先天性绕核性白内障家系的疾病相关候选基因定位及功能研究

发布时间：2018-03-27 15:38

本文选题：先天性白内障　切入点：基因突变　出处：《浙江大学》2014年博士论文

【摘要】：目的：先天性白内障是儿童期视力缺损的首要病因之一。遗传突变是最常见的致病因素。迄今为止,已发现与遗传性先天性白内障相关约37条致病基因、数百个突变位点。本实验对两个绕核性表型的先天性白内障家系进行疾病相关候选基因定位及功能研究。方法：本实验拟研究在浙江大学附属第二医院眼科中心收集的两个先天性白内障家系。所有家系成员均接受病史调查和眼科检查,包括视力、裂隙灯、散瞳眼底检查等。抽取外周血样本,提取基因组DNA。聚合酶链反应(PCR)扩增相关的候选基因的编码区及内含子、外显子交界区。扩增产物双向测序,并进行序列分析。SIFT, PolyPhen-2和疏水性分析预测突变对蛋白质结构、功能的影响。从人眼cDNA文库中获得目的基因,通过DNA重组技术克隆至真核表达载体pEGFP-N1,构建野生型质粒。利用定点突变技术构建携带突变基因的质粒。将野生组(Cx46wt-EGFP)、突变组质粒(Cx46H95Y-EGFP、Cx46R76H-EGFP)及空白质粒(pEGFP-N1)转染入Hek293细胞,G418抗生素筛选,建立稳定转染细胞系,观察蛋白亚细胞定位和缝隙连接形成情况。在低钙、生理钙及缝隙连接通道抑制剂环境下,运用极性染料染核技术,检测野生组与突变组半通道功能。结果：裂隙灯下检查两个家系表型皆为绕核性。分析家系图,遗传方式均为常染色体显性遗传。测序结果：家系一：发现CX46基因编码区一条等位基因的第283位碱基由胞嘧啶变为胸腺嘧啶(c.283CT),导致了第95位氨基酸由高保守的组氨酸变为酪氨酸(p.H95Y)。家系二：发现CX46基因编码区一条等位基因的第227位碱基由鸟嘌呤变为腺嘌呤(c.227GA),导致了第76位氨基酸由高保守的精氨酸变为组氨酸(p.R76H)。这两个碱基改变在相应的家系患者中共分离,而不存在于健康的家族成员及对照组100名中国汉族健康者。SIFT, PolyPhen-2均提示Cx46-R76H、H95Y为可影响蛋白质结构和功能的有意义的突变。疏水性分析结果显示H95Y突变蛋白在突变区域的疏水性高于野生型缝隙连接蛋白46,而R76H突变蛋白疏水性低于野生型。在突变的功能研究部分,我们成功建立了稳定转染Cx46wt-EGFP, Cx46H95Y-EGFP, Cx46R76H-EGFP, pEGFP-N1质粒的Hek293细胞系。在野生组和H95Y突变组细胞中都能观察到缝隙连接的形成,但H95Y突变组的缝隙连接数量明显少于野生组。而R76H突变组极少观察到缝隙连接的形成。Cx46H95Y-EGFP, Cx46R76H-EGFP两组突变细胞质中均可见高荧光聚集。在半通道功能检测中,当低钙环境下,野生组细胞因有正常缝隙连接半通道的存在,可摄取PI和DAPI两种染料,而Cx46H95Y-EGFP, Cx46R76H-EGFP突变组和pEGFP-N1空白组细胞不能摄取；当在生理钙和缝隙连接蛋白通道抑制剂环境下,四组细胞均不能摄取染料。结论：本研究发现了与绕核性先天性白内障相关的两个CX46基因错义突变：其中,c.283CT为首次报道的新突变,c.227GA为已报道的突变。Cx46蛋白H95Y和R76H突变可导致蛋白胞质内聚集,降低缝隙连接的表达,影响缝隙连接半通道的正常开放。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机制,为先天性白内障的预防和基因治疗提供理论基础。
[Abstract]:Purpose :

Congenital cataract is one of the primary causes of visual impairment in children . Genetic mutations are the most common pathogenic factors . So far , about 37 pathogenic genes and hundreds of mutation sites associated with hereditary congenital cataract have been found .

Method :

In this study , two congenital cataract families collected at the Second Affiliated Hospital of Zhejiang University were studied . All of the members received medical history investigation and ophthalmologic examination , including vision , slit lamp , mydriatic fundus examination and so on . Peripheral blood samples were taken to extract genomic DNA . Polymerase chain reaction ( PCR ) was used to amplify the coding region of candidate genes and intron and exon boundary region . The amplification products were sequenced in two directions and sequenced . The effects of mutation on protein structure and function were analyzed by SIFT , PolyPhen - 2 and hydrophobic analysis .

The target gene was obtained from human eye cDNA library , and the wild type plasmid was constructed by cloning the plasmid containing the mutant gene by DNA recombination technique . The wild group ( Cx46wt - EGFP ) , the mutant group plasmid ( Cx46H95Y - EGFP , Cx46R76H - EGFP ) and the blank plasmid were transfected into Hek293 cells .

Results :

The results of the sequencing showed that the 283rd base of one allele of CX46 gene coding region was changed from cytosine to adenine ( c.283CT ) .

In this study , we successfully established Hek293 cell line stably transfected with Cx46wt - EGFP , Cx46H95Y - EGFP , Cx46R76H - EGFP and eukaryotic expression plasmids .
When in physiological calcium and gap junction protein channel inhibitor environment , four groups of cells do not uptake dye .

Conclusion :

The mutation of Cx46 protein H95Y and R76H can lead to intracellular accumulation of protein , decrease the expression of gap junction and affect the normal opening of gap junction half - channel . This study extends the gene mutation spectrum of congenital cataract , and reveals the mechanism of connexin mutation leading to congenital cataract . It provides a theoretical basis for preventing and gene therapy of congenital cataract .

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R776.1

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