Association Analysis Of Genetic Variants Of Synaptic Gene NRXN1?NLGN3?NLGN4X?CNTNAP2?SHANK3 With Autism Spectrum Disorders In A Chinese Han Cohort

Objective:Autism spectrum disorders (ASDs) are severe neurodevelopmental syndrome with a complex genetic etiology, which characterized by impaired reciprocal social interactions, deficient communication, restricted interests and stereotyped activity patterns. ASDs are acknowledged to be among the most heritable neuropsychiatric disorders and etiologically heterogeneous, probably associated with a combination of the effects of multiple genes and environmental factors, and the role of genetic factors in the pathogenesis of ASDs has been definite established. Many independent genome-wide scans for ASDs susceptibility loci have been carried out. The region on chromosome 2q?7q?22q?Xq and Xp stands out as the region of suggestive linkage to atiology of ASDs in many independent genome-wide scans. The synaptic hypothesis was also proposed that alteration of synaptic homeostasis and/or impairments in synapse development is thought to be a major cause of brain disorders such as autism and mental retardation. A genetics topic in ASDs has emerged focusing on identification of the synaptic genes contributing to the formation and function of the synapse. Of particular interests are synaptic genes of the NRXNs-NLGNs-SHANK3 pathway in ASDs pathogenesis. Five synaptic associated genes NRXN1?NLGN3?NLGN4X?CNTNAP2 and SHANK3 selected in this study, are located within these regions. By using the case-control association analysis with 31 tagging SNPs of these five syanptic genes, we will investigate the relationship between ASDs and the sigle SNP and the haplotypes, also analysis with gender stratification. As a result, we will try to find out if these five genes are susceptibility genes of the ASDs.Moreover, ASDs are hereditary heterogeneous with a combination of the effects of multiple genes. So it is importance to analysis the gene-gene interaction of multiple genes for etiology of ASDs.It has also been considered that genes contributing to ASDs are likely a combination of rare variants in fewer individuals with a dramatic effect and common variants in majority individuals with small increments of risk. So we attempted to replicate rare mutations in genes of this pathway, which were previously described to be associated with ASDs, to validate the initial findings and evaluate if they also play a potentially role for ASDs in Chinese Han population.Part?Association analysis of SNPs of synaptic gene NRXN1?NLGN3?NLGN4X, CNTNAP2?SHANK3 with autism spectrum disorders in a Chinese Han cohortMethods:1. We studied a Chinese sample of Han origin consisting of 229 unrelated patients with ASDs and 191 ethnically and geographically matched controls in this study. All affected subjects were diagnosed according to the diagnostic and statistical manual of mental disorders (DSM-IV) criteria or International Classification of Diseases-10 (ICD-10) from outpatients of Children's Hospital, Zhejiang University School of Medicine and from several special autism-training schools. All controls were randomly drawn from out-patients of our hospital with no personal history of psychiatric disorders.2. SNPs selection and genotyping:Based on the genotype data from the SNP databases and International HapMap Project,31 tagging SNPs within these five candidate genes with a minor allele frequency (MAF) greater than 5% in the Han_Chinese or in the HCB_Asian population were selected to capture the majority of the common variations. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF MS) was used for SNP genotyping.3. Statistic Analysis:The Hardy-Weinberg equilibrium (HWE) was assessed with chi-square test. SNPs were used in case-control study for allelic, genotypic and haplotypic association and were also performed with genders stratification.Results:1. Association analysis of SNPs of NRXN1 with autism spectrum disorders(1) A significant genetic association between an intronic SNP rs1421589 and female ASDs was observed, with T-allele having an increased risk to develop to autism(P=0.038, OR=1.955,95%CI:1.035-3.69), and the log-additive model was accepted as the best inhericance model fitting this data(P=0.023, OR=2.28,95%CI: 1.09-4.74). Haplotype-specific association analysis revealed that the haplotype T-T (rs1421589-rs1563018) showed significant association with female ASDs and has increased risk to develop to ASDs (P=0.039, OR=1.925,95%CI:1.029-3.604).