Genome-wide methylation profiling of cell-free DNA in maternal plasma using Methylated DNA Sequencing (MeD-seq)

BackgroundPlacental-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. MethodsWe collected cfDNA from non-pregnant 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 non-pregnant 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. ResultsWe identified 436 DMRs between cfDNA from pregnant and non-pregnant women which were validated using male cfDNA. RASSF1 promoter methylation was higher in maternal cfDNA (fold change 2.87, unpaired t-test p<0.0001). Differential methylation of Y-chromosomal sequences could determine fetal sex. DMRs in maternal cfDNA showed large overlap with DNA methylation of these regions in placentas and buffy coats, indicating a placental and immune-cell contribution to the pregnancy-specific cfDNA methylation signature. Sixteen DMRs in maternal cfDNA were specifically found only in placentas. These novel potential placental-specific DMRs were more prominent than RASSF1. ConclusionsMeD-seq can detect (novel) genome-wide placental DNA methylation marks and determine fetal sex in maternal cfDNA. This study supports future research into maternal cfDNA methylation using MeD-seq. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/610227v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@b8fd55org.highwire.dtl.DTLVardef@ffe942org.highwire.dtl.DTLVardef@12afbf4org.highwire.dtl.DTLVardef@1036a6a_HPS_FORMAT_FIGEXP M_FIG C_FIG Studies investigating the maternal cell-free DNA (cfDNA) methylome are scarce and generally technologically challenging. We identified 436 autosomal differentially methylated regions (DMRs) between cfDNA from pregnant and non-pregnant women, using the innovative methylated DNA sequencing (MeD-seq) technique. Y-chromosomal methylation could determine fetal sex, we show hypermethylation of the placental-marker RASSF1, and identify 16 novel placental-specific markers in maternal cfDNA including DMRs related to TMEM240, DHRS3, and PCMTD2. This pilot study supports future research into the maternal cfDNA methylome using MeD-seq.