Polymorphism, and Copy Number Variant Markers.

PubMed

Jung, Seung-Hyun; Cho, Sung-Min; Yim, Seon-Hee; Kim, So-Hee; Park, Hyeon-Chun; Cho, Mi-La; Shim, Seung-Cheol; Kim, Tae-Hwan; Park, Sung-Hwan; Chung, Yeun-Jun

2016-12-01

To develop a genotype-based ankylosing spondylitis (AS) risk prediction model that is more sensitive and specific than HLA-B27 typing. To develop the AS genetic risk scoring (AS-GRS) model, 648 individuals (285 cases and 363 controls) were examined for 5 copy number variants (CNV), 7 single-nucleotide polymorphisms (SNP), and an HLA-B27 marker by TaqMan assays. The AS-GRS model was developed using logistic regression and validated with a larger independent set (576 cases and 680 controls). Through logistic regression, we built the AS-GRS model consisting of 5 genetic components: HLA-B27, 3 CNV (1q32.2, 13q13.1, and 16p13.3), and 1 SNP (rs10865331). All significant associations of genetic factors in the model were replicated in the independent validation set. The discriminative ability of the AS-GRS model measured by the area under the curve was excellent: 0.976 (95% CI 0.96-0.99) in the model construction set and 0.951 (95% CI 0.94-0.96) in the validation set. The AS-GRS model showed higher specificity and accuracy than the HLA-B27-only model when the sensitivity was set to over 94%. When we categorized the individuals into quartiles based on the AS-GRS scores, OR of the 4 groups (low, intermediate-1, intermediate-2, and high risk) showed an increasing trend with the AS-GRS scores (r 2 = 0.950) and the highest risk group showed a 494× higher risk of AS than the lowest risk group (95% CI 237.3-1029.1). Our AS-GRS could be used to identify individuals at high risk for AS before major symptoms appear, which may improve the prognosis for them through early treatment.

Genomic profile in gestational and non-gestational choriocarcinomas.

PubMed

Mello, Julia Bette Homem de; Ramos Cirilo, Priscila Daniele; Michelin, Odair Carlito; Custódio Domingues, Maria Aparecida; Cunha Rudge, Marilza Vieira; Rogatto, Silvia Regina; Maestá, Izildinha

2017-02-01

Gestational (GC) (derived from the placenta) and non-gestational (NGC) choriocarcinomas are trophoblastic diseases originated from abnormal proliferation of trophoblastic cells. These rare tumors share similar morphology and pathological features and differ on chemotherapy response, genetic origin and prognosis. In this study, the genomic profile of choriocarcinomas was performed according to their origin (GC or NGC) aiming to better understand these poorly characterized diseases. Thirteen patients were included in this study; 10 presented previous history of hydatidiform mole and six developed metastasis. Twelve polymorphic microsatellite markers (D15S659, APOC2, D5S816, BAT25, D3S1614, D3S1311, D1S1656, APC-D5S346, D3S1601, D18S70, D8S1110 and D11S1999) were investigated to distinguish GC from NGC. All choriocarcinomas were evaluated by copy number alterations using array CGH. Eight cases were classified as GC and five as NGC. Although potentially polymorphic, NGC exhibited significant gain of 21p11. Rare copy number alterations (CNA) were detected as a frequent event in GC including gains of 1p36.33-p36.32 (3 cases), 17q25.3 (4 cases), and losses of 9q33.1 (5 cases), 17q21.3 (3 cases) and 18q22.1 (4 cases) (varying from 724 to 3,053 Kb). Two tumor suppressor genes are candidates to be involved in GC: TRIM32 (9q33.1) and CDH19 (18q22.1). Gains of CBX2, CBX4 and CBX8 were frequently found in high risk prognostic score in GC. The in silico functional interaction analysis revealed the involvement of PTEN and PI3K-Akt signaling pathways. These data pointed out significant genomic alterations in GC, opening new avenues to better characterize the pathobiology of this disease. Copyright © 2016 Elsevier Ltd. All rights reserved.

Genetic Determinants of 1,3-Butadiene Metabolism and Detoxification in Three Populations of Smokers with Different Risks of Lung Cancer.

PubMed

Boldry, Emily J; Patel, Yesha M; Kotapati, Srikanth; Esades, Amanda; Park, Sungshim L; Tiirikainen, Maarit; Stram, Daniel O; Le Marchand, Loïc; Tretyakova, Natalia

2017-07-01

Background: 1,3-Butadiene (BD) is an important carcinogen in tobacco smoke that undergoes metabolic activation to DNA-reactive epoxides. These species can be detoxified via glutathione conjugation and excreted in urine as the corresponding N-acetylcysteine conjugates. We hypothesize that single nucleotide polymorphisms (SNPs) in BD-metabolizing genes may change the balance of BD bioactivation and detoxification in White, Japanese American, and African American smokers, potentially contributing to ethnic differences in lung cancer risk. Methods: We measured the levels of BD metabolites, 1- and 2-( N -acetyl-L-cysteine-S-yl)-1-hydroxybut-3-ene (MHBMA) and N -acetyl- S -(3,4-dihydroxybutyl)-L-cysteine (DHBMA), in urine samples from a total of 1,072 White, Japanese American, and African American smokers and adjusted these values for body mass index, age, batch, and total nicotine equivalents. We also conducted a genome-wide association study to identify genetic determinants of BD metabolism. Results: We found that mean urinary MHBMA concentrations differed significantly by ethnicity ( P = 4.0 × 10 -25 ). African Americans excreted the highest levels of MHBMA followed by Whites and Japanese Americans. MHBMA levels were affected by GSTT1 gene copy number ( P < 0.0001); conditional on GSTT1 , no other polymorphisms showed a significant association. Urinary DHBMA levels also differed between ethnic groups ( P = 3.3 × 10 -4 ), but were not affected by GSTT1 copy number ( P = 0.226). Conclusions: GSTT1 gene deletion has a strong effect on urinary MHBMA levels, and therefore BD metabolism, in smokers. Impact: Our results show that the order of MHBMA levels among ethnic groups is consistent with their respective lung cancer risk and can be partially explained by GSTT1 genotype. Cancer Epidemiol Biomarkers Prev; 26(7); 1034-42. ©2017 AACR . ©2017 American Association for Cancer Research.

Functional effects of CCL3L1 copy number

PubMed Central

Carpenter, Danielle; McIntosh, Richard S; Pleass, Richard J; Armour, John AL

2012-01-01

Copy number variation (CNV) is becoming increasingly important as a feature of human variation in disease susceptibility studies. However, the consequences of copy number variation are not so well understood. Here we present data exploring the functional consequences of copy number variation of CCL3L1 in 55 independent UK samples with no known clinical phenotypes. Copy number of CCL3L1 was determined by the paralogue ratio test (PRT), and expression levels of MIP-1α and mRNA from stimulated monocytes were measured and analysed. The data show no statistically significant association of MIP-1α protein levels with copy number. However, there was a significant correlation between copy number and CCL3L1:CCL3 mRNA ratio. The data also provide evidence that expression of CCL3 predominates in both protein and mRNA, and therefore the observed variation of CCL3 is potentially more important biologically than that of copy number variation of CCL3L1. PMID:22476153

Five Polymorphisms and Breast Cancer Risk: Results from the Breast Cancer Association Consortium

PubMed Central

Gaudet, Mia M.; Milne, Roger L.; Cox, Angela; Camp, Nicola J.; Goode, Ellen L.; Humphreys, Manjeet K.; Dunning, Alison M.; Morrison, Jonathan; Giles, Graham G.; Severi, Gianluca; Baglietto, Laura; English, Dallas R.; Couch, Fergus J.; Olson, Janet E.; Wang, Xianshu; Chang-Claude, Jenny; Flesch-Janys, Dieter; Abbas, Sascha; Salazar, Ramona; Mannermaa, Arto; Kataja, Vesa; Kosma, Veli-Matti; Lindblom, Annika; Margolin, Sara; Heikkinen, Tuomas; Kämpjärvi, Kati; Aaltonen, Kirsimari; Nevanlinna, Heli; Bogdanova, Natalia; Coinac, Irina; Schürmann, Peter; Dörk, Thilo; Bartram, Claus R.; Schmutzler, Rita K.; Tchatchou, Sandrine; Burwinkel, Barbara; Brauch, Hiltrud; Torres, Diana; Hamann, Ute; Justenhoven, Christina; Ribas, Gloria; Arias, José I.; Benitez, Javier; Bojesen, Stig E.; Nordestgaard, Børge G.; Flyger, Henrik L.; Peto, Julian; Fletcher, Olivia; Johnson, Nichola; Silva, Isabel dos Santos; Fasching, Peter A.; Beckmann, Matthias W.; Strick, Reiner; Ekici, Arif B.; Broeks, Annegien; Schmidt, Marjanka K.; van Leeuwen, Flora E.; Van’t Veer, Laura J.; Southey, Melissa C.; Hopper, John L.; Apicella, Carmel; Haiman, Christopher A.; Henderson, Brian E.; Le Marchand, Loic; Kolonel, Laurence N.; Kristensen, Vessela; Alnæs, Grethe Grenaker; Hunter, David J.; Kraft, Peter; Cox, David G.; Hankinson, Susan E.; Seynaeve, Caroline; Vreeswijk, Maaike P.G.; Tollenaar, Rob A.E.M.; Devilee, Peter; Chanock, Stephen; Lissowska, Jolanta; Brinton, Louise; Peplonska, Beata; Czene, Kamila; Hall, Per; Li, Yuqing; Liu, Jianjun; Balasubramanian, Sabapathy; Rafii, Saeed; Reed, Malcolm W.R.; Pooley, Karen A.; Conroy, Don; Baynes, Caroline; Kang, Daehee; Yoo, Keun-Young; Noh, Dong-Young; Ahn, Sei-Hyun; Shen, Chen-Yang; Wang, Hui-Chun; Yu, Jyh-Cherng; Wu, Pei-Ei; Anton-Culver, Hoda; Ziogoas, Argyrios; Egan, Kathleen; Newcomb, Polly; Titus-Ernstoff, Linda; Dietz, Amy Trentham; Sigurdson, Alice J.; Alexander, Bruce H.; Bhatti, Parveen; Allen-Brady, Kristina; Cannon-Albright, Lisa A.; Wong, Jathine; Chenevix-Trench, Georgia; Spurdle, Amanda B.; Beesley, Jonathan; Pharoah, Paul D.P.; Easton, Doug F.; Garcia-Closas, Montserrat

2009-01-01

Previous studies have suggested that minor alleles for ERCC4 rs744154, TNF rs361525, CASP10 rs13010627, PGR rs1042838, and BID rs8190315 may influence breast cancer risk, but the evidence is inconclusive due to their small sample size. These polymorphisms were genotyped in more than 30,000 breast cancer cases and 30,000 controls, primarily of European descent, from 30 studies in the Breast Cancer Association Consortium. We calculated odds ratios (OR) and 95% confidence intervals (95% CI) as a measure of association. We found that the minor alleles for these polymorphisms were not related to invasive breast cancer risk overall in women of European descent: ECCR4 per-allele OR (95% CI) = 0.99 (0.97–1.02), minor allele frequency = 27.5%; TNF 1.00 (0.95–1.06), 5.0%; CASP10 1.02 (0.98–1.07), 6.5%; PGR 1.02 (0.99–1.06), 15.3%; and BID 0.98 (0.86–1.12), 1.7%. However, we observed significant between-study heterogeneity for associations with risk for single-nucleotide polymorphisms (SNP) in CASP10, PGR, and BID. Estimates were imprecise for women of Asian and African descent due to small numbers and lower minor allele frequencies (with the exception of BID SNP). The ORs for each copy of the minor allele were not significantly different by estrogen or progesterone receptor status, nor were any significant interactions found between the polymorphisms and age or family history of breast cancer. In conclusion, our data provide persuasive evidence against an overall association between invasive breast cancer risk and ERCC4 rs744154, TNF rs361525, CASP10 rs13010627, PGR rs1042838, and BID rs8190315 genotypes among women of European descent. PMID:19423537

Pooled Enrichment Sequencing Identifies Diversity and Evolutionary Pressures at NLR Resistance Genes within a Wild Tomato Population.

PubMed

Stam, Remco; Scheikl, Daniela; Tellier, Aurélien

2016-06-02

Nod-like receptors (NLRs) are nucleotide-binding domain and leucine-rich repeats containing proteins that are important in plant resistance signaling. Many of the known pathogen resistance (R) genes in plants are NLRs and they can recognize pathogen molecules directly or indirectly. As such, divergence and copy number variants at these genes are found to be high between species. Within populations, positive and balancing selection are to be expected if plants coevolve with their pathogens. In order to understand the complexity of R-gene coevolution in wild nonmodel species, it is necessary to identify the full range of NLRs and infer their evolutionary history. Here we investigate and reveal polymorphism occurring at 220 NLR genes within one population of the partially selfing wild tomato species Solanum pennellii. We use a combination of enrichment sequencing and pooling ten individuals, to specifically sequence NLR genes in a resource and cost-effective manner. We focus on the effects which different mapping and single nucleotide polymorphism calling software and settings have on calling polymorphisms in customized pooled samples. Our results are accurately verified using Sanger sequencing of polymorphic gene fragments. Our results indicate that some NLRs, namely 13 out of 220, have maintained polymorphism within our S. pennellii population. These genes show a wide range of πN/πS ratios and differing site frequency spectra. We compare our observed rate of heterozygosity with expectations for this selfing and bottlenecked population. We conclude that our method enables us to pinpoint NLR genes which have experienced natural selection in their habitat. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Integrating common and rare genetic variation in diverse human populations.

PubMed

Altshuler, David M; Gibbs, Richard A; Peltonen, Leena; Altshuler, David M; Gibbs, Richard A; Peltonen, Leena; Dermitzakis, Emmanouil; Schaffner, Stephen F; Yu, Fuli; Peltonen, Leena; Dermitzakis, Emmanouil; Bonnen, Penelope E; Altshuler, David M; Gibbs, Richard A; de Bakker, Paul I W; Deloukas, Panos; Gabriel, Stacey B; Gwilliam, Rhian; Hunt, Sarah; Inouye, Michael; Jia, Xiaoming; Palotie, Aarno; Parkin, Melissa; Whittaker, Pamela; Yu, Fuli; Chang, Kyle; Hawes, Alicia; Lewis, Lora R; Ren, Yanru; Wheeler, David; Gibbs, Richard A; Muzny, Donna Marie; Barnes, Chris; Darvishi, Katayoon; Hurles, Matthew; Korn, Joshua M; Kristiansson, Kati; Lee, Charles; McCarrol, Steven A; Nemesh, James; Dermitzakis, Emmanouil; Keinan, Alon; Montgomery, Stephen B; Pollack, Samuela; Price, Alkes L; Soranzo, Nicole; Bonnen, Penelope E; Gibbs, Richard A; Gonzaga-Jauregui, Claudia; Keinan, Alon; Price, Alkes L; Yu, Fuli; Anttila, Verneri; Brodeur, Wendy; Daly, Mark J; Leslie, Stephen; McVean, Gil; Moutsianas, Loukas; Nguyen, Huy; Schaffner, Stephen F; Zhang, Qingrun; Ghori, Mohammed J R; McGinnis, Ralph; McLaren, William; Pollack, Samuela; Price, Alkes L; Schaffner, Stephen F; Takeuchi, Fumihiko; Grossman, Sharon R; Shlyakhter, Ilya; Hostetter, Elizabeth B; Sabeti, Pardis C; Adebamowo, Clement A; Foster, Morris W; Gordon, Deborah R; Licinio, Julio; Manca, Maria Cristina; Marshall, Patricia A; Matsuda, Ichiro; Ngare, Duncan; Wang, Vivian Ota; Reddy, Deepa; Rotimi, Charles N; Royal, Charmaine D; Sharp, Richard R; Zeng, Changqing; Brooks, Lisa D; McEwen, Jean E

2010-09-02

Despite great progress in identifying genetic variants that influence human disease, most inherited risk remains unexplained. A more complete understanding requires genome-wide studies that fully examine less common alleles in populations with a wide range of ancestry. To inform the design and interpretation of such studies, we genotyped 1.6 million common single nucleotide polymorphisms (SNPs) in 1,184 reference individuals from 11 global populations, and sequenced ten 100-kilobase regions in 692 of these individuals. This integrated data set of common and rare alleles, called 'HapMap 3', includes both SNPs and copy number polymorphisms (CNPs). We characterized population-specific differences among low-frequency variants, measured the improvement in imputation accuracy afforded by the larger reference panel, especially in imputing SNPs with a minor allele frequency of

Single nucleotide polymorphism array karyotyping: a diagnostic and prognostic tool in myelodysplastic syndromes with unsuccessful conventional cytogenetic testing.

PubMed

Arenillas, Leonor; Mallo, Mar; Ramos, Fernando; Guinta, Kathryn; Barragán, Eva; Lumbreras, Eva; Larráyoz, María-José; De Paz, Raquel; Tormo, Mar; Abáigar, María; Pedro, Carme; Cervera, José; Such, Esperanza; José Calasanz, María; Díez-Campelo, María; Sanz, Guillermo F; Hernández, Jesús María; Luño, Elisa; Saumell, Sílvia; Maciejewski, Jaroslaw; Florensa, Lourdes; Solé, Francesc

2013-12-01

Cytogenetic aberrations identified by metaphase cytogenetics (MC) have diagnostic, prognostic, and therapeutic implications in myelodysplastic syndromes (MDS). However, in some MDS patients MC study is unsuccesful. Single nucleotide polymorphism array (SNP-A) based karyotyping could be helpful in these cases. We performed SNP-A in 62 samples from bone marrow or peripheral blood of primary MDS with an unsuccessful MC study. SNP-A analysis enabled the detection of aberrations in 31 (50%) patients. We used the copy number alteration information to apply the International Prognostic Scoring System (IPSS) and we observed differences in survival between the low/intermediate-1 and intermediate-2/high risk patients. We also saw differences in survival between very low/low/intermediate and the high/very high patients when we applied the revised IPSS (IPSS-R). In conclusion, SNP-A can be used successfully in PB samples and the identification of CNA by SNP-A improve the diagnostic and prognostic evaluation of this group of MDS patients. Copyright © 2013 Wiley Periodicals, Inc.

GEOGRAPHIC DISTRIBUTION OF MOLECULAR VARIANCE WITHIN THE BLUE MARLIN (MAKAIRA NIGRICANS): A HIERARCHICAL ANALYSIS OF ALLOZYME, SINGLE-COPY NUCLEAR DNA, AND MITOCHONDRIAL DNA MARKERS.

PubMed

Buonaccorsi, Vincent P; Reece, Kimberly S; Morgan, Lee W; Graves, John E

1999-04-01

This study presents a comparative hierarchical analysis of variance applied to three classes of molecular markers within the blue marlin (Makaira nigricans). Results are reported from analyses of four polymorphic allozyme loci, four polymorphic anonymously chosen single-copy nuclear DNA (scnDNA) loci, and previously reported restriction fragment length polymorphisms (RFLPs) of mitochondrial DNA (mtDNA). Samples were collected within and among the Atlantic and Pacific Oceans over a period of several years. Although moderate levels of genetic variation were detected at both polymorphic allozyme (H = 0.30) and scnDNA loci (H = 0.37), mtDNA markers were much more diverse (h = 0.85). Allele frequencies were significantly different between Atlantic and Pacific Ocean samples at three of four allozyme loci and three of four scnDNA loci. Estimates of allozyme genetic differentiation (θ O ) ranged from 0.00 to 0.15, with a mean of 0.08. The θ O values for scnDNA loci were similar to those of allozymes, ranging from 0.00 to 0.12 with a mean of 0.09. MtDNA RFLP divergence between oceans (θ O = 0.39) was significantly greater than divergence detected at nuclear loci (95% nuclear confidence interval = 0.04-0.11). The fourfold smaller effective population size of mtDNA and male-mediated gene flow may account for the difference observed between nuclear and mitochondrial divergence estimates. © 1999 The Society for the Study of Evolution.

The cost of copy number in a selfish genetic element: the 2-μm plasmid of Saccharomyces cerevisiae.

PubMed

Harrison, Ellie; Koufopanou, V; Burt, A; MacLean, R C

2012-11-01

Many autonomously replicating genetic elements exist as multiple copies within the cell. The copy number of these elements is often assumed to have important fitness consequences for both element and host, yet the forces shaping its evolution are not well understood. The 2 μm is a multicopy plasmid of Saccharomyces yeasts, encoding just four genes that are solely involved in plasmid replication. One simple model for the fitness relationship between yeasts and 2 μm is that plasmid copy number evolves as a trade-off between selection for increased vertical transmission, favouring high copy number, and selection for decreased virulence, favouring low copy number. To test this model, we experimentally manipulated the copy number of the plasmid and directly measured the fitness cost, in terms of growth rate reduction, associated with high plasmid copy number. We find that the fitness burden imposed by the 2 μm increases with plasmid copy number, such that each copy imposes a fitness burden of 0.17% (± 0.008%), greatly exceeding the cost expected for it to be stably maintained in yeast populations. Our results demonstrate the crucial importance of copy number in the evolution of yeast per 2 μm associations and pave the way for future studies examining how selection can shape the cost of multicopy elements. © 2012 The Authors. Journal of Evolutionary Biology © 2012 European Society For Evolutionary Biology.

Transformation of Chloroplast Ribosomal RNA Genes in Chlamydomonas: Molecular and Genetic Characterization of Integration Events

PubMed Central

Newman, S. M.; Boynton, J. E.; Gillham, N. W.; Randolph-Anderson, B. L.; Johnson, A. M.; Harris, E. H.

1990-01-01

Transformation of chloroplast ribosomal RNA (rRNA) genes in Chlamydomonas has been achieved by the biolistic process using cloned chloroplast DNA fragments carrying mutations that confer antibiotic resistance. The sites of exchange employed during the integration of the donor DNA into the recipient genome have been localized using a combination of antibiotic resistance mutations in the 16S and 23S rRNA genes and restriction fragment length polymorphisms that flank these genes. Complete or nearly complete replacement of a region of the chloroplast genome in the recipient cell by the corresponding sequence from the donor plasmid was the most common integration event. Exchange events between the homologous donor and recipient sequences occurred preferentially near the vector:insert junctions. Insertion of the donor rRNA genes and flanking sequences into one inverted repeat of the recipient genome was followed by intramolecular copy correction so that both copies of the inverted repeat acquired identical sequences. Increased frequencies of rRNA gene transformants were achieved by reducing the copy number of the chloroplast genome in the recipient cells and by decreasing the heterology between donor and recipient DNA sequences flanking the selectable markers. In addition to producing bona fide chloroplast rRNA transformants, the biolistic process induced mutants resistant to low levels of streptomycin, typical of nuclear mutations in Chlamydomonas. PMID:1981764

Genome wide analysis of rare copy number variations in alcohol abuse or dependence.

PubMed

Rodríguez-López, Julio; Flórez, Gerardo; Blanco, Vanessa; Pereiro, César; Fernández, José Manuel; Fariñas, Emilio; Estévez, Valentín; Gómez-Trigo, Jesús; Gurriarán, Xaquín; Calvo, Raquel; Sáiz, Pilar; Vázquez, Fernando Lino; Arrojo, Manuel; Costas, Javier

2018-06-02

Genetics plays an important role in alcohol abuse/dependence. Its heritability has been estimated as 45-65%. Rare copy number variations (CNVs) have been confirmed as relevant genetic factors in other neuropsychiatric disorders, such as autism spectrum disorders, schizophrenia, epilepsy, or Tourette syndrome. In the present study, we analyzed the role of rare CNVs affecting exons of coding genes in a sample from Northwest Spain genotyped using the Illumina Infinium PsychArray Beadchip. After rigorous genotyping quality control procedure, 712 patients with alcohol abuse or dependence and 804 controls were used for CNV detection. CNV calling was performed using PennCNV and cnvPartition, and analyses were restricted to CNVs of at least 100 kb and including at least 10 single nucleotide polymorphisms. Logistic regression was used to test for the effect of CNV as well as number of genes affected by CNVs on case/control status, after adjustment for demographic and experimental covariates. We have found an excess of deletions (p = 0.008) and genes affected by deletions (p = 0.017) in cases. This effect was restricted to the 14.8% of affected genes that are intolerant to loss-of-function mutations (gene count p = 0.009). The importance of this subset of genes is emerging in other psychiatric disorders of neurodevelopmental origin, suggesting that disturbance in neurodevelopment mediated by genetic alterations may be a risk factor for alcohol use disorder. Copyright © 2018 Elsevier Ltd. All rights reserved.

Validation of copy number variants associated with prostate cancer risk and prognosis.

PubMed

Blackburn, August; Wilson, Desiree; Gelfond, Jonathan; Yao, Li; Hernandez, Javier; Thompson, Ian M; Leach, Robin J; Lehman, Donna M

2014-01-01

Two recent studies have reported novel heritable copy number variants on chromosomes 2p, 15q, and 12q to be associated with prostate cancer (PCa) risk in non-Hispanic Caucasians. The goal of this study was to determine whether these findings could be independently confirmed in the Caucasian population from the South Texas area. The study subjects consisted of participants of the San Antonio Biomarkers of Risk for PCa cohort and additional cases ascertained in the same metropolitan area. We genotyped all 7 of the reported copy number variants using real-time quantitative polymerase chain reaction in 1,536 (317 cases and 1,219 controls) non-Hispanic Caucasian men, and additionally, we genotyped 632 (191 cases and 441 controls) Hispanic Caucasian men for one of these variants, a deletion on 2p24.3. Association of the deletion on 2p24.3 with overall PCa risk did not meet our significance criteria but was consistent with previous reports (odds ratio, 1.40; 95% confidence interval 0.99-2.00; P = 0.06). Among Hispanic Caucasians, this deletion is much less prevalent (minor allele frequencies of 0.059 and 0.024 in non-Hispanic and Hispanic Caucasians, respectively) and did not show evidence of association with risk for PCa. Interestingly, among non-Hispanic Caucasians, carrying a homozygous deletion of 2p24.3 was significantly associated with high-grade PCa as defined by Gleason score sum ≥8 (odds ratio, 27.99; 95% confidence interval 1.99-392.6; P = 0.007 [the Fisher exact test]). The remaining 6 copy number variable regions either were not polymorphic in our cohort of non-Hispanic Caucasians or showed no evidence of association. Our findings are consistent with the reported observation that a heritable deletion on 2p24.3 is associated with PCa risk in non-Hispanic Caucasians. Additionally, our observations indicate that the 2p24.3 variant is associated with risk for high-grade PCa in a recessive manner. We were unable to replicate any association with PCa for the variants on chromosomes 15q and 12q, which may be explained by regional population differences in low frequency variants and disease heterogeneity. Published by Elsevier Inc.

Data-driven approach to detect common copy-number variations and frequency profiles in a population-based Korean cohort.

PubMed

Moon, Sanghoon; Kim, Young Jin; Hong, Chang Bum; Kim, Dong-Joon; Lee, Jong-Young; Kim, Bong-Jo

2011-11-01

To date, hundreds of thousands of copy-number variation (CNV) data have been reported using various platforms. The proportion of Asians in these data is, however, relatively small as compared with that of other ethnic groups, such as Caucasians and Yorubas. Because of limitations in platform resolution and the high noise level in signal intensity, in most CNV studies (particularly those using single nucleotide polymorphism arrays), the average number of CNVs in an individual is less than the number of known CNVs. In this study, we ascertained reliable, common CNV regions (CNVRs) and identified actual frequency rates in the Korean population to provide more CNV information. We performed two-stage analyses for detecting structural variations with two platforms. We discovered 576 common CNVRs (88 CNV segments on average in an individual), and 87% (501 of 576) of these CNVRs overlapped by ≥1 bp with previously validated CNV events. Interestingly, from the frequency analysis of CNV profiles, 52 of 576 CNVRs had a frequency rate of <1% in the 8842 individuals. Compared with other common CNV studies, this study found six common CNVRs that were not reported in previous CNV studies. In conclusion, we propose the data-driven detection approach to discover common CNVRs including those of unreported in the previous Korean CNV study while minimizing false positives. Through our approach, we successfully discovered more common CNVRs than previous Korean CNV study and conducted frequency analysis. These results will be a valuable resource for the effective level of CNVs in the Korean population.

40 CFR 262.22 - Number of copies.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 26 2011-07-01 2011-07-01 false Number of copies. 262.22 Section 262...) STANDARDS APPLICABLE TO GENERATORS OF HAZARDOUS WASTE The Manifest § 262.22 Number of copies. The manifest consists of at least the number of copies which will provide the generator, each transporter, and the owner...

40 CFR 262.22 - Number of copies.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 40 Protection of Environment 27 2012-07-01 2012-07-01 false Number of copies. 262.22 Section 262...) STANDARDS APPLICABLE TO GENERATORS OF HAZARDOUS WASTE The Manifest § 262.22 Number of copies. The manifest consists of at least the number of copies which will provide the generator, each transporter, and the owner...

40 CFR 262.22 - Number of copies.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 25 2010-07-01 2010-07-01 false Number of copies. 262.22 Section 262...) STANDARDS APPLICABLE TO GENERATORS OF HAZARDOUS WASTE The Manifest § 262.22 Number of copies. The manifest consists of at least the number of copies which will provide the generator, each transporter, and the owner...

DNA copy number, including telomeres and mitochondria, assayed using next-generation sequencing.

PubMed

Castle, John C; Biery, Matthew; Bouzek, Heather; Xie, Tao; Chen, Ronghua; Misura, Kira; Jackson, Stuart; Armour, Christopher D; Johnson, Jason M; Rohl, Carol A; Raymond, Christopher K

2010-04-16

DNA copy number variations occur within populations and aberrations can cause disease. We sought to develop an improved lab-automatable, cost-efficient, accurate platform to profile DNA copy number. We developed a sequencing-based assay of nuclear, mitochondrial, and telomeric DNA copy number that draws on the unbiased nature of next-generation sequencing and incorporates techniques developed for RNA expression profiling. To demonstrate this platform, we assayed UMC-11 cells using 5 million 33 nt reads and found tremendous copy number variation, including regions of single and homogeneous deletions and amplifications to 29 copies; 5 times more mitochondria and 4 times less telomeric sequence than a pool of non-diseased, blood-derived DNA; and that UMC-11 was derived from a male individual. The described assay outputs absolute copy number, outputs an error estimate (p-value), and is more accurate than array-based platforms at high copy number. The platform enables profiling of mitochondrial levels and telomeric length. The assay is lab-automatable and has a genomic resolution and cost that are tunable based on the number of sequence reads.

DNA copy number, including telomeres and mitochondria, assayed using next-generation sequencing

PubMed Central

2010-01-01

Background DNA copy number variations occur within populations and aberrations can cause disease. We sought to develop an improved lab-automatable, cost-efficient, accurate platform to profile DNA copy number. Results We developed a sequencing-based assay of nuclear, mitochondrial, and telomeric DNA copy number that draws on the unbiased nature of next-generation sequencing and incorporates techniques developed for RNA expression profiling. To demonstrate this platform, we assayed UMC-11 cells using 5 million 33 nt reads and found tremendous copy number variation, including regions of single and homogeneous deletions and amplifications to 29 copies; 5 times more mitochondria and 4 times less telomeric sequence than a pool of non-diseased, blood-derived DNA; and that UMC-11 was derived from a male individual. Conclusion The described assay outputs absolute copy number, outputs an error estimate (p-value), and is more accurate than array-based platforms at high copy number. The platform enables profiling of mitochondrial levels and telomeric length. The assay is lab-automatable and has a genomic resolution and cost that are tunable based on the number of sequence reads. PMID:20398377

Recombinant SINEs are formed at high frequency during induced retrotransposition in vivo.

PubMed

Yadav, Vijay Pal; Mandal, Prabhat Kumar; Bhattacharya, Alok; Bhattacharya, Sudha

2012-05-22

Non-long terminal repeat Retrotransposons are referred to as long interspersed nuclear elements (LINEs) and their non-autonomous partners are short interspersed nuclear elements (SINEs). It is believed that an active SINE copy, upon retrotransposition, generates near identical copies of itself, which subsequently accumulate mutations resulting in sequence polymorphism. Here we show that when a retrotransposition-competent cell line of the parasitic protist Entamoeba histolytica, transfected with a marked SINE copy, is induced to retrotranspose, >20% of the newly retrotransposed copies are neither identical to the marked SINE nor to the mobilized resident SINEs. Rather they are recombinants of resident SINEs and the marked SINE. They are a consequence of retrotransposition and not DNA recombination, as they are absent in cells not expressing the retrotransposition functions. This high-frequency recombination provides a new explanation for the existence of mosaic SINEs, which may impact on genetic analysis of SINE lineages, and measurement of phylogenetic distances.

Mate-choice copying: A fitness-enhancing behavior that evolves by indirect selection.

PubMed

Santos, Mauro; Sapage, Manuel; Matos, Margarida; Varela, Susana A M

2017-06-01

A spatially explicit, individual-based simulation model is used to study the spread of an allele for mate-choice copying (MCC) through horizontal cultural transmission when female innate preferences do or do not coevolve with a male viability-increasing trait. Evolution of MCC is unlikely when innate female preferences coevolve with the trait, as copier females cannot express a higher preference than noncopier females for high-fitness males. However, if a genetic polymorphism for innate preference persists in the population, MCC can evolve by indirect selection through hitchhiking: the copying allele hitchhikes on the male trait. MCC can be an adaptive behavior-that is, a behavior that increases a population's average fitness relative to populations without MCC-even though the copying allele itself may be neutral or mildly deleterious. © 2017 The Author(s). Evolution © 2017 The Society for the Study of Evolution.

Development of single-copy nuclear intron markers for species-level phylogenetics: Case study with Paullinieae (Sapindaceae).

PubMed

Chery, Joyce G; Sass, Chodon; Specht, Chelsea D

2017-09-01

We developed a bioinformatic pipeline that leverages a publicly available genome and published transcriptomes to design primers in conserved coding sequences flanking targeted introns of single-copy nuclear loci. Paullinieae (Sapindaceae) is used to demonstrate the pipeline. Transcriptome reads phylogenetically closer to the lineage of interest are aligned to the closest genome. Single-nucleotide polymorphisms are called, generating a "pseudoreference" closer to the lineage of interest. Several filters are applied to meet the criteria of single-copy nuclear loci with introns of a desired size. Primers are designed in conserved coding sequences flanking introns. Using this pipeline, we developed nine single-copy nuclear intron markers for Paullinieae. This pipeline is highly flexible and can be used for any group with available genomic and transcriptomic resources. This pipeline led to the development of nine variable markers for phylogenetic study without generating sequence data de novo.

Dietary starch intake modifies the relation between copy number variation in the salivary amylase gene and BMI.

PubMed

Rukh, Gull; Ericson, Ulrika; Andersson-Assarsson, Johanna; Orho-Melander, Marju; Sonestedt, Emily

2017-07-01

Background: Studies have shown conflicting associations between the salivary amylase gene ( AMY1 ) copy number and obesity. Salivary amylase initiates starch digestion in the oral cavity; starch is a major source of energy in the diet. Objective: We investigated the association between AMY1 copy number and obesity traits, and the effect of the interaction between AMY1 copy number and starch intake on these obesity traits. Design: We first assessed the association between AMY1 copy number (genotyped by digital droplet polymerase chain reaction) and obesity traits in 4800 individuals without diabetes (mean age: 57 y; 60% female) from the Malmö Diet and Cancer Cohort. Then we analyzed interactions between AMY1 copy number and energy-adjusted starch intake (obtained by a modified diet history method) on body mass index (BMI) and body fat percentage. Results: AMY1 copy number was not associated with BMI ( P = 0.80) or body fat percentage ( P = 0.38). We observed a significant effect of the interaction between AMY1 copy number and starch intake on BMI ( P -interaction = 0.007) and body fat percentage ( P -interaction = 0.03). Upon stratification by dietary starch intake, BMI tended to decrease with increasing AMY1 copy numbers in the low-starch intake group ( P = 0.07) and tended to increase with increasing AMY1 copy numbers in the high-starch intake group ( P = 0.08). The lowest mean BMI was observed in the group of participants with a low AMY1 copy number and a high dietary intake of starch. Conclusions: Our findings suggest an effect of the interaction between starch intake and AMY1 copy number on obesity. Individuals with high starch intake but low genetic capacity to digest starch had the lowest BMI, potentially because larger amounts of undigested starch are transported through the gastrointestinal tract, contributing to fewer calories extracted from ingested starch. © 2017 American Society for Nutrition.

ABCB1 genetic variability and methadone dosage requirements in opioid-dependent individuals.

PubMed

Coller, Janet K; Barratt, Daniel T; Dahlen, Karianne; Loennechen, Morten H; Somogyi, Andrew A

2006-12-01

The most common treatment for opioid dependence is substitution therapy with another opioid such as methadone. The methadone dosage is individualized but highly variable, and program retention rates are low due in part to nonoptimal dosing resulting in withdrawal symptoms and further heroin craving and use. Methadone is a substrate for the P-glycoprotein transporter, encoded by the ABCB1 gene, which regulates central nervous system exposure. This retrospective study aimed to investigate the influence of ABCB1 genetic variability on methadone dose requirements. Genomic deoxyribonucleic acid was isolated from opioid-dependent subjects (n = 60) and non-opioid-dependent control subjects (n = 60), and polymerase chain reaction-restriction fragment length polymorphism and allele-specific polymerase chain reaction were used to determine the presence of single nucleotide polymorphisms at positions 61, 1199, 1236, 2677, and 3435. ABCB1 haplotypes were inferred with PHASE software (version 2.1). There were no significant differences in the allele or genotype frequencies of the individual single nucleotide polymorphisms or haplotypes between the 2 populations. ABCB1 genetic variability influenced daily methadone dose requirements, such that subjects carrying 2 copies of the wild-type haplotype required higher doses compared with those with 1 copy and those with no copies (98.3 +/- 10.4, 58.6 +/- 20.9, and 55.4 +/- 26.1 mg/d, respectively; P = .029). In addition, carriers of the AGCTT haplotype required significantly lower doses than noncarriers (38.0 +/- 16.8 and 61.3 +/- 24.6 mg/d, respectively; P = .04). Although ABCB1 genetic variability is not related to the development of opioid dependence, identification of variant haplotypes may, after larger prospective studies have been performed, provide clinicians with a tool for methadone dosage individualization.

Epidermal growth factor receptor and AKT1 gene copy numbers by multi-gene fluorescence in situ hybridization impact on prognosis in breast cancer.

PubMed

Li, Jiao; Su, Wei; Zhang, Sheng; Hu, Yunhui; Liu, Jingjing; Zhang, Xiaobei; Bai, Jingchao; Yuan, Weiping; Hu, Linping; Cheng, Tao; Zetterberg, Anders; Lei, Zhenmin; Zhang, Jin

2015-05-01

The epidermal growth factor receptor (EGFR)/PI3K/AKT signaling pathway aberrations play significant roles in breast cancer occurrence and development. However, the status of EGFR and AKT1 gene copy numbers remains unclear. In this study, we showed that the rates of EGFR and AKT1 gene copy number alterations were associated with the prognosis of breast cancer. Among 205 patients, high EGFR and AKT1 gene copy numbers were observed in 34.6% and 27.8% of cases by multi-gene fluorescence in situ hybridization, respectively. Co-heightened EGFR/AKT1 gene copy numbers were identified in 11.7% cases. No changes were found in 49.3% of patients. Although changes in EGFR and AKT1 gene copy numbers had no correlation with patients' age, tumor stage, histological grade and the expression status of other molecular makers, high EGFR (P = 0.0002) but not AKT1 (P = 0.1177) gene copy numbers correlated with poor 5-year overall survival. The patients with co-heightened EGFR/AKT1 gene copy numbers displayed a poorer prognosis than those with tumors with only high EGFR gene copy numbers (P = 0.0383). Both Univariate (U) and COX multivariate (C) analyses revealed that high EGFR and AKT1 gene copy numbers (P = 0.000 [U], P = 0.0001 [C]), similar to histological grade (P = 0.001 [U], P = 0.012 [C]) and lymph node metastasis (P = 0.046 [U], P = 0.158 [C]), were independent prognostic indicators of 5-year overall survival. These results indicate that high EGFR and AKT1 gene copy numbers were relatively frequent in breast cancer. Co-heightened EGFR/AKT1 gene copy numbers had a worse outcome than those with only high EGFR gene copy numbers, suggesting that evaluation of these two genes together may be useful for selecting patients for anti-EGFR-targeted therapy or anti-EGFR/AKT1-targeted therapy and for predicting outcomes. © 2015 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

Dynamics in copy numbers of five plasmids of a dairy Lactococcus lactis in dairy-related conditions including near-zero growth rates.

PubMed

van Mastrigt, Oscar; Lommers, Marcel M A N; de Vries, Yorick C; Abee, Tjakko; Smid, Eddy J

2018-03-23

Lactic acid bacteria can carry multiple plasmids affecting their performance in dairy fermentations. The expression of plasmid-encoded genes and the activity of the corresponding proteins is severely affected by changes in the number of plasmid copies. We studied the impact of growth rate on dynamics of plasmid copy numbers at high growth rates in chemostat cultures and down to near-zero growth rates in retentostat cultures. Five plasmids of the dairy strain Lactococcus lactis FM03-V1 were selected which varied in size (3 to 39 kb), in replication mechanism (theta or rolling-circle) and in putative (dairy-associated) functions. Copy numbers ranged from 1.5 to 40.5 and the copy number of theta-type replicating plasmids were negatively correlated to the plasmid size. Despite the extremely wide range of growth rates (0.0003 h -1 to 0.6 h -1 ), copy numbers of the five plasmids were stable and only slightly increased at near-zero growth rates showing that the plasmid replication rate was strictly controlled. One low-copy number plasmid, carrying a large exopolysaccharide gene cluster, was segregationally unstable during retentostat cultivations reflected in complete loss of the plasmid in one of the retentostat cultures. The copy number of the five plasmids was also hardly affected by varying the pH value, nutrient limitation or presence of citrate (maximum 2.2-fold) signifying the stability in copy number of the plasmids. Importance Lactococcus lactis is extensively used in starter cultures for dairy fermentations. Important traits for growth and survival of L. lactis in dairy fermentations are encoded by genes located on plasmids, such as genes involved in lactose and citrate metabolism, protein degradation and oligopeptide uptake and bacteriophage resistance. Because the number of plasmid copies could affect the expression of plasmid-encoded genes, it is important to know the factors that influence the plasmid copy numbers. We monitored plasmid copy numbers of L. lactis at near-zero growth rates, characteristic for cheese ripening. Moreover, we analysed the effect of pH, nutrient limitation and presence of citrate. This showed that plasmid copy numbers were stable giving insight into plasmid copy number dynamics in dairy fermentations. Copyright © 2018 American Society for Microbiology.

Genome-Wide Detection of Allele Specific Copy Number Variation Associated with Insulin Resistance in African Americans from the HyperGEN Study

PubMed Central

Pajewski, Nicholas M.; Kabagambe, Edmond K.; Gu, Charles C.; Pankow, Jim; North, Kari E.; Wilk, Jemma B.; Freedman, Barry I.; Franceschini, Nora; Broeckel, Uli; Tiwari, Hemant K.; Arnett, Donna K.

2011-01-01

African Americans have been understudied in genome wide association studies of diabetes and related traits. In the current study, we examined the joint association of single nucleotide polymorphisms (SNPs) and copy number variants (CNVs) with fasting insulin and an index of insulin resistance (HOMA-IR) in the HyperGEN study, a family based study with proband ascertainment for hypertension. This analysis is restricted to 1,040 African Americans without diabetes. We generated allele specific CNV genotypes at 872,243 autosomal loci using Birdsuite, a freely available multi-stage program. Joint tests of association for SNPs and CNVs were performed using linear mixed models adjusting for covariates and familial relationships. Our results highlight SNPs associated with fasting insulin and HOMA-IR (rs6576507 and rs8026527, 3.7*10−7≤P≤1.1*10−5) near ATPase, class V, type 10A (ATP10A), and the L Type voltage dependent calcium channel (CACNA1D, rs1401492, P≤5.2*10−6). ATP10A belongs to a family of aminophospholipid-transporting ATPases and has been associated with type 2 diabetes in mice. CACNA1D has been linked to pancreatic beta cell generation in mice. The two most significant copy variable markers (rs10277702 and rs361367; P<2.0*10−4) were in the beta variable region of the T-cell receptor gene (TCRVB). Human and mouse TCR has been shown to mimic insulin and its receptor and could contribute to insulin resistance. Our findings differ from genome wide association studies of fasting insulin and other diabetes related traits in European populations, highlighting the continued need to investigate unique genetic influences for understudied populations such as African Americans. PMID:21901158

Genome-wide detection of allele specific copy number variation associated with insulin resistance in African Americans from the HyperGEN study.

PubMed

Irvin, Marguerite R; Wineinger, Nathan E; Rice, Treva K; Pajewski, Nicholas M; Kabagambe, Edmond K; Gu, Charles C; Pankow, Jim; North, Kari E; Wilk, Jemma B; Freedman, Barry I; Franceschini, Nora; Broeckel, Uli; Tiwari, Hemant K; Arnett, Donna K

2011-01-01

African Americans have been understudied in genome wide association studies of diabetes and related traits. In the current study, we examined the joint association of single nucleotide polymorphisms (SNPs) and copy number variants (CNVs) with fasting insulin and an index of insulin resistance (HOMA-IR) in the HyperGEN study, a family based study with proband ascertainment for hypertension. This analysis is restricted to 1,040 African Americans without diabetes. We generated allele specific CNV genotypes at 872,243 autosomal loci using Birdsuite, a freely available multi-stage program. Joint tests of association for SNPs and CNVs were performed using linear mixed models adjusting for covariates and familial relationships. Our results highlight SNPs associated with fasting insulin and HOMA-IR (rs6576507 and rs8026527, 3.7*10(-7)≤P≤1.1*10(-5)) near ATPase, class V, type 10A (ATP10A), and the L Type voltage dependent calcium channel (CACNA1D, rs1401492, P≤5.2*10(-6)). ATP10A belongs to a family of aminophospholipid-transporting ATPases and has been associated with type 2 diabetes in mice. CACNA1D has been linked to pancreatic beta cell generation in mice. The two most significant copy variable markers (rs10277702 and rs361367; P<2.0*10(-4)) were in the beta variable region of the T-cell receptor gene (TCRVB). Human and mouse TCR has been shown to mimic insulin and its receptor and could contribute to insulin resistance. Our findings differ from genome wide association studies of fasting insulin and other diabetes related traits in European populations, highlighting the continued need to investigate unique genetic influences for understudied populations such as African Americans.

Rapid detection of pathological mutations and deletions of the haemoglobin beta gene (HBB) by High Resolution Melting (HRM) analysis and Gene Ratio Analysis Copy Enumeration PCR (GRACE-PCR).

