Blood DNA Methylation Across Pregnancy Offers New Clues to Preeclampsia Risk

Preeclampsia remains one of the most feared complications of pregnancy, striking roughly five to seven percent of expectant mothers and standing among the leading causes of pregnancy-related illness and death in the United States. Despite decades of intensive research, clinicians still cannot reliably predict who will develop the condition, and the only definitive treatment remains delivery of the dysfunctional placenta. Now, a team of researchers led by investigators at the University of Pittsburgh has taken one of the most detailed looks yet at the epigenetic fingerprints that precede the disease, tracking chemical modifications to DNA in maternal blood across all three trimesters of pregnancy. Their findings, published in Epigenetics Communications, reveal tantalizing hints of molecular differences between women who developed preeclampsia and those who experienced uncomplicated pregnancies, while also underscoring just how difficult it is to pin down reliable early warning signs for this notoriously heterogeneous syndrome.

The study focused on DNA methylation, a chemical modification in which methyl groups are attached to cytosine bases at specific locations in the genome, known as CpG sites. Methylation patterns act as a dynamic layer of gene regulation, influencing which genes are switched on or off without altering the underlying genetic code. Because these patterns can shift in response to disease processes, researchers have long suspected that methylation signatures in peripheral blood might reveal the biological pathways that go awry in preeclampsia, or even serve as biomarkers for predicting risk, identifying disease subtypes, or monitoring response to therapy. What set this investigation apart was its longitudinal design. Most previous epigenetic studies of preeclampsia sampled blood only after symptoms had appeared, capturing the aftermath of disease rather than its origins. By collecting blood samples in each trimester from women before any signs of illness, the team hoped to catch the earliest molecular ripples of the disorder.

The discovery phase drew on participants from the Prenatal Exposures and Preeclampsia Prevention Project, a NICHD-funded cohort recruited at UPMC Magee-Women’s Hospital between 2008 and 2014. The researchers assembled 56 women, half of whom went on to develop preeclampsia and half of whom remained normotensive throughout pregnancy, delivered at term, and birthed normally grown infants without proteinuria. Critically, each case was matched one-to-one with a control on self-identified race, pre-pregnancy body mass index, smoking history, and gestational age at sample collection, minimizing the influence of factors known to confound methylation measurements. Genome-wide methylation data were generated from the longitudinal blood samples using the Illumina Infinium MethylationEPIC Beadchip, an array that interrogates more than 850,000 CpG sites across the genome. After rigorous quality control removed problematic probes and samples, more than 700,000 sites remained available for analysis.

To squeeze signal out of noisy high-dimensional data, the team employed surrogate variable analysis, a statistical technique that identifies and removes unwanted sources of variation such as batch effects and the shifting proportions of different white blood cell types across pregnancy. This cell-type heterogeneity is a notorious pitfall in blood-based epigenetic studies, because methylation patterns differ sharply between immune cell populations, and pregnancy itself remodels the maternal immune system. The epigenome-wide association study was conducted separately within each trimester, regressing methylation values on preeclampsia status while adjusting for the surrogate variables. The results were sobering but not empty: no individual CpG site reached the stringent genome-wide significance threshold of 9 multiplied by 10 to the power of negative 8, yet 16 sites, distributed as five in the first trimester, one in the second, and ten in the third, met the suggestive threshold of 1 multiplied by 10 to the power of negative 5.

Recognizing that testing each CpG in isolation sacrifices statistical power, the researchers also searched for differentially methylated regions, contiguous stretches of the genome where neighboring CpG sites show consistent methylation differences between cases and controls. Using the dmrff method, which aggregates association statistics across nearby sites, they identified three significant regions in each trimester, nine in total, all surviving strict Bonferroni correction for multiple testing. In the first trimester, the top region mapped to RAB18 on chromosome 10; in the second, the strongest signal fell within NCOA4, a gene involved in iron metabolism, on the same chromosome; and in the third trimester, the leading region encompassed TRAF3IP2 and its antisense long noncoding RNA TRAF3IP2-AS1 on chromosome 6. That final region contained the two strongest individual hits from the third-trimester analysis, making it the natural candidate to carry forward into replication testing.

