Methylation status of the p15(INK4B) gene in hematopoietic progenitors and peripheral blood cells in myelodysplastic syndromes

We previously reported that the hypermethylation of the p15(INK4B) gene promoter was frequently observed in myelodysplastic syndromes (MDS), and that it may be associated with disease progression. An unanswered question is whether p15(INK4B) gene methylation is restricted to undifferentiated blastic cells, or whether differentiated cells such as granulocytes or erythrocytes of MDS origin also harbor this epigenetic alteration. In this study, we analyzed the methylation status of the p15(INK4B) gene in MDS by the methylation-specific PCR (MSP) method, which is more sensitive than Southern blotting. The bone marrow mononuclear cells (BM-MNCs) of 23 MDS patients were analyzed, and six of them showed p15(INK4B) methylation. Progenitor assay with methylcellulose medium was also performed in all patients. In two of the six patients with p15(INK4B)-methylated BM-MNCs, erythroid and/or non-erythroid colonies formed were subjected to molecular analysis. Colonies with and without p15(INK4B) methylation were detected in both patients. Furthermore, X-chromosome inactivation (XCI) pattern of each colony was simultaneously determined by MSP-based human androgen receptor gene analysis (HUMARA-MSP), and all p15(INK4B)-methylated colonies showed the same XCI pattern, which was dominant among the colonies, while p15(INK4B)-unmethylated colonies showed both patterns of XCI, in each of the two patients. We then examined the methylation status of the p15(INK4B) gene of granulocyte (PB-PMN) fractions from 10 patients with available peripheral blood cells, in all four patients with p15(INK4B)-methylated BM-MNCs, their PB-PMNs showed p15(INK4B) methylation. These results suggest that p15(INK4B) methylation in hematopoietic cells in MDS patients is restricted to the MDS clone but not necessarily to blast cells.