Hypomethylation preferentially affects regulatory regions of the genome in GC B cells. (A) Four genes from the GC B-cell signature were selected for validation by MassArray. The results are represented as heatmaps in which the columns correspond to individual samples, while rows represent individual CpGs with color reflecting methylation value. P values are from moderated t test comparing methylation values from all tested CpGs between GC B cells and NBs. The location of MassArray amplicons (blue) and HELP probesets (red) relative to the transcriptional start site (TSS) of each gene (black) is illustrated below each heatmap. (B) The relative transcript abundance of the same 4 genes was measured by QPCR in 3 additional NB and GC B-cell specimens. The y-axis depicts fold expression difference in GC B-cells versus NBs calculated using ddCT method. All 4 genes are expressed at higher levels in GC B-cells, concordant with their hypomethylation. (C) LUMA assays performed on 3 NB and 2 GC B-cell specimens show a mild increase in the abundance of hypomethylated HpaII sites. The y-axis depicts relative signal of HpaII vs MspI signals. (D) Liquid chromatography-mass spectrometry was performed in 3 NB and 2 GC B-cell specimens. The y-axis depicts the percentage of methylcytosine versus total cytosines in each specimen.