Type Of Copy Number Variation Research Articles

Large Inverted Duplications in the Human Genome Form via a Fold-Back Mechanism

Inverted duplications are a common type of copy number variation (CNV) in germline and somatic genomes. Large duplications that include many genes can lead to both neurodevelopmental phenotypes in children and gene amplifications in tumors. There are several models for inverted duplication formation, most of which include a dicentric chromosome intermediate followed by breakage-fusion-bridge (BFB) cycles, but the mechanisms that give rise to the inverted dicentric chromosome in most inverted duplications remain unknown. Here we have combined high-resolution array CGH, custom sequence capture, next-generation sequencing, and long-range PCR to analyze the breakpoints of 50 nonrecurrent inverted duplications in patients with intellectual disability, autism, and congenital anomalies. For half of the rearrangements in our study, we sequenced at least one breakpoint junction. Sequence analysis of breakpoint junctions reveals a normal-copy disomic spacer between inverted and non-inverted copies of the duplication. Further, short inverted sequences are present at the boundary of the disomic spacer and the inverted duplication. These data support a mechanism of inverted duplication formation whereby a chromosome with a double-strand break intrastrand pairs with itself to form a “fold-back” intermediate that, after DNA replication, produces a dicentric inverted chromosome with a disomic spacer corresponding to the site of the fold-back loop. This process can lead to inverted duplications adjacent to terminal deletions, inverted duplications juxtaposed to translocations, and inverted duplication ring chromosomes.

Gene Network Analysis of Candidate Loci for Human Anorectal Malformations

Anorectal malformations (ARMs) are birth defects that require surgery and carry significant chronic morbidity. Our earlier genome-wide copy number variation (CNV) study had provided a wealth of candidate loci. To find out whether these candidate loci are related to important developmental pathways, we have performed an extensive literature search coupled with the currently available bioinformatics tools. This has allowed us to assign both genic and non-genic CNVs to interrelated pathways known to govern the development of the anorectal region. We have linked 11 candidate genes to the WNT signalling pathway and 17 genes to the cytoskeletal network. Interestingly, candidate genes with similar functions are disrupted by the same type of CNV. The gene network we discovered provides evidence that rare mutations in different interrelated genes may lead to similar phenotypes, accounting for genetic heterogeneity in ARMs. Classification of patients according to the affected pathway and lesion type should eventually improve the diagnosis and the identification of common genes/molecules as therapeutic targets.

Widespread Divergence of the CEACAM/PSG Genes in Vertebrates and Humans Suggests Sensitivity to Selection

In mammals, carcinoembryonic antigen cell adhesion molecules (CEACAMs) and pregnancy-specific glycoproteins (PSGs) play important roles in the regulation of pathogen transmission, tumorigenesis, insulin signaling turnover, and fetal–maternal interactions. However, how these genes evolved and to what extent they diverged in humans remain to be investigated specifically. Based on syntenic mapping of chordate genomes, we reveal that diverging homologs with a prototypic CEACAM architecture–including an extracellular domain with immunoglobulin variable and constant domain-like regions, and an intracellular domain containing ITAM motif–are present from cartilaginous fish to humans, but are absent in sea lamprey, cephalochordate or urochordate. Interestingly, the CEACAM/PSG gene inventory underwent radical divergence in various vertebrate lineages: from zero in avian species to dozens in therian mammals. In addition, analyses of genetic variations in human populations showed the presence of various types of copy number variations (CNVs) at the CEACAM/PSG locus. These copy number polymorphisms have 3–80% frequency in select populations, and encompass single to more than six PSG genes. Furthermore, we found that CEACAM/PSG genes contain a significantly higher density of nonsynonymous single nucleotide polymorphism (SNP) compared to the chromosome average, and many CEACAM/PSG SNPs exhibit high population differentiation. Taken together, our study suggested that CEACAM/PSG genes have had a more dynamic evolutionary history in vertebrates than previously thought. Given that CEACAM/PSGs play important roles in maternal–fetal interaction and pathogen recognition, these data have laid the groundwork for future analysis of adaptive CEACAM/PSG genotype-phenotypic relationships in normal and complicated pregnancies as well as other etiologies.

