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.
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 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.
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.
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 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
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
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
Autism spectrum disorders (ASD) are a heterogeneous group of neurodevelopmental disorders with a complex inheritance pattern. While many rare variants in synaptic proteins have been identified in patients with ASD, little is known about their effects at the synapse and their interactions with other genetic variations. Here, following the discovery of two de novo SHANK2 deletions by the Autism Genome Project, we identified a novel 421 kb de novo SHANK2 deletion in a patient with autism. We then sequenced SHANK2 in 455 patients with ASD and 431 controls and integrated these results with those reported by Berkel et al. 2010 (n = 396 patients and n = 659 controls). We observed a significant enrichment of variants affecting conserved amino acids in 29 of 851 (3.4%) patients and in 16 of 1,090 (1.5%) controls (P = 0.004, OR = 2.37, 95% CI = 1.23–4.70). In neuronal cell cultures, the variants identified in patients were associated with a reduced synaptic density at dendrites compared to the variants only detected in controls (P = 0.0013). Interestingly, the three patients with de novo SHANK2 deletions also carried inherited CNVs at 15q11–q13 previously associated with neuropsychiatric disorders. In two cases, the nicotinic receptor CHRNA7 was duplicated and in one case the synaptic translation repressor CYFIP1 was deleted. These results strengthen the role of synaptic gene dysfunction in ASD but also highlight the presence of putative modifier genes, which is in keeping with the “multiple hit model” for ASD. A better knowledge of these genetic interactions will be necessary to understand the complex inheritance pattern of ASD.
Autism spectrum disorders (ASD) are a heterogeneous group of neurodevelopmental disorders with a complex inheritance pattern. While mutations in several genes have been identified in patients with ASD, little is known about their effects on neuronal function and their interaction with other genetic variations. Using a combination of genetic and functional approaches, we identified novel SHANK2 mutations including a de novo loss of one copy of the SHANK2 gene in a patient with autism and several mutations observed in patients that reduced neuronal cell contacts in vitro. Further genomic analysis of three patients carrying de novo SHANK2 deletions identified additional rare genomic imbalances previously associated with neuropsychiatric disorders. Taken together, these results strengthen the role of synaptic gene dysfunction in ASD but also highlight the presence of putative modifier genes, which is in keeping with the “multiple hit model” for ASD. A better knowledge of these genetic interactions will be necessary to understand the complex inheritance pattern of ASD.
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
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
The 15q24 microdeletion syndrome has been recently described as a recurrent, submicroscopic genomic imbalance found in individuals with intellectual disability, typical facial appearance, hypotonia, and digital and genital abnormalities. Gene dosage abnormalities, including copy number variations (CNVs), have been identified in a significant fraction of individuals with autism spectrum disorders (ASDs). In this study we surveyed two ASD cohorts for 15q24 abnormalities to assess the frequency of genomic imbalances in this interval.
We screened 173 unrelated subjects with ASD from the Central Valley of Costa Rica and 1336 subjects with ASD from 785 independent families registered with the Autism Genetic Resource Exchange (AGRE) for CNVs across 15q24 using oligonucleotide arrays. Rearrangements were confirmed by array comparative genomic hybridization and quantitative PCR.
Among the patients from Costa Rica, an atypical de novo deletion of 3.06 Mb in 15q23-q24.1 was detected in a boy with autism sharing many features with the other 13 subjects with the 15q24 microdeletion syndrome described to date. He exhibited intellectual disability, constant smiling, characteristic facial features (high anterior hairline, broad medial eyebrows, epicanthal folds, hypertelorism, full lower lip and protuberant, posteriorly rotated ears), single palmar crease, toe syndactyly and congenital nystagmus. The deletion breakpoints are atypical and lie outside previously characterized low copy repeats (69,838-72,897 Mb). Genotyping data revealed that the deletion had occurred in the paternal chromosome. Among the AGRE families, no large 15q24 deletions were observed.
