Mutational and Functional Analysis in Human Ras/MAP Kinase Genetic Syndromes

  • William E. Tidyman
  • Katherine A. Rauen
Part of the Methods in Molecular Biology book series (MIMB, volume 661)


The Ras/mitogen-activated protein kinase (MAPK) pathway is essential in regulation of the cell cycle, cell differentiation, growth, and cell senescence, each of which are critical to normal development. A class of developmental disorders, the “RASopathies,” is caused by germline mutations in genes that encode protein components of the Ras/MAPK pathway which result in dysregulation of the pathway and profound deleterious effects on development. One of these syndromes, cardiofaciocutaneous (CFC) syndrome, is caused by germline mutations in BRAF, MAP2K1 (MEK1) and MAP2K2 (MEK2), and possibly KRAS genes. Here, we describe the laboratory protocols and methods that we used to identify mutations in BRAF and MEK1/2 genes as causative for CFC syndrome. In addition, we present the techniques used to determine the effect these mutations have on activity of the Ras/MAPK pathway through Western blot analysis of the phosphorylation of endogenous ERK1/2, as well as through the use of an in vitro kinase assay that measures the phosphorylation of Elk-1.

Key words

RASopathies Cardio-facio-cutaneous syndrome (CFC) BRAF MAP2K1 (MEK1) MAP2K2 (MEK2) Ras/MAPK Signal transduction pathway ERK1 ERK2 Elk-1 Kinase assay 



We thank Anne Estep and Clarice Bunag for their expert technical assistance. Special thanks are due to Pablo Rodriguez-Viciana for his expert assistance in the development of some of these protocols.


  1. 1.
    Tidyman, W.E. and K.A. Rauen, (2009) The RASopathies: developmental syndromes of Ras/MAPK pathway dysregulation. Curr Opin Genet Dev. 19, 230–236.PubMedCrossRefGoogle Scholar
  2. 2.
    Tartaglia, M., et al., (2001) Mutations in PTPN11, encoding the protein tyrosine phosphatase SHP-2, cause Noonan syndrome. Nat Genet. 29, 465–468.PubMedCrossRefGoogle Scholar
  3. 3.
    Roberts, A.E., et al., (2007) Germline gain-of-function mutations in SOS1 cause Noonan syndrome. Nat Genet. 39, 70–74.PubMedCrossRefGoogle Scholar
  4. 4.
    Tartaglia, M., et al., (2007) Gain-of-function SOS1 mutations cause a distinctive form of Noonan syndrome. Nat Genet. 39, 75–79.PubMedCrossRefGoogle Scholar
  5. 5.
    Razzaque, M.A., et al., (2007) Germline gain-of-function mutations in RAF1 cause Noonan syndrome. Nat Genet. 39, 1013–1017.PubMedCrossRefGoogle Scholar
  6. 6.
    Pandit, B., et al., (2007) Gain-of-function RAF1 mutations cause Noonan and LEOPARD syndromes with hypertrophic cardiomyopathy. Nat Genet. 39, 1007–1012.PubMedCrossRefGoogle Scholar
  7. 7.
    Schubbert, S., et al., (2006) Germline KRAS mutations cause Noonan syndrome. Nat Genet. 38, 331–336.PubMedCrossRefGoogle Scholar
  8. 8.
    Cawthon, R.M., et al., (1990) Identification and characterization of transcripts from the neurofibromatosis 1 region: the sequence and genomic structure of EVI2 and mapping of other transcripts. Genomics. 7, 555–565.PubMedCrossRefGoogle Scholar
  9. 9.
    Viskochil, D., et al., (1990) Deletions and a translocation interrupt a cloned gene at the neurofibromatosis type 1 locus. Cell. 62, 187–192.PubMedCrossRefGoogle Scholar
  10. 10.
    Wallace, M.R., et al., (1990) Type 1 neurofibromatosis gene: identification of a large transcript disrupted in three NF1 patients. Science. 249, 181–186.PubMedCrossRefGoogle Scholar
  11. 11.
    Digilio, M.C., et al., (2002) Grouping of multiple-lentigines/LEOPARD and Noonan syndromes on the PTPN11 gene. Am J Hum Genet. 71, 389–394.PubMedCrossRefGoogle Scholar
  12. 12.
    Hart, T.C., et al., (2002) A mutation in the SOS1 gene causes hereditary gingival fibromatosis type 1. Am J Hum Genet. 70, 943–954.PubMedCrossRefGoogle Scholar
  13. 13.
    Eerola, I., et al., (2003) Capillary malformation-arteriovenous malformation, a new clinical and genetic disorder caused by RASA1 mutations. Am J Hum Genet. 73, 1240–1249.PubMedCrossRefGoogle Scholar
  14. 14.
    Aoki, Y., et al., (2005) Germline mutations in HRAS proto-oncogene cause Costello syndrome. Nat Genet. 37, 1038–1040.PubMedCrossRefGoogle Scholar
  15. 15.
    Niihori, T., et al., (2006) Germline KRAS and BRAF mutations in cardio-facio-cutaneous syndrome. Nat Genet. 38, 294–296.PubMedCrossRefGoogle Scholar
  16. 16.
    Rodriguez-Viciana, P., et al., (2006) Germline mutations in genes within the MAPK pathway cause cardio-facio-cutaneous syndrome. Science. 311, 1287–1290.PubMedCrossRefGoogle Scholar
  17. 17.
    Brems, H., et al., (2007) Germline loss-of-function mutations in SPRED1 cause a neurofibromatosis 1-like phenotype. Nat Genet. 39, 1120–1126.PubMedCrossRefGoogle Scholar
  18. 18.
    Oishi, K., et al., (2009) Phosphatase-defective LEOPARD syndrome mutations in PTPN11 gene have gain-of-function effects during Drosophila development. Hum Mol Genet. 18, 193–201.PubMedCrossRefGoogle Scholar
  19. 19.
    Hmitou, I., et al., (2007) Differential regulation of B-raf isoforms by phosphorylation and autoinhibitory mechanisms. Mol Cell Biol. 27, 31–43.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Meida, LLC 2010

Authors and Affiliations

  • William E. Tidyman
    • 1
  • Katherine A. Rauen
    • 2
  1. 1.Department of Pediatrics, Division of Medical GeneticsUniversity of California San FranciscoSan FranciscoUSA
  2. 2.Department of Pediatrics, Division of Medical Genetics, UCSF Helen Diller Family Comprehensive Cancer CenterUniversity of California San FranciscoSan FranciscoUSA

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