Sample records for 16q copy number

  1. Measuring human salivary amylase copy number variation

    Dhar, Sugandha


    Copy number variations represent large scale genomic alterations varying from 1kb to 3Mb and are proposed as a driving force for genome evolution and variation. One such locus exhibiting copy number variation and genome evolution is salivary amylase, which is responsible for the digestion of starch in the human parotid glands. It was reported that since human salivary amylase gene (AMY1) copy numbers are correlated positively with protein levels, and also due to the correlation of high gene c...

  2. Variable copy number DNA sequences in rice.

    Kikuchi, S; Takaiwa, F; Oono, K


    We have cloned two types of variable copy number DNA sequences from the rice embryo genome. One of these sequences, which was cloned in pRB301, was amplified about 50-fold during callus formation and diminished in copy number to the embryonic level during regeneration. The other clone, named pRB401, showed the reciprocal pattern. The copy numbers of both sequences were changed even in the early developmental stage and eliminated from nuclear DNA along with growth of the plant. Sequencing analysis of the pRB301 insert revealed some open reading frames and direct repeat structures, but corresponding sequences were not identified in the EMBL and LASL DNA databases. Sequencing of the nuclear genomic fragment cloned in pRB401 revealed the presence of the 3'rps12-rps7 region of rice chloroplast DNA. Our observations suggest that during callus formation (dedifferentiation), regeneration and the growth process the copy numbers of some DNA sequences are variable and that nuclear integrated chloroplast DNA acts as a variable copy number sequence in the rice genome. Based on data showing a common sequence in mitochondria and chloroplast DNA of maize (Stern and Lonsdale 1982) and that the rps12 gene of tobacco chloroplast DNA is a divided gene (Torazawa et al. 1986), it is suggested that the sequence on the inverted repeat structure of chloroplast DNA may have the character of a movable genetic element. PMID:3481021

  3. Rare copy number variation in cerebral palsy

    McMichael, Gai; Girirajan, Santhosh; Moreno-De-Luca, Andres; Gecz, Jozef; Shard, Chloe; Nguyen, Lam Son; Nicholl, Jillian; Gibson, Catherine; Haan, Eric; Eichler, Evan; Martin, Christa Lese; MacLennan, Alastair


    Recent studies have established the role of rare copy number variants (CNVs) in several neurological disorders but the contribution of rare CNVs to cerebral palsy (CP) is not known. Fifty Caucasian families having children with CP were studied using two microarray designs. Potentially pathogenic, rare (

  4. Getting DNA copy numbers without control samples

    Ortiz-Estevez Maria


    Full Text Available Abstract 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

  5. Identification of copy number variants in horses

    Doan, R.


    Copy number variants (CNVs) represent a substantial source of genetic variation in mammals. However, the occurrence of CNVs in horses and their subsequent impact on phenotypic variation is unknown. We performed a study to identify CNVs in 16 horses representing 15 distinct breeds (Equus caballus) and an individual gray donkey (Equus asinus) using a whole-exome tiling array and the array comparative genomic hybridization methodology. We identified 2368 CNVs ranging in size from 197 bp to 3.5 Mb. Merging identical CNVs from each animal yielded 775 CNV regions (CNVRs), involving 1707 protein- and RNA-coding genes. The number of CNVs per animal ranged from 55 to 347, with median and mean sizes of CNVs of 5.3 kb and 99.4 kb, respectively. Approximately 6% of the genes investigated were affected by a CNV. Biological process enrichment analysis indicated CNVs primarily affected genes involved in sensory perception, signal transduction, and metabolism. CNVs also were identified in genes regulating blood group antigens, coat color, fecundity, lactation, keratin formation, neuronal homeostasis, and height in other species. Collectively, these data are the first report of copy number variation in horses and suggest that CNVs are common in the horse genome and may modulate biological processes underlying different traits observed among horses and horse breeds.

  6. Profiling genomic copy number changes in retinoblastoma beyond loss of RB1.

    Bowles, Ella; Corson, Timothy W; Bayani, Jane; Squire, Jeremy A; Wong, Nathalie; Lai, Paul B-S; Gallie, Brenda L


    Loss of both RB1 alleles is rate limiting for development of retinoblastoma (RB), but genomic copy number gain or loss may impact oncogene(s) and tumor suppressor genes, facilitating tumor progression. We used quantitative multiplex polymerase chain reaction to profile "hot spot" genomic copy number changes for gain at 1q32.1, 6p22, and MYCN, and loss at 16q22 in 87 primary RB and 7 cell lines. Loss at 16q22 (48%) negatively associated with MYCN gain (18%) (Fisher's exact P = 0.031), gain at 1q32.1 (62%) positively associated with 6p "hot spot" gain (43%) (P = 0.033), and there was a trend for positive association between 1q and MYCN gain (P = 0.095). Cell lines had a higher frequency of MYCN amplification than primary tumors (29% versus 3%; P = 0.043). Novel high-level amplification of 1q32.1 in one primary tumor, confirmed by fluorescence in situ hybridization, strongly supports the presence of oncogene(s) in this region, possibly the mitotic kinesin, KIF14. Gene-specific quantitative multiplex polymerase chain reaction of candidate oncogenes at 1q32.1 (KIF14), 6p22 (E2F3 and DEK), and tumor suppressor genes at 16q22 (CDH11) and 17q21 (NGFR) showed the most common gene gains in RB to be KIF14 in cell lines (80%) and E2F3 in primary tumors (70%). The patterns of gain/loss were qualitatively different in 25 RB compared with 12 primary hepatocellular carcinoma and 12 breast cancer cell lines. Gene specific analysis of one bone marrow metastasis of RB, prechemotherapy and postchemotherapy, showed the typical genomic changes of RB pretreatment, which normalized after chemotherapy. PMID:17099872

  7. Copy number variation in the bovine genome

    Bendixen Christian


    Full Text Available Abstract 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.

  8. Copy number variation in the bovine genome

    Fadista, João; Thomsen, Bo; Holm, Lars-Erik;


    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...

  9. Sociobiological control of plasmid copy number in bacteria.

    Mukta M Watve

    Full Text Available All genes critical for plasmid replication regulation are located on the plasmid rather than on the host chromosome. It is possible therefore that there can be copy-up "cheater" mutants. In spite of this possibility, low copy number plasmids appear to exist stably in host populations. We examined this paradox using a multilevel selection model. Simulations showed that, a slightly higher copy number mutant could out-compete the wild type. Consequently, another mutant with still higher copy number could invade the first invader. However, the realized benefit of increasing intra-host fitness was saturating whereas that of inter-host fitness was exponential. As a result, above a threshold, intra-host selection was overcompensated by inter-host selection and the low copy number wild type plasmid could back invade a very high copy number plasmid. This led to a rock-paper-scissor (RPS like situation that allowed the coexistence of plasmids with varied copy numbers. Furthermore, another type of cheater that had lost the genes required for conjugation but could hitchhike on a conjugal plasmid, could further reduce the advantage of copy-up mutants. These sociobiological interactions may compliment molecular mechanisms of replication regulation in stabilizing the copy numbers.

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

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


    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. PMID:26258394

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

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


    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

  12. Copy Number Variants and Segmental Duplications Show Different Formation Signatures

    Kim, Philip M.; Korbel, Jan O.; Chen, Xueying; Gerstein, Mark B.


    In addition to variation in terms of single nucleotide polymorphisms (SNPs), whole regions ranging from several kilobases up to a megabase in length differ in copy number among individuals. These differences are referred to as Copy Number Variants (CNVs) and extensive mapping of these is underway. Recent studies have highlighted their great prevalence in the human genome. Segmental Duplications (SDs) are long (>1kb) stretches of duplicated DNA with high sequence identity. First, we analyzed t...

  13. Partitioning of copy-number genotypes in pedigrees

    Andelfinger Gregor U


    Full Text Available Abstract Background Copy number variations (CNVs and polymorphisms (CNPs have only recently gained the genetic community's attention. Conservative estimates have shown that CNVs and CNPs might affect more than 10% of the genome and that they may be at least as important as single nucleotide polymorphisms in assessing human variability. Widely used tools for CNP analysis have been implemented in Birdsuite and PLINK for the purpose of conducting genetic association studies based on the unpartitioned total number of CNP copies provided by the intensities from Affymetrix's Genome-Wide Human SNP Array. Here, we are interested in partitioning copy number variations and polymorphisms in extended pedigrees for the purpose of linkage analysis on familial data. Results We have developed CNGen, a new software for the partitioning of copy number polymorphism using the integrated genotypes from Birdsuite with the Affymetrix platform. The algorithm applied to familial trios or extended pedigrees can produce partitioned copy number genotypes with distinct parental alleles. We have validated the algorithm using simulations on a complex pedigree structure using frequencies calculated from a real dataset of 300 genotyped samples from 42 pedigrees segregating a congenital heart defect phenotype. Conclusions CNGen is the first published software for the partitioning of copy number genotypes in pedigrees, making possible the use CNPs and CNVs for linkage analysis. It was implemented with the Python interpreter version 2.5.2. It was successfully tested on current Linux, Windows and Mac OS workstations.

  14. Copy-number variants in neurodevelopmental disorders: promises and challenges.

    Merikangas, Alison K


    Copy-number variation (CNV) is the most prevalent type of structural variation in the human genome. There is emerging evidence that copy-number variants (CNVs) provide a new vista on understanding susceptibility to neuropsychiatric disorders. Some challenges in the interpretation of current CNV studies include the use of overlapping samples, differing phenotypic definitions, an absence of population norms for CNVs and a lack of consensus in methods for CNV detection and analysis. Here, we review current CNV association study methods and results in autism spectrum disorders (ASD) and schizophrenia, and provide suggestions for design approaches to future studies that might maximize the translation of this work to etiological understanding.

  15. Mapping cattle copy number variations in water buffalo

    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 ...

  16. Copy number variation of KIR genes influences HIV-1 control

    Pelak, Kimberly; Need, Anna C; Fellay, Jacques;


    A genome-wide screen for large structural variants showed that a copy number variant (CNV) in the region encoding killer cell immunoglobulin-like receptors (KIR) associates with HIV-1 control as measured by plasma viral load at set point in individuals of European ancestry. This CNV encompasses t...

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

    Wrede, Joanna E; Mengel-From, Jonas; Buchwald, Dedra;


    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...... 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...

  18. Analysis of copy number variation in the bovine genome

    We initiated a systematic study of the copy number variation (CNV) within the Bovine HapMap cattle population using array comparative genomic hybridization (array CGH). Oligonucleotide CGH arrays were designed and fabricated to provide a genome-wide coverage with an average interval of 6 kb using t...

  19. Quantum state discrimination using the minimum average number of copies

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


    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 consider minimizing the average resources for a fixed admissible error probability. We derive a detection scheme optimized for the latter task, and experimentally test it, along with schemes previously considered for the former task. We show that, for our new task, our new scheme outperforms all previously considered schemes.

  20. Visualization of ribosomal RNA operon copy number distribution

    DasGupta Indrani; Wu Martin; Rastogi Rajat; Fox George E


    Abstract Background Results of microbial ecology studies using 16S rRNA sequence information can be deceiving due to differences in rRNA operon copy number and genome size of the detected organisms. It therefore will be useful for investigators to have a better understanding of how these two parameters differ in various organism types. In this study, the number of ribosomal operons and genome size were separately mapped onto a Bacterial phylogenetic tree. Results A representative Bacterial tr...

  1. Copy Number Variation at the APOL1 Locus.

    Rupam Ruchi

    Full Text Available Two coding variants in the APOL1 gene (G1 and G2 explain most of the high rate of kidney disease in African Americans. APOL1-associated kidney disease risk inheritance follows an autosomal recessive pattern: The relative risk of kidney disease associated with inheritance of two high-risk variants is 7-30 fold, depending on the specific kidney phenotype. We wished to determine if the variability in phenotype might in part reflect structural differences in APOL1 gene. We analyzed sequence coverage from 1000 Genomes Project Phase 3 samples as well as exome sequencing data from African American kidney disease cases for copy number variation. 8 samples sequenced in the 1000 Genomes Project showed increased coverage over a ~100kb region that includes APOL2, APOL1 and part of MYH9, suggesting the presence of APOL1 copy number greater than 2. We reasoned that such duplications should be enriched in apparent G1 heterozygotes with kidney disease. Using a PCR-based assay, we observed the presence of this duplication in additional samples from apparent G0G1 or G0G2 individuals. The frequency of this APOL1 duplication was compared among cases (n = 123 and controls (n = 255 with apparent G0G1 heterozygosity. The presence of APOL1 duplication was observed in 4.06% of cases and 0.78% controls, preliminary evidence that this APOL1 duplication may alter susceptibility to kidney disease (p = 0.03. Taqman-based copy number assays confirmed the presence of 3 APOL1 copies in individuals positive for this specific duplication by PCR assay, but also identified a small number of individuals with additional APOL1 copies of presumably different structure. These observations motivate further studies to better assess the contribution of APOL1 copy number on kidney disease risk and on APOL1 function. Investigators and clinicians genotyping APOL1 should also consider whether the particular genotyping platform used is subject to technical errors when more than two copies of

  2. Comparative Copy Number Variation From Whole Genome Sequencing

    Janevski, A.; Varadan, V.; Kamalakaran, S.; Banerjee, N.; Dimitrova, D


    Whole genome sequencing enables a high resolution view of the humangenome and enables unique insights into copy number variations in anunprecedented scale. Numerous tools and studies have already been introduced that provide confirmatory and new genomic variability datain individuals and across populations. We investigate two such methods, CNV-seq and FREEC and compare their outputs when applied to five whole genome sequences representing four populations. We focus onthe ability of these tool...

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

    Tomita-Mitchell, Aoy; 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.


    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 appro...

  4. Clinically relevant copy number variations detected in cerebral palsy

    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.; Stephen W Scherer


    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 variat...

  5. Copy number alterations in small intestinal neuroendocrine tumors determined by array comparative genomic hybridization

    Small intestinal neuroendocrine tumors (SI-NETs) are typically slow-growing tumors that have metastasized already at the time of diagnosis. The purpose of the present study was to further refine and define regions of recurrent copy number (CN) alterations (CNA) in SI-NETs. Genome-wide CNAs was determined by applying array CGH (a-CGH) on SI-NETs including 18 primary tumors and 12 metastases. Quantitative PCR analysis (qPCR) was used to confirm CNAs detected by a-CGH as well as to detect CNAs in an extended panel of SI-NETs. Unsupervised hierarchical clustering was used to detect tumor groups with similar patterns of chromosomal alterations based on recurrent regions of CN loss or gain. The log rank test was used to calculate overall survival. Mann–Whitney U test or Fisher’s exact test were used to evaluate associations between tumor groups and recurrent CNAs or clinical parameters. The most frequent abnormality was loss of chromosome 18 observed in 70% of the cases. CN losses were also frequently found of chromosomes 11 (23%), 16 (20%), and 9 (20%), with regions of recurrent CN loss identified in 11q23.1-qter, 16q12.2-qter, 9pter-p13.2 and 9p13.1-11.2. Gains were most frequently detected in chromosomes 14 (43%), 20 (37%), 4 (27%), and 5 (23%) with recurrent regions of CN gain located to 14q11.2, 14q32.2-32.31, 20pter-p11.21, 20q11.1-11.21, 20q12-qter, 4 and 5. qPCR analysis confirmed most CNAs detected by a-CGH as well as revealed CNAs in an extended panel of SI-NETs. Unsupervised hierarchical clustering of recurrent regions of CNAs revealed two separate tumor groups and 5 chromosomal clusters. Loss of chromosomes 18, 16 and 11 and again of chromosome 20 were found in both tumor groups. Tumor group II was enriched for alterations in chromosome cluster-d, including gain of chromosomes 4, 5, 7, 14 and gain of 20 in chromosome cluster-b. Gain in 20pter-p11.21 was associated with short survival. Statistically significant differences were observed between primary

  6. Regulation of mitochondrial DNA copy number during spermatogenesis.

    Rantanen, A; Larsson, N G


    The nuclear genome is physically compacted during spermatogenesis by replacing histones with protamines and transition proteins. This altered nuclear protein context may make gene regulation at the transcriptional level less efficient and could explain why post-transcriptional regulation is prominent in haploid male germ cells. Mitochondria and mitochondrial (mt) DNA are maternally inherited, whereas the transmission of paternal mtDNA is blocked in mammals. The paternal mtDNA enters the oocyte but is no longer detectable in the preimplantation embryo. Several mechanisms could be responsible for preventing the transmission of paternal mtDNA, including the down-regulation of mtDNA copy number during spermatogenesis, specific elimination of paternal mitochondria in fertilized oocytes, and the suspension of mtDNA replication in the fertilized oocyte. It is the first of these that is the subject of the present review. Mitochondrial transcription factor A (mtTFA, or Tfam) is a key regulator of mtDNA copy number in mammals. Germ cell-specific Tfam transcript isoforms are expressed during spermatogenesis in mice and humans. These alternative Tfam transcript isoforms have a structure that could prevent protein translation; their expression coincides with down-regulation of the mitochondrial Tfam protein values. We propose that this down-regulation of mitochondrial Tfam protein levels in turn down-regulates mtDNA copy number during mammalian spermatogenesis. PMID:11041516

  7. Mitochondrial DNA copy number variation across human cancers.

    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


    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. PMID:26901439

  8. An integrated analysis of miRNA and gene copy numbers in xenografts of Ewing's sarcoma

    Mosakhani Neda


    Full Text Available Abstract Background Xenografts have been shown to provide a suitable source of tumor tissue for molecular analysis in the absence of primary tumor material. We utilized ES xenograft series for integrated microarray analyses to identify novel biomarkers. Method Microarray technology (array comparative genomic hybridization (aCGH and micro RNA arrays was used to screen and identify copy number changes and differentially expressed miRNAs of 34 and 14 passages, respectively. Incubated cells used for xenografting (Passage 0 were considered to represent the primary tumor. Four important differentially expressed miRNAs (miR-31, miR-31*, miR-145, miR-106 were selected for further validation by real time polymerase chain reaction (RT-PCR. Integrated analysis of aCGH and miRNA data was performed on 14 xenograft passages by bioinformatic methods. Results The most frequent losses and gains of DNA copy number were detected at 9p21.3, 16q and at 8, 15, 17q21.32-qter, 1q21.1-qter, respectively. The presence of these alterations was consistent in all tumor passages. aCGH profiles of xenograft passages of each series resembled their corresponding primary tumors (passage 0. MiR-21, miR-31, miR-31*, miR-106b, miR-145, miR-150*, miR-371-5p, miR-557 and miR-598 showed recurrently altered expression. These miRNAS were predicted to regulate many ES-associated genes, such as genes of the IGF1 pathway, EWSR1, FLI1 and their fusion gene (EWS-FLI1. Twenty differentially expressed miRNAs were pinpointed in regions carrying altered copy numbers. Conclusion In the present study, ES xenografts were successfully applied for integrated microarray analyses. Our findings showed expression changes of miRNAs that were predicted to regulate many ES associated genes, such as IGF1 pathway genes, FLI1, EWSR1, and the EWS-FLI1 fusion genes.

  9. Copy number variation of KIR genes influences HIV-1 control

    Pelak, Kimberly; Need, Anna C; Fellay, Jacques;


    KIR3DL1-KIR3DS1 locus, encoding receptors that interact with specific HLA-Bw4 molecules to regulate the activation of lymphocyte subsets including natural killer (NK) cells. We quantified the number of copies of KIR3DS1 and KIR3DL1 in a large HIV-1 positive cohort, and showed that an increase in KIR3...... individuals with multiple copies of KIR3DL1, in the presence of KIR3DS1 and the appropriate ligands, inhibit HIV-1 replication more robustly, and associated with a significant expansion in the frequency of KIR3DS1+, but not KIR3DL1+, NK cells in their peripheral blood. Our results suggest that the relative...

  10. Visualization of ribosomal RNA operon copy number distribution

    DasGupta Indrani


    Full Text Available Abstract Background Results of microbial ecology studies using 16S rRNA sequence information can be deceiving due to differences in rRNA operon copy number and genome size of the detected organisms. It therefore will be useful for investigators to have a better understanding of how these two parameters differ in various organism types. In this study, the number of ribosomal operons and genome size were separately mapped onto a Bacterial phylogenetic tree. Results A representative Bacterial tree was constructed using 31 marker genes found in 578 bacterial genome sequences. Organism names are displayed on the trees using graduations of color such that similar colors indicate similar numbers of operons or genome size. The resulting images provide an intuitive understanding of how copy number and genome size vary in different Bacterial phyla. Conclusion Once the phylogenetic position of a novel organism is known the number of rRNA operons, and to a lesser extent the genome size, can be estimated by examination of the colored maps. Further detail can then be obtained for members of relevant taxa from the rrnDB database.

  11. Mitochondrial DNA Copy Number in Spermatozoa of Fertile Stallions.

    Orsztynowicz, M; Pawlak, P; Podstawski, Z; Nizanski, W; Partyka, A; Gotowiecka, M; Kosiniak-Kamysz, K; Lechniak, D


    Predicting male fertility on non-invasive sperm traits is of big importance to human and animal reproduction strategies. Combining the wide range of parameters monitored by computer-assisted sperm analysis (CASA) with some molecular traits (e.g. mtDNA content) may help to identify markers of the male fertility. The aim of this study was to characterize variation in the mtDNA copy number in equine sperm and to investigate whether mtDNA content is correlated with quality traits of stallion spermatozoa and the age of the male. Ejaculates collected from 53 fertile stallions were divided into four age groups (3-5, 6-10, 11-14 and >15 years) and were subjected to a complex investigation including conventional analysis, CASA, flow cytometry and mtDNA content (real-time PCR). The mean (±SD) number of mtDNA copies equalled 14 ± 9 and varied from 3 to 64. Considering the great number of sperm parameters monitored in this study, only few of them were correlated with the mtDNA content: ejaculate volume (a positive correlation), the amplitude of lateral head displacement (ALH; a negative correlation) and the high mitochondrial activity index (a negative correlation). The stallion age was not correlated with the mtDNA copy number. This study provides the first set of data on mtDNA content in equine sperm and confirms phenomena previously described for humans and dog on associations between sperm mtDNA content and selected motility parameters monitored by the CASA. Basing our study on spermatozoa from fertile stallions could however limit the extent of detected associations. PMID:27037507

  12. Breast tumor copy number aberration phenotypes and genomic instability

    Genomic DNA copy number aberrations are frequent in solid tumors, although the underlying causes of chromosomal instability in tumors remain obscure. Genes likely to have genomic instability phenotypes when mutated (e.g. those involved in mitosis, replication, repair, and telomeres) are rarely mutated in chromosomally unstable sporadic tumors, even though such mutations are associated with some heritable cancer prone syndromes. We applied array comparative genomic hybridization (CGH) to the analysis of breast tumors. The variation in the levels of genomic instability amongst tumors prompted us to investigate whether alterations in processes/genes involved in maintenance and/or manipulation of the genome were associated with particular types of genomic instability. We discriminated three breast tumor subtypes based on genomic DNA copy number alterations. The subtypes varied with respect to level of genomic instability. We find that shorter telomeres and altered telomere related gene expression are associated with amplification, implicating telomere attrition as a promoter of this type of aberration in breast cancer. On the other hand, the numbers of chromosomal alterations, particularly low level changes, are associated with altered expression of genes in other functional classes (mitosis, cell cycle, DNA replication and repair). Further, although loss of function instability phenotypes have been demonstrated for many of the genes in model systems, we observed enhanced expression of most genes in tumors, indicating that over expression, rather than deficiency underlies instability. Many of the genes associated with higher frequency of copy number aberrations are direct targets of E2F, supporting the hypothesis that deregulation of the Rb pathway is a major contributor to chromosomal instability in breast tumors. These observations are consistent with failure to find mutations in sporadic tumors in genes that have roles in maintenance or manipulation of the genome

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


    Full Text Available 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

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

    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


    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

  15. Keratoconus is associated with increased copy number of mitochondrial DNA

    Abu-Amero, Khaled K; Altaf A. Kondkar; Azad, Taif Anwar; Sultan, Tahira; Kalantan, Hatem; Al-Muammar, Abdulrahman M.


    Purpose To investigate the possible association of oxidative stress with keratoconus (KC), we estimated the changes in relative mitochondrial DNA (mtDNA) content. Methods The study included 119 patients with KC and 208 controls matched for gender, ethnicity, and systemic disease status. We selected controls who were older than the patients since the mtDNA copy number tends to increase with age. The age mean (standard deviation) was 26.4(7.6) and 54.5(14.4) years for the patients and controls,...

  16. Copy number variation of KIR genes influences HIV-1 control.

    Kimberly Pelak


    Full Text Available A genome-wide screen for large structural variants showed that a copy number variant (CNV in the region encoding killer cell immunoglobulin-like receptors (KIR associates with HIV-1 control as measured by plasma viral load at set point in individuals of European ancestry. This CNV encompasses the KIR3DL1-KIR3DS1 locus, encoding receptors that interact with specific HLA-Bw4 molecules to regulate the activation of lymphocyte subsets including natural killer (NK cells. We quantified the number of copies of KIR3DS1 and KIR3DL1 in a large HIV-1 positive cohort, and showed that an increase in KIR3DS1 count associates with a lower viral set point if its putative ligand is present (p = 0.00028, as does an increase in KIR3DL1 count in the presence of KIR3DS1 and appropriate ligands for both receptors (p = 0.0015. We further provide functional data that demonstrate that NK cells from individuals with multiple copies of KIR3DL1, in the presence of KIR3DS1 and the appropriate ligands, inhibit HIV-1 replication more robustly, and associated with a significant expansion in the frequency of KIR3DS1+, but not KIR3DL1+, NK cells in their peripheral blood. Our results suggest that the relative amounts of these activating and inhibitory KIR play a role in regulating the peripheral expansion of highly antiviral KIR3DS1+ NK cells, which may determine differences in HIV-1 control following infection.

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

    Mona S. Jahromi


    Full Text Available 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.

  18. Reduced mitochondrial DNA copy number in Chinese patients with osteosarcoma.

    Yu, Man; Wan, Yanfang; Zou, Qinghua


    A plethora of somatic mutations and germline variations in mitochondrial DNA (mtDNA) have been increasingly reported in numerous cancer entities including osteosarcoma. However, it remains largely unclear whether mtDNA copy number changes occur during the multistep process of osteosarcoma carcinogenesis. For this purpose, we determined quantitative mtDNA levels in 31 primary osteosarcoma specimens and 5 normal bone tissue samples using a real-time polymerase chain reaction assay. Our data showed that the average mtDNA amount was significantly reduced in osteosarcoma tissues compared with normal bone controls. The copy number of mtDNA was statistically associated with tumor metastasis. There was an approximately 2-fold decrease of mtDNA quantity in tumors with metastasis than that in low-grade tumors without metastasis. Furthermore, change in mtDNA content was linked with somatic mutations in the D-loop regulatory region. Tumors carrying somatic D-loop mutations, at the polycytidine stretch between nucleotide positions 303 and 309 or close to the replication origin sites of the heavy strand, had significantly lowered mtDNA levels in comparison with those without mutations. Taken together, these results provide evidence for the first time that reduced mtDNA content may be critically implicated in the development and/or progression of osteosarcoma. Somatic D-loop mutation is likely one key factor among others leading to altered mtDNA amount in osteosarcoma. PMID:23177796

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

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


    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. PMID:27222811

  20. Detection of common copy number variation with application to population clustering from next generation sequencing data*

    Duan, Junbo; Zhang, Ji-Gang; Deng, Hong-Wen; Wang, Yu-Ping


    Copy number variation (CNV) is a structural variation in human genome that has been associated with many complex diseases. In this paper we present a method to detect common copy number variation from next generation sequencing data. First, copy number variations are detected from each individual sample, which is formulated as a total variation penalized least square problem. Second, the common copy number discovery from multiple samples is obtained using source separation techniques such as ...

  1. [Association of common copy number variations with diseases].

    Yang, Fei; Cao, Pengbo; Zhou, Gangqiao


    Genomic polymorphisms come in various forms including single nucleotide variations, translocations, insertions and copy number variations (CNVs). As a form of structural variation, the CNVs comprise common and rare forms based on their populational frequencies. Studies have demonstrated that certain CNVs are associated with risks for neuro-developmental diseases, viral infections, chronic inflammations, and cancers. With the development of high-resolution genome typing technologies such as microarrays and whole genome sequencing, the human genomic CNVs map has been continuously improved and refined. In-depth study of CNVs not only can provide comprehensive understanding for their structural variations and genetic evolution, but also provide new insights into genetic factors contributing to such diseases. In this paper, the general characteristics, pathogenesis and detection methods for the CNVs, as well as their association with human diseases are reviewed. PMID:27264828

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

    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. 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. 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. 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

  3. Variation in CCL3L1 copy number in rhesus macaques (Macaca mulatta).

    Taormina, Patrick L; Satkoski Trask, Jessica A; Smith, David G; Kanthaswamy, Sreetharan


    We used real-time quantitative PCR (qPCR) methodology to examine copy number variation (CNV) of the CCL3L1 gene among pure Indian-origin, pure Chinese-origin, and hybrid Indian-Chinese rhesus macaques (Macaca mulatta). CNV among purebred macaques fell within expected ranges, with Indian macaques having lower copy numbers than those of Chinese macaques. Compared with the purebred macaques, Indian-Chinese hybrid rhesus macaques showed much greater variance in copy number and an intermediate average copy number. Copy numbers of CCL3L1 in rhesus macaque trios (sire, dam, and offspring) were consistent with Mendelian inheritance. PMID:22776055

  4. Determination of beta-defensin genomic copy number in different populations

    Fode, Peder; Jespersgaard, Cathrine; Hardwick, Robert J; Bogle, Helen; Theisen, Michael; Dodoo, Daniel; Lenicek, Martin; Vitek, Libor; Vieira, Ana; Freitas, Joao; Andersen, Paal Skytt; Hollox, Edward J


    There have been conflicting reports in the literature on association of gene copy number with disease, including CCL3L1 and HIV susceptibility, and ß-defensins and Crohn's disease. Quantification of precise gene copy numbers is important in order to define any association of gene copy number with...... disease. At present, real-time quantitative PCR (QPCR) is the most commonly used method to determine gene copy number, however the Paralogue Ratio Test (PRT) is being used in more and more laboratories.......There have been conflicting reports in the literature on association of gene copy number with disease, including CCL3L1 and HIV susceptibility, and ß-defensins and Crohn's disease. Quantification of precise gene copy numbers is important in order to define any association of gene copy number with...

  5. Determination of beta-defensin genomic copy number in different populations: a comparison of three methods.

    Peder Fode

    Full Text Available BACKGROUND: There have been conflicting reports in the literature on association of gene copy number with disease, including CCL3L1 and HIV susceptibility, and β-defensins and Crohn's disease. Quantification of precise gene copy numbers is important in order to define any association of gene copy number with disease. At present, real-time quantitative PCR (QPCR is the most commonly used method to determine gene copy number, however the Paralogue Ratio Test (PRT is being used in more and more laboratories. FINDINGS: In this study we compare a Pyrosequencing-based Paralogue Ratio Test (PPRT for determining beta-defensin gene copy number with two currently used methods for gene copy number determination, QPCR and triplex PRT by typing five different cohorts (UK, Danish, Portuguese, Ghanaian and Czech of DNA from a total of 576 healthy individuals. We found a systematic measurement bias between DNA cohorts revealed by QPCR, but not by the PRT-based methods. Using PRT, copy number ranged from 2 to 9 copies, with a modal copy number of 4 in all populations. CONCLUSIONS: QPCR is very sensitive to quality of the template DNA, generating systematic biases that could produce false-positive or negative disease associations. Both triplex PRT and PPRT do not show this systematic bias, and type copy number within the correct range, although triplex PRT appears to be a more precise and accurate method to type beta-defensin copy number.

  6. Rare copy number deletions predict individual variation in intelligence.

    Ronald A Yeo

    Full Text Available Phenotypic variation in human intellectual functioning shows substantial heritability, as demonstrated by a long history of behavior genetic studies. Many recent molecular genetic studies have attempted to uncover specific genetic variations responsible for this heritability, but identified effects capture little variance and have proven difficult to replicate. The present study, motivated an interest in "mutation load" emerging from evolutionary perspectives, examined the importance of the number of rare (or infrequent copy number variations (CNVs, and the total number of base pairs included in such deletions, for psychometric intelligence. Genetic data was collected using the Illumina 1MDuoBeadChip Array from a sample of 202 adult individuals with alcohol dependence, and a subset of these (N = 77 had been administered the Wechsler Abbreviated Scale of Intelligence (WASI. After removing CNV outliers, the impact of rare genetic deletions on psychometric intelligence was investigated in 74 individuals. The total length of the rare deletions significantly and negatively predicted intelligence (r = -.30, p = .01. As prior studies have indicated greater heritability in individuals with relatively higher parental socioeconomic status (SES, we also examined the impact of ethnicity (Anglo/White vs. Other, as a proxy measure of SES; these groups did not differ on any genetic variable. This categorical variable significantly moderated the effect of length of deletions on intelligence, with larger effects being noted in the Anglo/White group. Overall, these results suggest that rare deletions (between 5% and 1% population frequency or less adversely affect intellectual functioning, and that pleotropic effects might partly account for the association of intelligence with health and mental health status. Significant limitations of this research, including issues of generalizability and CNV measurement, are discussed.

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

    Zhang, Yanping; Xu, Hongen; Frishman, Dmitrij


    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. PMID:26732428

  8. Association tests and software for copy number variant data

    Plagnol Vincent


    Full Text Available Abstract Recent studies have suggested that copy number variation (CNV significantly contributes to genetic predisposition to several common disorders. These findings, combined with the imperfect tagging of CNVs by single nucleotide polymorphisms (SNPs, have motivated the development of association studies directly targeting CNVs. Several assays, including comparative genomic hybridisation arrays, SNP genotyping arrays, or DNA quantification through real-time polymerase chain reaction analysis, allow direct assessment of CNV status in cohorts sufficiently large to provide adequate statistical power for association studies. When analysing data provided by these assays, association tests for CNV data are not fundamentally different from SNP-based association tests. The main difference arises when the quality of the CNV assay is not sufficient to convert unequivocally the raw measurement into discrete calls -- a common issue, given the technological limitations of current CNV assays. When this is the case, association tests are more appropriately based on the raw continuous measurement provided by the CNV assay, instead of potentially inaccurate discrete calls, thus motivating the development of new statistical methods. Here, the programs available for CNV association testing for case control or family data are reviewed, using either discrete calls or raw continuous data.

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

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


    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. PMID:27327179

  10. Copy number variants in German patients with schizophrenia.

    Lutz Priebe

    Full Text Available Large rare copy number variants (CNVs have been recognized as significant genetic risk factors for the development of schizophrenia (SCZ. However, due to their low frequency (1∶150 to 1∶1000 among patients, large sample sizes are needed to detect an association between specific CNVs and SCZ. So far, the majority of genome-wide CNV analyses have focused on reporting only CNVs that reached a significant P-value within the study cohort and merely confirmed the frequency of already-established risk-carrying CNVs. As a result, CNVs with a very low frequency that might be relevant for SCZ susceptibility are lost for secondary analyses. In this study, we provide a concise collection of high-quality CNVs in a large German sample consisting of 1,637 patients with SCZ or schizoaffective disorder and 1,627 controls. All individuals were genotyped on Illumina's BeadChips and putative CNVs were identified using QuantiSNP and PennCNV. Only those CNVs that were detected by both programs and spanned ≥30 consecutive SNPs were included in the data collection and downstream analyses (2,366 CNVs, 0.73 CNVs per individual. The genome-wide analysis did not reveal a specific association between a previously unknown CNV and SCZ. However, the group of CNVs previously reported to be associated with SCZ was more frequent in our patients than in the controls. The publication of our dataset will serve as a unique, easily accessible, high-quality CNV data collection for other research groups. The dataset could be useful for the identification of new disease-relevant CNVs that are currently overlooked due to their very low frequency and lack of power for their detection in individual studies.

  11. SNP and gene networks construction and analysis from classification of copy number variations data

    Liu, Yang; Lee, Yiu Fai; Ng, Michael K.


    Background Detection of genomic DNA copy number variations (CNVs) can provide a complete and more comprehensive view of human disease. It is interesting to identify and represent relevant CNVs from a genome-wide data due to high data volume and the complexity of interactions. Results In this paper, we incorporate the DNA copy number variation data derived from SNP arrays into a computational shrunken model and formalize the detection of copy number variations as a case-control classification ...

  12. Copy number variation in the genomes of twelve natural isolates of Caenorhabditis elegans

    Flibotte Stephane; Edgley Mark L; Lorch Adam; Maydan Jason S; Moerman Donald G


    Abstract Background Copy number variation is an important component of genetic variation in higher eukaryotes. The extent of natural copy number variation in C. elegans is unknown outside of 2 highly divergent wild isolates and the canonical N2 Bristol strain. Results We have used array comparative genomic hybridization (aCGH) to detect copy number variation in the genomes of 12 natural isolates of Caenorhabditis elegans. Deletions relative to the canonical N2 strain are more common in these ...

  13. Identification of genes with a correlation between copy number and expression in gastric cancer

    Cheng Lei; Wang Ping; Yang Sheng; Yang Yanqing; Zhang Qing; Zhang Wen; Xiao Huasheng; Gao Hengjun; Zhang Qinghua


    Abstract Background To elucidate gene expression associated with copy number changes, we performed a genome-wide copy number and expression microarray analysis of 25 pairs of gastric tissues. Methods We applied laser capture microdissection (LCM) to obtain samples for microarray experiments and profiled DNA copy number and gene expression using 244K CGH Microarray and Human Exon 1.0 ST Microarray. Results Obviously, gain at 8q was detected at the highest frequency (70%) and 20q at the second ...

  14. Extensive Copy-Number Variation of the Human Olfactory Receptor Gene Family

    Janet M Young; Endicott, RaeLynn M.; Parghi, Sean S; Walker, Megan; Kidd, Jeffrey M.; Trask, Barbara J.


    As much as a quarter of the human genome has been reported to vary in copy number between individuals, including regions containing about half of the members of the olfactory receptor (OR) gene family. We have undertaken a detailed study of copy-number variation of ORs to elucidate the selective and mechanistic forces acting on this gene family and the true impact of copy-number variation on human OR repertoires. We argue that the properties of copy-number variants (CNVs) and other sets of la...

  15. Causative Factors for ADHD: Role of Copy Number Variants in ADHD

    J Gordon Millichap; John J Millichap


    Investigators from Brazil determined if copy number variants (CNVs) in glutamate metabotropic receptor genes (GRM) were overrepresented in 1038 individuals with ADHD compared to 1057 subjects without ADHD.

  16. Mitochondrial DNA copy number and lung cancer risk in a prospective cohort study

    Hosgood, H Dean; Liu, Chin-San; Rothman, Nathaniel; Weinstein, Stephanie J.; Bonner, Matthew R; Shen, Min; Lim, Unhee; Virtamo, Jarmo; Cheng, Wen-Ling; Albanes, Demetrius; Lan, Qing


    Mitochondria are eukaryotic organelles responsible for energy production. Mitochondrial DNA (mtDNA) lack introns and protective histones, have limited DNA repair capacity and compensate for damage by increasing the number of mtDNA copies. As a consequence, mitochondria are more susceptible to reactive oxygen species, an important determinant of cancer risk, and it is hypothesized that increased mtDNA copy number may be associated with carcinogenesis. We assessed the association of mtDNA copy ...

  17. High-Resolution Analysis of Gene Copy Number Alterations in Human Prostate Cancer Using CGH on cDNA Microarrays: Impact of Copy Number on Gene Expression

    Maija Wolf


    Full Text Available Identification of target genes for genetic rearrangements in prostate cancer and the impact of copy number changes on gene expression are currently not well understood. Here, we applied high-resolution comparative genomic hybridization (CGH on cDNA microarrays for analysis of prostate cancer cell lines. CGH microarrays identified most of the alterations detected by classical chromosomal CGH, as well as a number of previously unreported alterations. Specific recurrent regions of gain (28 and loss (18 were found, their boundaries defined with sub-megabasepair accuracy. The most common changes included copy number decreases at 13% and gains at iq and 5p. Refined mapping identified several sites, such as at 13q (33-44, 49-51, 74-76 Mbp from the p-telomere, which matched with minimal regions of loss seen in extensive loss of heterozygosity mapping studies of large numbers of tumors. Previously unreported recurrent changes were found at 2p, 2q, 3p, 17q (losses, at 3q, 5p, 6p (gains. Integration of genomic and transcriptomic data revealed the role of individual candidate target genes for genomic alterations as well as a highly significant (P < .0001 overall association between copy number levels and the percentage of differentially expressed genes. Across the genome, the overall impact of copy number on gene expression levels was, to a large extent, attributable to low-level gains and losses of copy number, corresponding to common deletions and gains of often large chromosomal regions.

  18. Copy number variations and cognitive phenotypes in unselected populations

    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


    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

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

    Westland, R.; Verbitsky, M.; Vukojevic, K.; Perry, B.J.; Fasel, D.A.; Zwijnenburg, P.J.; Bokenkamp, A.; Gille, J.J.P.; Saraga-Babic, M.; Ghiggeri, G.M.; D'Agati, V.D.; Schreuder, M.F.; Gharavi, A.G.; Wijk, J.A. van; Sanna-Cherchi, S.


    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 dr

  20. 47 CFR 1.742 - Place of filing, fees, and number of copies.


    ... 47 Telecommunication 1 2010-10-01 2010-10-01 false Place of filing, fees, and number of copies. 1.742 Section 1.742 Telecommunication FEDERAL COMMUNICATIONS COMMISSION GENERAL PRACTICE AND PROCEDURE Complaints, Applications, Tariffs, and Reports Involving Common Carriers Applications § 1.742 Place of filing, fees, and number of copies....

  1. 5 CFR 2429.25 - Number of copies and paper size.


    ... 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...

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

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


    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. PMID:25477383

  3. Peripheral blood mitochondrial DNA copy number is associated with prostate cancer risk and tumor burden.

    Zhou, Weimin; Zhu, Min; Gui, Ming; Huang, Lihua; Long, Zhi; Wang, Li; Chen, Hui; Yin, Yinghao; Jiang, Xianzhen; Dai, Yingbo; Tang, Yuxin; He, Leye; Zhong, Kuangbiao


    Alterations of mitochondrial DNA (mtDNA) have been associated with the risk of a number of human cancers; however, the relationship between mtDNA copy number in peripheral blood leukocytes (PBLs) and the risk of prostate cancer (PCa) has not been investigated. In a case-control study of 196 PCa patients and 196 age-paired healthy controls in a Chinese Han population, the association between mtDNA copy number in PBLs and PCa risk was evaluated. The relative mtDNA copy number was measured using quantitative real-time PCR; samples from three cases and two controls could not be assayed, leaving 193 cases and 194 controls for analysis. PCa patients had significantly higher mtDNA copy numbers than controls (medians 0.91 and 0.82, respectively; P<0.001). Dichotomized at the median value of mtDNA copy number in the controls, high mtDNA copy number was significantly associated with an increased risk of PCa (adjusted odds ratio= 1.85, 95% confidence interval: 1.21-2.83). A significant dose-response relationship was observed between mtDNA copy number and risk of PCa in quartile analysis (Ptrend = 0.011). Clinicopathological analysis showed that high mtDNA copy numbers in PCa patients were significantly associated with high Gleason score and advanced tumor stage, but not serum prostate-specific antigen level (P = 0.002, 0.012 and 0.544, respectively). These findings of the present study indicate that increased mtDNA copy number in PBLs is significantly associated with an increased risk of PCa and may be a reflection of tumor burden. PMID:25279731

  4. Peripheral blood mitochondrial DNA copy number is associated with prostate cancer risk and tumor burden.

    Weimin Zhou

    Full Text Available Alterations of mitochondrial DNA (mtDNA have been associated with the risk of a number of human cancers; however, the relationship between mtDNA copy number in peripheral blood leukocytes (PBLs and the risk of prostate cancer (PCa has not been investigated. In a case-control study of 196 PCa patients and 196 age-paired healthy controls in a Chinese Han population, the association between mtDNA copy number in PBLs and PCa risk was evaluated. The relative mtDNA copy number was measured using quantitative real-time PCR; samples from three cases and two controls could not be assayed, leaving 193 cases and 194 controls for analysis. PCa patients had significantly higher mtDNA copy numbers than controls (medians 0.91 and 0.82, respectively; P<0.001. Dichotomized at the median value of mtDNA copy number in the controls, high mtDNA copy number was significantly associated with an increased risk of PCa (adjusted odds ratio= 1.85, 95% confidence interval: 1.21-2.83. A significant dose-response relationship was observed between mtDNA copy number and risk of PCa in quartile analysis (Ptrend = 0.011. Clinicopathological analysis showed that high mtDNA copy numbers in PCa patients were significantly associated with high Gleason score and advanced tumor stage, but not serum prostate-specific antigen level (P = 0.002, 0.012 and 0.544, respectively. These findings of the present study indicate that increased mtDNA copy number in PBLs is significantly associated with an increased risk of PCa and may be a reflection of tumor burden.

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

    Jackson Stuart


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

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

    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


    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. PMID:26953680

  7. Use of Quantitative Polymerase Chain Reaction for Determining Copy Numbers of Transgenes in Lesquerella fendleri

    Grace Q. Chen


    Full Text Available Problem statement: In transgenic plants, the number of transgene copies could greatly influence the level of expression and genetic stability of the target gene, thus it is important to develop an efficient method for accurate estimation of transgene copies. The quantitative Polymerase Chain Reaction (qPCR technique is becoming more efficient nowadays to determine copy numbers of transgenes in transgenic plants, being used here, for the first time in quantifying copy numbers of transgenes in Lesquerella fendleri. Approach: The system utilized a known one copy gene, LfKCS4/5, from L. fendleri as an endogenous calibrator and the threshold Crossing point (Ct measured by Applied Biosystem 7500 system to calculate the copy numbers of transgenes in primary transgenic lines (T0 generation. Results: The qPCR condition was optimized and each primer set had a PCR efficiency of 0.99 or 1.01. Our data demonstrated unambiguous 2-fold discrimination of the copy number of β-glucuronidase gene (gusA and hygromycine phosphotransferase II (hptII genes in 12 T0 lines. Most of the lines contained one or two copies of each gene. Eight out of 12 samples (66.7% showed more copies of gusA gene than that of hptII gene, suggesting rearrangements of the Transferred (T-DNA. Possible modifications of the T-DNA cassette in L. fendleri are discussed based on main models of T-DNA integration in the plant genome. Conclusion: The qPCR described in this study is an efficient method and it is particularly useful in identification and selection of transgenic plants with desirable copy numbers at early stage.

  8. DNA Extraction Procedures Meaningfully Influence qPCR-Based mtDNA Copy Number Determination

    Guo, Wen; Jiang, Lan; Bhasin, Shalender; Khan, Shaharyar M.; Russell H. Swerdlow


    Quantitative real time PCR (qPCR) is commonly used to determine cell mitochondrial DNA (mtDNA) copy number. This technique involves obtaining the ratio of an unknown variable (number of copies of an mtDNA gene) to a known parameter (number of copies of a nuclear DNA gene) within a genomic DNA sample. We considered the possibility that mtDNA: nuclear DNA (nDNA) ratio determinations could vary depending on the method of genomic DNA extraction used, and that these differences could substantively...

  9. A prospective study of mitochondrial DNA copy number and risk of non-Hodgkin lymphoma

    Lan, Qing; Lim, Unhee; Liu, Chin-San; Weinstein, Stephanie J.; Chanock, Stephen; Bonner, Matthew R; Virtamo, Jarmo; Albanes, Demetrius; Rothman, Nathaniel


    Mitochondrial DNA (mtDNA) copy number is increased in patients with chronic lymphocytic leukemia (CLL), in Burkitt lymphoma and Epstein-Barr virus–transformed lymphoblastoid cell lines, and in T cells activated via the T-cell receptor. We hypothesized that having a higher mtDNA copy number in peripheral white blood cell DNA from healthy subjects would be associated with future risk of non-Hodgkin lymphoma (NHL). We analyzed mtDNA copy number in 104 incident male NHL cases and 104 matched cont...

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

    Verginelli, Fabio; De Lellis, Laura; Capelli, Cristian; Verzilli, Delfina; Chiarelli, Francesco; Mohn, Angelika; Cama, Alessandro


    Background 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. Methods 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. Results 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. Conclusions 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. PMID:27149670

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

    M Loredana Marcovecchio

    Full Text Available 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.

  12. Bayesian estimation of genomic copy number with single nucleotide polymorphism genotyping arrays

    Davis Caleb


    Full Text Available Abstract Background The identification of copy number aberration in the human genome is an important area in cancer research. We develop a model for determining genomic copy numbers using high-density single nucleotide polymorphism genotyping microarrays. The method is based on a Bayesian spatial normal mixture model with an unknown number of components corresponding to true copy numbers. A reversible jump Markov chain Monte Carlo algorithm is used to implement the model and perform posterior inference. Results The performance of the algorithm is examined on both simulated and real cancer data, and it is compared with the popular CNAG algorithm for copy number detection. Conclusions We demonstrate that our Bayesian mixture model performs at least as well as the hidden Markov model based CNAG algorithm and in certain cases does better. One of the added advantages of our method is the flexibility of modeling normal cell contamination in tumor samples.

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

    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.

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

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


    Common multi-allelic copy number variants (CNVs) appear enriched for phenotypic associations compared to their biallelic counterparts. Here we investigated the influence of gene dosage effects on adiposity through a CNV association study of gene expression levels in adipose tissue. We identified significant association of a multi-allelic CNV encompassing the salivary amylase gene (AMY1) with body mass index (BMI) and obesity, and we replicated this finding in 6,200 subjects. Increased AMY1 copy number was positively associated with both amylase gene expression (P = 2.31 × 10(-14)) and serum enzyme levels (P 9) and bottom (copy number < 4) 10% of the copy number distribution. Our study provides a first genetic link between carbohydrate metabolism and BMI and demonstrates the power of integrated genomic approaches beyond genome-wide association studies. PMID:24686848

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

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


    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

  16. Using GWAS Data to Identify Copy Number Variants Contributing to Common Complex Diseases

    Zöllner, Sebastian; Teslovich, Tanya M


    Copy number variants (CNVs) account for more polymorphic base pairs in the human genome than do single nucleotide polymorphisms (SNPs). CNVs encompass genes as well as noncoding DNA, making these polymorphisms good candidates for functional variation. Consequently, most modern genome-wide association studies test CNVs along with SNPs, after inferring copy number status from the data generated by high-throughput genotyping platforms. ¶ Here we give an overview of CNV genomics in humans, ...

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

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


    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' (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. PMID:26679989

  18. Reducing system noise in copy number data using principal components of self-self hybridizations

    Lee, Yoon-ha; Ronemus, Michael; Kendall, Jude; Lakshmi, B.; Leotta, Anthony; Levy, Dan; Esposito, Diane; Grubor, Vladimir; Ye, Kenny; Wigler, Michael; Yamrom, Boris


    Genomic copy number variation underlies genetic disorders such as autism, schizophrenia, and congenital heart disease. Copy number variations are commonly detected by array based comparative genomic hybridization of sample to reference DNAs, but probe and operational variables combine to create correlated system noise that degrades detection of genetic events. To correct for this we have explored hybridizations in which no genetic signal is expected, namely “self-self” hybridizations (SSH) co...

  19. Population-specific GSTM1 copy number variation

    Huang, R Stephanie; Chen, Peixian; Wisel, Steve; Duan, Shiwei; Zhang, Wei; Cook, Edwin H.; Das, Soma; Cox, Nancy J.; Dolan, M. Eileen


    As one of the major glutathione conjugation enzymes, GSTM1 detoxifies a number of drugs and xenobiotics. Its expression and activity have been shown to correlate both with cancer risks and drug resistance. Through a genome-wide association study, we identified a significant association between HapMap SNP rs366631 and GSTM1 expression. In this study, utilizing lymphoblastoid cell lines derived from International HapMap Consortium CEU and YRI populations, we designed and performed site-specific...

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


    Background 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. Results 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. Conclusions 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

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

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


    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. PMID:26176207

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

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


    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.

  3. Directed gene copy number amplification in Pichia pastoris by vector integration into the ribosomal DNA locus.

    Marx, Hans; Mecklenbräuker, Astrid; Gasser, Brigitte; Sauer, Michael; Mattanovich, Diethard


    The yeast Pichia pastoris is a widely used host organism for heterologous protein production. One of the basic steps for strain improvement is to ensure a sufficient level of transcription of the heterologous gene, based on promoter strength and gene copy number. To date, high-copy-number integrants of P. pastoris are achievable only by screening of random events or by cloning of gene concatemers. Methods for rapid and reliable multicopy integration of the expression cassette are therefore desirable. Here we present such a method based on vector integration into the rDNA locus and post-transformational vector amplification by repeated selection on increased antibiotic concentrations. Data are presented for two exemplary products: human serum albumin, which is secreted into the supernatant, and human superoxide dismutase, which is accumulated in the cytoplasm of the cells. The striking picture evolving is that intracellular protein production is tightly correlated with gene copy number, while use of the secretory pathway introduces a high clonal variability and the correlation with gene copy number is valid only for low gene copy numbers. PMID:19799640

  4. Relationships between cell cycle regulator gene copy numbers and protein expression levels in Schizosaccharomyces pombe.

    Ayako Chino

    Full Text Available We previously determined the copy number limits of overexpression for cell division cycle (cdc regulatory genes in the fission yeast Schizosaccharomyces pombe using the "genetic tug-of-war" (gTOW method. In this study, we measured the levels of tandem affinity purification (TAP-tagged target proteins when their copy numbers are increased in gTOW. Twenty analyzed genes showed roughly linear correlations between increased protein levels and gene copy numbers, which suggested a general lack of compensation for gene dosage in S. pombe. Cdc16 and Sid2 protein levels but not their mRNA levels were much lower than that expected by their copy numbers, which suggested the existence of a post-transcriptional down regulation of these genes. The cyclin Cig1 protein level and its mRNA level were much higher than that expected by its copy numbers, which suggested a positive feedback mechanism for its expression. A higher Cdc10 protein level and its mRNA level, probably due to cloning its gene into a plasmid, indicated that Cdc10 regulation was more robust than that previously predicted.

  5. Relationships between cell cycle regulator gene copy numbers and protein expression levels in Schizosaccharomyces pombe.

    Chino, Ayako; Makanae, Koji; Moriya, Hisao


    We previously determined the copy number limits of overexpression for cell division cycle (cdc) regulatory genes in the fission yeast Schizosaccharomyces pombe using the "genetic tug-of-war" (gTOW) method. In this study, we measured the levels of tandem affinity purification (TAP)-tagged target proteins when their copy numbers are increased in gTOW. Twenty analyzed genes showed roughly linear correlations between increased protein levels and gene copy numbers, which suggested a general lack of compensation for gene dosage in S. pombe. Cdc16 and Sid2 protein levels but not their mRNA levels were much lower than that expected by their copy numbers, which suggested the existence of a post-transcriptional down regulation of these genes. The cyclin Cig1 protein level and its mRNA level were much higher than that expected by its copy numbers, which suggested a positive feedback mechanism for its expression. A higher Cdc10 protein level and its mRNA level, probably due to cloning its gene into a plasmid, indicated that Cdc10 regulation was more robust than that previously predicted. PMID:24019917

  6. Copy number variations in the amylase gene (AMY2B) in Japanese native dog breeds.

    Tonoike, A; Hori, Y; Inoue-Murayama, M; Konno, A; Fujita, K; Miyado, M; Fukami, M; Nagasawa, M; Mogi, K; Kikusui, T


    A recent study suggested that increased copy numbers of the AMY2B gene might be a crucial genetic change that occurred during the domestication of dogs. To investigate AMY2B expansion in ancient breeds, which are highly divergent from modern breeds of presumed European origins, we analysed copy numbers in native Japanese dog breeds. Copy numbers in the Akita and Shiba, two ancient breeds in Japan, were higher than those in wolves. However, compared to a group of various modern breeds, Akitas had fewer copy numbers, whereas Shibas exhibited the same level of expansion as modern breeds. Interestingly, average AMY2B copy numbers in the Jomon-Shiba, a unique line of the Shiba that has been bred to maintain their appearance resembling ancestors of native Japanese dogs and that originated in the same region as the Akita, were lower than those in the Shiba. These differences may have arisen from the earlier introduction of rice farming to the region in which the Shiba originated compared to the region in which the Akita and the Jomon-Shiba originated. Thus, our data provide insights into the relationship between the introduction of agriculture and AMY2B expansion in dogs. PMID:26358734

  7. Dynamic changes in functional gene copy numbers and microbial communities during degradation of pyrene in soils

    This study investigates the dynamics of pyrene degradation rates, microbial communities, and functional gene copy numbers during the incubation of pyrene-spiked soils. Spiking pyrene to the soil was found to have negligible effects on the bacterial community present. Our results demonstrated that there was a significant difference in nidA gene copy numbers between sampling dates in QZ soil. Mycobacterium 16S rDNA clone libraries showed that more than 90% mycobacteria detected were closely related to fast-growing PAH-degrading Mycobacterium in pyrene-spiked soil, while other sequences related to slow-growing Mycobacterium were only detected in the control soil. It is suggested that nidA gene copy number and fast-growing PAH-degrading Mycobacterium could be used as indicators to predict pyrene contamination and its degradation activity in soils. - nidA gene and fast-growing PAH-degrading Mycobacterium can serve as indicators for pyrene contamination.

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

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


    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. PMID:23395647

  9. Relative Copy Number Variations of CYP2C19 in South Indian Population

    Anichavezhi Devendran


    Full Text Available CYP2C19 is a polymorphic enzyme involved in the metabolism of clinically important drugs. Genotype-phenotype association studies of CYP2C19 have reported wide ranges in the metabolic ratios of its substrates. These discrepancies could be attributed to the variations in the promoter region and this aspect has been reported recently. The observations in the recent reports on the influence of promoter region variants on the metabolism of CYP2C19 substrates might also have been influenced by the copy number variations of CYP2C19. In this paper, we describe copy number variations of CYP2C19 using real-time polymerase chain reaction by comparative Ct method. No copy number variations were observed in the south Indian population indicating the observed discrepancies in genotype-phenotype association studies might be due to the regulatory region polymorphisms as reported earlier.

  10. Hardy-Weinberg equilibrium revisited for inferences on genotypes featuring allele and copy-number variations.

    Recke, Andreas; Recke, Klaus-Günther; Ibrahim, Saleh; Möller, Steffen; Vonthein, Reinhard


    Copy number variations represent a substantial source of genetic variation and are associated with a plethora of physiological and pathophysiological conditions. Joint copy number and allelic variations (CNAVs) are difficult to analyze and require new strategies to unravel the properties of genotype distributions. We developed a Bayesian hidden Markov model (HMM) approach that allows dissecting intrinsic properties and metastructures of the distribution of CNAVs within populations, in particular haplotype phases of genes with varying copy numbers. As a key feature, this approach incorporates an extension of the Hardy-Weinberg equilibrium, allowing both a comprehensive and parsimonious model design. We demonstrate the quality of performance and applicability of the HMM approach with a real data set describing the Fcγ receptor (FcγR) gene region. Our concept, using a dynamic process to analyze a static distribution, establishes the basis for a novel understanding of complex genomic data sets. PMID:25765626

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

    Bantong Xue


    Full Text Available 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.

  12. Urinary Mitochondrial DNA Copy Number Identifies Chronic Renal Injury in Hypertensive Patients.

    Eirin, Alfonso; Saad, Ahmed; Tang, Hui; Herrmann, Sandra M; Woollard, John R; Lerman, Amir; Textor, Stephen C; Lerman, Lilach O


    Mitochondrial injury contributes to renal dysfunction in several models of renal disease, but its involvement in human hypertension remains unknown. Fragments of the mitochondrial genome released from dying cells are considered surrogate markers of mitochondrial injury. We hypothesized that hypertension would be associated with increased urine mitochondrial DNA (mtDNA) copy numbers. We prospectively measured systemic and urinary copy number of the mtDNA genes cytochrome-c oxidase-3 and NADH dehydrogenase subunit-1 by quantitative polymerase chain reaction in essential (n=25) and renovascular (RVH, n=34) hypertensive patients and compared them with healthy volunteers (n=22). Urinary kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin served as indices of renal injury. Renal blood flow and oxygenation were assessed by multidetector computed tomography and blood oxygen level-dependent magnetic resonance imaging. Blood pressure, urinary neutrophil gelatinase-associated lipocalin, and kidney injury molecule-1 were similarly elevated in essential hypertension and RVH, and estimated glomerular filtration rate was lower in RVH versus healthy volunteers and essential hypertension. Renal blood flow was lower in RVH compared with essential hypertension. Urinary mtDNA copy number was higher in hypertension compared with healthy volunteers, directly correlated with urinary neutrophil gelatinase-associated lipocalin and kidney injury molecule-1 and inversely with estimated glomerular filtration rate. In RVH, urinary mtDNA copy number correlated directly with intrarenal hypoxia. Furthermore, in an additional validation cohort, urinary mtDNA copy number was higher in RVH compared with healthy volunteers (n=10 each). The change in serum creatinine levels and estimated glomerular filtration rate 3 months after medical therapy without or with revascularization correlated with the change in urinary mtDNA. Therefore, elevated urinary mtDNA copy numbers in

  13. High copy number of mitochondrial DNA (mtDNA) predicts good prognosis in glioma patients

    Zhang, Yanfang; Qu, Yiping; Gao, Ke; Yang, Qi; Shi, Bingyin; Hou, Peng; Ji, Meiju


    Alterations in mitochondrial DNA (mtDNA) copy number have been widely identified in many types of human cancers and are considered a common cancer hallmark. However, the prognostic value of altered mtDNA content in gliomas remains largely unknown. The aim of this study was to investigate mtDNA copy number in a cohort of gliomas (n = 124) and non-neoplastic brain tissues (control subjects; n = 27) and to explore the association between variable mtDNA content and clinical outcomes in glioma pat...

  14. DNA copy number concentration measured by digital and droplet digital quantitative PCR using certified reference materials.

    Corbisier, Philippe; Pinheiro, Leonardo; Mazoua, Stéphane; Kortekaas, Anne-Marie; Chung, Pui Yan Jenny; Gerganova, Tsvetelina; Roebben, Gert; Emons, Hendrik; Emslie, Kerry


    The value assignment for properties of six certified reference materials (ERM-AD623a-f), each containing a plasmid DNA solution ranging from 1 million to 10 copies per μL, by using digital PCR (dPCR) with the BioMark™ HD System (Fluidigm) has been verified by applying droplet digital PCR (ddPCR) using the QX100 system (Bio-Rad). One of the critical factors in the measurement of copy number concentrations by digital PCR is the partition volume. Therefore, we determined the average droplet volume by optical microscopy, revealing an average droplet volume that is 8 % smaller than the droplet volume used as the defined parameter in the QuantaSoft software version (Bio-Rad) to calculate the copy number concentration. This observation explains why copy number concentrations estimated with ddPCR and using an average droplet volume predefined in the QuantaSoft software were systematically lower than those measured by dPCR, creating a significant bias between the values obtained by these two techniques. The difference was not significant anymore when the measured droplet volume of 0.834 nL was used to estimate copy number concentrations. A new version of QuantaSoft software (version, which has since been released with Bio-Rad's new QX200 systems and QX100 upgrades, uses a droplet volume of 0.85 nL as a defined parameter to calculate copy number concentration. PMID:25600685

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

    Kolacsek Orsolya


    Full Text Available Abstract 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.

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

    Wongsrichanalai Chansuda


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

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

    Fatemeh Bahreini


    Full Text Available 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 abnormalities 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 possible 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 Cambridge 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 difference 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 identified as neutral variants in controls. There was an inverse association between mitochondrial 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.

  18. Measurement of the copy number of the master quorum-sensing regulator of a bacterial cell.

    Teng, Shu-Wen; Wang, Yufang; Tu, Kimberly C; Long, Tao; Mehta, Pankaj; Wingreen, Ned S; Bassler, Bonnie L; Ong, N P


    Quorum-sensing is the mechanism by which bacteria communicate and synchronize group behaviors. Quantitative information on parameters such as the copy number of particular quorum-sensing proteins should contribute strongly to understanding how the quorum-sensing network functions. Here, we show that the copy number of the master regulator protein LuxR in Vibrio harveyi can be determined in vivo by exploiting small-number fluctuations of the protein distribution when cells undergo division. When a cell divides, both its volume and LuxR protein copy number, N, are partitioned with slight asymmetries. We measured the distribution functions describing the partitioning of the protein fluorescence and the cell volume. The fluorescence distribution is found to narrow systematically as the LuxR population increases, whereas the volume partitioning is unchanged. Analyzing these changes statistically, we determined that N = 80-135 dimers at low cell density and 575 dimers at high cell density. In addition, we measured the static distribution of LuxR over a large (3000) clonal population. Combining the static and time-lapse experiments, we determine the magnitude of the Fano factor of the distribution. This technique has broad applicability as a general in vivo technique for measuring protein copy number and burst size. PMID:20441767

  19. Effective Normalization for Copy Number Variation Detection from Whole Genome Sequencing

    Janevski Angel; Varadan Vinay; Kamalakaran Sitharthan; Banerjee Nilanjana; Dimitrova Nevenka


    Abstract Background Whole genome sequencing enables a high resolution view of the human genome and provides unique insights into genome structure at an unprecedented scale. There have been a number of tools to infer copy number variation in the genome. These tools, while validated, also include a number of parameters that are configurable to genome data being analyzed. These algorithms allow for normalization to account for individual and population-specific effects on individual genome CNV e...

