Autism spectrum disorders (ASDs) are highly heritable, yet relatively few associated genetic loci have been replicated. Copy number variations (CNVs) have been implicated in autism; however, the majority of loci contribute to <1% of the disease population. Therefore, independent studies are important to refine associated CNV regions and discover novel susceptibility genes. In this study, a genome-wide SNP array was utilized for CNV detection by two distinct algorithms in a European ancestry case–control data set. We identify a significantly higher burden in the number and size of deletions, and disrupting more genes in ASD cases. Moreover, 18 deletions larger than 1 Mb were detected exclusively in cases, implicating novel regions at 2q22.1, 3p26.3, 4q12 and 14q23. Case-specific CNVs provided further evidence for pathways previously implicated in ASDs, revealing new candidate genes within the GABAergic signaling and neural development pathways. These include DBI, an allosteric binder of GABA receptors, GABARAPL1, the GABA receptor-associated protein, and SLC6A11, a postsynaptic GABA transporter. We also identified CNVs in COBL, deletions of which cause defects in neuronal cytoskeleton morphogenesis in model vertebrates, and DNER, a neuron-specific Notch ligand required for cerebellar development. Moreover, we found evidence of genetic overlap between ASDs and other neurodevelopmental and neuropsychiatric diseases. These genes include glutamate receptors (GRID1, GRIK2 and GRIK4), synaptic regulators (NRXN3, SLC6A8 and SYN3), transcription factor (ZNF804A) and RNA-binding protein FMR1. Taken together, these CNVs may be a few of the missing pieces of ASD heritability and lead to discovering novel etiological mechanisms.
Autism spectrum disorder (ASD) is highly heritable, yet genome-wide association studies (GWAS), copy number variation screens, and candidate gene association studies have found no single factor accounting for a large percentage of genetic risk. ASD trio exome sequencing studies have revealed genes with recurrent de novo loss-of-function variants as strong risk factors, but there are relatively few recurrently affected genes while as many as 1000 genes are predicted to play a role. As such, it is critical to identify the remaining rare and low-frequency variants contributing to ASD.
We have utilized an approach of prioritization of genes by GWAS and follow-up with massively parallel sequencing in a case-control cohort. Using a previously reported ASD noise reduction GWAS analyses, we prioritized 837 RefSeq genes for custom targeting and sequencing. We sequenced the coding regions of those genes in 2071 ASD cases and 904 controls of European white ancestry. We applied comprehensive annotation to identify single variants which could confer ASD risk and also gene-based association analysis to identify sets of rare variants associated with ASD.
We identified a significant over-representation of rare loss-of-function variants in genes previously associated with ASD, including a de novo premature stop variant in the well-established ASD candidate gene RBFOX1. Furthermore, ASD cases were more likely to have two damaging missense variants in candidate genes than controls. Finally, gene-based rare variant association implicates genes functioning in excitatory neurotransmission and neurite outgrowth and guidance pathways including CACNAD2, KCNH7, and NRXN1.
We find suggestive evidence that rare variants in synaptic genes are associated with ASD and that loss-of-function mutations in ASD candidate genes are a major risk factor, and we implicate damaging mutations in glutamate signaling receptors and neuronal adhesion and guidance molecules. Furthermore, the role of de novo mutations in ASD remains to be fully investigated as we identified the first reported protein-truncating variant in RBFOX1 in ASD. Overall, this work, combined with others in the field, suggests a convergence of genes and molecular pathways underlying ASD etiology.
Electronic supplementary material
The online version of this article (doi:10.1186/s13229-015-0034-z) contains supplementary material, which is available to authorized users.
Significant evidence exists for the association between copy number variants (CNVs) and Autism Spectrum Disorder (ASD); however, most of this work has focused solely on the diagnosis of ASD. There is limited understanding of the impact of CNVs on the ‘sub-phenotypes' of ASD. The objective of this paper is to evaluate associations between CNVs in differentially brain expressed (DBE) genes or genes previously implicated in ASD/intellectual disability (ASD/ID) and specific sub-phenotypes of ASD. The sample consisted of 1590 cases of European ancestry from the Autism Genome Project (AGP) with a diagnosis of an ASD and at least one rare CNV impacting any gene and a core set of phenotypic measures, including symptom severity, language impairments, seizures, gait disturbances, intelligence quotient (IQ) and adaptive function, as well as paternal and maternal age. Classification analyses using a non-parametric recursive partitioning method (random forests) were employed to define sets of phenotypic characteristics that best classify the CNV-defined groups. There was substantial variation in the classification accuracy of the two sets of genes. The best variables for classification were verbal IQ for the ASD/ID genes, paternal age at birth for the DBE genes and adaptive function for de novo CNVs. CNVs in the ASD/ID list were primarily associated with communication and language domains, whereas CNVs in DBE genes were related to broader manifestations of adaptive function. To our knowledge, this is the first study to examine the associations between sub-phenotypes and CNVs genome-wide in ASD. This work highlights the importance of examining the diverse sub-phenotypic manifestations of CNVs in ASD, including the specific features, comorbid conditions and clinical correlates of ASD that comprise underlying characteristics of the disorder.
