Science.gov

Sample records for absolute copy numbers

  1. BACOM2.0 facilitates absolute normalization and quantification of somatic copy number alterations in heterogeneous tumor

    NASA Astrophysics Data System (ADS)

    Fu, Yi; Yu, Guoqiang; Levine, Douglas A.; Wang, Niya; Shih, Ie-Ming; Zhang, Zhen; Clarke, Robert; Wang, Yue

    2015-09-01

    Most published copy number datasets on solid tumors were obtained from specimens comprised of mixed cell populations, for which the varying tumor-stroma proportions are unknown or unreported. The inability to correct for signal mixing represents a major limitation on the use of these datasets for subsequent analyses, such as discerning deletion types or detecting driver aberrations. We describe the BACOM2.0 method with enhanced accuracy and functionality to normalize copy number signals, detect deletion types, estimate tumor purity, quantify true copy numbers, and calculate average-ploidy value. While BACOM has been validated and used with promising results, subsequent BACOM analysis of the TCGA ovarian cancer dataset found that the estimated average tumor purity was lower than expected. In this report, we first show that this lowered estimate of tumor purity is the combined result of imprecise signal normalization and parameter estimation. Then, we describe effective allele-specific absolute normalization and quantification methods that can enhance BACOM applications in many biological contexts while in the presence of various confounders. Finally, we discuss the advantages of BACOM in relation to alternative approaches. Here we detail this revised computational approach, BACOM2.0, and validate its performance in real and simulated datasets.

  2. Rare Copy Number Variants

    PubMed Central

    Grozeva, Detelina; Kirov, George; Ivanov, Dobril; Jones, Ian R.; Jones, Lisa; Green, Elaine K.; St Clair, David M.; Young, Allan H.; Ferrier, Nicol; Farmer, Anne E.; McGuffin, Peter; Holmans, Peter A.; Owen, Michael J.; O’Donovan, Michael C.; Craddock, Nick

    2015-01-01

    Context Recent studies suggest that copy number variation in the human genome is extensive and may play an important role in susceptibility to disease, including neuropsychiatric disorders such as schizophrenia and autism. The possible involvement of copy number variants (CNVs) in bipolar disorder has received little attention to date. Objectives To determine whether large (>100 000 base pairs) and rare (found in <1% of the population) CNVs are associated with susceptibility to bipolar disorder and to compare with findings in schizophrenia. Design A genome-wide survey of large, rare CNVs in a case-control sample using a high-density microarray. Setting The Wellcome Trust Case Control Consortium. Participants There were 1697 cases of bipolar disorder and 2806 nonpsychiatric controls. All participants were white UK residents. Main Outcome Measures Overall load of CNVs and presence of rare CNVs. Results The burden of CNVs in bipolar disorder was not increased compared with controls and was significantly less than in schizophrenia cases. The CNVs previously implicated in the etiology of schizophrenia were not more common in cases with bipolar disorder. Conclusions Schizophrenia and bipolar disorder differ with respect to CNV burden in general and association with specific CNVs in particular. Our data are consistent with the possibility that possession of large, rare deletions may modify the phenotype in those at risk of psychosis: those possessing such events are more likely to be diagnosed as having schizophrenia, and those without them are more likely to be diagnosed as having bipolar disorder. PMID:20368508

  3. Counting copy number and calories.

    PubMed

    White, Stefan

    2015-08-01

    Copy number variation (CNV) at several genomic loci has been associated with different human traits and diseases, but in many cases the findings could not be replicated. A new study provides insights into the degree of variation present at the amylase locus and calls into question a previous association between amylase copy number and body mass index.

  4. Rawcopy: Improved copy number analysis with Affymetrix arrays

    PubMed Central

    Mayrhofer, Markus; Viklund, Björn; Isaksson, Anders

    2016-01-01

    Microarray data is subject to noise and systematic variation that negatively affects the resolution of copy number analysis. We describe Rawcopy, an R package for processing of Affymetrix CytoScan HD, CytoScan 750k and SNP 6.0 microarray raw intensities (CEL files). Noise characteristics of a large number of reference samples are used to estimate log ratio and B-allele frequency for total and allele-specific copy number analysis. Rawcopy achieves better signal-to-noise ratio and higher proportion of validated alterations than commonly used free and proprietary alternatives. In addition, Rawcopy visualizes each microarray sample for assessment of technical quality, patient identity and genome-wide absolute copy number states. Software and instructions are available at http://rawcopy.org. PMID:27796336

  5. Mechanisms of change in gene copy number.

    PubMed

    Hastings, P J; Lupski, James R; Rosenberg, Susan M; Ira, Grzegorz

    2009-08-01

    Deletions and duplications of chromosomal segments (copy number variants, CNVs) are a major source of variation between individual humans and are an underlying factor in human evolution and in many diseases, including mental illness, developmental disorders and cancer. CNVs form at a faster rate than other types of mutation, and seem to do so by similar mechanisms in bacteria, yeast and humans. Here we review current models of the mechanisms that cause copy number variation. Non-homologous end-joining mechanisms are well known, but recent models focus on perturbation of DNA replication and replication of non-contiguous DNA segments. For example, cellular stress might induce repair of broken replication forks to switch from high-fidelity homologous recombination to non-homologous repair, thus promoting copy number change.

  6. Gene copy number and malaria biology

    PubMed Central

    Anderson, Tim J.C.; Patel, Jigar; Ferdig, Michael T.

    2010-01-01

    Alteration in gene copy number provides a simple way to change expression levels and alter phenotype. This was fully appreciated by bacteriologists more than 25 years ago, but the extent and implications of copy number polymorphism (CNP) have only recently become apparent in other organisms. New methods demonstrate the ubiquity of CNPs in eukaryotes and their medical importance in humans. CNP is also widespread in the Plasmodium falciparum genome and has an important and underappreciated role in determining phenotype. In this review, we summarize the distribution of CNP, its evolutionary dynamics within populations, its functional importance and its mode of evolution. PMID:19559648

  7. Genomic characteristics of cattle copy number variations

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We performed a systematic analysis of cattle copy number variations (CNVs) using the Bovine HapMap SNP genotyping data, including 539 animals of 21 modern cattle breeds and 6 outgroups. After correcting genomic waves and considering the trio information, we identified 682 candidate CNV regions (CNVR...

  8. 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

  9. Modeling genetic inheritance of copy number variations

    PubMed Central

    Wang, Kai; Chen, Zhen; Tadesse, Mahlet G.; Glessner, Joseph; Grant, Struan F. A.; Hakonarson, Hakon; Bucan, Maja

    2008-01-01

    Copy number variations (CNVs) are being used as genetic markers or functional candidates in gene-mapping studies. However, unlike single nucleotide polymorphism or microsatellite genotyping techniques, most CNV detection methods are limited to detecting total copy numbers, rather than copy number in each of the two homologous chromosomes. To address this issue, we developed a statistical framework for intensity-based CNV detection platforms using family data. Our algorithm identifies CNVs for a family simultaneously, thus avoiding the generation of calls with Mendelian inconsistency while maintaining the ability to detect de novo CNVs. Applications to simulated data and real data indicate that our method significantly improves both call rates and accuracy of boundary inference, compared to existing approaches. We further illustrate the use of Mendelian inheritance to infer SNP allele compositions in each of the two homologous chromosomes in CNV regions using real data. Finally, we applied our method to a set of families genotyped using both the Illumina HumanHap550 and Affymetrix genome-wide 5.0 arrays to demonstrate its performance on both inherited and de novo CNVs. In conclusion, our method produces accurate CNV calls, gives probabilistic estimates of CNV transmission and builds a solid foundation for the development of linkage and association tests utilizing CNVs. PMID:18832372

  10. CONTRA: copy number analysis for targeted resequencing

    PubMed Central

    Li, Jason; Lupat, Richard; Amarasinghe, Kaushalya C.; Thompson, Ella R.; Doyle, Maria A.; Ryland, Georgina L.; Tothill, Richard W.; Halgamuge, Saman K.; Campbell, Ian G.; Gorringe, Kylie L.

    2012-01-01

    Motivation: In light of the increasing adoption of targeted resequencing (TR) as a cost-effective strategy to identify disease-causing variants, a robust method for copy number variation (CNV) analysis is needed to maximize the value of this promising technology. Results: We present a method for CNV detection for TR data, including whole-exome capture data. Our method calls copy number gains and losses for each target region based on normalized depth of coverage. Our key strategies include the use of base-level log-ratios to remove GC-content bias, correction for an imbalanced library size effect on log-ratios, and the estimation of log-ratio variations via binning and interpolation. Our methods are made available via CONTRA (COpy Number Targeted Resequencing Analysis), a software package that takes standard alignment formats (BAM/SAM) and outputs in variant call format (VCF4.0), for easy integration with other next-generation sequencing analysis packages. We assessed our methods using samples from seven different target enrichment assays, and evaluated our results using simulated data and real germline data with known CNV genotypes. Availability and implementation: Source code and sample data are freely available under GNU license (GPLv3) at http://contra-cnv.sourceforge.net/ Contact: Jason.Li@petermac.org Supplementary information: Supplementary data are available at Bioinformatics online. PMID:22474122

  11. Number matters: control of mammalian mitochondrial DNA copy number.

    PubMed

    Clay Montier, Laura L; Deng, Janice J; Bai, Yidong

    2009-03-01

    Regulation of mitochondrial biogenesis is essential for proper cellular functioning. Mitochondrial DNA (mtDNA) depletion and the resulting mitochondrial malfunction have been implicated in cancer, neurodegeneration, diabetes, aging, and many other human diseases. Although it is known that the dynamics of the mammalian mitochondrial genome are not linked with that of the nuclear genome, very little is known about the mechanism of mtDNA propagation. Nevertheless, our understanding of the mode of mtDNA replication has advanced in recent years, though not without some controversies. This review summarizes our current knowledge of mtDNA copy number control in mammalian cells, while focusing on both mtDNA replication and turnover. Although mtDNA copy number is seemingly in excess, we reason that mtDNA copy number control is an important aspect of mitochondrial genetics and biogenesis and is essential for normal cellular function.

  12. Why to compare absolute numbers of mitochondria.

    PubMed

    Schmitt, Sabine; Schulz, Sabine; Schropp, Eva-Maria; Eberhagen, Carola; Simmons, Alisha; Beisker, Wolfgang; Aichler, Michaela; Zischka, Hans

    2014-11-01

    Prompted by pronounced structural differences between rat liver and rat hepatocellular carcinoma mitochondria, we suspected these mitochondrial populations to differ massively in their molecular composition. Aiming to reveal these mitochondrial differences, we came across the issue on how to normalize such comparisons and decided to focus on the absolute number of mitochondria. To this end, fluorescently stained mitochondria were quantified by flow cytometry. For rat liver mitochondria, this approach resulted in mitochondrial protein contents comparable to earlier reports using alternative methods. We determined similar protein contents for rat liver, heart and kidney mitochondria. In contrast, however, lower protein contents were determined for rat brain mitochondria and for mitochondria from the rat hepatocellular carcinoma cell line McA 7777. This result challenges mitochondrial comparisons that rely on equal protein amounts as a typical normalization method. Exemplarily, we therefore compared the activity and susceptibility toward inhibition of complex II of rat liver and hepatocellular carcinoma mitochondria and obtained significant discrepancies by either normalizing to protein amount or to absolute mitochondrial number. Importantly, the latter normalization, in contrast to the former, demonstrated a lower complex II activity and higher susceptibility toward inhibition in hepatocellular carcinoma mitochondria compared to liver mitochondria. These findings demonstrate that solely normalizing to protein amount may obscure essential molecular differences between mitochondrial populations.

  13. Copy Number Heterogeneity of JC Virus Standards

    PubMed Central

    Bateman, Allen C.; Atienza, Ederlyn E.; Wendt, Sharon; Makhsous, Negar; Jerome, Keith R.; Cook, Linda

    2016-01-01

    ABSTRACT Quantitative PCR is a diagnostic mainstay of clinical virology, and accurate quantitation of viral load among labs requires the use of international standards. However, the use of multiple passages of viral isolates to obtain sufficient material for international standards may result in genomic changes that complicate their use as quantitative standards. We performed next-generation sequencing to obtain single-nucleotide resolution and relative copy number of JC virus (JCV) clinical standards. Strikingly, the WHO international standard and the Exact v1/v2 prototype standards for JCV showed 8-fold and 4-fold variation in genomic coverage between different loci in the viral genome, respectively, due to large deletions in the large T antigen region. Intriguingly, several of the JCV standards sequenced in this study with large T antigen deletions were cultured in cell lines immortalized using simian virus 40 (SV40) T antigen, suggesting the possibility of transcomplementation in cell culture. Using a cutoff 5% allele fraction for junctional reads, 7 different rearrangements were present in the JC virus sequences present in the WHO standard across multiple library preparations and sequencing runs. Neither the copy number differences nor the rearrangements were observed in a clinical sample with a high copy number of JCV or a plasmid control. These results were also confirmed by the quantitative real-time PCR (qPCR), droplet digital PCR (ddPCR), and Sanger sequencing of multiple rearrangements. In summary, targeting different regions of the same international standard can result in up to an 8-fold difference in quantitation. We recommend the use of next-generation sequencing to validate standards in clinical virology. PMID:27974546

  14. Getting DNA copy numbers without control samples

    PubMed Central

    2012-01-01

    Background The selection of the reference to scale the data in a copy number analysis has paramount importance to achieve accurate estimates. Usually this reference is generated using control samples included in the study. However, these control samples are not always available and in these cases, an artificial reference must be created. A proper generation of this signal is crucial in terms of both noise and bias. We propose NSA (Normality Search Algorithm), a scaling method that works with and without control samples. It is based on the assumption that genomic regions enriched in SNPs with identical copy numbers in both alleles are likely to be normal. These normal regions are predicted for each sample individually and used to calculate the final reference signal. NSA can be applied to any CN data regardless the microarray technology and preprocessing method. It also finds an optimal weighting of the samples minimizing possible batch effects. Results Five human datasets (a subset of HapMap samples, Glioblastoma Multiforme (GBM), Ovarian, Prostate and Lung Cancer experiments) have been analyzed. It is shown that using only tumoral samples, NSA is able to remove the bias in the copy number estimation, to reduce the noise and therefore, to increase the ability to detect copy number aberrations (CNAs). These improvements allow NSA to also detect recurrent aberrations more accurately than other state of the art methods. Conclusions NSA provides a robust and accurate reference for scaling probe signals data to CN values without the need of control samples. It minimizes the problems of bias, noise and batch effects in the estimation of CNs. Therefore, NSA scaling approach helps to better detect recurrent CNAs than current methods. The automatic selection of references makes it useful to perform bulk analysis of many GEO or ArrayExpress experiments without the need of developing a parser to find the normal samples or possible batches within the data. The method is

  15. 40 CFR 262.22 - Number of copies.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...) 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... returned to the generator....

  16. 40 CFR 262.22 - Number of copies.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ...) 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... returned to the generator....

  17. 40 CFR 262.22 - Number of copies.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...) 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... returned to the generator....

  18. 40 CFR 262.22 - Number of copies.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...) 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... returned to the generator....

  19. 40 CFR 262.22 - Number of copies.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...) 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... returned to the generator....

  20. 12 CFR 269b.730 - Number of copies; form.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 12 Banks and Banking 4 2013-01-01 2013-01-01 false Number of copies; form. 269b.730 Section 269b... SYSTEM (CONTINUED) CHARGES OF UNFAIR LABOR PRACTICES General Rules § 269b.730 Number of copies; form... filed with four copies in addition to the original. All matters filed shall be printed, typed,...

  1. 12 CFR 269b.730 - Number of copies; form.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 12 Banks and Banking 3 2011-01-01 2011-01-01 false Number of copies; form. 269b.730 Section 269b... SYSTEM CHARGES OF UNFAIR LABOR PRACTICES General Rules § 269b.730 Number of copies; form. Except as... copies in addition to the original. All matters filed shall be printed, typed, or otherwise...

  2. Schizophrenia copy number variants and associative learning.

    PubMed

    Clifton, N E; Pocklington, A J; Scholz, B; Rees, E; Walters, J T R; Kirov, G; O'Donovan, M C; Owen, M J; Wilkinson, L S; Thomas, K L; Hall, J

    2017-02-01

    Large-scale genomic studies have made major progress in identifying genetic risk variants for schizophrenia. A key finding from these studies is that there is an increased burden of genomic copy number variants (CNVs) in schizophrenia cases compared with controls. The mechanism through which these CNVs confer risk for the symptoms of schizophrenia, however, remains unclear. One possibility is that schizophrenia risk CNVs impact basic associative learning processes, abnormalities of which have long been associated with the disorder. To investigate whether genes in schizophrenia CNVs impact on specific phases of associative learning we combined human genetics with experimental gene expression studies in animals. In a sample of 11 917 schizophrenia cases and 16 416 controls, we investigated whether CNVs from patients with schizophrenia are enriched for genes expressed during the consolidation, retrieval or extinction of associative memories. We show that CNVs from cases are enriched for genes expressed during fear extinction in the hippocampus, but not genes expressed following consolidation or retrieval. These results suggest that CNVs act to impair inhibitory learning in schizophrenia, potentially contributing to the development of core symptoms of the disorder.

  3. Schizophrenia copy number variants and associative learning

    PubMed Central

    Clifton, N E; Pocklington, A J; Scholz, B; Rees, E; Walters, J T R; Kirov, G; O'Donovan, M C; Owen, M J; Wilkinson, L S; Thomas, K L; Hall, J

    2017-01-01

    Large-scale genomic studies have made major progress in identifying genetic risk variants for schizophrenia. A key finding from these studies is that there is an increased burden of genomic copy number variants (CNVs) in schizophrenia cases compared with controls. The mechanism through which these CNVs confer risk for the symptoms of schizophrenia, however, remains unclear. One possibility is that schizophrenia risk CNVs impact basic associative learning processes, abnormalities of which have long been associated with the disorder. To investigate whether genes in schizophrenia CNVs impact on specific phases of associative learning we combined human genetics with experimental gene expression studies in animals. In a sample of 11 917 schizophrenia cases and 16 416 controls, we investigated whether CNVs from patients with schizophrenia are enriched for genes expressed during the consolidation, retrieval or extinction of associative memories. We show that CNVs from cases are enriched for genes expressed during fear extinction in the hippocampus, but not genes expressed following consolidation or retrieval. These results suggest that CNVs act to impair inhibitory learning in schizophrenia, potentially contributing to the development of core symptoms of the disorder. PMID:27956746

  4. Copy number variation in the horse genome.

    PubMed

    Ghosh, Sharmila; Qu, Zhipeng; Das, Pranab J; Fang, Erica; Juras, Rytis; Cothran, E Gus; McDonell, Sue; Kenney, Daniel G; Lear, Teri L; Adelson, David L; Chowdhary, Bhanu P; Raudsepp, Terje

    2014-10-01

    We constructed a 400K WG tiling oligoarray for the horse and applied it for the discovery of copy number variations (CNVs) in 38 normal horses of 16 diverse breeds, and the Przewalski horse. Probes on the array represented 18,763 autosomal and X-linked genes, and intergenic, sub-telomeric and chrY sequences. We identified 258 CNV regions (CNVRs) across all autosomes, chrX and chrUn, but not in chrY. CNVs comprised 1.3% of the horse genome with chr12 being most enriched. American Miniature horses had the highest and American Quarter Horses the lowest number of CNVs in relation to Thoroughbred reference. The Przewalski horse was similar to native ponies and draft breeds. The majority of CNVRs involved genes, while 20% were located in intergenic regions. Similar to previous studies in horses and other mammals, molecular functions of CNV-associated genes were predominantly in sensory perception, immunity and reproduction. The findings were integrated with previous studies to generate a composite genome-wide dataset of 1476 CNVRs. Of these, 301 CNVRs were shared between studies, while 1174 were novel and require further validation. Integrated data revealed that to date, 41 out of over 400 breeds of the domestic horse have been analyzed for CNVs, of which 11 new breeds were added in this study. Finally, the composite CNV dataset was applied in a pilot study for the discovery of CNVs in 6 horses with XY disorders of sexual development. A homozygous deletion involving AKR1C gene cluster in chr29 in two affected horses was considered possibly causative because of the known role of AKR1C genes in testicular androgen synthesis and sexual development. While the findings improve and integrate the knowledge of CNVs in horses, they also show that for effective discovery of variants of biomedical importance, more breeds and individuals need to be analyzed using comparable methodological approaches.

  5. Copy Number Profiling of Brazilian Astrocytomas

    PubMed Central

    Bidinotto, Lucas Tadeu; Torrieri, Raul; Mackay, Alan; Almeida, Gisele Caravina; Viana-Pereira, Marta; Cruvinel-Carloni, Adriana; Spina, Maria Luisa; Campanella, Nathalia Cristina; Pereira de Menezes, Weder; Clara, Carlos Afonso; Becker, Aline Paixão; Jones, Chris; Reis, Rui Manuel

    2016-01-01

    Copy number alterations (CNA) are one of the driving mechanisms of glioma tumorigenesis, and are currently used as important biomarkers in the routine setting. Therefore, we performed CNA profiling of 65 astrocytomas of distinct malignant grades (WHO grade I–IV) of Brazilian origin, using array-CGH and microsatellite instability analysis (MSI), and investigated their correlation with TERT and IDH1 mutational status and clinico-pathological features. Furthermore, in silico analysis using the Oncomine database was performed to validate our findings and extend the findings to gene expression level. We found that the number of genomic alterations increases in accordance with glioma grade. In glioblastomas (GBM), the most common alterations were gene amplifications (PDGFRA, KIT, KDR, EGFR, and MET) and deletions (CDKN2A and PTEN). Log-rank analysis correlated EGFR amplification and/or chr7 gain with better survival of the patients. MSI was observed in 11% of GBMs. A total of 69% of GBMs presented TERT mutation, whereas IDH1 mutation was most frequent in diffuse (85.7%) and anaplastic (100%) astrocytomas. The combination of 1p19q deletion and TERT and IDH1 mutational status separated tumor groups that showed distinct age of diagnosis and outcome. In silico validation pointed to less explored genes that may be worthy of future investigation, such as CDK2, DMRTA1, and MTAP. Herein, using an extensive integrated analysis, we indicated potentially important genes, not extensively studied in gliomas, that could be further explored to assess their biological and clinical impact in astrocytomas. PMID:27172220

  6. Comparison of Copy Number of HSF Genes in Two Buffalo Genomes.

    PubMed

    Lal, Shardul Vikram; Mukherjee, Ayan; Brahma, Biswajit; Gohain, Moloya; Patra, Mahesh Chandra; Saini, Sushil Kumar; Mishra, Purushottam; Ahlawat, Sonika; Upadhyaya, Ramesh C; Datta, Tirtha K; De, Sachinandan

    2016-01-01

    The copy number variation (CNV) is the number of copies of a particular gene in the genotype of an individual. Recent evidences show that the CNVs can vary in frequency and occurrence between breeds. These variations reportedly allowed different breeds to adapt to different environments. As copy number variations follow Mendelian pattern of inheritance, identification and distribution of these variants between populations can be used to infer the evolutionary history of the species. In this study, we have examined the absolute copy number of four Heat shock factor genes viz. HSF-1, 2, 4, and 5 in two different breeds of buffalo species using real-time PCR. Here, we report that the absolute copy number of HSF2 varies between the two breeds. In contrast no significant difference was observed in the copy number for HSF-1, 4, and 5 between the two breeds. Our results provide evidence for the presence of breed specific differences in HSF2 genomic copy number. This seems to be the first step in delineating the genetic factors underlying environmental adaptation between the two breeds. Nevertheless, a more detailed study is needed to characterize the functional consequence of this variation.

  7. RECONSTRUCTING DNA COPY NUMBER BY PENALIZED ESTIMATION AND IMPUTATION.

    PubMed

    Zhang, Zhongyang; Lange, Kenneth; Ophoff, Roel; Sabatti, Chiara

    2010-12-01

    Recent advances in genomics have underscored the surprising ubiquity of DNA copy number variation (CNV). Fortunately, modern genotyping platforms also detect CNVs with fairly high reliability. Hidden Markov models and algorithms have played a dominant role in the interpretation of CNV data. Here we explore CNV reconstruction via estimation with a fused-lasso penalty as suggested by Tibshirani and Wang [Biostatistics 9 (2008) 18-29]. We mount a fresh attack on this difficult optimization problem by the following: (a) changing the penalty terms slightly by substituting a smooth approximation to the absolute value function, (b) designing and implementing a new MM (majorization-minimization) algorithm, and (c) applying a fast version of Newton's method to jointly update all model parameters. Together these changes enable us to minimize the fused-lasso criterion in a highly effective way.We also reframe the reconstruction problem in terms of imputation via discrete optimization. This approach is easier and more accurate than parameter estimation because it relies on the fact that only a handful of possible copy number states exist at each SNP. The dynamic programming framework has the added bonus of exploiting information that the current fused-lasso approach ignores. The accuracy of our imputations is comparable to that of hidden Markov models at a substantially lower computational cost.

  8. Copy number variation in Thai population.

    PubMed

    Suktitipat, Bhoom; Naktang, Chaiwat; Mhuantong, Wuttichai; Tularak, Thitima; Artiwet, Paramita; Pasomsap, Ekawat; Jongjaroenprasert, Wallaya; Fuchareon, Suthat; Mahasirimongkol, Surakameth; Chantratita, Wasan; Yimwadsana, Boonsit; Charoensawan, Varodom; Jinawath, Natini

    2014-01-01

    Copy number variation (CNV) is a major genetic polymorphism contributing to genetic diversity and human evolution. Clinical application of CNVs for diagnostic purposes largely depends on sufficient population CNV data for accurate interpretation. CNVs from general population in currently available databases help classify CNVs of uncertain clinical significance, and benign CNVs. Earlier studies of CNV distribution in several populations worldwide showed that a significant fraction of CNVs are population specific. In this study, we characterized and analyzed CNVs in 3,017 unrelated Thai individuals genotyped with the Illumina Human610, Illumina HumanOmniexpress, or Illumina HapMap550v3 platform. We employed hidden Markov model and circular binary segmentation methods to identify CNVs, extracted 23,458 CNVs consistently identified by both algorithms, and cataloged these high confident CNVs into our publicly available Thai CNV database. Analysis of CNVs in the Thai population identified a median of eight autosomal CNVs per individual. Most CNVs (96.73%) did not overlap with any known chromosomal imbalance syndromes documented in the DECIPHER database. When compared with CNVs in the 11 HapMap3 populations, CNVs found in the Thai population shared several characteristics with CNVs characterized in HapMap3. Common CNVs in Thais had similar frequencies to those in the HapMap3 populations, and all high frequency CNVs (>20%) found in Thai individuals could also be identified in HapMap3. The majorities of CNVs discovered in the Thai population, however, were of low frequency, or uniquely identified in Thais. When performing hierarchical clustering using CNV frequencies, the CNV data were clustered into Africans, Europeans, and Asians, in line with the clustering performed with single nucleotide polymorphism (SNP) data. As CNV data are specific to origin of population, our population-specific reference database will serve as a valuable addition to the existing resources for

  9. Copy Number Variation in Familial Parkinson Disease

    PubMed Central

    Pankratz, Nathan; Dumitriu, Alexandra; Hetrick, Kurt N.; Sun, Mei; Latourelle, Jeanne C.; Wilk, Jemma B.; Halter, Cheryl; Doheny, Kimberly F.; Gusella, James F.; Nichols, William C.; Myers, Richard H.; Foroud, Tatiana; DeStefano, Anita L.

    2011-01-01

    Copy number variants (CNVs) are known to cause Mendelian forms of Parkinson disease (PD), most notably in SNCA and PARK2. PARK2 has a recessive mode of inheritance; however, recent evidence demonstrates that a single CNV in PARK2 (but not a single missense mutation) may increase risk for PD. We recently performed a genome-wide association study for PD that excluded individuals known to have either a LRRK2 mutation or two PARK2 mutations. Data from the Illumina370Duo arrays were re-clustered using only white individuals with high quality intensity data, and CNV calls were made using two algorithms, PennCNV and QuantiSNP. After quality assessment, the final sample included 816 cases and 856 controls. Results varied between the two CNV calling algorithms for many regions, including the PARK2 locus (genome-wide p = 0.04 for PennCNV and p = 0.13 for QuantiSNP). However, there was consistent evidence with both algorithms for two novel genes, USP32 and DOCK5 (empirical, genome-wide p-values<0.001). PARK2 CNVs tended to be larger, and all instances that were molecularly tested were validated. In contrast, the CNVs in both novel loci were smaller and failed to replicate using real-time PCR, MLPA, and gel electrophoresis. The DOCK5 variation is more akin to a VNTR than a typical CNV and the association is likely caused by artifact due to DNA source. DNA for all the cases was derived from whole blood, while the DNA for all controls was derived from lymphoblast cell lines. The USP32 locus contains many SNPs with low minor allele frequency leading to a loss of heterozygosity that may have been spuriously interpreted by the CNV calling algorithms as support for a deletion. Thus, only the CNVs within the PARK2 locus could be molecularly validated and associated with PD susceptibility. PMID:21829596

  10. Performance of Molecular Inversion Probes (MIP) in Allele CopyNumber Determination

    SciTech Connect

    Wang, Yuker; Moorhead, Martin; Karlin-Neumann, George; Wang,Nicolas J.; Ireland, James; Lin, Steven; Chen, Chunnuan; Heiser, LauraM.; Chin, Koei; Esserman, Laura; Gray, Joe W.; Spellman, Paul T.; Faham,Malek

    2007-05-14

    We have developed a new protocol for using MolecularInversion Probes (MIP) to accurately and specifically measure allele copynumber (ACN). The new protocol provides for significant improvementsincluding the reduction of input DNA (from 2?g) by more than 25 fold (to75ng total genomic DNA), higher overall precision resulting in one orderof magnitude lower false positive rate, and greater dynamic range withaccurate absolute copy number up to 60 copies.

  11. Mitochondrial DNA Copy Number Is Associated with Breast Cancer Risk

    PubMed Central

    Thyagarajan, Bharat; Wang, Renwei; Nelson, Heather; Barcelo, Helene; Koh, Woon-Puay; Yuan, Jian-Min

    2013-01-01

    Mitochondrial DNA (mtDNA) copy number in peripheral blood is associated with increased risk of several cancers. However, data from prospective studies on mtDNA copy number and breast cancer risk are lacking. We evaluated the association between mtDNA copy number in peripheral blood and breast cancer risk in a nested case-control study of 183 breast cancer cases with pre-diagnostic blood samples and 529 individually matched controls among participants of the Singapore Chinese Health Study. The mtDNA copy number was measured using real time PCR. Conditional logistic regression analyses showed that there was an overall positive association between mtDNA copy number and breast cancer risk (Ptrend = 0.01). The elevated risk for higher mtDNA copy numbers was primarily seen for women with <3 years between blood draw and cancer diagnosis; ORs (95% CIs) for 2nd, 3rd, 4th, and 5th quintile of mtDNA copy number were 1.52 (0.61, 3.82), 2.52 (1.03, 6.12), 3.12 (1.31, 7.43), and 3.06 (1.25, 7.47), respectively, compared with the 1st quintile (Ptrend = 0.004). There was no association between mtDNA copy number and breast cancer risk among women who donated a blood sample ≥3 years before breast cancer diagnosis (Ptrend = 0.41). This study supports a prospective association between increased mtDNA copy number and breast cancer risk that is dependent on the time interval between blood collection and breast cancer diagnosis. Future studies are warranted to confirm these findings and to elucidate the biological role of mtDNA copy number in breast cancer risk. PMID:23776581

  12. 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

  13. Mitochondrial DNA copy number in peripheral blood and melanoma risk.

    PubMed

    Shen, Jie; Gopalakrishnan, Vancheswaran; Lee, Jeffrey E; Fang, Shenying; Zhao, Hua

    2015-01-01

    Mitochondrial DNA (mtDNA) copy number in peripheral blood has been suggested as risk modifier in various types of cancer. However, its influence on melanoma risk is unclear. We evaluated the association between mtDNA copy number in peripheral blood and melanoma risk in 500 melanoma cases and 500 healthy controls from an ongoing melanoma study. The mtDNA copy number was measured using real-time polymerase chain reaction. Overall, mean mtDNA copy number was significantly higher in cases than in controls (1.15 vs 0.99, P<0.001). Increased mtDNA copy number was associated with a 1.45-fold increased risk of melanoma (95% confidence interval: 1.12-1.97). Significant joint effects between mtDNA copy number and variables related to pigmentation and history of sunlight exposure were observed. This study supports an association between increased mtDNA copy number and melanoma risk that is independent on the known melanoma risk factors (pigmentation and history of sunlight exposure).

  14. 47 CFR 3.25 - Number of copies.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... AUTHORITIES IN MARITIME AND MARITIME MOBILE-SATELLITE RADIO SERVICES Application Procedures § 3.25 Number of copies. One original and one copy of FCC Form 44, “Application For Certification As An Accounting Authority” will be required. Only applications mailed to the Commission on official, Commission...

  15. 47 CFR 3.25 - Number of copies.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... AUTHORITIES IN MARITIME AND MARITIME MOBILE-SATELLITE RADIO SERVICES Application Procedures § 3.25 Number of copies. One original and one copy of FCC Form 44, “Application For Certification As An Accounting Authority” will be required. Only applications mailed to the Commission on official, Commission...

  16. Human copy number variation and complex genetic disease.

    PubMed

    Girirajan, Santhosh; Campbell, Catarina D; Eichler, Evan E

    2011-01-01

    Copy number variants (CNVs) play an important role in human disease and population diversity. Advancements in technology have allowed for the analysis of CNVs in thousands of individuals with disease in addition to thousands of controls. These studies have identified rare CNVs associated with neuropsychiatric diseases such as autism, schizophrenia, and intellectual disability. In addition, copy number polymorphisms (CNPs) are present at higher frequencies in the population, show high diversity in copy number, sequence, and structure, and have been associated with multiple phenotypes, primarily related to immune or environmental response. However, the landscape of copy number variation still remains largely unexplored, especially for smaller CNVs and those embedded within complex regions of the human genome. An integrated approach including characterization of single nucleotide variants and CNVs in a large number of individuals with disease and normal genomes holds the promise of thoroughly elucidating the genetic basis of human disease and diversity.

  17. Molecular methods for genotyping complex copy number polymorphisms.

    PubMed

    Cantsilieris, Stuart; Baird, Paul N; White, Stefan J

    2013-02-01

    Genome structural variation shows remarkable complexity with respect to copy number, sequence content and distribution. While the discovery of copy number polymorphisms (CNP) has increased exponentially in recent years, the transition from discovery to genotyping has proved challenging, particularly for CNPs embedded in complex regions of the genome. CNPs that are collectively common in the population and possess a dynamic range of copy numbers have proved the most difficult to genotype in association studies. This is in some part due to technical limitations of genotyping assays and the sequence properties of the genomic region being analyzed. Here we describe in detail the basis of a number of molecular techniques used to genotype complex CNPs, compare and contrast these approaches for determination of multi-allelic copy number, and discuss the potential application of these techniques in genetic studies.

  18. Copy number variation and evolution in humans and chimpanzees

    PubMed Central

    Perry, George H.; Yang, Fengtang; Marques-Bonet, Tomas; Murphy, Carly; Fitzgerald, Tomas; Lee, Arthur S.; Hyland, Courtney; Stone, Anne C.; Hurles, Matthew E.; Tyler-Smith, Chris; Eichler, Evan E.; Carter, Nigel P.; Lee, Charles; Redon, Richard

    2008-01-01

    Copy number variants (CNVs) underlie many aspects of human phenotypic diversity and provide the raw material for gene duplication and gene family expansion. However, our understanding of their evolutionary significance remains limited. We performed comparative genomic hybridization on a single human microarray platform to identify CNVs among the genomes of 30 humans and 30 chimpanzees as well as fixed copy number differences between species. We found that human and chimpanzee CNVs occur in orthologous genomic regions far more often than expected by chance and are strongly associated with the presence of highly homologous intrachromosomal segmental duplications. By adapting population genetic analyses for use with copy number data, we identified functional categories of genes that have likely evolved under purifying or positive selection for copy number changes. In particular, duplications and deletions of genes with inflammatory response and cell proliferation functions may have been fixed by positive selection and involved in the adaptive phenotypic differentiation of humans and chimpanzees. PMID:18775914

  19. Hotspots for copy number variation in chimpanzees and humans

    PubMed Central

    Perry, George H.; Tchinda, Joelle; McGrath, Sean D.; Zhang, Junjun; Picker, Simon R.; Cáceres, Angela M.; Iafrate, A. John; Tyler-Smith, Chris; Scherer, Stephen W.; Eichler, Evan E.; Stone, Anne C.; Lee, Charles

    2006-01-01

    Copy number variation is surprisingly common among humans and can be involved in phenotypic diversity and variable susceptibility to complex diseases, but little is known of the extent of copy number variation in nonhuman primates. We have used two array-based comparative genomic hybridization platforms to identify a total of 355 copy number variants (CNVs) in the genomes of 20 wild-born chimpanzees (Pan troglodytes) and have compared the identified chimpanzee CNVs to known human CNVs from previous studies. Many CNVs were observed in the corresponding regions in both chimpanzees and humans; especially those CNVs of higher frequency. Strikingly, these loci are enriched 20-fold for ancestral segmental duplications, which may facilitate CNV formation through nonallelic homologous recombination mechanisms. Therefore, some of these regions may be unstable “hotspots” for the genesis of copy number variation, with recurrent duplications and deletions occurring across and within species. PMID:16702545

  20. Evolution of ribosomal RNA gene copy number on the sex chromosomes of Drosophila melanogaster.

    PubMed

    Lyckegaard, E M; Clark, A G

    1991-07-01

    A diverse array of cellular and evolutionary forces--including unequal crossing-over, magnification, compensation, and natural selection--is at play modulating the number of copies of ribosomal RNA (rRNA) genes on the X and Y chromosomes of Drosophila. Accurate estimates of naturally occurring distributions of copy numbers on both the X and Y chromosomes are needed in order to explore the evolutionary end result of these forces. Estimates of relative copy numbers of the ribosomal DNA repeat, as well as of the type I and type II inserts, were obtained for a series of 96 X chromosomes and 144 Y chromosomes by using densitometric measurements of slot blots of genomic DNA from adult D. melanogaster bearing appropriate deficiencies that reveal chromosome-specific copy numbers. Estimates of copy number were put on an absolute scale with slot blots having serial dilutions both of the repeat and of genomic DNA from nonpolytene larval brain and imaginal discs. The distributions of rRNA copy number are decidedly skewed, with a long tail toward higher copy numbers. These distributions were fitted by a population genetic model that posits three different types of exchange events--sister-chromatid exchange, intrachromatid exchange, and interchromosomal crossing-over. In addition, the model incorporates natural selection, because experimental evidence shows that there is a minimum number of functional elements necessary for survival. Adequate fits of the model were found, indicating that either natural selection also eliminates chromosomes with high copy number or that the rate of intrachromatid exchange exceeds the rate of interchromosomal exchange.

  1. Evolution vs the number of gene copies per primitive cell.

    PubMed

    Koch, A L

    1984-01-01

    Computer simulations are presented of the rate at which an advantageous mutant would displace the prototype in a replicating system without an accurate segregation mechanism. If the number of gene copies in the system is indefinitely large, Darwinian evolution is essentially stopped because there is no coupling of phenotype with genotype, i.e., there is no growth advantage to the advantageous gene relative to the prototype and therefore no "survival of the fittest." The inhibition of evolution due to a number of gene copies less than 100 would have been not insurmountable. Although the presence of multiple copies would have allowed replacement by an advantageous mutant, it provided a way for the primitive cell to conserve less immediately useful genes that could evolve into different or more effective genes. This possibility was lost as accurate segregation mechanisms evolved and cells with few copies of each gene, such as modern procaryotes, arose.

  2. Copy number variations exploration of multiple genes in Graves’ disease

    PubMed Central

    Song, Rong-hua; Shao, Xiao-qing; Li, Ling; Wang, Wen; Zhang, Jin-an

    2017-01-01

    Abstract Background: Few previous published papers reported copy number variations of genes could affect the predisposition of Graves’ disease (GD). Herein, the aim of this study was to explore the association between copy number variations (CNV) profile and GD. Methods: The preliminary copy number microarray used to screen copy number variant genes was performed in 6 GD patients. Five CNV candidate genes (CFH, CFHR1, KIAA0125, UGT2B15, and UGT2B17) were then validated in an independent set of samples (50 GD patients and 50 matched healthy ones) by the Accucopy assay method. The CNV of the other 2 genes TRY6 and CCL3L1 was investigated in 144 GD patients and 144 healthy volunteers by the definitive genotyping technique using the Taqman quantitative polymerase-chain-reaction (Taqman qPCR). TRY6 gene-associated single nucleotide polymorphism (SNP), rs13230029, was genotyped by the PCR-ligase detection reaction (LDR) in 675 GD patients and 898 healthy controls. Results: There were no correlation of the gene copy number (GCN) of CFH, CFHR1, KIAA0125, UGT2B15, and UGT2B17 with GD. In comparison with that of controls, the GCN distribution of TRY6 and CCL3L1 in GD patients did not show significantly differ (P > 0.05). Furthermore, TRY6-related polymorphism (rs13230029) showed no difference between GD patients and controls. No correlation was found between CNV or SNP genotype and clinical phenotypes. Generally, there were no link of the copy numbers of several genes, including CFH, CFHR1, KIAA0125, UGT2B15, UGT2B17, TRY6, and CCL3L1 to GD. Conclusion: Our results clearly indicated that the copy number variations of multiple genes, namely CFH, CFHR1, KIAA0125, UGT2B15, UGT2B17, TRY6, and CCL3L1, were not associated with the development of GD. PMID:28121931

  3. Reconstructing DNA copy number by joint segmentation of multiple sequences

    PubMed Central

    2012-01-01

    Background Variations in DNA copy number carry information on the modalities of genome evolution and mis-regulation of DNA replication in cancer cells. Their study can help localize tumor suppressor genes, distinguish different populations of cancerous cells, and identify genomic variations responsible for disease phenotypes. A number of different high throughput technologies can be used to identify copy number variable sites, and the literature documents multiple effective algorithms. We focus here on the specific problem of detecting regions where variation in copy number is relatively common in the sample at hand. This problem encompasses the cases of copy number polymorphisms, related samples, technical replicates, and cancerous sub-populations from the same individual. Results We present a segmentation method named generalized fused lasso (GFL) to reconstruct copy number variant regions. GFL is based on penalized estimation and is capable of processing multiple signals jointly. Our approach is computationally very attractive and leads to sensitivity and specificity levels comparable to those of state-of-the-art specialized methodologies. We illustrate its applicability with simulated and real data sets. Conclusions The flexibility of our framework makes it applicable to data obtained with a wide range of technology. Its versatility and speed make GFL particularly useful in the initial screening stages of large data sets. PMID:22897923

  4. Genetically complex epilepsies, copy number variants and syndrome constellations.

    PubMed

    Mefford, Heather C; Mulley, John C

    2010-10-05

    Epilepsy is one of the most common neurological disorders, with a prevalence of 1% and lifetime incidence of 3%. There are numerous epilepsy syndromes, most of which are considered to be genetic epilepsies. Despite the discovery of more than 20 genes for epilepsy to date, much of the genetic contribution to epilepsy is not yet known. Copy number variants have been established as an important source of mutation in other complex brain disorders, including intellectual disability, autism and schizophrenia. Recent advances in technology now facilitate genome-wide searches for copy number variants and are beginning to be applied to epilepsy. Here, we discuss what is currently known about the contribution of copy number variants to epilepsy, and how that knowledge is redefining classification of clinical and genetic syndromes.

  5. Systematic biases in DNA copy number originate from isolation procedures

    PubMed Central

    2013-01-01

    Background The ability to accurately detect DNA copy number variation in both a sensitive and quantitative manner is important in many research areas. However, genome-wide DNA copy number analyses are complicated by variations in detection signal. Results While GC content has been used to correct for this, here we show that coverage biases are tissue-specific and independent of the detection method as demonstrated by next-generation sequencing and array CGH. Moreover, we show that DNA isolation stringency affects the degree of equimolar coverage and that the observed biases coincide with chromatin characteristics like gene expression, genomic isochores, and replication timing. Conclusion These results indicate that chromatin organization is a main determinant for differential DNA retrieval. These findings are highly relevant for germline and somatic DNA copy number variation analyses. PMID:23618369

  6. Histotype-specific copy-number alterations in ovarian cancer

    PubMed Central

    2012-01-01

    Background Epithelial ovarian cancer is characterized by multiple genomic alterations; most are passenger alterations which do not confer tumor growth. Like many cancers, it is a heterogeneous disease and can be broadly categorized into 4 main histotypes of clear cell, endometrioid, mucinous, and serous. To date, histotype-specific copy number alterations have been difficult to elucidate. The difficulty lies in having sufficient sample size in each histotype for statistical analyses. Methods To dissect the heterogeneity of ovarian cancer and identify histotype-specific alterations, we used an in silico hypothesis-driven approach on multiple datasets of epithelial ovarian cancer. Results In concordance with previous studies on global copy number alterations landscape, the study showed similar alterations. However, when the landscape was de-convoluted into histotypes, distinct alterations were observed. We report here significant histotype-specific copy number alterations in ovarian cancer and showed that there is genomic diversity amongst the histotypes. 76 cancer genes were found to be significantly altered with several as potential copy number drivers, including ERBB2 in mucinous, and TPM3 in endometrioid histotypes. ERBB2 was found to have preferential alterations, where it was amplified in mucinous (28.6%) but deleted in serous tumors (15.1%). Validation of ERBB2 expression showed significant correlation with microarray data (p=0.007). There also appeared to be reciprocal relationship between KRAS mutation and copy number alterations. In mucinous tumors where KRAS mutation is common, the gene was not significantly altered. However, KRAS was significantly amplified in serous tumors where mutations are rare in high grade tumors. Conclusions The study demonstrates that the copy number landscape is specific to the histotypes and identification of these alterations can pave the way for targeted drug therapy specific to the histotypes. PMID:23078675

  7. Selection of suitable endogenous reference genes for relative copy number detection in sugarcane.

    PubMed

    Xue, Bantong; Guo, Jinlong; Que, Youxiong; Fu, Zhiwei; Wu, Luguang; Xu, Liping

    2014-05-19

    Transgene copy number has a great impact on the expression level and stability of exogenous gene in transgenic plants. Proper selection of endogenous reference genes is necessary for detection of genetic components in genetically modification (GM) crops by quantitative real-time PCR (qPCR) or by qualitative PCR approach, especially in sugarcane with polyploid and aneuploid genomic structure. qPCR technique has been widely accepted as an accurate, time-saving method on determination of copy numbers in transgenic plants and on detection of genetically modified plants to meet the regulatory and legislative requirement. In this study, to find a suitable endogenous reference gene and its real-time PCR assay for sugarcane (Saccharum spp. hybrids) DNA content quantification, we evaluated a set of potential "single copy" genes including P4H, APRT, ENOL, CYC, TST and PRR, through qualitative PCR and absolute quantitative PCR. Based on copy number comparisons among different sugarcane genotypes, including five S. officinarum, one S. spontaneum and two S. spp. hybrids, these endogenous genes fell into three groups: ENOL-3--high copy number group, TST-1 and PRR-1--medium copy number group, P4H-1, APRT-2 and CYC-2--low copy number group. Among these tested genes, P4H, APRT and CYC were the most stable, while ENOL and TST were the least stable across different sugarcane genotypes. Therefore, three primer pairs of P4H-3, APRT-2 and CYC-2 were then selected as the suitable reference gene primer pairs for sugarcane. The test of multi-target reference genes revealed that the APRT gene was a specific amplicon, suggesting this gene is the most suitable to be used as an endogenous reference target for sugarcane DNA content quantification. These results should be helpful for establishing accurate and reliable qualitative and quantitative PCR analysis of GM sugarcane.

  8. Estimation of copy number alterations from exome sequencing data.

    PubMed

    Valdés-Mas, Rafael; Bea, Silvia; Puente, Diana A; López-Otín, Carlos; Puente, Xose S

    2012-01-01

    Exome sequencing constitutes an important technology for the study of human hereditary diseases and cancer. However, the ability of this approach to identify copy number alterations in primary tumor samples has not been fully addressed. Here we show that somatic copy number alterations can be reliably estimated using exome sequencing data through a strategy that we have termed exome2cnv. Using data from 86 paired normal and primary tumor samples, we identified losses and gains of complete chromosomes or large genomic regions, as well as smaller regions affecting a minimum of one gene. Comparison with high-resolution comparative genomic hybridization (CGH) arrays revealed a high sensitivity and a low number of false positives in the copy number estimation between both approaches. We explore the main factors affecting sensitivity and false positives with real data, and provide a side by side comparison with CGH arrays. Together, these results underscore the utility of exome sequencing to study cancer samples by allowing not only the identification of substitutions and indels, but also the accurate estimation of copy number alterations.

  9. Self consistent, absolute calibration technique for photon number resolving detectors.

    PubMed

    Avella, A; Brida, G; Degiovanni, I P; Genovese, M; Gramegna, M; Lolli, L; Monticone, E; Portesi, C; Rajteri, M; Rastello, M L; Taralli, E; Traina, P; White, M

    2011-11-07

    Well characterized photon number resolving detectors are a requirement for many applications ranging from quantum information and quantum metrology to the foundations of quantum mechanics. This prompts the necessity for reliable calibration techniques at the single photon level. In this paper we propose an innovative absolute calibration technique for photon number resolving detectors, using a pulsed heralded photon source based on parametric down conversion. The technique, being absolute, does not require reference standards and is independent upon the performances of the heralding detector. The method provides the results of quantum efficiency for the heralded detector as a function of detected photon numbers. Furthermore, we prove its validity by performing the calibration of a Transition Edge Sensor based detector, a real photon number resolving detector that has recently demonstrated its effectiveness in various quantum information protocols.

  10. Genomic Copy Number Variation in Disorders of Cognitive Development

    ERIC Educational Resources Information Center

    Morrow, Eric M.

    2010-01-01

    Objective: To highlight recent discoveries in the area of genomic copy number variation in neuropsychiatric disorders including intellectual disability, autism, and schizophrenia. To emphasize new principles emerging from this area, involving the genetic architecture of disease, pathophysiology, and diagnosis. Method: Review of studies published…

  11. Mapping cattle copy number variations in water buffalo

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Copy number variation (CNV) is abundant in livestock, differing from SNPs in extent, origin and functional impact. Despite progress in CNV discovery, the nucleotide resolution architecture of most CNVs remains elusive. Using modified forms of open-source variant detection software packages, we have ...

  12. 10 CFR 205.372 - Filing procedures; number of copies.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 10 Energy 3 2010-01-01 2010-01-01 false Filing procedures; number of copies. 205.372 Section 205.372 Energy DEPARTMENT OF ENERGY OIL ADMINISTRATIVE PROCEDURES AND SANCTIONS Electric Power System... Electric Facilities and the Transfer of Electricity to Alleviate An Emergency Shortage of Electric...

  13. Genomic and evolutionary characteristics of cattle copy number variations

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We performed a systematic analysis of cattle copy number variations (CNVs) using the Bovine HapMap SNP genotyping data, including 539 animals of 21 modern cattle breeds and 6 outgroups. After correcting genomic waves and considering the trio information, we identified 682 candidate CNV regions (CNVR...

  14. Analysis of copy number variations reveals differences among cattle breeds

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genomic structural variation is an important and abundant source of genetic and phenotypic variation. Here we describe the first systematic and genome-wide analysis of copy number variations (CNVs) in the modern domesticated cattle using array comparative genomic hybridization (array CGH) and quanti...

  15. Analysis of copy number variations among cattle breeds

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genomic structural variation is an important and abundant source of genetic and phenotypic variation. Here we describe the first systematic and genome-wide analysis of copy number variations (CNVs) in the modern domesticated cattle using array comparative genomic hybridization (array CGH) and quanti...

  16. Quantum State Discrimination Using the Minimum Average Number of Copies

    NASA Astrophysics Data System (ADS)

    Slussarenko, Sergei; Weston, Morgan M.; Li, Jun-Gang; Campbell, Nicholas; Wiseman, Howard M.; Pryde, Geoff J.

    2017-01-01

    In the task of discriminating between nonorthogonal quantum states from multiple copies, the key parameters are the error probability and the resources (number of copies) used. Previous studies have considered the task of minimizing the average error probability for fixed resources. Here we introduce a new state discrimination task: minimizing the average resources for a fixed admissible error probability. We show that this new task is not performed optimally by previously known strategies, and derive and experimentally test a detection scheme that performs better.

  17. High Resolution Analysis of Copy Number Mutation in Breast Cancer

    DTIC Science & Technology

    2005-05-01

    U.S. Army Medical Research and Materiel Command Fort Detrick, Maryland 21702-5012 DISTRIBUTION STATEMENT: Approved for Public Release; Distribution...SPONSORING i MONITORING AGENCY NAME(S) AND ADDRESS(ES) AGENCY REPORT NUMBER U.S. Army Medical Research and Materiel Command Fort Detrick, Maryland 21702...Fellow ofthe Watson School of Biological Sciences and a predoctoral fel- stable copy number and map location, low of the Howard Hughes Medical Institute

  18. Selection of Suitable Endogenous Reference Genes for Relative Copy Number Detection in Sugarcane

    PubMed Central

    Xue, Bantong; Guo, Jinlong; Que, Youxiong; Fu, Zhiwei; Wu, Luguang; Xu, Liping

    2014-01-01

    Transgene copy number has a great impact on the expression level and stability of exogenous gene in transgenic plants. Proper selection of endogenous reference genes is necessary for detection of genetic components in genetically modification (GM) crops by quantitative real-time PCR (qPCR) or by qualitative PCR approach, especially in sugarcane with polyploid and aneuploid genomic structure. qPCR technique has been widely accepted as an accurate, time-saving method on determination of copy numbers in transgenic plants and on detection of genetically modified plants to meet the regulatory and legislative requirement. In this study, to find a suitable endogenous reference gene and its real-time PCR assay for sugarcane (Saccharum spp. hybrids) DNA content quantification, we evaluated a set of potential “single copy” genes including P4H, APRT, ENOL, CYC, TST and PRR, through qualitative PCR and absolute quantitative PCR. Based on copy number comparisons among different sugarcane genotypes, including five S. officinarum, one S. spontaneum and two S. spp. hybrids, these endogenous genes fell into three groups: ENOL-3—high copy number group, TST-1 and PRR-1—medium copy number group, P4H-1, APRT-2 and CYC-2—low copy number group. Among these tested genes, P4H, APRT and CYC were the most stable, while ENOL and TST were the least stable across different sugarcane genotypes. Therefore, three primer pairs of P4H-3, APRT-2 and CYC-2 were then selected as the suitable reference gene primer pairs for sugarcane. The test of multi-target reference genes revealed that the APRT gene was a specific amplicon, suggesting this gene is the most suitable to be used as an endogenous reference target for sugarcane DNA content quantification. These results should be helpful for establishing accurate and reliable qualitative and quantitative PCR analysis of GM sugarcane. PMID:24857916

  19. High-resolution copy number arrays in cancer and the problem of normal genome copy number variation.

    PubMed

    Gorringe, Kylie L; Campbell, Ian G

    2008-11-01

    High-resolution techniques for analysis of genome copy number (CN) enable the analysis of complex cancer somatic genetics. However, the analysis of these data is difficult, and failure to consider a number of issues in depth may result in false leads or unnecessary rejection of true positives. First, segmental duplications may falsely generate CN breakpoints in aneuploid samples. Second, even when tumor data were each normalized to matching lymphocyte DNA, we still observed copy number polymorphisms masquerading as somatic alterations due to allelic imbalance. We investigated a number of different solutions and determined that evaluating matching normal DNA, or at least using locally derived normal baseline data, were preferable to relying on current online databases because of poor cross-platform compatibility and the likelihood of excluding genuine small somatic alterations.

  20. Mitochondrial DNA copy number variation across human cancers

    PubMed Central

    Reznik, Ed; Miller, Martin L; Şenbabaoğlu, Yasin; Riaz, Nadeem; Sarungbam, Judy; Tickoo, Satish K; Al-Ahmadie, Hikmat A; Lee, William; Seshan, Venkatraman E; Hakimi, A Ari; Sander, Chris

    2016-01-01

    Mutations, deletions, and changes in copy number of mitochondrial DNA (mtDNA), are observed throughout cancers. Here, we survey mtDNA copy number variation across 22 tumor types profiled by The Cancer Genome Atlas project. We observe a tendency for some cancers, especially of the bladder, breast, and kidney, to be depleted of mtDNA, relative to matched normal tissue. Analysis of genetic context reveals an association between incidence of several somatic alterations, including IDH1 mutations in gliomas, and mtDNA content. In some but not all cancer types, mtDNA content is correlated with the expression of respiratory genes, and anti-correlated to the expression of immune response and cell-cycle genes. In tandem with immunohistochemical evidence, we find that some tumors may compensate for mtDNA depletion to sustain levels of respiratory proteins. Our results highlight the extent of mtDNA copy number variation in tumors and point to related therapeutic opportunities. DOI: http://dx.doi.org/10.7554/eLife.10769.001 PMID:26901439

  1. A method for calling copy number polymorphism using haplotypes

    PubMed Central

    Ho Jang, Gun; Christie, Jason D.; Feng, Rui

    2013-01-01

    Single nucleotide polymorphism (SNP) and copy number variation (CNV) are both widespread characteristic of the human genome, but are often called separately on common genotyping platforms. To capture integrated SNP and CNV information, methods have been developed for calling allelic specific copy numbers or so called copy number polymorphism (CNP), using limited inter-marker correlation. In this paper, we proposed a haplotype-based maximum likelihood method to call CNP, which takes advantage of the valuable multi-locus linkage disequilibrium (LD) information in the population. We also developed a computationally efficient algorithm to estimate haplotype frequencies and optimize individual CNP calls iteratively, even at presence of missing data. Through simulations, we demonstrated our model is more sensitive and accurate in detecting various CNV regions, compared with commonly-used CNV calling methods including PennCNV, another hidden Markov model (HMM) using CNP, a scan statistic, segCNV, and cnvHap. Our method often performs better in the regions with higher LD, in longer CNV regions, and in common CNV than the opposite. We implemented our method on the genotypes of 90 HapMap CEU samples and 23 patients with acute lung injury (ALI). For each ALI patient the genotyping was performed twice. The CNPs from our method show good consistency and accuracy comparable to others. PMID:24069028

  2. Copy number variation of individual cattle genomes using next-generation sequencing

    PubMed Central

    Bickhart, Derek M.; Hou, Yali; Schroeder, Steven G.; Alkan, Can; Cardone, Maria Francesca; Matukumalli, Lakshmi K.; Song, Jiuzhou; Schnabel, Robert D.; Ventura, Mario; Taylor, Jeremy F.; Garcia, Jose Fernando; Van Tassell, Curtis P.; Sonstegard, Tad S.; Eichler, Evan E.; Liu, George E.

    2012-01-01

    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 Holstein, and one Hereford) and one indicine (Nelore) cattle. Within mapped chromosomal sequence, we identified 1265 CNV regions comprising ∼55.6-Mbp sequence—476 of which (∼38%) have not previously been reported. We validated this sequence-based CNV call set with array comparative genomic hybridization (aCGH), quantitative PCR (qPCR), and fluorescent in situ hybridization (FISH), achieving a validation rate of 82% and a false positive rate of 8%. We further estimated absolute copy numbers for genomic segments and annotated genes in each individual. Surveys of the top 25 most variable genes revealed that the Nelore individual had the lowest copy numbers in 13 cases (∼52%, χ2 test; P-value <0.05). In contrast, genes related to pathogen- and parasite-resistance, such as CATHL4 and ULBP17, were highly duplicated in the Nelore individual relative to the taurine cattle, while genes involved in lipid transport and metabolism, including APOL3 and FABP2, were highly duplicated in the beef breeds. These CNV regions also harbor genes like BPIFA2A (BSP30A) and WC1, suggesting that some CNVs may be associated with breed-specific differences in adaptation, health, and production traits. By providing the first individualized cattle CNV and segmental duplication maps and genome-wide gene copy number estimates, we enable future CNV studies into highly duplicated regions in the cattle genome. PMID:22300768

  3. Genome Architecture and Its Roles in Human Copy Number Variation

    PubMed Central

    Chen, Lu; Zhou, Weichen; Zhang, Ling

    2014-01-01

    Besides single-nucleotide variants in the human genome, large-scale genomic variants, such as copy number variations (CNVs), are being increasingly discovered as a genetic source of human diversity and the pathogenic factors of diseases. Recent experimental findings have shed light on the links between different genome architectures and CNV mutagenesis. In this review, we summarize various genomic features and discuss their contributions to CNV formation. Genomic repeats, including both low-copy and high-copy repeats, play important roles in CNV instability, which was initially known as DNA recombination events. Furthermore, it has been found that human genomic repeats can also induce DNA replication errors and consequently result in CNV mutations. Some recent studies showed that DNA replication timing, which reflects the high-order information of genomic organization, is involved in human CNV mutations. Our review highlights that genome architecture, from DNA sequence to high-order genomic organization, is an important molecular factor in CNV mutagenesis and human genomic instability. PMID:25705150

  4. Bias of selection on human copy-number variants.

    PubMed

    Nguyen, Duc-Quang; Webber, Caleb; Ponting, Chris P

    2006-02-01

    Although large-scale copy-number variation is an important contributor to conspecific genomic diversity, whether these variants frequently contribute to human phenotype differences remains unknown. If they have few functional consequences, then copy-number variants (CNVs) might be expected both to be distributed uniformly throughout the human genome and to encode genes that are characteristic of the genome as a whole. We find that human CNVs are significantly overrepresented close to telomeres and centromeres and in simple tandem repeat sequences. Additionally, human CNVs were observed to be unusually enriched in those protein-coding genes that have experienced significantly elevated synonymous and nonsynonymous nucleotide substitution rates, estimated between single human and mouse orthologues. CNV genes encode disproportionately large numbers of secreted, olfactory, and immunity proteins, although they contain fewer than expected genes associated with Mendelian disease. Despite mouse CNVs also exhibiting a significant elevation in synonymous substitution rates, in most other respects they do not differ significantly from the genomic background. Nevertheless, they encode proteins that are depleted in olfactory function, and they exhibit significantly decreased amino acid sequence divergence. Natural selection appears to have acted discriminately among human CNV genes. The significant overabundance, within human CNVs, of genes associated with olfaction, immunity, protein secretion, and elevated coding sequence divergence, indicates that a subset may have been retained in the human population due to the adaptive benefit of increased gene dosage. By contrast, the functional characteristics of mouse CNVs either suggest that advantageous gene copies have been depleted during recent selective breeding of laboratory mouse strains or suggest that they were preferentially fixed as a consequence of the larger effective population size of wild mice. It thus appears that CNV

  5. Copy number variation plays an important role in clinical epilepsy

    PubMed Central

    Olson, Heather; Shen, Yiping; Avallone, Jennifer; Sheidley, Beth R.; Pinsky, Rebecca; Bergin, Ann M.; Berry, Gerard T.; Duffy, Frank H.; Eksioglu, Yaman; Harris, David J.; Hisama, Fuki M.; Ho, Eugenia; Irons, Mira; Jacobsen, Christina M.; James, Philip; Kothare, Sanjeev; Khwaja, Omar; Lipton, Jonathan; Loddenkemper, Tobias; Markowitz, Jennifer; Maski, Kiran; Megerian, J. Thomas; Neilan, Edward; Raffalli, Peter C.; Robbins, Michael; Roberts, Amy; Roe, Eugene; Rollins, Caitlin; Sahin, Mustafa; Sarco, Dean; Schonwald, Alison; Smith, Sharon E.; Soul, Janet; Stoler, Joan M.; Takeoka, Masanori; Tan, Wen-Han; Torres, Alcy R.; Tsai, Peter; Urion, David K.; Weissman, Laura; Wolff, Robert; Wu, Bai-Lin; Miller, David T.; Poduri, Annapurna

    2015-01-01

    Objective To evaluate the role of copy number abnormalities detectable by chromosomal microarray (CMA) testing in patients with epilepsy at a tertiary care center. Methods We identified patients with ICD-9 codes for epilepsy or seizures and clinical CMA testing performed between October 2006 and February 2011 at Boston Children’s Hospital. We reviewed medical records and included patients meeting criteria for epilepsy. We phenotypically characterized patients with epilepsy-associated abnormalities on CMA. Results Of 973 patients who had CMA and ICD-9 codes for epilepsy or seizures, 805 patients satisfied criteria for epilepsy. We observed 437 copy number variants (CNVs) in 323 patients (1–4 per patient), including 185 (42%) deletions and 252 (58%) duplications. Forty (9%) were confirmed de novo, 186 (43%) were inherited, and parental data were unavailable for 211 (48%). Excluding full chromosome trisomies, CNV size ranged from 18 kb to 142 Mb, and 34% were over 500 kb. In at least 40 cases (5%), the epilepsy phenotype was explained by a CNV, including 29 patients with epilepsy-associated syndromes and 11 with likely disease-associated CNVs involving epilepsy genes or “hotspots.” We observed numerous recurrent CNVs including 10 involving loss or gain of Xp22.31, a region described in patients with and without epilepsy. Interpretation Copy number abnormalities play an important role in patients with epilepsy. Given that the diagnostic yield of CMA for epilepsy patients is similar to the yield in autism spectrum disorders and in prenatal diagnosis, for which published guidelines recommend testing with CMA, we recommend the implementation of CMA in the evaluation of unexplained epilepsy. PMID:24811917

  6. Eclipse period of R1 plasmids during downshift from elevated copy number: Nonrandom selection of copies for replication.

    PubMed

    Olsson, Jan A; Berg, Otto; Nordström, Kurt; Dasgupta, Santanu

    2012-03-01

    The classical Meselson-Stahl density-shift method was used to study replication of pOU71, a runaway-replication derivative of plasmid R1 in Escherichia coli. The miniplasmid maintained the normal low copy number of R1 during steady growth at 30°C, but as growth temperatures were raised above 34°C, the copy number of the plasmid increased to higher levels, and at 42°C, it replicated without control in a runaway replication mode with lethal consequences for the host. The eclipse periods (minimum time between successive replication of the same DNA) of the plasmid shortened with rising copy numbers at increasing growth temperatures (Olsson et al., 2003). In this work, eclipse periods were measured during downshifts in copy number of pOU71 after it had replicated at 39 and 42°C, resulting in 7- and 50-fold higher than normal plasmid copy number per cell, respectively. Eclipse periods for plasmid replication, measured during copy number downshift, suggested that plasmid R1, normally selected randomly for replication, showed a bias such that a newly replicated DNA had a higher probability of replication compared to the bulk of the R1 population. However, even the unexpected nonrandom replication followed the copy number kinetics such that every generation, the plasmids underwent the normal inherited number of replication, n, independent of the actual number of plasmid copies in a newborn cell.

  7. Reconstructing Breakage Fusion Bridge Architectures Using Noisy Copy Numbers

    PubMed Central

    Bafna, Vineet

    2015-01-01

    Abstract The Breakage Fusion Bridge (BFB) process is a key marker for genomic instability, producing highly rearranged genomes in relatively small numbers of cell cycles. While the process itself was observed during the late 1930s, little is known about the extent of BFB in tumor genome evolution. Moreover, BFB can dramatically increase copy numbers of chromosomal segments, which in turn hardens the tasks of both reference-assisted and ab initio genome assembly. Based on available data such as Next Generation Sequencing (NGS) and Array Comparative Genomic Hybridization (aCGH) data, we show here how BFB evidence may be identified, and how to enumerate all possible evolutions of the process with respect to observed data. Specifically, we describe practical algorithms that, given a chromosomal arm segmentation and noisy segment copy number estimates, produce all segment count vectors supported by the data that can be produced by BFB, and all corresponding BFB architectures. This extends the scope of analyses described in our previous work, which produced a single count vector and architecture per instance. We apply these analyses to a comprehensive human cancer dataset, demonstrate the effectiveness and efficiency of the computation, and suggest methods for further assertions of candidate BFB samples. Source code of our tool can be found online. PMID:26020441

  8. Plasmid copy number noise in monoclonal populations of bacteria

    NASA Astrophysics Data System (ADS)

    Wong Ng, Jérôme; Chatenay, Didier; Robert, Jérôme; Poirier, Michael Guy

    2010-01-01

    Plasmids are extra chromosomal DNA that can confer to their hosts’ supplementary characteristics such as antibiotic resistance. Plasmids code for their copy number through their own replication frequency. Even though the biochemical networks underlying the plasmid copy number (PCN) regulation processes have been studied and modeled, no measurement of the heterogeneity in PCN within a whole population has been done. We have developed a fluorescent-based measurement system, which enables determination of the mean and noise in PCN within a monoclonal population of bacteria. Two different fluorescent protein reporters were inserted: one on the chromosome and the other on the plasmid. The fluorescence of these bacteria was measured with a microfluidic flow cytometry device. We show that our measurements are consistent with known plasmid characteristics. We find that the partitioning system lowers the PCN mean and standard deviation. Finally, bacterial populations were allowed to grow without selective pressure. In this case, we were able to determine the plasmid loss rate and growth inhibition effect.

  9. Confirmed rare copy number variants implicate novel genes in schizophrenia.

    PubMed

    Tam, Gloria W C; van de Lagemaat, Louie N; Redon, Richard; Strathdee, Karen E; Croning, Mike D R; Malloy, Mary P; Muir, Walter J; Pickard, Ben S; Deary, Ian J; Blackwood, Douglas H R; Carter, Nigel P; Grant, Seth G N

    2010-04-01

    Understanding how cognitive processes including learning, memory, decision making and ideation are encoded by the genome is a key question in biology. Identification of sets of genes underlying human mental disorders is a path towards this objective. Schizophrenia is a common disease with cognitive symptoms, high heritability and complex genetics. We have identified genes involved with schizophrenia by measuring differences in DNA copy number across the entire genome in 91 schizophrenia cases and 92 controls in the Scottish population. Our data reproduce rare and common variants observed in public domain data from >3000 schizophrenia cases, confirming known disease loci as well as identifying novel loci. We found copy number variants in PDE10A (phosphodiesterase 10A), CYFIP1 [cytoplasmic FMR1 (Fragile X mental retardation 1)-interacting protein 1], K(+) channel genes KCNE1 and KCNE2, the Down's syndrome critical region 1 gene RCAN1 (regulator of calcineurin 1), cell-recognition protein CHL1 (cell adhesion molecule with homology with L1CAM), the transcription factor SP4 (specificity protein 4) and histone deacetylase HDAC9, among others (see http://www.genes2cognition.org/SCZ-CNV). Integrating the function of these many genes into a coherent model of schizophrenia and cognition is a major unanswered challenge.

  10. 17 CFR 230.402 - Number of copies; binding; signatures.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... copy shall be bound, in one or more parts, without stiff covers. The binding shall be made on the side... filed with the Commission. Each copy shall be bound, in one or more parts, without stiff covers....

