Functional analysis of pathogenic variants with a Noonan-like phenotype.

RRAS2, a member of the R-Ras subfamily of Ras-like low-molecular-weight GTPases, is considered to regulate cell proliferation and differentiation via the RAS/MAPK signaling pathway. Seven pathogenic variants have been reported in patients with Noonan syndrome; however, few functional analyses have been conducted. Herein, we report two patients who presented with a Noonan-like phenotype with recurrent and novel pathogenic variants (p.Gly23Val and p.Gly24Glu, respectively) and the results of their functional analysis. Wild-type (WT) and mutant genes were transiently expressed in Human Embryonic Kidney293 cells. Expression of RRAS2 and phosphorylation of ERK1/2 were confirmed by Western blotting, and the RAS signaling pathway activity was measured using a reporter assay system with the serum response element-luciferase construct. WT and p.Gly23Val RRAS2 were expressed in eye using the glass multiple reporter-Gal4 driver. Mutant mRNA microinjection into zebrafish embryos was performed, and the embryo jaws were observed. No obvious differences in the expression of proteins WT, p.Gly23Val, and p.Gly24Glu were observed. The luciferase reporter assay showed that the activity of p.Gly23Val was 2.45 ± 0.95-fold higher than WT, and p.Gly24Glu was 3.06 ± 1.35-fold higher than WT. For transgenic flies, the p.Gly23Val expression resulted in no adults flies emerging, indicating lethality. For mutant mRNA-injected zebrafish embryos, an oval shape and delayed jaw development were observed compared with WT mRNA-injected embryos. These indicated hyperactivity of the RAS signaling pathway. Recurrent and novel variants that we reported showed increased or RAS signaling pathway activity because of gain-of-function variants. Clinical features are similar to those previously reported, suggesting that gain-of-function variants cause this disease in patients.