From the current and previous studies, deletions in the 15q24 region represent rare causes of ASDs with an estimated frequency of 0.1 to 0.2% in individuals ascertained for ASDs, although the proportion might be higher in sporadic cases. These rates compare with a frequency of about 0.3% in patients ascertained for unexplained intellectual disability and congenital anomalies. This atypical deletion reduces the minimal interval for the syndrome from 1.75 Mb to 766 kb, implicating a reduced number of genes (15 versus 38). Sequencing of genes in the 15q24 interval in large ASD and intellectual disability samples may identify mutations of etiologic importance in the development of these disorders.
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
Autism spectrum disorder (ASD) is reported in 30 to 60% of patients with tuberous sclerosis complex (TSC) but shared genetic mechanisms that exist between TSC-associated ASD and idiopathic ASD have yet to be determined. Through the small G-protein Rheb, the TSC proteins, hamartin and tuberin, negatively regulate mammalian target of rapamycin complex 1 (mTORC1) signaling. It is well established that mTORC1 plays a pivotal role in neuronal translation and connectivity, so dysregulation of mTORC1 signaling could be a common feature in many ASDs. Pam, an E3 ubiquitin ligase, binds to TSC proteins and regulates mTORC1 signaling in the CNS, and the FBXO45-Pam ubiquitin ligase complex plays an essential role in neurodevelopment by regulating synapse formation and growth. Since mounting evidence has established autism as a disorder of the synapses, we tested whether rare genetic variants in TSC1, TSC2, MYCBP2, RHEB and FBXO45, genes that regulate mTORC1 signaling and/or play a role in synapse development and function, contribute to the pathogenesis of idiopathic ASD.
Exons and splice junctions of TSC1, TSC2, MYCBP2, RHEB and FBXO45 were resequenced for 300 ASD trios from the Simons Simplex Collection (SSC) using a pooled PCR amplification and next-generation sequencing strategy, targeted to the discovery of deleterious coding variation. These detected, potentially functional, variants were confirmed by Sanger sequencing of the individual samples comprising the pools in which they were identified.
We identified a total of 23 missense variants in MYCBP2, TSC1 and TSC2. These variants exhibited a near equal distribution between the proband and parental pools, with no statistical excess in ASD cases (P > 0.05). All proband variants were inherited. No putative deleterious variants were confirmed in RHEB and FBXO45. Three intronic variants, identified as potential splice defects in MYCBP2 did not show aberrant splicing upon RNA assay. Overall, we did not find an over-representation of ASD causal variants in the genes studied to support them as contributors to autism susceptibility.
We did not observe an enrichment of rare functional variants in TSC1 and TSC2 genes in our sample set of 300 trios.
Autism spectrum disorder; Tuberous sclerosis complex; Mammalian target of rapamycin; Next-generation sequencing; Rare variants
Obesity is an increasingly common disorder that predisposes to several medical conditions, including type 2 diabetes. We investigated whether large and rare copy-number variations (CNVs) differentiate moderate to extreme obesity from never-overweight control subjects.
RESEARCH DESIGN AND METHODS
Using single nucleotide polymorphism (SNP) arrays, we performed a genome-wide CNV survey on 430 obese case subjects (BMI >35 kg/m2) and 379 never-overweight control subjects (BMI <25 kg/m2). All subjects were of European ancestry and were genotyped on the Illumina HumanHap550 arrays with ∼550,000 SNP markers. The CNV calls were generated by PennCNV software.
CNVs >1 Mb were found to be overrepresented in case versus control subjects (odds ratio [OR] = 1.5 [95% CI 0.5–5]), and CNVs >2 Mb were present in 1.3% of the case subjects but were absent in control subjects (OR = infinity [95% CI 1.2–infinity]). When focusing on rare deletions that disrupt genes, even more pronounced effect sizes are observed (OR = 2.7 [95% CI 0.5–27.1] for CNVs >1 Mb). Interestingly, obese case subjects who carry these large CNVs have moderately high BMI and do not appear to be extreme cases. Several CNVs disrupt known candidate genes for obesity, such as a 3.3-Mb deletion disrupting NAP1L5 and a 2.1-Mb deletion disrupting UCP1 and IL15.