  11. Copy number analysis of NIPBL in a cohort of 510 patients reveals rare copy number variants and a mosaic deletion

    PubMed Central

    Cheng, Yu-Wei; Tan, Christopher A; Minor, Agata; Arndt, Kelly; Wysinger, Latrice; Grange, Dorothy K; Kozel, Beth A; Robin, Nathaniel H; Waggoner, Darrel; Fitzpatrick, Carrie; Das, Soma; del Gaudio, Daniela

    2014-01-01

    Cornelia de Lange syndrome (CdLS) is a genetically heterogeneous disorder characterized by growth retardation, intellectual disability, upper limb abnormalities, hirsutism, and characteristic facial features. In this study we explored the occurrence of intragenic NIPBL copy number variations (CNVs) in a cohort of 510 NIPBL sequence-negative patients with suspected CdLS. Copy number analysis was performed by custom exon-targeted oligonucleotide array-comparative genomic hybridization and/or MLPA. Whole-genome SNP array was used to further characterize rearrangements extending beyond the NIPBL gene. We identified NIPBL CNVs in 13 patients (2.5%) including one intragenic duplication and a deletion in mosaic state. Breakpoint sequences in two patients provided further evidence of a microhomology-mediated replicative mechanism as a potential predominant contributor to CNVs in NIPBL. Patients for whom clinical information was available share classical CdLS features including craniofacial and limb defects. Our experience in studying the frequency of NIBPL CNVs in the largest series of patients to date widens the mutational spectrum of NIPBL and emphasizes the clinical utility of performing NIPBL deletion/duplication analysis in patients with CdLS. PMID:24689074

  12. DNA Copy Number Control Through Inhibition of Replication Fork Progression

    PubMed Central

    Nordman, Jared T.; Kozhevnikova, Elena N.; Verrijzer, C. Peter; Pindyurin, Alexey V.; Andreyeva, Evgeniya N.; Shloma, Victor V.; Zhimulev, Igor F.; Orr-Weaver, Terry L.

    2014-01-01

    Summary Proper control of DNA replication is essential to ensure faithful transmission of genetic material and to prevent chromosomal aberrations that can drive cancer progression and developmental disorders. DNA replication is regulated primarily at the level of initiation and is under strict cell cycle regulation. Importantly, DNA replication is highly influenced by developmental cues. In Drosophila, specific regions of the genome are repressed for DNA replication during differentiation by the SNF2 domain-containing protein SUUR through an unknown mechanism. We demonstrate that SUUR is recruited to active replication forks and mediates repression of DNA replication by directly inhibiting replication fork progression instead of functioning as a replication fork barrier. Mass-spec identification of SUUR associated proteins identified the replicative helicase member CDC45 as a SUUR-associated protein, supporting a role for SUUR directly at replication forks. Our results reveal that control of eukaryotic DNA copy number can occur through inhibition of replication fork progression. PMID:25437540

  13. Regional copy number-independent deregulation of transcription in cancer.

    PubMed

    Stransky, Nicolas; Vallot, Céline; Reyal, Fabien; Bernard-Pierrot, Isabelle; de Medina, Sixtina Gil Diez; Segraves, Rick; de Rycke, Yann; Elvin, Paul; Cassidy, Andrew; Spraggon, Carolyn; Graham, Alexander; Southgate, Jennifer; Asselain, Bernard; Allory, Yves; Abbou, Claude C; Albertson, Donna G; Thiery, Jean Paul; Chopin, Dominique K; Pinkel, Daniel; Radvanyi, François

    2006-12-01

    Genetic and epigenetic alterations have been identified that lead to transcriptional deregulation in cancers. Genetic mechanisms may affect single genes or regions containing several neighboring genes, as has been shown for DNA copy number changes. It was recently reported that epigenetic suppression of gene expression can also extend to a whole region; this is known as long-range epigenetic silencing. Various techniques are available for identifying regional genetic alterations, but no large-scale analysis has yet been carried out to obtain an overview of regional epigenetic alterations. We carried out an exhaustive search for regions susceptible to such mechanisms using a combination of transcriptome correlation map analysis and array CGH data for a series of bladder carcinomas. We validated one candidate region experimentally, demonstrating histone methylation leading to the loss of expression of neighboring genes without DNA methylation.

  14. [Copy number variation: markers and predictors for type 2 diabetes].

    PubMed

    Ramírez-Valverde, Alan Gilberto; Antúnez-Ortiz, Diana Lizzete; Méndez-Beleche, Alberto; Flores-Alfaro, Eugenia; Ascencio-Montiel, Iván Jesús; Cruz, Miguel

    2015-01-01

    Type 2 diabetes (T2D) is a disease characterized by a deficiency in production or action of insulin. It is the result mainly of the interaction of the environment, lifestyle, as well as genetic factors. It is considered as one of the major health issues in the world because it affects severely the psychological well-being and overall life quality. Recently it has been shown that DNA copy number variations (CNVs) are associated with several diseases, including obesity and T2D. The CNVs are present from 9 to 18 % of the genome and can modify the expression levels of mRNA and proteins encoded by genes located near their localization. Less is known about their contribution to the pathogenesis of metabolic diseases, which is necessary to characterize so that these variations can be potentially used as biomarkers of genetic risk CNVs of T2D.

  15. Detection of Copy Number Alterations Using Single Cell Sequencing.

    PubMed

    Knouse, Kristin A; Wu, Jie; Hendricks, Austin

    2017-02-17

    Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and microbial populations. Traditional methods for assessing genetic heterogeneity have been limited by low resolution, low sensitivity, and/or low specificity. Single cell sequencing has emerged as a powerful tool for detecting genetic heterogeneity with high resolution, high sensitivity and, when appropriately analyzed, high specificity. Here we provide a protocol for the isolation, whole genome amplification, sequencing, and analysis of single cells. Our approach allows for the reliable identification of megabase-scale copy number variants in single cells. However, aspects of this protocol can also be applied to investigate other types of genetic alterations in single cells.

  16. Copy Number Variants in Alzheimer’s Disease

    PubMed Central

    Cuccaro, Denis; De Marco, Elvira Valeria; Cittadella, Rita; Cavallaro, Sebastiano

    2016-01-01

    Alzheimer’s disease (AD) is a devastating disease mainly afflicting elderly people, characterized by decreased cognition, loss of memory, and eventually death. Although risk and deterministic genes are known, major genetics research programs are underway to gain further insights into the inheritance of AD. In the last years, in particular, new developments in genome-wide scanning methodologies have enabled the association of a number of previously uncharacterized copy number variants (CNVs, gain or loss of DNA) in AD. Because of the exceedingly large number of studies performed, it has become difficult for geneticists as well as clinicians to systematically follow, evaluate, and interpret the growing number of (sometime conflicting) CNVs implicated in AD. In this review, after a brief introduction of this type of structural variation, and a description of available databases, computational analyses, and technologies involved, we provide a systematic review of all published data showing statistical and scientific significance of pathogenic CNVs and discuss the role they might play in AD. PMID:27662298

  17. Analysis of genetic copy number changes in cervical disease progression

    PubMed Central

    2010-01-01

    Background Cervical dysplasia and tumorigenesis have been linked with numerous chromosomal aberrations. The goal of this study was to evaluate 35 genomic regions associated with cervical disease and to select those which were found to have the highest frequency of aberration for use as probes in fluorescent in-situ hybridization. Methods The frequency of gains and losses using fluorescence in-situ hybridization were assessed in these 35 regions on 30 paraffin-embedded cervical biopsy specimens. Based on this assessment, 6 candidate fluorescently labeled probes (8q24, Xp22, 20q13, 3p14, 3q26, CEP15) were selected for additional testing on a set of 106 cervical biopsy specimens diagnosed as Normal, CIN1, CIN2, CIN3, and SCC. The data were analyzed on the basis of signal mean, % change of signal mean between histological categories, and % positivity. Results The study revealed that the chromosomal regions with the highest frequency of copy number gains and highest combined sensitivity and specificity in high-grade cervical disease were 8q24 and 3q26. The cytological application of these two probes was then evaluated on 118 ThinPrep™ samples diagnosed as Normal, ASCUS, LSIL, HSIL and Cancer to determine utility as a tool for less invasive screening. Using gains of either 8q24 or 3q26 as a positivity criterion yielded specificity (Normal +LSIL+ASCUS) of 81.0% and sensitivity (HSIL+Cancer) of 92.3% based on a threshold of 4 positive cells. Conclusions The application of a FISH assay comprised of chromosomal probes 8q24 and 3q26 to cervical cytology specimens confirms the positive correlation between increasing dysplasia and copy gains and shows promise as a marker in cervical disease progression. PMID:20712890

  18. Decoding NF1 Intragenic Copy-Number Variations.

    PubMed

    Hsiao, Meng-Chang; Piotrowski, Arkadiusz; Callens, Tom; Fu, Chuanhua; Wimmer, Katharina; Claes, Kathleen B M; Messiaen, Ludwine

    2015-08-06

    Genomic rearrangements can cause both Mendelian and complex disorders. Currently, several major mechanisms causing genomic rearrangements, such as non-allelic homologous recombination (NAHR), non-homologous end joining (NHEJ), fork stalling and template switching (FoSTeS), and microhomology-mediated break-induced replication (MMBIR), have been proposed. However, to what extent these mechanisms contribute to gene-specific pathogenic copy-number variations (CNVs) remains understudied. Furthermore, few studies have resolved these pathogenic alterations at the nucleotide-level. Accordingly, our aim was to explore which mechanisms contribute to a large, unique set of locus-specific non-recurrent genomic rearrangements causing the genetic neurocutaneous disorder neurofibromatosis type 1 (NF1). Through breakpoint-spanning PCR as well as array comparative genomic hybridization, we have identified the breakpoints in 85 unrelated individuals carrying an NF1 intragenic CNV. Furthermore, we characterized the likely rearrangement mechanisms of these 85 CNVs, along with those of two additional previously published NF1 intragenic CNVs. Unlike the most typical recurrent rearrangements mediated by flanking low-copy repeats (LCRs), NF1 intragenic rearrangements vary in size, location, and rearrangement mechanisms. We propose the DNA-replication-based mechanisms comprising both FoSTeS and/or MMBIR and serial replication stalling to be the predominant mechanisms leading to NF1 intragenic CNVs. In addition to the loop within a 197-bp palindrome located in intron 40, four Alu elements located in introns 1, 2, 3, and 50 were also identified as intragenic-rearrangement hotspots within NF1.

  19. Decoding NF1 Intragenic Copy-Number Variations

    PubMed Central

    Hsiao, Meng-Chang; Piotrowski, Arkadiusz; Callens, Tom; Fu, Chuanhua; Wimmer, Katharina; Claes, Kathleen B.M.; Messiaen, Ludwine

    2015-01-01

    Genomic rearrangements can cause both Mendelian and complex disorders. Currently, several major mechanisms causing genomic rearrangements, such as non-allelic homologous recombination (NAHR), non-homologous end joining (NHEJ), fork stalling and template switching (FoSTeS), and microhomology-mediated break-induced replication (MMBIR), have been proposed. However, to what extent these mechanisms contribute to gene-specific pathogenic copy-number variations (CNVs) remains understudied. Furthermore, few studies have resolved these pathogenic alterations at the nucleotide-level. Accordingly, our aim was to explore which mechanisms contribute to a large, unique set of locus-specific non-recurrent genomic rearrangements causing the genetic neurocutaneous disorder neurofibromatosis type 1 (NF1). Through breakpoint-spanning PCR as well as array comparative genomic hybridization, we have identified the breakpoints in 85 unrelated individuals carrying an NF1 intragenic CNV. Furthermore, we characterized the likely rearrangement mechanisms of these 85 CNVs, along with those of two additional previously published NF1 intragenic CNVs. Unlike the most typical recurrent rearrangements mediated by flanking low-copy repeats (LCRs), NF1 intragenic rearrangements vary in size, location, and rearrangement mechanisms. We propose the DNA-replication-based mechanisms comprising both FoSTeS and/or MMBIR and serial replication stalling to be the predominant mechanisms leading to NF1 intragenic CNVs. In addition to the loop within a 197-bp palindrome located in intron 40, four Alu elements located in introns 1, 2, 3, and 50 were also identified as intragenic-rearrangement hotspots within NF1. PMID:26189818

  20. Family-Based Benchmarking of Copy Number Variation Detection Software.

    PubMed

    Nutsua, Marcel Elie; Fischer, Annegret; Nebel, Almut; Hofmann, Sylvia; Schreiber, Stefan; Krawczak, Michael; Nothnagel, Michael

    2015-01-01

    The analysis of structural variants, in particular of copy-number variations (CNVs), has proven valuable in unraveling the genetic basis of human diseases. Hence, a large number of algorithms have been developed for the detection of CNVs in SNP array signal intensity data. Using the European and African HapMap trio data, we undertook a comparative evaluation of six commonly used CNV detection software tools, namely Affymetrix Power Tools (APT), QuantiSNP, PennCNV, GLAD, R-gada and VEGA, and assessed their level of pair-wise prediction concordance. The tool-specific CNV prediction accuracy was assessed in silico by way of intra-familial validation. Software tools differed greatly in terms of the number and length of the CNVs predicted as well as the number of markers included in a CNV. All software tools predicted substantially more deletions than duplications. Intra-familial validation revealed consistently low levels of prediction accuracy as measured by the proportion of validated CNVs (34-60%). Moreover, up to 20% of apparent family-based validations were found to be due to chance alone. Software using Hidden Markov models (HMM) showed a trend to predict fewer CNVs than segmentation-based algorithms albeit with greater validity. PennCNV yielded the highest prediction accuracy (60.9%). Finally, the pairwise concordance of CNV prediction was found to vary widely with the software tools involved. We recommend HMM-based software, in particular PennCNV, rather than segmentation-based algorithms when validity is the primary concern of CNV detection. QuantiSNP may be used as an additional tool to detect sets of CNVs not detectable by the other tools. Our study also reemphasizes the need for laboratory-based validation, such as qPCR, of CNVs predicted in silico.

  1. Copy number gain of VCX, X-linked multi-copy gene, leads to cell proliferation and apoptosis during spermatogenesis

    PubMed Central

    Huang, Zhenyao; Zhang, Yan; Zhou, Ran; Song, Ling; Ling, Xiufeng; Hu, Zhibin; Miao, Dengshun; Shen, Hongbing; Xia, Yankai; Wang, Xinru; Lu, Chuncheng

    2016-01-01

    Male factor infertility affects one-sixth of couples worldwide, and non-obstructive azoospermia (NOA) is one of the most severe forms. In recent years there has been increasing evidence to implicate the participation of X chromosome in the process of spermatogenesis. To uncover the roles of X-linked multi-copy genes in spermatogenesis, we performed systematic analysis of X-linked gene copy number variations (CNVs) and Y chromosome haplogrouping in 447 idiopathic NOA patients and 485 healthy controls. Interestingly, the frequency of individuals with abnormal level copy of Variable charge, X-linked (VCX) was significantly different between cases and controls after multiple test correction (p = 5.10 × 10−5). To discriminate the effect of gain/loss copies in these genes, we analyzed the frequency of X-linked multi-copy genes in subjects among subdivided groups. Our results demonstrated that individuals with increased copy numbers of Nuclear RNA export factor 2 (NXF2) (p = 9.21 × 10−8) and VCX (p = 1.97 × 10−4) conferred the risk of NOA. In vitro analysis demonstrated that increasing copy number of VCX could upregulate the gene expression and regulate cell proliferation and apoptosis. Our study establishes a robust association between the VCX CNVs and NOA risk. PMID:27705943

  2. 18 CFR 32.4 - Form and style; number of copies.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 18 Conservation of Power and Water Resources 1 2010-04-01 2010-04-01 false Form and style; number of copies. 32.4 Section 32.4 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY... style; number of copies. An original and six conformed copies of an application under §§ 32.1 to...

  3. Clinically relevant copy number variations detected in cerebral palsy

    PubMed Central

    Oskoui, Maryam; Gazzellone, Matthew J.; Thiruvahindrapuram, Bhooma; Zarrei, Mehdi; Andersen, John; Wei, John; Wang, Zhuozhi; Wintle, Richard F.; Marshall, Christian R.; Cohn, Ronald D.; Weksberg, Rosanna; Stavropoulos, Dimitri J.; Fehlings, Darcy; Shevell, Michael I.; Scherer, Stephen W.

    2015-01-01

    Cerebral palsy (CP) represents a group of non-progressive clinically heterogeneous disorders that are characterized by motor impairment and early age of onset, frequently accompanied by co-morbidities. The cause of CP has historically been attributed to environmental stressors resulting in brain damage. While genetic risk factors are also implicated, guidelines for diagnostic assessment of CP do not recommend for routine genetic testing. Given numerous reports of aetiologic copy number variations (CNVs) in other neurodevelopmental disorders, we used microarrays to genotype a population-based prospective cohort of children with CP and their parents. Here we identify de novo CNVs in 8/115 (7.0%) CP patients (∼1% rate in controls). In four children, large chromosomal abnormalities deemed likely pathogenic were found, and they were significantly more likely to have severe neuromotor impairments than those CP subjects without such alterations. Overall, the CNV data would have impacted our diagnosis or classification of CP in 11/115 (9.6%) families. PMID:26236009

  4. Analysis of rare copy number variation in absence epilepsies

    PubMed Central

    Rosch, Richard E.; Valentin, Antonio; Makoff, Andrew; Robinson, Robert; Everett, Kate V.; Nashef, Lina; Pal, Deb K.

    2016-01-01

    Objective: To identify shared genes and pathways between common absence epilepsy (AE) subtypes (childhood absence epilepsy [CAE], juvenile absence epilepsy [JAE], and unclassified absence epilepsy [UAE]) that may indicate common mechanisms for absence seizure generation and potentially a diagnostic continuum. Methods: We used high-density single-nucleotide polymorphism arrays to analyze genome-wide rare copy number variation (CNV) in a cohort of 144 children with AEs (95 CAE, 26 UAE, and 23 JAE). Results: We identified CNVs that are known risk factors for AE in 4 patients, including 3x 15q11.2 deletion. We also expanded the phenotype at 4 regions more commonly identified in other neurodevelopmental disorders: 1p36.33 duplication, 1q21.1 deletion, 22q11.2 duplication, and Xp22.31 deletion and duplication. Fifteen patients (10.5%) were found to carry rare CNVs that disrupt genes associated with neuronal development and function (8 CAE, 2 JAE, and 5 UAE). Four categories of protein are each disrupted by several CNVs: (1) synaptic vesicle membrane or vesicle endocytosis, (2) synaptic cell adhesion, (3) synapse organization and motility via actin, and (4) gap junctions. CNVs within these categories are shared across the AE subtypes. Conclusions: Our results have reinforced the complex and heterogeneous nature of the AEs and their potential for shared genetic mechanisms and have highlighted several pathways that may be important in epileptogenesis of absence seizures. PMID:27123475

  5. Mapping copy number variation by population-scale genome sequencing.

    PubMed

    Mills, Ryan E; Walter, Klaudia; Stewart, Chip; Handsaker, Robert E; Chen, Ken; Alkan, Can; Abyzov, Alexej; Yoon, Seungtai Chris; Ye, Kai; Cheetham, R Keira; Chinwalla, Asif; Conrad, Donald F; Fu, Yutao; Grubert, Fabian; Hajirasouliha, Iman; Hormozdiari, Fereydoun; Iakoucheva, Lilia M; Iqbal, Zamin; Kang, Shuli; Kidd, Jeffrey M; Konkel, Miriam K; Korn, Joshua; Khurana, Ekta; Kural, Deniz; Lam, Hugo Y K; Leng, Jing; Li, Ruiqiang; Li, Yingrui; Lin, Chang-Yun; Luo, Ruibang; Mu, Xinmeng Jasmine; Nemesh, James; Peckham, Heather E; Rausch, Tobias; Scally, Aylwyn; Shi, Xinghua; Stromberg, Michael P; Stütz, Adrian M; Urban, Alexander Eckehart; Walker, Jerilyn A; Wu, Jiantao; Zhang, Yujun; Zhang, Zhengdong D; Batzer, Mark A; Ding, Li; Marth, Gabor T; McVean, Gil; Sebat, Jonathan; Snyder, Michael; Wang, Jun; Ye, Kenny; Eichler, Evan E; Gerstein, Mark B; Hurles, Matthew E; Lee, Charles; McCarroll, Steven A; Korbel, Jan O

    2011-02-03

    Genomic structural variants (SVs) are abundant in humans, differing from other forms of variation in extent, origin and functional impact. Despite progress in SV characterization, the nucleotide resolution architecture of most SVs remains unknown. We constructed a map of unbalanced SVs (that is, copy number variants) based on whole genome DNA sequencing data from 185 human genomes, integrating evidence from complementary SV discovery approaches with extensive experimental validations. Our map encompassed 22,025 deletions and 6,000 additional SVs, including insertions and tandem duplications. Most SVs (53%) were mapped to nucleotide resolution, which facilitated analysing their origin and functional impact. We examined numerous whole and partial gene deletions with a genotyping approach and observed a depletion of gene disruptions amongst high frequency deletions. Furthermore, we observed differences in the size spectra of SVs originating from distinct formation mechanisms, and constructed a map of SV hotspots formed by common mechanisms. Our analytical framework and SV map serves as a resource for sequencing-based association studies.

  6. The Role of Constitutional Copy Number Variants in Breast Cancer.

    PubMed

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

    2015-09-08

    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.

  7. Amplification ratio control system for copy number variation genotyping

    PubMed Central

    Guthrie, Philip A. I.; Gaunt, Tom R.; Abdollahi, Mohammed R.; Rodriguez, Santiago; Lawlor, Debbie A.; Smith, George Davey; Day, Ian N. M.

    2011-01-01

    We describe a generic design for ratiometric analysis suitable for determination of copy number variation (CNV) class of a gene. Following two initial sequence-specific PCR priming cycles, both ends of both amplicons (one test and one reference) in a duplex reaction, are all primed by the same universal primer (UP). Following each amplification denaturation step, the UP target and its reverse complement (UP′) in each strand form a hairpin. The bases immediately beyond the 3′-end of the UP and 5′ of UP′ are chosen such as not to base pair in the hairpin (otherwise priming is ablated). This hairpin creates a single constant environment for priming events and chaperones free 3′-ends of amplicon strands. The resultant ‘amplification ratio control system’ (ARCS) permits ratiometric representation of amplicons relative to the original template into PCR plateau phase. These advantages circumvent the need for real-time PCR for quantitation. Choice of different %(G+C) content for the target and reference amplicons allows liquid phase thermal melt discrimination and quantitation of amplicons. The design is generic, simple to set up and economical. Comparisons with real-time PCR and other techniques are made and CNV assays demonstrated for haptoglobin duplicon and ‘chemokine (C-C motif) ligand 3-like 1’ gene. PMID:21300641

  8. 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

  9. Bayesian hierarchical mixture modeling to assign copy number from a targeted CNV array.

    PubMed

    Cardin, Niall; Holmes, Chris; Donnelly, Peter; Marchini, Jonathan

    2011-09-01

    Accurate assignment of copy number at known copy number variant (CNV) loci is important for both increasing understanding of the structural evolution of genomes as well as for carrying out association studies of copy number with disease. As with calling SNP genotypes, the task can be framed as a clustering problem but for a number of reasons assigning copy number is much more challenging. CNV assays have lower signal-to-noise ratios than SNP assays, often display heavy tailed and asymmetric intensity distributions, contain outlying observations and may exhibit systematic technical differences among different cohorts. In addition, the number of copy-number classes at a CNV in the population may be unknown a priori. Due to these complications, automatic and robust assignment of copy number from array data remains a challenging problem. We have developed a copy number assignment algorithm, CNVCALL, for a targeted CNV array, such as that used by the Wellcome Trust Case Control Consortium's recent CNV association study. We use a Bayesian hierarchical mixture model that robustly identifies both the number of different copy number classes at a specific locus as well as relative copy number for each individual in the sample. This approach is fully automated which is a critical requirement when analyzing large numbers of CNVs. We illustrate the methods performance using real data from the Wellcome Trust Case Control Consortium's CNV association study and using simulated data.

  10. Human gene copy number spectra analysis in congenital heart malformations

    PubMed Central

    Mahnke, Donna K.; Struble, Craig A.; Tuffnell, Maureen E.; Stamm, Karl D.; Hidestrand, Mats; Harris, Susan E.; Goetsch, Mary A.; Simpson, Pippa M.; Bick, David P.; Broeckel, Ulrich; Pelech, Andrew N.; Tweddell, James S.; Mitchell, Michael E.

    2012-01-01

    The clinical significance of copy number variants (CNVs) in congenital heart disease (CHD) continues to be a challenge. Although CNVs including genes can confer disease risk, relationships between gene dosage and phenotype are still being defined. Our goal was to perform a quantitative analysis of CNVs involving 100 well-defined CHD risk genes identified through previously published human association studies in subjects with anatomically defined cardiac malformations. A novel analytical approach permitting CNV gene frequency “spectra” to be computed over prespecified regions to determine phenotype-gene dosage relationships was employed. CNVs in subjects with CHD (n = 945), subphenotyped into 40 groups and verified in accordance with the European Paediatric Cardiac Code, were compared with two control groups, a disease-free cohort (n = 2,026) and a population with coronary artery disease (n = 880). Gains (≥200 kb) and losses (≥100 kb) were determined over 100 CHD risk genes and compared using a Barnard exact test. Six subphenotypes showed significant enrichment (P ≤ 0.05), including aortic stenosis (valvar), atrioventricular canal (partial), atrioventricular septal defect with tetralogy of Fallot, subaortic stenosis, tetralogy of Fallot, and truncus arteriosus. Furthermore, CNV gene frequency spectra were enriched (P ≤ 0.05) for losses at: FKBP6, ELN, GTF2IRD1, GATA4, CRKL, TBX1, ATRX, GPC3, BCOR, ZIC3, FLNA and MID1; and gains at: PRKAB2, FMO5, CHD1L, BCL9, ACP6, GJA5, HRAS, GATA6 and RUNX1. Of CHD subjects, 14% had causal chromosomal abnormalities, and 4.3% had likely causal (significantly enriched), large, rare CNVs. CNV frequency spectra combined with precision phenotyping may lead to increased molecular understanding of etiologic pathways. PMID:22318994

  11. Population Structure Shapes Copy Number Variation in Malaria Parasites

    PubMed Central

    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.

    2016-01-01

    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. PMID:26613787

  12. 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.

  13. Copy number variations related to reproduction traits in Holstein cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Daughter pregnancy rate (DPR) is one of important reproduction traits that affect overall profitability in dairy industry. However, historical selection for production and conformation rather than reproduction has resulted in a decline in cow fertility. Genomic structural variation including copy nu...

  14. Endometriosis Is Associated with Rare Copy Number Variants

    PubMed Central

    Chettier, Rakesh; Ward, Kenneth; Albertsen, Hans M.

    2014-01-01

    Endometriosis is a complex gynecological condition that affects 6–10% of women in their reproductive years and is defined by the presence of endometrial glands and stroma outside the uterus. Twin, family, and genome-wide association (GWA) studies have confirmed a genetic role, yet only a small part of the genetic risk can be explained by SNP variation. Copy number variants (CNVs) account for a greater portion of human genetic variation than SNPs and include more recent mutations of large effect. CNVs, likely to be prominent in conditions with decreased reproductive fitness, have not previously been examined as a genetic contributor to endometriosis. Here we employ a high-density genotyping microarray in a genome-wide survey of CNVs in a case-control population that includes 2,126 surgically confirmed endometriosis cases and 17,974 population controls of European ancestry. We apply stringent quality filters to reduce the false positive rate common to many CNV-detection algorithms from 77.7% to 7.3% without noticeable reduction in the true positive rate. We detected no differences in the CNV landscape between cases and controls on the global level which showed an average of 1.92 CNVs per individual with an average size of 142.3 kb. On the local level we identify 22 CNV-regions at the nominal significance threshold (P<0.05), which is greater than the 8.15 CNV-regions expected based on permutation analysis (P<0.001). Three CNV's passed a genome-wide P-value threshold of 9.3×10−4; a deletion at SGCZ on 8p22 (P = 7.3×10−4, OR = 8.5, Cl = 2.3–31.7), a deletion in MALRD1 on 10p12.31 (P = 5.6×10−4, OR = 14.1, Cl = 2.7–90.9), and a deletion at 11q14.1 (P = 5.7×10−4, OR = 33.8, Cl = 3.3–1651). Two SNPs within the 22 CNVRs show significant genotypic association with endometriosis after adjusting for multiple testing; rs758316 in DPP6 on 7q36.2 (P = 0.0045) and rs4837864 in ASTN2 on 9q33.1 (P = 0.0002). Together

  15. Copy number variations and cognitive phenotypes in unselected populations

    PubMed Central

    Männik, Katrin; Mägi, Reedik; Macé, Aurélien; Cole, Ben; Guyatt, Anna; Shihab, Hashem A.; Maillard, Anne M.; Alavere, Helene; Kolk, Anneli; Reigo, Anu; Mihailov, Evelin; Leitsalu, Liis; Ferreira, Anne-Maud; Nõukas, Margit; Teumer, Alexander; Salvi, Erika; Cusi, Daniele; McGue, Matt; Iacono, William G.; Gaunt, Tom R.; Beckmann, Jacques S.; Jacquemont, Sébastien; Kutalik, Zoltán; Pankratz, Nathan; Timpson, Nicholas; Metspalu, Andres; Reymond, Alexandre

    2015-01-01

    Importance The association of rare copy number variants (CNVs) with complex disorders is almost exclusively evaluated using clinically ascertained cohorts. As a result, the contribution of these genetic variants to cognitive phenotypes in the general population remains unclear. Objectives - To investigate the clinical features of genomic disorders in adult carriers without clinical pre-selection. - To assess the genome-wide burden of rare CNVs on carriers’ educational attainment and intellectual disability prevalence in the general population. Design, Setting, and Participants The population biobank of Estonia (EGCUT) contains 52,000 participants, or 5% of the Estonian adults, enrolled in 2002-2010. General practitioners examined participants and filled out a questionnaire of health- and lifestyle-related questions, as well as reported diagnoses. As EGCUT is representative of the country's population, we investigated a random sample of 7877 individuals for CNV analysis and genotype-phenotype associations with education and disease traits. Main Outcomes and Measures Phenotypes of genomic disorders in the general population, prevalence of autosomal CNVs, and association of the latter variants with decreased educational attainment and increased prevalence of intellectual disability. Results We identified 56 carriers of genomic disorders. Their phenotypes are reminiscent of those described for carriers of identical rearrangements ascertained in clinical cohorts. We also generated a genome-wide map of rare (frequency ≤0.05%) autosomal CNVs and identified 10.5% of the screened general population (n=831) as carriers of CNVs ≥250kb. Carriers of deletions ≥250kb or duplications ≥1Mb show, compared to the Estonian population, a greater prevalence of intellectual disability (P=0.0015, OR=3.16, (95%CI: 1.51-5.98); P=0.0083, OR=3.67, (95%CI: 1.29-8.54), respectively), reduced mean education attainment (a proxy for intelligence; P=1.06e-04; P=5.024e-05, respectively

  16. Copy number ratios determined by two digital polymerase chain reaction systems in genetically modified grains

    NASA Astrophysics Data System (ADS)

    Pérez Urquiza, M.; Acatzi Silva, A. I.

    2014-02-01

    Three certified reference materials produced from powdered seeds to measure the copy number ratio sequences of p35S/hmgA in maize containing MON 810 event, p35S/Le1 in soybeans containing GTS 40-3-2 event and DREB1A/acc1 in wheat were produced according to the ISO Guides 34 and 35. In this paper, we report digital polymerase chain reaction (dPCR) protocols, performance parameters and results of copy number ratio content of genetically modified organisms (GMOs) in these materials using two new dPCR systems to detect and quantify molecular deoxyribonucleic acid: the BioMark® (Fluidigm) and the OpenArray® (Life Technologies) systems. These technologies were implemented at the National Institute of Metrology in Mexico (CENAM) and in the Reference Center for GMO Detection from the Ministry of Agriculture (CNRDOGM), respectively. The main advantage of this technique against the more-used quantitative polymerase chain reaction (qPCR) is that it generates an absolute number of target molecules in the sample, without reference to standards or an endogenous control, which is very useful when not much information is available for new developments or there are no standard reference materials in the market as in the wheat case presented, or when it was not possible to test the purity of seeds as in the maize case presented here. Both systems reported enhanced productivity, increased reliability and reduced instrument footprint. In this paper, the performance parameters and uncertainty of measurement obtained with both systems are presented and compared.

  17. Mutant Kras copy number defines metabolic reprogramming and therapeutic susceptibilities.

    PubMed

    Kerr, Emma M; Gaude, Edoardo; Turrell, Frances K; Frezza, Christian; Martins, Carla P

    2016-03-03

    The RAS/MAPK (mitogen-activated protein kinase) signalling pathway is frequently deregulated in non-small-cell lung cancer, often through KRAS activating mutations. A single endogenous mutant Kras allele is sufficient to promote lung tumour formation in mice but malignant progression requires additional genetic alterations. We recently showed that advanced lung tumours from Kras(G12D/+);p53-null mice frequently exhibit Kras(G12D) allelic enrichment (Kras(G12D)/Kras(wild-type) > 1) (ref. 7), implying that mutant Kras copy gains are positively selected during progression. Here we show, through a comprehensive analysis of mutant Kras homozygous and heterozygous mouse embryonic fibroblasts and lung cancer cells, that these genotypes are phenotypically distinct. In particular, Kras(G12D/G12D) cells exhibit a glycolytic switch coupled to increased channelling of glucose-derived metabolites into the tricarboxylic acid cycle and glutathione biosynthesis, resulting in enhanced glutathione-mediated detoxification. This metabolic rewiring is recapitulated in mutant KRAS homozygous non-small-cell lung cancer cells and in vivo, in spontaneous advanced murine lung tumours (which display a high frequency of Kras(G12D) copy gain), but not in the corresponding early tumours (Kras(G12D) heterozygous). Finally, we demonstrate that mutant Kras copy gain creates unique metabolic dependences that can be exploited to selectively target these aggressive mutant Kras tumours. Our data demonstrate that mutant Kras lung tumours are not a single disease but rather a heterogeneous group comprising two classes of tumours with distinct metabolic profiles, prognosis and therapeutic susceptibility, which can be discriminated on the basis of their relative mutant allelic content. We also provide the first, to our knowledge, in vivo evidence of metabolic rewiring during lung cancer malignant progression.

  18. 5 CFR 2429.25 - Number of copies and paper size.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 5 Administrative Personnel 3 2010-01-01 2010-01-01 false Number of copies and paper size. 2429.25... Requirements § 2429.25 Number of copies and paper size. Unless otherwise provided by the Authority or the... the exception of any prescribed forms, any document or paper filed with the Authority, General...

  19. 5 CFR 2429.25 - Number of copies and paper size.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 5 Administrative Personnel 3 2011-01-01 2011-01-01 false Number of copies and paper size. 2429.25... Requirements § 2429.25 Number of copies and paper size. Unless otherwise provided by the Authority or the... the exception of any prescribed forms, any document or paper filed with the Authority, General...

  20. 40 CFR 761.209 - Number of copies of a manifest.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 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..., AND USE PROHIBITIONS PCB Waste Disposal Records and Reports § 761.209 Number of copies of a...

  1. 40 CFR 761.209 - Number of copies of a manifest.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 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..., AND USE PROHIBITIONS PCB Waste Disposal Records and Reports § 761.209 Number of copies of a...

  2. 5 CFR 2429.25 - Number of copies and paper size.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 5 Administrative Personnel 3 2013-01-01 2013-01-01 false Number of copies and paper size. 2429.25... Requirements § 2429.25 Number of copies and paper size. (a) General rule. Except as discussed in paragraph (b... attachments, must be on 81/2 by 11 inch size paper, using normal margins and font sizes. You must file...

  3. 5 CFR 2429.25 - Number of copies and paper size.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 5 Administrative Personnel 3 2014-01-01 2014-01-01 false Number of copies and paper size. 2429.25... Requirements § 2429.25 Number of copies and paper size. (a) General rule. Except as discussed in paragraph (b... attachments, must be on 81/2 by 11 inch size paper, using normal margins and font sizes. You must file...

  4. qPCR for quantification of transgene expression and determination of transgene copy number.

    PubMed

    Fletcher, Stephen J

    2014-01-01

    Quantitative real-time PCR (qPCR) is a mature technology that can be used to accurately quantify the number of copies of a target nucleic acid in a sample. Here, we describe a method for using this technology to determine the copy number of a transgene stably integrated into a plant's genome and to ascertain the level of transgene expression.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  6. 10 CFR 205.307 - Form and style; number of copies

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 10 Energy 3 2013-01-01 2013-01-01 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...

  7. 10 CFR 205.307 - Form and style; number of copies

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 10 Energy 3 2014-01-01 2014-01-01 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...

  8. 10 CFR 205.324 - Form and style; number of copies.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 10 Energy 3 2014-01-01 2014-01-01 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...

  9. 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...

  10. 10 CFR 205.324 - Form and style; number of copies.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 10 Energy 3 2012-01-01 2012-01-01 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...

  11. 10 CFR 205.324 - Form and style; number of copies.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 10 Energy 3 2013-01-01 2013-01-01 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...

  12. 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...

  13. 10 CFR 205.307 - Form and style; number of copies

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 10 Energy 3 2012-01-01 2012-01-01 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...

  14. 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...

  15. 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...

  16. Clinical Significance of MET Gene Copy Number in Patients with Curatively Resected Gastric Cancer

    PubMed Central

    Kang, Byung Woog; Park, Heyoung; Park, Bo Eun; Jeon, Seong Woo; Bae, Han Ik; Kwon, Oh-kyoung; Chung, Ho Young; Yu, Wansik

    2015-01-01

    The present study analyzed the prognostic impact of MET gene copy number in patients with curatively resected gastric cancer who received a combination regimen of cisplatin and S-1. The MET gene copy number was analyzed by use of quantitative real-time polymerase chain reaction. From January 2006 to July 2010, 70 tumor samples from 74 patients enrolled in a pilot study were analyzed. According to a cutoff MET gene copy number of ≥2 copies, a high MET gene copy number was observed in 38 patients (54.3%). The characteristics of the 2 groups divided according to MET gene copy number were similar. With a median follow-up duration of 26.4 months (range, 2.6-73.2 months), the estimated 3-year relapse-free survival and overall survival rates were 54.3% and 77.4%, respectively. No significant association was observed between the MET gene copy number and survival in a multivariate analysis. The MET gene copy number investigated in this study was not found to be associated with prognosis in patients with curatively resected gastric cancer. PMID:26306302

  17. Increased leukocyte mitochondrial DNA copy number is associated with oral premalignant lesions: an epidemiology study.

    PubMed

    He, Yonggang; Gong, Yilei; Gu, Jian; Lee, J Jack; Lippman, Scott M; Wu, Xifeng

    2014-08-01

    Although changes in the mitochondrial DNA (mtDNA) copy number in peripheral blood leukocytes (PBLs) have been linked to increased susceptibility to several cancers, the relationship between the mtDNA copy number in PBLs and the risk of cancer precursors has not been investigated. In this study, we measured the relative mtDNA copy number in PBLs of 143 patients with histologically confirmed oral premalignant lesions (OPLs) and of 357 healthy controls that were frequency-matched to patients according to age, sex and race. OPL patients had a significantly higher mtDNA copy number than the controls (1.36 ± 0.74 versus 1.11 ± 0.32; P < 0.001). In analyses stratified by sex, race, alcohol consumption and smoking status, the mtDNA copy number was higher in the OPL patients than in the controls in all the strata. Using the median mtDNA copy number in the control group as a cutoff, we found that individuals with a high mtDNA copy number had significantly higher risk of having OPLs than individuals with a low mtDNA copy number (adjusted odds ratio, 1.93; 95% confidence interval, 1.23-3.05, P = 0.004). Analysis of the joint effect of alcohol consumption and smoking revealed even greater risk for OPLs. Our results suggest that high mtDNA copy number in PBLs is significantly associated with having OPLs. To our knowledge, this is the first epidemiologic study to show that the mtDNA copy number may indicate the risk of cancer precursors.

  18. High EGFR gene copy number predicts poor outcome in triple-negative breast cancer.

    PubMed

    Park, Heae Surng; Jang, Min Hye; Kim, Eun Joo; Kim, Hyun Jeong; Lee, Hee Jin; Kim, Yu Jung; Kim, Jee Hyun; Kang, Eunyoung; Kim, Sung-Won; Kim, In Ah; Park, So Yeon

    2014-09-01

    Epidermal growth factor receptor (EGFR) is frequently overexpressed in triple-negative breast cancer and is emerging as a therapeutic target. EGFR gene copy number alteration and mutation are highly variable and scientists have been challenged to define their prognostic significance in triple-negative breast cancer. We examined EGFR protein expression, EGFR gene copy number alteration and mutation of exon 18 to 21 in 151 cases of triple-negative breast cancer and correlated these findings with clinical outcomes. In addition, intratumoral agreement of EGFR protein overexpression and gene copy number alteration was evaluated. EGFR overexpression was found in 97 of 151 cases (64%) and high EGFR gene copy number was detected in 50 cases (33%), including 3 gene amplification (2%) and 47 high polysomy (31%). Five EGFR mutations were detected in 4 of 151 cases (3%) and included G719A in exon 18 (n=1), V786M in exon 20 (n=1), and L858R in exon 21 (n=3). One case had two mutations (G719A and L858R). High EGFR copy number, but not EGFR mutation, correlated with EGFR protein overexpression. Intratumoral heterogeneity of EGFR protein overexpression and EGFR copy number alteration was not significant. In survival analyses, high EGFR copy number was found to be an independent prognostic factor for poor disease-free survival in patients with triple-negative breast cancer. Our findings showed that EGFR mutation was a rare event, but high EGFR copy number was relatively frequent and correlated with EGFR overexpression in triple-negative breast cancer. Moreover, high EGFR copy number was associated with poor clinical outcome in triple-negative breast cancer, suggesting that evaluation of EGFR copy number may be useful for predicting outcomes in patients with triple-negative breast cancer and for selecting patients for anti-EGFR-targeted therapy.

  19. Upregulation of TFAM and mitochondria copy number in human lymphoblastoid cells.

    PubMed

    Chakrabarty, Sanjiban; D'Souza, Reena Reshma; Kabekkodu, Shama Prasada; Gopinath, Puthiya M; Rossignol, Rodrigue; Satyamoorthy, Kapaettu

    2014-03-01

    Mitochondria are central to several physiological and pathological conditions in humans. In the present study, we performed copy number analysis of nuclear encoded mitochondrial genes, in peripheral blood mononuclear cells (PBMCs) and its representative lymphoblastoid cells (LCLs). We have observed hyper diploid copies of mitochondrial transcription factor A (TFAM) gene in the LCLs along with increased mtDNA copy number, mitochondrial mass, intracellular ROS and mitochondrial membrane potential, suggesting elevated mitochondrial biogenesis in LCLs. Gene expression analysis confirmed TFAM over-expression in LCLs when compared to PBMC. Based on our observation, we suggest that increased copy number of TFAM gene upregulates its expression, increases mtDNA copy numbers and protects it from oxidative stress induced damage in the transformed LCLs.

  20. Increased pfmdr1 copy number in Plasmodium falciparum isolates from Suriname.

    PubMed

    Labadie-Bracho, Mergiory; Adhin, Malti R

    2013-07-01

    Amplification of the pfmdr1 gene is associated with clinical failures and reduced in vivo and in vitro sensitivity to both mefloquine and artemether-lumefantrine in South-East Asia. Several African countries have reported the absence or very low prevalence of increased copy number, whilst South American reports are limited to Peru without and Venezuela with increased pfmdr1 multiplication. The relative pfmdr1 copy numbers were assessed in 68 isolates from Suriname collected from different endemic villages (2005) and from mining areas (2009). 11% of the isolates harbour multiple copies of the pfmdr1 gene. Isolates originating from mining areas do not yet display a higher tendency for increased copy number and no significant differences could be registered within a time span of 4 years, but the mere presence of increased copy number warrants caution and should be considered as an early warning sign for emerging drug resistance in Suriname and South America.

  1. TSPY1 Copy Number Variation Influences Spermatogenesis and Shows Differences among Y Lineages

    PubMed Central

    Giachini, Claudia; Nuti, Francesca; Turner, Daniel J.; Laface, Ilaria; Xue, Yali; Daguin, Fabrice; Forti, Gianni; Tyler-Smith, Chris; Krausz, Csilla

    2012-01-01

    Context TSPY1 is a tandemly-repeated gene on the human Y chromosome forming an array of approximately 21–35 copies. The testicular expression pattern and the inferred function of the TSPY1 protein suggest possible involvement in spermatogenesis. However, data are scarce on TSPY1 copy number variation in different Y lineages and its role in spermatogenesis. Objectives We sought to define: 1) the extent of TSPY1 copy number variation within and among Y chromosome haplogroups; and 2) the role of TSPY1 dosage in spermatogenic efficiency. Materials and Methods A total of 154 idiopathic infertile men and 130 normozoospermic controls from Central Italy were analyzed. We used a quantitative PCR assay to measure TSPY1 copy number and also defined Y haplogroups in all subjects. Results We provide evidence that TSPY1 copy number shows substantial variation among Y haplogroups and thus that population stratification does represent a potential bias in case-control association studies. We also found: 1) a significant positive correlation between TSPY1 copy number and sperm count (P < 0.001); 2) a significant difference in mean TSPY1 copy number between patients and controls (28.4 ± 8.3 vs. 33.9 ± 10.7; P < 0.001); and 3) a 1.5-fold increased risk of abnormal sperm parameters in men with less than 33 copies (P < 0.001). Conclusions TSPY copy number variation significantly influences spermatogenic efficiency. Low TSPY1 copy number is a new risk factor for male infertility with potential clinical consequences. PMID:19773397

  2. Allele-specific copy number profiling by next-generation DNA sequencing.

    PubMed

    Chen, Hao; Bell, John M; Zavala, Nicolas A; Ji, Hanlee P; Zhang, Nancy R

    2015-02-27

    The progression and clonal development of tumors often involve amplifications and deletions of genomic DNA. Estimation of allele-specific copy number, which quantifies the number of copies of each allele at each variant loci rather than the total number of chromosome copies, is an important step in the characterization of tumor genomes and the inference of their clonal history. We describe a new method, falcon, for finding somatic allele-specific copy number changes by next generation sequencing of tumors with matched normals. falcon is based on a change-point model on a bivariate mixed Binomial process, which explicitly models the copy numbers of the two chromosome haplotypes and corrects for local allele-specific coverage biases. By using the Binomial distribution rather than a normal approximation, falcon more effectively pools evidence from sites with low coverage. A modified Bayesian information criterion is used to guide model selection for determining the number of copy number events. Falcon is evaluated on in silico spike-in data and applied to the analysis of a pre-malignant colon tumor sample and late-stage colorectal adenocarcinoma from the same individual. The allele-specific copy number estimates obtained by falcon allows us to draw detailed conclusions regarding the clonal history of the individual's colon cancer.

  3. Decreases in average bacterial community rRNA operon copy number during succession.

    PubMed

    Nemergut, Diana R; Knelman, Joseph E; Ferrenberg, Scott; Bilinski, Teresa; Melbourne, Brett; Jiang, Lin; Violle, Cyrille; Darcy, John L; Prest, Tiffany; Schmidt, Steven K; Townsend, Alan R

    2016-05-01

    Trait-based studies can help clarify the mechanisms driving patterns of microbial community assembly and coexistence. Here, we use a trait-based approach to explore the importance of rRNA operon copy number in microbial succession, building on prior evidence that organisms with higher copy numbers respond more rapidly to nutrient inputs. We set flasks of heterotrophic media into the environment and examined bacterial community assembly at seven time points. Communities were arrayed along a geographic gradient to introduce stochasticity via dispersal processes and were analyzed using 16 S rRNA gene pyrosequencing, and rRNA operon copy number was modeled using ancestral trait reconstruction. We found that taxonomic composition was similar between communities at the beginning of the experiment and then diverged through time; as well, phylogenetic clustering within communities decreased over time. The average rRNA operon copy number decreased over the experiment, and variance in rRNA operon copy number was lowest both early and late in succession. We then analyzed bacterial community data from other soil and sediment primary and secondary successional sequences from three markedly different ecosystem types. Our results demonstrate that decreases in average copy number are a consistent feature of communities across various drivers of ecological succession. Importantly, our work supports the scaling of the copy number trait over multiple levels of biological organization, ranging from cells to populations and communities, with implications for both microbial ecology and evolution.

  4. Decreases in average bacterial community rRNA operon copy number during succession

    PubMed Central

    Nemergut, Diana R; Knelman, Joseph E; Ferrenberg, Scott; Bilinski, Teresa; Melbourne, Brett; Jiang, Lin; Violle, Cyrille; Darcy, John L; Prest, Tiffany; Schmidt, Steven K; Townsend, Alan R

    2016-01-01

    Trait-based studies can help clarify the mechanisms driving patterns of microbial community assembly and coexistence. Here, we use a trait-based approach to explore the importance of rRNA operon copy number in microbial succession, building on prior evidence that organisms with higher copy numbers respond more rapidly to nutrient inputs. We set flasks of heterotrophic media into the environment and examined bacterial community assembly at seven time points. Communities were arrayed along a geographic gradient to introduce stochasticity via dispersal processes and were analyzed using 16 S rRNA gene pyrosequencing, and rRNA operon copy number was modeled using ancestral trait reconstruction. We found that taxonomic composition was similar between communities at the beginning of the experiment and then diverged through time; as well, phylogenetic clustering within communities decreased over time. The average rRNA operon copy number decreased over the experiment, and variance in rRNA operon copy number was lowest both early and late in succession. We then analyzed bacterial community data from other soil and sediment primary and secondary successional sequences from three markedly different ecosystem types. Our results demonstrate that decreases in average copy number are a consistent feature of communities across various drivers of ecological succession. Importantly, our work supports the scaling of the copy number trait over multiple levels of biological organization, ranging from cells to populations and communities, with implications for both microbial ecology and evolution. PMID:26565722

  5. Copy number variations in IL22 gene are associated with Psoriasis vulgaris.

    PubMed

    Prans, Ele; Kingo, Külli; Traks, Tanel; Silm, Helgi; Vasar, Eero; Kõks, Sulev

    2013-06-01

    Psoriasis vulgaris (PsV) is a frequent, chronically relapsing, immune-mediated systemic disease with characteristic skin changes. IL22 is a cytokine of IL10 family, with significant proliferative effect on different cell lines. Copy number variations (CNV) have been discovered to have phenotypic consequences and are associated with various types of diseases. In the work presented here we analyzed the copy number variations in IL22 gene of exon1 and exon5. Our results showed that the IL22 gene exon1 was significantly associated with psoriasis severity (P<0.0001). However, the association between IL22 gene exon5 copy numbers and psoriasis was not detected.

  6. Methodological strategies for transgene copy number quantification in goats (Capra hircus) using real-time PCR.

    PubMed

    Batista, Ribrio I T P; Luciano, Maria C S; Teixeira, Dárcio I A; Freitas, Vicente J F; Melo, Luciana M; Andreeva, Lyudmila E; Serova, Irina A; Serov, Oleg L

    2014-01-01

    Taking into account the importance of goats as transgenic models, as well as the rarity of copy number (CN) studies in farm animals, the present work aimed to evaluate methodological strategies for accurate and precise transgene CN quantification in goats using quantitative polymerase chain reaction (qPCR). Mouse and goat lines transgenic for human granulocyte-colony stimulating factor were used. After selecting the best genomic DNA extraction method to be applied in mouse and goat samples, intra-assay variations, accuracy and precision of CN quantifications were assessed. The optimized conditions were submitted to mathematical strategies and used to quantify CN in goat lines. The findings were as follows: validation of qPCR conditions is required, and amplification efficiency is the most important. Absolute and relative quantifications are able to produce similar results. For normalized absolute quantification, the same plasmid fragment used to generate goat lines must be mixed with wild-type goat genomic DNA, allowing the choice of an endogenous reference gene for data normalization. For relative quantifications, a resin-based genomic DNA extraction method is strongly recommended when using mouse tail tips as calibrators to avoid tissue-specific inhibitors. Efficient qPCR amplifications (≥95%) allow reliable CN measurements with SYBR technology. TaqMan must be used with caution in goats if the nucleotide sequence of the endogenous reference gene is not yet well understood. Adhering to these general guidelines can result in more exact CN determination in goats. Even when working under nonoptimal circumstances, if assays are performed that respect the minimum qPCR requirements, good estimations of transgene CN can be achieved.

  7. Association between TLR7 copy number variations and hepatitis B virus infection outcome in Chinese

    PubMed Central

    Li, Fang; Li, Xu; Zou, Gui-Zhou; Gao, Yu-Feng; Ye, Jun

    2017-01-01

    AIM To explore whether copy number variations (CNVs) of toll-like receptor 7 (TLR7) are associated with susceptibility to chronic hepatitis B virus (HBV) infection. METHODS This study included 623 patients (495 males and 128 females) with chronic hepatitis B virus infection (CHB) and 300 patients (135 females and 165 males) with acute hepatitis B virus infection (AHB) as controls. All CHB patients were further categorized according to disease progression after HBV infection (CHB, liver cirrhosis, or hepatocellular carcinoma). Copy numbers of the TLR7 gene were measured using the AccuCopy method. χ2 tests were used to evaluate the association between TLR7 CNVs and infection type. P values, odds ratios, and 95% confidence intervals (CIs) were used to estimate the effects of risk. RESULTS Among male patients, there were significant differences between the AHB group and CHB group in the distribution of TLR7 CNVs. Low copy number of TLR7 was significantly associated with chronic HBV infection (OR = 0.329, 95%CI: 0.229-0.473, P < 0.001). Difference in TLR7 copy number was also found between AHB and CHB female patients, with low copy number again associated with an increased risk of chronic HBV infection (OR = 0.292, 95%CI: 0.173-0.492, P < 0.001). However, there were no significant differences in TLR7 copy number among the three types of chronic HBV infection (CHB, liver cirrhosis, or hepatocellular carcinoma). In addition, there was no association between TLR7 copy number and titer of the HBV e antigen. CONCLUSION Low TLR7 copy number is a risk factor for chronic HBV infection but is not associated with later stages of disease progression. PMID:28321161

  8. The most appropriate position and number for absolute anchorages for orthodontic tooth movements.

    PubMed

    Yu, Jian-Hong; Takakuda, Kazuo; Miyairi, Hiroo; Soma, Kunimichi

    2003-04-01

    Absolute anchorages proved to be very effective for orthodontic tooth movements. We used a 3D digitizer to record each tooth on pre-treatment diagnostic and post-treatment predictive setup models and then 3D coordinate system conversion was performed to make the coordinate values comparable. An arithmetic calculation of vector and moment based on the orthodontic forces and the tooth displacement under preliminary premises undertaken to decide the most favorable position and number for absolute anchorages. Position--For two-dimensional and three-dimensional calculations, the most appropriate positions for absolute anchorages should theoretically be on the line of resultant force (2D) and the plane (3D) where the total moment effect tends to be zero. Number--As for the number of the absolute anchorages needed, it depends on the number of target teeth. Different combinations of target teeth provide different sets of results.

  9. CNVkit: Genome-Wide Copy Number Detection and Visualization from Targeted DNA Sequencing

    PubMed Central

    Shain, A. Hunter; Botton, Thomas; Bastian, Boris C.

    2016-01-01

    Germline copy number variants (CNVs) and somatic copy number alterations (SCNAs) are of significant importance in syndromic conditions and cancer. Massively parallel sequencing is increasingly used to infer copy number information from variations in the read depth in sequencing data. However, this approach has limitations in the case of targeted re-sequencing, which leaves gaps in coverage between the regions chosen for enrichment and introduces biases related to the efficiency of target capture and library preparation. We present a method for copy number detection, implemented in the software package CNVkit, that uses both the targeted reads and the nonspecifically captured off-target reads to infer copy number evenly across the genome. This combination achieves both exon-level resolution in targeted regions and sufficient resolution in the larger intronic and intergenic regions to identify copy number changes. In particular, we successfully inferred copy number at equivalent to 100-kilobase resolution genome-wide from a platform targeting as few as 293 genes. After normalizing read counts to a pooled reference, we evaluated and corrected for three sources of bias that explain most of the extraneous variability in the sequencing read depth: GC content, target footprint size and spacing, and repetitive sequences. We compared the performance of CNVkit to copy number changes identified by array comparative genomic hybridization. We packaged the components of CNVkit so that it is straightforward to use and provides visualizations, detailed reporting of significant features, and export options for integration into existing analysis pipelines. CNVkit is freely available from https://github.com/etal/cnvkit. PMID:27100738

  10. 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.

  11. Identification of candidate growth promoting genes in ovarian cancer through integrated copy number and expression analysis.

    PubMed

    Ramakrishna, Manasa; Williams, Louise H; Boyle, Samantha E; Bearfoot, Jennifer L; Sridhar, Anita; Speed, Terence P; Gorringe, Kylie L; Campbell, Ian G

    2010-04-08

    Ovarian cancer is a disease characterised by complex genomic rearrangements but the majority of the genes that are the target of these alterations remain unidentified. Cataloguing these target genes will provide useful insights into the disease etiology and may provide an opportunity to develop novel diagnostic and therapeutic interventions. High resolution genome wide copy number and matching expression data from 68 primary epithelial ovarian carcinomas of various histotypes was integrated to identify genes in regions of most frequent amplification with the strongest correlation with expression and copy number. Regions on chromosomes 3, 7, 8, and 20 were most frequently increased in copy number (> 40% of samples). Within these regions, 703/1370 (51%) unique gene expression probesets were differentially expressed when samples with gain were compared to samples without gain. 30% of these differentially expressed probesets also showed a strong positive correlation (r > or =0.6) between expression and copy number. We also identified 21 regions of high amplitude copy number gain, in which 32 known protein coding genes showed a strong positive correlation between expression and copy number. Overall, our data validates previously known ovarian cancer genes, such as ERBB2, and also identified novel potential drivers such as MYNN, PUF60 and TPX2.

  12. Identification of Candidate Growth Promoting Genes in Ovarian Cancer through Integrated Copy Number and Expression Analysis

    PubMed Central

    Ramakrishna, Manasa; Williams, Louise H.; Boyle, Samantha E.; Bearfoot, Jennifer L.; Sridhar, Anita; Speed, Terence P.; Gorringe, Kylie L.; Campbell, Ian G.

    2010-01-01

    Ovarian cancer is a disease characterised by complex genomic rearrangements but the majority of the genes that are the target of these alterations remain unidentified. Cataloguing these target genes will provide useful insights into the disease etiology and may provide an opportunity to develop novel diagnostic and therapeutic interventions. High resolution genome wide copy number and matching expression data from 68 primary epithelial ovarian carcinomas of various histotypes was integrated to identify genes in regions of most frequent amplification with the strongest correlation with expression and copy number. Regions on chromosomes 3, 7, 8, and 20 were most frequently increased in copy number (>40% of samples). Within these regions, 703/1370 (51%) unique gene expression probesets were differentially expressed when samples with gain were compared to samples without gain. 30% of these differentially expressed probesets also showed a strong positive correlation (r≥0.6) between expression and copy number. We also identified 21 regions of high amplitude copy number gain, in which 32 known protein coding genes showed a strong positive correlation between expression and copy number. Overall, our data validates previously known ovarian cancer genes, such as ERBB2, and also identified novel potential drivers such as MYNN, PUF60 and TPX2. PMID:20386695

  13. Copy number analysis of ductal carcinoma in situ with and without recurrence.

    PubMed

    Gorringe, Kylie L; Hunter, Sally M; Pang, Jia-Min; Opeskin, Ken; Hill, Prue; Rowley, Simone M; Choong, David Y H; Thompson, Ella R; Dobrovic, Alexander; Fox, Stephen B; Mann, G Bruce; Campbell, Ian G

    2015-09-01

    Ductal carcinoma in situ (DCIS) is a non-obligate precursor of invasive breast cancer and a frequent mammographic finding requiring treatment. Up to 25% of DCIS can recur and half of recurrences are invasive, but there are no reliable biomarkers for recurrence. We hypothesised that copy number aberrations could predict likelihood of recurrence. We analysed a cohort of pure DCIS cases treated only with wide local excision for genome-wide copy number and loss of heterozygosity using Affymetrix OncoScan MIP arrays. Cases included those without recurrence within 7 years (n = 25) and with recurrence between 1 and 5 years after diagnosis (n = 15). Pure DCIS were broadly similar in copy number changes compared with invasive breast cancer, with the consistent exception of a greater frequency of ERBB2 amplification in DCIS. There were no significant differences in age or ER status between the cases with a recurrence vs those without. Overall, the DCIS cases with recurrence had more copy number events than the DCIS without recurrence. The increased copy number appeared non-random with several genomic regions showing an increase in frequency in recurrent cases, including 20 q gain, ERBB2 amplification and 15q loss. Copy number changes may provide prognostic information for DCIS recurrence, but validation in additional cohorts is required.

  14. Replicated linear association between DUF1220 copy number and severity of social impairment in autism.

    PubMed

    Davis, J M; Searles Quick, V B; Sikela, J M

    2015-06-01

    Sequences encoding DUF1220 protein domains exhibit an exceptional human-specific increase in copy number and have been associated with several phenotypes related to brain size. Autism is a highly heritable and heterogeneous condition characterized behaviorally by social and communicative impairments, and increased repetitive and stereotyped behavior. Given the accelerated brain growth pattern observed in many individuals with autism, and the association between DUF1220 subtype CON1 copy number and brain size, we previously investigated associations between CON1 copy number and autism-related symptoms. We determined that CON1 copy number increase is associated with increasing severity of all three behavioral features of autism. The present study sought to replicate these findings in an independent population (N = 166). Our results demonstrate a replication of the linear relationship between CON1 copy number and the severity of social impairment in individuals with autism as measured by Autism Diagnostic Interview-Revised Social Diagnostic Score, such that with each additional copy of CON1 Social Diagnostic Score increased 0.24 points (SE = 0.11, p = 0.036). We also identified an analogous trend between CON1 copy number and Communicative Diagnostic Score, but did not replicate the relationship between CON1 copy number and Repetitive Behavior Diagnostic Score. Interestingly, these associations appear to be most pronounced in multiplex children. These results, representing the first replication of a gene dosage relationship with the severity of a primary symptom of autism, lend further support to the possibility that the same protein domain family implicated in the evolutionary expansion of the human brain may also be involved in autism severity.

  15. Ribosome Dwell Times and the Protein Copy Number Distribution

    NASA Astrophysics Data System (ADS)

    Gorissen, Mieke; Vanderzande, Carlo

    2012-09-01

    Translation is the cellular process in which ribosomes make proteins from information encoded on messenger RNA (mRNA). We model translation with an exclusion process taking into account the experimentally determined, non-exponential, waiting time between steps of a ribosome. From numerical simulations using realistic parameter values, we determine the distribution P( E) of the number of proteins E produced by one mRNA. We find that for small E this distribution is not geometric. We present a simplified and analytically solvable model that relates P( E) to the distributions of the times to produce the first E proteins.

  16. Mutant allele specific imbalance in oncogenes with copy number alterations: Occurrence, mechanisms, and potential clinical implications.

    PubMed

    Yu, Chih-Chieh; Qiu, Wanglong; Juang, Caroline S; Mansukhani, Mahesh M; Halmos, Balazs; Su, Gloria H

    2017-01-01

    Mutant allele specific imbalance (MASI) was initially coined to describe copy number alterations associated with the mutant allele of an oncogene. The copy number gain (CNG) specific to the mutant allele can be readily observed in electropherograms. With the development of genome-wide analyses at base-pair resolution with copy number counts, we can now further differentiate MASI into those with CNG, with copy neutral alteration (also termed acquired uniparental disomy; UPD), or with loss of heterozygosity (LOH) due to the loss of the wild-type (WT) allele. Here we summarize the occurrence of MASI with CNG, aUPD, or MASI with LOH in some major oncogenes (such as EGFR, KRAS, PIK3CA, and BRAF). We also discuss how these various classifications of MASI have been demonstrated to impact tumorigenesis, progression, metastasis, prognosis, and potentially therapeutic responses in cancer, notably in lung, colorectal, and pancreatic cancers.

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

    PubMed Central

    Paolella, Brenton R; Gibson, William J; Urbanski, Laura M; Alberta, John A; Zack, Travis I; Bandopadhayay, Pratiti; Nichols, Caitlin A; Agarwalla, Pankaj K; Brown, Meredith S; Lamothe, Rebecca; Yu, Yong; Choi, Peter S; Obeng, Esther A; Heckl, Dirk; Wei, Guo; Wang, Belinda; Tsherniak, Aviad; Vazquez, Francisca; Weir, Barbara A; Root, David E; Cowley, Glenn S; Buhrlage, Sara J; Stiles, Charles D; Ebert, Benjamin L; Hahn, William C; Reed, Robin; Beroukhim, Rameen

    2017-01-01

    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. One of these, the pre-mRNA splicing factor SF3B1, is also frequently mutated in cancer. We validated SF3B1 as a CYCLOPS gene and found that human cancer cells harboring partial SF3B1 copy-loss lack a reservoir of SF3b complex that protects cells with normal SF3B1 copy number from cell death upon partial SF3B1 suppression. These data provide a catalog of copy-number associated gene dependencies and identify partial copy-loss of wild-type SF3B1 as a novel, non-driver cancer gene dependency. DOI: http://dx.doi.org/10.7554/eLife.23268.001 PMID:28177281

  18. HaplotypeCN: copy number haplotype inference with Hidden Markov Model and localized haplotype clustering.

    PubMed

    Lin, Yen-Jen; Chen, Yu-Tin; Hsu, Shu-Ni; Peng, Chien-Hua; Tang, Chuan-Yi; Yen, Tzu-Chen; Hsieh, Wen-Ping

    2014-01-01

    Copy number variation (CNV) has been reported to be associated with disease and various cancers. Hence, identifying the accurate position and the type of CNV is currently a critical issue. There are many tools targeting on detecting CNV regions, constructing haplotype phases on CNV regions, or estimating the numerical copy numbers. However, none of them can do all of the three tasks at the same time. This paper presents a method based on Hidden Markov Model to detect parent specific copy number change on both chromosomes with signals from SNP arrays. A haplotype tree is constructed with dynamic branch merging to model the transition of the copy number status of the two alleles assessed at each SNP locus. The emission models are constructed for the genotypes formed with the two haplotypes. The proposed method can provide the segmentation points of the CNV regions as well as the haplotype phasing for the allelic status on each chromosome. The estimated copy numbers are provided as fractional numbers, which can accommodate the somatic mutation in cancer specimens that usually consist of heterogeneous cell populations. The algorithm is evaluated on simulated data and the previously published regions of CNV of the 270 HapMap individuals. The results were compared with five popular methods: PennCNV, genoCN, COKGEN, QuantiSNP and cnvHap. The application on oral cancer samples demonstrates how the proposed method can facilitate clinical association studies. The proposed algorithm exhibits comparable sensitivity of the CNV regions to the best algorithm in our genome-wide study and demonstrates the highest detection rate in SNP dense regions. In addition, we provide better haplotype phasing accuracy than similar approaches. The clinical association carried out with our fractional estimate of copy numbers in the cancer samples provides better detection power than that with integer copy number states.

  19. 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

  20. Ribosomal DNA and Stellate gene copy number variation on the Y chromosome of Drosophila melanogaster.

    PubMed

    Lyckegaard, E M; Clark, A G

    1989-03-01

    Multigene families on the Y chromosome face an unusual array of evolutionary forces. Both ribosomal DNA and Stellate, the two families examined here, have multiple copies of similar sequences on the X and Y chromosomes. Although the rate of sequence divergence on the Y chromosome depends on rates of mutation, gene conversion and exchange with the X chromosome, as well as purifying selection, the regulation of gene copy number may also depend on other pleiotropic functions, such as maintenance of chromosome pairing. Gene copy numbers were estimated for a series of 34 Y chromosome replacement lines using densitometric measurements of slot blots of genomic DNA from adult Drosophila melanogaster. Scans of autoradiographs of the same blots probed with the cloned alcohol dehydrogenase gene, a single copy gene, served as internal standards. Copy numbers span a 6-fold range for ribosomal DNA and a 3-fold range for Stellate DNA. Despite this magnitude of variation, there was no association between copy number and segregation variation of the sex chromosomes.

  1. Association between Leukocyte Mitochondrial DNA Copy Number and Regular Exercise in Postmenopausal Women

    PubMed Central

    Chang, Yu Kyung; Kim, Da Eun; Cho, Soo Hyun

    2016-01-01

    Background Previous studies suggest that habitual exercise can improve skeletal mitochondrial function; however, to date, the association between exercise and mitochondrial function in peripheral leukocytes has not been reported. The aim of this study was to evaluate the relationship between regular exercise and mitochondrial function by measuring leukocyte mitochondrial DNA (mtDNA) copy number in postmenopausal women. Methods This cross-sectional study included 144 relatively healthy, non-diabetic, non-smoking, postmenopausal women. Clinical parameters, including anthropometric measurements and cardio-metabolic parameters, were assessed. Regular exercise was defined as at least 150 minutes per week of moderate-intensity activity, or an equivalent combination of moderate and vigorous-intensity activity, over a duration of at least 6 months. Leukocyte mtDNA copy numbers were measured using real-time polymerase chain reaction assays, and these were normalized to the β-globin copy number to give the relative mtDNA copy number. Results The mtDNA copy number of peripheral leukocytes was significantly greater in the exercise group (1.33±0.02) than in the no exercise group (1.05±0.02, P<0.01). Stepwise multiple regression analysis showed that regular exercise was independently associated with mtDNA copy number (β=0.25, P<0.01) after adjusting for the variables age, body mass index, waist-to-hip ratio, systolic and diastolic blood pressure, homeostasis model assessment of insulin resistance value, and levels of high-density lipoprotein cholesterol, triglycerides, and homocysteine. Conclusion Regular exercise is associated with greater leukocyte mtDNA copy number in postmenopausal women. PMID:27900071

  2. Sparse representation and Bayesian detection of genome copy number alterations from microarray data

    PubMed Central

    Pique-Regi, Roger; Monso-Varona, Jordi; Ortega, Antonio; Seeger, Robert C.; Triche, Timothy J.; Asgharzadeh, Shahab

    2008-01-01

    Motivation: Genomic instability in cancer leads to abnormal genome copy number alterations (CNA) that are associated with the development and behavior of tumors. Advances in microarray technology have allowed for greater resolution in detection of DNA copy number changes (amplifications or deletions) across the genome. However, the increase in number of measured signals and accompanying noise from the array probes present a challenge in accurate and fast identification of breakpoints that define CNA. This article proposes a novel detection technique that exploits the use of piece wise constant (PWC) vectors to represent genome copy number and sparse Bayesian learning (SBL) to detect CNA breakpoints. Methods: First, a compact linear algebra representation for the genome copy number is developed from normalized probe intensities. Second, SBL is applied and optimized to infer locations where copy number changes occur. Third, a backward elimination (BE) procedure is used to rank the inferred breakpoints; and a cut-off point can be efficiently adjusted in this procedure to control for the false discovery rate (FDR). Results: The performance of our algorithm is evaluated using simulated and real genome datasets and compared to other existing techniques. Our approach achieves the highest accuracy and lowest FDR while improving computational speed by several orders of magnitude. The proposed algorithm has been developed into a free standing software application (GADA, Genome Alteration Detection Algorithm). Availability: http://biron.usc.edu/~piquereg/GADA Contact: jpei@chop.swmed.edu and rpique@ieee.org Supplementary information: Supplementary data are available at Bioinformatics online. PMID:18203770

  3. BIOFILTER AS A FUNCTIONAL ANNOTATION PIPELINE FOR COMMON AND RARE COPY NUMBER BURDEN

    PubMed Central

    KIM, DOKYOON; LUCAS, ANASTASIA; GLESSNER, JOSEPH; VERMA, SHEFALI S.; BRADFORD, YUKI; LI, RUOWANG; FRASE, ALEX T.; HAKONARSON, HAKON; PEISSIG, PEGGY; BRILLIANT, MURRAY; RITCHIE, MARYLYN D.

