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Sample records for copy number shuttle

  1. 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.

  2. Hacking DNA copy number for circuit engineering.

    PubMed

    Wu, Feilun; You, Lingchong

    2017-07-27

    DNA copy number represents an essential parameter in the dynamics of synthetic gene circuits but typically is not explicitly considered. A new study demonstrates how dynamic control of DNA copy number can serve as an effective strategy to program robust oscillations in gene expression circuits.

  3. Copy Number Variation across European Populations

    PubMed Central

    Chen, Wanting; Hayward, Caroline; Wright, Alan F.; Hicks, Andrew A.; Vitart, Veronique; Knott, Sara; Wild, Sarah H.; Pramstaller, Peter P.; Wilson, James F.; Rudan, Igor; Porteous, David J.

    2011-01-01

    Genome analysis provides a powerful approach to test for evidence of genetic variation within and between geographical regions and local populations. Copy number variants which comprise insertions, deletions and duplications of genomic sequence provide one such convenient and informative source. Here, we investigate copy number variants from genome wide scans of single nucleotide polymorphisms in three European population isolates, the island of Vis in Croatia, the islands of Orkney in Scotland and the South Tyrol in Italy. We show that whereas the overall copy number variant frequencies are similar between populations, their distribution is highly specific to the population of origin, a finding which is supported by evidence for increased kinship correlation for specific copy number variants within populations. PMID:21829696

  4. 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.

  5. 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

  6. Genomic characteristics of cattle copy number variations

    USDA-ARS?s Scientific Manuscript database

    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...

  7. 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

  8. 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

  9. 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.

  10. PCR-Based Detection of DNA Copy Number Variation.

    PubMed

    Mehrotra, Meenakshi

    2016-01-01

    Copy number variations are important polymorphisms that can influence gene expression within and close to the rearranged region, and results in phenotypic variation. Techniques that detect abnormalities in DNA copy number are therefore useful for studying the associations between DNA aberrations and disease phenotype and for locating critical genes. PCR-based detection of copy number of target gene using TaqMan copy number assay offers a reliable method to measure copy number variation in human genome.

  11. Gene copy number and cell cycle arrest

    NASA Astrophysics Data System (ADS)

    Ghosh, Bhaswar; Bose, Indrani

    2006-03-01

    The cell cycle is an orderly sequence of events which ultimately lead to the division of a single cell into two daughter cells. In the case of DNA damage by radiation or chemicals, the damage checkpoints in the G1 and G2 phases of the cell cycle are activated. This results in an arrest of the cell cycle so that the DNA damage can be repaired. Once this is done, the cell continues with its usual cycle of activity. We study a mathematical model of the DNA damage checkpoint in the G2 phase which arrests the transition from the G2 to the M (mitotic) phase of the cell cycle. The tumor suppressor protein p53 plays a key role in activating the pathways leading to cell cycle arrest in mammalian systems. If the DNA damage is severe, the p53 proteins activate other pathways which bring about apoptosis, i.e., programmed cell death. Loss of the p53 gene results in the proliferation of cells containing damaged DNA, i.e., in the growth of tumors which may ultimately become cancerous. There is some recent experimental evidence which suggests that the mutation of a single copy of the p53 gene (in the normal cell each gene has two identical copies) is sufficient to trigger the formation of tumors. We study the effect of reducing the gene copy number of the p53 and two other genes on cell cycle arrest and obtain results consistent with experimental observations.

  12. Copy Number Heterogeneity of JC Virus Standards.

    PubMed

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

    2017-03-01

    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. Copyright © 2017 Greninger et al.

  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. Getting DNA copy numbers without control samples.

    PubMed

    Ortiz-Estevez, Maria; Aramburu, Ander; Rubio, Angel

    2012-08-16

    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. 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. 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 available in the open-source R package

  16. 18 CFR 33.8 - 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 Number of copies. 33.8... Number of copies. An original and eight copies of the application under this part must be submitted. If... for privileged treatment), the original and at least three of the eight copies must be of the non...

  17. Copy number variation in the bovine genome

    PubMed Central

    2010-01-01

    Background Copy number variations (CNVs), which represent a significant source of genetic diversity in mammals, have been shown to be associated with phenotypes of clinical relevance and to be causative of disease. Notwithstanding, little is known about the extent to which CNV contributes to genetic variation in cattle. Results We designed and used a set of NimbleGen CGH arrays that tile across the assayable portion of the cattle genome with approximately 6.3 million probes, at a median probe spacing of 301 bp. This study reports the highest resolution map of copy number variation in the cattle genome, with 304 CNV regions (CNVRs) being identified among the genomes of 20 bovine samples from 4 dairy and beef breeds. The CNVRs identified covered 0.68% (22 Mb) of the genome, and ranged in size from 1.7 to 2,031 kb (median size 16.7 kb). About 20% of the CNVs co-localized with segmental duplications, while 30% encompass genes, of which the majority is involved in environmental response. About 10% of the human orthologous of these genes are associated with human disease susceptibility and, hence, may have important phenotypic consequences. Conclusions Together, this analysis provides a useful resource for assessment of the impact of CNVs regarding variation in bovine health and production traits. PMID:20459598

  18. 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. ...

  19. 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. ...

  20. 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. ...

  1. 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. ...

  2. 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. ...

  3. Genomic characteristics of cattle copy number variations

    PubMed Central

    2011-01-01

    Background Copy number variation (CNV) represents another important source of genetic variation complementary to single nucleotide polymorphism (SNP). High-density SNP array data have been routinely used to detect human CNVs, many of which have significant functional effects on gene expression and human diseases. In the dairy industry, a large quantity of SNP genotyping results are becoming available and can be used for CNV discovery to understand and accelerate genetic improvement for complex traits. Results We performed a systematic analysis of CNV 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 pedigree information, we identified 682 candidate CNV regions, which represent 139.8 megabases (~4.60%) of the genome. Selected CNVs were further experimentally validated and we found that copy number "gain" CNVs were predominantly clustered in tandem rather than existing as interspersed duplications. Many CNV regions (~56%) overlap with cattle genes (1,263), which are significantly enriched for immunity, lactation, reproduction and rumination. The overlap of this new dataset and other published CNV studies was less than 40%; however, our discovery of large, high frequency (> 5% of animals surveyed) CNV regions showed 90% agreement with other studies. These results highlight the differences and commonalities between technical platforms. Conclusions We present a comprehensive genomic analysis of cattle CNVs derived from SNP data which will be a valuable genomic variation resource. Combined with SNP detection assays, gene-containing CNV regions may help identify genes undergoing artificial selection in domesticated animals. PMID:21345189

  4. 12 CFR 269b.730 - Number of copies; form.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 12 Banks and Banking 3 2010-01-01 2010-01-01 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 legibly...

  5. 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...

  6. Copy Number Variations in Tilapia Genomes.

    PubMed

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

    2017-02-01

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

  7. 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.

  8. 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

  9. Genome-wide copy number analysis using copy number inferring tool (CNIT) and DNA pooling.

    PubMed

    Lin, Chien-hsing; Huang, Mei-chu; Li, Ling-hui; Wu, Jer-yuarn; Chen, Yuan-tsong; Fann, Cathy S J

    2008-08-01

    Copy number variation (CNV) has become an important genomic structure element in the human population, and some CNVs are related to specific traits and diseases. Moreover, analysis of human genomes has been potentiated by the use of high-resolution microarrays that assess single nucleotide polymorphisms (SNPs). Although many programs have been designed to analyze data from Affymetrix SNP microarrays, they all have high false-positive rates (FPRs) in copy number (CN) analyses. Copy number analysis tool (CNAT) 4.0 is a recently developed program that offers improved CN estimation, but small amplifications and deletions are lost when using the smoothing procedure. Here, we propose a copy number inferring tool (CNIT) algorithm for the 100K SNP microarray to investigate CNVs at 29.6-kb resolution. CNIT estimated SNP allelic and total CN with reliable P values based on intensity data. In addition, the hidden Markov model (HMM) method was applied to predict regions having altered CN by considering contiguous SNPs. Based on a CN analysis of 23 unrelated Taiwanese and 30 HapMap Centre d'Etude du Polymorphisme Humain (CEPH) trios, CNIT showed higher accuracy and power than other programs. The FPRs and false-negative rates (FNRs) of CNIT were 0.1% and 0.16%, respectively. CNIT also showed better sensitivity for detecting small amplifications and deletions. Furthermore, DNA pooling of 10 and 30 normal unrelated individuals were applied to the 100K SNP microarray, respectively, and 12 common CN-variable regions were identified, suggesting that DNA pooling can be applied to discover common CNVs.

  10. Copy Number Variation in the Horse Genome

    PubMed Central

    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

    2014-01-01

    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. PMID:25340504

  11. 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

  12. 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.

  13. Ribosomal DNA copy number loss and sequence variation in cancer

    PubMed Central

    Xu, Baoshan; Li, Hua; Perry, John M.; Singh, Vijay Pratap; Yu, Zulin; Zakari, Musinu; Li, Linheng

    2017-01-01

    Ribosomal DNA is one of the most variable regions in the human genome with respect to copy number. Despite the importance of rDNA for cellular function, we know virtually nothing about what governs its copy number, stability, and sequence in the mammalian genome due to challenges associated with mapping and analysis. We applied computational and droplet digital PCR approaches to measure rDNA copy number in normal and cancer states in human and mouse genomes. We find that copy number and sequence can change in cancer genomes. Counterintuitively, human cancer genomes show a loss of copies, accompanied by global copy number co-variation. The sequence can also be more variable in the cancer genome. Cancer genomes with lower copies have mutational evidence of mTOR hyperactivity. The PTEN phosphatase is a tumor suppressor that is critical for genome stability and a negative regulator of the mTOR kinase pathway. Surprisingly, but consistent with the human cancer genomes, hematopoietic cancer stem cells from a Pten-/- mouse model for leukemia have lower rDNA copy number than normal tissue, despite increased proliferation, rRNA production, and protein synthesis. Loss of copies occurs early and is associated with hypersensitivity to DNA damage. Therefore, copy loss is a recurrent feature in cancers associated with mTOR activation. Ribosomal DNA copy number may be a simple and useful indicator of whether a cancer will be sensitive to DNA damaging treatments. PMID:28640831

  14. Ribosomal DNA copy number loss and sequence variation in cancer.

    PubMed

    Xu, Baoshan; Li, Hua; Perry, John M; Singh, Vijay Pratap; Unruh, Jay; Yu, Zulin; Zakari, Musinu; McDowell, William; Li, Linheng; Gerton, Jennifer L

    2017-06-01

    Ribosomal DNA is one of the most variable regions in the human genome with respect to copy number. Despite the importance of rDNA for cellular function, we know virtually nothing about what governs its copy number, stability, and sequence in the mammalian genome due to challenges associated with mapping and analysis. We applied computational and droplet digital PCR approaches to measure rDNA copy number in normal and cancer states in human and mouse genomes. We find that copy number and sequence can change in cancer genomes. Counterintuitively, human cancer genomes show a loss of copies, accompanied by global copy number co-variation. The sequence can also be more variable in the cancer genome. Cancer genomes with lower copies have mutational evidence of mTOR hyperactivity. The PTEN phosphatase is a tumor suppressor that is critical for genome stability and a negative regulator of the mTOR kinase pathway. Surprisingly, but consistent with the human cancer genomes, hematopoietic cancer stem cells from a Pten-/- mouse model for leukemia have lower rDNA copy number than normal tissue, despite increased proliferation, rRNA production, and protein synthesis. Loss of copies occurs early and is associated with hypersensitivity to DNA damage. Therefore, copy loss is a recurrent feature in cancers associated with mTOR activation. Ribosomal DNA copy number may be a simple and useful indicator of whether a cancer will be sensitive to DNA damaging treatments.

  15. Copy number variation in familial Parkinson disease.

    PubMed

    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.

  16. 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

  17. 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

  18. 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.730 Banks and Banking FEDERAL RESERVE SYSTEM (CONTINUED) BOARD OF GOVERNORS OF THE FEDERAL RESERVE SYSTEM (CONTINUED) CHARGES OF UNFAIR LABOR PRACTICES General Rules § 269b.730 Number of copies; form...

  19. 22 CFR 1429.25 - Number of copies.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 22 Foreign Relations 2 2011-04-01 2009-04-01 true Number of copies. 1429.25 Section 1429.25 Foreign Relations FOREIGN SERVICE LABOR RELATIONS BOARD; FEDERAL LABOR RELATIONS AUTHORITY; GENERAL... AND GENERAL REQUIREMENTS General Requirements § 1429.25 Number of copies. Unless otherwise provided by...

  20. 22 CFR 1429.25 - Number of copies.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 22 Foreign Relations 2 2010-04-01 2010-04-01 true Number of copies. 1429.25 Section 1429.25 Foreign Relations FOREIGN SERVICE LABOR RELATIONS BOARD; FEDERAL LABOR RELATIONS AUTHORITY; GENERAL... AND GENERAL REQUIREMENTS General Requirements § 1429.25 Number of copies. Unless otherwise provided by...

  1. 14 CFR 221.92 - Number of copies required.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 4 2010-01-01 2010-01-01 false Number of copies required. 221.92 Section 221.92 Aeronautics and Space OFFICE OF THE SECRETARY, DEPARTMENT OF TRANSPORTATION (AVIATION PROCEEDINGS) ECONOMIC REGULATIONS TARIFFS Filing Tariff Publications With Department § 221.92 Number of copies...

  2. 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

  3. CNARA: reliability assessment for genomic copy number profiles.

    PubMed

    Ai, Ni; Cai, Haoyang; Solovan, Caius; Baudis, Michael

    2016-10-12

    DNA copy number profiles from microarray and sequencing experiments sometimes contain wave artefacts which may be introduced during sample preparation and cannot be removed completely by existing preprocessing methods. Besides, large derivative log ratio spread (DLRS) of the probes correlating with poor DNA quality is sometimes observed in genome screening experiments and may lead to unreliable copy number profiles. Depending on the extent of these artefacts and the resulting misidentification of copy number alterations/variations (CNA/CNV), it may be desirable to exclude such samples from analyses or to adapt the downstream data analysis strategy accordingly. Here, we propose a method to distinguish reliable genomic copy number profiles from those containing heavy wave artefacts and/or large DLRS. We define four features that adequately summarize the copy number profiles for reliability assessment, and train a classifier on a dataset of 1522 copy number profiles from various microarray platforms. The method can be applied to predict the reliability of copy number profiles irrespective of the underlying microarray platform and may be adapted for those sequencing platforms from which copy number estimates could be computed as a piecewise constant signal. Further details can be found at https://github.com/baudisgroup/CNARA . We have developed a method for the assessment of genomic copy number profiling data, and suggest to apply the method in addition to and after other state-of-the-art noise correction and quality control procedures. CNARA could be instrumental in improving the assessment of data used for genomic data mining experiments and support the reliable functional attribution of copy number aberrations especially in cancer research.

  4. No association between mitochondrial DNA copy number and colorectal adenomas.

    PubMed

    Thyagarajan, Bharat; Guan, Weihua; Fedirko, Veronika; Barcelo, Helene; Tu, Huakang; Gross, Myron; Goodman, Michael; Bostick, Roberd M

    2016-08-01

    Despite previously reported associations between peripheral blood mtDNA copy number and colorectal cancer, it remains unclear whether altered mtDNA copy number in peripheral blood is a risk factor for colorectal cancer or a biomarker for undiagnosed colorectal cancer. Though colorectal adenomas are well-recognized precursor lesions to colorectal cancer, no study has evaluated an association between mtDNA copy number and colorectal adenoma risk. Hence, we investigated an association between peripheral blood mtDNA copy number and incident, sporadic colorectal adenoma in 412 colorectal adenoma cases and 526 cancer-free controls pooled from three colonoscopy-based case-control studies that used identical methods for case ascertainment, risk factor determination, and biospecimen collection. We also evaluated associations between relative mtDNA copy number and markers of oxidative stress, including circulating F2 -isoprostanes, carotenoids, and fluorescent oxidation products. We measured mtDNA copy number using a quantitative real time polymerase chain reaction (PCR). We used unconditional logistic regression to analyze the association between mtDNA copy number and colorectal adenoma risk after multivariable adjustment. We found no association between logarithmically transformed relative mtDNA copy number, analyzed as a continuous variable, and colorectal adenoma risk (odds ratio = 1.02, 95%CI: 0.82-1.27; P = 0.86). There were no statistically significant associations between relative mtDNA copy number and other markers of oxidative stress. Our findings, taken together with those from previous studies, suggest that relative mtDNA copy number in peripheral blood may more likely be a marker of early colorectal cancer than of risk for the disease or of in vivo oxidative stress. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

  5. 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

  6. 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).

  7. 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...

  8. 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 approved...

  9. Plasmid Copy Number Determination by Quantitative Polymerase Chain Reaction.

    PubMed

    Anindyajati; Artarini, A Anita; Riani, Catur; Retnoningrum, Debbie S

    2016-01-01

    Recombinant therapeutic proteins are biopharmaceutical products that develop rapidly for years. Recombinant protein production in certain hosts requires vector expression harboring the gene encoding the corresponding protein. Escherichia coli is the prokaryote organism mostly used in recombinant protein production, commonly using a plasmid as the expression vector. Recombinant protein production is affected by plasmid copy number harboring the encoded gene, hence the determination of plasmid copy number also plays an important role in establishing a recombinant protein production system. On the industrial scale, a low copy number of plasmids are more suitable due to their better stability. In the previous study we constructed pCAD, a plasmid derived from the low copy number pBR322 plasmid. This study was aimed to confirm pCAD's copy number by quantitative polymerase chain reaction (qPCR). Plasmid copy number was determined by comparing the quantification signal from the plasmid to those from the chromosome. Copy number was then calculated by using a known copy number plasmid as a standard. Two pairs of primers, called tdk and ori, were designed for targeting a single gene tdk in the chromosome and a conserved domain in the plasmid's ori, respectively. Primer quality was analyzed in silico using PrimerSelect DNASTAR and PraTo software prior to in vitro evaluation on primer specificity and efficiency as well as optimization of qPCR conditions. Plasmid copy number determination was conducted on E. coli lysates harboring each plasmid, with the number of cells ranging from 10(2)-10(5) cells/μL. Cells were lysed by incubation at 95ºC for 10 minutes, followed by immediate freezing at -4°C. pBR322 plasmid with the copy number of ~19 copies/cell was used as the standard, while pJExpress414-sod plasmid possessing the high copy number pUC ori was also determined to test the method being used. In silico analysis based on primer-primer and primer-template interactions showed

  10. Copy number alterations and copy number variation in cancer: close encounters of the bad kind.

    PubMed

    Speleman, F; Kumps, C; Buysse, K; Poppe, B; Menten, B; De Preter, K

    2008-01-01

    Recent studies have unveiled copy number variants (CNVs) as an important source of genetic variation. Many of these CNVs contain coding sequences, which have been shown to be dosage sensitive. Evidence is accumulating that certain CNVs have impact on susceptibility to human diseases such as HIV infection and autoimmune diseases, as well as on adaptability to environmental conditions or nutrition. The possible role and impact of CNVs on cancer development and progression is only now emerging. In this review we look into the role of CNVs and their associated genomic structural features in relation to the formation of chromosome alterations in cancer cells and evolutionary genomic plasticity, as well as the de novo occurrence of known or putative CNVs as somatic events during oncogenesis. The role of germline CNVs in cancer predisposition is still largely unexplored. A number of observations seem to warrant the importance of further studies to elucidate the impact of these variants in the early steps of carcinogenesis. Copyright 2009 S. Karger AG, Basel.

  11. 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.

  12. 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.

  13. 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

  14. 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

  15. DNA replication stress restricts ribosomal DNA copy number

    PubMed Central

    Salim, Devika; Bradford, William D.; Freeland, Amy; Cady, Gillian; Wang, Jianmin

    2017-01-01

    Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100–200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how “normal” copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a “normal” rDNA copy number. PMID:28915237

  16. Mitochondrial DNA Copy Number in Sleep Duration Discordant Monozygotic Twins.

    PubMed

    Wrede, Joanna E; Mengel-From, Jonas; Buchwald, Dedra; Vitiello, Michael V; Bamshad, Michael; Noonan, Carolyn; Christiansen, Lene; Christensen, Kaare; Watson, Nathaniel F

    2015-10-01

    Mitochondrial DNA (mtDNA) copy number is an important component of mitochondrial function and varies with age, disease, and environmental factors. We aimed to determine whether mtDNA copy number varies with habitual differences in sleep duration within pairs of monozygotic twins. Academic clinical research center. 15 sleep duration discordant monozygotic twin pairs (30 twins, 80% female; mean age 42.1 years [SD 15.0]). Sleep duration was phenotyped with wrist actigraphy. Each twin pair included a "normal" (7-9 h/24) and "short" (< 7 h/24) sleeping twin. Fasting peripheral blood leukocyte DNA was assessed for mtDNA copy number via the n-fold difference between qPCR measured mtDNA and nuclear DNA creating an mtDNA measure without absolute units. We used generalized estimating equation linear regression models accounting for the correlated data structure to assess within-pair effects of sleep duration on mtDNA copy number. Mean within-pair sleep duration difference per 24 hours was 94.3 minutes (SD 62.6 min). We found reduced sleep duration (β = 0.06; 95% CI 0.004, 0.12; P < 0.05) and sleep efficiency (β = 0.51; 95% CI 0.06, 0.95; P < 0.05) were significantly associated with reduced mtDNA copy number within twin pairs. Thus every 1-minute decrease in actigraphy-defined sleep duration was associated with a decrease in mtDNA copy number of 0.06. Likewise, a 1% decrease in actigraphy-defined sleep efficiency was associated with a decrease in mtDNA copy number of 0.51. Reduced sleep duration and sleep efficiency were associated with reduced mitochondrial DNA copy number in sleep duration discordant monozygotic twins offering a potential mechanism whereby short sleep impairs health and longevity through mitochondrial stress. © 2015 Associated Professional Sleep Societies, LLC.

  17. DNA replication stress restricts ribosomal DNA copy number.

    PubMed

    Salim, Devika; Bradford, William D; Freeland, Amy; Cady, Gillian; Wang, Jianmin; Pruitt, Steven C; Gerton, Jennifer L

    2017-09-15

    Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100-200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how "normal" copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen the yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a "normal" rDNA copy number.

  18. A tremendous expansion of copy number in crossbred bulls ( × ).

    PubMed

    Zhang, G W; Guan, J Q; Luo, Z G; Zhang, W X; Wang, L; Luo, X L; Zuo, F Y

    2016-04-01

    Crossbreeding between cattle () and yak () exhibits significant hybrid advantages in milk yield and meat production. By contrast, cattle-yak F hybrid bulls are sterile. Copy number variations (CNV) of multicopy gene families in male-specific regions of the mammalian Y chromosome (MSY) affect human and animal fertility. The present study investigated CNV of (), (), (), and () in 5 yak breed bulls ( = 63), cattle-yak F ( = 22) and F ( = 2) hybrid bulls, and Chinese Yellow (CY) cattle bulls ( = 10) by quantitative real-time PCR. showed restricted amplification in yak bulls in that the average geometric mean copy number (CN) was estimated to be 4 copies. The most compelling finding is that there is a tremendous expansion of CN in F hybrids (385 copies; 95% confidence interval [CI] = 351-421) and F hybrids (356 copies) compared with the male parent breed CY cattle (142 copies; 95% CI = 95-211). Copy numbers of and were also extensively expanded on the Y chromosome in yak and CY cattle bulls. The geometric mean CN of and were estimated to be 123 (95% CI = 114-132) and 250 copies (95% CI = 233-268) in yak bulls and 71 (95% CI = 61-82) and 133 (95% CI = 107-164) copies in CY cattle, respectively. Yak and CY cattle have 2 copies of the gene on the Y chromosome. Similarly to gene, the F and F hybrid bulls have higher CN of , , and than CY cattle ( < 0.01). These results indicated that the MSY of yak and cattle-yak crossbred hybrids was fundamentally different from cattle MSY in the context of genomic organization. Based on the model of cattle-yak F and F hybrid bull sterility, the CNV of may serve as a potential risk factor for crossbred bull ( × ) infertility. To our knowledge, this is the first study to examine differences in multicopy genes in MSY between yak and cattle-yak bulls.

  19. SHC2 gene copy number in multiple system atrophy (MSA).

    PubMed

    Ferguson, Marcus C; Garland, Emily M; Hedges, Lora; Womack-Nunley, Bethany; Hamid, Rizwan; Phillips, John A; Shibao, Cyndya A; Raj, Satish R; Biaggioni, Italo; Robertson, David

    2014-02-01

    Multiple system atrophy (MSA) is a sporadic, late onset, rapidly progressing neurodegenerative disorder, which is characterized by autonomic failure, together with Parkinsonian, cerebellar, and pyramidal motor symptoms. The pathologic hallmark is the glial cytoplasmic inclusion with α-synuclein aggregates. MSA is thus an α-synucleinopathy. Recently, Sasaki et al. reported that heterozygosity for copy number loss of Src homology 2 domain containing-transforming protein 2 (SHC2) genes (heterozygous SHC2 gene deletions) occurred in DNAs from many Japanese individuals with MSA. Because background copy number variation can be distinct in different human populations, we assessed SHC2 allele copy number in DNAs from a US cohort of individuals with MSA, to determine the contribution of SHC2 gene copy number variation in an American cohort followed at a US referral center for MSA. Our cohort included 105 carefully phenotyped individuals with MSA. We studied 105 well-characterized patients with MSA and 5 control subjects with reduced SHC2 gene copy number. We used two TaqMan Gene Copy Number Assays, to determine the copy number of two segments of the SHC2 gene that are separated by 27 kb. Assay results of DNAs from all of our 105 subjects with MSA showed 2 copies of both segments of their SHC2 genes. Our results indicate that SHC2 gene deletions underlie few, if any, cases of well-characterized MSA in the US population. This is in contrast to the Japanese experience reported by Sasaki et al., likely reflecting heterogeneity of the disease in different genetic backgrounds.

  20. 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.

  1. 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

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

    PubMed

    Song, Rong-Hua; Shao, Xiao-Qing; Li, Ling; Wang, Wen; Zhang, Jin-An

    2017-01-01

    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. 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. 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. 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.

  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. 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

  5. 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

  6. A portrait of copy-number polymorphism in Drosophila melanogaster.

    PubMed

    Dopman, Erik B; Hartl, Daniel L

    2007-12-11

    Thomas Hunt Morgan and colleagues identified variation in gene copy number in Drosophila in the 1920s and 1930s and linked such variation to phenotypic differences [Bridges CB (1936) Science 83:210]. Yet the extent of variation in the number of chromosomes, chromosomal regions, or gene copies, and the importance of this variation within species, remain poorly understood. Here, we focus on copy-number variation in Drosophila melanogaster. We characterize copy-number polymorphism (CNP) across genomic regions, and we contrast patterns to infer the evolutionary processes acting on this variation. Copy-number variation in D. melanogaster is nonrandomly distributed, presumably because of a mutational bias produced by tandem repeats or other mechanisms. Comparisons of coding and noncoding CNPs, however, reveal a strong effect of purifying selection in the removal of structural variation from functionally constrained regions. Most patterns of CNP in D. melanogaster suggest that negative selection and mutational biases are the primary agents responsible for shaping structural variation.

  7. Increased Sox2 copy number in lung squamous cell carcinomas

    PubMed Central

    SASAKI, HIDEFUMI; YOKOTA, KEISUKE; HIKOSAKA, YU; MORIYAMA, SATORU; YANO, MOTOKI; FUJII, YOSHITAKA

    2012-01-01

    The transcription factor Sox2 is necessary for foregut morphogenesis. Sox2 is also required for the normal development of the trachea and lung. Recently, Sox2 amplifications were investigated using large-scale single nucleotide polymorphism arrays in esophageal and lung cancer. We hypothesized that Sox2 overexpression might be correlated with clinicopathological features of lung cancers. The increased copy number of the Sox2 gene was analyzed by real-time polymerase chain reaction amplifications in 127 surgically treated non-small cell lung cancer cases from Nagoya City University Hospital, Japan. A total of 87 squamous cell carcinoma (SCC) cases were involved. An increased Sox2 gene copy number was found in 42 (33.1%) lung cancer patients. Increased Sox2 copy number status was significantly correlated with gender (females, 9.5% vs. males, 34.1%; p=0.0026), smoking status (never smoker, 4.8% vs. smoker, 32.9%; p=0.0003) and pathological subtypes (squamous cell carcinoma, 44.8% vs. non-squamous cell carcinoma, 7.5%; p<0.0001). However, among the SCCs, the Sox2 copy number status was not significantly correlated with gender, smoking status, pathological stage or differentiation status. An increased Sox2 copy number is common within SCC. PMID:22969842

  8. 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

  9. Genome wide copy number analysis of single cells

    PubMed Central

    Baslan, Timour; Kendall, Jude; Rodgers, Linda; Cox, Hilary; Riggs, Mike; Stepansky, Asya; Troge, Jennifer; Ravi, Kandasamy; Esposito, Diane; Lakshmi, B.; Wigler, Michael; Navin, Nicholas; Hicks, James

    2016-01-01

    Summary Copy number variation (CNV) is increasingly recognized as an important contributor to phenotypic variation in health and disease. Most methods for determining CNV rely on admixtures of cells, where information regarding genetic heterogeneity is lost. Here, we present a protocol that allows for the genome wide copy number analysis of single nuclei isolated from mixed populations of cells. Single nucleus sequencing (SNS), combines flow sorting of single nuclei based on DNA content, whole genome amplification (WGA), followed by next generation sequencing to quantize genomic intervals in a genome wide manner. Multiplexing of single cells is discussed. Additionally, we outline informatic approaches that correct for biases inherent in the WGA procedure and allow for accurate determination of copy number profiles. All together, the protocol takes ~3 days from flow cytometry to sequence-ready DNA libraries. PMID:22555242

  10. 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.

