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.

Allelic methylation levels of the noncoding VTRNA2-1 located on chromosome 5q31.1 predict outcome in AML.

Deletions of chromosome 5q are associated with poor outcomes in acute myeloid leukemia (AML) suggesting the presence of tumor suppressor(s) at the locus. However, definitive identification of putative tumor suppressor genes remains controversial. Here we show that a 106-nucleotide noncoding RNA vault RNA2-1 (vtRNA2-1), previously misannotated as miR886, could potentially play a role in the biology and prognosis of AML. vtRNA2-1 is transcribed by polymerase III and is monoallelically methylated in 75% of healthy individuals whereas the remaining 25% of the population have biallelic hypomethylation. AML patients without methylation of VTRNA2-1 have a considerably better outcome than those with monoallelic or biallelic methylation (n = 101, P = .001). We show that methylation is inversely correlated with vtRNA2-1 expression, and that 5-azanucleosides induce vtRNA2-1 and down-regulate the phosphorylated RNA-dependent protein kinase (pPKR), whose activity has been shown to be modulated by vtRNA2-1. Because pPKR promotes cell survival in AML, the data are consistent with vtRNA2-1 being a tumor suppressor in AML. This is the first study to show that vtRNA2-1 might play a significant role in AML, that it is either mono- or biallelically expressed in the blood cells of healthy individuals, and that its methylation state predicts outcome in AML.

Equitoxic doses of 5-azacytidine and 5-aza-2'deoxycytidine induce diverse immediate and overlapping heritable changes in the transcriptome.

BACKGROUND: The hypomethylating agent 5-Azacytidine (5-Aza-CR) is the first drug to prolong overall survival in patients with myelodysplastic syndrome (MDS). Surprisingly, the deoxyribonucleoside analog 5-Aza-2'deoxycytidine (5-Aza-CdR) did not have a similar effect on survival in a large clinical trial. Both drugs are thought to exert their effects after incorporation into DNA by covalent binding of DNA methyltransferase (DNMT). While 5-Aza-CdR is incorporated into only DNA, 5-Aza-CR is also incorporated into RNA. Here, we have analyzed whether this difference in nucleic acid incorporation may influence the capacities of these drugs to regulate the expression of mRNA and microRNAs (miRNA), which may potentially affect the activities of the drugs in patients. METHODOLOGY/PRINCIPAL FINDINGS: A hematopoietic (HL-60; acute myeloid leukemia) and a solid (T24; transitional cell carcinoma) cancer cell line were treated with equitoxic doses of 5-Aza-CR and 5-Aza-CdR for 24 hrs, and the immediate (day 2) and lasting (day 8) effects on RNA expression examined. There was considerable overlap between the RNAs heritably upregulated by both drugs on day 8 but more RNAs were stably induced by the deoxy analog. Both drugs strongly induced expression of cancer testis antigens. On day 2 more RNAs were downregulated by 5-Aza-CR, particularly at higher doses. A remarkable downregulation of miRNAs and a significant upregulation of tRNA synthetases and other genes involved in amino acid metabolism was observed in T24 cells. CONCLUSIONS/SIGNIFICANCE: Overall, this suggests that significant differences exist in the immediate action of the two drugs, however the dominant pattern of the lasting, and possible heritable changes, is overlapping.