In silico versus functional characterization of genetic variants: lessons from muscle channelopathies.

Accurate determination of the pathogenicity of missense genetic variants of uncertain significance is a huge challenge for implementing genetic data in clinical practice. In silico predictive tools are used to score variants' pathogenicity. However, their value in clinical settings is often unclear, as they have not usually been validated against robust functional assays. We compared nine widely used in silico predictive tools, including more recently developed tools (EVE and REVEL) with detailed cell-based electrophysiology, for 126 CLCN1 variants discovered in patients with the skeletal muscle channelopathy myotonia congenita. We found poor accuracy for most tools. The highest accuracy was obtained with MutationTaster (84.58%) and REVEL (82.54%). Both of these scores showed poor specificity, although specificity was better using EVE. Combining methods based on concordance improved performance overall but still lacked specificity. Our calculated statistics for the predictive tools were different to reported values for other genes in the literature, suggesting that the utility of the tools varies between genes. Overall, current predictive tools for this chloride channel are not reliable for clinical use, and tools with better specificity are urgently required. Improving the accuracy of predictive tools is a wider issue and a huge challenge for effective clinical implementation of genetic data.

De novo KCNA6 variants with attenuated KV 1.6 channel deactivation in patients with epilepsy.

OBJECTIVE: Mutations in the genes encoding neuronal ion channels are a common cause of Mendelian neurological diseases. We sought to identify novel de novo sequence variants in cases with early infantile epileptic phenotypes and neurodevelopmental anomalies. METHODS: Following clinical diagnosis, we performed whole exome sequencing of the index cases and their parents. Identified channel variants were expressed in Xenopus oocytes and their functional properties assessed using two-electrode voltage clamp. RESULTS: We identified novel de novo variants in KCNA6 in four unrelated individuals variably affected with neurodevelopmental disorders and seizures with onset in the first year of life. Three of the four identified mutations affect the pore-lining S6 α-helix of KV 1.6. A prominent finding of functional characterization in Xenopus oocytes was that the channel variants showed only minor effects on channel activation but slowed channel closure and shifted the voltage dependence of deactivation in a hyperpolarizing direction. Channels with a mutation affecting the S6 helix display dominant effects on channel deactivation when co-expressed with wild-type KV 1.6 or KV 1.1 subunits. SIGNIFICANCE: This is the first report of de novo nonsynonymous variants in KCNA6 associated with neurological or any clinical features. Channel variants showed a consistent effect on channel deactivation, slowing the rate of channel closure following normal activation. This specific gain-of-function feature is likely to underlie the neurological phenotype in our patients. Our data highlight KCNA6 as a novel channelopathy gene associated with early infantile epileptic phenotypes and neurodevelopmental anomalies.

Translating genetic and functional data into clinical practice: a series of 223 families with myotonia.

High-throughput DNA sequencing is increasingly employed to diagnose single gene neurological and neuromuscular disorders. Large volumes of data present new challenges in data interpretation and its useful translation into clinical and genetic counselling for families. Even when a plausible gene is identified with confidence, interpretation of the clinical significance and inheritance pattern of variants can be challenging. We report our approach to evaluating variants in the skeletal muscle chloride channel ClC-1 identified in 223 probands with myotonia congenita as an example of these challenges. Sequencing of CLCN1, the gene that encodes CLC-1, is central to the diagnosis of myotonia congenita. However, interpreting the pathogenicity and inheritance pattern of novel variants is notoriously difficult as both dominant and recessive mutations are reported throughout the channel sequence, ClC-1 structure-function is poorly understood and significant intra- and interfamilial variability in phenotype is reported. Heterologous expression systems to study functional consequences of CIC-1 variants are widely reported to aid the assessment of pathogenicity and inheritance pattern. However, heterogeneity of reported analyses does not allow for the systematic correlation of available functional and genetic data. We report the systematic evaluation of 95 CIC-1 variants in 223 probands, the largest reported patient cohort, in which we apply standardized functional analyses and correlate this with clinical assessment and inheritance pattern. Such correlation is important to determine whether functional data improves the accuracy of variant interpretation and likely mode of inheritance. Our data provide an evidence-based approach that functional characterization of ClC-1 variants improves clinical interpretation of their pathogenicity and inheritance pattern, and serve as reference for 34 previously unreported and 28 previously uncharacterized CLCN1 variants. In addition, we identify novel pathogenic mechanisms and find that variants that alter voltage dependence of activation cluster in the first half of the transmembrane domains and variants that yield no currents cluster in the second half of the transmembrane domain. None of the variants in the intracellular domains were associated with dominant functional features or dominant inheritance pattern of myotonia congenita. Our data help provide an initial estimate of the anticipated inheritance pattern based on the location of a novel variant and shows that systematic functional characterization can significantly refine the assessment of risk of an associated inheritance pattern and consequently the clinical and genetic counselling.