    2015-01-01

    Recent studies on copy number variation (CNV) have suggested that an increasing burden of CNVs is associated with susceptibility or resistance to disease. A large number of genes or genomic loci contribute to complex diseases such as autism. Thus, total genomic copy number burden, as an accumulation of copy number change, is a meaningful measure of genomic instability to identify the association between global genetic effects and phenotypes of interest. However, no systematic annotation pipeline has been developed to interpret biological meaning based on the accumulation of copy number change across the genome associated with a phenotype of interest. In this study, we develop a comprehensive and systematic pipeline for annotating copy number variants into genes/genomic regions and subsequently pathways and other gene groups using Biofilter – a bioinformatics tool that aggregates over a dozen publicly available databases of prior biological knowledge. Next we conduct enrichment tests of biologically defined groupings of CNVs including genes, pathways, Gene Ontology, or protein families. We applied the proposed pipeline to a CNV dataset from the Marshfield Clinic Personalized Medicine Research Project (PMRP) in a quantitative trait phenotype derived from the electronic health record – total cholesterol. We identified several significant pathways such as toll-like receptor signaling pathway and hepatitis C pathway, gene ontologies (GOs) of nucleoside triphosphatase activity (NTPase) and response to virus, and protein families such as cell morphogenesis that are associated with the total cholesterol phenotype based on CNV profiles (permutation p-value < 0.01). Based on the copy number burden analysis, it follows that the more and larger the copy number changes, the more likely that one or more target genes that influence disease risk and phenotypic severity will be affected. Thus, our study suggests the proposed enrichment pipeline could improve the

  4. BIOFILTER AS A FUNCTIONAL ANNOTATION PIPELINE FOR COMMON AND RARE COPY NUMBER BURDEN.

    PubMed

    Kim, Dokyoon; Lucas, Anastasia; Glessner, Joseph; Verma, Shefali S; Bradford, Yuki; Li, Ruowang; Frase, Alex T; Hakonarson, Hakon; Peissig, Peggy; Brilliant, Murray; Ritchie, Marylyn D

    2016-01-01

    Recent studies on copy number variation (CNV) have suggested that an increasing burden of CNVs is associated with susceptibility or resistance to disease. A large number of genes or genomic loci contribute to complex diseases such as autism. Thus, total genomic copy number burden, as an accumulation of copy number change, is a meaningful measure of genomic instability to identify the association between global genetic effects and phenotypes of interest. However, no systematic annotation pipeline has been developed to interpret biological meaning based on the accumulation of copy number change across the genome associated with a phenotype of interest. In this study, we develop a comprehensive and systematic pipeline for annotating copy number variants into genes/genomic regions and subsequently pathways and other gene groups using Biofilter - a bioinformatics tool that aggregates over a dozen publicly available databases of prior biological knowledge. Next we conduct enrichment tests of biologically defined groupings of CNVs including genes, pathways, Gene Ontology, or protein families. We applied the proposed pipeline to a CNV dataset from the Marshfield Clinic Personalized Medicine Research Project (PMRP) in a quantitative trait phenotype derived from the electronic health record - total cholesterol. We identified several significant pathways such as toll-like receptor signaling pathway and hepatitis C pathway, gene ontologies (GOs) of nucleoside triphosphatase activity (NTPase) and response to virus, and protein families such as cell morphogenesis that are associated with the total cholesterol phenotype based on CNV profiles (permutation p-value < 0.01). Based on the copy number burden analysis, it follows that the more and larger the copy number changes, the more likely that one or more target genes that influence disease risk and phenotypic severity will be affected. Thus, our study suggests the proposed enrichment pipeline could improve the interpretability of

  5. Quantification of protein copy number in single mitochondria: The Bcl-2 family proteins.

    PubMed

    Chen, Chaoxiang; Zhang, Xiang; Zhang, Shuyue; Zhu, Shaobin; Xu, Jingyi; Zheng, Yan; Han, Jinyan; Zeng, Jin-Zhang; Yan, Xiaomei

    2015-12-15

    Bcl-2 family proteins, represented by antiapoptotic protein Bcl-2 and proapoptotic protein Bax, are key regulators of mitochondria-mediated apoptosis pathway. To build a quantitative model of how Bcl-2 family protein interactions control mitochondrial outer membrane permeabilization and subsequent cytochrome c release, it is essential to know the number of proteins in individual mitochondria. Here, we report an effective method to quantify the copy number and distribution of proteins in single mitochondria via immunofluorescent labeling and sensitive detection by a laboratory-built high sensitivity flow cytometer (HSFCM). Mitochondria isolated from HeLa cells were stained with Alexa Fluor 488 (AF488)-labeled monoclonal antibodies specifically targeting Bcl-2 or Bax and with nucleic acid dye. A series of fluorescent nanospheres with fluorescence intensity calibrated in the unit of molecules of equivalent soluble fluorochrome (MESF)-AF488 were used to construct a calibration curve for converting the immunofluorescence of a single mitochondrion to the number of antibodies bound to it and then to the number of proteins per mitochondrion. Under the normal condition, the measured mean copy numbers were 1300 and 220 per mitochondrion for Bcl-2 and Bax, respectively. A significant variation in protein copy number was identified, which ranged from 130 to 6000 (2.5-97.5%) for Bcl-2 and from 65 to 700 (2.5-97.5%) for Bax, respectively. We observed an approximately 4.4 fold increase of Bax copy number per mitochondrion upon 9h of apoptosis stimulation while the abundance of Bcl-2 remained almost unchanged. To the best of our knowledge, this is the first report of Bcl-2 family protein copy number and variance in single mitochondria. Collectively, we demonstrate that the HSFCM-based immunoassay provides a rapid and sensitive method for determining protein copy number distribution in single mitochondria.

  6. Integration of DNA Copy Number Alterations and Transcriptional Expression Analysis in Human Gastric Cancer

    PubMed Central

    Coral, Ho; Yuen, Siu Tsan; Chu, Kent Man; Law, Simon; Zhang, Lianhai; Ji, Jiafu; Leung, Suet Yi; Chen, Xin

    2012-01-01

    Background Genomic instability with frequent DNA copy number alterations is one of the key hallmarks of carcinogenesis. The chromosomal regions with frequent DNA copy number gain and loss in human gastric cancer are still poorly defined. It remains unknown how the DNA copy number variations contributes to the changes of gene expression profiles, especially on the global level. Principal Findings We analyzed DNA copy number alterations in 64 human gastric cancer samples and 8 gastric cancer cell lines using bacterial artificial chromosome (BAC) arrays based comparative genomic hybridization (aCGH). Statistical analysis was applied to correlate previously published gene expression data obtained from cDNA microarrays with corresponding DNA copy number variation data to identify candidate oncogenes and tumor suppressor genes. We found that gastric cancer samples showed recurrent DNA copy number variations, including gains at 5p, 8q, 20p, 20q, and losses at 4q, 9p, 18q, 21q. The most frequent regions of amplification were 20q12 (7/72), 20q12–20q13.1 (12/72), 20q13.1–20q13.2 (11/72) and 20q13.2–20q13.3 (6/72). The most frequent deleted region was 9p21 (8/72). Correlating gene expression array data with aCGH identified 321 candidate oncogenes, which were overexpressed and showed frequent DNA copy number gains; and 12 candidate tumor suppressor genes which were down-regulated and showed frequent DNA copy number losses in human gastric cancers. Three networks of significantly expressed genes in gastric cancer samples were identified by ingenuity pathway analysis. Conclusions This study provides insight into DNA copy number variations and their contribution to altered gene expression profiles during human gastric cancer development. It provides novel candidate driver oncogenes or tumor suppressor genes for human gastric cancer, useful pathway maps for the future understanding of the molecular pathogenesis of this malignancy, and the construction of new therapeutic

  7. A HIERARCHICAL BAYESIAN MODEL FOR INFERENCE OF COPY NUMBER VARIANTS AND THEIR ASSOCIATION TO GENE EXPRESSION

    PubMed Central

    Cassese, Alberto; Guindani, Michele; Tadesse, Mahlet G.; Falciani, Francesco; Vannucci, Marina

    2014-01-01

    A number of statistical models have been successfully developed for the analysis of high-throughput data from a single source, but few methods are available for integrating data from different sources. Here we focus on integrating gene expression levels with comparative genomic hybridization (CGH) array measurements collected on the same subjects. We specify a measurement error model that relates the gene expression levels to latent copy number states which, in turn, are related to the observed surrogate CGH measurements via a hidden Markov model. We employ selection priors that exploit the dependencies across adjacent copy number states and investigate MCMC stochastic search techniques for posterior inference. Our approach results in a unified modeling framework for simultaneously inferring copy number variants (CNV) and identifying their significant associations with mRNA transcripts abundance. We show performance on simulated data and illustrate an application to data from a genomic study on human cancer cell lines. PMID:24834139

  8. 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

  9. Gyrase activity and number of copies of the gyrase B subunit gene in Haemophilus influenzae.

    PubMed Central

    Cabrera-Juárez, E; Setlow, J K

    1985-01-01

    Gyrase activities in extracts of various strains of Haemophilus influenzae can differ by more than an order of magnitude (J. K. Setlow, E. Cabrera-Juárez, W. L. Albritton, D. Spikes, and A. Mutschler, J. Bacteriol. 164:525-534, 1985). Measurements of in vitro activity and copy number indicated that most of these differences arose from variations in the number of copies of the gene for the gyrase B subunit, with some strains containing multicopy plasmids coding for that subunit. The quantitative relationship between gyrase and copy number depended on the mutations in the plasmids and in the host. The gyrase and copy number were considerably lower in plasmid-bearing strains carrying the prophage HP1c1. Two mutations affecting gyrase that are apparently regulatory caused an increase in gyrase without a concomitant increase in copy number. The possibility that the in vivo gyrase activity did not reflect the in vitro data was explored by measurement of alkaline phosphatase and ATPase activity in the extracts. Alkaline phosphatase activity increased with increasing gyrase activity measured in vitro, but ATPase activity did not. We conclude that extra supercoiling enhanced transcription of the alkaline phosphatase gene but not the ATPase gene and that it is unlikely that there is much discrepancy between gyrase activity assayed in vitro and the activity in the cell. PMID:2997116

  10. Inferring Haplotypes of Copy Number Variations From High-Throughput Data With Uncertainty

    PubMed Central

    Kato, Mamoru; Yoon, Seungtai; Hosono, Naoya; Leotta, Anthony; Sebat, Jonathan; Tsunoda, Tatsuhiko; Zhang, Michael Q.

    2011-01-01

    Accurate information on haplotypes and diplotypes (haplotype pairs) is required for population-genetic analyses; however, microarrays do not provide data on a haplotype or diplotype at a copy number variation (CNV) locus; they only provide data on the total number of copies over a diplotype or an unphased sequence genotype (e.g., AAB, unlike AB of single nucleotide polymorphism). Moreover, such copy numbers or genotypes are often incorrectly determined when microarray signal intensities derived from different copy numbers or genotypes are not clearly separated due to noise. Here we report an algorithm to infer CNV haplotypes and individuals’ diplotypes at multiple loci from noisy microarray data, utilizing the probability that a signal intensity may be derived from different underlying copy numbers or genotypes. Performing simulation studies based on known diplotypes and an error model obtained from real microarray data, we demonstrate that this probabilistic approach succeeds in accurate inference (error rate: 1–2%) from noisy data, whereas previous deterministic approaches failed (error rate: 12–18%). Applying this algorithm to real microarray data, we estimated haplotype frequencies and diplotypes in 1486 CNV regions for 100 individuals. Our algorithm will facilitate accurate population-genetic analyses and powerful disease association studies of CNVs. PMID:22384316

  11. Copy number variation analysis identifies novel CAKUT candidate genes in children with a solitary functioning kidney

    PubMed Central

    Westland, Rik; Verbitsky, Miguel; Vukojevic, Katarina; Perry, Brittany J.; Fasel, David A.; Zwijnenburg, Petra J.G.; Bökenkamp, Arend; Gille, Johan J.P.; Saraga-Babic, Mirna; Ghiggeri, Gian Marco; D’Agati, Vivette D.; Schreuder, Michiel F.; Gharavi, Ali G.; van Wijk, Joanna A.E.; Sanna-Cherchi, Simone

    2016-01-01

    Copy number variations associate with different developmental phenotypes and represent a major cause of congenital anomalies of the kidney and urinary tract (CAKUT). Because rare pathogenic copy number variations are often large and contain multiple genes, identification of the underlying genetic drivers has proven to be difficult. Here we studied the role of rare copy number variations in 80 patients from the KIMONO-study cohort for which pathogenic mutations in three genes commonly implicated in CAKUT were excluded. In total, 13 known or novel genomic imbalances in 11 of 80 patients were absent or extremely rare in 23,362 population controls. To identify the most likely genetic drivers for the CAKUT phenotype underlying these rare copy number variations, we used a systematic in silico approach based on frequency in a large dataset of controls, annotation with publicly available databases for developmental diseases, tolerance and haploinsufficiency scores, and gene expression profile in the developing kidney and urinary tract. Five novel candidate genes for CAKUT were identified that showed specific expression in the human and mouse developing urinary tract. Among these genes, DLG1 and KIF12 are likely novel susceptibility genes for CAKUT in humans. Thus, there is a significant role of genomic imbalance in the determination of kidney developmental phenotypes. Additionally, we defined a systematic strategy to identify genetic drivers underlying rare copy number variations. PMID:26352300

  12. Dietary Variation and Evolution of Gene Copy Number among Dog Breeds

    PubMed Central

    Reiter, Taylor; Jagoda, Evelyn; Capellini, Terence D.

    2016-01-01

    Prolonged human interactions and artificial selection have influenced the genotypic and phenotypic diversity among dog breeds. Because humans and dogs occupy diverse habitats, ecological contexts have likely contributed to breed-specific positive selection. Prior to the advent of modern dog-feeding practices, there was likely substantial variation in dietary landscapes among disparate dog breeds. As such, we investigated one type of genetic variant, copy number variation, in three metabolic genes: glucokinase regulatory protein (GCKR), phytanol-CoA 2-hydroxylase (PHYH), and pancreatic α-amylase 2B (AMY2B). These genes code for proteins that are responsible for metabolizing dietary products that originate from distinctly different food types: sugar, meat, and starch, respectively. After surveying copy number variation among dogs with diverse dietary histories, we found no correlation between diet and positive selection in either GCKR or PHYH. Although it has been previously demonstrated that dogs experienced a copy number increase in AMY2B relative to wolves during or after the dog domestication process, we demonstrate that positive selection continued to act on amylase copy number in dog breeds that consumed starch-rich diets in time periods after domestication. Furthermore, we found that introgression with wolves is not responsible for deterioration of positive selection on AMY2B among diverse dog breeds. Together, this supports the hypothesis that the amylase copy number expansion is found universally in dogs. PMID:26863414

  13. The 2-micron plasmid as a nonselectable, stable, high copy number yeast vector

    NASA Technical Reports Server (NTRS)

    Ludwig, D. L.; Bruschi, C. V.

    1991-01-01

    The endogenous 2-microns plasmid of Saccharomyces cerevisiae has been used extensively for the construction of yeast cloning and expression plasmids because it is a native yeast plasmid that is able to be maintained stably in cells at high copy number. Almost invariably, these plasmid constructs, containing some or all 2-microns sequences, exhibit copy number levels lower than 2-microns and are maintained stably only under selective conditions. We were interested in determining if there was a means by which 2-microns could be utilized for vector construction, without forfeiting either copy number or nonselective stability. We identified sites in the 2-microns plasmid that could be used for the insertion of genetic sequences without disrupting 2-microns coding elements and then assessed subsequent plasmid constructs for stability and copy number in vivo. We demonstrate the utility of a previously described 2-microns recombination chimera, pBH-2L, for the manipulation and transformation of 2-microns as a pure yeast plasmid vector. We show that the HpaI site near the STB element in the 2-microns plasmid can be utilized to clone yeast DNA of at least 3.9 kb with no loss of plasmid stability. Additionally, the copy number of these constructs is as high as levels reported for the endogenous 2-microns.

  14. 47 CFR 1.51 - Number of copies of pleadings, briefs, and other papers.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 47 Telecommunication 1 2014-10-01 2014-10-01 false Number of copies of pleadings, briefs, and other papers. 1.51 Section 1.51 Telecommunication FEDERAL COMMUNICATIONS COMMISSION GENERAL PRACTICE AND PROCEDURE General Rules of Practice and Procedure Pleadings, Briefs, and Other Papers § 1.51 Number...

  15. 47 CFR 1.51 - Number of copies of pleadings, briefs, and other papers.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 47 Telecommunication 1 2012-10-01 2012-10-01 false Number of copies of pleadings, briefs, and other papers. 1.51 Section 1.51 Telecommunication FEDERAL COMMUNICATIONS COMMISSION GENERAL PRACTICE AND PROCEDURE General Rules of Practice and Procedure Pleadings, Briefs, and Other Papers § 1.51 Number...

  16. 47 CFR 1.51 - Number of copies of pleadings, briefs, and other papers.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 47 Telecommunication 1 2011-10-01 2011-10-01 false Number of copies of pleadings, briefs, and other papers. 1.51 Section 1.51 Telecommunication FEDERAL COMMUNICATIONS COMMISSION GENERAL PRACTICE AND PROCEDURE General Rules of Practice and Procedure Pleadings, Briefs, and Other Papers § 1.51 Number...

  17. 47 CFR 1.51 - Number of copies of pleadings, briefs and other papers.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 47 Telecommunication 1 2010-10-01 2010-10-01 false Number of copies of pleadings, briefs and other papers. 1.51 Section 1.51 Telecommunication FEDERAL COMMUNICATIONS COMMISSION GENERAL PRACTICE AND PROCEDURE General Rules of Practice and Procedure Pleadings, Briefs, and Other Papers § 1.51 Number...

  18. 47 CFR 1.51 - Number of copies of pleadings, briefs, and other papers.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 47 Telecommunication 1 2013-10-01 2013-10-01 false Number of copies of pleadings, briefs, and other papers. 1.51 Section 1.51 Telecommunication FEDERAL COMMUNICATIONS COMMISSION GENERAL PRACTICE AND PROCEDURE General Rules of Practice and Procedure Pleadings, Briefs, and Other Papers § 1.51 Number...

  19. Chloroplast DNA Copy Number Changes during Plant Development in Organelle DNA Polymerase Mutants

    PubMed Central

    Morley, Stewart A.; Nielsen, Brent L.

    2016-01-01

    Chloroplast genome copy number is very high in leaf tissue, with upwards of 10,000 or more copies of the chloroplast DNA (ctDNA) per leaf cell. This is often promoted as a major advantage for engineering the plastid genome, as it provides high gene copy number and thus is expected to result in high expression of foreign proteins from integrated genes. However, it is also known that ctDNA copy number and ctDNA integrity decrease as cells age. Quantitative PCR (qPCR) allows measurement of organelle DNA levels relative to a nuclear gene target. We have used this approach to determine changes in copy number of ctDNA relative to the nuclear genome at different ages of Arabidopsis plant growth and in organellar DNA polymerase mutants. The mutant plant lines have T-DNA insertions in genes encoding the two organelle localized DNA polymerases (PolIA and PolIB). Each of these mutant lines exhibits some delay in plant growth and development as compared to wild-type plants, with the PolIB plants having a more pronounced delay. Both mutant lines develop to maturity and produce viable seeds. Mutants for both proteins were observed to have a reduction in ctDNA and mtDNA copy number relative to wild type plants at all time points as measured by qPCR. Both DNA polymerase mutants had a fairly similar decrease in ctDNA copy number, while the PolIB mutant had a greater effect of reduction in mtDNA levels. However, despite similar decreases in genome copy number, RT-PCR analysis of PolIA mutants show that PolIB expression remains unchanged, suggesting that PolIA may not be essential to plant survival. Furthermore, genotypic analysis of plants from heterozygous parents display a strong pressure to maintain two functioning copies of PolIB. These results indicate that the two DNA polymerases are both important in ctDNA replication, and they are not fully redundant to each other, suggesting each has a specific function in plant organelles. PMID:26870072

  20. Diet and the evolution of human amylase gene copy number variation

    PubMed Central

    Perry, George H.; Dominy, Nathaniel J.; Claw, Katrina G.; Lee, Arthur S.; Fiegler, Heike; Redon, Richard; Werner, John; Villanea, Fernando A.; Mountain, Joanna L.; Misra, Rajeev; Carter, Nigel P.; Lee, Charles; Stone, Anne C.

    2008-01-01

    Starch consumption is a prominent characteristic of agricultural societies and hunter-gatherers in arid environments. In contrast, rainforest and circum-arctic hunter-gatherers and some pastoralists consume much less starch1-3. This behavioral variation raises the possibility that different selective pressures have acted on amylase, the enzyme responsible for starch hydrolysis4. We found that salivary amylase gene (AMY1) copy number is correlated positively with salivary amylase protein levels, and that individuals from populations with high-starch diets have on average more AMY1 copies than those with traditionally low-starch diets. Comparisons with other loci in a subset of these populations suggest that the level of AMY1 copy number differentiation is unusual. This example of positive selection on a copy number variable gene is one of the first in the human genome. Higher AMY1 copy numbers and protein levels likely improve the digestion of starchy foods, and may buffer against the fitness-reducing effects of intestinal disease. PMID:17828263

  1. Use of competitive PCR to assay copy number of repetitive elements in banana.

    PubMed

    Baurens, F C; Noyer, J L; Lanaud, C; Lagoda, P J

    1996-11-27

    Banana is one of the most important subtropical fruit crops. Genetic improvement by traditional breeding strategies is difficult and better knowledge of genomic structure is needed. Repeated sequences are powerful markers for genetic fingerprinting. The method proposed here to determine the copy number of nuclear repetitive elements is based on competitive reverse transcription-polymerase chain reaction and can also be used for quantifying cytosolic sequences. The reliability of this method was investigated on crude preparations of total DNA. Variations due to the heterogeneity of crude DNA extracts showed that a single locus reference is needed for accurate quantification. A mapped microsatellite locus was used to normalize copy number measurements. Copy number assay of repetitive elements using this method clearly distinguishes between the two banana subspecies investigated: Musa acuminata spp. banskii and M. acuminata spp. malaccensis. Two repetitive sequence families, pMaCIR1115 and pA9-26, were assayed that cover up to 1% of the M. acuminata genome. Their copy number varied up to six fold between the two subspecies. Furthermore, sequence quantification showed that mitochondrial genomes are present in crude leaf-extracted banana DNA at up to 40 copies per cell.

  2. Enhancing yields of low and single copy number plasmid DNAs from Escherichia coli cells.

    PubMed

    Wood, Whitney N; Smith, Kyle D; Ream, Jennifer A; Kevin Lewis, L

    2017-02-01

    Many plasmids used for gene cloning and heterologous protein expression in Escherichia coli cells are low copy number or single copy number plasmids. The extraction of these types of plasmids from small bacterial cell cultures produces low DNA yields. In this study, we have quantitated yields of low copy and single copy number plasmid DNAs after growth of cells in four widely used broths (SB, SOC, TB, and 2xYT) and compared results to those obtained with LB, the most common E. coli cell growth medium. TB (terrific broth) consistently generated the greatest amount of plasmid DNA, in agreement with its ability to produce higher cell titers. The superiority of TB was primarily due to its high levels of yeast extract (24g/L) and was independent of glycerol, a unique component of this broth. Interestingly, simply preparing LB with similarly high levels of yeast extract (LB24 broth) resulted in plasmid yields that were equivalent to those of TB. By contrast, increasing ampicillin concentration to enhance plasmid retention did not improve plasmid DNA recovery. These experiments demonstrate that yields of low and single copy number plasmid DNAs from minipreps can be strongly enhanced using simple and inexpensive media.

  3. Evaluating quantitative methods for measuring plasmid copy numbers in single cells

    PubMed Central

    Tal, Shay; Paulsson, Johan

    2013-01-01

    The life of plasmids is a constant battle against fluctuations: failing to correct copy number fluctuations can increase the plasmid loss rate by many orders of magnitude, as can a failure to more evenly divide the copies between daughters at cell division. Plasmids are therefore long-standing model systems for stochastic processes in cells, much thanks to the efforts of Kurt Nordström to whose memory this issue is dedicated. Here we analyze a range of experimental methods for measuring plasmid copy numbers in single cells, focusing on challenges, trade-offs and necessary experimental controls. In particular we analyze published and unpublished strategies to infer copy numbers from expression of plasmid-encoded reporters, direct labeling of plasmids with fluorescent probes or DNA binding proteins fused to fluorescent reporters, PCR based methods applied to single cell lysates, and plasmid-specific replication arrest. We conclude that no method currently exists to measure plasmid copy numbers in single cells, and that most methods instead inadvertently measure various types of experimental noise. We also discuss how accurate methods can be developed. PMID:22305922

  4. De Novo Chromosome Copy Number Variation in Fanconi Anemia-Associated Hematopoietic Defects

    DTIC Science & Technology

    2014-08-01

    in Fanconi Anemia -Associated Hematopoietic Defects " PRINCIPAL INVESTIGATOR: Niall George Howlett, Ph.D. CONTRACTING ORGANIZATION...Number Variation in Fanconi Anemia -Associated 5a. CONTRACT NUMBER Hematopoietic Defects 5b. GRANT NUMBER W81XWH-11-1-0318 5c. PROGRAM ELEMENT...the molecular mechanism(s) by which FANCD2 performs this key function. 15. SUBJECT TERMS Fanconi anemia , Copy number variation, Monoclonal cell

  5. EPSPS Gene Copy Number and Whole-Plant Glyphosate Resistance Level in Kochia scoparia.

    PubMed

    Gaines, Todd A; Barker, Abigail L; Patterson, Eric L; Westra, Philip; Westra, Eric P; Wilson, Robert G; Jha, Prashant; Kumar, Vipan; Kniss, Andrew R

    2016-01-01

    Glyphosate-resistant (GR) Kochia scoparia has evolved in dryland chemical fallow systems throughout North America and the mechanism of resistance involves 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene duplication. Agricultural fields in four states were surveyed for K. scoparia in 2013 and tested for glyphosate-resistance level and EPSPS gene copy number. Glyphosate resistance was confirmed in K. scoparia populations collected from sugarbeet fields in Colorado, Wyoming, and Nebraska, and Montana. Glyphosate resistance was also confirmed in K. scoparia accessions collected from wheat-fallow fields in Montana. All GR samples had increased EPSPS gene copy number, with median population values up to 11 from sugarbeet fields and up to 13 in Montana wheat-fallow fields. The results indicate that glyphosate susceptibility can be accurately diagnosed using EPSPS gene copy number.

  6. EPSPS Gene Copy Number and Whole-Plant Glyphosate Resistance Level in Kochia scoparia

    PubMed Central

    Gaines, Todd A.; Barker, Abigail L.; Patterson, Eric L.; Westra, Philip; Westra, Eric P.; Wilson, Robert G.; Jha, Prashant; Kumar, Vipan

    2016-01-01

    Glyphosate-resistant (GR) Kochia scoparia has evolved in dryland chemical fallow systems throughout North America and the mechanism of resistance involves 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene duplication. Agricultural fields in four states were surveyed for K. scoparia in 2013 and tested for glyphosate-resistance level and EPSPS gene copy number. Glyphosate resistance was confirmed in K. scoparia populations collected from sugarbeet fields in Colorado, Wyoming, and Nebraska, and Montana. Glyphosate resistance was also confirmed in K. scoparia accessions collected from wheat-fallow fields in Montana. All GR samples had increased EPSPS gene copy number, with median population values up to 11 from sugarbeet fields and up to 13 in Montana wheat-fallow fields. The results indicate that glyphosate susceptibility can be accurately diagnosed using EPSPS gene copy number. PMID:27992501

  7. Punctuated Copy Number Evolution and Clonal Stasis in Triple-Negative Breast Cancer

    PubMed Central

    Gao, Ruli; Davis, Alexander; McDonald, Thomas O.; Sei, Emi; Shi, Xiuqing; Wang, Yong; Tsai, Pei-Ching; Casasent, Anna; Waters, Jill; Zhang, Hong; Meric-Bernstam, Funda; Michor, Franziska; Navin, Nicholas E.

    2016-01-01

    Aneuploidy is a hallmark of breast cancer; however, our knowledge of how these complex genomic rearrangements evolve during tumorigenesis is limited. In this study we developed a highly multiplexed single-nucleus-sequencing method to investigate copy number evolution in triple-negative breast cancer patients. We sequenced 1000 single cells from 12 patients and identified 1–3 major clonal subpopulations in each tumor that shared a common evolutionary lineage. We also identified a minor subpopulation of non-clonal cells that were classified as: 1) metastable, 2) pseudo-diploid, or 3) chromazemic. Phylogenetic analysis and mathematical modeling suggest that these data are unlikely to be explained by the gradual accumulation of copy number events over time. In contrast, our data challenge the paradigm of gradual evolution, showing that the majority of copy number aberrations are acquired at the earliest stages of tumor evolution, in short punctuated bursts, followed by stable clonal expansions that form the tumor mass. PMID:27526321

  8. Dosage sensitivity is a major determinant of human copy number variant pathogenicity

    PubMed Central

    Rice, Alan M.; McLysaght, Aoife

    2017-01-01

    Human copy number variants (CNVs) account for genome variation an order of magnitude larger than single-nucleotide polymorphisms. Although much of this variation has no phenotypic consequences, some variants have been associated with disease, in particular neurodevelopmental disorders. Pathogenic CNVs are typically very large and contain multiple genes, and understanding the cause of the pathogenicity remains a major challenge. Here we show that pathogenic CNVs are significantly enriched for genes involved in development and genes that have greater evolutionary copy number conservation across mammals, indicative of functional constraints. Conversely, genes found in benign CNV regions have more variable copy number. These evolutionary constraints are characteristic of genes in pathogenic CNVs and can only be explained by dosage sensitivity of those genes. These results implicate dosage sensitivity of individual genes as a common cause of CNV pathogenicity. These evolutionary metrics suggest a path to identifying disease genes in pathogenic CNVs. PMID:28176757

  9. Sequential Model Selection based Segmentation to Detect DNA Copy Number Variation

    PubMed Central

    Hu, Jianhua; Zhang, Liwen; Wang, Huixia Judy

    2016-01-01

    Summary Array-based CGH experiments are designed to detect genomic aberrations or regions of DNA copy-number variation that are associated with an outcome, typically a state of disease. Most of the existing statistical methods target on detecting DNA copy number variations in a single sample or array. We focus on the detection of group effect variation, through simultaneous study of multiple samples from multiple groups. Rather than using direct segmentation or smoothing techniques, as commonly seen in existing detection methods, we develop a sequential model selection procedure that is guided by a modified Bayesian information criterion. This approach improves detection accuracy by accumulatively utilizing information across contiguous clones, and has computational advantage over the existing popular detection methods. Our empirical investigation suggests that the performance of the proposed method is superior to that of the existing detection methods, in particular, in detecting small segments or separating neighboring segments with differential degrees of copy-number variation. PMID:26954760

  10. The potential role for use of mitochondrial DNA copy number as predictive biomarker in presbycusis

    PubMed Central

    Falah, Masoumeh; Houshmand, Massoud; Najafi, Mohammad; Balali, Maryam; Mahmoudian, Saeid; Asghari, Alimohamad; Emamdjomeh, Hessamaldin; Farhadi, Mohammad

    2016-01-01

    Objectives Age-related hearing impairment, or presbycusis, is the most common communication disorder and neurodegenerative disease in the elderly. Its prevalence is expected to increase, due to the trend of growth of the elderly population. The current diagnostic test for detection of presbycusis is implemented after there has been a change in hearing sensitivity. Identification of a pre-diagnostic biomarker would raise the possibility of preserving hearing sensitivity before damage occurs. Mitochondrial dysfunction, including the production of reactive oxygen species and induction of expression of apoptotic genes, participates in the progression of presbycusis. Mitochondrial DNA sequence variation has a critical role in presbycusis. However, the nature of the relationship between mitochondrial DNA copy number, an important biomarker in many other diseases, and presbycusis is undetermined. Methods Fifty-four subjects with presbycusis and 29 healthy controls were selected after ear, nose, throat examination and pure-tone audiometry. DNA was extracted from peripheral blood samples. The copy number of mitochondrial DNA relative to the nuclear genome was measured by quantitative real-time polymerase chain reaction. Results Subjects with presbycusis had a lower median mitochondrial DNA copy number than healthy subjects and the difference was statistically significant (P=0.007). Mitochondrial DNA copy number was also significantly associated with degree of hearing impairment (P=0.025) and audiogram configuration (P=0.022). Conclusion The findings of this study suggest that lower mitochondrial DNA copy number is responsible for presbycusis through alteration of mitochondrial function. Moreover, the significant association of mitochondrial DNA copy number in peripheral blood samples with the degree of hearing impairment and audiogram configuration has potential for use as a standard test for presbycusis, providing the possibility of the development of an easy

  11. Association of Copy Number Variants With Specific Ultrasonographically Detected Fetal Anomalies

    PubMed Central

    Donnelly, Jennifer C; Platt, Lawrence D; Rebarber, Andrei; Zachary, Julia; Grobman, William A; Wapner, Ronald J

    2014-01-01

    Objective To evaluate the association of other-than-common benign copy number variants with specific fetal abnormalities detected by ultrasonogram. Methods Fetuses with structural anomalies were compared to fetuses without detected abnormalities for the frequency of other-than-common benign copy number variants. This is a secondary analysis from the previously published National Institute of Child Health and Human Development microarray trial. Ultrasound reports were reviewed and details of structural anomalies were entered into a nonhierarchical web-based database. The frequency of other-than-common benign copy number variants (ie, either pathogenic or variants of uncertain significance) not detected by karyotype was calculated for each anomaly in isolation and in the presence of other anomalies and compared to the frequency in fetuses without detected abnormalities. Results Of 1,082 fetuses with anomalies detected on ultrasound, 752 had a normal karyotype. Other-than-common benign copy number variants were present in 61 (8.1%) of these euploid fetuses. Fetuses with anomalies in more than one system had a 13.0% frequency of other-than-common benign copy number variants, which was significantly higher (p<0.001) than the frequency (3.6%) in fetuses without anomalies (n = 1966). Specific organ systems in which isolated anomalies were nominally significantly associated with other-than-common benign copy number variants were the renal (p= 0.036) and cardiac systems (p=0.012) but did not meet the adjustment for multiple comparisons. Conclusions When a fetal anomaly is detected on ultrasonogram, chromosomal microarray offers additional information over karyotype, the degree of which depends on the organ system involved. PMID:24901266

  12. Copy number variations of 11 macronuclear chromosomes and their gene expression in Oxytricha trifallax.

    PubMed

    Xu, Ke; Doak, Thomas G; Lipps, Hans J; Wang, Jingmei; Swart, Estienne C; Chang, Wei-Jen

    2012-08-15

    Ciliated protozoa are peculiar for their nuclear dimorphism, wherein two types of nuclei divide nuclear functions: a germline micronucleus (MIC) is transcriptionally inert during vegetative growth, but serves as the genetic blueprint for the somatic macronucleus (MAC), which is responsible for all transcripts supporting cell growth and reproduction. While all the advantages/disadvantages associated with nuclear dimorphism are not clear, an essential advantage seems to be the ability to produce a highly polyploid MAC, which then allows for the maintenance of extremely large single cells - many ciliate cells are larger than small metazoa. In some ciliate classes, chromosomes in the MAC are extensively fragmented to create extremely short chromosomes that often carry single genes, and these chromosomes may be present in different copy numbers, resulting in different ploidies. While using gene copy number to regulate gene expression is limited in most eukaryotic systems, the extensive fragmentation in some ciliate classes provides this opportunity to every MAC gene. However, it is still unclear if this mechanism is in fact used extensively in these ciliates. To address this, we have quantified copy numbers of 11 MAC chromosomes and their gene expression in Oxytricha trifallax (CI: Spirotrichea). We compared copy numbers between two subpopulations of O. trifallax, and copy numbers of 7 orthologous genes between O. trifallax and the closely related Stylonychia lemnae. We show that copy numbers of MAC chromosomes are variable, dynamic, and positively correlated to gene expression. These features might be conserved in all spirotrichs, and might exist in other classes of ciliates with heavily fragmented MAC chromosomes.

  13. Interactive analysis and assessment of single-cell copy-number variations

    PubMed Central

    Garvin, Tyler; Aboukhalil, Robert; Kendall, Jude; Baslan, Timour; Atwal, Gurinder S.; Hicks, James; Wigler, Michael; Schatz, Michael C.

    2015-01-01

    We present an open-source web platform, Ginkgo (http://qb.cshl.edu/ginkgo), for the analysis and assessment of single-cell copy-number variations (CNVs). Ginkgo automatically constructs copy-number profiles of cells from mapped reads and constructs phylogenetic trees of related cells. We validate Ginkgo by reproducing the results of five major studies and examine the characteristics of three commonly used single-cell amplification techniques to conclude degenerate oligonucleotide-primed PCR to be the most consistent for CNV analysis. PMID:26344043

  14. EXCAVATOR: detecting copy number variants from whole-exome sequencing data.

    PubMed

    Magi, Alberto; Tattini, Lorenzo; Cifola, Ingrid; D'Aurizio, Romina; Benelli, Matteo; Mangano, Eleonora; Battaglia, Cristina; Bonora, Elena; Kurg, Ants; Seri, Marco; Magini, Pamela; Giusti, Betti; Romeo, Giovanni; Pippucci, Tommaso; De Bellis, Gianluca; Abbate, Rosanna; Gensini, Gian Franco

    2013-01-01

    We developed a novel software tool, EXCAVATOR, for the detection of copy number variants (CNVs) from whole-exome sequencing data. EXCAVATOR combines a three-step normalization procedure with a novel heterogeneous hidden Markov model algorithm and a calling method that classifies genomic regions into five copy number states. We validate EXCAVATOR on three datasets and compare the results with three other methods. These analyses show that EXCAVATOR outperforms the other methods and is therefore a valuable tool for the investigation of CNVs in largescale projects, as well as in clinical research and diagnostics. EXCAVATOR is freely available at http://sourceforge.net/projects/excavatortool/.

  15. Importance of rare gene copy number alterations for personalized tumor characterization and survival analysis.

    PubMed

    Seifert, Michael; Friedrich, Betty; Beyer, Andreas

    2016-10-03

    It has proven exceedingly difficult to ascertain rare copy number alterations (CNAs) that may have strong effects in individual tumors. We show that a regulatory network inferred from gene expression and gene copy number data of 768 human cancer cell lines can be used to quantify the impact of patient-specific CNAs on survival signature genes. A focused analysis of tumors from six tissues reveals that rare patient-specific gene CNAs often have stronger effects on signature genes than frequent gene CNAs. Further comparison to a related network-based approach shows that the integration of indirectly acting gene CNAs significantly improves the survival analysis.

  16. Copy Number Variation Analysis by Array Analysis of Single Cells Following Whole Genome Amplification.

    PubMed

    Dimitriadou, Eftychia; Zamani Esteki, Masoud; Vermeesch, Joris Robert

    2015-01-01

    Whole genome amplification is required to ensure the availability of sufficient material for copy number variation analysis of a genome deriving from an individual cell. Here, we describe the protocols we use for copy number variation analysis of non-fixed single cells by array-based approaches following single-cell isolation and whole genome amplification. We are focusing on two alternative protocols, an isothermal and a PCR-based whole genome amplification method, followed by either comparative genome hybridization (aCGH) or SNP array analysis, respectively.

  17. Variation in topoisomerase I gene copy number as a mechanism for intrinsic drug sensitivity.

    PubMed Central

    McLeod, H. L.; Keith, W. N.

    1996-01-01

    DNA topoisomerase I (topo I) is the principle target for camptothecin and its derivatives such as SN38. Levels of topo I expression vary widely between and within tumour types and the basis for this is poorly understood. We have used fluorescence in situ hybridisation to detect the topo I locus in a panel of breast and colon cancer cell lines. This approach has identified a range of topo I gene copies from 1 to 6 between the cell lines as a result of DNA amplification, polysomy and isochromosome formation. Topo I gene copy number was highly correlated with topo I expression, (rs = 0.92), and inversely correlated to sensitivity to a 1 h exposure to SN38 (rs = -0.904). This illustrates the significant impact of altered topo I gene copy number on intrinsic drug sensitivity and influences potential mechanisms for acquisition of drug resistance. Images Figure 1 Figure 2 PMID:8761363

  18. HSFY and ZNF280BY show copy number variations within 17 water buffalo populations.

    PubMed

    Zhang, X; Han, H; Zhang, T; Sun, T; Xi, Y; Chen, N; Huang, Y; Dang, R; Lan, X; Chen, H; Lei, C

    2017-04-01

    Recent transcriptomic analysis of the bovine Y chromosome revealed abundant presence of multi-copy protein coding gene families on the male-specific region of the Y chromosome (MSY). Copy number variations (CNVs) of several MSY genes are closely related to semen quality and male reproduction in cattle. However, the CNVs of MSY genes in water buffalo are largely unknown. Therefore, this study aimed to investigate the CNVs of HSFY and ZNF280BY of 298 buffaloes from 17 populations distributed in China, Vietnam and Laos using quantitative PCR. Our results revealed that the median copy numbers of the HSFY and ZNF280BY genes were 47 (ranging from 20 to 145) and 269 (ranging from 73 to 974) respectively. In conclusion, this study indicated that HSFY and ZNF280BY showed abundant CNVs within swamp buffalo populations.

  19. Inducible Amplification of Gene Copy Number and Heterologous Protein Production in the Yeast Kluyveromyces lactis

    PubMed Central

    Morlino, Giovanni B.; Tizzani, Lorenza; Fleer, Reinhard; Frontali, Laura; Bianchi, Michele M.

    1999-01-01

    Heterologous protein production can be doubled by increasing the copy number of the corresponding heterologous gene. We constructed a host-vector system in the yeast Kluyveromyces lactis that was able to induce copy number amplification of pKD1 plasmid-based vectors upon expression of an integrated copy of the plasmid recombinase gene. We increased the production and secretion of two heterologous proteins, glucoamylase from the yeast Arxula adeninivorans and mammalian interleukin-1β, following gene dosage amplification when the heterologous genes were carried by pKD1-based vectors. The choice of the promoters for expression of the integrated recombinase gene and of the episomal heterologous genes are critical for the mitotic stability of the host-vector system. PMID:10543790

  20. Inducible amplification of gene copy number and heterologous protein production in the yeast Kluyveromyces lactis.

    PubMed

    Morlino, G B; Tizzani, L; Fleer, R; Frontali, L; Bianchi, M M

    1999-11-01

    Heterologous protein production can be doubled by increasing the copy number of the corresponding heterologous gene. We constructed a host-vector system in the yeast Kluyveromyces lactis that was able to induce copy number amplification of pKD1 plasmid-based vectors upon expression of an integrated copy of the plasmid recombinase gene. We increased the production and secretion of two heterologous proteins, glucoamylase from the yeast Arxula adeninivorans and mammalian interleukin-1beta, following gene dosage amplification when the heterologous genes were carried by pKD1-based vectors. The choice of the promoters for expression of the integrated recombinase gene and of the episomal heterologous genes are critical for the mitotic stability of the host-vector system.

  1. 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.

  2. Mitochondrial Copy Number and D-Loop Variants in Pompe Patients

    PubMed Central

    Bahreini, Fatemeh; Houshmand, Massoud; Modaresi, Mohammad Hossein; Tonekaboni, Hassan; Nafissi, Shahriar; Nazari, Ferdoss; Akrami, Seyed Mohammad

    2016-01-01

    Objective Pompe disease is a rare neuromuscular genetic disorder and is classified into two forms of early and late-onset. Over the past two decades, mitochondrial abnor- malities have been recognized as an important contributor to an array of neuromuscular diseases. We therefore aimed to compare mitochondrial copy number and mitochondrial displacement-loop sequence variation in infantile and adult Pompe patients. Materials and Methods In this retrospective study, the mitochondrial D-loop sequence was analyzed by polymerase chain reaction (PCR) and direct sequencing to detect pos- sible variation in 28 Pompe patients (17 infants and 11 adults). Results were compared with 100 healthy controls and sequences of all individuals were compared with the Cam- bridge reference sequence. Real-time PCR was used to quantify mitochondrial DNA copy number. Results Among 59 variants identified, 37(62.71%) were present in the infant group, 14(23.333%) in the adult group and 8(13.333%) in both groups. Mitochondrial copy number in infant patients was lower than adults (P<0.05). A significant frequency differ- ence was seen between the two groups for 12 single nucleotide polymorphism (SNP). A novel insertion (317-318 ins CCC) was observed in patients and six SNPs were iden- tified as neutral variants in controls. There was an inverse association between mito- chondrial copy number and D-loop variant number (r=0.54). Conclusion The 317-318 ins CCC was detected as a new mitochondrial variant in Pompe patients. PMID:27602323

  3. Accurate measurement of transgene copy number in crop plants using droplet digital PCR.

    PubMed

    Collier, Ray; Dasgupta, Kasturi; Xing, Yan-Ping; Hernandez, Bryan Tarape; Shao, Min; Rohozinski, Dominica; Kovak, Emma; Lin, Jeanie; de Oliveira, Maria Luiza P; Stover, Ed; McCue, Kent F; Harmon, Frank G; Blechl, Ann; Thomson, James G; Thilmony, Roger

    2017-02-23

    Genetic transformation is a powerful means for the improvement of crop plants, but requires labor and resource intensive methods. An efficient method for identifying single copy transgene insertion events from a population of independent transgenic lines is desirable. Currently transgene copy number is estimated by either Southern blot hybridization analyses or quantitative polymerase chain reaction (qPCR) experiments. Southern hybridization is a convincing and reliable method, but it also is expensive, time-consuming and often requires a large amount of genomic DNA and radioactively labeled probes. Alternatively, qPCR requires less DNA and is potentially simpler to perform, but its results can lack the accuracy and precision needed to confidently distinguish between one and two copy events in transgenic plants with large genomes. To address this need, we developed a droplet digital PCR (dPCR)-based method for transgene copy number measurement in an array of crops: rice, citrus, potato, maize, tomato, and wheat. The method utilizes specific primers to amplify target transgenes, and endogenous reference genes in a single duplexed reaction containing thousands of droplets. Endpoint amplicon production in the droplets is detected and quantified using sequence-specific fluorescently labeled probes. The results demonstrate that this approach can generate confident copy number measurements in independent transgenic lines in these crop species. This method and the compendium of probes and primers will be a useful resource for the plant research community, enabling the simple and accurate determination of transgene copy number in these six important crop species. This article is protected by copyright. All rights reserved.

  4. Validation of a digital PCR method for quantification of DNA copy number concentrations by using a certified reference material.

    PubMed

    Deprez, Liesbet; Corbisier, Philippe; Kortekaas, Anne-Marie; Mazoua, Stéphane; Beaz Hidalgo, Roxana; Trapmann, Stefanie; Emons, Hendrik

    2016-09-01

    Digital PCR has become the emerging technique for the sequence-specific detection and quantification of nucleic acids for various applications. During the past years, numerous reports on the development of new digital PCR methods have been published. Maturation of these developments into reliable analytical methods suitable for diagnostic or other routine testing purposes requires their validation for the intended use. Here, the results of an in-house validation of a droplet digital PCR method are presented. This method is intended for the quantification of the absolute copy number concentration of a purified linearized plasmid in solution with a nucleic acid background. It has been investigated which factors within the measurement process have a significant effect on the measurement results, and the contribution to the overall measurement uncertainty has been estimated. A comprehensive overview is provided on all the aspects that should be investigated when performing an in-house method validation of a digital PCR method.

  5. Exploiting rRNA Operon Copy Number to Investigate Bacterial Reproductive Strategies

    PubMed Central

    Roller, Benjamin R.K.; Stoddard, Steven F.; Schmidt, Thomas M.

    2016-01-01

    Summary The potential for rapid reproduction is a hallmark of microbial life, but microbes in nature must also survive and compete when growth is constrained by resource availability. Successful reproduction requires different strategies when resources are scarce compared to when they are abundant1,2, but a systematic framework for predicting these reproductive strategies in bacteria has not been available. Here we show that the number of ribosomal RNA operons (rrn) in bacterial genomes predicts two important components of reproduction – growth rate and growth efficiency – which are favored under contrasting regimes of resource availability3,4. We find that the maximum reproductive rate of bacteria doubles with a doubling of rrn copy number, while the efficiency of carbon use is inversely related to maximal growth rate and rrn copy number. We also identify a feasible explanation for these patterns: the rate and yield of protein synthesis mirror the overall pattern in maximum growth rate and growth efficiency. Furthermore, comparative analysis of genomes from 1,167 bacterial species reveals that rrn copy number predicts traits associated with resource availability, including chemotaxis and genome streamlining. Genome-wide patterns of orthologous gene content covary with rrn copy number, suggesting convergent evolution in response to resource availability. Our findings indicate that basic cellular processes adapt in contrasting ways to long-term differences in resource availability. They also establish a basis for predicting changes in bacterial community composition in response to resource perturbations using rrn copy number measurements5 or inferences6,7. PMID:27617693

  6. Real-time PCR for the detection of precise transgene copy number in durum wheat.

    PubMed

    Gadaleta, Agata; Giancaspro, Angelica; Cardone, Maria Francesca; Blanco, Antonio

    2011-12-01

    Recent results obtained in various crops indicate that real-time PCR could be a powerful tool for the detection and characterization of transgene locus structures. The determination of transgenic locus number through real-time PCR overcomes the problems linked to phenotypic segregation analysis (i.e. lack of detectable expression even when the transgenes are present) and can analyse hundreds of samples in a day, making it an efficient method for estimating gene copy number. Despite these advantages, many authors speak of "estimating" copy number by real-time PCR, and this is because the detection of a precise number of transgene depends on how well real-time PCR performs.This study was conducted to determine transgene copy number in transgenic wheat lines and to investigate potential variability in sensitivity and resolution of real-time chemistry by TaqMan probes. We have applied real-time PCR to a set of four transgenic durum wheat lines previously obtained. A total of 24 experiments (three experiments for two genes in each transgenic line) were conducted and standard curves were obtained from serial dilutions of the plasmids containing the genes of interest. The correlation coefficients ranged from 0.95 to 0.97. By using TaqMan quantitative real-time PCR we were able to detect 1 to 41 copies of transgenes per haploid genome in the DNA of homozygous T4 transformants. Although a slight variability was observed among PCR experiments, in our study we found real-time PCR to be a fast, sensitive and reliable method for the detection of transgene copy number in durum wheat, and a useful adjunct to Southern blot and FISH analyses to detect the presence of transgenic DNA in plant material.

  7. Pervasive gene content variation and copy number variation in maize and its undomesticated progenitor

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Different individuals of the same species are generally thought to have very similar genomes. However, there is growing evidence that structural variation in the form of copy number variation (CNV) and presence-absence variation (PAV) can lead to variation in the genome content of individuals withi...

  8. Structural and functional impacts of copy number variations on the cattle genome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Although there have been significant advances in resolving the pattern and nature of single nucleotide polymorphisms (SNPs), similar realizations for larger, more complex forms of genetic variation have just emerged. Several recent publications reveal that copy number variations (CNVs) are common an...

  9. Distribution of Disease-Associated Copy Number Variants across Distinct Disorders of Cognitive Development

    ERIC Educational Resources Information Center

    Pescosolido, Matthew F.; Gamsiz, Ece D.; Nagpal, Shailender; Morrow, Eric M.

    2013-01-01

    Objective: The purpose of the present study was to discover the extent to which distinct "DSM" disorders share large, highly recurrent copy number variants (CNVs) as susceptibility factors. We also sought to identify gene mechanisms common to groups of diagnoses and/or specific to a given diagnosis based on associations with CNVs. Method:…

  10. Concurrent Whole-Genome Haplotyping and Copy-Number Profiling of Single Cells

    PubMed Central

    Zamani Esteki, Masoud; Dimitriadou, Eftychia; Mateiu, Ligia; Melotte, Cindy; Van der Aa, Niels; Kumar, Parveen; Das, Rakhi; Theunis, Koen; Cheng, Jiqiu; Legius, Eric; Moreau, Yves; Debrock, Sophie; D’Hooghe, Thomas; Verdyck, Pieter; De Rycke, Martine; Sermon, Karen; Vermeesch, Joris R.; Voet, Thierry

    2015-01-01

    Methods for haplotyping and DNA copy-number typing of single cells are paramount for studying genomic heterogeneity and enabling genetic diagnosis. Before analyzing the DNA of a single cell by microarray or next-generation sequencing, a whole-genome amplification (WGA) process is required, but it substantially distorts the frequency and composition of the cell’s alleles. As a consequence, haplotyping methods suffer from error-prone discrete SNP genotypes (AA, AB, BB) and DNA copy-number profiling remains difficult because true DNA copy-number aberrations have to be discriminated from WGA artifacts. Here, we developed a single-cell genome analysis method that reconstructs genome-wide haplotype architectures as well as the copy-number and segregational origin of those haplotypes by employing phased parental genotypes and deciphering WGA-distorted SNP B-allele fractions via a process we coin haplarithmisis. We demonstrate that the method can be applied as a generic method for preimplantation genetic diagnosis on single cells biopsied from human embryos, enabling diagnosis of disease alleles genome wide as well as numerical and structural chromosomal anomalies. Moreover, meiotic segregation errors can be distinguished from mitotic ones. PMID:25983246

  11. Evolutionary and functional features of copy number variation in the cattle genome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genomic structural variations are an important source of genetic diversity. Copy number variations (CNVs), gains and losses of large regions of genomic sequence between individuals of a species, are known to be associated with both diseases and phenotypic traits. In cattle, as well as many other spe...

  12. Rare DNA copy number variants in cardiovascular malformations with extracardiac abnormalities

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clinically significant cardiovascular malformations (CVMs) occur in 5-8 per 1000 live births. Recurrent copy number variations (CNVs) are among the known causes of syndromic CVMs, accounting for an important fraction of cases. We hypothesized that many additional rare CNVs also cause CVMs and can be...

  13. Investigation of Copy Number Variation in Children with Conotruncal Heart Defects

    PubMed Central

    Campos, Carla Marques Rondon; Zanardo, Evelin Aline; Dutra, Roberta Lelis; Kulikowski, Leslie Domenici; Kim, Chong Ae

    2015-01-01

    Background Congenital heart defects (CHD) are the most prevalent group of structural abnormalities at birth and one of the main causes of infant morbidity and mortality. Studies have shown a contribution of the copy number variation in the genesis of cardiac malformations. Objectives Investigate gene copy number variation (CNV) in children with conotruncal heart defect. Methods Multiplex ligation-dependent probe amplification (MLPA) was performed in 39 patients with conotruncal heart defect. Clinical and laboratory assessments were conducted in all patients. The parents of the probands who presented abnormal findings were also investigated. Results Gene copy number variation was detected in 7/39 patients: 22q11.2 deletion, 22q11.2 duplication, 15q11.2 duplication, 20p12.2 duplication, 19p deletion, 15q and 8p23.2 duplication with 10p12.31 duplication. The clinical characteristics were consistent with those reported in the literature associated with the encountered microdeletion/microduplication. None of these changes was inherited from the parents. Conclusions Our results demonstrate that the technique of MLPA is useful in the investigation of microdeletions and microduplications in conotruncal congenital heart defects. Early diagnosis of the copy number variation in patients with congenital heart defect assists in the prevention of morbidity and decreased mortality in these patients. PMID:25387403

  14. Copy number polymorphisms in new HapMap III and Singapore populations.

    PubMed

    Ku, Chee-Seng; Teo, Shu-Mei; Naidoo, Nasheen; Sim, Xueling; Teo, Yik-Ying; Pawitan, Yudi; Seielstad, Mark; Chia, Kee-Seng; Salim, Agus

    2011-08-01

    Copy number variations can be identified using newer genotyping arrays with higher single nucleotide polymorphisms (SNPs) density and copy number probes accompanied by newer algorithms. McCarroll et al. (2008) applied these to the HapMap II samples and identified 1316 copy number polymorphisms (CNPs). In our study, we applied the same approach to 859 samples from three Singapore populations and seven HapMap III populations. Approximately 50% of the 1291 autosomal CNPs were found to be polymorphic only in populations of non-African ancestry. Pairwise comparisons among the 10 populations showed substantial differences in the CNPs frequencies. Additionally, 698 CNPs showed significant differences with false discovery rate (FDR)<0.01 among the 10 populations and these loci overlap with known disease-associated or pharmacogenetic-related genes such as CFHR3 and CFHR1 (age related macular degeneration), GSTTI (metabolism of various carcinogenic compounds and cancers) and UGT2B17 (prostate cancer and graft-versus-host disease). The correlations between CNPs and genome-wide association studies-SNPs were investigated and several loci, which were previously unreported, that may potentially be implicated in complex diseases and traits were found; for example, childhood acute lymphoblastic leukaemia, age-related macular degeneration, breast cancer, response to antipsychotic treatment, rheumatoid arthritis and type-1 diabetes. Additionally, we also found 5014 novel copy number loci that have not been reported previously by McCarroll et al. (2008) in the 10 populations.

  15. 18 CFR 156.3 - Applications; number of copies; general requirements.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... improvement of transportation facilities, physical connection of facilities or service of natural gas together... ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY REGULATIONS UNDER NATURAL GAS ACT APPLICATIONS FOR ORDERS UNDER SECTION 7(a) OF THE NATURAL GAS ACT § 156.3 Applications; number of copies;...

  16. Subtelomeric Rearrangements and Copy Number Variations in People with Intellectual Disabilities

    ERIC Educational Resources Information Center

    Christofolini, D. M.; De Paula Ramos, M. A.; Kulikowski, L. D.; Da Silva Bellucco, F. T.; Belangero, S. I. N.; Brunoni, D.; Melaragno, M. I.

    2010-01-01

    Background: The most prevalent type of structural variation in the human genome is represented by copy number variations that can affect transcription levels, sequence, structure and function of genes. Method: In the present study, we used the multiplex ligation-dependent probe amplification (MLPA) technique and quantitative PCR for the detection…

  17. Somatic Copy Number Alterations at Oncogenic Loci Show Diverse Correlations with Gene Expression

    NASA Astrophysics Data System (ADS)

    Roszik, Jason; Wu, Chang-Jiun; Siroy, Alan E.; Lazar, Alexander J.; Davies, Michael A.; Woodman, Scott E.; Kwong, Lawrence N.

    2016-01-01

    Somatic copy number alterations (SCNAs) affecting oncogenic drivers have a firmly established role in promoting cancer. However, no agreed-upon standard exists for calling locus-specific amplifications and deletions in each patient sample. Here, we report the correlative analysis of copy number amplitude and length with gene expression across 6,109 samples from The Cancer Genome Atlas (TCGA) dataset across 16 cancer types. Using specificity, sensitivity, and precision-based scores, we assigned optimized amplitude and length cutoffs for nine recurrent SCNAs affecting known oncogenic drivers, using mRNA expression as a functional readout. These cutoffs captured the majority of SCNA-driven, highly-expression-altered samples. The majority of oncogenes required only amplitude cutoffs, as high amplitude samples were almost invariably focal; however, CDKN2A and PTEN uniquely required both amplitude and length cutoffs as primary predictors. For PTEN, these extended to downstream AKT activation. In contrast, SCNA genes located peri-telomerically or in fragile sites showed poor expression-copy number correlations. Overall, our analyses identify optimized amplitude and length cutoffs as efficient predictors of gene expression changes for specific oncogenic SCNAs, yet warn against one-size-fits-all interpretations across all loci. Our results have implications for cancer data analyses and the clinic, where copy number and mutation data are increasingly used to personalize cancer therapy.

  18. Whole-genome sequencing reveals the diversity of cattle copy number variations and multicopy genes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Structural and functional impacts of copy number variations (CNVs) on livestock genomes are not yet well understood. We identified 1853 CNV regions using population-scale sequencing data generated from 75 cattle representing 8 breeds (Angus, Brahman, Gir, Holstein, Jersey, Limousin, Nelore, Romagnol...

  19. Diversity and population-genetic properties of copy number variations and multicopy genes in cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The diversity and population-genetics of copy number variation (CNV) in domesticated animals are not well understood. In this study, we analyzed 75 genomes of major taurine and indicine cattle breeds (including Angus, Brahman, Gir, Holstein, Jersey, Limousin, Nelore, Romagnola), sequenced to 11-fold...

  20. Identification of copy number variable gene families in Holstein and Jersey cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Copy number variants (CNV) represent a large proportion of genetic variation within the cattle genome that has yet to be accurately characterized by SNP genotyping arrays. While significant progress has been made in the identification of CNVs within individual animals using next generation sequence ...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Besides single nucleotide polymorphism (SNP), copy number variation (CNV) which comprise insertions, deletions and duplications of genomic sequence, is a new informative type of genetic variations. CNVs have been shown to be both common in mammals and important for understanding relationship between...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Copy number variations (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...

  3. Somatic Copy Number Alterations at Oncogenic Loci Show Diverse Correlations with Gene Expression

    PubMed Central

    Roszik, Jason; Wu, Chang-Jiun; Siroy, Alan E.; Lazar, Alexander J.; Davies, Michael A; Woodman, Scott E; Kwong, Lawrence N

    2016-01-01

    Somatic copy number alterations (SCNAs) affecting oncogenic drivers have a firmly established role in promoting cancer. However, no agreed-upon standard exists for calling locus-specific amplifications and deletions in each patient sample. Here, we report the correlative analysis of copy number amplitude and length with gene expression across 6,109 samples from The Cancer Genome Atlas (TCGA) dataset across 16 cancer types. Using specificity, sensitivity, and precision-based scores, we assigned optimized amplitude and length cutoffs for nine recurrent SCNAs affecting known oncogenic drivers, using mRNA expression as a functional readout. These cutoffs captured the majority of SCNA-driven, highly-expression-altered samples. The majority of oncogenes required only amplitude cutoffs, as high amplitude samples were almost invariably focal; however, CDKN2A and PTEN uniquely required both amplitude and length cutoffs as primary predictors. For PTEN, these extended to downstream AKT activation. In contrast, SCNA genes located peri-telomerically or in fragile sites showed poor expression-copy number correlations. Overall, our analyses identify optimized amplitude and length cutoffs as efficient predictors of gene expression changes for specific oncogenic SCNAs, yet warn against one-size-fits-all interpretations across all loci. Our results have implications for cancer data analyses and the clinic, where copy number and mutation data are increasingly used to personalize cancer therapy. PMID:26787600

  4. Fine mapping of copy number variations on two cattle genome assemblies using high density SNP array

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Btau_4.0 and UMD3.1 are two distinct cattle reference genome assemblies. In our previous study using the low density BovineSNP50 array, we reported a copy number variation (CNV) analysis on Btau_4.0 with 521 animals of 21 cattle breeds, yielding 682 CNV regions with a total length of 139.8 megabases...

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

    PubMed Central

    2014-01-01

    Background 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. Results 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. Conclusions 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. PMID:25077862

  6. Compact, robust, and spectrally pure diode-laser system with a filtered output and a tunable copy for absolute referencing

    NASA Astrophysics Data System (ADS)

    Kirilov, E.; Mark, M. J.; Segl, M.; Nägerl, H.-C.

    2015-05-01

    We report on a design of a compact laser system composed of an extended-cavity diode laser with high passive stability and a pre-filter Fabry-Perot cavity. The laser is frequency-stabilized relative to the cavity using a serrodyne technique with a correction bandwidth of ≥6 MHz and a dynamic range of ≥700 MHz. The free-running laser system has a power spectral density (PSD) ≤100 Hz2/Hz centered mainly in the acoustic frequency range. A highly tunable, 0.5-1.3 GHz copy of the spectrally pure output beam is provided, which can be used for further stabilization of the laser system to an ultra-stable reference. We demonstrate a simple one-channel lock to such a reference that brings down the PSD to the sub-Hz level. The tuning, frequency stabilization, and sideband imprinting are achieved by a minimum number of key elements comprising a fibered electro-optic modulator, acousto-optic modulator, and a nonlinear transmission line. The system is easy to operate, scalable, and highly applicable to atomic/molecular experiments demanding high spectral purity, long-term stability, and robustness.

  7. Amy2B copy number variation reveals starch diet adaptations in ancient European dogs

    PubMed Central

    Tresset, Anne; Bastian, Fabiola; Lagoutte, Laetitia; Arendt, Maja-Louise; Bălăşescu, Adrian; Marshour, Marjan; Sablin, Mikhail V.; Salanova, Laure; Vigne, Jean-Denis; Hitte, Christophe; Hänni, Catherine

    2016-01-01

    Extant dog and wolf DNA indicates that dog domestication was accompanied by the selection of a series of duplications on the Amy2B gene coding for pancreatic amylase. In this study, we used a palaeogenetic approach to investigate the timing and expansion of the Amy2B gene in the ancient dog populations of Western and Eastern Europe and Southwest Asia. Quantitative polymerase chain reaction was used to estimate the copy numbers of this gene for 13 ancient dog samples, dated to between 15 000 and 4000 years before present (cal. BP). This evidenced an increase of Amy2B copies in ancient dogs from as early as the 7th millennium cal. BP in Southeastern Europe. We found that the gene expansion was not fixed across all dogs within this early farming context, with ancient dogs bearing between 2 and 20 diploid copies of the gene. The results also suggested that selection for the increased Amy2B copy number started 7000 years cal. BP, at the latest. This expansion reflects a local adaptation that allowed dogs to thrive on a starch rich diet, especially within early farming societies, and suggests a biocultural coevolution of dog genes and human culture. PMID:28018628

  8. The Association of Mitochondrial Potential and Copy Number with Pig Oocyte Maturation and Developmental Potential

    PubMed Central

    LEE, Seul-Ki; ZHAO, Ming-Hui; KWON, Jung-Woo; LI, Ying-Hua; LIN, Zi-Li; JIN, Yong-Xun; KIM, Nam-Hyung; CUI, Xiang-Shun

    2014-01-01

    ATP is critical for oocyte maturation, fertilization, and subsequent embryo development. Both mitochondrial membrane potential and copy number expand during oocyte maturation. In order to differentiate the roles of mitochondrial metabolic activity and mtDNA copy number during oocyte maturation, we used two inhibitors, FCCP (carbonyl cyanide p-(tri-fluromethoxy)phenyl-hydrazone) and ddC (2’3-dideoxycytidine), to deplete the mitochondrial membrane potential (Δφm) and mitochondrial copy number, respectively. FCCP (2000 nM) reduced ATP production by affecting mitochondrial Δφm, decreased the mRNA expression of Bmp15 (bone morphogenetic protein 15), and shortened the poly(A) tails of Bmp15, Gdf9 (growth differentiation factor 9), and Cyclin B1 transcripts. FCCP (200 and 2000 nM) also affected p34cdc2 kinase activity. By contrast, ddC did not alter ATP production. Instead, ddC significantly decreased mtDNA copy number (P < 0.05). FCCP (200 and 2000 nM) also decreased extrusion of the first polar body, whereas ddC at all concentrations did not affect the ability of immature oocytes to reach metaphase II. Both FCCP (200 and 2000 nM) and ddC (200 and 2000 µM) reduced parthenogenetic blastocyst formation compared with untreated oocytes. However, these inhibitors did not affect total cell number and apoptosis. These findings suggest that mitochondrial metabolic activity is critical for oocyte maturation and that both mitochondrial metabolic activity and replication contribute to the developmental competence of porcine oocytes. PMID:24492657

  9. DNA copy number aberrations associated with lymphovascular invasion in upper urinary tract urothelial carcinoma.

    PubMed

    Misumi, Taku; Yamamoto, Yoshiaki; Miyachika, Yoshihiro; Eguchi, Satoshi; Chochi, Yasuyo; Nakao, Motonao; Nagao, Kazuhiro; Hara, Takahiko; Sakano, Shigeru; Furuya, Tomoko; Oga, Atsunori; Kawauchi, Shigeto; Sasaki, Kohsuke; Matsuyama, Hideyasu

    2012-06-01

    Recent studies have reported that lymphovascular invasion (LVI) is a predictor of patient prognosis in upper urinary tract urothelial carcinoma (UUTUC). DNA copy number aberrations (DCNAs) identified by array-based comparative genomic hybridization (aCGH) had not previously been examined in UUTUC. We therefore examined DCNAs in UUTUC and compared them with DCNAs in LVI. We applied aCGH technology using DNA chips spotted with 4,030 BAC clones to 32 UUTUC patients. Frequent copy number gains were detected on chromosomal regions 8p23.1 and 20q13.12, whereas frequent copy number losses were detected on chromosomal regions 13q21.1, 17p13.1, 6q16.3, and 17p11.2. DCNAs occurred more frequently in tumors with LVI than in those without it (P = 0.0002), and this parameter was more closely associated with LVI than with the tumor grade or pT stage. Disease-specific survival rate was higher in tumors without LVI than in those with it (P = 0.0120); however, tumor grade and stage were not significant prognostic factors of patient outcome. These data support our hypothesis that tumors with LVI have more genetic alterations in terms of total numbers of DCNAs than those without, and provide proof that aggressive adjuvant therapy should be considered for UUTUC patients with LVI.

