Contribution of SHANK3 mutations to autism spectrum disorder

Mutations in SHANK3, which encodes a synaptic scaffolding protein, have been described in subjects with an autism spectrum disorder (ASD). To assess the quantitative contribution of SHANK3 to the pathogenesis of autism, we determined the frequency of DNA sequence and copy-number variants in this gene in 400 ASD-affected subjects ascertained in Canada. One de novo mutation and two gene deletions were discovered, indicating a contribution of 0.75% in this cohort. One additional SHANK3 deletion was characterized in two ASD-affected siblings from another collection, which brings the total number of published mutations in unrelated ASD-affected families to seven. The combined data provide support that haploinsufficiency of SHANK3 can cause a monogenic form of autism in sufficient frequency to warrant consideration in clinical diagnostic testing.     

SHANK1 Deletions in Males with Autism Spectrum Disorder

Recent studies have highlighted the involvement of rare (<1% frequency) copy-number variations and point mutations in the genetic etiology of autism spectrum disorder (ASD); these variants particularly affect genes involved in the neuronal synaptic complex. The SHANK gene family consists of three members (SHANK1, SHANK2, and SHANK3), which encode scaffolding proteins required for the proper formation and function of neuronal synapses. Although SHANK2 and SHANK3 mutations have been implicated in ASD and intellectual disability, the involvement of SHANK1 is unknown. Here, we assess microarray data from 1,158 Canadian and 456 European individuals with ASD to discover microdeletions at the SHANK1 locus on chromosome 19. We identify a hemizygous SHANK1 deletion that segregates in a four-generation family in which male carriers--but not female carriers--have ASD with higher functioning. A de novo SHANK1 deletion was also detected in an unrelated male individual with ASD with higher functioning, and no equivalent SHANK1 mutations were found in >15,000 controls (p = 0.009). The discovery of apparent reduced penetrance of ASD in females bearing inherited autosomal SHANK1 deletions provides a possible contributory model for the male gender bias in autism. The data are also informative for clinical-genetics interpretations of both inherited and sporadic forms of ASD involving SHANK1.     

Meta-analysis of SHANK Mutations in Autism Spectrum Disorders: A Gradient of Severity in Cognitive Impairments

SHANK genes code for scaffold proteins located at the post-synaptic density of glutamatergic synapses. In neurons, SHANK2 and SHANK3 have a positive effect on the induction and maturation of dendritic spines, whereas SHANK1 induces the enlargement of spine heads. Mutations in SHANK genes have been associated with autism spectrum disorders (ASD), but their prevalence and clinical relevance remain to be determined. Here, we performed a new screen and a meta-analysis of SHANK copy-number and coding-sequence variants in ASD. Copy-number variants were analyzed in 5,657 patients and 19,163 controls, coding-sequence variants were ascertained in 760 to 2,147 patients and 492 to 1,090 controls (depending on the gene), and, individuals carrying de novo or truncating SHANK mutations underwent an extensive clinical investigation. Copy-number variants and truncating mutations in SHANK genes were present in ∼1% of patients with ASD: mutations in SHANK1 were rare (0.04%) and present in males with normal IQ and autism; mutations in SHANK2 were present in 0.17% of patients with ASD and mild intellectual disability; mutations in SHANK3 were present in 0.69% of patients with ASD and up to 2.12% of the cases with moderate to profound intellectual disability. In summary, mutations of the SHANK genes were detected in the whole spectrum of autism with a gradient of severity in cognitive impairment. Given the rare frequency of SHANK1 and SHANK2 deleterious mutations, the clinical relevance of these genes remains to be ascertained. In contrast, the frequency and the penetrance of SHANK3 mutations in individuals with ASD and intellectual disability—more than 1 in 50—warrant its consideration for mutation screening in clinical practice.     

