中国西北部分地区0～7岁听障儿童常见致聋基因及扩展基因的突变研究

发布时间：2018-01-19 09:45

本文关键词： 听力损失 GJB2 SLC26A4 MTRNRl 基因突变单核苷酸多态性拷贝数变异 Sanger测序 CDH23 MYO15A　出处：《兰州大学》2014年博士论文　论文类型：学位论文

【摘要】：听力损失是造成人类残疾的最常见感官障碍,60%以上的听力损失由遗传因素所致。目前已发现与听力损失相关的基因有200余个,表现出明显的遗传异质性。尽管如此,GJB2、SLC26A4和MTRNR1这三个基因及其突变在相当一部分遗传性耳聋的发病中起到至关重要的作用。0-7岁是儿童言语习得的关键阶段,若在该时期出现听力障碍,会导致语言障碍、智力发育迟缓等一系列问题,对儿童今后的生活和学习产生众多弊端。为掌握西北部分地区0-7岁听障患儿的致聋基因突变频谱,本课题重点进行了该地区0-7岁人群的常见基因突变研究；在此基础上建立了快速SNP位点和CNV片段检测体系；并对未找到常见致聋基因突变的患儿进行聋病扩展基因研究,旨在提高基因突变检出率,为下一步提供遗传咨询和分子诊断提供坚实的理论依据。第一部分中国西北部分地区0-7岁昕障儿童常见聋病基因GJB2、SLC26A、MTRNR1突变研究目的：研究常见聋病基因GJB2、SLC26A4、MTNRN1在西北部分地区0-7岁听障儿童中的分布频率及突变热点,为遗传咨询和建立新的检测方法提供理论依据。方法：纳入444例西北部分地区0-7岁听障患儿,收集其临床听力学资料,抽取外周静脉血并提取基因组DNA。对常见致聋基因GJB2、SLC26A4和MTRNR1分别采用编码区测序、突变热点区域测序和等位基因特异性PCR方法进行突变检测。另纳入500例听力正常者作对照,应用STATA11.0软件进行统计学分析,组间或组内率的比较采用卡方检验,检验水准设为0.05。结果：444例患儿中,共发现79例患儿携带GJB2基因突变,其中31例患儿携带纯合突变、19例患儿携带复合杂合突变,突变携带率为17.79%,致病突变率为11.26%；共检测到14种GJB2基因突变位点,其中c.235delC占所有突变的60%,为该地区的热点突变形式。有66例患儿携带SLC26A4基因突变,其中21例患儿携带纯合突变、7例患儿携带复合杂合突变,突变携带率为14.86%,致病突变率为6.31%；共检测到6种SLC26A4基因突变位点,其中c.919-2AG占所有突变的84%,为该地区的热点突变形式。共检测到19例MTRNR1基因m.1555AG均质性突变,突变频率为4.28%。检出突变的患儿均有氨基糖甙类抗生素用药史,占所有使用药物患儿的21%。500例对照组儿童中未检到GJB2和MTRNR1基因突变,仅检到6例SLC26A4基因c.919-2AG杂合突变。结论：通过对常见致聋基因进行突变检测,为该地区21.9%的患儿明确了耳聋病因,对于在西北部分地区开展遗传咨询和建立下一部分新的检测方法提供了理论指导和依据。第二部分应用进行听障儿童常见聋病基因突变快速检测目的：探讨多重SNP分型和基因拷贝数变异技术在昕障儿童常见聋病基因突变快速检测的可行性。方法：纳入538例0-7岁非综合征型感音神经性听障患儿,收集临床资料,抽取外周静脉血样。利用SNPscanTM和CNVplexTM技术,建立常见致聋突变检测体系,进行上述患儿常见聋病基因突变检测。与测序方法比较,评价该方法的准确性。另纳入800例听力正常儿童作为对照,应用STATA11.0软件进行统计学分析,组间或组内率的比较采用卡方检验,检验水准设为0.05。结果：建立可检测三个常见致聋基因的117个SNP位点和9个CNV片段的反应体系。538例0-7岁听障患儿中,共检测到288例携带至少一种常见致聋基因突变,其中纯合突变68例、复合杂合突变89例,突变携带率53.5%,致病突变率29.2%；共检测到29个SNP位点,致病突变有24个,等位基因携带率为32.16%,其中GJB2基因的热点突变为c.235delC、SLC26A4基因的热点突变为c.919-2AG、MTRNR1基因的热点突变为m.1555AG。对照组800例听力正常儿童中,共发现35例携带常见致聋基因杂合突变,突变率为4.38%,与病例组比较,差异有显著统计学意义(P0.05)。结论：应用SNPscanTM和CNVplexTM技术进行听障儿童常见致聋基因突变检测,具有准确性高、检测速度快、费用低廉、操作简单的特点。鉴于上述优势,该技术符合临床耳聋基因检测和分子诊断的要求,适合大规模推广应用。第三部分中国西北部分地区0-7岁昕障儿童聋病扩展基因突变研究目的：通过对常见致聋基因检测阴性的患儿进行聋病扩展基因检测,旨在提高基因突变检出率,为更多的听障患儿找到其致聋原因。方法：纳入51例未找到常见致聋基因突变的患儿,应用Sanger测序进行聋病扩展基因CDH23和MY015A的突变检测。另纳入100例听力正常儿童作为对照,应用STATA11.0软件进行统计学分析,组间或组内率的比较采用卡方检验,检验水准设为0.05。结果：共发现6例患儿携带CDH23基因突变,携带率为11.76%；检测到2种致病突变形式,分别为第12外显子上的p.D428N和第23外显子上的p.D1040N,等位基因突变频率为5.88%。共检到10例患儿携带MYO15A基因突变,其中5例患儿携带双等位基因突变,突变携带率为19.61%,致病突变率为9.80%；发现7种致病突变形式,等位基因突变频率为14.71%,其中p.P1009H占所有突变的53%,为该地区0-7岁听障患儿MYO15A基因的热点突变。对照组100例儿童中未检到CDH23和MYO15A基因的任何突变。结论：通过对扩展基因CDH23和MYO15A进行突变检测,为该地区约10%的患儿明确了致聋原因。在常见致聋基因检测的基础上,融入聋病扩展基因检测,提高了基因突变检出率,能够为约40%的听障患儿找到其致聋原因,对下一步的分子诊断和三级预防奠定了坚实的理论基础。
[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.

【学位授予单位】：兰州大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R764.43

【共引文献】