Functional study of a novel SCN4A variant c.611C>T identified in a Japanese patient with myasthenia

Recent advances in sequencing technologies have significantly contributed to the identification of disease-associated gene variants. However, a substantial number of patients, particularly those presenting with atypical neuromuscular phenotypes, remain genetically undiagnosed. Herein, we report a case of a Japanese patient with myasthenic symptoms in the eyelids and limbs rather than periodic paralysis having a novel heterozygous variant (c.611C>T) located at the 3′ end of exon 4 in SCN4A. The analysis of the proband’s SCN4A mRNA showed that this variant causes an alanine-to-valine missense change at the amino acid position of 204 (p.A204V, 39%) and a disruption of the splicing of exons 4 and 5 leading to the production of truncated Nav1.4 variant protein (p.A204Vfs*94, 4%). We anticipated that the p.A204V missense change would impair Nav1.4 function; however, the ion channel activity and membrane targeting of p.A204V Nav1.4 were found to be wild-type (WT)-like. We also examined the cytotoxicities of the p.A204V and p.A204Vfs*94 variants; however, the cell lines heterologously overexpressing these Nav1.4 variant proteins did not induce cell death any more than the WT control. Although the loss or gain of anomalous ion channel function that is commonly suspected in channelopathies was ruled out in the present case, the precise mechanism of the pathogenic role of c.611C>T SCN4A remains to be elucidated.

Introduction

Skeletal muscle channelopathies are genetic disorders caused by mutations in the genes coding for the voltage-gated ion channels regulating skeletal muscle excitability. SCN4A that encodes for the voltage-gated sodium channel Nav1.4 is one of the causative genes for such disorders, and its variants have been associated with several clinical phenotypes, such as sodium channel myotonia (SCM), paramyotonia congenita (PMC), hyperkalemic periodic paralysis (HyperPP), and hypokalemic periodic paralysis type 2 (HypoPP2) (Cannon, 2015). Electrophysiological analysis of the Nav1.4 channel variants using heterologous expression systems like HEK293T cells as well as computational simulations based on the Hodgkin–Huxley model have significantly contributed to elucidating the pathological mechanisms of SCM, PMC, and HyperPP associated with gain-of-function in the mutant Nav1.4 (Cannon, 2015). Moreover, the discovery of aberrant leak currents, referred to as “gating pore currents,” caused by mutations in the voltage-sensing domain of Nav1.4 have shed light on the pathological mechanisms of HypoPP2 (Sokolov et al., 2007; Struyk and Cannon, 2007; Cannon, 2010). However, there are several atypical phenotypes of skeletal muscle channelopathies associated with Nav1.4; for example, compound heterozygous SCN4A loss-of-function variants are reportedly associated with congenital myasthenic syndrome (CMS) and/or congenital myopathy (CM) (Zaharieva et al., 2016; Nicole and Lory, 2021; Tsujino et al., 2003; Arnold et al., 2015; Habbout et al., 2016; Hadjipanteli et al., 2024; Kubota et al., 2025; Elia et al., 2019). A few cases presenting with both myotonia and periodic paralysis have also been reported (Sugiura et al., 2003; Kokunai et al., 2018). These atypical phenotypes indicate the complexity of electrical excitability of the skeletal muscles.

From the genotype-phenotype perspective, most SCN4A missense variants are typically associated with SCM, PMC, HyperPP, and HypoPP2 (Cannon, 2015; Kubota and Takahashi, 2025), while a few SCN4A nonsense variants are associated with CMS/CM (Zaharieva et al., 2016; Nicole and Lory, 2021). SCN4A variants that are predicted to affect splicing are very rare, and only one case of an indel variant has been reported thus far (Kubota et al., 2011).

Herein, we present the case of a Japanese patient with myasthenic symptoms in the eyelids and limbs rather than periodic paralysis, and these changes are linked to a novel heterozygous variant c.611C>T located at the 3′ end of exon 4 in SCN4A. Our polymerase chain reaction (PCR)-based analysis of the patient’s skeletal muscle specimen showed transcripts coding for two types of SCN4A variants, namely, a missense (p.A204V) and an alternative splicing (p.A204Vfs*94) variants. To explore the associated pathological mechanisms, we examined the functional impacts of p.A204V on membrane targeting and ion channel functions. We also examined the cytotoxicities of the p.A204V and p.A204Vfs*94 Nav1.4 proteins. Although these efforts did not provide definite experimental evidence supporting the pathogenicity of the c.611C>T SCN4A, our study excludes the possibility that a gain of anomalous ion channel function or cytotoxicity underlies the atypical paralytic phenotype, highlighting the broad spectrum of pathological reasons causing channelopathies.