Andersen-Tawil syndrome: deep phenotyping reveals significant cardiac and neuromuscular morbidity.

Andersen-Tawil syndrome is a neurological channelopathy caused by mutations in the KCNJ2 gene that encodes the ubiquitously expressed Kir2.1 potassium channel. The syndrome is characterized by episodic weakness, cardiac arrythmias and dysmorphic features. However, the full extent of the multisystem phenotype is not well described. In-depth, multisystem phenotyping is required to inform diagnosis and guide management. We report our findings following deep multimodal phenotyping across all systems in a large case series of 69 total patients, with comprehensive data for 52. As a national referral centre, we assessed point prevalence and showed it is higher than previously reported, at 0.105 per 100 000 population in England. While the classical phenotype of episodic weakness is recognized, we found that a quarter of our cohort have fixed myopathy and 13.5% required a wheelchair or gait aid. We identified frequent fat accumulation on MRI and tubular aggregates on muscle biopsy, emphasizing the active myopathic process underpinning the potential for severe neuromuscular disability. Long exercise testing was not reliable in predicting neuromuscular symptoms. A normal long exercise test was seen in five patients, of whom four had episodic weakness. Sixty-seven per cent of patients treated with acetazolamide reported a good neuromuscular response. Thirteen per cent of the cohort required cardiac defibrillator or pacemaker insertion. An additional 23% reported syncope. Baseline electrocardiograms were not helpful in stratifying cardiac risk, but Holter monitoring was. A subset of patients had no cardiac symptoms, but had abnormal Holter monitor recordings which prompted medication treatment. We describe the utility of loop recorders to guide management in two such asymptomatic patients. Micrognathia was the most commonly reported skeletal feature; however, 8% of patients did not have dysmorphic features and one-third of patients had only mild dysmorphic features. We describe novel phenotypic features including abnormal echocardiogram in nine patients, prominent pain, fatigue and fasciculations. Five patients exhibited executive dysfunction and slowed processing which may be linked to central expression of KCNJ2. We report eight new KCNJ2 variants with in vitro functional data. Our series illustrates that Andersen-Tawil syndrome is not benign. We report marked neuromuscular morbidity and cardiac risk with multisystem involvement. Our key recommendations include proactive genetic screening of all family members of a proband. This is required, given the risk of cardiac arrhythmias among asymptomatic individuals, and a significant subset of Andersen-Tawil syndrome patients have no (or few) dysmorphic features or negative long exercise test. We discuss recommendations for increased cardiac surveillance and neuropsychometry testing.

Progressive myoclonus epilepsy KCNC1 variant causes a developmental dendritopathy.

