Nav1.7 P610T mutation in two siblings with persistent ocular pain after corneal axon transection: impaired slow inactivation and hyperexcitable trigeminal neurons.

Despite extensive study, the mechanisms underlying pain after axonal injury remain incompletely understood. Pain after corneal refractive surgery provides a model, in humans, of the effect of injury to trigeminal afferent nerves. Axons of trigeminal ganglion neurons that innervate the cornea are transected by laser-assisted in situ keratomileusis (LASIK). Although most patients do not experience postoperative pain, a small subgroup develop persistent ocular pain. We previously carried out genomic analysis and determined that some patients with persistent pain after axotomy of corneal axons during refractive surgery carry mutations in genes that encode the electrogenisome of trigeminal ganglion neurons, the ensemble of ion channels and receptors that regulate excitability within these cells, including SCN9A, which encodes sodium channel Nav1.7, a threshold channel abundantly expressed in sensory neurons that has been implicated in a number of pain-related disorders. Here, we describe the biophysical and electrophysiological profiling of the P610T Nav1.7 mutation found in two male siblings with persistent ocular pain after refractive surgery. Our results indicate that this mutation impairs the slow inactivation of Nav1.7. As expected from this proexcitatory change in channel function, we also demonstrate that this mutation produces increased spontaneous activity in trigeminal ganglion neurons. These findings suggest that this gain-of-function mutation in Nav1.7 may contribute to pain after injury to the axons of trigeminal ganglion neurons.NEW & NOTEWORTHY Mechanisms underlying pain after axonal injury remain elusive. A small subgroup of patients experience pain after corneal refractive surgery, providing a human pain model after well-defined injury to axons. Here we analyze a mutation (P610T) in Nav1.7, a threshold sodium channel expressed in nociceptors, found in two siblings with persistent ocular pain after refractive surgery. We show that it impairs channel slow inactivation, thereby triggering inappropriate repetitive activity in trigeminal ganglion axons that signal eye pain.

KCNQ variants and pain modulation: a missense variant in Kv7.3 contributes to pain resilience.

There is a pressing need for understanding of factors that confer resilience to pain. Gain-of-function mutations in sodium channel Nav1.7 produce hyperexcitability of dorsal root ganglion neurons underlying inherited erythromelalgia, a human genetic model of neuropathic pain. While most individuals with erythromelalgia experience excruciating pain, occasional outliers report more moderate pain. These differences in pain profiles in blood-related erythromelalgia subjects carrying the same pain-causative Nav1.7 mutation and markedly different pain experience provide a unique opportunity to investigate potential genetic factors that contribute to inter-individual variability in pain. We studied a patient with inherited erythromelalgia and a Nav1.7 mutation (c.4345T>G, p. F1449V) with severe pain as is characteristic of most inherited erythromelalgia patients, and her mother who carries the same Nav1.7 mutation with a milder pain phenotype. Detailed six-week daily pain diaries of pain episodes confirmed their distinct pain profiles. Electrophysiological studies on subject-specific induced pluripotent stem cell-derived sensory neurons from each of these patients showed that the excitability of these cells paralleled their pain phenotype. Whole-exome sequencing identified a missense variant (c.2263C>T, p. D755N) in KCNQ3 (Kv7.3) in the pain resilient mother. Voltage-clamp recordings showed that co-expression of Kv7.2-wild type (WT)/Kv7.3-D755N channels produced larger M-currents than that of Kv7.2-WT/Kv7.3-WT. The difference in excitability of the patient-specific induced pluripotent stem cell-derived sensory neurons was mimicked by modulating M-current levels using the dynamic clamp and a model of the mutant Kv7.2-WT/Kv7.3-D755N channels. These results show that a 'pain-in-a-dish' model can be used to explicate genetic contributors to pain, and confirm that KCNQ variants can confer pain resilience via an effect on peripheral sensory neurons.

Genomic analysis of 21 patients with corneal neuralgia after refractive surgery.

