南宁市孕期女性耳聋基因筛查结果分析

作者:未知

   【摘要】 目的:對南宁市1 001例听力正常的孕期女性进行耳聋基因检测,了解本市常见耳聋基因突变类型和携带情况,为遗传咨询和优生优育提供数据。方法:在知情同意情况下,抽取孕妇外周血提取DNA,用微阵列芯片法检测GJB2基因c.35delG、c.235delC、c.176dell6和c.299-300delAT位点,GJB3基因c.538C>T位点、SLC26A4基因c.2168A>G和c.IVS7-2A>G位点、线粒体12S rRNA m.1555A>G和m.1494C>T位点。并对GJB2进行全基因组测序,以便发现更多与耳聋相关的突变位点。结果:1 001例孕期女性中微阵列芯片法共检测出18例(1.80%)致病性耳聋基因突变位点,其中GJB2突变c.235delC位点6例(0.60%),c.299-300delAT位点2例(0.20%),c.176dell6和c.35delG位点未检出突变;GJB3基因突变c.538C>T位点3例(0.30%);SLC26A4基因突变c.2168A>G位点1例(0.10%),c.IVS7-2A>G位点5例(0.50%);线粒体12S rRNA检出m.1555A>G突变1例(0.10%)。GJB2全基因测序发现318例(31.77%)孕妇携带致病性基因突变,常见的致病突变位点:c.109G>A 295例(29.47%)、c.11G>A突变15例(1.50%)、c.-23+1G>A突变7例(0.70%)。结论:南宁市孕妇GJB2基因突变携带最高,其次GJB3、SLC26A4、线粒体12S rRNA,致病性突变位点有GJB2基因:c.109G>A、c.11G>A、c.-23+1G>A、c.235delC和c.299-300delAT;GJB3基因:c.538C>T;SLC26A4:c.2168A>G和c.IVS7-2A>G;线粒体12S rRNA m.1555A>G。因此,孕期女性进行耳聋基因筛查,在预防遗传性耳聋患儿出生中有重要价值。
   【关键词】 耳聋基因 孕妇 产前筛查
   Analysis of the Screening Results of Deafness Genes in Pregnant Women in Nanning/QIN Weijuan, HUANG Weitong, YE Zhiling. //Medical Innovation of China, 2020, 17(04): 0-080
   [Abstract] Objective: The detection of deafness genes in 1001 pregnant women with normal hearing in Nanning City was conducted to understand the mutation types and carrying status of common deafness genes in this city, and provide data for genetic counseling and prenatal and postnatal care. Method: DNA was extracted from the peripheral blood of pregnant women with informed consent. Microarray chip was used to detect the c.35delG, c.235delC, c.176dell6 and c.299-300delAT loci of GJB2 gene, c.538C>T loci of GJB3 gene, c.2168A>G and c.IVS7-2A>G loci of SLC26A4 gene, m.1555A>G and m.1494C>T loci of mitochondrial 12S rRNA. The whole genome of GJB2 was sequenced in order to find more mutation sites associated with deafness. Result: A total of 18 cases (1.80%) of pathogenic deafness mutations were detected by Microarray chip in 1001 pregnant women, Among them, GJB2 gene mutations were found in 6(0.60%) cases at c.235delC site, 2 cases (0.20%) at c.299-300delAT site, and no mutations were found at c.176del6 and c.35delG site; gjb3 gene mutations were found in 3 cases (0.30%) at c.538c > T site; SLC26A4 gene mutations were found in 1 case (0.10%) at c.2168A > G site and 5 cases (0.50%) at c.IVS7-2A> G site; mitochondrial 12SRNA mutations were found in 1 case (0.10%) at m.1555a > G site. GJB2 whole-genome sequencing found that 318 pregnant women (31.77%) carry pathogenic gene mutations. The common pathogenic mutation sites are c.109G>A with 295 cases (29.47%), c.11G>A with 15 cases (1.50%) and c.-23+1G>A with 7 cases (0.70%). Conclusion: GJB2 gene mutation is the highest in Nanning pregnant women, followed by GJB3, SLC26A4 and mitochondrial 12S rRNA. Pathogenic mutation sites are GJB2 gene: c.109G>A,c.11G>A, c.-23+1G>A, c.235delC and c.299-300delAT; GJB3 gene: c.538C>T; SLC26A4 :c.2168A>G and c.IVS7-2A>G; mitochondrial 12S rRNA m.1555A>G. Therefore, the screening of deafness gene in pregnant women is of great value in preventing the birth of hereditary deafness.    [Key words] Deafness gene Pregnant women Prenatal screening
   First-author’s address: Nanning Maternal and Child Health Hospital, Nanning 530011, China
   doi:10.3969/j.issn.1674-4985.2020.04.019
   目前国内外研究已证实50%以上的耳聋人群是遗传因素导致[1],与耳聋相关的遗传基因有GJB2、JGB6、GJB3、MYO7A、MYO15A、OTOF、SLC26A4等。我国常见的非综合征型耳聋基因位点是GJB2的c.235delC,c.299-300delAT,c.176del16,c.35delG位点;SLC26A4的c.919-2A>G和c.2168A>G位点;线粒体12S rRNA的m.1555A>G和m.1494C>T位点;GJB3的c.538C>T位点[2],近5年来国内有报道采用基因芯片法對新生儿进行GJB2、GJB3、SLC26A4、线粒体12S rRNA筛查,发现遗传性耳聋基因携带率约为4.7%[3],尤其是GJB2致病性突变最高,有显著的种族区域差异。如果孕期女性进行耳聋基因筛查,可以发现遗传基因突变位点,可做预防和早期干预,能有效地降低耳聋患儿的发病率。本研究采用微阵列芯片对南宁市1 001例听力正常的孕期女性进行GJB2、GJB3、SLC26A4、线粒体12S rRNA基因9个位点检测,并对GJB2进行全基因测序,旨在了解南宁市孕期女性耳聋基因突变携带情况,扩大GJB2基因突变位点检测,以便发现更多的致病位点,为临床提供更可靠的预防和诊断依据。
  1 对象与方法
  1.1 研究对象 收集2017年6-12月来自南宁市妇幼保健院产前门诊的孕期女性。纳入标准:孕周7~20周,听力正常的孕期女性。排除标准:直系亲属有听力障碍者。本研究遵循医学伦理学相关规定,取得了所有研究对象的知情同意,并得到南宁市妇幼保健院的伦理委员会许可批准。
  1.2 芯片法 采集孕期女性外周血2 mL,提取DNA,检测浓度及纯度。使用微阵列芯片法晶芯九项遗传性耳聋基因检测试剂盒(成都博奥生物有限公司)对1 001例孕妇进行遗传性耳聋基因检测,检测基因位点:GJB2(c.35delG、c.235delC、c.176dell6和c.299-300delAT),GJB3(c.538C>T),SLC26A4(c.2168A>G、c.IVS7-2A>G),线粒体12S rRNA(m.1555A>G、m.1494C>T)。按照试剂盒说明书进行PCR扩增、杂交、洗片、芯片扫描及判读。