OBJECTIVE: Mutations in KCNC1 can cause severe neurological dysfunction, including intellectual disability, epilepsy, and ataxia. The Arg320His variant, which occurs in the voltage-sensing domain of the channel, causes a highly penetrant and specific form of progressive myoclonus epilepsy with severe ataxia, designated myoclonus epilepsy and ataxia due to potassium channel mutation (MEAK). KCNC1 encodes the voltage-gated potassium channel KV 3.1, a channel that is important for enabling high-frequency firing in interneurons, raising the possibility that MEAK is associated with reduced interneuronal function. METHODS: To determine how this variant triggers MEAK, we expressed KV 3.1bR320H in cortical interneurons in vitro and investigated the effects on neuronal function and morphology. We also performed electrophysiological recordings of oocytes expressing KV 3.1b to determine whether the mutation introduces gating pore currents. RESULTS: Expression of the KV 3.1bR320H variant profoundly reduced excitability of mature cortical interneurons, and cells expressing these channels were unable to support high-frequency firing. The mutant channel also had an unexpected effect on morphology, severely impairing neurite development and interneuron viability, an effect that could not be rescued by blocking KV 3 channels. Oocyte recordings confirmed that in the adult KV 3.1b isoform, R320H confers a dominant negative loss-of-function effect by slowing channel activation, but does not introduce potentially toxic gating pore currents. SIGNIFICANCE: Overall, our data suggest that, in addition to the regulation of high-frequency firing, KV 3.1 channels play a hitherto unrecognized role in neuronal development. MEAK may be described as a developmental dendritopathy.

Spider toxin inhibits gating pore currents underlying periodic paralysis.

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.

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

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.

A novel ATP1A2 mutation in a patient with hypokalaemic periodic paralysis and CNS symptoms.

Hypokalaemic periodic paralysis is a rare genetic neuromuscular disease characterized by episodes of skeletal muscle paralysis associated with low serum potassium. Muscle fibre inexcitability during attacks of paralysis is due to an aberrant depolarizing leak current through mutant voltage sensing domains of either the sarcolemmal voltage-gated calcium or sodium channel. We report a child with hypokalaemic periodic paralysis and CNS involvement, including seizures, but without mutations in the known periodic paralysis genes. We identified a novel heterozygous de novo missense mutation in the ATP1A2 gene encoding the α2 subunit of the Na+/K+-ATPase that is abundantly expressed in skeletal muscle and in brain astrocytes. Pump activity is crucial for Na+ and K+ homeostasis following sustained muscle or neuronal activity and its dysfunction is linked to the CNS disorders hemiplegic migraine and alternating hemiplegia of childhood, but muscle dysfunction has not been reported. Electrophysiological measurements of mutant pump activity in Xenopus oocytes revealed lower turnover rates in physiological extracellular K+ and an anomalous inward leak current in hypokalaemic conditions, predicted to lead to muscle depolarization. Our data provide important evidence supporting a leak current as the major pathomechanism underlying hypokalaemic periodic paralysis and indicate ATP1A2 as a new hypokalaemic periodic paralysis gene.