Our results suggest that large CNVs, especially rare deletions, confer risk of obesity in patients with moderate obesity and that genes impacted by large CNVs represent intriguing candidates for obesity that warrant further study.
Autism spectrum disorders (ASDs) are a group of highly heritable neurodevelopmental disorders which are characteristically comprised of impairments in social interaction, communication and restricted interests/behaviours. Several cell adhesion transmembrane leucine-rich repeat (LRR) proteins are highly expressed in the nervous system and are thought to be key regulators of its development. Here we present an association study analysing the roles of four promising candidate genes - LRRTM1 (2p), LRRTM3 (10q), LRRN1 (3p) and LRRN3 (7q) - in order to identify common genetic risk factors underlying ASDs.
In order to gain a better understanding of how the genetic variation within these four gene regions may influence susceptibility to ASDs, a family-based association study was undertaken in 661 families of European ancestry selected from four different ASD cohorts. In addition, a case-control study was undertaken across the four LRR genes, using logistic regression in probands with ASD of each population against 295 ECACC controls.
Significant results were found for LRRN3 and LRRTM3 (P < 0.005), using both single locus and haplotype approaches. These results were further supported by a case-control analysis, which also highlighted additional SNPs in LRRTM3.
Overall, our findings implicate the neuronal leucine-rich genes LRRN3 and LRRTM3 in ASD susceptibility.
Autism spectrum disorders (ASDs) comprise a distinct entity of neurodevelopmental disorders with a strong genetic component. Despite the identification of several candidate genes and causative genomic copy number variations (CNVs), the majority of ASD cases still remain unresolved. We have applied microarray-based comparative genomic hybridization (array-CGH) using Agilent 400K custom array in the first Cyprus population screening for identification of ASD-associated CNVs. A cohort of 50 ASD patients (G1), their parents (G2), 50 ethnically matched normal controls (G3), and 80 normal individuals having children with various developmental and neurological conditions (G4) were tested. As a result, 14 patients were found to carry 20 potentially causative aberrations, two of which were de novo. Comparison of the four population groups revealed an increased rate of rare disease-associated variants in normal parents of children with autism. The above data provided additional evidence, supporting the complexity of ASD aetiology in comparison to other developmental disorders involving cognitive impairment. Furthermore, we have demonstrated the rationale of a more targeted approach combining accurate clinical description with high-resolution population-oriented genomic screening for defining the role of CNVs in autism and identifying meaningful associations on the molecular level.
We have recently identified the nuclear hormone receptor RORA (retinoic acid-related orphan receptor-alpha) as a novel candidate gene for autism spectrum disorder (ASD). Our independent cohort studies have consistently demonstrated the reduction of RORA transcript and/or protein levels in blood-derived lymphoblasts as well as in the postmortem prefrontal cortex and cerebellum of individuals with ASD. Moreover, we have also shown that RORA has the potential to be under negative and positive regulation by androgen and estrogen, respectively, suggesting the possibility that RORA may contribute to the male bias of ASD. However, little is known about transcriptional targets of this nuclear receptor, particularly in humans.
Here we identify transcriptional targets of RORA in human neuronal cells on a genome-wide level using chromatin immunoprecipitation (ChIP) with an anti-RORA antibody followed by whole-genome promoter array (chip) analysis. Selected potential targets of RORA were then validated by an independent ChIP followed by quantitative PCR analysis. To further demonstrate that reduced RORA expression results in reduced transcription of RORA targets, we determined the expression levels of the selected transcriptional targets in RORA-deficient human neuronal cells, as well as in postmortem brain tissues from individuals with ASD who exhibit reduced RORA expression.
