Abstract

Germline mutations in PTPN11, the gene encoding the protein tyrosine phosphatase SHP-2, cause Noonan syndrome (NS) and the clinically related LEOPARD syndrome (LS), whereas somatic mutations in the same gene contribute to leukemogenesis. On the basis of our previously gathered genetic and biochemical data, we proposed a model that splits NS- and leukemia-associated PTPN11 mutations into two major classes of activating lesions with differential perturbing effects on development and hematopoiesis. To test this model, we investigated further the diversity of germline and somatic PTPN11 mutations, delineated the association of those mutations with disease, characterized biochemically a panel of mutant SHP-2 proteins recurring in NS, LS, and leukemia, and performed molecular dynamics simulations to determine the structural effects of selected mutations. Our results document a strict correlation between the identity of the lesion and disease and demonstrate that NS-causative mutations have less potency for promoting SHP-2 gain of function than do leukemia-associated ones. Furthermore, we show that the recurrent LS-causing Y279C and T468M amino acid substitutions engender loss of SHP-2 catalytic activity, identifying a previously unrecognized behavior for this class of missense PTPN11 mutations. Germline mutations in PTPN11, the gene encoding the protein tyrosine phosphatase SHP-2, cause Noonan syndrome (NS) and the clinically related LEOPARD syndrome (LS), whereas somatic mutations in the same gene contribute to leukemogenesis. On the basis of our previously gathered genetic and biochemical data, we proposed a model that splits NS- and leukemia-associated PTPN11 mutations into two major classes of activating lesions with differential perturbing effects on development and hematopoiesis. To test this model, we investigated further the diversity of germline and somatic PTPN11 mutations, delineated the association of those mutations with disease, characterized biochemically a panel of mutant SHP-2 proteins recurring in NS, LS, and leukemia, and performed molecular dynamics simulations to determine the structural effects of selected mutations. Our results document a strict correlation between the identity of the lesion and disease and demonstrate that NS-causative mutations have less potency for promoting SHP-2 gain of function than do leukemia-associated ones. Furthermore, we show that the recurrent LS-causing Y279C and T468M amino acid substitutions engender loss of SHP-2 catalytic activity, identifying a previously unrecognized behavior for this class of missense PTPN11 mutations. The PTPN11 gene (MIM 176876) encodes SHP-2, a cytoplasmic protein tyrosine phosphatase (PTP) characterized by two tandemly arranged Src homology 2 (SH2) domains at the N-terminus, a catalytic domain, and a C-terminal tail containing a proline-rich region and two tyrosyl residues that undergo reversible phosphorylation (Neel et al. Neel et al., 2003Neel BG Gu H Pao L The ‘Shp'ing news: SH2 domain-containing tyrosine phosphatases in cell signaling.Trends Biochem Sci. 2003; 28: 284-293Abstract Full Text Full Text PDF PubMed Scopus (957) Google Scholar). SHP-2 is a critical component of signal transduction for several growth factor–, hormone-, and cytokine-signaling pathways controlling developmental processes (Tang et al. 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Consistent with the crucial role of SHP-2 in development, germline missense mutations in PTPN11 cause Noonan syndrome (NS [MIM 163950]) (Tartaglia et al. Tartaglia et al., 2001Tartaglia M Mehler EL Goldberg R Zampino G Brunner HG Kremer H van der Burgt I Crosby AH Ion A Jeffery S Kalidas K Patton MA Kucherlapati RS Gelb BD Mutations in PTPN11, encoding the protein tyrosine phosphatase SHP-2, cause Noonan syndrome.Nat Genet. 