Analysis of a rare pathogenic variant of the SCN4A gene (c.4307T>C, L1436P): from clinic to patch-clamp

Analysis of a rare pathogenic variant of the SCN4A gene (c.4307T>C, L1436P): from clinic to patch-clamp | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Analysis of a rare pathogenic variant of the SCN4A gene (c.4307T>C, L1436P): from clinic to patch-clamp François Charles WANG, Olivier BOUQUIAUX, Isabelle LIEVENS, Bernard LAKAYE, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6382511/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract We report nine index cases with a rare mutation in the SCN4A gene, NM_000334.4( SCN4A ).4307T > C (p.Leu1436Pro), which codes for the muscle Na v 1.4 channel. Patients were evaluated clinically and by an electrodiagnostic study. In addition, the biophysical characteristics of the mutant channels were compared to those of wild type channels and a better-known mutant, R1448H, using whole-cell patch clamp recordings of hNa v 1.4 currents in stably transfected HEK293 cells, at near physiological temperature (32°C), room temperature (22°C) and cold temperature (15°C). The phenotypes associated with this SCN4A mutation included one sodium channel myotonia (SCM), six paramyotonia congenita , and one SCM worsened by cold. Regarding the phenotype of hyperkalemic periodic paralysis, three probands described episodes of muscle weakness. Whole-cell recordings showed that the L1436P mutation induced a significant slowing down of fast inactivation of the Na v current at several voltages, but this effect was less marked than in R1448H. The L1436P mutation also tended to induce a right shift in the steady-state inactivation curve, but only at cold temperature. On the other hand, a leftward shift in the activation curve was seen at cold and room temperatures with R1448H, but not L1436P. Recovery from fast inactivation was slowed down in both mutantsat cold temperature. In conclusion, this report confirms that the L1436P mutation of the SCN4A gene leads to different clinical phenotypes. Epigenetic alterations, modifying genes or environmental factors may influence clinical expression. Our experimental data for L1436P reveals a biophysical phenotype consistent with the clinical phenotype of a majority of patients. Neurology Cellular & Molecular Neuroscience Medical Genetics Case report myotonia paramyotonia SCN4A gene whole-cell patch-clamp Figures Figure 1 Figure 2 Figure 3 Introduction Sodium channel myotonia (SCM) and paramyotonia congenita (PMC) are rare non-dystrophic muscle disorders with muscle hyperexcitability caused by gain-of-function mutations in the voltage-gated skeletal muscle sodium channel gene ( SCN4A) (Stunnenberg et al. 2020 ). Clinically, they manifest as myotonia, delayed muscle relaxation after voluntary contraction, resulting in stiffness, pain, fatigue and weakness. SCM is characterized by myotonia at the beginning of muscle activity, which improves with repeated muscle effort (warm-up phenomenon), whereas in PMC, originally described by Eulenburg, myotonia worsens with repeated muscle activity (paradoxical myotonia) and with cold (Eulenburg 1886 ). The precise reason why PMC mutations induce symptoms in these specific conditions is so far unclear. Non-dystrophic myotonias (NDMs) due to SCN4A mutations have autosomal dominant inheritance. About a hundred mutations of the gene are described in the literature (Morales and Pusch ( 2020 ). These mutations are responsible for the PMC or SCM phenotypes. Among them, the variant c.4307T > C (p.Leu1436Pro or L1436P) has rarely been reported in the literature (Matthews et al. 2008 ; Bissay et al. 2011 ). In a recent retrospective epidemiological study on the incidence and prevalence of NDMs in France, the L1436P mutation was found in only eight index cases (out of 1005 patients) and eleven relatives (out of 1624 relatives) (data not yet published, but reported as an abstract at the “23 es Journées Francophones d’ElectroNeuroMyographie” by Sternberg et al. 2024). We retrospectively report the clinical and electrophysiological data of nine apparently unrelated families (nine probands) seen in the Clinical Electrophysiology Department of the University Hospital of Liège (Belgium) between 2005 and 2023, all carrying the c.4307T > C mutation of the SCN4A gene. Furthermore, to clarify the functional and cold-related deficit associated with this mutant, we report results from an in vitro electrophysiological study (whole-cell patch-clamp) conducted on HEK-293 (Human Embryonic Kidney) cells transfected with the L1436P mutant. The properties of this channel were compared to those of the NM_000334.4(SCN4A):c.4343G > A (p.Arg1448His) mutation (R1448H), which is more commonly found in PMC patients (Jurkat-Rott et al. 2010 ; Sasaki et al. 2020 ), as well as those of wild type (WT) channels. Methods Clinical and electrophysiological data (report of cases) The medical history and neurological examination of the nine probands are reported (Cases 1– 9 ). A standard needle-electrode electromyography was performed for each of these patients. The muscles, typically the anterior tibialis , quadriceps, common extensor of the fingers, and deltoid, were studied both at rest and during increasing efforts of voluntary contraction. For seven of them (Cases 1–7), these data were completed by an electrophysiological evaluation according to the recommendations of Fournier (Fig. 1 ) (Fournier et al. 2004 ). The amplitude variations of the CMAP were analyzed both after a long exercise (5 minutes) and after short exercises (10 seconds) repeated three times (at one-minute intervals) at room temperature and after 7 minutes of muscle cooling (Fournier et al. 2006 ). Furthermore, for cases 1–7, repetitive stimulation at 10 Hz for 10 seconds of the ulnar nerve at the wrist, with recording from the abductor digiti minimi muscle, was performed to document a possible decrement characteristic of certain muscle channelopathies (Michel et al. 2007 ). Genetic analysis data The nine probands benefited from a polymerase chain reaction (PCR) amplification and sequencing of the 24 exons and parts of the flanking introns of the SCN4A gene. Whole-cell patch-clamp experiments Experiments were performed on stable Flp-In™ T-Rex™ 293 cell lines co-expressing SCN4A and SCN1B. Plasmid pcDNA3.1 bearing the human SCN4A cDNA ORF tagged at the c-terminus by a FLAG epitope was obtained from Genscript (clone OHu27269). The whole coding sequence (SCN4A + Flag) was subcloned into pcDNA5/FRT/TO (Invitrogen, Waltham, MA). A DNA fragment comprising the T2A self-cleaving peptide and the human SCN1B ORF (coding for the b1 subunit of the channel and provided by Prof. S.C. Cannon, UCLA, United States) was inserted downstream of the SCN4A-FLAG to allow the simultaneous production of the alpha and beta subunits of the Nav1.4 channel. Mutations were introduced in SCN4A sequence by using the Q5 Site-Directed Mutagenesis kit (New England Biolabs, Massachusetts, USA). Sequencing of all constructs was used to confirm the accuracy of the WT channels and of the mutants that were created. For stable cell line generation, the pOG44 plasmid (Invitrogen) was co-transfected with pcDNA5/FRT/TO co-expressing of Nav1.4 a and b1 subunits and stable clones were selected based on their hygromycin resistance as described by the manufacturer. To induce channel expression, Flp-In™ T-Rex™ 293 cell lines were incubated for 24 hours with 1 µg/ml tetracycline before patch-clamp experiments. For the patch clamp experiments, the composition of the extracellular solution, which was superfused at 2 ml/min, was (in mM): 145 NaCl, 4 KCl, 2 CaCl 2 , 1 MgCl 2 , 5 glucose and 10 HEPES, with the pH adjusted to 7.2–7.4 using NaOH. The composition of the internal solution was (in mM): 120 CsF, 10 CsCl, 10 NaCl, 5 EGTA, 5 HEPES, pH adjusted to 7.3 with CsOH. The osmolarity of internal and extracellular solutions was ~ 290 and ~ 305 mOsm/l, respectively. Experiments were run at various temperatures using an Accel 500 LC temperature controller from Thermo Fisher Scientific (Waltham, MA, USA) coupled to a home-made heat exchanger. The accuracy of the temperature was checked at the beginning and at the end of each experiment by measuring the temperature in the dish. It was within 0.5°C of the expected value. We used temperatures of 15 (cold), 22 (room temperature, RT) and 32° C (near physiological). Whole-cell patch-clamp experiments were performed using classical methods. Data was acquired using a Multiclamp 700B amplifier and PClamp 11.1.0.23 software (both from Molecular Devices, Winnersh, England). Low-resistance pipettes (1–3 MΩ) were pulled from filamented borosilicate glass tubing (2.0 mm outer diameter, 0.42 mm wall thickness; Hilgenberg, Malsfeld, Germany) with a P87 puller (Sutter Instruments, Novato, CA, USA). In order to optimize voltage control of fast Na v currents, electrode shanks were coated with wax (H00827, Coltène/Whaledent, Cuyahoga Falls, OH, USA) prior to filling. After break-in, the cell was allowed to stabilize for ≥ 10 min. Access resistances were below 6 MΩ in most cases and varied by less than 20% during the experiments. Quality control was performed by inspecting the traces during the first activation protocols. We selected recordings where a progressive increase in speed of activation and inactivation occurred between − 40 and 0 mV, as expected from the theory. We also excluded cells which had an input resistance below 400 MΩ. Liquid junction potentials and series resistance were not corrected for. First a classical activation protocol was used, starting at -120 mV and depolarizing the membrane during 30 ms in 10 mV increments until + 30 mV. For steady-state inactivation, the membrane was maintained at various voltages between − 120 and 0 mV during a step of 100 ms, immediately followed by a test pulse to 0 mV. This allowed us to measure the fraction of channels available for activation at different voltages (as compared to -120 mV). We also measured recovery from fast inactivation as follows: after an initial 30 ms pulse from − 120 to 0 mV, the membrane was repolarized to -120 mV for various durations, followed by a test pulse to 0 mV. This allowed us to assess the kinetics of recovery from inactivation by dividing the amplitude of the current obtained during each test pulse by the amplitude of the current evoked by the initial pulse Data analysis Data analysis and curve fitting were performed using Python (version 3.12.7) within a Jupyter Notebook environment and Stimfit 0.15 (Guzman et al. 2014 ). Inactivation and activation curves were fitted with classical Boltzmann-type equations, allowing to extract k (the slope factor) and V 50 . For activation, current-voltage plots were converted to conductance-voltage plots using the equation G = I/(V-E Na ), the reversal potential for Na + being + 69 mV at RT in our conditions. In this analysis we considered the values between − 120 and + 30 mV, because values at voltages close to E Na tended to introduce too much variability. Fast inactivation was analyzed by fitting a monoexponential to the data, allowing us to extract the time constant t. Fits were considered acceptable when R was > 0.98. Statistical analysis was performed using SAS software (SAS University Edition, Cary, NC). Descriptive statistics are reported as medians and interquartile ranges (IQR). Given the small and variable sample sizes, only non-parametric tests were used: the Kruskal-Wallis (KW) test for comparisons of more than two groups ; if the KW showed a significant difference between the various groups, pairwise comparisons were performed using the Dwass, Steel, Critchlow-Fligner (DSCF) method ; the effect size for the KW test was measured using eta ² = (H − k + 1)/(n − k), where H = KW test statistic, k = number of groups, and n = total number of observations (≥ 0.14: large effect) ; and the effect size r for DSCF was calculated by dividing the Z statistic (obtained from the Wilcoxon test) by the square root of the total number of observations (≥ 0.50: large effect). A p-value < 0.05 was considered statistically significant. Results Report of cases Genetically, the nine cases exhibited, in the heterozygous state, the missense mutation L1436P in exon 24 of the SCN4A gene, changing a highly conserved leucine to a proline. Case 9 was unique in presenting, in addition to the L1436P mutation, a c.5211dup (p.Tyr1738LeufsTer27) duplication of the SCN4A gene of indeterminate significance. The L1436P mutation was also found in six relatives of four probands (a niece and a daughter of case 6, the mother of cases 3 and 5, and 2 daughters of case 1). Table 1 presents the clinical and electrophysiological data regarding the nine probands with the L1436P mutation. Table 1 clinical and electrophysiological