High-resolution SNP microarray investigation of copy number variations on chromosome 18 in a control cohort

Copy number variations (CNVs) as described in the healthy population are purported to contribute significantly to genetic heterogeneity. Recent studies have described CNVs using lymphoblastoid cell lines or by application of specifically developed algorithms to interrogate previously described data. However, the full extent of CNVs remains unclear. Using high-density SNP array, we have undertaken a comprehensive investigation of chromosome 18 for CNV discovery and characterisation of distribution and association with chromosome architecture. We identified 399 CNVs, of which loss represents 98%, 58% are less than 2.5 kb in size and 71% are intergenic. Intronic deletions account for the majority of copy number changes with gene involvement. Furthermore, one-third of CNVs do not have putative breakpoints within repetitive sequences. We conclude that replicative processes, mediated either by repetitive elements or microhomology, account for the majority of CNVs in the healthy population. Genomic instability involving the formation of a non-B structure is demonstrated in one region.

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

Acknowledgements We would like to thank all of the patients, relatives and control individuals who participated in the study. We are indebted to the late Prof. Walter Muir, Chair of Developmental Psychiatry and Honorary Consultant in Learning Disability Psychiatry, University of Edinburgh, who initiated these studies and whose work was dedicated to the welfare of the patients who generously participated. We are also grateful to Mrs. Pat Malloy for her assistance with DNA collection and MAQ assays screening of the Scottish samples. The Scottish sample collection was supported by a grant from the Chief Scientist Office (CSO), part of the Scottish Government Health and Social Care Directorates. This research was funded by grants from the CSO to B.S.P. (grant CZB/4/610), The Academy of Medical Sciences/Wellcome Trust to M.J. (grant R41455) and The RS Macdonald Charitable Trust (grant D21419 together with J.H.), the Swedish Research Council (grants 2003-5158 and 2006-4472), the Medical Faculty, Umeå University, and the County Councils of Västerbotten and Norrbotten, Sweden, as well as by grants from the Fund for Scientific Research Flanders (FWO-F), the Industrial Research Fund (IWT) and the Special Research Fund of the University of Antwerp, Belgium. M.J. is funded by a Wellcome Trust Clinical Research Fellowship for MB PhD graduates (R42811). We acknowledge the contribution of the personnel of the VIB Genetic Service Facility (http://www.vibgeneticservicefacility.be/) for the genetic analysis of the Swedish samples. Research nurses Gunnel Johansson, Lotta Kronberg, Tage Johansson and Lisbeth Bertilsson are thankfully acknowledged for their help and expertise. The Betula Study was funded by the Swedish Research Council (grants 345-2003-3883 and 315-2004-6977). We also acknowledge the contribution by the staff in the Betula project

Integration of miRNA and mRNA expression with DNA copy number of Ewing sarcoma cell lines

Ewing sarcoma is an aggressive and poorly differentiated malignancy of bone and soft tissue. It primarily affects children, adolescents, and young adults, with a slight male predominance. It is characterized by a translocation between chromosomes 11 and 22 resulting in the EWSR1-FLI1fusion transcription factor. The aim of this study is to identify putative Ewing sarcoma target genes through an integrative analysis of three microarray data sets. Array comparative genomic hybridization is used to measure changes in DNA copy number, and analyzed to detect common chromosomal aberrations. mRNA and miRNA microarrays are used to measure expression of protein-coding and miRNA genes, and these results integrated with the copy number data. Chromosomal aberrations typically contain also bystanders in addition to the driving tumor suppressor and oncogenes, and integration with expression helps to identify the true targets. Correlation between expression of miRNAs and their predicted target mRNAs is also evaluated to assess the results of post-transcriptional miRNA regulation on mRNA levels. The highest frequencies of copy number gains were identified in chromosome 8, 1q, and X. Losses were most frequent in 9p21.3, which also showed an enrichment of copy number breakpoints relative to the rest of the genome. Copy number losses in 9p21.3 were found have a statistically significant effect on the expression of MTAP, but not on CDKN2A, which is a known tumor-suppressor in the same locus. MTAP was also down-regulated in the Ewing sarcoma cell lines compared to mesenchymal stem cells. Genes exhibiting elevated expression in association with copy number gains and up-regulation compared to the reference samples included DCAF7, ENO2, MTCP1, andSTK40. Differentially expressed miRNAs were detected by comparing Ewing sarcoma cell lines against mesenchymal stem cells. 21 up-regulated and 32 down-regulated miRNAs were identified, includingmiR-145, which has been previously linked to Ewing sarcoma. The EWSR1-FLI1 fusion gene represses miR-145, which in turn targets FLI1 forming a mutually repressive feedback loop. In addition higher expression linked to copy number gains and compared to mesenchymal stem cells, STK40 was also found to be a target of four different miRNAs that were all down-regulated in Ewing sarcoma cell lines compared to the reference samples. SLCO5A1 was identified as the only up-regulated gene within a frequently gained region in chromosome 8. This region was gained in over 90 % of the cell lines, and also with a higher frequency than the neighboring regions. In addition, SLCO5A1 was found to be a target of three miRNAs that were down-regulated compared to the mesenchymal stem cells.

