Differential methylation of genes associated with cell adhesion in preeclamptic placentas

Abstract

Preeclampsia (PE), a hypertensive disorder of pregnancy, is hypothesized to be associated with, if not mechanistically related to abnormal placental function. However, the exact mechanisms regulating the pathogenesis of PE remain unclear. While many studies have investigated changes in gene expression in the PE placenta, the role of epigenetics in PE associated placental dysfunction remains unclear. Using the genome-wide Illumina Infinium Methylation 450 BeadChip array, we analyzed gene-specific alterations in DNA methylation in placental biopsies collected from normal pregnant women delivering at term (n = 14), with term PE (≥37 weeks; n = 19) or with preterm PE (<37 weeks, n = 12). Of the 485,582 gene loci on the array, compared to controls, 229 loci were differentially methylated in PE placentas and 3411 loci were differentially methylated in preterm PE (step up p-value <0.05 and >5% methylation difference). Functional annotation of the differentially methylated genes in preterm PE placentas revealed a 32 gene cluster in the cadherin and cell adhesion functional groups (Benjamini p<0.00001). Hypermethylation of CDH11 (p = 0.0143), COL5A1 (p = 0.0127) and TNF (p = 0.0098) and hypomethylation of NCAM1 (p = 0.0158) was associated with altered mRNA expression in preterm PE placentas. Demethylation of first trimester extravillous trophoblast cells resulted in altered CDH11 (p = 0.0087), COL5A1 (p = 0.0043), NCAM1 (p = 0.0260) and TNF (p = 0.0022) mRNA expression. These studies demonstrate aberrant methylation, correlating with disease severity, in PE placentas. Furthermore, we provide evidence that disruption of gene-specific methylation in preterm PE placentas and first trimester trophoblasts is significantly associated with altered gene expression demonstrating that epigenetic modifications early in pregnancy can have effects on trophoblast function contributing to PE.

Figure 1. Principle component analysis (PCA) plots of control vs term and preterm preeclamptic placentas.

PCA plots show the results of the Illumina Infinium Methylation 450 BeadChip Array comparing (A) control placentas to preterm preeclamptic placentas and (B) control placentas to term and preterm preeclamptic placentas. The PCA plot was created prior to eliminating gene probes based on p-value or changes in percent methylation. Distinct clustering is seen between normal controls and preterm preeclamptic placentas indicating significant alterations in methylation status in preterm preeclamptic placentas. Each dot represents a single patient sample. Blue dots designate control placentas, green dots designate preterm preeclamptic placentas and red dots designate term preeclamptic placentas.

Figure 2. Altered methylation status of four genes of interest in term and preterm preeclamptic placentas.

Scatter plots of β values (representing percent methylation) from the Illumina Infinium Methylation 450 BeadChip of array probe CpGs associated with four genes of interest in control versus term and preterm preeclamptic placentas. β values in preterm preeclamptic placentas were significantly altered in (A) Cadherin 11 (CDH11) (cg26624576), (B) Collagen, type V, alpha 1 (COL5A1) (cg14237069), (C) Neuronal Cell Adhesion Molecule 1 (NCAM1) (cg20857767) and (D) Tissue Necrosis Factor (TNF) (cg04425624). Each dot represents a placental sample from each enrolled study patient. Solid bars represent mean of each group. * Statistical significance is based on Step-up (false discovery rate) p-values <0.01.

Figure 3. Altered methylation of cell adhesion genes is associated with changes in mRNA expression.

Changes in methylation status in term and preterm preeclamptic placentas resulted in altered mRNA expression of (A) Cadherin 11 (CDH11), (B) Collagen, type V, alpha 1 (COL5A1), (C) Neuronal Cell Adhesion Molecule 1 (NCAM1) and (D) Tissue Necrosis Factor (TNF). Each dot represents a placental sample from each enrolled study patient. Solid bars represent mean of each group. *p<0.05.

Figure 4. In vitro demethylation of primary first trimester extravillous trophoblast cells results in alterations of mRNA expression.

First trimester extravillous trophoblast cells treated with the demethylating agent 5-aza-2′-deoxycytidine (AZA) versus dimethyl sulfoxide (DMSO, vehicle control) showed significant alterations in mRNA expression of (A) Cadherin 11 (CDH11), (B) Collagen, type V, alpha 1 (COL5A1), (C) Neuronal Cell Adhesion Molecule 1 (NCAM1) and (D) Tissue Necrosis Factor (TNF). Values are mean ± SEM. n = 6 *p<0.05.