Delineation of Promoter Methylation in Patients with Myelodysplastic Syndromes: Widespread and Concurrent Hypermethylation of Multiple Genes and Increased mRNA Expression of the DNA Methyltransferases DNMT1, 3A and 3B.

Transcriptional silencing of tumor suppressor genes by promoter hypermethylation is associated with hematological malignancy, including myelodysplastic syndromes (MDS). Specifically, hypermethylation of the p15 gene has previously been associated with MDS and seems to be acquired with disease progression. The methylation status of other genes associated with the development of myeloid malignancies is, however, largely unknown. We have elucidated this in total bone marrow mononuclear cells (BM-MNC) as well as CD34 enriched cells from 17 patients with different stages of MDS by determining the promoter methylation status in four genes believed to be associated with the development of acute myeloid leukemia (AML), namely those encoding for the p15, HIC, E-cadherin (ECAD), and the Estrogen receptor (ER). Employing Bisulfite-Denaturing Gradient Gel Electrophoresis (Bisulfite-DGGE) we found all four genes to be hypermethylated in MDS, albeit with varying frequencies (19/37, 12/37, 10/37, and 7/37, respectively). Since the Bisulfite-DGGE allows for the detection of heterogeneous methylation patterns we were able to compare these patterns. Similar Bisulfite-DGGE band patterns were observed in total BM-MNC and CD34 cells in all positive patients suggesting that methylation is a process encompassing both immature and end-stage hematopoietic cells. In a total of 37 patients (14 RA, 3 RAS, 11 RAEB, 4 RAEB-t and 5 with AML secondary to MDS) analyzed, we found promoter hypermethylation of the HIC gene to be significantly increased in advanced MDS (p<0.05). To determine the kinetics of promoter methylation in the single patient, bone marrow from eleven MDS patients, where two or more samples were available, were analyzed (observation time between sampling ranged from 43 to 1132 days, median 284 days). These analyses showed that the patterns of methylation are surprisingly stable over time. When two or more samples were analyzed we found that the number of methylated genes only changed in three of the eleven patients. To delineate the process of hypermethylation we determined the mRNA levels of the DNA methyltransferases (DNMT) 1, 3A, and 3B. Interestingly, all three DNMTs were up-regulated in MDS compared to normal bone marrow (DNMT1 and DNMT3A: p<0.01; DNMT3B: p<0.0001) and DNMT3A and 3B were up-regulated in advanced MDS compared to low-risk MDS (p<0.01). Taken together, these results imply an involvement of promoter hypermethylation in MDS disease progression, and DNMT overexpression as a contributing factor for the aberrant methylation in these patients.

Figure: Promoter hypermethylation status over time. A) Development in methylation status in 11 patients. Arrows indicate patients where methylation status changes over time. a) RA, b) RAS, c) RAEB, d) RAEB-t, e) sAML. Black quarters: methylated; white quarters: unmethylated. B) Bisulfite-DGGE experiments for methylation in the p15, HIC , and ER promoters in UPN 2.

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