Identification of Rare Recurrent Copy Number Variants in High-Risk Autism Families and Their Prevalence in a Large ASD Population

Structural variation is thought to play a major etiological role in the development of autism spectrum disorders (ASDs), and numerous studies documenting the relevance of copy number variants (CNVs) in ASD have been published since 2006. To determine if large ASD families harbor high-impact CNVs that may have broader impact in the general ASD population, we used the Affymetrix genome-wide human SNP array 6.0 to identify 153 putative autism-specific CNVs present in 55 individuals with ASD from 9 multiplex ASD pedigrees. To evaluate the actual prevalence of these CNVs as well as 185 CNVs reportedly associated with ASD from published studies many of which are insufficiently powered, we designed a custom Illumina array and used it to interrogate these CNVs in 3,000 ASD cases and 6,000 controls. Additional single nucleotide variants (SNVs) on the array identified 25 CNVs that we did not detect in our family studies at the standard SNP array resolution. After molecular validation, our results demonstrated that 15 CNVs identified in high-risk ASD families also were found in two or more ASD cases with odds ratios greater than 2.0, strengthening their support as ASD risk variants. In addition, of the 25 CNVs identified using SNV probes on our custom array, 9 also had odds ratios greater than 2.0, suggesting that these CNVs also are ASD risk variants. Eighteen of the validated CNVs have not been reported previously in individuals with ASD and three have only been observed once. Finally, we confirmed the association of 31 of 185 published ASD-associated CNVs in our dataset with odds ratios greater than 2.0, suggesting they may be of clinical relevance in the evaluation of children with ASDs. Taken together, these data provide strong support for the existence and application of high-impact CNVs in the clinical genetic evaluation of children with ASD.     

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.     

The contribution of de novo and rare inherited copy number changes to congenital heart disease in an unselected sample of children with conotruncal defects or hypoplastic left heart disease

Congenital heart disease (CHD) is the most common congenital malformation, with evidence of a strong genetic component. We analyzed data from 223 consecutively ascertained families, each consisting of at least one child affected by a conotruncal defect (CNT) or hypoplastic left heart disease (HLHS) and both parents. The NimbleGen HD2-2.1 comparative genomic hybridization platform was used to identify de novo and rare inherited copy number variants (CNVs). Excluding 10 cases with 22q11.2 DiGeorge deletions, we validated de novo CNVs in 8 % of 148 probands with CNTs, 12.7 % of 71 probands with HLHS and none in 4 probands with both. Only 2 % of control families showed a de novo CNV. We also identified a group of ultra-rare inherited CNVs that occurred de novo in our sample, contained a candidate gene for CHD, recurred in our sample or were present in an affected sibling. We confirmed the contribution to CHD of copy number changes in genes such as GATA4 and NODAL and identified several genes in novel recurrent CNVs that may point to novel CHD candidate loci. We also found CNVs previously associated with highly variable phenotypes and reduced penetrance, such as dup 1q21.1, dup 16p13.11, dup 15q11.2-13, dup 22q11.2, and del 2q23.1. We found that the presence of extra-cardiac anomalies was not related to the frequency of CNVs, and that there was no significant difference in CNV frequency or specificity between the probands with CNT and HLHS. In agreement with other series, we identified likely causal CNVs in 5.6 % of our total sample, half of which were de novo.     

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.     