  20. Detection of erbB2 copy number variations in plasma of patients with esophageal carcinoma

    Mortality is high in patients with esophageal carcinoma as tumors are rarely detected before the disease has progressed to an advanced stage. Here, we sought to isolate cell-free DNA released into the plasma of patients with esophageal carcinoma, to analyze copy number variations of marker genes in the search for early detection of tumor progression. Plasma of 41 patients with esophageal carcinoma was prospectively collected before tumor resection and chemotherapy. Our dataset resulted heterogeneous for clinical data, resembling the characteristics of the tumor. DNA from the plasma was extracted to analyze copy number variations of the erbB2 gene using real-time PCR assays. The real-time PCR assays for erbB2 gene showed significant (P = 0.001) copy number variations in the plasma of patients with esophageal carcinoma, as compared to healthy controls with high sensitivity (80%) and specificity (95%). These variations in erbB2 were negatively correlated to the progression free survival of these patients (P = 0.03), and revealed a further risk category stratification of patients with low VEGF expression levels. The copy number variation of erbB2 gene from plasma can be used as prognostic marker for early detection of patients at risk of worse clinical outcome in esophageal cancer

  1. Copy number variation analysis of matched ovarian primary tumors and peritoneal metastasis.

    Joel A Malek

    Full Text Available Ovarian cancer is the most deadly gynecological cancer. The high rate of mortality is due to the large tumor burden with extensive metastatic lesion of the abdominal cavity. Despite initial chemosensitivity and improved surgical procedures, abdominal recurrence remains an issue and results in patients' poor prognosis. Transcriptomic and genetic studies have revealed significant genome pathologies in the primary tumors and yielded important information regarding carcinogenesis. There are, however, few studies on genetic alterations and their consequences in peritoneal metastatic tumors when compared to their matched ovarian primary tumors. We used high-density SNP arrays to investigate copy number variations in matched primary and metastatic ovarian cancer from 9 patients. Here we show that copy number variations acquired by ovarian tumors are significantly different between matched primary and metastatic tumors and these are likely due to different functional requirements. We show that these copy number variations clearly differentially affect specific pathways including the JAK/STAT and cytokine signaling pathways. While many have shown complex involvement of cytokines in the ovarian cancer environment we provide evidence that ovarian tumors have specific copy number variation differences in many of these genes.

  2. Identification of copy number variants defining genomic differences among major human groups.

    Lluís Armengol

    Full Text Available 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.

  3. Copy number variants in a hospital-based cohort of children with epilepsy

    Vlaskamp, D.R.M.; Callenbach, P.M.C.; Rump, P.; Van Ravenswaaij-Arts, C.M.A.; Brouwer, O.F.


    Purpose: Copy number variants (CVNs), detected with chromosomal microarray, have been shown to cause or predispose to epilepsy. We aimed to evaluate the diagnostic yield of microarray in a large cohort of children with epilepsy and to identify novel genes and regions for epilepsy. Method: From a sin

  4. Decreased mtDNA Copy Number of Gastric Cancer: a New Tumor Marker?

    FanLi; XiaosongWang; ChengboHan; JieLin


    OBJECTIVE To explore the relationship between mtDNA (mitochondrial DNA) and gastric cancer by comparing the difference of mtDNA copy number in gastric cancers and paracancerous tissues.METHODS The HV1 (hypervariable region) and HV2 of the mitochondrial Dloop region from 20 cases of gastric cancer and 20 paracancerous tissues were amplified by PCR with 13-actin serving as a quantitative standard marker. The products were separated by polyacrylamide gel electrophoresis (PAGE) and silver stained in order to compare the difference in mtDNA copy number between gastric cancers and paracancerous tissues. The mtDNA copy number was determined for gastric cancer shaving various pathological characteristics and the results compared with previous immunohistochemical staininq of the tumors,RESULTS There was a significantly quantitative difference in HV1, HV2 (standardized with β-actin) between gastric cancers and paracancerous tissues (P0.05).CONCLUSION The occurrence of gastric cancer was closely associated with decreased mtDNA copy number, which may be a new tumor marker.

  5. Using expression arrays for copy number detection: an example from E. coli

    Stitzer Michael E


    Full Text Available Abstract Background The sequencing of many genomes and tiling arrays consisting of millions of DNA segments spanning entire genomes have made high-resolution copy number analysis possible. Microarray-based comparative genomic hybridization (array CGH has enabled the high-resolution detection of DNA copy number aberrations. While many of the methods and algorithms developed for the analysis microarrays have focused on expression analysis, the same technology can be used to detect genetic alterations, using for example standard commercial Affymetrix arrays. Due to the nature of the resultant data, standard techniques for processing GeneChip expression experiments are inapplicable. Results We have developed a robust and flexible methodology for high-resolution analysis of DNA copy number of whole genomes, using Affymetrix high-density expression oligonucleotide microarrays. Copy number is obtained from fluorescence signals after processing with novel normalization, spatial artifact correction, data transformation and deletion/duplication detection. We applied our approach to identify deleted and amplified regions in E. coli mutants obtained after prolonged starvation. Conclusion The availability of Affymetrix expression chips for a wide variety of organisms makes the proposed array CGH methodology useful more generally.

  6. Prediction of a deletion copy number variant by a dense SNP panel

    Kadri, N.K.; Koks, P.D.; Meuwissen, T.H.E.


    Background: A newly recognized type of genetic variation, Copy Number Variation (CNV), is detected in mammalian genomes, e.g. the cattle genome. This form of variation can potentially cause phenotypic variation. Our objective was to determine whether dense SNP (single nucleotide polymorphisms) panel

  7. High copy number of mitochondrial DNA (mtDNA) predicts good prognosis in glioma patients.

    Zhang, Yanfang; Qu, Yiping; Gao, Ke; Yang, Qi; Shi, Bingyin; Hou, Peng; Ji, Meiju


    Alterations in mitochondrial DNA (mtDNA) copy number have been widely identified in many types of human cancers and are considered a common cancer hallmark. However, the prognostic value of altered mtDNA content in gliomas remains largely unknown. The aim of this study was to investigate mtDNA copy number in a cohort of gliomas (n = 124) and non-neoplastic brain tissues (control subjects; n = 27) and to explore the association between variable mtDNA content and clinical outcomes in glioma patients. Using real-time quantitative PCR assay, we demonstrated that glioma patients had an increased mtDNA content as compared with control subjects. In addition, our data showed that increased mtDNA copy number was significantly negatively associated with tumor grade, recurrence and cancer-related death, whereas there was a significantly positively relationship between increased mtDNA content and seizures. More importantly, increased mtDNA content were closely relevant to longer survival in glioma patients. Taken together, our data provide the strong evidences that high copy number of mtDNA may be a useful good prognostic factor in glioma patients. PMID:26045999

  8. Quantitative Analysis of Copy Number Variants Based on Real-Time LightCycler PCR

    Ma, Lijiang; Chung, Wendy K.


    Quantitative real-time PCR is PCR visualized in real time by the use of fluorescent or intercalating dyes used to measure gene expression or gene quantification including including contiguous gene deletions or duplications. A simple method is described to quantify DNA copy number from human samples.

  9. Mapping cattle copy number variation by population-scale genome sequencing

    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. As a pilot population study of cattle CNV, we sequenced 100 representative cattle...

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

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


    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.

  11. Alpha-defensin DEFA1A3 gene copy number elevation in Danish Crohn's disease patients

    Jespersgaard, Cathrine; Fode, Peder; Dybdahl, Marianne;


    BACKGROUND AND PURPOSE OF STUDY: Extensive copy number variation is observed for the DEFA1A3 gene encoding alpha-defensins 1-3. The objective of this study was to determine the involvement of alpha-defensins in colonic tissue from Crohn's disease (CD) patients and the possible genetic association...

  12. TOP1 gene copy numbers are increased in cancers of the bile duct and pancreas

    Grunnet, Mie; Calatayud, Dan; Schultz, Nicolai Aa.;


    ) poison. Top1 protein, TOP1 gene copy number and mRNA expression, respectively, have been proposed as predictive biomarkers of response to irinotecan in other cancers. Here we investigate the occurrence of TOP1 gene aberrations in cancers of the bile ducts and pancreas. Material and methods. TOP1...

  13. A high-resolution map of copy number variation in the cattle genome

    We conducted a systematic study of the cattle copy number variation (CNV) using array comparative genomic hybridization (array CGH). Oligonucleotide CGH arrays were designed and fabricated to provide a genome-wide coverage with an average interval of 6 kb using the Bta3.1 genome assembly. Dual-lab...

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

    Campos, Carla Marques Rondon, E-mail: [Universidade Federal de Mato Grosso, Cuiabá, MT (Brazil); Zanardo, Evelin Aline; Dutra, Roberta Lelis [Departamento de Patologia - Laboratório de Citogenômica - LIM 03 - Universidade de São Paulo, São Paulo, SP (Brazil); Kulikowski, Leslie Domenici [Universidade de São Paulo, São Paulo, SP (Brazil); Departamento de Patologia - Laboratório de Citogenômica - LIM 03 - Universidade de São Paulo, São Paulo, SP (Brazil); Kim, Chong Ae [Universidade de São Paulo, São Paulo, SP (Brazil)


    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. Investigate gene copy number variation (CNV) in children with conotruncal heart defect. 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. 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. 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.

  15. Copy number variants involving components of the glutamatergic synaptic pathway in ASD patients

    Oliveira, B.A.; Conceição, I.C.; Correia, C.A.; Café, C.; De Almeida, J.; Mouga, S; Duque, F; G. Oliveira; Vicente, A. M.


    Copy Number Variants (CNVs) play an important role in susceptibility to Autism Spectrum Disorders (ASD), in particular when deleting or duplicating genes involved in synaptic structure and function such as glutamatergic synapse genes. Identifying CNVs of etiologic relevance for ASD that include glutamatergic genes may contribute to the understanding of glutamate-related pathogenic mechanisms in this disorder.

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

    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. Investigate gene copy number variation (CNV) in children with conotruncal heart defect. 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. 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. 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

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

    Carla Marques Rondon Campos


    Full Text Available 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.

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

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

  19. Selective constraint on copy number variation in human piwi-interacting RNA Loci.

    David W Gould

    Full Text Available Piwi-interacting RNAs (piRNAs are a recently discovered class of small non-coding RNA found in animals. PiRNAs are primarily expressed in the germline where their best understood function is to repress transposable elements. Unlike previous studies that investigated the evolution of piRNA-generating loci at the level of nucleotide substitutions, here we studied the evolution of piRNA-generating loci at the level of copy number variation (i.e. duplications and deletions using genome-wide copy number variation data from three human populations. Our analysis shows that at the level of copy number variation there is strong selective constraint and a very high mutation rate in human piRNA-generating loci. Our results differ from a model of positive selection on copy number variation in piRNA-generating loci previously proposed in rodents. We discuss possible reasons for this difference based on the transposable element insertion histories in the rodent and primate lineages.

  20. Social responsiveness scale-aided analysis of the clinical impact of copy number variations in autism

    van Daalen, Emma; Kemner, Chantal; Verbeek, Nienke E.; van der Zwaag, Bert; Dijkhuizen, Trijnie; Rump, Patrick; Houben, Renske; van 't Slot, Ruben; de Jonge, Maretha V.; Staal, Wouter G.; Beemer, Frits A.; Vorstman, Jacob A. S.; Burbach, J. Peter H.; van Amstel, Hans Kristian Ploos; Hochstenbach, Ron; Brilstra, Eva H.; Poot, Martin


    Recent array-based studies have detected a wealth of copy number variations (CNVs) in patients with autism spectrum disorders (ASD). Since CNVs also occur in healthy individuals, their contributions to the patient's phenotype remain largely unclear. In a cohort of children with symptoms of ASD, diag

  1. Social Responsiveness Scale-aided analysis of the clinical impact of copy number variations in autism.

    Daalen, E. van; Kemner, C.; Verbeek, N.E.; Zwaag, B. van der; Dijkhuizen, T.; Rump, P.; Houben, R.; Slot, R. van 't; Jonge, M.V. de; Staal, W.G.; Beemer, F.A.; Vorstman, J.A.; Burbach, J.P.H.; Amstel, H.K. van; Hochstenbach, R.; Brilstra, E.H.; Poot, M.


    Recent array-based studies have detected a wealth of copy number variations (CNVs) in patients with autism spectrum disorders (ASD). Since CNVs also occur in healthy individuals, their contributions to the patient's phenotype remain largely unclear. In a cohort of children with symptoms of ASD, diag

  2. DNA copy number concentration measured by digital and droplet digital quantitative PCR using certified reference materials

    CORBISIER Philippe; Pinheiro, Leonardo; Mazoua, Stephane; KORTEKAAS Anna Maria; CHUNG PUI YAN JENNY; GERGANOVA TSVETELINA IVANOVA; Roebben, Gert; Emons, Hendrik; Emslie, K


    The value assignment for properties of six certified reference materials (ERM-AD623a–f), each containing a plasmid DNA solution ranging from 1 million to 10 copies per μL, by using digital PCR (dPCR) with the BioMark™ HD System (Fluidigm) has been verified by applying droplet digital PCR (ddPCR) using the QX100 system (Bio-Rad). One of the critical factors in the measurement of copy number concentrations by digital PCR is the partition volume. Therefore, we determined the average droplet volu...

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

    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.


    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

  4. Genomic DNA copy-number alterations of the let-7 family in human cancers.

    Yanling Wang

    Full Text Available In human cancer, expression of the let-7 family is significantly reduced, and this is associated with shorter survival times in patients. However, the mechanisms leading to let-7 downregulation in cancer are still largely unclear. Since an alteration in copy-number is one of the causes of gene deregulation in cancer, we examined copy number alterations of the let-7 family in 2,969 cancer specimens from a high-resolution SNP array dataset. We found that there was a reduction in the copy number of let-7 genes in a cancer-type specific manner. Importantly, focal deletion of four let-7 family members was found in three cancer types: medulloblastoma (let-7a-2 and let-7e, breast cancer (let-7a-2, and ovarian cancer (let-7a-3/let-7b. For example, the genomic locus harboring let-7a-3/let-7b was deleted in 44% of the specimens from ovarian cancer patients. We also found a positive correlation between the copy number of let-7b and mature let-7b expression in ovarian cancer. Finally, we showed that restoration of let-7b expression dramatically reduced ovarian tumor growth in vitro and in vivo. Our results indicate that copy number deletion is an important mechanism leading to the downregulation of expression of specific let-7 family members in medulloblastoma, breast, and ovarian cancers. Restoration of let-7 expression in tumor cells could provide a novel therapeutic strategy for the treatment of cancer.

  5. Optimizing sparse sequencing of single cells for highly multiplex copy number profiling

    Baslan, Timour; Kendall, Jude; Ward, Brian; Cox, Hilary; Leotta, Anthony; Rodgers, Linda; Riggs, Michael; D'Italia, Sean; Sun, Guoli; Yong, Mao; Miskimen, Kristy; Gilmore, Hannah; Saborowski, Michael; Dimitrova, Nevenka; Krasnitz, Alexander


    Genome-wide analysis at the level of single cells has recently emerged as a powerful tool to dissect genome heterogeneity in cancer, neurobiology, and development. To be truly transformative, single-cell approaches must affordably accommodate large numbers of single cells. This is feasible in the case of copy number variation (CNV), because CNV determination requires only sparse sequence coverage. We have used a combination of bioinformatic and molecular approaches to optimize single-cell DNA...

  6. Change and Significance of Mitochondrial DNA Copy Number in Esophageal Squamous Cell Carcinoma

    Zongwen Liu; Zhihua Zhao; Qiumin Zhao; Shenglei Li; Dongling Gao; Xia Pang; Kuisheng Chen; Yunhan Zhang


    OBJECTIVE To compare the differences of mitochondrial DNA (mtDNA)copies among the tissues of esophageal squamous cell carcinoma (ESCC),para-neoplastic tissue and normal mucous membrane of the esophagus,and to study the relationship between the mtDNA and the occurrence and development of esophageal squamous cell carcinoma.METHODS The mtDNA copies of 42 specimens with the ESCC,paraneoplastic mucous tissue and normal mucous membrane of the esophagus were determined using real-time fluorescence quantitative PCR.The mtDNA was analyzed using agarose gel electrophoresis.RESULTS The mtDNA from all of the tissues (42/42) from the ESCC,para-neoplastic tissue and normal esophageal mucous membranes was analyzed.showing thal there were an average mtDNA copy number of 27.1894x106 μg DNA.9.4102x106 μg DNA and 5.9347x106 μg DNA,from the respective tissues.There were significant differences (F=27.83,P<0.05) in mtDNA copy number among the three.A positive band was shown at 403 bp after qel electrophoresis of the PCR products.and the lane where the ESCC mtDNA located was rather bright.which was in accordance with the result of the real-time PCR determination.CONCLUSION An increase in the mtDNA copy number is related to the occurrence and development of ESCC.

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

    Walker Susan


    Full Text Available Abstract 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.

  8. Analysis of Mitochondrial DNA Copy Number and Its Regulation Through DNA Methylation of POLGA.

    Sun, Xin; Lee, William; Vaghjiani, Vijesh; St John, Justin C


    Replication of mitochondrial DNA (mtDNA) is important for ensuring that cells have sufficient mtDNA copy number to meet their specific requirements for the generation of cellular energy through oxidative phosphorylation. A number of transcription and replication factors are required for this process, with a key factor being the nuclear-encoded mtDNA-specific DNA polymerase γ. DNA polymerase γ has a catalytic subunit (POLGA), whose gene has been shown to be DNA methylated at exon 2. This methylation is considered to be one of the key mechanisms that regulate mtDNA copy number. These findings have made it of great importance to establish optimal methods for investigating the effects of DNA methylation on mtDNA replication. Here, we provide methods to determine the extent of DNA methylation at exon 2 of POLGA as well as other gene targets of interest. We also show how mtDNA copy number is assessed and, from these two outputs, define the efficiency of mtDNA replication by calculating the mtDNA-replicative efficiency index. PMID:26530679

  9. A robust penalized method for the analysis of noisy DNA copy number data

    Huang Jian


    Full Text Available Abstract Background Deletions and amplifications of the human genomic DNA copy number are the causes of numerous diseases, such as, various forms of cancer. Therefore, the detection of DNA copy number variations (CNV is important in understanding the genetic basis of many diseases. Various techniques and platforms have been developed for genome-wide analysis of DNA copy number, such as, array-based comparative genomic hybridization (aCGH and high-resolution mapping with high-density tiling oligonucleotide arrays. Since complicated biological and experimental processes are often associated with these platforms, data can be potentially contaminated by outliers. Results We propose a penalized LAD regression model with the adaptive fused lasso penalty for detecting CNV. This method contains robust properties and incorporates both the spatial dependence and sparsity of CNV into the analysis. Our simulation studies and real data analysis indicate that the proposed method can correctly detect the numbers and locations of the true breakpoints while appropriately controlling the false positives. Conclusions The proposed method has three advantages for detecting CNV change points: it contains robustness properties; incorporates both spatial dependence and sparsity; and estimates the true values at each marker accurately.

  10. The Orphan Gene dauerless Regulates Dauer Development and Intraspecific Competition in Nematodes by Copy Number Variation.

    Melanie G Mayer


    Full Text Available Many nematodes form dauer larvae when exposed to unfavorable conditions, representing an example of phenotypic plasticity and a major survival and dispersal strategy. In Caenorhabditis elegans, the regulation of dauer induction is a model for pheromone, insulin, and steroid-hormone signaling. Recent studies in Pristionchus pacificus revealed substantial natural variation in various aspects of dauer development, i.e. pheromone production and sensing and dauer longevity and fitness. One intriguing example is a strain from Ohio, having extremely long-lived dauers associated with very high fitness and often forming the most dauers in response to other strains' pheromones, including the reference strain from California. While such examples have been suggested to represent intraspecific competition among strains, the molecular mechanisms underlying these dauer-associated patterns are currently unknown. We generated recombinant-inbred-lines between the Californian and Ohioan strains and used quantitative-trait-loci analysis to investigate the molecular mechanism determining natural variation in dauer development. Surprisingly, we discovered that the orphan gene dauerless controls dauer formation by copy number variation. The Ohioan strain has one dauerless copy causing high dauer formation, whereas the Californian strain has two copies, resulting in strongly reduced dauer formation. Transgenic animals expressing multiple copies do not form dauers. dauerless is exclusively expressed in CAN neurons, and both CAN ablation and dauerless mutations increase dauer formation. Strikingly, dauerless underwent several duplications and acts in parallel or downstream of steroid-hormone signaling but upstream of the nuclear-hormone-receptor daf-12. We identified the novel or fast-evolving gene dauerless as inhibitor of dauer development. Our findings reveal the importance of gene duplications and copy number variations for orphan gene function and suggest daf-12 as

  11. The Orphan Gene dauerless Regulates Dauer Development and Intraspecific Competition in Nematodes by Copy Number Variation.

    Mayer, Melanie G; Rödelsperger, Christian; Witte, Hanh; Riebesell, Metta; Sommer, Ralf J


    Many nematodes form dauer larvae when exposed to unfavorable conditions, representing an example of phenotypic plasticity and a major survival and dispersal strategy. In Caenorhabditis elegans, the regulation of dauer induction is a model for pheromone, insulin, and steroid-hormone signaling. Recent studies in Pristionchus pacificus revealed substantial natural variation in various aspects of dauer development, i.e. pheromone production and sensing and dauer longevity and fitness. One intriguing example is a strain from Ohio, having extremely long-lived dauers associated with very high fitness and often forming the most dauers in response to other strains' pheromones, including the reference strain from California. While such examples have been suggested to represent intraspecific competition among strains, the molecular mechanisms underlying these dauer-associated patterns are currently unknown. We generated recombinant-inbred-lines between the Californian and Ohioan strains and used quantitative-trait-loci analysis to investigate the molecular mechanism determining natural variation in dauer development. Surprisingly, we discovered that the orphan gene dauerless controls dauer formation by copy number variation. The Ohioan strain has one dauerless copy causing high dauer formation, whereas the Californian strain has two copies, resulting in strongly reduced dauer formation. Transgenic animals expressing multiple copies do not form dauers. dauerless is exclusively expressed in CAN neurons, and both CAN ablation and dauerless mutations increase dauer formation. Strikingly, dauerless underwent several duplications and acts in parallel or downstream of steroid-hormone signaling but upstream of the nuclear-hormone-receptor daf-12. We identified the novel or fast-evolving gene dauerless as inhibitor of dauer development. Our findings reveal the importance of gene duplications and copy number variations for orphan gene function and suggest daf-12 as major target for

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

    John P Didion


    Full Text Available 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.

  13. A Genome-wide Study Reveals Copy Number Variants Exclusive to Childhood Obesity Cases

    Joseph T. Glessner; Bradfield, Jonathan P.; Wang, Kai; Takahashi, Nagahide; Zhang, Haitao; Sleiman, Patrick M.; Mentch, Frank D.; Kim, Cecilia E; Hou, Cuiping; Thomas, Kelly A.; Garris, Maria L.; Deliard, Sandra; Frackelton, Edward C; Otieno, F. George; Zhao, Jianhua


    The prevalence of obesity in children and adults in the United States has increased dramatically over the past decade. Genomic copy number variations (CNVs) have been strongly implicated in subjects with extreme obesity and coexisting developmental delay. To complement these previous studies, we addressed CNVs in common childhood obesity by examining children with a BMI in the upper 5th percentile but excluding any subject greater than three standard deviations from the mean in order to reduc...

  14. Exploring Cancer's Fractured Genomic Landscape: Searching for Cancer Drivers and Vulnerabilities in Somatic Copy Number Alterations

    Zack, Travis Ian


    Somatic copy number alterations (SCNAs) are a class of alterations that lead to deviations from diploidy in developing and established tumors. A feature that distinguishes SCNAs from other alterations is their genomic footprint. The large genomic footprint of SCNAs in a typical cancer's genome presents both a challenge and an opportunity to find targetable vulnerabilities in cancer. Because a single event affects many genes, it is often challenging to identify the tumorigenic targets of SCNAs...

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

    Farideh eShadravan


    Gender plays a pivotal role in the human genetic identity and is also manifested in many genetic disorders particularly mental retardation. In this study its effect on copy number variation (CNV), known to cause genetic disorders was explored. As the olfactory receptor (OR) repertoire comprises the largest human gene family, it was selected for this study, which was carried out within and between three populations, derived from 150 individuals from the 1000 Genome Project. Analysis of 3872 CN...

  16. Performance of case-control rare copy number variation annotation in classification of autism

    Engchuan, Worrawat; Dhindsa, Kiret; Lionel, Anath C.; Scherer, Stephen W.; Chan, Jonathan H; Merico, Daniele


    Background A substantial proportion of Autism Spectrum Disorder (ASD) risk resides in de novo germline and rare inherited genetic variation. In particular, rare copy number variation (CNV) contributes to ASD risk in up to 10% of ASD subjects. Despite the striking degree of genetic heterogeneity, case-control studies have detected specific burden of rare disruptive CNV for neuronal and neurodevelopmental pathways. Here, we used machine learning methods to classify ASD subjects and controls, ba...

  17. De novo copy number variations in cloned dogs from the same nuclear donor

    Jung, Seung-Hyun; Yim, Seon-Hee; Oh, Hyun Ju; Park, Jung Eun; Kim, Min Jung; Kim, Geon A; Kim, Tae-Min; Kim, Jin-Soo; Lee, Byeong Chun; Chung, Yeun-Jun


    Background Somatic mosaicism of copy number variants (CNVs) in human body organs and de novo CNV event in monozygotic twins suggest that de novo CNVs can occur during mitotic recombination. These de novo CNV events are important for understanding genetic background of evolution and diverse phenotypes. In this study, we explored de novo CNV event in cloned dogs with identical genetic background. Results We analyzed CNVs in seven cloned dogs using the nuclear donor genome as reference by array-...

  18. The landscape of somatic copy-number alteration across human cancers

    Beroukhim, Rameen; Mermel, Craig H.; Porter, Dale; Wei, Guo; Raychaudhuri, Soumya; Donovan, Jerry; Barretina, Jordi; Boehm, Jesse S.; Dobson, Jennifer; Urashima, Mitsuyoshi; Mc Henry, Kevin T.; Pinchback, Reid M.; Ligon, Azra H.; Cho, Yoon-Jae; Haery, Leila


    A powerful way to discover key genes playing causal roles in oncogenesis is to identify genomic regions that undergo frequent alteration in human cancers. Here, we report high-resolution analyses of somatic copy-number alterations (SCNAs) from 3131 cancer specimens, belonging largely to 26 histological types. We identify 158 regions of focal SCNA that are altered at significant frequency across multiple cancer types, of which 122 cannot be explained by the presence of a known cancer target ge...

  19. A robust statistical method for case-control association testing with copy number variation

    Barnes, Chris; Plagnol, Vincent; Fitzgerald, Tomas; Redon, Richard; Marchini, Jonathan; Clayton, David; Hurles, Matthew E.


    Copy number variation (CNV) is pervasive in the human genome and can play a causal role in genetic diseases. The functional impact of CNV cannot be fully captured through linkage disequilibrium with SNPs. These observations motivate the development of statistical methods for performing direct CNV association studies. We show through simulation that current tests for CNV association are prone to false-positive associations in the presence of differential errors between cases and controls, espe...

  20. Copy-Number Variations Involving the IHH Locus Are Associated with Syndactyly and Craniosynostosis

    Klopocki, Eva; Lohan, Silke; Brancati, Francesco; Koll, Randi; Brehm, Anja; Seemann, Petra; Dathe, Katarina; Stricker, Sigmar; Hecht, Jochen; Bosse, Kristin; Betz, Regina C.; Garaci, Francesco Giuseppe; Dallapiccola, Bruno; Jain, Mahim; Muenke, Maximilian


    Indian hedgehog (IHH) is a secreted signaling molecule of the hedgehog family known to play important roles in the regulation of chondrocyte differentiation, cortical bone formation, and the development of joints. Here, we describe that copy-number variations of the IHH locus involving conserved noncoding elements (CNEs) are associated with syndactyly and craniosynostosis. These CNEs are able to drive reporter gene expression in a pattern highly similar to wild-type Ihh expression. We postula...

  1. A Comprehensive Analysis of Common Copy-Number Variations in the Human Genome

    Wong, Kendy K. ; deLeeuw, Ronald J. ; Dosanjh, Nirpjit S. ; Kimm, Lindsey R. ; Cheng, Ze ; Horsman, Douglas E. ; MacAulay, Calum ; Ng, Raymond T. ; Brown, Carolyn J. ; Eichler, Evan E. ; Lam, Wan L. 


    Segmental copy-number variations (CNVs) in the human genome are associated with developmental disorders and susceptibility to diseases. More importantly, CNVs may represent a major genetic component of our phenotypic diversity. In this study, using a whole-genome array comparative genomic hybridization assay, we identified 3,654 autosomal segmental CNVs, 800 of which appeared at a frequency of at least 3%. Of these frequent CNVs, 77% are novel. In the 95 individuals analyzed, the two most div...

  2. Short Copy Number Variations Potentially Associated with Tonic Immobility Responses in Newly Hatched Chicks

    Hideaki Abe; Kenji Nagao; Miho Inoue-Murayama


    INTRODUCTION: Tonic immobility (TI) is fear-induced freezing that animals may undergo when confronted by a threat. It is principally observed in prey species as defence mechanisms. In our preliminary research, we detected large inter-individual variations in the frequency and duration of freezing behavior among newly hatched domestic chicks (Gallus gallus). In this study we aim to identify the copy number variations (CNVs) in the genome of chicks as genetic candidates that underlie the behavi...

  3. Leveraging wall-sized high-resolution displays for comparative genomics analyses of copy number variation

    Ruddle, RA; Fateen, W; Treanor, D; Quirke, P.; Sondergeld, P


    The scale of comparative genomics data frequently overwhelms current data visualization methods on conventional (desktop) displays. This paper describes two types of solution that take advantage of wall-sized high-resolution displays (WHirDs), which have orders of magnitude more display real estate (i.e., pixels) than desktop displays. The first allows users to view detailed graphics of copy number variation (CNV) that were output by existing software. A WHirD's resolution allowed a 10× incre...

  4. Human beta-defensin gene copy number variation and consequences in disease and evolution

    Pala, Raquel Rodrigues


    Research on human genetic variation has shown that the human genome is not a fixed, invariant framework, but that there can be extensive structural variation. This variation includes copy number variation (CNV), which can lead to changes in DNA dosage contributing significantly to variation between individual human genomes and heritable traits. Human beta-defensins are small, secreted antimicrobial peptides encoded by DEFB genes located in a cluster of at least seven genes on 8p23.1. These...

  5. Stability-Based Comparison of Class Discovery Methods for DNA Copy Number Profiles

    Brito, Isabel; Hupe, Philippe; Neuvial, Pierre; Barillot, Emmanuel


    Motivation: Array-CGH can be used to determine DNA copy number, imbalances in which are a fundamental factor in the genesis and progression of tumors. The discovery of classes with similar patterns of array-CGH profiles therefore adds to our understanding of cancer and the treatment of patients. Various input data representations for array-CGH, dissimilarity measures between tumor samples and clustering algorithms may be used for this purpose. The choice between procedures is often difficult....

  6. Multi-factor data normalization enables the detection of copy number aberrations in amplicon sequencing data

    Boeva, Valentina; Popova, Tatiana; Lienard, Maxime; Toffoli, Sebastien; Kamal, Maud; Le Tourneau, Christophe; Gentien, David; Servant, Nicolas; Gestraud, Pierre; Rio Frio, Thomas; Hupé, Philippe; Barillot, Emmanuel; Laes, Jean-François


    Motivation: Because of its low cost, amplicon sequencing, also known as ultra-deep targeted sequencing, is now becoming widely used in oncology for detection of actionable mutations, i.e. mutations influencing cell sensitivity to targeted therapies. Amplicon sequencing is based on the polymerase chain reaction amplification of the regions of interest, a process that considerably distorts the information on copy numbers initially present in the tumor DNA. Therefore, additional experiments such...

  7. Indexing Effects of Copy Number Variation on Genes Involved in Developmental Delay

    Mohammed Uddin; Giovanna Pellecchia; Bhooma Thiruvahindrapuram; Lia D’Abate; Daniele Merico; Ada Chan; Mehdi Zarrei; Kristiina Tammimies; Susan Walker; Gazzellone, Matthew J.; Thomas Nalpathamkalam; Yuen, Ryan K.C.; Koenraad Devriendt; Géraldine Mathonnet; Emmanuelle Lemyre


    A challenge in clinical genomics is to predict whether copy number variation (CNV) affecting a gene or multiple genes will manifest as disease. Increasing recognition of gene dosage effects in neurodevelopmental disorders prompted us to develop a computational approach based on critical-exon (highly expressed in brain, highly conserved) examination for potential etiologic effects. Using a large CNV dataset, our updated analyses revealed significant (P 

  8. Large transcription units unify copy number variants and common fragile sites arising under replication stress

    Wilson, Thomas E.; Arlt, Martin F.; Park, So Hae; Rajendran, Sountharia; Paulsen, Michelle; Ljungman, Mats; Glover, Thomas W.


    Copy number variants (CNVs) resulting from genomic deletions and duplications and common fragile sites (CFSs) seen as breaks on metaphase chromosomes are distinct forms of structural chromosome instability precipitated by replication inhibition. Although they share a common induction mechanism, it is not known how CNVs and CFSs are related or why some genomic loci are much more prone to their occurrence. Here we compare large sets of de novo CNVs and CFSs in several experimental cell systems ...

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

    Stadler, Zsofia K.; Esposito, Diane; Shah, Sohela; Vijai, Joseph; Yamrom, Boris; Levy, Dan; Lee, Yoon-ha; Kendall, Jude; Leotta, Anthony; Ronemus, Michael; Hansen, Nichole; Sarrel, Kara; Rau-Murthy, Rohini; Schrader, Kasmintan; Kauff, Noah


    Although heritable factors are an important determinant of risk of early-onset cancer, the majority of these malignancies appear to occur sporadically without identifiable risk factors. Germline de novo copy-number variations (CNVs) have been observed in sporadic neurocognitive and cardiovascular disorders. We explored this mechanism in 382 genomes of 116 early-onset cancer case-parent trios and unaffected siblings. Unique de novo germline CNVs were not observed in 107 breast or colon cancer ...

  10. Social responsiveness scale-aided analysis of the clinical impact of copy number variations in autism

    van Daalen, Emma; Kemner, Chantal; Verbeek, Nienke E; van der Zwaag, Bert; Dijkhuizen, Trijnie; Rump, Patrick; Houben, Renske; van ‘t Slot, Ruben; de Jonge, Maretha V.; Staal, Wouter G; Beemer, Frits A.; Vorstman, Jacob A. S.; J. Peter H. Burbach; van Amstel, Hans Kristian Ploos; Hochstenbach, Ron


    Recent array-based studies have detected a wealth of copy number variations (CNVs) in patients with autism spectrum disorders (ASD). Since CNVs also occur in healthy individuals, their contributions to the patient’s phenotype remain largely unclear. In a cohort of children with symptoms of ASD, diagnosis of the index patient using ADOS-G and ADI-R was performed, and the Social Responsiveness Scale (SRS) was administered to the index patients, both parents, and all available siblings. CNVs wer...

  11. Identification of genes with a correlation between copy number and expression in gastric cancer

    Cheng Lei


    Full Text Available Abstract Background To elucidate gene expression associated with copy number changes, we performed a genome-wide copy number and expression microarray analysis of 25 pairs of gastric tissues. Methods We applied laser capture microdissection (LCM to obtain samples for microarray experiments and profiled DNA copy number and gene expression using 244K CGH Microarray and Human Exon 1.0 ST Microarray. Results Obviously, gain at 8q was detected at the highest frequency (70% and 20q at the second (63%. We also identified molecular genetic divergences for different TNM-stages or histological subtypes of gastric cancers. Interestingly, the C20orf11 amplification and gain at 20q13.33 almost separated moderately differentiated (MD gastric cancers from poorly differentiated (PD type. A set of 163 genes showing the correlations between gene copy number and expression was selected and the identified genes were able to discriminate matched adjacent noncancerous samples from gastric cancer samples in an unsupervised two-way hierarchical clustering. Quantitative RT-PCR analysis for 4 genes (C20orf11, XPO5, PUF60, and PLOD3 of the 163 genes validated the microarray results. Notably, some candidate genes (MCM4 and YWHAZ and its adjacent genes such as PRKDC, UBE2V2, ANKRD46, ZNF706, and GRHL2, were concordantly deregulated by genomic aberrations. Conclusions Taken together, our results reveal diverse chromosomal region alterations for different TNM-stages or histological subtypes of gastric cancers, which is helpful in researching clinicopathological classification, and highlight several interesting genes as potential biomarkers for gastric cancer.

  12. Relationship between EGFR expression, copy number and mutation in lung adenocarcinomas

    This study was designed to investigate EGFR protein expression, EGFR copy number and EGFR mutations in lung adenocarcinomas, to explore the relationship of the three markers. EGFR status was analyzed in surgically resected lung adenocarcinoma samples from 133 Chinese patients by three methods: protein expression (n = 133) by standardized immunohistochemistry (IHC), gene copy number (n = 133) by fluorescence in situ hybridization (FISH), and mutation analysis using the Scorpion amplification refractory mutation system (ARMS) (n = 133). The results showed that 68.4% of the samples were positive by IHC, 42.1% were positive by FISH, and 63.9% contained activating kinase domain mutations. EGFR mutations were more frequent in non-smoking patients (p = 0.008), and EGFR mutations were associated with EGFR FISH positivity (p < 0.0001). When using 10% positivity and 2+ as cutoffs, EGFR protein expression was significantly correlated with EGFR FISH positivity (p = 0.012) and EGFR mutations (p = 0.008) after Bonferroni correction. EGFR protein expression, EGFR copy number and EGFR mutations were closely related to each other. Standard methods and interpretation criteria need to be established

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

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


    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

  14. Chromosomal instability selects gene copy number variants encoding core regulators of proliferation in ER+ breast cancer

    Endesfelder, David; McGranahan, Nicholas; Howell, Mike; Parker, Peter J.; Downward, Julian; Swanton, Charles; Kschischo, Maik


    Chromosomal instability (CIN) is associated with poor outcome in epithelial malignancies including breast carcinomas. Evidence suggests that prognostic signatures in estrogen receptor-positive (ER+) breast cancer define tumors with CIN and high proliferative potential. Intriguingly, CIN induction in lower eukaryotic cells and human cells is context-dependent, typically resulting in a proliferation disadvantage but conferring a fitness benefit under strong selection pressures. We hypothesised that CIN permits accelerated genomic evolution through the generation of diverse DNA copy number events that may be selected during disease development. In support of this hypothesis, we found evidence for selection of gene amplification of core regulators of proliferation in CIN-associated cancer genomes. Stable DNA copy number amplifications of the core regulators TPX2 and UBE2C were associated with expression of a gene module involved in proliferation. The module genes were enriched within prognostic signature gene sets for ER+ breast cancer, providing a logical connection between CIN and prognostic signature expression. Our results provide a framework to decipher the impact of intratumor heterogeneity on key cancer phenotypes, and they suggest that CIN provides a permissive landscape for selection of copy number alterations which drive cancer proliferation. PMID:24970479

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

    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

  16. Proteomic changes resulting from gene copy number variations in cancer cells.

    Tamar Geiger


    Full Text Available Along the transformation process, cells accumulate DNA aberrations, including mutations, translocations, amplifications, and deletions. Despite numerous studies, the overall effects of amplifications and deletions on the end point of gene expression--the level of proteins--is generally unknown. Here we use large-scale and high-resolution proteomics combined with gene copy number analysis to investigate in a global manner to what extent these genomic changes have a proteomic output and therefore the ability to affect cellular transformation. We accurately measure expression levels of 6,735 proteins and directly compare them to the gene copy number. We find that the average effect of these alterations on the protein expression is only a few percent. Nevertheless, by using a novel algorithm, we find the combined impact that many of these regional chromosomal aberrations have at the protein level. We show that proteins encoded by amplified oncogenes are often overexpressed, while adjacent amplified genes, which presumably do not promote growth and survival, are attenuated. Furthermore, regulation of biological processes and molecular complexes is independent of general copy number changes. By connecting the primary genome alteration to their proteomic consequences, this approach helps to interpret the data from large-scale cancer genomics efforts.

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

    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


    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

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

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


    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 PMID:25883151

  19. Fast detection of MYCN copy number alterations in brain neuronal tumors by real-time PCR.

    Malakho, S G; Korshunov, A; Stroganova, A M; Poltaraus, A B


    Increased MYCN gene copy number is a characteristic property of neurogenic tumors. Fluorescence in situ hybridization (FISH) and array-based comparative genomic hybridization (array-CGH) are traditionally used to determine MYCN amplification for tumor stratification. A unique ability of real-time quantitative polymerase chain reaction (qPCR) to determine gene copy number, even within a small percent of observed tumor cells, and can be more appropriate. MYCN genomic copy number from 44 human brain tumors (22 medulloblastomas and 22 neurocytomas) was determined by means of FISH, array-CGH, and qPCR. By qPCR, with the original set of oligonucleotides, 17 out of 44 (38.6%) tumors were found to contain a 1.3- to 2.9-fold increase of MYCN defined as low-level gain. An absolute qPCR method was used to get high accuracy of results. Strong correlation was observed between the three methods: for medulloblastomas, r=1 (Pgain and amplification). PMID:18348317

  20. ReadDepth: a parallel R package for detecting copy number alterations from short sequencing reads.

    Christopher A Miller

    Full Text Available Copy number alterations are important contributors to many genetic diseases, including cancer. We present the readDepth package for R, which can detect these aberrations by measuring the depth of coverage obtained by massively parallel sequencing of the genome. In addition to achieving higher accuracy than existing packages, our tool runs much faster by utilizing multi-core architectures to parallelize the processing of these large data sets. In contrast to other published methods, readDepth does not require the sequencing of a reference sample, and uses a robust statistical model that accounts for overdispersed data. It includes a method for effectively increasing the resolution obtained from low-coverage experiments by utilizing breakpoint information from paired end sequencing to do positional refinement. We also demonstrate a method for inferring copy number using reads generated by whole-genome bisulfite sequencing, thus enabling integrative study of epigenomic and copy number alterations. Finally, we apply this tool to two genomes, showing that it performs well on genomes sequenced to both low and high coverage. The readDepth package runs on Linux and MacOSX, is released under the Apache 2.0 license, and is available at

  1. CCL3L gene copy number and survival in an HIV-1 infected Zimbabwean population

    Larsen, Margit Hørup; Wegner, Lise Thørner; Zinyama, Rutendo;


    The C-C motif chemokine ligand 3-like (CCL3L) protein is a potent chemoattractant which by binding to C-C chemokine receptor type 5 (CCR5) inhibits human immunodeficiency virus (HIV) entry. Copy number variation (CNV) of the CCL3L has been shown to be associated with HIV susceptibility and progre.......9), viral load (P=0.9), or CCL3 protein levels (P=1.0). Survival among the HIV infected individuals did not differ according to CCL3L copy number. In this cohort, CCL3L CNV did not affect HIV status, pathogenesis, or survival.......The C-C motif chemokine ligand 3-like (CCL3L) protein is a potent chemoattractant which by binding to C-C chemokine receptor type 5 (CCR5) inhibits human immunodeficiency virus (HIV) entry. Copy number variation (CNV) of the CCL3L has been shown to be associated with HIV susceptibility and...... progression to AIDS, but these results have been inconsistent. We examined a Zimbabwean study population for an association of CCL3L CNV with HIV status, progression (CD4 T-cells and viral load), and survival. Another aim was to investigate the possible effects of CCL3L CNV on CCL3 protein concentration. A...

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

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


    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

  3. Copy-number gains of HUWE1 due to replication- and recombination-based rearrangements.

    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


    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

  4. Copy number variation associates with mortality in long-lived individuals

    Nygaard, Marianne; Debrabant, Birgit; Tan, Qihua;


    Study (mean age 93.2 years, range 88.9-103.4 years). First, we assessed the association between the CNV burden of each individual (the number of CNVs, the average CNV length, and the total CNV length) and mortality and found a significant increase in mortality per 10 kb increase in the average CNV......Copy number variants (CNVs) represent a significant source of genetic variation in the human genome and have been implicated in numerous diseases and complex traits. To date, only a few studies have investigated the role of CNVs in human lifespan. To investigate the impact of CNVs on prospective...

  5. Integration of transcript expression, copy number and LOH analysis of infiltrating ductal carcinoma of the breast

    Hawthorn Lesleyann


    Full Text Available Abstract Background A major challenge in the interpretation of genomic profiling data generated from breast cancer samples is the identification of driver genes as distinct from bystander genes which do not impact tumorigenesis. One way to assess the relative importance of alterations in the transcriptome profile is to combine parallel analyses that assess changes in the copy number alterations (CNAs. This integrated analysis permits the identification of genes with altered expression that map within specific chromosomal regions which demonstrate copy number alterations, providing a mechanistic approach to identify the 'driver genes'. Methods We have performed whole genome analysis of CNAs using the Affymetrix 250K Mapping array on 22 infiltrating ductal carcinoma samples (IDCs. Analysis of transcript expression alterations was performed using the Affymetrix U133 Plus2.0 array on 16 IDC samples. Fourteen IDC samples were analyzed using both platforms and the data integrated. We also incorporated data from loss of heterozygosity (LOH analysis to identify genes showing altered expression in LOH regions. Results Common chromosome gains and amplifications were identified at 1q21.3, 6p21.3, 7p11.2-p12.1, 8q21.11 and 8q24.3. A novel amplicon was identified at 5p15.33. Frequent losses were found at 1p36.22, 8q23.3, 11p13, 11q23, and 22q13. Over 130 genes were identified with concurrent increases or decreases in expression that mapped to these regions of copy number alterations. LOH analysis revealed three tumors with whole chromosome or p arm allelic loss of chromosome 17. Genes were identified that mapped to copy neutral LOH regions. LOH with accompanying copy loss was detected on Xp24 and Xp25 and genes mapping to these regions with decreased expression were identified. Gene expression data highlighted the PPARα/RXRα Activation Pathway as down-regulated in the tumor samples. Conclusion We have demonstrated the utility of the application of

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

    Cheng Yuanyuan


    Full Text Available Abstract 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.

  7. Landscape of somatic single-nucleotide and copy-number mutations in uterine serous carcinoma.

    Zhao, Siming; Choi, Murim; Overton, John D; Bellone, Stefania; Roque, Dana M; Cocco, Emiliano; Guzzo, Federica; English, Diana P; Varughese, Joyce; Gasparrini, Sara; Bortolomai, Ileana; Buza, Natalia; Hui, Pei; Abu-Khalaf, Maysa; Ravaggi, Antonella; Bignotti, Eliana; Bandiera, Elisabetta; Romani, Chiara; Todeschini, Paola; Tassi, Renata; Zanotti, Laura; Carrara, Luisa; Pecorelli, Sergio; Silasi, Dan-Arin; Ratner, Elena; Azodi, Masoud; Schwartz, Peter E; Rutherford, Thomas J; Stiegler, Amy L; Mane, Shrikant; Boggon, Titus J; Schlessinger, Joseph; Lifton, Richard P; Santin, Alessandro D


    Uterine serous carcinoma (USC) is a biologically aggressive subtype of endometrial cancer. We analyzed the mutational landscape of USC by whole-exome sequencing of 57 cancers, most of which were matched to normal DNA from the same patients. The distribution of the number of protein-altering somatic mutations revealed that 52 USC tumors had fewer than 100 (median 36), whereas 5 had more than 3,000 somatic mutations. The mutations in these latter tumors showed hallmarks of defects in DNA mismatch repair. Among the remainder, we found a significantly increased burden of mutation in 14 genes. In addition to well-known cancer genes (i.e., TP53, PIK3CA, PPP2R1A, KRAS, FBXW7), there were frequent mutations in CHD4/Mi2b, a member of the NuRD-chromatin-remodeling complex, and TAF1, an element of the core TFIID transcriptional machinery. Additionally, somatic copy-number variation was found to play an important role in USC, with 13 copy-number gains and 12 copy-number losses that occurred more often than expected by chance. In addition to loss of TP53, we found frequent deletion of a small segment of chromosome 19 containing MBD3, also a member of the NuRD-chromatin-modification complex, and frequent amplification of chromosome segments containing PIK3CA, ERBB2 (an upstream activator of PIK3CA), and CCNE1 (a target of FBXW7-mediated ubiquitination). These findings identify frequent mutation of DNA damage, chromatin remodeling, cell cycle, and cell proliferation pathways in USC and suggest potential targets for treatment of this lethal variant of endometrial cancer. PMID:23359684

  8. CNV-seq, a new method to detect copy number variation using high-throughput sequencing

    Tammi Martti T


    Full Text Available Abstract Background DNA copy number variation (CNV has been recognized as an important source of genetic variation. Array comparative genomic hybridization (aCGH is commonly used for CNV detection, but the microarray platform has a number of inherent limitations. Results Here, we describe a method to detect copy number variation using shotgun sequencing, CNV-seq. The method is based on a robust statistical model that describes the complete analysis procedure and allows the computation of essential confidence values for detection of CNV. Our results show that the number of reads, not the length of the reads is the key factor determining the resolution of detection. This favors the next-generation sequencing methods that rapidly produce large amount of short reads. Conclusion Simulation of various sequencing methods with coverage between 0.1× to 8× show overall specificity between 91.7 – 99.9%, and sensitivity between 72.2 – 96.5%. We also show the results for assessment of CNV between two individual human genomes.

  9. Clinical Omics Analysis of Colorectal Cancer Incorporating Copy Number Aberrations and Gene Expression Data

    Tsuyoshi Yoshida


    Full Text Available Background: Colorectal cancer (CRC is one of the most frequently occurring cancers in Japan, and thus a wide range of methods have been deployed to study the molecular mechanisms of CRC. In this study, we performed a comprehensive analysis of CRC, incorporating copy number aberration (CRC and gene expression data. For the last four years, we have been collecting data from CRC cases and organizing the information as an “omics” study by integrating many kinds of analysis into a single comprehensive investigation. In our previous studies, we had experienced difficulty in finding genes related to CRC, as we observed higher noise levels in the expression data than in the data for other cancers. Because chromosomal aberrations are often observed in CRC, here, we have performed a combination of CNA analysis and expression analysis in order to identify some new genes responsible for CRC. This study was performed as part of the Clinical Omics Database Project at Tokyo Medical and Dental University. The purpose of this study was to investigate the mechanism of genetic instability in CRC by this combination of expression analysis and CNA, and to establish a new method for the diagnosis and treatment of CRC. Materials and methods: Comprehensive gene expression analysis was performed on 79 CRC cases using an Affymetrix Gene Chip, and comprehensive CNA analysis was performed using an Affymetrix DNA Sty array. To avoid the contamination of cancer tissue with normal cells, laser micro-dissection was performed before DNA/RNA extraction. Data analysis was performed using original software written in the R language. Result: We observed a high percentage of CNA in colorectal cancer, including copy number gains at 7, 8q, 13 and 20q, and copy number losses at 8p, 17p and 18. Gene expression analysis provided many candidates for CRC-related genes, but their association with CRC did not reach the level of statistical significance. The combination of CNA and gene

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

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


    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

  11. Genomic profiling reveals extensive heterogeneity in somatic DNA copy number aberrations of canine hemangiosarcoma.

    Thomas, Rachael; Borst, Luke; Rotroff, Daniel; Motsinger-Reif, Alison; Lindblad-Toh, Kerstin; Modiano, Jaime F; Breen, Matthew


    Canine hemangiosarcoma is a highly aggressive vascular neoplasm associated with extensive clinical and anatomical heterogeneity and a grave prognosis. Comprehensive molecular characterization of hemangiosarcoma may identify novel therapeutic targets and advanced clinical management strategies, but there are no published reports of tumor-associated genome instability and disrupted gene dosage in this cancer. We performed genome-wide microarray-based somatic DNA copy number profiling of 75 primary intra-abdominal hemangiosarcomas from five popular dog breeds that are highly predisposed to this disease. The cohort exhibited limited global genomic instability, compared to other canine sarcomas studied to date, and DNA copy number aberrations (CNAs) were predominantly of low amplitude. Recurrent imbalances of several key cancer-associated genes were evident; however, the global penetrance of any single CNA was low and no distinct hallmark aberrations were evident. Copy number gains of dog chromosomes 13, 24, and 31, and loss of chromosome 16, were the most recurrent CNAs involving large chromosome regions, but their relative distribution within and between cases suggests they most likely represent passenger aberrations. CNAs involving CDKN2A, VEGFA, and the SKI oncogene were identified as potential driver aberrations of hemangiosarcoma development, highlighting potential targets for therapeutic modulation. CNA profiles were broadly conserved between the five breeds, although subregional variation was evident, including a near twofold lower incidence of VEGFA gain in Golden Retrievers versus other breeds (22 versus 40 %). These observations support prior transcriptional studies suggesting that the clinical heterogeneity of this cancer may reflect the existence of multiple, molecularly distinct subtypes of canine hemangiosarcoma. PMID:24599718

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

    Zhang, Liangzhi


    Background: Copy number variations (CNVs) are a main source of genomic structural variations underlying animal evolution and production traits. Here, with one pure-blooded Angus bull as reference, we describe a genome-wide analysis of CNVs based on comparative genomic hybridization arrays in 29 Chinese domesticated bulls and examined their effects on gene expression and cattle growth traits.Results: We identified 486 copy number variable regions (CNVRs), covering 2.45% of the bovine genome, in 24 taurine (Bos taurus), together with 161 ones in 2 yaks (Bos grunniens) and 163 ones in 3 buffaloes (Bubalus bubalis). Totally, we discovered 605 integrated CNVRs, with more " loss" events than both " gain" and " both" ones, and clearly clustered them into three cattle groups. Interestingly, we confirmed their uneven distributions across chromosomes, and the differences of mitochondrion DNA copy number (gain: taurine, loss: yak & buffalo). Furthermore, we confirmed approximately 41.8% (253/605) and 70.6% (427/605) CNVRs span cattle genes and quantitative trait loci (QTLs), respectively. Finally, we confirmed 6 CNVRs in 9 chosen ones by using quantitative PCR, and further demonstrated that CNVR22 had significantly negative effects on expression of PLA2G2D gene, and both CNVR22 and CNVR310 were associated with body measurements in Chinese cattle, suggesting their key effects on gene expression and cattle traits.Conclusions: The results advanced our understanding of CNV as an important genomic structural variation in taurine, yak and buffalo. This study provides a highly valuable resource for Chinese cattle\\'s evolution and breeding researches. 2014 Zhang et al.; licensee BioMed Central Ltd.

  13. Focal DNA copy number changes in neuroblastoma target MYCN regulated genes.

    Candy Kumps

    Full Text Available Neuroblastoma is an embryonic tumor arising from immature sympathetic nervous system cells. Recurrent genomic alterations include MYCN and ALK amplification as well as recurrent patterns of gains and losses of whole or large partial chromosome segments. A recent whole genome sequencing effort yielded no frequently recurring mutations in genes other than those affecting ALK. However, the study further stresses the importance of DNA copy number alterations in this disease, in particular for genes implicated in neuritogenesis. Here we provide additional evidence for the importance of focal DNA copy number gains and losses, which are predominantly observed in MYCN amplified tumors. A focal 5 kb gain encompassing the MYCN regulated miR-17~92 cluster as sole gene was detected in a neuroblastoma cell line and further analyses of the array CGH data set demonstrated enrichment for other MYCN target genes in focal gains and amplifications. Next we applied an integrated genomics analysis to prioritize MYCN down regulated genes mediated by MYCN driven miRNAs within regions of focal heterozygous or homozygous deletion. We identified RGS5, a negative regulator of G-protein signaling implicated in vascular normalization, invasion and metastasis, targeted by a focal homozygous deletion, as a new MYCN target gene, down regulated through MYCN activated miRNAs. In addition, we expand the miR-17~92 regulatory network controlling TGFß signaling in neuroblastoma with the ring finger protein 11 encoding gene RNF11, which was previously shown to be targeted by the miR-17~92 member miR-19b. Taken together, our data indicate that focal DNA copy number imbalances in neuroblastoma (1 target genes that are implicated in MYCN signaling, possibly selected to reinforce MYCN oncogene addiction and (2 serve as a resource for identifying new molecular targets for treatment.

  14. Copy number variations of chromosome 16p13.1 region associated with schizophrenia

    Ingason, A; Rujescu, D; Cichon, S;


    Deletions and reciprocal duplications of the chromosome 16p13.1 region have recently been reported in several cases of autism and mental retardation (MR). As genomic copy number variants found in these two disorders may also associate with schizophrenia, we examined 4345 schizophrenia patients an...... hyperactivity disorder and dyslexia. Candidate genes in the region include NTAN1 and NDE1. We conclude that duplications and perhaps also deletions of chromosome 16p13.1, previously reported to be associated with autism and MR, also confer risk of schizophrenia....

  15. Copy-number variations involving the IHH locus are associated with syndactyly and craniosynostosis.

    Klopocki, Eva; Lohan, Silke; Brancati, Francesco; Koll, Randi; Brehm, Anja; Seemann, Petra; Dathe, Katarina; Stricker, Sigmar; Hecht, Jochen; Bosse, Kristin; Betz, Regina C; Garaci, Francesco Giuseppe; Dallapiccola, Bruno; Jain, Mahim; Muenke, Maximilian; Ng, Vivian C W; Chan, Wilson; Chan, Danny; Mundlos, Stefan


    Indian hedgehog (IHH) is a secreted signaling molecule of the hedgehog family known to play important roles in the regulation of chondrocyte differentiation, cortical bone formation, and the development of joints. Here, we describe that copy-number variations of the IHH locus involving conserved noncoding elements (CNEs) are associated with syndactyly and craniosynostosis. These CNEs are able to drive reporter gene expression in a pattern highly similar to wild-type Ihh expression. We postulate that the observed duplications lead to a misexpression and/or overexpression of IHH and by this affect the complex regulatory signaling network during digit and skull development. PMID:21167467

  16. Distribution and functional impact of DNA copy number variation in the rat

    Guryev, V.; Saar, K.; Adamovic, T.; Verheul, M.; Van Heesch, S.A.A.C.; Cook, S.; Pravenec, Michal; Aitman, T.; Jacob, H.; Shull, J.D.; Hubner, N.; Cuppen, E.


    Roč. 40, č. 5 (2008), s. 538-545. ISSN 1061-4036 R&D Projects: GA MŠk(CZ) 1P05ME791; GA MŠk(CZ) 1M0520 Grant ostatní: HHMI(US) 55005624; -(XE) LSHG-CT-2005-019015 Institutional research plan: CEZ:AV0Z50110509 Source of funding: N - neverejné zdroje ; R - rámcový projekt EK Keywords : copy number variation * rat * recombinant inbred strains Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 30.259, year: 2008

  17. Selective Constraint on Copy Number Variation in Human Piwi-Interacting RNA Loci

    Gould, David W.; Sergio Lukic; Chen, Kevin C


    Piwi-interacting RNAs (piRNAs) are a recently discovered class of small non-coding RNA found in animals. PiRNAs are primarily expressed in the germline where their best understood function is to repress transposable elements. Unlike previous studies that investigated the evolution of piRNA-generating loci at the level of nucleotide substitutions, here we studied the evolution of piRNA-generating loci at the level of copy number variation (i.e. duplications and deletions) using genome-wide cop...

  18. Reduction in mitochondrial DNA copy number in peripheral leukocytes after onset of Huntington's disease

    Petersen, Maria Hvidberg; Budtz-Jørgensen, Esben; Sørensen, Sven Asger;


    Huntington's disease (HD) is an inherited neurodegenerative disorder characterised by movement disorder, cognitive symptoms and psychiatric symptoms with predominantly adult-onset. The mutant huntingtin protein leads to mitochondrial dysfunction in blood leukocytes. This discovery led to the...... investigation of the mitochondrial DNA (mtDNA) copy number relative to nuclear DNA (nDNA) in leukocytes from carriers of the HD mutation compared to healthy individuals. We found significantly reduced mtDNA/nDNA in HD mutation carriers compared to controls. A longitudinal study of archive DNA sample pairs from...

  19. High Glucose-Induced Oxidative Stress Increases the Copy Number of Mitochondrial DNA in Human Mesangial Cells

    Jamal Golbahar; Ghada Al-Kafaji


    Oxidative damage to mitochondrial DNA (mtDNA) has been linked to the pathogenicity of diabetic nephropathy. We tested the hypothesis that mtDNA copy number may be increased in human mesangial cells in response to high glucose-induced reactive oxygen species (ROS) to compensate for damaged mtDNA. The effect of manganese superoxide dismutase mimetic (MnTBAP) on glucose-induced mtDNA copy number was also examined. The copy number of mtDNA was determined by real-time PCR in human mesangial cells ...

  20. Low copy number of mitochondrial DNA (mtDNA) predicts worse prognosis in early-stage laryngeal cancer patients

    Dang, Siwen; Qu, Yiping; Wei, Jing; Shao, Yuan; Yang, Qi; Ji, Meiju; Shi, Bingyin; Hou, Peng


    Objectives Alterations in mitochondrial DNA (mtDNA) copy number have been widely reported in various human cancers, and been considered to be an important hallmark of cancers. However, little is known about the value of copy number variations of mtDNA in the prognostic evaluation of laryngeal cancer. Design and methods Using real-time quantitative PCR method, we investigated mtDNA copy number in a cohort of laryngeal cancers (n =204) and normal laryngeal tissues (n =40), and explored the asso...

  1. Germline DNA copy number aberrations identified as potential prognostic factors for breast cancer recurrence.

    Yadav Sapkota

    Full Text Available Breast cancer recurrence (BCR is a common treatment outcome despite curative-intent primary treatment of non-metastatic breast cancer. Currently used prognostic and predictive factors utilize tumor-based markers, and are not optimal determinants of risk of BCR. Germline-based copy number aberrations (CNAs have not been evaluated as determinants of predisposition to experience BCR. In this study, we accessed germline DNA from 369 female breast cancer subjects who received curative-intent primary treatment following diagnosis. Of these, 155 experienced BCR and 214 did not, after a median duration of follow up after breast cancer diagnosis of 6.35 years (range = 0.60-21.78 and 8.60 years (range = 3.08-13.57, respectively. Whole genome CNA genotyping was performed on the Affymetrix SNP array 6.0 platform. CNAs were identified using the SNP-Fast Adaptive States Segmentation Technique 2 algorithm implemented in Nexus Copy Number 6.0. Six samples were removed due to poor quality scores, leaving 363 samples for further analysis. We identified 18,561 CNAs with ≥1 kb as a predefined cut-off for observed aberrations. Univariate survival analyses (log-rank tests identified seven CNAs (two copy number gains and five copy neutral-loss of heterozygosities, CN-LOHs showing significant differences (P<2.01×10(-5 in recurrence-free survival (RFS probabilities with and without CNAs.We also observed three additional but distinct CN-LOHs showing significant differences in RFS probabilities (P<2.86×10(-5 when analyses were restricted to stratified cases (luminal A, n = 208 only. After adjusting for tumor stage and grade in multivariate analyses (Cox proportional hazards models, all the CNAs remained strongly associated with the phenotype of BCR. Of these, we confirmed three CNAs at 17q11.2, 11q13.1 and 6q24.1 in representative samples using independent genotyping platforms. Our results suggest further investigations on the potential use of germline DNA

  2. The Porcine TSPY Gene Is Tricopy but Not a Copy Number Variant.

    Anh T Quach

    Full Text Available The testis-specific protein Y-encoded (TSPY gene is situated on the mammalian Y-chromosome and exhibits some remarkable biological characteristics. It has the highest known copy number (CN of all protein coding genes in the human and bovine genomes (up to 74 and 200, respectively and also shows high individual variability. Although the biological function of TSPY has not yet been elucidated, its specific expression in the testis and several identified binding domains within the protein suggests roles in male reproduction. Here we describe the porcine TSPY, as a multicopy gene with three copies located on the short arm of the Y-chromosome with no variation at three exon loci among 20 animals of normal reproductive health from four breeds of domestic pigs (Piétrain, Landrace, Duroc and Yorkshire. To further investigate the speculation that porcine TSPY is not a copy number variant, we have included five Low-fertility boars and five boars with exceptional High-fertility records. Interestingly, there was no difference between the High- and Low-fertile groups, but we detected slightly lower TSPY CN at all three exons (2.56-2.85 in both groups, as compared to normal animals, which could be attributed to technical variability or somatic mosaicism. The results are based on both relative quantitative real-time PCR (qPCR and droplet digital PCR (ddPCR. Chromosomal localization of the porcine TSPY was done using fluorescence in situ hybridization (FISH with gene specific PCR probes.

  3. Whole genome distribution and ethnic differentiation of copy number variation in Caucasian and Asian populations.

    Jian Li

    Full Text Available Although copy number variation (CNV has recently received much attention as a form of structure variation within the human genome, knowledge is still inadequate on fundamental CNV characteristics such as occurrence rate, genomic distribution and ethnic differentiation. In the present study, we used the Affymetrix GeneChip(R Mapping 500K Array to discover and characterize CNVs in the human genome and to study ethnic differences of CNVs between Caucasians and Asians. Three thousand and nineteen CNVs, including 2381 CNVs in autosomes and 638 CNVs in X chromosome, from 985 Caucasian and 692 Asian individuals were identified, with a mean length of 296 kb. Among these CNVs, 190 had frequencies greater than 1% in at least one ethnic group, and 109 showed significant ethnic differences in frequencies (p<0.01. After merging overlapping CNVs, 1135 copy number variation regions (CNVRs, covering approximately 439 Mb (14.3% of the human genome, were obtained. Our findings of ethnic differentiation of CNVs, along with the newly constructed CNV genomic map, extend our knowledge on the structural variation in the human genome and may furnish a basis for understanding the genomic differentiation of complex traits across ethnic groups.

