NLGN3和NLGN4X与中国人群儿童孤独症的相关性研究

发布时间：2018-04-13 15:19

  本文选题：孤独症 + NLGN3　； 参考：《中南大学》2013年博士论文

【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。
[Abstract]:Autism is a neurodevelopmental disorder that affects children ' s health . The main clinical features include social interaction disorders , verbal communication disorders , narrow interests and repetitive stereotypes . The prevalence of autism is about 4 : 1 - 10 : 1 . The prevalence of autism has risen sharply in recent years . Recent epidemiological studies have found that the incidence of autism is up to 1.14 - 2.6 % . The etiology and pathogenesis of autism have not yet been elucidated . Genetic studies have found that some of the more specific susceptibility genes of autism are involved in neurodevelopmental processes , such as synaptic transmission and synaptic plasticity .

The most extensive research on the pathogenesis of autism is neurexin family protein and neuroligin family protein . The difference of male and female in autism makes the study of NLGN3 and NLGN4X located on X chromosome particularly notable . The proteins encoded by NLGN3 and NLGN4X belong to the postsynaptic cell adhesion factor of mammalian brain . The interaction of NLGN3 and NLGN4X plays an important role in synaptic structure formation , neurotransmitter release , synaptic identification , synaptic maturation and information transmission .

Methods : The exon sequences of NLGN3 and NLGN4X exon sequences and exon flanking sequences were analyzed by direct sequencing in 318 autism patients and 453 normal controls , and we found 10 known SNPs in NLGN3 and NLGN4X and four unreported ( p . G426S - NLGN3 , p . G84R - NLGN4X , P . Q162K - NLGN4X and p . A283T - NLGN4X ) in the patient group , and 13 SNPs were found in the normal control group ( including 10 SNPs found in patients ) .

In order to clarify their roles and mechanisms in the pathogenesis of autism , we hope to clarify the role and mechanism of NLGN3 and NLGN4X in the pathogenesis of autism .
The possible degradation pathways of neurotrophin - 3 and neurotrophin 4X were blocked by proteasome inhibitor ( MG132 ) .
determining the degradation rate of the relevant mutant by CHX inhibiting protein synthesis ;
The formation of neuroligin - neurexin complex was influenced by neuroligin and IgG - 鈻，

本文编号：1745065