Convergence of Genes and Cellular Pathways Dysregulated in Autism Spectrum Disorders

Rare copy-number variation (CNV) is an important source of risk for autism spectrum disorders (ASDs). We analyzed 2,446 ASD-affected families and confirmed an excess of genic deletions and duplications in affected versus control groups (1.41-fold, p = 1.0 × 10⁻⁵) and an increase in affected subjects carrying exonic pathogenic CNVs overlapping known loci associated with dominant or X-linked ASD and intellectual disability (odds ratio = 12.62, p = 2.7 × 10⁻¹⁵, ∼3% of ASD subjects). Pathogenic CNVs, often showing variable expressivity, included rare de novo and inherited events at 36 loci, implicating ASD-associated genes (CHD2, HDAC4, and GDI1) previously linked to other neurodevelopmental disorders, as well as other genes such as SETD5, MIR137, and HDAC9. Consistent with hypothesized gender-specific modulators, females with ASD were more likely to have highly penetrant CNVs (p = 0.017) and were also overrepresented among subjects with fragile X syndrome protein targets (p = 0.02). Genes affected by de novo CNVs and/or loss-of-function single-nucleotide variants converged on networks related to neuronal signaling and development, synapse function, and chromatin regulation.     

High-resolution copy-number variation map reflects human olfactory receptor diversity and evolution

Olfactory receptors (ORs), which are involved in odorant recognition, form the largest mammalian protein superfamily. The genomic content of OR genes is considerably reduced in humans, as reflected by the relatively small repertoire size and the high fraction ( approximately 55%) of human pseudogenes. Since several recent low-resolution surveys suggested that OR genomic loci are frequently affected by copy-number variants (CNVs), we hypothesized that CNVs may play an important role in the evolution of the human olfactory repertoire. We used high-resolution oligonucleotide tiling microarrays to detect CNVs across 851 OR gene and pseudogene loci. Examining genomic DNA from 25 individuals with ancestry from three populations, we identified 93 OR gene loci and 151 pseudogene loci affected by CNVs, generating a mosaic of OR dosages across persons. Our data suggest that approximately 50% of the CNVs involve more than one OR, with the largest CNV spanning 11 loci. In contrast to earlier reports, we observe that CNVs are more frequent among OR pseudogenes than among intact genes, presumably due to both selective constraints and CNV formation biases. Furthermore, our results show an enrichment of CNVs among ORs with a close human paralog or lacking a one-to-one ortholog in chimpanzee. Interestingly, among the latter we observed an enrichment in CNV losses over gains, a finding potentially related to the known diminution of the human OR repertoire. Quantitative PCR experiments performed for 122 sampled ORs agreed well with the microarray results and uncovered 23 additional CNVs. Importantly, these experiments allowed us to uncover nine common deletion alleles that affect 15 OR genes and five pseudogenes. Comparison to the chimpanzee reference genome revealed that all of the deletion alleles are human derived, therefore indicating a profound effect of human-specific deletions on the individual OR gene content. Furthermore, these deletion alleles may be used in future genetic association studies of olfactory inter-individual differences.     

The fine-scale and complex architecture of human copy-number variation

Despite considerable excitement over the potential functional significance of copy-number variants (CNVs), we still lack knowledge of the fine-scale architecture of the large majority of CNV regions in the human genome. In this study, we used a high-resolution array-based comparative genomic hybridization (aCGH) platform that targeted known CNV regions of the human genome at approximately 1 kb resolution to interrogate the genomic DNAs of 30 individuals from four HapMap populations. Our results revealed that 1020 of 1153 CNV loci (88%) were actually smaller in size than what is recorded in the Database of Genomic Variants based on previously published studies. A reduction in size of more than 50% was observed for 876 CNV regions (76%). We conclude that the total genomic content of currently known common human CNVs is likely smaller than previously thought. In addition, approximately 8% of the CNV regions observed in multiple individuals exhibited genomic architectural complexity in the form of smaller CNVs within larger ones and CNVs with interindividual variation in breakpoints. Future association studies that aim to capture the potential influences of CNVs on disease phenotypes will need to consider how to best ascertain this previously uncharacterized complexity.     