Case presentation

The proband was a 54-year-old Japanese female patient experiencing severe fatigue in her extremities, double vision, and bilateral ptosis, which deteriorated especially in the evenings since the age of 47. Her symptoms, including fatigue, fluctuated throughout the day and improved with rest and cold exposure. The symptoms also worsened at night and improved by the following morning. Ice pack application improved her ptosis, indicating that the ice pack test was positive. Her symptoms further deteriorated in 6 months, and a neurological assessment was performed. The patient’s height was 152 cm and weight was 45 kg (body mass index: 19.4 kg/m²). Clinical examination revealed bilateral ptosis but all other cranial nerves were intact. Muscle strength in the extremities was assessed as Grade 5. There was no evidence of muscle atrophy or hypertrophy; however, her movements appeared sluggish and clumsy owing to fatigue. Her electrocardiography (ECG) was normal, and chest X-ray was unremarkable except for slight scoliosis with a leftward convex curvature. It was found that her symptoms and episodes are indicative of the autoimmune neuromuscular disorder myasthenia gravis (MG). However, the pathological autoantibodies, such as antiacetylcholine receptor (ACh-R) antibody, anti-muscle-specific tyrosine kinase (MuSK) antibody, and anti-low-density lipoprotein receptor-related protein 4 (Lrp4) antibody, were undetectable in her serum. The repetitive nerve stimulation test, which is one of the clinical neurophysiological examinations for detecting conduction failure of the neuromuscular junction, was negative. Although the specific marker for MG was negative, the patient was clinically diagnosed with seronegative MG and administered oral prednisolone along with tacrolimus as an immunosuppressive agent. Despite these immunological treatments, her symptoms worsened. She was hospitalized and received three rounds of intravenous immunoglobulin therapy over the duration of a year, but this did not relieve the symptoms. During the third hospitalization, the possibility of a rare genetic muscle disorder rather than MG was suspected because one of her sons had similar but milder symptoms (Figure 1A). The prolonged exercise test, which is a specific neurophysiological examination to detect paralytic symptoms in skeletal muscle channelopathies, was positive in the proband, suggesting that the proband might have familial periodic paralysis (Figure 1B) (Fournier et al., 2004; Tan et al., 2011). To elucidate the pathogenic mechanisms underlying the proband’s atypical paralytic phenotype, we performed a genetic analysis subsequently.

Figure 1

Figure 1. Clinical and genetic analyses of the case. (A) Family tree of the proband. The filled symbols indicate family members who present similar muscular symptoms, including fatigue and ptosis. The proband is indicated as “p” with an arrow. (B) Prolonged exercise test in the proband revealed exercise-induced decrement of the compound muscle action potentials (CMAPs). The y axis indicates normalized amplitude of the CMAP, and the x axis indicates time. The shaded area indicates the period of the exercise load. (C) Schematic representation of the c.611C>T variant in the SCN4A exon. The arrows indicate the positions of the polymerase chain reaction (PCR) primers. (D) Electrophoresis of the PCR products using the primers in (C) for the proband (left lane) along with a patient with myotonic dystrophy (DM) and a patient with amyotrophic lateral sclerosis (ALS) as the disease controls. (E) Theoretical illustration of the alternative splicing caused by c.611C>T is shown in the upper panel. The sequencing results of the PCR products are shown in the bottom panel: normal, c.611_612deletion resulting in p.A204Vfs*94, and p.A204V. The frequencies of these isoforms are shown in Table 2.

Materials and methods

Genetic analysis

Following the approval of the study protocol by the Institutional Review Committee at the University of Osaka (approval #721), we obtained written informed consent from the proband, which included permission to conduct genetic analysis and present the findings in an academic journal and at meetings. Consent for the genetic testing could not be obtained from the proband’s family members. We extracted the genomic DNA from the proband’s peripheral blood and performed genetic analysis using next-generation sequencing (NGS) at Kazusa DNA Research Institute (https://www.kazusa.or.jp/en/). The protein-coding regions of the voltage-dependent Cl⁻ channel gene (CLCN1) and voltage-dependent Na⁺ channel gene (SCN4A) that are associated with myotonia due to skeletal muscle channel disease as well as the boundary regions between their exons and introns (up to 10 bases within the intron) were analyzed using targeted NGS through the hybrid capture method. We compared the obtained nucleotide sequences with the publicly available human genome reference sequence (GRCh38/hg38) and analyzed the low-frequency base substitutions as well as presence or absence of short nucleotide sequence deletions or insertions. Furthermore, to exclude other genetic abnormalities related to “muscle weakness” in the Human Phenotype Ontology (HPO), we performed whole-exome sequencing using NovaSeq 6000 (Illumina, San Diego, CA, United States) with a read length of 100 bp paired-end with the SureSelect Human All Exon V8 kit (Agilent Technology, Santa Clara, CA, United States). The FASTQ files were checked for quality using FastQC, and the low-quality reads were removed using Trimmomatic-0.36. The quality-checked reads were aligned to GRCh38 using the Burrows–Wheeler aligner. The variants were identified using HaplotypeCaller in the Genome Analysis Toolkit (GATK v.4.1.0) and annotated using ANNOVAR. The pathogenicity of each variant was scored using Sorting Intolerant From Tolerant (SIFT) (Sim et al., 2012; https://sift.bii.a-star.edu.sg/), Polymorphism Phenotyping version 2 (PolyPhen2) (Adzhubei et al., 2010; http://genetics.bwh.harvard.edu/pph2/), Protein Variation Effect Analyzer (PROVEAN) (Choi and Chan, 2015; http://provean.jcvi.org/seq_submit.php), and Combined Annotation-Dependent Depletion (CADD) (Kircher et al., 2014; https://cadd.gs.washington.edu/). The variants were filtered on the basis of these scores, genotypes, and minor allele frequency (MAF) to determine those responsible for causing the disease. The MAF cutoff values were as follows: autosomal dominant 0.03, autosomal recessive 0.05, de novo 0.03, X-linked 0.05, and compound heterozygous 0.05.

