Chromosomal microarray analysis of consecutive individuals with autism spectrum disorders or learning disability presenting for genetic services

Chromosomal microarray analysis is now commonly used in clinical practice to identify copy number variants (CNVs) in the human genome. We report our experience with the use of the 105 K and 180 K oligonucleotide microarrays in 215 consecutive patients referred with either autism or autism spectrum disorders (ASD) or developmental delay/learning disability for genetic services at the University of Kansas Medical Center during the past 4 years (2009–2012). Of the 215 patients [140 males and 75 females (male/female ratio = 1.87); 65 with ASD and 150 with learning disability], abnormal microarray results were seen in 45 individuals (21%) with a total of 49 CNVs. Of these findings, 32 represented a known diagnostic CNV contributing to the clinical presentation and 17 represented non-diagnostic CNVs (variants of unknown significance). Thirteen patients with ASD had a total of 14 CNVs, 6 CNVs recognized as diagnostic and 8 as non-diagnostic. The most common chromosome involved in the ASD group was chromosome 15. For those with a learning disability, 32 patients had a total of 35 CNVs. Twenty-six of the 35 CNVs were classified as a known diagnostic CNV, usually a deletion (n = 20). Nine CNVs were classified as an unknown non-diagnostic CNV, usually a duplication (n = 8). For the learning disability subgroup, chromosomes 2 and 22 were most involved. Thirteen out of 65 patients (20%) with ASD had a CNV compared with 32 out of 150 patients (21%) with a learning disability. The frequency of chromosomal microarray abnormalities compared by subject group or gender was not statistically different. A higher percentage of individuals with a learning disability had clinical findings of seizures, dysmorphic features and microcephaly, but not statistically significant. While both groups contained more males than females, a significantly higher percentage of males were present in the ASD group.     

Genome arrays for the detection of copy number variations in idiopathic mental retardation, idiopathic generalized epilepsy and neuropsychiatric disorders: lessons for diagnostic workflow and research

We review the contributions and limitations of genome-wide array-based identification of copy number variants (CNVs) in the clinical diagnostic evaluation of patients with mental retardation (MR) and other brain-related disorders. In unselected MR referrals a causative genomic gain or loss is detected in 14-18% of cases. Usually, such CNVs arise de novo, are not found in healthy subjects, and have a major impact on the phenotype by altering the dosage of multiple genes. This high diagnostic yield justifies array-based segmental aneuploidy screening as the initial genetic test in these patients. This also pertains to patients with autism (expected yield about 5-10% in nonsyndromic and 10-20% in syndromic patients) and schizophrenia (at least 5% yield). CNV studies in idiopathic generalized epilepsy, attention-deficit hyperactivity disorder, major depressive disorder and Tourette syndrome indicate that patients have, on average, a larger CNV burden as compared to controls. Collectively, the CNV studies suggest that a wide spectrum of disease-susceptibility variants exists, most of which are rare (<0.1%) and of variable and usually small effect. Notwithstanding, a rare CNV can have a major impact on the phenotype. Exome sequencing in MR and autism patients revealed de novo mutations in protein coding genes in 60 and 20% of cases, respectively. Therefore, it is likely that arrays will be supplanted by next-generation sequencing methods as the initial and perhaps ultimate diagnostic tool in patients with brain-related disorders, revealing both CNVs and mutations in a single test.     

Copy Number Variation in Thai Population

Copy number variation (CNV) is a major genetic polymorphism contributing to genetic diversity and human evolution. Clinical application of CNVs for diagnostic purposes largely depends on sufficient population CNV data for accurate interpretation. CNVs from general population in currently available databases help classify CNVs of uncertain clinical significance, and benign CNVs. Earlier studies of CNV distribution in several populations worldwide showed that a significant fraction of CNVs are population specific. In this study, we characterized and analyzed CNVs in 3,017 unrelated Thai individuals genotyped with the Illumina Human610, Illumina HumanOmniexpress, or Illumina HapMap550v3 platform. We employed hidden Markov model and circular binary segmentation methods to identify CNVs, extracted 23,458 CNVs consistently identified by both algorithms, and cataloged these high confident CNVs into our publicly available Thai CNV database. Analysis of CNVs in the Thai population identified a median of eight autosomal CNVs per individual. Most CNVs (96.73%) did not overlap with any known chromosomal imbalance syndromes documented in the DECIPHER database. When compared with CNVs in the 11 HapMap3 populations, CNVs found in the Thai population shared several characteristics with CNVs characterized in HapMap3. Common CNVs in Thais had similar frequencies to those in the HapMap3 populations, and all high frequency CNVs (>20%) found in Thai individuals could also be identified in HapMap3. The majorities of CNVs discovered in the Thai population, however, were of low frequency, or uniquely identified in Thais. When performing hierarchical clustering using CNV frequencies, the CNV data were clustered into Africans, Europeans, and Asians, in line with the clustering performed with single nucleotide polymorphism (SNP) data. As CNV data are specific to origin of population, our population-specific reference database will serve as a valuable addition to the existing resources for the investigation of clinical significance of CNVs in Thais and related ethnicities.     

