Heterozygous Germline Mutations in the CBL Tumor-Suppressor Gene Cause a Noonan Syndrome-like Phenotype
2010; Elsevier BV; Volume: 87; Issue: 2 Linguagem: Inglês
10.1016/j.ajhg.2010.06.015
ISSN1537-6605
AutoresSimone Martinelli, Alessandro De Luca, Emilia Stellacci, Cesare Rossi, Saula Checquolo, Francesca Romana Lepri, Viviana Caputo, Marianna Silvano, Francesco Buscherini, Federica Consoli, Grazia Ferrara, M. Cristina Digilio, Maria Luigia Cavaliere, Johanna M. van Hagen, Giuseppe Zampino, Ineke van der Burgt, Giovanni Battista Ferrero, Laura Mazzanti, Isabella Screpanti, Helger G. Yntema, Willy M. Nillesen, Ravi Savarirayan, Martin Zenker, Bruno Dallapiccola, Bruce D. Gelb, Marco Tartaglia,
Tópico(s)Peptidase Inhibition and Analysis
ResumoRAS signaling plays a key role in controlling appropriate cell responses to extracellular stimuli and participates in early and late developmental processes. Although enhanced flow through this pathway has been established as a major contributor to oncogenesis, recent discoveries have revealed that aberrant RAS activation causes a group of clinically related developmental disorders characterized by facial dysmorphism, a wide spectrum of cardiac disease, reduced growth, variable cognitive deficits, ectodermal and musculoskeletal anomalies, and increased risk for certain malignancies. Here, we report that heterozygous germline mutations in CBL, a tumor-suppressor gene that is mutated in myeloid malignancies and encodes a multivalent adaptor protein with E3 ubiquitin ligase activity, can underlie a phenotype with clinical features fitting or partially overlapping Noonan syndrome (NS), the most common condition of this disease family. Independent CBL mutations were identified in two sporadic cases and two families from among 365 unrelated subjects who had NS or suggestive features and were negative for mutations in previously identified disease genes. Phenotypic heterogeneity and variable expressivity were documented. Mutations were missense changes altering evolutionarily conserved residues located in the RING finger domain or the linker connecting this domain to the N-terminal tyrosine kinase binding domain, a known mutational hot spot in myeloid malignancies. Mutations were shown to affect CBL-mediated receptor ubiquitylation and dysregulate signal flow through RAS. These findings document that germline mutations in CBL alter development to cause a clinically variable condition that resembles NS and that possibly predisposes to malignancies. RAS signaling plays a key role in controlling appropriate cell responses to extracellular stimuli and participates in early and late developmental processes. Although enhanced flow through this pathway has been established as a major contributor to oncogenesis, recent discoveries have revealed that aberrant RAS activation causes a group of clinically related developmental disorders characterized by facial dysmorphism, a wide spectrum of cardiac disease, reduced growth, variable cognitive deficits, ectodermal and musculoskeletal anomalies, and increased risk for certain malignancies. Here, we report that heterozygous germline mutations in CBL, a tumor-suppressor gene that is mutated in myeloid malignancies and encodes a multivalent adaptor protein with E3 ubiquitin ligase activity, can underlie a phenotype with clinical features fitting or partially overlapping Noonan syndrome (NS), the most common condition of this disease family. Independent CBL mutations were identified in two sporadic cases and two families from among 365 unrelated subjects who had NS or suggestive features and were negative for mutations in previously identified disease genes. Phenotypic heterogeneity and