Sequencing analysis of the SCN4A gene transcripts

The total RNA was extracted from frozen skeletal muscle biopsy specimens of the proband’s biceps brachii using Maxwell® RSC simplyRNA Cells and simplyRNA Tissue kits (Promega, Tokyo, Japan). The extracted RNA was next reverse transcribed into cDNA using the iScript™ cDNA synthesis kit (Bio-Rad, Tokyo, Japan). The cDNA was then used as a template for PCR amplification with Platinum™ PCR SuperMix High Fidelity™ (Invitrogen™ by Thermo Fisher Scientific™, Tokyo, Japan) using the following primers: [forward primer] 5′-GCT​GTT​CAG​CAT​GTT​CAT​CAT​GAT​C-3′ and [reverse primer] 5′-CCA​TGA​AGA​GCT​GCA​GTC​CT-3′. As disease controls, we also amplified the cDNA from tongue muscle samples of patients with myotonic dystrophy type 1 (DM1) and amyotrophic lateral sclerosis (ALS) that had been preserved in the laboratory previously. We obtained informed consent from these patients for the use of their specimens for other research purposes. The size of the PCR product, presumed to be 427 bp, was confirmed by agarose gel electrophoresis. The PCR products were cloned into the pTA2 vector using the TArget Clone™ Plus kit (TOYOBO, Osaka, Japan), and the resulting plasmid was transformed into Escherichia coli DH5α competent cells (Takara, Shiga, Japan). Of the 54 colonies obtained, the TA vector was extracted and purified from each colony. After verifying the inserts, Sanger sequencing was performed.

Generation of stable cell lines

We used HEK293T-based stable cell lines coexpressing the α (wild type (WT) or p.A204V) and β (SCN1B) subunits of human Nav1.4 for the membrane targeting assay and whole-cell patch-clamp experiments. In these cell lines, the full-length Nav1.4 α subunit (WT or p.A204V) with N-terminal mTurquoise 2 (mTq2) tag and β subunit were translated from the same transcript but produced separately using the P2A self-cleaving peptide (GSGATNFSLLKQAGDVEENPGP). The cDNAs coding for these P2A-linked constructs were cloned into the pSBtet-Pur vector (Kowarz et al., 2015) (Addgene plasmid # 60497) using NEBuilder HiFi DNA Assembly (NEBuilder HiFi DNA Assembly Master Mix, E2621; New England Biolabs, Japan) and co-transfected with pCMV (CAT) T7-SB100 (Addgene plasmid # 34879) in the HEK293T cells using ViaFect transfection agent (Promega, Madison, WI, United States). Twenty-four hours after the transfection, the Dulbecco’s modified Eagle medium (DMEM)-based culture medium was replaced with fresh medium containing 1 μg/mL puromycin for selection. The sequences of the mutagenic primers used for generating the p.A204V Nav1.4 construct were as follows: 5′-TGT​ACC​TGA​CAG​AGT​TTG​TGG​ACT​TG-3′ (forward); 5′-CCA​TCA​TGA​TGA​CAC​TGA​AGT​CCA​G-3′ (reverse). A site-directed mutagenesis kit (KOD-Plus mutagenesis kit, SMK-101, TOYOBO) was used in this step.

Membrane protein targeting assay

The HEK293T-based stable cells (same as above) were seeded on 35-mm dishes, and expression of the Nav1.4 constructs or mTq2 alone (negative control) was induced by doxycycline (Dox; 3 μg/mL). On the next day, the cells were washed once with phosphate-buffered saline (PBS) and incubated with 100 µM of sulfo-cyanine3 NHS ester (Lumiprobe) dissolved in 2 mL of ice-cold PBS for 40 min on a nutator at 4 °C. The reaction was stopped by the addition of 200 µL of 100 mM glycine. The cells were then collected into a tube, washed with Tris-buffered saline (TBS), and centrifuged at 500×g for 3 min. After removing the supernatant, the cells were resuspended and lysed in 600 µL of a lysis buffer (150 mM of NaCl, 20 mM of HEPES at pH 7.5, 1 mM of EDTA, 20 mM of DDM, 1 mM of DTT, and 50 μg/mL of leupeptin) on ice. The lysate was centrifuged at 15,000×g for 10 min at 4 °C, and approximately 3–5 µL of green fluorescent protein (GFP) selector slurry (NanoTag Biotechnologies) was added to the supernatant and incubated for 30 min at 4 °C with gentle tapping every 5 min. The bound proteins were collected alongside the GFP selector by brief centrifugation. A portion of the GFP selector containing solution was transferred to a glass slide and observed under a fluorescence microscope (ORCA Spark, Hamamatsu Photonics K.K., Shizuoka, Japan). The fluorescence intensity of cyanine 3 (F_Cy3) reflects the amount of protein expressed on the cell membrane, while that of mTq2 (F_mTq2) reflects the total amount of the mTq2-tagged protein. The ratio of Cy3 to mTq2 fluorescence intensities (F_Cy3/F_mTq2) thus indicates the membrane targeting efficiency.

Measurement of sodium currents

Measurement of the sodium currents was performed using the conventional whole-cell recording approach described previously (Kubota et al., 2011; Horie et al., 2020). We used Axopatch 200B (Molecular Devices, San Jose, CA, United States) for recording and Digidata 1550B (Molecular Devices) for the data acquisition and analysis. Heat-polished electrodes made from borosilicate glass tubes (Sutter, Novato, CA, United States) using the P-97 Flaming/Brown Micropipette Puller (Sutter) were used for the patch-clamp recordings. The resistances of the electrodes filled with the internal recording solution were in the range of 1.6–2.1 MΩ in the bath solution. The series resistance after whole-cell compensation (70% prediction) was ∼1.0 MΩ. We excluded cells with peak currents less than 2 nA or exceeding 10 nA elicited by stepped depolarization from −120 mV to −10 mV. The composition of the internal solution was as follows: 105 mM of CsF, 35 mM of NaCl, 10 mM of ethylene glycol tetra-acetic acid (EGTA), and HEPES (pH 7.4) with 10 mM of CsOH. The composition of the bath solution was as follows: 140 mM of NaCl, 4 mM of KCl, 2 mM of CaCl₂, 1 mM of MgCl₂, 5 mM of glucose, and Na-HEPES (pH 7.4) with 10 mM of NaOH.

