| PartⅠGenetic screening of mutation about GJB2、SLC26A4 and mtDNA12S rRNA for 1201patients with non-syndromic hearingloss in ShanxiObjective:Hearing impairment is one of the most frequent sensorineural disorder with high genetic heterogeneity.The incidence of neonatal morbidity is about 1/1000,more than half of them are caused by genetic defects.Among them,approximately 70%are non-syndromic,in which the hearing impairment is the only distinctive clinical feature in these patients.Many researches have shown that hereditary deafness is mainly related to 4genes（GJB2,GJB3,SLC26A4,mt DNA）in China.Therefore,the clinical genetic diagnosis of deafness mainly carries on the analysis of high frequent mutation about the four genes mentioned above.At present,the screening of deafness gene have not systematically undergone in many regions in China.In this study,after detecting the deafness gene mutations,the molecular etiology investigation was completed and the deafness gene hot-spot mutation was analyzed in Shanxi.This study will perfect the deafness gene mutation database in Shanxi area.It will provide the reference for the early diagnosis and treatment for deafness.It also provides the experimental basis for the research and development of the rapid diagnostic kit for deafness in Shanxi.Methods:In total 1201 non-syndromic deafness subjects from Shanxi Province,of which were enrolled from 11 schools for deaf-mute and special school in 10 cities in Shanxi Province,were recruited by the second hospital of Shanxi medical university and Taiyuan city center hospital.The 1201 subjects were screening from 1685 cases of deafness on the basis of the inclusion and exclusion criteria.After signing the informed consent by patients or guardians,the comprehensive blood was collected for subsequently DNA extraction using EDTA-K2 anticoagulation,and saved in-20℃.Whole blood genomic DNA was extracted and the common deafness genes were analyzed by PCR technology and Sanger sequencing technology.Subsequently,the frequency of mutations was counted and analyzed by using SPSS 22.0 software to compare the frequency of deafness gene mutation.Finally,the deafness gene hot-spot mutation was screened in Shanxi province.Results:A total of 1201 patient with non-syndromic deafness were screening from 1685 cases of patients on the basis of the inclusion and exclusion criteria,all of whom were Han Chinese.Among them,there were 698 males and 503 females,and the ratio was 1.387:1.Using polymerase chain reaction（PCR）technology and Sanger sequencing technology to analyze the high frequency genes of deafness,it was found that the gene mutation rate was 31.97%（384/1201）.1.No GJB3 gene mutation was detected in 1201 patients with NSHL.2.Among 1201 patients with non-syndromic deafness,GJB2 mutations were found in255 patients.The mutations accounted for 21.23%.Among them,c.235delC,c.176-191del16,c.299-300delAT,c.109G>A and c.35delG were the hot-spot mutations.c.235delC was the hotspot mutation of GJB2 in NSHL patients in Shanxi,it accounted for 10.99%（132/1201）,followed by 299-300delAT with a frequency of 2.67%（32/1201）.There were203 cases with a single allele mutation and 52 cases with two allele mutations.At the same time,11 new mutations in GJB2 were found in the study.3.In all patients with non-syndromic hearing loss,112 patients were found carrying mutations in the hot regions of SLC26A4,with a carrying rate of 9.33%（112/1201）.Mutations in SLC26A4 accounted for 9.33%（112/1201）of the patients detected.IVS7-2A>G was the most prevalent mutation in our patient group.IVS7-2A>G was identified in 57 patients（57/1201,4.75%）.Also,the study revealed that 2.66%（32/1201）patients had two mutated alleles（homozygote or compound heterozygote）.4.Seventeen patients were found carrying mt DNA 12S rRNA mutation in the study.Among them,15 cases were found to carry 1555A>G,and 2 cases were detected to carry1494 C>T.Conclusion:PCR and Sanger sequencing technology were efficient for analyzing common gene mutation of hearing loss,they have the high detection rate.The rate was similar to that in other parts in China,but there was a large difference,which is obviously regional.The main pathogenic genes of deafness in Shanxi Province were GJB2,SLC26A4 and mtDNA.c.235delC,c.299-300delat,c.176-191del16 and c.109G>A is the high frequency mutations of GJB2,IVS7-2A>G and c.2168A>G were the hot-spot mutations of SLC26A4,and1555A>G is the hot-spot mutation of mitochondrial 12S rRNA.Among them,c.235delC and IVS7-2A>G were the most frequent deafness mutations.Part Ⅱ Pathogenic site analysis in the deafness family with Usher syndromeObjective:Usher syndrome is one of the most common sensorineural hearing loss along with retinal pigment degeneration,which is mainly diagnosed by family history and clinical symptoms.Because its clinical phenotype is not specific,gene diagnosis is a rapid and accurate diagnostic method.16 genes have been identified in Usher syndrome.In this study,whole exome sequencing technology was used to analyze the pathogenic mutation in a family with Usher syndrome.The novel mutations were expected to found.At the same time,the clinical diagnosis method was established,which whole exome sequencing technology was used for diagnosing the hereditary disease involved in more pathogenic genes.Also,it contributed to improve the diagnostic sensitivity and specificity,and to shorten the diagnosis time.Methods:Among the 1685 cases of deafness collected in the first part,a case suspected Usher syndrome was detected based on clinical characteristics.After asking family history and signing the informed consent by the patient or guardian,the comprehensive blood was collected for subsequently DNA extraction using EDTA-K2 anticoagulation,and stored in-20℃.At the same time,300 individuals of control group without family history were collected as control.Whole blood genomic DNA was extracted and the gene was detected by whole exome sequencing technology.After analyzing using 1000 G database,db SNP42 database,noflag SNP138 database,gwas Catalog database and OMIM database,mutations were screened.All members in the family and the control group were detected by Sanger sequencing technology.It is also searched in HGMD database and Pubmed database.Results:Usher syndrome was caused by autosomal recessive inheritance and there were two patients in this pedigree,but a patient has died.7 candidate mutations were found after detected by whole exome sequencing and data analysis using 1000 G and other databases.Eight members collected blood in the family and the control group were detected by Sanger sequencing technology,and only c.G373A（p.A125T）in USH2 A was shared by all the patients and not in control group.This mutation has been reported in the HGMD database and Pubmed database.Conclusion:1.This study reported a rare Usher syndrome pedigree involving 21 healthy people and 2 patients,but a patient has died.2.The pathogenic mutation in the hereditary family was c.G373A（p.A125T）.3.WES technology can be used for the clinical genetic diagnosis in Usher syndrome,which improves the diagnostic sensitivity and specificity of the disease and reduces the time of diagnosis. |