Autism spectrum disorders (ASDs) are highly heritable and characterised by deficits in social interaction and communication, as well as restricted and repetitive behaviours. Although a number of highly penetrant ASD gene variants have been identified, there is growing evidence to support a causal role for combinatorial effects arising from the contributions of multiple loci. By examining synaptic and circadian neurological phenotypes resulting from the dosage variants of unique human:fly orthologues in Drosophila, we observe numerous synergistic interactions between pairs of informatically-identified candidate genes whose orthologues are jointly affected by large de novo copy number variants (CNVs). These CNVs were found in the genomes of individuals with autism, including a patient carrying a 22q11.2 deletion. We first demonstrate that dosage alterations of the unique Drosophila orthologues of candidate genes from de novo CNVs that harbour only a single candidate gene display neurological defects similar to those previously reported in Drosophila models of ASD-associated variants. We then considered pairwise dosage changes within the set of orthologues of candidate genes that were affected by the same single human de novo CNV. For three of four CNVs with complete orthologous relationships, we observed significant synergistic effects following the simultaneous dosage change of gene pairs drawn from a single CNV. The phenotypic variation observed at the Drosophila synapse that results from these interacting genetic variants supports a concordant phenotypic outcome across all interacting gene pairs following the direction of human gene copy number change. We observe both specificity and transitivity between interactors, both within and between CNV candidate gene sets, supporting shared and distinct genetic aetiologies. We then show that different interactions affect divergent synaptic processes, demonstrating distinct molecular aetiologies. Our study illustrates mechanisms through which synergistic effects resulting from large structural variation can contribute to human disease.
Autism spectrum disorders (ASDs), which are characterised by poor social interaction and repetitive behaviours, are in part caused by genetic variation. A number of genes that vary in copy number in ASD patients have been identified, many of which were known to function at the neuronal synapse. We theorised that in some cases the dosage change of multiple genes simultaneously, rather than singularly, may lead to faulty neuronal development, and contribute to ASD. To test this, we asked whether alterations in these candidate genes would cause neuronal synapse and sleep/rest changes using the fruit fly Drosophila, and validated this model using single-gene models. We considered the simultaneous change of pairs of genes that were jointly affected by a large human copy number variant (CNVs), which are structural changes in the genome. In three of four CNVs, mutations in subsets of genes synergistically interacted to cause neuronal changes comparable to the single gene candidates. We also observed that the changes in synapse size followed the direction of the human gene copy number change. Finally, we show that different interactions affect the development of the synapse through different mechanisms, allowing us to identify distinct molecular alterations that illuminate the etiological heterogeneity of ASD.
The identification of rare inherited and de novo copy number variations (CNVs) in human subjects has proven a productive approach to highlight risk genes for autism spectrum disorder (ASD). A variety of microarrays are available to detect CNVs, including single-nucleotide polymorphism (SNP) arrays and comparative genomic hybridization (CGH) arrays. Here, we examine a cohort of 696 unrelated ASD cases using a high-resolution one-million feature CGH microarray, the majority of which were previously genotyped with SNP arrays. Our objective was to discover new CNVs in ASD cases that were not detected by SNP microarray analysis and to delineate novel ASD risk loci via combined analysis of CGH and SNP array data sets on the ASD cohort and CGH data on an additional 1000 control samples. Of the 615 ASD cases analyzed on both SNP and CGH arrays, we found that 13,572 of 21,346 (64%) of the CNVs were exclusively detected by the CGH array. Several of the CGH-specific CNVs are rare in population frequency and impact previously reported ASD genes (e.g., NRXN1, GRM8, DPYD), as well as novel ASD candidate genes (e.g., CIB2, DAPP1, SAE1), and all were inherited except for a de novo CNV in the GPHN gene. A functional enrichment test of gene-sets in ASD cases over controls revealed nucleotide metabolism as a potential novel pathway involved in ASD, which includes several candidate genes for follow-up (e.g., DPYD, UPB1, UPP1, TYMP). Finally, this extensively phenotyped and genotyped ASD clinical cohort serves as an invaluable resource for the next step of genome sequencing for complete genetic variation detection.
rare variants; gene copy number; chromosomal abnormalities; cytogenetics; molecular pathways
Autism spectrum disorders (ASD) represent a group of neurodevelopmental disorders characterized by a core set of social-communicative and behavioral impairments. Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the brain, acting primarily via the GABA receptors (GABR). Multiple lines of evidence, including altered GABA and GABA receptor expression in autistic patients, indicate that the GABAergic system may be involved in the etiology of autism.