  4. A classification model for distinguishing copy number variants from cancer-related alterations

    Olshen Adam B


    Full Text Available Abstract Background Both somatic copy number alterations (CNAs and germline copy number variants (CNVs that are prevalent in healthy individuals can appear as recurrent changes in comparative genomic hybridization (CGH analyses of tumors. In order to identify important cancer genes CNAs and CNVs must be distinguished. Although the Database of Genomic Variants (DGV contains a list of all known CNVs, there is no standard methodology to use the database effectively. Results We develop a prediction model that distinguishes CNVs from CNAs based on the information contained in the DGV and several other variables, including segment's length, height, closeness to a telomere or centromere and occurrence in other patients. The models are fitted on data from glioblastoma and their corresponding normal samples that were collected as part of The Cancer Genome Atlas project and hybridized to Agilent 244 K arrays. Conclusions Using the DGV alone CNVs in the test set can be correctly identified with about 85% accuracy if the outliers are removed before segmentation and with 72% accuracy if the outliers are included, and additional variables improve the prediction by about 2-3% and 12%, respectively. Final models applied to data from ovarian tumors have about 90% accuracy with all the variables and 86% accuracy with the DGV alone.

  5. Genome-wide analysis of copy number variation in type 1 diabetes.

    Britney L Grayson

    Full Text Available Type 1 diabetes (T1D tends to cluster in families, suggesting there may be a genetic component predisposing to disease. However, a recent large-scale genome-wide association study concluded that identified genetic factors, single nucleotide polymorphisms, do not account for overall familiality. Another class of genetic variation is the amplification or deletion of >1 kilobase segments of the genome, also termed copy number variations (CNVs. We performed genome-wide CNV analysis on a cohort of 20 unrelated adults with T1D and a control (Ctrl cohort of 20 subjects using the Affymetrix SNP Array 6.0 in combination with the Birdsuite copy number calling software. We identified 39 CNVs as enriched or depleted in T1D versus Ctrl. Additionally, we performed CNV analysis in a group of 10 monozygotic twin pairs discordant for T1D. Eleven of these 39 CNVs were also respectively enriched or depleted in the Twin cohort, suggesting that these variants may be involved in the development of islet autoimmunity, as the presently unaffected twin is at high risk for developing islet autoimmunity and T1D in his or her lifetime. These CNVs include a deletion on chromosome 6p21, near an HLA-DQ allele. CNVs were found that were both enriched or depleted in patients with or at high risk for developing T1D. These regions may represent genetic variants contributing to development of islet autoimmunity in T1D.

  6. Association of Copy Number Variations in Autism Spectrum Disorders: A Systematic Review

    Elif Funda Sener


    Full Text Available Autism spectrum disorders (ASDs are characterized by language impairments, social deficits, and repetitive behaviors. The onset of symptoms occurs by the age of 3 and shows a lifelong persistence. Genetics plays a major role in the etiology of ASD. Except genetics, several potential risk factors (environmental factors and epigenetics may contribute to ASD. Copy number variations (CNVs are the most widespread structural variations in the human genome. These variations can alter the genome structure either by deletion or by duplication. CNVs can be de novo or inherited. Chromosomal rearrangements have been detected in 5–10% of the patients with ASD and recently copy number changes ranging from a few kilobases (kb to several megabases (Mb in size have been reported. Recent data have also revealed that submicroscopic CNVs can have a role in ASD, and de novo CNVs seem to be a more common risk factor in sporadic compared with inherited forms of ASD. CNVs are being implicated as a contributor to the pathophysiology of complex neurodevelopmental disorders and they can affect a wide range of human phenotypes including mental retardation (MR, autism, neuropsychiatric disorders, and susceptibility to other complex traits such as HIV, Crohn’s disease, and psoriasis. This review emphasizes the major CNVs reported to date in ASD.

  7. Downregulation of Tfam and mtDNA copy number during mammalian spermatogenesis.

    Rantanen, A; Jansson, M; Oldfors, A; Larsson, N G


    Mitochondrial transcription factor A (Tfam) is required for mtDNA maintenance, and mitochondrial Tfam protein levels directly affect mtDNA copy number. Previous studies have shown significant reduction of Tfam protein levels in mitochondria together with the appearance of abundant testis-specific Tfam mRNA isoforms as spermatogenesis proceeds in both mouse and man. Interestingly, an abundant testis-specific nuclear Tfam protein isoform of unknown function is found in the mouse, but not in humans. We have now characterized Tfam expression in rat testis to identify conserved features in mammalian spermatogenesis. The nuclear Tfam protein isoform is absent in the rat and is thus dispensable for mammalian spermatogenesis. Similar to mice and humans, we found expression of alternate Tfam transcripts, downregulation of mitochondrial Tfam protein levels, and downregulation of mtDNA copy number during rat spermatogenesis. These features are thus common to all mammals and may provide one of several mechanisms preventing paternal mtDNA transmission. PMID:11668394

  8. CLAMMS: a scalable algorithm for calling common and rare copy number variants from exome sequencing data

    Packer, Jonathan S.; Maxwell, Evan K.; O’Dushlaine, Colm; Lopez, Alexander E.; Dewey, Frederick E.; Chernomorsky, Rostislav; Baras, Aris; Overton, John D.; Habegger, Lukas; Reid, Jeffrey G.


    Motivation: Several algorithms exist for detecting copy number variants (CNVs) from human exome sequencing read depth, but previous tools have not been well suited for large population studies on the order of tens or hundreds of thousands of exomes. Their limitations include being difficult to integrate into automated variant-calling pipelines and being ill-suited for detecting common variants. To address these issues, we developed a new algorithm—Copy number estimation using Lattice-Aligned Mixture Models (CLAMMS)—which is highly scalable and suitable for detecting CNVs across the whole allele frequency spectrum. Results: In this note, we summarize the methods and intended use-case of CLAMMS, compare it to previous algorithms and briefly describe results of validation experiments. We evaluate the adherence of CNV calls from CLAMMS and four other algorithms to Mendelian inheritance patterns on a pedigree; we compare calls from CLAMMS and other algorithms to calls from SNP genotyping arrays for a set of 3164 samples; and we use TaqMan quantitative polymerase chain reaction to validate CNVs predicted by CLAMMS at 39 loci (95% of rare variants validate; across 19 common variant loci, the mean precision and recall are 99% and 94%, respectively). In the Supplementary Materials (available at the CLAMMS Github repository), we present our methods and validation results in greater detail. Availability and implementation: (implemented in C). Contact: Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26382196

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

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


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

  10. Genomic Copy Number Variations in the Genomes of Leukocytes Predict Prostate Cancer Clinical Outcomes.

    Yan P Yu

    Full Text Available Accurate prediction of prostate cancer clinical courses remains elusive. In this study, we performed whole genome copy number analysis on leukocytes of 273 prostate cancer patients using Affymetrix SNP6.0 chip. Copy number variations (CNV were found across all chromosomes of the human genome. An average of 152 CNV fragments per genome was identified in the leukocytes from prostate cancer patients. The size distributions of CNV in the genome of leukocytes were highly correlative with prostate cancer aggressiveness. A prostate cancer outcome prediction model was developed based on large size ratio of CNV from the leukocyte genomes. This prediction model generated an average prediction rate of 75.2%, with sensitivity of 77.3% and specificity of 69.0% for prostate cancer recurrence. When combined with Nomogram and the status of fusion transcripts, the average prediction rate was improved to 82.5% with sensitivity of 84.8% and specificity of 78.2%. In addition, the leukocyte prediction model was 62.6% accurate in predicting short prostate specific antigen doubling time. When combined with Gleason's grade, Nomogram and the status of fusion transcripts, the prediction model generated a correct prediction rate of 77.5% with 73.7% sensitivity and 80.1% specificity. To our knowledge, this is the first study showing that CNVs in leukocyte genomes are predictive of clinical outcomes of a human malignancy.

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

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


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

  12. Gene Copy Number Analysis for Family Data Using Semiparametric Copula Model

    Ao Yuan


    Full Text Available Gene copy number changes are common characteristics of many genetic disorders. A new technology, array comparative genomic hybridization (a-CGH, is widely used today to screen for gains and losses in cancers and other genetic diseases with high resolution at the genome level or for specific chromosomal region. Statistical methods for analyzing such a-CGH data have been developed. However, most of the existing methods are for unrelated individual data and the results from them provide explanation for horizontal variations in copy number changes. It is potentially meaningful to develop a statistical method that will allow for the analysis of family data to investigate the vertical kinship effects as well. Here we consider a semiparametric model based on clustering method in which the marginal distributions are estimated nonparametrically, and the familial dependence structure is modeled by copula. The model is illustrated and evaluated using simulated data. Our results show that the proposed method is more robust than the commonly used multivariate normal model. Finally, we demonstrated the utility of our method using a real dataset.

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

    Chen-Sung Lin


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

  14. High frequency of rare copy number variants affecting functionally related genes in patients with structural brain malformations

    Kariminejad, Roxana; Lind-Thomsen, Allan; Tümer, Zeynep;


    investigate copy number variants (CNVs) in a cohort of 169 patients with various structural brain malformations including lissencephaly, polymicrogyria, focal cortical dysplasia, and corpus callosum agenesis. The majority of the patients had intellectual disabilities (ID) and suffered from symptomatic...

  15. PCR-Based Analysis of Mitochondrial DNA Copy Number, Mitochondrial DNA Damage, and Nuclear DNA Damage.

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


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

  16. Mitochondrial DNA copy number in peripheral blood cells declines with age and is associated with general health among elderly

    Mengel-From, Jonas; Thinggaard, Mikael; Dalgård, Christine;


    The role of the mitochondria in disease, general health and aging has drawn much attention over the years. Several attempts have been made to describe how the numbers of mitochondria correlate with age, although with inconclusive results. In this study, the relative quantity of mitochondrial DNA...... compared to nuclear DNA, i.e. the mitochondrial DNA copy number, was measured by PCR technology and used as a proxy for the content of mitochondria copies. In 1,067 Danish twins and singletons (18-93 years of age), with the majority being elderly individuals, the estimated mean mitochondrial DNA copy...

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

    Heli Viljakainen

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

  18. CNTN6 copy number variations in 14 patients: a possible candidate gene for neurodevelopmental and neuropsychiatric disorders

    Hu, Jie; Liao, Jun; Sathanoori, Malini; Kochmar, Sally; Sebastian, Jessica; Yatsenko, Svetlana A.; Surti, Urvashi


    Background Neurodevelopmental disorders are impairments of brain function that affect emotion, learning, and memory. Copy number variations of contactin genes (CNTNs), including CNTN3, CNTN4, CNTN5, and CNTN6, have been suggested to be associated with these disorders. However, phenotypes have been reported in only a handful of patients with copy number variations involving CNTNs. Methods From January 2009 to January 2013, 3724 patients ascertained through the University of Pittsburgh Medical ...

  19. Variable copy number of mitochondrial DNA (mtDNA) predicts worse prognosis in advanced gastric cancer patients

    Zhang, Guanjun; Qu, Yiping; Dang, Siwen; Yang, Qi; Shi, Bingyin; Hou, Peng


    Background Change of mitochondrial DNA (mtDNA) copy number is widely reported in various human cancers, including gastric cancer, and is considered to be an important hallmark of cancers. However, there is remarkably little consensus on the value of variable mtDNA content in the prognostic evaluation of this cancer. Methods Using real-time quantitative PCR approach, we examined mtDNA copy number in a cohort of gastric cancers and normal gastric tissues, and explored the association of variabl...

  20. A temperature cline in copy number for 412 but not roo/B104 retrotransposons in populations of Drosophila simulans.

    Vieira, C; Aubry, P.; Lepetit, D; Biémont, C.


    The copy number of the retrotransposable element 412 of Drosophila simulans from populations collected worldwide shows a negative correlation with minimum temperature. No association was detected for the roo/B104 element. The possibility that selective pressures might regulate the 412 copy number in these natural populations is supported by detection of selection against the detrimental effects of 412 insertions (estimated by the proportion of insertions on the X chromosome in comparison with...

  1. Analysis of changes in DNA sequence copy number by comparative genomic hybridization in archival paraffin-embedded tumor samples.

    Isola, J; DeVries, S; Chu, L; Ghazvini, S.; Waldman, F.


    Analysis of previously unknown genetic aberrations in solid tumors has become possible through the use of comparative genomic hybridization (CGH), which is based on competitive binding of tumor and control DNA to normal metaphase chromosomes. CGH allows detection of DNA sequence copy number changes (deletions, gains, and amplifications) on a genome-wide scale in a single hybridization. We describe here an improved CGH technique, which enables reliable detection of copy number changes in archi...

  2. GAPDH as a control gene to estimate genome copy number in Great Tits, with cross-amplification in Blue Tits

    Atema, Els; van Oers, Kees; Verhulst, Simon; Prop, Jouke


    Estimating the number of genome copies in a tissue sample can serve various purposes. For example, such an estimate serves as a scaling variable when measuring telomeres with quantitative PCR. We describe the primer development and evaluation for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene in the Great Tit Parus major, as a control gene to estimate genome copy number. We demonstrate specific amplification with negligible variation in 48 Great Tits and cross-amplification in 53 B...

  3. The Number of Herpes Simplex Virus-Infected Neurons and the Number of Viral Genome Copies per Neuron Correlate with the Latent Viral Load in Ganglia*

    Hoshino, Yo; Qin, Jing; Follmann, Dean; Cohen, Jeffrey I.; Straus, Stephen E


    The latent viral load is the most important factor that predicts reactivation rates of animals latently infected with herpes simplex virus (HSV). To estimate the latent viral load, individual latently infected mouse trigeminal ganglia were dispersed into single cell suspensions and plated into 96-well real-time PCR plates, and HSV-2 genome copies were measured. By assuming a Poisson distribution for both the number of HSV-2 infected cells per well and the number of HSV-2 genome copies per inf...

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

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


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

  5. Mitochondrial DNA copy number and risk of oral cancer: a report from Northeast India.

    Rosy Mondal

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

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

    Ammar F. Hameed


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

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

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


    Stable transformation of plants is a powerful tool for hypothesis testing. A rapid and reliable evaluation method of the transgenic allele for copy number and homozygosity is vital in analysing these transformations. Here the suitability of Southern blot analysis, thermal asymmetric interlaced (TAIL-)PCR, quantitative (q)PCR and digital droplet (dd)PCR to estimate T-DNA copy number, locus complexity and homozygosity were compared in transgenic tobacco. Southern blot analysis and ddPCR on three generations of transgenic offspring with contrasting zygosity and copy number were entirely consistent, whereas TAIL-PCR often underestimated copy number. qPCR deviated considerably from the Southern blot results and had lower precision and higher variability than ddPCR. Comparison of segregation analyses and ddPCR of T1 progeny from 26 T0 plants showed that at least 19% of the lines carried multiple T-DNA insertions per locus, which can lead to unstable transgene expression. Segregation analyses failed to detect these multiple copies, presumably because of their close linkage. This shows the importance of routine T-DNA copy number estimation. Based on our results, ddPCR is the most suitable method, because it is as reliable as Southern blot analysis yet much faster. A protocol for this application of ddPCR to large plant genomes is provided. PMID:26670088

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

    Jun Ding


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

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

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


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

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

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


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

  11. A pilot study on commonality and specificity of copy number variants in schizophrenia and bipolar disorder.

    Chen, J; Calhoun, V D; Perrone-Bizzozero, N I; Pearlson, G D; Sui, J; Du, Y; Liu, J


    Schizophrenia (SZ) and bipolar disorder (BD) are known to share genetic risks. In this work, we conducted whole-genome scanning to identify cross-disorder and disorder-specific copy number variants (CNVs) for these two disorders. The Database of Genotypes and Phenotypes (dbGaP) data were used for discovery, deriving from 2416 SZ patients, 592 BD patients and 2393 controls of European Ancestry, as well as 998 SZ patients, 121 BD patients and 822 controls of African Ancestry. PennCNV and Birdsuite detected high-confidence CNVs that were aggregated into CNV regions (CNVRs) and compared with the database of genomic variants for confirmation. Then, large (size⩾500 kb) and small common CNVRs (size models. PMID:27244233

  12. Improved determination of plasmid copy number using quantitative real-time PCR for monitoring fermentation processes

    Štrukelj Borut


    Full Text Available Abstract Background Recombinant protein production in Escherichia coli cells is a complex process, where among other parameters, plasmid copy number, structural and segregational stability of plasmid have an important impact on the success of productivity. It was recognised that a method for accurate and rapid quantification of plasmid copy number is necessary for optimization and better understanding of this process. Lately, qPCR is becoming the method of choice for this purpose. In the presented work, an improved qPCR method adopted for PCN determination in various fermentation processes was developed. Results To avoid experimental errors arising from irreproducible DNA isolation, whole cells, treated by heating at 95°C for 10 minutes prior to storage at -20°C, were used as a template source. Relative quantification, taking into account different amplification efficiencies of amplicons for chromosome and plasmid, was used in the PCN calculation. The best reproducibility was achieved when the efficiency estimated for specific amplicon, obtained within one run, was averaged. It was demonstrated that the quantification range of 2 log units (100 to 10000 bacteria per well enable quantification in each time point during fermentation. The method was applied to study PCN variation in fermentation at 25°C and the correlation between PCN and protein accumulation was established. Conclusion Using whole cells as a template source and relative quantification considering different PCR amplification efficiencies are significant improvements of the qPCR method for PCN determination. Due to the approaches used, the method is suitable for PCN determination in fermentation processes using various media and conditions.

  13. Copy number and targeted mutational analysis reveals novel somatic events in metastatic prostate tumors.

    Robbins, Christiane M; Tembe, Waibov A; Baker, Angela; Sinari, Shripad; Moses, Tracy Y; Beckstrom-Sternberg, Stephen; Beckstrom-Sternberg, James; Barrett, Michael; Long, James; Chinnaiyan, Arul; Lowey, James; Suh, Edward; Pearson, John V; Craig, David W; Agus, David B; Pienta, Kenneth J; Carpten, John D


    Advanced prostate cancer can progress to systemic metastatic tumors, which are generally androgen insensitive and ultimately lethal. Here, we report a comprehensive genomic survey for somatic events in systemic metastatic prostate tumors using both high-resolution copy number analysis and targeted mutational survey of 3508 exons from 577 cancer-related genes using next generation sequencing. Focal homozygous deletions were detected at 8p22, 10q23.31, 13q13.1, 13q14.11, and 13q14.12. Key genes mapping within these deleted regions include PTEN, BRCA2, C13ORF15, and SIAH3. Focal high-level amplifications were detected at 5p13.2-p12, 14q21.1, 7q22.1, and Xq12. Key amplified genes mapping within these regions include SKP2, FOXA1, and AR. Furthermore, targeted mutational analysis of normal-tumor pairs has identified somatic mutations in genes known to be associated with prostate cancer including AR and TP53, but has also revealed novel somatic point mutations in genes including MTOR, BRCA2, ARHGEF12, and CHD5. Finally, in one patient where multiple independent metastatic tumors were available, we show common and divergent somatic alterations that occur at both the copy number and point mutation level, supporting a model for a common clonal progenitor with metastatic tumor-specific divergence. Our study represents a deep genomic analysis of advanced metastatic prostate tumors and has revealed candidate somatic alterations, possibly contributing to lethal prostate cancer. PMID:21147910

  14. Copy number variants including RAS pathway genes-How much RASopathy is in the phenotype?

    Lissewski, Christina; Kant, Sarina G; Stark, Zornitza; Schanze, Ina; Zenker, Martin


    The RASopathies comprise a group of clinically overlapping developmental syndromes the common pathogenetic basis of which is dysregulated signal flow through the RAS-MAPK pathway. Mutations in several components or modifiers of the pathway have been identified in Noonan syndrome and related disorders. Over the past years copy number variants (CNVs) encompassing RAS pathway genes (PTPN11, RAF1, MEK2, or SHOC2) have been reported in children with developmental syndromes. These observations raised speculations that the associated phenotypes represent RASopathies, implying that the increased or reduced expression of the respective RAS pathway component and a consecutive dysregulation of RAS pathway signalling is responsible for the clinical picture. Herein, we present two individuals and three of their relatives harboring duplications of either 3p25.2 including the RAF1 locus or 19p13.3 including the MEK2 locus. Duplication carriers exhibited variable clinical phenotypes including non-specific facial dysmorphism, short stature, and learning difficulties. A careful review of the literature supported the impression that phenotypes associated with CNVs including RAS pathway genes commonly share non-specific symptoms with RASopathies, while the characteristic "gestalt" is lacking. Considering the known molecular pathogenesis of RASopathies, it is questionable that a modest increase in the expression of a functionally normal signaling component can mimic the effects of a qualitatively abnormal (hyperactive) mutant protein. We thus argue that current empirical and biological evidence is still insufficient to allow the conclusion that an altered copy number of a RAS pathway component is indeed the mechanism that is critical for the phenotype associated with CNVs including RASopathy genes. PMID:25974318

  15. Rare de novo copy number variants in patients with congenital pulmonary atresia.

    Li Xie

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

  16. Copy number variants in patients with severe oligozoospermia and Sertoli-cell-only syndrome.

    Frank Tüttelmann

    Full Text Available A genetic origin is estimated in 30% of infertile men with the common phenotypes of oligo- or azoospermia, but the pathogenesis of spermatogenic failure remains frequently obscure. To determine the involvement of Copy Number Variants (CNVs in the origin of male infertility, patients with idiopathic severe oligozoospermia (N = 89, Sertoli-cell-only syndrome (SCOS, N = 37 and controls with normozoospermia (N = 100 were analysed by array-CGH using the 244A/400K array sets (Agilent Technologies. The mean number of CNVs and the amount of DNA gain/loss were comparable between all groups. Ten recurring CNVs were only found in patients with severe oligozoospermia, three only in SCOS and one CNV in both groups with spermatogenic failure but not in normozoospermic men. Sex-chromosomal, mostly private CNVs were significantly overrepresented in patients with SCOS. CNVs found several times in all groups were analysed in a case-control design and four additional candidate genes and two regions without known genes were associated with SCOS (P<1×10(-3. In conclusion, by applying array-CGH to study male infertility for the first time, we provide a number of candidate genes possibly causing or being risk factors for the men's spermatogenic failure. The recurring, patient-specific and private, sex-chromosomal CNVs as well as those associated with SCOS are candidates for further, larger case-control and re-sequencing studies.

  17. Variations in CCL3L gene cluster sequence and non-specific gene copy numbers

    Edberg Jeffrey C


    Full Text Available Abstract Background Copy number variations (CNVs of the gene CC chemokine ligand 3-like1 (CCL3L1 have been implicated in HIV-1 susceptibility, but the association has been inconsistent. CCL3L1 shares homology with a cluster of genes localized to chromosome 17q12, namely CCL3, CCL3L2, and, CCL3L3. These genes are involved in host defense and inflammatory processes. Several CNV assays have been developed for the CCL3L1 gene. Findings Through pairwise and multiple alignments of these genes, we have shown that the homology between these genes ranges from 50% to 99% in complete gene sequences and from 70-100% in the exonic regions, with CCL3L1 and CCL3L3 being identical. By use of MEGA 4 and BioEdit, we aligned sense primers, anti-sense primers, and probes used in several previously described assays against pre-multiple alignments of all four chemokine genes. Each set of probes and primers aligned and matched with overlapping sequences in at least two of the four genes, indicating that previously utilized RT-PCR based CNV assays are not specific for only CCL3L1. The four available assays measured median copies of 2 and 3-4 in European and African American, respectively. The concordance between the assays ranged from 0.44-0.83 suggesting individual discordant calls and inconsistencies with the assays from the expected gene coverage from the known sequence. Conclusions This indicates that some of the inconsistencies in the association studies could be due to assays that provide heterogenous results. Sequence information to determine CNV of the three genes separately would allow to test whether their association with the pathogenesis of a human disease or phenotype is affected by an individual gene or by a combination of these genes.

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

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


    Uniparental disomy (UPD) for imprinted chromosomes can cause abnormal phenotypes due to absent or overexpression of imprinted genes. UPD(14)pat causes a unique constellation of features including thoracic skeletal anomalies, polyhydramnios, placentomegaly, and limited survival; its hypothesized cause is overexpression of paternally expressed RTL1, due to absent regulatory effects of maternally expressed RTL1as. UPD(14)mat causes a milder condition with hypotonia, growth failure, and precocious puberty; its hypothesized cause is absence of paternally expressed DLK1. To more clearly establish how gains and losses of imprinted genes can cause disease, we report six individuals with copy number variations of the imprinted 14q32 region identified through clinical microarray-based comparative genomic hybridization. Three individuals presented with UPD(14)mat-like phenotypes (Temple syndrome) and had apparently de novo deletions spanning the imprinted region, including DLK1. One of these deletions was shown to be on the paternal chromosome. Two individuals with UPD(14)pat-like phenotypes had 122-154kb deletions on their maternal chromosomes that included RTL1as but not the differentially methylated regions that regulate imprinted gene expression, providing further support for RTL1 overexpression as a cause for the UPD(14)pat phenotype. The sixth individual is tetrasomic for a 1.7Mb segment, including the imprinted region, and presents with intellectual disability and seizures but lacks significant phenotypic overlap with either UPD(14) syndrome. Therefore, the 14q32 imprinted region is dosage sensitive, with deletions of different critical regions causing UPD(14)mat- and UPD(14)pat-like phenotypes, while copy gains are likely insufficient to recapitulate these phenotypes. PMID:25756153

  19. Array-based comparative genomic hybridization for the detection of DNA sequence copy number changes in Barrett's adenocarcinoma.

    Albrecht, Bettina; Hausmann, Michael; Zitzelsberger, Horst; Stein, Hubert; Siewert, Jörg Rüdiger; Hopt, Ulrich; Langer, Rupert; Höfler, Heinz; Werner, Martin; Walch, Axel


    Array-based comparative genomic hybridization (aCGH) allows the identification of DNA sequence copy number changes at high resolution by co-hybridizing differentially labelled test and control DNAs to a micro-array of genomic clones. The present study has analysed a series of 23 formalin-fixed, paraffin wax-embedded tissue samples of Barrett's adenocarcinoma (BCA, n = 18) and non-neoplastic squamous oesophageal (n = 2) and gastric cardia mucosa (n = 3) by aCGH. The micro-arrays used contained 287 genomic targets covering oncogenes, tumour suppressor genes, and DNA sequences localized within chromosomal regions previously reported to be altered in BCA. DNA sequence copy number changes for a panel of approximately 50 genes were identified, most of which have not been previously described in BCA. DNA sequence copy number gains (mean 41 +/- 25/BCA) were more frequent than DNA sequence copy number losses (mean 20 +/- 15/BCA). The highest frequencies for DNA sequence copy number gains were detected for SNRPN (61%); GNLY (44%); NME1 (44%); DDX15, ABCB1 (MDR), ATM, LAMA3, MYBL2, ZNF217, and TNFRSF6B (39% each); and MSH2, TERC, SERPINE1, AFM137XA11, IGF1R, and PTPN1 (33% each). DNA sequence copy number losses were identified for PDGFB (44%); D17S125 (39%); AKT3 (28%); and RASSFI, FHIT, CDKN2A (p16), and SAS (CDK4) (28% each). In all non-neoplastic tissue samples of squamous oesophageal and gastric cardia mucosa, the measured mean ratios were 1.00 (squamous oesophageal mucosa) or 1.01 (gastric mucosa), indicating that no DNA sequence copy number chances were present. For validation, the DNA sequence copy number changes of selected clones (SNRPN, CMYC, HER2, ZNF217) detected by aCGH were confirmed by fluorescence in situ hybridization (FISH). These data show the sensitivity of aCGH for the identification of DNA sequence copy number changes at high resolution in BCA. The newly identified genes may include so far unknown biomarkers in BCA and are therefore a starting point for

  20. Association testing of copy number variants in schizophrenia and autism spectrum disorders

    Crespi Bernard J


    Full Text Available Abstract Background Autism spectrum disorders and schizophrenia have been associated with an overlapping set of copy number variant loci, but the nature and degree of overlap in copy number variants (deletions compared to duplications between these two disorders remains unclear. Methods We systematically evaluated three lines of evidence: (1 the statistical bases for associations of autism spectrum disorders and schizophrenia with a set of the primary CNVs thus far investigated, from previous studies; (2 data from case series studies on the occurrence of these CNVs in autism spectrum disorders, especially among children, and (3 data on the extent to which the CNVs were associated with intellectual disability and developmental, speech, or language delays. We also conducted new analyses of existing data on these CNVs in autism by pooling data from seven case control studies. Results Four of the CNVs considered, dup 1q21.1, dup 15q11-q13, del 16p11.2, and dup 22q11.21, showed clear statistical evidence as autism risk factors, whereas eight CNVs, del 1q21.1, del 3q29, del 15q11.2, del 15q13.3, dup 16p11.2, dup 16p13.1, del 17p12, and del 22q11.21, were strongly statistically supported as risk factors for schizophrenia. Three of the CNVs, dup 1q21.1, dup 16p11.2, and dup 16p13.1, exhibited statistical support as risk factors for both autism and schizophrenia, although for each of these CNVs statistical significance was nominal for tests involving one of the two disorders. For the CNVs that were statistically associated with schizophrenia but were not statistically associated with autism, a notable number of children with the CNVs have been diagnosed with autism or ASD; children with these CNVs also demonstrate a high incidence of intellectual disability and developmental, speech, or language delays. Conclusions These findings suggest that although CNV loci notably overlap between autism and schizophrenia, the degree of strongly statistically

  1. mtDNA copy number in oocytes of different sizes from individual pre- and post-pubertal pigs

    Pedersen, Hanne Skovsgaard; Løvendahl, Peter; Larsen, Knud Erik;


    Oocyte competence has been related to mtDNA copy number, but a large variation in mtDNA copy number between oocytes has been observed, caused by, e.g. oocyte donor and oocyte size (Sato et al. 2014 PLOS ONE 9, e94488; Cotterill et al. 2013 Mol. Hum. Reprod. 19, 444–450; El Shourbagy et al. 2006...... Reproduction 131, 233–245). However, the correlation between size and mtDNA copy number in single oocytes has not been determined. This study describes the relation between oocytes of defined diameters from individual pre- and postpubertal pigs and mtDNA copy number. Cumulus-oocyte complexes were aspirated...... from ovaries of 10 pre- and 10 post-pubertal pigs. Cumulus cells were removed and the oocytes were measured (inside-ZP-diameter). Oocytes were transferred to DNAase-free tubes, snap-frozen, and stored at –80°C. The genes ND1 and COX1 were used to determine the mtDNA copy number. Plasmid preparations...

  2. Mutations in the D-loop region and increased copy number of mitochondrial DNA in human laryngeal squamous cell carcinoma.

    Guo, Wei; Yang, Denghua; Xu, Hongbo; Zhang, Yang; Huang, Junwei; Yang, Zheng; Chen, Xiaohong; Huang, Zhigang


    The effects of D-loop mutations and the mtDNA copy number alterations in LSCC are poorly understood. Herein, we investigated the features and roles of somatic mutations of the D-loop region and copy number alterations in mtDNA of LSCC. Using direct sequencing and real-time quantitative PCR, we examined D-loop mutations and mtDNA copy number in LSCC tissues, paracancerous normal tissues and peripheral vein blood samples from 40 LSCC patients. A student's t test, ANOVA test and χ(2) test were used to analyze association among mutations, mtDNA copy number alterations with clinicopathologic parameters. The results revealed that 21 tumors (52.5 %) had somatic mtDNA D-loop mutations with a total of 34 mutations. Among them, 28 (82.4 %) and 6 (17.6 %) were located in HVII and HVI, respectively. D-loop mutations correlated with tumor differentiation and p53 mutation (P D-loop mutation was significantly higher than that of the negative cases (P D-loop in LSCC is an unstable region with a high frequency of somatic mutation and polymorphisms. Together with the increase in mtDNA copy number, these factors may play a role in carcinogenesis of the larynx. PMID:23114912

  3. Towards an evidence-based process for the clinical interpretation of copy number variation

    Riggs, ER; Church, DM; Hanson, K; Horner, VL; Kaminsky, EB; Kuhn, RM; Wain, KE; Williams, ES; Aradhya, S; Kearney, HM; Ledbetter, DH; South, ST; Thorland, EC; Martin, CL


    The evidence-based review (EBR) process has been widely used to develop standards for medical decision-making and to explore complex clinical questions. This approach can be applied to genetic tests, such as chromosomal microarrays, in order to assist in the clinical interpretation of certain copy number variants (CNVs), particularly those that are rare, and guide array design for optimal clinical utility. To address these issues, the International Standards for Cytogenomic Arrays Consortium has established an EBR Work Group charged with building a framework to systematically assess the potential clinical relevance of CNVs throughout the genome. This group has developed a rating system enumerating the evidence supporting or refuting dosage sensitivity for individual genes and regions that considers the following criteria: number of causative mutations reported; patterns of inheritance; consistency of phenotype; evidence from large-scale case-control studies; mutational mechanisms; data from public genome variation databases; and expert consensus opinion. The system is designed to be dynamic in nature, with regions being reevaluated periodically to incorporate emerging evidence. The evidence collected will be displayed within a publically available database, and can be used in part to inform clinical laboratory CNV interpretations as well as to guide array design. PMID:22097934

  4. Copy number variations in Hanwoo and Yanbian cattle genomes using the massively parallel sequencing data.

    Choi, Jung-Woo; Chung, Won-Hyong; Lim, Kyu-Sang; Lim, Won-Jun; Choi, Bong-Hwan; Lee, Seung-Hwan; Kim, Hyeong-Cheol; Lee, Seung-Soo; Cho, Eun-Seok; Lee, Kyung-Tai; Kim, Namshin; Kim, Jeong-Dae; Kim, Jong-Bok; Chai, Han-Ha; Cho, Yong-Min; Kim, Tae-Hun; Lim, Dajeong


    Hanwoo is an indigenous Korean beef cattle breed, and it shared an ancestor with Yanbian cattle that are found in the Northeast provinces in China until the last century. During recent decades, those cattle breeds experienced different selection pressures. Here, we present genome-wide copy number variations (CNVs) by comparing Hanwoo and Yanbian cattle sequencing data. We used ~3.12 and ~3.07 billion sequence reads from Hanwoo and Yanbian cattle, respectively. A total of 901 putative CNV regions (CNVRs) were identified throughout the genome, representing 5,513,340bp. This is a smaller number than has been reported in previous studies, indicating that Hanwoo are genetically close to Yanbian cattle. Of the CNVRs, 53.2% and 46.8% were found to be gains and losses in Hanwoo. Potential functional roles of each CNVR were assessed by annotating all CNVRs and gene ontology (GO) enrichment analysis. We found that 278 CNVRs overlapped with cattle gene-sets (genic-CNVRs) that could be promising candidates to account for economically important traits in cattle. The enrichment analysis indicated that genes were significantly over-represented in GO terms, including developmental process, multicellular organismal process, reproduction, and response to stimulus. These results provide a valuable genomic resource for determining how CNVs are associated with cattle traits. PMID:27188257

  5. An initial map of chromosomal segmental copy number variations in the chicken

    Bohannon-Stewart Ann


    Full Text Available Abstract Background Chromosomal segmental copy number variation (CNV has been recently recognized as a very important source of genetic variability. Some CNV loci involve genes or conserved regulatory elements. Compelling evidence indicates that CNVs impact genome functions. The chicken is a very important farm animal species which has also served as a model for biological and biomedical research for hundreds of years. A map of CNVs in chickens could facilitate the identification of chromosomal regions that segregate for important agricultural and disease phenotypes. Results Ninety six CNVs were identified in three lines of chickens (Cornish Rock broiler, Leghorn and Rhode Island Red using whole genome tiling array. These CNVs encompass 16 Mb (1.3% of the chicken genome. Twenty six CNVs were found in two or more animals. Whereas most small sized CNVs reside in none coding sequences, larger CNV regions involve genes (for example prolactin receptor, aldose reductase and zinc finger proteins. These results suggest that chicken CNVs potentially affect agricultural or disease related traits. Conclusion An initial map of CNVs for the chicken has been described. Although chicken genome is approximately one third the size of a typical mammalian genome, the pattern of chicken CNVs is similar to that of mammals. The number of CNVs detected per individual was also similar to that found in dogs, mice, rats and macaques. A map of chicken CNVs provides new information on genetic variations for the understanding of important agricultural traits and disease.

  6. Copy number variation, chromosome rearrangement, and their association with recombination during avian evolution

    Völker, Martin; Backström, Niclas; Skinner, Benjamin M.; Langley, Elizabeth J.; Bunzey, Sydney K.; Ellegren, Hans; Griffin, Darren K.


    Chromosomal rearrangements and copy number variants (CNVs) play key roles in genome evolution and genetic disease; however, the molecular mechanisms underlying these types of structural genomic variation are not fully understood. The availability of complete genome sequences for two bird species, the chicken and the zebra finch, provides, for the first time, an ideal opportunity to analyze the relationship between structural genomic variation (chromosomal and CNV) and recombination on a genome-wide level. The aims of this study were therefore threefold: (1) to combine bioinformatics, physical mapping to produce comprehensive comparative maps of the genomes of chicken and zebra finch. In so doing, this allowed the identification of evolutionary chromosomal rearrangements distinguishing them. The previously reported interchromosomal conservation of synteny was confirmed, but a larger than expected number of intrachromosomal rearrangements were reported; (2) to hybridize zebra finch genomic DNA to a chicken tiling path microarray and identify CNVs in the zebra finch genome relative to chicken; 32 interspecific CNVs were identified; and (3) to test the hypothesis that there is an association between CNV, chromosomal rearrangements, and recombination by correlating data from (1) and (2) with recombination rate data from a high-resolution genetic linkage map of the zebra finch. We found a highly significant association of both chromosomal rearrangements and CNVs with elevated recombination rates. The results thus provide support for the notion of recombination-based processes playing a major role in avian genome evolution. PMID:20357050

  7. 17 CFR 230.497 - Filing of investment company prospectuses-number of copies.


    ... citations affecting § 230.497, see the List of CFR Sections Affected, which appears in the Finding Aids... copies. (a) Five copies of every form of prospectus sent or given to any person prior to the effective... investment company advertisement under § 230.482 shall be filed under this paragraph (a) (but not as part...

  8. Copy number variation of scavenger-receptor cysteine-rich domains within DMBT1 and Crohn's disease

    Polley, Shamik; Prescott, Natalie; Nimmo, Elaine;


    the number of bacteria-binding domains, different alleles may alter host-microbe interactions in the gut. Our aim was to investigate the role of this complex variation in susceptibility to Crohn's disease by assessing the previously reported association. We analysed the association of both copy number...

  9. Human PTCHD3 nulls: rare copy number and sequence variants suggest a non-essential gene

    Lionel Anath C


    Full Text Available Abstract Background Copy number variations (CNVs can contribute to variable degrees of fitness and/or disease predisposition. Recent studies show that at least 1% of any given genome is copy number variable when compared to the human reference sequence assembly. Homozygous deletions (or CNV nulls that are found in the normal population are of particular interest because they may serve to define non-essential genes in human biology. Results In a genomic screen investigating CNV in Autism Spectrum Disorders (ASDs we detected a heterozygous deletion on chromosome 10p12.1, spanning the Patched-domain containing 3 (PTCHD3 gene, at a frequency of ~1.4% (6/427. This finding seemed interesting, given recent discoveries on the role of another Patched-domain containing gene (PTCHD1 in ASD. Screening of another 177 ASD probands yielded two additional heterozygous deletions bringing the frequency to 1.3% (8/604. The deletion was found at a frequency of ~0.73% (27/3,695 in combined control population from North America and Northern Europe predominately of European ancestry. Screening of the human genome diversity panel (HGDP-CEPH covering worldwide populations yielded deletions in 7/1,043 unrelated individuals and those detected were confined to individuals of European/Mediterranean/Middle Eastern ancestry. Breakpoint mapping yielded an identical 102,624 bp deletion in all cases and controls tested, suggesting a common ancestral event. Interestingly, this CNV occurs at a break of synteny between humans and mouse. Considering all data, however, no significant association of these rare PTCHD3 deletions with ASD was observed. Notwithstanding, our RNA expression studies detected PTCHD3 in several tissues, and a novel shorter isoform for PTCHD3 was characterized. Expression in transfected COS-7 cells showed PTCHD3 isoforms colocalize with calnexin in the endoplasmic reticulum. The presence of a patched (Ptc domain suggested a role for PTCHD3 in various biological

  10. Lack of topoisomerase copy number changes in patients with de novo and relapsed diffuse large B-cell lymphoma

    Pedersen, Mette Ø; Poulsen, Tim S; Gang, Anne O;


    Topoisomerase (TOP) gene copy number changes may predict response to treatment with TOP-targeting drugs in cancer treatment. This was first described in patients with breast cancer and is currently being investigated in other malignant diseases. TOP-targeting drugs may induce TOP gene copy number...... chemotherapy regimens including TOP2-targeting drugs (n = 16). No TOP1 or TOP2A copy number changes were found. Polysomy of chromosomes 20 and 17 was seen in 3 of 25 patients (12%) and 2 of 32 patients (6%) with de novo DLBCL. Among relapsed patients, chromosome polysomy was more frequently observed in 5 of 13......-targeting drugs. Increased polyploidy of chromosomes 17 and 20 among patients with relapsed DLBCL may reflect genetic compensation in the tumor cells after TOP2 inhibition, but is more likely due to the increased genetic instability often seen in progressed cancers. Therefore, it is unlikely that TOP1 and...

  11. TOP1 gene copy number and TOP1/CEN-20 ratio in stage III colorectal cancer samples

    Rømer, Maria Unni Koefoed; Nygård, Sune Boris; Christensen, Ib Jarle;

    fixed paraffin embedded (FFPE) primary tumor tissue has been suggested as a predictive biomarker of the effect of irinotecan in the treatment of metastatic CRC. Quantification of TOP1 protein levels in FFPE tissue may be difficult and calls for alternative methods.We have recently reported on TOP1 FISH......AIM OF STUDY To investigate if TOP1 gene copy number and/or the TOP1/CEN-20 ratio in colorectal cancer (CRC) areassociated with prognosis. BACKGROUND TOP1, localized on chromosome 20, encodes topoisomerase I (TOP1), which is the sole molecular target of irinotecan. TOP1 immunoreactivity in formalin...... gene copy number/cell and OS exists. A continuous relationship between TOP1 gene copy number/cell and LR exists. A continuous relationship exists between TOP1/CEN-20 ratio and LR CONCLUSION Our data suggest that TOP1 and TOP1/CEN-20 ratio are associated with prognosis in colorectal cancer. Future...

  12. EGFR-activating mutations, DNA copy number abundance of ErbB family, and prognosis in lung adenocarcinoma

    Chen, Hsuan-Yu; Liu, Chia-Hsin; Chang, Ya-Hsuan; Yu, Sung-Liang; Ho, Bing-Ching; Hsu, Chung-Ping; Yang, Tsung-Ying; Chen, Kun-Chieh; Hsu, Kuo-Hsuan; Tseng, Jeng-Sen; Hsia, Jiun-Yi; Chuang, Cheng-Yen; Chang, Chi-Sheng; Li, Yu-Cheng; Li, Ker-Chau; Chang, Gee-Chen; Yang, Pan-Chyr


    In this study, EGFR-activating mutation status and DNA copy number abundances of members of ErbB family were measured in 261 lung adenocarcinomas. The associations between DNA copy number abundances of ErbB family, EGFR-activating mutation status, and prognosis were explored. Results showed that DNA copy number abundances of EGFR, ERBB2, ERBB3, and ERBB4 had associations with overall survival in lung adenocarcinoma with EGFR-activating mutations. In the stratification analysis, only ERBB2 showed significant discrepancy in patients carrying wild type EGFR and other members of ErbB family in patients carrying EGFR-activating mutation. This indicated that CNAs of ErbB family had effect modifications of EGFR-activating mutation status. Findings of this study demonstrate potential molecular guidance of patient management of lung adenocarcinoma with or without EGFR-activating mutations. PMID:26824984

  13. Overexpression of TFAM or twinkle increases mtDNA copy number and facilitates cardioprotection associated with limited mitochondrial oxidative stress.

    Masataka Ikeda

    Full Text Available Mitochondrial DNA (mtDNA copy number decreases in animal and human heart failure (HF, yet its role in cardiomyocytes remains to be elucidated. Thus, we investigated the cardioprotective function of increased mtDNA copy number resulting from the overexpression of human transcription factor A of mitochondria (TFAM or Twinkle helicase in volume overload (VO-induced HF.Two strains of transgenic (TG mice, one overexpressing TFAM and the other overexpressing Twinkle helicase, exhibit an approximately 2-fold equivalent increase in mtDNA copy number in heart. These TG mice display similar attenuations in eccentric hypertrophy and improved cardiac function compared to wild-type (WT mice without any deterioration of mitochondrial enzymatic activities in response to VO, which was accompanied by a reduction in matrix-metalloproteinase (MMP activity and reactive oxygen species after 8 weeks of VO. Moreover, acute VO-induced MMP-2 and MMP-9 upregulation was also suppressed at 24 h in both TG mice. In isolated rat cardiomyocytes, mitochondrial reactive oxygen species (mitoROS upregulated MMP-2 and MMP-9 expression, and human TFAM (hTFAM overexpression suppressed mitoROS and their upregulation. Additionally, mitoROS were equally suppressed in H9c2 rat cardiomyoblasts that overexpress hTFAM or rat Twinkle, both of which exhibit increased mtDNA copy number. Furthermore, mitoROS and mitochondrial protein oxidation from both TG mice were suppressed compared to WT mice.The overexpression of TFAM or Twinkle results in increased mtDNA copy number and facilitates cardioprotection associated with limited mitochondrial oxidative stress. Our findings suggest that increasing mtDNA copy number could be a useful therapeutic strategy to target mitoROS in HF.

  14. Genome-wide gene copy number and expression analysis of primary gastric tumors and gastric cancer cell lines

    Gastric cancer is one of the most common malignancies worldwide and the second most common cause of cancer related death. Gene copy number alterations play an important role in the development of gastric cancer and a change in gene copy number is one of the main mechanisms for a cancer cell to control the expression of potential oncogenes and tumor suppressor genes. To highlight genes of potential biological and clinical relevance in gastric cancer, we carried out a systematic array-based survey of gene expression and copy number levels in primary gastric tumors and gastric cancer cell lines and validated the results using an affinity capture based transcript analysis (TRAC assay) and real-time qRT-PCR. Integrated microarray analysis revealed altogether 256 genes that were located in recurrent regions of gains or losses and had at least a 2-fold copy number- associated change in their gene expression. The expression levels of 13 of these genes, ALPK2, ASAP1, CEACAM5, CYP3A4, ENAH, ERBB2, HHIPL2, LTB4R, MMP9, PERLD1, PNMT, PTPRA, and OSMR, were validated in a total of 118 gastric samples using either the qRT-PCR or TRAC assay. All of these 13 genes were differentially expressed between cancerous samples and nonmalignant tissues (p < 0.05) and the association between copy number and gene expression changes was validated for nine (69.2%) of these genes (p < 0.05). In conclusion, integrated gene expression and copy number microarray analysis highlighted genes that may be critically important for gastric carcinogenesis. TRAC and qRT-PCR analyses validated the microarray results and therefore the role of these genes as potential biomarkers for gastric cancer

  15. Genomic Copy Number Dictates a Gene-Independent Cell Response to CRISPR/Cas9 Targeting | Office of Cancer Genomics

    The CRISPR/Cas9 system enables genome editing and somatic cell genetic screens in mammalian cells. We performed genome-scale loss-of-function screens in 33 cancer cell lines to identify genes essential for proliferation/survival and found a strong correlation between increased gene copy number and decreased cell viability after genome editing. Within regions of copy-number gain, CRISPR/Cas9 targeting of both expressed and unexpressed genes, as well as intergenic loci, led to significantly decreased cell proliferation through induction of a G2 cell-cycle arrest.

  16. Copy Number Variants in Short Children Born Small for Gestational Age

    Wit, Jan M.; van Duyvenvoorde, Hermine A.; van Klinken, Jan B.; Caliebe, Janina; Bosch, Cathy A.J.; Lui, Julian C.; Gijsbers, Antoinet C.J.; Bakker, Egbert; Breuning, Martijn H.; Oostdijk, Wilma; Losekoot, Monique; Baron, Jeffrey; Binder, Gerhard; Ranke, Michael B.; Ruivenkamp, Claudia A.L.


    Background/aims In addition to Genome-Wide Association studies (GWAS) height-associated genes may be uncovered by studying individuals with extreme short or tall stature. Methods Genome-wide analysis for copy number variants (CNVs), using Single Nucleotide Polymorphism (SNP) arrays, was performed in 49 index cases born small for gestational age (SGA) with persistent short stature. Segregation analysis was performed, and genes in CNVs were compared with information from GWAS, gene expression in rodents’ growth plates, and published information. Results CNVs were detected in 13 cases. In 5 children a known cause of short stature was found: UPD7, UPD14, a duplication of the SHOX enhancer region, an IGF1R deletion, and a 22q11.21 deletion. In the remaining 8 cases potential pathogenic CNVs were detected, either de novo (n=1), segregating (n=2), or not segregating with short stature (n=5). Bioinformatic analysis of the de novo and segregating CNVs suggested that HOXD4, AGPS, PDE11A, OSBPL6, PRKRA and PLEKHA3, and possibly DGKB and TNFRSF11B are potential candidate genes. A SERPINA7 or NRK defect may be associated with an X-linked form of short stature. Conclusion SNP arrays detected 5 known causes of short stature with prenatal onset and suggested several potential candidate genes. PMID:25300501

  17. Fast MCMC sampling for hidden markov models to determine copy number variations

    Mahmud Md Pavel


    Full Text Available Abstract Background Hidden Markov Models (HMM are often used for analyzing Comparative Genomic Hybridization (CGH data to identify chromosomal aberrations or copy number variations by segmenting observation sequences. For efficiency reasons the parameters of a HMM are often estimated with maximum likelihood and a segmentation is obtained with the Viterbi algorithm. This introduces considerable uncertainty in the segmentation, which can be avoided with Bayesian approaches integrating out parameters using Markov Chain Monte Carlo (MCMC sampling. While the advantages of Bayesian approaches have been clearly demonstrated, the likelihood based approaches are still preferred in practice for their lower running times; datasets coming from high-density arrays and next generation sequencing amplify these problems. Results We propose an approximate sampling technique, inspired by compression of discrete sequences in HMM computations and by kd-trees to leverage spatial relations between data points in typical data sets, to speed up the MCMC sampling. Conclusions We test our approximate sampling method on simulated and biological ArrayCGH datasets and high-density SNP arrays, and demonstrate a speed-up of 10 to 60 respectively 90 while achieving competitive results with the state-of-the art Bayesian approaches. Availability: An implementation of our method will be made available as part of the open source GHMM library from

  18. DNA Copy-Number Control through Inhibition of Replication Fork Progression

    Jared T. Nordman


    Full Text Available Proper control of DNA replication is essential to ensure faithful transmission of genetic material and 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 the repression of DNA replication by directly inhibiting replication fork progression instead of functioning as a replication fork barrier. Mass spectrometry 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 the inhibition of replication fork progression.

  19. Mutation dependance of the mitochondrial DNA copy number in the first stages of human embryogenesis.

    Monnot, Sophie; Samuels, David C; Hesters, Laetitia; Frydman, Nelly; Gigarel, Nadine; Burlet, Philippe; Kerbrat, Violaine; Lamazou, Frédéric; Frydman, René; Benachi, Alexandra; Feingold, Josué; Rotig, Agnes; Munnich, Arnold; Bonnefont, Jean-Paul; Steffann, Julie


    Mitochondrial DNA (mtDNA) content is thought to remain stable over the preimplantation period of human embryogenesis that is, therefore, suggested to be entirely dependent on ooplasm mtDNA capital. We have explored the impact of two disease-causing mutations [m.3243A>G myopathy, encephalopathy, lactic acidosis and stroke-like syndrome (MELAS) and m.8344A>G myoclonic epilepsy associated with ragged-red fibers (MERRF)] on mtDNA amounts in human oocytes and day 4-5 preimplantation embryos. The mtDNA amount was stable in MERRF and control materials, whereas gradually increasing from the germinal vesicle of oogenesis to the blastocyst stage of embryogenesis in MELAS cells, MELAS embryos carrying ∼3-fold higher mtDNA amount than control embryos (P = 0.0003). A correlation between mtDNA copy numbers and mutant loads was observed in MELAS embryos (R(2) = 0.42, P < 0.0013), suggestive of a compensation for the respiratory chain defect resulting from high mutation levels. These results suggest that mtDNA can replicate in early embryos and emphasize the need for sufficient amount of wild-type mtDNA to sustain embryonic development in humans. PMID:23390135

  20. Phenotypic Association Analyses With Copy Number Variation in Recurrent Depressive Disorder

    Rucker, James J.H.; Tansey, Katherine E.; Rivera, Margarita; Pinto, Dalila; Cohen-Woods, Sarah; Uher, Rudolf; Aitchison, Katherine J.; Craddock, Nick; Owen, Michael J.; Jones, Lisa; Jones, Ian; Korszun, Ania; Barnes, Michael R.; Preisig, Martin; Mors, Ole; Maier, Wolfgang; Rice, John; Rietschel, Marcella; Holsboer, Florian; Farmer, Anne E.; Craig, Ian W.; Scherer, Stephen W.; McGuffin, Peter; Breen, Gerome


    Background Defining the molecular genomic basis of the likelihood of developing depressive disorder is a considerable challenge. We previously associated rare, exonic deletion copy number variants (CNV) with recurrent depressive disorder (RDD). Sex chromosome abnormalities also have been observed to co-occur with RDD. Methods In this reanalysis of our RDD dataset (N = 3106 cases; 459 screened control samples and 2699 population control samples), we further investigated the role of larger CNVs and chromosomal abnormalities in RDD and performed association analyses with clinical data derived from this dataset. Results We found an enrichment of Turner’s syndrome among cases of depression compared with the frequency observed in a large population sample (N = 34,910) of live-born infants collected in Denmark (two-sided p = .023, odds ratio = 7.76 [95% confidence interval = 1.79–33.6]), a case of diploid/triploid mosaicism, and several cases of uniparental isodisomy. In contrast to our previous analysis, large deletion CNVs were no more frequent in cases than control samples, although deletion CNVs in cases contained more genes than control samples (two-sided p = .0002). Conclusions After statistical correction for multiple comparisons, our data do not support a substantial role for CNVs in RDD, although (as has been observed in similar samples) occasional cases may harbor large variants with etiological significance. Genetic pleiotropy and sample heterogeneity suggest that very large sample sizes are required to study conclusively the role of genetic variation in mood disorders. PMID:25861698

  1. GROM-RD: resolving genomic biases to improve read depth detection of copy number variants

    Sean D. Smith


    Full Text Available Amplifications or deletions of genome segments, known as copy number variants (CNVs, have been associated with many diseases. Read depth analysis of next-generation sequencing (NGS is an essential method of detecting CNVs. However, genome read coverage is frequently distorted by various biases of NGS platforms, which reduce predictive capabilities of existing approaches. Additionally, the use of read depth tools has been somewhat hindered by imprecise breakpoint identification. We developed GROM-RD, an algorithm that analyzes multiple biases in read coverage to detect CNVs in NGS data. We found non-uniform variance across distinct GC regions after using existing GC bias correction methods and developed a novel approach to normalize such variance. Although complex and repetitive genome segments complicate CNV detection, GROM-RD adjusts for repeat bias and uses a two-pipeline masking approach to detect CNVs in complex and repetitive segments while improving sensitivity in less complicated regions. To overcome a typical weakness of RD methods, GROM-RD employs a CNV search using size-varying overlapping windows to improve breakpoint resolution. We compared our method to two widely used programs based on read depth methods, CNVnator and RDXplorer, and observed improved CNV detection and breakpoint accuracy for GROM-RD. GROM-RD is available at

  2. Copy number variants calling for single cell sequencing data by multi-constrained optimization.

    Xu, Bo; Cai, Hongmin; Zhang, Changsheng; Yang, Xi; Han, Guoqiang


    Variations in DNA copy number carry important information on genome evolution and regulation of DNA replication in cancer cells. The rapid development of single-cell sequencing technology allows one to explore gene expression heterogeneity among single-cells, thus providing important cancer cell evolution information. Single-cell DNA/RNA sequencing data usually have low genome coverage, which requires an extra step of amplification to accumulate enough samples. However, such amplification will introduce large bias and makes bioinformatics analysis challenging. Accurately modeling the distribution of sequencing data and effectively suppressing the bias influence is the key to success variations analysis. Recent advances demonstrate the technical noises by amplification are more likely to follow negative binomial distribution, a special case of Poisson distribution. Thus, we tackle the problem CNV detection by formulating it into a quadratic optimization problem involving two constraints, in which the underling signals are corrupted by Poisson distributed noises. By imposing the constraints of sparsity and smoothness, the reconstructed read depth signals from single-cell sequencing data are anticipated to fit the CNVs patterns more accurately. An efficient numerical solution based on the classical alternating direction minimization method (ADMM) is tailored to solve the proposed model. We demonstrate the advantages of the proposed method using both synthetic and empirical single-cell sequencing data. Our experimental results demonstrate that the proposed method achieves excellent performance and high promise of success with single-cell sequencing data. PMID:26923213

  3. MHC gene copy number variation in Tasmanian devils: implications for the spread of a contagious cancer.

    Siddle, Hannah V; Marzec, Jolanta; Cheng, Yuanyuan; Jones, Menna; Belov, Katherine


    Tasmanian devils face extinction owing to the emergence of a contagious cancer. Devil facial tumour disease (DFTD) is a clonal cancer spread owing to a lack of major histocompatibility complex (MHC) barriers in Tasmanian devil populations. We present a comprehensive screen of MHC diversity in devils and identify 25 MHC types and 53 novel sequences, but conclude that overall levels of MHC diversity at the sequence level are low. The majority of MHC Class I variation can be explained by allelic copy number variation with two to seven sequence variants identified per individual. MHC sequences are divided into two distinct groups based on sequence similarity. DFTD cells and most devils have sequences from both groups. Twenty per cent of individuals have a restricted MHC repertoire and contain only group I or only group II sequences. Counterintuitively, we postulate that the immune system of individuals with a restricted MHC repertoire may recognize foreign MHC antigens on the surface of the DFTD cell. The implication of these results for management of DFTD and this endangered species are discussed. PMID:20219742

  4. Is mitochondrial DNA copy number associated with clinical characteristics and prognosis in gastric cancer?

    Lee, Hyunsu; Lee, Jae-Ho; Kim, Dong-Choon; Hwang, IlSeon; Kang, Yu-Na; Gwon, Gi-Jeong; Choi, In-Jang; Kim, Shin


    Alterations in mitochondrial DNA (mtDNA) have been studied in various cancers. However, the clinical value of mtDNA copy number (mtCN) alterations in gastric cancer (GC) is poorly understood. In the present study, we investigated whether alterations in mtCNs might be associated with clinicopathological parameters in GC cases. mtCN was measured in 109 patients with GC by quantitative real-time PCR. Then, correlations with clinicopathological characteristics were analyzed. mtCN was elevated in 64.2% of GC tissues compared with paired, adjacent, non-cancerous tissue. However, the observed alterations in mtCN were not associated with any clinicopathological characteristics, including age, gender, TN stage, Lauren classification, lymph node metastasis, and depth of invasion. Moreover, Kaplan-Meier survival curves revealed that mtCN was not significantly associated with the survival of GC patients. In this study, we demonstrated that mtCN was not a significant marker for predicting clinical characteristics or prognosis in GC. PMID:25640396

  5. Tropically adapted cattle of Africa: perspectives on potential role of copy number variations.

    Wang, M D; Dzama, K; Rees, D J G; Muchadeyi, F C


    Africa is host to diverse and locally adapted cattle breeds that are expected to survive the harsh and extreme tropical environments associated with diseases and parasite infections, heat stress and episodes of feed and water scarcity. Genomic copy number variations (CNVs) are considered to be primary role players in cattle breed formation and adaptation where isolation and genetic drift together with subsequent mutations have created an enormous diversity of local populations. CNVs are modifications in DNA structure comprising deletions, duplications and insertions that are >1 kb in size. Despite attracting much attention, the frequency and pattern of bovine CNV events, especially in African cattle breeds, are for the most part largely unknown. Characterization of genetic variation in the indigenous cattle of Africa will be a vital step toward dissecting the molecular mechanisms underlying phenotypic variation and local adaptation. This review therefore aims to describe the current knowledge regarding bovine CNVs and the implications and potentials they encompass for dissecting genetic adaptation and the genotypic skeleton of tropical African cattle populations. PMID:26644080

  6. Copy Number Variations in DISC1 and DISC1-Interacting Partners in Major Mental Illness

    Johnstone, Mandy; Maclean, Alan; Heyrman, Lien; Lenaerts, An-Sofie; Nordin, Annelie; Nilsson, Lars-Göran; De Rijk, Peter; Goossens, Dirk; Adolfsson, Rolf; St. Clair, David M.; Hall, Jeremy; Lawrie, Stephen M.; McIntosh, Andrew M.; Del-Favero, Jurgen; Blackwood, Douglas H.R.; Pickard, Benjamin S.


    Robust statistical, genetic and functional evidence supports a role for DISC1 in the aetiology of major mental illness. Furthermore, many of its protein-binding partners show evidence for involvement in the pathophysiology of a range of neurodevelopmental and psychiatric disorders. Copy number variants (CNVs) are suspected to play an important causal role in these disorders. In this study, CNV analysis of DISC1 and its binding partners PAFAH1B1, NDE1, NDEL1, FEZ1, MAP1A, CIT and PDE4B in Scottish and Northern Swedish population-based samples was carried out using multiplex amplicon quantification. Here, we report the finding of rare CNVs in DISC1, NDE1 (together with adjacent genes within the 16p13.11 duplication), NDEL1 (including the overlapping MYH10 gene) and CIT. Our findings provide further evidence for involvement of DISC1 and its interaction partners in neuropsychiatric disorders and also for a role of structural variants in the aetiology of these devastating diseases.

  7. ROS1 copy number alterations are frequent in non-small cell lung cancer

    Clavé, Sergi; Gimeno, Javier; Muñoz-Mármol, Ana M.; Vidal, Joana; Reguart, Noemí; Carcereny, Enric; Pijuan, Lara; Menéndez, Sílvia; Taus, Álvaro; Mate, José Luís; Serrano, Sergio; Albanell, Joan; Espinet, Blanca; Arriola, Edurne; Salido, Marta


    Objectives We aimed to determine the prevalence and partners of ROS1 rearrangements, to explore the correlation between FISH and IHC assays, and to investigate clinical implications of ROS1 copy number alterations (CNAs). Methods A total of 314 NSCLC patients were screened using ROS1 FISH break-apart probes. Of these, 47 surgical tumors were included in TMAs to analyze ROS1 heterogeneity assessed either by FISH and IHC, and chromosome 6 aneusomy. To characterize ROS1 partners, probes for CD74, EZR, SLC34A2 and SDC3 genes were developed. ROS1 positive FISH cases were screened also by IHC. Results Five patients were ROS1 positive (1.8%). We identified two known fusion partners in three patients: CD74 and SLC34A2. Four out of five ROS1 rearranged patients were female, never smokers and with adenocarcinoma histology. Rearranged cases were also positive by IHC as well. According to ROS1 CNAs, we found a prevalence of 37.8% gains/amplifications and 25.1% deletions. Conclusions This study point out the high prevalence of ROS1 CNAs in a large series of NSCLC. ROS1 gains, amplifications and deletions, most of them due to chromosome 6 polysomy or monosomy, were heterogeneous within a tumor and had no impact on overall survival. PMID:26783962

  8. Shared Copy Number Variation in Simultaneous Nephroblastoma and Neuroblastoma due to Fanconi Anemia

    Serra, A.; Eirich, K.; Winkler, A.K.; Mrasek, K.; Göhring, G.; Barbi, G.; Cario, H.; Schlegelberger, B.; Pokora, B.; Liehr, T.; Leriche, C.; Henne-Bruns, D.; Barth, T.F.; Schindler, D.


