本文關鍵詞： Bietti樣萎縮 視網(wǎng)膜色素變性 CYP4V2基因 Best病 BEST1基因 突變 BEST1基因 突變 hBest1 功能　出處：《北京協(xié)和醫(yī)學院》2015年博士論文　論文類型：學位論文

【摘要】：目的:Bietti結晶樣視網(wǎng)膜色素變性。ietti crystalline comeoretinal dystrophy,BCD)是一種W視網(wǎng)膜結晶和視網(wǎng)膜色素上皮萎縮為特征的罕見常染色體單基因隱性遺傳性視網(wǎng)膜變性疾病。己證實CF鮮�；蛲蛔兪菍е翨CD的原因。本研巧旨在分析鑒定5個中國BCD家系的CZP4F2基因突變W及患者的臨床特征。方法:對所有患者進行詳細眼科學檢查。采集患者外周血血樣并提取基因組DNA。通過聚合酶鏈式反應對CyP4F2基因的所有外顯子和外顯子內(nèi)含子交界區(qū)域序列進行擴增,并通過DNA直接測序技術檢測突變位點。同時對100條對照染色體進行基因突變檢測1^^排除非致病性基因多態(tài)位點。結果:眼底檢查發(fā)現(xiàn)所有患者眼底后極部均存在細小黃色結晶和視網(wǎng)膜色素上皮萎縮,一例患者并發(fā)脈絡膜新生血管。共檢出5種不同CyP4松基因突變位點,包括兩個錯義突變(P.F73L,P.R400H),2個剪切位點突變(c.802-8_810dell7insGC,c.l091-2AG)和1個單堿基對缺失突變(p.T479TfsX7orc.l437delC),其中3例患者中均檢出送2個剪切位點突變。突變位點p.T479TfsX7是首次報道的新發(fā)突變位點,在100條正常對照染色體中未檢出。結論:剪切位點突變c.802-8_810d過17insGC和c.l091-2AG是中國BCD患者常見突變。本研巧結果擴展了Bietti結晶樣視網(wǎng)膜色素變性的等位基因異質(zhì)性。目的:人類公公S77基因突變與至少4種不同的視網(wǎng)膜變性疾病相關,它們被統(tǒng)稱為B的煽,包括:B的t卵黃樣黃斑營養(yǎng)不良(B VMD),常染色體隱性遺傳Best病(ARB),成人型卵黃樣黃斑營養(yǎng)不良和常染色體顯性遺傳玻璃體視網(wǎng)膜脈絡膜病變。本研究的目的是對中國人群中Bes偏患者的公U_77基因突變進行分析并描述這些患者的臨床特征。方法;本研究共納入來自12個無關中國家系的13例Best病患者。對送些患者進巧詳細臨床檢查,包括最佳矯正視力、裂隙燈檢查、眼底檢查和彩照、光學相干斷層掃描、眼底自發(fā)英光檢查、視網(wǎng)膜電流圖檢查和眼電生理檢查。采集受試者血樣并提取基因組DNA,通過直接測序方法對公怎義口基因突變進行分析。同時對100條對照染色體進行基因突變檢測W排除非致病性基因多態(tài)位點。結果:7例患者表現(xiàn)為BVMD表型,并攜帶與常染色體顯性遺傳相符的度扮77基因雜合突變。在這些患著中共檢出2個化晵77基因新發(fā)突變位點(p.T4I,p.A291V)和3個己報道突變位點(P.R218C,P.Q293H,P.D301G)。6例患者攜帶與常染色體隱性遺傳相符的公妨77基因雙突變,其中4例BVMD表型患者攜帶公ES77基因雜合雙突變,2例ARB表型患者攜帶公ES77基因純合雙突變。在這些患者中共檢出8個公^僑77基因突變位點,包括4個新發(fā)突變位點(P.Y33H,P.R130L,P.M163民,c.519delA)和4個己報道突變位點(P.民13W'P.A195V,P.R255W,P.W287*)。結論:攜帶公勘77基因雙突變的患者在中國Best病患者中常見,其臨床表型具有一定異質(zhì)性。公^僑77基因突變位點的不同巧質(zhì)、不同組合W及非等位基因的上位效應均可能影響患者表型。