  11. Global variation in copy number in the human genome

    PubMed Central

    Redon, Richard; Ishikawa, Shumpei; Fitch, Karen R.; Feuk, Lars; Perry, George H.; Andrews, T. Daniel; Fiegler, Heike; Shapero, Michael H.; Carson, Andrew R.; Chen, Wenwei; Cho, Eun Kyung; Dallaire, Stephanie; Freeman, Jennifer L.; Gonzalez, Juan R.; Gratacos, Monica; Huang, Jing; Kalaitzopoulos, Dimitrios; Komura, Daisuke; MacDonald, Jeffrey R.; Marshall, Christian R.; Mei, Rui; Montgomery, Lyndal; Nishimura, Kunihiro; Okamura, Kohji; Shen, Fan; Somerville, Martin J.; Tchinda, Joelle; Valsesia, Armand; Woodwark, Cara; Yang, Fengtang; Zhang, Junjun; Zerjal, Tatiana; Zhang, Jane; Armengol, Lluis; Conrad, Donald F.; Estivill, Xavier; Tyler-Smith, Chris; Carter, Nigel P.; Aburatani, Hiroyuki; Lee, Charles; Jones, Keith W.; Scherer, Stephen W.; Hurles, Matthew E.

    2009-01-01

    Copy number variation (CNV) of DNA sequences is functionally significant but has yet to be fully ascertained. We have constructed a first-generation CNV map of the human genome through the study of 270 individuals from four populations with ancestry in Europe, Africa or Asia (the HapMap collection). DNA from these individuals was screened for CNV using two complementary technologies: single nucleotide polymorphism (SNP) genotyping arrays, and clone-based comparative genomic hybridization. 1,447 copy number variable regions covering 360 megabases (12% of the genome) were identified in these populations; these CNV regions contained hundreds of genes, disease loci, functional elements and segmental duplications. Strikingly, these CNVs encompassed more nucleotide content per genome than SNPs, underscoring the importance of CNV in genetic diversity and evolution. The data obtained delineate linkage disequilibrium patterns for many CNVs, and reveal dramatic variation in copy number among populations. We also demonstrate the utility of this resource for genetic disease studies. PMID:17122850

  12. 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

  13. 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.

  14. Complexity and algorithms for copy-number evolution problems.

    PubMed

    El-Kebir, Mohammed; Raphael, Benjamin J; Shamir, Ron; Sharan, Roded; Zaccaria, Simone; Zehavi, Meirav; Zeira, Ron

    2017-01-01

    Cancer is an evolutionary process characterized by the accumulation of somatic mutations in a population of cells that form a tumor. One frequent type of mutations is copy number aberrations, which alter the number of copies of genomic regions. The number of copies of each position along a chromosome constitutes the chromosome's copy-number profile. Understanding how such profiles evolve in cancer can assist in both diagnosis and prognosis. We model the evolution of a tumor by segmental deletions and amplifications, and gauge distance from profile [Formula: see text] to [Formula: see text] by the minimum number of events needed to transform [Formula: see text] into [Formula: see text]. Given two profiles, our first problem aims to find a parental profile that minimizes the sum of distances to its children. Given k profiles, the second, more general problem, seeks a phylogenetic tree, whose k leaves are labeled by the k given profiles and whose internal vertices are labeled by ancestral profiles such that the sum of edge distances is minimum. For the former problem we give a pseudo-polynomial dynamic programming algorithm that is linear in the profile length, and an integer linear program formulation. For the latter problem we show it is NP-hard and give an integer linear program formulation that scales to practical problem instance sizes. We assess the efficiency and quality of our algorithms on simulated instances. https://github.com/raphael-group/CNT-ILP.

  15. 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…

  16. Mapping cattle copy number variations in water buffalo

    USDA-ARS?s Scientific Manuscript database

    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 ...

  17. Genomic and evolutionary characteristics of cattle copy number variations

    USDA-ARS?s Scientific Manuscript database

    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...

  18. 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…

  19. Copy of drawing number 1 (of 58 shop drawings), of ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Copy of drawing number 1 (of 58 shop drawings), of the erection plan for the center street bridge, prepared by the King Bridge Co. of Cleveland. Drawing courtesy Engineering Department, City of Cleveland - Center Street Swing Bridge, Southwest of Public Square, Cleveland, Cuyahoga County, OH

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

    USDA-ARS?s Scientific Manuscript database

    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...

  1. Analysis of copy number variations among cattle breeds

    USDA-ARS?s Scientific Manuscript database

    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...

  2. 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.

  3. Endogenous RNA interference is driven by copy number

    PubMed Central

    Cruz, Cristina; Houseley, Jonathan

    2014-01-01

    A plethora of non-protein coding RNAs are produced throughout eukaryotic genomes, many of which are transcribed antisense to protein-coding genes and could potentially instigate RNA interference (RNAi) responses. Here we have used a synthetic RNAi system to show that gene copy number is a key factor controlling RNAi for transcripts from endogenous loci, since transcripts from multi-copy loci form double stranded RNA more efficiently than transcripts from equivalently expressed single-copy loci. Selectivity towards transcripts from high-copy DNA is therefore an emergent property of a minimal RNAi system. The ability of RNAi to selectively degrade transcripts from high-copy loci would allow suppression of newly emerging transposable elements, but such a surveillance system requires transcription. We show that low-level genome-wide pervasive transcription is sufficient to instigate RNAi, and propose that pervasive transcription is part of a defense mechanism capable of directing a sequence-independent RNAi response against transposable elements amplifying within the genome. DOI: http://dx.doi.org/10.7554/eLife.01581.001 PMID:24520161

  4. 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.

  5. Industrial Relevance of Chromosomal Copy Number Variation in Saccharomyces Yeasts

    PubMed Central

    Gorter de Vries, Arthur R.; Pronk, Jack T.

    2017-01-01

    ABSTRACT Chromosomal copy number variation (CCNV) plays a key role in evolution and health of eukaryotes. The unicellular yeast Saccharomyces cerevisiae is an important model for studying the generation, physiological impact, and evolutionary significance of CCNV. Fundamental studies of this yeast have contributed to an extensive set of methods for analyzing and introducing CCNV. Moreover, these studies provided insight into the balance between negative and positive impacts of CCNV in evolutionary contexts. A growing body of evidence indicates that CCNV not only frequently occurs in industrial strains of Saccharomyces yeasts but also is a key contributor to the diversity of industrially relevant traits. This notion is further supported by the frequent involvement of CCNV in industrially relevant traits acquired during evolutionary engineering. This review describes recent developments in genome sequencing and genome editing techniques and discusses how these offer opportunities to unravel contributions of CCNV in industrial Saccharomyces strains as well as to rationally engineer yeast chromosomal copy numbers and karyotypes. PMID:28341679

  6. Decreased mitochondrial copy numbers in oral squamous cell carcinoma.

    PubMed

    Takeda, Daisuke; Hasegawa, Takumi; Ueha, Takeshi; Sakakibara, Akiko; Kawamoto, Teruya; Minamikawa, Tsutomu; Sakai, Yoshitada; Komori, Takahide

    2016-08-01

    Mitochondrial dysfunction and altered respiration have long been suspected to affect the development and progression of cancer. Although quantitative changes in mitochondrial DNA (mtDNA) have been reported in head and neck squamous cell carcinoma (SCC), differences in mtDNA copy numbers between normal and cancerous tissues from same patients have not been assessed. We compared mtDNA copy numbers and expressions of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) and mitochondrial transcription factor A (TFAM) between normal mucous membrane and cancerous tissues resected from 35 patients with oral SCC, using TaqMan quantitative real-time polymerase chain reaction (PCR) and immunohistochemical staining. We found mtDNA copy numbers and expressions of PGC-1α and TFAM were decreased in cancerous tissues compared with normal tissues from the same patients. The PGC-1α-TFAM mitochondrial pathway may be associated with malignant potential in human oral SCC, and could be an attractive therapeutic target. © 2016 Wiley Periodicals, Inc. Head Neck 38:1170-1175, 2016. © 2016 Wiley Periodicals, Inc.

  7. 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

  8. 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

  9. Large multi-allelic copy number variations in humans

    PubMed Central

    Handsaker, Robert E.; Van Doren, Vanessa; Berman, Jennifer R.; Genovese, Giulio; Kashin, Seva; Boettger, Linda M.; McCarroll, Steven A.

    2015-01-01

    Thousands of genome segments appear to be present in widely varying copy number in different human genomes. We developed ways to use increasingly abundant whole genome sequence data to identify the copy numbers, alleles and haplotypes present at most large, multi-allelic CNVs (mCNVs). We analyzed 849 genomes sequenced by the 1000 Genomes Project to identify most large (>5 kb) mCNVs, including 3,878 duplications, of which 1,356 appear to have three or more segregating alleles. We find that mCNVs give rise to most human gene-dosage variation – exceeding sevenfold the contribution of deletions and biallelic duplications – and that this variation in gene dosage generates abundant variation in gene expression. We describe “runaway duplication haplotypes” in which genes, including HPR and ORM1, have mutated to high copy number on specific haplotypes. We describe partially successful initial strategies for analyzing mCNVs via imputation and provide an initial data resource to support such analyses. PMID:25621458

  10. 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

  11. 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

  12. 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

  13. 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.

  14. 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

  15. Copy Number Alterations and Methylation in Ewing's Sarcoma.

    PubMed

    Jahromi, Mona S; Jones, Kevin B; Schiffman, Joshua D

    2011-01-01

    Ewing's sarcoma is the second most common bone malignancy affecting children and young adults. The prognosis is especially poor in metastatic or relapsed disease. The cell of origin remains elusive, but the EWS-FLI1 fusion oncoprotein is present in the majority of cases. The understanding of the molecular basis of Ewing's sarcoma continues to progress slowly. EWS-FLI1 affects gene expression, but other factors must also be at work such as mutations, gene copy number alterations, and promoter methylation. This paper explores in depth two molecular aspects of Ewing's sarcoma: copy number alterations (CNAs) and methylation. While CNAs consistently have been reported in Ewing's sarcoma, their clinical significance has been variable, most likely due to small sample size and tumor heterogeneity. Methylation is thought to be important in oncogenesis and balanced karyotype cancers such as Ewing's, yet it has received only minimal attention in prior studies. Future CNA and methylation studies will help to understand the molecular basis of this disease.

  16. 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.

  17. 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.

  18. 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.

  19. Copy number gains in EGFR and copy number losses in PTEN are common events in osteosarcoma tumors.

    PubMed

    Freeman, Serena S; Allen, Steven W; Ganti, Ramapriya; Wu, Jianrong; Ma, Jing; Su, Xiaoping; Neale, Geoff; Dome, Jeffrey S; Daw, Najat C; Khoury, Joseph D

    2008-09-15

    Osteosarcoma cell lines and tumors have been shown to express epidermal growth factor receptor (EGFR) and harbor amplifications at the EGFR locus. In this study, the authors further analyzed the genomic features of EGFR in osteosarcoma tumors and investigated whether they correlate with phosphatase and tensin homolog (PTEN) expression and copy number status. EGFR and PTEN expression was assessed by immunohistochemistry (n = 28), and copy number alterations at the EGFR and PTEN loci were surveyed using Affymetrix (Santa Clara, Calif) 50K single nucleotide polymorphism (SNP) arrays (n = 31) and fluorescence in situ hybridization (FISH) (n = 27). The EGFR tyrosine kinase domain was sequenced to survey for activating mutations (n = 34). In addition, EGFRvIII expression was assessed using reverse transcriptase polymerase chain reaction (n = 24). Results were correlated with available clinical information on 59 patients, with a median age of 14.1 years (range, 5-23 years) and median follow-up of 4.4 years. EGFR expression was detected in the majority of osteosarcoma tumors surveyed (23 of 28; 82%). SNP arrays revealed evidence of frequent copy number gains at 7p11.2 and losses at 10q23.21. A sizeable subset (16 of 27 cases; 59%) showed gains at the EGFR locus using FISH (amplification in 4 of 27 [15%] and balanced chromosome 7 polysomy in 12 of 27 [44%]), and 12 cases showed deletions at the PTEN locus (biallelic deletions in 4 of 27 [15%] and monoallelic deletion in 9 of 27 [33%]). No activating mutations in the EGFR tyrosine kinase domain, EGFRvIII expression, or association with clinical findings were detected. EGFR expression and genomic gains at the EGFR locus are prevalent in osteosarcoma tumors, which also commonly harbor deletions at the PTEN locus. (c) 2008 American Cancer Society.

  20. Environmental change drives accelerated adaptation through stimulated copy number variation.

    PubMed

    Hull, Ryan M; Cruz, Cristina; Jack, Carmen V; Houseley, Jonathan

    2017-06-01

    Copy number variation (CNV) is rife in eukaryotic genomes and has been implicated in many human disorders, particularly cancer, in which CNV promotes both tumorigenesis and chemotherapy resistance. CNVs are considered random mutations but often arise through replication defects; transcription can interfere with replication fork progression and stability, leading to increased mutation rates at highly transcribed loci. Here we investigate whether inducible promoters can stimulate CNV to yield reproducible, environment-specific genetic changes. We propose a general mechanism for environmentally-stimulated CNV and validate this mechanism for the emergence of copper resistance in budding yeast. By analysing a large cohort of individual cells, we directly demonstrate that CNV of the copper-resistance gene CUP1 is stimulated by environmental copper. CNV stimulation accelerates the formation of novel alleles conferring enhanced copper resistance, such that copper exposure actively drives adaptation to copper-rich environments. Furthermore, quantification of CNV in individual cells reveals remarkable allele selectivity in the rate at which specific environments stimulate CNV. We define the key mechanistic elements underlying this selectivity, demonstrating that CNV is regulated by both promoter activity and acetylation of histone H3 lysine 56 (H3K56ac) and that H3K56ac is required for CUP1 CNV and efficient copper adaptation. Stimulated CNV is not limited to high-copy CUP1 repeat arrays, as we find that H3K56ac also regulates CNV in 3 copy arrays of CUP1 or SFA1 genes. The impact of transcription on DNA damage is well understood, but our research reveals that this apparently problematic association forms a pathway by which mutations can be directed to particular loci in particular environments and furthermore that this mutagenic process can be regulated through histone acetylation. Stimulated CNV therefore represents an unanticipated and remarkably controllable pathway

  1. Environmental change drives accelerated adaptation through stimulated copy number variation

    PubMed Central

    Hull, Ryan M.; Cruz, Cristina; Jack, Carmen V.

    2017-01-01

    Copy number variation (CNV) is rife in eukaryotic genomes and has been implicated in many human disorders, particularly cancer, in which CNV promotes both tumorigenesis and chemotherapy resistance. CNVs are considered random mutations but often arise through replication defects; transcription can interfere with replication fork progression and stability, leading to increased mutation rates at highly transcribed loci. Here we investigate whether inducible promoters can stimulate CNV to yield reproducible, environment-specific genetic changes. We propose a general mechanism for environmentally-stimulated CNV and validate this mechanism for the emergence of copper resistance in budding yeast. By analysing a large cohort of individual cells, we directly demonstrate that CNV of the copper-resistance gene CUP1 is stimulated by environmental copper. CNV stimulation accelerates the formation of novel alleles conferring enhanced copper resistance, such that copper exposure actively drives adaptation to copper-rich environments. Furthermore, quantification of CNV in individual cells reveals remarkable allele selectivity in the rate at which specific environments stimulate CNV. We define the key mechanistic elements underlying this selectivity, demonstrating that CNV is regulated by both promoter activity and acetylation of histone H3 lysine 56 (H3K56ac) and that H3K56ac is required for CUP1 CNV and efficient copper adaptation. Stimulated CNV is not limited to high-copy CUP1 repeat arrays, as we find that H3K56ac also regulates CNV in 3 copy arrays of CUP1 or SFA1 genes. The impact of transcription on DNA damage is well understood, but our research reveals that this apparently problematic association forms a pathway by which mutations can be directed to particular loci in particular environments and furthermore that this mutagenic process can be regulated through histone acetylation. Stimulated CNV therefore represents an unanticipated and remarkably controllable pathway

  2. 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....

  3. Copy number analysis of the low-copy repeats at the primate NPHP1 locus by array comparative genomic hybridization.

    PubMed

    Yuan, Bo; Liu, Pengfei; Rogers, Jeffrey; Lupski, James R

    2016-06-01

    Array comparative genomic hybridization (aCGH) has been widely used to detect copy number variants (CNVs) in both research and clinical settings. A customizable aCGH platform may greatly facilitate copy number analyses in genomic regions with higher-order complexity, such as low-copy repeats (LCRs). Here we present the aCGH analyses focusing on the 45 kb LCRs [1] at the NPHP1 region with diverse copy numbers in humans. Also, the interspecies aCGH analysis comparing human and nonhuman primates revealed dynamic copy number transitions of the human 45 kb LCR orthologues during primate evolution and therefore shed light on the origin of complexity at this locus. The original aCGH data are available at GEO under GSE73962.

  4. 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

  5. [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.

  6. 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.

  7. 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

  8. 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.

  9. Atrazine exposure elicits copy number alterations in the zebrafish genome.

    PubMed

    Wirbisky, Sara E; Freeman, Jennifer L

    2017-04-01

    Atrazine is an agricultural herbicide used throughout the Midwestern United States that frequently contaminates potable water supplies resulting in human exposure. Using the zebrafish model system, an embryonic atrazine exposure was previously reported to decrease spawning rates with an increase in progesterone and ovarian follicular atresia in adult females. In addition, alterations in genes associated with distinct molecular pathways of the endocrine system were observed in brain and gonad tissue of the adult females and males. Current hypotheses for mechanistic changes in the developmental origins of health and disease include genetic (e.g., copy number alterations) or epigenetic (e.g., DNA methylation) mechanisms. As such, in the current study we investigated whether an atrazine exposure would generate copy number alterations (CNAs) in the zebrafish genome. A zebrafish fibroblast cell line was used to limit detection to CNAs caused by the chemical exposure. First, cells were exposed to a range of atrazine concentrations and a crystal violet assay was completed, showing confluency decreased by ~60% at 46.3μM. Cells were then exposed to 0, 0.463, 4.63, or 46.3μM atrazine and array comparative genomic hybridization completed. Results showed 34, 21, and 44 CNAs in the 0.463, 4.63, and 46.3μM treatments, respectively. Furthermore, CNAs were associated with previously reported gene expression alterations in adult male and female zebrafish. This study demonstrates that atrazine exposure can generate CNAs that are linked to gene expression alterations observed in adult zebrafish exposed to atrazine during embryogenesis providing a mechanism of the developmental origins of atrazine endocrine disruption. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. 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

  11. Contrasting mechanisms of de novo copy number mutagenesis suggest the existence of different classes of environmental copy number mutagens.

    PubMed

    Conover, Hailey N; Argueso, Juan Lucas

    2016-01-01

    While gene copy number variations (CNVs) are abundant in the human genome, and often are associated with disease consequences, the mutagenic pathways and environmental exposures that cause these large structural mutations are understudied relative to conventional nucleotide substitutions in DNA. The members of the environmental mutagenesis community are currently seeking to remedy this deficiency, and there is a renewed interest in the development of mutagenicity assays to identify and characterize compounds that may induce de novo CNVs in humans. To achieve this goal, it is critically important to acknowledge that CNVs exist in two very distinct classes: nonrecurrent and recurrent CNVs. The goal of this commentary is to emphasize the deep contrasts that exist between the proposed pathways that lead to these two mutation classes. Nonrecurrent de novo CNVs originate primarily in mitotic cells through replication-dependent DNA repair pathways that involve microhomologies (<10 bp), and are detected at higher frequency in children of older fathers. In contrast, recurrent de novo CNVs are most often formed in meiotic cells through homologous recombination between nonallelic large low-copy repeats (>10,000 bp), without an associated paternal age effect. Given the biological differences between the two CNV classes, it is our belief that nonrecurrent and recurrent CN mutagens will probably differ substantially in their modes of action. Therefore, each CNV class may require their own uniquely designed assays, so that we as a field may succeed in uncovering the broadest possible spectrum of environmental CN mutagens. © 2015 Wiley Periodicals, Inc.

  12. Cyanobacteria Maintain Constant Protein Concentration despite Genome Copy-Number Variation.

    PubMed

    Zheng, Xiao-Yu; O'Shea, Erin K

    2017-04-18

    The cyanobacterium Synechococcus elongatus PCC 7942 has multiple copies of its single chromosome, and the copy number varies in individual cells, providing an ideal system to study the effect of genome copy-number variation on cell size and gene expression. Using single-cell fluorescence imaging, we found that protein concentration remained constant across individual cells regardless of genome copy number. Cell volume and the total protein amount from a single gene were both positively, linearly correlated with genome copy number, suggesting that changes in cell volume play an important role in buffering genome copy-number variance. This study provides a quantitative examination of gene expression regulation in cells with variable genome copies and sheds light on the compensation mechanisms for variance in genome copy number. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  13. 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.

  14. Copy number variation in potato - an asexually propagated autotetraploid species.

    PubMed

    Iovene, Marina; Zhang, Tao; Lou, Qunfeng; Buell, C Robin; Jiang, Jiming

    2013-07-01

    Copy number variation (CNV) has been revealed as a significant contributor to the genetic variation in humans. Although CNV has been reported in several model animal and plant species, the presence of CNV and its biological impact in polyploid species has not yet been documented. We conducted a fluorescence in situ hybridization (FISH)-based CNV survey in potato, a vegetatively propagated autotetraploid species (2n = 4x = 48). We conducted FISH analysis using 18 randomly selected potato bacterial artificial chromosome (BAC) clones in a set of 16 potato cultivars with diverse breeding backgrounds. Six BACs (33%) with insert sizes of 137-145 kb were found to be associated with large CNV events detectable at the cytological level. We demonstrate that the large CNVs associated with two specific BACs (RH102I10 and RH83C08) were widespread among potato cultivars developed in North America and Europe. We measured the transcript abundance of four genes associated with the CNV spanned by BAC RH102I10. All four genes displayed a dosage effect in transcription. Although potato is vegetatively propagated, we observed that female gametes lacking the RH102I10-associated CNV were inferior to those with at least one copy of this CNV, indicating that the RH102I10-associated CNV can impact on the growth and development of the potato plants. Our results show that CNV is highly abundant in the potato genome and may play a significant role in genetic variation of this important food crop. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

  15. 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

  16. Copy Number Variants in the Kallikrein Gene Cluster

    PubMed Central

    Lindahl, Pernilla; Säll, Torbjörn; Bjartell, Anders; Johansson, Anna M.; Lilja, Hans; Halldén, Christer

    2013-01-01

    The kallikrein gene family (KLK1-KLK15) is the largest contiguous group of protease genes within the human genome and is associated with both risk and outcome of cancer and other diseases. We searched for copy number variants in all KLK genes using quantitative PCR analysis and analysis of inheritance patterns of single nucleotide polymorphisms. Two deletions were identified: one 2235-bp deletion in KLK9 present in 1.2% of alleles, and one 3394-bp deletion in KLK15 present in 4.0% of alleles. Each deletion eliminated one complete exon and created out-of-frame coding that eliminated the catalytic triad of the resulting truncated gene product, which therefore likely is a non-functional protein. Deletion breakpoints identified by DNA sequencing located the KLK9 deletion breakpoint to a long interspersed element (LINE) repeated sequence, while the deletion in KLK15 is located in a single copy sequence. To search for an association between each deletion and risk of prostate cancer (PC), we analyzed a cohort of 667 biopsied men (266 PC cases and 401 men with no evidence of PC at biopsy) using short deletion-specific PCR assays. There was no association between evidence of PC in this cohort and the presence of either gene deletion. Haplotyping revealed a single origin of each deletion, with most recent common ancestor estimates of 3000-8000 and 6000-14 000 years for the deletions in KLK9 and KLK15, respectively. The presence of the deletions on the same haplotypes in 1000 Genomes data of both European and African populations indicate an early origin of both deletions. The old age in combination with homozygous presence of loss-of-function variants suggests that some kallikrein-related peptidases have non-essential functions. PMID:23894413

  17. 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

  18. The Effect of Algorithms on Copy Number Variant Detection

    PubMed Central

    Ely, Benjamin; Chi, Peter; Wang, Kenneth; Raskind, Wendy H.; Kim, Sulgi; Brkanac, Zoran; Yu, Chang-En

    2010-01-01

    Background The detection of copy number variants (CNVs) and the results of CNV-disease association studies rely on how CNVs are defined, and because array-based technologies can only infer CNVs, CNV-calling algorithms can produce vastly different findings. Several authors have noted the large-scale variability between CNV-detection methods, as well as the substantial false positive and false negative rates associated with those methods. In this study, we use variations of four common algorithms for CNV detection (PennCNV, QuantiSNP, HMMSeg, and cnvPartition) and two definitions of overlap (any overlap and an overlap of at least 40% of the smaller CNV) to illustrate the effects of varying algorithms and definitions of overlap on CNV discovery. Methodology and Principal Findings We used a 56 K Illumina genotyping array enriched for CNV regions to generate hybridization intensities and allele frequencies for 48 Caucasian schizophrenia cases and 48 age-, ethnicity-, and gender-matched control subjects. No algorithm found a difference in CNV burden between the two groups. However, the total number of CNVs called ranged from 102 to 3,765 across algorithms. The mean CNV size ranged from 46 kb to 787 kb, and the average number of CNVs per subject ranged from 1 to 39. The number of novel CNVs not previously reported in normal subjects ranged from 0 to 212. Conclusions and Significance Motivated by the availability of multiple publicly available genome-wide SNP arrays, investigators are conducting numerous analyses to identify putative additional CNVs in complex genetic disorders. However, the number of CNVs identified in array-based studies, and whether these CNVs are novel or valid, will depend on the algorithm(s) used. Thus, given the variety of methods used, there will be many false positives and false negatives. Both guidelines for the identification of CNVs inferred from high-density arrays and the establishment of a gold standard for validation of CNVs are needed

  19. Molecular analysis of complement component C4 gene copy number.

    PubMed

    Castley, Alison S L; Martinez, O Patricia

    2012-01-01

    Classical, alternative, or lectin pathways may activate the complement system cascade. The classical pathway includes the C4 protein and functions in the prevention of immune complex precipitation and in clearance of immune complexes.Two isotypes of C4-C4A and C4B-are coded by genes located at two loci within the major histocompatibility complex (MHC) on chromosome 6. While these isotypes share over 99% amino acid sequence homology, five nucleotide differences located in exon 26 are responsible for major structural and functional differences between the C4 isotypes.C4A and C4B are highly polymorphic with over 40 alleles, gene duplications, and "null alleles". C4 genes may be short (14.6 kb) or long (21 kb), due to the absence or presence of an endogenous retroviral sequence-HERV-K(C4)-in intron 9, respectively. The C4 gene copy number (GCN) can vary from 1-3 per haplotype or 2-6 per diploid genome. The variation in GCN leads to a range of C4 plasma protein concentrations among healthy subjects. In subjects with equal numbers of C4 genes, subjects with short genes have C4 plasma levels relatively higher than subjects with long genes.Variation of the C4 GCN, the gene size (long or short) and the C4 isotypes (C4A and C4B) may also lead to susceptibility to autoimmune disease. Therefore, in subjects with autoimmune disease, a low serum C4 level may be due to ongoing disease activity associated with complement activation and consumption or it may be due to genetic factors. Distinguishing between these will have clinical implications.Exact determination of GCN can be difficult, at least in part due to the high degree of homology between C4A and C4B and a variety of techniques has been described. This chapter describes a quantitative TaqMan real-time PCR (qPCR) copy number assay, based on our laboratory experience using this assay.

  20. Copy number variations and cognitive phenotypes in unselected populations.

    PubMed

    Männik, Katrin; Mägi, Reedik; Macé, Aurélien; Cole, Ben; Guyatt, Anna L; 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

    2015-05-26

    The association of copy number variations (CNVs), differing numbers of copies of genetic sequence at locations in the genome, with phenotypes such as intellectual disability has been almost exclusively evaluated using clinically ascertained cohorts. The contribution of these genetic variants to cognitive phenotypes in the general population remains unclear. To investigate the clinical features conferred by CNVs associated with known syndromes in adult carriers without clinical preselection and to assess the genome-wide consequences of rare CNVs (frequency ≤0.05%; size ≥250 kilobase pairs [kb]) on carriers' educational attainment and intellectual disability prevalence in the general population. The population biobank of Estonia contains 52,000 participants enrolled from 2002 through 2010. General practitioners examined participants and filled out a questionnaire of health- and lifestyle-related questions, as well as reported diagnoses. Copy number variant analysis was conducted on a random sample of 7877 individuals and genotype-phenotype associations with education and disease traits were evaluated. Our results were replicated on a high-functioning group of 993 Estonians and 3 geographically distinct populations in the United Kingdom, the United States, and Italy. Phenotypes of genomic disorders in the general population, prevalence of autosomal CNVs, and association of these variants with educational attainment (from less than primary school through scientific degree) and prevalence of intellectual disability. Of the 7877 in the Estonian cohort, we identified 56 carriers of CNVs associated with known syndromes. Their phenotypes, including cognitive and psychiatric problems, epilepsy, neuropathies, obesity, and congenital malformations are similar to those described for carriers of identical rearrangements ascertained in clinical cohorts. A genome-wide evaluation of rare autosomal CNVs (frequency, ≤0.05%; ≥250 kb) identified 831 carriers (10.5%) of the

  1. 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.

