Molecular Etiology Of Hereditary Deafness Based On The Next Generation Sequencing

Objective To analyze the molecular etiology of 98 hereditary deafness families with a new generation of targeted sequence capture sequencing technology,to clarify the cause of deafness and explore the mechanism of deafness caused by more known genes and found new pathogenic sites and lay a molecular etiology foundation for the prevention,diagnosis and treatment of deafness.Methods We analyzed the medical history,audiology,imaging and physical examination data of the 98 proband and family members which were selected from the hereditary deafness database with no detectable GJB2,SLC26A4 common mutation sites and mitochondrial DNA 1555 and 1494 mutation sites.Genomic DNA was extracted using the Blood Genomic Kit,and the known deafness genes（including 245 nuclear genes and mitochondrial genes）were reported to be captured and enriched using a personalized deafness gene,and then next generation sequencing were performed on the Illumina platform.Finally,Bioinformatics analysis was utilized to screen for possible pathogenic mutations.To verify the results of gene chip capture and high throughput sequencing,primers were designed upstream and downstream of all fragments found in the pathogenic site of variation.PCR amplification and identify responsibility genes and mutations.A total of 207 Clinical data and blood samples from normal hearing polationgs were collected as a control group.Results1.Clinical medical data analysis results: Among the 98 probands,there were 1 case of syndrome-type deafness and 97 cases of non-syndromic deafness.There were 8 cases of autosomal dominant family and 90 cases of autosomal recessive family.Analysis of audiological examination results: there were 4 cases of moderate sensorineural deafness,6 cases of moderate to severe sensorineural deafness,8 cases of severe sensorineural deafness,80 cases of severe sensorineural deafness,including 90 cases of pre-lingual deafness and 8cases of post-lingual deafness.2.A total of 1127 non-synonymous or splice site variants of 181 genes were found in 98 samples including 1011 non-synonymous variants,30 nonsense variants and 86 types of frameshift insertion deletion mutations.Of these,129 genes are currently known to be associated with deafness,with a total of 937 mutations;the other 52 genes have been reported to be associated with deafness in other species but have not been reported in the deafness population or have been reported to be associated with mitochondrial function in other diseases.A total of 190 mutations were detected in these 52 genes.3.Of the 98 samples,35 homozygous variants were identified in 32 autosomal recessive hereditary deafness syndromes,of which 7 were reported,including: CDH23: c.C719T（p.Pro240Leu）;LHFPL5: c.C494T（p.Thr165Met）;MYO15A: c.6340G>A（p.Val2114Met）;OTOF:c.2977₂978del（p.L937delinsFL）;SLC26A4:c.T716A（p.Val239Asp）and TMPRSS3:c.G727A（p.Gly243Arg）has been proved its pathogenic variation with research evidence.PCDH15:c.1195A>C（p.Ser399Arg）is pathogenic inaccurate in the results of other investigators and requires more research data to determine its pathogenicity.Of the 29 newly discovered mutations,15 were nonsense mutations or frameshift insertion deletion mutations,and 14 were missense mutations.The above mutations were found to be in protein functional conserved regions by homologous alignment;At least one of the predicted results in the Mutation Taster,Polyphen2_HVAR and SIFT prediction programs was pathogenic,and the above mutations were not found in 207 normal hearing controls,suggesting that they may alter the structure or function of the encoded protein,thereby causing deafness.4.Of the 98 samples,three pathogenic mutations were identified in eight dominant hereditary deafness progenitors,of which SOX10:c.342G>A（p.Trp114Ter）was an unreported new pathogenic mutation.It resulted in Waardenburg syndrome type 2E,and this mutation was not found in 207 normal hearing controls.The other two variants,GJB2: c.551G>A（p.Arg184Gln）,WFS1:c.2051C>T（p.Ala684Val）,were previously reported to be associated with autosomal dominant deafness type 3A,autosomal display Sexual hereditary deafness 6/14/38.Conclusion1.In 98 samples,32 autosomal recessive hereditary deafness probands and 8 dominant hereditary deafness probands were identified.The results will provide clear molecular etiological diagnosis and effective genetic counseling for hereditary deafness in these families,and provide strong theoretical guidance for prevention,diagnosis and treatment of deafness.2.In this study,29 new mutations were found in the known deafness genes,and the possibility of their pathogenesis was studied by bioinformatics.These results provide a theoretical basis for the subsequently functional studies.At the same time,they not only provide a clear molecular etiological diagnosis for more deafness families,but also add new members to the deaf gene pool.3.The 190 variants found in this study require more research（including case screening and functional verification）to determine their pathogenicity.4.The results of this study suggest that in patients with autosomal recessive deafness,in addition to GJB2,SLC26A4 and mitochondrial DNA m.1555A>G and m.1494C>T mutations are common cariogenic genes,MYO15 A gene,CDH23 gene OTOF gene and FGF3 gene can also be used as candidate genes for conventional cariogenic genes,providing more choices for clinical diagnosis of deafness,and providing more theoretical support for the design of genetic markers for neonatal deafness in Northwest China.