Homozygous deletion and methylation of 5′ CpG islands of p16 and p15 in MM. Lanes 1 through 12 in (B) and (C) correspond to patients no. 1 through 12 as described in the tables. (A) Southern blot analysis with the p16 exon 1 probe for normal samples (blood and BM aspirate). Lane e is normal blood DNA restricted with EcoRI alone (the 4.3-kb band simulates the fragment size seen as if the SacII sites are methylated). Lanes 1 through 12 (normal blood) and lane 13 (marrow) are DNA all restricted with EcoRI plus SacII. All lanes show no methylation band but two smaller fragments of 3.3 and 0.3 kb, which indicate absence of methylation. (B) Southern blot analysis with the p16 exon 1 probe for MM. For reference, lanes a and b are, respectively, normal blood DNA restricted with EcoRI alone (lane a, a 4.3-kb fragment simulates the fragment size seen as if the SacII sites are methylated) and EcoRI plus SacII (lane b, two smaller fragments of 3.3 and 0.3 kb, representing unmethylation status). Lanes 1 through 12 are MM DNA all restricted with EcoRI and SacII. Lanes 4, 7, and 9 show no retention of the 4.3-kb methylation bands and hence unmethylated states. All others show aberrant methylation with mixtures of 4.3-, 3.3-, and 0.3-kb fragments. (C) Southern blot analysis with the p15 exon 1 probe for MM. Similarly, lanes c and d are, respectively, normal blood DNA restricted with HindIII alone (lane c, a 2.8-kb fragment simulates the fragment size seen as if the Eag I site is methylated) and HindIII plus Eag I (lane d, two smaller fragments of 2.2 and 0.5 kb, represent unmethylation status). *An unidentified fragment recognized by the p15 exon 1 probe. Lanes 1 through 12 are MM DNA all restricted with HindIII and Eag I. Lanes 4, 7, 9, and 10 show 2.2- and 0.5-kb fragments and, hence, unmethylated states. All other lanes show the 2.8-, 2.2-, and 0.5-kb fragments and, hence, the presence of aberrant methylation.