As copy number variations (CNVs), particularly rare and de novo CNVs, have now been implicated in ASD risk, we examined the GABA receptors and genes in related pathways for structural variation that may be associated with autism. We further extended our candidate gene set to include 19 genes and regions that had either been directly implicated in the autism literature or were directly related (via function or ancestry) to these primary candidates. For the high resolution CNV screen we employed custom-designed 244 k comparative genomic hybridization (CGH) arrays. Collectively, our probes spanned a total of 11 Mb of GABA-related and additional candidate regions with a density of approximately one probe every 200 nucleotides, allowing a theoretical resolution for detection of CNVs of approximately 1 kb or greater on average. One hundred and sixty-eight autism cases and 149 control individuals were screened for structural variants. Prioritized CNV events were confirmed using quantitative PCR, and confirmed loci were evaluated on an additional set of 170 cases and 170 control individuals that were not included in the original discovery set. Loci that remained interesting were subsequently screened via quantitative PCR on an additional set of 755 cases and 1,809 unaffected family members.
Results include rare deletions in autistic individuals at JAKMIP1, NRXN1, Neuroligin4Y, OXTR, and ABAT. Common insertion/deletion polymorphisms were detected at several loci, including GABBR2 and NRXN3. Overall, statistically significant enrichment in affected vs. unaffected individuals was observed for NRXN1 deletions.
These results provide additional support for the role of rare structural variation in ASD.
AUTISM; CGH; CNV; GABA; NRXN1
Autism spectrum disorders (ASDs) are a group of neurodevelopmental conditions with a demonstrated genetic etiology. Rare (<1% frequency) copy number variations (CNVs) account for a proportion of the genetic events involved, but the contribution of these events in non-European ASD populations has not been well studied. Here, we report on rare CNVs detected in a cohort of individuals with ASD of Han Chinese background.
DNA samples were obtained from 104 ASD probands and their parents who were recruited from Harbin, China. Samples were genotyped on the Affymetrix CytoScan HD platform. Rare CNVs were identified by comparing data with 873 technology-matched controls from Ontario and 1,235 additional population controls of Han Chinese ethnicity.
Of the probands, 8.6% had at least 1 de novo CNV (overlapping the GIGYF2, SPRY1, 16p13.3, 16p11.2, 17p13.3-17p13.2, DMD, and NAP1L6 genes/loci). Rare inherited CNVs affected other plausible neurodevelopmental candidate genes including GRID2, LINGO2, and SLC39A12. A 24-kb duplication was also identified at YWHAE, a gene previously implicated in ASD and other developmental disorders. This duplication is observed at a similar frequency in cases and in population controls and is likely a benign Asian-specific copy number polymorphism.
Our findings help define genomic features relevant to ASD in the Han Chinese and emphasize the importance of using ancestry-matched controls in medical genetic interpretations.
Autism spectrum disorder (ASD); Copy number variations (CNVs); Microarray diagnostic testing; Han Chinese
Copy number variations (CNVs) are a major cause of genetic disruption in the human genome with far more nucleotides being altered by duplications and deletions than by single nucleotide polymorphisms (SNPs). In the multifaceted etiology of autism spectrum disorders (ASDs), CNVs appear to contribute significantly to our understanding of the pathogenesis of this complex disease. A unique resource of 42 extended ASD families was genotyped for over 1 million SNPs to detect CNVs that may contribute to ASD susceptibility. Each family has at least one avuncular or cousin pair with ASD. Families were then evaluated for co-segregation of CNVs in ASD patients. We identified a total of five deletions and seven duplications in eleven families that co-segregated with ASD. Two of the CNVs overlap with regions on 7p21.3 and 15q24.1 that have been previously reported in ASD individuals and two additional CNVs on 3p26.3 and 12q24.32 occur near regions associated with schizophrenia. These findings provide further evidence for the involvement of ICA1 and NXPH1 on 7p21.3 in ASD susceptibility and highlight novel ASD candidates, including CHL1, FGFBP3 and POUF41. These studies highlight the power of using extended families for gene discovery in traits with a complex etiology.
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.
One genetic mechanism known to be associated with autism spectrum disorders (ASD) is chromosomal abnormalities. The identification of copy number variants (CNV) i.e. microdeletions and microduplications that are undetectable at the level of traditional cytogenetic analysis allows the potential association of submicroscopic chromosomal imbalances and human disease.
