A novel gain-of-function sodium channel ß2 subunit mutation in idiopathic small fiber neuropathy.

Small fiber neuropathy (SFN) is a common condition affecting thinly myelinated Aδ and unmyelinated C fibers, often resulting in excruciating pain and dysautonomia. SFN has been associated with several conditions, but a significant number of cases have no discernible cause. Recent genetic studies have identified potentially pathogenic gain-of-function mutations in several pore-forming voltage-gated sodium channel α subunits (NaV) in a subset of patients with SFN, but the auxiliary sodium channel ß subunits have been less implicated in the development of the disease. ß subunits modulate NaV trafficking and gating, and several mutations have been linked to epilepsy and cardiac dysfunction. Recently, we provided the first evidence for the contribution of a mutation in the ß2 subunit to pain in human painful diabetic neuropathy. Here, we provide the first evidence for the involvement of a sodium channel ß subunit mutation in the pathogenesis of SFN with no other known causes. We show, through current-clamp analysis, that the newly identified Y69H variant of the ß2 subunit induces neuronal hyperexcitability in dorsal root ganglion neurons, lowering the threshold for action potential firing and allowing for increased repetitive action potential spiking. Underlying the hyperexcitability induced by the ß2-Y69H variant, we demonstrate an upregulation in tetrodotoxin-sensitive, but not tetrodotoxin-resistant sodium currents. This provides the first evidence for the involvement of ß2 subunits in SFN and strengthens the link between sodium channel ß subunits and the development of neuropathic pain in humans.NEW & NOTEWORTHY Small fiber neuropathy (SFN) often has no discernible cause, although mutations in the voltage-gated sodium channel α subunits have been implicated in some cases. We identify a patient suffering from SFN with a mutation in the auxiliary ß2 subunit and no other discernible causes for SFN. Functional assessment confirms this mutation renders dorsal root ganglion neurons hyperexcitable and upregulates tetrodotoxin-sensitive sodium currents. This study strengthens a newly emerging link between sodium channel ß2 subunit mutations and human pain disorders.

The small fiber neuropathy NaV1.7 I228M mutation: impaired neurite integrity via bioenergetic and mitotoxic mechanisms, and protection by dexpramipexole.

Gain-of-function variants in voltage-gated sodium channel NaV1.7 that increase firing frequency and spontaneous firing of dorsal root ganglion (DRG) neurons have recently been identified in 5-10% of patients with idiopathic small fiber neuropathy (I-SFN). Our previous in vitro observations suggest that enhanced sodium channel activity can contribute to a decrease in length of peripheral sensory axons. We have hypothesized that sustained sodium influx due to the expression of SFN-associated sodium channel variants may trigger an energetic deficit in neurons that contributes to degeneration and loss of nerve fibers in SFN. Using an ATP FRET biosensor, we now demonstrate reduced steady-state levels of ATP and markedly faster ATP decay in response to membrane depolarization in cultured DRG neurons expressing an SFN-associated variant NaV1.7, I228M, compared with wild-type neurons. We also observed that I228M neurons show a significant reduction in mitochondrial density and size, indicating dysfunctional mitochondria and a reduced bioenergetic capacity. Finally, we report that exposure to dexpramipexole, a drug that improves mitochondrial energy metabolism, increases the neurite length of I228M-expressing neurons. Our data suggest that expression of gain-of-function variants of NaV1.7 can damage mitochondria and compromise cellular capacity for ATP production. The resulting bioenergetic crisis can consequently contribute to loss of axons in SFN. We suggest that, in addition to interventions that reduce ionic disturbance caused by mutant NaV1.7 channels, an alternative therapeutic strategy might target the bioenergetic burden and mitochondrial damage that occur in SFN associated with NaV1.7 gain-of-function mutations.NEW & NOTEWORTHY Sodium channel NaV1.7 mutations that increase dorsal root ganglion (DRG) neuron excitability have been identified in small fiber neuropathy (SFN). We demonstrate reduced steady-state ATP levels, faster depolarization-evoked ATP decay, and reduced mitochondrial density and size in cultured DRG neurons expressing SFN-associated variant NaV1.7 I228M. Dexpramipexole, which improves mitochondrial energy metabolism, has a protective effect. Because gain-of-function NaV1.7 variants can compromise bioenergetics, therapeutic strategies that target bioenergetic burden and mitochondrial damage merit study in SFN.

