Molecular and clinical analysis of RAF1 in Noonan syndrome and related disorders: dephosphorylation of serine 259 as the essential mechanism for mutant activation

Noonan syndrome (NS) and related disorders are autosomal dominant disorders characterized by heart defects, facial dysmorphism, ectodermal abnormalities, and mental retardation. The dysregulation of the RAS/MAPK pathway appears to be a common molecular pathogenesis of these disorders: mutations in PTPN11, KRAS, and SOS1 have been identified in patients with NS, those in KRAS, BRAF, MAP2K1, and MAP2K2 in patients with CFC syndrome, and those in HRAS mutations in Costello syndrome patients. Recently, mutations in RAF1 have been also identified in patients with NS and two patients with LEOPARD (multiple lentigines, electrocardiographic conduction abnormalities, ocular hypertelorism, pulmonary stenosis, abnormal genitalia, retardation of growth, and sensorineural deafness) syndrome. In the current study, we identified eight RAF1 mutations in 18 of 119 patients with NS and related conditions without mutations in known genes. We summarized clinical manifestations in patients with RAF1 mutations as well as those in NS patients withPTPN11, SOS1, or KRAS mutations previously reported. Hypertrophic cardiomyopathy and short stature were found to be more frequently observed in patients with RAF1 mutations. Mutations in RAF1 were clustered in the conserved region 2 (CR2) domain, which carries an inhibitory phosphorylation site (serine at position 259; S259). Functional studies revealed that the RAF1 mutants located in the CR2 domain resulted in the decreased phosphorylation of S259, and that mutant RAF1 then dissociated from 14‐3‐3, leading to a partial ERK activation. Our results suggest that the dephosphorylation of S259 is the primary pathogenic mechanism in the activation of RAF1 mutants located in the CR2 domain as well as of downstream ERK. Hum Mutat 30:1–11, 2010. © 2010 Wiley‐Liss, Inc.

[1]  Koichi Tabayashi,et al.  Clinical manifestations in patients with SOS1 mutations range from Noonan syndrome to CFC syndrome , 2008, Journal of Human Genetics.

[2]  B. Gelb,et al.  Germline missense mutations affecting KRAS Isoform B are associated with a severe Noonan syndrome phenotype. , 2006, American journal of human genetics.

[3]  J. Ko,et al.  PTPN11, SOS1, KRAS, and RAF1 gene analysis, and genotype–phenotype correlation in Korean patients with Noonan syndrome , 2008, Journal of Human Genetics.

[4]  M. Digilio,et al.  Grouping of multiple-lentigines/LEOPARD and Noonan syndromes on the PTPN11 gene. , 2002, American journal of human genetics.

[5]  J. Hussain,et al.  CRAF autophosphorylation of serine 621 is required to prevent its proteasome-mediated degradation. , 2008, Molecular cell.

[6]  G. Thomas,et al.  Germline loss-of-function mutations in SPRED1 cause a neurofibromatosis 1–like phenotype , 2007, Nature Genetics.

[7]  R. Hennekam.  Costello syndrome: An overview , 2003, American journal of medical genetics. Part C, Seminars in medical genetics.

[8]  Kam Y. J. Zhang,et al.  Germline KRAS mutations cause Noonan syndrome , 2006, Nature Genetics.

[9]  G. Ferrero,et al.  Clinical and molecular characterization of 40 patients with Noonan syndrome. , 2008, European journal of medical genetics.

[10]  Judith G. Hall,et al.  Noonan syndrome: the changing phenotype. , 1985, American journal of medical genetics.

[11]  W. Kress,et al.  Genotype-phenotype correlations in Noonan syndrome. , 2004, The Journal of pediatrics.

[12]  Yukichi Tanaka,et al.  Germline mutations in HRAS proto-oncogene cause Costello syndrome , 2005, Nature Genetics.

[13]  Li Li,et al.  Germline gain-of-function mutations in SOS1 cause Noonan syndrome , 2007, Nature Genetics.

[14]  Wendy Schackwitz,et al.  Gain-of-function SOS1 mutations cause a distinctive form of Noonan syndrome , 2006, Nature Genetics.

[15]  Pablo Rodriguez-Viciana,et al.  Germline Mutations in Genes Within the MAPK Pathway Cause Cardio-facio-cutaneous Syndrome , 2006, Science.

[16]  F. Lo,et al.  Noonan syndrome caused by germline KRAS mutation in Taiwan: report of two patients and a review of the literature , 2009, European Journal of Pediatrics.

[17]  J. Opitz,et al.  New multiple congenital anomalies/mental retardation syndrome with cardio-facio-cutaneous involvement--the CFC syndrome. , 1986, American journal of medical genetics.

[18]  B. Neel,et al.  Stops along the RAS pathway in human genetic disease , 2006, Nature Medicine.

[19]  Walter Kolch,et al.  Phosphatase and Feedback Regulation of Raf-1 Signaling , 2007, Cell cycle.

[20]  M. Schimek,et al.  Two transforming C-RAF germ-line mutations identified in patients with therapy-related acute myeloid leukemia. , 2006, Cancer research.

[21]  H. Ropers,et al.  Spectrum of mutations in PTPN11 and genotype–phenotype correlation in 96 patients with Noonan syndrome and five patients with cardio-facio-cutaneous syndrome , 2003, European Journal of Human Genetics.

[22]  C. Pritchard,et al.  Raf proteins and cancer: B-Raf is identified as a mutational target. , 2003, Biochimica et biophysica acta.

[23]  Michael A. Patton,et al.  Mutations in PTPN11, encoding the protein tyrosine phosphatase SHP-2, cause Noonan syndrome , 2001, Nature Genetics.

[24]  R. Marais,et al.  14-3-3 Antagonizes Ras-Mediated Raf-1 Recruitment to the Plasma Membrane To Maintain Signaling Fidelity , 2002, Molecular and Cellular Biology.

[25]  D. Horn,et al.  SOS1 is the second most common Noonan gene but plays no major role in cardio-facio-cutaneous syndrome , 2007, Journal of Medical Genetics.

[26]  Bruce D Gelb,et al.  PTPN11 mutations in Noonan syndrome: molecular spectrum, genotype-phenotype correlation, and phenotypic heterogeneity. , 2002, American journal of human genetics.

[27]  J. Opitz,et al.  Noonan syndrome: a review. , 1985, American journal of medical genetics.

[28]  M. Patton,et al.  Genotypic and phenotypic characterization of Noonan syndrome: New data and review of the literature , 2005, American journal of medical genetics. Part A.

[29]  R. Hennekam,et al.  Germline KRAS and BRAF mutations in cardio-facio-cutaneous syndrome , 2006, Nature Genetics.

[30]  Michael J Ackerman,et al.  Gain-of-function RAF1 mutations cause Noonan and LEOPARD syndromes with hypertrophic cardiomyopathy , 2007, Nature Genetics.

[31]  Y. Matsubara,et al.  The RAS/MAPK syndromes: novel roles of the RAS pathway in human genetic disorders , 2008, Human mutation.

[32]  Stefan Mundlos,et al.  Expansion of the genotypic and phenotypic spectrum in patients with KRAS germline mutations , 2006, Journal of Medical Genetics.