variable expressivity were documented. Mutations were missense changes altering evolutionarily conserved residues located in the RING finger domain or the linker connecting this domain to the N-terminal tyrosine kinase binding domain, a known mutational hot spot in myeloid malignancies. Mutations were shown to affect CBL-mediated receptor ubiquitylation and dysregulate signal flow through RAS. These findings document that germline mutations in CBL alter development to cause a clinically variable condition that resembles NS and that possibly predisposes to malignancies. RAS signaling plays a crucial role in cell proliferation, migration, survival, and cell fate determination and differentiation and participates in early and late developmental processes, including organogenesis, morphology determination, and growth.1Karnoub A.E. Weinberg R.A. Ras oncogenes: Split personalities.Nat. Rev. Mol. Cell Biol. 2008; 9: 517-531Crossref PubMed Scopus (1126) Google Scholar Because of its nodal role in signal transduction, signal traffic through RAS is tightly controlled, and enhanced flow through it contributes to oncogenesis.2Rajalingam K. Schreck R. Rapp U.R. Albert S. Ras oncogenes and their downstream targets.Biochim. Biophys. Acta. 2007; 1773: 1177-1195Crossref PubMed Scopus (343) Google Scholar, 3Schubbert S. Shannon K. Bollag G. Hyperactive Ras in developmental disorders and cancer.Nat. Rev. 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Genet. 2006; 38: 331-336Crossref PubMed Scopus (600) Google Scholar NRAS (MIM 164790),14Cirstea I.C. Kutsche K. Dvorsky R. Gremer L. Carta C. Horn D. Roberts A.E. Lepri F. Merbitz-Zahradnik T. König R. et al.A restricted spectrum of NRAS mutations causes Noonan syndrome.Nat. Genet. 2010; 42: 27-29Crossref PubMed Scopus (239) Google Scholar RAF1 (MIM 164760),15Pandit B. Sarkozy A. Pennacchio L.A. Carta C. Oishi K. Martinelli S. Pogna E.A. Schackwitz W. Ustaszewska A. Landstrom A. et al.Gain-of-function RAF1 mutations cause Noonan and LEOPARD syndromes with hypertrophic cardiomyopathy.Nat. Genet. 2007; 39: 1007-1012Crossref PubMed Scopus (540) Google Scholar, 16Razzaque M.A. Nishizawa T. Komoike Y. Yagi H. Furutani M. Amo R. Kamisago M. Momma K. Katayama H. Nakagawa M. et al.Germline gain-of-function mutations in RAF1 cause Noonan syndrome.Nat. Genet. 2007; 39: 1013-1017Crossref PubMed Scopus (389) Google Scholar BRAF (MIM 164757),17Sarkozy A. Carta C. Moretti S. Zampino G. Digilio M.C. 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Genet. 2007; 44: 763-771Crossref PubMed Scopus (206) Google Scholar (MIM 176872) genes have been documented to account for 70%–75% of affected individuals. These genes encode transducers that positively contribute to RAS-MAPK signaling. Although some (i.e., NRAS, KRAS, and BRAF) had previously been recognized as proto-oncogenes, the finding of the causal link to NS provided the basis for the discovery of PTPN11's involvement in leukemogenesis.20Tartaglia M. Niemeyer C.M. Fragale A. Song X. Buechner J. Jung A. Hählen K. Hasle H. Licht J.D. Gelb B.D. Somatic mutations in PTPN11 in juvenile myelomonocytic leukemia, myelodysplastic syndromes and acute myeloid leukemia.Nat. Genet. 