Association Study Of NRXN1Gene And Identification Of Two CNVs Associated With Autism In Chinese Han Population

SECTION1Association Study of NRXN1with autism in Chinese Han PopulationAutism, a brain developmental disorder characterized by impaired social interaction and communication as well as restricted and repetitive behaviors, forms the core of the autism spectrum disorders (ASDs). Autism is the most important sub-categories of the Pervasive Developmental Disorder (PDD). The diagnosis of autism was lack of objective indicators, which mainly accorded to the behavioral deficits. The treatment of autism lacked special efficacy, mainly relyed on the behavioral training, and the prognosis of autism was poor, about60%-70%of autism patients unabled to live independently. Epidemiological studies have shown that the incidence of autism increased rapidly. About1in88children has been identified with an autism spectrum disorder (ASD) according to estimates from CDC's Autism and Developmental Disabilities Monitoring (ADDM) Network in2012. And autism are almost5times more common among boys (1in54) than among girls (1in252). According to the estimate of World Health Organization (WHO), there were0.6-1.8million of autism patients in China, which bright a great deal of burden to society. On the basis of familial relative risk and twin studies, autism is considered as a multi-factorial disorder, whereas the genetic factors are predominant in the pathogenesis. Previous studies indicated that the abnormal synaptic homeostasis and neural plasticity might represent a risk factor to autism. The synapse-related genes, including NRXN1, NLGN3and NLGN4, SHANK3, CNTNAP2, PCD9and PCD10, might associated with autism. The neurexin-1{NRXN1) gene mapped on chromosome2p16.3encodes neurexin, a cell adhesion molecule and receptor in the vertebrate nervous system. The binding of neurexins located on pre-synaptic membranes with neuroligins on post-synaptic membranes plays an important role during synaptic formation, which implies a role in cell adhesion. The interactions between neurexins and neuroligins ensured pre-synaptic neurones align correctly with the post-synaptic neuron, thereby permitting interaction between matching neurotransmitter release sites and receptors. And the abnormal synapses may lead to the cause of autism. In2006, Friedman et al firstly reported the heterozygosity deletion of NRXN-1α associated with neurodevelopmental disorder. Subsequently, a lot of research detected deletions of NRXN-1exons in multiple psychiatric disorders such as schizophrenia, autism and mental retardation patients in different population. In2008, rare missense mutations of NRXN1were detected in autism patients, suggesting that NRXN1involved in autism pathogenesis. The neurological behavior defects of NRXN-1α null mice and Drosophila model also provide evidence that NRXN-1α is involved in autism pathogenesis. All these data has implicated neurexin-1as a candidate gene for susceptibility to autism. However, no any data of the association between NRXN1and Chinese Han population with autism is reported. In this study, we performed the mutation analysis and association analysis on NRXN1gene in autism patients of Chinese Han population.By direct sequencing, we analyzed the entire coding regions and associated splice junctions of NRXN1in313Chinese autism patients. For exons in which non-synonymous variants were identified, sequencing was performed in500healthy controls. In the meantime of the identification of rare de novo mutations, association studies based on Case-Control was performed.We identified22variants in the NRXN1coding regions, including7missense variants,3deletions, and12synonymous mutations. Among them,3missense and3synonymous variants were not reported in the dbSNP database and absent in500control subjects; whereas4missense variants,3deletions and3synonymous mutations were not reported in the dbSNP database but were identified in the control subjects. However, there is no significant association of these mutations with autism risk. Interestingly, there was a statistically significant association of NRXN1SNP (rs2303298) with risk of autism (26.2%vs.13.8%;χ2=22.487; p= 3.45E-006; OR=2.152(1.559-2.970).Our data suggest a possible association of NRXN1with autism risk in Chinese Han population, warranting further large-scale and function study on this gene. SECTION2Identification of Two CNVs Associated with AutismIt has been reported that the chromosomal abnormalities and rare gene variants are associated with autism. Copy Number Variations (CNVs) are considered to be associated with autism. The study of high-density chip-based copy number variation (CNVs) has made great progress in the identification of the autism susceptibility locus.Autism is one of the earliest diseases studied by CNVs genome-wide association analysis. In2007, Sebat et al first reported the association of the de novo CNVs and autism. Subsequently, a study based on autism pedigree found a593kb deletion located in16p11.2, which was validated by other three study groups. CNVs regions associated with autism contains a number of novel autism-related genes, such as SHANK2, SYNGAP1,DLGAP2and X-linked DDX53-PTCHD1. Up to now, studies have shown that the disease-related CNVs were detected in about7%-20%autism patients, At least chromosome1,3,5,7,15,16and22are involved. These studies provided important evidence for CNVs in pathogenesis of highly genetic heterogeneity disease, such as autism. All the CNVs founded might affect different genes in different patients, which means that although different autism patients has similar symptoms, the symptoms might be caused by different genetic defects. Therefore, the identification of de novo CNVs will provide a research base for the location of the possible susceptibility genes involved in CNVs.To clarify the genetic etiology of autism in Chinese Han population, we used Human CNV370-Duo chip to perform genome-wide genotyping for455autism samples. The genome-wide CNVs analysis was performed with the software cnv Partition v2.4.4. The results were compared with the existed autism genetic variation databases, and were between patients and controls. Then, we classified the reported CNVs and the newly discovered rare CNVs. Subsequently, Real-time fluorescence quantitative PCR (Real-time PCR) technology was performed to authenticate the rare CNVs. Finally, we screened the genes located in the area of rare CNVs by bioinformatics analysis, evaluated the relevance of this locus and disease, as well as the contribution of disease risk. Autism is a neurodevelo-pmental-related diseases, genetic factors leading to abnormal expression of neural development-related proteins, enzymes, receptors, neurotransmitters, leading to the abnormal of proliferation and differentiation of neurons, which provided biological basis for autism susceptibility. Therefore, when screening the candidate genes, we should focus on the genes expressed in the nervous system, especially those genes related to neural development.Our study identified hundreds of CNVs in455autism patients. The data from the patients was compared with the controls and the DGV database, two rare de novo CNVs respectively found in two autism patients were further analyzed. The one was a8Mb heterozygous deletion located on chromosome5p15.33-p15.2region, which was a de novo CNV with the reduction change of copy number, the SLC6A3gene located in5p15.33was reported involved in a variety of neurodevelopment abnormalities, mental and nervous system disease; The second is a1Mb heterozygous duplications located on chromosome10p12.33region, which was the increased change of copy number. The CACNB2gene located in the CNV region belongs to the Ca2+channel genes. It has been reported that mutations in Ca2+channel genes might be responsible for ASD. Moreover, the PTPLA and STAM gene located in the CNV involved in signal transduction, is considered to be associated with neurodegenerative diseases. These genes might affect the expression of protein related to neurogenesis or development. Our results suggest that the two CNVs might be associated with autism.De novo CNVs associated with autism were identified in455autism patients. Our future study will focus on the genes located in the two CNVs regions. Our study provided molecular research foundation to the further localization and clone research of autism susceptibility genes.