findings of the 9 probants with L1436P variant EMG: electromyography ; CMAP: compound muscle action potential ; NA: not applicable ; ND: not done ; *: pseudo-athletic aspect ; **: calf hypertrophy Case 1 Case 2 Case 3 Case 4 Case 5 Case 6 Case 7 Case 8 Case 9 Gender female female female male female female female female female Age at onset (year) 36 34 36 20 46 60 37 66 50 Clinical myotonia - history suggestive - mechanical myotonia Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No Yes Episodic weakness No Yes No Yes No No No No Yes Warm-up phenomenon Yes No No No No No No No No Exacerbation during exercise No NA Yes Yes No NA Yes Yes Yes Exacerbation after exercise NA Yes Yes No NA NA No No No Cold exacerbation No Yes Yes Yes Yes Yes Yes Yes No Stress exacerbation NA NA NA Yes NA NA NA NA NA Pain - severity - distribution Yes - moderate - arms, hands, feet, thighs Yes - moderate - hands, thighs, calves Yes - mild - diffuse Yes - mild - face, hands No Yes - mild - hands No NA Yes - moderate - lower > upper limbs Muscle bulk Hypertrophy* Normal Normal Hypertrophy** Normal Normal Normal Normal Normal Myotonic discharges on needle EMG Yes Yes Yes Yes Yes Yes Yes Yes Yes Myogenic traces No No No No No No No No Yes CMAP amplitude - after short exercise - idem after cooling - after long exercise - postexercise myotonic potential - No decrease - No decrease - No change - No - No decrease - Decrease - No change - No - No decrease - Decrease - No change - No - No decrease - Decrease - No change - No - No decrease - Decrease - No change - No - No decrease - No decrease - No change - No - No decrease - Decrease - No change - No ND ND 10 Hz decrement No No No No No No No ND ND The sex was predominantly female (8 out of 9 cases). All six related cases were also female. The age of onset of symptoms ranged from 20 to 66 years (average 42.8; standard deviation: 14.2). The nine cases exhibited clinical myotonias: either patients reported episodes of muscle stiffness and rigidity (8 out of 9 cases) during their medical history, or mechanical myotonias were identified upon extensor digitorum muscle percussion during the clinical examination (9 out of 9 cases), see the Supplementary Video . Only cases 2, 4 and 9 reported episodes of muscle weakness. Muscle pain was frequently reported (6 out of 9 cases). The intensity of this pain was either mild (cases 3, 4, 6) or moderate (cases 1, 2, 9 ), but never severe. The distribution of this pain was either diffuse (cases 3, 9 ) or more localized, with pain reported in the hands in four cases (1, 2, 4, 6), thighs in two cases (1, 2), arms and feet in case 1, and calves in case 2. Except for cases 1 and 9 , symptoms were exacerbated by exposure to cold. Only case 1 reported a warm-up phenomenon, a feature of SCM, not PMC. The exacerbation of symptoms was often related to exercise (6 out of 8 cases), either during the repetition of the exercise (cases 3, 4, 7, 8, 9 ), or at rest after exercise (cases 2, 3). Only case 4 spontaneously reported an exacerbation of symptoms related to stress. Muscle trophicity was mostly normal (7 out of 9 cases). Case 1 had a pseudo-athletic appearance, while case 4 presented with calf hypertrophy. Electrophysiologically, the nine cases had very abundant myotonic discharges, characteristic “dive bomber” spontaneous discharges, during the needle-electrode examination of the skeletal musculature. The electromyographic (EMG) recordings did not show any specific abnormalities, except for case 9 where myopathic-like traces were recorded, with EMG traces reduced in amplitude and too early interference pattern during right quadriceps muscle contraction of increasing intensity. The study of the amplitude variation of the compound muscle action potential (CMAP) of the abductor digiti minimi muscle following repetitive nerve stimulation of the ulnar nerve at 10 Hz never revealed a significant decrement at room temperature. For cases 1–7, the CMAP amplitude was never significantly reduced after short exercises performed at room temperature. No significant change in CMAP amplitude was observed after a long exercise. No post-exercise myotonic potentials were recorded. However, after muscle cooling, the CMAP amplitude was significantly reduced after short exercises in cases 2, 3, 4, 5, and 7, with the amplitude reduction becoming more pronounced with each repetition of the short exercise (Fig. 1 ). Biophysical data To evaluate the degree to which the Na v channel function was altered in our patients, we carried out patch clamp experiments in transfected HEK293 cells (see methods). To have relevant comparison points, we studied WT, L1436P and R1448H channels. The reason for choosing the latter is that it is one of the most frequent variants (Sasaki et al. 2020 ) Due to the cold sensitivity of many of the patients’ symptoms, we performed experiments at three temperatures: cold (15°C), room temperature (RT) (22°C) and near physiological (32°C). We first studied the activation of the channels with a classical protocol starting from − 120 mV. Examples of raw traces for WT and each mutation are shown in Fig. 2 a. The data was converted to conductance-voltage curves (see Methods) and the voltages of half maximal activation (V 50 ) were extracted. Two examples of such curves are displayed in Fig. 2 b,c and the numerical data is provided in Table 2 . The L1436P mutant activated at the same voltages as the WT channels, whereas the R1448H mutant activated at significantly more negative voltages in cold and room temperature, but not at 32°C (see Tables 2 and 3 for the statistics). Table 2 Biophysical parameters of the three types of Nav1.4 channels that were studied. 15°C 22°C 32°C WT LP RH WT LP RH WT LP RH V 50 of fast activation (mV) Median -32.13 (8) -30.55 (6) -38.59 (11) -24.35 (6) -24.41 (10) -31.49 (6) -25.36 (6) -25.71 (7) -25.41 (6) IQR 6.15 1.33 6.85 2.88 2.47 1.23 4.12 3.70 2.81 KW (η 2 ) p = 0.0017 (0.49) p = 0.0052 (0.45) p = 0.5714 Time constant of fast inactivation at -10 mV (ms) Median 0.93 (8) 1.76 (5) 4.42 (11) 0.41 (6) 0.69 (9) 1.76 (6) 0.18 (6) 0.33 (7) 0.95 (6) IQR 0.11 0.37 0.46 0.03 0.16 0.46 0.03 0.03 0.11 KW (η 2 ) p < 0.0001 (0.85) p = 0.0002 (0.86) p = 0.0005 (0.83) V 50 of steady-state inactivation (mV) Median -70.17 (7) -59.96 (6) -67.37 (11) -59.82 (5) -54.10 (10) -58.95 (6) -52.11 (6) -51.29 (7) -54.55 (6) IQR 7.36 2.59 3.99 2.96 3.02 3.16 7.93 2.88 7.45 KW (η 2 ) p = 0.0152 (0.30) p = 0.0057 (0.46) p = 0.9806 Time constant of recovery from fast inactivation at -120 mV (ms) Median 3.07 (8) 1.58 (5) 2.19 (10) 0.64 (6) 0.47 (9) 0.66 (6) 0.36 (6) 0.39 (7) 0.33 (6) IQR 0.56 0.44 0.66 0.28 0.24 0.13 0.09 0.23 0.15 KW (η 2 ) p = 0.0005 (0.67) p = 0.1693 p = 0.5461 WT = wild type ; LP = L1436P mutant ; RH = R1448H mutant. Values are represented as median and interquartile range (IQR). The number of experiments is indicated in brackets. Statistically significant differences between WT and mutant channels (L1436P and R1448H) were determined using a Kruskal-Wallis (KW) analysis. Effect size for the KW test is measured by eta 2 (η 2 ) = (H − k + 1)/(n − k) with H = KW test statistic, k = number of groups, n = total number of observations (≥ 0.14: Large effect). Table 3 Pairwise comparisons by the Dwass, Steel, Critchlow-Fligner method. 15°C 22°C 32°C V 50 of fast activation (mV) WT versus LP p = 0.9207 p = 0.9935 p = 0.9972 WT versus RH p = 0.0083 (0.67) p = 0.0281 (0.72) p = 0.7026 LP versus RH p = 0.0100 (0.69) p = 0.0068 (0.75) p = 0.5766 Time constant of fast inactivation at -10 mV (ms) WT versus LP p = 0.0096 (0.79) p = 0.0042 (0.81) p = 0.0182 (0.73) WT versus RH p = 0.0008 (0.82) p = 0.0110 (0.81) p = 0.0110 (0.81) LP versus RH p = 0.0052 (0.76) p = 0.0042 (0.76) p = 0.0076 (0.81) V 50 of steady-state inactivation (mV) WT versus LP p = 0.1122 p = 0.0604 p = 0.9888 WT versus RH p = 0.7216 p = 1.000 p = 0.8806 LP versus RH p = 0.0100 (0.69) p = 0.0095 (0.72) p = 0.9037 Time constant of recovery from fast inactivation at -120 mV (ms) WT versus LP p = 0.0096 (0.79) p = 0.4660 p = 0.7550 WT versus RH p = 0.0053 (0.72) p = 0.9860 p = 0.9860 LP versus RH p = 0.0521 p = 0.1428 p = 0.4877 WT = wild type ; LP = L1436P mutant ; RH = R1448H mutant. Effect size r (in brackets) is calculated by dividing the Z statistic (obtained from the Wilcoxon test) by the square root of the total number of observations (≥ 0.50: Large effect). We next turned our attention to the fast inactivation of the channels following their activation. The R1448H mutant is known to have a much slower inactivation than WT channels and this feature is considered as the main contributing factor to the induction of myotonic runs (Chahine et al. 1994 ; Richmond et al. 1997 ). We confirmed this observation, as shown in Fig. 2 a and 3a,b. Indeed, values of the time constant of inactivation were much larger in R1448H than in WT (Table 3 and Fig. 3a,b). For example, at -10 mV, the median value was 0.95 ms in R1448H and 0.18 ms in WT at 32°C and remained much larger in R1448H throughout temperatures. The L1436P mutant’s behavior was intermediate in this respect, with a value of 0.33 ms at the same voltage at 32°C. The time constant became significantly higher at lower temperatures in all genotypes (Fig. 3a,b and Table 2 ). At 22°C, which corresponds to moderate cold, values were 0.41, 0.69 and 1.76 ms for WT, L1436P and R1448H, respectively. Overall, the L1436P mutant inactivated significantly slowlier than the WT. However, its effect was also significantly smaller than the one of R1448H across all voltages and temperatures (see Fig. 3b and Table 3 for pairwise comparisons). One other important aspect of Na v function is the recovery from inactivation upon repolarization of the membrane. This parameter was also evaluated and showed a faster recovery in the L1436P and R1448H mutants as compared to the WT channels, but only at cold temperature (see Tables 2 and 3 ). Steady-state inactivation, which reflects the availability of the channels at various voltages, was studied next. For this purpose, the membrane was clamped for 100 ms at various voltages before giving a pulse to 0 mV. The amount of current measured at this voltage reflects the percentage of channels that are not inactivated. The fitting of the curves allowed us to extract the V 50 of steady-state inactivation (see Methods). The L1436P mutant significantly differed from the R1448H mutant in terms of steady-state inactivation (Fig. 3c, Table 3 ), but not from the WT channels, although a trend was noted (p = 0.06 at 22°C). Thus, the V 50 was more depolarized in the L1436P mutant than in the R1448H mutant, both at RT and cold temperatures (by 4–7 mV), but not at near physiological temperature (Fig. 3d). The consequence of this is that a higher percentage of L1436P channels are available in the region of the resting membrane potential of myocytes ( ~ − 85 mV) than is the case for the R1448H mutant. Discussion The L1436P mutation was first mentioned in 2008 by a London team (Matthews et al. 2008 ). The case reported by these authors presented a typical clinical phenotype of paramyotonia with myotonias exacerbated by exercise and exposure to cold, as well as muscle pain. The second time the L1436P mutation was mentioned in the literature was by Belgian neurologists (Bissay et al. 2011 ), who described this particular variant in 3 unrelated families (3 probands and 8 relatives) from the Brussels region. The clinical phenotype of these patients supported a diagnosis of SCM. Indeed, they met the main criteria for SCM, specifically exercise-induced delayed-onset myotonia (8 out of 11 cases) and a warm-up phenomenon (symptoms relieved by repetitive muscle contraction) without weakness (10 out of 11 cases). Additionally, clinical electrophysiological data were more indicative of SCM than of PMC, as short exercises performed at room temperature and after muscle cooling did not significantly reduce the CMAP amplitude (7 out of 7 cases). Indeed, their electrophysiological data corresponded with Pattern III of the Fournier classification, both at room temperature and after muscle cooling (Fournier et al. 2004 , 2006 ). However, surprisingly, the authors noted that the phenotype of their families differed from classic SCM in that the myotonia did worsen with cold exposure and the frequency and severity of muscle pain. For these reasons, the authors proposed adding a fourth category to the fluctuans , permanent and acetazolamide-responsive myotonias, which they named cold-aggravated myotonias. Therefore, in the original article concerning the L1436P mutant, the described phenotype was PMC (Matthews et al. 2008 ), and in the second article (Bissay et al. 2011 ), the reported phenotype in the three families was SCM that worsened with cold. Additionally, the