Ion Channel Genes in Painful Neuropathies.

Neuropathic pain (NP) is a typical symptom of peripheral nerve disorders, including painful neuropathy. The biological mechanisms that control ion channels are important for many cell activities and are also therapeutic targets. Disruption of the cellular mechanisms that govern ion channel activity can contribute to pain pathophysiology. The voltage-gated sodium channel (VGSC) is the most researched ion channel in terms of NP; however, VGSC impairment is detected in only <20% of painful neuropathy patients. Here, we discuss the potential role of the other peripheral ion channels involved in sensory signaling (transient receptor potential cation channels), neuronal excitation regulation (potassium channels), involuntary action potential generation (hyperpolarization-activated cyclic nucleotide-gated channels), thermal pain (anoctamins), pH modulation (acid sensing ion channels), and neurotransmitter release (calcium channels) related to pain and their prospective role as therapeutic targets for painful neuropathy.

Genetic Profiling of Sodium Channels in Diabetic Painful and Painless and Idiopathic Painful and Painless Neuropathies.

Neuropathic pain is a frequent feature of diabetic peripheral neuropathy (DPN) and small fiber neuropathy (SFN). Resolving the genetic architecture of these painful neuropathies will lead to better disease management strategies, counselling and intervention. Our aims were to profile ten sodium channel genes (SCG) expressed in a nociceptive pathway in painful and painless DPN and painful and painless SFN patients, and to provide a perspective for clinicians who assess patients with painful peripheral neuropathy. Between June 2014 and September 2016, 1125 patients with painful-DPN (n = 237), painless-DPN (n = 309), painful-SFN (n = 547) and painless-SFN (n = 32), recruited in four different centers, were analyzed for SCN3A, SCN7A-SCN11A and SCN1B-SCN4B variants by single molecule Molecular inversion probes-Next Generation Sequence. Patients were grouped based on phenotype and the presence of SCG variants. Screening of SCN3A, SCN7A-SCN11A, and SCN1B-SCN4B revealed 125 different (potential) pathogenic variants in 194 patients (17.2%, n = 194/1125). A potential pathogenic variant was present in 18.1% (n = 142/784) of painful neuropathy patients vs. 15.2% (n = 52/341) of painless neuropathy patients (17.3% (n = 41/237) for painful-DPN patients, 14.9% (n = 46/309) for painless-DPN patients, 18.5% (n = 101/547) for painful-SFN patients, and 18.8% (n = 6/32) for painless-SFN patients). Of the variants detected, 70% were in SCN7A, SCN9A, SCN10A and SCN11A. The frequency of SCN9A and SCN11A variants was the highest in painful-SFN patients, SCN7A variants in painful-DPN patients, and SCN10A variants in painless-DPN patients. Our findings suggest that rare SCG genetic variants may contribute to the development of painful neuropathy. Genetic profiling and SCG variant identification should aid in a better understanding of the genetic variability in patients with painful and painless neuropathy, and may lead to better risk stratification and the development of more targeted and personalized pain treatments.

Peripheral Ion Channel Genes Screening in Painful Small Fiber Neuropathy.

Neuropathic pain is a characteristic feature of small fiber neuropathy (SFN), which in 18% of the cases is caused by genetic variants in voltage-gated sodium ion channels. In this study, we assessed the role of fifteen other ion channels in neuropathic pain. Patients with SFN (n = 414) were analyzed for ANO1, ANO3, HCN1, KCNA2, KCNA4, KCNK18, KCNN1, KCNQ3, KCNQ5, KCNS1, TRPA1, TRPM8, TRPV1, TRPV3 and TRPV4 variants by single-molecule molecular inversion probes-next-generation sequencing. These patients did not have genetic variants in SCN3A, SCN7A-SCN11A and SCN1B-SCN4B. In twenty patients (20/414, 4.8%), a potentially pathogenic heterozygous variant was identified in an ion-channel gene (ICG). Variants were present in seven genes, for two patients (0.5%) in ANO3, one (0.2%) in KCNK18, two (0.5%) in KCNQ3, seven (1.7%) in TRPA1, three (0.7%) in TRPM8, three (0.7%) in TRPV1 and two (0.5%) in TRPV3. Variants in the TRP genes were the most frequent (n = 15, 3.6%), partly in patients with high mean maximal pain scores VAS = 9.65 ± 0.7 (n = 4). Patients with ICG variants reported more severe pain compared to patients without such variants (VAS = 9.36 ± 0.72 vs. VAS = 7.47 ± 2.37). This cohort study identified ICG variants in neuropathic pain in SFN, complementing previous findings of ICG variants in diabetic neuropathy. These data show that ICG variants are central in neuropathic pain of different etiologies and provides promising gene candidates for future research.

