Janneke G. J. Hoeijmakers

Gain of function NaV1.7 mutations in idiopathic small fiber neuropathy

Small nerve fiber neuropathy (SFN) often occurs without apparent cause, but no systematic genetic studies have been performed in patients with idiopathic SFN (I‐SFN). We sought to identify a genetic basis for I‐SFN by screening patients with biopsy‐confirmed idiopathic SFN for mutations in the SCN9A gene, encoding voltage‐gated sodium channel NaV1.7, which is preferentially expressed in small diameter peripheral axons.

Gain-of-function Nav1.8 mutations in painful neuropathy

Painful peripheral neuropathy often occurs without apparent underlying cause. Gain-of-function variants of sodium channel Nav1.7 have recently been found in ∼30% of cases of idiopathic painful small-fiber neuropathy. Here, we describe mutations in Nav1.8, another sodium channel that is specifically expressed in dorsal root ganglion (DRG) neurons and peripheral nerve axons, in patients with painful neuropathy. Seven Nav1.8 mutations were identified in 9 subjects within a series of 104 patients with painful predominantly small-fiber neuropathy. Three mutations met criteria for potential pathogenicity based on predictive algorithms and were assessed by voltage and current clamp. Functional profiling showed that two of these three Nav1.8 mutations enhance the channel’s response to depolarization and produce hyperexcitability in DRG neurons. These observations suggest that mutations of Nav1.8 contribute to painful peripheral neuropathy.

Gain-of-function mutations in sodium channel Na V 1.9 in painful neuropathy

Sodium channel Nav1.9 is expressed in peripheral nociceptive neurons, as well as visceral afferents, and has been shown to act as a threshold channel. Painful peripheral neuropathy represents a significant public health challenge and may involve gain-of-function variants in sodium channels that are preferentially expressed in peripheral sensory neurons. Although gain-of-function variants of peripheral sodium channels Nav1.7 and Nav1.8 have recently been found in painful small fibre neuropathy, the aetiology of peripheral neuropathy in many cases remains unknown. We evaluated 459 patients who were referred for possible painful peripheral neuropathy, and confirmed the diagnosis of small fibre neuropathy in a cohort of 393 patients (369 patients with pure small fibre neuropathy, and small fibre neuropathy together with large fibre involvement in an additional 24 patients). From this cohort of 393 patients with peripheral neuropathy, we sequenced SCN11A in 345 patients without mutations in SCN9A and SCN10A, and found eight variants in 12 patients. Functional profiling by electrophysiological recordings showed that these Nav1.9 mutations confer gain-of-function attributes to the channel, depolarize resting membrane potential of dorsal root ganglion neurons, enhance spontaneous firing, and increase evoked firing of these neurons. Our data show, for the first time, missense mutations of Nav1.9 in individuals with painful peripheral neuropathy. These genetic and functional observations identify missense mutations of Nav1.9 as a cause of painful peripheral neuropathy.

Small-fibre neuropathies—advances in diagnosis, pathophysiology and management

Small-fibre neuropathy (SFN), a disorder of thinly myelinated Aδ-fibres and unmyelinated C-fibres, is clinically characterized by neuropathic pain symptoms and autonomic complaints. Diagnosis of SFN is challenging as the clinical picture can be difficult to interpret and results from nerve conduction studies are often normal. In cases of suspected SFN, measurement of intraepidermal nerve fibre density and/or analysis of quantitative sensory testing can enable diagnosis. New diagnostic techniques (including measurement of nerve fibre density using corneal confocal microscopy, and nociceptive evoked potentials) may contribute to the diagnostic work-up. SFN can be associated with systemic diseases such as immune-mediated disorders, but remains idiopathic in a substantial proportion of patients. Gain-of-function variants in the Nav1.7 sodium channel have recently been found in nearly 30% of patients with idiopathic SFN, but the mechanisms of axonal degeneration in the disorder remain under investigation. Identification of the systemic diseases underlying SFN will enable development of drugs that target affected pathways to improve the management of neuropathic pain and autonomic dysfunction. In this Review, we discuss recent advances in the diagnosis and pathophysiology of SFN, highlighting how improved understanding of these aspects of the disorder will contribute to better patient management.

Intra- and interfamily phenotypic diversity in pain syndromes associated with a gain-of-function variant of NaV1.7