We performed array comparative genomic hybridization (aCGH) utilizing a 19K whole genome tiling path bacterial artificial chromosome (BAC) microarray on 397 unrelated subjects with autism spectrum disorder (ASD). Common CNV were excluded using a control group comprised of 372 individuals from the NIMH Genetics Initiative Control samples. Confirmation studies were performed on all remaining CNV using FISH (Fluorescence In Situ Hybridization), microsatellite analysis and/or quantitative PCR analysis.
A total of 51 CNV were confirmed in 46 ASD subjects. Three maternal interstitial duplications of 15q11-q13 known to be associated with ASD were identified. The other 48 CNV ranged in size from 189 kb to 5.5 Mb and contained from 0 to ~40 RefSeq genes. Seven CNV were de novo and 44 were inherited.
51 autism-specific CNV were identified in 46/397 ASD patients using a 19K BAC microarray for an overall rate of 11.6%. These microdeletions and microduplications cause gene dosage imbalance in 272 genes many of which could be considered as candidate genes for autism.
autism; array comparative genomic hybridization; microdeletions; microduplications
Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by deficits in social communication, absence or delay in language development, and stereotyped or repetitive behaviors. Genetic studies show that neurexin-neuroligin (NRXN-NLGN) pathway genes contribute susceptibility to ASD, which include cell adhesion molecules NLGN3, NLGN4 and scaffolding proteins SHANK2 and SHANK3. Neuroligin proteins play an important role in synaptic function and trans-synaptic signaling by interacting with presynaptic neurexins. Shank proteins are scaffolding molecules of excitatory synapses, which function as central organizers of the postsynaptic density. Sequence level mutations and structural variations in these genes have been identified in ASD cases, while few studies were performed in Chinese population. In this study, we examined the copy numbers of four genes NLGN4, NLGN3, SHANK2, and SHANK3 in 285 ASD cases using multiplex fluorescence competitive polymerase chain reaction (PCR). We also screened the regulatory region including the promoter region and 5′/3′ untranslated regions (UTR) and the entire coding region of NLGN4 in a cohort of 285 ASD patients and 384 controls by direct sequencing of genomic DNA using the Sanger method. DNA copy number calculation in four genes showed no deletion or duplication in our cases. No missense mutations in NLGN4 were identified in our cohort. Association analysis of 6 common SNPs in NLGN4 did not find significant difference between ASD cases and controls. These findings showed that these genes may not be major disease genes in Chinese ASD cases.
The autism spectrum disorders (ASDs) are a group of conditions characterized by impairments in reciprocal social interaction and communication, and the presence of restricted and repetitive behaviors1. Individuals with an ASD vary greatly in cognitive development, which can range from above average to intellectual disability (ID)2. While ASDs are known to be highly heritable (~90%)3, the underlying genetic determinants are still largely unknown. Here, we analyzed the genome-wide characteristics of rare (<1% frequency) copy number variation (CNV) in ASD using dense genotyping arrays. When comparing 996 ASD individuals of European ancestry to 1,287 matched controls, cases were found to carry a higher global burden of rare, genic CNVs (1.19 fold, P= 0.012), especially so for loci previously implicated in either ASD and/or intellectual disability (1.69 fold, P= 3.4×10−4). Among the CNVs, there were numerous de novo and inherited events, sometimes in combination in a given family, implicating many novel ASD genes like SHANK2, SYNGAP1, DLGAP2 and the X-linked DDX53-PTCHD1 locus. We also discovered an enrichment of CNVs disrupting functional gene-sets involved in cellular proliferation, projection and motility, and GTPase/Ras signaling. Our results reveal many new genetic and functional targets in ASD that may lead to final connected pathways.
Copy number variations (CNVs) can contribute to variable degrees of fitness and/or disease predisposition. Recent studies show that at least 1% of any given genome is copy number variable when compared to the human reference sequence assembly. Homozygous deletions (or CNV nulls) that are found in the normal population are of particular interest because they may serve to define non-essential genes in human biology.
