Diverse biophysical mechanisms in voltage-gated sodium channel Nav1.4 variants associated with myotonia.

Mutations in SCN4A gene encoding Nav1.4 channel α-subunit, are known to cause neuromuscular disorders such as myotonia or paralysis. Here, we study the effect of two amino acid replacements, K1302Q and G1306E, in the DIII-IV loop of the channel, corresponding to mutations found in patients with myotonia. We combine clinical, electrophysiological, and molecular modeling data to provide a holistic picture of the molecular mechanisms operating in mutant channels and eventually leading to pathology. We analyze the existing clinical data for patients with the K1302Q substitution, which was reported for adults with or without myotonia phenotypes, and report two new unrelated patients with the G1306E substitution, who presented with severe neonatal episodic laryngospasm and childhood-onset myotonia. We provide a functional analysis of the mutant channels by expressing Nav1.4 α-subunit in Xenopus oocytes in combination with ß1 subunit and recording sodium currents using two-electrode voltage clamp. The K1302Q variant exhibits abnormal voltage dependence of steady-state fast inactivation, being the likely cause of pathology. K1302Q does not lead to decelerated fast inactivation, unlike several other myotonic mutations such as G1306E. For both mutants, we observe increased window currents corresponding to a larger population of channels available for activation. To elaborate the structural rationale for our experimental data, we explore the contacts involving K/Q1302 and E1306 in the AlphaFold2 model of wild-type Nav1.4 and Monte Carlo-minimized models of mutant channels. Our data provide the missing evidence to support the classification of K1302Q variant as likely pathogenic and may be used by clinicians.

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

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

Next generation sequencing panel as an effective approach to genetic testing in patients with a highly variable phenotype of neuromuscular disorders.

Neuromuscular disorders (NMDs) include a wide range of diseases affecting the peripheral nervous system. The genetic diagnoses are increasingly obtained with using the next generation sequencing (NGS). We applied the custom-design targeted NGS panel including 89 genes, together with genotyping and multiplex ligation-dependent probe amplification (MLPA) to identify a genetic spectrum of NMDs in 52 Polish patients. As a result, the genetic diagnosis was determined by NGS panel in 29 patients so its diagnostic utility is estimated at 55.8%. The most pathogenic variants were found in CLCN1, followed by CAPN3, SCN4A, and SGCA genes. Genotyping of myotonic dystrophy type 1 and 2 (DM1 and DM2) as a secondary approach has been performed. The co-occurrence of CAPN3 and CNBP mutations in one patient as well as DYSF and CNBP mutations in another suggests possibly more complex inheritance as well as expression of a phenotype. In 7 individuals with single nucleotide variant found in NGS testing, the MLPA of the CAPN3 gene was performed detecting the deletion encompassing exons 2-8 in the CAPN3 gene in one patient, confirming recessive limb-girdle muscular dystrophy type 1 (LGMDR1). Thirty patients obtained a genetic diagnosis (57.7%) after using NGS testing, genotyping and MLPA analysis. The study allowed for the identification of 27 known and 4 novel pathogenic/likely pathogenic variants and variants of uncertain significance (VUS) associated with NMDs.In conclusion, the diagnostic approach with diverse molecular techniques enables to broaden the mutational spectrum and maximizes the diagnostic yield. Furthermore, the co-occurrence of DM2 and LGMD has been detected in 2 individuals.

Genetic analysis of 37 cases with primary periodic paralysis in Chinese patients.

BACKGROUND: Primary periodic paralysis (PPP) is an inherited disorders of ion channel dysfunction characterized by recurrent episodes of flaccid muscle weakness, which can classified as hypokalemic (HypoPP), normokalemic (NormoPP), or hyperkalemic (HyperPP) according to the potassium level during the paralytic attacks. However, PPP is charactered by remarkable clinical and genetic heterogeneity, and the diagnosis of suspected patients is based on the characteristic clinical presentation then confirmed by genetic testing. At present, there are only limited cohort studies on PPP in the Chinese population. RESULTS: We included 37 patients with a clinical diagnosis of PPP. Eleven (29.7%) patients were tested using a specific gene panel and 26 (70.3%) by the whole-exome sequencing (WES). Twenty-two cases had a genetic variant identified, representing a diagnostic rate of 59.5% (22/37). All the identified mutations were either in the SCN4A or the CACNA1S gene. The overall detection rate was comparable between the panel (54.5%: 6/11) and WES (61.5%: 16/26). The remaining patients unresolved through panel sequencing were further analyzed by WES, without the detection of any mutation. The novel atypical splicing variant c.2020-5G > A affects the normal splicing of the SCN4A mRNA, which was confirmed by minigene splicing assay. Among 21 patients with HypoPP, 15 patients were classified as HypoPP-2 with SCN4A variants, and 6 HypoPP-1 patients had CACNA1S variants. CONCLUSIONS: Our results suggest that SCN4A alleles are the main cause in our cohort, with the remainder caused by CACNA1S alleles, which are the predominant cause in Europe and the United States. Additionally, this study identified 3 novel SCN4A and 2 novel CACNA1S variants, broadening the mutation spectrum of genes associated with PPP.

