| 其他摘要 | 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 |
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