Characterization of ClC-1 chloride channels in zebrafish: a new model to study myotonia.

The function of the chloride channel ClC-1 is crucial for the control of muscle excitability. Thus, reduction of ClC-1 functions by CLCN1 mutations leads to myotonia congenita. Many different animal models have contributed to understanding the myotonia pathophysiology. However, these models do not allow in vivo screening of potentially therapeutic drugs, as the zebrafish model does. In this work, we identified and characterized the two zebrafish orthologues (clc-1a and clc-1b) of the ClC-1 channel. Both channels are mostly expressed in the skeletal muscle as revealed by RT-PCR, western blot, and electrophysiological recordings of myotubes, and clc-1a is predominantly expressed in adult stages. Characterization in Xenopus oocytes shows that the zebrafish channels display similar anion selectivity and voltage dependence to their human counterparts. However, they show reduced sensitivity to the inhibitor 9-anthracenecarboxylic acid (9-AC), and acidic pH inverts the voltage dependence of activation. Reduction of clc-1a/b expression hampers spontaneous and mechanically stimulated movement, which could be reverted by expression of human ClC-1 but not by some ClC-1 containing myotonia mutations. Treatment of clc-1-depleted zebrafish with mexiletine, a typical drug used in human myotonia, improves the motor behaviour. Our work extends the repertoire of ClC channels to evolutionary structure-function studies and proposes the zebrafish clcn1 crispant model as a simple tool to find novel therapies for myotonia. KEY POINTS: We have identified two orthologues of ClC-1 in zebrafish (clc-1a and clc-1b) which are mostly expressed in skeletal muscle at different developmental stages. Functional characterization of the activity of these channels reveals many similitudes with their mammalian counterparts, although they are less sensitive to 9-AC and acidic pH inverts their voltage dependence of gating. Reduction of clc-1a/b expression hampers spontaneous and mechanically stimulated movement which could be reverted by expression of human ClC-1. Myotonia-like symptoms caused by clc-1a/b depletion can be reverted by mexiletine, suggesting that this model could be used to find novel therapies for myotonia.

Reduced K+ build-up in t-tubules contributes to resistance of the diaphragm to myotonia.

Patients with myotonia congenita suffer from slowed muscle relaxation caused by hyperexcitability. The diaphragm is only mildly affected in myotonia congenita; discovery of the mechanism underlying its resistance to myotonia could identify novel therapeutic targets. Intracellular recordings from two mouse models of myotonia congenita revealed the diaphragm had less myotonia than either the extensor digitorum longus (EDL) or the soleus muscles. A mechanism contributing to resistance of the diaphragm to myotonia was reduced depolarization of the interspike membrane potential during repetitive firing of action potentials, a process driven by build-up of K+ in small invaginations of muscle membrane known as t-tubules. We explored differences between diaphragm and EDL that might underlie reduction of K+ build-up in diaphragm t-tubules. Smaller size of diaphragm fibres, which promotes diffusion of K+ out of t-tubules, was identified as a contributor. Intracellular recording revealed slower repolarization of action potentials in diaphragm suggesting reduced Kv conductance. Higher resting membrane conductance was identified suggesting increased Kir conductance. Computer simulation found that a reduction of Kv conductance had little effect on K+ build-up whereas increased Kir conductance lessened build-up, although the effect was modest. Our data and computer simulation suggest opening of K+ channels during action potentials has little effect on K+ build-up whereas opening of K+ channels during the interspike interval slightly lessens K+ build-up. We conclude that activation of K+ channels may lessen myotonia by opposing depolarization to action potential threshold without worsening K+ build-up in t-tubules. KEY POINTS: In mouse models of the muscle disease myotonia congenita, the diaphragm has much less myotonia (muscle stiffness) than the extensor digitorum longus or soleus muscles. Identifying why the diaphragm is resistant to myotonia may help in developing novel therapy. We found the reason the diaphragm has less myotonia is that it is less prone to depolarization caused by K+ build-up in t-tubules during repetitive firing of action potentials. Smaller fibre size contributes to resistance to K+ build-up with differences in K+ currents playing little role. Our data suggest drugs that open K+ channels may be effective in treating myotonia as they may lessen excitability without worsening K+ build-up in t-tubules.

BK channels promote action potential repolarization in skeletal muscle but contribute little to myotonia.

