Phenotype Analysis Of Hearing Loss Related To PCDH15 And Characteristics Study Of Aifm1 Auditory Neuropathy Mice

Part 1:Analysis of Genetic and Hearing Clinical Characteristics of Hearing loss Related to Variants of PCDH15Objective This study aims to identify PCDH15 gene variant-related deafness families,and explore the genotype and phenotype of PCDH15 gene variant.Then the patients with PCDH15 gene variant need to be followed up to observe the changes of visual function and vestibular function.Methods 1.Use WES to identify PCDH15 gene variants in autosomal recessive deafness families,and perform Sanger sequencing verification in family members.2.Collect the medical history and imaging examination of patients,and conduct audiological examinations,including pure tone audiometry,ABR,AS SR,40 Hz AERP,immittance test,and DPOAE.3.Follow up the patients,and focuse on the changes of visual function and vestibular function of the patients.Results 1.6 patients from 4 deaf families were all PCDH15 biallelic variants.5 unreported PCDH15 variant sites and 2 known PCDH15 variant sites were identified.The 5 newly discovered variant sites include:2 frameshift variations(c.3441 del A,c.2756 del T),2 splice variations(c.1997+1G>A,c.157+1G>A),1 Copy number variation(Ex19-21 del).2 reported PCDH15 gene mutation sites include:1 nonsense mutation(c.733C>T),1 missense mutation(c.2528C>A).2.The 6 patients were all congenital severe bilateral hearing loss,including 3 adults and 3 children.Follow-up founded that all 3 children had motor retardation,2 children had hyperopic astigmatism in both eyes,and 1 of them had night blindness.The vestibular function and visual function of 3 adult patients were within the normal range.In addition,all3 children underwent unilateral cochlear implantation within 11 months.They had good speech development and are able to communicate with others.They were all enrolled in normal schools.One adult patient underwent unilateral cochlear implantation at the age of 13,but the effect was not good,and the cochlear implant was no longer used.Conclusion WES can effectively detect USH1F and DFNB23 patients.Both USH1F and DFNB23 are congenital severe bilateral hearing loss.USH1F show motor retardation in the early stage,and then gradually develop abnormal visual symptoms such as hyperopia astigmatism and night blindness.For congenital deafness patients with PCD1H5 gene variants,early implantation of cochlear implants is beneficial to speech development.Part 2:Analysis of Auditory Function and Cochlear Characteristics after Noise Exposure in Aifm1R450Q/Y Mutant MiceObjective This study aims to explore the phenotype of Aifm1R450Q/Y(Aifm1+/Y)gene mutation male CBA mice related to auditory neuropathy(AN)and to study the characteristics of auditory and cochlear function after moderate noise exposure,clarifying the mechanism of AIFM1 related hearing impairment.Methods In this study,1-month-old Aifm1R450Q/Y male mice(Aifm1+/Y group)and wild-type male mice(WT group)in the same cage were selected for experiments.Both groups were exposed to 100 dB SPL white noise for 2 hours.According to the noise exposure time,they were divided into before exposure,after exposure 1,7,and 14 days.The following experiments were carried out at these 4 time points:1.ABR detection:ABR(Click-ABR;Tone Burst-ABR:8k Hz,16k Hz,24k Hz,32k Hz)was performed on two groups of mice,and ABR threshold and ABR wave I amplitude at 80 dB SPL were recorded.2.Immunofluorescence:Using Anti-CtBP2 and Anti-Neurofilament,the synapses and nerve fibers of inner hair cells were observed.3.Western Blot:two groups of mouse cochlear protein samples were prepared,and Western Blot was used to detect the changes in mouse Aif protein content.4.RNA sequencing:cochlear RNA from the two groups of mice were extracted,RNA sequencing and bioinformatics analysis were conducted,exploring the differential changes and key signaling pathways of cochlear mRNA and LncRNA in the two groups.Results 1.In terms of auditory function,the ABR threshold of the Aifm1+/Y group was slightly higher than the WT group before exposure,but there was no statistical difference.With the increase of age,compared with 1 month old,the ABR thresholds of group at 7 and 13 months were increased.At 13 months,the ABR threshold increases were especially at 8k Hz,24k Hz and 32k Hz,while the ABR threshold increase relatively little at 16k Hz.After noise exposure,the ABR thresholds in both groups increased,and the Aifm1+/Y group had a larger threshold shift(the average threshold shift of the Aifm1+/Y group was 31 dB SPL,and the average threshold shift of the WT group was 19 dB SPL).The ABR thresholds of the two groups subsequently recovered,and after 14 days of noise exposure,the WT group returned to normal levels,but the Aifm1+/Y group still had an average threshold shift of 17 dB SPL.In addition,except for 32k Hz,all frequencies of the ABR wave I amplitude at 80 dB SPL decreased significantly after noise intervention.And the amplitude of Aifm1+/Y group was continued to decrease as the number of days increases at 32k Hz,and showed a significant decrease after 14 days of noise exposure.2.In terms of cochlear tissue morphology,regardless of noise exposure,the CtBP2 counts in hair cell synapses in the Aifm1+/Y group were lower than those in the WT group.After noise exposure,the number of synapses in both groups were decreased,and the nuclei of inner hair cells in the Aifm1+/Y group became flat and irregularly arranged.The number of synapses subsequently recovered,and after 14 days of noise exposure,synapses in the WT group recovered to 63%of the normal level.Aifm1+/Y group recovered to 55%of normal level.3.In terms of RNA and protein expression,Aifm1+/Y group Aif protein expression was significantly lower than WT group,Aif protein of WT group had no significant change,while Aifm1+/Y group Aif protein expression increase after noise exposure for 7days.The amount increased significantly,suggesting that oxidative stress may occur in cells,leading to apoptosis.The results of RNA sequencing showed that there were significant differences between the two groups on mRNA and LncRNA,and the multicellular organismal homeostasis pathway may be the key signaling pathway after noise exposure.Conclusion Aifm1+/Y gene mutation reduces the number of synapses and decreases the expression of Aif protein.The defect of Aif protein increases the sensitivity of Aifm1+/Y mice to noise.After noise exposure,the ABR threshold cannot be recovered within 14 days,the amplitude of wave I at 80 dB SPL is reduced.The number of synapses is greatly reduced and cannot be recovered within 14 days.Aif protein is up-regulated in response to cellular oxidative stress after noise exposure.