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

Congenital myopathies are a clinically and genetically heterogeneous group of muscle disorders characterized by congenital or early-onset hypotonia and muscle weakness, and specific pathological features on muscle biopsy. The phenotype ranges from foetal akinesia resulting in in utero or neonatal mortality, to milder disorders that are not life-limiting. Over the past decade, more than 20 new congenital myopathy genes have been identified. Most encode proteins involved in muscle contraction; however, mutations in ion channel-encoding genes are increasingly being recognized as a cause of this group of disorders. SCN4A encodes the α-subunit of the skeletal muscle voltage-gated sodium channel (Nav1.4). This channel is essential for the generation and propagation of the muscle action potential crucial to muscle contraction. Dominant SCN4A gain-of-function mutations are a well-established cause of myotonia and periodic paralysis. Using whole exome sequencing, we identified homozygous or compound heterozygous SCN4A mutations in a cohort of 11 individuals from six unrelated kindreds with congenital myopathy. Affected members developed in utero- or neonatal-onset muscle weakness of variable severity. In seven cases, severe muscle weakness resulted in death during the third trimester or shortly after birth. The remaining four cases had marked congenital or neonatal-onset hypotonia and weakness associated with mild-to-moderate facial and neck weakness, significant neonatal-onset respiratory and swallowing difficulties and childhood-onset spinal deformities. All four surviving cohort members experienced clinical improvement in the first decade of life. Muscle biopsies showed myopathic features including fibre size variability, presence of fibrofatty tissue of varying severity, without specific structural abnormalities. Electrophysiology suggested a myopathic process, without myotonia. In vitro functional assessment in HEK293 cells of the impact of the identified SCN4A mutations showed loss-of-function of the mutant Nav1.4 channels. All, apart from one, of the mutations either caused fully non-functional channels, or resulted in a reduced channel activity. Each of the affected cases carried at least one full loss-of-function mutation. In five out of six families, a second loss-of-function mutation was present on the trans allele. These functional results provide convincing evidence for the pathogenicity of the identified mutations and suggest that different degrees of loss-of-function in mutant Nav1.4 channels are associated with attenuation of the skeletal muscle action potential amplitude to a level insufficient to support normal muscle function. The results demonstrate that recessive loss-of-function SCN4A mutations should be considered in patients with a congenital myopathy.

Successful transfer to sulfonylureas in KCNJ11 neonatal diabetes is determined by the mutation and duration of diabetes.

AIMS/HYPOTHESIS: The finding that patients with diabetes due to potassium channel mutations can transfer from insulin to sulfonylureas has revolutionised the management of patients with permanent neonatal diabetes. The extent to which the in vitro characteristics of the mutation can predict a successful transfer is not known. Our aim was to identify factors associated with successful transfer from insulin to sulfonylureas in patients with permanent neonatal diabetes due to mutations in KCNJ11 (which encodes the inwardly rectifying potassium channel Kir6.2). METHODS: We retrospectively analysed clinical data on 127 patients with neonatal diabetes due to KCNJ11 mutations who attempted to transfer to sulfonylureas. We considered transfer successful when patients completely discontinued insulin whilst on sulfonylureas. All unsuccessful transfers received ≥0.8 mg kg(-1) day(-1) glibenclamide (or the equivalent) for >4 weeks. The in vitro response of mutant Kir6.2/SUR1 channels to tolbutamide was assessed in Xenopus oocytes. For some specific mutations, not all individuals carrying the mutation were able to transfer successfully; we therefore investigated which clinical features could predict a successful transfer. RESULTS: In all, 112 out of 127 (88%) patients successfully transferred to sulfonylureas from insulin with an improvement in HbA1c from 8.2% (66 mmol/mol) on insulin, to 5.9% (41 mmol/mol) on sulphonylureas (p = 0.001). The in vitro response of the mutation to tolbutamide determined the likelihood of transfer: the extent of tolbutamide block was <63% for the p.C166Y, p.I296L, p.L164P or p.T293N mutations, and no patients with these mutations successfully transferred. However, most individuals with mutations for which tolbutamide block was >73% did transfer successfully. The few patients with these mutations who could not transfer had a longer duration of diabetes than those who transferred successfully (18.2 vs 3.4 years, p = 0.032). There was no difference in pre-transfer HbA1c (p = 0.87), weight-for-age z scores (SD score; p = 0.12) or sex (p = 0.17). CONCLUSIONS/INTERPRETATION: Transfer from insulin is successful for most KCNJ11 patients and is best predicted by the in vitro response of the specific mutation and the duration of diabetes. Knowledge of the specific mutation and of diabetes duration can help predict whether successful transfer to sulfonylureas is likely. This result supports the early genetic testing and early treatment of patients with neonatal diabetes aged under 6 months.

A novel KCNA1 mutation in a family with episodic ataxia and malignant hyperthermia.