Patients with myotonia congenita suffer from slowed relaxation of muscle (myotonia), due to hyperexcitability caused by loss-of-function mutations in the ClC-1 chloride channel. A recent study suggested that block of large-conductance voltage- and Ca2+- activated K+ channels (BK) may be effective as therapy. The mechanism underlying efficacy was suggested to be lessening of the depolarizing effect of build-up of K+ in t-tubules of muscle during repetitive firing. BK channels are widely expressed in the nervous system and have been shown to play a central role in regulation of excitability, but their contribution to muscle excitability has not been determined. We performed intracellular recordings as well as force measurements in both wild type and BK-/- mouse extensor digitorum longus muscles. Action potential width was increased in BK-/- muscle due to slowing of repolarization, consistent with the possibility K+ build-up in t-tubules is lessened by block of BK channels in myotonic muscle. However, there was no difference in the severity of myotonia triggered by block of muscle Cl- channels with 9-anthracenecarboxylic acid (9AC) in wild type and BK-/- muscle fibers. Further study revealed no difference in the interspike membrane potential during repetitive firing suggesting there was no reduction in K+ build-up in t-tubules of BK-/- muscle. Force recordings following block of muscle Cl- channels demonstrated little reduction in myotonia in BK-/- muscle. In contrast, the current standard of care, mexiletine, significantly reduced myotonia. Our data suggest BK channels regulate muscle excitability, but are not an attractive target for therapy of myotonia.

Myotonia congenita in a Greek cohort: Genotype spectrum and impact of the CLCN1:c.501C > G variant as a genetic modifier.

INTRODUCTION/AIMS: Myotonia congenita (MC) is the most common hereditary channelopathy in humans. Characterized by muscle stiffness, MC may be transmitted as either an autosomal dominant (Thomsen) or a recessive (Becker) disorder. MC is caused by variants in the voltage-gated chloride channel 1 (CLCN1) gene, important for the normal repolarization of the muscle action potential. More than 250 disease-causing variants in the CLCN1 gene have been reported. This study provides an MC genotype-phenotype spectrum in a large cohort of Greek patients and focuses on novel variants and disease epidemiology, including additional insights for the variant CLCN1:c.501C > G. METHODS: Sanger sequencing for the entire coding region of the CLCN1 gene was performed. Targeted segregation analysis of likely candidate variants in additional family members was performed. Variant classification was based on American College of Medical Genetics (ACMG) guidelines. RESULTS: Sixty-one patients from 47 unrelated families were identified, consisting of 51 probands with Becker MC (84%) and 10 with Thomsen MC (16%). Among the different variants detected, 11 were novel and 16 were previously reported. The three most prevalent variants were c.501C > G, c.2680C > T, and c.1649C > G. Additionally, c.501C > G was detected in seven Becker cases in-cis with the c.1649C > G. DISCUSSION: The large number of patients in whom a diagnosis was established allowed the characterization of genotype-phenotype correlations with respect to both previously reported and novel findings. For the c.501C > G (p.Phe167Leu) variant a likely nonpathogenic property is suggested, as it only seems to act as an aggravating modifying factor in cases in which a pathogenic variant triggers phenotypic expression.

Clinical and molecular characterization of myotonia congenita using whole-exome sequencing in Egyptian patients.

BACKGROUND: Myotonia Congenita (MC) is a rare disease classified into two major forms; Thomsen and Becker disease caused by mutations in the CLCN1 gene, which affects muscle excitability and encodes voltage-gated chloride channels (CLC-1). While, there are no data regarding the clinical and molecular characterization of myotonia in Egyptian patients. METHODS: Herein, we report seven Egyptian MC patients from six unrelated families. Following the clinical diagnosis, whole-exome sequencing (WES) was performed for genetic diagnosis. Various in silico prediction tools were utilized to interpret variant pathogenicity. The candidate variants were then validated using Sanger sequencing technique. RESULTS: In total, seven cases were recruited. The ages at the examination were ranged from eight months to nineteen years. Clinical manifestations included warm-up phenomenon, hand grip, and percussion myotonia. Electromyography was performed in all patients and revealed myotonic discharges. Molecular genetic analysis revealed five different variants. Of them, we identified two novel variants in the CLCN1 gene ( c.1583G > C; p.Gly528Ala and c.2203_2216del;p.Thr735ValfsTer57) and three known variants in the CLCN1 and SCN4A gene. According to in silico tools, the identified novel variants were predicted to have deleterious effects. CONCLUSIONS: As the first study to apply WES among Egyptian MC patients, our findings reported two novel heterozygous variants that expand the CLCN1 mutational spectrum for MC diagnosis. These results further confirm that genetic testing is essential for early diagnosis of MC, which affects follow-up treatment and prognostic assessment in clinical practice.

