METHODS: A four-generation Chinese family affected with autosomal dominant congenital cataract was recruited. Nineteen individuals took part in this study including 5 affected and 14 unaffected individuals. Sanger sequencing targeted hot-spot regions of 27 congenital cataract-causing genes for variant discovery. The pathogenicity of the variant was evaluated by the guidelines of American College of Medical Genetics and InterVar software. Confocal microscopy was applied to detect the subcellular localization of fluorescence-labeled ephrin type-A receptor 2 (EPHA2). Co-immunoprecipitation assay was implemented to estimate the interaction between EphA2 and other lens membrane proteins. The mRNA and protein expression were analyzed by reverse transcription-polymerase chain reaction (qRT-PCR) and Western blotting assay, respectively. The cell migration was analyzed by wound healing assay. Zebrafish model was generated by ectopic expression of human EPHA2/p.R957P mutant to demonstrate whether the mutant could cause lens opacity in vivo.
RESULTS: A novel missense and pathogenic variant c.2870G>C was identified in the sterile alpha motif (SAM) domain of EPHA2. Functional studies demonstrated the variant\'s impact: reduced EPHA2 protein expression, altered subcellular localization, and disrupted interactions with other lens membrane proteins. This mutant notably enhanced human lens epithelial cell migration, and induced a central cloudy region and roughness in zebrafish lenses with ectopic expression of human EPHA2/p.R957P mutant under differential interference contrast (DIC) optics.
CONCLUSIONS: Novel pathogenic c.2870G>C variant of EPHA2 in a Chinese congenital cataract family contributes to disease pathogenesis.
方法:招募一个患有常染色体显性遗传先天性白内障的四代中国家庭。19个人参加了这项研究,其中包括5名受影响的个人和14名未受影响的个人。Sanger测序针对27个先天性白内障致病基因的热点区域进行变异发现。变异体的致病性通过美国医学遗传学学会和InterVar软件的指南进行评估。应用共聚焦显微镜检测荧光标记的ephrinA型受体2(EPHA2)的亚细胞定位。实施免疫共沉淀测定以估计EphA2与其他晶状体膜蛋白之间的相互作用。通过逆转录-聚合酶链反应(qRT-PCR)和蛋白质印迹分析mRNA和蛋白表达,分别。通过伤口愈合测定分析细胞迁移。通过人EPHA2/p的异位表达产生斑马鱼模型。R957P突变体证明突变体是否可以在体内引起晶状体混浊。
结果:在EPHA2的无菌α基序(SAM)结构域中鉴定出一种新的错义和致病性变体c.2870G>C。功能研究表明变体的影响:降低EPHA2蛋白表达,亚细胞定位改变,并破坏了与其他晶状体膜蛋白的相互作用。这种突变体显著增强了人晶状体上皮细胞的迁移,并在斑马鱼晶状体中引起中心混浊区域和粗糙度,并异位表达人EPHA2/p。微分干涉对比(DIC)光学下的R957P突变体。
结论:中国先天性白内障家族中EPHA2的新型致病c.2870G>C变体参与了疾病的发病机制。