  10. Detection of Clinically Relevant Exonic Copy-Number Changes by Array CGH

    PubMed Central

    Boone, Philip M.; Bacino, Carlos A.; Shaw, Chad A.; Eng, Patricia A.; Hixson, Patricia M.; Pursley, Amber N.; Kang, Sung-Hae L.; Yang, Yaping; Wiszniewska, Joanna; Nowakowska, Beata A.; Gaudio, Daniela del; Xia, Zhilian; Simpson-Patel, Gayle; Immken, LaDonna L.; Gibson, James B.; Tsai, Anne C.-H.; Bowers, Jennifer A.; Reimschisel, Tyler E.; Schaaf, Christian P.; Potocki, Lorraine; Scaglia, Fernando; Gambin, Tomasz; Sykulski, Maciej; Bartnik, Magdalena; Derwinska, Katarzyna; Wisniowiecka-Kowalnik, Barbara; Lalani, Seema R.; Probst, Frank J.; Bi, Weimin; Beaudet, Arthur L.; Patel, Ankita; Lupski, James R.; Cheung, Sau Wai; Stankiewicz, Pawel

    2011-01-01

    Array comparative genomic hybridization (aCGH) is a powerful tool for the molecular elucidation and diagnosis of disorders resulting from genomic copy-number variation (CNV). However, intragenic deletions or duplications—those including genomic intervals of a size smaller than a gene—have remained beyond the detection limit of most clinical aCGH analyses. Increasing array probe number improves genomic resolution, although higher cost may limit implementation, and enhanced detection of benign CNV can confound clinical interpretation. We designed an array with exonic coverage of selected disease and candidate genes and used it clinically to identify losses or gains throughout the genome involving at least one exon and as small as several hundred base pairs in size. In some patients, the detected copy-number change occurs within a gene known to be causative of the observed clinical phenotype, demonstrating the ability of this array to detect clinically relevant CNVs with subkilobase resolution. In summary, we demonstrate the utility of a custom-designed, exon-targeted oligonucleotide array to detect intragenic copy-number changes in patients with various clinical phenotypes. PMID:20848651

  11. MSRV pol sequence copy number as a potential marker of multiple sclerosis.

    PubMed

    Zawada, Mariola; Liwień, Izabela; Pernak, Monika; Januszkiewicz-Lewandowska, Danuta; Nowicka-Kujawska, Karina; Rembowska, Jolanta; Lewandowski, Krzysztof; Hertmanowska, Hanna; Wender, Mieczysław; Nowak, Jerzy

    2003-01-01

    Multiple sclerosis (MS) is a neurological disease in which demyelination in the brain and spinal cord is observed. The causal influence of bacterial/viral infections and genetic/immune factors in the etiology of multiple sclerosis is suggested. Multiple sclerosis-related retrovirus (MSRV) is one of the potential agents, which can lead to development of the disease. The aim of cytogenetic studies was assessment of MSRV pol sequence copy number in patients with MS compared to normal individuals. Cytogenetic slides with interphase nuclei and extended chromatin fibers were prepared from peripheral blood of 16 patients with MS and 10 healthy individuals. Fluorescence in situ hybridization (FISH) with biotinylated product of polymerase chain reaction was used in order to analyze MSRV pol sequence copy number in the examined material. Detection of MSRV pol probe was carried out by immunological reaction with avidin-fluorescein and biotinylated anti-avidin. MSRV pol sequence copy number was significantly greater in MS patients than in normal individuals. Using FISH technique to extended chromatin fibers, it was observed that MSRV pol exists as tandem repeats on various chromosomes. The increased number of MSRV pol sequence has been found on chromatin fibers of MS patients as compared to healthy controls.

  12. Copy number analysis identifies novel interactions between genomic loci in ovarian cancer.

    PubMed

    Gorringe, Kylie L; George, Joshy; Anglesio, Michael S; Ramakrishna, Manasa; Etemadmoghadam, Dariush; Cowin, Prue; Sridhar, Anita; Williams, Louise H; Boyle, Samantha E; Yanaihara, Nozomu; Okamoto, Aikou; Urashima, Mitsuyoshi; Smyth, Gordon K; Campbell, Ian G; Bowtell, David D L

    2010-09-10

    Ovarian cancer is a heterogeneous disease displaying complex genomic alterations, and consequently, it has been difficult to determine the most relevant copy number alterations with the scale of studies to date. We obtained genome-wide copy number alteration (CNA) data from four different SNP array platforms, with a final data set of 398 ovarian tumours, mostly of the serous histological subtype. Frequent CNA aberrations targeted many thousands of genes. However, high-level amplicons and homozygous deletions enabled filtering of this list to the most relevant. The large data set enabled refinement of minimal regions and identification of rare amplicons such as at 1p34 and 20q11. We performed a novel co-occurrence analysis to assess cooperation and exclusivity of CNAs and analysed their relationship to patient outcome. Positive associations were identified between gains on 19 and 20q, gain of 20q and loss of X, and between several regions of loss, particularly 17q. We found weak correlations of CNA at genomic loci such as 19q12 with clinical outcome. We also assessed genomic instability measures and found a correlation of the number of higher amplitude gains with poorer overall survival. By assembling the largest collection of ovarian copy number data to date, we have been able to identify the most frequent aberrations and their interactions.

  13. Copy Number Analysis Identifies Novel Interactions Between Genomic Loci in Ovarian Cancer

    PubMed Central

    Anglesio, Michael S.; Ramakrishna, Manasa; Etemadmoghadam, Dariush; Cowin, Prue; Sridhar, Anita; Williams, Louise H.; Boyle, Samantha E.; Yanaihara, Nozomu; Okamoto, Aikou; Urashima, Mitsuyoshi; Smyth, Gordon K.; Campbell, Ian G.; Bowtell, David D. L.

    2010-01-01

    Ovarian cancer is a heterogeneous disease displaying complex genomic alterations, and consequently, it has been difficult to determine the most relevant copy number alterations with the scale of studies to date. We obtained genome-wide copy number alteration (CNA) data from four different SNP array platforms, with a final data set of 398 ovarian tumours, mostly of the serous histological subtype. Frequent CNA aberrations targeted many thousands of genes. However, high-level amplicons and homozygous deletions enabled filtering of this list to the most relevant. The large data set enabled refinement of minimal regions and identification of rare amplicons such as at 1p34 and 20q11. We performed a novel co-occurrence analysis to assess cooperation and exclusivity of CNAs and analysed their relationship to patient outcome. Positive associations were identified between gains on 19 and 20q, gain of 20q and loss of X, and between several regions of loss, particularly 17q. We found weak correlations of CNA at genomic loci such as 19q12 with clinical outcome. We also assessed genomic instability measures and found a correlation of the number of higher amplitude gains with poorer overall survival. By assembling the largest collection of ovarian copy number data to date, we have been able to identify the most frequent aberrations and their interactions. PMID:20844748

  14. Absolute calibration of photon-number-resolving detectors with an analog output using twin beams

    SciTech Connect

    Peřina, Jan; Haderka, Ondřej; Allevi, Alessia; Bondani, Maria

    2014-01-27

    A method for absolute calibration of a photon-number resolving detector producing analog signals as the output is developed using a twin beam. The method gives both analog-to-digital conversion parameters and quantum detection efficiency for the photon fields. Characteristics of the used twin beam are also obtained. A simplified variant of the method applicable to fields with high signal to noise ratios and suitable for more intense twin beams is suggested.

  15. Whole-exome sequencing of muscle-invasive bladder cancer identifies recurrent copy number variation in IPO11 and prognostic significance of importin-11 overexpression on poor survival

    PubMed Central

    He, Minghui; Zhang, Zhensheng; Zeng, Shuxiong; Ma, Chong; Sun, Yinghao; Xu, Chuanliang

    2016-01-01

    Non-muscle-invasive bladder cancer (NMIBC) often has a worse prognosis following its progression to muscle-invasive bladder cancer (MIBC), despite radical cystectomy with pelvic lymph node dissection combined with chemotherapy. Therefore, the discovery of novel biomarkers for predicting the progression of this disease and of therapeutic targets for preventing it is crucial. We performed whole-exome sequencing to analyze superficial tumor tissues (Tsup) and basal tumor tissues (Tbas) from 3 MIBC patients and identified previously unreported copy number variations in IPO11 that warrants further investigation as a molecular target. In addition, we identified a significant association between the absolute copy number and mRNA expression of IPO11 and found that high importin-11 expression was correlated with poor 3-year overall survival (OS), cancer-specific survival (CSS) and cancer-free survival (CFS) compared with low expression in the BCa patients. Importin-11 overexpression was also an independent risk factor for CSS and CFS in the BCa patients. Our study has revealed that IPO11 copy number amplification contributes to its overexpression and that these changes are unfavorable prognostic factors in NMIBC. Thus, IPO11 copy number amplification and importin-11 overexpression are promising biomarkers for predicting the progression and poor prognosis of patients with NMIBC. PMID:27689332

  16. An initiator protein for plasmid R6K DNA replication. Mutations affecting the copy-number control.

    PubMed

    Inuzuka, M; Wada, Y

    1988-02-08

    Two kinds of mutations affecting the copy-number control of plasmid R6K were isolated and identified in an initiator pi protein by DNA sequencing. Firstly, a temperature-sensitive replication mutation, ts22, with decreased copy number results in a substitution of threonine to isoleucine at position 138 of the 305-amino-acid pi protein. Secondly, a high-copy-number (cop21) mutant was isolated from this ts mutant and was identified by an alteration of alanine to serine at position 162. This cop21 mutation suppressed the Ts character and was recessive to the wild-type allele in the copy control.

  17. CoNCoS: copy number estimation in cancer with controlled support.

    PubMed

    Abdallah, Ali T; Fischer, Matthias; Nürnberg, Peter; Nothnagel, Michael; Frommolt, Peter

    2015-10-01

    Somatic copy number (CN) alterations are major drivers of tumorigenesis and growth. Although next-generation sequencing (NGS) technologies enable a deep genomic analysis of cancers, the analysis of the data remains subject to biases and multiple sources of error, including varying local read coverage. The currently existing algorithms for NGS-based detection of CN abberations do not incorporate information on the local coverage quality. We have developed a new algorithm, copy number estimation with controlled support (CoNCoS) that increases the accuracy of CN estimation in paired tumor/normal exome sequencing data sets by assessing and optimizing the support for a site-specific CN estimate. We show by simulations and in a benchmarking study against single nucleotide polymorphism (SNP) microarray data that our approach outperforms the commonly used methods CNAnorm and VarScan2. Our algorithm is suitable to increase the accuracy of somatic CN analysis by a support-optimized estimation approach.

  18. Correcting Transcription Factor Gene Sets for Copy Number and Promoter Methylation Variations

    PubMed Central

    Rathi, Komal S.; Gaykalova, Daria A.; Hennesey, Patrick; Califano, Joseph A.; Ochs, Michael F.

    2014-01-01

    Gene set analysis provides a method to generate statistical inferences across sets of linked genes, primarily using high-throughput expression data. Common gene sets include biological pathways, operons, and targets of transcriptional regulators. In higher eukaryotes, especially when dealing with diseases with strong genetic and epigenetic components such as cancer, copy number loss and gene silencing through promoter methylation can eliminate the possibility that a gene is transcribed. This, in turn, can adversely affect the estimation of transcription factor or pathway activity from a set of target genes, since some of the targets may not be responsive to transcriptional regulation. Here we introduce a simple filtering approach that removes genes from consideration if they show copy number loss or promoter methylation and demonstrate the improvement in inference of transcription factor activity in a simulated data set based on the background expression observed in normal head and neck tissue. PMID:25195578

  19. Correcting transcription factor gene sets for copy number and promoter methylation variations.

    PubMed

    Rathi, Komal S; Gaykalova, Daria A; Hennessey, Patrick; Califano, Joseph A; Ochs, Michael F

    2014-09-01

    Gene set analysis provides a method to generate statistical inferences across sets of linked genes, primarily using high-throughput expression data. Common gene sets include biological pathways, operons, and targets of transcriptional regulators. In higher eukaryotes, especially when dealing with diseases with strong genetic and epigenetic components such as cancer, copy number loss and gene silencing through promoter methylation can eliminate the possibility that a gene is transcribed. This, in turn, can adversely affect the estimation of transcription factor or pathway activity from a set of target genes, as some of the targets may not be responsive to transcriptional regulation. Here we introduce a simple filtering approach that removes genes from consideration if they show copy number loss or promoter methylation, and demonstrate the improvement in inference of transcription factor activity in a simulated dataset based on the background expression observed in normal head and neck tissue.

  20. Copy-number variation and false positive prenatal aneuploidy screening results.

    PubMed

    Snyder, Matthew W; Simmons, LaVone E; Kitzman, Jacob O; Coe, Bradley P; Henson, Jessica M; Daza, Riza M; Eichler, Evan E; Shendure, Jay; Gammill, Hilary S

    2015-04-23

    Investigations of noninvasive prenatal screening for aneuploidy by analysis of circulating cell-free DNA (cfDNA) have shown high sensitivity and specificity in both high-risk and low-risk cohorts. However, the overall low incidence of aneuploidy limits the positive predictive value of these tests. Currently, the causes of false positive results are poorly understood. We investigated four pregnancies with discordant prenatal test results and found in two cases that maternal duplications on chromosome 18 were the likely cause of the discordant results. Modeling based on population-level copy-number variation supports the possibility that some false positive results of noninvasive prenatal screening may be attributable to large maternal copy-number variants. (Funded by the National Institutes of Health and others.).

  1. Copy-Number Variation and False Positive Prenatal Aneuploidy Screening Results

    PubMed Central

    Snyder, Matthew W.; Simmons, LaVone E.; Kitzman, Jacob O.; Coe, Bradley P.; Henson, Jessica M.; Daza, Riza M.; Eichler, Evan E.; Shendure, Jay; Gammill, Hilary S.

    2015-01-01

    SUMMARY Investigations of noninvasive prenatal screening for aneuploidy by analysis of circulating cell-free DNA (cfDNA) have shown high sensitivity and specificity in both high-risk and low-risk cohorts. However, the overall low incidence of aneuploidy limits the positive predictive value of these tests. Currently, the causes of false positive results are poorly understood. We investigated four pregnancies with discordant prenatal test results and found in two cases that maternal duplications on chromosome 18 were the likely cause of the discordant results. Modeling based on population-level copy-number variation supports the possibility that some false positive results of noninvasive prenatal screening may be attributable to large maternal copy-number variants. (Funded by the National Institutes of Health and others.) PMID:25830323

  2. Fast Bayesian Inference of Copy Number Variants using Hidden Markov Models with Wavelet Compression

    PubMed Central

    Wiedenhoeft, John; Brugel, Eric; Schliep, Alexander

    2016-01-01

    By integrating Haar wavelets with Hidden Markov Models, we achieve drastically reduced running times for Bayesian inference using Forward-Backward Gibbs sampling. We show that this improves detection of genomic copy number variants (CNV) in array CGH experiments compared to the state-of-the-art, including standard Gibbs sampling. The method concentrates computational effort on chromosomal segments which are difficult to call, by dynamically and adaptively recomputing consecutive blocks of observations likely to share a copy number. This makes routine diagnostic use and re-analysis of legacy data collections feasible; to this end, we also propose an effective automatic prior. An open source software implementation of our method is available at http://schlieplab.org/Software/HaMMLET/ (DOI: 10.5281/zenodo.46262). This paper was selected for oral presentation at RECOMB 2016, and an abstract is published in the conference proceedings. PMID:27177143

  3. Copy Number Variants Associated with 14 Cases of Self-Injurious Behavior.

    PubMed

    Shirley, Matthew D; Frelin, Laurence; López, José Soria; Jedlicka, Anne; Dziedzic, Amanda; Frank-Crawford, Michelle A; Silverman, Wayne; Hagopian, Louis; Pevsner, Jonathan

    2016-01-01

    Copy number variants (CNVs) were detected and analyzed in 14 probands with autism and intellectual disability with self-injurious behavior (SIB) resulting in tissue damage. For each proband we obtained a clinical history and detailed behavioral descriptions. Genetic anomalies were observed in all probands, and likely clinical significance could be established in four cases. This included two cases having novel, de novo copy number variants and two cases having variants likely to have functional significance. These cases included segmental trisomy 14, segmental monosomy 21, and variants predicted to disrupt the function of ZEB2 (encoding a transcription factor) and HTR2C (encoding a serotonin receptor). Our results identify variants in regions previously implicated in intellectual disability and suggest candidate genes that could contribute to the etiology of SIB.

  4. ascatNgs: Identifying Somatically Acquired Copy-Number Alterations from Whole-Genome Sequencing Data.

    PubMed

    Raine, Keiran M; Van Loo, Peter; Wedge, David C; Jones, David; Menzies, Andrew; Butler, Adam P; Teague, Jon W; Tarpey, Patrick; Nik-Zainal, Serena; Campbell, Peter J

    2016-12-08

    We have developed ascatNgs to aid researchers in carrying out Allele-Specific Copy number Analysis of Tumours (ASCAT). ASCAT is capable of detecting DNA copy number changes affecting a tumor genome when comparing to a matched normal sample. Additionally, the algorithm estimates the amount of tumor DNA in the sample, known as Aberrant Cell Fraction (ACF). ASCAT itself is an R-package which requires the generation of many file types. Here, we present a suite of tools to help handle this for the user. Our code is available on our GitHub site (https://github.com/cancerit). This unit describes both 'one-shot' execution and approaches more suitable for large-scale compute farms. © 2016 by John Wiley & Sons, Inc.

  5. Focal chromosomal copy number aberrations in cancer-Needles in a genome haystack.

    PubMed

    Krijgsman, Oscar; Carvalho, Beatriz; Meijer, Gerrit A; Steenbergen, Renske D M; Ylstra, Bauke

    2014-11-01

    The extent of focal chromosomal copy number aberrations (CNAs) in cancer has been uncovered through technical innovations, and this discovery has been critical for the identification of new cancer driver genes in genomics projects such as TCGA and ICGC. Unlike constitutive copy number variations (CNVs), focal CNAs are the result of many selection events during the evolution of cancer genomes. Therefore, it is possible that a single gene in a focal CNA gives the tumor a selective growth advantage. This concept has been instrumental in the discovery of new cancer driver genes. However, focal CNAs lack a consensus definition; therefore, we propose one based on pragmatic considerations. We also describe different strategies to identify focal CNAs and procedures to distinguish them from large CNAs and CNVs.

  6. Copy Number Variants Associated with 14 Cases of Self-Injurious Behavior

    PubMed Central

    Shirley, Matthew D.; Frelin, Laurence; López, José Soria; Jedlicka, Anne; Dziedzic, Amanda; Frank-Crawford, Michelle A.; Silverman, Wayne; Hagopian, Louis; Pevsner, Jonathan

    2016-01-01

    Copy number variants (CNVs) were detected and analyzed in 14 probands with autism and intellectual disability with self-injurious behavior (SIB) resulting in tissue damage. For each proband we obtained a clinical history and detailed behavioral descriptions. Genetic anomalies were observed in all probands, and likely clinical significance could be established in four cases. This included two cases having novel, de novo copy number variants and two cases having variants likely to have functional significance. These cases included segmental trisomy 14, segmental monosomy 21, and variants predicted to disrupt the function of ZEB2 (encoding a transcription factor) and HTR2C (encoding a serotonin receptor). Our results identify variants in regions previously implicated in intellectual disability and suggest candidate genes that could contribute to the etiology of SIB. PMID:26933844

  7. A Multi-Megabase Copy Number Gain Causes Maternal Transmission Ratio Distortion on Mouse Chromosome 2

    PubMed Central

    Didion, John P.; Morgan, Andrew P.; Clayshulte, Amelia M.-F.; Mcmullan, Rachel C.; Yadgary, Liran; Petkov, Petko M.; Bell, Timothy A.; Gatti, Daniel M.; Crowley, James J.; Hua, Kunjie; Aylor, David L.; Bai, Ling; Calaway, Mark; Chesler, Elissa J.; French, John E.; Geiger, Thomas R.; Gooch, Terry J.; Garland, Theodore; Harrill, Alison H.; Hunter, Kent; McMillan, Leonard; Holt, Matt; Miller, Darla R.; O'Brien, Deborah A.; Paigen, Kenneth; Pan, Wenqi; Rowe, Lucy B.; Shaw, Ginger D.; Simecek, Petr; Sullivan, Patrick F.; Svenson, Karen L; Weinstock, George M.; Threadgill, David W.; Pomp, Daniel; Churchill, Gary A.; Pardo-Manuel de Villena, Fernando

    2015-01-01

    Significant departures from expected Mendelian inheritance ratios (transmission ratio distortion, TRD) are frequently observed in both experimental crosses and natural populations. TRD on mouse Chromosome (Chr) 2 has been reported in multiple experimental crosses, including the Collaborative Cross (CC). Among the eight CC founder inbred strains, we found that Chr 2 TRD was exclusive to females that were heterozygous for the WSB/EiJ allele within a 9.3 Mb region (Chr 2 76.9 – 86.2 Mb). A copy number gain of a 127 kb-long DNA segment (designated as responder to drive, R2d) emerged as the strongest candidate for the causative allele. We mapped R2d sequences to two loci within the candidate interval. R2d1 is located near the proximal boundary, and contains a single copy of R2d in all strains tested. R2d2 maps to a 900 kb interval, and the number of R2d copies varies from zero in classical strains (including the mouse reference genome) to more than 30 in wild-derived strains. Using real-time PCR assays for the copy number, we identified a mutation (R2d2WSBdel1) that eliminates the majority of the R2d2WSB copies without apparent alterations of the surrounding WSB/EiJ haplotype. In a three-generation pedigree segregating for R2d2WSBdel1, the mutation is transmitted to the progeny and Mendelian segregation is restored in females heterozygous for R2d2WSBdel1, thus providing direct evidence that the copy number gain is causal for maternal TRD. We found that transmission ratios in R2d2WSB heterozygous females vary between Mendelian segregation and complete distortion depending on the genetic background, and that TRD is under genetic control of unlinked distorter loci. Although the R2d2WSB transmission ratio was inversely correlated with average litter size, several independent lines of evidence support the contention that female meiotic drive is the cause of the distortion. We discuss the implications and potential applications of this novel meiotic drive system. PMID

  8. Copy number expansion of the STX17 duplication in melanoma tissue from Grey horses

    PubMed Central

    2012-01-01

    Background Greying with age in horses is an autosomal dominant trait, associated with loss of hair pigmentation, melanoma and vitiligo-like depigmentation. We recently identified a 4.6 kb duplication in STX17 to be associated with the phenotype. The aims of this study were to investigate if the duplication in Grey horses shows copy number variation and to exclude that any other polymorphism is uniquely associated with the Grey mutation. Results We found little evidence for copy number expansion of the duplicated sequence in blood DNA from Grey horses. In contrast, clear evidence for copy number expansions was indicated in five out of eight tested melanoma tissues or melanoma cell lines. A tendency of a higher copy number in aggressive tumours was also found. Massively parallel resequencing of the ~350 kb Grey haplotype did not reveal any additional mutations perfectly associated with the phenotype, confirming the duplication as the true causative mutation. We identified three SNP alleles that were present in a subset of Grey haplotypes within the 350 kb region that shows complete linkage disequilibrium with the causative mutation. Thus, these three nucleotide substitutions must have occurred subsequent to the duplication, consistent with our interpretation that the Grey mutation arose more than 2,000 years before present. Conclusions These results suggest that the mutation acts as a melanoma-driving regulatory element. The elucidation of the mechanistic features of the duplication will be of considerable interest for the characterization of these horse melanomas as well as for the field of human melanoma research. PMID:22857264

  9. Alterations of Mitochondrial DNA Copy Number and Telomere Length with Early Adversity and Psychopathology

    PubMed Central

    Tyrka, Audrey R.; Parade, Stephanie H.; Price, Lawrence H.; Kao, Hung-Teh; Porton, Barbara; Philip, Noah S.; Welch, Emma S.; Carpenter, Linda L.

    2015-01-01

    Background Telomere shortening and alterations of mitochondrial biogenesis are involved in cellular aging. Childhood adversity is associated with telomere shortening, and several investigations have shown short telomeres in psychiatric disorders. Recent studies have examined whether mitochondria might be involved in neuropsychiatric conditions; findings are limited and no prior work has examined this in relation to stress exposure. Methods Two-hundred and ninety healthy adults provided information on childhood parental loss and maltreatment and completed diagnostic interviews. Participants were categorized into four groups based upon the presence or absence of childhood adversity and the presence or absence of lifetime psychopathology (depressive, anxiety, and substance use disorders). Telomere length and mtDNA copy number were measured from leukocyte DNA by qPCR. Results Childhood adversity and lifetime psychopathology were each associated with shorter telomeres (p < .01) and higher mtDNA copy numbers (p < .001). Significantly higher mtDNA copy numbers and shorter telomeres were seen in individuals with major depression, depressive disorders, and anxiety disorders, as well as those with parental loss and childhood maltreatment. A history of substance disorders was also associated with significantly higher mtDNA copy numbers. Conclusion This study provides the first evidence of an alteration of mitochondrial biogenesis with early life stress and with anxiety and substance use disorders. We replicate prior work on telomere length and psychopathology, and show that this effect is not secondary to medication use or comorbid medical illness. Finally, we show that early life stress and psychopathology are each associated with these markers of cellular aging. PMID:25749099

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

    DTIC Science & Technology

    2015-08-01

    and select 330 samples for CNV analysis. Months :1 - 2 • Subtask 2 Prepare sections (10 μm) for microdissection to ensure>80% tumor. Months 3 - 8...Subtask 3 DNA preparation from microdissected specimens. Months 3 - 8 Major Task 2: To determine the copy number gain and loss for early stage high...to prepare chip compatible samples. Months : 9 - 18 • Subtask 2 Genomic abnormality analysis by IlluminaHumanOmniExpress-FFPE BeadChip system. Months

  11. Genome Wide Distributions and Functional Characterization of Copy Number Variations between Chinese and Western Pigs

    PubMed Central

    Wang, Hongyang; Wang, Chao; Yang, Kui; Liu, Jing; Zhang, Yu; Wang, Yanan; Xu, Xuewen; Michal, Jennifer J.; Jiang, Zhihua; Liu, Bang

    2015-01-01

    Copy number variations (CNVs) refer to large insertions, deletions and duplications in the genomic structure ranging from one thousand to several million bases in size. Since the development of next generation sequencing technology, several methods have been well built for detection of copy number variations with high credibility and accuracy. Evidence has shown that CNV occurring in gene region could lead to phenotypic changes due to the alteration in gene structure and dosage. However, it still remains unexplored whether CNVs underlie the phenotypic differences between Chinese and Western domestic pigs. Based on the read-depth methods, we investigated copy number variations using 49 individuals derived from both Chinese and Western pig breeds. A total of 3,131 copy number variation regions (CNVRs) were identified with an average size of 13.4 Kb in all individuals during domestication, harboring 1,363 genes. Among them, 129 and 147 CNVRs were Chinese and Western pig specific, respectively. Gene functional enrichments revealed that these CNVRs contribute to strong disease resistance and high prolificacy in Chinese domestic pigs, but strong muscle tissue development in Western domestic pigs. This finding is strongly consistent with the morphologic characteristics of Chinese and Western pigs, indicating that these group-specific CNVRs might have been preserved by artificial selection for the favored phenotypes during independent domestication of Chinese and Western pigs. In this study, we built high-resolution CNV maps in several domestic pig breeds and discovered the group specific CNVs by comparing Chinese and Western pigs, which could provide new insight into genomic variations during pigs’ independent domestication, and facilitate further functional studies of CNV-associated genes. PMID:26154170

  12. Incorporating 16S gene copy number information improves estimates of microbial diversity and abundance.

    PubMed

    Kembel, Steven W; Wu, Martin; Eisen, Jonathan A; Green, Jessica L

    2012-01-01

    The abundance of different SSU rRNA ("16S") gene sequences in environmental samples is widely used in studies of microbial ecology as a measure of microbial community structure and diversity. However, the genomic copy number of the 16S gene varies greatly - from one in many species to up to 15 in some bacteria and to hundreds in some microbial eukaryotes. As a result of this variation the relative abundance of 16S genes in environmental samples can be attributed both to variation in the relative abundance of different organisms, and to variation in genomic 16S copy number among those organisms. Despite this fact, many studies assume that the abundance of 16S gene sequences is a surrogate measure of the relative abundance of the organisms containing those sequences. Here we present a method that uses data on sequences and genomic copy number of 16S genes along with phylogenetic placement and ancestral state estimation to estimate organismal abundances from environmental DNA sequence data. We use theory and simulations to demonstrate that 16S genomic copy number can be accurately estimated from the short reads typically obtained from high-throughput environmental sequencing of the 16S gene, and that organismal abundances in microbial communities are more strongly correlated with estimated abundances obtained from our method than with gene abundances. We re-analyze several published empirical data sets and demonstrate that the use of gene abundance versus estimated organismal abundance can lead to different inferences about community diversity and structure and the identity of the dominant taxa in microbial communities. Our approach will allow microbial ecologists to make more accurate inferences about microbial diversity and abundance based on 16S sequence data.

  13. 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

  14. Genome-Wide Association of Copy Number Polymorphisms and Kidney Function

    PubMed Central

    Li, Man; Carey, Jacob; Cristiano, Stephen; Susztak, Katalin; Coresh, Josef; Boerwinkle, Eric; Beaty, Terri H.; Köttgen, Anna; Scharpf, Robert B.

    2017-01-01

    Genome-wide association studies (GWAS) using single nucleotide polymorphisms (SNPs) have identified more than 50 loci associated with estimated glomerular filtration rate (eGFR), a measure of kidney function. However, significant SNPs account for a small proportion of eGFR variability. Other forms of genetic variation have not been comprehensively evaluated for association with eGFR. In this study, we assess whether changes in germline DNA copy number are associated with GFR estimated from serum creatinine, eGFRcrea. We used hidden Markov models (HMMs) to identify copy number polymorphic regions (CNPs) from high-throughput SNP arrays for 2,514 African (AA) and 8,645 European ancestry (EA) participants in the Atherosclerosis Risk in Communities (ARIC) study. Separately for the EA and AA cohorts, we used Bayesian Gaussian mixture models to estimate copy number at regions identified by the HMM or previously reported in the HapMap Project. We identified 312 and 464 autosomal CNPs among individuals of EA and AA, respectively. Multivariate models adjusted for SNP-derived covariates of population structure identified one CNP in the EA cohort near genome-wide statistical significance (Bonferroni-adjusted p = 0.067) located on chromosome 5 (876–880kb). Overall, our findings suggest a limited role of CNPs in explaining eGFR variability. PMID:28135296

  15. c-myc copy number gain is a powerful prognosticator of disease outcome in cervical dysplasia.

    PubMed

    Kübler, Kirsten; Heinenberg, Sally; Rudlowski, Christian; Keyver-Paik, Mignon-Denise; Abramian, Alina; Merkelbach-Bruse, Sabine; Büttner, Reinhard; Kuhn, Walther; Schildhaus, Hans-Ulrich

    2015-01-20

    Cervical carcinoma develops from preneoplasia by a multistep process. Although most low-grade dysplastic lesions will regress without intervention and even high-grade changes exhibit a substantial rate of regression, a small percentage of dysplasia will progress over time. Thus, indicators are needed to estimate the biological risk and to help avoid overtreatment in women who desire to preserve fertility. In addition to the classical biomarkers, PCR-ELISA-determined HPV genotype and immunohistochemically assessed p16INK4a and Ki-67 expression, cells with integrated HPV and copy number gain of TERC and c-myc were quantified in a panel of 104 benign, intraepithelial neoplastic (CIN I, II, III) and cancerous lesions using fluorescence in situ hybridization. Optimal cut-off values were calculated; Kaplan-Meier curves and a Cox proportional hazard regression model were used to evaluate prognostic signatures. The assay reliably identified HPV integration, TERC and c-myc copy number gain as determined by comparisons with established biomarkers. All biomarker levels increased with the progression of the disease. However, only c-myc copy number gain independently prognosticated a low probability of dysplastic regression. Our results suggest that c-myc plays a key role in the process of dysplastic transformation and might thus be exploited for treatment and follow-up decision-making of cervical dysplasia.

  16. Clinical relevance of copy number profiling in oral and oropharyngeal squamous cell carcinoma.

    PubMed

    van Kempen, Pauline M W; Noorlag, Rob; Braunius, Weibel W; Moelans, Cathy B; Rifi, Widad; Savola, Suvi; Koole, Ronald; Grolman, Wilko; van Es, Robert J J; Willems, Stefan M

    2015-10-01

    Current conventional treatment modalities in head and neck squamous cell carcinoma (HNSCC) are nonselective and have shown to cause serious side effects. Unraveling the molecular profiles of head and neck cancer may enable promising clinical applications that pave the road for personalized cancer treatment. We examined copy number status in 36 common oncogenes and tumor suppressor genes in a cohort of 191 oropharyngeal squamous cell carcinomas (OPSCC) and 164 oral cavity squamous cell carcinomas (OSCC) using multiplex ligation probe amplification. Copy number status was correlated with human papillomavirus (HPV) status in OPSCC, with occult lymph node status in OSCC and with patient survival. The 11q13 region showed gain or amplifications in 59% of HPV-negative OPSCC, whereas this amplification was almost absent in HPV-positive OPSCC. Additionally, in clinically lymph node-negative OSCC (Stage I-II), gain of the 11q13 region was significantly correlated with occult lymph node metastases with a negative predictive value of 81%. Multivariate survival analysis revealed a significantly decreased disease-free survival in both HPV-negative and HPV-positive OPSCC with a gain of Wnt-induced secreted protein-1. Gain of CCND1 showed to be an independent predictor for worse survival in OSCC. These results show that copy number aberrations, mainly of the 11q13 region, may be important predictors and prognosticators which allow for stratifying patients for personalized treatment of HNSCC.

  17. Rare Copy Number Variants Identified Suggest the Regulating Pathways in Hypertension-Related Left Ventricular Hypertrophy

    PubMed Central

    Marshall, Christian R.; Majid, Fadhlina; Danuri, Norlaila; Basir, Fashieha; Thiruvahindrapuram, Bhooma; Scherer, Stephen W.; Yusoff, Khalid

    2016-01-01

    Left ventricular hypertrophy (LVH) is an independent risk factor for cardiovascular morbidity and mortality, and a powerful predictor of adverse cardiovascular outcomes in the hypertensive patients. It has complex multifactorial and polygenic basis for its pathogenesis. We hypothesized that rare copy number variants (CNVs) contribute to the LVH pathogenesis in hypertensive patients. Copy number variants (CNV) were identified in 258 hypertensive patients, 95 of whom had LVH, after genotyping with a high resolution SNP array. Following stringent filtering criteria, we identified 208 rare, or private CNVs that were only present in our patients with hypertension related LVH. Preliminary findings from Gene Ontology and pathway analysis of this study confirmed the involvement of the genes known to be functionally involved in cardiac development and phenotypes, in line with previously reported transcriptomic studies. Network enrichment analyses suggested that the gene-set was, directly or indirectly, involved in the transcription factors regulating the “foetal cardiac gene programme” which triggered the hypertrophic cascade, confirming previous reports. These findings suggest that multiple, individually rare copy number variants altering genes may contribute to the pathogenesis of hypertension-related LVH. In summary, we have provided further supporting evidence that rare CNV could potentially impact this common and complex disease susceptibility with lower heritability. PMID:26930585

  18. A novel satellite DNA sequence in the Peromyscus genome (PMSat): Evolution via copy number fluctuation.

    PubMed

    Louzada, Sandra; Vieira-da-Silva, Ana; Mendes-da-Silva, Ana; Kubickova, Svatava; Rubes, Jiri; Adega, Filomena; Chaves, Raquel

    2015-11-01

    Satellite DNAs (satDNA) are tandemly arrayed repeated sequences largely present in eukaryotic genomes, which play important roles in genome evolution and function, and therefore, their analysis is vital. Here, we describe the isolation of a novel satellite DNA family (PMSat) from the rodent Peromyscus eremicus (Cricetidae, Rodentia), which is located in pericentromeric regions and exhibits a typical satellite DNA genome organization. Orthologous PMSat sequences were isolated and characterized from three species belonging to Cricetidae: Cricetus cricetus, Phodopus sungorus and Microtus arvalis. In these species, PMSat is highly conserved, with the absence of fixed species-specific mutations. Strikingly, different numbers of copies of this sequence were found among the species, suggesting evolution by copy number fluctuation. Repeat units of PMSat were also found in the Peromyscus maniculatus bairdii BioProject, but our results suggest that these repeat units are from genome regions outside the pericentromere. The remarkably high evolutionary sequence conservation along with the preservation of a few numbers of copies of this sequence in the analyzed genomes may suggest functional significance but a different sequence nature/organization. Our data highlight that repeats are difficult to analyze due to the limited tools available to dissect genomes and the fact that assemblies do not cover regions of constitutive heterochromatin.

  19. A Highly Polymorphic Copy Number Variant in the NSF Gene is Associated with Cocaine Dependence

    PubMed Central

    Cabana-Domínguez, Judit; Roncero, Carlos; Grau-López, Lara; Rodríguez-Cintas, Laia; Barral, Carmen; Abad, Alfonso C.; Erikson, Galina; Wineinger, Nathan E.; Torrico, Bàrbara; Arenas, Concepció; Casas, Miquel; Ribasés, Marta; Cormand, Bru; Fernàndez-Castillo, Noèlia

    2016-01-01

    Cocaine dependence is a complex psychiatric disorder involving both genetic and environmental factors. Several neurotransmitter systems mediate cocaine’s effects, dependence and relapse, being the components of the neurotransmitter release machinery good candidates for the disorder. Previously, we identified a risk haplotype for cocaine dependence in the NSF gene, encoding the protein N-Ethylmaleimide-Sensitive Factor essential for synaptic vesicle turnover. Here we examined the possible contribution to cocaine dependence of a large copy number variant (CNV) that encompasses part of the NSF gene. We performed a case-control association study in a discovery sample (359 cases and 356 controls) and identified an association between cocaine dependence and the CNV (P = 0.013), that was confirmed in the replication sample (508 cases and 569 controls, P = 7.1e-03) and in a pooled analysis (P = 1.8e-04), with an over-representation of low number of copies in cases. Subsequently, we studied the functional impact of the CNV on gene expression and found that the levels of two NSF transcripts were significantly increased in peripheral blood mononuclear cells (PBMC) along with the number of copies of the CNV. These results, together with a previous study from our group, support the role of NSF in the susceptibility to cocaine dependence. PMID:27498889

  20. Estimation of transgene copy number in transformed citrus plants by quantitative multiplex real-time PCR.

    PubMed

    Omar, Ahmad A; Dekkers, Marty G H; Graham, James H; Grosser, Jude W

    2008-01-01

    Quantitative real-time PCR (qRT-PCR) was adapted to estimate transgene copy number of the rice Xa21 gene in transgenic citrus plants. This system used TaqMan qRT-PCR and the endogenous citrus gene encoding for lipid transfer protein (LTP). Transgenic "Hamlin" sweet orange plants were generated using two different protoplast-GFP transformation systems: cotransformation and single plasmid transformation. A dilution series of genomic DNA from one of the transgenic lines was used to generate a standard curve for the endogenous LTP and the transgene Xa21. This standard curve was used for relative quantification of the endogenous gene and the transgene. Copy numbers of the transgene Xa21 detected from qRT-PCR analysis correlated with that from Southern blot analysis (r = 0.834). Thus, qRT-PCR is an efficient means of estimating copy number in transgenic citrus plants. This analysis can be performed at much earlier stages of transgenic plant development than southern blot analysis, which expedites investigation of transgenes in slow-growing woody plants.

  1. Genes and small RNA transcripts exhibit dosage-dependent expression pattern in maize copy-number alterations

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Copy-number alterations are widespread in animal and plant genomes, but their immediate impact on gene expression is still unclear. In animals, copy-number alterations usually exhibit dosage effects, except for sex chromosomes that tend to be dosage compensated. In plants, genes within small duplica...

  2. 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

  3. Bacterial expression system with tightly regulated gene expression and plasmid copy number.

    PubMed

    Bowers, Lisa M; Lapoint, Kathleen; Anthony, Larry; Pluciennik, Anna; Filutowicz, Marcin

    2004-09-29

    A new Escherichia coli host/vector system has been engineered to allow tight and uniform modulation of gene expression and gamma origin (ori) plasmid copy number. Regulation of gamma ori plasmid copy number is achieved through arabinose-inducible expression of the necessary Rep protein, pi, whose gene was integrated into the chromosome of the host strain under control of the P(BAD) promoter. gamma ori replication can be uniformly modulated over 100-fold by changing the concentration of l-arabinose in the growth medium. This strain avoids the problem of all-or-nothing induction of P(BAD) because it is deficient in both arabinose uptake and degradation genes. Arabinose enters the cell by a mutant LacY transporter, LacYA177C, which is expressed from the host chromosome. Although this strain could be compatible with any gamma ori plasmid, we describe the utility of a gamma ori expression vector that allows especially tight regulation of gene expression. With this host/vector system, it is possible to independently modulate gene expression and gene dosage, facilitating the cloning and overproduction of toxic gene products. We describe the successful use of this system for cloning a highly potent toxin, Colicin E3, in the absence of its cognate immunity protein. This system could be useful for cloning genes encoding other potent toxins, screening libraries for potential toxins, and maintaining any gamma ori vector at precise copy levels in a cell.

  4. Copy number gain at Xp22.31 includes complex duplication rearrangements and recurrent triplications.

    PubMed

    Liu, Pengfei; Erez, Ayelet; Nagamani, Sandesh C Sreenath; Bi, Weimin; Carvalho, Claudia M B; Simmons, Alexandra D; Wiszniewska, Joanna; Fang, Ping; Eng, Patricia A; Cooper, M Lance; Sutton, V Reid; Roeder, Elizabeth R; Bodensteiner, John B; Delgado, Mauricio R; Prakash, Siddharth K; Belmont, John W; Stankiewicz, Pawel; Berg, Jonathan S; Shinawi, Marwan; Patel, Ankita; Cheung, Sau Wai; Lupski, James R

    2011-05-15

    Genomic instability is a feature of the human Xp22.31 region wherein deletions are associated with X-linked ichthyosis, mental retardation and attention deficit hyperactivity disorder. A putative homologous recombination hotspot motif is enriched in low copy repeats that mediate recurrent deletion at this locus. To date, few efforts have focused on copy number gain at Xp22.31. However, clinical testing revealed a high incidence of duplication of Xp22.31 in subjects ascertained and referred with neurobehavioral phenotypes. We systematically studied 61 unrelated subjects with rearrangements revealing gain in copy number, using multiple molecular assays. We detected not only the anticipated recurrent and simple nonrecurrent duplications, but also unexpectedly identified recurrent triplications and other complex rearrangements. Breakpoint analyses enabled us to surmise the mechanisms for many of these rearrangements. The clinical significance of the recurrent duplications and triplications were assessed using different approaches. We cannot find any evidence to support pathogenicity of the Xp22.31 duplication. However, our data suggest that the Xp22.31 duplication may serve as a risk factor for abnormal phenotypes. Our findings highlight the need for more robust Xp22.31 triplication detection in that such further gain may be more penetrant than the duplications. Our findings reveal the distribution of different mechanisms for genomic duplication rearrangements at a given locus, and provide insights into aspects of strand exchange events between paralogous sequences in the human genome.

  5. Quantitation of DNA copy number in individual mitochondrial particles by capillary electrophoresis.

    PubMed

    Navratil, Marian; Poe, Bobby G; Arriaga, Edgar A

    2007-10-15

    Here, we present a direct method for determining mitochondrial DNA (mtDNA) copy numbers in individual mitochondrial particles, isolated from cultured cells, by means of capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection. We demonstrate that this method can detect a single molecule of PicoGreen-stained mtDNA in intact DsRed2-labeled mitochondrial particles isolated from human osteosarcoma 143B cells. This ultimate limit of mtDNA detection made it possible to confirm that an individual mitochondrial nucleoid, the genetic unit of mitochondrial inheritance, can contain a single copy of mtDNA. The validation of this approach was achieved via monitoring chemically induced mtDNA depletion and comparing the CE-LIF results to those obtained by quantitative microscopy imaging and multiplex real-time PCR analysis. Owing to its sensitivity, the CE-LIF method may become a powerful tool for investigating the copy number and organization of mtDNA in mitochondrial disease and aging, and in molecular biology techniques requiring manipulation and quantitation of DNA molecules such as plasmids.

  6. Copy Number Variation of Mitochondrial DNA Genes in Pneumocystis jirovecii According to the Fungal Load in BAL Specimens

    PubMed Central

    Valero, Clara; Buitrago, María José; Gits-Muselli, Maud; Benazra, Marion; Sturny-Leclère, Aude; Hamane, Samia; Guigue, Nicolas; Bretagne, Stéphane; Alanio, Alexandre

    2016-01-01

    Pneumocystis jirovecii is an unculturable fungus and the causative agent of Pneumocystis pneumonia, a life-threatening opportunistic infection. Although molecular diagnosis is often based on the detection of mtLSU rRNA mitochondrial gene, the number of copies of mitochondrial genes had not been investigated. We developed and optimized six real-time PCR assays in order to determine the copy number of four mitochondrial genes (mtSSU rRNA, mtLSU rRNA, NAD1, and CYTB) in comparison to nuclear genome (DHPS and HSP70) and tested 84 bronchoalveolar fluids of patients at different stages of the infection. Unexpectedly, we found that copy number of mitochondrial genes varied from gene to gene with mtSSU rRNA gene being more represented (37 copies) than NAD1 (23 copies), mtLSU rRNA (15 copies) and CYTB (6 copies) genes compared to nuclear genome. Hierarchical clustering analysis (HCA) allowed us to define five major clusters, significantly associated with fungal load (p = 0.029), in which copy number of mitochondrial genes was significantly different among them. More importantly, copy number of mtLSU rRNA, NAD1, and CYTB but not mtSSU rRNA differed according to P. jirovecii physiological state with a decreased number of copies when the fungal load is low. This suggests the existence of a mixture of various subspecies of mtDNA that can harbor different amplification rates. Overall, we revealed here an unexpected variability of P. jirovecii mtDNA copy number that fluctuates according to P. jirovecii’s physiological state, except for mtSSU that is the most stable and the most present mitochondrial gene. PMID:27672381

  7. Fluorescent in situ hybridization (FISH) assessment of chromosome copy number in sperm

    SciTech Connect

    Sheu, M.; Sigman, M.; Mark, H.F.L.

    1994-09-01

    Approximately 15% of all recognized pregnancies end in spontaneous abortions. The overall frequency of chromosome abnormalities in spontaneous abortions is approximately 50%. Thus aneuploidy is a significant cause of fetal wastage. In addition, structural and numerical abnormalities of chromosomes can also lead to birth defects, developmental delay, mental retardation and infertility. Conventional cytogenetic analysis via GTG- and other banding techniques is a powerful tool in the elucidation of the nature of chromosomal abnormalities. Fluorescent in situ hybridization (FISH) enables detection of numerical chromosomal abnormalities, especially trisomies, in intact cells. Using FISH and commercially available biotin-labeled probes, we have initiated a prospective study to assess specific chromosome copy number of preparations of unstained smears from men referred for a male infertility evaluation as well as smears from normal control males chosen randomly from the sample of sperm donors. A total of approximately 19,000 sperm nuclei have been examined thus far. Of those suitable for analysis, 7382 (38.75%) were normal possessing one copy of chromosome 8, 155 (0.81%) were disomic, and 15 (0.079%) had more than two copies of chromosome 8. Comparisons with data available in the literature will be discussed. Work is ongoing to increase the efficiency of hybridization using both reported and previously untried pretreatment and fixation protocols. We have also initiated studies using multicolor FISH with various chromosome enumeration probes. The assay described here is a potentially powerful tool for detecting rare events such as spontaneous germ cell aneuploidy, aneuploidy detected in semen from men with carcinoma in situ of the testis and aneuploidy induced by potential environmental genotoxicants. It can also be utilized for segregation analysis and for correlating chromosome copy number with germ cell morphology.

  8. MLPA-Based Analysis of Copy Number Variation in Plant Populations

    PubMed Central

    Samelak-Czajka, Anna; Marszalek-Zenczak, Malgorzata; Marcinkowska-Swojak, Malgorzata; Kozlowski, Piotr; Figlerowicz, Marek; Zmienko, Agnieszka

    2017-01-01

    Copy number variants (CNVs) are intraspecies duplications/deletions of large DNA segments (>1 kb). A growing number of reports highlight the functional and evolutionary impact of CNV in plants, increasing the need for appropriate tools that enable locus-specific CNV genotyping on a population scale. Multiplex ligation-dependent probe amplification (MLPA) is considered a gold standard in genotyping CNV in humans. Consequently, numerous commercial MLPA assays for CNV-related human diseases have been created. We routinely genotype complex multiallelic CNVs in human and plant genomes using the modified MLPA procedure based on fully synthesized oligonucleotide probes (90–200 nt), which greatly simplifies the design process and allows for the development of custom assays. Here, we present a step-by-step protocol for gene-specific MLPA probe design, multiplexed assay setup and data analysis in a copy number genotyping experiment in plants. As a case study, we present the results of a custom assay designed to genotype the copy number status of 12 protein coding genes in a population of 80 Arabidopsis accessions. The genes were pre-selected based on whole genome sequencing data and are localized in the genomic regions that display different levels of population-scale variation (non-variable, biallelic, or multiallelic, as well as CNVs overlapping whole genes or their fragments). The presented approach is suitable for population-scale validation of the CNV regions inferred from whole genome sequencing data analysis and for focused analysis of selected genes of interest. It can also be very easily adopted for any plant species, following optimization of the template amount and design of the appropriate control probes, according to the general guidelines presented in this paper. PMID:28270823

  9. Host Genetic Variants and Gene Expression Patterns Associated with Epstein-Barr Virus Copy Number in Lymphoblastoid Cell Lines

    PubMed Central

    Houldcroft, Charlotte J.; Petrova, Velislava; Liu, Jimmy Z.; Frampton, Dan; Anderson, Carl A.; Gall, Astrid; Kellam, Paul

    2014-01-01

    Lymphoblastoid cell lines (LCLs) are commonly used in molecular genetics, supplying DNA for the HapMap and 1000 Genomes Projects, used to test chemotherapeutic agents, and informing the basis of a number of population genetics studies of gene expression. The process of transforming human B cells into LCLs requires the presence of Epstein-Barr virus (EBV), a double-stranded DNA virus which through B-cell immortalisation maintains an episomal virus genome in every cell of an LCL at variable copy numbers. Previous studies have reported that EBV alters host-gene expression and EBV copy number may be under host genetic control. We performed a genome-wide association study of EBV genome copy number in LCLs and found the phenotype to be highly heritable, although no individual SNPs achieved a significant association with EBV copy number. The expression of two host genes (CXCL16 and AGL) was positively correlated and expression of ADARB2 was negatively correlated with EBV copy number in a genotype-independent manner. This study shows an association between EBV copy number and the gene expression profile of LCLs, and suggests that EBV copy number should be considered as a covariate in future studies of host gene expression in LCLs. PMID:25290448

  10. Post-polyploidisation morphotype diversification associates with gene copy number variation

    PubMed Central

    Schiessl, Sarah; Huettel, Bruno; Kuehn, Diana; Reinhardt, Richard; Snowdon, Rod

    2017-01-01

    Genetic models for polyploid crop adaptation provide important information relevant for future breeding prospects. A well-suited model is Brassica napus, a recent allopolyploid closely related to Arabidopsis thaliana. Flowering time is a major adaptation trait determining life cycle synchronization with the environment. Here we unravel natural genetic variation in B. napus flowering time regulators and investigate associations with evolutionary diversification into different life cycle morphotypes. Deep sequencing of 35 flowering regulators was performed in 280 diverse B. napus genotypes. High sequencing depth enabled high-quality calling of single-nucleotide polymorphisms (SNPs), insertion-deletions (InDels) and copy number variants (CNVs). By combining these data with genotyping data from the Brassica 60 K Illumina® Infinium SNP array, we performed a genome-wide marker distribution analysis across the 4 ecogeographical morphotypes. Twelve haplotypes, including Bna.FLC.A10, Bna.VIN3.A02 and the Bna.FT promoter on C02_random, were diagnostic for the diversification of winter and spring types. The subspecies split between oilseed/kale (B. napus ssp. napus) and swedes/rutabagas (B. napus ssp. napobrassica) was defined by 13 haplotypes, including genomic rearrangements encompassing copies of Bna.FLC, Bna.PHYA and Bna.GA3ox1. De novo variation in copies of important flowering-time genes in B. napus arose during allopolyploidisation, enabling sub-functionalisation that allowed different morphotypes to appropriately fine-tune their lifecycle. PMID:28165502

  11. The positioning logic and copy number control of genes in bacteria under stress

    NASA Astrophysics Data System (ADS)

    Zhang, Qiucen; Austin, Robert; Vyawahare, Saurabh; Lau, Alexandra

    2013-03-01

    Escherichia coli (E. coli) cells when challenged with sublethal concentrations of the genotoxic antibiotic ciprofloxacin cease to divide and form long filaments which contain multiple bacterial chromosomes. These filaments are individual mesoscopic environmental niches which provide protection for a community of chromosomes (as opposed to cells) under mutagenic stress and can provide an evolutionary fitness advantage within the niche. We use comparative genomic hybridization to show that the mesoscopic niche evolves within 20 minutes of ciprofloxacin exposure via replication of multiple copies of genes expressing ATP dependent transporters. We show that this rapid genomic amplification is done in a time efficient manner via placement of the genes encoding the pumps near the origin of replication on the bacterial chromosome. The de-amplification of multiple copies back to the wild type number is a function of the duration is a function of the ciprofloxacin exposure duration: the longer the exposure, the slower the removal of the multiple copies. The project described was supported by the National Science Foundation and the National Cancer Institute

  12. Epigenetics, copy number variation, and other molecular mechanisms underlying neurodevelopmental disabilities: new insights and diagnostic approaches.

    PubMed

    Gropman, Andrea L; Batshaw, Mark L

    2010-09-01

    The diagnostic evaluation of children with intellectual disability (ID) and other neurodevelopmental disabilities (NDD) has become increasingly complex in recent years owing to a number of newly recognized genetic mechanisms and sophisticated methods to diagnose them. Previous studies have attempted to address the diagnostic yield of finding a genetic cause in ID. The results have varied widely from 10% to 81%, with the highest percentage being found in studies using new array comparative genomic hybridization methodology especially in autism. Although many cases of ID/NDD result from chromosomal aneuploidy or structural rearrangements, single gene disorders and new categories of genome modification, including epigenetics and copy number variation play an increasingly important role in diagnosis and testing. Epigenetic mechanisms, such as DNA methylation and modifications to histone proteins, regulate high-order DNA structure and gene expression. Aberrant epigenetic and copy number variation mechanisms are involved in several neurodevelopmental and neurodegenerative disorders including Rett syndrome, fragile X syndrome, and microdeletion syndromes. This review will describe a number of the molecular genetic mechanisms that play a role in disorders leading to ID/NDD and will discuss the categories and technologies for diagnostic testing of these conditions.

  13. Comparison of methods to detect copy number alterations in cancer using simulated and real genotyping data

    PubMed Central

    2012-01-01

    Background The detection of genomic copy number alterations (CNA) in cancer based on SNP arrays requires methods that take into account tumour specific factors such as normal cell contamination and tumour heterogeneity. A number of tools have been recently developed but their performance needs yet to be thoroughly assessed. To this aim, a comprehensive model that integrates the factors of normal cell contamination and intra-tumour heterogeneity and that can be translated to synthetic data on which to perform benchmarks is indispensable. Results We propose such model and implement it in an R package called CnaGen to synthetically generate a wide range of alterations under different normal cell contamination levels. Six recently published methods for CNA and loss of heterozygosity (LOH) detection on tumour samples were assessed on this synthetic data and on a dilution series of a breast cancer cell-line: ASCAT, GAP, GenoCNA, GPHMM, MixHMM and OncoSNP. We report the recall rates in terms of normal cell contamination levels and alteration characteristics: length, copy number and LOH state, as well as the false discovery rate distribution for each copy number under different normal cell contamination levels. Assessed methods are in general better at detecting alterations with low copy number and under a little normal cell contamination levels. All methods except GPHMM, which failed to recognize the alteration pattern in the cell-line samples, provided similar results for the synthetic and cell-line sample sets. MixHMM and GenoCNA are the poorliest performing methods, while GAP generally performed better. This supports the viability of approaches other than the common hidden Markov model (HMM)-based. Conclusions We devised and implemented a comprehensive model to generate data that simulate tumoural samples genotyped using SNP arrays. The validity of the model is supported by the similarity of the results obtained with synthetic and real data. Based on these results and

  14. From DNA Copy Number to Gene Expression: Local aberrations, Trisomies and Monosomies

    NASA Astrophysics Data System (ADS)

    Shay, Tal

    The goal of my PhD research was to study the effect of DNA copy number changes on gene expression. DNA copy number aberrations may be local, encompassing several genes, or on the level of an entire chromosome, such as trisomy and monosomy. The main dataset I studied was of Glioblastoma, obtained in the framework of a collaboration, but I worked also with public datasets of cancer and Down's Syndrome. The molecular basis of expression changes in Glioblastoma. Glioblastoma is the most common and aggressive type of primary brain tumors in adults. In collaboration with Prof. Hegi (CHUV, Switzerland), we analyzed a rich Glioblastoma dataset including clinical information, DNA copy number (array CGH) and expression profiles. We explored the correlation between DNA copy number and gene expression at the level of chromosomal arms and local genomic aberrations. We detected known amplification and over expression of oncogenes, as well as deletion and down-regulation of tumor suppressor genes. We exploited that information to map alterations of pathways that are known to be disrupted in Glioblastoma, and tried to characterize samples that have no known alteration in any of the studied pathways. Identifying local DNA aberrations of biological significance. Many types of tumors exhibit chromosomal losses or gains and local amplifications and deletions. A region that is aberrant in many tumors, or whose copy number change is stronger, is more likely to be clinically relevant, and not just a by-product of genetic instability. We developed a novel method that defines and prioritizes aberrations by formalizing these intuitions. The method scores each aberration by the fraction of patients harboring it, its length and its amplitude, and assesses the significance of the score by comparing it to a null distribution obtained by permutations. This approach detects genetic locations that are significantly aberrant, generating a 'genomic aberration profile' for each sample. The 'genomic

  15. Genome-wide detection of copy number variations among diverse horse breeds by array CGH.

    PubMed

    Wang, Wei; Wang, Shenyuan; Hou, Chenglin; Xing, Yanping; Cao, Junwei; Wu, Kaifeng; Liu, Chunxia; Zhang, Dong; Zhang, Li; Zhang, Yanru; Zhou, Huanmin

    2014-01-01

    Recent studies have found that copy number variations (CNVs) are widespread in human and animal genomes. CNVs are a significant source of genetic variation, and have been shown to be associated with phenotypic diversity. However, the effect of CNVs on genetic variation in horses is not well understood. In the present study, CNVs in 6 different breeds of mare horses, Mongolia horse, Abaga horse, Hequ horse and Kazakh horse (all plateau breeds) and Debao pony and Thoroughbred, were determined using aCGH. In total, seven hundred CNVs were identified ranging in size from 6.1 Kb to 0.57 Mb across all autosomes, with an average size of 43.08 Kb and a median size of 15.11 Kb. By merging overlapping CNVs, we found a total of three hundred and fifty-three CNV regions (CNVRs). The length of the CNVRs ranged from 6.1 Kb to 1.45 Mb with average and median sizes of 38.49 Kb and 13.1 Kb. Collectively, 13.59 Mb of copy number variation was identified among the horses investigated and accounted for approximately 0.61% of the horse genome sequence. Five hundred and eighteen annotated genes were affected by CNVs, which corresponded to about 2.26% of all horse genes. Through the gene ontology (GO), genetic pathway analysis and comparison of CNV genes among different breeds, we found evidence that CNVs involving 7 genes may be related to the adaptation to severe environment of these plateau horses. This study is the first report of copy number variations in Chinese horses, which indicates that CNVs are ubiquitous in the horse genome and influence many biological processes of the horse. These results will be helpful not only in mapping the horse whole-genome CNVs, but also to further research for the adaption to the high altitude severe environment for plateau horses.

  16. Integrative Analysis of Transcriptional Regulatory Network and Copy Number Variation in Intrahepatic Cholangiocarcinoma

    PubMed Central

    Li, Ling; Lian, Baofeng; Li, Chao; Li, Wei; Li, Jing; Zhang, Yuannv; He, Xianghuo; Li, Yixue; Xie, Lu

    2014-01-01

    Background Transcriptional regulatory network (TRN) is used to study conditional regulatory relationships between transcriptional factors and genes. However few studies have tried to integrate genomic variation information such as copy number variation (CNV) with TRN to find causal disturbances in a network. Intrahepatic cholangiocarcinoma (ICC) is the second most common hepatic carcinoma with high malignancy and poor prognosis. Research about ICC is relatively limited comparing to hepatocellular carcinoma, and there are no approved gene therapeutic targets yet. Method We first constructed TRN of ICC (ICC-TRN) using forward-and-reverse combined engineering method, and then integrated copy number variation information with ICC-TRN to select CNV-related modules and constructed CNV-ICC-TRN. We also integrated CNV-ICC-TRN with KEGG signaling pathways to investigate how CNV genes disturb signaling pathways. At last, unsupervised clustering method was applied to classify samples into distinct classes. Result We obtained CNV-ICC-TRN containing 33 modules which were enriched in ICC-related signaling pathways. Integrated analysis of the regulatory network and signaling pathways illustrated that CNV might interrupt signaling through locating on either genomic sites of nodes or regulators of nodes in a signaling pathway. In the end, expression profiles of nodes in CNV-ICC-TRN were used to cluster the ICC patients into two robust groups with distinct biological function features. Conclusion Our work represents a primary effort to construct TRN in ICC, also a primary effort to try to identify key transcriptional modules based on their involvement of genetic variations shown by gene copy number variations (CNV). This kind of approach may bring the traditional studies of TRN based only on expression data one step further to genetic disturbance. Such kind of approach can easily be extended to other disease samples with appropriate data. PMID:24897108

  17. Detection of MET Gene Copy Number in Cancer Samples Using the Droplet Digital PCR Method

    PubMed Central

    Zhang, Yanni; Tang, En-Tzu; Du, Zhiqiang

    2016-01-01

    Purpose The analysis of MET gene copy number (CN) has been considered to be a potential biomarker to predict the response to MET-targeted therapies in various cancers. However, the current standard methods to determine MET CN are SNP 6.0 in the genomic DNA of cancer cell lines and fluorescence in situ hybridization (FISH) in tumor models, respectively, which are costly and require advanced technical skills and result in relatively subjective judgments. Therefore, we employed a novel method, droplet digital PCR (ddPCR), to determine the MET gene copy number with high accuracy and precision. Methods The genomic DNA of cancer cell lines or tumor models were tested and compared with the MET gene CN and MET/CEN-7 ratio determined by SNP 6.0 and FISH, respectively. Results In cell lines, the linear association of the MET CN detected by ddPCR and SNP 6.0 is strong (Pearson correlation = 0.867). In tumor models, the MET CN detected by ddPCR was significantly different between the MET gene amplification and non-amplification groups according to FISH (mean: 15.4 vs 2.1; P = 0.044). Given that MET gene amplification is defined as MET CN >5.5 by ddPCR, the concordance rate between ddPCR and FISH was 98.0%, and Cohen's kappa coefficient was 0.760 (95% CI, 0.498–1.000; P <0.001). Conclusions The results demonstrated that the ddPCR method has the potential to quantify the MET gene copy number with high precision and accuracy as compared with the results from SNP 6.0 and FISH in cancer cell lines and tumor samples, respectively. PMID:26765781

  18. High-resolution analysis of copy number alterations and associated expression changes in ovarian tumors

    PubMed Central

    Haverty, Peter M; Hon, Lawrence S; Kaminker, Joshua S; Chant, John; Zhang, Zemin

    2009-01-01

    Background DNA copy number alterations are frequently observed in ovarian cancer, but it remains a challenge to identify the most relevant alterations and the specific causal genes in those regions. Methods We obtained high-resolution 500K SNP array data for 52 ovarian tumors and identified the most statistically significant minimal genomic regions with the most prevalent and highest-level copy number alterations (recurrent CNAs). Within a region of recurrent CNA, comparison of expression levels in tumors with a given CNA to tumors lacking that CNA and to whole normal ovary samples was used to select genes with CNA-specific expression patterns. A public expression array data set of laser capture micro-dissected (LCM) non-malignant fallopian tube epithelia and LCM ovarian serous adenocarcinoma was used to evaluate the effect of cell-type mixture biases. Results Fourteen recurrent deletions were detected on chromosomes 4, 6, 9, 12, 13, 15, 16, 17, 18, 22 and most prevalently on X and 8. Copy number and expression data suggest several apoptosis mediators as candidate drivers of the 8p deletions. Sixteen recurrent gains were identified on chromosomes 1, 2, 3, 5, 8, 10, 12, 15, 17, 19, and 20, with the most prevalent gains localized to 8q and 3q. Within the 8q amplicon, PVT1, but not MYC, was strongly over-expressed relative to tumors lacking this CNA and showed over-expression relative to normal ovary. Likewise, the cell polarity regulators PRKCI and ECT2 were identified as putative drivers of two distinct amplicons on 3q. Co-occurrence analyses suggested potential synergistic or antagonistic relationships between recurrent CNAs. Genes within regions of recurrent CNA showed an enrichment of Cancer Census genes, particularly when filtered for CNA-specific expression. Conclusion These analyses provide detailed views of ovarian cancer genomic changes and highlight the benefits of using multiple reference sample types for the evaluation of CNA-specific expression changes

  19. Genome-Wide Detection of Copy Number Variations among Diverse Horse Breeds by Array CGH

    PubMed Central

    Hou, Chenglin; Xing, Yanping; Cao, Junwei; Wu, Kaifeng; Liu, Chunxia; Zhang, Dong; Zhang, Li; Zhang, Yanru; Zhou, Huanmin

    2014-01-01

    Recent studies have found that copy number variations (CNVs) are widespread in human and animal genomes. CNVs are a significant source of genetic variation, and have been shown to be associated with phenotypic diversity. However, the effect of CNVs on genetic variation in horses is not well understood. In the present study, CNVs in 6 different breeds of mare horses, Mongolia horse, Abaga horse, Hequ horse and Kazakh horse (all plateau breeds) and Debao pony and Thoroughbred, were determined using aCGH. In total, seven hundred CNVs were identified ranging in size from 6.1 Kb to 0.57 Mb across all autosomes, with an average size of 43.08 Kb and a median size of 15.11 Kb. By merging overlapping CNVs, we found a total of three hundred and fifty-three CNV regions (CNVRs). The length of the CNVRs ranged from 6.1 Kb to 1.45 Mb with average and median sizes of 38.49 Kb and 13.1 Kb. Collectively, 13.59 Mb of copy number variation was identified among the horses investigated and accounted for approximately 0.61% of the horse genome sequence. Five hundred and eighteen annotated genes were affected by CNVs, which corresponded to about 2.26% of all horse genes. Through the gene ontology (GO), genetic pathway analysis and comparison of CNV genes among different breeds, we found evidence that CNVs involving 7 genes may be related to the adaptation to severe environment of these plateau horses. This study is the first report of copy number variations in Chinese horses, which indicates that CNVs are ubiquitous in the horse genome and influence many biological processes of the horse. These results will be helpful not only in mapping the horse whole-genome CNVs, but also to further research for the adaption to the high altitude severe environment for plateau horses. PMID:24497987

  20. Copy number variations alter methylation and parallel IGF2 overexpression in adrenal tumors

    PubMed Central

    Nielsen, Helene Myrtue; How-Kit, Alexandre; Guerin, Carole; Castinetti, Frederic; Vollan, Hans Kristian Moen; De Micco, Catherine; Daunay, Antoine; Taieb, David; Van Loo, Peter; Besse, Celine; Kristensen, Vessela N; Hansen, Lise Lotte; Barlier, Anne; Sebag, Frederic; Tost, Jörg

    2015-01-01

    Overexpression of insulin growth factor 2 (IGF2) is a hallmark of adrenocortical carcinomas and pheochromocytomas. Previous studies investigating the IGF2/H19 locus have mainly focused on a single molecular level such as genomic alterations or altered DNA methylation levels and the causal changes underlying IGF2 overexpression are still not fully established. In the current study, we analyzed 62 tumors of the adrenal gland from patients with Conn's adenoma (CA, n=12), pheochromocytomas (PCC, n=10), adrenocortical benign tumors (ACBT, n=20), and adrenocortical carcinomas (ACC, n=20). Gene expression, somatic copy number variation of chr11p15.5, and DNA methylation status of three differential methylated regions of the IGF2/H19 locus including the H19 imprinting control region were integratively analyzed. IGF2 overexpression was found in 85% of the ACCs and 100% of the PCCs compared to 23% observed in CAs and ACBTs. Copy number aberrations of chr11p15.5 were abundant in both PCCs and ACCs but while PCCs retained a diploid state, ACCs were frequently tetraploid (7/19). Loss of either a single allele or loss of two alleles of the same parental origin in tetraploid samples resulted in a uniparental disomy-like genotype. These copy number changes correlated with hypermethylation of the H19 ICR suggesting that the lost alleles were the unmethylated maternal alleles. Our data provide conclusive evidence that loss of the maternal allele correlates with IGF2 overexpression in adrenal tumors and that hypermethylation of the H19 ICR is a consequence thereof. PMID:26400872

  1. DNA copy-number alterations underlie gene expression differences between microsatellite stable and unstable colorectal cancers

    PubMed Central

    Jorissen, Robert N.; Lipton, Lara; Gibbs, Peter; Chapman, Matthew; Desai, Jayesh; Jones, Ian T.; Yeatman, Timothy J.; East, Philip; Tomlinson, Ian P.M.; Verspaget, Hein W.; Aaltonen, Lauri A.; Kruhøffer, Mogens; Ørntoft, Torben F.; Andersen, Claus Lindbjerg; Sieber, Oliver M.

    2008-01-01

    Purpose About 15% of colorectal cancers (CRCs) harbor microsatellite instability (MSI). MSI-associated gene expression changes have been identified in CRCs, but little overlap exists between signatures hindering an assessment of overall consistency. Little is known about the causes and downstream effects of differential gene expression. Experimental Design DNA microarray data on 89 MSI and 140 MSS CRCs from this study, and 58 MSI and 77 MSS cases from three published reports were randomly divided into test and training sets. MSI-associated gene expression changes were assessed for cross-study consistency using training samples, and validated as MSI classifier using test samples. Differences in biological pathways were identified by functional category analysis. Causation of differential gene expression was investigated by comparison to DNA copy-number data. Results MSI-associated gene expression changes in CRCs were found to be highly consistent across multiple studies of primary tumors and cancer cell lines from patients of different ethnicities (P<0.001). Clustering based on consistent changes separated additional test cases by MSI status, and classification of individual samples predicted MSI status with a sensitivity of 96% and specificity of 85%. Genes associated with immune response were up-regulated in MSI cancers, whereas genes associated with cell-cell adhesion, ion-binding and regulation of metabolism were down-regulated. Differential gene expression was shown to reflect systematic differences in DNA copy-number aberrations between MSI and MSS tumors (P<0.001). Conclusions Our results demonstrate cross-study consistency of MSI-associated gene expression changes in CRCs. DNA copy-number alterations partly cause the differences in gene expression between MSI and MSS cancers. PMID:19088021

  2. ITS1 copy number varies among Batrachochytrium dendrobatidis strains: implications for qPCR estimates of infection intensity from field-collected amphibian skin swabs.

