Preliminary Study On The Pathogenicity And Pathogenic Mechanism Of A Variant In 5’UTR Of Novel Deafness Gene EPHA10

Objective: To elucidate the pathogenicity and pathogenic mechanism of a novel deafness gene EPHA10 and its variant in 5’UTR from the aspects of causative gene identification,gene function and molecular mechanism.Methods:(1)Causative gene identification: Investigation of candidate causative genes and their variants was conducted by whole exome sequencing combining with linkage analysis.Referring to the clinical characteristics of the patients,study of the pathogenicity of variants was conducted by co-segregation analysis,searching the population frequency in different databases and conservation analysis.(2)Gene function:(1)Detection of EPHA10 expression in cochlea was conducted by RT-PCR,Western blot,immunohistochemistry and immunofluorescence staining.(2)The effects which the variant in 5’UTR exerted on EPHA10 were investigated by dual luciferase assay,RNA secondary structure prediction and EPHA10 expression level detection on lymphoblastoid cell lines.(3)The upregulation of EPHA10 was simulated by Eph overexpression from mating of Gal4 and UAS flies.The structure and function of fly chordotonal organ were evaluated by phalloidin staining and negative geotaxis assay.(4)The 5’UTR of Epha10 of C57BL/6J mice was edited using CRISPR/Cas9 to knock the variant in.The hearing of mice was evaluated by auditory brainstem response.(3)Molecular mechanism: HEI-OC1 cells were infected by lentivirus to over-express Epha10 and then RNA-seq was performed.Known deafness genes which may be relevant to Epha10 were investigated from the differentially expressed genes.The correlation between Epha10 and known deafness genes was explained by the results of enrichment analysis and protein-protein interaction prediction.Results:(1)Causative gene identification: The variant in 5’UTR NM＿001099439.1: c.-81＿-73 delins AGC co-segregated with deaf phenotype in the family.It was rarely seen in population.The DNA sequence of variant site was conservative in evolution.(2)Gene function:(1)EPHA10 expression was found in the organ of Corti,spiral ganglion and stria vascularis,and the expression level was very low.(2)The variant in 5’UTR altered the predicted secondary structure of EPHA10 mRNA.The variant in 5’UTR enhanced the activity of a putative promoter of EPHA10,and upregulated EPHA10 m RNA and protein expression.(3)Eph over-expression resulted in looser and less orderly arrangement of scolopale rods of fly chordotonal organ Johnston’s organ.Eph over-expression also resulted in lower pass rate in negative geotaxis assay.(4)Knock in of the variant in 5’UTR of Epha10 led to hearing loss of mice which resembled the deaf phenotype of the patients.(3)Molecular mechanism: Epha10 over-expression caused downregulation of known deafness gene Tnc and other basilar membrane related genes including Col4a1,Lama5 and Lamb3.EPHA10 may interacted with Tenascin-C through Wnt/RYK signaling.Conclusion: EPHA10 has had several typical characteristics of deafness gene,and the variant in 5’UTR is pathogenic.Tenascin-C may participate in the pathophysiological process of hereditary hearing loss caused by EPHA10 upregualtion.