High Copy Number Variation Research Articles

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

BackgroundBtau_4.0 and UMD3.1 are two distinct cattle reference genome assemblies. In our previous study using the low density BovineSNP50 array, we reported a copy number variation (CNV) analysis on Btau_4.0 with 521 animals of 21 cattle breeds, yielding 682 CNV regions with a total length of 139.8 megabases.ResultsIn this study using the high density BovineHD SNP array, we performed high resolution CNV analyses on both Btau_4.0 and UMD3.1 with 674 animals of 27 cattle breeds. We first compared CNV results derived from these two different SNP array platforms on Btau_4.0. With two thirds of the animals shared between studies, on Btau_4.0 we identified 3,346 candidate CNV regions representing 142.7 megabases (~4.70%) of the genome. With a similar total length but 5 times more event counts, the average CNVR length of current Btau_4.0 dataset is significantly shorter than the previous one (42.7 kb vs. 205 kb). Although subsets of these two results overlapped, 64% (91.6 megabases) of current dataset was not present in the previous study. We also performed similar analyses on UMD3.1 using these BovineHD SNP array results. Approximately 50% more and 20% longer CNVs were called on UMD3.1 as compared to those on Btau_4.0. However, a comparable result of CNVRs (3,438 regions with a total length 146.9 megabases) was obtained. We suspect that these results are due to the UMD3.1 assembly's efforts of placing unplaced contigs and removing unmerged alleles. Selected CNVs were further experimentally validated, achieving a 73% PCR validation rate, which is considerably higher than the previous validation rate. About 20-45% of CNV regions overlapped with cattle RefSeq genes and Ensembl genes. Panther and IPA analyses indicated that these genes provide a wide spectrum of biological processes involving immune system, lipid metabolism, cell, organism and system development.ConclusionWe present a comprehensive result of cattle CNVs at a higher resolution and sensitivity. We identified over 3,000 candidate CNV regions on both Btau_4.0 and UMD3.1, further compared current datasets with previous results, and examined the impacts of genome assemblies on CNV calling.

Cn.MOPS: mixture of Poissons for discovering copy number variations in next-generation sequencing data with a low false discovery rate

Quantitative analyses of next-generation sequencing (NGS) data, such as the detection of copy number variations (CNVs), remain challenging. Current methods detect CNVs as changes in the depth of coverage along chromosomes. Technological or genomic variations in the depth of coverage thus lead to a high false discovery rate (FDR), even upon correction for GC content. In the context of association studies between CNVs and disease, a high FDR means many false CNVs, thereby decreasing the discovery power of the study after correction for multiple testing. We propose ‘Copy Number estimation by a Mixture Of PoissonS’ (cn.MOPS), a data processing pipeline for CNV detection in NGS data. In contrast to previous approaches, cn.MOPS incorporates modeling of depths of coverage across samples at each genomic position. Therefore, cn.MOPS is not affected by read count variations along chromosomes. Using a Bayesian approach, cn.MOPS decomposes variations in the depth of coverage across samples into integer copy numbers and noise by means of its mixture components and Poisson distributions, respectively. The noise estimate allows for reducing the FDR by filtering out detections having high noise that are likely to be false detections. We compared cn.MOPS with the five most popular methods for CNV detection in NGS data using four benchmark datasets: (i) simulated data, (ii) NGS data from a male HapMap individual with implanted CNVs from the X chromosome, (iii) data from HapMap individuals with known CNVs, (iv) high coverage data from the 1000 Genomes Project. cn.MOPS outperformed its five competitors in terms of precision (1–FDR) and recall for both gains and losses in all benchmark data sets. The software cn.MOPS is publicly available as an R package at http://www.bioinf.jku.at/software/cnmops/ and at Bioconductor.

