Clinical Phenotype And Identification Of The Causative Genes For Three Families With Hearing Loss And Correlation Analysis On NsSNPs Of WFS1 Gene And Diseases

Objective: Hearing impairment is one of the common sensory sensorineural disabilities.Hereditary hearing loss may be caused by a variety of unknown genes and mutations.We recruited three Chinese families with hearing loss in order to summarize the clinical characteristics and screen out their pathogenic mutation.The pathogenic mutation of WFS1 c.2421C>G(p.Ser807Arg)was further analyzed to explore its possible pathogenic mechanism,and the relationship among the ns SNPs of WFS1 gene and disease was explored in depth.Methods: Three Chinese families with deafness were recruited in clinic.A detailed medical history inquiry and related examinations of the participants were performed.The next-generation sequencing was used to confirm the gene mutation in the proband,and the Sanger sequencing was used to verify the co-segregation of the pathogenic mutations and the hearing phenotype within the family.Further analysis of the pathogenic mechanisms of WFS1 c.2421C>G(p.Ser807Arg)and WFS1 ns SNPs were performed by using multiple bioinformatics software.MUpro,I-Mutant 2.0,INPS-MD and i Stable were used to analyze the stability of protein after variation.The evolutionary conservation analysis of ns SNPs was estimated by Consurf server.SOPMA and TMHMM Server 2.0 were used to predict the secondary structure and transmembrane structure of protein separately.Structural presentation of wild and mutant proteins was made by using Py MOL programs.HOPE analyzed the impact of a given mutation on the protein structure.The Accessible Surface Area and Accessibility Calculation for Protein(ver.1.2)online server calculated the solvent-accessible surface areas of 890 amino acids of wolframin protein.STRING was used to explore the WFS1 gene interaction network with other proteins.Results: HBSJZ-1 and HBSJZ-2 belonged to the non-syndromic deafness family,while HBSJZ-3 belonged to the auditory neuropathy family.The auditory characteristics of the HBSJZ-1 family were symmetrical binaural sensorineural hearing loss,especially in the low frequency.The WFS1 c.2421C>G(p.Ser807Arg)was the pathogenic mutation.The audiological characteristics of the HBSJZ-2 family were symmetric binaural sensorineural hearing loss,especially occurring at medium or full frequency.The POU4F3 c.932T>C(p.Leu311Pro)was the pathogenic mutation.The pathogenic mutation in HBSJZ-3 family was not found.The bioinformatics software analysis showed that WFS1 c.2421 C > G(p.Ser807Arg)was highly conservative in evolution and predicted that the mutation led to an increase of protein stability,the mutant residue was larger,the charge of mutant residue changed from neutral to positive,the mutant residues was less hydrophobic,the proportion of core amino acids in the mutant protein decreased,and the protein structure and hydrogen bonds changed.15660 ns SNPs of WFS1 were retrieved from the three databases.The pathogenicity of 1782 ns SNPs were predicted by fourteen pathogenicity prediction tools,and 13 ns SNPs were considered high risk and selected for in-depth analysis.All the 13 high-risk mutations were highly conserved residues with a conservative score of 9 or 8and they mostly cause decrease in protein stability.There were nine transmembrane regions in Wolframin protein and none of the mutations resulted in changes in transmembrane structure.The pathogenic mutations had an important effect on not only amino acid size,charge and hydrophobicity,but also protein’s spatial structure.The proportion of protein core amino acids was decreased in all 13 WFS1 high-risk pathogenic ns SNPs mutations.Among these,11 ns SNPs had been previously published or cited and 2ns SNPs(Gly695Ser and Glu776Lys)had not been reported.The two novel variants increased or decreased hydrogen bonds.The protein-protein interaction network of WFS1 could be exported form the STRING.It was shown that WFS1 could participate the regulatory of Na/K-ATPase,endoplasmic reticulum stress,unfolded protein response,Ca2+ homeostasis,insulin synthesis and release.Conclusions: WFS1 is a common pathogenic gene for low-frequency sensorineural hearing loss,the mutations related to NSHL are mainly concentrated in exon 8,and mainly concentrated in the C-terminal domain of Wolframin protein(amino acids 652 – 890),which indicates that this region is very important for the normal protein function in the inner ear.Most of the pathogenic ns SNPs of WFS1 gene will cause changes in protein stability,secondary structure,tertiary structure and hydrogen bond,and affect the normal binding of Wolframin protein with other proteins,leading to disease phenotype.In conclusion,the bioinformatics analysis is useful to efficiently identify harmful ns SNPs.But computer analysis lacks clinical evidence and experimental support,further vivo and vitro functional studies are required to verify the accuracy of our methods.