High resolution array in the clinical approach to chromosomal phenotypes

Array genomic hybridization (AGH) has recently been implemented as a diagnostic tool for the detection of submicroscopic copy number variants (CNVs) in patients with developmental disorders. However, there is no consensus regarding the choice of the platform, the minimal resolution needed and systematic interpretation of CNVs. We report our experience in the clinical diagnostic use of high resolution AGH up to 100 kb on 131 patients with chromosomal phenotypes but previously normal karyotype. We evaluated the usefulness in our clinics and laboratories by the detection rate of causal CNVs and CNVs of unknown clinical significance and to what extent their interpretation would challenge the systematic use of high-resolution arrays in clinical application. Prioritizing phenotype-genotype correlation in our interpretation strategy to criteria previously described, we identified 33 (25.2%) potentially pathogenic aberrations. 16 aberrations were confirmed pathogenic (16.4% syndromic, 8.5% non-syndromic patients); 9 were new and individual aberrations, 3 of them were pathogenic although inherited and one is as small as approx 200 kb. 13 of 16 further CNVs of unknown significance were classified likely benign, for 3 the significance remained unclear. High resolution array allows the detection of up to 12.2% of pathogenic aberrations in a diagnostic clinical setting. Although the majority of aberrations are larger, the detection of small causal aberrations may be relevant for family counseling. The number of remaining unclear CNVs is limited. Careful phenotype-genotype correlations of the individual CNVs and clinical features are challenging but remain a hallmark for CNV interpretation.     

Novel origins of copy number variation in the dog genome

ABSTRACT: BACKGROUND: Copy number variants (CNVs) account for substantial variation between genomes and are a major source of normal and pathogenic phenotypic differences. The dog is an ideal model to investigate mutational mechanisms that generate CNVs as its genome lacks a functional ortholog of the PRDM9 gene implicated in recombination and CNV formation in humans. Here we comprehensively assay CNVs using high-density array comparative genomic hybridization in 50 dogs from 17 dog breeds and 3 gray wolves. RESULTS: We use a stringent new method to identify a total of 430 high-confidence CNV loci, that range in size from 9 kb to 1.6 Mb and span 26.4 Mb, or 1.08%, of the assayed dog genome, overlapping 413 annotated genes. 98% of CNVs observed in each breed are also observed in multiple breeds. CNVs predicted to disrupt gene function are significantly less common than expected by chance. We identify a significant overrepresentation of peaks of GC content, previously shown to be enriched in dog recombination hotspots, in the vicinity of CNV breakpoints. CONCLUSIONS: A number of the CNVs identified by this study are candidates for generating breed-specific phenotypes. Purifying selection seems to be a major factor shaping structural variation in the dog genome, suggesting that many CNVs are deleterious. Localized peaks of GC content appear to be novel sites of CNV formation in the dog genome by non-allelic homologous recombination, potentially activated by the loss of PRDM9. These sequence features may have driven genome instability and chromosomal rearrangements throughout canid evolution.     

Comprehensive assessment of array-based platforms and calling algorithms for detection of copy number variants

We have systematically compared copy number variant (CNV) detection on eleven microarrays to evaluate data quality and CNV calling, reproducibility, concordance across array platforms and laboratory sites, breakpoint accuracy and analysis tool variability. Different analytic tools applied to the same raw data typically yield CNV calls with <50% concordance. Moreover, reproducibility in replicate experiments is <70% for most platforms. Nevertheless, these findings should not preclude detection of large CNVs for clinical diagnostic purposes because large CNVs with poor reproducibility are found primarily in complex genomic regions and would typically be removed by standard clinical data curation. The striking differences between CNV calls from different platforms and analytic tools highlight the importance of careful assessment of experimental design in discovery and association studies and of strict data curation and filtering in diagnostics. The CNV resource presented here allows independent data evaluation and provides a means to benchmark new algorithms.     