Cryptogenic small-fiber neuropathies: Serum autoantibody binding to trisulfated heparan disaccharide and fibroblast growth factor receptor-3.

INTRODUCTION: Causes of small-fiber peripheral neuropathies (SFN) are often undefined. In this study we investigated associations of serum autoantibodies, immunoglobulin G (IgG) vs fibroblast growth factor receptor-3 (FGFR-3), and immunoglobulin M (IgM) vs trisulfated heparan disaccharide (TS-HDS) in cryptogenic SFN. METHODS: One hundred fifty-five patients with biopsy-proven SFN and no identified cause for their neuropathy were blindly tested for serum IgM vs TS-HDS and IgG vs FGFR-3. RESULTS: Forty-eight percent of SFN patients had serum antibodies, 37% with IgM vs TS-HDS and 15% with IgG vs FGFR-3. TS-HDS antibodies were more frequent in SFN patients than in controls (P = .0012). Both antibodies were more common in females, and with non-length-dependent nerve pathology. Nintey-two percent of patients with acute-onset SFN had serum IgM vs TS-HDS. DISCUSSION: Autoantibodies directed against TS-HDS and FGFR-3 suggest an immune disorder in otherwise idiopathic SFN. Serum IgM vs TS-HDS may be a marker for SFN with an acute onset.

Ganglioside GM3 synthase depletion reverses neuropathic pain and small fiber neuropathy in diet-induced diabetic mice.

BACKGROUND: Small fiber neuropathy is a well-recognized complication of type 2 diabetes and has been shown to be responsible for both neuropathic pain and impaired wound healing. In previous studies, we have demonstrated that ganglioside GM3 depletion by knockdown of GM3 synthase fully reverses impaired wound healing in diabetic mice. However, the role of GM3 in neuropathic pain and small fiber neuropathy in diabetes is unknown. PURPOSE: Determine whether GM3 depletion is able to reverse neuropathic pain and small fibers neuropathy and the mechanism of the reversal. RESULTS: We demonstrate that GM3 synthase knockout and the resultant GM3 depletion rescues the denervation in mouse footpad skin and fully reverses the neuropathic pain in diet-induced obese diabetic mice. In cultured dorsal root ganglia from diet-induced diabetic mice, GM3 depletion protects against increased intracellular calcium influx in vitro. CONCLUSIONS: These studies establish ganglioside GM3 as a new candidate responsible for neuropathic pain and small fiber neuropathy in diabetes. Moreover, these observations indicate that systemic or topically applied interventions aimed at depleting GM3 may improve both the painful neuropathy and the wound healing impairment in diabetes by protecting against nerve end terminal degeneration, providing a disease-modifying approach to this common, currently intractable medical issue.

Generation of induced pluripotent stem cell line (UKWNLi008) derived from a patient carrying a c.1678C>G variant in the transient receptor potential cation channel subfamily A member (TRPA1) gene potentially associated with small fiber neuropathy.

Using human dermal fibroblasts (hdF) derived from a patient carrying a c.1678C>G variant located in the TRPA1 gene, induced pluripotent stem cells (iPSC) were generated. Cells were reprogrammed via non-modified (NM) RNA-based transfection resulting in three clones. All three clones showed typical embryonic stem cell-like properties including expression of pluripotency markers, morphology, normal karyotype, and potential differentiation in all three germ layers. With this novel cell line, we offer an in vitro option to study TRPA1 gene variant c.1678C>G and its potential involvement in the development of neuropathic pain as a symptom of small fiber neuropathy (SFN).

Characterisation of the pathophysiology of neuropathy and sensory dysfunction in a mouse model of recessive dystrophic epidermolysis bullosa.

