Article

Germline KRAS Mutations Cause Aberrant Biochemical and Physical Properties Leading to Developmental Disorders

Institute of Biochemistry and Molecular Biology II, Heinrich-Heine University, Düsseldorf, Germany.
Human Mutation (Impact Factor: 5.14). 01/2011; 32(1):33-43. DOI: 10.1002/humu.21377
Source: PubMed

ABSTRACT

The KRAS gene is the most common locus for somatic gain-of-function mutations in human cancer. Germline KRAS mutations were shown recently to be associated with developmental disorders, including Noonan syndrome (NS), cardio-facio-cutaneous syndrome (CFCS), and Costello syndrome (CS). The molecular basis of this broad phenotypic variability has in part remained elusive so far. Here, we comprehensively analyzed the biochemical and structural features of ten germline KRAS mutations using physical and cellular biochemistry. According to their distinct biochemical and structural alterations, the mutants can be grouped into five distinct classes, four of which markedly differ from RAS oncoproteins. Investigated functional alterations comprise the enhancement of intrinsic and guanine nucleotide exchange factor (GEF) catalyzed nucleotide exchange, which is alternatively accompanied by an impaired GTPase-activating protein (GAP) stimulated GTP hydrolysis, an overall loss of functional properties, and a deficiency in effector interaction. In conclusion, our data underscore the important role of RAS in the pathogenesis of the group of related disorders including NS, CFCS, and CS, and provide clues to the high phenotypic variability of patients with germline KRAS mutations.

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    • "Therefore, " gain-of-function " and " hyperactivation " concerning HRAS downstream signaling pathways are widely used to explain the molecular basis of Costello syndrome. However, the RAS-dependent signaling pathway's intricate regulation defies a simple one dimensional characterization of up and down regulation, as exemplified by the complex effects caused by KRAS sequence changes [Gremer et al., 2010b]. In line with this, functional characterization of rarer HRAS germline mutations revealed additional molecular consequences, including altered GDP/GTP nucleotide affinities (p.Lys117Arg) [Denayer et al., 2008] and inefficient effector binding (p.Glu37dup) [Gremer et al., 2010a]. "
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    ABSTRACT: Heterozygous germline mutations in the proto-oncogene HRAS cause Costello syndrome (CS), an intellectual disability condition with severe failure to thrive, cardiac abnormalities, predisposition to tumors, and neurologic abnormalities. More than 80% of patients share the HRAS mutation c.34G>A (p.Gly12Ser) associated with the typical, relatively homogeneous phenotype. Rarer mutations occurred in individuals with an attenuated phenotype and less characteristic facial features. Most pathogenic HRAS alterations affect hydrolytic HRAS activity resulting in constitutive activation. "Gain-of-function" and "hyperactivation" concerning downstream pathways are widely used to explain the molecular basis and dysregulation of the RAS-MAPK pathway is the biologic mechanism shared amongst rasopathies. Panel testing for rasopathies identified a novel HRAS mutation (c.179G>A; p.Gly60Asp) in three individuals with attenuated features of Costello syndrome. De novo paternal origin occurred in two, transmission from a heterozygous mother in the third. Individuals showed subtle facial features; curly hair and relative macrocephaly were seen in three; atrial tachycardia and learning difficulties in two, and pulmonic valve dysplasia and mildly thickened left ventricle in one. None had severe failure to thrive, intellectual disability or cancer, underscoring the need to consider HRAS mutations in individuals with an unspecific rasopathy phenotype. Functional studies revealed strongly increased HRAS(Gly60Asp) binding to RAF1, but not to other signaling effectors. Hyperactivation of the MAPK downstream signaling pathways was absent. Our results indicate that an increase in the proportion of activated RAS downstream signaling components does not entirely explain the molecular basis of CS. We conclude that the phenotypic variability in CS recapitulates variable qualities of molecular dysfunction. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.
    Full-text · Article · Apr 2015 · American Journal of Medical Genetics Part A
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    • "The cancer mutations were extracted from the OMIM database , which lists all mutations even if not confirmed to be diseasecausing [in cancer often > 200 proteins are mutated and not all are expected to be disease-causing (Vogelstein et al, 2013)]. To exclude possible cancer passenger mutations, we generated a 'gold set' of RASopathy and cancer mutations by including only those mutations that have been already studied experimentally, and for which there is evidence of their transforming potential, or for which sustained increased kinase activity has been demonstrated (Wan et al, 2004; Gremer et al, 2011) (Supplementary Table S2). Using this gold set, the FoldX energy values for germline and cancer mutants separate "
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    ABSTRACT: The Ras/MAPK syndromes (‘RASopathies’) are a class of developmental disorders caused by germline mutations in 15 genes encoding proteins of the Ras/mitogen-activated protein kinase (MAPK) pathway frequently involved in cancer. Little is known about the molecular mechanisms underlying the differences in mutations of the same protein causing either cancer or RASopathies. Here, we shed light on 956 RASopathy and cancer missense mutations by combining protein network data with mutational analyses based on 3D structures. Using the protein design algorithm FoldX, we predict that most of the missense mutations with destabilising energies are in structural regions that control the activation of proteins, and only a few are predicted to compromise protein folding. We find a trend that energy changes are higher for cancer compared to RASopathy mutations. Through network modelling, we show that partly compensatory mutations in RASopathies result in only minor downstream pathway deregulation. In summary, we suggest that quantitative rather than qualitative network differences determine the phenotypic outcome of RASopathy compared to cancer mutations.
    Full-text · Article · May 2014 · Molecular Systems Biology
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    • "Effector binding assays were performed in 30 mm Tris–HCl, pH 7.5, 100 mm NaCl, 5 mm MgCl2, 3 mm DTE at 25°C using a Fluoromax 4 fluorimeter in polarization mode. Increasing amounts of GST-tagged RAS-binding domains (RBD) of RAS effectors were titrated to 0.3 µm mantGppNHp-bound RAS proteins resulting in an increase of polarization (64). The dissociation constants (Kd) were calculated by fitting the concentration-dependent binding curve using a quadratic ligand binding equation. "
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    ABSTRACT: RASopathies, a family of disorders characterized by cardiac defects, defective growth, facial dysmorphism, variable cognitive deficits and predisposition to certain malignancies, are caused by constitutional dysregulation of RAS signalling predominantly through the RAF/MEK/ERK (MAPK) cascade. We report on two germline mutations (p.Gly39dup and p.Val55Met) in RRAS, a gene encoding a small monomeric GTPase controlling cell adhesion, spreading and migration, underlying a rare (2 subjects among 504 individuals analysed) and variable phenotype with features partially overlapping Noonan syndrome, the most common RASopathy. We also identified somatic RRAS mutations (p.Gly39dup and p.Gln87Leu) in 2 of 110 cases of non-syndromic juvenile myelomonocytic leukaemia, a childhood myeloproliferative/myelodysplastic disease caused by upregulated RAS signalling, defining an atypical form of this haematological disorder rapidly progressing to acute myeloid leukaemia. Two of the three identified mutations affected known oncogenic hotspots of RAS genes and conferred variably enhanced RRAS function and stimulus-dependent MAPK activation. Expression of an RRAS mutant homolog in Caenorhabditis elegans enhanced RAS signalling and engendered protruding vulva, a phenotype previously linked to the RASopathy-causing SHOC2S2G mutant. Overall, these findings provide evidence of a functional link between RRAS and MAPK signalling and reveal an unpredicted role of enhanced RRAS function in human disease.
    Full-text · Article · Apr 2014 · Human Molecular Genetics
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