Epigenetic changes in myelofibrosis:Distinct methylation changes in the myeloid compartments and in cases with <i>ASXL1</i> 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

Whole-exome sequencing and genome-wide methylation analyses identify novel disease associated mutations and methylation patterns in idiopathic hypereosinophilic syndrome

A thorough understanding of the idiopathic hypereosinophilic syndrome (IHES) and further optimization of diagnostic work-up procedures are warranted. We analyzed purified eosinophils from patients with IHES by next-generation whole-exome sequencing and compared DNA methylation profiles from reactive eosinophilic conditions to known clonal and suspected clonal eosinophilia. Somatic missense mutations in cancer-related genes were detected in three IHES patients. These included the spliceosome gene PUF60 and the cadherin gene CDH17. Furthermore, reactive eosinophilia samples could be differentiated from known- and suspected clonal eosinophilia samples based on 285 differentially methylated CpG sites corresponding to 128 differentially methylated genes. Using Ingenuity pathway analysis, we found that differentially methylated genes were highly enriched in functional pathways such as cancer, cell death and survival, and hematological disease. Our data show that a subset of IHES may be of clonal origin not related to the classical molecular aberrations of FGFR, PDGFRA/B, or T-cells, and that the initiating hits could be point mutations in a variety of genes, including spliceosome mutations or hypermethylated tumor suppressor genes. In addition, we identified a DNA methylation signature that is relevant for distinguishing clonal and suspected clonal eosinophilia from reactive eosinophilia per se, which may be useful in daily clinical work

Investigation of MGMT and DAPK1 methylation patterns in diffuse large B-cell lymphoma using allelic MSP-pyrosequencing

The tumor suppressor genes MGMT and DAPK1 become methylated in several cancers including diffuse large B-cell lymphoma (DLBCL). However, allelic methylation patterns have not been investigated in DLBCL. We developed a fast and cost-efficient method for the analysis of allelic methylation based on pyrosequencing of methylation specific PCR (MSP) products including a SNP. Allelic methylation patterns were reliably analyzed in standards of known allelic methylation status even when diluted in unmethylated DNA to below 1% methylation. When studying 148 DLBCL patients MGMT and DAPK1 methylation was observed in 19% and 89%, respectively, and among methylated and heterozygous patients 29% and 55%, respectively, were biallelically methylated. An association between the T-allele of the rs16906252 SNP and MGMT methylation was observed (p-value = 0.04), and DAPK1 methylation of the A-allele was associated with shorter overall survival (p-value = 0.006). In future cancer research allelic MSP-pyrosequencing may be used to study a wide range of other loci