In a genomic screen investigating CNV in Autism Spectrum Disorders (ASDs) we detected a heterozygous deletion on chromosome 10p12.1, spanning the Patched-domain containing 3 (PTCHD3) gene, at a frequency of ~1.4% (6/427). This finding seemed interesting, given recent discoveries on the role of another Patched-domain containing gene (PTCHD1) in ASD. Screening of another 177 ASD probands yielded two additional heterozygous deletions bringing the frequency to 1.3% (8/604). The deletion was found at a frequency of ~0.73% (27/3,695) in combined control population from North America and Northern Europe predominately of European ancestry. Screening of the human genome diversity panel (HGDP-CEPH) covering worldwide populations yielded deletions in 7/1,043 unrelated individuals and those detected were confined to individuals of European/Mediterranean/Middle Eastern ancestry. Breakpoint mapping yielded an identical 102,624 bp deletion in all cases and controls tested, suggesting a common ancestral event. Interestingly, this CNV occurs at a break of synteny between humans and mouse. Considering all data, however, no significant association of these rare PTCHD3 deletions with ASD was observed. Notwithstanding, our RNA expression studies detected PTCHD3 in several tissues, and a novel shorter isoform for PTCHD3 was characterized. Expression in transfected COS-7 cells showed PTCHD3 isoforms colocalize with calnexin in the endoplasmic reticulum. The presence of a patched (Ptc) domain suggested a role for PTCHD3 in various biological processes mediated through the Hedgehog (Hh) signaling pathway. However, further investigation yielded one individual harboring a homozygous deletion (PTCHD3 null) without ASD or any other overt abnormal phenotype. Exon sequencing of PTCHD3 in other individuals with deletions revealed compound point mutations also resulting in a null state.
Our data suggests that PTCHD3 may be a non-essential gene in some humans and characterization of this novel CNV at 10p12.1 will facilitate population and disease studies.
Recent array-based studies have detected a wealth of copy number variations (CNVs) in patients with autism spectrum disorders (ASD). Since CNVs also occur in healthy individuals, their contributions to the patient’s phenotype remain largely unclear. In a cohort of children with symptoms of ASD, diagnosis of the index patient using ADOS-G and ADI-R was performed, and the Social Responsiveness Scale (SRS) was administered to the index patients, both parents, and all available siblings. CNVs were identified using SNP arrays and confirmed by FISH or array CGH. To evaluate the clinical significance of CNVs, we analyzed three families with multiple affected children (multiplex) and six families with a single affected child (simplex) in which at least one child carried a CNV with a brain-transcribed gene. CNVs containing genes that participate in pathways previously implicated in ASD, such as the phosphoinositol signaling pathway (PIK3CA, GIRDIN), contactin-based networks of cell communication (CNTN6), and microcephalin (MCPH1) were found not to co-segregate with ASD phenotypes. In one family, a loss of CNTN5 co-segregated with disease. This indicates that most CNVs may by themselves not be sufficient to cause ASD, but still may contribute to the phenotype by additive or epistatic interactions with inherited (transmitted) mutations or non-genetic factors. Our study extends the scope of genome-wide CNV profiling beyond de novo CNVs in sporadic patients and may aid in uncovering missing heritability in genome-wide screening studies of complex psychiatric disorders.
Autism; Social Responsiveness Scale (SRS); SNP array-based CNV profiling; Gene prioritization; Phosphoinositol signaling; Contactin genes
Although autism spectrum disorder (ASD) shows a high degree of heritability, only a few mutated genes and mostly de novo copy number variations (CNVs) with a high phenotypic impact have as yet been identified. In families with multiple ASD patients, transmitted CNVs often do not appear to cosegregate with disease. Therefore, also transmitted single nucleotide variants which escape detection if genetic analyses were limited to CNVs may contribute to disease risk. In several studies of ASD patients, CNVs covering at least one gene of the contactin gene family were found. To determine whether there is evidence for a contribution of transmitted variants in contactin genes, a cohort of 67 ASD patients and a population-based reference of 117 healthy individuals, who were not related to the ASD families, were compared. In total, 1,648 SNPs, spanning 12.1 Mb of genomic DNA, were examined. After Bonferroni correction for multiple testing, the strongest signal was found for a SNP located within the CNTN5 gene (rs6590473 [G], p = 4.09 × 10-7; OR = 3.117; 95% CI = 1.603-6.151). In the ASD cohort, a combination of risk alleles of SNPs in CNTN6 (rs9878022 [A]; OR = 3.749) and in CNTNAP2 (rs7804520 [G]; OR = 2.437) was found more frequently than would be expected under random segregation, albeit this association was not statistically significant. The latter finding is consistent with a polygenic disease model in which multiple mutagenic mechanisms, operating concomitantly, elicit the ASD phenotype. Altogether, this study corroborates the possible involvement of contactins in ASD, which has been indicated by earlier studies of CNVs.
Autism; Candidate gene association study; Contactin gene family; CNTN5; CNTN6; CNTNAP2; Copy number variation
Reading and language skills have overlapping genetic bases, most of which are still unknown. Part of the missing heritability may be caused by copy number variants (CNVs).
In a dataset of children recruited for a history of reading disability (RD, also known as dyslexia) or attention deficit hyperactivity disorder (ADHD) and their siblings, we investigated the effects of CNVs on reading and language performance. First, we called CNVs with PennCNV using signal intensity data from Illumina OmniExpress arrays (~723,000 probes). Then, we computed the correlation between measures of CNV genomic burden and the first principal component (PC) score derived from several continuous reading and language traits, both before and after adjustment for performance IQ. Finally, we screened the genome, probe-by-probe, for association with the PC scores, through two complementary analyses: we tested a binary CNV state assigned for the location of each probe (i.e., CNV+ or CNV−), and we analyzed continuous probe intensity data using FamCNV.
