DNA methylation mediates the effect of maternal cognitive appraisal of a disaster in pregnancy on the child’s C-peptide secretion in adolescence: Project Ice Storm

<div><p>Animal and human studies suggest that prenatal exposure to stress is associated with adverse health outcomes such as type 2 diabetes. Epigenetic modification, such as DNA methylation, is considered one possible underlying mechanism. The 1998 Quebec ice storm provides a unique opportunity to study an independent prenatal stressor on child outcomes. C-peptide is the best measure of endogenous insulin secretion and is widely used in the clinical management of patients with diabetes. The objectives of this study are to determine 1) the extent to which prenatal exposure to disaster-related stress (maternal objective hardship and maternal cognitive appraisal) influences children’s C-peptide secretion, and 2) whether DNA methylation of diabetes-related genes mediates the effects of prenatal stress on C-peptide secretion. Children’s (n = 30) C-peptide secretion in response to an oral glucose tolerance test were assessed in blood at 13½ years. DNA methylation levels of selected type 1 and 2 diabetes-related genes were chosen based upon the genes associated with prenatal maternal objective hardship and/or cognitive appraisal levels. Bootstrapping analyses were performed to determine the mediation effect of DNA methylation. We found that children whose mothers experienced higher objective hardship exhibited higher C-peptide secretion. Cognitive appraisal was not directly associated with C-peptide secretion. DNA methylation of diabetes-related genes had a positive mediation effect of objective hardship on C-peptide secretion: higher objective hardship predicted higher C-peptide secretion through DNA methylation. Negative mediation effects of cognitive appraisal were observed: negative cognitive appraisal predicted higher C-peptide secretion through DNA methylation. However, only one gene, <i>LTA</i>, remained a significant mediator of cognitive appraisal on C-peptide secretion after the conservative Bonferroni multiple corrections. Our findings suggest that DNA methylation could act as an intervening variable between prenatal stress and metabolic outcomes, highlighting the importance of epigenetic mechanisms in response to environmental factors.</p></div

The characteristics of the mothers and children (N = 30).

<p>The characteristics of the mothers and children (N = 30).</p

Results of DNA methylation of genes that significantly mediated the effect of cognitive appraisal on C-peptide outcome in initial analyses.

<p>(The mediation effect that remained significant after multiple correction is highlighted in bold).</p

Mediation analysis on the relationship between exposure cognitive appraisal and C-peptide secretion.

<p>The effect of cognitive appraisal on the DNA methylation of cg16280132 from LTA is -0.548 (path a); the effect of the DNA methylation of cg16280132 on C-peptide level, controlling for cognitive appraisal is 1.144 (path b); the direct effect of cognitive appraisal on C-peptide level, controlling for the DNA methylation of cg16280132, is 0.661 (path c’); the mediating effect of cognitive appraisal on C-peptide level through DNA methylation of cg16280132 is -0.627 (path a*b). Together, the combination of cognitive appraisal and DNA methylation of cg16280132 explained 26.14% of the variance in C-peptide secretion.</p

DNA Methylation Signatures Triggered by Prenatal Maternal Stress Exposure to a Natural Disaster: Project Ice Storm

<div><p>Background</p><p>Prenatal maternal stress (PNMS) predicts a wide variety of behavioral and physical outcomes in the offspring. Although epigenetic processes may be responsible for PNMS effects, human research is hampered by the lack of experimental methods that parallel controlled animal studies. Disasters, however, provide natural experiments that can provide models of prenatal stress.</p><p>Methods</p><p>Five months after the 1998 Quebec ice storm we recruited women who had been pregnant during the disaster and assessed their degrees of objective hardship and subjective distress. Thirteen years later, we investigated DNA methylation profiling in T cells obtained from 36 of the children, and compared selected results with those from saliva samples obtained from the same children at age 8.</p><p>Results</p><p>Prenatal maternal objective hardship was correlated with DNA methylation levels in 1675 CGs affiliated with 957 genes predominantly related to immune function; maternal subjective distress was uncorrelated. DNA methylation changes in <i>SCG5</i> and <i>LTA</i>, both highly correlated with maternal objective stress, were comparable in T cells, peripheral blood mononuclear cells (PBMCs) and saliva cells.</p><p>Conclusions</p><p>These data provide first evidence in humans supporting the conclusion that PNMS results in a lasting, broad, and functionally organized DNA methylation signature in several tissues in offspring. By using a natural disaster model, we can infer that the epigenetic effects found in Project Ice Storm are due to objective levels of hardship experienced by the pregnant woman rather than to her level of sustained distress.</p></div

The effect of DNA methylation on <i>SCG5</i> promoter activity.

