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Quantification of Mitochondrial DNA with the A1555G Mutation in Deaf Patients Using Real-Time Amplification Refractory Mutation System-Quantitative PCR.

The objective of this study was to develop a real-time amplification refractory mutation system-quantitative PCR (RT-ARMS-qPCR) assay for quantitation of mitochondrial DNA (mtDNA) with a mutated 1555 site and to explore the relationship between the degree of mtDNA1555 mutation and the clinical phenotype of mitochondrial deafness. An amplified mtDNA fragment containing the 1555 site was ligated into a vector to construct a plasmid DNA standard. An RT-ARMS-qPCR system was then used to measure the mtDNA copy number containing wild-type and mutant sequences in a cohort of 96 deaf patients. The variation coefficient in the cohort was 1.34%; the interassay variation coefficient was 1.96%; and the linear range was 10(2) to 10(8) copies/mul for detecting a recombinant, wild-type plasmid. The primers amplified only the intended sequences. Mutation copy number correlated with the degree of deafness in sporadic cases with heteroplasmic mutations of mtDNA A1555G (r = 0.771, P = 0.003), but not in sporadic cases with homoplasmic mutations (r = 0.001, P = 0.997). Furthermore, the copy number of homoplasmic or heteroplasmic mutations of mtDNA A1555G in familial cases correlated with degree of deafness (r = 0.341, P = 0.022 and r = 0.85, P = 0.015, respectively). The RT-ARMS-qPCR system was therefore suitable for determining the copy number of mtDNA fragments containing the A1555G mutation, and mtDNA A1555G copy number correlates with severity of hearing loss.