摘要
Background:Leber’s Hereditary Optic Neuropathy(LHON;MIM 535000)is one of the most commonly inherited optic neuropathies and it results in significant visual morbidity among young adults with a peak age of onset between the ages of 15–30.The worldwide incidence of LHON is approximately 1 in 31,000.95%of LHON patients will have one of 3 primary mitochondrial mutations,G3460A(A52T of ND1),G11778A(R340H of ND4)and T14484C(M64V of ND6).There is incomplete penetrance and a marked gender bias in the development of visual morbidity with approximately 50%of male carriers and 10%of female carriers developing optic neuropathy.Visual recovery can occur but is dependent on the mutation present with the highest level of visual recovery seen in patients who have the T14484C mutation.The 3 primary mutations are typically identified by individual end-point PCR-restriction fragment length polymorphism(RFLP)or individual targeted bi-directional Sanger sequencing reactions.The purpose of this study was to design a simple multiplex PCR-RFLP that could detect these 3 primary LHON mutations in one assay.Methods:PCR primers were designed to incorporate a MaeIII restriction site in the presence of 3460A and 14484C mutations with the 11778A mutation naturally incorporating a MaeIII site.A multiplex PCR-RFLP assay was developed to detect the 3 common mutations in a single assay.Synthetic LHON controls based on the mitochondrial genome harbouring the 3 common mutations were synthesized and cloned into plasmids to act as reliable assay controls.DNA from previously tested patients and the synthetic LHON controls were subjected to the multiplex PCR-RFLP assay.The RFLP products were detected by agarose gel electrophoresis.Results:The novel PCR-RFLP assay accurately detects the 3 primary mutations both in patient DNA and in synthesized DNA control samples with a simple visual mutation detection procedure.The synthesized DNA was demonstrated to be a robust control for the detection of LHON Mutations.Conclusion:In this paper,we describe a novel,robust and simple PCR-RFLP based method for the detection of mutations causing LHON,and report the generation of a series of LHON DNA controls suitable for all currently published assays.
