FIGURE 4

Assessment of SMAD3 VUS pathogenicity using a zebrafish embryonic assay. (A) Diagram of the spatial distribution of specific VUS mutations examined. (B) Confocal images of the tail aorta/vein labeled in Tg[kdrl:mCherry] in 48hpf embryos. White dashed boxes indicate the enlargement regions used to highlight differences in diameter (white dashed lines). Embryos imaged were either control, injected with smad3a^WT mRNA or smad3a^P124T, smad3a^R287W, smad3a^L296P or smad3a^A349P. (C) Quantification of diameter counts. Each point represents an individual embryo measured. Standard deviation is depicted in red. Variants were compared to WT: *p < 0.05, **p < 0.01. (D) Summary of VUS classification changes based on the zebrafish embryonic assay results. P, pathogenic, LP, likely pathogenic.