Behavioral phenotypes in males with XYY and possible role of increased NLGN4Y expression in autism features

The male sex chromosome disorder, 47,XYY syndrome (XYY), is associated with increased risk for social‐emotional difficulties, attention‐deficit hyperactivity disorder (ADHD) and autism spectrum disorder (ASD). We hypothesize that increased Y chromosome gene copy number in XYY leads to overexpression of Y‐linked genes related to brain development and function, thereby increasing risk for these phenotypes. We measured expression in blood of two Y genes NLGN4Y and RPS4Y in 26 boys with XYY and 11 male controls and evaluated whether NLGN4Y expression correlates with anxiety, ADHD, depression and autistic behaviors (from questionnaires) in boys with XYY. The XYY cohort had increased risk of ASD behaviors on the social responsiveness scale (SRS) and increased attention deficits on the Conners' DSM‐IV inattention and hyperactive scales. In contrast, there was no increase in reported symptoms of anxiety or depression by the XYY group. Peripheral expression of two Y genes in boys with XYY vs. typically developing controls was increased twofold in the XYY group. Results from the SRS total and autistic mannerisms scales, but not from the attention, anxiety or depression measures, correlated with peripheral expression of NLGN4Y in boys with XYY. Males with XYY have social phenotypes that include increased risk for autism‐related behaviors and ADHD. Expression of NLGN4Y, a gene that may be involved in synaptic function, is increased in boys with XYY, and the level of expression correlates with overall social responsiveness and autism symptoms. Thus, further investigation of NLGN4Y as a plausible ASD risk gene in XYY is warranted.     

Variations analysis of NLGN3 and NLGN4X gene in Chinese autism patients

Autism is a neurodevelopmental disorder clinically characterized by impairment of social interaction, deficits in verbal communication, as well as stereotypic and repetitive behaviors. Several studies have implicated that abnormal synaptogenesis was involved in the incidence of autism. Neuroligins are postsynaptic cell adhesion molecules and interacted with neurexins to regulate the fine balance between excitation and inhibition of synapses. Recently, mutation analysis, cellular and mice models hinted neuroligin mutations probably affected synapse maturation and function. In this study, four missense variations [p.G426S (NLGN3), p.G84R (NLGN4X), p.Q162 K (NLGN4X) and p.A283T (NLGN4X)] in four different unrelated patients have been identified by PCR and direct sequencing. These four missense variations were absent in the 453 controls and have not been reported in 1000 Genomes Project. Bioinformatic analysis of the four missense variations revealed that p.G84R and p.A283T were “Probably Damaging”. The variations may cause abnormal synaptic homeostasis and therefore trigger the patients more predisposed to autism. By case–control analysis, we identified the common SNPs (rs3747333 and rs3747334) in the NLGN4X gene significantly associated with risk for autism [p = 5.09E-005; OR 4.685 (95 % CI 2.073–10.592)]. Our data provided a further evidence for the involvement of NLGN3 and NLGN4X gene in the pathogenesis of autism in Chinese population.     

SHANK1 and autism spectrum disorders

Autism spectrum disorders(ASD) are highly heterogeneous pediatric developmental disorders with estimated heritability more than 70%. Although the genetic factors in ASD are mainly unknown, a large number of gene mutations have been found, especially in genes involved in neurogenesis. The Neurexin-Neuroligin-Shank(NRXN-NLGN-SHANK) pathway plays a key role in the formation, maturation and maintenance of synapses, consistent with the hypothesis of neurodevelopmental abnormality in ASD. Presynaptic NRXNs interact with postsynaptic NLGNs in excitatory glutamatergic synapses. SHANK proteins function as core components of the postsynaptic density(PSD) by interacting with multiple proteins. Recently, deletions and point mutations of the SHANK1 gene have been detected in ASD individuals, indicating the involvement of SHANK1 in ASD. This review focuses on the function of SHANK1 protein, Shank1 mouse models, and the molecular genetics of the SHANK1 gene in human ASD.     

A commonly carried genetic variant,rs9616915, in SHANK3 gene is associated with a reduced risk of autism spectrum disorder: replication in a Chinese population

Autism spectrum disorder (ASD) is one of neurodevelopmental disorders with highly heritability. Recently, abnormality at the synapse is found to be important etiology of ASD. SHANK3 gene is suggested as a strong candidate gene for the pathogenesis of ASD, because it is essential for normally synaptic structure and function. We performed a case–control study to identify association between rs9616915 of the SHANK3 gene and ASD in a Chinese population. Genomic DNA was extracted from oral swabs samples of 212 patients and 636 controls and the SNP genotypes were determined by a polymerase chain reaction–restriction fragment length polymerase assay. Significant difference in genotype distribution of rs9616915 was observed between cases and controls by Pearson’s χ ² test (χ ² = 6.92, P = 0.031). Genetic analysis of heterozygous model, dominant model and additive model showed an association of the C allele of the rs9616915 with ASD (e.g., additive model, OR 0.582, 95 % CI 0.359–0.942, P = 0.028). In conclusion, our results suggested that this commonly genetic variant in SHANK3 gene strikingly decreased the risk of ASD in China.     

