靶向捕获技术在先天性白内障遗传诊断中的应用和EPHA2基因G668D突变致病机理研究

发布时间：2018-05-19 15:12

本文选题：先天性白内障 + 靶向捕获　；参考：《浙江大学》2016年博士论文

【摘要】：研究目的:本研究拟对28个先天性白内障家系中进行致病突变的筛查。并通过目标基因体外克隆合成、质粒构建、蛋白定位与定量等方法,对检测到的EPHA2受体蛋白基因G668D突变的致病功能进行探索,尝试理解该突变导致先天性白内障发生的机制。研究方法:在浙江大学医学院附属第二医院眼科收集到了 28个先天性白内障家系,抽取各个家系中先证者的外周血样本,提取基因组DNA。通过靶向捕获联合高通量技术在48个先天性白内障候选致病基因中筛选潜在致病突变。结果对比基因组数据库,并使用SIFT、Polyphen、MutationTaster等生物信息学方法分析,以获得疾病候选致病突变。利用PCR联合Sanger测序法在家系中明确与疾病共分离的真正致病突变,并以100名健康中国汉族人DNA样本作为阴性对照进行验证。对EPHA2基因G668D突变所引起的先天性白内障家系中所有家族成员进行眼部裂隙灯检查与全身体格检查,明确疾病表型与遗传模式。为探索EPHA2基因G668D突变引起的功能性改变,我们首先检测了各个配体ephrin在人晶状体上皮前囊膜中的表达情况。我们体外合成了 EPHA2基因cDNA,通过克隆重组与定点突变技术,构建了野生型及突变型的EPHA2过表达GV358质粒与pEGFP-N1 EPHA2-GFP融合蛋白质粒。使用野生型、突变型质粒与空白GV358载体分别转染HLE B3细胞系(人晶状体上皮细胞系),并通过划痕愈合实验检测细胞迁移能力,Hoechst33342检测细胞凋亡情况。使用野生型、突变型质粒和空白pEGFP-N1载体分别转染Hek293T细胞,利用荧光显微镜观察蛋白在细胞中的表达量与细胞中定位。使用免疫印迹法检测野生型与突变型蛋白的表达量,并使用蛋白酶体途径抑制剂MG-132处理细胞,探索突变型蛋白的降解途径。研究结果:利用靶向捕获技术,在28个家系中的14个家系中找到了先天性白内障致病突变,检出率达到50%。此14个突变在家系健康成员与100名健康中国汉族人中均未检测到。其中有10个突变为未曾报道过的新先天性白内障致病突变,4个突变为此前别的研究组报道过的致病突变。眼科检查与体格检查提示,靶向捕获技术捕获到的EPHA2 G668D引起的先天性白内障为常染色体显性遗传的先天性后极性白内障。功能实验提示,ephrin配体在人晶状体上皮前囊膜中高表达。G668D突变引起晶状体上皮细胞系迁移能力增强,对于细胞凋亡无显著性影响。荧光显微镜下EphA2突变蛋白更倾向于形成蛋白聚集,且突变蛋白表达量较野生型蛋白显著降低。蛋白免疫印迹结果提示,突变蛋白在胞内的水平显著降低,但使用蛋白酶体抑制剂MG-132处理后,突变蛋白水平显著回升。研究结论:靶向捕获联合高通量技术是高效可行的先天性白内障遗传诊断技术,值得在科研及临床中广泛推广。晶状体蛋白基因突变仍是引起先天性白内障的主要致病突变。Eph/ephrin受体配体系统在人晶状体功能与白内障发生中同样发挥重要作用,值得更深入的研究。本研究首次报道的EPHA2激酶区突变G668D可能通过增加细胞的迁移能力,从而导致先天性后极性白内障的发生。且这一迁移力的增加可能由于突变型蛋白更容易被蛋白酶体途径降解,导致突变杂合子晶状体内部的EphA2蛋白含量降低,蛋白单倍剂量不足所致。
[Abstract]:The purpose of this study is to screen the pathogenic mutations in 28 congenital cataract families and to explore the pathogeny function of the detected EPHA2 receptor gene G668D mutation by cloning, plasmid construction, protein localization and quantitative analysis of the target gene in vitro, and trying to understand the mutation leading to congenital cataract. The mechanism. Research methods: 28 congenital cataract families were collected at the Second Affiliated Hospital of Zhejiang University Medical College, and the peripheral blood samples were extracted from the first evidence of each family. The genomic DNA. was extracted by target capture combined high flux technique to screen the potential pathogenic mutation among the 48 congenital cataract candidate genes. The results were compared with the genomic database, and using SIFT, Polyphen, MutationTaster and other bioinformatics methods to obtain the disease candidate mutations. Using PCR combined with Sanger sequencing, the real pathogenic mutation was clearly separated from the disease in the family, and the DNA samples of 100 healthy Chinese Han people were tested as negative controls. To EPHA2 In order to explore the functional changes of the EPHA2 gene G668D mutation, we first detected the expression of each ligand ephrin in the anterior capsule of the human lens epithelial cell by detecting all the family members of the family of congenital cataract caused by the mutation of the gene G668D. We synthesized the EPHA2 gene cDNA in vitro, and constructed wild and mutant EPHA2 overexpressed GV358 plasmids and pEGFP-N1 EPHA2-GFP fusion proteins by cloned