J. G. J. H. reports no disclosures. I. S. J. M. served on a scientific advisory board for CSL Behring; has received funding for travel from Talecris Biotherapeutics; and has received research support from the GBS/CIDP International Foundation, the Talents Program foundation, and the Peripheral Nerve Society. M. M. G. reports no disclosures. S. G. W. has consulted for Cardiome Pharm, Bristol Myers Squibb, Vertex Pharmaceuticals, ChromaCell and DaiNippon Sumitomo Pharm; inventor of a patent on sodium channel Nav1.9 (Yale holds patent); grants from Pfizer and TransMolecular Inc, C. G. F. reports no disclosures.
Genetic aspects of sodium channelopathy in small fiber neuropathy
Article first published online: 7 AUG 2012
© 2012 John Wiley & Sons A/S
Volume 82, Issue 4, pages 351–358, October 2012
How to Cite
Genetic aspects of sodium channelopathy in small fiber neuropathy., , , , .
- Issue published online: 11 SEP 2012
- Article first published online: 7 AUG 2012
- Accepted manuscript online: 13 JUL 2012 11:47AM EST
- Manuscript Revised: 11 JUL 2012
- Manuscript Accepted: 11 JUL 2012
- Manuscript Received: 20 JUN 2012
- small fiber neuropathy;
- sodium channelopathy
Small fiber neuropathy (SFN) is a disorder typically dominated by neuropathic pain and autonomic dysfunction, in which the thinly myelinated Aδ-fibers and unmyelinated C-fibers are selectively injured. The diagnosis SFN is based on a reduced intraepidermal nerve fiber density and/or abnormal thermal thresholds in quantitative sensory testing. The etiologies of SFN are diverse, although no apparent cause is frequently seen.
Recently, SCN9A-gene variants (single amino acid substitutions) have been found in ∼30% of a cohort of idiopathic SFN patients, producing gain-of-function changes in sodium channel NaV1.7, which is preferentially expressed in small diameter peripheral axons. Functional testing showed that these variants altered fast inactivation, slow inactivation or resurgent current and rendered dorsal root ganglion neurons hyperexcitable. In this review, we discuss the role of NaV1.7 in pain and highlight the molecular genetics and pathophysiology of SCN9A-gene variants in SFN. With increasing knowledge regarding the underlying pathophysiology in SFN, the development of specific treatment in these patients seems a logical target for future studies.
Small fiber neuropathy (SFN) is a disorder of the thinly myelinated Aδ-fibers and unmyelinated C-fibers and is typically dominated by neuropathic pain and autonomic dysfunction [1-6]. Clinical signs of small-fiber damage include loss of pinprick sensation, thermal sensory loss, allodynia or hyperalgesia. In pure SFN, large diameter fibers are spared, reflected by preservation of muscle strength, tendon reflexes, light touch, proprioceptive and vibratory sense in combination with normal nerve conduction studies [4-7]. In addition to the clinical picture, the diagnosis of SFN can be confirmed by demonstration of a reduced intraepidermal nerve fiber density in skin biopsy and/or abnormal thermal thresholds in quantitative sensory testing [7, 8]. After the diagnosis is made, an underlying cause for SFN has to be searched for, as some of these are potentially treatable. Potential causes for SFN are, among others, diabetes mellitus, impaired glucose tolerance, drugs and other toxins, Fabry disease, celiac disease, sarcoidosis, human immunodeficiency virus (HIV), and other systemic illnesses [4, 6, 7].
There is growing evidence for a genetic origin in various pain syndromes: an autosomal dominant inheritance has been shown in family members with burning feet syndrome, suggesting a genetic base for the small nerve fiber involvement in this disorder, although the locus responsible for the neuropathy has not been elucidated yet [9, 10]. Gain-of-function mutations in the SCN9A-gene, encoding the NaV1.7 sodium channel, have been found in inherited erythromelalgia (IEM) [11, 12], paroxysmal extreme pain disorder (PEPD) , and recently in a substantial proportion of patients diagnosed with idiopathic SFN . These findings imply a pivotal role of NaV1.7 mutations in painful diseases and suggest an etiological basis for idiopathic SFN, whereby expression of gain-of-function sodium channel variants in small diameter peripheral axons produces pain and may cause these fibers to degenerate. In this review, the significance of NaV1.7 in pain is discussed focusing on the role of SCN9A-gene variants in patients diagnosed with SFN, highlighting the molecular genetics and known pathophysiological evidence. In the near future, this new knowledge will result in the development of specific treatment for these patients.
