【作者】 陈强；

【导师】 郑芳；

【作者基本信息】 武汉大学 ， 临床检验诊断学， 2010， 博士

【摘要】 背景：白内障是全球首要致盲眼病。其中,先天性白内障是一种常见的儿童眼病。环境和遗传因素是先天性白内障的两大病因,其中遗传因素在白内障的发生和发展中起着重要作用。随着分子遗传学技术的发展,越来越多的先天性白内障相关候选基因被定位和克隆。然而,先天性白内障的发病机制还研究得不是很清楚。本研究拟对一个先天性白内障家系进行致病基因的筛查及致病机制研究,以期找到先天性白内障相关的突变位点,揭示先天性白内障的发病机理。方法：采用直接测序的方法对一个来自湖北省的常染色体显性遗传先天性白内障家系进行基因突变检测。应用生物信息学工具Expasy蛋白组学服务器(http://www.expasy.org)对aB-晶体蛋白及其突变体进行生物物理学预测。应用基因重组技术在大肠杆菌E. coli BL21(DE3)中表达野生型和R11H突变型aB-晶状体蛋白。应用圆二色光谱技术对野生型和R11H突变型aB-晶状体蛋白的结构进行分析；应用bis-ANS荧光探针结合实验分析aB-晶状体蛋白的表面疏水区域；分别以胰岛素和乙醇脱氢酶(ADH)为底物比较它们的分子伴侣活性。此外利用脂质体方法分别将野生型和R11H突变型CRYAB基因转入HeLa细胞和人晶状体上皮细胞(HLE),应用激光共聚焦显微镜和流式细胞仪对野生型和R11H突变型aB-晶状体蛋白的特征进行了比较分析。通过优化载体,诱导时间,诱导温度,诱导剂浓度等条件摸索aA-晶状体蛋白在大肠杆菌中的表达条件。结果：候选基因直接测序在aB-晶状体蛋白基因(CRYAB)第一个外显子上发现了一个新的突变位点,该突变导致第11位密码子由精氨酸变成了组氨酸(R11H)。在家系中正常人和200例正常对照及40例老年性白内障患者中均未发现此突变,排除了多态性的可能。aB-晶状体蛋白计算机模型的构建和分析提示突变型aB-晶状体蛋白基因改变了该蛋白的结构及理化性质。R11H突变型和野生型aB-晶状体蛋白的近紫外和远紫外圆二色光谱呈现较大差异。bis-ANS荧光光谱实验显示突变型蛋白的表面疏水区域减少。而热稳定性测定表明R11H突变并没有改变蛋白的稳定性。分子伴侣活性测定表明突变型aB-晶状体蛋白的分子伴侣活性升高。激光共聚焦显微镜分析表明野生型和R11H突变型aB-晶状体蛋白都主要分布在细胞质里面,两者在细胞内的定位无明显差异。流式细胞术分析表明R11H突变型aB-晶状体蛋白能诱导晶状体上皮细胞的早期凋亡。利用pET-32a(+)重组表达菌株,在0.5 mM IPTG,37℃下诱导5 h成功表达并纯化得到aA-晶状体蛋白。结论：在一常染色体显性遗传先天性白内障家系中发现了一个新的CRYAB基因突变(R11H)。突变型aB-晶状体蛋白基因改变了蛋白的结构,导致aB-晶状体蛋白的分子伴侣活性升高。同时突变基因能诱导晶状体上皮细胞的早期凋亡。这种结构与功能上的改变可能与白内障的发生相关。更多还原

【Abstract】 Background Cataract is a leading cause of blindness worldwide and congenital cataract is a common eye disease that a child is born with or develops soon after birth. Genetic factors as well as environmental factors play a significant part in cataract aetiology and about one third of all congenital cataracts are inherited. With the development of advanced molecular biological techniques, more and more genes causing congenital cataracts have been identified; however, the underlying mechanisms are not fully understood. The aim of this study was to identify the causative genetic mutation among the known cataract candidate genes in a Chinese family and to investigate the molecular mechanism of cataract that may develop due to the mutation.Methods Because of the small family size, we used the functional candidate gene analysis approach. We screened the Chinese family, clinically documented to have congenital nuclear cataracts, for mutation in the candidate genes CRYG (C & D), GJA8, CRY (AA & AB), CRYBA and CRY (BB1 & BB2) through polymerase chain reaction analyses and sequencing. Biophysical predictions of the altered protein were analyzed using the Bioinformatics tool of the Expasy Proteomics server (http://www.expasy.org). Recombinant wild-type and R11H mutant aB-crystallin were expressed in E.coli BL21 (DE3) and purified to homogeneity. The recombinant wild-type and mutant aB-crystallin were characterized by UV circular dichroism, bis-ANS fluorescence and thermal stability. The chaperone activities were measured using insulin and alcohol dehydrogenase (ADH) as substrates. Furthermore, wild-type and R11HαB-crystallin were expressed in HeLa and human lens epithelial (HLE) cells. The functional characteristics of mutant aB-crystallin were examined using confocal fluorescence microscopy and flow cytometry in comparison with wild-type aB-crystallin. The optimal condition for the expression of aA-crystallin in E.coli was studied. Results Sequencing of the exons of the CRYAB gene identified a sequence variation in exon 1 (32 G>A) with a substitution of Arg to His at position 11. All affected family members revealed this change but it was not observed in any of the unaffected members of the family or in the randomly selected 200 DNA samples from ophthalmologically normal individuals and in 40 unrelated senile cataract patients of the same ethnic background of the family members. Computer-assisted prediction suggested that this mutation affected the biochemical properties as well as structure of aB-crystallin. The R11H mutant exhibited altered structures, decreased surface hydrophobicity and enhanced chaperone-like activity compared with the wild-type aB-crystallin. It was remarkably similar to the wild-type protein in its subcellular distribution and thermal stability, but it resulted in increased cell apoptosis. After optimization of aA-crystallin expression in E.coli, recombinant aA-crystallin was purified by Ni-NTA sepharose resin affinity chromatography under natural conditions.Conclusions This study identified a novel mutation in CRYAB gene (R11H) in a Chinese family with autosomal dominant congenital cataract. The mutation in aB-crystallin resulted in altered folding, decrease in hydrophobicity and abnormal ability to induce cell death which could contribute to turbidity and loss of transparency of the lens.更多还原