【作者】 李战梅；

【导师】 贺翔鸽；

【作者基本信息】 第三军医大学 ， 眼科学， 2002， 硕士

【摘要】 青光眼是世界上第二大致盲眼病，但其发病原因尚不清楚。国内外研究均认为青光眼有遗传倾向。自TIGR基因被定位于1q23-24(GLC1A)内而成为青光眼的致病基因后，先后有很多研究致力于TIGR基因的突变研究。目前发现在TIGR基因的编码区有30多种序列变化，其中至少16种与青光眼密切相关。而在不同的人种、不同的地区的研究中，报道了多种不同的突变位点、突变率。在中国人原发性开角型青光眼中TIGR基因突变位点与突变率尚不清楚。本课题拟研究中国西南部基因库相对稳定的重庆地区原发性开角型青光眼患者与TIGR基因突变的关系，以便进一步揭示TIGR基因突变在中国人原发性开角型青光眼发病中的作用。同时，还可进一步利用人类基因组计划研究成果及现代分子生物学、分子遗传学理论与技术深入研究原发性开角型青光眼发病的分子机理，从而为临床防治该致盲性眼病探索一条新的途径。方法：本实验以重庆地区原发性开角型青光眼患者以及其一级亲属、正常人外周血白细胞DNA为模板，用PCR的方法扩增TIGR基因的三个外显子（包含了部分启动子及部分内含子），扩增产物以单链空间构象多态性及基因测序等方法进行分析，同时对检测到的突变结果进行生物信息学分析。结果：1、本研究在重庆地区15例原发性开角型青光眼患者中，共发现TIGR基因突变者5例（5/15），在10例亲属中发现2例（2/10），正常对照的20例中未发现TIGR基因突变。2、本研究共发现4个序列改变，其中编码区两个，非编码区两个。编码区的两个突变位点（Ser55Thr、Asp247Stop）为新发现的突变位点，另外在第二内含子区bp35c--t的突变，均未见报道。而在启动子区域bp-83c---t的变化有文献报道为一个多态位<WP=7>点。3、生物信息学分析发现编码区的突变可导致氨基酸序列、蛋白质的二级结构及等电点、抗原结合位点等发生改变。结论：本研究中发现在青光眼患者及其一级亲属中均可检测出TIGR基因的突变，而正常人未检测到，同时检测到突变的五例患者中有四例为青少年型开角型青光眼，说明TIGR基因突变与青少年开角型青光眼的发生密切相关，也可由此推测青光眼患者的亲属发病率较正常人高。同时对本研究中发现的突变基因编码蛋白质的氨基酸序列和二级结构及等电点、抗原结合位点等进行生物信息学分析，推测该基因的突变可引起TIGR蛋白的结构及理化特性的变化，这些变化可能是患者发生青光眼的原因之一。更多还原

【Abstract】 It is not yet wholly understood the mechanism of glaucoma, which is the second eye disease leading to blindness. A lot of efforts have been made on trabecular-meshwork inducible glucocorticoid response (TIGR) gene mutations, since TIGR gene located on chromosome 1q23-24 was selected as a gene causing primary open angle glaucoma (POAG). TIGR gene contains more than 30 sorts of DNA sequence variants including at least 16 variants closely related to POAG. Studies on people from different human races or from different living regions have reported there were varied mutation sites and prevalence of TIGR gene. Besides, it is still not known about the role of TIGR gene mutation in the occurrence of POAG among Chinese. The purpose of our study was to investigate the relationship between TIGR gene mutation and POAG in Chongqing where inheriting informations in population were stable. It will help us to understand the role of TIGR gene mutation in the occurrence of POAG among Chinese. Furthermore, with development of the human genome project and by advanced technique of modern molecular biology and molecular genetics, a new way for effective prevention and treatment of POAG might be found by deeply understanding the molecular mechanism of POAG.METHODS：In this study, genomic DNA was extracted from 1 ml whole blood of patients with POAG, their relatives and normal controls. The coding sequence of TIGR was screened for sequence alterations using polymerase chain reaction(PCR) followed by single-strand conformation polymorphism (SSCP).Samples corresponding to bands of altered mobility were sequenced.<WP=5>The sequence alterations were analyzed by bioinformatics.RESULTS：1、While TIGR gene mutations were screened in 5 patients among 15 patients with POAG in Chongqing, 2 cases of TIGR gene mutation were found in 10 persons whose family happened POAG. However, no TIGR gene mutation was detected in 20 people of control group.2、Four altered sequences were identified in our study. The mutation sites Ser55Thr, Asp247Stop and in 49021 c-t alteration were newly discovered in our study. The 63155 c-t alternation was reported as a polymorphism site.3、Bioinformatics analysis indicated that the mutations in structural domain might lead to the changes of the amino acid sequence coded, second structure of protein, isoelectric point and antigen binding site.CONCLUSIONS：The mutations of TIGR gene only were observed in patients with POAG and their relatives. There were four JOAG (Juvenile Onset Open Angle Glaucoma) in five patients occurred TIGR gene mutation. It indicated that TIGR gene mutations were closely related to the JOAG in Chongqing. The mutation frequency of TIGR in the relative of patient with POAG was higher than that of in control subjects. Furthermore, after analyzing the amino acid sequence, second structure of protein, isoelectric point and antigen binding site of altered gene, we suggested that the alteration of structure and biological activity of TIGR protein resulted from gene mutation might be one of the important factors involved in the pathogenesis of POAG.更多还原