Table 1.
Patient characteristics for cases assessed by bisulfite pyrosequencing.
Figure 1.
CpG sites on the Illumina Infinium HumanMethylation450 BeadChip array across the NR3C1 gene region.
Upper graph: DNA methylation at each CpG site in control (n = 19) and early onset pre-eclampsia (EOPET; n = 19) placentae. Lower graph: enlargement of region associated with CpG island containing multiple alternative first exons (black boxes). The Illumina CpG probe identifier is indicated by cg# and the position of CpG sites on the graphs are not to scale. *P<0.05.
Table 2.
Illumina Infinium HumanMethylation450 BeadChip array data.
Figure 2.
Placental DNA methylation of genes involved in cortisol signalling and bioavailability.
DNA methylation at CpG sites within A) exon 1D promoter of the nuclear receptor subfamily 3, group C, member 1 (NR3C1), B) corticotropin releasing hormone (CRH) and C) CRH binding protein (CRHBP) genes. Control, early onset pre-eclampsia (EOPET), late onset pre-eclampsia (LOPET) and normotensive intrauterine growth restriction (nIUGR) placentae are compared. Median and interquartile ranges are given based on average assay CpG methylation measured by bisulfite pyrosequencing. *P<0.05, **P<0.01.
Figure 3.
Placental DNA methylation of genes involved in steroidogenesis.
DNA methylation at CpG sites within A) CYP11A1, B) 3β-hydroxl-delta-5-steroid dehydrogenase type I (HSD3B1), C) TEA domain family member 3 (TEAD3) and D) CYP19 genes. Control, early onset pre-eclampsia (EOPET), late onset pre-eclampsia (LOPET) and normotensive intrauterine growth restriction (nIUGR) placentae are compared. Median and interquartile ranges are given based on average assay CpG methylation measured by bisulfite pyrosequencing. ***P<0.001.
Figure 4.
Placental DNA methylation of the 11β-hydroxysteroid dehydrogenase type 2 (HSD11B2) promoter.
Control, early onset pre-eclampsia (EOPET), late onset pre-eclampsia (LOPET) and normotensive intrauterine growth restriction (nIUGR) placentae are compared. Mean ± SD DNA methylation values are given for consecutive CpG sites in HSD11B2 assay 1 (A) and assay 2 (B) measured by bisulfite pyrosequencing.
Table 3.
Illumina HT-12v4 Expression BeadChip gene expression array data for genes followed up by bisulfite pyrosequencing.
Figure 5.
NR3C1 isoform expression in the human placenta.
Expression of NR3C1 exon 1C and 1D alternative transcripts in the placenta across gestation. A cDNA reaction prepared from adult male hippocampus RNA was used as a positive control. The negative control consisted of a cDNA reaction omitting reverse transcriptase. NR3C1 exon 1C and 1D PCR amplicons were 288 base pairs (bp) and 159 bp in length, respectively.
Figure 6.
Model of altered stress and hormonal signalling gene-pathways in pre-eclampsia.
Left hand side: summary of altered DNA methylation at specific CpG sites across candidate genes, where black and white circles indicate gain or loss of methylation, respectively. Coding regions are indicated by black rectangles and transcription factor (TF) binding motifs are represented by grey circles and include yin yang 1 (YY1; putative), activated protein 2 (AP2; known) and transcription enhancer factor 5 (TEF5; known). Bent lines represent transcription start sites: arrow or blunt heads hypothesise increased or decreased expression, respectively. Green bars represent CpG islands. Annotations are of approximate location and distance. Right hand side: hypothesised model of the downstream effects of altered placental DNA methylation at candidate genes. NR3C1: nuclear receptor subfamily 3, group C, member 1, CRH: corticotropin releasing hormone, CRHBP: CRH binding protein, HSD3B1∶3β-hydroxy-delta-5-steroid dehydrogenase type 1, TEAD3: TEA domain family member 3, GR: glucocorticoid receptor, HSD11B2∶11β-hydroxysteroid dehydrogenase type 2, P450scc: P450 side chain cleavage, 3βHSD1∶3β-hydroxysteroid dehydrogenase type 1.