DNA Methylation Signatures of Breastfeeding in Buccal Cells Collected in Mid-Childhood

Abstract

Breastfeeding has long-term benefits for children that may be mediated via the epigenome. This pathway has been hypothesized, but the number of empirical studies in humans is small and mostly done by using peripheral blood as the DNA source. We performed an epigenome-wide association study (EWAS) in buccal cells collected around age nine (mean = 9.5) from 1006 twins recruited by the Netherlands Twin Register (NTR). An age-stratified analysis examined if effects attenuate with age (median split at 10 years; n<10 = 517, mean age = 7.9; n>10 = 489, mean age = 11.2). We performed replication analyses in two independent cohorts from the NTR (buccal cells) and the Avon Longitudinal Study of Parents and Children (ALSPAC) (peripheral blood), and we tested loci previously associated with breastfeeding in epigenetic studies. Genome-wide DNA methylation was assessed with the Illumina Infinium MethylationEPIC BeadChip (Illumina, San Diego, CA, USA) in the NTR and with the HumanMethylation450 Bead Chip in the ALSPAC. The duration of breastfeeding was dichotomized (‘never‘ vs. ‘ever’). In the total sample, no robustly associated epigenome-wide significant CpGs were identified (α = 6.34 × 10–8). In the sub-group of children younger than 10 years, four significant CpGs were associated with breastfeeding after adjusting for child and maternal characteristics. In children older than 10 years, methylation differences at these CpGs were smaller and non-significant. The findings did not replicate in the NTR sample (n = 98; mean age = 7.5 years), and no nearby sites were associated with breastfeeding in the ALSPAC study (n = 938; mean age = 7.4). Of the CpG sites previously reported in the literature, three were associated with breastfeeding in children younger than 10 years, thus showing that these CpGs are associated with breastfeeding in buccal and blood cells. Our study is the first to show that breastfeeding is associated with epigenetic variation in buccal cells in children. Further studies are needed to investigate if methylation differences at these loci are caused by breastfeeding or by other unmeasured confounders, as well as what mechanism drives changes in associations with age.

DNA methylation of blood cells is associated with prevalent type 2 diabetes in a meta-analysis of four European cohorts

“…This method allows to exclude outliers of DNA methylation caused by technical artefacts or rare genetic variants that can bias the analysis [ 65 ]. This method has been widely implemented in other studies [ 65 – 67 ].…”

Clinical insights gained through metabolomic analysis of human breast milk

“…The recent findings of Odintsova et al, investigating methylation pathways in buccal cells of children, allows to conclude that breastfeeding, and potentially the metabolic composition of FM, could influence epigenetic pathways in middle-infancy[81].…”

Epigenome-Wide Association of Infant Feeding and Changes in DNA Methylation from Birth to 10 Years

“…Reports that used breastfeeding as a confounder, e.g., analyzed in tumor samples or studies that did not provide CpG sites information were excluded. The final list includes a total of 4308 CpGs, i.e., 12 CpGs from the study of Odintsova et al [30], 13 CpGs from the study of Sherwood et al [27,32], 7 CpG from Hartwig et al [28], and 4276 CpGs from Naumova et al [29]. We then used this list to look for the overlap of CpGs or their mapped genes with our findings.…”

“…A recent systematic review suggests that findings from previous association studies on breastfeeding and DNAm are inconclusive and recommends more epigenome-wide association studies (EWASs) with proper measures of relevant covariates to be performed [ 20 ]. Our literature search identified ten association studies on breastfeeding and DNAm in humans (two candidate gene studies [ 21 , 22 ], two EWASs with breastfeeding as a covariate [ 23 , 24 ], and six EWASs with breastfeeding as an exposure variable [ 25 , 26 , 27 , 28 , 29 , 30 ]) ( Table S1 ). It is important to note that these studies were highly diverse in terms of the age of assessment, tissue examined, array platform, DNAm targets and respective methods, and the categorization of breastfeeding.…”

“…Besides the limitations of prior association studies discussed in the recent systematic review [ 20 ], we identified two additional limitations in their study designs. First, most of the studies when assessing effects of breastfeeding duration on the epigenome were conducted irrespective of formula or solid feeding [ 25 , 26 , 27 , 28 ], which can be confounded by effects of breastfeeding. For example, infants could be mostly formula fed but still classified as breastfeeding in categorizations that have been widely investigated.…”