常染色体显性视神经萎缩患者OPA1基因突变分析及功能验证

Autosomal dominant optic atrophy (ADOA) is one of the most common hereditary optic neuropathies, which mainly lead to vision loss in adolescents and young adults. Over 60% ADOA patients are caused by mutations in the OPA1 gene which encodes a fusion protein of mitochondrial inner membrane. Hitherto, the exact pathogenic mechanism of ADOA remains to be further explored. There some common clinical features for ADOA patients, such as mtDNA damage, decrease of mtDNA copy number and mitochondrial dysfunction. All these defects suggested that OPA1 gene mutations might cause ADOA via mitochondrial damage and dysfunction. We have analyzed the full exons of the OPA1 gene in 30 patients with ADOA or suspected ADOA and identified 7 pathogenic mutations (including 4 novel mutations). Our results suggest that there is a remarkable difference in the OPA1 gene mutation spectra between Chinese and European patients. In this study, we will detect mutations in exons and tagSNPs of the OPA1 gene in 200 patients with ADOA or suspected ADOA. We will validate the pathogenic mutations by constructing different mutant OPA1 expression vectors and systematically evaluate their deleterious effect on mitochondrial function. We will focus on measurement of reactive oxygen species (ROS), mtDNA damage, mitochondrial respiration rate and other related parameters. This study will uncover mutation spectra of the OPA1 gene and elucidate the molecular pathogenic mechanisms of mitochondrial dysfunction caused by these OPA1 mutations. We believe our study will provide essential information for genetic counseling and clinical diagnosis and treatment of patients with ADOA.

常染色体显性视神经萎缩（ADOA）是最常见的遗传性视神经病变之一，多发于青少年时期，60%以上的病例由编码线粒体融合蛋白的OPA1基因突变导致，但其具体发病机理尚未阐明。已有研究报道ADOA患者存在mtDNA缺失、拷贝数降低和线粒体功能障碍，提示OPA1基因突变可通过线粒体损伤途径致病。我们前期针对30例ADOA患者和疑似患者进行了OPA1基因全部外显子测序分析，发现7个突变（包括4个新突变），提示我国病人突变频谱和报道的欧洲人群很可能存在差别。本研究拟扩大病例数到200例，进行OPA1基因全部外显子区域突变/变异分析，同时构建突变体进行体外表达，在细胞层次验证这些突变/变异与ADOA的相关性。我们将集中检测各种OPA1基因突变导致的线粒体功能损伤。本研究有望得到我国ADOA患者OPA1基因突变谱，并阐明不同突变导致线粒体损伤的致病机理，为患者的遗传咨询和临床诊治提供理论依据。

遗传性视神经萎缩患者主要由线粒体DNA突变致病，但临床上还存在很多无线粒体DNA突变的遗传性视神经萎缩患者（称为疑似视神经萎缩患者），表明这些患者的发病与核基因有关。通过本项目的支持，我们对疑似视神经萎缩患者的OPA1基因进行检测，该基因是显性遗传性视神经萎缩患者的主要致病基因。我们对105个中国汉族疑似视神经萎缩患者OPA1基因的29个外显子进行了测序，检测了存在OPA1基因突变和不存在OPA1基因突变的疑似视神经萎缩患者的线粒体DNA拷贝数，同时以无眼部疾病的正常人群作为对照人群。我们在8个患者中发现了9个可能的OPA1基因致病突变（其中6个突变位于外显子区，3个突变位于剪切区），对这些突变的致病性用软件预测、进化分析和细胞功能实验进行证实。发现c.1172T >G是我们首次报道的致病突变。通过比较我国汉族疑似视神经萎缩患者（我们的105例和已报到的193例）与普通对照人群（4327例）的OPA1基因的突变频率，发现该基因在视神经萎缩患者中具有较高的突变率。细胞实验证实突变c.869G>A和c.2708_2711de可以改变OPA1蛋白的剪切形式，从而影响线粒体的形态和自由氧含量，导致患者发病。我们的研究成果提示临床上有必要对疑似视神经萎缩患者进行OPA1基因突变谱检测，从而为这类患者的诊断和治疗提供依据和参考。