Expression of SHANK3 in the Temporal Neocortex of Patients with Intractable Temporal Epilepsy and Epilepsy Rat Models

SH3 and multiple ankyrin (ANK) repeat domain 3 (SHANK3) is a synaptic scaffolding protein enriched in the postsynaptic density of excitatory synapses. SHANK3 plays an important role in the formation and maturation of excitatory synapses. In the brain, SHANK3 directly or indirectly interacts with various synaptic molecules including N-methyl-D-aspartate receptor, the metabotropic glutamate receptor (mGluR), and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor. Previous studies have shown that Autism spectrum disorder is a result of mutations of the main SHANK3 isoforms, which may be due to deficit in excitatory synaptic transmission and plasticity. Recently, accumulating evidence has demonstrated that overexpression of SHANK3 could induce seizures in vivo. However, little is known about the role of SHANK3 in refractory temporal lobe epilepsy (TLE). Therefore, we investigated the expression pattern of SHANK3 in patients with intractable temporal lobe epilepsy and in pilocarpine-induced models of epilepsy. Immunofluorescence, immunohistochemistry, and western blot analysis were used to locate and determine the expression of SHANK3 in the temporal neocortex of patients with epilepsy, and in the hippocampus and temporal lobe cortex of rats in a pilocarpine-induced epilepsy model. Double-labeled immunofluorescence showed that SHANK3 was mainly expressed in neurons. Western blot analysis confirmed that SHANK3 expression was increased in the neocortex of TLE patients and rats. These results indicate that SHANK3 participates in the pathology of epilepsy.     

Genetic and functional analyses of SHANK2 mutations suggest a multiple hit model of autism spectrum disorders

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.     

Functional phenotypic rescue of Caenorhabditis elegans neuroligin-deficient mutants by the human and rat NLGN1 genes

Neuroligins are cell adhesion proteins that interact with neurexins at the synapse. This interaction may contribute to differentiation, plasticity and specificity of synapses. In humans, single mutations in neuroligin encoding genes lead to autism spectrum disorder and/or mental retardation. Caenorhabditis elegans mutants deficient in nlg-1, an orthologue of human neuroligin genes, have defects in different behaviors. Here we show that the expression of human NLGN1 or rat Nlgn1 cDNAs in C. elegans nlg-1 mutants rescues the fructose osmotic strength avoidance and gentle touch response phenotypes. Two specific point mutations in NLGN3 and NLGN4 genes, involved in autistic spectrum disorder, were further characterized in this experimental system. The R451C allele described in NLGN3, was analyzed with both human NLGN1 (R453C) and worm NLG-1 (R437C) proteins, and both were not functional in rescuing the osmotic avoidance behavior and the gentle touch response phenotype. The D396X allele described in NLGN4, which produces a truncated protein, was studied with human NLGN1 (D432X) and they did not rescue any of the behavioral phenotypes analyzed. In addition, RNAi feeding experiments measuring gentle touch response in wild type strain and worms expressing SID-1 in neurons (which increases the response to dsRNA), both fed with bacteria expressing dsRNA for nlg-1, provided evidence for a postsynaptic in vivo function of neuroligins both in muscle cells and neurons, equivalent to that proposed in mammals. This finding was further confirmed generating transgenic nlg-1 deficient mutants expressing NLG-1 under pan-neuronal (nrx-1) or pan-muscular (myo-3) specific promoters. All these results suggest that the nematode could be used as an in vivo model for studying particular synaptic mechanisms with proteins orthologues of humans involved in pervasive developmental disorders.     

