Postnatal stability, tissue, and time specific effects of AHRR methylation change in response to maternal smoking in pregnancy

Abstract

The intrauterine environment has the potential to "program" the developing fetus in a way that can be potentially deleterious to later health. While in utero environmental/stochastic factors are known to influence DNA methylation profile at birth, it has been difficult to assign specific examples of epigenetic variation to specific environmental exposures. Recently, several studies have linked exposure to smoking with DNA methylation change in the aryl hydrocarbon receptor repressor (AHRR) gene in blood. This includes hypomethylation of AHRR in neonatal blood in response to maternal smoking in pregnancy. The role of AHRR as a negative regulator of pathways involved in pleiotropic responses to environmental contaminants raises the possibility that smoking-induced hypomethylation is an adaptive response to an adverse in utero environmental exposure. However, the tissue specificity of the response to maternal smoking, and the stability of the methylation changes early in life remain to be determined. In this study we analyzed AHRR methylation in three cell types-cord blood mononuclear cells (CBMCs), buccal epithelium, and placenta tissue-from newborn twins of mothers who smoked throughout pregnancy and matched controls. Further, we explored the postnatal stability of this change at 18 months. Our results confirm the previous association between maternal smoking and AHRR methylation in neonatal blood. In addition, this study expands the region of AHRR methylation altered in response to maternal smoking during pregnancy and reveals the tissue-specific nature of epigenetic responses to environmental exposures in utero. Further, the evidence for postnatal stability of smoking-induced epigenetic change supports a role for epigenetics as a mediator of long-term effects of specific in utero exposures in humans. Longitudinal analysis of further specific exposures in larger cohorts is required to examine the extent of this phenomenon in humans.

Keywords: Aryl hydrocarbon receptor repressor (AHRR); Cord blood mononuclear cells; DNA methylation; DOHaD; early life epigenetics; intrauterine environment; smoking; twins.

Figure 1. Tissue specific DNA methylation patterns within AHRR. (A) Map of the EpiTYPER assays (blue rectangles) covering the AHRR region of interest, showing analyzable CpG sites. Assay A and B cover the CpG site of interest (CpG_A7 - cg05575921) and surrounding CpG sites. The CGI is represented as a green rectangle. (B) DNA methylation level in CBMCs, buccal epithelium, and placenta show tissue-specific differences at CpG_A7. Grey shaded box = “cg05575921” CpG site of interest.

Figure 2. Association between maternal smoking and AHRR methylation in CBMCs. Maternal smoking throughout pregnancy is associated with lower methylation at (A) CpG_A7, (B) across assay A and (C) across assay B. Maternal smoking early in pregnancy was not associated with lower AHRR methylation. (D) 8 CpG sites, contained within 6 CpG units, show lower methylation (dB > 0.05, P < 0.05) in response to maternal smoking. The difference in methylation is limited to the CpG Island shore, whereas the CGI is hypomethylated in all CBMC samples. Y-axis = DNA methylation level. Grey shaded box = “cg05575921” CpG site of interest. Error bars = 95% CI.

Figure 3. AHRR expression in CBMCs exposed and not exposed to smoke during pregnancy. (A) AHRR expression is higher in CBMCs compared with placenta tissue. (B) AHRR expression in CBMCs exposed to smoking during pregnancy (n = 5) is slightly higher than in non-smoking controls (n = 10), P = 0.11. Bars represent standard deviation.

Figure 4. Maternal smoking associated DNA methylation change is maintained in peripheral blood mononuclear cells at 18 mo of age. DNA methylation levels (y-axis) across assay A and B at birth and 18 mo in smokers and controls. There is a general increase in methylation, at the majority smoking-associated CpG sites, from birth to 18 mo in both groups. DNA methylation differences between smoked throughout and never smoked groups remain significant at several CpG sites, however the absolute difference in methylation between the groups is lower at 18 mo.

Figure 5. Twin model analysis suggests a genetic component to AHRR methylation. The classic twin model analysis was performed to determine the genetic contribution to AHRR methylation at CpG_A7 and across assay A. Twin “1” methylation level is shown on the x axis, and twin “2” methylation level on the y-axis. The within-pair correlation (R²) was higher in MZ twins (B and D) compared with DZ twins (A and C). This suggests that MZ twins are generally more similar in their AHRR DNA methylation level than DZ twins, indicating a genetic control of methylation at this locus.