2001; 29: 465-468Crossref PubMed Scopus (1327) Google Scholar), a developmental disorder characterized by short stature, facial dysmorphisms, skeletal and hematological defects, and cardiovascular abnormalities (Noonan Noonan, 1968Noonan JA Hypertelorism with Turner phenotype: a new syndrome with associated congenital heart disease.Am J Dis Child. 1968; 116: 373-380Crossref PubMed Scopus (453) Google Scholar; Allanson Allanson, 1987Allanson JE Noonan syndrome.J Med Genet. 1987; 24: 9-13Crossref PubMed Scopus (316) Google Scholar). Accumulating genetic, modeling, and biochemical data indicate that PTPN11 mutations account for ∼50% of affected individuals and promote SHP-2 gain of function (Tartaglia and Gelb Tartaglia and Gelb, 2005bTartaglia M Gelb BD Noonan syndrome and related disorders: genetics and pathogenesis.Annu Rev Genomics Hum Genet. 2005; 6: 45-68Crossref PubMed Scopus (275) Google Scholar). Germline lesions in PTPN11 have also been identified in the clinically related LEOPARD syndrome (LS [MIM 151100]) (Digilio et al. Digilio et al., 2002Digilio MC Conti E Sarkozy A Mingarelli R Dottorini T Marino B Pizzuti A Dallapiccola B Grouping of multiple-lentigines/LEOPARD and Noonan syndromes on the PTPN11 gene.Am J Hum Genet. 2002; 71: 389-394Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar; Legius et al. Legius et al., 2002Legius E Schrander-Stumpel C Schollen E Pulles-Heintzberger C Gewillig M Fryns JP PTPN11 mutations in LEOPARD syndrome.J Med Genet. 2002; 39: 571-574Crossref PubMed Scopus (203) Google Scholar), with two amino acid substitutions (Y279C and T468M) occurring in the vast majority of subjects (Tartaglia and Gelb Tartaglia and Gelb, 2005bTartaglia M Gelb BD Noonan syndrome and related disorders: genetics and pathogenesis.Annu Rev Genomics Hum Genet. 2005; 6: 45-68Crossref PubMed Scopus (275) Google Scholar). Before the study described in this article, there was no information regarding the consequences of LS-causing PTPN11 mutations on the function of SHP-2. Children with NS are prone to develop malignancies—most commonly, juvenile myelomonocytic leukemia (JMML [MIM 607785]), a myeloproliferative disorder of childhood (Emanuel Emanuel, 2004Emanuel PD Juvenile myelomonocytic leukemia.Curr Hematol Rep. 2004; 3: 203-209PubMed Google Scholar). Since excessive signaling through the RAS/MAPK pathway has been implicated in a wide variety of cancers and because SHP-2 is a positive modulator of RAS signaling (Neel et al. Neel et al., 2003Neel BG Gu H Pao L The ‘Shp'ing news: SH2 domain-containing tyrosine phosphatases in cell signaling.Trends Biochem Sci. 2003; 28: 284-293Abstract Full Text Full Text PDF PubMed Scopus (957) Google Scholar; Tartaglia et al. Tartaglia et al., 2004bTartaglia M Niemeyer CM Shannon KM Loh ML SHP-2 and myeloid malignancies.Curr Opin Hematol. 2004; 11: 44-50Crossref PubMed Scopus (91) Google Scholar), it was consistent that germline PTPN11 mutations were present in children with NS and JMML (NS/JMML) (Tartaglia et al. Tartaglia et al., 2003Tartaglia M Niemeyer CM Fragale A Song X Buechner J Jung A Hahlen K Hasle H Licht JD Gelb BD Somatic mutations in PTPN11 in juvenile myelomonocytic leukemia, myelodysplastic syndromes and acute myeloid leukemia.Nat Genet. 2003; 34: 148-150Crossref PubMed Scopus (821) Google Scholar). Moreover, somatic missense mutations in PTPN11 have been documented with variable prevalence in a heterogeneous group of hematologic malignancies (Tartaglia et al. Tartaglia et al., 2003Tartaglia M Niemeyer CM Fragale A Song X Buechner J Jung A Hahlen K Hasle H Licht JD Gelb BD Somatic mutations in PTPN11 in juvenile myelomonocytic leukemia, myelodysplastic syndromes and acute myeloid leukemia.Nat Genet. 