非综合征型耳聋患者五个耳聋相关线粒体候选基因的检测及临床表型分析
发布时间：2018-09-09 17:47
【摘要】：目的:通过对西北地区97例非综合征型耳聋患者进行线粒体DNA全序列扩增测序,进而分析可能致聋的mtDNA突变位点。方法:采集中国西北五省、有家族史、无亲缘关系的97例中度至极重度非综合征型耳聋先证者的血样,并收集相关的临床及家系资料,另外收集376例听力正常人群作为对照组。用传统酚氯仿法提取相关基因组DNA,并用24对有部分重叠的正反向引物进行线粒体DNA全序列PCR扩增,扩增后测序,对结果进行分析,查找所有的线粒体DNA突变位点,确定线粒体单体型,分析突变位点保守性、突变位点的突变频率、突变可能对基因二级结构的影响,从而查找致聋的线粒体候选基因。结果:1、在病例组,男女患者比例为0.9:1(46:51),平均检测年龄为22±0.10岁,平均发病年龄3±0.83岁,听力损失从中度到极重度不等,其中10例先证者具有氨基糖苷类用药史。对照组男女比例为0.9:1(181:195),平均采集年龄25±4.12岁。2、病例组筛查到706个线粒体突变位点,其中D-loop区180个,12S rRNA基因27个(1个新位点和26个已报道位点),16S rRNA基因29个(7个新位点和22个已报道位点),tRNA基因29个(3个新位点和26个已报道位点),蛋白编码区434个,包含122个错义突变(4个新位点和108个已报道位点)和312个同义突变(20个新位点和292个已报道位点),非编码区7个。3、在病例组中,单体型D,G,M7,M8,M10,M11,M12,A,B4,F,H,N和R的频率分别为24.74%,7.22%,5.15%,10.31%,1.03%,1.03%,1.03%,8.25%,5.15%,13.40%,7.22%,7.22%和8.25%,而在对照组中,它们的频率分别为21.54%,4.26%,6.91%,10.64%,1.33%,0.53%,0.00%,6.65%,18.62%,15.96%,0.80%,8.78%和2.39%。其中单体型B、H、T在病例组和对照组之间的频数差异具有统计学意义。结论:通过本研究发现了五个与耳聋相关的线粒体候选基因。1、12S rRNA基因上新突变位点1473CT,可能改变了12S rRNA的三级或四级结构,影响线粒体蛋白质的合成,从而影响线粒体的功能。2、tRNA基因上新突变位点614 AC位于tRNAPhe反密码子环上(A37),突变可能影响密码子识别的精确性,从而影响tRNA结构和功能的稳定性;新突变位点8339AG位于tRNALys的T臂上(A50),突变破坏了原有的A-U配对,tRNA结构和功能稳定性受到影响。3、5656AG位于轻链t RNAAla和轻链tRNAAsn之间,突变可影响tRNAAla和tRNAAsn前体的处理,易导致临床表型异常。4、线粒体ND1 3866TC使NADH脱氢酶亚单位1上的极性疏水性异亮氨酸向苏氨酸过渡,影响NADH脱氢酶活性和破坏线粒体的正常功能,进而造成内耳的毛细胞损伤。5、单体型H、T在病组和对照组之间的频数差异具有统计学意义,考虑与耳聋的发病具有相关性。
[Abstract]:Objective: to analyze the mtDNA mutation sites in 97 patients with non-syndromic deafness in Northwest China by mitochondrial DNA amplification and sequencing. Methods: the blood samples of 97 patients with moderate to very severe non-syndromic hearing loss were collected from five provinces of Northwest China with family history and no relationship. The clinical and family data were collected and 376 cases of normal hearing were collected as control group. Genomic DNA, was extracted by traditional phenol chloroform method and 24 pairs of positive and negative primers with partial overlap were used to amplify the whole mitochondrial DNA sequence PCR. The results were sequenced and the results were analyzed to find all the mitochondrial DNA mutation sites. To determine mitochondrial haplotype, analyze the conservation of mutation sites, the mutation frequency of mutation sites, mutation may affect the secondary structure of the gene, so as to find the candidate genes for deafness mitochondria. Results in the case group, the ratio of male to female was 0.9: 1 (46:51), the mean age of detection was 22 卤0.10 years, the mean onset age was 3 卤0.83 years, and the hearing loss ranged from moderate to very severe. Among them, 10 proband patients had a history of aminoglycoside. The ratio of male to female in the control group was 0.9: 1 (181: 195). The mean sampling age was 25 卤4.12 years. 706 mitochondrial mutation sites were screened in the case group. Among them, there are 27 (1 new and 26 reported) rRNA genes in 180 D-loop regions, 29 (7 new and 22 reported) rRNA genes and 29 (3 new and 26 reported) rRNA genes, and 434 protein coding regions. It contains 122 missense mutations (4 new and 108 reported) and 312 synonymous mutations (20 new and 292 reported). 鍗曚綋鍨婦,G,M7,M8,M10,M11,M12,A,B4,F,H,N鍜孯鐨勯鐜囧垎鍒负24.74%,7.22%,5.15%,10.31%,1.03%,1.03%,1.03%,8.25%,5.15%,13.40%,7.22%,7.22%鍜，
本文编号：2233116
[Abstract]:Objective: to analyze the mtDNA mutation sites in 97 patients with non-syndromic deafness in Northwest China by mitochondrial DNA amplification and sequencing. Methods: the blood samples of 97 patients with moderate to very severe non-syndromic hearing loss were collected from five provinces of Northwest China with family history and no relationship. The clinical and family data were collected and 376 cases of normal hearing were collected as control group. Genomic DNA, was extracted by traditional phenol chloroform method and 24 pairs of positive and negative primers with partial overlap were used to amplify the whole mitochondrial DNA sequence PCR. The results were sequenced and the results were analyzed to find all the mitochondrial DNA mutation sites. To determine mitochondrial haplotype, analyze the conservation of mutation sites, the mutation frequency of mutation sites, mutation may affect the secondary structure of the gene, so as to find the candidate genes for deafness mitochondria. Results in the case group, the ratio of male to female was 0.9: 1 (46:51), the mean age of detection was 22 卤0.10 years, the mean onset age was 3 卤0.83 years, and the hearing loss ranged from moderate to very severe. Among them, 10 proband patients had a history of aminoglycoside. The ratio of male to female in the control group was 0.9: 1 (181: 195). The mean sampling age was 25 卤4.12 years. 706 mitochondrial mutation sites were screened in the case group. Among them, there are 27 (1 new and 26 reported) rRNA genes in 180 D-loop regions, 29 (7 new and 22 reported) rRNA genes and 29 (3 new and 26 reported) rRNA genes, and 434 protein coding regions. It contains 122 missense mutations (4 new and 108 reported) and 312 synonymous mutations (20 new and 292 reported). 鍗曚綋鍨婦,G,M7,M8,M10,M11,M12,A,B4,F,H,N鍜孯鐨勯鐜囧垎鍒负24.74%,7.22%,5.15%,10.31%,1.03%,1.03%,1.03%,8.25%,5.15%,13.40%,7.22%,7.22%鍜，
本文编号：2233116
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