大前庭水管综合征患者基因芯片法与DNA测序法基因诊断的对比研究

发布时间：2018-02-16 18:55

本文关键词： 大前庭水管综合征基因芯片突变 SLC26A4基因 FOXI1基因　出处：《中南大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的：为了研究大前庭水管综合征(Enlarged vestibular aqueduct syndrome, EVAS)在中国人中的基因型和分子流行病学特点,我们应用耳聋基因芯片联合DNA测序法对30例大前庭水管综合征患者进行了相关基因SLC26A4、FOXI1的突变检测,探讨适合中国人大前庭水管综合征患者的基因诊断策略。方法：采集30例EVAS患者和50例听力检测正常者的外周血,提取基因组DNA,用耳聋基因芯片检测国人中SLC26A4基因的2个热点突变IVS7-2AG和2168AG。同时应用DNA测序法对EVAS患者的SLC26A4基因7-8号外显子和19号外显子基因编码区序列进行检测,以验证基因芯片检测这两个位点的准确性。如检测发现纯合突变或复合杂合突变即停止筛查,如未发现突变或发现单纯杂合突变则用DNA测序法筛查剩余外显子,直至发现另一个突变或筛查完全部外显子。同时对30例EVAS患者FOXI1的2个外显子及相邻的内含子区域进行突变检测。采用DNAstar软件分析所有测序结果,判断有无突变。结果：30例EVAS患者组中,基因芯片方法共检出SLC26A4基因突变25例,检出率为83.33%。正常人对照组发现一例IVS7-2AG单杂合突变,EVAS患者组和正常人对照组的检出率差异有显著统计学意义(X2检验,P0.01)。基因芯片发现突变的行DNA测序进行验证,符合率为100%。对未发现突变或发现单杂合突变的患者,联合DNA测序法进一步检测,有28例患者检测出了SLC26A4基因突变,检出率为93.33%。但基因芯片法检出率与DNA测序法检出率的差异无统计学意义(X2检验,P0.05)。我们共发现了16种突变类型,其中4种为新的突变类型(G368X, IVS8-1G＞T, IVS13+9C＞T和Q696X)。在所有的突变中,IVS7-2AG突变的发生率最高,其次为H723R和T410M。对FOXI1基因检测发现两个多态：279GA,1044TC。结论：SLC26A4基因突变是导致大前庭水管综合征的主要原因,其中IVS7-2AG突变的发生率最高,其次为H723R和T410M。耳聋基因芯片对大前庭水管综合征患者SLC26A4基因的两个热点突变检出率高,能够适用于一般的基因筛查。但对基因芯片未发现这两种突变或仅发现单个突变的仍有必要采用DNA测序法进一步检测。发现的4种新突变类型对大前庭水管综合征的分子病因研究和基因诊断具有重要意义。本研究中只检测到FOXI1的两个多态,未发现有意义的突变,证明其在大前庭水管综合征患者中的发生率低。此外,EVAS的发生可能还存在其他的致病因素。
[Abstract]:Objective: to study the genotypic and molecular epidemiological characteristics of large vestibular aqueduct syndrome (EVAS) in Chinese. We used deafness gene chip combined with DNA sequencing to detect the mutation of the associated gene SLC26A4 and FOXI1 in 30 patients with large vestibular aqueduct syndrome, and to explore the strategy of gene diagnosis for Chinese patients with large vestibular aqueduct syndrome. Methods: peripheral blood samples were collected from 30 patients with EVAS and 50 patients with normal hearing test. Genomic DNA was extracted and two hot spot mutations IVS7-2AG and 2168AG of SLC26A4 gene were detected by deafness gene chip in Chinese. Meanwhile, the sequence of exon 7-8 and exon 19 of SLC26A4 gene in EVAS patients was detected by DNA sequencing. If homozygous mutation or complex heterozygous mutation was detected, the screening would be stopped. If no mutation was found or a simple heterozygous mutation was found, the remaining exons would be screened by DNA sequencing. Until another mutation was found or all exons were screened, two exons and adjacent intron regions of FOXI1 were detected in 30 patients with EVAS. All sequencing results were analyzed by DNAstar software to determine whether there were mutations. Results in 30 patients with EVAS, 25 cases of SLC26A4 gene mutation were detected by gene chip method. The detectable rate was 83.33. The difference of the detection rate of a single heterozygous mutation of IVS7-2AG between the normal control group and the normal control group was statistically significant (P < 0.01). DNA sequencing was performed to verify the mutation found by gene chip. The coincidence rate was 100%. For those patients with no mutation or single heterozygosity mutation, 28 patients with SLC26A4 gene mutations were further detected by DNA sequencing. The detection rate was 93.33%. But there was no significant difference between the detection rate of gene chip method and DNA sequencing method (P 0.05). We found 16 mutation types. Four of them were new mutation types: G368X, IVS8-1G > T, IVS13 9C > T and Q696X.Of all the mutations, the frequency of mutation was the highest in IVS7-2AG, followed by H723R and T410M.The detection of FOXI1 gene revealed two polymorphisms: 279GA1044TC. Conclusion the main cause of the large vestibular aqueduct syndrome is the mutation of the gene of 1: SLC26A4, in which the incidence of IVS7-2AG mutation is the highest, followed by H723R and T410M.The detection rate of the two hot spot mutations in the SLC26A4 gene of the patients with large vestibular aqueduct syndrome is high by deafness gene chip. However, it is necessary to use DNA sequencing method to further detect the two mutations or only a single mutation in the microarray. Four new mutation types have been found for vestibular aqueduct synthesis. In this study, only two polymorphisms of FOXI1 were detected. No significant mutation was found, which indicated that the incidence of EVAS was low in patients with great vestibular aqueduct syndrome. In addition, there may be other pathogenic factors in the occurrence of EVAS.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R764

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