References 1 Sellick GS, Catovsky D, Houlston RS. Familial chronic lymphocytic leukemia. Semin Oncol (2006); 33: 195–201. 2 Sellick GS, Webb EL, Allinson R, Matutes E, Dyer MJ, Jonsson V et al. A high-density SNP genomewide linkage scan for chronic lymphocytic leukemia-susceptibility loci. Am J Hum Genet 2005; 77: 420–429. 3 Rosenbauer F, Owens BM, Yu L, Tumang JR, Steidl U, Kutok JL et al. Lymphoid cell growth transformation are suppressed by a key regulatory element of the gene-encoding PU.1. Nat Genet 2006; 38: 27–37. 4 Okuno Y, Huang G, Rosenbauer F, Evans EK, Radomska HS, Iwasaki H et al. Potential autoregulation of transcription factor PU.1 by an upstream regulatory element. Mol Cell Biol 2005; 25: 2832– 2845. 5 Mueller BU, Pabst T, Osato M, Asou N, Johansen LM, Minden MD et al. Heterozygous PU.1 mutations are associated with acute myeloid leukemia. Blood 2002; 100: 998–1007. 6 Schievella AR, Chen JH, Graham JR, Lin LL. MADD a novel death domain protein that interacts with the type 1 tumor necrosis factor receptor activates mitogen-activated protein kinase. J Biol Chem 1997; 272: 12069–12075. 7 Lim KM, Yeo WS, Chow VT. Antisense abrogation of DENN expression induces apoptosis of leukemia cells in vitro causes tumor regression in vivo alters the transcription of genes involved in apoptosis the cell cycle. Int J Cancer 2004; 109: 24–37. 8 Mu ¨ller-Hermelink H, Montserrat E, Catovsky D, Harris N. Chronic lymphocytic leukaemia/small lymphocytic lymphoma.In: Jaffe ES, Harris NL, Stein H, Vardiman JW (eds) World Health Organization classification of tumours: Pathology and genetics of tumours of haematopoietic and lymphoid tissues. IARC Press: Lyon, 2001, pp 127–130. 9 Al-Zoubi AM, Efimova EV, Kaithamana S, Martinez O, El-Idrissi Mel A, Dogan RE et al. Contrasting effects of IG20 and its splice isoforms, MADD and DENN-SV, on tumor necrosis factor alpha- induced apoptosis and activation of caspase-8 and -3. J Biol Chem 2001; 276: 47202–47211. High-throughput, sensitive and quantitative assay for the detection of BCR-ABL kinase domain mutations Leukemia (2007) 21, 1318–1321. doi:10.1038/sj.leu.2404635; published online 1 March 2007 The causative molecular event in chronic myeloid leukemia (CML) is the genetic transposition of ABL and BCR sequences to form a BCR-ABL fusion gene, leading to the expression of a constitutively active tyrosine kinase. 1 Inhibition of BCR-ABL Kinase activity with the small molecule drug imatinib (IM) results in a rapid hematologic response and has revolutionized the therapy of CML patients. 2 Resistance to IM is a growing clinical problem, resulting in many cases from the acquisition of mutations in the BCR-ABL kinase domain (KD). Detection of specific mutations directs changes in management such as the use of new tyrosine kinase inhibitors or stem cell transplanta- tion. For this reason, periodic genotyping of the BCR-ABL KD to screen for drug-resistant mutations may play an important role in the management of CML patients. 3 We developed a sensitive and quantitative high-throughput assay using SEQUENOM MassARRAY system that enables fast and reliable detection of mutations in the BCR-ABL KD. One hundred and two cDNA samples obtained from peripheral blood of 40 patients diagnosed with Philadelphia- positive CML were analyzed, 26 patients had chronic phase, 10 accelerated and four blast crisis CML. Twenty-nine patients were IM resistant (mean disease duration 47 months) and 11 were IM intolerant (mean disease duration 28 months). MassARRAY genotyping was carried out using a chip-based matrix-assisted laser desorption-time-of-flight mass spectrometer (Figure 1a) (Sequenom, San Diego, CA, USA). Multiplex SNP assays were designed (Supplementary File 1 online) (Mass- ARRAY assay design v 3.0, Sequenom) for 27 previously reported different mutations that confer IM resistance (Table 1). The reactions were carried out according to the manufacturer’s instructions 4 (Figure 1b). The BCR-ABL KD mutational status was also determined by direct sequencing as previously Table 1 ABL KD mutations analyzed Amino-acid change Nucleotide change Amino-acid change Nucleotide change 1 a A1094G M244V 15 b C1308T T315I 2 C1106G L248V 16 C1308A T315N 3 a G1113A G250E 17 a C1315G F317L 4 G1112A G250R 18 T1392C M343T 5 G1120C Q252H 19 T1416C M351T 6 G1120T Q252H 20 a T1439G F359V 7 a T1121C Y253H 21 T1440G F359C 8 A1122T Y253F 22 G1499A V379I 9 G1127A E255K 23 T1508C F382L 10 A1128T E255V 24 a T1523A L387M 11 A1191G D276G 25 G1525C L387F 12 A1194G T277A 26 A1551C H396P 13 T1230C V289A 27 A1551G H396R 14 T1295C F311L a Detected mutations. b Mutation was identified in two different patients. Letters to the Editor 1318 Leukemia