Roope Männikkö

Loss-of-function mutations in SCN4A cause severe foetal hypokinesia or 'classical' congenital myopathy

See Cannon (doi: 10.1093/brain/awv400 ) for a scientific commentary on this article. Dominant gain-of-function mutations in SCN4A , which encodes the α-subunit of the voltage-gated sodium channel, are a common cause of myotonia and periodic paralysis. Zaharieva et al. now report recessive loss-of-function SCN4A mutations in 11 patents with congenital myopathy. The mutations cause fully non-functional channels or result in reduced channel activity.

Muscle channelopathies: recent advances in genetics, pathophysiology and therapy.

PURPOSE OF REVIEW This article reviews recent advances in clinical, genetic, diagnostic and pathophysiological aspects of the skeletal muscle channelopathies. RECENT FINDINGS Genetic advances include the use of the minigene assay to confirm pathogenicity of splice site mutations of CLC-1 chloride channels and a new gene association for Andersen-Tawil syndrome. Mutations causing a gating pore current have been established as a pathomechanism for hypokalaemic periodic paralysis. Mutations in nonchannel genes, including the mitochondrial mATP6/8 genes, have been linked to channelopathy-like episodic weakness. Advances in diagnostic tools include the use of MRI and muscle velocity recovery cycles to evaluate myotonia congenita patients. Specific neonatal presentations of sodium channel myotonia are now well documented. An international multicentre placebo-controlled randomized clinical trial established that mexiletine is an effective therapy in the nondystrophic myotonias. This is the first evidence-based treatment for a skeletal muscle channelopathy. Recent evidence in mouse models indicated that bumetanide can prevent attacks of hypokalaemic periodic paralysis, but this has not yet been tested in patient trials. SUMMARY Advances in genetic, clinical, diagnostic and pathomechanistic understanding of skeletal muscle channelopathies are being translated into improved therapies. Mexiletine is the first evidence-based treatment for nondystrophic myotonias. Bumetanide is effective in preventing attacks in mouse models of hypokalaemic periodic paralysis and now needs to be tested in patients.

A recessive Nav1.4 mutation underlies congenital myasthenic syndrome with periodic paralysis

Objective: To determine the molecular basis of a complex phenotype of congenital muscle weakness observed in an isolated but consanguineous patient. Methods: The proband was evaluated clinically and neurophysiologically over a period of 15 years. Genetic testing of candidate genes was performed. Functional characterization of the candidate mutation was done in mammalian cell background using whole cell patch clamp technique. Results: The proband had fatigable muscle weakness characteristic of congenital myasthenic syndrome with acute and reversible attacks of most severe muscle weakness as observed in periodic paralysis. We identified a novel homozygous SCN4A mutation (p.R1454W) linked to this recessively inherited phenotype. The p.R1454W substitution induced an important enhancement of fast and slow inactivation, a slower recovery for these inactivated states, and a frequency-dependent regulation of Nav1.4 channels in the heterologous expression system. Conclusion: We identified a novel loss-of-function mutation of Nav1.4 that leads to a recessive phenotype combining clinical symptoms and signs of congenital myasthenic syndrome and periodic paralysis, probably by decreasing channel availability for muscle action potential genesis at the neuromuscular junction and propagation along the sarcolemma.

Novel mutations in human and mouse SCN4A implicate AMPK in myotonia and periodic paralysis

Corrochano Sanchez et al. identify a novel mutation (I588V) in SCN4A, which encodes the Nav1.4 voltage-gated sodium channel, in a patient with myotonia and periodic paralysis. By generating and characterizing a mouse model (‘draggen’) carrying the equivalent point mutation (I582V), they uncover novel pathological and metabolic features of SCN4A channelopathies.

