Robinow syndrome skeletal phenotypes caused by the WNT5AC83S variant are due to dominant interference with chondrogenesis

Abstract

Heterozygous missense mutations in several genes in the WNT5A signaling pathway cause autosomal dominant Robinow syndrome 1 (DRS1). Our objective was to clarify the functional impact of a missense mutation in WNT5A on the skeleton, one of the main affected tissues in RS. We delivered avian replication competent retroviruses (RCAS) containing human wild-type WNT5A (wtWNT5A), WNT5AC83S variant or GFP/AlkPO4 control genes to the chicken embryo limb. Strikingly, WNT5AC83S consistently caused a delay in ossification and bones were more than 50% shorter and 200% wider than controls. In contrast, bone dimensions in wtWNT5A limbs were slightly affected (20% shorter, 25% wider) but ossification occurred on schedule. The dysmorphology of bones was established during cartilage differentiation. Instead of stereotypical stacking of chondrocytes, the WNT5AC83S-infected cartilage was composed of randomly oriented chondrocytes and that had diffuse, rather than concentrated Prickle staining, both signs of disrupted planar cell polarity (PCP) mechanisms. Biochemical assays revealed that C83S variant was able to activate the Jun N-terminal kinase-PCP pathway similar to wtWNT5A; however, the activity of the variant ligand was influenced by receptor availability. Unexpectedly, the C83S change caused a reduction in the amount of protein being synthesized and secreted, compared to wtWNT5A. Thus, in the chicken and human, RS phenotypes are produced from the C83S mutation, even though the variant protein is less abundant than wtWNT5A. We conclude the variant protein has dominant-negative effects on chondrogenesis leading to limb abnormalities.

Craniofacial studies in chicken embryos confirm the pathogenicity of Frizzled2 variants associated with Robinow syndrome

“…In this study, misexpression of wt or variant hFZD2 in embryonic face did not cause morphological defects in the upper beak but inhibited patterning and ossification. These decreased ossification phenotypes are comparable to the effect of misexpression of ADRS hWNT5A 248G>C variant that caused short and wide limbs and delay in ossification (Gignac et al, 2019). Likewise, in mouse model, an inducible transgene containing Wnt5a inhibited cranial bone formation (van Amerongen et al, 2012).…”

“…This design suitable for studying autosomal dominant mutations that are thought to cause an interference between mutant and wild-type protein. Indeed, our previous work on an autosomal dominant DVL1 frameshift mutations (Gignac et al, 2023) and WNT5A missense mutations (Gignac et al 2019;Hosseini-Farahabadi et al 2017) found evidence for dominant interference that reduced bone size and changed bone shape. FZD2 is ubiquitously expressed in the face in chicken and mouse (Saal et al, 2015;Yu et al, 2012;Yu et al, 2010;Geetha-Loganathan et al, 2009).…”

“…In humans, the frontonasal mass contributes to midline structures of the face encompassing the bridge of the nose, the central region of the nose, the nasal septum, the philtrum, and the central portion of the upper lip, including the premaxilla (Chen et al, 2013). Previous studies from our lab have demonstrated that ADRS-WNT5A (Gignac et al, 2019;Hosseini-Farahabadi et al, 2017) and DVL1 variants (Gignac et al, 2023) have dominant-negative effects on chondrogenesis leading to abnormal bone morphology mimicking ADRS. In this study, we uncover functional evidence that both 425C>T and 1301G>T weakly activate the canonical WNT pathway, have dominant negative effects on activity of wt FZD2 and inhibit skeletogenesis.…”

“…RCAS virus containing GFP insert (kindly provided by A. Gaunt) served as a control in virus overexpression studies, as published (Geetha-Loganathan et al, 2014;Gignac et al, 2019;Gignac et al, 2023;Hosseini-Farahabadi et al, 2013;Hosseini-Farahabadi et al, 2017). DF1 cells were cultured at 37°C and 5% CO 2 in DMEM (ThermoFisher#1967497) medium supplemented with 10% fetal bovine serum (FBS; Sigma #F1051) and 1% penicillin/streptomycin (ThermoFisher #15070-063).…”

Symmetry and fluctuation of cell movements in neural crest-derived facial mesenchyme

“…The JNK signal results in cytoskeletal changes that affect cell shape, orientation, polarity and directed cell migration ( Butler and Wallingford, 2017 ). WNT5A protein induces JNK-PCP signaling activity as shown in facial and limb mesenchyme ( Geetha-Loganathan et al, 2014 ; Gignac et al, 2019 ; Hosseini-Farahabadi et al, 2013 , 2017 ). WNT5A is expressed in a horizontal band across the frontonasal mass at the stages we imaged here ( Geetha-Loganathan et al, 2009 ) ( Fig.…”