    Concurrent emergence of nephroblastoma (Wilms Tumor; WT) and neuroblastoma (NB) is rare and mostly observed in patients with severe subtypes of Fanconi anemia (FA) with or without VACTER-L association (VL). We investigated the hypothesis that early consequences of genomic instability result in shared regions with copy number variation in different precursor cells that originate distinct embryonal tumors. We observed a newborn girl with FA and VL (aplasia of the thumbs, cloacal atresia (urogenital sinus), tethered cord at L3/L4, muscular ventricular septum defect, and horseshoe-kidney with a single ureter) who simultaneously acquired an epithelial-type WT in the left portion of the kidney and a poorly differentiated adrenal NB in infancy. A novel homozygous germline frameshift mutation in PALB2 (c.1676_c1677delAAinsG) leading to protein truncation (pGln526ArgfsX1) inherited from consanguineous parents formed the genetic basis of FA-N. Spontaneous and induced chromosomal instability was detected in the majority of cells analyzed from peripheral lymphocytes, bone marrow, and cultured fibroblasts. Bone marrow cells also showed complex chromosome rearrangements consistent with the myelodysplastic syndrome at 11 months of age. Array-comparative genomic hybridization analyses of both WT and NB showed shared gains or amplifications within the chromosomal regions 11p15.5 and 17q21.31-q25.3, including genes that are reportedly implicated in tumor development such as IGF2, H19, WT2, BIRC5, and HRAS. PMID:23112754

  9. A genome-wide association study of copy number variations with umbilical hernia in swine.

    Long, Yi; Su, Ying; Ai, Huashui; Zhang, Zhiyan; Yang, Bin; Ruan, Guorong; Xiao, Shijun; Liao, Xinjun; Ren, Jun; Huang, Lusheng; Ding, Nengshui


    Umbilical hernia (UH) is one of the most common congenital defects in pigs, leading to considerable economic loss and serious animal welfare problems. To test whether copy number variations (CNVs) contribute to pig UH, we performed a case-control genome-wide CNV association study on 905 pigs from the Duroc, Landrace and Yorkshire breeds using the Porcine SNP60 BeadChip and penncnv algorithm. We first constructed a genomic map comprising 6193 CNVs that pertain to 737 CNV regions. Then, we identified eight CNVs significantly associated with the risk for UH in the three pig breeds. Six of seven significantly associated CNVs were validated using quantitative real-time PCR. Notably, a rare CNV (CNV14:13030843-13059455) encompassing the NUGGC gene was strongly associated with UH (permutation-corrected P = 0.0015) in Duroc pigs. This CNV occurred exclusively in seven Duroc UH-affected individuals. SNPs surrounding the CNV did not show association signals, indicating that rare CNVs may play an important role in complex pig diseases such as UH. The NUGGC gene has been implicated in human omphalocele and inguinal hernia. Our finding supports that CNVs, including the NUGGC CNV, contribute to the pathogenesis of pig UH. PMID:27028052

  10. Refining analyses of copy number variation identifies specific genes associated with developmental delay.

    Coe, Bradley P; Witherspoon, Kali; Rosenfeld, Jill A; van Bon, Bregje W M; Vulto-van Silfhout, Anneke T; Bosco, Paolo; Friend, Kathryn L; Baker, Carl; Buono, Serafino; Vissers, Lisenka E L M; Schuurs-Hoeijmakers, Janneke H; Hoischen, Alex; Pfundt, Rolph; Krumm, Nik; Carvill, Gemma L; Li, Deana; Amaral, David; Brown, Natasha; Lockhart, Paul J; Scheffer, Ingrid E; Alberti, Antonino; Shaw, Marie; Pettinato, Rosa; Tervo, Raymond; de Leeuw, Nicole; Reijnders, Margot R F; Torchia, Beth S; Peeters, Hilde; O'Roak, Brian J; Fichera, Marco; Hehir-Kwa, Jayne Y; Shendure, Jay; Mefford, Heather C; Haan, Eric; Gécz, Jozef; de Vries, Bert B A; Romano, Corrado; Eichler, Evan E


    Copy number variants (CNVs) are associated with many neurocognitive disorders; however, these events are typically large, and the underlying causative genes are unclear. We created an expanded CNV morbidity map from 29,085 children with developmental delay in comparison to 19,584 healthy controls, identifying 70 significant CNVs. We resequenced 26 candidate genes in 4,716 additional cases with developmental delay or autism and 2,193 controls. An integrated analysis of CNV and single-nucleotide variant (SNV) data pinpointed 10 genes enriched for putative loss of function. Follow-up of a subset of affected individuals identified new clinical subtypes of pediatric disease and the genes responsible for disease-associated CNVs. These genetic changes include haploinsufficiency of SETBP1 associated with intellectual disability and loss of expressive language and truncations of ZMYND11 in individuals with autism, aggression and complex neuropsychiatric features. This combined CNV and SNV approach facilitates the rapid discovery of new syndromes and genes involved in neuropsychiatric disease despite extensive genetic heterogeneity. PMID:25217958

  11. Origins and functional impact of copy number variation in the human genome.

    Conrad, Donald F; Pinto, Dalila; Redon, Richard; Feuk, Lars; Gokcumen, Omer; Zhang, Yujun; Aerts, Jan; Andrews, T Daniel; Barnes, Chris; Campbell, Peter; Fitzgerald, Tomas; Hu, Min; Ihm, Chun Hwa; Kristiansson, Kati; Macarthur, Daniel G; Macdonald, Jeffrey R; Onyiah, Ifejinelo; Pang, Andy Wing Chun; Robson, Sam; Stirrups, Kathy; Valsesia, Armand; Walter, Klaudia; Wei, John; Tyler-Smith, Chris; Carter, Nigel P; Lee, Charles; Scherer, Stephen W; Hurles, Matthew E


    Structural variations of DNA greater than 1 kilobase in size account for most bases that vary among human genomes, but are still relatively under-ascertained. Here we use tiling oligonucleotide microarrays, comprising 42 million probes, to generate a comprehensive map of 11,700 copy number variations (CNVs) greater than 443 base pairs, of which most (8,599) have been validated independently. For 4,978 of these CNVs, we generated reference genotypes from 450 individuals of European, African or East Asian ancestry. The predominant mutational mechanisms differ among CNV size classes. Retrotransposition has duplicated and inserted some coding and non-coding DNA segments randomly around the genome. Furthermore, by correlation with known trait-associated single nucleotide polymorphisms (SNPs), we identified 30 loci with CNVs that are candidates for influencing disease susceptibility. Despite this, having assessed the completeness of our map and the patterns of linkage disequilibrium between CNVs and SNPs, we conclude that, for complex traits, the heritability void left by genome-wide association studies will not be accounted for by common CNVs. PMID:19812545

  12. TAGCNA: a method to identify significant consensus events of copy number alterations in cancer.

    Yuan, Xiguo; Zhang, Junying; Yang, Liying; Zhang, Shengli; Chen, Baodi; Geng, Yaojun; Wang, Yue


    Somatic copy number alteration (CNA) is a common phenomenon in cancer genome. Distinguishing significant consensus events (SCEs) from random background CNAs in a set of subjects has been proven to be a valuable tool to study cancer. In order to identify SCEs with an acceptable type I error rate, better computational approaches should be developed based on reasonable statistics and null distributions. In this article, we propose a new approach named TAGCNA for identifying SCEs in somatic CNAs that may encompass cancer driver genes. TAGCNA employs a peel-off permutation scheme to generate a reasonable null distribution based on a prior step of selecting tag CNA markers from the genome being considered. We demonstrate the statistical power of TAGCNA on simulated ground truth data, and validate its applicability using two publicly available cancer datasets: lung and prostate adenocarcinoma. TAGCNA identifies SCEs that are known to be involved with proto-oncogenes (e.g. EGFR, CDK4) and tumor suppressor genes (e.g. CDKN2A, CDKN2B), and provides many additional SCEs with potential biological relevance in these data. TAGCNA can be used to analyze the significance of CNAs in various cancers. It is implemented in R and is freely available at PMID:22815924

  13. A Bayesian Analysis for Identifying DNA Copy Number Variations Using a Compound Poisson Process

    Yiğiter Ayten


    Full Text Available To study chromosomal aberrations that may lead to cancer formation or genetic diseases, the array-based Comparative Genomic Hybridization (aCGH technique is often used for detecting DNA copy number variants (CNVs. Various methods have been developed for gaining CNVs information based on aCGH data. However, most of these methods make use of the log-intensity ratios in aCGH data without taking advantage of other information such as the DNA probe (e.g., biomarker positions/distances contained in the data. Motivated by the specific features of aCGH data, we developed a novel method that takes into account the estimation of a change point or locus of the CNV in aCGH data with its associated biomarker position on the chromosome using a compound Poisson process. We used a Bayesian approach to derive the posterior probability for the estimation of the CNV locus. To detect loci of multiple CNVs in the data, a sliding window process combined with our derived Bayesian posterior probability was proposed. To evaluate the performance of the method in the estimation of the CNV locus, we first performed simulation studies. Finally, we applied our approach to real data from aCGH experiments, demonstrating its applicability.

  14. Measurement of lentiviral vector titre and copy number by cross-species duplex quantitative PCR.

    Christodoulou, I; Patsali, P; Stephanou, C; Antoniou, M; Kleanthous, M; Lederer, C W


    Lentiviruses are the vectors of choice for many preclinical studies and clinical applications of gene therapy. Accurate measurement of biological vector titre before treatment is a prerequisite for vector dosing, and the calculation of vector integration sites per cell after treatment is as critical to the characterisation of modified cell products as it is to long-term follow-up and the assessment of risk and therapeutic efficiency in patients. These analyses are typically based on quantitative real-time PCR (qPCR), but as yet compromise accuracy and comparability between laboratories and experimental systems, the former by using separate simplex reactions for the detection of endogene and lentiviral sequences and the latter by designing different PCR assays for analyses in human cells and animal disease models. In this study, we validate in human and murine cells a qPCR system for the single-tube assessment of lentiviral vector copy numbers that is suitable for analyses in at least 33 different mammalian species, including human and other primates, mouse, pig, cat and domestic ruminants. The established assay combines the accuracy of single-tube quantitation by duplex qPCR with the convenience of one-off assay optimisation for cross-species analyses and with the direct comparability of lentiviral transduction efficiencies in different species. PMID:26202078

  15. A genome-wide copy number variant study of suicidal behavior.

    Jeffrey A Gross

    Full Text Available Suicide and suicide attempts are complex behaviors that result from the interaction of different factors, including genetic variants that increase the predisposition to suicidal behaviors. Copy number variations (CNVs are deletions or duplications of a segment of DNA usually larger than one kilobase. These structural genetic changes, although quite rare, have been associated with genetic liability to mental disorders, such as autism, schizophrenia, and bipolar disorder. No genome-wide level studies have been published investigating the potential role of CNVs in suicidal behaviors. Based on single-nucleotide polymorphism array data, we followed the Penn-CNV standards to detect CNVs in 1,608 subjects, comprising 475 suicide and suicide attempt cases and 1,133 controls. Although the initial algorithms determined the presence of CNVs on chromosomes 6 and 12 in seven and eight cases, respectively, compared with none of the controls, visual inspection of the raw data did not support this finding. Furthermore we were unable to validate these findings by CNV-specific real-time polymerase chain reaction. Additionally, rare CNV burden analysis did not find an association between the frequency or length of rare CNVs and suicidal behavior in our sample population. Although our findings suggest CNVs do not play an important role in the etiology of suicidal behaviors, they are not inconsistent with the strong evidence from the literature suggesting that other genetic variants account for a portion of the total phenotypic variability in suicidal behavior.

  16. Spectrum of EGFR gene copy number changes and KRAS gene mutation status in Korean triple negative breast cancer patients.

    Yoonjung Kim

    Full Text Available Anti-epidermal growth factor receptor (EGFR therapy has been tried in triple negative breast cancer (TNBC patients without evaluation of molecular and clinical predictors in several randomized clinical studies. Only fewer than 20% of metastatic TNBCs showed response to anti-EGFR therapy. In order to increase the overall response rate, first step would be to classify TNBC into good or poor responders according to oncogenic mutation profiles. This study provides the molecular characteristics of TNBCs including EGFR gene copy number changes and mutation status of EGFR and KRAS gene in Korean TNBC patients. Mutation analysis for EGFR, KRAS, BRAF and TP53 from a total of 105 TNBC tissue samples was performed by direct sequencing, peptide nucleic acid-mediated PCR clamping method and real-time PCR. Copy number changes of EGFR gene were evaluated using multiplex ligation-dependent probe amplification. Out of all 105 TNBCs, 15.2% (16/105 showed EGFR copy number changes. Among them, increased or decreased EGFR copy number was detected in 13 (5 single copy gain, 2 amplification and 4 high-copy number amplification and 3 cases (3 hemizygous deletion, respectively. The mutation frequencies of KRAS, EGFR and TP53 gene were 1.9% (G12V and G12D, 1.0% (exon 19 del and 31.4%, respectively. There was no BRAF V600E mutation found. Future studies are needed to evaluate the clinical outcomes of TNBC patients who undergo anti-EGFR therapy according to the genetic status of EGFR.

  17. Integrated analysis of copy number variation and genome-wide expression profiling in colorectal cancer tissues.

    Nur Zarina Ali Hassan

    Full Text Available Integrative analyses of multiple genomic datasets for selected samples can provide better insight into the overall data and can enhance our knowledge of cancer. The objective of this study was to elucidate the association between copy number variation (CNV and gene expression in colorectal cancer (CRC samples and their corresponding non-cancerous tissues. Sixty-four paired CRC samples from the same patients were subjected to CNV profiling using the Illumina HumanOmni1-Quad assay, and validation was performed using multiplex ligation probe amplification method. Genome-wide expression profiling was performed on 15 paired samples from the same group of patients using the Affymetrix Human Gene 1.0 ST array. Significant genes obtained from both array results were then overlapped. To identify molecular pathways, the data were mapped to the KEGG database. Whole genome CNV analysis that compared primary tumor and non-cancerous epithelium revealed gains in 1638 genes and losses in 36 genes. Significant gains were mostly found in chromosome 20 at position 20q12 with a frequency of 45.31% in tumor samples. Examples of genes that were associated at this cytoband were PTPRT, EMILIN3 and CHD6. The highest number of losses was detected at chromosome 8, position 8p23.2 with 17.19% occurrence in all tumor samples. Among the genes found at this cytoband were CSMD1 and DLC1. Genome-wide expression profiling showed 709 genes to be up-regulated and 699 genes to be down-regulated in CRC compared to non-cancerous samples. Integration of these two datasets identified 56 overlapping genes, which were located in chromosomes 8, 20 and 22. MLPA confirmed that the CRC samples had the highest gains in chromosome 20 compared to the reference samples. Interpretation of the CNV data in the context of the transcriptome via integrative analyses may provide more in-depth knowledge of the genomic landscape of CRC.

  18. A danger of low copy numbers for inferring incorrect cooperativity degree

    Konkoli Zoran


    Full Text Available Abstract Background A dose-response curve depicts the fraction of bound proteins as a function of unbound ligands. Dose-response curves are used to measure the cooperativity degree of a ligand binding process. Frequently, the Hill function is used to fit the experimental data. The Hill function is parameterized by the value of the dissociation constant and the Hill coefficient, which describes the cooperativity degree. The use of Hill's model and the Hill function has been heavily criticised in this context, predominantly the assumption that all ligands bind at once, which resulted in further refinements of the model. In this work, the validity of the Hill function has been studied from an entirely different point of view. In the limit of low copy numbers the dynamics of the system becomes noisy. The goal was to asses the validity of the Hill function in this limit, and to see in what ways the effects of the fluctuations change the form of the dose-response curves. Results Dose-response curves were computed taking into account effects of fluctuations. The effects of fluctuations were described at the lowest order (the second moment of the particle number distribution by using the previously developed Pair Approach Reaction Noise EStimator (PARNES method. The stationary state of the system is described by nine equations with nine unknowns. To obtain fluctuation-corrected dose-response curves the equations have been investigated numerically. Conclusions The Hill function cannot describe dose-response curves in a low particle limit. First, dose-response curves are not solely parameterized by the dissociation constant and the Hill coefficient. In general, the shape of a dose-response curve depends on the variables that describe how an experiment (ensemble is designed. Second, dose-response curves are multi-valued in a rather non-trivial way.

  19. Low C4 gene copy numbers are associated with superior graft survival in patients transplanted with a deceased donor kidney

    Bay, Jakob T; Schejbel, Lone; Madsen, Hans O; Sørensen, Søren S; Hansen, Jesper M; Garred, Peter


    rejection, but a relationship between graft survival and serum C4 concentration as well as C4 genetic variation has not been established. We evaluated this using a prospective study design of 676 kidney transplant patients and 211 healthy individuals as controls. Increasing C4 gene copy numbers...... significantly correlated with the C4 serum concentration in both patients and controls. Patients with less than four total copies of C4 genes transplanted with a deceased donor kidney experienced a superior 5-year graft survival (hazard ratio 0.46, 95% confidence interval: 0.25-0.84). No significant association...... was observed in patients transplanted with a living donor. Thus, low C4 copy numbers are associated with increased kidney graft survival in patients receiving a kidney from a deceased donor. Hence, the degree of ischemia may influence the clinical impact of complement....

  20. A novel rare copy number variant of the ABCF1 gene identified among dengue fever patients from Peninsular Malaysia.

    Hoh, B P; Sam, S S; Umi, S H; Mahiran, M; Nik Khairudin, N Y; Rafidah Hanim, S; Abubakar, S


    Copy number variation (CNV) is a form of genetic variation in addition to single nucleotide polymorphisms. The significance of CNV in the manifestation of a number of diseases is only recently receiving considerable attention. We genotyped 163 dengue patients from Peninsular Malaysia for genes possibly linked to dengue infection using quantitative real-time PCR. Here, we report a serendipitous discovery of a novel rare CNV of the ABCF1 gene among the dengue patients. Among these patients, two had a gain of 1 copy (CN = 3) and one had lost 1 copy (CN = 1), indicating that a rare CNV of the ABCF1 gene was detected among dengue patients from Peninsular Malaysia. Although the gene is suspected to regulate inflammatory responses and pathogen-induced cytokine storm, its relevance to dengue requires further investigation. PMID:24634119

  1. Change in the plasmid copy number in acetic acid bacteria in response to growth phase and acetic acid concentration.

    Akasaka, Naoki; Astuti, Wiwik; Ishii, Yuri; Hidese, Ryota; Sakoda, Hisao; Fujiwara, Shinsuke


    Plasmids pGE1 (2.5 kb), pGE2 (7.2 kb), and pGE3 (5.5 kb) were isolated from Gluconacetobacter europaeus KGMA0119, and sequence analyses revealed they harbored 3, 8, and 4 genes, respectively. Plasmid copy numbers (PCNs) were determined by real-time quantitative PCR at different stages of bacterial growth. When KGMA0119 was cultured in medium containing 0.4% ethanol and 0.5% acetic acid, PCN of pGE1 increased from 7 copies/genome in the logarithmic phase to a maximum of 12 copies/genome at the beginning of the stationary phase, before decreasing to 4 copies/genome in the late stationary phase. PCNs for pGE2 and pGE3 were maintained at 1-3 copies/genome during all phases of growth. Under a higher concentration of ethanol (3.2%) the PCN for pGE1 was slightly lower in all the growth stages, and those of pGE2 and pGE3 were unchanged. In the presence of 1.0% acetic acid, PCNs were higher for pGE1 (10 copies/genome) and pGE3 (6 copies/genome) during the logarithmic phase. Numbers for pGE2 did not change, indicating that pGE1 and pGE3 increase their PCNs in response to acetic acid. Plasmids pBE2 and pBE3 were constructed by ligating linearized pGE2 and pGE3 into pBR322. Both plasmids were replicable in Escherichia coli, Acetobacter pasteurianus and G. europaeus, highlighting their suitability as vectors for acetic acid bacteria. PMID:25575969

  2. Role of Rom protein in copy number control of plasmid pBR322 at different growth rates in Escherichia coli K-12

    Tove, Atlung; Christensen, Bjarke Bak; Hansen, Flemming G.


    The copy number per cell mass of plasmid pBR322 and a rom(-) derivative was measured as a function of generation time. In fast growing cells the copy number per cell mass was virtually identical for rom(+) and rom(-) derivatives. However, the copy number of pBR322 only increased 3- to 4-fold from...... itself, respectively, at fast and slow growth. We conclude that the rom gene product-the Rom protein-is an important element in copy number control of Co1E1-type plasmids especially in slowly growing cells. (C) 1999 Academic Press.......The copy number per cell mass of plasmid pBR322 and a rom(-) derivative was measured as a function of generation time. In fast growing cells the copy number per cell mass was virtually identical for rom(+) and rom(-) derivatives. However, the copy number of pBR322 only increased 3- to 4-fold from a...... effect on the copy number of pBR322 rom(-) at fast growth, but it decreased its copy number at slow growth to the same level as found for pBR322, i.e., complemented the pBR322 rom(-) plasmid. The pACYC184 plasmid and its rom(+) derivatives showed copy numbers similar to those of pBR322 rom(-) and pBR322...

  3. Mitochondrial DNA copy number and biogenesis in different tissues of early- and late-lactating dairy cows.

    Laubenthal, L; Hoelker, M; Frahm, J; Dänicke, S; Gerlach, K; Südekum, K-H; Sauerwein, H; Häussler, S


    Energy balance in dairy cows changes during the course of lactation due to alterations in voluntary feed intake and energy required for milk synthesis. To adapt to the demands of lactation, energy metabolism needs to be regulated and coordinated in key organs such as adipose tissue (AT), liver, and mammary gland. Mitochondria are the main sites of energy production in mammalian cells and their number varies depending on age, organ, and physiological condition. The copy number of the mitochondrial genome, the mitochondrial DNA (mtDNA), reflects the abundance of mitochondria within a cell and is regulated by transcriptional and translational factors. Environmental, physiological, and energetic conditions change during lactation and we thus hypothesized that these changes may influence the mtDNA copy number and the abundance of genes regulating mitochondrial biogenesis. Therefore, we aimed to provide an overview of mitochondrial biogenesis in liver, subcutaneous (sc)AT, mammary gland, and peripheral blood cells during early and late lactation in dairy cows. German Holstein cows (n=21) were fed according to their requirements, and biopsies from scAT, liver, mammary gland, and blood were collected in early and late lactation and assayed for relative mtDNA copy numbers and the mRNA abundance of genes regulating mitochondrial biogenesis, such as nuclear-respiratory factor 1 and 2 (NRF-1, NRF-2), mitochondrial transcription factor A (TFAM), and peroxisome proliferator-activated receptor-gamma coactivator 1-α (PGC-1α). The number of mtDNA copies increased from early to late lactation in all tissues, whereas that in peripheral blood cells was greater in early compared with late lactation. Moreover, mitochondrial activity enzymes (i.e., citrate synthase and cytochrome c oxidase) increased from early to late lactation in scAT. Comparing the number of mtDNA copies between tissues and blood in dairy cows, the highest mtDNA content was observed in liver. The mRNA abundance of

  4. Genomic and functional characteristics of copy number variations in Angus cattle selected for resistance or susceptibility to gastrointestinal nematodes

    Genomic structural variation is an important and abundant source of genetic and phenotypic variation. We previously reported an initial analysis of copy number variations (CNVs) in Angus cattle selected for resistance or susceptibility to intestinal nematodes. In this study, we performed a large sca...

  5. Recurrent copy number changes in mentally retarded children harbour genes involved in cellular localization and the glutamate receptor complex

    Poot, Martin; Eleveld, Marc J.; van't Slot, Ruben; van Amstel, Hans Kristian Ploos; Hochstenbach, Ron


    To determine the phenotypic significance of copy number changes (CNCs) in the human genome, we performed genome-wide segmental aneuploidy profiling by BAC-based array-CGH of 278 unrelated patients with multiple congenital abnormalities and mental retardation (MCAMR) and in 48 unaffected family membe

  6. Genome-wide copy number variant analysis in inbred chicken lines with different susceptibility to Marek’s disease

    Breeding of genetically resistant chickens to Marek’s disease (MD) is a vital strategy to poultry health. To find the markers underlying the genetic resistance to MD, copy number variation (CNV) was examined in inbred MD-resistant and -susceptible chicken lines. A total of 45 CNVs were found in four...

  7. Copy number variants in a sample of patients with psychotic disorders: Is standard screening relevant for actual clinical practice?

    N.W.A. van de Kerkhof (Noortje); I. Feenstra (Ilse); F.M.M.A. van der Heijden (Frank); N. de Leeuw (Nicole); R. Pfundt; G. Stöber (Gerald); J.I.M. Egger (Jos); W.M.A. Verhoeven (Wim)


    textabstractWith the introduction of new genetic techniques such as genome-wide array comparative genomic hybridization, studies on the putative genetic etiology of schizophrenia have focused on the detection of copy number variants (CNVs), ie, microdeletions and/or microduplications, that are estim

  8. Mitochondrial DNA copy number, but not haplogroup, confers a genetic susceptibility to leprosy in Han Chinese from Southwest China.

    Dong Wang

    Full Text Available BACKGROUND: Leprosy is a chronic infectious disease caused by Mycobacterium leprae, an unculturable pathogen with an exceptionally eroded genome. The high level of inactivation of gene function in M. leprae, including many genes in its metabolic pathways, has led to a dependence on host energy production and nutritional products. We hypothesized that host cellular powerhouse--the mitochondria--may affect host susceptibility to M. leprae and the onset of clinical leprosy, and this may be reflected by mitochondrial DNA (mtDNA background and mtDNA copy number. METHODS: We analyzed the mtDNA sequence variation of 534 leprosy patients and 850 matched controls from Yunnan Province and classified each subject by haplogroup. mtDNA copy number, taken to be proportional to mtDNA content, was measured in a subset of these subjects (296 patients and 231 controls and 12 leprosy patients upon diagnosis. RESULTS: Comparison of matrilineal components of the case and control populations revealed no significant difference. However, measurement of mtDNA copy number showed that lepromatous leprosy patients had a significantly higher mtDNA content than controls (P = 0.008. Past medical treatments had no effect on the alteration of mtDNA copy number. CONCLUSIONS: Our results suggested that mtDNA content, but not haplogroup, affects leprosy and this influence is limited to the clinical subtype of lepromatous leprosy.

  9. High-resolution copy number analysis of paired normal-tumor samples from diffuse large B cell lymphoma.

    Sebastián, Elena; Alcoceba, Miguel; Martín-García, David; Blanco, Óscar; Sanchez-Barba, Mercedes; Balanzategui, Ana; Marín, Luis; Montes-Moreno, Santiago; González-Barca, Eva; Pardal, Emilia; Jiménez, Cristina; García-Álvarez, María; Clot, Guillem; Carracedo, Ángel; Gutiérrez, Norma C; Sarasquete, M Eugenia; Chillón, Carmen; Corral, Rocío; Prieto-Conde, M Isabel; Caballero, M Dolores; Salaverria, Itziar; García-Sanz, Ramón; González, Marcos


    Copy number analysis can be useful for assessing prognosis in diffuse large B cell lymphoma (DLBCL). We analyzed copy number data from tumor samples of 60 patients diagnosed with DLBCL de novo and their matched normal samples. We detected 63 recurrent copy number alterations (CNAs), including 33 gains, 30 losses, and nine recurrent acquired copy number neutral loss of heterozygosity (CNN-LOH). Interestingly, 20 % of cases acquired CNN-LOH of 6p21 locus, which involves the HLA region. In normal cells, there were no CNAs but we observed CNN-LOH involving some key lymphoma regions such as 6p21 and 9p24.1 (5 %) and 17p13.1 (2.5 %) in DLBCL patients. Furthermore, a model with some specific CNA was able to predict the subtype of DLBCL, 1p36.32 and 10q23.31 losses being restricted to germinal center B cell-like (GCB) DLBCL. In contrast, 8p23.3 losses and 11q24.3 gains were strongly associated with the non-GCB subtype. A poor prognosis was associated with biallelic inactivation of TP53 or 18p11.32 losses, while prognosis was better in cases carrying 11q24.3 gains. In summary, CNA abnormalities identify specific DLBCL groups, and we describe CNN-LOH in germline cells from DLBCL patients that are associated with genes that probably play a key role in DLBCL development. PMID:26573278

  10. Genome-wide assessment of the association of rare and common copy number variations to testicular germ cell cancer

    Edsgard, Stefan Daniel; Dalgaard, Marlene Danner; Weinhold, Nils; Wesolowska, Agata; Rajpert-De Meyts, Ewa; Ottesen, Anne Marie; Juul, Anders; Skakkebæk, Niels Erik; Jensen, Thomas Skøt; Gupta, Ramneek; Leffers, Henrik; Brunak, Søren


    Testicular germ cell cancer (TGCC) is one of the most heritable forms of cancer. Previous genome-wide association studies have focused on single nucleotide polymorphisms, largely ignoring the influence of copy number variants (CNVs). Here we present a genome-wide study of CNV on a cohort of 212...

  11. Copy number variations due to large genomic deletion in X-linked chronic granulomatous disease.

    Takashi Arai

    Full Text Available Mutations in genes for any of the six subunits of NADPH oxidase cause chronic granulomatous disease (CGD, but almost 2/3 of CGD cases are caused by mutations in the X-linked CYBB gene, also known as NAD (P H oxidase 2. Approximately 260 patients with CGD have been reported in Japan, of whom 92 were shown to have mutations of the CYBB gene and 16 to have chromosomal deletions. However, there has been very little detailed analysis of the range of the deletion or close understanding of the disease based on this. We therefore analyzed genomic rearrangements in X-linked CGD using array comparative genomic hybridization analysis, revealing the extent and the types of the deletion genes. The subjects were five Japanese X-linked CGD patients estimated to have large base deletions of 1 kb or more in the CYBB gene (four male patients, one female patient and the mothers of four of those patients. The five Japanese patients were found to range from a patient exhibiting deletions only of the CYBB gene to a female patient exhibiting an extensive DNA deletion and the DMD and CGD phenotype manifested. Of the other three patients, two exhibited CYBB, XK, and DYNLT3 gene deletions. The remaining patient exhibited both a deletion encompassing DNA subsequent to the CYBB region following intron 2 and the DYNLT3 gene and a complex copy number variation involving the insertion of an inverted duplication of a region from the centromere side of DYNLT3 into the deleted region.

  12. Connecting Anxiety and Genomic Copy Number Variation: A Genome-Wide Analysis in CD-1 Mice.

    Julia Brenndörfer

    Full Text Available Genomic copy number variants (CNVs have been implicated in multiple psychiatric disorders, but not much is known about their influence on anxiety disorders specifically. Using next-generation sequencing (NGS and two additional array-based genotyping approaches, we detected CNVs in a mouse model consisting of two inbred mouse lines showing high (HAB and low (LAB anxiety-related behavior, respectively. An influence of CNVs on gene expression in the central (CeA and basolateral (BLA amygdala, paraventricular nucleus (PVN, and cingulate cortex (Cg was shown by a two-proportion Z-test (p = 1.6 x 10-31, with a positive correlation in the CeA (p = 0.0062, PVN (p = 0.0046 and Cg (p = 0.0114, indicating a contribution of CNVs to the genetic predisposition to trait anxiety in the specific context of HAB/LAB mice. In order to confirm anxiety-relevant CNVs and corresponding genes in a second mouse model, we further examined CD-1 outbred mice. We revealed the distribution of CNVs by genotyping 64 CD 1 individuals using a high-density genotyping array (Jackson Laboratory. 78 genes within those CNVs were identified to show nominally significant association (48 genes, or a statistical trend in their association (30 genes with the time animals spent on the open arms of the elevated plus-maze (EPM. Fifteen of them were considered promising candidate genes of anxiety-related behavior as we could show a significant overlap (permutation test, p = 0.0051 with genes within HAB/LAB CNVs. Thus, here we provide what is to our knowledge the first extensive catalogue of CNVs in CD-1 mice and potential corresponding candidate genes linked to anxiety-related behavior in mice.

  13. Inference of haplotypic phase and missing genotypes in polyploid organisms and variable copy number genomic regions

    Balding David J


    Full Text Available Abstract Background The power of haplotype-based methods for association studies, identification of regions under selection, and ancestral inference, is well-established for diploid organisms. For polyploids, however, the difficulty of determining phase has limited such approaches. Polyploidy is common in plants and is also observed in animals. Partial polyploidy is sometimes observed in humans (e.g. trisomy 21; Down's syndrome, and it arises more frequently in some human tissues. Local changes in ploidy, known as copy number variations (CNV, arise throughout the genome. Here we present a method, implemented in the software polyHap, for the inference of haplotype phase and missing observations from polyploid genotypes. PolyHap allows each individual to have a different ploidy, but ploidy cannot vary over the genomic region analysed. It employs a hidden Markov model (HMM and a sampling algorithm to infer haplotypes jointly in multiple individuals and to obtain a measure of uncertainty in its inferences. Results In the simulation study, we combine real haplotype data to create artificial diploid, triploid, and tetraploid genotypes, and use these to demonstrate that polyHap performs well, in terms of both switch error rate in recovering phase and imputation error rate for missing genotypes. To our knowledge, there is no comparable software for phasing a large, densely genotyped region of chromosome from triploids and tetraploids, while for diploids we found polyHap to be more accurate than fastPhase. We also compare the results of polyHap to SATlotyper on an experimentally haplotyped tetraploid dataset of 12 SNPs, and show that polyHap is more accurate. Conclusion With the availability of large SNP data in polyploids and CNV regions, we believe that polyHap, our proposed method for inferring haplotypic phase from genotype data, will be useful in enabling researchers analysing such data to exploit the power of haplotype-based analyses.

  14. p53 regulates mtDNA copy number and mitocheckpoint pathway

    Kulawiec Mariola


    Full Text Available Background: We previously hypothesized a role for mitochondria damage checkpoint (mito-checkpoint in maintaining the mitochondrial integrity of cells. Consistent with this hypothesis, defects in mitochondria have been demonstrated to cause genetic and epigenetic changes in the nuclear DNA, resistance to cell-death and tumorigenesis. In this paper, we describe that defects in mitochondria arising from the inhibition of mitochondrial oxidative phosphorylation (mtOXPHOS induce cell cycle arrest, a response similar to the DNA damage checkpoint response. Materials and Methods: Primary mouse embryonic fibroblasts obtained from p53 wild-type and p53-deficient mouse embryos (p53 -/- were treated with inhibitors of electron transport chain and cell cycle analysis, ROS production, mitochondrial content analysis and immunoblotting was performed. The expression of p53R2 was also measured by real time quantitative PCR. Results: We determined that, while p53 +/+ cells arrest in the cell cycle, p53 -/- cells continued to divide after exposure to mitochondrial inhibitors, showing that p53 plays an important role in the S-phase delay in the cell cycle. p53 is translocated to mitochondria after mtOXPHOS inhibition. Our study also revealed that p53-dependent induction of reactive oxygen species acts as a major signal triggering a mito-checkpoint response. Furthermore our study revealed that loss of p53 results in down regulation of p53R2 that contributes to depletion of mtDNA in primary MEF cells. Conclusions: Our study suggests that p53 1 functions as mito-checkpoint protein and 2 regulates mtDNA copy number and mitochondrial biogenesis. We describe a conceptual organization of the mito-checkpoint pathway in which identified roles of p53 in mitochondria are incorporated.

  15. Flexible and accurate detection of genomic copy-number changes from aCGH.

    Oscar M Rueda


    Full Text Available Genomic DNA copy-number alterations (CNAs are associated with complex diseases, including cancer: CNAs are indeed related to tumoral grade, metastasis, and patient survival. CNAs discovered from array-based comparative genomic hybridization (aCGH data have been instrumental in identifying disease-related genes and potential therapeutic targets. To be immediately useful in both clinical and basic research scenarios, aCGH data analysis requires accurate methods that do not impose unrealistic biological assumptions and that provide direct answers to the key question, "What is the probability that this gene/region has CNAs?" Current approaches fail, however, to meet these requirements. Here, we introduce reversible jump aCGH (RJaCGH, a new method for identifying CNAs from aCGH; we use a nonhomogeneous hidden Markov model fitted via reversible jump Markov chain Monte Carlo; and we incorporate model uncertainty through Bayesian model averaging. RJaCGH provides an estimate of the probability that a gene/region has CNAs while incorporating interprobe distance and the capability to analyze data on a chromosome or genome-wide basis. RJaCGH outperforms alternative methods, and the performance difference is even larger with noisy data and highly variable interprobe distance, both commonly found features in aCGH data. Furthermore, our probabilistic method allows us to identify minimal common regions of CNAs among samples and can be extended to incorporate expression data. In summary, we provide a rigorous statistical framework for locating genes and chromosomal regions with CNAs with potential applications to cancer and other complex human diseases.

  16. Indexing Effects of Copy Number Variation on Genes Involved in Developmental Delay.

    Uddin, Mohammed; Pellecchia, Giovanna; Thiruvahindrapuram, Bhooma; D'Abate, Lia; Merico, Daniele; Chan, Ada; Zarrei, Mehdi; Tammimies, Kristiina; Walker, Susan; Gazzellone, Matthew J; Nalpathamkalam, Thomas; Yuen, Ryan K C; Devriendt, Koenraad; Mathonnet, Géraldine; Lemyre, Emmanuelle; Nizard, Sonia; Shago, Mary; Joseph-George, Ann M; Noor, Abdul; Carter, Melissa T; Yoon, Grace; Kannu, Peter; Tihy, Frédérique; Thorland, Erik C; Marshall, Christian R; Buchanan, Janet A; Speevak, Marsha; Stavropoulos, Dimitri J; Scherer, Stephen W


    A challenge in clinical genomics is to predict whether copy number variation (CNV) affecting a gene or multiple genes will manifest as disease. Increasing recognition of gene dosage effects in neurodevelopmental disorders prompted us to develop a computational approach based on critical-exon (highly expressed in brain, highly conserved) examination for potential etiologic effects. Using a large CNV dataset, our updated analyses revealed significant (P < 1.64 × 10(-15)) enrichment of critical-exons within rare CNVs in cases compared to controls. Separately, we used a weighted gene co-expression network analysis (WGCNA) to construct an unbiased protein module from prenatal and adult tissues and found it significantly enriched for critical exons in prenatal (P < 1.15 × 10(-50), OR = 2.11) and adult (P < 6.03 × 10(-18), OR = 1.55) tissues. WGCNA yielded 1,206 proteins for which we prioritized the corresponding genes as likely to have a role in neurodevelopmental disorders. We compared the gene lists obtained from critical-exon and WGCNA analysis and found 438 candidate genes associated with CNVs annotated as pathogenic, or as variants of uncertain significance (VOUS), from among 10,619 developmental delay cases. We identified genes containing CNVs previously considered to be VOUS to be new candidate genes for neurodevelopmental disorders (GIT1, MVB12B and PPP1R9A) demonstrating the utility of this strategy to index the clinical effects of CNVs. PMID:27363808

  17. A Poisson hierarchical modelling approach to detecting copy number variation in sequence coverage data

    Sepúlveda, Nuno


    Background: The advent of next generation sequencing technology has accelerated efforts to map and catalogue copy number variation (CNV) in genomes of important micro-organisms for public health. A typical analysis of the sequence data involves mapping reads onto a reference genome, calculating the respective coverage, and detecting regions with too-low or too-high coverage (deletions and amplifications, respectively). Current CNV detection methods rely on statistical assumptions (e.g., a Poisson model) that may not hold in general, or require fine-tuning the underlying algorithms to detect known hits. We propose a new CNV detection methodology based on two Poisson hierarchical models, the Poisson-Gamma and Poisson-Lognormal, with the advantage of being sufficiently flexible to describe different data patterns, whilst robust against deviations from the often assumed Poisson model.Results: Using sequence coverage data of 7 Plasmodium falciparum malaria genomes (3D7 reference strain, HB3, DD2, 7G8, GB4, OX005, and OX006), we showed that empirical coverage distributions are intrinsically asymmetric and overdispersed in relation to the Poisson model. We also demonstrated a low baseline false positive rate for the proposed methodology using 3D7 resequencing data and simulation. When applied to the non-reference isolate data, our approach detected known CNV hits, including an amplification of the PfMDR1 locus in DD2 and a large deletion in the CLAG3.2 gene in GB4, and putative novel CNV regions. When compared to the recently available FREEC and cn.MOPS approaches, our findings were more concordant with putative hits from the highest quality array data for the 7G8 and GB4 isolates.Conclusions: In summary, the proposed methodology brings an increase in flexibility, robustness, accuracy and statistical rigour to CNV detection using sequence coverage data. 2013 Seplveda et al.; licensee BioMed Central Ltd.

  18. High-resolution copy-number variation map reflects human olfactory receptor diversity and evolution.

    Yehudit Hasin


    Full Text Available Olfactory receptors (ORs, which are involved in odorant recognition, form the largest mammalian protein superfamily. The genomic content of OR genes is considerably reduced in humans, as reflected by the relatively small repertoire size and the high fraction ( approximately 55% of human pseudogenes. Since several recent low-resolution surveys suggested that OR genomic loci are frequently affected by copy-number variants (CNVs, we hypothesized that CNVs may play an important role in the evolution of the human olfactory repertoire. We used high-resolution oligonucleotide tiling microarrays to detect CNVs across 851 OR gene and pseudogene loci. Examining genomic DNA from 25 individuals with ancestry from three populations, we identified 93 OR gene loci and 151 pseudogene loci affected by CNVs, generating a mosaic of OR dosages across persons. Our data suggest that approximately 50% of the CNVs involve more than one OR, with the largest CNV spanning 11 loci. In contrast to earlier reports, we observe that CNVs are more frequent among OR pseudogenes than among intact genes, presumably due to both selective constraints and CNV formation biases. Furthermore, our results show an enrichment of CNVs among ORs with a close human paralog or lacking a one-to-one ortholog in chimpanzee. Interestingly, among the latter we observed an enrichment in CNV losses over gains, a finding potentially related to the known diminution of the human OR repertoire. Quantitative PCR experiments performed for 122 sampled ORs agreed well with the microarray results and uncovered 23 additional CNVs. Importantly, these experiments allowed us to uncover nine common deletion alleles that affect 15 OR genes and five pseudogenes. Comparison to the chimpanzee reference genome revealed that all of the deletion alleles are human derived, therefore indicating a profound effect of human-specific deletions on the individual OR gene content. Furthermore, these deletion alleles may be used

  19. Stability-based comparison of class discovery methods for DNA copy number profiles.

    Isabel Brito

    Full Text Available MOTIVATION: Array-CGH can be used to determine DNA copy number, imbalances in which are a fundamental factor in the genesis and progression of tumors. The discovery of classes with similar patterns of array-CGH profiles therefore adds to our understanding of cancer and the treatment of patients. Various input data representations for array-CGH, dissimilarity measures between tumor samples and clustering algorithms may be used for this purpose. The choice between procedures is often difficult. An evaluation procedure is therefore required to select the best class discovery method (combination of one input data representation, one dissimilarity measure and one clustering algorithm for array-CGH. Robustness of the resulting classes is a common requirement, but no stability-based comparison of class discovery methods for array-CGH profiles has ever been reported. RESULTS: We applied several class discovery methods and evaluated the stability of their solutions, with a modified version of Bertoni's [Formula: see text]-based test [1]. Our version relaxes the assumption of independency required by original Bertoni's [Formula: see text]-based test. We conclude that Minimal Regions of alteration (a concept introduced by [2] for input data representation, sim [3] or agree [4] for dissimilarity measure and the use of average group distance in the clustering algorithm produce the most robust classes of array-CGH profiles. AVAILABILITY: The software is available from It has also been partly integrated into "Visualization and analysis of array-CGH"(VAMP[5]. The data sets used are publicly available from ACTuDB [6].

  20. The landscape of copy number variations in Finnish families with autism spectrum disorders.

    Kanduri, Chakravarthi; Kantojärvi, Katri; Salo, Paula M; Vanhala, Raija; Buck, Gemma; Blancher, Christine; Lähdesmäki, Harri; Järvelä, Irma


    Rare de novo and inherited copy number variations (CNVs) have been implicated in autism spectrum disorder (ASD) risk. However, the genetic underpinnings of ASD remain unknown in more than 80% of cases. Therefore, identification of novel candidate genes and corroboration of known candidate genes may broaden the horizons of determining genetic risk alleles, and subsequent development of diagnostic testing. Here, using genotyping arrays, we characterized the genetic architecture of rare CNVs (1 Mb) CNVs and rare, exonic CNVs. The exonic rare de novo CNV rate (∼22.5%) seemed higher compared to previous reports. We identified several CNVs in well-known ASD regions including GSTM1-5, DISC1, FHIT, RBFOX1, CHRNA7, 15q11.2, 15q13.2-q13.3, 17q12, and 22q11.21. Additionally, several novel candidate genes (BDKRB1, BDKRB2, AP2M1, SPTA1, PTH1R, CYP2E1, PLCD3, F2RL1, UQCRC2, LILRB3, RPS9, and COL11A2) were identified through gene prioritization. The majority of these genes belong to neuroactive ligand-receptor interaction pathways, and calcium signaling pathways, thus suggesting that a subset of these novel candidate genes may contribute to ASD risk. Furthermore, several metabolic pathways like caffeine metabolism, drug metabolism, retinol metabolism, and calcium-signaling pathway were found to be affected by the rare exonic ASD CNVs. Additionally, biological processes such as bradykinin receptor activity, endoderm formation and development, and oxidoreductase activity were enriched among the rare exonic ASD CNVs. Overall, our findings may add data about new genes and pathways that contribute to the genetic architecture of ASD. PMID:26052927

  1. High quality copy number and genotype data from FFPE samples using Molecular Inversion Probe (MIP) microarrays

    Wang, Yuker; Carlton, Victoria E.H.; Karlin-Neumann, George; Sapolsky, Ronald; Zhang, Li; Moorhead, Martin; Wang, Zhigang C.; Richardson, Andrea L.; Warren, Robert; Walther, Axel; Bondy, Melissa; Sahin, Aysegul; Krahe, Ralf; Tuna, Musaffe; Thompson, Patricia A.; Spellman, Paul T.; Gray, Joe W.; Mills, Gordon B.; Faham, Malek


    A major challenge facing DNA copy number (CN) studies of tumors is that most banked samples with extensive clinical follow-up information are Formalin-Fixed Paraffin Embedded (FFPE). DNA from FFPE samples generally underperforms or suffers high failure rates compared to fresh frozen samples because of DNA degradation and cross-linking during FFPE fixation and processing. As FFPE protocols may vary widely between labs and samples may be stored for decades at room temperature, an ideal FFPE CN technology should work on diverse sample sets. Molecular Inversion Probe (MIP) technology has been applied successfully to obtain high quality CN and genotype data from cell line and frozen tumor DNA. Since the MIP probes require only a small ({approx}40 bp) target binding site, we reasoned they may be well suited to assess degraded FFPE DNA. We assessed CN with a MIP panel of 50,000 markers in 93 FFPE tumor samples from 7 diverse collections. For 38 FFPE samples from three collections we were also able to asses CN in matched fresh frozen tumor tissue. Using an input of 37 ng genomic DNA, we generated high quality CN data with MIP technology in 88% of FFPE samples from seven diverse collections. When matched fresh frozen tissue was available, the performance of FFPE DNA was comparable to that of DNA obtained from matched frozen tumor (genotype concordance averaged 99.9%), with only a modest loss in performance in FFPE. MIP technology can be used to generate high quality CN and genotype data in FFPE as well as fresh frozen samples.

  2. A genome-wide investigation of copy number variation in patients with sporadic brain arteriovenous malformation.

    Nasrine Bendjilali

    Full Text Available BACKGROUND: Brain arteriovenous malformations (BAVM are clusters of abnormal blood vessels, with shunting of blood from the arterial to venous circulation and a high risk of rupture and intracranial hemorrhage. Most BAVMs are sporadic, but also occur in patients with Hereditary Hemorrhagic Telangiectasia, a Mendelian disorder caused by mutations in genes in the transforming growth factor beta (TGFβ signaling pathway. METHODS: To investigate whether copy number variations (CNVs contribute to risk of sporadic BAVM, we performed a genome-wide association study in 371 sporadic BAVM cases and 563 healthy controls, all Caucasian. Cases and controls were genotyped using the Affymetrix 6.0 array. CNVs were called using the PennCNV and Birdsuite algorithms and analyzed via segment-based and gene-based approaches. Common and rare CNVs were evaluated for association with BAVM. RESULTS: A CNV region on 1p36.13, containing the neuroblastoma breakpoint family, member 1 gene (NBPF1, was significantly enriched with duplications in BAVM cases compared to controls (P = 2.2×10(-9; NBPF1 was also significantly associated with BAVM in gene-based analysis using both PennCNV and Birdsuite. We experimentally validated the 1p36.13 duplication; however, the association did not replicate in an independent cohort of 184 sporadic BAVM cases and 182 controls (OR = 0.81, P = 0.8. Rare CNV analysis did not identify genes significantly associated with BAVM. CONCLUSION: We did not identify common CNVs associated with sporadic BAVM that replicated in an independent cohort. Replication in larger cohorts is required to elucidate the possible role of common or rare CNVs in BAVM pathogenesis.

  3. Analysis of genomic copy number variation in equine recurrent airway obstruction (heaves).

    Ghosh, S; Das, P J; McQueen, C M; Gerber, V; Swiderski, C E; Lavoie, J-P; Chowdhary, B P; Raudsepp, T


    We explored the involvement of genomic copy number variants (CNVs) in susceptibility to recurrent airway obstruction (RAO), or heaves-an asthmalike inflammatory disease in horses. Analysis of 16 RAO-susceptible (cases) and six RAO-resistant (control) horses on a custom-made whole-genome 400K equine tiling array identified 245 CNV regions (CNVRs), 197 previously known and 48 new, distributed on all horse autosomes and the X chromosome. Among the new CNVRs, 30 were exclusively found in RAO cases and were further analyzed by quantitative PCR, including additional cases and controls. Suggestive association (P = 0.03; corrected P = 0.06) was found between RAO and a loss on chromosome 5 involving NME7, a gene necessary for ciliary functions in lungs and involved in primary ciliary dyskinesia in humans. The CNVR could be a potential marker for RAO susceptibility but needs further study in additional RAO cohorts. Other CNVRs were not associated with RAO, although several involved genes of interest, such as SPI2/SERPINA1 from the serpin gene family, which are associated with chronic obstructive pulmonary disease and asthma in humans. The SPI2/SERPINA1 CNVR showed striking variation among horses, but it was not significantly different between RAO cases and controls. The findings provide baseline information on the relationship between CNVs and RAO susceptibility. Discovery of new CNVs and the use of a larger population of RAO-affected and control horses are needed to shed more light on their significance in modulating this complex and heterogeneous disease. PMID:26932307

  4. Social Responsiveness Scale-aided analysis of the clinical impact of copy number variations in autism.

    van Daalen, Emma; Kemner, Chantal; Verbeek, Nienke E; van der Zwaag, Bert; Dijkhuizen, Trijnie; Rump, Patrick; Houben, Renske; van 't Slot, Ruben; de Jonge, Maretha V; Staal, Wouter G; Beemer, Frits A; Vorstman, Jacob A S; Burbach, J Peter H; van Amstel, Hans Kristian Ploos; Hochstenbach, Ron; Brilstra, Eva H; Poot, Martin


    Recent array-based studies have detected a wealth of copy number variations (CNVs) in patients with autism spectrum disorders (ASD). Since CNVs also occur in healthy individuals, their contributions to the patient's phenotype remain largely unclear. In a cohort of children with symptoms of ASD, diagnosis of the index patient using ADOS-G and ADI-R was performed, and the Social Responsiveness Scale (SRS) was administered to the index patients, both parents, and all available siblings. CNVs were identified using SNP arrays and confirmed by FISH or array CGH. To evaluate the clinical significance of CNVs, we analyzed three families with multiple affected children (multiplex) and six families with a single affected child (simplex) in which at least one child carried a CNV with a brain-transcribed gene. CNVs containing genes that participate in pathways previously implicated in ASD, such as the phosphoinositol signaling pathway (PIK3CA, GIRDIN), contactin-based networks of cell communication (CNTN6), and microcephalin (MCPH1) were found not to co-segregate with ASD phenotypes. In one family, a loss of CNTN5 co-segregated with disease. This indicates that most CNVs may by themselves not be sufficient to cause ASD, but still may contribute to the phenotype by additive or epistatic interactions with inherited (transmitted) mutations or non-genetic factors. Our study extends the scope of genome-wide CNV profiling beyond de novo CNVs in sporadic patients and may aid in uncovering missing heritability in genome-wide screening studies of complex psychiatric disorders. PMID:21837366

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

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


    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)...

  6. Molecular Characterization of Mycobacterium bovis Isolates with High Copy Number of IS6110 by Restriction Fragment Length Polymorphism

    Behnam Rafiee


    Full Text Available Background: Mycobacterium bovis is the causative agent of bovine tuberculosis and belongs to the Mycobacterium tuberculosis complex. M. bovis usually carries only one or a few copies of the insertion sequence IS6110 in its genome. The aim of this study was evaluation of copy number of IS6110 in M. bovis isolates by restriction fragment length polymorphism (RFLP method. Materials and Methods: In this experimental study, 25 lymph node specimens of tuberculin-positive cattle were collected and cultured by standard methods, afterward genomic DNA was extracted by chloroform-isoamyl alcohol. Genetic studies were conducted by Pvull and DNA hybridization with IS6110. Results: Two isolates displayed more than of 4 copies of IS6110 by RFLP (IS6110-RFLP method. Conclusion: The results of this study are unique and specific in Iran but reported in the world rarely. Therefore the new strains of M. bovis imported to Iran from other countries of the world.

  7. An initial comparative map of copy number variations in the goat (Capra hircus genome

    Casadio Rita


    Full Text Available Abstract Background The goat (Capra hircus represents one of the most important farm animal species. It is reared in all continents with an estimated world population of about 800 million of animals. Despite its importance, studies on the goat genome are still in their infancy compared to those in other farm animal species. Comparative mapping between cattle and goat showed only a few rearrangements in agreement with the similarity of chromosome banding. We carried out a cross species cattle-goat array comparative genome hybridization (aCGH experiment in order to identify copy number variations (CNVs in the goat genome analysing animals of different breeds (Saanen, Camosciata delle Alpi, Girgentana, and Murciano-Granadina using a tiling oligonucleotide array with ~385,000 probes designed on the bovine genome. Results We identified a total of 161 CNVs (an average of 17.9 CNVs per goat, with the largest number in the Saanen breed and the lowest in the Camosciata delle Alpi goat. By aggregating overlapping CNVs identified in different animals we determined CNV regions (CNVRs: on the whole, we identified 127 CNVRs covering about 11.47 Mb of the virtual goat genome referred to the bovine genome (0.435% of the latter genome. These 127 CNVRs included 86 loss and 41 gain and ranged from about 24 kb to about 1.07 Mb with a mean and median equal to 90,292 bp and 49,530 bp, respectively. To evaluate whether the identified goat CNVRs overlap with those reported in the cattle genome, we compared our results with those obtained in four independent cattle experiments. Overlapping between goat and cattle CNVRs was highly significant (P Conclusions We describe a first map of goat CNVRs. This provides information on a comparative basis with the cattle genome by identifying putative recurrent interspecies CNVs between these two ruminant species. Several goat CNVs affect genes with important biological functions. Further studies are needed to evaluate the

  8. Capsulation and Gene Copy Number at the cap Locus of Haemophilus influenzae Type b


    Although more than 98% of natural isolates of Haemophilus influenzae type b carry a duplication of 17 kilobases (kb) of DNA at the chromosomal capsulation locus, only one copy is required for capsulation. In one laboratory-derived and two clinical type b strains, the capsulation locus had a single copy of this 17-kb segment, together with 1.3 kb of DNA identified as lying between the repeats of the duplicated locus. This 1.3 kb appears to be crucial for capsule production, since strains lacki...

  9. SNP array-based copy number and genotype analyses for preimplantation genetic diagnosis of human unbalanced translocations

    van Uum, Chris MJ; Stevens, Servi JC; Dreesen, Joseph CFM; Drüsedau, Marion; Smeets, Hubert J.; Hollanders-Crombach, Bertien; Die-Smulders, Christine EM de; Geraedts, Joep PM; Engelen, John JM; Coonen, Edith


    Preimplantation genetic diagnosis (PGD) for chromosomal rearrangements (CR) is mainly based on fluorescence in situ hybridisation (FISH). Application of this technique is limited by the number of available fluorochromes, the extensive preclinical work-up and technical and interpretative artefacts. We aimed to develop a universal, off-the-shelf protocol for PGD by combining single-nucleotide polymorphism (SNP) array-derived copy number (CN) determination and genotyping for detection of unbalan...

  10. Genomic copy number variants: evidence for association with antibody response to anthrax vaccine adsorbed.

    Michael I Falola

    Full Text Available BACKGROUND: Anthrax and its etiologic agent remain a biological threat. Anthrax vaccine is highly effective, but vaccine-induced IgG antibody responses vary widely following required doses of vaccinations. Such variation can be related to genetic factors, especially genomic copy number variants (CNVs that are known to be enriched among genes with immunologic function. We have tested this hypothesis in two study populations from a clinical trial of anthrax vaccination. METHODS: We performed CNV-based genome-wide association analyses separately on 794 European Americans and 200 African-Americans. Antibodies to protective antigen were measured at week 8 (early response and week 30 (peak response using an enzyme-linked immunosorbent assay. We used DNA microarray data (Affymetrix 6.0 and two CNV detection algorithms, hidden markov model (PennCNV and circular binary segmentation (GeneSpring to determine CNVs in all individuals. Multivariable regression analyses were used to identify CNV-specific associations after adjusting for relevant non-genetic covariates. RESULTS: Within the 22 autosomal chromosomes, 2,943 non-overlapping CNV regions were detected by both algorithms. Genomic insertions containing HLA-DRB5, DRB1 and DQA1/DRA genes in the major histocompatibility complex (MHC region (chromosome 6p21.3 were moderately associated with elevated early antibody response (β = 0.14, p = 1.78×10(-3 among European Americans, and the strongest association was observed between peak antibody response and a segmental insertion on chromosome 1, containing NBPF4, NBPF5, STXMP3, CLCC1, and GPSM2 genes (β = 1.66, p = 6.06×10(-5. For African-Americans, segmental deletions spanning PRR20, PCDH17 and PCH68 genes on chromosome 13 were associated with elevated early antibody production (β = 0.18, p = 4.47×10(-5. Population-specific findings aside, one genomic insertion on chromosome 17 (containing NSF, ARL17 and LRRC37A genes was associated

  11. Detection of copy number variants reveals association of cilia genes with neural tube defects.

    Xiaoli Chen

    Full Text Available BACKGROUND: Neural tube defects (NTDs are one of the most common birth defects caused by a combination of genetic and environmental factors. Currently, little is known about the genetic basis of NTDs although up to 70% of human NTDs were reported to be attributed to genetic factors. Here we performed genome-wide copy number variants (CNVs detection in a cohort of Chinese NTD patients in order to exam the potential role of CNVs in the pathogenesis of NTDs. METHODS: The genomic DNA from eighty-five NTD cases and seventy-five matched normal controls were subjected for whole genome CNVs analysis. Non-DGV (the Database of Genomic Variants CNVs from each group were further analyzed for their associations with NTDs. Gene content in non-DGV CNVs as well as participating pathways were examined. RESULTS: Fifty-five and twenty-six non-DGV CNVs were detected in cases and controls respectively. Among them, forty and nineteen CNVs involve genes (genic CNV. Significantly more non-DGV CNVs and non-DGV genic CNVs were detected in NTD patients than in control (41.2% vs. 25.3%, p<0.05 and 37.6% vs. 20%, p<0.05. Non-DGV genic CNVs are associated with a 2.65-fold increased risk for NTDs (95% CI: 1.24-5.87. Interestingly, there are 41 cilia genes involved in non-DGV CNVs from NTD patients which is significantly enriched in cases compared with that in controls (24.7% vs. 9.3%, p<0.05, corresponding with a 3.19-fold increased risk for NTDs (95% CI: 1.27-8.01. Pathway analyses further suggested that two ciliogenesis pathways, tight junction and protein kinase A signaling, are top canonical pathways implicated in NTD-specific CNVs, and these two novel pathways interact with known NTD pathways. CONCLUSIONS: Evidence from the genome-wide CNV study suggests that genic CNVs, particularly ciliogenic CNVs are associated with NTDs and two ciliogenesis pathways, tight junction and protein kinase A signaling, are potential pathways involved in NTD pathogenesis.

  12. Identification of chloroquine resistance Pfcrt-K76T and determination of Pfmdr1-N86Y copy number by SYBR Green I qPCR

    Addimas; Tajebe; Mulugeta; Aemero; Kimani; Francis; Gabriel; Magoma


    Objective:To identify prevalence of chloroquine resistance point mutation at(Pfcrt,K76T)and(Pfindr1.N86Y) copy number variation.Methods:SYBR Green I based real time PCR was used.One hundred and thirty-three samples were analyzed for(Pfcrt,K76T) and(Pfmdr1.N86Y) copy number from dried blood spot.Parasite DNA was extracted using high pure DNA preparation kit.The amplification of DNA was done by using AccuPower 2* GreenStar ’’ qPCR Master mix.For quantification purpose a new primer pair was designed for 178 base pair template from complete genome sequence of Plasmodium falciparum strain 3D7 at NCBI.Absolute quantification method was used to determine the Pfmdr1-N86 Y copy number variations.Standard curve was built from strain3D7 gDNA since it has single copy of Pfindr1 per haploid genome.The known positive controls with single and multi-copy number of Pfindr1 gene were included in each experiment.The copy number ratio of the samples to the standard calibrator was made to obtain the fold difference among the samples with respect to copy number variation.Results:Out of 133 samples 73(54.89%) were confirmed as mutant(Pfcrt,76T) and the remaining 60(45.11%) were genotyped as wild type(Pfcrt,K76).The(Pfindr1.N86Y) copy number variation was determined for 133 clinical samples.Out of these samples 61(45.86%)had single copy and the remaining 72(54.14%) had multi-copy numbers higher than 1.5 copies per genome.Thirty-four(25.56%) multi-copies were between 1.5 and 2.5 copies per genome while 38(28.57%) were more than 2.5 copies per genome.The minimum and maximum copies per genome were 0.474 and 4.741.respectively.Conclusions:The study showed high prevalence level and fixation of Pfcrt.76 T mutation after chloroquine withdrawal.The prevalence of Pfindr1 copy number variant suggested that the presence of modulating factor for emergence of Plasmodium falciparum strains with higher copy numbers.However,the prevalence level was not statistically significant.

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

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


    Lipocalins are involved in the binding of small molecules like sex steroids. We show here that the previously reported tilapia male-specific protein (MSP) is a lipocalin encoded by a variety of paralogous and homologous genes in different tilapia species. Exon-intron boundaries of MSP genes were typical of the six-exon genomic structure of lipocalins, and the transcripts were capable of encoding 200 amino-acid polypeptides that consisted of a putative signal peptide and a lipocalin domain. Cysteine residues are conserved in positions analogous to those forming the three disulfide bonds characteristic of the ligand pocket. The calculated molecular mass of the secreted MSP (20.4 kDa) was less than half of that observed, suggesting that it is highly glycosylated like its homologue tributyltin-binding protein. Analysis of sequence variations revealed three types of paralogs MSPA, MSPB and MSPC. Expression of both MSPA and MSPB was detected in testis. In haploid Oreochromis niloticus embryos, each of these types consisted of two closely related paralogs, and asymmetry between MSP copy numbers on the maternal (six copies) and the paternal (three copies) chromosomes was observed. Using this polymorphism we mapped MSPA and MSPC to linkage group 12 of an F(2) mapping family derived from a cross between O. niloticus and Oreochromis aureus. Females with high MSP copy number were more frequent by more than twofold than males. Gender-MSPC combinations showed significant deviation from expected Mendelian segregation (P=0.009) suggesting elimination of males with MSPC copies. We discuss different hypotheses to explain this elimination, including possibility for allelic conflict resulted by the hybridization. PMID:18648387

  14. CCL3L1 gene copy number in individuals with and without HIV-associated neurocognitive disorder

    Brown A


    Full Text Available Amanda Brown1, Ned Sacktor1, Karen Marder2, Bruce Cohen3, Giovanni Schifitto4, Richard L Skolasky1, Jason Creighton1, Liping Guo1, Justin C McArthur11Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD, 2Department of Neurology, Psychiatry, Sergievsky Center and Taub Institute on Alzheimers Disease and the Aging Brain, New York Presbyterian Hospital, Columbia University College of Physicians and Surgeons, New York, NY, 3Department of Neurology, Northwestern University Feinberg School of Medicine, Chicago, IL, 4Department of Neurology, University of Rochester, School of Medicine and Dentistry, Rochester, NY, USABackground: CCL3L1 copy number variation has been implicated as a marker for susceptibility and immunity to human immunodeficiency virus (HIV-1 infection and its pathogenic sequelae. Some of these findings have been confirmed in several, but not all, subsequent independent cohort studies. A three-fold risk for the development of HIV-associated dementia was reported in individuals possessing a CCL3L1 copy number below the ethnic group median combined with a detrimental CCR5 genotype. With the availability of antiretroviral therapy since 1996, there has been a significant decline in HIV-associated dementia, and milder forms of HIV-associated neurocognitive impairment (HAND are now most prevalent. Moreover, patients are living longer with HIV-1 infection and it is recognized that aging may be a contributory factor to the development of cognitive disorder. Thus, the need for biomarkers that can be used in clinical practice to identify and provide optimal treatment for those at increased risk for HAND is great. HAND affects 20%–30% of HIV-infected individuals, and several genetic loci which have been shown to confer susceptibility to HIV infection may also modulate the development of neurocognitive disorder. The aim of this study was to determine whether CCL3L1 chemokine gene copy number in self-defined ethnic

  15. The partitioning and copy number control systems of the selfish yeast plasmid: an optimized molecular design for stable persistence in host cells

    Yen-Ting-Liu,; Sau, Saumitra; Ma, Chien-Hui; Kachroo, Aashiq H.; Rowley, Paul A.; Chang, Keng-Ming; Fan, Hsiu-Fang; Jayaram, Makkuni


    The multi-copy 2 micron plasmid of Saccharomyces cerevisiae, a resident of the nucleus, is remarkable for its high chromosome-like stability. The plasmid does not appear to contribute to the fitness of the host, nor does it impose a significant metabolic burden on the host at its steady state copy number. The plasmid may be viewed as a highly optimized selfish DNA element whose genome design is devoted entirely towards efficient replication, equal segregation and copy number maintenance. A pa...

  16. Cancer gene prioritization by integrative analysis of mRNA expression and DNA copy number data: a comparative review

    Lahti, Leo; Klein, Hans-Ulrich; Bicciato, Silvio; Dugas, Martin


    A variety of genome-wide profiling techniques are available to probe complementary aspects of genome structure and function. Integrative analysis of heterogeneous data sources can reveal higher-level interactions that cannot be detected based on individual observations. A standard integration task in cancer studies is to identify altered genomic regions that induce changes in the expression of the associated genes based on joint analysis of genome-wide gene expression and copy number profiling measurements. In this review, we provide a comparison among various modeling procedures for integrating genome-wide profiling data of gene copy number and transcriptional alterations and highlight common approaches to genomic data integration. A transparent benchmarking procedure is introduced to quantitatively compare the cancer gene prioritization performance of the alternative methods. The benchmarking algorithms and data sets are available at

  17. S-SCAM, A Rare Copy Number Variation Gene, Induces Schizophrenia-Related Endophenotypes in Transgenic Mouse Model

    Zhang, Nanyan; Zhong, PENG; Shin, Seung Min; Metallo, Jacob; Danielson, Eric; Olsen, Christopher M.; Liu, Qing-song; Lee, Sang H.


    Accumulating genetic evidence suggests that schizophrenia (SZ) is associated with individually rare copy number variations (CNVs) of diverse genes, often specific to single cases. However, the causality of these rare mutations remains unknown. One of the rare CNVs found in SZ cohorts is the duplication of Synaptic Scaffolding Molecule (S-SCAM, also called MAGI-2), which encodes a postsynaptic scaffolding protein controlling synaptic AMPA receptor levels, and thus the strength of excitatory sy...

