视网膜色素变性及相关疾病RetNet基因变异频谱分析

发布时间：2018-05-31 11:06

本文选题：全外显子组测序 + RetNet　；参考：《中山大学》2016年博士论文

【摘要】：遗传性视网膜变性是一类常见难治的遗传性致盲眼病,这其中最常见的有视网膜色素变性(retinitis pigmentosa,RP),最严重的是Leber先天性黑朦(Leber congenital amaurosis,LCA),具有高度遗传异质性和临床表型多样性。迄今报道至少有253个基因被发现与遗传性视网膜变性相关(Ret Net网站:https://www.sph.uth.edu/Ret Net),其中82个与RP有关。由于致病基因众多,以及视网膜变性遗传学和表型的高度异质性,目前此类疾病的基因诊断存在极大困难。测序技术的限制使以往遗传性眼病的分子遗传学研究以单基因家系研究为主,鲜有对所有遗传性视网膜变性已知基因全面系统分析的研究。随着测序技术的迅猛发展,越来越多的研究者使用高通量测序分析遗传病的发病原因。虽然高通量测序可以克服技术瓶颈在短时间内获得大量序列变异,但测序得到的数据量和复杂性也随之增加,给变异的注释和致病突变的确定带来了新的挑战。本研究旨在全面系统分析常见遗传性视网膜变性患者Ret Net基因变异,可以对疾病的分子遗传学病因有一个系统的了解。本研究基于高通量测序大数据的分析发现并总结以往研究忽视的一些可能不致病的基因和变异,提醒后续的基因研究注意。本课题在课题组已有病例的全外显子组测序数据基础上,一方面系统分析了157例RP患者Ret Net基因的突变情况;另一方面,在既往系列研究基础上,系统总结了一组LCA病例Ret Net基因突变频谱,同时对个别基因变异进行了进一步分析。在近60%的RP和LCA先证者中发现潜在致病突变,总结了Ret Net基因在这两种常见的遗传性视网膜变性疾病中的突变频谱和频率。发现ALMS1基因不仅与Alstr?m综合征相关,还可以作为早发重型视网膜变性先证者的候选基因。基于对近千例遗传性视网膜变性患者外显子测序结果的分析,总结了高通量测序在眼遗传病致病基因突变寻找过程中遇到的常见问题,包括在眼遗传眼病先证者全外显子组测序结果中发现频率大于1%且Human Gene Mutation Database报道致病的突变;经生物信息学预测致病的变异,也可以同时并存于不同遗传眼病中,或无法在家系共分离分析中得到验证。研究提示了人类基因的复杂性,以及分析眼遗传病高通量测序的结果时需要结合临床多方面多角度综合分析,确定变异致病性时需要谨慎。本研究为进一步开展遗传性视网膜变性疾病的基因诊断奠定基础,也为这类疾病的预防、治疗措施,及新基因的寻找提供线索。第一部分Ret Net基因突变在视网膜色素变性先证者中的频谱分析目的:本研究基于157例视网膜色素变性先证者全外显子组测序数据,系统分析了Ret Net基因的突变频谱和频率。总结在高通量测序分析遗传性视网膜变性致病基因突变过程中面临的困难和解决策略。方法:分析157例RP先证者DNA全外显子组测序数据,对Ret Net(2014年1月)中191个基因(包括62个RP已知基因和129个其他类型遗传性视网膜变性基因)的变异通过频率、功能预测、对照分析和家系共分离分析等一系列方法分析确定可能的致病突变并由Sanger测序验证。结合课题组先前的研究,总结Ret Net基因突变在157例RP先证者中突变频谱和频率。结果:分析157个RP先证者全外显子组测序数据中的Ret Net基因突变,共在90个(57.3%,90/157)家系中发现潜在致病突变,其中在79个家系中发现28个RP已知基因的突变,以及在11个家系中发现5个其他类型视网膜变性基因突变。检出突变的90个家系中包括31(34.4%,31/90)个家系携带常染色体显性基因的杂合突变,41(45.6%,41/90)个家系携带常染色体隐性基因的纯合突变(10)和复合杂合突变(31),18(20.0%,18/90)个家系携带X连锁基因的半合突变。在分析828个遗传眼病全外显子组测序数据时,发现Human Gene Mutation Database列出的致病突变中,有27个在我们所有遗传眼病人群中等位基因频率超过1%,分布在24个基因(12个综合征基因和12和Ret Net基因)中。14个显性Ret Net基因生物信息学预测致病的变异同时在不同疾病患者中发现。Ret Net基因预测致病变异在16个家系中发现不符合家系共分离。结论:通过全外显子组测序,有57.3%的RP先证者在Ret Net基因中发现潜在致病突变。本研究不仅系统分析了中国RP患者全部视网膜变性已知基因突变频谱和频率,而且提供了中国RP患者分子病因的概述。对已知基因的分析为进一步发现新的RP致病基因提供了基础和线索。本研究总结了高通量测序分析遗传性视网膜变性致病基因突变过程中面临的困难和解决策略,列举了Ret Net基因突变可能不致病的基因和变异,提示在分析高通量测序突变数据时需要结合临床多方面多角度分析。第二部分Ret Net基因突变在视网膜色素变性相关疾病先证者中的频谱分析目的:全面分析159个中国LCA先证者Ret Net基因的突变频谱和频率。进一步发现LCA新的致病基因。另外,在46个中国高度远视先证者中筛查高度远视合并RP的致病基因——MFRP的突变。方法:基于159例LCA先证者全外显子组测序数据,对191个Ret Net基因(截止于2014年1月,包括62个RP已知基因和129个其他类型遗传性视网膜变性基因)存在的变异,进行频率、功能预测、对照分析和家系共分离分析等一系列分析,确定可能的致病突变并由Sanger测序验证。由于意外发现ALMS1存在高频无效突变,所以进一步扩大样本量,分析了1220个遗传性眼病患者的ALMS1基因变异,发现了多个ALMS1无效突变,进行Sanger测序验证、家系共分离分析和对照分析,在回访中检查患者其他全身综合征症状表现。在42个高度远视先证者利用全外显子组测序分析MFRP突变,另外4个先证者通过Sanger测序分析MFRP编码序列,进一步在家系成员和192个正常对照中进行候选变异分析。结果:159个LCA先证者中,有90个发现了Ret Net基因的可能致病突变,占总人数的56.6%。其中72个只进行全外线组测序分析的患者中,有45个(62.5%)发现携带突变。突变频率最高的几个基因分别是GUCY2D(10.7%)、CRB1(7.5%)、CEP290(6.9%)、RPGRIP1(5.0%)、IQCB1(4.4%)、ALMS1(3.8%)和CRX(3.8%)。在1220个遗传眼病患者中,11个LCA和早发重型锥杆细胞营养不良(cone-rod dystrophy,CORD)先证者发现携带13个ALMS1无效突变,这11个携带ALMS1无效突变的先证者在总数为215例的LCA和CORD中占5.1%(11/215)。15个患者中的9个通过回访检查发现Alstr?m综合征的全身症状缺失或表现轻微。在3个高度远视先证者中发现可能致病的复合杂合突变。临床资料显示这些患者屈光度至少有+13.50D或眼轴最长为16.78mm。视网膜电图显示携带错义突变的患者视锥视杆细胞反应正常,另一个携带错义和截短突变的患者视杆细胞反应降低,并伴有黄斑囊样变性。结论:一系列的突变研究分析发现Ret Net已知基因突变可以解释56.6%的中国LCA先证者患病原因。本研究对中国LCA患者种族特异性的已知基因突变频谱和频率进行了全面的分子遗传学分析。提示ALMS1应该作为LCA或早发重型CORD的先证者的候选基因,眼部异常可能是综合征最早发最明显的症状。本研究扩展了MFRP的突变频谱和其相关表型,这是在中国人群中首次报道MFRP突变。
