本文關(guān)鍵詞：中國漢族原發(fā)性圓錐角膜致病基因的定位研究　出處：《浙江大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 圓錐角膜 二代測序 基因突變 ZNF469 COL5A1 COL4A4 TIMP3 LOXMPDZ TGFBI FNDC3B

【摘要】：研究背景:圓錐角膜是一種以進(jìn)行性的角膜中央變薄、局部前凸形成圓錐狀,繼發(fā)高度近視及不規(guī)則散光為特征的角膜變性疾病。早期可通過配戴框架眼鏡及合適的角膜接觸鏡緩解癥狀,后期因視力嚴(yán)重下降需行角膜移植手術(shù)治療。據(jù)文獻(xiàn)報(bào)道,相較于白種人,亞洲人發(fā)病率更高,發(fā)病年齡更早,病情更嚴(yán)重,然而目前亞洲人群的相關(guān)研究較少,且病因及發(fā)病機(jī)制尚不明確。在諸多學(xué)說中,隨著精準(zhǔn)醫(yī)療及靶向治療概念的提出及基因測序技術(shù)的迅猛發(fā)展,基因?qū)W說的重要性日益受到關(guān)注,全世界范圍內(nèi)已有40余種基因相繼被定位和克隆,然而,遺憾的是,圓錐角膜具有明顯的遺傳異質(zhì)性,即同一基因的突變可形成不同的臨床表現(xiàn),而同一臨床表現(xiàn)可源于不同的基因突變。目前尚無一種基因可以解釋大多數(shù)病例發(fā)病機(jī)制,基于上述原因,本研究擬在中國漢族圓錐角膜人群中探究原發(fā)性圓錐角膜的致病基因,以期擴(kuò)大突變基因譜并在中國人群中進(jìn)行重復(fù)性驗(yàn)證,為圓錐角膜的早期診斷及靶向治療提供一定的理論依據(jù)。研究方法:共收集散發(fā)性圓錐角膜患者53例及種族匹配的正常人群100例作為對照納入研究。所有入組者均進(jìn)行詳細(xì)的病史及臨床資料的采集,并由同一有經(jīng)驗(yàn)的醫(yī)師進(jìn)行全面的眼科檢查及全身檢查。抽取外周靜脈血后提取DNA,病例組利用基因芯片聯(lián)合二代測序技術(shù)進(jìn)行候選基因的突變篩查后,應(yīng)用一代Sanger測序進(jìn)行驗(yàn)證;陽性位點(diǎn)隨后在100例正常對照中進(jìn)行突變篩查。借助生物信息學(xué)方法對突變基因的結(jié)構(gòu)和功能進(jìn)行分析。研究結(jié)果:在二代測序中得出的76個(gè)陽性位點(diǎn),排除單核苷酸多態(tài)性位點(diǎn)后,選擇最小等位基因0.1%(根據(jù)2012年5月發(fā)表的千人基因組計(jì)劃和dbSNP數(shù)據(jù)庫)、未出現(xiàn)在220個(gè)無眼部疾病的漢族人群的全外顯子測序數(shù)據(jù)庫中的15個(gè)位點(diǎn)進(jìn)行了一代測序驗(yàn)證。除去1個(gè)為假陽性外,共發(fā)現(xiàn)14個(gè)新的位點(diǎn)突變:缺失突變1個(gè),COL4A4 基因(c.3636_3637del:p.R1212fs);插入突變 1 個(gè),TGFBI 基因(c.624+7-A);錯義突變12個(gè),包括ZNF469基因的7個(gè)位點(diǎn)(c.2059GA:p.E687K;c.2137CA:p.P713T;c.3466GA:p.A1 156T;c.3749CT:p.P1250L;c.4300GA:p.D1434N;c.4684GA:p.E1562K;c.7262GA:p.R2421H),FNDC3B 基因 1 個(gè)位點(diǎn)(c.455CT:p.P152L),LOX 基因1 個(gè)位點(diǎn)(c.95CT:p.P32L),MPDZ 基因 1 個(gè)位點(diǎn)(c.28GA:p.A10T),COL5A1基因 1 個(gè)位點(diǎn)(c.1372CT:p.P458S),TIMP3基因 1 個(gè)位點(diǎn)(c.476CT.p.S159F),所有的位點(diǎn)均為雜合性突變,未在100例正常對照中發(fā)現(xiàn)這些位點(diǎn)的突變。研究結(jié)論:1.本研究是迄今為止第一次利用二代測序?qū)A錐角膜候選基因進(jìn)行大范圍的篩查研究;2.經(jīng)過一代測序驗(yàn)證后,發(fā)現(xiàn)14個(gè)新的位點(diǎn)突變,涉及8個(gè)基因,擴(kuò)大了基因突變譜;3.證實(shí)了圓錐角膜的遺傳異質(zhì)性,為進(jìn)一步探討圓錐角膜的發(fā)病機(jī)制提供了新的突破口,為圓錐角膜的早期診斷及基因治療提供了理論依據(jù)。
[Abstract]:Background: keratoconus is a central cornea with progressive thinning of the local lordosis conification, characterized by degeneration of cornea diseases with high myopia and irregular astigmatism. Early can alleviate the symptoms through wearing glasses and a suitable contact lens, late because of a serious decline in vision for corneal transplantation. It is reported that compared to Caucasians, Asians have higher incidence, younger age, more serious illness, but the current Asian population less relevant research, and the etiology and pathogenesis is still not clear. In many theories, along with the rapid development of medical and precise targeting and gene sequencing technology put forward the concept of treatment. The importance of gene theory has attracted increasing attention worldwide, has more than 40 kinds of genes have been cloned and