Whole Genome Sequencing Reveals a De Novo SHANK3 Mutation in Familial Autism Spectrum Disorder

IntroductionClinical genomics promise to be especially suitable for the study of etiologically heterogeneous conditions such as Autism Spectrum Disorder (ASD). Here we present three siblings with ASD where we evaluated the usefulness of Whole Genome Sequencing (WGS) for the diagnostic approach to ASD.MethodsWe identified a family segregating ASD in three siblings with an unidentified cause. We performed WGS in the three probands and used a state-of-the-art comprehensive bioinformatic analysis pipeline and prioritized the identified variants located in genes likely to be related to ASD. We validated the finding by Sanger sequencing in the probands and their parents.ResultsThree male siblings presented a syndrome characterized by severe intellectual disability, absence of language, autism spectrum symptoms and epilepsy with negative family history for mental retardation, language disorders, ASD or other psychiatric disorders. We found germline mosaicism for a heterozygous deletion of a cytosine in the exon 21 of the SHANK3 gene, resulting in a missense sequence of 5 codons followed by a premature stop codon ({"type":"entrez-nucleotide","attrs":{"text":"NM_033517","term_id":"380748962","term_text":"NM_033517"}}NM_033517:c.3259_3259delC, p.Ser1088Profs*6).ConclusionsWe reported an infrequent form of familial ASD where WGS proved useful in the clinic. We identified a mutation in SHANK3 that underscores its relevance in Autism Spectrum Disorder.     

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

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

Learning and reaction times in mouse touchscreen tests are differentially impacted by mutations in genes encoding postsynaptic interacting proteins SYNGAP1, NLGN3, DLGAP1, DLGAP2 and SHANK2

The postsynaptic terminal of vertebrate excitatory synapses contains a highly conserved multiprotein complex that comprises neurotransmitter receptors, cell-adhesion molecules, scaffold proteins and enzymes, which are essential for brain signalling and plasticity underlying behaviour. Increasingly, mutations in genes that encode postsynaptic proteins belonging to the PSD-95 protein complex, continue to be identified in neurodevelopmental disorders (NDDs) such as autism spectrum disorder, intellectual disability and epilepsy. These disorders are highly heterogeneous, sharing genetic aetiology and comorbid cognitive and behavioural symptoms. Here, by using genetically engineered mice and innovative touchscreen-based cognitive testing, we sought to investigate whether loss-of-function mutations in genes encoding key interactors of the PSD-95 protein complex display shared phenotypes in associative learning, updating of learned associations and reaction times. Our genetic dissection of mice with loss-of-function mutations in Syngap1, Nlgn3, Dlgap1, Dlgap2 and Shank2 showed that distinct components of the PSD-95 protein complex differentially regulate learning, cognitive flexibility and reaction times in cognitive processing. These data provide insights for understanding how human mutations in these genes lead to the manifestation of diverse and complex phenotypes in NDDs.     

Synaptic Dysfunction in Neurodevelopmental Disorders Associated with Autism and Intellectual Disabilities