Peripheral Ion Channel Gene Screening in Painful- and Painless-Diabetic Neuropathy.

Neuropathic pain is common in diabetic peripheral neuropathy (DN), probably caused by pathogenic ion channel gene variants. Therefore, we performed molecular inversion probes-next generation sequencing of 5 transient receptor potential cation channels, 8 potassium channels and 2 calcium-activated chloride channel genes in 222 painful- and 304 painless-DN patients. Twelve painful-DN (5.4%) patients showed potentially pathogenic variants (five nonsense/frameshift, seven missense, one out-of-frame deletion) in ANO3 (n = 3), HCN1 (n = 1), KCNK18 (n = 2), TRPA1 (n = 3), TRPM8 (n = 3) and TRPV4 (n = 1) and fourteen painless-DN patients (4.6%-three nonsense/frameshift, nine missense, one out-of-frame deletion) in ANO1 (n = 1), KCNK18 (n = 3), KCNQ3 (n = 1), TRPA1 (n = 2), TRPM8 (n = 1), TRPV1 (n = 3) and TRPV4 (n = 3). Missense variants were present in both conditions, presumably with loss- or gain-of-functions. KCNK18 nonsense/frameshift variants were found in painless/painful-DN, making a causal role in pain less likely. Surprisingly, premature stop-codons with likely nonsense-mediated RNA-decay were more frequent in painful-DN. Although limited in number, painful-DN patients with ion channel gene variants reported higher maximal pain during the night and day. Moreover, painful-DN patients with TRP variants had abnormal thermal thresholds and more severe pain during the night and day. Our results suggest a role of ion channel gene variants in neuropathic pain, but functional validation is required.

Hydropathicity-based prediction of pain-causing NaV1.7 variants.

BACKGROUND: Mutation-induced variations in the functional architecture of the NaV1.7 channel protein are causally related to a broad spectrum of human pain disorders. Predicting in silico the phenotype of NaV1.7 variant is of major clinical importance; it can aid in reducing costs of in vitro pathophysiological characterization of NaV1.7 variants, as well as, in the design of drug agents for counteracting pain-disease symptoms. RESULTS: In this work, we utilize spatial complexity of hydropathic effects toward predicting which NaV1.7 variants cause pain (and which are neutral) based on the location of corresponding mutation sites within the NaV1.7 structure. For that, we analyze topological and scaling hydropathic characteristics of the atomic environment around NaV1.7's pore and probe their spatial correlation with mutation sites. We show that pain-related mutation sites occupy structural locations in proximity to a hydrophobic patch lining the pore while clustering at a critical hydropathic-interactions distance from the selectivity filter (SF). Taken together, these observations can differentiate pain-related NaV1.7 variants from neutral ones, i.e., NaV1.7 variants not causing pain disease, with 80.5[Formula: see text] sensitivity and 93.7[Formula: see text] specificity [area under the receiver operating characteristics curve = 0.872]. CONCLUSIONS: Our findings suggest that maintaining hydrophobic NaV1.7 interior intact, as well as, a finely-tuned (dictated by hydropathic interactions) distance from the SF might be necessary molecular conditions for physiological NaV1.7 functioning. The main advantage for using the presented predictive scheme is its negligible computational cost, as well as, hydropathicity-based biophysical rationalization.

