Functional impact of the TRPA1 H560D variant on neuropathic pain.

Small fiber neuropathy (SFN) is characterized by acral burning pain and may be associated with genetic variants of the transient receptor potential ankyrin 1 (TRPA1) channel. We studied a 43-year-old male patient with SFN with severe acral and abdominal pain carrying the TRPA1 variant NM_007332.3:c.1678C>G (His560Asp). The patient underwent a complete neurological work-up including a skin punch biopsy for the generation of induced pluripotent stem cells. Sensory-like neurons (iSN) were differentiated and characterized by immunocytochemistry, qRT-PCR, and patch-clamp recordings. hTRPA1_H560D was further transfected into human embryonic kidney 293 (HEK293) cells and characterized using Ca 2+ -flux assay, Ca 2+ -imaging, and patch-clamp under TRPA1-agonist/-antagonist conditions. Channel localization was assessed by immunocytochemistry and western blot, and cell viability by a CellTiter-Glo Assay. The clinical work-up confirmed SFN. Sensory-like neurons were electrically excitable and expressed characteristic neuronal and peripheral markers. Ca 2+ -flux assay and Ca 2+ -imaging revealed reduced sensitivity to allyl isothiocyanate in iSN ( P < 0.001) and transfected HEK293 cells ( P < 0.05). Patch-clamp recordings showed diminished agonist-evoked currents in hTRPA1_H560D-expressing cells compared with hTRPA1_WT ( P < 0.05). TRPA1_H560D reduced TRPA1 expression ( P < 0.001) and impaired membrane localization ( P < 0.05) and channel glycosylation, without affecting HEK293 cell viability. Our 2 complementary in vitro models present TRPA1_H560D as a loss-of-function variant despite its association with clinical pain. This paradox suggests pain mechanisms beyond channel hyperactivity, such as impaired membrane trafficking, e.g. because of altered glycosylation. Our findings further emphasize the complexity of genotype-phenotype relationships in SFN and highlight the need for cautious interpretation of genetic variants of uncertain significance in clinical practice.