Fig. 1

DNA methylation analyses for H19-DMR and IGF2-DMRs by COBRA and pyrosequencing assays. a Location of CpG sites within H19-DMR, IGF2-DMR0, and IGF2-DMR2 analyzed. Exons of IGF2 and H19 genes are shown as blue and red squares, respectively. Transcriptional start sites of IGF2 (P0-P4) and H19 are indicated by arrows. The approximate positions of the genomic intervals subjected to DNA methylation analyses within H19-DMR, IGF2-DMR0, and IGF2-DMR2 are shown by light green two-headed arrows. The genomic intervals of IGF2-DMR0 and IGF2-DMR2 were described previously [17]. Closed circles represent CpG sites included in the bisulfite-PCR amplicons subjected to COBRA, pyrosequencing, and bisulfite sequencing analyses. Downward arrowheads in the r1 and r3 subregions within H19-DMR indicate TaqI sites used in COBRA assays. The CpG sites whose methylation level was assessed by pyrosequencing are numbered underneath the closed circles. b, c DNA methylation levels of H19-DMR (r1 and r3 subregions) and IGF2-DMRs in FGR and/or PIH placentas determined by COBRA. The methylation levels were measured for chorionic plate of case group I (n = 140) and control group I (n = 39) (b) and for chorionic villi of case group II (n = 62) and control group II (n = 27) (c). Methylation levels corresponding to ± 3SD of the mean of control samples, our criterion for aberrant DNA methylation, are shown by blue horizontal dashed lines in each panel. The dots for cases 1 and 2 (in b) and for case 3 (in c) are shown in black and with the corresponding number. d, e Methylation levels at H19-DMR and IGF2-DMRs in the placenta of cases 1–3 examined by pyrosequencing. The methylation levels in case 1 (closed triangle), case 2 (closed rectangle), and the average of five controls from the control set I (chorionic plate samples) (open circle) are shown in d. The methylation levels in case 3 (black circle) and the average of five controls from the control set II (chorionic villi samples) (grey circle) in H19-DMR, IGF2-DMR0, and IGF2-DMR2 are shown in e. The numbers for CpG sites shown underneath the plots (d, e) correspond to those depicted in a