基于高通量测序和连锁分析鉴定先天性白内障致病基因

Congenital cataract is a leading cause of visual disability in children, and approximately half of them are genetically determined. A total of 18 causative genes associated with autosomal dominant congenital cataracts have been identified to date. However, we have previously found that all of the 18 causative genes can only account for 20% of the congenital cataract cases, which indicates that more important genes remain to be discovered. In this study, we captured exomes of eight individuals from two independent Chinese families with congenital cataracts using the platform of Nimblegen exome capture, and performed high-throughput sequencing with a mean coverage of 50× and sufficient depth to call variants at 99% of each targeted exome. We will exclude the candidate genes by bioinformatics analysis in the next step. The possible causative genes will be identified using a combined strategy of exome sequencing and linkage analysis in the two families. To further confirm it, 300 control individuals of matched geographical ancestry and 50 additional unrelated autosomal dominant congenital cataract families where the known causative gene mutations have been excluded will be examined using Sanger sequencing or MassARRAY. Recombinant wildtype and mutant constructs of the novel causative gene will be synthesized. Triton X-100 solubility, cell viability and subcellular localization of the recombinant proteins will be analyzed in lens epithelial B3 cells. The further functional study will further validate the causative gene. The identification of the novel causative gene will not only improve our understanding of the mechanism of congenital cataract formation, but will lay the foundation for prenatal diagnosis, prevention and new treatment of congenital cataracts.

先天性白内障是儿童首位致盲性眼病，已有研究表明50%与遗传有关，截止目前已发现18个致病基因与常染色体显性遗传性白内障有关。但是，我们在前期工作中发现目前已知的致病基因仅能解释20%先天性白内障的发生，说明存在更重要的基因在先天性白内障发病中起作用。我们基于2个已排除已知致病基因的先天性白内障家系，通过NimbleGen 液相捕获平台进行外显子组测序，目标外显子区域测序深度50X以上，覆盖度高达99%。本研究拟进一步对测序数据进行高级生物信息学分析筛选候选基因，利用高通量测序和连锁分析相结合的策略，在家系内、另外50个先天性白内障家系及300正常对照对候选基因进行验证。构建真核表达体系，运用细胞生物学手段研究突变对蛋白质溶解性、亚细胞定位及细胞凋亡的影响，对其进行功能验证，最终确定致病基因。研究成果将为阐明先天性白内障发病机制、开展其产前诊断、预防和基因治疗奠定基础。

先天性白内障是儿童首位致盲性眼病，已有研究表明50%与遗传有关，截止目前已发现18个致病基因与常染色体显性遗传性白内障有关。但是，我们在前期工作中发现目前已知的致病基因仅能解释20%先天性白内障的发生，说明存在更重要的基因在先天性白内障发病中起作用。我们基于2 个已排除已知致病基因的先天性白内障家系，通过NimbleGen 液相捕获平台进行外显子组测序，目标外显子区域测序深度50X 以上，覆盖度高达99%。本研究进一步对测序数据进行高级生物信息学分析筛选候选基因，通过高通量测序和连锁分析相结合的策略，在两个家系中分别发现新的致病基因A和CRYGD P24T，连锁分析显示，最大LOD值均>2，同时在另外64个先天性白内障家系及300正常对照对候选基因进行进一步验证。利用斑马鱼模型进行体内功能验证，通过吗啉寡聚核苷酸敲低致病基因A的表达，结果显示野生型和MO型在光学显微镜和投射电镜下晶状体透明度无明显差别，该结果进一步证实了该突变的功能不是失去功能而是获得功能，下一步我们将利用基因突变敲入小鼠模型进一步研究。本研究首次发现新的致病基因A与先天性白内障有关，将为阐明先天性白内障发病机制、开展其产前诊断、预防和基因治疗奠定基础。