Germline KRAS mutations cause Noonan syndrome Suzanne Schubbert 1 , Martin Zenker 2 , Sara L Rowe 1 , Silke Bo ¨ll 3 , Cornelia Klein 3 , Gideon Bollag 4 , Ineke van der Burgt 5 , Luciana Musante 6 , Vera Kalscheuer 6 , Lars-Erik Wehner 7 , Hoa Nguyen 4 , Brian West 4 , Kam Y J Zhang 4 , Erik Sistermans 5 , Anita Rauch 2 , Charlotte M Niemeyer 3 , Kevin Shannon 1,8 & Christian P Kratz 3 Noonan syndrome (MIM 163950) is characterized by short stature, facial dysmorphism and cardiac defects 1 . Heterozygous mutations in PTPN11, which encodes SHP-2, cause B50% of cases of Noonan syndrome 1,2 . The SHP-2 phosphatase relays signals from activated receptor complexes to downstream effectors, including Ras 3 . We discovered de novo germline KRAS mutations that introduce V14I, T58I or D153V amino acid substitutions in five individuals with Noonan syndrome and a P34R alteration in a individual with cardio-facio- cutaneous syndrome (MIM 115150), which has overlapping features with Noonan syndrome 1,4 . Recombinant V14I and T58I K-Ras proteins show defective intrinsic GTP hydrolysis and impaired responsiveness to GTPase activating proteins, render primary hematopoietic progenitors hypersensitive to growth factors and deregulate signal transduction in a cell lineage–specific manner. These studies establish germline KRAS mutations as a cause of human disease and infer that the constellation of developmental abnormalities seen in Noonan syndrome spectrum is, in large part, due to hyperactive Ras. Ras proteins regulate cell fates by cycling between active guanosine triphosphate (GTP)-bound and inactive guanosine diphosphate (GDP)-bound conformations (Ras-GTP and Ras-GDP) (reviewed in refs. 5,6). The competing activities of guanosine nucleotide exchange factors (GNEFs) and GTPase activating proteins (GAPs) regulate Ras- GTP levels. Activated growth factor receptors recruit signal relay proteins that stimulate GNEFs, which displace guanine nucleotides from Ras and permit passive binding to GTP. Ras-GTP interacts productively with Raf1, phosphatidylinositol 3-kinase, Ral-GDS and other effectors. The intrinsic Ras GTPase terminates signaling by hydrolyzing Ras-GTP to Ras-GDP. This slow ‘off’ reaction is greatly augmented by GAPs 5,6 . The somatic KRAS and NRAS mutations found in myeloid malignancies and other cancers, which introduce amino acid substitutions at codons 12, 13 and 61, encode proteins that accumulate in the GTP-bound conformation owing to defective intrinsic GTPase activity and resistance to GAPs 5–7 . Juvenile myelomonocytic leukemia (JMML) is a myeloproliferative disorder (MPD) characterized by leukocytosis with tissue infiltration and in vitro hypersensitivity of myeloid progenitors to granulocyte- macrophage colony stimulating factor (GM-CSF; reviewed in ref. 8). The incidence of JMML is increased 200- to 500-fold in children with neurofibromatosis type 1 (NF1), a familial cancer syndrome caused by inactivating mutations in the NF1 tumor suppressor. Neurofibromin, the NF1 gene product, is a GAP for Ras (reviewed in refs. 6,8). JMML cells from children with NF1 show biallelic NF1 inactivation and elevated Ras-GTP levels 9,10 . Approximately 25% of JMMLs demon- strate somatic NRAS or KRAS mutations, which are restricted to individuals who do not have NF1 (ref. 8). The discovery of hetero- zygous PTPN11 mutations as a major cause of Noonan syndrome 2 revealed a novel molecular lesion in JMML cases without RAS or NF1 mutations 11,12 . SHP-2, the PTPN11 gene product, is a non–receptor protein tyrosine phosphatase that has a positive role in transducing signals to Ras-GTP and other effectors (reviewed in ref. 3). The PTPN11 mutations identified in Noonan syndrome are gain-of- function alleles that encode SHP-2 proteins that variably deregulate phosphatase activity, the affinity of the SH2 domains for phosphotyr- osyl ligands and/or substrate specificities 1,13 . Infants with Noonan syndrome show a spectrum of hematologic abnormalities and are predisposed to JMML 8 . Specific germline PTPN11 mutations are associated with JMML, and previously unknown somatic mutations were unexpectedly identified in B35% of de novo JMMLs 11,12 . Leukemia-associated PTPN11 alleles encode strong gain-of-function SHP-2 proteins that deregulate Ras signaling and cause aberrant myeloid proliferation in vivo and in vitro 14–16 . Here we show that germline KRAS mutations are a cause of both Noonan syndrome and cardio-facio-cutaneous (CFC) syndrome and describe previously unknown biochemical and functional properties of two mutant K-Ras proteins. We found several novel KRAS mutations in individuals with Noonan and CFC syndromes. A 3-month-old female with Noonan syndrome and a severe clinical phenotype presented with a JMML-like MPD (individual 1, Table 1). We did not identify a PTPN11 mutation Received 18 November 2005; accepted 13 January 2006; published online 12 February 2006; doi:10.1038/ng1748 1 Department of Pediatrics, University of California, 513 Parnassus Avenue, San Francisco, California 94143, USA. 2 Institute of Human Genetics, University of Erlangen-Nuremberg, 91054 Erlangen, Germany. 3 Division of Pediatric Hematology and Oncology, Department of Pediatrics and Adolescent Medicine, University of Freiburg, Mathildenstrasse 1, 79106 Freiburg, Germany. 4 Plexxikon, Inc., 91 Bolivar Dr., Berkeley, California 94710, USA. 5 Department of Human Genetics, University Medical Center Nijmegen, 6500 HB Nijmegen, The Netherlands. 6 Max Planck Institute of Molecular Genetics, 14195 Berlin, Germany. 7 Institute of Human Genetics, University of Go ¨ttingen, 37075 Germany. 8 Comprehensive Cancer Center, University of California, San Francisco, California 94115, USA. Correspondence should be addressed to C.P.K. (christian.kratz@uniklinik-freiburg.de ) or K.S. (shannonk@peds.ucsf.edu). NATURE GENETICS VOLUME 38 [ NUMBER 3 [ MARCH 2006 331 LETTERS © 2006 Nature Publishing Group http://www.nature.com/naturegenetics