Fig. 2

The results of methylation and expression analyses. a Methylation indices (MIs, the ratios of methylated CpGs at each CpG site) obtained by pyrosequencing for 49 CpGs. Black circles represent the mean MIs calculated after four times of analyses in this patient, and gray vertical bars indicate the reference ranges (minimum–maximum) obtained from 50 control subjects. b Heatmap indicating the Δβ values for 753 CpG sites examined by the HumanMethylation450 BeadChip. A single row indicates a single CpG site. The methylation levels of CpG sites are classified into nine categories based on Δβ values. For the formal nomenclature of examined DMRs/loci, see Monk et al. [22]. c Bisulfite sequencing analysis for the MEG3/DLK1:IG-DMR (CG4) and MEG3:TSS-DMR (CG7). Each line indicates each clone, and filled and open circles represent methylated and unmethylated cytosines at the CpG dinucleotides, respectively. The four CpGs at the MEG3/DLK1:IG-DMR and five CpGs at the MEG3:TSS-DMR highlighted in orange have also been examined by pyrosequencing. Since the CG4 region contains a G/A SNP (rs12437020), genotyping data for this SNP are also shown; the leukocytes and placental samples are derived from different control subjects as indicated by different genotyping data. d Quantitative real-time PCR analysis using immortalized lymphocytes. Shown are relative mRNA expression levels of MEG3, H19, GNAS-A/B, and miR134-5p (mean ± SE). The expression studies were performed three times for each sample