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

    PubMed Central

    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. PMID:26197066

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

    PubMed

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

    2016-11-29

    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.

  4. 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

  5. 17 CFR 240.12b-11 - Number of copies; signatures; binding.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Number of copies; signatures... Under the Securities Exchange Act of 1934 Formal Requirements § 240.12b-11 Number of copies; signatures; binding. (a) Except as provided in a particular form, three complete copies of each statement or report...

  6. 17 CFR 260.10a-3 - Number of copies-Filing-Signatures.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Number of copies-Filing... Number of copies—Filing—Signatures. (a) Three copies of every application pursuant to rule 10a-1 (§ 260... principal office. (b) One copy shall be manually signed by the applicant's duly authorized officer (or...

  7. 17 CFR 260.5b-3 - Number of copies-Filing-Signatures.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Number of copies-Filing... Number of copies—Filing—Signatures. (a) Three copies of every application pursuant to rule 5b-1 (§ 260.5b... calculated to result in filing with the Commission by that date. (b) One copy shall be manually signed by the...

  8. 17 CFR 270.8b-11 - Number of copies; signatures; binding.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Number of copies; signatures... (CONTINUED) RULES AND REGULATIONS, INVESTMENT COMPANY ACT OF 1940 § 270.8b-11 Number of copies; signatures; binding. (a) Three complete copies of each registration statement or report, including exhibits and all...

  9. 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 32.4...

  10. 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

  11. 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

  12. 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.

  13. Copy Number Variation in Hereditary Non-Polyposis Colorectal Cancer

    PubMed Central

    Masson, Amy L.; Talseth-Palmer, Bente A.; Evans, Tiffany-Jane; Grice, Desma M.; Duesing, Konsta; Hannan, Garry N.; Scott, Rodney J.

    2013-01-01

    Hereditary non-polyposis colorectal cancer (HNPCC) is the commonest form of inherited colorectal cancer (CRC) predisposition and by definition describes families which conform to the Amsterdam Criteria or reiterations thereof. In ~50% of patients adhering to the Amsterdam criteria germline variants are identified in one of four DNA Mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2. Loss of function of any one of these genes results in a failure to repair DNA errors occurring during replication which can be most easily observed as DNA microsatellite instability (MSI)—a hallmark feature of this disease. The remaining 50% of patients without a genetic diagnosis of disease may harbour more cryptic changes within or adjacent to MLH1, MSH2, MSH6 or PMS2 or elsewhere in the genome. We used a high density cytogenetic array to screen for deletions or duplications in a series of patients, all of whom adhered to the Amsterdam/Bethesda criteria, to determine if genomic re-arrangements could account for a proportion of patients that had been shown not to harbour causative mutations as assessed by standard diagnostic techniques. The study has revealed some associations between copy number variants (CNVs) and HNPCC mutation negative cases and further highlights difficulties associated with CNV analysis. PMID:24705261

  14. Hydroxyurea induces de novo copy number variants in human cells

    PubMed Central

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

    2011-01-01

    Copy number variants (CNVs) are widely distributed throughout the human genome, where they contribute to genetic variation and phenotypic diversity. Spontaneous CNVs are also a major cause of genetic and developmental disorders and arise frequently in cancer cells. As with all mutation classes, genetic and environmental factors almost certainly increase the risk for new and deleterious CNVs. However, despite the importance of CNVs, there is limited understanding of these precipitating risk factors and the mechanisms responsible for a large percentage of CNVs. Here we report that low doses of hydroxyurea, an inhibitor of ribonucleotide reductase and an important drug in the treatment of sickle cell disease and other diseases induces a high frequency of de novo CNVs in cultured human cells that resemble pathogenic and aphidicolin-induced CNVs in size and breakpoint structure. These CNVs are distributed throughout the genome, with some hotspots of de novo CNV formation. Sequencing revealed that CNV breakpoint junctions are characterized by short microhomologies, blunt ends, and short insertions. These data provide direct experimental support for models of replication-error origins of CNVs and suggest that any agent or condition that leads to replication stress has the potential to induce deleterious CNVs. In addition, they point to a need for further study of the genomic consequences of the therapeutic use of hydroxyurea. PMID:21987784

  15. The importance of copy number variation in congenital heart disease

    PubMed Central

    Costain, Gregory; Silversides, Candice K; Bassett, Anne S

    2017-01-01

    Congenital heart disease (CHD) is the most common class of major malformations in humans. The historical association with large chromosomal abnormalities foreshadowed the role of submicroscopic rare copy number variations (CNVs) as important genetic causes of CHD. Recent studies have provided robust evidence for these structural variants as genome-wide contributors to all forms of CHD, including CHD that appears isolated without extra-cardiac features. Overall, a CNV-related molecular diagnosis can be made in up to one in eight patients with CHD. These include de novo and inherited variants at established (chromosome 22q11.2), emerging (chromosome 1q21.1), and novel loci across the genome. Variable expression of rare CNVs provides support for the notion of a genetic spectrum of CHD that crosses traditional anatomic classification boundaries. Clinical genetic testing using genome-wide technologies (e.g., chromosomal microarray analysis) is increasingly employed in prenatal, paediatric and adult settings. CNV discoveries in CHD have translated to changes to clinical management, prognostication and genetic counselling. The convergence of findings at individual gene and at pathway levels is shedding light on the mechanisms that govern human cardiac morphogenesis. These clinical and research advances are helping to inform whole-genome sequencing, the next logical step in delineating the genetic architecture of CHD. PMID:28706735

  16. Gene copy number variation throughout the Plasmodium falciparum genome.

    PubMed

    Cheeseman, Ian H; Gomez-Escobar, Natalia; Carret, Celine K; Ivens, Alasdair; Stewart, Lindsay B; Tetteh, Kevin K A; Conway, David J

    2009-08-04

    Gene copy number variation (CNV) is responsible for several important phenotypes of the malaria parasite Plasmodium falciparum, including drug resistance, loss of infected erythrocyte cytoadherence and alteration of receptor usage for erythrocyte invasion. Despite the known effects of CNV, little is known about its extent throughout the genome. We performed a whole-genome survey of CNV genes in P. falciparum using comparative genome hybridisation of a diverse set of 16 laboratory culture-adapted isolates to a custom designed high density Affymetrix GeneChip array. Overall, 186 genes showed hybridisation signals consistent with deletion or amplification in one or more isolate. There is a strong association of CNV with gene length, genomic location, and low orthology to genes in other Plasmodium species. Sub-telomeric regions of all chromosomes are strongly associated with CNV genes independent from members of previously described multigene families. However, approximately 40% of CNV genes were located in more central regions of the chromosomes. Among the previously undescribed CNV genes, several that are of potential phenotypic relevance are identified. CNV represents a major form of genetic variation within the P. falciparum genome; the distribution of gene features indicates the involvement of highly non-random mutational and selective processes. Additional studies should be directed at examining CNV in natural parasite populations to extend conclusions to clinical settings.

  17. 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.

  18. 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

  19. 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

  20. Genomic determinants of somatic copy number alterations across human cancers.

    PubMed

    Zhang, Yanping; Xu, Hongen; Frishman, Dmitrij

    2016-03-01

    Somatic copy number alterations (SCNAs) play an important role in carcinogenesis. However, the impact of genomic architecture on the global patterns of SCNAs in cancer genomes remains elusive. In this work, we conducted multiple linear regression (MLR) analyses of the pooled SCNA data from The Cancer Genome Atlas (TCGA) Pan-Cancer project. We performed MLR analyses for 11 individual cancer types and three different kinds of SCNAs--amplifications and deletions, telomere-bound and interstitial SCNAs and local SCNAs. Our MLR model explains >30% of the pooled SCNA breakpoint variation, with the explanatory power ranging from 13 to 32% for different cancer types and SCNA types. In addition to confirming previously identified features [e.g. long interspersed element-1 (L1) and short interspersed nuclear elements], we also identified several novel informative features, including distance to telomere, distance to centromere and low-complexity repeats. The results of the MLR analyses were additionally confirmed on an independent SCNA data set obtained from the catalogue of somatic mutations in cancer database. Using a rare-event logistic regression model and an extremely randomized tree classifier, we revealed that genomic features are informative for defining common SCNA breakpoint hotspots. Our findings shed light on the molecular mechanisms of SCNA generation in cancer. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  1. Elevated Gene Copy Number Does Not Always Explain Elevated Amylase Activities in Fishes.

    PubMed

    German, Donovan P; Foti, Dolly M; Heras, Joseph; Amerkhanian, Hooree; Lockwood, Brent L

    2016-01-01

    Amylase activity variation in the guts of several model organisms appears to be explained by amylase gene copy number variation. We tested the hypothesis that amylase gene copy number is always elevated in animals with high amylolytic activity. We therefore sequenced the amylase genes and examined amylase gene copy number in prickleback fishes (family Stichaeidae) with different diets including two species of convergently evolved herbivores with the elevated amylase activity phenotype. We found elevated amylase gene copy number (six haploid copies) with sequence variation among copies in one herbivore (Cebidichthys violaceus) and modest gene copy number (two to three haploid copies) with little sequence variation in the remaining taxa, which included herbivores, omnivores, and a carnivore. Few functional differences in amylase biochemistry were observed, and previous investigations showed similar digestibility among the convergently evolved herbivores with differing amylase genetics. Hence, the phenotype of elevated amylase activity can be achieved by different mechanisms (i.e., elevated expression of fewer genes, increased gene copy number, or expression of more efficient amylase proteins) with similar results. Phylogenetic and comparative genomic analyses of available fish amylase genes show mostly lineage-specific duplication events leading to gene copy number variation, although a whole-genome duplication event or chromosomal translocation may have produced multiple amylase copies in the Ostariophysi, again showing multiple routes to the same result.

  2. 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.

  3. 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

  4. 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

  5. Analysis of copy number variations among diverse cattle breeds

    PubMed Central

    Liu, George E.; Hou, Yali; Zhu, Bin; Cardone, Maria Francesca; Jiang, Lu; Cellamare, Angelo; Mitra, Apratim; Alexander, Leeson J.; Coutinho, Luiz L.; Dell'Aquila, Maria Elena; Gasbarre, Lou C.; Lacalandra, Gianni; Li, Robert W.; Matukumalli, Lakshmi K.; Nonneman, Dan; de A. Regitano, Luciana C.; Smith, Tim P.L.; Song, Jiuzhou; Sonstegard, Tad S.; Van Tassell, Curt P.; Ventura, Mario; Eichler, Evan E.; McDaneld, Tara G.; Keele, John W.

    2010-01-01

    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 modern domesticated cattle using array comparative genomic hybridization (array CGH), quantitative PCR (qPCR), and fluorescent in situ hybridization (FISH). The array CGH panel included 90 animals from 11 Bos taurus, three Bos indicus, and three composite breeds for beef, dairy, or dual purpose. We identified over 200 candidate CNV regions (CNVRs) in total and 177 within known chromosomes, which harbor or are adjacent to gains or losses. These 177 high-confidence CNVRs cover 28.1 megabases or ∼1.07% of the genome. Over 50% of the CNVRs (89/177) were found in multiple animals or breeds and analysis revealed breed-specific frequency differences and reflected aspects of the known ancestry of these cattle breeds. Selected CNVs were further validated by independent methods using qPCR and FISH. Approximately 67% of the CNVRs (119/177) completely or partially span cattle genes and 61% of the CNVRs (108/177) directly overlap with segmental duplications. The CNVRs span about 400 annotated cattle genes that are significantly enriched for specific biological functions, such as immunity, lactation, reproduction, and rumination. Multiple gene families, including ULBP, have gone through ruminant lineage-specific gene amplification. We detected and confirmed marked differences in their CNV frequencies across diverse breeds, indicating that some cattle CNVs are likely to arise independently in breeds and contribute to breed differences. Our results provide a valuable resource beyond microsatellites and single nucleotide polymorphisms to explore the full dimension of genetic variability for future cattle genomic research. PMID:20212021

  6. Copy number variations in three children with sudden infant death.

    PubMed

    Toruner, G A; Kurvathi, R; Sugalski, R; Shulman, L; Twersky, S; Pearson, P G; Tozzi, R; Schwalb, M N; Wallerstein, R

    2009-07-01

    Sudden death of an infant is a devastating event that needs an explanation. When an explanation cannot be found, the case is labeled as sudden infant death syndrome or unclassified sudden infant death. The influence of genetic factors has been recognized for sudden infant death, but copy number variations (CNVs) as potential risk factors have not been evaluated yet. Twenty-seven families were enrolled in this study. The tissue specimens from deceased children were obtained and array-based comparative genomic hybridization (array-CGH) experiments were performed on the genomic DNA isolated from these specimens using Agilent Technologies Custom 4 x 44K arrays. Quantitative polymerase chain reaction experiments were performed to confirm the overlapping duplication and deletion region in two different cases. A de novo CNV is detected in 3 of 27 cases (11%). In case 1, an approximately 3-Mb (chr 8: 143,211,215-qter) duplication on 8q24.3-qter and a 4.4-Mb deletion on the 22q13.3-qter (chr 22: 45,047,068-qter) were detected. Subtelomeric chromosome analysis of the father and the surviving sibling of case 1 showed a balanced reciprocal translocation, 46,XY,t(8;22)(q24.3;q13.3). A 240-kb (chr 6: 26,139,810-26,380,787) duplication and a 1.9-Mb deletion (chr 6: 26,085,971-27,966,150) at chromosome 6p22 were found in cases 2 and 3, respectively. Array-CGH and conventional cytogenetic studies did not reveal the observed CNVs in the parents and the siblings of cases 2 and 3. The detected CNVs in cases 2 and 3 encompassed several genes including the major histone cluster genes. Array-CGH analysis may be beneficial during the investigations after sudden infant death.

  7. DNA Copy Number Variations Characterize Benign and Malignant Thyroid Tumors

    PubMed Central

    Liu, Yan; Cope, Leslie; Sun, Wenyue; Wang, Yongchun; Prasad, Nijaguna; Sangenario, Lauren; Talbot, Kristen; Somervell, Helina; Westra, William; Bishop, Justin; Califano, Joseph; Zeiger, Martha

    2013-01-01

    Context: Fine-needle aspiration (FNA) is the best diagnostic tool for preoperative evaluation of thyroid nodules but is often inconclusive as a guide for surgical management. Objective: Our hypothesis was that thyroid tumor subtypes may show characteristic DNA copy number variation (CNV) patterns, which may further improve the preoperative classification. Design: Our study cohorts included benign follicular adenomas (FAs), classic papillary thyroid carcinomas (PTCs), and follicular variant PTCs (FVPTCs), the three subtypes most commonly associated with inconclusive preoperative cytopathology. Setting: Tissue and FNA samples were obtained at an academic tertiary referral center. Patients: Cases were identified that underwent partial or complete thyroidectomy for malignant or indeterminate thyroid lesions between 2000 and 2008 and had adequate snap-frozen tissue. Interventions: Pairs of tumor tissue and matching normal thyroid tissue-derived DNA were compared using 550K single-nucleotide polymorphism arrays. Main Outcome Measure: Statistically significant differences in CNV patterns between tumor subtypes were identified. Results: Segmental amplifications in chromosomes (Ch) 7 and 12 were more common in FAs than in PTCs or FVPTCs. Additionally, a subset of FAs and FVPTCs showed deletions in Ch22. We identified the 5 CNV-associated genes best at discriminating between FAs and PTCs/FVPTCs, which correctly classified 90% of cases. These 5 Ch12 genes were validated by quantitative genomic PCR and gene expression array analyses on the same patient cohort. The 5-gene signature was then successfully validated against an independent test cohort of benign and malignant tumor samples. Finally, we performed a feasibility study on matched FA-derived intraoperative FNA samples and were able to correctly identify FAs harboring the Ch12 amplification signature, whereas FAs without amplification showed a normal Ch12 signature. Conclusions: Thyroid tumor subtypes possess

  8. A Map of Copy Number Variations in Chinese Populations

    PubMed Central

    Yang, Yajun; Kang, Longli; Zhang, Xin; Jin, Wenfei; Wu, Bailin; Jin, Li; Xu, Shuhua

    2011-01-01

    It has been shown that the human genome contains extensive copy number variations (CNVs). Investigating the medical and evolutionary impacts of CNVs requires the knowledge of locations, sizes and frequency distribution of them within and between populations. However, CNV study of Chinese minorities, which harbor the majority of genetic diversity of Chinese populations, has been underrepresented considering the same efforts in other populations. Here we constructed, to our knowledge, a first CNV map in seven Chinese populations representing the major linguistic groups in China with 1,440 CNV regions identified using Affymetrix SNP 6.0 Array. Considerable differences in distributions of CNV regions between populations and substantial population structures were observed. We showed that ∼35% of CNV regions identified in minority ethnic groups are not shared by Han Chinese population, indicating that the contribution of the minorities to genetic architecture of Chinese population could not be ignored. We further identified highly differentiated CNV regions between populations. For example, a common deletion in Dong and Zhuang (44.4% and 50%), which overlaps two keratin-associated protein genes contributing to the structure of hair fibers, was not observed in Han Chinese. Interestingly, the most differentiated CNV deletion between HapMap CEU and YRI containing CCL3L1 gene reported in previous studies was also the highest differentiated regions between Tibetan and other populations. Besides, by jointly analyzing CNVs and SNPs, we found a CNV region containing gene CTDSPL were in almost perfect linkage disequilibrium between flanking SNPs in Tibetan while not in other populations except HapMap CHD. Furthermore, we found the SNP taggability of CNVs in Chinese populations was much lower than that in European populations. Our results suggest the necessity of a full characterization of CNVs in Chinese populations, and the CNV map we constructed serves as a useful resource in

  9. 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.

  10. Using the R Package crlmm for Genotyping and Copy Number Estimation

    PubMed Central

    Scharpf, Robert B.; Irizarry, Rafael A.; Ritchie, Matthew E.; Carvalho, Benilton; Ruczinski, Ingo

    2011-01-01

    Genotyping platforms such as Affymetrix can be used to assess genotype-phenotype as well as copy number-phenotype associations at millions of markers. While genotyping algorithms are largely concordant when assessed on HapMap samples, tools to assess copy number changes are more variable and often discordant. One explanation for the discordance is that copy number estimates are susceptible to systematic differences between groups of samples that were processed at different times or by different labs. Analysis algorithms that do not adjust for batch effects are prone to spurious measures of association. The R package crlmm implements a multilevel model that adjusts for batch effects and provides allele-specific estimates of copy number. This paper illustrates a workflow for the estimation of allele-specific copy number and integration of the marker-level estimates with complimentary Bioconductor software for inferring regions of copy number gain or loss. All analyses are performed in the statistical environment R. PMID:22523482

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

    USDA-ARS?s Scientific Manuscript database

    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...

  12. Mitochondrial DNA Copy Number and Exposure to Polycyclic Aromatic Hydrocarbons

    PubMed Central

    Pavanello, Sofia; Dioni, Laura; Hoxha, Mirjam; Fedeli, Ugo; Mielzynska-Švach, Danuta; Baccarelli, Andrea A.

    2013-01-01

    Background Increased mitochondrial DNA copy number (mtDNAcn) is a biological response to mtDNA damage and dysfunction predictive of lung cancer risk. Polycyclic aromatic hydrocarbons (PAHs) are established lung carcinogens and may cause mitochondrial toxicity. Whether PAH exposure and PAH-related nuclear DNA (nDNA) genotoxic effects are linked with increased mtDNAcn has never been evaluated. Methods We investigated the effect of chronic exposure to PAHs on mtDNAcn in peripheral blood lymphocytes (PBLs) of 46 Polish male non-current smoking cokeoven workers and 44 matched controls, who were part of a group of 94 study individuals examined in our previous work. Subjects PAH exposure and genetic alterations were characterized through measures of internal dose (urinary 1-pyrenol), target dose [anti-benzo[a]pyrene diolepoxide (anti-BPDE)-DNA adduct], genetic instability (micronuclei, MN and telomere length [TL]) and DNA methylation [p53 promoter] in PBLs. mtDNAcn (MT/S) was measured using a validated real-time PCR method. Results Workers with PAH exposure above the median value (>3 µmol 1-pyrenol/mol creatinine) showed higher mtDNAcn [geometric means (GM) of 1.06 (unadjusted) and 1.07 (age-adjusted)] compared to controls [GM 0.89 (unadjusted); 0.89 (age-adjusted)] (p=0.029 and 0.016), as well as higher levels of genetic and chromosomal [i.e. anti-BPDE-DNA adducts (p<0.001), MN (p<0.001) and TL (p=0.053)] and epigenetic [i.e., p53 gene-specific promoter methylation (p<0.001)] alterations in the nDNA. In the whole study population, unadjusted and age-adjusted mtDNAcn was positively correlated with 1-pyrenol (p=0.043 and 0.032) and anti-BPDE-DNA adducts (p=0.046 and 0.049). Conclusions PAH exposure and PAH-related nDNA genotoxicity are associated with increased mtDNAcn. Impact The present study is suggestive of potential roles of mtDNAcn in PAH-induced carcinogenesis. PMID:23885040

  13. 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

  14. Macronuclear Actin copy number variations in single cells of different Pseudokeronopsis (Alveolata, Ciliophora) populations.

    PubMed

    Huang, Lijuan; Lu, Xuefen; Zhu, Changyu; Lin, Xiaofeng; Yi, Zhenzhen

    2017-06-01

    Macronuclear chromosomes of ciliates, especially those of Spirotrichea, Armophorea and Phyllopharyngea, are extensively fragmented and their copy numbers vary significantly. A recent study suggested that parental RNA molecules regulate macronuclear copy number in offspring cells after conjugation. However, variations in patterns of macronuclear copy number during vegetative growth are not clear. Previous studies have reported macronuclear copy numbers of population averages, potentially masking individual variation. In the present investigation, we studied copy number variations among closely related species of Pseudokeronopsis and among individual cells during vegetative growth. We found that macronuclear copy numbers of Actin I, II in our Pseudokeronopsis populations are in the same range as in other spirotrichean species, but no close relationship is detected among morphologically related Pseudokeronopsis species. Copy numbers of three cells within each Pseudokeronopsis population range from 1.01 to 4.55 fold, suggesting that stochastic influences copy number during vegetative growth. Furthermore, the absence of a relationship between macronuclear copy numbers of Actin I and Actin II within Pseudokeronopsis is consistent with the fact that these genes are located on different gene-sized macronuclear chromosomes. Additionally, Actin II might have disappeared in P. carnea during evolution within the Actin gene family. Copyright © 2017 Elsevier GmbH. All rights reserved.

  15. Gene copy number effects in the mer operon of plasmid NR1.

    PubMed Central

    Nakahara, H; Kinscherf, T G; Silver, S; Miki, T; Easton, A M; Rownd, R H

    1979-01-01

    The level of resistance to Hg2+ determined by the inducible mer operon of plasmid NR1 was essentially the same for three gene copy number variants in Escherichia coli, less in Proteus mirabilis, and intermediate in P. mirabilis "transitioned" to a high r-determinant gene copy number. Cell-free volatilization rates of radioactive mercury indicated increasing levels of intracellular mercuric reductase enzyme from low- to high-gene copy number forms in P. mirabilis and from low- to high-copy number forms in E. coli, but the additional enzyme in E. coli was effectively cryptic. PMID:374374

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed

    Sun, Yue; Joyce, Priya Aiyar

    2017-08-28

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

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

    PubMed Central

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

    2013-01-01

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

  19. 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

  20. 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.

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

    USDA-ARS?s Scientific Manuscript database

    Copy Number Variations (CNVs) affect a wide range of phenotypic traits; however, CNVs in or near segmental duplication regions are often difficult to track. Using a read depth approach based on next generation sequencing, we examined genome-wide copy number differences among five taurine (three Angu...

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

    USDA-ARS?s Scientific Manuscript database

    Copy number variations (CNVs) affect a wide range of phenotypic traits; however, CNVs in or near segmental duplication regions are often intractable. Using a read depth approach based on next-generation sequencing, we examined genome-wide copy number differences among five taurine (three Angus, one ...

  3. Individualized cattle copy number and segmental duplication maps using next generation sequencing

    USDA-ARS?s Scientific Manuscript database

    Copy Number Variations (CNVs) affect a wide range of phenotypic traits; however, CNVs in or near segmental duplication regions are often intractable. Using a read depth approach based on next generation sequencing, we examined genome-wide copy number differences among five taurine (three Angus, one ...

  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. 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 an...

  6. 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 Counsel...

  7. 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 an...

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

    USDA-ARS?s Scientific Manuscript database

    Genome structure variation, including copy-number (CNV) and presence/absence variation (PAV), comprise a large extent of maize genetic diversity but their effect on phenotypes remains largely unexplored. Here we describe how copy-number variation in a major aluminum (Al) tolerance locus contributes ...

  9. 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... SUBSTANCES CONTROL ACT POLYCHLORINATED BIPHENYLS (PCBs) MANUFACTURING, PROCESSING, DISTRIBUTION IN COMMERCE, AND USE PROHIBITIONS PCB Waste Disposal Records and Reports § 761.209 Number of copies of a manifest...

  10. 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...

  11. 18 CFR 34.7 - Number of copies to be filed.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 18 Conservation of Power and Water Resources 1 2010-04-01 2010-04-01 false Number of copies to be filed. 34.7 Section 34.7 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION... SECURITIES OR THE ASSUMPTION OF LIABILITIES § 34.7 Number of copies to be filed. Link to an amendment...

  12. 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...

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

    Cancer.gov

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

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

    DTIC Science & Technology

    2015-08-01

    AWARD NUMBER: W81XWH-14-1-0194 TITLE: DNA Copy Number Signature to Predict Recurrence in Early-Stage Ovarian Cancer PRINCIPAL INVESTIGATOR...SUBTITLE 5a. CONTRACT NUMBER DNA Copy Number Signature to Predict Recurrence in Early Stage Ovarian Cancer 5b. GRANT NUMBER W81XWH-14-1-0194 5c...tasks Major Task 1: Obtain DNA samples from consortium specimens • Subtask 1 Pathological review of 592 early-stage high-grade ovarian cancer specimens

  15. 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

  16. Lower mitochondrial DNA copy number in peripheral blood leukocytes increases the risk of endometrial cancer.

    PubMed

    Sun, Yuhui; Zhang, Liren; Ho, Simon S; Wu, Xifeng; Gu, Jian

    2016-06-01

    Mitochondria are the primary source of energy generation in human cells. Low mitochondrial DNA (mtDNA) copy number in peripheral blood leukocytes (PBLs) has been associated with obesity and increased risks of several cancers. Since obesity is a significant risk factor for endometrial cancer, we hypothesize that low mtDNA copy number in PBLs is associated with an increased susceptibility to endometrial cancer. Using a Caucasian case-control study, we measured mtDNA copy number in PBLs from 139 endometrial cancer patients and 139 age-matched controls and determined the association of mtDNA copy number with the risk of endometrial cancer using multivariate logistic regression analysis. The normalized mtDNA copy number was significantly lower in endometrial cancer cases (median, 0.84; range, 0.24-2.00) than in controls (median, 1.06; range, 0.64-1.96) (P < 0.001). Dichotomized into high and low groups based on the median mtDNA copy number value in the controls, individuals with low mtDNA copy number had a significantly increased risk of endometrial cancer (adjusted OR, 5.59; 95%CI, 3.05-10.25; P < 0.001) compared to those with high mtDNA copy number. There was a significant dose-response association in tertile analysis. In addition, there was a significant joint effect between lower mtDNA copy number and never smoking, hypertension, diabetes, and obesity in elevating the risk of endometrial cancer. Low mtDNA copy number in PBLs is significantly associated with an increased risk of endometrial cancer in Caucasians. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

  17. 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.

  18. 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.

  19. Abundant copy-number loss of CYCLOPS and STOP genes in gastric adenocarcinoma.

    PubMed

    Cutcutache, Ioana; Wu, Alice Yingting; Suzuki, Yuka; McPherson, John Richard; Lei, Zhengdeng; Deng, Niantao; Zhang, Shenli; Wong, Wai Keong; Soo, Khee Chee; Chan, Weng Hoong; Ooi, London Lucien; Welsch, Roy; Tan, Patrick; Rozen, Steven G

    2016-04-01

    Gastric cancer, a leading cause of cancer death worldwide, has been little studied compared with other cancers that impose similar health burdens. Our goal is to assess genomic copy-number loss and the possible functional consequences and therapeutic implications thereof across a large series of gastric adenocarcinomas. We used high-density single-nucleotide polymorphism microarrays to determine patterns of copy-number loss and allelic imbalance in 74 gastric adenocarcinomas. We investigated whether suppressor of tumorigenesis and/or proliferation (STOP) genes are associated with genomic copy-number loss. We also analyzed the extent to which copy-number loss affects Copy-number alterations Yielding Cancer Liabilities Owing to Partial losS (CYCLOPS) genes-genes that may be attractive targets for therapeutic inhibition when partially deleted. The proportion of the genome subject to copy-number loss varies considerably from tumor to tumor, with a median of 5.5 %, and a mean of 12 % (range 0-58.5 %). On average, 91 STOP genes were subject to copy-number loss per tumor (median 35, range 0-452), and STOP genes tended to have lower copy-number compared with the rest of the genes. Furthermore, on average, 1.6 CYCLOPS genes per tumor were both subject to copy-number loss and downregulated, and 51.4 % of the tumors had at least one such gene. The enrichment of STOP genes in regions of copy-number loss indicates that their deletion may contribute to gastric carcinogenesis. Furthermore, the presence of several deleted and downregulated CYCLOPS genes in some tumors suggests potential therapeutic targets in these tumors.