Genome-wide scan of copy number variation in late-onset Alzheimer's disease.

Alzheimer's disease is a complex and progressive neurodegenerative disease leading to loss of memory, cognitive impairment, and ultimately death. To date, six large-scale genome-wide association studies have been conducted to identify SNPs that influence disease predisposition. These studies have confirmed the well-known APOE epsilon4 risk allele, identified a novel variant that influences disease risk within the APOE epsilon4 population, found a SNP that modifies the age of disease onset, as well as reported the first sex-linked susceptibility variant. Here we report a genome-wide scan of Alzheimer's disease in a set of 331 cases and 368 controls, extending analyses for the first time to include assessments of copy number variation. In this analysis, no new SNPs show genome-wide significance. We also screened for effects of copy number variation, and while nothing was significant, a duplication in CHRNA7 appears interesting enough to warrant further investigation.

Comparative analyses of seven algorithms for copy number variant identification from single nucleotide polymorphism arrays.

Determination of copy number variants (CNVs) inferred in genome wide single nucleotide polymorphism arrays has shown increasing utility in genetic variant disease associations. Several CNV detection methods are available, but differences in CNV call thresholds and characteristics exist. We evaluated the relative performance of seven methods: circular binary segmentation, CNVFinder, cnvPartition, gain and loss of DNA, Nexus algorithms, PennCNV and QuantiSNP. Tested data included real and simulated Illumina HumHap 550 data from the Singapore cohort study of the risk factors for Myopia (SCORM) and simulated data from Affymetrix 6.0 and platform-independent distributions. The normalized singleton ratio (NSR) is proposed as a metric for parameter optimization before enacting full analysis. We used 10 SCORM samples for optimizing parameter settings for each method and then evaluated method performance at optimal parameters using 100 SCORM samples. The statistical power, false positive rates, and receiver operating characteristic (ROC) curve residuals were evaluated by simulation studies. Optimal parameters, as determined by NSR and ROC curve residuals, were consistent across datasets. QuantiSNP outperformed other methods based on ROC curve residuals over most datasets. Nexus Rank and SNPRank have low specificity and high power. Nexus Rank calls oversized CNVs. PennCNV detects one of the fewest numbers of CNVs.

Discovering subgroups of patients from DNA copy number data using NMF on compacted matrices