Voltage-gated sodium channels
Just 60 years ago, Hodgkin and Huxley discovered the role of sodium channels in action potentials . In the decades that followed the primary structure and functional characteristics of these channels have been elucidated . These channels are large integral membrane polypeptides that are comprised of a large α subunit, which forms the voltage-sensitive and ion-selective pore, and smaller auxiliary β subunit(s) that can regulate channel density in the cell membrane, and modulate the kinetics and voltage dependence of channel gating [16, 17]. The α subunit folds into four domains (I–IV), each of which contain six transmembrane segments, linked by three loops. In mammals, nine distinct voltage-gated sodium channels (VGSCs) α isoforms have been identified (NaV1.1–NaV1.9), encoded by SCN1A-SCN5A and SCN8A-SCN11A genes. Besides their kinetics and voltage-dependent properties, they can be defined by their sensitivity to the sodium-channel blocker tetrodotoxin (TTX). VGSCs play a fundamental role in different types of excitable cells, including nerve, muscle and neuroendocrine cells [16, 18].
Voltage-gated sodium channels and pain
Dorsal root ganglia (DRG) and trigeminal neurons are the primary sensory neurons. Small DRG neurons give rise to the thinly myelinated Aδ-fibers and unmyelinated C-fibers which extend to the cutaneous layer. They provide the cold and warm sense, nociception and autonomic functions [19, 20]. In SFN these fibers are specifically involved.
More than one century ago, Charles Sherrington was the first who described the existence of nociceptors . Physiologically, in response to noxious stimuli the nociceptive neurons transmit pain signals to the central nervous system. Usually these cells are relatively quiescent and have high thresholds for activation . The excitability of the nociceptors seems to be controlled by different types of ion channels [22, 23]. NaV1.3, NaV1.7, NaV1.8 and NaV1.9 are preferentially expressed in peripheral sensory neurons, whereby the last three are particularly located in nociceptive neurons [12, 18, 24-26]. Dysregulated expression of the channels initiated by trauma, inflammation or metabolic disorders has been linked to chronic pain [27-33].
NaV1.3 is only expressed in embryonic DRG, but is upregulated following axotomy in adults . Knockdown of NaV1.3 with antisense oligonucleotides and targeted shRNA has been reported to abrogate pain in rodent nerve injury models [35, 36]. On the other hand, knockout of NaV1.3 has been reported not to influence pain behavior after nerve injury . Despite clear upregulation of NaV1.3 expression in DRG, dorsal horn, and thalamic neurons after axonal injury, the role of NaV1.3 in pain remains controversial.
NaV1.7, NaV1.8, and NaV1.9 may show changes in the level of expression within injured human DRG neuron, although accumulation of NaV1.7 and NaV1.8 within injured axons in painful human neuromas was also described [38-43]. The biophysical properties of NaV1.8, its critical role in repetitive firing , and its presence in free nerve endings , where pain-signaling is initiated, suggest that NaV1.8 can significantly influence nociceptor excitability, thus contributing to pain . Experimental evidence supports a role for NaV1.9 in inflammatory and diabetic neuropathy pain, although a role in neuropathic pain is less clear . Expression of NaV1.9 has been shown to increase in DRG neurons innervating inflamed rat hindpaw . Although expression levels of NaV1.9 do not appear to be altered in small DRG neurons from diabetic rats, increased NaV1.9 levels in large-diameter neurons suggest a contribution to painful diabetic neuropathy . In contrast, NaV1.9 mRNA and protein levels and current density are downregulated in several animal models of neuropathic pain [48-52]. An early study using NaV1.9 antisense oligodeoxynucleotide (ODN) treatment did not report amelioration of neuropathic pain .