Dysfunction of NaV1.4, a skeletal muscle voltage-gated sodium channel, in sudden infant death syndrome: a case-control study

Summary Background Sudden infant death syndrome (SIDS) is the leading cause of post-neonatal infant death in high-income countries. Central respiratory system dysfunction seems to contribute to these deaths. Excitation that drives contraction of skeletal respiratory muscles is controlled by the sodium channel NaV1.4, which is encoded by the gene SCN4A. Variants in NaV1.4 that directly alter skeletal muscle excitability can cause myotonia, periodic paralysis, congenital myopathy, and myasthenic syndrome. SCN4A variants have also been found in infants with life-threatening apnoea and laryngospasm. We therefore hypothesised that rare, functionally disruptive SCN4A variants might be over-represented in infants who died from SIDS. Methods We did a case-control study, including two consecutive cohorts that included 278 SIDS cases of European ancestry and 729 ethnically matched controls without a history of cardiovascular, respiratory, or neurological disease. We compared the frequency of rare variants in SCN4A between groups (minor allele frequency <0·00005 in the Exome Aggregation Consortium). We assessed biophysical characterisation of the variant channels using a heterologous expression system. Findings Four (1·4%) of the 278 infants in the SIDS cohort had a rare functionally disruptive SCN4A variant compared with none (0%) of 729 ethnically matched controls (p=0·0057). Interpretation Rare SCN4A variants that directly alter NaV1.4 function occur in infants who had died from SIDS. These variants are predicted to significantly alter muscle membrane excitability and compromise respiratory and laryngeal function. These findings indicate that dysfunction of muscle sodium channels is a potentially modifiable risk factor in a subset of infant sudden deaths. Funding UK Medical Research Council, the Wellcome Trust, National Institute for Health Research, the British Heart Foundation, Biotronik, Cardiac Risk in the Young, Higher Education Funding Council for England, Dravet Syndrome UK, the Epilepsy Society, the Eunice Kennedy Shriver National Institute of Child Health & Human Development of the National Institutes of Health, and the Mayo Clinic Windland Smith Rice Comprehensive Sudden Cardiac Death Program.

Spider toxin inhibits gating pore currents underlying periodic paralysis

Significance Voltage-gated ion channels contain domains that have discrete functionalities. The central pore domain allows current flow and provides ion selectivity, whereas peripherally located voltage-sensing domains (VSDs) are needed for voltage-dependent gating. Certain mutations trigger a leak current through VSDs, known as gating pore current. Hypokalemic periodic paralysis (HypoPP) type 2 is caused by mutations in the skeletal muscle voltage-gated sodium channel NaV1.4 that neutralize positive charges in S4 voltage-sensing segments of VSDs. We show that Hm-3 toxin from the crab spider Heriaeus melloteei inhibits gating pore currents through such mutant channels. We propose that Hm-3 and similar toxins may constitute useful hits in developing gating pore current inhibitors and HypoPP therapy. Gating pore currents through the voltage-sensing domains (VSDs) of the skeletal muscle voltage-gated sodium channel NaV1.4 underlie hypokalemic periodic paralysis (HypoPP) type 2. Gating modifier toxins target ion channels by modifying the function of the VSDs. We tested the hypothesis that these toxins could function as blockers of the pathogenic gating pore currents. We report that a crab spider toxin Hm-3 from Heriaeus melloteei can inhibit gating pore currents due to mutations affecting the second arginine residue in the S4 helix of VSD-I that we have found in patients with HypoPP and describe here. NMR studies show that Hm-3 partitions into micelles through a hydrophobic cluster formed by aromatic residues and reveal complex formation with VSD-I through electrostatic and hydrophobic interactions with the S3b helix and the S3–S4 extracellular loop. Our data identify VSD-I as a specific binding site for neurotoxins on sodium channels. Gating modifier toxins may constitute useful hits for the treatment of HypoPP.

