Study On Mechanism Of Hearing Loss Induced By New Deafness Genes And Analysis Of LOXHD1 Mutation In Chinese Deafness Population

Genetic factors account for more than 50% proportion of the causes of hearing loss.It is of great importance to clarify the molecular causes of hereditary hearing loss for the corresponding clinical diagnosis and treatment.With the development of next generation sequencing(NGS)technology and the gradual promotion of whole exome sequencing(WES),more and more deafness genes are being discovered.In this study,the mechanism of hearing loss induced by new deafness genes was further discussed on the basis of our previous work.We also analyzed the prevalence of LOXHD1 mutation in Chinese patients with non-syndromic hearing loss.PART ONE: Study on mechanism of hearing loss induced by new deafness genesIn this study,we selected a family of autosomal recessive non-syndromic auditory neuropathy,and finally screened out two suspected candidate pathogenic genes,SH3GLB1 and TWNK by use of whole exome sequencing and Sanger verification.In order to clarify the genetic causes of hearing loss in this family,we first studied the expression and localization of related proteins in the inner ear of mice.The results showed that Sh3glb1 was expressed in the inner ear of mice of different ages,and the inner hair cells and spiral ganglion neurons could be located,which was consistent with the manifestations of auditory neuropathy.However,the expression of Twnk in the inner ear of mice was low,and weak signal localization could be seen in spiral ganglion neurons.But the pathogenicity of TWNK could not be completely excluded.On the other hand,we constructed a Sh3glb1-knockout mouse model and a sh3glb1a-knockdown zebrafish model for SH3GLB1,and the corresponding audiological phenotype did not appear in the mouse model,suggesting a possible compensatory mechanism.While in the zebrafish model appeared reduced number of neuromasts.In view of the above results,we believe SH3GLB1 mutation was most likely the molecular causes for this family,while the possibility of TWNK participated in pathogenesis cannot be completely ruled out.Subsequent intends are to further clarify the molecular etiology and pathogenic mechanism from the perspective of the cytology study.A missense mutation of IFNLR1 [c.296G>A(p.Arg99His)] was identified for the first time in the world in our previous work.There was no report about this gene associated with deafness before.Immunofluorescence localization showed that Ifnlr1 was widely expressed in the inner ear of wild-type C57 mouse,and zebrafish with abnormal Ifnlr1 function showed multisystem dysplasia,including lateral line system.In order to further clarify the effects of this mutation on hearing loss and further study its deafness-causing mechanism,a mouse model of Ifnlr1-knockin at Arg99 His locus was constructed to evaluate the hearing phenotype,and the results showed that there was no corresponding audiological phenotype.At the same time,the transcriptome analysis results of ifnlr1 abnormal zebrafish were further analyzed.The results suggest that immune response was involved in the mechanism of hearing impairment,which can guide the study of its pathogenesis to a certain extent.PART TWO: Study on mechanism of hearing loss induced by new deafness genes Analysis ofLOXHD1 mutation in Chinese deafness populationNon-syndromic hearing loss(NSHL)is a common neurosensory disease with an extreme genetic heterogeneity which has been linked to variants in over 120 genes.The LOXHD1 gene(DFNB77),encoding lipoxygenase homology domain 1,is a rare hearing loss gene found in several populations.The prevalence of mutations on this gene in the Chinese NSHL population remains unclear.To evaluate the importance of LOXHD1 variants in Chinese patients with NSHL,we performed genetic analysis on LOXHD1 in 2901 sporadic Chinese patients to identify the aspect and frequency of LOXHD1 causative variants.Next-generation sequencing using a custom gene panel of hearing loss was conducted on2641 unrelated patients and whole exome sequencing on the remaining 260 patients.Results showed a total of 33 likely causative variants were identified in 21 patients,including 20 novel variants and 13 previously reported pathogenic variants.Each of the 20 novel variants was evaluated according to ACMG criteria.These findings showed that causative variants in LOXHD1 were found in about 0.72%(21/2901)of Chinese NSHL patients.Phenotypic assessment of 15 patients with available hearing data showed that 66.7%(10/15)of the patients had bilateral symmetrical sloping hearing loss.This study is by far the largest number of novel variants identified in this gene expanding the range of pathogenic variants in LOXHD1,and suggests that variants in this gene occur relatively commonly in Chinese NSHL patients.This extensive investigation of LOXHD1 in Chinese NSHL patients proposed six recurrent LOXHD1 variants.These findings may assist in both molecular diagnosis and genetic counseling.