ABSTRACT: Recessive dystrophic epidermolysis bullosa (RDEB) is a rare genetic condition in which mutations in the type VII collagen gene ( COL7A1 ) lead to decreased expression of this anchoring protein of the skin, causing the loss of stability at the dermo-epidermal junction. Most patients with RDEB experience neuropathic pain and itch due to the development of a small fibre neuropathy, characterised by decreased intraepidermal innervation and thermal hypoaesthesia. To understand the physiopathology of this neuropathy, we used a mouse model of RDEB (Col7a1 flNeo/flNeo ) and performed a detailed characterisation of the somatosensory system. Col7a1 flNeo/flNeo mice showed a decrease in heat sensitivity, an increase in spontaneous scratching, and a significant decrease in intraepidermal nerve fibre density in the hindpaw; these changes were distal because there was no significant loss of unmyelinated or myelinated fibres in the nerve trunk. Of interest, we observed a decrease in axon diameter in both myelinated and unmyelinated fibres. This axonal damage was not associated with inflammation of the dorsal root ganglion or central projection targets at the time of assessment. These results suggest that in RDEB, there is a distal degeneration of axons produced by exclusive damage of small fibres in the epidermis, and in contrast with traumatic and acute neuropathies, it does not induce sustained neuroinflammation. Thus, this animal model emphasizes the importance of a healthy cutaneous environment for maintenance of epidermal innervation and faithfully replicates the pathology in humans, offering the opportunity to use this model in the development of treatments for pain for patients with RDEB.

Peripheral synucleinopathy in Parkinson disease with LRRK2 G2385R variants.

OBJECTIVE: Recent studies demonstrated cutaneous phosphorylated α synuclein (p-syn) deposition in idiopathic and some monogenetic Parkinson disease (PD) patients, suggesting synucleinopathy identical to that in the brain. Although the LRRK2 Gly2385Arg (G2385R) variant is a common PD risk factor in the Chinese population, the pathogenesis of PD with G2385R variant has not been reported. We investigated whether synucleinopathy and small fiber neuropathy (SFN) are associated with the G2385R variant. METHODS: We performed genotyping in 59 PD patients and 30 healthy controls from the skin biopsy database. The scale of SFN was assessed, as well as bright-field immunohistochemistry against antiprotein gene product 9.5 (PGP9.5) and double-labeling immunofluorescence with anti-PGP9.5 and anti-p-syn. RESULTS: (1) p-syn deposited in the skin nerve fibers of G2385R carrier PD patients, which was a different pattern from noncarriers, without no difference observed between proximal and distal regions; (2) decreased distal intraepidermal nerve fiber density was found in both the G2385R carrier and the noncarrier PD group, and was negatively correlated with composite autonomic symptom score-31 item (COMPASS-31) scores; (3) PD patients with the G2385R variant showed a more peculiar clinical profile than noncarriers with a higher nonmotor symptoms scale, COMPASS-31 score, and levodopa equivalent dose, in addition to an increased prevalence of certain autonomic symptoms or rapid eye movement sleep behavior disorders. INTERPRETATION: Synucleinopathy is related to the LRRK2 G2385R genotype and implies a different pathogenesis in G2385R variant carriers and noncarriers. This study also extended the clinical profiles of PD patients with the G2385R variant.

Expression of pathogenic SCN9A mutations in the zebrafish: A model to study small-fiber neuropathy.

Small-fiber neuropathy (SFN) patients experience a spectrum of sensory abnormalities, including attenuated responses to non-noxious temperatures in combination with a decreased density of the small-nerve fibers. Gain-of-function mutations in the voltage-gated sodium channels SCN9A, SCN10A and SCN11A have been identified as an underlying genetic cause in a subpopulation of patients with SFN. Based on clinical-diagnostic tests for SFN, we have set up a panel of two read-outs reflecting SFN in zebrafish, being nerve density and behavioral responses. Nerve density was studied using a transgenic line in which the sensory neurons are GFP-labelled. For the behavioral experiments, a temperature-controlled water compartment was developed. This device allowed quantification of the behavioral response to temperature changes. By using these read-outs we demonstrated that zebrafish embryos transiently overexpressing the pathogenic human SCN9A p.(I228M) or p.(G856D) mutations both have a significantly decreased density of the small-nerve fibers. Additionally, larvae overexpressing the p.(I228M) mutation displayed a significant increase in activity induced by temperature change. As these features closely resemble the clinical hallmarks of SFN, our data suggest that transient overexpression of mutant human mRNA provides a model for SFN in zebrafish. This disease model may provide a basis for testing the pathogenicity of novel genetic variants identified in SFN patients. Furthermore, this model could be used for studying SFN pathophysiology in an in vivo model and for testing therapeutic interventions.

Small fiber neuropathy and phosphorylated alpha-synuclein in the skin of E46K-SNCA mutation carriers.

