Novel Germ Line Mutations in the KRAS2 Gene Cause Noonan Syndrome and Deregulate Hematopoietic Cell Growth.

Abstract

RAS genes encode 21-kDa signal switch molecules that regulate cell fates by cycling between inactive GDP-bound (Ras-GDP) and active GTP-bound (Ras-GTP) conformations. Cancer-associated somatic mutations lead to substitutions at codons G12, G13, or Q61 that impair the intrinsic Ras GTPase and confer resistance to GTPase-activating proteins (GAPs). Noonan syndrome (NS) is an autosomal dominant developmental disorder characterized by short stature, facial dysmorphism, and variable congenital cardiac defects. Infants with NS are predisposed to hematologic disorders including juvenile myelomonocytic leukemia (JMML). In ~50% of patients, NS is caused by mutations in the PTPN11 gene. PTPN11 encodes SHP-2, a non receptor protein-tyrosine phosphatase (PTPase) that relays signals from growth factor receptors to Ras and other signaling molecules. Interestingly, somatic PTPN11 mutations that cause aberrant PTPase activity are found in ~35% of JMML bone marrows from patients without NS and occur at lower frequency in other hematologic malignancies (Tartaglia and Niemeyer et alia, Nat Genet 34, 148, 2003; Loh et alia, Blood 103, 2325, 2004). We identified a patient with NS and JMML who did not have a PTPN11 mutation. Instead, molecular analysis revealed a novel de novo germ line mutation in KRAS2, c.173C>T, which introduces a T58I substitution within the K-Ras protein. We then screened DNA samples from 124 NS patients that did not show PTPN11 mutations for alterations in HRAS, NRAS, and KRAS2. These studies uncovered a second novel de novo KRAS2 mutation (c.40G>A) in 3 unrelated cases, which predicts a V14I substitution in the phosphate-binding loop of the protein. We expressed the mutant K-Ras T58I and V14I proteins in COS-7 cells and measured Ras-GTP levels and the activation of Ras effectors. Whereas K-Ras V14I-accumulated in the GTP-bound conformation in both basal and starved conditions and was associated with hyper-phosphorylation of MEK, the T58I mutant protein did not dramatically alter Ras-GTP levels in this system. By contrast, expressing T58I and V14I K-Ras in primary murine hematopoietic cells induced a hypersensitive pattern of granuocyte-macrophage colony forming unit (CFU-GM) growth in response to granulocyte-macrophage colony stimulating factor (GM-CSF), which was more pronounced for the T58I mutant protein. T58I-expressing cells also exhibited enhanced erythroid progenitor colony growth. These studies establish germ line KRAS2 mutations as a cause of NS. While the more severe effects of T58I K-Ras on hematopoietic growth are consistent with the occurrence of JMML in the proband, the stronger biochemical phenotype of V14I K-Ras in COS-7 cells suggests that cell context strongly modulates the activation state of these mutant proteins. Ongoing in vitro experiments in which we are measuring the intrinsic rates of GTP hydrolysis of recombinant T58I and V14I K-Ras and comparing their sensitivities to GAPs will further elucidate the functional consequences of these novel disease-associated mutations.