Studies On Mutations Of Deafness Causative Genes In Children Aged0～7Years From Part Of Northwest China

Hearing loss is the most common sensory disorder that leads to human disability. More than60%of the hearing loss is caused by genetic factors. At present, over200genes have been identified to associate with hearing loss and it showed obvious genetic heterogeneity. Whereas, several studies indicate that a large proportion of deafness are caused by GJB2, SLC26A4, and MTRNR1genes. The period of0～7years old is the important stage for children’s language acquisition. If the hearing loss occurred in this period, it will cause the language barrier, mental retardation and so on. And it has many disadvantages for children’s future life and study. To grasp the spectrum of deafness causative gene mutations, this study focused on the study of common deafness causative gene mutations; On the basis of the above study, we set up the detection system of SNPs and CNVs; In order to improve the mutant detection rate and provide the theoretical evidence for genetic counseling and molecular diagnosis.Part one Study on mutations of GJB2, SLC26A4, and MTRNR1genes in hearing loss children aged0-7years from part of Northwest ChinaObjective:To provide the theory basis for developing genetic counseling and establishing a new detection method, we investigate the frequency and hotspot mutation of GJB2, SLC26A4, and MTRNR1genes in hearing loss children aged0-7years from part of Northwest China.Methods:The clinical materials and DNA samples were collected from the444hearing loss children aged0～7years from part of Northwest China. GJB2gene was detected with sequencing of coding area, SLC26A4gene was detected with sequencing of hotspot mutation area, and MTRNR1gene was detected with allele specific PCR. As the control group,500DNA samples were also collected from the children aged0～7years with normal hearing. The statistical analyses were carried out using STATA version11.0. The inter-or intra-group difference in frequency was compared using2-tailed chi-square test. If the p-value less than0.05, differences were considered as statistically significant.Results:A total of79children had molecular defects in GJB2gene. Among them,31carried homozygotes mutations and19carried compound heterozygotes mutations. The mutation carrier rate of GJB2gene was17.79%, and the pathogenic mutation rate was11.26%. There were totally fourteen different kinds of variations. Among them, c.235deIC accounted for60%in total mutations and it is the commonest mutation in this area. A total of66children had molecular defects in SLC26A4gene. Among them,21carried homozygotes mutations and7carried compound heterozygotes mutations. The mutation carrier rate of SLC26A4gene was14.86%, and the pathogenic mutation rate was6.31%. There were totally six different kinds of variations. Among them, c.919-2A>G accounted for84%in total mutations and it is the commonest mutation in this area. A total of19children had the m.1555A>G mutation in MTRNR1gene, and the mutation carrier rate was4.28%. All of them had an obvious history of aminoglycoside use. And it accounted for21%in total children with aminoglycoside use. In the control group, none of the mutations were detected in GJB2and MTRNR1genes and6children were detected the c.919-2A>G heterozygote mutation in SLC26A4gene.Conclusions:Through the detection of common causative deafness genes,21.9%of the children were identified to have the confirmed hereditary hearing loss caused by mutations in GJB2, SLC26A4, and MT-RNR1genes. It will provide the theoretical guidance for genetic counseling and the establishment of a new technique.Part two Applying the multiple SNP classification and copy number variation for rapid mutation detection of common causative deafness genes in hearing loss childrenObjective:To explore the feasibility of multiple SNP classification and copy number variation technique in the rapid mutation detection of common causative deafness genes for hearing loss children.Methods:The clinical materials and DNA samples were collected from the538nonsyndromic sensorineural hearing loss children. We use the SNPscanTM and CNVplexTM technology to set up the detection system of common causative deafness mutation for the above children. The veracity of this technology was evaluated using the Sanger sequencing. As the control group,800DNA samples were also collected from the children aged0～7years with normal hearing. The statistical analyses were carried out using STATA version11.0. The inter-or intra-group difference in frequency was compared using2-tailed chi-square test. If the p-value less than0.05, differences were considered as statistically significant.Results:We set up a detection system including117SNP mutations and9copy number variations. A total of288children had molecular defects in the three common causative deafness genes. Among them,68carried homozygotes mutations and89carried compound heterozygotes mutations. The mutation carrier rate of three genes was53.5%, and the pathogenic mutation rate was29.2%. There were totally29different kinds of SNP mutations (24pathogenic mutations, allele carrying rate was32.16%). Among them, c.235delC is the commonest mutation in GJB2gene, c.919-2A>G is the commonest mutation in SLC26A4gene, and m.1555A>G is the commonest mutation in MTRNR1gene.35children were detected the heterozygote mutations in common causative deafness genes for the control group, and the carrier rate was4.38%. There were significant difference between test group and control group (P<0.05).Conclusions:The SNPscanTM and CNVplexTM technology has the characteristics of high accuracy, rapid speed, low price, and simple operation. In terms of the above advantages, this technology meets the requirement of clinical deafness gene detection and molecular diagnosis, and it also suitable for large-scale application.Part three Study on mutations of extensional causative deafness genes in hearing loss children aged0～7years from part of Northwest ChinaObjective:In order to improve the detection rate and find out the more pathogenic cause of hearing loss children, we detected the extensional deafness genes in children with no common mutations.Methods:The clinical materials and DNA samples were collected from the51hearing loss children with no common mutations. CDH23and MYO15A genes were detected with Sanger sequencing. As the control group,100DNA samples were also collected from the children aged0-7years with normal hearing. The statistical analyses were carried out using STATA version11.0. The inter-and intra-group difference in frequency was compared using2-tailed chi-square test. If the p-value less than0.05, differences were considered as statistically significant.Results:A total of6children had molecular defects in CDH23gene, and the mutation carrier rate was11.76%. There were two different kinds of variations:p.D428N in exon12and p.D1040N in exon23, and the allele mutation frequency was5.88%. A total of10children had molecular defects in MYO15A4gene, and the mutation carrier rate was19.61%. Among them,5children carried compound heterozygous mutations and the detection ratio was9.80%. There were seven different kinds of variations. The allele mutation frequency was14.71%. And p.P1009H mutation which was the commonest mutation in this area accounted for53%of total mutations. None of mutations were detected in the control group.Conclusions:Through the detection of extensional causative deafness genes,10%of the children were identified to have the confirmed hereditary hearing loss caused by mutations in CDH23and MYO15A genes. On the basis of the common deafness genes detection, we integrated the extensional genes detection. It may improve the mutation detection rate, and may find out40%causes of hearing loss. These results provided the theoretical basis for the molecular diagnostics and tertiary prevention.