Genome-wide DNA methylation association study of predominant breastfeeding

Abstract

Introduction: Breastfeeding is considered a healthy habit with clear benefits at population level. It has been associated with lower risk of infections, asthma, obesity, diabetes or malocclusion, with increased survival, improved neurodevelopment and better immune functioning. However, the molecular bases of the benefits are yet to be completely established. This benefits have been linked to breast milk biologically active components: fatty acids, growth and immunological factors, nucleic acids, live maternal cells, and oligosaccharides for gut bacteria, among others. These compounds could have a final effect on the epigenome of the offspring. Aim: The aim of this study was to perform an Epigenome-Wide Association Study (EWAS) to identify DNA methylation signatures associated with breastfeeding and gain some mechanistic insights of the processes underlying the benefits of breastfeeding on child health. Material and Methods: Predominant breastfeeding (Never, ≤4 months and >4 months) was assessed through questionnaires administrated to the mothers from three European cohorts. DNA methylation in blood samples collected at birth and at age 4/5y was measured with the Illumina 450k array. Differential methylation was analyzed using robust linear regressions adjusting for covariates. Two EWAS were performed: 1) methylation data at age 4/5y (N=597); and 2) paired methylation data at 0y and age 4/5y (N=330). Functional enrichment analyses were performed with ConsensusPathDB tool. Methylation levels at top CpG sites were associated with gene expression levels in whole blood at age 4/5y. Results: At a 10% False Discovery Rate (FDR), 4 CpG sites were significantly associated with predominant breastfeeding, 1 at age 4/5y (cg13463516) and 3 in the paired analysis (cg24713259, cg09254049, cg08576185). The maximum difference in methylation was around 2%. Functional enrichment analysis revealed pathways related to apoptosis, immunity (TNF-alpha and NFKB), and phosphatidylcholine remodeling, amongst others. The expression levels of HLA-DRB5, HLA-DQB1 and HLA-DQB1-AS genes were negatively related to the methylation levels at cg24713259. 8 out of the 70 transcripts tested were related to higher or lower levels of methylation at cg08576185. Conclusion: Four CpG sites were associated with predominant breastfeeding. However, differences in methylation were minimal and probably due to technical noise. Increasing the sample size and testing different definitions of predominant breastfeeding are two points to be addressed in future studies.