2003; 34: 148-150Crossref PubMed Scopus (824) Google Scholar, 21Tartaglia M. Martinelli S. Cazzaniga G. Cordeddu V. Iavarone I. Spinelli M. Palmi C. Carta C. Pession A. Aricò M. et al.Genetic evidence for lineage-related 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 Here, we provide evidence that germline mutations in the Cas-Br-M (murine) ecotropic retroviral transforming sequence (CBL [MIM 165360]) gene, which encodes a multivalent adaptor protein with an established role in myeloid malignancies, can underlie a clinically variable condition with features fitting or partially overlapping NS. CBL is a member of a small family of E3 ubiquitin ligases that negatively regulate intracellular signaling downstream of receptor tyrosine kinases (RTKs), but it also contributes to signal traffic through its adaptor function.22Dikic I. Schmidt M.H. Malfunctions within the Cbl interactome uncouple receptor tyrosine kinases from destructive transport.Eur. J. Cell Biol. 2007; 86: 505-512Crossref PubMed Scopus (28) Google Scholar CBL is ubiquitously expressed and characterized by an N-terminal tyrosine kinase-binding (TKB) domain that is involved in protein-protein interaction and is connected by a short linker to a zinc-binding RING-finger domain mediating the E3 ubiquitin ligase activity (Figure 1A ). The C-terminal portion of the protein includes an extensive proline-rich region containing a number of putative SH3-binding motifs, and this region is followed by multiple SH2-binding tyrosine phosphorylation sites and a ubiquitin-associated (UBA) domain overlapping with a leucine zipper (LZ) motif involved in ubiquitin binding and protein dimerization, respectively.23Swaminathan G. Tsygankov A.Y. The Cbl family proteins: ring leaders in regulation of cell signaling.J. Cell. Physiol. 2006; 209: 21-43Crossref PubMed Scopus (235) Google Scholar CBL mediates the conjugation of ubiquitin to activated RTKs; this conjugation is required for receptor internalization, endocytic sorting, and switching off signaling via receptor degradation or recycling.24Schmidt M.H. Dikic I. The Cbl interactome and its functions.Nat. Rev. Mol. Cell Biol. 2005; 6: 907-918Crossref PubMed Scopus (327) Google Scholar Somatically acquired, mostly homozygous CBL mutations have recently been found to occur with variable prevalence in myeloproliferative disorders, including JMML, and myeloid leukemias.25Caligiuri M.A. Briesewitz R. Yu J. Wang L. Wei M. Arnoczky K.J. Marburger T.B. Wen J. Perrotti D. Bloomfield C.D. Whitman S.P. Novel c-CBL and CBL-b ubiquitin ligase mutations in human acute myeloid leukemia.Blood. 2007; 110: 1022-1024Crossref PubMed Scopus (126) Google Scholar, 26Sargin B. Choudhary C. Crosetto N. Schmidt M.H. Grundler R. Rensinghoff M. Thiessen C. Tickenbrock L. Schwäble J. Brandts C. et al.Flt3-dependent transformation by inactivating c-Cbl mutations in AML.Blood. 2007; 110: 1004-1012Crossref PubMed Scopus (162) Google Scholar, 27Loh M.L. Sakai D.S. Flotho C. Kang M. Fliegauf M. Archambeault S. Mullighan C.G. Chen L. Bergstraesser E. Bueso-Ramos C.E. et al.Mutations in CBL occur frequently in juvenile myelomonocytic leukemia.Blood. 2009; 114: 1859-1863Crossref PubMed Scopus (223) Google Scholar, 28Makishima H. Cazzolli H. Szpurka H. Dunbar A. Tiu R. Huh J. Muramatsu H. O'Keefe C. Hsi E. Paquette R.L. et al.Mutations of e3 ubiquitin ligase cbl family members constitute a novel common pathogenic lesion in myeloid malignancies.J. Clin. Oncol. 2009; 27: 6109-6116Crossref PubMed Scopus (173) Google Scholar, 29Muramatsu H. Makishima H. Jankowska A.M. Cazzolli H. O'Keefe C. Yoshida N. Xu Y. Nishio N. Hama A. Yagasaki H. et al.Mutations of an E3 ubiquitin ligase c-Cbl but not TET2 mutations are pathogenic in juvenile myelomonocytic leukemia.Blood. 2010; 115: 1969-1975Crossref PubMed Scopus (83) Google Scholar, 30Reindl C. Quentmeier H. Petropoulos K. Greif P.A. Benthaus T. Argiropoulos B. Mellert G. Vempati S. Duyster J. Buske C. et al.CBL exon 8/9 mutants activate the FLT3 pathway and cluster in core binding factor/11q deletion acute myeloid leukemia/myelodysplastic syndrome subtypes.Clin. Cancer Res. 2009; 15: 2238-2247Crossref PubMed Scopus (93) Google Scholar, 31Sanada M. Suzuki T. Shih L.Y. Otsu M. Kato M. Yamazaki S. Tamura A. Honda H. Sakata-Yanagimoto M. Kumano K. et al.Gain-of-function of mutated C-CBL tumour suppressor in myeloid neoplasms.Nature. 