Toxic to the touch: The makings of lethal mantles in pitohui birds and poison dart frogs.

How do chemically defended animals resist their own toxins? This intriguing question on the concept of autotoxicity is at the heart of how species interactions evolve. In this issue of Molecular Ecology (Molecular Ecology, 2024, 33), Bodawatta and colleagues report on how Papua New Guinean birds coopted deadly neurotoxins to create lethal mantles that protect against predators and parasites. Combining chemical screening of the plumage of a diverse collection of passerine birds with genome sequencing, the researchers unlocked a deeper understanding of how some birds sequester deadly batrachotoxin (BTX) from their food without poisoning themselves. They identified that birds impervious to BTX bear amino acid substitutions in the toxin-binding site of the voltage-gated sodium channel Nav1.4, whose function is essential for proper contraction and relaxation of vertebrate muscles. Comparative genetic and molecular docking analyses show that several of the substitutions associated with insensitivity to BTX may have become prevalent among toxic birds through positive selection. Intriguingly, poison dart frogs that also co-opted BTX in their lethal mantles were found to harbour similar toxin insensitivity substitutions in their Nav1.4 channels. Taken together, this sets up a powerful model system for studying the mechanisms behind convergent molecular evolution and how it may drive biological diversity.

A c.1775C > T Point Mutation of Sodium Channel Alfa Subunit Gene (SCN4A) in a Three-Generation Sardinian Family with Sodium Channel Myotonia.

Background: The nondystrophic myotonias are rare muscle hyperexcitability disorders caused by gain-of-function mutations in the SCN4A gene or loss-of-function mutations in the CLCN1 gene. Clinically, they are characterized by myotonia, defined as delayed muscle relaxation after voluntary contraction, which leads to symptoms of muscle stiffness, pain, fatigue, and weakness. Diagnosis is based on history and examination findings, the presence of electrical myotonia on electromyography, and genetic confirmation. Methods: Next-generation sequencing including the CLCN1 and SCN4A genes was performed in patients with clinical neuromuscular disorders. Electromyography, Short Exercise Test, in vivo and in vitro electrophysiology, site-directed mutagenesis and heterologous expression were collected. Results: A heterozygous point mutation (c.1775C > T, p.Thr592Ile) of muscle voltage-gated sodium channel α subunit gene (SCN4A) has been identified in five female patients over three generations, in a family with non-dystrophic myotonia. The muscle stiffness and myotonia involve mainly the face and hands, but also affect walking and running, appearing early after birth and presenting a clear cold sensitivity. Very hot temperatures, menstruation and pregnancy also exacerbate the symptoms; muscle pain and a warm-up phenomenon are variable features. Neither paralytic attacks nor post-exercise weakness has been reported. Muscle hypertrophy with cramp-like pain and increased stiffness developed during pregnancy. The symptoms were controlled with both mexiletine and acetazolamide. The Short Exercise Test after muscle cooling revealed two different patterns, with moderate absolute changes of compound muscle action potential amplitude. Conclusions: The p.Thr592Ile mutation in the SCN4A gene identified in this Sardinian family was responsible of clinical phenotype of myotonia.

Idiopathic generalized epilepsy in a family with SCN4A-related myotonia.

OBJECTIVES: Myotonia is a clinical sign typical of a group of skeletal muscle channelopathies, the non-dystrophic myotonias. These disorders are electrophysiologically characterized by altered membrane excitability, due to specific genetic variants in known causative genes (CLCN1 and SCN4A). Juvenile Myoclonic Epilepsy (JME) is an epileptic syndrome identified as idiopathic generalized epilepsy, its genetics is complex and still unclarified. The co-occurrence of these two phenotypes is rare and the causes likely have a genetic background. In this study, we have genetically investigated an Italian family in which co-segregates myotonia, JME, or abnormal EEG without seizures was observed. METHODS: All six individuals of the family, 4 affected and 2 unaffected, were clinically evaluated; EMG and EEG examinations were performed. For genetic testing, Exome Sequencing was performed for the six family members and Sanger sequencing was used to confirm the candidate variant. RESULTS: Four family members, the mother and three siblings, were affected by myotonia. Moreover, EEG recordings revealed interictal generalized sharp-wave discharges in all affected individuals, and two siblings were affected by JME. All four affected members share the same identified variant, c.644 T > C, p.Ile215Thr, in SCN4A gene. Variants that could account for the epileptic phenotype alone, separately from the myotonic one, were not identified. SIGNIFICANCE: These results provide supporting evidence that both myotonic and epileptic phenotypes could share a common genetic background, due to variants in SCN4A gene. SCN4A pathogenic variants, already known to be causative of myotonia, likely increase the susceptibility to epilepsy in our family. PLAIN LANGUAGE SUMMARY: This study analyzed all members of an Italian family, in which the mother and three siblings had myotonia and epilepsy. Genetic analysis allowed to identify a variant in the SCN4A gene, which appears to be the cause of both clinical signs in this family.