DNA promoter hypermethylation contributes to the epigenetic silencing of the PRDX2 gene in AML. (A) MSP shows promoter methylation in patients with AML no. 4 and no. 7. In total, 105 AML samples and 49 controls (normal BM, nonmalignant controls) were analyzed by MSP. (B) Overall, 18% of the AML samples was methylated at the PRDX2 promoter as analyzed by MSP. No DNA methylation was detected in nonleukemic controls. (C) Low expression of PRDX2 mRNA transcript in AML (n = 61) compared with normal BM specimens (n = 15) as assessed by real-time RT-PCR analysis on BM specimens obtained from patients with AML and healthy persons. Relative gene expression levels were calculated with the use of standard curves with GAPDH being used as internal standard. Statistical analyses were done with SPSS 12.0 (SPSS Inc), and the nonparametric Mann-Whitney U test was used to compare the expression levels of PRDX2 between AML and controls (P = .01, Mann-Whitney U test). Primer sequences are given in the supplemental information for methods. (D) Western blot analysis shows reduction of PRDX2 protein levels in primary AML samples compared with normal CD34⁺ cells. Actin was used as a loading control. (E) The mRNA expression of different PRDX genes was analyzed in a published microarray dataset with 542 AML and 73 nonleukemic BM specimens. P values were calculated with the Mann-Whitney U test. The Affymetrix probe IDs are indicated as well. Two probes of similar quality were present for each PRDX3 and PRDX5. Both led to similar results so only one each was shown in the figure.