孤独症疾病相关突变位点高通量筛选及致病机制研究

Autism is a neurodevelopmental disorder. Early twin studies produced heritability estimated approaching 90%. However, it is a multigenic common disorder. The basis of this heritability remains largely unexplained. To data, genetic association studies have had limited success in identifying genetic risk for autism. The lack of common predisposition genes has led to the suggestion that rare variants constitute the majority of autism risk. The Rare Allele-Major Effects (RAME) model postulates that rare penetrant variants are the key to the genetic etiology of common disease. Functional mutations that lead to an altered amino acid are often deleterious and potentially disease causing. Although whole exome sequencing studies were performed in autism with Caucasine, the results of these studies were not consistent. As for the different genetic backgrouds between different ethnic popultiaons, the heterogeneity was obvious in different ethnic popultiaons. Therefore we will perform whole exome sequencing in autism with Han Chinese population. First, we will sequence 10 autism twin families (40 individuals). Second, to decrease the false postive results, we will sequence the mutations found in the autism twin families using Sanger sequencing and Miseq sequence platform in 1000 autism trios and 1000 health controls. The aim of this study is to seek the de novo mutations in patients with autism. Finally, the function of these de novo mutations and the effects of them on neurodevelopment will be explored in experiment in vivo and in vitro. The results of this study will provide the clues and evidence to investigate the pathogenesis of autism.

孤独症是一种高遗传度的广泛发育障碍，属于多基因复杂疾病。针对复杂疾病，罕见等位基因-重大效应(Rare Allele-Major Effects)理论认为，罕见突变是常见复杂疾病遗传病因的关键，功能性突变位点导致所编码氨基酸改变，可能是疾病潜在的病因。尽管国外已有外显子组测序的研究，但结果并不一致；由于不同种族遗传背景不同，也存在人群异质性差异。本研究将在中国汉族人群中进行高通量测序工作，对10个孤独症双生子家系(共40人)进行全外显子组测序；为减少假阳性结果，继而采用Sanger 和MiSeq测序方法，对全外显子组测序结果在另外的1000个孤独症核心家系和1000例正常对照中进一步验证，明确孤独症新发(de novo)且影响氨基酸改变的突变位点；最后通过在体及离体实验开展功能研究，探讨突变位点对疾病发生发展的影响。本项目预期结果将为深入研究易感基因在孤独症的致病机制提供实验线索。

孤独症是一种高遗传度的广泛发育障碍，属于多基因复杂疾病。罕见突变是常见复杂疾病遗传病因的关键，功能性突变位点导致所编码氨基酸改变，可能是疾病潜在的病因。目前尚缺乏中国汉族人群外显子组测序的研究。本项目共完成24个孤独症家系的全外显子测序，包括14个孤独症双生子家系，10个孤独症患病或未患病同胞家系。高通量筛选孤独症基因突变位点，进一步在扩大的孤独症家系和正常对照中进行验证。SNV和Indel位于156个基因，其中ADRB2，FOXP1，MUC4，OPRM1，PCDH15，TTN，ZNF292也存在于SFARI孤独症风险基因库。针对孤独症双生子及同胞家系外显子测序线索，对染色体15q11-q13区段的孤独症风险基因突变、孤独症易感基因AUTS2基因、少突胶质细胞分化调控基因DUSP15进行了突变位点筛选。测序发现孤独症患者携带GABRG3基因2个罕见变异即rs201602655（p.Val233Met）和rs201427468（p.Pro365Ser），功能预测提示为有害突变。本项目也发现孤独症患儿携带AUTS2基因和DUSP15基因的罕见突变。功能研究发现，孤独症易感基因EZH2通过表观遗传调控Reelin，影响Reelin蛋白的表达进而影响神经元迁移，可能导致大脑皮层板层形成异常。对孤独症易感基因MEGF10启动子区域风险等位基因和单体型，通过双荧光素酶报告系统和凝胶阻滞试验，发现该孤独症风险位点影响MEGF10基因与转录因子的结合，影响MEGF10基因转录水平，可能导致突触修剪异常增加了孤独症的患病风险。本项目结果为深入研究易感基因在孤独症的致病机制提供实验线索。