Fig. 4

Functional characterization of human IRF6 missense gene variant protein functions with the zebrafish irf6 model.

(A) Experimental approach for characterizing protein functions of human IRF6 missense gene variants. Variant mRNAs were synthesized and microinjected into maternal-null irf6 ^-/- embryos at the one-cell stage and assessed for phenotypic rescue at 24 hpf. (B-C) Protein modeling of the protein-binding domain and C-terminus of IRF6 using ExPASy with crystalline structures of IRF1. (B) is mapped with missense variant amino acid residues (green) whose mRNA rescued the periderm rupture phenotype, while (C) is mapped with missense variant amino acid residues (red) whose mRNA failed to rescue. (D-F) Results for functional rescue of periderm rupture with maternal-null irf6 ^-/- embryos for representative human IRF6 missense gene variants. Results were classified as rescued if any maternal-null irf6 ^-/- embryos injected with variant mRNA remained alive and phenotypically wild type at 24 hpf (50 embryos/round, n = 3). Missense gene variants were categorized by location within the IRF6 protein, and by computational results from PolyPhen-2 and SIFT on whether the in silico predictions agreed on the deleterious effects of the missense gene variants on protein function. Further shown are ACMG guideline pathogenicity predictions (pathogenic, likely pathogenic, uncertain, and benign), and the number of families identified for each variant (all gene variant annotations were based on NM_006147.3).