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Promoter Hypermethylation Silences Expression of the Hoxa4 Gene and Correlates with Igvh Mutational Status in CLL

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Promoter Hypermethylation Silences Expression of the Hoxa4 Gene and Correlates with Igvh Mutational Status in CLL Letters to the Editor 1326 using antigen-specific, antibody-bound microbeads (Mittenyi It remains unclear as to how a single somatic mutation can Biotech, Survey, UK) on an AutoMacs cell separator (Mittenyi give rise to such a heterogeneous clinical presentation, and it Biotech). In addition, haematopoietic progenitor cell-derived seems likely that additional changes in either the signalling colonies were generated from BM aspirate low-density cells. cascade or other genetic alterations may contribute to the Cultures were supplemented with granulocyte–macrophage disease phenotype. Our report adds to the observation that JAK2 colony-stimulating factor (GM-CSF) alone, GM-CSF with Epo mutation is not an infrequent event in CNL. All patients with and GM-CSF, Epo IL-3 and stem cell factor. Individual colony- newly diagnosed CNL should be screened to determine the true forming units granulocyte/macrophage (CFU-GM)-derived co- incidence of the mutation. Given the poor outcome of this lonies from GM-CSF-supplemented and fully supplemented disease, patients with CNL and JAK2 mutation should be consi- cultures were harvested for JAK2 analysis by polymerase chain dered as potential candidates for molecular-targeted therapy. reaction (PCR). For PCR performed on haematopoietic colonies, colonies were placed directly into the PCR reaction without NC Lea1,4, Z Lim1,2,4, NB Westwood1, MJ Arno3,JGa¨ken1, prior DNA extraction. Cell separation kits were used according A Mohamedali1 and GJ Mufti1,2 to the manufacturer’s instructions. The presence of the mutant 1Department of Haematological Medicine, Kings College JAK2 allele was detected using an allele-specific PCR (AS-PCR).4 London, London, UK; The presence of the mutation and the ratio of mutant to wild- 2Department of Haematological Medicine, Guy’s King’s, type JAK2 allele were confirmed by pyrosequencing. Polymer- Thomas’ School of Medicine, Denmark Hill Campus, Kings College Hospital, London, UK and ase chain reaction products were generated using the AS-PCR 3 primer sequences. Sequences were read from a reverse Genomics Center, School of Biomedical and Health 0 0 Sciences, Kings College London, London, UK sequencing primer 5 -TCTCGTCTCCACAGA-3 . Pyrosequen- E-mail: [email protected] cing reactions were run on a Biotage PSQ HS 96 pyrosequencer. 4These authors contributed equally to this work Allele-specific polymerase chain reaction was used to detect the JAK2 V617F mutation in whole-blood, sub-fractionated BM and CFU-GM-derived haematopoietic colonies. The presence of the JAK2 V617F mutation was detected in whole peripheral blood and BM and was also found in all BM cell lineages with References the exception of the lymphoid compartment (CD3- and CD19- positive cells). Day 14 cultures contained only CFU-GM-derived 1 Imbert M, Bain B, Pierre R. Chronic neutrophilic leukaemia. In: Jaffe colonies. BFU-E-derived and mixed lineage colonies were not ES, Harris NL, Stein H et al. (eds). World Health Organisation detected. All colonies picked and amplified by AS-PCR were Classification of Tumours. Pathology and Genetics of Tumours of positive for the V617F mutation (data not shown). Pyrosequen- and Lymphoid Tissues. IARC Press: Lyon, 2001, pp. 27–28. 2 Elliot MA, Hanson CA, Dewald GW, Smoley SA, Lasho TL, Tefferi cing of subfractionated BM cells confirms the presence of the A. WHO-defined chronic neutrophilia leukaemia: a long-term JAK2 mutation in CD14-, CD15-, CD34-, CD61- and GPA- analysis of 12 cases and a critical review of the literature. positive cells and its absence in CD3- and CD19-positive cells Leukaemia 2005; 19: 313–317. (Figure 1). In addition, homo/heterozygosity calling by the 3 James C, Ugo V, LeCouedic JP, Staerk J, Delhommeau F, Lacout C Biotage software indicates that the mutation is homozygous and et al. A unique clonal JAK2 mutation leading to constitutive that the BM subcellular fractions are almost 100% clonal with signaling causes polycythaemia vera. Nature 2005; 434: 1144– 1148. respect to the JAK2 mutant allele. 