Copy number variations (CNVs) in human pluripotent cell-derived neuroprogenitors

Results from the analysis of copy number variations (CNVs) in human pluripotent cell-derived neuroprogenitor cell lines (hiPSC and hESC-derived NPC) are presented. Two different types of CNVs were detected: a) CNVs inherited from the original source of pluripotent cells (hESC and hiPSC) and b) CNVs detected either in the original source of pluripotent cells or in the derived NPC cell lines but not in both at the same time.Our data suggest that submicroscopic chromosomal changes happened during culture and manipulation of cells and those differentiation procedures could result in gains and losses of genomic regions in pluripotent cell-derived neuroprogenitors. Overall, the results indicate that even chromosomally stable stem cell lines would need to be analyzed in detail by high resolution methodologies before their clinical use.

Genovar: a detection and visualization tool for genomic variants

BackgroundAlong with single nucleotide polymorphisms (SNPs), copy number variation (CNV) is considered an important source of genetic variation associated with disease susceptibility. Despite the importance of CNV, the tools currently available for its analysis often produce false positive results due to limitations such as low resolution of array platforms, platform specificity, and the type of CNV. To resolve this problem, spurious signals must be separated from true signals by visual inspection. None of the previously reported CNV analysis tools support this function and the simultaneous visualization of comparative genomic hybridization arrays (aCGH) and sequence alignment. The purpose of the present study was to develop a useful program for the efficient detection and visualization of CNV regions that enables the manual exclusion of erroneous signals.ResultsA JAVA-based stand-alone program called Genovar was developed. To ascertain whether a detected CNV region is a novel variant, Genovar compares the detected CNV regions with previously reported CNV regions using the Database of Genomic Variants (DGV, http://projects.tcag.ca/variation) and the Single Nucleotide Polymorphism Database (dbSNP). The current version of Genovar is capable of visualizing genomic data from sources such as the aCGH data file and sequence alignment format files.ConclusionsGenovar is freely accessible and provides a user-friendly graphic user interface (GUI) to facilitate the detection of CNV regions. The program also provides comprehensive information to help in the elimination of spurious signals by visual inspection, making Genovar a valuable tool for reducing false positive CNV results. Availability: http://genovar.sourceforge.net/.

Lipoprotein(a): Reloaded

Lipoprotein(a) is a macromolecular complex of enigmatic function in human plasma. Concentrations of Lp(a) constitute a quantitative genetic trait. They are primarily determined by the LPA locus on chromosome 6q26-27, which is characterized by a unique type of copy number variation (CNV), the kringle-IV type 2 (K-IV-2) repeats. Meta-analysis of prospective studies as well as old and new genetic evidence have identified Lp(a) as an independent risk factor for atherothrombotic disease. The K-IV-2 CNV in LPA is the strongest known common genetic variation predicting risk for CHD: small isoforms of this CNV are associated with a doubling of risk for CHD. Together with observations that removal of Lp(a) from plasma may be beneficial in individuals with severe CHD, the strong genetic evidence justifies the consideration of Lp(a) in clinical practice.