Predominantly myalgic phenotype caused by the c.3466G>A p.A1156T mutation in SCN4A gene

Objective: To characterize the clinical phenotype in patients with p.A1156T sodium channel mutation. Methods: Twenty-nine Finnish patients identified with the c.3466G>A p.A1156T mutation in the SCN4A gene were extensively examined. In a subsequent study, 63 patients with similar myalgic phenotype and with negative results in myotonic dystrophy type 2 genetic screening (DM2-neg group) and 93 patients diagnosed with fibromyalgia were screened for the mutation. Functional consequences of the p.A1156T mutation were studied in HEK293 cells with whole-cell patch clamp. Results: The main clinical manifestation in p.A1156T patients was not myotonia or periodic paralysis but exercise- and cold-induced muscle cramps, muscle stiffness, and myalgia. EMG myotonic discharges were detected in most but not all. Electrophysiologic compound muscle action potentials exercise test showed variable results. The p.A1156T mutation was identified in one patient in the DM2-neg group but not in the fibromyalgia group, making a total of 30 patients so far identified. Functional studies of the p.A1156T mutation showed mild attenuation of channel fast inactivation. Conclusions: The unspecific symptoms of myalgia stiffness and exercise intolerance without clinical myotonia or periodic paralysis in p.A1156T patients make the diagnosis challenging. The symptoms of milder SCN4A mutations may be confused with other similar myalgic syndromes, including fibromyalgia and myotonic dystrophy type 2.

De novo KCNA2 mutations cause hereditary spastic paraplegia

grounds, and most of them were recruited through the CanHSP network, which includes 7 centers across Canada. All patients were diagnosed by neurologists with expertise in motor neuron diseases, and all provided informed consent before participating in the study, which was approved by the respective institutional review boards. The average coverage of KCNA2 was 3 217, with a range of 358 to 3 379 across all samples, and in all samples 100% of the coding nucleotides of KCNA2 were covered by>310. No nonsynonymous, splice-site, or stop mutations were identified in any of the HSP patients. Only 3 synonymous variants were found. One is a rare variant c.G372A (p.E124E), with allele frequency (AF) of 0.0002 in ExAC (exac.broadinstitute. org), but it was found in only 1 patient of 6 affected with HSP from the same family. Two more common variants were also identified, c.G1185C (p.A395A, AF 5 0.05, rs78349687) and c.T1026C (p.D342D, AF 5 0.14, rs12407942), which also did not segregate with the HSP phenotype, and the allele frequencies are too common to be considered likely pathogenic. Our data suggest that HSP patients with KCNA2 mutations are rare, at least in our ethnically diverse population. We suggest that the various phenotypes reported in individuals with KCNA2 mutations in the recent studies, including epilepsy, intellectual disability, ataxia, and spasticity, represent a spectrum of the same disorder with the same underlying mechanism, especially because some of the recently reported cases had phenotypes similar to those previously reported. Although novel phenotypes associated with known genes are well recognized, sufficient data that support the proposed genotype–phenotype correlation is necessary. The identification of more families with clinically confirmed HSP and KCNA2 mutations would support this mutation being a separate entity causing HSP.

Hypokalaemic periodic paralysis and myotonia in a patient with homozygous mutation p.R1451L in Na V 1.4

Dominantly inherited channelopathies of the skeletal muscle voltage-gated sodium channel NaV1.4 include hypokalaemic and hyperkalaemic periodic paralysis (hypoPP and hyperPP) and myotonia. HyperPP and myotonia are caused by NaV1.4 channel overactivity and overlap clinically. Instead, hypoPP is caused by gating pore currents through the voltage sensing domains (VSDs) of NaV1.4 and seldom co-exists clinically with myotonia. Recessive loss-of-function NaV1.4 mutations have been described in congenital myopathy and myasthenic syndromes. We report two families with the NaV1.4 mutation p.R1451L, located in VSD-IV. Heterozygous carriers in both families manifest with myotonia and/or hyperPP. In contrast, a homozygous case presents with both hypoPP and myotonia, but unlike carriers of recessive NaV1.4 mutations does not manifest symptoms of myopathy or myasthenia. Functional analysis revealed reduced current density and enhanced closed state inactivation of the mutant channel, but no evidence for gating pore currents. The rate of recovery from inactivation was hastened, explaining the myotonia in p.R1451L carriers and the absence of myasthenic presentations in the homozygous proband. Our data suggest that recessive loss-of-function NaV1.4 variants can present with hypoPP without congenital myopathy or myasthenia and that myotonia can present even in carriers of homozygous NaV1.4 loss-of-function mutations.

