中国人群两种遗传性视网膜变性疾病的基因型和表现型研究

发布时间：2018-01-23 02:25

  本文关键词： Bietti样萎缩 视网膜色素变性 CYP4V2基因 Best病 BEST1基因 突变 BEST1基因 突变 hBest1 功能　出处：《北京协和医学院》2015年博士论文　论文类型：学位论文

【摘要】：目的:Bietti结晶样视网膜色素变性。ietti crystalline comeoretinal dystrophy,BCD)是一种W视网膜结晶和视网膜色素上皮萎缩为特征的罕见常染色体单基因隐性遗传性视网膜变性疾病。己证实CF鲜。基因突变是导致BCD的原因。本研巧旨在分析鉴定5个中国BCD家系的CZP4F2基因突变W及患者的临床特征。方法:对所有患者进行详细眼科学检查。采集患者外周血血样并提取基因组DNA。通过聚合酶链式反应对CyP4F2基因的所有外显子和外显子内含子交界区域序列进行扩增,并通过DNA直接测序技术检测突变位点。同时对100条对照染色体进行基因突变检测1^^排除非致病性基因多态位点。结果:眼底检查发现所有患者眼底后极部均存在细小黄色结晶和视网膜色素上皮萎缩,一例患者并发脉络膜新生血管。共检出5种不同CyP4松基因突变位点,包括两个错义突变(P.F73L,P.R400H),2个剪切位点突变(c.802-8_810dell7insGC,c.l091-2AG)和1个单碱基对缺失突变(p.T479TfsX7orc.l437delC),其中3例患者中均检出送2个剪切位点突变。突变位点p.T479TfsX7是首次报道的新发突变位点,在100条正常对照染色体中未检出。结论:剪切位点突变c.802-8_810d过17insGC和c.l091-2AG是中国BCD患者常见突变。本研巧结果扩展了Bietti结晶样视网膜色素变性的等位基因异质性。目的:人类公公S77基因突变与至少4种不同的视网膜变性疾病相关,它们被统称为B的煽,包括:B的t卵黄样黄斑营养不良(B VMD),常染色体隐性遗传Best病(ARB),成人型卵黄样黄斑营养不良和常染色体显性遗传玻璃体视网膜脉络膜病变。本研究的目的是对中国人群中Bes偏患者的公U_77基因突变进行分析并描述这些患者的临床特征。方法;本研究共纳入来自12个无关中国家系的13例Best病患者。对送些患者进巧详细临床检查,包括最佳矫正视力、裂隙灯检查、眼底检查和彩照、光学相干断层扫描、眼底自发英光检查、视网膜电流图检查和眼电生理检查。采集受试者血样并提取基因组DNA,通过直接测序方法对公怎义口基因突变进行分析。同时对100条对照染色体进行基因突变检测W排除非致病性基因多态位点。结果:7例患者表现为BVMD表型,并携带与常染色体显性遗传相符的度扮77基因杂合突变。在这些患着中共检出2个化晵77基因新发突变位点(p.T4I,p.A291V)和3个己报道突变位点(P.R218C,P.Q293H,P.D301G)。6例患者携带与常染色体隐性遗传相符的公妨77基因双突变,其中4例BVMD表型患者携带公ES77基因杂合双突变,2例ARB表型患者携带公ES77基因纯合双突变。在这些患者中共检出8个公^僑77基因突变位点,包括4个新发突变位点(P.Y33H,P.R130L,P.M163民,c.519delA)和4个己报道突变位点(P.民13W'P.A195V,P.R255W,P.W287*)。结论:携带公勘77基因双突变的患者在中国Best病患者中常见,其临床表型具有一定异质性。公^僑77基因突变位点的不同巧质、不同组合W及非等位基因的上位效应均可能影响患者表型。本研究结果拓展了公^僑J7基因的突变谱。目的:常染色体隐目的：Bietti结晶样视网膜色素变性(Bietti crystalline corneoretinal dystrophy, BCD)是一种以视网膜结晶和视网膜色素上皮萎缩为特征的罕见常染色体单基因隐性遗传性视网膜变性疾病。已证实CYP4V2基因突变是导致BCD的原因。本研究旨在分析鉴定5个中国BCD家系的CYP4V2基因突变以及患者的临床特征。方法：对所有患者进行详细眼科学检查。采集患者外周血血样并提取基因组DNA。通过聚合酶链式反应对CYP4V2基因的所有外显子和外显子内含子交界区域序列进行扩增,并通过DNA直接测序技术检测突变位点。同时对100条对照染色体进行基因突变检测以排除非致病性基因多态位点。结果：眼底检查发现所有患者眼底后极部均存在细小黄色结晶和视网膜色素上皮萎缩,一例患者并发脉络膜新生血管。共检出5种不同CYP4V2基因突变位点,包括两个错义突变(p.F73L,p.R400H),2个剪切位点突变(c.802-8_810del17insGC, c.1091-2AG)和1个单碱基对缺失突变(p.T479TfsX7 or c.1437delC),其中3例患者中均检出这2个剪切位点突变。突变位点p.T479TfsX7是首次报道的新发突变位点,在100条正常对照染色体中未检出。结论：剪切位点突变c.802-8_810del17insGC和c.1091-2AG是中国BCD患者常见突变。本研究结果扩展了Bietti结晶样视网膜色素变性的等位基因异质性。目的：人类BEST1基因突变与至少4种不同的视网膜变性疾病相关,它们被统称为Best病,包括：Best卵黄样黄斑营养不良(BVMD),常染色体隐性遗传Best病(ARB),成人型卵黄样黄斑营养不良和常染色体显性遗传玻璃体视网膜脉络膜病变。本研究的目的是对中国人群中Best病患者的BEST1基因突变进行分析并描述这些患者的临床特征。方法：本研究共纳入来自12个无关中国家系的13例Best病患者。对这些患者进行详细临床检查,包括最佳矫正视力、裂隙灯检查、眼底检查和彩照、光学相干断层扫描、眼底自发荧光检查、视网膜电流图检查和眼电生理检查。采集受试者血样并提取基因组DNA,通过直接测序方法对BEST1基因突变进行分析。同时对100条对照染色体进行基因突变检测以排除非致病性基因多态位点。结果：7例患者表现为BVMD表型,并携带与常染色体显性遗传相符的BEST1基因杂合突变。在这些患者中共检出2个BEST1基因新发突变位点(p.T4I,p.A291V)和3个已报道突变位点(p.R218C, p.Q293H, p.D301G).6例患者携带与常染色体隐性遗传相符的BEST1基因双突变,其中4例BVMD表型患者携带BEST1基因杂合双突变,2例ARB表型患者携带BEST1基因纯合双突变。