Abstract
Purpose :
Wnt signaling is an evolutionarily conserved pathway that plays a crucial role in various developmental processes, including cell fate determination, cell migration, cell polarity, neural patterning, and organogenesis. WNT2B is one of 19 known human WNT proteins and has recently been associated with Congenital Diarrheas and Enteropathies (CODE). Several patients demonstrated ocular involvement, including coloboma and microcornea. We hypothesize that WNT2B plays a role in eye development independent of its role in intestinal homeostasis/association with CODE.
Methods :
To gain insight into the role of WNT2B in eye development we explored zebrafish as a model system and recruited additional patients. At the protein level, zebrafish wnt2bb shares 78% identity with human WNT2B and putative pathogenic residues were conserved in zebrafish. Using CRISPR/Cas9, we generated crispant alleles in a wnt2bb zebrafish model to study wnt2bb loss of function (LOF) and pathogenic variants in WNT2B. We injected Cas9 protein, wnt2bb sgRNA and single stranded template DNA encoding wnt2bb (p.Tyr274Cys) into 1-cell stage zebrafish zygotes. These embryos were grown to adulthood and genotyped. Lastly, we documented more patients exhibiting ocular manifestations.
Results :
The adult F0 fish with variants predicted to result in wnt2bb LOF did not exhibit an abnormal ocular phenotype and will be crossed to generate homozygous lines. We found two patients with eye defects and novel WNT2B variants. A patient with microphthalmia/anophthalmia was heterozygous for a missense variant, WNT2B(NM_024494): c.806A>G;(p.Tyr269Cys)(inheritance not known) and a patient with colobomatous microphthalmia was homozygous for WNT2B(NM_024494.3): c.757C>G;(p.Arg253Gly). There are two previously documented nonsense variants in WNT2B, one frameshift variant and 3 missense variants that have been associated with colobomatous microphthalmia. The missense variants all occurred within a span of 30 AA residues (241-269) and this distribution strongly implies the involvement of this region of WNT2B in mediating crucial protein interactions.
Conclusions :
The data from these two patients further strengthen our notion that WNT2B plays a role in eye development independent of CODE. We are continuing our research on zebrafish models of loss- and gain of function for wnt2bb so that we can better understand the pathogenesis of the eye defects.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.