中国福建线粒体耳聋患者线粒体DNA A1555G突变检测及其与耳聋严重程度的关系

背景 已知突变型与野生型线粒体DNA的比例与耳聋的临床表型有关。本研究建立高灵敏度的RT-ARMS-qPCR（real time-amplification refractory mutation system-quantitative PCR）系统定量测定含A1555G位点突变的线粒体DNA（mitochondrial DNA, mtDNA），探讨突变型mtDNA的比例变化与中国福建线粒体耳聋（mitochondrial deafness, MD）患者耳聋严重程度的关系。 方法 以PCR扩增含mtDNA 1555位点的片段，并将其克隆到pGEMT Easy载体上，构建质粒标准品，建立RT-ARMS-qPCR系统定量检测126个中国福建MD患者含突变型和野生型mtDNA 1555位点的片段的拷贝数。结合患者的临床资料，分析耳聋严重程度与突变型mtDNA所占比例的关系。 结果　RT-ARMS-qPCR系统在检测1个含野生型mtDNA 1555的重组质粒DNA模板时，其批内变异系数（CV）为1.21%，批间CV为1.78%，线性范围为102～108拷贝数/μl；突变型或野生型引物只特异扩增相对应的序列，特异性好；散发组mtDNA A1555G同质性突变的患者中，突变拷贝数与耳聋轻重程度无关（R=0.007，P=0.989）；散发组mtDNA A1555G异质性突变的患者中，突变型与野生型的比例与耳聋轻重程度相关（R=0.811，P=0.003）；家系组mtDNA A1555G同质性突变的拷贝数与耳聋轻重程度相关（R=0.352,P=0.023）；家系组mtDNA A1555G异质性突变的拷贝数与耳聋轻重程度相关（R=0.90，P=0.012）。 结论　RT-ARMS-qPCR系统适合于定量检测mtDNA A1555G点突变的线粒体DNA片段，结果特异、稳定、准确。线粒体耳聋的严重程度与突变型mtDNA 1555所占比例有关。

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

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 develop a high sensitive real-time amplification refractory mutation system-quantitative PCR (RT-ARMS-qPCR) assay for quantitation of the mitochondrial DNA (mtDNA) with a mutated 1555 site, and explore 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 explore 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 102-108 copies/µl 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 was 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.