  1.3 测序法 使用全基因测序法检测1 001例孕妇GJB2基因。(1)文库构建,PCR扩增根据样品量计算需配制的PCR扩增体系,选取目的片段扩增程序进行扩增;(2)文库纯化;(3)文库质检:参照KAPA Library Quantification Kit Illumina? platforms TDS对文库进行定量;(4)测序:将文库pooling,变性最终以1.8 pM的文库上机测序。生物信息学分析:利用GATK工具判读SNP和Indel位点;利用Snpeff工具注释SNP,最后利用dbSNP、千人基因组数据库、HGMD等数据库、clinvar数据库进行注释和比较分析。
  2 结果
  2.1 芯片法和测序法基因突变情况 (1)1 001例孕期女性中使用基因芯片共检测出18例(1.80%)耳聋基因突变,均为杂合子。其中GJB2突变c.235delC位点6例(0.60%)、c.299-300delAT位点2例(0.20%),c.176dell6和c.35delG位点未检出突变;GJB3基因c.538C>T位点突变3例(0.30%);SLC26A4基因突变c.2168A>G位点1例(0.10%)、c.IVS7-2A>G位点5例(0.50%);线粒体12S rRNA检出m.1555A>G突变1例(0.10%),m.1494C>T位点未检出突变。(2)对GJB2进行测序共发现607例(60.64%)孕期女性携带有突变,其中致病性突变318例(31.77%),318例携带GJB2致病性突变的孕妇中,c.109G>A位点突变295例,占致病性突变的92.77%。GJB2测序结果有32种基因型,其中致病性基因型18种,多态性和未分类基因型有14种。18种致病性基因型中纯合子有21例(2.10%)均为c.109G>A突变;杂合子290例(28.97%),分别是:c.109G>A/wt 188例,c.109G>A/wt、c.217C>A/wt 8例,c.109G>A/wt、c.608T>C/wt 30例,c.109G>A/wt、c.79G>A/wt 5例,c.109G>A/wt、c.-121G>A/wt 1例,c.109G>A/wt、c.444C>T/wt 1例,c.109G>A/wt、c.79G>A/wt、c.341A>G/wt 36例,c.109G>A/wt、c.79G>A/wt、c.217C>A/wt 5例,c.11G>A/wt 8例,c.235delC/wt 4例,c.235delC/wt、c.79G>A/wt、c.341A>G/c.341A>G 2例,c.299-300delAT/wt 2例;c.109G>A/wt单杂合子是最常见的基因型共188例(18.78%);复合杂合子7例(0.70%),测序结果发现的复合杂合子7例(0.70%)是c.11G>A和c.-23+1G>A复合杂合突变,分别是c.11G>A/wt、c.-23+1G>A/wt、c.217C>A/wt基因型5例,c.11G>A/wt、c.-23+1G>A/wt、c.608T>C/wt基因型2例;测序同时还发现c.11G>A突变杂合子8例(0.79%),c.235delC突变杂合子6例(0.60%),c.299-300delAT突变杂合子2例,c.176dell6和c.35delG突变未检出,与芯片法检测结果一致。GJB2测序结果中最常见的多态性基因型是c.79G>A/wt、c.341A>G/wt共99例(9.89%)。见表1。   2.2 致病基因突变位点 本研究中GJB2测序共检出5个致病性位点,其中c.109G>A位点突变有295例(29.47%)是最常见的致病突变位点,等位基因频率为15.78%。GJB2基因其他4个致病性突变位点分别是c.11G>A位点15例(1.50%)、c.-23+1G>A位点7例(0.70%)、c.235delC位点6例(0.60%)、c.299-300delAT位点2例(0.20%)。本研究中GJB2多态性突变位点有4个:c.79G>A、c.608T>C、c.341A>G、c.444C>T,其中c.79G>A位点突变是最常见的多态性位点,等位基因频率为12.39%,其次是c.341A>G位点等位基因频率为10.04%。同时发现了3个GJB2突变新位点:c.553C>T、c.217C>A、c.-121G>A;1个未分类的位点:c.368C>A。见表2。
  3 讨论
  3.1 孕期女性的耳聋基因携带率 本研究结果显示,使用微陣列芯片法检测耳聋基因携带率为1.80%,其中携带GJB2、GJB3、SLC26A4、12S rRNA突变基因分别为0.80%、0.30%、0.60%、0.10%。采用芯片检测出GJB2基因突变率比较低,采用测序法检测出GJB2突变携带率比较高,致病性突变318例(31.77%);318例携带GJB2致病性突变的孕妇中,c.109G>A位点突变295例,占致病性突变的92.77%。说明本市孕妇GJB2突变率比较高,并以c.109G>A位点突变为主,致病位点比其他城市有明显差别。采用测序法比芯片法能筛查出GJB2更多的致病性突变位点,从而为临床提供更准确的遗传咨询。
