A comprehensive functional analysis on the pathogenesis of novel TSPAN12 and NDP variants in familial exudative vitreoretinopathy

Familial exudative vitreoretinopathy (FEVR) is an inherited blinding disorder; however, the known FEVR-associated variants account for approximately only 50% cases. Currently, the pathogenesis of most reported variants is not well studied, we aim to identify novel variants from FEVR-associated genes and perform a comprehensive functional analysis to uncover the pathogenesis of variants that cause FEVR. Using targeted gene panel and Sanger sequencing, we identified six novel and three known variants in TSPAN12 and NDP. These variants were demonstrated to cause significant inhibition of Norrin/β-catenin pathway by dual-luciferase reporter assay and western blot analysis. Structural analysis and co-immunoprecipitation revealed compromised interactions between missense variants and binding partners in the Norrin/β-catenin pathway. Immunofluorescence and subcellular protein extraction were performed to reveal the abnormal subcellular trafficking. Additionally, over-expression of TSPAN12 successfully enhanced the Norrin/β-catenin signaling activity by strengthening the binding affinity of mutant Norrin with FZD4 or LRP5. Together, these observations expanded the spectrum of FEVR-associated variants for the genetic counseling and prenatal diagnosis of FEVR, as well providing a potential therapeutic strategy for the treatment of FEVR.