本文選題：先天性白內(nèi)障 + 連鎖分析��； 參考：《天津醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的確定所收集的中國(guó)一先天性白內(nèi)障家系的臨床表型,利用連鎖分析方法對(duì)該家系進(jìn)行已知致病基因的排除定位,并應(yīng)用全外顯子測(cè)序法尋找該家系的致病基因。方法1.臨床研究:經(jīng)患者及家屬知情同意后獲取家系成員血液樣本,并對(duì)所有成員進(jìn)行病史采集和詳細(xì)的臨床檢查。2.連鎖分析:在已知先天性白內(nèi)障致病基因位置的上、下游各尋找一個(gè)物理位置與候選基因緊密連鎖的微衛(wèi)星標(biāo)記,利用通用引物M13進(jìn)行PCR擴(kuò)增,擴(kuò)增產(chǎn)物經(jīng)ABI3130自動(dòng)測(cè)序儀分離目的片段,然后使用Gene Mapper軟件在內(nèi)參下讀出目的片段的大小,再利用家系遺傳圖譜觀察是否存在與家系表型共分離的片段,并找出對(duì)應(yīng)的候選基因,最后對(duì)候選基因進(jìn)行直接測(cè)序?qū)ふ彝蛔凕c(diǎn),堿基序列比對(duì)使用DNAStar軟件。3.全外顯子測(cè)序:本研究采用Agilent的液相芯片捕獲系統(tǒng),對(duì)待測(cè)DNA樣品的全部外顯子區(qū)域進(jìn)行建庫(kù)、富集和捕獲,之后在Illumina平臺(tái)上進(jìn)行高通量、高深度的測(cè)序。測(cè)序結(jié)束后對(duì)結(jié)果數(shù)據(jù)進(jìn)行生物信息分析,首先參考人類遺傳數(shù)據(jù)庫(kù)信息來(lái)逐步過(guò)濾篩除數(shù)據(jù)排除非致病性的多態(tài)性位點(diǎn),之后根據(jù)家系遺傳模式、突變類型來(lái)篩選致病基因及突變點(diǎn),并使用SIFT、Polyphen-2等軟件進(jìn)行突變蛋白的有害性預(yù)測(cè),同時(shí)將已知的先天性白內(nèi)障致病基因帶入全外顯子測(cè)序結(jié)果中進(jìn)行篩查,最終對(duì)篩選出的可能致病突變進(jìn)行Sanger測(cè)序驗(yàn)證,堿基序列比對(duì)同樣使用DNAStar軟件。結(jié)果1.該家系為常染色體顯性遺傳性先天性白內(nèi)障,臨床表型均為后囊下型白內(nèi)障,家系成員除白內(nèi)障以外無(wú)其他眼病及全身疾病。2.本研究共尋找到38個(gè)已知的先天性白內(nèi)障候選基因,分別對(duì)位于1、2、3、4、6、8、10、11、12、13、14、15、16、17、18、19、20、21、22號(hào)染色體上的66個(gè)微衛(wèi)星標(biāo)記進(jìn)行了連鎖分析。連鎖分析后除發(fā)現(xiàn)19號(hào)染色體上的D19S902和D19S904兩個(gè)微衛(wèi)星標(biāo)記可能與家系致病基因連鎖外未發(fā)現(xiàn)其余微衛(wèi)星標(biāo)記與家系致病基因連鎖,對(duì)兩個(gè)可能連鎖的微衛(wèi)星標(biāo)記所對(duì)應(yīng)的OPA3和FTL基因進(jìn)行全部外顯子的直接測(cè)序,測(cè)序結(jié)果經(jīng)與正�；蛐蛄斜葘�(duì)后未發(fā)現(xiàn)任何突變點(diǎn)。3.對(duì)本家系兩個(gè)患者成員進(jìn)行WES后經(jīng)生物信息篩選得到6個(gè)可疑致病基因及其突變點(diǎn),分別是BFSP2(c.G1219A)、WFS1(c.A41G)、RPE65(c.C1154T)、CRYBG3(c.A8620C)、EPHA2(c.C1532T)和FYCO1(c.C2036T),直接測(cè)序后經(jīng)與數(shù)據(jù)庫(kù)基因?qū)?yīng)位點(diǎn)的正常堿基序列比對(duì)后證實(shí)所篩選突變不為家系的致病突變。全外顯子測(cè)序結(jié)果未在先天性白內(nèi)障已知候選基因中發(fā)現(xiàn)本研究家系的致病突變,與連鎖分析結(jié)果相符合。結(jié)論1.該先天性性白內(nèi)障家系為常染色體顯性遺傳的后囊下型白內(nèi)障;2.對(duì)該家系成員進(jìn)行連鎖分析排除了1、2、3、4、6、8、10、11、12、13、14、15、16、17、18、19、20、21、22號(hào)染色體上已知的38個(gè)先天性白內(nèi)障候選基因?yàn)樵撓忍煨园變?nèi)障家系致病基因的可能性,顯示本研究家系的致病基因可能為新的基因;3.本研究縮小了該家系致病基因的尋找范圍,為該家系的后續(xù)研究提供了基礎(chǔ)和思路,同時(shí)也為其他先天性白內(nèi)障致病基因的研究提供了一條新的研究方法。
[Abstract]:Objective to determine the clinical phenotype of a Chinese family with congenital cataract, and to use the method of linkage analysis to locate the known pathogenicity genes of the family, and to find out the pathogenic genes of the family by exon sequencing. Method 1. clinical study: the blood samples of family members were obtained after informed consent of the patients and their families, and .2. linkage analysis of all members of the disease history collection and detailed clinical examination: in the known location of the congenital cataract disease gene, the downstream each seeks a microsatellite marker closely linked to the physical location and candidate genes, PCR amplification by universal primer M13, and the amplified products to separate the target fragments by the ABI3130 automatic sequencer. Then Gene Mapper software was used to read the size of the target fragments under the internal reference, and then the family genetic map was used to observe whether there was a common fragment separated from the family phenotype, and to find the corresponding candidate