A mutation in FRIZZLED2 impairs Wnt signaling and causes autosomal dominant omodysplasia

Abstract

Autosomal dominant omodysplasia is a rare skeletal dysplasia characterized by short humeri, radial head dislocation, short first metacarpals, facial dysmorphism and genitourinary anomalies. We performed next-generation whole-exome sequencing and comparative analysis of a proband with omodysplasia, her unaffected parents and her affected daughter. We identified a de novo mutation in FRIZZLED2 (FZD2) in the proband and her daughter that was not found in unaffected family members. The FZD2 mutation (c.1644G>A) changes a tryptophan residue at amino acid 548 to a premature stop (p.Trp548*). This altered protein is still produced in vitro, but we show reduced ability of this mutant form of FZD2 to interact with its downstream target DISHEVELLED. Furthermore, expressing the mutant form of FZD2 in vitro is not able to facilitate the cellular response to canonical Wnt signaling like wild-type FZD2. We therefore conclude that the FRIZZLED2 mutation is a de novo, novel cause for autosomal dominant omodysplasia.

Figure 1.

Phenotyping of proband and pedigree of patients with AD omodysplasia. Proband at 10 (A) and 25 years of age (B) presenting with skeletal dysplasia and craniofacial phenotypes. The proband's parents and her daughter, represented in the pedigree in (C), provided specimens for exome sequencing.

Figure 2.

Patient 2 with AD omodysplasia. Patient 2 at 3 months (A) and 6 years (B). Skeletal phenotypes include abnormalities of the vertebrae (C), humerus (D) and hands (E).

Figure 3.

Mutations in FRIZZLED2 cause AD omodysplasia. (A) Sanger sequencing confirms findings from next-generation sequencing of heterozygous FZD2 mutations in the proband and daughter, but not the proband's parents. (B) FZD2 mutation creates a premature stop. (C) This is predicted to create an FZD2 protein lacking the majority of the intracellular cytoplasmic tail partially responsible for Dishevelled binding (gray).

Figure 4.

Fzd2 expression in developing craniofacial and limb tissues. RNA in situ hybridization indicates the expression of Fzd2 in HH stage 30 chicken embryos in the developing face (A and B) and limbs (C). Image in B is higher magnification view of image shown in the inset. Digoxigenin-labeled riboprobes were used according to published protocols. (D) Western immunoblotting for FZD2 protein confirms expression in these tissues from HH stage 25 embryos. c-cartilage, e-eye, hl-hindlimb, ncc-neural crest cells, oc-oral cavity, te-telencephalon and t-tongue.

Figure 5.

The p.W458X mutation does not alter protein levels or subcellular localization. Expression of either Fzd2^WT-GFP (A and C) or Fzd2^p.TRP548*-GFP (B and D) in HEK293T (A and B) or NIH3T3 (C and D) cells does not significantly differ in abundance or subcellular localization. Scale bar is equal to 20 μm.

Figure 6.

Dvl2–Fzd2 interactions are perturbed in cells expressing truncated FZD2. Co-expression of Dvl2-FLAG and either Fzd2^WT-GFP (A–H) or Fzd2^p.TRP548*-GFP (I–P) in unstimulated HEK293T cells resulted in accumulations of Dvl2-FLAG puncta throughout the cell. Wnt treatment in Fzd2^WT-GFP cells (E–H) resulted in increased colocalization of the Fzd and Dvl after immunocytochemistry and dissipation of most Dvl2-positive puncta, indicating successful recruitment to the plasma membrane. Treatment of Fzd2^p.TRP548*-GFP-mutant cells, however, did not show a robust recruitment of Dvl2-FLAG (M–P). This was quantified as proportion of cells with ‘no’, ‘some’ or ‘significant’ colocalization and loss of puncta (Q) and as an average colocalization value for each cell (R). Number of double-transfected cells analyzed for each condition is indicated in R. Scale bar is equal to 10 μm. **P = 0.001 versus untreated cells.

Figure 7.

Expression of truncated FZD2 attenuates canonical Wnt-signaling. STF Wnt reporter cells were transfected with control plasmids or Fzd2^p.TRP548*-GFP (mut-Fzd2) and treated with Wnt-conditioned media. Expression of increasing amounts of Fzd2^p.TRP548*-GFP reduced the cellular response to Wnt ligand as measured by luciferase activity. Wnt luciferase activity was normalized to the treated, non-transfected cells.**P = 0.002 versus control transfection.