Drug repurposing in skeletal muscle ion channelopathies.

Skeletal muscle ion channelopathies are rare genetic diseases mainly characterized by myotonia (muscle stiffness) or periodic paralysis (muscle weakness). Here, we reviewed the available therapeutic options in non-dystrophic myotonias (NDM) and periodic paralyses (PP), which consists essentially in drug repositioning to address stiffness or weakness attacks. Empirical use followed by successful randomized clinical trials eventually led to the orphan drug designation and marketing authorization granting of mexiletine for NDM and dichlorphenamide for PP. Yet, these treatments neither consider the genetic cause of the diseases nor address the individual variability in drug response. Thus, ongoing research aims at the identification of repurposed drugs alternative to mexiletine and dichlorphenamide to allow personalization of treatment. This review highlights how drug repurposing may represent an efficient strategy in rare diseases, allowing reduction of drug development time and costs in a context in which the return on investment may be particularly challenging.

Clinical and genetic spectrum of a Chinese cohort with SCN4A gene mutations.

Skeletal muscle sodium channelopathies due to SCN4A gene mutations have a broad clinical spectrum. However, each phenotype has been reported in few cases of Chinese origin. We present detailed phenotype and genotype data from a cohort of 40 cases with SCN4A gene mutations seen in neuromuscular diagnostic service in Huashan hospital, Fudan University. Cases were referred from 6 independent provinces from 2010 to 2018. A questionnaire covering demographics, precipitating factors, episodes of paralysis and myotonia was designed to collect the clinical information. Electrodiagnostic studies and muscle MRI were retrospectively analyzed. The clinical spectrum of patients included: 6 Hyperkalemic periodic paralysis (15%), 18 Hypokalemic periodic paralysis (45%), 7 sodium channel myotonia (17.5%), 4 paramyotonia congenita (10%) and 5 heterozygous asymptomatic mutation carriers (12.5%). Review of clinical information highlights a significant delay to diagnosis (median 15 years), reports of pain and myalgia in the majority of patients, male predominance, circadian rhythm and common precipitating factors. Electrodiagnostic studies revealed subclinical myotonic discharges and a positive long exercise test in asymptomatic carriers. Muscle MRI identified edema and fatty infiltration in gastrocnemius and soleus. A total of 13 reported and 2 novel SCN4A mutations were identified with most variants distributed in the transmembrane helix S4 to S6, with a hotspot mutation p.Arg675Gln accounting for 32.5% (13/40) of the cohort. Our study revealed a higher proportion of periodic paralysis in SCN4A-mutated patients compared with cohorts from England and the Netherlands. It also highlights the importance of electrodiagnostic studies in diagnosis and segregation studies.

Pain as a significant symptom in patients with periodic paralysis-A cross-sectional survey.

INTRODUCTION: Periodic paralysis (PP) is thought to be limited to episodes of muscle weakness, but there are reports of fibromyalgia-like pain in PP. We aimed to evaluate pain and comorbid sleep, fatigue, and mood disorders in PP patients. METHODS: We administered a cross-sectional survey to PP patients at the 2019 Periodic Paralysis Conference. The survey consisted of the Brief Pain Inventory, Widespread Pain Index, Pittsburgh Sleep Quality Index, Modified Fatigue Impact Scale, and ten-question Center for Epidemiologic Studies Depression Scale (CESD-10). Descriptive statistics for PP patients were calculated and compared with earlier studies. RESULTS: Forty-four individuals with PP took the survey. Of these patients, 52.3% reported a moderate to severe interference of pain on their lives, and 45.5% met the study criteria for fibromyalgia. Patients with SCN4A mutations had higher rates of fibromyalgia than the next most prevalent gene mutation, CACNA1S. In patients with pain, there were increased rates of comorbid fatigue, depression, and poor sleep quality. DISCUSSION: Pain, akin to fibromyalgia, is a significant symptom of PP and can affect quality of life. Pain in PP was more prevalent than in the general population, at a rate comparable with other chronic neuromuscular disease groups. PP patients could benefit from a multidisciplinary approach to assess their pain, sleep, fatigue, and mood.

