FYCO1 功能与白内障发病的分子机制研究

FYCO1 plays a role in autophagy but its exact function is not still very clear. Our previous study showed that mutations in FYCO1 cause congenital cataract (Am J Hum Genet, published). Congenital cataract is clinically and hereditarily heterogeneous. Therefore its pathogenesis should still be worth studying. Recently it is generally accepted that autophagy modulation may be as a potential therapeutic target for diverse diseases. We plan to explore the function of FYCO1 and its role in the pathogenesis of cataract. We will construct expression recombinant of FYCO1 gene and siRNA targeting against FYCO1, transfect it into Human lens epithelial cell (HLE) expressing LC3 and make transfected cell under starvation to test the effect of siRNA mediated FYCO1 knockdown. Further, we will test whether abnormal FYCO1 expression affect protein and organelles degradation by autophagy like mitochondria. we will perform yeast two-hybrid screens of cDNA library from mouse using full-length FYCO1 as bait to identify FYCO1 partners. Following, we perform GST pull down or Co-immunoprecipitation plus colocalization to ensure protein-protein interaction. And we will construct mutants with the mutations found in cataract families to test whether protein-protein interaction is impaired or not. In addition, FYCO1 knock out mice will be established and their phenotype will be analyzed. Autophagy activity will detected in cells from different tissue. The change of histology will be checked in mouse whole eye. Changes in lens expression especially of cataract genes and key genes of different stages in autophagy will be detected. And those genes that will have been found changed and organelles markers will be detected in in mouse whole eye specimens from different stages to elucidate FYCO1 in the pathogenesis of cataract.This study may provide a new cellular and molecular entry point for understanding lens transparency and eventually better cataract prevention or non-surgery treatment. Meanwhile, it may be helpful to characterize machinery in autophagy pathway.

FYCO1参与自噬，但具体功能尚不清楚。申请人首次在多个先天性白内障(CC）家系中发现FYCO1缺陷是其致病原因（已发表于Am J Hum Genet）。CC高度遗传及临床异质性，其致病分子研究仍必要。调节自噬已被认为是多种疾病治疗的潜在靶点。本研究拟建立FYCO1表达异常的人晶体上皮细胞(HLE),在饥饿状态或试剂诱导下诱发自噬，观察FYCO1对HLE自噬活动的影响，然后进一步具体研究对蛋白自噬和细胞器自噬的影响。借助酵母双杂交系统筛选并鉴定小鼠晶状体cDNA文库中FYCO1结合蛋白，构建突变体并检测突变是否影响蛋白间结合。建立FYCO1基因敲除小鼠并分析表型，观察体内组织细胞自噬情况及FYCO1缺陷对自噬影响；晶状体切片病理并检测晶状体白内障基因、细胞器标志性基因及自噬过程关键性基因表达变化，揭示FYCO1缺陷在白内障发病的机制。为白内障非手术治疗奠定基础，同时丰富自噬通路分子机制。

先天性白内障的主要因素之一是遗传，它是一组具有高度遗传和临床异质性的单基因遗传病。课题负责人在前期研究中首次发现FYCO1缺陷是先天性白内障致病原因，但FYCO1的分子发病机制仍不清楚。我们采用CRISPR-Cas9编辑技术完成Fyco1基因敲除小鼠的建立，课题按照既定方案，围绕课题宗旨（探讨FYCO1的分子发病机制）对获得的Fyco1基因敲除的小鼠进行一系列视力分析、组织切片分析等，揭示FYCO1缺陷在白内障发病的机制；同时，对已收集到的100例先天性白内障散发病例和或家系中先证者进行FYCO1突变筛查，在一个家系中鉴定了FYCO1突变，这将是在中国人群中首次发现。通过研究FYCO1分子的互作蛋白，我们发现一个在晶状体发育过程中起重要作用的基因，并且发现该基因的突变在一个白内障家系中共分离，已用靶向高通量测序排除该家系已知白内障致病基因的可能，并通过外显子测序进一步验证该基因突变是这个家系的致病原因。