Introduction: 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 RRAS2 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 RRAS2 pathogenic variants (p.Gly23Val and p.Gly24Glu, respectively) and the results of their functional analysis. Materials and methods: Wild-type (WT) and mutant RRAS2 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 Drosophila eye using the glass multiple reporter-Gal4 driver. Mutant mRNA microinjection into zebrafish embryos was performed, and the embryo jaws were observed. Results: 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. Discussion: Recurrent and novel RRAS2 variants that we reported showed increased in vitro or in vivo RAS signaling pathway activity because of gain-of-function RRAS2 variants. Clinical features are similar to those previously reported, suggesting that RRAS2 gain-of-function variants cause this disease in patients.
Rare diseases are estimated to affect 3.5%-5.9% of the population worldwide and are difficult to diagnose. Genome analysis is useful for diagnosis. However, since some variants, especially missense variants, are also difficult to interpret, tools to accurately predict the effect of missense variants are very important and needed. Here we developed a method, "VarMeter", to predict whether a missense variant is damaging based on Gibbs free energy and solvent-accessible surface area calculated from the AlphaFold 3D protein model. We applied this method to the whole-exome sequencing data of 900 individuals with rare or undiagnosed disease in our in-house database, and identified four who were hemizygous for missense variants of arylsulfatase L (ARSL; known as the genetic cause of chondrodysplasia punctata 1, CPDX1). Two individuals had a novel Ser89 to Asn (Ser89Asn) or Arg469 to Trp (Arg469Trp) substitution, respectively predicted as "damaging" or "benign"; the other two had an Arg111 to His (Arg111His) or Gly117 to Arg (Gly117Arg) substitution, respectively predicted as "damaging" or "possibly damaging" and previously reported in patients showing clinical manifestations of CDPX1. Expression and analysis of the missense variant proteins showed that the predicted pathogenic variants (Ser89Asn, Arg111His, and Gly117Arg) had complete loss of sulfatase activity and reduced protease resistance due to destabilization of protein structure, while the predicted benign variant (Arg469Trp) had activity and protease resistance comparable to those of wild-type ARSL. The individual with the novel pathogenic Ser89Asn variant exhibited characteristics of CDPX1, while the individual with the benign Arg469Trp variant exhibited no such characteristics. These findings demonstrate that VarMeter may be used to predict the deleteriousness of variants found in genome sequencing data and thereby support disease diagnosis.
