孕妇mtDNA A1555G突变位点的筛查

发布时间：2018-01-31 17:06

本文关键词： 线粒体DNA A1555G突变孕妇氨基糖甙类抗生素非综合征型耳聋　出处：《天津医科大学》2012年硕士论文　论文类型：学位论文

【摘要】：背景听力损伤是最常见的感觉器官障碍之一,在新生儿中的发病率为1%0,50%的耳聋患者是由遗传因素引起的,其中约有1%的患者是由线粒体基因突变引起。线粒体基因突变可以导致个体对致聋环境因素易感性增加,其中以对氨基糖甙类抗生素易感性增加的线粒体DNA12S rRNA基因上的A1555G突变最为常见。该突变位点携带者在没有使用氨基糖甙类抗生素时可以表现为正常听力,在使用氨基糖甙类抗生素之后表现为对此类药物敏感性增加,导致患者在使用正常剂量甚至微小剂量的药物时就可以发生重度甚至极重度的听力损失。所以在孕妇中进行mtDNA A1555G突变位点的筛查具有重要的意义。目的通过对孕妇mtDNA A1555G突变位点的筛查,评估A1555G突变在孕妇中的携带频率,为在我国孕妇中进行氨基糖甙类抗生素致聋相关的线粒体基因突变位点的筛查提供依据,对筛查阳性的孕妇检测胎儿羊水细胞线粒体A1555G突变情况,确定胎儿基因型,为其今后用药提供临床指导。方法对2009年到2010年来天津市中心妇产科医院做产前检查的520名孕妇进行线粒体DNA12S rRNA基因上的1555位点突变情况进行检测：应用酚-氯仿法提取全血DNA, PCR扩增目的片段,限制性内切酶酶切扩增产物,对酶切阳性的产物直接测序,测序结果与标准线粒体DNA序列比较。对筛查阳性的患者在B超引导下行羊膜腔穿刺抽取羊水提取胎儿DNA,进行A1555G突变位点的检测,确定胎儿基因型。结果520名受检孕妇中有2名孕妇的PCR-酶切产物提示扩增目的片段酶切位点消失,酶切产物琼脂糖凝胶电泳显示741bp一条带,PCR产物直接测序证实2名孕妇为A1555G同质性突变携带者,孕妇中A1555G突变携带者检出率为0.38%,均为同质性突变,低于我国正常人群中0.7%的突变携带率,经胎儿羊水细胞mtDNA检测发现胎儿基因型与母亲基因型完全相同。结论1.本研究通过对孕妇mtDNA A1555G位点突变的筛查在国内首次得出该位点突变的携带频率,且均为同质性突变,填补了该领域研究的空白。2.对在产前筛查出阳性孕妇的胎儿mtDNA基因型检测发现其基因型与其母亲基因型完全相同,从而得出在我国孕妇中进行该突变位点的检测对指导后代临床用药具有重要意义,对于减少由于mtDNAA1555G突变引起的耳聋患儿的出现具有重要的意义。
[Abstract]:Background hearing impairment is one of the most common sensory organ disorders. The incidence of hearing loss in newborns is 10% and 50% of deaf patients are caused by genetic factors. About 1% of the patients were caused by mitochondrial gene mutations, which could lead to increased susceptibility to environmental factors of deafness. Mitochondrial DNA12S with increased susceptibility to p-aminoglycoside antibiotics. The A1555G mutation in the rRNA gene is the most common. The mutation locus carriers can present normal hearing without the use of aminoglycoside antibiotics. The use of aminoglycoside antibiotics showed an increased sensitivity to such drugs. This can lead to severe or even severe hearing loss in patients with normal or even small doses of drugs. Therefore, mtDNA is performed in pregnant women. Objective to screen mtDNA A1555G mutation sites in pregnant women. To evaluate the frequency of A1555G mutation in pregnant women, and to provide evidence for screening amino glycoside antibiotic induced deafness related mitochondrial gene mutation in pregnant women in China. The mtDNA A1555G mutation of amniotic fluid cells in pregnant women with positive screening was detected and the fetal genotypes were determined to provide clinical guidance for the future use of amniotic fluid. Methods from 2009 to 2010, 520 pregnant women who came to Tianjin central obstetrics and gynecology hospital for prenatal examination were treated with mitochondrial DNA12S. The mutation of 1555 locus on rRNA gene was detected. The whole blood DNA was extracted by phenol-chloroform method. PCR was used to amplify the target fragment, and the restriction endonuclease digestion products were sequenced directly. The results of sequencing were compared with the standard mitochondrial DNA sequence. Amniotic fluid was extracted from amniotic fluid by amniocentesis guided by B-ultrasound to detect the mutation site of A1555G in patients with positive screening. Results the PCR- digestion products of 2 of 520 pregnant women showed that the restriction site of the amplified fragment disappeared, and the agarose gel electrophoresis showed a band of 741 BP. Direct sequencing of PCR products confirmed that 2 pregnant women were A1555G homogenous mutation carriers, the detection rate of A1555G mutation carriers in pregnant women was 0.38%, all of them were homogenous mutations. The mutation carrying rate of 0.7% was lower than that of normal population in China. MtDNA analysis of fetal amniotic fluid cells revealed that the fetal genotype was identical to that of the mother. 1. The frequency of A1555G mutation was obtained for the first time in China. All of them were homogenous mutations, which filled the blank in this field. 2. The fetal mtDNA genotypes of prenatal screening positive pregnant women were found to be exactly the same as that of their mothers. It is concluded that the detection of the mutation site in pregnant women in China is of great significance in guiding the clinical drug use of offspring. It is of great significance to reduce the occurrence of deafness caused by mtDNAA1555G mutation.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R764;R440

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