Correction: Evaluation of molecular inversion probe versus TruSeq® custom methods for targeted next-generation sequencing.

[This corrects the article DOI: 10.1371/journal.pone.0238467.].

Evaluation of molecular inversion probe versus TruSeq® custom methods for targeted next-generation sequencing.

Resolving the genetic architecture of painful neuropathy will lead to better disease management strategies. We aimed to develop a reliable method to re-sequence multiple genes in a large cohort of painful neuropathy patients at low cost. In this study, we compared sensitivity, specificity, targeting efficiency, performance and cost effectiveness of Molecular Inversion Probes-Next generation sequencing (MIPs-NGS) and TruSeq® Custom Amplicon-Next generation sequencing (TSCA-NGS). Capture probes were designed to target nine sodium channel genes (SCN3A, SCN8A-SCN11A, and SCN1B-SCN4B). One hundred sixty-six patients with diabetic and idiopathic neuropathy were tested by both methods, 70 patients were validated by Sanger sequencing. Sensitivity, specificity and performance of both techniques were comparable, and in agreement with Sanger sequencing. The average targeted regions coverage for MIPs-NGS was 97.3% versus 93.9% for TSCA-NGS. MIPs-NGS has a more versatile assay design and is more flexible than TSCA-NGS. The cost of MIPs-NGS is >5 times cheaper than TSCA-NGS when 500 or more samples are tested. In conclusion, MIPs-NGS is a reliable, flexible, and relatively inexpensive method to detect genetic variations in a large cohort of patients. In our centers, MIPs-NGS is currently implemented as a routine diagnostic tool for screening of sodium channel genes in painful neuropathy patients.

Parental repeat length instability in myotonic dystrophy type 1 pre- and protomutations.

Myotonic dystrophy type 1 (DM1) is caused by a CTG trinucleotide repeat expansion on chromosome 19q13.3. While DM1 premutation (36-50 repeats) and protomutation (51-80 repeats) allele carriers are mostly asymptomatic, offspring is at risk of inheriting expanded, symptom-associated, (CTG)n repeats of n > 80. In this study we aimed to evaluate the intergenerational instability of DM1 pre- and protomutation alleles, focussing on the influence of parental gender. One hundred and forty-six parent-child pairs (34 parental premutations, 112 protomutations) were retrospectively selected from the DM1 patient cohort of the Maastricht University Medical Center+. CTG repeat size of parents and children was determined by (triplet-primed) PCR followed by fragment length analysis and Southern blot analysis. Fifty-eight out of eighty-one (71.6%) paternal transmissions led to a (CTG)n repeat of n > 80 in offspring, compared with 15 out of 65 (23.1%) maternal transmissions (p < 0.001). Repeat length instability occurred for paternal (CTG)n repeats of n ≥ 45, while maternal instability did not occur until (CTG)n repeats reached a length of n ≥ 71. Transmission of premutations caused (CTG)n repeats of n > 80 in offspring only when paternally transmitted (two cases), while protomutations caused (CTG)n repeats of n > 80 in offspring in 71 cases, of which 56 (78.9%) were paternally transmitted. In conclusion, our data show that paternally transmitted pre- and protomutations were more unstable than maternally transmitted pre- and protomutations. For genetic counseling, this implies that males with a small DMPK mutation have a higher risk of symptomatic offspring compared with females. Consequently, we suggest addressing sex-dependent factors in genetic counseling of small-sized CTG repeat carriers.

Differential effect of lacosamide on Nav1.7 variants from responsive and non-responsive patients with small fibre neuropathy.

