LRP5Mutations In Familial Exudative Vitreoretinopathy And The Gene Expression Profile Of Human Retinal Endothelium After LRP5Down-expression

PurposeTo describe the clinical features and identify the disease-causing mutations in two Chinesefamilies with familial exudative vitreoretinopathy (FEVR) and to further explore thedifference of gene expressions in human retinal endothelial cell (hREC）after LRP5RNAinterferon using RNA sequencing to discuss the potential mechanisms of LRP5in retinalangiogenesis.Methods1. We collected the clinical informations of the patients with FEVR. Genomic DNAfrom the patients was also obtained and Sanger sequencing was performed to detect thecoding exons and adjacent intronic regions of4known causative genes (FZD4, LRP5,TSPAN12and NDP). Wild-type and mutant LRP5proteins were assayed forNorrin/β-catenin pathway by luciferase reporter assays to evaluate the pathogenesis of thenovel mutations.2. LRP5siRNA was transfected into hREC and the total RNA was purified andsubjected to Illumina Hi-seq RNA sequencing. Differentially expressed genes were furtheranalyzed by GO and KEGG pathway analysis.Results1. Two novel heterozygous mutations in LRP5gene were identified in two relatives:p.A422T and p.L540P, both of which were cosegregated in their families. Luciferase assayimplied that both p.A422T and p.L540P mutants may be pathogenic. Both of the patientshad an additional LRP5sequence change (p.Q816P in Patient1from the unaffected mother and p.T852M in Patient2verified as a new mutation). Luciferase assay suggestedp.Q816P may be not pathogenic but p.T852M may be pathogenic. The probandsmanifested severe symptoms compared with the affected father or mother, which may bedue to the combination of gene mutations. Mild reduced bone mineral density (BMD) wasfound in the two patients and the affected father or mother.2. RNA sequencing showed there were87up-regulated genes and52down-regulatedgenes by two folds (p<0.05),28up-regulated genes and22down-regulated genes by twofolds (p<0.01) after LRP5down-expression. Nineteen differentially expressedgenes(p<0.01) were related to6GO terms, including protein amino acid phosphorylation,positive regulation of steroid metabolic process, cholesterol efflux, plasma membrane part,cell junction and enzyme binding. KEGG Pathway analysis indicated that31differentiallyexpressed genes were evolved in13pathways. Focal adhesion, mitogen-activated proteinkinase (MAPK), ECM-receptor interaction, JAK-STAT and ErbB signaling pathway maybe implicated to be related to the retinal angiogenesis.Conclusions1. Our findings provide two novel LRP5mutations in Chinese patients with FEVR andmild reduced bone mineral density (BMD). The combination of mutations may causesevere phenotype.2. Differentially expressed genes in hREC after LRP5gene down-expression and therelated biological processes and pathways, such as protein amino acid phosphorylationand focal adhesion, may be potential mediators of Wnt-driven regulation in retinal bloodvessel growth.