Introduction
Sodium channel Na
V1.7 is preferentially and abundantly expressed within dorsal root ganglia (DRG) [
1,
2], trigeminal ganglia [
3] and sympathetic ganglion neurons [
1,
2], and their fine-diameter axons [
4]. The physiological attributes of Na
V1.7 include slow closed-state inactivation, which permits activation of the channel in response to small, slow depolarizations close to resting potential [
5]. Na
V1.7 thus acts as a threshold channel, amplifying stimuli such as generator potentials in nociceptors, thereby setting their gain [
6].
Gain-of-function mutations and variants (single amino acid substitutions) of Na
V1.7 have been linked to three pain syndromes. Inherited erythromelalgia (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 Na
V1.7 mutations that hyperpolarize activation, slow deactivation, and enhance the channel ramp response [
7]. Paroxysmal extreme pain disorder (PEPD) is characterized by perirectal, periocular or perimandibular pain, often triggered by defecation or lower body stimulation [
8], and has been linked to Na
V1.7 mutations that severely impair fast-inactivation [
9]. Small Fiber Neuropathy (SFN), which involves thinly myelinated and unmyelinated peripheral nerve fibers [
10,
11], presents a clinical picture that is characteristically dominated by neuropathic pain and autonomic symptoms [
12], together with preservation of normal strength, tendon reflexes, and vibration sense, and normal nerve conduction studies (NCS), which rule out large fiber involvement. The diagnosis of SFN can be confirmed by demonstration of reduced intraepidermal nerve fiber density (IENFD) on skin biopsy and/or abnormal quantitative sensory testing (QST) [
13,
14]. No apparent cause for SFN can be identified in 24% to 93% of cases in published patient series, and these cases are termed idiopathic I-SFN [
10,
15,
16]. Faber
et al., recently reported that gain-of-function variants (single amino acid substitutions) of voltage-gated sodium channel Na
V1.7 are present in approximately 30% of patients with biopsy-confirmed I-SFN [
17].
Distal (feet, and in some cases, hands) burning or stabbing pain or paraesthesias are the initial symptoms in most patients with I-SFN, and facial pain is rare. Most of the eight patients with SFN described earlier by Faber
et al., [
17] fit this clinical picture, and presented with pain in the feet and in some cases the hands early in their course, but did not manifest facial pain [
17]. In contrast, one patient in this series presented with severe pain in the teeth, jaw, and behind the eyes. This patient (patient 8 in Faber
et al., 2011) harbored the Na
V1.7 variant c.684C > G (I228M) [
17]; functional properties of this variant have not been previously reported. We subsequently studied the sister of this patient, who houses the same variant (c.684C > G (I228M) in Na
V1.7) and suffers from a different syndrome of pain and redness of the hands and feet triggered by warmth, and have encountered an additional patient housing the same Na
V1.7 variant with pain over the scalp. In this study we report these three different clinical presentations of the I228M Na
V1.7 variant, and demonstrate the effects of the Na
V1.7/I228M channels on excitability in both trigeminal ganglion and DRG neurons.
Discussion
In this study we describe three patients (two siblings, and a third, unrelated patient) housing the I228M variant of sodium channel Na
V1.7. One of these patients displayed a clinical phenotype that included pain in the face as well as in other parts of the body together with autonomic symptoms, with the diagnosis of SFN confirmed by demonstration of reduced IENFD on skin biopsy, and abnormal QST. The second patient gave a history of distal extremity pain and redness, triggered by warmth and relieved by cooling. While these symptoms are commonly reported in IEM [
7,
21], she also reported autonomic symptoms including increased perspiration, gastrointestinal complaints and hot flashes, which are not characteristic of IEM. The third patient initially experienced discomfort and vasomotor instability over the occiput, which progressed to involve the distal extremities, together with abnormal perspiration, intermittent difficulties with micturition; skin biopsy and QST in this patient were both abnormal, confirming the diagnosis of SFN.
Because facial pain was a prominent part of the clinical picture in one of the patients described in this paper, we assessed the effect of the I228M mutation on excitability of trigeminal ganglion neurons. Our current-clamp analysis demonstrated that the I228M variant depolarizes resting membrane potential, reduces current threshold and enhances repetitive firing in these cells. The effect of only one other Na
V1.7 mutation has been assessed in trigeminal ganglion neurons. We previously reported that the A1632E Na
V1.7 mutation, from a patient who displayed a mixed clinical phenotype with features of both IEM and PEPD, produces hyperexcitability in trigeminal ganglion neurons [
22]. The A1632E mutation, however, produced hyperexcitability in these cells over the entire range of stimulus intensities, while I228M produces hyperexcitability only at low stimulus intensities. Whether other gain-of-function mutations of Na
V1.7 have similar effects on trigeminal ganglion neurons remains to be determined.