Small fibre neuropathy is a common pain disorder, which in many cases fails to respond to treatment with existing medications. Gain-of-function mutations of voltage-gated sodium channel Nav1.7 underlie dorsal root ganglion neuronal hyperexcitability and pain in a subset of patients with small fibre neuropathy. Recent clinical studies have demonstrated that lacosamide, which blocks sodium channels in a use-dependent manner, attenuates pain in some patients with Nav1.7 mutations; however, only a subgroup of these patients responded to the drug. Here, we used voltage-clamp recordings to evaluate the effects of lacosamide on five Nav1.7 variants from patients who were responsive or non-responsive to treatment. We show that, at the clinically achievable concentration of 30 µM, lacosamide acts as a potent sodium channel inhibitor of Nav1.7 variants carried by responsive patients, via a hyperpolarizing shift of voltage-dependence of both fast and slow inactivation and enhancement of use-dependent inhibition. By contrast, the effects of lacosamide on slow inactivation and use-dependence in Nav1.7 variants from non-responsive patients were less robust. Importantly, we found that lacosamide selectively enhances fast inactivation only in variants from responders. Taken together, these findings begin to unravel biophysical underpinnings that contribute to responsiveness to lacosamide in patients with small fibre neuropathy carrying select Nav1.7 variants.

A gain-of-function sodium channel ß2-subunit mutation in painful diabetic neuropathy.

Diabetes mellitus is a global challenge with many diverse health sequelae, of which diabetic peripheral neuropathy is one of the most common. A substantial number of patients with diabetic peripheral neuropathy develop chronic pain, but the genetic and epigenetic factors that predispose diabetic peripheral neuropathy patients to develop neuropathic pain are poorly understood. Recent targeted genetic studies have identified mutations in α-subunits of voltage-gated sodium channels (Navs) in patients with painful diabetic peripheral neuropathy. Mutations in proteins that regulate trafficking or functional properties of Navs could expand the spectrum of patients with Nav-related peripheral neuropathies. The auxiliary sodium channel ß-subunits (ß1-4) have been reported to increase current density, alter inactivation kinetics, and modulate subcellular localization of Nav. Mutations in ß-subunits have been associated with several diseases, including epilepsy, cancer, and diseases of the cardiac conducting system. However, mutations in ß-subunits have never been shown previously to contribute to neuropathic pain. We report here a patient with painful diabetic peripheral neuropathy and negative genetic screening for mutations in SCN9A, SCN10A, and SCN11A-genes encoding sodium channel α-subunit that have been previously linked to the development of neuropathic pain. Genetic analysis revealed an aspartic acid to asparagine mutation, D109N, in the ß2-subunit. Functional analysis using current-clamp revealed that the ß2-D109N rendered dorsal root ganglion neurons hyperexcitable, especially in response to repetitive stimulation. Underlying the hyperexcitability induced by the ß2-subunit mutation, as evidenced by voltage-clamp analysis, we found a depolarizing shift in the voltage dependence of Nav1.7 fast inactivation and reduced use-dependent inhibition of the Nav1.7 channel.

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.

Yield of peripheral sodium channels gene screening in pure small fibre neuropathy.

BACKGROUND: Neuropathic pain is common in peripheral neuropathy. Recent genetic studies have linked pathogenic voltage-gated sodium channel (VGSC) variants to human pain disorders. Our aims are to determine the frequency of SCN9A, SCN10A and SCN11A variants in patients with pure small fibre neuropathy (SFN), analyse their clinical features and provide a rationale for genetic screening. METHODS: Between September 2009 and January 2017, 1139 patients diagnosed with pure SFN at our reference centre were screened for SCN9A, SCN10A and SCN11A variants. Pathogenicity of variants was classified according to established guidelines of the Association for Clinical Genetic Science and frequencies were determined. Patients with SFN were grouped according to the VGSC variants detected, and clinical features were compared. RESULTS: Among 1139 patients with SFN, 132 (11.6%) patients harboured 73 different (potentially) pathogenic VGSC variants, of which 50 were novel and 22 were found in ≥ 1 patient. The frequency of (potentially) pathogenic variants was 5.1% (n=58/1139) for SCN9A, 3.7% (n=42/1139) for SCN10A and 2.9% (n=33/1139) for SCN11A. Only erythromelalgia-like symptoms and warmth-induced pain were significantly more common in patients harbouring VGSC variants. CONCLUSION: (Potentially) pathogenic VGSC variants are present in 11.6% of patients with pure SFN. Therefore, genetic screening of SCN9A, SCN10A and SCN11A should be considered in patients with pure SFN, independently of clinical features or underlying conditions.