Higher Copy Number Variation and Diverse X Chromosome Inactivation in Parthenote-derived Human Embryonic Stem Cells

Parthenote-derived human embryonic stem cells (phESCs) have many advantages over conventionally derived human embryonic stem cells (hESCs), but a more thorough investigation of these cells is needed before they can be implemented in cell therapies. In this work, we used a Cytogenetics Whole-Genome Array to study the copy number variation (CNV) status in phESCs and hESCs. We also investigated X chromosome inactivation (XCI) and expression levels of marker genes in these cells. More CNVs were found in phESCs than in hESCs in the present study, and gene expression appeared to be associated with the gain or loss of CNVs. In addition, a variable XCI status and different expression pattern of paternally expressed imprinted gene were also found in phESCs. In conclusion, although phESCs had a similar pluripotent profile to conventionally derived hESCs, these cells differed in imprinted gene expression, XCI status and number of CNVs. Our work highlights the need for a deeper investigation to elucidate the genetic and epigenetic characteristics of these cells.

Evidence of novel fine-scale structural variation at autism spectrum disorder candidate loci

BackgroundAutism spectrum disorders (ASD) represent a group of neurodevelopmental disorders characterized by a core set of social-communicative and behavioral impairments. Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the brain, acting primarily via the GABA receptors (GABR). Multiple lines of evidence, including altered GABA and GABA receptor expression in autistic patients, indicate that the GABAergic system may be involved in the etiology of autism.MethodsAs copy number variations (CNVs), particularly rare and de novo CNVs, have now been implicated in ASD risk, we examined the GABA receptors and genes in related pathways for structural variation that may be associated with autism. We further extended our candidate gene set to include 19 genes and regions that had either been directly implicated in the autism literature or were directly related (via function or ancestry) to these primary candidates. For the high resolution CNV screen we employed custom-designed 244 k comparative genomic hybridization (CGH) arrays. Collectively, our probes spanned a total of 11 Mb of GABA-related and additional candidate regions with a density of approximately one probe every 200 nucleotides, allowing a theoretical resolution for detection of CNVs of approximately 1 kb or greater on average. One hundred and sixty-eight autism cases and 149 control individuals were screened for structural variants. Prioritized CNV events were confirmed using quantitative PCR, and confirmed loci were evaluated on an additional set of 170 cases and 170 control individuals that were not included in the original discovery set. Loci that remained interesting were subsequently screened via quantitative PCR on an additional set of 755 cases and 1,809 unaffected family members.ResultsResults include rare deletions in autistic individuals at JAKMIP1, NRXN1, Neuroligin4Y, OXTR, and ABAT. Common insertion/deletion polymorphisms were detected at several loci, including GABBR2 and NRXN3. Overall, statistically significant enrichment in affected vs. unaffected individuals was observed for NRXN1 deletions.ConclusionsThese results provide additional support for the role of rare structural variation in ASD.

CCL4L Polymorphisms and CCL4/CCL4L Serum Levels Are Associated with Psoriasis Severity

Psoriasis is a common inflammatory skin disease with key immunological and genetic components. Recruitment of leukocytes into the skin is a central step in its pathogenesis, mediated by cytokines. Among the cytokines expressed in psoriatic lesions, C-C chemokine ligand 4 (CCL4) and C-C chemokine ligand 4-like (CCL4L) chemokines appear to be pivotal elements for the skin recruitment of proinflammatory cells. The aim of this study is to evaluate the relationship between CCL4L polymorphisms (including single-nucleotide polymorphisms (SNPs) and copy number variation (CNV)) and the course and prognosis of psoriasis. We analyzed the CNV and the rs4796195 SNP in 211 psoriatic patients and 234 controls; sera from both populations were also quantified for CCL4/CCL4L protein. Our results showed that a high CNV (≥3 copies) is associated with psoriasis severity, whereas moderate disease correlated with a lower CNV (≤2 copies); specifically, the CCL4L1 allele frequency is higher in severe psoriasis, whereas CCL4L2 is more frequent in patients with a milder disease. In addition, we found a positive correlation between the CNV and sera protein levels. Our results suggest that CCL4L genotyping could not only allow a better understanding of the psoriatic pathogenesis but could also be used as a prognostic tool, even helping to modulate the efficacy of treatments.