No significant correlation was found between measures of CNV burden and PC scores, and no genome-wide significant associations were detected in probe-by-probe screening. Nominally significant associations were detected (p~10−2–10−3) within CNTN4 (contactin 4) and CTNNA3 (catenin alpha 3). These genes encode cell adhesion molecules with a likely role in neuronal development, and they have been previously implicated in autism and other neurodevelopmental disorders. A further, targeted assessment of candidate CNV regions revealed associations with the PC score (p~0.026–0.045) within CHRNA7 (cholinergic nicotinic receptor alpha 7), which encodes a ligand-gated ion channel and has also been implicated in neurodevelopmental conditions and language impairment. FamCNV analysis detected a region of association (p~10−2–10−4) within a frequent deletion ~6 kb downstream of ZNF737 (zinc finger protein 737, uncharacterized protein), which was also observed in the association analysis using CNV calls.
These data suggest that CNVs do not underlie a substantial proportion of variance in reading and language skills. Analysis of additional, larger datasets is warranted to further assess the potential effects that we found and to increase the power to detect CNV effects on reading and language.
Electronic supplementary material
The online version of this article (doi:10.1186/s11689-016-9147-8) contains supplementary material, which is available to authorized users.
Reading disability; Developmental dyslexia; Language; Reading; Copy number variants; Family-based GWAS; Meta-analysis; CLDRC
Genetic mutations in NLGN4X (neuroligin 4), including point mutations and copy number variants (CNVs), have been associated with susceptibility to autism spectrum disorders (ASDs). However, it is unclear how mutations in NLGN4X result in neurodevelopmental defects. Here, we used neural stem cells (NSCs) as in vitro models to explore the impacts of NLGN4X knockdown on neurodevelopment. Using two shRNAmir-based vectors targeting NLGN4X and one control shRNAmir vector, we modulated NLGN4X expression and differentiated these NSCs into mature neurons. We monitored the neurodevelopmental process at Weeks 0, 0.5, 1, 2, 4 and 6, based on morphological analysis and whole-genome gene expression profiling. At the cellular level, in NSCs with NLGN4X knockdown, we observed increasingly delayed neuronal development and compromised neurite formation, starting from Week 2 through Week 6 post differentiation. At the molecular level, we identified multiple pathways, such as neurogenesis, neuron differentiation and muscle development, which are increasingly disturbed in cells with NLGN4X knockdown. Notably, several postsynaptic genes, including DLG4, NLGN1 and NLGN3, also have decreased expression. Based on in vitro models, NLGN4X knockdown directly impacts neurodevelopmental process during the formation of neurons and their connections. Our functional genomics study highlights the utility of NSCs models in understanding the functional roles of CNVs in affecting neurodevelopment and conferring susceptibility to neurodevelopmental diseases.
There is strong evidence that rare copy number variants (CNVs) have a role in susceptibility to autism spectrum disorders (ASDs). Much research has focused on how CNVs mediate a phenotypic effect by altering gene expression levels. We investigated an alternative mechanism whereby CNVs combine the 5′ and 3′ ends of two genes, creating a ‘fusion gene'. Any resulting mRNA with an open reading frame could potentially alter the phenotype via a gain-of-function mechanism. We examined 2382 and 3096 rare CNVs from 996 individuals with ASD and 1287 controls, respectively, for potential to generate fusion transcripts. There was no increased burden in individuals with ASD; 122/996 cases harbored at least one rare CNV of this type, compared with 179/1287 controls (P=0.89). There was also no difference in the overall frequency distribution between cases and controls. We examined specific examples of such CNVs nominated by case–control analysis and a candidate approach. Accordingly, a duplication involving REEP1-POLR1A (found in 3/996 cases and 0/1287 controls) and a single occurrence CNV involving KIAA0319-TDP2 were tested. However, no fusion transcripts were detected by RT-PCR. Analysis of additional samples based on cell line availability resulted in validation of a MAPKAPK5-ACAD10 fusion transcript in two probands. However, this variant was present in controls at a similar rate and is unlikely to influence ASD susceptibility. In summary, although we find no evidence that fusion-gene generating CNVs lead to ASD susceptibility, discovery of a MAPKAPK5-ACAD10 transcript with an estimated frequency of ∼1/200 suggests that gain-of-function mechanisms should be considered in future CNVs studies.
