【摘要】　目的　研究我国原发性开角型青光眼(primary open angle glaucoma, POAG)患者小梁网糖皮质激素诱导反应蛋白（trabecular meshwork induced glucocorticoid response protein, TIGR）基因的突变情况。方法　(1)应用聚合酶链式反应(polymerase chain reaction, PCR)方法扩增70例POAG患者、20例正常对照组的TIGR 基因3个外显子（7对引物）的各个片段，应用单链构像多态(single- stranded conformation polymorphism, SSCP)筛选可能突变的PCR产物。(2)将筛选出的样本（PCR产物），克隆到PT-Adv载体，以Ecor I酶切鉴定重组质粒，然后用ABI-373自动测序仪行双向测序。结果　(1)用SSCP方法筛选的2例POAG患者在TIGR基因第3外显子中部（第6对引物）片段出现单链带型异常；正常对照组未见异常。(2)克隆测序的2例样品，1例在388密码子位置出现由GAT突变为AAT，氨基酸由天冬氨酸替换为天冬酰胺，即Asp388Asn；另1例碱基序列无变化。提示我国POAG患者TIGR基因突变仅为1.4%（1/70），远较国外为低。结论　我国POAG患者的发病机制可能与TIGR基因突变有关，但突变率较国外低，提示POAG发病存在地区或种族之间的差异。

To screen, clone and sequence TIGR gene mutation in Chinese patients with primary open- angle glaucoma

ZHUO Yehong, GE Jian, GUO Yan, et al.

　　(Zhongshan Ophthalmic Center, Sun Yat-sen University of Medical Sciences, Guangzhou 510060，China)

　　【Abstract】　Objective　To study trabecular meshwork induced glucocorticoid response protein (TIGR) gene mutation in Chinese patients with primary open-angle glaucoma (POAG). Methods　(1) From 70 patients with POAG and 20 normal controls, TIGR gene which consists of three exons (7 pairs of primer) was amplified by polymerase chain reaction (PCR). The mutation of PCR amplification products was evaluated by single-stranded conformation polymorphism (SSCP). (2) After the above PCR products were cloned into the PT-Adv vectors, the construction plasmids were evaluated by Ecor I endonuclease and direct sequence was performed. Results　(1) With SSCP screening, the single strand band abnormality was found in the middle fragments of the third exon (the sixth pair of primer) of TIGR gene in two patients with POAG, but the control group appeared normal. (2) The two samples using clone and sequence showed that one had a 'GAT'- to- 'AAT' transition at amino acid Asp 338 Asn mutation; the base sequence of another one had no change. These results suggested that mutation rate of TIGR gene in Chinese patients with POAG be only 1.4% (1/70), being lower than that of foreigners. Conclusion　The pathogenesis of Chinese patients with POAG may be related with TIGR gene mutation, but the mutation rate is lower than that in foreigners, indicating that the mechanism of POAG in China and aboard is different and the pathogenesis of POAG has difference in regions and races.

　　【Key words】　Primary open-angle glaucoma；　Glucocorticoid response protein, trabecular meshwork induced；　Genes；　DNA mutation；　Cloning, molecular

　　青光眼的遗传倾向早已为人们所关注。近年研究表明原发性开角型青光眼(primary open- angle glaucoma，POAG)致病的相关基因位于1q21至1q 31［1］， 进一步的连锁分析将这个区域缩短在DIS3665 至DIS3664区间［2］。位于该区域的小梁网糖皮质激素诱导反应蛋白（trabecular meshwork induced glucocorticoid response protein, TIGR）基因也被证实与青少年发病的POAG及部分非选择性POAG患者的发病有关，并发现多个突变点［3］。为探讨TIGR基因突变与我国POAG患者发病的关系，我们对70 例进展期POAG患者的TIGR 基因进行分析，现将结果报告如下。

资料与方法

　　一、诊断标准

　　参照1987年全国青光眼学组推荐的诊断标准，选择确诊为POAG(包括青少年发病和成年发病)的进展期或晚期而拟行抗青光眼手术的住院患者70例作为研究对象，其中男48 例，女22例，年龄14～40岁，所有患者均无明显青光眼家族史。对照组为排除青光眼及其他眼病的正常人20例。

　　二、实验方法

　　1.提取DNA:抽取每人的外周血1或2 ml，以Qiagen血样试剂盒进行基因组DNA的提取。

　　2.聚合酶链式反应(polymerase chain reaction, PCR)：以基因DNA为模板，用PCR反应扩增TIGR基因的3个外显子片段，包含各外显子的7对引物（表1）：

表1　用PCR反应扩增TIGR基因的3个

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