一个氨基糖甙类药物致聋家系线粒体DNA全序列分析

Sequence analysis of total mitochondrial genome in a pedigree with aminoglycoside-induced hearing loss

目的评估线粒体单倍型对一个氨基糖甙类药物致聋家系中12S rRNA A827G突变表型的影响。方法用基因组DNA抽提试剂盒提取外周血DNA,PCR扩增家系成员mtDNA 12S rRNA基因进行A827G突变的测序验证,对携带A827G突变的家系先证者进行mtDNA全序列PCR扩增和测序分析,结果与修正的剑桥参考序列比对,识别除A827G以外的突变位点。结果 5名母系成员mtDNA 12S rRNA序列分析均检测到A827G突变。与修正的剑桥参考序列及家系配偶相比,先证者的mtDNA全序列分析显示出独特的多态性改变,除已知的12S rRNA A827G突变外,另检测到24个碱基变异,其中12S rRNA基因A783C、G786C以及ND4基因C11720A突变(L319A)为首次发现。系统进化法分析显示,上述多态性位点均位于mtDNA非保守区域。结论线粒体单倍型对该家系mtDNA A827G突变的表型表达无明显影响。

Objective To understand the role of mitochondrial haplotypes in the phenotypic expression of the 12S rRNA A827G mutation in a family with aminoglycoside-induced and nonsyndromic hearing loss.Methods Genomic DNA was isolated from whole blood of participants using the Puregene DNA Isolation Kits.The subject＇s DNA fragments spanning the entire mitochondrial 12S rRNA gene were amplified by PCR and sequenced to identify A827G mutation.The total mitochondrial genomes of the proband carrying the A827G mutation were then PCR amplified and submitted for sequence analysis.The resultant sequence data were compared with the revised Cambridge reference sequence.Results PCR amplifications were successfully conducted in all the subjects.Mitochondrial 12S rRNA A827G mutation was detected in 5 maternal members which was absent in the two spouses of the family.Sequence analysis of the complete mitochondrial genomes in the proband showed the distinct sets of mtDNA polymorphism（24 other nucleotide changes）,in addition to the identical 12S rRNA A827G mutation.These variants were further evaluated by phylogenetic analysis with mtDNAs from other organisms,and none of the sites of these variants are evolutionarily conserved.Conclusion Mitochondrial haplotypes are unlikely to affect phenotypic expression of the 12S rRNA A827G mutation in this family.