BACKGROUND AND OBJECTIVE: In 2004 we described the E46K mutation in alpha-synuclein gene (E46K-SNCA), a rare point mutation causing an aggressive Lewy body disease with early prominent non-motor features and small fiber denervation of myocardium. Considering the potential interest of the skin as a target for the development of biomarkers in Parkinson's Disease (PD), in this work we aimed to evaluate structural and functional integrity of small autonomic nerve fibers and phosphorylated alpha-synuclein (p-synuclein) deposition in the skin of E46K-SNCA carriers as compared to those observed in parkin gene mutation (PARK2) carriers and healthy controls. PATIENTS AND METHODS: We studied 7 E46K-SNCA carriers (3 dementia with Lewy bodies, 2 pure autonomic failure, 1 PD and 1 asymptomatic), 2 PARK2 carriers and 2 healthy controls to quantify intraepidermal nerve fiber density and p-synuclein deposition with cervical skin punch biopsies (immunohistochemistry against anti PGP9.5/UCHL-1, TH and p-synuclein) and sudomotor function with electrochemical skin conductance (ESC) (SudoScan). RESULTS: All E46K-SNCA carriers had moderate to severe p-synuclein deposits and small fiber neurodegeneration in different epidermal and dermal structures including nerve fascicles and glands, especially in carriers with Pure Autonomic Failure, while p-synuclein aggregates where absent in healthy controls and in one of two PARK2 carriers. The severity of the latter skin abnormalities in E46K-SNCA were correlated with sudomotor dysfunction (lower ESC) in hands (p = 0.035). INTERPRETATION: These results together with our previous findings support the relevance of E46K-SNCA mutation as a suitable model to study small fiber neuropathy in Lewy body diseases.

Generation of two induced pluripotent stem cell lines from skin fibroblasts of sisters carrying a c.1094C>A variation in the SCN10A gene potentially associated with small fiber neuropathy.

Induced pluripotent stem cells (iPSC) were derived from human dermal fibroblasts (HDF) of two siblings with small fiber neuropathy (SFN) potentially based on the same variation in SCN10A but exhibiting diverse disease phenotypes. HDF were reprogrammed using a non-integrating mRNA approach and showed robust expression of pluripotency markers. iPSC displayed no chromosomal aberrations and were differentiated into all three germ-layers. These two cell lines with a familial genetic background may provide a useful in vitro tool to investigate the underlying mechanisms leading to different phenotypes caused by the same variation.

Tumor necrosis factor-α mediated pain hypersensitivity through Ret receptor in resiniferatoxin neuropathy.

Neurogenic inflammation is an onset characteristic of small fiber neuropathy (SFN), which is attributed to neuropathic manifestations. Tumor necrosis factor-α (TNFα) is a cytokine that mainly mediates neurogenic inflammation through the ligand receptor TNF receptor 1 (TNFR1), and targeting TNFα/TNFR1 signaling is a direction toward treating inflammatory diseases and injury-induced neuropathy. However, the relationships between TNFα/TNFR1 signaling and Ret signaling, which mediates pain hypersensitivity, remains elusive. This study used resiniferatoxin (RTX), an ultrapotent analog of capsaicin, to generate a mouse model of SFN, leading to marked hindpaw edema (p = 0.013) and parallel the release of TNFα (p = 0.014), which was associated with the upregulation of Ret(+) neurons (p = 0.0043) and partial depletion of TNFR1 caused by colocalization with TRPV1 depleted by RTX. Pharmacological intervention of TNFα with etanercept (Enbrel®, Wyeth), a clinical application of TNFα blockers, relieved neurogenic inflammation and caused a reduction in hindpaw thickness (p = 0.03) and TNFα releases (p = 0.01), which were determined to be associated with the normalization of mechanical allodynia (p = 0.22). The extraction of either TNFR1(+) or Ret(+) neurons from total of TNFR1(+):Ret(+) neurons indicated that TNFR1(-)/Ret(+) neurons correlated with the mechanical threshold in an antiparallel fashion (r = -0.84, p < 0.0001) but had no relationship with thermal latencies. This study confirmed that TNFα rather than TNFα mediated neuropathic manifestation through the Ret receptor, specifically mechanical allodynia in RTX neuropathy.

Distinct TrkA and Ret modulated negative and positive neuropathic behaviors in a mouse model of resiniferatoxin-induced small fiber neuropathy.

