The identification of those genes that are frequently mutated in malignancies is essential for a full understanding of the molecular pathogenesis of these disorders, and often for the provision of markers for the study of disease progression. Recently, mutation of the ASXL1 (additional sex combs 1) gene has been reported in 4 out of 35 patients (11%) with myelodysplastic syndromes (MDS) and in 17 out of 39 patients (43%) with chronic myelomonocytic leukemia (CMML), a disease classified as MDS/myeloproliferative disorder. 1 The mutations were all found in exon 12 of the gene and are predicted to lead to the truncation of the C-terminus of the protein, which contains a PHD finger. 1 The PHD finger is a structural motif found in nuclear proteins involved in chromatin modifications. The function of the human ASXL1 protein is poorly understood, but there is some evidence to suggest that it may represent a component of DNA-and/or histone-modifying complexes. 2 ASXL1 belongs to a family of three members that encode polycomb and mixed-lineage leukemia/trithorax chromatin modifier complexes. 2 Most recently, mutations of ASXL1 have been reported in 5 out of 64 (approximately 8%) patients with the myeloproliferative disorders essential thrombocythemia and primary myelofibrosis, and in 11 out of 63 (17%) patients with acute myeloid leukemia (AML). 3, 4 These data suggest that ASXL1 represents a new tumor suppressor gene, the inactivation of which may have an important role in the molecular pathogenesis of malignant myeloid disorders. We have screened ASXL1 for mutations in a large group of patients with MDS, CMML and AML, in order to determine their frequency in these malignant myeloid disorders and to determine any association with specific MDS subtypes, karyotypic groups and disease progression. Moreover, we have determined the effect of these mutations on global gene expression patterns and gene pathways. A total of 300 patients with MDS, AML or CMML and 111 healthy controls were included in this study. The study was approved by the ethics committees of the institutes involved (Oxford C00. 196, Bournemouth 9991/03/E, Duisburg 2283/03, Stockholm 410/03, Pavia 26264/2002, PamplonaFapproval by the Investigational Review Boards at the University of Navarra) and informed consent was obtained. Genomic DNA was isolated from patient bone marrow or peripheral blood samples by phenol/chloroform extraction using standard methods and evaluated for quality and concentration using an ND-1000 spectrophotometer (Nanodrop, Wilmington, DE, USA). All previously reported mutations of ASXL1 in hematological malignancies were found exclusively in exon 12 of the gene. 1, 3

Sequences spanning exon 12 of the ASXL1 gene were amplified by PCR from the DNA of 300 patient samples and 111 normal controls. Primers and amplification conditions were as previously published. 1 PCR products were purified and directly sequenced using the BigDye Terminator v1. 1 cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) and an ABI 3100 Genetic analyzer. Sequence data were analyzed using Mutation Surveyor V3. 25 (Softgenetics, State College, PA, USA). ASXL1 mutations were observed in 62 out of the 300 patients (21%; Table 1). ASXL1 mutations were found in 5 out of 79 MDS patients with refractory anemia (RA; 6%), in 17 out of 55 patients with either refractory anemia with excess blasts 1 (RAEB-1) or RAEB-2 (31%), and in 17 out of 67 patients with AML (25%; Table 1). Thus, the mutation frequency is relatively high in late MDS and AML, with a lower frequency across the spectrum of early MDS (Table 1 …