2009; 460: 904-908Crossref PubMed Scopus (333) Google Scholar, 32Grand F.H. Hidalgo-Curtis C.E. Ernst T. Zoi K. Zoi C. McGuire C. Kreil S. Jones A. Score J. Metzgeroth G. et al.Frequent CBL mutations associated with 11q acquired uniparental disomy in myeloproliferative neoplasms.Blood. 2009; 113: 6182-6192Crossref PubMed Scopus (308) Google Scholar, 33Sugimoto Y. Muramatsu H. Makishima H. Prince C. Jankowska A.M. Yoshida N. Xu Y. Nishio N. Hama A. Yagasaki H. et al.Spectrum of molecular defects in juvenile myelomonocytic leukaemia includes ASXL1 mutations.Br. J. Haematol. 2010; (in press)PubMed Google Scholar They are largely small in-frame deletions or missense changes affecting the RING-finger domain and/or the adjacent linker region, and they appear to act in a dominant-negative fashion by uncoupling CBL binding to activated RTKs from their ubiquitylation and degradation. Given the link between the functions of CBL and RAS,34Moghal N. Sternberg P.W. A component of the transcriptional mediator complex inhibits RAS-dependent vulval fate specification in C. elegans.Development. 2003; 130: 57-69Crossref PubMed Scopus (47) Google Scholar CBL's ubiquitous expression, and the negative modulatory role on signaling elicited by several RTKs—including the epidermal growth factor receptor (EGFR [MIM 131550])—that have roles in developmental processes,35Grøvdal L.M. Stang E. Sorkin A. Madshus I.H. Direct interaction of Cbl with pTyr 1045 of the EGF receptor (EGFR) is required to sort the EGFR to lysosomes for degradation.Exp. Cell Res. 2004; 300: 388-395Crossref PubMed Scopus (141) Google Scholar, 36Visser G.D. Lill N.L. The Cbl RING finger C-terminal flank controls epidermal growth factor receptor fate downstream of receptor ubiquitination.Exp. Cell Res. 2005; 311: 281-293Crossref PubMed Scopus (12) Google Scholar, 37Umebayashi K. Stenmark H. Yoshimori T. Ubc4/5 and c-Cbl continue to ubiquitinate EGF receptor after internalization to facilitate polyubiquitination and degradation.Mol. Biol. Cell. 2008; 19: 3454-3462Crossref PubMed Scopus (84) Google Scholar as well as the contribution of impaired CBL function to leukemogenesis, particularly JMML, we hypothesized that this gene might be implicated in the pathogenesis of NS or related phenotypes. Three hundred and sixty-five subjects with NS or a phenotype suggestive of this disorder and without mutation in most of the previously identified disease genes (M.T., B.D., C.R., M.Z., and H.Y., unpublished data) were included in the study. The clinical features in most individuals satisfied the diagnostic criteria for NS,8van der Burgt I. Noonan syndrome.Orphanet J. Rare Dis. 2007; 2: 4Crossref PubMed Scopus (265) Google Scholar but subjects lacking sufficient characteristics to allow a definitive diagnosis were also considered. DNA samples and clinical data were collected under research projects approved by an institutional review board, and informed consent for genetic analyses was obtained from all subjects included in the study. Genomic DNA was isolated from peripheral blood leukocytes, and the entire CBL coding sequence (NM_005188.2) was scanned for mutations. Primer pairs designed to amplify exons and their intron boundaries are listed in Table S1. PCR amplifications were carried out with the high-fidelity Optimase Polymerase (Transgenomic) at conditions indicated by Optimase ProtocolWriter software (Transgenomic). Mutation analysis of the amplimers was performed via denaturing high-performance liquid chromatography (DHPLC) with the Wave 2100 