<p>A) Schematic representation of the location of CGs investigated in the <i>SCG5</i> promoter. The CGs are denoted as lollipops and the +1 position indicates the transcription start site (TSS). White bar indicates the region that contains the 4 differentially methylated CGs. Gray bar indicates the luciferase reporter gene in pCpGL-reporter. The two fragments of 650 bp and 692 bp from the SCG5 promoter region were cloned into the <i>Bgl</i>II and <i>Nco</i>I restriction sites in pCpGL-reporter in sense and anti-sense orientation, respectively. B) Relative luciferase activity of two promoters region before and after mock methylation (−) or complete in vitro methylation with CpG methyltransferase (M.<i>Sss</i>I) (+) and transient transfection (48 h) into in HEK293 cell line (***P<0.001). Promoter activity was normalized to protein concentration. The values are the averages of at least three independent experiments. Data are mean ± SEM.</p

The molecular and cellular functions of the 957 genes analyzed with IPA.

<p>A) Top 10 functions of the 957 differentially methylated genes. The y-axis shows functions while the x-axis shows -log(p-value). The yellow line indicates the threshold value of p<0.05. B) The most significant canonical pathway: CD28 Signaling in T helper cells. Genes whose methylation levels are positively correlated with objective PNMS are colored in red and those whose methylation levels are negatively correlated with objective PNMS are colored in blue. CD247: CD247 molecule; FYN: a membrane-associated tyrosine kinase; CD3E: CD3-epsilon polypeptide; CSK: C-Src Tyrosine Kinase; PLCG1: Phospholipase C, Gamma 1; NFATC1: Nuclear Factor Of Activated T-Cells, Cytoplasmic, Calcineurin-Dependent 1; HLA-DMB: Major Histocompatibility Complex, Class II, DM Beta; ITPR1: inositol 1,4,5-trisphosphate receptor, type 1; CD3D: CD3d Molecule, Delta; CTLA4: cytotoxic T-lymphocyte-associated protein 4; CD3G: CD3-gamma polypeptide; CD28: CD28 Molecule; LCK: lymphocyte-specific protein tyrosine kinase; ACTR3: ARP3 Actin-Related Protein 3 Homolog (Yeast); NFKBIA: nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha; BCL10: B-Cell CLL/Lymphoma 10; SYK: spleen tyrosine kinase; ZAP70: zeta-chain (TCR) associated protein kinase 70 kDa; ARPC4: actin related protein 2/3 complex, subunit 4; MAPK10: mitogen-activated protein kinase 10; HLA-DOB: Major Histocompatibility Complex, Class II, DO Beta; PIK3CD: phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit delta; PIK3R2: phosphoinositide-3-kinase, regulatory subunit 2 (beta); LCP2: Lymphocyte Cytosolic Protein 2; ITK: IL2-inducible T-cell kinase.</p

Correlations between objective PNMS and methylation levels of CGs in <i>SCG5</i> and <i>LTA</i> in 3 cell types.

<p>**. Correlation is significant at the 0.01 level (2-tailed); *. Correlation is significant at the 0.05 level (2-tailed).</p><p>Correlations between objective PNMS and methylation levels of CGs in <i>SCG5</i> and <i>LTA</i> in 3 cell types.</p

Differentially methylated CGs responding to objective PNMS (Storm32 score).

<p>Heatmap represents the DNA methylation levels of 500 CGs most significantly associated with objective PNMS (Storm32 score) in 34 donors. Each column represents an individual and each row a single CG. Each cell represents the CG methylation level for one site in one sample. A color gradient intensity scale at the lower right-hand corner of the Heatmap expresses methylation changes. The darkest green indicates the lowest methylation level (Beta-value = 0), the gray indicates the median score (Beta-value = 0.5) and the darkest red indicates the highest methylation level (Beta-value = 1). The color bar on the top of the Heatmap indicates subjects' categorization by their mother's objective PNMS. A color gradient intensity scale at the higher right-hand corner of the Heatmap shows the level of objective PNMS. The darkest blue indicates the lowest objective PNMS (Storm32 score = 5), the gray indicates the median objective PNMS (Storm32 score = 11) and the darkest red indicates the highest objective PNMS (Storm32 score = 21).</p