  3.2 GJB2基因突变与耳聋相关性分析 GJB2位于13q12的DFNB1基因座上,编码CX26间隙连接通道蛋白,在钾循环、细胞间钙离子信号传导和支持耳蜗电活动等方面发挥重要生理作用,是耳蜗发育期间和成熟耳蜗功能中获得听觉必不可少的物质[4]。GJB2基因携带者的听觉表型具有明显的多样性,听力损失的程度从生时的重度耳聋到儿童晚期出现的轻度进行性耳聋不等,并且与基因型高度相关[5-6]。本研究中孕期女性GJB2基因致病性突变318例,携带率为31.77%,c.109G>A位点是最常见的突变位点,突变率为29.47%;其次是c.11G>A和c.-23+1G>A位点,突变率分别为1.50%、0.7%,而c.235delC突变率仅为0.60%,与国内其他城市报道有明显的种族区域差异。c.109G>A位点最初被认为良性多态性位点,然而近年大量的研究、荟萃分析都证实了GJB2基因c.109G>A位点与常染色体隐性遗传非综合征性耳聋相关[7-10],2019年出版的临床听力损失专家指南增添了GJB2基因c.109G>A位点[11]。c.109G>A在台湾有高达8.5%的等位基因频率[12],国内c.109G>A在广东、四川和山东的检出率分别为11.61%、10.44%和2.88%,具有明显地域差异,南方人群明显高于北方人群[13],而北京地区孕期女性的c.109G>A位点的携带率仅为为2.00%[14]。本研究中还发现7例(0.70%)GJB2基因c.11G>A和c.-23+1G>A复合杂合突变。c.-23+1G>A位点位于GJB2基因非编码区外显子1区,突变可引起常染色体隐性遗传听力损失,主要分布在南亚和中东地区的蒙古人、亚库特人[15]。本研究中孕期女性c.-23+1G>A位点携带率为0.7%,因c.-23+1G>A位点位于GJB2非编码区,在我国比较少有相关的报道。c.11G>A突变可在耳聋患者中检出,但致病性仍有争议[16-17],本研究中GJB2基因c.11G>A突变15例,携带率1.50%。本研究中广西南宁地区孕妇耳聋基因GJB2常见的致病突变位点以c.109G>A、c.11G>A和c.-23+1G>A突变为主,与国内其他地区有明显差异,可能是国内其他地区检测GJB2基因时未检测以上3个位点。
  3.3 GJB3基因突变与耳聋的相关性分析 GJB3基因位于染色体1p34.3,由2个外显子组成,编码连接蛋白31(Cx31),是一个广泛表达的蛋白家族的成员,它形成细胞间跨膜通道或缝隙连接,最近的研究发现GJB3与非综合征性常染色体隐性和显性耳聋有关[18]。我国GJB3基因突变引起的非综合性耳聋主要是c.538C>T和c.547C>T位点,本研究芯片法检测有3例孕妇携带GJB3基因c.538C>T位点突变,携带率0.30%,与文献[19]相近;可扩大筛查GJB3基因c.547C>T位点,选择适合本地区的筛查位点。
  3.4 SLC26A4基因突变与耳聋的相关性分析 SLC26A4突变引起的耳聋临床特征是前庭导水管综合征,先天性的内耳畸形[20-21],SLC26A4突变的个体应避免头部和耳朵的损伤,可显著降低耳聋的发生率。SLC26A4突变频率和基因型在不同的区域种族人群中差异显著,据有显著的遗传异质性。本研究中芯片法检测出SLC26A4突变6例(0.60%),其中c.2168A>G位点突变1例(0.10%),c.IVS7-2A>G位点突变5例(0.50%),c.IVS7-2A>G突变是SLC26A4基因突变主要位点,携带率比较低,可通过测序法发现更多SLC26A4基因致病性突变位点,筛选适合本地区的突变热点。
  3.5 线粒体12S rRNA基因突变与耳聋的相关性分析 线粒体12S rRNA m.1555A>G和m.1494C>T突变可致氨基糖苷类抗生素(AmAn)耳聋。在非综合征性耳聋人群中,线粒体12S rRNA基因以m.1555A>G突变为主,而1494C>T突变的频率要低得多,荟萃分析显示m.1555A>G和m.1494C>T在我国新生儿携带率分别为0.20%和0.03%[22]。本研究中共检出1例线粒体12S rRNA基因突变的孕妇,m.1555A>G突变1例(0.10%),m.1494C>T突变未检出。对于线粒体12S rRNA基因突变的携带者应避免使用氨基糖苷类药物,并使用替代药物进行抗生素治疗,以避免诱发耳毒性听力损失的发生。    综上所述,南宁市孕妇GJB2携带較高,其次为GJB3、SLC26A4、线粒体12S rRNA,其中致病性基因突变位点有GJB2基因c.109G>A、c.11G>A、c.-23+1G>A、c.299-300delAT和c.235delC;GJB3基因c.538C>T;SLC26A4基因c.2168A>G和c.IVS7-2A>G;线粒体12S rRNA基因 m.1555A>G。因此,孕期女性进行产前耳聋基因筛查,在预防遗传性耳聋患儿出生中有重要价值。
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  (收稿日期:2019-12-02) (本文编辑:张爽)
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