本文關(guān)鍵詞： 聽力損失 GJB2 SLC26A4 MTRNRl 基因突變 單核苷酸多態(tài)性 拷貝數(shù)變異 Sanger測序 CDH23 MYO15A　出處：《蘭州大學(xué)》2014年博士論文　論文類型：學(xué)位論文

【摘要】：聽力損失是造成人類殘疾的最常見感官障礙,60%以上的聽力損失由遺傳因素所致。目前已發(fā)現(xiàn)與聽力損失相關(guān)的基因有200余個,表現(xiàn)出明顯的遺傳異質(zhì)性。盡管如此,GJB2、SLC26A4和MTRNR1這三個基因及其突變在相當(dāng)一部分遺傳性耳聾的發(fā)病中起到至關(guān)重要的作用。0-7歲是兒童言語習(xí)得的關(guān)鍵階段,若在該時期出現(xiàn)聽力障礙,會導(dǎo)致語言障礙、智力發(fā)育遲緩等一系列問題,對兒童今后的生活和學(xué)習(xí)產(chǎn)生眾多弊端。為掌握西北部分地區(qū)0-7歲聽障患兒的致聾基因突變頻譜,本課題重點進行了該地區(qū)0-7歲人群的常見基因突變研究；在此基礎(chǔ)上建立了快速SNP位點和CNV片段檢測體系；并對未找到常見致聾基因突變的患兒進行聾病擴展基因研究,旨在提高基因突變檢出率,為下一步提供遺傳咨詢和分子診斷提供堅實的理論依據(jù)。 第一部分中國西北部分地區(qū)0-7歲昕障兒童常見聾病基因GJB2、SLC26A、MTRNR1突變研究 目的：研究常見聾病基因GJB2、SLC26A4、MTNRN1在西北部分地區(qū)0-7歲聽障兒童中的分布頻率及突變熱點,為遺傳咨詢和建立新的檢測方法提供理論依據(jù)。 方法：納入444例西北部分地區(qū)0-7歲聽障患兒,收集其臨床聽力學(xué)資料,抽取外周靜脈血并提取基因組DNA。對常見致聾基因GJB2、SLC26A4和MTRNR1分別采用編碼區(qū)測序、突變熱點區(qū)域測序和等位基因特異性PCR方法進行突變檢測。另納入500例聽力正常者作對照,應(yīng)用STATA11.0軟件進行統(tǒng)計學(xué)分析,組間或組內(nèi)率的比較采用卡方檢驗,檢驗水準設(shè)為0.05。 結(jié)果：444例患兒中,共發(fā)現(xiàn)79例患兒攜帶GJB2基因突變,其中31例患兒攜帶純合突變、19例患兒攜帶復(fù)合雜合突變,突變攜帶率為17.79%,致病突變率為11.26%；共檢測到14種GJB2基因突變位點,其中c.235delC占所有突變的60%,為該地區(qū)的熱點突變形式。有66例患兒攜帶SLC26A4基因突變,其中21例患兒攜帶純合突變、7例患兒攜帶復(fù)合雜合突變,突變攜帶率為14.86%,致病突變率為6.31%；共檢測到6種SLC26A4基因突變位點,其中c.919-2AG占所有突變的84%,為該地區(qū)的熱點突變形式。共檢測到19例MTRNR1基因m.1555AG均質(zhì)性突變,突變頻率為4.28%。檢出突變的患兒均有氨基糖甙類抗生素用藥史,占所有使用藥物患兒的21%。500例對照組兒童中未檢到GJB2和MTRNR1基因突變,僅檢到6例SLC26A4基因c.919-2AG雜合突變。 結(jié)論：通過對常見致聾基因進行突變檢測,為該地區(qū)21.9%的患兒明確了耳聾病因,對于在西北部分地區(qū)開展遺傳咨詢和建立下一部分新的檢測方法提供了理論指導(dǎo)和依據(jù)。 第二部分應(yīng)用進行聽障兒童常見聾病基因突變快速檢測 目的：探討多重SNP分型和基因拷貝數(shù)變異技術(shù)在昕障兒童常見聾病基因突變快速檢測的可行性。 方法：納入538例0-7歲非綜合征型感音神經(jīng)性聽障患兒,收集臨床資料,抽取外周靜脈血樣。利用SNPscanTM和CNVplexTM技術(shù),建立常見致聾突變檢測體系,進行上述患兒常見聾病基因突變檢測。與測序方法比較,評價該方法的準確性。另納入800例聽力正常兒童作為對照,應(yīng)用STATA11.0軟件進行統(tǒng)計學(xué)分析,組間或組內(nèi)率的比較采用卡方檢驗,檢驗水準設(shè)為0.05。 結(jié)果：建立可檢測三個常見致聾基因的117個SNP位點和9個CNV片段的反應(yīng)體系。538例0-7歲聽障患兒中,共檢測到288例攜帶至少一種常見致聾基因突變,其中純合突變68例、復(fù)合雜合突變89例,突變攜帶率53.5%,致病突變率29.2%；共檢測到29個SNP位點,致病突變有24個,等位基因攜帶率為32.16%,其中GJB2基因的熱點突變?yōu)閏.235delC、SLC26A4基因的熱點突變?yōu)閏.919-2AG、MTRNR1基因的熱點突變?yōu)閙.1555AG。對照組800例聽力正常兒童中,共發(fā)現(xiàn)35例攜帶常見致聾基因雜合突變,突變率為4.38%,與病例組比較,差異有顯著統(tǒng)計學(xué)意義(P0.05)。 結(jié)論：應(yīng)用SNPscanTM和CNVplexTM技術(shù)進行聽障兒童常見致聾基因突變檢測,具有準確性高、檢測速度快、費用低廉、操作簡單的特點。鑒于上述優(yōu)勢,該技術(shù)符合臨床耳聾基因檢測和分子診斷的要求,適合大規(guī)模推廣應(yīng)用。 第三部分中國西北部分地區(qū)0-7歲昕障兒童聾病擴展基因突變研究 目的：通過對常見致聾基因檢測陰性的患兒進行聾病擴展基因檢測,旨在提高基因突變檢出率,為更多的聽障患兒找到其致聾原因。 方法：納入51例未找到常見致聾基因突變的患兒,應(yīng)用Sanger測序進行聾病擴展基因CDH23和MY015A的突變檢測。另納入100例聽力正常兒童作為對照,應(yīng)用STATA11.0軟件進行統(tǒng)計學(xué)分析,組間或組內(nèi)率的比較采用卡方檢驗,檢驗水準設(shè)為0.05。 