Identification Of LRP5 Mutations In Families With Familial Exudative Vitreoretinopathy

Familial Exudative Vitreoretinopathy(FEVR)is a hereditary ophthalmic disease characterized by retinal vascular abnormalities and lesions.FEVR Can be expressed as retinal blood vessels bleeding,leakage,traction,etc.,and even cause retinal detachment and eventually lead to blindness.The pathogenesis of the disease is complex.Previous research showed that genetic factors plays a very critical role in the pathogenesis of the disease.At least five genes has been shown to be associated with FEVR: NDP,FZD4,LRP5,TSPAN12 and ZNF408.However,Mutations in these genes can only explain about 50% of FEVR cases.Therefore,it is important to find new pathogenic mutations or pathogenic genes and understand the pathogenesis of the disease.Such studies are essential for development of proper clinical diagnosis and intervention.FEVR is a progressive retinal disease with no effective treatment.This study identified more genetic mutations of FEVR disease in Chinese Han population,which can provide useful information for molecular diagnosis and genetic counseling of the disease.In this study,we investigated the relationship between pathogenic mutations and disease phenotypes by studying four cases of FEVR in China.PCR primers were designed to amplify the exon regions and exon-intron boundary regions of 4 FEVR pathogenic genes FZD4,LRP5,NDP and TSPAN12.Amplified DNA samples were sequenced by Sanger method to identify the mutation sites.The effect of mutations on protein function was also studied by using bioinformatics method.Mutations were introduced into the coding region of LRP5.The changes of protein expression were investigated by Western blotting,and the function of mutated proteins was evaluated by TOP-flash luciferase assay using SFT cells.FEVR patients were diagnosed by fundus examination,fundus fluorescein angiography(FFA)and OCT.Five mutations in LRP5 were identified by Sanger sequencing results: c.542T> G,p.M181 R,c.1197G> T,p.R399 S and c.1507 G > A,p.G503 R,c.1481G>A,p.R494 Q and c.2626G> A,p.G876 S.All five amino acid residues affected by these five mutations are highly conserved.The results of Western blotting showed that xx mutated proteins were unstable.Luciferase assay demonstrated that all mutated proteins lost their ability to activate Norrin/FZD4/ ?-catenin signaling pathway.Three heterozygous mutations were not been reported in the dbSNP database and the thousands of human genome project database in the LRP5 gene:542T> G: p.M181 R,c.1197G> T: p.R399 S and c.1507 G > A: p.G503 R.Two heterozygous mutations c.1481G>A,p.R494 Q and c.2626G> A: p.G876 S,were reported in other diseases but not reported in FEVR.Bioinformatics analysis showed that all five mutations were harmful mutations,and the amino acid residues affected by these mutations were highly conserved.Further luciferase reporter experiments demonstrated that none of these five mutations could activate the Norrin / ?-catenin signaling pathway,demonstrating the pathogenicity of these five mutations.