An integrated approach for measuring copy number variation at the FCGR3 (CD16) locus

Copy number variation (CNV) is an important source of genomic diversity in humans, and influences disease susceptibility. The immunoglobulin‐receptor genes FCGR3A and FCGR3B on chromosome 1q23.3 show CNV, and CNV of the FCGR3B gene is associated with glomerulonephritis in systemic lupus erythematosus and organ‐specific autoimmunity. Large‐scale case‐control association studies of CNV require technologies that are amenable to high‐throughput analysis with low error rates. Here we propose an integrated suite of five assays, four of them duplexed to reduce DNA usage, that assays for CNV at FCGR3A and FCGR3B, and genotype the polymorphic neutrophil antigen HNA1. We show how a maximum‐likelihood (ML) approach to combining the results from these five assays allows estimation of statistical confidence for each individual copy number, and therefore an appropriate significance threshold to be set, controlling the error rate. This approach results in a high‐throughput copy number genotyping system, with demonstrable precision and accuracy, that can be applied to large case‐control cohort studies. We demonstrate Mendelian inheritance of this CNV, variation in frequency between Europeans and East Asians, and a lack of strong association between the CNV and flanking SNP genotypes, with important consequences for genome‐wide association studies. Hum Mutat 0, 1–8, 2008. © 2008 Wiley‐Liss, Inc.     

Genomic disorders 20 years on—mechanisms for clinical manifestations

Genomic disorders result from copy‐number variants (CNVs) or submicroscopic rearrangements of the genome rather than from single nucleotide variants (SNVs). Diverse technologies, including array comparative genomic hybridization (aCGH) and single nucleotide polymorphism (SNP) microarrays, and more recently, whole genome sequencing and whole‐exome sequencing, have enabled robust genome‐wide unbiased detection of CNVs in affected individuals and in reportedly healthy controls. Sequencing of breakpoint junctions has allowed for elucidation of upstream mechanisms leading to genomic instability and resultant structural variation, whereas studies of the association between CNVs and specific diseases or susceptibility to morbid traits have enhanced our understanding of the downstream effects. In this review, we discuss the hallmarks of genomic disorders as they were defined during the first decade of the field, including genomic instability and the mechanism for rearrangement defined as nonallelic homologous recombination (NAHR); recurrent vs nonrecurrent rearrangements; and gene dosage sensitivity. Moreover, we highlight the exciting advances of the second decade of this field, including a deeper understanding of genomic instability and the mechanisms underlying complex rearrangements, mechanisms for constitutional and somatic chromosomal rearrangements, structural intra‐species polymorphisms and susceptibility to NAHR, the role of CNVs in the context of genome‐wide copy number and single nucleotide variation, and the contribution of noncoding CNVs to human disease.     

Frequency distribution of autoimmunity associated FCGR3B gene copy number in Indian population

Amongst several human genome variations, copy number variations (CNVs) are considered as an important source of variability contributing to susceptibility to wide range of diseases. Although CNV is scattered for genes throughout the human genome, several of autoimmunity related genes have CN variation and therefore play an important role in susceptibility to autoimmune diseases. The association of the Fc gamma receptor 3B (FCGR3B) gene copy number in autoimmunity is well characterized in various populations studied. The Fc gamma receptor is a low affinity, glycosylphosphatidylinositol‐linked receptor for IgG molecule predominantly expressed on human neutrophils. The variable gene copy number of FCGR3B is found to be involved in the impaired clearance of immune complexes, which significantly contribute to the pathogenesis of several autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), type‐1 diabetes and others. The FCGR3B copy number ranged from 0 to ≥2 copies per diploid genome in other populations, but yet not explored in Indian population. Hence, this study aims to evaluate the variation in the frequency distribution of FCGR3B CNV in Indian population. FCGR3B gene copy number varied significantly when compared to other population of the world. This observation will help us in exploring the potential role of CNV in FCGR3B gene and its association to autoimmune disorders in Indian population.     

