缝隙连接蛋白50基因V44A突变致先天性白内障的机制研究

发布时间：2019-02-27 10:28

【摘要】：研究目的：本研究拟对一先天性特殊表型白内障大家系进行疾病相关候选基因的定位及目标基因体外克隆,通过对蛋白定位、缝隙连接半通道功能及缝隙连接通道功能的检测,探究该突变导致先天性白内障发生的机制。研究方法：基因筛查部分：收集一先天性遗传性白内障家系,所有家族成员接受眼部裂隙灯检查,抽取外周血样本,提取基因组DNA。白内障术中收集患者前囊膜及晶状体样本,制作电镜标本并观察晶状体超微结构改变。聚合酶链反应(PCR)扩增与该家系表型相关的候选基因并进行序列分析。以100位健康中国人基因组DNA为阴性对照。根据上述结果,运用Antheprot4.3和Swiss-model软件对野生组及突变组蛋白二级结构及三级结构进行分析。基因功能研究部分：从人晶状体cDNA文库中获得GJA8基因,克隆并重组至pEGFP-N1质粒中,构建野生型质粒Cx50-EGFP。利用定点突变技术,构建突变型质粒Cx50V44A-EGFP。野生型质粒、突变型质粒及空白质粒(pEGFP-N1)分别转染Hek-293细胞,利用G418筛选,获得稳定转染各组质粒的细胞系。在稳定转染细胞中观察蛋白定位及缝隙连接形成。在低钙、生理钙及Connexin通道抑制剂环境下,运用极性染料染核技术及膜片钳技术,检测各组细胞半通道功能区别。在生理钙环境下,利用单细胞注射及染料弥散试验,检测各组细胞缝隙连接通道功能区别。研究结果：该家系患者白内障表型为核性混浊,但不累及“Y”字缝,不伴有其他眼部或系统性疾病,遗传方式为常染色体显性遗传。基因测序发现该家系患者GJA8基因cDNA第131位碱基胸腺嘧啶转换为胞嘧啶(c.131TC),该突变导致GJA8基因所表达Cx50蛋白的44位氨基酸由缬氨酸转变为丙氨酸(p.V44A),而在家系正常人及100个中国正常汉族人中均未发现该突变。成功建立稳定转染Cx50-EGFP、 Cx50V44A-EGFP、pEGFP-N1质粒的Hek293细胞系。在野生组细胞及突变组细胞中皆观察到缝隙连接的形成,数量上无统计学差异。在半通道功能检测中,低钙环境下,野生组细胞可摄取DAPI和PI两种染料,而突变组及空白组细胞不能摄取；而在生理钙和Cx通道抑制剂环境下,三组细胞皆不能摄取染料。全细胞膜片钳检测发现低钙环境下野生组细胞表面电流显著高于突变组细胞。缝隙连接通道功能检测中,4%neurobiotin可在野生组及突变组细胞中弥散,而5%Lucifer yellow仅在野生组细胞中弥散。研究结论：本研究首次发现GJA8基因c.131TC突变导致一特殊表型先天性白内障的发生,其表型为核性浑浊但不累及“Y”字缝。基因功能研究发现Connexin50蛋白V44A突变不影响细胞中缝隙连接的表达,但影响缝隙连接半通道及缝隙连接通道的开放,通道功能改变直接导致先天性白内障的发生。
[Abstract]:Objective: the aim of this study is to locate the disease-related candidate genes and to clone the target genes in vitro in order to locate the proteins in a congenital special phenotypic cataract family. Detection of gap junction half-channel function and gap junction channel function to explore the mechanism of congenital cataract caused by this mutation. Methods: gene screening: a family with congenital cataract was collected. All family members underwent eye slit lamp examination, peripheral blood samples were taken and genomic DNA. was extracted. The anterior capsule and lens samples were collected, and the ultrastructural changes of lens were observed. The candidate genes associated with phenotypes of the family were amplified by polymerase chain reaction (PCR) and sequenced. A total of 100 healthy Chinese were used as negative controls for genomic DNA. According to the above results, the secondary structure and tertiary structure of wild and mutant histone were analyzed by Antheprot4.3 and Swiss-model software. Gene function research: GJA8 gene was obtained from human lens cDNA library, cloned and recombined into pEGFP-N1 plasmid to construct wild type plasmid Cx50-EGFP.. Construction of Mutant plasmid Cx50V44A-EGFP. by site-directed Mutagenesis Wild-type plasmid, mutant plasmid and blank plasmid (pEGFP-N1) were transfected into Hek-293 cells respectively. The cell lines stably transfected with each group of plasmids were obtained by G418 screening. Protein localization and gap junction formation were observed in stable transfected cells. Under the condition of low calcium, physiological calcium and Connexin channel inhibitor, polar dye staining and patch clamp technique were used to detect the difference of half-channel function in each group. In physiological calcium environment, single cell injection and dye diffusion test were used to detect the function difference of gap junctional channels in each group. Results: the cataract phenotype of this family is nuclear opacification, but does not involve the "Y" suture, without other eye diseases or systemic diseases, the genetic pattern is autosomal dominant inheritance. Gene sequencing revealed that thymine was converted into cytosine (c.131TC) at position 131 of GJA8 gene cDNA in this family. The mutation resulted in the conversion of 44 amino acids of Cx50 protein expressed in GJA8 gene from valine to alanine (p.V44A). However, the mutation was not found in 100 Chinese normal Chinese and normal controls. A Hek293 cell line stably transfected with Cx50-EGFP, Cx50V44A-EGFP,pEGFP-N1 plasmid was successfully established. Gap junctions were observed in wild cells and mutant cells, and there was no significant difference in the number of gap junctions. In the half-channel function test, the wild group cells could absorb DAPI and PI dyes in low calcium environment, but the mutant group and blank group cells could not, while in the physiological calcium and Cx channel inhibitor environment, all the three groups cells could not absorb the dyes. Whole-cell patch clamp assay showed that the cell surface current in wild group was significantly higher than that in mutant group in low calcium environment. In the detection of gap junction channel function, 4%neurobiotin was diffused in wild group and mutant group, while 5%Lucifer yellow was only diffused in wild group. Conclusion: in this study, we first found that the c.131TC mutation of GJA8 gene leads to a special phenotype of congenital cataract, which is nuclear turbid but does not involve the "Y" suture. Gene function studies showed that the mutation of Connexin50 protein V44A did not affect the expression of gap junctions, but affected the opening of gap junction half-channel and gap junction channel, and the alteration of channel function directly led to the occurrence 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】：2013
【分类号】：R776.1

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