  20. Determining Fungi rRNA Copy Number by PCR

    PubMed Central

    Black, Jonathan; Dean, Timothy; Byfield, Grace; Foarde, Karin; Menetrez, Marc

    2013-01-01

    The goal of this project is to improve the quantification of indoor fungal pollutants via the specific application of quantitative PCR (qPCR). Improvement will be made in the controls used in current qPCR applications. This work focuses on the use of two separate controls within a standard qPCR reaction. The first control developed was the internal standard control gene, benA. This gene encodes for β-tubulin and was selected based on its single-copy nature. The second control developed was the standard control plasmid, which contained a fragment of the ribosomal RNA (rRNA) gene and produced a specific PCR product. The results confirm the multicopy nature of the rRNA region in several filamentous fungi and show that we can quantify fungi of unknown genome size over a range of spore extractions by inclusion of these two standard controls. Advances in qPCR have led to extremely sensitive and quantitative methods for single-copy genes; however, it has not been well established that the rRNA can be used to quantitate fungal contamination. We report on the use of qPCR, combined with two controls, to identify and quantify indoor fungal contaminants with a greater degree of confidence than has been achieved previously. Advances in indoor environmental health have demonstrated that contamination of the built environment by the filamentous fungi has adverse impacts on the health of building occupants. This study meets the need for more accurate and reliable methods for fungal identification and quantitation in the indoor environment. PMID:23543828

  1. 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.

  2. 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.

  3. 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

  4. 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.

  5. 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

  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. An efficient method for measuring copy number variation applied to improvement of nematode resistance in soybean.

    PubMed

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

    2016-10-01

    Copy number variation (CNV) is implicated in important traits in multiple crop plants, but can be challenging to genotype using conventional methods. The Rhg1 locus of soybean, which confers resistance to soybean cyst nematode (SCN), is a CNV of multiple 31.2-kb genomic units each containing four genes. Reliable, high-throughput methods to quantify Rhg1 and other CNVs for selective breeding were developed. The CNV genotyping assay described here uses a homeologous gene copy within the paleopolyploid soybean genome to provide the internal control for a single-tube TaqMan copy number assay. Using this assay, CNV in breeding populations can be tracked with high precision. We also show that extensive CNV exists within Fayette, a released, inbred SCN-resistant soybean cultivar with a high copy number at Rhg1 derived from a single donor parent. Copy number at Rhg1 is therefore unstable within a released variety over a relatively small number of generations. Using this assay to select for individuals with altered copy number, plants were obtained with both increased copy number and increased SCN resistance relative to control plants. Thus, CNV genotyping technologies can be used as a new type of marker-assisted selection to select for desirable traits in breeding populations, and to control for undesirable variation within cultivars. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

  8. Mitochondrial DNA copy number variation as a potential predictor of renal cell carcinoma.

    PubMed

    Elsayed, Eman T; Hashad, Mohamed M; Elgohary, Iman E

    2017-07-24

    Peripheral blood mitochondrial DNA (mtDNA) copy number alteration has been suggested as a risk factor for several types of cancer. The aim of the present study was to assess the role of peripheral blood mtDNA copy number variation as a noninvasive biomarker in the prediction and early detection of renal cell carcinoma (RCC) in a cohort of Egyptian patients. Quantitative real-time polymerase chain reaction (qPCR) was used to measure peripheral blood mtDNA copy numbers in 57 patients with newly diagnosed, early-stage localized RCC and 60 age- and sex-matched healthy individuals as a control group. Median mtDNA copy number was significantly higher in RCC cases than in controls (166 vs. 91, p<0.001). Increased mtDNA copy number was associated with an 18-fold increased risk of RCC (95% confidence interval: 5.065-63.9). On receiver operating characteristic curve analysis, it was found that mtDNA could distinguish between RCC patients and healthy controls, with 86% sensitivity, 80% specificity, 80.3% positive predictive value and 85.7% negative predictive value at a cutoff value of 108.5. Our results showed that increased peripheral blood mtDNA copy number was associated with increased risk of RCC. Therefore, RCC might be considered as part of a range of potential tumors in cases with elevated blood mtDNA copy number.

  9. 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.

  10. Mitochondrial DNA copy number in whole blood and glioma risk: A case control study.

    PubMed

    Shen, Jie; Song, Renduo; Lu, Zhimin; Zhao, Hua

    2016-12-01

    Alterations in mitochondrial DNA (mtDNA) copy number are observed in human gliomas. However, whether variations in mtDNA copy number in whole blood play any role in glioma carcinogenesis is still largely unknown. In current study with 395 glioma patients and 425 healthy controls, we intended to investigate the association between mtDNA copy number in whole blood and glioma risk. Overall, we found that levels of mtDNA copy number were significantly higher in glioma cases than healthy controls (mean: 1.48 vs. 1.32, P < 0.01). In both cases and controls, levels of mtDNA copy number were inversely correlated with age (P < 0.01, respectively). And in cases, newly diagnosed, glioblastoma (GBM), and high grade glioma patients had significantly lower mtDNA copy number than their counterparts (P = 0.02, P < 0.01, and P = 0.04, respectively). In the multivariate analysis, elevated mtDNA copy number levels were associated with a 1.63-fold increased risk of glioma (adjusted odds ratio (OR) = 1.63, 95% confidence interval (CI) = 1.23-2.14). In further quartile analysis, study subjects who had highest levels of mtNDA copy number had 1.75-fold increased risk of gliomas (adjOR = 1.75, 95%CI = 1.18-2.61). In brief, our findings support the role of mtDNA copy number in the glioma carcinogenesis. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  11. 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.

  12. 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

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

    PubMed

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

    2017-03-07

    To explore whether copy number variations (CNVs) of toll-like receptor 7 (TLR7) are associated with susceptibility to chronic hepatitis B virus (HBV) infection. 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. χ(2) 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. 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. Low TLR7 copy number is a risk factor for chronic HBV infection but is not associated with later stages of disease progression.

  14. Low AMY1 Gene Copy Number Is Associated with Increased Body Mass Index in Prepubertal Boys.

    PubMed

    Marcovecchio, M Loredana; Florio, Rosalba; Verginelli, Fabio; De Lellis, Laura; Capelli, Cristian; Verzilli, Delfina; Chiarelli, Francesco; Mohn, Angelika; Cama, Alessandro

    2016-01-01

    Genome-wide association studies have identified more than 60 single nucleotide polymorphisms associated with Body Mass Index (BMI). Additional genetic variants, such as copy number variations (CNV), have also been investigated in relation to BMI. Recently, the highly polymorphic CNV in the salivary amylase (AMY1) gene, encoding an enzyme implicated in the first step of starch digestion, has been associated with obesity in adults and children. We assessed the potential association between AMY1 copy number and a wide range of BMI in a population of Italian school-children. 744 children (354 boys, 390 girls, mean age (±SD): 8.4±1.4years) underwent anthropometric assessments (height, weight) and collection of saliva samples for DNA extraction. AMY1 copies were evaluated by quantitative PCR. A significant increase of BMI z-score by decreasing AMY1 copy number was observed in boys (β: -0.117, p = 0.033), but not in girls. Similarly, waist circumference (β: -0.155, p = 0.003, adjusted for age) was negatively influenced by AMY1 copy number in boys. Boys with 8 or more AMY1 copy numbers presented a significant lower BMI z-score (p = 0.04) and waist circumference (p = 0.01) when compared to boys with less than 8 copy numbers. In this pediatric-only, population-based study, a lower AMY1 copy number emerged to be associated with increased BMI in boys. These data confirm previous findings from adult studies and support a potential role of a higher copy number of the salivary AMY1 gene in protecting from excess weight gain.

  15. The Mu Transposable Elements of Maize: Evidence for Transposition and Copy Number Regulation during Development

    PubMed Central

    Alleman, Mary; Freeling, Michael

    1986-01-01

    The Mu transposon of maize exists in a highly mutagenic strain called Robertson's Mutator. Plants of this strain contain 10–50 copies of the Mu element, whereas most maize strains and other plants have none. When Mutator plants are crossed to plants of the inbred line 1S2P, which does not have copies of Mu, the progeny plants have approximately the same number of Mu sequences as did their Mutator parent. Approximately one-half of these copies have segregated from their parent and one-half have arisen by transposition and are integrated into new positions in the genome. This maintenance of copy number can be accounted for by an extremely high rate of transposition of the Mu elements (10–15 transpositions per gamete per generation). When Mutator plants are self-pollinated, the progeny double their Mu copy number in the first generation, but maintain a constant number of Mu sequences with subsequent self-pollinations. Transposition of Mu and the events that lead to copy number maintenance occur very late in the development of the germ cells but before fertilization. A larger version of the Mu element transposes but is not necessary for transposition of the Mu sequences. The progeny of crosses with a Mutator plant occasionally lack Mutator activity; these strains retain copies of the Mu element, but these elements no longer transpose. PMID:3002907

  16. High resolution measurement of DUF1220 domain copy number from whole genome sequence data.

    PubMed

    Astling, David P; Heft, Ilea E; Jones, Kenneth L; Sikela, James M

    2017-08-14

    DUF1220 protein domains found primarily in Neuroblastoma BreakPoint Family (NBPF) genes show the greatest human lineage-specific increase in copy number of any coding region in the genome. There are 302 haploid copies of DUF1220 in hg38 (~160 of which are human-specific) and the majority of these can be divided into 6 different subtypes (referred to as clades). Copy number changes of specific DUF1220 clades have been associated in a dose-dependent manner with brain size variation (both evolutionarily and within the human population), cognitive aptitude, autism severity, and schizophrenia severity. However, no published methods can directly measure copies of DUF1220 with high accuracy and no method can distinguish between domains within a clade. Here we describe a novel method for measuring copies of DUF1220 domains and the NBPF genes in which they are found from whole genome sequence data. We have characterized the effect that various sequencing and alignment parameters and strategies have on the accuracy and precision of the method and defined the parameters that lead to optimal DUF1220 copy number measurement and resolution. We show that copy number estimates obtained using our read depth approach are highly correlated with those generated by ddPCR for three representative DUF1220 clades. By simulation, we demonstrate that our method provides sufficient resolution to analyze DUF1220 copy number variation at three levels: (1) DUF1220 clade copy number within individual genes and groups of genes (gene-specific clade groups) (2) genome wide DUF1220 clade copies and (3) gene copy number for DUF1220-encoding genes. To our knowledge, this is the first method to accurately measure copies of all six DUF1220 clades and the first method to provide gene specific resolution of these clades. This allows one to discriminate among the ~300 haploid human DUF1220 copies to an extent not possible with any other method. The result is a greatly enhanced capability to analyze the

  17. 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.

  18. RUBIC identifies driver genes by detecting recurrent DNA copy number breaks

    PubMed Central

    van Dyk, Ewald; Hoogstraat, Marlous; ten Hoeve, Jelle; Reinders, Marcel J. T.; Wessels, Lodewyk F. A.

    2016-01-01

    The frequent recurrence of copy number aberrations across tumour samples is a reliable hallmark of certain cancer driver genes. However, state-of-the-art algorithms for detecting recurrent aberrations fail to detect several known drivers. In this study, we propose RUBIC, an approach that detects recurrent copy number breaks, rather than recurrently amplified or deleted regions. This change of perspective allows for a simplified approach as recursive peak splitting procedures and repeated re-estimation of the background model are avoided. Furthermore, we control the false discovery rate on the level of called regions, rather than at the probe level, as in competing algorithms. We benchmark RUBIC against GISTIC2 (a state-of-the-art approach) and RAIG (a recently proposed approach) on simulated copy number data and on three SNP6 and NGS copy number data sets from TCGA. We show that RUBIC calls more focal recurrent regions and identifies a much larger fraction of known cancer genes. PMID:27396759

  19. 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

  20. Psoriasis is associated with increased beta-defensin genomic copy number

    PubMed Central

    Hollox, Edward J.; Huffmeier, Ulrike; Zeeuwen, Patrick L.J.M.; Palla, Raquel; Lascorz, Jesús; Rodijk-Olthuis, Diana; van de Kerkhof, Peter C.M.; Traupe, Heiko; de Jongh, Gys; den Heijer, Martin; Reis, André; Armour, John A.L.; Schalkwijk, Joost

    2008-01-01

    Psoriasis is a common inflammatory skin disease with a strong genetic component. We have analysed the genomic copy number polymorphism of the beta-defensin region on human chromosome 8 in 179 Dutch psoriasis patients and 272 controls, and in 319 German psoriasis patients and 305 controls. Comparisons in both cohorts show a significant association between higher genomic copy number for beta-defensin genes and the risk of psoriasis. PMID:18059266

  1. Reduced mtDNA copy number increases the sensitivity of tumor cells to chemotherapeutic drugs

    PubMed Central

    Mei, H; Sun, S; Bai, Y; Chen, Y; Chai, R; Li, H

    2015-01-01

    Many cancer drugs are toxic to cells by activating apoptotic pathways. Previous studies have shown that mitochondria have key roles in apoptosis in mammalian cells, but the role of mitochondrial DNA (mtDNA) copy number variation in the pathogenesis of tumor cell apoptosis remains largely unknown. We used the HEp-2, HNE2, and A549 tumor cell lines to explore the relationship between mtDNA copy number variation and cell apoptosis. We first induced apoptosis in three tumor cell lines and one normal adult human skin fibroblast cell line (HSF) with cisplatin (DDP) or doxorubicin (DOX) treatment and found that the mtDNA copy number significantly increased in apoptotic tumor cells, but not in HSF cells. We then downregulated the mtDNA copy number by transfection with shRNA-TFAM plasmids or treatment with ethidium bromide and found that the sensitivity of tumor cells to DDP or DOX was significantly increased. Furthermore, we observed that levels of reactive oxygen species (ROS) increased significantly in tumor cells with lower mtDNA copy numbers, and this might be related to a low level of antioxidant gene expression. Finally, we rescued the increase of ROS in tumor cells with lipoic acid or N-acetyl-L-cysteine and found that the apoptosis rate decreased. Our studies suggest that the increase of mtDNA copy number is a self-protective mechanism of tumor cells to prevent apoptosis and that reduced mtDNA copy number increases ROS levels in tumor cells, increases the tumor cells' sensitivity to chemotherapeutic drugs, and increases the rate of apoptosis. This research provides evidence that mtDNA copy number variation might be a promising new therapeutic target for the clinical treatment of tumors. PMID:25837486

  2. Porcine oocyte mtDNA copy number is high or low depending on the donor.

    PubMed

    Pedersen, Hanne Skovsgaard; Løvendahl, Peter; Larsen, Knud; Madsen, Lone Bruhn; Callesen, Henrik

    2016-08-01

    Oocyte capacity is relevant in understanding decreasing female fertility and in the use of assisted reproductive technologies in human and farm animals. Mitochondria are important to the development of a functionally good oocyte and the oocyte mtDNA copy number has been introduced as a useful parameter for prediction of oocyte competence. The aim of this study was to investigate: (i) if the oocyte donor has an influence on its oocyte's mtDNA copy number; and (ii) the relation between oocyte size and mtDNA copy number using pre- and postpubertal pig oocytes. Cumulus-oocyte complexes were collected from individual donor pigs. The oocytes were allocated into different size-groups, snap-frozen and single-oocyte mtDNA copy number was estimated by quantitative real-time PCR using the genes ND1 and COX1. Results showed that mean mtDNA copy number in oocytes from any individual donor could be categorized as either 'high' (≥100,000) or 'low' (<100,000) with no difference in threshold between pre- and postpubertal oocytes. No linear correlation was detected between oocyte size and mtDNA copy number within pre- and postpubertal oocytes. This study demonstrates the importance of the oocyte donor in relation to oocyte mtDNA copy number, irrespectively of the donor's puberty status and the oocyte's growth stage. Observations from this study facilitate both further investigations of the importance of mtDNA copy number and the unravelling of relations between different mitochondrial parameters and oocyte competence.

  3. ALK gene copy number gain and immunohistochemical expression status using three antibodies in neuroblastoma.

    PubMed

    Kim, Eun Kyung; Kim, Sewha

    2016-03-17

    Anaplastic lymphoma kinase (ALK) gene aberrations-such as mutations, amplifications, and copy number gains-represent a major genetic predisposition to neuroblastoma (NB). This study aimed to evaluate the correlation between ALK gene copy number status, ALK protein expression, and clinicopathological parameters. We retrospectively retrieved 30 cases of poorly differentiated NB and constructed tissue microarrays (TMAs). ALK copy number changes were assessed by fluorescence in situ hybridization (FISH) assays, and ALK immunohistochemistry (IHC) testing was performed using three different antibodies (ALK1, D5F3, and 5A4 clones). ALK amplification and copy number gain were observed in 10% (3/30) and 53.3% (16/30) of the cohort, respectively. There were positive correlations between ALK copy number and IHC positive rate in ALK1 and 5A4 antibodies (p= < 0.001 and 0.019, respectively). ALK1, D5F3, and 5A4 antibodies equally showed 100% sensitivity in detecting ALK amplification. However, the sensitivity for detecting copy number gain differed among the three antibodies, with 75% sensitivity in D5F3 and 0% sensitivity in ALK1. ALK-amplified NBs were correlated with synchronous MYCN amplification and chromosome 1p deletion. ALK IHC positivity was frequently observed in INSS stage IV and high-risk group patients. In conclusion, this study identified that an increase in the ALK copy number is a frequent genetic alteration in poorly differentiated NB. ALK-amplified NBs showed consistent ALK IHC positivity with all kinds of antibodies. In contrast, the detection performance of ALK copy number gain was antibody dependent, with the D5F3 antibody showing the best sensitivity.

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

    PubMed

    Talevich, Eric; Shain, A Hunter; Botton, Thomas; Bastian, Boris C

    2016-04-01

    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.

  5. 47 CFR 1.1408 - Number of copies and form of pleadings.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 47 Telecommunication 1 2010-10-01 2010-10-01 false Number of copies and form of pleadings. 1.1408... Attachment Complaint Procedures § 1.1408 Number of copies and form of pleadings. (a) An original and three... the complaint proceeding must be drawn in conformity with the requirements of §§ 1.49, 1.50 and 1.52. ...

  6. 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.

  7. Estimating Copy Number and Allelic Variation at the Immunoglobulin Heavy Chain Locus Using Short Reads.

    PubMed

    Luo, Shishi; Yu, Jane A; Song, Yun S

    2016-09-01

    The study of genomic regions that contain gene copies and structural variation is a major challenge in modern genomics. Unlike variation involving single nucleotide changes, data on the variation of copy number is difficult to collect and few tools exist for analyzing the variation between individuals. The immunoglobulin heavy variable (IGHV) locus, which plays an integral role in the adaptive immune response, is an example of a complex genomic region that varies in gene copy number. Lack of standard methods to genotype this region prevents it from being included in association studies and is holding back the growing field of antibody repertoire analysis. Here we develop a method that takes short reads from high-throughput sequencing and outputs a genetic profile of the IGHV locus with the read coverage depth and a putative nucleotide sequence for each operationally defined gene cluster. Our operationally defined gene clusters aim to address a major challenge in studying the IGHV locus: the high sequence similarity between gene segments in different genomic locations. Tests on simulated data demonstrate that our approach can accurately determine the presence or absence of a gene cluster from reads as short as 70 bp. More detailed resolution on the copy number of gene clusters can be obtained from read coverage depth using longer reads (e.g., ≥ 100 bp). Detail at the nucleotide resolution of single copy genes (genes present in one copy per haplotype) can be determined with 250 bp reads. For IGHV genes with more than one copy, accurate nucleotide-resolution reconstruction is currently beyond the means of our approach. When applied to a family of European ancestry, our pipeline outputs genotypes that are consistent with the family pedigree, confirms existing multigene variants and suggests new copy number variants. This study paves the way for analyzing population-level patterns of variation in IGHV gene clusters in larger diverse datasets and for quantitatively

  8. Clonal expansion of a globally disseminated lineage of Mycobacterium tuberculosis with low IS6110 copy numbers.

    PubMed

    Warren, R M; Victor, T C; Streicher, E M; Richardson, M; van der Spuy, G D; Johnson, R; Chihota, V N; Locht, C; Supply, P; van Helden, P D

    2004-12-01

    Knowledge of the clonal expansion of Mycobacterium tuberculosis and accurate identification of predominant evolutionary lineages in this species remain limited, especially with regard to low-IS6110-copy-number strains. In this study, 170 M. tuberculosis isolates with number tandem repeat (VNTR) typing. These analyses indicated that all but one of the isolates analyzed were members of principal genetic group 2 and of the same low-IS6110-copy-number lineage. The remaining isolate was a member of principal genetic group 1 and a different low-IS6110-copy-number lineage. Phylogenetic reconstruction suggests clonal expansion through sequential acquisition of additional IS6110 copies, expansion and contraction of VNTR sequences, and the deletion of specific direct-variable-repeat sequences. Furthermore, comparison of the genotypic data of 91 representative low-IS6110-copy-number isolates from Cape Town, other southern African regions, Europe, and the United States suggests that certain low-IS6110-copy-number strain spoligotypes and IS6110 fingerprints were acquired in the distant past. These clones have subsequently become widely disseminated and now play an important role in the global tuberculosis epidemic.

  9. 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.

  10. 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

  11. 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.

  12. Accurate, high-throughput typing of copy number variation using paralogue ratios from dispersed repeats

    PubMed Central

    Armour, John A. L.; Palla, Raquel; Zeeuwen, Patrick L. J. M.; den Heijer, Martin; Schalkwijk, Joost; Hollox, Edward J.

    2007-01-01

    Recent work has demonstrated an unexpected prevalence of copy number variation in the human genome, and has highlighted the part this variation may play in predisposition to common phenotypes. Some important genes vary in number over a high range (e.g. DEFB4, which commonly varies between two and seven copies), and have posed formidable technical challenges for accurate copy number typing, so that there are no simple, cheap, high-throughput approaches suitable for large-scale screening. We have developed a simple comparative PCR method based on dispersed repeat sequences, using a single pair of precisely designed primers to amplify products simultaneously from both test and reference loci, which are subsequently distinguished and quantified via internal sequence differences. We have validated the method for the measurement of copy number at DEFB4 by comparison of results from >800 DNA samples with copy number measurements by MAPH/REDVR, MLPA and array-CGH. The new Paralogue Ratio Test (PRT) method can require as little as 10 ng genomic DNA, appears to be comparable in accuracy to the other methods, and for the first time provides a rapid, simple and inexpensive method for copy number analysis, suitable for application to typing thousands of samples in large case-control association studies. PMID:17175532

  13. Telomere length is correlated with mitochondrial DNA copy number in intestinal, but not diffuse, gastric cancer.

    PubMed

    Jung, Soo-Jung; Cho, Ji-Hyoung; Park, Won-Jin; Heo, Yu-Ran; Lee, Jae-Ho

    2017-07-01

    A positive correlation between telomere length and mitochondrial DNA (mtDNA) copy number has previously been observed in healthy individuals, and in patients with psychiatric disorders. In the present study, telomere length and mtDNA copy number were evaluated in gastric cancer (GC) tissue samples. DNA was extracted from 109 GC samples (including 82 intestinal, and 27 diffuse cases), and the telomere length and mtDNA copy number were analyzed using a quantitative-polymerase chain reaction assay. The relative telomere length and mtDNA copy number in tumor tissue, as compared with in normal tissue, (mean ± standard deviation) in all GC samples were 11.48±1.14 and 14.86±1.35, respectively. Telomere length and mtDNA copy number were not identified as exhibiting clinical or prognostic value for GC. However, positive correlations between telomere length and mitochondrial DNA copy number were identified in GC (r=0.408, P<0.001) and in the adjacent normal mucosa (r=0.363; P<0.001). When stratified by Lauren classification, the correlation was identified in intestinal type GC samples (r=0.461; P<0.001), but not in diffuse type GC samples (r=0.225; P=0.260). This result indicated that loss of the correlation of telomeres and mitochondrial function may induce the initiation or progression of GC pathogenesis.

  14. CCL3L1 copy number, HIV load, and immune reconstitution in sub-Saharan Africans

    PubMed Central

    2013-01-01

    Background The role of copy number variation of the CCL3L1 gene, encoding MIP1α, in contributing to the host variation in susceptibility and response to HIV infection is controversial. Here we analyse a sub-Saharan African cohort from Tanzania and Ethiopia, two countries with a high prevalence of HIV-1 and a high co-morbidity of HIV with tuberculosis. Methods We use a form of quantitative PCR called the paralogue ratio test to determine CCL3L1 gene copy number in 1134 individuals and validate our copy number typing using array comparative genomic hybridisation and fiber-FISH. Results We find no significant association of CCL3L1 gene copy number with HIV load in antiretroviral-naïve patients prior to initiation of combination highly active anti-retroviral therapy. However, we find a significant association of low CCL3L1 gene copy number with improved immune reconstitution following initiation of highly active anti-retroviral therapy (p = 0.012), replicating a previous study. Conclusions Our work supports a role for CCL3L1 copy number in immune reconstitution following antiretroviral therapy in HIV, and suggests that the MIP1α -CCR5 axis might be targeted to aid immune reconstitution. PMID:24219137

  15. Genomic copy number alterations of primary and secondary metastasizing pleomorphic adenomas.

    PubMed

    Mariano, Fernanda Viviane; Gondak, Rogério de Oliveira; Martins, Antonio Santos; Coletta, Ricardo Della; Paes de Almeida, Oslei; Kowalski, Luiz Paulo; Krepischi, Ana Cristina Victorino; Altemani, Albina

    2015-09-01

    Metastasizing pleomorphic adenoma (MPA) is a rare tumour, and its mechanism of metastasis still is unknown. To date, there has been no study on MPA genomics. We analysed primary and secondary MPAs with array comparative genomic hybridization to identify somatic copy number alterations and affected genes. Tumour DNA samples from primary (parotid salivary gland) and secondary (scalp skin) MPAs were subjected to array comparative genomic hybridization investigation, and the data were analysed with NEXUS COPY NUMBER DISCOVERY. The primary MPA showed copy number losses affecting 3p22.2p14.3 and 19p13.3p123, and a complex pattern of four different deletions at chromosome 6. The 3p deletion encompassed several genes: CTNNB1, SETD2, BAP1, and PBRM1, among others. The secondary MPA showed a genomic profile similar to that of the primary MPA, with acquisition of additional copy number changes affecting 9p24.3p13.1 (loss), 19q11q13.43 (gain), and 22q11.1q13.33 (gain). Our findings indicated a clonal origin of the secondary MPA, as both tumours shared a common profile of genomic copy number alterations. Furthermore, we were able to detect in the primary tumour a specific pattern of copy number alterations that could explain the metastasizing characteristic, whereas the secondary MPA showed a more unbalanced genome. © 2015 John Wiley & Sons Ltd.

  16. Transcription reporters that shuttle cloned DNA between high-copy Escherichia coli plasmids and low-copy broad-host-range plasmids.

    PubMed

    Ouimet, M C; Marczynski, G T

    2000-09-01

    We describe and apply lacZ transcription reporter plasmids designed for both biochemical analyses requiring high DNA yield and physiological studies requiring low gene dosage. Standard DNA ligations are performed at seven unique restriction sites 5' to the lacZ gene on high-copy ColE1 plasmids suitable for double- or single-strand DNA sequencing. A divergent gusA transcription reporter is included and serves as an internal control. Rec(+) Escherichia coli cells readily shuttle DNA placed between gusA and lacZ by allelic exchange with pRK290-based plasmids that subsequently conjugate and replicate in most gram-negative bacteria. We applied this system to study Caulobacter crescentus cell cycle promoters directed by the CtrA response-regulator protein. Synthetic oligonucleotides were ligated to create altered CtrA binding sites and corresponding promoters with varied transcription strength. We also document the phenomenon of long-range promoter interference. A strong promoter can repress up to twofold the transcription from a divergent promoter located 100 bp away. However, the cell cycle timing of both promoters is not changed. Additional applications of our system and theoretical aspects of promoter organization are discussed. Copyright 2000 Academic Press.

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

    PubMed

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

    2017-05-01

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

  18. 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.

  19. 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.