Neurotrophic factors and their corresponding receptors play key roles in the maintenance of different phenotypic dorsal root ganglion (DRG) neurons, the axons of which degenerate in small fiber neuropathy, leading to various neuropathic manifestations. Mechanisms underlying positive and negative symptoms of small fiber neuropathy have not been systematically explored. This study investigated the molecular basis of these seemingly paradoxical neuropathic behaviors according to the profiles of TrkA and Ret with immunohistochemical and pharmacological interventions in a mouse model of resiniferatoxin (RTX)-induced small fiber neuropathy. Mice with RTX neuropathy exhibited thermal hypoalgesia and mechanical allodynia, reduced skin innervation, and altered DRG expression profiles with decreased TrkA(+) neurons and increased Ret(+) neurons. RTX neuropathy induced the expression of activating transcription factor 3 (ATF3), and ATF3(+) neurons were colocalized with Ret but not with TrkA (P<0.001). As a neuroprotectant, 4-Methylcatechol (4MC) promoted skin reinnervation partially with correlated reversal of the neuropathic behaviors and altered neurochemical expression. Gambogic amide, a selective TrkA agonist, normalized thermal hypoalgesia, and GW441756, a TrkA kinase inhibitor, induced thermal hypoalgesia, which was already reversed by 4MC. Mechanical allodynia was reversed by a Ret kinase inhibitor, AST487, which induced thermal hyperalgesia in naïve mice. The activation of Ret signaling by XIB4035 induced mechanical allodynia and thermal hypoalgesia in RTX neuropathy mice in which the neuropathic behaviors were previously normalized by 4MC. Distinct neurotrophic factor receptors, TrkA and Ret, accounted for negative and positive neuropathic behaviors in RTX-induced small fiber neuropathy, respectively: TrkA for thermal hypoalgesia and Ret for mechanical allodynia and thermal hypoalgesia.

Downregulation of adenosine and adenosine A1 receptor contributes to neuropathic pain in resiniferatoxin neuropathy.

The neurochemical effects of adenosine signaling in small-fiber neuropathy leading to neuropathic pain are yet to be explored in a direct manner. This study examined this system at the level of ligand (through the ectonucleotidase activity of prostatic acid phosphatase [PAP]) and adenosine A1 receptors (A1Rs) in resiniferatoxin (RTX) neuropathy, a peripheral neurodegenerative disorder that specifically affects nociceptive nerves expressing transient receptor potential vanilloid type 1 (TRPV1). We conducted immunohistochemistry on dorsal root ganglion (DRG) neurons, high-performance liquid chromatography for functional assays, and pharmacological interventions to alter PAP and A1Rs in mice with RTX neuropathy. In DRG of RTX neuropathy, PAP(+) neurons were reduced compared with vehicle-treated mice (P = 0.002). Functionally, PAP ectonucleotidase activity was consequently reduced (ie, the content of adenosine in DRG, P = 0.012). PAP(+) neuronal density was correlated with the degree of mechanical allodynia, which was reversed by intrathecal (i.t.) lumbar puncture injection of recombinant PAP with a dose-dependent effect. Furthermore, A1Rs were downregulated (P = 0.002), and this downregulation was colocalized with the TRPV1 receptor (31.0% ± 2.8%). Mechanical allodynia was attenuated in a dose-dependent response by i.t. injection of the A1R ligand, adenosine; however, no analgesia was evident when an exogenous adenosine was blocked by A1R antagonist. This study demonstrated dual mechanisms of neuropathic pain in TRPV1-induced neuropathy, involving a reduced adenosine system at both the ligand (adenosine) and receptor (A1Rs) levels.

Skin globotriaosylceramide 3 deposits are specific to Fabry disease with classical mutations and associated with small fibre neuropathy.

