采用新方法分析中国福建省聋病患者线粒体 DNA A1555G 突变与野生型的比例及其与听力损失严重程度的关系。

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.

机构信息

Department of Laboratory Medicine, First Affiliated Hospital, Fujian Medical University, Fuzhou, Fujian 350005, China.

出版信息

Chin Med J (Engl). 2011 Oct;124(20):3347-52.

PMID:22088533

Abstract

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 10(2) - 10(8) 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 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.

摘要

背景

有人提出，突变型和野生型线粒体 DNA 的比例可能与其临床表型有关。在这项研究中，我们开发了一种高灵敏度的实时扩增阻滞突变系统-定量聚合酶链反应（RT-ARMS-qPCR）检测方法，用于定量含有突变 1555 位的线粒体 DNA（mtDNA），并探讨了中国福建省线粒体聋（MD）患者突变 mtDNA 比例与听力损失严重程度的关系。

方法

将含有 1555 位的扩增 mtDNA 片段连接到载体上，构建质粒 DNA 标准品。采用 RT-ARMS-qPCR 系统测量中国福建 126 例 MD 患者mtDNA 中野生型和突变型序列的拷贝数。结合临床资料，探讨了突变 mtDNA 比例与 MD 听力损失严重程度的关系。

结果

该队列的变异系数为 1.21%，批间变异系数为 1.78%，检测重组野生型质粒的线性范围为 10(2)-10(8)拷贝/μl。引物仅扩增预期序列。在散发性 mtDNA A1555G 异质性突变患者中，突变拷贝数与耳聋程度相关（R = 0.811，P = 0.003），但在散发性 mtDNA A1555G 同质性突变患者中不相关（R = 0.007，P = 0.989）。家族性病例 mtDNA A1555G 同质性或异质性突变的拷贝数与耳聋程度相关（R = 0.352，P = 0.023 和 R = 0.90，P = 0.012）。