Diagnostic efficacy and new variants in isolated and complex autism spectrum disorder using molecular karyotyping

Autism spectrum disorder (ASD) is a group of the neurodevelopment disorders presenting as an isolated ASD or more complex forms, where a broader clinical phenotype comprised of developmental delay and intellectual disability is present. Both the isolated and complex forms have a significant causal genetic component and submicroscopic genomic copy number variations (CNV) are the most common identifiable genetic factor in these patients. The data on microarray testing in ASD cohorts are still accumulating and novel loci are often identified; therefore, we aimed to evaluate the diagnostic efficacy of the method and the relevance of implementing it into routine genetic testing in ASD patients. A genome-wide CNV analysis using the Agilent microarrays was performed in a group of 150 individuals with an isolated or complex ASD. Altogether, 11 (7.3%) pathogenic CNVs and 15 (10.0%) variants of unknown significance (VOUS) were identified, with the highest proportion of pathogenic CNVs in the subgroup of the complex ASD patients (14.3%). An interesting case of previously unreported partial UPF3B gene deletion was identified among the pathogenic CNVs. Among the CNVs with unknown significance, four VOUS involved genes with possible correlation to ASD, namely genes SNTG2, PARK2, CADPS2 and NLGN4X. The diagnostic efficacy of aCGH in our cohort was comparable with those of the previously reported and identified an important proportion of genetic ASD cases. Despite the continuum of published studies on the CNV testing in ASD cohorts, a considerable number of VOUS CNVs is still being identified, namely 10.0% in our study.     

Considering specific clinical features as evidence of pathogenic copy number variants

Since the introduction of high-resolution microarray technologies, it has become apparent that structural chromosomal rearrangements can lead to a wide variety of clinical manifestations, including developmental delay/intellectual disability (DD/ID). It has been shown previously that the diagnostic yield of genome-wide array-based identification of submicroscopic alterations in patients with ID varies widely and depends on the patient selection criteria. More attempts have recently been made to define the phenotypic clues of pathogenic copy number variants (CNVs). The aim of this study was to investigate a well-phenotyped cohort of patients with DD/ID and determine whether certain clinical features may serve as indicators for pathogenic CNVs. A retrospective analysis was conducted for patients with DD/ID (n?=?211) who were tested using genome-wide chromosomal microarray technologies and a review of the clinical data was performed. Pathogenic CNVs were detected in 29 patients. In comparison with individuals who had normal molecular karyotyping results (n?=?182), malformations of the musculoskeletal system; congenital malformations of the CNS (particularly hydrocephalus and congenital malformations of the corpus callosum); minor anomalies of the eye, face, and neck subgroup (particularly downward-slanting palpebral fissures, minor anomalies of the ear, and micrognathia); brachydactyly; and umbilical hernia were more common in patients with chromosomal alterations. A multivariate logistic regression analysis allowed the identification of three independent pathogenic CNV predictors: congenital malformations of the corpus callosum, minor anomalies of the ear, and brachydactyly. Insights into the chromosomal phenotype may help to increase the diagnostic yield of microarray technologies and sharpen the distinction between chromosomal alterations and other conditions.     

Copy number variants in pharmacogenetic genes

Variation in drug efficacy and toxicity remains an important clinical concern. Presently, single nucleotide polymorphisms (SNPs) only explain a portion of this problem, even in situations where the pharmacological trait is clearly heritable. The Human CNV Project identified copy number variations (CNVs) across approximately 12% of the human genome, and these CNVs were considered causes of diseases. Although the contribution of CNVs to the pathogenesis of many common diseases is questionable, CNVs play a clear role in drug-related genes by altering drug metabolizing and drug response. In this review, we provide a comprehensive evaluation of the clinical relevance of CNVs to drug efficacy, toxicity, and disease prevalence in world populations, and discuss the implication of using CNVs as a diagnostic tool in clinical intervention.     