We conducted the patch-clamp experiments at room temperature (approximately 25 °C). After achieving the whole-cell configuration, the membrane potentials were maintained at −120 mV (for measurement of currents during activation and fast inactivation) or −100 mV (for measurement of currents during slow inactivation) for 5 min so that the Nav channels transitioned from the slow inactivated state to resting state. Then, the sodium currents were measured using the corresponding protocols shown in the insets of Figures 3B, D, E. The details of the pulse protocols are as follows. After achieving whole-cell configuration, the membrane potential was held at −120 mV for 5 min to recover from slow inactivation before starting the measurements. The activation current was measured by applying a 10-ms pulse in steps of 5 mV from −80 mV to +80 mV starting from a holding potential of −120 mV. The steady-state fast inactivation was measured as the peak current at −10 mV after applying a 300-ms conditioning pulse in the range of −120 mV to −10 mV. The voltage-dependent kinetics of fast inactivation was measured using the following three-step approach. Recovery from fast inactivation was measured by applying a 30-ms pulse at −10 mV, followed by recovery at a hyperpolarized potential (−110 to −80 mV) with a duration of 0.05–900 ms, and a 10-ms test pulse at −10 mV. The obtained time course of recovery (recovery curve) was fitted with a single exponential increment to determine the time constant for each cell (Figure 3C). The entry to fast inactivation was measured by applying 0–300 ms conditioning pulses at various potentials (−70 to −40 mV), followed by a 10-ms test pulse at −10 mV. The obtained entry curve was fitted with a single exponential decay to determine the time constant for each cell. The fast inactivation kinetics from the open state (−20 to +20 mV) was obtained from the time constant of the sodium current decay measured in the activation protocol. The steady-state slow inactivation was measured as the peak current at −10 mV pulse after applying a 60-s conditioning pulse in the range of −120 mV to +10 mV. The peak current was normalized with the reference peak current at −10 mV pulse from a holding potential of −100 mV. Between the conditioning and test pulses, a 20-ms pulse was applied at −100 mV to recover from fast inactivation.

Data analysis of sodium currents

Origin software (OriginLab, Northampton, MA, United States) was used to fit the curves manually. The conductance of the sodium channels was calculated as G(V) = I_peak (V)/(V - E_rev), where the reversal potential (E_rev) was obtained from the individual experiments. The voltage dependence of activation was quantified by determining the conductance fitted to the Boltzmann function as G(V) = G_max/[1 + exp (-(V - V_1/2)/k)], where V_1/2 is the half-maximum voltage, and k is a slope factor. The steady-state fast inactivation and activation were fitted as well. The steady-state slow inactivation was fitted using a baseline (I₀) in the Boltzmann function and calculated as I/I_max = {(1 - I₀)/[ 1 + exp((V-V_1/2)/k)] + I₀} because the availability of the channel fraction did not decrease to zero in slow inactivation. The error bars indicate the standard error of the mean (SEM). Statistical significance was determined by the Student’s t-test, where a p-value <0.05 was considered to be statistically significant.

Cytotoxicity assays

We established the HEK293T-based stable cell lines for the cytotoxicity assays by introducing the pSBtet-Pur vector with the α subunit (WT, p.A204V, or pA204Vfs*94) of the human Nav1.4 fused to the mCherry and P2A sequences at their N-terminus (Figure 4A). Stable cells carrying these Dox-inducible Nav1.4 constructs were seeded in a 96-well plate at 2.5 × 10⁴ cells/well and cultured overnight in DMEM (cat. no. 11965092, Gibco) supplemented with 10% fetal bovine serum (FBS) at 37 °C in 5% CO₂. On the next day, a medium containing CellTox™ Green dye (cat. no. G8741, Promega) and Dox (0–10 μg/mL) was added to each well. The plate was immediately transferred to a plate reader (Synergy Neo2, Agilent/BioTek) to initiate the cell death assay as described previously (Kojima et al., 2021). The CellTox Green (indicates cell death) and mCherry (indicates Nav1.4 expression) fluorescence intensities were monitored every 5 min for 72 h and plotted offline using GraphPad Prism 10.6.