In silico versus functional characterization of genetic variants: lessons from muscle channelopathies.

Accurate determination of the pathogenicity of missense genetic variants of uncertain significance is a huge challenge for implementing genetic data in clinical practice. In silico predictive tools are used to score variants' pathogenicity. However, their value in clinical settings is often unclear, as they have not usually been validated against robust functional assays. We compared nine widely used in silico predictive tools, including more recently developed tools (EVE and REVEL) with detailed cell-based electrophysiology, for 126 CLCN1 variants discovered in patients with the skeletal muscle channelopathy myotonia congenita. We found poor accuracy for most tools. The highest accuracy was obtained with MutationTaster (84.58%) and REVEL (82.54%). Both of these scores showed poor specificity, although specificity was better using EVE. Combining methods based on concordance improved performance overall but still lacked specificity. Our calculated statistics for the predictive tools were different to reported values for other genes in the literature, suggesting that the utility of the tools varies between genes. Overall, current predictive tools for this chloride channel are not reliable for clinical use, and tools with better specificity are urgently required. Improving the accuracy of predictive tools is a wider issue and a huge challenge for effective clinical implementation of genetic data.

Effectiveness and safety of mexiletine versus placebo in patients with myotonia: a systematic review and meta-analysis.

BACKGROUND: The rare nature of dystrophic and non-dystrophic myotonia has limited the available evidence on the efficacy of mexiletine as a potential treatment. To address this gap, we conducted a systematic review and meta-analysis to evaluate the effectiveness and safety of mexiletine for both dystrophic and non-dystrophic myotonic patients. METHODS: The search was conducted on various electronic databases up to March 2023, for randomized clinical trials (RCTs) comparing mexiletine versus placebo in myotonic patients. A risk of bias assessment was carried out, and relevant data was extracted manually into an online sheet. RevMan software (version 5.4) was employed for analysis. RESULTS: A total of five studies, comprising 186 patients, were included in the meta-analysis. Our findings showed that mexiletine was significantly more effective than placebo in improving stiffness score (SMD = - 1.19, 95% CI [- 1.53, - 0.85]), as well as in reducing hand grip myotonia (MD = - 1.36 s, 95% CI [- 1.83, - 0.89]). Mexiletine also significantly improved SF-36 Physical and Mental Component Score in patients with non-dystrophic myotonia only. Regarding safety, mexiletine did not significantly alter ECG parameters but was associated with greater gastrointestinal symptoms (GIT) compared to placebo (RR 3.7, 95% CI [1.79, 7.64]). Other adverse events showed no significant differences. CONCLUSION: The results support that mexiletine is effective and safe in myotonic patients; however, it is associated with a higher risk of GIT symptoms. Due to the scarcity of published RCTs and the prevalence of GIT symptoms, we recommend further well-designed RCTs testing various drug combinations to reduce GIT symptoms.

Self-reported outcomes and quality of life of patients with non-dystrophic myotonia: The French IMPACT 2022 survey.

Non-dystrophic myotonias (NDM) are disabling genetic diseases that impact quality of life. To reduce the impact of NDM, patients develop coping strategies such as lifestyle adaptation and avoiding key triggers. To understand how myotonia affects patients' lives, the IMPACT survey, an online questionnaire on patient-reported outcomes, was developed based on international IMPACT questionnaire. The French IMPACT 2022 survey was completed by 47 NDM French patients. Besides muscle stiffness (98%), patients reported muscle pain (83%), falls (70%) and anxiety (77%). These issues negatively impacted abilities to work/study (49%), daily life at home (49%) and overall mobility outside (49%). Most patients (96%) reported ongoing pharmacological treatment (mexiletine, 91%) associated with improvement in muscle stiffness (100%) and reduction in falls (94%), muscle pain (87%) and anxiety (80%). Patients were moderately satisfied (19.1%), satisfied (42.6%) and very satisfied (29.8%) with the current management; 32% rated their quality of life positively (≥ 8 on 10-point scale). In conclusion, this French survey confirms the impact of myotonia on daily life and quality of life. The improvement in patient-reported outcomes in treated participants highlights the importance of managing myotonia with effective treatments. More work should be initiated to assess the importance of NDM symptom management and patients' adherence and compliance to treatment.

