Widespread DNA hypomethylation at gene enhancer regions in placentas associated with early-onset pre-eclampsia

Abstract

Pre-eclampsia is a serious complication of pregnancy that can affect both maternal and fetal outcomes. Early-onset pre-eclampsia (EOPET) is a severe form of pre-eclampsia that is associated with altered physiological characteristics and gene expression in the placenta. DNA methylation is a relatively stable epigenetic modification to DNA that can reflect gene expression, and can provide insight into the mechanisms underlying such expression changes. This case-control study focused on DNA methylation and gene expression of whole chorionic villi samples from 20 EOPET placentas and 20 gestational age-matched controls from pre-term births. DNA methylation was also assessed in placentas affected by late-onset pre-eclampsia (LOPET) and normotensive intrauterine growth restriction (nIUGR). The Illumina HumanMethylation450 BeadChip was used to assess DNA methylation at >480 000 cytosine-guanine dinucleotide (CpG) sites. The Illumina HT-12v4 Expression BeadChip was used to assess gene expression of >45 000 transcripts in a subset of cases and controls. DNA methylation analysis by pyrosequencing was used to follow-up the initial findings in four genes with a larger cohort of cases and controls, including nIUGR and LOPET placentas. Bioinformatic analysis was used to identify overrepresentation of gene ontology categories and transcription factor binding motifs. We identified 38 840 CpG sites with significant (false discovery rate <0.01) DNA methylation alterations in EOPET, of which 282 had >12.5% methylation difference compared with the controls. Significant sites were enriched at the enhancers and low CpG density regions of the associated genes and the majority (74.5%) of these sites were hypomethylated in EOPET. EOPET, but not associated clinical features, such as intrauterine growth restriction (IUGR), presented a distinct DNA methylation profile. CpG sites from four genes relevant to pre-eclampsia (INHBA, BHLHE40, SLC2A1 and ADAM12) showed different extent of changes in LOPET and nIUGR. Genome-wide expression in a subset of samples showed that some of the gene expression changes were negatively correlated with DNA methylation changes, particularly for genes that are responsible for angiogenesis (such as EPAS1 and FLT1). Results could be confounded by altered cell populations in abnormal placentas. Larger sample sizes are needed to fully address the possibility of sub-profiles of methylation within the EOPET cohort. Based on DNA methylation profiling, we conclude that there are widespread DNA methylation alterations in EOPET that may be associated with changes in placental function. This property may provide a useful tool for early screening of such placentas. This study identifies DNA methylation changes at many loci previously reported to have altered gene expression in EOPET placentas, as well as in novel biologically relevant genes we confirmed to be differentially expressed. These results may be useful for DNA- methylation-based non-invasive prenatal diagnosis of at-risk pregnancies.

Keywords: 450 K array; DNA methylation; placenta; pre-eclampsia.

Figure 1

Principle component analysis based upon values of the 282 significant differentially methylated probes in this study. EOPET and control samples form distinct clusters (circled), with no additional clustering based on the presence of IUGR, severe proteinuria, hemolysis, elevated liver enzymes and low platelet count (HELLP) or gestational diabetes. Sex of sample is indicated by ^m or ^f.

Figure 2

Boxplots from pyrosequencing follow-up of candidate CpGs associated with four genes. (A) INHBA (cg11079619); (B) BHLHE40 (cg20971407); (C) SLC2A1 (cg01924561) and (D) ADAM12 (cg02494582). Percent methylation is indicated on the Y-axis. Analysis of covariance (ANCOVA) adjusting for gestational age followed by Bonferroni corrected pair-wise post hoc analysis was used to determine significance between individual groups *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure 3

DNA methylation levels for cases and controls according to genomic location. Genomic order of CpGs is given on the X-axis. CpG islands are assigned according to UCSC Genome Browser delineations. Each plot represents one candidate gene: INHBA (A), BHLHE40 (B), SLC2A1 (C) and ADAM12 (D). Asterisk indicates candidate CpG site. Candidate CpGs fell into a variety of genomic regions relative to the gene structure.

Figure 4

Methylation at candidate sites is compared with gene expression. Results for four genes are given: INHBA (A), BHLHE40 (B), SLC2A1 (C) and ADAM12 (D). The expected negative correlation in methylation and gene expression exists for BHLHE40, with similar trends in SLC2A1 and ADAM12.

Figure 5

Principle component analysis using CpG sites that are significantly altered in cultured cytotrophoblast exposed to hypoxic conditions for 24 h (Yuen et al., 2013). Control and pre-eclampsia samples had a tendency to cluster apart from each other. Sex of sample is indicated by ^m or ^f.

Figure 6

Comparison of proportions of significant (candidate) CpGs relative to the array as a whole that fall into different genomic elements. The elements compared include high CpG density islands (HC), intermediate CpG density islands (IC), intermediate density CpG islands that are on the shore of an HC (ICshore), low CpG density regions (LC) and annotated genomic enhancers. **P < 0.01 and ***P < 0.001.