【作者】 饶艳；

【导师】 郑芳；

【作者基本信息】 武汉大学 ， 临床医学 临床检验诊断学， 2017， 博士

【摘要】 目的:先天性白内障是目前世界范围内儿童视力缺损的主要病因之一。遗传变异是最常见的先天性白内障致病因素。本研究旨在对两例先天性核性白内障家系进行疾病相关候选基因的定位以及进一步的蛋白功能研究。方法:本实验拟研究在武汉大学中南医院眼科和东莞市妇幼保健院儿保科收集的两个先天性白内障家系。所有家系成员均接受详细的病史调查和眼科检查,包括视力、裂隙灯、散瞳眼底检查等。采集家系患者和健康成员的外周血样本,提取基因组DNA。在湖北石首白内障家系中采用候选热点基因聚合酶链反应(PCR)-直接测序的方法进行致病基因筛查。发现致病基因突变后进一步构建野生型和突变型原核表达载体,利用生物化学和生物物理学方法对纯化的蛋白进行理化特性的研究。运用靶向外显子组重测序的策略对广东东莞白内障家系进行致病基因定位研究,借助SIFT、PolyPhen-2等生物信息学工具对突变型蛋白进行结构和功能的预测分析。结果:在湖北石首白内障家系中,患者临床表型为核性白内障,伴有眼球震颤,该家系为常染色体显性遗传。我们在该家系中发现了 CRYBB1基因一个新的无义突变c.C679T,p.Q227X,此突变导致第227位进化高度保守的谷氨酰胺被终止密码子所取代,进而截短野生型βB1晶状体蛋白(βB1 crystalline)使其C末端结构域缺失26个氨基酸。野生型和突变型蛋白的理化性质研究表明p.Q227X突变可以引起突变蛋白的构象改变,可溶性下降,稳定性被破坏。同时突变蛋白在热应激状态下对于αA晶状体(αAcrystalline)蛋白的分子伴侣保护效应也有所降低。在广东东莞白内障家系中患者临床表型也表现为核性白内障,不伴其他眼部或系统性疾病,该家系的疾病遗传方式为常染色体显性遗传。靶向重测序结果提示在该家系中发现了 GJA8基因的一个新的错义突变c.C565G,p.P189A,此突变导致缝隙连接蛋白50(Connexin50,Cx50)第189位进化高度保守的脯氨酸突变为丙氨酸。SIFT,PolyPhen-2预测结果均表明p.P189A为可影响蛋白质结构和功能的有意义的突变。结论:本研究中我们发现了两个与常染色体显性遗传核性白内障相关的新突变(CRYBB1 基因 c.C679T,p.Q227X 和 GJA8 基因 c.C565G,p.P189A),p.Q227X突变的功能研究表明βB1晶状体蛋白的C末端在维持蛋白的稳定和功能中发挥着重要作用,揭示了该突变引发白内障的分子机制。本研究进一步丰富了先天性白内障的致病基因突变谱表,为先天性白内障分子致病机制研究奠定了一定的理论基础。更多还原

【Abstract】 Purpose:Congenital cataract,one of the leading causes of treatable blindness in children worldwide.Genetic mutation is the most common cause for congenital cataract,we aim to identify the disease-causing gene in two families with autosomal dominat congenital nuclear cataract and explore the molecular mechanism of cataractogensis.Method:Two families affected with congenital cataracts were recruited in Zhongnan hospital,Wuhan university and Dongguan maternal and child care hospital.All the family members were determined by history records and ophthalmic examination including visual acuity,slit-lamp examination,fundus examination.The genomic DNA samples were extracted from peripheral blood of all the members.We applie the PCR-direct sequencing method to screen the candidate genes associated with the cataract family in Shishou,Hubei.The wild type and mutant type expression plasmids(pET-28a-WT and pET-28a-MU)were constructed for further Biophysical studies of the recombinant full-length βB1(WT)and mutant(MU)crystalline.We utilize targeted exome sequencing(TES)to screen all known cataract genes or locus for the disease-causing mutation in the proband and Sanger sequencing was used to confirm the potential pathogenic variants in the family in Dongguan,Guangdong.Computational analysis,including SIFT,PolyPhen2 and conservative analysis are used to predict whether the amino acid substitution of the protein sequence is deleterious to the function of the protein.Results:The phenotype of the cataract of the affected individuals in the family in Shishou was characterized as nuclear opacity with nystagmus.The inheritance pattern of the family is autosomal dominant.We identified a c.C749T(p.Q227X)transversion in exon 6 of CRYBB1,a cataract-causative gene.This nonsense mutation changes a phylogenetically conserved glutamine to a stop codon and is predicted to truncate the C-terminus of the wild-type protein by 26 amino acids.Comparison of the biophysical and biochemical properties of the recombinant full-length and truncated βB1 crystallins revealed that the mutation led to the insolubility and the phase separation phenomenon of the truncated protein with a changed conformation.Meanwhile,the thermal stability of the truncated βB1-crystallin was significantly decreased,and the mutation diminished the chaperoning ability of aA-crystallin with the mutant under heating stress.The phenotype of the cataract of the affected individuals in the family in Dongguan also was nuclear cataract.We successfully identified a novel c.C565G mutation in exon 2 of GJA8 gene.The mutation showed perfect co-segregation with the disease phenotype in the family but was not observed in 100 controls using Sanger sequencing.The heterozygous c.C565G mutation lead to the substitution of a highly conserved proline by alanine at codon 189(p.P189A)in the coding region of connexin50(Cx50).Moreover,the mutation was predicted be deleterious by both PolyPhen-2 and SIFT with consistent results.Conclusions:This study has identified two novel mutation(c.C679T,p.Q227X of CRYBB1 gene and c.C565G,p.P189A of GJA8 gene).Our findings highlight the importance of the C-terminus in βB1 crystallin in maintaining the crystalline function and stability,and provide a novel insight into the molecular mechanism underlying the pathogenesis of human autosomal dominant congenital cataract.更多还原