PubMed

Turner, Andrew; Sasse, Jurgen; Varadi, Aniko

2016-10-19

Inherited disorders of haemoglobin are the world's most common genetic diseases, resulting in significant morbidity and mortality. The large number of mutations associated with the haemoglobin beta gene (HBB) makes gene scanning by High Resolution Melting (HRM) PCR an attractive diagnostic approach. However, existing HRM-PCR assays are not able to detect all common point mutations and have only a very limited ability to detect larger gene rearrangements. The aim of the current study was to develop a HBB assay, which can be used as a screening test in highly heterogeneous populations, for detection of both point mutations and larger gene rearrangements. The assay is based on a combination of conventional HRM-PCR and a novel Gene Ratio Analysis Copy Enumeration (GRACE) PCR method. HRM-PCR was extensively optimised, which included the use of an unlabelled probe and incorporation of universal bases into primers to prevent interference from common non-pathological polymorphisms. GRACE-PCR was employed to determine HBB gene copy numbers relative to a reference gene using melt curve analysis to detect rearrangements in the HBB gene. The performance of the assay was evaluated by analysing 410 samples. A total of 44 distinct pathological genotypes were detected. In comparison with reference methods, the assay has a sensitivity of 100 % and a specificity of 98 %. We have developed an assay that detects both point mutations and larger rearrangements of the HBB gene. This assay is quick, sensitive, specific and cost effective making it suitable as an initial screening test that can be used for highly heterogeneous cohorts.

Copy number variation at the 7q11.23 segmental duplications is a susceptibility factor for the Williams-Beuren syndrome deletion

PubMed Central

Cuscó, Ivon; Corominas, Roser; Bayés, Mònica; Flores, Raquel; Rivera-Brugués, Núria; Campuzano, Victoria; Pérez-Jurado, Luis A.

2008-01-01

Large copy number variants (CNVs) have been recently found as structural polymorphisms of the human genome of still unknown biological significance. CNVs are significantly enriched in regions with segmental duplications or low-copy repeats (LCRs). Williams-Beuren syndrome (WBS) is a neurodevelopmental disorder caused by a heterozygous deletion of contiguous genes at 7q11.23 mediated by nonallelic homologous recombination (NAHR) between large flanking LCRs and facilitated by a structural variant of the region, a ∼2-Mb paracentric inversion present in 20%–25% of WBS-transmitting progenitors. We now report that eight out of 180 (4.44%) WBS-transmitting progenitors are carriers of a CNV, displaying a chromosome with large deletion of LCRs. The prevalence of this CNV among control individuals and non-transmitting progenitors is much lower (1%, n = 600), thus indicating that it is a predisposing factor for the WBS deletion (odds ratio 4.6-fold, P = 0.002). LCR duplications were found in 2.22% of WBS-transmitting progenitors but also in 1.16% of controls, which implies a non–statistically significant increase in WBS-transmitting progenitors. We have characterized the organization and breakpoints of these CNVs, encompassing ∼100–300 kb of genomic DNA and containing several pseudogenes but no functional genes. Additional structural variants of the region have also been defined, all generated by NAHR between different blocks of segmental duplications. Our data further illustrate the highly dynamic structure of regions rich in segmental duplications, such as the WBS locus, and indicate that large CNVs can act as susceptibility alleles for disease-associated genomic rearrangements in the progeny. PMID:18292220

The relationship between mitochondrial DNA copy number and stallion sperm function.

PubMed

Darr, Christa R; Moraes, Luis E; Connon, Richard E; Love, Charles C; Teague, Sheila; Varner, Dickson D; Meyers, Stuart A

2017-05-01

Mitochondrial DNA (mtDNA) copy number has been utilized as a measure of sperm quality in several species including mice, dogs, and humans, and has been suggested as a potential biomarker of fertility in stallion sperm. The results of the present study extend this recent discovery using sperm samples from American Quarter Horse stallions of varying age. By determining copy number of three mitochondrial genes, cytochrome b (CYTB), NADH dehydrogenase 1 (ND1) and NADH dehydrogenase 4 (ND4), instead of a single gene, we demonstrate an improved understanding of mtDNA fate in stallion sperm mitochondria following spermatogenesis. Sperm samples from 37 stallions ranging from 3 to 24 years old were collected at four breeding ranches in north and central Texas during the 2015 breeding season. Samples were analyzed for sperm motion characteristics, nuclear DNA denaturability and mtDNA copy number. Mitochondrial DNA content in individual sperm was determined by real-time qPCR and normalized with a single copy nuclear gene, Beta actin. Exploratory correlation analysis revealed that total motility was negatively correlated with CYTB copy number and sperm chromatin structure. Stallion age did not have a significant effect on copy number for any of the genes. Copy number differences existed between the three genes with CYTB having the greatest number of copies (20.6 ± 1.2 copies, range: 6.0 to 41.1) followed by ND4 (15.5 ± 0.8 copies, range: 6.7 to 27.8) and finally ND1 (12.0 ± 1.0 copies, range: 0.4 to 26.6) (P < 0.05). Varying copy number across mitochondrial genes is likely to be a result of mtDNA fragmentation and degradation since downregulation of sperm mtDNA occurs during spermatogenesis and may be important for normal sperm function. Beta regression analysis suggested that for every unit increase in mtDNA copy number of CYTB, there was a 4% decrease in the odds of sperm movement (P = 0.001). Influential analysis suggested that results are robust and not highly influenced by data from individual stallions despite the low number of stallions sampled with low sperm motility. Further genome sequencing is necessary to investigate if mutations or deletions are the underlying causes of inconsistent copy numbers across mitochondrial genes. In conclusion, we show, for the first time, that increased mtDNA copy number is associated with decreased total sperm motility in stallions. We therefore suggest that mtDNA copy number may be an indicator of defective spermatogenesis in stallions. Copyright © 2017 Elsevier Inc. All rights reserved.

Comparing CNV detection methods for SNP arrays.

PubMed

Winchester, Laura; Yau, Christopher; Ragoussis, Jiannis

2009-09-01

Data from whole genome association studies can now be used for dual purposes, genotyping and copy number detection. In this review we discuss some of the methods for using SNP data to detect copy number events. We examine a number of algorithms designed to detect copy number changes through the use of signal-intensity data and consider methods to evaluate the changes found. We describe the use of several statistical models in copy number detection in germline samples. We also present a comparison of data using these methods to assess accuracy of prediction and detection of changes in copy number.

Impact of recombination on polymorphism of genes encoding Kunitz-type protease inhibitors in the genus Solanum.

PubMed

Speranskaya, Anna S; Krinitsina, Anastasia A; Kudryavtseva, Anna V; Poltronieri, Palmiro; Santino, Angelo; Oparina, Nina Y; Dmitriev, Alexey A; Belenikin, Maxim S; Guseva, Marina A; Shevelev, Alexei B

2012-08-01

The group of Kunitz-type protease inhibitors (KPI) from potato is encoded by a polymorphic family of multiple allelic and non-allelic genes. The previous explanations of the KPI variability were based on the hypothesis of random mutagenesis as a key factor of KPI polymorphism. KPI-A genes from the genomes of Solanum tuberosum cv. Istrinskii and the wild species Solanum palustre were amplified by PCR with subsequent cloning in plasmids. True KPI sequences were derived from comparison of the cloned copies. "Hot spots" of recombination in KPI genes were independently identified by DnaSP 4.0 and TOPALi v2.5 software. The KPI-A sequence from potato cv. Istrinskii was found to be 100% identical to the gene from Solanum nigrum. This fact illustrates a high degree of similarity of KPI genes in the genus Solanum. Pairwise comparison of KPI A and B genes unambiguously showed a non-uniform extent of polymorphism at different nt positions. Moreover, the occurrence of substitutions was not random along the strand. Taken together, these facts contradict the traditional hypothesis of random mutagenesis as a principal source of KPI gene polymorphism. The experimentally found mosaic structure of KPI genes in both plants studied is consistent with the hypothesis suggesting recombination of ancestral genes. The same mechanism was proposed earlier for other resistance-conferring genes in the nightshade family (Solanaceae). Based on the data obtained, we searched for potential motifs of site-specific binding with plant DNA recombinases. During this work, we analyzed the sequencing data reported by the Potato Genome Sequencing Consortium (PGSC), 2011 and found considerable inconsistence of their data concerning the number, location, and orientation of KPI genes of groups A and B. The key role of recombination rather than random point mutagenesis in KPI polymorphism was demonstrated for the first time. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Increased vitamin D receptor gene expression and rs11568820 and rs4516035 promoter polymorphisms in autistic disorder.

PubMed

Balta, Burhan; Gumus, Hakan; Bayramov, Ruslan; Korkmaz Bayramov, Keziban; Erdogan, Murat; Oztop, Didem Behice; Dogan, Muhammet Ensar; Taheri, Serpil; Dundar, Munis

2018-05-18

Although there are a large number of sequence variants of different genes and copy number variations at various loci identified in autistic disorder (AD) patients, the pathogenesis of AD has not been elucidated completely. Recently, in AD patients, a large number of expression array and transcriptome studies have shown an increase in the expression of genes especially related to innate immune response. Antimicrobial effects of vitamin D and VDR are exerted through Toll-Like-Receptors (TLR) which have an important role in the innate immune response, are expressed by antigen presenting cells and recognize foreign microorganisms. In this study, age and gender matched 30 patients diagnosed with AD and 30 healthy controls were included in the study. Comparatively whole blood VDR gene expression and rs11568820 and rs4516035 SNP profile of the promoter region of the VDR gene were investigated by real time PCR. Whole blood VDR gene expression was significantly higher in the AD group compared to control subjects (p < 0.0001). There were no significant differences among allele and genotype distribution of rs11568820 and rs4516035 polymorphisms between AD patients and controls. The increase of VDR gene expression in patients with AD may be in accordance with an increase in the innate immune response in patients with AD. Furthermore, this study will stimulate new studies in order to clarify the relationship among AD, vitamin D, VDR, and innate immunity.

Genome-wide DNA polymorphism in the indica rice varieties RGD-7S and Taifeng B as revealed by whole genome re-sequencing.

PubMed

Fu, Chong-Yun; Liu, Wu-Ge; Liu, Di-Lin; Li, Ji-Hua; Zhu, Man-Shan; Liao, Yi-Long; Liu, Zhen-Rong; Zeng, Xue-Qin; Wang, Feng

2016-03-01

Next-generation sequencing technologies provide opportunities to further understand genetic variation, even within closely related cultivars. We performed whole genome resequencing of two elite indica rice varieties, RGD-7S and Taifeng B, whose F1 progeny showed hybrid weakness and hybrid vigor when grown in the early- and late-cropping seasons, respectively. Approximately 150 million 100-bp pair-end reads were generated, which covered ∼86% of the rice (Oryza sativa L. japonica 'Nipponbare') reference genome. A total of 2,758,740 polymorphic sites including 2,408,845 SNPs and 349,895 InDels were detected in RGD-7S and Taifeng B, respectively. Applying stringent parameters, we identified 961,791 SNPs and 46,640 InDels between RGD-7S and Taifeng B (RGD-7S/Taifeng B). The density of DNA polymorphisms was 256.8 SNPs and 12.5 InDels per 100 kb for RGD-7S/Taifeng B. Copy number variations (CNVs) were also investigated. In RGD-7S, 1989 of 2727 CNVs were overlapped in 218 genes, and 1231 of 2010 CNVs were annotated in 175 genes in Taifeng B. In addition, we verified a subset of InDels in the interval of hybrid weakness genes, Hw3 and Hw4, and obtained some polymorphic InDel markers, which will provide a sound foundation for cloning hybrid weakness genes. Analysis of genomic variations will also contribute to understanding the genetic basis of hybrid weakness and heterosis.

Prediction of peripheral neuropathy in multiple myeloma patients receiving bortezomib and thalidomide: a genetic study based on a single nucleotide polymorphism array.

PubMed

García-Sanz, Ramón; Corchete, Luis Antonio; Alcoceba, Miguel; Chillon, María Carmen; Jiménez, Cristina; Prieto, Isabel; García-Álvarez, María; Puig, Noemi; Rapado, Immaculada; Barrio, Santiago; Oriol, Albert; Blanchard, María Jesús; de la Rubia, Javier; Martínez, Rafael; Lahuerta, Juan José; González Díaz, Marcos; Mateos, María Victoria; San Miguel, Jesús Fernando; Martínez-López, Joaquín; Sarasquete, María Eugenia

2017-12-01

Bortezomib- and thalidomide-based therapies have significantly contributed to improved survival of multiple myeloma (MM) patients. However, treatment-induced peripheral neuropathy (TiPN) is a common adverse event associated with them. Risk factors for TiPN in MM patients include advanced age, prior neuropathy, and other drugs, but there are conflicting results about the role of genetics in predicting the risk of TiPN. Thus, we carried out a genome-wide association study based on more than 300 000 exome single nucleotide polymorphisms in 172 MM patients receiving therapy involving bortezomib and thalidomide. We compared patients developing and not developing TiPN under similar treatment conditions (GEM05MAS65, NCT00443235). The highest-ranking single nucleotide polymorphism was rs45443101, located in the PLCG2 gene, but no significant differences were found after multiple comparison correction (adjusted P = .1708). Prediction analyses, cytoband enrichment, and pathway analyses were also performed, but none yielded any significant findings. A copy number approach was also explored, but this gave no significant results either. In summary, our study did not find a consistent genetic component associated with TiPN under bortezomib and thalidomide therapies that could be used for prediction, which makes clinical judgment essential in the practical management of MM treatment. Copyright © 2016 John Wiley & Sons, Ltd.

A critical analysis of disease-associated DNA polymorphisms in the genes of cattle, goat, sheep, and pig

PubMed Central

Ibeagha-Awemu, Eveline M.; Kgwatalala, Patrick; Ibeagha, Aloysius E.

2008-01-01

Genetic variations through their effects on gene expression and protein function underlie disease susceptibility in farm animal species. The variations are in the form of single nucleotide polymorphisms, deletions/insertions of nucleotides or whole genes, gene or whole chromosomal rearrangements, gene duplications, and copy number polymorphisms or variants. They exert varying degrees of effects on gene action, such as substitution of an amino acid for another, shift in reading frame and premature termination of translation, and complete deletion of entire exon(s) or gene(s) in diseased individuals. These factors influence gene function by affecting mRNA splicing pattern or by altering/eliminating protein function. Elucidating the genetic bases of diseases under the control of many genes is very challenging, and it is compounded by several factors, including host × pathogen × environment interactions. In this review, the genetic variations that underlie several diseases of livestock (under monogenic and polygenic control) are analyzed. Also, factors hampering research efforts toward identification of genetic influences on animal disease identification and control are highlighted. A better understanding of the factors analyzed could be better harnessed to effectively identify and control, genetically, livestock diseases. Finally, genetic control of animal diseases can reduce the costs associated with diseases, improve animal welfare, and provide healthy animal products to consumers, and should be given more attention. PMID:18350334

Heme Oxygenase-1 and 2 Common Genetic Variants and Risk for Multiple Sclerosis

PubMed Central

Agúndez, José A. G.; García-Martín, Elena; Martínez, Carmen; Benito-León, Julián; Millán-Pascual, Jorge; Díaz-Sánchez, María; Calleja, Patricia; Pisa, Diana; Turpín-Fenoll , Laura; Alonso-Navarro, Hortensia; Pastor, Pau; Ortega-Cubero, Sara; Ayuso-Peralta, Lucía; Torrecillas, Dolores; García-Albea, Esteban; Plaza-Nieto, José Francisco; Jiménez-Jiménez, Félix Javier

2016-01-01

Several neurochemical, neuropathological, and experimental data suggest a possible role of oxidative stress in the ethiopathogenesis of multiple sclerosis(MS). Heme-oxygenases(HMOX) are an important defensive mechanism against oxidative stress, and HMOX1 is overexpressed in the brain and spinal cord of MS patients and in experimental autoimmune encephalomyelitis(EAE). We analyzed whether common polymorphisms affecting the HMOX1 and HMOX2 genes are related with the risk to develop MS. We analyzed the distribution of genotypes and allelic frequencies of the HMOX1 rs2071746, HMOX1 rs2071747, HMOX2 rs2270363, and HMOX2 rs1051308 SNPs, as well as the presence of Copy number variations(CNVs) of these genes in 292 subjects MS and 533 healthy controls, using TaqMan assays. The frequencies of HMOX2 rs1051308AA genotype and HMOX2 rs1051308A and HMOX1 rs2071746A alleles were higher in MS patients than in controls, although only that of the SNP HMOX2 rs1051308 in men remained as significant after correction for multiple comparisons. None of the studied polymorphisms was related to the age at disease onset or with the MS phenotype. The present study suggests a weak association between HMOX2 rs1051308 polymorphism and the risk to develop MS in Spanish Caucasian men and a trend towards association between the HMOX1 rs2071746A and MS risk. PMID:26868429

Heme Oxygenase-1 and 2 Common Genetic Variants and Risk for Multiple Sclerosis.

PubMed

Agúndez, José A G; García-Martín, Elena; Martínez, Carmen; Benito-León, Julián; Millán-Pascual, Jorge; Díaz-Sánchez, María; Calleja, Patricia; Pisa, Diana; Turpín-Fenoll, Laura; Alonso-Navarro, Hortensia; Pastor, Pau; Ortega-Cubero, Sara; Ayuso-Peralta, Lucía; Torrecillas, Dolores; García-Albea, Esteban; Plaza-Nieto, José Francisco; Jiménez-Jiménez, Félix Javier

2016-02-12

Several neurochemical, neuropathological, and experimental data suggest a possible role of oxidative stress in the ethiopathogenesis of multiple sclerosis(MS). Heme-oxygenases(HMOX) are an important defensive mechanism against oxidative stress, and HMOX1 is overexpressed in the brain and spinal cord of MS patients and in experimental autoimmune encephalomyelitis(EAE). We analyzed whether common polymorphisms affecting the HMOX1 and HMOX2 genes are related with the risk to develop MS. We analyzed the distribution of genotypes and allelic frequencies of the HMOX1 rs2071746, HMOX1 rs2071747, HMOX2 rs2270363, and HMOX2 rs1051308 SNPs, as well as the presence of Copy number variations(CNVs) of these genes in 292 subjects MS and 533 healthy controls, using TaqMan assays. The frequencies of HMOX2 rs1051308AA genotype and HMOX2 rs1051308A and HMOX1 rs2071746A alleles were higher in MS patients than in controls, although only that of the SNP HMOX2 rs1051308 in men remained as significant after correction for multiple comparisons. None of the studied polymorphisms was related to the age at disease onset or with the MS phenotype. The present study suggests a weak association between HMOX2 rs1051308 polymorphism and the risk to develop MS in Spanish Caucasian men and a trend towards association between the HMOX1 rs2071746A and MS risk.

The transcription factor titration effect dictates level of gene expression.

PubMed

Brewster, Robert C; Weinert, Franz M; Garcia, Hernan G; Song, Dan; Rydenfelt, Mattias; Phillips, Rob

2014-03-13

Models of transcription are often built around a picture of RNA polymerase and transcription factors (TFs) acting on a single copy of a promoter. However, most TFs are shared between multiple genes with varying binding affinities. Beyond that, genes often exist at high copy number-in multiple identical copies on the chromosome or on plasmids or viral vectors with copy numbers in the hundreds. Using a thermodynamic model, we characterize the interplay between TF copy number and the demand for that TF. We demonstrate the parameter-free predictive power of this model as a function of the copy number of the TF and the number and affinities of the available specific binding sites; such predictive control is important for the understanding of transcription and the desire to quantitatively design the output of genetic circuits. Finally, we use these experiments to dynamically measure plasmid copy number through the cell cycle. Copyright © 2014 Elsevier Inc. All rights reserved.

The distribution and impact of common copy-number variation in the genome of the domesticated apple, Malus x domestica Borkh.

PubMed

Boocock, James; Chagné, David; Merriman, Tony R; Black, Michael A

2015-10-23

Copy number variation (CNV) is a common feature of eukaryotic genomes, and a growing body of evidence suggests that genes affected by CNV are enriched in processes that are associated with environmental responses. Here we use next generation sequence (NGS) data to detect copy-number variable regions (CNVRs) within the Malus x domestica genome, as well as to examine their distribution and impact. CNVRs were detected using NGS data derived from 30 accessions of M. x domestica analyzed using the read-depth method, as implemented in the CNVrd2 software. To improve the reliability of our results, we developed a quality control and analysis procedure that involved checking for organelle DNA, not repeat masking, and the determination of CNVR identity using a permutation testing procedure. Overall, we identified 876 CNVRs, which spanned 3.5 % of the apple genome. To verify that detected CNVRs were not artifacts, we analyzed the B- allele-frequencies (BAF) within a single nucleotide polymorphism (SNP) array dataset derived from a screening of 185 individual apple accessions and found the CNVRs were enriched for SNPs having aberrant BAFs (P < 1e-13, Fisher's Exact test). Putative CNVRs overlapped 845 gene models and were enriched for resistance (R) gene models (P < 1e-22, Fisher's exact test). Of note was a cluster of resistance gene models on chromosome 2 near a region containing multiple major gene loci conferring resistance to apple scab. We present the first analysis and catalogue of CNVRs in the M. x domestica genome. The enrichment of the CNVRs with R gene models and their overlap with gene loci of agricultural significance draw attention to a form of unexplored genetic variation in apple. This research will underpin further investigation of the role that CNV plays within the apple genome.

Copy number variations of E2F1: a new genetic risk factor for testicular cancer.

PubMed

Rocca, Maria Santa; Di Nisio, Andrea; Marchiori, Arianna; Ghezzi, Marco; Opocher, Giuseppe; Foresta, Carlo; Ferlin, Alberto

2017-03-01

Testicular germ cell tumor (TGCT) is one of the most heritable forms of cancer. In last years, many evidence suggested that constitutional genetic factors, mainly single nucleotide polymorphisms, can increase its risk. However, the possible contribution of copy number variations (CNVs) in TGCT susceptibility has not been substantially addressed. Indeed, an increasing number of studies have focused on the effect of CNVs on gene expression and on the role of these structural genetic variations as risk factors for different forms of cancer. E2F1 is a transcription factor that plays an important role in regulating cell growth, differentiation, apoptosis and response to DNA damage. Therefore, deficiency or overexpression of this protein might significantly influence fundamental biological processes involved in cancer development and progression, including TGCT. We analyzed E2F1 CNVs in 261 cases with TGCT and 165 controls. We found no CNVs in controls, but 17/261 (6.5%) cases showed duplications in E2F1 Blot analysis demonstrated higher E2F1 expression in testicular samples of TGCT cases with three copies of the gene. Furthermore, we observed higher phosphorylation of Akt and mTOR in samples with E2F1 duplication. Interestingly, normal, non-tumoral testicular tissue in patient with E2F1 duplication showed lower expression of E2F1 and lower AKT/mTOR phosphorylation with respect to adjacent tumor tissue. Furthermore, increased expression of E2F1 obtained in vitro in NTERA-2 testicular cell line induced increased AKT/mTOR phosphorylation. This study suggests for the first time an involvement of E2F1 CNVs in TGCT susceptibility and supports previous preliminary data on the importance of AKT/mTOR signaling pathway in this cancer. © 2017 Society for Endocrinology.

DNA Copy Number Signature to Predict Recurrence in Early Stage Ovarian Cancer

DTIC Science & Technology

2016-08-01

AWARD NUMBER: W81XWH-14-1-0194 TITLE: DNA Copy Number Signature to Predict Recurrence in Early-Stage Ovarian Cancer PRINCIPAL INVESTIGATOR...SUBTITLE 5a. CONTRACT NUMBER DNA Copy Number Signature to Predict Recurrence in Early-Stage Ovarian Cancer 5b. GRANT NUMBER W81XWH-14-1-0194 5c. PROGRAM...determine the copy number gain and loss for early stage high grade ovarian cancers through IlluminaHumanOmniExpress-FFPE BeadChip system • Subtask 1 DNA

Age-related decline in mitochondrial DNA copy number in isolated human pancreatic islets.

PubMed

Cree, L M; Patel, S K; Pyle, A; Lynn, S; Turnbull, D M; Chinnery, P F; Walker, M

2008-08-01

Pancreatic beta cell function has been shown to decline with age in man. Depletion of mitochondrial DNA (mtDNA) copy number is associated with impaired insulin secretion in pancreatic beta cell lines, and decreased mtDNA copy number has been observed with age in skeletal muscle in man. We investigated whether mtDNA copy number decreases with age in human pancreatic beta cells, which might in turn contribute to the age-related decline in insulin secretory capacity. We quantified mtDNA copy number in isolated human islet preparations from 15 pancreas donors aged between 17 and 75 years. Islets (n = 20) were individually hand-picked and pooled from each donor isolate for the quantification of mtDNA copy number and deleted mtDNA (%), which were determined using real-time PCR methods. There was a significant negative correlation between mtDNA copy number and islet donor age (r = -0.53, p = 0.044). mtDNA copy number was significantly decreased in islet preparations from donors aged > or =50 years (n = 8) compared with those aged <50 years (n = 7) (median [interquartile range]: 418 [236-503] vs 596 [554-729] mtDNA copy number/diploid genome; p = 0.032). None of the islet preparations harboured high levels of deleted mtDNA affecting the major arc. Given the correlation between mtDNA content and respiratory chain activity, the age-related decrease in mtDNA copy number that we observed in human pancreatic islet preparations may contribute to the age-dependent decline in pancreatic beta cell insulin secretory capacity.

Allelic recombination between distinct genomic locations generates copy number diversity in human β-defensins

PubMed Central

Bakar, Suhaili Abu; Hollox, Edward J.; Armour, John A. L.

2009-01-01

β-Defensins are small secreted antimicrobial and signaling peptides involved in the innate immune response of vertebrates. In humans, a cluster of at least 7 of these genes shows extensive copy number variation, with a diploid copy number commonly ranging between 2 and 7. Using a genetic mapping approach, we show that this cluster is at not 1 but 2 distinct genomic loci ≈5 Mb apart on chromosome band 8p23.1, contradicting the most recent genome assembly. We also demonstrate that the predominant mechanism of change in β-defensin copy number is simple allelic recombination occurring in the interval between the 2 distinct genomic loci for these genes. In 416 meiotic transmissions, we observe 3 events creating a haplotype copy number not found in the parent, equivalent to a germ-line rate of copy number change of ≈0.7% per gamete. This places it among the fastest-changing copy number variants currently known. PMID:19131514

Characterizing the cancer genome in lung adenocarcinoma

PubMed Central

Weir, Barbara A.; Woo, Michele S.; Getz, Gad; Perner, Sven; Ding, Li; Beroukhim, Rameen; Lin, William M.; Province, Michael A.; Kraja, Aldi; Johnson, Laura A.; Shah, Kinjal; Sato, Mitsuo; Thomas, Roman K.; Barletta, Justine A.; Borecki, Ingrid B.; Broderick, Stephen; Chang, Andrew C.; Chiang, Derek Y.; Chirieac, Lucian R.; Cho, Jeonghee; Fujii, Yoshitaka; Gazdar, Adi F.; Giordano, Thomas; Greulich, Heidi; Hanna, Megan; Johnson, Bruce E.; Kris, Mark G.; Lash, Alex; Lin, Ling; Lindeman, Neal; Mardis, Elaine R.; McPherson, John D.; Minna, John D.; Morgan, Margaret B.; Nadel, Mark; Orringer, Mark B.; Osborne, John R.; Ozenberger, Brad; Ramos, Alex H.; Robinson, James; Roth, Jack A.; Rusch, Valerie; Sasaki, Hidefumi; Shepherd, Frances; Sougnez, Carrie; Spitz, Margaret R.; Tsao, Ming-Sound; Twomey, David; Verhaak, Roel G. W.; Weinstock, George M.; Wheeler, David A.; Winckler, Wendy; Yoshizawa, Akihiko; Yu, Soyoung; Zakowski, Maureen F.; Zhang, Qunyuan; Beer, David G.; Wistuba, Ignacio I.; Watson, Mark A.; Garraway, Levi A.; Ladanyi, Marc; Travis, William D.; Pao, William; Rubin, Mark A.; Gabriel, Stacey B.; Gibbs, Richard A.; Varmus, Harold E.; Wilson, Richard K.; Lander, Eric S.; Meyerson, Matthew

2008-01-01

Somatic alterations in cellular DNA underlie almost all human cancers1. The prospect of targeted therapies2 and the development of high-resolution, genome-wide approaches3–8 are now spurring systematic efforts to characterize cancer genomes. Here we report a large-scale project to characterize copy-number alterations in primary lung adenocarcinomas. By analysis of a large collection of tumors (n = 371) using dense single nucleotide polymorphism arrays, we identify a total of 57 significantly recurrent events. We find that 26 of 39 autosomal chromosome arms show consistent large-scale copy-number gain or loss, of which only a handful have been linked to a specific gene. We also identify 31 recurrent focal events, including 24 amplifications and 7 homozygous deletions. Only six of these focal events are currently associated with known mutations in lung carcinomas. The most common event, amplification of chromosome 14q13.3, is found in ~12% of samples. On the basis of genomic and functional analyses, we identify NKX2-1 (NK2 homeobox 1, also called TITF1), which lies in the minimal 14q13.3 amplification interval and encodes a lineage-specific transcription factor, as a novel candidate proto-oncogene involved in a significant fraction of lung adenocarcinomas. More generally, our results indicate that many of the genes that are involved in lung adenocarcinoma remain to be discovered. PMID:17982442

Respiratory chain complex III deficiency in patients with tRNA-leu mutation.

PubMed

Jiang, J; Wang, X L; Ma, Y Y

2015-12-29

The aim of this study was to investigate the clinical and genetic profiles of mitochondrial disease resulting from deficiencies in the respiratory chain complex III. Three patients, aged between 8 months and 12 years, were recruited for this study. The activities of mitochondrial respiratory chain complexes in the peripheral leucocytes were spectrophotometrically measured. The entire mitochondrial DNA (mtDNA) sequence was analyzed. Samples obtained from the three patients and their families were subjected to restriction fragment length polymorphism and gene sequencing analyses. mtDNA copy numbers of all patients and their mothers were analyzed. The patients displayed nervous system impairment, including motor and mental developmental delay, hypotonia, and motor regression. Two patients also suffered from Leigh syndrome. Assay of the mitochondrial respiratory chain enzymes revealed an isolated complex III deficiency in the three patients. The m.3243 A>G mutation was detected in all patients and their mothers. The mutation loads were 48.3, 57.2, and 45.5% in the patients, and 20.5, 16.4, and 23.6% in their respective mothers. The leukocyte mtDNA copy numbers of the patients and their mothers were within the control range. The clinical manifestation and genetics were observed to be very heterogeneous. Patient carrying an m.3243 A>G mutation may biochemically display a deficiency in the mitochondrial respiratory chain complex III.

Impact of Parental Bos taurus and Bos indicus Origins on Copy Number Variation in Traditional Chinese Cattle Breeds.

PubMed

Zhang, Liangzhi; Jia, Shangang; Plath, Martin; Huang, Yongzhen; Li, Congjun; Lei, Chuzhao; Zhao, Xin; Chen, Hong

2015-08-10

Copy number variation (CNV) is an important component of genomic structural variation and plays a role not only in evolutionary diversification but also in domestication. Chinese cattle were derived from Bos taurus and Bos indicus, and several breeds presumably are of hybrid origin, but the evolution of CNV regions (CNVRs) has not yet been examined in this context. Here, we of CNVRs, mtDNA D-loop sequence variation, and Y-chromosomal single nucleotide polymorphisms to assess the impact of maternal and paternal B. taurus and B. indicus origins on the distribution of CNVRs in 24 Chinese domesticated bulls. We discovered 470 genome-wide CNVRs, only 72 of which were shared by all three Y-lineages (B. taurus: Y1, Y2; B. indicus: Y3), whereas 265 were shared by inferred taurine or indicine paternal lineages, and 228 when considering their maternal taurine or indicine origins. Phylogenetic analysis uncovered eight taurine/indicine hybrids, and principal component analysis on CNVs corroborated genomic exchange during hybridization. The distribution patterns of CNVRs tended to be lineage-specific, and correlation analysis revealed significant positive or negative co-occurrences of CNVRs across lineages. Our study suggests that CNVs in Chinese cattle partly result from selective breeding during domestication, but also from hybridization and introgression. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Copy number variants in patients with short stature

PubMed Central

van Duyvenvoorde, Hermine A; Lui, Julian C; Kant, Sarina G; Oostdijk, Wilma; Gijsbers, Antoinet CJ; Hoffer, Mariëtte JV; Karperien, Marcel; Walenkamp, Marie JE; Noordam, Cees; Voorhoeve, Paul G; Mericq, Verónica; Pereira, Alberto M; Claahsen-van de Grinten, Hedi L; van Gool, Sandy A; Breuning, Martijn H; Losekoot, Monique; Baron, Jeffrey; Ruivenkamp, Claudia AL; Wit, Jan M

2014-01-01

Height is a highly heritable and classic polygenic trait. Recent genome-wide association studies (GWAS) have revealed that at least 180 genetic variants influence adult height. However, these variants explain only about 10% of the phenotypic variation in height. Genetic analysis of short individuals can lead to the discovery of novel rare gene defects with a large effect on growth. In an effort to identify novel genes associated with short stature, genome-wide analysis for copy number variants (CNVs), using single-nucleotide polymorphism arrays, in 162 patients (149 families) with short stature was performed. Segregation analysis was performed if possible, and genes in CNVs were compared with information from GWAS, gene expression in rodents' growth plates and published information. CNVs were detected in 40 families. In six families, a known cause of short stature was found (SHOX deletion or duplication, IGF1R deletion), in two combined with a de novo potentially pathogenic CNV. Thirty-three families had one or more potentially pathogenic CNVs (n=40). In 24 of these families, segregation analysis could be performed, identifying three de novo CNVs and nine CNVs segregating with short stature. Four were located near loci associated with height in GWAS (ADAMTS17, TULP4, PRKG2/BMP3 and PAPPA). Besides six CNVs known to be causative for short stature, 40 CNVs with possible pathogenicity were identified. Segregation studies and bioinformatics analysis suggested various potential candidate genes. PMID:24065112

Single-Nucleotide Polymorphism Array-Based Karyotyping of Acute Promyelocytic Leukemia

PubMed Central

Gómez-Seguí, Inés; Sánchez-Izquierdo, Dolors; Barragán, Eva; Such, Esperanza; Luna, Irene; López-Pavía, María; Ibáñez, Mariam; Villamón, Eva; Alonso, Carmen; Martín, Iván; Llop, Marta; Dolz, Sandra; Fuster, Óscar; Montesinos, Pau; Cañigral, Carolina; Boluda, Blanca; Salazar, Claudia

2014-01-01

Acute promyelocytic leukemia (APL) is characterized by the t(15;17)(q22;q21), but additional chromosomal abnormalities (ACA) and other rearrangements can contribute in the development of the whole leukemic phenotype. We hypothesized that some ACA not detected by conventional techniques may be informative of the onset of APL. We performed the high-resolution SNP array (SNP-A) 6.0 (Affymetrix) in 48 patients diagnosed with APL on matched diagnosis and remission sample. Forty-six abnormalities were found as an acquired event in 23 patients (48%): 22 duplications, 23 deletions and 1 Copy-Neutral Loss of Heterozygocity (CN-LOH), being a duplication of 8(q24) (23%) and a deletion of 7(q33-qter) (6%) the most frequent copy-number abnormalities (CNA). Four patients (8%) showed CNAs adjacent to the breakpoints of the translocation. We compared our results with other APL series and found that, except for dup(8q24) and del(7q33-qter), ACA were infrequent (≤3%) but most of them recurrent (70%). Interestingly, having CNA or FLT3 mutation were mutually exclusive events. Neither the number of CNA, nor any specific CNA was associated significantly with prognosis. This study has delineated recurrent abnormalities in addition to t(15;17) that may act as secondary events and could explain leukemogenesis in up to 40% of APL cases with no ACA by conventional cytogenetics. PMID:24959826

Single-nucleotide polymorphism array-based karyotyping of acute promyelocytic leukemia.

PubMed

Gómez-Seguí, Inés; Sánchez-Izquierdo, Dolors; Barragán, Eva; Such, Esperanza; Luna, Irene; López-Pavía, María; Ibáñez, Mariam; Villamón, Eva; Alonso, Carmen; Martín, Iván; Llop, Marta; Dolz, Sandra; Fuster, Oscar; Montesinos, Pau; Cañigral, Carolina; Boluda, Blanca; Salazar, Claudia; Cervera, Jose; Sanz, Miguel A

2014-01-01

Acute promyelocytic leukemia (APL) is characterized by the t(15;17)(q22;q21), but additional chromosomal abnormalities (ACA) and other rearrangements can contribute in the development of the whole leukemic phenotype. We hypothesized that some ACA not detected by conventional techniques may be informative of the onset of APL. We performed the high-resolution SNP array (SNP-A) 6.0 (Affymetrix) in 48 patients diagnosed with APL on matched diagnosis and remission sample. Forty-six abnormalities were found as an acquired event in 23 patients (48%): 22 duplications, 23 deletions and 1 Copy-Neutral Loss of Heterozygocity (CN-LOH), being a duplication of 8(q24) (23%) and a deletion of 7(q33-qter) (6%) the most frequent copy-number abnormalities (CNA). Four patients (8%) showed CNAs adjacent to the breakpoints of the translocation. We compared our results with other APL series and found that, except for dup(8q24) and del(7q33-qter), ACA were infrequent (≤3%) but most of them recurrent (70%). Interestingly, having CNA or FLT3 mutation were mutually exclusive events. Neither the number of CNA, nor any specific CNA was associated significantly with prognosis. This study has delineated recurrent abnormalities in addition to t(15;17) that may act as secondary events and could explain leukemogenesis in up to 40% of APL cases with no ACA by conventional cytogenetics.

Rare Germline Copy Number Variations and Disease Susceptibility in Familial Melanoma.

PubMed

Shi, Jianxin; Zhou, Weiyin; Zhu, Bin; Hyland, Paula L; Bennett, Hunter; Xiao, Yanzi; Zhang, Xijun; Burke, Laura S; Song, Lei; Hsu, Chih Hao; Yan, Chunhua; Chen, Qingrong; Meerzaman, Daoud; Dagnall, Casey L; Burdette, Laurie; Hicks, Belynda; Freedman, Neal D; Chanock, Stephen J; Yeager, Meredith; Tucker, Margaret A; Goldstein, Alisa M; Yang, Xiaohong R

2016-12-01

Mounting evidence suggests that copy number variations (CNVs) can contribute to cancer susceptibility. The main goal of this study was to evaluate the role of germline CNVs in melanoma predisposition in high-risk melanoma families. We used genome-wide tiling comparative genomic hybridization and single nucleotide polymorphism arrays to characterize CNVs in 335 individuals (240 melanoma cases) from American melanoma-prone families (22 with germline CDKN2A or CDK4 mutations). We found that the global burden of overall CNVs (or deletions or duplications separately) was not significantly associated with case-control or CDKN2A/CDK4 mutation status after accounting for the familial dependence. However, we identified several rare CNVs that either involved known melanoma genes (e.g., PARP1, CDKN2A) or cosegregated with melanoma (duplication on 10q23.23, 3p12.2 and deletions on 8q424.3, 2q22.1) in families without mutations in known melanoma high-risk genes. Some of these CNVs were correlated with expression changes in disrupted genes based on RNASeq data from a subset of melanoma cases included in the CNV study. These results suggest that rare cosegregating CNVs may influence melanoma susceptibility in some melanoma-prone families and genes found in our study warrant further evaluation in future genetic analyses of melanoma. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Analysis of chromosome 22q11 copy number variations by multiplex ligation-dependent probe amplification for prenatal diagnosis of congenital heart defect.

PubMed

Zhang, Jingjing; Ma, Dingyuan; Wang, Yan; Cao, Li; Wu, Yun; Qiao, Fengchang; Liu, An; Li, Li; Lin, Ying; Liu, Gang; Liu, Cuiyun; Hu, Ping; Xu, Zhengfeng

2015-01-01

Congenital heart defects (CHD) represent one of the most common birth defects. This study aimed to evaluate the value of multiplex ligation-dependent probe amplification (MLPA) as a tool to detect the copy number variations (CNVs) of 22q11 in fetuses with CHD. A large cohort of 225 fetuses with CHD was screened by fetal echocardiography. Once common chromosome abnormalities in 30 fetuses were screened out by conventional G-banding analysis, the CNVs of chromosome 22q11 in the remaining 195 fetuses were determined by MLPA for prenatal genetic counseling. In 195 CHD fetuses with normal karyotype, 11 cases had pathological CNVs, including 22q11.2 deletion (seven cases), the deletion of 22q11 cat eye syndrome (CES) region (one case), 22q11.2 duplication (one case), 22q13.3 deletion (one case) and 17p13.3 deletion (one case). In total, our findings from MLPA screening represented 4.9 % in our cohort. Among these, three cases were inherited CNVs, and eight cases were de novo. These CNVs were further verified by single nucleotide polymorphism (SNP)-array analysis, and their chromosomal location was refined. This study indicated that MLPA could serve as an effective test for routine prenatal diagnosis of 22q11 in fetuses with CHD.

Low copy number of the salivary amylase gene predisposes to obesity.

PubMed

Falchi, Mario; El-Sayed Moustafa, Julia Sarah; Takousis, Petros; Pesce, Francesco; Bonnefond, Amélie; Andersson-Assarsson, Johanna C; Sudmant, Peter H; Dorajoo, Rajkumar; Al-Shafai, Mashael Nedham; Bottolo, Leonardo; Ozdemir, Erdal; So, Hon-Cheong; Davies, Robert W; Patrice, Alexandre; Dent, Robert; Mangino, Massimo; Hysi, Pirro G; Dechaume, Aurélie; Huyvaert, Marlène; Skinner, Jane; Pigeyre, Marie; Caiazzo, Robert; Raverdy, Violeta; Vaillant, Emmanuel; Field, Sarah; Balkau, Beverley; Marre, Michel; Visvikis-Siest, Sophie; Weill, Jacques; Poulain-Godefroy, Odile; Jacobson, Peter; Sjostrom, Lars; Hammond, Christopher J; Deloukas, Panos; Sham, Pak Chung; McPherson, Ruth; Lee, Jeannette; Tai, E Shyong; Sladek, Robert; Carlsson, Lena M S; Walley, Andrew; Eichler, Evan E; Pattou, Francois; Spector, Timothy D; Froguel, Philippe

2014-05-01

Common multi-allelic copy number variants (CNVs) appear enriched for phenotypic associations compared to their biallelic counterparts. Here we investigated the influence of gene dosage effects on adiposity through a CNV association study of gene expression levels in adipose tissue. We identified significant association of a multi-allelic CNV encompassing the salivary amylase gene (AMY1) with body mass index (BMI) and obesity, and we replicated this finding in 6,200 subjects. Increased AMY1 copy number was positively associated with both amylase gene expression (P = 2.31 × 10(-14)) and serum enzyme levels (P < 2.20 × 10(-16)), whereas reduced AMY1 copy number was associated with increased BMI (change in BMI per estimated copy = -0.15 (0.02) kg/m(2); P = 6.93 × 10(-10)) and obesity risk (odds ratio (OR) per estimated copy = 1.19, 95% confidence interval (CI) = 1.13-1.26; P = 1.46 × 10(-10)). The OR value of 1.19 per copy of AMY1 translates into about an eightfold difference in risk of obesity between subjects in the top (copy number > 9) and bottom (copy number < 4) 10% of the copy number distribution. Our study provides a first genetic link between carbohydrate metabolism and BMI and demonstrates the power of integrated genomic approaches beyond genome-wide association studies.

DNA methylation polymorphism in a set of elite rice cultivars and its possible contribution to inter-cultivar differential gene expression.

PubMed

Wang, Yongming; Lin, Xiuyun; Dong, Bo; Wang, Yingdian; Liu, Bao

2004-01-01

RAPD (randomly amplified polymorphic DNA) and ISSR (inter-simple sequence repeat) fingerprinting on HpaII/MspI-digested genomic DNA of nine elite japonica rice cultivars implies inter-cultivar DNA methylation polymorphism. Using both DNA fragments isolated from RAPD or ISSR gels and selected low-copy sequences as probes, methylation-sensitive Southern blot analysis confirms the existence of extensive DNA methylation polymorphism in both genes and DNA repeats among the rice cultivars. The cultivar-specific methylation patterns are stably maintained, and can be used as reliable molecular markers. Transcriptional analysis of four selected sequences (RdRP, AC9, HSP90 and MMR) on leaves and roots from normal and 5-azacytidine-treated seedlings of three representative cultivars shows an association between the transcriptional activity of one of the genes, the mismatch repair (MMR) gene, and its CG methylation patterns.

Association of STin2 Variable Number of Tandem Repeat (VNTR) Polymorphism of Serotonin Transporter Gene with Lifelong Premature Ejaculation: A Case-Control Study in Han Chinese Subjects

PubMed Central

Huang, Yuanyuan; Zhang, Xiansheng; Gao, Jingjing; Tang, Dongdong; Gao, Pan; Peng, Dangwei; Liang, Chaozhao

2016-01-01

Background The STin2 VNTR polymorphism has a variable number of tandem repeats in intron 2 of the serotonin transporter gene. We aimed to explore the relationship between STin2 VNTR polymorphism and lifelong premature ejaculation (LPE). Material/Methods We recruited a total of 115 outpatients who complained of ejaculating prematurely and who were diagnosed as LPE, and 101 controls without PE complaint. Allelic variations of STin2 VNTR were genotyped using PCR-based technology. We evaluated the associations between STin2 VNTR allelic and genotypic frequencies and LPE, as well as the intravaginal ejaculation latency time (IELT) of different STin2 VNTR genotypes among LPE patients. Results The patients and controls did not differ significantly in terms of any characteristic except age. A significantly higher frequency of STin2.12/12 genotype was found among LPE patients versus controls (P=0.026). Frequency of patients carrying at least 1 copy of the 10-repeat allele was significantly lower compared to the control group (28.3% vs. 41.8%, OR=0.55; 95%CI=0.31–0.97, P=0.040). In the LPE group, the mean IELT showed significant difference in STin2.12/12 genotype when compared to those with STin2.12/10 and STin2.10/10 genotypes. The mean IELT in10-repeat allele carriers was 50% longer compared to homozygous carriers of the STin2.12 allele. Conclusions Our results indicate the presence of STin2.10 allele is a protective factor for LPE. Men carrying the higher expression genotype STin2. 12/12 have shorter IELT than 10-repeat allele carriers. PMID:27713390

Aluminum tolerance in maize is associated with higher MATE1 gene copy number

PubMed Central

Maron, Lyza G.; Guimarães, Claudia T.; Kirst, Matias; Albert, Patrice S.; Birchler, James A.; Bradbury, Peter J.; Buckler, Edward S.; Coluccio, Alison E.; Danilova, Tatiana V.; Kudrna, David; Magalhaes, Jurandir V.; Piñeros, Miguel A.; Schatz, Michael C.; Wing, Rod A.; Kochian, Leon V.

