Rare TRPM2 Variants in Painful Small Fiber Neuropathy: NGS Analysis and Functional Insights

Background & Aims

Small fiber neuropathy (SFN) is a heterogeneous group of disorders affecting thin myelinated A? and unmyelinated C fibers, in which neuropathic pain typically dominates the clinical picture, along with possible loss of thermal and nociceptive sensation, and autonomic disturbances [3]. The aetiology of SFN is complex, involving both inherited and acquired factors, Genetic studies in painful SFN revealed that Voltage Gated Sodium Channels (VGSC) genes, particularly SCN9A, are involved in pain amplification [1,2]. Besides VGSC genes, Transient Receptor Potential (TRP) channel genes have also been associated with neuropathic pain because of their pivotal role in nociception [6]. The aim of this study was to further broaden the genetic knowledge of painful SFN by performing whole-exome sequencing (WES) analysis on Italian families with at least one member with diagnosis of SFN, and to investigate experimentally the impact of the identified mutations on channel function.

Methods

Twelve families with painful SFN were selected having at least one affected member, positive neurological examination and pain questionnaire result with numerical rating score >=4. After performing WES, Variants were filtered, keeping only the ones mapping to a manually curated panel of pain related genes (n=592), with minor allele frequency ?5% in population databases [4]; conservation and computational predictors were also considered in the filtering. Segregation analysis was performed in each pedigree. The impact of selected causative variants was assessed through electrophysiological patch-clamp recordings on HEK cell line cultures transfected with wildtype and mutant expression vectors [5].

Results

Among the selected variants, we focused on two rare missense mutations mapping in TRP Melastatin 2 (TRPM2), p.Arg411Trp and p.Pro1102Ser, identified in two unrelated patients from two different pedigrees. Affected individuals in the families report neuropathic pain and decreased intra-epidermal fiber density. The TRPM2:p.Arg411Trp mutation is found in the N-terminal segment IQ-like motif domain (406-416) where calmodulin binds and regulates channel activation; the second mutation p.Pro1102Ser is near the pore region, between segment 5 and 6 that is involved in channel sensitization through calcium ion flux. Whole-cell patch-clamp recordings revealed functional expression of both mutant channels but a significant reduction in TRPM2 currents compared to wild-type. The p.Arg411Trp mutation resulted in a 83.1% reduction in peak TRPM2 current (p-value =0.0067 at +80 mV), while the p.Pro1102Ser mutation resulted in a 93.3% reduction (p-value =0.001 at +80 mV) in comparison to wildtype.

Conclusions

This study reports two novel missense mutations in TRPM2 segregating with painful SFN. Functional analysis revealed that both mutations affect the normal function of the TRPM2 channel, leading to a significant reduction in their currents. Whether this is a consequence of channel disfunction or impaired membrane expression is under investigation. TRPM2 channel was previously linked to neuropathic pain in animal models [6] but not in human genetic studies on neuropathic pain or SFN. These findings suggest that these mutations are likely causative and contribute to painful SFN development. Further investigations are warranted to elucidate the precise mechanisms by which these mutations lead to painful SFN and to speculate on TRPM2 as a potential pharmacological druggable target in future studies.

References

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