National Center for Biotechnology Information (NCBI) classifies the L1436P variant as hyperkalemic periodic paralysis. The cases described in our work confirm the heterogeneity of the phenotype linked to the L1436P mutation. Indeed, among the seven probands for whom clinical and electrophysiological data were available, the phenotype was either SCM (case 1 with warm-up phenomenon, no exacerbation by cold, and no electrophysiological pattern suggestive of paramyotonia ), or PMC (cases 2, 3, 4, 5, 7 with absence of warm-up phenomenon, exacerbation by cold, and a typical electrophysiological pattern of paramyotonia ). Case 6 could be related to SCM aggravated by cold (exacerbation by cold without a typical electrophysiological pattern of paramyotonia ), but the patient showed no changes in symptoms related to exercise, neither warm-up phenomenon nor exacerbation during exercise. Case 8 was clinically suggestive of PMC (with absence of the warm-up phenomenon and exacerbation by cold and during exercise), but the Fournier protocol was not performed. Regarding the phenotype of hyperkalemic periodic paralysis, three probands described episodes of muscle weakness (cases 2, 4, 9 ), while the long exercise test, performed for cases 1–7, was negative due to the absence of significant and characteristic changes in CMAP amplitude, either the immediate increase after exercise or the late reduction after exercise (Fig. 1 ) (Fournier et al. 2004 ). The clinical data confirm that the L1436P mutation is frequently associated with muscle pain, which often constituted the reason for consultation (cases 1, 2, 3, 4, 6, 9 ) and sometimes led to an initial misdiagnosis of fibromyalgia. Muscle hypertrophy was mostly absent. Nevertheless, case 1 with an SCM phenotype presented with a pseudo-athletic appearance, while case 4 ( paramyotonia phenotype and the only male in our series) had calf hypertrophy. Thus, we confirm that the L1436P mutation of the SCN4A gene can cause different phenotypes. So far unknown epigenetic alterations, modifying genes and/or environmental factors may influence the clinical expression within and between families with the same SCN4A mutation. The male-to-female ratio found in this work (only one male among 9 probands and 6 relatives) suggests that hormonal climate could play a role in the phenotypic expression of this genetic disease. This gender-related peculiarity was not reported by Bissay whose cases included 4 women and 7 men (Bissay et al. 2011 ). Case 9 was somewhat unusual in that the medical history was not suggestive of myotonia. It was the clinical examination and EMG that indicated a myotonic syndrome by demonstrating clinical myotonia and typical myotonic bursts. The patient complained of muscle pain and weakness unrelated to cold exposure. The EMG recorded myogenic-like features in the right quadriceps muscle. This somewhat unusual presentation may be related to the presence of a second variant of uncertain significance, a c.5211dup (p.Tyr1738LeufsTer27) duplication of the SCN4A gene. The 26 reported cases of the L1436P mutation of the SCN4A gene in Belgium—specifically, three probands and eight relatives in the Brussels region (Bissay et al. 2011 ), and nine probands and six relatives in the Liège region—contrast with the limited data available in the literature and with the 17 cases (8 probands and 11 relatives) found in a large epidemiological study by Sternberg (not yet published) on the incidence and prevalence of NDMs in France (a country six times more populous than Belgium). It is therefore possible that there is a founder effect in Belgium related to this mutant. Our patch clamp results can be summarized as follows: we show that the L1436P mutant induces several abnormalities, including a significant, yet modest slowing down of fast inactivation across several voltages and a mildly depolarized steady-state inactivation curve, the latter meaning a slightly higher availability of channels. On the other hand, voltage-dependence of activation of the channels remains similar to the WT. Overall, its biophysical phenotype seems less severe than the one of the R1448H mutant. In addition, a striking feature of both mutants is that most abnormalities were observed at relatively cold or very cold temperatures (except for the slowing of the fast inactivation). In this regard, our results emphasize the usefulness of recording currents at various temperatures. Do these biophysical abnormalities explain the myotonic phenotype of the L1436P patients? This is hard to say. First, we have not yet tested all biophysical parameters. For example, some Na v mutants exhibit defects in slow inactivation (Webb et al. 2008 ; Carle et al. 2009 ), i.e. an inactivation process that is different from the one that occurs immediately after activation and proceeds over a time scale of seconds or minutes (Hayward et al. 1997 ). It may be that L1436P differs from WT channels in this respect. In addition, there is a clear need to incorporate these different abnormalities in a realistic myocyte model to be able to make predictions. This will allow us to check whether the modest increases in fast inactivation time constants are sufficient to explain the electromyographic phenotype. We are currently developing a new conductance-based computer model of a myocyte based on the one by Cannon (Cannon et al. 1993 ) with improved features such as incorporation of recent data on ClC-1 and K ir 2.1 biophysics, as well as the possibility to include two types of Na v currents, a normal one and a pathological one (thereby mimicking the heterozygosity of the patients). This will hopefully help us to answer this question. One interesting point is that both clinical and biophysical evaluation of the L1436P mutant shows that it manifests itself predominantly at cold temperatures with overall less alterations at physiological temperature. Conclusion In conclusion, we have described the clinical and biophysical characteristics of a rare Na v 1.4 mutant, L1436P. The patients bearing this mutation show a rather variable phenotype. Our patch clamp analysis shows biophysical alterations that worsen at infraphysiological temperatures. This is consistent with what was observed clinically in a majority of, but not all our patients. Abbreviations ANOVA: Analysis of Variance CMAP: Compound Muscle Action Potential DSCF: Dwass, Steel, Critchlow-Finger method HEK: Human Embryonic Kidney KW: Kruskal-Wallis L1436P: NM_000334.4(SCN4A):c.4307T>C (p.Leu1436Pro) NDM: Non-Dystrophic Myotonia PMC: Paramyotonia Congenita R1448H: NM_000334.4(SCN4A):c.4343G>A (p.Arg1448His) SCM: Sodium Channel Myotonia SCN4A : Sodium Voltage-Gated Channel Alpha Subunit 4 WT: Wild Type Declarations Funding A patch-clamp setup (including a Zeiss microscope) was partially purchased thanks to a scientific research investment fund (2022): Fonds d’Investissements de Recherche Scientifique (FIRS) from the University Hospital of Liège (Belgium) to FCW. BL is research associate at the FRS-FNRS. NI was supported by a grant from the University of Liège and more recently a grant from the FRIA (FNRS, Belgium). Study sponsors played no role in data collection, analysis, interpretation and in drafting of the manuscript. Competing Interests The authors have no relevant financial or non-financial interests to disclose Author contributions All authors contributed to the study conception and design. The patients in this study were clinically managed by François Charles WANG, Olivier BOUQUIAUX, Isabelle LIEVENS, and Margaux POLEUR. The experimental data were recorded by François Charles WANG, Vincent SEUTIN, Nurcan INCI, and Samira ABDOU IDE. Bernard LAKAYE helped with the creation of the mutations and stable lines. The first draft of the manuscript was written by François Charles WANG and Vincent SEUTIN. Nurcan INCI analyzed the experimental data and created the figures. All authors read and approved the final manuscript. Data Availability François Charles WANG takes full responsibility for the data, the analysis and interpretation and the conduct of the research. He has full access to all of the data and has the right to publish any and all data separate and apart from any sponsor. Data reported in this paper is fully accessible to interested readers from François Charles WANG. Ethics approval This research study was conducted retrospectively from data obtained for clinical purposes. We consulted extensively with the Ethics Committee of the University of Liège and of the University Hospital of Liège (“Comité d’éthique hospitalo-facultaire Universitaire de Liège”) who determined that our study did not need ethical approval. An official waiver of ethical approval was granted by the Ethics Committee. Consent to participate Not applicable Consent to publish Not applicable References Bissay V, Keymolen K, Lissens W, Laureys G, Schmedding E, De Keyser J (2011) Late onset painful cold-aggravated myotonia: three families with SCN4A L1436P mutation. Neuromuscul Disord 21(8):590–593. 10.1016/j.nmd.2011.05.006 Cannon SC, Brown RH Jr, Corey DP (1993) Theoretical reconstruction of myotonia and paralysis caused by incomplete inactivation of sodium channels. Biophys J 65(1):270–288. 10.1016/S0006-3495(93)81045-2 Carle T, Fournier E, Sternberg D, Fontaine B, Tabti N (2009) Cold-induced disruption of Na + channel slow inactivation underlies paralysis in highly thermosensitive paramyotonia. J Physiol 587(Pt 8):1705–1714. 10.1113/jphysiol.2008.165787 Chahine M, George AL, Zhou M, Ji S, Sun W, Barchi RL, Horn R (1994) Sodium channel mutations in paramyotonia congenita uncouple inactivation from activation. Neuron 12(2):281–294. 10.1016/0896-6273(94)90271-2 Eulenburg A (1886) Über eine familiäre, durch sechs Generationen verfolgbare congenitaler Paramyotonia. Neurol Centralblb 12:265–272 Fournier E, Arzel M, Sternberg D, Vicart S, Laforet P, Eymard B, Willer JC, Tabti N, Fontaine B (2004) Electromyography guides toward subgroups of mutations in muscle channelopathies. Ann Neurol 56(5):650–661. 10.1002/ana.20241 Fournier E, Viala K, Gervais H, Sternberg D, Arzel-Hézode M, Laforêt P, Eymard B, Tabti N, Willer JC, Vial C, Fontaine B (2006) Cold extends electromyography distinction between ion channel mutations causing myotonia. Ann Neurol 60(3):356–365. 10.1002/ana.20905 Guzman SJ, Schlögl A, Schmidt-Hieber C (2014) Stimfit: quantifying electrophysiological data with Python. Front Neuroinform 8:16. 10.3389/fninf.2014.00016 Hayward LJ, Brown RH Jr, Cannon SC (1997) Slow inactivation differs among mutant Na channels associated with myotonia and periodic paralysis. Biophys J 72(3):1204–1219. 10.1016/S0006-3495(97)78768-X Jurkat-Rott K, Holzherr B, Fauler M, Lehmann-Horn F (2010) Sodium channelopathies of skeletal muscle result from gain or loss of function. Pflugers Arch - Eur J Physiol 460:239–248. https://doi.org/10.1007/s00424-010-0814-4 Matthews E, Tan SV, Fialho D, Sweeney MG, Sud R, Haworth A, Stanley E, Cea G, Davis MB, Hanna MG (2008) What causes paramyotonia in the United Kingdom? Common and new SCN4A mutations revealed. Neurology 70(1):50–53. 10.1212/01.wnl.0000287069.21162.94 Michel P, Sternberg D, Jeannet PY, Dunand M, Thonney F, Kress W, Fontaine B, Fournier E, Kuntzer T (2007) Comparative efficacy of repetitive nerve stimulation, exercise, and cold in differentiating myotonic disorders. Muscle Nerve 36(5):643–650. 10.1002/mus.20856 Morales F, Pusch M (2020) An Up-to-Date Overview of the Complexity of Genotype-Phenotype Relationships in Myotonic Channelopathies. Front Neurol 10:1404. 10.3389/fneur.2019.01404 Richmond JE, Featherstone DE, Ruben PC (1997) Human Na + channel fast and slow inactivation in paramyotonia congenita mutants expressed in Xenopus laevis oocytes. J Physiol 499(Pt 3):589–600. 10.1113/jphysiol.1997.sp021952 Sasaki Y, Nakaza M, Furuta M, Fujino H, Kubota T, Takahashi MP (2020) Mutation spectrum and health status in skeletal muscle channelopathies in Japan. Neuromuscul Disord 30(7):546–553. 10.1016/j.nmd.2020.06.001 Stunnenberg BC, LoRusso S, Arnold WD, Barohn RJ, Cannon SC, Fontaine B, Griggs RC, Hanna MG, Matthews E, Meola G, Sansone VA, Trivedi JR, van Engelen BGM, Vicart S, Statland JM (2020) Guidelines on clinical presentation and management of nondystrophic myotonias. Muscle Nerve 62(4):430–444. 10.1002/mus.26887 Webb J, Cannon SC (2008) Cold-induced defects of sodium channel gating in atypical periodic paralysis plus myotonia. Neurology 70(10):755–761. 