2013-01-01

Genome structure variation, including copy number variation and presence/absence variation, comprises a large extent of maize genetic diversity; however, its effect on phenotypes remains largely unexplored. Here, we describe how copy number variation underlies a rare allele that contributes to maize aluminum (Al) tolerance. Al toxicity is the primary limitation for crop production on acid soils, which make up 50% of the world’s potentially arable lands. In a recombinant inbred line mapping population, copy number variation of the Al tolerance gene multidrug and toxic compound extrusion 1 (MATE1) is the basis for the quantitative trait locus of largest effect on phenotypic variation. This expansion in MATE1 copy number is associated with higher MATE1 expression, which in turn results in superior Al tolerance. The three MATE1 copies are identical and are part of a tandem triplication. Only three maize inbred lines carrying the three-copy allele were identified from maize and teosinte diversity panels, indicating that copy number variation for MATE1 is a rare, and quite likely recent, event. These maize lines with higher MATE1 copy number are also Al-tolerant, have high MATE1 expression, and originate from regions of highly acidic soils. Our findings show a role for copy number variation in the adaptation of maize to acidic soils in the tropics and suggest that genome structural changes may be a rapid evolutionary response to new environments. PMID:23479633

Isolation and characterization of novel mutations in the pSC101 origin that increase copy number

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

Thompson, Mitchell G.; Sedaghatian, Nima; Barajas, Jesus F.

pSC101 is a narrow host range, low-copy plasmid commonly used for genetically manipulating Escherichia coli. As a byproduct of a genetic screen for a more sensitive lactam biosensor, we identified multiple novel mutations that increase the copy number of plasmids with the pSC101 origin. All mutations identified in this study occurred on plasmids which also contained at least one mutation localized to the RepA protein encoded within the origin. Homology modelling predicts that many of these mutations occur within the dimerization interface of RepA. Mutant RepA resulted in plasmid copy numbers between ~31 and ~113 copies/cell, relative to ~5 copies/cellmore » in wild-type pSC101 plasmids. Combining the mutations that were predicted to disrupt multiple contacts on the dimerization interface resulted in copy numbers of ~500 copies/cell, while also attenuating growth in host strains. Fluorescent protein production expressed from an arabinose-inducible promoter on mutant origin derived plasmids did correlate with copy number. Plasmids harboring RepA with one of two mutations, E83K and N99D, resulted in fluorescent protein production similar to that from p15a- (~20 copies/cell) and ColE1- (~31 copies/cell) based plasmids, respectively. The mutant copy number variants retained compatibility with p15a, pBBR, and ColE1 origins of replication. Thus, these pSC101 variants may be useful in future metabolic engineering efforts that require medium or high-copy vectors compatible with p15a- and ColE1-based plasmids.« less

Isolation and characterization of novel mutations in the pSC101 origin that increase copy number

DOE PAGES

Thompson, Mitchell G.; Sedaghatian, Nima; Barajas, Jesus F.; ...

2018-01-25

pSC101 is a narrow host range, low-copy plasmid commonly used for genetically manipulating Escherichia coli. As a byproduct of a genetic screen for a more sensitive lactam biosensor, we identified multiple novel mutations that increase the copy number of plasmids with the pSC101 origin. All mutations identified in this study occurred on plasmids which also contained at least one mutation localized to the RepA protein encoded within the origin. Homology modelling predicts that many of these mutations occur within the dimerization interface of RepA. Mutant RepA resulted in plasmid copy numbers between ~31 and ~113 copies/cell, relative to ~5 copies/cellmore » in wild-type pSC101 plasmids. Combining the mutations that were predicted to disrupt multiple contacts on the dimerization interface resulted in copy numbers of ~500 copies/cell, while also attenuating growth in host strains. Fluorescent protein production expressed from an arabinose-inducible promoter on mutant origin derived plasmids did correlate with copy number. Plasmids harboring RepA with one of two mutations, E83K and N99D, resulted in fluorescent protein production similar to that from p15a- (~20 copies/cell) and ColE1- (~31 copies/cell) based plasmids, respectively. The mutant copy number variants retained compatibility with p15a, pBBR, and ColE1 origins of replication. Thus, these pSC101 variants may be useful in future metabolic engineering efforts that require medium or high-copy vectors compatible with p15a- and ColE1-based plasmids.« less

Heterogeneous genetic diversity pattern in Plasmodium vivax genes encoding merozoite surface proteins (MSP) -7E, -7F and -7L.

PubMed

Garzón-Ospina, Diego; Forero-Rodríguez, Johanna; Patarroyo, Manuel A

2014-12-13

The msp-7 gene has become differentially expanded in the Plasmodium genus; Plasmodium vivax has the highest copy number of this gene, several of which encode antigenic proteins in merozoites. DNA sequences from thirty-six Colombian clinical isolates from P. vivax (pv) msp-7E, -7F and -7L genes were analysed for characterizing and studying the genetic diversity of these pvmsp-7 members which are expressed during the intra-erythrocyte stage; natural selection signals producing the variation pattern so observed were evaluated. The pvmsp-7E gene was highly polymorphic compared to pvmsp-7F and pvmsp-7L which were seen to have limited genetic diversity; pvmsp-7E polymorphism was seen to have been maintained by different types of positive selection. Even though these copies seemed to be species-specific duplications, a search in the Plasmodium cynomolgi genome (P. vivax sister taxon) showed that both species shared the whole msp-7 repertoire. This led to exploring the long-term effect of natural selection by comparing the orthologous sequences which led to finding signatures for lineage-specific positive selection. The results confirmed that the P. vivax msp-7 family has a heterogeneous genetic diversity pattern; some members are highly conserved whilst others are highly diverse. The results suggested that the 3'-end of these genes encode MSP-7 proteins' functional region whilst the central region of pvmsp-7E has evolved rapidly. The lineage-specific positive selection signals found suggested that mutations occurring in msp-7s genes during host switch may have succeeded in adapting the ancestral P. vivax parasite population to humans.

Direct isolation of differentially expressed genes from a specific chromosome region of common wheat: application of the amplified fragment length polymorphism-based mRNA fingerprinting (AMF) method in combination with a deletion line of wheat.

PubMed

Kojima, T; Habu, Y; Iida, S; Ogihara, Y

2000-05-01

The amplified restriction fragment length polymorphism (AFLP)-based mRNA fingerprinting (AMF) method makes it possible systematically and conveniently to identify differentially expressed cDNAs with high reproducibility. We have applied the AMF method to the cloning of the Q gene of common wheat, which is located on the long arm of chromosome 5A and pleiotropically controls the spike morphology and the threshing character of seeds. Using the AMF method, we compared the fingerprints of mRNA samples extracted from the young spikes of Triticum aestivum cv. Chinese Spring (CS) carrying the Q gene to those of a chromosome deletion line of CS, namely, q5, which lacks 15% of 5AL including the Q gene. Approximately 12,200 fragments were produced after PCR with 256 primer combinations. Of these, 92 fragments were differentially expressed between CS and q5. Northern and Southern analyses showed that 16 fragments gave specific or relatively stronger transcript signals in CS, and these clones were present in single copy or in low copy numbers in the wheat genome. Four clones were genetically mapped to the region deleted in q5. Subsequently, one clone, pTaQ22, was mapped at the same locus as the Q gene, indicating that pTaQ22 corresponds to the Q gene or is tightly linked to it. DNA sequence data showed that pTaQ22 had no homology to any known genes, thus suggesting a novel function for this gene in flower morphogenesis. This AMF method might provide a straightforward method for isolating genes in the hexaploid background of common wheat.

Detection of clonal evolution in hematopoietic malignancies by combining comparative genomic hybridization and single nucleotide polymorphism arrays.

PubMed

Hartmann, Luise; Stephenson, Christine F; Verkamp, Stephanie R; Johnson, Krystal R; Burnworth, Bettina; Hammock, Kelle; Brodersen, Lisa Eidenschink; de Baca, Monica E; Wells, Denise A; Loken, Michael R; Zehentner, Barbara K

2014-12-01

Array comparative genomic hybridization (aCGH) has become a powerful tool for analyzing hematopoietic neoplasms and identifying genome-wide copy number changes in a single assay. aCGH also has superior resolution compared with fluorescence in situ hybridization (FISH) or conventional cytogenetics. Integration of single nucleotide polymorphism (SNP) probes with microarray analysis allows additional identification of acquired uniparental disomy, a copy neutral aberration with known potential to contribute to tumor pathogenesis. However, a limitation of microarray analysis has been the inability to detect clonal heterogeneity in a sample. This study comprised 16 samples (acute myeloid leukemia, myelodysplastic syndrome, chronic lymphocytic leukemia, plasma cell neoplasm) with complex cytogenetic features and evidence of clonal evolution. We used an integrated manual peak reassignment approach combining analysis of aCGH and SNP microarray data for characterization of subclonal abnormalities. We compared array findings with results obtained from conventional cytogenetic and FISH studies. Clonal heterogeneity was detected in 13 of 16 samples by microarray on the basis of log2 values. Use of the manual peak reassignment analysis approach improved resolution of the sample's clonal composition and genetic heterogeneity in 10 of 13 (77%) patients. Moreover, in 3 patients, clonal disease progression was revealed by array analysis that was not evident by cytogenetic or FISH studies. Genetic abnormalities originating from separate clonal subpopulations can be identified and further characterized by combining aCGH and SNP hybridization results from 1 integrated microarray chip by use of the manual peak reassignment technique. Its clinical utility in comparison to conventional cytogenetic or FISH studies is demonstrated. © 2014 American Association for Clinical Chemistry.

Different Facets of Copy Number Changes: Permanent, Transient, and Adaptive

PubMed Central

Mishra, Sweta

2016-01-01

Chromosomal copy number changes are frequently associated with harmful consequences and are thought of as an underlying mechanism for the development of diseases. However, changes in copy number are observed during development and occur during normal biological processes. In this review, we highlight the causes and consequences of copy number changes in normal physiologic processes as well as cover their associations with cancer and acquired drug resistance. We discuss the permanent and transient nature of copy number gains and relate these observations to a new mechanism driving transient site-specific copy gains (TSSGs). Finally, we discuss implications of TSSGs in generating intratumoral heterogeneity and tumor evolution and how TSSGs can influence the therapeutic response in cancer. PMID:26755558

Peripheral artery disease, calf skeletal muscle mitochondrial DNA copy number, and functional performance.

PubMed

McDermott, Mary M; Peterson, Charlotte A; Sufit, Robert; Ferrucci, Luigi; Guralnik, Jack M; Kibbe, Melina R; Polonsky, Tamar S; Tian, Lu; Criqui, Michael H; Zhao, Lihui; Stein, James H; Li, Lingyu; Leeuwenburgh, Christiaan

2018-05-01

In people without lower extremity peripheral artery disease (PAD), mitochondrial DNA copy number declines with aging, and this decline is associated with declines in mitochondrial activity and functional performance. However, whether lower extremity ischemia is associated with lower mitochondrial DNA copy number and whether mitochondrial DNA copy number is associated with the degree of functional impairment in people with PAD is unknown. In people with and without PAD, age 65 years and older, we studied associations of the ankle-brachial index (ABI) with mitochondrial DNA copy number and associations of mitochondrial DNA copy number with functional impairment. Calf muscle biopsies were obtained from 34 participants with PAD (mean age: 73.5 years (SD 6.4), mean ABI: 0.67 (SD 0.15), mean 6-minute walk distance: 1191 feet (SD 223)) and 10 controls without PAD (mean age: 73.1 years (SD 4.7), mean ABI: 1.14 (SD 0.07), mean 6-minute walk distance: 1387 feet (SD 488)). Adjusting for age and sex, lower ABI values were associated with higher mitochondrial DNA copy number, measured in relative copy number (ABI<0.60: 914, ABI 0.60-0.90: 731, ABI 0.90-1.50: 593; p trend=0.016). The association of mitochondrial DNA copy number with the 6-minute walk distance and 4-meter walking velocity differed significantly between participants with versus without PAD ( p-value for interaction=0.001 and p=0.015, respectively). The correlation coefficient between mitochondrial DNA copy number and the 6-minute walk distance was 0.653 ( p=0.056) among people without PAD and -0.254 ( p=0.154) among people with PAD and ABI < 0.90. In conclusion, lower ABI values are associated with increased mitochondrial DNA copy number. Associations of mitochondrial DNA copy number with the 6-minute walk distance and 4-meter walking velocity significantly differed between people with versus without PAD, with stronger positive associations observed in people without PAD than in people with PAD. The cross-sectional and exploratory nature of the analyses precludes conclusions regarding causal inferences. ClinicalTrials.gov Identifier: NCT02246660.

17 CFR 270.8b-11 - Number of copies; signatures; binding.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Number of copies; signatures... (CONTINUED) RULES AND REGULATIONS, INVESTMENT COMPANY ACT OF 1940 § 270.8b-11 Number of copies; signatures... manner prescribed by the appropriate form. Unsigned copies shall be conformed. If the signature of any...

SMN1 and SMN2 copy numbers in cell lines derived from patients with spinal muscular atrophy as measured by array digital PCR.

PubMed

Stabley, Deborah L; Harris, Ashlee W; Holbrook, Jennifer; Chubbs, Nicholas J; Lozo, Kevin W; Crawford, Thomas O; Swoboda, Kathryn J; Funanage, Vicky L; Wang, Wenlan; Mackenzie, William; Scavina, Mena; Sol-Church, Katia; Butchbach, Matthew E R

2015-07-01

Proximal spinal muscular atrophy (SMA) is an early-onset motor neuron disease characterized by loss of α-motor neurons and associated muscle atrophy. SMA is caused by deletion or other disabling mutation of survival motor neuron 1 (SMN1). In the human genome, a large duplication of the SMN-containing region gives rise to a second copy of this gene (SMN2) that is distinguishable by a single nucleotide change in exon 7. Within the SMA population, there is substantial variation in SMN2 copy number; in general, those individuals with SMA who have a high SMN2 copy number have a milder disease. Because SMN2 functions as a disease modifier, its accurate copy number determination may have clinical relevance. In this study, we describe the development of an assay to assess SMN1 and SMN2 copy numbers in DNA samples using an array-based digital PCR (dPCR) system. This dPCR assay can accurately and reliably measure the number of SMN1 and SMN2 copies in DNA samples. In a cohort of SMA patient-derived cell lines, the assay confirmed a strong inverse correlation between SMN2 copy number and disease severity. Array dPCR is a practical technique to determine, accurately and reliably, SMN1 and SMN2 copy numbers from SMA samples.

Glyoxalase 1 copy number variation in patients with well differentiated gastro-entero-pancreatic neuroendocrine tumours (GEP-NET)

PubMed Central

Xue, Mingzhan; Shafie, Alaa; Qaiser, Talha; Rajpoot, Nasir M.; Kaltsas, Gregory; James, Sean; Gopalakrishnan, Kishore; Fisk, Adrian; Dimitriadis, Georgios K.; Grammatopoulos, Dimitris K.; Rabbani, Naila; Thornalley, Paul J.; Weickert, Martin O.

2017-01-01

Background The glyoxalase-1 gene (GLO1) is a hotspot for copy-number variation (CNV) in human genomes. Increased GLO1 copy-number is associated with multidrug resistance in tumour chemotherapy, but prevalence of GLO1 CNV in gastro-entero-pancreatic neuroendocrine tumours (GEP-NET) is unknown. Methods GLO1 copy-number variation was measured in 39 patients with GEP-NET (midgut NET, n = 25; pancreatic NET, n = 14) after curative or debulking surgical treatment. Primary tumour tissue, surrounding healthy tissue and, where applicable, additional metastatic tumour tissue were analysed, using real time qPCR. Progression and survival following surgical treatment were monitored over 4.2 ± 0.5 years. Results In the pooled GEP-NET cohort, GLO1 copy-number in healthy tissue was 2.0 in all samples but significantly increased in primary tumour tissue in 43% of patients with pancreatic NET and in 72% of patients with midgut NET, mainly driven by significantly higher GLO1 copy-number in midgut NET. In tissue from additional metastases resection (18 midgut NET and one pancreatic NET), GLO1 copy number was also increased, compared with healthy tissue; but was not significantly different compared with primary tumour tissue. During mean 3 - 5 years follow-up, 8 patients died and 16 patients showed radiological progression. In midgut NET, a high GLO1 copy-number was associated with earlier progression. In NETs with increased GLO1 copy number, there was increased Glo1 protein expression compared to non-malignant tissue. Conclusions GLO1 copy-number was increased in a large percentage of patients with GEP-NET and correlated positively with increased Glo1 protein in tumour tissue. Analysis of GLO1 copy-number variation particularly in patients with midgut NET could be a novel prognostic marker for tumour progression. PMID:29100361

The transposon Galileo generates natural chromosomal inversions in Drosophila by ectopic recombination.

PubMed

Delprat, Alejandra; Negre, Bàrbara; Puig, Marta; Ruiz, Alfredo

2009-11-18

Transposable elements (TEs) are responsible for the generation of chromosomal inversions in several groups of organisms. However, in Drosophila and other Dipterans, where inversions are abundant both as intraspecific polymorphisms and interspecific fixed differences, the evidence for a role of TEs is scarce. Previous work revealed that the transposon Galileo was involved in the generation of two polymorphic inversions of Drosophila buzzatii. To assess the impact of TEs in Drosophila chromosomal evolution and shed light on the mechanism involved, we isolated and sequenced the two breakpoints of another widespread polymorphic inversion from D. buzzatii, 2z(3). In the non inverted chromosome, the 2z(3) distal breakpoint was located between genes CG2046 and CG10326 whereas the proximal breakpoint lies between two novel genes that we have named Dlh and Mdp. In the inverted chromosome, the analysis of the breakpoint sequences revealed relatively large insertions (2,870-bp and 4,786-bp long) including two copies of the transposon Galileo (subfamily Newton), one at each breakpoint, plus several other TEs. The two Galileo copies: (i) are inserted in opposite orientation; (ii) present exchanged target site duplications; and (iii) are both chimeric. Our observations provide the best evidence gathered so far for the role of TEs in the generation of Drosophila inversions. In addition, they show unequivocally that ectopic recombination is the causative mechanism. The fact that the three polymorphic D. buzzatii inversions investigated so far were generated by the same transposon family is remarkable and is conceivably due to Galileo's unusual structure and current (or recent) transpositional activity.

Prediction of Response to Therapy and Clinical Outcome through a Pilot Study of Complete Genetic Assessment of Ovarian Cancer

DTIC Science & Technology

2015-12-01

Oncology program supported by this grant consented patients to 11-104. OncoPanel is a cancer genomic assay that detects somatic mutations, copy number...KMT2D, EP300, FANCD2 Sertoli Leydig cell DICER1 Copy number variants: In addition, 219 patients were analyzed for copy-number variations ( CNV ) in...OncoPanel genes. >12,000 total CNV were reported in the cohort (Figure 2). Single- copy deletions (n=5558) and copy-number gains (low amplification) (n

High and uneven levels of 45S rDNA site-number variation across wild populations of a diploid plant genus (Anacyclus, Asteraceae)

PubMed Central

Rosato, Marcela; Álvarez, Inés; Nieto Feliner, Gonzalo

2017-01-01

The nuclear genome harbours hundreds to several thousand copies of ribosomal DNA. Despite their essential role in cellular ribogenesis few studies have addressed intrapopulation, interpopulation and interspecific levels of rDNA variability in wild plants. Some studies have assessed the extent of rDNA variation at the sequence and copy-number level with large sampling in several species. However, comparable studies on rDNA site number variation in plants, assessed with extensive hierarchical sampling at several levels (individuals, populations, species) are lacking. In exploring the possible causes for ribosomal loci dynamism, we have used the diploid genus Anacyclus (Asteraceae) as a suitable system to examine the evolution of ribosomal loci. To this end, the number and chromosomal position of 45S rDNA sites have been determined in 196 individuals from 47 populations in all Anacyclus species using FISH. The 45S rDNA site-number has been assessed in a significant sample of seed plants, which usually exhibit rather consistent features, except for polyploid plants. In contrast, the level of rDNA site-number variation detected in Anacyclus is outstanding in the context of angiosperms particularly regarding populations of the same species. The number of 45S rDNA sites ranged from four to 11, accounting for 14 karyological ribosomal phenotypes. Our results are not even across species and geographical areas, and show that there is no clear association between the number of 45S rDNA loci and the life cycle in Anacyclus. A single rDNA phenotype was detected in several species, but a more complex pattern that included intra-specific and intra-population polymorphisms was recorded in A. homogamos, A. clavatus and A. valentinus, three weedy species showing large and overlapping distribution ranges. It is likely that part of the cytogenetic changes and inferred dynamism found in these species have been triggered by genomic rearrangements resulting from contemporary hybridisation. PMID:29088249

High and uneven levels of 45S rDNA site-number variation across wild populations of a diploid plant genus (Anacyclus, Asteraceae).

PubMed

Rosato, Marcela; Álvarez, Inés; Nieto Feliner, Gonzalo; Rosselló, Josep A

2017-01-01

The nuclear genome harbours hundreds to several thousand copies of ribosomal DNA. Despite their essential role in cellular ribogenesis few studies have addressed intrapopulation, interpopulation and interspecific levels of rDNA variability in wild plants. Some studies have assessed the extent of rDNA variation at the sequence and copy-number level with large sampling in several species. However, comparable studies on rDNA site number variation in plants, assessed with extensive hierarchical sampling at several levels (individuals, populations, species) are lacking. In exploring the possible causes for ribosomal loci dynamism, we have used the diploid genus Anacyclus (Asteraceae) as a suitable system to examine the evolution of ribosomal loci. To this end, the number and chromosomal position of 45S rDNA sites have been determined in 196 individuals from 47 populations in all Anacyclus species using FISH. The 45S rDNA site-number has been assessed in a significant sample of seed plants, which usually exhibit rather consistent features, except for polyploid plants. In contrast, the level of rDNA site-number variation detected in Anacyclus is outstanding in the context of angiosperms particularly regarding populations of the same species. The number of 45S rDNA sites ranged from four to 11, accounting for 14 karyological ribosomal phenotypes. Our results are not even across species and geographical areas, and show that there is no clear association between the number of 45S rDNA loci and the life cycle in Anacyclus. A single rDNA phenotype was detected in several species, but a more complex pattern that included intra-specific and intra-population polymorphisms was recorded in A. homogamos, A. clavatus and A. valentinus, three weedy species showing large and overlapping distribution ranges. It is likely that part of the cytogenetic changes and inferred dynamism found in these species have been triggered by genomic rearrangements resulting from contemporary hybridisation.

Exonic duplication CNV of NDRG1 associated with autosomal-recessive HMSN-Lom/CMT4D.

PubMed

Okamoto, Yuji; Goksungur, Meryem Tuba; Pehlivan, Davut; Beck, Christine R; Gonzaga-Jauregui, Claudia; Muzny, Donna M; Atik, Mehmed M; Carvalho, Claudia M B; Matur, Zeliha; Bayraktar, Serife; Boone, Philip M; Akyuz, Kaya; Gibbs, Richard A; Battaloglu, Esra; Parman, Yesim; Lupski, James R

2014-05-01

Copy-number variations as a mutational mechanism contribute significantly to human disease. Approximately one-half of the patients with Charcot-Marie-Tooth (CMT) disease have a 1.4 Mb duplication copy-number variation as the cause of their neuropathy. However, non-CMT1A neuropathy patients rarely have causative copy-number variations, and to date, autosomal-recessive disease has not been associated with copy-number variation as a mutational mechanism. We performed Agilent 8 × 60 K array comparative genomic hybridization on DNA from 12 recessive Turkish families with CMT disease. Additional molecular studies were conducted to detect breakpoint junctions and to evaluate gene expression levels in a family in which we detected an intragenic duplication copy-number variation. We detected an ~6.25 kb homozygous intragenic duplication in NDRG1, a gene known to be causative for recessive HMSNL/CMT4D, in three individuals from a Turkish family with CMT neuropathy. Further studies showed that this intragenic copy-number variation resulted in a homozygous duplication of exons 6-8 that caused decreased mRNA expression of NDRG1. Exon-focused high-resolution array comparative genomic hybridization enables the detection of copy-number variation carrier states in recessive genes, particularly small copy-number variations encompassing or disrupting single genes. In families for whom a molecular diagnosis has not been elucidated by conventional clinical assays, an assessment for copy-number variations in known CMT genes might be considered.

rDNA Copy Number Variants Are Frequent Passenger Mutations in Saccharomyces cerevisiae Deletion Collections and de Novo Transformants

PubMed Central

Kwan, Elizabeth X.; Wang, Xiaobin S.; Amemiya, Haley M.; Brewer, Bonita J.; Raghuraman, M. K.

2016-01-01

The Saccharomyces cerevisiae ribosomal DNA (rDNA) locus is known to exhibit greater instability relative to the rest of the genome. However, wild-type cells preferentially maintain a stable number of rDNA copies, suggesting underlying genetic control of the size of this locus. We performed a screen of a subset of the Yeast Knock-Out (YKO) single gene deletion collection to identify genetic regulators of this locus and to determine if rDNA copy number correlates with yeast replicative lifespan. While we found no correlation between replicative lifespan and rDNA size, we identified 64 candidate strains with significant rDNA copy number differences. However, in the process of validating candidate rDNA variants, we observed that independent isolates of our de novo gene deletion strains had unsolicited but significant changes in rDNA copy number. Moreover, we were not able to recapitulate rDNA phenotypes from the YKO yeast deletion collection. Instead, we found that the standard lithium acetate transformation protocol is a significant source of rDNA copy number variation, with lithium acetate exposure being the treatment causing variable rDNA copy number events after transformation. As the effects of variable rDNA copy number are being increasingly reported, our finding that rDNA is affected by lithium acetate exposure suggested that rDNA copy number variants may be influential passenger mutations in standard strain construction in S. cerevisiae. PMID:27449518

rDNA Copy Number Variants Are Frequent Passenger Mutations in Saccharomyces cerevisiae Deletion Collections and de Novo Transformants.

PubMed

Kwan, Elizabeth X; Wang, Xiaobin S; Amemiya, Haley M; Brewer, Bonita J; Raghuraman, M K

2016-09-08

The Saccharomyces cerevisiae ribosomal DNA (rDNA) locus is known to exhibit greater instability relative to the rest of the genome. However, wild-type cells preferentially maintain a stable number of rDNA copies, suggesting underlying genetic control of the size of this locus. We performed a screen of a subset of the Yeast Knock-Out (YKO) single gene deletion collection to identify genetic regulators of this locus and to determine if rDNA copy number correlates with yeast replicative lifespan. While we found no correlation between replicative lifespan and rDNA size, we identified 64 candidate strains with significant rDNA copy number differences. However, in the process of validating candidate rDNA variants, we observed that independent isolates of our de novo gene deletion strains had unsolicited but significant changes in rDNA copy number. Moreover, we were not able to recapitulate rDNA phenotypes from the YKO yeast deletion collection. Instead, we found that the standard lithium acetate transformation protocol is a significant source of rDNA copy number variation, with lithium acetate exposure being the treatment causing variable rDNA copy number events after transformation. As the effects of variable rDNA copy number are being increasingly reported, our finding that rDNA is affected by lithium acetate exposure suggested that rDNA copy number variants may be influential passenger mutations in standard strain construction in S. cerevisiae. Copyright © 2016 Kwan et al.

Gene copy number variation and its significance in cyanobacterial phylogeny

PubMed Central

2012-01-01

Background In eukaryotes, variation in gene copy numbers is often associated with deleterious effects, but may also have positive effects. For prokaryotes, studies on gene copy number variation are rare. Previous studies have suggested that high numbers of rRNA gene copies can be advantageous in environments with changing resource availability, but further association of gene copies and phenotypic traits are not documented. We used one of the morphologically most diverse prokaryotic phyla to test whether numbers of gene copies are associated with levels of cell differentiation. Results We implemented a search algorithm that identified 44 genes with highly conserved copies across 22 fully sequenced cyanobacterial taxa. For two very basal cyanobacterial species, Gloeobacter violaceus and a thermophilic Synechococcus species, distinct phylogenetic positions previously found were supported by identical protein coding gene copy numbers. Furthermore, we found that increased ribosomal gene copy numbers showed a strong correlation to cyanobacteria capable of terminal cell differentiation. Additionally, we detected extremely low variation of 16S rRNA sequence copies within the cyanobacteria. We compared our results for 16S rRNA to three other eubacterial phyla (Chroroflexi, Spirochaetes and Bacteroidetes). Based on Bayesian phylogenetic inference and the comparisons of genetic distances, we could confirm that cyanobacterial 16S rRNA paralogs and orthologs show significantly stronger conservation than found in other eubacterial phyla. Conclusions A higher number of ribosomal operons could potentially provide an advantage to terminally differentiated cyanobacteria. Furthermore, we suggest that 16S rRNA gene copies in cyanobacteria are homogenized by both concerted evolution and purifying selection. In addition, the small ribosomal subunit in cyanobacteria appears to evolve at extraordinary slow evolutionary rates, an observation that has been made previously for morphological characteristics of cyanobacteria. PMID:22894826

Measurement methods and accuracy in copy number variation: failure to replicate associations of beta-defensin copy number with Crohn's disease.

PubMed

Aldhous, Marian C; Abu Bakar, Suhaili; Prescott, Natalie J; Palla, Raquel; Soo, Kimberley; Mansfield, John C; Mathew, Christopher G; Satsangi, Jack; Armour, John A L

2010-12-15

The copy number variation in beta-defensin genes on human chromosome 8 has been proposed to underlie susceptibility to inflammatory disorders, but presents considerable challenges for accurate typing on the scale required for adequately powered case-control studies. In this work, we have used accurate methods of copy number typing based on the paralogue ratio test (PRT) to assess beta-defensin copy number in more than 1500 UK DNA samples including more than 1000 cases of Crohn's disease. A subset of 625 samples was typed using both PRT-based methods and standard real-time PCR methods, from which direct comparisons highlight potentially serious shortcomings of a real-time PCR assay for typing this variant. Comparing our PRT-based results with two previous studies based only on real-time PCR, we find no evidence to support the reported association of Crohn's disease with either low or high beta-defensin copy number; furthermore, it is noteworthy that there are disagreements between different studies on the observed frequency distribution of copy number states among European controls. We suggest safeguards to be adopted in assessing and reporting the accuracy of copy number measurement, with particular emphasis on integer clustering of results, to avoid reporting of spurious associations in future case-control studies.

A comprehensive profile of DNA copy number variations in a Korean population: identification of copy number invariant regions among Koreans.

PubMed

Jeon, Jae Pil; Shim, Sung Mi; Jung, Jong Sun; Nam, Hye Young; Lee, Hye Jin; Oh, Berm Seok; Kim, Kuchan; Kim, Hyung Lae; Han, Bok Ghee

2009-09-30

To examine copy number variations among the Korean population, we compared individual genomes with the Korean reference genome assembly using the publicly available Korean HapMap SNP 50 k chip data from 90 individuals. Korean individuals exhibited 123 copy number variation regions (CNVRs) covering 27.2 mb, equivalent to 1.0% of the genome in the copy number variation (CNV) analysis using the combined criteria of P value (P<0.01) and standard deviation of copy numbers (SD>or= 0.25) among study subjects. In contrast, when compared to the Affymetrix reference genome assembly from multiple ethnic groups, considerably more CNVRs (n=643) were detected in larger proportions (5.0%) of the genome covering 135.1 mb even by more stringent criteria (P<0.001 and SD>or=0.25), reflecting ethnic diversity of structural variations between Korean and other populations. Some CNVRs were validated by the quantitative multiplex PCR of short fluorescent fragment (QMPSF) method, and then copy number invariant regions were detected among the study subjects. These copy number invariant regions would be used as good internal controls for further CNV studies. Lastly, we demonstrated that the CNV information could stratify even a single ethnic population with a proper reference genome assembly from multiple heterogeneous populations.

Real-time PCR assays for monitoring anaerobic fungal biomass and population size in the rumen.

PubMed

Lwin, Khin Ohnmar; Hayakawa, Mika; Ban-Tokuda, Tomomi; Matsui, Hiroki

2011-04-01

The relationship between copy numbers of internal transcribed spacer 1 (ITS1) and biomass or zoospore count of anaerobic fungi was studied to develop a quantitative real-time PCR-based monitoring method for fungal biomass or population in the rumen. Nine fungal strains were used to determine the relationship between ITS1 copy number and fungal biomass. Rumen fluid from three sheep and a cow were used to determine the relationship between ITS1 copy number and fungal population. ITS1 copy number was determined by real-time PCR with a specific primer set for anaerobic fungi. Freeze-dried fungal cells were weighed for fungal biomass. Zoospore counts were determined by the roll-tube method. A positive correlation was observed between both ITS1 copy number and dry weight and ITS1 copy number and zoospore counts, suggesting that the use of ITS1 copy numbers is effective for estimating fungal biomass and population density. On the basis of ITS1 copy numbers, fluctuations in the fungal population in sheep rumen showed that although the values varied among individual animals, the fungal population tended to decrease after feeding. In the present study, a culture-independent method was established that will provide a powerful tool for understanding the ecology of anaerobic fungi in the rumen.

Measurement methods and accuracy in copy number variation: failure to replicate associations of beta-defensin copy number with Crohn's disease

PubMed Central

Aldhous, Marian C.; Abu Bakar, Suhaili; Prescott, Natalie J.; Palla, Raquel; Soo, Kimberley; Mansfield, John C.; Mathew, Christopher G.; Satsangi, Jack; Armour, John A.L.

2010-01-01

The copy number variation in beta-defensin genes on human chromosome 8 has been proposed to underlie susceptibility to inflammatory disorders, but presents considerable challenges for accurate typing on the scale required for adequately powered case–control studies. In this work, we have used accurate methods of copy number typing based on the paralogue ratio test (PRT) to assess beta-defensin copy number in more than 1500 UK DNA samples including more than 1000 cases of Crohn's disease. A subset of 625 samples was typed using both PRT-based methods and standard real-time PCR methods, from which direct comparisons highlight potentially serious shortcomings of a real-time PCR assay for typing this variant. Comparing our PRT-based results with two previous studies based only on real-time PCR, we find no evidence to support the reported association of Crohn's disease with either low or high beta-defensin copy number; furthermore, it is noteworthy that there are disagreements between different studies on the observed frequency distribution of copy number states among European controls. We suggest safeguards to be adopted in assessing and reporting the accuracy of copy number measurement, with particular emphasis on integer clustering of results, to avoid reporting of spurious associations in future case–control studies. PMID:20858604

17 CFR 260.7a-3 - Number of copies; filing; signatures; binding.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Number of copies; filing; signatures; binding. 260.7a-3 Section 260.7a-3 Commodity and Securities Exchanges SECURITIES AND EXCHANGE... § 260.7a-3 Number of copies; filing; signatures; binding. (a) Three copies of the complete application...

17 CFR 260.4c-3 - Number of copies; filing; signatures; binding.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Number of copies; filing; signatures; binding. 260.4c-3 Section 260.4c-3 Commodity and Securities Exchanges SECURITIES AND EXCHANGE... § 260.4c-3 Number of copies; filing; signatures; binding. (a) Three copies of every application and of...

17 CFR 260.5a-3 - Number of copies; filing; signatures; binding.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Number of copies; filing; signatures; binding. 260.5a-3 Section 260.5a-3 Commodity and Securities Exchanges SECURITIES AND EXCHANGE... § 260.5a-3 Number of copies; filing; signatures; binding. (a) Three copies of each statement of...

Human Transposon Tectonics

PubMed Central

Burns, Kathleen H.; Boeke, Jef D.

2012-01-01

Mobile DNAs have had a central role in shaping our genome. More than half of our DNA is comprised of interspersed repeats resulting from replicative copy and paste events of retrotransposons. Although most are fixed, incapable of templating new copies, there are important exceptions to retrotransposon quiescence. De novo insertions cause genetic diseases and cancers, though reliably detecting these occurrences has been difficult. New technologies aimed at uncovering polymorphic insertions reveal that mobile DNAs provide a substantial and dynamic source of structural variation. Key questions going forward include the how and how much new transposition events affect human health and disease. PMID:22579280

48 CFR 1852.246-72 - Material inspection and receiving report.

Code of Federal Regulations, 2010 CFR

2010-10-01

... Receiving Report (DD Form 250 series) prepared in __ [Insert number of copies, including original] copies, an original and __ copies [Insert number of copies]. (b) The Contractor shall prepare the DD Form 250 in accordance with NASA FAR Supplement 1846.6. The Contractor shall enclose the copies of the DD Form...

Interpretation of clinical relevance of X-chromosome copy number variations identified in a large cohort of individuals with cognitive disorders and/or congenital anomalies.

PubMed

Willemsen, Marjolein H; de Leeuw, Nicole; de Brouwer, Arjan P M; Pfundt, Rolph; Hehir-Kwa, Jayne Y; Yntema, Helger G; Nillesen, Willy M; de Vries, Bert B A; van Bokhoven, Hans; Kleefstra, Tjitske

2012-11-01

Genome-wide array studies are now routinely being used in the evaluation of patients with cognitive disorders (CD) and/or congenital anomalies (CA). Therefore, inevitably each clinician is confronted with the challenging task of the interpretation of copy number variations detected by genome-wide array platforms in a diagnostic setting. Clinical interpretation of autosomal copy number variations is already challenging, but assessment of the clinical relevance of copy number variations of the X-chromosome is even more complex. This study provides an overview of the X-Chromosome copy number variations that we have identified by genome-wide array analysis in a large cohort of 4407 male and female patients. We have made an interpretation of the clinical relevance of each of these copy number variations based on well-defined criteria and previous reports in literature and databases. The prevalence of X-chromosome copy number variations in this cohort was 57/4407 (∼1.3%), of which 15 (0.3%) were interpreted as (likely) pathogenic. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Assessment of copy number variation in genes related to drug resistance in Plasmodium vivax and Plasmodium falciparum isolates from the Brazilian Amazon and a systematic review of the literature.

PubMed

Costa, Gabriel Luíz; Amaral, Lara Cotta; Fontes, Cor Jesus Fernandes; Carvalho, Luzia Helena; de Brito, Cristiana Ferreira Alves; de Sousa, Taís Nóbrega

2017-04-19

Parasite resistance to anti-malarials represents a great obstacle for malaria elimination. The majority of studies have investigated the association between single-nucleotide polymorphisms (SNPs) and drug resistance; however, it is becoming clear that the copy number variation (CNV) is also associated with this parasite phenotype. To provide a baseline for molecular surveillance of anti-malarial drug resistance in the Brazilian Amazon, the present study characterized the genetic profile of both markers in the most common genes associated with drug resistance in Plasmodium falciparum and Plasmodium vivax isolates. Additionally, these data were compared to data published elsewhere applying a systematic review of the literature published over a 20-year time period. The genomic DNA of 67 patients infected by P. falciparum and P. vivax from three Brazilian States was obtained between 2002 and 2012. CNV in P. falciparum multidrug resistance gene-1 (pfmdr1), GTP cyclohydrolase 1 (pfgch1) and P. vivax multidrug resistance gene-1 (pvmdr1) were assessed by real-time PCR assays. SNPs in the pfmdr1 and pfcrt genes were assessed by PCR-RFLP. A literature search for studies that analysed CNP in the same genes of P. falciparum and P. vivax was conducted between May 2014 and March 2017 across four databases. All analysed samples of P. falciparum carried only one copy of pfmdr1 or pfgch1. Although the pfcrt K76T polymorphism, a determinant of CQ resistance, was present in all samples genotyped, the pfmdr1 N86Y was absent. For P. vivax isolates, an amplification rate of 20% was found for the pvmdr1 gene. The results of the study are in agreement with the low amplification rates for pfmdr1 gene evidenced in the Americas and Africa, while higher rates have been described in Southeast Asia. For P. vivax, very low rates of amplification for pvmdr1 have been described worldwide, with exceptions in French Guiana, Cambodia, Thailand and Brazil. The present study was the first to evaluate gch1 CNV in P. falciparum isolates from Brazil, showing an absence of amplification of this gene more than 20 years after the withdrawal of the Brazilian antifolates therapeutic scheme. Furthermore, the rate of pvmdr1 amplification was significantly higher than that previously reported for isolates circulating in Northern Brazil.

Statistical tools for transgene copy number estimation based on real-time PCR.

PubMed

Yuan, Joshua S; Burris, Jason; Stewart, Nathan R; Mentewab, Ayalew; Stewart, C Neal

2007-11-01

As compared with traditional transgene copy number detection technologies such as Southern blot analysis, real-time PCR provides a fast, inexpensive and high-throughput alternative. However, the real-time PCR based transgene copy number estimation tends to be ambiguous and subjective stemming from the lack of proper statistical analysis and data quality control to render a reliable estimation of copy number with a prediction value. Despite the recent progresses in statistical analysis of real-time PCR, few publications have integrated these advancements in real-time PCR based transgene copy number determination. Three experimental designs and four data quality control integrated statistical models are presented. For the first method, external calibration curves are established for the transgene based on serially-diluted templates. The Ct number from a control transgenic event and putative transgenic event are compared to derive the transgene copy number or zygosity estimation. Simple linear regression and two group T-test procedures were combined to model the data from this design. For the second experimental design, standard curves were generated for both an internal reference gene and the transgene, and the copy number of transgene was compared with that of internal reference gene. Multiple regression models and ANOVA models can be employed to analyze the data and perform quality control for this approach. In the third experimental design, transgene copy number is compared with reference gene without a standard curve, but rather, is based directly on fluorescence data. Two different multiple regression models were proposed to analyze the data based on two different approaches of amplification efficiency integration. Our results highlight the importance of proper statistical treatment and quality control integration in real-time PCR-based transgene copy number determination. These statistical methods allow the real-time PCR-based transgene copy number estimation to be more reliable and precise with a proper statistical estimation. Proper confidence intervals are necessary for unambiguous prediction of trangene copy number. The four different statistical methods are compared for their advantages and disadvantages. Moreover, the statistical methods can also be applied for other real-time PCR-based quantification assays including transfection efficiency analysis and pathogen quantification.

Multiplex digital PCR: breaking the one target per color barrier of quantitative PCR.

PubMed

Zhong, Qun; Bhattacharya, Smiti; Kotsopoulos, Steven; Olson, Jeff; Taly, Valérie; Griffiths, Andrew D; Link, Darren R; Larson, Jonathan W

2011-07-07

Quantitative polymerase chain reactions (qPCR) based on real-time PCR constitute a powerful and sensitive method for the analysis of nucleic acids. However, in qPCR, the ability to multiplex targets using differently colored fluorescent probes is typically limited to 4-fold by the spectral overlap of the fluorophores. Furthermore, multiplexing qPCR assays requires expensive instrumentation and most often lengthy assay development cycles. Digital PCR (dPCR), which is based on the amplification of single target DNA molecules in many separate reactions, is an attractive alternative to qPCR. Here we report a novel and easy method for multiplexing dPCR in picolitre droplets within emulsions-generated and read out in microfluidic devices-that takes advantage of both the very high numbers of reactions possible within emulsions (>10(6)) as well as the high likelihood that the amplification of only a single target DNA molecule will initiate within each droplet. By varying the concentration of different fluorogenic probes of the same color, it is possible to identify the different probes on the basis of fluorescence intensity. Adding multiple colors increases the number of possible reactions geometrically, rather than linearly as with qPCR. Accurate and precise copy numbers of up to sixteen per cell were measured using a model system. A 5-plex assay for spinal muscular atrophy was demonstrated with just two fluorophores to simultaneously measure the copy number of two genes (SMN1 and SMN2) and to genotype a single nucleotide polymorphism (c.815A>G, SMN1). Results of a pilot study with SMA patients are presented. This journal is © The Royal Society of Chemistry 2011

Genetics of Congenital Heart Disease: Past and Present.

PubMed

Muntean, Iolanda; Togănel, Rodica; Benedek, Theodora

2017-04-01

Congenital heart disease is the most common congenital anomaly, representing an important cause of infant morbidity and mortality. Congenital heart disease represents a group of heart anomalies that include septal defects, valve defects, and outflow tract anomalies. The exact genetic, epigenetic, or environmental basis of congenital heart disease remains poorly understood, although the exact mechanism is likely multifactorial. However, the development of new technologies including copy number variants, single-nucleotide polymorphism, next-generation sequencing are accelerating the detection of genetic causes of heart anomalies. Recent studies suggest a role of small non-coding RNAs, micro RNA, in congenital heart disease. The recently described epigenetic factors have also been found to contribute to cardiac morphogenesis. In this review, we present past and recent genetic discoveries in congenital heart disease.

The Molecular Genetics of Autism Spectrum Disorders: Genomic Mechanisms, Neuroimmunopathology, and Clinical Implications

PubMed Central

Guerra, Daniel J.

2011-01-01

Autism spectrum disorders (ASDs) have become increasingly common in recent years. The discovery of single-nucleotide polymorphisms and accompanying copy number variations within the genome has increased our understanding of the architecture of the disease. These genetic and genomic alterations coupled with epigenetic phenomena have pointed to a neuroimmunopathological mechanism for ASD. Model animal studies, developmental biology, and affective neuroscience laid a foundation for dissecting the neural pathways impacted by these disease-generating mechanisms. The goal of current autism research is directed toward a systems biological approach to find the most basic genetic and environmental causes to this severe developmental disease. It is hoped that future genomic and neuroimmunological research will be directed toward finding the road toward prevention, treatment, and cure of ASD. PMID:22937247

Genetic diversity of the DBLalpha region in Plasmodium falciparum var genes among Asia-Pacific isolates.

PubMed

Fowler, Elizabeth V; Peters, Jennifer M; Gatton, Michelle L; Chen, Nanhua; Cheng, Qin

2002-03-01

In Plasmodium falciparum a highly polymorphic multi-copy gene family, var, encodes the variant surface antigen P. falciparum erythrocyte membrane protein 1 (PfEMP1), which has an important role in cytoadherence and immune evasion. Using previously described universal PCR primers for the first Duffy binding-like domain (DBLalpha) of var we analysed the DBLalpha repertoires of Dd2 (originally from Thailand) and eight isolates from the Solomon Islands (n=4), Philippines (n=2), Papua New Guinea (n=1) and Africa (n=1). We found 15-32 unique DBLalpha sequence types among these isolates and estimated detectable DBLalpha repertoire sizes ranging from 33-38 to 52-57 copies per genome. Our data suggest that var gene repertoires generally consist of 40-50 copies per genome. Eighteen DBLalpha sequences appeared in more than one Asia-Pacific isolate with the number of sequences shared between any two isolates ranging from 0 to 6 (mean=2.0 +/-1.6). At the amino acid level DBLalpha sequence similarity within isolates ranged from 45.2 +/- 7.1 to 50.2 +/- 6.9%, and was not significantly different from the DBLalpha amino acid sequence similarity among isolates (P>0.1). Comparisons with published sequences also revealed little overlap among DBLalpha sequences from different regions. High DBLalpha sequence diversity and minimal overlap among these isolates suggest that the global var gene repertoire is immense, and may potentially be selected for by the host's protective immune response to the var gene products, PfEMP1.