The discovery of the genetic causes of syndromic autism spectrum disorders and intellectual disabilities has greatly informed our understanding of the molecular pathways critical for normal synaptic function. The top-down approaches using human phenotypes and genetics helped identify causative genes and uncovered the broad spectrum of neuropsychiatric features that can result from various mutations in the same gene. Importantly, the human studies unveiled the exquisite sensitivity of cognitive function to precise levels of many diverse proteins. Bottom-up approaches applying molecular, biochemical, and neurophysiological studies to genetic models of these disorders revealed unsuspected pathogenic mechanisms and identified potential therapeutic targets. Moreover, studies in model organisms showed that symptoms of these devastating disorders can be reversed, which brings hope that affected individuals might benefit from interventions even after symptoms set in. Scientists predict that insights gained from studying these rare syndromic disorders will have an impact on the more common nonsyndromic autism and mild cognitive deficits.It is estimated that ∼1% of the human population has an autism spectrum disorder (ASD). ASD has widely varied behavioral manifestations, severity, and comorbid conditions (hence the term “spectrum”), but those diagnosed with autism are characterized by impaired communication and reciprocal social interactions, and restricted and repetitive patterns of activities and interests (Baird et al. 2006). Approximately 70% of those diagnosed with autism also have intellectual disability (ID), and 25% have a seizure disorder (Tuchman and Rapin 2002). There is a strong genetic basis for autism, but the risk architecture is highly heterogeneous, and a large number of genes have been implicated (Abrahams and Geschwind 2008). This daunting phenotypic and etiologic complexity, shared by other major psychiatric illnesses, has slowed progress toward developing new therapies.However, autism researchers are optimistic that the possibility of substantial progress may soon be realized (Krueger and Bear 2011). First, the genes have been discovered for numerous syndromic disorders that prominently feature ASD and ID. Second, these gene mutations have been reproduced in animal models that allow detailed examination of the underlying brain pathophysiology. Third, animal research has converged on altered synaptic function as a likely basis for impaired cognition and possibly ASD. Fourth, insights gained on how synapses function differently in the face of these mutations have suggested novel therapeutic interventions validated in preclinical models and that have shown promise in preliminary human clinical trials. Fifth, the fact that ASD and ID can be diagnosed in early childhood maximizes potential benefits of therapy because it can be started at a time when the brain is most plastic. Finally, animal studies using gene reactivation or pharmacological interventions suggest that substantial improvements can be seen even when treatments begin in adulthood (Ehninger et al. 2008b). Thus, a genetic diagnosis of a developmental brain disorder need not be a “life sentence” of permanent and inexorable mental disability.The path from gene discovery to novel treatment is outlined in Figure 1. This process often begins with astute clinical observations that some patients can be distinguished by a common set of phenotypic traits, thus defining a syndrome. Molecular genetic studies can then be undertaken to test the hypothesis that the syndrome has a genetic cause. In the event that disruption of a single gene or DNA segment causes the disease (i.e., a “highly penetrant” mutation), then it is possible to create an animal model (usually a mouse) that carries the same genetic disruption. Although the effects of the genetic lesion will likely manifest differently at the behavioral level in animals and humans because of differences in the complexity of the brains, it is reasonable to postulate that disruptions in elementary neuronal functions are likely to be shared. Understanding this neuronal pathophysiology is critical for identifying potential therapeutic targets. If these targets can be validated in the animal models, then chemistry ensues to generate molecules that can engage the target and satisfy the pharmacodynamic and pharmacokinetic drug requirements. If they are shown to be safe, drug candidates may then advance to human clinical trials. There are currently clinical trials ongoing in several single-gene syndromic disorders associated with ASD and ID. Most of these target alterations in synaptic signaling.Open in a separate windowFigure 1.The promise of molecular medicine in genetically defined disorders of brain development.The notion that some ASD and associated ID represent “synapsopathies” (or “synaptopathies”) is supported by the preponderance of penetrant mutations in genes associated with synaptic structure and function. The most common single-gene mutations in ASD with ID are associated with fragile X syndrome (FMR1), tuberous sclerosis (TSC1, TSC2), neurofibromatosis (NF1), Angelman syndrome (UBE3A), Rett syndrome (MECP2), the PTEN hamartoma tumor syndrome, and Phelan-McDermid syndrome (SHANK3) (for review, see Betancur 2011). Rare mutations in the neuroligin (NLGN3, NLGN2) and neurexin (NRXN1) genes also cause autism (Jamain et al. 2003). Although this is by no means an exhaustive list of genes implicated in autism (and many await discovery), it is notable nonetheless that these highly penetrant mutations occur in genes that are critical regulators of synaptic function, and further, illuminate biochemical pathways that might be pathogenic in ASD and ID (Fig. 2).Open in a separate windowFigure 2.(A) Schematic of a neuron and axonal-dendritic synapse that depict examples of cellular localization of the various types of defects in ASD/ID. (B) A signaling pathway at the excitatory synapses that couples activity as registered by the release of glutamate to local control of protein synthesis. Disruption of the gene products indicated in the colored boxes greatly increases the risk of ASD/ID. Syndromic disorders with increased prevalence of ASD include Phelan-McDermid Syndrome (SHANK3); Noonan syndrome (RAF1, MEK1); Neurofibromatosis type 1 (NF1); Costello syndrome (H-Ras, MEK1); Cowden syndrome (PTEN); Cardio-facio-cutaneous (CFC) syndrome (MEK1/2); Tuberous sclerosis complex (TSC1/2); Fragile X syndrome (FMRP); Angelman syndrome (AS UBE3a); Rett syndrome (RTT–MeCP2); and Rubinstein-Taybi syndrome (RTS–CREB binding protein, p300). Rare, nonsyndromic ASDs include NLGN3/4 and NRXN1; ID/ASD: SHANK2.In this article, we focus on a few syndromic disorders associated with ASD and ID that are characterized by penetrant mutations in genes that have been shown in animal models to disrupt synaptic function. Our goal is to highlight the similarities and differences in these syndromes and their underlying synaptic pathophysiology. Optimal synaptic function occurs within a narrow dynamic range along many dimensions, and it is not surprising that pathophysiology occurs at the edges of these spectra. What has come as a surprise, however, is that ASD and ID appear to be common consequences of disruptive mutations that cause synaptic pathophysiology at both ends of a spectrum. In other words, both “gain-of-function” and “loss-of-function” mutations can manifest in similar ways. Insights into the pathophysiology of ASD and ID have raised the possibility of therapeutic interventions to bring synapses into a normal operating range.     