  18. A hybrid qPCR/SNP array approach allows cost efficient assessment of KIR gene copy numbers in large samples

    Pontikos, Nikolas; Smyth, Deborah J.; Schuilenburg, Helen; Howson, Joanna MM; Walker, Neil M.; Burren, Oliver S.; Guo, Hui; Onengut-Gumuscu, Suna; Chen, Wei-Min; Concannon, Patrick; Rich, Stephen S.; Jayaraman, Jyothi; Jiang, Wei; James A. Traherne; Trowsdale, John


    Background Killer Immunoglobulin-like Receptors (KIRs) are surface receptors of natural killer cells that bind to their corresponding Human Leukocyte Antigen (HLA) class I ligands, making them interesting candidate genes for HLA-associated autoimmune diseases, including type 1 diabetes (T1D). However, allelic and copy number variation in the KIR region effectively mask it from standard genome-wide association studies: single nucleotide polymorphism (SNP) probes targeting the region are often ...

  19. Computational methods for detecting copy number variations in cancer genome using next generation sequencing: principles and challenges

    Liu, Biao; Morrison, Carl D.; Johnson, Candace S.; Trump, Donald L.; Qin, Maochun; Conroy, Jeffrey C.; Wang, Jianmin; Liu, Song


    Accurate detection of somatic copy number variations (CNVs) is an essential part of cancer genome analysis, and plays an important role in oncotarget identifications. Next generation sequencing (NGS) holds the promise to revolutionize somatic CNV detection. In this review, we provide an overview of current analytic tools used for CNV detection in NGS-based cancer studies. We summarize the NGS data types used for CNV detection, decipher the principles for data preprocessing, segmentation, and ...

  20. Analysis of copy number variants by three detection algorithms and their association with body size in horses

    Metzger, Julia; Philipp, Ute; Lopes, Maria Susana; da Camara Machado, Artur; Felicetti, Michela; Silvestrelli, Maurizio; Distl, Ottmar


    Background Copy number variants (CNVs) have been shown to play an important role in genetic diversity of mammals and in the development of many complex phenotypic traits. The aim of this study was to perform a standard comparative evaluation of CNVs in horses using three different CNV detection programs and to identify genomic regions associated with body size in horses. Results Analysis was performed using the Illumina Equine SNP50 genotyping beadchip for 854 horses. CNVs were detected by th...

  1. A High-Throughput Computational Framework for Identifying Significant Copy Number Aberrations from Array Comparative Genomic Hybridisation Data

    Ian Roberts; Carter, Stephanie A; Scarpini, Cinzia G.; Konstantina Karagavriilidou; Barna, Jenny C. J.; Mark Calleja; Nicholas Coleman


    Reliable identification of copy number aberrations (CNA) from comparative genomic hybridization data would be improved by the availability of a generalised method for processing large datasets. To this end, we developed swatCGH, a data analysis framework and region detection heuristic for computational grids. swatCGH analyses sequentially displaced (sliding) windows of neighbouring probes and applies adaptive thresholds of varying stringency to identify the 10% of each chromosome that contain...

  2. Detection of pathogenic copy number variants in children with idiopathic intellectual disability using 500 K SNP array genomic hybridization

    Li H Irene; Lemyre Emmanuelle; Langlois Sylvie; Gibson William T; Flibotte Stephane; Delaney Allen D; Chai David; Chan Susanna; Boerkoel Cornelius; Birch Patricia; Baross Agnes; Armstrong Linlea; Arbour Laura; Adam Shelin; Friedman JM


    Abstract Background Array genomic hybridization is being used clinically to detect pathogenic copy number variants in children with intellectual disability and other birth defects. However, there is no agreement regarding the kind of array, the distribution of probes across the genome, or the resolution that is most appropriate for clinical use. Results We performed 500 K Affymetrix GeneChip® array genomic hybridization in 100 idiopathic intellectual disability trios, each comprised of a chil...

  3. Identification by full-coverage array CGH of human DNA copy number increases relative to chimpanzee and gorilla

    Wilson, Gary M.; Flibotte, Stephane; Missirlis, Perseus I.; Marra, Marco A.; Jones, Steven; Thornton, Kevin; Clark, Andrew G.; Holt, Robert A.


    Duplication of chromosomal segments and associated genes is thought to be a primary mechanism for generating evolutionary novelty. By comparative genome hybridization using a full-coverage (tiling) human BAC array with 79-kb resolution, we have identified 63 chromosomal segments, ranging in size from 0.65 to 1.3 Mb, that have inferred copy number increases in human relative to chimpanzee. These segments span 192 Ensembl genes, including 82 gene duplicates (41 reciprocal best BLAST matches). S...

  4. Copy number variation analysis implicates the cell polarity gene glypican 5 as a human spina bifida candidate gene

    Bassuk, Alexander G.; Muthuswamy, Lakshmi B.; Boland, Riley; Smith, Tiffany L.; Hulstrand, Alissa M.; Northrup, Hope; Hakeman, Matthew; Dierdorff, Jason M.; Yung, Christina K.; Long, Abby; Brouillette, Rachel B.; Au, Kit Sing; Gurnett, Christina; Houston, Douglas W.; Cornell, Robert A


    Neural tube defects (NTDs) are common birth defects of complex etiology. Family and population-based studies have confirmed a genetic component to NTDs. However, despite more than three decades of research, the genes involved in human NTDs remain largely unknown. We tested the hypothesis that rare copy number variants (CNVs), especially de novo germline CNVs, are a significant risk factor for NTDs. We used array-based comparative genomic hybridization (aCGH) to identify rare CNVs in 128 Cauca...

  5. High Fidelity Copy Number Analysis of Formalin-Fixed and Paraffin-Embedded Tissues Using Affymetrix Cytoscan HD Chip

    Yu, Yan P.; Amantha Michalopoulos; Ying Ding; George Tseng; Jian-Hua Luo


    Detection of human genome copy number variation (CNV) is one of the most important analyses in diagnosing human malignancies. Genome CNV detection in formalin-fixed and paraffin-embedded (FFPE) tissues remains challenging due to suboptimal DNA quality and failure to use appropriate baseline controls for such tissues. Here, we report a modified method in analyzing CNV in FFPE tissues using microarray with Affymetrix Cytoscan HD chips. Gel purification was applied to select DNA with good qualit...

  6. Copy number variations associated with obesity related traits in African Americans: a joint analysis between GENOA and HyperGEN

    Zhao, Wei; Wineinger, Nathan E.; Tiwari, Hemant K.; Mosley, Thomas H (Jr); Broeckel, Ulrich; Arnett, Donna K.; Kardia, Sharon L. R.; Kabagambe, Edmond K.; Sun, Yan V


    Obesity is a highly heritable trait and a growing public health problem. African Americans are a genetically diverse, yet understudied population with a high prevalence of obesity (body mass index (BMI) greater than 30 kg/m2). Recent studies based upon single nucleotide polymorphisms (SNPs) have identified genetic markers associated with obesity. However, a large proportion of the heritability of obesity remains unexplained. Copy number variation (CNV) has been cited as a possible source of m...

  7. Fluctuations in Tat copy number when it counts the most: a possible mechanism to battle the HIV latency

    Konkoli, Zoran; Jesorka, Aldo


    The HIV-1 virus can enter a dormant state and become inactive, which reduces accessibility by antiviral drugs. We approach this latency problem from an unconventional point of view, with the focus on understanding how intrinsic chemical noise (copy number fluctuations of the Tat protein) can be used to assist the activation process of the latent virus. Several phase diagrams have been constructed in order to visualize in which regions of the parameter space noise can drive the activation proc...

  8. Genome-Wide Copy Number Variant Analysis in Inbred Chickens Lines With Different Susceptibility to Marek’s Disease

    Luo, Juan; Yu, Ying; Mitra, Apratim; Chang, Shuang; Zhang, Huanmin; LIU, GEORGE; Yang, Ning; Song, Jiuzhou


    Breeding of genetically resistant chickens to Marek’s disease (MD) is a vital strategy to poultry health. To find the markers underlying the genetic resistance to MD, copy number variation (CNV) was examined in inbred MD-resistant and -susceptible chicken lines. A total of 45 CNVs were found in four lines of chickens, and 28 were potentially involved in immune response and cell proliferation, etc. Importantly, two CNVs related with MD resistance were transmitted to descendent recombinant cong...

  9. Copy Numbers of Telomeric Repeat Sequences of Human Herpesvirus 6B in Clinical Isolates: Possibility of Mixed Infections

    KATO, Yuri; Ihira, Masaru; Umeda, Mami; Higashimoto, Yuki; Kawamura, Yoshiki; Ohashi, Masahiro; Ishi, Junichi; Yoshikawa, Tetsushi


    In order to determine whether mixed infections of human herpesvirus 6B (HHV-6B) occur in immunocompetent and immunocompromised individuals, we examined the copy numbers of telomeric repeat sequences (TRS) of clinical isolates. In clinical isolates obtained from patients with exanthem subitum caused by primary HHV-6B infection, PCR products with HHV-6B TRS ranging between 400 and 800 bp were amplified. PCR products of various sizes were amplified in four clinical isolates from drug-induced hyp...

  10. Gene copy number variations in the leukocyte genome of hepatocellular carcinoma patients with integrated hepatitis B virus DNA

    Xu, Guixia; Cheng, Kai; Cao, Guangwen; Wu, Mengchao; Cheng, Shuqun; Liu, Shanrong


    Integration of hepatitis B virus (HBV) DNA into the human liver cell genome is believed to promote HBV-related carcinogenesis. This study aimed to quantify the integration of HBV DNA into the leukocyte genome in hepatocellular carcinoma (HCC) patients in order to identify potential biomarkers for HBV-related diseases. Whole-genome comparative genomic hybridization (CGH) chip array analyses were performed to screen gene copy number variations (CNV) in the leukocyte genome, and the results were confirmed by quantitative polymerase chain reaction (qPCR). The commonly detected regions included chromosome arms 19p, 5q, 1q and 15p, where 200 copy number gain events and 270 copy number loss events were noted. In particular, gains were observed in 5q35.3 (OR4F3) and 19p13.3 (OR4F17) in 90% of the samples. Successful homologous recombination of OR4F3 and the HBV P gene was demonstrated, and the amplification at 5q35.3 is potentially associated with the integration of HBV P gene into natural killer cells isolated from peripheral blood mononuclear cells (PBMCs). Receiver operating characteristic (ROC) curve analysis indicated that the combination of OR4F3 and OR4F17 a novel potential biomarker of HBV-related diseases. PMID:26769853

  11. Chromosomal instability selects gene copy-number variants encoding core regulators of proliferation in ER+ breast cancer.

    Endesfelder, David; Burrell, Rebecca A; Kanu, Nnennaya; McGranahan, Nicholas; Howell, Mike; Parker, Peter J; Downward, Julian; Swanton, Charles; Kschischo, Maik


    Chromosomal instability (CIN) is associated with poor outcome in epithelial malignancies, including breast carcinomas. Evidence suggests that prognostic signatures in estrogen receptor-positive (ER(+)) breast cancer define tumors with CIN and high proliferative potential. Intriguingly, CIN induction in lower eukaryotic cells and human cells is context dependent, typically resulting in a proliferation disadvantage but conferring a fitness benefit under strong selection pressures. We hypothesized that CIN permits accelerated genomic evolution through the generation of diverse DNA copy-number events that may be selected during disease development. In support of this hypothesis, we found evidence for selection of gene amplification of core regulators of proliferation in CIN-associated cancer genomes. Stable DNA copy-number amplifications of the core regulators TPX2 and UBE2C were associated with expression of a gene module involved in proliferation. The module genes were enriched within prognostic signature gene sets for ER(+) breast cancer, providing a logical connection between CIN and prognostic signature expression. Our results provide a framework to decipher the impact of intratumor heterogeneity on key cancer phenotypes, and they suggest that CIN provides a permissive landscape for selection of copy-number alterations that drive cancer proliferation. PMID:24970479

  12. Copy number and loss of heterozygosity detected by SNP array of formalin-fixed tissues using whole-genome amplification.

    Angela Stokes

    Full Text Available The requirement for large amounts of good quality DNA for whole-genome applications prohibits their use for small, laser capture micro-dissected (LCM, and/or rare clinical samples, which are also often formalin-fixed and paraffin-embedded (FFPE. Whole-genome amplification of DNA from these samples could, potentially, overcome these limitations. However, little is known about the artefacts introduced by amplification of FFPE-derived DNA with regard to genotyping, and subsequent copy number and loss of heterozygosity (LOH analyses. Using a ligation adaptor amplification method, we present data from a total of 22 Affymetrix SNP 6.0 experiments, using matched paired amplified and non-amplified DNA from 10 LCM FFPE normal and dysplastic oral epithelial tissues, and an internal method control. An average of 76.5% of SNPs were called in both matched amplified and non-amplified DNA samples, and concordance was a promising 82.4%. Paired analysis for copy number, LOH, and both combined, showed that copy number changes were reduced in amplified DNA, but were 99.5% concordant when detected, amplifications were the changes most likely to be 'missed', only 30% of non-amplified LOH changes were identified in amplified pairs, and when copy number and LOH are combined ∼50% of gene changes detected in the unamplified DNA were also detected in the amplified DNA and within these changes, 86.5% were concordant for both copy number and LOH status. However, there are also changes introduced as ∼20% of changes in the amplified DNA are not detected in the non-amplified DNA. An integrative network biology approach revealed that changes in amplified DNA of dysplastic oral epithelium localize to topologically critical regions of the human protein-protein interaction network, suggesting their functional implication in the pathobiology of this disease. Taken together, our results support the use of amplification of FFPE-derived DNA, provided sufficient samples are used

  13. Applying microsatellite multiplex PCR analysis (MMPA for determining allele copy-number status and percentage of normal cells within tumors.

    Carles Garcia-Linares

    Full Text Available The study of somatic genetic alterations in tumors contributes to the understanding and management of cancer. Genetic alterations, such us copy number or copy neutral changes, generate allelic imbalances (AIs that can be determined using polymorphic markers. Here we report the development of a simple set of calculations for analyzing microsatellite multiplex PCR data from control-tumor pairs that allows us to obtain accurate information not only regarding the AI status of tumors, but also the percentage of tumor-infiltrating normal cells, the locus copy-number status and the mechanism involved in AI. We validated this new approach by re-analyzing a set of Neurofibromatosis type 1-associated dermal neurofibromas and comparing newly generated data with results obtained for the same tumors in a previous study using MLPA, Paralog Ratio Analysis and SNP-array techniques.Microsatellite multiplex PCR analysis (MMPA should be particularly useful for analyzing specific regions of the genome containing tumor suppressor genes and also for determining the percentage of infiltrating normal cells within tumors allowing them to be sorted before they are analyzed by more expensive techniques.

  14. Male-Biased Autosomal Effect of 16p13.11 Copy Number Variation in Neurodevelopmental Disorders

    Maria Tropeano; Joo Wook Ahn; Dobson, Richard J. B.; Gerome Breen; James Rucker; Abhishek Dixit; Pal, Deb K; Peter McGuffin; Anne Farmer; White, Peter S.; Joris Andrieux; Evangelos Vassos; Caroline Mackie Ogilvie; Sarah Curran; Collier, David A


    Copy number variants (CNVs) at chromosome 16p13.11 have been associated with a range of neurodevelopmental disorders including autism, ADHD, intellectual disability and schizophrenia. Significant sex differences in prevalence, course and severity have been described for a number of these conditions but the biological and environmental factors underlying such sex-specific features remain unclear. We tested the burden and the possible sex-biased effect of CNVs at 16p13.11 in a sample of 10,397 ...

  15. Copy number variation analysis of the 17q21.31 region and its role in neurodegenerative diseases.

    Cervera-Carles, Laura; Pagonabarraga, Javier; Pascual-Sedano, Berta; Pastor, Pau; Campolongo, Antonia; Fortea, Juan; Blesa, Rafael; Alcolea, Daniel; Morenas-Rodríguez, Estrella; Sala, Isabel; Lleó, Alberto; Kulisevsky, Jaime; Clarimón, Jordi


    The H1 haplotype of the 17q21.31 inversion polymorphism has been consistently associated with progressive supranuclear palsy, corticobasal degeneration, and Parkinson's disease in Caucasians. Recently, large polymorphic segmental duplications resulting into complex rearrangements at this locus with a high diversity range in human populations have been revealed. We sought to explore whether the two multi-allelic copy number variants that are present in the H1 clade (with segmental duplications of 300 and 218 kilobases in length) could be responsible for the known H1-related risk of developing these neurodegenerative disorders. A total of 857 Spanish subjects including 330 patients with Parkinson's disease, 96 with progressive supranuclear palsy, 55 with corticobasal degeneration, 51 dementia with Lewy bodies, and 325 neurologically healthy controls, were genotyped for the H1/H2 haplotype. Subsequently, the two copy number variants that are characteristic of the H1 haplotype were evaluated through a high-resolution approach based on droplet digital polymerase chain reaction, in all H1 homozygous subjects. The H1 allele was significantly overrepresented in all diagnostic groups compared with controls (Parkinson's disease, P = 0.0001; progressive supranuclear palsy, P = 1.22 × 10(-6) ; corticobasal degeneration, P = 0.0002; and dementia with Lewy bodies, P = 0.032). However, no dosage differences were found for any of the two copy number variants analyzed. The H1 haplotype is associated with the risk of several neurodegenerative disorders, including dementia with Lewy bodies. However, common structural diversity within the 17q21.31-H1 clade does not explain this genetic association. © 2015 Wiley Periodicals, Inc. PMID:26453547

  16. Advances in the Research of Copy Number Variation%拷贝数目变异研究进展

    钱源; 褚嘉祐


    DNA copy number variation has been associated with variable susceptibility to complex diseases and genomic disorders,but its ubiquity in human genomes was not fully realized until recently with the progress of Hapmap.Many CNVs are observed in the corresponding regions in both chimpanzees and humans with high frequency.It seems likely that at least in humans,copy number variants account for a substantial amount of genetic variation.This review disusses the recent advances in the research of CNVs in normal individual and the relationship with gene disorder,potential mechanisms of CNVs formation and evolution.%人类基因组中的DNA拷贝数目变异(copy number variation,CNVs)一直以来都被认为分布频率较低,并与疾病的发生以及不同个体对于疾病的易感性相关.随着Hapmap研究计划的顺利进行,研究者逐渐发现CNVs广泛分布于人类基因组中.黑猩猩和实验室近交系的小鼠基因组也存在CNVs的广泛分布.目前已有多项研究证明了CNVs是人类基因组变异的主要原因,本综述将从CNVs的定义及其在健康人群的分布研究以及与疾病的相关性研究、CNVs的形成机制和CNVs的进化等方面对CNVs研究进展作较为全面的概述.

  17. Anaplastic lymphoma kinase gene copy number gain in inflammatory breast cancer (IBC: prevalence, clinicopathologic features and prognostic implication.

    Min Hwan Kim

    Full Text Available Inflammatory breast cancer (IBC is the most aggressive form of breast cancer, and its molecular pathogenesis still remains to be elucidated. This study aimed to evaluate the prevalence and implication of anaplastic lymphoma kinase (ALK copy number change in IBC patients.We retrospectively collected formalin-fixed, paraffin-embedded tumor tissues and medical records of IBC patients from several institutes in Korea. ALK gene copy number change and rearrangement were assessed by fluorescence in situ hybridization (FISH assay, and ALK expression status was evaluated by immunohistochemical (IHC staining.Thirty-six IBC patients including those with HER2 (+ breast cancer (16/36, 44.4% and triple-negative breast cancer (13/36, 36.1% were enrolled in this study. ALK copy number gain (CNG was observed in 47.2% (17/36 of patients, including one patient who harbored ALK gene amplification. ALK CNG (+ patients showed significantly worse overall survival compared to ALK CNG (- patients in univariate analysis (24.9 months vs. 38.1 months, p = 0.033. Recurrence free survival (RFS after curative mastectomy was also significantly shorter in ALK CNG (+ patients than in ALK CNG (- patients (n = 22, 12.7 months vs. 43.3 months, p = 0.016. Multivariate Cox regression analysis with adjustment for HER2 and ER statuses showed significantly poorer RFS for ALK CNG (+ patients (HR 5.63, 95% CI 1.11-28.44, p = 0.037.This study shows a significant presence of ALK CNG in IBC patients, and ALK CNG was associated with significantly poorer RFS.

  18. Determination of Cytochrome P450 2D6 (CYP2D6 Gene Copy Number by Real-Time Quantitative PCR

    Laurent Bodin


    Full Text Available Gene dosage by real-time quantitative PCR has proved to be accurate for measuring gene copy number. The aim of this study was to apply this approach to the CYP2D6 gene to allow for rapid identification of poor and ultrarapid metabolizers (0, 1, or more than 2 gene copy number. Using the 2−ΔΔCt calculation method and a duplex reaction, the number of CYP2D6 gene copies was determined. Quantitative PCR was performed on 43 samples previously analyzed by Southern blotting and long PCR including 20 samples with a heterozygous deletion, 11 with normal copy number (2 copies, and 12 samples with duplicated genes. The average ratio ranged from 1.02 to 1.28, 1.85 to 2.21, and 2.55 to 3.30, respectively, for the samples with 1 copy, 2 copies, and 3 copies. This study shows that this method is sensitive enough to detect either a heterozygous gene deletion or duplication.

  19. A Sequence That Affects the Copy Number and Stability of pSW200 and ColE1 ▿

    Wu, Ying-Chung; Liu, Shih-Tung


    Pantoea stewartii SW2 contains 13 plasmids. One of these plasmids, pSW200, has a replicon that resembles that of ColE1. This study demonstrates that pSW200 contains a 9-bp UP element, 5′-AAGATCTTC, which is located immediately upstream of the −35 box in the RNAII promoter. A transcriptional fusion study reveals that substituting this 9-bp sequence reduces the activity of the RNAII promoter by 78%. The same mutation also reduced the number of plasmid copies from 13 to 5, as well as the plasmid...

  20. Kid cleaves specific mRNAs at UUACU sites to rescue the copy number of plasmid R1

    Pimentel, Belén; Madine, Mark A.; de la Cueva-Méndez, Guillermo


    Stability and copy number of extra-chromosomal elements are tightly regulated in prokaryotes and eukaryotes. Toxin Kid and antitoxin Kis are the components of the parD stability system of prokaryotic plasmid R1 and they can also function in eukaryotes. In bacteria, Kid was thought to become active only in cells that lose plasmid R1 and to cleave exclusively host mRNAs at UA(A/C/U) trinucleotide sites to eliminate plasmid-free cells. Instead, we demonstrate here that Kid becomes active in plas...

  1. Allelic distributions of CYP2D6 gene copy number variation in the Eastern Han Chinese population

    Hai-hui SHENG; Yun-lan DU; Jian SUN; Hua-sheng XIAO; Ai-ping ZENG; Wen-xiang ZHU; Ren-fang ZHU; Hong-mei LI; Zhi-dong ZHU; Ying QIN; Wei JIN; Yan LIU


    Aim: The human cytochrome P450 2D6 (CYP2D6) gene copy number variation, involving CYP2D6 gene deletion (CYP2D6*5) and duplication or multiduplication (CYP2D6*×N), can result in reduced or increased metabolism of many clinically used drugs. The identification of CYP2D6*5 and CYP2D6*×N and the investigation of their allelic distributions in ethnic populations can be important in deter-mining the right drug and dosage for each patient. Methods: The CYP2D6*5 andCYP2D6 genes, and CYP2D6 gene duplication were identified by 2 modified long PCR, respectively. To determine duplicated alleles, a novel long PCR was developed to amplify the entire duplicated CYP2D6 gene which was used as template for subsequent PCR amplification. A total of 363 unrelated Eastern Han Chinese individuals were analyzed for CYP2D6 gene copy number variation. Results: The frequency of CYP2D6*5 and CYP2D6*×N were 4.82% (n=35) and 0.69% (n=5) in the Eastern Han Chinese population, respectively. Of the 5 duplicated alleles, 3were CYP2D6*1×N and 2 were CYP2D6*10×N. One individual was a carrier of both CYP2D6*5 and CYP2D6*1×N. Taken together, the CYP2D6 gene rear-rangements were present in 10.74% of subjects. Conclusion: Allelic distributions of the CYP2D6 gene copy number variation differ among Chinese from different regions, indicating ethnic variety in Chinese. Long PCR are convenient, cost effective, specific and semiquantitative for the detection of the CYP2D6 gene copy number variation, and amplification of the entire duplicated CYP2D6 gene is necessary for the accurate identification of duplicated alleles.

  2. SNPs and real-time quantitative PCR method for constitutional allelic copy number determination, the VPREB1 marker case

    Costa Elena; Donnangelo Anita; Carminati Mario; Valaperta Rea; Rusconi Daniela; de Filippis Tiziana; Passeri Elena; Frigerio Marcello; Persani Luca; Finelli Palma; Corbetta Sabrina


    Abstract Background 22q11.2 microdeletion is responsible for the DiGeorge Syndrome, characterized by heart defects, psychiatric disorders, endocrine and immune alterations and a 1 in 4000 live birth prevalence. Real-time quantitative PCR (qPCR) approaches for allelic copy number determination have recently been investigated in 22q11.2 microdeletions detection. The qPCR method was performed for 22q11.2 microdeletions detection as a first-level screening approach in a genetically unknown series...

  3. Relationship of increased aurora kinase A gene copy number, prognosis and response to chemotherapy in patients with metastatic colorectal cancer

    Dotan, E; Meropol, N J; Zhu, F; Zambito, F; Bove, B; Cai, K Q; Godwin, A. K.; Golemis, E A; Astsaturov, I; Cohen, S. J.


    Background: Increased Aurora kinase A gene copy number (AURKA-CN) has been reported in metastatic colorectal cancer (mCRC), with unknown relationship to clinical outcome. We correlated increased AURKA-CN in mCRC tumours with KRAS mutation status, overall and progression-free survival (OS, PFS). Methods: Sixty-one mCRC tumours were analysed for AURKA-CN using q-PCR, and KRAS mutation status by direct sequencing. Expression of AURKA protein was analysed by immunohistochemistry. Cox-proportional...

  4. Copy number variations are a rare cause of non-CMT1A Charcot-Marie-Tooth disease

    Huang, Jia; Wu, Xingyao; Montenegro, Gladys; Price, Justin; Wang, GaoFeng; Vance, Jeffery M.; Shy, Michael E.; Züchner, Stephan


    Hereditary peripheral neuropathies present a group of clinically and genetically heterogeneous entities. All known forms, including the various forms of Charcot-Marie-Tooth disease (CMT) are characterized as Mendelian traits and over 35 genes have been identified thus far. The mutational mechanism of the most common CMT type, CMT1A, is a 1.5 Mb chromosomal duplication at 17p12 that contains the gene PMP22. Only recently it has been realized that such copy number variants (CNV) are a widesprea...

  5. Simultaneous quantification of mitochondrial DNA copy number and deletion ratio: a multiplex real-time PCR assay.

    Phillips, Nicole R; Sprouse, Marc L; Roby, Rhonda K


    Mitochondrial dysfunction is implicated in a vast array of diseases and conditions, such as Alzheimer's disease, cancer, and aging. Alterations in mitochondrial DNA (mtDNA) may provide insight into the processes that either initiate or propagate this dysfunction. Here, we describe a unique multiplex assay which simultaneously provides assessments of mtDNA copy number and the proportion of genomes with common large deletions by targeting two mitochondrial sites and one nuclear locus. This probe-based, single-tube multiplex provides high specificity while eliminating well-to-well variability that results from assaying nuclear and mitochondrial targets individually. PMID:24463429

  6. Topoisomerase-1 and -2A gene copy numbers are elevated in mismatch repair-proficient colorectal cancers

    Sønderstrup, Ida Marie Heeholm; Nygård, Sune Boris; Poulsen, Tim Svenstrup;


    . MATERIAL AND METHODS: Test cohort: FISH analysis with an in-house TOP1/CEN20 probe mix and a commercially available TOP2A/CEN17 (Dako, Glostrup, Denmark) probe mix was performed on archival formalin fixed paraffin embedded (FFPE) tissue samples from 18 patients with proficient MMR (pMMR) CRC and 18...... patients with deficient MMR (dMMR) CRC. TOP1 and TOP2A gene copy numbers and their ratios per nucleus were correlated with MMR status using the Mann-Whitney test. Validation cohort: FFPE samples from 154 patients with primary stage III CRC (originally included in the RANX05 study) were classified according...

  7. Insights on the Functional Impact of MicroRNAs Present in Autism-Associated Copy Number Variants

    Vaishnavi, Varadarajan; Manikandan, Mayakannan; Tiwary, Basant K.; Munirajan, Arasambattu Kannan


    Autism spectrum disorder is a complex neurodevelopmental disorder that appears during the first three years of infancy and lasts throughout a person’s life. Recently a large category of genomic structural variants, denoted as copy number variants (CNVs), were established to be a major contributor of the pathophysiology of autism. To date almost all studies have focussed only on the genes present in the CNV loci, but the impact of non-coding regulatory microRNAs (miRNAs) present in these regio...

  8. Impact of copy number of distinct SV40PolyA segments on expression of a GFP reporter gene


    The presence of Alu repeats downregulates the expression of the green fluorescent protein(GFP) gene.We found that SV40PolyA(PolyA,240 bp),in either orientation,eliminated the inhibition of GFP gene expression induced by Alu repeats when it was placed between the GFP gene and the Alu repeats.In this study,4 different segments(each 60 bp) were amplified from antisense PolyA(PolyAas) by PCR,and inserted upstream of Alu14 in pAlu14 plasmid(14 Alu repeats inserted downstream of the GFP gene in vector pEGFP-C1 in a head-tail tandem manner).Segments 1F1R(the first 60 bp segment at the 5’ end of PolyAas) and 4F4R(the fourth 60 bp segment from the 5’ end of PolyAas) did not activate GFP gene expression,whereas 2F2R and 3F3R(the middle two segments) did(as detected by Northern blot analysis and fluorescent microscopy).Different copy numbers of 2F2R and 3F3R segments,in a head and tail tandem manner,were inserted downstream of the GFP gene in pAlu14.p2F2R*4-Alu28,p3F3R*4-Alu18 and p3F3R*4-Alu28 were used as length controls to verify that the decrease in the expression of GFP was not due to the increased length of the inserted segment in the expression vectors.We found that 2 and 4 copies of 2F2R or 3F3R activated the GFP gene more strongly than one copy of them.However,more than 8 copies of 2F2R or 3F3R reduced the activation of the GFP gene.We concluded that SV40PolyAas contained at least two gene-activating elements(2F2R and 3F3R) and 2-4 copies of 2F2R or 3F3R were optimal for the expression of the GFP gene.

  9. A sequence that affects the copy number and stability of pSW200 and ColE1.

    Wu, Ying-Chung; Liu, Shih-Tung


    Pantoea stewartii SW2 contains 13 plasmids. One of these plasmids, pSW200, has a replicon that resembles that of ColE1. This study demonstrates that pSW200 contains a 9-bp UP element, 5'-AAGATCTTC, which is located immediately upstream of the -35 box in the RNAII promoter. A transcriptional fusion study reveals that substituting this 9-bp sequence reduces the activity of the RNAII promoter by 78%. The same mutation also reduced the number of plasmid copies from 13 to 5, as well as the plasmid stability. When a similar sequence in a ColE1 derivative, pYCW301, is mutated, the copy number of the plasmid also declines from 34 to 16 per cell. Additionally, inserting this 9-bp sequence stabilizes an unstable pSW100 derivative, pSW142K, which also contains a replicon resembling that of ColE1, indicating the importance of this sequence in maintaining the stability of the plasmid. In conclusion, the 9-bp sequence upstream of the -35 box in the RNAII promoter is required for the efficient synthesis of RNAII and maintenance of the stability of the plasmids in the ColE1 family. PMID:20494993

  10. Genetic mechanisms and age-related macular degeneration: common variants, rare variants, copy number variations, epigenetics, and mitochondrial genetics

    Liu Melissa M


    Full Text Available Abstract Age-related macular degeneration (AMD is a complex and multifaceted disease involving contributions from both genetic and environmental influences. Previous work exploring the genetic contributions of AMD has implicated numerous genomic regions and a variety of candidate genes as modulators of AMD susceptibility. Nevertheless, much of this work has revolved around single-nucleotide polymorphisms (SNPs, and it is apparent that a significant portion of the heritability of AMD cannot be explained through these mechanisms. In this review, we consider the role of common variants, rare variants, copy number variations, epigenetics, microRNAs, and mitochondrial genetics in AMD. Copy number variations in regulators of complement activation genes (CFHR1 and CFHR3 and glutathione S transferase genes (GSTM1 and GSTT1 have been associated with AMD, and several additional loci have been identified as regions of potential interest but require further evaluation. MicroRNA dysregulation has been linked to the retinal pigment epithelium degeneration in geographic atrophy, ocular neovascularization, and oxidative stress, all of which are hallmarks in the pathogenesis of AMD. Certain mitochondrial DNA haplogroups and SNPs in mitochondrially encoded NADH dehydrogenase genes have also been associated with AMD. The role of these additional mechanisms remains only partly understood, but the importance of their further investigation is clear to elucidate more completely the genetic basis of AMD.

  11. SLC26A4 gene copy number variations in Chinese patients with non-syndromic enlarged vestibular aqueduct

    Zhao Jiandong


    Full Text Available Abstract Background Many patients with enlarged vestibular aqueduct (EVA have either only one allelic mutant of the SLC26A4 gene or lack any detectable mutation. In this study, multiplex ligation-dependent probe amplification (MLPA was used to screen for copy number variations (CNVs of SLC26A4 and to reveal the pathogenic mechanisms of non-syndromic EVA (NSEVA. Methods Between January 2003 and March 2010, 923 Chinese patients (481 males, 442 females with NSEVA were recruited. Among these, 68 patients (7.4% were found to carry only one mutant allele of SLC26A4 and 39 patients (4.2% lacked any detectable mutation in SLC26A4; these 107 patients without double mutant alleles were assigned to the patient group. Possible copy number variations in SLC26A4 were detected by SALSA MLPA. Results Using GeneMapper, no significant difference was observed between the groups, as compared with the standard probe provided in the assay. The results of the capillary electrophoresis showed no significant difference between the patients and controls. Conclusion Our results suggest that CNVs and the exon deletion in SLC26A4 are not important factors in NSEVA. However, it would be premature to conclude that CNVs have no role in EVA. Genome-wide studies to explore CNVs within non-coding regions of the SLC26A4 gene and neighboring regions are warranted, to elucidate their roles in NSEVA etiology.

  12. Significance of genome-wide analysis of copy number alterations and UPD in myelodysplastic syndromes using combined CGH - SNP arrays.

    Ahmad, Ausaf; Iqbal, M Anwar


    Genetic information is an extremely valuable data source in characterizing the personal nature of cancer. Chromosome instability is a hallmark of most cancer cells. Chromosomal abnormalities are correlated with poor prognosis, disease classification, risk stratification, and treatment selection. Copy number alterations (CNAs) are an important molecular signature in cancer initiation, development, and progression. Recent application of whole-genome tools to characterize normal and cancer genomes provides the powerful molecular cytogenetic means to enumerate the multiple somatic, genetic and epigenetic alterations that occur in cancer. Combined array comparative genomic hybridization (aCGH) with single nucleotide polymorphism (SNP) array is a useful technique allowing detection of CNAs and loss of heterozygosity (LOH) or uni-parental disomy (UPD) together in a single experiment. It also provides allelic information on deletions, duplications, and amplifications. UPD can result in an abnormal phenotype when the chromosomes involved are imprinted. Myelodysplastic syndromes (MDS) are the most common clonal stem cell hematologic malignancy characterized by ineffective hematopoiesis, which leads to rapid progression into acute myeloid leukemia. UPD that occurs without concurrent changes in the gene copy number is a common chromosomal defect in hematologic malignancies, especially in MDS. Approximately 40-50% of MDS patients do not have karyotypic abnormalities that are detectable using classical metaphase cytogenetic techniques (MC) because of inherent limitations of MC, low resolution and the requirement of having dividing cells. In this review, we highlight advances in the clinical application of microarray technology in MDS and discuss the clinical potential of microarray. PMID:22680919

  13. Allelic and copy-number variations of FcγRs affect granulocyte function and susceptibility for autoimmune blistering diseases.

    Recke, Andreas; Vidarsson, Gestur; Ludwig, Ralf J; Freitag, Miriam; Möller, Steffen; Vonthein, Reinhard; Schellenberger, Julia; Haase, Ozan; Görg, Siegfried; Nebel, Almut; Flachsbart, Friederike; Schreiber, Stefan; Lieb, Wolfgang; Gläser, Regine; Benoit, Sandrine; Sárdy, Miklós; Eming, Rüdiger; Hertl, Michael; Zillikens, Detlef; König, Inke R; Schmidt, Enno; Ibrahim, Saleh


    Low-affinity Fcγ receptors (FcγR) bridge innate and adaptive immune responses. In many autoimmune diseases, these receptors act as key mediators of the pathogenic effects of autoantibodies. Genes encoding FcγR exhibit frequent variations in sequence and gene copy number that influence their functional properties. FcγR variations also affect the susceptibility to systemic autoimmunity, e.g. systemic lupus erythematosus and rheumatoid arthritis. This raises the question whether FcγR variations are also associated with organ-specific autoimmunity, particularly autoantibody-mediated diseases, such as subepidermal autoimmune blistering diseases (AIBD). A multitude of evidence suggests a pathogenic role of neutrophil granulocyte interaction with autoantibodies via FcγR. In a two-stage study, we analyzed whether the FcγR genotype affects neutrophil function and mRNA expression, and consequently, bullous pemphigoid (BP) disease risk. We compared this to findings in pemphigus vulgaris/foliaceus (PV/PF), two Fc-independent AIBDs. Our results indicate that both allele and copy number variation of FcγR genes affect FcγR mRNA expression and reactive oxygen species (ROS) release by granulocytes. Susceptibility of BP was associated with FcγR genotypes that led to a decreased ROS release by neutrophils, indicating an unexpected protective role for these cells. BP and PV/PF differed substantially regarding the FcγR genotype association patterns, pointing towards different disease etiologies. PMID:26032265

  14. Human GPR42 is a transcribed multisite variant that exhibits copy number polymorphism and is functional when heterologously expressed.

    Puhl, Henry L; Won, Yu-Jin; Lu, Van B; Ikeda, Stephen R


    FFAR3 (GPR41) is a G-protein coupled receptor for which short-chain fatty acids serve as endogenous ligands. The receptor is found on gut enteroendocrine L-cells, pancreatic β-cells, and sympathetic neurons, and is implicated in obesity, diabetes, allergic airway disease, and altered immune function. In primates, FFAR3 is segmentally duplicated resulting in GPR42, a gene currently classified as a suspected pseudogene. In this study, we sequenced FFAR3 and GPR42 open reading frames from 56 individuals and found an unexpectedly high frequency of polymorphisms contributing to several complex haplotypes. We also identified a frequent (18.8%) structural variation that results in GPR42 copy number polymorphism. Finally, sequencing revealed that 50.6% of GPR42 haplotypes differed from FFAR3 by only a single non-synonymous substitution and that the GPR42 reference sequence matched only 4.4% of the alleles. Sequencing of cDNA from human sympathetic ganglia and colon revealed processed transcripts matching the GPR42 genotype. Expression of several GPR42 haplotypes in rat sympathetic neurons revealed diverse pharmacological phenotypes that differed in potency and efficacy. Our data suggest that GPR42 be reclassified as a functioning gene and that recognition of sequence and copy number polymorphism of the FFAR3/GPR42 complex be considered during genetic and pharmacological investigation of these receptors. PMID:26260360

  15. Copy number variation of a gene cluster encoding endopolygalacturonase mediates flesh texture and stone adhesion in peach.

    Gu, Chao; Wang, Lu; Wang, Wei; Zhou, Hui; Ma, Baiquan; Zheng, Hongyu; Fang, Ting; Ogutu, Collins; Vimolmangkang, Sornkanok; Han, Yuepeng


    Texture is an important attribute affecting consumer perception of fruit quality. Peach melting flesh and flesh adhesion to stone (endocarp) are simply inherited and controlled by the F-M locus on linkage group (LG) 4. Here, we report that two genes encoding endopolygalacturonase (endoPG) in the F-M locus, designated PpendoPGF and PpendoPGM, are associated with the melting flesh and stone adhesion traits. PpendoPGM controls melting flesh while PpendoPGF has pleiotropic effects on both melting flesh and stone adhesion. The F-M locus has three allelic copy number variants of endoPG, H1 (PpendoPGF and PpendoPGM), H2 (PpendoPGM), and H3 (null). The H2 haplotype represents the ancestral one while the H1 and H3 haplotypes are two variants due to duplication and deletion of PpendoPGM, respectively. Accessions with H1H1, H1H2, or H1H3 genotypes show the freestone or semi-freestone and melting flesh phenotype, while both H2H2 and H2H3 accessions have the clingstone and melting flesh phenotype. The H3H3 accessions have the clingstone and non-melting flesh phenotype. Our study not only demonstrates a driving role of gene copy number variations in flesh texture diversification in fruit trees, but also provides a useful diagnostic tool for early seedling selection in peach breeding programmes. PMID:26850878

  16. Association of activating KIR copy number variation of NK cells with containment of SIV replication in rhesus monkeys.

    Ina Hellmann


    Full Text Available While the contribution of CD8⁺ cytotoxic T lymphocytes to early containment of HIV-1 spread is well established, a role for NK cells in controlling HIV-1 replication during primary infection has been uncertain. The highly polymorphic family of KIR molecules expressed on NK cells can inhibit or activate these effector cells and might therefore modulate their activity against HIV-1-infected cells. In the present study, we investigated copy number variation in KIR3DH loci encoding the only activating KIR receptor family in rhesus monkeys and its effect on simian immunodeficiency virus (SIV replication during primary infection in rhesus monkeys. We observed an association between copy numbers of KIR3DH genes and control of SIV replication in Mamu-A*01⁻ rhesus monkeys that express restrictive TRIM5 alleles. These findings provide further evidence for an association between NK cells and the early containment of SIV replication, and underscore the potential importance of activating KIRs in stimulating NK cell responses to control SIV spread.

  17. Genetic changes during laboratory propagation: copy number At the reticulocyte-binding protein 1 locus of Plasmodium falciparum.

    Nair, Shalini; Nkhoma, Standwell; Nosten, François; Mayxay, Mayfong; French, Neil; Whitworth, Jim; Anderson, Tim


    Comparative genomic hybridization studies have revealed elevated copy number (CN) at the reticulocyte-binding protein 1 gene (PfRh1) in fast growing lab-adapted parasites, while genetic manipulation demonstrates a causal link between cell invasion and PfRh1 CN. We therefore examined PfRh1 copy number variation (CNV) in 202 single clone parasite isolates from four countries to quantify the extent of CNV within natural populations. Surprisingly, we found that no natural parasite infections showed elevated CN. In contrast, 4/28 independent laboratory reference strains show elevated CN. One possibility is that amplification of PfRh1 (or neighboring loci) is selected during laboratory culture. In the case of FCR3 group of parasites, clone trees show that PfRh1 amplification arose in laboratory lines following establishment in culture. These data show that CNV at PfRh1 is rare or non-existent in natural populations, but can arise during laboratory propagation. We conclude that PfRh1 CNV is not an important determinant of gene expression, cell invasion or growth rate in natural parasite populations. PMID:20363264

  18. Characterization of a Large, Stable, High-Copy-Number Streptomyces Plasmid That Requires Stability and Transfer Functions for Heterologous Polyketide Overproduction▿

    Fong, Ryan; Hu, Zhihao; Hutchinson, C. Richard; Huang, Jianqiang; Cohen, Stanley; Kao, Camilla


    A major limitation to improving small-molecule pharmaceutical production in streptomycetes is the inability of high-copy-number plasmids to tolerate large biosynthetic gene cluster inserts. A recent finding has overcome this barrier. In 2003, Hu et al. discovered a stable, high-copy-number, 81-kb plasmid that significantly elevated production of the polyketide precursor to the antibiotic erythromycin in a heterologous Streptomyces host (J. Ind. Microbiol. Biotechnol. 30:516-522, 2003). Here, ...

  19. Copy Number Amplification of the PIK3CA Gene Is Associated with Poor Prognosis in Non-lymph node metastatic Head and Neck Squamous Cell Carcinoma

    Deregulation of the EGFR signaling pathway is one of the most frequently observed genetic abnormalities that drives cancer development. Although mutations in the downstream components of the EGFR signaling pathway, including KRAS, BRAF and PIK3CA, have been reported in numerous cancers, extensive mutation and copy number analysis of these genes in clinical samples has not been performed for head and neck squamous cell carcinoma (HNSCC). We examined the mutations and copy number alterations of KRAS, BRAF and PIK3CA in 115 clinical specimens of HNSCC obtained from surgically treated patients. We used DNA sequencing to detect mutations and the copy number changes were evaluated by qPCR and array comparative genomic hybridization (CGH) analysis. We examined the mutations and copy number alterations of KRAS, BRAF and PIK3CA in 115 clinical specimens of HNSCC obtained from surgically treated patients. We identified 3 mutations (2.6%) in K-RAS and 3 mutations (2.6%) in PIK3CA. Copy number amplification was found in 37 cases (32.2%) for PIK3CA, 10 cases (8.7%) for K-RAS and 2 cases (1.7%) for BRAF. Kaplan-Meier survival analysis revealed that copy-number amplification of PIK3CA was markedly associated with cancer relapse in patients without lymph node metastasis. (Log-rank test, p = 0.026) Copy number amplification of the PIK3CA gene is associated with poor prognosis in HNSCC patients without lymph node metastasis. The PIK3CA copy number status will serve as a marker of poor prognosis in patients with HNSCC

  20. Genomic Pathology of SLE-Associated Copy-Number Variation at the FCGR2C/FCGR3B/FCGR2B Locus

    Mueller, Michael; Barros, Paula; Witherden, Abigail S.; Roberts, Amy L.; Zhang, Zhou; Schaschl, Helmut; Yu, Chack-Yung; Hurles, Matthew E.; Schaffner, Catherine; Floto, R. Andres; Game, Laurence; Steinberg, Karyn Meltz; Wilson, Richard K.; Graves, Tina A.; Eichler, Evan E.


    Reduced FCGR3B copy number is associated with increased risk of systemic lupus erythematosus (SLE). The five FCGR2/FCGR3 genes are arranged across two highly paralogous genomic segments on chromosome 1q23. Previous studies have suggested mechanisms for structural rearrangements at the FCGR2/FCGR3 locus and have proposed mechanisms whereby altered FCGR3B copy number predisposes to autoimmunity, but the high degree of sequence similarity between paralogous segments has prevented precise definit...

  1. Accuracy and differential bias in copy number measurement of CCL3L1 in association studies with three auto-immune disorders

    Carpenter Danielle


    Full Text Available Abstract Background Copy number variation (CNV contributes to the variation observed between individuals and can influence human disease progression, but the accurate measurement of individual copy numbers is technically challenging. In the work presented here we describe a modification to a previously described paralogue ratio test (PRT method for genotyping the CCL3L1/CCL4L1 copy variable region, which we use to ascertain CCL3L1/CCL4L1 copy number in 1581 European samples. As the products of CCL3L1 and CCL4L1 potentially play a role in autoimmunity we performed case control association studies with Crohn's disease, rheumatoid arthritis and psoriasis clinical cohorts. Results We evaluate the PRT methodology used, paying particular attention to accuracy and precision, and highlight the problems of differential bias in copy number measurements. Our PRT methods for measuring copy number were of sufficient precision to detect very slight but systematic differential bias between results from case and control DNA samples in one study. We find no evidence for an association between CCL3L1 copy number and Crohn's disease, rheumatoid arthritis or psoriasis. Conclusions Differential bias of this small magnitude, but applied systematically across large numbers of samples, would create a serious risk of false positive associations in copy number, if measured using methods of lower precision, or methods relying on single uncorroborated measurements. In this study the small differential bias detected by PRT in one sample set was resolved by a simple pre-treatment by restriction enzyme digestion.

  2. Association between the SMN2 gene copy number and clinical characteristics of patients with spinal muscular atrophy with homozygous deletion of exon 7 of the SMN1 gene

    Žarkov Marija


    Full Text Available Background/Aim. Spinal muscular atrophy (SMA is an autosomal recessive disease characterized by degeneration of alpha motor neurons in the spinal cord and the medulla oblongata, causing progressive muscle weakness and atrophy. The aim of this study was to determine association between the SMN2 gene copy number and disease phenotype in Serbian patients with SMA with homozygous deletion of exon 7 of the SMN1 gene. Methods. The patients were identified using regional Serbian hospital databases. Investigated clinical characteristics of the disease were: patients’ gender, age at disease onset, achieved and current developmental milestones, disease duration, current age, and the presence of the spinal deformities and joint contractures. The number of SMN1 and SMN2 gene copies was determined using real-time polymerase chain reaction (PCR. Results. Among 43 identified patients, 37 (86.0% showed homozygous deletion of SMN1 exon 7. One (2.7% of 37 patients had SMA type I with 3 SMN2 copies, 11 (29.7% patients had SMA type II with 3.1 ± 0.7 copies, 17 (45.9% patients had SMA type III with 3.7 ± 0.9 copies, while 8 (21.6% patients had SMA type IV with 4.2 ± 0.9 copies. There was a progressive increase in the SMN2 gene copy number from type II towards type IV (p < 0.05. A higher SMN2 gene copy number was associated with better current motor performance (p < 0.05. Conclusion. In the Serbian patients with SMA, a higher SMN2 gene copy number correlated with less severe disease phenotype. A possible effect of other phenotype modifiers should not be neglected.

  3. Characterization of a large, stable, high-copy-number Streptomyces plasmid that requires stability and transfer functions for heterologous polyketide overproduction.

    Fong, Ryan; Vroom, Jonathan A; Hu, Zhihao; Hutchinson, C Richard; Huang, Jianqiang; Cohen, Stanley N; Cohen, Stanley; Kao, Camilla M; Kao, Camilla


    A major limitation to improving small-molecule pharmaceutical production in streptomycetes is the inability of high-copy-number plasmids to tolerate large biosynthetic gene cluster inserts. A recent finding has overcome this barrier. In 2003, Hu et al. discovered a stable, high-copy-number, 81-kb plasmid that significantly elevated production of the polyketide precursor to the antibiotic erythromycin in a heterologous Streptomyces host (J. Ind. Microbiol. Biotechnol. 30:516-522, 2003). Here, we have identified mechanisms by which this SCP2*-derived plasmid achieves increased levels of metabolite production and examined how the 45-bp deletion mutation in the plasmid replication origin increased plasmid copy number. A plasmid intramycelial transfer gene, spd, and a partition gene, parAB, enhance metabolite production by increasing the stable inheritance of large plasmids containing biosynthetic genes. Additionally, high product titers required both activator (actII-ORF4) and biosynthetic genes (eryA) at high copy numbers. DNA gel shift experiments revealed that the 45-bp deletion abolished replication protein (RepI) binding to a plasmid site which, in part, supports an iteron model for plasmid replication and copy number control. Using the new information, we constructed a large high-copy-number plasmid capable of overproducing the polyketide 6-deoxyerythronolide B. However, this plasmid was unstable over multiple culture generations, suggesting that other SCP2* genes may be required for long-term, stable plasmid inheritance. PMID:17142363

  4. Copy number variation of CCL3-like genes affects rate of progression to simian-AIDS in Rhesus Macaques (Macaca mulatta.

    Jeremiah D Degenhardt


    Full Text Available Variation in genes underlying host immunity can lead to marked differences in susceptibility to HIV infection among humans. Despite heavy reliance on non-human primates as models for HIV/AIDS, little is known about which host factors are shared and which are unique to a given primate lineage. Here, we investigate whether copy number variation (CNV at CCL3-like genes (CCL3L, a key genetic host factor for HIV/AIDS susceptibility and cell-mediated immune response in humans, is also a determinant of time until onset of simian-AIDS in rhesus macaques. Using a retrospective study of 57 rhesus macaques experimentally infected with SIVmac, we find that CCL3L CNV explains approximately 18% of the variance in time to simian-AIDS (p<0.001 with lower CCL3L copy number associating with more rapid disease course. We also find that CCL3L copy number varies significantly (p<10(-6 among rhesus subpopulations, with Indian-origin macaques having, on average, half as many CCL3L gene copies as Chinese-origin macaques. Lastly, we confirm that CCL3L shows variable copy number in humans and chimpanzees and report on CCL3L CNV within and among three additional primate species. On the basis of our findings we suggest that (1 the difference in population level copy number may explain previously reported observations of longer post-infection survivorship of Chinese-origin rhesus macaques, (2 stratification by CCL3L copy number in rhesus SIV vaccine trials will increase power and reduce noise due to non-vaccine-related differences in survival, and (3 CCL3L CNV is an ancestral component of the primate immune response and, therefore, copy number variation has not been driven by HIV or SIV per se.

  5. Rubinstein Taybi Syndrome: A Case Report 46 XY del 16 q2 3

    Narlı, Nejat; Atıcı, Aytuğ; Satar, Mehmet; Süleymanova, Dilara


    Rubinstein Taybi syndrome is a condition characterized by typical facial appearance broad thumbs broad great toes and motormental retardation So far 600 cases have been reported in the literature The most common chromosomal abnormality was identified as microdeletion of 16p13 3 in this syndrome In this paper a case of Rubinstein Taybi Syndrome with multiple cardiac abnormalities and unusual chromosomal abnormality [46 XY del 16 q2 3 ] is presented Key words: Rubinstein Taybi syndrome

  6. Obesity, starch digestion and amylase: association between copy number variants at human salivary (AMY1) and pancreatic (AMY2) amylase genes.

    Carpenter, Danielle; Dhar, Sugandha; Mitchell, Laura M; Fu, Beiyuan; Tyson, Jess; Shwan, Nzar A A; Yang, Fengtang; Thomas, Mark G; Armour, John A L


    The human salivary amylase genes display extensive copy number variation (CNV), and recent work has implicated this variation in adaptation to starch-rich diets, and in association with body mass index. In this work, we use paralogue ratio tests, microsatellite analysis, read depth and fibre-FISH to demonstrate that human amylase CNV is not a smooth continuum, but is instead partitioned into distinct haplotype classes. There is a fundamental structural distinction between haplotypes containing odd or even numbers of AMY1 gene units, in turn coupled to CNV in pancreatic amylase genes AMY2A and AMY2B. Most haplotypes have one copy each of AMY2A and AMY2B and contain an odd number of copies of AMY1; consequently, most individuals have an even total number of AMY1. In contrast, haplotypes carrying an even number of AMY1 genes have rearrangements leading to CNVs of AMY2A/AMY2B. Read-depth and experimental data show that different populations harbour different proportions of these basic haplotype classes. In Europeans, the copy numbers of AMY1 and AMY2A are correlated, so that phenotypic associations caused by variation in pancreatic amylase copy number could be detected indirectly as weak association with AMY1 copy number. We show that the quantitative polymerase chain reaction (qPCR) assay previously applied to the high-throughput measurement of AMY1 copy number is less accurate than the measures we use and that qPCR data in other studies have been further compromised by systematic miscalibration. Our results uncover new patterns in human amylase variation and imply a potential role for AMY2 CNV in functional associations. PMID:25788522

  7. The gut fungus Basidiobolus ranarum has a large genome and different copy numbers of putatively functionally redundant elongation factor genes.

    Daniel A Henk

    Full Text Available Fungal genomes range in size from 2.3 Mb for the microsporidian Encephalitozoon intestinalis up to 8000 Mb for Entomophaga aulicae, with a mean genome size of 37 Mb. Basidiobolus, a common inhabitant of vertebrate guts, is distantly related to all other fungi, and is unique in possessing both EF-1α and EFL genes. Using DNA sequencing and a quantitative PCR approach, we estimated a haploid genome size for Basidiobolus at 350 Mb. However, based on allelic variation, the nuclear genome is at least diploid, leading us to believe that the final genome size is at least 700 Mb. We also found that EFL was in three times the copy number of its putatively functionally overlapping paralog EF-1α. This suggests that gene or genome duplication may be an important feature of B. ranarum evolution, and also suggests that B. ranarum may have mechanisms in place that favor the preservation of functionally overlapping genes.

  8. Genome-wide copy number analysis of cerebrospinal fluid tumor cells and their corresponding archival primary tumors

    Mark Jesus M. Magbanua


    Full Text Available A debilitating complication of breast cancer is the metastatic spread of tumor cells to the leptomeninges or cerebrospinal fluid (CSF. Patients diagnosed with this aggressive clinical syndrome, known as leptomeningeal carcinomatosis, have very poor prognosis. Despite improvements in detecting cerebrospinal fluid tumor cells (CSFTCs, information regarding their molecular biology is extremely limited. In our recent work, we utilized a protocol previously used for circulating tumor cell isolation to purify tumor cells from the CSF. We then performed genomic characterization of CSFTCs as well as archival tumors from the same patient. Here, we describe the microarray data and quality controls associated with our study published in the Cancer Research journal in 2013 [1]. We also provide an R script containing code for quality control of microarray data and assessment of copy number calls. The microarray data has been deposited into Gene Expression Omnibus under accession # GSE46068.

  9. Computational methods for detecting copy number variations in cancer genome using next generation sequencing: principles and challenges

    Liu, Biao; Morrison, Carl D.; Johnson, Candace S.; Trump, Donald L.; Qin, Maochun; Conroy, Jeffrey C.; Wang, Jianmin; Liu, Song


    Accurate detection of somatic copy number variations (CNVs) is an essential part of cancer genome analysis, and plays an important role in oncotarget identifications. Next generation sequencing (NGS) holds the promise to revolutionize somatic CNV detection. In this review, we provide an overview of current analytic tools used for CNV detection in NGS-based cancer studies. We summarize the NGS data types used for CNV detection, decipher the principles for data preprocessing, segmentation, and interpretation, and discuss the challenges in somatic CNV detection. This review aims to provide a guide to the analytic tools used in NGS-based cancer CNV studies, and to discuss the important factors that researchers need to consider when analyzing NGS data for somatic CNV detections. PMID:24240121

  10. Insights on the Functional Interactions between miRNAs and Copy Number Variations in the Aging Brain

    Ronald Bontrop


    Full Text Available MicroRNAs (miRNAs are regulatory genetic elements that coordinate the expression of thousands of genes and play important roles in brain aging and neurodegeneration. DNA polymorphisms affecting miRNA biogenesis, dosage and gene targeting may represent potentially functional variants. The consequences of single nucleotide polymorphisms (SNPs affecting miRNA function were previously demonstrated by both experimental and computational methods. However, little is known about how copy number variations (CNVs influence miRNA metabolism and regulatory networks. We discuss potential mechanisms of CNVs-mediated effects on miRNA function and regulation that might have consequences for brain aging. We argue that CNVs, which potentially can alter miRNA expression, regulation or target gene recognition, are possible functional variants and should be considered high priority candidates in genotype-phenotype mapping studies of brain-related disorders.

  11. Variation of PRLR gene copy number in duck%鸭PRLR基因拷贝数的变异

    叶炎; 汪稳; 张金耀; 黄守婷; 钟志新; 周世业; 肖天放


    Copy number variation ( CNV) merges to be an effective tool in poultry breeding. To elucidate the correlation between productive performance and polymorphism, gene of Brown Tsaiya ducks were amplified by fluorescence quantitative PCR, and fol-lowed by being transferred into CNV via 2-ΔΔCt method. Subsequent production traits responses to different CNV of PRLR gene were investigated. Results showed that correlation coefficients of standard curve for PRLR and Ldh-B were 0.991 and 0.990, with slopes of standard curves being-3.070 and-3.135, respectively. Amplification efficiency of PRLR and Ldh-B target gene were 111.68% and 108.429%, which were approximately at the same level with reference gene. Traits of Brown Tsaiya ducks were correlated with CNV ( P0.05).因此,PRLR基因拷贝变异区域可能影响蛋壳厚度和蛋形指数.

  12. Multiplex Ligation-Dependent Probe Amplification Technique for Copy Number Analysis on Small Amounts of DNA Material

    Sørensen, Karina; Andersen, Paal; Larsen, Lars; Schwartz, Marianne; Schouten, Jan; Nygren, Anders


    The multiplex ligation-dependent probe amplification (MLPA) technique is a sensitive technique for relative quantification of up to 50 different nucleic acid sequences in a single reaction, and the technique is routinely used for copy number analysis in various syndromes and diseases. The aim of...... the study was to exploit the potential of MLPA when the DNA material is limited. The DNA concentration required in standard MLPA analysis is not attainable from dried blood spot samples (DBSS) often used in neonatal screening programs. A novel design of MLPA probes has been developed to permit for...... MLPA analysis on small amounts of DNA. Six patients with congenital adrenal hyperplasia (CAH) were used in this study. DNA was extracted from both whole blood and DBSS and subjected to MLPA analysis using normal and modified probes. Results were analyzed using GeneMarker and manual Excel analysis. A...

  13. Identification of rare high-risk copy number variants affecting the dopamine transporter gene in mental disorders

    Hoeffding, Louise K; Duong, Linh T T; Ingason, Andrés;


    rare high-risk variants of psychiatric disorders. METHODS: We performed a systematic screening for CNVs affecting SLC6A3 in 761 healthy controls, 672 schizophrenia patients, and 194 patients with bipolar disorder in addition to 253 family members from six large pedigrees affected by mental disorders...... affective disorders. Recently, copy number variants (CNVs) in SLC6A3 have been identified in healthy subjects but so far, the implication of CNVs affecting this gene in psychiatric diseases has not been addressed. AIMS: In the present study, we aimed to investigate whether CNVs affecting SLC6A3 represent...... sizes and two affected several genes in addition to SLC6A3. CONCLUSION: Our findings suggest that rare high-risk CNVs affecting the gene encoding the dopamine transporter contribute to the pathogenesis of schizophrenia and affective disorders....

  14. Individual differences in AMY1 gene copy number, salivary α-amylase levels, and the perception of oral starch.

    Abigail L Mandel

    Full Text Available BACKGROUND: The digestion of dietary starch in humans is initiated by salivary α-amylase, an endo-enzyme that hydrolyzes starch into maltose, maltotriose and larger oligosaccharides. Salivary amylase accounts for 40 to 50% of protein in human saliva and rapidly alters the physical properties of starch. Importantly, the quantity and enzymatic activity of salivary amylase show significant individual variation. However, linking variation in salivary amylase levels with the oral perception of starch has proven difficult. Furthermore, the relationship between copy number variations (CNVs in the AMY1 gene, which influence salivary amylase levels, and starch viscosity perception has not been explored. PRINCIPAL FINDINGS: Here we demonstrate that saliva containing high levels of amylase has sufficient activity to rapidly hydrolyze a viscous starch solution in vitro. Furthermore, we show with time-intensity ratings, which track the digestion of starch during oral manipulation, that individuals with high amylase levels report faster and more significant decreases in perceived starch viscosity than people with low salivary amylase levels. Finally, we demonstrate that AMY1 CNVs predict an individual's amount and activity of salivary amylase and thereby, ultimately determine their perceived rate of oral starch viscosity thinning. CONCLUSIONS: By linking genetic variation and its consequent salivary enzymatic differences to the perceptual sequellae of these variations, we show that AMY1 copy number relates to salivary amylase concentration and enzymatic activity level, which, in turn, account for individual variation in the oral perception of starch viscosity. The profound individual differences in salivary amylase levels and salivary activity may contribute significantly to individual differences in dietary starch intake and, consequently, to overall nutritional status.

  15. Copy number variation analysis implicates the cell polarity gene glypican 5 as a human spina bifida candidate gene.

    Bassuk, Alexander G; Muthuswamy, Lakshmi B; Boland, Riley; Smith, Tiffany L; Hulstrand, Alissa M; Northrup, Hope; Hakeman, Matthew; Dierdorff, Jason M; Yung, Christina K; Long, Abby; Brouillette, Rachel B; Au, Kit Sing; Gurnett, Christina; Houston, Douglas W; Cornell, Robert A; Manak, J Robert


    Neural tube defects (NTDs) are common birth defects of complex etiology. Family and population-based studies have confirmed a genetic component to NTDs. However, despite more than three decades of research, the genes involved in human NTDs remain largely unknown. We tested the hypothesis that rare copy number variants (CNVs), especially de novo germline CNVs, are a significant risk factor for NTDs. We used array-based comparative genomic hybridization (aCGH) to identify rare CNVs in 128 Caucasian and 61 Hispanic patients with non-syndromic lumbar-sacral myelomeningocele. We also performed aCGH analysis on the parents of affected individuals with rare CNVs where parental DNA was available (42 sets). Among the eight de novo CNVs that we identified, three generated copy number changes of entire genes. One large heterozygous deletion removed 27 genes, including PAX3, a known spina bifida-associated gene. A second CNV altered genes (PGPD8, ZC3H6) for which little is known regarding function or expression. A third heterozygous deletion removed GPC5 and part of GPC6, genes encoding glypicans. Glypicans are proteoglycans that modulate the activity of morphogens such as Sonic Hedgehog (SHH) and bone morphogenetic proteins (BMPs), both of which have been implicated in NTDs. Additionally, glypicans function in the planar cell polarity (PCP) pathway, and several PCP genes have been associated with NTDs. Here, we show that GPC5 orthologs are expressed in the neural tube, and that inhibiting their expression in frog and fish embryos results in NTDs. These results implicate GPC5 as a gene required for normal neural tube development. PMID:23223018

  16. High-throughput sequencing and copy number variation detection using formalin fixed embedded tissue in metastatic gastric cancer.