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

Analysis of X chromosome genomic DNA sequence copy number variation associated with premature ovarian failure (POF)

PubMed Central

Quilter, C.R.; Karcanias, A.C.; Bagga, M.R.; Duncan, S.; Murray, A.; Conway, G.S.; Sargent, C.A.; Affara, N.A.

2013-01-01

BACKGROUND Premature ovarian failure (POF) is a heterogeneous disease defined as amenorrhoea for >6 months before age 40, with an FSH serum level >40 mIU/ml (menopausal levels). While there is a strong genetic association with POF, familial studies have also indicated that idiopathic POF may also be genetically linked. Conventional cytogenetic analyses have identified regions of the X chromosome that are strongly associated with ovarian function, as well as several POF candidate genes. Cryptic chromosome abnormalities that have been missed might be detected by array comparative genomic hybridization. METHODS In this study, samples from 42 idiopathic POF patients were subjected to a complete end-to-end X/Y chromosome tiling path array to achieve a detailed copy number variation (CNV) analysis of X chromosome involvement in POF. The arrays also contained a 1 Mb autosomal tiling path as a reference control. Quantitative PCR for selected genes contained within the CNVs was used to confirm the majority of the changes detected. The expression pattern of some of these genes in human tissue RNA was examined by reverse transcription (RT)–PCR. RESULTS A number of CNVs were identified on both Xp and Xq, with several being shared among the POF cases. Some CNVs fall within known polymorphic CNV regions, and others span previously identified POF candidate regions and genes. CONCLUSIONS The new data reported in this study reveal further discrete X chromosome intervals not previously associated with the disease and therefore implicate new clusters of candidate genes. Further studies will be required to elucidate their involvement in POF. PMID:20570974

Accuracy and differential bias in copy number measurement of CCL3L1 in association studies with three auto-immune disorders.

PubMed

Carpenter, Danielle; Walker, Susan; Prescott, Natalie; Schalkwijk, Joost; Armour, John Al

2011-08-18

Copy number variation (CNV) contributes to the variation observed between individuals and can influence human disease progression, but the accurate measurement of individual copy numbers is technically challenging. In the work presented here we describe a modification to a previously described paralogue ratio test (PRT) method for genotyping the CCL3L1/CCL4L1 copy variable region, which we use to ascertain CCL3L1/CCL4L1 copy number in 1581 European samples. As the products of CCL3L1 and CCL4L1 potentially play a role in autoimmunity we performed case control association studies with Crohn's disease, rheumatoid arthritis and psoriasis clinical cohorts. We evaluate the PRT methodology used, paying particular attention to accuracy and precision, and highlight the problems of differential bias in copy number measurements. Our PRT methods for measuring copy number were of sufficient precision to detect very slight but systematic differential bias between results from case and control DNA samples in one study. We find no evidence for an association between CCL3L1 copy number and Crohn's disease, rheumatoid arthritis or psoriasis. Differential bias of this small magnitude, but applied systematically across large numbers of samples, would create a serious risk of false positive associations in copy number, if measured using methods of lower precision, or methods relying on single uncorroborated measurements. In this study the small differential bias detected by PRT in one sample set was resolved by a simple pre-treatment by restriction enzyme digestion.

Accuracy and differential bias in copy number measurement of CCL3L1 in association studies with three auto-immune disorders

PubMed Central

2011-01-01

Background Copy number variation (CNV) contributes to the variation observed between individuals and can influence human disease progression, but the accurate measurement of individual copy numbers is technically challenging. In the work presented here we describe a modification to a previously described paralogue ratio test (PRT) method for genotyping the CCL3L1/CCL4L1 copy variable region, which we use to ascertain CCL3L1/CCL4L1 copy number in 1581 European samples. As the products of CCL3L1 and CCL4L1 potentially play a role in autoimmunity we performed case control association studies with Crohn's disease, rheumatoid arthritis and psoriasis clinical cohorts. Results We evaluate the PRT methodology used, paying particular attention to accuracy and precision, and highlight the problems of differential bias in copy number measurements. Our PRT methods for measuring copy number were of sufficient precision to detect very slight but systematic differential bias between results from case and control DNA samples in one study. We find no evidence for an association between CCL3L1 copy number and Crohn's disease, rheumatoid arthritis or psoriasis. Conclusions Differential bias of this small magnitude, but applied systematically across large numbers of samples, would create a serious risk of false positive associations in copy number, if measured using methods of lower precision, or methods relying on single uncorroborated measurements. In this study the small differential bias detected by PRT in one sample set was resolved by a simple pre-treatment by restriction enzyme digestion. PMID:21851606

Mitochondrial DNA copy number in peripheral blood cell and hypertension risk among mining workers: a case-control study in Chinese coal miners.

PubMed

Lei, L; Guo, J; Shi, X; Zhang, G; Kang, H; Sun, C; Huang, J; Wang, T

2017-09-01

Alteration of mitochondrial DNA (mtDNA) copy number, which reflects oxidant-induced cell damage, has been observed in a wide range of human diseases. However, whether it correlates with hypertension has not been elucidated. We aimed to explore the association between mtDNA copy number and the risk of hypertension in Chinese coal miners. A case-control study was performed with 378 hypertension patients and 325 healthy controls in a large coal mining group located in North China. Face-to-face interviews were conducted by trained staffs with necessary medical knowledge. The mtDNA copy number was measured by a quantitative real-time PCR assay using DNA extracted from peripheral blood. No significant differences in mtDNA copy number were observed between hypertension patients and healthy controls. However, in both case and control groups, the mtDNA copy number was statistically significantly lower in the elder population (≥45 years old) compared with the younger subjects (<45 years old; 7.17 vs 6.64, P=0.005 and 7.21 vs 6.84, P=0.036). A significantly higher mtDNA copy number could be found in hypertension patients consuming alcohol regularly compared with no alcohol consumption patients (7.09 vs 6.69); mtDNA copy number was also positively correlated with age and alcohol consumption. Hypertension was found significantly correlated with factors such as age, work duration, monthly family income and drinking status. Our results suggest that the mtDNA copy number is not associated with hypertension in coal miners.

Amylase activity is associated with AMY2B copy numbers in dog: implications for dog domestication, diet and diabetes

PubMed Central

Arendt, Maja; Fall, Tove; Lindblad-Toh, Kerstin; Axelsson, Erik

2014-01-01

High amylase activity in dogs is associated with a drastic increase in copy numbers of the gene coding for pancreatic amylase, AMY2B, that likely allowed dogs to thrive on a relatively starch-rich diet during early dog domestication. Although most dogs thus probably digest starch more efficiently than do wolves, AMY2B copy numbers vary widely within the dog population, and it is not clear how this variation affects the individual ability to handle starch nor how it affects dog health. In humans, copy numbers of the gene coding for salivary amylase, AMY1, correlate with both salivary amylase levels and enzyme activity, and high amylase activity is related to improved glycemic homeostasis and lower frequencies of metabolic syndrome. Here, we investigate the relationship between AMY2B copy numbers and serum amylase activity in dogs and show that amylase activity correlates with AMY2B copy numbers. We then describe how AMY2B copy numbers vary in individuals from 20 dog breeds and find strong breed-dependent patterns, indicating that the ability to digest starch varies both at the breed and individual level. Finally, to test whether AMY2B copy number is strongly associated with the risk of developing diabetes mellitus, we compare copy numbers in cases and controls as well as in breeds with varying diabetes susceptibility. Although we see no such association here, future studies using larger cohorts are needed before excluding a possible link between AMY2B and diabetes mellitus. PMID:24975239

TEGS-CN: A Statistical Method for Pathway Analysis of Genome-wide Copy Number Profile.

PubMed

Huang, Yen-Tsung; Hsu, Thomas; Christiani, David C

2014-01-01

The effects of copy number alterations make up a significant part of the tumor genome profile, but pathway analyses of these alterations are still not well established. We proposed a novel method to analyze multiple copy numbers of genes within a pathway, termed Test for the Effect of a Gene Set with Copy Number data (TEGS-CN). TEGS-CN was adapted from TEGS, a method that we previously developed for gene expression data using a variance component score test. With additional development, we extend the method to analyze DNA copy number data, accounting for different sizes and thus various numbers of copy number probes in genes. The test statistic follows a mixture of X (2) distributions that can be obtained using permutation with scaled X (2) approximation. We conducted simulation studies to evaluate the size and the power of TEGS-CN and to compare its performance with TEGS. We analyzed a genome-wide copy number data from 264 patients of non-small-cell lung cancer. With the Molecular Signatures Database (MSigDB) pathway database, the genome-wide copy number data can be classified into 1814 biological pathways or gene sets. We investigated associations of the copy number profile of the 1814 gene sets with pack-years of cigarette smoking. Our analysis revealed five pathways with significant P values after Bonferroni adjustment (<2.8 × 10(-5)), including the PTEN pathway (7.8 × 10(-7)), the gene set up-regulated under heat shock (3.6 × 10(-6)), the gene sets involved in the immune profile for rejection of kidney transplantation (9.2 × 10(-6)) and for transcriptional control of leukocytes (2.2 × 10(-5)), and the ganglioside biosynthesis pathway (2.7 × 10(-5)). In conclusion, we present a new method for pathway analyses of copy number data, and causal mechanisms of the five pathways require further study.

Copy number neutral loss of heterozygosity at 17p and homozygous mutations of TP53 are associated with complex chromosomal aberrations in patients newly diagnosed with myelodysplastic syndromes.

PubMed

Svobodova, Karla; Zemanova, Zuzana; Lhotska, Halka; Novakova, Milena; Podskalska, Lucie; Belickova, Monika; Brezinova, Jana; Sarova, Iveta; Izakova, Silvia; Lizcova, Libuse; Berkova, Adela; Siskova, Magda; Jonasova, Anna; Cermak, Jaroslav; Michalova, Kyra

2016-03-01

Complex karyotypes are seen in approximately 20% of patients with myelodysplastic syndromes (MDS) and are associated with a high risk of transformation to acute myeloid leukemia and poor outcomes in patients. Copy number neutral loss of heterozygosity (CN-LOH, i.e., both copies of a chromosomal pair or their parts originate from one parent) might contribute to increased genomic instability in the bone-marrow cells of patients with MDS. The pathological potential of CN-LOH, which arises as a clonal aberration in a proportion of somatic cells, consists of tumor suppressor gene and oncogene homozygous mutations. The aim of our study was to evaluate the frequency of CN-LOH at 17p in bone-marrow cells of newly diagnosed MDS patients with complex chromosomal aberrations and to assess its correlation with mutations in the TP53 gene (17p13.1). CN-LOH was detected in 40 chromosomal regions in 21 (29%) of 72 patients analyzed. The changes in 27 of the 40 regions identified were sporadic. The most common finding was CN-LOH of the short arm of chromosome 17, which was detected in 13 (18%) of 72 patients. A mutational analysis confirmed the homozygous mutation of TP53 in all CN-LOH 17p patients, among which two frameshift mutations are not registered in the International Agency for Research on Cancer TP53 Database. CN-LOH 17p correlated with aggressive disease (median overall survival 4 months) and was strongly associated with a complex karyotype in the cohort studied, which might cause rapid disease progression in high-risk MDS. No other CN-LOH region previously recorded in MDS or AML patients (1p, 4q, 7q, 11q, 13q, 19q, 21q) was detected in our cohort of patients with complex karyotype examined at the diagnosis of MDS. The LOH region appeared to be balanced (i.e., with no DNA copy number change) when examined with conventional and molecular cytogenetic methods. Therefore, a microarray that detects single-nucleotide polymorphisms is an ideal method with which to identify and further characterize CN-LOH. Our data should specify the prognosis and should lead to the identification of potential targets for therapeutic interventions. Copyright © 2016 Elsevier Ltd. All rights reserved.

Genetic variation in the Solanaceae fruit bearing species lulo and tree tomato revealed by Conserved Ortholog (COSII) markers

PubMed Central

2010-01-01

The Lulo or naranjilla (Solanum quitoense Lam.) and the tree tomato or tamarillo (Solanum betaceum Cav. Sendt.) are both Andean tropical fruit species with high nutritional value and the potential for becoming premium products in local and export markets. Herein, we present a report on the genetic characterization of 62 accessions of lulos (n = 32) and tree tomatoes (n = 30) through the use of PCR-based markers developed from single-copy conserved orthologous genes (COSII) in other Solanaceae (Asterid) species. We successfully PCR amplified a set of these markers for lulos (34 out of 46 initially tested) and tree tomatoes (26 out of 41) for molecular studies. Six polymorphic COSII markers were found in lulo with a total of 47 alleles and five polymorphic markers in tree tomato with a total of 39 alleles in the two populations. Further genetic analyses indicated a high population structure (with FST > 0.90), which may be a result of low migration between populations, adaptation to various niches and the number of markers evaluated. We propose COSII markers as sound tools for molecular studies, conservation and the breeding of these two fruit species. PMID:21637482

Indels, structural variation, and recombination drive genomic diversity in Plasmodium falciparum

PubMed Central

Miles, Alistair; Iqbal, Zamin; Vauterin, Paul; Pearson, Richard; Campino, Susana; Theron, Michel; Gould, Kelda; Mead, Daniel; Drury, Eleanor; O'Brien, John; Ruano Rubio, Valentin; MacInnis, Bronwyn; Mwangi, Jonathan; Samarakoon, Upeka; Ranford-Cartwright, Lisa; Ferdig, Michael; Hayton, Karen; Su, Xin-zhuan; Wellems, Thomas; Rayner, Julian; McVean, Gil; Kwiatkowski, Dominic

2016-01-01

The malaria parasite Plasmodium falciparum has a great capacity for evolutionary adaptation to evade host immunity and develop drug resistance. Current understanding of parasite evolution is impeded by the fact that a large fraction of the genome is either highly repetitive or highly variable and thus difficult to analyze using short-read sequencing technologies. Here, we describe a resource of deep sequencing data on parents and progeny from genetic crosses, which has enabled us to perform the first genome-wide, integrated analysis of SNP, indel and complex polymorphisms, using Mendelian error rates as an indicator of genotypic accuracy. These data reveal that indels are exceptionally abundant, being more common than SNPs and thus the dominant mode of polymorphism within the core genome. We use the high density of SNP and indel markers to analyze patterns of meiotic recombination, confirming a high rate of crossover events and providing the first estimates for the rate of non-crossover events and the length of conversion tracts. We observe several instances of meiotic recombination within copy number variants associated with drug resistance, demonstrating a mechanism whereby fitness costs associated with resistance mutations could be compensated and greater phenotypic plasticity could be acquired. PMID:27531718

Integrative pipeline for profiling DNA copy number and inferring tumor phylogeny.

PubMed

Urrutia, Eugene; Chen, Hao; Zhou, Zilu; Zhang, Nancy R; Jiang, Yuchao

2018-06-15

Copy number variation is an important and abundant source of variation in the human genome, which has been associated with a number of diseases, especially cancer. Massively parallel next-generation sequencing allows copy number profiling with fine resolution. Such efforts, however, have met with mixed successes, with setbacks arising partly from the lack of reliable analytical methods to meet the diverse and unique challenges arising from the myriad experimental designs and study goals in genetic studies. In cancer genomics, detection of somatic copy number changes and profiling of allele-specific copy number (ASCN) are complicated by experimental biases and artifacts as well as normal cell contamination and cancer subclone admixture. Furthermore, careful statistical modeling is warranted to reconstruct tumor phylogeny by both somatic ASCN changes and single nucleotide variants. Here we describe a flexible computational pipeline, MARATHON, which integrates multiple related statistical software for copy number profiling and downstream analyses in disease genetic studies. MARATHON is publicly available at https://github.com/yuchaojiang/MARATHON. Supplementary data are available at Bioinformatics online.

Survival differences of CIMP subtypes integrated with CNA information in human breast cancer.

PubMed

Wang, Huihan; Yan, Weili; Zhang, Shumei; Gu, Yue; Wang, Yihan; Wei, Yanjun; Liu, Hongbo; Wang, Fang; Wu, Qiong; Zhang, Yan

2017-07-25

CpG island methylator phenotype of breast cancer is associated with widespread aberrant methylation at specified CpG islands and distinct patient outcomes. However, the influence of copy number contributing to the prognosis of tumors with different CpG island methylator phenotypes is still unclear. We analyzed both genetic (copy number) and epigenetic alterations in 765 breast cancers from The Cancer Genome Atlas data portal and got a panel of 15 biomarkers for copy number and methylation status evaluation. The gene panel identified two groups corresponding to distinct copy number profiles. In status of mere-loss copy number, patients were faced with a greater risk if they presented a higher CpG islands methylation pattern in biomarker panels. But for samples presenting merely-gained copy number, higher methylation level of CpG islands was associated with improved viability. In all, the integration of copy number alteration and methylation information enhanced the classification power on prognosis. Moreover, we found the molecular subtypes of breast cancer presented different distributions in two CpG island methylation phenotypes. Generated by the same set of human methylation 450K data, additional copy number information could provide insights into survival prediction of cancers with less heterogeneity and might help to determine the biomarkers for diagnosis and treatment for breast cancer patients in a more personalized approach.

Survival differences of CIMP subtypes integrated with CNA information in human breast cancer

PubMed Central

Wang, Huihan; Yan, Weili; Zhang, Shumei; Gu, Yue; Wang, Yihan; Wei, Yanjun; Liu, Hongbo; Wang, Fang; Wu, Qiong; Zhang, Yan

2017-01-01

CpG island methylator phenotype of breast cancer is associated with widespread aberrant methylation at specified CpG islands and distinct patient outcomes. However, the influence of copy number contributing to the prognosis of tumors with different CpG island methylator phenotypes is still unclear. We analyzed both genetic (copy number) and epigenetic alterations in 765 breast cancers from The Cancer Genome Atlas data portal and got a panel of 15 biomarkers for copy number and methylation status evaluation. The gene panel identified two groups corresponding to distinct copy number profiles. In status of mere-loss copy number, patients were faced with a greater risk if they presented a higher CpG islands methylation pattern in biomarker panels. But for samples presenting merely-gained copy number, higher methylation level of CpG islands was associated with improved viability. In all, the integration of copy number alteration and methylation information enhanced the classification power on prognosis. Moreover, we found the molecular subtypes of breast cancer presented different distributions in two CpG island methylation phenotypes. Generated by the same set of human methylation 450K data, additional copy number information could provide insights into survival prediction of cancers with less heterogeneity and might help to determine the biomarkers for diagnosis and treatment for breast cancer patients in a more personalized approach. PMID:28415743

Integrated genome-wide DNA copy number and expression analysis identifies distinct mechanisms of primary chemoresistance in ovarian carcinomas.

PubMed

Etemadmoghadam, Dariush; deFazio, Anna; Beroukhim, Rameen; Mermel, Craig; George, Joshy; Getz, Gad; Tothill, Richard; Okamoto, Aikou; Raeder, Maria B; Harnett, Paul; Lade, Stephen; Akslen, Lars A; Tinker, Anna V; Locandro, Bianca; Alsop, Kathryn; Chiew, Yoke-Eng; Traficante, Nadia; Fereday, Sian; Johnson, Daryl; Fox, Stephen; Sellers, William; Urashima, Mitsuyoshi; Salvesen, Helga B; Meyerson, Matthew; Bowtell, David

2009-02-15

A significant number of women with serous ovarian cancer are intrinsically refractory to platinum-based treatment. We analyzed somatic DNA copy number variation and gene expression data to identify key mechanisms associated with primary resistance in advanced-stage serous cancers. Genome-wide copy number variation was measured in 118 ovarian tumors using high-resolution oligonucleotide microarrays. A well-defined subset of 85 advanced-stage serous tumors was then used to relate copy number variation to primary resistance to treatment. The discovery-based approach was complemented by quantitative-PCR copy number analysis of 12 candidate genes as independent validation of previously reported associations with clinical outcome. Likely copy number variation targets and tumor molecular subtypes were further characterized by gene expression profiling. Amplification of 19q12, containing cyclin E (CCNE1), and 20q11.22-q13.12, mapping immediately adjacent to the steroid receptor coactivator NCOA3, was significantly associated with poor response to primary treatment. Other genes previously associated with copy number variation and clinical outcome in ovarian cancer were not associated with primary treatment resistance. Chemoresistant tumors with high CCNE1 copy number and protein expression were associated with increased cellular proliferation but so too was a subset of treatment-responsive patients, suggesting a cell-cycle independent role for CCNE1 in modulating chemoresponse. Patients with a poor clinical outcome without CCNE1 amplification overexpressed genes involved in extracellular matrix deposition. We have identified two distinct mechanisms of primary treatment failure in serous ovarian cancer, involving CCNE1 amplification and enhanced extracellular matrix deposition. CCNE1 copy number is validated as a dominant marker of patient outcome in ovarian cancer.

A Simulation Model for Purchasing Duplicate Copies in a Library

ERIC Educational Resources Information Center

Arms, W. Y.; Walter, T. P.

1974-01-01

A method of estimating the number of duplicate copies of books needed based on a computer simulation which takes into account number of copies available, number of loans, total underlying demand, satisfaction level, percentage time on shelf. (LS)

A selective sweep of >8 Mb on chromosome 26 in the Boxer genome.

PubMed

Quilez, Javier; Short, Andrea D; Martínez, Verónica; Kennedy, Lorna J; Ollier, William; Sanchez, Armand; Altet, Laura; Francino, Olga

2011-07-01

Modern dog breeds display traits that are either breed-specific or shared by a few breeds as a result of genetic bottlenecks during the breed creation process and artificial selection for breed standards. Selective sweeps in the genome result from strong selection and can be detected as a reduction or elimination of polymorphism in a given region of the genome. Extended regions of homozygosity, indicative of selective sweeps, were identified in a genome-wide scan dataset of 25 Boxers from the United Kingdom genotyped at ~20,000 single-nucleotide polymorphisms (SNPs). These regions were further examined in a second dataset of Boxers collected from a different geographical location and genotyped using higher density SNP arrays (~170,000 SNPs). A selective sweep previously associated with canine brachycephaly was detected on chromosome 1. A novel selective sweep of over 8 Mb was observed on chromosome 26 in Boxer and for a shorter region in English and French bulldogs. It was absent in 171 samples from eight other dog breeds and 7 Iberian wolf samples. A region of extended increased heterozygosity on chromosome 9 overlapped with a previously reported copy number variant (CNV) which was polymorphic in multiple dog breeds. A selective sweep of more than 8 Mb on chromosome 26 was identified in the Boxer genome. This sweep is likely caused by strong artificial selection for a trait of interest and could have inadvertently led to undesired health implications for this breed. Furthermore, we provide supporting evidence for two previously described regions: a selective sweep on chromosome 1 associated with canine brachycephaly and a CNV on chromosome 9 polymorphic in multiple dog breeds.

Inferring mechanisms of copy number change from haplotype structures at the human DEFA1A3 locus.

PubMed

Black, Holly A; Khan, Fayeza F; Tyson, Jess; Al Armour, John

2014-07-21

The determination of structural haplotypes at copy number variable regions can indicate the mechanisms responsible for changes in copy number, as well as explain the relationship between gene copy number and expression. However, obtaining spatial information at regions displaying extensive copy number variation, such as the DEFA1A3 locus, is complex, because of the difficulty in the phasing and assembly of these regions. The DEFA1A3 locus is intriguing in that it falls within a region of high linkage disequilibrium, despite its high variability in copy number (n = 3-16); hence, the mechanisms responsible for changes in copy number at this locus are unclear. In this study, a region flanking the DEFA1A3 locus was sequenced across 120 independent haplotypes with European ancestry, identifying five common classes of DEFA1A3 haplotype. Assigning DEFA1A3 class to haplotypes within the 1000 Genomes project highlights a significant difference in DEFA1A3 class frequencies between populations with different ancestry. The features of each DEFA1A3 class, for example, the associated DEFA1A3 copy numbers, were initially assessed in a European cohort (n = 599) and replicated in the 1000 Genomes samples, showing within-class similarity, but between-class and between-population differences in the features of the DEFA1A3 locus. Emulsion haplotype fusion-PCR was used to generate 61 structural haplotypes at the DEFA1A3 locus, showing a high within-class similarity in structure. Structural haplotypes across the DEFA1A3 locus indicate that intra-allelic rearrangement is the predominant mechanism responsible for changes in DEFA1A3 copy number, explaining the conservation of linkage disequilibrium across the locus. The identification of common structural haplotypes at the DEFA1A3 locus could aid studies into how DEFA1A3 copy number influences expression, which is currently unclear.

Interpreting aCGH-defined karyotypic changes in gliomas using copy number status, loss of heterozygosity and allelic ratios

PubMed Central

Cowell, John K; Lo, Ken C; Luce, Jesse; Hawthorn, Lesleyann

2009-01-01

We have used SNP mapping arrays to simultaneously record copy number changes, loss of heterozygosity and allele ratios (ploidy) in a series of 13 gliomas. This combined analysis has defined novel amplification events in this tumor type involving chr1:241544532-243005121 and chr18:54716681-54917277 which contain the AKT3 and ZNF532 genes respectively. The high resolution of this analysis has also identified homozygous deletions involving chr17:25600031-26490848 and Chr19:53883612-55061878. Throughout the karyotypes of these tumors, the combined analysis revealed counter intuitive relationships between copy number and LOH that requires reinterpretation of the significance of copy number gains and losses. It was not uncommon to observe copy number gains that were associated with loss of heterozygosity as well as copy number losses that were not. These events appeared to be related to ploidy status in the tumors as determined using allelic ratio calculations. Overall, this analysis of gliomas provides evidence for the need to perform more comprehensive interpretation of the CGH data beyond copy number analysis alone to evaluate the significance of individual events in the karyotypes. PMID:19818351

Selective sweep on human amylase genes postdates the split with Neanderthals

PubMed Central

Inchley, Charlotte E.; Larbey, Cynthia D. A.; Shwan, Nzar A. A.; Pagani, Luca; Saag, Lauri; Antão, Tiago; Jacobs, Guy; Hudjashov, Georgi; Metspalu, Ene; Mitt, Mario; Eichstaedt, Christina A.; Malyarchuk, Boris; Derenko, Miroslava; Wee, Joseph; Abdullah, Syafiq; Ricaut, François-Xavier; Mormina, Maru; Mägi, Reedik; Villems, Richard; Metspalu, Mait; Jones, Martin K.; Armour, John A. L.; Kivisild, Toomas

2016-01-01

Humans have more copies of amylase genes than other primates. It is still poorly understood, however, when the copy number expansion occurred and whether its spread was enhanced by selection. Here we assess amylase copy numbers in a global sample of 480 high coverage genomes and find that regions flanking the amylase locus show notable depression of genetic diversity both in African and non-African populations. Analysis of genetic variation in these regions supports the model of an early selective sweep in the human lineage after the split of humans from Neanderthals which led to the fixation of multiple copies of AMY1 in place of a single copy. We find evidence of multiple secondary losses of copy number with the highest frequency (52%) of a deletion of AMY2A and associated low copy number of AMY1 in Northeast Siberian populations whose diet has been low in starch content. PMID:27853181

Selective sweep on human amylase genes postdates the split with Neanderthals.

PubMed

Inchley, Charlotte E; Larbey, Cynthia D A; Shwan, Nzar A A; Pagani, Luca; Saag, Lauri; Antão, Tiago; Jacobs, Guy; Hudjashov, Georgi; Metspalu, Ene; Mitt, Mario; Eichstaedt, Christina A; Malyarchuk, Boris; Derenko, Miroslava; Wee, Joseph; Abdullah, Syafiq; Ricaut, François-Xavier; Mormina, Maru; Mägi, Reedik; Villems, Richard; Metspalu, Mait; Jones, Martin K; Armour, John A L; Kivisild, Toomas

2016-11-17

Humans have more copies of amylase genes than other primates. It is still poorly understood, however, when the copy number expansion occurred and whether its spread was enhanced by selection. Here we assess amylase copy numbers in a global sample of 480 high coverage genomes and find that regions flanking the amylase locus show notable depression of genetic diversity both in African and non-African populations. Analysis of genetic variation in these regions supports the model of an early selective sweep in the human lineage after the split of humans from Neanderthals which led to the fixation of multiple copies of AMY1 in place of a single copy. We find evidence of multiple secondary losses of copy number with the highest frequency (52%) of a deletion of AMY2A and associated low copy number of AMY1 in Northeast Siberian populations whose diet has been low in starch content.

Cytochrome P450 1B1 and catechol-O-methyltransferase genetic polymorphisms and breast cancer risk in Chinese women: results from the shanghai breast cancer study and a meta-analysis.

PubMed

Wen, Wanqing; Cai, Qiuyin; Shu, Xiao-Ou; Cheng, Jia-Rong; Parl, Fritz; Pierce, Larry; Gao, Yu-Tang; Zheng, Wei

2005-02-01

Cytochrome P450 1B1 (CYP1B1) and catechol-O-methyltransferase (COMT) are important estrogen-metabolizing enzymes and, thus, genetic polymorphisms of these enzymes may affect breast cancer risk. A population-based case-control study was conducted to assess the association of breast cancer risk with CYP1B1 and COMT polymorphisms. A meta-analysis was done to summarize the findings from this and previous studies. Included in this study were 1,135 incident breast cancer cases diagnosed from August 1996 through March 1998 among female residents of Shanghai and 1,235 randomly selected, age frequency-matched controls from the same general population. The common alleles of the CYP1B1 gene were Arg (79.97%) in codon 48, Ala (80.53%) in codon 119, and Leu (86.57%) in codon 432. The Val allele accounted for 72.46% of the total alleles identified in codon 108/158 of the COMT gene. No overall associations of breast cancer risk were found with any of the single nucleotide polymorphisms described above. This finding was supported by a meta-analysis of all previous published studies. No gene-gene interactions were observed between CYP1B1 and COMT genotypes. The associations of breast cancer risk with factors related to endogenous estrogen exposure, such as years of menstruation and body mass index, were not significantly modified by the CYP1B1 and COMT genotypes. We observed, however, that women who carried one copy of the variant allele in CYP1B1 codons 48 or 119 were less likely to have estrogen receptor-positive breast cancer than those who carried two copies of the corresponding wild-type alleles. The results from this study were consistent with those from most previous studies, indicating no major associations of breast cancer risk with CYP1B1 and COMT polymorphisms.

DNA repair gene XRCC1 polymorphisms, smoking, and bladder cancer risk.

PubMed

Stern, M C; Umbach, D M; van Gils, C H; Lunn, R M; Taylor, J A

2001-02-01

Bladder cancer is the sixth most common cancer in the United States. The main identified risk factor is cigarette smoking, which is estimated to contribute to up to 50% of new cases in men and 20% in women. Besides containing other carcinogens, cigarette smoke is a rich source of reactive oxygen species (ROS) that can induce a variety of DNA damage, some of which is repaired by the base excision repair (BER) pathway. The XRCC1 gene protein plays an important role in BER by serving as a scaffold for other repair enzymes and by recognizing single-strand DNA breaks. Three polymorphisms that induce amino acid changes have been found in codon 194 (exon 6), codon 280 (exon 9), and codon 399 (exon 10) of this gene. We tested whether polymorphisms in XRCC1 were associated with bladder cancer risk and whether this association was modified by cigarette smoking. Therefore, we genotyped for the three polymorphisms in 235 bladder cancer cases and 213 controls who had been frequency matched to cases on age, sex, and ethnicity. We found no evidence of an association between the codon 280 variant and bladder cancer risk [odds ratio (OR), 1.2; 95% confidence interval (CI), 0.6-2.6]. We found some evidence of a protective effect for subjects that carried at least one copy of the codon 194 variant allele relative to those homozygous for the common allele (OR, 0.59; 95% CI, 0.3-1.0). The combined analysis with smoking history suggested a possible gene-exposure interaction; however, the results were not statistically significant. Similarly, for the codon 399 polymorphism, our data suggested a protective effect of the homozygous variant genotype relative to carriers of either one or two copies of the common allele (OR, 0.70; 95% CI, 0.4-1.3), and provided limited evidence, albeit not statistically significant, for a gene-smoking interaction.

Amylase activity is associated with AMY2B copy numbers in dog: implications for dog domestication, diet and diabetes.

PubMed

Arendt, Maja; Fall, Tove; Lindblad-Toh, Kerstin; Axelsson, Erik

2014-10-01

High amylase activity in dogs is associated with a drastic increase in copy numbers of the gene coding for pancreatic amylase, AMY2B, that likely allowed dogs to thrive on a relatively starch-rich diet during early dog domestication. Although most dogs thus probably digest starch more efficiently than do wolves, AMY2B copy numbers vary widely within the dog population, and it is not clear how this variation affects the individual ability to handle starch nor how it affects dog health. In humans, copy numbers of the gene coding for salivary amylase, AMY1, correlate with both salivary amylase levels and enzyme activity, and high amylase activity is related to improved glycemic homeostasis and lower frequencies of metabolic syndrome. Here, we investigate the relationship between AMY2B copy numbers and serum amylase activity in dogs and show that amylase activity correlates with AMY2B copy numbers. We then describe how AMY2B copy numbers vary in individuals from 20 dog breeds and find strong breed-dependent patterns, indicating that the ability to digest starch varies both at the breed and individual level. Finally, to test whether AMY2B copy number is strongly associated with the risk of developing diabetes mellitus, we compare copy numbers in cases and controls as well as in breeds with varying diabetes susceptibility. Although we see no such association here, future studies using larger cohorts are needed before excluding a possible link between AMY2B and diabetes mellitus. © 2014 The Authors. Animal Genetics published by John Wiley & Sons Ltd on behalf of Stichting International Foundation for Animal Genetics.

Reduced mtDNA copy number increases the sensitivity of tumor cells to chemotherapeutic drugs.

PubMed

Mei, H; Sun, S; Bai, Y; Chen, Y; Chai, R; Li, H

2015-04-02

Many cancer drugs are toxic to cells by activating apoptotic pathways. Previous studies have shown that mitochondria have key roles in apoptosis in mammalian cells, but the role of mitochondrial DNA (mtDNA) copy number variation in the pathogenesis of tumor cell apoptosis remains largely unknown. We used the HEp-2, HNE2, and A549 tumor cell lines to explore the relationship between mtDNA copy number variation and cell apoptosis. We first induced apoptosis in three tumor cell lines and one normal adult human skin fibroblast cell line (HSF) with cisplatin (DDP) or doxorubicin (DOX) treatment and found that the mtDNA copy number significantly increased in apoptotic tumor cells, but not in HSF cells. We then downregulated the mtDNA copy number by transfection with shRNA-TFAM plasmids or treatment with ethidium bromide and found that the sensitivity of tumor cells to DDP or DOX was significantly increased. Furthermore, we observed that levels of reactive oxygen species (ROS) increased significantly in tumor cells with lower mtDNA copy numbers, and this might be related to a low level of antioxidant gene expression. Finally, we rescued the increase of ROS in tumor cells with lipoic acid or N-acetyl-L-cysteine and found that the apoptosis rate decreased. Our studies suggest that the increase of mtDNA copy number is a self-protective mechanism of tumor cells to prevent apoptosis and that reduced mtDNA copy number increases ROS levels in tumor cells, increases the tumor cells' sensitivity to chemotherapeutic drugs, and increases the rate of apoptosis. This research provides evidence that mtDNA copy number variation might be a promising new therapeutic target for the clinical treatment of tumors.

ALK gene copy number gain and immunohistochemical expression status using three antibodies in neuroblastoma.

PubMed

Kim, Eun Kyung; Kim, Sewha

2016-03-17

Anaplastic lymphoma kinase (ALK) gene aberrations-such as mutations, amplifications, and copy number gains-represent a major genetic predisposition to neuroblastoma (NB). This study aimed to evaluate the correlation between ALK gene copy number status, ALK protein expression, and clinicopathological parameters. We retrospectively retrieved 30 cases of poorly differentiated NB and constructed tissue microarrays (TMAs). ALK copy number changes were assessed by fluorescence in situ hybridization (FISH) assays, and ALK immunohistochemistry (IHC) testing was performed using three different antibodies (ALK1, D5F3, and 5A4 clones). ALK amplification and copy number gain were observed in 10% (3/30) and 53.3% (16/30) of the cohort, respectively. There were positive correlations between ALK copy number and IHC positive rate in ALK1 and 5A4 antibodies (p= < 0.001 and 0.019, respectively). ALK1, D5F3, and 5A4 antibodies equally showed 100% sensitivity in detecting ALK amplification. However, the sensitivity for detecting copy number gain differed among the three antibodies, with 75% sensitivity in D5F3 and 0% sensitivity in ALK1. ALK-amplified NBs were correlated with synchronous MYCN amplification and chromosome 1p deletion. ALK IHC positivity was frequently observed in INSS stage IV and high-risk group patients. In conclusion, this study identified that an increase in the ALK copy number is a frequent genetic alteration in poorly differentiated NB. ALK-amplified NBs showed consistent ALK IHC positivity with all kinds of antibodies. In contrast, the detection performance of ALK copy number gain was antibody dependent, with the D5F3 antibody showing the best sensitivity.

ALK Gene Copy Number Gain and Immunohistochemical Expression Status Using Three Antibodies in Neuroblastoma.

PubMed

Kim, Eun Kyung; Kim, Sewha

2017-01-01

Anaplastic lymphoma kinase ( ALK) gene aberrations-such as mutations, amplifications, and copy number gains-represent a major genetic predisposition to neuroblastoma (NB). This study aimed to evaluate the correlation between ALK gene copy number status, ALK protein expression, and clinicopathological parameters. We retrospectively retrieved 30 cases of poorly differentiated NB and constructed tissue microarrays (TMAs). ALK copy number changes were assessed by fluorescence in situ hybridization (FISH) assays, and ALK immunohistochemistry (IHC) testing was performed using three different antibodies (ALK1, D5F3, and 5A4 clones). ALK amplification and copy number gain were observed in 10% (3/30) and 53.3% (16/30) of the cohort, respectively. There were positive correlations between ALK copy number and IHC-positive rate in ALK1 and 5A4 antibodies ( P < 0.001 and P = 0.019, respectively). ALK1, D5F3, and 5A4 antibodies equally showed 100% sensitivity in detecting ALK amplification. However, the sensitivity for detecting copy number gain differed among the three antibodies, with 75% sensitivity in D5F3 and 0% sensitivity in ALK1. ALK-amplified NBs were correlated with synchronous MYCN amplification and chromosome 1p deletion. ALK IHC positivity was frequently observed in INSS stage IV and high-risk group patients. In conclusion, this study identified that an increase in the ALK copy number is a frequent genetic alteration in poorly differentiated NB. ALK-amplified NBs showed consistent ALK IHC positivity with all kinds of antibodies. In contrast, the detection performance of ALK copy number gain was antibody dependent, with the D5F3 antibody showing the best sensitivity.

Association of mitochondrial DNA in peripheral blood with depression, anxiety and stress- and adjustment disorders in primary health care patients.

PubMed

Wang, Xiao; Sundquist, Kristina; Rastkhani, Hamideh; Palmér, Karolina; Memon, Ashfaque A; Sundquist, Jan

2017-08-01

Mitochondrial dysfunction may result in a variety of diseases. The objectives here were to examine possible differences in mtDNA copy number between healthy controls and patients with depression, anxiety or stress- and adjustment disorders; the association between mtDNA copy number and disease severity at baseline; and the association between mtDNA copy number and response after an 8-week treatment (mindfulness, cognitive based therapy). A total of 179 patients in primary health care (age 20-64 years) with depression, anxiety and stress- and adjustment disorders, and 320 healthy controls (aged 19-70 years) were included in the study. Relative mtDNA copy number was measured using quantitative real-time PCR on peripheral blood samples. We found that the mean mtDNA copy number was significantly higher in patients compared to controls (84.9 vs 75.9, p<0.0001) at baseline. The difference in mtDNA copy number between patients and controls remained significant after controlling for age and sex (ß=8.13, p<0.0001; linear regression analysis). The mtDNA copy number was significantly associated with Patient Health Questionnaire (PHQ-9) scores (β=0.57, p=0.02) at baseline. After treatment, the change in mtDNA copy number was significantly associated with the treatment response, i.e., change in Hospital Anxiety and Depression Scale (HADS-D) and PHQ-9 scores (ß=1.00, p=0.03 and ß=0.65, p=0.04, respectively), after controlling for baseline scores, age, sex, BMI, smoking status, alcohol drinking and medication. Our findings show that mtDNA copy number is associated with symptoms of depression, anxiety and stress- and adjustment disorders and treatment response in these disorders. Copyright © 2017 Elsevier B.V. and ECNP. All rights reserved.

Mitochondrial DNA Copy Number in Sleep Duration Discordant Monozygotic Twins.

PubMed

Wrede, Joanna E; Mengel-From, Jonas; Buchwald, Dedra; Vitiello, Michael V; Bamshad, Michael; Noonan, Carolyn; Christiansen, Lene; Christensen, Kaare; Watson, Nathaniel F

2015-10-01

Mitochondrial DNA (mtDNA) copy number is an important component of mitochondrial function and varies with age, disease, and environmental factors. We aimed to determine whether mtDNA copy number varies with habitual differences in sleep duration within pairs of monozygotic twins. Academic clinical research center. 15 sleep duration discordant monozygotic twin pairs (30 twins, 80% female; mean age 42.1 years [SD 15.0]). Sleep duration was phenotyped with wrist actigraphy. Each twin pair included a "normal" (7-9 h/24) and "short" (< 7 h/24) sleeping twin. Fasting peripheral blood leukocyte DNA was assessed for mtDNA copy number via the n-fold difference between qPCR measured mtDNA and nuclear DNA creating an mtDNA measure without absolute units. We used generalized estimating equation linear regression models accounting for the correlated data structure to assess within-pair effects of sleep duration on mtDNA copy number. Mean within-pair sleep duration difference per 24 hours was 94.3 minutes (SD 62.6 min). We found reduced sleep duration (β = 0.06; 95% CI 0.004, 0.12; P < 0.05) and sleep efficiency (β = 0.51; 95% CI 0.06, 0.95; P < 0.05) were significantly associated with reduced mtDNA copy number within twin pairs. Thus every 1-minute decrease in actigraphy-defined sleep duration was associated with a decrease in mtDNA copy number of 0.06. Likewise, a 1% decrease in actigraphy-defined sleep efficiency was associated with a decrease in mtDNA copy number of 0.51. Reduced sleep duration and sleep efficiency were associated with reduced mitochondrial DNA copy number in sleep duration discordant monozygotic twins offering a potential mechanism whereby short sleep impairs health and longevity through mitochondrial stress. © 2015 Associated Professional Sleep Societies, LLC.

DNA replication stress restricts ribosomal DNA copy number.

PubMed

Salim, Devika; Bradford, William D; Freeland, Amy; Cady, Gillian; Wang, Jianmin; Pruitt, Steven C; Gerton, Jennifer L

2017-09-01

Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100-200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how "normal" copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a "normal" rDNA copy number.

AlleleCoder: a PERL script for coding codominant polymorphism data for PCA analysis

USDA-ARS?s Scientific Manuscript database

A useful biological interpretation of diploid heterozygotes is in terms of the dose of the common allele (0, 1 or 2 copies). We have developed a PERL script that converts FASTA files into coded spreadsheets suitable for Principal Component Analysis (PCA). In combination with R and R Commander, two- ...

Quantification of Plasmid Copy Number with Single Colour Droplet Digital PCR.

PubMed

Plotka, Magdalena; Wozniak, Mateusz; Kaczorowski, Tadeusz

2017-01-01

Bacteria can be considered as biological nanofactories that manufacture a cornucopia of bioproducts most notably recombinant proteins. As such, they must perfectly match with appropriate plasmid vectors to ensure successful overexpression of target genes. Among many parameters that correlate positively with protein productivity plasmid copy number plays pivotal role. Therefore, development of new and more accurate methods to assess this critical parameter will result in optimization of expression of plasmid-encoded genes. In this study, we present a simple and highly accurate method for quantifying plasmid copy number utilizing an EvaGreen single colour, droplet digital PCR. We demonstrate the effectiveness of this method by examining the copy number of the pBR322 vector within Escherichia coli DH5α cells. The obtained results were successfully validated by real-time PCR. However, we observed a strong dependency of the plasmid copy number on the method chosen for isolation of the total DNA. We found that application of silica-membrane-based columns for DNA purification or DNA isolation with use of bead-beating, a mechanical cell disruption lead to determination of an average of 20.5 or 7.3 plasmid copies per chromosome, respectively. We found that recovery of the chromosomal DNA from purification columns was less efficient than plasmid DNA (46.5 ± 1.9% and 87.4 ± 5.5%, respectively) which may lead to observed differences in plasmid copy number. Besides, the plasmid copy number variations dependent on DNA template isolation method, we found that droplet digital PCR is a very convenient method for measuring bacterial plasmid content. Careful determination of plasmid copy number is essential for better understanding and optimization of recombinant proteins production process. Droplet digital PCR is a very precise method that allows performing thousands of individual PCR reactions in a single tube. The ddPCR does not depend on running standard curves and is a straightforward and reliable method to quantify the plasmid copy number. Therefore we believe that the ddPCR designed in this study will be widely used for any plasmid copy number calculation in the future.

Quantification of Plasmid Copy Number with Single Colour Droplet Digital PCR

PubMed Central

Plotka, Magdalena; Wozniak, Mateusz; Kaczorowski, Tadeusz

2017-01-01

Bacteria can be considered as biological nanofactories that manufacture a cornucopia of bioproducts most notably recombinant proteins. As such, they must perfectly match with appropriate plasmid vectors to ensure successful overexpression of target genes. Among many parameters that correlate positively with protein productivity plasmid copy number plays pivotal role. Therefore, development of new and more accurate methods to assess this critical parameter will result in optimization of expression of plasmid-encoded genes. In this study, we present a simple and highly accurate method for quantifying plasmid copy number utilizing an EvaGreen single colour, droplet digital PCR. We demonstrate the effectiveness of this method by examining the copy number of the pBR322 vector within Escherichia coli DH5α cells. The obtained results were successfully validated by real-time PCR. However, we observed a strong dependency of the plasmid copy number on the method chosen for isolation of the total DNA. We found that application of silica-membrane-based columns for DNA purification or DNA isolation with use of bead-beating, a mechanical cell disruption lead to determination of an average of 20.5 or 7.3 plasmid copies per chromosome, respectively. We found that recovery of the chromosomal DNA from purification columns was less efficient than plasmid DNA (46.5 ± 1.9% and 87.4 ± 5.5%, respectively) which may lead to observed differences in plasmid copy number. Besides, the plasmid copy number variations dependent on DNA template isolation method, we found that droplet digital PCR is a very convenient method for measuring bacterial plasmid content. Careful determination of plasmid copy number is essential for better understanding and optimization of recombinant proteins production process. Droplet digital PCR is a very precise method that allows performing thousands of individual PCR reactions in a single tube. The ddPCR does not depend on running standard curves and is a straightforward and reliable method to quantify the plasmid copy number. Therefore we believe that the ddPCR designed in this study will be widely used for any plasmid copy number calculation in the future. PMID:28085908

Polymorphisms in carcinogen metabolism enzymes, fish intake, and risk of prostate cancer

PubMed Central

Stern, Mariana C.

