High Frequency of Tumor Suppressor Genes Promoter Hypermethylation in Peripheral Blood of Patients with Myelodysplastic Syndromes

Myelodysplastic syndromes (MDS) are a heterogeneous group of hematopoietic stem cell disorders characterized by ineffective hematopoiesis and peripheral blood cytopenias, with very little established about their pathogenesis. DNA methylation is a common modification thought to be relevant to carcinogenesis. The epigenetic silencing, through methylation of CpG islands within the promoter regions, is one of the mechanisms by which numerous genes are inactivated in MDS and acute myeloid leukemias (AML). In particular, the promoter of CDKN2B (encoding p15INK4b), has been shown to be hypermethylated in poor-risk subtypes of MDS and often predicts transformation to AML. In this study, we examined the promoter methylation status of 7 genes in peripheral blood (PB) cells collected from 19 patients affected by untreated MDS. We investigated genes involved in cell cycle regulation (p14ARF, p15INK4b, p16INK4a), apoptosis (SPARC, DAPK1), differentiation (RARb) and response to growth factors (RASSF1A) previously reported as being abnormally hypermethylated in acute leukemia and in a variety of solid malignancies. DNA was extracted from PB low-density mononuclear cells and promoters methylation status was analyzed by means of methylation-specific PCR assay (MSP). MSP distinguishes unmethylated from methylated alleles in a given gene based on sequence changes produced after bisulfite treatment of DNA, which converts unmethylated (but not methylated) cytosines to uracil, and subsequent PCR using primers designed for either methylated or unmethylated DNA. Sodium bisulfite modification was performed using the Methylamp DNA Modification Kit (Epigentek) following manufacturer‘s instructions. Our series included 4 RA, 3 RARS, 3 RCMD, 2 RCMD/RS, 5 RAEB-1, 1 RAEB-2 and 1 CMML-1; according to the IPSS, 17 patients (89%) were classified as either low- or intermediate-1 risk. Median age was 72 years (range 49–86). Cytogenetics were normal in 12 patients (63%), while in 3 patients showed the 5q- abnormality. Median CBC counts were: WBC 3.1 × 10⁹/L (0.9–12.5), Hb 10 g/dl (6.4–15), PLT 106 × 10⁹/L (9–406). The cell cycle regulator p15INK4b was found to be the most frequently hypermethylated gene (14/19 pts, 74%) in our MDS series, followed by the p14ARF (13/19 pts, 68%). The latter was hypermethylated in 100% of our 5q- cases. Ninety-five percent (18/19 pts) of the MDS samples were found to be hypermethylated in at least one of the seven genes, with the only exception being the patient with CMML (unmethylated in all the tested genes). Hypermethylation of the cell cycle regulator p16INK4a was detected in 47% of samples. In 3 patients (16%) we found hypermethylation in all genes but RASSF1A, which indeed was found unmethylated in all but one of the samples analyzed (5%). The tumor suppressor gene SPARC was hypermethylated in 58% of patients (including 2 out of the 3 cases of 5q-), whereas 42% and 47% of samples, respectively, showed hypermethylation of the RARb and DAPK1 genes promoter. Although in a limited number of samples, we detected a differential rate of hypermethylation of the tested genes in peripheral blood of MDS patients. Altogether, our results seem to confirm high frequency of hypermethylation genes involved in regulation of cell cycle and apoptosis in MDS. We conclude that mononuclear cells from peripheral blood represents an easily accessible sample for detection and monitoring of genes promoter hypermethylation in myelodysplastic syndromes.