Association Analysis Of NLGN3and NLGN4X Gene With Autism In Chinese Population

Backgroud:Autism is a severe neurodevelopmental disorder with high clinical and genetic heterogeneity, clinically characterized by three core deficits:impairment of social interaction, deficits in verbal communication, as well as stereotypic and repetitive behaviors. The onset of autism is usually before the age of3years old. Autism shows a remarkable sex bias, with a male-to-female ratio of4:1to10:1. According to the epidemiological study, the worldwide prevalence of autism has reached1.14-2.6%. The etiology of autism is still unknown. However, autism related genes, which involve in the process of neural developments including synaptogenesis and synaptic plasticity, have been discovered in recent autism studies.Previous studies about molecular pathogenic mechanism of autism mainly focused on neurexin and neuroligin family proteins. However, remarkable sex bias of autism makes NLGN3and NLGN4X which locus on X chromosomes studied more frequently. Neuroligin3and neuroligin4X are postsynaptic cell-adhesion molecules that interact with neurexin to connect pre and postsynaptic neurons at synapses, mediate transsynaptic signaling, and shape neural network properties by specifying synaptic functions.Methods:In the previous study, we analyzed the whole exons (excluding the3’UTR) and the exon flanking intron sequences of NLGN3gene and NLGN4X gene in318unrelated sporadic autism cases and453controls. In the patient cohort, we identified10reported SNPs and4unreported missense mutations(p.G426S-NLGN3, p.G84R-NLGN4X, p.Q162K-NLGN4X and p.A283T-NLGN4X). In the control cohort, we identified another thirteen reported SNPs including the10reported SNPs found in patient cohort.In this proposal,1.based on the10SNPs, we aim to make case-control anslysis to study the association between NLGN3as well as NLGN4X and autism in Chinese population;2.based on the4missense mutations, we aim to use HEK293cells stably expressing neuroligin3or neuroligin4X to study the function about wildtype and mutant protein:(1) To determine the subcellular localization of mutant protein, the HEK293cells stably expressing neuroligin3or neuroligin4X was immunostained with ER marker calnexin or nogo and anti-GFP or anti-Flag antibodies.(2)To clarify the degradation pathway of mutant protein, the HEK293cells stably expressing neuroligin3or neuroligin4X were treated with proteasomal inhibitor MG132and lysosomal inhibitor CQ, respectively, followed by detecting protein level of neuroligin3or neuroligin4X using Western Blot.(3)To determine half-life of mutant protein, HEK293cells stably expressing neuroligin3or neuroligin4X were treated with CHX for various times periods followed by detecting proteins level of neuroligin3or neuroligin4X using western bolt.(4) To analyze the form of neurexin-neuroligin complex, proteins of neuroligin3or neuroligin4X were co-immunoprecipitated with IgG-△neurexin(-SS4) fusion protein followed by detecting proteins level of neuroligin3or neuroligin4X using western bolt.Result:1.The family association studies based on case-control analysis showed that the rs3747333and rs3747334of NLGN4X gene is associated with the increased risk for autism(OR=4.685,95%CI=2.073-10.592, p=5.09E-05), indicate that significant association of NLGN4X and autism in Chinese population.2.The functional analysis showed:(1)Neuroligin3and neuroligin4X wildtype and mutant proteins localized on the plasma membrane;(2) Neuroligin3and neuroligin4X wildtype and mutant proteins are mainly degraded by the Ubiqitin-proteasome;(3) The half-life of the4mutant proteins of neuroligin3and neuroligin4X are as the same as the wildtype;(4)Both Neuroligin3and neuroligin4X wildtype as well as the mutant proteins are co-immunoprecipitated by the IgG-△neurexin(-SS4) fusion protein.Conclusion:Our data suggest a possible association of rs3747333and rs3747334of NLGN4X gene with autism risk in Chinese Han population, which support the NLGN4X is susceptible gene of autism. However, the subcellular localization, degradation pathway,half-life and interaction with neurexin of4mutant protein are not changed by the related missense mutation. Therefore, we speculate that the4missense mutations (p.G426S-NLGN3, p.G84R-NLGN4X, p.Q162K-NLGN4X and p.A283T-NLGN4X) may contribute the disease by other unclarified biological pathway.