  20. Software comparison for evaluating genomic copy number variation for Affymetrix 6.0 SNP array platform.

    PubMed

    Eckel-Passow, Jeanette E; Atkinson, Elizabeth J; Maharjan, Sooraj; Kardia, Sharon L R; de Andrade, Mariza

    2011-05-31

    Copy number data are routinely being extracted from genome-wide association study chips using a variety of software. We empirically evaluated and compared four freely-available software packages designed for Affymetrix SNP chips to estimate copy number: Affymetrix Power Tools (APT), Aroma.Affymetrix, PennCNV and CRLMM. Our evaluation used 1,418 GENOA samples that were genotyped on the Affymetrix Genome-Wide Human SNP Array 6.0. We compared bias and variance in the locus-level copy number data, the concordance amongst regions of copy number gains/deletions and the false-positive rate amongst deleted segments. APT had median locus-level copy numbers closest to a value of two, whereas PennCNV and Aroma.Affymetrix had the smallest variability associated with the median copy number. Of those evaluated, only PennCNV provides copy number specific quality-control metrics and identified 136 poor CNV samples. Regions of copy number variation (CNV) were detected using the hidden Markov models provided within PennCNV and CRLMM/VanillaIce. PennCNV detected more CNVs than CRLMM/VanillaIce; the median number of CNVs detected per sample was 39 and 30, respectively. PennCNV detected most of the regions that CRLMM/VanillaIce did as well as additional CNV regions. The median concordance between PennCNV and CRLMM/VanillaIce was 47.9% for duplications and 51.5% for deletions. The estimated false-positive rate associated with deletions was similar for PennCNV and CRLMM/VanillaIce. If the objective is to perform statistical tests on the locus-level copy number data, our empirical results suggest that PennCNV or Aroma.Affymetrix is optimal. If the objective is to perform statistical tests on the summarized segmented data then PennCNV would be preferred over CRLMM/VanillaIce. Specifically, PennCNV allows the analyst to estimate locus-level copy number, perform segmentation and evaluate CNV-specific quality-control metrics within a single software package. PennCNV has relatively small bias

  1. Statistical tools for transgene copy number estimation based on real-time PCR.

    PubMed

    Yuan, Joshua S; Burris, Jason; Stewart, Nathan R; Mentewab, Ayalew; Stewart, C Neal

    2007-11-01

    As compared with traditional transgene copy number detection technologies such as Southern blot analysis, real-time PCR provides a fast, inexpensive and high-throughput alternative. However, the real-time PCR based transgene copy number estimation tends to be ambiguous and subjective stemming from the lack of proper statistical analysis and data quality control to render a reliable estimation of copy number with a prediction value. Despite the recent progresses in statistical analysis of real-time PCR, few publications have integrated these advancements in real-time PCR based transgene copy number determination. Three experimental designs and four data quality control integrated statistical models are presented. For the first method, external calibration curves are established for the transgene based on serially-diluted templates. The Ct number from a control transgenic event and putative transgenic event are compared to derive the transgene copy number or zygosity estimation. Simple linear regression and two group T-test procedures were combined to model the data from this design. For the second experimental design, standard curves were generated for both an internal reference gene and the transgene, and the copy number of transgene was compared with that of internal reference gene. Multiple regression models and ANOVA models can be employed to analyze the data and perform quality control for this approach. In the third experimental design, transgene copy number is compared with reference gene without a standard curve, but rather, is based directly on fluorescence data. Two different multiple regression models were proposed to analyze the data based on two different approaches of amplification efficiency integration. Our results highlight the importance of proper statistical treatment and quality control integration in real-time PCR-based transgene copy number determination. These statistical methods allow the real-time PCR-based transgene copy number estimation

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

    PubMed

    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

    2017-02-08

    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.

  3. 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

  4. 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.

  5. A prospective study of mitochondrial DNA copy number and the risk of prostate cancer.

    PubMed

    Moore, Amy; Lan, Qing; Hofmann, Jonathan N; Liu, Chin-San; Cheng, Wen-Ling; Lin, Ta-Tsung; Berndt, Sonja I

    2017-06-01

    Evidence suggests that mitochondrial DNA (mtDNA) copy number increases in response to DNA damage. Increased mtDNA copy number has been observed in prostate cancer (PCa) cells, suggesting a role in PCa development, but this association has not yet been investigated prospectively. We conducted a nested case-control study (793 cases and 790 controls) of men randomized to the screening arm of the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO) to evaluate the association between pre-diagnosis mtDNA copy number, measured in peripheral blood leukocytes, and the risk of PCa. We used logistic regression to estimate odds ratios (OR) and 95% confidence intervals (CI) and polytomous logistic regression to analyze differences in associations by non-aggressive (Stage I/II AND Gleason grade < 8) or aggressive (Stage III/IV OR Gleason grade ≥ 8) PCa. Although mtDNA copy number was not significantly associated with PCa risk overall (OR 1.23, 95% CI 0.97-1.55, p = 0.089), increasing mtDNA copy number was associated with an increased risk of non-aggressive PCa (OR 1.29, 95% CI 1.01-1.65, p = 0.044) compared to controls. No association was observed with aggressive PCa (OR 1.02, 95% CI 0.64-1.63, p = 0.933). Higher mtDNA copy number was also associated with increased PSA levels among controls (p = 0.014). These results suggest that alterations in mtDNA copy number may reflect disruption of the normal prostate glandular architecture seen in early-stage disease, as opposed to reflecting the large number of tumor cells seen with advanced PCa.

  6. HaplotypeCN: Copy Number Haplotype Inference with Hidden Markov Model and Localized Haplotype Clustering

    PubMed Central

    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. PMID:24849202

  7. 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.

  8. 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

  9. Quantification of Plasmid Copy Number with Single Colour Droplet Digital PCR.

    PubMed

    Plotka, Magdalena; Wozniak, Mateusz; Kaczorowski, Tadeusz

    2017-01-01

    Bacteria can be considered as biological nanofactories that manufacture a cornucopia of bioproducts most notably recombinant proteins. As such, they must perfectly match with appropriate plasmid vectors to ensure successful overexpression of target genes. Among many parameters that correlate positively with protein productivity plasmid copy number plays pivotal role. Therefore, development of new and more accurate methods to assess this critical parameter will result in optimization of expression of plasmid-encoded genes. In this study, we present a simple and highly accurate method for quantifying plasmid copy number utilizing an EvaGreen single colour, droplet digital PCR. We demonstrate the effectiveness of this method by examining the copy number of the pBR322 vector within Escherichia coli DH5α cells. The obtained results were successfully validated by real-time PCR. However, we observed a strong dependency of the plasmid copy number on the method chosen for isolation of the total DNA. We found that application of silica-membrane-based columns for DNA purification or DNA isolation with use of bead-beating, a mechanical cell disruption lead to determination of an average of 20.5 or 7.3 plasmid copies per chromosome, respectively. We found that recovery of the chromosomal DNA from purification columns was less efficient than plasmid DNA (46.5 ± 1.9% and 87.4 ± 5.5%, respectively) which may lead to observed differences in plasmid copy number. Besides, the plasmid copy number variations dependent on DNA template isolation method, we found that droplet digital PCR is a very convenient method for measuring bacterial plasmid content. Careful determination of plasmid copy number is essential for better understanding and optimization of recombinant proteins production process. Droplet digital PCR is a very precise method that allows performing thousands of individual PCR reactions in a single tube. The ddPCR does not depend on running standard curves and is a

  10. Prognostic significance of centromere 17 copy number gain in breast cancer depends on breast cancer subtype.

    PubMed

    Lee, Kyuongyul; Jang, Min Hye; Chung, Yul Ri; Lee, Yangkyu; Kang, Eunyoung; Kim, Sung-Won; Kim, Yu Jung; Kim, Jee Hyun; Kim, In Ah; Park, So Yeon

    2017-03-01

    Increased copy number of chromosome enumeration probe (CEP) targeting centromere 17 is frequently encountered during HER2 in situ hybridization (ISH) in breast cancer. The aim of this study was to clarify the clinicopathologic significance of CEP17 copy number gain in a relatively large series of breast cancer patients. We analyzed 945 cases of invasive breast cancers whose HER2 fluorescence ISH reports were available from 2004 to 2011 at a single institution and evaluated the association of CEP17 copy number gain with clinicopathologic features of tumors and patient survival. We detected 186 (19.7%) cases of CEP17 copy number gain (CEP17≥3.0) among 945 invasive breast cancers. In survival analysis, CEP17 copy number gain was not associated with disease-free survival of the patients in the whole group. Nonetheless, it was found to be an independent adverse prognostic factor in the HER2-negative group but not in the HER2-positive group. In further subgroup analyses, CEP17 copy number gain was revealed as an independent poor prognostic factor in HER2-negative and hormone receptor-positive breast cancers, and it was associated with aggressive histologic variables including high T stage, high histologic grade, lymphovascular invasion, p53 overexpression, and high Ki-67 proliferative index. In conclusion, we found that elevated CEP17 count can serve as a prognostic marker in luminal/HER2-negative subtype of invasive breast cancer. We advocate the use of the dual-colored fluorescence ISH using CEP17 rather than the single-colored one because it gives additional valuable information on CEP17 copy number alterations. Copyright © 2016 Elsevier Inc. All rights reserved.

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

    PubMed

    Chang, Yu Kyung; Kim, Da Eun; Cho, Soo Hyun; Kim, Jung-Ha

    2016-11-01

    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. 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. 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. Regular exercise is associated with greater leukocyte mtDNA copy number in postmenopausal women.

  12. Modulation of Mitochondrial DNA Copy Number to Induce Hepatocytic Differentiation of Human Amniotic Epithelial Cells.

    PubMed

    Vaghjiani, Vijesh; Cain, Jason E; Lee, William; Vaithilingam, Vijayaganapathy; Tuch, Bernard E; St John, Justin C

    2017-09-05

    Mitochondrial deoxyribonucleic acid (mtDNA) copy number is tightly regulated during pluripotency and differentiation. There is increased demand of cellular adenosine triphosphate (ATP) during differentiation for energy-intensive cell types such as hepatocytes and neurons to meet the cell's functional requirements. During hepatocyte differentiation, mtDNA copy number should be synchronously increased to generate sufficient ATP through oxidative phosphorylation. Unlike bone marrow mesenchymal cells, mtDNA copy number failed to increase by 28 days of differentiation of human amniotic epithelial cells (hAEC) into hepatocyte-like cells (HLC) despite their expression of some end-stage hepatic markers. This was due to higher levels of DNA methylation at exon 2 of POLGA, the mtDNA-specific replication factor. Treatment with a DNA demethylation agent, 5-azacytidine, resulted in increased mtDNA copy number, reduced DNA methylation at exon 2 of POLGA, and reduced hepatic gene expression. Depletion of mtDNA followed by subsequent differentiation did not increase mtDNA copy number, but reduced DNA methylation at exon 2 of POLGA and increased expression of hepatic and pluripotency genes. We encapsulated hAEC in barium alginate microcapsules and subsequently differentiated them into HLC. Encapsulation resulted in no net increase of mtDNA copy number but a significant reduction in DNA methylation of POLGA. RNAseq analysis showed that differentiated HLC express hepatocyte-specific genes but also increased expression of inflammatory interferon genes. Differentiation in encapsulated cells showed suppression of inflammatory genes as well as increased expression of genes associated with hepatocyte function pathways and networks. This study demonstrates that an increase in classical hepatic gene expression can be achieved in HLC through encapsulation, although they fail to effectively regulate mtDNA copy number.

  13. 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

  14. 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.

  15. 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

  16. Simple screening method for copy number variations associated with physical features.

    PubMed

    Ueki, Misuzu; Takeshita, Haruo; Fujihara, Junko; Kimura-Kataoka, Kaori; Iida, Reiko; Yasuda, Toshihiro

    2017-03-01

    Recent studies of copy number variations (CNVs) associated with physical features, such as body mass index, body height or bone length, have suggested that such CNVs could serve as markers in forensic cases involving unidentified individuals. However, the process of cataloging CNVs has been slow because of the cumbersome nature and low reliability of the procedures involved. Here we describe a simple quantitative real-time PCR (Q-PCR) method for screening of medicolegally useful CNVs, which does not require reference DNA with known copy number. The first step is to prepare a chimeric plasmid vector including one copy each of the single-copy gene-specific sequence as the internal standard, and the target CNV-specific sequence. To assess the validity of this new method, we analyzed CNVs in the LTBP1 and ETV6 gene regions, both of which are candidate CNVs associated with body height. The PCR efficiencies for the single-copy (reference) gene and the target CNV were similar, indicating that quantitation was reliable. Furthermore, simulated analysis of the LTBP1 CNV using mock samples prepared by mixing vectors in varying proportions showed that this analytical method allowed correct determination of the LTBP1 copy number. These results demonstrated that our simple method has considerable potential for screening of trait-related CNVs that would be useful for forensic casework. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. 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

  18. 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

  19. 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.

  20. Trans-complementable copy-number mutants of plasmid ColE1.

    PubMed

    Twigg, A J; Sherratt, D

    1980-01-10

    Plasmid ColE1, like many other small non-conjugative plasmids, is present in multiple copies (about 15 per chromsome equivalent) in Escherichia coli cells. Because of their high copy number, the replication of such plasmids has been described as 'relaxed', even though there is good evidence that it is strictly controlled: ColE1 derivatives have characteristic but different copy numbers and ColE1 copy-number mutants have been characterised. No plasmid-specified protein is essential for the replication of ColE1 and related plasmids, as extensive replication can occur in chloramphenicol-treated cells, in plasmid-free chloramphenicol-treated cells transfected with a hybrid ColE1/phage replicon and in vitro in extracts derived from plasmid-free cells. Nevertheless, it is possible that a plasmid-specified protein is involved in ColE1 replication control in viable cells. Here we show that deletion of a given non-essential region from ColE1-like plasmids results in a raised copy number. Such plasmids are stably maintained and have their copy number returned to normal when a complementing plasmid is present in the same cell, indicating that a plasmid-specified diffusible gene product regulates the plasmid content of ColE1-containing cells. Deletion of the equivalent region from the cloning vector pBR322 gives a derivative which has a raised copy number and which has also lost its origin for conjugal transfer; unlike pBR322, it cannot be mobilised.

  1. 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.

  2. 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

  3. 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

  4. Copy number variations in urine cell free DNA as biomarkers in advanced prostate cancer.

    PubMed

    Xia, Yun; Huang, Chiang-Ching; Dittmar, Rachel; Du, Meijun; Wang, Yuan; Liu, Hongyan; Shenoy, Niraj; Wang, Liang; Kohli, Manish

    2016-06-14

    Genetic profiling of urine cell free DNA (cfDNA) has not been evaluated in advanced prostate cancer. We performed whole genome sequencing of urine cfDNAs to identify tumor-associated copy number variations in urine before and after initiating androgen deprivation therapy in HSPC stage and docetaxel chemotherapy in CRPC stage. A log2 ratio-based copy number analysis detected common genomic abnormalities in prostate cancer including AR amplification in 5/10 CRPC patients. Other abnormalities identified included TMPRSS2-ERG fusion, PTEN gene deletion, NOTCH1 locus amplification along with genomic amplifications at 8q24.3, 9q34.3, 11p15.5 and 14q11.2, and deletions at 4q35.2, 5q31.3, 7q36.3, 12q24.33, and 16p11.2. By comparing copy number between pre- and post-treatment, we found significant copy number changes in 34 genomic loci. To estimate the somatic tumor DNA fraction in urine cfDNAs, we developed a Urine Genomic Abnormality (UGA) score algorithm that summed the top ten most significant segments with copy number changes. The UGA scores correlated with tumor burden and the change in UGA score after stage-specific therapies reflected disease progression status and overall survival. The study demonstrates the potential clinical utility of urine cfDNAs in predicting treatment response and monitoring disease progression.

  5. 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.

  6. 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

  7. Mitochondrial DNA copy number, but not haplogroup is associated with keratoconus in Han Chinese population.

    PubMed

    Hao, Xiao-Dan; Chen, Peng; Wang, Ye; Li, Su-Xia; Xie, Li-Xin

    2015-03-01

    Oxidative stress may play a role in the pathogenesis of keratoconus (KC). Mitochondrial DNA (mtDNA) is closely related to mitochondrion function, and variations may affect the generation of reactive oxygen species (ROS) and be involved in the pathogenesis of KC. To test whether mtDNA background and copy number confer genetic susceptibility to KC in the Han Chinese population, we performed this association study. We analyzed mtDNA sequence variations in 210 KC patients and 309 matched individuals from China, and classified each subject by haplogroup. Mitochondrial DNA copy number was measured in a subset of these subjects (193 patients and 103 controls). Comparison of matrilineal components of the cases and control populations revealed no significant difference. However, measurement of mtDNA copy number showed that KC patients had significantly lower mtDNA copy numbers than controls (P = 0.0002), even when age, gender, and mtDNA background were considered. Our results suggest that mtDNA copy number, but not haplogroup, is associated with keratoconus, and may contribute to its pathogenesis. Copyright © 2015 Elsevier Ltd. All rights reserved.

  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. Low-level copy number changes of MYC genes have a prognostic impact in medulloblastoma.

    PubMed

    Zitterbart, Karel; Filkova, Hana; Tomasikova, Lenka; Necesalova, Eva; Zambo, Iva; Kantorova, Dagmar; Slamova, Iva; Vranova, Vladimira; Zezulkova, Dita; Pesakova, Martina; Pavelka, Zdenek; Veselska, Renata; Kuglik, Petr; Sterba, Jaroslav

    2011-03-01

    High-level amplifications of MYC genes are associated with poor outcomes in childhood medulloblastoma (MB). However, the occurrence of MYCN and MYCC copy number increases below the intense amplification pattern is rarely reported, and its clinical impact has not yet been determined. Here, we describe this phenomenon and its prognostic significance in a cohort of 29 MB patients. Using interphase fluorescence in situ hybridization (I-FISH), low-level copy number alterations, i.e. gain of MYCN, were shown in 5/27 (19%) samples, whereas amplification was revealed in only 1/27 (4%) samples. MYCC gain was revealed in 6/29 (21%) MB, while amplification was disclosed in only 2/29 (7%). Hyperploidy and co-incidence of gains in both MYC loci were frequently observed in samples with copy number aberrations. Survival analysis has clearly shown that MYC copy number increases are associated with lowered event-free survival and overall survival in MB. In the case of MYCN, this negative correlation was statistically significant. We conclude that limited numerical alterations in loci 2p24 (MYCN) and 8q24 (MYCC), as assessed by I-FISH, are present in MB with a higher frequency than high-level amplifications. Poor prognoses were observed in patients with copy number increases in MYC genes. Our data illustrate the importance of further investigations in multicenter trials to better refine the emerging genomic-based prognostic stratification in MB.

  10. ALK gene copy number in lung cancer: Unspecific polyploidy versus specific amplification visible as double minutes.

    PubMed

    Caliò, Anna; Bria, Emilio; Pilotto, Sara; Gilioli, Eliana; Nottegar, Alessia; Eccher, Albino; Cima, Luca; Santo, Antonio; Pedron, Serena; Turri, Giona; Knuutila, Sakari; Chilosi, Marco; Vanzo, Francesca; Bogina, Giuseppe; Terzi, Alberto; Tortora, Giampaolo; Scarpa, Aldo; Loda, Massimo; Martignoni, Guido; Brunelli, Matteo

    2017-01-01

    Gains of a gene due to DNA polyploidy versus amplification of the specific locus are distinct molecular alterations in tumors. We quantified copy number gains of ALK gene due to unspecific polyploidy versus amplifications of the specific locus in a series of non-small cell lung cancers. The locus specific ALK copy (LSI) number status was evaluated in 205 cases by FISH. Ratio LSI ALK copy number corrected for control probes CEP2, CEP3 and CEP17 (CEPs) was scored. Amplification of the specific ALK locus was defined when ratio set to ≥ 2 while polyploidy was interpreted when the increase in gene copy resulted < 2 in ratio (LSI/control CEPs). Twenty one cases (10.2%) showed ≥ 8 ALK signals, 68 cases (33.2%) 3-7 signals and 116 cases (56.6%) a mean of 2 signals. Only 2/21 cases of the cohort harboring ≥ 8 signals showed a ratio ≥ 2 after CEPs correction interpretable as amplified, showing numerous doubled fluorescent spots. All the remaining cases showed a mirrored number of fluorescent spots per each CEPs, interpretable as polyploidy. We detected a high prevalence of ALK gene copy number usually due to polyploidy rather than ALK locus amplification, the latter visible prevalently as double minutes.

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

    PubMed

    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.

  12. 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

  13. 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

  14. Probe-free allele-specific copy number detection and analysis of tumors.

    PubMed

    Zhu, Ailin; Guan, Xiaowei; Gu, Xinbin; Xie, Guiqin

    2016-03-15

    Cancer development and progression frequently involve nucleotide mutations as well as amplifications and deletions of genomic segments. Quantification of allele-specific copy number is an important step in characterizing tumor genomes for precision medicine. Despite advances in approaches to high-throughput genomic DNA analysis, inexpensive and simple methods for analyzing complex nucleotide and copy number variants are still needed. Real-time polymerase chain reaction (PCR) methods for discovering and genotyping single nucleotide polymorphisms are becoming increasingly important in genetic analysis. In this study, we describe a simple, single-tube, probe-free method that combines SYBR Green I-based quantitative real-time PCR and quantitative melting curve analysis both to detect specific nucleotide variants and to quantify allele-specific copy number variants of tumors. The approach is based on the quantification of the targets of interest and the relative abundance of two alleles in a single tube. The specificity, sensitivity, and utility of the assay were demonstrated in detecting allele-specific copy number changes critical for carcinogenesis and therapeutic intervention. Our approach would be useful for allele-specific copy number analysis or precise genotyping. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Cardiometabolic phenotypes and mitochondrial DNA copy number in two cohorts of UK women.

    PubMed

    Guyatt, Anna L; Burrows, Kimberley; Guthrie, Philip A I; Ring, Sue; McArdle, Wendy; Day, Ian N M; Ascione, Raimondo; Lawlor, Debbie A; Gaunt, Tom R; Rodriguez, Santiago

    2017-08-15

    The mitochondrial genome is present at variable copy number between individuals. Mitochondria are vulnerable to oxidative stress, and their dysfunction may be associated with cardiovascular disease. The association of mitochondrial DNA copy number with cardiometabolic risk factors (lipids, glycaemic traits, inflammatory markers, anthropometry and blood pressure) was assessed in two independent cohorts of European origin women, one in whom outcomes were measured at mean (SD) age 30 (4.3) years (N=2278) and the second at 69.4 (5.5) years (N=2872). Mitochondrial DNA copy number was assayed by quantitative polymerase chain reaction. Associations were adjusted for smoking, sociodemographic status, laboratory factors and white cell traits. Out of a total of 12 outcomes assessed in both cohorts, mitochondrial DNA copy number showed little or no association with the majority (point estimates were close to zero and nearly all p-values were >0.01). The strongest evidence was for an inverse association in the older cohort with insulin (standardised beta [95%CI]: -0.06, [-0.098, -0.022], p=0.002), but this association did not replicate in the younger cohort. Our findings do not provide support for variation in mitochondrial DNA copy number having an important impact on cardio-metabolic risk factors in European origin women. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  16. DiNAMIC: a method to identify recurrent DNA copy number aberrations in tumors

    PubMed Central

    Walter, Vonn; Nobel, Andrew B.; Wright, Fred A.

    2011-01-01

    Motivation: DNA copy number gains and losses are commonly found in tumor tissue, and some of these aberrations play a role in tumor genesis and development. Although high resolution DNA copy number data can be obtained using array-based techniques, no single method is widely used to distinguish between recurrent and sporadic copy number aberrations. Results: Here we introduce Discovering Copy Number Aberrations Manifested In Cancer (DiNAMIC), a novel method for assessing the statistical significance of recurrent copy number aberrations. In contrast to competing procedures, the testing procedure underlying DiNAMIC is carefully motivated, and employs a novel cyclic permutation scheme. Extensive simulation studies show that DiNAMIC controls false positive discoveries in a variety of realistic scenarios. We use DiNAMIC to analyze two publicly available tumor datasets, and our results show that DiNAMIC detects multiple loci that have biological relevance. Availability: Source code implemented in R, as well as text files containing examples and sample datasets are available at http://www.bios.unc.edu/research/genomic_software/DiNAMIC. Contact: vwalter@email.unc.edu; fwright@bios.unc.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:21183584

  17. 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

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

    PubMed

    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

    2015-12-01

    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 data set 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.

  19. 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.

  20. 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

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

    PubMed

    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.

  2. Optimal design of oligonucleotide microarrays for measurement of DNA copy-number.

    PubMed

    Sharp, Andrew J; Itsara, Andy; Cheng, Ze; Alkan, Can; Schwartz, Stuart; Eichler, Evan E

    2007-11-15

    Copy-number variants (CNVs) occur frequently within the human genome, and may be associated with many human phenotypes. If disease association studies of CNVs are to be performed routinely, it is essential that the copy-number status be accurately genotyped. We systematically assessed the dynamic range response of an oligonucleotide microarray platform to accurately predict copy-number in a set of seven patients who had previously been shown to carry between 1 and 6 copies of an approximately 4 Mb region of 15q12.2-q13.1. We identify probe uniqueness, probe length, uniformity of probe melting temperature, overlap with SNPs and common repeats (particularly Alu elements) and guanine homopolymer content as parameters that significantly affect probe performance. Further, we prove the influence of these criteria on array performance by using these parameters to prospectively filter data from a second array design covering an independent genomic region and observing significant improvements in data quality. The informed selection of probes which have superior performance characteristics allows the prospective design of oligonucleotide arrays which show increased sensitivity and specificity compared with current designs. Although based on the analysis of data from comparative genomic hybridization experiments, we anticipate that our results are relevant to the design of improved oligonucleotide arrays for high-throughput copy-number genotyping of complex regions of the human genome.

  3. BRAF, GNAQ, and GNA11 mutations and copy number in pediatric low-grade glioma.

    PubMed

    Laviv, Yosef; Toledano, Helen; Michowiz, Shalom; Dratviman-Storobinsky, Olga; Turm, Yuval; Fichman-Horn, Suzana; Kagnovski, Ella; Goldenberg-Cohen, Nitza

    2012-01-01

    Fifty-two samples of pediatric low-grade glioma (48 primary, 4 recurrent) were analyzed for BRAF copy number variation (digital PCR analysis, CopyCaller) and point mutations of BRAF V600E, and exon 5 Q209 in GNAQ, and GNA11, using the MALDI-TOF mass spectrometer with validation by direct sequencing. An increased BRAF copy number was found in 18/47 primary samples tested; 15 of them (83.3%) were pilocytic astrocytomas. A BRAF mutation was found in 3/48 primary tumors, all with a normal BRAF copy number and no GNAQ mutation. One sample had a GNAQ209 mutation (Q209P626) with a normal BRAF gene; none of the tumors had a GNA11Q209 mutation. Recurrent or progressive tumors, analyzed in four patients, had the same molecular genotype as their primary. Increased BRAF copy number and activating BRAF mutations may be involved in the development of low-grade glioma via overactivation of the Ras/Raf pathway. This is the first report of a mutation in GNAQ209 in pediatric low-grade glioma. Understanding the molecular mechanisms underlying glioma initiation and growth may assist in the development of targeted therapies.

  4. 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

  5. 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.

  6. Pyruvate Kinase and Fcγ Receptor Gene Copy Numbers Associated With Malaria Phenotypes.

    PubMed

    Faik, Imad; van Tong, Hoang; Lell, Bertrand; Meyer, Christian G; Kremsner, Peter G; Velavan, Thirumalaisamy P

    2017-07-15

    Genetic factors are associated with susceptibility to many infectious diseases and may be determinants of clinical progression. Gene copy number variation (CNV) has been shown to be associated with phenotypes of numerous diseases, including malaria. We quantified gene copy numbers of the pyruvate kinase, liver, and red blood cell (PKLR) gene as well as of the Fcγ receptor 2A and Fcγ receptor 2C (FCGR2A, FCGR2C) and Fcγ receptor 3 (FCGR3) genes using real-time quantitative polymerase chain reaction (RT-qPCR) assays in Gabonese children with severe (n = 184) or and mild (n = 189) malaria and in healthy Gabonese and white individuals (n = 76 each). The means of PKLR, FCGR2A, FCGR2C, and FCGR3 copy numbers were significantly higher among children with severe malaria compared to those with mild malaria (P < .002), indicating that a surplus of copies of those genes is significantly associated with malaria severity. Copy numbers of the FCGR2A and FCGR2C genes were significantly lower (P = .005) in Gabonese individuals compared with white individuals. In conclusion, CNV of the PKLR, FCGR2A, FCGR2C, and FCGR3 genes is associated with malaria severity, and our results provide evidence for a role of CNV in host responses to malaria. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

  7. 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.

  8. 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

  9. Copy number alterations of the polycomb gene BMI1 in gliomas.

    PubMed

    Häyry, Valtteri; Tanner, Minna; Blom, Tea; Tynninen, Olli; Roselli, Annariikka; Ollikainen, Miina; Sariola, Hannu; Wartiovaara, Kirmo; Nupponen, Nina N

    2008-07-01

    Gliomas are heterogeneous tumours that grow in an uninhibited fashion, and these brain tumour cells share numerous characteristics with neural stem cells. The BMI1 gene encodes a component of the polycomb protein complex regulating epigenetically gene activity via histone modification. It functions for instance during the development of the central nervous system and maturation of neural cells. BMI-1 protein expression is deregulated in several forms of cancer and gene amplification has been identified in mantle cell lymphomas. Since BMI1 is located at chromosome 10p, a region implicated frequently in brain tumourigenesis, we investigated the genetic status and the corresponding expression patterns of BMI1 in a series of 100 low- and high-grade primary and recurrent gliomas. Chromogenic in situ hybridisation (CISH) with probes directed against BMI1 at 10p13 and the centromere of chromosome 10 was used in the analyses. Of all gliomas, 59% demonstrated aberrant copy numbers of BMI1. Deletions of the BMI1 locus were found in most types of tumours, and in a univariate survival analysis these cases had poor prognosis. Increased copy numbers of the BMI1 locus (3-5 copies) were found in all histological types, especially in high-grade astrocytomas. No difference in prognosis between cases with normal copy numbers and cases with increased copy numbers could be observed. This data suggests that BMI1 gene is aberrant at the chromosomal level in a subset of gliomas, and possibly contributes to brain tumour pathogenesis.

  10. 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

  11. High-resolution analysis of DNA copy number alterations in patients with isolated sporadic keratoconus

    PubMed Central

    Hellani, Ali M.; Al Mansouri, Sameer M.; Kalantan, Hatem; Al-Muammar, Abdulrahman M.

