Supporting data for "Cord Blood DNA methylation and Cell Type composition are not significantly associated with severe preeclampsia, after confounding adjustment"

Preeclampsia is a severe pregnancy complication that threatens maternal and neonatal health and well-being. Previous studies on epigenome-wide association analysis (EWAS) of preeclampsia produced inconsistent results in cord blood tissues, and one possible explanation is their failure to rigorously adjust for cell proportions, gestational age, or other necessary variables. Here, we calculated the DNA methylation change in cord blood from newborns affected by preeclampsia, using a multi-ethnic cohort from the Hawaii population (24 cases, 38 controls). We comprehensively adjusted for variables such as maternal age, body mass index (BMI), parity, and estimated the cell proportions. We also re-analyzed two previous datasets with adjustments to estimated cell proportions and conducted a pooled analysis by merging all three datasets together to increase the statistical power (58 cases, 71 controls). Lastly, we include idiopathic preterm (preterm delivery with no known reasons) cord blood samples (n=11) to disentangle the effect of severe preeclampsia and small gestational age. We showed that after adjusting cell type proportions and patient clinical characteristics, most of the so-called statistically significant CpG methylation changes associated with severe preeclampsia disappeared in our own data, two public datasets, and the pooled analysis combining all three datasets. This result still holds after including idiopathic preterm samples in the control group. Rather, we found that gestation progression is accompanied by statistically significant proportion changes in several cell types, such as granulocytes, nRBCs, CD8Ts, and B cells, which contribute to most DNA methylation differences between case and control groups. Preeclampsia has interactions on cell proportion changes in granulocytes, monocytes, and nRBCs. In summary, our study shows that the previously reported differentially methylated patterns in cord blood are actually artifacts due to not properly adjusting for cell type heterogeneity, gestational age, and clinical covariates. Severe preeclampsia is not associated with statistically significant DNA methylation changes but changes in cell proportion. This finding alerts to the scientific rigor needed in EWAS.