The cardiovascular and hypothalamus-pituitary-adrenal axis response to stress is controlled by glucocorticoid receptor sequence variants and promoter methylation

Abstract

Background: Gender, genetic makeup, and prior experience interact to determine physiological responses to an external perceived stressor. Here, we investigated the contribution of both genetic variants and promoter methylation of the NR3C1 (glucocorticoid receptor) gene to the cardiovascular and hypothalamus-pituitary-adrenal (HPA) axis response to the socially evaluated cold pressor test (seCPT).

Results: Two hundred thirty-two healthy participants were recruited and underwent the experiment. They were randomly assigned to either the seCPT group (cold water) or a control group (warm water). The seCPT group had a clear stress reaction; salivary cortisol levels and peak systolic and diastolic blood pressure all increased significantly compared to the control group. GR genotype (TthIIII, NR3C1-I, 1H, E22E, R23K, BclI and 9beta) and methylation data were obtained from 218 participants. Haplotypes were built from the GR genotypes, and haplotype 2 (minor allele of BclI) carriers had a higher cortisol response to the seCPT in comparison to non-carriers (20.77 ± 13.22; 14.99 ± 8.42; p = 0.034), as well as independently of the experimental manipulation, higher baseline heart rate (72.44 ± 10.99; 68.74 ± 9.79; p = 0.022) and blood pressure (115.81 ± 10.47; 111.61 ± 10.74; p = 0.048). Average methylation levels throughout promoter 1F and 1H were low (2.76 and 1.69 %, respectively), but there was a strong correlation between individual CpGs and the distance separating them (Pearson's correlation r = 0.725, p = 3.03 × 10(-26)). Higher promoter-wide methylation levels were associated with decreased baseline blood pressure, and when incorporated into a linear mixed effect model significantly predicted lower systolic and diastolic blood pressure evolution over time in response to the experimental manipulation. The underlying genotype significantly predicted methylation levels; particularly, the homozygous BclI minor allele was associated with higher methylation in promoter 1H (p = 0.042).

Conclusions: This is one of the first studies linking epigenetic modifications of the GR promoter, receptor genotype and physiological measures of the stress response. At baseline, there were clear genetic and epigenetic effects on blood pressure. The seCPT induced a strong cardiovascular and HPA axis response, and both systems were affected by the functional genetic variants, although methylation also predicted blood pressure reactivity. The return to baseline was predominantly influenced by the genomic sequence. Overall, the physiological response to the seCPT is controlled by an exquisite mix of genetic and epigenetic factors.

Keywords: Alternative promoter; Glucocorticoid receptor; Methylation; Single nucleotide polymorphism.

Fig. 1

Recruitment summary for all donors contacted, participating, exiting and analysed after completion of the study

Fig. 2

The genomic organisation, sequence variants and haplotype structure of the glucocorticoid receptor gene (NR3C1). a A schematic representation of the NR3C1 genomic organisation. Rectangles represent transcribed exons. Exons 1A–1I are alternatively spliced to a common acceptor site at the start of exon 2. White exons are non-coding, grey exons represent the coding sequence. The lower section of the panel shows the six haplotypes observed, their constituent variants and frequencies. Minor alleles are represented by bold red letters. b The linkage disequilibrium (LD) structure of the NR3C1. LD between two variants are given by colour, blue/grey no LD; white, limited LD; light red to dark red, medium to strong LD. Numbers within the LD diamonds represent the value of D prime (D’) between the two loci. D’ is statistic normalised parameters of disequilibrium

Fig. 3

Haplotype 2 (BclI alone) effects of the warm (control) or ice-cold water (seCPT) condition on SBP, heart rate and cortisol over the course of the experiment. a Systolic blood pressure in millimeter of mercury after the control (left panel) or cold water (right panel). b Heart rate in beats per minute after the control (left panel) or cold water (right panel). c Salivary cortisol levels in the control (left panel) or cold water (right panel). The seCPT or warm water was administered at 23 min and lasted 3 min. In all panels, filled circles are homozygous wild-type (CC) participants and open circles are homozygous minor allele (GG) participants. Data are the mean ± the standard error of the mean

Fig. 4

Methylation of the NR3C1 promoters 1F and 1H. a Frequency distribution of the sum of the methylation throughout promoter 1F. Female donors, open circles; male donors, open triangles. b Frequency distribution of the sum of the methylation throughout promoter 1H. Female donors, open circles; male donors, open triangles. c Pearson’s correlation coefficients were calculated for all CpG pairs and subsequently plotted against the physical distance measured in nucleotides, demonstrating that the closer two CpG nucleotides are, the stronger their correlation in methylation levels. Each data point represents Pearson’s correlation coefficient for one pair of CpGs from all donors. d Pearson’s correlation in methylation levels between sum methylation levels in promoter 1F and 1H. Each data point represents one participant

Fig. 5

NR3C1 promoter methylation effects on the systolic blood pressure response to the warm (control) or ice-cold water (seCPT) over the course of the experiment. Donors were split by median sum methylation levels. Systolic blood pressure in millimeter of mercury after the control (a) or cold water (b) was administered at 23 min and lasted 3 min. In both panels: filled circles, low methylation group; filled triangles, high methylation group. Data are the mean ± standard deviation. c Correlation between the mean baseline SBP and sum promoter 1F and 1H methylation levels. All participants are included, and each data point represents one participant. d Correlation between the mean baseline DBP and sum promoter 1F and 1H methylation levels. All participants are included, and each data point represents one participant. Baseline SBP and DBP mean of the three time-points immediately preceding the warm or cold water exposure

Fig. 6

Statistical interpretation of the link between haplotype 2 (BclI alone) and promoter methylation. a Mean methylation level of donors separated by BclI genotype. b Summary linear association test results