122: Genome-Wide Mapping of 5-methylcytosine and 5-hydroxymethylcytosine in Acute Myeloid Leukemia Primary Samples & Cord Blood Cells

Statement of Purpose: DNA methylome studies does not differentiate between the two epigenetic marks 5-metyhlcytosine (5mC) and its oxidized form 5-hydroxymethylcytosine (5hmC) due to limitations in the available techniques. The inability to differentiate between the two marks results in overestimation of the 5mC level. Additionally, 5mC and 5hmC were shown to have different impact on gene expression. Accordingly, it is essential to differentiate between both marks at different genomic regions. The objective of this study is to conduct a genome-wide methylome analysis using oxidative bisulfite sequencing (OxBS), which can discriminate between 5mC and 5hmC in primary human acute myeloid leukemia (AML) samples and in human cord blood cells.

Description of Methods & Materials: Six de-identified AML patient samples and four human cord blood cells samples were obtained from the leukemia tissue bank at Ohio State Univ. Genomic DNA was extracted from the samples and digested with MspI enzyme. Next Generation Sequencing (NGS) was performed using a modified method of Reduced Representation Bisulfite Sequencing (RRBS), where the samples were oxidized before bisulfite treatment. The oxidation step converted 5hmC into 5-fluorocytosine (5fC), which was converted into thymine after bisulfite treatment. On the other hand, 5mC was not affected by the oxidation step and discrimination between the two marks was achieved. Bioinformatics analysis was performed using GREAT tool.

Data & Results: The 5mC mark was more abundant than the 5hmC mark in both AML and normal samples. The ratio of 5mC/5hmC was approximately 3:1 in both normal and AML samples. Differentially methylated regions (DMR) in CpG islands were identified, where 3427 regions in AML samples showed an increase in 5mC and 2058 regions showed a decrease in 5mC relative to normal cells (Figure 1).

Interpretation, Conclusion or Significance: The methylome of AML cells demonstrated significant differences from that of the normal cells as evidenced by the DMR. However, the percentage of both 5mC and 5hmC were not significantly different between AML and normal cells. The genes associated with the observed DMR are currently under analysis to identify their biological function and their relevance to AML.

Disclosures: None