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Background:  We aimed to assess the extent to which the buffy coat DNA methylome is representative of methylation patterns in constitutive white blood cell (WBC) types in normal pregnancy.   Methods:  A comparison of differential methylation of buffy coat DNA vs DNA isolated from polymorphonuclear (PMN) and lymphocytic fractions was performed for each blood sample obtained within 24 h prior to delivery from 29 normotensive pregnant women. Methylation profiles were obtained using an Illumina Human Methylation 450 BeadChip and CHaMP bioinformatics pipeline. A subset of differentially methylated probes (DMPs) showing discordant methylation were further investigated using statistical modeling and enrichment analysis.   Results:  The smallest number of DMPs was found between the buffy coat and the PMN fraction (2.96%). Pathway enrichment analysis of the DMPs identified biological pathways involved in the particular leukocyte lineage, consistent with perturbations during isolation. The comparisons between the buffy coat and the isolated fractions as a group using linear modeling yielded a small number of probes (∼29,000) with discordant methylation. Demethylation of probes in the buffy coat compared to derived cell lines was more common and was prevalent in shelf and open sea regions.   Conclusion:  Buffy coat is representative of methylation patterns in WBC types in normal pregnancy. The differential methylations are consistent with perturbations during isolation of constituent cells and likely originate  in vitro  due to the physical stress during cell separation and are of no physiological relevance. These findings help the interpretation of DNA methylation profiling in pregnancy and numerous other conditions.

Buffy Coat DNA Methylation Profile Is Representative of Methylation Patterns in White Blood Cell Types in Normal Pregnancy