【作者】 李海峰；

【导师】 邢光前；

【作者基本信息】 南京医科大学 ， 耳鼻咽喉科学， 2011， 硕士

【摘要】 目的:探讨2个氨基糖甙类药物所致非综合征型耳聋中国家系的分子遗传学特征。方法:从2个氨基糖甙类药物性耳聋及非综合征型耳聋家系选择部分成员,包括7名母系成员及1名听力正常配偶,取外周血,提取基因组DNA。用遗传性耳聋基因检测芯片检测4个中国人中常见的耳聋相关基因中的9个热点突变,包括GJB2 ( 35delG,176del16bp,235delC及299delAT ),GJB3 ( C538T ),SLC26A4( IVS7-2A>G,A2168G )和线粒体DNA 12S rRNA ( A1555G,C1494T )。PCR扩增阳性突变基因并测序验证检测结果。然后,采用特定引物对两家系的先证者分别进行线粒体DNA全序列及核基因TRMU和MTO1编码区的PCR扩增,扩增产物经纯化后直接用ABI 3730型测序仪测序,并将测序结果与标准序列比对,识别有意义的碱基变异。结果:芯片检测发现,两家系的7名母系成员均存在同质性线粒体DNA12S rRNA基因C1494T突变。与修正的剑桥参考序列相比,两名先证者的线粒体DNA全序列分析共发现了53个碱基变异,但除已知的12S rRNA C1494T突变外,其余52个碱基变异均为已报道的多态性位点;两家系先证者线粒体单体型分别是D4和D5a。TRMU和MTO1基因序列分析无异常发现。结论:线粒体DNA 12S rRNA C1494T突变是这两个家系耳聋发生的主要分子基础,而氨基糖甙类抗生素的应用增强了该突变的表型表达;未能证实线粒体单体型以及核基因TRMU和MTO1对家系成员C1494T突变的表型具有修饰作用。更多还原

【Abstract】 Objective:To explore the molecular genetic characterization of two Chinese families withaminoglycoside-induced and nonsyndromic sensorineural hearing loss.Methods:Blood samples were obtained from 7 maternal members and 1 married-in spouseof the two Chinese families. Genomic DNA was isolated from the peripheralleukocytes of the subjects using the Puregene DNA Isolation Kits. Firstly, 9 hot spotsfor mutations in four most common pathologic genes, GJB2 (35delG, 176del16bp,235delC and 299delAT), GJB3 (C538T ), SLC26A4 (IVS7-2A>G, A2168G) andmitochondria 12S rRNA (A1555G, C1494T), were screened with the DNA microarrayto detect the deafness-associated mutations. The whole mitochondrial genomes andthe coding sequence of TRMU and MTO1 genes of two probands were then PCRamplified using specific primers. Each fragment was purified and subsequentlyanalyzed by direct sequencing in an Applied Biosystems 3730 automated DNAsequencer. The resultant sequence data were compared with the standard sequence toidentify base changes.Results:Mitochondrial 12S rRNA C1494T mutation was detected in all 7 maternalmembers of the two families. Sequence analysis of the complete mitochondrialgenomes in the two probands revealed the distinct sets of mitochondrial DNApolymorphism (52 other nucleotide changes), in addition to the identical 12S rRNAC1494T mutation. None of these 52 variants, however, were shown to be pathogenic.The whole mitochondrial genome of proband from each of the two families wasestablished that they belong to mitochondrial haplogroups D4 and D5a respectively.No mutations were identified in either TRMU gene or MTO1 gene. Conclusions:C1494T mutation in the mitochondrial 12S rRNA gene is the main molecularmechanism responsible for the hearing loss in the two pedigrees, and the use ofaminoglycoside antibiotics may enhance the phenotypic manifestation of deafness-associated mitochondrial C1494T mutation. Mitochondrial haplogroups and nucleargenes of TRMU and MTO1, however, seem not contribute to the phenotypicexpression of C1494T mutation in these two families.更多还原