2003; 34: 148-150Crossref PubMed Scopus (821) Google Scholar, Tartaglia et al., 2004aTartaglia M Martinelli S Cazzaniga G Cordeddu V Iavarone I Spinelli M Palmi C Carta C Pession A Arico M Masera G Basso G Sorcini M Gelb BD Biondi A Genetic evidence for lineage- and differentiation stage-related contribution of somatic PTPN11 mutations to leukemogenesis in childhood acute leukemia.Blood. 2004; 104: 307-313Crossref PubMed Scopus (233) Google Scholar, Tartaglia et al., 2005Tartaglia M Martinelli S Iavarone I Cazzaniga G Spinelli M Giarin E Petrangeli V Carta C Masetti R Aricò M Locatelli F Basso G Sorcini M Pession A Biondi A Somatic PTPN11 mutations in childhood acute myeloid leukaemia.Br J Haematol. 2005; 129: 333-339Crossref PubMed Scopus (58) Google Scholar; Loh et al. 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Bentires-Alj et al., 2004Bentires-Alj M Paez JG David FS Keilhack H Halmos B Naoki K Maris JM Richardson A Bardelli A Sugarbaker DJ Richards WG Du J Girard L Minna JD Loh ML Fisher DE Velculescu VE Vogelstein B Meyerson M Sellers WR Neel BG Activating mutations of the noonan syndrome-associated SHP2/PTPN11 gene in human solid tumors and adult acute myelogenous leukemia.Cancer Res. 2004; 64: 8816-8820Crossref PubMed Scopus (417) Google Scholar). Similar to what was observed for NS, genetic, biochemical, and functional data support the view that acquired lesions contributing to leukemia promote SHP-2 gain of function (Tartaglia et al. Tartaglia et al., 2003Tartaglia M Niemeyer CM Fragale A Song X Buechner J Jung A Hahlen K Hasle H Licht JD Gelb BD Somatic mutations in PTPN11 in juvenile myelomonocytic leukemia, myelodysplastic syndromes and acute myeloid leukemia.Nat Genet. 2003; 34: 148-150Crossref PubMed Scopus (821) Google Scholar; Chan et al. Chan et al., 2005Chan RJ Leedy MB Munugalavadla V Voorhorst CS Li Y Yu M Kapur R Human somatic PTPN11 mutations induce hematopoietic cell hypersensitivity to granulocyte-macrophage colony stimulating factor.Blood. 2005; 105: 3737-3742Crossref PubMed Scopus (133) Google Scholar; Keilhack et al. Keilhack et al., 2005Keilhack H David FS McGregor M Cantley LC Neel BG Diverse biochemical properties of Shp2 mutants: implications for disease phenotypes.J Biol Chem. 2005; 280: 30984-30993Crossref PubMed Scopus (234) Google Scholar; Mohi et al. Mohi et al., 2005Mohi MG Williams IR Dearolf CR Chan G Kutok JL Cohen S Morgan K Boulton C Shigematsu H Keilhack H Akashi K Gilliland DG Neel BG Prognostic, therapeutic, and mechanistic implications of a mouse model of leukemia evoked by Shp2 (PTPN11) mutations.Cancer Cell. 2005; 7: 179-191Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar; Shubbert et al. Shubbert et al., 2005Shubbert S Lieuw K Rowe SL Lee CM Li X Loh ML Clapp DW Shannon KM Functional analysis of leukemia-associated PTPN11 mutations in primary hematopoietic cells.Blood. 2005; 106: 311-317Crossref PubMed Scopus (127) Google Scholar), providing the first evidence of a mutated PTP acting as an oncoprotein in cancer. Our initial studies indicated that mutations identified in NS (germline origin) and leukemia (somatic origin) rarely overlap (Tartaglia et al. Tartaglia et al., 2003Tartaglia M Niemeyer CM Fragale A Song X Buechner J Jung A Hahlen K Hasle H Licht JD Gelb BD Somatic mutations in PTPN11 in juvenile myelomonocytic leukemia, myelodysplastic syndromes and acute myeloid leukemia.Nat Genet. 