The I228M substitution is located within the fourth transmembrane segment (S4) within domain I of the Na
V1.7 channel. The S4 in each of the domains of sodium channels is an amphiphatic helix which is characterized by a repeat motif of positively charged amino acids at every third position [
19]. Non-charge-conserved mutations, S211P and F216S, in DI/S4 have been linked to IEM, and have been shown to shift voltage-dependence of activation in a hyperpolarizing direction, making it easier to open the mutant channels [
23,
24]. The I228M substitution does not change the number of charges in the S4 segment, and reasonably conserves the hydrophobic nature of the side-chain of this residue, and thus might not have been predicted to have a functional effect. A link to function, however, is suggested by the conservation of the I228 residue at the equivalent position in all voltage-gated sodium channels sequenced to date (Figure
1); I228 is substituted by the other branched side-chain residue, valine, in Na
V1.9. The functional effect of I228M might be related to the proximity of the I228 residue to the cytoplasmic end of the S4 segment, which could alter the local structure of the helix in a subtle manner affecting slow-inactivation but not activation. Notably, while the I228M variant produced hyperexcitability in both DRG and trigeminal ganglion neurons, only two of the three patients described here reported cranial pain, and it was experienced in the jaw and eyes in one, while it was focused on the scalp in the other.
Our results demonstrate phenotypic diversity in the pain syndromes associated with the I228M substitution in the Na
V1.7 channel in three different patients. Two of these patients were from the same family, which also includes patient 1's two asymptomatic sons who carry the I228M Na
V1.7 variant. Both of these asymptomatic carriers are younger than the age of onset of the three patients presented, and whether they will develop pain in the future is unclear. We have previously noted different ages of onset and different degrees of pain, and an asymptomatic carrier in members of a single family, all housing the G616R Na
V1.7 mutation [
25]. Whether this phenotypic variability is due to modifier genes, epigenetic factors, and/or environmental factors is not yet clear. The minor allele of the Na
V1.7 R1150W variant, which is known to produce hyperexcitability in DRG neurons [
26], has been associated with increased pain scores in a number of acquired pain syndromes (osteoarthritis, compressive radiculopathies, traumatic limb amputation), suggesting that environmental factors may, at least in some individuals, act as triggers or increase risk of developing pain [
27].
Most peripheral neuropathies present in a "stocking glove" distribution with sensory abnormalities and pain first appearing in the most distal parts of the limbs (feet, then hands). It has traditionally been held that longer nerve fibers, or the cells giving rise to them, are affected before shorter fibers or the cells giving rise to them. A number of potential mechanisms have been invoked for this length-dependent mode of progression of neuropathy, including impairment of axoplasmic transport [
28], increased probability of demyelination along longer nerve fibers [
29], or a higher probability of impairment of calcium homeostasis along longer nerve fibers [
30,
31]. However, the present results show that the Na
V1.7 I228M variant, which impairs slow-inactivation, produces physiological changes in primary afferent neurons (trigeminal ganglion neurons) that innervate the relatively proximal sensory field of the face and scalp, as well as DRG neurons. While we do not know whether there was degeneration of small fibers innervating the face or scalp in these patients, both exhibited degeneration of the relatively long axons, as demonstrated by reduced IENFD on skin biopsy from the leg.
In summary, our results demonstrate phenotypic diversity in pain syndromes associated with the I228M gain-of-function variant of NaV1.7. Importantly, variability in clinical presentation was present not only when comparing patients from different families, but also for patients within a single family. Our findings also demonstrate that the I228M variant can increase excitability of trigeminal ganglion as well as DRG neurons. While the mechanism(s) responsible for this phenotypic diversity remain unexplained, our findings suggest that clinical studies, in patients who are carriers of functional variants of sodium channels, should be designed to take phenotypic variability, even within single families, into account.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
ME acquired electrophysiological data, completed data analysis, and participated in writing the manuscript. CH acquired electrophysiological data, completed data analysis, and participated in writing the manuscript. JSC acquired electrophysiological data and completed data analysis. JGJH provided clinical assessment of patients. GL participated in study design and manuscript editing. JPHD provided genomic assessment of patients. MMG provided genetic analysis of patients. SDDH participated in study design, data analysis and manuscript editing. CGF provided overall project management, participated in study design, and writing of the manuscript. ISJM provided overall project management, participated in study design, and writing of the manuscript. SGW provided overall project management, participated in study design, data analysis, and writing of the manuscript. All authors have read and approved the final manuscript.