Study On Mutation Of Mitochondrial DNA Of In Patients Of Mitochondrial Myopathy And Encephalomyopathy

Objectives:.To screen the mtDNA mutations of mitochondrial myopathy and encephalomyopathy, and discuss relationship between the genotype and phenotype. AND to establish and assess the quantitative technique of real-time amplification refractory mutation system quantitative PCR(RT ARMS-qPCR)in the detection of the mitochondrial DNA A3243G mutation load,to investigate the mutation load in different tissues of mitochondrial myopathy and encephalomyopathy.Methods: The mtDNA hot-spots mutations A3302G, A3243G, T3271C, G3460A, A8344G, T8356C, T8993G, G11778A, T14484C, G15257A were screened in 45 patients of mitochondrial mitochondrial myopathy and encephalomyopathy by the polymerase reaction(PCR) combined with restriction fragment length polymorphism(PCR-RFLP) and sequencing, then the clinical features of patients with mutations were analysed. We construceted two kinds of cloned plasmids with mitochondrial A3243G point mutation of wild-type and mutant sequence respectively,then maked up 13 standard controls which had different mutation loads by mixing known amounts of cloned plasmid DNA. The mutation loads of the standard controls and different tissues from 7 patients and 1 carrier with A3243G point mutation. Then we sequenced the whole mtDNA of three patients who have been clinically diagnosised of mitochondrial myopathy and encephalomyopathy .Results: The A3243G mutation existed in eight cases of the 45 suspected mitochondrial myopathy and encephalomyopathy, accounting for 17.78% ,all the 8 cases were MELAS, and the mutation load of the muscle tissue ,hair, urine was higher than in peripheral blood. None of the 45 mitochondrial myopathy and encephalomyopathy cases was with the other hot-spots mutations. There was linear correlation between the results detected and expected values in the quantitative detection of standard controls. A determination coefficient of 0.991 between the expected and observed values by RT ARMS-qPCR was higher than that of 0.885 by PCR-RFLP. The mutation loads varied in different tissues from the 7 patients. The mutation loads of the patients detected by RT ARMS-qPCR were higher than by PCR-RFLP,the RT ARMS-qPCR can be more sensitive than PCR-RFLP to detect the lower A3243G mutation. The mutation load in muscle, hair follicles or urinary sediment is higher than that in leukocytes. After sequenced the whole mtDNA of three patients who have been Clinical diagnosis of mitochondrial encephalomyopathy and myopathy, one heteroplasmic variation and five homoplasmic variations were founded.120 normal controls were without these variations.Conclusions:The A3243G accounts for the most conment mutaions in mitochondrial myopathy and encephalomyopathy in China. The proportion of heteroplasmic mutations such as the A3243G variaties in different tissues. The RT ARMS-qPCR provides convenient, rapid, sensitive, reliable quantitative detection of heteroplasmic mutant mtDNA A3243G in different tissues. Urine and hair are tissues of choice for the diagnosis of mtDNA mutations, as they are easy to obtain and the mutation load is almost always greater than in blood. The C5691T is likely a new pathogenic mutation of MERRF,T1520C may be the polymorphism associated with mitochondrial myopathy and encephalomyopathy, G7600A, G7604A, A14071G, C14632T are polymorphisms.