Association of maternal and nutrient supply line factors with DNA methylation at the imprinted IGF2/H19 locus in multiple tissues of newborn twins

Abstract

Epigenetic events are crucial for early development, but can be influenced by environmental factors, potentially programming the genome for later adverse health outcomes. The insulin-like growth factor 2 (IGF2)/H19 locus is crucial for prenatal growth and the epigenetic state at this locus is environmentally labile. Recent studies have implicated maternal factors, including folate intake and smoking, in the regulation of DNA methylation at this locus, although data are often conflicting in the direction and magnitude of effect. Most studies have focused on single tissues and on one or two differentially-methylated regions (DMRs) regulating IGF2/H19 expression. In this study, we investigated the relationship between multiple shared and non-shared gestational/maternal factors and DNA methylation at four IGF2/H19 DMRs in five newborn cell types from 67 pairs of monozygotic and 49 pairs of dizygotic twins. Data on maternal and non-shared supply line factors were collected during the second and third trimesters of pregnancy and DNA methylation was measured via mass spectrometry using Sequenom MassArray EpiTyper analysis. Our exploratory approach showed that the site of umbilical cord insertion into the placenta in monochorionic twins has the strongest positive association with methylation in all IGF2/H19 DMRs (p<0.05). Further, evidence for tissue- and locus-specific effects were observed, emphasizing that responsiveness to environmental exposures in utero cannot be generalized across genes and tissues, potentially accounting for the lack of consistency in previous findings. Such complexity in responsiveness to environmental exposures in utero has implications for all epigenetic studies investigating the developmental origins of health and disease.

Keywords: DNA methylation; developmental origins of health and disease (DOHaD); imprinted genes; maternal factors; twins.

Figure 1. Structure of the IGF2/H19 locus with DMRs. The IGF2/H19 locus is shown with direction of transcription, exon locations (gray boxes) and DMRs (white boxes). DMRs assayed for DNA methylation in this study are indicated in bold.

Figure 2. Association of IGF2/H19 methylation and nutrition factors across all cell types and DMRs. Light gray bars represent methylation changes in the IGF2 DMRs, and the dark gray bars represent changes in H19 DMRs. 95% confidence intervals of the coefficient change are represented by a horizontal black line through each bar. The mean methylation differences corresponding to serum vitamin B12, serum homocysteine, and macronutrient are for a 1 standard deviation increase in the respective factor’s z-score. Macronutrient score is obtained as the average of protein, carbohydrate and energy z-scores.

Figure 3. Association of IGF2/H19 methylation and lifestyle factors across all cell types and DMRs. Legend as for Figure 2. The mean methylation difference corresponding to stress is for a 1 standard deviation increase in the stress z-scores.

Figure 4. Association of IGF2/H19 methylation and supply line factors across all cell types and DMRs. Legend as for Figure 2. Mean methylation differences are regression coefficients obtained from multivariable regression analysis, whereby regression coefficients are converted to percentage difference by multiplying each regression coefficient with average standard deviation. The mean methylation corresponding to placenta weight is for a 1 standard deviation increase in placenta weight z-score.