本研究結果拓展了公^僑J7基因的突變譜。目的:常染色體隱目的：Bietti結晶樣視網(wǎng)膜色素變性(Bietti crystalline corneoretinal dystrophy, BCD)是一種以視網(wǎng)膜結晶和視網(wǎng)膜色素上皮萎縮為特征的罕見常染色體單基因隱性遺傳性視網(wǎng)膜變性疾病。已證實CYP4V2基因突變是導致BCD的原因。本研究旨在分析鑒定5個中國BCD家系的CYP4V2基因突變以及患者的臨床特征。方法：對所有患者進行詳細眼科學檢查。采集患者外周血血樣并提取基因組DNA。通過聚合酶鏈式反應對CYP4V2基因的所有外顯子和外顯子內(nèi)含子交界區(qū)域序列進行擴增,并通過DNA直接測序技術檢測突變位點。同時對100條對照染色體進行基因突變檢測以排除非致病性基因多態(tài)位點。結果：眼底檢查發(fā)現(xiàn)所有患者眼底后極部均存在細小黃色結晶和視網(wǎng)膜色素上皮萎縮,一例患者并發(fā)脈絡膜新生血管。共檢出5種不同CYP4V2基因突變位點,包括兩個錯義突變(p.F73L,p.R400H),2個剪切位點突變(c.802-8_810del17insGC, c.1091-2AG)和1個單堿基對缺失突變(p.T479TfsX7 or c.1437delC),其中3例患者中均檢出這2個剪切位點突變。突變位點p.T479TfsX7是首次報道的新發(fā)突變位點,在100條正常對照染色體中未檢出。結論：剪切位點突變c.802-8_810del17insGC和c.1091-2AG是中國BCD患者常見突變。本研究結果擴展了Bietti結晶樣視網(wǎng)膜色素變性的等位基因異質(zhì)性。目的：人類BEST1基因突變與至少4種不同的視網(wǎng)膜變性疾病相關,它們被統(tǒng)稱為Best病,包括：Best卵黃樣黃斑營養(yǎng)不良(BVMD),常染色體隱性遺傳Best病(ARB),成人型卵黃樣黃斑營養(yǎng)不良和常染色體顯性遺傳玻璃體視網(wǎng)膜脈絡膜病變。本研究的目的是對中國人群中Best病患者的BEST1基因突變進行分析并描述這些患者的臨床特征。方法：本研究共納入來自12個無關中國家系的13例Best病患者。對這些患者進行詳細臨床檢查,包括最佳矯正視力、裂隙燈檢查、眼底檢查和彩照、光學相干斷層掃描、眼底自發(fā)熒光檢查、視網(wǎng)膜電流圖檢查和眼電生理檢查。采集受試者血樣并提取基因組DNA,通過直接測序方法對BEST1基因突變進行分析。同時對100條對照染色體進行基因突變檢測以排除非致病性基因多態(tài)位點。結果：7例患者表現(xiàn)為BVMD表型,并攜帶與常染色體顯性遺傳相符的BEST1基因雜合突變。在這些患者中共檢出2個BEST1基因新發(fā)突變位點(p.T4I,p.A291V)和3個已報道突變位點(p.R218C, p.Q293H, p.D301G).6例患者攜帶與常染色體隱性遺傳相符的BEST1基因雙突變,其中4例BVMD表型患者攜帶BEST1基因雜合雙突變,2例ARB表型患者攜帶BEST1基因純合雙突變。在這些患者中共檢出8個BEST1基因突變位點,包括4個新發(fā)突變位點(p.Y33H, p.R130L, p.M163R, c.519delA)和4個已報道突變位點(p.R13H, p.A195V, p.R255W, p.W287*)結論：攜帶BEST1基因雙突變的患者在中國Best病患者中常見,其臨床表型具有一定異質(zhì)性。BEST1基因突變位點的不同性質(zhì)、不同組合以及非等位基因的上位效應均可能影響患者表型。本研究結果拓展了BEST1基因的突變譜。目的：常染色體隱性遺傳Best病(ARB)與雙雜合或純合BEST1基因突變有關,是一種與典型卵黃樣黃斑營養(yǎng)不良(BVMD)臨床表現(xiàn)不同的疾病。