Abstract 3211: FGFR1 and FGFR2 gene copy number variations in prostate cancer

Abstract Patients with hormone refractory prostate cancer (HRPC) have very few clinical options. A bottle neck in development of new therapies is the poor understanding of the molecular basis of hormone independency. A pathway thought to be important in this progression is FGFR. Amplification of FGFR1 has been observed in breast cancer and has led to the hypothesis that it is involved in Tamoxifen resistance (Turner et al., 2010). Here we evaluated the copy number variations (CNV) of the gene loci of FGFR1 (8p12) and FGFR2 (10q26) by dual color FISH in FFPE tissue of recurrent PC patients (n=28), non-recurrent PC patients (n=15), PC patients undergoing palliative RPE (n=15), patients with benign prostate hyperplasia (n=17) and tumor free tissue samples (n=12). We analyzed CNV between different clinical patient subgroups. Two types of CNV for the gene loci of FGFR1 and FGFR2 were observed. Either the gene dose of the target gene locus occurred to be amplified or the gene dose was elevated due to polysomy of the respective chromosome. Furthermore patients with CNV of FGFR1 or FGFR2 exhibited in 40-50% intra tumor heterogeneity by presenting normal cells and cells with CNV. FGFR1 gene dose elevations (either due to gene locus amplification or polysomy of chromosome 8) were observed in 7% (1/15) of patients without recurrence but in 49% (17/35) of the patients with recurrence. Also, in patients undergoing palliative RPE 75% (12/16) of the patients exhibited FGFR1 gene dose elevation. In the case of FGFR2, gene dose elevations (gene locus amplification or polysomy of chromosome 10) were observed in none of the patients without recurrence but in 13% of the patients with recurrence and in 20% of patients undergoing palliative RPE. In the control groups such variations were not found. Interestingly, the gene dose variations differed in the prostate cancer patients depending on their hormone sensitivity. 47% (7/15) of hormone refractory patients and 30% (6/20) of the hormone sensitive patients showed elevated FGFR1 gene dose, for FGFR2, 30% and 20% of the respective subgroups were affected. In summary we conclude that this is first evidence that CNV of FGFR1 and FGFR2 might have implications for hormone resistance in PC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3211. doi:10.1158/1538-7445.AM2011-3211

Copy number variation of microRNA genes in the human genome

BackgroundMicroRNAs (miRNAs) are important genetic elements that regulate the expression of thousands of human genes. Polymorphisms affecting miRNA biogenesis, dosage and target recognition may represent potentially functional variants. The functional consequences of single nucleotide polymorphisms (SNPs) within critical miRNA sequences and outside of miRNA genes were previously demonstrated using both experimental and computational methods. However, little is known about how copy number variations (CNVs) affect miRNA genes.ResultsIn this study, we analyzed the co-localization of all miRNA loci with known CNV regions. Using bioinformatic tools we identified and validated 209 copy number variable miRNA genes (CNV-miRNAs) in CNV regions deposited in Database of Genomic Variations (DGV) and 11 CNV-miRNAs in two sets of CNVs defined as highly polymorphic. We propose potential mechanisms of CNV-mediated variation of functional copies of miRNAs (dosage) for different types of CNVs overlapping miRNA genes. We also showed that, consistent with their essential biological functions, miRNA loci are underrepresented in highly polymorphic and well-validated CNV regions.ConclusionWe postulate that CNV-miRNAs are potential functional variants and should be considered high priority candidate variants in genotype-phenotype association studies.

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

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

On the analysis of copy‐number variations in genome‐wide association studies: a translation of the family‐based association test

Though there is an increasing support for an important contribution of copy number variation (CNV) to the genetic architecture of complex disease, few methods have been developed for the analysis of such variation in the context of genetic association studies. In this paper, we propose a generalization of family-based association tests (FBATs) to allow for the analysis of CNVs at a genome-wide level. We translate the popular FBAT approach so that, instead of genotypes, raw intensity values that reflect copy number are used directly in the test statistic, thereby bypassing the need for a CNV genotyping algorithm. Moreover, both inherited and de novo CNVs can be analyzed without any prior knowledge about the type of CNV, making it easily applicable to large-scale association studies. All robustness properties of the genotype FBAT approach are maintained and all previously developed FBAT extensions, including FBATs for time-to-onset, multivariate FBATs, and FBAT-testing strategies, can be directly transferred to the analysis of CNVs. Using simulation studies, we evaluate the power and the robustness of the new approach. Furthermore, for those CNVs that can be genotyped, we compare FBATs based on genotype calls with FBATs that are directly based on the intensity data. An application to one of the first CNV genome-wide-association studies of asthma identifies a very plausible candidate gene. A software implementation of the approach is freely available at http://www.hsph.harvard.edu/research/iuliana-ionita/software. The approach has also been completely integrated in the PBAT software package.