α晶状体蛋白基因多态与年龄相关性白内障易感性及其机制的研究

Age-related cataract (ARC) is the leading cause of blindness in the world. The study of the gene polymorphism in the pathogenesis of this disease is just start. Our previous study showed that the chaperone function and phosphorylation of α crystallin are closely associated with the development of ARC, and gene polymorphisms will impact the protein function. Accordingly, we speculate that α crystallin gene polymorphism may play an important role in the pathogenesis of ARC. This study will detect the αA and αB crystallin gene polymorphism in ARC patients and control subjects using gene exon sequencing technique. Furthermore, plasmids of polymorphism will be constructed for clinically significant sites. Promoter efficiency, molecular chaperone function, antioxidant, anti-apoptosis and other biological phenotype will be detected in molecular, cellular, and population level respectively. The aim of this study is to detect the association between α crystallin gene polymorphism and the risk of ARC, to clarify the function of those polymorphisms, and to provide a theoretical basis of prevention and treatment of ARC.

年龄相关性白内障(ARC)是全球首位致盲性眼病，遗传因素与ARC发病关系的研究尚处于起步阶段。本项目组前期研究发现α晶状体蛋白分子基因多态性在ARC患者与对照间存在差异，同时发现基因多态性对蛋白质功能有重要影响，由此推测α晶状体蛋白基因多态性可能在ARC发病机制中发挥了重要作用。本研究拟提取ARC患者与对照静脉血基因组DNA，对αA和αB晶状体蛋白基因外显子、5'UTR、3'UTR区域进行测序，尽可能全面地检测出基因多态。并进一步构建ARC发病相关位点的等位基因质粒，对启动子区多态进行启动效率的检测，对3'UTR区多态进行miRNA分析，对外显子区改变氨基酸编码的多态进行分子伴侣功能、抗氧化能力、抗凋亡等生物学表型检测，分别在分子、细胞以及人群水平进行基因多态的功能验证。通过上述研究，力图阐明α晶状体蛋白基因多态性与ARC发病风险的关系，为早期干预与个体化治疗提供临床与实验依据。

通过对比年龄相关性白内障和对照组血液DNA序列，成功鉴定出CRYAA基因多态性位点rs3761381, rs13053109, rs7278468，rs76740365及CRYAB多态性位点-628位（A/G）、-224位(C/G)和-223位（A/G）。其中CRYAA基因中rs3761381和rs7278468与核性年龄相关性白内障显著相关、rs76740365与后囊下型年龄相关性白内障显著相关；CRYAB中多态性位点未显示与年龄相关性白内障存在相关关系。构建年龄相关性白内障（ARC）Haplotype分型发病风险模型（rs3761381-rs13053109-rs7278468）：发现分型T-G-T 是ARC的危险因素、分型 T-G-G是ARC的保护性因素。证实CRYAA基因rs7278468位点突变可影响启动子与转录因子Sp1结合强度， T>G突变增强Sp1与CRYAA启动子的结合强度，进而增强转录活性。rs76740365位点突变则是通过激活ERK和Src通路，增高细胞内活性氧水平，增加细胞凋亡及晶状体上皮细胞迁移能力，导致后发型白内障的发生发展。