中国汉族原发性圆锥角膜致病基因的定位研究

发布时间：2018-01-07 16:08

  本文关键词：中国汉族原发性圆锥角膜致病基因的定位研究　出处：《浙江大学》2017年硕士论文　论文类型：学位论文

  更多相关文章： 圆锥角膜 二代测序 基因突变 ZNF469 COL5A1 COL4A4 TIMP3 LOXMPDZ TGFBI FNDC3B

【摘要】：研究背景:圆锥角膜是一种以进行性的角膜中央变薄、局部前凸形成圆锥状,继发高度近视及不规则散光为特征的角膜变性疾病。早期可通过配戴框架眼镜及合适的角膜接触镜缓解症状,后期因视力严重下降需行角膜移植手术治疗。据文献报道,相较于白种人,亚洲人发病率更高,发病年龄更早,病情更严重,然而目前亚洲人群的相关研究较少,且病因及发病机制尚不明确。在诸多学说中,随着精准医疗及靶向治疗概念的提出及基因测序技术的迅猛发展,基因学说的重要性日益受到关注,全世界范围内已有40余种基因相继被定位和克隆,然而,遗憾的是,圆锥角膜具有明显的遗传异质性,即同一基因的突变可形成不同的临床表现,而同一临床表现可源于不同的基因突变。目前尚无一种基因可以解释大多数病例发病机制,基于上述原因,本研究拟在中国汉族圆锥角膜人群中探究原发性圆锥角膜的致病基因,以期扩大突变基因谱并在中国人群中进行重复性验证,为圆锥角膜的早期诊断及靶向治疗提供一定的理论依据。研究方法:共收集散发性圆锥角膜患者53例及种族匹配的正常人群100例作为对照纳入研究。所有入组者均进行详细的病史及临床资料的采集,并由同一有经验的医师进行全面的眼科检查及全身检查。抽取外周静脉血后提取DNA,病例组利用基因芯片联合二代测序技术进行候选基因的突变筛查后,应用一代Sanger测序进行验证;阳性位点随后在100例正常对照中进行突变筛查。借助生物信息学方法对突变基因的结构和功能进行分析。研究结果:在二代测序中得出的76个阳性位点,排除单核苷酸多态性位点后,选择最小等位基因0.1%(根据2012年5月发表的千人基因组计划和dbSNP数据库)、未出现在220个无眼部疾病的汉族人群的全外显子测序数据库中的15个位点进行了一代测序验证。除去1个为假阳性外,共发现14个新的位点突变:缺失突变1个,COL4A4 基因(c.3636_3637del:p.R1212fs);插入突变 1 个,TGFBI 基因(c.624+7-A);错义突变12个,包括ZNF469基因的7个位点(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 个位点(c.455CT:p.P152L),LOX 基因1 个位点(c.95CT:p.P32L),MPDZ 基因 1 个位点(c.28GA:p.A10T),COL5A1基因 1 个位点(c.1372CT:p.P458S),TIMP3基因 1 个位点(c.476CT.p.S159F),所有的位点均为杂合性突变,未在100例正常对照中发现这些位点的突变。研究结论:1.本研究是迄今为止第一次利用二代测序对圆锥角膜候选基因进行大范围的筛查研究;2.经过一代测序验证后,发现14个新的位点突变,涉及8个基因,扩大了基因突变谱;3.证实了圆锥角膜的遗传异质性,为进一步探讨圆锥角膜的发病机制提供了新的突破口,为圆锥角膜的早期诊断及基因治疗提供了理论依据。
[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.

【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R772.2
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本文编号：1393264