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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Available from: Martin Zenker, Sep 26, 2015
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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.
    Molecular Systems Biology 05/2014; 10(5). DOI:10.1002/msb.20145092 · 10.87 Impact Factor
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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.
    Human Molecular Genetics 04/2014; 23(16). DOI:10.1093/hmg/ddu148 · 6.39 Impact Factor
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    • "Described KRAS mutations (p.Lys5Asn, p.Val14Ile, p.Gln22Glu, p.Gln22Arg, p.Pro34Leu, p.Pro34Arg, p.Thr58Ile, p.Gly60Arg, p.Asp134Val, p.Phe156Leu, p.Val152Gly, and p.Asp153Val) are regularly associated with CHD (6-10). Extremely severe phenotype cases of NS and other RASopathies are typically caused only by specific mutations, resulting in overall signal flow dysregulation of the RAS-MAPK pathway (9,11,12). "
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    ABSTRACT: Noonan syndrome is a relatively common and heterogeneous genetic disorder, including congenital heart defect in more than half of the cases. If the defect is not large, life expectancy is normal. Here we report on a case of an infant with Noonan syndrome and rapidly progressive hypertrophic cardiomyopathy with lethal outcome, in whom we identified a novel mutation in the KRAS gene. This heterozygous unclassified missense variant in exon 3: c.179G> T (p.Gly60Val) might be associated with a lethal form of Noonan syndrome. The malignant clinical course of the disease and the lethal outcome in an infant only a few months old might be connected to RAS-mitogen-activated protein kinase pathway hyperactivation, consequently promoting cell growth and proliferation, leading to rapidly progressive hypertrophic cardiomyopathy. Further biochemical and functional studies are needed to confirm this hypothesis.
    Croatian Medical Journal 12/2013; 54(6):574-8. DOI:10.3325/cmj.2013.54.574 · 1.31 Impact Factor
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