10.1212/01.wnl.0000265397.70057.d8 Additional Declarations The authors declare no competing interests. Supplementary Files MYOTONIECLINIQUE.mp4 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6382511","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":438839306,"identity":"0d438708-fe78-442c-a4d4-ac88d7bd59e2","order_by":0,"name":"François Charles WANG","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5klEQVRIiWNgGAWjYBAC9gYILccgwcB4IAHElCCghecAhDYGqSRNS2IDSAsDUVrYD7BJ/NxRm94/u8fgwIOKWjkG6eYD+LXwJLBJ9p45njvjzhmDAwlnjhszyBxLwKvFXoKB7QZv27HcDRI5BgcS244BXZhjgN8WoJabf9uOpRuAtfw7Vt8gkf+BoJbbvG01CRAtDTUJDBI5eHUA/ZLY/lu27YDhjBtpBQcSjh0wbJM5RsBh7IcPG75tq5Pnn5G88eGPGiBDuvkBfmsYGBuAxGEY7zADGwH1MFCHwRgFo2AUjIJRAAcAUOFKKe5XDFIAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-7283-8687","institution":"University of Liège","correspondingAuthor":true,"prefix":"","firstName":"François","middleName":"Charles","lastName":"WANG","suffix":""},{"id":438839307,"identity":"8942a571-f879-4fb0-b0fc-75b35fb4b662","order_by":1,"name":"Olivier BOUQUIAUX","email":"","orcid":"","institution":"University of Liège","correspondingAuthor":false,"prefix":"","firstName":"Olivier","middleName":"","lastName":"BOUQUIAUX","suffix":""},{"id":438839308,"identity":"cc356129-1aa4-44fa-a728-187e70b1fd38","order_by":2,"name":"Isabelle LIEVENS","email":"","orcid":"","institution":"University of Liège","correspondingAuthor":false,"prefix":"","firstName":"Isabelle","middleName":"","lastName":"LIEVENS","suffix":""},{"id":438839309,"identity":"a845f439-35be-41ba-9c09-954b8278384a","order_by":3,"name":"Bernard LAKAYE","email":"","orcid":"","institution":"University of Liège","correspondingAuthor":false,"prefix":"","firstName":"Bernard","middleName":"","lastName":"LAKAYE","suffix":""},{"id":438839310,"identity":"95a4d7c3-35ff-48c8-bae4-c5fad12b0ccb","order_by":4,"name":"Margaux POLEUR","email":"","orcid":"","institution":"University of Liège","correspondingAuthor":false,"prefix":"","firstName":"Margaux","middleName":"","lastName":"POLEUR","suffix":""},{"id":438839311,"identity":"8d759a4e-7e1d-4619-9a25-b3135a930d97","order_by":5,"name":"Samira ABDOU IDE","email":"","orcid":"","institution":"University of Liège","correspondingAuthor":false,"prefix":"","firstName":"Samira","middleName":"ABDOU","lastName":"IDE","suffix":""},{"id":438839312,"identity":"92a4e707-5d4f-4e51-a3da-91be00ecc702","order_by":6,"name":"Nurcan INCI","email":"","orcid":"","institution":"University of Liège","correspondingAuthor":false,"prefix":"","firstName":"Nurcan","middleName":"","lastName":"INCI","suffix":""},{"id":438839313,"identity":"bd9914a7-ed38-4aa2-9dee-9a491a98ca15","order_by":7,"name":"Vincent SEUTIN","email":"","orcid":"https://orcid.org/0000-0002-8046-8142","institution":"University of Liège","correspondingAuthor":false,"prefix":"","firstName":"Vincent","middleName":"","lastName":"SEUTIN","suffix":""}],"badges":[],"createdAt":"2025-04-05 14:11:19","currentVersionCode":1,"declarations":{"humanSubjects":true,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":true,"humanSubjectConsent":true,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":true,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-6382511/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6382511/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":80144920,"identity":"62159104-1b49-4d2f-886e-3b3c1ad1bb1c","added_by":"auto","created_at":"2025-04-08 12:20:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":134598,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCompound muscle action potential (CMAP) amplitude related to time after short and long exercises (Case 3)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe CMAP of the \u003cem\u003eabductor digiti minimi\u003c/em\u003emuscle is recorded following stimulation of the ulnar nerve at the wrist. A) The short exercises consist of a maximal voluntary muscle contraction against resistance for 10 seconds (represented by the two black vertical lines), repeated three times at one-minute intervals. Prior to this, the supramaximal intensity of nerve stimulation is determined by setting it at 150% of the intensity at which the CMAP amplitude no longer increases. The first 10-second exercise is performed, and the CMAP amplitude is measured immediately after this effort and then four more times every 10 seconds. The CMAP amplitude is normalized (%) relative to the maximal CMAP amplitude established before the exercises. The same sequence is repeated twice. B) The same test is repeated on the same muscle after 7 minutes of muscle cooling (cold pack). C) The long exercise is performed on the other \u003cem\u003eabductor digiti minimi\u003c/em\u003e muscle to avoid interference from the short exercises and muscle cooling. This exercise consists of a maximal voluntary muscle contraction against resistance for 25 seconds, followed by 5 seconds of rest, repeated 10 times (represented by the two black vertical lines). The CMAP amplitude is measured immediately after this effort, then after 1 minute, 4 minutes later, and then every 5 minutes for at least 35 minutes. The CMAP amplitude is normalized (%) relative to the maximal CMAP amplitude established before the long exercise.\u003c/p\u003e\n\u003cp\u003eIn this example, during the exercises performed at room temperature (A), the CMAP amplitude slightly increases immediately after each short exercise and then returns to its baseline value, which is most often observed in healthy controls. This slight increase in amplitude reflects post-exercise potentiation due to the excess acetylcholine in the synaptic clefts. After muscle cooling (B), the CMAP amplitude collapses, and this effect becomes more pronounced as the short exercise is repeated, to the point that after the third short exercise, the CMAP amplitude can no longer be reliably measured. During the long exercise (C), the changes in CMAP amplitude are those typically observed in a healthy controls, namely a slight increase in amplitude immediately after the long effort and a moderate and progressive reduction afterward, not exceeding 40% compared to the motor response elicited immediately after the long effort.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6382511/v1/98750a47e18f7d2c61a0e8a5.png"},{"id":80144922,"identity":"b93e8ccc-d653-437d-bbc3-55aa46a24412","added_by":"auto","created_at":"2025-04-08 12:20:46","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":459486,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eActivation and fast inactivation of L1436P, R1448H and WT channels\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea) Raw traces of Na\u003csub\u003ev\u003c/sub\u003e currents that were recorded at 22°C in HEK293T cells expressing WT or mutant hNa\u003csub\u003ev\u003c/sub\u003e1.4 channels. One representative cell is shown for each subtype. Ten mV steps from -120 to 30 mV evoked a rapidly inactivating current with slower inactivation kinetics in the mutants (especially R1448H). Traces have been leak-subtracted (see Methods)\u003c/p\u003e\n\u003cp\u003eb) and c) Normalized conductance (G/Gmax) versus voltage curves obtained for L1436P (n = 10); R1448H (n = 6), and WT channels (n = 6) using the equation G = I/(V-V\u003csub\u003erev\u003c/sub\u003e), with V\u003csub\u003erev\u003c/sub\u003e being + 69 mV under our conditions). R1448H channels activated at more negative potentials than the other species at 22°C, but not at 32°C\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6382511/v1/a43f95a98dcae4d2137a878e.jpeg"},{"id":80144932,"identity":"5d871e70-d6de-40e2-bc56-5dfc33bc6e99","added_by":"auto","created_at":"2025-04-08 12:20:46","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":711964,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of temperature on fast inactivation of the various channels and steady-state inactivation\u003cbr\u003e\n \u003c/strong\u003ea) Representative raw traces recorded at −10 mV of WT, L1436P, and R1448H currents at 15 °C, 22 °C and 32 °C. Note the marked slowing down of fast inactivation (and of activation) in the three channel species when temperature was lower\u003c/p\u003e\n\u003cp\u003eb) Summary of voltage- and temperature-dependence of inactivation time constants. The decay of the current at depolarized voltages was best described by a single exponential fit, with the resulting time constants (τ) plotted as a function of voltage. Note the differences in the vertical scales at the three temperatures\u003c/p\u003e\n\u003cp\u003ec) and d) The steady-state inactivation curve of the L1436P mutant was slightly more depolarized than the one of the other species at 22°C, but not at 32°C\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6382511/v1/a55da002ee3e90e19b1bb958.jpeg"},{"id":80146071,"identity":"a0de188a-42dc-4bfd-825c-a62d9dc5d8ef","added_by":"auto","created_at":"2025-04-08 12:36:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2302784,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6382511/v1/af946a73-d950-4d9a-b274-428e479a7bc3.pdf"},{"id":80144925,"identity":"9ebbb540-39c6-4a76-9dc5-88495ff1bdf8","added_by":"auto","created_at":"2025-04-08 12:20:46","extension":"mp4","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1078229,"visible":true,"origin":"","legend":"","description":"","filename":"MYOTONIECLINIQUE.mp4","url":"https://assets-eu.researchsquare.com/files/rs-6382511/v1/add3089630660de04dca9358.mp4"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eAnalysis of a rare pathogenic variant of the \u003cem\u003eSCN4A\u003c/em\u003e gene (c.4307T\u0026gt;C, L1436P): from clinic to patch-clamp\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSodium channel myotonia (SCM) and \u003cem\u003eparamyotonia congenita\u003c/em\u003e (PMC) are rare non-dystrophic muscle disorders with muscle hyperexcitability caused by gain-of-function mutations in the voltage-gated skeletal muscle sodium channel gene (\u003cem\u003eSCN4A)\u003c/em\u003e (Stunnenberg et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Clinically, they manifest as myotonia, delayed muscle relaxation after voluntary contraction, resulting in stiffness, pain, fatigue and weakness. SCM is characterized by myotonia at the beginning of muscle activity, which improves with repeated muscle effort (warm-up phenomenon), whereas in PMC, originally described by Eulenburg, myotonia worsens with repeated muscle activity (paradoxical myotonia) and with cold (Eulenburg \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1886\u003c/span\u003e). The precise reason why PMC mutations induce symptoms in these specific conditions is so far unclear.\u003c/p\u003e \u003cp\u003eNon-dystrophic myotonias (NDMs) due to \u003cem\u003eSCN4A\u003c/em\u003e mutations have autosomal dominant inheritance. About a hundred mutations of the gene are described in the literature (Morales and Pusch (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). These mutations are responsible for the PMC or SCM phenotypes. Among them, the variant c.4307T \u0026gt; C (p.Leu1436Pro or L1436P) has rarely been reported in the literature (Matthews et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2008\u003c/span\u003e ; Bissay et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In a recent retrospective epidemiological study on the incidence and prevalence of NDMs in France, the L1436P mutation was found in only eight index cases (out of 1005 patients) and eleven relatives (out of 1624 relatives) (data not yet published, but reported as an abstract at the “23\u003csup\u003ees\u003c/sup\u003e Journées Francophones d’ElectroNeuroMyographie” by Sternberg et al. 2024).\u003c/p\u003e \u003cp\u003eWe retrospectively report the clinical and electrophysiological data of nine apparently unrelated families (nine probands) seen in the Clinical Electrophysiology Department of the University Hospital of Liège (Belgium) between 2005 and 2023, all carrying the c.4307T \u0026gt; C mutation of the \u003cem\u003eSCN4A\u003c/em\u003e gene. Furthermore, to clarify the functional and cold-related deficit associated with this mutant, we report results from an \u003cem\u003ein vitro\u003c/em\u003e electrophysiological study (whole-cell patch-clamp) conducted on HEK-293 (Human Embryonic Kidney) cells transfected with the L1436P mutant. The properties of this channel were compared to those of the NM_000334.4(SCN4A):c.4343G \u0026gt; A (p.Arg1448His) mutation (R1448H), which is more commonly found in PMC patients (Jurkat-Rott et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2010\u003c/span\u003e ; Sasaki et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), as well as those of wild type (WT) channels.