    PubMed

    Longo, Ana V; Rodriguez, David; da Silva Leite, Domingos; Toledo, Luís Felipe; Mendoza Almeralla, Cinthya; Burrowes, Patricia A; Zamudio, Kelly R

    2013-01-01

    Genomic studies of the amphibian-killing fungus (Batrachochytrium dendrobatidis, [Bd]) identified three highly divergent genetic lineages, only one of which has a global distribution. Bd strains within these linages show variable genomic content due to differential loss of heterozygosity and recombination. The current quantitative polymerase chain reaction (qPCR) protocol to detect the fungus from amphibian skin swabs targets the intergenic transcribed spacer 1 (ITS1) region using a TaqMan fluorescent probe specific to Bd. We investigated the consequences of genomic differences in the quantification of ITS1 from eight distinct Bd strains, including representatives from North America, South America, the Caribbean, and Australia. To test for potential differences in amplification, we compared qPCR standards made from Bd zoospore counts for each strain, and showed that they differ significantly in amplification rates. To test potential mechanisms leading to strain differences in qPCR reaction parameters (slope and y-intercept), we: a) compared standard curves from the same strains made from extracted Bd genomic DNA in equimolar solutions, b) quantified the number of ITS1 copies per zoospore using a standard curve made from PCR-amplicons of the ITS1 region, and c) cloned and sequenced PCR-amplified ITS1 regions from these same strains to verify the presence of the probe site in all haplotypes. We found high strain variability in ITS1 copy number, ranging from 10 to 144 copies per single zoospore. Our results indicate that genome size might explain strain differences in ITS1 copy number, but not ITS1 sequence variation because the probe-binding site and primers were conserved across all haplotypes. For standards constructed from uncharacterized Bd strains, we recommend the use of single ITS1 PCR-amplicons as the absolute standard in conjunction with current quantitative assays to inform on copy number variation and provide universal estimates of pathogen zoospore loads

  3. Fast detection of de novo copy number variants from SNP arrays for case-parent trios

    PubMed Central

    2012-01-01

    Background In studies of case-parent trios, we define copy number variants (CNVs) in the offspring that differ from the parental copy numbers as de novo and of interest for their potential functional role in disease. Among the leading array-based methods for discovery of de novo CNVs in case-parent trios is the joint hidden Markov model (HMM) implemented in the PennCNV software. However, the computational demands of the joint HMM are substantial and the extent to which false positive identifications occur in case-parent trios has not been well described. We evaluate these issues in a study of oral cleft case-parent trios. Results Our analysis of the oral cleft trios reveals that genomic waves represent a substantial source of false positive identifications in the joint HMM, despite a wave-correction implementation in PennCNV. In addition, the noise of low-level summaries of relative copy number (log R ratios) is strongly associated with batch and correlated with the frequency of de novo CNV calls. Exploiting the trio design, we propose a univariate statistic for relative copy number referred to as the minimum distance that can reduce technical variation from probe effects and genomic waves. We use circular binary segmentation to segment the minimum distance and maximum a posteriori estimation to infer de novo CNVs from the segmented genome. Compared to PennCNV on simulated data, MinimumDistance identifies fewer false positives on average and is comparable to PennCNV with respect to false negatives. Genomic waves contribute to discordance of PennCNV and MinimumDistance for high coverage de novo calls, while highly concordant calls on chromosome 22 were validated by quantitative PCR. Computationally, MinimumDistance provides a nearly 8-fold increase in speed relative to the joint HMM in a study of oral cleft trios. Conclusions Our results indicate that batch effects and genomic waves are important considerations for case-parent studies of de novo CNV, and that the

  4. Global copy number profiling of cancer genomes | Office of Cancer Genomics

    Cancer.gov

    In this article, we introduce a robust and efficient strategy for deriving global and allele-specific copy number alternations (CNA) from cancer whole exome sequencing data based on Log R ratios and B-allele frequencies. Applying the approach to the analysis of over 200 skin cancer samples, we demonstrate its utility for discovering distinct CNA events and for deriving ancillary information such as tumor purity. Availability and implementation: https://github.com/xfwang/CLOSE CONTACT: xuefeng.wang@stonybrook.edu or michael.krauthammer@yale.edu. (Publication Abstract)

  5. GENETIC ASSOCIATION ANALYSIS OF COPY NUMBER VARIATION (CNVs) IN HUMAN DISEASE PATHOGENESIS

    PubMed Central

    Ionita-Laza, Iuliana; Rogers, Angela J.; Lange, Christoph; Raby, Benjamin A.; Lee, Charles

    2009-01-01

    Structural genetic variation, including copy number variation (CNV), constitutes a substantial fraction of total genetic variability and the importance of structural genetic variants in modulating human disease is increasingly being recognized. Early successes in identifying disease-associated CNVs via a candidate gene approach mandate that future disease association studies need to include structural genetic variation. Such analyses should not rely on previously developed methodologies that were designed to evaluate single nucleotide polymorphisms (SNPs). Instead, development of novel technical, statistical, and epidemiologic methods will be necessary to optimally capture this newly-appreciated form of genetic variation in a meaningful manner. PMID:18822366

  6. Plasticity of the Leishmania genome leading to gene copy number variations and drug resistance

    PubMed Central

    Papadopoulou, Barbara; Ouellette, Marc

    2016-01-01

    Leishmania has a plastic genome, and drug pressure can select for gene copy number variation (CNV). CNVs can apply either to whole chromosomes, leading to aneuploidy, or to specific genomic regions. For the latter, the amplification of chromosomal regions occurs at the level of homologous direct or inverted repeated sequences leading to extrachromosomal circular or linear amplified DNAs. This ability of Leishmania to respond to drug pressure by CNVs has led to the development of genomic screens such as Cos-Seq, which has the potential of expediting the discovery of drug targets for novel promising drug candidates. PMID:27703673

  7. Novel population specific autosomal copy number variation and its functional analysis amongst Negritos from Peninsular Malaysia.

    PubMed

    Mokhtar, Siti Shuhada; Marshall, Christian R; Phipps, Maude E; Thiruvahindrapuram, Bhooma; Lionel, Anath C; Scherer, Stephen W; Peng, Hoh Boon

    2014-01-01

    Copy number variation (CNV) has been recognized as a major contributor to human genome diversity. It plays an important role in determining phenotypes and has been associated with a number of common and complex diseases. However CNV data from diverse populations is still limited. Here we report the first investigation of CNV in the indigenous populations from Peninsular Malaysia. We genotyped 34 Negrito genomes from Peninsular Malaysia using the Affymetrix SNP 6.0 microarray and identified 48 putative novel CNVs, consisting of 24 gains and 24 losses, of which 5 were identified in at least 2 unrelated samples. These CNVs appear unique to the Negrito population and were absent in the DGV, HapMap3 and Singapore Genome Variation Project (SGVP) datasets. Analysis of gene ontology revealed that genes within these CNVs were enriched in the immune system (GO:0002376), response to stimulus mechanisms (GO:0050896), the metabolic pathways (GO:0001852), as well as regulation of transcription (GO:0006355). Copy number gains in CNV regions (CNVRs) enriched with genes were significantly higher than the losses (P value <0.001). In view of the small population size, relative isolation and semi-nomadic lifestyles of this community, we speculate that these CNVs may be attributed to recent local adaptation of Negritos from Peninsular Malaysia.

  8. NF1 single and multi-exons copy number variations in neurofibromatosis type 1.

    PubMed

    Imbard, Apolline; Pasmant, Eric; Sabbagh, Audrey; Luscan, Armelle; Soares, Magali; Goussard, Philippe; Blanché, Hélène; Laurendeau, Ingrid; Ferkal, Salah; Vidaud, Michel; Pinson, Stéphane; Bellanne-Chantelot, Christine; Vidaud, Dominique; Wolkenstein, Pierre; Parfait, Béatrice

    2015-04-01

    Neurofibromatosis type 1 (NF1) is caused by dominant loss-of-function mutations of the tumor suppressor NF1 containing 57 constitutive coding exons. A huge number of different pathogenic NF1 alterations has been reported. The aim of the present study was to evaluate the usefulness of a multiplex ligation-dependent probe amplification (MLPA) approach in NF1 patients to detect single and multi-exon NF1 gene copy number variations. A genotype-phenotype correlation was then performed in NF1 patients carrying these types of genetic alterations. Among 565 NF1 index cases from the French NF1 cohort, single and multi-exon deletions/duplications screening identified NF1 partial deletions/duplications in 22 patients (~4%) using MLPA analysis. Eight single exon deletions, 11 multiple exons deletions, 1 complex rearrangement and 2 duplications were identified. All results were confirmed using a custom array-CGH. MLPA and custom array-CGH allowed the identification of rearrangements that were missed by cDNA/DNA sequencing or microsatellite analysis. We then performed a targeted next-generation sequencing of NF1 that allowed confirmation of all 22 rearrangements. No clear genotype-phenotype correlations were found for the most clinically significant disease features of NF1 in patients with single and multi-exons NF1 gene copy number changes.

  9. Copy number variation upstream of PMP22 in Charcot–Marie–Tooth disease

    PubMed Central

    Weterman, Marian AJ; van Ruissen, Fred; de Wissel, Marit; Bordewijk, Lou; Samijn, Johnny PA; van der Pol, W Ludo; Meggouh, Farid; Baas, Frank

    2010-01-01

    In several individuals with a Charcot–Marie–Tooth (CMT) phenotype, we found a copy number variation (CNV) on chromosome 17p12 in the direct vicinity of the peripheral myelin protein 22 (PMP22) gene. The exact borders and size of this CNV were determined by Southern blot analysis, MLPA, vectorette PCR, and microarray hybridization analyses. All patients from six apparently unrelated families carried an identical 186-kb duplication different from the commonly reported 1.5-Mb duplication associated with CMT1A. This ancestral mutation that was not reported in the human structural variation database was only detected in affected individuals and family members. It was absent in 2124 control chromosomes and 40 patients with a chronic inflammatory demyelinating polyneuropathy (CIDP) and therefore should be regarded as causative for the disease. This variant escapes most routine diagnostic screens for CMT1A, because copy numbers of PMP22 probes were all normal. No indications were found for the involvement of the genes that are located within this duplication. A possible association of this duplication with a mutation in the PMP22 coding regions was also excluded. We suggest that this CNV proximal of the PMP22 gene leads to CMT through an unknown mechanism affecting PMP22 expression. PMID:19888301

  10. DNA copy number amplifications in human neoplasms: review of comparative genomic hybridization studies.

    PubMed Central

    Knuutila, S.; Björkqvist, A. M.; Autio, K.; Tarkkanen, M.; Wolf, M.; Monni, O.; Szymanska, J.; Larramendy, M. L.; Tapper, J.; Pere, H.; El-Rifai, W.; Hemmer, S.; Wasenius, V. M.; Vidgren, V.; Zhu, Y.

    1998-01-01

    This review summarizes reports of recurrent DNA sequence copy number amplifications in human neoplasms detected by comparative genomic hybridization. Some of the chromosomal areas with recurrent DNA copy number amplifications (amplicons) of 1p22-p31, 1p32-p36, 1q, 2p13-p16, 2p23-p25, 2q31-q33, 3q, 5p, 6p12-pter, 7p12-p13, 7q11.2, 7q21-q22, 8p11-p12, 8q, 11q13-q14, 12p, 12q13-q21, 13q14, 13q22-qter, 14q13-q21, 15q24-qter, 17p11.2-p12, 17q12-q21, 17q22-qter, 18q, 19p13.2-pter, 19cen-q13.3, 20p11.2-p12, 20q, Xp11.2-p21, and Xp11-q13 and genes therein are presented in more detail. The paper with more than 150 references and two tables can be accessed from our web site http://www.helsinki.fi/lglvwww/CMG.html. The data will be updated biannually until the year 2001. PMID:9588877

  11. Diversity and population-genetic properties of copy number variations and multicopy genes in cattle

    PubMed Central

    Bickhart, Derek M.; Xu, Lingyang; Hutchison, Jana L.; Cole, John B.; Null, Daniel J.; Schroeder, Steven G.; Song, Jiuzhou; Garcia, Jose Fernando; Sonstegard, Tad S.; Van Tassell, Curtis P.; Schnabel, Robert D.; Taylor, Jeremy F.; Lewin, Harris A.; Liu, George E.

    2016-01-01

    The diversity and population genetics of copy number variation (CNV) in domesticated animals are not well understood. In this study, we analysed 75 genomes of major taurine and indicine cattle breeds (including Angus, Brahman, Gir, Holstein, Jersey, Limousin, Nelore, and Romagnola), sequenced to 11-fold coverage to identify 1,853 non-redundant CNV regions. Supported by high validation rates in array comparative genomic hybridization (CGH) and qPCR experiments, these CNV regions accounted for 3.1% (87.5 Mb) of the cattle reference genome, representing a significant increase over previous estimates of the area of the genome that is copy number variable (∼2%). Further population genetics and evolutionary genomics analyses based on these CNVs revealed the population structures of the cattle taurine and indicine breeds and uncovered potential diversely selected CNVs near important functional genes, including AOX1, ASZ1, GAT, GLYAT, and KRTAP9-1. Additionally, 121 CNV gene regions were found to be either breed specific or differentially variable across breeds, such as RICTOR in dairy breeds and PNPLA3 in beef breeds. In contrast, clusters of the PRP and PAG genes were found to be duplicated in all sequenced animals, suggesting that subfunctionalization, neofunctionalization, or overdominance play roles in diversifying those fertility-related genes. These CNV results provide a new glimpse into the diverse selection histories of cattle breeds and a basis for correlating structural variation with complex traits in the future. PMID:27085184

  12. Diversity and population-genetic properties of copy number variations and multicopy genes in cattle.

    PubMed

    Bickhart, Derek M; Xu, Lingyang; Hutchison, Jana L; Cole, John B; Null, Daniel J; Schroeder, Steven G; Song, Jiuzhou; Garcia, Jose Fernando; Sonstegard, Tad S; Van Tassell, Curtis P; Schnabel, Robert D; Taylor, Jeremy F; Lewin, Harris A; Liu, George E

    2016-06-01

    The diversity and population genetics of copy number variation (CNV) in domesticated animals are not well understood. In this study, we analysed 75 genomes of major taurine and indicine cattle breeds (including Angus, Brahman, Gir, Holstein, Jersey, Limousin, Nelore, and Romagnola), sequenced to 11-fold coverage to identify 1,853 non-redundant CNV regions. Supported by high validation rates in array comparative genomic hybridization (CGH) and qPCR experiments, these CNV regions accounted for 3.1% (87.5 Mb) of the cattle reference genome, representing a significant increase over previous estimates of the area of the genome that is copy number variable (∼2%). Further population genetics and evolutionary genomics analyses based on these CNVs revealed the population structures of the cattle taurine and indicine breeds and uncovered potential diversely selected CNVs near important functional genes, including AOX1, ASZ1, GAT, GLYAT, and KRTAP9-1 Additionally, 121 CNV gene regions were found to be either breed specific or differentially variable across breeds, such as RICTOR in dairy breeds and PNPLA3 in beef breeds. In contrast, clusters of the PRP and PAG genes were found to be duplicated in all sequenced animals, suggesting that subfunctionalization, neofunctionalization, or overdominance play roles in diversifying those fertility-related genes. These CNV results provide a new glimpse into the diverse selection histories of cattle breeds and a basis for correlating structural variation with complex traits in the future.

  13. Somatic copy number mosaicism in human skin revealed by induced pluripotent stem cells.

    PubMed

    Abyzov, Alexej; Mariani, Jessica; Palejev, Dean; Zhang, Ying; Haney, Michael Seamus; Tomasini, Livia; Ferrandino, Anthony F; Rosenberg Belmaker, Lior A; Szekely, Anna; Wilson, Michael; Kocabas, Arif; Calixto, Nathaniel E; Grigorenko, Elena L; Huttner, Anita; Chawarska, Katarzyna; Weissman, Sherman; Urban, Alexander Eckehart; Gerstein, Mark; Vaccarino, Flora M

    2012-12-20

    Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) has been suspected of causing de novo copy number variation. To explore this issue, here we perform a whole-genome and transcriptome analysis of 20 human iPSC lines derived from the primary skin fibroblasts of seven individuals using next-generation sequencing. We find that, on average, an iPSC line manifests two copy number variants (CNVs) not apparent in the fibroblasts from which the iPSC was derived. Using PCR and digital droplet PCR, we show that at least 50% of those CNVs are present as low-frequency somatic genomic variants in parental fibroblasts (that is, the fibroblasts from which each corresponding human iPSC line is derived), and are manifested in iPSC lines owing to their clonal origin. Hence, reprogramming does not necessarily lead to de novo CNVs in iPSCs, because most of the line-manifested CNVs reflect somatic mosaicism in the human skin. Moreover, our findings demonstrate that clonal expansion, and iPSC lines in particular, can be used as a discovery tool to reliably detect low-frequency CNVs in the tissue of origin. Overall, we estimate that approximately 30% of the fibroblast cells have somatic CNVs in their genomes, suggesting widespread somatic mosaicism in the human body. Our study paves the way to understanding the fundamental question of the extent to which cells of the human body normally acquire structural alterations in their DNA post-zygotically.

  14. Long insert whole genome sequencing for copy number variant and translocation detection

    PubMed Central

    Liang, Winnie S.; Aldrich, Jessica; Tembe, Waibhav; Kurdoglu, Ahmet; Cherni, Irene; Phillips, Lori; Reiman, Rebecca; Baker, Angela; Weiss, Glen J.; Carpten, John D.; Craig, David W.

    2014-01-01

    As next-generation sequencing continues to have an expanding presence in the clinic, the identification of the most cost-effective and robust strategy for identifying copy number changes and translocations in tumor genomes is needed. We hypothesized that performing shallow whole genome sequencing (WGS) of 900–1000-bp inserts (long insert WGS, LI-WGS) improves our ability to detect these events, compared with shallow WGS of 300–400-bp inserts. A priori analyses show that LI-WGS requires less sequencing compared with short insert WGS to achieve a target physical coverage, and that LI-WGS requires less sequence coverage to detect a heterozygous event with a power of 0.99. We thus developed an LI-WGS library preparation protocol based off of Illumina’s WGS library preparation protocol and illustrate the feasibility of performing LI-WGS. We additionally applied LI-WGS to three separate tumor/normal DNA pairs collected from patients diagnosed with different cancers to demonstrate our application of LI-WGS on actual patient samples for identification of somatic copy number alterations and translocations. With the evolution of sequencing technologies and bioinformatics analyses, we show that modifications to current approaches may improve our ability to interrogate cancer genomes. PMID:24071583

  15. Extensive Copy Number Variations in Admixed Indian Population of African Ancestry: Potential Involvement in Adaptation

    PubMed Central

    Dash, Debasis; Mukerji, Mitali

    2014-01-01

    Admixture mapping has been enormously resourceful in identifying genetic variations linked to phenotypes, adaptation, and diseases. In this study through analysis of copy number variable regions (CNVRs), we report extensive restructuring in the genomes of the recently admixed African-Indian population (OG-W-IP) that inhabits a highly saline environment in Western India. The study included subjects from OG-W-IP (OG), five different Indian and three HapMap populations that were genotyped using Affymetrix version 6.0 arrays. Copy number variations (CNVs) detected using Birdsuite were used to define CNVRs. Population structure with respect to CNVRs was delineated using random forest approach. OG genomes have a surprising excess of CNVs in comparison to other studied populations. Individual ancestry proportions computed using STRUCTURE also reveals a unique genetic component in OGs. Population structure analysis with CNV genotypes indicates OG to be distant from both the African and Indian ancestral populations. Interestingly, it shows genetic proximity with respect to CNVs to only one Indian population IE-W-LP4, which also happens to reside in the same geographical region. We also observe a significant enrichment of molecular processes related to ion binding and receptor activity in genes encompassing OG-specific CNVRs. Our results suggest that retention of CNVRs from ancestral natives and de novo acquisition of CNVRs could accelerate the process of adaptation especially in an extreme environment. Additionally, this population would be enormously useful for dissecting genes and delineating the involvement of CNVs in salt adaptation. PMID:25398783

  16. Evolutionary and Functional Features of Copy Number Variation in the Cattle Genome

    PubMed Central

    Keel, Brittney N.; Lindholm-Perry, Amanda K.; Snelling, Warren M.

    2016-01-01

    Genomic structural variations are an important source of genetic diversity. Copy number variations (CNVs), gains and losses of large regions of genomic sequence between individuals of a species, have been associated with a wide variety of phenotypic traits. However, in cattle, as well as many other species, relatively little is understood about CNV, including frequency of CNVs in the genome, sizes, and locations, chromosomal properties, and evolutionary processes acting to shape CNV. In this work, we focused on copy number variation in the bovine genome, with the aim to detect CNVs in Bos taurus coding sequence and explore potential evolutionary mechanisms shaping these CNV. We identified and characterized CNV regions by utilizing exome sequence from 175 influential sires used in the Germplasm Evaluation project, representing 10 breeds. We examined various evolutionary and functional aspects of these CNVs, including selective constraint on CNV-overlapped genes, centrality of CNV genes in protein-protein interaction networks, and tissue-specific expression of CNV genes. Patterns of CNV in the Bos taurus genome reveal that reduced functional constraint and mutational bias may play a prominent role in shaping this type of structural variation. PMID:27920798

  17. CNV-TV: A robust method to discover copy number variation from short sequencing reads

    PubMed Central

    2013-01-01

    Background Copy number variation (CNV) is an important structural variation (SV) in human genome. Various studies have shown that CNVs are associated with complex diseases. Traditional CNV detection methods such as fluorescence in situ hybridization (FISH) and array comparative genomic hybridization (aCGH) suffer from low resolution. The next generation sequencing (NGS) technique promises a higher resolution detection of CNVs and several methods were recently proposed for realizing such a promise. However, the performances of these methods are not robust under some conditions, e.g., some of them may fail to detect CNVs of short sizes. There has been a strong demand for reliable detection of CNVs from high resolution NGS data. Results A novel and robust method to detect CNV from short sequencing reads is proposed in this study. The detection of CNV is modeled as a change-point detection from the read depth (RD) signal derived from the NGS, which is fitted with a total variation (TV) penalized least squares model. The performance (e.g., sensitivity and specificity) of the proposed approach are evaluated by comparison with several recently published methods on both simulated and real data from the 1000 Genomes Project. Conclusion The experimental results showed that both the true positive rate and false positive rate of the proposed detection method do not change significantly for CNVs with different copy numbers and lengthes, when compared with several existing methods. Therefore, our proposed approach results in a more reliable detection of CNVs than the existing methods. PMID:23634703

  18. Identification of copy number alterations associated with the progression of DCIS to invasive ductal carcinoma.

    PubMed

    Johnson, Clint E; Gorringe, Kylie L; Thompson, Ella R; Opeskin, Ken; Boyle, Samantha E; Wang, Yuker; Hill, Prue; Mann, G Bruce; Campbell, Ian G

    2012-06-01

    Ductal carcinoma in situ (DCIS) is a non-obligate precursor to invasive ductal carcinoma (IDC). Annotation of the genetic differences between the two lesions may assist in the identification of genes that promote the invasive phenotype. Synchronous DCIS and IDC cells were microdissected from FFPE tissue and analysed by molecular inversion probe (MIP) copy number arrays. Matched IDC and DCIS showed highly similar copy number profiles (average of 83% of the genome shared) indicating a common clonal origin although there is evidence that the DCIS continues to evolve in parallel with the co-existing IDC. Four chromosomal regions of loss (3q, 6q, 8p and 11q) and four regions of gain (5q, 16p, 19q and 20) were recurrently affected in IDC but not in DCIS. CCND1 and MYC showed increased amplitude of gain in IDC. One region of loss (17p11.2) was specific to DCIS. IDC-specific regions include genes with previous links to breast cancer progression and potential therapeutic targets such as AXL, SPHK1 and PLAUR.

  19. Blattabacteria, the endosymbionts of cockroaches, have small genome sizes and high genome copy numbers.

    PubMed

    López-Sánchez, María José; Neef, Alexander; Patiño-Navarrete, Rafael; Navarro, Lara; Jiménez, Ricardo; Latorre, Amparo; Moya, Andrés

    2008-12-01

    Blattabacteria are intracellular endosymbionts of cockroaches and primitive termites that belong to the class Flavobacteria and live only in specialized cells in the abdominal fat body of their hosts. In the present study we determined genome sizes as well as genome copy numbers for the endosymbionts of three cockroach species, Blattella germanica, Periplaneta americana and Blatta orientalis. The sole presence of blattabacteria in the fat body was demonstrated by rRNA-targeting techniques. The genome sizes of the three blattabacteria were determined by pulsed field gel electrophoresis. The resulting total genome sizes for the three symbionts were all approximately 650 +/- 15 kb. Comparison of the genome sizes with those of free-living Bacteroidetes shows extended reduction, as occurs in other obligatory insect endosymbionts. Genome copy numbers were determined based on cell counts and determination of DNA amounts via quantitative PCR. Values between 10.2 and 18.3 and between 323 and 353 were found for the symbionts of P. americana and B. orientalis respectively. Polyploidy in intracellular bacteria may play a significant role in the genome reduction process.

  20. Identification of copy number variants associated with BPES-like phenotypes.

    PubMed

    Gijsbers, Antoinet C J; D'haene, Barbara; Hilhorst-Hofstee, Yvonne; Mannens, Marcel; Albrecht, Beate; Seidel, Joerg; Witt, David R; Maisenbacher, Melissa K; Loeys, Bart; van Essen, Ton; Bakker, Egbert; Hennekam, Raoul; Breuning, Martijn H; De Baere, Elfride; Ruivenkamp, Claudia A L

    2008-12-01

    Blepharophimosis-Ptosis-Epicanthus inversus syndrome (BPES) is a well-characterized rare syndrome that includes an eyelid malformation associated with (type I) or without premature ovarian failure (type II). Patients with typical BPES have four major characteristics: blepharophimosis, ptosis, epicanthus inversus and telecanthus. Mutations in the FOXL2 gene, encoding a forkhead transcription factor, are responsible for the majority of both types of BPES. However, many patients with BPES-like features, i.e., having at least two major characteristics of BPES, have an unidentified cause. Here, we report on a group of 27 patients with BPES-like features, but without an identified genetic defect in the FOXL2 gene or flanking region. These patients were analyzed with whole-genome high-density arrays in order to identify copy number variants (CNVs) that might explain the BPES-like phenotype. In nine out of 27 patients (33%) CNVs not previously described as polymorphisms were detected. Four of these patients displayed psychomotor retardation as an additional clinical characteristic. In conclusion, we demonstrate that BPES-like phenotypes are frequently caused by CNVs, and we emphasize the importance of whole-genome copy number screening to identify the underlying genetic causes of these phenotypes.

  1. 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

  2. Gene copy number variation spanning 60 million years of human and primate evolution

    PubMed Central

    Dumas, Laura; Kim, Young H.; Karimpour-Fard, Anis; Cox, Michael; Hopkins, Janet; Pollack, Jonathan R.; Sikela, James M.

    2007-01-01

    Given the evolutionary importance of gene duplication to the emergence of species-specific traits, we have extended the application of cDNA array-based comparative genomic hybridization (aCGH) to survey gene duplications and losses genome-wide across 10 primate species, including human. Using human cDNA arrays that contained 41,126 cDNAs, corresponding to 24,473 unique human genes, we identified 4159 genes that likely represent most of the major lineage-specific gene copy number gains and losses that have occurred in these species over the past 60 million years. We analyzed 1,233,780 gene-to-gene data points and found that gene gains typically outnumbered losses (ratio of gains/losses = 2.34) and these frequently cluster in complex and dynamic genomic regions that are likely to serve as gene nurseries. Almost one-third of all human genes (6696) exhibit an aCGH- predicted change in copy number in one or more of these species, and within-species gene amplification is also evident. Many of the genes identified here are likely to be important to lineage-specific traits including, for example, human-specific duplications of the AQP7 gene, which represent intriguing candidates to underlie the key physiological adaptations in thermoregulation and energy utilization that permitted human endurance running. PMID:17666543

  3. Chromosomal copy number changes of locally advanced rectal cancers treated with preoperative chemoradiotherapy

    PubMed Central

    Grade, Marian; Gaedcke, Jochen; Wangsa, Danny; Varma, Sudhir; Beckmann, Jaje; Liersch, Torsten; Hess, Clemens; Becker, Heinz; Difilippantonio, Michael J.; Ried, Thomas; Ghadimi, B. Michael

    2009-01-01

    Introduction Standard treatment of rectal cancer patients comprises preoperative chemoradiotherapy followed by radical surgery. However, clinicians are faced with the problem that response rates vary from one individual to another. Predictive biomarkers would therefore be helpful. Materials and Methods In order to identify genomic imbalances that might assist in stratifying tumors into responsive or non-responsive, we used metaphase comparative genomic hybridization to prospectively analyze pre-therapeutic biopsies from 42 patients with locally advanced rectal cancers. These patients were subsequently treated with 5-FU based preoperative chemoradiotherapy. Results Based on downsizing of the T-category, 21 rectal cancers were later classified as responsive, while 21 were non-responsive. Comparing these two groups, we could show that gains of chromosomal regions 7q32-q36 and 7q11-q31, and amplifications of 20q11-q13 were significantly associated with responsiveness to preoperative chemoradiotherapy (P<0.05). However, the probability to detect these copy number changes by chance is high (P=0.21). Conclusion Our primary results suggest that pre-therapeutic evaluation of chromosomal copy number changes may be of value for response prediction of rectal cancers to preoperative chemoradiotherapy. This will require validation in a larger cohort of patients. PMID:19602460

  4. Genome-wide analysis of copy number variants in age-related macular degeneration

    PubMed Central

    Meyer, Kacie J.; Davis, Lea K.; Schindler, Emily I.; Beck, John S.; Rudd, Danielle S.; Grundstad, A. Jason; Scheetz, Todd E.; Braun, Terry A.; Fingert, John H.; Alward, Wallace L.; Kwon, Young H.; Folk, James C.; Russell, Stephen R.; Stone, Edwin M.; Sheffield, Val C.

    2012-01-01

    Age-related macular degeneration (AMD) is a complex genetic disease, with many loci demonstrating appreciable attributable disease risk. Despite significant progress toward understanding the genetic and environmental etiology of AMD, identification of additional risk factors is necessary to fully appreciate and treat AMD pathology. In this study, we investigated copy number variants (CNVs) as potential AMD risk variants in a cohort of 400 AMD patients and 500 AMD-free controls ascertained at the University of Iowa. We used three publicly available copy number programs to analyze signal intensity data from Affymetrix® GeneChip SNP Microarrays. CNVs were ranked based on prevalence in the disease cohort and absence from the control group; high interest CNVs were subsequently confirmed by qPCR. While we did not observe a single-locus “risk CNV” that could account for a major fraction of AMD, we identified several rare and overlapping CNVs containing or flanking compelling candidate genes such as NPHP1 and EFEMP1. These and other candidate genes highlighted by this study deserve further scrutiny as sources of genetic risk for AMD. PMID:20981449

  5. Genotype copy number variations using Gaussian mixture models: theory and algorithms.

    PubMed

    Lin, Chang-Yun; Lo, Yungtai; Ye, Kenny Q

    2012-10-12

    Copy number variations (CNVs) are important in the disease association studies and are usually targeted by most recent microarray platforms developed for GWAS studies. However, the probes targeting the same CNV regions could vary greatly in performance, with some of the probes carrying little information more than pure noise. In this paper, we investigate how to best combine measurements of multiple probes to estimate copy numbers of individuals under the framework of Gaussian mixture model (GMM). First we show that under two regularity conditions and assume all the parameters except the mixing proportions are known, optimal weights can be obtained so that the univariate GMM based on the weighted average gives the exactly the same classification as the multivariate GMM does. We then developed an algorithm that iteratively estimates the parameters and obtains the optimal weights, and uses them for classification. The algorithm performs well on simulation data and two sets of real data, which shows clear advantage over classification based on the equal weighted average.

  6. Functional impact of global rare copy number variation in autism spectrum disorders.

    PubMed

    Pinto, Dalila; Pagnamenta, Alistair T; Klei, Lambertus; Anney, Richard; Merico, Daniele; Regan, Regina; Conroy, Judith; Magalhaes, Tiago R; Correia, Catarina; Abrahams, Brett S; Almeida, Joana; Bacchelli, Elena; Bader, Gary D; Bailey, Anthony J; Baird, Gillian; Battaglia, Agatino; Berney, Tom; Bolshakova, Nadia; Bölte, Sven; Bolton, Patrick F; Bourgeron, Thomas; Brennan, Sean; Brian, Jessica; Bryson, Susan E; Carson, Andrew R; Casallo, Guillermo; Casey, Jillian; Chung, Brian H Y; Cochrane, Lynne; Corsello, Christina; Crawford, Emily L; Crossett, Andrew; Cytrynbaum, Cheryl; Dawson, Geraldine; de Jonge, Maretha; Delorme, Richard; Drmic, Irene; Duketis, Eftichia; Duque, Frederico; Estes, Annette; Farrar, Penny; Fernandez, Bridget A; Folstein, Susan E; Fombonne, Eric; Freitag, Christine M; Gilbert, John; Gillberg, Christopher; Glessner, Joseph T; Goldberg, Jeremy; Green, Andrew; Green, Jonathan; Guter, Stephen J; Hakonarson, Hakon; Heron, Elizabeth A; Hill, Matthew; Holt, Richard; Howe, Jennifer L; Hughes, Gillian; Hus, Vanessa; Igliozzi, Roberta; Kim, Cecilia; Klauck, Sabine M; Kolevzon, Alexander; Korvatska, Olena; Kustanovich, Vlad; Lajonchere, Clara M; Lamb, Janine A; Laskawiec, Magdalena; Leboyer, Marion; Le Couteur, Ann; Leventhal, Bennett L; Lionel, Anath C; Liu, Xiao-Qing; Lord, Catherine; Lotspeich, Linda; Lund, Sabata C; Maestrini, Elena; Mahoney, William; Mantoulan, Carine; Marshall, Christian R; McConachie, Helen; McDougle, Christopher J; McGrath, Jane; McMahon, William M; Merikangas, Alison; Migita, Ohsuke; Minshew, Nancy J; Mirza, Ghazala K; Munson, Jeff; Nelson, Stanley F; Noakes, Carolyn; Noor, Abdul; Nygren, Gudrun; Oliveira, Guiomar; Papanikolaou, Katerina; Parr, Jeremy R; Parrini, Barbara; Paton, Tara; Pickles, Andrew; Pilorge, Marion; Piven, Joseph; Ponting, Chris P; Posey, David J; Poustka, Annemarie; Poustka, Fritz; Prasad, Aparna; Ragoussis, Jiannis; Renshaw, Katy; Rickaby, Jessica; Roberts, Wendy; Roeder, Kathryn; Roge, Bernadette; Rutter, Michael L; Bierut, Laura J; Rice, John P; Salt, Jeff; Sansom, Katherine; Sato, Daisuke; Segurado, Ricardo; Sequeira, Ana F; Senman, Lili; Shah, Naisha; Sheffield, Val C; Soorya, Latha; Sousa, Inês; Stein, Olaf; Sykes, Nuala; Stoppioni, Vera; Strawbridge, Christina; Tancredi, Raffaella; Tansey, Katherine; Thiruvahindrapduram, Bhooma; Thompson, Ann P; Thomson, Susanne; Tryfon, Ana; Tsiantis, John; Van Engeland, Herman; Vincent, John B; Volkmar, Fred; Wallace, Simon; Wang, Kai; Wang, Zhouzhi; Wassink, Thomas H; Webber, Caleb; Weksberg, Rosanna; Wing, Kirsty; Wittemeyer, Kerstin; Wood, Shawn; Wu, Jing; Yaspan, Brian L; Zurawiecki, Danielle; Zwaigenbaum, Lonnie; Buxbaum, Joseph D; Cantor, Rita M; Cook, Edwin H; Coon, Hilary; Cuccaro, Michael L; Devlin, Bernie; Ennis, Sean; Gallagher, Louise; Geschwind, Daniel H; Gill, Michael; Haines, Jonathan L; Hallmayer, Joachim; Miller, Judith; Monaco, Anthony P; Nurnberger, John I; Paterson, Andrew D; Pericak-Vance, Margaret A; Schellenberg, Gerard D; Szatmari, Peter; Vicente, Astrid M; Vieland, Veronica J; Wijsman, Ellen M; Scherer, Stephen W; Sutcliffe, James S; Betancur, Catalina

    2010-07-15

    The autism spectrum disorders (ASDs) are a group of conditions characterized by impairments in reciprocal social interaction and communication, and the presence of restricted and repetitive behaviours. Individuals with an ASD vary greatly in cognitive development, which can range from above average to intellectual disability. Although ASDs are known to be highly heritable ( approximately 90%), the underlying genetic determinants are still largely unknown. Here we analysed the genome-wide characteristics of rare (<1% frequency) copy number variation in ASD using dense genotyping arrays. When comparing 996 ASD individuals of European ancestry to 1,287 matched controls, cases were found to carry a higher global burden of rare, genic copy number variants (CNVs) (1.19 fold, P = 0.012), especially so for loci previously implicated in either ASD and/or intellectual disability (1.69 fold, P = 3.4 x 10(-4)). Among the CNVs there were numerous de novo and inherited events, sometimes in combination in a given family, implicating many novel ASD genes such as SHANK2, SYNGAP1, DLGAP2 and the X-linked DDX53-PTCHD1 locus. We also discovered an enrichment of CNVs disrupting functional gene sets involved in cellular proliferation, projection and motility, and GTPase/Ras signalling. Our results reveal many new genetic and functional targets in ASD that may lead to final connected pathways.

  7. Development of small high-copy-number plasmid vectors for gene expression in Caulobacter crescentus.

    PubMed

    Umelo-Njaka, E; Nomellini, J F; Yim, H; Smit, J

    2001-07-01

    Caulobacter crescentus is a bacterium with a distinctive life cycle and so it is studied as a cell development model. In addition, we have adapted this bacterium for recombinant protein production and display based on the crystalline surface protein (S)-layer and its C-terminal secretion signal. We report here the development of small, high-copy-number plasmid vectors and methods for producing an obligate expression host. The vectors are based on a narrow-host-range colE1-replicon-based plasmid commonly used in Escherichia coli, to which was added the replication origin of the IncQ plasmid RSF1010. C. crescentus strains were modified to enable plasmid replication by introduction of the RSF1010 repBAC genes at the recA locus. The small (4.0-4.5 kb) plasmids were in high copy numbers in both C. crescentus and E. coli and amenable to rapid methods for plasmid isolation and DNA sequencing. The method for introducing repBAC is suitable for other C. crescentus strains or any bacterium with an adequately homologous recA gene. Application of the vector for protein expression, based on the type I secretion system of the S-layer protein, when compared to constructs in broad-host-range plasmids, resulted in reduced time and steps required from clone construction to recombinant protein recovery and increased protein yield.

  8. Role of Mitochondrial DNA Copy Number Alteration in Human Renal Cell Carcinoma †

    PubMed Central

    Lin, Chen-Sung; Lee, Hui-Ting; Lee, Ming-Huei; Pan, Siao-Cian; Ke, Chen-Yeh; Chiu, Allen Wen-Hsiang; Wei, Yau-Huei

    2016-01-01

    We investigated the role of mitochondrial DNA (mtDNA) copy number alteration in human renal cell carcinoma (RCC). The mtDNA copy numbers of paired cancer and non-cancer parts from five resected RCC kidneys after radical nephrectomy were determined by quantitative polymerase chain reaction (Q-PCR). An RCC cell line, 786-O, was infected by lentiviral particles to knock down mitochondrial transcriptional factor A (TFAM). Null target (NT) and TFAM-knockdown (TFAM-KD) represented the control and knockdown 786-O clones, respectively. Protein or mRNA expression levels of TFAM; mtDNA-encoded NADH dehydrogenase subunit 1 (ND1), ND6 and cytochrome c oxidase subunit 2 (COX-2); nuclear DNA (nDNA)-encoded succinate dehydrogenase subunit A (SDHA); v-akt murine thymoma viral oncogene homolog 1 gene (AKT)-encoded AKT and v-myc myelocytomatosis viral oncogene homolog gene (c-MYC)-encoded MYC; glycolytic enzymes including hexokinase II (HK-II), glucose 6-phosphate isomerase (GPI), phosphofructokinase (PFK), and lactate dehydrogenase subunit A (LDHA); and hypoxia-inducible factors the HIF-1α and HIF-2α, pyruvate dehydrogenase kinase 1 (PDK1), and pyruvate dehydrogenase E1 component α subunit (PDHA1) were analyzed by Western blot or Q-PCR. Bioenergetic parameters of cellular metabolism, basal mitochondrial oxygen consumption rate (mOCRB) and basal extracellular acidification rate (ECARB), were measured by a Seahorse XFe-24 analyzer. Cell invasiveness was evaluated by a trans-well migration assay and vimentin expression. Doxorubicin was used as a chemotherapeutic agent. The results showed a decrease of mtDNA copy numbers in resected RCC tissues (p = 0.043). The TFAM-KD clone expressed lower mtDNA copy number (p = 0.034), lower mRNA levels of TFAM (p = 0.008), ND1 (p = 0.007), and ND6 (p = 0.017), and lower protein levels of TFAM and COX-2 than did the NT clone. By contrast, the protein levels of HIF-2α, HK-II, PFK, LDHA, AKT, MYC and vimentin; trans-well migration activity (p = 0

  9. PCR-based analysis of mitochondrial DNA copy number, mitochondrial DNA damage, and nuclear DNA damage

    PubMed Central

    Gonzalez-Hunt, Claudia P.; Rooney, John P.; Ryde, Ian T.; Anbalagan, Charumathi; Joglekar, Rashmi

    2016-01-01

    Because of the role DNA damage and depletion play in human disease, it is important to develop and improve tools to assess these endpoints. This unit describes PCR-based methods to measure nuclear and mitochondrial DNA damage and copy number. Long amplicon quantitative polymerase chain reaction (LA-QPCR) is used to detect DNA damage by measuring the number of polymerase-inhibiting lesions present based on the amount of PCR amplification; real-time PCR (RT-PCR) is used to calculate genome content. In this unit we provide step-by-step instructions to perform these assays in Homo sapiens, Mus musculus, Rattus norvegicus, Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, Oryzias latipes, Fundulus grandis, and Fundulus heteroclitus, and discuss the advantages and disadvantages of these assays. PMID:26828332

  10. On the origins of tandemly repeated genes: does histone gene copy number in Drosophila reflect chromosomal location?

    PubMed

    Fitch, D H; Strausbaugh, L D; Barrett, V

    1990-04-01

    Widely regarded beliefs about Drosophila histone gene copy numbers and developmental requirements have been generalized from fairly limited data since studies on histone gene arrangements and copy numbers have been largely confined to a single species, D. melanogaster. Histone gene copy numbers and chromosomal locations were examined in three species: D. melangaster, D. hydei and D. hawaiiensis. Quantitative whole genome blot analysis of DNA from diploid tissues revealed a tenfold variability in histone gene copy numbers for these three species. In situ hybridization to polytene chromosomes showed that the histone DNA (hDNA) chromosomal location is different in all three species. These observations lead us to propose a relationship between histone gene reiteration and chromosomal position.

  11. High interindividual restriction fragment length and copy number of polymorphism of a TVRI family in moderate human DNA repeats

    SciTech Connect

    Rogaev, E.I.; Shapiro, Yu.A.

    1987-06-01

    The authors describe the selection of cloned human DNA sequences, with a copy number not exceeding 1000 copies per diploid genome, and their testing for interindividual restriction fragment lengths and copy number of polymorphism (RFLCP). As a result of the investigation a DNA clone was found (TVRI-6), about 2.8 kilobase-pairs in size, for which an unusually high level of interindividual RFLCP was discovered. The TVRI-6 sequence was obtained from a bank of Pst I restriction fragments of human placental nuclear DNA cloned in pBR 322. The bank was analyzed by hybridization of colonies with phosphorus 32-labelled human nuclear DNA.

  12. Mathematical Analysis of Copy Number Variation in a DNA Sample Using Digital PCR on a Nanofluidic Device

    PubMed Central

    Dube, Simant; Qin, Jian; Ramakrishnan, Ramesh

    2008-01-01

    Copy Number Variations (CNVs) of regions of the human genome have been associated with multiple diseases. We present an algorithm which is mathematically sound and computationally efficient to accurately analyze CNV in a DNA sample utilizing a nanofluidic device, known as the digital array. This numerical algorithm is utilized to compute copy number variation and the associated statistical confidence interval and is based on results from probability theory and statistics. We also provide formulas which can be used as close approximations. PMID:18682853

  13. Low Copy Number of the AMY1 Locus Is Associated with Early-Onset Female Obesity in Finland

    PubMed Central

    Armenio, Miriam; Pekkinen, Minna; Pettersson, Maria; Valta, Helena; Lipsanen-Nyman, Marita; Mäkitie, Outi; Lindstrand, Anna

    2015-01-01

    Background The salivary α-amylase locus (AMY1) is located in a highly polymorphic multi allelic copy number variable chromosomal region. A recent report identified an association between AMY1 copy numbers and BMI in common obesity. The present study investigated the relationship between AMY1 copy number, BMI and serum amylase in childhood-onset obesity. Patients Sixty-one subjects with a history of childhood-onset obesity (mean age 19.1 years, 54% males) and 71 matched controls (19.8 yrs, 45% males) were included. All anthropometric measures were greater in the obese; their mean BMI was 40 kg/m2 (range 25-62 kg/m2) compared with 23 kg/m2 in the controls (15-32 kg/m2). Results Mean AMY1 copy numbers did not differ between the obese and control subjects, but gender differences were observed; obese men showed the highest and obese women the lowest number of AMY1 copies (p=0.045). Further, only in affected females, AMY1 copy number correlated significantly with whole body fat percent (r=-0.512, p=0.013) and BMI (r=-0.416, p=0.025). Finally, a clear linear association between AMY1 copy number and serum salivary amylase was observed in all subgroups but again differences existed between obese males and females. Conclusions In conclusion, our findings suggest that AMY1 copy number differences play a role in childhood-onset obesity but the effect differs between males and females. Further studies in larger cohorts are needed to confirm these observations. PMID:26132294

  14. DUF1220 copy number is associated with schizophrenia risk and severity: implications for understanding autism and schizophrenia as related diseases

    PubMed Central

    Searles Quick, V B; Davis, J M; Olincy, A; Sikela, J M

    2015-01-01

    The copy number of DUF1220, a protein domain implicated in human brain evolution, has been linearly associated with autism severity. Given the possibility that autism and schizophrenia are related disorders, the present study examined DUF1220 copy number variation in schizophrenia severity. There are notable similarities between autism symptoms and schizophrenia negative symptoms, and divergence between autism symptoms and schizophrenia positive symptoms. We therefore also examined DUF1220 copy number in schizophrenia subgroups defined by negative and positive symptom features, versus autistic individuals and controls. In the schizophrenic population (N=609), decreased DUF1220 copy number was linearly associated with increasing positive symptom severity (CON1 P=0.013, HLS1 P=0.0227), an association greatest in adult-onset schizophrenia (CON1 P=0.00155, HLS1 P=0.00361). In schizophrenic males, DUF1220 CON1 subtype copy number increase was associated with increased negative symptom severity (P=0.0327), a finding similar to that seen in autistic populations. Subgroup analyses demonstrated that schizophrenic individuals with predominantly positive symptoms exhibited reduced CON1 copy number compared with both controls (P=0.0237) and schizophrenic individuals with predominantly negative symptoms (P=0.0068). These findings support the view that (1) autism and schizophrenia exhibit both opposing and partially overlapping phenotypes and may represent a disease continuum, (2) variation in DUF1220 copy number contributes to schizophrenia disease risk and to the severity of both disorders, and (3) schizophrenia and autism may be, in part, a harmful by-product of the rapid and extreme evolutionary increase in DUF1220 copy number in the human species. PMID:26670282

  15. 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

    2016-12-30

    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.

  16. Measurement and relevance of neuroblastoma DNA copy number changes in the post-genome era.

    PubMed

    Mosse, Yael P; Greshock, Joel; Weber, Barbara L; Maris, John M

    2005-10-18

    The completion of the human genome sequence and the development of high throughput technology present exciting opportunities for the study of cancer cells. High-resolution analysis of chromosomal aberrations provides a global framework for understanding complex patterns in cancer cells, allowing us to ask hypothesis-driven questions. Genome-wide analysis of amplification and deletion of genomic regions is a critical step to resolving the mechanisms of neuroblastoma tumorigenesis. We used a high-resolution aCGH system that has over 4000 human BAC clones, resulting in an average coverage of 1Mb across the genome, to define whole genome copy number aberrations (CNAs) in a panel of human neuroblastoma-derived cell lines. By combining the aCGH data with meticulous regional validation studies, we showed that array CGH could reliably detect known aberrations including single copy gain or loss, that data correlate well with standard techniques used for the detection of these genetic changes, and that this technique can be used to identify novel regions of genomic imbalance.

  17. High-throughput quantitative analysis with cell growth kinetic curves for low copy number mutant cells.

    PubMed

    Xing, James Z; Gabos, Stephan; Huang, Biao; Pan, Tianhong; Huang, Min; Chen, Jie

    2012-10-01

    The mutation rate in cells induced by environmental genotoxic hazards is very low and difficult to detect using traditional cell counting assays. The established genetic toxicity tests currently recognized by regulatory authorities, such as conventional Ames and hypoxanthine guanine phosphoribosyl-transferase (HPRT) assays, are not well suited for higher-throughput screening as they require large amounts of test compounds and are very time consuming. In this study, we developed a novel cell-based assay for quantitative analysis of low numbers of cell copies with HPRT mutation induced by an environmental mutagen. The HPRT gene mutant cells induced by the mutagen were selected by 6-thioguanine (6-TG) and the cell's kinetic growth curve monitored by a real-time cell electronic sensor (RT-CES) system. When a threshold is set at a certain cell index (CI) level, samples with different initial mutant cell copies take different amounts of time in order for their growth (or CI accumulation) to cross this threshold. The more cells that are initially seeded in the test well, the faster the cell accumulation and therefore the shorter the time required to cross this threshold. Therefore, the culture time period required to cross the threshold of each sample corresponds to the original number of cells in the sample. A mutant cell growth time threshold (MT) value of each sample can be calculated to predict the number of original mutant cells. For mutagenesis determination, the RT-CES assay displayed an equal sensitivity (p > 0.05) and coefficients of variation values with good correlation to conventional HPRT mutagenic assays. Most importantly, the RT-CES mutation assay has a higher throughput than conventional cellular assays.

  18. 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.

  19. Mitochondrial DNA Copy Number and Risk of Oral Cancer: A Report from Northeast India

    PubMed Central

    Mondal, Rosy; Ghosh, Sankar Kumar; Choudhury, Javed Hussain; Seram, Anil; Sinha, Kavita; Hussain, Marine; Laskar, Ruhina Shirin; Rabha, Bijuli; Dey, Pradip; Ganguli, Sabitri; NathChoudhury, Monisha; Talukdar, Fazlur Rahman; Chaudhuri, Biswadeep; Dhar, Bishal

    2013-01-01

    Background Oral squamous cell carcinoma (OSCC) is the sixth most common cancer globally. Tobacco consumption and HPV infection, both are the major risk factor for the development of oral cancer and causes mitochondrial dysfunction. Genetic polymorphisms in xenobiotic-metabolizing enzymes modify the effect of environmental exposures, thereby playing a significant role in gene–environment interactions and hence contributing to the individual susceptibility to cancer. Here, we have investigated the association of tobacco - betel quid chewing, HPV infection, GSTM1-GSTT1 null genotypes, and tumour stages with mitochondrial DNA (mtDNA) content variation in oral cancer patients. Methodology/Principal Findings The study comprised of 124 cases of OSCC and 140 control subjects to PCR based detection was done for high-risk HPV using a consensus primer and multiplex PCR was done for detection of GSTM1-GSTT1 polymorphism. A comparative ΔCt method was used for determination of mtDNA content. The risk of OSCC increased with the ceased mtDNA copy number (Ptrend = 0.003). The association between mtDNA copy number and OSCC risk was evident among tobacco – betel quid chewers rather than tobacco – betel quid non chewers; the interaction between mtDNA copy number and tobacco – betel quid was significant (P = 0.0005). Significant difference was observed between GSTM1 - GSTT1 null genotypes (P = 0.04, P = 0.001 respectively) and HPV infection (P<0.001) with mtDNA content variation in cases and controls. Positive correlation was found with decrease in mtDNA content with the increase in tumour stages (P<0.001). We are reporting for the first time the association of HPV infection and GSTM1-GSTT1 null genotypes with mtDNA content in OSCC. Conclusion Our results indicate that the mtDNA content in tumour tissues changes with tumour stage and tobacco-betel quid chewing habits while low levels of mtDNA content suggests invasive thereby serving as a biomarker in detection

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

    PubMed Central

    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

  1. Dynamic Expansion and Contraction of cagA Copy Number in Helicobacter pylori Impact Development of Gastric Disease

    PubMed Central

    Su, Hanfu; Blum, Faith C.; Bae, Sarang; Choi, Yun Hui; Kim, Aeryun; Hong, Youngmin A.; Kim, Jinmoon; Kim, Ji-Hye; Gunawardhana, Niluka; Jeon, Yeong-Eui; Yoo, Yun-Jung; Merrell, D. Scott

    2017-01-01

    ABSTRACT Infection with Helicobacter pylori is a major risk factor for development of gastric disease, including gastric cancer. Patients infected with H. pylori strains that express CagA are at even greater risk of gastric carcinoma. Given the importance of CagA, this report describes a new molecular mechanism by which the cagA copy number dynamically expands and contracts in H. pylori. Analysis of strain PMSS1 revealed a heterogeneous population in terms of numbers of cagA copies; strains carried from zero to four copies of cagA that were arranged as direct repeats within the chromosome. Each of the multiple copies of cagA was expressed and encoded functional CagA; strains with more cagA repeats exhibited higher levels of CagA expression and increased levels of delivery and phosphorylation of CagA within host cells. This concomitantly resulted in more virulent phenotypes as measured by cell elongation and interleukin-8 (IL-8) induction. Sequence analysis of the repeat region revealed three cagA homologous areas (CHAs) within the cagA repeats. Of these, CHA-ud flanked each of the cagA copies and is likely important for the dynamic variation of cagA copy numbers. Analysis of a large panel of clinical isolates showed that 7.5% of H. pylori strains isolated in the United States harbored multiple cagA repeats, while none of the tested Korean isolates carried more than one copy of cagA. Finally, H. pylori strains carrying multiple cagA copies were differentially associated with gastric disease. Thus, the dynamic expansion and contraction of cagA copy numbers may serve as a novel mechanism by which H. pylori modulates gastric disease development. PMID:28223454

  2. 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

  3. An evaluation of new and established methods to determine T-DNA copy number and homozygosity in transgenic plants.

    PubMed

    Głowacka, Katarzyna; Kromdijk, Johannes; Leonelli, Lauriebeth; Niyogi, Krishna K; Clemente, Tom E; Long, Stephen P

    2016-04-01

    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.

  4. Assessing Mitochondrial DNA Variation and Copy Number in Lymphocytes of ~2,000 Sardinians Using Tailored Sequencing Analysis Tools

    PubMed Central

    Ding, Jun; Sidore, Carlo; Butler, Thomas J.; Wing, Mary Kate; Qian, Yong; Meirelles, Osorio; Busonero, Fabio; Tsoi, Lam C.; Maschio, Andrea; Angius, Andrea; Kang, Hyun Min; Nagaraja, Ramaiah; Cucca, Francesco; Abecasis, Gonçalo R.; Schlessinger, David

    2015-01-01

    DNA sequencing identifies common and rare genetic variants for association studies, but studies typically focus on variants in nuclear DNA and ignore the mitochondrial genome. In fact, analyzing variants in mitochondrial DNA (mtDNA) sequences presents special problems, which we resolve here with a general solution for the analysis of mtDNA in next-generation sequencing studies. The new program package comprises 1) an algorithm designed to identify mtDNA variants (i.e., homoplasmies and heteroplasmies), incorporating sequencing error rates at each base in a likelihood calculation and allowing allele fractions at a variant site to differ across individuals; and 2) an estimation of mtDNA copy number in a cell directly from whole-genome sequencing data. We also apply the methods to DNA sequence from lymphocytes of ~2,000 SardiNIA Project participants. As expected, mothers and offspring share all homoplasmies but a lesser proportion of heteroplasmies. Both homoplasmies and heteroplasmies show 5-fold higher transition/transversion ratios than variants in nuclear DNA. Also, heteroplasmy increases with age, though on average only ~1 heteroplasmy reaches the 4% level between ages 20 and 90. In addition, we find that mtDNA copy number averages ~110 copies/lymphocyte and is ~54% heritable, implying substantial genetic regulation of the level of mtDNA. Copy numbers also decrease modestly but significantly with age, and females on average have significantly more copies than males. The mtDNA copy numbers are significantly associated with waist circumference (p-value = 0.0031) and waist-hip ratio (p-value = 2.4×10-5), but not with body mass index, indicating an association with central fat distribution. To our knowledge, this is the largest population analysis to date of mtDNA dynamics, revealing the age-imposed increase in heteroplasmy, the relatively high heritability of copy number, and the association of copy number with metabolic traits. PMID:26172475

  5. DUF1220 copy number is linearly associated with increased cognitive function as measured by total IQ and mathematical aptitude scores.

    PubMed

    Davis, Jonathon M; Searles, Veronica B; Anderson, Nathan; Keeney, Jonathon; Raznahan, Armin; Horwood, L John; Fergusson, David M; Kennedy, Martin A; Giedd, Jay; Sikela, James M

    2015-01-01

    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 (R (2) = 0.13, p = 0.02), which may be driven by males aged 6-11 (R (2) = 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 (R (2) = 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 (R (2) = 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.

  6. CopyRighter: a rapid tool for improving the accuracy of microbial community profiles through lineage-specific gene copy number correction

    PubMed Central

    2014-01-01

    Background Culture-independent molecular surveys targeting conserved marker genes, most notably 16S rRNA, to assess microbial diversity remain semi-quantitative due to variations in the number of gene copies between species. Results Based on 2,900 sequenced reference genomes, we show that 16S rRNA gene copy number (GCN) is strongly linked to microbial phylogenetic taxonomy, potentially under-representing Archaea in amplicon microbial profiles. Using this relationship, we inferred the GCN of all bacterial and archaeal lineages in the Greengenes database within a phylogenetic framework. We created CopyRighter, new software which uses these estimates to correct 16S rRNA amplicon microbial profiles and associated quantitative (q)PCR total abundance. CopyRighter parses microbial profiles and, because GCN estimates are pre-computed for all taxa in the reference taxonomy, rapidly corrects GCN bias. Software validation with in silico and in vitro mock communities indicated that GCN correction results in more accurate estimates of microbial relative abundance and improves the agreement between metagenomic and amplicon profiles. Analyses of human-associated and anaerobic digester microbiomes illustrate that correction makes tangible changes to estimates of qPCR total abundance, α and β diversity, and can significantly change biological interpretation. For example, human gut microbiomes from twins were reclassified into three rather than two enterotypes after GCN correction. Conclusions The CopyRighter bioinformatic tools permits rapid correction of GCN in microbial surveys, resulting in improved estimates of microbial abundance, α and β diversity. PMID:24708850

  7. A Novel Graph-based Algorithm to Infer Recurrent Copy Number Variations in Cancer

    PubMed Central

    Chi, Chen; Ajwad, Rasif; Kuang, Qin; Hu, Pingzhao

    2016-01-01

    Many cancers have been linked to copy number variations (CNVs) in the genomic DNA. Although there are existing methods to analyze CNVs from individual samples, cancer-causing genes are more frequently discovered in regions where CNVs are common among tumor samples, also known as recurrent CNVs. Integrating multiple samples and locating recurrent CNV regions remain a challenge, both computationally and conceptually. We propose a new graph-based algorithm for identifying recurrent CNVs using the maximal clique detection technique. The algorithm has an optimal solution, which means all maximal cliques can be identified, and guarantees that the identified CNV regions are the most frequent and that the minimal regions have been delineated among tumor samples. The algorithm has successfully been applied to analyze a large cohort of breast cancer samples and identified some breast cancer-associated genes and pathways. PMID:27773988

  8. Gene discovery and functional assessment of rare copy-number variants in neurodevelopmental disorders.

    PubMed

    Iyer, Janani; Girirajan, Santhosh

    2015-09-01

    Rare copy-number variants (CNVs) are a significant cause of neurodevelopmental disorders. The sequence architecture of the human genome predisposes certain individuals to deletions and duplications within specific genomic regions. While assessment of individuals with different breakpoints has identified causal genes for certain rare CNVs, deriving gene-phenotype correlations for rare CNVs with similar breakpoints has been challenging. We present a comprehensive review of the literature related to genetic architecture that is predisposed to recurrent rearrangements, and functional evaluation of deletions, duplications and candidate genes within rare CNV intervals using mouse, zebrafish and fruit fly models. It is clear that phenotypic assessment and complete genetic evaluation of large cohorts of individuals carrying specific CNVs and functional evaluation using multiple animal models are necessary to understand the molecular genetic basis of neurodevelopmental disorders.

  9. Rare De Novo Germline Copy-Number Variation in Testicular Cancer

    PubMed Central

    Stadler, Zsofia K.; Esposito, Diane; Shah, Sohela; Vijai, Joseph; Yamrom, Boris; Levy, Dan; Lee, Yoon-ha; Kendall, Jude; Leotta, Anthony; Ronemus, Michael; Hansen, Nichole; Sarrel, Kara; Rau-Murthy, Rohini; Schrader, Kasmintan; Kauff, Noah; Klein, Robert J.; Lipkin, Steven M.; Murali, Rajmohan; Robson, Mark; Sheinfeld, Joel; Feldman, Darren; Bosl, George; Norton, Larry; Wigler, Michael; Offit, Kenneth

    2012-01-01

    Although heritable factors are an important determinant of risk of early-onset cancer, the majority of these malignancies appear to occur sporadically without identifiable risk factors. Germline de novo copy-number variations (CNVs) have been observed in sporadic neurocognitive and cardiovascular disorders. We explored this mechanism in 382 genomes of 116 early-onset cancer case-parent trios and unaffected siblings. Unique de novo germline CNVs were not observed in 107 breast or colon cancer trios or controls but were indeed found in 7% of 43 testicular germ cell tumor trios; this percentage exceeds background CNV rates and suggests a rare de novo genetic paradigm for susceptibility to some human malignancies. PMID:22863192

  10. Mitochondrial DNA detection and copy number determination in the spermatozoa of the sea urchin Arbacia lixula.

    PubMed

    De Giorgi, C; D'Alessandro, A; Saccone, C

    1992-02-14

    The Polymerase Chain reaction technique has been used in order to detect and amplify a specific region of mtDNA, in a total DNA preparation extracted from the sperm of the sea urchin Arbacia lixula. The amplified fragment is the D-loop region which hybridizes with the homologous region extracted from the egg mtDNA. The results demonstrate that mtDNA is present in sperm cell, and, since the replication origin is present it is potentially able to replicate in the zygote. Furthermore, the technique used allowed us to estimate mtDNA copy number in sea urchin sperm, which has never been done before. Our results are that sea urchin sperm cell contains between 4 and 28 mtDNA molecules.

  11. A Novel Graph-based Algorithm to Infer Recurrent Copy Number Variations in Cancer.

    PubMed

    Chi, Chen; Ajwad, Rasif; Kuang, Qin; Hu, Pingzhao

    2016-01-01

    Many cancers have been linked to copy number variations (CNVs) in the genomic DNA. Although there are existing methods to analyze CNVs from individual samples, cancer-causing genes are more frequently discovered in regions where CNVs are common among tumor samples, also known as recurrent CNVs. Integrating multiple samples and locating recurrent CNV regions remain a challenge, both computationally and conceptually. We propose a new graph-based algorithm for identifying recurrent CNVs using the maximal clique detection technique. The algorithm has an optimal solution, which means all maximal cliques can be identified, and guarantees that the identified CNV regions are the most frequent and that the minimal regions have been delineated among tumor samples. The algorithm has successfully been applied to analyze a large cohort of breast cancer samples and identified some breast cancer-associated genes and pathways.

  12. Copy number variation at the GL7 locus contributes to grain size diversity in rice.

    PubMed

    Wang, Yuexing; Xiong, Guosheng; Hu, Jiang; Jiang, Liang; Yu, Hong; Xu, Jie; Fang, Yunxia; Zeng, Longjun; Xu, Erbo; Xu, Jing; Ye, Weijun; Meng, Xiangbing; Liu, Ruifang; Chen, Hongqi; Jing, Yanhui; Wang, Yonghong; Zhu, Xudong; Li, Jiayang; Qian, Qian

    2015-08-01

    Copy number variants (CNVs) are associated with changes in gene expression levels and contribute to various adaptive traits. Here we show that a CNV at the Grain Length on Chromosome 7 (GL7) locus contributes to grain size diversity in rice (Oryza sativa L.). GL7 encodes a protein homologous to Arabidopsis thaliana LONGIFOLIA proteins, which regulate longitudinal cell elongation. Tandem duplication of a 17.1-kb segment at the GL7 locus leads to upregulation of GL7 and downregulation of its nearby negative regulator, resulting in an increase in grain length and improvement of grain appearance quality. Sequence analysis indicates that allelic variants of GL7 and its negative regulator are associated with grain size diversity and that the CNV at the GL7 locus was selected for and used in breeding. Our work suggests that pyramiding beneficial alleles of GL7 and other yield- and quality-related genes may improve the breeding of elite rice varieties.

  13. Global diversity, population stratification, and selection of human copy number variation

    PubMed Central

    Sudmant, Peter H.; Mallick, Swapan; Nelson, Bradley J.; Hormozdiari, Fereydoun; Krumm, Niklas; Huddleston, John; Coe, Bradley P.; Baker, Carl; Nordenfelt, Susanne; Bamshad, Michael; Jorde, Lynn B.; Posukh, Olga L.; Sahakyan, Hovhannes; Watkins, W. Scott; Yepiskoposyan, Levon; Abdullah, M. Syafiq; Bravi, Claudio M.; Capelli, Cristian; Hervig, Tor; Wee, Joseph T. S.; Tyler-Smith, Chris; van Driem, George; Romero, Irene Gallego; Jha, Aashish R.; Karachanak-Yankova, Sena; Toncheva, Draga; Comas, David; Henn, Brenna; Kivisild, Toomas; Ruiz-Linares, Andres; Sajantila, Antti; Metspalu, Ene; Parik, Jüri; Villems, Richard; Starikovskaya, Elena B.; Ayodo, George; Beall, Cynthia M.; Di Rienzo, Anna; Hammer, Michael; Khusainova, Rita; Khusnutdinova, Elza; Klitz, William; Winkler, Cheryl; Labuda, Damian; Metspalu, Mait; Tishkoff, Sarah A.; Dryomov, Stanislav; Sukernik, Rem; Patterson, Nick; Reich, David; Eichler, Evan E.

    2015-01-01

    In order to explore the diversity and selective signatures of duplication and deletion human copy number variants (CNVs), we sequenced 236 individuals from 125 distinct human populations. We observed that duplications exhibit fundamentally different population genetic and selective signatures than deletions and are more likely to be stratified between human populations. Through reconstruction of the ancestral human genome, we identify megabases of DNA lost in different human lineages and pinpoint large duplications that introgressed from the extinct Denisova lineage now found at high frequency exclusively in Oceanic populations. We find that the proportion of CNV base pairs to single nucleotide variant base pairs is greater among non-Africans than it is among African populations, but we conclude that this difference is likely due to unique aspects of non-African population history as opposed to differences in CNV load. PMID:26249230

  14. Assessment of megabase-scale somatic copy number variation using single-cell sequencing

    PubMed Central

    Knouse, Kristin A.; Wu, Jie; Amon, Angelika

    2016-01-01

    Megabase-scale copy number variants (CNVs) can have profound phenotypic consequences. Germline CNVs of this magnitude are associated with disease and experience negative selection. However, it is unknown whether organismal function requires that every cell maintain a balanced genome. It is possible that large somatic CNVs are tolerated or even positively selected. Single-cell sequencing is a useful tool for assessing somatic genomic heterogeneity, but its performance in CNV detection has not been rigorously tested. Here, we develop an approach that allows for reliable detection of megabase-scale CNVs in single somatic cells. We discover large CNVs in 8%–9% of cells across tissues and identify two recurrent CNVs. We conclude that large CNVs can be tolerated in subpopulations of cells, and particular CNVs are relatively prevalent within and across individuals. PMID:26772196

  15. Assessment of megabase-scale somatic copy number variation using single-cell sequencing.

    PubMed

    Knouse, Kristin A; Wu, Jie; Amon, Angelika

    2016-03-01

    Megabase-scale copy number variants (CNVs) can have profound phenotypic consequences. Germline CNVs of this magnitude are associated with disease and experience negative selection. However, it is unknown whether organismal function requires that every cell maintain a balanced genome. It is possible that large somatic CNVs are tolerated or even positively selected. Single-cell sequencing is a useful tool for assessing somatic genomic heterogeneity, but its performance in CNV detection has not been rigorously tested. Here, we develop an approach that allows for reliable detection of megabase-scale CNVs in single somatic cells. We discover large CNVs in 8%-9% of cells across tissues and identify two recurrent CNVs. We conclude that large CNVs can be tolerated in subpopulations of cells, and particular CNVs are relatively prevalent within and across individuals.

  16. Low copy number DNA profiling from isolated sperm using the aureka®-micromanipulation system.

    PubMed

    Schneider, C; Müller, U; Kilper, R; Siebertz, B

    2012-07-01

    A new cell isolation technique linked to the aureka® micromanipulation system (aureka®) was used to pick sperm from mixed samples containing sperm and epithelial cells. Both cell types were stained using the HY-LITER™ high-resolution, fluorescent staining kit. To isolate a single sperm of interest under a fluorescent microscope, a specific microsphere picking technique was used. This sensitive and reliable cell identification and isolation technique enables low-copy-number (LCN) DNA profiling, as few as 20 sperm are sufficient for obtaining a full short tandem repeat (STR) profile without any allelic drop out. The presented protocol covers the whole workflow, from sample staining and cell pick up to STR analysis.