BACKGROUND: Refractive surgery, specifically laser-assisted in situ keratomileusis and photorefractive keratectomy, are widely applied procedures to treat myopia, hyperopia, and astigmatism. After surgery, a subgroup of cases suffers from persistent and intractable pain of obscure etiology, thought to be neuropathic. We aimed to investigate the contribution of genomic factors in the pathogenesis of these patients with corneal neuralgia. METHODS: We enrolled 21 cases (6 males and 15 females) from 20 unrelated families, who reported persistent pain (>3 months), after refractive surgery (20 laser-assisted in situ keratomileusis and 1 photorefractive keratectomy patients). Whole-exome sequencing and gene-based association test were performed. RESULTS: Whole-exome sequencing demonstrated low-frequency variants (allele frequency < 0.05) in electrogenisome-related ion channels and cornea-expressed collagens, most frequently in SCN10A (5 cases), SCN9A (4 cases), TRPV1 (4 cases), CACNA1H and CACNA2D2 (5 cases each), COL5A1 (6 cases), COL6A3 (5 cases), and COL4A2 (4 cases). Two variants, p.K655R of SCN9A and p.Q85R of TRPV1, were previously characterized as gain-of-function. Gene-based association test assessing "damaging" missense variants against gnomAD exome database (non-Finnish European or global), identified a gene, SLC9A3R1, with statistically significant effect (odds ratio = 17.09 or 17.04; Bonferroni-corrected P-value < 0.05). CONCLUSION: These findings in a small patient cohort did not identify a common gene/variant among most of these cases, as found in other disorders, for example small-fiber neuropathy. Further studies of these candidate genes/variants might enhance understanding of the role of genetic factors in the pathogenesis of corneal neuralgia.

A Novel Gain-of-Function Nav1.9 Mutation in a Child With Episodic Pain.

Voltage-gated sodium channel Nav1.9 is a threshold channel that regulates action potential firing. Nav1.9 is preferentially expressed in myenteric neurons, and small-diameter dorsal root ganglion (DRG) and trigeminal ganglion neurons including nociceptors. Recent studies have demonstrated a monogenic Mendelian link of Nav1.9 to human pain disorders. Gain-of-function variants in Nav1.9, which cause smaller depolarizations of RMP, have been identified in patients with familial episodic pain type 3 (FEPS3) and the more common pain disorder small fiber neuropathy. To explore the phenotypic spectrum of Nav1.9 channelopathy, here we report a new Nav1.9 mutation, N816K, in a child with early-onset episodic pain in both legs, episodic abdominal pain, and chronic constipation. Sequencing of further selected pain genes was normal. N816K alters a residue at the N-terminus of loop 2, proximal to the cytoplasmic terminus of transmembrane segment 6 in domain II. Voltage-clamp recordings demonstrate that Nav1.9-N816K significantly increases current density and hyperpolarizes voltage-dependence of activation by 10 mV, enabling a larger window current. Current-clamp recordings in DRG neurons shows that N816K channels depolarize RMP of small DRG neurons by 7 mV, reduce current threshold of firing an action potential and render DRG neurons hyperexcitable. Taken together these data demonstrate gain-of-function attributes of the newly described N816K mutation at the channel and cellular levels, which are consistent with a pain phenotype in the carrier of this mutation.

Resilience to Pain: A Peripheral Component Identified Using Induced Pluripotent Stem Cells and Dynamic Clamp.

Pain is a complex process that involves both detection in the peripheral nervous system and perception in the CNS. Individual-to-individual differences in pain are well documented, but not well understood. Here we capitalized on inherited erythromelalgia (IEM), a well characterized human genetic model of chronic pain, and studied a unique family containing related IEM subjects with the same disease-causing NaV1.7 mutation, which is known to make dorsal root ganglion (DRG) neurons hyperexcitable, but different pain profiles (affected son with severe pain, affected mother with moderate pain, and an unaffected father). We show, first, that, at least in some cases, relative sensitivity to pain can be modeled in subject-specific induced pluripotent stem cell (iPSC)-derived sensory neurons in vitro; second, that, in some cases, mechanisms operating in peripheral sensory neurons contribute to interindividual differences in pain; and third, using whole exome sequencing (WES) and dynamic clamp, we show that it is possible to pinpoint a specific variant of another gene, KCNQ in this particular kindred, that modulates the excitability of iPSC-derived sensory neurons in this family. While different gene variants may modulate DRG neuron excitability and thereby contribute to interindividual differences in pain in other families, this study shows that subject-specific iPSCs can be used to model interindividual differences in pain. We further provide proof-of-principle that iPSCs, WES, and dynamic clamp can be used to investigate peripheral mechanisms and pinpoint specific gene variants that modulate pain signaling and contribute to interindividual differences in pain.SIGNIFICANCE STATEMENT Individual-to-individual differences in pain are well documented, but not well understood. In this study, we show, first, that, at least in some cases, relative sensitivity to pain can be modeled in subject-specific induced pluripotent stem cell-derived sensory neurons in vitro; second, that, in some cases, mechanisms operating in peripheral sensory neurons contribute to interindividual differences in pain; and third, using whole exome sequencing and dynamic clamp, we show that it is possible to pinpoint a specific gene variant that modulates pain signaling and contributes to interindividual differences in pain.

