本文關(guān)鍵詞： 大前庭水管綜合征 基因芯片 突變 SLC26A4基因 FOXI1基因　出處：《中南大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：為了研究大前庭水管綜合征(Enlarged vestibular aqueduct syndrome, EVAS)在中國人中的基因型和分子流行病學(xué)特點(diǎn),我們應(yīng)用耳聾基因芯片聯(lián)合DNA測序法對30例大前庭水管綜合征患者進(jìn)行了相關(guān)基因SLC26A4、FOXI1的突變檢測,探討適合中國人大前庭水管綜合征患者的基因診斷策略。 方法：采集30例EVAS患者和50例聽力檢測正常者的外周血,提取基因組DNA,用耳聾基因芯片檢測國人中SLC26A4基因的2個(gè)熱點(diǎn)突變IVS7-2AG和2168AG。同時(shí)應(yīng)用DNA測序法對EVAS患者的SLC26A4基因7-8號外顯子和19號外顯子基因編碼區(qū)序列進(jìn)行檢測,以驗(yàn)證基因芯片檢測這兩個(gè)位點(diǎn)的準(zhǔn)確性。如檢測發(fā)現(xiàn)純合突變或復(fù)合雜合突變即停止篩查,如未發(fā)現(xiàn)突變或發(fā)現(xiàn)單純雜合突變則用DNA測序法篩查剩余外顯子,直至發(fā)現(xiàn)另一個(gè)突變或篩查完全部外顯子。同時(shí)對30例EVAS患者FOXI1的2個(gè)外顯子及相鄰的內(nèi)含子區(qū)域進(jìn)行突變檢測。采用DNAstar軟件分析所有測序結(jié)果,判斷有無突變。 結(jié)果：30例EVAS患者組中,基因芯片方法共檢出SLC26A4基因突變25例,檢出率為83.33%。正常人對照組發(fā)現(xiàn)一例IVS7-2AG單雜合突變,EVAS患者組和正常人對照組的檢出率差異有顯著統(tǒng)計(jì)學(xué)意義(X2檢驗(yàn),P0.01)�；蛐酒l(fā)現(xiàn)突變的行DNA測序進(jìn)行驗(yàn)證,符合率為100%。對未發(fā)現(xiàn)突變或發(fā)現(xiàn)單雜合突變的患者,聯(lián)合DNA測序法進(jìn)一步檢測,有28例患者檢測出了SLC26A4基因突變,檢出率為93.33%。但基因芯片法檢出率與DNA測序法檢出率的差異無統(tǒng)計(jì)學(xué)意義(X2檢驗(yàn),P0.05)。我們共發(fā)現(xiàn)了16種突變類型,其中4種為新的突變類型(G368X, IVS8-1G＞T, IVS13+9C＞T和Q696X)。在所有的突變中,IVS7-2AG突變的發(fā)生率最高,其次為H723R和T410M。對FOXI1基因檢測發(fā)現(xiàn)兩個(gè)多態(tài)：279GA,1044TC。 結(jié)論：SLC26A4基因突變是導(dǎo)致大前庭水管綜合征的主要原因,其中IVS7-2AG突變的發(fā)生率最高,其次為H723R和T410M。耳聾基因芯片對大前庭水管綜合征患者SLC26A4基因的兩個(gè)熱點(diǎn)突變檢出率高,能夠適用于一般的基因篩查。但對基因芯片未發(fā)現(xiàn)這兩種突變或僅發(fā)現(xiàn)單個(gè)突變的仍有必要采用DNA測序法進(jìn)一步檢測。發(fā)現(xiàn)的4種新突變類型對大前庭水管綜合征的分子病因研究和基因診斷具有重要意義。本研究中只檢測到FOXI1的兩個(gè)多態(tài),未發(fā)現(xiàn)有意義的突變,證明其在大前庭水管綜合征患者中的發(fā)生率低。此外,EVAS的發(fā)生可能還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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R764

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