4 Baxter EJ, Scott LM, Campbell PJ, East C, Fourouclas N, Swanton S Two other cases have recently described the concurrent et al. Acquired mutation of the tyrosine kinase JAK2 in human presence of the JAK2 V17F mutation in the context of CNL with myeloproliferative disorders. Lancet 2005; 365: 1054–1061. one case diagnosed with concurrent B-cell non-Hodgkin’s 5 Kralovics R, Pssamonti F, Buser A, Soon-Siong Teo BS, lymphoma.7,8 Both case reports described the homozygous Tiedt R, Passweg JR et al. A gain-of-function mutation of presence of the JAK2 mutation within granulocytes as observed JAK2 in myeloproliferative disorders. N Engl J Med 2005; 352: 1779–1790. in our case. In addition, we have also performed a lineage- 6 Jones AV, Kriel S, Zoi K, Waghorn K, Curtis C, Zhang L et al. specific analysis and our data demonstrate that the mutation is Widespread occurrence of the JAK2 V617F mutation in chronic present in all myeloid and erythroid cells tested, but is absent myeloproliferative disorders. Blood 2005; 106: 2162–2168. from the lymphoid compartment. We have also found homo- 7 Steensma DP, Dewald GW, Lasho TL, Powell HL, McClure RF, zygous-mutated JAK2 present in 100% of CFU-GM-derived Levine RL et al. The JAK2 V617F activating tyrosine kinase mutation haematopoietic colonies. These observations concur with separate is an infrequent event in both ‘atypical’ myeloproliferative disorders and myelodysplastic syndromes. Blood 2005; 106: 1207–1209. studies on patients with MPDs, which have shown that the JAK2 8 McLornan DP, Percy MJ, Jones AV, Cross NCP, McMullin MF. mutation is likely to occur as a myeloid lineage-specific Chronic neutrophilic leukaemia with an associated V617F Jak2 mutation. tyrosine kinase mutation. Haematologica 2005; 90: 1696–1697. Promoter hypermethylation silences expression of the HoxA4 gene and correlates with IgVh mutational status in CLL Leukemia (2006) 20, 1326–1329. doi:10.1038/sj.leu.2404254; HoxD). They each encode homeodomain containing transcrip- published online 11 May 2006 tion factors, which are known to be key regulators of embryonic development.1 The Hox genes are also expressed in adult cells The human Hox genes are a large gene family, consisting of 39 where they play important roles in the control of cellular members, which are all located in four gene clusters (HoxA– differentiation, particularly during haematopoiesis.2 Several Hox Leukemia Letters to the Editor 1327 gene family members (particularly those in the 50 end of clusters, paralogs 9–13) are involved in leukaemia-associated transloca- tions2 and aberrant expression of Hox genes is a common event in leukaemogenesis. Indeed overexpression of multiple Hox genes (particularly within the HoxA cluster) has been identified in both lymphoid and myeloid malignancies.2 Although the potential mechanisms leading to increased Hox gene expression have been widely studied, aberrant suppression of Hox genes (which are expressed in normal haematopoietic progenitors) has received little attention. Alterations in the patterns of DNA methylation are critical in the development of all types of cancer,3 and transcriptional repression owing to hypermethylation of promoter-associated CpG islands has been shown to inactivate many genes whose downregulation is known to be important in tumour development, both in haematological malignancies and in solid tumours.4,5 All but one of the HoxA cluster genes (HoxA3) have CpG islands asso- Figure 1 Examples of HoxA4 COBRA analysis in CLL patient ciated with their transcriptional start site (sequence derived from samples. Examples of COBRA analysis for samples which were clones RP1-170O19 (AC004080), CTD-2536K9 (AC010990), unmutated at IgVh (a) or mutated at IgVh (b), using the upstream (upper panel) and downstream (lower panel) primer sets. Upper RP1-167F23 (AC004079) in GenBank), suggesting that altered (unmethylated) and lower (methylated) bands are indicated. The DNA methylation is a potential candidate mechanism for altered presence ( þ ) or absence (À) of hypermethylation is indicated below Hox gene expression in leukaemia. each sample. IVM, 100% in vitro methylated positive control. B cells, We chose to investigate the methylation status of the HoxA4 purified normal B-lymphocytes. gene, which is the most 30 HoxA gene previously identified as expressed in haematopoietic progenitor cells.6 Although Hox genes at the 50 end of the cluster, particularly HoxA9, have been implicated as oncogenes,7 the role of the more 30 genes is less clear. Initially, a test panel of 21 chronic lymphocytic leukaemia (CLL) samples were analysed for HoxA4 methylation using the COBRA assay, which allows semiquantitative assessment of methylation levels at specific CpG sites (the percentage of COBRA PCR product digested in this assay is equivalent to the percentage of methylated DNA in the sample). Quantitation of methylation levels was carried out by measurement of methylated and unmethylated band intensities using the GeneTools system (Syngene). The values were then corrected for the smaller size (and thus lower ethidium bromide binding capacity) of the methylated band and plotted against those Figure 2 Hypermethylation of the HoxA4 CpG island correlates obtained using samples, derived from in vitro methylated DNA, with loss of gene expression. Real time qRT–PCR analysis (using the of known methylation status (100, 66 and 33% methylated DyNAmo SYBR Green qPCR kit (Finnzymes) and an Opticon 2 DNA controls were used). 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