Structural variation mutagenesis of the human genome: Impact on disease and evolution

Watson‐Crick base‐pair changes, or single‐nucleotide variants (SNV), have long been known as a source of mutations. However, the extent to which DNA structural variation, including duplication and deletion copy number variants (CNV) and copy number neutral inversions and translocations, contribute to human genome variation and disease has been appreciated only recently. Moreover, the potential complexity of structural variants (SV) was not envisioned; thus, the frequency of complex genomic rearrangements and how such events form remained a mystery. The concept of genomic disorders, diseases due to genomic rearrangements and not sequence‐based changes for which genomic architecture incite genomic instability, delineated a new category of conditions distinct from chromosomal syndromes and single‐gene Mendelian diseases. Nevertheless, it is the mechanistic understanding of CNV/SV formation that has promoted further understanding of human biology and disease and provided insights into human genome and gene evolution. Environ. Mol. Mutagen. 56:419–436, 2015. © 2015 Wiley Periodicals, Inc.     

Analysis of copy number variation in the rhesus macaque genome identifies candidate loci for evolutionary and human disease studies

Copy number variants (CNVs) are heritable gains and losses of genomic DNA in normal individuals. While copy number variation is widely studied in humans, our knowledge of CNVs in other mammalian species is more limited. We have designed a custom array-based comparative genomic hybridization (aCGH) platform with 385 000 oligonucleotide probes based on the reference genome sequence of the rhesus macaque (Macaca mulatta), the most widely studied non-human primate in biomedical research. We used this platform to identify 123 CNVs among 10 unrelated macaque individuals, with 24% of the CNVs observed in multiple individuals. We found that segmental duplications were significantly enriched at macaque CNV loci. We also observed significant overlap between rhesus macaque and human CNVs, suggesting that certain genomic regions are prone to recurrent CNV formation and instability, even across a total of approximately 50 million years of primate evolution ( approximately 25 million years in each lineage). Furthermore, for eight of the CNVs that were observed in both humans and macaques, previous human studies have reported a relationship between copy number and gene expression or disease susceptibility. Therefore, the rhesus macaque offers an intriguing, non-human primate outbred model organism with which hypotheses concerning the specific functions of phenotypically relevant human CNVs can be tested.     

Large duplication in LMBR1 gene in a large Chinese pedigree with triphalangeal thumb polysyndactyly syndrome

Polydactyly and syndactyly are digital abnormalities in limb‐associated birth defects usually caused by genetic disorders. In this study, a five‐generation Chinese pedigree was found with triphalangeal thumb polysyndactyly syndrome (TPTPS), showing an autosomal dominant pattern of inheritance. We utilized linkage analysis and whole genome sequencing (WGS) for the genetic diagnosis of this pedigree. Linkage analysis was performed using a genome‐wide single nucleotide polymorphism (SNP) chip and three genomic regions were identified in chromosomes 2, 6, and 7 with significant linkage signals. WGS discovered a copy number variation (CNV) mutation caused by a large duplication region at the tail of chromosome 7 located in exons 1–5 of the LMBR1 gene, including the zone of polarizing activity regulatory sequence (ZRS), with a length of approximately 180 kb. A real‐time polymerase chain reaction (PCR) assay confirmed the duplication. The findings of our study supported the notion that large duplications including the ZRS caused TPTPS. Our study showed that linkage analysis in combination with WGS could successfully identify the disease locus and causative mutation in TPTPS, which could help elucidate the molecular mechanisms and genotype–phenotype correlations in polydactyly.     