在这些患者中共检出8个BEST1基因突变位点,包括4个新发突变位点(p.Y33H, p.R130L, p.M163R, c.519delA)和4个已报道突变位点(p.R13H, p.A195V, p.R255W, p.W287*)结论：携带BEST1基因双突变的患者在中国Best病患者中常见,其临床表型具有一定异质性。BEST1基因突变位点的不同性质、不同组合以及非等位基因的上位效应均可能影响患者表型。本研究结果拓展了BEST1基因的突变谱。目的：常染色体隐性遗传Best病(ARB)与双杂合或纯合BEST1基因突变有关,是一种与典型卵黄样黄斑营养不良(BVMD)临床表现不同的疾病。本研究的目的在于通过研究第二部分所检出的与ARB相关的BEST1基因新发错义突变位点p.R130L和p.M163R编码突变型蛋白的细胞定位和稳定性,对其功能及致病机制进行初步探讨。方法：在野生型BEST1基因真核表达载体BEST1-pCMV6的基础上,利用定点诱变技术,构建p.R130L和p.M163R突变型BEST1基因真核表达载体。体外培养MDCKⅡ (Madin-Darby canine kidney II)细胞,将野生型以及突变型BEST1基因真核表达载体分别转染至MDCKⅡ细胞,经免疫荧光染色,激光扫描共聚焦显微镜观察比较野生型以及突变型蛋白在MDCKⅡ细胞中的表达和分布。同时将溶酶体抑制剂或蛋白酶体抑制剂与MDCKⅡ细胞共同孵育,细胞裂解产物进行Western blot检测,对野生型以及突变型蛋白的稳定性进行评估。结果：成功构建突变型BEST1基因真核表达载体,体外转染至MDCKⅡ细胞后,野生型hBestl蛋白和突变型蛋白hBest1R130L主要分布于细胞膜,而突变型蛋白hBest1M163R则主要分布于胞浆中。突变型蛋白hBest1M163R可被蛋白酶体快速降解,溶酶体抑制剂对于突变型蛋白hBest1R130L和hBest1M163R的降解无显著影响。结论：突变型蛋白hBest1M163R加工折叠和细胞定位错误是导致发病的分子机制。不同致病机制的突变型蛋白可导致同一临床表型。对于BEST1基因突变位点致病机制的研究有助于加深对于ARB病理机制的理解。性遗传Best病(ARJB)与双杂合或纯合公基因突变有关,是一种与典型卵黄样黄斑营养不良(BVMD)临床表现不同的疾病。本研巧的目的在于通过研巧第二部分所检出的与ARB相关的公c汿/基因新发措义突变位点P义130L和P.M163民编码突变型蛋白的细胞定位和稳定性,对其功能及致病机制进行初步探讨。方法:在野生型公邸77基因真核表达载体化晵77-PCMV6的基础上,利用定点诱变技术,构建P.R130L和P.M163R突变型公做77基因真核表达载体。体外培养MDCKII(Madin-Darby canine kidn巧II)细胞,将野生型W及突变型公尼S7V基因真核表达载体分别转染至MDCKII细胞,经免疫巧光染色,激光扫描共聚焦显微镜观察比较野生型W及突变型蛋白在MDCKII细胞中的表达和分布。同时将溶酶体抑制剂或蛋白酶体抑制剂与MDCKII细胞共同解育,细胞裂解产物进行Western blot检测,对野生型W及突变型蛋白的稳定性进行评估。结果:成功构建突变型S城77基因真核表达载体,体外转染至MDCKII细胞后,野生型hBestl蛋白和突变型蛋白hBestlKiwL主要分布于细胞膜,而突变型蛋白陆estlMiMK则主要分布于胞浆中。突变型蛋白姐estlMiwK可被蛋白酶体快速降解,溶酶体抑制剂对于突变型蛋白hBestlKiwt和hfiestlMWK的降解无显著影响。结论:突变型蛋白hBestlMiwK加工折叠和细胞定位错误是导致发病的分子机制。不同致病机制的突变型蛋白可导致同一临床表型。对于基因突变位点致病机制的研究有助于加深对于ARB病理机制的理解。
[Abstract]:Objective: Bietti.Ietti crystalline retinitis pigmentosa comeoretinal dystrophy, BCD W) is a kind of crystal retinal and retinal pigment epithelial atrophy characterized by rare autosomal recessive single gene hereditary retinal degeneration diseases. It has been proved that the CF gene mutation is fresh. The causes for BCD. The research and clinical features of W patients with CZP4F2 mutation how to analyze gene identification of 5 China BCD families. Methods: a detailed ophthalmologic inspection of all patients were collected. Peripheral blood samples and extracted genomic DNA. by polymerase chain reaction based on CyP4F2 for all the exons and exon intron junction region were amplified and detected by DNA direct sequence. Sequencing mutation. At the same time, 100 control chromosomes gene mutation 1^^ to exclude non pathogenic gene polymorphism. Results: the fundus examination revealed all Patients with posterior pole are tiny yellow crystal and retinal pigment epithelial atrophy, patients with choroidal neovascularization. There were 5 kinds of different pine CyP4 gene mutations, including two missense mutations (P.F73L, P.R400H), 2 splice site mutations (c.802-8_810dell7insGC, c.l091-2AG) and 1 single base pair deletion mutation (p.T479TfsX7orc.l437delC), in which 3 cases were detected and sent the 2 splice site mutations. Mutations of p.T479TfsX7 are reported for the first time in the new mutation sites in 100 normal control chromosomes were not detected. Conclusion: 17insGC and c.802-8_810d splice site mutation c.l091-2AG Chinese is common in patients with BCD mutation. The research results by extension Bietti crystalline retinitis pigmentosa allelic heterogeneity. Objective: human S77 gene mutation father-in-law is associated with at least 4 different retinal degenerative diseases, they are unified Called B fans, including: B t vitelliform macular dystrophy (B VMD), autosomal recessive Best disease (ARB), adult vitelliform macular dystrophy and autosomal dominant hereditary vitreous body chorioretinopathy. The purpose of this study is to analyze the U_77 gene in patients with partial Bes the mutation Chinese crowd and describe the clinical features of these patients were included in this study. Methods; from 12 unrelated China families in 13 cases of Best patients. To send some patients with skillful clinical examination, including best corrected visual acuity, slit lamp examination, fundus examination and color, optical coherence tomography, fundus spontaneous the ray of electroretinogram and visual electrophysiological examination. Samples were collected blood samples and genomic DNA was extracted by direct sequencing method of the public how Yi Kou mutation analysis was performed. At the same time, 100 control chromosomes gene mutation Detection of W to exclude non pathogenic gene polymorphism. Results: 7 patients showed BVMD phenotype, and carrying is consistent with autosomal dominant inheritance of a 77 gene mutation. In these patients were detected in 2 of 77 new mutations of a surname gene (p.T4I, p.A291V) and 3 have been reported mutations (P.R218C, P.Q293H, P.D301G).6 patient is consistent with autosomal recessive male. Double mutation of 77 genes, including 4 cases of BVMD patients carrying ES77 gene phenotype in heterozygous mutation, 2 cases of ARB patients carrying the male phenotype of homozygous ES77 gene mutation. Among these patients there were 8 males overseas Chinese ^ 77 gene mutation, including 4 new mutations (P.Y33H, P.R130L, P.M163 people, c.519delA) and 4 reported mutations (P. and 13W'P.A195V, P.R255W, P.W287*). Conclusion: the male carrying 77 gene mutation in patients with double exploration Chinese common in Best patients and its clinical Has a certain phenotypic heterogeneity. The Chinese ^ 77 gene mutation in different Qiao matter, epistatic effects of different combinations of W and non allelic genes can affect the patients. The