本文關(guān)鍵詞： 線粒體DNA A1555G突變 孕婦 氨基糖甙類(lèi)抗生素 非綜合征型耳聾　出處：《天津醫(yī)科大學(xué)》2012年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：背景聽(tīng)力損傷是最常見(jiàn)的感覺(jué)器官障礙之一,在新生兒中的發(fā)病率為1%0,50%的耳聾患者是由遺傳因素引起的,其中約有1%的患者是由線粒體基因突變引起。線粒體基因突變可以導(dǎo)致個(gè)體對(duì)致聾環(huán)境因素易感性增加,其中以對(duì)氨基糖甙類(lèi)抗生素易感性增加的線粒體DNA12S rRNA基因上的A1555G突變最為常見(jiàn)。該突變位點(diǎn)攜帶者在沒(méi)有使用氨基糖甙類(lèi)抗生素時(shí)可以表現(xiàn)為正常聽(tīng)力,在使用氨基糖甙類(lèi)抗生素之后表現(xiàn)為對(duì)此類(lèi)藥物敏感性增加,導(dǎo)致患者在使用正常劑量甚至微小劑量的藥物時(shí)就可以發(fā)生重度甚至極重度的聽(tīng)力損失。所以在孕婦中進(jìn)行mtDNA A1555G突變位點(diǎn)的篩查具有重要的意義。目的通過(guò)對(duì)孕婦mtDNA A1555G突變位點(diǎn)的篩查,評(píng)估A1555G突變?cè)谠袐D中的攜帶頻率,為在我國(guó)孕婦中進(jìn)行氨基糖甙類(lèi)抗生素致聾相關(guān)的線粒體基因突變位點(diǎn)的篩查提供依據(jù),對(duì)篩查陽(yáng)性的孕婦檢測(cè)胎兒羊水細(xì)胞線粒體A1555G突變情況,確定胎兒基因型,為其今后用藥提供臨床指導(dǎo)。 方法對(duì)2009年到2010年來(lái)天津市中心婦產(chǎn)科醫(yī)院做產(chǎn)前檢查的520名孕婦進(jìn)行線粒體DNA12S rRNA基因上的1555位點(diǎn)突變情況進(jìn)行檢測(cè)：應(yīng)用酚-氯仿法提取全血DNA, PCR擴(kuò)增目的片段,限制性內(nèi)切酶酶切擴(kuò)增產(chǎn)物,對(duì)酶切陽(yáng)性的產(chǎn)物直接測(cè)序,測(cè)序結(jié)果與標(biāo)準(zhǔn)線粒體DNA序列比較。對(duì)篩查陽(yáng)性的患者在B超引導(dǎo)下行羊膜腔穿刺抽取羊水提取胎兒DNA,進(jìn)行A1555G突變位點(diǎn)的檢測(cè),確定胎兒基因型。結(jié)果520名受檢孕婦中有2名孕婦的PCR-酶切產(chǎn)物提示擴(kuò)增目的片段酶切位點(diǎn)消失,酶切產(chǎn)物瓊脂糖凝膠電泳顯示741bp一條帶,PCR產(chǎn)物直接測(cè)序證實(shí)2名孕婦為A1555G同質(zhì)性突變攜帶者,孕婦中A1555G突變攜帶者檢出率為0.38%,均為同質(zhì)性突變,低于我國(guó)正常人群中0.7%的突變攜帶率,經(jīng)胎兒羊水細(xì)胞mtDNA檢測(cè)發(fā)現(xiàn)胎兒基因型與母親基因型完全相同。結(jié)論1.本研究通過(guò)對(duì)孕婦mtDNA A1555G位點(diǎn)突變的篩查在國(guó)內(nèi)首次得出該位點(diǎn)突變的攜帶頻率,且均為同質(zhì)性突變,填補(bǔ)了該領(lǐng)域研究的空白。2.對(duì)在產(chǎn)前篩查出陽(yáng)性孕婦的胎兒mtDNA基因型檢測(cè)發(fā)現(xiàn)其基因型與其母親基因型完全相同,從而得出在我國(guó)孕婦中進(jìn)行該突變位點(diǎn)的檢測(cè)對(duì)指導(dǎo)后代臨床用藥具有重要意義,對(duì)于減少由于mtDNAA1555G突變引起的耳聾患兒的出現(xiàn)具有重要的意義。
[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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R764;R440

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