SCNVSim: somatic copy number variation and structure variation simulator

Background

Somatically acquired structure variations (SVs) and copy number variations (CNVs) can induce genetic changes that are directly related to tumor genesis. Somatic SV/CNV detection using next-generation sequencing (NGS) data still faces major challenges introduced by tumor sample characteristics, such as ploidy, heterogeneity, and purity. A simulated cancer genome with known SVs and CNVs can serve as a benchmark for evaluating the performance of existing somatic SV/CNV detection tools and developing new methods.

Results

SCNVSim is a tool for simulating somatic CNVs and structure variations SVs. Other than multiple types of SV and CNV events, the tool is capable of simulating important features related to tumor samples including aneuploidy, heterogeneity and purity.

Conclusions

SCNVSim generates the genomes of a cancer cell population with detailed information of copy number status, loss of heterozygosity (LOH), and event break points, which is essential for developing and evaluating somatic CNV and SV detection methods in cancer genomics studies.     

Gene copy number variations in adaptive evolution: The genomic distribution of gene copy number variations revealed by genetic mapping and their adaptive role in an undomesticated species,white spruce (Picea glauca)

Gene copy number variation (CNV) has been associated with phenotypic variability in animals and plants, but a genomewide understanding of their impacts on phenotypes is largely restricted to human and agricultural systems. As such, CNVs have rarely been considered in investigations of the genomic architecture of adaptation in wild species. Here, we report on the genetic mapping of gene CNVs in white spruce, which lacks a contiguous assembly of its large genome (~20 Gb), and their relationships with adaptive phenotypic variation. We detected 3,911 gene CNVs including de novo structural variations using comparative genome hybridization on arrays (aCGH) in a large progeny set. We inferred the heterozygosity at CNV loci within parents by comparing haploid and diploid tissues and genetically mapped 82 gene CNVs. Our analysis showed that CNVs were distributed over 10 linkage groups and identified four CNV hotspots that we predict to occur in other species of the Pinaceae. Significant relationships were found between 29 of the gene CNVs and adaptive traits based on regression analyses with timings of bud set and bud flush, and height growth, suggesting a role for CNVs in climate adaptation. The importance of CNVs in adaptive evolution of white spruce was also indicated by functional gene annotations and the clustering of 31% of the mapped adaptive gene CNVs in CNV hotspots. Taken together, these results illustrate the feasibility of studying CNVs in undomesticated species and represent a major step towards a better understanding of the roles of CNVs in adaptive evolution.     

Copy number variation (CNV) identification,interpretation, and database from Brazilian patients

Copy number variations (CNVs) constitute an important class of variation in the human genome and the interpretation of their pathogenicity considering different frequencies across populations is still a challenge for geneticists. Since the CNV databases are predominantly composed of European and non-admixed individuals, and Brazilian genetic constitution is admixed and ethnically diverse, diagnostic screenings on Brazilian variants are greatly difficulted by the lack of populational references. We analyzed a clinical sample of 268 Brazilian individuals, including patients with neurodevelopment disorders and/or congenital malformations. The pathogenicity of CNVs was classified according to their gene content and overlap with known benign and pathogenic variants. A total of 1,504 autosomal CNVs (1,207 gains and 297 losses) were classified as benign (92.9%), likely benign (1.6%), VUS (2.6%), likely pathogenic (0.2%) and pathogenic (2.7%). Some of the CNVs were recurrent and with frequency increased in our sample, when compared to populational open resources of structural variants: 14q32.33, 22q11.22, 1q21.1, and 1p36.32 gains. Thus, these highly recurrent CNVs classified as likely benign or VUS were considered non-pathogenic in our Brazilian sample. This study shows the relevance of introducing CNV data from diverse cohorts to improve on the interpretation of clinical impact of genomic variations.     

Refinement of primate copy number variation hotspots identifies candidate genomic regions evolving under positive selection

Background

Copy number variants (CNVs), defined as losses and gains of segments of genomic DNA, are a major source of genomic variation.

Results

In this study, we identified over 2,000 human CNVs that overlap with orthologous chimpanzee or orthologous macaque CNVs. Of these, 170 CNVs overlap with both chimpanzee and macaque CNVs, and these were collapsed into 34 hotspot regions of CNV formation. Many of these hotspot regions of CNV formation are functionally relevant, with a bias toward genes involved in immune function, some of which were previously shown to evolve under balancing selection in humans. The genes in these primate CNV formation hotspots have significant differential expression levels between species and show evidence for positive selection, indicating that they have evolved under species-specific, directional selection.

