Genetic mapping in Diversity Outbred mice identifies a Trpa1 variant influencing late-phase formalin response

Abstract

Identification of genetic variants that influence susceptibility to pain is key to identifying molecular mechanisms and targets for effective and safe therapeutic alternatives to opioids. To identify genes and variants associated with persistent pain, we measured late-phase response to formalin injection in 275 male and female Diversity Outbred mice genotyped for over 70,000 single nucleotide polymorphisms. One quantitative trait locus reached genome-wide significance on chromosome 1 with a support interval of 3.1 Mb. This locus, Nociq4 (nociceptive sensitivity quantitative trait locus 4; MGI: 5661503), harbors the well-known pain gene Trpa1 (transient receptor potential cation channel, subfamily A, member 1). Trpa1 is a cation channel known to play an important role in acute and chronic pain in both humans and mice. Analysis of Diversity Outbred founder strain allele effects revealed a significant effect of the CAST/EiJ allele at Trpa1, with CAST/EiJ carrier mice showing an early, but not late, response to formalin relative to carriers of the 7 other inbred founder alleles (A/J, C57BL/6J, 129S1/SvImJ, NOD/ShiLtJ, NZO/HlLtJ, PWK/PhJ, and WSB/EiJ). We characterized possible functional consequences of sequence variants in Trpa1 by assessing channel conductance, TRPA1-TRPV1 interactions, and isoform expression. The phenotypic differences observed in CAST/EiJ relative to C57BL/6J carriers were best explained by Trpa1 isoform expression differences, implicating a splice junction variant as the causal functional variant. This study demonstrates the utility of advanced, high-precision genetic mapping populations in resolving specific molecular mechanisms of variation in pain sensitivity.

Figure 1.. Late phase formalin response has a significant QTL on mouse chromosome 1 (Nociq4).

A. Genome-wide scan for late phase response to formalin injection reveals a QTL with a peak LOD score of 5.71 at 14.29 Mb. Permutation-derived significance thresholds are marked by horizontal lines: 0.63 (bottom), 0.1 (middle), 0.05 (top). B. The founder allele effects show that the CAST/EiJ allele contributes to lower late phase formalin response sensitivity. Each line represents the effect of one of the eight founder alleles in DO mice. The differences between strains are significant when the LOD score in panel C is high. C. Genome scan for sensitivity to late phase formalin response on chromosome 1.

Figure 2.. Trpa1 lies within a genetically mapped region of chromosome 1 significantly correlated with late phase behavioral response to formalin injection.

A. Minor allele frequency of the SNPs with the highest LOD score and shown in red in panel B. Strains: A, A/J; B, C57BL/6J; C, 129S1/SvImJ; D, NOD/ShiLtJ; E, NZO/HlLtJ; F, CAST/EiJ; G, PWK/PhJ; H, WSB/EiJ. SNPs for which only CAST/EiJ (F) contributes the alternate allele have the highest LOD scores at the Nociq4 locus. B. LOD scores of SNP association mapping in the chromosome 1 QTL interval. Each point represents the LOD score from one SNP. Red SNPs represent a 1-LOD drop from the maximum LOD. C. Candidate protein coding genes underlying the Nociq4 locus relative to mouse genome build GRCm38 and Ensembl annotation version 75 [73]. Top candidate gene Trpa1 is circled in blue (Chr1:14.87-14.91 [−]).

Figure 3.. GeneWeaver analysis corroborates Trpa1 as most likely Nociq4 candidate gene.

The gene set bipartite graph shows Trpa1 as the most highly connected gene among pain related genes within the QTL interval. Genes are represented by oval shaped nodes, edges represent gene set membership, and the rectangular nodes represent gene sets retrieved from the GeneWeaver database. Other highly connected genes include Eya1, Tram1, Lactb2, Slo5a1, Sulf1, Ncoa2.

Figure 4.. CAST/EiJ mice exhibit a strain-specific response to formalin injection at Nociq4.

Averaging founder strain allele effects over time relative to Nociq4 indicates a unique contribution by the CAST/EiJ allele to lower phenotypic response during late phase formalin injection. Each line represents the effect of one (or more) of the eight founder alleles in DO mice. Sample size per allele at the QTL variant (n) is shown in panel B. Standard error confidence bands are shown as shading around each line in panel C. All 8 DO founder strains respond similarly during the early (acute pain) phase following formalin injection (0-10 mins) (A, C, D). All 8 strains exhibit a drop in phenotypic response ~10 mins post-injection, which signifies the shift from the acute pain response (early phase) to the (late phase) tonic pain response (A, C, D). Only CAST/EiJ does not develop the late phase behavior typically associated with tonic response to formalin injection (10-60 mins) (panel D).

Figure 5.. Schematic representation of potential functional effects of Trpa1 candidate SNPs in C57BL/6J and CAST/EiJ.

A. SNP rs32035600 induces a Valine 115 to Isoleucine shift (a difference of one methyl group) between the strains in the ankyrin repeat domain of Trpa1. The slight side chain change may alter the structure and thus functional properties of the protein. B. The same amino acid changing SNP, rs32035600, could affect TRPA1 function in an allele specific manner by altering the TRPV1-TRPA1 interaction. C. SNP rs239908314 in intron 21 regulates the expression and/or alternative splicing of the Trpa1 transcript, affecting levels of Trpa1a and Trpa1b on the cell membrane. Trpa1a and Trpa1b expression levels could also be influenced by 3’ UTR SNP rs215479411 – if the same differential expression pattern is observed for both isoforms.

Figure 6.. The Trpa1 CAST/EiJ variant rs32035600 does not significantly alter TRPA1 channel conductance in HEK293T cells.

A. Both C57BL/6J and CAST/EiJ alleles of Trpa1 variant rs32035600 showed robust responses to the application of mustard oil (MO) during whole-cell patch clamp recording in HEK293T cells 48–72h after transfection. B. There was no difference between C57BL/6J (n=15 cells) and CAST/EiJ (n=19 cells) TRPA1 in current amplitude (p = 0.56). There was no difference between C57BL/6J and CAST/EiJ TRPA1 in the rise time (C.) (10-90% of peak; p = 0.34) or decay time (D.) (p = 0.47) of MO-induced currents.

Figure 7.. Quantification of western blot intensity of TRPA1-TRPV1 coupling shows a sex × genotype effect.

CAST/EiJ males have a heightened response to formalin that results in increased receptor co-IP and C57BL/6J females decrease receptor co-IP in response at 30 mins post-formalin injection.

Figure 8.. Quantitative-PCR shows higher average concentration of Trpa1a in the dorsal root ganglia (DRG) of naïve male and female CAST/EiJ compared to C57BL/6J mice.

Total Trpa1 expression is approximately doubled in CAST/EiJ mice compared to age- and sex-matched C57BL/6J controls. When both Trpa1a and Trpa1b isoforms are considered, only Trpa1a is differentially expressed between the strains, expressed nearly 3x higher in CAST/EiJ DRG.