Open Collections

Tophkhane, Shruti Sanjay

University of British Columbia

Robinow syndrome (RS) is a rare skeletal disorder caused by variants in seven genes in the Wingless-related Int-1 (WNT) signalling pathway. RS is primarily characterized by short stature and face anomalies. Here, we studied two missense FZD2 variants and one frameshift DVL1 variant using the chicken embryo model. Avian-specific replication-competent retroviruses containing human genes were overexpressed in the frontonasal mass over the endogenous chicken genome. Effects of the variant viruses were compared to wild-type (wt) to distinguish variant-specific effects from those due to over-expression. In vivo, the hFZD2 and hDVL1 variants disrupted upper beak patterning and inhibited frontonasal mass narrowing, recapitulating clinically wider faces in RS individuals. The increased width was not due to increased proliferation. hFZD2 and hDVL1 variants expressed in primary frontonasal mass mesenchyme inhibited chondrogenesis, unlike the wt genes. The DVL1 frameshift causes an abnormal C-terminal peptide. We made two additional DVL1 constructs - hDVL1¹⁵¹⁹* and hDVL1¹⁴³¹* to delete part or all of the C-terminus. hDVL1¹⁵¹⁹* did not affect beak morphology in vivo or chondrogenesis in vitro, suggesting that the C-terminus is needed for the gain-of-function affecting beak morphology seen in wtDVL1 expressing embryos. Also, the inhibition of chondrogenesis is due to the abnormal peptide in the frame-shifted variants. In contrast, hDVL1¹⁴³¹* caused embryo lethality and external beak shortening. Thus, the presence of the C terminus is needed for normal development. Luciferase assays on primary cells showed weak activation of both canonical (Super Topflash, STF) and non-canonical JNK/PCP (ATF2) pathways by hFZD2 and hDVL1 variants. The hDVL1¹⁵¹⁹* or the entire C-terminal deletion (hDVL1¹⁴³¹*) retained some WNT signalling activity, showing neither construct is a complete loss-of-function. The DVL1 variant protein and the hDVL1¹⁴³¹* truncated protein were also unable to traffic normally in the cell. Our model clarified the pathogenicity of hFZD2 variants and shed light on the role of the abnormal C-terminus in ADRS. These discoveries broaden our understanding of the WNT pathway and suggest a mechanism for the facial phenotypes. One day, this work will lead to novel therapeutic interventions for patients with skeletal dysplasias.

Text

2024-04-26

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/88032

Cell and Developmental Biology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/