本文選題：視網(wǎng)膜色素變性 + 常染色體顯性�。� 參考：《寧夏醫(yī)科大學(xué)》2012年碩士論文

【摘要】：目的視網(wǎng)膜色素變性（RP）是最常見的遺傳性、致盲性眼底病。目前已經(jīng)發(fā)現(xiàn)至少63個基因與其有關(guān)。我國已經(jīng)對20個RP相關(guān)基因進(jìn)行了研究，但沒有任何一個基因能夠單獨(dú)解釋超過5%的RP發(fā)病原因。本次研究的目的是研究視錐-桿細(xì)胞同源盒（cone-rod homebox gene,CRX）基因在寧夏地區(qū)RP患者中的突變頻率及特征，并探討其在RP發(fā)病機(jī)制中的作用。 方法按國際通用的視網(wǎng)膜色素變性診斷標(biāo)準(zhǔn)對所收集到的100例視網(wǎng)膜色素變性患者（包括18例ADRP患者，15例ARRP患者，67例SRP患者），抽取外周靜脈血3～5ml，EDTA抗凝，采用北京天根基因公司生產(chǎn)的試劑盒抽提紅細(xì)胞DNA，按已發(fā)表的CRX基因序列資料，參照Dryja等報道的方法進(jìn)行PCR引物設(shè)計，在設(shè)計引物時使其擴(kuò)增區(qū)域覆蓋CRX基因的4個外顯子及其附近拼接位點中的內(nèi)含子，共設(shè)計合成了4對引物。聚合酶鏈反應(yīng)（polymerase chain reaction, PCR）擴(kuò)增CRX基因第1～4外顯子基因片段，反應(yīng)結(jié)束后將其產(chǎn)物進(jìn)行瓊脂糖凝膠（1.5%）電泳，證實PCR擴(kuò)增的目的片段（大小在253bp~665bp之間）。將所有的PCR產(chǎn)物進(jìn)行DNA測序并進(jìn)行序列分析；并隨機(jī)收集100例正常人進(jìn)行對照檢測。采用Polyphen分析突變導(dǎo)致的氨基酸改變對CRX蛋白結(jié)構(gòu)和功能的影響，運(yùn)用多因素分析研究CRX基因突變位點對RP的作用。 結(jié)果在100例RP患者的CRX基因上共檢測出了5個變異位點，2個為同義突變（p.Leu78Leu，p.Ala92Ala），3個為錯義突變（p.Ala112Val，p.Gly122Asp andp.Thr187Ile）,其中p.Leu78Leu，p.Ala92Ala和p.Thr187Ile為新發(fā)現(xiàn)的突變。其余突變位點均證實為CRX基因的多態(tài)性。p.Thr187Ile突變位點僅在2例ARRP患者身上檢出，在正常對照組未發(fā)現(xiàn)該位點突變。對此位點進(jìn)行PSIC預(yù)測，為非致病性突變，多因素Logistic回歸進(jìn)一步分析p.Thr187Ile與RP的發(fā)生無相關(guān)性（P＞0.05）。 結(jié)論寧夏地區(qū)RP患者中，CRX基因的突變率小于1%。p.Thr187Ile不是RP的致病性突變，但它是否可能通過影響其它基因的表達(dá)，，增加常染色體顯性遺傳視網(wǎng)膜色素變性（ARRP）的發(fā)生。有待于進(jìn)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.
【學(xué)位授予單位】：寧夏醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R774.1

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