System (Transgenomic) at column temperatures recommended by Navigator software, version 1.6.4.12 (Transgenomic). Amplimers with abnormal denaturing profiles were purified (Microcon PCR [Millipore]) and then sequenced bidirectionally with the ABI BigDye Terminator Sequencing Kit v.1.1 (Applied Biosystems) and ABI Prism 3100 and 3730 Genetic Analyzers (Applied Biosystems). Mutation analysis allowed the identification of heterozygosity for a CBL mutation in four unrelated individuals (Figure 1B). The c.1100A > C, c.1144A > G and c.1168G > T missense changes, predicting the Gln367Pro, Lys382Glu, and Asp390Tyr amino acid substitutions, respectively, were identified in three apparently sporadic cases. Parental DNA sequencing of the relevant exon demonstrated the de novo origin of the c.1100A > C and c.1168G > T transversions, and STR genotyping (AmpF/STR Identifier PCR Amplification Kit [Applied Biosystems]) confirmed paternity. In these cases, the defects were documented in hair bulb (c.1168G > T) and buccal epithelial (c.1100A > C and c.1168G > T) cell specimens, which excluded a somatic event restricted to hematopoietic cells (Figure S1A available online). In case BO5149, the c.1144A > G change was documented as having been inherited from the father, who was originally deemed unaffected. Review of his clinical status, however, revealed minor signs and Chiari type 1 malformation (see below), the latter known to recur in NCFCS, which suggested markedly variable expressivity of the mutation. In the family transmitting the trait, the c.1259G > A transition, predicting the Arg420Gln substitution, cosegregated with disease. The inherited c.1144A > G and c.1259G > A changes were unobserved in more than 400 population-matched unaffected individuals scanned by DHPLC analysis and direct sequencing, strongly arguing against the possibility that these variants were disease-unrelated polymorphisms occurring in the population. As expected, the two de novo CBL missense changes were not observed in the controls, further evidence that these variants were mutations. The four mutations affected residues evolutionarily conserved in CBL orthologs and paralogs (Figure S1B) that were located within the RING finger domain (Lys382, Asp390, and Arg420) or the adjacent linker connecting this domain to the N-terminal TKB domain (Gln367), a region that is known to represent the mutational hot spot for somatically acquired lesions occurring in malignancies.25Caligiuri M.A. Briesewitz R. Yu J. Wang L. Wei M. Arnoczky K.J. Marburger T.B. Wen J. Perrotti D. Bloomfield C.D. Whitman S.P. Novel c-CBL and CBL-b ubiquitin ligase mutations in human acute myeloid leukemia.Blood. 2007; 110: 1022-1024Crossref PubMed Scopus (126) Google Scholar, 26Sargin B. Choudhary C. Crosetto N. Schmidt M.H. Grundler R. Rensinghoff M. Thiessen C. Tickenbrock L. Schwäble J. Brandts C. et al.Flt3-dependent transformation by inactivating c-Cbl mutations in AML.Blood. 2007; 110: 1004-1012Crossref PubMed Scopus (162) Google Scholar, 27Loh M.L. Sakai D.S. Flotho C. Kang M. Fliegauf M. Archambeault S. Mullighan C.G. Chen L. Bergstraesser E. Bueso-Ramos C.E. et al.Mutations in CBL occur frequently in juvenile myelomonocytic leukemia.Blood. 2009; 114: 1859-1863Crossref PubMed Scopus (223) Google Scholar, 29Muramatsu H. Makishima H. Jankowska A.M. Cazzolli H. O'Keefe C. Yoshida N. Xu Y. Nishio N. Hama A. Yagasaki H. et al.Mutations of an E3 ubiquitin ligase c-Cbl but not TET2 mutations are pathogenic in juvenile myelomonocytic leukemia.Blood. 