\u003c/p\u003e "},{"header":"Methods","content":"\u003cp\u003eClinical and electrophysiological data (report of cases)\u003c/p\u003e\u003cp\u003eThe medical history and neurological examination of the nine probands are reported (Cases 1–\u003cspan refid=\"FPar1\" class=\"InternalRef\"\u003e9\u003c/span\u003e). A standard needle-electrode electromyography was performed for each of these patients. The muscles, typically the \u003cem\u003eanterior tibialis\u003c/em\u003e, quadriceps, common extensor of the fingers, and deltoid, were studied both at rest and during increasing efforts of voluntary contraction. For seven of them (Cases 1–7), these data were completed by an electrophysiological evaluation according to the recommendations of Fournier (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) (Fournier et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). The amplitude variations of the CMAP were analyzed both after a long exercise (5 minutes) and after short exercises (10 seconds) repeated three times (at one-minute intervals) at room temperature and after 7 minutes of muscle cooling (Fournier et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Furthermore, for cases 1–7, repetitive stimulation at 10 Hz for 10 seconds of the ulnar nerve at the wrist, with recording from the \u003cem\u003eabductor digiti minimi\u003c/em\u003e muscle, was performed to document a possible decrement characteristic of certain muscle channelopathies (Michel et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eGenetic analysis data\u003c/p\u003e\u003cp\u003eThe nine probands benefited from a polymerase chain reaction (PCR) amplification and sequencing of the 24 exons and parts of the flanking introns of the \u003cem\u003eSCN4A\u003c/em\u003e gene.\u003c/p\u003e\u003cp\u003eWhole-cell patch-clamp experiments\u003c/p\u003e\u003cp\u003eExperiments were performed on stable Flp-In™ T-Rex™ 293 cell lines co-expressing SCN4A and SCN1B. Plasmid pcDNA3.1 bearing the human SCN4A cDNA ORF tagged at the c-terminus by a FLAG epitope was obtained from Genscript (clone OHu27269). The whole coding sequence (SCN4A + Flag) was subcloned into pcDNA5/FRT/TO (Invitrogen, Waltham, MA). A DNA fragment comprising the T2A self-cleaving peptide and the human SCN1B ORF (coding for the b1 subunit of the channel and provided by Prof. S.C. Cannon, UCLA, United States) was inserted downstream of the SCN4A-FLAG to allow the simultaneous production of the alpha and beta subunits of the Nav1.4 channel. Mutations were introduced in SCN4A sequence by using the Q5 Site-Directed Mutagenesis kit (New England Biolabs, Massachusetts, USA). Sequencing of all constructs was used to confirm the accuracy of the WT channels and of the mutants that were created. For stable cell line generation, the pOG44 plasmid (Invitrogen) was co-transfected with pcDNA5/FRT/TO co-expressing of Nav1.4 a and b1 subunits and stable clones were selected based on their hygromycin resistance as described by the manufacturer. To induce channel expression, Flp-In™ T-Rex™ 293 cell lines were incubated for 24 hours with 1 µg/ml tetracycline before patch-clamp experiments. For the patch clamp experiments, the composition of the extracellular solution, which was superfused at 2 ml/min, was (in mM): 145 NaCl, 4 KCl, 2 CaCl\u003csub\u003e2\u003c/sub\u003e, 1 MgCl\u003csub\u003e2\u003c/sub\u003e, 5 glucose and 10 HEPES, with the pH adjusted to 7.2–7.4 using NaOH. The composition of the internal solution was (in mM): 120 CsF, 10 CsCl, 10 NaCl, 5 EGTA, 5 HEPES, pH adjusted to 7.3 with CsOH. The osmolarity of internal and extracellular solutions was ~ 290 and ~ 305 mOsm/l, respectively.\u003c/p\u003e\u003cp\u003eExperiments were run at various temperatures using an Accel 500 LC temperature controller from Thermo Fisher Scientific (Waltham, MA, USA) coupled to a home-made heat exchanger. The accuracy of the temperature was checked at the beginning and at the end of each experiment by measuring the temperature in the dish. It was within 0.5°C of the expected value. We used temperatures of 15 (cold), 22 (room temperature, RT) and 32° C (near physiological).\u003c/p\u003e\u003cp\u003eWhole-cell patch-clamp experiments were performed using classical methods. Data was acquired using a Multiclamp 700B amplifier and PClamp 11.1.0.23 software (both from Molecular Devices, Winnersh, England). Low-resistance pipettes (1–3 MΩ) were pulled from filamented borosilicate glass tubing (2.0 mm outer diameter, 0.42 mm wall thickness; Hilgenberg, Malsfeld, Germany) with a P87 puller (Sutter Instruments, Novato, CA, USA). In order to optimize voltage control of fast Na\u003csub\u003ev\u003c/sub\u003e currents, electrode shanks were coated with wax (H00827, Coltène/Whaledent, Cuyahoga Falls, OH, USA) prior to filling. After break-in, the cell was allowed to stabilize for ≥ 10 min. Access resistances were below 6 MΩ in most cases and varied by less than 20% during the experiments. Quality control was performed by inspecting the traces during the first activation protocols. We selected recordings where a progressive increase in speed of activation and inactivation occurred between − 40 and 0 mV, as expected from the theory. We also excluded cells which had an input resistance below 400 MΩ. Liquid junction potentials and series resistance were not corrected for. First a classical activation protocol was used, starting at -120 mV and depolarizing the membrane during 30 ms in 10 mV increments until + 30 mV. For steady-state inactivation, the membrane was maintained at various voltages between − 120 and 0 mV during a step of 100 ms, immediately followed by a test pulse to 0 mV. This allowed us to measure the fraction of channels available for activation at different voltages (as compared to -120 mV). We also measured recovery from fast inactivation as follows: after an initial 30 ms pulse from − 120 to 0 mV, the membrane was repolarized to -120 mV for various durations, followed by a test pulse to 0 mV. This allowed us to assess the kinetics of recovery from inactivation by dividing the amplitude of the current obtained during each test pulse by the amplitude of the current evoked by the initial pulse\u003c/p\u003e\u003ch2\u003eData analysis\u003c/h2\u003e\u003cp\u003eData analysis and curve fitting were performed using Python (version 3.12.7) within a Jupyter Notebook environment and Stimfit 0.15 (Guzman et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Inactivation and activation curves were fitted with classical Boltzmann-type equations, allowing to extract k (the slope factor) and V\u003csub\u003e50\u003c/sub\u003e. For activation, current-voltage plots were converted to conductance-voltage plots using the equation G = I/(V-E\u003csub\u003eNa\u003c/sub\u003e), the reversal potential for Na\u003csup\u003e+\u003c/sup\u003e being + 69 mV at RT in our conditions. In this analysis we considered the values between − 120 and + 30 mV, because values at voltages close to E\u003csub\u003eNa\u003c/sub\u003e tended to introduce too much variability. Fast inactivation was analyzed by fitting a monoexponential to the data, allowing us to extract the time constant t. Fits were considered acceptable when R was \u0026gt; 0.98.\u003c/p\u003e\u003cp\u003eStatistical analysis was performed using SAS software (SAS University Edition, Cary, NC). Descriptive statistics are reported as medians and interquartile ranges (IQR). Given the small and variable sample sizes, only non-parametric tests were used: the Kruskal-Wallis (KW) test for comparisons of more than two groups ; if the KW showed a significant difference between the various groups, pairwise comparisons were performed using the Dwass, Steel, Critchlow-Fligner (DSCF) method ; the effect size for the KW test was measured using \u003cem\u003eeta\u003c/em\u003e² = (H − k + 1)/(n − k), where H = KW test statistic, k = number of groups, and n = total number of observations (≥ 0.14: large effect) ; and the effect size r for DSCF was calculated by dividing the Z statistic (obtained from the Wilcoxon test) by the square root of the total number of observations (≥ 0.50: large effect). A p-value \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eReport of cases\u003c/p\u003e \u003cp\u003eGenetically, the nine cases exhibited, in the heterozygous state, the missense mutation L1436P in exon 24 of the \u003cem\u003eSCN4A\u003c/em\u003e gene, changing a highly conserved leucine to a proline. Case \u003cspan refid=\"FPar1\" class=\"InternalRef\"\u003e9\u003c/span\u003e was unique in presenting, in addition to the L1436P mutation, a c.5211dup (p.Tyr1738LeufsTer27) duplication of the \u003cem\u003eSCN4A\u003c/em\u003e gene of indeterminate significance. The L1436P mutation was also found in six relatives of four probands (a niece and a daughter of case 6, the mother of cases 3 and 5, and 2 daughters of case 1).\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e presents the clinical and electrophysiological data regarding the nine probands with the L1436P mutation.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eclinical and electrophysiological findings of the 9 probants with L1436P variant\u003c/b\u003e EMG: electromyography ; CMAP: compound muscle action potential ; NA: not applicable ; ND: not done ; *: pseudo-athletic aspect ; **: calf hypertrophy\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCase 1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCase 2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCase 3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCase 4\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCase 5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCase 6\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCase 7\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eCase 8\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eCase \u003cspan refid=\"FPar1\" class=\"InternalRef\"\u003e9\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGender\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003efemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003efemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003efemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003emale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003efemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003efemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003efemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003efemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003efemale\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge at onset (year)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClinical myotonia\u003c/p\u003e \u003cp\u003e- history suggestive\u003c/p\u003e \u003cp\u003e- mechanical myotonia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003cp\u003eYes\u003c/p\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003cp\u003eYes\u003c/p\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003cp\u003eYes\u003c/p\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003cp\u003eYes\u003c/p\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003cp\u003eYes\u003c/p\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003cp\u003eYes\u003c/p\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003cp\u003eYes\u003c/p\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003cp\u003eYes\u003c/p\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003cp\u003eNo\u003c/p\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEpisodic weakness\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWarm-up phenomenon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExacerbation during exercise\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExacerbation after exercise\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCold exacerbation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStress exacerbation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePain\u003c/p\u003e \u003cp\u003e- severity\u003c/p\u003e \u003cp\u003e- distribution\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003cp\u003e- moderate\u003c/p\u003e \u003cp\u003e- arms, hands, feet, thighs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003cp\u003e- moderate\u003c/p\u003e \u003cp\u003e- hands, thighs, calves\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003cp\u003e- mild\u003c/p\u003e \u003cp\u003e- diffuse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003cp\u003e- mild\u003c/p\u003e \u003cp\u003e- face, hands\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003cp\u003e- mild\u003c/p\u003e \u003cp\u003e- hands\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003cp\u003e- moderate\u003c/p\u003e \u003cp\u003e- lower\u0026thinsp;\u0026gt;\u0026thinsp;upper limbs\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMuscle bulk\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHypertrophy*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNormal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNormal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHypertrophy**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNormal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNormal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNormal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNormal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eNormal\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMyotonic discharges on needle EMG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMyogenic traces\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCMAP amplitude\u003c/p\u003e \u003cp\u003e- after short exercise\u003c/p\u003e \u003cp\u003e- idem after cooling\u003c/p\u003e \u003cp\u003e- after long exercise\u003c/p\u003e \u003cp\u003e- postexercise myotonic potential\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e- No decrease\u003c/p\u003e \u003cp\u003e- No decrease\u003c/p\u003e \u003cp\u003e- No change\u003c/p\u003e \u003cp\u003e- No\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e- No decrease\u003c/p\u003e \u003cp\u003e- Decrease\u003c/p\u003e \u003cp\u003e- No change\u003c/p\u003e \u003cp\u003e- No\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e- No decrease \u003c/p\u003e \u003cp\u003e- Decrease\u003c/p\u003e \u003cp\u003e- No change\u003c/p\u003e \u003cp\u003e- No\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e- No decrease\u003c/p\u003e \u003cp\u003e- Decrease\u003c/p\u003e \u003cp\u003e- No change\u003c/p\u003e \u003cp\u003e- No\u003c/p\u003e\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e- No decrease\u003c/p\u003e \u003cp\u003e- Decrease\u003c/p\u003e\u003cp\u003e- No change\u003c/p\u003e\u003cp\u003e- No\u003c/p\u003e\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e- No decrease\u003c/p\u003e \u003cp\u003e- No decrease\u003c/p\u003e \u003cp\u003e- No change\u003c/p\u003e \u003cp\u003e- No\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e- No decrease\u003c/p\u003e \u003cp\u003e- Decrease\u003c/p\u003e\u003cp\u003e- No change\u003c/p\u003e\u003cp\u003e- No\u003c/p\u003e\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10 Hz decrement\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe sex was predominantly female (8 out of 9 cases). All six related cases were also female. The age of onset of symptoms ranged from 20 to 66 years (average 42.8; standard deviation: 14.2).\u003c/p\u003e \u003cp\u003eThe nine cases exhibited clinical myotonias: either patients reported episodes of muscle stiffness and rigidity (8 out of 9 cases) during their medical history, or mechanical myotonias were identified upon \u003cem\u003eextensor digitorum\u003c/em\u003e muscle percussion during the clinical examination (9 out of 9 cases), \u003cb\u003esee the Supplementary Video\u003c/b\u003e. Only cases 2, 4 and \u003cspan refid=\"FPar1\" class=\"InternalRef\"\u003e9\u003c/span\u003e reported episodes of muscle weakness. Muscle pain was frequently reported (6 out of 9 cases). The intensity of this pain was either mild (cases 3, 4, 6) or moderate (cases 1, 2, \u003cspan refid=\"FPar1\" class=\"InternalRef\"\u003e9\u003c/span\u003e), but never severe. The distribution of this pain was either diffuse (cases 3, \u003cspan refid=\"FPar1\" class=\"InternalRef\"\u003e9\u003c/span\u003e) or more localized, with pain reported in the hands in four cases (1, 2, 4, 6), thighs in two cases (1, 2), arms and feet in case 1, and calves in case 2. Except for cases 1 and \u003cspan refid=\"FPar1\" class=\"InternalRef\"\u003e9\u003c/span\u003e, symptoms were exacerbated by exposure to cold. Only case 1 reported a warm-up phenomenon, a feature of SCM, not PMC. The exacerbation of symptoms was often related to exercise (6 out of 8 cases), either during the repetition of the exercise (cases 3, 4, 7, 8, \u003cspan refid=\"FPar1\" class=\"InternalRef\"\u003e9\u003c/span\u003e), or at rest after exercise (cases 2, 3). Only case 4 spontaneously reported an exacerbation of symptoms related to stress. Muscle trophicity was mostly normal (7 out of 9 cases). Case 1 had a pseudo-athletic appearance, while case 4 presented with calf hypertrophy.\u003c/p\u003e \u003cp\u003eElectrophysiologically, the nine cases had very abundant myotonic discharges, characteristic \u0026ldquo;dive bomber\u0026rdquo; spontaneous discharges, during the needle-electrode examination of the skeletal musculature. The electromyographic (EMG) recordings did not show any specific abnormalities, except for case \u003cspan refid=\"FPar1\" class=\"InternalRef\"\u003e9\u003c/span\u003e where myopathic-like traces were recorded, with EMG traces reduced in amplitude and too early interference pattern during right quadriceps muscle contraction of increasing intensity. The study of the amplitude variation of the compound muscle action potential (CMAP) of the \u003cem\u003eabductor digiti minimi\u003c/em\u003e muscle following repetitive nerve stimulation of the ulnar nerve at 10 Hz never revealed a significant decrement at room temperature. For cases 1\u0026ndash;7, the CMAP amplitude was never significantly reduced after short exercises performed at room temperature. No significant change in CMAP amplitude was observed after a long exercise. No post-exercise myotonic potentials were recorded. However, after muscle cooling, the CMAP amplitude was significantly reduced after short exercises in cases 2, 3, 4, 5, and 7, with the amplitude reduction becoming more pronounced with each repetition of the short exercise (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBiophysical data\u003c/p\u003e \u003cp\u003eTo evaluate the degree to which the Na\u003csub\u003ev\u003c/sub\u003e channel function was altered in our patients, we carried out patch clamp experiments in transfected HEK293 cells (see methods). To have relevant comparison points, we studied WT, L1436P and R1448H channels. The reason for choosing the latter is that it is one of the most frequent variants (Sasaki et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eDue to the cold sensitivity of many of the patients\u0026rsquo; symptoms, we performed experiments at three temperatures: cold (15\u0026deg;C), room temperature (RT) (22\u0026deg;C) and near physiological (32\u0026deg;C). We first studied the activation of the channels with a classical protocol starting from \u0026minus;\u0026thinsp;120 mV. Examples of raw traces for WT and each mutation are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea. The data was converted to conductance-voltage curves (see Methods) and the voltages of half maximal activation (V\u003csub\u003e50\u003c/sub\u003e) were extracted. Two examples of such curves are displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb,c and the numerical data is provided in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The L1436P mutant activated at the same voltages as the WT channels, whereas the R1448H mutant activated at significantly more negative voltages in cold and room temperature, but not at 32\u0026deg;C (see Tables\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e for the statistics).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBiophysical parameters of the three types of Nav1.4 channels that were studied.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"11\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003e15\u0026deg;C\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e \u003cp\u003e22\u0026deg;C\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c11\" namest=\"c9\"\u003e \u003cp\u003e32\u0026deg;C\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eWT\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eLP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eRH\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003eWT\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003eLP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003eRH\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003eWT\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003eLP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003eRH\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eV\u003c/b\u003e\u003csub\u003e\u003cb\u003e50\u003c/b\u003e\u003c/sub\u003e \u003cb\u003eof fast\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eactivation (mV)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMedian\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-32.13 (8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-30.55 (6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-38.59 (11)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-24.35 (6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-24.41 (10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-31.49 (6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-25.36 (6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-25.71 (7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-25.41 (6)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIQR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e4.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e3.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e2.81\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKW (η\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.0017 (0.49)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.0052 (0.45)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c11\" namest=\"c9\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.5714\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eTime constant of fast inactivation at -10 mV (ms)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMedian\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.93 (8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.76 (5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.42 (11)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.41 (6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.69 (9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.76 (6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.18 (6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.33 (7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.95 (6)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIQR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKW (η\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 (0.85)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.0002 (0.86)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c11\" namest=\"c9\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.0005 (0.83)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eV\u003c/b\u003e\u003csub\u003e\u003cb\u003e50\u003c/b\u003e\u003c/sub\u003e \u003cb\u003eof steady-state inactivation (mV)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMedian\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-70.17 (7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-59.96 (6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-67.37 (11)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-59.82 (5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-54.10 (10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-58.95 (6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-52.11 (6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-51.29 (7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-54.55 (6)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIQR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e7.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e2.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e7.45\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKW (η\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.0152 (0.30)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.0057 (0.46)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c11\" namest=\"c9\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.9806\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eTime constant of recovery from fast inactivation at -120 mV (ms)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMedian\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.07 (8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.58 (5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.19 (10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.64 (6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.47 (9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.66 (6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.36 (6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.39 (7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.33 (6)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIQR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKW (η\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.0005 (0.67)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.1693\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c11\" namest=\"c9\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.5461\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"11\"\u003eWT\u0026thinsp;=\u0026thinsp;wild type ; LP\u0026thinsp;=\u0026thinsp;L1436P mutant ; RH\u0026thinsp;=\u0026thinsp;R1448H mutant. Values are represented as median and interquartile range (IQR). The number of experiments is indicated in brackets. Statistically significant differences between WT and mutant channels (L1436P and R1448H) were determined using a Kruskal-Wallis (KW) analysis. Effect size for the KW test is measured by \u003cem\u003eeta\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e (η\u003csup\u003e2\u003c/sup\u003e) = (H\u0026thinsp;\u0026minus;\u0026thinsp;k\u0026thinsp;+\u0026thinsp;1)/(n\u0026thinsp;\u0026minus;\u0026thinsp;k) with H\u0026thinsp;=\u0026thinsp;KW test statistic, k\u0026thinsp;=\u0026thinsp;number of groups, n\u0026thinsp;=\u0026thinsp;total number of observations (\u0026ge;\u0026thinsp;0.14: Large effect).