BACKGROUND: Fabry Disease (FD) is characterized by globotriaosylceramide-3 (Gb3) accumulation in several tissues and a small fibre neuropathy (SFN), however the underlying mechanisms are poorly known. This study aimed to: 1) ascertain the presence of Gb3 deposits in skin samples, by an immunofluorescence method collected from FD patients with classical GLA mutations or late-onset FD variants or GLA polymorphisms; 2) correlate skin GB3 deposits with skin innervation. METHODS: we studied 52 genetically-defined FD patients (32 with classical GLA mutations and 20 with late-onset variants or GLA polymorphisms), 15 patients with SFN associated with a specific cause and 22 healthy controls. Subjects underwent skin biopsy to evaluate Gb3 deposits and epi-dermal innervation. RESULTS: Skin Gb3 deposits were found in all FD patients with classical GLA mutations but never in FD patients with late-onset variants or GLA polymorphisms or in patients with SFN and healthy controls. Abnormal deposits were found inside different skin structures but never inside axons. FD patients with GB3 deposits showed lower skin innervation than FD patients with late-onset variants or polymorphisms. CONCLUSIONS: 1) Skin Gb3 deposits are specific to FD patients with classical GLA mutations; 2) Gb3 deposits were associated with lower skin innervation but they were not found inside axons, suggesting an indirect damage on peripheral small fibre innervation.

Candesartan prevents resiniferatoxin-induced sensory small-fiber neuropathy in mice by promoting angiotensin II-mediated AT2 receptor stimulation.

Sensory defects associated with small-fiber neuropathy (SFN) can lead to profound disabilities. The relationship between the sensory nervous system and modulation of the renin-angiotensin system (RAS) has been described and focused on pain and neurodegeneration in several animal models. We have recently developed an experimental model of functional sensory neuropathy showing thermal hypoalgesia and neuropeptide depletion without nerve fiber degeneration. Here, we aimed to determine whether the modulation of angiotensin II (Ang II) activity could prevent sensory neuropathy induced by RTX. Control and RTX mice received ramipril, an Ang II converting enzyme (ACE) inhibitor, (0.5 mg/kg/day) or candesartan, an Ang II type 1 receptor (AT1R) blocker (0.5 mg/kg/day), one day before vehicle or RTX administration, and each day for the next seven days. Ramipril did not have a beneficial effect in RTX mice, whereas candesartan prevented thermal hypoalgesia and reduced neuropeptide depletion in intraepidermal nerve fibers and dorsal root ganglion neurons. The preventive effect of candesartan was not observed in mice deficient for the Ang II type 2 receptor (AT2R) and was counteracted in wild type mice by EMA200, an AT2R antagonist (3 mg/kg/day). Thus, candesartan may promote AT2R activation by blocking AT1R and increasing Ang II production and enhance its mechanisms of neuroprotection in our RTX model. Our finding that candesartan prevents nociception deficits and neuropeptide depletion encourages the evaluation of its therapeutic potential in patients presenting SFN, particularly those who experience chemotherapy-induced SFN.

Automated PGP9.5 immunofluorescence staining: a valuable tool in the assessment of small fiber neuropathy?

BACKGROUND: In this study we explored the possibility of automating the PGP9.5 immunofluorescence staining assay for the diagnosis of small fiber neuropathy using skin punch biopsies. The laboratory developed test (LDT) was subjected to a validation strategy as required by good laboratory practice guidelines and compared to the well-established gold standard method approved by the European Federation of Neurological Societies (EFNS). To facilitate automation, the use of thinner sections. (16 µm) was evaluated. Biopsies from previously published studies were used. The aim was to evaluate the diagnostic performance of the LDT compared to the gold standard. We focused on technical aspects to reach high-quality standardization of the PGP9.5 assay and finally evaluate its potential for use in large scale batch testing. RESULTS: We first studied linear nerve fiber densities in skin of healthy volunteers to establish reference ranges, and compared our LDT using the modifications to the EFNS counting rule to the gold standard in visualizing and quantifying the epidermal nerve fiber network. As the LDT requires the use of 16 µm tissue sections, a higher incidence of intra-epidermal nerve fiber fragments and a lower incidence of secondary branches were detected. Nevertheless, the LDT showed excellent concordance with the gold standard method. Next, the diagnostic performance and yield of the LDT were explored and challenged to the gold standard using skin punch biopsies of capsaicin treated subjects, and patients with diabetic polyneuropathy. The LDT reached good agreement with the gold standard in identifying small fiber neuropathy. The reduction of section thickness from 50 to 16 µm resulted in a significantly lower visualization of the three-dimensional epidermal nerve fiber network, as expected. However, the diagnostic performance of the LDT was adequate as characterized by a sensitivity and specificity of 80 and 64 %, respectively. CONCLUSIONS: This study, designed as a proof of principle, indicated that the LDT is an accurate, robust and automated assay, which adequately and reliably identifies patients presenting with small fiber neuropathy, and therefore has potential for use in large scale clinical studies.