Next-generation sequencing application to investigate skeletal muscle channelopathies in a large cohort of Italian patients.

Non-dystrophic myotonias and periodic paralyses are a heterogeneous group of disabling diseases classified as skeletal muscle channelopathies. Their genetic characterization is essential for prognostic and therapeutic purposes; however, several genes are involved. Sanger-based sequencing of a single gene is time-consuming, often expensive; thus, we designed a next-generation sequencing panel of 56 putative candidate genes for skeletal muscle channelopathies, codifying for proteins involved in excitability, excitation-contraction coupling, and metabolism of muscle fibres. We analyzed a large cohort of 109 Italian patients with a suspect of NDM or PP by next-generation sequencing. We identified 24 patients mutated in CLCN1 gene, 15 in SCN4A, 3 in both CLCN1 and SCN4A, 1 in ATP2A1, 1 in KCNA1 and 1 in CASQ1. Eight were novel mutations: p.G395Cfs*32, p.L843P, p.V829M, p.E258E and c.1471+4delTCAAGAC in CLCN1, p.K1302R in SCN4A, p.L208P in ATP2A1 and c.280-1G>C in CASQ1 genes. This study demonstrated the utility of targeted next generation sequencing approach in molecular diagnosis of skeletal muscle channelopathies and the importance of the collaboration between clinicians and molecular geneticists and additional methods for unclear variants to make a conclusive diagnosis.

Familial periodic paralysis associated with a rare KCNJ5 variant that supposed to have incomplete penetrance.

BACKGROUND: The periodic paralyses are a group of skeletal muscle channelopathies caused by variants in several ion channel genes. Potassium Inwardly Rectifying Channel Subfamily J Member 5 (KCNJ5) encodes the G-protein-activated inwardly rectifying potassium channel 4 (Kir3.4) and the heterozygous KCNJ5 variants cause familial hyperaldosteronism and long QT syndrome (LQTS). Recent studies suggested that variants in KCNJ5 are also causative for Andersen-Tawil syndrome, which showed periodic paralysis and characteristic electrocardiogram features. CLINICAL REPORT: We found a heterozygous KCNJ5 variant c.1159G > C, p.(Gly387Arg) in an individual with familial periodic paralysis using exome sequencing. Sanger sequencing revealed that this variant was inherited from his affected mother. The same variant had been previously found in two cases of familial LQTS or Andersen-Tawil syndrome, and functional analysis suggested that this variant might have loss of function effect on channel activity. However, the allele frequency of c.1159G > C variant in an East Asian population of public databases ranged from 0.21% to 0.25%, indicating possible incomplete penetrance. CONCLUSIONS: Our two patients expand the phenotypic spectrum associated with the c.1159G > C KCNJ5 variant, though the variant has very low penetrance.

Genome-wide meta-analysis reveals novel susceptibility loci for thyrotoxic periodic paralysis.

OBJECTIVE: Thyrotoxic periodic paralysis (TPP) is a rare and potentially fatal complication of hyperthyroidism. By meta-analysis of genome-wide association studies, we aim to discover novel susceptibility loci and understand the pathogenesis of TPP. METHODS: This meta-analysis comprised 319 TPP cases and 3516 healthy controls from three independent cohorts (two from Hong Kong; one from Shanghai). Genetic variants in each cohort were separately genotyped, imputed and analyzed for association with TPP. Fixed-effect meta-analysis was performed to combine the data. Using the three independent genome-wide significant variants, a weighted genetic risk score (GRS) was developed. RESULTS: Of 7 077 246 variants tested for association with TPP, 260 variants reached genome-wide significance and were represented by independent variants from four distinct genomic loci, but a risk locus for Graves' disease at 6p21.33-p21.22 was excluded from subsequent analyses. Two novel loci near TRIM2 (4q31.3; rs6827197: OR = 4.075; P = 3.46 × 10-9) and AC140912.1 (16q22.3; rs6420387: OR = 1.861; P = 2.66 × 10-8) were identified. Together with previously reported KCNJ2 (17q24.3; rs312743: OR = 2.564; P = 1.15 × 10-21), the three susceptibility variants explained 4.36% of the genetic liability. Expression quantitative trait loci analyses showed the variants altered expression of TRIM2 in nerve and KCNJ2 in skeletal muscle. The weighted GRS had an area under curve of 0.827 and 0.682 in the derivation and validation cohorts in Hong Kong. CONCLUSIONS: We identified two novel TPP risk loci near TRIM2 and AC140912.1. While rare mutations in TRIM2 and KCNJ2 were implicated in monogenic disorders characterized by muscle paralysis, our study suggested common variants near these genes might dysregulate gene expression and lead to milder phenotypes.

