视网膜色素变性患者视锥杆细胞同源盒基因突变的研究

发布时间：2018-06-02 16:02

本文选题：视网膜色素变性 + 常染色体显性　；参考：《宁夏医科大学》2012年硕士论文

【摘要】：目的视网膜色素变性（RP）是最常见的遗传性、致盲性眼底病。目前已经发现至少63个基因与其有关。我国已经对20个RP相关基因进行了研究，但没有任何一个基因能够单独解释超过5%的RP发病原因。本次研究的目的是研究视锥-杆细胞同源盒（cone-rod homebox gene,CRX）基因在宁夏地区RP患者中的突变频率及特征，并探讨其在RP发病机制中的作用。方法按国际通用的视网膜色素变性诊断标准对所收集到的100例视网膜色素变性患者（包括18例ADRP患者，15例ARRP患者，67例SRP患者），抽取外周静脉血3～5ml，EDTA抗凝，采用北京天根基因公司生产的试剂盒抽提红细胞DNA，按已发表的CRX基因序列资料，参照Dryja等报道的方法进行PCR引物设计，在设计引物时使其扩增区域覆盖CRX基因的4个外显子及其附近拼接位点中的内含子，共设计合成了4对引物。聚合酶链反应（polymerase chain reaction, PCR）扩增CRX基因第1～4外显子基因片段，反应结束后将其产物进行琼脂糖凝胶（1.5%）电泳，证实PCR扩增的目的片段（大小在253bp~665bp之间）。将所有的PCR产物进行DNA测序并进行序列分析；并随机收集100例正常人进行对照检测。采用Polyphen分析突变导致的氨基酸改变对CRX蛋白结构和功能的影响，运用多因素分析研究CRX基因突变位点对RP的作用。结果在100例RP患者的CRX基因上共检测出了5个变异位点，2个为同义突变（p.Leu78Leu，p.Ala92Ala），3个为错义突变（p.Ala112Val，p.Gly122Asp andp.Thr187Ile）,其中p.Leu78Leu，p.Ala92Ala和p.Thr187Ile为新发现的突变。其余突变位点均证实为CRX基因的多态性。p.Thr187Ile突变位点仅在2例ARRP患者身上检出，在正常对照组未发现该位点突变。对此位点进行PSIC预测，为非致病性突变，多因素Logistic回归进一步分析p.Thr187Ile与RP的发生无相关性（P＞0.05）。结论宁夏地区RP患者中，CRX基因的突变率小于1%。p.Thr187Ile不是RP的致病性突变，但它是否可能通过影响其它基因的表达，，增加常染色体显性遗传视网膜色素变性（ARRP）的发生。有待于进一步研究。
[Abstract]:Objective retinal pigmentosa (RP) is the most common hereditary, blinding fundus disease. At least 63 genes have been found to be involved. Twenty RP-related genes have been studied in China, but none of them can explain more than 5% of the causes of RP alone. The aim of this study was to study the mutation frequency and characteristics of cone-rod homebox gene in RP patients in Ningxia, and to explore its role in the pathogenesis of RP. Methods according to the international standard for the diagnosis of retinal pigmentosa, 100 cases of retinal pigmentosa were collected, including 18 cases of ADRP, 15 cases of ARRP and 67 cases of SRP. The red blood cell DNA was extracted by the kit produced by Beijing Tianjingyin Company. According to the published CRX gene sequence data, the PCR primers were designed according to the methods reported by Dryja et al. Four pairs of primers were designed and synthesized to cover the four exons of the CRX gene and the introns in the adjacent splicing sites when the primers were designed. Polymerase chain reaction (PCR) polymerase chain reaction, PCR) was used to amplify the exon 1 of CRX gene. After the reaction, the product was amplified by agarose gel electrophoresis. It was confirmed that the target fragment of PCR amplification was between 253bp~665bp and 253bp~665bp. All PCR products were sequenced and sequenced. The effect of amino acid mutation on the structure and function of CRX protein was analyzed by Polyphen, and the effect of mutation site of CRX gene on RP was studied by multivariate analysis. Results A total of 5 mutation sites were detected in the CRX gene of 100 patients with RP, two were synonymous mutation, two were synonymous mutation. Leu78 Leup.Ala92AlaA, and three were missense mutations. The new mutations were P. Leu78 Leup.Ala92Ala and P. Thr187Ilean.Three mutations were found in P. Leu78 Leup.Ala92Ala and p.Thr187Ile. All the other mutation sites were confirmed to be the polymorphism of CRX gene. P. Thr187Ile mutation was detected only in 2 patients with ARRP, but not in the normal control group. PSIC was used to predict this locus as a non-pathogenic mutation. The multivariate Logistic regression analysis showed that there was no correlation between p.Thr187Ile and RP (P > 0.05). Conclusion the mutation rate of CRX gene in RP patients in Ningxia area is lower than that in 1%.p.Thr187Ile patients, but whether it may increase the occurrence of autosomal dominant retinitis pigmentosa by affecting the expression of other genes. Further study is needed.
【学位授予单位】：宁夏医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R774.1

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