Methylation status of the p15^INK4Bgene in patients with PNH. (A) Methylation status of thep15^INK4B gene in a patient (UPN 14) with acute myelogenous leukemia (AML) evolved from PNH. (a) Unmethylated DNA-specific and methylated DNA-specific MSP primers produced 162-bp and 154-bp products, respectively. Bone marrow (BM) samples at PNH (BM-P, polymorphonuclear cells; BM-M, mononuclear cells), his colon cancer, and normal colon tissue resected 1 year after PNH presentation showed unmethylated p15^INK4B gene. In contrast, leukemic blasts showed hypermethylation of thep15^INK4B gene. (b) Southern blotting also showed the methylated status in leukemic blasts⁴; lanes 1 and 2, control B lymphocytes. Lane 3, patient’s leukemic blasts. (B) Methylation status of the p15^INK4B gene in patients with PNH. All samples obtained from peripheral blood (PB) showed unmethylated pattern. PB-P, polymorphonuclear cells; PB-M, mononuclear cells; PB-T, T lymphocytes; SM, size marker (ØX174/HaeIII). UPNs are common to those in our previous report.5 (C) Methylation status of the p15^INK4B gene in control samples. Two healthy volunteers (HV) showed unmethylated pattern. A patient with overt leukemia evolved from MDS (MDS-OL) showed intense methylation in both PB-M and PB-P populations, whereas a patient with refractory anemia (RA) showed faint methylation. ML1 and HL60 were completely methylated and unmethylated, respectively, as previously reported.4