本文選題：全外顯子組測序 + RetNet��； 參考：《中山大學》2016年博士論文

【摘要】：遺傳性視網(wǎng)膜變性是一類常見難治的遺傳性致盲眼病,這其中最常見的有視網(wǎng)膜色素變性(retinitis pigmentosa,RP),最嚴重的是Leber先天性黑朦(Leber congenital amaurosis,LCA),具有高度遺傳異質性和臨床表型多樣性。迄今報道至少有253個基因被發(fā)現(xiàn)與遺傳性視網(wǎng)膜變性相關(Ret Net網(wǎng)站:https://www.sph.uth.edu/Ret Net),其中82個與RP有關。由于致病基因眾多,以及視網(wǎng)膜變性遺傳學和表型的高度異質性,目前此類疾病的基因診斷存在極大困難。測序技術的限制使以往遺傳性眼病的分子遺傳學研究以單基因家系研究為主,鮮有對所有遺傳性視網(wǎng)膜變性已知基因全面系統(tǒng)分析的研究。隨著測序技術的迅猛發(fā)展,越來越多的研究者使用高通量測序分析遺傳病的發(fā)病原因。雖然高通量測序可以克服技術瓶頸在短時間內獲得大量序列變異,但測序得到的數(shù)據(jù)量和復雜性也隨之增加,給變異的注釋和致病突變的確定帶來了新的挑戰(zhàn)。本研究旨在全面系統(tǒng)分析常見遺傳性視網(wǎng)膜變性患者Ret Net基因變異,可以對疾病的分子遺傳學病因有一個系統(tǒng)的了解。本研究基于高通量測序大數(shù)據(jù)的分析發(fā)現(xiàn)并總結以往研究忽視的一些可能不致病的基因和變異,提醒后續(xù)的基因研究注意。本課題在課題組已有病例的全外顯子組測序數(shù)據(jù)基礎上,一方面系統(tǒng)分析了157例RP患者Ret Net基因的突變情況;另一方面,在既往系列研究基礎上,系統(tǒng)總結了一組LCA病例Ret Net基因突變頻譜,同時對個別基因變異進行了進一步分析。在近60%的RP和LCA先證者中發(fā)現(xiàn)潛在致病突變,總結了Ret Net基因在這兩種常見的遺傳性視網(wǎng)膜變性疾病中的突變頻譜和頻率。發(fā)現(xiàn)ALMS1基因不僅與Alstr?m綜合征相關,還可以作為早發(fā)重型視網(wǎng)膜變性先證者的候選基因。基于對近千例遺傳性視網(wǎng)膜變性患者外顯子測序結果的分析,總結了高通量測序在眼遺傳病致病基因突變尋找過程中遇到的常見問題,包括在眼遺傳眼病先證者全外顯子組測序結果中發(fā)現(xiàn)頻率大于1%且Human Gene Mutation Database報道致病的突變;經(jīng)生物信息學預測致病的變異,也可以同時并存于不同遺傳眼病中,或無法在家系共分離分析中得到驗證。研究提示了人類基因的復雜性,以及分析眼遺傳病高通量測序的結果時需要結合臨床多方面多角度綜合分析,確定變異致病性時需要謹慎。本研究為進一步開展遺傳性視網(wǎng)膜變性疾病的基因診斷奠定基礎,也為這類疾病的預防、治療措施,及新基因的尋找提供線索。第一部分Ret Net基因突變在視網(wǎng)膜色素變性先證者中的頻譜分析目的:本研究基于157例視網(wǎng)膜色素變性先證者全外顯子組測序數(shù)據(jù),系統(tǒng)分析了Ret Net基因的突變頻譜和頻率�？偨Y在高通量測序分析遺傳性視網(wǎng)膜變性致病基因突變過程中面臨的困難和解決策略。方法:分析157例RP先證者DNA全外顯子組測序數(shù)據(jù),對Ret Net(2014年1月)中191個基因(包括62個RP已知基因和129個其他類型遺傳性視網(wǎng)膜變性基因)的變異通過頻率、功能預測、對照分析和家系共分離分析等一系列方法分析確定可能的致病突變并由Sanger測序驗證。結合課題組先前的研究,總結Ret Net基因突變在157例RP先證者中突變頻譜和頻率。結果:分析157個RP先證者全外顯子組測序數(shù)據(jù)中的Ret Net基因突變,共在90個(57.3%,90/157)家系中發(fā)現(xiàn)潛在致病突變,其中在79個家系中發(fā)現(xiàn)28個RP已知基因的突變,以及在11個家系中發(fā)現(xiàn)5個其他類型視網(wǎng)膜變性基因突變。檢出突變的90個家系中包括31(34.4%,31/90)個家系攜帶常染色體顯性基因的雜合突變,41(45.6%,41/90)個家系攜帶常染色體隱性基因的純合突變(10)和復合雜合突變(31),18(20.0%,18/90)個家系攜帶X連鎖基因的半合突變。在分析828個遺傳眼病全外顯子組測序數(shù)據(jù)時,發(fā)現(xiàn)Human Gene Mutation Database列出的致病突變中,有27個在我們所有遺傳眼病人群中等位基因頻率超過1%,分布在24個基因(12個綜合征基因和12和Ret Net基因)中。