results of this study extends the phenotypic spectrum of mutations in the J7 gene of the male ^ overseas. Objective: Objective: autosomal recessive Bietti crystalline retinal pigment degeneration (Bietti crystalline corneoretinal dystrophy, BCD) is a kind of crystal retinal and retinal pigment epithelial atrophy features rare autosomal recessive single gene hereditary retinal degeneration diseases. It has been confirmed that CYP4V2 gene mutations cause BCD CYP4V2 gene. This study aims to analyze the identification of 5 Chinese pedigree of BCD mutation and the clinical characteristics of patients methods: the detailed ophthalmologic inspection of all patients were collected. Peripheral blood samples and extracted genomic DNA. by polymerase chain reaction CYP4V2 gene exon of all The exons and introns were amplified at the junction region sequence was detected by direct sequencing of DNA mutations on chromosome 100. At the same time the control of gene mutation detection to exclude non pathogenic gene polymorphism. Results: the fundus examination showed all patients posterior pole are tiny yellow crystal and retinal pigment epithelial atrophy. One patient with choroidal neovascularization. There were 5 kinds of different CYP4V2 gene mutations, including two missense mutations (p.F73L, p.R400H), 2 splice site mutations (c.802-8_810del17insGC, c.1091-2AG) and 1 single base pair deletion mutation (p.T479TfsX7 or c.1437delC), in which 3 cases were detected in the 2 splice site mutations. Mutations of p.T479TfsX7 are reported for the first time the new mutation was not detected in 100 normal control chromosomes. Conclusion: c.802-8_810del splice site mutation 17insGC and c.1091-2AG Chinese is common in patients with BCD mutation. The results of this study extends the Bietti crystalline retinitis pigmentosa allelic heterogeneity. Objective: mutations in the human BEST1 gene is associated with at least 4 different retinal degenerative diseases, including they are collectively referred to as Best disease, Best vitelliform macular dystrophy (BVMD). Autosomal recessive Best disease (ARB), adult vitelliform macular dystrophy and autosomal dominant hereditary vitreous body chorioretinopathy. The purpose of this study is to BEST1 gene in patients with Best disease China crowd mutation analysis and describe the clinical features of these patients. Methods: This study included 12 unrelated from China family of 13 cases of Best patients. The detailed clinical examination for these patients, including best corrected visual acuity, slit lamp examination, fundus examination and color, optical phase stem tomography Description of fundus autofluorescence examination of electroretinogram and visual electrophysiological examination. Samples were