2012-01-01

Cooking fish at high temperature can produce potent carcinogens such as heterocyclic amines and polycyclic aromatic hydrocarbons. The effects of these carcinogens may undergo modification by the enzymes responsible for their detoxification and/or activation. In this study, we investigated genetic polymorphisms in nine carcinogen metabolism enzymes and their modifying effects on the association between white or dark fish consumption and prostate cancer (PCA) risk. We genotyped 497 localized and 936 advanced PCA cases and 760 controls from the California Collaborative Case–Control Study of Prostate Cancer. Three polymorphisms, EPHX1 Tyr113His, CYP1B1 Leu432Val and GSTT1 null/present, were associated with localized PCA risk. The PTGS2 765 G/C polymorphism modified the association between white fish consumption and advanced PCA risk (interaction P 5 0.002), with high white fish consumption being positively associated with risk only among carriers of the C allele. This effect modification by PTGS2 genotype was stronger when restricted to consumption of well-done white fish (interaction P 5 0.021). These findings support the hypotheses that changes in white fish brought upon by high-temperature cooking methods, such as carcinogen accumulation and/or fatty acid composition changes, may contribute to prostate carcinogenesis. However, the gene–diet interactions should be interpreted with caution given the limited sample size. Thus, our findings require further validation with additional studies. Abbreviations: AA African American; BMI body mass index; CI confidence interval; CNV copy number variant; EPIC European Prospective Investigation into Cancer and Nutrition; HCA heterocyclic amine; HCFA Health Care Financing Administration; LAC Los Angeles county; MAF minor allele frequency; NHW non-Hispanic White; OR odds ratio; PAH polycyclic aromatic hydrocarbon; PCA prostate cancer; PTGS2 prostaglandin- endoperoxide synthase 2; PUFA polyunsaturated fatty acids; RDD random-digit dialing; SEER Surveillance, Epidemiology, and End Result; SES socio-economic status; SFBA San Francisco Bay Area; SNP single-nucleotide polymorphism PMID:22610071

A comparative molecular characterization of AMDV strains isolated from cases of clinical and subclinical infection.

PubMed

Kowalczyk, Marek; Jakubczak, Andrzej; Horecka, Beata; Kostro, Krzysztof

2018-05-29

The Aleutian mink disease virus (AMDV) is one of the most serious threats to modern mink breeding. The disease can have various courses, from progressive to subclinical infections. The objective of the study was to provide a comparative molecular characterization of isolates of AMDV from farms with a clinical and subclinical course of the disease. The qPCR analysis showed a difference of two orders of magnitude between the number of copies of the viral DNA on the farm with the clinical course of the disease (10 5 ) and the farm with the subclinical course (10 3 ). The sequencing results confirm a high level of homogeneity within each farm and variation between them. The phylogenetic analysis indicates that the variants belonging to different farms are closely related and occupy different branches of the same clade. The in silico analysis of the effect of differences in the sequence encoding the VP2 protein between the farms revealed no effect of the polymorphism on its functionality. The close phylogenetic relationship between the isolates from the two farms, the synonymous nature of most of the polymorphisms and the potentially minor effect on the functionality of the protein indicate that the differences in the clinical picture may be due not only to polymorphisms in the nucleotide and amino acid sequences, but also to the stage of infection on the farm and the degree of stabilization of the pathogen-host relationship.

Understanding the role of epigenomic, genomic and genetic alterations in the development of endometriosis (review).

PubMed

Kobayashi, Hiroshi; Imanaka, Shogo; Nakamura, Haruki; Tsuji, Ayumi

2014-05-01

Endometriosis is a complex disease influenced by genetic, epigenetic and environmental factors. The aim of the present study was to describe genomic instability, genetic polymorphisms and their haplotype, epigenetic alterations associated with predisposition to endometriosis, and the key factors associated with endometriosis-related ovarian neoplasms. Focus has been given on the developing paradigm that epigenetic alterations or genetic mutations in endometriosis may start in utero or in adolescent and young adults. A search was conducted between 1966 and 2010 through the English language literature (online Medline PubMed database) using the keywords endometriosis combined with epigenetic, genetic and environment. Genetic/epigenetic alterations include single‑nucleotide polymorphisms (SNPs), copy number variation, loss of heterozygosity (LOH), and promoter methylation. Several genes with genetic polymorphisms analyzed in the present study tended to overlap previously reported endometriosis susceptibility genes. Retrograde menstruation leads to iron overload, which facilitates the accumulation of somatic mutations through Fenton reaction-mediated oxidative stress. The epigenetic disruption of gene expression plays an important role in the development of endometriosis through interaction with environmental changes. There seems to be at least three spatiotemporally distinct phases of the development of endometriosis: the initial phase of genetic background inherited from parents; followed by epigenetic modifications in the female offspring; and iron overload, which is subject to dynamic modulation later in life. In conclusion, the marked regulation of endometriosis susceptibility genes may stem from a mechanism responsible for epigenetic and genetic mutations based on the microenvironmental changes.

Detection of mandarin in orange juice by single-nucleotide polymorphism qPCR assay.

PubMed

Aldeguer, Miriam; López-Andreo, María; Gabaldón, José A; Puyet, Antonio

2014-02-15

A dual-probe real time PCR (qPCR) DNA-based analysis was devised for the identification of mandarin in orange juice. A single nucleotide polymorphism at the trnL-trnF intergenic region of the chloroplast chromosome was confirmed in nine orange (Citrus sinensis) and thirteen commercial varieties of mandarin, including Citrus reticulata and Citrus unshiu species and a mandarin × tangelo hybrid. Two short minor-groove binding fluorescent probes targeting the polymorphic sequence were used in the dual-probe qPCR, which allowed the detection of both species in single-tube reactions. The similarity of PCR efficiencies allowed a simple estimation of the ratio mandarin/orange in the juice samples, which correlated to the measured difference of threshold cycle values for both probes. The limit of detection of the assay was 5% of mandarin in orange juice, both when the juice was freshly prepared (not from concentrate) or reconstituted from concentrate, which would allow the detection of fraudulently added mandarin juice. The possible use of the dual-probe system for quantitative measurements was also tested on fruit juice mixtures. qPCR data obtained from samples containing equal amounts of mandarin and orange juice revealed that the mandarin target copy number was approximately 2.6-fold higher than in orange juice. The use of a matrix-adapted control as calibrator to compensate the resulting C(T) bias allowed accurate quantitative measurements to be obtained. Copyright © 2013 Elsevier Ltd. All rights reserved.

Genome comparison of two Magnaporthe oryzae field isolates reveals genome variations and potential virulence effectors

PubMed Central

2013-01-01

Background Rice blast caused by the fungus Magnaporthe oryzae is an important disease in virtually every rice growing region of the world, which leads to significant annual decreases of grain quality and yield. To prevent disease, resistance genes in rice have been cloned and introduced into susceptible cultivars. However, introduced resistance can often be broken within few years of release, often due to mutation of cognate avirulence genes in fungal field populations. Results To better understand the pattern of mutation of M. oryzae field isolates under natural selection forces, we used a next generation sequencing approach to analyze the genomes of two field isolates FJ81278 and HN19311, as well as the transcriptome of FJ81278. By comparing the de novo genome assemblies of the two isolates against the finished reference strain 70–15, we identified extensive polymorphisms including unique genes, SNPs (single nucleotide polymorphism) and indels, structural variations, copy number variations, and loci under strong positive selection. The 1.75 MB of isolate-specific genome content carrying 118 novel genes from FJ81278, and 0.83 MB from HN19311 were also identified. By analyzing secreted proteins carrying polymorphisms, in total 256 candidate virulence effectors were found and 6 were chosen for functional characterization. Conclusions We provide results from genome comparison analysis showing extensive genome variation, and generated a list of M. oryzae candidate virulence effectors for functional characterization. PMID:24341723

aCGH Local Copy Number Aberrations Associated with Overall Copy Number Genomic Instability in Colorectal Cancer: Coordinate Involvement of the Regions Including BCR and ABL

PubMed Central

Bartos, Jeremy D.; Gaile, Daniel P.; McQuaid, Devin E.; Conroy, Jeffrey M.; Darbary, Huferesh; Nowak, Norma J.; Block, Annemarie; Petrelli, Nicholas J.; Mittelman, Arnold; Stoler, Daniel L.; Anderson, Garth R.

2007-01-01

In order to identify small regions of the genome whose specific copy number alteration is associated with high genomic instability in the form of overall genome-wide copy number aberrations, we have analyzed array-based comparative genomic hybridization (aCGH) data from 33 sporadic colorectal carcinomas. Copy number changes of a small number of specific regions were significantly correlated with elevated overall amplifications and deletions scattered throughout the entire genome. One significant region at 9q34 includes the c-ABL gene Another region spanning 22q11–13 includes the breakpoint cluster region (BCR) of the Philadelphia chromosome Coordinate 22q11–13 alterations were observed in nine of eleven tumors with the 9q34 alteration Additional regions on 1q and 14q were associated with overall genome-wide copy number changes, while copy number aberrations on chromosome 7p, 7q, and 13q21.1–31.3 were found associated with this instability only in tumors from patients with a smoking history Our analysis demonstrates there are a small number of regions of the genome where gain or loss is commonly associated with a tumor’s overall level of copy number aberrations Our finding BCR and ABL located within two of the instability-associated regions, and the involvement of these two regions occurring coordinately, suggests a system akin to the BCR-ABL translocation of CML may be involved in genomic instability in about one-third of human colorectal carcinomas. PMID:17196995

Negative Enrichment and Isolation of Circulating Tumor Cells for Whole Genome Amplification.

PubMed

Kanwar, Nisha; Done, Susan J

2017-01-01

Circulating tumor cells (CTCs) are a rare population of cells found in the peripheral blood of patients with many types of cancer such as breast, prostate, colon, and lung cancers. Higher numbers of these cells in blood are associated with a poorer prognosis of patients. Genomic profiling of CTCs would help characterize markers specific for the identification of these cells in blood, and also define genomic alterations that give these cells a metastatic advantage over other cells in the primary tumor. Here, we describe an immunomagnetic method to enrich CTCs from the blood of patients with breast cancer, followed by single-cell laser capture microdissection to isolate single CTCs. Whole genome amplification of isolated CTCs allows for many downstream applications to be performed to aide in their characterization, such as whole genome or exome sequencing, Single Nucleotide Polymorphism (SNP) and copy number analysis, and targeted sequencing or quantitative Polymerase Chain Reaction (qPCR) for genomic analyses.

PGen: large-scale genomic variations analysis workflow and browser in SoyKB.

PubMed

Liu, Yang; Khan, Saad M; Wang, Juexin; Rynge, Mats; Zhang, Yuanxun; Zeng, Shuai; Chen, Shiyuan; Maldonado Dos Santos, Joao V; Valliyodan, Babu; Calyam, Prasad P; Merchant, Nirav; Nguyen, Henry T; Xu, Dong; Joshi, Trupti

2016-10-06

With the advances in next-generation sequencing (NGS) technology and significant reductions in sequencing costs, it is now possible to sequence large collections of germplasm in crops for detecting genome-scale genetic variations and to apply the knowledge towards improvements in traits. To efficiently facilitate large-scale NGS resequencing data analysis of genomic variations, we have developed "PGen", an integrated and optimized workflow using the Extreme Science and Engineering Discovery Environment (XSEDE) high-performance computing (HPC) virtual system, iPlant cloud data storage resources and Pegasus workflow management system (Pegasus-WMS). The workflow allows users to identify single nucleotide polymorphisms (SNPs) and insertion-deletions (indels), perform SNP annotations and conduct copy number variation analyses on multiple resequencing datasets in a user-friendly and seamless way. We have developed both a Linux version in GitHub ( https://github.com/pegasus-isi/PGen-GenomicVariations-Workflow ) and a web-based implementation of the PGen workflow integrated within the Soybean Knowledge Base (SoyKB), ( http://soykb.org/Pegasus/index.php ). Using PGen, we identified 10,218,140 single-nucleotide polymorphisms (SNPs) and 1,398,982 indels from analysis of 106 soybean lines sequenced at 15X coverage. 297,245 non-synonymous SNPs and 3330 copy number variation (CNV) regions were identified from this analysis. SNPs identified using PGen from additional soybean resequencing projects adding to 500+ soybean germplasm lines in total have been integrated. These SNPs are being utilized for trait improvement using genotype to phenotype prediction approaches developed in-house. In order to browse and access NGS data easily, we have also developed an NGS resequencing data browser ( http://soykb.org/NGS_Resequence/NGS_index.php ) within SoyKB to provide easy access to SNP and downstream analysis results for soybean researchers. PGen workflow has been optimized for the most efficient analysis of soybean data using thorough testing and validation. This research serves as an example of best practices for development of genomics data analysis workflows by integrating remote HPC resources and efficient data management with ease of use for biological users. PGen workflow can also be easily customized for analysis of data in other species.

The Transposon Galileo Generates Natural Chromosomal Inversions in Drosophila by Ectopic Recombination

PubMed Central

Delprat, Alejandra; Ruiz, Alfredo

2009-01-01

Background Transposable elements (TEs) are responsible for the generation of chromosomal inversions in several groups of organisms. However, in Drosophila and other Dipterans, where inversions are abundant both as intraspecific polymorphisms and interspecific fixed differences, the evidence for a role of TEs is scarce. Previous work revealed that the transposon Galileo was involved in the generation of two polymorphic inversions of Drosophila buzzatii. Methodology/Principal Findings To assess the impact of TEs in Drosophila chromosomal evolution and shed light on the mechanism involved, we isolated and sequenced the two breakpoints of another widespread polymorphic inversion from D. buzzatii, 2z 3. In the non inverted chromosome, the 2z 3 distal breakpoint was located between genes CG2046 and CG10326 whereas the proximal breakpoint lies between two novel genes that we have named Dlh and Mdp. In the inverted chromosome, the analysis of the breakpoint sequences revealed relatively large insertions (2,870-bp and 4,786-bp long) including two copies of the transposon Galileo (subfamily Newton), one at each breakpoint, plus several other TEs. The two Galileo copies: (i) are inserted in opposite orientation; (ii) present exchanged target site duplications; and (iii) are both chimeric. Conclusions/Significance Our observations provide the best evidence gathered so far for the role of TEs in the generation of Drosophila inversions. In addition, they show unequivocally that ectopic recombination is the causative mechanism. The fact that the three polymorphic D. buzzatii inversions investigated so far were generated by the same transposon family is remarkable and is conceivably due to Galileo's unusual structure and current (or recent) transpositional activity. PMID:19936241

Hacking DNA copy number for circuit engineering.

PubMed

Wu, Feilun; You, Lingchong

2017-07-27

DNA copy number represents an essential parameter in the dynamics of synthetic gene circuits but typically is not explicitly considered. A new study demonstrates how dynamic control of DNA copy number can serve as an effective strategy to program robust oscillations in gene expression circuits.

22 CFR 1429.25 - Number of copies.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 22 Foreign Relations 2 2010-04-01 2010-04-01 true Number of copies. 1429.25 Section 1429.25 Foreign Relations FOREIGN SERVICE LABOR RELATIONS BOARD; FEDERAL LABOR RELATIONS AUTHORITY; GENERAL... AND GENERAL REQUIREMENTS General Requirements § 1429.25 Number of copies. Unless otherwise provided by...

DNA copy number changes define spatial patterns of heterogeneity in colorectal cancer

PubMed Central

Mamlouk, Soulafa; Childs, Liam Harold; Aust, Daniela; Heim, Daniel; Melching, Friederike; Oliveira, Cristiano; Wolf, Thomas; Durek, Pawel; Schumacher, Dirk; Bläker, Hendrik; von Winterfeld, Moritz; Gastl, Bastian; Möhr, Kerstin; Menne, Andrea; Zeugner, Silke; Redmer, Torben; Lenze, Dido; Tierling, Sascha; Möbs, Markus; Weichert, Wilko; Folprecht, Gunnar; Blanc, Eric; Beule, Dieter; Schäfer, Reinhold; Morkel, Markus; Klauschen, Frederick; Leser, Ulf; Sers, Christine

2017-01-01

Genetic heterogeneity between and within tumours is a major factor determining cancer progression and therapy response. Here we examined DNA sequence and DNA copy-number heterogeneity in colorectal cancer (CRC) by targeted high-depth sequencing of 100 most frequently altered genes. In 97 samples, with primary tumours and matched metastases from 27 patients, we observe inter-tumour concordance for coding mutations; in contrast, gene copy numbers are highly discordant between primary tumours and metastases as validated by fluorescent in situ hybridization. To further investigate intra-tumour heterogeneity, we dissected a single tumour into 68 spatially defined samples and sequenced them separately. We identify evenly distributed coding mutations in APC and TP53 in all tumour areas, yet highly variable gene copy numbers in numerous genes. 3D morpho-molecular reconstruction reveals two clusters with divergent copy number aberrations along the proximal–distal axis indicating that DNA copy number variations are a major source of tumour heterogeneity in CRC. PMID:28120820

Transmission of H7N9 Influenza Viruses with a Polymorphism at PB2 Residue 627 in Chickens and Ferrets.

PubMed

Luk, Geraldine S M; Leung, Connie Y H; Sia, Sin Fun; Choy, Ka-Tim; Zhou, Jie; Ho, Candy C K; Cheung, Peter P H; Lee, Elaine F; Wai, Chris K L; Li, Pamela C H; Ip, Sin-Ming; Poon, Leo L M; Lindsley, William G; Peiris, Malik; Yen, Hui-Ling

2015-10-01

Poultry exposure is a major risk factor for human H7N9 zoonotic infections, for which the mode of transmission remains unclear. We studied the transmission of genetically related poultry and human H7N9 influenza viruses differing by four amino acids, including the host determinant PB2 residue 627. A/Silkie chicken/HK/1772/2014 (SCk1772) and A/HK/3263/14 (HK3263) replicated to comparable titers in chickens, with superior oropharyngeal over cloacal shedding; both viruses transmitted efficiently among chickens via direct contact but inefficiently via the airborne route. Interspecies transmission via the airborne route was observed for ferrets exposed to the SCk1772- or HK3263-infected chickens, while low numbers of copies of influenza viral genome were detected in the air, predominantly at particle sizes larger than 4 μm. In ferrets, the human isolate HK3263 replicated to higher titers and transmitted more efficiently via direct contact than SCk1772. We monitored "intrahost" and "interhost" adaptive changes at PB2 residue 627 during infection and transmission of the Sck1772 that carried E627 and HK3263 that carried V/K/E polymorphism at 60%, 20%, and 20%, respectively. For SCk1772, positive selection for K627 over E627 was observed in ferrets during the chicken-to-ferret or ferret-to-ferret transmission. For HK3263 that contained V/K/E polymorphism, mixed V627 and E627 genotypes were transmitted among chickens while either V627 or K627 was transmitted to ferrets with a narrow transmission bottleneck. Overall, our results suggest direct contact as the main mode for H7N9 transmission and identify the PB2-V627 genotype with uncompromised fitness and transmissibility in both avian and mammalian species. We studied the modes of H7N9 transmission, as this information is crucial for developing effective control measures for prevention. Using chicken (SCk1772) and human (HK3263) H7N9 isolates that differed by four amino acids, including the host determinant PB2 residue 627, we observed that both viruses transmitted efficiently among chickens via direct contact but inefficiently via the airborne route. Chicken-to-ferret transmission via the airborne route was observed, along with the detection of viral genome in the air at low copy numbers. In ferrets, HK3263 transmitted more efficiently than SCk1772 via direct contact. During the transmission of SCk1772 that contained E and HK3263 that contained V/K/E polymorphism at PB2 residue 627, positive selections of E627 and K627 were observed in chickens and ferrets, respectively. In addition, PB2-V627 was transmitted and stably maintained in both avian and mammalian species. Our results support applying intervention strategies that minimize direct and indirect contact at the poultry markets during epidemics. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Transmission of H7N9 Influenza Viruses with a Polymorphism at PB2 Residue 627 in Chickens and Ferrets

PubMed Central

Luk, Geraldine S. M.; Leung, Connie Y. H.; Sia, Sin Fun; Choy, Ka-Tim; Zhou, Jie; Ho, Candy C. K.; Cheung, Peter P. H.; Lee, Elaine F.; Wai, Chris K. L.; Li, Pamela C. H.; Ip, Sin-Ming; Poon, Leo L. M.; Lindsley, William G.

2015-01-01

ABSTRACT Poultry exposure is a major risk factor for human H7N9 zoonotic infections, for which the mode of transmission remains unclear. We studied the transmission of genetically related poultry and human H7N9 influenza viruses differing by four amino acids, including the host determinant PB2 residue 627. A/Silkie chicken/HK/1772/2014 (SCk1772) and A/HK/3263/14 (HK3263) replicated to comparable titers in chickens, with superior oropharyngeal over cloacal shedding; both viruses transmitted efficiently among chickens via direct contact but inefficiently via the airborne route. Interspecies transmission via the airborne route was observed for ferrets exposed to the SCk1772- or HK3263-infected chickens, while low numbers of copies of influenza viral genome were detected in the air, predominantly at particle sizes larger than 4 μm. In ferrets, the human isolate HK3263 replicated to higher titers and transmitted more efficiently via direct contact than SCk1772. We monitored “intrahost” and “interhost” adaptive changes at PB2 residue 627 during infection and transmission of the Sck1772 that carried E627 and HK3263 that carried V/K/E polymorphism at 60%, 20%, and 20%, respectively. For SCk1772, positive selection for K627 over E627 was observed in ferrets during the chicken-to-ferret or ferret-to-ferret transmission. For HK3263 that contained V/K/E polymorphism, mixed V627 and E627 genotypes were transmitted among chickens while either V627 or K627 was transmitted to ferrets with a narrow transmission bottleneck. Overall, our results suggest direct contact as the main mode for H7N9 transmission and identify the PB2-V627 genotype with uncompromised fitness and transmissibility in both avian and mammalian species. IMPORTANCE We studied the modes of H7N9 transmission, as this information is crucial for developing effective control measures for prevention. Using chicken (SCk1772) and human (HK3263) H7N9 isolates that differed by four amino acids, including the host determinant PB2 residue 627, we observed that both viruses transmitted efficiently among chickens via direct contact but inefficiently via the airborne route. Chicken-to-ferret transmission via the airborne route was observed, along with the detection of viral genome in the air at low copy numbers. In ferrets, HK3263 transmitted more efficiently than SCk1772 via direct contact. During the transmission of SCk1772 that contained E and HK3263 that contained V/K/E polymorphism at PB2 residue 627, positive selections of E627 and K627 were observed in chickens and ferrets, respectively. In addition, PB2-V627 was transmitted and stably maintained in both avian and mammalian species. Our results support applying intervention strategies that minimize direct and indirect contact at the poultry markets during epidemics. PMID:26202239

[Relationship between mitochondrial DNA copy number, membrane potential of human embryo and embryo morphology].

PubMed

Zhao, H; Teng, X M; Li, Y F

2017-11-25

Objective: To explore the relationship between the embryo with the different morphological types in the third day and its mitochondrial copy number, the membrane potential. Methods: Totally 117 embryos with poor development after normal fertilization and were not suitable transferred in the fresh cycle and 106 frozen embryos that were discarded voluntarily by infertility patients with in vitro fertilization-embryo transfer after successful pregnancy were selected. According to evaluation of international standard in embryos, all cleavage stage embryos were divided into class Ⅰ frozen embryo group ( n= 64), class Ⅱ frozen embryo group ( n= 42) and class Ⅲ fresh embryonic group (not transplanted embryos; n= 117). Real-time PCR and confocal microscopy methods were used to detect mitochondrial DNA (mtDNA) copy number and the mitochondrial membrane potential of a single embryo. The differences between embryo quality and mtDNA copy number and membrane potential of each group were compared. Results: The copy number of mtDNA and the mitochondrial membrane potential in class Ⅲ fresh embryonic group [(1.7±1.0)×10(5) copy/μl, 1.56±0.32] were significantly lower than those in class Ⅰ frozen embryo group [(3.4±1.7)×10(5) copy/μl, 2.66±0.21] and class Ⅱ frozen embryo group [(2.6±1.2)×10(5) copy/μl, 1.80±0.32; all P< 0.05]. The copy number of mtDNA and the mitochondrial membrane potential in classⅠ frozen embryo group were significantly higher than those in classⅡ frozen embryo group (both P< 0.05). Conclusion: The mtDNA copy number and the mitochondrial membrane potential of embryos of the better quality embryo are higher.

MC3R gene polymorphisms are associated with early childhood adiposity gain and infant appetite in an Asian population.

PubMed

Aris, I M; Tint, M T; Teh, A L; Holbrook, J D; Quah, P L; Chong, M F-F; Lin, X; Soh, S E; Saw, S-M; Kwek, K; Godfrey, K M; Gluckman, P D; Chong, Y S; Lek, N; Yap, F; Lee, Y S

2016-12-01

Polymorphic variants within human melanocortin-3 receptor gene (MC3R) gene have been associated with obesity. However, its influence on infancy and early childhood adiposity has not been reported before. We assessed associations between genotype at polymorphic sites within MC3R with early childhood adiposity and interaction with early childhood appetitive traits. We studied 1090 singletons in an Asian mother-offspring cohort genotyped for MC3R and in a subgroup (n = 422) who had completed Child Eating Behaviour Questionnaires (CEBQ) at 12 months. Children were followed from birth to 48 months, and up to 10 measurements of body mass index and five measures of triceps and subscapular skin-folds were obtained. Independent of potential confounders, each additional MC3R minor allele copy was associated with greater body mass index standard deviation score [B{95% confidence interval}: 0.004 units/month {0.001,0.007}; p = 0.007], triceps [0.009 mm/month {0.001,0.02}; p = 0.021] and subscapular skin-fold [0.008 mm/month {0.002,0.01}; p = 0.011] gain velocity in the first 48 months. Each additional MC3R minor allele copy was also associated with increased odds of overweight [odds ratio {95% confidence interval}: 1.48{1.17-1.88}] and obesity [1.58{1.10-2.28}] in the first 48 months. Every additional copy of MC3R minor allele was positively associated with 'slowness-in-eating' appetitive trait [0.24{0.06,0.39}, p = 0.006]; however, the relationship between 'slowness-in-eating' with adiposity gain was not statistically significant. Our findings support the role of MC3R genetic variants in adiposity gain during early childhood. © 2015 The Authors. Pediatric Obesity published by John Wiley & Sons Ltd on behalf of World Obesity Federation.

14 CFR 221.92 - Number of copies required.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 14 Aeronautics and Space 4 2014-01-01 2014-01-01 false Number of copies required. 221.92 Section 221.92 Aeronautics and Space OFFICE OF THE SECRETARY, DEPARTMENT OF TRANSPORTATION (AVIATION PROCEEDINGS) ECONOMIC REGULATIONS TARIFFS Filing Tariff Publications With Department § 221.92 Number of copies...

The CAG polymorphism in androgen receptor (AR) gene impacts the moral permissibility of harmful behavior in females.

PubMed

Gong, Pingyuan; Fang, Pengpeng; Yang, Xing; Ru, Wenzhao; Wang, Bei; Gao, Xiaocai; Liu, Jinting

2017-06-01

The moral permissibility of harm is strikingly varied among individuals. In light of the connection between testosterone levels and utilitarian moral judgment, this study examined to what extent a CAG polymorphism in the androgen receptor gene, a genetic polymorphism with the ability to regulate testosterone function, contributes to individual differences in moral judgment. Four hundred and thirty-nine Chinese Han participants completed permissibility ratings of harm in moral dilemmas and moral transgression scenarios. Results showed a significant association between the CAG polymorphism and moral permissibility of harm in females. Females with more copies of the S allele, which is associated with higher availability of testosterone, were more likely to judge harmful utilitarian acts and unintentionally harmful acts as permissible, while these effects were absent in males. The findings provide the first evidence for a link between the androgen receptor gene and moral judgment and highlight the role of androgens in moral foundations. Copyright © 2017 Elsevier Ltd. All rights reserved.

Extensive Copy Number Variation in Fermentation-Related Genes Among Saccharomyces cerevisiae Wine Strains.

PubMed

Steenwyk, Jacob; Rokas, Antonis

2017-05-05

Due to the importance of Saccharomyces cerevisiae in wine-making, the genomic variation of wine yeast strains has been extensively studied. One of the major insights stemming from these studies is that wine yeast strains harbor low levels of genetic diversity in the form of single nucleotide polymorphisms (SNPs). Genomic structural variants, such as copy number (CN) variants, are another major type of variation segregating in natural populations. To test whether genetic diversity in CN variation is also low across wine yeast strains, we examined genome-wide levels of CN variation in 132 whole-genome sequences of S. cerevisiae wine strains. We found an average of 97.8 CN variable regions (CNVRs) affecting ∼4% of the genome per strain. Using two different measures of CN diversity, we found that gene families involved in fermentation-related processes such as copper resistance ( CUP ), flocculation ( FLO ), and glucose metabolism ( HXT ), as well as the SNO gene family whose members are expressed before or during the diauxic shift, showed substantial CN diversity across the 132 strains examined. Importantly, these same gene families have been shown, through comparative transcriptomic and functional assays, to be associated with adaptation to the wine fermentation environment. Our results suggest that CN variation is a substantial contributor to the genomic diversity of wine yeast strains, and identify several candidate loci whose levels of CN variation may affect the adaptation and performance of wine yeast strains during fermentation. Copyright © 2017 Steenwyk and Rokas.

Complex and multi-allelic copy number variation in human disease

PubMed Central

McCarroll, Steven A.

2015-01-01

Hundreds of copy number variants are complex and multi-allelic, in that they have many structural alleles and have rearranged multiple times in the ancestors who contributed chromosomes to current humans. Not only are the relationships of these multi-allelic CNVs (mCNVs) to phenotypes generally unknown, but many mCNVs have not yet been described at the basic levels—alleles, allele frequencies, structural features—that support genetic investigation. To date, most reported disease associations to these variants have been ascertained through candidate gene studies. However, only a few associations have reached the level of acceptance defined by durable replications in many cohorts. This likely stems from longstanding challenges in making precise molecular measurements of the alleles individuals have at these loci. However, approaches for mCNV analysis are improving quickly, and some of the unique characteristics of mCNVs may assist future association studies. Their various structural alleles are likely to have different magnitudes of effect, creating a natural allelic series of growing phenotypic impact and giving investigators a set of natural predictions and testable hypotheses about the extent to which each allele of an mCNV predisposes to a phenotype. Also, mCNVs’ low-to-modest correlation to individual single-nucleotide polymorphisms (SNPs) may make it easier to distinguish between mCNVs and nearby SNPs as the drivers of an association signal, and perhaps, make it possible to preliminarily screen candidate loci, or the entire genome, for the many mCNV–disease relationships that remain to be discovered. PMID:26163405

Clonal evolution and clinical significance of copy number neutral loss of heterozygosity of chromosome arm 6p in acquired aplastic anemia.

PubMed

Betensky, Marisol; Babushok, Daria; Roth, Jacquelyn J; Mason, Philip J; Biegel, Jaclyn A; Busse, Tracy M; Li, Yimei; Lind, Curt; Papazoglou, Anna; Monos, Dimitri; Podsakoff, Gregory; Bessler, Monica; Olson, Timothy S

2016-01-01

Acquired aplastic anemia (aAA) results from the T cell-mediated autoimmune destruction of hematopoietic stem cells. Factors predicting response to immune suppression therapy (IST) or development of myelodysplastic syndrome (MDS) are beginning to be elucidated. Our recent data suggest most patients with aAA treated with IST develop clonal somatic genetic alterations in hematopoietic cells. One frequent acquired abnormality is copy-number neutral loss of heterozygosity on chromosome 6p (6p CN-LOH) involving the human leukocyte antigen (HLA) locus. We hypothesized that because 6p CN-LOH clones may arise from selective pressure to escape immune surveillance through deletion of HLA alleles, the development of 6p CN-LOH may affect response to IST. We used single nucleotide polymorphism array genotyping and targeted next-generation sequencing of HLA alleles to assess frequency of 6p CN-LOH, identity of HLA alleles lost through 6p CN-LOH, and impact of 6p CN-LOH on response to IST. 6p CN-LOH clones were present in 11.3% of patients, remained stable over time, and were not associated with development of MDS-defining cytogenetic abnormalities. Notably, no patient with 6p CN-LOH treated with IST achieved a complete response. In summary, clonal 6p CN-LOH in aAA defines a unique subgroup of patients that may provide insights into hematopoietic clonal evolution. Copyright © 2016 Elsevier Inc. All rights reserved.

Clonal Evolution and Clinical Significance of Copy Number Neutral Loss of Heterozygosity of Chromosome Arm 6p in Acquired Aplastic Anemia

PubMed Central

Betensky, Marisol; Babushok, Daria; Roth, Jacquelyn J.; Mason, Philip J; Biegel, Jaclyn A.; Busse, Tracy M; Li, Yimei; Lind, Curt; Papazoglou, Anna; Monos, Dimitri; Podsakoff, Gregory; Bessler, Monica; Olson, Timothy S.

2015-01-01

Acquired aplastic anemia (aAA) results from the T cell-mediated autoimmune destruction of hematopoietic stem cells. Factors predicting response to immune suppression therapy (IST) or development of myelodysplastic syndrome (MDS) are beginning to be elucidated. Our recent data suggest most patients with aAA treated with IST develop clonal somatic genetic alterations in hematopoietic cells. One frequent acquired abnormality is copy-number neutral loss of heterozygosity on chromosome 6p (6p CN-LOH) involving the human leukocyte antigen (HLA) locus. We hypothesized that because 6p CN-LOH clones may arise from selective pressure to escape immune surveillance through deletion of HLA alleles, the development of 6p CN-LOH may affect response to IST. We used single nucleotide polymorphism array genotyping and targeted next-generation sequencing of HLA alleles to assess frequency of 6p CN-LOH, identity of HLA alleles lost through 6p CN-LOH, and impact of 6p CN-LOH on response to IST. 6p CN-LOH clones were present in 11.3% of patients, remained stable over time, and were not associated with development of MDS-defining cytogenetic abnormalities. Notably, no patient with 6p CN-LOH treated with IST achieved a complete response. In summary, clonal 6p CN-LOH in aAA defines a unique subgroup of patients that may provide insights into hematopoietic clonal evolution. PMID:26702937

Copy Number Variations of TBK1 in Australian Patients With Primary Open-Angle Glaucoma

PubMed Central

AWADALLA, MONA S.; FINGERT, JOHN H.; ROOS, BENJAMIN E.; CHEN, SIMON; HOLMES, RICHARD; GRAHAM, STUART L.; CHEHADE, MARK; GALANOPOLOUS, ANNA; RIDGE, BRONWYN; SOUZEAU, EMMANUELLE; ZHOU, TIGER; SIGGS, OWEN M.; HEWITT, ALEX W.; MACKEY, DAVID A.; BURDON, KATHRYN P.; CRAIG, JAMIE E.

2015-01-01

PURPOSE To investigate the presence of TBK1 copy number variations in a large, well-characterized Australian cohort of patients with glaucoma comprising both normal-tension glaucoma and high-tension glaucoma cases. DESIGN A retrospective cohort study. METHODS DNA samples from patients with normal-tension glaucoma and high-tension glaucoma and unaffected controls were screened for TBK1 copy number variations using real-time quantitative polymerase chain reaction. Samples with additional copies of the TBK1 gene were further tested using custom comparative genomic hybridization arrays. RESULTS Four out of 334 normal-tension glaucoma cases (1.2%) were found to carry TBK1 copy number variations using quantitative polymerase chain reaction. One extra dose of the TBK1 gene (duplication) was detected in 3 normal-tension glaucoma patients, while 2 extra doses of the gene (triplication) were detected in a fourth normal-tension glaucoma patient. The results were further confirmed by custom comparative genomic hybridization arrays. Further, the TBK1 copy number variation segregated with normal-tension glaucoma in the family members of the probands, showing an autosomal dominant pattern of inheritance. No TBK1 copy number variations were detected in 1045 Australian patients with high-tension glaucoma or in 254 unaffected controls. CONCLUSION We report the presence of TBK1 copy number variations in our Australian normal-tension glaucoma cohort, including the first example of more than 1 extra copy of this gene in glaucoma patients (gene triplication). These results confirm TBK1 to be an important cause of normal-tension glaucoma, but do not suggest common involvement in high-tension glaucoma. PMID:25284765

Copy number rather than epigenetic alterations are the major dictator of imprinted methylation in tumors.

PubMed

Martin-Trujillo, Alex; Vidal, Enrique; Monteagudo-Sa Nchez, Ana; Sanchez-Delgado, Marta; Moran, Sebastian; Hernandez Mora, Jose Ramon; Heyn, Holger; Guitart, Miriam; Esteller, Manel; Monk, David

2017-09-07

It has been postulated that imprinting aberrations are common in tumors. To understand the role of imprinting in cancer, we have characterized copy-number and methylation in over 280 cancer cell lines and confirm our observations in primary tumors. Imprinted differentially methylated regions (DMRs) regulate parent-of-origin monoallelic expression of neighboring transcripts in cis. Unlike single-copy CpG islands that may be prone to hypermethylation, imprinted DMRs can either loose or gain methylation during tumorigenesis. Here, we show that methylation profiles at imprinted DMRs often not represent genuine epigenetic changes but simply the accumulation of underlying copy-number aberrations (CNAs), which is independent of the genome methylation state inferred from cancer susceptible loci. Our results reveal that CNAs also influence allelic expression as loci with copy-number neutral loss-of-heterozygosity or amplifications may be expressed from the appropriate parental chromosomes, which is indicative of maintained imprinting, although not observed as a single expression foci by RNA FISH.Altered genomic imprinting is frequently reported in cancer. Here, the authors analyze copy number and methylation in cancer cell lines and primary tumors to show that imprinted methylation profiles represent the accumulation of copy number alteration, rather than epigenetic alterations.

Genomic analysis of the chromosome 15q11-q13 Prader-Willi syndrome region and characterization of transcripts for GOLGA8E and WHCD1L1 from the proximal breakpoint region.

PubMed

Jiang, Yong-Hui; Wauki, Kekio; Liu, Qian; Bressler, Jan; Pan, Yanzhen; Kashork, Catherine D; Shaffer, Lisa G; Beaudet, Arthur L

2008-01-28

Prader-Willi syndrome (PWS) is a neurobehavioral disorder characterized by neonatal hypotonia, childhood obesity, dysmorphic features, hypogonadism, mental retardation, and behavioral problems. Although PWS is most often caused by a paternal interstitial deletion of a 6-Mb region of chromosome 15q11-q13, the identity of the exact protein coding or noncoding RNAs whose deficiency produces the PWS phenotype is uncertain. There are also reports describing a PWS-like phenotype in a subset of patients with full mutations in the FMR1 (fragile X mental retardation 1) gene. Taking advantage of the human genome sequence, we have performed extensive sequence analysis and molecular studies for the PWS candidate region. We have characterized transcripts for the first time for two UCSC Genome Browser predicted protein-coding genes, GOLGA8E (golgin subfamily a, 8E) and WHDC1L1 (WAS protein homology region containing 1-like 1) and have further characterized two previously reported genes, CYF1P1 and NIPA2; all four genes are in the region close to the proximal/centromeric deletion breakpoint (BP1). GOLGA8E belongs to the golgin subfamily of coiled-coil proteins associated with the Golgi apparatus. Six out of 16 golgin subfamily proteins in the human genome have been mapped in the chromosome 15q11-q13 and 15q24-q26 regions. We have also identified more than 38 copies of GOLGA8E-like sequence in the 15q11-q14 and 15q23-q26 regions which supports the presence of a GOLGA8E-associated low copy repeat (LCR). Analysis of the 15q11-q13 region by PFGE also revealed a polymorphic region between BP1 and BP2. WHDC1L1 is a novel gene with similarity to mouse Whdc1 (WAS protein homology region 2 domain containing 1) and human JMY protein (junction-mediating and regulatory protein). Expression analysis of cultured human cells and brain tissues from PWS patients indicates that CYFIP1 and NIPA2 are biallelically expressed. However, we were not able to determine the allele-specific expression pattern for GOLGA8E and WHDC1L1 because these two genes have highly related sequences that might also be expressed. We have presented an updated version of a sequence-based physical map for a complex chromosomal region, and we raise the possibility of polymorphism in the genomic orientation of the BP1 to BP2 region. The identification of two new proteins GOLGA8E and WHDC1L1 encoded by genes in the 15q11-q13 region may extend our understanding of the molecular basis of PWS. In terms of copy number variation and gene organization, this is one of the most polymorphic regions of the human genome, and perhaps the single most polymorphic region of this type.

Copy number variation of individual cattle genomes using next-generation sequencing

USDA-ARS?s Scientific Manuscript database

Copy number variations (CNVs) affect a wide range of phenotypic traits; however, CNVs in or near segmental duplication regions are often intractable. Using a read depth approach based on next-generation sequencing, we examined genome-wide copy number differences among five taurine (three Angus, one ...

Individualized cattle copy number and segmental duplication maps using next generation sequencing

USDA-ARS?s Scientific Manuscript database

Copy Number Variations (CNVs) affect a wide range of phenotypic traits; however, CNVs in or near segmental duplication regions are often intractable. Using a read depth approach based on next generation sequencing, we examined genome-wide copy number differences among five taurine (three Angus, one ...

Copy number variation of individual cattle genomes using next-generation sequencing

USDA-ARS?s Scientific Manuscript database

Copy Number Variations (CNVs) affect a wide range of phenotypic traits; however, CNVs in or near segmental duplication regions are often difficult to track. Using a read depth approach based on next generation sequencing, we examined genome-wide copy number differences among five taurine (three Angu...

40 CFR 761.209 - Number of copies of a manifest.

Code of Federal Regulations, 2014 CFR

2014-07-01

..., AND USE PROHIBITIONS PCB Waste Disposal Records and Reports § 761.209 Number of copies of a manifest... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Number of copies of a manifest. 761.209 Section 761.209 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) TOXIC...

40 CFR 761.209 - Number of copies of a manifest.

Code of Federal Regulations, 2013 CFR

2013-07-01

..., AND USE PROHIBITIONS PCB Waste Disposal Records and Reports § 761.209 Number of copies of a manifest... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Number of copies of a manifest. 761.209 Section 761.209 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) TOXIC...

Aluminum tolerance is associated with higher MATE1 gene copy-number in maize

USDA-ARS?s Scientific Manuscript database

Genome structure variation, including copy-number (CNV) and presence/absence variation (PAV), comprise a large extent of maize genetic diversity but their effect on phenotypes remains largely unexplored. Here we describe how copy-number variation in a major aluminum (Al) tolerance locus contributes ...

GeneCount: genome-wide calculation of absolute tumor DNA copy numbers from array comparative genomic hybridization data

PubMed Central

Lyng, Heidi; Lando, Malin; Brøvig, Runar S; Svendsrud, Debbie H; Johansen, Morten; Galteland, Eivind; Brustugun, Odd T; Meza-Zepeda, Leonardo A; Myklebost, Ola; Kristensen, Gunnar B; Hovig, Eivind; Stokke, Trond

2008-01-01

Absolute tumor DNA copy numbers can currently be achieved only on a single gene basis by using fluorescence in situ hybridization (FISH). We present GeneCount, a method for genome-wide calculation of absolute copy numbers from clinical array comparative genomic hybridization data. The tumor cell fraction is reliably estimated in the model. Data consistent with FISH results are achieved. We demonstrate significant improvements over existing methods for exploring gene dosages and intratumor copy number heterogeneity in cancers. PMID:18500990

Characterization of the Copy Number and Variants of Deformed Wing Virus (DWV) in the Pairs of Honey Bee Pupa and Infesting Varroa destructor or Tropilaelaps mercedesae.

PubMed

Wu, Yunfei; Dong, Xiaofeng; Kadowaki, Tatsuhiko

2017-01-01

Recent honey bee colony losses, particularly during the winter, have been shown to be associated with the presence of both ectoparasitic mites and Deformed Wing Virus (DWV). Whilst the role of Varroa destructor mites as a viral vector is well established, the role of Tropilaelaps mercedesae mites in viral transmission has not been fully investigated. In this study, we tested the effects that V. destructor and T. mercedesae infestation have on fluctuation of the DWV copy number and alteration of the virus variants in honey bees by characterizing individual pupae and their infesting mites. We observed that both mite species were associated with increased viral copy number in honey bee pupae. We found a positive correlation between DWV copy number in pupae and copy number in infesting mites, and the same DWV type A variant was present in either low or high copy number in both honey bee pupae and infesting V. destructor . These data also suggest that variant diversity is similar between honey bee pupae and the mites that infest them. These results support a previously proposed hypothesis that DWV suppresses the honey bee immune system when virus copy number reaches a specific threshold, promoting greater replication.

Characterization of the Copy Number and Variants of Deformed Wing Virus (DWV) in the Pairs of Honey Bee Pupa and Infesting Varroa destructor or Tropilaelaps mercedesae

PubMed Central

Wu, Yunfei; Dong, Xiaofeng; Kadowaki, Tatsuhiko

2017-01-01

Recent honey bee colony losses, particularly during the winter, have been shown to be associated with the presence of both ectoparasitic mites and Deformed Wing Virus (DWV). Whilst the role of Varroa destructor mites as a viral vector is well established, the role of Tropilaelaps mercedesae mites in viral transmission has not been fully investigated. In this study, we tested the effects that V. destructor and T. mercedesae infestation have on fluctuation of the DWV copy number and alteration of the virus variants in honey bees by characterizing individual pupae and their infesting mites. We observed that both mite species were associated with increased viral copy number in honey bee pupae. We found a positive correlation between DWV copy number in pupae and copy number in infesting mites, and the same DWV type A variant was present in either low or high copy number in both honey bee pupae and infesting V. destructor. These data also suggest that variant diversity is similar between honey bee pupae and the mites that infest them. These results support a previously proposed hypothesis that DWV suppresses the honey bee immune system when virus copy number reaches a specific threshold, promoting greater replication. PMID:28878743

Accurate, high-throughput typing of copy number variation using paralogue ratios from dispersed repeats

PubMed Central

Armour, John A. L.; Palla, Raquel; Zeeuwen, Patrick L. J. M.; den Heijer, Martin; Schalkwijk, Joost; Hollox, Edward J.