In the study of complex genetic diseases, the identification of subgroups of patients sharing similar genetic characteristics represents a challenging task, for example, to improve treatment decision. One type of genetic lesion, frequently investigated in such disorders, is the change of the DNA copy number (CN) at specific genomic traits. Non-negative Matrix Factorization (NMF) is a standard technique to reduce the dimensionality of a data set and to cluster data samples, while keeping its most relevant information in meaningful components. Thus, it can be used to discover subgroups of patients from CN profiles. It is however computationally impractical for very high dimensional data, such as CN microarray data. Deciding the most suitable number of subgroups is also a challenging problem. The aim of this work is to derive a procedure to compact high dimensional data, in order to improve NMF applicability without compromising the quality of the clustering. This is particularly important for analyzing high-resolution microarray data. Many commonly used quality measures, as well as our own measures, are employed to decide the number of subgroups and to assess the quality of the results. Our measures are based on the idea of identifying robust subgroups, inspired by biologically/clinically relevance instead of simply aiming at well-separated clusters. We evaluate our procedure using four real independent data sets. In these data sets, our method was able to find accurate subgroups with individual molecular and clinical features and outperformed the standard NMF in terms of accuracy in the factorization fitness function. Hence, it can be useful for the discovery of subgroups of patients with similar CN profiles in the study of heterogeneous diseases.

Análise do gene TUBB3 e de Copy Number Variations em pacientes com Polimicrogiria

A polimicrogiria (PMG) é uma malformação do córtex cerebral causada por falhas no seu desenvolvimento, caracterizando-se por um número excessivo de pequenos giros e laminação anormal, dando à superfície cortical uma aparência irregular e grosseira. A gravidade de suas manifestações clínicas se relaciona diretamente com a extensão da malformação e das regiões cerebrais afetadas, sendo que a presença de lesões bilaterais ou unilaterais extensas indica um pior prognóstico. Uma das síndromes de polimicrogiria mais freqüentes e, conseqüentemente, mais bem descritas clinicamente, é a polimicrogiria perisylviana bilateral (PPB). Essa forma de PMG atinge a região que tange a fenda Sylviana, podendo apresentar-se tanto unilateralmente quanto em ambos os hemisférios. Vários genes têm sido relacionados a diferentes formas de polimicrogiria, são eles AFF2,TUBA1A, TUBB2B e TUBA8, SRPX2 e WDR62. Estes genes já foram estudados pelo nosso grupo de pesquisa em um grupo de pacientes compostos de casos familiares e esporádicos, acometidos em sua maioria pela forma perisylviana de PMG. Nenhuma variante deletéria foi identificada nestes genes. Recentemente um novo gene foi implicado na etiologia molecular das PMG, o TUBB3. O gene em questão pertence à mesma família de TUBA1A, TUBB2B e TUBA8 e codifica uma proteína de ligação aos microtúbulos, tendo importante papel na formação do fuso. Além deste gene, também tem sido descritas alterações genômicas, denominadas de Copy Number Variations (CNV), estas variações estruturais tem sido associadas com diversos distúrbios neurológicos, que vão desde transtornos psiquiátricos até malformações do córtex cerebral como a PMG. Desta forma, o objetivo deste trabalho foi analisar a existência de alterações de ponto deletérias no gene TUBB3 em pacientes com PMG e também, o envolvimento de CNVsna etiologia deste tipo de malformação ...

Germline DNA copy number variation in familial and early-onset breast cancer

Introduction: Genetic factors predisposing individuals to cancer remain elusive in the majority of patients with a familial or clinical history suggestive of hereditary breast cancer. Germline DNA copy number variation (CNV) has recently been implicated in predisposition to cancers such as neuroblastomas as well as prostate and colorectal cancer. We evaluated the role of germline CNVs in breast cancer susceptibility, in particular those with low population frequencies (rare CNVs), which are more likely to cause disease." Methods: Using whole-genome comparative genomic hybridization on microarrays, we screened a cohort of women fulfilling criteria for hereditary breast cancer who did not carry BRCA1/BRCA2 mutations. Results: The median numbers of total and rare CNVs per genome were not different between controls and patients. A total of 26 rare germline CNVs were identified in 68 cancer patients, however, a proportion that was significantly different (P = 0.0311) from the control group (23 rare CNVs in 100 individuals). Several of the genes affected by CNV in patients and controls had already been implicated in cancer. Conclusions: This study is the first to explore the contribution of germline CNVs to BRCA1/2-negative familial and early-onset breast cancer. The data suggest that rare CNVs may contribute to cancer predisposition in this small cohort of patients, and this trend needs to be confirmed in larger population samples.

