视神经萎缩患者OPA1基因突变与功能验证
文献类型：学位论文
| 作者 | 胡秋香 |
| 学位类别 | 硕士 |
| 答辩日期 | 2014-03 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 姚永刚 |
| 关键词 | 常染色体显性视神经萎缩 OPA1基因 突变 线粒体 |
| 其他题名 | Mutation screening and functional characterization of the OPA1 gene in Chinese patients with optic atrophy |
| 学位专业 | 遗传学 |
| 中文摘要 | 背景 常染色体显性视神经萎缩（autosomal dominant optic atrophy, ADOA），也称Kjer型视神经萎缩，是一种最常见的遗传性视神经病变，主要表现为在青少年和青壮年时期视力丧失。约60%的ADOA由OPA1（Optic Atrophy 1）基因突变引起，OPA1蛋白位于线粒体内膜，在线粒体融合以及凋亡通路中发挥着重要作用，其功能异常会引起线粒体DNA（mitochondrial DNA，mtDNA）以及线粒体功能的变化。迄今为止，ADOA的致病机制仍然不清楚，mtDNA损伤、mtDNA拷贝数的变化以及线粒体功能紊乱等都被报道和ADOA相关。 方法 我们收集了106例疑似ADOA患者，对这些患者进行OPA1基因的全外显子测序，检测OPA1基因的突变频谱。同时，为了探索这些突变在ADOA中的潜在致病作用，我们构建了野生型和突变型的OPA1过表达载体，转染Hela细胞，检测OPA1蛋白不同剪切体表达的变化，并进行了一系列线粒体相关功能检测，包括线粒体形态、mtDNA拷贝数、氧化自由基水平（Reactive oxygen species，ROS）以及线粒体质量（mitochondrial mass）等变化情况。 结果 在106例疑似ADOA患者中，我们在8例患者中发现9个潜在的致病突变。其中有有3个变异位于内含子和外显子的交接区域（c.556+2T>G、c.2819-2A>G和c.2614-2A>G），可能影响OPA1的外显子剪接；有5个突变导致了氨基酸的改变（c.565G>A、c.575C>T、c.869G>A、c.1172T>G和c.1465A>G）；有一个4碱基缺失突变c.2708_2711del导致OPA1蛋白翻译提前终止而形成截短蛋白。这些突变中有4个（c.565G>A、c.1172T>G、c.1465A>G和c.556+2T>G）是本研究中新发现的，其中有一例患者同时携带两个潜在的致病突变（c.575C>T 和 c.1465A>G）。 我们构建过表达载体对以上突变进行功能研究。通过免疫荧光检测，我们发现c.565G>A和c.2708_2711del突变会改变线粒体的网状结构。通过蛋白免疫印迹（western blot）检测，我们发现c.565G>A、c.575C>T 和c.869G>A突变会改变OPA1异构体的剪切方式，而c.2708_2711del缺失突变导致35KDa左右的OPA1蛋白积累。 通过检测三组人群（携带OPA1突变的ADOA患者、不携带OPA1突变的疑似ADOA患者和正常人群）的血液中mtDNA的拷贝数，我们发现携带OPA1突变的ADOA患者mtDNA拷贝数比其他两组都低，但没有达到统计学显著水平，而两个对照组之间mtDNA拷贝数差异不大。该结果提示OPA1突变会导致mtDNA拷贝数下降；在Hela细胞中过表达OPA1突变载体的检测结果显示不同的OPA1突变对mtDNA拷贝数的影响不同。我们也对细胞内的ROS和线粒体质量变化进行了检测，我们的结果显示：OPA1突变导致细胞ROS水平均有所升高，同时OPA1蛋白过表达还导致线粒体质量有所升高。 结论 我们的结果表明OPA1基因突变在中国的ADOA病人中比较常见。不同的OPA1突变可能对mtDNA拷贝数影响不同；转染OPA1突变的Hela细胞的ROS升高。过表达OPA1蛋白的细胞线粒体质量也有所升高，可能是细胞内的代偿性反应以弥补线粒体的损伤。我们的结果也提示不同的OPA1突变可能通过不同的途径影响ADOA的发病，同时可能还有一些其他的因素共同影响着ADOA的发病，有待于我们进一步的研究。 关键词：常染色体显性视神经萎缩，OPA1基因，突变，线粒体 |
| 英文摘要 | Background Autosomal dominant optic atrophy (ADOA), Kjer type, is one of the most common hereditary optic neuropathies, which mainly leads to vision loss in adolescents and young adults. Over 60% ADOA cases are caused by mutations in the OPA1 gene which encodes a fusion protein of mitochondrial inner membrane. OPA1 protein plays an important role in mitochondrial fusion and apoptosis pathway. Mitochondrial DNA (mtDNA) and function can be changed by haploinsufficiency of OPA1 protein. Hitherto, the exact pathogenic mechanism of ADOA remains to be further explored. There are some common features for ADOA patients, such as mtDNA damage, alterations of mtDNA copy number and mitochondrial dysfunction. All these defects suggested that OPA1 gene mutations might cause ADOA via mitochondrial damage and dysfunction. Methods We collected 106 suspected ADOA patients and screened mutant spectra of the OPA1 gene by sequencing all 31 exons. To evaluate the role of the identified mutations, wild-type and mutant OPA1 were over-expressed in Hela cells. Western blot was performed to detect the change of