BackgroundSodium channel NaV1.7 is preferentially expressed within dorsal root ganglia (DRG), trigeminal ganglia and sympathetic ganglion neurons and their fine-diamter axons, where it acts as a threshold channel, amplifying stimuli such as generator potentials in nociceptors. Gain-of-function mutations and variants (single amino acid substitutions) of NaV1.7 have been linked to three pain syndromes: Inherited Erythromelalgia (IEM), Paroxysmal Extreme Pain Disorder (PEPD), and Small Fiber Neuropathy (SFN). IEM is characterized clinically by burning pain and redness that is usually focused on the distal extremities, precipitated by mild warmth and relieved by cooling, and is caused by mutations that hyperpolarize activation, slow deactivation, and enhance the channel ramp response. PEPD is characterized by perirectal, periocular or perimandibular pain, often triggered by defecation or lower body stimulation, and is caused by mutations that severely impair fast-inactivation. SFN presents a clinical picture dominated by neuropathic pain and autonomic symptoms; gain-of-function variants have been reported to be present in approximately 30% of patients with biopsy-confirmed idiopathic SFN, and functional testing has shown altered fast-inactivation, slow-inactivation or resurgent current. In this paper we describe three patients who house the NaV1.7/I228M variant.MethodsWe have used clinical assessment of patients, quantitative sensory testing and skin biopsy to study these patients, including two siblings in one family, in whom genomic screening demonstrated the I228M NaV1.7 variant. Electrophysiology (voltage-clamp and current-clamp) was used to test functional effects of the variant channel.ResultsWe report three different clinical presentations of the I228M NaV1.7 variant: presentation with severe facial pain, presentation with distal (feet, hands) pain, and presentation with scalp discomfort in three patients housing this NaV1.7 variant, two of which are from a single family. We also demonstrate that the NaV1.7/I228M variant impairs slow-inactivation, and produces hyperexcitability in both trigeminal ganglion and DRG neurons.ConclusionOur results demonstrate intra- and interfamily phenotypic diversity in pain syndromes produced by a gain-of-function variant of NaV1.7.

Small-Fiber Neuropathy Nav1.8 Mutation Shifts Activation to Hyperpolarized Potentials and Increases Excitability of Dorsal Root Ganglion Neurons

Idiopathic small-fiber neuropathy (I-SFN), clinically characterized by burning pain in distal extremities and autonomic dysfunction, is a disorder of small-caliber nerve fibers of unknown etiology with limited treatment options. Functional variants of voltage-gated sodium channel Nav1.7, encoded by SCN9A, have been identified in approximately one-third of I-SFN patients. These variants render dorsal root ganglion (DRG) neurons hyperexcitable. Sodium channel Nav1.8, encoded by SCN10A, is preferentially expressed in small-diameter DRG neurons, and produces most of the current underlying the upstroke of action potentials in these neurons. We previously demonstrated two functional variants of Nav1.8 that either enhance ramp current or shift activation in a hyperpolarizing direction, and render DRG neurons hyperexcitable, in I-SFN patients with no mutations of SCN9A. We have now evaluated additional I-SFN patients with no mutations in SCN9A, and report a novel I-SFN-related Nav1.8 mutation I1706V in a patient with painful I-SFN. Whole-cell voltage-clamp recordings in small DRG neurons demonstrate that the mutation hyperpolarizes activation and the response to slow ramp depolarizations. However, it decreases fractional channels resistant to fast inactivation and reduces persistent currents. Current-clamp studies reveal that mutant channels decrease current threshold and increase the firing frequency of evoked action potentials within small DRG neurons. These observations suggest that the effects of this mutation on activation and ramp current are dominant over the reduced persistent current, and show that these pro-excitatory gating changes confer hyperexcitability on peripheral sensory neurons, which may contribute to pain in this individual with I-SFN.

Nav1.7-related small fiber neuropathy: Impaired slow-inactivation and DRG neuron hyperexcitability

Objectives: Although small fiber neuropathy (SFN) often occurs without apparent cause, the molecular etiology of idiopathic SFN (I-SFN) has remained enigmatic. Sodium channel Nav1.7 is preferentially expressed within dorsal root ganglion (DRG) and sympathetic ganglion neurons and their small-diameter peripheral axons. We recently reported the presence of Nav1.7 variants that produce gain-of-function changes in channel properties in 28% of patients with painful I-SFN and demonstrated impaired slow-inactivation in one of these mutations after expression within HEK293 cells. Here we show that the I739V Nav1.7 variant in a patient with biopsy-confirmed I-SFN impairs slow-inactivation within DRG neurons and increases their excitability. Methods: A patient with SFN symptoms including pain, and no identifiable underlying cause, was evaluated by skin biopsy, quantitative sensory testing, nerve conduction studies, screening of genomic DNA for variants in SCN9A, and functional analysis. Results: Voltage-clamp analysis following expression within DRG neurons revealed that the Nav1.7/I739V substitution impairs slow-inactivation, depolarizing the midpoint (V1/2) by 5.6 mV, and increasing the noninactivating component at 10 mV from 16.5% to 22.2%. Expression of I739V channels within DRG neurons rendered these cells hyperexcitable, reducing current threshold and increasing the frequency of firing evoked by graded suprathreshold stimuli. Conclusions: These observations provide support, from a patient with biopsy-confirmed SFN, for the suggestion that functional variants of Nav1.7 that impair slow-inactivation can produce DRG neuron hyperexcitability that contributes to pain in SFN. Nav1.7 channelopathy-associated SFN should be considered in the differential diagnosis of cases of SFN in which no other cause is found.