Conclusions

These hotspots of primate CNV formation provide a novel perspective on divergence and selective pressures acting on these genomic regions.     

A map of copy number variations in Chinese populations

It has been shown that the human genome contains extensive copy number variations (CNVs). Investigating the medical and evolutionary impacts of CNVs requires the knowledge of locations, sizes and frequency distribution of them within and between populations. However, CNV study of Chinese minorities, which harbor the majority of genetic diversity of Chinese populations, has been underrepresented considering the same efforts in other populations. Here we constructed, to our knowledge, a first CNV map in seven Chinese populations representing the major linguistic groups in China with 1,440 CNV regions identified using Affymetrix SNP 6.0 Array. Considerable differences in distributions of CNV regions between populations and substantial population structures were observed. We showed that ～35% of CNV regions identified in minority ethnic groups are not shared by Han Chinese population, indicating that the contribution of the minorities to genetic architecture of Chinese population could not be ignored. We further identified highly differentiated CNV regions between populations. For example, a common deletion in Dong and Zhuang (44.4% and 50%), which overlaps two keratin-associated protein genes contributing to the structure of hair fibers, was not observed in Han Chinese. Interestingly, the most differentiated CNV deletion between HapMap CEU and YRI containing CCL3L1 gene reported in previous studies was also the highest differentiated regions between Tibetan and other populations. Besides, by jointly analyzing CNVs and SNPs, we found a CNV region containing gene CTDSPL were in almost perfect linkage disequilibrium between flanking SNPs in Tibetan while not in other populations except HapMap CHD. Furthermore, we found the SNP taggability of CNVs in Chinese populations was much lower than that in European populations. Our results suggest the necessity of a full characterization of CNVs in Chinese populations, and the CNV map we constructed serves as a useful resource in further evolutionary and medical studies.     

Challenges of interpreting copy number variation in syndromic and non-syndromic congenital heart defects

Array comparative genomic hybridization (aCGH) has led to an increased detection of causal chromosomal imbalances in individuals with congenital heart defects (CHD). The introduction of aCGH as a diagnostic tool in a clinical cardiogenetic setting entails numerous challenges. Based on our own experience as well as those of others described in the literature, we outline the state of the art and attempt to answer a number of outstanding questions such as the detection frequency of causal imbalances in different patient populations, the added value of higher-resolution arrays, and the existence of predictive factors in syndromic cases. We introduce a step-by-step approach for clinical interpretation of copy number variants (CNV) detected in CHD, which is primarily based on gene content and overlap with known chromosomal syndromes, rather than on CNV inheritance and size. Based on this algorithm, we have reclassified the detected aberrations in aCGH studies for their causality for syndromic and non-syndromic CHD. From this literature overview, supplemented with own investigations in a cohort of 46 sporadic patients with severe non-syndromic CHD, it seems clear that the frequency of causal CNVs in non-syndromic CHD populations is lower than that in syndromic CNV populations (3.6 vs. 19%). Moreover, causal CNVs in non-syndromic CHD mostly involve imbalances with a moderate effect size and reduced penetrance, whereas the majority of causal imbalances in syndromic CHD consistently affects human development and significantly reduces reproductive fitness.     

Application of high resolution SNP arrays in patients with congenital oral clefts in south China