CNV; MAPKAPK5; ACAD10; ALDH2; KIAA0319; dyslexia
Autism spectrum disorders (ASDs) are a heterogeneous group of neurodevelopmental disorders, including childhood autism, atypical autism, and Asperger syndrome, with an estimated prevalence of 1.0–2.5% in the general population. ASDs have a complex multifactorial etiology, with genetic causes being recognized in only 10–20% of cases. Recently, copy-number variants (CNVs) have been shown to contribute to over 10% of ASD cases. We have applied a custom-designed oligonucleotide array comparative genomic hybridization with an exonic coverage of over 1700 genes, including 221 genes known to cause autism and autism candidate genes, in a cohort of 145 patients with ASDs. The patients were classified according to ICD-10 standards and the Childhood Autism Rating Scale protocol into three groups consisting of 45 individuals with and 69 individuals without developmental delay/intellectual disability (DD/ID), and 31 patients, in whom DD/ID could not be excluded. In 12 patients, we have identified 16 copy-number changes, eight (5.5%) of which likely contribute to ASDs. In addition to known recurrent CNVs such as deletions 15q11.2 (BP1-BP2) and 3q13.31 (including DRD3 and ZBTB20), and duplications 15q13.3 and 16p13.11, our analysis revealed two novel genes clinically relevant for ASDs: ARHGAP24 (4q21.23q21.3) and SLC16A7 (12q14.1). Our results further confirm the diagnostic importance of array CGH in detection of CNVs in patients with ASDs and demonstrate that CNVs are an important cause of ASDs as a heterogeneous condition with a variety of contributory genes.
autism; copy-number variation; comparative genomic hybridization
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.
Copy number variations (CNVs) and DNA sequence alterations affecting specific neuronal genes are established risk factors for Autism Spectrum Disorder (ASD). In what is largely considered a genetic condition, so far, these mutations account for ~20% of individuals having an ASD diagnosis. However, non-coding genomic sequence also contains functional elements introducing additional disease risk loci for investigation.
We have performed genome-wide analyses and identified rare inherited CNVs affecting non-genic intervals in 41 of 1491 (3%) of ASD cases examined. Examples of such intergenic CNV regions include 16q21 and 2p16.3 near known ASD risk genes CDH8 and NRXN1 respectively, as well as novel loci contiguous with ZHX2, MOCS1, LRRC4C, SEMA3C, and other genes.
Rare variants in intergenic regions may implicate new risk loci and genes in ASD and also present useful data for comparison with coming whole genome sequence datasets.
Autism spectrum disorder; Copy number variation; Non-coding DNA
Infantile spasms (IS) is a specific type of epileptic encephalopathy associated with severe developmental disabilities. Genetic factors are strongly implicated in IS, however, the exact genetic defects remain unknown in the majority of cases. Rare mutations in a single gene or in copy number variants (CNVs) have been implicated in IS of children in Western countries. The objective of this study was to dissect the role of copy number variations in Chinese children with infantile spasms.
We used the Agilent Human Genome CGH microarray 180 K for genome-wide detection of CNVs. Real-time qPCR was used to validate the CNVs. We performed genomic and medical annotations for individual CNVs to determine the pathogenicity of CNVs related to IS.
We report herein the first genome-wide CNV analysis in children with IS, detecting a total of 14 CNVs in a cohort of 47 Chinese children with IS. Four CNVs (4/47 = 8.5%) (1q21.1 gain; 1q44, 2q31.1, and 17p13 loss) are considered to be pathogenic. The CNV loss at 17p13.3 contains PAFAH1B1 (LIS1), a causative gene for lissencephaly. Although the CNVs at 1q21.1, 1q44, and 2q23.1 have been previously implicated in a wide spectrum of clinical features including autism spectrum disorders (ASD) and generalized seizure, our study is the first report identifying them in individuals with a primary diagnosis of IS. The CNV loss in the 1q44 region contains HNRNPU, a strong candidate gene recently suggested in IS by the whole exome sequencing of children with IS. The CNV loss at 2q23.1 includes MBD5, a methyl-DNA binding protein that is a causative gene of ASD and a candidate gene for epileptic encephalopathy. We also report a distinct clinical presentation of IS, microcephaly, intellectual disability, and absent hallux in a case with the 2q23.1 deletion.
Our findings strongly support the role of CNVs in infantile spasms and expand the clinical spectrum associate with 2q23.1 deletion. In particular, our study implicates the HNRNPU and MBD5 genes in Chinese children with IS. Our study also supports that the molecular mechanisms of infantile spasms appear conserved among different ethnic backgrounds.
Infantile spasms; Copy number variants; Array CGH; Autism spectrum disorders; MBD5; HNRNPU
The purpose of the present study is to discover the extent to which distinct DSM disorders share large, highly recurrent copy number variants (CNVs) as susceptibility factors. We also seek to identify gene mechanisms common to groups of diagnoses and/or specific to a given diagnosis based on associations with CNVs.