Neutral lipid storage disease with myopathy and myotonia associated to pathogenic variants on PNPLA2 and CLCN1 genes: case report.

BACKGROUND: Neutral lipid storage disease with myopathy (NLSD-M) is an autosomal recessive disease that manifests itself around the 3rd to 4th decade with chronic myopathy predominantly proximal in the shoulder girdle. Clinical myotonia is uncommon. We will report a rare case of association of pathogenic variants on PNPLA2 and CLCN1 genes with a mixed phenotype of NLSD-M and a subclinical form of Thomsen's congenital myotonia. CASE PRESENTATION: We describe a patient with chronic proximal myopathy, subtle clinical myotonia and electrical myotonia on electromyography (EMG). Serum laboratory analysis disclosure hyperCKemia (CK 1280 mg/dL). A blood smear analysis showed Jordan's anomaly, a hallmark of NLSD-M. A genetic panel was collected using next-generation sequencing (NGS) technique, which identified two pathogenic variants on genes supporting two different diagnosis: NLSD-M and Thomsen congenital myotonia, whose association has not been previously described. CONCLUSIONS: Although uncommon, it is important to remember the possibility of association of pathogenic variants to explain a specific neuromuscular disease phenotype. The use of a range of complementary methods, including myopathy genetic panels, may be essential to diagnostic definition in such cases.

Translating genetic and functional data into clinical practice: a series of 223 families with myotonia.

High-throughput DNA sequencing is increasingly employed to diagnose single gene neurological and neuromuscular disorders. Large volumes of data present new challenges in data interpretation and its useful translation into clinical and genetic counselling for families. Even when a plausible gene is identified with confidence, interpretation of the clinical significance and inheritance pattern of variants can be challenging. We report our approach to evaluating variants in the skeletal muscle chloride channel ClC-1 identified in 223 probands with myotonia congenita as an example of these challenges. Sequencing of CLCN1, the gene that encodes CLC-1, is central to the diagnosis of myotonia congenita. However, interpreting the pathogenicity and inheritance pattern of novel variants is notoriously difficult as both dominant and recessive mutations are reported throughout the channel sequence, ClC-1 structure-function is poorly understood and significant intra- and interfamilial variability in phenotype is reported. Heterologous expression systems to study functional consequences of CIC-1 variants are widely reported to aid the assessment of pathogenicity and inheritance pattern. However, heterogeneity of reported analyses does not allow for the systematic correlation of available functional and genetic data. We report the systematic evaluation of 95 CIC-1 variants in 223 probands, the largest reported patient cohort, in which we apply standardized functional analyses and correlate this with clinical assessment and inheritance pattern. Such correlation is important to determine whether functional data improves the accuracy of variant interpretation and likely mode of inheritance. Our data provide an evidence-based approach that functional characterization of ClC-1 variants improves clinical interpretation of their pathogenicity and inheritance pattern, and serve as reference for 34 previously unreported and 28 previously uncharacterized CLCN1 variants. In addition, we identify novel pathogenic mechanisms and find that variants that alter voltage dependence of activation cluster in the first half of the transmembrane domains and variants that yield no currents cluster in the second half of the transmembrane domain. None of the variants in the intracellular domains were associated with dominant functional features or dominant inheritance pattern of myotonia congenita. Our data help provide an initial estimate of the anticipated inheritance pattern based on the location of a novel variant and shows that systematic functional characterization can significantly refine the assessment of risk of an associated inheritance pattern and consequently the clinical and genetic counselling.

Clinical score for early diagnosis of myotonic dystrophy type 2.

