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Mutations in PTPN11 are uncommon in adult myelodysplastic syndromes and acute myeloid leukaemia

2004; Wiley; Volume: 124; Issue: 6 Linguagem: Inglês

10.1111/j.1365-2141.2004.04862.x

1365-2141

Muhammad Farid Johan, David Bowen, Marion E. Frew, Anne Goodeve, G. A. Wilson, I. R. Peake, John T. Reilly,

Ubiquitin and proteasome pathways

The PTPN11 gene encodes the ubiquitously expressed non-receptor-type protein tyrosine phosphatase SHP-2 (src homology region 2-domain phosphatase-2) (Neel, 1993). SHP-2 is a key molecule in the cellular response to growth factors, hormones, cytokines, and cell adhesion molecules and is required for the activation of the RAS/MEK/ERK kinase cascade (Cunnick et al, 2002). Recently, PTPN11 has been identified as the Noonan syndrome disease gene, through use of a positional candidacy approach (Tartaglia et al, 2001). As a result of the rare association of juvenile myelomonocytic leukaemia (JMML) and Noonan syndrome, Tartaglia et al (2003) screened non-syndromic JMML patients for PTPN11 mutations and documented somatic changes in 34% of cases. Furthermore, the same group reported mutations in 10% and 4% of children with myelodysplasic syndromes (MDS) and de novo acute myeloid leukaemia (AML) respectively. These acquired mutations were predicted to cause gain-of-function of SHP-2 through preferential occupation of the activated state of the phosphatase. To assess the pathogenetic relevance of PTPN11 mutations in adult myeloid disorders, we have undertaken mutational analysis of exons 2, 3, 4, 7, 8 and 13 of PTPN11 in MDS and AML. Genomic DNA was obtained from peripheral blood or bone marrow from 107 cases of MDS, refractory anaemia (RA; n = 20), RA with ringed sideroblasts (RARS; n = 20), RA with excess blasts (RAEB; n = 30), RAEB in transformation (RAEB-t; n = 2) and chronic myelomonocytic leukaemia (n = 35). Genomic DNA was also obtained at presentation of 64 cases of AML entered into the Medical Research council (MRC) AML X and XII Trials. The cases were classified according to the French–American–British (FAB) criteria, as: M0 (n = 4), M1 (n = 8), M2 (n = 13), M3 (n = 10), M4 (n = 13), M5 (n = 10), M6 (n = 6). Genomic DNA was also prepared from five cases of JMML [four de novo, one associated with neurofibromatosis type 1 (NF1)], and from the peripheral blood of 35 normal individuals using the Nucleon Biosciences BACC II kit. Genomic DNA was amplified using polymerase chain reaction and the coding sequences and intron/exon boundaries corresponding to exons 2, 3, 4, 7, 8 and 13 were screened as described by Tartaglia et al (2001). Conformation sensitive gel electrophoresis (CSGE) was used to screen for mutations and samples displaying abnormal CSGE profiles were purified (Qiagen) and sequenced (MWG Biotech). Screening 64 cases of AML revealed a single missense mutation (1·5%). The patient, classified as M2 with normal cytogenetics, exhibited an exon 3 C → G transition at nucleotide 218 that is predicted to result in a threonine to isoleucine substitution at codon 73. Interestingly, this individual did not possess a RAS (N-, or Ki), c-FMS, FLT3-ITD, FLT3835 or c-KIT816 mutation. A silent change was identified in exon 2 in another AML patient (nucleotide 48A → G; Ala16Ala). None of the 107 MDS cases exhibited a PTPN11 mutation. However, one of the four de novo JMML cases possessed an exon 3 G → A transversion at position 226, which is predicted to result in a glutamic acid to lysine substitution at codon 76. A further de novo JMML case possessed an N-RAS12 mutation but was negative for a PTPN11 mutation, while the NF1-associated JMML was negative for both RAS and PTPN11 mutations. All reported mutations affect residues located at the N-SH2 and PTP interacting surfaces and result in gain-of-function of SHP-2 through preferential occupation of the phosphatase's activated state (Tartaglia et al, 2001, 2003). The two mutations identified in this study, which are located in the N-SH2 domain, have previously been associated with de novo and Noonan syndrome-associated JMML, as well as with paediatric MDS (Tartaglia et al, 2003). Our results suggest that PTPN11 mutations are not common in adult AML, occurring in approximately 1·5% of cases. Interestingly, the Thr73Ile mutation, which occurred in a case of AML M2, was not associated with a RAS, c-FMS, FLT3 or c-KIT mutation, supporting the concept of mutual exclusivity of RTK/RAS pathway mutations. In contrast to the 10% reported frequency of PTPN11 mutations in paediatric MDS (Tartaglia et al, 2003), none of the 107 adult MDS cases possessed a mutation, suggesting a difference in the pathogenesis of adult and paediatric MDS. Finally, the results from our small study of JMML patients supports the findings of Tartaglia et al (2003), that defects in the regulatory components of the mitogen-activated protein kinase cascade, namely RAS, neurofibromin and SHP-2, appear to be mutually exclusive.