先天性后囊下型白内障一家系致病基因的研究

发布时间：2018-05-21 09:14

本文选题：先天性白内障 + 连锁分析　；参考：《天津医科大学》2017年硕士论文

【摘要】：目的确定所收集的中国一先天性白内障家系的临床表型,利用连锁分析方法对该家系进行已知致病基因的排除定位,并应用全外显子测序法寻找该家系的致病基因。方法1.临床研究:经患者及家属知情同意后获取家系成员血液样本,并对所有成员进行病史采集和详细的临床检查。2.连锁分析:在已知先天性白内障致病基因位置的上、下游各寻找一个物理位置与候选基因紧密连锁的微卫星标记,利用通用引物M13进行PCR扩增,扩增产物经ABI3130自动测序仪分离目的片段,然后使用Gene Mapper软件在内参下读出目的片段的大小,再利用家系遗传图谱观察是否存在与家系表型共分离的片段,并找出对应的候选基因,最后对候选基因进行直接测序寻找突变点,碱基序列比对使用DNAStar软件。3.全外显子测序:本研究采用Agilent的液相芯片捕获系统,对待测DNA样品的全部外显子区域进行建库、富集和捕获,之后在Illumina平台上进行高通量、高深度的测序。测序结束后对结果数据进行生物信息分析,首先参考人类遗传数据库信息来逐步过滤筛除数据排除非致病性的多态性位点,之后根据家系遗传模式、突变类型来筛选致病基因及突变点,并使用SIFT、Polyphen-2等软件进行突变蛋白的有害性预测,同时将已知的先天性白内障致病基因带入全外显子测序结果中进行筛查,最终对筛选出的可能致病突变进行Sanger测序验证,碱基序列比对同样使用DNAStar软件。结果1.该家系为常染色体显性遗传性先天性白内障,临床表型均为后囊下型白内障,家系成员除白内障以外无其他眼病及全身疾病。2.本研究共寻找到38个已知的先天性白内障候选基因,分别对位于1、2、3、4、6、8、10、11、12、13、14、15、16、17、18、19、20、21、22号染色体上的66个微卫星标记进行了连锁分析。连锁分析后除发现19号染色体上的D19S902和D19S904两个微卫星标记可能与家系致病基因连锁外未发现其余微卫星标记与家系致病基因连锁,对两个可能连锁的微卫星标记所对应的OPA3和FTL基因进行全部外显子的直接测序,测序结果经与正常基因序列比对后未发现任何突变点。3.对本家系两个患者成员进行WES后经生物信息筛选得到6个可疑致病基因及其突变点,分别是BFSP2(c.G1219A)、WFS1(c.A41G)、RPE65(c.C1154T)、CRYBG3(c.A8620C)、EPHA2(c.C1532T)和FYCO1(c.C2036T),直接测序后经与数据库基因对应位点的正常碱基序列比对后证实所筛选突变不为家系的致病突变。全外显子测序结果未在先天性白内障已知候选基因中发现本研究家系的致病突变,与连锁分析结果相符合。结论1.该先天性性白内障家系为常染色体显性遗传的后囊下型白内障;2.对该家系成员进行连锁分析排除了1、2、3、4、6、8、10、11、12、13、14、15、16、17、18、19、20、21、22号染色体上已知的38个先天性白内障候选基因为该先天性白内障家系致病基因的可能性,显示本研究家系的致病基因可能为新的基因;3.本研究缩小了该家系致病基因的寻找范围,为该家系的后续研究提供了基础和思路,同时也为其他先天性白内障致病基因的研究提供了一条新的研究方法。
[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.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R776.1

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