Assessment of copy number variation using the Illumina Infinium 1M SNP-array: a comparison of methodological approaches in the Spanish Bladder Cancer/EPICURO study

High-throughput single nucleotide polymorphism (SNP)-array technologies allow to investigate copy number variants (CNVs) in genome-wide scans and specific calling algorithms have been developed to determine CNV location and copy number. We report the results of a reliability analysis comparing data from 96 pairs of samples processed with CNVpartition, PennCNV, and QuantiSNP for Infinium Illumina Human 1Million probe chip data. We also performed a validity assessment with multiplex ligation-dependent probe amplification (MLPA) as a reference standard. The number of CNVs per individual varied according to the calling algorithm. Higher numbers of CNVs were detected in saliva than in blood DNA samples regardless of the algorithm used. All algorithms presented low agreement with mean Kappa Index (KI) <66. PennCNV was the most reliable algorithm (KI(w=) 98.96) when assessing the number of copies. The agreement observed in detecting CNV was higher in blood than in saliva samples. When comparing to MLPA, all algorithms identified poorly known copy aberrations (sensitivity = 0.19-0.28). In contrast, specificity was very high (0.97-0.99). Once a CNV was detected, the number of copies was truly assessed (sensitivity >0.62). Our results indicate that the current calling algorithms should be improved for high performance CNV analysis in genome-wide scans. Further refinement is required to assess CNVs as risk factors in complex diseases.

CCL4L polymorphisms and serum levels are associated with psoriasis severity

Psoriasis is a common inflammatory skin disease with key immunological and genetic components. Recruitment of leukocytes into the skin is a central step in its pathogenesis, and it is mediated by cytokines. Among the cytokines expressed in psoriatic lesions, CCL4 and CCL4L chemokines appear to be pivotal elements for the skin recruitment of proinflammatory cells. The aim of this study is to evaluate the relationship between CCL4L polymorphisms [including Single Nucleotide Polymorphisms (SNPs) and Copy Number Variation (CNV)] and the course and prognosis of psoriasis. We analyzed the CNV and the rs4796195 SNP in 211 psoriatic patients and 234 controls; sera from both populations were also quantified for CCL4/CCL4L protein. Our results showed that a high CNV (≥3 copies) is associated with psoriasis severity, while moderate disease is more frequent in patients with lower CNV (≤2 copies); specifically CCL4L1 allele is more present in patients with severe psoriasis, while CCL4L2 correlates with a milder disease. In addition we found a positive correlation between the CNV and sera protein levels. Our results suggest that CCL4L genotyping could not only allow a better understanding of the psoriatic pathogenesis, but could also be used as a prognostic tool, even helping to modulate the efficacy of treatments.

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

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

Distinct high resolution genome profiles of early onset and late onset colorectal cancer integrated with gene expression data identify candidate susceptibility loci

BackgroundEstimates suggest that up to 30% of colorectal cancers (CRC) may develop due to an increased genetic risk. The mean age at diagnosis for CRC is about 70 years. Time of disease onset 20 years younger than the mean age is assumed to be indicative of genetic susceptibility. We have compared high resolution tumor genome copy number variation (CNV) (Roche NimbleGen, 385 000 oligo CGH array) in microsatellite stable (MSS) tumors from two age groups, including 23 young at onset patients without known hereditary syndromes and with a median age of 44 years (range: 28-53) and 17 elderly patients with median age 79 years (range: 69-87). Our aim was to identify differences in the tumor genomes between these groups and pinpoint potential susceptibility loci. Integration analysis of CNV and genome wide mRNA expression data, available for the same tumors, was performed to identify a restricted candidate gene list.ResultsThe total fraction of the genome with aberrant copy number, the overall genomic profile and the TP53 mutation spectrum were similar between the two age groups. However, both the number of chromosomal aberrations and the number of breakpoints differed significantly between the groups. Gains of 2q35, 10q21.3-22.1, 10q22.3 and 19q13.2-13.31 and losses from 1p31.3, 1q21.1, 2q21.2, 4p16.1-q28.3, 10p11.1 and 19p12, positions that in total contain more than 500 genes, were found significantly more often in the early onset group as compared to the late onset group. Integration analysis revealed a covariation of DNA copy number at these sites and mRNA expression for 107 of the genes. Seven of these genes, CLC, EIF4E, LTBP4, PLA2G12A, PPAT, RG9MTD2, and ZNF574, had significantly different mRNA expression comparing median expression levels across the transcriptome between the two groups.ConclusionsTen genomic loci, containing more than 500 protein coding genes, are identified as more often altered in tumors from early onset versus late onset CRC. Integration of genome and transcriptome data identifies seven novel candidate genes with the potential to identify an increased risk for CRC.

