Abstract
OBJECTIVES: To investigate the molecular mechanism underlying Leber hereditary optic neuropathy (LHON) caused by the m.3472T>C (p.Phe56Leu) mutation. METHODS: Three large pedigrees (WZL122, WZ676, WZ706) carrying the m.3472T>C mutation were identified and selected from a cohort of 1397 LHON patients. Immortalized lymphoblast cell lines were established from peripheral blood samples obtained from two affected individuals carrying homoplasmic m.3472T>C mutation (derived from one Han Chinese LHON pedigree) and two genetically unrelated, matched healthy controls. The MitoTool software was used to analyze the complete mitochondrial DNA sequence to determine the haplogroup background, and the conservation of the mitochondrial DNA (mtDNA) mutation site was evaluated using the MitoMap database. The secondary structure of the NADH dehydrogenase subunit 1 (ND1) protein was predicted and analyzed using online prediction software. The three-dimensional structure and molecular interaction changes of wild-type and mutant ND1 proteins were visualized and analyzed using PyMOL. Western blotting was performed to determine the expression levels of key proteins. Cellular adenosine triphosphate (ATP) levels were measured using a chemiluminescence assay. Mitochondrial membrane potential and intracellular reactive oxygen species (ROS) levels were assessed by flow cytometry. RESULTS: mtDNA haplotype analysis showed that all the subjects belonged to the East Asian mitochondrial haplogroup D4. The m.3472T>C mutation results in the substitution of a highly conserved phenylalanine with leucine (p.Phe56Leu) in the ND1 of mitochondrial complex Ⅰ, which may alter the structure and function of ND1. In lymphoblast cell lines carrying the m.3472T>C mutation, manifestations of mutant ND1 instability were observed, including reduced ND1 protein levels, diminished mitochondrial ATP production, decreased mitochondrial membrane potential, and increased mitochondrial ROS production. Concurrently, the expression levels of antioxidant enzymes catalase and superoxide dismutase 2 were elevated (all P<0.01). These findings collectively suggest impaired complex Ⅰ function. CONCLUSIONS: The m.3472T>C mutation contributes to the pathogenesis of LHON by disrupting the structural stability of ND1 in complex Ⅰ, leading to mitochondrial dysfunction.