Global Epigenomic Profiling Demonstrates That Myelodysplasia Is Characterized by a Distinct Epigenetic Signature with Aberrant DNA Methylation Changes Involving Various Malignant and Hematopoietic Pathways.

Myelodysplasia (MDS) is a clonal hematopoietic disorder that leads to ineffective hematopoiesis and peripheral cytopenias. DNMT inhibitors such as azacytidine have led to clinical responses in patients, though the genes affected by epigenetic alterations are not well known. Whole genome DNA methylation was analyzed by a recently described novel method, The HELP assay (HpaII tiny fragment Enrichment by Ligation-mediated PCR; Khulan et al, Genome Res. 2006 Aug;16(8)) that uses differential methylation specific restriction digestion by HpaII and MspI followed by amplification, two color labeling and cohybridization to quantitatively determine individual promoter island methylation. A whole genome human promoter array (Nimblegen) was used to determine the level of methylation of 25626 gene promoters by calculating HpaII/MspI cut fragment intensity ratio. Peripheral blood leucocytes from 13 patients with MDS were compared to 9 age matched normal and anemic controls. Gene expression analysis was performed using 37K oligo maskless arrays on cDNA obtained from the same samples. Analysis showed that whole genome methylation profiling has greater discriminatory power in separating clusters of MDS samples from normal and anemic controls when compared to gene expression analysis. Unsupervised clustering based on epigenetic profiling demonstrated that only two cases of early MDS clustered with normals as compared to absolutely no separation between MDS and normals with clustering based on gene expression patterns. A high correlation (r=0.88–0.96) was observed between global methylation profiles of matched sets of bone marrow and peripheral blood leucocyte samples from selected patients demonstrating that peripheral blood leucocytes can be a valid surrogate for epigenomic analysis. Further analysis showed that genes consistently aberrantly methylated in MDS included Syk kinase, HOXB3, several histone acetyltranferases and others. Functional analysis by Ingenuity showed that cancer and cell signaling pathways were the most affected by epigenetic silencing. Most interestingly, a large proportion of gene promoters were also aberrantly hypomethylated. These included genes from Ras oncogene family, the CDC42 GTPase, various methyl binding proteins and other proteins mainly encoding for cancer and hematopoiesis functional pathways, thus biologically validating our analysis. Therefore, our data demonstrates that MDS is characterized by distinct epigenetic aberrations that are preserved in peripheral blood leucocytes. These can be the basis of future studies on pathogenesis and diagnosis for this disease and can potentially uncover a new set of therapeutic gene targets.