Post Transplant Allogeneic Antibody Responses Form against Annexin 8

Allogeneic hematopoietic cell transplantation can cure hematologic malignancies through graft versus leukemia effects but are limited by graft versus host disease. Antibodies against human Y chromosome antigens are associated with graft versus host disease and we hypothesized that antibodies could conversely be associated with post transplant graft versus leukemia effects. We identified an index male patient with chronic lymphocytic leukemia who received a female PBSC graft following reduced intensity conditioning allogeneic hematopoietic cell transplantation. This F→M had developed allogeneic antibodies against H-Y antigen UTY. We hypothesized allogeneic antibodies develop against autosomal minor histocompatibility antigens and could be identified as targets of allo-antibodies. Protein microarrays (Invitrogen Protoarray) featuring 8000 baculovirus expressed human proteins with N-terminal GST fusions were screened with plasma samples from the index patient and antibodies binding to potential graft versus leukemia antigens were detected with fluorescently tagged secondary antibodies. We compared post-transplant with pre-transplant and donor plasma fluorescent intensities and identified new post transplant antibody responses against annexin 8 which were not present prior to transplant or in the donor. Our finding of new antibody responses against annexin 8 was confirmed by Western blot against E.coli expressed affinity purified recombinant V5-His6 tagged annexin 8.Annexin 8 is a membrane associated protein postulated to play a role in cellular proliferation. Recent genomic analyses have identified three loci coding for three separate annexin 8 genes clustered on chromosome 10 in a region of high frequency copy number variation (CNV). In addition, the annexin 8 mRNA transcript undergoes alternative splicing with one variant being identified from a choriocarcinoma cell line. Both CNV and alternative splicing may result in immunologic disparities between donor and recipient inducing alloimmunity.. Therefore, we tested these possibilities by synthesizing recombinant V5-His6 annexin 8 proteins corresponding to the three possible isoforms and using them in an ELISA to screen 187 patients with a variety of malignancies for post-allogeneic transplant antibodies. The recombinant proteins included the alternative splice variant from choriocarcinoma which differed from the common variant by the insertion of a 38 amino acid sequence after the second exon and the deletion of two 57 and 32 amino acid sequences from exons 6, 7, and 10 of the common variant, as well as two versions of the common variant which differed by a G to C nonsynonymous single nucleotide polymorphism resulting in a change from glycine to alanine. These two recombinant common variant isoforms correspond to the reported sequences of two of the annexin 8 loci affected by copy number variation. Thus reactions to either, but not both, might indicate disparities in annexin 8 copy number between recipient and donor. Of 187 patients, 22 had significant antibody reactions (defined as greater than 1 standard deviation above the mean in normal controls) to either of the common variant isoforms but only 4 had reactions to both isoforms suggesting that antibodies were differentially recognizing annexin 8 isoforms based on a single nonsynonymous single nucleotide polymorphism. Six of 187 patients had significant antibody reactions against the splice variant isoform alone, suggesting that the alternatively inserted exon may be the antibody target.Taken together, our findings suggest that annexin 8 may be a target of the post-transplant humoral response due to immune disparities between recipient and donor caused by copy number variation as well as alternative splicing.