本研究的目的在于通過研究第二部分所檢出的與ARB相關的BEST1基因新發(fā)錯義突變位點p.R130L和p.M163R編碼突變型蛋白的細胞定位和穩(wěn)定性,對其功能及致病機制進行初步探討。方法：在野生型BEST1基因真核表達載體BEST1-pCMV6的基礎上,利用定點誘變技術,構建p.R130L和p.M163R突變型BEST1基因真核表達載體。體外培養(yǎng)MDCKⅡ (Madin-Darby canine kidney II)細胞,將野生型以及突變型BEST1基因真核表達載體分別轉染至MDCKⅡ細胞,經(jīng)免疫熒光染色,激光掃描共聚焦顯微鏡觀察比較野生型以及突變型蛋白在MDCKⅡ細胞中的表達和分布。同時將溶酶體抑制劑或蛋白酶體抑制劑與MDCKⅡ細胞共同孵育,細胞裂解產(chǎn)物進行Western blot檢測,對野生型以及突變型蛋白的穩(wěn)定性進行評估。結果：成功構建突變型BEST1基因真核表達載體,體外轉染至MDCKⅡ細胞后,野生型hBestl蛋白和突變型蛋白hBest1R130L主要分布于細胞膜,而突變型蛋白hBest1M163R則主要分布于胞漿中。突變型蛋白hBest1M163R可被蛋白酶體快速降解,溶酶體抑制劑對于突變型蛋白hBest1R130L和hBest1M163R的降解無顯著影響。結論：突變型蛋白hBest1M163R加工折疊和細胞定位錯誤是導致發(fā)病的分子機制。不同致病機制的突變型蛋白可導致同一臨床表型。對于BEST1基因突變位點致病機制的研究有助于加深對于ARB病理機制的理解。性遺傳Best病(ARJB)與雙雜合或純合公基因突變有關,是一種與典型卵黃樣黃斑營養(yǎng)不良(BVMD)臨床表現(xiàn)不同的疾病。本研巧的目的在于通過研巧第二部分所檢出的與ARB相關的公c汿/基因新發(fā)措義突變位點P義130L和P.M163民編碼突變型蛋白的細胞定位和穩(wěn)定性,對其功能及致病機制進行初步探討。方法:在野生型公邸77基因真核表達載體化晵77-PCMV6的基礎上,利用定點誘變技術,構建P.R130L和P.M163R突變型公做77基因真核表達載體。體外培養(yǎng)MDCKII(Madin-Darby canine kidn巧II)細胞,將野生型W及突變型公尼S7V基因真核表達載體分別轉染至MDCKII細胞,經(jīng)免疫巧光染色,激光掃描共聚焦顯微鏡觀察比較野生型W及突變型蛋白在MDCKII細胞中的表達和分布。同時將溶酶體抑制劑或蛋白酶體抑制劑與MDCKII細胞共同解育,細胞裂解產(chǎn)物進行Western blot檢測,對野生型W及突變型蛋白的穩(wěn)定性進行評估。結果:成功構建突變型S城77基因真核表達載體,體外轉染至MDCKII細胞后,野生型hBestl蛋白和突變型蛋白hBestlKiwL主要分布于細胞膜,而突變型蛋白陸estlMiMK則主要分布于胞漿中。突變型蛋白姐estlMiwK可被蛋白酶體快速降解,溶酶體抑制劑對于突變型蛋白hBestlKiwt和hfiestlMWK的降解無顯著影響。結論:突變型蛋白hBestlMiwK加工折疊和細胞定位錯誤是導致發(fā)病的分子機制。不同致病機制的突變型蛋白可導致同一臨床表型。對于基因突變位點致病機制的研究有助于加深對于ARB病理機制的理解。
[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?yōu)鍚?閲庣敓鍨媓Bestl铔嬬櫧鍜岀獊鍙樺瀷铔嬬櫧hBest1R130L涓昏鍒嗗竷浜庣粏鑳?yōu)鑶?鑰岀獊鍙樺瀷铔嬬櫧hBest1M163R鍒欎富瑕佸垎甯冧簬鑳?yōu)娴嗕福?

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