(M0930-04-27) High-Resolution Sequencing Comparison of the DNA Methylome and Hydroxymethylome in Leukemia and Normal Blood Cells

Purpose: 5-Methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are common epigenetic marks found in the mammalian genome and play a role in regulating gene expression. The role of aberrant DNA methylation in hematologic malignancies and leukemia is well established. However, the changes in 5mC and 5hmC associated with leukemia at the single-base resolution are not clear. Moreover, quantitative analysis of 5mC using DNA bisulfite sequencing does not differentiate between the 5mC and 5hmC marks and results in over-estimation of 5mC. The purpose of this study is to quantitate both 5mC and 5hmC in acute myeloid leukemia (AML) primary patient samples and normal cord blood cells and identify the differentially methylated regions (DMR) in both cell types.

Methods: Four human cord blood cells and six de-identified AML patient samples were collected from the leukemia tissue bank at Ohio State University. Enzyme digestion of genomic DNA by the methylation insensitive MspI, which recognizes the sequence CCGG, was performed. A modified method of Reduced Representation Bisulfite Sequencing (RRBS) was used to sequence the DNA fragments, where each sample was split equally into two subsamples. The first subsample was oxidized before bisulfite treatment and the second one skipped the oxidation step (mock oxidation) and was directly bisulfite treated. The oxidation step converted 5hmC into 5-fluorocytosine, which was converted into thymine (T) after bisulfite treatment. On the other hand, 5mC was not affected by the oxidation step and consequently discrimination between the two marks was achieved. Bioinformatics analysis and DMR analysis were performed using Genomic Regions Enrichment of Annotations Tool (GREAT) and CGmap Tools.

Results: DMR of 5mC 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. illustrates the increase (red) and decrease (blue) in 5 mC. 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 AML samples and normal cells (Table 1).

Conclusion: The percentage of both 5mC and 5hmC epigenetic marks were not significantly different between AML and normal cells. However, DMR of both 5mC and 5hmC in AML patient samples were identified. The impact of these DMR on the expression of AML-relevant genes is currently under investigation.

Acknowledgements: Research reported in this abstract was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Number P20GM103434 to the West Virginia IDeA Network of Biomedical Research Excellence (WV-INBRE). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Figure 1. DMR in AML primary cells. AML-Normal>0.1 indicates that regions with greater than 10% difference only are included.

Table 1. Percentage of 5mC and 5hmC in AML and normal cord blood cells.