As a second approach, probe level analysis was used to observe higher-resolution methylation patterns in distal regions without diluting signals over the core promoter. This analysis extended to the entire set of gene promoters and CpG islands covered by the array. DNA regions with increased or decreased methylation levels were based on signal intensities using a two consecutive probe analysis method, where adjacent probes (on the same DNA strand with each probe covering 50 bp and typically 50 bp apart) were used to assess methylation. Adjacent genes were considered to be hyper- or hypomethylated if they had no fewer than 2 consecutive and consistently differentially methylated primers. A linear mixed model data analysis was used to account for response variability (population average, subject specific effects) as well as random effects. Following adjacent-probe analysis, 1,054 (FDR, ≤30%) regions showed a significant difference in DNA methylation, with 681 having decreased methylation, and 373 with increased methylation.