genes. Finally, the candidate genes were sequenced directly to find the mutation points, and the base sequence alignment was sequenced using the DNAStar software.3. total exon: our research The Agilent liquid chip capture system was used to build a library, enrich and capture all the exons of DNA samples, and then carry out high flux and high depth sequencing on the Illumina platform. After sequencing the data, the results were analyzed by biological information. First, the information of human genetic database was used to filter the screening data gradually. The non pathogenic polymorphic loci were excluded, then the pathogenic genes and mutations were screened according to the family genetic pattern, the mutation type, and the SIFT, Polyphen-2 and other software were used to predict the harmful effects of the mutant proteins, and the known congenital cataract pathogenic genes were screened in the result of the exon sequencing. The possible pathogenic mutation was verified by Sanger sequencing, and the base sequence alignment was also used by DNAStar software. Results 1. the family was autosomal dominant congenital cataract, the clinical phenotype was posterior subcapsular cataract. The family members had no other ophthalmopathy except for cataract and the whole body disease.2.. This study found 38 known congenital diseases. A linkage analysis of 66 microsatellite markers on chromosome 1,2,3,4,6,8,10,11,12,13,14,15,16,17,18,19,20,21,22 was carried out for the candidate genes of sexual cataract. After linkage analysis, there were no other microsatellite markers that may be linked to the family gene of two microsatellite markers on chromosome 19 and D19S904. The OPA3 and FTL genes corresponding to two possible linked microsatellite markers were sequenced directly from the family related genes, and the sequencing results were not found at any mutation point.3. after comparison with normal gene sequences, and 6 suspected pathogenic genes were screened by biological information after WES of the two members of the family. The mutation points are BFSP2 (c.G1219A), WFS1 (c.A41G), RPE65 (c.C1154T), CRYBG3 (c.A8620C), EPHA2 (c.C1532T), and FYCO1 (c.C2036T). After direct sequencing, the mutation is confirmed by the normal base sequence alignment with the corresponding loci of the database. The result of the exon sequencing is not known in congenital cataract. The candidate genes were found to be in accordance with the results of linkage analysis. Conclusion 1. the congenital cataract family is autosomal dominant posterior subcapsular cataract; 2. the linkage analysis of the family members excludes 38 known 1,2,3,4,6,8,10,11,12,13,14,15,16,17,18,19,20,21,22 chromosomes. The possibility of the congenital cataract candidate because of the gene of the congenital cataract family shows that the pathogenetic gene of the family may be a new gene. 3. this study narrowed the range of finding the pathogenic genes in the family, provided a basis and thought for the follow-up study of the family, and also for other congenital cataract pathogeny. The study of the cause provides a new method of research.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R776.1

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