Genome-wide analysis of rare copy number variations reveals PARK2 as a candidate gene for attention-deficit/hyperactivity disorder

Attention-deficit/hyperactivity disorder (ADHD) is a common, highly heritable neurodevelopmental disorder. Genetic loci have not yet been identified by genome-wide association studies. Rare copy number variations (CNVs), such as chromosomal deletions or duplications, have been implicated in ADHD and other neurodevelopmental disorders. To identify rare (frequency 1%) CNVs that increase the risk of ADHD, we performed a whole-genome CNV analysis based on 489 young ADHD patients and 1285 adult population-based controls and identified one significantly associated CNV region. In tests for a global burden of large (>500 kb) rare CNVs, we observed a nonsignificant (P=0.271) 1.126-fold enriched rate of subjects carrying at least one such CNV in the group of ADHD cases. Locus-specific tests of association were used to assess if there were more rare CNVs in cases compared with controls. Detected CNVs, which were significantly enriched in the ADHD group, were validated by quantitative (q)PCR. Findings were replicated in an independent sample of 386 young patients with ADHD and 781 young population-based healthy controls. We identified rare CNVs within the parkinson protein 2 gene (PARK2) with a significantly higher prevalence in ADHD patients than in controls (P=2.8 × 10(-4) after empirical correction for genome-wide testing). In total, the PARK2 locus (chr 6: 162 659 756-162 767 019) harboured three deletions and nine duplications in the ADHD patients and two deletions and two duplications in the controls. By qPCR analysis, we validated 11 of the 12 CNVs in ADHD patients (P=1.2 × 10(-3) after empirical correction for genome-wide testing). In the replication sample, CNVs at the PARK2 locus were found in four additional ADHD patients and one additional control (P=4.3 × 10(-2)). Our results suggest that copy number variants at the PARK2 locus contribute to the genetic susceptibility of ADHD. Mutations and CNVs in PARK2 are known to be associated with Parkinson disease.Molecular Psychiatry advance online publication, 20 November 2012; doi:10.1038/mp.2012.161.

Detection of cryptic pathogenic copy number variations and constitutional loss of heterozygosity using high resolution SNP microarray analysis in 117 patients referred for cytogenetic analysis and impact on clinical practice

BACKGROUND: Microarray genome analysis is realising its promise for improving detection of genetic abnormalities in individuals with mental retardation and congenital abnormality. Copy number variations (CNVs) are now readily detectable using a variety of platforms and a major challenge is the distinction of pathogenic from ubiquitous, benign polymorphic CNVs. The aim of this study was to investigate replacement of time consuming, locus specific testing for specific microdeletion and microduplication syndromes with microarray analysis, which theoretically should detect all known syndromes with CNV aetiologies as well as new ones. METHODS: Genome wide copy number analysis was performed on 117 patients using Affymetrix 250K microarrays. RESULTS: 434 CNVs (195 losses and 239 gains) were found, including 18 pathogenic CNVs and 9 identified as "potentially pathogenic". Almost all pathogenic CNVs were larger than 500 kb, significantly larger than the median size of all CNVs detected. Segmental regions of loss of heterozygosity larger than 5 Mb were found in 5 patients. CONCLUSIONS: Genome microarray analysis has improved diagnostic success in this group of patients. Several examples of recently discovered "new syndromes" were found suggesting they are more common than previously suspected and collectively are likely to be a major cause of mental retardation. The findings have several implications for clinical practice. The study revealed the potential to make genetic diagnoses that were not evident in the clinical presentation, with implications for pretest counselling and the consent process. The importance of contributing novel CNVs to high quality databases for genotype-phenotype analysis and review of guidelines for selection of individuals for microarray analysis is emphasised.

CopywriteR: DNA copy number detection from off-target sequence data.

Current methods for detection of copy number variants (CNV) and aberrations (CNA) from targeted sequencing data are based on the depth of coverage of captured exons. Accurate CNA determination is complicated by uneven genomic distribution and non-uniform capture efficiency of targeted exons. Here we present CopywriteR, which eludes these problems by exploiting 'off-target' sequence reads. CopywriteR allows for extracting uniformly distributed copy number information, can be used without reference, and can be applied to sequencing data obtained from various techniques including chromatin immunoprecipitation and target enrichment on small gene panels. CopywriteR outperforms existing methods and constitutes a widely applicable alternative to available tools.