COL6A5 variants in familial neuropathic chronic itch.

Itch is thought to represent the peculiar response to stimuli conveyed by somatosensory pathways shared with pain through the activation of specific neurons and receptors. It can occur in association with dermatological, systemic and neurological diseases, or be the side effect of certain drugs. However, some patients suffer from chronic idiopathic itch that is frequently ascribed to psychological distress and for which no biomarker is available to date. We investigated three multigenerational families, one of which diagnosed with joint hypermobility syndrome/Ehlers-Danlos syndrome hypermobility type (JHS/EDS-HT), characterized by idiopathic chronic itch with predominantly proximal distribution. Skin biopsy was performed in all eight affected members and revealed in six of them reduced intraepidermal nerve fibre density consistent with small fibre neuropathy. Whole exome sequencing identified two COL6A5 rare variants co-segregating with chronic itch in eight affected members and absent in non-affected members, and in one unrelated sporadic patient with type 1 painless diabetic neuropathy and chronic itch. Two families and the diabetic patient carried the nonsense c.6814G>T (p.Glu2272*) variant and another family carried the missense c.6486G>C (p.Arg2162Ser) variant. Both variants were predicted as likely pathogenic by in silico analyses. The two variants were rare (minor allele frequency < 0.1%) in 6271 healthy controls and absent in 77 small fibre neuropathy and 167 JHS/EDS-HT patients without itch. Null-allele test on cDNA from patients' fibroblasts of both families carrying the nonsense variant demonstrated functional haploinsufficiency due to activation of nonsense mediated RNA decay. Immunofluorescence microscopy and western blotting revealed marked disorganization and reduced COL6A5 synthesis, respectively. Indirect immunofluorescence showed reduced COL6A5 expression in the skin of patients carrying the nonsense variant. Treatment with gabapentinoids provided satisfactory itch relief in the patients carrying the mutations. Our findings first revealed an association between COL6A5 gene and familiar chronic itch, suggesting a new contributor to the pathogenesis of neuropathic itch and identifying a new candidate therapeutic target.

No Fabry Disease in Patients Presenting with Isolated Small Fiber Neuropathy.

OBJECTIVE: Screening for Fabry disease in patients with small fiber neuropathy has been suggested, especially since Fabry disease is potentially treatable. However, the diagnostic yield of testing for Fabry disease in isolated small fiber neuropathy patients has never been systematically investigated. Our aim is to determine the presence of Fabry disease in patients with small fiber neuropathy. METHODS: Patients referred to our institute, who met the criteria for isolated small fiber neuropathy were tested for Fabry disease by measurement of alpha-Galactosidase A activity in blood, lysosomal globotriaosylsphingosine in urine and analysis on possible GLA gene mutations. RESULTS: 725 patients diagnosed with small fiber neuropathy were screened for Fabry disease. No skin abnormalities were seen except for redness of the hands or feet in 30.9% of the patients. Alfa-Galactosidase A activity was tested in all 725 patients and showed diminished activity in eight patients. Lysosomal globotriaosylsphingosine was examined in 509 patients and was normal in all tested individuals. Screening of GLA for mutations was performed for 440 patients, including those with diminished α-Galactosidase A activity. Thirteen patients showed a GLA gene variant. One likely pathogenic variant was found in a female patient. The diagnosis Fabry disease could not be confirmed over time in this patient. Eventually none of the patients were diagnosed with Fabry disease. CONCLUSIONS: In patients with isolated small fiber neuropathy, and no other signs compatible with Fabry disease, the diagnostic yield of testing for Fabry disease is extremely low. Testing for Fabry disease should be considered only in cases with additional characteristics, such as childhood onset, cardiovascular disease, renal failure, or typical skin lesions.

The Domain II S4-S5 Linker in Nav1.9: A Missense Mutation Enhances Activation, Impairs Fast Inactivation, and Produces Human Painful Neuropathy.

