Differential methylation of the TRPA1 promoter in pain sensitivity

Abstract

Chronic pain is a global public health problem, but the underlying molecular mechanisms are not fully understood. Here we examine genome-wide DNA methylation, first in 50 identical twins discordant for heat pain sensitivity and then in 50 further unrelated individuals. Whole-blood DNA methylation was characterized at 5.2 million loci by MeDIP sequencing and assessed longitudinally to identify differentially methylated regions associated with high or low pain sensitivity (pain DMRs). Nine meta-analysis pain DMRs show robust evidence for association (false discovery rate 5%) with the strongest signal in the pain gene TRPA1 (P=1.2 × 10(-13)). Several pain DMRs show longitudinal stability consistent with susceptibility effects, have similar methylation levels in the brain and altered expression in the skin. Our approach identifies epigenetic changes in both novel and established candidate genes that provide molecular insights into pain and may generalize to other complex traits.

Figure 1. DNA methylation in MZ twins.

CpG-density-weighted DNA methylation levels in 25 MZ discovery twin pairs showing. (a) DNA methylation (blue inner circle), within-pair MZ intraclass correlation (ICC) (red middle circle) and ASM levels (green outer circle). Methylation levels using running medians in 1-Mb windows are shown from low (AMS=0, light blue) to high (AMS=1,000, blue), and MZ ICCs are plotted from −0.25 (inner red circle radius) to 0.3 (outer red circle radius). ASM green circle represents the proportion of individuals that show evidence for ASM from 0.8 (light green) to 1 (green) at the 106 SNPs. (b) Genome-wide distribution of MZ ICCs and (c) correlations within MZ twins and unrelated pairs in the discovery MZ twin cohort (box shows the 25 and 75% quantiles and whiskers extend to 1.5 times the inner quartile range (IQR)). (d) DNA methylation levels across genomic annotations. (e) Within-individual MeDIP-seq and Illumina-450k DNA methylation comparison, showing the density of points in blue.

Figure 2. Meta-analysis pain EWAS results.

(a) EWAS results and FDR 5% threshold (red line). (b,c) Differential methylation in TRPA1 in pain sensitivity, showing (b) MeDIP-seq hypermethylation (blue) effects in the discovery set (co-twins linked by lines). (c) Pain DMR validates in the bisulphite sequencing data from CpG site at chr8:73151235, bp (pain-DMR rank correlation=0.22 and association taking into account twin structure, P=0.03). (d) Gene expression increases (LMER P=0.03) with higher pain thresholds in 341 twins.

Figure 3. Longitudinal stability of pain DMRs.

(a) DNA methylation differences (MEDIPS AMS) over 2–3 years in 33 discovery set individuals (columns), ordered by longitudinal differences in HPST scores (bar plot). Heatmap rows correspond to the 100 top-ranked MAP-DMRs, where the top 25% are highly variable and show loss (blue) and gain (red) of methylation over time. (b) Eighteen MAP-DMRs with greater HPST differences in individuals (n≥6) with variable methylation (change 30–100%) over time (green), compared with HPST differences in individuals with stable methylation (change 0–30%) over time (grey). Box shows the 25 and 75% quantiles and whiskers extend to 1.5 times the IQR.