The ChIP-on-chip analysis reveals that RORA1, a major isoform of RORA protein in human brain, can be recruited to as many as 2,764 genomic locations corresponding to promoter regions of 2,544 genes across the human genome. Gene ontology analysis of this dataset of genes that are potentially directly regulated by RORA1 reveals statistically significant enrichment in biological functions negatively impacted in individuals with ASD, including neuronal differentiation, adhesion and survival, synaptogenesis, synaptic transmission and plasticity, and axonogenesis, as well as higher level functions such as development of the cortex and cerebellum, cognition, memory, and spatial learning. Independent ChIP-quantitative PCR analyses confirm binding of RORA1 to promoter regions of selected ASD-associated genes, including A2BP1, CYP19A1, ITPR1, NLGN1, and NTRK2, whose expression levels (in addition to HSD17B10) are also decreased in RORA1-repressed human neuronal cells and in prefrontal cortex tissues from individuals with ASD.
Findings from this study indicate that RORA transcriptionally regulates A2BP1, CYP19A1, HSD17B10, ITPR1, NLGN1, and NTRK2, and strongly suggest that reduction of this sex hormone-sensitive nuclear receptor in the brain causes dysregulated expression of these ASD-relevant genes as well as their associated pathways and functions which, in turn, may contribute to the underlying pathobiology of ASD.
RORA; Autism; Nuclear hormone receptor; Transcriptional targets; Chromatin immunoprecipitation; Promoter microarray
Autism Spectrum Disorders (ASD) are highly heritable and characterised by impairments in social interaction and communication, and restricted and repetitive behaviours. Considering four sets of de novo copy number variants (CNVs) identified in 181 individuals with autism and exploiting mouse functional genomics and known protein-protein interactions, we identified a large and significantly interconnected interaction network. This network contains 187 genes affected by CNVs drawn from 45% of the patients we considered and 22 genes previously implicated in ASD, of which 192 form a single interconnected cluster. On average, those patients with copy number changed genes from this network possess changes in 3 network genes, suggesting that epistasis mediated through the network is extensive. Correspondingly, genes that are highly connected within the network, and thus whose copy number change is predicted by the network to be more phenotypically consequential, are significantly enriched among patients that possess only a single ASD-associated network copy number changed gene (p = 0.002). Strikingly, deleted or disrupted genes from the network are significantly enriched in GO-annotated positive regulators (2.3-fold enrichment, corrected p = 2×10−5), whereas duplicated genes are significantly enriched in GO-annotated negative regulators (2.2-fold enrichment, corrected p = 0.005). The direction of copy change is highly informative in the context of the network, providing the means through which perturbations arising from distinct deletions or duplications can yield a common outcome. These findings reveal an extensive ASD-associated molecular network, whose topology indicates ASD-relevant mutational deleteriousness and that mechanistically details how convergent aetiologies can result extensively from CNVs affecting pathways causally implicated in ASD.
Autism Spectrum Disorders (ASD) are characterised by impairments in social interaction and communication, and restricted and repetitive behaviours. ASD are highly heritable and many different stretches of DNA have been found to be duplicated or deleted in individuals with ASD. We found that an unusually high number of genes affected by these DNA deletions/duplications are associated with the functioning of synaptic transmission between nerve cells. The proteins made by many of these genes are known to interact with each other and, together with proteins from other deleted/duplicated genes, form a large interlinked biological network. This network was affected by almost 50% of the deletions/duplications in the ASD patients considered. Many individual ASD patients had deletions or duplications of multiple genes within this network, but for those patients with just a single gene from the network changed, that single gene appeared to play an important role. Furthermore, the network predicts that the effects arising from the genes in the deletions are similar to the effects arising from the genes in the duplications. Thus, the way that this ASD-associated network is wired together contributes to the understanding of the impact of these DNA deletions and duplications.