【摘要】：目的:通過對西北地區(qū)97例非綜合征型耳聾患者進行線粒體DNA全序列擴增測序,進而分析可能致聾的mtDNA突變位點。方法:采集中國西北五省、有家族史、無親緣關系的97例中度至極重度非綜合征型耳聾先證者的血樣,并收集相關的臨床及家系資料,另外收集376例聽力正常人群作為對照組。用傳統(tǒng)酚氯仿法提取相關基因組DNA,并用24對有部分重疊的正反向引物進行線粒體DNA全序列PCR擴增,擴增后測序,對結果進行分析,查找所有的線粒體DNA突變位點,確定線粒體單體型,分析突變位點保守性、突變位點的突變頻率、突變可能對基因二級結構的影響,從而查找致聾的線粒體候選基因。結果:1、在病例組,男女患者比例為0.9:1(46:51),平均檢測年齡為22±0.10歲,平均發(fā)病年齡3±0.83歲,聽力損失從中度到極重度不等,其中10例先證者具有氨基糖苷類用藥史。對照組男女比例為0.9:1(181:195),平均采集年齡25±4.12歲。2、病例組篩查到706個線粒體突變位點,其中D-loop區(qū)180個,12S rRNA基因27個(1個新位點和26個已報道位點),16S rRNA基因29個(7個新位點和22個已報道位點),tRNA基因29個(3個新位點和26個已報道位點),蛋白編碼區(qū)434個,包含122個錯義突變(4個新位點和108個已報道位點)和312個同義突變(20個新位點和292個已報道位點),非編碼區(qū)7個。3、在病例組中,單體型D,G,M7,M8,M10,M11,M12,A,B4,F,H,N和R的頻率分別為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%,而在對照組中,它們的頻率分別為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%。其中單體型B、H、T在病例組和對照組之間的頻數(shù)差異具有統(tǒng)計學意義。結論:通過本研究發(fā)現(xiàn)了五個與耳聾相關的線粒體候選基因。1、12S rRNA基因上新突變位點1473CT,可能改變了12S rRNA的三級或四級結構,影響線粒體蛋白質的合成,從而影響線粒體的功能。2、tRNA基因上新突變位點614 AC位于tRNAPhe反密碼子環(huán)上(A37),突變可能影響密碼子識別的精確性,從而影響tRNA結構和功能的穩(wěn)定性;新突變位點8339AG位于tRNALys的T臂上(A50),突變破壞了原有的A-U配對,tRNA結構和功能穩(wěn)定性受到影響。3、5656AG位于輕鏈t RNAAla和輕鏈tRNAAsn之間,突變可影響tRNAAla和tRNAAsn前體的處理,易導致臨床表型異常。4、線粒體ND1 3866TC使NADH脫氫酶亞單位1上的極性疏水性異亮氨酸向蘇氨酸過渡,影響NADH脫氫酶活性和破壞線粒體的正常功能,進而造成內(nèi)耳的毛細胞損傷。5、單體型H、T在病組和對照組之間的頻數(shù)差異具有統(tǒng)計學意義,考慮與耳聾的發(fā)病具有相關性。
[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 鹵0.10 years, the mean onset age was 3 鹵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 鹵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負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，

本文編號：2233116