Identification Of Combination Mutation Of G3460A And G9804A Causing Leber Hereditary Optic Neuropath By Mitochondrial DNA Sequencing

Objective:To detect the mutations of G3460A and G9804A in mtDNA of LHON and to explore the pathogenic mechanism about the mutations of G3460A and G9804A causing LHON and the effect of the maternally inherited mitochondria in LHON.Method:There are11people in a pedigree of LHON including the proband and his grandmother, mother and father, two uncles (one is single), two cousins, and a brother and a sister. One of his uncles is also LHON and others are normal. Ten ml anticoagulant blood of each object was obtained and peripheral blood mononuclear cells (PBMC) were isolated by Ficoll. One part of PBMC was used to separate the mitochondria and mtDNA. The PCR primers were designed and synthesized according to the sequences of mutations related with LHON. And the products of PCR were sequenced. Other part of PBMC was applied to construction of DNA library. The imbricated probes of mitochondrial genes were designed and used to capture the mitochondrial genes in above DNA library. The captured DNA were amplified with Illumina primers after they were purified and the purified PCR products were sequenced by Hisiq2500. The sequencing results were analyzed by bioinformatics softwares to investigate the mtDNA mutations associated with LHON.Results:G3460A was found in the mitochondria DNA of the proband, his uncle with LHON, grandmother and mother after detecting common mitochondrial mutation genes with PCR. The proband’s brother is normal although he carries G3460A. The mitochondria DNA of each object were sequenced to investigate the cause of LHON in this pedigree. And another mutation point, G9804A was found. It occurs not only in the proband and his uncle with LHON, but also in the normal uncle. Other39 non-hotspotted mutations were also found in this pedigree. The references and bioinformatics analysis show that G3460A leads to decrease of NADH dehydrogenase in the complex I of the oxidative phosphorylation chain in mitochondria which is critical to light-induced neural pathways. And G9804A causes the defects in cytochrome c oxidase subunit III (MTC03) in respiratory Complex IV, resulting in cell dysfunction.Conclusion:1. The combination of G3460A and G9804A causes Leber hereditary optic neuropath;2. The individual differences in offspring exist in the maternally inherited mitochondria. And the new mutations may be produced in the offspring during mitochondrial inheritance.