A novel gain-of-function Nav1.7 mutation in a carbamazepine-responsive patient with adult-onset painful peripheral neuropathy.

Voltage-gated sodium channel Nav1.7 is a threshold channel in peripheral dorsal root ganglion (DRG), trigeminal ganglion, and sympathetic ganglion neurons. Gain-of-function mutations in Nav1.7 have been shown to increase excitability in DRG neurons and have been linked to rare Mendelian and more common pain disorders. Discovery of Nav1.7 variants in patients with pain disorders may expand the spectrum of painful peripheral neuropathies associated with a well-defined molecular target, thereby providing a basis for more targeted approaches for treatment. We screened the genome of a patient with adult-onset painful peripheral neuropathy characterized by severe burning pain and report here the new Nav1.7-V810M variant. Voltage-clamp recordings were used to assess the effects of the mutation on biophysical properties of Nav1.7 and the response of the mutant channel to treatment with carbamazepine (CBZ), and multi-electrode array (MEA) recordings were used to assess the effects of the mutation on the excitability of neonatal rat pup DRG neurons. The V810M variant increases current density, shifts activation in a hyperpolarizing direction, and slows kinetics of deactivation, all gain-of-function attributes. We also show that DRG neurons that express the V810M variant become hyperexcitable. The patient responded to treatment with CBZ. Although CBZ did not depolarize activation of the mutant channel, it enhanced use-dependent inhibition. Our results demonstrate the presence of a novel gain-of-function variant of Nav1.7 in a patient with adult-onset painful peripheral neuropathy and the responsiveness of that patient to treatment with CBZ, which is likely due to the classical mechanism of use-dependent inhibition.

Brain activity associated with pain in inherited erythromelalgia: stimulus-free pain engages brain areas involved in valuation and learning.

Inherited erythromelalgia (IEM) is a chronic pain disorder caused by gain-of-function mutations of peripheral sodium channel Nav1.7, in which warmth triggers severe pain. Little is known about the brain representation of pain in IEM. Here we study two subjects with the IEM Nav1.7-S241T mutation using functional brain imaging (fMRI). Subjects were scanned during each of five visits. During each scan, pain was first triggered using a warming boot and subjects rated their thermal-heat pain. Next, the thermal stimulus was terminated and subjects rated stimulus-free pain. Last, subjects performed a control visual rating task. Thermal-heat induced pain mapped to the frontal gyrus, ventro-medial prefrontal cortex, superior parietal lobule, supplementary motor area, insula, primary and secondary somato-sensory motor cortices, dorsal and ventral striatum, amygdala, and hippocampus. Stimulus-free pain, by contrast, mapped mainly to the frontal cortex, including dorsal, ventral and medial prefrontal cortex, and supplementary motor area. Examination of time periods when stimulus-free pain was changing showed further activations in the valuation network including the rostral anterior cingulate cortex, striatum and amygdala, in addition to brainstem, thalamus, and insula. We conclude that, similar to other chronic pain conditions, the brain representation of stimulus-free pain during an attack in subjects with IEM engages brain areas involved in acute pain as well as valuation and learning.

Nav1.7-A1632G Mutation from a Family with Inherited Erythromelalgia: Enhanced Firing of Dorsal Root Ganglia Neurons Evoked by Thermal Stimuli.

