Genome-wide methylation profiling of maternal cell-free DNA using methylated DNA sequencing (MeD-seq) indicates a placental and immune-cell signature

Abstract

Background: Placental-originated cell-free DNA (cfDNA) provides unique opportunities to study (epi)genetic placental programming remotely, but studies investigating the cfDNA methylome are scarce and usually technologically challenging. Methylated DNA sequencing (MeD-seq) is well compatible with low cfDNA concentrations and has a high genome-wide coverage. We therefore aim to investigate the feasibility of genome-wide methylation profiling of first trimester maternal cfDNA using MeD-seq, by identifying placental-specific methylation marks in cfDNA. Methods: We collected cfDNA from nonpregnant controls (female n = 6, male n = 12) and pregnant women (n = 10), first trimester placentas (n = 10), and paired preconceptional and first trimester buffy coats (total n = 20). Differentially methylated regions (DMRs) were identified between pregnant and nonpregnant women. We investigated placental-specific markers in maternal cfDNA, including RASSF1 promoter and Y-chromosomal methylation, and studied overlap with placental and buffy coat DNA methylation. Results: We identified 436 DMRs between cfDNA from pregnant and nonpregnant women, which were validated using male cfDNA. RASSF1 promoter methylation was higher in maternal cfDNA (fold change 2.87, unpaired t-test p