located, however, unfortunately, keratoconus has significant genetic heterogeneity, i.e. Mutations in the same gene can form different clinical manifestations, clinical manifestations and the same can be derived from different gene mutations. There is not a gene can explain the pathogenesis of most of the cases, based on the above reasons, this study intends to explore the pathogenic gene of idiopathic keratoconus in Han Chinese keratoconus population, in order to expand mutation spectrum and repeatability validation in Chinese population, provide a theoretical basis for early diagnosis and target treatment of keratoconus. To study methods: collected 53 cases of patients with sporadic keratoconus and normal ethnically matched group in 100 cases as control were enrolled in the study. All subjects were performed and clinical data a detailed history collection, and by the same experienced physicians ophthalmologic examination and systemic examination comprehensive. Peripheral venous blood samples from DNA patients using gene chip combined with two generation test Mutation screening of candidate gene sequencing technology after the application of generation Sanger sequencing verified; positive loci followed by mutation screening in 100 cases of normal control. Methods of gene structure and function were analyzed by bioinformatics. Results: 76 positive loci obtained in the two generation sequencing, single nucleotide polymorphism elimination site, select the minimum allele 0.1% (according to the May 2012 publication of the 1000 Genomes Project and dbSNP), did not appear in all 220 without eye disease in the Han population. 15 loci were sub sequence database generation sequencing. Except for the 1 false positive, 14 new mutations were found: 1 deletion mutation, COL4A4 gene (c.3636_3637del:p.R1212fs); insertion mutation 1, TGFBI gene (c.624+7-A); 12 missense mutations, including 7 loci of ZNF469 gene (c.2059GA:p .E687K; c.2137CA:p.P713T; c.3466GA:p.A1 156T; c.3749CT:p.P1250L; c.4300GA:p.D1434N; c.4684GA:p.E1562K; c.7262GA:p.R2421H), 1 SNPs of FNDC3B gene (c.455CT:p.P152L), LOX gene 1 loci (c.95CT:p.P32L, MPDZ) gene 1 loci (c.28GA:p.A10T, COL5A1) gene 1 loci (c.1372CT:p.P458S, TIMP3) gene 1 loci (c.476CT.p.S159F), all sites are all heterozygous mutation, no mutation was found in these loci in 100 cases of normal control. Conclusion: 1. this study is by far the first screening study using two generation sequencing to keratoconus candidate genes; 2. after generation after sequencing, discovered 14 new mutations, involving 8 genes, expand the spectrum of mutations; 3. confirmed the genetic heterogeneity of keratoconus, provides a new breakthrough to further explore the pathogenesis of keratoconus, It provides a theoretical basis for the early diagnosis and gene therapy of keratoconus.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R772.2
，

本文編號：1393264