collected blood samples and genomic DNA, mutation of BEST1 gene were analyzed by direct sequencing method. At the same time on the 100 control chromosomes gene mutation detection to exclude non pathogenic gene polymorphism. Results: 7 patients showed the BVMD phenotype, and carrying BEST1 and autosomal dominant gene with heterozygous mutation. Among these patients there were 2 new mutations of BEST1 gene (p.T4I, p.A291V) and 3 reported mutations (p.R218C, p.Q293H, p.D301G).6 patients with double mutations and autosomal recessive BEST1. Genes, including 4 cases of BVMD patients with BEST1 gene heterozygous phenotype of the double mutant, 2 cases of ARB patients with BEST1 gene phenotype of homozygous double mutant. Among these patients there were 8 mutations in the BEST1 gene locus, Including 4 new mutations (p.Y33H, p.R130L, p.M163R, c.519delA) and 4 reported mutations (p.R13H, p.A195V, p.R255W, p.W287*) conclusion: carrying two mutations in the BEST1 gene in patients with common China in patients with Best disease and its clinical phenotype with different nature of certain heterogeneity in.BEST1 gene mutation. The effects of different combinations and non allelic genes can affect the phenotype of patients. The results of this study expands the spectrum of mutations in the BEST1 gene. Objective: autosomal recessive Best disease (ARB) and double heterozygous or homozygous mutations of BEST1 gene, is a kind of typical vitelliform macular dystrophy (BVMD) the clinical manifestations of different diseases. The purpose of this study is that cellular localization and ARB of BEST1 gene related to the study of the second part of detection of novel missense mutation p.R130L and p.M163R encoding mutant protein and stability, A preliminary discussion on its function and pathogenesis. Methods: the wild type BEST1 gene eukaryotic expression vector BEST1-pCMV6, using site directed mutagenesis, constructed p.R130L and p.M163R mutant BEST1 gene eukaryotic expression vector. MDCK II (Madin-Darby canine kidney II in vitro cultured cells), wild type and mutant BEST1 gene eukaryotic expression vectors were transfected into MDCK II cells by immunofluorescence staining, confocal laser scanning microscopy in comparison to wild-type and mutant proteins in MDCK II cells in the expression and distribution. The lysosome inhibitor or proteasome inhibitor and MDCK II cells were incubated with cell lysates of Western blot detection to evaluate, and the stability of the wild type of mutant protein. Results: the successful construction of mutant BEST1 gene eukaryotic expression vector, transfection of MDCK II to fine 鑳炲悗,閲庣敓鍨媓Bestl铔嬬櫧鍜岀獊鍙樺瀷铔嬬櫧hBest1R130L涓昏�佸垎甯冧簬缁嗚優鑶�,鑰岀獊鍙樺瀷铔嬬櫧hBest1M163R鍒欎富瑕佸垎甯冧簬鑳炴祮涓，

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