Mutations in the gene encoding CADM1 are associated with autism spectrum disorder

The unified idea on the molecular pathogenesis of Autism Spectrum Disorder (ASD) is still unknown although mutations in genes encoding neuroligins and SHANK3 have been shown in a small part of the patients. RA175/SynCAM1/CADM1(CADM1), a member of immunoglobulin superfamily, is another synaptic cell adhesion molecule. To clarify the idea that impaired synaptogenesis underlies the pathogenesis of ASD, we examined the relationship between mutations in the CADM1 gene and ASD. We found two missense mutations, C739A(H246N) and A755C(Y251S), in the CADM1 gene of male Caucasian ASD patients and their family members. Both mutations were located in the third immunoglobulin domain, which is essential for trans-active interaction. The mutated CADM1 exhibited less amount of high molecular weight with the matured oligosaccharide, defective trafficking to the cell surface, and more susceptibility to the cleavage and or degradation. Our findings provide key support for the unified idea that impaired synaptogenesis underlies the pathogenesis of ASD.     

Evidence for Association of Cell Adhesion Molecules Pathway and NLGN1 Polymorphisms with Schizophrenia in Chinese Han Population

Multiple risk variants of schizophrenia have been identified by Genome-wide association studies (GWAS). As a complement for GWAS, previous pathway-based analysis has indicated that cell adhesion molecules (CAMs) pathway might be involved in the pathogenesis of schizophrenia. However, less replication studies have been reported. Our objective was to investigate the association between CAMs pathway and schizophrenia in the Chinese Han population. We first performed a pathway analysis utilizing our previous GWAS data. The CAMs pathway (hsa04514) was significantly associated with schizophrenia using hybrid gene set-based test (P = 1.03×10⁻¹⁰) and hypergeometric test (P = 5.04×10⁻⁶). Moreover, 12 genes (HLA-A, HLA-C, HLA-DOB, HLA-DPB1, HLA-DQA2, HLA-DRB1, MPZ, CD276, NLGN1, NRCAM, CLDN1 and ICAM3) were modestly significantly associated with schizophrenia (P<0.01). Then, we selected one promising gene neuroligin 1 (NLGN1) to further investigate the association between eight significant SNPs and schizophrenia in an independent sample (1814 schizophrenia cases and 1487 healthy controls). Our study showed that seven SNPs of NLGN1 and two haplotype blocks were significantly associated with schizophrenia. This association was confirmed by the results of combined analysis. Among them, SNP rs9835385 had the most significant association with schizophrenia (P = 2.83×10⁻⁷). Furthermore, in silico analysis we demonstrated that NLGN1 is preferentially expressed in human brain and SNP rs1488547 was related to the expression level. We validated the association of CAMs pathway with schizophrenia in pathway-level and identified one susceptibility gene NLGN1. Further investigation of the roles of CAMs pathway in the pathogenesis of schizophrenia is warranted.     

High proportion of 22q13 deletions and SHANK3 mutations in Chinese patients with intellectual disability

Intellectual disability (ID) is a heterogeneous disorder caused by chromosomal abnormalities, monogenic factors and environmental factors. 22q13 deletion syndrome is a genetic disorder characterized by severe ID. Although the frequency of 22q13 deletions in ID is unclear, it is believed to be largely underestimated. To address this issue, we used Affymetrix Human SNP 6.0 array to detect the 22q13 deletions in 234 Chinese unexplained ID patients and 103 controls. After the Quality Control (QC) test of raw data, 22q13 deletions were found in four out of 230 cases (1.7%), while absent in parents of the cases and 101 controls. A review of genome-wide microarray studies in ID was performed and the frequency of 22q13 deletions from the literatures was 0.24%, much lower than our report. The overlapping region shared by all 4 cases encompasses the gene SHANK3. A heterozygous de novo nonsense mutation Y1015X of SHANK3 was identified in one ID patient. Cortical neurons were prepared from embryonic mice and were transfected with a control plasmid, shank3 wild-type (WT) or mutant plasmids. Overexpression of the Y1015 mutant in neurons significantly affected neurite outgrowth compared with shank3 WT. These findings suggest that 22q13 deletions may be a more frequent cause for Chinese ID patients than previously thought, and the SHANK3 gene is involved in the neurite development.     

