Screening And Functional Characterization Of The WFS1 Gene Mutations Identified From Wolfram Syndrome Pedigrees And Bioinformatic Analysis Of WFS1 Gene

Objective:Wolfram syndrome is a specific type of diabetes mellitus,which is characterized by diabetes insipidus,juvenile-onset diabetes mellitus,optic atrophy,and sensorineural deafness.The WFS1 gene mutation is the main cause of Wolfram syndrome.The protein encoded by WFS1 is named wolframin.We sequenced the WFS1 gene of a Wolfram syndrome patient to identify the WFS1 gene mutation.After that,we cloned the mutant WFS1 gene and other mutants(F417de1,Y534D)identified in Wolfram syndrome patients by our laboratory before in vitro to perform relative functional analysis of these WFS1 mutants.To get a further insight into the function of the WFS1 gene,bioinformatic analysis of the DEG profiles was performed in the liver tissues of Wfsl knockout mice.Methods:Sequencing was conducted with genomic DNA prepared from patient and parental peripheral blood to identify the WFS1 gene mutation.The mutations were introduced into the WFS1 cDNA by site-directed mutagenesis.The expression plasmids carrying the wild type or the WFS1 mutants were transiently transfected into cell lines and the expression of WFS1 genes were evaluated by western blot and real-time PCR.We also examined the roles of proteasome inhibitor and lysosome inhibitor in the degradation of mutant wolframin.We then determined the cellular localization of wild-type and mutant wolframin by immunofluorescence.Luciferase reporter assay was used to investigate the level of endoplasmic reticulum stress.Furthermore,we downloaded the hepatic gene expression profile of wild-type and Wfs1-knockout mice from GEO database to analyse the DEG.We then conducted GO and KEGG enrichment analyses and established protein-protein interaction network.Results:The patient was found to be compound heterozygotes for G526D and W648X mutations.WFS1 gene mutations(F417del,Y534D,G526D,W648X,G526D-W648X)led to the decrease of wolframin,which was prevented by proteasome inhibitor.Wild-type and mutants localized with endoplasmic reticulum but mutants did not affect protein cellular distribution.All of the WFS1 mutants(F417del,Y534D,G526D,W648X,G526D-W648X)could enhance GRP78 promoter reporter activity.A total of 198 DEG were obtained from the hepatic gene expression profiles.GO analyses showed that DEG were significantly enriched in sodium-independent organic anion transmembrane transporter activity and aromatase activity.KEGG analyses showed that DEG were significantly enriched in fatty acid biosynthesis and primary bile acid biosynthesis.26 hub genes were discovered in protein-protein interaction network.Conclusion:G526D/W648X mutation occurred in WFS1 gene is the main cause of Wolfram syndrome in this patient and G526D mutation has not been reported yet.Mutants(F417del,Y534D,G526D,W648X)showed lower protein levels probably resulting from the increase of proteasome degradation.All of the WFS1 mutants could increase endoplasmic reticulum stress which probably is the main cause of Wolfram syndrome.Knockout Wfsl gene in mice could probably affect fatty acid biosynthesis and primary bile acid biosynthesis.