[Abstract]:Hereditary retinal degeneration is a common type of refractory hereditary blinding ophthalmopathy, the most common of which are retinitis pigmentosa (RP), and the most serious Leber congenital amamamus (Leber congenital amaurosis, LCA), with high genetic heterogeneity and clinical phenotypic diversity. At least 253 genes have been reported to date. It was found to be associated with hereditary retinal degeneration (Ret Net website: https://www.sph.uth.edu/Ret Net), 82 of which are associated with RP. The genetic diagnosis of such diseases is extremely difficult because of the numerous pathogenic genes, and the high heterogeneity of the genetic and phenotypes of the retina. With the rapid development of sequencing technology, more and more researchers have used high throughput sequencing to analyze the causes of genetic diseases. Although high throughput sequencing can overcome technical bottlenecks in a short period of time, high throughput sequencing can be achieved in a short time. A large number of sequences were mutated, but the amount and complexity of the data obtained by sequencing also increased, which brought new challenges to the annotation of mutation and the determination of the pathogenic mutation. The aim of this study is to systematically analyze the Ret Net gene mutations in the common hereditary retinal degeneration patients, which can have a systematic understanding of the molecular genetic cause of the disease. On the basis of the exon sequencing data of the existing cases, the mutation of the Ret Net gene in 157 RP patients was systematically analyzed. On the one hand, on the basis of a series of previous studies, a series of mutations in the Ret Net gene of LCA cases were systematically summarized, and a further analysis of individual gene mutations was carried out. Potential pathogenic mutations were found in nearly 60% of the RP and LCA precursor, and the mutation spectrum of the Ret Net gene in these two common genetic retinal degeneration diseases was summarized. It is found that the ALMS1 gene is not only associated with the Alstr? M syndrome, but also as a candidate gene for early onset heavy retinal degeneration. Based on the analysis of the exon sequencing results of nearly 1000 cases of hereditary retinal degeneration, the common problems encountered by high throughput sequencing in the search for genetic mutations in ophthalmopathy are summarized. It was found that frequencies greater than 1% and Human Gene Mutation Database reported pathogenic mutations in the whole exon group of ocular genetic ophthalmopathy, and the prediction of pathogenic variation by bioinformatics could also coexist in different genetic ophthalmopathy, or could not be verified in family co separation analysis. The complexity, and the analysis of the results of high throughput sequencing of ophthalmic hereditary diseases, need to be combined with clinical multifaceted and multidimensional analysis to determine the variability of pathogenicity. This study lays the foundation for further genetic diagnosis of genetic