2003; 34: 148-150Crossref PubMed Scopus (821) Google Scholar). On the basis of those results, we proposed a model requiring at least two classes of activating mutations of PTPN11 with different roles in development and leukemogenesis. A third class, as-yet functionally uncharacterized, would include a few mutations, including the recurrent LS-causing lesions, which affect residues clustering within or in proximity to the active site of the protein and are predicted to impair catalysis. To test this model, we investigated the diversity and structural consequences of germline and somatic PTPN11 mutations, delineated their association with disease, and characterized biochemically and structurally a panel of amino acid changes recurring in NS, LS, and leukemia that affect distinct functional domains of the protein. Two large cohorts containing subjects with NS or LS who might have germline PTPN11 mutations (N=425) and those with hematologic malignancies (N=303) who might have somatic ones were included in the study. For the NS/LS cohort, there were two groups. For one (N=116), subjects were enrolled in research protocols. Clinical features for the majority of these individuals satisfied the diagnostic criteria reported by van der Burgt et al. (van der Burgt et al., 1994van der Burgt I Berends E Lommen E van Beersum S Hamel B Mariman E Clinical and molecular studies in a large Dutch family with Noonan syndrome.Am J Med Genet. 1994; 53: 187-191Crossref PubMed Scopus (218) Google Scholar) or Voron et al. (Voron et al., 1976Voron DA Hatfield HH Kalkhoff MD Multiple lentigines syndrome: case report and review of the literature.Am J Med. 1976; 60: 447-456Abstract Full Text PDF PubMed Scopus (174) Google Scholar), but individuals who lacked sufficient features to receive a definitive diagnosis were also included in the study. The second group (N=309) comprised mutation data from anonymous samples from individuals with phenotypes suggestive of NS or LS and prompting commercial DNA diagnostic testing at GeneDx (Gaithersburg, MD); no phenotype data were available for these persons. In the leukemia cohort, which included 63 children with JMML, 147 adult subjects with acute myeloid leukemia (AML), and 93 adult subjects with acute lymphoblastic leukemia (ALL), diagnosis was established according to standard morphologic, cytochemical, and immunological criteria (Bene et al. Bene et al., 1995Bene MC Castoldi G Knapp W Ludwig WD Matutes E Orfao A van't Veer MB Proposals for the immunological classification of acute leukemias.Leukemia. 1995; 9: 1783-1786PubMed Google Scholar; Emanuel Emanuel, 2004Emanuel PD Juvenile myelomonocytic leukemia.Curr Hematol Rep. 2004; 3: 203-209PubMed Google Scholar) and was centrally reviewed (R.F., C.S., and P.D.E.). All nonanonymous samples were collected under institutional review board–approved protocols and with informed consent. Genomic DNA was isolated from peripheral blood lymphocytes (NS/LS) or from bone marrow aspirates (leukemias) obtained at the time of diagnosis, before therapy, as well as during follow-up. PTPN11 mutational screening was performed by denaturing high-performance liquid chromatography analysis with the use of the Wave 2100 System (Transgenomics) at column temperatures recommended by the Navigator version 1.5.4.23 software (Transgenomics), as described elsewhere (Tartaglia et al. Tartaglia et al., 2002Tartaglia M Kalidas K Shaw A Song X Musat DL van der Burgt I Brunner HG Bertola DR Crosby A Ion A Kucherlapati RS Jeffery S Patton MA Gelb BD PTPN11 mutations in Noonan syndrome: molecular spectrum, genotype-phenotype correlation, and phenotypic heterogeneity.Am J Hum Genet. 2002; 70: 1555-1563Abstract Full Text Full Text PDF PubMed Scopus (602) Google Scholar, Tartaglia et al., 2004aTartaglia M Martinelli S Cazzaniga G Cordeddu V Iavarone I Spinelli M Palmi C Carta C Pession A Arico M Masera G Basso G Sorcini M Gelb BD Biondi A Genetic evidence for lineage- and differentiation stage-related contribution of somatic PTPN11 mutations to leukemogenesis in childhood acute leukemia.Blood. 