    2011-01-01

    Purpose To determine whether patients with sporadic, non-familial keratoconus and no pathogenic mutations in the visual system homeobox 1 (VSX1) gene have evidence of chromosomal copy number alterations. Methods Twenty Saudi Arabian patients with isolated keratoconus, no family history of the disease and no mutations in VSX1 were recruited. Additionally, 10 ethnically-matched healthy controls were also recruited for this study. We screened patients for chromosomal copy number aberrations using the Agilent Human Genome CGH 244A Oligo Microarray Chip. Results None of the keratoconus patients screened had evidence of chromosomal copy number alterations when compared to normal ethnically matched controls. Conclusions Chromosomal deletions and/or duplications were not detected in any of the patients tested here. Other chromosomal imbalances such as translocations, inversions, and some ploidies cannot be detected by current array CGH technology and other nuclear genetic or epigenetic factors cannot be excluded as a possible contributing factor to keratoconus pathogenesis. PMID:21528002

  12. High-resolution analysis of DNA copy number alterations in patients with isolated sporadic keratoconus.

    PubMed

    Abu-Amero, Khaled K; Hellani, Ali M; Al Mansouri, Sameer M; Kalantan, Hatem; Al-Muammar, Abdulrahman M

    2011-03-30

    To determine whether patients with sporadic, non-familial keratoconus and no pathogenic mutations in the visual system homeobox 1 (VSX1) gene have evidence of chromosomal copy number alterations. Twenty Saudi Arabian patients with isolated keratoconus, no family history of the disease and no mutations in VSX1 were recruited. Additionally, 10 ethnically-matched healthy controls were also recruited for this study. We screened patients for chromosomal copy number aberrations using the Agilent Human Genome CGH 244A Oligo Microarray Chip. None of the keratoconus patients screened had evidence of chromosomal copy number alterations when compared to normal ethnically matched controls. Chromosomal deletions and/or duplications were not detected in any of the patients tested here. Other chromosomal imbalances such as translocations, inversions, and some ploidies cannot be detected by current array CGH technology and other nuclear genetic or epigenetic factors cannot be excluded as a possible contributing factor to keratoconus pathogenesis.

  13. 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.

  14. 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

  15. 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

  16. Detection of DNA copy number alterations in cancer by array comparative genomic hybridization.

    PubMed

    Michels, Evi; De Preter, Katleen; Van Roy, Nadine; Speleman, Frank

    2007-09-01

    Over the past few years, various reliable platforms for high-resolution detection of DNA copy number changes have become widely available. Together with optimized protocols for labeling and hybridization and algorithms for data analysis and representation, this has lead to a rapid increase in the application of this technology in the study of copy number variation in the human genome in normal cells and copy number imbalances in genetic diseases, including cancer. In this review, we briefly discuss specific technical issues relevant for array comparative genomic hybridization analysis in cancer tissues. We specifically focus on recent successes of array comparative genomic hybridization technology in the progress of our understanding of oncogenesis in a variety of cancer types. A third section highlights the potential of sensitive genome-wide detection of patterns of DNA imbalances or molecular portraits for class discovery and therapeutic stratification.

  17. 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

  18. 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

  19. 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

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

    PubMed

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

    2015-01-01

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

  1. Loss of the Association between Telomere Length and Mitochondrial DNA Copy Number Contribute to Colorectal Carcinogenesis.

    PubMed

    Lee, Hyunsu; Cho, Ji-Hyoung; Park, Won-Jin; Jung, Soo-Jung; Choi, In-Jang; Lee, Jae-Ho

    2017-05-09

    Positive association between telomere length and mitochondrial DNA (mtDNA) copy number were introduced in healthy and patients with psychiatric disorder. Based on frequent genetic changes of telomere and mitochondria in colorectal carcinomas (CRC), we studied their clinical characteristics and their association in colorectal carcinogenesis. DNA was extracted from 109 CRCs, 64 colorectal tubular adenomas (TAs), and 28 serrated polyps (SPs), and then, telomere length and mtDNA copy number were analyzed in these legions by using a real-time PCR assay. Telomere length and mtDNA copy number (mean ± S.D) in CRCs was 1.87 ± 1.52 and 1.61 ± 1.37, respectively. In TAs and SPs, relative mtDNA copy number was 0.92 ± 0.71 and 1.84 ± 1.06, respectively, shoing statistical difference (p = 0.017). However, telomere length was similar in these precancerous legions. Telomere length and mtDNA copy number did not show clinical and prognostic values in CRCs, however, positive correlation between telomere length and mitochondrial DNA copy number were found in CRC (r = 0.408, p < 0.001). However, this association was not shown in precancerous lesions (r = -0.031, p = 0.765). This result suggests that loss of co-regulation between telomeres and mitochondrial function may induce the initiation or play a role as trigger factor of colorectal carcinogenesis.

  2. 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

  3. 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.

  4. 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/.

  5. Allele-specific copy number analysis of tumor samples with aneuploidy and tumor heterogeneity

    PubMed Central

    2011-01-01

    We describe a bioinformatic tool, Tumor Aberration Prediction Suite (TAPS), for the identification of allele-specific copy numbers in tumor samples using data from Affymetrix SNP arrays. It includes detailed visualization of genomic segment characteristics and iterative pattern recognition for copy number identification, and does not require patient-matched normal samples. TAPS can be used to identify chromosomal aberrations with high sensitivity even when the proportion of tumor cells is as low as 30%. Analysis of cancer samples indicates that TAPS is well suited to investigate samples with aneuploidy and tumor heterogeneity, which is commonly found in many types of solid tumors. PMID:22023820

  6. 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.

  7. 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

  8. Reliable transgene-independent method for determining Sleeping Beauty transposon copy numbers

    PubMed Central

    2011-01-01

    Background The transposon-based gene delivery technique is emerging as a method of choice for gene therapy. The Sleeping Beauty (SB) system has become one of the most favored methods, because of its efficiency and its random integration profile. Copy-number determination of the delivered transgene is a crucial task, but a universal method for measuring this is lacking. In this paper, we show that a real-time quantitative PCR-based, transgene-independent (qPCR-TI) method is able to determine SB transposon copy numbers regardless of the genetic cargo. Results We designed a specific PCR assay to amplify the left inverted repeat-direct repeat region of SB, and used it together with the single-copy control gene RPPH1 and a reference genomic DNA of known copy number. The qPCR-TI method allowed rapid and accurate determination of SB transposon copy numbers in various cell types, including human embryonic stem cells. We also found that this sensitive, rapid, highly reproducible and non-radioactive method is just as accurate and reliable as the widely used blotting techniques or the transposon display method. Because the assay is specific for the inverted repeat region of the transposon, it could be used in any system where the SB transposon is the genetic vehicle. Conclusions We have developed a transgene-independent method to determine copy numbers of transgenes delivered by the SB transposon system. The technique is based on a quantitative real-time PCR detection method, offering a sensitive, non-radioactive, rapid and accurate approach, which has a potential to be used for gene therapy. PMID:21371313

  9. Effects of reduced chloroplast gene copy number on chloroplast gene expression in maize.

    PubMed

    Udy, Dylan B; Belcher, Susan; Williams-Carrier, Rosalind; Gualberto, José M; Barkan, Alice

    2012-11-01

    Chloroplasts and other members of the plastid organelle family contain a small genome of bacterial ancestry. Young chloroplasts contain hundreds of genome copies, but the functional significance of this high genome copy number has been unclear. We describe molecular phenotypes associated with mutations in a nuclear gene in maize (Zea mays), white2 (w2), encoding a predicted organellar DNA polymerase. Weak and strong mutant alleles cause a moderate (approximately 5-fold) and severe (approximately 100-fold) decrease in plastid DNA copy number, respectively, as assayed by quantitative PCR and Southern-blot hybridization of leaf DNA. Both alleles condition a decrease in most chloroplast RNAs, with the magnitude of the RNA deficiencies roughly paralleling that of the DNA deficiency. However, some RNAs are more sensitive to a decrease in genome copy number than others. The rpoB messenger RNA (mRNA) exhibited a unique response, accumulating to dramatically elevated levels in response to a moderate reduction in plastid DNA. Subunits of photosynthetic enzyme complexes were reduced more severely than were plastid mRNAs, possibly because of impaired translation resulting from limiting ribosomal RNA, transfer RNA, and ribosomal protein mRNA. These results indicate that chloroplast genome copy number is a limiting factor for the expression of a subset of chloroplast genes in maize. Whereas in Arabidopsis (Arabidopsis thaliana) a pair of orthologous genes function redundantly to catalyze DNA replication in both mitochondria and chloroplasts, the w2 gene is responsible for virtually all chloroplast DNA replication in maize. Mitochondrial DNA copy number was reduced approximately 2-fold in mutants harboring strong w2 alleles, suggesting that w2 also contributes to mitochondrial DNA replication.

  10. 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. © 2016 Stichting International Foundation for Animal Genetics.

  11. 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

  12. 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

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. Saccharomyces cerevisiae Shuttle vectors.

    PubMed

    Gnügge, Robert; Rudolf, Fabian

    2017-01-10

    Yeast shuttle vectors are indispensable tools in yeast research. They enable cloning of defined DNA sequences in Escherichia coli and their direct transfer into Saccharomyces cerevisiae cells. There are three types of commonly used yeast shuttle vectors: centromeric plasmids, episomal plasmids and integrating plasmids. In this review, we discuss the different plasmid systems and their characteristic features. We focus on their segregational stability and copy number and indicate how to modify these properties. Copyright © 2017 John Wiley & Sons, Ltd.

  18. 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

  19. 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.

  20. 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:…

  1. 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:…

  2. 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 Reports; Applications; Administrative Procedures and Sanctions Application for Authorization to Transmit...

  3. 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 Reports; Applications; Administrative Procedures and Sanctions Application for Authorization to Transmit...

  4. 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 Reports; Applications; Administrative Procedures and Sanctions Application for Presidential Permit...

  5. 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 Reports; Applications; Administrative Procedures and Sanctions Application for Presidential Permit...

  6. 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 Reports; Applications; Administrative Procedures and Sanctions Application for Presidential Permit...

  7. 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 Reports; Applications; Administrative Procedures and Sanctions Application for Authorization to Transmit...

  8. 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 Reports; Applications; Administrative Procedures and Sanctions Application for Presidential Permit...

  9. 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 Reports; Applications; Administrative Procedures and Sanctions Application for Authorization to Transmit...

  10. 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 Reports; Applications; Administrative Procedures and Sanctions Application for Presidential Permit...

  11. 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 Reports; Applications; Administrative Procedures and Sanctions Application for Authorization to Transmit...

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

    USDA-ARS?s Scientific Manuscript database

    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...

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

    USDA-ARS?s Scientific Manuscript database

    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...

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

    PubMed

    Sundström, Elisabeth; Imsland, Freyja; Mikko, Sofia; Wade, Claire; Sigurdsson, Snaevar; Pielberg, Gerli Rosengren; Golovko, Anna; Curik, Ino; Seltenhammer, Monika H; Sölkner, Johann; Lindblad-Toh, Kerstin; Andersson, Leif

    2012-08-02

    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. 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. 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.

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

    USDA-ARS?s Scientific Manuscript database

    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...

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

    USDA-ARS?s Scientific Manuscript database

    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...

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

    USDA-ARS?s Scientific Manuscript database

    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...

  18. Genome-wide copy number variations using SNP genotyping in a mixed breed swine population

    USDA-ARS?s Scientific Manuscript database

    Copy number variations (CNVs) are increasingly understood to affect phenotypic variation. This study uses SNP genotyping of trios of mixed breed swine to add to the catalog of known genotypic variation in an important agricultural animal. Porcine SNP60 BeadChip genotypes were collected from 1802 pi...

  19. 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…

  20. 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

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

    USDA-ARS?s Scientific Manuscript database

    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...

  2. 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…

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

    USDA-ARS?s Scientific Manuscript database

    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...

  4. 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;...

  5. 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.

  6. 47 CFR 25.110 - Filing of applications, fees, and number of copies.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ..., (2) The call sign of the space station or earth station, and (3) The file number of the application. (d) Copies. Applications must be filed electronically though IBFS. The Commission will not accept any... must print out the filed application, obtain the proper signatures, and keep the original in its files...

  7. 47 CFR 25.110 - Filing of applications, fees, and number of copies.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ..., (2) The call sign of the space station or earth station, and (3) The file number of the application. (d) Copies. Applications must be filed electronically though IBFS. The Commission will not accept any... must print out the filed application, obtain the proper signatures, and keep the original in its files...

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

    USDA-ARS?s Scientific Manuscript database

    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 ...

  9. 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.

  10. Extensive Copy-Number Variation of Young Genes across Stickleback Populations

    PubMed Central

    Eizaguirre, Christophe; Samonte, Irene E.; Kalbe, Martin; Lenz, Tobias L.; Stoll, Monika; Bornberg-Bauer, Erich; Milinski, Manfred; Reusch, Thorsten B. H.

    2014-01-01

    Duplicate genes emerge as copy-number variations (CNVs) at the population level, and remain copy-number polymorphic until they are fixed or lost. The successful establishment of such structural polymorphisms in the genome plays an important role in evolution by promoting genetic diversity, complexity and innovation. To characterize the early evolutionary stages of duplicate genes and their potential adaptive benefits, we combine comparative genomics with population genomics analyses to evaluate the distribution and impact of CNVs across natural populations of an eco-genomic model, the three-spined stickleback. With whole genome sequences of 66 individuals from populations inhabiting three distinct habitats, we find that CNVs generally occur at low frequencies and are often only found in one of the 11 populations surveyed. A subset of CNVs, however, displays copy-number differentiation between populations, showing elevated within-population frequencies consistent with local adaptation. By comparing teleost genomes to identify lineage-specific genes and duplications in sticklebacks, we highlight rampant gene content differences among individuals in which over 30% of young duplicate genes are CNVs. These CNV genes are evolving rapidly at the molecular level and are enriched with functional categories associated with environmental interactions, depicting the dynamic early copy-number polymorphic stage of genes during population differentiation. PMID:25474574

  11. Candidate predisposing germline copy number variants in early onset colorectal cancer patients.

    PubMed

    Brea-Fernandez, A J; Fernandez-Rozadilla, C; Alvarez-Barona, M; Azuara, D; Ginesta, M M; Clofent, J; de Castro, L; Gonzalez, D; Andreu, M; Bessa, X; Llor, X; Xicola, R; Jover, R; Castells, A; Castellvi-Bel, S; Capella, G; Carracedo, A; Ruiz-Ponte, C

    2017-05-01

    A great proportion of the heritability of colorectal cancer (CRC) still remains unexplained, and rare variants, as well as copy number changes, have been proposed as potential candidates to explain the so-called 'missing heritability'. We aimed to identify rare high-to-moderately penetrant copy number variants (CNVs) in patients suspected of having hereditary CRC due to an early onset. We have selected for genome-wide copy number analysis, 27 MMR-proficient early onset CRC patients (<50 years) without identifiable germline mutations in Mendelian genes related to this phenotype. Rare CNVs were selected by removing all CNVs detected at MAF >1% in the in-house control CNV database (n = 629 healthy controls). Copy number assignment was checked by duplex real-time quantitative PCR or multiplex ligation probe amplification. Somatic mutation analysis in candidate genes included: loss of heterozygosity studies, point mutation screening, and methylation status of the promoter. We have identified two rare germline deletions involving the AK3 and SLIT2 genes in two patients. The search for a second somatic mutational event in the corresponding CRC tumors showed loss of heterozygosity in AK3, and promoter hypermethylation in SLIT2. Both genes have been previously related to colorectal carcinogenesis. These findings suggest that AK3 and SLIT2 may be potential candidates involved in genetic susceptibility to CRC.

  12. 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

  13. Identification of Copy Number Variants Defining Genomic Differences among Major Human Groups

    PubMed Central

    Armengol, Lluís; Villatoro, Sergi; González, Juan R.; Pantano, Lorena; García-Aragonés, Manel; Rabionet, Raquel; Cáceres, Mario; Estivill, Xavier

    2009-01-01

    Background Understanding the genetic contribution to phenotype variation of human groups is necessary to elucidate differences in disease predisposition and response to pharmaceutical treatments in different human populations. Methodology/Principal Findings We have investigated the genome-wide profile of structural variation on pooled samples from the three populations studied in the HapMap project by comparative genome hybridization (CGH) in different array platforms. We have identified and experimentally validated 33 genomic loci that show significant copy number differences from one population to the other. Interestingly, we found an enrichment of genes related to environment adaptation (immune response, lipid metabolism and extracellular space) within these regions and the study of expression data revealed that more than half of the copy number variants (CNVs) translate into gene-expression differences among populations, suggesting that they could have functional consequences. In addition, the identification of single nucleotide polymorphisms (SNPs) that are in linkage disequilibrium with the copy number alleles allowed us to detect evidences of population differentiation and recent selection at the nucleotide variation level. Conclusions Overall, our results provide a comprehensive view of relevant copy number changes that might play a role in phenotypic differences among major human populations, and generate a list of interesting candidates for future studies. PMID:19789632

  14. A High-Resolution Map of Segmental DNA Copy Number Variation in the Mouse Genome

    PubMed Central

    Graubert, Timothy A; Selzer, Rebecca R; Richmond, Todd A; Eis, Peggy S; Shannon, William D; Li, Xia; McLeod, Howard L; Cheverud, James M; Ley, Timothy J

    2007-01-01

    Submicroscopic (less than 2 Mb) segmental DNA copy number changes are a recently recognized source of genetic variability between individuals. The biological consequences of copy number variants (CNVs) are largely undefined. In some cases, CNVs that cause gene dosage effects have been implicated in phenotypic variation. CNVs have been detected in diverse species, including mice and humans. Published studies in mice have been limited by resolution and strain selection. We chose to study 21 well-characterized inbred mouse strains that are the focus of an international effort to measure, catalog, and disseminate phenotype data. We performed comparative genomic hybridization using long oligomer arrays to characterize CNVs in these strains. This technique increased the resolution of CNV detection by more than an order of magnitude over previous methodologies. The CNVs range in size from 21 to 2,002 kb. Clustering strains by CNV profile recapitulates aspects of the known ancestry of these strains. Most of the CNVs (77.5%) contain annotated genes, and many (47.5%) colocalize with previously mapped segmental duplications in the mouse genome. We demonstrate that this technique can identify copy number differences associated with known polymorphic traits. The phenotype of previously uncharacterized strains can be predicted based on their copy number at these loci. Annotation of CNVs in the mouse genome combined with sequence-based analysis provides an important resource that will help define the genetic basis of complex traits. PMID:17206864

  15. Detection of copy number variations and their effects in Chinese bulls

    USDA-ARS?s Scientific Manuscript database

    Copy number variations (CNVs) are a main source of genomic structural variations underlying animal evolution and production traits. Here, we describe a genome-wide analysis of CNVs in Chinese domesticated bulls and examination of their effects on gene expression and cattle growth traits. We identifi...

  16. Highly dynamic temporal changes of TSPY gene copy number in aging bulls.

    PubMed

    Oluwole, Olutobi A; Mahboubi, Kiana; Favetta, Laura A; Revay, Tamas; Kroetsch, Tom; King, William Allan

    2017-01-01

    The Y-chromosomal TSPY gene is one of the highest copy number mammalian protein coding gene and represents a unique biological model to study various aspects of genomic copy number variations. This study investigated the age-related copy number variability of the bovine TSPY gene, a new and unstudied aspect of the biology of TSPY that has been shown to vary among cattle breeds, individual bulls and somatic tissues. The subjects of this prospective 30-month long study were 25 Holstein bulls, sampled every six months. Real-time quantitative PCR was used to determine the relative TSPY copy number (rTSPY CN) and telomere length in the DNA samples extracted from blood. Twenty bulls showed an altered rTSPY CN after 30 months, although only 9 bulls showed a significant change (4 significant increase while 5 significant decrease, P<0.01). The sequential sampling provided the flow of rTSPY CN over six observations in 30 months and wide-spread variation of rTSPY CN was detected. Although a clear trend of the direction of change was not identifiable, the highly dynamic changes of individual rTSPY CN in aging bulls were observed here for the first time. In summary we have observed a highly variable rTSPY CN in bulls over a short period of time. Our results suggest the importance of further long term studies of the dynamics of rTSPY CN variablility.

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

    USDA-ARS?s Scientific Manuscript database

    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...

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

    USDA-ARS?s Scientific Manuscript database

    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...

  19. 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

  20. 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

  1. DUF1220-domain copy number implicated in human brain-size pathology and evolution.

    PubMed

    Dumas, Laura J; O'Bleness, Majesta S; Davis, Jonathan M; Dickens, C Michael; Anderson, Nathan; Keeney, J G; Jackson, Jay; Sikela, Megan; Raznahan, Armin; Giedd, Jay; Rapoport, Judith; Nagamani, Sandesh S C; Erez, Ayelet; Brunetti-Pierri, Nicola; Sugalski, Rachel; Lupski, James R; Fingerlin, Tasha; Cheung, Sau Wai; Sikela, James M

    2012-09-07

    DUF1220 domains show the largest human-lineage-specific increase in copy number of any protein-coding region in the human genome and map primarily to 1q21, where deletions and reciprocal duplications have been associated with microcephaly and macrocephaly, respectively. Given these findings and the high correlation between DUF1220 copy number and brain size across primate lineages (R(2) = 0.98; p = 1.8 × 10(-6)), DUF1220 sequences represent plausible candidates for underlying 1q21-associated brain-size pathologies. To investigate this possibility, we used specialized bioinformatics tools developed for scoring highly duplicated DUF1220 sequences to implement targeted 1q21 array comparative genomic hybridization on individuals (n = 42) with 1q21-associated microcephaly and macrocephaly. We show that of all the 1q21 genes examined (n = 53), DUF1220 copy number shows the strongest association with brain size among individuals with 1q21-associated microcephaly, particularly with respect to the three evolutionarily conserved DUF1220 clades CON1(p = 0.0079), CON2 (p = 0.0134), and CON3 (p = 0.0116). Interestingly, all 1q21 DUF1220-encoding genes belonging to the NBPF family show significant correlations with frontal-occipital-circumference Z scores in the deletion group. In a similar survey of a nondisease population, we show that DUF1220 copy number exhibits the strongest correlation with brain gray-matter volume (CON1, p = 0.0246; and CON2, p = 0.0334). Notably, only DUF1220 sequences are consistently significant in both disease and nondisease populations. Taken together, these data strongly implicate the loss of DUF1220 copy number in the etiology of 1q21-associated microcephaly and support the view that DUF1220 domains function as general effectors of evolutionary, pathological, and normal variation in brain size. Copyright © 2012 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

  2. DUF1220-Domain Copy Number Implicated in Human Brain-Size Pathology and Evolution

    PubMed Central

    Dumas, Laura J.; O’Bleness, Majesta S.; Davis, Jonathan M.; Dickens, C. Michael; Anderson, Nathan; Keeney, J.G.; Jackson, Jay; Sikela, Megan; Raznahan, Armin; Giedd, Jay; Rapoport, Judith; Nagamani, Sandesh S.C.; Erez, Ayelet; Brunetti-Pierri, Nicola; Sugalski, Rachel; Lupski, James R.; Fingerlin, Tasha; Cheung, Sau Wai; Sikela, James M.

    2012-01-01

    DUF1220 domains show the largest human-lineage-specific increase in copy number of any protein-coding region in the human genome and map primarily to 1q21, where deletions and reciprocal duplications have been associated with microcephaly and macrocephaly, respectively. Given these findings and the high correlation between DUF1220 copy number and brain size across primate lineages (R2 = 0.98; p = 1.8 × 10−6), DUF1220 sequences represent plausible candidates for underlying 1q21-associated brain-size pathologies. To investigate this possibility, we used specialized bioinformatics tools developed for scoring highly duplicated DUF1220 sequences to implement targeted 1q21 array comparative genomic hybridization on individuals (n = 42) with 1q21-associated microcephaly and macrocephaly. We show that of all the 1q21 genes examined (n = 53), DUF1220 copy number shows the strongest association with brain size among individuals with 1q21-associated microcephaly, particularly with respect to the three evolutionarily conserved DUF1220 clades CON1(p = 0.0079), CON2 (p = 0.0134), and CON3 (p = 0.0116). Interestingly, all 1q21 DUF1220-encoding genes belonging to the NBPF family show significant correlations with frontal-occipital-circumference Z scores in the deletion group. In a similar survey of a nondisease population, we show that DUF1220 copy number exhibits the strongest correlation with brain gray-matter volume (CON1, p = 0.0246; and CON2, p = 0.0334). Notably, only DUF1220 sequences are consistently significant in both disease and nondisease populations. Taken together, these data strongly implicate the loss of DUF1220 copy number in the etiology of 1q21-associated microcephaly and support the view that DUF1220 domains function as general effectors of evolutionary, pathological, and normal variation in brain size. PMID:22901949

  3. 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

  4. Copy number loss or silencing of apoptosis-effector genes in cancer.

    PubMed

    Mauro, James A; Butler, Shanitra N; Ramsamooj, Michael; Blanck, George

    2015-01-01

    Cancer cells undergo a variety of DNA copy number gains and losses (CNV), raising two important questions related to cancer development: (i) Which genes are affected? (ii) And how do CNVs, that do not represent complete deletions but do represent gene-dosage alterations, impact cancer cell functions? Recent studies have indicated that CNVs in cancer can impact genes for regulatory proteins long known to be associated with cancer development, but less is understood about CNVs affecting effector genes. Also, we have recently indicated the likely importance of transcription factor binding site (TFBS) copies in effector genes, in regulating the transition from a proliferative to an apoptotic state. Here we report data-mining analyses that indicate that copies of apoptosis-effector genes are commonly lost in cancer development, in comparison to proliferation-effector genes, and when not, apoptosis effector genes have silenced chromatin structures. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. [Detection of the exogenous gene copy number of the transgenic tomato anti-caries vaccine].

    PubMed

    Bai, Guo-hui; Liu, Jian-guo; Tian, Yuan; Chen, Zhu; Bai, Peng-yuan; Han, Qi; Gu, Yu; Guan, Xiao-yan; Wang, Hai-hui

    2013-12-01

    To detect the exogenous gene copy number of the transgenic tomato anti-caries vaccine by using the SYBR Green real-time PCR. Recombinant plasmid pEAC10 and pEPC10 were used as standard to detect genome samples of exogenous gene pacA-ctxB and pacP-ctxB by SYBR green fluorescent quantitation, then the average value was calculated as gene copy number. The copy number of the transgenic tomato carrying pacA-ctxB was 1.3 and the pacP-ctxB was 3.2. The transgenic tomato plants which have high stability are low-copy transgenic plants. Supported by National Natural Science Foundation of China (30160086, 81260164), Science and Technical Fund of Guizhou Province (LKZ[2011]41), Project of Technology Innovation Team in Guizhou Province, Leading Academic Discipline Construction Project in Guizhou Province and Excellent Scientific Research Team Cultivation Project in Zunyi Medical College ([2012]12).

  6. Systematic Inference of Copy-Number Genotypes from Personal Genome Sequencing Data Reveals Extensive Olfactory Receptor Gene Content Diversity

    PubMed Central

    Waszak, Sebastian M.; Hasin, Yehudit; Zichner, Thomas; Olender, Tsviya; Keydar, Ifat; Khen, Miriam; Stütz, Adrian M.; Schlattl, Andreas; Lancet, Doron; Korbel, Jan O.

    2010-01-01

    Copy-number variations (CNVs) are widespread in the human genome, but comprehensive assignments of integer locus copy-numbers (i.e., copy-number genotypes) that, for example, enable discrimination of homozygous from heterozygous CNVs, have remained challenging. Here we present CopySeq, a novel computational approach with an underlying statistical framework that analyzes the depth-of-coverage of high-throughput DNA sequencing reads, and can incorporate paired-end and breakpoint junction analysis based CNV-analysis approaches, to infer locus copy-number genotypes. We benchmarked CopySeq by genotyping 500 chromosome 1 CNV regions in 150 personal genomes sequenced at low-coverage. The assessed copy-number genotypes were highly concordant with our performed qPCR experiments (Pearson correlation coefficient 0.94), and with the published results of two microarray platforms (95–99% concordance). We further demonstrated the utility of CopySeq for analyzing gene regions enriched for segmental duplications by comprehensively inferring copy-number genotypes in the CNV-enriched >800 olfactory receptor (OR) human gene and pseudogene loci. CopySeq revealed that OR loci display an extensive range of locus copy-numbers across individuals, with zero to two copies in some OR loci, and two to nine copies in others. Among genetic variants affecting OR loci we identified deleterious variants including CNVs and SNPs affecting ∼15% and ∼20% of the human OR gene repertoire, respectively, implying that genetic variants with a possible impact on smell perception are widespread. Finally, we found that for several OR loci the reference genome appears to represent a minor-frequency variant, implying a necessary revision of the OR repertoire for future functional studies. CopySeq can ascertain genomic structural variation in specific gene families as well as at a genome-wide scale, where it may enable the quantitative evaluation of CNVs in genome-wide association studies involving high

  7. Chromosome and gene copy number variation allow major structural change between species and strains of Leishmania.

    PubMed

    Rogers, Matthew B; Hilley, James D; Dickens, Nicholas J; Wilkes, Jon; Bates, Paul A; Depledge, Daniel P; Harris, David; Her, Yerim; Herzyk, Pawel; Imamura, Hideo; Otto, Thomas D; Sanders, Mandy; Seeger, Kathy; Dujardin, Jean-Claude; Berriman, Matthew; Smith, Deborah F; Hertz-Fowler, Christiane; Mottram, Jeremy C

    2011-12-01

    Leishmania parasites cause a spectrum of clinical pathology in humans ranging from disfiguring cutaneous lesions to fatal visceral leishmaniasis. We have generated a reference genome for Leishmania mexicana and refined the reference genomes for Leishmania major, Leishmania infantum, and Leishmania braziliensis. This has allowed the identification of a remarkably low number of genes or paralog groups (2, 14, 19, and 67, respectively) unique to one species. These were found to be conserved in additional isolates of the same species. We have predicted allelic variation and find that in these isolates, L. major and L. infantum have a surprisingly low number of predicted heterozygous SNPs compared with L. braziliensis and L. mexicana. We used short read coverage to infer ploidy and gene copy numbers, identifying large copy number variations between species, with 200 tandem gene arrays in L. major and 132 in L. mexicana. Chromosome copy number also varied significantly between species, with nine supernumerary chromosomes in L. infantum, four in L. mexicana, two in L. braziliensis, and one in L. major. A significant bias against gene arrays on supernumerary chromosomes was shown to exist, indicating that duplication events occur more frequently on disomic chromosomes. Taken together, our data demonstrate that there is little variation in unique gene content across Leishmania species, but large-scale genetic heterogeneity can result through gene amplification on disomic chromosomes and variation in chromosome number. Increased gene copy number due to chromosome amplification may contribute to alterations in gene expression in response to environmental conditions in the host, providing a genetic basis for disease tropism.

