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. 2012;7(10):e46705.
doi: 10.1371/journal.pone.0046705. Epub 2012 Oct 5.

Heterogeneity in white blood cells has potential to confound DNA methylation measurements

Bjorn T Adalsteinsson  1 , Haukur Gudnason, Thor Aspelund, Tamara B Harris, Lenore J Launer, Gudny Eiriksdottir, Albert V Smith, Vilmundur Gudnason
Affiliations

Heterogeneity in white blood cells has potential to confound DNA methylation measurements

Bjorn T Adalsteinsson et al. PLoS One. 2012.

Abstract

Epigenetic studies are commonly conducted on DNA from tissue samples. However, tissues are ensembles of cells that may each have their own epigenetic profile, and therefore inter-individual cellular heterogeneity may compromise these studies. Here, we explore the potential for such confounding on DNA methylation measurement outcomes when using DNA from whole blood. DNA methylation was measured using pyrosequencing-based methodology in whole blood (n = 50-179) and in two white blood cell fractions (n = 20), isolated using density gradient centrifugation, in four CGIs (CpG Islands) located in genes HHEX (10 CpG sites assayed), KCNJ11 (8 CpGs), KCNQ1 (4 CpGs) and PM20D1 (7 CpGs). Cellular heterogeneity (variation in proportional white blood cell counts of neutrophils, lymphocytes, monocytes, eosinophils and basophils, counted by an automated cell counter) explained up to 40% (p<0.0001) of the inter-individual variation in whole blood DNA methylation levels in the HHEX CGI, but not a significant proportion of the variation in the other three CGIs tested. DNA methylation levels in the two cell fractions, polymorphonuclear and mononuclear cells, differed significantly in the HHEX CGI; specifically the average absolute difference ranged between 3.4-15.7 percentage points per CpG site. In the other three CGIs tested, methylation levels in the two fractions did not differ significantly, and/or the difference was more moderate. In the examined CGIs, methylation levels were highly correlated between cell fractions. In summary, our analysis detects region-specific differential DNA methylation between white blood cell subtypes, which can confound the outcome of whole blood DNA methylation measurements. Finally, by demonstrating the high correlation between methylation levels in cell fractions, our results suggest a possibility to use a proportional number of a single white blood cell type to correct for this confounding effect in analyses.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Percent DNA methylation in whole blood samples.
Percent DNA methylation (y-axis) in whole blood DNA per CpG site (x-axis) in four CGIs located in the HHEX (n = 169), KCNJ11 (n = 54), KCNQ1 (n = 49) and PM20D1 (n = 59) genes respectively. Data for each CGI are depicted in a separate boxplot. Below each boxplot is a gene-map which roughly indicates the position of the analyzed CpG sites (adapted from the UCSC genome browser) . Genes are depicted in blue, the exons as blocks, the introns as thin lines connecting the blocks, and the 5′ and 3′ untranslated regions as thin blocks at each end. CGIs are shown as green blocks. The genomic position depicted for each CGI is; 10:94,439,661–94,445,388 (chromosome:first base-last base) for the HHEX CGI, chr11:17,363,372–17,366,783 for the KCNJ11 CGI, chr11:2,422,797–2,826,916 for the KCNQ1 CGI and chr1:204,063,776–204,085,881 for the PM20D1 CGI. The gene map for KCNQ1 includes the KCNQ1OT1 (KCNQ1 overlapping transcript 1) gene, which appears as a large exon roughly in the middle of the map. Arrows indicate the direction of transcription and the position of the transcription start site.
Figure 2
Figure 2. Percent DNA methylation in mononuclear and polymorphonuclear cells.
Percent DNA methylation (y-axis) in mononuclear and polymorphonuclear cells (MNCs and PMNCs) per CpG site (x-axis) in four CGIs located in the HHEX, KCNJ11, KCNQ1 and PM20D1 genes respectively (n = 20 each). Data for each CGI are depicted in a separate boxplot where measurements for MNCs are shown in red and for PMNCs in blue. The dotted lines separating the boxes indicate that at each CpG site a pair of data are being compared (i.e., for MNCs and PMNCs). Significantly (p<0.01) differentially methylated CpG sites (MNCs versus PMNCs DNA methylation) are indicated with an asterisk.
Figure 3
Figure 3. Correlation between DNA methylation in mononuclear and polymorphonuclear cells.
Comparison of DNA methylation levels measured in two cell fractions, mononuclear cells (MNCs) and polymorphonuclear cells (PMNCs). Percent methylation in PMNCs (y-axis) is plotted against percent methylation in MNCs (x-axis). Each dot represents the two measurements for a single CpG per individual. The Spearman ρ for correlation between measurements in MNCs and PMNCs for each CGI is shown in the legend.
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