Functional analysis of a duplication (p.E63_D69dup) in the switch II region of HRAS: New aspects of the molecular pathogenesis underlying Costello syndrome

Costello syndrome is a congenital disorder comprising a characteristic face, severe feeding difficulties, skeletal, cardiac and skin abnormalities, intellectual disability and predisposition to malignancies. It is caused by heterozygous germline HRAS mutations mostly affecting Gly¹² or Gly¹³, which impair HRAS-GTPase activity and result in increased downstream signal flow independent of incoming signals. Functional analyses of rarer HRAS mutations identified in individuals with attenuated Costello syndrome phenotypes revealed altered GDP/GTP nucleotide affinities (p.K117R) and inefficient effector binding (p.E37dup). Thus, both phenotypic and functional variability associated with HRAS mutations are evident. Here, we report on a novel heterozygous HRAS germline mutation (c.187_207dup, p.E63_D69dup) in a girl presenting with a phenotype at the milder end of the Costello syndrome spectrum. The p.E63_D69dup mutation impaired co-precipitation of recombinant HRAS with NF1 GTPase-activating protein (GAP) suggesting constitutive HRAS^E63_D69dup activation due to GAP insensitivity. Indeed, we identified strongly augmented active HRAS^E63_D69dup that co-precipitated with effectors RAF1, RAL guanine nucleotide dissociation stimulator and phospholipase C1. However, we could not pull down active HRAS^E63_D69dup using the target protein PIK3CA, indicating a compromised association between active HRAS^E63_D69dup and PIK3CA. Accordingly, overexpression of HRAS^E63_D69dup increased steady-state phosphorylation of MEK1/2 and ERK1/2 downstream of RAF, whereas AKT phosphorylation downstream of phosphoinositide 3-kinase (PI3K) was not enhanced. By analyzing signaling dynamics, we found that HRAS^E63_D69dup has impaired reagibility to stimuli resulting in reduced and disrupted capacity to transduce incoming signals to the RAF-MAPK and PI3K-AKT cascade, respectively. We suggest that disrupted HRAS reagibility, as we demonstrate for the p.E63_D69dup mutation, is a previously unappreciated molecular pathomechanism underlying Costello syndrome.