错配修复基因MSH3突变导致先天性白内障的分子机制研究

Cataract is the most common ocular disease leading to blindness, and congenital cataract is the leading cause of blindness and visual impairment in children, which is usually caused by genetic defects, accounting for about 25% of all the patients. We identified a missense variant p.R1061G in MSH3 gene in a Chinese nuclear congenital cataract family by using whole exome sequencing, then we thereafter explore the molecular mechanism and the function of MSH3 in this project. First, in order to verify the association between MSH3 gene mutation and congenital cataract, we perform high-throughput sequencing on additional congenital cataract patients by using a custom congenital cataract panel targeting 81 candidate genes including MSH3, and whole exome sequencing will be performed on the patients without known mutations, thus novel causative genes may be identified. Then, we study the DNA damage repair ability of MSH3 mutation and its effect on the differentiation of embryonic stem cells into lens fiber cells and explore the molecular mechanisms as well. Finally, We study the directed differentiation of patients' induced pluripotent stem cells into lens fiber cells and analyze the role of MSH3 in lens development and the molecular mechanism of MSH3 mutation associated with congenital cataract, and our study will provide theoretical basis for the prevention and treatment of cataract.

白内障是世界第一位致盲性疾病，先天性白内障是造成儿童失明和弱视的重要原因，其发生与遗传因素密切相关。本项目是在一个中国先天性核性白内障家系中通过全外显子组测序技术发现一个新的白内障致病基因MSH3突变的基础上，深入开展突变致病性验证和功能研究。首先，利用定制的白内障基因panel捕获靶基因进行高通量测序，检测更多先天性白内障患者基因变异情况，未发现致病基因者进一步行全外显子组测序，以期发现新的致病基因；然后，考察MSH3突变DNA损伤修复功能和对胚胎干细胞向晶状体纤维细胞分化的影响及其分子机制；最后，研究患者诱导性多能干细胞定向分化为晶状体纤维细胞，探讨MSH3在晶状体发育中的作用及其突变导致先天性白内障的分子机制，为白内障的防治新策略提供理论基础。

白内障是世界第一位致盲性疾病，先天性白内障是造成儿童失明和弱视的重要原因，其发生与遗传因素密切相关。.收集了61例先天性白内障家系，其中37个有家族史，24个为散发核心家系，55个是单纯性白内障，6例有眼外症状，智力迟缓、耳廓前倾、牙齿异常和房缺等。55个先证者进行了白内障panel靶向测序，6个家系进行了WES，3个家系进行了WGS，2个家系进行了CGH分析。37个有家族史的患者中，26个发现了已知白内障基因可疑变异，其中11个位点未见报道。24个散发患者中，11个发现了可疑变异，其中7个为未报道的位点。.全基因组连锁及单体型分析确定9q34.3区域是与家系F-#17和F-#18疾病表型唯一连锁区域，在该区域家系患者单体型共享，两点连锁分析最大LOD分别为3.68和3.61，支持9q34.3与疾病连锁。在三个无亲缘关系家系F-#17、F-#18和F-#19中发现9q34.3区域EXD3基因c.C112T, p.(Arg38Trp)与患者疾病表型共分离，正常人数据库中均未见该变异位点，全基因组测序未在该区域发现其他可疑变异。为了验证EXD3基因c.C112T,p.(Arg38Trp)致病性，利用CRISPR/Cas9技术制备了EXD3 p.(Arg38Trp)基因条件性敲入小鼠，包含“CAG promoter-loxP-3*SV40pA-loxP-Kozak-Mutant human EXD3 CDS-rBGpA”供体载体,和Cas9 mRNA共注射到C57BL6受精卵，经PCR鉴定敲入成功的F0代与野生型小鼠繁育得到F1代小鼠两只:2#和4#。经过繁育目前已经得到2#纯合雄性和雌性子代各4只，4#纯合雄性子代4只，纯合雌性子代2只。纯合实验鼠与cre工具鼠合笼，繁育杂合EXD3+/-cre+/-或EXD3+/-cre+/0子代，后续将进行晶状体表型分析工作。.此外，全基因组连锁及单体型分析确定5q14.3区域是与家系F-#20疾病表型唯一连锁区域，在该区域家系患者单体型共享，两点连锁分析最大LOD为2.7，全基因组测序检测到RASA1和MEF2C基因内含子区点突变，与疾病表型共分离，其致病性有待后续功能研究证实。.研究结果对揭示白内障发生的分子基础，阐明其病理生理学机制，丰富晶状体发育的理论内容和指导白内障的预防等都具有重要意义。