2007-01-01

Recent work has demonstrated an unexpected prevalence of copy number variation in the human genome, and has highlighted the part this variation may play in predisposition to common phenotypes. Some important genes vary in number over a high range (e.g. DEFB4, which commonly varies between two and seven copies), and have posed formidable technical challenges for accurate copy number typing, so that there are no simple, cheap, high-throughput approaches suitable for large-scale screening. We have developed a simple comparative PCR method based on dispersed repeat sequences, using a single pair of precisely designed primers to amplify products simultaneously from both test and reference loci, which are subsequently distinguished and quantified via internal sequence differences. We have validated the method for the measurement of copy number at DEFB4 by comparison of results from >800 DNA samples with copy number measurements by MAPH/REDVR, MLPA and array-CGH. The new Paralogue Ratio Test (PRT) method can require as little as 10 ng genomic DNA, appears to be comparable in accuracy to the other methods, and for the first time provides a rapid, simple and inexpensive method for copy number analysis, suitable for application to typing thousands of samples in large case-control association studies. PMID:17175532

Obesity, starch digestion and amylase: association between copy number variants at human salivary (AMY1) and pancreatic (AMY2) amylase genes

PubMed Central

Carpenter, Danielle; Dhar, Sugandha; Mitchell, Laura M.; Fu, Beiyuan; Tyson, Jess; Shwan, Nzar A.A.; Yang, Fengtang; Thomas, Mark G.; Armour, John A.L.

2015-01-01

The human salivary amylase genes display extensive copy number variation (CNV), and recent work has implicated this variation in adaptation to starch-rich diets, and in association with body mass index. In this work, we use paralogue ratio tests, microsatellite analysis, read depth and fibre-FISH to demonstrate that human amylase CNV is not a smooth continuum, but is instead partitioned into distinct haplotype classes. There is a fundamental structural distinction between haplotypes containing odd or even numbers of AMY1 gene units, in turn coupled to CNV in pancreatic amylase genes AMY2A and AMY2B. Most haplotypes have one copy each of AMY2A and AMY2B and contain an odd number of copies of AMY1; consequently, most individuals have an even total number of AMY1. In contrast, haplotypes carrying an even number of AMY1 genes have rearrangements leading to CNVs of AMY2A/AMY2B. Read-depth and experimental data show that different populations harbour different proportions of these basic haplotype classes. In Europeans, the copy numbers of AMY1 and AMY2A are correlated, so that phenotypic associations caused by variation in pancreatic amylase copy number could be detected indirectly as weak association with AMY1 copy number. We show that the quantitative polymerase chain reaction (qPCR) assay previously applied to the high-throughput measurement of AMY1 copy number is less accurate than the measures we use and that qPCR data in other studies have been further compromised by systematic miscalibration. Our results uncover new patterns in human amylase variation and imply a potential role for AMY2 CNV in functional associations. PMID:25788522

Obesity, starch digestion and amylase: association between copy number variants at human salivary (AMY1) and pancreatic (AMY2) amylase genes.

PubMed

Carpenter, Danielle; Dhar, Sugandha; Mitchell, Laura M; Fu, Beiyuan; Tyson, Jess; Shwan, Nzar A A; Yang, Fengtang; Thomas, Mark G; Armour, John A L

2015-06-15

The human salivary amylase genes display extensive copy number variation (CNV), and recent work has implicated this variation in adaptation to starch-rich diets, and in association with body mass index. In this work, we use paralogue ratio tests, microsatellite analysis, read depth and fibre-FISH to demonstrate that human amylase CNV is not a smooth continuum, but is instead partitioned into distinct haplotype classes. There is a fundamental structural distinction between haplotypes containing odd or even numbers of AMY1 gene units, in turn coupled to CNV in pancreatic amylase genes AMY2A and AMY2B. Most haplotypes have one copy each of AMY2A and AMY2B and contain an odd number of copies of AMY1; consequently, most individuals have an even total number of AMY1. In contrast, haplotypes carrying an even number of AMY1 genes have rearrangements leading to CNVs of AMY2A/AMY2B. Read-depth and experimental data show that different populations harbour different proportions of these basic haplotype classes. In Europeans, the copy numbers of AMY1 and AMY2A are correlated, so that phenotypic associations caused by variation in pancreatic amylase copy number could be detected indirectly as weak association with AMY1 copy number. We show that the quantitative polymerase chain reaction (qPCR) assay previously applied to the high-throughput measurement of AMY1 copy number is less accurate than the measures we use and that qPCR data in other studies have been further compromised by systematic miscalibration. Our results uncover new patterns in human amylase variation and imply a potential role for AMY2 CNV in functional associations. © The Author 2015. Published by Oxford University Press.

14 CFR 221.92 - Number of copies required.

Code of Federal Regulations, 2010 CFR

2010-01-01

... PROCEEDINGS) ECONOMIC REGULATIONS TARIFFS Filing Tariff Publications With Department § 221.92 Number of copies required. Two copies of each paper tariff, tariff revision and adoption notice to be filed shall be sent to...

[Copy number variation of trinucleotide repeat in dynamic mutation sites of autosomal dominant cerebellar ataxias related genes].

PubMed

Chen, Pu; Ma, Mingyi; Shang, Huifang; Su, Dan; Zhang, Sizhong; Yang, Yuan

2009-12-01

To standardize the experimental procedure of the gene test for autosomal dominant cerebellar ataxias (ADCA), and provide the basis for quantitative criteria of the dynamic mutation of spinocerebellar ataxia (SCA) genes in Chinese population. Genotyping of the dynamic mutation loci of the SCA1, SCA2, SCA3, SCA6 and SCA7 genes was performed, using florescence PCR-capillary electrophoresis followed by DNA sequencing, to investigate the variation range of copy number of CAG tandem repeat of the genes in 263 probands of ADCA pedigrees and 261 non-related normal controls. Based on the sequencing result, the bias of the CAG copy number estimation using capillary electrophoresis with different DNA controls was compared to analyze the technical detailes of the electrophresis method in testing the dynamic mutation sites. PCR products containing dynamic mutation loci of the SCA genes showed significantly higher mobility than that of molecular weigh marker with relatively balanced GC content. This was particularly obvious in the SCA2, SCA 6 and SCA7 genes whereas the deviation of copy number could be corrected to +/-1 when known CAG copy number fragments were used as controls. The mobility of PCR products was primarily related to the copy number of CAG repeat when the fragments contained normal CAG repeat. In the 263 ADCA pedigrees, 6 (2.28%) carried SCA1 gene mutation, 8 (3.04%) had SCA2 mutation and 81 (30.80%) harbored SCA3 mutation. The gene mutation of SCA6 and SCA7 was not found. The normal variation range of the CAG repeat was 17-36 copies in SCA1 gene, 13-30 copies in SCA2, 14-39 copies in SCA3, 6-16 copies in SCA6 and 6-13 copies in SCA7. The heterozygosity was 76.1%, 17.7%, 74.4%, 72.1% and 41.3%, respectively. The mutation range of the CAG repeat was 49-56 copies in SCA1 gene, 36-41 copies in SCA2, 59-81 copies in SCA3. Neither homozygous mutation of an SCA gene nor double heterozygous mutation of the SCA genes was observed in the study. The copy number of the CAG repeat in SCA genes could be calculated accurately based on the result of florescence PCR-capillary electrophoresis when limited amount of known repeat copy number controls were used. Our result supported that the notion that SCA3 gene mutation was the most common cause for ADCA, and the obtained data would be helpful for establishing quantitative criteria of the dynamic mutation of the SCA genes in Chinese.

Mitochondrial DNA Copy Number in Sleep Duration Discordant Monozygotic Twins

PubMed Central

Wrede, Joanna E.; Mengel-From, Jonas; Buchwald, Dedra; Vitiello, Michael V.; Bamshad, Michael; Noonan, Carolyn; Christiansen, Lene; Christensen, Kaare; Watson, Nathaniel F.

2015-01-01

Study Objectives: Mitochondrial DNA (mtDNA) copy number is an important component of mitochondrial function and varies with age, disease, and environmental factors. We aimed to determine whether mtDNA copy number varies with habitual differences in sleep duration within pairs of monozygotic twins. Setting: Academic clinical research center. Participants: 15 sleep duration discordant monozygotic twin pairs (30 twins, 80% female; mean age 42.1 years [SD 15.0]). Design: Sleep duration was phenotyped with wrist actigraphy. Each twin pair included a “normal” (7–9 h/24) and “short” (< 7 h/24) sleeping twin. Fasting peripheral blood leukocyte DNA was assessed for mtDNA copy number via the n-fold difference between qPCR measured mtDNA and nuclear DNA creating an mtDNA measure without absolute units. We used generalized estimating equation linear regression models accounting for the correlated data structure to assess within-pair effects of sleep duration on mtDNA copy number. Measurements and Results: Mean within-pair sleep duration difference per 24 hours was 94.3 minutes (SD 62.6 min). We found reduced sleep duration (β = 0.06; 95% CI 0.004, 0.12; P < 0.05) and sleep efficiency (β = 0.51; 95% CI 0.06, 0.95; P < 0.05) were significantly associated with reduced mtDNA copy number within twin pairs. Thus every 1-minute decrease in actigraphy-defined sleep duration was associated with a decrease in mtDNA copy number of 0.06. Likewise, a 1% decrease in actigraphy-defined sleep efficiency was associated with a decrease in mtDNA copy number of 0.51. Conclusions: Reduced sleep duration and sleep efficiency were associated with reduced mitochondrial DNA copy number in sleep duration discordant monozygotic twins offering a potential mechanism whereby short sleep impairs health and longevity through mitochondrial stress. Citation: Wrede JE, Mengel-From J, Buchwald D, Vitiello MV, Bamshad M, Noonan C, Christiansen L, Christensen K, Watson NF. Mitochondrial DNA copy number in sleep duration discordant monozygotic twins. SLEEP 2015;38(10):1655–1658. PMID:26039967

Toward an integrated knowledge environment to support modern oncology.

PubMed

Blake, Patrick M; Decker, David A; Glennon, Timothy M; Liang, Yong Michael; Losko, Sascha; Navin, Nicholas; Suh, K Stephen

2011-01-01

Around the world, teams of researchers continue to develop a wide range of systems to capture, store, and analyze data including treatment, patient outcomes, tumor registries, next-generation sequencing, single-nucleotide polymorphism, copy number, gene expression, drug chemistry, drug safety, and toxicity. Scientists mine, curate, and manually annotate growing mountains of data to produce high-quality databases, while clinical information is aggregated in distant systems. Databases are currently scattered, and relationships between variables coded in disparate datasets are frequently invisible. The challenge is to evolve oncology informatics from a "systems" orientation of standalone platforms and silos into an "integrated knowledge environments" that will connect "knowable" research data with patient clinical information. The aim of this article is to review progress toward an integrated knowledge environment to support modern oncology with a focus on supporting scientific discovery and improving cancer care.

A variant c-KIT mutation, D816H, fundamental to the sequential development of an ovarian mixed germ cell tumor and systemic mastocytosis with chronic myelomonocytic leukemia.

PubMed

Mitchell, Sarah G; Bunting, Silvia T; Saxe, Debra; Olson, Thomas; Keller, Frank G

2017-04-01

An activating point mutation of the c-KIT tyrosine kinase receptor gene, D816H, has been described in germ cell tumors (GCTs). We report an adolescent diagnosed with an ovarian mixed GCT and systemic mastocytosis with chronic myelomonocytic leukemia (SM-CMML). The teratoma and dysgerminoma differed by copy number aberrations via single nucleotide polymorphism (SNP) microarray, but were inclusive of the same c-KIT D816H point mutation (c.2446G>C) also identified in blood and bone marrow mast cells. These findings indicate not only a clonal origin of the GCT and hematologic malignancy, but also suggest a rare KIT mutation may be playing a fundamental role in malignancy development. © 2016 Wiley Periodicals, Inc.

FAS Gene Copy Numbers are Associated with Susceptibility to Behçet Disease and VKH Syndrome in Han Chinese.

PubMed

Yu, Hongsong; Luo, Le; Wu, Lili; Zheng, Minming; Zhang, Lijun; Liu, Yunjia; Li, Hua; Cao, Qingfeng; Kijlstra, Aize; Yang, Peizeng

2015-11-01

Previous studies have identified that disturbed apoptosis was involved in the pathogenesis of Behçet disease (BD) and Vogt-Koyanagi-Harada (VKH) syndrome. This study aims to investigate whether copy number variations of apoptosis-related genes, including FAS, CASPASE8, CASPASE3, and BCL2, are associated with BD and VKH syndrome in Han Chinese. A two-stage association study was performed in 1,014 BD patients, 1,051 VKH syndrome patients, and 2,076 healthy controls. TaqMan(®) Copy Number Assays and real-time PCR were performed. The first-stage study showed that increased frequency of high FAS copy number (>2) was found in BD (P = 1.05 × 10(-3) ) and VKH syndrome (P = 2.56 × 10(-3) ). Replication and combined study confirmed the association of high copy number (>2) of FAS with BD (P = 3.35 × 10(-8) ) and VKH syndrome (P = 9.77 × 10(-8) ). A significant upregulated mRNA expression of FAS was observed in anti-CD3/CD28 antibodies-stimulated CD4(+) T cells from individuals carrying a high gene copy number (>2) as compared to normal diploid 2 copy number carriers (P = 0.004). Moreover, the mRNA expression of FAS both in active patients with BD and VKH syndrome was significantly higher than that in controls (P = 0.001 and P = 0.007, respectively). Our findings suggest that a high copy number of FAS gene confers risk for BD and VKH syndrome. © 2015 WILEY PERIODICALS, INC.

10 CFR 205.324 - Form and style; number of copies.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 10 Energy 3 2010-01-01 2010-01-01 false Form and style; number of copies. 205.324 Section 205.324 Energy DEPARTMENT OF ENERGY OIL ADMINISTRATIVE PROCEDURES AND SANCTIONS Electric Power System Permits and... Electric Energy at International Boundaries § 205.324 Form and style; number of copies. All applicants...

DETECTION OF STACHYBOTRYS CHARTARUM USING rRNA, tri5, AND Β-TUBULIN PRIMERS AND DETERMINING THEIR RELATIVE COPY NUMBER BY REAL TIME PCR

EPA Science Inventory

This research utilizes the quantitative polymerase chain reaction (qPCR) to determine ribosomal copy number of fungal organisms found in unhealthy indoor environments. Knowing specific copy numbers will allow for greater accuracy in quantification when utilizing current pQCR tec...

10 CFR 205.324 - Form and style; number of copies.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 10 Energy 3 2011-01-01 2011-01-01 false Form and style; number of copies. 205.324 Section 205.324 Energy DEPARTMENT OF ENERGY OIL ADMINISTRATIVE PROCEDURES AND SANCTIONS Electric Power System Permits and... Electric Energy at International Boundaries § 205.324 Form and style; number of copies. All applicants...

Copy number of the Adenomatous Polyposis Coli gene is not always neutral in sporadic colorectal cancers with loss of heterozygosity for the gene.

PubMed

Zauber, Peter; Marotta, Stephen; Sabbath-Solitare, Marlene

2016-03-12

Changes in the number of alleles of a chromosome may have an impact upon gene expression. Loss of heterozygosity (LOH) indicates that one allele of a gene has been lost, and knowing the exact copy number of the gene would indicate whether duplication of the remaining allele has occurred. We were interested to determine the copy number of the Adenomatous Polyposis Coli (APC) gene in sporadic colorectal cancers with LOH. We selected 38 carcinomas with LOH for the APC gene region of chromosome 5, as determined by amplification of the CA repeat region within the D5S346 loci. The copy number status of APC was ascertained using the SALSA® MLPA® P043-B1 APC Kit. LOH for the DCC gene, KRAS gene mutation, and microsatellite instability were also evaluated for each tumor, utilizing standard polymerase chain reaction methods. No tumor demonstrated microsatellite instability. LOH of the DCC gene was also present in 33 of 36 (91.7%) informative tumors. A KRAS gene mutation was present in 16 of the 38 (42.1%) tumors. Twenty-four (63.2%) of the tumors were copy number neutral, 10 (26.3%) tumors demonstrated major loss, while two (5.3%) showed partial loss. Two tumors (5.3%) had copy number gain. Results of APC and DCC LOH, KRAS and microsatellite instability indicate our colorectal cancer cases were typical of sporadic cancers following the 'chromosomal instability' pathway. The majority of our colorectal carcinomas with LOH for APC gene are copy number neutral. However, one-third of our cases showed copy number loss, suggesting that duplication of the remaining allele is not required for the development of a colorectal carcinoma.

DUF1220 copy number is linearly associated with increased cognitive function as measured by total IQ and mathematical aptitude scores

PubMed Central

Davis, Jonathon M.; Searles, Veronica B.; Anderson, Nathan; Keeney, Jonathon; Raznahan, Armin; Horwood, L. John; Fergusson, David M.; Kennedy, Martin A.; Giedd, Jay

2014-01-01

DUF1220 protein domains exhibit the greatest human lineage-specific copy number expansion of any protein-coding sequence in the genome, and variation in DUF1220 copy number has been linked to both brain size in humans and brain evolution among primates. Given these findings, we examined associations between DUF1220 subtypes CON1 and CON2 and cognitive aptitude. We identified a linear association between CON2 copy number and cognitive function in two independent populations of European descent. In North American males, an increase in CON2 copy number corresponded with an increase in WISC IQ (R2 = 0.13, p = 0.02), which may be driven by males aged 6–11 (R2 = 0.42, p = 0.003). We utilized ddPCR in a subset as a confirmatory measurement. This group had 26–33 copies of CON2 with a mean of 29, and each copy increase of CON2 was associated with a 3.3-point increase in WISC IQ (R2 = 0.22, p = 0.045). In individuals from New Zealand, an increase in CON2 copy number was associated with an increase in math aptitude ability (R2 = 0.10 p = 0.018). These were not confounded by brain size. To our knowledge, this is the first study to report a replicated association between copy number of a gene coding sequence and cognitive aptitude. Remarkably, dosage variations involving DUF1220 sequences have now been linked to human brain expansion, autism severity and cognitive aptitude, suggesting that such processes may be genetically and mechanistically inter-related. The findings presented here warrant expanded investigations in larger, well-characterized cohorts. PMID:25287832

The sheep growth hormone gene polymorphism and its effects on milk traits.

PubMed

Dettori, Maria Luisa; Pazzola, Michele; Pira, Emanuela; Paschino, Pietro; Vacca, Giuseppe Massimo

2015-05-01

Growth hormone (GH) is encoded by the GH gene, which may be single copy or duplicate in sheep. The two copies of the sheep GH gene (GH1/GH2-N and GH2-Z) were entirely sequenced in one 106 ewes of Sarda breed, in order to highlight sequence polymorphisms and investigate possible association between genetic variants and milk traits. Milk traits included milk yield, fat, protein, casein and lactose percentage. We evidenced 75 nucleotide changes. Transcription factor binding site prediction revealed two sequences potentially recognised by the pituitary-specific transcription factor POU1FI at the GH1/GH2-N gene, which were lost at the promoter of GH2-Z, which might explain the different tissues of expression of GH1/GH2-N (pituitary) and GH2-Z (placenta). Significant differences in milk traits were observed among genotypes at polymorphic loci only for the GH2-Z gene. Sheep with homozygote genotype ss748770547 CC had higher fat percentage (P < 0.01) than TT. SNP ss748770547 was part of a potential transcription factor binding site for C/EBP alpha (CCAAT/Enhancer Binding Protein), which is involved in the regulation of adipogenesis and adipoblast differentiation. SNP ss748770547, located in the GH2-Z gene 5' flanking region, may be a causal mutation affecting milk fat content. These findings might contribute to the knowledge of the sheep GH locus and might be useful in selection processes in sheep.

SNP array profiling of childhood adrenocortical tumors reveals distinct pathways of tumorigenesis and highlights candidate driver genes.

PubMed

Letouzé, Eric; Rosati, Roberto; Komechen, Heloisa; Doghman, Mabrouka; Marisa, Laetitia; Flück, Christa; de Krijger, Ronald R; van Noesel, Max M; Mas, Jean-Christophe; Pianovski, Mara A D; Zambetti, Gerard P; Figueiredo, Bonald C; Lalli, Enzo

2012-07-01

Childhood adrenocortical tumors (ACT) are rare malignancies, except in southern Brazil, where a higher incidence rate is associated to a high frequency of the founder R337H TP53 mutation. To date, copy number alterations in these tumors have only been analyzed by low-resolution comparative genomic hybridization. We analyzed an international series of 25 childhood ACT using high-resolution single nucleotide polymorphism arrays to: 1) detect focal copy number alterations highlighting candidate driver genes; and 2) compare genetic alterations between Brazilian patients carrying the R337H TP53 mutation and non-Brazilian patients. We identified 16 significantly recurrent chromosomal alterations (q-value < 0.05), the most frequent being -4q34, +9q33-q34, +19p, loss of heterozygosity (LOH) of chromosome 17 and 11p15. Focal amplifications and homozygous deletions comprising well-known oncogenes (MYC, MDM2, PDGFRA, KIT, MCL1, BCL2L1) and tumor suppressors (TP53, RB1, RPH3AL) were identified. In addition, eight focal deletions were detected at 4q34, defining a sharp peak region around the noncoding RNA LINC00290 gene. Although non-Brazilian tumors with a mutated TP53 were similar to Brazilian tumors, those with a wild-type TP53 displayed distinct genomic profiles, with significantly fewer rearrangements (P = 0.019). In particular, three alterations (LOH of chromosome 17, +9q33-q34, and -4q34) were significantly more frequent in TP53-mutated samples. Finally, two of four TP53 wild-type tumors displayed as sole rearrangement a copy-neutral LOH of the imprinted region at 11p15, supporting a major role for this region in ACT development. Our findings highlight potential driver genes and cellular pathways implicated in childhood ACT and demonstrate the existence of different oncogenic routes in this pathology.

[Analysis of Microbial Community in the Membrane Bio-Reactor (MBR) Rural Sewage Treatment System].

PubMed

Kong, Xiao; Cui, Bing-jian; Jin, De-cai; Wu, Shang-hua; Yang, Bo; Deng, Ye; Zhuang, Guo-qiang; Zhuang, Xu-liang

2015-09-01

Uncontrolled release and arbitrary irrigation reuse of rural wastewater may lead to water pollution, and the microbial pathogens could threaten the safety of freshwater resources and public health. To understand the microbial community structure of rural wastewater and provide the theory for microbial risk assessment of wastewater irrigation, microbial community diversities in the Membrane Bio-Reactor (MBR) process for rural wastewater treatment was studied by terminal restriction fragment length polymorphism (T-RFLP) and 16S rDNA gene clone library. Meanwhile, changes of Arcobacter spp. and total bacteria before and after treatment were detected through real-time quantitative PCR. The clone library results showed that there were 73 positive clones included Proteobacteria (91. 80%), Firmicutes (2. 70%), Bacteroidetes (1. 40%), and uncultured bacteria (4. 10%) in the untreated wastewater. The typical pathogenic genus Arcobacter belonging to e-Proteobacteria was the dominant component of the library, accounting for 68. 5% of all clones. The main groups and their abundance in different treatments were significantly distinct. The highest values of species abundance (S), Shannon-Wiener (H) and Evenness (E) were observed in the adjusting tank, which were 43. 0, 3. 56 and 0. 95, respectively. The real-time quantitative PCR results showed that the copy number of Arcobacter spp. was (1. 09 ± 0. 064 0) x 10(11) copies.L-1 in the untreated sewage, which was consistent with the result of 16S rDNA gene clone library. Compared to untreated wastewater, bacterial copy number in the treated effluent decreased 100 to 1 000 times, respectively, suggesting that MBR treatment system could remove the microbial quantity in such scale. In the recycled water, the physicochemical parameters and indicator bacteria met the water quality standard of farmland irrigation. However, further research is needed to estimate the potential health risks caused by residual pathogenic microorganisms in future.

Stoichiometric balance of protein copy numbers is measurable and functionally significant in a protein-protein interaction network for yeast endocytosis

PubMed Central

2018-01-01

Stoichiometric balance, or dosage balance, implies that proteins that are subunits of obligate complexes (e.g. the ribosome) should have copy numbers expressed to match their stoichiometry in that complex. Establishing balance (or imbalance) is an important tool for inferring subunit function and assembly bottlenecks. We show here that these correlations in protein copy numbers can extend beyond complex subunits to larger protein-protein interactions networks (PPIN) involving a range of reversible binding interactions. We develop a simple method for quantifying balance in any interface-resolved PPINs based on network structure and experimentally observed protein copy numbers. By analyzing such a network for the clathrin-mediated endocytosis (CME) system in yeast, we found that the real protein copy numbers were significantly more balanced in relation to their binding partners compared to randomly sampled sets of yeast copy numbers. The observed balance is not perfect, highlighting both under and overexpressed proteins. We evaluate the potential cost and benefits of imbalance using two criteria. First, a potential cost to imbalance is that ‘leftover’ proteins without remaining functional partners are free to misinteract. We systematically quantify how this misinteraction cost is most dangerous for strong-binding protein interactions and for network topologies observed in biological PPINs. Second, a more direct consequence of imbalance is that the formation of specific functional complexes depends on relative copy numbers. We therefore construct simple kinetic models of two sub-networks in the CME network to assess multi-protein assembly of the ARP2/3 complex and a minimal, nine-protein clathrin-coated vesicle forming module. We find that the observed, imperfectly balanced copy numbers are less effective than balanced copy numbers in producing fast and complete multi-protein assemblies. However, we speculate that strategic imbalance in the vesicle forming module allows cells to tune where endocytosis occurs, providing sensitive control over cargo uptake via clathrin-coated vesicles. PMID:29518071

Stoichiometric balance of protein copy numbers is measurable and functionally significant in a protein-protein interaction network for yeast endocytosis.

PubMed

Holland, David O; Johnson, Margaret E

2018-03-01

Stoichiometric balance, or dosage balance, implies that proteins that are subunits of obligate complexes (e.g. the ribosome) should have copy numbers expressed to match their stoichiometry in that complex. Establishing balance (or imbalance) is an important tool for inferring subunit function and assembly bottlenecks. We show here that these correlations in protein copy numbers can extend beyond complex subunits to larger protein-protein interactions networks (PPIN) involving a range of reversible binding interactions. We develop a simple method for quantifying balance in any interface-resolved PPINs based on network structure and experimentally observed protein copy numbers. By analyzing such a network for the clathrin-mediated endocytosis (CME) system in yeast, we found that the real protein copy numbers were significantly more balanced in relation to their binding partners compared to randomly sampled sets of yeast copy numbers. The observed balance is not perfect, highlighting both under and overexpressed proteins. We evaluate the potential cost and benefits of imbalance using two criteria. First, a potential cost to imbalance is that 'leftover' proteins without remaining functional partners are free to misinteract. We systematically quantify how this misinteraction cost is most dangerous for strong-binding protein interactions and for network topologies observed in biological PPINs. Second, a more direct consequence of imbalance is that the formation of specific functional complexes depends on relative copy numbers. We therefore construct simple kinetic models of two sub-networks in the CME network to assess multi-protein assembly of the ARP2/3 complex and a minimal, nine-protein clathrin-coated vesicle forming module. We find that the observed, imperfectly balanced copy numbers are less effective than balanced copy numbers in producing fast and complete multi-protein assemblies. However, we speculate that strategic imbalance in the vesicle forming module allows cells to tune where endocytosis occurs, providing sensitive control over cargo uptake via clathrin-coated vesicles.

DNA replication stress restricts ribosomal DNA copy number

PubMed Central

Salim, Devika; Bradford, William D.; Freeland, Amy; Cady, Gillian; Wang, Jianmin

2017-01-01

Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100–200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how “normal” copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a “normal” rDNA copy number. PMID:28915237

Integrated Copy Number and Expression Analysis Identifies Profiles of Whole-Arm Chromosomal Alterations and Subgroups with Favorable Outcome in Ovarian Clear Cell Carcinomas

PubMed Central

Uehara, Yuriko; Oda, Katsutoshi; Ikeda, Yuji; Koso, Takahiro; Tsuji, Shingo; Yamamoto, Shogo; Asada, Kayo; Sone, Kenbun; Kurikawa, Reiko; Makii, Chinami; Hagiwara, Otoe; Tanikawa, Michihiro; Maeda, Daichi; Hasegawa, Kosei; Nakagawa, Shunsuke; Wada-Hiraike, Osamu; Kawana, Kei; Fukayama, Masashi; Fujiwara, Keiichi; Yano, Tetsu; Osuga, Yutaka; Fujii, Tomoyuki; Aburatani, Hiroyuki

2015-01-01

Ovarian clear cell carcinoma (CCC) is generally associated with chemoresistance and poor clinical outcome, even with early diagnosis; whereas high-grade serous carcinomas (SCs) and endometrioid carcinomas (ECs) are commonly chemosensitive at advanced stages. Although an integrated genomic analysis of SC has been performed, conclusive views on copy number and expression profiles for CCC are still limited. In this study, we performed single nucleotide polymorphism analysis with 57 epithelial ovarian cancers (31 CCCs, 14 SCs, and 12 ECs) and microarray expression analysis with 55 cancers (25 CCCs, 16 SCs, and 14 ECs). We then evaluated PIK3CA mutations and ARID1A expression in CCCs. SNP array analysis classified 13% of CCCs into a cluster with high frequency and focal range of copy number alterations (CNAs), significantly lower than for SCs (93%, P < 0.01) and ECs (50%, P = 0.017). The ratio of whole-arm to all CNAs was higher in CCCs (46.9%) than SCs (21.7%; P < 0.0001). SCs with loss of heterozygosity (LOH) of BRCA1 (85%) also had LOH of NF1 and TP53, and LOH of BRCA2 (62%) coexisted with LOH of RB1 and TP53. Microarray analysis classified CCCs into three clusters. One cluster (CCC-2, n = 10) showed more favorable prognosis than the CCC-1 and CCC-3 clusters (P = 0.041). Coexistent alterations of PIK3CA and ARID1A were more common in CCC-1 and CCC-3 (7/11, 64%) than in CCC-2 (0/10, 0%; P < 0.01). Being in cluster CCC-2 was an independent favorable prognostic factor in CCC. In conclusion, CCC was characterized by a high ratio of whole-arm CNAs; whereas CNAs in SC were mainly focal, but preferentially caused LOH of well-known tumor suppressor genes. As such, expression profiles might be useful for sub-classification of CCC, and might provide useful information on prognosis. PMID:26043110

Beta-defensin genomic copy number is not a modifier locus for cystic fibrosis

PubMed Central

Hollox, Edward J; Davies, Jane; Griesenbach, Uta; Burgess, Juliana; Alton, Eric WFW; Armour, John AL

2005-01-01

Human beta-defensin 2 (DEFB4, also known as DEFB2 or hBD-2) is a salt-sensitive antimicrobial protein that is expressed in lung epithelia. Previous work has shown that it is encoded in a cluster of beta-defensin genes at 8p23.1, which varies in copy number between 2 and 12 in different individuals. We determined the copy number of this locus in 355 patients with cystic fibrosis (CF), and tested for correlation between beta-defensin cluster genomic copy number and lung disease associated with CF. No significant association was found. PMID:16336654

Evaluation of copy-number variants as modifiers of breast and ovarian cancer risk for BRCA1 pathogenic variant carriers

PubMed Central

Walker, Logan C; Marquart, Louise; Pearson, John F; Wiggins, George A R; O'Mara, Tracy A; Parsons, Michael T; Barrowdale, Daniel; McGuffog, Lesley; Dennis, Joe; Benitez, Javier; Slavin, Thomas P; Radice, Paolo; Frost, Debra; Godwin, Andrew K; Meindl, Alfons; Schmutzler, Rita Katharina; Isaacs, Claudine; Peshkin, Beth N; Caldes, Trinidad; Hogervorst, Frans BL; Lazaro, Conxi; Jakubowska, Anna; Montagna, Marco; Chen, Xiaoqing; Offit, Kenneth; Hulick, Peter J; Andrulis, Irene L; Lindblom, Annika; Nussbaum, Robert L; Nathanson, Katherine L; Chenevix-Trench, Georgia; Antoniou, Antonis C; Couch, Fergus J; Spurdle, Amanda B

2017-01-01

Genome-wide studies of patients carrying pathogenic variants (mutations) in BRCA1 or BRCA2 have reported strong associations between single-nucleotide polymorphisms (SNPs) and cancer risk. To conduct the first genome-wide association analysis of copy-number variants (CNVs) with breast or ovarian cancer risk in a cohort of 2500 BRCA1 pathogenic variant carriers, CNV discovery was performed using multiple calling algorithms and Illumina 610k SNP array data from a previously published genome-wide association study. Our analysis, which focused on functionally disruptive genomic deletions overlapping gene regions, identified a number of loci associated with risk of breast or ovarian cancer for BRCA1 pathogenic variant carriers. Despite only including putative deletions called by at least two or more algorithms, detection of selected CNVs by ancillary molecular technologies only confirmed 40% of predicted common (>1% allele frequency) variants. These include four loci that were associated (unadjusted P<0.05) with breast cancer (GTF2H2, ZNF385B, NAALADL2 and PSG5), and two loci associated with ovarian cancer (CYP2A7 and OR2A1). An interesting finding from this study was an association of a validated CNV deletion at the CYP2A7 locus (19q13.2) with decreased ovarian cancer risk (relative risk=0.50, P=0.007). Genomic analysis found this deletion coincides with a region displaying strong regulatory potential in ovarian tissue, but not in breast epithelial cells. This study highlighted the need to verify CNVs in vitro, but also provides evidence that experimentally validated CNVs (with plausible biological consequences) can modify risk of breast or ovarian cancer in BRCA1 pathogenic variant carriers. PMID:28145423

Complex structure of knob DNA on maize chromosome 9. Retrotransposon invasion into heterochromatin.

PubMed Central

Ananiev, E V; Phillips, R L; Rines, H W

1998-01-01

The recovery of maize (Zea mays L.) chromosome addition lines of oat (Avena sativa L.) from oat x maize crosses enables us to analyze the structure and composition of specific regions, such as knobs, of individual maize chromosomes. A DNA hybridization blot panel of eight individual maize chromosome addition lines revealed that 180-bp repeats found in knobs are present in each of these maize chromosomes, but the copy number varies from approximately 100 to 25, 000. Cosmid clones with knob DNA segments were isolated from a genomic library of an oat-maize chromosome 9 addition line with the help of the 180-bp knob-associated repeated DNA sequence used as a probe. Cloned knob DNA segments revealed a complex organization in which blocks of tandemly arranged 180-bp repeating units are interrupted by insertions of other repeated DNA sequences, mostly represented by individual full size copies of retrotransposable elements. There is an obvious preference for the integration of retrotransposable elements into certain sites (hot spots) of the 180-bp repeat. Sequence microheterogeneity including point mutations and duplications was found in copies of 180-bp repeats. The 180-bp repeats within an array all had the same polarity. Restriction maps constructed for 23 cloned knob DNA fragments revealed the positions of polymorphic sites and sites of integration of insertion elements. Discovery of the interspersion of retrotransposable elements among blocks of tandem repeats in maize and some other organisms suggests that this pattern may be basic to heterochromatin organization for eukaryotes. PMID:9691055

Tank-Binding Kinase 1 (TBK1) Gene and Open-Angle Glaucomas (An American Ophthalmological Society Thesis)

PubMed Central

Fingert, John H.; Robin, Alan L.; Scheetz, Todd E.; Kwon, Young H.; Liebmann, Jeffrey M.; Ritch, Robert; Alward, Wallace L.M.

2016-01-01

Purpose To investigate the role of TANK-binding kinase 1 (TBK1) gene copy-number variations (ie, gene duplications and triplications) in the pathophysiology of various open-angle glaucomas. Methods In previous studies, we discovered that copy-number variations in the TBK1 gene are associated with normal-tension glaucoma. Here, we investigated the prevalence of copy-number variations in cohorts of patients with other open-angle glaucomas—juvenile-onset open-angle glaucoma (n=30), pigmentary glaucoma (n=209), exfoliation glaucoma (n=225), and steroid-induced glaucoma (n=79)—using a quantitative polymerase chain reaction assay. Results No TBK1 gene copy-number variations were detected in patients with juvenile-onset open-angle glaucoma, pigmentary glaucoma, or steroid-induced glaucoma. A TBK1 gene duplication was detected in one (0.44%) of the 225 exfoliation glaucoma patients. Conclusions TBK1 gene copy-number variations (gene duplications and triplications) have been previously associated with normal-tension glaucoma. An exploration of other open-angle glaucomas detected a TBK1 copy-number variation in a patient with exfoliation glaucoma, which is the first example of a TBK1 mutation in a glaucoma patient with a diagnosis other than normal-tension glaucoma. A broader phenotypic range may be associated with TBK1 copy-number variations, although mutations in this gene are most often detected in patients with normal-tension glaucoma. PMID:27881886

Higher DEFB4 genomic copy number in SLE and ANCA-associated small vasculitis.

PubMed

Zhou, Xu-Jie; Cheng, Fa-Juan; Lv, Ji-Cheng; Luo, Huan; Yu, Feng; Chen, Min; Zhao, Ming-Hui; Zhang, Hong

2012-06-01

Evidence shows that defensins are involved in the pathogenesis of SLE and ANCA-associated small vasculitis (AASV). The copy number variation of DEFB4 has been proposed to be susceptible to inflammatory disorders. This study aims to investigate whether the DEFB4 genomic copy number variations associate with the susceptibility to these two autoimmune diseases. A total of 1178 Chinese people were enrolled, including panel 1 comprising 240 SLE patients and 275 matched controls, panel 2 comprising 303 SLE patients and 248 matched controls and panel 3 with 112 AASV patients. The DEFB4 copy number was typed by a paralogue ratio test (PRT), and all the subjects in panel 1 were also typed using the restriction enzyme digest variant ratio (REDVR) for validation. The results from PRT and REDVR were highly concordant (R = 0.911, P = 3.85 × 10(-199)) and allowed copy numbers to be assigned into integer classes with high confidence. Comparison of mean DEFB4 copy number revealed a small increase in cases with SLE both in Panel 1 (P = 0.063) and Panel 2 (P = 0.017). When pooling panels 1 and 2 together, the association was reinforced (P = 0.002) in SLE. Such association was also observed in AASV (P = 0.009). We found that a higher DEFB4 gene copy number was associated with both SLE and AASV.

Tank-Binding Kinase 1 (TBK1) Gene and Open-Angle Glaucomas (An American Ophthalmological Society Thesis).

PubMed

Fingert, John H; Robin, Alan L; Scheetz, Todd E; Kwon, Young H; Liebmann, Jeffrey M; Ritch, Robert; Alward, Wallace L M

2016-08-01

To investigate the role of TANK-binding kinase 1 ( TBK1 ) gene copy-number variations (ie, gene duplications and triplications) in the pathophysiology of various open-angle glaucomas. In previous studies, we discovered that copy-number variations in the TBK1 gene are associated with normal-tension glaucoma. Here, we investigated the prevalence of copy-number variations in cohorts of patients with other open-angle glaucomas-juvenile-onset open-angle glaucoma (n=30), pigmentary glaucoma (n=209), exfoliation glaucoma (n=225), and steroid-induced glaucoma (n=79)-using a quantitative polymerase chain reaction assay. No TBK1 gene copy-number variations were detected in patients with juvenile-onset open-angle glaucoma, pigmentary glaucoma, or steroid-induced glaucoma. A TBK1 gene duplication was detected in one (0.44%) of the 225 exfoliation glaucoma patients. TBK1 gene copy-number variations (gene duplications and triplications) have been previously associated with normal-tension glaucoma. An exploration of other open-angle glaucomas detected a TBK1 copy-number variation in a patient with exfoliation glaucoma, which is the first example of a TBK1 mutation in a glaucoma patient with a diagnosis other than normal-tension glaucoma. A broader phenotypic range may be associated with TBK1 copy-number variations, although mutations in this gene are most often detected in patients with normal-tension glaucoma.

A novel approach for copy number variation analysis by combining multiplex PCR with matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

PubMed

Gao, Yonghui; Chen, Xiaoli; Wang, Jianhua; Shangguan, Shaofang; Dai, Yaohua; Zhang, Ting; Liu, Junling

2013-06-20

With the increasing interest in copy number variation as it pertains to human genomic variation, common phenotypes, and disease susceptibility, there is a pressing need for methods to accurately identify copy number. In this study, we developed a simple approach that combines multiplex PCR with matrix-assisted laser desorption ionization time-of-flight mass spectrometry for submicroscopic copy number variation detection. Two pairs of primers were used to simultaneously amplify query and endogenous control regions in the same reaction. Using a base extension reaction, the two amplicons were then distinguished and quantified in a mass spectrometry map. The peak ratio between the test region and the endogenous control region was manually calculated. The relative copy number could be determined by comparing the peak ratio between the test and control samples. This method generated a copy number measurement comparable to those produced by two other commonly used methods - multiplex ligation-dependent probe amplification and quantitative real-time PCR. Furthermore, it can discriminate a wide range of copy numbers. With a typical 384-format SpectroCHIP, at least six loci on 384 samples can be analyzed simultaneously in a hexaplex assay, making this assay adaptable for high throughput, and potentially applicable for large-scale association studies. Copyright © 2013 Elsevier B.V. All rights reserved.

Development of a high-copy plasmid for enhanced production of recombinant proteins in Leuconostoc citreum.

PubMed

Son, Yeon Jeong; Ryu, Ae Jin; Li, Ling; Han, Nam Soo; Jeong, Ki Jun

2016-01-15

Leuconostoc is a hetero-fermentative lactic acid bacteria, and its importance is widely recognized in the dairy industry. However, due to limited genetic tools including plasmids for Leuconostoc, there has not been much extensive research on the genetics and engineering of Leuconostoc yet. Thus, there is a big demand for high-copy-number plasmids for useful gene manipulation and overproduction of recombinant proteins in Leuconostoc. Using an existing low-copy plasmid, the copy number of plasmid was increased by random mutagenesis followed by FACS-based high-throughput screening. First, a random library of plasmids was constructed by randomizing the region responsible for replication in Leuconostoc citreum; additionally, a superfolder green fluorescent protein (sfGFP) was used as a reporter protein. With a high-speed FACS sorter, highly fluorescent cells were enriched, and after two rounds of sorting, single clone exhibiting the highest level of sfGFP was isolated. The copy number of the isolated plasmid (pCB4270) was determined by quantitative PCR (qPCR). It was found that the isolated plasmid has approximately a 30-fold higher copy number (approx. 70 copies per cell) than that of the original plasmid. From the sequence analysis, a single mutation (C→T) at position 4690 was found, and we confirmed that this single mutation was responsible for the increased plasmid copy number. The effectiveness of the isolated high-copy-number plasmid for the overproduction of recombinant proteins was successfully demonstrated with two protein models Glutathione-S-transferase (GST) and α-amylase. The high-copy number plasmid was successfully isolated by FACS-based high-throughput screening of a plasmid library in L. citreum. The isolated plasmid could be a useful genetic tool for high-level gene expression in Leuconostoc, and for extending the applications of this useful bacteria to various areas in the dairy and pharmaceutical industries.

A Simple PCR Method for Rapid Genotype Analysis of Mycobacterium ulcerans

PubMed Central

Stinear, Timothy; Davies, John K.; Jenkin, Grant A.; Portaels, Françoise; Ross, Bruce C.; OppEdIsano, Frances; Purcell, Maria; Hayman, John A.; Johnson, Paul D. R.

2000-01-01

Two high-copy-number insertion sequences, IS2404 and IS2606, were recently identified in Mycobacterium ulcerans and were shown by Southern hybridization to possess restriction fragment length polymorphism between strains from different geographic origins. We have designed a simple genotyping method that captures these differences by PCR amplification of the region between adjacent copies of IS2404 and IS2606. We have called this system 2426 PCR. The method is rapid, reproducible, sensitive, and specific for M. ulcerans, and it has confirmed previous studies suggesting a clonal population structure of M. ulcerans within a geographic region. M. ulcerans isolates from Australia, Papua New Guinea, Malaysia, Surinam, Mexico, Japan, China, and several countries in Africa were easily differentiated based on an array of 4 to 14 PCR products ranging in size from 200 to 900 bp. Numerical analysis of the banding patterns suggested a close evolutionary link between M. ulcerans isolates from Africa and southeast Asia. The application of 2426 PCR to total DNA, extracted directly from M. ulcerans-infected tissue specimens without culture, demonstrated the sensitivity and specificity of this method and confirmed for the first time that both animal and human isolates from areas of endemicity in southeast Australia have the same genotype. PMID:10747130

17 CFR 260.10a-3 - Number of copies-Filing-Signatures.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Number of copies-Filing-Signatures. 260.10a-3 Section 260.10a-3 Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION... Number of copies—Filing—Signatures. (a) Three copies of every application pursuant to rule 10a-1 (§ 260...

17 CFR 240.12b-11 - Number of copies; signatures; binding.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Number of copies; signatures... Under the Securities Exchange Act of 1934 Formal Requirements § 240.12b-11 Number of copies; signatures... bound on the left side in such a manner as to leave the reading matter legible. (d) Signatures. Where...

17 CFR 260.5b-3 - Number of copies-Filing-Signatures.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Number of copies-Filing-Signatures. 260.5b-3 Section 260.5b-3 Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION... Number of copies—Filing—Signatures. (a) Three copies of every application pursuant to rule 5b-1 (§ 260.5b...

10 CFR 205.307 - Form and style; number of copies

Code of Federal Regulations, 2010 CFR

2010-01-01

... 10 Energy 3 2010-01-01 2010-01-01 false Form and style; number of copies 205.307 Section 205.307 Energy DEPARTMENT OF ENERGY OIL ADMINISTRATIVE PROCEDURES AND SANCTIONS Electric Power System Permits and... Electric Energy to A Foreign Country § 205.307 Form and style; number of copies An original and two...

47 CFR 1.742 - Place of filing, fees, and number of copies.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 47 Telecommunication 1 2010-10-01 2010-10-01 false Place of filing, fees, and number of copies. 1..., fees, and number of copies. All applications which do not require a fee shall be filed at the... then forwarded to the Wireline Competition Bureau. All applications accompanied by a fee payment should...

12 CFR 116.40 - Where do I file my application?

Code of Federal Regulations, 2014 CFR

2014-01-01

... application? (a) OCC Office. (1) You must file the original application and the number of copies indicated on.... If the form does not indicate the number of copies you must file or if the OCC has not prescribed a... OCC licensing office at headquarters. You must file the number of copies indicated on the applicable...

12 CFR 516.40 - Where do I file my application?

Code of Federal Regulations, 2014 CFR

2014-01-01

... application? (a) Regional Office. (1) You must file the original application and the number of copies... in paragraph (a)(2) of this section. If the form does not indicate the number of copies you must file...., Washington, DC 20552. You must file the number of copies indicated on the applicable form. If the form does...

12 CFR 116.40 - Where do I file my application?

Code of Federal Regulations, 2012 CFR

2012-01-01

... application? (a) OCC Office. (1) You must file the original application and the number of copies indicated on.... If the form does not indicate the number of copies you must file or if the OCC has not prescribed a... OCC licensing office at headquarters. You must file the number of copies indicated on the applicable...