Voltage-gated sodium channel NaV1.7
NaV1.7 was first isolated in 1995 as human neuroendocrine sodium channel (hNE-Na) from a human medullary thyroid carcinoma cell line . The suggestion that the channel is solely expressed in neuroendocrine cells was soon rejected by the isolation of the channel from rabbit Schwann cells (NaS) . Subsequently, the rat homologue, also known as peripheral nerve type 1 or PN1, was cloned and showed to be preferentially expressed in DRG and sympathetic ganglion neurons [56, 57]. NaV1.7 is encoded by the SCN9A-gene on chromosome 2 . The human SCN9A-gene promoter has been identified and is located ∼64,000 nucleotides upstream to the ATG translation start site. There is a high degree of sequence conservation between human and mouse . NaV1.7 produces a fast activating and inactivating current that is TTX-sensitive (TTX-S) [54, 56] and is slow-repriming from fast inactivation [60, 61]. Another electrophysiological feature of NaV1.7 is the slow closed-state inactivation that generates a substantial current (ramp current) in response to small, slow depolarization [60, 61]. This ability to respond to ramp stimuli permits the channel to act as a threshold channel for firing action potentials, setting the gain in nociceptive neurons [62, 63].
NaV1.7 and pain in animal studies
Several animal models have been performed to study the contribution of NaV1.7 to acquired channelopathies. In rats, peripheral tissue inflammation induced by carrageenan or Freund's complete adjuvant, showed upregulation of NaV1.7 transcripts and protein, parallel with a significant increase in the level of sodium current in small DRG neurons [64-66]. Inflammatory cytokines, such as nerve growth factor (NGF), also appear to upregulate NaV1.7 and might contribute to neuronal hyperexcitability [57, 67, 68]. In accordance with these results, knockdown of NaV1.7 in primary afferents or DRG-neurons counteract the inflammation-induced mechanical and thermal hyperalgesia [69, 70] and plays a major role in pain after burn injury . In a recent study in experimental rat sciatic nerve neuromas, 2 weeks after nerve ligation and transaction, increased levels of NaV1.7 were shown and elevated levels of phosphorylated ERK1/2 within individual neuroma axons that exhibit NaV1.7 accumulation were shown . Neuropathic pain seems to persist after knockdown of NaV1.7 within DRG neurons , but is attenuated after knockdown of NaV1.7 in both DRG and sympathetic ganglion neurons .
Alterations of sodium channel expression have also been linked to the development of painful diabetic neuropathy [47, 74-76]. One study showed dysregulation of several sodium channels in streptozotocin (STZ)-induced diabetic neuropathy, although without change of NaV1.7 expression . In contrast, other studies just reported increased levels of NaV1.7 protein [74, 76]. In addition, normalization of NaV1.7 levels in STZ-diabetic rats, achieved by a non-replicating herpes simplex virus-based vector, substantially reduced pain-related behavior .
NaV1.7 and pain in human studies
The contribution of NaV1.7 in traumatic injury has also been studied in human patients. Peripheral nerve injury showed reduction of NaV1.7 in DRG neurons . Upregulation has been shown in painful neuromas [40, 41, 43].