Painful small fiber neuropathy is a challenging medical condition with no effective treatment. Non-genetic causes can be identified in one half of the subjects. Gain-of-function variants of sodium channels Nav1.7 and Nav1.8 have recently been associated with painful small fiber neuropathy. More recently, mutations of sodium channel Nav1.9 have been linked to human pain disorders, with two gain-of-function mutations found in patients with painful small fiber neuropathy. Here we report a novel Nav1.9 mutation, a glycine 699 substitution by arginine (G699R) in the domain II S4-S5 linker, identified in a patient with painful small fiber neuropathy. In this study, we assayed the mutant channels by voltage-clamp in superior cervical ganglion neurons, which do not produce endogenous Nav1.8 or Nav1.9 currents, and provide a novel platform where Nav1.9 is expressed at relatively high levels. Voltage-clamp analysis showed that the mutation hyperpolarizes (-10.1 mV) channel activation, depolarizes (+6.3 mV) steady-state fast inactivation, slows deactivation, and enhances ramp responses compared with wild-type Nav1.9 channels. Current-clamp analysis showed that the G699R mutant channels render dorsal root ganglion neurons hyperexcitable, via depolarized resting membrane potential, reduced current threshold and increased evoked firing. These observations show that the domain II S4-S5 linker plays an important role in the gating of Nav1.9 and demonstrates that a mutation in this linker is linked to a common pain disorder.

Sodium channel genes in pain-related disorders: phenotype-genotype associations and recommendations for clinical use.

Human studies have firmly implicated voltage-gated sodium channels in human pain disorders, and targeted and massively parallel genomic sequencing is beginning to be used in clinical practice to determine which sodium channel variants are involved. Missense substitutions of SCN9A, the gene encoding sodium channel NaV1.7, SCN10A, the gene encoding sodium channel NaV1.8, and SCN11A, the gene encoding sodium channel NaV1.9, produce gain-of-function changes that contribute to pain in many human painful disorders. Genomic sequencing might help to establish a diagnosis, and in the future might support individualisation of therapeutic approaches. However, in many cases, and especially in sodium channelopathies, the results from genomic sequencing can only be appropriately interpreted in the context of an extensive functional assessment, or family segregation analysis of phenotype and genotype.

Painful neuropathies: the emerging role of sodium channelopathies.

Pain is a frequent debilitating feature reported in peripheral neuropathies with involvement of small nerve (Aδ and C) fibers. Voltage-gated sodium channels are responsible for the generation and conduction of action potentials in the peripheral nociceptive neuronal pathway where NaV 1.7, NaV 1.8, and NaV 1.9 sodium channels (encoded by SCN9A, SCN10A, and SCN11A) are preferentially expressed. The human genetic pain conditions inherited erythromelalgia and paroxysmal extreme pain disorder were the first to be linked to gain-of-function SCN9A mutations. Recent studies have expanded this spectrum with gain-of-function SCN9A mutations in patients with small fiber neuropathy and in a new syndrome of pain, dysautonomia, and small hands and small feet (acromesomelia). In addition, painful neuropathies have been recently linked to SCN10A mutations. Patch-clamp studies have shown that the effect of SCN9A mutations is dependent upon the cell-type background. The functional effects of a mutation in dorsal root ganglion (DRG) neurons and sympathetic neuron cells may differ per mutation, reflecting the pattern of expression of autonomic symptoms in patients with painful neuropathies who carry the mutation in question. Peripheral neuropathies may not always be length-dependent, as demonstrated in patients with initial facial and scalp pain symptoms with SCN9A mutations showing hyperexcitability in both trigeminal ganglion and DRG neurons. There is some evidence suggesting that gain-of-function SCN9A mutations can lead to degeneration of peripheral axons. This review will focus on the emerging role of sodium channelopathies in painful peripheral neuropathies, which could serve as a basis for novel therapeutic strategies.

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.

The G1662S NaV1.8 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.

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.