Detection and Characterization of Copy Number Variation in Autism Spectrum Disorder

Copy number variants (CNVs) have a major role in the etiology of autism spectrum disorders (ASD), and several of these have reached statistical significance in case–control analyses. Nevertheless, current ASD cohorts are not large enough to detect very rare CNVs that may be causative or contributory (that is, risk alleles). Here, we use a tiered approach, in which clinically significant CNVs are first identified in large clinical cohorts of neurodevelopmental disorders (including but not specific to ASD), after which these CNVs are then systematically identified within well-characterized ASD cohorts. We focused our initial analysis on 48 recurrent CNVs (segmental duplication-mediated ‘hotspots’) from 24 loci in 31 516 published clinical cases with neurodevelopmental disorders and 13 696 published controls, which yielded a total of 19 deletion CNVs and 11 duplication CNVs that reached statistical significance. We then investigated the overlap of these 30 CNVs in a combined sample of 3955 well-characterized ASD cases from three published studies. We identified 73 deleterious recurrent CNVs, including 36 deletions from 11 loci and 37 duplications from seven loci, for a frequency of 1 in 54; had we considered the ASD cohorts alone, only 58 CNVs from eight loci (24 deletions from three loci and 34 duplications from five loci) would have reached statistical significance. In conclusion, until there are sufficiently large ASD research cohorts with enough power to detect very rare causative or contributory CNVs, data from larger clinical cohorts can be used to infer the likely clinical significance of CNVs in ASD.

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.

SU16 - THE IMPACT OF CNVs ON ASD/ADHD RISK IN MULTIPLEX FAMILIES

In this issue of JAMA, Mannik and colleagues1 report that large (>250 kilobase [kb] pairs) structural variants in the genome, specifically deletion and duplication copy number variations (CNVs), were associated with cognitive phenotypes including intellectual disability and reduced educational achievement. These CNVs are deletions and duplications of DNA sequences in the human genome that can be considered as deviations from the normal diploid state at a given location in the genome and represent differing numbers of copies of genetic sequences. The authors also identified and examined the phenotypic consequences of genomic structural variation associated with known genomic disorders2 among individuals in whom the genomic disorders were not initially clinically recognized.

Rare chromosomal deletions and duplications in attention-deficit hyperactivity disorder: a genome-wide analysis

Cancer is usually considered to be an acquired disease caused by the progressive accumulation of somatic deoxyribonucleic acid (DNA) changes. It is clear, however, that inherited factors may also play a significant role in the initiation of this disease. Some of these factors represent loss‐of‐function mutations in tumour suppressor genes, resulting in an increased cancer risk among carriers. Such an increased risk may also be attributed to the presence of multiple polymorphic variants such as single nucleotide polymorphisms (SNPs), each of which may convey a mild effect upon cancer susceptibility. DNA copy number variation (CNV) and other structural variations in the human genome are increasingly recognised as an alternative source of genetic variation that may influence cancer risk. Specifically, rare CNVs may affect important cancer‐associated genes and/or pathways and, thus, provide an explanation for moderate‐ to high‐risk cancer families. Therefore, it is anticipated that the identification of rare germline CNVs in unexplained familial and early onset cancer patients will contribute to our understanding of cancer predisposition and development. Key Concepts: Copy number variation (CNV) is a common form of normal variation affecting a significant proportion of the human genome. CNVs frequently encompass annotated genes. Disruption and/or silencing of critical genes located in rare CNVs may result in increased risk of disease, including cancer. Genome‐wide copy number analysis can be used as a strategy to identify novel moderate‐ to high‐penetrant cancer predisposing genes and/or mechanisms. Discovery of cancer‐predisposing CNVs may reveal new cancer syndromes that exhibit additional clinical features.

This Month in Genetics

FunVar: A systematic pipeline to unravel the convergence patterns of genetic variants in ASD, a paradigmatic complex disease.