    Seokhwi Kim

    Full Text Available In the era of targeted therapy, mutation profiling of cancer is a crucial aspect of making therapeutic decisions. To characterize cancer at a molecular level, the use of formalin-fixed paraffin-embedded tissue is important. We tested the Ion AmpliSeq Cancer Hotspot Panel v2 and nCounter Copy Number Variation Assay in 89 formalin-fixed paraffin-embedded gastric cancer samples to determine whether they are applicable in archival clinical samples for personalized targeted therapies. We validated the results with Sanger sequencing, real-time quantitative PCR, fluorescence in situ hybridization and immunohistochemistry. Frequently detected somatic mutations included TP53 (28.17%, APC (10.1%, PIK3CA (5.6%, KRAS (4.5%, SMO (3.4%, STK11 (3.4%, CDKN2A (3.4% and SMAD4 (3.4%. Amplifications of HER2, CCNE1, MYC, KRAS and EGFR genes were observed in 8 (8.9%, 4 (4.5%, 2 (2.2%, 1 (1.1% and 1 (1.1% cases, respectively. In the cases with amplification, fluorescence in situ hybridization for HER2 verified gene amplification and immunohistochemistry for HER2, EGFR and CCNE1 verified the overexpression of proteins in tumor cells. In conclusion, we successfully performed semiconductor-based sequencing and nCounter copy number variation analyses in formalin-fixed paraffin-embedded gastric cancer samples. High-throughput screening in archival clinical samples enables faster, more accurate and cost-effective detection of hotspot mutations or amplification in genes.

  17. Variation in chromosome copy number influences the virulence of Cryptococcus neoformans and occurs in isolates from AIDS patients

    Hu Guanggan


    Full Text Available Abstract Background The adaptation of pathogenic fungi to the host environment via large-scale genomic changes is a poorly characterized phenomenon. Cryptococcus neoformans is the leading cause of fungal meningoencephalitis in HIV/AIDS patients, and we recently discovered clinical strains of the fungus that are disomic for chromosome 13. Here, we examined the genome plasticity and phenotypes of monosomic and disomic strains, and compared their virulence in a mouse model of cryptococcosis Results In an initial set of strains, melanin production was correlated with monosomy at chromosome 13, and disomic variants were less melanized and attenuated for virulence in mice. After growth in culture or passage through mice, subsequent strains were identified that varied in melanin formation and exhibited copy number changes for other chromosomes. The correlation between melanin and disomy at chromosome 13 was observed for some but not all strains. A survey of environmental and clinical isolates maintained in culture revealed few occurrences of disomic chromosomes. However, an examination of isolates that were freshly collected from the cerebrospinal fluid of AIDS patients and minimally cultured provided evidence for infections with multiple strains and copy number variation. Conclusions Overall, these results suggest that the genome of C. neoformans exhibits a greater degree of plasticity than previously appreciated. Furthermore, the expression of an essential virulence factor and the severity of disease are associated with genome variation. The occurrence of chromosomal variation in isolates from AIDS patients, combined with the observed influence of disomy on virulence, indicates that genome plasticity may have clinical relevance.

  18. Genome-wide association study identifies a maternal copy-number deletion in PSG11 enriched among preeclampsia patients

    Zhao Linlu


    Full Text Available Abstract Background Specific genetic contributions for preeclampsia (PE are currently unknown. This genome-wide association study (GWAS aims to identify maternal single nucleotide polymorphisms (SNPs and copy-number variants (CNVs involved in the etiology of PE. Methods A genome-wide scan was performed on 177 PE cases (diagnosed according to National Heart, Lung and Blood Institute guidelines and 116 normotensive controls. White female study subjects from Iowa were genotyped on Affymetrix SNP 6.0 microarrays. CNV calls made using a combination of four detection algorithms (Birdseye, Canary, PennCNV, and QuantiSNP were merged using CNVision and screened with stringent prioritization criteria. Due to limited DNA quantities and the deleterious nature of copy-number deletions, it was decided a priori that only deletions would be selected for assay on the entire case-control dataset using quantitative real-time PCR. Results The top four SNP candidates had an allelic or genotypic p-value between 10-5 and 10-6, however, none surpassed the Bonferroni-corrected significance threshold. Three recurrent rare deletions meeting prioritization criteria detected in multiple cases were selected for targeted genotyping. A locus of particular interest was found showing an enrichment of case deletions in 19q13.31 (5/169 cases and 1/114 controls, which encompasses the PSG11 gene contiguous to a highly plastic genomic region. All algorithm calls for these regions were assay confirmed. Conclusions CNVs may confer risk for PE and represent interesting regions that warrant further investigation. Top SNP candidates identified from the GWAS, although not genome-wide significant, may be useful to inform future studies in PE genetics.

  19. Comparative analyses of gene copy number and mRNA expression in GBM tumors and GBM xenografts

    Hodgson, J. Graeme; Yeh, Ru-Fang; Ray, Amrita; Wang, Nicholas J.; Smirnov, Ivan; Yu, Mamie; Hariono, Sujatmi; Silber, Joachim; Feiler, Heidi S.; Gray, Joe W.; Spellman, Paul T.; Vandenberg, Scott R.; Berger, Mitchel S.; James, C. David


    Development of model systems that recapitulate the molecular heterogeneity observed among glioblastoma multiforme (GBM) tumors will expedite the testing of targeted molecular therapeutic strategies for GBM treatment. In this study, we profiled DNA copy number and mRNA expression in 21 independent GBM tumor lines maintained as subcutaneous xenografts (GBMX), and compared GBMX molecular signatures to those observed in GBM clinical specimens derived from the Cancer Genome Atlas (TCGA). The predominant copy number signature in both tumor groups was defined by chromosome-7 gain/chromosome-10 loss, a poor-prognosis genetic signature. We also observed, at frequencies similar to that detected in TCGA GBM tumors, genomic amplification and overexpression of known GBM oncogenes, such as EGFR, MDM2, CDK6, and MYCN, and novel genes, including NUP107, SLC35E3, MMP1, MMP13, and DDX1. The transcriptional signature of GBMX tumors, which was stable over multiple subcutaneous passages, was defined by overexpression of genes involved in M phase, DNA replication, and chromosome organization (MRC) and was highly similar to the poor-prognosis mitosis and cell-cycle module (MCM) in GBM. Assessment of gene expression in TCGA-derived GBMs revealed overexpression of MRC cancer genes AURKB, BIRC5, CCNB1, CCNB2, CDC2, CDK2, and FOXM1, which form a transcriptional network important for G2/M progression and/or checkpoint activation. Our study supports propagation of GBM tumors as subcutaneous xenografts as a useful approach for sustaining key molecular characteristics of patient tumors, and highlights therapeutic opportunities conferred by this GBMX tumor panel for testing targeted therapeutic strategies for GBM treatment.

  20. Integrated analysis of copy number and loss of heterozygosity in primary breast carcinomas using high-density SNP array.

    Ching, Ho Ching; Naidu, Rakesh; Seong, Mun Kein; Har, Yip Cheng; Taib, Nur Aishah Mohd


    Breast cancer is a heterogeneous disease, marked by extensive chromosomal aberrations. In this study, we aimed to explicate the underlying chromosomal copy number (CN) alterations and loss of heterozygosity (LOH) implicated in a cohort of Malaysian hospital-based primary breast carcinoma samples using a single nucleotide polymorphism (SNP) array platform. The analysis was conducted by hybridizing the extracted DNA of 70 primary breast carcinomas and 37 normal peripheral blood samples to the Affymetrix 250K Sty SNP arrays. Locus-specific CN aberrations and LOH were statistically summarized using the binary segmentation algorithm and hidden Markov model. Selected genes from the SNP array analysis were also validated using quantitative real-time PCR. The merging of CN and LOH data fabricated distinctive integrated alteration profiles, which were comprised of finely demarcated minimal sites of aberrations. The most prevalent gains (≥ 30%) were detected at the 8q arm: 8q23.1, 8q23.3, 8q24.11, 8q24.13, 8q24.21, 8q24.22, 8q24.23 and 8q24.3, whilst the most ubiquitous losses (≥ 20%) were noted at the 8p12, 8p21.1, 8p21.2, 8p21.1-p21.2, 8p21.3, 8p22, 8p23.1, 8p23.1‑p23.2, 8p23.3, 17p11.2, 17p12, 17p11.2-p12, 17p13.1 and 17p13.2 regions. Copy-neutral LOH was characterized as the most prevailing LOH event, in which the most frequent distributions (≥ 30%) were revealed at 3p21.31, 5q33.2, 12q24.12, 12q24.12‑q24.13 and 14q23.1. These findings offer compre-hensive genome-wide views on breast cancer genomic changes, where the most recurrent gain, loss and copy-neutral LOH events were harboured within the 8q24.21, 8p21.1 and 14q23.1 loci, respectively. This will facilitate the uncovering of true driver genes pertinent to breast cancer biology and the develop-ment of prospective therapeutics. PMID:21687935

  1. Heterogeneous EGFR gene copy number increase is common in colorectal cancer and defines response to anti-EGFR therapy.

    Ålgars, Annika; Avoranta, Tuulia; Österlund, Pia; Lintunen, Minnamaija; Sundström, Jari; Jokilehto, Terhi; Ristimäki, Ari; Ristamäki, Raija; Carpén, Olli


    Anti-EGFR therapy is commonly used to treat colorectal cancer (CRC), although only a subset of patients benefit from the treatment. While KRAS mutation predicts non-responsiveness, positive predictive markers are not in clinical practice. We previously showed that immunohistochemistry (IHC)-guided EGFR gene copy number (GCN) analysis may identify CRC patients benefiting from anti-EGFR treatment. Here we tested the predictive value of such analysis in chemorefractory metastatic CRC, elucidated EGFR GCN heterogeneity within the tumors, and evaluated the association between EGFR GCN, KRAS status, and anti-EGFR antibody response in CRC cell lines. The chemorefractory patient cohort consisted of 54 KRAS wild-type (WT) metastatic CRC patients. EGFR GCN status was analyzed by silver in situ hybridization using a cut-off value of 4.0 EGFR gene copies/cell. KRAS-WT and KRAS mutant CRC cell lines with different EGFR GCN were used in in vitro studies. The chemorefractory CRC tumors with EGFR GCN increase (≥4.0) responded better to anti-EGFR therapy than EGFR GCN (<4.0) tumors (clinical benefit, P = 0.0004; PFS, HR = 0.23, 95% CI 0.12-0.46). EGFR GCN counted using EGFR IHC guidance was significantly higher than the value from randomly selected areas verifying intratumoral EGFR GCN heterogeneity. In CRC cell lines, EGFR GCN correlated with EGFR expression. Best anti-EGFR response was seen with KRAS-WT, EGFR GCN = 4 cells and poorest response with KRAS-WT, EGFR GCN = 2 cells. Anti-EGFR response was associated with AKT and ERK1/2 phosphorylation, which was effectively inhibited only in cells with KRAS-WT and increased EGFR GCN. In conclusion, IHC-guided EGFR GCN is a promising predictor of anti-EGFR treatment efficacy in chemorefractory CRC. PMID:24940619

  2. Heterogeneous EGFR Gene Copy Number Increase Is Common in Colorectal Cancer and Defines Response to Anti-EGFR Therapy

    Ålgars, Annika; Lintunen, Minnamaija; Sundström, Jari; Jokilehto, Terhi; Ristimäki, Ari; Ristamäki, Raija; Carpén, Olli


    Anti-EGFR therapy is commonly used to treat colorectal cancer (CRC), although only a subset of patients benefit from the treatment. While KRAS mutation predicts non-responsiveness, positive predictive markers are not in clinical practice. We previously showed that immunohistochemistry (IHC)-guided EGFR gene copy number (GCN) analysis may identify CRC patients benefiting from anti-EGFR treatment. Here we tested the predictive value of such analysis in chemorefractory metastatic CRC, elucidated EGFR GCN heterogeneity within the tumors, and evaluated the association between EGFR GCN, KRAS status, and anti-EGFR antibody response in CRC cell lines. The chemorefractory patient cohort consisted of 54 KRAS wild-type (WT) metastatic CRC patients. EGFR GCN status was analyzed by silver in situ hybridization using a cut-off value of 4.0 EGFR gene copies/cell. KRAS-WT and KRAS mutant CRC cell lines with different EGFR GCN were used in in vitro studies. The chemorefractory CRC tumors with EGFR GCN increase (≥4.0) responded better to anti-EGFR therapy than EGFR GCN (<4.0) tumors (clinical benefit, P = 0.0004; PFS, HR = 0.23, 95% CI 0.12–0.46). EGFR GCN counted using EGFR IHC guidance was significantly higher than the value from randomly selected areas verifying intratumoral EGFR GCN heterogeneity. In CRC cell lines, EGFR GCN correlated with EGFR expression. Best anti-EGFR response was seen with KRAS-WT, EGFR GCN = 4 cells and poorest response with KRAS-WT, EGFR GCN = 2 cells. Anti-EGFR response was associated with AKT and ERK1/2 phosphorylation, which was effectively inhibited only in cells with KRAS-WT and increased EGFR GCN. In conclusion, IHC-guided EGFR GCN is a promising predictor of anti-EGFR treatment efficacy in chemorefractory CRC. PMID:24940619

  3. Male-biased autosomal effect of 16p13.11 copy number variation in neurodevelopmental disorders.

    Maria Tropeano

    Full Text Available Copy number variants (CNVs at chromosome 16p13.11 have been associated with a range of neurodevelopmental disorders including autism, ADHD, intellectual disability and schizophrenia. Significant sex differences in prevalence, course and severity have been described for a number of these conditions but the biological and environmental factors underlying such sex-specific features remain unclear. We tested the burden and the possible sex-biased effect of CNVs at 16p13.11 in a sample of 10,397 individuals with a range of neurodevelopmental conditions, clinically referred for array comparative genomic hybridisation (aCGH; cases were compared with 11,277 controls. In order to identify candidate phenotype-associated genes, we performed an interval-based analysis and investigated the presence of ohnologs at 16p13.11; finally, we searched the DECIPHER database for previously identified 16p13.11 copy number variants. In the clinical referral series, we identified 46 cases with CNVs of variable size at 16p13.11, including 28 duplications and 18 deletions. Patients were referred for various phenotypes, including developmental delay, autism, speech delay, learning difficulties, behavioural problems, epilepsy, microcephaly and physical dysmorphisms. CNVs at 16p13.11 were also present in 17 controls. Association analysis revealed an excess of CNVs in cases compared with controls (OR = 2.59; p = 0.0005, and a sex-biased effect, with a significant enrichment of CNVs only in the male subgroup of cases (OR = 5.62; p = 0.0002, but not in females (OR = 1.19, p = 0.673. The same pattern of results was also observed in the DECIPHER sample. Interval-based analysis showed a significant enrichment of case CNVs containing interval II (OR = 2.59; p = 0.0005, located in the 0.83 Mb genomic region between 15.49-16.32 Mb, and encompassing the four ohnologs NDE1, MYH11, ABCC1 and ABCC6. Our data confirm that duplications and deletions at 16p13

  4. Genome-wide mapping of copy number variation in humans: comparative analysis of high resolution array platforms.

    Rajini R Haraksingh

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

  5. Application of whole-exome sequencing for detecting copy number variants in CMT1A/HNPP.

    Jo, H-Y; Park, M-H; Woo, H-M; Han, M H; Kim, B-Y; Choi, B-O; Chung, K W; Koo, S K


    Large insertions and deletions (indels), including copy number variations (CNVs), are commonly seen in many diseases. Standard approaches for indel detection rely on well-established methods such as qPCR or short tandem repeat (STR) markers. Recently, a number of tools for CNV detection based on next-generation sequencing (NGS) data have also been developed; however, use of these methods is limited. Here, we used whole-exome sequencing (WES) in patients previously diagnosed with CMT1A or HNPP using STR markers to evaluate the ability of WES to improve the clinical diagnosis. Patients were evaluated utilizing three CNV detection tools including CONIFER, ExomeCNV and CEQer, and array comparative genomic hybridization (aCGH). We identified a breakpoint region at 17p11.2-p12 in patients with CMT1A and HNPP. CNV detection levels were similar in both 6 Gb (mean read depth = 80×) and 17 Gb (mean read depth = 190×) data. Taken together, these data suggest that 6 Gb WES data are sufficient to reveal the genetic causes of various diseases and can be used to estimate single mutations, indels, and CNVs simultaneously. Furthermore, our data strongly indicate that CNV detection by NGS is a rapid and cost-effective method for clinical diagnosis of genetically heterogeneous disorders such as CMT neuropathy. PMID:26662885

  6. Epidermal growth factor receptor gene copy number in 101 advanced colorectal cancer patients treated with chemotherapy plus cetuximab

    Zeuli Massimo


    Full Text Available Abstract Background Responsiveness to Cetuximab alone can be mediated by an increase of Epidermal Growth factor Receptor (EGFR Gene Copy Number (GCN. Aim of this study was to assess the role of EGFR-GCN in advanced colorectal cancer (CRC patients receiving chemotherapy plus Cetuximab. Methods One hundred and one advanced CRC patients (43 untreated- and 58 pre-treated were retrospectively studied by fluorescence in situ hybridization (FISH to assess EGFR-GCN and by immunohistochemistry (IHC to determine EGFR expression. Sixty-one out of 101 patients were evaluated also for k-ras status by direct sequencing. Clinical end-points were response rate (RR, progression-free survival (PFS and overall survival (OS. Results Increased EGFR-GCN was found in 60/101 (59% tumor samples. There was no correlation between intensity of EGFR-IHC and EGFR-GCN (p = 0.43. Patients receiving chemotherapy plus Cetuximab as first line treatment had a RR of 70% (30/43 while it was 18% (10/56 in the group with previous lines of therapy (p Conclusion In metastatic CRC patients treated with chemotherapy plus Cetuximab number of chemotherapy lines and increased EGFR-GCN were significantly associated with a better clinical outcome, independent of k-ras status.

  7. A simple screening method for detection of Klinefelter syndrome and other X-chromosome aneuploidies based on copy number of the androgen receptor gene

    Ottesen, A M; Garn, I D; Aksglaede, L; Juul, A; Rajpert-De Meyts, E


    copy number assessment of the androgen receptor (AR) gene, located to Xq11.2-q12. We analysed samples from 50 individuals, including a healthy male and female controls and patients with Klinefelter syndrome (47,XXY; 48,XXXY) (n = 28), mosaicisms (46,XX/47,XXY/48XXYY; 45,X/46,XY) (n = 3), other sex...... chromosome abnormalities (46,XX males; 47,XYY)(n = 4) and normal karyotypes (46,XY) (n = 13). The reference range for the AR-copy number was established as 0.8-1.2 for one copy and 1.7-2.3 for two copies. The qPCR results were within the reference range in 17/18 samples (94%) or 30/31 (97%) samples with one...... or two copies of the AR gene, respectively. None of the Klinefelter patients were misdiagnosed as having a karyotype with only one X-chromosome, and in none of the 46,XY males were two copies demonstrated. We systematically compared qPCR results with those obtained with another PCR-based method, the...

  8. Comparison of the Agilent, ROMA/NimbleGen and Illumina platforms for classification of copy number alterations in human breast tumors

    Naume B; Gunderson K; Bruhn L; Sun H; Hicks J; Johansen FE; Aarøe J; Baumbusch LO; Kristensen VN; Liestøl K; Børresen-Dale A-L; Lingjærde OC


    Background Microarray Comparative Genomic Hybridization (array CGH) provides a means to examine DNA copy number aberrations. Various platforms, brands and underlying technologies are available, facing the user with many choices regarding platform sensitivity and number, localization, and density distribution of probes. Results We evaluate three different platforms presenting different nature and arran...

  9. Structural Effects of L16Q, S20G, and L16Q-S20G Mutations on hlAPP: A Comparative Molecular Dynamics Study%Structural Effects of L16Q, S20G, and L16Q-S20G Mutations on hlAPP: A Comparative Molecular Dynamics Study

    Wang, Mian; Yang, Jipeng; Wang, Jianyi; Wang, Xiaojuan


    The conformation change picture of human islet amyloid polypeptide (hlAPP) is outlined using molecular dynamics simulation, and the structural influences of L16Q, S20G, and L16Q-S20G mutations on the conformation of hlAPP are analyzed. Particularly, the conformational changes of the amyloidogenic-related regions of residues 15-- 17 and 20--29 are emphasized. Our studies find that, for WT hlAPP, residues 15--17 always maintain a stable a-helix structure, residues 20--25 structurally fluctuate between turn and 5-helix, and residues 26--29 mainly adopt coil and bend structures. The hydrogen bonds between the polar groups of hlAPP, long-rang van der Waals forces between the residues, and hydrophobic interactions between the residues of hlAPP are important driving forces to maintain the secondary structure of hlAPP. The replacement of leucine 16 by glutamine stabilizes the helix structure of residues 15--17 and 20--23 of hlAPP monomer, and the structure of residues 24--29 fluctuates be- tween helix and turn. The relatively stable helix structures of residues 15--17 and 20--29 are supposed to be beneficial for L16Q hlAPP to resist the aggregation as observed in the experiment. The substitution of serine20 by glycinc drives residues 15--17 and 20--29 of hlAPP to transform from helix structure to β-strands or coil structures with higher extension and flexibility, which may promote the aggregation of hlAPP as the experiments reported. These results are significant to understand the aggregation mechanism of hlAPP monomer into the dimer, trimer, oligomers and fibrils associated with the type 2 diabetes at the atomic level.

  10. Scaling up Copy Detection

    Li, Xian; Dong, Xin Luna; Lyons, Kenneth B.; Meng, Weiyi; Srivastava, Divesh


    Recent research shows that copying is prevalent for Deep-Web data and considering copying can significantly improve truth finding from conflicting values. However, existing copy detection techniques do not scale for large sizes and numbers of data sources, so truth finding can be slowed down by one to two orders of magnitude compared with the corresponding techniques that do not consider copying. In this paper, we study {\\em how to improve scalability of copy detection on structured data}. Ou...

  11. Array-based comparative genomic hybridization for genomic-wide screening of DNA copy number alterations in aggressive bone tumors

    Kanamori Masahiko


    Full Text Available Abstract Background The genetic pathways of aggressive changes of bone tumors are still poorly understood. It is very important to analyze DNA copy number alterations (DCNAs, to identify the molecular events in the step of progression to the aggressive change of bone tissue. Methods Genome-wide array-based comparative genomic hybridization (array CGH was used to investigate DCNAs of 14 samples from 13 aggressive bone tumors, such as giant cell tumors (GCTs and osteosarcoma (OS, etc. Results Primary aggressive bone tumors had copy number gains of 17.8±12.7% in the genome, and losses of 17.3±11.4% in 287 target clones (threshold for each DCNA: ≦085, 1.15≦. Genetic unstable cases, which were defined by the total DCNAs aberration ≧30%, were identified in 9 of 13 patients (3 of 7 GCTs and all malignant tumors. High-level amplification of TGFβ2, CCND3, WI-6509, SHGC-5557, TCL1A, CREBBP, HIC1, THRA, AFM217YD10, LAMA3, RUNX1 and D22S543, were commonly observed in aggressive bone tumors. On the other hand, NRAS, D2S447, RAF1, ROBO1, MYB, MOS, FGFR2, HRAS, D13S319, D13S327, D18S552, YES1 and DCC, were commonly low. We compared genetic instability between a primary OS and its metastatic site in Case #13. Metastatic lesion showed increased 9 DCNAs of remarkable change (m/p ratio ≧1.3 folds, compared to a primary lesion. D1S214, D1S1635, EXT1, AFM137XA11, 8 M16/SP6, CCND2, IGH, 282 M15/SP6, HIC1 and LAMA3, were overexpressed. We gave attention to HIC1 (17p13.3, which was common high amplification in this series. Conclusion Our results may provide several entry points for the identification of candidate genes associated with aggressive change of bone tumors. Especially, the locus 17p11-13 including HIC1 close to p53 was common high amplification in this series and review of the literature.

  12. Copy number variants in a sample of patients with psychotic disorders: is standard screening relevant for actual clinical practice?

    Van de Kerkhof NW


    Full Text Available Noortje WA Van de Kerkhof,1 Ilse Feenstra,2 Jos IM Egger,1,3,4 Nicole de Leeuw,2 Rolph Pfundt,2 Gerald Stöber,5 Frank MMA van der Heijden,1 Willem MA Verhoeven1,61Vincent van Gogh Institute for Psychiatry, Centre of Excellence for Neuropsychiatry, Venray, The Netherlands; 2Radboud University Nijmegen Medical Centre, Department of Human Genetics, Nijmegen, The Netherlands; 3Donders Institute for Brain, Cognition and Behaviour, 4Behavioural Science Institute, Radboud University, Nijmegen, The Netherlands; 5University of Würzburg, Department of Psychiatry, Psychosomatics and Psychotherapy, Würzburg, Germany; 6Erasmus University Medical Centre, Department of Psychiatry, Rotterdam, The NetherlandsAbstract: With the introduction of new genetic techniques such as genome-wide array comparative genomic hybridization, studies on the putative genetic etiology of schizophrenia have focused on the detection of copy number variants (CNVs, ie, microdeletions and/or microduplications, that are estimated to be present in up to 3% of patients with schizophrenia. In this study, out of a sample of 100 patients with psychotic disorders, 80 were investigated by array for the presence of CNVs. The assessment of the severity of psychiatric symptoms was performed using standardized instruments and ICD-10 was applied for diagnostic classification. In three patients, a submicroscopic CNV was demonstrated, one with a loss in 1q21.1 and two with a gain in 1p13.3 and 7q11.2, respectively. The association between these or other CNVs and schizophrenia or schizophrenia-like psychoses and their clinical implications still remain equivocal. While the CNV affected genes may enhance the vulnerability for psychiatric disorders via effects on neuronal architecture, these insights have not resulted in major changes in clinical practice as yet. Therefore, genome-wide array analysis should presently be restricted to those patients in whom psychotic symptoms are paired with other

  13. Screening for copy-number alterations and loss of heterozygosity in chronic lymphocytic leukemia--a comparative study of four differently designed, high resolution microarray platforms

    Gunnarsson, R.; Staaf, J.; Jansson, M.;


    Screening for gene copy-number alterations (CNAs) has improved by applying genome-wide microarrays, where SNP arrays also allow analysis of loss of heterozygozity (LOH). We here analyzed 10 chronic lymphocytic leukemia (CLL) samples using four different high-resolution platforms: BAC arrays (32K...... of 32 additional regions present in 2-3 platforms illustrated a discrepancy in detection of small CNAs, which often involved reported copy-number variations. LOH analysis using dChip revealed concordance of mainly large regions, but showed numerous, small nonoverlapping regions and LOH escaping...... detection. Evaluation of baseline variation and copy-number ratio response showed the best performance for the Agilent platform and confirmed the robustness of BAC arrays. Accordingly, these platforms demonstrated a higher degree of platform-specific CNAs. The SNP arrays displayed higher technical variation...

  14. Original Copies

    Sørensen, Tim Flohr


    This article explores inter-artefactual relations in the Nordic Bronze Age. Notions of copying and imitation have been dominant in the description of a number of bronze and flint artefacts from period I of the Nordic Bronze Age (ca. 1700–1500 BC). It has been argued that local bronze manufacturer...

  15. Escherichia coli MW005: lambda Red-mediated recombineering and copy-number induction of oriV-equipped constructs in a single host

    Hope Ian A


    Full Text Available Abstract Background Escherichia coli strain EL350 contains chromosomally integrated phage lambda Red recombinase genes enabling this strain to be used for modifying the sequence of resident clones via recombineering. BAC and fosmid clones are highly suitable for modification by recombineering but, because they are present at low (1-2 copies per cell, the DNA is difficult to isolate in high yield and purity. To overcome this limitation vectors, e.g. pCC1FOS, have been constructed that contain the additional replication origin, oriV, which permits copy-number to be induced transiently when propagated in a suitable host strain, e.g. EPI300, that supplies the cognate trans-replication protein TrfA. Previously, we used EL350 and EPI300 sequentially to recombineer oriV-equipped fosmid genomic clones and, subsequently, to induce copy-number of the resulting recombinant clone. To eliminate these intervening DNA isolation and transformation steps we retrofitted EL350 with a PBAD-driven trfA gene generating strain MW005 that supports, independently, both recombineering and copy-number induction. Results The PBAD-driven copy of cre in EL350 was replaced seamlessly with a copy of trfA, PCR-amplified from EPI300 chromosomal DNA, to generate MW005. This new strain has been used to both generate, via recombineering, a number of reporter gene fusions directly from pCC1FOS-based Caenorhabditis elegans genomic clones and to transiently induce copy-number of fosmid and BAC clones prior to DNA preparation. Conclusions By retrofitting EL350, an established 'recombineering' E. coli strain, with a tightly regulated copy of trfA we have produced a new strain, MW005, which combines recombineering capacity with the useful ability to transiently induce copy-number of oriV-equipped clones. By coupling these two steps in a single strain, use of MW005 will enable the more rapid recombineering-mediated production of recombinant clones in the yield and quality necessary for

  16. S-SCAM, a rare copy number variation gene, induces schizophrenia-related endophenotypes in transgenic mouse model.

    Zhang, Nanyan; Zhong, Peng; Shin, Seung Min; Metallo, Jacob; Danielson, Eric; Olsen, Christopher M; Liu, Qing-song; Lee, Sang H


    Accumulating genetic evidence suggests that schizophrenia (SZ) is associated with individually rare copy number variations (CNVs) of diverse genes, often specific to single cases. However, the causality of these rare mutations remains unknown. One of the rare CNVs found in SZ cohorts is the duplication of Synaptic Scaffolding Molecule (S-SCAM, also called MAGI-2), which encodes a postsynaptic scaffolding protein controlling synaptic AMPA receptor levels, and thus the strength of excitatory synaptic transmission. Here we report that, in a transgenic mouse model simulating the duplication conditions, elevation of S-SCAM levels in excitatory neurons of the forebrain was sufficient to induce multiple SZ-related endophenotypes. S-SCAM transgenic mice showed an increased number of lateral ventricles and a reduced number of parvalbumin-stained neurons. In addition, the mice exhibited SZ-like behavioral abnormalities, including hyperlocomotor activity, deficits in prepulse inhibition, increased anxiety, impaired social interaction, and working memory deficit. Notably, the S-SCAM transgenic mice showed a unique sex difference in showing these behavioral symptoms, which is reminiscent of human conditions. These behavioral abnormalities were accompanied by hyperglutamatergic function associated with increased synaptic AMPA receptor levels and impaired long-term potentiation. Importantly, reducing glutamate release by the group 2 metabotropic glutamate receptor agonist LY379268 ameliorated the working memory deficits in the transgenic mice, suggesting that hyperglutamatergic function underlies the cognitive functional deficits. Together, these results contribute to validate a causal relationship of the rare S-SCAM CNV and provide supporting evidence for the rare CNV hypothesis in SZ pathogenesis. Furthermore, the S-SCAM transgenic mice provide a valuable new animal model for studying SZ pathogenesis. PMID:25653350

  17. A nested case-control study of leukocyte mitochondrial DNA copy number and renal cell carcinoma in the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial.

    Hofmann, Jonathan N; Hosgood, H Dean; Liu, Chin-San; Chow, Wong-Ho; Shuch, Brian; Cheng, Wen-Ling; Lin, Ta-Tsung; Moore, Lee E; Lan, Qing; Rothman, Nathaniel; Purdue, Mark P


    Mitochondrial DNA (mtDNA) is vulnerable to mutations, and the number of copies of mtDNA per cell may increase to compensate for DNA damage. Case-control studies have reported associations between altered mtDNA copy number and risk of renal cell carcinoma (RCC); however, this association has not been investigated prospectively. We conducted a nested case-control study (252 cases and 504 controls) of RCC risk in relation to pre-diagnostic leukocyte mtDNA copy number in the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial. mtDNA copy number was measured in triplicate using a fluorescence-based quantitative PCR assay; samples from 22 cases and 36 controls could not be assayed, leaving 230 cases and 468 controls for analysis. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated using unconditional logistic regression. High mtDNA copy number was associated with an increased risk of RCC, both overall (highest quartile versus lowest: OR = 2.0, 95% CI = 1.2-3.2; P trend = 0.002) and among cases diagnosed ≥6 years after blood collection (OR = 2.6, 95% CI = 1.4-5.0; P trend = 0.003). These findings did not differ significantly by sex, body mass index, history of hypertension or smoking status (P interaction ≥ 0.3). Results of this study suggest that high pre-diagnostic leukocyte mtDNA copy number, a suspected marker of oxidative DNA damage and mitochondrial dysfunction, is associated with increased future RCC risk. PMID:24398668

  18. Analysis of the terminator region after the deoCABD operon of Escherichia coli K-12 using a new class of single copy number operon-fusion vectors.

    Larsen, J E; Albrechtsen, B; Valentin-Hansen, P


    We describe the construction of low copy number operon-fusion vectors, and use one of these vectors for the cloning and transcriptional analysis of the terminator region after the deo operon of Escherichia coli K-12. The new vectors are miniderivatives of plasmid R1 containing the parB stability locus of this plasmid and the lac genes as a selectable marker. Since the copy number of the vectors is only one per genome-equivalent at temperatures below 37 degrees C this system is ideally suited ...

  19. Estimation of 16S rRNA gene copy number in several probiotic Lactobacillus strains isolated from the gastrointestinal tract of chicken

    Lee, Chin Mei; Sieo, Chin Chin; Abdullah, Norhani; Ho, Yin Wan


    The copy numbers of 16S rRNA genes in 12 probiotic Lactobacillus strains of poultry origin were analyzed. Genomic DNA of the strains was digested with restriction endonucleases that do not cut within the 16S rRNA gene of the strains. This was followed by Southern hybridization with a biotinylated probe complementary to the 16S rRNA gene. The copy number of the 16S rRNA gene within a Lactobacillus species was found to be conserved. From the hybridization results, Lactobacillus salivarius I 24 ...

  20. Mouse Lymphoblastic Leukemias Induced by Aberrant Prdm14 Expression Demonstrate Widespread Copy Number Alterations Also Found in Human ALL

    Aberrant expression and activation of oncogenes in somatic cells has been associated with cancer initiation. Required for reacquisition of pluripotency in the developing germ cell, PRDM14 initiates lymphoblastic leukemia when misexpressed in murine bone marrow. Activation of pluripotency in somatic cells can lead to aneuploidy and copy number alterations during iPS cell generation, and we hypothesized that PRDM14-induced lymphoblastic leukemias would demonstrate significant chromosomal damage. High-resolution oligo array comparative genomic hybridization demonstrated infrequent aneuploidy but frequent amplification and deletion, with amplifications occurring in a 5:1 ratio with deletions. Many deletions (i.e., Cdkn2a, Ebf1, Pax5, Ikzf1) involved B-cell development genes and tumor suppressor genes, recapitulating deletions occurring in human leukemia. Pathways opposing senescence were frequently deactivated via Cdkn2a deletion or Tbx2 amplification, with corollary gene expression. Additionally, gene expression studies of abnormal pre-leukemic B-precursors showed downregulation of genes involved in chromosomal stability (i.e., Xrcc6) and failure to upregulate DNA repair pathways. We propose a model of leukemogenesis, triggered by pluripotency genes like Prdm14, which involves ongoing DNA damage and failure to activate non-homologous end-joining secondary to aberrant gene expression

  1. β-Defensin Genomic Copy Number Does Not Influence the Age of Onset in Huntington’s Disease

    Vittori, Angelica; Orth, Michael; Roos, Raymund A. C.; Outeiro, Tiago F.; Giorgini, Flaviano; Hollox, Edward J.


    Background Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by the abnormal expansion of a CAG triplet repeat tract in the huntingtin gene. While the length of this CAG expansion is the major determinant of the age of onset (AO), other genetic factors have also been shown to play a modulatory role. Recent evidence suggests that neuroinflammation is a pivotal factor in the pathogenesis of HD, and that targeting this process may have important therapeutic ramifications. The human β-defensin 2 (hBD2) – encoded by DEFB4 – is an antimicrobial peptide that exhibits inducible expression in astrocytes during inflammation and is an important regulator of innate and adaptive immune response. Therefore, DEFB4 may contribute to the neuroinflammatory processes observed in HD. Objective In this study we tested the hypothesis that copy number variation (CNV) of the β-defensin region, including DEFB4, modifies the AO in HD. Methods and results We genotyped β-defensin CNV in 490 HD individuals using the paralogue ratio test and found no association between β-defensin CNV and onset of HD. Conclusions We conclude that it is unlikely that DEFB4 plays a role in HD pathogenesis. PMID:24587836

  2. Rare copy number variants and congenital heart defects in the 22q11.2 deletion syndrome

    Mlynarski, Elisabeth E.; Xie, Michael; Taylor, Deanne; Sheridan, Molly B.; Guo, Tingwei; Racedo, Silvia E.; McDonald-McGinn, Donna M.; Chow, Eva W. C.; Vorstman, Jacob; Swillen, Ann; Devriendt, Koen; Breckpot, Jeroen; Digilio, Maria Cristina; Marino, Bruno; Dallapiccola, Bruno; Philip, Nicole; Simon, Tony J.; Roberts, Amy E.; Piotrowicz, Małgorzata; Bearden, Carrie E.; Eliez, Stephan; Gothelf, Doron; Coleman, Karlene; Kates, Wendy R.; Devoto, Marcella; Zackai, Elaine; Heine-Suñer, Damian; Goldmuntz, Elizabeth; Bassett, Anne S.; Morrow, Bernice E.


    The 22q11.2 deletion syndrome (22q11DS; velocardiofacial/DiGeorge syndrome; VCFS/DGS; MIM #192430; 188400) is the most common microdeletion syndrome. The phenotypic presentation of 22q11DS is highly variable; approximately 60–75 % of 22q11DS patients have been reported to have a congenital heart defect (CHD), mostly of the conotruncal type, and/or aortic arch defect. The etiology of the cardiac phenotypic variability is not currently known for the majority of patients. We hypothesized that rare copy number variants (CNVs) outside the 22q11.2 deleted region may modify the risk of being born with a CHD in this sensitized population. Rare CNV analysis was performed using Affymetrix SNP Array 6.0 data from 946 22q11DS subjects with CHDs (n = 607) or with normal cardiac anatomy (n = 339). Although there was no significant difference in the overall burden of rare CNVs, an overabundance of CNVs affecting cardiac-related genes was detected in 22q11DS individuals with CHDs. When the rare CNVs were examined with regard to gene interactions, specific cardiac networks, such as Wnt signaling, appear to be overrepresented in 22q11DS CHD cases but not 22q11DS controls with a normal heart. Collectively, these data suggest that CNVs outside the 22q11.2 region may contain genes that modify risk for CHDs in some 22q11DS patients. PMID:26742502

  3. Absence of Substantial Copy Number Differences in a Pair of Monozygotic Twins Discordant for Features of Autism Spectrum Disorder

    Marina Laplana


    Full Text Available Autism spectrum disorder (ASD is a highly heritable disease (~0.9 with a complex genetic etiology. It is initially characterized by altered cognitive ability which commonly includes impaired language and communication skills as well as fundamental deficits in social interaction. Despite the large amount of studies described so far, the high clinical diversity affecting the autism phenotype remains poorly explained. Recent studies suggest that rare genomic variations, in particular copy number variation (CNV, may account for a significant proportion of the genetic basis of ASD. The use of disease-discordant monozygotic twins represents a powerful strategy to identify de novo and inherited CNV in the disorder. Here we present the results of a comparative genome hybridization (CGH analysis with a pair of monozygotic twins affected of ASD with significant differences in their clinical manifestations that specially affect speech language impairment and communication skills. Array CGH was performed in three different tissues: blood, saliva, and hair follicle, in an attempt to identify germinal and somatic CNV regions that may explain these differences. Our results argue against a role of large CNV rearrangements as a molecular etiology of the observed differences. This forwards future research to explore de novo point mutation and epigenomic alterations as potential explanations of the observed clinical differences.

  4. Genome-Wide Mapping of Structural Variations Reveals a Copy Number Variant That Determines Reproductive Morphology in Cucumber.

    Zhang, Zhonghua; Mao, Linyong; Chen, Huiming; Bu, Fengjiao; Li, Guangcun; Sun, Jinjing; Li, Shuai; Sun, Honghe; Jiao, Chen; Blakely, Rachel; Pan, Junsong; Cai, Run; Luo, Ruibang; Van de Peer, Yves; Jacobsen, Evert; Fei, Zhangjun; Huang, Sanwen


    Structural variations (SVs) represent a major source of genetic diversity. However, the functional impact and formation mechanisms of SVs in plant genomes remain largely unexplored. Here, we report a nucleotide-resolution SV map of cucumber (Cucumis sativas) that comprises 26,788 SVs based on deep resequencing of 115 diverse accessions. The largest proportion of cucumber SVs was formed through nonhomologous end-joining rearrangements, and the occurrence of SVs is closely associated with regions of high nucleotide diversity. These SVs affect the coding regions of 1676 genes, some of which are associated with cucumber domestication. Based on the map, we discovered a copy number variation (CNV) involving four genes that defines the Female (F) locus and gives rise to gynoecious cucumber plants, which bear only female flowers and set fruit at almost every node. The CNV arose from a recent 30.2-kb duplication at a meiotically unstable region, likely via microhomology-mediated break-induced replication. The SV set provides a snapshot of structural variations in plants and will serve as an important resource for exploring genes underlying key traits and for facilitating practical breeding in cucumber. PMID:26002866

  5. TMPRSS2-ERG Gene Fusion Causing ERG Overexpression Precedes Chromosome Copy Number Changes in Prostate Carcinomas, Paired HGPIN Lesions

    Nuno Cerveira


    Full Text Available TMPRSS2-ETS gene fusions have been found recurrently in prostate carcinomas, but not in the presumed precursor lesion, high-grade prostatic intraepithelial neoplasia (HGPIN. However, HGPIN lesions may share chromosomal changes with prostate cancer. To determine the relative order of genetic events in prostate carcinogenesis, we have analyzed 34 prostate carcinomas, 19 paired HGPIN lesions, 14 benign prostate hyperplasias, 11 morphologically normal prostatic tissues for TMPRSS2-ERG, TMPRSS2-ETV1 rearrangements, genomic imbalances. TMPRSS2 exon 1 was fused in-frame with ERG exon 4 in 17 of 34 (50% prostate carcinomas, in 4 of 19 (21% HGPIN lesions, but in none of controls. The findings were further validated by sequencing analysis, by the real-time polymerase chain reaction quantification of TMPRSS2-ERG fusion transcript, the ERG exons 5/6:exons 1/2 expression ratio. Chromosome copy number changes were detected by comparative genomic hybridization in 42% of clinically confined carcinomas, in none of the 16 HGPIN lesions analyzed. We demonstrate for the first time that the TMPRSS2-ERG fusion gene can be detected in a proportion of HGPIN lesions, that this molecular rearrangement is an early event that may precede chromosome-level alterations in prostate carcinogenesis.

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

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


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

  7. Genome-wide copy number variation study associates metabotropic glutamate receptor gene networks with attention deficit hyperactivity disorder

    Elia, Josephine; Glessner, Joseph T; Wang, Kai; Takahashi, Nagahide; Shtir, Corina J; Hadley, Dexter; Sleiman, Patrick M A; Zhang, Haitao; Kim, Cecilia E; Robison, Reid; Lyon, Gholson J; Flory, James H; Bradfield, Jonathan P; Imielinski, Marcin; Hou, Cuiping; Frackelton, Edward C; Chiavacci, Rosetta M; Sakurai, Takeshi; Rabin, Cara; Middleton, Frank A; Thomas, Kelly A; Garris, Maria; Mentch, Frank; Freitag, Christine M; Steinhausen, Hans-Christoph; Todorov, Alexandre A; Reif, Andreas; Rothenberger, Aribert; Franke, Barbara; Mick, Eric O; Roeyers, Herbert; Buitelaar, Jan; Lesch, Klaus-Peter; Banaschewski, Tobias; Ebstein, Richard P; Mulas, Fernando; Oades, Robert D; Sergeant, Joseph; Sonuga-Barke, Edmund; Renner, Tobias J; Romanos, Marcel; Romanos, Jasmin; Warnke, Andreas; Walitza, Susanne; Meyer, Jobst; Pálmason, Haukur; Seitz, Christiane; Loo, Sandra K; Smalley, Susan L; Biederman, Joseph; Kent, Lindsey; Asherson, Philip; Anney, Richard J L; Gaynor, J William; Shaw, Philip; Devoto, Marcella; White, Peter S; Grant, Struan F A; Buxbaum, Joseph D; Rapoport, Judith L; Williams, Nigel M; Nelson, Stanley F; Faraone, Stephen V; Hakonarson, Hakon


    Attention deficit hyperactivity disorder (ADHD) is a common, heritable neuropsychiatric disorder of unknown etiology. We performed a whole-genome copy number variation (CNV) study on 1,013 cases with ADHD and 4,105 healthy children of European ancestry using 550,000 SNPs. We evaluated statistically significant findings in multiple independent cohorts, with a total of 2,493 cases with ADHD and 9,222 controls of European ancestry, using matched platforms. CNVs affecting metabotropic glutamate receptor genes were enriched across all cohorts (P = 2.1 × 10−9). We saw GRM5 (encoding glutamate receptor, metabotropic 5) deletions in ten cases and one control (P = 1.36 × 10−6). We saw GRM7 deletions in six cases, and we saw GRM8 deletions in eight cases and no controls. GRM1 was duplicated in eight cases. We experimentally validated the observed variants using quantitative RT-PCR. A gene network analysis showed that genes interacting with the genes in the GRM family are enriched for CNVs in ~10% of the cases (P = 4.38 × 10−10) after correction for occurrence in the controls. We identified rare recurrent CNVs affecting glutamatergic neurotransmission genes that were overrepresented in multiple ADHD cohorts. PMID:22138692

  8. Copy number variations of genes involved in stress responses reflect the redox state and DNA damage in brewing yeasts.

    Adamczyk, Jagoda; Deregowska, Anna; Skoneczny, Marek; Skoneczna, Adrianna; Natkanska, Urszula; Kwiatkowska, Aleksandra; Rawska, Ewa; Potocki, Leszek; Kuna, Ewelina; Panek, Anita; Lewinska, Anna; Wnuk, Maciej


    The yeast strains of the Saccharomyces sensu stricto complex involved in beer production are a heterogeneous group whose genetic and genomic features are not adequately determined. Thus, the aim of the present study was to provide a genetic characterization of selected group of commercially available brewing yeasts both ale top-fermenting and lager bottom-fermenting strains. Molecular karyotyping revealed that the diversity of chromosome patterns and four strains with the most accented genetic variabilities were selected and subjected to genome-wide array-based comparative genomic hybridization (array-CGH) analysis. The differences in the gene copy number were found in five functional gene categories: (1) maltose metabolism and transport, (2) response to toxin, (3) siderophore transport, (4) cellular aldehyde metabolic process, and (5) L-iditol 2-dehydrogenase activity (p stress stimuli compared to other strains. We suggest that compromised stress response may not only promote oxidant-based changes in the nucleolus state that may affect fermentation performance but also provide novel directions for future strain improvement. PMID:27299603

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

    Rocco Piazza

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

  10. Cloning of the promoter of NDE1, a gene implicated in psychiatric and neurodevelopmental disorders through copy number variation.

    Bradshaw, N J


    Copy number variation at 16p13.11 has been associated with a range of neurodevelopmental and psychiatric conditions, with duplication of this region being more common in individuals with schizophrenia. A prominent candidate gene within this locus is NDE1 (Nuclear Distribution Element 1) given its known importance for neurodevelopment, previous associations with mental illness and its well characterized interaction with the Disrupted in Schizophrenia 1 (DISC1) protein. In order to accurately model the effect of NDE1 duplication, it is important to first gain an understanding of how the gene is expressed. The complex promoter system of NDE1, which produces three distinct transcripts, each encoding for the same full-length NDE1 protein (also known as NudE), was therefore cloned and tested in human cell lines. The promoter for the longest of these three NDE1 transcripts was found to be responsible for the majority of expression in these systems, with its extended 5' untranslated region (UTR) having a limiting effect on its expression. These results thus highlight and clone the promoter elements required to generate systems in which the NDE1 protein is exogenously expressed under its native promoter, providing a biologically relevant model of 16p13.11 duplication in major mental illness. PMID:26975893

  11. Identification of copy number variations in three Chinese horse breeds using 70K single nucleotide polymorphism BeadChip array.

    Kader, Adiljan; Liu, Xuexue; Dong, Kunzhe; Song, Shen; Pan, Jianfei; Yang, Min; Chen, Xiaofei; He, Xiaohong; Jiang, Lin; Ma, Yuehui


    Copy number variation (CNV), an essential form of genetic variation, has been increasingly recognized as one promising genetic marker in the analysis of animal genomes. Here, we used the Equine 70K single nucleotide polymorphism genotyping array for the genome-wide detection of CNVs in 96 horses from three diverse Chinese breeds: Debao pony (DB), Mongolian horse (MG) and Yili horse (YL). A total of 287 CNVs were determined and merged into 122 CNV regions (CNVRs) ranging from 199 bp to 2344 kb in size and distributed in a heterogeneous manner on chromosomes. These CNVRs were integrated with seven existing reports to generate a composite genome-wide dataset of 1558 equine CNVRs, revealing 69 (56.6%) novel CNVRs. The majority (69.7%) of the 122 CNVRs overlapped with 438 genes, whereas 30.3% were located in intergenic regions. Most of these genes were associated with common CNVRs, which were shared by divergent horse breeds. As many as 60, 42 and 91 genes overlapping with the breed-specific ss were identified in DB, MG and YL respectively. Among these genes, FGF11, SPEM1, PPARG, CIDEB, HIVEP1 and GALR may have potential relevance to breed-specific traits. These findings provide valuable information for understanding the equine genome and facilitating association studies of economically important traits with equine CNVRs in the future. PMID:27440410

  12. A DNA target-enrichment approach to detect mutations, copy number changes and immunoglobulin translocations in multiple myeloma.

    Bolli, N; Li, Y; Sathiaseelan, V; Raine, K; Jones, D; Ganly, P; Cocito, F; Bignell, G; Chapman, M A; Sperling, A S; Anderson, K C; Avet-Loiseau, H; Minvielle, S; Campbell, P J; Munshi, N C


    Genomic lesions are not investigated during routine diagnostic workup for multiple myeloma (MM). Cytogenetic studies are performed to assess prognosis but with limited impact on therapeutic decisions. Recently, several recurrently mutated genes have been described, but their clinical value remains to be defined. Therefore, clinical-grade strategies to investigate the genomic landscape of myeloma samples are needed to integrate new and old prognostic markers. We developed a target-enrichment strategy followed by next-generation sequencing (NGS) to streamline simultaneous analysis of gene mutations, copy number changes and immunoglobulin heavy chain (IGH) translocations in MM in a high-throughput manner, and validated it in a panel of cell lines. We identified 548 likely oncogenic mutations in 182 genes. By integrating published data sets of NGS in MM, we retrieved a list of genes with significant relevance to myeloma and found that the mutational spectrum of primary samples and MM cell lines is partially overlapping. Gains and losses of chromosomes, chromosomal segments and gene loci were identified with accuracy comparable to conventional arrays, allowing identification of lesions with known prognostic significance. Furthermore, we identified IGH translocations with high positive and negative predictive value. Our approach could allow the identification of novel biomarkers with clinical relevance in myeloma. PMID:27588520

  13. Mucopolysaccharidosis IV A: Assignment of the human N-acetylgalactosamine-6-sulfate sulfatase (GALNS) gene to chromosome 16q24

    Masuno, Mitsuo; Tomatsu, Shunji; Nakashima, Yoshihiro; Hori, Toshinori; Fukuda, Seiji; Masue, Michiya; Sukegawa, Kazuko; Orii, Tadao (Gifu Univ. School of Medicine (Japan))


    Plasmid clones of three independent genomic fragments of the gene of human N-acetylgalactosamine-6-sulfate sulfatase (GALNS; EC were utilized in a fluorescence in situ suppression hybridization study to assign the locus to chromosome 16q24. Enzyme assay for GALNS in a patient with del(16)(q22.1) confirmed this finding. 8 refs., 1 fig., 1 tab.

  14. Detection of copy number variation from array intensity and sequencing read depth using a stepwise Bayesian model

    Gerstein Mark B


    Full Text Available Abstract Background Copy number variants (CNVs have been demonstrated to occur at a high frequency and are now widely believed to make a significant contribution to the phenotypic variation in human populations. Array-based comparative genomic hybridization (array-CGH and newly developed read-depth approach through ultrahigh throughput genomic sequencing both provide rapid, robust, and comprehensive methods to identify CNVs on a whole-genome scale. Results We developed a Bayesian statistical analysis algorithm for the detection of CNVs from both types of genomic data. The algorithm can analyze such data obtained from PCR-based bacterial artificial chromosome arrays, high-density oligonucleotide arrays, and more recently developed high-throughput DNA sequencing. Treating parameters--e.g., the number of CNVs, the position of each CNV, and the data noise level--that define the underlying data generating process as random variables, our approach derives the posterior distribution of the genomic CNV structure given the observed data. Sampling from the posterior distribution using a Markov chain Monte Carlo method, we get not only best estimates for these unknown parameters but also Bayesian credible intervals for the estimates. We illustrate the characteristics of our algorithm by applying it to both synthetic and experimental data sets in comparison to other segmentation algorithms. Conclusions In particular, the synthetic data comparison shows that our method is more sensitive than other approaches at low false positive rates. Furthermore, given its Bayesian origin, our method can also be seen as a technique to refine CNVs identified by fast point-estimate methods and also as a framework to integrate array-CGH and sequencing data with other CNV-related biological knowledge, all through informative priors.

  15. Oncogene mutations, copy number gains and mutant allele specific imbalance (MASI frequently occur together in tumor cells.

    Junichi Soh

    Full Text Available BACKGROUND: Activating mutations in one allele of an oncogene (heterozygous mutations are widely believed to be sufficient for tumorigenesis. However, mutant allele specific imbalance (MASI has been observed in tumors and cell lines harboring mutations of oncogenes. METHODOLOGY/PRINCIPAL FINDINGS: We determined 1 mutational status, 2 copy number gains (CNGs and 3 relative ratio between mutant and wild type alleles of KRAS, BRAF, PIK3CA and EGFR genes by direct sequencing and quantitative PCR assay in over 400 human tumors, cell lines, and xenografts of lung, colorectal, and pancreatic cancers. Examination of a public database indicated that homozygous mutations of five oncogenes were frequent (20% in 833 cell lines of 12 tumor types. Our data indicated two major forms of MASI: 1 MASI with CNG, either complete or partial; and 2 MASI without CNG (uniparental disomy; UPD, due to complete loss of wild type allele. MASI was a frequent event in mutant EGFR (75% and was due mainly to CNGs, while MASI, also frequent in mutant KRAS (58%, was mainly due to UPD. Mutant: wild type allelic ratios at the genomic level were precisely maintained after transcription. KRAS mutations or CNGs were significantly associated with increased ras GTPase activity, as measured by ELISA, and the two molecular changes were synergistic. Of 237 lung adenocarcinoma tumors, the small number with both KRAS mutation and CNG were associated with shortened survival. CONCLUSIONS: MASI is frequently present in mutant EGFR and KRAS tumor cells, and is associated with increased mutant allele transcription and gene activity. The frequent finding of mutations, CNGs and MASI occurring together in tumor cells indicates that these three genetic alterations, acting together, may have a greater role in the development or maintenance of the malignant phenotype than any individual alteration.

  16. Copy number variation identification and analysis of the chicken genome using a 60K SNP BeadChip.

    Rao, Y S; Li, J; Zhang, R; Lin, X R; Xu, J G; Xie, L; Xu, Z Q; Wang, L; Gan, J K; Xie, X J; He, J; Zhang, X Q


    Copy number variation (CNV) is an important source of genetic variation in organisms and a main factor that affects phenotypic variation. A comprehensive study of chicken CNV can provide valuable information on genetic diversity and facilitate future analyses of associations between CNV and economically important traits in chickens. In the present study, an F2 full-sib chicken population (554 individuals), established from a cross between Xinghua and White Recessive Rock chickens, was used to explore CNV in the chicken genome. Genotyping was performed using a chicken 60K SNP BeadChip. A total of 1,875 CNV were detected with the PennCNV algorithm, and the average number of CNV was 3.42 per individual. The CNV were distributed across 383 independent CNV regions (CNVR) and covered 41 megabases (3.97%) of the chicken genome. Seven CNVR in 108 individuals were validated by quantitative real-time PCR, and 81 of these individuals (75%) also were detected with the PennCNV algorithm. In total, 274 CNVR (71.54%) identified in the current study were previously reported. Of these, 147 (38.38%) were reported in at least 2 studies. Additionally, 109 of the CNVR (28.46%) discovered here are novel. A total of 709 genes within or overlapping with the CNVR was retrieved. Out of the 2,742 quantitative trait loci (QTL) collected in the chicken QTL database, 43 QTL had confidence intervals overlapping with the CNVR, and 32 CNVR encompassed one or more functional genes. The functional genes located in the CNVR are likely to be the QTG that are associated with underlying economic traits. This study considerably expands our insight into the structural variation in the genome of chickens and provides an important resource for genomic variation, especially for genomic structural variation related to economic traits in chickens. PMID:27118864

  17. High-resolution array copy number analyses for detection of deletion, gain, amplification and copy-neutral LOH in primary neuroblastoma tumors: Four cases of homozygous deletions of the CDKN2A gene

    Kogner Per; Abrahamsson Jonas; Sjöberg Rose-Marie; Enerbäck Charlotta; Olsson Linda; Erichsen Jennie; Carén Helena; Martinsson Tommy


    Abstract Background Neuroblastoma is a very heterogeneous pediatric tumor of the sympathetic nervous system showing clinically significant patterns of genetic alterations. Favorable tumors usually have near-triploid karyotypes with few structural rearrangements. Aggressive stage 4 tumors often have near-diploid or near-tetraploid karyotypes and structural rearrangements. Whole genome approaches for analysis of genome-wide copy number have been used to analyze chromosomal abnormalities in tumo...

  18. Copy number variation in glutathione S-transferases M1 and T1 and ischemic vascular disease: four studies and meta-analyses

    Nørskov, Marianne S; Frikke-Schmidt, Ruth; Loft, Steffen;


    Glutathione S-transferases (GSTs) M1 and T1 detoxify products of oxidative stress and may protect against atherosclerosis and ischemic vascular disease (IVD). We tested the hypothesis that copy number variation (CNV) in GSTM1 and GSTT1 genes, known to be associated with stepwise decreases in...

  19. DNA copy number analysis of fresh and formalin-fixed specimens by shallow whole-genome sequencing with identification and exclusion of problematic regions in the genome assembly

    Scheinin, I.; Sie, D.; Bengtsson, H.; Wiel, M.A. van de; Olshen, A.B.; Thuijl, H.F. van; Essen, H.F. van; Eijk, P.P.; Rustenburg, F.; Meijer, G.A.; Reijneveld, J.C.; Wesseling, P.; Pinkel, D.; Albertson, D.G.; Ylstra, B.


    Detection of DNA copy number aberrations by shallow whole-genome sequencing (WGS) faces many challenges, including lack of completion and errors in the human reference genome, repetitive sequences, polymorphisms, variable sample quality, and biases in the sequencing procedures. Formalin-fixed paraff

  20. Recurrent copy number gains of ACVR1 and corresponding transcript overexpression are associated with survival in head and neck squamous cell carcinomas

    Ambrosio, Eliane P; Drigo, Sandra A; Bérgamo, Nádia A;


    AIMS: This study aimed to evaluate the copy number alteration on 2q24, its association with ACVR1 transcript expression and the prognostic value of these data in head and neck squamous cell carcinomas. METHODS AND RESULTS: Twenty-eight samples of squamous cell carcinoma were evaluated by fluoresc...

  1. Assessment of the topoisomerase I gene copy number as a predictive biomarker of objective response to irinotecan in metastatic colorectal cancer

    Nygård, Sune Boris; Christensen, Ib Jarle; Nielsen, Signe Lykke;


    (TOP1) copy number and objective response following irinotecan treatment in patients with metastatic colorectal cancer. Materials and methods. Formalin-fixed, paraffin-embedded tumor samples from 78 patients, who received irinotecan monotherapy in second line, were included. TOP1 was assessed by...

  2. High-throughput genotyping of copy number variation in glutathione S-transferases M1 and T1 using real-time PCR in 20,687 individuals

    Norskov, M.S.; Frikke-Schmidt, R.; Loft, S.;


    OBJECTIVES: Characteristic for the genes encoding glutathione S-transferase (GST) M1 and GSTT1 is a null allele, suggested to increase susceptibility to chronic diseases. We report an optimized method for the determination of copy number variation (CNV) in GST genes. DESIGN AND METHODS: Real-time...

  3. SNP microarray analyses reveal copy number alterations and progressive genome reorganization during tumor development in SVT/t driven mice breast cancer

    Tumor development is known to be a stepwise process involving dynamic changes that affect cellular integrity and cellular behavior. This complex interaction between genomic organization and gene, as well as protein expression is not yet fully understood. Tumor characterization by gene expression analyses is not sufficient, since expression levels are only available as a snapshot of the cell status. So far, research has mainly focused on gene expression profiling or alterations in oncogenes, even though DNA microarray platforms would allow for high-throughput analyses of copy number alterations (CNAs). We analyzed DNA from mouse mammary gland epithelial cells using the Affymetrix Mouse Diversity Genotyping array (MOUSEDIVm520650) and calculated the CNAs. Segmental copy number alterations were computed based on the probeset CNAs using the circular binary segmentation algorithm. Motif search was performed in breakpoint regions (inter-segment regions) with the MEME suite to identify common motif sequences. Here we present a four stage mouse model addressing copy number alterations in tumorigenesis. No considerable changes in CNA were identified for non-transgenic mice, but a stepwise increase in CNA was found during tumor development. The segmental copy number alteration revealed informative chromosomal fragmentation patterns. In inter-segment regions (hypothetical breakpoint sides) unique motifs were found. Our analyses suggest genome reorganization as a stepwise process that involves amplifications and deletions of chromosomal regions. We conclude from distinctive fragmentation patterns that conserved as well as individual breakpoints exist which promote tumorigenesis

  4. DNA Topoisomerase I Gene Copy Number and mRNA Expression Assessed as Predictive Biomarkers for Adjuvant Irinotecan in Stage II/III Colon Cancer

    Nygård, Sune Boris; Vainer, Ben; Nielsen, Signe L;


    PURPOSE: Prospective-retrospective assessment of the TOP1 gene copy number and TOP1 mRNA expression as predictive biomarkers for adjuvant irinotecan in stage II/III colon cancer (CC). EXPERIMENTAL DESIGN: Formalin-fixed, paraffin-embedded tissue microarrays were obtained from an adjuvant CC trial...

  5. A single cell level based method for copy number variation analysis by low coverage massively parallel sequencing.

    Chunlei Zhang

    Full Text Available Copy number variations (CNVs, a common genomic mutation associated with various diseases, are important in research and clinical applications. Whole genome amplification (WGA and massively parallel sequencing have been applied to single cell CNVs analysis, which provides new insight for the fields of biology and medicine. However, the WGA-induced bias significantly limits sensitivity and specificity for CNVs detection. Addressing these limitations, we developed a practical bioinformatic methodology for CNVs detection at the single cell level using low coverage massively parallel sequencing. This method consists of GC correction for WGA-induced bias removal, binary segmentation algorithm for locating CNVs breakpoints, and dynamic threshold determination for final signals filtering. Afterwards, we evaluated our method with seven test samples using low coverage sequencing (4∼9.5%. Four single-cell samples from peripheral blood, whose karyotypes were confirmed by whole genome sequencing analysis, were acquired. Three other test samples derived from blastocysts whose karyotypes were confirmed by SNP-array analysis were also recruited. The detection results for CNVs of larger than 1 Mb were highly consistent with confirmed results reaching 99.63% sensitivity and 97.71% specificity at base-pair level. Our study demonstrates the potential to overcome WGA-bias and to detect CNVs (>1 Mb at the single cell level through low coverage massively parallel sequencing. It highlights the potential for CNVs research on single cells or limited DNA samples and may prove as a promising tool for research and clinical applications, such as pre-implantation genetic diagnosis/screening, fetal nucleated red blood cells research and cancer heterogeneity analysis.

  6. Chromosomal copy number variation, selection and uneven rates of recombination reveal cryptic genome diversity linked to pathogenicity.

    Rhys A Farrer

    Full Text Available Pathogenic fungi constitute a growing threat to both plant and animal species on a global scale. Despite a clonal mode of reproduction dominating the population genetic structure of many fungi, putatively asexual species are known to adapt rapidly when confronted by efforts to control their growth and transmission. However, the mechanisms by which adaptive diversity is generated across a clonal background are often poorly understood. We sequenced a global panel of the emergent amphibian pathogen, Batrachochytrium dendrobatidis (Bd, to high depth and characterized rapidly changing features of its genome that we believe hold the key to the worldwide success of this organism. Our analyses show three processes that contribute to the generation of de novo diversity. Firstly, we show that the majority of wild isolates manifest chromosomal copy number variation that changes over short timescales. Secondly, we show that cryptic recombination occurs within all lineages of Bd, leading to large regions of the genome being in linkage equilibrium, and is preferentially associated with classes of genes of known importance for virulence in other pathosystems. Finally, we show that these classes of genes are under directional selection, and that this has predominantly targeted the Global Panzootic Lineage (BdGPL. Our analyses show that Bd manifests an unusually dynamic genome that may have been shaped by its association with the amphibian host. The rates of variation that we document likely explain the high levels of phenotypic variability that have been reported for Bd, and suggests that the dynamic genome of this pathogen has contributed to its success across multiple biomes and host-species.