recombinant and site directed mutagenesis, and transfected the HLE B3 cell line (human lens epithelial cell line) with the wild type, the mutant plasmid and the blank GV358 vector, and passed through the transfection of the HLE B3 cell line (human lens epithelial cell line). The cell migration ability was detected by the scratch healing test, and the apoptosis of the cells was detected by Hoechst33342. The Hek293T cells were transfected with the wild type, the mutant plasmid and the blank pEGFP-N1 vector respectively. The expression of the protein in the cells and the cells were observed by the fluorescence microscope. The expression of the wild type and the mutant protein was detected by the immunoblotting method. In addition, the proteasome pathway inhibitor MG-132 was used to treat the cells and explore the degradation pathway of the mutant protein. The results of the study showed that the incidence of congenital cataract was found in 14 families of 28 families with the target capture technique, and the detection rate reached to 50%., the 14 mutations in family health members and 100 healthy Chinese Han people. 10 of the mutations were unreported new congenital cataract mutations and 4 mutations were previously reported by other research groups. Ophthalmology and physical examination suggested that the congenital cataract caused by EPHA2 G668D captured by the target capture technique was an autosomal dominant congenital posterior polarity. The functional experiment showed that the high expression of.G668D mutation in the anterior capsule of human lens epithelial cells caused by ephrin ligand increased the migration ability of the lens epithelial cell line, and had no significant effect on the apoptosis. The EphA2 mutant protein was more inclined to form protein aggregation under the fluorescence microscope, and the expression of mutant protein was significantly lower than that of the wild type protein. The results of protein immunoblotting showed that the level of mutant protein decreased significantly in the intracellular level, but the level of mutant protein increased significantly after the use of proteasome inhibitor MG-132. Conclusion: the target capture combined high flux technique is a highly effective and feasible genetic diagnosis technique for congenital cataract, which is worth popularizing in scientific research and clinical practice. The mutation of the body protein gene is still the main cause of congenital cataract, the.Eph/ephrin receptor ligand system plays an important role in the human lens function and cataract, and it is worth further research. The first reported EPHA2 kinase region mutation G668D may lead to the increase of cell migration ability, thus leading to the result of this study. The occurrence of congenital posterior polar cataract, and the increase of this mobility may be more easily degraded by the proteasome pathway, resulting in the decrease of the EphA2 protein content in the mutant heterozygote and the inadequacy of the protein dose.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R776.1

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