GATA1 mutational analysis in chronic myeloid leukaemia (2007)

Chronic myeloid leukaemia (CML) is characterized by the presence of a reciprocal t(9;22)(q34;q11) translocation. This leads to production of the bcr-abl oncoprotein with enhanced tyrosine kinase activity. Thus, aberrant expression of an enzyme involved in cell signaling (type 1 mutation) leads to immortalization of a haematopoietic stem cell. Key genetic changes associated with progression to accelerated phase and blast crisis are not well understood but would be predicted to involve mutations in transcription factors or other genes involved in haematopoietic differentiation (type II mutations). The haematopoietic transcription factor GATA1 plays an essential role in the commitment to, and subsequent differentiation within, erythroid, megakaryocyte, eosinophil and mast cell lineages. The observation of frequent eosinophilia and thrombocytosis alongside neutrophilia in CML suggests that GATA1 is expressed in the chronic phase of the disease. GATA1 mutations have been described in the acute megakaryoblastic leukaemia of Down syndrome (Wechsler et al, 2002). GATA1 mutations are also associated with transient abnormal myelopoiesis seen in up to 10% of infants with Down syndrome, this disease can morphologically resemble CML with basophilia and eosinophilia in addition to circulating blast cells (Zipursky, 2003). The expressed sequence tag (EST) and nucleotide database for Human GATA1 contains five sequences cloned from CML sources. In four of five cases (BF212679, BF211536, BF210926, BF183877) these ESTs showed multiple mutations when compared with the established GATA1 mRNA (NM-002049). In the fifth case (BC009797) the transcript corresponded to an alternative splice variant. Given the plausible association between perturbations in GATA1 function and the molecular pathogenesis of CML, along with suggestive EST evidence, we hypothesized that mutations in GATA1 are involved in progression from chronic phase to blast crisis in CML. Following informed consent and ethical approval we performed GATA1 mutational analysis. Peripheral blood or bone marrow samples were obtained from 24 patients with CML at the time of diagnosis of accelerated phase (n = 1), myeloid blast crisis (n = 15) or lymphoid blast crisis (n = 8). Leucocytes were isolated following ammonium chloride red cell lysis and some samples underwent CD34 selection to enrich for blast populations. Genomic DNA was extracted and initial screening for mutations used Denaturing High-performance Liquid Chromatographic (DHPLC) profiling (WAVE; Transgenomics, Omaha, NE, USA). This technique was chosen because of its sensitivity. In our experience, clone sizes as small as 2·5% can be detected, allowing identification of mutations even in small populations of blast cells. Details of the method for mutation analysis are outlined in Fig 1. Twenty-one of 23 samples appeared normal by WAVE analysis. Two of 24 samples had abnormal migration patterns (Fig 1A). Sequencing these abnormal products revealed single nucleotide substitutions (c>t) in the intronic sequence 40 bp upstream of exon 5 in sample M84BC and 37 bp upstream of exon 5 in sample M949BC (Fig 1B). These sequence variations do not correspond to any known single nucleotide polymorphisms. As these two mutations do not arise in coding or known regulatory regions of the gene it seems unlikely that they are of functional significance. Genomic DNA was used as a template to generate polymerase chain reaction (PCR) products encompassing each coding exon of GATA1 and about 50 bp of flanking sequence. Exons 3 and 6 were divided into two fragments each for analysis. PCR products were heteroduplexed on a thermocycler using standard conditions and screened for mutations in GATA1 using High Performance Liquid Chromatograph (WAVE, Transgenomics). (A) Exon 5 migration profile showing wild-type peak and faster eluting peaks due to mismatched heteroduplexes from two samples with suspected mutations M84 and M949. (B) Sequence chromatograms from wild type, M84 and M949, the abnormal migration profiles were due to single base pair changes in the flanking 5′-intron of exon 5 (see text). Abnormal regulation of GATA1 activity either at the transcriptional or post-translational level may still play an important part in the pathogenesis of the disease. The mitogen-activated protein kinase (MAPK) pathway has recently been shown to be important in controlling the oncogenic potential of bcr-abl (Notari et al, 2006). MAPK-mediated phosphorylation of GATA1 at critical serine residues promotes cell survival via increased bcl-XL expression (Yu et al, 2005). In addition, an investigation of upregulation of inducible heat shock protein 70 (Hsp70) in CML showed that the major p210 bcr-abl responsive element upstream of the Hsp70 promoter was mapped to a GATA-binding motif (Ray et al, 2004). These findings suggest that GATA1 mutations are not frequent occurrences in the evolution of blast crisis from CML chronic phase. However, this study does not rule out an important role for dysregulated GATA1 activity in the molecular pathogenesis of CML.