Genomic landscapes of Chinese sporadic autism spectrum disorders revealed by whole-genome sequencing

Autism spectrum disorder (ASD) is a neurodevelopmental disorder with considerable clinical and genetic heterogeneity.In this study,we identified all classes of genomic variants from whole-genome sequencing (WGS) dataset of 32 Chinese trios with ASD,including de novo mutations,inherited variants,copy number variants (CNVs) and genomic structural variants.A higher mutation rate (Poisson test,P2.2×10~(-16)) in exonic (1.37×10~(-8)) and 3'-UTR regions (1.42×10~(-8)) was revealed in comparison with that of whole genome (1.05×10~(-8)).Using an integrated model,we identified 87 potentially risk genes (P0.01) from 4832 genes harboring various rare deleterious variants,including CHD8 and NRXN2,implying that the disorders may be in favor to multiple-hit.In particular,frequent rare inherited mutations of several microcephaly-associated genes (ASPM,WDR62,and ZNF335)were found in ASD.In chromosomal structure analyses,we found four de novo CNVs and one de novo chromosomal rearrangement event,including a de novo duplication of UBE3A-containing region at 15q11.2-q13.1,which causes Angelman syndrome and microcephaly,and a disrupted TNR due to de novo chromosomal translocation t (1;5) (q25.1;q33.2).Taken together,our results suggest that abnormalities of centrosomal function and chromatin remodeling of the microcephaly-associated genes may be implicated in pathogenesis of ASD.Adoption of WGS as a new yet efficient technique to illustrate the full genetic spectrum in complex disorders,such as ASD,could provide novel insights into pathogenesis,diagnosis and treatment.     

Osmotic Avoidance in Caenorhabditis elegans: Synaptic Function of Two Genes,Orthologues of Human NRXN1 and NLGN1, as Candidates for Autism

Neurexins and neuroligins are cell adhesion molecules present in excitatory and inhibitory synapses, and they are required for correct neuron network function¹. These proteins are found at the presynaptic and postsynaptic membranes ². Studies in mice indicate that neurexins and neurologins have an essential role in synaptic transmission ¹. Recent reports have shown that altered neuronal connections during the development of the human nervous system could constitute the basis of the etiology of numerous cases of autism spectrum disorders ³. Caenorhabditis elegans could be used as an experimental tool to facilitate the study of the functioning of synaptic components, because of its simplicity for laboratory experimentation, and given that its nervous system and synaptic wiring has been fully characterized. In C. elegansnrx-1 and nlg-1 genes are orthologous to human NRXN1 and NLGN1 genes which encode alpha-neurexin-1 and neuroligin-1 proteins, respectively. In humans and nematodes, the organization of neurexins and neuroligins is similar in respect to functional domains.The head of the nematode contains the amphid, a sensory organ of the nematode, which mediates responses to different stimuli, including osmotic strength. The amphid is made of 12 sensory bipolar neurons with ciliated dendrites and one presynaptic terminal axon ⁴. Two of these neurons, named ASHR and ASHL are particularly important in osmotic sensory function, detecting water-soluble repellents with high osmotic strength ⁵. The dendrites of these two neurons lengthen to the tip of the mouth and the axons extend to the nerve ring, where they make synaptic connections with other neurons determining the behavioral response ⁶.To evaluate the implications of neurexin and neuroligin in high osmotic strength avoidance, we show the different response of C. elegans mutants defective in nrx-1 and nlg-1 genes, using a method based on a 4M fructose ring ⁷. The behavioral phenotypes were confirmed using specific RNAi clones ⁸. In C. elegans, the dsRNA required to trigger RNAi can be administered by feeding ⁹. The delivery of dsRNA through food induces the RNAi interference of the gene of interest thus allowing the identification of genetic components and network pathways.