Functional analysis of RRAS2 pathogenic variants with a Noonan-like phenotype

Area of Science:

• Molecular Biology
• Genetics
• Developmental Biology

Background:

• RRAS2, a GTPase regulating cell proliferation and differentiation via the RAS/MAPK pathway, has been implicated in Noonan syndrome.
• Previous reports identified seven pathogenic RRAS2 variants, but limited functional analysis exists.

Purpose of the Study:

• To investigate the functional impact of novel and recurrent RRAS2 variants (p.Gly23Val and p.Gly24Glu) in patients with a Noonan-like phenotype.
• To elucidate the role of RRAS2 gain-of-function variants in RAS signaling pathway hyperactivity and disease pathogenesis.

Main Methods:

• Transient expression of wild-type (WT) and mutant RRAS2 in HEK293 cells, followed by Western blotting and luciferase reporter assays to assess RAS signaling activity.
• Functional analysis in Drosophila melanogaster and zebrafish (Danio rerio) models to evaluate the in vivo effects of RRAS2 variants.

Main Results:

• Mutant RRAS2 variants (p.Gly23Val and p.Gly24Glu) exhibited significantly increased RAS signaling pathway activity compared to WT.
• In vivo studies demonstrated RRAS2 variant-induced lethality in Drosophila and developmental defects (oval shape, delayed jaw development) in zebrafish embryos.
• These findings indicate hyperactivity of the RAS signaling pathway due to RRAS2 gain-of-function variants.

Conclusions:

• Recurrent and novel RRAS2 variants identified in patients with a Noonan-like phenotype are gain-of-function mutations.
• These RRAS2 variants lead to increased RAS signaling pathway activity, both in vitro and in vivo.
• The study confirms that RRAS2 gain-of-function variants are causative of Noonan syndrome-like phenotypes.