孤独症易感基因GRIN2B在神经元突触可塑性形成的功能研究

Autism is sever childhood neurodevelopmental disorders. By investigating, the prevalence is up to 1.0% worldwidely The etiology of autism is still unknown. Further more, autism is lack of effective therapy and the prognosis is not good. Epidemiological studies have shown that the the genetic factors are predominant in the pathogenesis of autism. Studies indicated that the Chromosome Abnormality, Copy Number Variations, rare mutation and so on are related to autism. More and more studies implied that GRIN2B was involved in the development of autism.It's reported that the GRIN2B mutations related to autism included the translocation breakpoint disrupting GRIN2B and the point mutation. In our study, we have found that GRIN2B is associated to autism by transmission disequilibrium test(TDT) analysis case-control analysis. Further we found two unreaported GRIN2B missense mutation. Our studied indicated that a possible role of GRIN2B as a candidate gene for the etiology of autism in Chinese Han population.GRIN2B is one of the NMDA subunits. Two NR1 subunits and two NR2 subunits form highly Ca2+ permeable cation channels which plays important roles in synaptic plasticity and in neuronal pattern formation during development. We suggest that the mutant NR2B may disrupt the neurotransmitter-gated ion channels which can control and regular the homeostasis of the ion related to release of neurotransmitter, change the excitatory postsynaptic potential(EPSP) and then lead to abnomal synaptic plasticity and other synaptic activity. These defective neuronal activities trigger development of autism. In this project, we plan to clarify the hypothesis by in vitro and in vivo experiments.

孤独症是一种严重影响儿童健康的神经发育性疾病。其发病率在全球高达1%。孤独症的病因学及发病机制尚未阐明，流行病学调查显示遗传因素是孤独症的主要病因，包括染色体异常、拷贝数变异、罕见基因变异等。GRIN2B是近年来新发现与孤独症相关的基因。现有研究发现与孤独症相关的GRIN2B突变主要包括GRIN2B基因断裂和点突变。本研究前期工作中，我们通过传递不平衡和case-control分析, 发现GRIN2B基因与中国孤独症显著相关，并通过测序在孤独症患者中鉴定2个未报道的GRIN2B错义突变，提示GRIN2B是中国人群孤独症易感基因。GRIN2B是NMDA受体家族亚基之一，其编码的蛋白NR2B与NR1形成神经元递质门控离子通道，参与突触可塑性及学习记忆。本项目拟阐明突变体NR2B是否导致突触可塑性异常及参与孤独症发生的机制。

目的： GRIN2B在孤独症发病中机制研究.方法：利用全基因组关联分析的方法和Sanger测序，分析了NR2B的常见变异与中国汉族人群自闭症的关联性。利用体外培养小鼠神经元细胞模型，研究GRIN2B对神经元发育及生长的影响。利用果蝇模型，研究GRIN2B相关神经环路。.结果：.（1）通过GWAS的方法，对两组中国人群（第一组为孤独症核心家系，共计825人；第二组为散发病例和正常对照，共计1120人）进行关联研究。经过meta分析和多重检验后发现11SNP微点与孤独症显著相关（P,comb<0.05）。通过SANGER测序的方法，首次发现NR2B的单体型与自闭症相关。.（2）GRIN2B突变体与野生型在HEK293细胞中的表达和定位无区别。.（3）在体外培养的神经元中干扰或者过表达GRIN2B，神经元轴突和总神经突的长度不变，轴突分支数目无显著变化，神经元树突棘密度棘密度无显著差异。.（4）在体外培养的神经元中干扰GRIN2B，神经元活化下降，钙释放频率下降。.（5）在果蝇中干扰Unr的表达，果蝇运动能力异常。在神经元特异性干扰后，果蝇节律表现出异常。.结论：.（1）GRIN2B与孤独症的发病相关。.（2）GRIN2B表达异常可能导致神经元功能性缺失。.（3）果蝇Unr基因表达量降低后会出现运动异常和节律异常。