Aims: Myelodysplastic syndromes (MDS) constitute a heterogeneous group of disorders with hematopoiesis in disarray and with preponderance to transformation to AML. In contrast to the situation in de novo AML patients, the hematopoiesis in MDS patients is often though to encompass most cell lineages with few non-malignant cells in the bone marrow due to displacement by the MDS clone. While the involvement of the myeloid sublineages has been relatively easy to verify in semi-solid CFU assays, that of the lymphoid lineages has required advanced flow sorting techniques in combination with FISH assays in the minority of MDS patients with a well-known genetic aberration such as 5q-, -7 or +8. Moreover, the latter studies have usually been restricted to the description of the involvement of the B-cell lineage. Transcriptional silencing of tumor suppressor genes by promoter hypermethylation is associated with hematological malignancy, including MDS. In a patient with a very immature MDS that was found to be hypermethylated in the p15, HIC, E-cadherin, and Estrogen receptor genes, we wanted to elucidate the methylation status in both myeloid and lymphoid cells.

Material and methods: Mononuclear cells from a 69-year-old female MDS patient with RAEB was analyzed. Using magnetic cell sorting, CD34 and CD3 positive cells were isolated from BM-MNC and PB-MNC, respectively. The purity of the isolated cell fractions was determined by flow cytometry (CD34+: 95% and CD3+: 84%). DNA isolated from the cells was treated with sodium bisulfite and analyzed for promoter hypermethylation in the p15, HIC, E-cadherin, and Estrogen receptor genes by means of Bisulfite-Denaturing Gradient Gel Electrophoresis (Bisulfite-DGGE).

Results: All cell fractions were found to be hypermethylated in the four genes analyzed. 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, total PB-MNC, CD34+ cells and CD3+ cells.

Conclusion: Because the methylation band patterns were the same in all cell fractions, it seems unlikely that methylation has occurred independently in progenitors committed for myeloid and lymphoid maturation, respectively. We therefore conclude that promoter hypermethylation in this patient has occurred in a stem cell with potential for both myeloid and lymphoid maturation. This methodology should provide an easy way to assess the extent of clonality of hematopoiesis in MDS patients.

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