Dkk1 inhibition normalizes limb phenotypes in a mouse model of Fzd2 associated omodysplasia Robinow syndromes

FRIZZLED-2 (FZD2) is a transmembrane Wnt ligand receptor. We previously identified a pathogenic human FZD2 variant, encoding for a protein with a premature stop and loss of 17 amino acids. This includes a portion of the consensus DISHEVELLED binding sequence required for Wnt signal transduction. Patients with this variant exhibited FZD2-associated autosomal dominant Robinow Syndrome. To model this variant, we utilized zygote microinjection and i-GONAD-based CRISPR/Cas9-mediated genome editing to generate an allelic series in the mouse. Embryos mosaic for humanized Fzd2W553* knock-in exhibited cleft palate and shortened limbs, consistent with FZD2W548* patient phenotypes. We also generated two germline mouse alleles with small deletions, Fzd2D3 and Fzd2D4. Homozygotes for each allele survive embryonic development at normal ratios but exhibit a highly penetrant cleft palate phenotype, shortened limbs compared to wild-type and perinatal lethality. Fzd2D4 craniofacial tissues indicated decreased canonical WNT signaling. In utero treatment with IIIC3a (DKK inhibitor) normalized the limb lengths in Fzd2D4 homozygotes. The in vivo replication represents an approach to further investigate the mechanism of FZD2 phenotypes and validates the utility of CRISPR knock-in mice as a tool for demonstrating pathogenicity of human genetic variants. We also present evidence for a potential therapeutic intervention.