The biology of TRAF3IP2 makes it a compelling suspect in preeclampsia. The gene encodes an adaptor protein that links two potent inflammatory signaling pathways, interleukin-17 and transforming growth factor beta, both of which have been implicated in the maladaptive immunomodulation thought to underlie the disease. Recent work has also shown that TRAF3IP2-AS1 regulates interleukin-17A signaling and TRAF3IP2 expression through recruitment of the splicing factor SRSF10. Adding weight to the connection, an earlier study found that TRAF3IP2 was differentially methylated in umbilical cord blood from pregnancies affected by early-onset preeclampsia. Other suggestive hits from the discovery scan also pointed toward genes with plausible roles in pregnancy biology, including NOTCH4, a regulator of placental vascular development; COQ10A, involved in coenzyme Q metabolism; AQP11, an aquaporin water channel; SEPN1, a selenoprotein; and TFF3, a trefoil factor. Four of these six genes had previously been linked to preeclampsia in earlier research.

Replication, however, proved elusive. Because only third-trimester samples were available from an independent cohort, the team focused their replication efforts on the two top third-trimester CpG sites, cg16155413 and cg21882990, within the TRAF3IP2 region. They first validated their discovery findings by re-measuring methylation at these sites in the original discovery samples using pyrosequencing, a different laboratory platform. The two technologies correlated well, with Pearson correlations of 0.86 and 0.82 for cg16155413 and its adjacent site, and the validation sample showed significantly higher methylation in preeclampsia cases at both sites, mirroring the discovery results. But when the researchers turned to the independent replication sample of 114 women from earlier iterations of the same cohort, the association vanished. Methylation levels were actually slightly lower, though not significantly so, in cases compared with controls, and the direction of effect reversed relative to the discovery sample.

The failure to replicate does not necessarily condemn the original finding as a false positive. The discovery and replication cohorts differed in ways that could plausibly affect methylation signals. The discovery sample was composed predominantly of women who self-identified as Black, roughly 72 to 75 percent, while the replication sample was overwhelmingly composed of women who self-identified as White, around 90 percent. The replication participants also had lower average body mass index, greater maternal age, and different distributions of gestational age at birth. Because DNA methylation is well documented to vary by self-reported race and ancestry, these demographic differences could easily mask or reverse a genuine signal. The researchers also noted that the replication analyses could not apply the same surrogate variable adjustment for cell-type heterogeneity, since genome-wide methylation data were not available for the independent samples, leaving a potentially important confounder uncorrected.

The study’s strengths are considerable. It is the largest investigation to date of blood-based DNA methylation in preeclampsia, and its longitudinal design, careful one-to-one matching, stringent quality control, and sophisticated statistical treatment of cell-type heterogeneity and multiple testing set a high methodological bar. Yet the same rigor carries a cost: strict correction for multiple testing may have obscured real but modest signals in a modestly sized sample. The authors also emphasize that preeclampsia is not one disease but a constellation of subtypes, including early-onset and late-onset forms with distinct underlying mechanisms. Pooling all cases into a single group, as this study necessarily did given its size, may dilute subtype-specific methylation signatures and complicate the search for clinically useful biomarkers. Future cohorts will need to be substantially larger and stratified by subtype to untangle this heterogeneity.

For now, the nine differentially methylated regions that survived Bonferroni correction stand as the study’s most durable contribution, a roadmap of genomic neighborhoods, spanning chromosomes 2, 5, 6, 10, 14, 15, 17, and 19, that deserve systematic follow-up in larger and more diverse populations. The TRAF3IP2 region in particular remains a biologically plausible candidate that warrants re-examination with genome-wide data in independent cohorts. The broader lesson may be that a single universal methylation biomarker for preeclampsia is unlikely to exist, and that progress will depend on embracing the syndrome’s complexity rather than averaging over it. As the researchers conclude, additional research is warranted not only for the suggestive hits in the first and second trimesters, which could not be tested in replication, but for the full set of significant regions identified across pregnancy. Each of these epigenetic waypoints may ultimately help illuminate the pathways that transform a healthy pregnancy into a hypertensive crisis, and perhaps, one day, allow clinicians to intervene before symptoms ever begin.

Subject of Research: Longitudinal epigenome-wide association study of blood DNA methylation in preeclamptic versus normotensive pregnancy

Article Title: A longitudinal epigenome-wide association study of preeclamptic and normotensive pregnancy

Article References: A longitudinal epigenome-wide association study of preeclamptic and normotensive pregnancy. (n.d.). https://doi.org/10.1186/s43682-022-00014-w

Image Credits: AI Generated

DOI: 10.1186/s43682-022-00014-w

Keywords: preeclampsia, DNA methylation, epigenetics, EWAS, pregnancy, TRAF3IP2, biomarkers, differentially methylated regions, longitudinal study, hypertensive disorders of pregnancy, surrogate variable analysis, maternal health