DNA Methylation Biomarkers in Myelodysplastic Syndromes
VT Nguyen, S Nalluri, MX Wang. University of Missouri School of Medicine, Columbia, MO
Background: Myelodysplastic syndrome (MDS) is defined as a clonal stem cell disorder characterized by single/multilineage dysplasia and ineffective hematopoiesis. Thirty percent of patients with MDS eventually progress to acute myeloid leukemia (AML), but the majority of patients die from marrow failure. Although half of all patients have clonal chromosomal abnormalities, yet none are specific for MDS. Emerging clinical and experimental evidence indicate the importance of epigenetic alterations, especially aberrant DNA hypermethylation, in the pathogenesis of MDS. Currently, there are no diagnostic molecular biomarkers available for MDS. In this study, we identified several hypermethylated loci in MDS patients, which have a potential use as diagnostic biomarkers.
Design: Genomic DNA was extracted from 21 MDS, 48 AML and 8 non-tumor patient bone marrow aspirates as well as 3 AML cell lines (KG1, KG1a and Kasumi-1). The DNA was subjected to digestion with 4 methylation sensitive restriction enzymes. The PCR target regions were carefully selected to ensure a complete digestion. Digested normal cell DNA will not be amplified by PCR. Conversely, hypermethylated regions in tumor cells, being resistant to methylation sensitive restriction enzymes, remain intact and therefore can be differentially amplified. Thus, the presence of PCR products is indicative of DNA methylation at those particular loci in the tumor cells.
Results: We analyzed 18 CGI DNA methylation hot spots, which have been implicated in leukemogenesis. The selected CGIs are contained within the genes involved in tumor suppression, cell cycle regulation, cellular proliferation and migration, DNA repair and apoptosis. Nine of 18 genes were found to be methylated in at least one AML cell line, while only 2 of 18 genes (SOCS-1 and PCDHGA12) were methylated in a subset of MDS patients' bone marrows. A strong methylation in PCDHGA12 gene was observed in 41 of 48 (85%) bone marrows from AML patients, while a weak PCDHGA12 methylation was also seen in 3 of 8 non-tumor bone marrow samples. None was observed in normal blood cell DNAs.
Conclusions: Using a novel PCR method, we were able to demonstrate aberrant DNA methylations in both AML and MDS patients' bone marrow samples. A greater number of methylated genes were detected in AML than in MDS samples. Both SOCS-1 and PCDHGA12 were methylated in MDS and AML. Epigenetic evolution with increased level of methylation may reflect the biological nature of the progression from MDS to AML.
Wednesday, March 11, 2009 1:00 PM
Poster Session VI # 193, Wednesday Afternoon