Functional genomics in chicken embryos reveal the pathogenicity of two missense FZD2 variants associated with dominant Robinow syndrome

Robinow syndrome (RS) is a rare disease caused by mutations in seven WNT pathway genes. Features include craniofacial widening and jaw hypoplasia. We used the chicken embryo to test two autosomal dominant RS (ADRS) missense FZD2 variants on the frontonasal mass, the affected region in RS. The wild-type (wt) and variant hFZD2 inhibited beak ossification. The bone hypoplasia was possibly mediated by decreased levels of WNT and BMP pathway genes. In primary cultures, hFZD2 variants inhibited chondrogenesis, increased nuclear shuttling of {beta}-catenin and increased expression of TWIST1, both known to suppress chondrogenesis. In luciferase reporter assays, proteins coding for 1301G>T and 425C>T FZD2 variants weakly activated canonical WNT reporter and dominantly interfered with wtFZD2. In the JNK-PCP WNT pathway luciferase assay, only the 425C>T showed a loss-of-function. The 1301G>T variant presumably acts through a JNK-independent pathway. This is the first study to demonstrate that the ADRS-FZD2 missense variants cause craniofacial and WNT signaling defects. Frontonasal mass width is increased by both hFZD2 variants which sheds light on the ontogeny of the broad facial features seen in individuals with RS. Summary StatementGain-of-function studies on FZD2 missense variants associated with Robinow syndrome led to increased facial width, altered Wnt signaling and inhibition of beak skeletogenesis in chicken embryos.