非综合征型遗传性耳聋两家系线粒体基因突变分析

Screening for mitochondrial DNA mutation in two pedigrees with nonsyndromic inherited sensorineural hearing loss

目的 探讨母系遗传非综合征型耳聋发病机理及 744 5 G点突变在这类家系及散发感音神经性耳聋病例中的发生率 ,为建立相应的基因诊断方法提供依据。方法 收集两个母系遗传非综合征型耳聋家系和 14个感音神经性耳聋散发病例 ;抽外周血标本 ,从白细胞中提取 DNA;聚合酶链反应扩增线粒体DNA(mitochondrial DNA,mt DNA)目的片段 ,分别以 Alw2 6 、Apa 及 Xba 限制性内切酶检测15 5 5 G、32 43G及 744 5 G点突变 ;行 mt DNA 12 S r RNA、t RNAL eu(UUR) 、t RNASer(UCN) 基因测序。结果 经酶切检测 ,两家系中 12例为 744 5 G点突变阳性 ,其余 6例及 14例散发病例均为阴性 ,所有病例 15 5 5 G、32 43G点突变均阴性 ;744 5 G点突变呈母系遗传。mt DNA测序显示 ,所有病例 15 5 5 G、32 43G点突变均阴性 ;酶切显示为 744 5 G突变阳性病例经基因测序均发现有 (nt) 744 5 A→ G替换。结论 744 5 G点突变在母系遗传非综合征型耳聋家系中有较高的发生率 ,而在散发病例中发生率很低 ;744 5 G 结合 15 5 5 G点突变筛查对这类耳聋的诊断有重要意义。

Objective. To investigate the genetic mechanism of maternal nonsyndromic inherited sensorineural hearing loss (SNHL), to identify the incidence of the 7445G mutation in such pedigrees and sporadic patients with SNHL, and to provide the theoretical evidence for the diagnosis of this disease. Methods. Blood samples were obtained from 2 pedigrees and 14 sporadic patients with SNHL. DNA was extracted from the isolated leukocytes. The mitochondrial DNA (mtDNA) fragments were amplified by PCR. The 1555G, 3243G and 7445G mutation was detected by Alw 26 I, Apa I and Xba 1 restriction endonuclease digestion respectively. The sequence of 12S rRNA, tRNALeu(UUR) and tRNASer(UCN) was examined. Results. Restriction endonuclease digestion analysis showed that 12 individuals from 2 pedigrees carried homoplasmic 7445G mutation, which was of maternal inheritance. Six individuals from 2 pedigrees and 14 sporadic patients did not have 7445G mutation. All individuals did not have 1555G and 3243G mutation. The sequence analysis further showed that none of them carried homoplasmic 1555G and 3243G mutation, 12 individuals had (nt)7445 A&rarrG substitution in tRNASer(UCN) gene. Conclusion. The incidence of 7445G mutation in such pedigrees is higher than that in sporadic patients. Screening for mtDNA 7445G mutation combined with 1555G examination is of much value to clinical use.