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Vietnamese patients (106) tentatively diagnosed with encephalomyopathy were screened for the presence of 15 common point mutations in mitochondria using PCR-RFLP. The screened mutations include A3243G, T3271C and T3291C for Mitochondrial Encephalopathy, Lactic Acidosis and Stroke-like episodes (MELAS); A8344G and T8356C for Myoclonus Epilepsy and Rag-Red Fibers (MERRF); G11778A, G3460A and T14484C for Leber's Hereditary Optic Neuropathy (LHON); T8993G/C and T9176G for Leigh syndrome; A1555G for deafness syndrome; G4298A, T10010C, T14728C and T14709C for neuromuscular syndrome. As a result, 6 cases of A3243G (5.7%) and 2 cases of T14727C (3.9%) were found. The 6 cases of A3243G mutation were heteroplasmic at different levels (4.23–80.85%). The T14727C change was discovered for the first time in the MTTE gene encoding for tRNAGluand showed homoplasmy. The T14727C change was probably a mutation because it was further confirmed as vertically inherited from the mother and not the result of isolated polymorphism. [ABSTRACT FROM AUTHOR]

The A3243G mitochondrial mutation is the major cause of mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS). The severity of the disease is correlated with the heteroplasmy level of the mutation. Here we describe for the first time the validation of a real-time polymerase chain reaction (PCR) assay with Taqman locked nucleic acid (LNA) fluorescent (FAM for mutant, HEX for wild type) probes for quantification of heteroplasmy levels in a total of 18 family members from 5 Vietnamese MELAS patients carrying A3243G. Almost no background of FAM signals was detected in normal samples, indicating that the probes were allele-specific. Standard curves indicate sensitive detection at 0.1% mutants and high reliability with R > 0.985. The correlation line between measured % mutant and expected % mutant was highly reliable, with a slope of 0.993 and R of 0.998. All positive A3243G mutant samples pre-screened by PCR-restriction fragment length polymorphism (RFLP) were confirmed, and their heteroplasmy levels quantified to be from 3.68 to 80.85%. The heteroplasmy levels in patients were higher than in their family members and generally correlated well with the severity of their clinical symptoms. Overall, this work is the first demonstration of the application of LNA probes for sensitive and highly reliable quantification of heteroplasmy levels in human mitochondria. [ABSTRACT FROM AUTHOR]