14個顯性Ret Net基因生物信息學預測致病的變異同時在不同疾病患者中發(fā)現(xiàn)。Ret Net基因預測致病變異在16個家系中發(fā)現(xiàn)不符合家系共分離。結論:通過全外顯子組測序,有57.3%的RP先證者在Ret Net基因中發(fā)現(xiàn)潛在致病突變。本研究不僅系統(tǒng)分析了中國RP患者全部視網(wǎng)膜變性已知基因突變頻譜和頻率,而且提供了中國RP患者分子病因的概述。對已知基因的分析為進一步發(fā)現(xiàn)新的RP致病基因提供了基礎和線索。本研究總結了高通量測序分析遺傳性視網(wǎng)膜變性致病基因突變過程中面臨的困難和解決策略,列舉了Ret Net基因突變可能不致病的基因和變異,提示在分析高通量測序突變數(shù)據(jù)時需要結合臨床多方面多角度分析。第二部分Ret Net基因突變在視網(wǎng)膜色素變性相關疾病先證者中的頻譜分析目的:全面分析159個中國LCA先證者Ret Net基因的突變頻譜和頻率。進一步發(fā)現(xiàn)LCA新的致病基因。另外,在46個中國高度遠視先證者中篩查高度遠視合并RP的致病基因——MFRP的突變。方法:基于159例LCA先證者全外顯子組測序數(shù)據(jù),對191個Ret Net基因(截止于2014年1月,包括62個RP已知基因和129個其他類型遺傳性視網(wǎng)膜變性基因)存在的變異,進行頻率、功能預測、對照分析和家系共分離分析等一系列分析,確定可能的致病突變并由Sanger測序驗證。由于意外發(fā)現(xiàn)ALMS1存在高頻無效突變,所以進一步擴大樣本量,分析了1220個遺傳性眼病患者的ALMS1基因變異,發(fā)現(xiàn)了多個ALMS1無效突變,進行Sanger測序驗證、家系共分離分析和對照分析,在回訪中檢查患者其他全身綜合征癥狀表現(xiàn)。在42個高度遠視先證者利用全外顯子組測序分析MFRP突變,另外4個先證者通過Sanger測序分析MFRP編碼序列,進一步在家系成員和192個正常對照中進行候選變異分析。結果:159個LCA先證者中,有90個發(fā)現(xiàn)了Ret Net基因的可能致病突變,占總人數(shù)的56.6%。其中72個只進行全外線組測序分析的患者中,有45個(62.5%)發(fā)現(xiàn)攜帶突變。突變頻率最高的幾個基因分別是GUCY2D(10.7%)、CRB1(7.5%)、CEP290(6.9%)、RPGRIP1(5.0%)、IQCB1(4.4%)、ALMS1(3.8%)和CRX(3.8%)。在1220個遺傳眼病患者中,11個LCA和早發(fā)重型錐桿細胞營養(yǎng)不良(cone-rod dystrophy,CORD)先證者發(fā)現(xiàn)攜帶13個ALMS1無效突變,這11個攜帶ALMS1無效突變的先證者在總數(shù)為215例的LCA和CORD中占5.1%(11/215)。15個患者中的9個通過回訪檢查發(fā)現(xiàn)Alstr?m綜合征的全身癥狀缺失或表現(xiàn)輕微。在3個高度遠視先證者中發(fā)現(xiàn)可能致病的復合雜合突變。臨床資料顯示這些患者屈光度至少有+13.50D或眼軸最長為16.78mm。視網(wǎng)膜電圖顯示攜帶錯義突變的患者視錐視桿細胞反應正常,另一個攜帶錯義和截短突變的患者視桿細胞反應降低,并伴有黃斑囊樣變性。結論:一系列的突變研究分析發(fā)現(xiàn)Ret Net已知基因突變可以解釋56.6%的中國LCA先證者患病原因。本研究對中國LCA患者種族特異性的已知基因突變頻譜和頻率進行了全面的分子遺傳學分析。提示ALMS1應該作為LCA或早發(fā)重型CORD的先證者的候選基因,眼部異�？赡苁蔷C合征最早發(fā)最明顯的癥狀。本研究擴展了MFRP的突變頻譜和其相關表型,這是在中國人群中首次報道MFRP突變。
[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.
【學位授予單位】：中山大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R774.1

【參考文獻】

相關期刊論文 前1條

1 Oscar Francisco Chacon-Camacho;Juan Carlos Zenteno;;Review and update on the molecular basis of Leber congenital amaurosis[J];World Journal of Clinical Cases;2015年02期

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本文編號：1959404