結(jié)果：共發(fā)現(xiàn)6例患兒攜帶CDH23基因突變,攜帶率為11.76%；檢測到2種致病突變形式,分別為第12外顯子上的p.D428N和第23外顯子上的p.D1040N,等位基因突變頻率為5.88%。共檢到10例患兒攜帶MYO15A基因突變,其中5例患兒攜帶雙等位基因突變,突變攜帶率為19.61%,致病突變率為9.80%；發(fā)現(xiàn)7種致病突變形式,等位基因突變頻率為14.71%,其中p.P1009H占所有突變的53%,為該地區(qū)0-7歲聽障患兒MYO15A基因的熱點突變。對照組100例兒童中未檢到CDH23和MYO15A基因的任何突變。 結(jié)論：通過對擴展基因CDH23和MYO15A進行突變檢測,為該地區(qū)約10%的患兒明確了致聾原因。在常見致聾基因檢測的基礎(chǔ)上,融入聾病擴展基因檢測,提高了基因突變檢出率,能夠為約40%的聽障患兒找到其致聾原因,對下一步的分子診斷和三級預(yù)防奠定了堅實的理論基礎(chǔ)。
[Abstract]:Hearing loss is the most common cause of human sensory disability disability, more than 60% of hearing loss caused by genetic factors. It has been found that the genes associated with hearing loss of more than 200, showed significant genetic heterogeneity. However, GJB2, the three genes SLC26A4 and MTRNR1 and its mutation in the pathogenesis of a considerable part of genetic deafness play a critical role in the age of.0-7 is the key stage of children's language acquisition, if hearing impairment in this period, will lead to a series of problems of language disorders, mental retardation, have many disadvantages on children's future life and learning. As the deafness gene mutation spectrum in the Northwest part of master 0-7 year old deaf in the study, this paper focuses on common gene mutations of the 0-7 age groups in the area; on the basis of a rapid SNP site and CNV fragment detection system; and was not found The children with common deafness gene mutations carry out the extended gene research of deaf patients, aiming to improve the detection rate of gene mutation, and provide a solid theoretical basis for the next step to provide genetic counseling and molecular diagnosis.
The first part of the GJB2, SLC26A, MTRNR1 mutation of the common deafness genes in children aged 0-7 in the northwest part of China
Objective: To study the distribution frequencies and mutation hotspots of gene GJB2, SLC26A4 and MTNRN1 in 0-7 years old hearing-impaired children in Northwest China, and provide theoretical basis for genetic counseling and establishing new detection methods.
Methods: a total of 444 cases of the northwestern parts of the hearing-impaired children 0-7 years old, collected the clinical audiology data, collected peripheral blood and extracted genomic DNA. of common deafness genes GJB2, SLC26A4 and MTRNR1 respectively encoding region sequencing, sequencing and mutation hotspot allele specific PCR method for mutation detection. The other 500 cases included in normal hearing subjects. The data were analyzed by STATA11.0 software, compared with the group or group in the rate of chi square test, the test level is 0.05.
