Research Of Mutant Genes In Patients With Familial Exudative Vitreoretinopathy

Objective: Familial exudative vitreoretinopathy(FEVR,OMIM 133780),characterized by incomplete retinal vascular development and pathological neovascularization,is a severe inherited retinal disorder.Mutations in ten genes have been reported to be associated with FEVR,but still leave ?50% of FEVR cases to be explained.The purpose of this study was to identify causative mutations in Chinese FEVR families and explore molecular mechanism of them.Methods: Whole-exome sequencing was performed to analyze the genomic DNA from 121 Chinese FEVR families.Sanger sequencing was carried out to verify all identified mutations.Western blotting method was utilized to assess the expression of TSPAN12 plasmids.Luciferase assay was used to test the activity of mutant protein in the Norrin-?-catenin signaling pathway.Immunocytochemical staining(ICC)was employed to analyze the location and expression of mutant protein.Result:1.Four novel heterozygous TSPAN12 mutations,including a initial-lost mutation,a single-base substitution mutation and two frameshift mutations,were discovered in these FEVR families: c.1A>G(p.0),c.614G>A(p.G205D),c.695 del T(p.V232Gfs*7)and c.833?842del(p.L278Qfs*25)2.The four TSPAN12 mutations c.1A>G(p.0),c.614G>A(p.G205D),c.695 del T(p.V232Gfs*7)and c.833?842del(p.L278Qfs*25)were confirmed by sanger sequencing.3.Western blotting method proved that the G205 D mutation TSPN12 protein can be normally expressed compared with WT TSPAN12 protein.4.Luciferase assays revealed that TSPAN12 mutation c.614G>A(p.G205D)could significantly reduce the activity of luciferase,indicating that the G205 D mutant protein lost its activity as a co-receptor of Norrin ligand.5.ICC showed that the fluorescence intensity of cells transfected with G205 D TSPAN12 plasmids was consistent with that of WT plasmids,demonstrating that the expressing level of G205 D mutation TSPN12 protein was equal to WT TSPAN12 protein.Conclusion:1.This study identified four new TSPAN12 mutations in 121 Chinese FEVR famlies,expanding the mutation spectrum of FEVR in Chinese.2.The G205 D mutation TSPAN12 protein can be express stably in cells and the expressing level is consistent with that of the wild type.However,the function of G205 D mutant protein is significantly affected,causing it to lose its activity as a coreceptor of Norrin ligand.