一个母系遗传非综合征型耳聋家系线粒体12SrRNAG709A位点的突变分析

Mutation analysis of mitochondrial 12S rRNA gene G709A in a maternally inherited pedigree with nonsyndromic deafness

目的通过对一个母系遗传非综合征型耳聋家系进行线粒体DNA（mitochondrialDNA，mtDNA）12SrRNA、tRNASer（UCN）以及核基因GJB2突变分析，研究mtDNA突变与遗传性耳聋的相关性。方法临床听力测试以明确诊断，收集非综合征型遗传性耳聋家系中18例母系成员和53名对照（包括6名父系亲属、7名配偶对照和40名当地无关对照）外周静脉血样本，采用聚合酶链反应和测序技术对mtDNA12SrRNA、浓NAser（UCN）和GJB2基因进行突变分析，并对发现的基因突变进行计算机辅助的二级结构模拟分析。结果测序结果表明，此家系线粒体DNA12SrRNA存在mtDNAG709A点突变，该突变未见报道；无tRNASer（UCN）基因突变；对GJB2突变分析发现4例具有299—300delAT。计算机分析显示12SrRNA的二级结构中第8、9茎环结构发生改变。结论家系中8例耳聋患者都具有线粒体12S水NAG709A位点的突变，该突变在正常人群中具有高度保守性，提示GT09A点突变与母系遗传家系成员的进行性耳聋具有相关性；10例具有G709A突变的母系遗传家系成员未出现耳聋的临床表现，提示G709A点突变可能在其他核修饰基因的协同作用下参与了听力损害的过程。

Objective To investigate the relationship of mitochondrial DNA mutations with inherited deafness in a maternally inherited pedigree with non-syndromic deafness. Methods The diagnosis was validated by hearing tests. Blood samples were collected from 18 maternal members of the family and 53 controls including 6 paternal members, 7 spouses and 40 unrelated individuals. DNA was extracted from the leukocytes in blood samples. The gene fragments of mitochondrial DNA 12S rRNA, tRNASer（UCN） and GJB2 gene were amplified by polymerase chain reaction （PCR）. PCR products were analyzed by sequencing. Computerized 12S rRNA secondary structure modeling was carried out to characterize the mutation found in the family. Results A novel mitochondrial DNA 12S rRNA 709 G to A transition was detected from all maternal members including 8 patients with hearing loss and other 10 symptom-free maternal members. Non-maternal members and other controls did not carry this mutation. In addition, the tRNASer（UCN） A7445G, 12S rRNA A1555G and GJI32 gene mutations were not observed in the study. Computerized modeling showed that this mutation changed the eighth and ninth loop-stem structure of the 12S rRNA secondary structure. Conclusion In this family, 8 deaf patients carried the mitochondrial DNA 12S rRNA 709 G to A mutation, which is highly conservative in healthy adults. It was confirmed that the mitochondrial DNA 12S rRNA gene G709A was associated with non syndromic inherited hearing loss. The other 10 maternal members carried the mutation, but they did not suffer from deafness, which might suggest that the G709A mutation may cause hearing impairment in combination with a synergistic effect of some other nuclear modifier genes.