  17. Excessive genomic DNA copy number variation in the Li–Fraumeni cancer predisposition syndrome

    PubMed Central

    Shlien, Adam; Tabori, Uri; Marshall, Christian R.; Pienkowska, Malgorzata; Feuk, Lars; Novokmet, Ana; Nanda, Sonia; Druker, Harriet; Scherer, Stephen W.; Malkin, David

    2008-01-01

    DNA copy number variations (CNVs) are a significant and ubiquitous source of inherited human genetic variation. However, the importance of CNVs to cancer susceptibility and tumor progression has not yet been explored. Li–Fraumeni syndrome (LFS) is an autosomal dominantly inherited disorder characterized by a strikingly increased risk of early-onset breast cancer, sarcomas, brain tumors and other neoplasms in individuals harboring germline TP53 mutations. Known genetic determinants of LFS do not fully explain the variable clinical phenotype in affected family members. As part of a wider study of CNVs and cancer, we conducted a genome-wide profile of germline CNVs in LFS families. Here, by examining DNA from a large healthy population and an LFS cohort using high-density oligonucleotide arrays, we show that the number of CNVs per genome is well conserved in the healthy population, but strikingly enriched in these cancer-prone individuals. We found a highly significant increase in CNVs among carriers of germline TP53 mutations with a familial cancer history. Furthermore, we identified a remarkable number of genomic regions in which known cancer-related genes coincide with CNVs, in both LFS families and healthy individuals. Germline CNVs may provide a foundation that enables the more dramatic chromosomal changes characteristic of TP53-related tumors to be established. Our results suggest that screening families predisposed to cancer for CNVs may identify individuals with an abnormally high number of these events. PMID:18685109

  18. Genome-wide copy number variation analysis in a Chinese autism spectrum disorder cohort

    PubMed Central

    Guo, Hui; Peng, Yu; Hu, Zhengmao; Li, Ying; Xun, Guanglei; Ou, Jianjun; Sun, Liangdan; Xiong, Zhimin; Liu, Yanling; Wang, Tianyun; Chen, Jingjing; Xia, Lu; Bai, Ting; Shen, Yidong; Tian, Qi; Hu, Yiqiao; Shen, Lu; Zhao, Rongjuan; Zhang, Xuejun; Zhang, Fengyu; Zhao, Jingping; Zou, Xiaobing; Xia, Kun

    2017-01-01

    Autism spectrum disorder (ASD) describes a group of neurodevelopmental disorders with high heritability, although the underlying genetic determinants of ASDs remain largely unknown. Large-scale whole-genome studies of copy number variation in Han Chinese samples are still lacking. We performed a genome-wide copy number variation analysis of 343 ASD trios, 203 patients with sporadic cases and 988 controls in a Chinese population using Illumina genotyping platforms to identify CNVs and related genes that may contribute to ASD risk. We identified 32 rare CNVs larger than 1 Mb in 31 patients. ASD patients were found to carry a higher global burden of rare, large CNVs than controls. Recurrent de novo or case-private CNVs were found at 15q11-13, Xp22.3, 15q13.1–13.2, 3p26.3 and 2p12. The de novo 15q11–13 duplication was more prevalent in this Chinese population than in those with European ancestry. Several genes, including GRAMD2 and STAM, were implicated as novel ASD risk genes when integrating whole-genome CNVs and whole-exome sequencing data. We also identified several CNVs that include known ASD genes (SHANK3, CDH10, CSMD1) or genes involved in nervous system development (NYAP2, ST6GAL2, GRM6). Besides, our study also implicated Contactins-NYAPs-WAVE1 pathway in ASD pathogenesis. Our findings identify ASD-related CNVs in a Chinese population and implicate novel ASD risk genes and related pathway for further study. PMID:28281572

  19. Characterization of copy number variation in genomic regions containing STR loci using array comparative genomic hybridization.

    PubMed

    Repnikova, Elena A; Rosenfeld, Jill A; Bailes, Andrea; Weber, Cecilia; Erdman, Linda; McKinney, Aimee; Ramsey, Sarah; Hashimoto, Sayaka; Lamb Thrush, Devon; Astbury, Caroline; Reshmi, Shalini C; Shaffer, Lisa G; Gastier-Foster, Julie M; Pyatt, Robert E

    2013-09-01

    Short tandem repeat (STR) loci are commonly used in forensic casework, familial analysis for human identification, and for monitoring hematopoietic cell engraftment after bone marrow transplant. Unexpected genetic variation leading to sequence and length differences in STR loci can complicate STR typing, and presents challenges in casework interpretation. Copy number variation (CNV) is a relatively recently identified form of genetic variation consisting of genomic regions present at variable copy numbers within an individual compared to a reference genome. Large scale population studies have demonstrated that likely all individuals carry multiple regions with CNV of 1kb in size or greater in their genome. To date, no study correlating genomic regions containing STR loci with CNV has been conducted. In this study, we analyzed results from 32,850 samples sent for clinical array comparative genomic hybridization (CGH) analysis for the presence of CNV at regions containing the 13 CODIS (Combined DNA Index System) STR, and the Amelogenin X (AMELX) and Amelogenin Y (AMELY) loci. Thirty-two individuals with CNV involving STR loci on chromosomes 2, 4, 7, 11, 12, 13, 16, and 21, and twelve with CNV involving the AMELX/AMELY loci were identified. These results were correlated with data from publicly available databases housing information on CNV identified in normal populations and additional clinical cases. These collective results demonstrate the presence of CNV in regions containing 9 of the 13 CODIS STR and AMELX/Y loci. Further characterization of STR profiles within regions of CNV, additional cataloging of these variants in multiple populations, and contributing such examples to the public domain will provide valuable information for reliable use of these loci.

  20. 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.

  1. Association of telomere length and mitochondrial DNA copy number in a community sample of healthy adults.

    PubMed

    Tyrka, Audrey R; Carpenter, Linda L; Kao, Hung-Teh; Porton, Barbara; Philip, Noah S; Ridout, Samuel J; Ridout, Kathryn K; Price, Lawrence H

    2015-06-01

    Cellular aging plays a role in longevity and senescence, and has been implicated in medical and psychiatric conditions, including heart disease, cancer, major depression and posttraumatic stress disorder. Telomere shortening and mitochondrial dysfunction are thought to be central to the cellular aging process. The present study examined the association between mitochondrial DNA (mtDNA) copy number and telomere length in a sample of medically healthy adults. Participants (total n=392) were divided into 4 groups based on the presence or absence of early life adversity and lifetime psychopathology: No Adversity/No Disorder, n=136; Adversity/No Disorder, n=91; No Adversity/Disorder, n=46; Adversity/Disorder, n=119. Telomere length and mtDNA copy number were measured using quantitative polymerase chain reaction. There was a positive correlation between mtDNA and telomere length in the entire sample (r=0.120, p<0.001) and in each of the four groups of participants (No Adversity/No Disorder, r=0.291, p=0.001; Adversity/No Disorder r=0.279, p=0.007; No Adversity/Disorder r=0.449, p=0.002; Adversity/Disorder, r=0.558, p<0.001). These correlations remained significant when controlling for age, smoking, and body mass index and establish an association between mtDNA and telomere length in a large group of women and men both with and without early adversity and psychopathology, suggesting co-regulation of telomeres and mitochondrial function. The mechanisms underlying this association may be important in the pathophysiology of age-related medical conditions, such as heart disease and cancer, as well as for stress-associated psychiatric disorders.

  2. Copy-number disorders are a common cause of congenital kidney malformations.

    PubMed

    Sanna-Cherchi, Simone; Kiryluk, Krzysztof; Burgess, Katelyn E; Bodria, Monica; Sampson, Matthew G; Hadley, Dexter; Nees, Shannon N; Verbitsky, Miguel; Perry, Brittany J; Sterken, Roel; Lozanovski, Vladimir J; Materna-Kiryluk, Anna; Barlassina, Cristina; Kini, Akshata; Corbani, Valentina; Carrea, Alba; Somenzi, Danio; Murtas, Corrado; Ristoska-Bojkovska, Nadica; Izzi, Claudia; Bianco, Beatrice; Zaniew, Marcin; Flogelova, Hana; Weng, Patricia L; Kacak, Nilgun; Giberti, Stefania; Gigante, Maddalena; Arapovic, Adela; Drnasin, Kristina; Caridi, Gianluca; Curioni, Simona; Allegri, Franca; Ammenti, Anita; Ferretti, Stefania; Goj, Vinicio; Bernardo, Luca; Jobanputra, Vaidehi; Chung, Wendy K; Lifton, Richard P; Sanders, Stephan; State, Matthew; Clark, Lorraine N; Saraga, Marijan; Padmanabhan, Sandosh; Dominiczak, Anna F; Foroud, Tatiana; Gesualdo, Loreto; Gucev, Zoran; Allegri, Landino; Latos-Bielenska, Anna; Cusi, Daniele; Scolari, Francesco; Tasic, Velibor; Hakonarson, Hakon; Ghiggeri, Gian Marco; Gharavi, Ali G

    2012-12-07

    We examined the burden of large, rare, copy-number variants (CNVs) in 192 individuals with renal hypodysplasia (RHD) and replicated findings in 330 RHD cases from two independent cohorts. CNV distribution was significantly skewed toward larger gene-disrupting events in RHD cases compared to 4,733 ethnicity-matched controls (p = 4.8 × 10(-11)). This excess was attributable to known and novel (i.e., not present in any database or in the literature) genomic disorders. All together, 55/522 (10.5%) RHD cases harbored 34 distinct known genomic disorders, which were detected in only 0.2% of 13,839 population controls (p = 1.2 × 10(-58)). Another 32 (6.1%) RHD cases harbored large gene-disrupting CNVs that were absent from or extremely rare in the 13,839 population controls, identifying 38 potential novel or rare genomic disorders for this trait. Deletions at the HNF1B locus and the DiGeorge/velocardiofacial locus were most frequent. However, the majority of disorders were detected in a single individual. Genomic disorders were detected in 22.5% of individuals with multiple malformations and 14.5% of individuals with isolated urinary-tract defects; 14 individuals harbored two or more diagnostic or rare CNVs. Strikingly, the majority of the known CNV disorders detected in the RHD cohort have previous associations with developmental delay or neuropsychiatric diseases. Up to 16.6% of individuals with kidney malformations had a molecular diagnosis attributable to a copy-number disorder, suggesting kidney malformations as a sentinel manifestation of pathogenic genomic imbalances. A search for pathogenic CNVs should be considered in this population for the diagnosis of their specific genomic disorders and for the evaluation of the potential for developmental delay.

  3. Rare De Novo Copy Number Variants in Patients with Congenital Pulmonary Atresia

    PubMed Central

    Xie, Li; Chen, Jin-Lan; Zhang, Wei-Zhi; Wang, Shou-Zheng; Zhao, Tian-Li; Huang, Can; Wang, Jian; Yang, Jin-Fu; Yang, Yi-Feng; Tan, Zhi-Ping

    2014-01-01

    Background Ongoing studies using genomic microarrays and next-generation sequencing have demonstrated that the genetic contributions to cardiovascular diseases have been significantly ignored in the past. The aim of this study was to identify rare copy number variants in individuals with congenital pulmonary atresia (PA). Methods and Results Based on the hypothesis that rare structural variants encompassing key genes play an important role in heart development in PA patients, we performed high-resolution genome-wide microarrays for copy number variations (CNVs) in 82 PA patient-parent trios and 189 controls with an Illumina SNP array platform. CNVs were identified in 17/82 patients (20.7%), and eight of these CNVs (9.8%) are considered potentially pathogenic. Five de novo CNVs occurred at two known congenital heart disease (CHD) loci (16p13.1 and 22q11.2). Two de novo CNVs that may affect folate and vitamin B12 metabolism were identified for the first time. A de novo 1-Mb deletion at 17p13.2 may represent a rare genomic disorder that involves mild intellectual disability and associated facial features. Conclusions Rare CNVs contribute to the pathogenesis of PA (9.8%), suggesting that the causes of PA are heterogeneous and pleiotropic. Together with previous data from animal models, our results might help identify a link between CHD and folate-mediated one-carbon metabolism (FOCM). With the accumulation of high-resolution SNP array data, these previously undescribed rare CNVs may help reveal critical gene(s) in CHD and may provide novel insights about CHD pathogenesis. PMID:24826987

  4. NDRG2 gene copy number is not altered in colorectal carcinoma

    PubMed Central

    Lorentzen, Anders; Mitchelmore, Cathy

    2017-01-01

    AIM To investigate if the down-regulation of N-myc Downstream Regulated Gene 2 (NDRG2) expression in colorectal carcinoma (CRC) is due to loss of the NDRG2 allele(s). METHODS The following were investigated in the human colorectal cancer cell lines DLD-1, LoVo and SW-480: NDRG2 mRNA expression levels using quantitative reverse transcription-polymerase chain reaction (qRT-PCR); interaction of the MYC gene-regulatory protein with the NDRG2 promoter using chromatin immunoprecipitation; and NDRG2 promoter methylation using bisulfite sequencing. Furthermore, we performed qPCR to analyse the copy numbers of NDRG2 and MYC genes in the above three cell lines, 8 normal colorectal tissue samples and 40 CRC tissue samples. RESULTS As expected, NDRG2 mRNA levels were low in the three colorectal cancer cell lines, compared to normal colon. Endogenous MYC protein interacted with the NDRG2 core promoter in all three cell lines. In addition, the NDRG2 promoter was heavily methylated in these cell lines, suggesting an epigenetic regulatory mechanism. Unaltered gene copy numbers of NDRG2 were observed in the three cell lines. In the colorectal tissues, one normal and three CRC samples showed partial or complete loss of one NDRG2 allele. In contrast, the MYC gene was amplified in one cell line and in more than 40% of the CRC cases. CONCLUSION Our study suggests that the reduction in NDRG2 expression observed in CRC is due to transcriptional repression by MYC and promoter methylation, and is not due to allelic loss. PMID:28246586

  5. Copy number variation in patients with disorders of sex development due to 46,XY gonadal dysgenesis.

    PubMed

    White, Stefan; Ohnesorg, Thomas; Notini, Amanda; Roeszler, Kelly; Hewitt, Jacqueline; Daggag, Hinda; Smith, Craig; Turbitt, Erin; Gustin, Sonja; van den Bergen, Jocelyn; Miles, Denise; Western, Patrick; Arboleda, Valerie; Schumacher, Valerie; Gordon, Lavinia; Bell, Katrina; Bengtsson, Henrik; Speed, Terry; Hutson, John; Warne, Garry; Harley, Vincent; Koopman, Peter; Vilain, Eric; Sinclair, Andrew

    2011-03-07

    Disorders of sex development (DSD), ranging in severity from mild genital abnormalities to complete sex reversal, represent a major concern for patients and their families. DSD are often due to disruption of the genetic programs that regulate gonad development. Although some genes have been identified in these developmental pathways, the causative mutations have not been identified in more than 50% 46,XY DSD cases. We used the Affymetrix Genome-Wide Human SNP Array 6.0 to analyse copy number variation in 23 individuals with unexplained 46,XY DSD due to gonadal dysgenesis (GD). Here we describe three discrete changes in copy number that are the likely cause of the GD. Firstly, we identified a large duplication on the X chromosome that included DAX1 (NR0B1). Secondly, we identified a rearrangement that appears to affect a novel gonad-specific regulatory region in a known testis gene, SOX9. Surprisingly this patient lacked any signs of campomelic dysplasia, suggesting that the deletion affected expression of SOX9 only in the gonad. Functional analysis of potential SRY binding sites within this deleted region identified five putative enhancers, suggesting that sequences additional to the known SRY-binding TES enhancer influence human testis-specific SOX9 expression. Thirdly, we identified a small deletion immediately downstream of GATA4, supporting a role for GATA4 in gonad development in humans. These CNV analyses give new insights into the pathways involved in human gonad development and dysfunction, and suggest that rearrangements of non-coding sequences disturbing gene regulation may account for significant proportion of DSD cases.

  6. SIRPB1 copy-number polymorphism as candidate quantitative trait locus for impulsive-disinhibited personality.

    PubMed

    Laplana, M; Royo, J L; García, L F; Aluja, A; Gomez-Skarmeta, J L; Fibla, J

    2014-09-01

    Impulsive-disinhibited personality (IDP) is a behavioral trait mainly characterized by seeking immediate gratification at the expense of more enduring or long-term gains. This trait has a major role in the development of several disinhibitory behaviors and syndromes, including psychopathy, attention-deficit and hyperactivity disorder, cluster-B personality disorders, criminality and alcoholism. Available data consistently support a strong heritable component, accounting for 30-60% of the observed variance in personality traits. A genome-wide analysis of copy-number variants was designed to identify novel genetic pathways associated with the IDP trait, using a series of 261 male participants with maximized opposite IDP scores. Quantitative trait locus analysis of candidate copy-number variants (CNVs) was conducted across the entire IDP continuum. Functional effects of associated variants were evaluated in zebrafish embryos. A common CNV mapping to the immune-related gene SIRPB1 was significantly associated with IDP scores in a dose-dependent manner (β=-0.172, P<0.017). Expression quantitative trait locus analysis of the critical region revealed higher SIRPB1 mRNA levels associated with the haplotype containing the deleted allele (P<0.0007). Epigenetic marks highlighted the presence of two potential insulators within the deleted region, confirmed by functional assays in zebrafish embryos, which suggests that SIRPB1 expression rates are affected by the presence/absence of the insulator regions. Upregulation of SIRPB1 has been described in prefrontal cortex of patients with schizophrenia, providing a link between SIRPB1 and diseases involving disinhibition and failure to control impulsivity. We propose SIRPB1 as a novel candidate gene to account for phenotypic differences observed in the IDP trait.

  7. Identification of copy number variants in whole-genome data using Reference Coverage Profiles.

    PubMed

    Glusman, Gustavo; Severson, Alissa; Dhankani, Varsha; Robinson, Max; Farrah, Terry; Mauldin, Denise E; Stittrich, Anna B; Ament, Seth A; Roach, Jared C; Brunkow, Mary E; Bodian, Dale L; Vockley, Joseph G; Shmulevich, Ilya; Niederhuber, John E; Hood, Leroy

    2015-01-01

    The identification of DNA copy numbers from short-read sequencing data remains a challenge for both technical and algorithmic reasons. The raw data for these analyses are measured in tens to hundreds of gigabytes per genome; transmitting, storing, and analyzing such large files is cumbersome, particularly for methods that analyze several samples simultaneously. We developed a very efficient representation of depth of coverage (150-1000× compression) that enables such analyses. Current methods for analyzing variants in whole-genome sequencing (WGS) data frequently miss copy number variants (CNVs), particularly hemizygous deletions in the 1-100 kb range. To fill this gap, we developed a method to identify CNVs in individual genomes, based on comparison to joint profiles pre-computed from a large set of genomes. We analyzed depth of coverage in over 6000 high quality (>40×) genomes. The depth of coverage has strong sequence-specific fluctuations only partially explained by global parameters like %GC. To account for these fluctuations, we constructed multi-genome profiles representing the observed or inferred diploid depth of coverage at each position along the genome. These Reference Coverage Profiles (RCPs) take into account the diverse technologies and pipeline versions used. Normalization of the scaled coverage to the RCP followed by hidden Markov model (HMM) segmentation enables efficient detection of CNVs and large deletions in individual genomes. Use of pre-computed multi-genome coverage profiles improves our ability to analyze each individual genome. We make available RCPs and tools for performing these analyses on personal genomes. We expect the increased sensitivity and specificity for individual genome analysis to be critical for achieving clinical-grade genome interpretation.

  8. Copy-Number Disorders Are a Common Cause of Congenital Kidney Malformations

    PubMed Central

    Sanna-Cherchi, Simone; Kiryluk, Krzysztof; Burgess, Katelyn E.; Bodria, Monica; Sampson, Matthew G.; Hadley, Dexter; Nees, Shannon N.; Verbitsky, Miguel; Perry, Brittany J.; Sterken, Roel; Lozanovski, Vladimir J.; Materna-Kiryluk, Anna; Barlassina, Cristina; Kini, Akshata; Corbani, Valentina; Carrea, Alba; Somenzi, Danio; Murtas, Corrado; Ristoska-Bojkovska, Nadica; Izzi, Claudia; Bianco, Beatrice; Zaniew, Marcin; Flogelova, Hana; Weng, Patricia L.; Kacak, Nilgun; Giberti, Stefania; Gigante, Maddalena; Arapovic, Adela; Drnasin, Kristina; Caridi, Gianluca; Curioni, Simona; Allegri, Franca; Ammenti, Anita; Ferretti, Stefania; Goj, Vinicio; Bernardo, Luca; Jobanputra, Vaidehi; Chung, Wendy K.; Lifton, Richard P.; Sanders, Stephan; State, Matthew; Clark, Lorraine N.; Saraga, Marijan; Padmanabhan, Sandosh; Dominiczak, Anna F.; Foroud, Tatiana; Gesualdo, Loreto; Gucev, Zoran; Allegri, Landino; Latos-Bielenska, Anna; Cusi, Daniele; Scolari, Francesco; Tasic, Velibor; Hakonarson, Hakon; Ghiggeri, Gian Marco; Gharavi, Ali G.

    2012-01-01

    We examined the burden of large, rare, copy-number variants (CNVs) in 192 individuals with renal hypodysplasia (RHD) and replicated findings in 330 RHD cases from two independent cohorts. CNV distribution was significantly skewed toward larger gene-disrupting events in RHD cases compared to 4,733 ethnicity-matched controls (p = 4.8 × 10−11). This excess was attributable to known and novel (i.e., not present in any database or in the literature) genomic disorders. All together, 55/522 (10.5%) RHD cases harbored 34 distinct known genomic disorders, which were detected in only 0.2% of 13,839 population controls (p = 1.2 × 10−58). Another 32 (6.1%) RHD cases harbored large gene-disrupting CNVs that were absent from or extremely rare in the 13,839 population controls, identifying 38 potential novel or rare genomic disorders for this trait. Deletions at the HNF1B locus and the DiGeorge/velocardiofacial locus were most frequent. However, the majority of disorders were detected in a single individual. Genomic disorders were detected in 22.5% of individuals with multiple malformations and 14.5% of individuals with isolated urinary-tract defects; 14 individuals harbored two or more diagnostic or rare CNVs. Strikingly, the majority of the known CNV disorders detected in the RHD cohort have previous associations with developmental delay or neuropsychiatric diseases. Up to 16.6% of individuals with kidney malformations had a molecular diagnosis attributable to a copy-number disorder, suggesting kidney malformations as a sentinel manifestation of pathogenic genomic imbalances. A search for pathogenic CNVs should be considered in this population for the diagnosis of their specific genomic disorders and for the evaluation of the potential for developmental delay. PMID:23159250

  9. 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.

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

    PubMed Central

    Koiffmann, Celia Priszkulnik

    2012-01-01

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

  11. Expression, tandem repeat copy number variation and stability of four macrosatellite arrays in the human genome

    PubMed Central

    2010-01-01

    Background Macrosatellites are some of the largest variable number tandem repeats in the human genome, but what role these unusual sequences perform is unknown. Their importance to human health is clearly demonstrated by the 4q35 macrosatellite D4Z4 that is associated with the onset of the muscle degenerative disease facioscapulohumeral muscular dystrophy. Nevertheless, many other macrosatellite arrays in the human genome remain poorly characterized. Results Here we describe the organization, tandem repeat copy number variation, transmission stability and expression of four macrosatellite arrays in the human genome: the TAF11-Like array located on chromosomes 5p15.1, the SST1 arrays on 4q28.3 and 19q13.12, the PRR20 array located on chromosome 13q21.1, and the ZAV array at 9q32. All are polymorphic macrosatellite arrays that at least for TAF11-Like and SST1 show evidence of meiotic instability. With the exception of the SST1 array that is ubiquitously expressed, all are expressed at high levels in the testis and to a lesser extent in the brain. Conclusions Our results extend the number of characterized macrosatellite arrays in the human genome and provide the foundation for formulation of hypotheses to begin assessing their functional role in the human genome. PMID:21078170

  12. ParseCNV integrative copy number variation association software with quality tracking.

    PubMed

    Glessner, Joseph T; Li, Jin; Hakonarson, Hakon

    2013-03-01

    A number of copy number variation (CNV) calling algorithms exist; however, comprehensive software tools for CNV association studies are lacking. We describe ParseCNV, unique software that takes CNV calls and creates probe-based statistics for CNV occurrence in both case-control design and in family based studies addressing both de novo and inheritance events, which are then summarized based on CNV regions (CNVRs). CNVRs are defined in a dynamic manner to allow for a complex CNV overlap while maintaining precise association region. Using this approach, we avoid failure to converge and non-monotonic curve fitting weaknesses of programs, such as CNVtools and CNVassoc, and although Plink is easy to use, it only provides combined CNV state probe-based statistics, not state-specific CNVRs. Existing CNV association methods do not provide any quality tracking information to filter confident associations, a key issue which is fully addressed by ParseCNV. In addition, uncertainty in CNV calls underlying CNV associations is evaluated to verify significant results, including CNV overlap profiles, genomic context, number of probes supporting the CNV and single-probe intensities. When optimal quality control parameters are followed using ParseCNV, 90% of CNVs validate by polymerase chain reaction, an often problematic stage because of inadequate significant association review. ParseCNV is freely available at http://parsecnv.sourceforge.net.

  13. Clinical features associated with copy number variations of the 14q32 imprinted gene cluster.

    PubMed

    Rosenfeld, Jill A; Fox, Joyce E; Descartes, Maria; Brewer, Fallon; Stroud, Tracy; Gorski, Jerome L; Upton, Sheila J; Moeschler, John B; Monteleone, Berrin; Neill, Nicholas J; Lamb, Allen N; Ballif, Blake C; Shaffer, Lisa G; Ravnan, J Britt

    2015-02-01

    Uniparental disomy (UPD) for imprinted chromosomes can cause abnormal phenotypes due to absent or overexpression of imprinted genes. UPD(14)pat causes a unique constellation of features including thoracic skeletal anomalies, polyhydramnios, placentomegaly, and limited survival; its hypothesized cause is overexpression of paternally expressed RTL1, due to absent regulatory effects of maternally expressed RTL1as. UPD(14)mat causes a milder condition with hypotonia, growth failure, and precocious puberty; its hypothesized cause is absence of paternally expressed DLK1. To more clearly establish how gains and losses of imprinted genes can cause disease, we report six individuals with copy number variations of the imprinted 14q32 region identified through clinical microarray-based comparative genomic hybridization. Three individuals presented with UPD(14)mat-like phenotypes (Temple syndrome) and had apparently de novo deletions spanning the imprinted region, including DLK1. One of these deletions was shown to be on the paternal chromosome. Two individuals with UPD(14)pat-like phenotypes had 122-154kb deletions on their maternal chromosomes that included RTL1as but not the differentially methylated regions that regulate imprinted gene expression, providing further support for RTL1 overexpression as a cause for the UPD(14)pat phenotype. The sixth individual is tetrasomic for a 1.7Mb segment, including the imprinted region, and presents with intellectual disability and seizures but lacks significant phenotypic overlap with either UPD(14) syndrome. Therefore, the 14q32 imprinted region is dosage sensitive, with deletions of different critical regions causing UPD(14)mat- and UPD(14)pat-like phenotypes, while copy gains are likely insufficient to recapitulate these phenotypes.

  14. Copy number variation at the breakpoint region of isochromosome 17q

    PubMed Central

    Carvalho, Claudia M.B.; Lupski, James R.

    2008-01-01

    Isochromosome 17q, or i(17q), is one of the most frequent nonrandom changes occurring in human neoplasia. Most of the i(17q) breakpoints cluster within a ∼240-kb interval located in the Smith-Magenis syndrome common deletion region in 17p11.2. The breakpoint cluster region is characterized by a complex architecture with large (∼38–49 kb), inverted and directly oriented, low-copy repeats (LCRs), known as REPA and REPB that apparently lead to genomic instability and facilitate somatic genetic rearrangements. Through the analysis of bacterial artificial chromosome (BAC) clones, pulsed-field gel electrophoresis (PFGE), and public array comparative genomic hybridization (array CGH) data, we show that the REPA/B structure is also susceptible to frequent meiotic rearrangements. It is a highly dynamic genomic region undergoing deletions, inversions, and duplications likely produced by non-allelic homologous recombination (NAHR) mediated by the highly identical SNORD3@, also known as U3, gene cluster present therein. We detected at least seven different REPA/B structures in samples from 29 individuals of which six represented potentially novel structures. Two polymorphic copy-number variation (CNV) variants, detected in 20% of samples, could be structurally described along with the likely underlying molecular mechanism for formation. Our data show the high susceptibility to rearrangements at the i(17q) breakpoint cluster region in the general population and exemplifies how large genomic regions laden with LCRs still represent a technical challenge for both determining specific structure and assaying population variation. The variant REPA/B structures identified may have different susceptibilities for inducing i(17q), thus potentially representing important risk alleles for tumor progression. PMID:18714090

  15. Copy number variations on chromosome 12q14 in patients with normal tension glaucoma

    PubMed Central

    Fingert, John H.; Robin, Alan L.; Stone, Jennifer L.; Roos, Ben R.; Davis, Lea K.; Scheetz, Todd E.; Bennett, Steve R.; Wassink, Thomas H.; Kwon, Young H.; Alward, Wallace L.M.; Mullins, Robert F.; Sheffield, Val C.; Stone, Edwin M.

    2011-01-01

    We report identification of a novel genetic locus (GLC1P) for normal tension glaucoma (NTG) on chromosome 12q14 using linkage studies of an African-American pedigree (maximum non-parametric linkage score = 19.7, max LOD score = 2.7). Subsequent comparative genomic hybridization and quantitative polymerase chain reaction (PCR) experiments identified a 780 kbp duplication within the GLC1P locus that is co-inherited with NTG in the pedigree. Real-time PCR studies showed that the genes within this duplication [TBK1 (TANK-binding kinase 1), XPOT, RASSF3 and GNS] are all expressed in the human retina. Cohorts of 478 glaucoma patients (including 152 NTG patients), 100 normal control subjects and 400 age-related macular degeneration patients were subsequently tested for copy number variation in GLC1P. Overlapping duplications were detected in 2 (1.3%) of the 152 NTG subjects, one of which had a strong family history of glaucoma. These duplications defined a 300 kbp critical region of GLC1P that spans two genes (TBK1 and XPOT). Microarray expression experiments and northern blot analysis using RNA obtained from human skin fibroblast cells showed that duplication of chromosome 12q14 results in increased TBK1 and GNS transcription. Finally, immunohistochemistry studies showed that TBK1 is expressed in the ganglion cells, nerve fiber layer and microvasculature of the human retina. Together, these data link the duplication of genes on chromosome 12q14 with familial NTG and suggest that an extra copy of the encompassed TBK1 gene is likely responsible for these cases of glaucoma. However, animal studies will be necessary to rule out a role for the other duplicated or neighboring genes. PMID:21447600

  16. Integrative analysis of copy number and transcriptional expression profiles in esophageal cancer to identify a novel driver gene for therapy

    PubMed Central

    Dong, Gaochao; Mao, Qixing; Yu, Decai; Zhang, Yi; Qiu, Mantang; Dong, Gaoyue; Chen, Qiang; Xia, Wenjie; Wang, Jie; Xu, Lin; Jiang, Feng

    2017-01-01

    An increasing amount of evidence has highlighted the critical roles that copy number variants play in cancer progression. Here, we systematically analyzed the copy number alterations and differentially transcribed genes. Integrative analysis of the association between copy number variants and differential gene expression suggested that copy number variants will lead to aberrant expression of the corresponding genes. We performed a KEGG pathway and GO analysis, which revealed that cell cycle may have an effective role in the progression of esophageal cancer. FAM60A was then screened out as a potential prognostic factor through survival analysis and correlation analysis with clinical-pathological parameters. We subsequently showed that silencing of FAM60A could inhibit esophageal carcinoma tumor cell growth, migration and invasion in vitro. Through the bioinformatic analysis, we predict that FAM60A may act as a transcriptional factor to regulate genes that are correlated with each cell cycle. In summary, we comprehensively analyzed copy number segments and transcriptional expression profiles, which provided a novel approach to identify clinical biomarkers and therapeutic targets of esophageal carcinoma. PMID:28169357

  17. Copy Number Variations in the Survival Motor Neuron Genes: Implications for Spinal Muscular Atrophy and Other Neurodegenerative Diseases

    PubMed Central

    Butchbach, Matthew E. R.

    2016-01-01

    Proximal spinal muscular atrophy (SMA), a leading genetic cause of infant death worldwide, is an early-onset, autosomal recessive neurodegenerative disease characterized by the loss of spinal α-motor neurons. This loss of α-motor neurons is associated with muscle weakness and atrophy. SMA can be classified into five clinical grades based on age of onset and severity of the disease. Regardless of clinical grade, proximal SMA results from the loss or mutation of SMN1 (survival motor neuron 1) on chromosome 5q13. In humans a large tandem chromosomal duplication has lead to a second copy of the SMN gene locus known as SMN2. SMN2 is distinguishable from SMN1 by a single nucleotide difference that disrupts an exonic splice enhancer in exon 7. As a result, most of SMN2 mRNAs lack exon 7 (SMNΔ7) and produce a protein that is both unstable and less than fully functional. Although only 10–20% of the SMN2 gene product is fully functional, increased genomic copies of SMN2 inversely correlates with disease severity among individuals with SMA. Because SMN2 copy number influences disease severity in SMA, there is prognostic value in accurate measurement of SMN2 copy number from patients being evaluated for SMA. This prognostic value is especially important given that SMN2 copy number is now being used as an inclusion criterion for SMA clinical trials. In addition to SMA, copy number variations (CNVs) in the SMN genes can affect the clinical severity of other neurological disorders including amyotrophic lateral sclerosis (ALS) and progressive muscular atrophy (PMA). This review will discuss how SMN1 and SMN2 CNVs are detected and why accurate measurement of SMN1 and SMN2 copy numbers is relevant for SMA and other neurodegenerative diseases. PMID:27014701

  18. New ideas in epilepsy genetics: novel epilepsy genes, copy number alterations, and gene regulation.

    PubMed

    Gurnett, Christina A; Hedera, Peter

    2007-03-01

    The majority of genes associated with epilepsy syndromes to date are ion channel genes. Selection bias may have allowed us to establish their role in epilepsy based on a priori knowledge of the significance of these proteins in regulating neuronal excitability. There are, however, more than 3000 genes expressed at the synapse, as well as many other genes expressed nearby in supporting cells and glia that can likewise regulate excitability. Identification of new genes involved in epilepsy may arise from studying the targets of anticonvulsant medications, ascertainment of an epileptic phenotype in mice, or as a result of positional cloning efforts. There are several loci for idiopathic focal and generalized epilepsies that lie in chromosomal regions that are devoid of known ion channels; therefore, the number of novel genes involved in epilepsy is likely to increase. Establishing the role of these novel genes in the pathogenesis of epilepsy has not been an easy task compared with the relative ease with which ion channel mutations can be studied. This review will describe several novel epilepsy genes and will then discuss other genetic causes of epilepsy, including alterations of chromosomal copy number and gene regulatory elements.

  19. Copy number variations in Hanwoo and Yanbian cattle genomes using the massively parallel sequencing data.

    PubMed

    Choi, Jung-Woo; Chung, Won-Hyong; Lim, Kyu-Sang; Lim, Won-Jun; Choi, Bong-Hwan; Lee, Seung-Hwan; Kim, Hyeong-Cheol; Lee, Seung-Soo; Cho, Eun-Seok; Lee, Kyung-Tai; Kim, Namshin; Kim, Jeong-Dae; Kim, Jong-Bok; Chai, Han-Ha; Cho, Yong-Min; Kim, Tae-Hun; Lim, Dajeong

    2016-09-01

    Hanwoo is an indigenous Korean beef cattle breed, and it shared an ancestor with Yanbian cattle that are found in the Northeast provinces in China until the last century. During recent decades, those cattle breeds experienced different selection pressures. Here, we present genome-wide copy number variations (CNVs) by comparing Hanwoo and Yanbian cattle sequencing data. We used ~3.12 and ~3.07 billion sequence reads from Hanwoo and Yanbian cattle, respectively. A total of 901 putative CNV regions (CNVRs) were identified throughout the genome, representing 5,513,340bp. This is a smaller number than has been reported in previous studies, indicating that Hanwoo are genetically close to Yanbian cattle. Of the CNVRs, 53.2% and 46.8% were found to be gains and losses in Hanwoo. Potential functional roles of each CNVR were assessed by annotating all CNVRs and gene ontology (GO) enrichment analysis. We found that 278 CNVRs overlapped with cattle gene-sets (genic-CNVRs) that could be promising candidates to account for economically important traits in cattle. The enrichment analysis indicated that genes were significantly over-represented in GO terms, including developmental process, multicellular organismal process, reproduction, and response to stimulus. These results provide a valuable genomic resource for determining how CNVs are associated with cattle traits.

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

    PubMed Central

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

    2016-01-01

    With rapid advances in DNA sequencing technologies, whole exome sequencing (WES) has become a popular approach for detecting somatic mutations in oncology studies. The initial intent of WES was to characterize single nucleotide variants, but it was observed that the number of sequencing reads that mapped to a genomic region correlated with the DNA copy number variants (CNVs). We propose a method RefCNV that uses a reference set to estimate the distribution of the coverage for each exon. The construction of the reference set includes an evaluation of the sources of variability in the coverage distribution. We observed that the processing steps had an impact on the coverage distribution. For each exon, we compared the observed coverage with the expected normal coverage. Thresholds for determining CNVs were selected to control the false-positive error rate. RefCNV prediction correlated significantly (r = 0.96–0.86) with CNV measured by digital polymerase chain reaction for MET (7q31), EGFR (7p12), or ERBB2 (17q12) in 13 tumor cell lines. The genome-wide CNV analysis showed a good overall correlation (Spearman’s coefficient = 0.82) between RefCNV estimation and publicly available CNV data in Cancer Cell Line Encyclopedia. RefCNV also showed better performance than three other CNV estimation methods in genome-wide CNV analysis. PMID:27147817

  1. 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

  2. Optimizing sparse sequencing of single cells for highly multiplex copy number profiling

    PubMed Central

    Baslan, Timour; Kendall, Jude; Ward, Brian; Cox, Hilary; Leotta, Anthony; Rodgers, Linda; Riggs, Michael; D'Italia, Sean; Sun, Guoli; Yong, Mao; Miskimen, Kristy; Gilmore, Hannah; Saborowski, Michael; Dimitrova, Nevenka; Krasnitz, Alexander; Harris, Lyndsay; Wigler, Michael; Hicks, James

    2015-01-01

    Genome-wide analysis at the level of single cells has recently emerged as a powerful tool to dissect genome heterogeneity in cancer, neurobiology, and development. To be truly transformative, single-cell approaches must affordably accommodate large numbers of single cells. This is feasible in the case of copy number variation (CNV), because CNV determination requires only sparse sequence coverage. We have used a combination of bioinformatic and molecular approaches to optimize single-cell DNA amplification and library preparation for highly multiplexed sequencing, yielding a method that can produce genome-wide CNV profiles of up to a hundred individual cells on a single lane of an Illumina HiSeq instrument. We apply the method to human cancer cell lines and biopsied cancer tissue, thereby illustrating its efficiency, reproducibility, and power to reveal underlying genetic heterogeneity and clonal phylogeny. The capacity of the method to facilitate the rapid profiling of hundreds to thousands of single-cell genomes represents a key step in making single-cell profiling an easily accessible tool for studying cell lineage. PMID:25858951

  3. Direct evidence for the adaptive role of copy number variation on antifolate susceptibility in Plasmodium falciparum

    PubMed Central

    Heinberg, Adina; Siu, Edwin; Stern, Chaya; Lawrence, Elizabeth A; Ferdig, Michael T; Deitsch, Kirk W; Kirkman, Laura A

    2013-01-01

    Summary Resistance to antimalarials targeting the folate pathway is widespread. GTP-cyclohydrolase (gch1), the first enzyme in this pathway, exhibits extensive copy number variation (CNV) in parasite isolates from areas with a history of longstanding antifolate use. Increased CN of gch1 is associated with a greater number of point mutations in enzymes targeted by the antifolates, pyrimethamine and sulfadoxine. While these observations suggest that increases in gch1 CN are an adaptation to drug pressure, changes in CN have not been experimentally demonstrated to directly alter drug susceptibility. To determine if changes in gch1 expression alone modify pyrimethamine sensitivity, we manipulated gch1 CN in several parasite lines to test the effect on drug sensitivity. We report that increases in gch1 CN alter pyrimethamine resistance in most parasites lines. However we find evidence of a detrimental effect of very high levels of gch1 overexpression in parasite lines with high endogenous levels of gch1 expression, revealing the importance of maintaining balance in the folate pathway and implicating changes in gch1 expression in preserving proper metabolic flux. This work expands our understanding of parasite adaptation to drug pressure and provides a possible mechanism for how specific mutations become fixed within parasite populations. PMID:23347134

  4. Discovering Recurrent Copy Number Aberrations in Complex Patterns via Non-Negative Sparse Singular Value Decomposition.

    PubMed

    Xi, Jianing; Li, Ao

    2016-01-01

    Recurrent copy number aberrations (RCNAs) in multiple cancer samples are strongly associated with tumorigenesis, and RCNA discovery is helpful to cancer research and treatment. Despite the emergence of numerous RCNA discovering methods, most of them are unable to detect RCNAs in complex patterns that are influenced by complicating factors including aberration in partial samples, co-existing of gains and losses and normal-like tumor samples. Here, we propose a novel computational method, called non-negative sparse singular value decomposition (NN-SSVD), to address the RCNA discovering problem in complex patterns. In NN-SSVD, the measurement of RCNA is based on the aberration frequency in a part of samples rather than all samples, which can circumvent the complexity of different RCNA patterns. We evaluate NN-SSVD on synthetic dataset by comparison on detection scores and Receiver Operating Characteristics curves, and the results show that NN-SSVD outperforms existing methods in RCNA discovery and demonstrate more robustness to RCNA complicating factors. Applying our approach on a breast cancer dataset, we successfully identify a number of genomic regions that are strongly correlated with previous studies, which harbor a bunch of known breast cancer associated genes.

  5. Impact of copy number variations burden on coding genome in humans using integrated high resolution arrays.

    PubMed

    Veerappa, Avinash M; Lingaiah, Kusuma; Vishweswaraiah, Sangeetha; Murthy, Megha N; Suresh, Raviraj V; Manjegowda, Dinesh S; Ramachandra, Nallur B

    2014-12-16

    Copy number variations (CNVs) alter the transcriptional and translational levels of genes by disrupting the coding structure and this burden of CNVs seems to be a significant contributor to phenotypic variations. Therefore it was necessary to assess the complexities of CNV burden on the coding genome. A total of 1715 individuals from 12 populations were used for CNV analysis in the present investigation. Analysis was performed using Affymetrix Genome-Wide Human SNP Array 6·0 chip and CytoScan High-Density arrays. CNVs were more frequently observed in the coding region than in the non-coding region. CNVs were observed vastly more frequently in the coding region than the non-coding region. CNVs were found to be enriched in the regions containing functional genes (83-96%) compared with the regions containing pseudogenes (4-17%). CNVs across the genome of an individual showed multiple hits across many genes, whose proteins interact physically and function under the same pathway. We identified varying numbers of proteins and degrees of interactions within protein complexes of single individual genomes. This study represents the first draft of a population-specific CNV genes map as well as a cross-populational map. The complex relationship of CNVs on genes and their physically interacting partners unravels many complexities involved in phenotype expression. This study identifies four mechanisms contributing to the complexities caused by the presence of multiple CNVs across many genes in the coding part of the genome.

  6. Copy number variation profile in the placental and parental genomes of recurrent pregnancy loss families

    PubMed Central

    Kasak, Laura; Rull, Kristiina; Sõber, Siim; Laan, Maris

    2017-01-01

    We have previously shown an extensive load of somatic copy number variations (CNVs) in the human placental genome with the highest fraction detected in normal term pregnancies. Hereby, we hypothesized that insufficient promotion of CNVs may impair placental development and lead to recurrent pregnancy loss (RPL). RPL affects ~3% of couples aiming at childbirth and idiopathic RPL represents ~50% of cases. We analysed placental and parental CNV profiles of idiopathic RPL trios (mother-father-placenta) and duos (mother-placenta). Consistent with the hypothesis, the placental genomes of RPL cases exhibited 2-fold less CNVs compared to uncomplicated 1st trimester pregnancies (P = 0.02). This difference mainly arose from lower number of duplications. Overall, 1st trimester control placentas shared only 5.3% of identified CNV regions with RPL cases, whereas the respective fraction with term placentas was 35.1% (P = 1.1 × 10−9). Disruption of the genes NUP98 (embryonic stem cell development) and MTRR (folate metabolism) was detected exclusively in RPL placentas, potentially indicative to novel loci implicated in RPL. Interestingly, genes with higher overall expression were prone to deletions (>3-fold higher median expression compared to genes unaffected by CNVs, P = 6.69 × 10−20). Additionally, large pericentromeric and subtelomeric CNVs in parental genomes emerged as a risk factor for RPL. PMID:28345611

  7. Optimizing sparse sequencing of single cells for highly multiplex copy number profiling.

    PubMed

    Baslan, Timour; Kendall, Jude; Ward, Brian; Cox, Hilary; Leotta, Anthony; Rodgers, Linda; Riggs, Michael; D'Italia, Sean; Sun, Guoli; Yong, Mao; Miskimen, Kristy; Gilmore, Hannah; Saborowski, Michael; Dimitrova, Nevenka; Krasnitz, Alexander; Harris, Lyndsay; Wigler, Michael; Hicks, James

    2015-05-01

    Genome-wide analysis at the level of single cells has recently emerged as a powerful tool to dissect genome heterogeneity in cancer, neurobiology, and development. To be truly transformative, single-cell approaches must affordably accommodate large numbers of single cells. This is feasible in the case of copy number variation (CNV), because CNV determination requires only sparse sequence coverage. We have used a combination of bioinformatic and molecular approaches to optimize single-cell DNA amplification and library preparation for highly multiplexed sequencing, yielding a method that can produce genome-wide CNV profiles of up to a hundred individual cells on a single lane of an Illumina HiSeq instrument. We apply the method to human cancer cell lines and biopsied cancer tissue, thereby illustrating its efficiency, reproducibility, and power to reveal underlying genetic heterogeneity and clonal phylogeny. The capacity of the method to facilitate the rapid profiling of hundreds to thousands of single-cell genomes represents a key step in making single-cell profiling an easily accessible tool for studying cell lineage.

  8. A sparse model based detection of copy number variations from exome sequencing data

    PubMed Central

    Duan, Junbo; Wan, Mingxi; Deng, Hong-Wen; Wang, Yu-Ping

    2016-01-01

    Goal Whole-exome sequencing provides a more cost-effective way than whole-genome sequencing for detecting genetic variants such as copy number variations (CNVs). Although a number of approaches have been proposed to detect CNVs from whole-genome sequencing, a direct adoption of these approaches to whole-exome sequencing will often fail because exons are separately located along a genome. Therefore, an appropriate method is needed to target the specific features of exome sequencing data. Methods In this paper a novel sparse model based method is proposed to discover CNVs from multiple exome sequencing data. First, exome sequencing data are represented with a penalized matrix approximation, and technical variability and random sequencing errors are assumed to follow a generalized Gaussian distribution. Second, an iteratively re-weighted least squares algorithm is used to estimate the solution. Results The method is tested and validated on both synthetic and real data, and compared with other approaches including CoNIFER, XHMM and cn.MOPS. The test demonstrates that the proposed method outperform other approaches. Conclusion The proposed sparse model can detect CNVs from exome sequencing data with high power and precision. Significance Sparse model can target the specific features of exome sequencing data. The software codes are freely available at http://www.tulane.edu/wyp/software/ExonCNV.m PMID:26258935

  9. Extensive variation in gene copy number at the killer immunoglobulin-like receptor locus in humans.

    PubMed

    Vendelbosch, Sanne; de Boer, Martin; Gouw, Remko A T W; Ho, Cynthia K Y; Geissler, Judy; Swelsen, Wendy T N; Moorhouse, Michael J; Lardy, Neubury M; Roos, Dirk; van den Berg, Timo K; Kuijpers, Taco W

    2013-01-01

    Killer immunoglobulin-like receptors (KIRs) are involved in the regulation of natural killer cell cytotoxicity. Within the human genome seventeen KIR genes are present, which all contain a large number of allelic variants. The high level of homology among KIR genes has hampered KIR genotyping in larger cohorts, and determination of gene copy number variation (CNV) has been difficult. We have designed a multiplex ligation-dependent probe amplification (MLPA) technique for genotyping and CNV determination in one single assay and validated the results by next-generation sequencing and with a KIR gene-specific short tandem repeat assay. In this way, we demonstrate in a cohort of 120 individuals a high level of CNV for all KIR genes except for the framework genes KIR3DL3 and KIR3DL2. Application of our MLPA assay in segregation analyses of families from the Centre d'Etude du Polymorphisme Humaine, previously KIR-genotyped by classical techniques, confirmed an earlier reported duplication and resulted in the identification of a novel duplication event in one of these families. In summary, our KIR MLPA assay allows rapid and accurate KIR genotyping and CNV detection, thus rendering improved transplantation programs and oncology treatment feasible, and enables more detailed studies on the role of KIRs in human (auto)immunity and infectious disease.

  10. 39 CFR 3001.10 - Form and number of copies of documents.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    .... If the document is bound, it shall be bound on the left side. Copies of documents for filing and service must be printed from a text-based pdf version of the document, where possible. Otherwise, they may... original and two hard copies, provided that the stored document is a file generated in either Acrobat...

  11. Segmental duplication and copy number variation of the patched domain containing 3 (PTCHD3) locus on pig chromosome 10

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mammalian genomes contain numerous blocks of highly homologous duplicated regions that can vary in copy number. We identified a segmental duplication encompassing the PTCHD3 gene, which has predicted hedgehog receptor activity, in a QTL region for nipple number on SSC10. A 3-fold coverage BAC screen...

  12. Hereditary breast and ovarian cancer: assessment of point mutations and copy number variations in Brazilian patients

    PubMed Central

    2014-01-01

    Background Germ line mutations in BRCA1 and BRCA2 (BRCA1/2) and other susceptibility genes have been identified as genetic causes of hereditary breast and ovarian cancer (HBOC). To identify the disease-causing mutations in a cohort of 120 Brazilian women fulfilling criteria for HBOC, we carried out a comprehensive screening of BRCA1/2, TP53 R337H, CHEK2 1100delC, followed by an analysis of copy number variations in 14 additional breast cancer susceptibility genes (PTEN, ATM, NBN, RAD50, RAD51, BRIP1, PALB2, MLH1, MSH2, MSH6, TP53, CDKN2A, CDH1 and CTNNB1). Methods Capillary sequencing and multiplex ligation-dependent probe amplification (MLPA) were used for detecting point mutations and copy number variations (CNVs), respectively, for the BRCA1 and BRCA2 genes; capillary sequencing was used for point mutation for both variants TP53 R337H and CHEK2 1100delC, and finally array comparative genomic hybridization (array-CGH) was used for identifying CNVs in the 14 additional genes. Results The positive detection rate in our series was 26%. BRCA1 pathogenic mutations were found in 20 cases, including two cases with CNVs, whereas BRCA2 mutations were found in 7 cases. We also found three patients with the TP53 R337H mutation and one patient with the CHEK2 1100delC mutation. Seven (25%) pathogenic mutations in BRCA1/2 were firstly described, including a splice-site BRCA1 mutation for which pathogenicity was confirmed by the presence of an aberrant transcript showing the loss of the last 62 bp of exon 7. Microdeletions of exon 4 in ATM and exon 2 in PTEN were identified in BRCA2-mutated and BRCA1/2-negative patients, respectively. Conclusions In summary, our results showed a high frequency of BRCA1/2 mutations and a higher prevalence of BRCA1 (64.5%) gene. Moreover, the detection of the TP53 R337H variant in our series and the fact that this variant has a founder effect in our population prompted us to suggest that all female breast cancer patients with clinical criteria

  13. High-resolution genomic copy number profiling of glioblastoma multiforme by single nucleotide polymorphism DNA microarray.

    PubMed

    Yin, Dong; Ogawa, Seishi; Kawamata, Norihiko; Tunici, Patrizia; Finocchiaro, Gaetano; Eoli, Marica; Ruckert, Christian; Huynh, Thien; Liu, Gentao; Kato, Motohiro; Sanada, Masashi; Jauch, Anna; Dugas, Martin; Black, Keith L; Koeffler, H Phillip

    2009-05-01

    Glioblastoma multiforme (GBM) is an extremely malignant brain tumor. To identify new genomic alterations in GBM, genomic DNA of tumor tissue/explants from 55 individuals and 6 GBM cell lines were examined using single nucleotide polymorphism DNA microarray (SNP-Chip). Further gene expression analysis relied on an additional 56 GBM samples. SNP-Chip results were validated using several techniques, including quantitative PCR (Q-PCR), nucleotide sequencing, and a combination of Q-PCR and detection of microsatellite markers for loss of heterozygosity with normal copy number [acquired uniparental disomy (AUPD)]. Whole genomic DNA copy number in each GBM sample was profiled by SNP-Chip. Several signaling pathways were frequently abnormal. Either the p16(INK4A)/p15(INK4B)-CDK4/6-pRb or p14(ARF)-MDM2/4-p53 pathways were abnormal in 89% (49 of 55) of cases. Simultaneous abnormalities of both pathways occurred in 84% (46 of 55) samples. The phosphoinositide 3-kinase pathway was altered in 71% (39 of 55) GBMs either by deletion of PTEN or amplification of epidermal growth factor receptor and/or vascular endothelial growth factor receptor/platelet-derived growth factor receptor alpha. Deletion of chromosome 6q26-27 often occurred (16 of 55 samples). The minimum common deleted region included PARK2, PACRG, QKI, and PDE10A genes. Further reverse transcription Q-PCR studies showed that PARK2 expression was decreased in another collection of GBMs at a frequency of 61% (34 of 56) of samples. The 1p36.23 region was deleted in 35% (19 of 55) of samples. Notably, three samples had homozygous deletion encompassing this site. Also, a novel internal deletion of a putative tumor suppressor gene, LRP1B, was discovered causing an aberrant protein. AUPDs occurred in 58% (32 of 55) of the GBM samples and five of six GBM cell lines. A common AUPD was found at chromosome 17p13.3-12 (included p53 gene) in 13 of 61 samples and cell lines. Single-strand conformational polymorphism and nucleotide

  14. Association of nsv823469 copy number loss with decreased risk of chronic obstructive pulmonary disease and pulmonary function in Chinese

    PubMed Central

    Chen, Xiaoliang; Lu, Xiaoxiao; Chen, Jiansong; Wu, Di; Qiu, Fuman; Xiong, Huali; Pan, Zihua; Yang, Lei; Yang, Binyao; Xie, Chenli; Zhou, Yifeng; Huang, Dongsheng; Zhou, Yumin; Lu, Jiachun

    2017-01-01

    It is highly possible that copy number variations (CNVs) in susceptible regions have effects on chronic obstructive pulmonary disease (COPD) development, while long noncoding RNA (lncRNAs) have been shown to cause COPD. We hypothesized that the common CNV, named nsv823469 located on 6p22.1, and covering lncRNAs (major histocompatibility complex, class I, A (HLA-A) and HLA complex group 4B (HCG4B)) has an effect on COPD risk. This association was assessed through a two-stage case-control study, and was further confirmed with COPD and pulmonary function-based family analyses, respectively. The copy number loss (0-copy/1-copy) of nsv823469 significantly decreased risk of COPD compared with normal (2-copy) (OR = 0.77, 95% CI = 0.69–0.85). The loss allele, inducing copy number loss of nsv823469, has a tendency to transmit to offspring or siblings (P = 0.010) and is associated with forced expiratory volume in 1 second (FEV1) (P = 0.030). Furthermore, the copy number loss of nsv823469 in normal pulmonary tissue decreases the expression levels of HCG4B (r = 0.315, P = 0.031) and HLA-A (r = 0.296, P = 0.044). Our data demonstrates that nsv823469 plays a role in COPD and pulmonary function inheritance by potentially altering expression of HCG4B. PMID:28079130

  15. EGFR-activating mutations, DNA copy number abundance of ErbB family, and prognosis in lung adenocarcinoma.

    PubMed

    Chen, Hsuan-Yu; Liu, Chia-Hsin; Chang, Ya-Hsuan; Yu, Sung-Liang; Ho, Bing-Ching; Hsu, Chung-Ping; Yang, Tsung-Ying; Chen, Kun-Chieh; Hsu, Kuo-Hsuan; Tseng, Jeng-Sen; Hsia, Jiun-Yi; Chuang, Cheng-Yen; Chang, Chi-Sheng; Li, Yu-Cheng; Li, Ker-Chau; Chang, Gee-Chen; Yang, Pan-Chyr

    2016-02-23

    In this study, EGFR-activating mutation status and DNA copy number abundances of members of ErbB family were measured in 261 lung adenocarcinomas. The associations between DNA copy number abundances of ErbB family, EGFR-activating mutation status, and prognosis were explored. Results showed that DNA copy number abundances of EGFR, ERBB2, ERBB3, and ERBB4 had associations with overall survival in lung adenocarcinoma with EGFR-activating mutations. In the stratification analysis, only ERBB2 showed significant discrepancy in patients carrying wild type EGFR and other members of ErbB family in patients carrying EGFR-activating mutation. This indicated that CNAs of ErbB family had effect modifications of EGFR-activating mutation status. Findings of this study demonstrate potential molecular guidance of patient management of lung adenocarcinoma with or without EGFR-activating mutations.

  16. Discovering subgroups of patients from DNA copy number data using NMF on compacted matrices.

    PubMed

    de Campos, Cassio P; Rancoita, Paola M V; Kwee, Ivo; Zucca, Emanuele; Zaffalon, Marco; Bertoni, Francesco

    2013-01-01

    In the study of complex genetic diseases, the identification of subgroups of patients sharing similar genetic characteristics represents a challenging task, for example, to improve treatment decision. One type of genetic lesion, frequently investigated in such disorders, is the change of the DNA copy number (CN) at specific genomic traits. Non-negative Matrix Factorization (NMF) is a standard technique to reduce the dimensionality of a data set and to cluster data samples, while keeping its most relevant information in meaningful components. Thus, it can be used to discover subgroups of patients from CN profiles. It is however computationally impractical for very high dimensional data, such as CN microarray data. Deciding the most suitable number of subgroups is also a challenging problem. The aim of this work is to derive a procedure to compact high dimensional data, in order to improve NMF applicability without compromising the quality of the clustering. This is particularly important for analyzing high-resolution microarray data. Many commonly used quality measures, as well as our own measures, are employed to decide the number of subgroups and to assess the quality of the results. Our measures are based on the idea of identifying robust subgroups, inspired by biologically/clinically relevance instead of simply aiming at well-separated clusters. We evaluate our procedure using four real independent data sets. In these data sets, our method was able to find accurate subgroups with individual molecular and clinical features and outperformed the standard NMF in terms of accuracy in the factorization fitness function. Hence, it can be useful for the discovery of subgroups of patients with similar CN profiles in the study of heterogeneous diseases.

  17. Validity of low copy number typing and applications to forensic science.

    PubMed

    Budowle, Bruce; Eisenberg, Arthur J; van Daal, Angela

    2009-06-01

    Low copy number (LCN) typing, particularly for current short tandem repeat (STR) typing, refers to the analysis of any sample that contains less than 200 pg of template DNA. Generally, LCN typing simply can be defined as the analysis of any DNA sample where the results are below the stochastic threshold for reliable interpretation. There are a number of methodologies to increase sensitivity of detection to enable LCN typing. These approaches encompass modifications during the polymerase chain reaction (PCR) and/or post-PCR manipulations. Regardless of the manipulations, when processing a small number of starting templates during the PCR exaggerated stochastic sampling effects will occur. The result is that several phenomena can occur: a substantial imbalance of 2 alleles at a given heterozygous locus, allelic dropout, or increased stutter. With increased sensitivity of detection there is a concomitant increased risk of contamination. Recently, a commission reviewed LCN typing and found it to be "robust" and "fit for purpose." Because LCN analysis by its nature is not reproducible, it cannot be considered as robust as that associated with conventional DNA typing. The findings of the commission seem inconsistent with the nature of LCN typing. While LCN typing is appropriate for identification of missing persons and human remains and for developing investigative leads, caution should be taken with its use in other endeavors until developments are made that overcome the vagaries of LCN typing. A more in-depth evaluation by the greater scientific community is warranted. The issues to consider include: training and education, evidence handling and collection procedures, the application or purpose for which the LCN result will be used, the reliability of current LCN methods, replicate analyses, interpretation and uncertainty, report writing, validation requirements, and alternate methodologies for better performance.

  18. Validity of Low Copy Number Typing and Applications to Forensic Science

    PubMed Central

    Budowle, Bruce; Eisenberg, Arthur J.; van Daal, Angela

    2009-01-01

    Low copy number (LCN) typing, particularly for current short tandem repeat (STR) typing, refers to the analysis of any sample that contains less than 200 pg of template DNA. Generally, LCN typing simply can be defined as the analysis of any DNA sample where the results are below the stochastic threshold for reliable interpretation. There are a number of methodologies to increase sensitivity of detection to enable LCN typing. These approaches encompass modifications during the polymerase chain reaction (PCR) and/or post-PCR manipulations. Regardless of the manipulations, when processing a small number of starting templates during the PCR exaggerated stochastic sampling effects will occur. The result is that several phenomena can occur: a substantial imbalance of 2 alleles at a given heterozygous locus, allelic dropout, or increased stutter. With increased sensitivity of detection there is a concomitant increased risk of contamination. Recently, a commission reviewed LCN typing and found it to be “robust” and “fit for purpose.” Because LCN analysis by its nature is not reproducible, it cannot be considered as robust as that associated with conventional DNA typing. The findings of the commission seem inconsistent with the nature of LCN typing. While LCN typing is appropriate for identification of missing persons and human remains and for developing investigative leads, caution should be taken with its use in other endeavors until developments are made that overcome the vagaries of LCN typing. A more in-depth evaluation by the greater scientific community is warranted. The issues to consider include: training and education, evidence handling and collection procedures, the application or purpose for which the LCN result will be used, the reliability of current LCN methods, replicate analyses, interpretation and uncertainty, report writing, validation requirements, and alternate methodologies for better performance. PMID:19480017

  19. Familial longevity study reveals a significant association of mitochondrial DNA copy number between centenarians and their offspring.

    PubMed

    He, Yong-Han; Chen, Xiao-Qiong; Yan, Dong-Jing; Xiao, Fu-Hui; Lin, Rong; Liao, Xiao-Ping; Liu, Yao-Wen; Pu, Shao-Yan; Yu, Qin; Sun, Hong-Peng; Jiang, Jian-Jun; Cai, Wang-Wei; Kong, Qing-Peng

    2016-11-01

    Reduced mitochondrial function is an important cause of aging and age-related diseases. We previously revealed a relatively higher level of mitochondrial DNA (mtDNA) content in centenarians. However, it is still unknown whether such an mtDNA content pattern of centenarians could be passed on to their offspring and how it was regulated. To address these issues, we recruited 60 longevity families consisting of 206 family members (cohort 1) and explored their mtDNA copy number. The results showed that the first generation of the offspring (F1 offspring) had a higher level of mtDNA copy number than their spouses (p < 0.05) independent of a gender effect. In addition, we found a positive association of mtDNA copy number in centenarians with that in F1 offspring (r = 0.54, p = 0.0008) but not with that in F1 spouses. These results were replicated in another independent cohort consisting of 153 subjects (cohort 2). RNA sequencing analysis suggests that the single-stranded DNA-binding protein 4 was significantly associated with mtDNA copy number and was highly expressed in centenarians as well as F1 offspring versus the F1 spouses, thus likely regulates the mtDNA copy number in the long-lived family members. In conclusion, our results suggest that the pattern of high mtDNA copy number is likely inheritable, which may act as a favorable factor to familial longevity through assuring adequate energy supply.

  20. Elevated Mitochondrial DNA Copy Number in Peripheral Blood and Tissue Predict the Opposite Outcome of Cancer: A Meta-Analysis

    PubMed Central

    Chen, Nan; Wen, Shu; Sun, Xiaoru; Fang, Qian; Huang, Lin; Liu, Shuai; Li, Wanling; Qiu, Meng

    2016-01-01

    Previous studies have suggested that mitochondrial DNA (mtDNA) copy number was associated with cancer risk. However, no solid conclusion revealed the potential predictive value of mtDNA copy number for cancer prognosis. The present meta-analysis was performed to clarify the problem. Hence, we performed a systematic search in PubMed, EmBase, Web of Science databases independently and a total of eighteen studies comprising 3961 cases satisfied the criteria and finally enrolled. Our results didn’t show the association between them but significant heterogeneity in overall analysis (OS: HR = 0.923, 95% CI: 0.653–1.306, p = 0.652; DFS: HR = 0.997, 95% CI: 0.599–1.659, p = 0.99). However, subgroup analysis stratified by sample came to the opposite conclusion. High level mitochondrial DNA copy number in peripheral blood predicted a poor cancer prognosis (OS: HR = 1.624, 95% CI: 1.211–2.177, p = 0.001; DFS: HR = 1.582, 95% CI: 1.026–2.439, p = 0.038) while patients with high level mitochondrial DNA copy number in tumor tissue exhibited better outcomes (OS: HR = 0.604 95% CI: 0.406–0.899, p = 0.013; DFS: HR = 0.593, 95% CI: 0.411–0.857, p = 0.005). These findings were further proved in detailed analyses in blood or tissue subgroup. In conclusion, our study suggested the elevated mtDNA copy number in peripheral blood predicted a poor cancer prognosis while the better outcome was presented among patients with elevated mtDNA copy number in tumor tissue. PMID:27857175

  1. Copy number analysis of 413 isolated talipes equinovarus patients suggests role for transcriptional regulators of early limb development

    PubMed Central

    Alvarado, David M; Buchan, Jillian G; Frick, Steven L; Herzenberg, John E; Dobbs, Matthew B; Gurnett, Christina A

    2013-01-01

    Talipes equinovarus is one of the most common congenital musculoskeletal anomalies and has a worldwide incidence of 1 in 1000 births. A genetic predisposition to talipes equinovarus is evidenced by the high concordance rate in twin studies and the increased risk to first-degree relatives. Despite the frequency of isolated talipes equinovarus and the strong evidence of a genetic basis for the disorder, few causative genes have been identified. To identify rare and/or recurrent copy number variants, we performed a genome-wide screen for deletions and duplications in 413 isolated talipes equinovarus patients using the Affymetrix 6.0 array. Segregation analysis within families and gene expression in mouse E12.5 limb buds were used to determine the significance of copy number variants. We identified 74 rare, gene-containing copy number variants that were present in talipes equinovarus probands and not present in 759 controls or in the Database of Genomic Variants. The overall frequency of copy number variants was similar between talipes equinovarus patients compared with controls. Twelve rare copy number variants segregate with talipes equinovarus in multiplex pedigrees, and contain the developmentally expressed transcription factors and transcriptional regulators PITX1, TBX4, HOXC13, UTX, CHD (chromodomain protein)1, and RIPPLY2. Although our results do not support a major role for recurrent copy number variations in the etiology of isolated talipes equinovarus, they do suggest a role for genes involved in early embryonic patterning in some families that can now be tested with large-scale sequencing methods. PMID:22892537

  2. Engineered ribosomal RNA operon copy-number variants of E. coli reveal the evolutionary trade-offs shaping rRNA operon number.

    PubMed

    Gyorfy, Zsuzsanna; Draskovits, Gabor; Vernyik, Viktor; Blattner, Frederick F; Gaal, Tamas; Posfai, Gyorgy

    2015-02-18

    Ribosomal RNA (rrn) operons, characteristically present in several copies in bacterial genomes (7 in E. coli), play a central role in cellular physiology. We investigated the factors determining the optimal number of rrn operons in E. coli by constructing isogenic variants with 5-10 operons. We found that the total RNA and protein content, as well as the size of the cells reflected the number of rrn operons. While growth parameters showed only minor differences, competition experiments revealed a clear pattern: 7-8 copies were optimal under conditions of fluctuating, occasionally rich nutrient influx and lower numbers were favored in stable, nutrient-limited environments. We found that the advantages of quick adjustment to nutrient availability, rapid growth and economic regulation of ribosome number all contribute to the selection of the optimal rrn operon number. Our results suggest that the wt rrn operon number of E. coli reflects the natural, 'feast and famine' life-style of the bacterium, however, different copy numbers might be beneficial under different environmental conditions. Understanding the impact of the copy number of rrn operons on the fitness of the cell is an important step towards the creation of functional and robust genomes, the ultimate goal of synthetic biology.

  3. Interactions between Obesity-Related Copy Number Variants and Dietary Behaviors in Childhood Obesity

    PubMed Central

    Zhang, Dandan; Li, Zhenli; Wang, Hao; Yang, Min; Liang, Li; Fu, Junfen; Wang, Chunling; Ling, Jie; Zhang, Yan; Zhang, Shuai; Xu, Yuyang; Zhu, Yimin; Lai, Maode

    2015-01-01

    Copy number variants (CNVs) have been implicated as an important genetic marker of obesity, and gene-environment interaction has been found to modulate risk of obesity. To evaluate the associations between CNVs and childhood obesity, as well as the interactions between CNVs and dietary behaviors, we recruited 534 obese children and 508 controls from six cities in China and six candidate CNVs were screened through published genome-wide studies (GWAS) on childhood obesity. We found three loci (10q11.22, 4q25 and 11q11) to be significantly associated with obesity after false discovery rate (FDR) correction (all the p ≤ 0.05). Cumulative effect of the three positive loci was measured by the genetic risk score (GRS), showing a significant relationship with the risk of obesity (Ptrend < 0.001). The OR of obesity increased to 21.38 (95% CI = 21.19–21.55) among the 10q11.22 deletion carriers who had meat-based diets, indicating prominent multiplicative interaction (MI) between deletions of 10q11.22 and preference for a meat-based diet. Simultaneous deletions of 5q13.2 and duplications of 6q14.1 had significant MI with a preference for salty foods. Our results suggested that CNVs may contribute to the genetic susceptibility of childhood obesity, and the CNV-diet interactions modulate the risk of obesity. PMID:25912042

  4. The genomic architecture of segmental duplications and associated copy number variants in dogs.

    PubMed

    Nicholas, Thomas J; Cheng, Ze; Ventura, Mario; Mealey, Katrina; Eichler, Evan E; Akey, Joshua M

    2009-03-01

    Structural variation is an important and abundant source of genetic and phenotypic variation. Here we describe the first systematic and genome-wide analysis of segmental duplications and associated copy number variants (CNVs) in the modern domesticated dog, Canis familiaris, which exhibits considerable morphological, physiological, and behavioral variation. Through computational analyses of the publicly available canine reference sequence, we estimate that segmental duplications comprise approximately 4.21% of the canine genome. Segmental duplications overlap 841 genes and are significantly enriched for specific biological functions such as immunity and defense and KRAB box transcription factors. We designed high-density tiling arrays spanning all predicted segmental duplications and performed aCGH in a panel of 17 breeds and a gray wolf. In total, we identified 3583 CNVs, approximately 68% of which were found in two or more samples that map to 678 unique regions. CNVs span 429 genes that are involved in a wide variety of biological processes such as olfaction, immunity, and gene regulation. Our results provide insight into mechanisms of canine genome evolution and generate a valuable resource for future evolutionary and phenotypic studies.

  5. A novel mechanistic spectrum underlies glaucoma-associated chromosome 6p25 copy number variation

    PubMed Central

    Chanda, Bhaskar; Asai-Coakwell, Mika; Ye, Ming; Mungall, Andrew J.; Barrow, Margaret; Dobyns, William B.; Behesti, Hourinaz; Sowden, Jane C.; Carter, Nigel P.; Walter, Michael A.; Lehmann, Ordan J.

