Gain-of-function RAF1 mutations cause Noonan and LEOPARD syndromes with hypertrophic cardiomyopathy

Article metrics


Noonan and LEOPARD syndromes are developmental disorders with overlapping features, including cardiac abnormalities, short stature and facial dysmorphia. Increased RAS signaling owing to PTPN11, SOS1 and KRAS mutations causes 60% of Noonan syndrome cases1,2,3,4,5,6, and PTPN11 mutations cause 90% of LEOPARD syndrome cases7. Here, we report that 18 of 231 individuals with Noonan syndrome without known mutations (corresponding to 3% of all affected individuals) and two of six individuals with LEOPARD syndrome without PTPN11 mutations have missense mutations in RAF1, which encodes a serine-threonine kinase that activates MEK1 and MEK2. Most mutations altered a motif flanking Ser259, a residue critical for autoinhibition of RAF1 through 14-3-3 binding. Of 19 subjects with a RAF1 mutation in two hotspots, 18 (or 95%) showed hypertrophic cardiomyopathy (HCM), compared with the 18% prevalence of HCM among individuals with Noonan syndrome in general. Ectopically expressed RAF1 mutants from the two HCM hotspots had increased kinase activity and enhanced ERK activation, whereas non–HCM-associated mutants were kinase impaired. Our findings further implicate increased RAS signaling in pathological cardiomyocyte hypertrophy.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: RAF1 domain structure and location of residues altered in Noonan and LEOPARD syndromes.
Figure 2: RAF1 kinase assays.
Figure 3: ERK activation assays.
Figure 4: 14-3-3 binding and phosphorylation status of Ser259 of RAF1.


  1. 1

    Carta, C. et al. Germline missense mutations affecting KRAS isoform B are associated with a severe Noonan syndrome phenotype. Am. J. Hum. Genet. 79, 129–135 (2006).

  2. 2

    Kontaridis, M.I., Swanson, K.D., David, F.S., Barford, D. & Neel, B.G. PTPN11 (Shp2) mutations in LEOPARD syndrome have dominant negative, not activating, effects. J. Biol. Chem. 281, 6785–6792 (2006).

  3. 3

    Schubbert, S. et al. Germline KRAS mutations cause Noonan syndrome. Nat. Genet. 38, 331–336 (2006).

  4. 4

    Tartaglia, M. et al. Mutations in PTPN11, encoding the protein tyrosine phosphatase SHP-2, cause Noonan syndrome. Nat. Genet. 29, 465–468 (2001).

  5. 5

    Roberts, A.E. et al. Germline gain-of-function mutations in SOS1 cause Noonan syndrome. Nat. Genet. 39, 70–74 (2007).

  6. 6

    Tartaglia, M. et al. Gain-of-function SOS1 mutations cause a distinctive form of Noonan syndrome. Nat. Genet. 39, 75–79 (2007).

  7. 7

    Digilio, M.C. et al. Grouping of multiple-lentigines/LEOPARD and Noonan syndromes on the PTPN11 Gene. Am. J. Hum. Genet. 71, 389–394 (2002).

  8. 8

    Aoki, Y. et al. Germline mutations in HRAS proto-oncogene cause Costello syndrome. Nat. Genet. 37, 1038–1040 (2005).

  9. 9

    Niihori, T. et al. Germline KRAS and BRAF mutations in cardio-facio-cutaneous syndrome. Nat. Genet. 38, 294–296 (2006).

  10. 10

    Rodriguez-Viciana, P. et al. Germline mutations in genes within the MAPK pathway cause cardio-facio-cutaneous syndrome. Science 311, 1287–1290 (2006).

  11. 11

    Tartaglia, M. et al. Diversity and functional consequences of germline and somatic PTPN11 mutations in human disease. Am. J. Hum. Genet. 78, 279–290 (2006).

  12. 12

    Tartaglia, M. et al. PTPN11 mutations in Noonan syndrome: molecular spectrum, genotype-phenotype correlation, and phenotypic heterogeneity. Am. J. Hum. Genet. 70, 1555–1563 (2002).

  13. 13

    Burch, M. et al. Cardiologic abnormalities in Noonan syndrome: phenotypic diagnosis and echocardiographic assessment of 118 patients. J. Am. Coll. Cardiol. 22, 1189–1192 (1993).

  14. 14

    Van Driest, S.L. et al. Myosin binding protein C mutations and compound heterozygosity in hypertrophic cardiomyopathy. J. Am. Coll. Cardiol. 44, 1903–1910 (2004).

  15. 15

    Wellbrock, C., Karasarides, M. & Marais, R. The RAF proteins take centre stage. Nat. Rev. Mol. Cell Biol. 5, 875–885 (2004).

  16. 16

    Emuss, V., Garnett, M., Mason, C. & Marais, R. Mutations of C-RAF are rare in human cancer because C-RAF has a low basal kinase activity compared with B-RAF. Cancer Res. 65, 9719–9726 (2005).

