【作者】 李世宏；

【导师】 贺翔鸽；

【作者基本信息】 第三军医大学 ， 眼科学， 2003， 硕士

【摘要】 目的：本课题对重庆地区一个POAG家系进行了详尽的遗传学调查以及系谱分析，拟阐明此家系POAG的发病与遗传的关系，并分析其遗传特征。进而通过分子生物学方法对家系中POAG患者、一级亲属的OPTN基因的编码外显子进行PCR扩增，以期发现是否存在OPTN基因的突变，并分析突变可能造成的影响。研究方法：1. 收集POAG家系，并根据孟德尔原则进行详尽的遗传学调查分析。2. 提取所有样本的基因组DNA，PCR 扩增的13个外显子，部分外显子周围的内含子，共4350bp。3. 对家系中所有POAG患者及一级亲属OPTN基因编码外显子直接测序。4. 根据测序结果，选择特异性内切酶在散发性正常对照组中进行限制性长度片段多态性分析。5. 统计分析家系POAG组和对照组中OPTN基因突变分布差异性。6. 对检测到的OPTN基因突变进行生物信息学分析。结果：1. 该家系可追踪到的有五代共54人，男性29人，女性25人。其中已确诊的POAG患者17 例，男性患者11人，女性患者6人， 6人已经死亡。家系中连续四代发病。POAG的患病率为31％。2. 患者IV-5、III-5、 III-4以及一级亲属IV-7中第4外显子412G>A.。患者II-5的第5外显子603T>A。患者III-5第10外显子1267-1268ins C/1271-1272ins C。III-3患者第12外显子1537-1538ins C。一级亲属V-3出现15外显子1878-1879ins A。3. 32例正常对照组中有1例出现412G>A突变，其他突变型在对照组中均未发现。4. 在不计算412G>A突变情况下，POAG家族发生OPTN基因编码区的突变概率为20%，而对照组中未发现。经X2分析，X2=6.93> X20.016.63，P<0.01, POAG家族与对照组相差显著。生物信息学分析表明除第4外显子突变不改变OPTN蛋白的结构外，其他外<WP=8>5. 显子突变均将导致结构的变化，影响OPTN的重要功能域。结论：1. 重庆忠县李某POAG家系遗传方式属常染色体显性遗传。在家系中存在导致POAG发病的突变的基因。2. OPTN基因突变与本家系POAG发病相关。3. 基因突变造成OPTN蛋白一二级结构和重要功能域异常，导致OPTN蛋白功能的异常可能是POAG发病的重要原因之一。4. 本研究中共发现5种类型的OPTN基因突变（412G>A、 603T>A 1267-1268ins C/1271-1272ins C、 1537-1538ins C、 1878-1879ins A）。除412G>A、 603T>A两种与报道相同外，其余均为本次研究中第一次发现。更多还原

【Abstract】 Glaucoma is the second leading cause of blindness which affect over 70 million people worldwide. Primary open angle glaucoma (POAG) is the most common subtype and the first reason of blindness of black man in America. Moreover, There are many subjects suffering from POAG in China. POAG is a kind of complex latent disease characterized by chronic progressive optic ganglion cells apoptosis and visual field injury. Thus, It is difficult to diagnoses and treat in early period. Although it is not yet wholly understood about the mechanism of glaucoma, more and more evidences suggest that there is an association between heredity and gene mutations with pathogenesis of glaucoma. In spite of great progress of glaucoma genetics, the hereditary form of POAG is not clear. on the other hand, about ten genes loci were found to associate with POAG after the discovery that TIGR gene links to POAG based on molecular genetics assay. The optineurin（optic neuropathy inducing, OPTN）, a novel gene linked to POAG, was found by Reiaze and his colleagues in 2002. Mutations in the OPTN gene were found in 16.7% of 54 families with autosomal dominant adult-onset POAG, and in 13.6% of the sporadic glaucoma cases. However, there is no report about mutations of OPTN gene in Chinese POAG cases.Purpose:To investigate the relationship between POAG and the mutations of OPTN gene in China, a POAG family in ChongQings was followed up, and the blood specimens of POAG individuals and controls were collected and processed by molecular biological strategy.Methods: 1. To analyze the genetic form of the pedigree according to Mandalian hereditary rules after we collect a great family with POAG. 2. The genomic DNA was extracted from the peripheral blood of all samples. A total of 4350bp of 13 encoding exons and introns beside exons were amplified by PCR using the sequence-specific primers.3. Automated sequencing method was used to screen and identify mutations of OPTN<WP=5>gene of both POAG subjects and first degree relatives in this family.4. Restriction endonuclease analysis was used to detect the same mutation in the controls samples.5. The frequency of genetypes between POAG group and controls were compared by statistics.6. The sequence alterations were analyzed by bioinfomatics.Results:1. This pedigree could be traced to 54 members in 5 generations, Including 29 males and 25 females. There are 17 patients with POAG, including 11 males and 6 females. Six patients had died in this family. The four continuous generations exist patients with POAG. The prevalence rate is 31%.2. The encoding exons of OPTN gene of all alive patients and collected first degree relatives were directly automatic- sequenced, it is found that 3 patients ( IV-5、III-5、 III-4) and one first degree relative(IV-7) exist the 412G>A mutation in 4th exon. II-5 patient has a 603T>A alteration. III-5 was found that two C pyrimidine insertions between 1267-1268 site and 1271-1272 of 10th exon, respectively. III-3 exist 1537-1538ins (insert) C alteration. 1878-1879ins A had been found in a first degree relative (V-3).3. No same mutations of OPTN gene were detected in 32 controls, except one person has a 412G>A mutation. 4. Rate of observed mutations in POAG family is 20%, no mutations were found in controls. The mutation rate of POAG family group is obviously higher than that in the controls group( X2=6.93> X20.016.63，P<0.01).5. Bioinformatics analysis indicated that all mutations will result in the alteration of OPTN protein structure which may underlies its abnormal functional domain except for 412G>A mutation.Conclusions:1. The form of heredity in this POAG family in Zhong county of Chongqing is autosomal dominant trait, and there must be a abnormal gene which result in POAG in this family.2. The mutation of OPTN gene is responsible for onset of POAG in this pedigree.3. Function abnormality of OPTN protein, which are induced by change of the structure and functional domains caused by mutation of OPTN, may be an important m更多还原