Identification And Functional Study Of A Novel Pathogenetic Gene TNN In A Pedigree Of Autosomal Recessive Hereditary Non-syndromic Auditory Neuropathy

PURPOSE:1.Identification of a novel causative gene TNN in the pedigree(AN-1)of autosomal recessive non-syndromic auditory neuropathy(AN).2.The study of tenascin-W expression in the cochleae of WT C57BL/6 mice at different stages of development.3.Identification of Tnn KO mice and its effect on the auditory function of Tnn KO mice.METHODS:1.Genetic analysis was performed on the pedigree,including hotspot variant screening and whole exome sequencing(WES).2.Six C57BL/6 mice were selected from different developmental stages of P1,P3,P14 and P28(P: postnatal day),nine were selected from P7.Expression and localization of tenascin-W in cochlea at each stage were observed by immunofluorescence.The relative expression level of Tnn mRNA in mice cochleae at each stage were detected by real-time quantitative polymerase chain reaction(RT-qPCR).Western blot tests were used to detect the expression of tenascin-W protein in mice cochleae.3.Male and female Tnn heterozygous KO C57BL/6mice were mated with each other,the newborns were marked and numbered,and their tails were taken to test for genotypic identification.8 mice were taken from Tnn KO mice and WT mice at P1 m,P2m and P3 m respectively and tested for ABR and DPOAE.The effects of tenascin-W on auditory function of mice were studied by comparing hearing level of ABR thresholds,ABR Ⅰ wave amplitudes and DPOAE amplitudes between Tnn KO mice and WT mice.RESULTS:1.WES detected a compound heterozygous missense variant,c.G736A(p.G246S)and c.C2954T(p.T985M)in TNN(tenascin-W,TN-W).Genetic variants were co-segregated with the disease in the pedigree.The patients were compound heterozygous missense variant,while both parents were carriers of monoallelic TNN gene mutations.The father was a monoallelic TNN gene mutation carrier of c.G736A(p.G246S),and the mother was a monoallelic TNN gene mutation carrier of c.C2954T(p.T985M).2.Immunofluorescence showed that tenascin-W was expressed in the cochleae of P1,P3,P7,P14 and P28 mice at different developmental stages.Tenascin-W was expressed in the spiral ganglion(SG)and organ of Corti(OC)of mice cochleae at P1,P3 and P7,while only highly expressed in the spiral ganglion,not in the OC at P14 and P28.Tenascin-W was expressed in the stria vascularis at all stages from P1 to P28.Double immunofluorescent staining revealed co-localization of tenascin-W and type Ⅲ β-tubulin in the cytoplasm of cell body of spiral ganglion neurons(SGNs).RT-qPCR showed the expression of Tnn mRNA was relatively high at p7.Western blot testified the expression of tenascin-W in mice cochleae at P7.3.Heterozygous Tnn C57BL/6 KO mice were mated but homozygous Tnn C57BL/6KO mice were not bred.Homozygous Tnn KO mice might be embryonic lethal.Heterozygous Tnn KO mice showed progressive hearing loss with increase of ABR thresholds,decrease of ABR Ⅰ wave amplitudes and DPOAE amplitudes comparing with WT mice.CONCLUSION:Tenascin-W plays an important role in the growth and development of mice,and its absence may cause the embryos failing to develop normally,thus leading to death.Tenascin-W is expressed in the mice cochleae and may play an essential part in the development and maintenance of auditory function.Abnormal expression of the protein may affect the neurite outgrowth or cell migration of SGNs,and furthermore,may affect the function of auditory system.