Much more is known about inherited pain disorders linked to mutations in the SCN9A-gene. Before the discovery of SCN9A-gene variants in SFN, NaV1.7 mutations have been identified in three pain syndromes (Fig. 1) of which the IEM is the most widely known. The clinical picture of IEM was first described in 1878  and is characterized by a red discoloration of the extremities, aggravated by warmth and exercise. In 2004, missense mutations in the SCN9A-gene leading to gain-of-function of NaV1.7 were identified in two Chinese families . Since then multiple additional cases have been described. Most of the mutations show hyperpolarized activation, slow deactivation and increase the ramp response of the channel [80-84], which contributes to DRG neuron hyperexcitability. The second inherited human pain syndrome with gain-of-function mutations of NaV1.7 is PEPD. This autosomal dominant condition was first described in 1959 . The clinical features consist of paroxysms of excruciating rectal, ocular and submaxillary pain associated with flushing of the buttocks, eyelid, periorbital skin and legs [13, 85]. Two years after the identification of the molecular basis of IEM, mutant NaV1.7 channels were found in PEPD . In addition to the difference in phenotype, the two conditions show differences in channel dysfunction, whereby mutant channels in PEPD show impaired fast inactivation, leading to prolonged action potentials and repetitive neuron firing in response to provoking stimuli, such as stretching and experiencing cold . In contrast with IEM and PEPD, the third syndrome, congenital insensitivity to pain (CIP) is an autosomal recessive condition characterized by loss-of-function NaV1.7 mutations leading to insensitivity to pain [86, 87]. CIP is a disorder linked to different genetically targets . The patients who show a SCN9A-gene mutation form a subset that was first described in 2006 , and can be differentiated from other underlying causes by anosmia and absence of autonomic dysfunction .
SFN shows some clinical similarities with IEM and PEPD. Burning pain is a common characteristic. Most SFN patients experience pain in the distal extremities such as IEM [14, 79], but pain throughout the entire body or facial pain has also been described, the latter also seen in PEPD [13, 14, 89]. Despite the similarities, thus far SFN is considered a distinctive entity. For example, autonomic dysfunction is a prominent feature of SFN, whereas it is nearly absent in IEM [7, 11, 90]. In addition, skin reddening is typical for IEM, but is only occasionally present in SFN [14, 91]. The same applies for aggravation of symptoms by warmth and relief by cold in IEM. This is seen in some cases of SFN [14, 91, 92], but sometimes the opposite pattern is reported [14, 89].
Given the shared characteristics of SFN with IEM and PEPD and the results of animal studies of VGSCs in pain as described above, the SCN9A-gene was considered a potential candidate gene to be involved in idiopathic SFN. Recent research has confirmed this assumption. In a cohort of 28 Dutch Caucasian patients who met the strict criteria for idiopathic SFN based on reduced intraepidermal nerve fiber density plus abnormal quantitative sensory testing, eight showed gain-of-function SCN9A-gene variants . Subsequently, in a kindred with SFN and acromesomelia (small hands and feet) a gain-of-funtion SCN9A-gene variant was also shown .
Increasing knowledge regarding pathophysiology may lead to novel therapeutic options.
Up to now, in idiopathic SFN 9 SCN9A missense variants (single amino acid substitutions) have been reported [14, 89-92] (Table 1; Fig. 2). All variants substitute a highly conserved residue. Except for variant M1532I which is located in domain IV of the channel, all other variants are situated in domain I or II [14, 91]. Two familial cases have been described [89, 91], suggesting an autosomal dominant inheritance. However, there appears to be a variable penetrance as the complaints vary in severity between affected family members. Three variants have been reported in unrelated cases (R185H, I739V and I228M) [14, 89, 90]. Variant I228M has been described in three patients, two being family members [14, 89]. While carrying the same mutation, the three patients present with different complaints, one with severe facial pain, the second with distal (feet, hands) pain, and the last with scalp discomfort. These patients show intra- and interfamily phenotypic diversity in patients with SFN produced by a gain-of-function variant of NaV1.7.
|Nucleotide change||Protein||Domain||Number of patients||Reference(s)||Allele frequency|
|dbSNP||1000 genomes project||EVS|
(n = 3/585)
(n = 13/2184)
(n = 68/11908)
|c.2215A>G||I739V||II||3||[14, 90, 92]||0.2%|
(n = 1/571)
(n = 2/2184)
(n = 36/11838)
(n = 1/9464)
(n = 2/11762)
(n = 82/2188)
(n = 81/2184)
(n = 44/13006)
(n = 82/2188)
(n = 81/2184)
(n = 42/11764)
(n = 1/2)d
(n = 1/2184)
(n = 15/12988)
One variant (G856D) not only resulted in SFN, but also caused a new syndrome of pain, dysautonomia and acromesomelia .
