Sex differences of leukocytes DNA methylation adjusted for estimated cellular proportions

Abstract

Background: DNA methylation, which is most frequently the transference of a methyl group to the 5-carbon position of the cytosine in a CpG dinucleotide, plays an important role in both normal development and diseases. To date, several genome-wide methylome studies have revealed sex-biased DNA methylation, yet no studies have investigated sex differences in DNA methylation by taking into account cellular heterogeneity. The aim of the present study was to investigate sex-biased DNA methylation on the autosomes in human blood by adjusting for estimated cellular proportions because cell-type proportions may vary by sex.

Methods: We performed a genome-wide DNA methylation profiling of the peripheral leukocytes in two sets of samples, a discovery set (49 males and 44 females) and a replication set (14 males and 10 females) using Infinium HumanMethylation450 BeadChips for 485,764 CpG dinucleotides and then examined the effect of sex on DNA methylation with a multiple linear regression analysis after adjusting for age, the estimated 6 cell-type proportions, and the covariates identified in a surrogate variable analysis.

Results: We identified differential DNA methylation between males and females at 292 autosomal CpG site loci in the discovery set (Bonferroni-adjusted p < 0.05). Of these 292 CpG sites, significant sex differences were also observed at 98 sites in the replication set (p < 0.05).

Conclusions: These findings provided further evidence that DNA methylation may play a role in the differentiation or maintenance of sexual dimorphisms. Our methylome mapping of the effects of sex may be useful to understanding the molecular mechanism involved in both normal development and diseases.

Keywords: Blood; Cell heterogeneity; DNA methylation; Epigenetics; Leukocyte; Microarray; Sex.

Fig. 1

Average estimated cellular proportions of male and female groups. The y axis is each of average estimated cellular proportions of CD8 + T cell, CD4 + T cell, CD56 + NK cell, CD19 + B cell, CD14 + monocyte, and granulocyte. Significant differences between the two groups were observed in 2 cell types (CD8 + T cell and CD56 + NK cell) (Welch’s t test p < 0.05)

Fig. 2

Volcano plots of differentially methylated CpG sites between males and females. This volcano plot shows the result of genome-wide DNA methylation differences between 49 males and 44 females after adjusting for the estimated cell mixture proportions. Average beta difference (males-females) is shown on the x axis. Log₁₀-converted p value is shown on the y-axis. CpG loci that showed a p value of less than 5 % after Bonferroni correction are colored red. Significant sex differences in DNA methylation were observed at 292 CpG sites (p < 1.44 × 10⁻⁷)

Fig. 3

Quantile-quantile (Q-Q) plot of DNA methylation between males and females. The x axis is the expected −log₁₀P value, and the y axis is the observed −log₁₀P value. This Q-Q plot shows a deviation of the observed from the expected, providing evidence of DNA methylation differences between males and females at numerous CpG sites