INTRODUCTION: Myotonic dystrophy type 2 (DM2) is a rare, multisystemic, autosomal dominant disease with highly variable clinical presentation. DM2 is considered to be highly underdiagnosed. OBJECTIVE: The aim of this study was to determine which symptoms, signs, and diagnostic findings in patients referred to neurological outpatient units are the most indicative to arouse suspicion of DM2. We tried to make a useful and easy-to-administer clinical scoring system for early diagnosis of DM2-DM2 early diagnosis score (DM2-EDS). PATIENTS AND METHODS: Two hundred ninety-one patients with a clinical suspicion of DM2 were included: 69 were genetically confirmed to have DM2, and 222 patients were DM2 negative. Relevant history, neurological, and paraclinical data were obtained from the electronic medical records. RESULTS: The following parameters appeared as significant predictors of DM2 diagnosis: cataracts (beta = 0.410, p < 0.001), myotonia on needle EMG (beta = 0.298, p < 0.001), hand tremor (beta = 0.211, p = 0.001), positive family history (beta = 0.171, p = 0.012), and calf hypertrophy (beta = 0.120, p = 0.043). In the final DM2-EDS, based on the beta values, symptoms were associated with the following values: cataracts (present 3.4, absent 0), myotonia (present 2.5, absent 0), tremor (present 1.7, absent 0), family history (positive 1.4, negative 0), and calf hypertrophy (present 1.0, absent 0). A cut-off value on DM2-EDS of 3.25 of maximum 10 points had a sensitivity of 84% and specificity of 81% to diagnose DM2. CONCLUSION: Significant predictors of DM2 diagnosis in the neurology outpatient unit were identified. We made an easy-to-administer DM2-EDS score for early diagnosis of DM2.

Blockers of Skeletal Muscle Nav1.4 Channels: From Therapy of Myotonic Syndrome to Molecular Determinants of Pharmacological Action and Back.

The voltage-gated sodium channels represent an important target for drug discovery since a large number of physiological processes are regulated by these channels. In several excitability disorders, including epilepsy, cardiac arrhythmias, chronic pain, and non-dystrophic myotonia, blockers of voltage-gated sodium channels are clinically used. Myotonia is a skeletal muscle condition characterized by the over-excitability of the sarcolemma, resulting in delayed relaxation after contraction and muscle stiffness. The therapeutic management of this disorder relies on mexiletine and other sodium channel blockers, which are not selective for the Nav1.4 skeletal muscle sodium channel isoform. Hence, the importance of deepening the knowledge of molecular requirements for developing more potent and use-dependent drugs acting on Nav1.4. Here, we review the available treatment options for non-dystrophic myotonia and the structure-activity relationship studies performed in our laboratory with a focus on new compounds with potential antimyotonic activity.

Therapeutic Targeting of the GSK3ß-CUGBP1 Pathway in Myotonic Dystrophy.

Myotonic Dystrophy type 1 (DM1) is a neuromuscular disease associated with toxic RNA containing expanded CUG repeats. The developing therapeutic approaches to DM1 target mutant RNA or correct early toxic events downstream of the mutant RNA. We have previously described the benefits of the correction of the GSK3ß-CUGBP1 pathway in DM1 mice (HSALR model) expressing 250 CUG repeats using the GSK3 inhibitor tideglusib (TG). Here, we show that TG treatments corrected the expression of ~17% of genes misregulated in DM1 mice, including genes involved in cell transport, development and differentiation. The expression of chloride channel 1 (Clcn1), the key trigger of myotonia in DM1, was also corrected by TG. We found that correction of the GSK3ß-CUGBP1 pathway in mice expressing long CUG repeats (DMSXL model) is beneficial not only at the prenatal and postnatal stages, but also during adulthood. Using a mouse model with dysregulated CUGBP1, which mimics alterations in DM1, we showed that the dysregulated CUGBP1 contributes to the toxicity of expanded CUG repeats by changing gene expression and causing CNS abnormalities. These data show the critical role of the GSK3ß-CUGBP1 pathway in DM1 muscle and in CNS pathologies, suggesting the benefits of GSK3 inhibitors in patients with different forms of DM1.

Acute myotonic reaction during succinylcholine anaesthesia.

A 21-year-old woman developed an acute myotonic reaction while undergoing anaesthesia using succinylcholine. Examination later showed she had shoulder, neck and calf hypertrophy, bilateral symmetrical ptosis and eyelid, handgrip and percussion myotonia. Peripheral neurophysiology studies identified significant, continuous myotonic discharges in both upper and lower limbs. Genetic analysis identified a c.3917G>A (p.Gly1306Glu) mutation in the SCN4A gene, confirming a diagnosis of sodium channel myotonia. Succinylcholine and other depolarising agents can precipitate life-threatening acute myotonic reactions when given to patients with myotonia. Patients with neuromuscular disorders are at an increased risk of perioperative anaesthetic complications. We report a woman who developed an acute myotonic reaction whilst undergoing anaesthesia, in the context of an unrecognised myotonic disorder. We then discuss an approach to the diagnosis of myotonic disorders.