Results

Genetic analysis

The proband’s genomic DNA was extracted from peripheral lymphocytes following written informed consent. Whole-exome sequencing was then conducted, which showed three heterozygous variants that were classified as “pathogenic” or “likely pathogenic” in InterVar: c.611C>T in SCN4A; c.1975G>C in SLC26A4; c.175A>G in PNLIP. We also searched for heterozygous variants known to be associated with “muscle weakness” in the HPO (Köhler et al., 2017; https://hpo.jax.org/)) and found a total of 50 variants in 33 genes; these were subsequently filtered to exclude those classified as “likely benign” in InterVar, having a recessive inheritance pattern, or having an allele frequency greater than 0.1% in gnomAD v4.1.0[all], gnomAD v4.1.0[east Asian] (https://gnomad.broadinstitute.org/), and 4.7KJPN (Takada S, et al., 2024; https://jmorp.megabank.tohoku.ac.jp/). Among all the variants detected initially, only c.611C>T in SCN4A and c.323T>C in TMEM43 passed these qualifications to be considered as potential causative variants. We did not identify any CLCN1 variants. TMEM43 is the causative gene for arrhythmogenic right ventricular cardiomyopathy type 5 (ARVC5), Emery–Dreifuss muscular dystrophy type 7 (EDMD7), and autosomal dominant auditory neuropathy type 3 (AUNA3). The c.323T>C TMEM43 variant changes an amino acid at the position of 108 from valine to alanine (p.V108A), and the widely used in silico pathogenicity predictors SIFT SIFT (Sim et al., 2012; https://sift.bii.a-star.edu.sg/), PolyPhen2 (Adzhubei et al., 2010; http://genetics.bwh.harvard.edu/pph2/), PROVEAN (Choi and Chan, 2015; http://provean.jcvi.org/seq_submit.php.), and CADD (Kircher et al., 2014; http://cadd.gs.washington.edu/) all classified this missense variant as “damaging” or “deleterious” (Table 1). However, the proband and her affected son had no cardiac complications, including arrhythmia, which are often seen in patients with ARVC5 or EDMD7. In addition, they did not suffer from hearing loss, unlike patients with AUNA3. The c.611C>T SCN4A variant located at the 3′ end of exon 4 was also predicted to be “damaging” or “deleterious” by the in silico pathogenicity predictors. Because the proband’s neurophysiological evaluation indicates a skeletal muscle channelopathy, we pursued the c.611C>T variant of SCN4A as the most likely to have a causal association in this case.

Table 1

Table 1. In silico pathogenicity prediction for variants identified in the genetic analysis.

Splicing isoform analysis

The c.611C>T variant of SCN4A is predicted to cause an alanine-to-valine missense change at the amino acid position of 204 (p.A204V, Figure 1C) or an alternative splicing owing to the location of the C-to-T change at the 3′ end of exon 4 (Figure 1E). The in silico splice-site predictors SpliceAI (de Sainte Agathe JM, et al., 2023; https://spliceailookup.broadinstitute.org/) and Pangolin (Zeng and Li., 2022; https://neurosnap.ai/service/Pangolin%20RNA%20Splicing%20Prediction) support this latter possibility (with scores of 0.87 and 0.64, respectively). To explore these possibilities, we conducted a PCR-based analysis to examine the SCN4A transcripts. Accordingly, cDNAs were synthesized using the total RNA extracted from the skeletal muscle specimen of the proband. The PCR products include the c.611 site shown in Figure 1C, as confirmed by electrophoresis (Figure 1D), which was inserted into TA vectors and transformed into competent E. coli cells for subsequent DNA sequencing analysis of the cloned inserts. Of the 54 clones collected, 31, 21, and 2 clones contained normal (57%), c.611C>T (39%), and c.611_612del (4%) variants of SCN4A, respectively (Table 2; Figure 1E). The results indicate that the primary consequence of the c.611C>T variant is the production of a Nav1.4 protein with the p.A204V missense change, although low production of the truncated and frame-shifted Nav1.4 protein p.A204Vfs*94 may be possible from the alternatively spliced c.611_612del mRNA.

Table 2

Table 2. Frequency of mRNA isoforms identified in the proband’s skeletal muscle samples.

Membrane trafficking efficiency of the mutant channel

Since the p.A204Vfs*94 Nav1.4 protein is highly unlikely to retain ion channel activity even if it were produced, we focused our attention on the p.A204V missense change; here, Ala²⁰⁴ is located at the extracellular side of segment 3 in domain I in Nav1.4 (Figures 2A, B). This alanine residue is conserved within the SCN gene family in both humans (Figure 2C) and non-human orthologs (Figure 2D), suggesting the essentiality of the alanine residue for functional expression of the channel proteins in the cell membrane. To define the functional impacts of p.A204V, we first examined the membrane trafficking efficacy of p.A204V Nav1.4 using the Cy3-based membrane protein targeting assay established recently (Kubota et al., 2025; Kubota et al., 2023). In brief, the membrane-impermeable Cy3 NHS ester labels only the extracellularly exposed primary amines of a membrane protein of interest, allowing fluorometric quantification of membrane targeting. We generated cell lines that expressed mTq2-tagged Nav1.4 proteins with and without p.A204V for this purpose. A cell line that expressed only the mTq2 moiety was also generated as a negative control. After treatment with the Cy3 NHS ester, the cells were lysed and Nav1.4 protein was immunoprecipitated using beads conjugated with the mTq2-binding nanobody (GFP selector). As expected, the membrane expression efficiency determined as the ratio of Cy3 to mTq2 fluorescence intensities (F_Cy3/F_mTq2) was negligibly small for the mTq2 control but significant for the WT Nav1.4 (Figure 2E). The F_Cy3/F_mTq2 ratio of p.A204V Nav1.4 was statistically indistinguishable from that of the WT case (p > 0.05, one-sample t-test) (Figures 2E, F), suggesting that the p.A204V missense change does not affect the protein folding and membrane expression of Nav1.4.

Figure 2

Figure 2. Illustration of the p.A204V variant of SCN4A and its impact on the membrane targeting efficiency. (A, B) Location of Ala²⁰⁴ in the voltage-sensing domain (VSD) of domain I in (A) a schematic representation and (B) the tertiary structure (PDB: 6AGF). (C, D) Ala²⁰⁴ is conserved among the (C) human SCN family members and (D) non-human SCN orthologs. (E) Fluorometric membrane targeting assay with depictions of representative Cy3 (F_Cy3) and mTq2 (F_mTq2) raw fluorescence data plots. The slopes (F_Cy3/F_mTq2) indicate the membrane targeting efficiencies of the wild type (WT; open squares), p.A204V (filled squares), and mTq2 alone (negative control, cross signs) channels. (F) Summary of the fluorometric membrane targeting assay. The membrane targeting efficiency of p.A204V (filled squares) is normalized to that of WT (thick black horizontal line). The membrane targeting efficiency of the mTq2 control (cross signs) is minimal as it does not target the cell membrane by itself. The thin horizontal lines indicate mean ± standard deviation values.