2010; 115: 1969-1975Crossref PubMed Scopus (83) Google Scholar, 30Reindl C. Quentmeier H. Petropoulos K. Greif P.A. Benthaus T. Argiropoulos B. Mellert G. Vempati S. Duyster J. Buske C. et al.CBL exon 8/9 mutants activate the FLT3 pathway and cluster in core binding factor/11q deletion acute myeloid leukemia/myelodysplastic syndrome subtypes.Clin. Cancer Res. 2009; 15: 2238-2247Crossref PubMed Scopus (93) Google Scholar, 31Sanada M. Suzuki T. Shih L.Y. Otsu M. Kato M. Yamazaki S. Tamura A. Honda H. Sakata-Yanagimoto M. Kumano K. et al.Gain-of-function of mutated C-CBL tumour suppressor in myeloid neoplasms.Nature. 2009; 460: 904-908Crossref PubMed Scopus (333) Google Scholar, 32Grand F.H. Hidalgo-Curtis C.E. Ernst T. Zoi K. Zoi C. McGuire C. Kreil S. Jones A. Score J. Metzgeroth G. et al.Frequent CBL mutations associated with 11q acquired uniparental disomy in myeloproliferative neoplasms.Blood. 2009; 113: 6182-6192Crossref PubMed Scopus (308) Google Scholar, 38Dunbar A.J. Gondek L.P. O'Keefe C.L. Makishima H. Rataul M.S. Szpurka H. Sekeres M.A. Wang X.F. McDevitt M.A. Maciejewski J.P. 250K single nucleotide polymorphism array karyotyping identifies acquired uniparental disomy and homozygous mutations, including novel missense substitutions of c-Cbl, in myeloid malignancies.Cancer Res. 2008; 68: 10349-10357Crossref PubMed Scopus (256) Google Scholar, 39Bacher U. Haferlach C. Schnittger S. Kohlmann A. Kern W. Haferlach T. Mutations of the TET2 and CBL genes: Novel molecular markers in myeloid malignancies.Ann. Hematol. 2010; 89: 643-652Crossref PubMed Scopus (54) Google Scholar According to the crystal structure of the TKB-linker-RING portion of CBL complexed with the ubiquitin-conjugating enzyme E2L 3 (UBE2L3 [MIM 603721]),40Zheng N. Wang P. Jeffrey P.D. Pavletich N.P. Structure of a c-Cbl-UbcH7 complex: RING domain function in ubiquitin-protein ligases.Cell. 2000; 102: 533-539Abstract Full Text Full Text PDF PubMed Scopus (721) Google Scholar Lys382 and Arg420 are positioned on the RING surface involved in binding to UBE2L3 (Figure 1C). These residues are adjacent or close to four of the invariant cysteine residues that participate in the stabilization of the domain structure by binding the two tetrahedrally coordinated zinc ions. Lys382 is adjacent to residues involved in UBE2L3 binding, and Arg420 directly participates in this binding network. Missense mutations affecting Arg420, including the Arg420Gln substitution, or residues flanking Lys382 (Leu380, Cys381, Ile383, and Cys384) had previously been documented in chronic myelomonocytic leukemia (CMML) and acute myeloid leukemia (AML) (Figure S2).26Sargin B. Choudhary C. Crosetto N. Schmidt M.H. Grundler R. Rensinghoff M. Thiessen C. Tickenbrock L. Schwäble J. Brandts C. et al.Flt3-dependent transformation by inactivating c-Cbl mutations in AML.Blood. 2007; 110: 1004-1012Crossref PubMed Scopus (162) Google Scholar, 27Loh M.L. Sakai D.S. Flotho C. Kang M. Fliegauf M. Archambeault S. Mullighan C.G. Chen L. Bergstraesser E. Bueso-Ramos C.E. et al.Mutations in CBL occur frequently in juvenile myelomonocytic leukemia.Blood. 2009; 114: 1859-1863Crossref PubMed Scopus (223) Google Scholar, 30Reindl C. Quentmeier H. Petropoulos K. Greif P.A. Benthaus T. Argiropoulos B. Mellert G. Vempati S. Duyster J. Buske C. et al.CBL exon 8/9 mutants activate the FLT3 pathway and cluster in core binding factor/11q deletion acute myeloid leukemia/myelodysplastic syndrome subtypes.Clin. Cancer Res. 2009; 15: 2238-2247Crossref PubMed Scopus (93) Google Scholar, 31Sanada M. Suzuki T. Shih L.Y. Otsu M. Kato M. Yamazaki S. Tamura A. Honda H. Sakata-Yanagimoto M. Kumano K. et al.Gain-of-function of mutated C-CBL tumour suppressor in myeloid neoplasms.Nature. 2009; 460: 904-908Crossref PubMed Scopus (333) Google Scholar, 32Grand F.H. Hidalgo-Curtis C.E. Ernst T. Zoi K. Zoi C. McGuire C. Kreil S. Jones A. Score J. Metzgeroth G. et al.Frequent CBL mutations associated with 11q acquired uniparental disomy in myeloproliferative neoplasms.Blood. 