TRPA1 rare variants in chronic neuropathic and nociplastic pain patients

Abstract

Missing aspects of the heritability of chronic neuropathic pain, as a complex adult-onset trait, may be hidden within rare variants with low effect on disease risk, unlikely to be resolved by a single-variant approach. To identify new risk genes, we performed a next-generation sequencing of 107 pain genes and collapsed the rare variants through gene-wise aggregation analysis. The optimal unified sequence kernel association test was applied to 169 patients with painful neuropathy, 223 patients with nociplastic pain (82 diagnosed with chronic widespread pain and 141 with fibromyalgia), and 216 healthy controls. Frequency and features of variants in TRPA1, which was the most significant gene, were further validated in 2 independent cohorts of 140 patients with chronic pain (90 with painful neuropathy and 50 with chronic widespread pain) and 34 with painless neuropathy. The effect of aminoacidic changes were modeled in silico according to physicochemical characteristics. TRPA1 was significantly enriched of rare variants which significantly discriminated chronic pain patients from healthy controls after Bonferroni correction (P = 6.7 × 10⁻⁴, ρ = 1), giving a risk of 4.8-fold higher based on the simple burden test (P = 0.0015, OR = 4.8). Among the 32 patients harboring TRPA1 variants, 24 (75%) were diagnosed with nociplastic pain, either fibromyalgia (12; 37.5%) or chronic widespread pain (12; 37.5%), whereas 8 (25%) with painful neuropathy. Irrespective of the clinical diagnosis, 12 patients (38%) complained of itch and 10 (31.3%) of cold-induced or cold-accentuated pain, mostly episodic. Our study widens the spectrum of channelopathy-related chronic pain disorders and contributes to bridging the gap between phenotype and targeted therapies based on patients' molecular profile.

Figure 1.

Schematic representation of human TRPA1 structure and genetic variants. Red dots indicate the variants exclusively carried by chronic pain patients, and black dots refer to variants identified also in healthy controls. Variants are mapped according to O75762 UniProtKB human template (https://www.uniprot.org/uniprotkb/O75762/entry). Created with BioRender.com (agreement number: XZ257JVKR).

Figure 2.

Residue substitutions on TRPA1 tetramer. (A) The residues are modelled on the solved structure (PDB ID: 6v9w, residues: 447-1079). In sticks the identified variants. The amino acidic changes are indicated with different colours according to their impact on the channel, taking into consideration the physical–chemical differences and the context of neighbour residues: red is used for a more deleterious predicted effect and green for substitutions with a milder effect, as indicated in Table S3, available at http://links.lww.com/PAIN/B804. (B) The double substitution carried by the same patient p.N373I and p.D412N, localized in a highly conserved loci; substitutions are visualized on the AF-O75762-F1 model; in grey sticks, the wild type side chains and in green, the mutants; in yellow is evidenced the H-bond lost in the mutant. (C) p.D495G localizes in the conserved ANK13 domain, wild type amino acid is in grey and mutant in light blue; in yellow is evidenced the H-bond lost in the mutant. (D) p.M626I is in the cytosol helixes, close to the residue C633, involved in disulphide bridge, whose position can be perturbed, and near to P622 and M634, involved in the activation by the scorpion wasabi receptor toxin; in grey sticks the wild type side chains and in green the mutants. (E) p.R919L localizes internally, in the pore lumen, and induces a change from a positively charged amino acid into a hydrophobic one, with a strong potential impact on the channel selectivity and permeability to Ca²⁺; in green the mutated side chains and in grey the wild type; distances between wild type side chains and mutated one are in evidence. PDB, Protein Data Bank.