Electrophysiological analysis

Next, we conducted whole-cell patch-clamp experiments to examine the impacts of the p.A204V missense change on the gating properties of Nav1.4, namely, activation, fast inactivation, and slow inactivation. The representative ionic current traces for the WT and p.A204V Nav1.4 channels expressed in the HEK293T cells are shown in Figure 3A. The voltage dependences of the conductance and steady-state fast inactivation are shown in Figure 3B. The voltage dependences of the conductance (V_1/2) and slope factor (k) of activation were similar for the WT (V_1/2 = −11.6 ± 1.3 mV; k = 5.4 ± 0.4 mV/e-fold (mean ± SEM, n = 12)) and p.A204V (V_1/2 = −10.5 ± 2.1 mV; k = 5.0 ± 0.3 mV/e-fold (mean ± SEM, n = 10)) cases (Table 3). The voltage dependences of V_1/2 and k of fast inactivation were also similar for WT (V_1/2 = −51.8 ± 1.6 mV; k = 5.7 ± 0.4 mV/e-fold (mean ± SEM, n = 12)) and p.A204V (V_1/2 = −50.6 ± 1.4 mV; k = 5.2 ± 0.3 mV/e-fold (mean ± SEM, n = 10)) (Table 3). The recovery curves from fast inactivation at −100 mV are shown in Figure 3C, and the time constants of fast inactivation ranging from −110 mV to +30 mV are shown in Figure 3D. The time constants for p.A204V were approximately 1.5 times faster than those of the WT channels between −50 mV and −110 mV, where there is a statistically significant difference only at −50 mV.

Figure 3

Figure 3. Whole-cell patch-clamp recordings of the HEK293T cells expressing WT or p.A204V Nav1.4. (A) Representative current traces for the WT and p.A204V human Nav1.4 channels expressed in the HEK293T cells. The voltage protocols used to elicit the currents are shown in the right inset of (B). (B) Voltage dependences of steady-state fast inactivation (circles) and activation (squares). The insets indicate the pulse protocols for activation (right) and steady-state fast inactivation (left). Open symbols, WT. Closed symbols, p.A204V. The solid (p.A204V) and broken (WT) lines indicate the corresponding Boltzmann fittings. (C) Time course of recovery at −100 mV from fast inactivation. The p.A204V Nav1.4 (filled squares: n = 7) recovered from fast inactivation slightly earlier than WT (open squares: n = 8). (D) Voltage-dependent fast inactivation kinetics of WT (open squares) and p.A204V Nav1.4 (filled squares). These data were obtained using three different protocols, namely, a recovery protocol (left upper inset), an entry protocol (left lower inset), and an activation protocol (B, right inset). Color was used to distinguish the fast inactivation protocols: recovery is shown in red, entry in blue, and ionic current decay in the open state in black. In the symbol legend, “rec” and “ent” denote data obtained using the recovery and entry protocols, respectively. The asterisk (*) indicates a statistically significant difference between WT and p.A204V (found only at −50 mV in D). (E) Voltage dependences of steady-state slow inactivation of WT (open squares) and p.A204V Nav1.4 (filled squares). The solid (p.A204V) and broken (WT) lines indicate the corresponding Boltzmann fittings.

Table 3

Table 3. Electrophysiological parameters of the WT and p.A204V human Nav1.4.

Determining the optimal stimulation rate is challenging in the case of use-dependent inactivation. Since the patient’s fatigue manifested several hours after exercising and resembled the phenotype observed in the prolonged exercise test (LET), a high-frequency stimulation protocol was deemed unsuitable. Therefore, we focused on exploring the defects in slow inactivation as a detailed analysis was deemed more likely to reveal any functional differences. The voltage dependence of steady-state slow inactivation is shown in Figure 3E; the voltage dependence (V_1/2), slope factor (k), and maximal residual fraction (I₀) of slow inactivation were similar for the WT (V_1/2 = −42.9 ± 1.0 mV; k = 8.7 ± 1.7 mV/e-fold; I₀ = 0.14 ± 0.02 (mean ± SEM, n = 6)) and p.A204V (V_1/2 = −39.4 ± 2.0 mV; k = 8.3 ± 0.7 mV/e-fold; I₀ = 0.14 ± 0.01 (mean ± SEM, n = 6)) cases (Table 3).

In summary, although the fast inactivation kinetics of p.A204V Nav1.4 at −50 mV was significantly faster than that of the WT channel (Figure 3D), the Na⁺ channel properties of p.A204V Nav1.4 were overall WT-like and hence unlikely to account for the proband’s symptoms.

