Reduction of p15 hypermethylation in a patient with high-risk MDS treated with decitabine.

Ms-SNuPE analysis was performed on bone marrow MNCs of patient 016 (RAEB-t, refractory anemia with excess of blasts in transformation) before (pre), after one (DAC1) and 4 courses (DAC4) of decitabine treatment, respectively. CML-145 (peripheral blood MNCs from a patient with blast crisis CML and known p15hypermethylation27), normal peripheral blood cells (white blood cells [WBCs]), and sperm DNA served as positive and negative controls, respectively. Using a 15% denaturing polyacrylamide gel (7 M urea), a band in the C lane resulting from the Ms-SNuPE labeling represents the amount of methylated cytosines (signal from quantitative incorporation of [³²P]dCTP), while a band in the T lane is the product of [³²P]dTTP incorporation at an unmethylated cytosine molecule at one of the 3 CpG sites examined (12-, 14-, and 17-bp bands represent the signals of the 3 oligonucleotides of respective lengths).27 p15methylation (given in percentages below the C lanes) was determined by quantifying the radioactive signal incorporated into the 3 labeled primers during the primer-extension step of Ms-SNuPE. Counts were measured using a phosphorimager. The average of all 3 CpG sites was calculated, subtracted from the lane background, and expressed as a C/C + T ratio as described in “Patients, materials, and methods.”