retinal degeneration, and also for the prevention, treatment, and new genes of this type of disease. The spectral analysis of the first part of the Ret Net gene mutation in the retinitis pigmentosa: Based on the sequencing data of the exon group of 157 cases of retinitis pigmentosa, the mutation spectrum and frequency of the Ret Net gene were systematically analyzed and the high throughput sequencing analysis of the pathogenicity of the hereditary retinal degeneration was summarized. Methods: analysis of the difficulties and solutions in the process of mutation. Methods: analysis of the sequence data of 157 RP precursor DNA exons and the frequency, function prediction, control analysis and family co analysis of the mutation of 191 genes (including 62 RP known and 129 other types of genetic retinal degeneration genes) in Ret Net (January 2014) A series of method analysis identified possible pathogenic mutations and was verified by Sanger sequencing. Combined with previous research in the group, the mutation frequency and frequency of the Ret Net gene mutation in 157 cases of RP precursor were summarized. Results: analysis of the Ret Net gene mutations in the sequencing data of 157 RP precursor exons, together in the 90 (57.3%, 90/157) families. Mutations in 28 RP known genes were found in 79 families, and 5 other types of retinal degeneration gene mutations were found in 11 families. 90 families with detected mutations included 31 (34.4%, 31/90) families with heterozygous mutations with autosomal dominant genes and 41 (45.6%, 41/90) families with constant staining. The homozygous mutation (10) and compound heterozygosity (31), 18 (20%, 18/90) families carry the semisexal mutation of the X linkage gene. In the analysis of the sequencing data of the total exon group of 828 genetic ophthalmopathy, it was found that of the Human Gene Mutation Database, there were 27 of the secondary gene frequencies in all our genetic ophthalmopathy. More than 1%, distributed in 24 genes (12 syndrome genes and 12 and Ret Net genes).14 dominant Ret Net gene in bioinformatics prediction of pathogenic variation in different disease patients and found that.Ret Net gene predicted pathogenetic variation in 16 families. Conclusion: there are 57.3% R through exon sequencing. The P precursor found the potential pathogenic mutation in the Ret Net gene. This study not only systematically analyzed the frequency and frequency of the known gene mutation of all retinal degeneration in Chinese RP patients, but also provided an overview of the molecular etiology of Chinese RP patients. This paper summarizes the difficulties and solutions in the process of high throughput sequencing analysis of genetic mutation of genetic retinal degeneration, lists the genes and variations that may not be pathogenic in the Ret Net gene mutation, suggesting that the analysis of high throughput sequencing mutation data needs to be combined with clinical multifaceted and multi angle analysis. The second part of the Ret Net gene mutation is in the case of high throughput sequencing. Spectral analysis in patients with retinitis pigmentosa related diseases: Objective: to comprehensively