Episodic ataxia type 1 (EA1) is an autosomal dominant channelopathy caused by mutations in KCNA1, which encodes the voltage-gated potassium channel, Kv1.1. Eleven members of an EA family were evaluated with molecular and functional studies. A novel c.746T>G (p.Phe249Cys) missense mutation of KCNA1 segregated in the family members with episodic ataxia, myokymia, and malignant hyperthermia susceptibility. No mutations were found in the known malignant hyperthermia genes RYR1 or CACNA1S. The Phe249Cys-Kv1.1 channels did not show any currents upon functional expression, confirming a pathogenic role of the mutation. Malignant hyperthermia may be a presentation of KCNA1 mutations, which has significant implications for the clinical care of these patients and illustrates the phenotypic heterogeneity of KCNA1 mutations.

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

Mutations in the skeletal muscle channel (SCN4A), encoding the Nav1.4 voltage-gated sodium channel, are causative of a variety of muscle channelopathies, including non-dystrophic myotonias and periodic paralysis. The effects of many of these mutations on channel function have been characterized both in vitro and in vivo. However, little is known about the consequences of SCN4A mutations downstream from their impact on the electrophysiology of the Nav1.4 channel. Here we report the discovery of a novel SCN4A mutation (c.1762A>G; p.I588V) in a patient with myotonia and periodic paralysis, located within the S1 segment of the second domain of the Nav1.4 channel. Using N-ethyl-N-nitrosourea mutagenesis, we generated and characterized a mouse model (named draggen), carrying the equivalent point mutation (c.1744A>G; p.I582V) to that found in the patient with periodic paralysis and myotonia. Draggen mice have myotonia and suffer from intermittent hind-limb immobility attacks. In-depth characterization of draggen mice uncovered novel systemic metabolic abnormalities in Scn4a mouse models and provided novel insights into disease mechanisms. We discovered metabolic alterations leading to lean mice, as well as abnormal AMP-activated protein kinase activation, which were associated with the immobility attacks and may provide a novel potential therapeutic target.

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.

Prevalence of genetically confirmed skeletal muscle channelopathies in the era of next generation sequencing.

We provide an up-to-date and accurate minimum point prevalence of genetically defined skeletal muscle channelopathies which is important for understanding the population impact, planning for treatment needs and future clinical trials. Skeletal muscle channelopathies include myotonia congenita (MC), sodium channel myotonia (SCM), paramyotonia congenita (PMC), hyperkalemic periodic paralysis (hyperPP), hypokalemic periodic paralysis (hypoPP) and Andersen- Tawil Syndrome (ATS). Patients referred to the UK national referral centre for skeletal muscle channelopathies and living in UK were included to calculate the minimum point prevalence using the latest data from the Office for National Statistics population estimate. We calculated a minimum point prevalence of all skeletal muscle channelopathies of 1.99/100 000 (95% CI 1.981-1.999). The minimum point prevalence of MC due to CLCN1 variants is 1.13/100 000 (95% CI 1.123-1.137), SCN4A variants which encode for PMC and SCM is 0.35/100 000 (95% CI 0.346 - 0.354) and for periodic paralysis (HyperPP and HypoPP) 0.41/100 000 (95% CI 0.406-0.414). The minimum point prevalence for ATS is 0.1/100 000 (95% CI 0.098-0.102). There has been an overall increase in point prevalence in skeletal muscle channelopathies compared to previous reports, with the biggest increase found to be in MC. This can be attributed to next generation sequencing and advances in clinical, electrophysiological and genetic characterisation of skeletal muscle channelopathies.

Interaction between mutations in the slide helix of Kir6.2 associated with neonatal diabetes and neurological symptoms.

