Analysis of the ratio of mitchondrial DNA with A1555G mutant to wild type in deaf patients of Fujian province in China by a new method and its relationship with the severity of hearing loss  

作　　者：OU Qi-shui CHENG Zu-jian YANG Bin JIANG Ling CHEN Jing 

机构地区：[1]Department of Laboratory Medicine, First Affiliated Hospital,Fujian Medical University, Fuzhou, Fujian 350005, China

出　　处：《Chinese Medical Journal》2011年第20期3347-3352,共6页中华医学杂志（英文版）

基　　金：This work was supported by grants from National Natural Science Foundation of China （No. 81041108） and Professor Development Foundation of Fujian Medical University （No. JS0014）.

摘　　要：Background It has been suggested that the ratio of mutant and wild type mitochondrial DNA may be related to its clinical phenotype. In this study, we developed a high sensitive real-time amplification refractory mutation system-quantitative polymerase chain reaction （RT-ARMS-qPCR） assay for quantitation of the mitochondrial DNA （mtDNA） with a mutated 1555 site, and explored the relationship between the ratio of mutated mtDNA and the severity of hearing loss of mitochondrial deafness （MD） patients of Fujian province in China. Methods 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 used to measure the mtDNA copy number containing wild-type and mutant sequences in a cohort of 126 MD patients of Fujian province in China. Combined with the clinical data, we explored the relationship between the ratio of mutated mtDNA and the severity of hearing loss of MD. Results The variation coefficient in the cohort was 1.21%, the interassay variation coefficient was 1.78%, and the linear range was 102-10e copies/pl 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=0.811, P=0.003）, but not in sporadic cases with homoplasmic mutations （R=0.007, P=0.989）. The copy number of homoplasmic or heteroplasmic mutations of mtDNA A1555G in familial cases correlated with degree of deafness （R=0.352, P=0.023 and R=0.90, P=0.012, respectively）. Conclusions The RT-ARMS-qPCR system is suitable for determining the copy number of mtDNA fragments containing the A1555G mutation. The ratio of mutated mtDNA correlates with the severity of hearing loss of MD.Background It has been suggested that the ratio of mutant and wild type mitochondrial DNA may be related to its clinical phenotype. In this study, we developed a high sensitive real-time amplification refractory mutation system-quantitative polymerase chain reaction （RT-ARMS-qPCR） assay for quantitation of the mitochondrial DNA （mtDNA） with a mutated 1555 site, and explored the relationship between the ratio of mutated mtDNA and the severity of hearing loss of mitochondrial deafness （MD） patients of Fujian province in China. Methods 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 used to measure the mtDNA copy number containing wild-type and mutant sequences in a cohort of 126 MD patients of Fujian province in China. Combined with the clinical data, we explored the relationship between the ratio of mutated mtDNA and the severity of hearing loss of MD. Results The variation coefficient in the cohort was 1.21%, the interassay variation coefficient was 1.78%, and the linear range was 102-10e copies/pl 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=0.811, P=0.003）, but not in sporadic cases with homoplasmic mutations （R=0.007, P=0.989）. The copy number of homoplasmic or heteroplasmic mutations of mtDNA A1555G in familial cases correlated with degree of deafness （R=0.352, P=0.023 and R=0.90, P=0.012, respectively）. Conclusions The RT-ARMS-qPCR system is suitable for determining the copy number of mtDNA fragments containing the A1555G mutation. The ratio of mutated mtDNA correlates with the severity of hearing loss of MD.

关 键 词：mitochondrial DNA A1555G real time-amplification refractory mutation system-quantitativepolymerase chain reaction DEAFNESS 

分 类 号：Q523[生物学—生物化学] TB53[理学—物理]