12 CFR 516.40 - Where do I file my application?

Code of Federal Regulations, 2012 CFR

2012-01-01

... application? (a) Regional Office. (1) You must file the original application and the number of copies... in paragraph (a)(2) of this section. If the form does not indicate the number of copies you must file...., Washington, DC 20552. You must file the number of copies indicated on the applicable form. If the form does...

12 CFR 516.40 - Where do I file my application?

Code of Federal Regulations, 2013 CFR

2013-01-01

... application? (a) Regional Office. (1) You must file the original application and the number of copies... in paragraph (a)(2) of this section. If the form does not indicate the number of copies you must file...., Washington, DC 20552. You must file the number of copies indicated on the applicable form. If the form does...

12 CFR 116.40 - Where do I file my application?

Code of Federal Regulations, 2013 CFR

2013-01-01

... application? (a) OCC Office. (1) You must file the original application and the number of copies indicated on.... If the form does not indicate the number of copies you must file or if the OCC has not prescribed a... OCC licensing office at headquarters. You must file the number of copies indicated on the applicable...

12 CFR 516.40 - Where do I file my application?

Code of Federal Regulations, 2011 CFR

2011-01-01

... application? (a) Regional Office. (1) You must file the original application and the number of copies... in paragraph (a)(2) of this section. If the form does not indicate the number of copies you must file...., Washington, DC 20552. You must file the number of copies indicated on the applicable form. If the form does...

12 CFR 516.40 - Where do I file my application?

Code of Federal Regulations, 2010 CFR

2010-01-01

... application? (a) Regional Office. (1) You must file the original application and the number of copies... in paragraph (a)(2) of this section. If the form does not indicate the number of copies you must file...., Washington, DC 20552. You must file the number of copies indicated on the applicable form. If the form does...

10 CFR 205.307 - Form and style; number of copies

Code of Federal Regulations, 2011 CFR

2011-01-01

... 10 Energy 3 2011-01-01 2011-01-01 false Form and style; number of copies 205.307 Section 205.307 Energy DEPARTMENT OF ENERGY OIL ADMINISTRATIVE PROCEDURES AND SANCTIONS Electric Power System Permits and... Electric Energy to A Foreign Country § 205.307 Form and style; number of copies An original and two...

Orthogonality and Burdens of Heterologous AND Gate Gene Circuits in E. coli

PubMed Central

2017-01-01

Synthetic biology approaches commonly introduce heterologous gene networks into a host to predictably program cells, with the expectation of the synthetic network being orthogonal to the host background. However, introduced circuits may interfere with the host’s physiology, either indirectly by posing a metabolic burden and/or through unintended direct interactions between parts of the circuit with those of the host, affecting functionality. Here we used RNA-Seq transcriptome analysis to quantify the interactions between a representative heterologous AND gate circuit and the host Escherichia coli under various conditions including circuit designs and plasmid copy numbers. We show that the circuit plasmid copy number outweighs circuit composition for their effect on host gene expression with medium-copy number plasmid showing more prominent interference than its low-copy number counterpart. In contrast, the circuits have a stronger influence on the host growth with a metabolic load increasing with the copy number of the circuits. Notably, we show that variation of copy number, an increase from low to medium copy, caused different types of change observed in the behavior of components in the AND gate circuit leading to the unbalance of the two gate-inputs and thus counterintuitive output attenuation. The study demonstrates the circuit plasmid copy number is a key factor that can dramatically affect the orthogonality, burden and functionality of the heterologous circuits in the host chassis. The results provide important guidance for future efforts to design orthogonal and robust gene circuits with minimal unwanted interaction and burden to their host. PMID:29240998

Diversity of human copy number variation and multicopy genes.

PubMed

Sudmant, Peter H; Kitzman, Jacob O; Antonacci, Francesca; Alkan, Can; Malig, Maika; Tsalenko, Anya; Sampas, Nick; Bruhn, Laurakay; Shendure, Jay; Eichler, Evan E

2010-10-29

Copy number variants affect both disease and normal phenotypic variation, but those lying within heavily duplicated, highly identical sequence have been difficult to assay. By analyzing short-read mapping depth for 159 human genomes, we demonstrated accurate estimation of absolute copy number for duplications as small as 1.9 kilobase pairs, ranging from 0 to 48 copies. We identified 4.1 million "singly unique nucleotide" positions informative in distinguishing specific copies and used them to genotype the copy and content of specific paralogs within highly duplicated gene families. These data identify human-specific expansions in genes associated with brain development, reveal extensive population genetic diversity, and detect signatures consistent with gene conversion in the human species. Our approach makes ~1000 genes accessible to genetic studies of disease association.

Modulation of Mitochondrial DNA Copy Number to Induce Hepatocytic Differentiation of Human Amniotic Epithelial Cells.

PubMed

Vaghjiani, Vijesh; Cain, Jason E; Lee, William; Vaithilingam, Vijayaganapathy; Tuch, Bernard E; St John, Justin C

2017-10-15

Mitochondrial deoxyribonucleic acid (mtDNA) copy number is tightly regulated during pluripotency and differentiation. There is increased demand of cellular adenosine triphosphate (ATP) during differentiation for energy-intensive cell types such as hepatocytes and neurons to meet the cell's functional requirements. During hepatocyte differentiation, mtDNA copy number should be synchronously increased to generate sufficient ATP through oxidative phosphorylation. Unlike bone marrow mesenchymal cells, mtDNA copy number failed to increase by 28 days of differentiation of human amniotic epithelial cells (hAEC) into hepatocyte-like cells (HLC) despite their expression of some end-stage hepatic markers. This was due to higher levels of DNA methylation at exon 2 of POLGA, the mtDNA-specific replication factor. Treatment with a DNA demethylation agent, 5-azacytidine, resulted in increased mtDNA copy number, reduced DNA methylation at exon 2 of POLGA, and reduced hepatic gene expression. Depletion of mtDNA followed by subsequent differentiation did not increase mtDNA copy number, but reduced DNA methylation at exon 2 of POLGA and increased expression of hepatic and pluripotency genes. We encapsulated hAEC in barium alginate microcapsules and subsequently differentiated them into HLC. Encapsulation resulted in no net increase of mtDNA copy number but a significant reduction in DNA methylation of POLGA. RNAseq analysis showed that differentiated HLC express hepatocyte-specific genes but also increased expression of inflammatory interferon genes. Differentiation in encapsulated cells showed suppression of inflammatory genes as well as increased expression of genes associated with hepatocyte function pathways and networks. This study demonstrates that an increase in classical hepatic gene expression can be achieved in HLC through encapsulation, although they fail to effectively regulate mtDNA copy number.

High BAALC copy numbers in peripheral blood prior to allogeneic transplantation predict early relapse in acute myeloid leukemia patients.

PubMed

Jentzsch, Madlen; Bill, Marius; Grimm, Juliane; Schulz, Julia; Goldmann, Karoline; Beinicke, Stefanie; Häntschel, Janine; Pönisch, Wolfram; Franke, Georg-Nikolaus; Vucinic, Vladan; Behre, Gerhard; Lange, Thoralf; Niederwieser, Dietger; Schwind, Sebastian

2017-10-20

High BAALC expression levels at acute myeloid leukemia diagnosis have been linked to adverse outcomes. Recent data indicate that high BAALC expression levels may also be used as marker for residual disease following acute myeloid leukemia treatment. Allogeneic hematopoietic stem cell transplantation (HSCT) offers a curative treatment for acute myeloid leukemia patients. However, disease recurrence remains a major clinical challenge and identification of high-risk patients prior to HSCT is crucial to improve outcomes. We performed absolute quantification of BAALC copy numbers in peripheral blood prior (median 7 days) to HSCT in complete remission (CR) or CR with incomplete peripheral recovery in 82 acute myeloid leukemia patients using digital droplet PCR (ddPCR) technology. An optimal cut-off of 0.14 BAALC / ABL1 copy numbers was determined and applied to define patients with high or low BAALC / ABL1 copy numbers. High pre-HSCT BAALC / ABL1 copy numbers significantly associated with higher cumulative incidence of relapse and shorter overall survival in univariable and multivariable models. Patients with high pre-HSCT BAALC / ABL1 copy numbers were more likely to experience relapse within 100 days after HSCT. Evaluation of pre-HSCT BAALC / ABL1 copy numbers in peripheral blood by ddPCR represents a feasible and rapid way to identify acute myeloid leukemia patients at high risk of early relapse after HSCT. The prognostic impact was also observed independently of other known clinical, genetic, and molecular prognosticators. In the future, prospective studies should evaluate whether acute myeloid leukemia patients with high pre-HSCT BAALC / ABL1 copy numbers benefit from additional treatment before or early intervention after HSCT.

High BAALC copy numbers in peripheral blood prior to allogeneic transplantation predict early relapse in acute myeloid leukemia patients

PubMed Central

Jentzsch, Madlen; Bill, Marius; Grimm, Juliane; Schulz, Julia; Goldmann, Karoline; Beinicke, Stefanie; Häntschel, Janine; Pönisch, Wolfram; Franke, Georg-Nikolaus; Vucinic, Vladan; Behre, Gerhard; Lange, Thoralf; Niederwieser, Dietger; Schwind, Sebastian

2017-01-01

High BAALC expression levels at acute myeloid leukemia diagnosis have been linked to adverse outcomes. Recent data indicate that high BAALC expression levels may also be used as marker for residual disease following acute myeloid leukemia treatment. Allogeneic hematopoietic stem cell transplantation (HSCT) offers a curative treatment for acute myeloid leukemia patients. However, disease recurrence remains a major clinical challenge and identification of high-risk patients prior to HSCT is crucial to improve outcomes. We performed absolute quantification of BAALC copy numbers in peripheral blood prior (median 7 days) to HSCT in complete remission (CR) or CR with incomplete peripheral recovery in 82 acute myeloid leukemia patients using digital droplet PCR (ddPCR) technology. An optimal cut-off of 0.14 BAALC/ABL1 copy numbers was determined and applied to define patients with high or low BAALC/ABL1 copy numbers. High pre-HSCT BAALC/ABL1 copy numbers significantly associated with higher cumulative incidence of relapse and shorter overall survival in univariable and multivariable models. Patients with high pre-HSCT BAALC/ABL1 copy numbers were more likely to experience relapse within 100 days after HSCT. Evaluation of pre-HSCT BAALC/ABL1 copy numbers in peripheral blood by ddPCR represents a feasible and rapid way to identify acute myeloid leukemia patients at high risk of early relapse after HSCT. The prognostic impact was also observed independently of other known clinical, genetic, and molecular prognosticators. In the future, prospective studies should evaluate whether acute myeloid leukemia patients with high pre-HSCT BAALC/ABL1 copy numbers benefit from additional treatment before or early intervention after HSCT. PMID:29152132

DNA Methylation Patterns in Normal Tissue Correlate more Strongly with Breast Cancer Status than Copy-Number Variants.

PubMed

Gao, Yang; Widschwendter, Martin; Teschendorff, Andrew E

2018-05-04

Normal tissue at risk of neoplastic transformation is characterized by somatic mutations, copy-number variation and DNA methylation changes. It is unclear however, which type of alteration may be more informative of cancer risk. We analyzed genome-wide DNA methylation and copy-number calls from the same DNA assay in a cohort of healthy breast samples and age-matched normal samples collected adjacent to breast cancer. Using statistical methods to adjust for cell type heterogeneity, we show that DNA methylation changes can discriminate normal-adjacent from normal samples better than somatic copy-number variants. We validate this important finding in an independent dataset. These results suggest that DNA methylation alterations in the normal cell of origin may offer better cancer risk prediction and early detection markers than copy-number changes. Copyright © 2018. Published by Elsevier B.V.

Molecular genetic heterogeneity in undifferentiated endometrial carcinomas.

PubMed

Rosa-Rosa, Juan M; Leskelä, Susanna; Cristóbal-Lana, Eva; Santón, Almudena; López-García, Ma Ángeles; Muñoz, Gloria; Pérez-Mies, Belen; Biscuola, Michele; Prat, Jaime; Esther, Oliva E; Soslow, Robert A; Matias-Guiu, Xavier; Palacios, Jose

2016-11-01

Undifferentiated and dedifferentiated endometrial carcinomas are rare and highly aggressive subtypes of uterine cancer, not well characterized at a molecular level. To investigate whether dedifferentiated carcinomas carry molecular genetic alterations similar to those of pure undifferentiated carcinomas, and to gain insight into the pathogenesis of these tumors, we selected a cohort of 18 undifferentiated endometrial carcinomas, 8 of them with a well-differentiated endometrioid carcinoma component (dedifferentiated endometrioid carcinomas), and studied them by immunohistochemistry and massive parallel and Sanger sequencing. Whole-exome sequencing of the endometrioid and undifferentiated components, as well as normal myometrium, was also carried out in one case. According to The Cancer Genome Atlas classification, we distributed 95% of the undifferentiated carcinomas in this series as follows: (a) hypermutated tumors with loss of any mismatch repair protein expression and microsatellite instability (eight cases, 45%); (b) ultramutated carcinomas carrying mutations in the exonuclease domain of POLE (two cases, 11%); (c) high copy number alterations (copy-number high) tumors group exhibiting only TP53 mutations and high number of alterations detected by FISH (two cases, 11%); and (d) low copy number alterations (copy-number low) tumors with molecular alterations typical of endometrioid endometrial carcinomas (five cases, 28%). Two of the latter cases, however, also had TP53 mutations and higher number of alterations detected by FISH and could have progressed to a copy-number high phenotype. Most dedifferentiated carcinomas belonged to the hypermutated group, whereas pure undifferentiated carcinomas shared molecular genetic alterations with copy-number low or copy-number high tumors. These results indicate that undifferentiated and dedifferentiated endometrial carcinomas are molecularly heterogeneous tumors, which may have prognostic value.

Molecular genetic heterogeneity in undifferentiated endometrial carcinomas

PubMed Central

Rosa-Rosa, J.M.; Leskelä, S.; Cristóbal-Lana, E.; Santón, A.; López-García, M.A.; Muñoz, G.; Pérez-Mies, B.; Biscuola, M; Prat, J.; Oliva, E.; Soslow, R.A.; Matias-Guiu, X.; Palacios, J.

2017-01-01

Undifferentiated and dedifferentiated endometrial carcinomas are rare and highly aggressive subtypes of uterine cancer, not well characterized at a molecular level. To investigate whether dedifferentiated carcinomas carry molecular genetic alterations similar to those of pure undifferentiated carcinomas, and to gain insight into the pathogenesis of these tumours, we selected a cohort of 18 undifferentiated endometrial carcinomas, 8 of them with a well differentiated endometrioid carcinoma component (dedifferentiated endometrioid carcinomas), and studied them by immunohistochemistry and massive parallel and Sanger sequencing. Whole exome sequencing of the endometrioid and undifferentiated components as well as normal myometrium, was also carried out in one case. According to The Cancer Genome Atlas classification, we distributed 95% of the undifferentiated carcinomas in this series as follows: a) hypermutated tumours with loss of any mismatch repair protein expression and microsatellite instability (eight cases, 45%); b) ultramutated carcinomas carrying mutations in the exonuclease domain of POLE (two cases, 11%); c) high copy number alterations (copy-number high) tumours group exhibiting only TP53 mutations and high number of alterations detected by FISH (two cases, 11%) ; and d) low copy number alterations (copy-number low) tumours with molecular alterations typical of endometrioid endometrial carcinomas (five cases, 28%). Two of the latter cases, however, also had TP53 mutations and higher number of alterations detected by FISH and could have progressed to a copy-number high phenotype. Most dedifferentiated carcinomas belonged to the hypermutated group whereas pure undifferentiated carcinomas shared molecular genetic alterations with copy-number low or copy-number high tumours. These results indicate that undifferentiated and dedifferentiated endometrial carcinomas are molecularly heterogeneous tumours, which may have prognostic value. PMID:27491810

Mitochondrial DNA in Residual Leukemia Cells in Cerebrospinal Fluid in Children with Acute Lymphoblastic Leukemia

PubMed Central

Egan, Kathryn; Kusao, Ian; Troelstrup, David; Agsalda, Melissa; Shiramizu, Bruce

2010-01-01

This feasibility study was designed to assess the ability to measure mitochondrial DNA (mtDNA) in cerebrospinal fluid (CSF) cells that contributed to minimal disease/persistent or residual disease (MD/PRD) from children with acute lymphoblastic leukemia (ALL). Increase in mtDNA copies in cancer cells has been suggested to play a role in MD/PRD. CSF as well as blood specimens from 6 children were assayed for MD/PRD and mtDNA copy numbers by quantitative real-time polymerase chain reaction. Of 7 MD/PRD-positive specimens, 6 had increased mtDNA copy numbers; while 11 MD/PRD-negative specimens had no increase in mtDNA copy numbers, p < 0.003. This is the first proof-of-concept study to measure mtDNA copy numbers in MD/PRD-positive CSF specimens from children with ALL. Increase of mtDNA copy numbers in MD/PRD childhood ALL cells and its significance as a mechanism for recurrence requires further investigation. Keywords Minimal residual disease; Acute lymphoblastic leukemia; Central nervous system; Cerebrospinal fluid; Mitochondria PMID:21331151

Molecular inversion probe assay for allelic quantitation

PubMed Central

Ji, Hanlee; Welch, Katrina

2010-01-01

Molecular inversion probe (MIP) technology has been demonstrated to be a robust platform for large-scale dual genotyping and copy number analysis. Applications in human genomic and genetic studies include the possibility of running dual germline genotyping and combined copy number variation ascertainment. MIPs analyze large numbers of specific genetic target sequences in parallel, relying on interrogation of a barcode tag, rather than direct hybridization of genomic DNA to an array. The MIP approach does not replace, but is complementary to many of the copy number technologies being performed today. Some specific advantages of MIP technology include: Less DNA required (37 ng vs. 250 ng), DNA quality less important, more dynamic range (amplifications detected up to copy number 60), allele specific information “cleaner” (less SNP crosstalk/contamination), and quality of markers better (fewer individual MIPs versus SNPs needed to identify copy number changes). MIPs can be considered a candidate gene (targeted whole genome) approach and can find specific areas of interest that otherwise may be missed with other methods. PMID:19488872

Use of autocorrelation scanning in DNA copy number analysis.

PubMed

Zhang, Liangcai; Zhang, Li

2013-11-01

Data quality is a critical issue in the analyses of DNA copy number alterations obtained from microarrays. It is commonly assumed that copy number alteration data can be modeled as piecewise constant and the measurement errors of different probes are independent. However, these assumptions do not always hold in practice. In some published datasets, we find that measurement errors are highly correlated between probes that interrogate nearby genomic loci, and the piecewise-constant model does not fit the data well. The correlated errors cause problems in downstream analysis, leading to a large number of DNA segments falsely identified as having copy number gains and losses. We developed a simple tool, called autocorrelation scanning profile, to assess the dependence of measurement error between neighboring probes. Autocorrelation scanning profile can be used to check data quality and refine the analysis of DNA copy number data, which we demonstrate in some typical datasets. lzhangli@mdanderson.org. Supplementary data are available at Bioinformatics online.

Copy number analysis reveals a novel multiexon deletion of the COLQ gene in congenital myasthenia.

PubMed

Wang, Wei; Wu, Yanhong; Wang, Chen; Jiao, Jinsong; Klein, Christopher J

2016-12-01

Congenital myasthenic syndrome (CMS) is genetically and clinically heterogeneous. 1 Despite a considerable number of causal genes discovered, many patients are left without a specific diagnosis after genetic testing. The presumption is that novel genes yet to be discovered will account for the majority of such patients. However, it is also possible that we are neglecting a type of genetic variation: copy number changes (>50 bp) as causal for some of these patients. Next-generation sequencing (NGS) can simultaneously screen all known causal genes 2 and is increasingly being validated to have a potential to identify copy number changes. 3 We present a CMS case who did not receive a genetic diagnosis from previous Sanger sequencing, but through a novel copy number analysis algorithm integrated into our targeted NGS panel, we discovered a novel copy number mutation in the COLQ gene and made a genetic diagnosis. This discovery expands the genotype-phenotype correlation of CMS, leads to improved genetic counsel, and allows for specific pharmacologic treatment. 1 .

Prognostic Impact of PHIP Copy Number in Melanoma: Linkage to Ulceration

PubMed Central

Nosrati, Mehdi; Tong, Schuyler; Wu, Clayton; Thummala, Suresh; Dar, Altaf A.; Leong, Stanley P.L.; Cleaver, James E.; Sagebiel, Richard W.; Miller, James R.; Kashani-Sabet, Mohammed

2013-01-01

Ulceration is an important prognostic factor in melanoma whose biologic basis is poorly understood. Here we assessed the prognostic impact of pleckstrin homology domain-interacting protein (PHIP) copy number and its relationship to ulceration. PHIP copy number was determined using fluorescence in situ hybridization (FISH) in a tissue microarray cohort of 238 melanomas. Elevated PHIP copy number was associated with significantly reduced DMFS (P = 0.01) and DSS (P = 0.009) by Kaplan-Meier analyses. PHIP FISH scores were independently predictive of DMFS (P = 0.03) and DSS (P = 0.03). Increased PHIP copy number was an independent predictor of ulceration status (P = 0.04). The combined impact of increased PHIP copy number and tumor vascularity on ulceration status was highly significant (P< 0.0001). Stable suppression of PHIP in human melanoma cells resulted in significantly reduced glycolytic activity in vitro, with lower expression of LDH5, HIF1A, and VEGF, and was accompanied by reduced microvessel density in vivo. These results provide further support for PHIP as a molecular prognostic marker of melanoma, and reveal a significant linkage between PHIP levels and ulceration. Moreover, they suggest that ulceration may be driven by increased glycolysis and angiogenesis. PMID:24005052

Chemiluminescent Detection for Estimating Relative Copy Numbers of Porcine Endogenous Retrovirus Proviruses from Chinese Minipigs Based on Magnetic Nanoparticles.

PubMed

Yang, Haowen; Liu, Ming; Zhou, Bingcong; Deng, Yan; He, Nongyue; Jiang, Hesheng; Guo, Yafen; Lan, Ganqiu; Jiang, Qinyang; Yang, Xiurong; Li, Zhiyang

2016-06-01

Chinese Bama minipigs could be potential donors for the supply of xenografts because they are genetically stable, highly inbred, and inexpensive. However, porcine endogenous retrovirus (PERV) is commonly integrated in pig genomes and could cause a cross-species infection by xenotransplantation. For screening out the pigs with low copy numbers of PERV proviruses, we have developed a novel semiquantitative analysis approach based on magnetic nanoparticles (MNPs) and chemiluminescence (CL) for estimating relative copy numbers (RCNs) of PERV proviruses in Chinese Bama minipigs. The CL intensities of PERV proviruses and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were respectively determined with this method, and the RCNs of PERV proviruses were calculated by the equation: RCN of PERV provirus = CL intensity of PERV provirus/CL intensity of GAPDH. The results showed that PERVs were integrated in the genomes of Bama minipigs at different copy numbers, and the copy numbers of PERV-C subtype were greatly low. Two Bama minipigs with low copy numbers of PERV proviruses were detected out and could be considered as xenograft donor candidates. Although only semiquantitation can be achieved, this approach has potential for screening out safe and suitable pig donors for xenotransplantation.

Insertional Polymorphisms of Endogenous Feline Leukemia Viruses

PubMed Central

Roca, Alfred L.; Nash, William G.; Menninger, Joan C.; Murphy, William J.; O'Brien, Stephen J.

2005-01-01

The number, chromosomal distribution, and insertional polymorphisms of endogenous feline leukemia viruses (enFeLVs) were determined in four domestic cats (Burmese, Egyptian Mau, Persian, and nonbreed) using fluorescent in situ hybridization and radiation hybrid mapping. Twenty-nine distinct enFeLV loci were detected across 12 of the 18 autosomes. Each cat carried enFeLV at only 9 to 16 of the loci, and many loci were heterozygous for presence of the provirus. Thus, an average of 19 autosomal copies of enFeLV were present per cat diploid genome. Only five of the autosomal enFeLV sites were present in all four cats, and at only one autosomal locus, B4q15, was enFeLV present in both homologues of all four cats. A single enFeLV occurred in the X chromosome of the Burmese cat, while three to five enFeLV proviruses occurred in each Y chromosome. The X chromosome and nine autosomal enFeLV loci were telomeric, suggesting that ectopic recombination between nonhomologous subtelomeres may contribute to enFeLV distribution. Since endogenous FeLVs may affect the infectiousness or pathogenicity of exogenous FeLVs, genomic variation in enFeLVs represents a candidate for genetic influences on FeLV leukemogenesis in cats. PMID:15767400

Heme Oxygenase-1 and 2 Common Genetic Variants and Risk for Restless Legs Syndrome

PubMed Central

García-Martín, Elena; Jiménez-Jiménez, Félix Javier; Alonso-Navarro, Hortensia; Martínez, Carmen; Zurdo, Martín; Turpín-Fenoll, Laura; Millán-Pascual, Jorge; Adeva-Bartolomé, Teresa; Cubo, Esther; Navacerrada, Francisco; Rojo-Sebastián, Ana; Rubio, Lluisa; Ortega-Cubero, Sara; Pastor, Pau; Calleja, Marisol; Plaza-Nieto, José Francisco; Pilo-de-la-Fuente, Belén; Arroyo-Solera, Margarita; García-Albea, Esteban; Agúndez, José A.G.

2015-01-01

Abstract Several neurochemical, neuropathological, neuroimaging, and experimental data, suggest that iron deficiency plays an important role in the pathophysiology of restless legs syndrome (RLS). Heme-oxygenases (HMOX) are an important defensive mechanism against oxidative stress, mainly through the degradation of heme to biliverdin, free iron, and carbon monoxide. We analyzed whether HMOX1 and HMOX2 genes are related with the risk to develop RLS. We analyzed the distribution of genotypes and allelic frequencies of the HMOX1 rs2071746, HMOX1 rs2071747, HMOX2 rs2270363, and HMOX2 rs1051308 SNPs, as well as the presence of Copy number variations (CNVs) of these genes in 205 subjects RLS and 445 healthy controls. The frequencies of rs2071746TT genotype and rs2071746T allelic variant were significantly lower in RLS patients than that in controls, although the other 3 studied SNPs did not differ between RLS patients and controls. None of the studied polymorphisms influenced the disease onset, severity of RLS, family history of RLS, serum ferritin levels, or response to dopaminergic agonist, clonazepam or GABAergic drugs. The present study suggests a weak association between HMOX1 rs2071746 polymorphism and the risk to develop RLS in the Spanish population. PMID:26313808

Heme Oxygenase-1 and 2 Common Genetic Variants and Risk for Restless Legs Syndrome.

PubMed

García-Martín, Elena; Jiménez-Jiménez, Félix Javier; Alonso-Navarro, Hortensia; Martínez, Carmen; Zurdo, Martín; Turpín-Fenoll, Laura; Millán-Pascual, Jorge; Adeva-Bartolomé, Teresa; Cubo, Esther; Navacerrada, Francisco; Rojo-Sebastián, Ana; Rubio, Lluisa; Ortega-Cubero, Sara; Pastor, Pau; Calleja, Marisol; Plaza-Nieto, José Francisco; Pilo-de-la-Fuente, Belén; Arroyo-Solera, Margarita; García-Albea, Esteban; Agúndez, José A G

2015-08-01

Several neurochemical, neuropathological, neuroimaging, and experimental data, suggest that iron deficiency plays an important role in the pathophysiology of restless legs syndrome (RLS). Heme-oxygenases (HMOX) are an important defensive mechanism against oxidative stress, mainly through the degradation of heme to biliverdin, free iron, and carbon monoxide. We analyzed whether HMOX1 and HMOX2 genes are related with the risk to develop RLS.We analyzed the distribution of genotypes and allelic frequencies of the HMOX1 rs2071746, HMOX1 rs2071747, HMOX2 rs2270363, and HMOX2 rs1051308 SNPs, as well as the presence of Copy number variations (CNVs) of these genes in 205 subjects RLS and 445 healthy controls.The frequencies of rs2071746TT genotype and rs2071746T allelic variant were significantly lower in RLS patients than that in controls, although the other 3 studied SNPs did not differ between RLS patients and controls. None of the studied polymorphisms influenced the disease onset, severity of RLS, family history of RLS, serum ferritin levels, or response to dopaminergic agonist, clonazepam or GABAergic drugs.The present study suggests a weak association between HMOX1 rs2071746 polymorphism and the risk to develop RLS in the Spanish population.

Organization and evolution of hsp70 clusters strikingly differ in two species of Stratiomyidae (Diptera) inhabiting thermally contrasting environments.

PubMed

Garbuz, David G; Yushenova, Irina A; Zatsepina, Olga G; Przhiboro, Andrey A; Bettencourt, Brian R; Evgen'ev, Michael B

2011-03-22

Previously, we described the heat shock response in dipteran species belonging to the family Stratiomyidae that develop in thermally and chemically contrasting habitats including highly aggressive ones. Although all species studied exhibit high constitutive levels of Hsp70 accompanied by exceptionally high thermotolerance, we also detected characteristic interspecies differences in heat shock protein (Hsp) expression and survival after severe heat shock. Here, we analyzed genomic libraries from two Stratiomyidae species from thermally and chemically contrasting habitats and determined the structure and organization of their hsp70 clusters. Although the genomes of both species contain similar numbers of hsp70 genes, the spatial distribution of hsp70 copies differs characteristically. In a population of the eurytopic species Stratiomys singularior, which exists in thermally variable and chemically aggressive (hypersaline) conditions, the hsp70 copies form a tight cluster with approximately equal intergenic distances. In contrast, in a population of the stenotopic Oxycera pardalina that dwells in a stable cold spring, we did not find hsp70 copies in tandem orientation. In this species, the distance between individual hsp70 copies in the genome is very large, if they are linked at all. In O. pardalina we detected the hsp68 gene located next to a hsp70 copy in tandem orientation. Although the hsp70 coding sequences of S. singularior are highly homogenized via conversion, the structure and general arrangement of the hsp70 clusters are highly polymorphic, including gross aberrations, various deletions in intergenic regions, and insertion of incomplete Mariner transposons in close vicinity to the 3'-UTRs. The hsp70 gene families in S. singularior and O. pardalina evolved quite differently from one another. We demonstrated clear evidence of homogenizing gene conversion in the S. singularior hsp70 genes, which form tight clusters in this species. In the case of the other species, O. pardalina, we found no clear trace of concerted evolution for the dispersed hsp70 genes. Furthermore, in the latter species we detected hsp70 pseudogenes, representing a hallmark of the birth-and-death process.

Organization and evolution of hsp70 clusters strikingly differ in two species of Stratiomyidae (Diptera) inhabiting thermally contrasting environments

PubMed Central

2011-01-01

Background Previously, we described the heat shock response in dipteran species belonging to the family Stratiomyidae that develop in thermally and chemically contrasting habitats including highly aggressive ones. Although all species studied exhibit high constitutive levels of Hsp70 accompanied by exceptionally high thermotolerance, we also detected characteristic interspecies differences in heat shock protein (Hsp) expression and survival after severe heat shock. Here, we analyzed genomic libraries from two Stratiomyidae species from thermally and chemically contrasting habitats and determined the structure and organization of their hsp70 clusters. Results Although the genomes of both species contain similar numbers of hsp70 genes, the spatial distribution of hsp70 copies differs characteristically. In a population of the eurytopic species Stratiomys singularior, which exists in thermally variable and chemically aggressive (hypersaline) conditions, the hsp70 copies form a tight cluster with approximately equal intergenic distances. In contrast, in a population of the stenotopic Oxycera pardalina that dwells in a stable cold spring, we did not find hsp70 copies in tandem orientation. In this species, the distance between individual hsp70 copies in the genome is very large, if they are linked at all. In O. pardalina we detected the hsp68 gene located next to a hsp70 copy in tandem orientation. Although the hsp70 coding sequences of S. singularior are highly homogenized via conversion, the structure and general arrangement of the hsp70 clusters are highly polymorphic, including gross aberrations, various deletions in intergenic regions, and insertion of incomplete Mariner transposons in close vicinity to the 3'-UTRs. Conclusions The hsp70 gene families in S. singularior and O. pardalina evolved quite differently from one another. We demonstrated clear evidence of homogenizing gene conversion in the S. singularior hsp70 genes, which form tight clusters in this species. In the case of the other species, O. pardalina, we found no clear trace of concerted evolution for the dispersed hsp70 genes. Furthermore, in the latter species we detected hsp70 pseudogenes, representing a hallmark of the birth-and-death process. PMID:21426536

An SNP resource for rice genetics and breeding based on subspecies indica and japonica genome alignments.

PubMed

Feltus, F Alex; Wan, Jun; Schulze, Stefan R; Estill, James C; Jiang, Ning; Paterson, Andrew H

2004-09-01

Dense coverage of the rice genome with polymorphic DNA markers is an invaluable tool for DNA marker-assisted breeding, positional cloning, and a wide range of evolutionary studies. We have aligned drafts of two rice subspecies, indica and japonica, and analyzed levels and patterns of genetic diversity. After filtering multiple copy and low quality sequence, 408,898 candidate DNA polymorphisms (SNPs/INDELs) were discerned between the two subspecies. These filters have the consequence that our data set includes only a subset of the available SNPs (in particular excluding large numbers of SNPs that may occur between repetitive DNA alleles) but increase the likelihood that this subset is useful: Direct sequencing suggests that 79.8% +/- 7.5% of the in silico SNPs are real. The SNP sample in our database is not randomly distributed across the genome. In fact, 566 rice genomic regions had unusually high (328 contigs/48.6 Mb/13.6% of genome) or low (237 contigs/64.7 Mb/18.1% of genome) polymorphism rates. Many SNP-poor regions were substantially longer than most SNP-rich regions, covering up to 4 Mb, and possibly reflecting introgression between the respective gene pools that may have occurred hundreds of years ago. Although 46.2% +/- 8.3% of the SNPs differentiate other pairs of japonica and indica genotypes, SNP rates in rice were not predictive of evolutionary rates for corresponding genes in another grass species, sorghum. The data set is freely available at http://www.plantgenome.uga.edu/snp.

An SNP Resource for Rice Genetics and Breeding Based on Subspecies Indica and Japonica Genome Alignments

PubMed Central

Feltus, F. Alex; Wan, Jun; Schulze, Stefan R.; Estill, James C.; Jiang, Ning; Paterson, Andrew H.

2004-01-01

Dense coverage of the rice genome with polymorphic DNA markers is an invaluable tool for DNA marker-assisted breeding, positional cloning, and a wide range of evolutionary studies. We have aligned drafts of two rice subspecies, indica and japonica, and analyzed levels and patterns of genetic diversity. After filtering multiple copy and low quality sequence, 408,898 candidate DNA polymorphisms (SNPs/INDELs) were discerned between the two subspecies. These filters have the consequence that our data set includes only a subset of the available SNPs (in particular excluding large numbers of SNPs that may occur between repetitive DNA alleles) but increase the likelihood that this subset is useful: Direct sequencing suggests that 79.8% ± 7.5% of the in silico SNPs are real. The SNP sample in our database is not randomly distributed across the genome. In fact, 566 rice genomic regions had unusually high (328 contigs/48.6 Mb/13.6% of genome) or low (237 contigs/64.7 Mb/18.1% of genome) polymorphism rates. Many SNP-poor regions were substantially longer than most SNP-rich regions, covering up to 4 Mb, and possibly reflecting introgression between the respective gene pools that may have occurred hundreds of years ago. Although 46.2% ± 8.3% of the SNPs differentiate other pairs of japonica and indica genotypes, SNP rates in rice were not predictive of evolutionary rates for corresponding genes in another grass species, sorghum. The data set is freely available at http://www.plantgenome.uga.edu/snp. PMID:15342564

Forensic strategy to ensure the quality of sequencing data of mitochondrial DNA in highly degraded samples.

PubMed

Adachi, Noboru; Umetsu, Kazuo; Shojo, Hideki

2014-01-01

Mitochondrial DNA (mtDNA) is widely used for DNA analysis of highly degraded samples because of its polymorphic nature and high number of copies in a cell. However, as endogenous mtDNA in deteriorated samples is scarce and highly fragmented, it is not easy to obtain reliable data. In the current study, we report the risks of direct sequencing mtDNA in highly degraded material, and suggest a strategy to ensure the quality of sequencing data. It was observed that direct sequencing data of the hypervariable segment (HVS) 1 by using primer sets that generate an amplicon of 407 bp (long-primer sets) was different from results obtained by using newly designed primer sets that produce an amplicon of 120-139 bp (mini-primer sets). The data aligned with the results of mini-primer sets analysis in an amplicon length-dependent manner; the shorter the amplicon, the more evident the endogenous sequence became. Coding region analysis using multiplex amplified product-length polymorphisms revealed the incongruence of single nucleotide polymorphisms between the coding region and HVS 1 caused by contamination with exogenous mtDNA. Although the sequencing data obtained using long-primer sets turned out to be erroneous, it was unambiguous and reproducible. These findings suggest that PCR primers that produce amplicons shorter than those currently recognized should be used for mtDNA analysis in highly degraded samples. Haplogroup motif analysis of the coding region and HVS should also be performed to improve the reliability of forensic mtDNA data. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Multiplex APLP System for High-Resolution Haplogrouping of Extremely Degraded East-Asian Mitochondrial DNAs

PubMed Central

Kakuda, Tsuneo; Shojo, Hideki; Tanaka, Mayumi; Nambiar, Phrabhakaran; Minaguchi, Kiyoshi; Umetsu, Kazuo; Adachi, Noboru

2016-01-01

Mitochondrial DNA (mtDNA) serves as a powerful tool for exploring matrilineal phylogeographic ancestry, as well as for analyzing highly degraded samples, because of its polymorphic nature and high copy numbers per cell. The recent advent of complete mitochondrial genome sequencing has led to improved techniques for phylogenetic analyses based on mtDNA, and many multiplex genotyping methods have been developed for the hierarchical analysis of phylogenetically important mutations. However, few high-resolution multiplex genotyping systems for analyzing East-Asian mtDNA can be applied to extremely degraded samples. Here, we present a multiplex system for analyzing mitochondrial single nucleotide polymorphisms (mtSNPs), which relies on a novel amplified product-length polymorphisms (APLP) method that uses inosine-flapped primers and is specifically designed for the detailed haplogrouping of extremely degraded East-Asian mtDNAs. We used fourteen 6-plex polymerase chain reactions (PCRs) and subsequent electrophoresis to examine 81 haplogroup-defining SNPs and 3 insertion/deletion sites, and we were able to securely assign the studied mtDNAs to relevant haplogroups. Our system requires only 1×10−13 g (100 fg) of crude DNA to obtain a full profile. Owing to its small amplicon size (<110 bp), this new APLP system was successfully applied to extremely degraded samples for which direct sequencing of hypervariable segments using mini-primer sets was unsuccessful, and proved to be more robust than conventional APLP analysis. Thus, our new APLP system is effective for retrieving reliable data from extremely degraded East-Asian mtDNAs. PMID:27355212

Distinctive Epstein-Barr virus variants associated with benign and malignant pediatric pathologies: LMP1 sequence characterization and linkage with other viral gene polymorphisms.

PubMed

Lorenzetti, Mario Alejandro; Gantuz, Magdalena; Altcheh, Jaime; De Matteo, Elena; Chabay, Paola Andrea; Preciado, María Victoria

2012-03-01

The ubiquitous Epstein-Barr virus (EBV) is related to the development of lymphoma and is also the etiological agent for infectious mononucleosis (IM). Sequence variations in the gene encoding LMP1 have been deeply studied in different pathologies and geographic regions. Controversial results propose the existence of tumor-related variants, while others argued in favor of a geographical distribution of these variants. Reports assessing EBV variants in IM were performed in adult patients who displayed multiple variant infections. In the present study, LMP1 variants in 15 pediatric patients with IM and 20 pediatric patients with EBV-associated lymphomas from Argentina were analyzed as representatives of benign and malignant infections in children, respectively. A 3-month follow-up study of LMP1 variants in peripheral blood cells and in oral secretions of patients with IM was performed. Moreover, an integrated linkage analysis was performed with variants of EBNA1 and the promoter region of BZLF1. Similar sequence polymorphisms were detected in both pathological conditions, IM and lymphoma, but these differ from those previously described in healthy donors from Argentina and Brazil. The results suggest that certain LMP1 polymorphisms, namely, the 30-bp deletion and high copy number of the 33-bp repeats, are associated with EBV-related pathologies, either benign or malignant, instead of just being tumor related. Additionally, this is the first study to describe the Alaskan variant in EBV-related lymphomas that previously was restricted to nasopharyngeal carcinomas from North America.

Distinctive Epstein-Barr Virus Variants Associated with Benign and Malignant Pediatric Pathologies: LMP1 Sequence Characterization and Linkage with Other Viral Gene Polymorphisms

PubMed Central

Gantuz, Magdalena; Altcheh, Jaime; De Matteo, Elena; Chabay, Paola Andrea; Preciado, María Victoria

2012-01-01

The ubiquitous Epstein-Barr virus (EBV) is related to the development of lymphoma and is also the etiological agent for infectious mononucleosis (IM). Sequence variations in the gene encoding LMP1 have been deeply studied in different pathologies and geographic regions. Controversial results propose the existence of tumor-related variants, while others argued in favor of a geographical distribution of these variants. Reports assessing EBV variants in IM were performed in adult patients who displayed multiple variant infections. In the present study, LMP1 variants in 15 pediatric patients with IM and 20 pediatric patients with EBV-associated lymphomas from Argentina were analyzed as representatives of benign and malignant infections in children, respectively. A 3-month follow-up study of LMP1 variants in peripheral blood cells and in oral secretions of patients with IM was performed. Moreover, an integrated linkage analysis was performed with variants of EBNA1 and the promoter region of BZLF1. Similar sequence polymorphisms were detected in both pathological conditions, IM and lymphoma, but these differ from those previously described in healthy donors from Argentina and Brazil. The results suggest that certain LMP1 polymorphisms, namely, the 30-bp deletion and high copy number of the 33-bp repeats, are associated with EBV-related pathologies, either benign or malignant, instead of just being tumor related. Additionally, this is the first study to describe the Alaskan variant in EBV-related lymphomas that previously was restricted to nasopharyngeal carcinomas from North America. PMID:22205789

Multiplex APLP System for High-Resolution Haplogrouping of Extremely Degraded East-Asian Mitochondrial DNAs.

PubMed

Kakuda, Tsuneo; Shojo, Hideki; Tanaka, Mayumi; Nambiar, Phrabhakaran; Minaguchi, Kiyoshi; Umetsu, Kazuo; Adachi, Noboru

2016-01-01

Mitochondrial DNA (mtDNA) serves as a powerful tool for exploring matrilineal phylogeographic ancestry, as well as for analyzing highly degraded samples, because of its polymorphic nature and high copy numbers per cell. The recent advent of complete mitochondrial genome sequencing has led to improved techniques for phylogenetic analyses based on mtDNA, and many multiplex genotyping methods have been developed for the hierarchical analysis of phylogenetically important mutations. However, few high-resolution multiplex genotyping systems for analyzing East-Asian mtDNA can be applied to extremely degraded samples. Here, we present a multiplex system for analyzing mitochondrial single nucleotide polymorphisms (mtSNPs), which relies on a novel amplified product-length polymorphisms (APLP) method that uses inosine-flapped primers and is specifically designed for the detailed haplogrouping of extremely degraded East-Asian mtDNAs. We used fourteen 6-plex polymerase chain reactions (PCRs) and subsequent electrophoresis to examine 81 haplogroup-defining SNPs and 3 insertion/deletion sites, and we were able to securely assign the studied mtDNAs to relevant haplogroups. Our system requires only 1×10-13 g (100 fg) of crude DNA to obtain a full profile. Owing to its small amplicon size (<110 bp), this new APLP system was successfully applied to extremely degraded samples for which direct sequencing of hypervariable segments using mini-primer sets was unsuccessful, and proved to be more robust than conventional APLP analysis. Thus, our new APLP system is effective for retrieving reliable data from extremely degraded East-Asian mtDNAs.

Intra-isolate genome variation in arbuscular mycorrhizal fungi persists in the transcriptome.

PubMed

Boon, E; Zimmerman, E; Lang, B F; Hijri, M

2010-07-01

Arbuscular mycorrhizal fungi (AMF) are heterokaryotes with an unusual genetic makeup. Substantial genetic variation occurs among nuclei within a single mycelium or isolate. AMF reproduce through spores that contain varying fractions of this heterogeneous population of nuclei. It is not clear whether this genetic variation on the genome level actually contributes to the AMF phenotype. To investigate the extent to which polymorphisms in nuclear genes are transcribed, we analysed the intra-isolate genomic and cDNA sequence variation of two genes, the large subunit ribosomal RNA (LSU rDNA) of Glomus sp. DAOM-197198 (previously known as G. intraradices) and the POL1-like sequence (PLS) of Glomus etunicatum. For both genes, we find high sequence variation at the genome and transcriptome level. Reconstruction of LSU rDNA secondary structure shows that all variants are functional. Patterns of PLS sequence polymorphism indicate that there is one functional gene copy, PLS2, which is preferentially transcribed, and one gene copy, PLS1, which is a pseudogene. This is the first study that investigates AMF intra-isolate variation at the transcriptome level. In conclusion, it is possible that, in AMF, multiple nuclear genomes contribute to a single phenotype.

An evaluation of new and established methods to determine T‐DNA copy number and homozygosity in transgenic plants.

PubMed Central

Głowacka, Katarzyna; Kromdijk, Johannes; Leonelli, Lauriebeth; Niyogi, Krishna K.; Clemente, Tom E.

2016-01-01

Abstract Stable transformation of plants is a powerful tool for hypothesis testing. A rapid and reliable evaluation method of the transgenic allele for copy number and homozygosity is vital in analysing these transformations. Here the suitability of Southern blot analysis, thermal asymmetric interlaced (TAIL‐)PCR, quantitative (q)PCR and digital droplet (dd)PCR to estimate T‐DNA copy number, locus complexity and homozygosity were compared in transgenic tobacco. Southern blot analysis and ddPCR on three generations of transgenic offspring with contrasting zygosity and copy number were entirely consistent, whereas TAIL‐PCR often underestimated copy number. qPCR deviated considerably from the Southern blot results and had lower precision and higher variability than ddPCR. Comparison of segregation analyses and ddPCR of T1 progeny from 26 T0 plants showed that at least 19% of the lines carried multiple T‐DNA insertions per locus, which can lead to unstable transgene expression. Segregation analyses failed to detect these multiple copies, presumably because of their close linkage. This shows the importance of routine T‐DNA copy number estimation. Based on our results, ddPCR is the most suitable method, because it is as reliable as Southern blot analysis yet much faster. A protocol for this application of ddPCR to large plant genomes is provided. PMID:26670088

Mitochondrial DNA copy numbers in pyramidal neurons are decreased and mitochondrial biogenesis transcriptome signaling is disrupted in Alzheimer's disease hippocampi.