Transmission Disequilibrium of Small CNVs in Simplex Autism

We searched for disruptive, genic rare copy-number variants (CNVs) among 411 families affected by sporadic autism spectrum disorder (ASD) from the Simons Simplex Collection by using available exome sequence data and CoNIFER (Copy Number Inference from Exome Reads). Compared to high-density SNP microarrays, our approach yielded ∼2× more smaller genic rare CNVs. We found that affected probands inherited more CNVs than did their siblings (453 versus 394, p = 0.004; odds ratio [OR] = 1.19) and that the probands’ CNVs affected more genes (921 versus 726, p = 0.02; OR = 1.30). These smaller CNVs (median size 18 kb) were transmitted preferentially from the mother (136 maternal versus 100 paternal, p = 0.02), although this bias occurred irrespective of affected status. The excess burden of inherited CNVs among probands was driven primarily by sibling pairs with discordant social-behavior phenotypes (p < 0.0002, measured by Social Responsiveness Scale [SRS] score), which contrasts with families where the phenotypes were more closely matched or less extreme (p > 0.5). Finally, we found enrichment of brain-expressed genes unique to probands, especially in the SRS-discordant group (p = 0.0035). In a combined model, our inherited CNVs, de novo CNVs, and de novo single-nucleotide variants all independently contributed to the risk of autism (p < 0.05). Taken together, these results suggest that small transmitted rare CNVs play a role in the etiology of simplex autism. Importantly, the small size of these variants aids in the identification of specific genes as additional risk factors associated with ASD.     

Rare copy number variations in adults with tetralogy of fallot implicate novel risk gene pathways

Structural genetic changes, especially copy number variants (CNVs), represent a major source of genetic variation contributing to human disease. Tetralogy of Fallot (TOF) is the most common form of cyanotic congenital heart disease, but to date little is known about the role of CNVs in the etiology of TOF. Using high-resolution genome-wide microarrays and stringent calling methods, we investigated rare CNVs in a prospectively recruited cohort of 433 unrelated adults with TOF and/or pulmonary atresia at a single centre. We excluded those with recognized syndromes, including 22q11.2 deletion syndrome. We identified candidate genes for TOF based on converging evidence between rare CNVs that overlapped the same gene in unrelated individuals and from pathway analyses comparing rare CNVs in TOF cases to those in epidemiologic controls. Even after excluding the 53 (10.7%) subjects with 22q11.2 deletions, we found that adults with TOF had a greater burden of large rare genic CNVs compared to controls (8.82% vs. 4.33%, p?=?0.0117). Six loci showed evidence for recurrence in TOF or related congenital heart disease, including typical 1q21.1 duplications in four (1.18%) of 340 Caucasian probands. The rare CNVs implicated novel candidate genes of interest for TOF, including PLXNA2, a gene involved in semaphorin signaling. Independent pathway analyses highlighted developmental processes as potential contributors to the pathogenesis of TOF. These results indicate that individually rare CNVs are collectively significant contributors to the genetic burden of TOF. Further, the data provide new evidence for dosage sensitive genes in PLXNA2-semaphorin signaling and related developmental processes in human cardiovascular development, consistent with previous animal models.     

Prioritization of Copy Number Variation Loci Associated with Autism from AutDB–An Integrative Multi-Study Genetic Database

Copy number variants (CNVs) are thought to play an important role in the predisposition to autism spectrum disorder (ASD). However, their relatively low frequency and widespread genomic distribution complicates their accurate characterization and utilization for clinical genetics purposes. Here we present a comprehensive analysis of multi-study, genome-wide CNV data from AutDB (http://mindspec.org/autdb.html), a genetic database that accommodates detailed annotations of published scientific reports of CNVs identified in ASD individuals. Overall, we evaluated 4,926 CNVs in 2,373 ASD subjects from 48 scientific reports, encompassing ∼2.12×10⁹ bp of genomic data. Remarkable variation was seen in CNV size, with duplications being significantly larger than deletions, (P  =  3×10⁻¹⁰⁵; Wilcoxon rank sum test). Examination of the CNV burden across the genome revealed 11 loci with a significant excess of CNVs among ASD subjects (P<7×10⁻⁷). Altogether, these loci covered 15,610 kb of the genome and contained 166 genes. Remarkable variation was seen both in locus size (20 - 4950 kb), and gene content, with seven multigenic (≥3 genes) and four monogenic loci. CNV data from control populations was used to further refine the boundaries of these ASD susceptibility loci. Interestingly, our analysis indicates that 15q11.2-13.3, a genomic region prone to chromosomal rearrangements of various sizes, contains three distinct ASD susceptibility CNV loci that vary in their genomic boundaries, CNV types, inheritance patterns, and overlap with CNVs from control populations. In summary, our analysis of AutDB CNV data provides valuable insights into the genomic characteristics of ASD susceptibility CNV loci and could therefore be utilized in various clinical settings and facilitate future genetic research of this disorder.     

