title: Functional analysis of a duplication (p.E63_D69dup) in the switch II region of HRAS: new aspects of the molecular pathogenesis underlying Costello syndrome.
abstract: 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 Gly(12) or Gly(13), 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.
authors: Institute of Human Genetics, University Medical Center Hamburg-Eppendorf, Hamburg 20246, Germany.
authors: Sybille
authors: S
authors: Lorenz
authors: Christina
authors: C
authors: Lissewski
authors: Pelin O
authors: PO
authors: Simsek-Kiper
authors: Yasemin
authors: Y
authors: Alanay
authors: Koray
authors: K
authors: Boduroglu
authors: Martin
authors: M
authors: Zenker
authors: Georg
authors: G
authors: Rosenberger
mesh_terms: D000015:Abnormalities, Multiple / Q000235:genetics / Q000473:pathology; D000293:Adolescent; D000818:Animals; D058534:Class I Phosphatidylinositol 3-Kinases; D056685:Costello Syndrome / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D005260:Female; D020690:GTPase-Activating Proteins / Q000235:genetics; D020440:Gene Duplication; D018095:Germ-Line Mutation; D006801:Humans; D008607:Intellectual Disability / Q000235:genetics / Q000473:pathology; D020935:MAP Kinase Signaling System / Q000235:genetics; D051379:Mice; D025542:Neurofibromin 1 / Q000235:genetics / Q000378:metabolism; D057089:Pathology, Molecular; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D020690:GTPase-Activating Proteins; D025542:Neurofibromin 1; D019869:Phosphatidylinositol 3-Kinases; D058534:Class I Phosphatidylinositol 3-Kinases; C484760:PIK3CA protein, human; D051057:Proto-Oncogene Proteins c-akt; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)