A novel variant p.Y250C of FZD4 influences Norrine/β-catenin signaling pathway that associates with familial exudative vitreoretinopathy (FEVR).

Frizzled-4 (FZD4) gene mutation is a known mechanism of familial exudative vitreoretinopathy (FEVR). To establish the pathogenicity of the novel FZD4 mutation c.A749G, functional studies are needed to connect this mutation to the patient's FEVR phenotypes. Fluorescence microscopy and co-immunoprecipitation (Co-IP) techniques were employed to determine the effect of FZD4 mutation on sub-cellular localization and interaction with partners. The activity of Norrin (NDP)/β-catenin pathway was assessed through the western blot and luciferase assays. Western blot was performed to evaluate the spatial and temporal expressions of the Fzd4 across various mouse tissues. The mutated [c.A749G (p.Y250C)] forms of Frizzled 4 (FZD4) constructs were successfully conducted. Wild-type FZD4 predominantly located in the cytoplasm and plasma membrane, while the mutant exhibited a tendency to aggregate at nuclear membrane and within the nucleus. Co-IP revealed preserved mutant FZD4-LRP5 (low-density lipoprotein receptor-related protein 5) binding, suggesting the formation of receptor complex was likely unaffected by this mutation. Overexpression of mutant FZD4 and NDP or activation with agonist R-Spordin 1 (RSPO1) reduced downstream signaling proteins, including phosphorylated β-catenin (p-β-catenin), and vascular endothelial growth factor (VEGF-A). β-catenin report activity was significant lower in mutant group (P < 0.05), aligning with attenuated pathway activation. Fzd4 exhibited broad tissue expression, and it was notably present during the early developmental stages of retinal and ocular formation in mice. The novel missense mutation c.A749G in the FZD4 gene may impair the Norrin/β-catenin signaling pathway and alter subcellular localization, thereby driving FEVR pathogenesis.