J. Burge

Refined exercise testing can aid dna‐based diagnosis in muscle channelopathies

To improve the accuracy of genotype prediction and guide genetic testing in patients with muscle channelopathies we applied and refined specialized electrophysiological exercise test parameters.

Prevalence study of genetically defined skeletal muscle channelopathies in England.

Objectives: To obtain minimum point prevalence rates for the skeletal muscle channelopathies and to evaluate the frequency distribution of mutations associated with these disorders. Methods: Analysis of demographic, clinical, electrophysiologic, and genetic data of all patients assessed at our national specialist channelopathy service. Only patients living in the United Kingdom with a genetically defined diagnosis of nondystrophic myotonia or periodic paralysis were eligible for the study. Prevalence rates were estimated for England, December 2011. Results: A total of 665 patients fulfilled the inclusion criteria, of which 593 were living in England, giving a minimum point prevalence of 1.12/100,000 (95% confidence interval [CI] 1.03–1.21). Disease-specific prevalence figures were as follows: myotonia congenita 0.52/100,000 (95% CI 0.46–0.59), paramyotonia congenita 0.17/100,000 (95% CI 0.13–0.20), sodium channel myotonias 0.06/100,000 (95% CI 0.04–0.08), hyperkalemic periodic paralysis 0.17/100,000 (95% CI 0.13–0.20), hypokalemic periodic paralysis 0.13/100,000 (95% CI 0.10–0.17), and Andersen-Tawil syndrome (ATS) 0.08/100,000 (95% CI 0.05–0.10). In the whole sample (665 patients), 15 out of 104 different CLCN1 mutations accounted for 60% of all patients with myotonia congenita, 11 out of 22 SCN4A mutations for 86% of paramyotonia congenita/sodium channel myotonia pedigrees, and 3 out of 17 KCNJ2 mutations for 42% of ATS pedigrees. Conclusion: We describe for the first time the overall prevalence of genetically defined skeletal muscle channelopathies in England. Despite the large variety of mutations observed in patients with nondystrophic myotonia and ATS, a limited number accounted for a large proportion of cases.

A new explanation for recessive myotonia congenita Exon deletions and duplications in CLCN1

Objective: To assess whether exon deletions or duplications in CLCN1 are associated with recessive myotonia congenita (MC). Methods: We performed detailed clinical and electrophysiologic characterization in 60 patients with phenotypes consistent with MC. DNA sequencing of CLCN1 followed by multiplex ligation-dependent probe amplification to screen for exon copy number variation was undertaken in all patients. Results: Exon deletions or duplications in CLCN1 were identified in 6% of patients with MC. Half had heterozygous exonic rearrangements. The other 2 patients (50%), with severe disabling infantile onset myotonia, were identified with both a homozygous mutation, Pro744Thr, which functional electrophysiology studies suggested was nonpathogenic, and a triplication/homozygous duplication involving exons 8–14, suggesting an explanation for the severe phenotype. Conclusions: These data indicate that copy number variation in CLCN1 may be an important cause of recessive MC. Our observations suggest that it is important to check for exon deletions and duplications as part of the genetic analysis of patients with recessive MC, especially in patients in whom sequencing identifies no mutations or only a single recessive mutation. These results also indicate that additional, as yet unidentified, genetic mechanisms account for cases not currently explained by either CLCN1 point mutations or exonic deletions or duplications.

Randomized, placebo-controlled trials of dichlorphenamide in periodic paralysis

Objective: To determine the short-term and long-term effects of dichlorphenamide (DCP) on attack frequency and quality of life in hyperkalemic (HYP) and hypokalemic (HOP) periodic paralysis. Methods: Two multicenter randomized, double-blind, placebo-controlled trials lasted 9 weeks (Class I evidence), followed by a 1-year extension phase in which all participants received DCP. Forty-four HOP and 21 HYP participants participated. The primary outcome variable was the average number of attacks per week over the final 8 weeks of the double-blind phase. Results: The median attack rate was lower in HOP participants on DCP than in participants on placebo (0.3 vs 2.4, p = 0.02). The 9-week mean change in the Physical Component Summary score of the Short Form–36 was also better in HOP participants receiving DCP (treatment effect = 7.29 points, 95% confidence interval 2.26 to 12.32, p = 0.006). The median attack rate was also lower in HYP participants on DCP (0.9 vs 4.8) than in participants on placebo, but the difference in median attack rate was not significant (p = 0.10). There were no significant effects of DCP on muscle strength or muscle mass in either trial. The most common adverse events in both trials were paresthesia (47% DCP vs 14% placebo, both trials combined) and confusion (19% DCP vs 7% placebo, both trials combined). Conclusions: DCP is effective in reducing the attack frequency, is safe, and improves quality of life in HOP periodic paralysis. Classification of evidence: These studies provide Class I evidence that DCP significantly reduces attack frequency in HOP but lacked the precision to support either efficacy or lack of efficacy of DCP in HYP.

New immunohistochemical method for improved myotonia and chloride channel mutation diagnostics

ABSTRACT Objective: The objective of this study was to validate the immunohistochemical assay for the diagnosis of nondystrophic myotonia and to provide full clarification of clinical disease to patients in whom basic genetic testing has failed to do so. Methods: An immunohistochemical assay of sarcolemmal chloride channel abundance using 2 different ClC1-specific antibodies. Results: This method led to the identification of new mutations, to the reclassification of W118G in CLCN1 as a moderately pathogenic mutation, and to confirmation of recessive (Becker) myotonia congenita in cases when only one recessive CLCN1 mutation had been identified by genetic testing. Conclusions: We have developed a robust immunohistochemical assay that can detect loss of sarcolemmal ClC-1 protein on muscle sections. This in combination with gene sequencing is a powerful approach to achieving a final diagnosis of nondystrophic myotonia.