UNLABELLED: Voltage-gated sodium channel Nav1.7 is a central player in human pain. Mutations in Nav1.7 produce several pain syndromes, including inherited erythromelalgia (IEM), a disorder in which gain-of-function mutations render dorsal root ganglia (DRG) neurons hyperexcitable. Although patients with IEM suffer from episodes of intense burning pain triggered by warmth, the effects of increased temperature on DRG neurons expressing mutant Nav1.7 channels have not been well documented. Here, using structural modeling, voltage-clamp, current-clamp, and multielectrode array recordings, we have studied a newly identified Nav1.7 mutation, Ala1632Gly, from a multigeneration family with IEM. Structural modeling suggests that Ala1632 is a molecular hinge and that the Ala1632Gly mutation may affect channel gating. Voltage-clamp recordings revealed that the Nav1.7-A1632G mutation hyperpolarizes activation and depolarizes fast-inactivation, both gain-of-function attributes at the channel level. Whole-cell current-clamp recordings demonstrated increased spontaneous firing, lower current threshold, and enhanced evoked firing in rat DRG neurons expressing Nav1.7-A1632G mutant channels. Multielectrode array recordings further revealed that intact rat DRG neurons expressing Nav1.7-A1632G mutant channels are more active than those expressing Nav1.7 WT channels. We also showed that physiologically relevant thermal stimuli markedly increase the mean firing frequencies and the number of active rat DRG neurons expressing Nav1.7-A1632G mutant channels, whereas the same thermal stimuli only increase these parameters slightly in rat DRG neurons expressing Nav1.7 WT channels. The response of DRG neurons expressing Nav1.7-A1632G mutant channels upon increase in temperature suggests a cellular basis for warmth-triggered pain in IEM. SIGNIFICANCE STATEMENT: Inherited erythromelalgia (IEM), a severe pain syndrome characterized by episodes of intense burning pain triggered by warmth, is caused by mutations in sodium channel Nav1.7, which are preferentially expressed in sensory and sympathetic neurons. More than 20 gain-of-function Nav1.7 mutations have been identified from IEM patients, but the question of how warmth triggers episodes of pain in IEM has not been well addressed. Combining multielectrode array, voltage-clamp, and current-clamp recordings, we assessed a newly identified IEM mutation (Nav1.7-A1632G) from a multigeneration family. Our data demonstrate gain-of-function attributes at the channel level and differential effects of physiologically relevant thermal stimuli on the excitability of DRG neurons expressing mutant and WT Nav1.7 channels, suggesting a cellular mechanism for warmth-triggered pain episodes in IEM patients.

Pharmacotherapy for Pain in a Family With Inherited Erythromelalgia Guided by Genomic Analysis and Functional Profiling.

IMPORTANCE: There is a need for more effective pharmacotherapy for chronic pain, including pain in inherited erythromelalgia (IEM) in which gain-of-function mutations of sodium channel NaV1.7 make dorsal root ganglion (DRG) neurons hyperexcitable. OBJECTIVE: To determine whether pain in IEM can be attenuated via pharmacotherapy guided by genomic analysis and functional profiling. DESIGN, SETTING, AND PARTICIPANTS: Pain in 2 patients with IEM due to the NaV1.7 S241T mutation, predicted by structural modeling and functional analysis to be responsive to carbamazepine, was assessed in a double-blind, placebo-controlled study conducted from September 2014 to April 21, 2015. Functional magnetic resonance imaging assessed patterns of brain activity associated with pain during treatment with placebo or carbamazepine. Multielectrode array technology was used to assess the effect of carbamazepine on firing of DRG neurons carrying S241T mutant channels. MAIN OUTCOMES AND MEASURES: Behavioral assessment of pain; functional magnetic resonance imaging; and assessment of firing in DRG neurons carrying S241T mutant channels. RESULTS: This study included 2 patients from the same family with IEM and the S241T NaV1.7 mutation. We showed that, as predicted by molecular modeling, thermodynamic analysis, and functional profiling, carbamazepine attenuated pain in patients with IEM due to the S241T NaV1.7 mutation. Patient 1 reported a reduction in mean time in pain (TIP) per day during the 15-day maintenance period, from 424 minutes while taking placebo to 231.9 minutes while taking carbamazepine (400 mg/day), and a reduction in total TIP over the 15-day maintenance period, from 6360 minutes while taking placebo to 3015 minutes while taking carbamazepine. Patient 2 reported a reduction in mean TIP per day during the maintenance period, from 61 minutes while taking placebo to 9.1 minutes while taking carbamazepine (400 mg then 200 mg/day), and a reduction in total TIP, from 915 minutes while taking placebo over the 15-day maintenance period to 136 minutes while taking carbamazepine. Patient 1 reported a reduction of mean episode duration, from 615 minutes while taking placebo to 274.1 minutes while taking carbamazepine, while patient 2 reported a reduction of the mean episode duration from 91.5 minutes while taking placebo to 45.3 minutes while taking carbamazepine. Patient 1, who had a history of night awakenings from pain, reported 101 awakenings owing to pain while taking placebo during the maintenance period and 32 awakenings while taking carbamazepine. Attenuation of pain was paralleled by a shift in brain activity from valuation and pain areas to primary and secondary somatosensory, motor, and parietal attention areas. Firing of DRG neurons expressing the S241T NaV1.7 mutant channel in response to physiologically relevant thermal stimuli was reduced by carbamazepine. CONCLUSIONS AND RELEVANCE: Our results demonstrate that pharmacotherapy guided by genomic analysis, molecular modeling, and functional profiling can attenuate neuropathic pain in patients carrying the S241T mutation.