Late sodium current: incomplete inactivation triggers seizures, myotonias, arrhythmias, and pain syndromes.

Acquired and inherited dysfunction in voltage-gated sodium channels underlies a wide range of diseases. In addition to defects in trafficking and expression, sodium channelopathies are caused by dysfunction in one or several gating properties, for instance activation or inactivation. Disruption of channel inactivation leads to increased late sodium current, which is a common defect in seizure disorders, cardiac arrhythmias skeletal muscle myotonia and pain. An increase in late sodium current leads to repetitive action potentials in neurons and skeletal muscles, and prolonged action potential duration in the heart. In this Topical Review, we compare the effects of late sodium current in brain, heart, skeletal muscle and peripheral nerves.

COVID-19 infection and vaccination in patients with skeletal muscle channelopathies.

INTRODUCTION/AIMS: Although we have gained insight into coronavirus disease-2019 (COVID-19) caused by severe acute respiratory syndrome-coronavirus 2 since the beginning of the pandemic, our understanding of the consequences for patients with neuromuscular disorders is evolving. In this study we aimed to study the impact of COVID-19 and COVID-19 vaccination on skeletal muscle channelopathies. METHODS: We conducted a survey of patients with genetically confirmed skeletal muscle channelopathies seen at the UK Nationally Commissioned Channelopathy Service. RESULTS: Thirty-eight patient responses were received. Six patients had COVID-19 infection leading to exacerbation of their underlying muscle channelopathy. No major complications were reported. Thirty-six patients had received one or two COVID-19 vaccinations and the majority (68%) had no worsening of their underlying channelopathy. Thirty-two percent reported worsening of their usual symptoms of their muscle channelopathy, but all reported recovery to baseline levels. No serious adverse events were reported. DISCUSSION: The overall rates of COVID-19 infection were low in our study and COVID-19 vaccine uptake rates were high. Our results have been useful to inform patients that a subset of patients have reversible worsening of their channelopathy post-COVID-19 vaccination. Our study provides information for giving advice to patients with skeletal muscle channelopathies regarding COVID-19 infection and vaccination.

Non-dystrophic myotonia: 2-year clinical and patient reported outcomes.

INTRODUCTION/AIMS: Consistency of differences between non-dystrophic myotonias over time measured by standardized clinical/patient-reported outcomes is lacking. Evaluation of longitudinal data could establish clinically relevant endpoints for future research. METHODS: Data from prospective observational study of 95 definite/clinically suspected non-dystrophic myotonia participants (six sites in the United States, United Kingdom, and Canada) between March 2006 and March 2009 were analyzed. Outcomes included: standardized symptom interview/exam, Short Form-36, Individualized Neuromuscular Quality of Life (INQoL), electrophysiological short/prolonged exercise tests, manual muscle testing, quantitative grip strength, modified get-up-and-go test. Patterns were assigned as described by Fournier et al. Comparisons were restricted to confirmed sodium channelopathies (SCN4A, baseline, year 1, year 2: n = 34, 19, 13), chloride channelopathies (CLCN1, n = 32, 26, 18), and myotonic dystrophy type 2 (DM2, n = 9, 6, 2). RESULTS: Muscle stiffness was the most frequent symptom over time (54.7%-64.7%). Eyelid myotonia and paradoxical handgrip/eyelid myotonia were more frequent in SCN4A. Grip strength and combined manual muscle testing remained stable. Modified get-up-and-go showed less warm up in SCN4A but remained stable. Median post short exercise decrement was stable, except for SCN4A (baseline to year 2 decrement difference 16.6% [Q1, Q3: 9.5, 39.2]). Fournier patterns type 2 (CLCN1) and 1 (SCN4A) were most specific; 40.4% of participants had a change in pattern over time. INQoL showed higher impact for SCN4A and DM2 with scores stable over time. DISCUSSION: Symptom frequency and clinical outcome assessments were stable with defined variability in myotonia measures supporting trial designs like cross over or combined n-of-1 as important for rare disorders.

