一种药物性耳聋相关的线粒体A1555G或C1494T突变快速检测的方法研究

目的 线粒体12S rRNA突变是药物性耳聋的重要分子基础,其中A1555G和C1494T突变被报道和非综合症性耳聋有关,本研究旨在建立一种药物性耳聋相关的线粒体12S rRNA A1555G或C1494T突变快速检测的新方法,为耳聋的早期诊断和预防提供理论依据。 方法 以3种基因型(野生型、A1555G突变型、C1494T突变型)的线粒体12S rRNA序列为模板,使用Primer 5.0软件设计4条引物进行PCR扩增,同时检测A1555G或C1494T突变,并初步用于200例非综合症性耳聋患者的临床基因突变检测分析,最后通过PCR-Sanger测序进行评估。 结果 携带A1555G突变的个体经过PCR扩增后可以电泳检测出两条带(226 bp和736 bp),携带C1494T突变的个体经过电泳检测出488 bp和736 bp两条条带,而野生型的12S rRNA使用该方法仅扩增出736 bp一条条带。200例非综合症性耳聋患者中的A1555G和C1494T突变的检出率为4%(8/200),其中A1555G突变个体4例,C1494T突变个体4例;DNA测序分析检测突变型检出率为4%(8/200)。2种方法具有极好的一致性(Kappa=1.000,P<0.01)。 结论 该方法是一种简便、经济、准确、有效的A1555G和C1494T突变检测方法,可用于鉴定药物性耳聋相关的线粒体12S rRNA基因突变,从而有效的预防药物性耳聋的发生。 

A novel method for detection the deafness-associated mitochondrial A1555G or C1494T mutation

Objective Mutations in mitochondrial 12 S rRNA gene are the molecular basis of hearing loss, of these, the A1555 G and C1494 T mutations have been found to be associated with nonsyndromic hearing loss in many families worldwide.This study aimed to establish a novel method for detection the deafness-associated mitochondrial 12 S rRNA A1555 G or C1494 T mutation. Methods We used 3 standard templates (wild type, A1555 G, C1494T mutation) of human mitochondrial 12 S rRNA to design four primers for PCR amplification by employing the Primer 5.0 software, then this method was applied to clinical screening of 200 non-syndromic hearing loss subjects and confirmed by Sanger sequencing. Results We found that samples with the A1555 G mutation can be detected by two specific bands (226 bp and 736 bp), samples with the C1494 T mutation can be detected by the 488 bp and 736 bp, while the samples without these mutations can be detected by only one band (736 bp).Using this method, we detected the 8 cases carrying these mutations (4%, 8/200), including 4 cases with A1555 G mutation, 4 cases with C1494 T mutation, which was well consistent with the results of DNA sequencing (Kappa=1.000, P < 0.01). Conclusion This is a convenience, cost-effective, accurate and useful method for detection the deafness-associated mitochondrial A1555 G or C1494 T mutation, which could identify the subjects at risk and effectively prevent the aminoglycoside induced hearing loss.