Identification of a SCN4A mutation in a large Chinese family with atypical normokalemic periodic paralysis using whole-exome sequencing.

OBJECTIVES: Normokalemic periodic paralysis (NormoKPP) of skeletal muscle is an autosomal dominant disorder caused by mutations in the gene encoding voltage-gated sodium channel protein type 4 subunit alpha (SCN4A), which leads to ion channel dysfunction. Little is known about the relationship between genotype and the clinical symptoms of NormoKPP. The present study aimed to evaluate the genetic variation in a large Chinese family with NormoKPP. The patients in this pedigree did not respond to saline treatment, but calcium gluconate treatment was effective. METHODS: We performed a series of clinical examinations and genetic analyses, using whole-exome and Sanger sequencing, to examine the mutation status of SCN4A in a Chinese family segregating for NormoKPP. RESULTS: Whole-exome sequencing revealed a c.2111C>T substitution in SCN4A in most of the affected family members. This mutation results in the amino acid substitution p.T704M. CONCLUSIONS: These results support a causative role of this mutation in SCN4A in NormoKPP, and provide information about the relationship between genotype and atypical clinical symptoms.

Improving genetic diagnostics of skeletal muscle channelopathies.

INTRODUCTION: Skeletal muscle channelopathies are rare inherited conditions that cause significant morbidity and impact on quality of life. Some subsets have a mortality risk. Improved genetic methodology and understanding of phenotypes have improved diagnostic accuracy and yield. AREAS COVERED: We discuss diagnostic advances since the advent of next-generation sequencing and the role of whole exome and genome sequencing. Advances in genotype-phenotype-functional correlations have improved understanding of inheritance and phenotypes. We outline new phenotypes, particularly in the pediatric setting and consider co-existing mutations that may act as genetic modifiers. We also discuss four newly identified genes associated with skeletal muscle channelopathies. EXPERT OPINION: Next-generation sequencing using gene panels has improved diagnostic rates, identified new mutations, and discovered patients with co-existing pathogenic mutations ('double trouble'). This field has previously focussed on single genes, but we are now beginning to understand interactions between co-existing mutations, genetic modifiers, and their role in pathomechanisms. New genetic observations in pediatric presentations of channelopathies broadens our understanding of the conditions. Genetic and mechanistic advances have increased the potential to develop treatments.

Family with primary periodic paralysis and a mutation in MCM3AP, a gene implicated in mRNA transport.

INTRODUCTION: Primary periodic paralyses (PPs) are rare genetic neuromuscular disorders commonly caused by mutations in genes related to ion channel function. However, 10%-20% of cases remain as genetically unexplained. Herein we present a family with PP with paralytic episodes generally lasting for 1-7 days at a time, associated with a drop in K+ levels. METHODS: Screening for mutations in known disease-causing genes was negative, hence we performed whole-exome sequencing of 5 family members. RESULTS: Minichromosome maintenance 3-associated protein (MCM3AP) c.2615G>A (p.C872Y) was found to cosegregate with disease in the family and was not present in control subjects. The mutation is novel, highly conserved across multiple species, and predicted to be damaging. DISCUSSION: MCM3AP encodes germinal center-associated nuclear protein (GANP), a protein involved in the export of certain messenger RNAs from the nucleus to the cytoplasm. Our findings suggest that a novel mutation in MCM3AP is associated with hypokalemic PP. Muscle Nerve, 2019.

