An essential role of the cysteine-rich domain of FZD4 in Norrin/Wnt signaling and familial exudative vitreoretinopathy.

The Wnt pathway plays important yet diverse roles in health and disease. Mutations in the Wnt receptor FZD4 gene have been confirmed to cause familial exudative vitreoretinopathy (FEVR). FEVR is characterized by incomplete vascularization of the peripheral retina, which can lead to vitreous bleeding, tractional retinal detachment, and blindness. We screened for mutations in the FZD4 gene in five families with FEVR and identified five mutations (C45Y, Y58C, W226X, C204R, and W496X), including three novel mutations (C45Y, Y58C, and W226X). In the retina, Norrin serves as a ligand and binds to FZD4 to activate the Wnt signaling pathway in normal angiogenesis and vascularization. The cysteine-rich domain (CRD) of FZD4 has been shown to play a critical role in Norrin-FZD4 binding. We investigated the effect of mutations in the FZD4 CRD in Norrin binding and signaling in vitro and in vivo. Wild-type and mutant FZD4 proteins were assayed for Norrin binding and Norrin-dependent activation of the canonical Wnt pathway by cell-surface and overlay binding assays and luciferase reporter assays. In HEK293 transfection studies, C45Y, Y58C, and C204R mutants did not bind to Norrin and failed to transduce FZD4-mediated Wnt/β-catenin signaling. In vivo studies using Xenopus embryos showed that these FZD4 mutations disrupt Norrin/β-catenin signaling as evidenced by decreased Siamois and Xnr3 expression. This study identified a new class of FZD4 gene mutations in human disease and demonstrates a critical role of the CRD in Norrin binding and activation of the β-catenin pathway.