【作者】 许晓娟；

【导师】 夏昆； 胡正茂；

【作者基本信息】 中南大学 ， 遗传学， 2013， 博士

【摘要】 孤独症是一种严重影响儿童健康的神经发育性疾病,其主要的临床特征包括社会交往障碍、言语交流障碍、狭隘兴趣和重复刻板行为等,通常3岁以内发病,男女比例约为4：1-10：1。孤独症患病率近年急剧上升,最新流行病学调查发现孤独症的发病率已高达1.14-2.6%。孤独症的病因学及发病机制尚未阐明,遗传学研究发现一些比较确定的孤独症易感基因,这些基因主要参与神经发育过程,如突触发生和突触可塑性等。对孤独症发病机制的研究中最为广泛的是neurexin家族蛋白和neuroligin家族蛋白。而孤独症患者的男女差异性使得位于X染色体上的NLGN3和NLGN4X的研究尤为引人注目。NLGN3和NLGN4X编码的蛋白属哺乳动物大脑突触后细胞粘附因子,通过和neurexin相互作用在突触结构形成、神经递质释放、突触的识别、突触成熟及信息传递等过程中发挥重要作用。方法：本研究前期工作中,通过直接测序对318名孤独症患者和453名正常对照的NLGN3和NLGN4X外显子序列及外显子侧翼序列进行分析,我们在患者组中发现了NLGN3和NLGN4X的10个已知SNPs和4个未报道的错义变异(p.G426S-NLGN3、p.G84R-NLGN4X、 P.Q162K-NLGN4X和p.A283T-NLGN4X),在正常对照组中发现了13个SNPs(包括患者中发现的10个SNPs)。本研究中针对这些变异,我们做了进一步的研究与分析,希望能够阐明他们在孤独症发病中的作用和机制。我们主要从两个方面进行研究。1.针对患者组和对照组共有的已知SNPs,通过基于病例-对照(Case-Control)的关联研究阐明NLGN3和NLGN4X与中国人群中孤独症的相关性。2.我们所发现的4个错义突变,建立稳定表达的HEK293细胞模型,通过与内质网标志蛋白免疫荧光染色,对NLGN3/4X野生型和相关突变体进行亚细胞定位研究；通过蛋白酶体抑制剂(MG132)阳溶酶体抑制剂(CQ)来阻断neuroligin3和neuroligin4X可能的降解途径确定相关突变体在细胞内的降解途径；通过CHX抑制蛋白合成,确定相关突变体的降解速率；通过neuroligin和IgG-△neurexin(-SS4)免疫共沉淀分析突变体是否影响neuroligin-neurexin复合体的形成。从而初步阐明NLGN突变蛋白能否正常行使其生理功能以及对突触的影响。结果：通过基于病例-对照(Case-Control)的关联研究,我们发现NLGN4X的2个连锁的常见SNP位点rs3747333和rs3747334等位基因频率和基因型频率在患者组和对照组之间的有显著统计学差异(OR=4.685,95%CI=2.073-10.592, p=5.09E-05)。对所发现的4个错义突变进行生物学功能分析,通过免疫荧光染色发现4个错义突变的相应突变蛋白在非神经元性的细胞HEK293细胞中呈现细胞膜分布,和neuroligin3或neuroligin4X野生型蛋白没有差异性；通过MG132和CQ药物作用于相应的细胞系,4个被检突变蛋白和野生型蛋白降解途径主要通过泛素介导的蛋白酶体降解途径进行降解；通过CHX药物作用于相应的细胞系,4个被检突变蛋白的降解速率和野生型蛋白没有明显差异；通过免疫共沉淀实验发现4个被检突变蛋白及野生型蛋白都能够与IgG-Aneurexin1β融合蛋白相结合而被沉淀,没有明显的差异。结论：本研究结果表明NLGN4X的2个连锁的常见NP位点rs3747333和rs3747334与中国汉族人群孤独症显著相关,NLGN4X是中国人群孤独症的易感基因。但是,在孤独症患者中发现的4个错义突变的相关突变蛋白的表达、亚细胞定位、降解过程以及和neurexin的相互作用在非神经元性的细胞HEK293细胞中没有发生明显的改变,可能通过其他尚未阐明的分子机制导致孤独症的发生。更多还原

【Abstract】 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.更多还原