Successful treatment of normokalemic periodic paralysis with hydrochlorothiazide.

BACKGROUND: Periodic paralysis (PP) is an autosomal dominant muscle disorder characterized by periodic muscle weakness attacks associated with serum potassium level variations. It is classified into hypokalemic (hypoKPP), hyperkalemic (hyperKPP), and normokalemic (normoKPP) forms based on the ictal serum potassium level. HyperKPP and normoKPP are caused by mutations of the same gene SCN4A, the gene encoding the skeletal muscle voltage-gated sodium channel. Prophylactic treatment with thiazide diuretics is highly effective in preventing attacks in hyperKPP. However, the efficacy and safety of such diuretics in normoKPP remain unclear. CASE: We describe a familial case of normoKPP wherein the affected individuals showed periodic muscle weakness attacks, with an early childhood onset, and a lack of serum potassium level variation during the paralytic attacks. Sequencing analysis of SCN4A gene revealed a heterozygous missense mutation (c. 2111C > T, p. Thr704Met) in all symptomatic family members. Oral administration of hydrochlorothiazide, a thiazide diuretic, markedly improved the paralytic attack frequency and duration in the affected individuals without adverse effects. CONCLUSION: Our case demonstrates the efficacy of hydrochlorothiazide in the prophylactic treatment of normoKPP caused by the SCN4A mutation of p.Thr704Met, the most frequent mutation of hyperKPP.

Structural basis for gating pore current in periodic paralysis.

Potassium-sensitive hypokalaemic and normokalaemic periodic paralysis are inherited skeletal muscle diseases characterized by episodes of flaccid muscle weakness1,2. They are caused by single mutations in positively charged residues ('gating charges') in the S4 transmembrane segment of the voltage sensor of the voltage-gated sodium channel Nav1.4 or the calcium channel Cav1.11,2. Mutations of the outermost gating charges (R1 and R2) cause hypokalaemic periodic paralysis1,2 by creating a pathogenic gating pore in the voltage sensor through which cations leak in the resting state3,4. Mutations of the third gating charge (R3) cause normokalaemic periodic paralysis 5 owing to cation leak in both activated and inactivated states 6 . Here we present high-resolution structures of the model bacterial sodium channel NavAb with the analogous gating-charge mutations7,8, which have similar functional effects as in the human channels. The R2G and R3G mutations have no effect on the backbone structures of the voltage sensor, but they create an aqueous cavity near the hydrophobic constriction site that controls gating charge movement through the voltage sensor. The R3G mutation extends the extracellular aqueous cleft through the entire length of the activated voltage sensor, creating an aqueous path through the membrane. Conversely, molecular modelling shows that the R2G mutation creates a continuous aqueous path through the membrane only in the resting state. Crystal structures of NavAb(R2G) in complex with guanidinium define a potential drug target site. Molecular dynamics simulations illustrate the mechanism of Na+ permeation through the mutant gating pore in concert with conformational fluctuations of the gating charge R4. Our results reveal pathogenic mechanisms of periodic paralysis at the atomic level and suggest designs of drugs that may prevent ionic leak and provide symptomatic relief from hypokalaemic and normokalaemic periodic paralysis.

Periodic paralysis.

The periodic paralyses are a group of skeletal muscle channelopathies characterizeed by intermittent attacks of muscle weakness often associated with altered serum potassium levels. The underlying genetic defects include mutations in genes encoding the skeletal muscle calcium channel Cav1.1, sodium channel Nav1.4, and potassium channels Kir2.1, Kir3.4, and possibly Kir2.6. Our increasing knowledge of how mutant channels affect muscle excitability has resulted in better understanding of many clinical phenomena which have been known for decades and sheds light on some of the factors that trigger attacks. Insights into the pathophysiology are also leading to new therapeutic approaches.

Atypical periodic paralysis and myalgia: A novel RYR1 phenotype.