2004; 104: 307-313Crossref PubMed Scopus (233) Google Scholar). Amplimers having abnormal denaturing profiles were purified (Microcon PCR [Millipore]) and were sequenced bidirectionally with the use of the ABI BigDye Terminator Sequencing Kit v.1.1 (Applied Biosystems) and an ABI Prism 310 Genetic Analyzer (Applied Biosystems). Length and dinucleotide mutations were determined by cloning purified PCR products in a pCR 2.1 TOPO vector (Invitrogen) and sequencing purified clones (Plasmid Mini Kit, Qiagen). The entire PTPN11 coding sequence was screened in the NS/LS cohort. On the basis of our previously generated data, exons 1, 2, 3, 4, 7, 8, 12, 13, and 14—encompassing all the PTPN11 lesions identified in NS, LS, and leukemias—were screened in the leukemia cohort. Germline and somatic PTPN11 mutations reported in studies published (or available online as preprint versions) before September 2005 were collected. In the resulting database, germline mutations referred to lesions identified in subjects with a developmental disorder (NS, LS, or a related condition), whereas somatic mutations referred to defects identified in subjects with isolated hematologic malignancies, preleukemic disorders, or solid tumors. The database and relative references are reported in table 1.Table 1SHP-2 Affected Residues and Amino Acid Changes Germinally Transmitted or Somatically AcquiredAmino Acid (Mutation Group) and ChangeGermline Origin (N=573)aReferences for germline mutations: Bentires-Alj et al. 2004; Bertola et al. 2004, 2005; Binder et al. 2005; Conti et al. 2003; Digilio et al. 2002, 2004; Ferreira et al. 2005; Jafarov et al. 2005; Jongmans et al. 2005; Kalidas et al. 2005; Keren et al. 2004; Kondoh et al. 2003; Kosaki et al. 2002; Kratz et al. 2005; Lee et al. 2005a, 2005b; Legius et al. 2002; Loh et al. 2004b; Maheshwari et al. 2002; Matsubara et al. 2005; Musante et al. 2003; Niihori et al. 2005; Sarkozy et al. 2003, 2004a, 2004b; Schluter et al. 2005; Schollen et al. 2003a, 2003b; Takahashi et al. 2005; Tartaglia et al. 2001, 2002, 2003; Yoshida et al. 2004a, 2004b, 2004c; Weismann et al. 2005; Zenker et al. 2004; present article.Somatic Origin (N=256)bReferences for somatic mutations: Bentires-Alj et al. 2004; Goemans et al. 2005; Hasegawa et al. 2005; Hugues et al. 2005; Johan et al. 2004; Kratz et al. 2005; Loh et al. 2004a, 2004b, 2005; Niihori et al. 2005; Nomdedeu et al. 2005; Shimada et al. 2004; Smith et al. 2005; Tartaglia et al. 2003, 2004a, 2005; present article.T2 (I): I3…T42 (V): A11…L43 (V): F1cIdentified in a subject with isolated congenital heart disease.…T52 (V): S…2H53 (V): Q…1N58 (I): H1… D6… K3… Y…1G60 (I): S1… A131 R…3 V…15 Deletion1…D61 (I): N163 G231 H…1 Y…19 V…15 Deletion2dIncluding one case with G60V+delD61.…Y62 (I): N1… D17… C1…Y63 (I): C441E69 (I): Q2… V…1 G…1 K…10F71 (I): L14 I1… K…2A72 (I): S18… G16… T…20 V…23 D…1T73 (I): I216E76 (I): D13… K…47 Q…6 V…6 G…23 A…5 M…1L77 (I): V…1Q79 (I): P2… R30…D106 (VI): A10…E110 (VI): A2…R138 (V): Q…1E139 (V): D161eDetailed clinical information was not available in this case (see Loh et al. 2004b).Q256 (I): R6… K1…G268 (IV): S1… C1…Y279 (II): S2… C33…I282 (II): V12…F285 (IV): L5… S7… C1…R289 (IV): G…1N308 (IV): D92… S21… T3…I309 (IV): V2…T411 (…): M1…A461 (II): T2…G464 (II): A3…T468 (IV): M29…P491 (IV): S41 L42R498 (IV): W2… L1…R501 (IV): K1…S502 (III): T91 A21 L22 P…3G503 (III): R141 L…1 E…2 V…6 A…10M504 (IV): V21…Q506 (II): P7…T507 (II): K…2Q510 (II): K…1 E3… P3… R1…L560 (…): F1…Note.—Database updated to August 2005.a References for germline mutations: Bentires-Alj et al. 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