發(fā)布時(shí)間：2018-05-31 11:06

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

【摘要】：遺傳性視網(wǎng)膜變性是一類(lèi)常見(jiàn)難治的遺傳性致盲眼病,這其中最常見(jiàn)的有視網(wǎng)膜色素變性(retinitis pigmentosa,RP),最嚴(yán)重的是Leber先天性黑朦(Leber congenital amaurosis,LCA),具有高度遺傳異質(zhì)性和臨床表型多樣性。迄今報(bào)道至少有253個(gè)基因被發(fā)現(xiàn)與遺傳性視網(wǎng)膜變性相關(guān)(Ret Net網(wǎng)站:https://www.sph.uth.edu/Ret Net),其中82個(gè)與RP有關(guān)。由于致病基因眾多,以及視網(wǎng)膜變性遺傳學(xué)和表型的高度異質(zhì)性,目前此類(lèi)疾病的基因診斷存在極大困難。測(cè)序技術(shù)的限制使以往遺傳性眼病的分子遺傳學(xué)研究以單基因家系研究為主,鮮有對(duì)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(cè)序技術(shù)的迅猛發(fā)展,越來(lái)越多的研究者使用高通量測(cè)序分析遺傳病的發(fā)病原因。雖然高通量測(cè)序可以克服技術(shù)瓶頸在短時(shí)間內(nèi)獲得大量序列變異,但測(cè)序得到的數(shù)據(jù)量和復(fù)雜性也隨之增加,給變異的注釋和致病突變的確定帶來(lái)了新的挑戰(zhàn)。本研究旨在全面系統(tǒng)分析常見(jiàn)遺傳性視網(wǎng)膜變性患者Ret Net基因變異,可以對(duì)疾病的分子遺傳學(xué)病因有一個(gè)系統(tǒng)的了解。本研究基于高通量測(cè)序大數(shù)據(jù)的分析發(fā)現(xiàn)并總結(jié)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(cè)序數(shù)據(jù)基礎(chǔ)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(chǔ)上,系統(tǒng)總結(jié)了一組LCA病例Ret Net基因突變頻譜,同時(shí)對(duì)個(gè)別基因變異進(jìn)行了進(jìn)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(jié)了Ret Net基因在這兩種常見(jiàn)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(guān),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(duì)近千例遺傳性視網(wǎng)膜變性患者外顯子測(cè)序結(jié)果的分析,總結(jié)了高通量測(cè)序在眼遺傳病致病基因突變尋找過(guò)程中遇到的常見(jiàn)問(wèn)題,包括在眼遺傳眼病先證者全外顯子組測(cè)序結(jié)果中發(fā)現(xiàn)頻率大于1%且Human Gene Mutation Database報(bào)道致病的突變;經(jīng)生物信息學(xué)預(yù)測(cè)致病的變異,也可以同時(shí)并存于不同遺傳眼病中,或無(wú)法在家系共分離分析中得到驗(yàn)證。研究提示了人類(lèi)基因的復(fù)雜性,以及分析眼遺傳病高通量測(cè)序的結(jié)果時(shí)需要結(jié)合臨床多方面多角度綜合分析,確定變異致病性時(shí)需要謹(jǐn)慎。本研究為進(jìn)一步開(kāi)展遺傳性視網(wǎng)膜變性疾病的基因診斷奠定基礎(chǔ),也為這類(lèi)疾病的預(yù)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?cè)谝暰W(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(cè)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(jié)在高通量測(cè)序分析遺傳性視網(wǎng)膜變性致病基因突變過(guò)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(cè)序數(shù)據(jù),對(duì)Ret Net(2014年1月)中191個(gè)基因(包括62個(gè)RP已知基因和129個(gè)其他類(lèi)型遺傳性視網(wǎng)膜變性基因)的變異通過(guò)頻率、功能預(yù)測(cè)、對(duì)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(cè)序驗(yàn)證。結(jié)合課題組先前的研究,總結(jié)Ret Net基因突變?cè)?57例RP先證者中突變頻譜和頻率。結(jié)果:分析157個(gè)RP先證者全外顯子組測(cè)序數(shù)據(jù)中的Ret Net基因突變,共在90個(gè)(57.3%,90/157)家系中發(fā)現(xiàn)潛在致病突變,其中在79個(gè)家系中發(fā)現(xiàn)28個(gè)RP已知基因的突變,以及在11個(gè)家系中發(fā)現(xiàn)5個(gè)其他類(lèi)型視網(wǎng)膜變性基因突變。檢出突變的90個(gè)家系中包括31(34.4%,31/90)個(gè)家系攜帶常染色體顯性基因的雜合突變,41(45.6%,41/90)個(gè)家系攜帶常染色體隱性基因的純合突變(10)和復(fù)合雜合突變(31),18(20.0%,18/90)個(gè)家系攜帶X連鎖基因的半合突變。