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePairwise comparisons by the Dwass, Steel, Critchlow-Fligner method.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15\u0026deg;C\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22\u0026deg;C\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e32\u0026deg;C\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eV\u003c/b\u003e\u003csub\u003e\u003cb\u003e50\u003c/b\u003e\u003c/sub\u003e \u003cb\u003eof fast\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eactivation (mV)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWT \u003cem\u003eversus\u003c/em\u003e LP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.9207\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.9935\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.9972\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWT \u003cem\u003eversus\u003c/em\u003e RH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.0083 (0.67)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.0281 (0.72)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.7026\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLP \u003cem\u003eversus\u003c/em\u003e RH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.0100 (0.69)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.0068 (0.75)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.5766\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eTime constant of fast inactivation at -10 mV (ms)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWT \u003cem\u003eversus\u003c/em\u003e LP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.0096 (0.79)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.0042 (0.81)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.0182 (0.73)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWT \u003cem\u003eversus\u003c/em\u003e RH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.0008 (0.82)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.0110 (0.81)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.0110 (0.81)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLP \u003cem\u003eversus\u003c/em\u003e RH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.0052 (0.76)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.0042 (0.76)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.0076 (0.81)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eV\u003c/b\u003e\u003csub\u003e\u003cb\u003e50\u003c/b\u003e\u003c/sub\u003e \u003cb\u003eof steady-state inactivation (mV)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWT \u003cem\u003eversus\u003c/em\u003e LP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.1122\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.0604\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.9888\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWT \u003cem\u003eversus\u003c/em\u003e RH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.7216\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;1.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.8806\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLP \u003cem\u003eversus\u003c/em\u003e RH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.0100 (0.69)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.0095 (0.72)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.9037\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eTime constant of recovery from fast inactivation at -120 mV (ms)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWT \u003cem\u003eversus\u003c/em\u003e LP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.0096 (0.79)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.4660\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.7550\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWT \u003cem\u003eversus\u003c/em\u003e RH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.0053 (0.72)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.9860\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.9860\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLP \u003cem\u003eversus\u003c/em\u003e RH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.0521\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.1428\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.4877\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eWT\u0026thinsp;=\u0026thinsp;wild type ; LP\u0026thinsp;=\u0026thinsp;L1436P mutant ; RH\u0026thinsp;=\u0026thinsp;R1448H mutant. Effect size r (in brackets) is calculated by dividing the Z statistic (obtained from the Wilcoxon test) by the square root of the total number of observations (\u0026ge;\u0026thinsp;0.50: Large effect).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eWe next turned our attention to the fast inactivation of the channels following their activation. The R1448H mutant is known to have a much slower inactivation than WT channels and this feature is considered as the main contributing factor to the induction of myotonic runs (Chahine et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1994\u003c/span\u003e ; Richmond et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). We confirmed this observation, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and 3a,b. Indeed, values of the time constant of inactivation were much larger in R1448H than in WT (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Fig.\u0026nbsp;3a,b). For example, at -10 mV, the median value was 0.95 ms in R1448H and 0.18 ms in WT at 32\u0026deg;C and remained much larger in R1448H throughout temperatures. The L1436P mutant\u0026rsquo;s behavior was intermediate in this respect, with a value of 0.33 ms at the same voltage at 32\u0026deg;C. The time constant became significantly higher at lower temperatures in all genotypes (Fig.\u0026nbsp;3a,b and Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). At 22\u0026deg;C, which corresponds to moderate cold, values were 0.41, 0.69 and 1.76 ms for WT, L1436P and R1448H, respectively. Overall, the L1436P mutant inactivated significantly slowlier than the WT. However, its effect was also significantly smaller than the one of R1448H across all voltages and temperatures (see Fig.\u0026nbsp;3b and Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e for pairwise comparisons).\u003c/p\u003e \u003cp\u003eOne other important aspect of Na\u003csub\u003ev\u003c/sub\u003e function is the recovery from inactivation upon repolarization of the membrane. This parameter was also evaluated and showed a faster recovery in the L1436P and R1448H mutants as compared to the WT channels, but only at cold temperature (see Tables\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSteady-state inactivation, which reflects the availability of the channels at various voltages, was studied next. For this purpose, the membrane was clamped for 100 ms at various voltages before giving a pulse to 0 mV. The amount of current measured at this voltage reflects the percentage of channels that are not inactivated. The fitting of the curves allowed us to extract the V\u003csub\u003e50\u003c/sub\u003e of steady-state inactivation (see Methods). The L1436P mutant significantly differed from the R1448H mutant in terms of steady-state inactivation (Fig.\u0026nbsp;3c, Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), but not from the WT channels, although a trend was noted (p\u0026thinsp;=\u0026thinsp;0.06 at 22\u0026deg;C). Thus, the V\u003csub\u003e50\u003c/sub\u003e was more depolarized in the L1436P mutant than in the R1448H mutant, both at RT and cold temperatures (by 4\u0026ndash;7 mV), but not at near physiological temperature (Fig.\u0026nbsp;3d). The consequence of this is that a higher percentage of L1436P channels are available in the region of the resting membrane potential of myocytes (\u0026thinsp;~\u0026thinsp;\u0026minus;\u0026thinsp;85 mV) than is the case for the R1448H mutant.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe L1436P mutation was first mentioned in 2008 by a London team (Matthews et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). The case reported by these authors presented a typical clinical phenotype of \u003cem\u003eparamyotonia\u003c/em\u003e with myotonias exacerbated by exercise and exposure to cold, as well as muscle pain.\u003c/p\u003e \u003cp\u003eThe second time the L1436P mutation was mentioned in the literature was by Belgian neurologists (Bissay et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), who described this particular variant in 3 unrelated families (3 probands and 8 relatives) from the Brussels region. The clinical phenotype of these patients supported a diagnosis of SCM. Indeed, they met the main criteria for SCM, specifically exercise-induced delayed-onset myotonia (8 out of 11 cases) and a warm-up phenomenon (symptoms relieved by repetitive muscle contraction) without weakness (10 out of 11 cases). Additionally, clinical electrophysiological data were more indicative of SCM than of PMC, as short exercises performed at room temperature and after muscle cooling did not significantly reduce the CMAP amplitude (7 out of 7 cases). Indeed, their electrophysiological data corresponded with Pattern III of the Fournier classification, both at room temperature and after muscle cooling (Fournier et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2004\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). However, surprisingly, the authors noted that the phenotype of their families differed from classic SCM in that the myotonia did worsen with cold exposure and the frequency and severity of muscle pain. For these reasons, the authors proposed adding a fourth category to the \u003cem\u003efluctuans\u003c/em\u003e, \u003cem\u003epermanent\u003c/em\u003e and acetazolamide-responsive myotonias, which they named cold-aggravated myotonias.\u003c/p\u003e \u003cp\u003eTherefore, in the original article concerning the L1436P mutant, the described phenotype was PMC (Matthews et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), and in the second article (Bissay et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), the reported phenotype in the three families was SCM that worsened with cold. Additionally, the National Center for Biotechnology Information (NCBI) classifies the L1436P variant as hyperkalemic periodic paralysis. The cases described in our work confirm the heterogeneity of the phenotype linked to the L1436P mutation. Indeed, among the seven probands for whom clinical and electrophysiological data were available, the phenotype was either SCM (case 1 with warm-up phenomenon, no exacerbation by cold, and no electrophysiological pattern suggestive of \u003cem\u003eparamyotonia\u003c/em\u003e), or PMC (cases 2, 3, 4, 5, 7 with absence of warm-up phenomenon, exacerbation by cold, and a typical electrophysiological pattern of \u003cem\u003eparamyotonia\u003c/em\u003e). Case 6 could be related to SCM aggravated by cold (exacerbation by cold without a typical electrophysiological pattern of \u003cem\u003eparamyotonia\u003c/em\u003e), but the patient showed no changes in symptoms related to exercise, neither warm-up phenomenon nor exacerbation during exercise. Case 8 was clinically suggestive of PMC (with absence of the warm-up phenomenon and exacerbation by cold and during exercise), but the Fournier protocol was not performed. Regarding the phenotype of hyperkalemic periodic paralysis, three probands described episodes of muscle weakness (cases 2, 4, \u003cspan refid=\"FPar1\" class=\"InternalRef\"\u003e9\u003c/span\u003e), while the long exercise test, performed for cases 1\u0026ndash;7, was negative due to the absence of significant and characteristic changes in CMAP amplitude, either the immediate increase after exercise or the late reduction after exercise (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) (Fournier et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). The clinical data confirm that the L1436P mutation is frequently associated with muscle pain, which often constituted the reason for consultation (cases 1, 2, 3, 4, 6, \u003cspan refid=\"FPar1\" class=\"InternalRef\"\u003e9\u003c/span\u003e) and sometimes led to an initial misdiagnosis of fibromyalgia. Muscle hypertrophy was mostly absent. Nevertheless, case 1 with an SCM phenotype presented with a pseudo-athletic appearance, while case 4 (\u003cem\u003eparamyotonia\u003c/em\u003e phenotype and the only male in our series) had calf hypertrophy. Thus, we confirm that the L1436P mutation of the \u003cem\u003eSCN4A\u003c/em\u003e gene can cause different phenotypes. So far unknown epigenetic alterations, modifying genes and/or environmental factors may influence the clinical expression within and between families with the same \u003cem\u003eSCN4A\u003c/em\u003e mutation. The male-to-female ratio found in this work (only one male among 9 probands and 6 relatives) suggests that hormonal climate could play a role in the phenotypic expression of this genetic disease. This gender-related peculiarity was not reported by Bissay whose cases included 4 women and 7 men (Bissay et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e Case 9 was somewhat unusual in that the medical history was not suggestive of myotonia. It was the clinical examination and EMG that indicated a myotonic syndrome by demonstrating clinical myotonia and typical myotonic bursts. The patient complained of muscle pain and weakness unrelated to cold exposure. The EMG recorded myogenic-like features in the right quadriceps muscle. This somewhat unusual presentation may be related to the presence of a second variant of uncertain significance, a c.5211dup (p.Tyr1738LeufsTer27) duplication of the \u003cem\u003eSCN4A\u003c/em\u003e gene.