Genomic alterations as mediators of miRNA dysregulation in ovarian cancer

Ovarian cancer is the fifth most common cause of cancer death in women worldwide. Serous epithelial ovarian cancer (SEOC) is the most common and aggressive histological subtype. Widespread genomic alterations go hand‐in‐hand with aberrant DNA damage signaling and are a hallmark of high‐grade SEOC. MicroRNAs (miRNAs) are a class of small noncoding RNA molecules that are nonrandomly distributed in the genome. They are frequently located in chromosomal regions susceptible to copy number variation (CNV) associated with malignancy that can influence their expression. Widespread changes in miRNA expression have been reported in multiple cancer types including ovarian cancer. This review examines CNV and single nucleotide polymorphisms, two common types of genomic alterations that occur in ovarian cancer, in the context of their influence on the expression of miRNA and the ability of miRNA to bind to and regulate their target genes. This includes genes encoding proteins involved in DNA repair and the maintenance of genomic stability. Improved understanding of mechanisms of miRNA dysregulation and the role of miRNA in ovarian cancer will provide further insight into the pathogenesis and treatment of this disease. © 2014 Wiley Periodicals, Inc.     

Implications of gene copy-number variation in health and diseases

Inter-individual genomic variations have recently become evident with advances in sequencing techniques and genome-wide array comparative genomic hybridization. Among such variations single nucleotide polymorphisms (SNPs) are widely studied and better defined because of availability of large-scale detection platforms. However, insertion-deletions, inversions, copy-number variations (CNVs) also populate our genomes. The large structural variations (>3?Mb) have been known for past 20 years, however, their link to health and disease remain ill-defined. CNVs are defined as the segment of DNA >1?kb in size, and compared with reference genome vary in its copy number. All these types of genomic variations are bound to have vital role in disease susceptibility and drug response. In this review, the discussion is confined to CNVs and their link to health, diseases and drug response. There are several CNVs reported till date, which have important roles in an individual's susceptibility to several complex and common disorders. This review compiles some of these CNVs and analyzes their involvement in diseases in different populations, analyses available evidence and rationalizes their involvement in the development of disease phenotype. Combined with SNP, additional genomic variations including CNV, will provide better correlations between individual genomic variations and health.     

Concurrent triplication and uniparental isodisomy: evidence for microhomology-mediated break-induced replication model for genomic rearrangements

Whole-genome oligonucleotide single-nucleotide polymorphism (oligo-SNP) arrays enable simultaneous interrogation of copy number variations (CNVs), copy neutral regions of homozygosity (ROH) and uniparental disomy (UPD). Structural variation in the human genome contributes significantly to genetic variation, and often has deleterious effects leading to disease causation. Co-occurrence of CNV and regions of allelic homozygosity in tandem involving the same chromosomal arm are extremely rare. Replication-based mechanisms such as microhomology-mediated break-induced replication (MMBIR) are recent models predicted to induce structural rearrangements and gene dosage aberrations; however, supportive evidence in humans for one-ended DNA break repair coupled with MMBIR giving rise to interstitial copy number gains and distal loss of heterozygosity has not been documented. We report on the identification and characterization of two cases with interstitial triplication followed by uniparental isodisomy (isoUPD) for remainder of the chromosomal arm. Case 1 has a triplication at 9q21.11–q21.33 and segmental paternal isoUPD for 9q21.33-qter, and presented with citrullinemia with a homozygous mutation in the argininosuccinate synthetase gene (ASS1 at 9q34.1). Case 2 has a triplication at 22q12.1–q12.2 and segmental maternal isoUPD 22q12.2-qter, and presented with hearing loss, mild dysmorphic features and bilateral iris coloboma. Interstitial triplication coupled with distal segmental isoUPD is a novel finding that provides human evidence for one-ended DNA break and replication-mediated repair. Both copy number gains and isoUPD may contribute to the phenotype. Significantly, these cases represent the first detailed genomic analysis that provides support for a MMBIR mechanism inducing copy number gains and segmental isoUPD in tandem.     

