A gain-of-function mutation in TRPA1 causes familial episodic pain syndrome

Abstract

Human monogenic pain syndromes have provided important insights into the molecular mechanisms that underlie normal and pathological pain states. We describe an autosomal-dominant familial episodic pain syndrome characterized by episodes of debilitating upper body pain, triggered by fasting and physical stress. Linkage and haplotype analysis mapped this phenotype to a 25 cM region on chromosome 8q12-8q13. Candidate gene sequencing identified a point mutation (N855S) in the S4 transmembrane segment of TRPA1, a key sensor for environmental irritants. The mutant channel showed a normal pharmacological profile but altered biophysical properties, with a 5-fold increase in inward current on activation at normal resting potentials. Quantitative sensory testing demonstrated normal baseline sensory thresholds but an enhanced secondary hyperalgesia to punctate stimuli on treatment with mustard oil. TRPA1 antagonists inhibit the mutant channel, promising a useful therapy for this disorder. Our findings provide evidence that variation in the TRPA1 gene can alter pain perception in humans.

Figure 1

Genetic Mapping of the Mutant Locus Underlying FEPS (A) Pedigree segregating FEPS. Microsatellite haplotypes spanning the linkage peak on chromosome 8q12.1–8q24.1 identified in the whole-genome scan. The haplotypes shown were reconstructed using markers from the whole-genome scan and fine-mapping stages. The haplotype cosegregating with the pain phenotype is shown in red. The minimal critical region spans 25 cM and is flanked by marker D8S1775 on the centromeric end and marker D8S1762 on the telomeric end (informative recombinants individuals being III:8 and II:5, respectively). Only individuals included in the linkage analysis are shown (individuals with an asterisk were unavailable for genotyping). (B) Multipoint LOD scores obtained on chromosome 8 (including fine-mapping markers) and location of the TRPA1 gene. Physical distance (in Mb) is shown at the top and genetic distance (in cM) at the bottom. The dotted lines indicate the LOD score thresholds of 3 and −2 (i.e., significant evidence for or against linkage, respectively).

Figure 2

Identification of a Point Mutation Underlying FEPS (A) Sequence chromatogram showing the TRPA1 mutation identified in the FEPS family. The arrow indicates the location of the mutation. Below is a selection of mammalian TRPA1 sequences showing that the mutation site region is evolutionarily conserved. (B) Schematic representation of the TRPA1 channel. The substitution (S) identified in the FEPS family occurs in asparagine (N) 855 located in putative transmembrane segment S4.

Figure 3

Increased Area of Punctate Hyperalgesia in Patients with FEPS after Topical Mustard Oil Application (A) The area of flare, secondary hyperalgesia to punctate stimuli, and brush-evoked allodynia 10 min after mustard oil application in a patient with FEPS and a family control. (B–E) Comparison of pain assessed by VAS during topical mustard oil application (B) and the area of flare (C), punctate hyperalgesia (D), and brush-evoked allodynia (E) at 10, 30, and 60 min after application in FEPS patients versus family controls. All assessments were performed in the same nine cases and eight family controls, except in (C) where only eight cases where examined. The boxes span the 25th to 75th percentile with the median represented as a solid line and the mean as a dotted line, the whiskers show the full range of the data. ^∗p < 0.05, unpaired t test.

Figure 4

Pharmacological and Biophysical Analysis of hTRPA1-WT and hTRPA1-N855S (A) Intracellular calcium response to 250 μM, 100 μM, 50 μM, and 10 μM CA and to 100 μM CA in the presence of 3 μM ruthenium red (RR). Results for HEK293 cells expressing hTRPA1-WT are shown on the left and for hTRPA1-N855S on the right. Horizontal bars at the top indicate the time of CA application. (B) Dose-response curve of CA-evoked calcium responses for hTRPA1-WT and hTRPA1-N855S. [Ca²⁺]_I normalized to maximum calcium response to 250 μM CA. Traces represent average [Ca²⁺]_I from 20–30 cells. Data were fit to the Hill equation. (C) HEK293 cells expressing hTRPA1-WT (left; n = 5) or hTRPA1-N855S (right; n = 6) show activation by CA (100 μM) and inhibition by ruthenium red (RR). Currents were recorded at +100 mV and –100 mV and are normalized to current at +100 mV. Letters denote time point at which voltage ramps (shown in D) were acquired to generate current-voltage relationships. (D) Average current-voltage relationship of hTRPA1-WT and hTRPA1-N855S in the presence of 100 μM CA. Currents are normalized to +100 mV. (E) Whole-cell current traces of HEK293 cells expressing hTRPA1-WT in response to the indicated voltage step protocol in the absence (left) and presence (right) of 100 μM CA. Bottom panel shows higher resolution of normalized tail current in response to a step to −140, 0, and +140 mV. Dotted line shows zero current level. (F) Same as (E) but in HEK293 cells expressing hTRPA1-N855S. (G) Mean steady-state activation curves obtained from tail currents (I_T) at −140 mV for hTRPA1-WT (n = 5) and hTRPA1-N855S (n = 6) in the presence of CA. The midpoints of voltage activation (V1/2) for the WT and mutant channels are indicated at the top. Error bars in all plots represent SEM across individual cell measurements.

Figure 5

Activation of hTRPA1-WT and hTRPA1-N855S with an Endogenous TRPA1 Ligand and Cold (A) HEK293 cells expressing hTRPA1-WT (left; n = 5) or hTRPA1-N855S (right; n = 7) show activation by 4-HNE and block HC-030031. Currents were recorded at +100 mV and –100 mV and are normalized to current at +100 mV. Letters denote time point at which voltage ramps (shown in B) were acquired to generate current-voltage relationships. (B) Average current-voltage relationship of hTRPA1-WT and hTRPA1-N855S in the presence of 100 μM 4-HNE (as shown by letters a and c, respectively, in A) and after perfusion of 100 μM 4-HNE + 10 μM HC-030031 (as shown by letters b and d, respectively, in A). Currents are normalized to +100 mV. (C) Whole-cell current traces of HEK293 cells expressing hTRPA1-WT (top) or hTRPA1-N855S (bottom) in response to the indicated voltage step protocol applied at 25°C (left) and 12°C (right). Dotted line shows zero current level. (D) Mean steady-state activation curves obtained from tail currents (I_T) at −140 mV for hTRPA1-WT (n = 5) and hTRPA1-N855S (n = 5) in response to 25°C and 12°C. Error bars in all plots represent SEM across individual cell measurements.