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. 2015 Mar 13;7(1):21.
doi: 10.1186/s13148-015-0052-x. eCollection 2015.

Genome-scale Hypomethylation in the Cord Blood DNAs Associated With Early Onset Preeclampsia

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Free PMC article

Genome-scale Hypomethylation in the Cord Blood DNAs Associated With Early Onset Preeclampsia

Travers Ching et al. Clin Epigenetics. .
Free PMC article

Abstract

Background: Preeclampsia is one of the leading causes of fetal and maternal morbidity and mortality worldwide. Preterm babies of mothers with early onset preeclampsia (EOPE) are at higher risks for various diseases later on in life, including cardiovascular diseases. We hypothesized that genome-wide epigenetic alterations occur in cord blood DNAs in association with EOPE and conducted a case control study to compare the genome-scale methylome differences in cord blood DNAs between 12 EOPE-associated and 8 normal births.

Results: Bioinformatics analysis of methylation data from the Infinium HumanMethylation450 BeadChip shows a genome-scale hypomethylation pattern in EOPE, with 51,486 hypomethylated CpG sites and 12,563 hypermethylated sites (adjusted P <0.05). A similar trend also exists in the proximal promoters (TSS200) associated with protein-coding genes. Using summary statistics on the CpG sites in TSS200 regions, promoters of 643 and 389 genes are hypomethylated and hypermethylated, respectively. Promoter-based differential methylation (DM) analysis reveals that genes in the farnesoid X receptor and liver X receptor (FXR/LXR) pathway are enriched, indicating dysfunction of lipid metabolism in cord blood cells. Additional biological functional alterations involve inflammation, cell growth, and hematological system development. A two-way ANOVA analysis among coupled cord blood and amniotic membrane samples shows that a group of genes involved in inflammation, lipid metabolism, and proliferation are persistently differentially methylated in both tissues, including IL12B, FAS, PIK31, and IGF1.

Conclusions: These findings provide, for the first time, evidence of prominent genome-scale DNA methylation modifications in cord blood DNAs associated with EOPE. They may suggest a connection between inflammation and lipid dysregulation in EOPE-associated newborns and a higher risk of cardiovascular diseases later in adulthood.

Keywords: Bioinformatics; Cord blood; DNA methylation; Epigenetics; Preeclampsia.

Figures

Figure 1
Figure 1
Global CpG hypomethylation in cord blood cells is associated with maternal preeclampsia. (A) 3D Principal Components Analysis (PCA) of the methylation M-values from cord blood samples. Preeclampsia-associated cases are shown in red, while controls are in blue; PCA1, PCA2, and PCA3 denote principal components 1, 2, and 3, respectively. The two groups are clearly separated when viewed using the first three principal components. (B) ANOVA plot of clinical factors using the methylation M-values in cord blood samples. Averaged ANOVA F-statistics are calculated for potential confounding factors, including preeclampsia, maternal age, gravida, parity, and baby gender. (C) Volcano plot of all CpG methylation M-values. The y-axis of the negative log10 transformation of the P values obtained from differential methylation analysis of the CpG sites is plotted against the x-axis of the difference in the average M-values between cases and controls. The dashed red line marks the α = 0.05 significance threshold after Benjamini-Hochberg correction of multiple hypothesis tests (MHT), whereas the dashed black line marks the unadjusted α = 0.05 significance threshold.
Figure 2
Figure 2
Technical validation of a set of DM CpG sites using pyrosequencing. (A) The overall correlation plot between the Beta values of seven DM CpG sites obtained from Illumina HumanMethylation450 BeadChip array (y-axis) and methylation percentage from pyrosequencing (x-axis). Five case and five control samples for each CpG site were selected for pyrosequencing validation. (B, C, D, E, F, G, H) Bar plots to compare the average Beta values and pyrosequencing methylation percentage for individual CpG sites in (A): (B) cg26478401, (C) cg26076905, (D) cg11671363, (E) cg06111285, (F) cg06652329, (G) cg08198483, and (H) cg08537847. Values are presented as mean ± standard deviation (s.d.). The CpG sites that have significant P < 0.05 between cases and controls are labeled by (*).
Figure 3
Figure 3
Comparison of CpG site methylation of cord-blood-cell-associated EOPE and normal mothers. Distributions of all CpG site categories on the Illumina HumanMethylation450 BeadChip array (background), all differentially methylated CpG sites, hypomethylated CpG sites, and hypermethylated CpG sites. Categories with significantly different proportions (P < 0.05, Chi-square test) of differential CpG sites vs. expected background (all CpG sites) are labeled by (*). Differences in categorical proportions between hypomethylation and hypermethylation are labeled the same. (A) CpG site proportions by location relative to CpG isle regions. As stated in the “Results” section, six groups are categorized according to Illumina 450 K annotation, namely north shelf, north shore, CpG island, south shore, south shelf, and open sea. (B) CpG site proportions by functional location relative to gene regions. Seven groups are categorized, namely the 200 to 1,500 bp upstream of transcription starting site (TSS1500), the transcription starting site to 200 bp upstream interval (TSS200), 5′ UTR, first exon, gene body, 3′ UTR, and the rest for integenic regions. DM, differential methylation.
Figure 4
Figure 4
Heatmaps of DM CpG sites located in CpG islands (A) and the TSS200 region of genes (B). The rows correspond to CpG sites, while the columns correspond to samples. Case samples are blue, and controls are green. The color of each cell in the heatmap is based on the Beta (β) value of the corresponding CpG site in the corresponding sample, with colors ranging from red (β = 0) to blue (β = 1). The ordering of CpG sites and samples in the heatmaps was determined via unsupervised hierarchical clustering.
Figure 5
Figure 5
Top networks in IPA and EpiMods analysis. (A) IPA network involved in the inflammatory response. (B) EpiMod validation network containing genes involved in an immune/inflammatory process. (C) IPA network involved in cellular development, growth and proliferation, and hematological system development and function. (D) IPA network involved in cell-to-cell signaling, hematological system development and function, and immune cell trafficking. Red and green colors represent hypermethylation and hypomethylation in the promoter of the gene, respectively.

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