【作者】 王玮；

【导师】 姚克；

【作者基本信息】 浙江大学 ， 眼科学， 2010， 博士

【摘要】 研究目的：先天性白内障是儿童盲的首要原因。大约1／3的先天性白内障由遗传突变引起,其中最常见的是常染色体显性遗传。目前我国进行了大量先天性白内障疾病相关候选基因及其突变方式的研究,研究成果丰富了疾病相关基因库。而有关突变基因功能的研究尚有待开展。本研究拟对一先天性核性粉尘样伴后极性白内障大家系进行疾病相关候选基因的定位及功能研究。在进行遗传方式分析后,提取家系成员血DNA并克隆疾病相关侯选基因,通过DNA测序明确突变基因及位点。通过DNA体外重组技术构建携带突变基因的真核表达质粒,转染真核细胞后,在体外水平研究基因突变引起的生物学效应,阐明其引起先天性白内障的分子机制。研究方法：家系全体成员经眼部及全身检查后,根据系谱行遗传方式分析。白内障手术中将先证者晶状体用灌吸方式吸出,使用透射电镜观察晶状体纤维细胞结构。提取家系全体成员静脉血并抽提基因组DNA,使用聚合酶链反应(PCR)克隆核性白内障相关基因,经DNA测序明确突变基因及位点后,利用高效液相色谱分析(DHPLC)鉴定基因突变。以正常人血基因组DNA为模板,通过PCR反应获取野生型目的基因并通过DNA重组技术克隆至真核表达载体pEGFPN1。利用定点突变技术构建携带突变基因的质粒,分别转染至人晶状体上皮细胞(HLEB-3)及宫颈癌上皮细胞(HeLa),激光共聚焦显微镜下观察蛋白定位；免疫荧光技术检测晶状体上皮细胞中Connexin43 (Cx43)的表达分布及缝隙连接形成情况；使用G418抗生素筛选,建立稳定表达突变蛋白的细胞系；流式细胞分析术检测转染细胞的凋亡情况；Western-blot法检测凋亡相关蛋白caspase3的表达。研究结果：该家系为常染色体显性遗传,所有患者晶状体均表现为特殊的核性粉尘样伴后极性浑浊。透射电镜观察发现,先证者晶状体纤维细胞中存在小球样物质沉积。DNA测序示GJA3基因第2号外显子第5个碱基发生了G→A的置换(c.5G>A),该突变使得GJA3编码的Connexin46(Cx46)蛋白N端第二个氨基酸发生甘氨酸到天冬酰胺的转变(p.G2N)。高效液相色谱分析(DHPLC)验证了该基因突变的存在。分别成功构建表达野生型Cx46蛋白的质粒pEGFPN1-wtCx46及表达突变蛋白的pEGFPN1-Cx46G2N,转染HeLa及HLEB-3细胞后发现突变蛋白呈团块样聚集,这种蛋白的聚集导致缝隙连接形成失败,并影响了Cx43形成缝隙连接的能力。G418筛选培养一周后,突变基因转染细胞全部死亡,提示该突变具有致死性。流式细胞分析证实了细胞凋亡比例增多,Western-blot发现总caspase3表达降低,而剪切的caspase3增多,提示了细胞凋亡通路的激活。结论：本研究首次发现GJA3基因N端的一个新的先天性白内障疾病相关点突变G2N。该突变可以导致蛋白的聚集、缝隙连接形成失败,进而引起细胞凋亡。本研究拓展了先天性白内障的基因突变谱,揭示了缝隙连接蛋白突变导致先天性白内障发病的新机制。同时提示Cx46蛋白N端在维持晶状体正常生理状态中担当重要作用,为先天性白内障的预防和基因治疗提供了理论基础。更多还原

【Abstract】 Purpose:Congenital cataract is the leading cause of visual disability in children worldwide. Approximately 1/3 of congenital cataracts are inherited, with the most common being the autosomal dominant form.Up to now, researchers in our courtury have done so much work on the localization of disease-associated genes of congenital cataract families. Functional detection of mutant genes is needed. Here, we studied on a family affected with congenital nuclear pulverulent and posterior polar cataract by localizaing and functional detecting of the disease- associated gene. Genomic DNA was extracted from the blood after the analysis of inheritance mode. Mutation was detected by gene cloning and DNA sequencing.Plasmid with the mutant gene was constructed, Molecular mechanisms by which this mutation caused congenital cataract were detected by gene transfection into cells. Method:The family with congenital cataracts was ascertained by ophthalmologic examination. Inherit mode was analyzed through the pedigree. Lens material was aspirated by irrigation and aspiration during cataract surgery from the proband. and was examined using transmission electron microscopy. Genomic DNA was extracted from the blood. Nuclear cataract-associated gene was amplified and sequenced directly.the mutation was verified by denaturing high-performance liquid chromatography (DHPLC). Wild type genes were PCR amplified from genomic DNA, then the amplifications were subcloned into pEGFP-N1.Quick change site directed mutagenesis was performed to construct the mutant plasmid. Behavior and cellular distribution of connexins were examined by expression in human lens epithelial cells(HLEB-3) and HeLa cells:protein distribution was observed by confocal fluorescence microscopy; distribution and function of Cx43 were detected by immunofiuorescene; Stably transfected clones were selected by their resistance to G418;cell apoptosis were detected by flow cytometry analysis and Western-blot.Results:We identified a four generation family with autosomal dominant congenital cataracts. The phenotype of the affected individuals’cataract was unique:not only were they nuclear pulverulent, but also exhibited posterior polar opacity. There were foci of dense, globular intracellular deposits in the proband’s lens fiber cells. Direct sequencing of the GJA3 gene revealed a novel heterozygous transition G→A at position 5(c.5G>A) in the affected individuals, resulting in the replacement of highly conserved Glycine by Aspaingine (p.G2N).DHPLC analysis confirmed this mutation. We successfully constructed plasmids with wt Cx46 and Cx46G2N. After transfection into HeLa and HLEB-3 cells, we detected protein aggregation, which inhibited the formation of gap junction plaques, and disrupted the activity of Cx43. After selected by G418 for about 1 week, all of the cells transfected with Cx46G2N died.This indicated that the mutation lead to cell apotosis. Flow cytometry analysis showed increased cell apoptosis. Immunoblotting showed a decreased level of caspase3 and increased level of cleaved caspase3 in the Cx46G2N group compared to wt Cx46. indicating the activity of caspase3.Conclusion:We identified a novel congenital cataract associated mutation, G2N. in the N terminal of the GJA3 gene. This mutation may cause protein aggregation, reduce gap junction plaques and influence intercellular communication, thus leading to cell apoptosis. Our study widened the mutation spectrum of congenital cataract, represented a novel mechanism by which mutant connexin can cause cataracts. It also suggested a potential contribution of the Cx46 NH2- terminal in maintaining lens homeostasis, provided more theoretical basis for the prevention and gene therapy of congenital cataracts.更多还原