P39 Prevalence study of skeletal muscle channelopathies in England

Introduction The non-dystrophic myotonias (NDM) and periodic paralyses (PP) are a group of skeletal muscle disorders caused by mutations in genes encoding ion channels. To date, very few studies have systematically evaluated the prevalence of these disorders, never in England, and most of them predate genetic diagnosis. Objective To obtain prevalence data on skeletal muscle channelopathies and to evaluate the relative frequency of common mutations. Methods The study covered all patients with NDM or PP living in the UK that were referred to the UK national reference centre for assessment. Inclusion criteria were clinical and electrophysiological features of NDM or PP, and confirmed mutations in genes encoding ion channels (96% of cases). England was selected as the geographical area for prevalence analysis. Results From a total of 582 patients identified, 286 had myotonia congenita, 70 paramyotonia congenita, 23 sodium-channel myotonias, 97 hypokalemic PP, 66 hyperkalemic PP, four normokalemic PP, and 36 Andersen-Tawil syndrome (ATS). 530 patients were from England, giving a point prevalence of 1/100 000. Significant allelic heterogeneity was associated with NDM and ATS. However, a limited number of mutations were responsible for most cases. Conclusion We have analysed the largest series of patients with skeletal muscle channelopathies reported so far, and documented for the first time their overall prevalence. The spectrum of mutations was similar to that previously reported.

G.P.101 New immunohistochemical method for improved myotonia and chloride channel mutation diagnostics

Abstract Most patients with myotonia have either congenital myotonia caused by mutated chloride or sodium channel, or progressive myotonic dystrophy (types 1 and 2). Currently, final diagnosis is frequently obtained by molecular genetic DNA testing. However, the increased use of genetic testing also results in many cases when the tests do not provide full clarification of the clinical disease. An immunohistochemical method using two different chloride channel protein specific antibodies for diagnostic purposes This method provided means to identify new mutations, to reclassify the W118G CLCN1 change as a moderately pathogenic mutation, and to clarify recessive Becker myotonia in cases when only one recessive mutation was identified by genetic testing. The developed immunohistochemical staining method for CLC-1 in muscle fibers proved to be a robust method for the detection of presence or loss of sarcolemmal CLC-1 protein on muscle sections. This in combination with gene sequencing is a powerful approach to achieving a final diagnosis of non-dystrophic myotonia.

1154 Skeletal muscle chloride channel gene (CLCN1) copy number variation can cause myotonia congenita

Myotonia Congenita (MC) is the commonest skeletal muscle channelopathy. It associates with recessive or dominant mutations in the chloride channel gene CLCN1. A significant number of patients from recessive pedigrees (14% in our series of 439 cases) only harbour a single recessive mutation despite full sequencing of CLCN1. The genetic basis of such cases remains unexplained; introducing uncertainties for diagnosis and genetic counselling. We hypothesised that CLCN1 copy number variations (CNV) may be a new genetic mechanism in such MC patients. We performed detailed clinical and neurophysiological characterisation in 60 patients with a MC phenotype. DNA sequencing of CLCN1 was followed by multiplex ligation dependent probe amplification (MLPA) to screen for CNV in CLCN1. We identified the first reported group of MC patients with CNV in CLCN1. Eight per cent of cases in our cohort carried a CNV in CLCN1. In two families, rearrangements affecting exons 8 to 14 had a very severe infantile onset myotonia. This indicates that CLCN1 CNV are an important previously unreported cause of recessive MC suggesting that MLPA should be done in the genetic work-up of patients with MC; especially in cases where direct DNA sequencing identifies no mutations or only a single recessive mutation. These data also indicate that additional unidentified genetic mechanisms exist to account for cases not currently explained by either exonic CLCN1 point mutations or copy number variation.

P25 Double-blind, placebo-controlled, parallel group, phase III study comparing dichlorphenamide vs. placebo for the treatment of periodic paralysis (HYP HOP trial)

the AChR subunits alter the channel kinetics by prolonging the burst duration, thereby causing cationic overload and leading to endplate myopathy. In order to test new therapies and analyse the mechanisms of existing therapies for slow channel syndrome, we generated a transgenic mouse expressing the slow channel mutation L221F in the human e-subunit of the AChR. EGFP was inserted into the large cytoplasmic loop, located between the third and fourth transmembrane domains, to allow visualisation of the mutant e subunit (eL221F-EGFP). Transgene transcription was driven from the AChR b-subunit promoter to restrict expression to the post-synaptic nuclei in skeletal muscle. Transgenic mice were back-crossed with mice deficient in endogenous AChR, to generate mice that expressed only mutated e-subunit. The mice have no obvious defects at birth, thrive and survive for longer than 12 months but show fatiguability. Analysis of endplates from EDL, soleus and diaphragm muscles using fluorescently tagged a-bungarotoxin showed that eL221F-EGFP is correctly incorporated into endplate AChR. Electron microscopy showed a large variability in endplate morphology within the same muscle. Electrophysiological analysis demonstrated prolonged decay of the EPPs and MEPPs, thereby confirming slow channel characteristics of the eL221F-EGFP receptors. Our model accurately reflects the disease in humans and is currently being used to test the efficacy and mechanisms of novel and existing slow channel therapies.