在分析828個(gè)遺傳眼病全外顯子組測(cè)序數(shù)據(jù)時(shí),發(fā)現(xiàn)Human Gene Mutation Database列出的致病突變中,有27個(gè)在我們所有遺傳眼病人群中等位基因頻率超過(guò)1%,分布在24個(gè)基因(12個(gè)綜合征基因和12和Ret Net基因)中。14個(gè)顯性Ret Net基因生物信息學(xué)預(yù)測(cè)致病的變異同時(shí)在不同疾病患者中發(fā)現(xiàn)。Ret Net基因預(yù)測(cè)致病變異在16個(gè)家系中發(fā)現(xiàn)不符合家系共分離。結(jié)論:通過(guò)全外顯子組測(cè)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(guó)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(guó)RP患者分子病因的概述。對(duì)已知基因的分析為進(jìn)一步發(fā)現(xiàn)新的RP致病基因提供了基礎(chǔ)和線索。本研究總結(jié)了高通量測(cè)序分析遺傳性視網(wǎng)膜變性致病基因突變過(guò)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(cè)序突變數(shù)據(jù)時(shí)需要結(jié)合臨床多方面多角度分析。第二部分Ret Net基因突變?cè)谝暰W(wǎng)膜色素變性相關(guān)疾病先證者中的頻譜分析目的:全面分析159個(gè)中國(guó)LCA先證者Ret Net基因的突變頻譜和頻率。進(jìn)一步發(fā)現(xiàn)LCA新的致病基因。另外,在46個(gè)中國(guó)高度遠(yuǎn)視先證者中篩查高度遠(yuǎn)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(cè)序數(shù)據(jù),對(duì)191個(gè)Ret Net基因(截止于2014年1月,包括62個(gè)RP已知基因和129個(gè)其他類(lèi)型遺傳性視網(wǎng)膜變性基因)存在的變異,進(jìn)行頻率、功能預(yù)測(cè)、對(duì)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(cè)序驗(yàn)證。由于意外發(fā)現(xiàn)ALMS1存在高頻無(wú)效突變,所以進(jìn)一步擴(kuò)大樣本量,分析了1220個(gè)遺傳性眼病患者的ALMS1基因變異,發(fā)現(xiàn)了多個(gè)ALMS1無(wú)效突變,進(jìn)行Sanger測(cè)序驗(yàn)證、家系共分離分析和對(duì)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(gè)高度遠(yuǎn)視先證者利用全外顯子組測(cè)序分析MFRP突變,另外4個(gè)先證者通過(guò)Sanger測(cè)序分析MFRP編碼序列,進(jìn)一步在家系成員和192個(gè)正常對(duì)照中進(jìn)行候選變異分析。結(jié)果:159個(gè)LCA先證者中,有90個(gè)發(fā)現(xiàn)了Ret Net基因的可能致病突變,占總?cè)藬?shù)的56.6%。其中72個(gè)只進(jìn)行全外線組測(cè)序分析的患者中,有45個(gè)(62.5%)發(fā)現(xiàn)攜帶突變。突變頻率最高的幾個(gè)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(gè)遺傳眼病患者中,11個(gè)LCA和早發(fā)重型錐桿細(xì)胞營(yíng)養(yǎng)不良(cone-rod dystrophy,CORD)先證者發(fā)現(xiàn)攜帶13個(gè)ALMS1無(wú)效突變,這11個(gè)攜帶ALMS1無(wú)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(gè)患者中的9個(gè)通過(guò)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(gè)高度遠(yuǎn)視先證者中發(fā)現(xiàn)可能致病的復(fù)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(zhǎng)為16.78mm。視網(wǎng)膜電圖顯示攜帶錯(cuò)義突變的患者視錐視桿細(xì)胞反應(yīng)正常,另一個(gè)攜帶錯(cuò)義和截短突變的患者視桿細(xì)胞反應(yīng)降低,并伴有黃斑囊樣變性。結(jié)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(guó)LCA先證者患病原因。本研究對(duì)中國(guó)LCA患者種族特異性的已知基因突變頻譜和頻率進(jìn)行了全面的分子遺傳學(xué)分析。提示ALMS1應(yīng)該作為L(zhǎng)CA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(kuò)展了MFRP的突變頻譜和其相關(guān)表型,這是在中國(guó)人群中首次報(bào)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(xué)位授予單位】：中山大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R774.1

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相關(guān)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(hào)：1959404