Rare copy number variants (CNVs) disrupting ASTN2 or both ASTN2 and TRIM32 have been reported at 9q33.1 by genome-wide studies in a few individuals with neurodevelopmental disorders (NDDs). The vertebrate-specific astrotactins, ASTN2 and its paralog ASTN1, have key roles in glial-guided neuronal migration during brain development. To determine the prevalence of astrotactin mutations and delineate their associated phenotypic spectrum, we screened ASTN2/TRIM32 and ASTN1 (1q25.2) for exonic CNVs in clinical microarray data from 89 985 individuals across 10 sites, including 64 114 NDD subjects. In this clinical dataset, we identified 46 deletions and 12 duplications affecting ASTN2. Deletions of ASTN1 were much rarer. Deletions near the 3' terminus of ASTN2, which would disrupt all transcript isoforms (a subset of these deletions also included TRIM32), were significantly enriched in the NDD subjects (P = 0.002) compared with 44 085 population-based controls. Frequent phenotypes observed in individuals with such deletions include autism spectrum disorder (ASD), attention deficit hyperactivity disorder (ADHD), speech delay, anxiety and obsessive compulsive disorder (OCD). The 3'-terminal ASTN2 deletions were significantly enriched compared with controls in males with NDDs, but not in females. Upon quantifying ASTN2 human brain RNA, we observed shorter isoforms expressed from an alternative transcription start site of recent evolutionary origin near the 3' end. Spatiotemporal expression profiling in the human brain revealed consistently high ASTN1 expression while ASTN2 expression peaked in the early embryonic neocortex and postnatal cerebellar cortex. Our findings shed new light on the role of the astrotactins in psychopathology and their interplay in human neurodevelopment.

Collaborative Genome-Wide Association and Copy Number Variation Analysis of Tourette Syndrome

BackgroundThe pathogenicity of copy number variations (CNV) in neurodevelopmental disorders is well supported by research literature. However, few studies have evaluated the utility and counseling challenges of CNV analysis in the clinic.MethodsWe analyzed the findings of CNV studies from a cohort referred for clinical genetics evaluation of autism spectrum disorders (ASD), developmental disability (DD), and intellectual disability (ID).ResultsTwenty-two CNV in 21 out of 115 probands are considered to be pathogenic (18.3%). Five CNV are likely pathogenic and 22 CNV are variants of unknown significance (VUS). We have found 7 cases with more than 2 CNV and 2 with a complex rearrangement of the 22q13.3 Phelan-McDermid syndrome region. We identified a new and de novo 1q21.3 deletion that encompasses SETDB1, a gene encoding methylates histone H3 on lysine-9 (H3K9) methyltransferase, in a case with typical ASD and ID.ConclusionsWe provide evidence to support the value of CNV analysis in etiological evaluation of neurodevelopmental disorders. However, interpretation of the clinical significance and counseling families are still challenging because of the variable penetrance and pleotropic expressivity of CNVs. In addition, the identification of a 1q21.3 deletion encompassing SETDB1 provides further support for the role of chromatin modifiers in the etiology of ASD.

Introduction: Copy number variations (CNVs) play a critical role in the pathogenesis of neurodevelopmental disorders (NDD) among children. In this study, we aim to identify clinically relevant CNVs, genes and their phenotypic characteristics in an ethnically underrepresented homogenous population of Bangladesh.Methods: We have conducted chromosomal microarray analysis (CMA) for 212 NDD patients with male to female ratio of 2.2:1.0 to identify rare CNVs. To identify candidate genes within the rare CNVs, gene constraint metrics [i.e., “Critical-Exon Genes (CEGs)”] were applied to the population data. Autism Diagnostic Observation Schedule-Second Edition (ADOS-2) was followed in a subset of 95 NDD patients to assess the severity of autism and all statistical tests were performed using the R package.Results: Of all the samples assayed, 12.26% (26/212) and 57.08% (121/212) patients carried pathogenic and variant of uncertain significance (VOUS) CNVs, respectively. While 2.83% (6/212) patients’ pathogenic CNVs were found to be located in the subtelomeric regions. Further burden test identified females are significant carriers of pathogenic CNVs compared to males (OR = 4.2; p = 0.0007). We have observed an increased number of Loss of heterozygosity (LOH) within cases with 23.85% (26/109) consanguineous parents. Our analyses on imprinting genes show, 36 LOH variants disrupting 69 unique imprinted genes and classified these variants as VOUS. ADOS-2 subset shows severe social communication deficit (p = 0.014) and overall ASD symptoms severity (p = 0.026) among the patients carrying duplication CNV compared to the CNV negative group. Candidate gene analysis identified 153 unique CEGs in pathogenic CNVs and 31 in VOUS. Of the unique genes, 18 genes were found to be in smaller (<1 MB) focal CNVs in our NDD cohort and we identified PSMC3 gene as a strong candidate gene for Autism Spectrum Disorder (ASD). Moreover, we hypothesized that KMT2B gene duplication might be associated with intellectual disability.Conclusion: Our results show the utility of CMA for precise genetic diagnosis and its integration into the diagnosis, therapy and management of NDD patients.