\u003c/p\u003e \u003c/p\u003e \u003cp\u003eThe 26 reported cases of the L1436P mutation of the \u003cem\u003eSCN4A\u003c/em\u003e gene in Belgium\u0026mdash;specifically, three probands and eight relatives in the Brussels region (Bissay et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), and nine probands and six relatives in the Li\u0026egrave;ge region\u0026mdash;contrast with the limited data available in the literature and with the 17 cases (8 probands and 11 relatives) found in a large epidemiological study by Sternberg (not yet published) on the incidence and prevalence of NDMs in France (a country six times more populous than Belgium). It is therefore possible that there is a founder effect in Belgium related to this mutant.\u003c/p\u003e \u003cp\u003eOur patch clamp results can be summarized as follows: we show that the L1436P mutant induces several abnormalities, including a significant, yet modest slowing down of fast inactivation across several voltages and a mildly depolarized steady-state inactivation curve, the latter meaning a slightly higher availability of channels. On the other hand, voltage-dependence of activation of the channels remains similar to the WT. Overall, its biophysical phenotype seems less severe than the one of the R1448H mutant. In addition, a striking feature of both mutants is that most abnormalities were observed at relatively cold or very cold temperatures (except for the slowing of the fast inactivation). In this regard, our results emphasize the usefulness of recording currents at various temperatures.\u003c/p\u003e \u003cp\u003eDo these biophysical abnormalities explain the myotonic phenotype of the L1436P patients? This is hard to say. First, we have not yet tested all biophysical parameters. For example, some Na\u003csub\u003ev\u003c/sub\u003e mutants exhibit defects in slow inactivation (Webb et al. 2008 ; Carle et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), i.e. an inactivation process that is different from the one that occurs immediately after activation and proceeds over a time scale of seconds or minutes (Hayward et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). It may be that L1436P differs from WT channels in this respect. In addition, there is a clear need to incorporate these different abnormalities in a realistic myocyte model to be able to make predictions. This will allow us to check whether the modest increases in fast inactivation time constants are sufficient to explain the electromyographic phenotype. We are currently developing a new conductance-based computer model of a myocyte based on the one by Cannon (Cannon et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1993\u003c/span\u003e) with improved features such as incorporation of recent data on ClC-1 and K\u003csub\u003eir\u003c/sub\u003e2.1 biophysics, as well as the possibility to include two types of Na\u003csub\u003ev\u003c/sub\u003e currents, a normal one and a pathological one (thereby mimicking the heterozygosity of the patients). This will hopefully help us to answer this question.\u003c/p\u003e \u003cp\u003eOne interesting point is that both clinical and biophysical evaluation of the L1436P mutant shows that it manifests itself predominantly at cold temperatures with overall less alterations at physiological temperature.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, we have described the clinical and biophysical characteristics of a rare Na\u003csub\u003ev\u003c/sub\u003e1.4 mutant, L1436P. The patients bearing this mutation show a rather variable phenotype. Our patch clamp analysis shows biophysical alterations that worsen at infraphysiological temperatures. This is consistent with what was observed clinically in a majority of, but not all our patients.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eANOVA: Analysis of Variance\u003c/p\u003e\n\u003cp\u003eCMAP: Compound Muscle Action Potential\u003c/p\u003e\n\u003cp\u003eDSCF: Dwass, Steel, Critchlow-Finger method\u003c/p\u003e\n\u003cp\u003eHEK: Human Embryonic Kidney\u003c/p\u003e\n\u003cp\u003eKW: Kruskal-Wallis\u003c/p\u003e\n\u003cp\u003eL1436P: NM_000334.4(SCN4A):c.4307T\u0026gt;C (p.Leu1436Pro)\u003c/p\u003e\n\u003cp\u003eNDM: Non-Dystrophic Myotonia\u003c/p\u003e\n\u003cp\u003ePMC: \u003cem\u003eParamyotonia Congenita\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eR1448H: NM_000334.4(SCN4A):c.4343G\u0026gt;A (p.Arg1448His)\u003c/p\u003e\n\u003cp\u003eSCM: Sodium Channel Myotonia\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSCN4A\u003c/em\u003e: Sodium Voltage-Gated Channel Alpha Subunit 4\u003c/p\u003e\n\u003cp\u003eWT: Wild Type\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch3\u003eFunding\u003c/h3\u003e\n\u003cp\u003eA patch-clamp setup (including a Zeiss microscope) was partially purchased thanks to a scientific research investment fund (2022): Fonds d\u0026rsquo;Investissements de Recherche Scientifique (FIRS) from the University Hospital of Liège (Belgium) to FCW. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBL is research associate at the FRS-FNRS.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNI was supported by a grant from the University of Li\u0026egrave;ge and more recently a grant from the FRIA (FNRS, Belgium).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eStudy sponsors played no role in data collection, analysis, interpretation and in drafting of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003eCompeting Interests\u003c/h3\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose\u003c/p\u003e\n\u003ch3\u003eAuthor contributions\u003c/h3\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. The patients in this study were clinically managed by Fran\u0026ccedil;ois Charles WANG, Olivier BOUQUIAUX, Isabelle LIEVENS, and Margaux POLEUR. The experimental data were recorded by Fran\u0026ccedil;ois Charles WANG, Vincent SEUTIN, Nurcan INCI, and Samira ABDOU IDE. Bernard LAKAYE helped with the creation of the mutations and stable lines. The first draft of the manuscript was written by Fran\u0026ccedil;ois Charles WANG and Vincent SEUTIN. Nurcan INCI analyzed the experimental data and created the figures. All authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003eData Availability\u0026nbsp;\u003c/h3\u003e\n\u003cp\u003eFran\u0026ccedil;ois Charles WANG\u0026nbsp;takes full responsibility for the data, the analysis and interpretation and the conduct of the research. He has full access to all of the data and has the right to publish any and all data separate and apart from any sponsor.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eData reported in this paper is fully accessible to interested readers from Fran\u0026ccedil;ois Charles WANG.\u003c/p\u003e\n\u003ch3\u003eEthics approval\u003c/h3\u003e\n\u003cp\u003eThis research study was conducted retrospectively from data obtained for clinical purposes. We consulted extensively with the Ethics Committee of the University of Li\u0026egrave;ge and of the University Hospital of Li\u0026egrave;ge (\u0026ldquo;Comit\u0026eacute; d\u0026rsquo;\u0026eacute;thique hospitalo-facultaire Universitaire de Li\u0026egrave;ge\u0026rdquo;)\u0026nbsp;who determined that our study did not need ethical approval. An official waiver of ethical approval was granted by the Ethics Committee.\u003c/p\u003e\n\u003ch3\u003eConsent to participate\u003c/h3\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003ch3\u003eConsent to publish\u003c/h3\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBissay V, Keymolen K, Lissens W, Laureys G, Schmedding E, De Keyser J (2011) Late onset painful cold-aggravated myotonia: three families with SCN4A L1436P mutation. 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Neurology 70(10):755\u0026ndash;761. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1212/01.wnl.0000265397.70057.d8\u003c/span\u003e\u003cspan address=\"10.1212/01.wnl.0000265397.70057.d8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"University of Liège","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Case report, myotonia, paramyotonia, SCN4A gene, whole-cell patch-clamp","lastPublishedDoi":"10.21203/rs.3.rs-6382511/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6382511/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWe report nine index cases with a rare mutation in the \u003cem\u003eSCN4A\u003c/em\u003e gene, NM_000334.4(\u003cem\u003eSCN4A\u003c/em\u003e).4307T\u0026thinsp;\u0026gt;\u0026thinsp;C (p.Leu1436Pro), which codes for the muscle Na\u003csub\u003ev\u003c/sub\u003e1.4 channel. Patients were evaluated clinically and by an electrodiagnostic study. In addition, the biophysical characteristics of the mutant channels were compared to those of wild type channels and a better-known mutant, R1448H, using whole-cell patch clamp recordings of hNa\u003csub\u003ev\u003c/sub\u003e1.4 currents in stably transfected HEK293 cells, at near physiological temperature (32\u0026deg;C), room temperature (22\u0026deg;C) and cold temperature (15\u0026deg;C). The phenotypes associated with this \u003cem\u003eSCN4A\u003c/em\u003e mutation included one sodium channel myotonia (SCM), six \u003cem\u003eparamyotonia congenita\u003c/em\u003e, and one SCM worsened by cold. Regarding the phenotype of hyperkalemic periodic paralysis, three probands described episodes of muscle weakness. Whole-cell recordings showed that the L1436P mutation induced a significant slowing down of fast inactivation of the Na\u003csub\u003ev\u003c/sub\u003e current at several voltages, but this effect was less marked than in R1448H. The L1436P mutation also tended to induce a right shift in the steady-state inactivation curve, but only at cold temperature. On the other hand, a leftward shift in the activation curve was seen at cold and room temperatures with R1448H, but not L1436P. Recovery from fast inactivation was slowed down in both mutantsat cold temperature. In conclusion, this report confirms that the L1436P mutation of the \u003cem\u003eSCN4A\u003c/em\u003e gene leads to different clinical phenotypes. Epigenetic alterations, modifying genes or environmental factors may influence clinical expression. Our experimental data for L1436P reveals a biophysical phenotype consistent with the clinical phenotype of a majority of patients.\u003c/p\u003e","manuscriptTitle":"Analysis of a rare pathogenic variant of the SCN4A gene (c.4307T\u0026gt;C, L1436P): from clinic to patch-clamp","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-08 12:20:41","doi":"10.21203/rs.3.rs-6382511/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b0913a91-1ffb-4fd0-9fdf-be393dbd9b8e","owner":[],"postedDate":"April 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":46727230,"name":"Neurology"},{"id":46727231,"name":"Cellular \u0026 Molecular Neuroscience"},{"id":46727232,"name":"Medical Genetics"}],"tags":[],"updatedAt":"2025-04-08T12:20:42+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-08 12:20:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6382511","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6382511","identity":"rs-6382511","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}