  7. Recurrent Rare Genomic Copy Number Variants and Bicuspid Aortic Valve Are Enriched in Early Onset Thoracic Aortic Aneurysms and Dissections

    Prakash, Siddharth; Kuang, Shao-Qing; Regalado, Ellen; Guo, Dongchuan; Milewicz, Dianna


    Thoracic Aortic Aneurysms and Dissections (TAAD) are a major cause of death in the United States. The spectrum of TAAD ranges from genetic disorders, such as Marfan syndrome, to sporadic isolated disease of unknown cause. We hypothesized that genomic copy number variants (CNVs) contribute causally to early onset TAAD (ETAAD). We conducted a genome-wide SNP array analysis of ETAAD patients of European descent who were enrolled in the National Registry of Genetically Triggered Thoracic Aortic Aneurysms and Cardiovascular Conditions (GenTAC). Genotyping was performed on the Illumina Omni-Express platform, using PennCNV, Nexus and CNVPartition for CNV detection. ETAAD patients (n = 108, 100% European American, 28% female, average age 20 years, 55% with bicuspid aortic valves) were compared to 7013 dbGAP controls without a history of vascular disease using downsampled Omni 2.5 data. For comparison, 805 sporadic TAAD patients with late onset aortic disease (STAAD cohort) and 192 affected probands from families with at least two affected relatives (FTAAD cohort) from our institution were screened for additional CNVs at these loci with SNP arrays. We identified 47 recurrent CNV regions in the ETAAD, FTAAD and STAAD groups that were absent or extremely rare in controls. Nine rare CNVs that were either very large (>1 Mb) or shared by ETAAD and STAAD or FTAAD patients were also identified. Four rare CNVs involved genes that cause arterial aneurysms when mutated. The largest and most prevalent of the recurrent CNVs were at Xq28 (two duplications and two deletions) and 17q25.1 (three duplications). The percentage of individuals harboring rare CNVs was significantly greater in the ETAAD cohort (32%) than in the FTAAD (23%) or STAAD (17%) cohorts. We identified multiple loci affected by rare CNVs in one-third of ETAAD patients, confirming the genetic heterogeneity of TAAD. Alterations of candidate genes at these loci may contribute to the pathogenesis of TAAD. PMID:27092555

  8. Risk stratification of T-cell Acute Lymphoblastic Leukemia patients based on gene expression, mutations and copy number variation.

    Mirji, Gauri; Bhat, Jaydeep; Kode, Jyoti; Banavali, Shripad; Sengar, Manju; Khadke, Prashant; Sait, Osama; Chiplunkar, Shubhada


    Gene expression, copy number variations (CNV), mutations and survival were studied to delineate TCRγδ+T-cell acute lymphoblastic leukemia (T-ALL) as a distinct subgroup from TCRαβ+T-ALL. Gene Ontology analysis showed that differential regulation of genes involved in pathways for leukemogenesis, apoptosis, cytokine-cytokine receptor interaction and antigen processing/presentation may offer a survival benefit to TCRγδ+T-ALL patients. Genes involved in disease biology and having equal expression in both the subgroups, were further analysed for mutations and CNV using droplet digital PCR. TCRγδ+T-ALL patients exhibited differential level of mutations for NOTCH1 and IKZF3; however BRAF mutations were detected at equal levels in both the subgroups. Although TCRγδ+T-ALL patients with these mutations demonstrated improved disease-free survival (DFS) as compared TCRαβ+T-ALL patients, it was not statistically significant. Patients with homozygous deletion of CDKN2A/CDKN2B showed poor DFS in each subgroup. TCRγδ+T-ALL patients with wild type/heterozygous deletion of CDKN2A/CDKN2B possess significantly better DFS over TCRαβ+T-ALL patients (p=0.017 and 0.045, respectively). Thus, the present study has for the first time demonstrated TCRγδ clonality and CDKN2A/CDKN2B CNV together as potential prognostic markers in management of T-ALL. Further understanding the functional significance of differentially regulated genes in T-ALL patients would aid in designing risk based treatment strategies in subset specific manner. PMID:27070758

  9. Association of copy number polymorphisms at the promoter and translated region of COMT with Japanese patients with schizophrenia.

    Higashiyama, Ryoko; Ohnuma, Tohru; Takebayashi, Yuto; Hanzawa, Ryo; Shibata, Nobuto; Yamamori, Hidenaga; Yasuda, Yuka; Kushima, Itaru; Aleksic, Branko; Kondo, Kenji; Ikeda, Masashi; Hashimoto, Ryota; Iwata, Nakao; Ozaki, Norio; Arai, Heii


    Chromosome 22q11.2 deletion syndrome and genetic variations including single-nucleotide polymorphism (SNP) and copy number variation (CNV) in catechol-O-methyltransferase (COMT) situated at 22q11.2 remains controversial. Here, the genetic relationship between COMT and Japanese patients with schizophrenia was investigated by examining whether the SNPs correlated with schizophrenia based on a common disease-common variant hypothesis. Additionally, 22q11.2DS were screened based on a common disease-rare variant hypothesis; low-frequency CNVs situated at two COMT promoters and exons were investigated based on the low-frequency variants with an intermediate effect; and positive findings from the first stage were reconfirmed using a second-stage replication study including a larger sample size. Eight SNPs and 10 CNVs were investigated using Taqman SNP and CNV quantitative real-time polymerase chain reaction method. For the first-stage analysis, 513 unrelated Japanese patients with schizophrenia and 705 healthy controls were examined. For the second-stage replication study, positive findings from the first stage were further investigated using a larger sample size, namely 1,854 patients with schizophrenia and 2,137 controls. The first-stage analysis showed significant associations among schizophrenia, intronic SNP rs165774, CNV6 situated at promoter 1, CNV8 at exon 6, and CNV9 at exon 7. The second-stage study showed that intronic SNP rs165774 (χ(2)  = 8.327, P = 0.0039), CNV6 (χ(2)  = 19.66, P = 0.00005), and CNV8 (χ(2)  = 16.57, P = 0.00025) were significantly associated with schizophrenia. Large and rare CNVs as well as low-frequency CNVs and relatively small CNVs, namely COMT, may be genetic risk factors for schizophrenia. © 2016 Wiley Periodicals, Inc. PMID:26852906

  10. Establishment of TaqMan Real-time Quantitative PCR Assay for Foreign Gene Copy Numbers in Transgenic Soybean

    Qiu You-wen; Gao Xue-jun; Qi Bang-ruo; Li Lu; Zhen Zhen


    TaqMan quantitative PCR technique was used to detect the copies of exogenous CaMV35S flanks sequence in transgenic soybean. With soybean lectin as the endogenous reference gene, and gene complex DNA in non-GMO soybeans as the endogenous reference standard, the gradient dilution method was used to separately calculate Ct value of endogenous reference gene and plasmid DNA and correlation standard curve equation of logarithm of copies, and then to calculate the copies of samples through substituting thus-obtained Ct into the standard curve equation. The standard curve equation of endogenous reference gene was y =–3.422x+35.201, R2=0.998; the standard curve equation of exogenous gene was y =–3.495x+35.303, R2=0.999. The sample copies was got by putting Ct value into the standard curve equation, and it was the ratio of exogenous gene and reference gene. We found that CaMV35S gene in transgenic soy was single copy.

  11. Genomic pathology of SLE-associated copy-number variation at the FCGR2C/FCGR3B/FCGR2B locus.

    Mueller, Michael; Barros, Paula; Witherden, Abigail S; Roberts, Amy L; Zhang, Zhou; Schaschl, Helmut; Yu, Chack-Yung; Hurles, Matthew E; Schaffner, Catherine; Floto, R Andres; Game, Laurence; Steinberg, Karyn Meltz; Wilson, Richard K; Graves, Tina A; Eichler, Evan E; Cook, H Terence; Vyse, Timothy J; Aitman, Timothy J


    Reduced FCGR3B copy number is associated with increased risk of systemic lupus erythematosus (SLE). The five FCGR2/FCGR3 genes are arranged across two highly paralogous genomic segments on chromosome 1q23. Previous studies have suggested mechanisms for structural rearrangements at the FCGR2/FCGR3 locus and have proposed mechanisms whereby altered FCGR3B copy number predisposes to autoimmunity, but the high degree of sequence similarity between paralogous segments has prevented precise definition of the molecular events and their functional consequences. To pursue the genomic pathology associated with FCGR3B copy-number variation, we integrated sequencing data from fosmid and bacterial artificial chromosome clones and sequence-captured DNA from FCGR3B-deleted genomes to establish a detailed map of allelic and paralogous sequence variation across the FCGR2/FCGR3 locus. This analysis identified two highly paralogous 24.5 kb blocks within the FCGR2C/FCGR3B/FCGR2B locus that are devoid of nonpolymorphic paralogous sequence variations and that define the limits of the genomic regions in which nonallelic homologous recombination leads to FCGR2C/FCGR3B copy-number variation. Further, the data showed evidence of swapping of haplotype blocks between these highly paralogous blocks that most likely arose from sequential ancestral recombination events across the region. Functionally, we found by flow cytometry, immunoblotting and cDNA sequencing that individuals with FCGR3B-deleted alleles show ectopic presence of FcγRIIb on natural killer (NK) cells. We conclude that FCGR3B deletion juxtaposes the 5'-regulatory sequences of FCGR2C with the coding sequence of FCGR2B, creating a chimeric gene that results in an ectopic accumulation of FcγRIIb on NK cells and provides an explanation for SLE risk associated with reduced FCGR3B gene copy number. PMID:23261299

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

    Ana V Longo

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

  13. High-resolution array copy number analyses for detection of deletion, gain, amplification and copy-neutral LOH in primary neuroblastoma tumors: Four cases of homozygous deletions of the CDKN2A gene

    Kogner Per


    Full Text Available Abstract Background Neuroblastoma is a very heterogeneous pediatric tumor of the sympathetic nervous system showing clinically significant patterns of genetic alterations. Favorable tumors usually have near-triploid karyotypes with few structural rearrangements. Aggressive stage 4 tumors often have near-diploid or near-tetraploid karyotypes and structural rearrangements. Whole genome approaches for analysis of genome-wide copy number have been used to analyze chromosomal abnormalities in tumor samples. We have used array-based copy number analysis using oligonucleotide single nucleotide polymorphisms (SNP arrays to analyze the chromosomal structure of a large number of neuroblastoma tumors of different clinical and biological subsets. Results Ninety-two neuroblastoma tumors were analyzed with 50 K and/or 250 K SNP arrays from Affymetrix, using CNAG3.0 software. Thirty percent of the tumors harbored 1p deletion, 22% deletion of 11q, 26% had MYCN amplification and 45% 17q gain. Most of the tumors with 1p deletion were found among those with MYCN amplification. Loss of 11q was most commonly seen in tumors without MYCN amplification. In the case of MYCN amplification, two types were identified. One type displayed simple continuous amplicons; the other type harbored more complex rearrangements. MYCN was the only common gene in all cases with amplification. Complex amplification on chromosome 12 was detected in two tumors and three different overlapping regions of amplification were identified. Two regions with homozygous deletions, four cases with CDKN2A deletions in 9p and one case with deletion on 3p (the gene RBMS3 were also detected in the tumors. Conclusion SNP arrays provide useful tools for high-resolution characterization of significant chromosomal rearrangements in neuroblastoma tumors. The mapping arrays from Affymetrix provide both copy number and allele-specific information at a resolution of 10–12 kb. Chromosome 9p, especially the gene

  14. Relationship of immunohistochemistry, copy number aberrations and epigenetic disorders with BRCAness pattern in hereditary and sporadic breast cancer.

    Murria Estal, Rosa; Palanca Suela, Sarai; de Juan Jiménez, Inmaculada; Alenda Gonzalez, Cristina; Egoavil Rojas, Cecilia; García-Casado, Zaida; López Guerrero, Jose Antonio; Juan Fita, María José; Sánchez Heras, Ana Beatriz; Segura Huerta, Ángel; Santaballa Bertrán, Ana; Chirivella González, Isabel; Llop García, Marta; Pérez Simó, Gema; Barragán González, Eva; Bolufer Gilabert, Pascual


    The study aims to identify the relevance of immunohistochemistry (IHC), copy number aberrations (CNA) and epigenetic disorders in BRCAness breast cancers (BCs). We studied 95 paraffin included BCs, of which 41 carried BRCA1/BRCA2 germline mutations and 54 were non hereditary (BRCAX/Sporadic). Samples were assessed for BRCA1ness and CNAs by Multiplex Ligation-dependent Probe Amplification (MLPA); promoter methylation (PM) was assessed by methylation-specific-MLPA and the expression of miR-4417, miR-423-3p, miR-590-5p and miR-187-3p by quantitative RT-PCR. IHC markers Ki67, ER, PR, HER2, CK5/6, EGFR and CK18 were detected with specific primary antibodies (DAKO, Denmark). BRCAness association with covariates was performed using multivariate binary logistic regression (stepwise backwards Wald option). BRCA1/2 mutational status (p = 0.027), large tumor size (p = 0.041) and advanced histological grade (p = 0.017) among clinic-pathological variables; ER (p < 0.001) among IHC markers; MYC (p < 0.001) among CNA; APC (p = 0.065), ATM (p = 0.014) and RASSF1 (p = 0.044) among PM; and miR-590-5p (p = 0.001), miR-4417 (p = 0.019) and miR-423 (p = 0.013) among microRNA expression, were the selected parameters significantly related with the BRCAness status. The logistic regression performed with all these parameters selected ER+ as linked with the lack of BRCAness (p = 0.001) and MYC CNA, APC PM and miR-590-5p expression with BRCAness (p = 0.014, 0.045 and 0.007, respectively). In conclusion, the parameters ER expression, APC PM, MYC CNA and miR-590-5p expression, allowed detection of most BRCAness BCs. The identification of BRCAness can help establish a personalized medicine addressed to predict the response to specific treatments. PMID:26723934

  15. Insights on the functional impact of microRNAs present in autism-associated copy number variants.

    Varadarajan Vaishnavi

    Full Text Available Autism spectrum disorder is a complex neurodevelopmental disorder that appears during the first three years of infancy and lasts throughout a person's life. Recently a large category of genomic structural variants, denoted as copy number variants (CNVs, were established to be a major contributor of the pathophysiology of autism. To date almost all studies have focussed only on the genes present in the CNV loci, but the impact of non-coding regulatory microRNAs (miRNAs present in these regions remain largely unexplored. Hence we attempted to elucidate the biological and functional significance of miRNAs present in autism-associated CNV loci and their target genes by using a series of computational tools. We demonstrate that nearly 11% of the CNV loci harbor miRNAs and a few of these miRNAs were previously reported to be associated with autism. A systematic analysis of the CNV-miRNAs based on their interactions with the target genes enabled the identification of top 10 miRNAs namely hsa-miR-590-3p, hsa-miR-944, hsa-miR-570, hsa-miR-34a, hsa-miR-124, hsa-miR-548f, hsa-miR-429, hsa-miR-200b, hsa-miR-195 and hsa-miR-497 as hub molecules. Further, the CNV-miRNAs formed a regulatory loop with transcription factors and their downstream target genes, and annotation of these target genes indicated their functional involvement in neurodevelopment and synapse. Moreover, miRNAs present in deleted and duplicated CNV loci may explain the difference in dosage of the crucial genes controlled by them. These CNV-miRNAs can also impair the global processing and biogenesis of all miRNAs by targeting key molecules in the miRNA pathway. To our knowledge, this is the first report to highlight the significance of CNV-microRNAs and their target genes to contribute towards the genetic heterogeneity and phenotypic variability of autism.

  16. Screening for copy number alterations in loci associated with autism spectrum disorders by two-color multiplex ligation-dependent probe amplification

    Bremer, Anna; Giacobini, Maibritt; Nordenskjöld, Magnus;


    identified in a subgroup of individuals with ASD using array technology. Adequate genetic testing for these genomic imbalances have not yet been widely implemented in the diagnostic setting due to lack of feasible and cost-effective methods as well as difficulties to interpret the clinical significance of...... these small copy number variants (CNVs). We developed a multiplex ligation-dependent probe amplification (MLPA) assay to investigate its usefulness for detection of copy number alterations (CNAs) in autistic patients. This test proved to be easy to perform, fast, cost-effective, and suitable for...... analysis of a continuously increasing number of CNAs associated with autism. Our result show that MLPA assay is an easy and cost-effective method for the identification of selected CNAs in diagnostic laboratories....

  17. Global repeat discovery and estimation of genomic copy number in a large, complex genome using a high-throughput 454 sequence survey

    Varala Kranthi


    Full Text Available Abstract Background Extensive computational and database tools are available to mine genomic and genetic databases for model organisms, but little genomic data is available for many species of ecological or agricultural significance, especially those with large genomes. Genome surveys using conventional sequencing techniques are powerful, particularly for detecting sequences present in many copies per genome. However these methods are time-consuming and have potential drawbacks. High throughput 454 sequencing provides an alternative method by which much information can be gained quickly and cheaply from high-coverage surveys of genomic DNA. Results We sequenced 78 million base-pairs of randomly sheared soybean DNA which passed our quality criteria. Computational analysis of the survey sequences provided global information on the abundant repetitive sequences in soybean. The sequence was used to determine the copy number across regions of large genomic clones or contigs and discover higher-order structures within satellite repeats. We have created an annotated, online database of sequences present in multiple copies in the soybean genome. The low bias of pyrosequencing against repeat sequences is demonstrated by the overall composition of the survey data, which matches well with past estimates of repetitive DNA content obtained by DNA re-association kinetics (Cot analysis. Conclusion This approach provides a potential aid to conventional or shotgun genome assembly, by allowing rapid assessment of copy number in any clone or clone-end sequence. In addition, we show that partial sequencing can provide access to partial protein-coding sequences.

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

    Zhang Rui


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

  19. Methods for accurate quantification of LTR-retrotransposon copy number using short-read sequence data: a case study in Sorghum.

    Ramachandran, Dhanushya; Hawkins, Jennifer S


    Transposable elements (TEs) are ubiquitous in eukaryotic genomes and their mobility impacts genome structure and function in myriad ways. Because of their abundance, activity, and repetitive nature, the characterization and analysis of TEs remain challenging, particularly from short-read sequencing projects. To overcome this difficulty, we have developed a method that estimates TE copy number from short-read sequences. To test the accuracy of our method, we first performed an in silico analysis of the reference Sorghum bicolor genome, using both reference-based and de novo approaches. The resulting TE copy number estimates were strikingly similar to the annotated numbers. We then tested our method on real short-read data by estimating TE copy numbers in several accessions of S. bicolor and its close relative S. propinquum. Both methods effectively identify and rank similar TE families from highest to lowest abundance. We found that de novo characterization was effective at capturing qualitative variation, but underestimated the abundance of some TE families, specifically families of more ancient origin. Also, interspecific reference-based mapping of S. propinquum reads to the S. bicolor database failed to fully describe TE content in S. propinquum, indicative of recent TE activity leading to changes in the respective repetitive landscapes over very short evolutionary timescales. We conclude that reference-based analyses are best suited for within-species comparisons, while de novo approaches are more reliable for evolutionarily distant comparisons. PMID:27295958

  20. Ribosomal Protein Genes S23 and L35 from Amphioxus Branchiostoma belcheri tsingtauense: Identification and Copy Number

    Xian LI; Shi-Cui ZHANG; Zhen-Hui LIU; Hong-Yan LI


    The complete cDNA and deduced amino acid sequences of the ribosomal proteins S23 (AmphiS23) and L35 (AmphiL35) from amphioxus Branchiostoma belcheri tsingtauense were identified in this study. AmphiS23 cDNA is 546 bp long and encodes a protein of 143 amino acids. It has a predicted molecular mass of 15,851 Da and a pI of 10.7. AmphiL35 cDNA comprises 473 bp, and codes for a protein of 123 amino acids with a predicted molecular mass of 14,543 Da and a pI of 10.8. AmphiS23 shares more than 83% identity with its homologues in the vertebrates and more than 84% identity with those in the invertebrates. AmphiL35 is more than 63% identical to its counterparts in the vertebrates and more than 52% identical to those in the invertebrates, Southern blot analysis demonstrated the existence of 1-2 copies of the S23 gene and 2-3 copies of the L35 gene in the genome of amphioxus B. belcheri tsingtauense. This is in sharp contrast to the presence of 6-13 copies of the S23 gene and 15-17 copies of the L35 gene in the rat genome. It is clear that the housekeeping genes like S23 and L35 underwent a large-scale duplication in the vertebrate lineage, reinforcing the gene/genome duplication hypothesis.

  1. Plasmid stability in immobilized and free recombinant Escherichia coli JM105(pKK223-200): importance of oxygen diffusion, growth rate, and plasmid copy number.

    de Taxis du Poët, P; Arcand, Y; Bernier, R.; Barbotin, J N; Thomas, D.


    Stability of the plasmid pKK223-200 in Escherichia coli JM105 was studied for both free and immobilized cells during continuous culture. The relationship between plasmid copy number, xylanase activity, which was coded for by the plasmid, and growth rate and culture conditions involved complex interactions which determined the plasmid stability. Generally, the plasmid stability was enhanced in cultured immobilized cells compared with free-cell cultures. This stability was associated with modif...

  2. A novel simple method for determining CYP2D6 gene copy number and identifying allele(s with duplication/multiplication.

    Taimour Langaee

    Full Text Available Cytochrome P450 2D6 (CYP2D6 gene duplication and multiplication can result in ultrarapid drug metabolism and therapeutic failure or excessive response in patients. Long range polymerase chain reaction (PCR, restriction fragment length polymorphism (RFLP and sequencing are usually used for genotyping CYP2D6 duplication/multiplications and identification, but are labor intensive, time consuming, and costly.We developed a simple allele quantification-based Pyrosequencing genotyping method that facilitates CYP2D6 copy number variation (CNV genotyping while also identifying allele-specific CYP2D6 CNV in heterozygous samples. Most routine assays do not identify the allele containing a CNV. A total of 237 clinical and Coriell DNA samples with different known CYP2D6 gene copy numbers were genotyped for CYP2D6 *2, *3, *4, *6, *10, *17, *41 polymorphisms and CNV determination.The CYP2D6 gene allele quantification/identification were determined simultaneously with CYP2D6*2, *3, *4, *6, *10, *17, *41 genotyping. We determined the exact CYP2D6 gene copy number, identified which allele had the duplication or multiplication, and assigned the correct phenotype and activity score for all samples.Our method can efficiently identify the duplicated CYP2D6 allele in heterozygous samples, determine its copy number in a fraction of time compared to conventional methods and prevent incorrect ultrarapid phenotype calls. It also greatly reduces the cost, effort and time associated with CYP2D6 CNV genotyping.

  3. Genomic copy number analysis of a spectrum of blue nevi identifies recurrent aberrations of entire chromosomal arms in melanoma ex blue nevus.

    Chan, May P; Andea, Aleodor A; Harms, Paul W; Durham, Alison B; Patel, Rajiv M; Wang, Min; Robichaud, Patrick; Fisher, Gary J; Johnson, Timothy M; Fullen, Douglas R


    Blue nevi may display significant atypia or undergo malignant transformation. Morphologic diagnosis of this spectrum of lesions is notoriously difficult, and molecular tools are increasingly used to improve diagnostic accuracy. We studied copy number aberrations in a cohort of cellular blue nevi, atypical cellular blue nevi, and melanomas ex blue nevi using Affymetrix's OncoScan platform. Cases with sufficient DNA were analyzed for GNAQ, GNA11, and HRAS mutations. Copy number aberrations were detected in 0 of 5 (0%) cellular blue nevi, 3 of 12 (25%) atypical cellular blue nevi, and 6 of 9 (67%) melanomas ex blue nevi. None of the atypical cellular blue nevi displayed more than one aberration, whereas complex aberrations involving four or more regions were seen exclusively in melanomas ex blue nevi. Gains and losses of entire chromosomal arms were identified in four of five melanomas ex blue nevi with copy number aberrations. In particular, gains of 1q, 4p, 6p, and 8q, and losses of 1p and 4q were each found in at least two melanomas. Whole chromosome aberrations were also common, and represented the sole finding in one atypical cellular blue nevus. When seen in melanomas, however, whole chromosome aberrations were invariably accompanied by partial aberrations of other chromosomes. Three melanomas ex blue nevi harbored aberrations, which were absent or negligible in their precursor components, suggesting progression in tumor biology. Gene mutations involving GNAQ and GNA11 were each detected in two of eight melanomas ex blue nevi. In conclusion, copy number aberrations are more common and often complex in melanomas ex blue nevi compared with cellular and atypical cellular blue nevi. Identification of recurrent gains and losses of entire chromosomal arms in melanomas ex blue nevi suggests that development of new probes targeting these regions may improve detection and risk stratification of these lesions. PMID:26743478

  4. High-Throughput Copy Number Analysis of 17q23 in 3520 Tissue Specimens by Fluorescence in Situ Hybridization to Tissue Microarrays

    Andersen, Claus L; Monni, Outi; Wagner, Urs; Kononen, Juha; Bärlund, Maarit; Bucher, Christoph; Haas, Philippe; Nocito, Antonio; Bissig, Heidi; Sauter, Guido; Kallioniemi, Anne


    The chromosomal region 17q23 has been shown to be commonly amplified in breast tumors, especially those with poor prognosis. In addition to breast cancer, studies by comparative genomic hybridization have implicated the involvement of 17q23 in other tumor types as well. Here we performed a large-scale survey on the distribution and frequency of the 17q23 copy number increases across different tumor types using fluorescence in situ hybridization on tissue microarrays containing 4788 specimens....

  5. Proteasome-Mediated Degradation of the Papillomavirus E2-TA Protein Is Regulated by Phosphorylation and Can Modulate Viral Genome Copy Number

    Penrose, Kerri J.; Alison A McBride


    The bovine papillomavirus E2 proteins regulate viral transcription, replication, and episomal genome maintenance. We have previously mapped the major phosphorylation sites of the E2 proteins to serine residues 298 and 301 and shown that mutation of serine residue 301 to alanine leads to a dramatic (10- to 20-fold) increase in viral DNA copy number. In this study we analyzed how phosphorylation regulates E2 protein function. S301 is located in a PEST sequence; these sequences are often found i...

  6. Using next-generation sequencing for high resolution multiplex analysis of copy number variation from nanogram quantities of DNA from formalin-fixed paraffin-embedded specimens

    Wood, HM; Belvedere,O; Conway, C; Daly, C; Chalkley, R; Bickerdike, M; McKinley, C; Egan, P; Ross, L; Hayward, B; Morgan, J.; Davidson, L; MacLennan, K; Ong, TK; Papagiannopoulos, K.


    The use of next-generation sequencing technologies to produce genomic copy number data has recently been described. Most approaches, however, reply on optimal starting DNA, and are therefore unsuitable for the analysis of formalin-fixed paraffin-embedded (FFPE) samples, which largely precludes the analysis of many tumour series. We have sought to challenge the limits of this technique with regards to quality and quantity of starting material and the depth of sequencing required. We confirm th...

  7. Autism-specific copy number variants further implicate the phosphatidylinositol signaling pathway and the glutamatergic synapse in the etiology of the disorder

    Cuscó, Ivon; Medrano, Andrés; Gener, Blanca; Vilardell, Mireia; Gallastegui, Fátima; Villa, Olaya; González, Eva; Rodríguez-Santiago, Benjamín; Vilella, Elisabet; Del Campo, Miguel; Pérez-Jurado, Luis A.


    Autism spectrum disorders (ASDs) constitute a group of severe neurodevelopmental conditions with complex multifactorial etiology. In order to explore the hypothesis that submicroscopic genomic rearrangements underlie some ASD cases, we have analyzed 96 Spanish patients with idiopathic ASD after extensive clinical and laboratory screening, by array comparative genomic hybridization (aCGH) using a homemade bacterial artificial chromosome (BAC) array. Only 13 of the 238 detected copy number alte...

  8. Beyond KRAS mutation status: influence of KRAS copy number status and microRNAs on clinical outcome to cetuximab in metastatic colorectal cancer patients

    Mekenkamp Leonie JM


    Full Text Available Abstract Background KRAS mutation is a negative predictive factor for treatment with anti-epidermal growth factor receptor (EGFR antibodies in metastatic colorectal cancer (mCRC. Novel predictive markers are required to further improve the selection of patients for this treatment. We assessed the influence of modification of KRAS by gene copy number aberration (CNA and microRNAs (miRNAs in correlation to clinical outcome in mCRC patients treated with cetuximab in combination with chemotherapy and bevacizumab. Methods Formalin-fixed paraffin-embedded primary tumour tissue was used from 34 mCRC patients in a phase III trial, who were selected based upon their good (n = 17 or poor (n = 17 progression-free survival (PFS upon treatment with cetuximab in combination with capecitabine, oxaliplatin, and bevacizumab. Gene copy number at the KRAS locus was assessed using high resolution genome-wide array CGH and the expression levels of 17 miRNAs targeting KRAS were determined by real-time PCR. Results Copy number loss of the KRAS locus was observed in the tumour of 5 patients who were all good responders including patients with a KRAS mutation. Copy number gains in two wild-type KRAS tumours were associated with a poor PFS. In KRAS mutated tumours increased miR-200b and decreased miR-143 expression were associated with a good PFS. In wild-type KRAS patients, miRNA expression did not correlate with PFS in a multivariate model. Conclusions Our results indicate that the assessment of KRAS CNA and miRNAs targeting KRAS might further optimize the selection of mCRC eligible for anti-EGFR therapy.

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

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


    Various positively selected adaptations to new nutrients have been identified. Lactase persistence is among the best known, conferring the ability for drinking milk at post weaning age. An augmented number of amylase gene (AMY1) copies, giving rise to higher salivary amylase activity, has been implicated in the consumption of starch-rich foods. Higher AMY1 copy numbers have been demonstrated in populations with recent histories of starchy-rich diets. It is however questionable whether the resulting polymorphisms have exerted positive selection only by providing easily available sources of macro and micronutrients. Humans have explored new environments more than any other animal. Novel environments challenge the host, but especially its immune system with new climatic conditions, food and especially pathogens. With the advent of the agricultural revolution and the concurrent domestication of cattle came new pathogens. We contend that specific new food ingredients (e.g., gluten) and novel pathogens drove selection for lactase persistence and higher AMY gene copy numbers. Both adaptations provide ample glucose for activating the sodium glucose-dependent co-transporter 1 (SGLT1), which is the principal glucose, sodium and water transporter in the gastro-intestinal tract. Their rapid uptake confers protection against potentially lethal dehydration, hyponatremia and ultimately multiple organ failure. Oral rehydration therapy aims at SGLT1 activity and is the current treatment of choice for chronic diarrhoea and vomiting. We hypothesize that lifelong lactase activity and rapid starch digestion should be looked at as the evolutionary covalent of oral rehydration therapy. PMID:24472865

  10. Phylogenetic analysis of multiple FISH markers in oral tongue squamous cell carcinoma suggests that a diverse distribution of copy number changes is associated with poor prognosis.

    Wangsa, Darawalee; Chowdhury, Salim Akhter; Ryott, Michael; Gertz, E Michael; Elmberger, Göran; Auer, Gert; Åvall Lundqvist, Elisabeth; Küffer, Stefan; Ströbel, Philipp; Schäffer, Alejandro A; Schwartz, Russell; Munck-Wikland, Eva; Ried, Thomas; Heselmeyer-Haddad, Kerstin


    Oral tongue squamous cell carcinoma (OTSCC) is associated with poor prognosis. To improve prognostication, we analyzed four gene probes (TERC, CCND1, EGFR and TP53) and the centromere probe CEP4 as a marker of chromosomal instability, using fluorescence in situ hybridization (FISH) in single cells from the tumors of sixty-five OTSCC patients (Stage I, n = 15; Stage II, n = 30; Stage III, n = 7; Stage IV, n = 13). Unsupervised hierarchical clustering of the FISH data distinguished three clusters related to smoking status. Copy number increases of all five markers were found to be correlated to non-smoking habits, while smokers in this cohort had low-level copy number gains. Using the phylogenetic modeling software FISHtrees, we constructed models of tumor progression for each patient based on the four gene probes. Then, we derived test statistics on the models that are significant predictors of disease-free and overall survival, independent of tumor stage and smoking status in multivariate analysis. The patients whose tumors were modeled as progressing by a more diverse distribution of copy number changes across the four genes have poorer prognosis. This is consistent with the view that multiple genetic pathways need to become deregulated in order for cancer to progress. PMID:26175310

  11. Inverse PCR and Quantitative PCR as Alternative Methods to Southern Blotting Analysis to Assess Transgene Copy Number and Characterize the Integration Site in Transgenic Woody Plants.

    Stefano, Biricolti; Patrizia, Bogani; Matteo, Cerboneschi; Massimo, Gori


    One of the major unanswered questions with respect to the commercial use of genetic transformation in woody plants is the stability of the transgene expression over several decades within the same individual. Gene expression is strongly affected by the copy number which has been integrated into the plant genome and by the local DNA features close to the integration sites. Because woody plants cannot be subjected to selfing or backcrossing to modify the transgenic allelic structure without affecting the valuable traits of the cultivar, molecular characterization of the transformation event is therefore crucial. After assessing the transgene copy number of a set of apple transgenic clones with Southern blotting, we describe two alternative methods: the first is based on inverse PCR (i-PCR) and the second on the quantitative PCR (q-PCR). The methods produced comparable results with the exception of the data regarding a high copy number clone, but while the q-PCR-based system is rapid and easily adaptable to high throughput systems, the i-PCR-based method can provide information regarding the transformation event and the characteristics of the sequences flanking the transgenic construct. PMID:26895172

  12. Copy number variation in glutathione-S-transferase T1 and M1 predicts incidence and 5-year survival from prostate and bladder cancer, and incidence of corpus uteri cancer in the general population

    Nørskov, M S; Frikke-Schmidt, R; Bojesen, S E;


    Glutathione-S-transferase T1 (GSTT1) and GSTM1 detoxify carcinogens and thus potentially contribute to inter-individual susceptibility to cancer. We determined the ability of GST copy number variation (CNV) to predict the risk of cancer in the general population. Exact copy numbers of GSTT1 and G...

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

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


    Frost tolerance is critical for wheat survival during cold winters. Natural variation for this trait is mainly associated with allelic differences at the VERNALIZATION 1 (VRN1) and FROST RESISTANCE 2 (FR2) loci. VRN1 regulates the transition between vegetative and reproductive stages and FR2, a locus including several tandemly duplicated C-REPEAT BINDING FACTOR (CBF) transcription factors, regulates the expression of Cold regulated genes. We identified sequence and copy number variation at these two loci among winter and spring wheat varieties and characterized their association with frost tolerance. We identified two FR-A2 haplotypes – ‘FR-A2-S’ and ‘FR-A2-T’ – distinguished by two insertion/deletions and ten single nucleotide polymorphisms within the CBF-A12 and CBF-A15 genes. Increased copy number of CBF-A14 was frequently associated with the FR-A2-T haplotypes and with higher CBF14 transcript levels in response to cold. Factorial ANOVAs revealed significant interactions between VRN1 and FR-A2 for frost tolerance in both winter and spring panels suggesting a crosstalk between vernalization and cold acclimation pathways. The model including these two loci and their interaction explained 32.0 and 20.7% of the variation in frost tolerance in the winter and spring panels, respectively. The interaction was validated in a winter wheat F4:5 population segregating for both genes. Increased VRN-A1 copy number was associated with improved frost tolerance among varieties carrying the FR-A2-T allele but not among those carrying the FR-A2-S allele. These results suggest that selection of varieties carrying the FR-A2-T allele and three copies of the recessive vrn-A1 allele would be a good strategy to improve frost tolerance in wheat. PMID:24626953

  14. High copy numbers and N terminal insertion position of influenza A M2E fused with hepatitis B core antigen enhanced immunogenicity.

    Sun, Xincheng; Wang, Yunlong; Dong, Caiwen; Hu, Jinqiang; Yang, Liping


    The extra domain of influenza M2 protein (M2e) is almost completely conserved among all influenza A virus subtypes. M2e is a promising candidate target for the development of a broad-spectrum recombinant influenza A vaccine. However, the immunogenicity of M2e needs to be improved. Copy numbers of M2e and its fusion expression with different carrier proteins may affect its immunopotency. In this study, we designed and created different constructs through genetic fusion of M2e (MSLLTEVETPTRSEWECRCSDSSD) (A/California/05/2009 (H1N1)) with the N-terminus (HBcAg1-149aa + Cys) by insertion in the N-terminus Hepatitis B Core (HBc) antigen 1-149aa and Middle 78-81aa of HBcAg1-149aa to construct a recombinant M2e-based vaccine candidate. These chimeric sequences were expressed in Escherichia coli. We constructed fusion proteins containing influenza A H1N1 influenza virus (2009), as well as one, two, and three copies of M2e and hepatitis B core antigen1-149aa amino acid-optimized codon inserted N and its intermediate. The recombinant protein was expressed and purified. Western blot analysis was employed to evaluate the expression of the M2e recombinant protein containing different copy numbers of M2e. Mice were immunized for two times with the purified fusion protein HBc/M2e BALB/c. Serum levels of M2e antibody gradually increased along with increase in immunity. The levels of different fusion protein M2e antibodies increase with increasing M2e copy number. In addition, the protein antibody level in the N terminal fusion protein is higher than that in intermediate fusion. PMID:26355223

  15. Lack of independent prognostic and predictive value of centromere 17 copy number changes in breast cancer patients with known HER2 and TOP2A status

    Nielsen, Kirsten Vang; Ejlertsen, Bent; Møller, Susanne;


    The clinical benefit of anthracyclines has been connected to HER2 status, TOP2A status and centromere 17 copy numbers (CEN-17). Data from a clinical trial randomizing patients to anthracyclines was used to assess whether the number of CEN-17 in breast cancers may predict incremental responsiveness...... determined in normal breast were applied to data on 767 patients with known HER2 and TOP2A status randomized to anthracyclines in the DBCG 89D trial. CEN-17 ploidy levels were in cut sections from the 767 breast cancer patients established as: Haploid: ≤1.25 (10%), diploid: 1.26-2.09 (60%), triploid: 2...

  16. Pan-cancer analysis of copy number changes in programmed death-ligand 1 (PD-L1, CD274) - associations with gene expression, mutational load, and survival.

    Budczies, Jan; Bockmayr, Michael; Denkert, Carsten; Klauschen, Frederick; Gröschel, Stefan; Darb-Esfahani, Silvia; Pfarr, Nicole; Leichsenring, Jonas; Onozato, Maristela L; Lennerz, Jochen K; Dietel, Manfred; Fröhling, Stefan; Schirmacher, Peter; Iafrate, A John; Weichert, Wilko; Stenzinger, Albrecht


    Inhibition of the PD-L1 (CD274) - PD-1 axis has emerged as a powerful cancer therapy that prevents evasion of tumor cells from the immune system. While immunohistochemical detection of PD-L1 was introduced as a predictive biomarker with variable power, much less is known about copy number alterations (CNA) affecting PD-L1 and their associations with expression levels, mutational load, and survival. To gain insight, we employed The Cancer Genome Atlas (TCGA) datasets to comprehensively analyze 22 major cancer types for PD-L1 CNAs. We observed a diverse landscape of PD-L1 CNAs, which affected focal regions, chromosome 9p or the entire chromosome 9. Deletions of PD-L1 were more frequent than gains (31% vs. 12%) with deletions being most prevalent in melanoma and non-small cell lung cancer. Copy number gains most frequently occurred in ovarian cancer, head and neck cancer, bladder cancer, cervical and endocervical cancer, sarcomas, and colorectal cancers. Fine-mapping of the genetic architecture revealed specific recurrently amplified and deleted core regions across cancers with putative biological and clinical consequences. PD-L1 CNAs correlated significantly with PD-L1 mRNA expression changes in many cancer types, and tumors with PD-L1 gains harbored significantly higher mutational load compared to non-amplified cases (median: 78 non-synonymous mutations vs. 40, P = 7.1e-69). Moreover, we observed that, in general, both PD-L1 amplifications and deletions were associated with dismal prognosis. In conclusion, PD-L1 CNAs, in particular PD-L1 copy number gains, represent frequent genetic alterations across many cancers, which influence PD-L1 expression levels, are associated with higher mutational loads, and may be exploitable as predictive biomarker for immunotherapy regimens. © 2016 Wiley Periodicals, Inc. PMID:27106868

  17. Optical Imaging of Paramagnetic Bead-DNA Aggregation Inhibition Allows for Low Copy Number Detection of Infectious Pathogens.

    Jacquelyn A DuVall

    Full Text Available DNA-paramagnetic silica bead aggregation in a rotating magnetic field facilitates the quantification of DNA with femtogram sensitivity, but yields no sequence-specific information. Here we provide an original description of aggregation inhibition for the detection of DNA and RNA in a sequence-specific manner following loop-mediated isothermal amplification (LAMP. The fragments generated via LAMP fail to induce chaotrope-mediated bead aggregation; however, due to their ability to passivate the bead surface, they effectively inhibit bead aggregation by longer 'trigger' DNA. We demonstrate the utility of aggregation inhibition as a method for the detection of bacterial and viral pathogens with sensitivity that approaches single copies of the target. We successfully use this methodology for the detection of notable food-borne pathogens Escherichia coli O157:H7 and Salmonella enterica, as well as Rift Valley fever virus, a weaponizable virus of national security concern. We also show the concentration dependence of aggregation inhibition, suggesting the potential for quantification of target nucleic acid in clinical and environmental samples. Lastly, we demonstrate the ability to rapidly detect infectious pathogens by utilizing a cell phone and custom-written application (App, making this novel detection modality fully portable for point-of-care use.

  18. Mechanisms of topoisomerase I (TOP1) gene copy number increase in a stage III colorectal cancer patient cohort

    David Hersi Smith; Ib Jarle Christensen; Niels Frank Jensen; Bo Markussen; Maria Unni Rømer; Sune Boris Nygård; Sven Müller; Hans Jørgen Nielsen; Nils Brünner; Kirsten Vang Nielsen


    BACKGROUND: Topoisomerase I (Top1) is the target of Top1 inhibitor chemotherapy. The TOP1 gene, located at 20q12-q13.1, is frequently detected at elevated copy numbers in colorectal cancer (CRC). The present study explores the mechanism, frequency and prognostic impact of TOP1 gene aberrations in stage III CRC and how these can be detected by fluorescent in situ hybridization (FISH). METHODS: Nine CRC cell line metaphase spreads were analyzed by FISH with a TOP1 probe in combination with a re...

  19. Studies on the plasmid stability, plasmid copy number and endo(1, 3)(1, 4) b-glucanase production by free and alginate immobilised recombinant saccharomyces cerevisiae cells

    Canavan, Peter D.


    A recombinant yeast strain, Saccharomyces cerevisiae DBY746, containing the plasmid pJG317, was grown in a variety of fermentation modes including batch, serial batch and chemostat culture incorporating a wide range of media types Plasmid pJG317 consists of a 2^-denved yeast episomal plasmid containing the gene which encodes for the bacterial enzyme endo (1,3)(1,4) P-glucanase. The concentration of enzyme produced appears to be proportional to the number of plasmid copies per cell. Specific e...

  20. Copy Number Analysis of 24 Oncogenes: MDM4 Identified as a Putative Marker for Low Recurrence Risk in Non Muscle Invasive Bladder Cancer

    Samanta Salvi; Daniele Calistri; Giorgia Gurioli; Elisa Carretta; Luigi Serra; Roberta Gunelli; Wainer Zoli; Valentina Casadio


    Patients with non-muscle invasive bladder cancer (NMIBC) generally have a high risk of relapsing locally after primary tumor resection. The search for new predictive markers of local recurrence thus represents an important goal for the management of this disease. We studied the copy number variations (CNVs) of 24 oncogenes (MDM4, MYCN, ALK, PDGFRA, KIT, KDR, DHFR, EGFR, MET, SMO, FGFR1, MYC, ABL1, RET, CCND1, CCND2, CDK4, MDM2, AURKB, ERBB2, TOP2A, AURKA, AR and BRAF) using multiplex ligati...

  1. An explorative analysis of ERCC1-19q13 copy number aberrations in a chemonaive stage III colorectal cancer cohort

    Smith, David Hersi; Christensen, Ib Jarle; Jensen, Niels Frank;


    Background: Platinum-based chemotherapy has long been used in the treatment of a variety of cancers and functions by inducing DNA damage. ERCC1 and ERCC4 are involved in the removal of this damage and have previously been implicated in resistance to platinum compounds. The aim of the current...... line panel and in tumor sections from 152 stage III CRC chemonaive patients. Relationships between biomarker status and clinical endpoints (overall survival, time to recurrence, and local recurrence in rectal cancer) were analyzed by survival statistics. Results: ERCC1-19q13 copy number alterations...

  2. The association of reduced folate carrier 80G>A polymorphism to outcome in childhood acute lymphoblastic leukemia interacts with chromosome 21 copy number

    Gregers, Jannie; Christensen, Ib Jarle; Dalhoff, Kim; Lausen, Birgitte Frederiksen; Schroeder, Henrik; Rosthoej, Steen; Carlsen, Niels; Schmiegelow, Kjeld; Peterson, Curt


    with chromosome 21 copy number in the leukemic clone. A total of 500 children with acute lymphoblastic leukemia treated according to the common Nordic treatment protocols were included, and we found that the RFC AA variant was associated with a 50% better chance of staying in remission compared with GG......The reduced folate carrier (RFC) is involved in the transport of methotrexate (MTX) across the cell membrane. The RFC gene (SLC19A1) is located on chromosome 21, and we hypothesized that the RFC80 G>A polymorphism would affect outcome and toxicity in childhood leukemia and that this could interact...

  3. Detection of Clonal and Subclonal Copy-Number Variants in Cell-Free DNA from Patients with Breast Cancer Using a Massively Multiplexed PCR Methodology

    Eser Kirkizlar; Bernhard Zimmermann; Tudor Constantin; Ryan Swenerton; Bin Hoang; Nicholas Wayham; Babiarz, Joshua E.; Zachary Demko; Robert J Pelham; Stephanie Kareht; Alexander L. Simon; Kristine N. Jinnett; Matthew Rabinowitz; Styrmir Sigurjonsson; Matthew Hill


    We demonstrate proof-of-concept for the use of massively multiplexed PCR and next-generation sequencing (mmPCR-NGS) to identify both clonal and subclonal copy-number variants (CNVs) in circulating tumor DNA. This is the first report of a targeted methodology for detection of CNVs in plasma. Using an in vitro model of cell-free DNA, we show that mmPCR-NGS can accurately detect CNVs with average allelic imbalances as low as 0.5%, an improvement over previously reported whole-genome sequencing a...

  4. SCRIB and PUF60 are primary drivers of the multisystemic phenotypes of the 8q24.3 copy-number variant

    Dauber, Andrew; Golzio, Christelle; Guenot, Cécile;


    Copy-number variants (CNVs) represent a significant interpretative challenge, given that each CNV typically affects the dosage of multiple genes. Here we report on five individuals with coloboma, microcephaly, developmental delay, short stature, and craniofacial, cardiac, and renal defects who...... phenotype, and the combinatorial suppression of both genes exacerbated some, but not all, phenotypic components. Consistent with these findings, we identified an individual with microcephaly, short stature, intellectual disability, and heart defects with a de novo c.505C>T variant leading to a p.His169Tyr...

  5. Chimera-free, high copy number YAC libraries and efficient methods of analysis. Final technical progress report



    The first experiment involved a low chimera YAC library in recombination deficient host strains. To determine if the genetic background of the yeast host strain contributes to the formation of chimeric YACs the same YAC ligation mixture was introduced into three isogenic yeast hosts differing only in their recombination abilities. To prepare YACs, human genomic DNA was partially digested with EcoR1 and then ligated to YAC vector pCGS966 arms. DNA was size fractionated before and after ligation by preparative pulsed field gel electrophoresis (CHEF), selecting for fragments greater than 400 kb, and introduced into competent spheroplasts. CHEF gel Southern blots of resulting colony-purified YACs were probed with human DNA to determine if multiple YACs or YAC fragments were present in the same cell. The frequency of chimeric YACs was measured by fluorescence in situ hybridization (FISH) of YACs to human prometaphase spreads. YACs that hybridized to more than one location were assumed to be chimeric. In the second experiment new YAC vectors featuring tags for capture of YACs and YAC inserts were constructed. Yeast Artificial Chromosomes (YACs) have been of tremendous value in the physical mapping of the human genome. Because they can carry very large inserts, YACs are likely not only to contain entire genes but also their control elements. However, the only mode of purification of YAC DNA from current commonly used YAC libraries such as the CEPH library is by pulsed field gel electrophoresis. This is an inefficient, time consuming process and due to the single copy nature of these YACs, often result in poor yields. The vector pCGS1000 was designed to test new efficient ways of YAC DNA purification.

  6. Copy number variation at the HvCBF4-HvCBF2 genomic segment is a major component of frost resistance in barley.

    Francia, Enrico; Morcia, Caterina; Pasquariello, Marianna; Mazzamurro, Valentina; Milc, Justyna Anna; Rizza, Fulvia; Terzi, Valeria; Pecchioni, Nicola


    A family of CBF transcription factors plays a major role in reconfiguring the plant transcriptome in response to low-freezing temperature in temperate cereals. In barley, more than 13 HvCBF genes map coincident with the major QTL FR-H2 suggesting them as candidates to explain the function of the locus. Variation in copy number (CNV) of specific HvCBFs was assayed in a panel of 41 barley genotypes using RT-qPCR. Taking advantage of an accurate phenotyping that combined Fv/Fm and field survival, resistance-associated variants within FR-H2 were identified. Genotypes with an increased copy number of HvCBF4 and HvCBF2 (at least ten and eight copies, respectively) showed greater frost resistance. A CAPS marker able to distinguish the CBF2A, CBF2B and CBF2A/B forms was developed and showed that all the higher-ranking genotypes in term of resistance harbour only CBF2A, while other resistant winter genotypes harbour also CBF2B, although at a lower CNV. In addition to the major involvement of the HvCBF4-HvCBF2 genomic segment in the proximal cluster of CBF elements, a negative role of HvCBF3 in the distal cluster was identified. Multiple linear regression models taking into account allelic variation at FR-H1/VRN-H1 explained 0.434 and 0.550 (both at p < 0.001) of the phenotypic variation for Fv/Fm and field survival respectively, while no interaction effect between CNV at the HvCBFs and FR-H1/VRN-H1 was found. Altogether our data suggest a major involvement of the CBF genes located in the proximal cluster, with no apparent involvement of the central cluster contrary to what was reported for wheat. PMID:27338258

  7. Haploinsufficiency of ANKRD11 (16q24.3) Is Not Obligatorily Associated with Cognitive Impairment but Shows a Clinical Overlap with Silver-Russell Syndrome

    Spengler, S.; Oehl-Jaschkowitz, B.; Begemann, M.; Hennes, P.; Zerres, K.; Eggermann, T.


    Microdeletions in 16q24.3 are associated with intellectual disability and a specific phenotype, e.g. short stature and a prominent forehead. The 16q24.3 microdeletion syndrome shows a broad phenotypic overlap with the KBG syndrome, which is caused by mutations within the ANKRD11 gene. Furthermore, both KBG and the 16q24.3 microdeletion syndromes show clinical findings reminiscent of Silver-Russell syndrome (SRS), an imprinting disorder characterized by severe primordial growth retardation. In a cohort of patients referred as SRS, we previously identified a 16q24.3 deletion, but at that time, only patients with larger imbalances in 16q24.3 and intellectual disability had been published. Considering the recent description of the ANKRD11 gene as the causative factor for the 2 16q24.3-associated disorders, we now classified our patient as a 16q24.3 microdeletion syndrome patient exhibiting some characteristic features but normal intelligence. Our case illustrates the broad clinical spectrum associated with microdeletions, and we confirm that the 16q24.3 microdeletion syndrome is a further microdeletion syndrome with very variable expressivity. Indeed, our case is the first 16q24.3 patient of normal intelligence, but we assume that this variant is present in further mentally healthy probands which have not yet been tested. In conclusion, the detection of the 16q24.3 deletion in a proband of unremarkable intellectual capacities once again illustrates the need to perform molecular karyotyping in dysmorphic patients with normal intelligence. PMID:23885231

  8. Genome-wide loss of heterozygosity and copy number alteration in esophageal squamous cell carcinoma using the Affymetrix GeneChip Mapping 10 K array

    Goldstein Alisa M


    Full Text Available Abstract Background Esophageal squamous cell carcinoma (ESCC is a common malignancy worldwide. Comprehensive genomic characterization of ESCC will further our understanding of the carcinogenesis process in this disease. Results Genome-wide detection of chromosomal changes was performed using the Affymetrix GeneChip 10 K single nucleotide polymorphism (SNP array, including loss of heterozygosity (LOH and copy number alterations (CNA, for 26 pairs of matched germ-line and micro-dissected tumor DNA samples. LOH regions were identified by two methods – using Affymetrix's genotype call software and using Affymetrix's copy number alteration tool (CNAT software – and both approaches yielded similar results. Non-random LOH regions were found on 10 chromosomal arms (in decreasing order of frequency: 17p, 9p, 9q, 13q, 17q, 4q, 4p, 3p, 15q, and 5q, including 20 novel LOH regions (10 kb to 4.26 Mb. Fifteen CNA-loss regions (200 kb to 4.3 Mb and 36 CNA-gain regions (200 kb to 9.3 Mb were also identified. Conclusion These studies demonstrate that the Affymetrix 10 K SNP chip is a valid platform to integrate analyses of LOH and CNA. The comprehensive knowledge gained from this analysis will enable improved strategies to prevent, diagnose, and treat ESCC.

  9. Performance Evaluation of NIPT in Detection of Chromosomal Copy Number Variants Using Low-Coverage Whole-Genome Sequencing of Plasma DNA.

    Hongtai Liu

    Full Text Available The aim of this study was to assess the performance of noninvasively prenatal testing (NIPT for fetal copy number variants (CNVs in clinical samples, using a whole-genome sequencing method.A total of 919 archived maternal plasma samples with karyotyping/microarray results, including 33 CNVs samples and 886 normal samples from September 1, 2011 to May 31, 2013, were enrolled in this study. The samples were randomly rearranged and blindly sequenced by low-coverage (about 7M reads whole-genome sequencing of plasma DNA. Fetal CNVs were detected by Fetal Copy-number Analysis through Maternal Plasma Sequencing (FCAPS to compare to the karyotyping/microarray results. Sensitivity, specificity and were evaluated.33 samples with deletions/duplications ranging from 1 to 129 Mb were detected with the consistent CNV size and location to karyotyping/microarray results in the study. Ten false positive results and two false negative results were obtained. The sensitivity and specificity of detection deletions/duplications were 84.21% and 98.42%, respectively.Whole-genome sequencing-based NIPT has high performance in detecting genome-wide CNVs, in particular >10Mb CNVs using the current FCAPS algorithm. It is possible to implement the current method in NIPT to prenatally screening for fetal CNVs.

  10. Association studies of the copy-number variable ß-defensin cluster on 8p23.1 in adenocarcinoma and chronic pancreatitis

    Taudien Stefan


    Full Text Available Abstract Background Human ß-defensins are a family of antimicrobial peptides located at the mucosal surface. Both sequence multi-site variations (MSV and copy-number variants (CNV of the defensin-encoding genes are associated with increased risk for various diseases, including cancer and inflammatory conditions such as psoriasis and acute pancreatitis. In a case–control study, we investigated the association between MSV in DEFB104 as well as defensin gene (DEF cluster copy number (CN, and pancreatic ductal adenocarcinoma (PDAC and chronic pancreatitis (CP. Results Two groups of PDAC (N=70 and CP (N=60 patients were compared to matched healthy control groups CARLA1 (N=232 and CARLA2 (N=160, respectively. Four DEFB104 MSV were haplotyped by PCR, cloning and sequencing. DEF cluster CN was determined by multiplex ligation-dependent probe amplification. Neither the PDAC nor the CP cohorts show significant differences in the DEFB104 haplotype distribution compared to the respective control groups CARLA1 and CARLA2, respectively. The diploid DEF cluster CN exhibit a significantly different distribution between PDAC and CARLA1 (Fisher’s exact test P=0.027, but not between CP and CARLA2 (P=0.867. Conclusion Different DEF cluster b CN distribution between PDAC patients and healthy controls indicate a potential protective effect of higher CNs against the disease.

  11. Porcine MAP3K5 analysis: molecular cloning, characterization, tissue expression pattern, and copy number variations associated with residual feed intake.

    Pu, L; Zhang, L C; Zhang, J S; Song, X; Wang, L G; Liang, J; Zhang, Y B; Liu, X; Yan, H; Zhang, T; Yue, J W; Li, N; Wu, Q Q; Wang, L X


    Mitogen-activated protein kinase kinase kinase 5 (MAP3K5) is essential for apoptosis, proliferation, differentiation, and immune responses, and is a candidate marker for residual feed intake (RFI) in pig. We cloned the full-length cDNA sequence of porcine MAP3K5 by rapid-amplification of cDNA ends. The 5451-bp gene contains a 5'-untranslated region (UTR) (718 bp), a coding region (3738 bp), and a 3'-UTR (995 bp), and encodes a peptide of 1245 amino acids, which shares 97, 99, 97, 93, 91, and 84% sequence identity with cattle, sheep, human, mouse, chicken, and zebrafish MAP3K5, respectively. The deduced MAP3K5 protein sequence contains two conserved domains: a DUF4071 domain and a protein kinase domain. Phylogenetic analysis showed that porcine MAP3K5 forms a separate branch to vicugna and camel MAP3K5. Tissue expression analysis using real-time quantitative polymerase chain reaction (qRT-PCR) revealed that MAP3K5 was expressed in the heart, liver, spleen, lung, kidney, muscle, fat, pancrea, ileum, and stomach tissues. Copy number variation was detected for porcine MAP3K5 and validated by qRT-PCR. Furthermore, a significant increase in average copy number was detected in the low RFI group when compared to the high RFI group in a Duroc pig population. These results provide useful information regarding the influence of MAP3K5 on RFI in pigs. PMID:27525933

  12. Frequently increased epidermal growth factor receptor (EGFR) copy numbers and decreased BRCA1 mRNA expression in Japanese triple-negative breast cancers

    Triple-negative breast cancer (estrogen receptor-, progesterone receptor-, and HER2-negative) (TNBC) is a high risk breast cancer that lacks specific therapy targeting these proteins. We studied 969 consecutive Japanese patients diagnosed with invasive breast cancer from January 1981 to December 2003, and selected TNBCs based on the immunohistochemical data. Analyses of epidermal growth factor receptor (EGFR) gene mutations and amplification, and BRCA1 mRNA expression were performed on these samples using TaqMan PCR assays. The prognostic significance of TNBCs was also explored. Median follow-up was 8.3 years. A total of 110 (11.3%) patients had TNBCs in our series. Genotyping of the EGFR gene was performed to detect 14 known EGFR mutations, but none was identified. However, EGFR gene copy number was increased in 21% of TNBCs, while only 2% of ER- and PgR-positive, HER2-negative tumors showed slightly increased EGFR gene copy numbers. Thirty-one percent of TNBCs stained positive for EGFR protein by immunohistochemistry. BRCA1 mRNA expression was also decreased in TNBCs compared with controls. Triple negativity was significantly associated with grade 3 tumors, TP53 protein accumulation, and high Ki67 expression. TNBC patients had shorter disease-free survival than non-TNBC in node-negative breast cancers. TNBCs have an aggressive clinical course, and EGFR and BRCA1 might be candidate therapeutic targets in this disease

  13. Cytogenetic abnormalities and genomic copy number variations in EPO (7q22) and SEC-61(7p11) genes in primary myelodysplastic syndromes.

    Mohanty, Purvi; Korgaonkar, Seema; Shanmukhaiah, Chandrakala; Ghosh, Kanjaksha; Vundinti, Babu Rao


    Myelodysplastic syndromes (MDSs) are heterogeneous clonal haematopoeitic stem cell disorders characterized by ineffective haematopoeisis, cytopenias and risk of progression to AML. We studied 150 MDS patients for cytogenetic aberrations and 60 patients with normal karyotype and 40 patients harboring cytogenetic abnormalities for copy number variations (CNVs). Cytogenetic abnormalities were detected in 46% of patients with a majority of patients harboring abnormalities of chromosome 7 and del (20q) at frequencies of 16% and 12% respectively. We explored the potential of quantitative multiplex PCR assay of short fluorescent fragments (QMPSF) to identify CNVs and correlated the findings with cytogenetic data and disease prognosis. CNVs (n=31) were detected in 28.3% of karyotypically normal and 23% patients with abnormal karyotype. Genetic losses or deletions (n=26) were more frequent than duplications (n=5). EPO (7q22) and SEC-61(7p11) emerged as new candidate genes susceptible to genetic losses with 57.7% deletions identified in regions on chromosome 7. The CNVs correlated with International Prognostic Scoring System (IPSS) intermediate disease risk group. Our integrative cytogenetic and copy number variation study suggests that abnormalities of chromosome 7 are predominant in Indian population and that they may play a secondary role in disease progression and should be evaluated further for asserting their clinical significance and influence on disease prognosis. PMID:27282568

  14. Array CGH identifies distinct DNA copy number profiles of oncogenes and tumor suppressor genes in chromosomal- and microsatellite-unstable sporadic colorectal carcinomas.

    Lassmann, Silke; Weis, Roland; Makowiec, Frank; Roth, Jasmine; Danciu, Mihai; Hopt, Ulrich; Werner, Martin


    DNA copy number changes represent molecular fingerprints of solid tumors and are as such relevant for better understanding of tumor development and progression. In this study, we applied genome-wide array comparative genomic hybridization (aCGH) to identify gene-specific DNA copy number changes in chromosomal (CIN)- and microsatellite (MIN)-unstable sporadic colorectal cancers (sCRC). Genomic DNA was extracted from microdissected, matching normal colorectal epithelium and invasive tumor cells of formalin-fixed and paraffin-embedded tissues of 22 cases with colorectal cancer (CIN = 11, MIN = 11). DNA copy number changes were determined by aCGH for 287 target sequences in tumor cell DNAs, using pooled normal DNAs as reference. aCGH data of tumor cell DNAs was confirmed by fluorescence in situ hybridization (FISH) for three genes on serial tissues as those used for aCGH. aCGH revealed DNA copy number changes previously described by metaphase CGH (gains 7, 8q, 13q, and 20q; losses 8p, 15q, 18q, and 17p). However, chromosomal regions 20q, 13q, 7, and 17p were preferentially altered in CIN-type tumors and included DNA amplifications of eight genes on chromosome 20q (TOP1, AIB1, MYBL2, CAS, PTPN1, STK15, ZNF217, and CYP24), two genes on chromosome 13q (BRCA2 and D13S25), and three genes on chromosome 7 (IL6, CYLN2, and MET) as well as DNA deletions of two genes on chromosome 17p (HIC1 and LLGL1). Finally, additional CIN-tumor-associated DNA amplifications were identified for EXT1 (8q24.11) and MYC (8q24.12) as well as DNA deletions for MAP2K5 (15q23) and LAMA3 (18q11.2). In contrast, distinct MIN-tumor-associated DNA amplifications were detected for E2F5 (8p22-q21.3), GARP (11q13.5-q14), ATM (11q22.3), KAL (Xp22.3), and XIST (Xq13.2) as well as DNA deletions for RAF1 (3p25), DCC (18q21.3), and KEN (21q tel). aCGH revealed distinct DNA copy number changes of oncogenes and tumor suppressor genes in CIN- and MIN-type sporadic colorectal carcinomas. The identified candidate

  15. Phenotype of transgenic mice carrying a very low copy number of the mutant human G93A superoxide dismutase-1 gene associated with amyotrophic lateral sclerosis.

    Jeffrey S Deitch

    Full Text Available Amyotrophic lateral sclerosis (ALS is a progressive neurodegenerative disease of the motor neuron. While most cases of ALS are sporadic, 10% are familial (FALS with 20% of FALS caused by a mutation in the gene that codes for the enzyme Cu/Zn superoxide dismutase (SOD1. There is variability in sporadic ALS as well as FALS where even within the same family some siblings with the same mutation do not manifest disease. A transgenic (Tg mouse model of FALS containing 25 copies of the mutant human SOD1 gene demonstrates motor neuron pathology and progressive weakness similar to ALS patients, leading to death at approximately 130 days. The onset of symptoms and survival of these transgenic mice are directly related to the number of copies of the mutant gene. We report the phenotype of a very low expressing (VLE G93A SOD1 Tg carrying only 4 copies of the mutant G93ASOD1 gene. While weakness can start at 9 months, only 74% of mice 18 months or older demonstrate disease. The VLE mice show decreased motor neurons compared to wild-type mice as well as increased cytoplasmic translocation of TDP-43. In contrast to the standard G93A SOD1 Tg mouse which always develops motor weakness leading to death, not all VLE animals manifested clinical disease or shortened life span. In fact, approximately 20% of mice older than 24 months had no motor symptoms and only 18% of VLE mice older than 22 months reached end stage. Given the variable penetrance of clinical phenotype, prolonged survival, and protracted loss of motor neurons the VLE mouse provides a new tool that closely mimics human ALS. This tool will allow the study of pathologic events over time as well as the study of genetic and environmental modifiers that may not be causative, but can exacerbate or accelerate motor neuron disease.

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

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


    Key Message: The loss of mini-exon 2 in the 1-FEH IIb glycosyl-hydrolase results in a putative non-functional allele. This loss of function has a strong impact on the susceptibility to post-harvest inulin depolymerization. Significant variation of copy number was identified in its close paralog 1-FEH IIa, but no quantitative effect of copy number on carbohydrates-related phenotypes was detected. Inulin polyfructan is the second most abundant storage carbohydrate in flowering plants. After ...

  17. Development, Optimization, and Evaluation of a Duplex Droplet Digital PCR Assay To Quantify the T-nos/hmg Copy Number Ratio in Genetically Modified Maize.

