Targeted bisulfite resequencing of differentially methylated cytosines in pre-eclampsia reveals a skewed dynamic balance in the DNA methylation of enhancers.

Pre-eclampsia (PE) is a major hypertensive disorder of pregnancy. Widespread differentially methylated cytosines (DMCs) with modest changes in methylation level are associated with PE, whereas their cause and biological significance remain unknown. We aimed to clarify DNA methylation patterns around DMCs in 103 placentas using MethylCap targeted bisulfite re-sequencing (MethylCap-seq) assays of 690 selected DMCs. We verified the MethylCap-seq method, then validated 677 (98.1%) of DMCs (vDMCs) in an independent cohort. The validated DMCs were strongly enriched in active placenta-specific enhancers and showed highly dynamic methylation levels. We found high epigenetic heterogeneity between vDMCs and adjacent CpG sites (r2 < 0.2) and a significant decrease in PE in the discovery and replication cohorts (P = 2.00 × 10-24 and 6.43 × 10-9, respectively). We replicated the methylation changes in a hypoxia/reoxygenation cell model. We constructed 112 methylation haplotype blocks and found that the frequencies of unmethylated haplotypes (UMHs) were dynamic with gestational age (GA) and were altered in maternal plasma of patients with PE. Our results uncovered additional DNA methylation features in PE placentas and suggested a model of skewed DNA methylation balance of enhancers in PE.