Application Of Next-generation Ion Torrent PGM^TM Platform On Diagnosis In Mitochondrial Diseases

Background and Objective: Mitochondrial diseases involving complex andvarious clinical manifestations can affect multiple systems. Lactate measurement,imaging, respiratory chain enzyme, sanger sequencing and so on can intimatediagnosis, but they are still lack of sufficient sensitivity or specificity and difficult todiagnose mitochondrial diseases. Next-generation sequencing technology appearingto detect a variety of levels of genes brings hopes to the gene researches. In this study,the use of Ion Torrent PGM sequencing technology on mitochondrial diseases inchildren detected clinically full mitochondrial genome and97complex I-associatednuclear genes’ mutations to find relevant mutations and discussed the geneticdiagnostic value of Ion Torrent PGM platform technology of mitochondrialdiseases.Methods:19cases were collected in the Department of Neurology of CapitalInstitute of Pediatrics from October in2011to May in2013, with approval from theirguardians or parents. Mitochondrial DNA was extracted from anticoagulatedperipheral blood. The products were mixed to prepare a library, and completed theentire sequencing by using Ion Torrent PGM platform. And when the enzymetesting of complex I was abnormal, blood samples of patients’ would be used tosequence97complex I-related nDNA genes. Ion Reporter software was used for dataanalysis.Results:(1) The total sequcing data from whole mitochondrial genome of19mitochondrial diseases`cases showed8.127×106bp,and8.082×106bp could bemapped on the reference(99.4%).(2)13cases of97complex Ⅰ-related nuclear genessequence report showed3.095×106bp data,2.903×106bp could be mapped on thereference(93.8%).(3)Ion Torrent Suite4.0.2and Torrent Browser software packagesused to analyze variants in samples got2357mutations(849mutations from mtDNAand1508from nDNA).1625variants were chosen by IgV software,including510mtDNA mutations and1115nDNA variants.Focusing on the mtDNA variants,5MD-related variants reported by other scholar articles were chosen by intercomparison of MITOMAP and Google sholar search engine，they are：A5514G、T12811C、T14502C、T14502C、G15927A. And T14502C and G15927A are morerelative with MD pathogenic genes.And there are13new variants founded in study.Conclusions:(1) In this study,there are5variants which are relative toMD,they are A5514G、T12811C、T14502C、A15924G、G15927A.And T14502Cmutation in the mitochondrial genome was reliable gene marker can help with Leber′s hereditary optic neuropathy(LHON)`s diagnosis;G15927A is a more pathogenicmutation associated with mitochondrial diseases.(2)There are13new variantsfounded in this study,and if they`re relative to MD need further research on them.(3)Next-generation on Torrent PGM platform has a broad and bright prospect ontesting pathogenic mutations of MD because of high sensitivity, rapid，cost-effectusing in sequencing.