Analysis Of Mitochondaral Mutation In Leber Hereditary Optic Neuropathy(LHON)

ObjectivesTo investigate the mitoehondrial factor related to Leber's hereditary neuropathy (LHON) through sequence analysis of the complete mtDNA and analysis the molecular pathogenetic mechanism of the disease.PatientsA six-generation Chinese family with 11 affected individuals clinically diagnosed LHON,including 5 affected individuals and 4 normal individuals,and 50 matched healthy controls was participated in this study.Methods(1)The patients and matched healthy controls's blood were collected;(2) Total genomic DNA was extracted from whole blood using the Wizard Genomic DNA Purification Kit (Promega, Beijing, China) according to the manufacturer's instructions.(3) Screening for the three primary LHON mutations (G3460A, G11778A, and T14484C) was done by multiplex allele-specific polymerase chain reaction(MAS-PCR).(4) The whole sequence were amplified using different two set PCR primers and the DNA sequences were analyzed.The sequencing results were compared with the updated consensus Cambridge sequence.ResultsThree primary LHON mtDNA mutations(G3460A, G11778A, and T14484C) were not detected in our patients .Sequencing analysis of the entire mtDNAs of the LHON family indicated the presence of a homoplasmic C4171A mutation and other 29 base changes relative to the standard Cambridge sequence. Conservation analysis and database search of the other 29 variants observed in our patients indicated that 27 of the 29 variants were known polymorphisms and the remaining two (nps 14841Gand 15095G in Cytb gene) were not.Mutations at the nucleotide positions (nps) 150T, 5231A, 12358G, 12372A,16257A, 16261T, 16129A, 4386C, 12007A, and 16111T partially defined the East Asian haplogroup N9a1.A novel heteroplasmic A14841G mutation,one of the variants with a serine substituted for a highly conserved asparagine at amino acid 32 of Cytochrome b(Cytb), may play a synergistic role with the C4171A mutation.Conclusion:The study further confirmed that homoplasmic C4171A mutation was a rare primary LHON mutation.In addition,we suggest that the novel heteroplasmic A14841G mutation in Cytb gene may as a second LHON mutation,may play a synergistic role with the C4171A mutation,leading to Chinese LHON/C4171A family with high penetrance of visual impairment and extremely low frequency of vision recovery.