    Félix-Urquídez, Dalmira; Pérez-Urquiza, Melina; Valdez Torres, José-Benigno; León-Félix, Josefina; García-Estrada, Raymundo; Acatzi-Silva, Abraham


    Certified reference materials (CRMs) are required to guarantee the reliability of analytical measurements. The CRMs available in the field of genetically modified organisms (GMOs) are characterized using real-time polymerase chain reaction (qPCR). This technology has limited application, because of its dependence on a calibrant. The objective of this study was to obtain a method with higher metrological quality, to characterize the CRMs for their contents of T-nos/hmg copy number ratio in maize. A duplex droplet digital PCR (ddPCR) assay was developed and optimized by a central composite design. The developed method achieved an absolute limit of detection (LOD) of 11 cP T-nos, a relative LOD of 0.034%, a limit of quantification (LOQ) of 23 cP (relative LOQ of 0.08%), and a dynamic range of 0.08%-100% T-nos/hmg ratio. The specificity and applicability of the assay were established for the analysis of low T-nos concentrations (0.9%) in several corn varieties. The convenience of DNA digestion to reduce measurement bias in the case of multiple-copy binding was confirmed through an enzymatic restriction assay. Given its overall performance, this method can be used to characterize CRM candidates for their contents of T-nos/hmg ratio. PMID:26605751

  18. Comparison of the Agilent, ROMA/NimbleGen and Illumina platforms for classification of copy number alterations in human breast tumors

    Naume B


    Full Text Available Abstract Background Microarray Comparative Genomic Hybridization (array CGH provides a means to examine DNA copy number aberrations. Various platforms, brands and underlying technologies are available, facing the user with many choices regarding platform sensitivity and number, localization, and density distribution of probes. Results We evaluate three different platforms presenting different nature and arrangement of the probes: The Agilent Human Genome CGH Microarray 44 k, the ROMA/NimbleGen Representational Oligonucleotide Microarray 82 k, and the Illumina Human-1 Genotyping 109 k BeadChip, with Agilent being gene oriented, ROMA/NimbleGen being genome oriented, and Illumina being genotyping oriented. We investigated copy number changes in 20 human breast tumor samples representing different gene expression subclasses, using a suite of graphical and statistical methods designed to work across platforms. Despite substantial differences in the composition and spatial distribution of probes, the comparison revealed high overall concordance. Notably however, some short amplifications and deletions of potential biological importance were not detected by all platforms. Both correlation and cluster analysis indicate a somewhat higher similarity between ROMA/NimbleGen and Illumina than between Agilent and the other two platforms. The programs developed for the analysis are available from Conclusion We conclude that platforms based on different technology principles reveal similar aberration patterns, although we observed some unique amplification or deletion peaks at various locations, only detected by one of the platforms. The correct platform choice for a particular study is dependent on whether the appointed research intention is gene, genome, or genotype oriented.

  19. Molecular Typing of Mycobacterium tuberculosis by Using Nine Novel Variable-Number Tandem Repeats across the Beijing Family and Low-Copy-Number IS6110 Isolates

    Scott Spurgiesz, R.; Quitugua, Teresa N.; Smith, Kimothy L.; Schupp, James; Palmer, Eldon G; Cox, Rebecca A.; Keim, Paul


    Molecular epidemiological tools for genotyping clinical isolates of Mycobacterium tuberculosis have been developed and used to help track and contain transmission of tuberculosis. We identified 87 short sequence repeat loci within the genome of the M. tuberculosis H37Rv strain. Nine tandem repeats were found to be variable (variable-number tandem repeats [VNTRs]) in a set of 91 isolates. Fifty-seven of the isolates had only four IS6110 bands. The other 34 isolates were members of the Beijing ...

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

    Hersoug, Lars-Georg; Brasch-Andersen, Charlotte; Husemoen, Lise Lotte Nystrup;


    Introduction: Exposure to particulate matter (PM) may induce inflammation and oxidative stress in the airways. Carriers of null polymorphisms of glutathione S-transferases (GSTs), which detoxify reactive oxygen species, may be particularly susceptible to the effects of PM. Objectives: To investig......Introduction: Exposure to particulate matter (PM) may induce inflammation and oxidative stress in the airways. Carriers of null polymorphisms of glutathione S-transferases (GSTs), which detoxify reactive oxygen species, may be particularly susceptible to the effects of PM. Objectives: To....... The relationship of glutathione-S-transferases copy number variation and indoor air pollution to symptoms and markers of respiratory disease. Clin Respir J 2011; DOI:10.1111/j.1752-699X.2011.00258.x....

  1. Minocycline resistance in an oral Streptococcus infantis isolate is encoded by tet(S) on a novel small, low copy number plasmid

    Ciric, Lena; Brouwer, Michael S M; Mullany, Peter; Roberts, Adam P


    We have determined the genetic basis of minocycline resistance in a strain of Streptococcus infantis isolated from a healthy human oral cavity. We demonstrate that tet(S), identical to tet(S) found on the enterococcal conjugative transposon Tn6000, is responsible for the observed resistance. The gene is located on a small, low copy number plasmid and is flanked by IS1216 elements. The tet(S) gene is capable of excising from the plasmid together with one of the IS1216 elements. The plasmid contains a putative toxin/antitoxin system related to relBE. Deletion of the toxin, relE, did not result in plasmid instability but did increase the fitness of the mutant compared to the wild-type strain. PMID:24605990

  2. Do Copy Number Changes in CACNA2D2, CACNA2D3, and CACNA1D Constitute a Predisposing Risk Factor for Alzheimer’s Disease?

    Villela, Darine; Suemoto, Claudia K.; Pasqualucci, Carlos A.; Grinberg, Lea T.; Rosenberg, Carla


    Dysregulation of calcium (Ca2+) homeostasis is now being recognized to be a key step in the pathogenesis of Alzheimer’s disease (AD). Data from the literature, in particular the association between AD and polymorphism that interfere with Ca2+ homeostasis indicates the presence of genetic factors in this process; further, presenilins mutations, which are known to cause the familial form of AD, are involved in the regulation of intracellular Ca2+ stores. Here, we wish to draw attention to rare DNA copy number variations identified in two subjects with late-onset AD that led to partial or full duplication of genes that encode different subunits of the same type of voltage-gated Ca2+ channels; these duplications of voltage-gated Ca2+ channel genes is consistent with the critical role of calcium signaling in molecular processes underlying memory as has been demonstrated by several studies. PMID:27379157

  3. Heterozygous deletion at the RLN1 locus in a family with testicular germ cell cancer identified by integrating copy number variation data with phenome and interactome information

    Edsgärd, D; Scheel, M; Hansen, N T;


    To search for disease-related copy number variations (CNVs) in families with a high frequency of germ cell tumours (GCT), we analysed 16 individuals from four families by array comparative genomic hybridization (aCGH) and applied an integrative systems biology algorithm that prioritizes risk......-associated genes among loci targeted by CNVs. The top-ranked candidate, RLN1, encoding a Relaxin-H1 peptide, although only detected in one of the families, was selected for further investigations. Validation of the CNV at the RLN1 locus was performed as an association study using qPCR with 106 sporadic testicular...... GCT patients and 200 healthy controls. Observed CNV frequencies of 1.9% among cases and 1.5% amongst controls were not significantly different and this was further confirmed by CNV data extracted from a genome-wide analysis of 189 cases and 380 controls, where similar frequencies of 2.2% were observed...

  4. Heterozygous deletion at the RLN1 locus in a family with testicular germ cell cancer identified by integrating copy number variation data with phenome and interactome information

    Edsgard, Stefan Daniel; Scheel, M.; Hansen, Niclas Tue;


    To search for disease‐related copy number variations (CNVs) in families with a high frequency of germ cell tumours (GCT), we analysed 16 individuals from four families by array comparative genomic hybridization (aCGH) and applied an integrative systems biology algorithm that prioritizes risk......‐associated genes among loci targeted by CNVs. The top‐ranked candidate, RLN1, encoding a Relaxin‐H1 peptide, although only detected in one of the families, was selected for further investigations. Validation of the CNV at the RLN1 locus was performed as an association study using qPCR with 106 sporadic testicular...... GCT patients and 200 healthy controls. Observed CNV frequencies of 1.9% among cases and 1.5% amongst controls were not significantly different and this was further confirmed by CNV data extracted from a genome‐wide analysis of 189 cases and 380 controls, where similar frequencies of 2.2% were observed...

  5. Comparative analyses across cattle genders and breeds reveal the pitfalls caused by false positive and lineage-differential copy number variations.

    Zhou, Yang; Utsunomiya, Yuri T; Xu, Lingyang; Hay, El Hamidi Abdel; Bickhart, Derek M; Sonstegard, Tad S; Van Tassell, Curtis P; Garcia, Jose Fernando; Liu, George E


    We compared CNV region (CNVR) results derived from 1,682 Nellore cattle with equivalent results derived from our previous analysis of Bovine HapMap samples. By comparing CNV segment frequencies between different genders and groups, we identified 9 frequent, false positive CNVRs with a total length of 0.8 Mbp that were likely caused by assembly errors. Although there was a paucity of lineage specific events, we did find one 54 kb deletion on chr5 significantly enriched in Nellore cattle. A few highly frequent CNVRs present in both datasets were detected within genomic regions containing olfactory receptor, ATP-binding cassette, and major histocompatibility complex genes. We further evaluated their impacts on downstream bioinformatics and CNV association analyses. Our results revealed pitfalls caused by false positive and lineage-differential copy number variations and will increase the accuracy of future CNV studies in both taurine and indicine cattle. PMID:27381368

  6. Susceptibility for Lupus Nephritis by Low Copy Number of the FCGR3B Gene Is Linked to Increased Levels of Pathogenic Autoantibodies

    Johannes C. Nossent


    Full Text Available Low copy number (CN of the FCGR3B gene reduces FCGR3B membrane expression on neutrophils and results in clearance of a smaller amount of immune complex. We investigated FCGR3B CN in relation to the clinical phenotype in a Caucasian SLE cohort (. FCGR3B CN was determined by three different qPCR parameter estimations (Ct−, Cy0, and cpD1 and confirmed by the FCGR2C/FCGR2A paralog ratio test. Clinical and serological data were then analyzed for their association with FCGR3B CN. Low FCGR3B CN (2. In multivariate analyses, LN was independently associated with anti-C1q-Ab levels ( and low FCGR3B CN (. We conclude that the susceptibility for LN in patients with low FCGR3B CN is linked to increased levels of pathogenic autoantibodies.

  7. Physical state & copy number of high risk human papillomavirus type 16 DNA in progression of cervical cancer

    Shirish Shukla


    Full Text Available Background & objectives: High-risk human papilloma virus (HR-HPV infection and its integration in host genome is a key event in malignant transformation of cervical cells. HPV16 being a dominant HR-HPV type, we undertook this study to analyze if viral load and physical state of the virus correlated with each other in the absence of other confounding variables and examined their potential as predictors of progressive cervical lesions. Methods: Both, viral load and integration status of HPV16 were determined by real time URR PCR and estimation of E2:E6 ratio in a total of 130 PGMY-RLB -confirmed, monotypic HPV16-infected cervical DNA samples from biopsies of cytology-confirmed low grade (LSIL, 30 and high grade (HSIL, 30, and invasive carcinoma, (squamous cell carcinoma SCC, 70 cases. Results: Investigation of DNA samples revealed a gradual increase in HPV16 viral load over several magnitudes and increased frequency of integration from LSIL to HSIL and HSIL to invasive cancer in relation to the severity of lesions in monotypic HPV16-infected cervical tissues. In a substantial number of precancer (11/60 and cancer cases (29/70, HPV16 was detected in concomitant mixed form. The concomitant form of HPV16 genome carried significantly higher viral load. Interpretation & conclusions: Overall, viral load and integration increased with disease severity and could be useful biomarkers in disease progression, at least, in HPV16-infected cervical pre-cancer and cancer lesions.

  8. Network-guided analysis of genes with altered somatic copy number and gene expression reveals pathways commonly perturbed in metastatic melanoma.

    Armand Valsesia

    Full Text Available Cancer genomes frequently contain somatic copy number alterations (SCNA that can significantly perturb the expression level of affected genes and thus disrupt pathways controlling normal growth. In melanoma, many studies have focussed on the copy number and gene expression levels of the BRAF, PTEN and MITF genes, but little has been done to identify new genes using these parameters at the genome-wide scale. Using karyotyping, SNP and CGH arrays, and RNA-seq, we have identified SCNA affecting gene expression ('SCNA-genes' in seven human metastatic melanoma cell lines. We showed that the combination of these techniques is useful to identify candidate genes potentially involved in tumorigenesis. Since few of these alterations were recurrent across our samples, we used a protein network-guided approach to determine whether any pathways were enriched in SCNA-genes in one or more samples. From this unbiased genome-wide analysis, we identified 28 significantly enriched pathway modules. Comparison with two large, independent melanoma SCNA datasets showed less than 10% overlap at the individual gene level, but network-guided analysis revealed 66% shared pathways, including all but three of the pathways identified in our data. Frequently altered pathways included WNT, cadherin signalling, angiogenesis and melanogenesis. Additionally, our results emphasize the potential of the EPHA3 and FRS2 gene products, involved in angiogenesis and migration, as possible therapeutic targets in melanoma. Our study demonstrates the utility of network-guided approaches, for both large and small datasets, to identify pathways recurrently perturbed in cancer.

  9. Regionalized pathology correlates with augmentation of mtDNA copy numbers in a patient with myoclonic epilepsy with ragged-red fibers (MERRF-syndrome).

    Brinckmann, Anja; Weiss, Claudia; Wilbert, Friederike; von Moers, Arpad; Zwirner, Angelika; Stoltenburg-Didinger, Gisela; Wilichowski, Ekkehard; Schuelke, Markus


    Human patients with myoclonic epilepsy with ragged-red fibers (MERRF) suffer from regionalized pathology caused by a mutation in the mitochondrial DNA (m.8344A→G). In MERRF-syndrome brain and skeletal muscles are predominantly affected, despite mtDNA being present in any tissue. In the past such tissue-specificity could not be explained by varying mtDNA mutation loads. In search for a region-specific pathology in human individuals we determined the mtDNA/nDNA ratios along with the mutation loads in 43 different post mortem tissue samples of a 16-year-old female MERRF patient and in four previously healthy victims of motor vehicle accidents. In brain and muscle we further determined the quantity of mitochondrial proteins (COX subunits II and IV), transcription factors (NRF1 and TFAM), and VDAC1 (Porin) as a marker for the mitochondrial mass. In the patient the mutation loads varied merely between 89-100%. However, mtDNA copy numbers were increased 3-7 fold in predominantly affected brain areas (e.g. hippocampus, cortex and putamen) and in skeletal muscle. Similar increases were absent in unaffected tissues (e.g. heart, lung, kidney, liver, and gastrointestinal organs). Such mtDNA copy number increase was not paralleled by an augmentation of mitochondrial mass in some investigated tissues, predominantly in the most affected tissue regions of the brain. We thus conclude that "futile" stimulation of mtDNA replication per se or a secondary failure to increase the mitochondrial mass may contribute to the regionalized pathology seen in MERRF-syndrome. PMID:20976001

  10. Multi-platform whole-genome microarray analyses refine the epigenetic signature of breast cancer metastasis with gene expression and copy number.

    Joseph Andrews

    Full Text Available BACKGROUND: We have previously identified genome-wide DNA methylation changes in a cell line model of breast cancer metastasis. These complex epigenetic changes that we observed, along with concurrent karyotype analyses, have led us to hypothesize that complex genomic alterations in cancer cells (deletions, translocations and ploidy are superimposed over promoter-specific methylation events that are responsible for gene-specific expression changes observed in breast cancer metastasis. METHODOLOGY/PRINCIPAL FINDINGS: We undertook simultaneous high-resolution, whole-genome analyses of MDA-MB-468GFP and MDA-MB-468GFP-LN human breast cancer cell lines (an isogenic, paired lymphatic metastasis cell line model using Affymetrix gene expression (U133, promoter (1.0R, and SNP/CNV (SNP 6.0 microarray platforms to correlate data from gene expression, epigenetic (DNA methylation, and combination copy number variant/single nucleotide polymorphism microarrays. Using Partek Software and Ingenuity Pathway Analysis we integrated datasets from these three platforms and detected multiple hypomethylation and hypermethylation events. Many of these epigenetic alterations correlated with gene expression changes. In addition, gene dosage events correlated with the karyotypic differences observed between the cell lines and were reflected in specific promoter methylation patterns. Gene subsets were identified that correlated hyper (and hypo methylation with the loss (or gain of gene expression and in parallel, with gene dosage losses and gains, respectively. Individual gene targets from these subsets were also validated for their methylation, expression and copy number status, and susceptible gene pathways were identified that may indicate how selective advantage drives the processes of tumourigenesis and metastasis. CONCLUSIONS/SIGNIFICANCE: Our approach allows more precisely profiling of functionally relevant epigenetic signatures that are associated with cancer

  11. Identification of Novel Breast Cancer Subtype-Specific Biomarkers by Integrating Genomics Analysis of DNA Copy Number Aberrations and miRNA-mRNA Dual Expression Profiling

    Dongguo Li


    Full Text Available Breast cancer is a heterogeneous disease with well-defined molecular subtypes. Currently, comparative genomic hybridization arrays (aCGH techniques have been developed rapidly, and recent evidences in studies of breast cancer suggest that tumors within gene expression subtypes share similar DNA copy number aberrations (CNA which can be used to further subdivide subtypes. Moreover, subtype-specific miRNA expression profiles are also proposed as novel signatures for breast cancer classification. The identification of mRNA or miRNA expression-based breast cancer subtypes is considered an instructive means of prognosis. Here, we conducted an integrated analysis based on copy number aberrations data and miRNA-mRNA dual expression profiling data to identify breast cancer subtype-specific biomarkers. Interestingly, we found a group of genes residing in subtype-specific CNA regions that also display the corresponding changes in mRNAs levels and their target miRNAs’ expression. Among them, the predicted direct correlation of BRCA1-miR-143-miR-145 pairs was selected for experimental validation. The study results indicated that BRCA1 positively regulates miR-143-miR-145 expression and miR-143-miR-145 can serve as promising novel biomarkers for breast cancer subtyping. In our integrated genomics analysis and experimental validation, a new frame to predict candidate biomarkers of breast cancer subtype is provided and offers assistance in order to understand the potential disease etiology of the breast cancer subtypes.

  12. Close correlation of copy number aberrations detected by next-generation sequencing with results from routine cytogenetics in acute myeloid leukemia.

    Vosberg, Sebastian; Herold, Tobias; Hartmann, Luise; Neumann, Martin; Opatz, Sabrina; Metzeler, Klaus H; Schneider, Stephanie; Graf, Alexander; Krebs, Stefan; Blum, Helmut; Baldus, Claudia D; Hiddemann, Wolfgang; Spiekermann, Karsten; Bohlander, Stefan K; Mansmann, Ulrich; Greif, Philipp A


    High throughput sequencing approaches, including the analysis of exomes or gene panels, are widely used and established to detect tumor-specific sequence variants such as point mutations or small insertions/deletions. Beyond single nucleotide resolution, sequencing data also contain information on changes in sequence coverage between samples and thus allow the detection of somatic copy number alterations (CNAs) representing gain or loss of genomic material in tumor cells arising from aneuploidy, amplifications, or deletions. To test the feasibility of CNA detection in sequencing data we analyzed the exomes of 25 paired leukemia/remission samples from acute myeloid leukemia (AML) patients with well-defined chromosomal aberrations, detected by conventional chromosomal analysis and/or molecular cytogenetics assays. Thereby, we were able to confirm chromosomal aberrations including trisomies, monosomies, and partial chromosomal deletions in 20 out of 25 samples. Comparison of CNA detection using exome, custom gene panel, and SNP array analysis showed equivalent results in five patients with variable clone size. Gene panel analysis of AML samples without matched germline control samples resulted in confirmation of cytogenetic findings in 18 out of 22 cases. In all cases with discordant findings, small clone size (AML samples including highly correlated clone size estimation (R = 0.85), while six out of 65 cytogenetically normal AML samples exhibited CNAs apparently missed by routine cytogenetics. Overall, our results show that high throughput targeted sequencing data can be reliably used to detect copy number changes in the dominant AML clone. © 2016 Wiley Periodicals, Inc. PMID:27015608

  13. Insulin-like growth factor-1 receptor protein expression and gene copy number alterations in non-small cell lung carcinomas.

    Tsuta, Koji; Mimae, Takahiro; Nitta, Hiroaki; Yoshida, Akihiko; Maeshima, Akiko M; Asamura, Hisao; Grogan, Thomas M; Furuta, Koh; Tsuda, Hitoshi


    Insulin-like growth factor-1 receptor (IGF-1R) is a tyrosine kinase receptor implicated in the pathogenesis of several malignancies and is potentially an attractive target for anticancer treatment. In this study, we included 379 patients who underwent surgical resection (179 diagnosed as having adenocarcinoma [ADC]; 150, squamous cell carcinoma [SCC]; 41, sarcomatoid carcinoma and 9, large cell carcinoma). IGF-1R expression and gene copy number were assessed by immunohistochemistry and bright-field in situ hybridization (BISH), respectively. IGF-1R expression in non-small cell lung carcinoma was observed in 41.4% of samples and was more prevalent in SCC (69.3%) than in ADC (25.1%), large cell carcinoma (33.3%), and sarcomatoid carcinoma (12.2%) (P < .001). Among ADCs, most mucinous ADCs (75%) showed strong membranous staining with the IGF-1R antibody. Compared with protein expression, IGF-1R gene alteration was rare (8.4%). A statistically significant correlation between IGF-1R expression and positive IGF-1R BISH was observed (γ = 0.762, P < .001). IGF-1R-positive tumors were more common in smokers (P = .004), and these tumors were larger (P = .006) than the IGF-1R-negative tumors. IGF-1R BISH positivity was not correlated with any clinicopathologic factor. IGF-1R expression and IGF-1R BISH positivity were not correlated with overall survival. IGF-1R is highly expressed in SCC and mucinous ADC, although copy number alterations in the IGF-1R gene were rare. These findings may have important implications for future anti-IGF-1R therapeutic approaches. PMID:23266446

  14. Systems biology analysis of hepatitis C virus infection reveals the role of copy number increases in regions of chromosome 1q in hepatocellular carcinoma metabolism.

    Elsemman, Ibrahim E; Mardinoglu, Adil; Shoaie, Saeed; Soliman, Taysir H; Nielsen, Jens


    Hepatitis C virus (HCV) infection is a worldwide healthcare problem; however, traditional treatment methods have failed to cure all patients, and HCV has developed resistance to new drugs. Systems biology-based analyses could play an important role in the holistic analysis of the impact of HCV on hepatocellular metabolism. Here, we integrated HCV assembly reactions with a genome-scale hepatocyte metabolic model to identify metabolic targets for HCV assembly and metabolic alterations that occur between different HCV progression states (cirrhosis, dysplastic nodule, and early and advanced hepatocellular carcinoma (HCC)) and healthy liver tissue. We found that diacylglycerolipids were essential for HCV assembly. In addition, the metabolism of keratan sulfate and chondroitin sulfate was significantly changed in the cirrhosis stage, whereas the metabolism of acyl-carnitine was significantly changed in the dysplastic nodule and early HCC stages. Our results explained the role of the upregulated expression of BCAT1, PLOD3 and six other methyltransferase genes involved in carnitine biosynthesis and S-adenosylmethionine metabolism in the early and advanced HCC stages. Moreover, GNPAT and BCAP31 expression was upregulated in the early and advanced HCC stages and could lead to increased acyl-CoA consumption. By integrating our results with copy number variation analyses, we observed that GNPAT, PPOX and five of the methyltransferase genes (ASH1L, METTL13, SMYD2, TARBP1 and SMYD3), which are all located on chromosome 1q, had increased copy numbers in the cancer samples relative to the normal samples. Finally, we confirmed our predictions with the results of metabolomics studies and proposed that inhibiting the identified targets has the potential to provide an effective treatment strategy for HCV-associated liver disorders. PMID:27040643

  15. High-resolution physical map for chromosome 16q12.1-q13, the Blau syndrome locus

    Bonavita Gina


    Full Text Available Abstract Background The Blau syndrome (MIM 186580, an autosomal dominant granulomatous disease, was previously mapped to chromosome 16p12-q21. However, inconsistent physical maps of the region and consequently an unknown order of microsatellite markers, hampered us from further refining the genetic locus for the Blau syndrome. To address this problem, we constructed our own high-resolution physical map for the Blau susceptibility region. Results We generated a high-resolution physical map that provides more than 90% coverage of a refined Blau susceptibility region. The map consists of four contigs of sequence tagged site-based bacterial artificial chromosomes with a total of 124 bacterial artificial chromosomes, and spans approximately 7.5 Mbp; however, three gaps still exist in this map with sizes of 425, 530 and 375 kbp, respectively, estimated from radiation hybrid mapping. Conclusions Our high-resolution map will assist genetic studies of loci in the interval from D16S3080, near D16S409, and D16S408 (16q12.1 to 16q13.

  16. Functional loss of E-cadherin and cadherin-11 alleles on chromosome 16q22 in colonic cancer.

    Braungart, E; Schumacher, C; Hartmann, E; Nekarda, H; Becker, K F; Höfler, H; Atkinson, M J


    Proteins of the cadherin family regulate cellular adhesion and motility and are believed to act as tumour suppressors. Previous studies have identified frequent mutation and allelic inactivation of the E-cadherin (cadherin-1) locus in diffuse gastric cancer. At least two other cadherin genes, P-cadherin (cadherin-3) and OB-cadherin (cadherin-11), have been mapped close to the E-cadherin gene on chromosome 16q22. As this region of the genome is frequently deleted in malignancy, multiple cadherin loci may be affected by losses of chromosome 16q22. The expression of mRNA transcripts from polymorphic alleles of the E-cadherin and cadherin-11 genes was examined in 30 cases of colonic, gastric, and renal carcinoma. In gastric cancer, loss of expression of one allele was restricted to the E-cadherin locus, whilst in renal carcinoma neither locus was affected. In colonic cancers, loss of expression of one E-cadherin allele was detected in 5 of 22 cases, whilst loss of a cadherin-11 allele was seen in 5 of 23 cases. This functional loss of cadherin gene expression may be due to gene deletion, inactivation or recombination. As no evidence of cadherin gene mutation was observed in the remaining transcripts, we can conclude that these two genes are only indirectly involved in the pathogenesis of colorectal cancer. PMID:10398117

  17. Transcriptome sequence and plasmid copy number analysis of the brewery isolate Pediococcus claussenii ATCC BAA-344 T during growth in beer.

    Vanessa Pittet

    Full Text Available Growth of specific lactic acid bacteria in beer leads to spoiled product and economic loss for the brewing industry. Microbial growth is typically inhibited by the combined stresses found in beer (e.g., ethanol, hops, low pH, minimal nutrients; however, certain bacteria have adapted to grow in this harsh environment. Considering little is known about the mechanisms used by bacteria to grow in and spoil beer, transcriptome sequencing was performed on a variant of the beer-spoilage organism Pediococcus claussenii ATCC BAA-344(T (Pc344-358. Illumina sequencing was used to compare the transcript levels in Pc344-358 growing mid-exponentially in beer to those in nutrient-rich MRS broth. Various operons demonstrated high gene expression in beer, several of which are involved in nutrient acquisition and overcoming the inhibitory effects of hop compounds. As well, genes functioning in cell membrane modification and biosynthesis demonstrated significantly higher transcript levels in Pc344-358 growing in beer. Three plasmids had the majority of their genes showing increased transcript levels in beer, whereas the two cryptic plasmids showed slightly decreased gene expression. Follow-up analysis of plasmid copy number in both growth environments revealed similar trends, where more copies of the three non-cryptic plasmids were found in Pc344-358 growing in beer. Transcriptome sequencing also enabled the addition of several genes to the P. claussenii ATCC BAA-344(T genome annotation, some of which are putatively transcribed as non-coding RNAs. The sequencing results not only provide the first transcriptome description of a beer-spoilage organism while growing in beer, but they also highlight several targets for future exploration, including genes that may have a role in the general stress response of lactic acid bacteria.

  18. A large scale survey reveals that chromosomal copy-number alterations significantly affect gene modules involved in cancer initiation and progression

    Cigudosa Juan C


    Full Text Available Abstract Background Recent observations point towards the existence of a large number of neighborhoods composed of functionally-related gene modules that lie together in the genome. This local component in the distribution of the functionality across chromosomes is probably affecting the own chromosomal architecture by limiting the possibilities in which genes can be arranged and distributed across the genome. As a direct consequence of this fact it is therefore presumable that diseases such as cancer, harboring DNA copy number alterations (CNAs, will have a symptomatology strongly dependent on modules of functionally-related genes rather than on a unique "important" gene. Methods We carried out a systematic analysis of more than 140,000 observations of CNAs in cancers and searched by enrichments in gene functional modules associated to high frequencies of loss or gains. Results The analysis of CNAs in cancers clearly demonstrates the existence of a significant pattern of loss of gene modules functionally related to cancer initiation and progression along with the amplification of modules of genes related to unspecific defense against xenobiotics (probably chemotherapeutical agents. With the extension of this analysis to an Array-CGH dataset (glioblastomas from The Cancer Genome Atlas we demonstrate the validity of this approach to investigate the functional impact of CNAs. Conclusions The presented results indicate promising clinical and therapeutic implications. Our findings also directly point out to the necessity of adopting a function-centric, rather a gene-centric, view in the understanding of phenotypes or diseases harboring CNAs.

  19. Age-dependent brain gene expression and copy number anomalies in autism suggest distinct pathological processes at young versus mature ages.

    Maggie L Chow

    Full Text Available Autism is a highly heritable neurodevelopmental disorder, yet the genetic underpinnings of the disorder are largely unknown. Aberrant brain overgrowth is a well-replicated observation in the autism literature; but association, linkage, and expression studies have not identified genetic factors that explain this trajectory. Few studies have had sufficient statistical power to investigate whole-genome gene expression and genotypic variation in the autistic brain, especially in regions that display the greatest growth abnormality. Previous functional genomic studies have identified possible alterations in transcript levels of genes related to neurodevelopment and immune function. Thus, there is a need for genetic studies involving key brain regions to replicate these findings and solidify the role of particular functional pathways in autism pathogenesis. We therefore sought to identify abnormal brain gene expression patterns via whole-genome analysis of mRNA levels and copy number variations (CNVs in autistic and control postmortem brain samples. We focused on prefrontal cortex tissue where excess neuron numbers and cortical overgrowth are pronounced in the majority of autism cases. We found evidence for dysregulation in pathways governing cell number, cortical patterning, and differentiation in young autistic prefrontal cortex. In contrast, adult autistic prefrontal cortex showed dysregulation of signaling and repair pathways. Genes regulating cell cycle also exhibited autism-specific CNVs in DNA derived from prefrontal cortex, and these genes were significantly associated with autism in genome-wide association study datasets. Our results suggest that CNVs and age-dependent gene expression changes in autism may reflect distinct pathological processes in the developing versus the mature autistic prefrontal cortex. Our results raise the hypothesis that genetic dysregulation in the developing brain leads to abnormal regional patterning, excess

  20. Copy number analysis of 24 oncogenes: MDM4 identified as a putative marker for low recurrence risk in non muscle invasive bladder cancer.

    Salvi, Samanta; Calistri, Daniele; Gurioli, Giorgia; Carretta, Elisa; Serra, Luigi; Gunelli, Roberta; Zoli, Wainer; Casadio, Valentina


    Patients with non-muscle invasive bladder cancer (NMIBC) generally have a high risk of relapsing locally after primary tumor resection. The search for new predictive markers of local recurrence thus represents an important goal for the management of this disease. We studied the copy number variations (CNVs) of 24 oncogenes (MDM4, MYCN, ALK, PDGFRA, KIT, KDR, DHFR, EGFR, MET, SMO, FGFR1, MYC, ABL1, RET, CCND1, CCND2, CDK4, MDM2, AURKB, ERBB2, TOP2A, AURKA, AR and BRAF) using multiplex ligation probe amplification technique to verify their role as predictive markers of recurrence. Formalin-fixed paraffin-embedded tissue samples from 43 patients who underwent transurethral resection of the bladder (TURB) were used; 23 patients had relapsed and 20 were disease-free after 5 years. Amplification frequencies were analyzed for all genes and MDM4 was the only gene that showed significantly higher amplification in non recurrent patients than in recurrent ones (0.65 vs. 0.3; Fisher's test p=0.023). Recurrence-free survival analysis confirmed the predictive role of MDM4 (log-rank test p=0.041). Our preliminary results indicate a putative role for the MDM4 gene in predicting local recurrence of bladder cancer. Confirmation of this hypothesis is needed in a larger cohort of NMIBC patients. PMID:25026175

  1. CEBPA copy number variations in normal karyotype acute myeloid leukemia: Possible role of breakpoint-associated microhomology and chromatin status in CEBPA mutagenesis.

    Libura, Marta; Pawełczyk, Marta; Florek, Izabella; Matiakowska, Karolina; Jaźwiec, Bożena; Borg, Katarzyna; Solarska, Iwona; Zawada, Magdalena; Czekalska, Sylwia; Libura, Jolanta; Salamanczuk, Zoriana; Jakóbczyk, Małgorzata; Mucha, Barbara; Duszeńko, Ewa; Soszyńska, Krystyna; Karabin, Karolina; Piątkowska-Jakubas, Beata; Całbecka, Małgorzata; Gajkowska-Kulig, Justyna; Gadomska, Grażyna; Kiełbiński, Marek; Ejduk, Anna; Kata, Dariusz; Grosicki, Sebastian; Kyrcz-Krzemień, Sławomira; Warzocha, Krzysztof; Kuliczkowski, Kazimierz; Skotnicki, Aleksander; Jęrzejczak, Wiesław Wiktor; Haus, Olga


    Copy number variations (CNV) in CEBPA locus represent heterogeneous group of mutations accompanying acute myeloid leukemia (AML). The aim of this study was to characterize different CEBPA mutation categories in regard to biological data like age, cytology, CD7, and molecular markers, and identify possible factors affecting their etiology. We report here the incidence of 12.6% of CEBPA mutants in the population of 262 normal karyotype AML (NK-AML) patients. We confirmed that double mutant AMLs presented uniform biological features when compared to single CEBPA mutations and accompanied mostly younger patients. We hypothesized that pathogenesis of distinct CEBPA mutation categories might be influenced by different factors. The detailed sequence analysis revealed frequent breakpoint-associated microhomologies of 2 to 12bp. The analysis of distribution of microhomology motifs along CEBPA gene showed that longer stretches of microhomology at the mutational junctions were relatively rare by chance which suggests their functional role in the CEBPA mutagenesis. Additionally, accurate quantification of CEBPA transcript levels showed that double CEBPA mutations correlated with high-level CEBPA expression, whereas single N-terminal CEBPA mutations were associated with low-level CEBPA expression. This might suggest that high-level CEBPA expression and/or accessibility of CEBPA locus contribute to B-ZIP in-frame duplications. PMID:26460249

  2. Clonal evolution and clinical significance of copy number neutral loss of heterozygosity of chromosome arm 6p in acquired aplastic anemia.

    Betensky, Marisol; Babushok, Daria; Roth, Jacquelyn J; Mason, Philip J; Biegel, Jaclyn A; Busse, Tracy M; Li, Yimei; Lind, Curt; Papazoglou, Anna; Monos, Dimitri; Podsakoff, Gregory; Bessler, Monica; Olson, Timothy S


    Acquired aplastic anemia (aAA) results from the T cell-mediated autoimmune destruction of hematopoietic stem cells. Factors predicting response to immune suppression therapy (IST) or development of myelodysplastic syndrome (MDS) are beginning to be elucidated. Our recent data suggest most patients with aAA treated with IST develop clonal somatic genetic alterations in hematopoietic cells. One frequent acquired abnormality is copy-number neutral loss of heterozygosity on chromosome 6p (6p CN-LOH) involving the human leukocyte antigen (HLA) locus. We hypothesized that because 6p CN-LOH clones may arise from selective pressure to escape immune surveillance through deletion of HLA alleles, the development of 6p CN-LOH may affect response to IST. We used single nucleotide polymorphism array genotyping and targeted next-generation sequencing of HLA alleles to assess frequency of 6p CN-LOH, identity of HLA alleles lost through 6p CN-LOH, and impact of 6p CN-LOH on response to IST. 6p CN-LOH clones were present in 11.3% of patients, remained stable over time, and were not associated with development of MDS-defining cytogenetic abnormalities. Notably, no patient with 6p CN-LOH treated with IST achieved a complete response. In summary, clonal 6p CN-LOH in aAA defines a unique subgroup of patients that may provide insights into hematopoietic clonal evolution. PMID:26702937

  3. Copy Number Analysis of 24 Oncogenes: MDM4 Identified as a Putative Marker for Low Recurrence Risk in Non Muscle Invasive Bladder Cancer

    Samanta Salvi


    Full Text Available Patients with non-muscle invasive bladder cancer (NMIBC generally have a high risk of relapsing locally after primary tumor resection. The search for new predictive markers of local recurrence thus represents an important goal for the management of this disease. We studied the copy number variations (CNVs of 24 oncogenes (MDM4, MYCN, ALK, PDGFRA, KIT, KDR, DHFR, EGFR, MET, SMO, FGFR1, MYC, ABL1, RET, CCND1, CCND2, CDK4, MDM2, AURKB, ERBB2, TOP2A, AURKA, AR and BRAF using multiplex ligation probe amplification technique to verify their role as predictive markers of recurrence. Formalin-fixed paraffin-embedded tissue samples from 43 patients who underwent transurethral resection of the bladder (TURB were used; 23 patients had relapsed and 20 were disease-free after 5 years. Amplification frequencies were analyzed for all genes and MDM4 was the only gene that showed significantly higher amplification in non recurrent patients than in recurrent ones (0.65 vs. 0.3; Fisher’s test p = 0.023. Recurrence-free survival analysis confirmed the predictive role of MDM4 (log-rank test p = 0.041. Our preliminary results indicate a putative role for the MDM4 gene in predicting local recurrence of bladder cancer. Confirmation of this hypothesis is needed in a larger cohort of NMIBC patients.

  4. Comprehensive Screening of Gene Copy Number Aberrations in Formalin-Fixed, Paraffin-Embedded Solid Tumors Using Molecular Inversion Probe-Based Single-Nucleotide Polymorphism Array.

    Singh, Rajesh R; Mehrotra, Meenakshi; Chen, Hui; Almohammedsalim, Alaa A; Sahin, Ayesagul; Bosamra, Alex; Patel, Keyur P; Routbort, Mark J; Lu, Xinyan; Ronald, Abraham; Mishra, Bal Mukund; Virani, Shumaila; Medeiros, L Jeffrey; Luthra, Rajyalakshmi


    Gene copy number aberrations (CNAs) represent a major class of cancer-related genomic alterations that drive solid tumors. Comprehensive and sensitive detection of CNAs is challenging because of often low quality and quantity of DNA isolated from the formalin-fixed, paraffin-embedded (FFPE) solid tumor samples. Here, in a clinical molecular diagnostic laboratory, we tested the utility and validated a molecular inversion probe-based (MIP) array to routinely screen for CNAs in solid tumors. Using low-input FFPE DNA, the array detects genome-wide CNAs with a special focus on 900 cancer-related genes. A cohort of 76 solid tumors of various types and tumor cellularity (20% to 100%), and four cancer cell lines were used. These harbored CNAs in clinically important genes (ERBB2, EGFR, FGFR1, KRAS, MYC) as detected by orthogonal techniques like next-generation sequencing or fluorescence in situ hybridization. Results of the MIP array were concordant with results from orthogonal techniques, and also provided additional information regarding the allelic nature of the CNAs. Limit-of-detection and assay reproducibility studies showed a high degree of sensitivity and reproducibility of detection, respectively. FFPE compatibility, ability to detect CNAs with high sensitivity, accuracy, and provide valuable information such as loss of heterozygosity along with relatively short turnaround times makes the MIP array a desirable clinical platform for routine screening of solid tumors in a clinical laboratory. PMID:27392636

  5. An integrative approach to investigate the respective roles of single-nucleotide variants and copy-number variants in Attention-Deficit/Hyperactivity Disorder.

    de Araújo Lima, Leandro; Feio-Dos-Santos, Ana Cecília; Belangero, Sintia Iole; Gadelha, Ary; Bressan, Rodrigo Affonseca; Salum, Giovanni Abrahão; Pan, Pedro Mario; Moriyama, Tais Silveira; Graeff-Martins, Ana Soledade; Tamanaha, Ana Carina; Alvarenga, Pedro; Krieger, Fernanda Valle; Fleitlich-Bilyk, Bacy; Jackowski, Andrea Parolin; Brietzke, Elisa; Sato, João Ricardo; Polanczyk, Guilherme Vanoni; Mari, Jair de Jesus; Manfro, Gisele Gus; do Rosário, Maria Conceição; Miguel, Eurípedes Constantino; Puga, Renato David; Tahira, Ana Carolina; Souza, Viviane Neri; Chile, Thais; Gouveia, Gisele Rodrigues; Simões, Sérgio Nery; Chang, Xiao; Pellegrino, Renata; Tian, Lifeng; Glessner, Joseph T; Hashimoto, Ronaldo Fumio; Rohde, Luis Augusto; Sleiman, Patrick M A; Hakonarson, Hakon; Brentani, Helena


    Many studies have attempted to investigate the genetic susceptibility of Attention-Deficit/Hyperactivity Disorder (ADHD), but without much success. The present study aimed to analyze both single-nucleotide and copy-number variants contributing to the genetic architecture of ADHD. We generated exome data from 30 Brazilian trios with sporadic ADHD. We also analyzed a Brazilian sample of 503 children/adolescent controls from a High Risk Cohort Study for the Development of Childhood Psychiatric Disorders, and also previously published results of five CNV studies and one GWAS meta-analysis of ADHD involving children/adolescents. The results from the Brazilian trios showed that cases with de novo SNVs tend not to have de novo CNVs and vice-versa. Although the sample size is small, we could also see that various comorbidities are more frequent in cases with only inherited variants. Moreover, using only genes expressed in brain, we constructed two "in silico" protein-protein interaction networks, one with genes from any analysis, and other with genes with hits in two analyses. Topological and functional analyses of genes in this network uncovered genes related to synapse, cell adhesion, glutamatergic and serotoninergic pathways, both confirming findings of previous studies and capturing new genes and genetic variants in these pathways. PMID:26947246

  6. Detection of Clonal and Subclonal Copy-Number Variants in Cell-Free DNA from Patients with Breast Cancer Using a Massively Multiplexed PCR Methodology

    Kirkizlar, Eser; Zimmermann, Bernhard; Constantin, Tudor; Swenerton, Ryan; Hoang, Bin; Wayham, Nicholas; Babiarz, Joshua E.; Demko, Zachary; Pelham, Robert J.; Kareht, Stephanie; Simon, Alexander L.; Jinnett, Kristine N.; Rabinowitz, Matthew; Sigurjonsson, Styrmir; Hill, Matthew


    We demonstrate proof-of-concept for the use of massively multiplexed PCR and next-generation sequencing (mmPCR-NGS) to identify both clonal and subclonal copy-number variants (CNVs) in circulating tumor DNA. This is the first report of a targeted methodology for detection of CNVs in plasma. Using an in vitro model of cell-free DNA, we show that mmPCR-NGS can accurately detect CNVs with average allelic imbalances as low as 0.5%, an improvement over previously reported whole-genome sequencing approaches. Our method revealed differences in the spectrum of CNVs detected in tumor tissue subsections and matching plasma samples from 11 patients with stage II breast cancer. Moreover, we showed that liquid biopsies are able to detect subclonal mutations that may be missed in tumor tissue biopsies. We anticipate that this mmPCR-NGS methodology will have broad applicability for the characterization, diagnosis, and therapeutic monitoring of CNV-enriched cancers, such as breast, ovarian, and lung cancer. PMID:26500031

  7. Identification of the first multi-exonic WDR72 deletion in isolated amelogenesis imperfecta, and generation of a WDR72-specific copy number screening tool.

    Hentschel, Julia; Tatun, Dana; Parkhomchuk, Dmitri; Kurth, Ingo; Schimmel, Bettina; Heinrich-Weltzien, Roswitha; Bertzbach, Sabine; Peters, Hartmut; Beetz, Christian


    Amelogenesis imperfecta (AI) is a clinically and genetically heterogeneous disorder of tooth development which is due to aberrant deposition or composition of enamel. Both syndromic and isolated forms exist; they may be inherited in an X-linked, autosomal recessive, or autosomal dominant manner. WDR72 is one of ten currently known genes for recessive isolated AI; nine WDR72 mutations affecting single nucleotides have been described to date. Based on whole exome sequencing in a large consanguineous AI pedigree, we obtained evidence for presence of a multi-exonic WDR72 deletion. A home-made multiplex ligation-dependent probe amplification assay was used to confirm the aberration, to narrow its extent, and to identify heterozygous carriers. Our study extends the mutational spectrum for WDR72 to include large deletions, and supports a relevance of the previously proposed loss-of-function mechanism. It also introduces an easy-to-use and highly sensitive tool for detecting WDR72 copy number alterations. PMID:27259663

  8. Multiplex Ligation-Dependent Probe Amplification Analysis of GATA4 Gene Copy Number Variations in Patients with Isolated Congenital Heart Disease

    Valentina Guida


    Full Text Available GATA4 mutations are found in patients with different isolated congenital heart defects (CHDs, mostly cardiac septal defects and tetralogy of Fallot. In addition, GATA4 is supposed to be the responsible gene for the CHDs in the chromosomal 8p23 deletion syndrome, which is recognized as a malformation syndrome with clinical symptoms of facial anomalies, microcephaly, mental retardation, and congenital heart defects. Thus far, no study has been carried out to investigate the role of GATA4 copy number variations (CNVs in non-syndromic CHDs. To explore the possible occurrence of GATA4 gene CNVs in isolated CHDs, we analyzed by multiplex ligation-dependent probe amplification (MLPA a cohort of 161 non-syndromic patients with cardiac anomalies previously associated with GATA4 gene mutations. The patients were mutation-negative for GATA4, NKX2.5, and FOG2 genes after screening with denaturing high performance liquid chromatography. MLPA analysis revealed that normalized MLPA signals were all found within the normal range values for all exons in all patients, excluding a major contribution of GATA4 gene CNVs in CHD pathogenesis.

  9. Comprehensive assessment of sequence variation within the copy number variable defensin cluster on 8p23 by target enriched in-depth 454 sequencing

    Zhang Xinmin


    Full Text Available Abstract Background In highly copy number variable (CNV regions such as the human defensin gene locus, comprehensive assessment of sequence variations is challenging. PCR approaches are practically restricted to tiny fractions, and next-generation sequencing (NGS approaches of whole individual genomes e.g. by the 1000 Genomes Project is confined by an affordable sequence depth. Combining target enrichment with NGS may represent a feasible approach. Results As a proof of principle, we enriched a ~850 kb section comprising the CNV defensin gene cluster DEFB, the invariable DEFA part and 11 control regions from two genomes by sequence capture and sequenced it by 454 technology. 6,651 differences to the human reference genome were found. Comparison to HapMap genotypes revealed sensitivities and specificities in the range of 94% to 99% for the identification of variations. Using error probabilities for rigorous filtering revealed 2,886 unique single nucleotide variations (SNVs including 358 putative novel ones. DEFB CN determinations by haplotype ratios were in agreement with alternative methods. Conclusion Although currently labor extensive and having high costs, target enriched NGS provides a powerful tool for the comprehensive assessment of SNVs in highly polymorphic CNV regions of individual genomes. Furthermore, it reveals considerable amounts of putative novel variations and simultaneously allows CN estimation.

  10. Copy Number Variation of Cytokinin Oxidase Gene Tackx4 Associated with Grain Weight and Chlorophyll Content of Flag Leaf in Common Wheat

    Chang, Cheng; Lu, Jie; Zhang, Hai-Ping; Ma, Chuan-Xi; Sun, Genlou


    As the main pigment in photosynthesis, chlorophyll significantly affects grain filling and grain weight of crop. Cytokinin (CTK) can effectively increase chlorophyll content and chloroplast stability, but it is irreversibly inactivated by cytokinin oxidase (CKX). In this study, therefore, twenty-four pairs of primers were designed to identify variations of wheat CKX (Tackx) genes associated with flag leaf chlorophyll content after anthesis, as well as grain weight in 169 recombinant inbred lines (RIL) derived from Triticum aestivum Jing 411 × Hongmangchun 21. Results indicated variation of Tackx4, identified by primer pair T19-20, was proven to significantly associate with chlorophyll content and grain weight in the RIL population. Here, two Tackx4 patterns were identified: one with two co-segregated fragments (Tackx4-1/Tackx4-2) containing 618 bp and 620 bp in size (as in Jing 411), and another with no PCR product. The two genotypes were designated as genotype-A and genotype-B, respectively. Grain weight and leaf chlorophyll content at 5~15 days after anthesis (DAA) were significantly higher in genotype-A lines than those in genotype-B lines. Mapping analysis indicated Tackx4 was closely linked to Xwmc169 on chromosome 3AL, as well as co-segregated with a major quantitative trait locus (QTL) for both grain weight and chlorophyll content of flag leaf at 5~15 DAA. This QTL explained 8.9~22.3% phenotypic variations of the two traits across four cropping seasons. Among 102 wheat varieties, a third genotype of Tackx4 was found and designated as genotype-C, also having two co-segregated fragments, Tackx4-2 and Tackx4-3 (615bp). The sequences of three fragments, Tackx4-1, Tackx4-2, and Tackx4-3, showed high identity (>98%). Therefore, these fragments could be considered as different copies at Tackx4 locus on chromosome 3AL. The effect of copy number variation (CNV) of Tackx4 was further validated. In general, genotype-A contains both significantly higher grain weight

  11. The DUB/USP17 deubiquitinating enzymes: A gene family within a tandemly repeated sequence, is also embedded within the copy number variable Beta-defensin cluster

    Scott Christopher J


    Full Text Available Abstract Background The DUB/USP17 subfamily of deubiquitinating enzymes were originally identified as immediate early genes induced in response to cytokine stimulation in mice (DUB-1, DUB-1A, DUB-2, DUB-2A. Subsequently we have identified a number of human family members and shown that one of these (DUB-3 is also cytokine inducible. We originally showed that constitutive expression of DUB-3 can block cell proliferation and more recently we have demonstrated that this is due to its regulation of the ubiquitination and activity of the 'CAAX' box protease RCE1. Results Here we demonstrate that the human DUB/USP17 family members are found on both chromosome 4p16.1, within a block of tandem repeats, and on chromosome 8p23.1, embedded within the copy number variable beta-defensin cluster. In addition, we show that the multiple genes observed in humans and other distantly related mammals have arisen due to the independent expansion of an ancestral sequence within each species. However, it is also apparent when sequences from humans and the more closely related chimpanzee are compared, that duplication events have taken place prior to these species separating. Conclusions The observation that the DUB/USP17 genes, which can influence cell growth and survival, have evolved from an unstable ancestral sequence which has undergone multiple and varied duplications in the species examined marks this as a unique family. In addition, their presence within the beta-defensin repeat raises the question whether they may contribute to the influence of this repeat on immune related conditions.

  12. Colorimetric In Situ Hybridization Identifies MYC Gene Signal Clusters Correlating With Increased Copy Number, mRNA, and Protein in Diffuse Large B-cell Lymphoma

    Valentino, Carlo; Kendrick, Samantha; Johnson, Nathalie; Gascoyne, Randy; Chan, Wing C.; Weisenburger, Dennis; Braziel, Rita; Cook, James R.; Tubbs, Raymond; Campo, Elias; Rosenwald, Andreas; Ott, German; Delabie, Jan; Jaffe, Elaine; Zhang, Wenjun


    Abnormalities of the MYC oncogene on chromosome 8 are characteristic of Burkitt lymphoma and other aggressive B-cell lymphomas, including diffuse large B-cell lymphoma (DLBCL). We recently described a colorimetric in situ hybridization (CISH) method for detecting extra copies of the MYC gene in DLBCL and the frequent occurrence of excess copies of discrete MYC signals in the context of diploidy or polyploidy of chromosome 8, which correlated with increased mRNA signals. We further observed en...

  13. Array-based comparative genomic hybridization analysis reveals chromosomal copy number aberrations associated with clinical outcome in canine diffuse large B-cell lymphoma.

    Arianna Aricò

    Full Text Available Canine Diffuse Large B-cell Lymphoma (cDLBCL is an aggressive cancer with variable clinical response. Despite recent attempts by gene expression profiling to identify the dog as a potential animal model for human DLBCL, this tumor remains biologically heterogeneous with no prognostic biomarkers to predict prognosis. The aim of this work was to identify copy number aberrations (CNAs by high-resolution array comparative genomic hybridization (aCGH in 12 dogs with newly diagnosed DLBCL. In a subset of these dogs, the genetic profiles at the end of therapy and at relapse were also assessed. In primary DLBCLs, 90 different genomic imbalances were counted, consisting of 46 gains and 44 losses. Two gains in chr13 were significantly correlated with clinical stage. In addition, specific regions of gains and losses were significantly associated to duration of remission. In primary DLBCLs, individual variability was found, however 14 recurrent CNAs (>30% were identified. Losses involving IGK, IGL and IGH were always found, and gains along the length of chr13 and chr31 were often observed (>41%. In these segments, MYC, LDHB, HSF1, KIT and PDGFRα are annotated. At the end of therapy, dogs in remission showed four new CNAs, whereas three new CNAs were observed in dogs at relapse compared with the previous profiles. One ex novo CNA, involving TCR, was present in dogs in remission after therapy, possibly induced by the autologous vaccine. Overall, aCGH identified small CNAs associated with outcome, which, along with future expression studies, may reveal target genes relevant to cDLBCL.

  14. Challenges in detecting genomic copy number aberrations using next-generation sequencing data and the eXome Hidden Markov Model: a clinical exome-first diagnostic approach

    Yamamoto, Toshiyuki; Shimojima, Keiko; Ondo, Yumiko; Imai, Katsumi; Chong, Pin Fee; Kira, Ryutaro; Amemiya, Mitsuhiro; Saito, Akira; Okamoto, Nobuhiko


    Next-generation sequencing (NGS) is widely used for the detection of disease-causing nucleotide variants. The challenges associated with detecting copy number variants (CNVs) using NGS analysis have been reported previously. Disease-related exome panels such as Illumina TruSight One are more cost-effective than whole-exome sequencing (WES) because of their selective target regions (~21% of the WES). In this study, CNVs were analyzed using data extracted through a disease-related exome panel analysis and the eXome Hidden Markov Model (XHMM). Samples from 61 patients with undiagnosed developmental delays and 52 healthy parents were included in this study. In the preliminary study to validate the constructed XHMM system (microarray-first approach), 34 patients who had previously been analyzed by chromosomal microarray testing were used. Among the five CNVs larger than 200 kb that were considered as non-pathogenic CNVs and were used as positive controls, four CNVs was successfully detected. The system was subsequently used to analyze different samples from 27 patients (NGS-first approach); 2 of these patients were successfully diagnosed as having pathogenic CNVs (an unbalanced translocation der(5)t(5;14) and a 16p11.2 duplication). These diagnoses were re-confirmed by chromosomal microarray testing and/or fluorescence in situ hybridization. The NGS-first approach generated no false-negative or false-positive results for pathogenic CNVs, indicating its high sensitivity and specificity in detecting pathogenic CNVs. The results of this study show the possible clinical utility of pathogenic CNV screening using disease-related exome panel analysis and XHMM.

  15. Genome-wide copy number variation analysis in extended families and unrelated individuals characterized for musical aptitude and creativity in music.

    Ukkola-Vuoti, Liisa; Kanduri, Chakravarthi; Oikkonen, Jaana; Buck, Gemma; Blancher, Christine; Raijas, Pirre; Karma, Kai; Lähdesmäki, Harri; Järvelä, Irma


    Music perception and practice represent complex cognitive functions of the human brain. Recently, evidence for the molecular genetic background of music related phenotypes has been obtained. In order to further elucidate the molecular background of musical phenotypes we analyzed genome wide copy number variations (CNVs) in five extended pedigrees and in 172 unrelated subjects characterized for musical aptitude and creative functions in music. Musical aptitude was defined by combination of the scores of three music tests (COMB scores): auditory structuring ability, Seashores test for pitch and for time. Data on creativity in music (herein composing, improvising and/or arranging music) was surveyed using a web-based questionnaire.Several CNVRs containing genes that affect neurodevelopment, learning and memory were detected. A deletion at 5q31.1 covering the protocadherin-α gene cluster (Pcdha 1-9) was found co-segregating with low music test scores (COMB) in both sample sets. Pcdha is involved in neural migration, differentiation and synaptogenesis. Creativity in music was found to co-segregate with a duplication covering glucose mutarotase gene (GALM) at 2p22. GALM has influence on serotonin release and membrane trafficking of the human serotonin transporter. Interestingly, genes related to serotonergic systems have been shown to associate not only with psychiatric disorders but also with creativity and music perception. Both, Pcdha and GALM, are related to the serotonergic systems influencing cognitive and motor functions, important for music perception and practice. Finally, a 1.3 Mb duplication was identified in a subject with low COMB scores in the region previously linked with absolute pitch (AP) at 8q24. No differences in the CNV burden was detected among the high/low music test scores or creative/non-creative groups. In summary, CNVs and genes found in this study are related to cognitive functions. Our result suggests new candidate genes for music perception

  16. Genome-wide copy number variation analysis in extended families and unrelated individuals characterized for musical aptitude and creativity in music.

    Liisa Ukkola-Vuoti

    Full Text Available Music perception and practice represent complex cognitive functions of the human brain. Recently, evidence for the molecular genetic background of music related phenotypes has been obtained. In order to further elucidate the molecular background of musical phenotypes we analyzed genome wide copy number variations (CNVs in five extended pedigrees and in 172 unrelated subjects characterized for musical aptitude and creative functions in music. Musical aptitude was defined by combination of the scores of three music tests (COMB scores: auditory structuring ability, Seashores test for pitch and for time. Data on creativity in music (herein composing, improvising and/or arranging music was surveyed using a web-based questionnaire.Several CNVRs containing genes that affect neurodevelopment, learning and memory were detected. A deletion at 5q31.1 covering the protocadherin-α gene cluster (Pcdha 1-9 was found co-segregating with low music test scores (COMB in both sample sets. Pcdha is involved in neural migration, differentiation and synaptogenesis. Creativity in music was found to co-segregate with a duplication covering glucose mutarotase gene (GALM at 2p22. GALM has influence on serotonin release and membrane trafficking of the human serotonin transporter. Interestingly, genes related to serotonergic systems have been shown to associate not only with psychiatric disorders but also with creativity and music perception. Both, Pcdha and GALM, are related to the serotonergic systems influencing cognitive and motor functions, important for music perception and practice. Finally, a 1.3 Mb duplication was identified in a subject with low COMB scores in the region previously linked with absolute pitch (AP at 8q24. No differences in the CNV burden was detected among the high/low music test scores or creative/non-creative groups. In summary, CNVs and genes found in this study are related to cognitive functions. Our result suggests new candidate genes for

  17. Whole exome sequencing is necessary to clarify ID/DD cases with de novo copy number variants of uncertain significance: Two proof-of-concept examples.

    Giorgio, Elisa; Ciolfi, Andrea; Biamino, Elisa; Caputo, Viviana; Di Gregorio, Eleonora; Belligni, Elga Fabia; Calcia, Alessandro; Gaidolfi, Elena; Bruselles, Alessandro; Mancini, Cecilia; Cavalieri, Simona; Molinatto, Cristina; Cirillo Silengo, Margherita; Ferrero, Giovanni Battista; Tartaglia, Marco; Brusco, Alfredo


    Whole exome sequencing (WES) is a powerful tool to identify clinically undefined forms of intellectual disability/developmental delay (ID/DD), especially in consanguineous families. Here we report the genetic definition of two sporadic cases, with syndromic ID/DD for whom array-Comparative Genomic Hybridization (aCGH) identified a de novo copy number variant (CNV) of uncertain significance. The phenotypes included microcephaly with brachycephaly and a distinctive facies in one proband, and hypotonia in the legs and mild ataxia in the other. WES allowed identification of a functionally relevant homozygous variant affecting a known disease gene for rare syndromic ID/DD in each proband, that is, c.1423C>T (p.Arg377*) in the Trafficking Protein Particle Complex 9 (TRAPPC9), and c.154T>C (p.Cys52Arg) in the Very Low Density Lipoprotein Receptor (VLDLR). Four mutations affecting TRAPPC9 have been previously reported, and the present finding further depicts this syndromic form of ID, which includes microcephaly with brachycephaly, corpus callosum hypoplasia, facial dysmorphism, and overweight. VLDLR-associated cerebellar hypoplasia (VLDLR-CH) is characterized by non-progressive congenital ataxia and moderate-to-profound intellectual disability. The c.154T>C (p.Cys52Arg) mutation was associated with a very mild form of ataxia, mild intellectual disability, and cerebellar hypoplasia without cortical gyri simplification. In conclusion, we report two novel cases with rare causes of autosomal recessive ID, which document how interpreting de novo array-CGH variants represents a challenge in consanguineous families; as such, clinical WES should be considered in diagnostic testing. © 2016 Wiley Periodicals, Inc. PMID:27108886

  18. Nuclear and mitochondrial DNAs microsatellite instability and mitochondrial DNA copy number in adenocarcinoma and squamous cell carcinoma of lung: a pilot study.

    Lee, Deok Heon; Lee, Jae-Ho; Kim, Dae-Kwang; Keum, Dong Yoon


    Mitochondrial genetic changes are considered as a key molecular step of mutations in various cancers. To clarify the role of genetic instability in lung cancer, we analyzed clinicopathological characteristics and frequencies of nuclear and mitochondrial microsatellite instability (nMSI and mtMSI), and alteration of mitochondrial DNA copy number (mtCN) in adenocarcinoma (ADC) and squamous cell carcinoma (SCC) of lung. DNA was isolated from 48 patients with ADC and 42 with SCC. Markers for nMSI, BAT 25 and 26, and markers for mtMSI, (C)n and (CA)n in mitochondrial D-loop region, were utilized. The mtCN were measured by real-time polymerase chain reaction. The nMSI was found in two patients (4.2%) of ADC and 6 (14.3%) of SCC. The mtMSI was detected in 10 patients (20.8%) of ADC and 8 (19.0%) of SCC. Mean mtCN was 5.05 ± 8.17 and 3.34 ± 5.14 in ADC and SCC respectively. The mtCN was increased in 35 patients (72.9%) of ADC and 30 (71.4%) of SCC. The mtMSI more frequently appeared in more advanced pathologic T stage in ADC (p = 0.003). Alterations of mtCN and a high frequency of mtMSI in our patient samples indicate that mitochondrial DNA is a potential molecular marker in lung cancers (ADC and SCC) correlating with their histological classification. PMID:26547371

  19. Identification of rare recurrent copy number variants in high-risk autism families and their prevalence in a large ASD population.

    Nori Matsunami

    Full Text Available Structural variation is thought to play a major etiological role in the development of autism spectrum disorders (ASDs, and numerous studies documenting the relevance of copy number variants (CNVs in ASD have been published since 2006. To determine if large ASD families harbor high-impact CNVs that may have broader impact in the general ASD population, we used the Affymetrix genome-wide human SNP array 6.0 to identify 153 putative autism-specific CNVs present in 55 individuals with ASD from 9 multiplex ASD pedigrees. To evaluate the actual prevalence of these CNVs as well as 185 CNVs reportedly associated with ASD from published studies many of which are insufficiently powered, we designed a custom Illumina array and used it to interrogate these CNVs in 3,000 ASD cases and 6,000 controls. Additional single nucleotide variants (SNVs on the array identified 25 CNVs that we did not detect in our family studies at the standard SNP array resolution. After molecular validation, our results demonstrated that 15 CNVs identified in high-risk ASD families also were found in two or more ASD cases with odds ratios greater than 2.0, strengthening their support as ASD risk variants. In addition, of the 25 CNVs identified using SNV probes on our custom array, 9 also had odds ratios greater than 2.0, suggesting that these CNVs also are ASD risk variants. Eighteen of the validated CNVs have not been reported previously in individuals with ASD and three have only been observed once. Finally, we confirmed the association of 31 of 185 published ASD-associated CNVs in our dataset with odds ratios greater than 2.0, suggesting they may be of clinical relevance in the evaluation of children with ASDs. Taken together, these data provide strong support for the existence and application of high-impact CNVs in the clinical genetic evaluation of children with ASD.

  20. Pediatric primary central nervous system germ cell tumors of different prognosis groups show characteristic miRNome traits and chromosome copy number variations

    Liang Muh-Lii


    Full Text Available Abstract Background Intracranial pediatric germ cell tumors (GCTs are rare and heterogeneous neoplasms and vary in histological differentiation, prognosis and clinical behavior. Germinoma and mature teratoma are GCTs that have a good prognosis, while other types of GCTs, termed nongerminomatous malignant germ cell tumors (NGMGCTs, are tumors with an intermediate or poor prognosis. The second group of tumors requires more extensive drug and irradiation treatment regimens. The mechanisms underlying the differences in incidence and prognosis of the various GCT subgroups are unclear. Results We identified a distinct mRNA profile correlating with GCT histological differentiation and prognosis, and also present in this study the first miRNA profile of pediatric primary intracranial GCTs. Most of the differentially expressed miRNAs were downregulated in germinomas, but miR-142-5p and miR-146a were upregulated. Genes responsible for self-renewal (such as POU5F1 (OCT4, NANOG and KLF4 and the immune response were abundant in germinomas, while genes associated with neuron differentiation, Wnt/β-catenin pathway, invasiveness and epithelial-mesenchymal transition (including SNAI2 (SLUG and TWIST2 were abundant in NGMGCTs. Clear transcriptome segregation based on patient survival was observed, with malignant NGMGCTs being closest to embryonic stem cells. Chromosome copy number variations (CNVs at cytobands 4q13.3-4q28.3 and 9p11.2-9q13 correlated with GCT malignancy and clinical risk. Six genes (BANK1, CXCL9, CXCL11, DDIT4L, ELOVL6 and HERC5 within 4q13.3-4q28.3 were more abundant in germinomas. Conclusions Our results integrate molecular profiles with clinical observations and provide insights into the underlying mechanisms causing GCT malignancy. The genes, pathways and microRNAs identified have the potential to be novel therapeutic targets.