Differential global DNA methylation between CN-AML with and without MLL-PTD. (A) Each RLGS autoradiograph was viewed independently by 3 trained individuals who catalogued each spot loss or gain in comparison to a reference RLGS autoradiograph generated using normal donor BM DNA. RLGS data were compiled for all patients and 321 spots were evaluable across all profiles. Global DNA methylation, that is, the number of methylation events observed, was determined for each patient and a comparison of global DNA methylation between MLL-WT and MLL-PTD AML groups, was made using the Wilcoxon rank sum test. Horizontal bars represent the medians. (B) Unsupervised hierarchical clustering of patients was carried out based on RLGS spots that were methylated in at least one patient (265 spots). Jaccard binary similarity metric was used. (C) The 18 RLGS spots with the strongest association, measured by Fisher exact test, between methylation and MLL AML groups are shown. Yellow squares represent a methylated locus; blue squares represent an unmethylated locus; gray squares indicate spot was not evaluable. Columns represent individual patients and rows represent RLGS spots. RLGS spot names are shown on right. Row 5 depicts results for RLGS spot 3D41, that is, a DNA fragment corresponding to a region of the SLC5A8 promoter and exon 1. The corresponding gene names and chromosomal locations, if known and as reported in Supplemental Table 5 from Smiraglia et al²⁷  are also shown. (D) RLGS was carried out as described in “Methods.” The area of the autoradiographs containing spot 3D41 (arrow) were scanned using a Storm 860 phosphorimager (Molecular Dynamics, Amersham Biosciences, Piscataway, NJ) and area with 3D41 enlarged (Photoshop v.8.0, Adobe Systems, San Jose, CA). Representative results are shown for the presence of 3D41 in a primary MLL-WT AML patient sample. (E) Representative RLGS results showing nearly complete loss of 3D41 in a primary MLL-PTD AML patient sample. (F) SLC5A8 mRNA detection in primary AML patient samples that exhibited loss or presence of RLGS spot 3D41.