Multiplex PCR-based real-time invader assay (mPCR-RETINA): a novel SNP-based method for detecting allelic asymmetries within copy number variation regions

We report the development of a real-time Invader assay combined with multiplex PCR (mPCR-RETINA), an SNP-based approach that can measure the allelic ratio in copy number variation (CNV) regions of a genome. RETINA monitors the real-time fluorescence intensity of each allele during the Invader assay and detects allelic asymmetries caused by genomic duplication/multiplication in heterozygous individuals. By combining mPCR-RETINA and real-time quantitative PCR that detects total copy number, we can estimate the copy number of each allele in CNV regions, which should be useful for investigating the functional significance of allele copy number with disease susceptibilities and drug responses. Also, mPCR-RETINA can efficiently refine the detailed structures of CNV regions. Due to the combination of RETINA with multiplex PCR, mPCR-RETINA requires a very small amount of genomic DNA for analysis (0.1-0.38 ng/locus). Additionally, mPCR-RETINA has clear advantages in its simple protocol and target-specific reaction, even in nonunique regions. We believe mPCR-RETINA will provide a significant contribution to identifying functional alleles in CNV regions.     

Searching genetic risk factors for schizophrenia and bipolar disorder: learn from the past and back to the future

For more than 20 years already, researchers from all over the world have tried to get insight into the genetic basis of psychiatric disorders such as schizophrenia (SZ) and bipolar (BP) disorder. Linkage and candidate gene association study results have led to a range of hypotheses about the pathogenesis of the disorders, but overall genetic findings have been inconsistent and not a single functional risk causing variant has yet been identified. Even genomewide association (GWA) studies in large samples, the most extensive and systematic interrogation of the genome thus far, seemingly have not brought the expected answers. A reasonable interpretation is that multiple rare variants, inherently linked with locus and allelic heterogeneity, confer a substantial proportion of susceptibility to the disorders. Also, structural variation might be an important factor and promising results are arising from copy‐number variation (CNV) analyses. In this review we shortly touch on “old” results from linkage and association studies and critically review the design and “new” results of GWA and CNV studies. We discuss what can be learned from the past and how this knowledge can be used in future study designs. Hum Mutat 30, 1139–1152, 2009. © 2009 Wiley‐Liss, Inc.     

The search for genetic variants and epigenetics related to asthma

For the past two decades, a huge number of genetic studies have been conducted to identify the genetic variants responsible for asthma risk. Several types of genetic and genomic approaches, including linkage analysis, candidate gene single nucleotide polymorphism studies, and whole genome-wide association studies have been applied. In this review article, the results of these approaches are summarized, and their limitations are discussed. Additionally, perspectives for applying upcoming new epigenetic or genomic technologies, such as copy number variation, are introduced to increase our understanding of new omic approaches to asthma genetics.     

Detection of clinically relevant exonic copy‐number changes by array CGH

Array comparative genomic hybridization (aCGH) is a powerful tool for the molecular elucidation and diagnosis of disorders resulting from genomic copy‐number variation (CNV). However, intragenic deletions or duplications—those including genomic intervals of a size smaller than a gene—have remained beyond the detection limit of most clinical aCGH analyses. Increasing array probe number improves genomic resolution, although higher cost may limit implementation, and enhanced detection of benign CNV can confound clinical interpretation. We designed an array with exonic coverage of selected disease and candidate genes and used it clinically to identify losses or gains throughout the genome involving at least one exon and as small as several hundred base pairs in size. In some patients, the detected copy‐number change occurs within a gene known to be causative of the observed clinical phenotype, demonstrating the ability of this array to detect clinically relevant CNVs with subkilobase resolution. In summary, we demonstrate the utility of a custom‐designed, exon‐targeted oligonucleotide array to detect intragenic copy‐number changes in patients with various clinical phenotypes. Hum Mutat 31:1–17, 2010. © 2010 Wiley‐Liss, Inc.     