Cytotoxicities of the Nav1.4 variants

Since the membrane targeting and ion channel function of the p.A204V Nav1.4 protein were found to be WT-like, we assessed p.A204Vfs*94 Nav1.4 to gain insights into the pathogenic role of the c.611C>T variant of SCN4A. Although the transcript coding for this truncated variant protein was low, it was found in the proband’s skeletal muscle specimen (see above). It is highly unlikely that p.A204Vfs*94 Nav1.4 retains its ion channel function even if translated; however, it may be cytotoxic if accumulated. To explore this possibility, we generated HEK293T-based stable cell lines expressing the Nav1.4 variants in a Dox-dependent manner. To fluorometrically monitor the expression of the Nav1.4 constructs, the red fluorescent protein mCherry was fused to the N-terminus of each construct via the ribosome-skipping sequence P2A (Figure 4A), which addresses the risk that direct covalent tagging could alter the cytotoxicities of the Nav1.4 protein constructs. Moreover, a pore-blocking mutation p.F1519S (Kubota et al., 2025) was introduced to the full-length Nav1.4 constructs because Nav1.4-mediated Na⁺ conductance is harmful to cells when overexpressed. Even with this pore-blocking mutation, the Dox-induced large expression of the Nav1.4 proteins (Figure 4B, lower left panel) induced significant cell death (Figure 4B, upper left panel). The Dox-induced large expression of p.A204V Nav1.4 (Figure 4B, lower middle panel) also induced significant cell death (Figure 4B, upper middle panel) but did not exceed that observed for the Nav1.4 control. Compared to these full-length Nav1.4 protein constructs, p.A204Vfs*94 Nav1.4 showed much greater Dox-induced expression (Figure 4B, lower right panel); however, the large expression of this truncated Nav1.4 protein variant barely induced any cell death (Figure 4B, upper right panel). These observations contradict the possibility that dominant inheritance of the c.611C>T variant of SCN4A may be attributed to the cytotoxicities of the p.A204V or p.A204Vfs*94 Nav1.4 proteins.

Figure 4

Figure 4. CellTox green cytotoxicity assay. (A) Schematic illustration of the Nav1.4 protein construct (control, p.A204V, or p.A204Vfs*94) expressed in the HEK293T-based stable cell line in a doxycycline (Dox)-inducible manner. (B) Representative traces of CellTox Green fluorescence (top panels, indicator of cell death) and mCherry fluorescence (bottom panels, indicator of protein expression).

Discussion

Herein, we present our findings on the identification of a Japanese patient with an atypical paralytic phenotype. Whole-exome sequencing analysis of the proband showed heterozygous variants in the SLC26A4 (c.1975G>C), PNLIP (c.175A>G), TMEM43 (c.323T>C), and SCN4A (c.611C>T) genes.

The vast majority of SLC26A4 variants are associated with Pendred syndrome or DFNB4, which are characterized by hearing loss and an enlarged vestibular aqueduct (OMIM: 274600; 600791). Congenital goiter is also observed in patients with Pendred syndrome; however, none of the SLC26A4 variants have been reported to be associated with skeletal muscle phenotypes. PNLIP is involved in pancreatic lipase synthesis, and its variants are often associated with abnormal lipid metabolism and pancreatic exocrine functions (OMIM: 246600; 614338); however, there are no reported PNLIP variants known to be associated with skeletal muscle phenotypes. Moreover, the SLC26A4 and PNLIP variants are typically inherited in an autosomal recessive manner, further opposing the possibility that the heterozygous c.1975G>C variant of SLC26A4 or heterozygous c.175A>G variant of PNLIP is causally associated with the proband’s observed phenotype. TMEM43 variants are inherited in an autosomal dominant manner and are associated with ARVC5, EDMD7, and AUNA3 (OMIM: 612048; 604400; 614302; 619832). EDMD is typically characterized by proximal muscle weakness and joint contractures from early childhood, contractures with varying onset times, and some overlaps in symptoms between these two conditions. In addition, EDMD frequently presents with cardiac conduction disorders. EDMD associated with TMEM43 is referred to as EDMD7 and was initially reported in two Japanese patients (Liang et al., 2011). In these prior reports, a 40-year-old male patient with the c.235G>A (p.Glu85Lys) variant of TMEM43 exhibited a typical EDMD phenotype, while a 68-year-old female patient with the c.271A>G (p.Ile91Val) variant of TMEM43 had arrhythmia, muscle atrophy, and slowly progressive muscle weakness. Because the proband in the present study did not exhibit muscle atrophy or cardiac conduction disorders at the time of examination, we ruled out the potential pathogenic contributions of the c.323T>C variant of TMEM43 to disease manifestation. Hence, we decided to pursue the c.611C>T variant of SCN4A as the most likely to have causal association in this clinical case as the proband’s neurophysiological evaluation indicates a skeletal muscle channelopathy.

The c.611C>T variant is novel and located at the 3′ end of exon 4 in the SCN4A gene. One of the consequences of this variant was found to be a missense change p.A204V located within segment 3 in domain I in hNav1.4. The alanine residue at this position is conserved among the human Nav isoforms and Nav1.4 orthologs in other species, implying its importance in the Na⁺ channel function. In fact, p.A204E was previously reported to alter the activation and inactivation of Nav1.4 protein, resulting in periodic paralysis (Kokunai et al., 2018). However, our functional analyses did not demonstrate notable changes to the voltage dependence in the p.A204V Nav1.4 channel, except that the inactivation kinetics at −50 mV was faster in p.A204V Nav1.4 compared to WT. Although this difference was statistically significant, it was small and therefore unlikely to account for the pathological condition of the patient. However, since cold exposure ameliorated the patient’s symptoms, the Nav1.4 channel kinetics may differ significantly between p.A204V and WT at normal body temperature (∼37 °C). We did not conduct whole-cell recording at 37 °C because the WT-like membrane targeting efficiency of p.A204V Nav1.4 (Figures 2E,F, with the cells cultured at 37 °C) suggests WT-like protein stability.

