Sex differences in DNA methylation assessed by 450 K BeadChip in newborns

Abstract

Background: DNA methylation is an important epigenetic mark that can potentially link early life exposures to adverse health outcomes later in life. Host factors like sex and age strongly influence biological variation of DNA methylation, but characterization of these relationships is still limited, particularly in young children.

Methods: In a sample of 111 Mexican-American subjects (58 girls , 53 boys), we interrogated DNA methylation differences by sex at birth using the 450 K BeadChip in umbilical cord blood specimens, adjusting for cell composition.

Results: We observed that ~3% of CpG sites were differentially methylated between girls and boys at birth (FDR P < 0.05). Of those CpGs, 3031 were located on autosomes, and 82.8% of those were hypermethylated in girls compared to boys. Beyond individual CpGs, we found 3604 sex-associated differentially methylated regions (DMRs) where the majority (75.8%) had higher methylation in girls. Using pathway analysis, we found that sex-associated autosomal CpGs were significantly enriched for gene ontology terms related to nervous system development and behavior. Among hits in our study, 35.9% had been previously reported as sex-associated CpG sites in other published human studies. Further, for replicated hits, the direction of the association with methylation was highly concordant (98.5-100%) with previous studies.

Conclusions: To our knowledge, this is the first reported epigenome-wide analysis by sex at birth that examined DMRs and adjusted for confounding by cell composition. We confirmed previously reported trends that methylation profiles are sex-specific even in autosomal genes, and also identified novel sex-associated CpGs in our methylome-wide analysis immediately after birth, a critical yet relatively unstudied developmental window.

Fig. 1

Manhattan plot for association between child sex and DNA methylation at all 450 K CpGs, adjusting for batch and cell composition by differential cell count (DCC). Associations where methylation was higher for girls relative to boys are plotted above the x-axis, while those with decreased methylation are plotted below. CpGs meeting FDR multiple testing threshold of (P < 0.05) shown in red

Fig. 2

Percent of 450 K CpGs (purple), and percent of all (blue), hypermethylated (dark green), and hypomethylated (light green) autosomal differentially methylated positions (DMPs) associated with sex (a & b). These percentages are given by island functional categories (island, shore, shelf, and open sea) in a, and gene functional categories (within 1500 bp of a transcription start site (TSS), 200 bp of a TSS, a 5′ untranslated region (UTR), first exon, gene body, 3′UTR, and intergenic) in b. * indicates that the proportion of sites significantly altered compared to the coverage on the 450 K BeadChip (P < 0.05)

Fig. 3

DNA methylation (β values) for CpG sites included in two top DMRs associated with child sex in newborns. One DMR (a) contains 7 CpG sites, is located on chromosome 6 and spans a 1763 bp region in the exon of PPP1R3G (chr6:5085986–5087749). The other (b) on chromosome 12 includes 8 CpGs over a 1365 bp region across the promoter and 1^st exon of PIWIL1 (chr12:130821453–130822818). Girls are shown with red circles, boys with blue triangles, and median methylation per CpG by sex is shown by red and blue lines. Green lines show the genomic coordinates of exon regions for each gene shown