Inherited erythromelalgia due to mutations in SCN9A: natural history, clinical phenotype and somatosensory profile.

Inherited erythromelalgia, the first human pain syndrome linked to voltage-gated sodium channels, is widely regarded as a genetic model of human pain. Because inherited erythromelalgia was linked to gain-of-function changes of sodium channel Na(v)1.7 only a decade ago, the literature has mainly consisted of reports of genetic and/or clinical characterization of individual patients. This paper describes the pattern of pain, natural history, somatosensory profile, psychosocial status and olfactory testing of 13 subjects with primary inherited erythromelalgia with mutations of SCN9A, the gene encoding Na(v)1.7. Subjects were clinically profiled using questionnaires, quantitative sensory testing and olfaction testing during the in-clinic phase of the study. In addition, a detailed pain phenotype for each subject was obtained over a 3-month period at home using diaries, enabling subjects to self-report pain attacks, potential triggers, duration and severity of pain. All subjects reported pain and heat in the extremities (usually feet and/or hands), with pain attacks triggered by heat or exercise and relieved mainly by non-pharmacological manoeuvres such as cooling. A large proportion of pain attacks (355/1099; 32%) did not involve a specific trigger. There was considerable variability in the number, duration and severity of pain attacks between subjects, even those carrying the same mutation within a family, and within individuals over the 12-13 week observation period. Most subjects (11/13) had pain between attacks. For these subjects, mean pain severity between pain attacks was usually lower than that during an attack. Olfaction testing using the Sniffin'T test did not demonstrate hyperosmia. One subject had evidence of orthostatic hypotension. Overall, there was a statistically significant correlation between total Hospital Anxiety and Depression Scale scores (P= 0.005) and pain between attacks and for Hospital Anxiety and Depression Scale Depression scores and pain between attacks (P= 0.001). Hospital Anxiety and Depression Scale scores for five subjects were below the threshold for mild anxiety or depression and none of the 13 subjects were severely anxious and/or depressed. Quantitative sensory testing revealed significantly increased detection thresholds for cold and warm stimuli at affected, compared to unaffected sites. By contrast, significantly decreased cold and heat pain thresholds were found at unaffected sites. Sensory profiles varied considerably between affected and unaffected sites, suggesting the existence of small fibre neuropathy in symptomatic sites. This in-depth clinical characterization of a well-defined inherited erythromelalgia population indicates the importance of characterizing the pain phenotype in individuals before undertaking clinical trials, given the inherent variability of pain both between and within inherited erythromelalgia subjects, even those within a family who carry the same mutation.

The let-7 microRNA target gene, Mlin41/Trim71 is required for mouse embryonic survival and neural tube closure.

In the nematode Caenorhabditis elegans, the let-7 microRNA (miRNA) controls the timing of key developmental events and terminal differentiation in part by directly regulating lin-41. C. elegans lin-41 mutants display precocious cell cycle exit and terminal differentiation of epidermal skin cells. lin-41 orthologues are found in more complex organisms including both mice and humans, but their roles are not known. We generated Mlin41 mouse mutants to ascertain a functional role for Mlin41. Strong loss of function Mlin41 gene-trap mutants demonstrated a striking neural tube closure defect during development, and embryonic lethality. Like C. elegans lin-41, Mlin41 also appears to be regulated by the let-7 and mir-125 miRNAs. Since Mlin41 is required for neural tube closure and survival it points to human lin-41 (HLIN41/TRIM71) as a potential human development and disease gene.

Reciprocal expression of lin-41 and the microRNAs let-7 and mir-125 during mouse embryogenesis.

In C. elegans, heterochronic genes control the timing of cell fate determination during development. Two heterochronic genes, let-7 and lin-4, encode microRNAs (miRNAs) that down-regulate a third heterochronic gene lin-41 by binding to complementary sites in its 3'UTR. let-7 and lin-4 are conserved in mammals. Here we report the cloning and sequencing of mammalian lin-41 orthologs. We find that mouse and human lin-41 genes contain predicted conserved complementary sites for let-7 and the lin-4 ortholog, mir-125, in their 3'UTRs. Mouse lin-41 (Mlin-41) is temporally expressed in developing mouse embryos, most dramatically in the limb buds. Mlin-41 is down-regulated during mid-embryogenesis at the time when mouse let-7c and mir-125 RNA levels are up-regulated. Our results suggest that mammalian lin-41 is temporally regulated by miRNAs in order to direct key developmental events such as limb formation.