Hypokalemic periodic paralysis: an omega pore mutation affects inactivation

Among the human diseases caused by ion channel mutations hypokalemic periodic paralysis (HypoPP) has thrown up more than its fair share of puzzles. Patients have attacks of skeletal muscle paralysis associated with low serum potassium, and harbor dominant mutations affecting the muscle calcium (CaV1.1) or sodium (NaV1.4) channels respectively. Why do mutations in either channel converge on a common phenotype, while other mutations in NaV1.4 lead to a quite different form of periodic paralysis associated with high or normal serum potassium, or muscle hyperexcitability manifesting as myotonia? A breakthrough in understanding the disease mechanisms came from the realization that the mutations affect arginine residues in the fourth transmembrane helix (S4) of the voltage-sensing domains (VSDs) of either channel. These mutations affect channel function by 2 distinct mechanisms. They can cause a loss-of-function of the channel by interfering with voltage-dependent activation and inactivation, which does not explain the phenotype, but they also open up an aberrant gating or ‘omega’ pore through the mutated VSD. The cation-mediated leak current through the gating pore is now thought to depolarize muscle fibers, inactivating sodium channels and reducing cell excitability, eventually leading to muscle paralysis. Both CaV1.1 and NaV1.4 channels contain 4 homologous domains (DI-DIV), each of which has a VSD consisting of 4 a helices (S1– S4). Each S4 contains 3 or more regularly spaced arginines which normally occlude a potential aqueous pore through the VSD. Charge neutralizing mutations of these arginines allow ions to leak through the gating pore. The HypoPP mutations tend to affect the most extracellular arginines (R1 or R2) of S4. Gating pore currents caused by these mutations are active in the hyperpolarized resting state of the VSD, when it is in the ‘down’ state. But what about arginine residues deeper in the S4? Groome and coworkers recently characterized the functional properties of the first 2 mutations, R1135H and R1135C, affecting the third arginine (R3) of DIII S4 associated with HypoPP. One of the mutations (R1135C) occurred in the homozygous state, which has not previously been reported for HypoPP. Biophysical analysis of these mutations brings new insights into the molecular rearrangements and roles of this domain in channel gating. Typical for HypoPP channels the R1135H and R1135C mutations cause loss-of-function of sodium currents and introduce gating pore currents. Atypical for HypoPP channels, these gating pore currents are activated by depolarization. A likely explanation is that the DIIIR3 normally occludes the gating pore in the ‘up’ state of the VSD. A mutation affecting another R3, DIIR3, similarly uncovers a gating pore activated by depolarization, but the R3 gating pore currents show important differences between domains II and III. In response to prolonged depolarization the DIIR3 gating pore is locked in an active state while the DIIIR3 gating pore seems to enter another state with reduced conductance. Repolarization eventually closes the DIIR3 gating pore, but re-activates the DIIIR3 gating pore. The significance of the unusual DIIIR3 gating pore currents for pathogenesis is unclear. However, the resting membrane potential of the R1135H muscle fibers is depolarized. Groome and co-workers now turn to the loss-of-function effects of DIIIR3, accounted for by a substantial enhancement of closed state inactivation. Groome et al. show that a fraction of the S4 gating charges of R1135C channels activate at more hyperpolarized voltages than the wild-type S4s. In addition, the sodium channel and the gating charge availabilities at sub-threshold voltages are both reduced in the mutant channel. Assuming that the R1135C mutation affects mainly the gating charge movement locally in DIII, the data would suggest that the enhanced activation of the DIII VSD is associated with enhanced closed-state inactivation. Enhanced mobility of the DIII VSD may provide a docking site for the inactivation particle. The altered mobility of DIII R3 mutants in NaV1.4 enhances inactivation and is likely to contribute to reduced action potential amplitude in the muscle. The gating pore currents are likely to underlie the periodic paralysis of the homozygous R1135C patient, and the role of loss of NaV1.4 function for the pathology remains to be elucidated. Thus far, there is only one report of loss-of-function mutation of Nav1.4 without gating pore currents causing a myasthenic syndrome. HypoPP still keeps some puzzles in store.