OBJECTIVE: To characterize the phenotype of patients with symptoms of periodic paralysis (PP) and ryanodine receptor (RYR1) gene mutations. METHODS: Cases with a possible diagnosis of PP but additional clinicopathologic findings previously associated with RYR1-related disorders were referred for a tertiary neuromuscular clinical assessment in which they underwent detailed clinical evaluation, including neurophysiologic assessment, muscle biopsy, and muscle MRI. Genetic analysis with next-generation sequencing and/or targeted Sanger sequencing was performed. RESULTS: Three cases with episodic muscle paralysis or weakness and additional findings compatible with a RYR1-related myopathy were identified. The McManis test, used in the diagnosis of PP, was positive in 2 of 3 cases. Genetic analysis of known PP genes was negative. RYR1 analysis confirmed likely pathogenic variants in all 3 cases. CONCLUSIONS: RYR1 mutations can cause late-onset atypical PP both with and without associated myopathy. Myalgia and cramps are prominent features. The McManis test may be a useful diagnostic tool to indicate RYR1-associated PP. We propose that clinicopathologic features suggestive of RYR1-related disorders should be sought in genetically undefined PP cases and that RYR1 gene testing be considered in those in whom mutations in SCN4A, CACNA1S, and KCNJ2 have already been excluded.

Kir2.2 p.Thr140Met: a genetic susceptibility to sporadic periodic paralysis.

INTRODUCTION: Periodic paralyses (PP) are recurrent episodes of flaccid limb muscle weakness. Next to autosomal dominant forms, sporadic PP (SPP) cases are known but their genetics are unclear. METHODS: In a patient with hypokalemic SPP, we performed exome sequencing to identify a candidate gene. We sequenced this gene in 263 unrelated PP patients without any known causative mutations. Then we performed functional analysis of all variants found and molecular modelling for interpretation. RESULTS: Exome sequencing in the proband yielded three heterozygous variants predicted to be linked to disease. These encoded p.Thr140Met in the Kir2.2 potassium channel, p.Asp229Asn in protein kinase C theta, and p.Thr15943Ile in titin. Since all hitherto known causative PP genes code for ion channels, we studied the Kir2.2-encoding gene, KCNJ12, for involvement in PP pathogenesis. KCNJ12 screening in 263 PP patients revealed three further variants, each in a single individual and coding for p.Gly419Ser, p.Cys75Tyr, and p.Ile283Val. All four Kir2.2 variants were functionally expressed. Only p.Thr140Met displayed relevant functional alterations, i.e. homo-tetrameric channels produced almost no current, and hetero-tetrameric channels suppressed co-expressed wildtype Kir2.1 in a dominant-negative manner. Molecular modelling showed Kir2.2 p.Thr140Met to reduce movement of potassium ions towards binding sites in the hetero-tetramer pore compatible with a reduced maximal current. MD simulations revealed loss of hydrogen bonding with the p.Thr140Met substitution. DISCUSSION: The electrophysiological findings of p.Thr140Met are similar to those found in thyrotoxic PP caused by Kir2.6 mutations. Also, the homologous Thr140 residue is mutated in Kir2.6. This supports the idea that Kir2.2 p.Thr140Met conveys susceptibility to SPP and should be included in genetic screening.

Effects of S906T polymorphism on the severity of a novel borderline mutation I692M in Nav 1.4 cause periodic paralysis.

Hyperkalemic periodic paralysis (HyperPP) is a dominantly inherited muscle disease caused by mutations in SCN4A gene encoding skeletal muscle voltage gated Nav 1.4 channels. We identified a novel Nav 1.4 mutation I692M in 14 families out of the 104 genetically identified HyperPP families in the Neuromuscular Centre Ulm and is therefore as frequent as I693T (13 families out of 14 HyperPP families) in Germany. Surprisingly, in 13 families, a known polymorphism S906T was also present. It was on the affected allele in at least 10 families compatible with a possible founder effect in central Europe. All affected members suffered from episodic weakness; myotonia was also common. Compared with I692M patients, I692M-S906T patients had longer weakness episodes, more affected muscles, CK elevation and presence of permanent weakness. Electrophysiological investigation showed that both mutants had incomplete slow inactivation and a hyperpolarizing shift of activation which contribute to membrane depolarization and weakness. Additionally, I692M-S906T significantly enhanced close-state fast inactivation compared with I692M alone, suggesting a higher proportion of inactivated I692M-S906T channels upon membrane depolarization which may facilitate the initiation of weakness episodes and therefore clinical manifestation. Our results suggest that polymorphism S906T has effects on the clinical phenotypic and electrophysiological severity of a novel borderline Nav 1.4 mutation I692M, making the borderline mutation fully penetrant.