  17. 17

    Zebisch, A. et al. Two transforming C-RAF germ-line mutations identified in patients with therapy-related acute myeloid leukemia. Cancer Res. 66, 3401–3408 (2006).

  18. 18

    Greenman, C. et al. Patterns of somatic mutation in human cancer genomes. Nature 446, 153–158 (2007).

  19. 19

    Muslin, A.J., Tanner, J.W., Allen, P.M. & Shaw, A.S. Interaction of 14–3-3 with signaling proteins is mediated by the recognition of phosphoserine. Cell 84, 889–897 (1996).

  20. 20

    Light, Y., Paterson, H. & Marais, R. 14–3-3 antagonizes Ras-mediated Raf-1 recruitment to the plasma membrane to maintain signaling fidelity. Mol. Cell. Biol. 22, 4984–4996 (2002).

  21. 21

    Wan, P.T. et al. Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF. Cell 116, 855–867 (2004).

  22. 22

    Muslin, A.J. Role of raf proteins in cardiac hypertrophy and cardiomyocyte survival. Trends Cardiovasc. Med. 15, 225–229 (2005).

  23. 23

    Bueno, O.F. et al. The MEK1–ERK1/2 signaling pathway promotes compensated cardiac hypertrophy in transgenic mice. EMBO J. 19, 6341–6350 (2000).

  24. 24

    Hunter, J.J., Tanaka, N., Rockman, H.A., Ross, J. Jr. & Chien, K.R. Ventricular expression of a MLC-2v-ras fusion gene induces cardiac hypertrophy and selective diastolic dysfunction in transgenic mice. J. Biol. Chem. 270, 23173–23178 (1995).

  25. 25

    Gripp, K.W. et al. HRAS mutation analysis in Costello syndrome: genotype and phenotype correlation. Am. J. Med. Genet. A. 140, 1–7 (2006).

  26. 26

    Yamaguchi, O. et al. Cardiac-specific disruption of the c-raf-1 gene induces cardiac dysfunction and apoptosis. J. Clin. Invest. 114, 937–943 (2004).

  27. 27

    Patel, R. et al. Simvastatin induces regression of cardiac hypertrophy and fibrosis and improves cardiac function in a transgenic rabbit model of human hypertrophic cardiomyopathy. Circulation 104, 317–324 (2001).

  28. 28

    Sarkozy, A. et al. Clinical and molecular analysis of 30 patients with multiple lentigines LEOPARD syndrome. J. Med. Genet. 41, e68 (2004).

  29. 29

    Ucar, C., Calyskan, U., Martini, S. & Heinritz, W. Acute myelomonocytic leukemia in a boy with LEOPARD syndrome (PTPN11 gene mutation positive). J. Pediatr. Hematol. Oncol. 28, 123–125 (2006).

  30. 30

    Merks, J.H., Caron, H.N. & Hennekam, R.C. High incidence of malformation syndromes in a series of 1,073 children with cancer. Am. J. Med. Genet. A. 134, 132–143 (2005).

Download references


We are indebted to the affected individuals and families who participated in the study, the physicians who referred the subjects and the Joint Genome Institute's production sequencing group. We thank D.K. Morrison (National Cancer Institute, US National Institutes of Health) for critical reagents and suggestions. We also thank G. Crisponi (Clinica Sant'Anna, Cagliari, Italy), F. Faravelli (Ospedale Galliera, Genova, Italy), L. Memo (ULLS9, Treviso, Italy) and F. Stanzial (Regional Hospital, Bolzano, Italy) for their valuable clinical assistance. This work was supported by Telethon-Italy grant GGP07115 (M.T.) and Programma di Collaborazione Italia-USA/malattie rare 2007 grants (M.T. and A.S.); US National Institutes of Health Grants HL71207, HD01294, HL074728 (B.D.G.), HD042569 (M.J.A.) and DK57683 (P.M.); the American Heart Association (M.J.A.); the Dr. Scholl Foundation (M.J.A.); the CJ Foundation for SIDS and the Mayo Foundation (M.J.A.) and Italian Ministry of Health Grant RC 2006-2007, Italian Ministry of University and Research C26A06KTTN and RBIP06PMF2_005 (B.D.). Research conducted at the E.O. Lawrence Berkeley National Laboratory and the Joint Genome Institute was performed under the Berkeley Program for Genomic Applications, funded by the US National Heart, Lung and Blood Institute (HL066681) and Department of Energy Contract DE-AC02-05CH11231 (University of California). We regret our inability to cite certain references describing work similar to references that were cited due to the limits of this journal.

Author information

Correspondence to Marco Tartaglia or Bruce D Gelb.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Table 1, Supplementary Table 2, Supplementary Figure 1 (PDF 517 kb)

Rights and permissions

Reprints and Permissions

About this article

Further reading