Clinical relevance of polymorphic DNA copy number variation (CNV) in African American women with stage I-II breast cancer

Objectives. The chief goal of this study was to analyze copy number variation (CNV) in breast cancer tumors from 25 African American women with early stage breast cancer (BC) using molecular inversion probes (MIP) in order to: (1) compare the degree of CNV in tumors compared to normal lymph nodes, and (2) determine whether gains and/or losses of genes in specific chromosomes differ between pathologic subtypes of breast cancer defined by known prognostic markers, (3) determine whether gains/losses in CN are associated with known oncogenes or tumor suppressor genes, and (4) determine whether increased gains/losses in CN for specific chromosomes were associated with differences in breast cancer recurrence. ^ Methods. Twenty to 37 nanograms of DNA extracted from 25 formalin-fixed paraffin embedded (FFPE) tumor samples and matched normal lymph nodes were added to individual tubes. Oligonucleotide probes with recognition sequences at each terminus were hybridized with a genomic target sequence to form a circular structure. Probes are released from genomic DNA obtained from FFPE samples, and those which have been correctly "circularized" in the proper allele/nucleotide reaction combination are amplified using polymerase chain reaction (PCR) primers. Amplicons were fluorescently labeled and the tag sequences released from the genome homology regions by treatment with uracil-N-glycosylase to cleave the probe at the site where uracils are present, and detected using a complementary tag array developed by Affymetrix. ^ Results. Analysis of CN gains and losses from tumors and normal tissues showed marked differences in tumors with numerous chromosomes affected. Similar changes were not observed in normal lymph nodes. When tumors were stratified into four groups based on expression or lack of expression of the estrogen receptor and HER2/neu, distinct patterns of CNV for different chromosomes were observed. Gains or losses in CN for specific chromosomes correlated with amplifications/deletions of particular oncogenes or tumor suppressor genes (i.e. such as found on chromosome 17) known to be associated with aggressive tumor phenotype and poor prognosis. There was a trend for increases in CN observed for chromosome 17 to correlate inversely with time to recurrence of BC (p=0.14 for trend). CNV was also observed for chromosomes 5, 8, 10, 11, and 16, which are known sites for several breast cancer susceptibility alleles. ^ Conclusions. This study is the first to validate the MIP technique, to correlate differences in gene expression with known prognostic tumor markers, and to correlate significant increases/decreases in CN with known tumor markers associated with prognosis. The results of this study may have far reaching public health implications towards identifying new high-risk groups based on genomic differences in CNP, both with respect to prognosis and response to therapy, and to eventually identify new therapeutic targets for prevention and treatment of this disease. ^

Simultaneous A8344G heteroplasmy and mitochondrial DNA copy number quantification in Myoclonus Epilepsy and Ragged-Red Fibers (MERRF) syndrome by a multiplex Molecular Beacon based real-time fluorescence PCR

The association of a particular mitochondrial DNA (mtDNA) mutation with different clinical phenotypes is a well-known feature of mitochondrial diseases. A simple genotype–phenotype correlation has not been found between mutation load and disease expression. Tissue and intercellular mosaicism as well as mtDNA copy number are thought to be responsible for the different clinical phenotypes. As disease expression of mitochondrial tRNA mutations is mostly in postmitotic tissues, studies to elucidate disease mechanisms need to be performed on patient material. Heteroplasmy quantitation and copy number estimation using small patient biopsy samples has not been reported before, mainly due to technical restrictions. In order to resolve this problem, we have developed a robust assay that utilizes Molecular Beacons to accurately quantify heteroplasmy levels and determine mtDNA copy number in small samples carrying the A8344G tRNALys mutation. It provides the methodological basis to investigate the role of heteroplasmy and mtDNA copy number in determining the clinical phenotypes.