The other consequence of the c.611C>T variant is disruption of the splice donor site, which results in an alternative splicing isoform c.611_612del (p.A204Vfs*94). Although the relative amount of this mis-spliced transcript was low (4%) compared to those coding for the full-length Nav1.4 proteins (WT and p.A204V), it was indeed detected in the proband’s skeletal muscles. It is highly unlikely that this truncating variant has any anomalous channel function or inhibitory effect on WT Nav1.4; however, it could be cytotoxic if translated and accumulated. We explored this possibility in a cell line heterologously expressing the p.A204Vfs*94 Nav1.4 protein. However, Dox-induced large expression of the truncated variant protein barely induced any cell death (Figure 4).

The presence of the p.A204Vfs*94 Nav1.4-coding transcript suggests that the c.611C>T allele of SCN4A is partially null, leading to reduced overall production of the functional Nav1.4 proteins (WT and p.A204V). Truncating variants that induce premature termination codons are typically removed by nonsense-mediated mRNA decay (NMD). In other words, the actual frequency of the mis-splicing event (before removal by NMD) would be much higher than 4%. Furthermore, p.A204V and p.A204Vfs*94 (if translated) may activate endoplasmic-reticulum-associated degeneration (ERAD) and reduce the overall expression of the functional Nav1.4 protein even for WT. Collectively, we tentatively conclude that SCN4A haploinsufficiency, owing to the cellular quality control mechanisms (Figure 5), may underlie the patient’s disease manifestation. It should be noted that the proband suffers from late-onset, but not congenital, myasthenia. It has been shown that mice harboring only one functional Scna4 allele are viable but suffer from latent myasthenia (Wu et al., 2016). Thus, it would not be far-fetched to entertain the possibility that the alternative splicing caused by the c.611C>T variant induces moderate haploinsufficiency, which becomes non-trivial over time. Cell-line-based forced protein expression systems, such as those used in the present study, have limitations in the exploration of haploinsufficiency-based pathogenic mechanisms. Future functional studies using more clinically relevant in vitro models, such as induced pluripotent stem cell-derived skeletal muscle cell lines (Fujiwara et al., 2022), may be needed to further experimental efforts on defining the pathogenic contributions of the c.611C>T variant of SCN4A. The potential fates of the c.611C>T variant of SCN4A are depicted in Figure 5.

Figure 5

Figure 5. Possible fates of the c.611C>T variant of SCN4A. The mechanisms explored in this study are shown in boldface black, such as “gating properties of A204V,” “trafficking efficiency of A204V,” and “cytotoxicity of A204Vfs*94.” The potential pathological mechanisms that have not been elucidated are shown in red squares, such as “nonsense-mediated mRNA decay (NMD)” and “endoplasmic-reticulum-associated degradation (ERAD).” In vivo, NMD is likely to degrade the SCN4A mRNA with c.611_612del to minimize production of the truncated p.A204Vfs*94 Nav1.4 protein. The p.A204V Nav1.4 protein is likely to be produced; however, the missense change may impair protein stability, leading to degradation by ERAD.

The phenotypic spectrum of SCN4A variants has been expanding, and functional studies on skeletal muscle channelopathies that have historically focused primarily on the ion channel functions of the variant proteins may pose limitations to fully define the pathogenic mechanisms underlying the atypical phenotype of the muscle weakness reported in the present study. At this point, the pathogenicity of the c.611C>T variant of SCN4A remains ambiguous, and we cannot exclude the possibility that the c.323T>C variant of TMEM43 is causally associated with the patient’s symptoms. We are presently following up with the patient to determine if she could develop an arrhythmia later on based on the previous study report of latent EDMD7 (Liang et al., 2011); if this were found to be the case, functional analysis of the c.323T>C variant of TMEM43 should be pursued.

Lastly, our continuing efforts to recruit the proband’s family members and other patients with similar disease manifestations for genetic testing would be crucial for definite identification of the pathogenic variant(s) underlying atypical neuromuscular phenotypes. whole genome sequencing analysis will be considered for these future efforts to address the possibility that the true pathogenic variant resides within the non-coding genomic region that was not examined in this study.

Data availability statement

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethics statement

The studies involving humans were approved by the Institutional Review Committee at the University of Osaka (approval #721). The studies were conducted in accordance with all local legislation and institutional requirements. The participants provided their written informed consent to participate in this study. Written informed consent was also obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.

Author contributions

NK: Data curation, Investigation, Writing – review and editing. KY: Resources, Writing – review and editing. ST: Data curation, Formal analysis, Investigation, Methodology, Supervision, Writing – original draft, Writing – review and editing. AY: Investigation, Writing – review and editing. TaK: Resources, Writing – review and editing. KH: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Supervision, Writing – original draft, Writing – review and editing. MT: Conceptualization, Funding acquisition, Supervision, Writing – review and editing. ToK: Conceptualization, Data curation, Formal analysis, Funding acquisition, Supervision, Writing – original draft, Writing – review and editing.

Funding

The author(s) declare that financial support was received for the research and/or publication of this article. This study was partly supported by the Research Grant for Intractable Disease from the Ministry of Health, Labour, and Welfare (no. 23FC1014a) to MT; Japan Agency for Medical Research and Development (no. JP24ek0109701) and Japan Society for the Promotion of Science (JSPS) KAKENHI (no. JP25K02581) to ToK; and NIH (no. DC017482) to KH.

Acknowledgements

All authors thank Akiko Ohno for the technical support. The authors also thank the Research Institute for Microbial Diseases (RIMD) Next Generation Sequencing (NGS) core facility at the University of Osaka and Kazusa DNA Research Institute for assistance with the genetic analyses.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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The authors declare that no Generative AI was used in the creation of this manuscript.

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