different OPA1 isoforms. Further assays for mitochondrial damage and parameters, including alterations of mtDNA copy number, reactive oxygen species (ROS) level and mitochondrial mass, were also performed. Results Nine potentially pathogenic mutations were found in eight of 106 suspected ADOA patients. Among them, three were located in splicing site between intron and exon (c.556+2T>G, c.2819-2A>G, and c.2614-2A>G), which could affect the splice and isoforms of OPA1; five led to change of amino acid (c.565G>A, c.575C>T, c.869G>A, c.1172T>G, c.1465A>G); one was a 4 bp deletion mutation c.2708_2711del, which would result in a truncated OPA1 protein. Four out of nine mutations (c.556+2T>G, c.565G>A, c.1172T>G and c.1465A>G) have not been reported before. Interestingly, one patient harbored two potentially pathogenic mutations (c.575C>T and c.1465A>G). We constructed overexpression vector of OPA1 for further detection. We found that the exogenous OPA1 proteins could be exactly located in mitochondria by using immunofluorescence, and overexpression of mutants c.565G>A and c.2708_2711del induced severe fragmentation of filamentous mitochondrial network. The result of western blot showed that mutants c.565G>A, c.575C>T, c.869G>A changed the level of defferent isoforms of OPA1 protein. Mutant c.2708_2711del led to accumulation of a 35 KDa protein. Furthermore, mtDNA copy number in the blood tissue of ADOA patients carrying OPA1 mutation was decreased when comparing with suspected ADOA patients without OPA1 mutation and normal population, and the level of mtDNA copy number between suspected ADOA patients without OPA1 mutation or normal population was similar. Hela cells overexpressing OPA1 mutants, the results showed different OPA1 mutants had different levels of mtDNA copy number. In the aspect of mitochondrial functional parameters, we observed a higher ROS level resulted from OPA1 mutants, and the increased mitochondrial mass level in cell overexressing OPA1 mutant protein. Conclusion Our results indicated that the OPA1 gene mutations were common in Chinese ADOA patients. Mitochondrial dysfunction was found in cells overexpressing OPA1 mutants. Different OPA1 mutants present different effects on mtDNA copy number. The increase of ROS level and mitochondrial mass level in cells overexpressing OPA1 mutants suggested compensation effects resulted from damaged mitochondrion. All these results suggested that different OPA1 mutants affected ADOA via different pathways. There may be other factors that could affect onset of ADOA together with OPA1, which shall be further studied in the future. Key words: Autosomal dominant optic atrophy, OPA1 gene, mutation, mitochondrion |
| 语种 | 中文 |
| 公开日期 | 2014-07-15 |
| 源URL | [http://159.226.149.42:8088/handle/152453/7958]  |
| 专题 | 昆明动物研究所_重大疾病机理的遗传学 |
| 推荐引用方式 GB/T 7714 | 胡秋香. 视神经萎缩患者OPA1基因突变与功能验证[D]. 北京. 中国科学院研究生院. 2014. |
入库方式： OAI收割
来源：昆明动物研究所
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