Fig. 3

Allelic DNA methylation and expression analyses and quantitative expression analysis of H19 and IGF2 for cases 1 to 3. a Placenta-specific paternal hypomethylation at H19-DMR in case 1 and case 2 revealed by clone-based bisulfite sequencing with allelic discrimination. Open and closed circles represent unmethylated and methylated cytosines, respectively. Each row of circles corresponds to an individual clone sequenced. Pla and CB denote placenta (chorionic plate) and cord blood, respectively. Parental alleles were distinguished based on the genotypes of rs2107425 and rs10732516 for the r1 subregion and of rs2251312 and rs568497589 for the r3 subregion. Cases and their parents were genotyped for four SNPs to find informative ones to distinguish parental alleles. b Biallelic expression of H19 in the chorionic plate of case 2. The upper sequencing electropherograms show the genotype at rs2067051 (an exonic A/G SNP within the H19 gene) in case 2, a control chorionic plate sample (from the control set I), and their parents. The bottom electropherograms show the sequencing results of RT-PCR amplicons spanning rs2067051. The nucleotide position corresponding to rs2067051 is marked by an asterisk in each panel. c Intra-individual correlation of expression levels of H19 and IGF2 in placenta. Normalized expression levels of H19 and IGF2 relative to those of a control are plotted for a subset of samples from the case/control set I (left) and from the case/control set II (right). The numbers of cases and controls are 27 controls (black), 17 FGR cases (blue), 2 FGR/PIH cases (pink), and 2 PIH cases including case 2 (red) from the case/control set I, and 6 controls, a FGR case, and a PIH case (case 3) from the case/control set II. Normalized expression levels were determined by the delta-delta Ct method using ACTB as a normalization control. Coefficient of determination calculated by Excel is shown in each plot (case 2 and case 3 were excluded from the calculation). d Relative expression ratios of H19/IGF2 in chorionic plate samples from the case/control set I (left) and in chorionic villi samples from the case/control set II (right). The expression ratios of H19 and IGF2 are shown for controls (n = 27, black), FGR cases (n = 17, blue), PIH/FGR cases (n = 2, pink), and PIH cases (n = 2 including case 2, red) from the case/control set I (left) and for controls (n = 6), a FGR case and a PIH case (Case 3) from the case/control set II. Ratios relative to one control sample shown leftmost are shown