analyze the mutation frequency and frequency of the Ret Net gene in 159 Chinese LCA presenters. Further identify the new LCA pathogenic gene. In addition, the mutation of the pathogenic gene of the highly hyperopia combined with RP is screened in 46 Chinese highly hyperopia presenters. Method: Based on 15 9 cases of LCA precursor total exon sequencing data, 191 Ret Net genes (including 62 RP known genes and 129 other types of genetic retinal degeneration genes) mutation, frequency, function prediction, control analysis and family co segregation analysis, and so on a series of analysis, determine the possible pathogenic mutation and Sang Er sequencing verification. Due to the accidental discovery of high frequency and ineffective mutations in ALMS1, the ALMS1 gene variation in 1220 patients with hereditary ophthalmopathy was further expanded, and multiple ALMS1 mutations were found, and multiple ALMS1 mutations were found to be verified by Sanger sequencing. Performance. The MFRP mutation was analyzed with total exon sequencing in 42 high hyperopia probands, and the other 4 precursor were analyzed by Sanger sequencing to analyze the MFRP coding sequence, further analysis of the candidate mutations in family members and 192 normal controls. Results: among the 159 LCA precursor, 90 found the possible pathogenic mutation of the Ret Net gene. Of the total number of 56.6%., 45 (62.5%) found a mutation in 72 full line sequencing analysis. The highest frequencies of the mutation were GUCY2D (10.7%), CRB1 (7.5%), CEP290 (6.9%), RPGRIP1 (5%), IQCB1 (4.4%), ALMS1 (3.8%) and CRX (3.8%). Among 1220 genetic ophthalmopathy, 11 LCA and early heavy-duty cones Cone-rod dystrophy (CORD) precursor found 13 ALMS1 ineffective mutations, and 11 of the 9 of the 215 cases of LCA and CORD in the total number of LCA and CORD in the total number of patients with 215 cases of null mutation found the absence or slight manifestation of general symptoms in Alstr? M syndrome. At 3 heights. The clinical data showed that the patients with at least +13.50D of diopter or the longest axis of the ocular axis were 16.78mm. electroretinogram showing that the conical optic rod cells in the patients with missense mutations were normal, and the other patients with missense and truncated mutations decreased the response to the rod cells and were accompanied by yellow. Conclusion: a series of mutation studies have found that the Ret Net gene mutation can explain 56.6% of the causes of the disease in Chinese LCA precursor. This study has carried out a comprehensive molecular genetic analysis of the spectrum and frequency of the mutation of the known gene mutations in Chinese LCA patients. It suggests that ALMS1 should be used as a LCA or an early severe CORD. The candidate gene of the precursor, the eye abnormalities may be the most obvious symptom of the earliest onset of the syndrome. This study extends the mutation spectrum of MFRP and its related phenotype, which is the first report of the MFRP mutation in the Chinese population.
【学位授予单位】：中山大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R774.1

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