Gene Expression Profiling Of Hematopoietic Cells From Patients With Myelodysplastic Syndrome

Myelodysplastic syndrome (MDS) represents a heterogeneous group of clonal disorders predominantly affecting hematopoietic stem cells, characterized by cytopenia in at least one lineage of peripheral blood and the presence of dysplastic features in marrow hematopoietic cells. MDS has been considered as a preleukemic condition for a high risk of transformation to acute leukemia. Based on the number of ring sideroblasts, excess blasts and presence of monocytes, the French-American-British cooperative study group has classified MDS into five categories: (1) refractory anemia (RA), (2) RA with ring sideroblasts (RAS), (3) RA with excess blasts (RAEB), (4) RAEB in transformation (RAEB-t), and (5) chronic myelomonocytic leukemia (CMML) Till now, Morphologic abnormality is still an important basis for the diagnosis of MDS. However, because hematopoiesis in each lineage is injured with varying degrees, some patients present with dysplasia in only one lineage such as idiopathic and unexplained macrocytic anemia, isolated neutropenia, thrombocytopenia or even no dysplasia. These 'early MDS' or 'not yet' MDS sometimes are confused with chonic aplastic anemia (CAA), megaloblastic anemia (MA) if lacking cytogenetic abnormalities before evolving into overt MDS. Cytogenetic abnormalities provide a convincing evidence for the clonaliry of the disorder. Unfortunately, the incidence of 30%~70% in cytogenetic abnormalities is relatively low.The pathogenesis of MDS and its frequent progression to acute leukemia is not well established. Increased apoptosis causing ineffective hematopoiesis has been demonstrated in MDS, while decreased apoptosis may be attributed to the course of progression. Some oncogenes and tumor suppressor genes such as Bcl2, c-Myc, Ras, p15INK4b, and TNF superfamily members, have been suggested to contribute to its molecular basis. Therefore, exploring the gene expression abnormalities not only help us understand the molecularbasis of MDS pathogenesis but also may provide useful biomarkers for diagnosis. To expand the understanding of genetic defects of hematopoietic cells in MDS and to find new gene changes related to the pathogenesis of MDS and the diagnosis of MDS, we applied cDNA microarray to analyze the clinical samples from MDS patients.We first mixed the equal amount of total RNA of bone marrow monoculear cells (BMNCs) from two MDS patients (one RA and one RAS) diagnosed according to FAB criteria. The Cy5-dCTP-labeled cDNA probes were prepared through reverse transcription with patients' RNA and Cy3-dCTP-labeled cDNA probes were also prepared with normal controls' total RNA. Two types of probes were mixed and hybridized with BioStar H141 microarray (United Gene Holdings Co. LTD, Shanghai, China) containing 13484 sequences. The results of duplicate hybridizations confirmed the feasibility of microarray assay in MDS study, however, the reproducibility was not so perfect that replication must be needed to reduce the expression bias caused by the experiment performances such as reverse transcription, labeling, hybridization, washing, and scanning processes. From our preliminary results and that of Biostar Company on 3 patients with acute myeloid leukemia (AML) (2 M2 and 1 M5), we performed the bioinformatic analysis and selected 500 genes with known functions and potential involvement in hematopoiesis regulation. Ten chips were customized in Biostar Company, in which a 17X34 spot array was printed in quadruplicate. We applied the custom-made microarray to explore the expression patterns of BMNCs from ten further MDS patients (4 RA, 1 RTC, 4 RAEB, and 1 RAEBt). Our results revealed that 95 genes were abnormally expressed in at least five MDS patients compared with normal controls, 68 down regulated and 27 up regulated. Among these 95 genes, bioinformatic analysis showed 59 were involved in the cell growth and differentiation, cell cycle regulation, signaling, redox regulation, including thrombospondin I (THBS1), phosphatase and tensin homolog (PTEN), MAD, DNA-damage-inducible transcript 3 (DDIT3), ets vari...