Complement receptor 1 coding variant p.Ser1610Thr in Alzheimer's disease and related endophenotypes

We previously described an intragenic functional copy number variation (CNV) in complement receptor 1 (CR1) that is associated with Alzheimer disease (AD) risk. A recent study, however, reported a rare CR1 coding variant p.Ser1610Thr (rs4844609) associated with AD susceptibility, explaining the effect of genome wide association (GWA) top single nucleotide polymorphism rs6656401. We assessed the role of the Ser1610Thr variant in AD pathogenesis and the effect on AD-related endophenotypes in a Flanders-Belgian cohort. We evaluated whether this rare variant rather than the CR1 CNV could explain the association of CR1 in our population. The Ser1610Thr variant was not associated with AD, memory impairment, total tau, amyloid β_1–42 or tau phosphorylated at threonine 181 levels. It did not explain (part of) the association of genome wide association top single-nucleotide polymorphisms rs3818361/rs6656401, nor of the CR1 CNV, with AD in our cohort, whereas the CR1 CNV and rs3818361/rs6656401 represented the same association signal. These findings question a role for the Ser1610Thr variant in AD risk and related endophenotypes, and reaffirm our previous observation that the CR1 CNV could be the true functional risk factor explaining the association between CR1 and AD.     

基因拷贝数变异与智力障碍病因学研究进展

基因拷贝数变异(copy number variations,CNVs)是指DNA片段大小从1 kb至Mb的亚微观结构变异,已成为探讨与疾病相关遗传变异的研究热点,特别是神经认知系统的遗传病,如精神分裂症、智力障碍等综合征、本文综述了基因拷贝数变异与智力障碍的关联关系和基因拷贝数变异检测技术进展在智力障碍诊断中的应用,这对揭示智力障碍发病机制有着重要意义.     

Gene copy number variation and common human disease

Fanciulli M, Petretto E, Aitman TJ. Gene copy number variation and common human disease. Variation in gene copy number is increasingly recognized as a common, heritable source of inter‐individual differences in genomic sequence. The role of copy number variation is well established in the pathogenesis of rare genomic disorders. More recently, germline and somatic copy number variation have been shown to be important pathogenic factors in a range of common diseases, including infectious, autoimmune and neuropsychiatric diseases and cancer. In this review, we describe the range of methods available for measuring copy number variants (CNVs) in individuals and populations, including the limitations of presently available assays, and highlight some key examples of common diseases in which CNVs have been shown clearly to have a pathogenic role. Although there has been major progress in this field in the last 5 years, understanding the full contribution of CNVs to the genetic basis of common diseases will require further studies, with more accurate CNV assays and larger cohorts than have presently been completed.     

基因拷贝数变异与智力障碍病因学研究进展

基因拷贝数变异(copy number variations,CNVs)是指DNA片段大小从1 kb至Mb的亚微观结构变异,已成为探讨与疾病相关遗传变异的研究热点,特别是神经认知系统的遗传病,如精神分裂症、智力障碍等综合征、本文综述了基因拷贝数变异与智力障碍的关联关系和基因拷贝数变异检测技术进展在智力障碍诊断中的应用,这对揭示智力障碍发病机制有着重要意义.     

Functional effects of CCL3L1 copy number

Copy number variation (CNV) is becoming increasingly important as a feature of human variation in disease susceptibility studies. However, the consequences of CNV are not so well understood. Here, we present data exploring the functional consequences of CNV of CCL3L1 in 55 independent UK samples with no known clinical phenotypes. The copy number of CCL3L1 was determined by the paralogue ratio test, and expression levels of macrophage inflammatory protein-1α (MIP-1α) and mRNA from stimulated monocytes were measured and analysed. The data show no statistically significant association of MIP-1α protein levels with copy number. However, there was a significant correlation between copy number and CCL3L1:CCL3 mRNA ratio. The data also provide evidence that expression of CCL3 predominates in both protein and mRNA, and therefore the observed variation of CCL3 is potentially more important biologically than that of CNV of CCL3L1.     

拷贝数目变异研究进展

人类基因组中的DNA 拷贝数目变异Copy Number Variation（CNVs）一直以来都认为分布频率较低,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的进化等方面对最新的CNVs研究进展做较为全面概述。