Voltage clamp and current clamp studies have been carried out to profile the effects of NaV1.7 variants in SFN on channel function and on DRG neuron firing properties [14, 93, 94]. Because functional properties of sodium channel can best be studied within the cells where they are normally expressed in, voltage clamp analysis was carried out after transfection into adult small DRG neurons , for some of the variants, when biophysical changes were not detected in a HEK293 cell background [14, 93, 94]. Current clamp analysis was carried out after transfection in DRG neurons [93, 94].
Voltage clamp analysis of the first published variant channels in idiopathic SFN showed that they were all gain-of-function mutations and impaired slow inactivation (I720K, M1532I, I228M, I739V), depolarized slow and fast inactivation (D623N), or enhanced resurgent currents (M932L/V991L, R185H) . They did not display the hyperpolarized activation and enhanced ramp responses characteristic of IEM mutations or the incomplete fast inactivation characteristic of PEPD mutations . After current clamp analysis all of these variants produced hyperexcitability and abnormal spontaneous activity within DRG neurons .
Several mutations show a distinct phenotype. Strikingly, the two patients housing variant R185H showed minimal autonomic dysfunction compared to the other patients [14, 90]. Therefore, the biophysical effects of this variant were also assessed by voltage clamp in sympathetic ganglion neurons (superior cervical ganglia). A variant associated with severe autonomic symptoms (I739V) was used as comparator [14, 90]. As would be expected on the basis of the clinic, the R185H variant did not produce detectable changes in properties of sympathetic ganglion neurons, in contrast with the I739V variant which had a profound effect on excitability of sympathetic ganglion neurons by increasing their current threshold and impairing their ability to generate repetitive activity , providing a biophysical basis for the clinical picture.
Current clamp analysis has also been carried out for some NaV1.7 variants in trigeminal ganglion neurons. One of the patients carrying variant I228M complained of severe facial pain [14, 89]. As predicted, the I228M variant produced hyperexcitability in trigeminal ganglion neurons .
Mechanisms by which some NaV1.7 variants produce, or increase risk for, axonal degeneration are under study, and may involve calcium loading, perhaps in response to a double hit by an additional stressor.
As noted above, variant G856D underlies an extraordinary phenotype consisting of SFN and acromesomelia. The NaV1.7 variant enhances activation, impairs fast inactivation and markedly enhances persistent current and the response to slow ramp stimuli . Limb underdevelopment has previously not been associated with NaV1.7. Multiple hypotheses might be considered to explain the small limbs in this kindred. NaV1.7 channel might be involved in the bone ontogenesis, similar to NaV1.2 sodium channels which are expressed in osteoblasts during embryogenesis . Another hypothesis suggests that dysfunction or injury of peripheral nerve fibers due to DRG hyperexcitability may have interfered with normal limb development. Consistent with this suggesting, brachial plexus neurectomy in newborn rabbits has been shown to result in impaired limb growth . Altered vasomotor function, as shown by the skin reddening in the G586D kindred, may also play a role. The variant may have an effect on sympathetic ganglion neurons . In addition, bones are innervated by small nerve fibers, some of which contain peptides which have been implicated as modulators of the metabolism of bone cells [98, 99]. Moreover, nerve fibers are often located in the epiphysial region suggesting a regulatory role for the epiphysial growth plate . Finally, some conditions are known to cause both neuropathy and limb defects in embryo. One of the most well-known examples is the limb teratogenesis produced by thalidomide . There is evidence suggesting that maternal varicella-zoster infection  or maternal diabetic  may also be teratogenic. Further work will be needed to delineate the role for the G856D NaV1.7 variant in this syndrome.
SCN9A-gene variants (single amino acid substitutions) have recently been found in patients with SFN. These variants produce gain-of-function changes in sodium channel NaV1.7, which is preferentially expressed in small diameter peripheral axons. These variants alter fast inactivation, slow inactivation or resurgent current and render the dorsal root ganglion neurons hyperexcitable. Selective NaV1.7 blockers are currently under development. Given the profound changes seen in DRG neurons carrying these NaV1.7 variants, targeted block of NaV1.7 may prove to be effective in SFN, especially in patients with SCN9A mutations.
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