【作者】 马志伟；

【导师】 孙慧敏；

【作者基本信息】 天津医科大学 ， 眼科学， 2006， 博士

【摘要】 目的:白内障是人类主要的致盲眼病,每10,000个新生儿就有1-6个先天性白内障,其中1/3是遗传性的。有三种不同的遗传方式:常染色体显性遗传,常染色体隐性遗传,性染色体连锁遗传,常染色体显性遗传是先天性白内障最常见的遗传方式。1963年,Renwick和Lawler在研究中发现一常染色体遗传性带样粉尘状白内障与杜非血型(Duffy blood group)位点发生共分离现象,1968杜非血型定位在1号染色体,先天性白内障也随之获得了第一个常染色体遗传位点。随着分子遗传学的发展,已不断发现几十个新的先天性白内障遗传位点,现在已明确18个白内障致病基因。但是基因型和表现型的关系尚不明确,遗传性和老年性白内障的关系还不清楚,对真正了解这一疾病的发病机制和防治还有很大的距离。我们通过对一个患皮质性进展性白内障的大家系,行致病基因定位和筛查的研究,希望发现可影响晶状体的相关基因,并探索其发病机制。 方法: 1.随访一个连续六代发病的白内障家系,签订知情同意书后,采集临床资料及血样标本,绘制系谱图,进行经典遗传学分析和细胞遗传学检查。 2.提取家系成员的基因组DNA,采用定位克隆的策略,首先在15个已知白内障基因所在的位点,每个位点选取3-4个微卫星标记排除已知基因。然后利用全基因组扫描试剂盒(ABI),共382个微卫星标记,每个间距约～10cM,初步定位。在定位区选取更多的微卫星标记,从Marshfield遗传数据库确定标记的顺序和位置。基因型和数据的收集利用ABI Prism GeneMapper(V3.0),利用LINKAGE(V5.10)中MLINK程序行两点连锁分析,计算Lod值。家系图和单体型的构建用Cyrillic(V2.1)软件。 3.根据物理图谱,选择定位区域内的候选致病基因,Genebank搜寻候选基因更多还原

【Abstract】 Objective: Cataract remains the leading cause of human blindness worldwide. Congenital cataracts have an estimated frequency of 1 -6 per 10,000 live births, with one third of cases are familial. Congenital cataracts have been reported with all three types of Mendelian inheritance, autosomal dominant, autosomal recessive, and X-linked. Autosomal dominant congenital cataracts with high penetrance appear to be the most common in affected individuals. The autosomal dominant zonular pulverulent cataract (CAE1) locus was initially linked with the Duffy blood-group locus (Renwick and Lawler 1963), and, in 1968, the Duffy bloodgroup locus was assigned to chromosome 1. This mapped the CAE1 locus to human chromosome 1 (Donahue et al. 1968). To date, more than twenty autosomal dominant congenital cataract loci have been mapped to different chromosomal. Recent work in molecular genetics has identified 18 genes involved in the pathogenesis of isolated inherited cataract. However both the relation of genotype and phenotype and of hereditary and age related cataract are unclear. There is a long way to understand the mechenism and precaution of cataract. We studied a family suffering form cortical progressive cataract and. We carried out gene mapping and mutation screening and tried to identify the pathogenic gene and mechenism of cataract. Moreover cataractogenesis and its eventual therapeutic amelioration were investigated. Methods:1. We investigated a six-generation cataract family. After obtaining informed consent Clinical information and blood specimens were obtained from familymembers. Then we performed Classic genetics and cytogenetics analysis.2. Genomic DNA was extracted from peripheral blood leucocytes. Firstly, we studied 11 loci for known candidate genes using three or four fluorescent microsatellite markers per locus and no evidence of linkage was detected. Subsequently a genomewide scan consisting of 382 microsatellite markers spaced at -10 cM intervals was performed using ABI PRISM Linkage Mapping sets. At last we performed fine mapping. The markers’ order and position were obtained from the Marshfield Genetic Database. Genotyping and data collection were conducted by ABI Prism GeneMapper v 3.0 software. We carried out two-point linkage analysis using the MLINK program from the LINKAGE v.5.10 software package. Pedigree and haplotype construction were performed by Cyrillic v.2.1 software.3. Candidate genes were selected in the physical map. The sequences of the genes were got from genebank database. The primer was designed by Primer 3 software. We screened the mutation of candidate genes by bidirectional sequencing PCR products.4. We perform bioinformatics analysis of mutant protein, including physical character, function, structure and et al.Results:1. We studied a Chinese six-generation cataract family composed of 119 individuals with a dominant pattern of inheritance. Clinical information and blood specimens were obtained from 64 family members, including 14 patients. The phenotype in this family was characterized by opacities in cortex. The opacities can be located in anterior cortex, posterior or peripheral cortex but no opacity was observed in the fetal nuclear region. Moreover this cataract is of a progressive nature, andcataractous changes were prominent in affected older individuals. Phenotypic variation in the size and density of opacities as well as the position was observed among the 14 affected members, even between the two lenses of an individual. From the pedigree, a autosome dominant pattern of inheritance was recognized. Chromosome2. After gene mapping and haplotype were performed, we identified a new autosomal dominant congenital cataract locus on chromosome 3q26.3-qter. The disease gene lay in the approximately 2.8 Mb physical intervals between D3S1571 and D3S3570. Linkage analysis gave a maximum two point LOD score 6.34 (9=0.00) for marker D3S1602.3. By sequencing CRYGS gene on two directions, we identified a 105G—>T (NMO17541) heterozygous mutation in exon 2 of CRYGS, resulting in a Gly-?Val substitution at codon 18 (NP060011) in 14 affected members. This mutation was not found in 50 unaffected members in this family, neither in 400 chromosomes of 200 unrelated control individuals, excluding the possibility of a rare polymorphism.4. we perform bioinformatics analysis of mutant protein, including function and structure. It has shown that the four-stranded greek key P-sheet peptide corresponding to crystallin fold forms an individual calcium-binding site, and the first calcium ligate at the residue next to the conserved aromatic amino acid of the sequence Y/E/WXXXXXXG, which is located at the end of the first p-strand. Gly is needed for forming a dihedral angle so it is irreplaceable. In this family just this Gly mutated, which may lead to structural alterations in the Ca2+-binding and -storing ability. Additionally, prediction of function sequence detect that "G[RK][RK]" is Amidation site. Gly mutatation may disturb this function, andthen effect the relation between crystallins. These might be novel mechanisms of cataract formation. Understanding non-structural properties of crystallins may be critical for understanding the malfunction in molecular cascades that lead to cataractogenesis and its eventual therapeutic amelioration. It may help to have an understanding of the disease etiology and contribute to better understanding of the function and properties of this gene. Perhaps this study of progressive phenotype and CRYGS mutation may provide insight into the cause of the more common sporadic form of age-related cataracts.Conclusion: Gamma-S crystalline gene (CRYGS) mutation causes hereditary cortical progressive cataract. This report is the first description of a mutation in CRYGS in humans and added another gene to growing list of genes involved in this heterogeneous monogenic disorder.更多还原