Coexistence of Charcot-Marie-Tooth 1A and nondystrophic myotonia due to PMP22 duplication and SCN4A pathogenic variants: a case report.

BACKGROUND: Charcot-Marie-Tooth disease (CMT) is a genetically heterogeneous hereditary neuropathy, and CMT1A is the most common form; it is caused by a duplication of the peripheral myelin protein 22 (PMP22) gene. Mutations in the transient sodium channel Nav1.4 alpha subunit (SCN4A) gene underlie a diverse group of dominantly inherited nondystrophic myotonias that run the spectrum from subclinical myopathy to severe muscle stiffness, disabling weakness, or frank episodes of paralysis. CASE PRESENTATION: We describe a Chinese family affected by both CMT1A and myotonia with concomitant alterations in both the PMP22 and SCN4A genes. In this family, the affected proband inherited the disease from his father in an autosomal dominant manner. Genetic analysis confirmed duplication of the PMP22 gene and a missense c.3917G > C (p. Gly1306Ala) mutation in SCN4A in both the proband and his father. The clinical phenotype in the proband showed the combined involvement of skeletal muscle and peripheral nerves. Electromyography showed myopathic changes, including myotonic discharges. MRI revealed the concurrence of neurogenic and myogenic changes in the lower leg muscles. Sural nerve biopsies revealed a chronic demyelinating and remyelinating process with onion bulb formations in the proband. The proband's father presented with confirmed subclinical myopathy, very mild distal atrophy and proximal hypertrophy of the lower leg muscles, pes cavus, and areflexia. CONCLUSION: This study reports the coexistence of PMP22 duplication and SCN4A mutation. The presenting features in this family suggested that both neuropathy and myopathy were inherited in an autosomal dominant manner. The proband had a typical phenotype of sodium channel myotonia (SCM) and CMT1A. However, his father with the same mutations presented a much milder clinical phenotype. Our study might expand the genetic and phenotypic spectra of neuromuscular disorders with concomitant mutations.

Value of short exercise and short exercise with cooling tests in diagnosis of recessive form of myotonia congenita (Becker disease) - are sex differences important?

INTRODUCTION: In myotonia congenita (MC), activation with exercise or cooling can induce transient changes in compound motor action potential (CMAP) parameters, thus providing a guide to genetic analysis. MATERIAL AND METHODS: We performed the short exercise test (SET) and the short exercise test with cooling (SETC) in 30 patients with genetically confirmed Becker disease (BMC) to estimate their utility in the diagnosis of BMC. RESULTS: Although we observed a significant decrease in CMAP amplitude immediately after maximal voluntary effort in both tests in the whole BMC group, in men this decline was significantly smaller than in women, especially in SET. Clinical implications/future directions: In men with a clinical suspicion of BMC, a small decrease in CMAP amplitude in SET together with a typical decline in SETC does not exclude the diagnosis of BMC. Our results show a sex-specific difference in chloride channel function in BMC, which needs further investigation.

Novel ORAI1 Mutation Disrupts Channel Trafficking Resulting in Combined Immunodeficiency.

Store-operated Ca2+ entry (SOCE) represents a predominant Ca2+ influx pathway in non-excitable cells. SOCE is required for immune cell activation and is mediated by the plasma membrane (PM) channel ORAI1 and the endoplasmic reticulum (ER) Ca2+ sensor STIM1. Mutations in the Orai1 or STIM1 genes abolish SOCE leading to combined immunodeficiency (CID), muscular hypotonia, and anhidrotic ectodermal dysplasia. Here, we identify a novel autosomal recessive mutation in ORAI1 in a child with CID. The patient is homozygous for p.C126R mutation in the second transmembrane domain (TM2) of ORAI1, a region with no previous loss-of-function mutations. SOCE is suppressed in the patient's lymphocytes, which is associated with impaired T cell proliferation and cytokine production. Functional analyses demonstrate that the p.C126R mutation does not alter protein expression but disrupts ORAI1 trafficking. Orai1-C126R does not insert properly into the bilayer resulting in ER retention. Insertion of an Arg on the opposite face of TM2 (L135R) also results in defective folding and trafficking. We conclude that positive side chains within ORAI1 TM2 are not tolerated and result in misfolding, defective bilayer insertion, and channel trafficking thus abolishing SOCE and resulting in CID.