Abstract

The myelodysplastic syndromes (MDS) are a heterogeneous group of haematopoietic malignancies, characterized by blood cytopenias, ineffective hematopoiesis and hypercellular bone marrow. MDS are believed to arise from the malignant transformation of a hematopoietic stem cell. We have used Affymetrix microarray technology to determine gene expression profiles in bone marrow CD34+ cells of 17 MDS patients and 6 healthy controls. Nine patients with RA and 8 patients with RAEB were included in the study. Eleven of 17 patients had a del(5q). CD34+ cells were isolated from bone marrow samples of MDS patients and controls using MACS magnetic cell separation columns (purity greater than 85%). Extracted total RNA was amplified using the Two-Cycle Target Labelling and Control Reagent package (Affymetrix). Biotin-labelled fragmented cRNA was hybridized to GeneChip Human Genome U133 Plus 2.0 arrays (Affymetrix), covering over 47,000 transcripts representing 39,000 human genes. Cell intensity calculation and scaling was performed using GeneChip Operating Software (GCOS) and data analysis using GeneSpring 6.2. Genes up-regulated by >2 fold in the majority of MDS patients include IFITM1, an interferon-inducible protein implicated in the control of cell growth, and KANGAI1, a metastasis suppressor gene. IFITM1 was up-regulated in 15 of 17 MDS patients. Up-regulation of DLK1, previously reported in MDS, was confirmed in 8 of 17 MDS patients. Genes down-regulated by >2 fold in the majority of MDS patients include FOSB (a transcription factor which regulates cell proliferation and differentiation), COX2 (anti-apoptotic, promotes cell survival), Gravin/AKAP12 (a putative tumor suppressor gene), CD24 and MME. FOSB was down-regulated in 16 of 17 MDS patients. The results for several genes have been confirmed by real-time quantitative PCR (TaqMan). MDS patients with a del(5q) could be discriminated from those without a del(5q), using a set of 12 genes obtained applying a parametric t-test with multiple testing correction (P<0.01). Five of the 12 genes (DCP2, HTGN29, ANKHD1, CSNK1A1 and RBM22) mapped to chromosome 5q and their expression levels were lower in MDS patients with del(5q) than those without del(5q). Using a parametric t-test with multiple testing correction (P<0.05), 221 genes were found to be significantly different between MDS patients and controls. These 221 genes were ranked by their power to discriminate the two classes. The eight genes with the highest prediction strength were used for leave-one-out cross-validation. Twenty-one of 23 samples (91%) were correctly classified as MDS or control and one was not assigned. This set of genes is being tested on an independent test set to perform class prediction. This study has identified genes differentially expressed in the CD34+ cells of MDS patients that may be important in the molecular pathogenesis of this disorder.

This study has been funded by Leukaemia Research Fund.

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