Results: in 444 cases, 79 cases with GJB2 mutations were found in 31 cases with homozygous mutations, 19 cases with compound heterozygous mutations, the mutation rate was 17.79%. The mutation rate was 11.26%; there were 14 mutations in GJB2 gene, the c.235delC accounted for 60% of all mutations mutation hotspots in the region, form. There were 66 cases of children with SLC26A4 mutations, including 21 cases with the homozygous mutation, 7 cases with compound heterozygous mutations, the mutation rate was 14.86%. The mutation rate was 6.31%; there were 6 mutations in SLC26A4 gene, which accounted for c.919-2AG of all mutations 84%, mutation hotspots in the region. 19 cases were detected MTRNR1 gene m.1555AG mutation heterogeneity, mutation frequency of 4.28%. mutations were detected aminoglycoside antibiotic medication history, accounting for all medication use with 21%.50 GJB2 and MTRNR1 mutations were not detected in 0 Children in the control group, and only 6 SLC26A4 gene c.919-2AG heterozygous mutations were detected.
Conclusion: the mutation detection of common deafness genes has identified the cause of deafness for 21.9% of the children in this area, and provided theoretical guidance and basis for developing genetic counseling in the northwest part of China and establishing the next part of the new detection methods.
Rapid detection of gene mutations in common deaf children with hearing impairment in the second part
Objective: To explore the feasibility of multiple SNP typing and gene copy number variation in rapid detection of gene mutation in common deaf children.
Methods: a total of 538 patients aged 0-7 with non syndromic sensorineural hearing impaired children, clinical data were collected, blood samples from peripheral vein. Using SNPscanTM and CNVplexTM technology, the establishment of common deafness mutation detection system, the detection of mutations in children with common deafness genes. Compared with the sequencing method, to evaluate the accuracy of the method. The other 800 cases were included in the normal hearing children as control, the application of STATA11.0 software for statistical analysis, compared with the group or group in the rate of chi square test, the test level is 0.05.
Results: to establish the detection of three common deafness genes of 117 SNP loci and 9 CNV fragment of the.538 reaction system were 0-7 years old hearing-impaired children, were detected in 288 patients with at least one kind of common deafness gene mutation, the homozygous mutation in 68 cases, 89 cases of compound heterozygous mutations, the mutation rate 53.5%, the pathogenic mutation rate of 29.2%; 29 SNP loci were detected, and 24 pathogenic mutations, allele carrying rate was 32.16%, the hot spot mutation of GJB2 gene is c.235delC hotspot mutation of SLC26A4 gene is c.919-2AG hotspot mutation of MTRNR1 gene m.1555AG. in 800 cases of control group in 35 cases of normal hearing children. With common deafness gene heterozygous mutations were found, the mutation rate was 4.38%, compared with the case group, the difference was statistically significant (P0.05).
Conclusion: the application of SNPscanTM and CNVplexTM technology for hearing-impaired children caused by mutations of common deafness genes, with high accuracy, fast detection speed, low cost, simple operation. In view of the above advantages, this technique can meet the clinical genetic testing and molecular diagnostic requirements, suitable for large scale application.
The third part of extended gene mutation of deaf children aged 0-7 years old in northwestern part of China
Objective: to detect the extended gene in deaf children by detecting the extended gene in deaf children with negative gene detection, so as to improve the detection rate of gene mutation and find the cause of hearing loss for more hearing-impaired children.
Methods: a total of 51 cases were found no common deafness gene mutations, using Sanger sequencing for mutation detection of deafness gene CDH23 and extended MY015A. The other 100 cases were included in the normal hearing children as control, the application of STATA11.0 software for statistical analysis, group or group rate compared with the chi square test, set the standard for inspection 0.05.
Results: 6 patients with CDH23 mutations were found. The positive rate is 11.76%; detecting 2 pathogenic mutations, respectively twelfth exon p.D428N and exon twenty-third of the p.D1040N mutant allele frequency with MYO15A gene mutation of 5.88%. were detected in 10 cases, of which 5 patients carrying biallelic mutations, the mutation rate was 19.61%, the pathogenic mutation rate was 9.80%; 7 pathogenic mutations, the allele frequencies of 14.71% p.P1009H, which accounted for 53% of all mutations, mutation gene 0-7 MYO15A hot hearing-impaired children in the region. The control group of 100 children without to check any mutations in the CDH23 gene and MYO15A gene.
Conclusion: the gene mutation was detected by CDH23 and MYO15A, the causes of deafness in the area about 10% of the children. On the basis of common deafness gene detection, into the expansion of deafness gene detection, improve the detection rate of gene mutation, for the hearing-impaired children about 40% to find the causes of deafness. Lay a solid theoretical foundation for the molecular diagnosis of the next step and three grade prevention.

【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R764.43

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