2009; 113: 6182-6192Crossref PubMed Scopus (308) Google Scholar, 38Dunbar A.J. Gondek L.P. O'Keefe C.L. Makishima H. Rataul M.S. Szpurka H. Sekeres M.A. Wang X.F. McDevitt M.A. Maciejewski J.P. 250K single nucleotide polymorphism array karyotyping identifies acquired uniparental disomy and homozygous mutations, including novel missense substitutions of c-Cbl, in myeloid malignancies.Cancer Res. 2008; 68: 10349-10357Crossref PubMed Scopus (256) Google Scholar, 39Bacher U. Haferlach C. Schnittger S. Kohlmann A. Kern W. Haferlach T. Mutations of the TET2 and CBL genes: Novel molecular markers in myeloid malignancies.Ann. Hematol. 2010; 89: 643-652Crossref PubMed Scopus (54) Google Scholar On the other hand, Asp390 is adjacent to a residue (Lys389) contributing to the intramolecular interaction involving the linker and RING domain with the TKB domain, which is critical for proper CBL function.40Zheng N. Wang P. Jeffrey P.D. Pavletich N.P. Structure of a c-Cbl-UbcH7 complex: RING domain function in ubiquitin-protein ligases.Cell. 2000; 102: 533-539Abstract Full Text Full Text PDF PubMed Scopus (721) Google Scholar, 41Kassenbrock C.K. Anderson S.M. Regulation of ubiquitin protein ligase activity in c-Cbl by phosphorylation-induced conformational change and constitutive activation by tyrosine to glutamate point mutations.J. Biol. Chem. 2004; 279: 28017-28027Crossref PubMed Scopus (112) Google Scholar Consistent with the hypothesis that the Asp390Tyr substitution affects the integrity of the RING-TKB interface, a missense change (Asp390Val) altering Asp390 has been reported in myeloid malignancies.39Bacher U. Haferlach C. Schnittger S. Kohlmann A. Kern W. Haferlach T. Mutations of the TET2 and CBL genes: Novel molecular markers in myeloid malignancies.Ann. Hematol. 2010; 89: 643-652Crossref PubMed Scopus (54) Google Scholar Similarly, Gln367 is located in a region of the linker that includes residues contributing to the intramolecular interaction with the TKB domain and UBE2L3 binding (Figure 1C).40Zheng N. Wang P. Jeffrey P.D. Pavletich N.P. Structure of a c-Cbl-UbcH7 complex: RING domain function in ubiquitin-protein ligases.Cell. 2000; 102: 533-539Abstract Full Text Full Text PDF PubMed Scopus (721) Google Scholar The Gln367Pro substitution and a nonconservative amino acid change Gln367Lys affecting the same residue have been identified in CMML.31Sanada M. Suzuki T. Shih L.Y. Otsu M. Kato M. Yamazaki S. Tamura A. Honda H. Sakata-Yanagimoto M. Kumano K. et al.Gain-of-function of mutated C-CBL tumour suppressor in myeloid neoplasms.Nature. 2009; 460: 904-908Crossref PubMed Scopus (333) Google Scholar Detailed clinical information was obtained for the six identified subjects harboring CBL mutations (Figure 1D). The patient heterozygous for the c.1100A > C transversion (NMC-NS076) was referred because of developmental delay and congenital heart disease. His parents were nonconsanguineous, apparently healthy, and had no features suggestive for NS. Clinical examination at 9 years showed a triangular face with hypertelorism, mild palpebral ptosis, as well as large, slightly low-set ears. He had normal stature, a thorax with widely spaced nipples, and pectus excavatum. He had several café-au-lait spots, but no other ectodermal abnormalities were apparent. Cardiologic evaluation revealed an enlarged left atrium and transient chaotic ventricular dysrhythmias, an uncommon feature in NS. A head MRI scan showed delayed myelinization without structural abnormalities. Overall, his
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