ATP-sensitive potassium (K(ATP)) channels regulate insulin secretion from pancreatic beta-cells. Gain-of-function mutations in the genes encoding the Kir6.2 and SUR1 subunits of this channel cause neonatal diabetes. We report two novel mutations on the same haplotype (cis), F60Y and V64L, in the slide helix of Kir6.2 in a patient with neonatal diabetes, developmental delay and epilepsy. Functional analysis revealed the F60Y mutation increases the intrinsic channel open probability (Po(0)), thereby indirectly producing a marked decrease in channel inhibition by ATP and an increase in whole-cell K(ATP) currents. When expressed alone, the V64L mutation caused a small reduction in apparent ATP inhibition, by enhancing the ability of MgATP to stimulate channel activity. The V64L mutation also ameliorated the deleterious effects on the F60Y mutation when it was expressed on the same (but not a different) subunit. These data indicate that F60Y is the pathogenic mutation and reveal that interactions between slide helix residues can influence K(ATP) channel gating.

A conserved tryptophan at the membrane-water interface acts as a gatekeeper for Kir6.2/SUR1 channels and causes neonatal diabetes when mutated.

We identified a novel heterozygous mutation, W68R, in the Kir6.2 subunit of the ATP-sensitive potassium (KATP) channel, in a patient with transient neonatal diabetes. This tryptophan is absolutely conserved in mammalian Kir channels. The functional effects of mutations at residue 68 of Kir6.2 were studied by heterologous expression in Xenopus oocytes, and by homology modelling. We found the Kir6.2-W68R mutation causes a small reduction in ATP inhibition in the heterozygous state and an increase in the whole-cell KATP current. This can explain the clinical phenotype of the patient. The effect of the mutation was not charge or size dependent, the order of potency for ATP inhibition being W

Ageing contributes to phenotype transition in a mouse model of periodic paralysis.

BACKGROUND: Periodic paralysis (PP) is a rare genetic disorder in which ion channel mutation causes episodic paralysis in association with hyper- or hypokalaemia. An unexplained but consistent feature of PP is that a phenotype transition occurs around the age of 40, in which the severity of potassium-induced muscle weakness declines but onset of fixed, progressive weakness is reported. This phenotype transition coincides with the age at which muscle mass and optimal motor function start to decline in healthy individuals. We sought to determine if the phenotype transition in PP is linked to the normal ageing phenotype transition and to explore the mechanisms involved. METHODS: A mouse model of hyperkalaemic PP was compared with wild-type littermates across a range of ages (13-104 weeks). Only male mice were used as penetrance is incomplete in females. We adapted the muscle velocity recovery cycle technique from humans to examine murine muscle excitability in vivo. We then examined changes in potassium-induced weakness or caffeine contracture force with age using ex vivo muscle tension testing. Muscles were further characterized by either Western blot, histology or energy charge measurement. For normally distributed data, a student's t-test (± Welch correction) or one- or two-way analysis of variance (ANOVA) was performed to determine significance. For data that were not normally distributed, Welch rank test, Mann Whitney U test or Kruskal-Wallis ANOVA was performed. When an ANOVA was significant (P < 0.05), post hoc Tukey testing was used. RESULTS: Both WT (P = 0.009) and PP (P = 0.007) muscles exhibit increased resistance to potassium-induced weakness with age. Our data suggest that healthy-old muscle develops mechanisms to maintain force despite sarcolemmal depolarization and sodium channel inactivation. In contrast, reduced caffeine contracture force (P = 0.00005), skeletal muscle energy charge (P = 0.004) and structural core pathology (P = 0.005) were specific to Draggen muscle, indicating that they are caused, or at least accelerated by, chronic genetic ion channel dysfunction. CONCLUSIONS: The phenotype transition with age is replicated in a mouse model of PP. Intrinsic muscle ageing protects against potassium-induced weakness in HyperPP mice. However, it also appears to accelerate impairment of sarcoplasmic reticulum calcium release, mitochondrial impairment and the development of core-like regions, suggesting acquired RyR1 dysfunction as the potential aetiology. This work provides a first description of mechanisms involved in phenotype transition with age in PP. It also demonstrates how studying phenotype transition with age in monogenic disease can yield novel insights into both disease physiology and the ageing process itself.