Prenatal maternal stress and wheeze in children: novel insights into epigenetic regulation

Abstract

Psychological stress during pregnancy increases the risk of childhood wheeze and asthma. However, the transmitting mechanisms remain largely unknown. Since epigenetic alterations have emerged as a link between perturbations in the prenatal environment and an increased disease risk we used whole genome bisulfite sequencing (WGBS) to analyze changes in DNA methylation in mothers and their children related to prenatal psychosocial stress and assessed its role in the development of wheeze in the child. We evaluated genomic regions altered in their methylation level due to maternal stress based of WGBS data of 10 mother-child-pairs. These data were complemented by longitudinal targeted methylation and transcriptional analyses in children from our prospective mother-child cohort LINA for whom maternal stress and wheezing information was available (n = 443). High maternal stress was associated with an increased risk for persistent wheezing in the child until the age of 5. Both mothers and children showed genome-wide alterations in DNA-methylation specifically in enhancer elements. Deregulated neuroendocrine and neurotransmitter receptor interactions were observed in stressed mothers and their children. In children but not in mothers, calcium- and Wnt-signaling required for lung maturation in the prenatal period were epigenetically deregulated and could be linked with wheezing later in children's life.

Figure 1. Concentration of homovanillic acid for different stress scores.

Homovanillic acid was measured in urine of mothers at 36^th weeks of gestation and normalized to creatinine. The concentration of the stress metabolite homovanillic acid is significantly higher in mothers with a high stress score compared to those with the lowest stress score supporting stress categorization based on our questionnaire. A significant relationship of homovanillic acid concentration and maternal stress score was determined by multiple regression analysis. Mean ratios were adjusted for maternal medication information derived from the same samples (box plots represent: mean +/− CI, whiskers +/− non-outlier range).

Figure 2. Global overview of differential methylation in children and mothers.

(A) Pie charts represent the number and percentage of DMRs observed in mothers and children. Two classes of DMRs were differentiated: gDMRs for which methylation significantly correlated with a meQTL-SNP and ngDMRs where the underlying sequence variation did not influence methylation (red: hypermethylation, blue: hypomethylation). (B) The table summarizes the functional genomic regions enriched for an overlap with these DMRs. Active regulatory genomic regions in particular enhancer elements were significantly enriched in overlapping DMRs irrespective of their type (gDMR or ngDMR).

Figure 3. DMR overlap with transcription factor binding sites and KEGG pathways.

(A) Z-scores of DMR overlaps with transcription factor binding peak sets from ENCODE. For each transcription factor, z-scores were calculated using the mean value and the standard deviation from the overlap distribution of randomly shuffled DMRs with the corresponding transcription factor binding factor binding peak set. The dashed line represents the 5% upper tail of the standard-normal distribution. Only z-scores are shown that are above the 10% upper tail threshold. (B) Enrichment of DMRs in KEGG pathways.

Figure 4. Pathways intertwined by stress induced differential methylation.

Figure 5. Neuromedin U Receptor 1 is differentially methylated and expressed in children experiencing prenatal maternal stress.

(A) Based on the histone modification data the DMR (WGBS: Δmeth = 28%) identified in the NMUR1 gene is located in an enhancer region (ENCODE/Active Elements (own ChIP-Seq data)). (B) Validation of the NMUR1 DMR (7 CpGs, chr2: 232,394,701–232,394,805) in the total cohort by MassARRAY shows that an increase of the maternal stress level leads to a significant elevation in the methylation level in children at time of birth. In highly stressed children this methylation increase corresponds to a significantly decreased mRNA expression of NMUR1 (mean +/− 95% CI, whiskers +/− non-outlier range). Relationship between NMUR1 methylation and maternal stress score were determined by multiple regression analysis adjusted for gender of the child, birth week, age of the mother, mode of delivery, maternal smoking/-medication during pregnancy, parental history of atopy and cell composition. (C) Relationship of interleukin-4, -5 and -6 secretion at time of birth and NMUR1 methylation. Data are presented as ratios of the mean (MR) and 95% confidence intervals. Models were adjusted for known confounders of interleukin concentrations in cord blood (month of birth, mode of delivery, gender of the child, parental history of atopy, smoking during pregnancy and cell composition).

Figure 6. G protein subunit alpha 11 is epigenetically perturbed in children by prenatal maternal stress.

(A) Although found in an intron the DMR (WGBS: Δmeth = 22%) in the GNA11 gene is located in an enhancer region according to our own ChIP-Seq data. ChIA-PET data suggest this enhancer regulates its host gene. (B) Maternal stress and the methylation level in the GNA11 DMR show a significant relationship at time of birth while at the same time a significant reverse relationship is observed for the expression of GNA11 when including all children in our cohort for whom persistent or never persistent wheeze was reported (mean +/− 95% CI, whiskers +/− non-outlier range, p-values adjusted for: gender of the child, birth week, age of the mother, mode of delivery, maternal smoking/-medication during pregnancy, parental history of atopy and cell composition). (C) In four-year-old children increased methylation in the GNA11 enhancer (chr19: 3110675) is associated with an increased risk for persistent wheeze. Corresponding results are observed for GNA11 expression. Odds ratios were calculated using logistic regression models adjusted for known confounders of wheezing (parental history of atopy, parental educational level, gender, siblings, smoking during pregnancy, ETS exposure after birth, cat keeping and maternal stress score).

Figure 7. Peripheral beta 4 subunit of voltage gated calcium channel in children is altered by maternal stress.

(A) Methylation could only be assessed for one CpG in the CACNB4 ngDMR. Amplification of the entire region yielded no PCR product therefore we could only consider two CpGs (chr2: 152898920–152899112). Since one of the two CpGs was a CpG destroying SNP further analysis was restricted to a single position. (A) At time of birth methylation of this CpG shows a significant relationship to the maternal stress score (considering all children either persistently wheezing or showing no respiratory symptoms), (B) while in the same children expression of CACNB4 is decreased to maternal stress. Adjusted p-values were calculated including gender of the child, birth week, age of the mother, mode of delivery, maternal smoking/-medication during pregnancy, parental history of atopy and cell composition as confounding variables.

Figure 8. Downstream targets of the Wnt5A/Ca2+ -signaling pathway are activated in prenatally stressed children.

mRNA expression in prenatally highly stressed children is significantly increased for the calcium binding beta subunit of calcineurin PPP3R1 and its downstream target NFATC3 in four year old children (given are p-values adjusted for: gender of the child, birth week, age of the mother, mode of delivery, maternal smoking/-medication during pregnancy and parental history of atopy; +/− 95% CI, whiskers +/− non-outlier range). A positive correlation of the stress score with NFATC3 expression was only observed in late and persistently wheezing children, no correlation of maternal stress score and transcription was observed in children without any respiratory symptoms for both PPP3R1 and NFATC3.