Chromosome microarray analysis (CMA) has proven to be a powerful tool in postnatal patients with intellectual disabilities. However, the diagnostic capability of CMA in patients with congenital oral clefts remain mysterious. Here, we present our clinical experience in implementing whole-genome high-resolution SNP arrays to investigate 33 patients with syndromic and nonsyndromic oral clefts in whom standard karyotyping analyses showed normal karyotypes. We aim to identify the genomic aetiology and candidate genes in patients with congenital oral clefts. CMA revealed copy number variants (CNVs) in every patient, which ranged from 2 to 9 per sample. The size of detected CNVs varied from 100 to 3.2 Mb. In 33 patients, we identified six clinically significant CNVs. The incidence of clinically significant CNVs was 18.2% (6/33). Three of these six CNVs were detected in patients with nonsyndromic clefts, including one who presented with isolated cleft lip with cleft palate (CLP) and two with cleft palate only (CPO). The remaining three CNVs were detected in patients with syndromic clefts. However, no CNV was detected in patients with cleft lip only (CLO). The six clinically significant CNVs were as follows: 8p23.1 microduplication (198 kb); 10q22.2-q22.3 microdeletion (1766 kb); 18q12.3 microduplication (638 kb); 20p12.1 microdeletion (184 kb); 6q26 microdeletion (389 kb); and 22q11.21-q11.23 microdeletion (3163 kb). In addition, two novel candidate genes for oral clefts, KAT6B and MACROD2, were putatively identified. We also found a CNV of unknown clinical significance with a detection rate of 3.0% (1/33). Our results further support the notion that CNVs significantly contributed to the genetic aetiology of oral clefts and emphasize the efficacy of whole-genome high-resolution SNP arrays to detect novel candidate genes in patients with syndromic and nonsyndromic clefts.     

Accurate Distinction of Pathogenic from Benign CNVs in Mental Retardation

Copy number variants (CNVs) have recently been recognized as a common form of genomic variation in humans. Hundreds of CNVs can be detected in any individual genome using genomic microarrays or whole genome sequencing technology, but their phenotypic consequences are still poorly understood. Rare CNVs have been reported as a frequent cause of neurological disorders such as mental retardation (MR), schizophrenia and autism, prompting widespread implementation of CNV screening in diagnostics. In previous studies we have shown that, in contrast to benign CNVs, MR-associated CNVs are significantly enriched in genes whose mouse orthologues, when disrupted, result in a nervous system phenotype. In this study we developed and validated a novel computational method for differentiating between benign and MR-associated CNVs using structural and functional genomic features to annotate each CNV. In total 13 genomic features were included in the final version of a Naïve Bayesian Tree classifier, with LINE density and mouse knock-out phenotypes contributing most to the classifier''s accuracy. After demonstrating that our method (called GECCO) perfectly classifies CNVs causing known MR-associated syndromes, we show that it achieves high accuracy (94%) and negative predictive value (99%) on a blinded test set of more than 1,200 CNVs from a large cohort of individuals with MR. These results indicate that this classification method will be of value for objectively prioritizing CNVs in clinical research and diagnostics.     

Identification of Genome-Wide Copy Number Variations among Diverse Pig Breeds Using SNP Genotyping Arrays

Copy number variations (CNVs) are important forms of genetic variation complementary to SNPs, and can be considered as promising markers for some phenotypic and economically important traits or diseases susceptibility in domestic animals. In the present study, we performed a genome-wide CNV identification in 14 individuals selected from diverse populations, including six types of Chinese indigenous breeds, one Asian wild boar population, as well as three modern commercial foreign breeds. We identified 63 CNVRs in total, which covered 9.98 Mb of polymorphic sequence and corresponded to 0.36% of the genome sequence. The length of these CNVRs ranged from 3.20 to 827.21 kb, with an average of 158.37 kb and a median of 97.85 kb. Functional annotation revealed these identified CNVR have important molecular function, and may play an important role in exploring the genetic basis of phenotypic variability and disease susceptibility among pigs. Additionally, to confirm these potential CNVRs, we performed qPCR for 12 randomly selected CNVRs and 8 of them (66.67%) were confirmed successfully. CNVs detected in diverse populations herein are essential complementary to the CNV map in the pig genome, which provide an important resource for studies of genomic variation and the association between various economically important traits and CNVs.     

Genome-wide mapping of copy number variations in commercial hybrid pigs using a high-density SNP genotyping array

Copy number variations (CNVs) are important forms of structural variation in human and animals and can be considered as a major genetic component of phenotypic diversity. Here we used the Illumina PorcineSNP60 BeadChip V2 and a DLY [Duroc × (Large White × Landrace)] commercial hybrid population to identify 272 CNVs belonging to 165 CNV regions (CNVRs), of which 66 are new. As CNVRs are specific to origin of population, our DLY-specific data is an important complementary to the existing CNV map in the pig genome. Eight CNVRs were selected for validation by quantitative real-time PCR (qRT-PCR) and the accurate rate was high (87.25%). Gene function analysis suggested that a common CNVR may play an important role in multiple traits, including growth rate and carcass quality.