Influences of Pregnancy on Different Genetic Subtypes of Non-Dystrophic Myotonia and Periodic Paralysis.

BACKGROUND: There are only few reports of pregnancy and delivery in non-dystrophic myotonia or periodic paralysis caused by CLCN1 or SCN4A gene mutations. METHODS: We report the medical histories and personal attitudes of 5 unrelated German patients, 2 following autosomal recessive inheritance (case 1; most likely and case 2; confirmed Becker disease) and 3 following autosomal dominant inheritance (case 3; CLCN1 mutation, cases 4-5; SCN4A mutations), who delivered a total of 9 children. RESULTS: Apart from case 5 with periodic paralysis, who had 5 early miscarriages and pre-eclampsia resulting in cesarean delivery, there was no evidence of increased obstetric complication rates, and neonatal outcome was favorable. In all patients, there was aggravation of myotonia or weakness in pregnancy, followed by a short-term improvement after delivery in cases 2 and 3. Mexiletine medication improved the clinical features significantly in case 2 but was unable to control pregnancy-related deterioration. In case 4 (and her sister) and case 5, there was a clear disease aggravation in pregnancy resulting in hospitalization or repeated neurological examinations. CONCLUSION: Pregnancy can be regarded as a strong triggering factor in inherited non-dystrophic myotonias and periodic paralysis, regardless of the underlying gene defect.

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.

Periodic Paralysis as a New Phenotype of Resistance to Thyroid Hormone Syndrome in a Chinese Male Adult.

CONTEXT: A majority of patients with resistance to thyroid hormone (RTH) are asymptomatic, whereas some patients show signs of hyperthyroidism, or hypothyroidism, or both. Thyrotoxic periodic paralysis is the most common form of acquired periodic paralysis. However, it has not been reported in a patient with RTH up to now. OBJECTIVE: We evaluated a 36-year-old male patient from China with elevated serum free T4 and free T3 and inappropriately high TSH who presented with periodic paralysis. STUDY DESIGN: Clinical, biochemical, and radiological assessments, as well as DNA sequencing, were performed. RESULTS: The patient's laboratory tests revealed the following: TSH, 6.14 mIU/L (0.27-4.2 mIU/L); free T3, 12.85 pmol/L (2.8-7.1 pmol/L); free T4, 33.62 pmol/L (9.05-25.5 pmol/L); and serum SHBG, 19.4 nmol/L (18.3-54.1 nmol/L). No significant suppression of TSH was observed in the rapid TSH suppression test with somatostatin analogs. Compound muscle action potential after exercise of the patient was reduced by 58%. Sequencing of thyroid hormone receptor genes confirmed a C446S mutation in the THRß gene. CONCLUSIONS: This is the first report of periodic paralysis as a new phenotype of RTH syndrome.

Successful treatment of periodic paralysis with coenzyme Q10: two case reports.

Primary periodic paralyses (PPs) are autosomal dominant ion channel disorders characterized by episodic flaccid weakness associated with variations in serum potassium level. The main prophylactic therapy of choice for PPsis carbonic anhydrase inhibitors that are not always effective. In this report, we described two PP patients who were successfully treated with coenzyme Q10. They remained asymptomatic since initiation of treatment, which may be associated with promotion of energy synthesis, anti-oxidant activity, influence of the fiber type composition and regulation of the expression of gene. To our knowledge, this is the first report of primary periodic paralyses which have been successfully treated with CoQ10. More observations need to substantiate this clinical finding in PPs.