Maternal Modifiers and Parent-of-Origin Bias of the Autism-Associated 16p11.2 CNV

Recurrent deletions and duplications at chromosomal region 16p11.2 are a major genetic contributor to autism but also associate with a wider range of pediatric diagnoses, including intellectual disability, coordination disorder, and language disorder. In order to investigate the potential genetic basis for phenotype variability, we assessed the parent of origin of the 16p11.2 copy-number variant (CNV) and the presence of additional CNVs in 126 families for which detailed phenotype data were available. Among de novo cases, we found a strong maternal bias for the origin of deletions (59/66, 89.4% of cases, p = 2.38 × 10⁻¹¹), the strongest such effect so far observed for a CNV associated with a microdeletion syndrome. In contrast to de novo events, we observed no transmission bias for inherited 16p11.2 CNVs, consistent with a female meiotic hotspot of unequal crossover driving this maternal bias. We analyzed this 16p11.2 CNV cohort for the presence of secondary CNVs and found a significant maternal transmission bias for secondary deletions (32 maternal versus 14 paternal, p = 1.14 × 10⁻²). Of the secondary deletions that disrupted a gene, 82% were either maternally inherited or de novo (p = 4.3 × 10⁻³). Nine probands carry secondary CNVs that disrupt genes associated with autism and/or intellectual disability risk variants. Our findings demonstrate a strong bias toward maternal origin of 16p11.2 de novo deletions as well as a maternal transmission bias for secondary deletions that contribute to the clinical outcome on a background sensitized by the 16p11.2 CNV.     

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.     

Rare De Novo Copy Number Variants in Patients with Congenital Pulmonary Atresia

Background

Ongoing studies using genomic microarrays and next-generation sequencing have demonstrated that the genetic contributions to cardiovascular diseases have been significantly ignored in the past. The aim of this study was to identify rare copy number variants in individuals with congenital pulmonary atresia (PA).

Methods and Results

Based on the hypothesis that rare structural variants encompassing key genes play an important role in heart development in PA patients, we performed high-resolution genome-wide microarrays for copy number variations (CNVs) in 82 PA patient-parent trios and 189 controls with an Illumina SNP array platform. CNVs were identified in 17/82 patients (20.7%), and eight of these CNVs (9.8%) are considered potentially pathogenic. Five de novo CNVs occurred at two known congenital heart disease (CHD) loci (16p13.1 and 22q11.2). Two de novo CNVs that may affect folate and vitamin B₁₂ metabolism were identified for the first time. A de novo 1-Mb deletion at 17p13.2 may represent a rare genomic disorder that involves mild intellectual disability and associated facial features.

Conclusions

Rare CNVs contribute to the pathogenesis of PA (9.8%), suggesting that the causes of PA are heterogeneous and pleiotropic. Together with previous data from animal models, our results might help identify a link between CHD and folate-mediated one-carbon metabolism (FOCM). With the accumulation of high-resolution SNP array data, these previously undescribed rare CNVs may help reveal critical gene(s) in CHD and may provide novel insights about CHD pathogenesis.     

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.     