    2008-01-01

    The factors that mediate chromosomal rearrangement remain incompletely defined. Among regions prone to structural variant formation, chromosome 6p25 is one of the few in which disease-associated segmental duplications and segmental deletions have been identified, primarily through gene dosage attributable ocular phenotypes. Using array comparative genome hybridization, we studied ten 6p25 duplication and deletion pedigrees and amplified junction fragments from each. Analysis of the breakpoint architecture revealed that all the rearrangements were non-recurrent, and in contrast to most previous examples the majority of the segmental duplications and deletions utilized coupled homologous and non-homologous recombination mechanisms. One junction fragment exhibited an unprecedented 367 bp insert derived from tandemly arranged breakpoint elements. While this accorded with a recently described replication-based mechanism, it differed from the previous example in being unassociated with template switching, and occurring in a segmental deletion. These results extend the mechanisms involved in structural variant formation, provide strong evidence that a spectrum of recombination, DNA repair and replication underlie 6p25 rearrangements, and have implications for genesis of copy number variations in other genomic regions. These findings highlight the benefits of undertaking the extensive studies necessary to characterize structural variants at the base pair level. PMID:18694899

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

    PubMed Central

    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.

    2016-01-01

    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. PMID:27005566

  7. CODEX: a normalization and copy number variation detection method for whole exome sequencing.

    PubMed

    Jiang, Yuchao; Oldridge, Derek A; Diskin, Sharon J; Zhang, Nancy R

    2015-03-31

    High-throughput sequencing of DNA coding regions has become a common way of assaying genomic variation in the study of human diseases. Copy number variation (CNV) is an important type of genomic variation, but detecting and characterizing CNV from exome sequencing is challenging due to the high level of biases and artifacts. We propose CODEX, a normalization and CNV calling procedure for whole exome sequencing data. The Poisson latent factor model in CODEX includes terms that specifically remove biases due to GC content, exon capture and amplification efficiency, and latent systemic artifacts. CODEX also includes a Poisson likelihood-based recursive segmentation procedure that explicitly models the count-based exome sequencing data. CODEX is compared to existing methods on a population analysis of HapMap samples from the 1000 Genomes Project, and shown to be more accurate on three microarray-based validation data sets. We further evaluate performance on 222 neuroblastoma samples with matched normals and focus on a well-studied rare somatic CNV within the ATRX gene. We show that the cross-sample normalization procedure of CODEX removes more noise than normalizing the tumor against the matched normal and that the segmentation procedure performs well in detecting CNVs with nested structures.

  8. Functional Impact of Global Rare Copy Number Variation in Autism Spectrum Disorder

    PubMed Central

    Pinto, Dalila; Pagnamenta, Alistair T.; Klei, Lambertus; Anney, Richard; Merico, Daniele; Regan, Regina; Conroy, Judith; Magalhaes, Tiago R.; Correia, Catarina; Abrahams, Brett S.; Almeida, Joana; Bacchelli, Elena; Bader, Gary D.; Bailey, Anthony J.; Baird, Gillian; Battaglia, Agatino; Berney, Tom; Bolshakova, Nadia; Bölte, Sven; Bolton, Patrick F.; Bourgeron, Thomas; Brennan, Sean; Brian, Jessica; Bryson, Susan E.; Carson, Andrew R.; Casallo, Guillermo; Casey, Jillian; Cochrane, Lynne; Corsello, Christina; Crawford, Emily L.; Crossett, Andrew; Dawson, Geraldine; de Jonge, Maretha; Delorme, Richard; Drmic, Irene; Duketis, Eftichia; Duque, Frederico; Estes, Annette; Farrar, Penny; Fernandez, Bridget A.; Filipa, Ana; Folstein, Susan E.; Fombonne, Eric; Freitag, Christine M.; Gilbert, John; Gillberg, Christopher; Glessner, Joseph T.; Goldberg, Jeremy; Green, Andrew; Green, Jonathan; Guter, Stephen J.; Hakonarson, Hakon; Heron, Elizabeth A.; Hill, Matthew; Holt, Richard; Howe, Jennifer L.; Hughes, Gillian; Hus, Vanessa; Igliozzi, Roberta; Kim, Cecilia; Klauck, Sabine M.; Kolevzon, Alexander; Korvatska, Olena; Kustanovich, Vlad; Lajonchere, Clara M.; Lamb, Janine A.; Laskawiec, Magdalena; Leboyer, Marion; Le Couteur, Ann; Leventhal, Bennett L.; Lionel, Anath C.; Liu, Xiao-Qing; Lord, Catherine; Lotspeich, Linda; Lund, Sabata C.; Maestrini, Elena; Mahoney, William; Mantoulan, Carine; Marshall, Christian R.; McConachie, Helen; McDougle, Christopher J.; McGrath, Jane; McMahon, William M.; Merikangas, Alison; Migita, Ohsuke; Minshew, Nancy J.; Mirza, Ghazala K.; Munson, Jeff; Nelson, Stanley F.; Noakes, Carolyn; Noor, Abdul; Nygren, Gudrun; Oliveira, Guiomar; Papanikolaou, Katerina; Parr, Jeremy R.; Parrini, Barbara; Paton, Tara; Pickles, Andrew; Pilorge, Marion; Piven, Joseph; Ponting, Chris P.; Posey, David J.; Poustka, Annemarie; Poustka, Fritz; Prasad, Aparna; Ragoussis, Jiannis; Renshaw, Katy; Rickaby, Jessica; Roberts, Wendy; Roeder, Kathryn; Roge, Bernadette; Rutter, Michael L.; Bierut, Laura J.; Rice, John P.; Consortium, SAGE; Salt, Jeff; Sansom, Katherine; Sato, Daisuke; Segurado, Ricardo; Senman, Lili; Shah, Naisha; Sheffield, Val C.; Soorya, Latha; Sousa, Inês; Stein, Olaf; Stoppioni, Vera; Strawbridge, Christina; Tancredi, Raffaella; Tansey, Katherine; Thiruvahindrapduram, Bhooma; Thompson, Ann P.; Thomson, Susanne; Tryfon, Ana; Tsiantis, John; Van Engeland, Herman; Vincent, John B.; Volkmar, Fred; Wallace, Simon; Wang, Kai; Wang, Zhouzhi; Wassink, Thomas H.; Webber, Caleb; Wing, Kirsty; Wittemeyer, Kerstin; Wood, Shawn; Wu, Jing; Yaspan, Brian L.; Zurawiecki, Danielle; Zwaigenbaum, Lonnie; Buxbaum, Joseph D.; Cantor, Rita M.; Cook, Edwin H.; Coon, Hilary; Cuccaro, Michael L.; Devlin, Bernie; Ennis, Sean; Gallagher, Louise; Geschwind, Daniel H.; Gill, Michael; Haines, Jonathan L.; Hallmayer, Joachim; Miller, Judith; Monaco, Anthony P.; Nurnberger, John I.; Paterson, Andrew D.; Pericak-Vance, Margaret A.; Schellenberg, Gerard D.; Szatmari, Peter; Vicente, Astrid M.; Vieland, Veronica J.; Wijsman, Ellen M.; Scherer, Stephen W.; Sutcliffe, James S.; Betancur, Catalina

    2010-01-01

    The autism spectrum disorders (ASDs) are a group of conditions characterized by impairments in reciprocal social interaction and communication, and the presence of restricted and repetitive behaviors1. Individuals with an ASD vary greatly in cognitive development, which can range from above average to intellectual disability (ID)2. While ASDs are known to be highly heritable (~90%)3, the underlying genetic determinants are still largely unknown. Here, we analyzed the genome-wide characteristics of rare (<1% frequency) copy number variation (CNV) in ASD using dense genotyping arrays. When comparing 996 ASD individuals of European ancestry to 1,287 matched controls, cases were found to carry a higher global burden of rare, genic CNVs (1.19 fold, P= 0.012), especially so for loci previously implicated in either ASD and/or intellectual disability (1.69 fold, P= 3.4×10−4). Among the CNVs, there were numerous de novo and inherited events, sometimes in combination in a given family, implicating many novel ASD genes like SHANK2, SYNGAP1, DLGAP2 and the X-linked DDX53-PTCHD1 locus. We also discovered an enrichment of CNVs disrupting functional gene-sets involved in cellular proliferation, projection and motility, and GTPase/Ras signaling. Our results reveal many new genetic and functional targets in ASD that may lead to final connected pathways. PMID:20531469

  9. Network modeling of the transcriptional effects of copy number aberrations in glioblastoma.

    PubMed

    Jörnsten, Rebecka; Abenius, Tobias; Kling, Teresia; Schmidt, Linnéa; Johansson, Erik; Nordling, Torbjörn E M; Nordlander, Bodil; Sander, Chris; Gennemark, Peter; Funa, Keiko; Nilsson, Björn; Lindahl, Linda; Nelander, Sven

    2011-04-26

    DNA copy number aberrations (CNAs) are a hallmark of cancer genomes. However, little is known about how such changes affect global gene expression. We develop a modeling framework, EPoC (Endogenous Perturbation analysis of Cancer), to (1) detect disease-driving CNAs and their effect on target mRNA expression, and to (2) stratify cancer patients into long- and short-term survivors. Our method constructs causal network models of gene expression by combining genome-wide DNA- and RNA-level data. Prognostic scores are obtained from a singular value decomposition of the networks. By applying EPoC to glioblastoma data from The Cancer Genome Atlas consortium, we demonstrate that the resulting network models contain known disease-relevant hub genes, reveal interesting candidate hubs, and uncover predictors of patient survival. Targeted validations in four glioblastoma cell lines support selected predictions, and implicate the p53-interacting protein Necdin in suppressing glioblastoma cell growth. We conclude that large-scale network modeling of the effects of CNAs on gene expression may provide insights into the biology of human cancer. Free software in MATLAB and R is provided.

  10. Copy Number Variations in a Population-Based Study of Charcot-Marie-Tooth Disease

    PubMed Central

    Høyer, Helle; Braathen, Geir J.; Eek, Anette K.; Nordang, Gry B. N.; Skjelbred, Camilla F.; Russell, Michael B.

    2015-01-01

    Copy number variations (CNVs) are important in relation to diversity and evolution but can sometimes cause disease. The most common genetic cause of the inherited peripheral neuropathy Charcot-Marie-Tooth disease is the PMP22 duplication; otherwise, CNVs have been considered rare. We investigated CNVs in a population-based sample of Charcot-Marie-Tooth (CMT) families. The 81 CMT families had previously been screened for the PMP22 duplication and point mutations in 51 peripheral neuropathy genes, and a genetic cause was identified in 37 CMT families (46%). Index patients from the 44 CMT families with an unknown genetic diagnosis were analysed by whole-genome array comparative genomic hybridization to investigate the entire genome for larger CNVs and multiplex ligation-dependent probe amplification to detect smaller intragenomic CNVs in MFN2 and MPZ. One patient had the pathogenic PMP22 duplication not detected by previous methods. Three patients had potentially pathogenic CNVs in the CNTNAP2, LAMA2, or SEMA5A, that is, genes related to neuromuscular or neurodevelopmental disease. Genotype and phenotype correlation indicated likely pathogenicity for the LAMA2 CNV, whereas the CNTNAP2 and SEMA5A CNVs remained potentially pathogenic. Except the PMP22 duplication, disease causing CNVs are rare but may cause CMT in about 1% (95% CI 0–7%) of the Norwegian CMT families. PMID:25648254

  11. ROS1 copy number alterations are frequent in non-small cell lung cancer

    PubMed Central

    Clavé, Sergi; Gimeno, Javier; Muñoz-Mármol, Ana M.; Vidal, Joana; Reguart, Noemí; Carcereny, Enric; Pijuan, Lara; Menéndez, Sílvia; Taus, Álvaro; Mate, José Luís; Serrano, Sergio; Albanell, Joan; Espinet, Blanca; Arriola, Edurne; Salido, Marta

    2016-01-01

    Objectives We aimed to determine the prevalence and partners of ROS1 rearrangements, to explore the correlation between FISH and IHC assays, and to investigate clinical implications of ROS1 copy number alterations (CNAs). Methods A total of 314 NSCLC patients were screened using ROS1 FISH break-apart probes. Of these, 47 surgical tumors were included in TMAs to analyze ROS1 heterogeneity assessed either by FISH and IHC, and chromosome 6 aneusomy. To characterize ROS1 partners, probes for CD74, EZR, SLC34A2 and SDC3 genes were developed. ROS1 positive FISH cases were screened also by IHC. Results Five patients were ROS1 positive (1.8%). We identified two known fusion partners in three patients: CD74 and SLC34A2. Four out of five ROS1 rearranged patients were female, never smokers and with adenocarcinoma histology. Rearranged cases were also positive by IHC as well. According to ROS1 CNAs, we found a prevalence of 37.8% gains/amplifications and 25.1% deletions. Conclusions This study point out the high prevalence of ROS1 CNAs in a large series of NSCLC. ROS1 gains, amplifications and deletions, most of them due to chromosome 6 polysomy or monosomy, were heterogeneous within a tumor and had no impact on overall survival. PMID:26783962

  12. Prospective diagnostic analysis of copy number variants using SNP microarrays in individuals with autism spectrum disorders

    PubMed Central

    Nava, Caroline; Keren, Boris; Mignot, Cyril; Rastetter, Agnès; Chantot-Bastaraud, Sandra; Faudet, Anne; Fonteneau, Eric; Amiet, Claire; Laurent, Claudine; Jacquette, Aurélia; Whalen, Sandra; Afenjar, Alexandra; Périsse, Didier; Doummar, Diane; Dorison, Nathalie; Leboyer, Marion; Siffroi, Jean-Pierre; Cohen, David; Brice, Alexis; Héron, Delphine; Depienne, Christel

    2014-01-01

    Copy number variants (CNVs) have repeatedly been found to cause or predispose to autism spectrum disorders (ASDs). For diagnostic purposes, we screened 194 individuals with ASDs for CNVs using Illumina SNP arrays. In several probands, we also analyzed candidate genes located in inherited deletions to unmask autosomal recessive variants. Three CNVs, a de novo triplication of chromosome 15q11–q12 of paternal origin, a deletion on chromosome 9p24 and a de novo 3q29 deletion, were identified as the cause of the disorder in one individual each. An autosomal recessive cause was considered possible in two patients: a homozygous 1p31.1 deletion encompassing PTGER3 and a deletion of the entire DOCK10 gene associated with a rare hemizygous missense variant. We also identified multiple private or recurrent CNVs, the majority of which were inherited from asymptomatic parents. Although highly penetrant CNVs or variants inherited in an autosomal recessive manner were detected in rare cases, our results mainly support the hypothesis that most CNVs contribute to ASDs in association with other CNVs or point variants located elsewhere in the genome. Identification of these genetic interactions in individuals with ASDs constitutes a formidable challenge. PMID:23632794

  13. Prospective diagnostic analysis of copy number variants using SNP microarrays in individuals with autism spectrum disorders.

    PubMed

    Nava, Caroline; Keren, Boris; Mignot, Cyril; Rastetter, Agnès; Chantot-Bastaraud, Sandra; Faudet, Anne; Fonteneau, Eric; Amiet, Claire; Laurent, Claudine; Jacquette, Aurélia; Whalen, Sandra; Afenjar, Alexandra; Périsse, Didier; Doummar, Diane; Dorison, Nathalie; Leboyer, Marion; Siffroi, Jean-Pierre; Cohen, David; Brice, Alexis; Héron, Delphine; Depienne, Christel

    2014-01-01

    Copy number variants (CNVs) have repeatedly been found to cause or predispose to autism spectrum disorders (ASDs). For diagnostic purposes, we screened 194 individuals with ASDs for CNVs using Illumina SNP arrays. In several probands, we also analyzed candidate genes located in inherited deletions to unmask autosomal recessive variants. Three CNVs, a de novo triplication of chromosome 15q11-q12 of paternal origin, a deletion on chromosome 9p24 and a de novo 3q29 deletion, were identified as the cause of the disorder in one individual each. An autosomal recessive cause was considered possible in two patients: a homozygous 1p31.1 deletion encompassing PTGER3 and a deletion of the entire DOCK10 gene associated with a rare hemizygous missense variant. We also identified multiple private or recurrent CNVs, the majority of which were inherited from asymptomatic parents. Although highly penetrant CNVs or variants inherited in an autosomal recessive manner were detected in rare cases, our results mainly support the hypothesis that most CNVs contribute to ASDs in association with other CNVs or point variants located elsewhere in the genome. Identification of these genetic interactions in individuals with ASDs constitutes a formidable challenge.

  14. Enhanced copy number variants detection from whole-exome sequencing data using EXCAVATOR2

    PubMed Central

    D'Aurizio, Romina; Pippucci, Tommaso; Tattini, Lorenzo; Giusti, Betti; Pellegrini, Marco; Magi, Alberto

    2016-01-01

    Copy Number Variants (CNVs) are structural rearrangements contributing to phenotypic variation that have been proved to be associated with many disease states. Over the last years, the identification of CNVs from whole-exome sequencing (WES) data has become a common practice for research and clinical purpose and, consequently, the demand for more and more efficient and accurate methods has increased. In this paper, we demonstrate that more than 30% of WES data map outside the targeted regions and that these reads, usually discarded, can be exploited to enhance the identification of CNVs from WES experiments. Here, we present EXCAVATOR2, the first read count based tool that exploits all the reads produced by WES experiments to detect CNVs with a genome-wide resolution. To evaluate the performance of our novel tool we use it for analysing two WES data sets, a population data set sequenced by the 1000 Genomes Project and a tumor data set made of bladder cancer samples. The results obtained from these analyses demonstrate that EXCAVATOR2 outperforms other four state-of-the-art methods and that our combined approach enlarge the spectrum of detectable CNVs from WES data with an unprecedented resolution. EXCAVATOR2 is freely available at http://sourceforge.net/projects/excavator2tool/. PMID:27507884

  15. Thin and thick primary cutaneous melanomas reveal distinct patterns of somatic copy number alterations

    PubMed Central

    Apollo, Alessandro; Pescucci, Chiara; Licastro, Danilo; Urso, Carmelo; Gerlini, Gianni; Borgognoni, Lorenzo; Luzzatto, Lucio; Stecca, Barbara

    2016-01-01

    Cutaneous melanoma is one of the most aggressive type of skin tumor. Early stage melanoma can be often cured by surgery; therefore current management guidelines dictate a different approach for thin (<1mm) versus thick (>4mm) melanomas. We have carried out whole-exome sequencing in 5 thin and 5 thick fresh-frozen primary cutaneous melanomas. Unsupervised hierarchical clustering analysis of somatic copy number alterations (SCNAs) identified two groups corresponding to thin and thick melanomas. The most striking difference between them was the much greater abundance of SCNAs in thick melanomas, whereas mutation frequency did not significantly change between the two groups. We found novel mutations and focal SCNAs in genes that are embryonic regulators of axon guidance, predominantly in thick melanomas. Analysis of publicly available microarray datasets provided further support for a potential role of Ephrin receptors in melanoma progression. In addition, we have identified a set of SCNAs, including amplification of BRAF and ofthe epigenetic modifier EZH2, that are specific for the group of thick melanomas that developed metastasis during the follow-up. Our data suggest that mutations occur early during melanoma development, whereas SCNAs might be involved in melanoma progression. PMID:27095580

  16. Tropically adapted cattle of Africa: perspectives on potential role of copy number variations.

    PubMed

    Wang, M D; Dzama, K; Rees, D J G; Muchadeyi, F C

    2016-04-01

    Africa is host to diverse and locally adapted cattle breeds that are expected to survive the harsh and extreme tropical environments associated with diseases and parasite infections, heat stress and episodes of feed and water scarcity. Genomic copy number variations (CNVs) are considered to be primary role players in cattle breed formation and adaptation where isolation and genetic drift together with subsequent mutations have created an enormous diversity of local populations. CNVs are modifications in DNA structure comprising deletions, duplications and insertions that are >1 kb in size. Despite attracting much attention, the frequency and pattern of bovine CNV events, especially in African cattle breeds, are for the most part largely unknown. Characterization of genetic variation in the indigenous cattle of Africa will be a vital step toward dissecting the molecular mechanisms underlying phenotypic variation and local adaptation. This review therefore aims to describe the current knowledge regarding bovine CNVs and the implications and potentials they encompass for dissecting genetic adaptation and the genotypic skeleton of tropical African cattle populations.

  17. Concerted copy number variation balances ribosomal DNA dosage in human and mouse genomes

    PubMed Central

    Gibbons, John G.; Branco, Alan T.; Godinho, Susana A.; Yu, Shoukai; Lemos, Bernardo

    2015-01-01

    Tandemly repeated ribosomal DNA (rDNA) arrays are among the most evolutionary dynamic loci of eukaryotic genomes. The loci code for essential cellular components, yet exhibit extensive copy number (CN) variation within and between species. CN might be partly determined by the requirement of dosage balance between the 5S and 45S rDNA arrays. The arrays are nonhomologous, physically unlinked in mammals, and encode functionally interdependent RNA components of the ribosome. Here we show that the 5S and 45S rDNA arrays exhibit concerted CN variation (cCNV). Despite 5S and 45S rDNA elements residing on different chromosomes and lacking sequence similarity, cCNV between these loci is strong, evolutionarily conserved in humans and mice, and manifested across individual genotypes in natural populations and pedigrees. Finally, we observe that bisphenol A induces rapid and parallel modulation of 5S and 45S rDNA CN. Our observations reveal a novel mode of genome variation, indicate that natural selection contributed to the evolution and conservation of cCNV, and support the hypothesis that 5S CN is partly determined by the requirement of dosage balance with the 45S rDNA array. We suggest that human disease variation might be traced to disrupted rDNA dosage balance in the genome. PMID:25583482

  18. FOXL2 copy number changes in the molecular pathogenesis of BPES: unique cohort of 17 deletions.

    PubMed

    D'haene, B; Nevado, J; Pugeat, M; Pierquin, G; Lowry, R B; Reardon, W; Delicado, A; García-Miñaur, S; Palomares, M; Courtens, W; Stefanova, M; Wallace, S; Watkins, W; Shelling, A N; Wieczorek, D; Veitia, R A; De Paepe, A; Lapunzina, P; De Baere, E

    2010-05-01

    Blepharophimosis Syndrome (BPES) is an autosomal dominant developmental disorder of the eyelids with or without ovarian dysfunction caused by FOXL2 mutations. Overall, FOXL2deletions represent 12% of all genetic defects in BPES. Here, we have identified and characterized 16 new and one known FOXL2 deletion combining multiplex ligation-dependent probe amplification (MLPA), custom-made quantitative PCR (qPCR) and/or microarray-based copy number screening. The deletion breakpoints could be localized for 13 out of 17 deletions. The deletion size is highly variable (29.8 kb - 11.5 Mb), indicating absence of a recombination hotspot. Although the heterogeneity of their size and breakpoints is not reflected in the uniform BPES phenotype, there is considerable phenotypic variability regarding associated clinical findings including psychomotor retardation (8/17), microcephaly (6/17), and subtle skeletal features (2/17). In addition, in all females in whom ovarian function could be assessed, FOXL2 deletions proved to be associated with variable degrees of ovarian dysfunction. In conclusion, we present the largest series of BPES patients with FOXL2 deletions and standardized phenotyping reported so far. Our genotype-phenotype data can be useful for providing a prognosis (i.e. occurrence of associated features) in newborns with BPES carrying a FOXL2 deletion.

  19. Identification of Medium-Sized Copy Number Alterations in Whole-Genome Sequencing

    PubMed Central

    Ozer, Hatice Gulcin; Usubalieva, Aisulu; Dorrance, Adrienne; Yilmaz, Ayse Selen; Caligiuri, Michael; Marcucci, Guido; Huang, Kun

    2014-01-01

    The genome-wide discoveries such as detection of copy number alterations (CNA) from high-throughput whole-genome sequencing data enabled new developments in personalized medicine. The CNAs have been reported to be associated with various diseases and cancers including acute myeloid leukemia. However, there are multiple challenges to the use of current CNA detection tools that lead to high false-positive rates and thus impede widespread use of such tools in cancer research. In this paper, we discuss these issues and propose possible solutions. First, since the entire genome cannot be mapped due to some regions lacking sequence uniqueness, current methods cannot be appropriately adjusted to handle these regions in the analyses. Thus, detection of medium-sized CNAs is also being directly affected by these mappability problems. The requirement for matching control samples is also an important limitation because acquiring matching controls might not be possible or might not be cost efficient. Here we present an approach that addresses these issues and detects medium-sized CNAs in cancer genomes by (1) masking unmappable regions during the initial CNA detection phase, (2) using pool of a few normal samples as control, and (3) employing median filtering to adjust CNA ratios to its surrounding coverage and eliminate false positives. PMID:25788829

  20. Reproducible copy number variation patterns among single circulating tumor cells of lung cancer patients

    PubMed Central

    Ni, Xiaohui; Zhuo, Minglei; Su, Zhe; Duan, Jianchun; Gao, Yan; Wang, Zhijie; Zong, Chenghang; Bai, Hua; Chapman, Alec R.; Zhao, Jun; Xu, Liya; An, Tongtong; Ma, Qi; Wang, Yuyan; Wu, Meina; Sun, Yu; Wang, Shuhang; Li, Zhenxiang; Yang, Xiaodan; Yong, Jun; Su, Xiao-Dong; Lu, Youyong; Bai, Fan; Xie, X. Sunney; Wang, Jie

    2013-01-01

    Circulating tumor cells (CTCs) enter peripheral blood from primary tumors and seed metastases. The genome sequencing of CTCs could offer noninvasive prognosis or even diagnosis, but has been hampered by low single-cell genome coverage of scarce CTCs. Here, we report the use of the recently developed multiple annealing and looping-based amplification cycles for whole-genome amplification of single CTCs from lung cancer patients. We observed characteristic cancer-associated single-nucleotide variations and insertions/deletions in exomes of CTCs. These mutations provided information needed for individualized therapy, such as drug resistance and phenotypic transition, but were heterogeneous from cell to cell. In contrast, every CTC from an individual patient, regardless of the cancer subtypes, exhibited reproducible copy number variation (CNV) patterns, similar to those of the metastatic tumor of the same patient. Interestingly, different patients with the same lung cancer adenocarcinoma (ADC) shared similar CNV patterns in their CTCs. Even more interestingly, patients of small-cell lung cancer have CNV patterns distinctly different from those of ADC patients. Our finding suggests that CNVs at certain genomic loci are selected for the metastasis of cancer. The reproducibility of cancer-specific CNVs offers potential for CTC-based cancer diagnostics. PMID:24324171

  1. Phenotypic Association Analyses With Copy Number Variation in Recurrent Depressive Disorder

    PubMed Central

    Rucker, James J.H.; Tansey, Katherine E.; Rivera, Margarita; Pinto, Dalila; Cohen-Woods, Sarah; Uher, Rudolf; Aitchison, Katherine J.; Craddock, Nick; Owen, Michael J.; Jones, Lisa; Jones, Ian; Korszun, Ania; Barnes, Michael R.; Preisig, Martin; Mors, Ole; Maier, Wolfgang; Rice, John; Rietschel, Marcella; Holsboer, Florian; Farmer, Anne E.; Craig, Ian W.; Scherer, Stephen W.; McGuffin, Peter; Breen, Gerome

    2016-01-01

    Background Defining the molecular genomic basis of the likelihood of developing depressive disorder is a considerable challenge. We previously associated rare, exonic deletion copy number variants (CNV) with recurrent depressive disorder (RDD). Sex chromosome abnormalities also have been observed to co-occur with RDD. Methods In this reanalysis of our RDD dataset (N = 3106 cases; 459 screened control samples and 2699 population control samples), we further investigated the role of larger CNVs and chromosomal abnormalities in RDD and performed association analyses with clinical data derived from this dataset. Results We found an enrichment of Turner’s syndrome among cases of depression compared with the frequency observed in a large population sample (N = 34,910) of live-born infants collected in Denmark (two-sided p = .023, odds ratio = 7.76 [95% confidence interval = 1.79–33.6]), a case of diploid/triploid mosaicism, and several cases of uniparental isodisomy. In contrast to our previous analysis, large deletion CNVs were no more frequent in cases than control samples, although deletion CNVs in cases contained more genes than control samples (two-sided p = .0002). Conclusions After statistical correction for multiple comparisons, our data do not support a substantial role for CNVs in RDD, although (as has been observed in similar samples) occasional cases may harbor large variants with etiological significance. Genetic pleiotropy and sample heterogeneity suggest that very large sample sizes are required to study conclusively the role of genetic variation in mood disorders. PMID:25861698

  2. Prioritizing Clinically Relevant Copy Number Variation from Genetic Interactions and Gene Function Data

    PubMed Central

    Foong, Justin; Girdea, Marta; Stavropoulos, James; Brudno, Michael

    2015-01-01

    It is becoming increasingly necessary to develop computerized methods for identifying the few disease-causing variants from hundreds discovered in each individual patient. This problem is especially relevant for Copy Number Variants (CNVs), which can be cheaply interrogated via low-cost hybridization arrays commonly used in clinical practice. We present a method to predict the disease relevance of CNVs that combines functional context and clinical phenotype to discover clinically harmful CNVs (and likely causative genes) in patients with a variety of phenotypes. We compare several feature and gene weighing systems for classifying both genes and CNVs. We combined the best performing methodologies and parameters on over 2,500 Agilent CGH 180k Microarray CNVs derived from 140 patients. Our method achieved an F-score of 91.59%, with 87.08% precision and 97.00% recall. Our methods are freely available at https://github.com/compbio-UofT/cnv-prioritization. Our dataset is included with the supplementary information. PMID:26437450

  3. Targeted deep sequencing of flowering regulators in Brassica napus reveals extensive copy number variation

    PubMed Central

    Schiessl, Sarah; Huettel, Bruno; Kuehn, Diana; Reinhardt, Richard; Snowdon, Rod J.

    2017-01-01

    Gene copy number variation (CNV) is increasingly implicated in control of complex trait networks, particularly in polyploid plants like rapeseed (Brassica napus L.) with an evolutionary history of genome restructuring. Here we performed sequence capture to assay nucleotide variation and CNV in a panel of central flowering time regulatory genes across a species-wide diversity set of 280 B. napus accessions. The genes were chosen based on prior knowledge from Arabidopsis thaliana and related Brassica species. Target enrichment was performed using the Agilent SureSelect technology, followed by Illumina sequencing. A bait (probe) pool was developed based on results of a preliminary experiment with representatives from different B. napus morphotypes. A very high mean target coverage of ~670x allowed reliable calling of CNV, single nucleotide polymorphisms (SNPs) and insertion-deletion (InDel) polymorphisms. No accession exhibited no CNV, and at least one homolog of every gene we investigated showed CNV in some accessions. Some CNV appear more often in specific morphotypes, indicating a role in diversification. PMID:28291231

  4. Prevalence of actionable mutations and copy number alterations and the price of a genomic testing panel

    PubMed Central

    Shen, Chan; Meric-Bernstam, Funda; Su, Xiaoping; Mendelsohn, John; Giordano, Sharon

    2016-01-01

    Interest in genomic testing for the selection of cancer therapy is growing. However, the cost of genomic testing has not been well studied. We sought to determine the price of identifying mutations and copy number alterations (CNAs) in theoretically actionable genes across multiple tumor types. We reviewed data from The Cancer Genome Atlas to determine the frequency of alterations in nine tumor types. We used price information from a commonly used commercial genomic testing platform (FoundationOne) to determine the price of detecting mutations and CNAs in different types of tumors. Although there are large variations in the prevalence by tumor type, when the detection of both mutations and CNAs was considered overall, most patients had at least one alteration in a potentially actionable gene (84% overall, range 51%- 98% among tumor types assessed). The corresponding average price of identifying at least one alteration per patient ranges from $5,897 to $11,572. Although the frequency of mutations and CNAs in actionable genes differs by tumor type, most patients have an actionable genomic alteration detectable by a commercially available panel. Determining CNAs as well as mutations improves actionability and reduces the price of detecting an alteration. PMID:27634896

  5. Gene copy number alteration profile and its clinical correlation in B-cell acute lymphoblastic leukemia.

    PubMed

    Gupta, Sanjeev Kumar; Bakhshi, Sameer; Kumar, Lalit; Kamal, Vineet Kumar; Kumar, Rajive

    2017-02-01

    The genes related to B-cell development are frequently altered in B-cell acute lymphoblastic leukemia (B-ALL). One hundred sixty-two newly diagnosed B-ALL cases, median age 8.5 years (2 months-67 years), were prospectively analyzed for copy number alterations (CNAs) in CDKN2A/B, IKZF1, PAX5, RB1, ETV6, BTG1, EBF1, and pseudoautosomal region genes (CRLF2, CSF2RA, IL3RA) using multiplex ligation-dependent probe amplification. The CNAs were detected in 114 (70.4%) cases; most commonly affected genes being CDKN2A/B-55 (34%), PAX5-51 (31.5%), and IKZF1-43 (26.5%). IKZF1 and RB1 deletions correlated with higher induction failure. Patients classified as good-risk, according to the integrated CNA profile and cytogenetic criteria, had lower induction failure [5 (8.6%) vs. 20 (25.3%); p = 0.012]. Those classified as good-risk, based on CNA profile irrespective of cytogenetics, also showed lower induction failure [6 (9.4%) vs. 19 (26%); p = 0.012]. The CNA profile identified patients with better induction outcome and has a potential role in better risk stratification of B-ALL.

  6. Copy Number Variants in Short Children Born Small for Gestational Age

    PubMed Central

    Wit, Jan M.; van Duyvenvoorde, Hermine A.; van Klinken, Jan B.; Caliebe, Janina; Bosch, Cathy A.J.; Lui, Julian C.; Gijsbers, Antoinet C.J.; Bakker, Egbert; Breuning, Martijn H.; Oostdijk, Wilma; Losekoot, Monique; Baron, Jeffrey; Binder, Gerhard; Ranke, Michael B.; Ruivenkamp, Claudia A.L.

    2014-01-01

    Background/aims In addition to Genome-Wide Association studies (GWAS) height-associated genes may be uncovered by studying individuals with extreme short or tall stature. Methods Genome-wide analysis for copy number variants (CNVs), using Single Nucleotide Polymorphism (SNP) arrays, was performed in 49 index cases born small for gestational age (SGA) with persistent short stature. Segregation analysis was performed, and genes in CNVs were compared with information from GWAS, gene expression in rodents’ growth plates, and published information. Results CNVs were detected in 13 cases. In 5 children a known cause of short stature was found: UPD7, UPD14, a duplication of the SHOX enhancer region, an IGF1R deletion, and a 22q11.21 deletion. In the remaining 8 cases potential pathogenic CNVs were detected, either de novo (n=1), segregating (n=2), or not segregating with short stature (n=5). Bioinformatic analysis of the de novo and segregating CNVs suggested that HOXD4, AGPS, PDE11A, OSBPL6, PRKRA and PLEKHA3, and possibly DGKB and TNFRSF11B are potential candidate genes. A SERPINA7 or NRK defect may be associated with an X-linked form of short stature. Conclusion SNP arrays detected 5 known causes of short stature with prenatal onset and suggested several potential candidate genes. PMID:25300501

  7. Mutation dependance of the mitochondrial DNA copy number in the first stages of human embryogenesis.

    PubMed

    Monnot, Sophie; Samuels, David C; Hesters, Laetitia; Frydman, Nelly; Gigarel, Nadine; Burlet, Philippe; Kerbrat, Violaine; Lamazou, Frédéric; Frydman, René; Benachi, Alexandra; Feingold, Josué; Rotig, Agnes; Munnich, Arnold; Bonnefont, Jean-Paul; Steffann, Julie

    2013-05-01

    Mitochondrial DNA (mtDNA) content is thought to remain stable over the preimplantation period of human embryogenesis that is, therefore, suggested to be entirely dependent on ooplasm mtDNA capital. We have explored the impact of two disease-causing mutations [m.3243A>G myopathy, encephalopathy, lactic acidosis and stroke-like syndrome (MELAS) and m.8344A>G myoclonic epilepsy associated with ragged-red fibers (MERRF)] on mtDNA amounts in human oocytes and day 4-5 preimplantation embryos. The mtDNA amount was stable in MERRF and control materials, whereas gradually increasing from the germinal vesicle of oogenesis to the blastocyst stage of embryogenesis in MELAS cells, MELAS embryos carrying ∼3-fold higher mtDNA amount than control embryos (P = 0.0003). A correlation between mtDNA copy numbers and mutant loads was observed in MELAS embryos (R(2) = 0.42, P < 0.0013), suggestive of a compensation for the respiratory chain defect resulting from high mutation levels. These results suggest that mtDNA can replicate in early embryos and emphasize the need for sufficient amount of wild-type mtDNA to sustain embryonic development in humans.

  8. Patterns of genic intolerance of rare copy number variation in 59,898 human exomes.

    PubMed

    Ruderfer, Douglas M; Hamamsy, Tymor; Lek, Monkol; Karczewski, Konrad J; Kavanagh, David; Samocha, Kaitlin E; Daly, Mark J; MacArthur, Daniel G; Fromer, Menachem; Purcell, Shaun M

    2016-10-01

    Copy number variation (CNV) affecting protein-coding genes contributes substantially to human diversity and disease. Here we characterized the rates and properties of rare genic CNVs (<0.5% frequency) in exome sequencing data from nearly 60,000 individuals in the Exome Aggregation Consortium (ExAC) database. On average, individuals possessed 0.81 deleted and 1.75 duplicated genes, and most (70%) carried at least one rare genic CNV. For every gene, we empirically estimated an index of relative intolerance to CNVs that demonstrated moderate correlation with measures of genic constraint based on single-nucleotide variation (SNV) and was independently correlated with measures of evolutionary conservation. For individuals with schizophrenia, genes affected by CNVs were more intolerant than in controls. The ExAC CNV data constitute a critical component of an integrated database spanning the spectrum of human genetic variation, aiding in the interpretation of personal genomes as well as population-based disease studies. These data are freely available for download and visualization online.

  9. Haptoglobin (HP) and Haptoglobin-related protein (HPR) copy number variation, natural selection, and trypanosomiasis.

    PubMed

    Hardwick, Robert J; Ménard, Anne; Sironi, Manuela; Milet, Jacqueline; Garcia, André; Sese, Claude; Yang, Fengtang; Fu, Beiyuan; Courtin, David; Hollox, Edward J

    2014-01-01

    Haptoglobin, coded by the HP gene, is a plasma protein that acts as a scavenger for free heme, and haptoglobin-related protein (coded by the HPR gene) forms part of the trypanolytic factor TLF-1, together with apolipoprotein L1 (ApoL1). We analyse the polymorphic small intragenic duplication of the HP gene, with alleles Hp1 and Hp2, in 52 populations, and find no evidence for natural selection either from extended haplotype analysis or from correlation with pathogen richness matrices. Using fiber-FISH, the paralog ratio test, and array-CGH data, we also confirm that the HPR gene is copy number variable, with duplication of the whole HPR gene at polymorphic frequencies in west and central Africa, up to an allele frequency of 15 %. The geographical distribution of the HPR duplication allele overlaps the region where the pathogen causing chronic human African trypanosomiasis, Trypanosoma brucei gambiense, is endemic. The HPR duplication has occurred on one SNP haplotype, but there is no strong evidence of extended homozygosity, a characteristic of recent natural selection. The HPR duplication shows a slight, non-significant undertransmission to human African trypanosomiasis-affected children of unaffected parents in the Democratic Republic of Congo. However, taken together with alleles of APOL1, there is an overall significant undertransmission of putative protective alleles to human African trypanosomiasis-affected children.

  10. Integrated small copy number variations and epigenome maps of disorders of sex development

    PubMed Central

    Amarillo, Ina E; Nievera, Isabelle; Hagan, Andrew; Huchthagowder, Vishwa; Heeley, Jennifer; Hollander, Abby; Koenig, Joel; Austin, Paul; Wang, Ting

    2016-01-01

    Small copy number variations (CNVs) have typically not been analyzed or reported in clinical settings and hence have remained underrepresented in databases and the literature. Here, we focused our investigations on these small CNVs using chromosome microarray analysis (CMA) data previously obtained from patients with atypical characteristics or disorders of sex development (DSD). Using our customized CMA track targeting 334 genes involved in the development of urogenital and reproductive structures and a less stringent analysis filter, we uncovered small genes with recurrent and overlapping CNVs as small as 1 kb, and small regions of homozygosity (ROHs), imprinting and position effects. Detailed analysis of these high-resolution data revealed CNVs and ROHs involving structural and functional domains, repeat elements, active transcription sites and regulatory regions. Integration of these genomic data with DNA methylation, histone modification and predicted RNA expression profiles in normal testes and ovaries suggested spatiotemporal and tissue-specific gene regulation. This study emphasized a DSD-specific and gene-targeted CMA approach that uncovered previously unanalyzed or unreported small genes and CNVs, contributing to the growing resources on small CNVs and facilitating the narrowing of the genomic gap for identifying candidate genes or regions. This high-resolution analysis tool could improve the diagnostic utility of CMA, not only in patients with DSD but also in other clinical populations. These integrated data provided a better genomic-epigenomic landscape of DSD and greater opportunities for downstream research. PMID:27340555

  11. Investigation of modifier genes within copy number variations in Rett syndrome.

    PubMed

    Artuso, Rosangela; Papa, Filomena T; Grillo, Elisa; Mucciolo, Mafalda; Yasui, Dag H; Dunaway, Keith W; Disciglio, Vittoria; Mencarelli, Maria A; Pollazzon, Marzia; Zappella, Michele; Hayek, Giuseppe; Mari, Francesca; Renieri, Alessandra; Lasalle, Janine M; Ariani, Francesca

    2011-07-01

    MECP2 mutations are responsible for two different phenotypes in females, classical Rett syndrome and the milder Zappella variant (Z-RTT). We investigated whether copy number variants (CNVs) may modulate the phenotype by comparison of array-CGH data from two discordant pairs of sisters and four additional discordant pairs of unrelated girls matched by mutation type. We also searched for potential MeCP2 targets within CNVs by chromatin immunopreceipitation microarray (ChIP-chip) analysis. We did not identify one major common gene/region, suggesting that modifiers may be complex and variable between cases. However, we detected CNVs correlating with disease severity that contain candidate modifiers. CROCC (1p36.13) is a potential MeCP2 target, in which a duplication in a Z-RTT and a deletion in a classic patient were observed. CROCC encodes a structural component of ciliary motility that is required for correct brain development. CFHR1 and CFHR3, on 1q31.3, may be involved in the regulation of complement during synapse elimination, and were found to be deleted in a Z-RTT but duplicated in two classic patients. The duplication of 10q11.22, present in two Z-RTT patients, includes GPRIN2, a regulator of neurite outgrowth and PPYR1, involved in energy homeostasis. Functional analyses are necessary to confirm candidates and to define targets for future therapies.

  12. Copy Number Variation in Subjects with Major Depressive Disorder Who Attempted Suicide

    PubMed Central

    Perlis, Roy H.; Ruderfer, Douglas; Hamilton, Steven P.; Ernst, Carl

    2012-01-01

    Background Suicide is one of the top ten leading causes of death in North America and represents a major public health burden, partcularly for people with Major Depressive disorder (MD). Many studies have suggested that suicidal behavior runs in families, however, identification of genomic loci that drive this efffect remain to be identified. Methodology/Principal Findings Using subjects collected as part of STAR*D, we genotyped 189 subjects with MD with history of a suicide attempt and 1073 subjects with Major Depressive disorder that had never attempted suicide. Copy Number Variants (CNVs) were called in Birdsuite and analyzed in PLINK. We found a set of CNVs present in the suicide attempter group that were not present in in the non-attempter group including in SNTG2 and MACROD2 – two brain expressed genes previously linked to psychopathology; however, these results failed to reach genome-wide signifigance. Conclusions These data suggest potential CNVs to be investigated further in relation to suicide attempts in MD using large sample sizes. PMID:23029476

  13. Measurement of lentiviral vector titre and copy number by cross-species duplex quantitative PCR.

    PubMed

    Christodoulou, I; Patsali, P; Stephanou, C; Antoniou, M; Kleanthous, M; Lederer, C W

    2016-01-01

    Lentiviruses are the vectors of choice for many preclinical studies and clinical applications of gene therapy. Accurate measurement of biological vector titre before treatment is a prerequisite for vector dosing, and the calculation of vector integration sites per cell after treatment is as critical to the characterisation of modified cell products as it is to long-term follow-up and the assessment of risk and therapeutic efficiency in patients. These analyses are typically based on quantitative real-time PCR (qPCR), but as yet compromise accuracy and comparability between laboratories and experimental systems, the former by using separate simplex reactions for the detection of endogene and lentiviral sequences and the latter by designing different PCR assays for analyses in human cells and animal disease models. In this study, we validate in human and murine cells a qPCR system for the single-tube assessment of lentiviral vector copy numbers that is suitable for analyses in at least 33 different mammalian species, including human and other primates, mouse, pig, cat and domestic ruminants. The established assay combines the accuracy of single-tube quantitation by duplex qPCR with the convenience of one-off assay optimisation for cross-species analyses and with the direct comparability of lentiviral transduction efficiencies in different species.

  14. Genome-Wide Association Study of Copy Number Variations in Patients with Familial Neurocardiogenic Syncope.

    PubMed

    Demir, Emre; Hasdemir, Can; Ak, Handan; Atay, Sevcan; Aydin, Hikmet Hakan

    2016-08-01

    Neurocardiogenic syncope (NCS) is the most frequent type of syncope characterized by a self-limited episode of systemic hypotension. In this study, we conducted the first genome-wide association study testing copy number variations for association with NCS. Study population consisted of 107 consecutive patients with recurrent syncope and positive head-up tilt table testing. Four families with NCS were selected for CNV analysis. Affymetrix GeneChip(®) SNP 6.0 array was used for CNV analysis. Data and statistical analysis were performed with Affymetrix genotyping console 4.0 and GraphPad Prism v6. Positive family history of NCS was present in 19.6 % (n = 21) in our study population (n = 107). Twenty-six CNV regions were found to be significantly altered in families with NCS (P < 0.05). Several CNVs were identified in families with NCS. Further studies comprising wider study population are required to determine the effect of these variations on NCS development.

  15. Copy number variants calling for single cell sequencing data by multi-constrained optimization.

    PubMed

    Xu, Bo; Cai, Hongmin; Zhang, Changsheng; Yang, Xi; Han, Guoqiang

    2016-08-01

    Variations in DNA copy number carry important information on genome evolution and regulation of DNA replication in cancer cells. The rapid development of single-cell sequencing technology allows one to explore gene expression heterogeneity among single-cells, thus providing important cancer cell evolution information. Single-cell DNA/RNA sequencing data usually have low genome coverage, which requires an extra step of amplification to accumulate enough samples. However, such amplification will introduce large bias and makes bioinformatics analysis challenging. Accurately modeling the distribution of sequencing data and effectively suppressing the bias influence is the key to success variations analysis. Recent advances demonstrate the technical noises by amplification are more likely to follow negative binomial distribution, a special case of Poisson distribution. Thus, we tackle the problem CNV detection by formulating it into a quadratic optimization problem involving two constraints, in which the underling signals are corrupted by Poisson distributed noises. By imposing the constraints of sparsity and smoothness, the reconstructed read depth signals from single-cell sequencing data are anticipated to fit the CNVs patterns more accurately. An efficient numerical solution based on the classical alternating direction minimization method (ADMM) is tailored to solve the proposed model. We demonstrate the advantages of the proposed method using both synthetic and empirical single-cell sequencing data. Our experimental results demonstrate that the proposed method achieves excellent performance and high promise of success with single-cell sequencing data.

  16. Copy Number Variation in Acetolactate Synthase Genes of Thifensulfuron-Methyl Resistant Alopecurus aequalis (Shortawn Foxtail) Accessions in Japan

    PubMed Central

    Iwakami, Satoshi; Shimono, Yoshiko; Manabe, Yohei; Endo, Masaki; Shibaike, Hiroyuki; Uchino, Akira; Tominaga, Tohru

    2017-01-01

    Severe infestations of Alopecurus aequalis (shortawn foxtail), a noxious weed in wheat and barley cropping systems in Japan, can occur even after application of thifensulfuron-methyl, a sulfonylurea (SU) herbicide. In the present study, nine accessions of A. aequalis growing in a single wheat field were tested for sensitivity to thifensulfuron-methyl. Seven of the nine accessions survived application of standard field rates of thifensulfuron-methyl, indicating that severe infestations likely result from herbicide resistance. Acetolactate synthase (ALS) is the target enzyme of SU herbicides. Full-length genes encoding ALS were therefore isolated to determine the mechanism of SU resistance. As a result, differences in ALS gene copy numbers among accessions were revealed. Two copies, ALS1 and ALS2, were conserved in all accessions, while some carried two additional copies, ALS3 and ALS4. A single-base deletion in ALS3 and ALS4 further indicated that they represent pseudogenes. No differences in ploidy level were observed between accessions with two or four copies of the ALS gene, suggesting that copy number varies. Resistant plants were found to carry a mutation in either the ALS1 or ALS2 gene, with all mutations causing an amino acid substitution at the Pro197 residue, which is known to confer SU resistance. Transcription of each ALS gene copy was confirmed by reverse transcription PCR, supporting involvement of these mutations in SU resistance. The information on the copy number and full-length sequences of ALS genes in A. aequalis will aid future analysis of the mechanism of resistance. PMID:28303143

  17. Genomic Copy Number Dictates a Gene-Independent Cell Response to CRISPR/Cas9 Targeting | Office of Cancer Genomics

    Cancer.gov

    The CRISPR/Cas9 system enables genome editing and somatic cell genetic screens in mammalian cells. We performed genome-scale loss-of-function screens in 33 cancer cell lines to identify genes essential for proliferation/survival and found a strong correlation between increased gene copy number and decreased cell viability after genome editing. Within regions of copy-number gain, CRISPR/Cas9 targeting of both expressed and unexpressed genes, as well as intergenic loci, led to significantly decreased cell proliferation through induction of a G2 cell-cycle arrest.

  18. MYC, FBXW7 and TP53 copy number variation and expression in Gastric Cancer

    PubMed Central

    2013-01-01

    Background MYC deregulation is a common event in gastric carcinogenesis, usually as a consequence of gene amplification, chromosomal translocations, or posttranslational mechanisms. FBXW7 is a p53-controlled tumor-suppressor that plays a role in the regulation of cell cycle exit and reentry via MYC degradation. Methods We evaluated MYC, FBXW7, and TP53 copy number, mRNA levels, and protein expression in gastric cancer and paired non-neoplastic specimens from 33 patients and also in gastric adenocarcinoma cell lines. We also determined the invasion potential of the gastric cancer cell lines. Results MYC amplification was observed in 51.5% of gastric tumor samples. Deletion of one copy of FBXW7 and TP53 was observed in 45.5% and 21.2% of gastric tumors, respectively. MYC mRNA expression was significantly higher in tumors than in non-neoplastic samples. FBXW7 and TP53 mRNA expression was markedly lower in tumors than in paired non-neoplastic specimens. Moreover, deregulated MYC and FBXW7 mRNA expression was associated with the presence of lymph node metastasis and tumor stage III-IV. Additionally, MYC immunostaining was more frequently observed in intestinal-type than diffuse-type gastric cancers and was associated with MYC mRNA expression. In vitro studies showed that increased MYC and reduced FBXW7 expression is associated with a more invasive phenotype in gastric cancer cell lines. This result encouraged us to investigate the activity of the gelatinases MMP-2 and MMP-9 in both cell lines. Both gelatinases are synthesized predominantly by stromal cells rather than cancer cells, and it has been proposed that both contribute to cancer progression. We observed a significant increase in MMP-9 activity in ACP02 compared with ACP03 cells. These results confirmed that ACP02 cells have greater invasion capability than ACP03 cells. Conclusion In conclusion, FBXW7 and MYC mRNA may play a role in aggressive biologic behavior of gastric cancer cells and may be a useful

  19. Genomewide profiling of copy-number alteration in monoclonal gammopathy of undetermined significance.

    PubMed

    Mikulasova, Aneta; Smetana, Jan; Wayhelova, Marketa; Janyskova, Helena; Sandecka, Viera; Kufova, Zuzana; Almasi, Martina; Jarkovsky, Jiri; Gregora, Evzen; Kessler, Petr; Wrobel, Marek; Walker, Brian A; Wardell, Christopher P; Morgan, Gareth J; Hajek, Roman; Kuglik, Petr

    2016-12-01

    Monoclonal gammopathy of undetermined significance (MGUS) is a benign condition with an approximate 1% annual risk of symptomatic plasma cell disorder development, mostly to multiple myeloma (MM). We performed genomewide screening of copy-number alterations (CNAs) in 90 MGUS and 33 MM patients using high-density DNA microarrays. We identified CNAs in a smaller proportion of MGUS (65.6%) than in MM (100.0%, P = 1.31 × 10(-5) ) and showed median number of CNAs is lower in MGUS (3, range 0-22) than in MM (13, range 4-38, P = 1.82 × 10(-10) ). In the MGUS cohort, the most frequent losses were located at 1p (5.6%), 6q (6.7%), 13q (30.0%), 14q (14.4%), 16q (8.9%), 21q (5.6%), and gains at 1q (23.3%), 2p (6.7%), 6p (13.3%), and Xq (7.8%). Hyperdiploidy was detected in 38.9% of MGUS cases, and the most frequent whole chromosome gains were 3 (25.6%), 5 (23.3%), 9 (37.8%), 15 (23.3%), and 19 (32.2%). We also identified CNAs such as 1p, 6q, 8p, 12p, 13q, 16q losses, 1q gain and hypodiploidy, which are potentially associated with an adverse prognosis in MGUS. In summary, we showed that MGUS is similar to MM in that it is a genetically heterogeneous disorder, but overall cytogenetic instability is lower than in MM, which confirms that genetic abnormalities play important role in monoclonal gammopathies.

  20. Detecting Large Copy Number Variants Using Exome Genotyping Arrays In a Large Swedish Schizophrenia Sample

    PubMed Central

    Szatkiewicz, Jin P.; Neale, Benjamin M.; O'Dushlaine, Colm; Fromer, Menachem; Goldstein, Jacqueline I.; Moran, Jennifer L.; Chambert, Kimberly; Kähler, Anna; Magnusson, Patrik KE; Hultman, Christina M.; Sklar, Pamela; Purcell, Shaun; McCarroll, Steven A.; Sullivan, Patrick F.

    2014-01-01

    Although copy number variants (CNVs) are important in genomic medicine, CNVs have not been systematically assessed for many complex traits. Several large rare CNVs increase risk for schizophrenia (SCZ) and autism and often demonstrate pleiotropic effects; however, their frequencies in the general population and other complex traits are unknown. Genotyping large numbers of samples is essential for progress. Large cohorts from many different diseases are being genotyped using exome-focused arrays designed to detect uncommon or rare protein-altering sequence variation. Although these arrays were not designed for CNV detection, the hybridization intensity data generated in each experiment could, in principle, be used for gene-focused CNV analysis. Our goal was to evaluate the extent to which CNVs can be detected using data from one particular exome array (the Illumina Human Exome Bead Chip). We genotyped 9, 100 Swedish subjects (3, 962 cases with SCZ and 5, 138 controls) using both standard GWAS arrays and exome arrays. In comparison to CNVs detected using GWAS arrays, we observed high sensitivity and specificity for detecting genic CNVs ≥400 kb including known pathogentic CNVs along with replicating the literature finding that cases with SCZ had greater enrichment for genic CNVs. Our data confirm the association of SCZ with 16p11.2 duplications and 22q11.2 deletions and suggest a novel association with deletions at 11q12.2. Our results suggest the utility of exome focused arrays in surveying large genic CNVs in very large samples; and thereby open the door for new opportunities such as conducting well-powered CNV assessment and comparisons between different diseases. The use of a single platform also minimizes potential confounding factors that could impact accurate detection. PMID:23938935

  1. Evaluation of copy number variations reveals novel candidate genes in autism spectrum disorder-associated pathways

    PubMed Central

    Griswold, Anthony J.; Ma, Deqiong; Cukier, Holly N.; Nations, Laura D.; Schmidt, Mike A.; Chung, Ren-Hua; Jaworski, James M.; Salyakina, Daria; Konidari, Ioanna; Whitehead, Patrice L.; Wright, Harry H.; Abramson, Ruth K.; Williams, Scott M.; Menon, Ramkumar; Martin, Eden R.; Haines, Jonathan L.; Gilbert, John R.; Cuccaro, Michael L.; Pericak-Vance, Margaret A.

    2012-01-01

    Autism spectrum disorders (ASDs) are highly heritable, yet relatively few associated genetic loci have been replicated. Copy number variations (CNVs) have been implicated in autism; however, the majority of loci contribute to <1% of the disease population. Therefore, independent studies are important to refine associated CNV regions and discover novel susceptibility genes. In this study, a genome-wide SNP array was utilized for CNV detection by two distinct algorithms in a European ancestry case–control data set. We identify a significantly higher burden in the number and size of deletions, and disrupting more genes in ASD cases. Moreover, 18 deletions larger than 1 Mb were detected exclusively in cases, implicating novel regions at 2q22.1, 3p26.3, 4q12 and 14q23. Case-specific CNVs provided further evidence for pathways previously implicated in ASDs, revealing new candidate genes within the GABAergic signaling and neural development pathways. These include DBI, an allosteric binder of GABA receptors, GABARAPL1, the GABA receptor-associated protein, and SLC6A11, a postsynaptic GABA transporter. We also identified CNVs in COBL, deletions of which cause defects in neuronal cytoskeleton morphogenesis in model vertebrates, and DNER, a neuron-specific Notch ligand required for cerebellar development. Moreover, we found evidence of genetic overlap between ASDs and other neurodevelopmental and neuropsychiatric diseases. These genes include glutamate receptors (GRID1, GRIK2 and GRIK4), synaptic regulators (NRXN3, SLC6A8 and SYN3), transcription factor (ZNF804A) and RNA-binding protein FMR1. Taken together, these CNVs may be a few of the missing pieces of ASD heritability and lead to discovering novel etiological mechanisms. PMID:22543975

  2. Integrated analysis of copy number variation and genome-wide expression profiling in colorectal cancer tissues.

    PubMed

    Ali Hassan, Nur Zarina; Mokhtar, Norfilza Mohd; Kok Sin, Teow; Mohamed Rose, Isa; Sagap, Ismail; Harun, Roslan; Jamal, Rahman

    2014-01-01

    Integrative analyses of multiple genomic datasets for selected samples can provide better insight into the overall data and can enhance our knowledge of cancer. The objective of this study was to elucidate the association between copy number variation (CNV) and gene expression in colorectal cancer (CRC) samples and their corresponding non-cancerous tissues. Sixty-four paired CRC samples from the same patients were subjected to CNV profiling using the Illumina HumanOmni1-Quad assay, and validation was performed using multiplex ligation probe amplification method. Genome-wide expression profiling was performed on 15 paired samples from the same group of patients using the Affymetrix Human Gene 1.0 ST array. Significant genes obtained from both array results were then overlapped. To identify molecular pathways, the data were mapped to the KEGG database. Whole genome CNV analysis that compared primary tumor and non-cancerous epithelium revealed gains in 1638 genes and losses in 36 genes. Significant gains were mostly found in chromosome 20 at position 20q12 with a frequency of 45.31% in tumor samples. Examples of genes that were associated at this cytoband were PTPRT, EMILIN3 and CHD6. The highest number of losses was detected at chromosome 8, position 8p23.2 with 17.19% occurrence in all tumor samples. Among the genes found at this cytoband were CSMD1 and DLC1. Genome-wide expression profiling showed 709 genes to be up-regulated and 699 genes to be down-regulated in CRC compared to non-cancerous samples. Integration of these two datasets identified 56 overlapping genes, which were located in chromosomes 8, 20 and 22. MLPA confirmed that the CRC samples had the highest gains in chromosome 20 compared to the reference samples. Interpretation of the CNV data in the context of the transcriptome via integrative analyses may provide more in-depth knowledge of the genomic landscape of CRC.

  3. Genomic copy number variation associated with clinical outcome in canine cutaneous mast cell tumors.

    PubMed

    Jark, Paulo C; Mundin, Deborah B P; de Carvalho, Marcio; Ferioli, Raquel B; Anai, Letícia A; Marchi, Fabio A; Rogatto, Silvia R; Laufer-Amorim, Renee; Tinucci-Costa, Mirela

    2017-04-01

    Mast cell tumors are the most common malignant cutaneous tumors in dogs. Although there are several prognostic factors involved, the clinical and biological behavior of this type of tumor varies greatly, making the best choice of treatment challenging. Molecular techniques can be used to evaluate a large number of genes involved in the neoplastic process and aid in the selection of candidate genes related to prognostic and predicting factors. Identification of the genes associated with tumor development and progression can be performed through the analysis of numerical and structural changes in DNA isolated from tumor cells by array comparative genomic hybridization (aCGH). The aim of this study was to compare copy number variations (CNVs) in cutaneous mast cell tumors of dogs that survived less than six (ST<6) and >12months (ST>12) from the date of diagnosis. Ten animals were used: four from Group ST>12 and six from Group ST<6. Genomic DNA was extracted, and aCGH was performed using Agilent Canine Genome CGH Microarray 4×180 (ID-252 552 - Agilent, USA). Data analysis was carried out using Nexus program version 5.0 (Biodiscovery, USA). The group ST>12 presented 11±3.3 CNVs, while the ST<6 group presented 85±38.5 CNVs. Regions of loss in PTEN and FAS as well as regions of gains in MAPK3, WNT5B, FGF, FOXM1 and RAD51 were detected in mast cell tumors with shorter survival times, and thus, worst prognoses, allowing for the identification of potential candidate genes for more detailed studies.

  4. Change in the plasmid copy number in acetic acid bacteria in response to growth phase and acetic acid concentration.

    PubMed

    Akasaka, Naoki; Astuti, Wiwik; Ishii, Yuri; Hidese, Ryota; Sakoda, Hisao; Fujiwara, Shinsuke

    2015-06-01

    Plasmids pGE1 (2.5 kb), pGE2 (7.2 kb), and pGE3 (5.5 kb) were isolated from Gluconacetobacter europaeus KGMA0119, and sequence analyses revealed they harbored 3, 8, and 4 genes, respectively. Plasmid copy numbers (PCNs) were determined by real-time quantitative PCR at different stages of bacterial growth. When KGMA0119 was cultured in medium containing 0.4% ethanol and 0.5% acetic acid, PCN of pGE1 increased from 7 copies/genome in the logarithmic phase to a maximum of 12 copies/genome at the beginning of the stationary phase, before decreasing to 4 copies/genome in the late stationary phase. PCNs for pGE2 and pGE3 were maintained at 1-3 copies/genome during all phases of growth. Under a higher concentration of ethanol (3.2%) the PCN for pGE1 was slightly lower in all the growth stages, and those of pGE2 and pGE3 were unchanged. In the presence of 1.0% acetic acid, PCNs were higher for pGE1 (10 copies/genome) and pGE3 (6 copies/genome) during the logarithmic phase. Numbers for pGE2 did not change, indicating that pGE1 and pGE3 increase their PCNs in response to acetic acid. Plasmids pBE2 and pBE3 were constructed by ligating linearized pGE2 and pGE3 into pBR322. Both plasmids were replicable in Escherichia coli, Acetobacter pasteurianus and G. europaeus, highlighting their suitability as vectors for acetic acid bacteria.

  5. Spectrum of large copy number variations in 26 diverse Indian populations: potential involvement in phenotypic diversity.

    PubMed

    Gautam, Pramod; Jha, Pankaj; Kumar, Dhirendra; Tyagi, Shivani; Varma, Binuja; Dash, Debasis; Mukhopadhyay, Arijit; Mukerji, Mitali

    2012-01-01

    Copy number variations (CNVs) have provided a dynamic aspect to the apparently static human genome. We have analyzed CNVs larger than 100 kb in 477 healthy individuals from 26 diverse Indian populations of different linguistic, ethnic and geographic backgrounds. These CNVRs were identified using the Affymetrix 50K Xba 240 Array. We observed 1,425 and 1,337 CNVRs in the deletion and amplification sets, respectively, after pooling data from all the populations. More than 50% of the genes encompassed entirely in CNVs had both deletions and amplifications. There was wide variability across populations not only with respect to CNV extent (ranging from 0.04-1.14% of genome under deletion and 0.11-0.86% under amplification) but also in terms of functional enrichments of processes like keratinization, serine proteases and their inhibitors, cadherins, homeobox, olfactory receptors etc. These did not correlate with linguistic, ethnic, geographic backgrounds and size of populations. Certain processes were near exclusive to deletion (serine proteases, keratinization, olfactory receptors, GPCRs) or duplication (homeobox, serine protease inhibitors, embryonic limb morphogenesis) datasets. Populations having same enriched processes were observed to contain genes from different genomic loci. Comparison of polymorphic CNVRs (5% or more) with those cataloged in Database of Genomic Variants revealed that 78% (2473) of the genes in CNVRs in Indian populations are novel. Validation of CNVs using Sequenom MassARRAY revealed extensive heterogeneity in CNV boundaries. Exploration of CNV profiles in such diverse populations would provide a widely valuable resource for understanding diversity in phenotypes and disease.

  6. Population clustering based on copy number variations detected from next generation sequencing data

    PubMed Central

    Duan, Junbo; Zhang, Ji-Gang; Wan, Mingxi; Deng, Hong-Wen; Wang, Yu-Ping

    2015-01-01

    Copy number variations (CNVs) can be used as significant bio-markers and next generation sequencing (NGS) provides a high resolution detection of these CNVs. But how to extract features from CNVs and further apply them to genomic studies such as population clustering have become a big challenge. In this paper, we propose a novel method for population clustering based on CNVs from NGS. First, CNVs are extracted from each sample to form a feature matrix. Then, this feature matrix is decomposed into the source matrix and weight matrix with non-negative matrix factorization (NMF). The source matrix consists of common CNVs that are shared by all the samples from the same group, and the weight matrix indicates the corresponding level of CNVs from each sample. Therefore, using NMF of CNVs one can differentiate samples from different ethnic groups, i.e. population clustering. To validate the approach, we applied it to the analysis of both simulation data and two real data set from the 1000 Genomes Project. The results on simulation data demonstrate that the proposed method can recover the true common CNVs with high quality. The results on the first real data analysis show that the proposed method can cluster two family trio with different ancestries into two ethnic groups and the results on the second real data analysis show that the proposed method can be applied to the whole-genome with large sample size consisting of multiple groups. Both results demonstrate the potential of the proposed method for population clustering. PMID:25152046

  7. A Discovery Resource of Rare Copy Number Variations in Individuals with Autism Spectrum Disorder

    PubMed Central

    Prasad, Aparna; Merico, Daniele; Thiruvahindrapuram, Bhooma; Wei, John; Lionel, Anath C.; Sato, Daisuke; Rickaby, Jessica; Lu, Chao; Szatmari, Peter; Roberts, Wendy; Fernandez, Bridget A.; Marshall, Christian R.; Hatchwell, Eli; Eis, Peggy S.; Scherer, Stephen W.

    2012-01-01

    The identification of rare inherited and de novo copy number variations (CNVs) in human subjects has proven a productive approach to highlight risk genes for autism spectrum disorder (ASD). A variety of microarrays are available to detect CNVs, including single-nucleotide polymorphism (SNP) arrays and comparative genomic hybridization (CGH) arrays. Here, we examine a cohort of 696 unrelated ASD cases using a high-resolution one-million feature CGH microarray, the majority of which were previously genotyped with SNP arrays. Our objective was to discover new CNVs in ASD cases that were not detected by SNP microarray analysis and to delineate novel ASD risk loci via combined analysis of CGH and SNP array data sets on the ASD cohort and CGH data on an additional 1000 control samples. Of the 615 ASD cases analyzed on both SNP and CGH arrays, we found that 13,572 of 21,346 (64%) of the CNVs were exclusively detected by the CGH array. Several of the CGH-specific CNVs are rare in population frequency and impact previously reported ASD genes (e.g., NRXN1, GRM8, DPYD), as well as novel ASD candidate genes (e.g., CIB2, DAPP1, SAE1), and all were inherited except for a de novo CNV in the GPHN gene. A functional enrichment test of gene-sets in ASD cases over controls revealed nucleotide metabolism as a potential novel pathway involved in ASD, which includes several candidate genes for follow-up (e.g., DPYD, UPB1, UPP1, TYMP). Finally, this extensively phenotyped and genotyped ASD clinical cohort serves as an invaluable resource for the next step of genome sequencing for complete genetic variation detection. PMID:23275889

  8. High-resolution copy number variation analysis of schizophrenia in Japan.

    PubMed

    Kushima, I; Aleksic, B; Nakatochi, M; Shimamura, T; Shiino, T; Yoshimi, A; Kimura, H; Takasaki, Y; Wang, C; Xing, J; Ishizuka, K; Oya-Ito, T; Nakamura, Y; Arioka, Y; Maeda, T; Yamamoto, M; Yoshida, M; Noma, H; Hamada, S; Morikawa, M; Uno, Y; Okada, T; Iidaka, T; Iritani, S; Yamamoto, T; Miyashita, M; Kobori, A; Arai, M; Itokawa, M; Cheng, M-C; Chuang, Y-A; Chen, C-H; Suzuki, M; Takahashi, T; Hashimoto, R; Yamamori, H; Yasuda, Y; Watanabe, Y; Nunokawa, A; Someya, T; Ikeda, M; Toyota, T; Yoshikawa, T; Numata, S; Ohmori, T; Kunimoto, S; Mori, D; Iwata, N; Ozaki, N

    2017-03-01

    Recent schizophrenia (SCZ) studies have reported an increased burden of de novo copy number variants (CNVs) and identified specific high-risk CNVs, although with variable phenotype expressivity. However, the pathogenesis of SCZ has not been fully elucidated. Using array comparative genomic hybridization, we performed a high-resolution genome-wide CNV analysis on a mainly (92%) Japanese population (1699 SCZ cases and 824 controls) and identified 7066 rare CNVs, 70.0% of which were small (<100 kb). Clinically significant CNVs were significantly more frequent in cases than in controls (odds ratio=3.04, P=9.3 × 10(-9), 9.0% of cases). We confirmed a significant association of X-chromosome aneuploidies with SCZ and identified 11 de novo CNVs (e.g., MBD5 deletion) in cases. In patients with clinically significant CNVs, 41.7% had a history of congenital/developmental phenotypes, and the rate of treatment resistance was significantly higher (odds ratio=2.79, P=0.0036). We found more severe clinical manifestations in patients with two clinically significant CNVs. Gene set analysis replicated previous findings (e.g., synapse, calcium signaling) and identified novel biological pathways including oxidative stress response, genomic integrity, kinase and small GTPase signaling. Furthermore, involvement of multiple SCZ candidate genes and biological pathways in the pathogenesis of SCZ was suggested in established SCZ-associated CNV loci. Our study shows the high genetic heterogeneity of SCZ and its clinical features and raises the possibility that genomic instability is involved in its pathogenesis, which may be related to the increased burden of de novo CNVs and variable expressivity of CNVs.