PubMed

Rice, Ann C; Keeney, Paula M; Algarzae, Norah K; Ladd, Amy C; Thomas, Ravindar R; Bennett, James P

2014-01-01

Alzheimer's disease (AD) is the major cause of adult-onset dementia and is characterized in its pre-diagnostic stage by reduced cerebral cortical glucose metabolism and in later stages by reduced cortical oxygen uptake, implying reduced mitochondrial respiration. Using quantitative PCR we determined the mitochondrial DNA (mtDNA) gene copy numbers from multiple groups of 15 or 20 pyramidal neurons, GFAP(+) astrocytes and dentate granule neurons isolated using laser capture microdissection, and the relative expression of mitochondrial biogenesis (mitobiogenesis) genes in hippocampi from 10 AD and 9 control (CTL) cases. AD pyramidal but not dentate granule neurons had significantly reduced mtDNA copy numbers compared to CTL neurons. Pyramidal neuron mtDNA copy numbers in CTL, but not AD, positively correlated with cDNA levels of multiple mitobiogenesis genes. In CTL, but not in AD, hippocampal cDNA levels of PGC1α were positively correlated with multiple downstream mitobiogenesis factors. Mitochondrial DNA copy numbers in pyramidal neurons did not correlate with hippocampal Aβ1-42 levels. After 48 h exposure of H9 human neural stem cells to the neurotoxic fragment Aβ25-35, mtDNA copy numbers were not significantly altered. In summary, AD postmortem hippocampal pyramidal neurons have reduced mtDNA copy numbers. Mitochondrial biogenesis pathway signaling relationships are disrupted in AD, but are mostly preserved in CTL. Our findings implicate complex alterations of mitochondria-host cell relationships in AD.

Copy number variation of human AMY1 is a minor contributor to variation in salivary amylase expression and activity.

PubMed

Carpenter, Danielle; Mitchell, Laura M; Armour, John A L

2017-02-20

Salivary amylase in humans is encoded by the copy variable gene AMY1 in the amylase gene cluster on chromosome 1. Although the role of salivary amylase is well established, the consequences of the copy number variation (CNV) at AMY1 on salivary amylase protein production are less well understood. The amylase gene cluster is highly structured with a fundamental difference between odd and even AMY1 copy number haplotypes. In this study, we aimed to explore, in samples from 119 unrelated individuals, not only the effects of AMY1 CNV on salivary amylase protein expression and amylase enzyme activity but also whether there is any evidence for underlying difference between the common haplotypes containing odd numbers of AMY1 and even copy number haplotypes. AMY1 copy number was significantly correlated with the variation observed in salivary amylase production (11.7% of variance, P < 0.0005) and enzyme activity (13.6% of variance, P < 0.0005) but did not explain the majority of observed variation between individuals. AMY1-odd and AMY1-even haplotypes showed a different relationship between copy number and expression levels, but the difference was not statistically significant (P = 0.052). Production of salivary amylase is correlated with AMY1 CNV, but the majority of interindividual variation comes from other sources. Long-range haplotype structure may affect expression, but this was not significant in our data.

Polycomb repressive complex 1 provides a molecular explanation for repeat copy number dependency in FSHD muscular dystrophy.

PubMed

Casa, Valentina; Runfola, Valeria; Micheloni, Stefano; Aziz, Arif; Dilworth, F Jeffrey; Gabellini, Davide

2017-02-15

Repression of repetitive elements is crucial to preserve genome integrity and has been traditionally ascribed to constitutive heterochromatin pathways. FacioScapuloHumeral Muscular Dystrophy (FSHD), one of the most common myopathies, is characterized by a complex interplay of genetic and epigenetic events. The main FSHD form is linked to a reduced copy number of the D4Z4 macrosatellite repeat on 4q35, causing loss of silencing and aberrant expression of the D4Z4-embedded DUX4 gene leading to disease. By an unknown mechanism, D4Z4 copy-number correlates with FSHD phenotype. Here we show that the DUX4 proximal promoter (DUX4p) is sufficient to nucleate the enrichment of both constitutive and facultative heterochromatin components and to mediate a copy-number dependent gene silencing. We found that both the CpG/GC dense DNA content and the repetitive nature of DUX4p arrays are important for their repressive ability. We showed that DUX4p mediates a copy number-dependent Polycomb Repressive Complex 1 (PRC1) recruitment, which is responsible for the copy-number dependent gene repression. Overall, we directly link genetic and epigenetic defects in FSHD by proposing a novel molecular explanation for the copy number-dependency in FSHD pathogenesis, and offer insight into the molecular functions of repeats in chromatin regulation. © The Author 2016. Published by Oxford University Press.

Mitochondrial DNA copy number is associated with risk of head and neck squamous cell carcinoma in Chinese population.

PubMed

Wang, Lihua; Lv, Hong; Ji, Pei; Zhu, Xun; Yuan, Hua; Jin, Guangfu; Dai, Juncheng; Hu, Zhibin; Su, Yuxiong; Ma, Hongxia

2018-04-19

Mitochondria show the special role in cellular bioenergy and many essential physiological activities. Previous researches have suggested that variations of mitochondrial DNA copy number contribute to development of different types of carcinomas. However, the relationship of mtDNA copy number in peripheral blood leukocytes (PBLs) with the risk of head and neck squamous cell carcinoma (HNSCC) is still inconclusive. We investigated the association of mtDNA with HNSCC risk through a case-control study including 570 HNSCC cases and 597 cancer-free controls. mtDNA copy number in PBLs was measured by real-time qPCR. Logistic regression was performed to estimate the association between the mtDNA copy number in PBLs and HNSCC risk. A U-shaped relation between the mtDNA copy number and HNSCC risk was found. Compared with those in the second quartile group, the adjusted odds ratios (ORs) and 95% confidence interval (CI) for those in the first and the forth quartile groups were 1.95 (1.37-2.76) and 2.16 (1.53-3.04), respectively. Using restricted cubic spline analysis, we confirmed such a significant U-shaped relation. Furthermore, the U-shaped association remained significant in different subgroups stratified by age, gender, tobacco smoking, and alcohol consumption. Both extremely low and high mtDNA copy numbers had significant associations with the increased HNSCC risk. © 2018 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Optical mapping and sequencing of the Escherichia coli KO11 genome reveal extensive chromosomal rearrangements, and multiple tandem copies of the Zymomonas mobilis pdc and adhB genes.

PubMed

Turner, Peter C; Yomano, Lorraine P; Jarboe, Laura R; York, Sean W; Baggett, Christy L; Moritz, Brélan E; Zentz, Emily B; Shanmugam, K T; Ingram, Lonnie O

2012-04-01

Escherichia coli KO11 (ATCC 55124) was engineered in 1990 to produce ethanol by chromosomal insertion of the Zymomonas mobilis pdc and adhB genes into E. coli W (ATCC 9637). KO11FL, our current laboratory version of KO11, and its parent E. coli W were sequenced, and contigs assembled into genomic sequences using optical NcoI restriction maps as templates. E. coli W contained plasmids pRK1 (102.5 kb) and pRK2 (5.4 kb), but KO11FL only contained pRK2. KO11FL optical maps made with AflII and with BamHI showed a tandem repeat region, consisting of at least 20 copies of a 10-kb unit. The repeat region was located at the insertion site for the pdc, adhB, and chloramphenicol-resistance genes. Sequence coverage of these genes was about 25-fold higher than average, consistent with amplification of the foreign genes that were inserted as circularized DNA. Selection for higher levels of chloramphenicol resistance originally produced strains with higher pdc and adhB expression, and hence improved fermentation performance, by increasing the gene copy number. Sequence data for an earlier version of KO11, ATCC 55124, indicated that multiple copies of pdc adhB were present. Comparison of the W and KO11FL genomes showed large inversions and deletions in KO11FL, mostly enabled by IS10, which is absent from W but present at 30 sites in KO11FL. The early KO11 strain ATCC 55124 had no rearrangements, contained only one IS10, and lacked most accumulated single nucleotide polymorphisms (SNPs) present in KO11FL. Despite rearrangements and SNPs in KO11FL, fermentation performance was equal to that of ATCC 55124.

The Origin of Mutants Under Selection: How Natural Selection Mimics Mutagenesis (Adaptive Mutation)

PubMed Central

Maisnier-Patin, Sophie; Roth, John R.

2015-01-01

Selection detects mutants but does not cause mutations. Contrary to this dictum, Cairns and Foster plated a leaky lac mutant of Escherichia coli on lactose medium and saw revertant (Lac+) colonies accumulate with time above a nongrowing lawn. This result suggested that bacteria might mutagenize their own genome when growth is blocked. However, this conclusion is suspect in the light of recent evidence that revertant colonies are initiated by preexisting cells with multiple copies the conjugative F′lac plasmid, which carries the lac mutation. Some plated cells have multiple copies of the simple F′lac plasmid. This provides sufficient LacZ activity to support plasmid replication but not cell division. In nongrowing cells, repeated plasmid replication increases the likelihood of a reversion event. Reversion to lac+ triggers exponential cell growth leading to a stable Lac+ revertant colony. In 10% of these plated cells, the high-copy plasmid includes an internal tandem lac duplication, which provides even more LacZ activity—sufficient to support slow growth and formation of an unstable Lac+ colony. Cells with multiple copies of the F′lac plasmid have an increased mutation rate, because the plasmid encodes the error-prone (mutagenic) DNA polymerase, DinB. Without DinB, unstable and stable Lac+ revertant types form in equal numbers and both types arise with no mutagenesis. Amplification and selection are central to behavior of the Cairns–Foster system, whereas mutagenesis is a system-specific side effect or artifact caused by coamplification of dinB with lac. Study of this system has revealed several broadly applicable principles. In all populations, gene duplications are frequent stable genetic polymorphisms, common near-neutral mutant alleles can gain a positive phenotype when amplified under selection, and natural selection can operate without cell division when variability is generated by overreplication of local genome subregions. PMID:26134316

Polymorphism in the GALNT1 Gene and Epithelial Ovarian Cancer in Non-Hispanic White Women: The Ovarian Cancer Association Consortium

PubMed Central

Phelan, Catherine M.; Tsai, Ya-Yu; Goode, Ellen L.; Vierkant, Robert A.; Fridley, Brooke L.; Beesley, Jonathan; Chen, Xiao Qing; Webb, Penelope M.; Chanock, Stephen; Cramer, Daniel W.; Moysich, Kirsten; Edwards, Robert P.; Chang-Claude, Jenny; Garcia-Closas, Montserrat; Yang, Hannah; Wang-Gohrke, Shan; Hein, Rebecca; Green, Adele C.; Lissowska, Jolanta; Carney, Michael E.; Lurie, Galina; Wilkens, Lynne R.; Ness, Roberta B.; Pearce, Celeste Leigh; Wu, Anna H.; Van Den Berg, David J.; Stram, Daniel O.; Terry, Kathryn L.; Whiteman, David C.; Whittemore, Alice S.; DiCioccio, Richard A.; McGuire, Valerie; Doherty, Jennifer A.; Rossing, Mary Anne; Anton-Culver, Hoda; Ziogas, Argyrios; Hogdall, Claus; Hogdall, Estrid; Kjaer, Susanne Krüger; Blaakaer, Jan; Quaye, Lydia; Ramus, Susan J.; Jacobs, Ian; Song, Honglin; Pharoah, Paul D.P.; Iversen, Edwin S.; Marks, Jeffrey R.; Pike, Malcolm C.; Gayther, Simon A.; Cunningham, Julie M.; Goodman, Marc T.; Schildkraut, Joellen M.; Chenevix-Trench, Georgia; Berchuck, Andrew; Sellers, Thomas A.

2010-01-01

Aberrant glycosylation is a well-described hallmark of cancer. In a previous ovarian cancer case control study that examined polymorphisms in 26 glycosylation-associated genes, we found strong statistical evidence (P = 0.00017) that women who inherited two copies of a single-nucleotide polymorphism in the UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferase, GALNT1, had decreased ovarian cancer risk. The current study attempted to replicate this observation. The GALNT1 single-nucleotide polymorphism rs17647532 was genotyped in 6,965 cases and 8,377 controls from 14 studies forming the Ovarian Cancer Association Consortium. The fixed effects estimate per rs17647532 allele was null (odds ratio, 0.99; 95% confidence interval, 0.92–1.07). When a recessive model was fit, the results were unchanged. Test for hetero geneity of the odds ratios revealed consistency across the 14 replication sites but significant differences compared with the original study population (P = 0.03). This study underscores the need for replication of putative findings in genetic association studies. PMID:20142253

17 CFR 260.7a-5 - Filing of amendments; number of copies.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Filing of amendments; number of copies. 260.7a-5 Section 260.7a-5 Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION (CONTINUED) GENERAL RULES AND REGULATIONS, TRUST INDENTURE ACT OF 1939 Rules Under Section 307 § 260.7a-5 Filing of amendments; number of copie...

17 CFR 260.7a-5 - Filing of amendments; number of copies.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 17 Commodity and Securities Exchanges 3 2013-04-01 2013-04-01 false Filing of amendments; number of copies. 260.7a-5 Section 260.7a-5 Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION (CONTINUED) GENERAL RULES AND REGULATIONS, TRUST INDENTURE ACT OF 1939 Rules Under Section 307 § 260.7a-5 Filing of amendments; number of copie...

17 CFR 260.7a-5 - Filing of amendments; number of copies.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 17 Commodity and Securities Exchanges 3 2012-04-01 2012-04-01 false Filing of amendments; number of copies. 260.7a-5 Section 260.7a-5 Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION (CONTINUED) GENERAL RULES AND REGULATIONS, TRUST INDENTURE ACT OF 1939 Rules Under Section 307 § 260.7a-5 Filing of amendments; number of copie...

17 CFR 260.7a-5 - Filing of amendments; number of copies.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 17 Commodity and Securities Exchanges 3 2011-04-01 2011-04-01 false Filing of amendments; number of copies. 260.7a-5 Section 260.7a-5 Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION (CONTINUED) GENERAL RULES AND REGULATIONS, TRUST INDENTURE ACT OF 1939 Rules Under Section 307 § 260.7a-5 Filing of amendments; number of copie...

17 CFR 260.7a-5 - Filing of amendments; number of copies.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 17 Commodity and Securities Exchanges 4 2014-04-01 2014-04-01 false Filing of amendments; number of copies. 260.7a-5 Section 260.7a-5 Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION (CONTINUED) GENERAL RULES AND REGULATIONS, TRUST INDENTURE ACT OF 1939 Rules Under Section 307 § 260.7a-5 Filing of amendments; number of copie...

Single Color Multiplexed ddPCR Copy Number Measurements and Single Nucleotide Variant Genotyping.

PubMed

Wood-Bouwens, Christina M; Ji, Hanlee P

2018-01-01

Droplet digital PCR (ddPCR) allows for accurate quantification of genetic events such as copy number variation and single nucleotide variants. Probe-based assays represent the current "gold-standard" for detection and quantification of these genetic events. Here, we introduce a cost-effective single color ddPCR assay that allows for single genome resolution quantification of copy number and single nucleotide variation.

Similar genomic proportions of copy number variation within gray wolves and modern dog breeds inferred from whole genome sequencing.

PubMed

Serres-Armero, Aitor; Povolotskaya, Inna S; Quilez, Javier; Ramirez, Oscar; Santpere, Gabriel; Kuderna, Lukas F K; Hernandez-Rodriguez, Jessica; Fernandez-Callejo, Marcos; Gomez-Sanchez, Daniel; Freedman, Adam H; Fan, Zhenxin; Novembre, John; Navarro, Arcadi; Boyko, Adam; Wayne, Robert; Vilà, Carles; Lorente-Galdos, Belen; Marques-Bonet, Tomas

2017-12-19

Whole genome re-sequencing data from dogs and wolves are now commonly used to study how natural and artificial selection have shaped the patterns of genetic diversity. Single nucleotide polymorphisms, microsatellites and variants in mitochondrial DNA have been interrogated for links to specific phenotypes or signals of domestication. However, copy number variation (CNV), despite its increasingly recognized importance as a contributor to phenotypic diversity, has not been extensively explored in canids. Here, we develop a new accurate probabilistic framework to create fine-scale genomic maps of segmental duplications (SDs), compare patterns of CNV across groups and investigate their role in the evolution of the domestic dog by using information from 34 canine genomes. Our analyses show that duplicated regions are enriched in genes and hence likely possess functional importance. We identify 86 loci with large CNV differences between dogs and wolves, enriched in genes responsible for sensory perception, immune response, metabolic processes, etc. In striking contrast to the observed loss of nucleotide diversity in domestic dogs following the population bottlenecks that occurred during domestication and breed creation, we find a similar proportion of CNV loci in dogs and wolves, suggesting that other dynamics are acting to particularly select for CNVs with potentially functional impacts. This work is the first comparison of genome wide CNV patterns in domestic and wild canids using whole-genome sequencing data and our findings contribute to study the impact of novel kinds of genetic changes on the evolution of the domestic dog.

Identification of copy number variation in French dairy and beef breeds using next-generation sequencing.

PubMed

Letaief, Rabia; Rebours, Emmanuelle; Grohs, Cécile; Meersseman, Cédric; Fritz, Sébastien; Trouilh, Lidwine; Esquerré, Diane; Barbieri, Johanna; Klopp, Christophe; Philippe, Romain; Blanquet, Véronique; Boichard, Didier; Rocha, Dominique; Boussaha, Mekki

2017-10-24

Copy number variations (CNV) are known to play a major role in genetic variability and disease pathogenesis in several species including cattle. In this study, we report the identification and characterization of CNV in eight French beef and dairy breeds using whole-genome sequence data from 200 animals. Bioinformatics analyses to search for CNV were carried out using four different but complementary tools and we validated a subset of the CNV by both in silico and experimental approaches. We report the identification and localization of 4178 putative deletion-only, duplication-only and CNV regions, which cover 6% of the bovine autosomal genome; they were validated by two in silico approaches and/or experimentally validated using array-based comparative genomic hybridization and single nucleotide polymorphism genotyping arrays. The size of these variants ranged from 334 bp to 7.7 Mb, with an average size of ~ 54 kb. Of these 4178 variants, 3940 were deletions, 67 were duplications and 171 corresponded to both deletions and duplications, which were defined as potential CNV regions. Gene content analysis revealed that, among these variants, 1100 deletions and duplications encompassed 1803 known genes, which affect a wide spectrum of molecular functions, and 1095 overlapped with known QTL regions. Our study is a large-scale survey of CNV in eight French dairy and beef breeds. These CNV will be useful to study the link between genetic variability and economically important traits, and to improve our knowledge on the genomic architecture of cattle.

CoNVaQ: a web tool for copy number variation-based association studies.

PubMed

Larsen, Simon Jonas; do Canto, Luisa Matos; Rogatto, Silvia Regina; Baumbach, Jan

2018-05-18

Copy number variations (CNVs) are large segments of the genome that are duplicated or deleted. Structural variations in the genome have been linked to many complex diseases. Similar to how genome-wide association studies (GWAS) have helped discover single-nucleotide polymorphisms linked to disease phenotypes, the extension of GWAS to CNVs has aided the discovery of structural variants associated with human traits and diseases. We present CoNVaQ, an easy-to-use web-based tool for CNV-based association studies. The web service allows users to upload two sets of CNV segments and search for genomic regions where the occurrence of CNVs is significantly associated with the phenotype. CoNVaQ provides two models: a simple statistical model using Fisher's exact test and a novel query-based model matching regions to user-defined queries. For each region, the method computes a global q-value statistic by repeated permutation of samples among the populations. We demonstrate our platform by using it to analyze a data set of HPV-positive and HPV-negative penile cancer patients. CoNVaQ provides a simple workflow for performing CNV-based association studies. It is made available as a web platform in order to provide a user-friendly workflow for biologists and clinicians to carry out CNV data analysis without installing any software. Through the web interface, users are also able to analyze their results to find overrepresented GO terms and pathways. In addition, our method is also available as a package for the R programming language. CoNVaQ is available at https://convaq.compbio.sdu.dk .

Systematic analysis of copy number variation associated with congenital diaphragmatic hernia.

PubMed

Zhu, Qihui; High, Frances A; Zhang, Chengsheng; Cerveira, Eliza; Russell, Meaghan K; Longoni, Mauro; Joy, Maliackal P; Ryan, Mallory; Mil-Homens, Adam; Bellfy, Lauren; Coletti, Caroline M; Bhayani, Pooja; Hila, Regis; Wilson, Jay M; Donahoe, Patricia K; Lee, Charles

2018-05-15

Congenital diaphragmatic hernia (CDH), characterized by malformation of the diaphragm and hypoplasia of the lungs, is one of the most common and severe birth defects, and is associated with high morbidity and mortality rates. There is growing evidence demonstrating that genetic factors contribute to CDH, although the pathogenesis remains largely elusive. Single-nucleotide polymorphisms have been studied in recent whole-exome sequencing efforts, but larger copy number variants (CNVs) have not yet been studied on a large scale in a case control study. To capture CNVs within CDH candidate regions, we developed and tested a targeted array comparative genomic hybridization platform to identify CNVs within 140 regions in 196 patients and 987 healthy controls, and identified six significant CNVs that were either unique to patients or enriched in patients compared with controls. These CDH-associated CNVs reveal high-priority candidate genes including HLX , LHX1 , and HNF1B We also discuss CNVs that are present in only one patient in the cohort but have additional evidence of pathogenicity, including extremely rare large and/or de novo CNVs. The candidate genes within these predicted disease-causing CNVs form functional networks with other known CDH genes and play putative roles in DNA binding/transcription regulation and embryonic development. These data substantiate the importance of CNVs in the etiology of CDH, identify CDH candidate genes and pathways, and highlight the importance of ongoing analysis of CNVs in the study of CDH and other structural birth defects. Copyright © 2018 the Author(s). Published by PNAS.

High mutation rates explain low population genetic divergence at copy-number-variable loci in Homo sapiens.

PubMed

Hu, Xin-Sheng; Yeh, Francis C; Hu, Yang; Deng, Li-Ting; Ennos, Richard A; Chen, Xiaoyang

2017-02-22

Copy-number-variable (CNV) loci differ from single nucleotide polymorphic (SNP) sites in size, mutation rate, and mechanisms of maintenance in natural populations. It is therefore hypothesized that population genetic divergence at CNV loci will differ from that found at SNP sites. Here, we test this hypothesis by analysing 856 CNV loci from the genomes of 1184 healthy individuals from 11 HapMap populations with a wide range of ancestry. The results show that population genetic divergence at the CNV loci is generally more than three times lower than at genome-wide SNP sites. Populations generally exhibit very small genetic divergence (G st  = 0.05 ± 0.049). The smallest divergence is among African populations (G st  = 0.0081 ± 0.0025), with increased divergence among non-African populations (G st  = 0.0217 ± 0.0109) and then among African and non-African populations (G st  = 0.0324 ± 0.0064). Genetic diversity is high in African populations (~0.13), low in Asian populations (~0.11), and intermediate in the remaining 11 populations. Few significant linkage disequilibria (LDs) occur between the genome-wide CNV loci. Patterns of gametic and zygotic LDs indicate the absence of epistasis among CNV loci. Mutation rate is about twice as large as the migration rate in the non-African populations, suggesting that the high mutation rates play dominant roles in producing the low population genetic divergence at CNV loci.

Target genes discovery through copy number alteration analysis in human hepatocellular carcinoma.

PubMed

Gu, De-Leung; Chen, Yen-Hsieh; Shih, Jou-Ho; Lin, Chi-Hung; Jou, Yuh-Shan; Chen, Chian-Feng

2013-12-21

High-throughput short-read sequencing of exomes and whole cancer genomes in multiple human hepatocellular carcinoma (HCC) cohorts confirmed previously identified frequently mutated somatic genes, such as TP53, CTNNB1 and AXIN1, and identified several novel genes with moderate mutation frequencies, including ARID1A, ARID2, MLL, MLL2, MLL3, MLL4, IRF2, ATM, CDKN2A, FGF19, PIK3CA, RPS6KA3, JAK1, KEAP1, NFE2L2, C16orf62, LEPR, RAC2, and IL6ST. Functional classification of these mutated genes suggested that alterations in pathways participating in chromatin remodeling, Wnt/β-catenin signaling, JAK/STAT signaling, and oxidative stress play critical roles in HCC tumorigenesis. Nevertheless, because there are few druggable genes used in HCC therapy, the identification of new therapeutic targets through integrated genomic approaches remains an important task. Because a large amount of HCC genomic data genotyped by high density single nucleotide polymorphism arrays is deposited in the public domain, copy number alteration (CNA) analyses of these arrays is a cost-effective way to reveal target genes through profiling of recurrent and overlapping amplicons, homozygous deletions and potentially unbalanced chromosomal translocations accumulated during HCC progression. Moreover, integration of CNAs with other high-throughput genomic data, such as aberrantly coding transcriptomes and non-coding gene expression in human HCC tissues and rodent HCC models, provides lines of evidence that can be used to facilitate the identification of novel HCC target genes with the potential of improving the survival of HCC patients.

Genetic Regulation of Charged Particle Mutagenesis in Human Cells

NASA Technical Reports Server (NTRS)

Kronenberg, Amy; Gauny, S.; Cherbonnel-Lasserre, C.; Liu, W.; Wiese, C.

1999-01-01

Our studies use a series of syngeneic, and where possible, isogenic human B-lymphoblastoid cell lines to assess the genetic factors that modulate susceptibility apoptosis and their impact on the mutagenic risks of low fluence exposures to 1 GeV Fe ions and 55 MeV protons. These ions are representative of the types of charged particle radiation that are of particular significance for human health in the space radiation environment. The model system employs cell lines derived from the male donor WIL-2. These cells have a single X chromosome and they are hemizygous for one mutation marker, hypoxanthine phosphoribosyltransferase (HPRT). TK6 and WTK1 cells were each derived from descendants of WIL-2 and were each selected as heterozygotes for a second mutation marker, the thymidine kinase (TK) gene located on chromosome 17q. The HPRT and TK loci can detect many different types of mutations, from single basepair substitutions up to large scale loss of heterozygosity (LOH). The single expressing copy of TK in the TK6 and WTKI cell lines is found on the same copy of chromosome 17, and this allele can be identified by a restriction fragment length polymorphism (RFLP) identified when high molecular weight DNA is digested by the SacI restriction endonuclease and hybridized against the cDNA probe for TK. A large series of polymorphic linked markers has been identified that span more than 60 cM of DNA (approx. 60 megabasepairs) and distinguish the copy of chromosome 17 bearing the initially active TK allele from the copy of chromosome 17 bearing the silent TK allele in both TK6 and WTKI cells. TK6 cells express normal p53 protein while WTKI cells express homozygous mutant p53. Expression of mutant p53 can increase susceptibility to x-ray-induced mutations. It's been suggested that the increased mutagenesis in p53 mutant cells might be due to reduced apoptosis.

Characterizing the genetic diversity of the monkey malaria parasite Plasmodium cynomolgi

PubMed Central

Sutton, Patrick L.; Luo, Zunping; Divis, Paul C. S.; Friedrich, Volney K.; Conway, David J.; Singh, Balbir; Barnwell, John W.; Carlton, Jane M.; Sullivan, Steven A.

2016-01-01

Plasmodium cynomolgi is a malaria parasite that typically infects Asian macaque monkeys, and humans on rare occasions. P. cynomolgi serves as a model system for the human malaria parasite Plasmodium vivax, with which it shares such important biological characteristics as formation of a dormant liver stage and a preference to invade reticulocytes. While genomes of three P. cynomolgi strains have been sequenced, genetic diversity of P. cynomolgi has not been widely investigated. To address this we developed the first panel of P. cynomolgi microsatellite markers to genotype eleven P. cynomolgi laboratory strains and 18 field isolates from Sarawak, Malaysian Borneo. We found diverse genotypes among most of the laboratory strains, though two nominally different strains were found to be genetically identical, We also investigated sequence polymorphism in two erythrocyte invasion gene families, the reticulocyte binding protein and Duffy binding protein genes, in these strains. We also observed copy number variation in rbp genes. PMID:26980604

Making Millennial Medicine More Meta.

PubMed

Turnbaugh, Peter J

2018-01-01

Although the importance of human genetic polymorphisms in therapeutic outcomes is well established, the role of specific genotypic or copy number variants in our "second genome" (the microbiome) has been largely overlooked. In this Perspective, I will discuss three major barriers to integrating metagenomics into pharmacology, highlighting ongoing research by us and others that has begun to shed light on the mechanisms that link the human microbiome to the efficacy and toxicity of small-molecule and biological therapies. The challenges for the next 5 years and beyond are many, requiring interdisciplinary scientific teams working at the interface of chemistry and biology, and a consideration of far more variables than traditionally included in pharmacological modeling. However, the potential benefits are immense. Continued progress could enable more precise tools for predicting patient responses and the development of a new generation of therapeutics based on, or targeted at, the human microbiome.

18 CFR 33.8 - Number of copies.

Code of Federal Regulations, 2010 CFR

2010-04-01

... ENERGY REGULATIONS UNDER THE FEDERAL POWER ACT APPLICATIONS UNDER FEDERAL POWER ACT SECTION 203 § 33.8 Number of copies. An original and eight copies of the application under this part must be submitted. If..., the applicant must submit all such information in electronic format (e.g., on computer diskette or on...

Great Genotypic and Phenotypic Diversities Associated with Copy-Number Variations of Complement C4 and RP-C4-CYP21-TNX (RCCX) Modules: a Comparison of Asian Indian and European American Populations

PubMed Central

Saxena, Kapil; Kitzmiller, Kathryn J.; Wu, Yee Ling; Zhou, Bi; Esack, Nazreen; Hiremath, Leena; Chung, Erwin K.; Yang, Yan; Yu, C. Yung

2009-01-01

Inter-individual gene copy-number variations (CNVs) probably afford human populations the flexibility to respond to a variety of environmental challenges, but also lead to differential disease predispositions. We investigated gene CNVs for complement component C4 and steroid 21-hydroxylase from the RP-C4-CYP21-TNX (RCCX) modules located in the major histocompatibility complex among healthy Asian-Indian Americans (AIA) and compared them to European Americans. A combination of definitive techniques that yielded cross-confirmatory results was used. The medium gene copy-numbers for C4 and its isotypes, acidic C4A and basic C4B, were 4, 2 and 2, respectively, but their frequencies were only 53–56%. The distribution patterns for total C4 and C4A are skewed towards the high copy-number side. For example, the frequency of AIA-subjects with three copies of C4A (30.7%) was 3.92-fold of those with a single copy (7.83%). The monomodular-short haplotype with a single C4B gene and the absence of C4A, which is in linkage- disequilibrium with HLA DRB1*0301 in Europeans and a strong risk factor for autoimmune diseases, has a frequency of 0.012 in AIA but 0.106 among healthy European Americans (p=6.6×10−8). The copy-number and the size of C4 genes strongly determine the plasma C4 protein concentrations. Parallel variations in copy-numbers of CYP21A (CYP21A1P) and TNXA with total C4 were also observed. Notably, 13.1% of AIA-subjects had three copies of the functional CYP21B, which were likely generated by recombinations between monomodular and bimodular RCCX haplotypes. The high copy-numbers of C4 and the high frequency of RCCX recombinants offer important insights to the prevalence of autoimmune and genetic diseases. PMID:19135723

Variability of rRNA Operon Copy Number and Growth Rate Dynamics of Bacillus Isolated from an Extremely Oligotrophic Aquatic Ecosystem

PubMed Central

Valdivia-Anistro, Jorge A.; Eguiarte-Fruns, Luis E.; Delgado-Sapién, Gabriela; Márquez-Zacarías, Pedro; Gasca-Pineda, Jaime; Learned, Jennifer; Elser, James J.; Olmedo-Alvarez, Gabriela; Souza, Valeria

2016-01-01

The ribosomal RNA (rrn) operon is a key suite of genes related to the production of protein synthesis machinery and thus to bacterial growth physiology. Experimental evidence has suggested an intrinsic relationship between the number of copies of this operon and environmental resource availability, especially the availability of phosphorus (P), because bacteria that live in oligotrophic ecosystems usually have few rrn operons and a slow growth rate. The Cuatro Ciénegas Basin (CCB) is a complex aquatic ecosystem that contains an unusually high microbial diversity that is able to persist under highly oligotrophic conditions. These environmental conditions impose a variety of strong selective pressures that shape the genome dynamics of their inhabitants. The genus Bacillus is one of the most abundant cultivable bacterial groups in the CCB and usually possesses a relatively large number of rrn operon copies (6–15 copies). The main goal of this study was to analyze the variation in the number of rrn operon copies of Bacillus in the CCB and to assess their growth-related properties as well as their stoichiometric balance (N and P content). We defined 18 phylogenetic groups within the Bacilli clade and documented a range of from six to 14 copies of the rrn operon. The growth dynamic of these Bacilli was heterogeneous and did not show a direct relation to the number of operon copies. Physiologically, our results were not consistent with the Growth Rate Hypothesis, since the copies of the rrn operon were decoupled from growth rate. However, we speculate that the diversity of the growth properties of these Bacilli as well as the low P content of their cells in an ample range of rrn copy number is an adaptive response to oligotrophy of the CCB and could represent an ecological mechanism that allows these taxa to coexist. These findings increase the knowledge of the variability in the number of copies of the rrn operon in the genus Bacillus and give insights about the physiology of this bacterial group under extreme oligotrophic conditions. PMID:26779143

Comparison of the copy numbers of bovine leukemia virus in the lymph nodes of cattle with enzootic bovine leukosis and cattle with latent infection.

PubMed

Somura, Yoshiko; Sugiyama, Emi; Fujikawa, Hiroshi; Murakami, Kenji

2014-10-01

To establish a diagnostic index for predicting enzootic bovine leukosis (EBL), proviral bovine leukemia virus (BLV) copies in whole blood, lymph nodes and spleen were examined by quantitative real-time PCR (qPCR). Cattle were divided into two groups, EBL and BLV-infected, based on meat inspection data. The number of BLV copies in all specimens of EBL cattle was significantly higher than those of BLV-infected cattle (p < 0.0001), and the number of BLV copies in the lymph nodes was particularly large. Over 70 % of the superficial cervical, medial iliac and jejunal lymph nodes from EBL cattle had more than 1,000 copies/10 ng DNA, whereas lymph nodes from BLV-infected cattle did not. These findings suggest that the cattle harboring more than 1,000 BLV copies may be diagnosed with EBL.

Genetic Control of L-a and L-(Bc) Dsrna Copy Number in Killer Systems of SACCHAROMYCES CEREVISIAE

PubMed Central

Ball, Steven G.; Tirtiaux, Catherine; Wickner, Reed B.

1984-01-01

M dsRNA in yeast encodes a toxin precursor and immunity protein, whereas L-A dsRNA encodes the 81,000-dalton major protein of the intracellular particles in which both L-A and M are found. L-(BC) dsRNA(s) are found in particles with different coat proteins. We find that M dsRNA lowers the copy number of L-A, but not L-(BC). The SKI gene products lower the copy number of L-(BC), L-A, M1 and M2. This is the first known interaction of L-(BC) with any element of the killer systems. The MAK3, MAK10 and PET18 gene products are necessary for L-A maintenance and replication, but mutations in these genes do not affect L-(BC) copy number. Mutations in MAK1, MAK4, MAK7, MAK17 and MAK24 do not detectably affect copy number of L-(BC) or L-A. PMID:17246214

Copy-number analysis and inference of subclonal populations in cancer genomes using Sclust.

PubMed

Cun, Yupeng; Yang, Tsun-Po; Achter, Viktor; Lang, Ulrich; Peifer, Martin

2018-06-01

The genomes of cancer cells constantly change during pathogenesis. This evolutionary process can lead to the emergence of drug-resistant mutations in subclonal populations, which can hinder therapeutic intervention in patients. Data derived from massively parallel sequencing can be used to infer these subclonal populations using tumor-specific point mutations. The accurate determination of copy-number changes and tumor impurity is necessary to reliably infer subclonal populations by mutational clustering. This protocol describes how to use Sclust, a copy-number analysis method with a recently developed mutational clustering approach. In a series of simulations and comparisons with alternative methods, we have previously shown that Sclust accurately determines copy-number states and subclonal populations. Performance tests show that the method is computationally efficient, with copy-number analysis and mutational clustering taking <10 min. Sclust is designed such that even non-experts in computational biology or bioinformatics with basic knowledge of the Linux/Unix command-line syntax should be able to carry out analyses of subclonal populations.

RUBIC identifies driver genes by detecting recurrent DNA copy number breaks

PubMed Central

van Dyk, Ewald; Hoogstraat, Marlous; ten Hoeve, Jelle; Reinders, Marcel J. T.; Wessels, Lodewyk F. A.

2016-01-01

The frequent recurrence of copy number aberrations across tumour samples is a reliable hallmark of certain cancer driver genes. However, state-of-the-art algorithms for detecting recurrent aberrations fail to detect several known drivers. In this study, we propose RUBIC, an approach that detects recurrent copy number breaks, rather than recurrently amplified or deleted regions. This change of perspective allows for a simplified approach as recursive peak splitting procedures and repeated re-estimation of the background model are avoided. Furthermore, we control the false discovery rate on the level of called regions, rather than at the probe level, as in competing algorithms. We benchmark RUBIC against GISTIC2 (a state-of-the-art approach) and RAIG (a recently proposed approach) on simulated copy number data and on three SNP6 and NGS copy number data sets from TCGA. We show that RUBIC calls more focal recurrent regions and identifies a much larger fraction of known cancer genes. PMID:27396759

Cancer vulnerabilities unveiled by genomic loss

PubMed Central

Nijhawan, Deepak; Zack, Travis I.; Ren, Yin; Strickland, Matthew R.; Lamothe, Rebecca; Schumacher, Steven E.; Tsherniak, Aviad; Besche, Henrike C.; Rosenbluh, Joseph; Shehata, Shyemaa; Cowley, Glenn S.; Weir, Barbara A.; Goldberg, Alfred L.; Mesirov, Jill P.; Root, David E.; Bhatia, Sangeeta N.; Beroukhim, Rameen; Hahn, William C.

2012-01-01

Summary Due to genome instability, most cancers exhibit loss of regions containing tumor suppressor genes and collateral loss of other genes. To identify cancer-specific vulnerabilities that are the result of copy-number losses, we performed integrated analyses of genome-wide copy-number and RNAi profiles and identified 56 genes for which gene suppression specifically inhibited the proliferation of cells harboring partial copy-number loss of that gene. These CYCLOPS (Copy-number alterations Yielding Cancer Liabilities Owing to Partial losS) genes are enriched for spliceosome, proteasome and ribosome components. One CYCLOPS gene, PSMC2, encodes an essential member of the 19S proteasome. Normal cells express excess PSMC2, which resides in a complex with PSMC1, PSMD2, and PSMD5 and acts as a reservoir protecting cells from PSMC2 suppression. Cells harboring partial PSMC2 copy-number loss lack this complex and die after PSMC2 suppression. These observations define a distinct class of cancer-specific liabilities resulting from genome instability. PMID:22901813

Hidden Markov Model-Based CNV Detection Algorithms for Illumina Genotyping Microarrays.

PubMed

Seiser, Eric L; Innocenti, Federico

2014-01-01

Somatic alterations in DNA copy number have been well studied in numerous malignancies, yet the role of germline DNA copy number variation in cancer is still emerging. Genotyping microarrays generate allele-specific signal intensities to determine genotype, but may also be used to infer DNA copy number using additional computational approaches. Numerous tools have been developed to analyze Illumina genotype microarray data for copy number variant (CNV) discovery, although commonly utilized algorithms freely available to the public employ approaches based upon the use of hidden Markov models (HMMs). QuantiSNP, PennCNV, and GenoCN utilize HMMs with six copy number states but vary in how transition and emission probabilities are calculated. Performance of these CNV detection algorithms has been shown to be variable between both genotyping platforms and data sets, although HMM approaches generally outperform other current methods. Low sensitivity is prevalent with HMM-based algorithms, suggesting the need for continued improvement in CNV detection methodologies.

Functional effects of CCL3L1 copy number.

PubMed

Carpenter, D; McIntosh, R S; Pleass, R J; Armour, J A L

2012-07-01

Copy number variation (CNV) is becoming increasingly important as a feature of human variation in disease susceptibility studies. However, the consequences of CNV are not so well understood. Here, we present data exploring the functional consequences of CNV of CCL3L1 in 55 independent UK samples with no known clinical phenotypes. The copy number of CCL3L1 was determined by the paralogue ratio test, and expression levels of macrophage inflammatory protein-1α (MIP-1α) and mRNA from stimulated monocytes were measured and analysed. The data show no statistically significant association of MIP-1α protein levels with copy number. However, there was a significant correlation between copy number and CCL3L1:CCL3 mRNA ratio. The data also provide evidence that expression of CCL3 predominates in both protein and mRNA, and therefore the observed variation of CCL3 is potentially more important biologically than that of CNV of CCL3L1.

[Detection of the exogenous gene copy number of the transgenic tomato anti-caries vaccine].

PubMed

Bai, Guo-hui; Liu, Jian-guo; Tian, Yuan; Chen, Zhu; Bai, Peng-yuan; Han, Qi; Gu, Yu; Guan, Xiao-yan; Wang, Hai-hui

2013-12-01

To detect the exogenous gene copy number of the transgenic tomato anti-caries vaccine by using the SYBR Green real-time PCR. Recombinant plasmid pEAC10 and pEPC10 were used as standard to detect genome samples of exogenous gene pacA-ctxB and pacP-ctxB by SYBR green fluorescent quantitation, then the average value was calculated as gene copy number. The copy number of the transgenic tomato carrying pacA-ctxB was 1.3 and the pacP-ctxB was 3.2. The transgenic tomato plants which have high stability are low-copy transgenic plants. Supported by National Natural Science Foundation of China (30160086, 81260164), Science and Technical Fund of Guizhou Province (LKZ[2011]41), Project of Technology Innovation Team in Guizhou Province, Leading Academic Discipline Construction Project in Guizhou Province and Excellent Scientific Research Team Cultivation Project in Zunyi Medical College ([2012]12).

PMS2 inactivation by a complex rearrangement involving an HERV retroelement and the inverted 100-kb duplicon on 7p22.1.

PubMed

Vogt, Julia; Wernstedt, Annekatrin; Ripperger, Tim; Pabst, Brigitte; Zschocke, Johannes; Kratz, Christian; Wimmer, Katharina

2016-11-01

Biallelic PMS2 mutations are responsible for more than half of all cases of constitutional mismatch repair deficiency (CMMRD), a recessively inherited childhood cancer predisposition syndrome. The mismatch repair gene PMS2 is partly embedded within one copy of an inverted 100-kb low-copy repeat (LCR) on 7p22.1. In an individual with CMMRD syndrome, PMS2 was found to be homozygously inactivated by a complex chromosomal rearrangement, which separates the 5'-part from the 3'-part of the gene. The rearrangement involves sequences of the inverted 100-kb LCR and a human endogenous retrovirus element and may be associated with an inversion that is indistinguishable from the known inversion polymorphism affecting the ~0.7-Mb sequence intervening the LCR. Its formation is best explained by a replication-based mechanism (RBM) such as fork stalling and template switching/microhomology-mediated break-induced replication (FoSTeS/MMBIR). This finding supports the hypothesis that the inverted LCR can not only facilitate the formation of the non-allelic homologous recombination-mediated inversion polymorphism but it also promotes the occurrence of more complex rearrangements that can be associated with a large inversion, as well, but are mediated by a RBM. This further suggests that among the inversion polymorphism on 7p22.1, more complex rearrangements might be hidden. Furthermore, as the locus is embedded in a common fragile site (CFS) region, this rearrangement also supports the recently raised hypothesis that CFS sequence motifs may facilitate replication-based rearrangement mechanisms.

PMS2 inactivation by a complex rearrangement involving an HERV retroelement and the inverted 100-kb duplicon on 7p22.1

PubMed Central

Vogt, Julia; Wernstedt, Annekatrin; Ripperger, Tim; Pabst, Brigitte; Zschocke, Johannes; Kratz, Christian; Wimmer, Katharina

2016-01-01

Biallelic PMS2 mutations are responsible for more than half of all cases of constitutional mismatch repair deficiency (CMMRD), a recessively inherited childhood cancer predisposition syndrome. The mismatch repair gene PMS2 is partly embedded within one copy of an inverted 100-kb low-copy repeat (LCR) on 7p22.1. In an individual with CMMRD syndrome, PMS2 was found to be homozygously inactivated by a complex chromosomal rearrangement, which separates the 5′-part from the 3′-part of the gene. The rearrangement involves sequences of the inverted 100-kb LCR and a human endogenous retrovirus element and may be associated with an inversion that is indistinguishable from the known inversion polymorphism affecting the ~0.7-Mb sequence intervening the LCR. Its formation is best explained by a replication-based mechanism (RBM) such as fork stalling and template switching/microhomology-mediated break-induced replication (FoSTeS/MMBIR). This finding supports the hypothesis that the inverted LCR can not only facilitate the formation of the non-allelic homologous recombination-mediated inversion polymorphism but it also promotes the occurrence of more complex rearrangements that can be associated with a large inversion, as well, but are mediated by a RBM. This further suggests that among the inversion polymorphism on 7p22.1, more complex rearrangements might be hidden. Furthermore, as the locus is embedded in a common fragile site (CFS) region, this rearrangement also supports the recently raised hypothesis that CFS sequence motifs may facilitate replication-based rearrangement mechanisms. PMID:27329736

Differential allelic expression of IL13 and CSF2 genes associated with asthma.

PubMed

Burkhardt, Jana; Kirsten, Holger; Wolfram, Grit; Quente, Elfi; Ahnert, Peter

2012-07-01

An important area of genetic research is the identification of functional mechanisms in polymorphisms associated with diseases. A highly relevant functional mechanism is the influence of polymorphisms on gene expression levels (differential allelic expression, DAE). The coding single nucleotide polymorphisms (SNPs) CSF2(rs25882) and IL13(rs20541) have been associated with asthma. In this work, we investigated whether the mRNA expression levels of CSF2 or IL13 were correlated with these SNPs. Samples were analyzed by mass spectrometry-based quantification of gene expression. Both SNPs influenced gene expression levels (CSF2(rs25882): p(overall) = 0.008 and p(DAE samples) = 0.00006; IL13(rs20541): p(overall) = 0.059 and p(DAE samples) = 0.036). For CSF2, the expression level was increased by 27.4% (95% CI: 18.5%-35.4%) in samples with significant DAE in the presence of one copy of risk variant CSF2(rs25882-T). The average expression level of IL13 was increased by 29.8% (95% CI: 3.1%-63.4%) in samples with significant DAE in the presence of one copy of risk variant IL13(rs20541-A). Enhanced expression of CSF2 could stimulate macrophages and neutrophils during inflammation and may be related to the etiology of asthma. For IL-13, higher expression could enhance the functional activity of the asthma-associated isoform. Overall, the analysis of DAE provides an efficient approach for identifying possible functional mechanisms that link disease-associated variants with altered gene expression levels.