Functional profiles of SCN9A variants in dorsal root ganglion neurons and superior cervical ganglion neurons correlate with autonomic symptoms in small fibre neuropathy

Patients with small fibre neuropathy typically manifest pain in distal extremities and severe autonomic dysfunction. However, occasionally patients present with minimal autonomic symptoms. The basis for this phenotypic difference is not understood. Sodium channel Na(v)1.7, encoded by the SCN9A gene, is preferentially expressed in the peripheral nervous system within sensory dorsal root ganglion and sympathetic ganglion neurons and their small diameter peripheral axons. We recently reported missense substitutions in SCN9A that encode functional Na(v)1.7 variants in 28% of patients with biopsy-confirmed small fibre neuropathy. Two patients with biopsy-confirmed small fibre neuropathy manifested minimal autonomic dysfunction unlike the other six patients in this series, and both of these patients carry the Na(v)1.7/R185H variant, presenting the opportunity to compare variants associated with extreme ends of a spectrum from minimal to severe autonomic dysfunction. Herein, we show by voltage-clamp that R185H variant channels enhance resurgent currents within dorsal root ganglion neurons and show by current-clamp that R185H renders dorsal root ganglion neurons hyperexcitable. We also show that in contrast, R185H variant channels do not produce detectable changes when studied by voltage-clamp within sympathetic neurons of the superior cervical ganglion, and have no effect on the excitability of these cells. As a comparator, we studied the Na(v)1.7 variant I739V, identified in three patients with small fibre neuropathy characterized by severe autonomic dysfunction as well as neuropathic pain, and show that this variant impairs channel slow inactivation within both dorsal root ganglion and superior cervical ganglion neurons, and renders dorsal root ganglion neurons hyperexcitable and superior cervical ganglion neurons hypoexcitable. Thus, we show that R185H, from patients with minimal autonomic dysfunction, does not produce detectable changes in the properties of sympathetic ganglion neurons, while I739V, from patients with severe autonomic dysfunction, has a profound effect on excitability of sympathetic ganglion neurons.

A painful neuropathy-associated Nav1.7 mutant leads to time-dependent degeneration of small-diameter axons associated with intracellular Ca2+ dysregulation and decrease in ATP levels:

Small fiber neuropathy is a painful sensory nervous system disorder characterized by damage to unmyelinated C- and thinly myelinated Aδ- nerve fibers, clinically manifested by burning pain in the distal extremities and dysautonomia. The clinical onset in adulthood suggests a time-dependent process. The mechanisms that underlie nerve fiber injury in small fiber neuropathy are incompletely understood, although roles for energetic stress have been suggested. In the present study, we report time-dependent degeneration of neurites from dorsal root ganglia neurons in culture expressing small fiber neuropathy-associated G856D mutant Nav1.7 channels and demonstrate a time-dependent increase in intracellular calcium levels [Ca2+]i and reactive oxygen species, together with a decrease in ATP levels. Together with a previous clinical report of burning pain in the feet and hands associated with reduced levels of Na+/K+-ATPase in humans with high altitude sickness, the present results link energetic stress and reactive oxygen species production with the development of a painful neuropathy that preferentially affects small-diameter axons.

The G1662S Na V 1.8 mutation in small fibre neuropathy: impaired inactivation underlying DRG neuron hyperexcitability

Objective Painful small fibre neuropathy (SFN) represents a significant public health problem, with no cause apparent in one-half of cases (termed idiopathic, I-SFN). Gain-of-function mutations of sodium channel NaV1.7 have recently been identified in nearly 30% of patients with biopsy-confirmed I-SFN. More recently, gain-of-function mutations of NaV1.8 have been found in patients with I-SFN. These NaV1.8 mutations accelerate recovery from inactivation, enhance the response to slow depolarisations, and enhance activation at the channel level, thereby producing hyperexcitability of small dorsal root ganglion (DRG) neurons, which include nociceptors, at the cellular level. Identification and functional profiling of additional NaV1.8 variants are necessary to determine the spectrum of changes in channel properties that underlie DRG neuron hyperexcitability in these patients. Methods Two patients with painful SFN were evaluated by skin biopsy, quantitative sensory testing, nerve conduction studies, screening of genomic DNA for mutations in SCN9A and SCN10A and electrophysiological functional analysis. Results A novel sodium channel NaV1.8 mutation G1662S was identified in both patients. Voltage-clamp analysis revealed that the NaV1.8/G1662S substitution impairs fast-inactivation, depolarising the midpoint (V1/2) by approximately 7 mV. Expression of G1662S mutant channels within DRG neurons rendered these cells hyperexcitable. Conclusions We report for the first time a mutation of NaV1.8 which impairs inactivation, in patients with painful I-SFN. Together with our earlier results, our observations indicate that an array of NaV1.8 mutations, which affect channel function in multiple ways, can contribute to the pathophysiology of painful peripheral neuropathy.