(2) One exonic SNP rs1045874 showed significant differences in genotypic frequencies between the individuals with ASDs and controls under overdominant model (P=0.016, OR:1.85,95%CI:1.11-3.07) by logistic regression analysis, although no significant difference in allelic frequencies was observed.2. Association analysis of SNPs of NLGN3 with autism spectrum disorders (1) Significant differences of allele frequencies were detected for 3 SNPs in NLGN3 gene contrasted between cases and controls for male and female samples separately (male:rs11795613, rs4844285 and rs5981079, female:rs11795613, rs4844285 and rs7051529). In addition, significant differences in genotype distributions of these three SNPs were also detected in female samples under a log-additive model: rs11795613, p=0.036; rs4844285, p=0.036; rs7051529, p=0.038.(2) Haplotype-specific association analysis revealed that 4-SNPs haplotype A-G-T-T,3-SNPs haplotype A-G-T,2-SNPs haplotype A-G and T-T play a role as a susceptibility factor for male ASDs (A-G-T-T:P=0.031, OR:1.633,95%CI: 1.046-2.549; A-G-T:P=0.015, OR:1.758,95%CI:1.112-2.778; A-G:P=0.0113, OR:1.803,95%CI:1.14-2.854; T-T:P=0.047, OR:1.571,95%CI:1.005-2.457), 2-SNPs haplotype G-A play a role as a protective factor for male ASDs(P=0.0184, OR: 0.575,95%CI:0.361-0.913). Moreover, the haplotype A-G-T and A-G play a role as a susceptibility factor for female ASDs (A-G-T:P=0.03, OR:2.16,95%CI: 1.071-4.357; A-G:P=0.035, OR:2.147,95%CI:1.048-4.400), the haplotype G-A and G-T play a role as a protective factor for female ASDs (G-A:P=0.035, OR:0.466, 95%CI:0.227-0.954; G-T:P=0.044, OR:0.383,95%CI:0.147-0.999)3. Association analysis of SNPs of NLGN4X with autism spectrum disorders(1) Significant difference in allelic and genotypic frequencies of SNP rs5961397 were detected between the female individuals with autism and controls (allelic:p=0.047, genotypic under log-additive model:p=0.039). The rs5961397-A allele was overrepresented in female ASDs (OR=2.133,95% CI=1.001-4.545), and the rs5961397-G was a protective allele. Haplotype G-G (rs6529901-sr5961397) showed significant association with female ASDs and has a protective haplotype (P=0.024, OR: 0.404,95%CI:0.180-0.904).(2) Haplotype-specific association analysis revealed that one haplotype block defined by two SNPs (rs1882409-rs5916352) showed a significant association with male ASDs and the haplotype A-C play a risk role for male ASDs (P=0.022, OR:2.551, 95%CI:1.121-5.804).4. Association analysis of SNPs of CNTNAP2 and SHANK3 with autisms spectrum disordersNeither single SNP nor haplotype in CNTNAP2 and SHANK3 was detected to show significant association with ASDs, even after gender stratification analysis.Conclusions:Our study suggest the possible involvement of NRXN1?NLGN3?NLGN4X genes in the suscepitibility to ASDs. Future replications are warrented before definitive conclusion can be drawn.Part?Gene-gene interaction of synaptic gene NRXN1?NLGN3?NLGN4X?CNTNAP2?SHANK3 with autism spectrum disorders in a Chinese Han cohortMethods:Twenty-nine SNPs of synaptic genes were involved in the analysis of gene-gene interaction using multifactor dimensionality reduction (MDR). Model with the highest CV consistency, the highest testing accuracy as well as the significant permutation test results was considered as the best interaction-model.Results:MDR analysis revealed that the two-loci genotypic combination (rs4844286-rs5916352) was the best gene-gene interaction model of synaptic genes in the etiology of autism(CV consistency=9/10, testing accuracy=0.617).Conclusions:Gene-gene interaction of NLGN3-NLGN4X may be involved in the etiology of autism in Chinese Han cohort. Part?Study of rare mutations of synaptic gene NRXN1?CNTNAP2?NLGN3?NLGN4X?SHANK3 with autism spectrum disoreders in a Chinese Han cohortMethods:Twenty-one rare variants in these synaptic genes (NRXN1, NLGN3, NLGN4X, CNTANP2 and SHANK3) previously reported to be associated with ASDs were selected to perform for mutations screening, using MALDI-TOF mass spectrometry and analyzed with the SpectroTYPER RT 2.0 software.Results:We successfully detected 21 rare variants previously described to be associated with ASDs in these five genes and three missense mutations were identified. One missense mutations, c.83G>C polymorphism in exonl in NRXN1 gene, were identified in both affected patients and control samples and the rare allele C was present in 13 cases (2.97%) and 4 controls (1.09%). The difference is not significant either for allelic frequencies (?2=3.269, p=0.118) or for genotypic (?2=3.339, p=0.114). Another missense mutation, c.3385G>C polymorphism in CNTNAP2 gene was only detected in a single control sample and absent from patients with ASDs. One missense mutation in SHANK3 gene, c.203T>C, was identified only in a female autism with serious language impairment, and was absent in normal control samples.Conclusion:It is for the first time, a specific mutation of SHANK3 gene (c.203T>C) is confirmed by a molecular screening in another ethinic population, suggesting this missense mutation may be a cause of ASDs. However, functional studies will be required to confirm that this mutation is indeed pathogenic.