Analysis of GATA-1 Mutations in Chronic Myeloid Leukaemia.

Despite extensive characterisation of the molecular pathogeneisis of chronic myeloid leukaemia (CML), little is known about the events underlying progression to accelerated phase and blast crisis. The haematopoietic transcription factor GATA1 plays an essential role in the commitment to, and differentiation within, erythroid, megakaryocyte, eosinophil and mast cell lineages. The observation of frequent eosinophilia and thrombocytosis alongside granulocytic hyperplasia suggests that GATA1 is actively expressed in CML chronic phase. GATA1 is mutated in the acute megakaryoblastic leukaemia of Down syndrome and in its preleukaemic form, transient abnormal myelopoiesis. The latter can morphologically resemble CML with basophilia and eosinophilia in addition to circulating blast cells.

Examination of the expressed sequence tag (EST) database for GATA1 shows 4 variant sequences. 3 of these are derived from CML patient bone marrow.

Given the plausible association between perturbations in GATA1 function and progression of CML, along with suggestive EST evidence, we hypothesized that mutations in GATA1 could be involved in the transition from chronic phase to blast crisis in CML.

Materials and methods: Following appropriate informed consent and ethical approval genomic DNA was extracted from blast cells from 24 patients with CML in accelerated phase (n=1), myeloid blast crisis (n=15) or lymphoid blast crisis (n=8). Polymerase chain reaction (PCR) products were generated encompassing each coding exon of GATA1 and about 50 base pairs of flanking sequence. Exons 3 and 6 were divided into 2 fragments each for analysis. Gel purified PCR products were screened for mutations in GATA1 using DHPLC (denaturing high pressure liquid chromatography, WAVE, Transgenomics, USA). Samples with abnormal migration profiles were directly sequenced by bi-directional sequencing using an ABI 3100 Capillary Array sequencer (Perkin Elmer, UK). Mutations detected by this method were confirmed by a repeat PCR reaction, Topo cloning into pCR4TOPO vector (Invitrogen) and commercial sequencing (Agowa, Germany)

Results and discussion 2/24 samples showed abnormal migration patterns by WAVE analysis. Sequencing confirmed 2 different single nucleotide changes in the intronic sequence flanking exon 5. These do not correspond to any known single nucleotide polymorphisms. Since these 2 mutations do not arise in coding or known regulatory regions of the gene it seems unlikely that they are of functional significance. 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. MAPK mediated phosphorylation of GATA1 at critical serine residues promotes cell survival via increased Bcl-XL expression. In addition study of the upregulation of inducible heat shock protein 70 (Hsp70) in CML has shown the major p210 bcr-abl responsive element upstream of the Hsp70 promoter to be a GATA binding motif.

Conclusion 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 chronic myeloid leukaemia.