Season of conception in rural gambia affects DNA methylation at putative human metastable epialleles

Abstract

Throughout most of the mammalian genome, genetically regulated developmental programming establishes diverse yet predictable epigenetic states across differentiated cells and tissues. At metastable epialleles (MEs), conversely, epigenotype is established stochastically in the early embryo then maintained in differentiated lineages, resulting in dramatic and systemic interindividual variation in epigenetic regulation. In the mouse, maternal nutrition affects this process, with permanent phenotypic consequences for the offspring. MEs have not previously been identified in humans. Here, using an innovative 2-tissue parallel epigenomic screen, we identified putative MEs in the human genome. In autopsy samples, we showed that DNA methylation at these loci is highly correlated across tissues representing all 3 embryonic germ layer lineages. Monozygotic twin pairs exhibited substantial discordance in DNA methylation at these loci, suggesting that their epigenetic state is established stochastically. We then tested for persistent epigenetic effects of periconceptional nutrition in rural Gambians, who experience dramatic seasonal fluctuations in nutritional status. DNA methylation at MEs was elevated in individuals conceived during the nutritionally challenged rainy season, providing the first evidence of a permanent, systemic effect of periconceptional environment on human epigenotype. At MEs, epigenetic regulation in internal organs and tissues varies among individuals and can be deduced from peripheral blood DNA. MEs should therefore facilitate an improved understanding of the role of interindividual epigenetic variation in human disease.

Figure 1. Two-tissue MSAM screen.

(A) Agarose gel images showing MSA product from PBL and HF genomic DNA of the 8 individuals included in the screen. MSA amplifies methylated SmaI/XmaI intervals ranging from <100 bp to 2 kb. (B) Results of the MSAM screen at chromosome 2 for one pair of PBL and HF interindividual cohybridizations. Each bar represents the average of all probes within a single SmaI/XmaI interval on chromosome 2. The y axis is a log₁₀ transformation of the P value of the interindividual signal ratio. Red and blue bars represent positive and negative interindividual differences, respectively, with P<10⁻⁴. SmaI/XmaI intervals showing concordant interindividual differences in PBL and HF are ME candidates; the location of the hit at PAX8 is indicated.

Figure 2. Candidate MEs, but not control genes, show systemic interindividual variation in DNA methylation.

(A–E) Scatter plots illustrating inter-tissue correlation of interindividual differences in DNA methylation at candidate MEs BOLA3, FLJ20433, PAX8, SLITRK1, and ZFYVE28. The top of each panel indicates the genomic region. Vertical lines represent CpG sites, and gray horizontal bars represent CpG islands. The CpG sites covered by each pyrosequencing assay are indicated by an asterisk. All 5 candidate MEs show significant inter-tissue correlation, indicating systemic interindividual variation in DNA methylation. (F–H) Control genes IGF2, GNASAS, and IL10 exhibit interindividual variation in DNA methylation comparable to that of the candidate MEs, but there is no significant inter-tissue correlation. (Correlation coefficients and P values for all regions are provided in Table S3.)

Figure 3. DNA methylation at putative MEs is influenced by season of conception in the Gambia.

(A) Percent methylation at putative MEs BOLA3, FLJ20433, PAX8, SLITRK1, and ZFYVE28 in PBL DNA of Gambian children, relative to season of conception. Each circle represents one individual, and the black lines represent group means (n = 25/group). At all 5 genomic regions, DNA methylation is higher in individuals conceived in the nutritionally challenged rainy season (BOLA3 P = 0.03, FLJ20433 P = 0.03, PAX8 P = 0.02, SLITRK1 P = 0.006, ZFYVE28 P = 0.002; overall P = 0.0001). (B) At generic LINE1 elements, as well as at 3 control genes (IGF2, GNASAS, and IL10), DNA methylation in these same individuals is not correlated with season of conception (overall P = 0.24), indicating that establishment of epigenotype at the regions we have identified is particularly labile to periconceptional environment.