Discovery and Statistical Genotyping of Copy-Number Variation from Whole-Exome Sequencing Depth

Sequencing of gene-coding regions (the exome) is increasingly used for studying human disease, for which copy-number variants (CNVs) are a critical genetic component. However, detecting copy number from exome sequencing is challenging because of the noncontiguous nature of the captured exons. This is compounded by the complex relationship between read depth and copy number; this results from biases in targeted genomic hybridization, sequence factors such as GC content, and batching of samples during collection and sequencing. We present a statistical tool (exome hidden Markov model [XHMM]) that uses principal-component analysis (PCA) to normalize exome read depth and a hidden Markov model (HMM) to discover exon-resolution CNV and genotype variation across samples. We evaluate performance on 90 schizophrenia trios and 1,017 case-control samples. XHMM detects a median of two rare (<1%) CNVs per individual (one deletion and one duplication) and has 79% sensitivity to similarly rare CNVs overlapping three or more exons discovered with microarrays. With sensitivity similar to state-of-the-art methods, XHMM achieves higher specificity by assigning quality metrics to the CNV calls to filter out bad ones, as well as to statistically genotype the discovered CNV in all individuals, yielding a trio call set with Mendelian-inheritance properties highly consistent with expectation. We also show that XHMM breakpoint quality scores enable researchers to explicitly search for novel classes of structural variation. For example, we apply XHMM to extract those CNVs that are highly likely to disrupt (delete or duplicate) only a portion of a gene.     

Contribution of global rare copy-number variants to the risk of sporadic congenital heart disease

Previous studies have shown that copy-number variants (CNVs) contribute to the risk of complex developmental phenotypes. However, the contribution of global CNV burden to the risk of sporadic congenital heart disease (CHD) remains incompletely defined. We generated genome-wide CNV data by using Illumina 660W-Quad SNP arrays in 2,256 individuals with CHD, 283 trio CHD-affected families, and 1,538 controls. We found association of rare genic deletions with CHD risk (odds ratio [OR] = 1.8, p = 0.0008). Rare deletions in study participants with CHD had higher gene content (p = 0.001) with higher haploinsufficiency scores (p = 0.03) than they did in controls, and they were enriched with Wnt-signaling genes (p = 1 × 10⁻⁵). Recurrent 15q11.2 deletions were associated with CHD risk (OR = 8.2, p = 0.02). Rare de novo CNVs were observed in ∼5% of CHD trios; 10 out of 11 occurred on the paternally transmitted chromosome (p = 0.01). Some of the rare de novo CNVs spanned genes known to be involved in heart development (e.g., HAND2 and GJA5). Rare genic deletions contribute ∼4% of the population-attributable risk of sporadic CHD. Second to previously described CNVs at 1q21.1, deletions at 15q11.2 and those implicating Wnt signaling are the most significant contributors to the risk of sporadic CHD. Rare de novo CNVs identified in CHD trios exhibit paternal origin bias.     

Multiple recurrent de novo CNVs, including duplications of the 7q11.23 Williams syndrome region, are strongly associated with autism

We have undertaken a genome-wide analysis of rare copy-number variation (CNV) in 1124 autism spectrum disorder (ASD) families, each comprised of a single proband, unaffected parents, and, in most kindreds, an unaffected sibling. We find significant association of ASD with de novo duplications of 7q11.23, where the reciprocal deletion causes Williams-Beuren syndrome, characterized by a highly social personality. We identify rare recurrent de novo CNVs at five additional regions, including 16p13.2 (encompassing genes USP7 and C16orf72) and Cadherin 13, and implement a rigorous approach to evaluating the statistical significance of these observations. Overall, large de novo CNVs, particularly those encompassing multiple genes, confer substantial risks (OR = 5.6; CI = 2.6-12.0, p = 2.4 × 10(-7)). We estimate there are 130-234 ASD-related CNV regions in the human genome and present compelling evidence, based on cumulative data, for association of rare de novo events at 7q11.23, 15q11.2-13.1, 16p11.2, and Neurexin 1.