Altered Epigenetic Profiles in the Placenta of Preeclamptic and Intrauterine Growth Restriction Patients

Abstract

Intrauterine growth restriction (IUGR) and preeclampsia (PE) are placental pathologies known to complicate pregnancy and cause neonatal disorders. To date, there is a limited number of studies on the genetic similarity of these conditions. DNA methylation is a heritable epigenetic process that can regulate placental development. Our objective was to identify methylation patterns in placental DNA from normal, PE and IUGR-affected pregnancies. DNA was extracted, and bisulfite was converted, prior to being hybridized for the methylation array. Methylation data were SWAN normalized and differently methylated regions were identified using applications within the USEQ program. UCSC's Genome browser and Stanford's GREAT analysis were used to identify gene promoters. The commonality among affected genes was confirmed by Western blot. We observed nine significantly hypomethylated regions, two being significantly hypomethylated for both PE and IGUR. Western blot confirmed differential protein expression of commonly regulated genes. We conclude that despite the uniqueness of methylation profiles for PE and IUGR, the similarity of some methylation alterations in pathologies could explain the clinical similarities observed with these obstetric complications. These results also provide insight into the genetic similarity between PE and IUGR and suggest possible gene candidates plausibly involved in the onset of both conditions.

Keywords: DNA methylation; epigenetics; intrauterine growth restriction; placenta; pre-eclampsia.

Figure 1

Comparative analyses of global profiles for PE, IUGR, and control placental tissues. Unsupervised cluster analysis of regionalized β-values for samples with a dendrogram indicating similarity (A). Scatter plots of regionalized β-values for IUGR vs Control, PE vs Control, and IUGR vs PE, respectively (B). Bar plots of average β-values at five global regions of interest (C).

Figure 2

Comparative methylation profiles and ontological analysis of placental tissues. Circos Plot indicating regions of significant hyper and hypomethylation (FDR > 13) across the genome in PE (outer ring) and IUGR (inner ring) (A). Height of region indicates the size of variance from control (log2ratio). Distance of each region from transcription start site of the most differentially methylated regions (FDR > 40) (B). GO term analysis of the most differentially methylated regions for PE (C).

Figure 3

Shared areas of hypomethylation at key regulatory regions in both PE and IUGR placental tissues. Bar plots indicating methylation beta-values at significantly hypomethylated regions (FDR > 40) in IUGR and PE samples compared with control (A). Venn diagram of each hypomethylated gene promoter region associated with PE and IUGR (B).

Figure 4

Linear regressions comparing FAN1/HLA-L gene promoter hypomethylation to maternal age (MA)/gestational age (GA). Plot legends include adjusted R² values, slopes, and p-values.

Figure 5

FAN1, CRABP1, HIST1H4L and NAPRT1 proteins in IUR, PE and control placentas. A representative western blot for these proteins is shown in (A,B). CRABP1 protein was decreased in the IUGR placenta (C). HIST1H4L protein was decrease while NAPRT1 protein was increased in the PE placenta (D,E). FAN1 protein was increased in IUGR and decreased in PE Placenta (F,G). Representative data are shown with ** p ≤ 0.05.