Array-based methylation analysis using EPIC

We conducted genome-wide methylation analysis using EPIC and obtained β values indicating the methylation levels for 842 CpGs on 78 imprinted DMRs as previously reported [11]. We defined aberrantly methylated DMR based on previous reports [12, 39] (analysis method 1). In brief, the median β value for each CpG within a DMR was determined as the MML (median methylation level) of the DMR. An aberrantly methylated DMR was defined as |MML|> 3 SD obtained from the mean of MML in 16 healthy child controls. The aberrantly methylated DMRs were further classified as mild (ΔMML < 0.1), moderate (0.2 ≥ ΔMML ≥ 0.1), and extreme (ΔMML > 0.2) according to the difference between the MML of each patient and the mean of MML in the controls. In addition, we re-analyzed methylation-array data applying more stringent bioinformatic parameters using the Crawford-Howell t-test [47] and defined the aberrantly methylated DMRs. In brief, we considered a probe as differentially methylated, with an absolute value of ∆β (|∆β|) > 0.1 and a false discovery rate < 0.05. When we detected two or more consecutive probes differentially methylated levels within a DMR (including at least four probes), we defined the DMR as aberrantly methylated (analysis method 2).