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

    PubMed

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

    2016-11-01

    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.

  9. 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

  10. Quadruplex MAPH: improvement of throughput in high-resolution copy number screening

    PubMed Central

    Tyson, Jess; Majerus, Tamsin MO; Walker, Susan; Armour, John AL

    2009-01-01

    Background Copy number variation (CNV) in the human genome is recognised as a widespread and important source of human genetic variation. Now the challenge is to screen for these CNVs at high resolution in a reliable, accurate and cost-effective way. Results Multiplex Amplifiable Probe Hybridisation (MAPH) is a sensitive, high-resolution technology appropriate for screening for CNVs in a defined region, for a targeted population. We have developed MAPH to a highly multiplexed format ("QuadMAPH") that allows the user a four-fold increase in the number of loci tested simultaneously. We have used this method to analyse a genomic region of 210 kb, including the MSH2 gene and 120 kb of flanking DNA. We show that the QuadMAPH probes report copy number with equivalent accuracy to simplex MAPH, reliably demonstrating diploid copy number in control samples and accurately detecting deletions in Hereditary Non-Polyposis Colorectal Cancer (HNPCC) samples. Conclusion QuadMAPH is an accurate, high-resolution method that allows targeted screening of large numbers of subjects without the expense of genome-wide approaches. Whilst we have applied this technique to a region of the human genome, it is equally applicable to the genomes of other organisms. PMID:19785739

  11. Copy Number of the Transposon, Pokey, in rDNA Is Positively Correlated with rDNA Copy Number in Daphnia obtusa

    PubMed Central

    LeRiche, Kaitlynn; Eagle, Shannon H. C.; Crease, Teresa J.

    2014-01-01

    Pokey is a class II DNA transposon that inserts into 28S ribosomal RNA (rRNA) genes and other genomic regions of species in the subgenus, Daphnia. Two divergent lineages, PokeyA and PokeyB have been identified. Recombination between misaligned rRNA genes changes their number and the number of Pokey elements. We used quantitative PCR (qPCR) to estimate rRNA gene and Pokey number in isolates from natural populations of Daphnia obtusa, and in clonally-propagated mutation accumulation lines (MAL) initiated from a single D. obtusa female. The change in direction and magnitude of Pokey and rRNA gene number did not show a consistent pattern across ∼87 generations in the MAL; however, Pokey and rRNA gene number changed in concert. PokeyA and 28S gene number were positively correlated in the isolates from both natural populations and the MAL. PokeyB number was much lower than PokeyA in both MAL and natural population isolates, and showed no correlation with 28S gene number. Preliminary analysis did not detect PokeyB outside rDNA in any isolates and detected only 0 to 4 copies of PokeyA outside rDNA indicating that Pokey may be primarily an rDNA element in D. obtusa. The recombination rate in this species is high and the average size of the rDNA locus is about twice as large as that in other Daphnia species such as D. pulicaria and D. pulex, which may have facilitated expansion of PokeyA to much higher numbers in D. obtusa rDNA than these other species. PMID:25490398

  12. 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

  13. Assessment of Complement C4 Gene Copy Number Using the Paralog Ratio Test

    PubMed Central

    Fernando, Michelle M.A.; Boteva, Lora; Morris, David L.; Zhou, Bi; Wu, Yee Ling; Lokki, Marja-Liisa; Yu, Chack Yung; Rioux, John D.; Hollox, Edward J.; Vyse, Timothy J.

    2013-01-01

    The complement C4 locus is in the class III region of the MHC, and exhibits copy number variation. Complement C4 null alleles have shown association with a number of diseases including systemic lupus erythematosus (SLE). However, most studies to date have used protein immunophenotyping and not direct interrogation of the genome to determine C4 null allele status. Moreover, a lack of accurate C4 gene copy number (GCN) estimation and tight linkage disequilibrium across the disease-associated MHC haplotypes has confounded attempts to establish whether or not these associations are causal. We have therefore developed a high through-put paralog ratio test (PRT) in association with two restriction enzyme digest variant ratio tests (REDVRs) to determine total C4 GCN, C4A GCN, and C4B GCN. In the densely genotyped CEU cohort we show that this method is accurate and reproducible when compared to gold standard Southern blot copy number estimation with a discrepancy rate of 9%. We find a broad range of C4 GCNs in the CEU and the 1958 British Birth Cohort populations under study. In addition, SNP-C4 CNV analyses show only moderate levels of correlation and therefore do not support the use of SNP genotypes as proxies for complement C4 GCN. PMID:20506482

  14. 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

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. 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.

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

    PubMed

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

    2017-07-01

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

  1. 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.

  2. 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.

  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. 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

  5. Inference of plasmid-copy-number mean and noise from single-cell gene expression data

    NASA Astrophysics Data System (ADS)

    Ghozzi, Stéphane; Wong Ng, Jérôme; Chatenay, Didier; Robert, Jérôme

    2010-11-01

    Plasmids are extrachromosomal DNA molecules which code for their own replication. We previously reported a setup using genes coding for fluorescent proteins of two colors that allowed us, using a simple model, to extract the plasmid-copy-number noise in a monoclonal population of bacteria [J. Wong Ng , Phys. Rev. E 81, 011909 (2010)10.1103/PhysRevE.81.011909]. Here we present a detailed calculation relating this noise to the measured levels of fluorescence, taking into account all sources of fluorescence fluctuations: not only the fluctuation of gene expression as in the simple model but also the growth and division of bacteria, the nonuniform distribution of their ages, the random partition of proteins at divisions, and the replication and partition of plasmids and chromosome. We show how to use the chromosome as a reference, which helps extracting the plasmid-copy-number noise in a self-consistent manner.

  6. 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.

  7. 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

  8. 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.

  9. 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

  10. 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.).

  11. 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

  12. High copy number of mitochondrial DNA predicts poor prognosis in patients with advanced stage colon cancer.

    PubMed

    Wang, Yun; He, Shuixiang; Zhu, Xingmei; Qiao, Wei; Zhang, Juan

    2016-12-23

    The aim of this investigation was to determine whether alterations in mitochondrial DNA (mtDNA) copy number in colon cancer were associated with clinicopathological parameters and postsurgical outcome. By quantitative real-time PCR assay, the mtDNA copy number was detected in a cohort of colon cancer and matched adjacent colon tissues (n = 162). The majority of patients had higher mtDNA content in colon cancer tissues than matched adjacent colon tissues. Moreover, high mtDNA content in tumor tissues was associated with larger tumor size, higher serum CEA level, advanced TNM stage, vascular emboli, and liver metastases. Further survival curve analysis showed that high mtDNA content was related to the worst survival in patients with colon cancer at advanced TNM stage. High mtDNA content is a potential effective factor of poor prognosis in patients with advanced stage colon cancer.

  13. Characterization of an inducible promoter in different DNA copy number conditions

    PubMed Central

    2012-01-01

    Background The bottom-up programming of living organisms to implement novel user-defined biological capabilities is one of the main goals of synthetic biology. Currently, a predominant problem connected with the construction of even simple synthetic biological systems is the unpredictability of the genetic circuitry when assembled and incorporated in living cells. Copy number, transcriptional/translational demand and toxicity of the DNA-encoded functions are some of the major factors which may lead to cell overburdening and thus to nonlinear effects on system output. It is important to disclose the linearity working boundaries of engineered biological systems when dealing with such phenomena. Results The output of an N-3-oxohexanoyl-L-homoserine lactone (HSL)-inducible RFP-expressing device was studied in Escherichia coli in different copy number contexts, ranging from 1 copy per cell (integrated in the genome) to hundreds (via multicopy plasmids). The system is composed by a luxR constitutive expression cassette and a RFP gene regulated by the luxI promoter, which is activated by the HSL-LuxR complex. System output, in terms of promoter activity as a function of HSL concentration, was assessed relative to the one of a reference promoter in identical conditions by using the Relative Promoter Units (RPU) approach. Nonlinear effects were observed in the maximum activity, which is identical in single and low copy conditions, while it decreases for higher copy number conditions. In order to properly compare the luxI promoter strength among all the conditions, a mathematical modeling approach was used to relate the promoter activity to the estimated HSL-LuxR complex concentration, which is the actual activator of transcription. During model fitting, a correlation between the copy number and the dissociation constant of HSL-LuxR complex and luxI promoter was observed. Conclusions Even in a simple inducible system, nonlinear effects are observed and non-trivial data

  14. 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

  15. Characterization of an inducible promoter in different DNA copy number conditions.

    PubMed

    Zucca, Susanna; Pasotti, Lorenzo; Mazzini, Giuliano; De Angelis, Maria Gabriella Cusella; Magni, Paolo

    2012-03-28

    The bottom-up programming of living organisms to implement novel user-defined biological capabilities is one of the main goals of synthetic biology. Currently, a predominant problem connected with the construction of even simple synthetic biological systems is the unpredictability of the genetic circuitry when assembled and incorporated in living cells. Copy number, transcriptional/translational demand and toxicity of the DNA-encoded functions are some of the major factors which may lead to cell overburdening and thus to nonlinear effects on system output. It is important to disclose the linearity working boundaries of engineered biological systems when dealing with such phenomena. The output of an N-3-oxohexanoyl-L-homoserine lactone (HSL)-inducible RFP-expressing device was studied in Escherichia coli in different copy number contexts, ranging from 1 copy per cell (integrated in the genome) to hundreds (via multicopy plasmids). The system is composed by a luxR constitutive expression cassette and a RFP gene regulated by the luxI promoter, which is activated by the HSL-LuxR complex. System output, in terms of promoter activity as a function of HSL concentration, was assessed relative to the one of a reference promoter in identical conditions by using the Relative Promoter Units (RPU) approach. Nonlinear effects were observed in the maximum activity, which is identical in single and low copy conditions, while it decreases for higher copy number conditions. In order to properly compare the luxI promoter strength among all the conditions, a mathematical modeling approach was used to relate the promoter activity to the estimated HSL-LuxR complex concentration, which is the actual activator of transcription. During model fitting, a correlation between the copy number and the dissociation constant of HSL-LuxR complex and luxI promoter was observed. Even in a simple inducible system, nonlinear effects are observed and non-trivial data processing is necessary to fully

  16. A multi-megabase copy number gain causes maternal transmission ratio distortion on mouse chromosome 2.

    PubMed

    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

    2015-02-01

    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.

  17. 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.

  18. 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

  19. A trans-Dominant Form of Gag Restricts Ty1 Retrotransposition and Mediates Copy Number Control

    PubMed Central

    Saha, Agniva; Mitchell, Jessica A.; Nishida, Yuri; Hildreth, Jonathan E.; Ariberre, Joshua A.; Gilbert, Wendy V.

    2015-01-01

    ABSTRACT Saccharomyces cerevisiae and Saccharomyces paradoxus lack the conserved RNA interference pathway and utilize a novel form of copy number control (CNC) to inhibit Ty1 retrotransposition. Although noncoding transcripts have been implicated in CNC, here we present evidence that a truncated form of the Gag capsid protein (p22) or its processed form (p18) is necessary and sufficient for CNC and likely encoded by Ty1 internal transcripts. Coexpression of p22/p18 and Ty1 decreases mobility more than 30,000-fold. p22/p18 cofractionates with Ty1 virus-like particles (VLPs) and affects VLP yield, protein composition, and morphology. Although p22/p18 and Gag colocalize in the cytoplasm, p22/p18 disrupts sites used for VLP assembly. Glutathione S-transferase (GST) affinity pulldowns also suggest that p18 and Gag interact. Therefore, this intrinsic Gag-like restriction factor confers CNC by interfering with VLP assembly and function and expands the strategies used to limit retroelement propagation. IMPORTANCE Retrotransposons dominate the chromosomal landscape in many eukaryotes, can cause mutations by insertion or genome rearrangement, and are evolutionarily related to retroviruses such as HIV. Thus, understanding factors that limit transposition and retroviral replication is fundamentally important. The present work describes a retrotransposon-encoded restriction protein derived from the capsid gene of the yeast Ty1 element that disrupts virus-like particle assembly in a dose-dependent manner. This form of copy number control acts as a molecular rheostat, allowing high levels of retrotransposition when few Ty1 elements are present and inhibiting transposition as copy number increases. Thus, yeast and Ty1 have coevolved a form of copy number control that is beneficial to both “host and parasite.” To our knowledge, this is the first Gag-like retrotransposon restriction factor described in the literature and expands the ways in which restriction proteins modulate

  20. Reduced purifying selection prevails over positive selection in human copy number variant evolution.

    PubMed

    Nguyen, Duc-Quang; Webber, Caleb; Hehir-Kwa, Jayne; Pfundt, Rolph; Veltman, Joris; Ponting, Chris P

    2008-11-01

    Copy number variation is a dominant contributor to genomic variation and may frequently underlie an individual's variable susceptibilities to disease. Here we question our previous proposition that copy number variants (CNVs) are often retained in the human population because of their adaptive benefit. We show that genic biases of CNVs are best explained, not by positive selection, but by reduced efficiency of selection in eliminating deleterious changes from the human population. Of four CNV data sets examined, three exhibit significant increases in protein evolutionary rates. These increases appear to be attributable to the frequent coincidence of CNVs with segmental duplications (SDs) that recombine infrequently. Furthermore, human orthologs of mouse genes, which, when disrupted, result in pre- or postnatal lethality, are unusually depleted in CNVs. Together, these findings support a model of reduced purifying selection (Hill-Robertson interference) within copy number variable regions that are enriched in nonessential genes, allowing both the fixation of slightly deleterious substitutions and increased drift of CNV alleles. Additionally, all four CNV sets exhibited increased rates of interspecies chromosomal rearrangement and nucleotide substitution and an increased gene density. We observe that sequences with high G+C contents are most prone to copy number variation. In particular, frequently duplicated human SD sequence, or CNVs that are large and/or observed frequently, tend to be elevated in G+C content. In contrast, SD sequences that appear fixed in the human population lie more frequently within low G+C sequence. These findings provide an overarching view of how CNVs arise and segregate in the human population.

  1. 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

  2. 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

  3. Incorporating 16S Gene Copy Number Information Improves Estimates of Microbial Diversity and Abundance

    PubMed Central

    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. PMID:23133348

  4. 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.

  5. 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

  6. 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

  7. A plasmid cloning vector with precisely regulatable copy number in Escherichia coli.

    PubMed

    Herman-Antosiewicz, A; Obuchowski, M; Wegrzyn, G

    2001-03-01

    We have developed a genetic system allowing for precise regulation of plasmid copy number in Escherichia coli cells. A cloning vector based on this system is described in this article. The pTC lambda 3 plasmid is a lambda replicon, but transcription controlling initiation of plasmid DNA replication starts from the PtetA promoter instead of phage lambda PR promoter. Additionally, activity of PtetA promoter is negatively controlled by the TetR repressor whose gene is located on the same plasmid vector and is induced by an analog of tetracycline, autoclaved chlortetracycline (aCT). Using different concentrations of the inducer it is possible to strictly regulate the copy number of pTC lambda 3 and thus the copy number of a cloned gene. The usefulness of the system for the regulatable production of a protein encoded by a gene inserted into pTC lambda 3 plasmid is demonstrated by dependence of beta-galactosidase activity on the lacZ gene dosage.

  8. 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.

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

    PubMed Central

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

    2015-01-01

    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. PMID:25596731

  10. 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.

  11. The Characterisation of Three Types of Genes that Overlie Copy Number Variable Regions

    PubMed Central

    Woodwark, Cara; Bateman, Alex

    2011-01-01

    Background Due to the increased accuracy of Copy Number Variable region (CNV) break point mapping, it is now possible to say with a reasonable degree of confidence whether a gene (i) falls entirely within a CNV; (ii) overlaps the CNV or (iii) actually contains the CNV. We classify these as type I, II and III CNV genes respectively. Principal Findings Here we show that although type I genes vary in copy number along with the CNV, most of these type I genes have the same expression levels as wild type copy numbers of the gene. These genes must, therefore, be under homeostatic dosage compensation control. Looking into possible mechanisms for the regulation of gene expression we found that type I genes have a significant paucity of genes regulated by miRNAs and are not significantly enriched for monoallelically expressed genes. Type III genes, on the other hand, have a significant excess of genes regulated by miRNAs and are enriched for genes that are monoallelically expressed. Significance Many diseases and genomic disorders are associated with CNVs so a better understanding of the different ways genes are associated with normal CNVs will help focus on candidate genes in genome wide association studies. PMID:21637334

  12. 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

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

    PubMed Central

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

    2015-01-01

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

  14. 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

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

    PubMed

    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

    2016-08-08

    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.

  16. Performance assessment of copy number microarray platforms using a spike-in experiment

    PubMed Central

    Halper-Stromberg, Eitan; Frelin, Laurence; Ruczinski, Ingo; Scharpf, Robert; Jie, Chunfa; Carvalho, Benilton; Hao, Haiping; Hetrick, Kurt; Jedlicka, Anne; Dziedzic, Amanda; Doheny, Kim; Scott, Alan F.; Baylin, Steve; Pevsner, Jonathan; Spencer, Forrest; Irizarry, Rafael A.

    2011-01-01

    Motivation: Changes in the copy number of chromosomal DNA segments [copy number variants (CNVs)] have been implicated in human variation, heritable diseases and cancers. Microarray-based platforms are the current established technology of choice for studies reporting these discoveries and constitute the benchmark against which emergent sequence-based approaches will be evaluated. Research that depends on CNV analysis is rapidly increasing, and systematic platform assessments that distinguish strengths and weaknesses are needed to guide informed choice. Results: We evaluated the sensitivity and specificity of six platforms, provided by four leading vendors, using a spike-in experiment. NimbleGen and Agilent platforms outperformed Illumina and Affymetrix in accuracy and precision of copy number dosage estimates. However, Illumina and Affymetrix algorithms that leverage single nucleotide polymorphism (SNP) information make up for this disadvantage and perform well at variant detection. Overall, the NimbleGen 2.1M platform outperformed others, but only with the use of an alternative data analysis pipeline to the one offered by the manufacturer. Availability: The data is available from http://rafalab.jhsph.edu/cnvcomp/. Contact: pevsner@jhmi.edu; fspencer@jhmi.edu; rafa@jhu.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:21478196

  17. 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.

  18. Genome-wide copy number variation (CNV) in patients with autoimmune Addison's disease

    PubMed Central

    2011-01-01

    Background Addison's disease (AD) is caused by an autoimmune destruction of the adrenal cortex. The pathogenesis is multi-factorial, involving genetic components and hitherto unknown environmental factors. The aim of the present study was to investigate if gene dosage in the form of copy number variation (CNV) could add to the repertoire of genetic susceptibility to autoimmune AD. Methods A genome-wide study using the Affymetrix GeneChip® Genome-Wide Human SNP Array 6.0 was conducted in 26 patients with AD. CNVs in selected genes were further investigated in a larger material of patients with autoimmune AD (n = 352) and healthy controls (n = 353) by duplex Taqman real-time polymerase chain reaction assays. Results We found that low copy number of UGT2B28 was significantly more frequent in AD patients compared to controls; conversely high copy number of ADAM3A was associated with AD. Conclusions We have identified two novel CNV associations to ADAM3A and UGT2B28 in AD. The mechanism by which this susceptibility is conferred is at present unclear, but may involve steroid inactivation (UGT2B28) and T cell maturation (ADAM3A). Characterization of these proteins may unravel novel information on the pathogenesis of autoimmunity. PMID:21851588

  19. Significant heterogeneity in Wolbachia copy number within and between populations of Onchocerca volvulus.

    PubMed

    Armoo, Samuel; Doyle, Stephen R; Osei-Atweneboana, Mike Y; Grant, Warwick N

    2017-04-18

    Wolbachia are intracellular bacteria found in arthropods and several filarial nematode species. The filarial Wolbachia have been proposed to be involved in the immunopathology associated with onchocerciasis. Higher Wolbachia-to-nematode ratios have been reported in the savannah-ecotype compared to the forest-ecotype, and have been interpreted as consistent with a correlation between Wolbachia density and disease severity. However, factors such as geographic stratification and ivermectin drug exposure can lead to significant genetic heterogeneity in the nematode host populations, so we investigated whether Wolbachia copy number variation is also associated with these underlying factors. Genomic DNA was prepared from single adult nematodes representing forest and savannah ecotypes sampled from Togo, Ghana, Côte d'Ivoire and Mali. A qPCR assay was developed to measure the number of Wolbachia genome(s) per nematode genome. Next-generation sequencing (NGS) was also used to measure relative Wolbachia copy number, and independently verify the qPCR assay. Significant variation was observed within the forest (range: 0.02 to 452.99; median: 10.58) and savannah (range: 0.01 to 1106.25; median: 9.10) ecotypes, however, no significant difference between ecotypes (P = 0.645) was observed; rather, strongly significant Wolbachia variation was observed within and between the nine study communities analysed (P = 0.021), independent of ecotype. Analysis of ivermectin-treated and untreated nematodes by qPCR showed no correlation (P = 0.869); however, an additional analysis of a subset of the nematodes by qPCR and NGS revealed a correlation between response to ivermectin treatment and Wolbachia copy number (P = 0.020). This study demonstrates that extensive within and between population variation exists in the Wolbachia content of individual adult O. volvulus. The origin and functional significance of such variation (up to ~ 100,000-fold between worms; ~10 to 100

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

    PubMed

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

    2015-02-01

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

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

    USDA-ARS?s Scientific Manuscript database

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

    PubMed

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

    2016-09-08

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

  3. Amylase activity is associated with AMY2B copy numbers in dog: implications for dog domestication, diet and diabetes

    PubMed Central

    Arendt, Maja; Fall, Tove; Lindblad-Toh, Kerstin; Axelsson, Erik

    2014-01-01

    High amylase activity in dogs is associated with a drastic increase in copy numbers of the gene coding for pancreatic amylase, AMY2B, that likely allowed dogs to thrive on a relatively starch-rich diet during early dog domestication. Although most dogs thus probably digest starch more efficiently than do wolves, AMY2B copy numbers vary widely within the dog population, and it is not clear how this variation affects the individual ability to handle starch nor how it affects dog health. In humans, copy numbers of the gene coding for salivary amylase, AMY1, correlate with both salivary amylase levels and enzyme activity, and high amylase activity is related to improved glycemic homeostasis and lower frequencies of metabolic syndrome. Here, we investigate the relationship between AMY2B copy numbers and serum amylase activity in dogs and show that amylase activity correlates with AMY2B copy numbers. We then describe how AMY2B copy numbers vary in individuals from 20 dog breeds and find strong breed-dependent patterns, indicating that the ability to digest starch varies both at the breed and individual level. Finally, to test whether AMY2B copy number is strongly associated with the risk of developing diabetes mellitus, we compare copy numbers in cases and controls as well as in breeds with varying diabetes susceptibility. Although we see no such association here, future studies using larger cohorts are needed before excluding a possible link between AMY2B and diabetes mellitus. PMID:24975239

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

    PubMed Central

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

    2016-01-01

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

  5. Copy-number variation of housekeeping gene rpl13a in rat strains selected for nervous system excitability.

    PubMed

    Kalendar, Ruslan; Belyayev, Alexander; Zachepilo, Tatiana; Vaido, Alexander; Maidanyuk, Dmitry; Schulman, Alan H; Dyuzhikova, Natalia

    2017-06-01

    We evaluated copy number variation (CNV) for four genes in rat strains differing in nervous system excitability. rpl13a copy number is significantly reduced in hippocampus and bone marrow in rats with a high excitability threshold and stress. The observed phenomenon may be associated with a role for rpl13a in lipid metabolism. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. 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

  7. Population-genetic nature of copy number variations in the human genome.

    PubMed

    Kato, Mamoru; Kawaguchi, Takahisa; Ishikawa, Shumpei; Umeda, Takayoshi; Nakamichi, Reiichiro; Shapero, Michael H; Jones, Keith W; Nakamura, Yusuke; Aburatani, Hiroyuki; Tsunoda, Tatsuhiko

    2010-03-01

    Copy number variations (CNVs) are universal genetic variations, and their association with disease has been increasingly recognized. We designed high-density microarrays for CNVs, and detected 3000-4000 CNVs (4-6% of the genomic sequence) per population that included CNVs previously missed because of smaller sizes and residing in segmental duplications. The patterns of CNVs across individuals were surprisingly simple at the kilo-base scale, suggesting the applicability of a simple genetic analysis for these genetic loci. We utilized the probabilistic theory to determine integer copy numbers of CNVs and employed a recently developed phasing tool to estimate the population frequencies of integer copy number alleles and CNV-SNP haplotypes. The results showed a tendency toward a lower frequency of CNV alleles and that most of our CNVs were explained only by zero-, one- and two-copy alleles. Using the estimated population frequencies, we found several CNV regions with exceptionally high population differentiation. Investigation of CNV-SNP linkage disequilibrium (LD) for 500-900 bi- and multi-allelic CNVs per population revealed that previous conflicting reports on bi-allelic LD were unexpectedly consistent and explained by an LD increase correlated with deletion-allele frequencies. Typically, the bi-allelic LD was lower than SNP-SNP LD, whereas the multi-allelic LD was somewhat stronger than the bi-allelic LD. After further investigation of tag SNPs for CNVs, we conclude that the customary tagging strategy for disease association studies can be applicable for common deletion CNVs, but direct interrogation is needed for other types of CNVs.

  8. Cell-free DNA copy number variations in plasma from colorectal cancer patients.

    PubMed

    Li, Jian; Dittmar, Rachel L; Xia, Shu; Zhang, Huijuan; Du, Meijun; Huang, Chiang-Ching; Druliner, Brooke R; Boardman, Lisa; Wang, Liang

    2017-08-01

    To evaluate the clinical utility of cell-free DNA (cfDNA), we performed whole-genome sequencing to systematically examine plasma cfDNA copy number variations (CNVs) in a cohort of patients with colorectal cancer (CRC, n = 80), polyps (n = 20), and healthy controls (n = 35). We initially compared cfDNA yield in 20 paired serum-plasma samples and observed significantly higher cfDNA concentration in serum (median = 81.20 ng, range 7.18-500 ng·mL(-1) ) than in plasma (median = 5.09 ng, range 3.76-62.8 ng·mL(-1) ) (P < 0.0001). However, tumor-derived cfDNA content was significantly lower in serum than in matched plasma samples tested. With ~10 million reads per sample, the sequencing-based copy number analysis showed common CNVs in multiple chromosomal regions, including amplifications on 1q, 8q, and 5q and deletions on 1p, 4q, 8p, 17p, 18q, and 22q. Copy number changes were also evident in genes critical to the cell cycle, DNA repair, and WNT signaling pathways. To evaluate whether cumulative copy number changes were associated with tumor stages, we calculated plasma genomic abnormality in colon cancer (PGA-C) score by summing the most significant CNVs. The PGA-C score showed predictive performance with an area under the curve from 0.54 to 0.84 for CRC stages I-IV. Locus-specific copy number analysis identified nine genomic regions where CNVs were significantly associated with survival in stage III-IV CRC patients. A multivariate model using six of nine genomic regions demonstrated a significant association of high-risk score with shorter survival (HR = 5.33, 95% CI = 6.76-94.44, P < 0.0001). Our study demonstrates the importance of using plasma (rather than serum) to test tumor-related genomic variations. Plasma cfDNA-based tests can capture tumor-specific genetic changes and may provide a measurable classifier for assessing clinical outcomes in advanced CRC patients. © 2017 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

  9. 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.

  10. 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.

  11. 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.

  12. BCRgt: a Bayesian cluster regression-based genotyping algorithm for the samples with copy number alterations

    PubMed Central

    2014-01-01

    Background Accurate genotype calling is a pre-requisite of a successful Genome-Wide Association Study (GWAS). Although most genotyping algorithms can achieve an accuracy rate greater than 99% for genotyping DNA samples without copy number alterations (CNAs), almost all of these algorithms are not designed for genotyping tumor samples that are known to have large regions of CNAs. Results This study aims to develop a statistical method that can accurately genotype tumor samples with CNAs. The proposed method adds a Bayesian layer to a cluster regression model and is termed a Bayesian Cluster Regression-based genotyping algorithm (BCRgt). We demonstrate that high concordance rates with HapMap calls can be achieved without using reference/training samples, when CNAs do not exist. By adding a training step, we have obtained higher genotyping concordance rates, without requiring large sample sizes. When CNAs exist in the samples, accuracy can be dramatically improved in regions with DNA copy loss and slightly improved in regions with copy number gain, comparing with the Bayesian Robust Linear Model with Mahalanobis distance classifier (BRLMM). Conclusions In conclusion, we have demonstrated that BCRgt can provide accurate genotyping calls for tumor samples with CNAs. PMID:24629125

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

    PubMed Central

    Liu, Pengfei; Erez, Ayelet; Sreenath Nagamani, Sandesh C.; 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.