Systematic review of 820 PubMed articles on autism spectrum disorder (ASD), intellectual disability (ID), schizophrenia, and epilepsy produced 54 CNVs associated with one or several disorders. Pathway analysis on genes implicated by CNVs in different groupings was conducted.
The majority of CNVs were found in ID with the other disorders somewhat subsumed, yet certain CNVs were associated with isolated or groups of disorders. Based on genes implicated by CNVs, ID encompassed 96.8% of genes in ASD, 92.8% of genes in schizophrenia, and 100.0% of genes in epilepsy. Pathway analysis revealed that synapse processes were enriched in ASD, ID, and schizophrenia. Disease-specific processes were identified in ID (actin cytoskeleton processes), schizophrenia (ubiquitin-related processes), and ASD (synaptic vesicle transport and exocytosis).
Intellectual disability may arise from the broadest range of genetic pathways, and specific subsets of these pathways appear relevant to other disorders or combinations of these disorders. It is clear that statistically significant CNVs across disorders of cognitive development are highly enriched for biological processes related to the synapse. There are also disorder-specific processes that may aid in understanding the distinct presentations and pathophysiology of these disorders.
autism; epilepsy; intellectual disability; schizophrenia; copy number variation
A substantial proportion of Autism Spectrum Disorder (ASD) risk resides in de novo germline and rare inherited genetic variation. In particular, rare copy number variation (CNV) contributes to ASD risk in up to 10% of ASD subjects. Despite the striking degree of genetic heterogeneity, case-control studies have detected specific burden of rare disruptive CNV for neuronal and neurodevelopmental pathways. Here, we used machine learning methods to classify ASD subjects and controls, based on rare CNV data and comprehensive gene annotations. We investigated performance of different methods and estimated the percentage of ASD subjects that could be reliably classified based on presumed etiologic CNV they carry.
We analyzed 1,892 Caucasian ASD subjects and 2,342 matched controls. Rare CNVs (frequency 1% or less) were detected using Illumina 1M and 1M-Duo BeadChips. Conditional Inference Forest (CF) typically performed as well as or better than other classification methods. We found a maximum AUC (area under the ROC curve) of 0.533 when considering all ASD subjects with rare genic CNVs, corresponding to 7.9% correctly classified ASD subjects and less than 3% incorrectly classified controls; performance was significantly higher when considering only subjects harboring de novo or pathogenic CNVs. We also found rare losses to be more predictive than gains and that curated neurally-relevant annotations (brain expression, synaptic components and neurodevelopmental phenotypes) outperform Gene Ontology and pathway-based annotations.
CF is an optimal classification approach for case-control rare CNV data and it can be used to prioritize subjects with variants potentially contributing to ASD risk not yet recognized. The neurally-relevant annotations used in this study could be successfully applied to rare CNV case-control data-sets for other neuropsychiatric disorders.
Copy number variation (CNV); Autism Spectrum Disorders (ASD); rare genetic variants; machine learning classification; Random Forest (RF)
Studies of copy number variation (CNV) have successfully characterized loci and molecular pathways involved in a range of neuropsychiatric conditions. We conducted an analysis of rare CNVs in Tourette Syndrome (TS) to identify novel risk regions and relevant molecular pathways, evaluate the burden of structural variation in cases versus controls, and to assess the overlap of identified variations with those implicated in other neuropsychiatric syndromes.
We conducted a case-control study of 460 individuals with TS, including 148 parent-child trios and 1131 controls. CNV analysis was undertaken using 370K to 1M probe arrays, and genome-wide genotyping data was used to match cases and controls for ancestry. Transmitted and de novo CNVs present in < 1% of the population were evaluated.
While there was no significant increase in the number of de novo or transmitted rare CNVs in cases versus controls, pathway analysis using multiple algorithms showed enrichment of genes within histamine receptor (H1R and H2R) signaling pathways (p=5.8×10-4-1.6×10-2) as well as “axon guidance”, “cell adhesion”, “nervous system development” and “synaptic structure and function” processes. Genes mapping within rare CNVs in TS showed significant overlap with those previously identified in autism spectrum disorders (ASD), but not intellectual disability or schizophrenia. Three large, likely-pathogenic, de novo events were identified, including one disrupting multiple gamma-Aminobutyric acid (GABA) receptor genes.
We identify further evidence supporting recent findings regarding the involvement of histaminergic and GABAergic mechanisms in the etiology of TS and show an overlap of rare CNVs in TS and ASD.
Tourette syndrome; copy number variation; CNV; histamine; GABA; autism