  9. Investigating the genetic basis of fever-associated syndromic epilepsies using copy number variation analysis.

    PubMed

    Hartmann, Corinna; von Spiczak, Sarah; Suls, Arvid; Weckhuysen, Sarah; Buyse, Gunnar; Vilain, Catheline; Van Bogaert, Patrick; De Jonghe, Peter; Cook, Joseph; Muhle, Hiltrud; Stephani, Ulrich; Helbig, Ingo; Mefford, Heather C

    2015-03-01

    Fever-associated syndromic epilepsies ranging from febrile seizures plus (FS+) to Dravet syndrome have a significant genetic component. However, apart from SCN1A mutations in >80% of patients with Dravet syndrome, the genetic underpinnings of these epilepsies remain largely unknown. Therefore, we performed a genome-wide screening for copy number variations (CNVs) in 36 patients with SCN1A-negative fever-associated syndromic epilepsies. Phenotypes included Dravet syndrome (n = 23; 64%), genetic epilepsy with febrile seizures plus (GEFS+) and febrile seizures plus (FS+) (n = 11; 31%) and unclassified fever-associated epilepsies (n = 2; 6%). Array comparative genomic hybridization (CGH) was performed using Agilent 4 × 180K arrays. We identified 13 rare CNVs in 8 (22%) of 36 individuals. These included known pathogenic CNVs in 4 (11%) of 36 patients: a 1q21.1 duplication in a proband with Dravet syndrome, a 14q23.3 deletion in a proband with FS+, and two deletions at 16p11.2 and 1q44 in two individuals with fever-associated epilepsy with concomitant autism and/or intellectual disability. In addition, a 3q13.11 duplication in a patient with FS+ and two de novo duplications at 7p14.2 and 18q12.2 in a patient with atypical Dravet syndrome were classified as likely pathogenic. Six CNVs were of unknown significance. The identified genomic aberrations overlap with known neurodevelopmental disorders, suggesting that fever-associated epilepsy syndromes may be a recurrent clinical presentation of known microdeletion syndromes.

  10. Autism genome-wide copy number variation reveals ubiquitin and neuronal genes

    PubMed Central

    Glessner, Joseph T.; Wang, Kai; Cai, Guiqing; Korvatska, Olena; Kim, Cecilia E.; Wood, Shawn; Zhang, Haitao; Estes, Annette; Brune, Camille W.; Bradfield, Jonathan P.; Imielinski, Marcin; Frackelton, Edward C.; Reichert, Jennifer; Crawford, Emily L.; Munson, Jeffrey; Sleiman, Patrick M. A.; Chiavacci, Rosetta; Annaiah, Kiran; Thomas, Kelly; Hou, Cuiping; Glaberson, Wendy; Flory, James; Otieno, Frederick; Garris, Maria; Soorya, Latha; Klei, Lambertus; Piven, Joseph; Meyer, Kacie J.; Anagnostou, Evdokia; Sakurai, Takeshi; Game, Rachel M.; Rudd, Danielle S.; Zurawiecki, Danielle; McDougle, Christopher J.; Davis, Lea K.; Miller, Judith; Posey, David J.; Michaels, Shana; Kolevzon, Alexander; Silverman, Jeremy M.; Bernier, Raphael; Levy, Susan E.; Schultz, Robert T.; Dawson, Geraldine; Owley, Thomas; McMahon, William M.; Wassink, Thomas H.; Sweeney, John A.; Nurnberger, John I.; Coon, Hilary; Sutcliffe, James S.; Minshew, Nancy J.; Grant, Struan F. A.; Bucan, Maja; Cook, Edwin H.; Buxbaum, Joseph D.; Devlin, Bernie; Schellenberg, Gerard D.; Hakonarson, Hakon

    2010-01-01

    Autism spectrum disorders (ASDs) are childhood neurodevelopmental disorders with complex genetic origins1–4. Previous studies focusing on candidate genes or genomic regions have identified several copy number variations (CNVs) that are associated with an increased risk of ASDs5–9. Here we present the results from a whole-genome CNV study on a cohort of 859 ASD cases and 1,409 healthy children of European ancestry who were genotyped with ~550,000 single nucleotide polymorphism markers, in an attempt to comprehensively identify CNVs conferring susceptibility to ASDs. Positive findings were evaluated in an independent cohort of 1,336 ASD cases and 1,110 controls of European ancestry. Besides previously reported ASD candidate genes, such as NRXN1 (ref. 10) and CNTN4 (refs 11, 12), several new susceptibility genes encoding neuronal cell-adhesion molecules, including NLGN1 and ASTN2, were enriched with CNVs in ASD cases compared to controls (P = 9.5 × 10−3). Furthermore, CNVs within or surrounding genes involved in the ubiquitin pathways, including UBE3A, PARK2, RFWD2 and FBXO40, were affected by CNVs not observed in controls (P = 3.3 × 10−3). We also identified duplications 55 kilobases upstream of complementary DNA AK123120 (P = 3.6 × 10−6). Although these variants may be individually rare, they target genes involved in neuronal cell-adhesion or ubiquitin degradation, indicating that these two important gene networks expressed within the central nervous system may contribute to the genetic susceptibility of ASD. PMID:19404257

  11. Using large clinical data sets to infer pathogenicity for rare copy number variants in autism cohorts

    PubMed Central

    Moreno-De-Luca, D; Sanders, S J; Willsey, A J; Mulle, J G; Lowe, J K; Geschwind, D H; State, M W; Martin, C L; Ledbetter, D H

    2013-01-01

    Copy number variants (CNVs) have a major role in the etiology of autism spectrum disorders (ASD), and several of these have reached statistical significance in case–control analyses. Nevertheless, current ASD cohorts are not large enough to detect very rare CNVs that may be causative or contributory (that is, risk alleles). Here, we use a tiered approach, in which clinically significant CNVs are first identified in large clinical cohorts of neurodevelopmental disorders (including but not specific to ASD), after which these CNVs are then systematically identified within well-characterized ASD cohorts. We focused our initial analysis on 48 recurrent CNVs (segmental duplication-mediated ‘hotspots') from 24 loci in 31 516 published clinical cases with neurodevelopmental disorders and 13 696 published controls, which yielded a total of 19 deletion CNVs and 11 duplication CNVs that reached statistical significance. We then investigated the overlap of these 30 CNVs in a combined sample of 3955 well-characterized ASD cases from three published studies. We identified 73 deleterious recurrent CNVs, including 36 deletions from 11 loci and 37 duplications from seven loci, for a frequency of 1 in 54; had we considered the ASD cohorts alone, only 58 CNVs from eight loci (24 deletions from three loci and 34 duplications from five loci) would have reached statistical significance. In conclusion, until there are sufficiently large ASD research cohorts with enough power to detect very rare causative or contributory CNVs, data from larger clinical cohorts can be used to infer the likely clinical significance of CNVs in ASD. PMID:23044707

  12. The clinical significance of small copy number variants in neurodevelopmental disorders

    PubMed Central

    Asadollahi, Reza; Oneda, Beatrice; Joset, Pascal; Azzarello-Burri, Silvia; Bartholdi, Deborah; Steindl, Katharina; Vincent, Marie; Cobilanschi, Joana; Sticht, Heinrich; Baldinger, Rosa; Reissmann, Regina; Sudholt, Irene; Thiel, Christian T; Ekici, Arif B; Reis, André; Bijlsma, Emilia K; Andrieux, Joris; Dieux, Anne; FitzPatrick, David; Ritter, Susanne; Baumer, Alessandra; Latal, Beatrice; Plecko, Barbara; Jenni, Oskar G; Rauch, Anita

    2014-01-01

    Background Despite abundant evidence for pathogenicity of large copy number variants (CNVs) in neurodevelopmental disorders (NDDs), the individual significance of genome-wide rare CNVs <500 kb has not been well elucidated in a clinical context. Methods By high-resolution chromosomal microarray analysis, we investigated the clinical significance of all rare non-polymorphic exonic CNVs sizing 1–500 kb in a cohort of 714 patients with undiagnosed NDDs. Results We detected 96 rare CNVs <500 kb affecting coding regions, of which 58 (60.4%) were confirmed. 6 of 14 confirmed de novo, one of two homozygous and four heterozygous inherited CNVs affected the known microdeletion regions 17q21.31, 16p11.2 and 2p21 or OMIM morbid genes (CASK, CREBBP, PAFAH1B1, SATB2; AUTS2, NRXN3, GRM8). Two further de novo CNVs affecting single genes (MED13L, CTNND2) were instrumental in delineating novel recurrent conditions. For the first time, we here report exonic deletions of CTNND2 causing low normal IQ with learning difficulties with or without autism spectrum disorder. Additionally, we discovered a homozygous out-of-frame deletion of ACOT7 associated with features comparable to the published mouse model. In total, 24.1% of the confirmed small CNVs were categorised as pathogenic or likely pathogenic (median size 130 kb), 17.2% as likely benign, 3.4% represented incidental findings and 55.2% remained unclear. Conclusions These results verify the diagnostic relevance of genome-wide rare CNVs <500 kb, which were found pathogenic in ∼2% (14/714) of cases (1.1% de novo, 0.3% homozygous, 0.6% inherited) and highlight their inherent potential for discovery of new conditions. PMID:25106414

  13. Rare DNA copy number variants in cardiovascular malformations with extracardiac abnormalities

    PubMed Central

    Lalani, Seema R; Shaw, Chad; Wang, Xueqing; Patel, Ankita; Patterson, Lance W; Kolodziejska, Katarzyna; Szafranski, Przemyslaw; Ou, Zhishuo; Tian, Qi; Kang, Sung-Hae L; Jinnah, Amina; Ali, Sophia; Malik, Aamir; Hixson, Patricia; Potocki, Lorraine; Lupski, James R; Stankiewicz, Pawel; Bacino, Carlos A; Dawson, Brian; Beaudet, Arthur L; Boricha, Fatima M; Whittaker, Runako; Li, Chumei; Ware, Stephanie M; Cheung, Sau Wai; Penny, Daniel J; Jefferies, John Lynn; Belmont, John W

    2013-01-01

    Clinically significant cardiovascular malformations (CVMs) occur in 5–8 per 1000 live births. Recurrent copy number variations (CNVs) are among the known causes of syndromic CVMs, accounting for an important fraction of cases. We hypothesized that many additional rare CNVs also cause CVMs and can be detected in patients with CVMs plus extracardiac anomalies (ECAs). Through a genome-wide survey of 203 subjects with CVMs and ECAs, we identified 55 CNVs >50 kb in length that were not present in children without known cardiovascular defects (n=872). Sixteen unique CNVs overlapping these variants were found in an independent CVM plus ECA cohort (n=511), which were not observed in 2011 controls. The study identified 12/16 (75%) novel loci including non-recurrent de novo 16q24.3 loss (4/714) and de novo 2q31.3q32.1 loss encompassing PPP1R1C and PDE1A (2/714). The study also narrowed critical intervals in three well-recognized genomic disorders of CVM, such as the cat-eye syndrome region on 22q11.1, 8p23.1 loss encompassing GATA4 and SOX7 and 17p13.3-p13.2 loss. An analysis of protein-interaction databases shows that the rare inherited and de novo CNVs detected in the combined cohort are enriched for genes encoding proteins that are direct or indirect partners of proteins known to be required for normal cardiac development. Our findings implicate rare variants such as 16q24.3 loss and 2q31.3-q32.1 loss, and delineate regions within previously reported structural variants known to cause CVMs. PMID:22929023

  14. High quality copy number and genotype data from FFPE samples using Molecular Inversion Probe (MIP) microarrays

    SciTech Connect

    Wang, Yuker; Carlton, Victoria E.H.; Karlin-Neumann, George; Sapolsky, Ronald; Zhang, Li; Moorhead, Martin; Wang, Zhigang C.; Richardson, Andrea L.; Warren, Robert; Walther, Axel; Bondy, Melissa; Sahin, Aysegul; Krahe, Ralf; Tuna, Musaffe; Thompson, Patricia A.; Spellman, Paul T.; Gray, Joe W.; Mills, Gordon B.; Faham, Malek

    2009-02-24

    A major challenge facing DNA copy number (CN) studies of tumors is that most banked samples with extensive clinical follow-up information are Formalin-Fixed Paraffin Embedded (FFPE). DNA from FFPE samples generally underperforms or suffers high failure rates compared to fresh frozen samples because of DNA degradation and cross-linking during FFPE fixation and processing. As FFPE protocols may vary widely between labs and samples may be stored for decades at room temperature, an ideal FFPE CN technology should work on diverse sample sets. Molecular Inversion Probe (MIP) technology has been applied successfully to obtain high quality CN and genotype data from cell line and frozen tumor DNA. Since the MIP probes require only a small ({approx}40 bp) target binding site, we reasoned they may be well suited to assess degraded FFPE DNA. We assessed CN with a MIP panel of 50,000 markers in 93 FFPE tumor samples from 7 diverse collections. For 38 FFPE samples from three collections we were also able to asses CN in matched fresh frozen tumor tissue. Using an input of 37 ng genomic DNA, we generated high quality CN data with MIP technology in 88% of FFPE samples from seven diverse collections. When matched fresh frozen tissue was available, the performance of FFPE DNA was comparable to that of DNA obtained from matched frozen tumor (genotype concordance averaged 99.9%), with only a modest loss in performance in FFPE. MIP technology can be used to generate high quality CN and genotype data in FFPE as well as fresh frozen samples.

  15. Complex and multi-allelic copy number variation in human disease.

    PubMed

    Usher, Christina L; McCarroll, Steven A

    2015-09-01

    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.

  16. 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

  17. Copy number variation of E3 ubiquitin ligase genes in peripheral blood leukocyte and colorectal cancer

    PubMed Central

    Bi, Haoran; Tian, Tian; Zhu, Lin; Zhou, Haibo; Hu, Hanqing; Liu, Yanhong; Li, Xia; Hu, Fulan; Zhao, Yashuang; Wang, Guiyu

    2016-01-01

    Given that E3 ubiquitin ligases (E3) regulate specific protein degradation in many cancer-related biological processes. E3 copy number variation (CNV) may affect the development and prognosis of colorectal cancer (CRC). Therefore, we detected CNVs of five E3 genes in 518 CRC patients and 518 age, gender and residence matched controls in China, and estimated the association between E3 gene CNVs and CRC risk and prognosis. We also estimated their interactions with environmental factors and CRC risk. We find a significant association between the CNVs of MDM2 and CRC risk (amp v.s. wt: odds ratio = 14.37, 95% confidence interval: 1.27, 163.74, P = 0.032), while SKP2 CNVs may significantly decrease CRC risk (del v.s. wt: odds ratio = 0.32, 95% confidence interval: 0.10, 1.00, P = 0.050). However, we find no significant association between the CNVs of other genes and CRC risk. The only significant gene-environment interaction effects are between SKP2 CNVs and consumption of fish and/or fruit (P = 0.014 and P = 0.035) and between FBXW7 CNVs and pork intake (P = 0.040). Finally, we find marginally significant association between β-TRCP CNVs and CRC prognosis (amp v.s. wt, hazard ratio = 0.42, 95% confidence interval: 0.19, 0.97, P = 0.050). PMID:27417709

  18. Genome Wide Association Analysis of Copy Number Variation in Recurrent Depressive Disorder

    PubMed Central

    Rucker, James J.H.; Breen, Gerome; Pinto, Dalila; Pedroso, Inti; Lewis, Cathryn M.; Cohen-Woods, Sarah; Uher, Rudolf; Schosser, Alexandra; Rivera, Margarita; Aitchison, Katherine J.; Craddock, Nick; Owen, Michael J.; Jones, Lisa; Jones, Ian; Korszun, Ania; Muglia, Pierandrea; Barnes, Michael R.; Preisig, Martin; Mors, Ole; Gill, Mike; Maier, Wolfgang; Rice, John; Rietschel, Marcella; Holsboer, Florian; Farmer, Anne E.; Craig, Ian W.; Scherer, Stephen W.; McGuffin, Peter

    2014-01-01

    Large, rare copy number variants (CNV) have been implicated in a variety of psychiatric disorders, but the role of CNVs in recurrent depression is unclear. We performed a genome-wide analysis of large, rare CNVs in 3,106 cases of recurrent depression, 459 controls screened for lifetime-absence of psychiatric disorder and 5,619 unscreened controls from phase 2 of the Wellcome Trust Case Control Consortium (WTCCC2). We compared the frequency of cases with CNVs against the frequency observed in each control group, analysing CNVs over the whole genome, genic, intergenic, intronic and exonic regions. We found that deletion CNVs were associated with recurrent depression while duplications were not. The effect was significant when comparing cases to WTCCC2 controls (p=7.7×10−6, OR =1.25 (95% CI 1.13 - 1.37)) and to screened controls (p=5.6×10−4, OR=1.52 (95% CI 1.20 - 1.93). Further analysis showed that CNVs deleting protein coding regions were largely responsible for the association. Within an analysis of regions previously implicated in schizophrenia, we found an overall enrichment of CNVs in our cases when compared to screened controls (p=0.019). We observe an ordered increase of samples with deletion CNVs, with the lowest proportion seen in screened controls, the next highest in unscreened controls and the highest in cases. This may suggest that the absence of deletion CNVs, especially in genes, is associated with resilience to recurrent depression. PMID:22042228

  19. A New Method for Detecting Associations with Rare Copy-Number Variants

    PubMed Central

    Tzeng, Jung-Ying; Magnusson, Patrik K. E.; Sullivan, Patrick F.; Szatkiewicz, Jin P.

    2015-01-01

    Copy number variants (CNVs) play an important role in the etiology of many diseases such as cancers and psychiatric disorders. Due to a modest marginal effect size or the rarity of the CNVs, collapsing rare CNVs together and collectively evaluating their effect serves as a key approach to evaluating the collective effect of rare CNVs on disease risk. While a plethora of powerful collapsing methods are available for sequence variants (e.g., SNPs) in association analysis, these methods cannot be directly applied to rare CNVs due to the CNV-specific challenges, i.e., the multi-faceted nature of CNV polymorphisms (e.g., CNVs vary in size, type, dosage, and details of gene disruption), and etiological heterogeneity (e.g., heterogeneous effects of duplications and deletions that occur within a locus or in different loci). Existing CNV collapsing analysis methods (a.k.a. the burden test) tend to have suboptimal performance due to the fact that these methods often ignore heterogeneity and evaluate only the marginal effects of a CNV feature. We introduce CCRET, a random effects test for collapsing rare CNVs when searching for disease associations. CCRET is applicable to variants measured on a multi-categorical scale, collectively modeling the effects of multiple CNV features, and is robust to etiological heterogeneity. Multiple confounders can be simultaneously corrected. To evaluate the performance of CCRET, we conducted extensive simulations and analyzed large-scale schizophrenia datasets. We show that CCRET has powerful and robust performance under multiple types of etiological heterogeneity, and has performance comparable to or better than existing methods when there is no heterogeneity. PMID:26431523

  20. Copy number variants are a common cause of non-syndromic hearing loss

    PubMed Central

    2014-01-01

    Background Copy number variants (CNVs) are a well-recognized cause of genetic disease; however, methods for their identification are often gene-specific, excluded as ‘routine’ in screens of genetically heterogeneous disorders, and not implemented in most next-generation sequencing pipelines. For this reason, the contribution of CNVs to non-syndromic hearing loss (NSHL) is most likely under-recognized. We aimed to incorporate a method for CNV identification as part of our standard analysis pipeline and to determine the contribution of CNVs to genetic hearing loss. Methods We used targeted genomic enrichment and massively parallel sequencing to isolate and sequence all exons of all genes known to cause NSHL. We completed testing on 686 patients with hearing loss with no exclusions based on type of hearing loss or any other clinical features. For analysis we used an integrated method for detection of single nucleotide changes, indels and CNVs. CNVs were identified using a previously published method that utilizes median read-depth ratios and a sliding-window approach. Results Of 686 patients tested, 15.2% (104) carried at least one CNV within a known deafness gene. Of the 38.9% (267) of individuals for whom we were able to determine a genetic cause of hearing loss, a CNV was implicated in 18.7% (50). We identified CNVs in 16 different genes including 7 genes for which no CNVs have been previously reported. CNVs of STRC were most common (73% of CNVs identified) followed by CNVs of OTOA (13% of CNVs identified). Conclusion CNVs are an important cause of NSHL and their detection must be included in comprehensive genetic testing for hearing loss. PMID:24963352

  1. Identification of specific gene copy number changes in asbestos-related lung cancer.

    PubMed

    Nymark, Penny; Wikman, Harriet; Ruosaari, Salla; Hollmén, Jaakko; Vanhala, Esa; Karjalainen, Antti; Anttila, Sisko; Knuutila, Sakari

    2006-06-01

    Asbestos is a well-known lung cancer-causing mineral fiber. In vitro and in vivo experiments have shown that asbestos can cause chromosomal damage and aberrations. Lung tumors, in general, have several recurrently amplified and deleted chromosomal regions. To investigate whether a distinct chromosomal aberration profile could be detected in the lung tumors of heavily asbestos-exposed patients, we analyzed the copy number profiles of 14 lung tumors from highly asbestos-exposed patients and 14 matched tumors from nonexposed patients using classic comparative genomic hybridization (CGH). A specific profile could lead to identification of the underlying genes that may act as mediators of tumor formation and progression. In addition, array CGH analyses on cDNA microarrays (13,000 clones) were carried out on 20 of the same patients. Classic CGH showed, on average, more aberrations in asbestos-exposed than in nonexposed patients, and an altered region in chromosome 2 seemed to occur more frequently in the asbestos-exposed patients. Array CGH revealed aberrations in 18 regions that were significantly associated with either of the two groups. The most significant regions were 2p21-p16.3, 5q35.3, 9q33.3-q34.11, 9q34.13-q34.3, 11p15.5, 14q11.2, and 19p13.1-p13.3 (P < 0.005). Furthermore, 11 fragile sites coincided with the 18 asbestos-associated regions (P = 0.08), which may imply preferentially caused DNA damage at these sites. Our findings are the first evidence, indicating that asbestos exposure may produce a specific DNA damage profile.

  2. Copy Number Variation in Obsessive-Compulsive Disorder and Tourette Syndrome: A Cross-Disorder Study

    PubMed Central

    McGrath, Lauren M.; Yu, Dongmei; Marshall, Christian; Davis, Lea K.; Thiruvahindrapuram, Bhooma; Li, Bingbin; Cappi, Carolina; Gerber, Gloria; Wolf, Aaron; Schroeder, Frederick A.; Osiecki, Lisa; O’Dushlaine, Colm; Kirby, Andrew; Illmann, Cornelia; Haddad, Stephen; Gallagher, Patience; Fagerness, Jesen A.; Barr, Cathy L.; Bellodi, Laura; Benarroch, Fortu; Bienvenu, O. Joseph; Black, Donald W.; Bloch, Michael H.; Bruun, Ruth D.; Budman, Cathy L.; Camarena, Beatriz; Cath, Danielle C.; Cavallini, Maria C.; Chouinard, Sylvain; Coric, Vladimir; Cullen, Bernadette; Delorme, Richard; Denys, Damiaan; Derks, Eske M.; Dion, Yves; Rosário, Maria C.; Eapen, Valsama; Evans, Patrick; Falkai, Peter; Fernandez, Thomas; Garrido, Helena; Geller, Daniel; Grabe, Hans J.; Grados, Marco A.; Greenberg, Benjamin D.; Gross-Tsur, Varda; Grünblatt, Edna; Heiman, Gary A.; Hemmings, Sian M.J.; Herrera, Luis D.; Hounie, Ana G.; Jankovic, Joseph; Kennedy, James L; King, Robert A.; Kurlan, Roger; Lanzagorta, Nuria; Leboyer, Marion; Leckman, James F.; Lennertz, Leonhard; Lochner, Christine; Lowe, Thomas L.; Lyon, Gholson J.; Macciardi, Fabio; Maier, Wolfgang; McCracken, James T.; McMahon, William; Murphy, Dennis L.; Naarden, Allan L; Neale, Benjamin M; Nurmi, Erika; Pakstis, Andrew J.; Pato, Michele T.; Pato, Carlos N.; Piacentini, John; Pittenger, Christopher; Pollak, Yehuda; Reus, Victor I.; Richter, Margaret A.; Riddle, Mark; Robertson, Mary M.; Rosenberg, David; Rouleau, Guy A.; Ruhrmann, Stephan; Sampaio, Aline S.; Samuels, Jack; Sandor, Paul; Sheppard, Brooke; Singer, Harvey S.; Smit, Jan H.; Stein, Dan J.; Tischfield, Jay A.; Vallada, Homero; Veenstra-VanderWeele, Jeremy; Walitza, Susanne; Wang, Ying; Wendland, Jens R.; Shugart, Yin Yao; Miguel, Euripedes C.; Nicolini, Humberto; Oostra, Ben A.; Moessner, Rainald; Wagner, Michael; Ruiz-Linares, Andres; Heutink, Peter; Nestadt, Gerald; Freimer, Nelson; Petryshen, Tracey; Posthuma, Danielle; Jenike, Michael A.; Cox, Nancy J.; Hanna, Gregory L.; Brentani, Helena; Scherer, Stephen W.; Arnold, Paul D.; Stewart, S. Evelyn; Mathews, Carol A.; Knowles, James A.; Cook, Edwin H.; Pauls, David L.; Wang, Kai; Scharf, Jeremiah M.

    2014-01-01

    Objective Obsessive-compulsive disorder (OCD) and Tourette syndrome (TS) are heritable, neurodevelopmental disorders with a partially shared genetic etiology. This study represents the first genome-wide investigation of large (>500kb), rare (<1%) copy number variants (CNVs) in OCD and the largest genome-wide CNV analysis in TS to date. Method The primary analyses utilized a cross-disorder design for 2,699 patients (1,613 ascertained for OCD, 1,086 ascertained for TS) and 1,789 controls. Parental data facilitated a de novo analysis in 348 OCD trios. Results Although no global CNV burden was detected in the cross-disorder analysis or in secondary, disease-specific analyses, there was a 3.3-fold increased burden of large deletions previously associated with other neurodevelopmental disorders (p=.09). Half of these neurodevelopmental deletions were located in a single locus, 16p13.11 (5 patient deletions: 0 control deletions, p=0.08 in current study, p=0.025 compared to published controls). Three 16p13.11 deletions were confirmed de novo, providing further support to the etiological significance of this region. The overall OCD de novo rate was 1.4%, which is intermediate between published rates in controls (0.7%) and in autism or schizophrenia (2–4%). Conclusion Several converging lines of evidence implicate 16p13.11 deletions in OCD, with weaker evidence for a role in TS. The trend toward increased overall neurodevelopmental CNV burden in TS and OCD suggests that deletions previously associated with other neurodevelopmental disorders may also contribute to these phenotypes. PMID:25062598

  3. Support for the involvement of large copy number variants in the pathogenesis of schizophrenia.

    PubMed

    Kirov, George; Grozeva, Detelina; Norton, Nadine; Ivanov, Dobril; Mantripragada, Kiran K; Holmans, Peter; Craddock, Nick; Owen, Michael J; O'Donovan, Michael C

    2009-04-15

    We investigated the involvement of rare (<1%) copy number variants (CNVs) in 471 cases of schizophrenia and 2792 controls that had been genotyped using the Affymetrix GeneChip 500K Mapping Array. Large CNVs >1 Mb were 2.26 times more common in cases (P = 0.00027), with the effect coming mostly from deletions (odds ratio, OR = 4.53, P = 0.00013) although duplications were also more common (OR = 1.71, P = 0.04). Two large deletions were found in two cases each, but in no controls: a deletion at 22q11.2 known to be a susceptibility factor for schizophrenia and a deletion on 17p12, at 14.0-15.4 Mb. The latter is known to cause hereditary neuropathy with liability to pressure palsies. The same deletion was found in 6 of 4618 (0.13%) cases and 6 of 36 092 (0.017%) controls in the re-analysed data of two recent large CNV studies of schizophrenia (OR = 7.82, P = 0.001), with the combined significance level for all three studies achieving P = 5 x 10(-5). One large duplication on 16p13.1, which has been previously implicated as a susceptibility factor for autism, was found in three cases and six controls (0.6% versus 0.2%, OR = 2.98, P = 0.13). We also provide the first support for a recently reported association between deletions at 15q11.2 and schizophrenia (P = 0.026). This study confirms the involvement of rare CNVs in the pathogenesis of schizophrenia and contributes to the growing list of specific CNVs that are implicated.

  4. Indexing Effects of Copy Number Variation on Genes Involved in Developmental Delay

    PubMed Central

    Uddin, Mohammed; Pellecchia, Giovanna; Thiruvahindrapuram, Bhooma; D’Abate, Lia; Merico, Daniele; Chan, Ada; Zarrei, Mehdi; Tammimies, Kristiina; Walker, Susan; Gazzellone, Matthew J.; Nalpathamkalam, Thomas; Yuen, Ryan K. C.; Devriendt, Koenraad; Mathonnet, Géraldine; Lemyre, Emmanuelle; Nizard, Sonia; Shago, Mary; Joseph-George, Ann M.; Noor, Abdul; Carter, Melissa T.; Yoon, Grace; Kannu, Peter; Tihy, Frédérique; Thorland, Erik C.; Marshall, Christian R.; Buchanan, Janet A.; Speevak, Marsha; Stavropoulos, Dimitri J.; Scherer, Stephen W.

    2016-01-01

    A challenge in clinical genomics is to predict whether copy number variation (CNV) affecting a gene or multiple genes will manifest as disease. Increasing recognition of gene dosage effects in neurodevelopmental disorders prompted us to develop a computational approach based on critical-exon (highly expressed in brain, highly conserved) examination for potential etiologic effects. Using a large CNV dataset, our updated analyses revealed significant (P < 1.64 × 10−15) enrichment of critical-exons within rare CNVs in cases compared to controls. Separately, we used a weighted gene co-expression network analysis (WGCNA) to construct an unbiased protein module from prenatal and adult tissues and found it significantly enriched for critical exons in prenatal (P < 1.15 × 10−50, OR = 2.11) and adult (P < 6.03 × 10−18, OR = 1.55) tissues. WGCNA yielded 1,206 proteins for which we prioritized the corresponding genes as likely to have a role in neurodevelopmental disorders. We compared the gene lists obtained from critical-exon and WGCNA analysis and found 438 candidate genes associated with CNVs annotated as pathogenic, or as variants of uncertain significance (VOUS), from among 10,619 developmental delay cases. We identified genes containing CNVs previously considered to be VOUS to be new candidate genes for neurodevelopmental disorders (GIT1, MVB12B and PPP1R9A) demonstrating the utility of this strategy to index the clinical effects of CNVs. PMID:27363808

  5. Formation of chimeric genes by copy-number variation as a mutational mechanism in schizophrenia.

    PubMed

    Rippey, Caitlin; Walsh, Tom; Gulsuner, Suleyman; Brodsky, Matt; Nord, Alex S; Gasperini, Molly; Pierce, Sarah; Spurrell, Cailyn; Coe, Bradley P; Krumm, Niklas; Lee, Ming K; Sebat, Jonathan; McClellan, Jon M; King, Mary-Claire

    2013-10-03

    Chimeric genes can be caused by structural genomic rearrangements that fuse together portions of two different genes to create a novel gene. We hypothesize that brain-expressed chimeras may contribute to schizophrenia. Individuals with schizophrenia and control individuals were screened genome wide for copy-number variants (CNVs) that disrupted two genes on the same DNA strand. Candidate events were filtered for predicted brain expression and for frequency < 0.001 in an independent series of 20,000 controls. Four of 124 affected individuals and zero of 290 control individuals harbored such events (p = 0.002); a 47 kb duplication disrupted MATK and ZFR2, a 58 kb duplication disrupted PLEKHD1 and SLC39A9, a 121 kb duplication disrupted DNAJA2 and NETO2, and a 150 kb deletion disrupted MAP3K3 and DDX42. Each fusion produced a stable protein when exogenously expressed in cultured cells. We examined whether these chimeras differed from their parent genes in localization, regulation, or function. Subcellular localizations of DNAJA2-NETO2 and MAP3K3-DDX42 differed from their parent genes. On the basis of the expression profile of the MATK promoter, MATK-ZFR2 is likely to be far more highly expressed in the brain during development than the ZFR2 parent gene. MATK-ZFR2 includes a ZFR2-derived isoform that we demonstrate localizes preferentially to neuronal dendritic branch sites. These results suggest that the formation of chimeric genes is a mechanism by which CNVs contribute to schizophrenia and that, by interfering with parent gene function, chimeras may disrupt critical brain processes, including neurogenesis, neuronal differentiation, and dendritic arborization.

  6. Systematic prioritization and integrative analysis of copy number variations in schizophrenia reveal key schizophrenia susceptibility genes.

    PubMed

    Luo, Xiongjian; Huang, Liang; Han, Leng; Luo, Zhenwu; Hu, Fang; Tieu, Roger; Gan, Lin

    2014-11-01

    Schizophrenia is a common mental disorder with high heritability and strong genetic heterogeneity. Common disease-common variants hypothesis predicts that schizophrenia is attributable in part to common genetic variants. However, recent studies have clearly demonstrated that copy number variations (CNVs) also play pivotal roles in schizophrenia susceptibility and explain a proportion of missing heritability. Though numerous CNVs have been identified, many of the regions affected by CNVs show poor overlapping among different studies, and it is not known whether the genes disrupted by CNVs contribute to the risk of schizophrenia. By using cumulative scoring, we systematically prioritized the genes affected by CNVs in schizophrenia. We identified 8 top genes that are frequently disrupted by CNVs, including NRXN1, CHRNA7, BCL9, CYFIP1, GJA8, NDE1, SNAP29, and GJA5. Integration of genes affected by CNVs with known schizophrenia susceptibility genes (from previous genetic linkage and association studies) reveals that many genes disrupted by CNVs are also associated with schizophrenia. Further protein-protein interaction (PPI) analysis indicates that protein products of genes affected by CNVs frequently interact with known schizophrenia-associated proteins. Finally, systematic integration of CNVs prioritization data with genetic association and PPI data identifies key schizophrenia candidate genes. Our results provide a global overview of genes impacted by CNVs in schizophrenia and reveal a densely interconnected molecular network of de novo CNVs in schizophrenia. Though the prioritized top genes represent promising schizophrenia risk genes, further work with different prioritization methods and independent samples is needed to confirm these findings. Nevertheless, the identified key candidate genes may have important roles in the pathogenesis of schizophrenia, and further functional characterization of these genes may provide pivotal targets for future therapeutics and

  7. Genomic regions showing copy number variations associate with resistance or susceptibility to gastrointestinal nematodes in Angus cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genomic structural variation is an important and abundant source of genetic and phenotypic variation. We previously reported an initial analysis of copy number variations (CNVs) in Angus cattle selected for resistance or susceptibility to gastrointestinal nematodes. In this study, we performed a lar...

  8. Genomic and functional characteristics of copy number variations in Angus cattle selected for resistance or susceptibility to gastrointestinal nematodes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genomic structural variation is an important and abundant source of genetic and phenotypic variation. We previously reported an initial analysis of copy number variations (CNVs) in Angus cattle selected for resistance or susceptibility to intestinal nematodes. In this study, we performed a large sca...

  9. Genomic regions showing copy number variations associate with resistance or susceptibility to gastrointestinal nematodes in Angus Cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genomic structural variation is an important and abundant source of genetic and phenotypic variation. We previously reported an initial analysis of copy number variations (CNVs) in Angus cattle selected for resistance or susceptibility to intestinal nematodes. In this study, we performed a large sca...

  10. High-resolution copy number analysis of paired normal-tumor samples from diffuse large B cell lymphoma.

    PubMed

    Sebastián, Elena; Alcoceba, Miguel; Martín-García, David; Blanco, Óscar; Sanchez-Barba, Mercedes; Balanzategui, Ana; Marín, Luis; Montes-Moreno, Santiago; González-Barca, Eva; Pardal, Emilia; Jiménez, Cristina; García-Álvarez, María; Clot, Guillem; Carracedo, Ángel; Gutiérrez, Norma C; Sarasquete, M Eugenia; Chillón, Carmen; Corral, Rocío; Prieto-Conde, M Isabel; Caballero, M Dolores; Salaverria, Itziar; García-Sanz, Ramón; González, Marcos

    2016-01-01

    Copy number analysis can be useful for assessing prognosis in diffuse large B cell lymphoma (DLBCL). We analyzed copy number data from tumor samples of 60 patients diagnosed with DLBCL de novo and their matched normal samples. We detected 63 recurrent copy number alterations (CNAs), including 33 gains, 30 losses, and nine recurrent acquired copy number neutral loss of heterozygosity (CNN-LOH). Interestingly, 20 % of cases acquired CNN-LOH of 6p21 locus, which involves the HLA region. In normal cells, there were no CNAs but we observed CNN-LOH involving some key lymphoma regions such as 6p21 and 9p24.1 (5 %) and 17p13.1 (2.5 %) in DLBCL patients. Furthermore, a model with some specific CNA was able to predict the subtype of DLBCL, 1p36.32 and 10q23.31 losses being restricted to germinal center B cell-like (GCB) DLBCL. In contrast, 8p23.3 losses and 11q24.3 gains were strongly associated with the non-GCB subtype. A poor prognosis was associated with biallelic inactivation of TP53 or 18p11.32 losses, while prognosis was better in cases carrying 11q24.3 gains. In summary, CNA abnormalities identify specific DLBCL groups, and we describe CNN-LOH in germline cells from DLBCL patients that are associated with genes that probably play a key role in DLBCL development.

  11. Exploring the feasibility of using copy number variants as genetic markers through large-scale whole genome sequencing experiments

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Copy number variants (CNV) are large scale duplications or deletions of genomic sequence that are caused by a diverse set of molecular phenomena that are distinct from single nucleotide polymorphism (SNP) formation. Due to their different mechanisms of formation, CNVs are often difficult to track us...

  12. Increased FGF19 copy number is frequently detected in hepatocellular carcinoma with a complete response after sorafenib treatment

    PubMed Central

    Ishizaki, Morihiko; Matsushima, Hideyuki; De Velasco, Marco A.; Matsui, Kosuke; Iida, Hiroya; Kitade, Hiroaki; Kwon, A-Hon; Nagano, Hiroaki; Wada, Hiroshi; Haji, Seiji; Tsukamoto, Tadashi; Kanazawa, Akishige; Takeda, Yutaka; Takemura, Shigekazu; Kubo, Shoji; Nishio, Kazuto

    2016-01-01

    The multi-kinase inhibitor sorafenib is clinically approved for the treatment of patients with advanced hepatocellular carcinoma (HCC). We previously reported that fibroblast growth factor 3 and 4 (FGF3/FGF4) amplification is a predictor of a response to sorafenib. This study aims to analyze the relationship between FGF-FGF receptor (FGFR) genetic alterations and the response to sorafenib. Formalin-fixed, paraffin-embedded tissue specimens from HCC patients who had achieved a complete response (CR, N=6) or non-CR (N=39) to sorafenib were collected and were examined for FGF-FGFR gene alterations using next generation sequencing and copy number assay. FGFR mutations were detected in 5 of 45 (11.1%) cases. There was no significant association between FGFR mutation status and the response to sorafenib. We detected no increase in the FGF3/FGF4 copy number in CR cases. An FGF19 copy number gain was detected more frequently among CR cases (2/6, 33.3%) than among non-CR cases (2/39, 5.1%) (P = 0.024, Chi-squared test). In conclusion, a copy number gain for FGF19 may be a predictor of a response to sorafenib, in addition to FGF3/FGF4 amplification. PMID:27384874

  13. Increased FGF19 copy number is frequently detected in hepatocellular carcinoma with a complete response after sorafenib treatment.

    PubMed

    Kaibori, Masaki; Sakai, Kazuko; Ishizaki, Morihiko; Matsushima, Hideyuki; De Velasco, Marco A; Matsui, Kosuke; Iida, Hiroya; Kitade, Hiroaki; Kwon, A-Hon; Nagano, Hiroaki; Wada, Hiroshi; Haji, Seiji; Tsukamoto, Tadashi; Kanazawa, Akishige; Takeda, Yutaka; Takemura, Shigekazu; Kubo, Shoji; Nishio, Kazuto

    2016-08-02

    The multi-kinase inhibitor sorafenib is clinically approved for the treatment of patients with advanced hepatocellular carcinoma (HCC). We previously reported that fibroblast growth factor 3 and 4 (FGF3/FGF4) amplification is a predictor of a response to sorafenib. This study aims to analyze the relationship between FGF-FGF receptor (FGFR) genetic alterations and the response to sorafenib. Formalin-fixed, paraffin-embedded tissue specimens from HCC patients who had achieved a complete response (CR, N=6) or non-CR (N=39) to sorafenib were collected and were examined for FGF-FGFR gene alterations using next generation sequencing and copy number assay. FGFR mutations were detected in 5 of 45 (11.1%) cases. There was no significant association between FGFR mutation status and the response to sorafenib. We detected no increase in the FGF3/FGF4 copy number in CR cases. An FGF19 copy number gain was detected more frequently among CR cases (2/6, 33.3%) than among non-CR cases (2/39, 5.1%) (P = 0.024, Chi-squared test). In conclusion, a copy number gain for FGF19 may be a predictor of a response to sorafenib, in addition to FGF3/FGF4 amplification.

  14. 29 CFR 500.226 - Service upon attorneys for the Department of Labor-number of copies.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 29 Labor 3 2010-07-01 2010-07-01 false Service upon attorneys for the Department of Labor-number of copies. 500.226 Section 500.226 Labor Regulations Relating to Labor (Continued) WAGE AND HOUR DIVISION, DEPARTMENT OF LABOR REGULATIONS MIGRANT AND SEASONAL AGRICULTURAL WORKER...

  15. Genome-wide DNA copy number analysis in pancreatic cancer using high-density single nucleotide polymorphism arrays

    PubMed Central

    Harada, T; Chelala, C; Bhakta, V; Chaplin, T; Caulee, K; Baril, P; Young, BD; Lemoine, NR

    2008-01-01

    To identify genomic abnormalities characteristic of pancreatic ductal adenocarcinoma (PDAC) in vivo, a panel of 27 microdissected PDAC specimens were analysed using high-density microarrays representing ∼116 000 single nucleotide polymorphism (SNP) loci. We detected frequent gains of 1q, 2, 3, 5, 7p, 8q, 11, 14q and 17q (≥78% of cases), and losses of 1p, 3p, 6, 9p, 13q, 14q, 17p and 18q (≥44%). Although the results were comparable with those from array CGH, regions of those genetic changes were defined more accurately by SNP arrays. Integrating the Ensembl public data, we have generated ‘gene’ copy number indices that facilitate the search for novel candidates involved in pancreatic carcinogenesis. Copy numbers in a subset of the genes were validated using quantitative real-time PCR. The SKAP2/SCAP2 gene (7p15.2), which belongs to the src family kinases, was most frequently (63%) amplified in our sample set and its recurrent overexpression (67%) was confirmed by reverse transcription–PCR. Furthermore, fluorescence in situ hybridization and in situ RNA hybridization analyses for this gene have demonstrated a significant correlation between DNA copy number and mRNA expression level in an independent sample set (P<0.001). These findings indicate that the dysregulation of SKAP2/SCAP2, which is mostly caused by its increased gene copy number, is likely to be associated with the development of PDAC. PMID:17952125

  16. 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

  17. Comparative analyses across cattle breeds reveal the pitfalls caused by artificial and lineage-differential copy number variations

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Copy number variations (CNV) are well known genomic variants, which often complicate structural and functional genomics studies. Here, we integrated the CNV region (CNVR) result detected from 1,682 Nellore cattle with the equivalent result derived from the Bovine HapMap samples. Through comparing CN...

  18. Genome-wide copy number variation in the bovine genome detected using low coverage sequence of popular beef breeds

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genomic structural variations are an important source of genetic diversity. Copy number variations (CNVs), gains and losses of large regions of genomic sequence between individuals of a species, are known to be associated with both diseases and phenotypic traits. Deeply sequenced genomes are often u...

  19. Electrical Noise and the Measurement of Absolute Temperature, Boltzmann's Constant and Avogadro's Number.

    ERIC Educational Resources Information Center

    Ericson, T. J.

    1988-01-01

    Describes an apparatus capable of measuring absolute temperatures of a tungsten filament bulb up to normal running temperature and measuring Botzmann's constant to an accuracy of a few percent. Shows that electrical noise techniques are convenient to demonstrate how the concept of temperature is related to the micro- and macroscopic world. (CW)

  20. Gene Copy-Number Variation and Associated Polymorphisms of Complement Component C4 in Human Systemic Lupus Erythematosus (SLE): Low Copy Number Is a Risk Factor for and High Copy Number Is a Protective Factor against SLE Susceptibility in European Americans

    PubMed Central

    Yang, Yan ; Chung, Erwin K. ; Wu, Yee Ling ; Savelli, Stephanie L. ; Nagaraja, Haikady N. ; Zhou, Bi ; Hebert, Maddie ; Jones, Karla N. ; Shu, Yaoling ; Kitzmiller, Kathryn ; Blanchong, Carol A. ; McBride, Kim L. ; Higgins, Gloria C. ; Rennebohm, Robert M. ; Rice, Robert R. ; Hackshaw, Kevin V. ; Roubey, Robert A. S. ; Grossman, Jennifer M. ; Tsao, Betty P. ; Birmingham, Daniel J. ; Rovin, Brad H. ; Hebert, Lee A. ; Yu, C. Yung 

    2007-01-01

    Interindividual gene copy-number variation (CNV) of complement component C4 and its associated polymorphisms in gene size (long and short) and protein isotypes (C4A and C4B) probably lead to different susceptibilities to autoimmune disease. We investigated the C4 gene CNV in 1,241 European Americans, including patients with systemic lupus erythematosus (SLE), their first-degree relatives, and unrelated healthy subjects, by definitive genotyping and phenotyping techniques. The gene copy number (GCN) varied from 2 to 6 for total C4, from 0 to 5 for C4A, and from 0 to 4 for C4B. Four copies of total C4, two copies of C4A, and two copies of C4B were the most common GCN counts, but each constituted only between one-half and three-quarters of the study populations. Long C4 genes were strongly correlated with C4A (R=0.695; P<.0001). Short C4 genes were correlated with C4B (R=0.437; P<.0001). In comparison with healthy subjects, patients with SLE clearly had the GCN of total C4 and C4A shifting to the lower side. The risk of SLE disease susceptibility significantly increased among subjects with only two copies of total C4 (patients 9.3%; unrelated controls 1.5%; odds ratio [OR] = 6.514; P=.00002) but decreased in those with ⩾5 copies of C4 (patients 5.79%; controls 12%; OR=0.466; P=.016). Both zero copies (OR=5.267; P=.001) and one copy (OR=1.613; P=.022) of C4A were risk factors for SLE, whereas ⩾3 copies of C4A appeared to be protective (OR=0.574; P=.012). Family-based association tests suggested that a specific haplotype with a single short C4B in tight linkage disequilibrium with the −308A allele of TNFA was more likely to be transmitted to patients with SLE. This work demonstrates how gene CNV and its related polymorphisms are associated with the susceptibility to a human complex disease. PMID:17503323

  1. Effect of Promoters and Plasmid Copy Number on Cyt1A Synthesis and Crystal Assembly in Bacillus thuringiensis.

    PubMed

    Park, Hyun-Woo; Hice, Robert H; Federici, Brian A

    2016-01-01

    Cyt1Aa is a major mosquitocidal protein synthesized during sporulation of Bacillus thuringiensis subsp. israelensis, composing more than 50% of its parasporal body. This high level of synthesis is due to several factors including three strong sporulation-dependent promoters, a strong transcription termination sequence, and an associated 20-kDa helper protein. Cyt1Aa's toxicity is low compared to the Cry proteins of this species, namely, Cry4Aa, Cry4Ba, and Cry11Aa, but it nevertheless plays an important role in the biology of B. thuringiensis subsp. israelensis in that it synergizes their mosquitocidal toxicity and suppresses the evolution of resistance. In the present study, the effects of using different cyt1Aa promoter combinations and plasmid copy number on synthesis of Cyt1Aa were evaluated. Using the 4Q7 (plasmid-cured) strain of B. thuringiensis subsp. israelensis as an experimental host, a plasmid copy number of two or three yielded no Cyt1Aa, whereas a copy number of four yielded only small crystals, even when expression was driven by one of the wild-type promoters. However, using all three wild-type promoters and a plasmid copy number of 20 yielded Cyt1A crystals tenfold larger than those produced by one promoter and a plasmid copy number of four. High levels of Cyt1Aa synthesis resulted in significantly fewer spores per unit medium and imperfectly formed crystals. Similar results were obtained when Cyt1Aa synthesis was evaluated using the same expression constructs in a mutant strain of B. thuringiensis subsp. israelensis that lacks the cyt1Aa gene.

  2. Interaction-Based Feature Selection for Uncovering Cancer Driver Genes Through Copy Number-Driven Expression Level.

    PubMed

    Park, Heewon; Niida, Atsushi; Imoto, Seiya; Miyano, Satoru

    2017-02-01

    Driver gene selection is crucial to understand the heterogeneous system of cancer. To identity cancer driver genes, various statistical strategies have been proposed, especially the L1-type regularization methods have drawn a large amount of attention. However, the statistical approaches have been developed purely from algorithmic and statistical point, and the existing studies have applied the statistical approaches to genomic data analysis without consideration of biological knowledge. We consider a statistical strategy incorporating biological knowledge to identify cancer driver gene. The alterations of copy number have been considered to driver cancer pathogenesis processes, and the region of strong interaction of copy number alterations and expression levels was known as a tumor-related symptom. We incorporate the influence of copy number alterations on expression levels to cancer driver gene-selection processes. To quantify the dependence of copy number alterations on expression levels, we consider [Formula: see text] and [Formula: see text] effects of copy number alterations on expression levels of genes, and incorporate the symptom of tumor pathogenesis to gene-selection procedures. We then proposed an interaction-based feature-selection strategy based on the adaptive L1-type regularization and random lasso procedures. The proposed method imposes a large amount of penalty on genes corresponding to a low dependency of the two features, thus the coefficients of the genes are estimated to be small or exactly 0. It implies that the proposed method can provide biologically relevant results in cancer driver gene selection. Monte Carlo simulations and analysis of the Cancer Genome Atlas (TCGA) data show that the proposed strategy is effective for high-dimensional genomic data analysis. Furthermore, the proposed method provides reliable and biologically relevant results for cancer driver gene selection in TCGA data analysis.

  3. The effects of mitochondrial DNA deletion and copy number variations on different exercise intensities in highly trained swimmers.

    PubMed

    Baykara, O; Sahin, S K; Akbas, F; Guven, M; Onaran, I

    2016-10-31

    It has been suggested that heavy exercise might increase oxidative stress, causing mitochondrial DNA (mtDNA) mutations as well as DNA mutations and changes in the mtDNA copy number in cells. mtDNA4977 deletion is one of the most common deletions seen on mitochondria. We hypothesize association between exercise induced oxidative stress and mtDNA damage in peripheral blood lymphocytes (PBLs) of highly trained swimmers. Therefore we studied the mtDNA4977 deletion level, mtDNA copy number and their relationship with cellular ATP and oxidative stress status in PBLs of swimmers. 8 highly trained and 8 normal trained swimmers and 8 non-athlete subjects were included in the study. The mtDNA4977 deletion and amount of mtDNA were measured using RT-PCR method whereas dichlorohydrofluoroscein (DCF) assay method was used to assess cellular oxidative stress and ATP levels were measured using bioluminescence method. Even though an increase in mtDNA4977 deletion was found in all study groups, the difference was not statistically significant (p=0.98). The mtDNA copy numbers were found to be surprisingly high in highly trained swimmers compared to normal trained swimmers and non-athlete subjects by 4.03 fold (p= 0.0002) and 5.58 fold (p=0.0003), respectively. No significant differences were found between groups by means of intracellular ATP levels (p=0.406) and oxidative stress (p=0.430).  No correlation was found between mtDNA copy number and intracellular ATP content of the PBLs (p=0.703). Our results suggest that heavy training does not have a specific effect on mtDNA4977 deletion but it may be affecting mitochondrial copy numbers which may act as a compensatory mechanism related to ATP levels in blood.

  4. Digital Genotyping of Macrosatellites and Multicopy Genes Reveals Novel Biological Functions Associated with Copy Number Variation of Large Tandem Repeats

    PubMed Central

    Quilez, Javier; Hasson, Dan; Borel, Christelle; Warburton, Peter; Sharp, Andrew J.

    2014-01-01

    Tandem repeats are common in eukaryotic genomes, but due to difficulties in assaying them remain poorly studied. Here, we demonstrate the utility of Nanostring technology as a targeted approach to perform accurate measurement of tandem repeats even at extremely high copy number, and apply this technology to genotype 165 HapMap samples from three different populations and five species of non-human primates. We observed extreme variability in copy number of tandemly repeated genes, with many loci showing 5–10 fold variation in copy number among humans. Many of these loci show hallmarks of genome assembly errors, and the true copy number of many large tandem repeats is significantly under-represented even in the high quality ‘finished’ human reference assembly. Importantly, we demonstrate that most large tandem repeat variations are not tagged by nearby SNPs, and are therefore essentially invisible to SNP-based GWAS approaches. Using association analysis we identify many cis correlations of large tandem repeat variants with nearby gene expression and DNA methylation levels, indicating that variations of tandem repeat length are associated with functional effects on the local genomic environment. This includes an example where expansion of a macrosatellite repeat is associated with increased DNA methylation and suppression of nearby gene expression, suggesting a mechanism termed “repeat induced gene silencing”, which has previously been observed only in transgenic organisms. We also observed multiple signatures consistent with altered selective pressures at tandemly repeated loci, suggesting important biological functions. Our studies show that tandemly repeated loci represent a highly variable fraction of the genome that have been systematically ignored by most previous studies, copy number variation of which can exert functionally significant effects. We suggest that future studies of tandem repeat loci will lead to many novel insights into their role in

  5. DNA copy number aberrations in small-cell lung cancer reveal activation of the focal adhesion pathway

    PubMed Central

    Ocak, S; Yamashita, H; Udyavar, AR; Miller, AN; Gonzalez, AL; Zou, Y; Jiang, A; Yi, Y; Shyr, Y; Estrada, L; Quaranta, V; Massion, PP

    2015-01-01

    Small-cell lung cancer (SCLC) is the most aggressive subtype of lung cancer in its clinical behavior, with a 5-year overall survival as low as 5%. Despite years of research in the field, molecular determinants of SCLC behavior are still poorly understood, and this deficiency has translated into an absence of specific diagnostics and targeted therapeutics. We hypothesized that tumor DNA copy number alterations would allow the identification of molecular pathways involved in SCLC progression. Array comparative genomic hybridization was performed on DNA extracted from 46 formalin-fixed paraffin-embedded SCLC tissue specimens. Genomic profiling of tumor and sex-matched control DNA allowed the identification of 70 regions of copy number gain and 55 regions of copy number loss. Using molecular pathway analysis, we found a strong enrichment in these regions of copy number alterations for 11 genes associated with the focal adhesion pathway. We verified these findings at the genomic, gene expression and protein level. Focal Adhesion Kinase (FAK), one of the central genes represented in this pathway, was commonly expressed in SCLC tumors and constitutively phosphorylated in SCLC cell lines. Those were poorly adherent to most substrates but not to laminin-322. Inhibition of FAK phosphorylation at Tyr397 by a small-molecule inhibitor, PF-573,228, induced a dose-dependent decrease of adhesion and an increase of spreading in SCLC cell lines on laminin-322. Cells that tended to spread also showed a decrease in focal adhesions, as demonstrated by a decreased vinculin expression. These results support the concept that pathway analysis of genes in regions of copy number alterations may uncover molecular mechanisms of disease progression and demonstrate a new role of FAK and associated adhesion pathways in SCLC. Further investigations of FAK at the functional level may lead to a better understanding of SCLC progression and may have therapeutic implications. PMID:20802517

  6. An initial comparative map of copy number variations in the goat (Capra hircus) genome

    PubMed Central

    2010-01-01

    Background The goat (Capra hircus) represents one of the most important farm animal species. It is reared in all continents with an estimated world population of about 800 million of animals. Despite its importance, studies on the goat genome are still in their infancy compared to those in other farm animal species. Comparative mapping between cattle and goat showed only a few rearrangements in agreement with the similarity of chromosome banding. We carried out a cross species cattle-goat array comparative genome hybridization (aCGH) experiment in order to identify copy number variations (CNVs) in the goat genome analysing animals of different breeds (Saanen, Camosciata delle Alpi, Girgentana, and Murciano-Granadina) using a tiling oligonucleotide array with ~385,000 probes designed on the bovine genome. Results We identified a total of 161 CNVs (an average of 17.9 CNVs per goat), with the largest number in the Saanen breed and the lowest in the Camosciata delle Alpi goat. By aggregating overlapping CNVs identified in different animals we determined CNV regions (CNVRs): on the whole, we identified 127 CNVRs covering about 11.47 Mb of the virtual goat genome referred to the bovine genome (0.435% of the latter genome). These 127 CNVRs included 86 loss and 41 gain and ranged from about 24 kb to about 1.07 Mb with a mean and median equal to 90,292 bp and 49,530 bp, respectively. To evaluate whether the identified goat CNVRs overlap with those reported in the cattle genome, we compared our results with those obtained in four independent cattle experiments. Overlapping between goat and cattle CNVRs was highly significant (P < 0.0001) suggesting that several chromosome regions might contain recurrent interspecies CNVRs. Genes with environmental functions were over-represented in goat CNVRs as reported in other mammals. Conclusions We describe a first map of goat CNVRs. This provides information on a comparative basis with the cattle genome by identifying putative

  7. Mapping IS6110 in high-copy number Mycobacterium tuberculosis strains shows specific insertion points in the Beijing genotype

    PubMed Central

    2013-01-01

    Background Mycobacterium tuberculosis Beijing strains are characterized by a large number of IS6110 copies, suggesting the potential implication of this element in the virulence and capacity for rapid dissemination characteristic of this family. This work studies the insetion points of IS6110 in high-copy clinical isolates specifically focusing on the Beijing genotype. Results In the present work we mapped the insertion points of IS6110 in all the Beijing strains available in the literature and in the DNA sequence databases. We generated a representative primer collection of the IS6110 locations, which was used to analyse 61 high-copy clinical isolates. A total of 440 points of insertion were identified and analysis of their flanking regions determined the exact location, the direct repeats (DRs), the orientation and the distance to neighboring genes of each copy of IS6110. We identified specific points of insertion in Beijing strains that enabled us to obtain a dendrogram that groups the Beijing genotype. Conclusions This work presents a detailed analysis of locations of IS6110 in high-copy clinical isolates, showing points of insertion present with high frequency in the Beijing family and absent in other strains. PMID:23800083

  8. Presequence-Independent Mitochondrial Import of DNA Ligase Facilitates Establishment of Cell Lines with Reduced mtDNA Copy Number.

    PubMed

    Spadafora, Domenico; Kozhukhar, Natalia; Alexeyev, Mikhail F

    2016-01-01

    Due to the essential role played by mitochondrial DNA (mtDNA) in cellular physiology and bioenergetics, methods for establishing cell lines with altered mtDNA content are of considerable interest. Here, we report evidence for the existence in mammalian cells of a novel, low- efficiency, presequence-independent pathway for mitochondrial protein import, which facilitates mitochondrial uptake of such proteins as Chlorella virus ligase (ChVlig) and Escherichia coli LigA. Mouse cells engineered to depend on this pathway for mitochondrial import of the LigA protein for mtDNA maintenance had severely (up to >90%) reduced mtDNA content. These observations were used to establish a method for the generation of mouse cell lines with reduced mtDNA copy number by, first, transducing them with a retrovirus encoding LigA, and then inactivating in these transductants endogenous Lig3 with CRISPR-Cas9. Interestingly, mtDNA depletion to an average level of one copy per cell proceeds faster in cells engineered to maintain mtDNA at low copy number. This makes a low-mtDNA copy number phenotype resulting from dependence on mitochondrial import of DNA ligase through presequence-independent pathway potentially useful for rapidly shifting mtDNA heteroplasmy through partial mtDNA depletion.

  9. Higher copy numbers of the potato RB transgene correspond to enhanced transcript and late blight resistance levels.

    PubMed

    Bradeen, James M; Iorizzo, Massimo; Mollov, Dimitre S; Raasch, John; Kramer, Lara Colton; Millett, Benjamin P; Austin-Phillips, Sandra; Jiang, Jiming; Carputo, Domenico

    2009-04-01

    Late blight of potato ranks among the costliest of crop diseases worldwide. Host resistance offers the best means for controlling late blight, but previously deployed single resistance genes have been short-lived in their effectiveness. The foliar blight resistance gene RB, previously cloned from the wild potato Solanum bulbocastanum, has proven effective in greenhouse tests of transgenic cultivated potato. In this study, we examined the effects of the RB transgene on foliar late blight resistance in transgenic cultivated potato under field production conditions. In a two-year replicated trial, the RB transgene, under the control of its endogenous promoter, provided effective disease resistance in various genetic backgrounds, including commercially prominent potato cultivars, without fungicides. RB copy numbers and transcript levels were estimated with transgene-specific assays. Disease resistance was enhanced as copy numbers and transcript levels increased. The RB gene, like many other disease resistance genes, is constitutively transcribed at low levels. Transgenic potato lines with an estimated 15 copies of the RB transgene maintain high RB transcript levels and were ranked among the most resistant of 57 lines tested. We conclude that even in these ultra-high copy number lines, innate RNA silencing mechanisms have not been fully activated. Our findings suggest resistance-gene transcript levels may have to surpass a threshold before triggering RNA silencing. Strategies for the deployment of RB are discussed in light of the current research.

  10. Copy number variation and microdeletions of the Y chromosome linked genes and loci across different categories of Indian infertile males

    PubMed Central

    Kumari, Anju; Yadav, Sandeep Kumar; Misro, Man Mohan; Ahmad, Jamal; Ali, Sher

    2015-01-01

    We analyzed 34 azoospermic (AZ), 43 oligospermic (OS), and 40 infertile males with normal spermiogram (INS) together with 55 normal fertile males (NFM) from the Indian population. AZ showed more microdeletions in the AZFa and AZFb regions whereas oligospermic ones showed more microdeletions in the AZFc region. Frequency of the AZF partial deletions was higher in males with spermatogenic impairments than in INS. Significantly, SRY, DAZ and BPY2 genes showed copy number variation across different categories of the patients and much reduced copies of the DYZ1 repeat arrays compared to that in normal fertile males. Likewise, INS showed microdeletions, sequence and copy number variation of several Y linked genes and loci. In the context of infertility, STS deletions and copy number variations both were statistically significant (p = 0.001). Thus, semen samples used during in vitro fertilization (IVF) and assisted reproductive technology (ART) must be assessed for the microdeletions of AZFa, b and c regions in addition to the affected genes reported herein. Present study is envisaged to be useful for DNA based diagnosis of different categories of the infertile males lending support to genetic counseling to the couples aspiring to avail assisted reproductive technologies. PMID:26638807

  11. Low D4Z4 copy number and gender difference in Korean patients with facioscapulohumeral muscular dystrophy type 1.

    PubMed

    Park, Hyung Jun; Hong, Ji-Man; Lee, Jung Hwan; Lee, Hyung Seok; Shin, Ha Young; Kim, Seung Min; Ki, Chang-Seok; Lee, Ji Hyun; Choi, Young-Chul

    2015-11-01

    The objective of this study was to investigate the clinical and genetic features of Korean patients with facioscapulohumeral muscular dystrophy type 1 (FSHD), and assessed the impact of molecular defects on phenotypic expression. We enrolled 104 FSHD patients from 87 unrelated Korean families with D4Z4 repeat array of less than 11 copies on 4q35. Sixty-one men and forty-three women were enrolled. Median D4Z4 copy number was 4 units and 99 (95%) Korean patients with FSHD carried 1-6 units. The median age at symptom onset was 13 [interquartile range: 8-17] years old. In 100 symptomatic patients, muscle weakness began in facial muscles in 58 patients, shoulder-girdle muscles in 37, and pelvic-girdle muscles in 5. Disease severity was significantly correlated with D4Z4 copy number. In addition, women were more severely affected than men even though there were no differences in age at examination or in D4Z4 copy number between the two genders. This gender difference among Korean patients was the opposite of analysis on individuals of European ancestry. In conclusion, the present study demonstrated the new diagnostic threshold for FSHD in Koreans based on the D4Z4 repeat array size distribution from 1 to 6 units and expanded the clinical spectrum.

  12. Presequence-Independent Mitochondrial Import of DNA Ligase Facilitates Establishment of Cell Lines with Reduced mtDNA Copy Number

    PubMed Central

    Spadafora, Domenico; Kozhukhar, Natalia; Alexeyev, Mikhail F.

    2016-01-01

    Due to the essential role played by mitochondrial DNA (mtDNA) in cellular physiology and bioenergetics, methods for establishing cell lines with altered mtDNA content are of considerable interest. Here, we report evidence for the existence in mammalian cells of a novel, low- efficiency, presequence-independent pathway for mitochondrial protein import, which facilitates mitochondrial uptake of such proteins as Chlorella virus ligase (ChVlig) and Escherichia coli LigA. Mouse cells engineered to depend on this pathway for mitochondrial import of the LigA protein for mtDNA maintenance had severely (up to >90%) reduced mtDNA content. These observations were used to establish a method for the generation of mouse cell lines with reduced mtDNA copy number by, first, transducing them with a retrovirus encoding LigA, and then inactivating in these transductants endogenous Lig3 with CRISPR-Cas9. Interestingly, mtDNA depletion to an average level of one copy per cell proceeds faster in cells engineered to maintain mtDNA at low copy number. This makes a low-mtDNA copy number phenotype resulting from dependence on mitochondrial import of DNA ligase through presequence-independent pathway potentially useful for rapidly shifting mtDNA heteroplasmy through partial mtDNA depletion. PMID:27031233

  13. 10 CFR 51.58 - Environmental report-number of copies; distribution.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... shall submit a copy to the Director of the Office of Nuclear Reactor Regulation, the Director of the Office of New Reactors, the Director of the Office of Nuclear Material Safety and Safeguards, as... instructions issued by the Director of the Office of New Reactors, the Director of the Office of...

  14. 10 CFR 51.58 - Environmental report-number of copies; distribution.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... shall submit a copy to the Director of the Office of Nuclear Reactor Regulation, the Director of the Office of New Reactors, the Director of the Office of Nuclear Material Safety and Safeguards, as... instructions issued by the Director of the Office of New Reactors, the Director of the Office of...

  15. 39 CFR 3001.10 - Form and number of copies of documents.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    .... If the document is bound, it shall be bound on the left side. Copies of documents for filing and service must be printed from a text-based pdf version of the document, where possible. Otherwise, they may... generated in either Acrobat (pdf), Word, or WordPerfect, or Rich Text Format (rtf)....

  16. 39 CFR 3001.10 - Form and number of copies of documents.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... which they are attached. If the document is bound, it shall be bound on the left side. Copies of documents for filing and service must be printed from a text-based pdf version of the document, where... generated in either Acrobat (pdf), Word, WordPerfect, or Rich Text Format (rtf). (d) Exception for...

  17. 39 CFR 3001.10 - Form and number of copies of documents.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... which they are attached. If the document is bound, it shall be bound on the left side. Copies of documents for filing and service must be printed from a text-based pdf version of the document, where... generated in either Acrobat (pdf), Word, WordPerfect, or Rich Text Format (rtf). (d) Exception for...

  18. 39 CFR 3001.10 - Form and number of copies of documents.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    .... If the document is bound, it shall be bound on the left side. Copies of documents for filing and service must be printed from a text-based pdf version of the document, where possible. Otherwise, they may... generated in either Acrobat (pdf), Word, or WordPerfect, or Rich Text Format (rtf)....

  19. Development, Optimization, and Evaluation of a Duplex Droplet Digital PCR Assay To Quantify the T-nos/hmg Copy Number Ratio in Genetically Modified Maize

    PubMed Central

    2015-01-01

    Certified reference materials (CRMs) are required to guarantee the reliability of analytical measurements. The CRMs available in the field of genetically modified organisms (GMOs) are characterized using real-time polymerase chain reaction (qPCR). This technology has limited application, because of its dependence on a calibrant. The objective of this study was to obtain a method with higher metrological quality, to characterize the CRMs for their contents of T-nos/hmg copy number ratio in maize. A duplex droplet digital PCR (ddPCR) assay was developed and optimized by a central composite design. The developed method achieved an absolute limit of detection (LOD) of 11 cP T-nos, a relative LOD of 0.034%, a limit of quantification (LOQ) of 23 cP (relative LOQ of 0.08%), and a dynamic range of 0.08%–100% T-nos/hmg ratio. The specificity and applicability of the assay were established for the analysis of low T-nos concentrations (0.9%) in several corn varieties. The convenience of DNA digestion to reduce measurement bias in the case of multiple-copy binding was confirmed through an enzymatic restriction assay. Given its overall performance, this method can be used to characterize CRM candidates for their contents of T-nos/hmg ratio. PMID:26605751

  20. Leukocyte mitochondrial DNA copy number, anthropometric indices, and weight change in US women

    PubMed Central

    Meng, Shasha; Wu, Shaowei; Liang, Liming; Liang, Geyu; Giovannucci, Edward; Vivo, Immaculata De; Nan, Hongmei

    2016-01-01

    Objectives To examine the association between leukocyte mitochondrial DNA copy number (mtCN) and different anthropometric indices as well as weight changes; and to compare mtCN and telomere length with respect to their associations with BMI and age. Design Population based cohort study. Setting Nurses' Health Study, an ongoing prospective cohort study of 121,700 nurses enrolled in 1976; in 1989-1990 a subset of 32,826 women provided blood samples. Participants 1,700 disease-free US women from case-control studies nested within the Nurses' Health Study with mtCN and telomere length measured who also have anthropometric measurements. Main outcome measure Relative mtCN and telomere lengths in peripheral blood leukocytes measured by quantitative real time polymerase chain reaction and various anthropometric measurements data from initial questionnaire. Results Leukocyte mtCN was inversely associated with current weight (LS means Q1-Q4: 0.07, 0.04, 0.03, −0.17; P trend =0.002), waist size (LS means Q1-Q4: 0.06, 0.05, −0.04, −0.06; P trend = 0.04), BMI (LS means normal light, normal heavy, overweight, pre-obese, obese: 0.11, −0.01, −0.04, 0.04, −0.25; P trend<0.0001), and waist-hip ratio (WHR) (LS means Q1-Q4: 0.06, 0.08, −0.04, −0.06; P trend = 0.03). A one-unit decrease in mtCN z score was equivalent to approximately 3.5 pounds of weight gain for an adult of 5′10″. In addition, weight gain was bi-directionally and inversely associated with mtCN. Moreover, mtCN was strongly positively correlated with telomere length (LS means Q1-Q4: −0.02, 0.09, 0.11, 0.33; P trend <0.0001). MtCN was inversely associated with BMI even after adjusting for telomere length (P trend =0.003), while telomere length was not associated with BMI. On the other hand, telomere length was inversely associated with age after adjusting for mtCN (P trend =0.04), while mtCN was not associated with age. Conclusions Our results provide compelling evidence for a potential bi