Enrichment of Genomic Pathways Based on Differential DNA Methylation Associated With Chronic Postsurgical Pain and Anxiety in Children: A Prospective, Pilot Study

Abstract

We have reported child anxiety sensitivity (Child Anxiety Sensitivity Index [CASI]) predicts chronic postsurgical pain (CPSP). Herein, we evaluated DNA methylation profiles to understand the gene-environment interactions underlying CPSP and CASI, to identify shared, enriched, genomic pathways. In 73 prospectively recruited adolescents undergoing spine fusion, preoperative CASI and pain data over 12 months after surgery were collected. DNA from the peripheral blood of evaluable subjects with (n = 16) and without CPSP (n = 40) were analyzed using MethylationEPIC arrays. We identified 637 and 2,445 differentially DNA methylated positions (DMPs) associated with CPSP and CASI, respectively (P ≤ .05). Ingenuity pathway analysis of 39 genes with DMPs for both CPSP and CASI revealed enrichment of several canonical pathways, including GABA receptor (P = .00016 for CPSP; P =.0008 for CASI) and dopamine-DARPP32 feedback in cyclic adenosine monophosphate (P = .004 for CPSP and P =.00003 for CASI) signaling. Gene-gene interaction network enrichment analysis revealed participation of pathways in cell signaling, molecular transport, metabolism, and neurologic diseases (P < 10^-8). Bioinformatic approaches to identify histone marks and transcription factor (TF) binding events underlying DMPs, showed their location in active regulatory regions in pain pathway relevant brain cells. Using Enrichr/Pinet enrichment and Library of Integrated Network-Based Cellular Signatures knockdown signatures, we identified TFs regulating genes with DMPs in association with CPSP and CASI. In conclusion, we identified epigenetically enriched pathways associated with CPSP and anxiety sensitivity in children undergoing surgery. Our findings support GABA hypofunction and the roles of the dopamine-DARPP32 pathway in emotion/reward and pain. This pilot study provides new epigenetic insights into the pathophysiology of CPSP and a basis for future studies in biomarker development and targetable interventions. PERSPECTIVE: Differential DNA methylation in regulatory genomic regions enriching shared neural pathways were associated with CPSP and CASI in adolescents undergoing spine surgery. Our findings support GABA hypofunction and the roles of the dopamine-DARPP32 pathway in emotion/reward contributing to behavioral maintenance of pain 10 to 12 months after surgery.

Keywords: Bioinformatics; DNA methylation; anxiety; chronic postsurgical pain; epigenetics; functional genomics.

Figure 1:

Workflow for the statistical analysis of MethylationEPIC array data for chronic postsurgical pain (CPSP) and anxiety sensitivity (CASI) outcomes. One sample did not pass quality control. DNA methylation beta and M values were modeled and CpG sites with methylation satisfying certain criteria were included in logistic (for CPSP) and linear (CASI) regression models adjusted for other covariates. Methylation at sites significantly associated with outcomes were then included in the pathway and functional analyses.

Figure 2:

Gene-gene interaction networks. Genes associated with the differentially methylated sites were uploaded to Ingenuity pathway analysis. Based on p-value cutoffs of 10⁻⁸, three networks were identified. Two of them were similar in function with several overlapping molecules. Hence, two of the different networks are presented here. The network in panel A is associated with Cell Signaling, Molecular Transport, Vitamin and Mineral Metabolism. It had a p-score of 33 and 14 focus molecules (including CACNA1A, CACNA1C, Calmodulin, ERK1/2, Histone h3, Histone h4, IkB-NfkB, NFkB (complex), miR-9–3p). The network in Panel B is associated with Neurological Disease, Organismal Injury and Abnormalities, Connective Tissue Development and Function; with a p-score of 12, and 6 focus molecules (including ESR1, KCNK6, PRIM2, TNF).

Figure 3:

The results of enrichment analysis using Enrichr are shown here, where for each transcription factor, e.g., REST, its (here ENCODE mapped) targets among genes identified in our study are indicated by red squares (and include in this case RIMS2, CDH13, SPTBN4 etc.). Note that statistical significance of the enrichment is indicated by red vertical bars associated with each transcription factor.