Epigenetic changes in myelofibrosis: Distinct methylation changes in the myeloid compartments and in cases with ASXL1 mutations

Abstract

This is the first study to compare genome-wide DNA methylation profiles of sorted blood cells from myelofibrosis (MF) patients and healthy controls. We found that differentially methylated CpG sites located to genes involved in 'cancer' and 'embryonic development' in MF CD34+ cells, in 'inflammatory disease' in MF mononuclear cells, and in 'immunological diseases' in MF granulocytes. Only few differentially methylated CpG sites were common among the three cell populations. Mutations in the epigenetic regulators ASXL1 (47%) and TET2 (20%) were not associated with a specific DNA methylation pattern using an unsupervised approach. However, in a supervised analysis of ASXL1 mutated versus wild-type cases, differentially methylated CpG sites were enriched in regions marked by histone H3K4me1, histone H3K27me3, and the bivalent histone mark H3K27me3 + H3K4me3 in human CD34+ cells. Hypermethylation of selected CpG sites was confirmed in a separate validation cohort of 30 MF patients by pyrosequencing. Altogether, we show that individual MF cell populations have distinct differentially methylated genes relative to their normal counterparts, which likely contribute to the phenotypic characteristics of MF. Furthermore, differentially methylated CpG sites in ASXL1 mutated MF cases are found in regulatory regions that could be associated with aberrant gene expression of ASXL1 target genes.

Figure 1

Venn diagram showing the overlap of differentially methylated CpG sites between MF cell populations. The CD34+ cell population is blue, the MF granulocyte population is yellow, and the MF mononuclear cell population is red. Five differentially methylated CpG sites overlapped between the three cell populations.

Figure 2

Validation of the methylated genes in a validation MF cohort. The DNA methylation level of 2–8 CpG sites annotated to four genes (ZNF577, WT1, LEP, and TRIM59) was validated in a validation MF cohort consisting of 30 MF patients where DNA had been isolated from whole blood. Hypermethylation of the ZNF577, LEP, and TRIM59 promoter regions and the WT1 gene body was verified using pyrosequencing (P ≤ 0.001 for all genes analyzed, Mann-Whitney test).

Figure 3

RPMM clustering of the granulocytes and their healthy age-matched counterparts with overlaid mutational status. Fifteen samples were analyzed for mutations in ASXL1, TET2, IDH1, IDH2, DNMT3A, CALR, JAK2, and MPL, while sample 13 was only analyzed for JAK2 mutations. The upper purple panel: Dynamic International Prognostic Scoring System (DIPSS). *The blast count was not available for MF patient F14. The middle black and red panel: Mutational status (black represents a mutation). Mutations were found for ASXL1, TET2, JAK2, CALR, and MPL. Lower panel: Hierarchical clustering of methylation levels in granulocytes from MF patients and healthy age-matched controls. β values range from 0 (blue; unmethylated) to 1 (red; methylated). Columns represent samples and rows represent differentially methylated CpG sites. Euclidean distance and complete linkage were used to study the cluster pattern of differential methylated probes. None of the mutations analyzed were associated with a DNA methylation-based subgrouping.

Figure 4

Hierarchical clustering of 308 differentially methylated CpG sites in MF CD34+ cells associated with ASXL1 mutations using Pearson correlation and Average distance. Green indicates hypomethylated CpG sites and red indicates hypermethylated CpG sites. Columns represent the 15 MF patients analyzed. Rows represent differentially methylated CpG sites in MF CD34+ cells in ASXL1 mutated (n = 7) and ASXL1 non-mutated (_NM) (n = 8) cases.

Figure 5

The relative distribution of the differentially methylated CpG sites associated with ASXL1 mutations in CD34 + MF cells. (A) Functional genomic distribution in gene body, 3’UTR, intergenic, and promoter; and (B) mapping according to the CpG density to islands, shelf, shore, and others/open sea. The majority of CpG sites were mapped to CpG shores (37%) and CpG islands (36%). (C) ASXL1 associated differentially methylated CpG sites were enriched in regions enriched for the histone marks H3K4me1 (P = 0.004), H3K27me3 (P = 1.10E-05), and the bivalent mark H3K27me3 plus H3K4me3 (P = 2.00E-03) in healthy CD34+ cells.