Autism spectrum disorder (ASD) is heterogeneous in symptom and etiology. Rare copy number variations (CNVs) are important genetic factors contributing to ASD. Currently chromosomal microarray (CMA) detecting CNVs is recommended as a first-tier diagnostic assay, largely based on research in North America and Europe. The feature of rare CNVs has not been well characterized in ASD cohorts from non-European ancestry. In this study, high resolution CMA was utilized to investigate rare CNVs in a Chinese cohort of ASD (n = 401, including 177 mildly/moderately and 224 severely affected individuals), together with an ancestry-matched control cohort (n = 197). Diagnostic yield was about 4.2%, with 17 clinically significant CNVs identified in ASD individuals, of which 12 CNVs overlapped with recurrent autism risk loci or genes. Autosomal rare CNV burden analysis showed an overrepresentation of rare loss events in ASD cohort, whereas the rate of rare gain events correlated with the phenotypic severity. Further analysis showed rare losses disrupting genes highly intolerant of loss-of-function variants were enriched in the ASD cohort. Among these highly constrained genes disrupted by rare losses, RIMS2 is a promising candidate contributing to ASD risk. This pilot study evaluated clinical utility of CMA and the feature of rare CNVs in Chinese ASD, with candidate genes identified as potential risk factors.

Advances in Our Understanding of Genetic Risk Factors for Autism Spectrum Disorders

Background/Objectives: Attention Deficit Hyperactivity Disorder (ADHD) is a common neurodevelopmental disorder frequently associated with other neuropsychiatric conditions, characterized by high clinical heterogeneity and a complex genetic background. Recent studies suggest that copy number variations (CNVs) may contribute to ADHD susceptibility, particularly when involving genes related to brain development, attention regulation, and impulse control. This study investigated the association between CNVs and ADHD phenotype by identifying patients with and without potential pathogenic CNVs. Methods: We evaluated 152 well-characterized ADHD pediatric patients through comprehensive clinical assessments, including dysmorphic features, brain MRI, EEG patterns, and cognitive testing. CNVs were identified using array Comparative Genomic Hybridization (array-CGH). Participants were classified as carrying potentially causative CNVs (PC-CNVs), non-causative CNVs (NC-CNVs), or without CNVs (W-CNVs) and statistically compared across clinical and neurodevelopmental measures. Results: CNVs were identified in 81 participants (53%), comprising 13 with PC-CNVs (8.5%) and 68 with NC-CNVs (44.7%). ADHD symptoms were pronounced across all groups, but PC-CNVs showed a higher burden of comorbidities, suggesting a stronger genetic contribution to ADHD complexity. Significant differences were observed in oppositional behavior, inattentive symptoms, brain MRI findings, and developmental language anomalies. Several CNVs involved genes previously implicated in neurodevelopmental disorders, supporting a potential genetic contribution to the clinical complexity of ADHD. Conclusions: This exploratory study supports the role of CNVs in ADHD susceptibility and highlights the value of genetic screening for understanding clinical variability. Larger studies are needed to clarify genotype–phenotype correlations in ADHD and to guide personalized clinical management.