    2011-01-01

    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. PMID:21355048

  14. K13 mutations and pfmdr1 copy number variation in Plasmodium falciparum malaria in Myanmar.

    PubMed

    Win, Aye A; Imwong, Mallika; Kyaw, Myat P; Woodrow, Charles J; Chotivanich, Kesinee; Hanboonkunupakarn, Borimas; Pukrittayakamee, Sasithon

    2016-02-24

    Artemisinin-based combination therapy has been first-line treatment for falciparum malaria in Myanmar since 2005. The wide extent of artemisinin resistance in the Greater Mekong sub-region and the presence of mefloquine resistance at the Myanmar-Thailand border raise concerns over resistance patterns in Myanmar. The availability of molecular markers for resistance to both drugs enables assessment even in remote malaria-endemic areas. A total of 250 dried blood spot samples collected from patients with Plasmodium falciparum malarial infection in five malaria-endemic areas across Myanmar were analysed for kelch 13 sequence (k13) and pfmdr1 copy number variation. K13 mutations in the region corresponding to amino acids 210-726 (including the propeller region of the protein) were detected by nested PCR amplification and sequencing, and pfmdr1 copy number variation by real-time PCR. In two sites, a sub-set of patients were prospectively followed up for assessment of day-3 parasite clearance rates after a standard course of artemether-lumefantrine. K13 mutations and pfmdr1 amplification were successfully analysed in 206 and 218 samples, respectively. Sixty-nine isolates (33.5 %) had mutations within the k13 propeller region with 53 of these (76.8 %) having mutations already known to be associated with artemisinin resistance. F446I (32 isolates) and P574L (15 isolates) were the most common examples. K13 mutation was less common in sites in western border regions (29 of 155 isolates) compared to samples from the east and north (40 of 51 isolates; p < 0.0001). The overall proportion of parasites with multiple pfmdr1 copies (greater than 1.5) was 5.5 %. Seven samples showed both k13 mutation and multiple copies of pfmdr1. Only one of 36 patients followed up after artemether-lumefantrine treatment still had parasites at day 3; molecular analysis indicated wild-type k13 and single copy pfmdr1. The proportion of P. falciparum isolates with mutations in the propeller region of k

  15. Adaptation of the osmotolerant yeast Zygosaccharomyces rouxii to an osmotic environment through copy number amplification of FLO11D.

    PubMed

    Watanabe, Jun; Uehara, Kenji; Mogi, Yoshinobu

    2013-10-01

    Copy number variations (CNVs) contribute to the adaptation process in two possible ways. First, they may have a direct role, in which a certain number of copies often provide a selective advantage. Second, CNVs can also indirectly contribute to adaptation because a higher copy number increases the so-called "mutational target size." In this study, we show that the copy number amplification of FLO11D in the osmotolerant yeast Zygosaccharomyces rouxii promotes its further adaptation to a flor-formative environment, such as osmostress static culture conditions. We demonstrate that a gene, which was identified as FLO11D, is responsible for flor formation and that its expression is induced by osmostress under glucose-free conditions, which confer unique characteristics to Z. rouxii, such as osmostress-dependent flor formation. This organism possesses zero to three copies of FLO11D, and it appears likely that the FLO11D copy number increased in a branch of the Z. rouxii tree. The cellular hydrophobicity correlates with the FLO11D copy number, and the strain with a higher copy number of FLO11D exhibits a fitness advantage compared to a reference strain under osmostress static culture conditions. Our data indicate that the FLO gene-related system in Z. rouxii has evolved remarkably to adapt to osmostress environments.

  16. Adaptation of the Osmotolerant Yeast Zygosaccharomyces rouxii to an Osmotic Environment Through Copy Number Amplification of FLO11D

    PubMed Central

    Watanabe, Jun; Uehara, Kenji; Mogi, Yoshinobu

    2013-01-01

    Copy number variations (CNVs) contribute to the adaptation process in two possible ways. First, they may have a direct role, in which a certain number of copies often provide a selective advantage. Second, CNVs can also indirectly contribute to adaptation because a higher copy number increases the so-called “mutational target size.” In this study, we show that the copy number amplification of FLO11D in the osmotolerant yeast Zygosaccharomyces rouxii promotes its further adaptation to a flor-formative environment, such as osmostress static culture conditions. We demonstrate that a gene, which was identified as FLO11D, is responsible for flor formation and that its expression is induced by osmostress under glucose-free conditions, which confer unique characteristics to Z. rouxii, such as osmostress-dependent flor formation. This organism possesses zero to three copies of FLO11D, and it appears likely that the FLO11D copy number increased in a branch of the Z. rouxii tree. The cellular hydrophobicity correlates with the FLO11D copy number, and the strain with a higher copy number of FLO11D exhibits a fitness advantage compared to a reference strain under osmostress static culture conditions. Our data indicate that the FLO gene-related system in Z. rouxii has evolved remarkably to adapt to osmostress environments. PMID:23893487

  17. Using XHMM software to detect copy number variation in whole-exome sequencing data

    PubMed Central

    Fromer, Menachem; Purcell, Shaun M.

    2014-01-01

    Copy number variation (CNV) has emerged as an important genetic component in human diseases, which are increasingly being studied for large numbers of samples by sequencing the coding regions of the genome, i.e., exome sequencing. Nonetheless, detecting this variation from such targeted sequencing data is a difficult task of sorting out signal from noise, for which we have recently developed a set of statistical and computational tools called XHMM. In this paper, we give detailed instructions on how to run XHMM and how to use the resulting CNV calls in biological analyses. PMID:24763994

  18. 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

  19. 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.

  20. The Tnt1 family member Retrosol copy number and structure disclose retrotransposon diversification in different Solanum species.

    PubMed

    Manetti, M E; Rossi, M; Nakabashi, M; Grandbastien, M A; Van Sluys, Marie Anne

    2009-03-01

    Eukaryotic genome expansion/retraction caused by LTR-retrotransposon activity is dependent on the expression of full length copies to trigger efficient transposition and recombination-driven events. The Tnt1 family of retrotransposons has served as a model to evaluate the diversity among closely related elements within Solanaceae species and found that members of the family vary mainly in their U3 region of the long terminal repeats (LTRs). Recovery of a full length genomic copy of Retrosol was performed through a PCR-based approach from wild potato, Solanum oplocense. Further characterization focusing on both LTR sequences of the amplified copy allowed estimating an approximate insertion time at 2 million years ago thus supporting the occurrence of transposition cycles after genus divergence. Copy number of Tnt1-like elements in Solanum species were determined through genomic quantitative PCR whereby results sustain that Retrosol in Solanum species is a low copy number retrotransposon (1-4 copies) while Retrolyc1 has an intermediate copy number (38 copies) in S. peruvianum. Comparative analysis of retrotransposon content revealed no correlation between genome size or ploidy level and Retrosol copy number. The tetraploid cultivated potato with a cellular genome size of 1,715 Mbp harbours similar copy number per monoploid genome than other diploid Solanum species (613-884 Mbp). Conversely, S. peruvianum genome (1,125 Mbp) has a higher copy number. These results point towards a lineage specific dynamic flux regarding the history of amplification/activity of Tnt1-like elements in the genome of Solanum species.

  1. Classification of human cancers based on DNA copy number amplification modeling

    PubMed Central

    Myllykangas, Samuel; Tikka, Jarkko; Böhling, Tom; Knuutila, Sakari; Hollmén, Jaakko

    2008-01-01

    Background DNA amplifications alter gene dosage in cancer genomes by multiplying the gene copy number. Amplifications are quintessential in a considerable number of advanced cancers of various anatomical locations. The aims of this study were to classify human cancers based on their amplification patterns, explore the biological and clinical fundamentals behind their amplification-pattern based classification, and understand the characteristics in human genomic architecture that associate with amplification mechanisms. Methods We applied a machine learning approach to model DNA copy number amplifications using a data set of binary amplification records at chromosome sub-band resolution from 4400 cases that represent 82 cancer types. Amplification data was fused with background data: clinical, histological and biological classifications, and cytogenetic annotations. Statistical hypothesis testing was used to mine associations between the data sets. Results Probabilistic clustering of each chromosome identified 111 amplification models and divided the cancer cases into clusters. The distribution of classification terms in the amplification-model based clustering of cancer cases revealed cancer classes that were associated with specific DNA copy number amplification models. Amplification patterns – finite or bounded descriptions of the ranges of the amplifications in the chromosome – were extracted from the clustered data and expressed according to the original cytogenetic nomenclature. This was achieved by maximal frequent itemset mining using the cluster-specific data sets. The boundaries of amplification patterns were shown to be enriched with fragile sites, telomeres, centromeres, and light chromosome bands. Conclusions Our results demonstrate that amplifications are non-random chromosomal changes and specifically selected in tumor tissue microenvironment. Furthermore, statistical evidence showed that specific chromosomal features co-localize with

  2. Antigen-presenting genes and genomic copy number variations in the Tasmanian devil MHC

    PubMed Central

    2012-01-01

    Background The Tasmanian devil (Sarcophilus harrisii) is currently under threat of extinction due to an unusual fatal contagious cancer called Devil Facial Tumour Disease (DFTD). DFTD is caused by a clonal tumour cell line that is transmitted between unrelated individuals as an allograft without triggering immune rejection due to low levels of Major Histocompatibility Complex (MHC) diversity in Tasmanian devils. Results Here we report the characterization of the genomic regions encompassing MHC Class I and Class II genes in the Tasmanian devil. Four genomic regions approximately 960 kb in length were assembled and annotated using BAC contigs and physically mapped to devil Chromosome 4q. 34 genes and pseudogenes were identified, including five Class I and four Class II loci. Interestingly, when two haplotypes from two individuals were compared, three genomic copy number variants with sizes ranging from 1.6 to 17 kb were observed within the classical Class I gene region. One deletion is particularly important as it turns a Class Ia gene into a pseudogene in one of the haplotypes. This deletion explains the previously observed variation in the Class I allelic number between individuals. The frequency of this deletion is highest in the northwestern devil population and lowest in southeastern areas. Conclusions The third sequenced marsupial MHC provides insights into the evolution of this dynamic genomic region among the diverse marsupial species. The two sequenced devil MHC haplotypes revealed three copy number variations that are likely to significantly affect immune response and suggest that future work should focus on the role of copy number variations in disease susceptibility in this species. PMID:22404855

  3. Antigen-presenting genes and genomic copy number variations in the Tasmanian devil MHC.

    PubMed

    Cheng, Yuanyuan; Stuart, Andrew; Morris, Katrina; Taylor, Robyn; Siddle, Hannah; Deakin, Janine; Jones, Menna; Amemiya, Chris T; Belov, Katherine

    2012-03-12

    The Tasmanian devil (Sarcophilus harrisii) is currently under threat of extinction due to an unusual fatal contagious cancer called Devil Facial Tumour Disease (DFTD). DFTD is caused by a clonal tumour cell line that is transmitted between unrelated individuals as an allograft without triggering immune rejection due to low levels of Major Histocompatibility Complex (MHC) diversity in Tasmanian devils. Here we report the characterization of the genomic regions encompassing MHC Class I and Class II genes in the Tasmanian devil. Four genomic regions approximately 960 kb in length were assembled and annotated using BAC contigs and physically mapped to devil Chromosome 4q. 34 genes and pseudogenes were identified, including five Class I and four Class II loci. Interestingly, when two haplotypes from two individuals were compared, three genomic copy number variants with sizes ranging from 1.6 to 17 kb were observed within the classical Class I gene region. One deletion is particularly important as it turns a Class Ia gene into a pseudogene in one of the haplotypes. This deletion explains the previously observed variation in the Class I allelic number between individuals. The frequency of this deletion is highest in the northwestern devil population and lowest in southeastern areas. The third sequenced marsupial MHC provides insights into the evolution of this dynamic genomic region among the diverse marsupial species. The two sequenced devil MHC haplotypes revealed three copy number variations that are likely to significantly affect immune response and suggest that future work should focus on the role of copy number variations in disease susceptibility in this species.

  4. 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

  5. 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

  6. 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

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

    PubMed

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

    2017-02-06

    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.

  8. Construction of plasmids with tunable copy numbers in Saccharomyces cerevisiae and their applications in pathway optimization and multiplex genome integration.

    PubMed

    Lian, Jiazhang; Jin, Run; Zhao, Huimin

    2016-11-01

    The CEN/ARS-based low-copy plasmids and 2 μ-based high-copy plasmids have been broadly used for both fundamental studies and practical applications in Saccharomyces cerevisiae. However, the relative low copy numbers and narrow dynamic range limit their applications in many cases. In this study, the expression level of the selection marker proteins was engineered to increase the plasmid copy numbers. A series of plasmids with step-wise increased copy numbers were constructed. The copy number of the plasmids with engineered dominant markers (5-100 copies per cell) showed a positive correlation with the concentration of antibiotics supplemented to the growth media. Based on this finding, we developed a simple yet highly efficient strategy, named Pathway Optimization by Tuning Antibiotic Concentrations (POTAC) to rapidly balance the flux of multi-gene pathways at the DNA level in S. cerevisiae. As proof of concept, POTAC was used to optimize the lycopene and n-butanol biosynthetic pathways, increasing the production of lycopene and n-butanol by 10- and 100-fold, respectively. Additionally, multiplex genome integration with controllable copy numbers was attempted by combining the engineered dominant markers with the CRISPR/Cas9 system. Biotechnol. Bioeng. 2016;113: 2462-2473. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  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. 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.

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. Chronic radiation exposure of neuroblastoma cells reduces nMYC copy number.

    PubMed

    Gnanamony, Manu; Antony, Reuben; Fernández, Karen S; Jaime, Libes; Lin, Julian; Joseph, Pushpa A; Gondi, Christopher S

    2017-09-01

    Neuroblastoma accounts for >15% of cancer-associated mortalities of children in the USA. Despite aggressive treatment regimens, the long-term survival for these children remains <40%. The identification of v-Myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (nMYC) gene amplification during diagnosis is associated with poor prognosis in neuroblastoma. There are limited studies examining changes in nMYC copy numbers in response to therapy and its biological effect on cancer cells. The aim of the present study was to evaluate the effect of radiation on nMYC expression and amplification status in high-risk neuroblastoma. The effect of acute (5 Gy) and chronic (25 Gy) radiation on two nMYC-amplified cell lines, SK-N-BE (2) and NB-1691, was investigated. The results demonstrate that, following chronic but not acute radiation, the two cell lines regained their proliferation potential similar to the controls. This increased proliferation was characterized by loss of nMYC mRNA and protein expression. It was also revealed that nMYC loss was accompanied by nuclear localization of c-Myc. Using fluorescent in situ hybridization and quantitative polymerase chain reaction analysis, the results of the present study demonstrated that chronic radiation causes a severe loss of nMYC gene copy number. The present study is the first to provide experimental evidence that prolonged radiation therapy affects nMYC gene copy number in high-risk neuroblastoma but does not significantly improve the prognostic outlook.

  18. 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.

  19. 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

  20. Effects of Biofilm Growth on Plasmid Copy Number and Expression of Antibiotic Resistance Genes in Enterococcus faecalis

    PubMed Central

    Cook, L. C.

    2013-01-01

    Biofilm growth causes increased average plasmid copy number as well as increased copy number heterogeneity in Enterococcus faecalis cells carrying plasmid pCF10. In this study, we examined whether biofilm growth affected the copy number and expression of antibiotic resistance determinants for several plasmids with diverse replication systems. Four different E. faecalis plasmids, unrelated to pCF10, demonstrated increased copy number in biofilm cells. In biofilm cells, we also observed increased transcription of antibiotic resistance genes present on these plasmids. The increase in plasmid copy number correlated with increased plating efficiency on high concentrations of antibiotics. Single-cell analysis of strains carrying two different plasmids suggested that the increase in plasmid copy number associated with biofilm growth was restricted to a subpopulation of biofilm cells. Regrowth of harvested biofilm cells in liquid culture resulted in a rapid reduction of plasmid copy number to that observed in the planktonic state. These results suggest a possible mechanism by which biofilm growth could reduce susceptibility to antibiotics in clinical settings. PMID:23380728

  1. Association between Salivary Mitochondrial DNA Copy Number and Chronic Fatigue according to Combined Symptoms in Korean Adults.

    PubMed

    Shin, Jinyoung; Kim, Kyong Chol; Lee, Duk Chul; Lee, Hye Ree; Shim, Jae Yong

    2017-07-01

    We examined the association between salivary mitochondrial DNA (mtDNA) copy number and chronic fatigue combined with depression and insomnia. This cross-sectional study included 58 healthy adults with moderate to severe fatigue (Brief Fatigue Inventory [BFI] ≥4) for longer than 6 months. Subjects were classified as those without combined symptoms, with either depression (Beck Depression Inventory [BDI] ≥13) or insomnia (Pittsburgh Sleep Quality Index [PSQI] ≥5), or with both depression and insomnia. Salivary mtDNA copy number was measured by real-time quantitative polymerase chain reaction. The association was evaluated using a general linear model. About 76% of participants had either depression or insomnia as additional symptoms. These subjects were predominately female, drank more alcohol, and exercised less than those without combined symptoms (P<0.05). The group with both depression and insomnia exhibited significantly higher BFI and lower mtDNA copy number than those without combined symptoms (P<0.05). After adjusting for confounding factors, significant negative associations between mtDNA copy number and usual fatigue were found in the group without combined symptoms, whereas the negative associations in the group with combined symptoms were attenuated. BDI and PSQI were not associated with mtDNA copy number. Chronic fatigue is negatively associated with salivary mtDNA copy number. Salivary mtDNA copy number may be a biological marker of fatigue with or without combined symptoms, indicating that a separate approach is necessary.

  2. A Double-Layered Mixture Model for the Joint Analysis of DNA Copy Number and Gene Expression Data

    PubMed Central

    Choi, Hyungwon; Qin, Zhaohui S.

    2010-01-01

    Abstract Copy number aberration is a common form of genomic instability in cancer. Gene expression is closely tied to cytogenetic events by the central dogma of molecular biology, and serves as a mediator of copy number changes in disease phenotypes. Accordingly, it is of interest to develop proper statistical methods for jointly analyzing copy number and gene expression data. This work describes a novel Bayesian inferential approach for a double-layered mixture model (DLMM) which directly models the stochastic nature of copy number data and identifies abnormally expressed genes due to aberrant copy number. Simulation studies were conducted to illustrate the robustness of DLMM under various settings of copy number aberration frequency, confounding effects, and signal-to-noise ratio in gene expression data. Analysis of a real breast cancer data shows that DLMM is able to identify expression changes specifically attributable to copy number aberration in tumors and that a sample-specific index built based on the selected genes is correlated with relevant clinical information. PMID:20170400

  3. 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

  4. The joint effect of air pollution exposure and copy number variation on risk for autism.

    PubMed

    Kim, Dokyoon; Volk, Heather; Girirajan, Santhosh; Pendergrass, Sarah; Hall, Molly A; Verma, Shefali S; Schmidt, Rebecca J; Hansen, Robin L; Ghosh, Debashis; Ludena-Rodriguez, Yunin; Kim, Kyoungmi; Ritchie, Marylyn D; Hertz-Picciotto, Irva; Selleck, Scott B

    2017-09-01

    Autism spectrum disorder is a complex trait with a high degree of heritability as well as documented susceptibility from environmental factors. In this study the contributions of copy number variation, exposure to air pollutants, and the interaction between the two on autism risk, were evaluated in the population-based case-control Childhood Autism Risks from Genetics and Environment (CHARGE) Study. For the current investigation, we included only those CHARGE children (a) who met criteria for autism or typical development and (b) for whom our team had conducted both genetic evaluation of copy number burden and determination of environmental air pollution exposures based on mapping addresses from the pregnancy and early childhood. This sample consisted of 158 cases of children with autism and 147 controls with typical development. Multiple logistic regression models were fit with and without environmental variable-copy number burden interactions. We found no correlation between average air pollution exposure from conception to age 2 years and the child's CNV burden. We found a significant interaction in which a 1SD increase in duplication burden combined with a 1SD increase in ozone exposure was associated with an elevated autism risk (OR 3.4, P < 0.005) much greater than the increased risks associated with either genomic duplication (OR 1.85, 95% CI 1.25-2.73) or ozone (OR 1.20, 95% CI 0.93-1.54) alone. Similar results were obtained when CNV and ozone were dichotomized to compare those in the top quartile relative to those having a smaller CNV burden and lower exposure to ozone, and when exposures were assessed separately for pregnancy, the first year of life, and the second year of life. No interactions were observed for other air pollutants, even those that demonstrated main effects; ozone tends to be negatively correlated with the other pollutants examined. While earlier work has demonstrated interactions between the presence of a pathogenic CNV and an

  5. Critical evaluation of HPV16 gene copy number quantification by SYBR green PCR.

    PubMed

    Roberts, Ian; Ng, Grace; Foster, Nicola; Stanley, Margaret; Herdman, Michael T; Pett, Mark R; Teschendorff, Andrew; Coleman, Nicholas

    2008-07-24

    Human papilloma virus (HPV) load and physical status are considered useful parameters for clinical evaluation of cervical squamous cell neoplasia. However, the errors implicit in HPV gene quantification by PCR are not well documented. We have undertaken the first rigorous evaluation of the errors that can be expected when using SYBR green qPCR for quantification of HPV type 16 gene copy numbers. We assessed a modified method, in which external calibration curves were generated from a single construct containing HPV16 E2, HPV16 E6 and the host gene hydroxymethylbilane synthase in a 1:1:1 ratio. When testing dilutions of mixed HPV/host DNA in replicate runs, we observed errors in quantifying E2 and E6 amplicons of 5-40%, with greatest error at the lowest DNA template concentration (3 ng/microl). Errors in determining viral copy numbers per diploid genome were 13-53%. Nevertheless, in cervical keratinocyte cell lines we observed reasonable agreement between viral loads determined by qPCR and Southern blotting. The mean E2/E6 ratio in episome-only cells was 1.04, but with a range of 0.76-1.32. In three integrant-only lines the mean E2/E6 ratios were 0.20, 0.72 and 2.61 (values confirmed by gene-specific Southern blotting). When E2/E6 ratios in fourteen HPV16-positive cervical carcinomas were analysed, conclusions regarding viral physical state could only be made in three cases, where the E2/E6 ratio was < or = 0.06. Run-to-run variation in SYBR green qPCR produces unavoidable inaccuracies that should be allowed for when quantifying HPV gene copy number. While E6 copy numbers can be considered to provide a useable indication of viral loads, the E2/E6 ratio is of limited value. Previous studies may have overestimated the frequency of mixed episomal/integrant HPV infections.

  6. YMAP: a pipeline for visualization of copy number variation and loss of heterozygosity in eukaryotic pathogens.

    PubMed

    Abbey, Darren A; Funt, Jason; Lurie-Weinberger, Mor N; Thompson, Dawn A; Regev, Aviv; Myers, Chad L; Berman, Judith

    2014-01-01

    The design of effective antimicrobial therapies for serious eukaryotic pathogens requires a clear understanding of their highly variable genomes. To facilitate analysis of copy number variations, single nucleotide polymorphisms and loss of heterozygosity events in these pathogens, we developed a pipeline for analyzing diverse genome-scale datasets from microarray, deep sequencing, and restriction site associated DNA sequence experiments for clinical and laboratory strains of Candida albicans, the most prevalent human fungal pathogen. The YMAP pipeline (http://lovelace.cs.umn.edu/Ymap/) automatically illustrates genome-wide information in a single intuitive figure and is readily modified for the analysis of other pathogens with small genomes.

  7. 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

  8. 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)

  9. 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

  10. Association between Mitochondrial DNA Copy Number in Peripheral Blood and Incident CKD in the Atherosclerosis Risk in Communities Study.

    PubMed

    Tin, Adrienne; Grams, Morgan E; Ashar, Foram N; Lane, John A; Rosenberg, Avi Z; Grove, Megan L; Boerwinkle, Eric; Selvin, Elizabeth; Coresh, Josef; Pankratz, Nathan; Arking, Dan E

    2016-08-01

    Mitochondrial dysfunction in kidney cells has been implicated in the pathogenesis of CKD. Mitochondrial DNA (mtDNA) copy number is a surrogate measure of mitochondrial function, and higher mtDNA copy number in peripheral blood has been associated with lower risk of two important risk factors for CKD progression, diabetes and microalbuminuria. We evaluated whether mtDNA copy number in peripheral blood associates with incident CKD in a population-based cohort of middle-aged adults. We estimated mtDNA copy number using 25 high-quality mitochondrial single nucleotide polymorphisms from the Affymetrix 6.0 array. Among 9058 participants, those with higher mtDNA copy number had a lower rate of prevalent diabetes and lower C-reactive protein levels and white blood cell counts. Baseline eGFR did not differ significantly by mtDNA copy number. Over a median follow-up of 19.6 years, 1490 participants developed CKD. Higher mtDNA copy number associated with lower risk of incident CKD (highest versus lowest quartile: hazard ratio 0.65; 95% confidence interval, 0.56 to 0.75; P<0.001) after adjusting for age, sex, and race. After adjusting for additional risk factors of CKD, including prevalent diabetes, hypertension, C-reactive protein level, and white blood cell count, this association remained significant (highest versus lowest quartile: hazard ratio 0.75; 95% confidence interval, 0.64 to 0.87; P<0.001). In conclusion, higher mtDNA copy number associated with lower incidence of CKD independent of traditional risk factors and inflammation biomarker levels in this cohort. Further research on modifiable factors influencing mtDNA copy number may lead to improvement in the prevention and treatment of CKD. Copyright © 2016 by the American Society of Nephrology.

  11. Assessment of epidermal growth factor receptor mutation/copy number and K-ras mutation in esophageal cancer.

    PubMed

    Guo, Kang; Wang, Wu-Ping; Jiang, Tao; Wang, Ju-Zheng; Chen, Zhao; Li, Yong; Zhou, Yong-An; Li, Xiao-Fei; Lu, Qiang; Zhang, Lan-Jun

    2016-07-01

    The molecular status of epidermal growth factor receptor (EGFR) in esophageal cancer has not been well elucidated. The purpose of the study was to investigate the prevalence of EGFR and K-ras mutation, and EGFR gene copy number status as well as its association with clinicopathologic characteristics, and also to identify the prognostic value of EGFR gene copy number in esophageal cancer. EGFR mutation in exon 19/exon 21 and K-ras mutation in codon 12/codon 13 were detected by real-time PCR method, while EGFR gene copy number status was analyzed by fluorescent in situ hybridization (FISH). EGFR gene amplification and high polysomy were defined as high EGFR gene copy number status (FISH-positive), and all else were defined as low EGFR gene copy number status (FISH-negative). The relationship between EGFR gene copy number status and clinicpathologic characteristics was analyzed. Kaplan-Meier method and Cox proportional hazards regression model were employed to evaluate the effects of EGFR gene copy number status on the patients' survival. A total of 57 esophageal squamous cell carcinoma (ESCC) patients and 9 esophageal adenocarcinoma (EADC) patients were enrolled in the study. EGFR mutation was identified in one patient who was diagnosed as ESCC with stage IIIC disease. K-ras mutation was identified in one patient who was diagnosed as EADC. In all, 34 of 66 (51.5%) samples were detected as FISH-positive, which includes 30 ESCC and 4 EADC tumor samples. The correlation analysis showed that FISH-positive was significantly associated with the tumor stage (P=0.019) and lymph node metastasis (P=0.005) in esophageal cancer patients, and FISH-positive was also significantly associated with the tumor stage (P=0.007) and lymph node metastasis (P=0.008) in ESCC patients. Cox regression analysis showed that high EGFR gene copy number was not a significant predictor of a poor outcome for esophageal cancer patients (P=0.251) or for ESCC patients (P=0.092), but esophageal cancer

  12. 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.

  13. Novel Population Specific Autosomal Copy Number Variation and Its Functional Analysis amongst Negritos from Peninsular Malaysia

    PubMed Central

    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. PMID:24956385

  14. 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.

  15. A method for generating new datasets based on copy number for cancer analysis.

    PubMed

    Kim, Shinuk; Kon, Mark; Kang, Hyunsik

    2015-01-01

    New data sources for the analysis of cancer data are rapidly supplementing the large number of gene-expression markers used for current methods of analysis. Significant among these new sources are copy number variation (CNV) datasets, which typically enumerate several hundred thousand CNVs distributed throughout the genome. Several useful algorithms allow systems-level analyses of such datasets. However, these rich data sources have not yet been analyzed as deeply as gene-expression data. To address this issue, the extensive toolsets used for analyzing expression data in cancerous and noncancerous tissue (e.g., gene set enrichment analysis and phenotype prediction) could be redirected to extract a great deal of predictive information from CNV data, in particular those derived from cancers. Here we present a software package capable of preprocessing standard Agilent copy number datasets into a form to which essentially all expression analysis tools can be applied. We illustrate the use of this toolset in predicting the survival time of patients with ovarian cancer or glioblastoma multiforme and also provide an analysis of gene- and pathway-level deletions in these two types of cancer.

  16. 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 t