Understanding the Pathophysiology of Skeletal Muscle Channelopathies

Skeletal muscle channelopathies are rare inherited neuromuscular conditions caused by ion channel gene mutations. They are grouped into the non-dystrophic myotonias and the periodic paralyses (PP). An intriguing but as yet unexplained phenomenon is that phenotype severity varies significantly with species, gender and age. The first part of my thesis explores my hypothesis that this phenotype variability in skeletal muscle channelopathies reflects physiological differences in skeletal muscle excitability. To do this, I reverse translated Muscle Velocity Recovery Cycles (MVRCs), an in vivo technique for assessing ion channel function, from humans to mice. My data suggest that murine skeletal muscle has increased chloride conductance compared to human skeletal muscle. This could explain the species difference seen between mouse models of Myotonia Congenita and humans with clinical disease. I also found gender differences in healthy murine muscle excitability that vary by muscle and are associated with gender difference in the skeletal muscle channelome. On the background of these physiological gender differences in murine muscle excitability, the effect of gain-of-function sodium channel mutation is more severe in male mice. Finally, I found that the phenotype change with age seen in patients with PP also occurs in a mouse model. Surprisingly, an increased resistance to potassium-induced weakness was most pronounced in old wild-type mice, suggesting it is a phenomenon of normal aging. In contrast, the onset of permanent progressive weakness was specific to PP mouse muscle. My experiments suggest this may be due to acquired ryanodine receptor dysfunction resulting in sarcoplasmic reticulum calcium leak and impaired mitochondrial oxidative capacity. The aim of the second part of my thesis was to extend our knowledge of ClC-1 structure-function and improve genetic counselling for patients with Myotonia Congenita (MC). I identified a novel molecular pathomechanism for MC and found that dominant mutations cluster in the first half of the channel sequence. Combining variant location with functional characterisation significantly improves the accuracy of genetic counselling we can provide patients

Clinical, morphological and genetic characterization of Brody disease: an international study of 40 patients

Brody disease is an autosomal recessive myopathy characterized by exercise-induced muscle stiffness due to mutations in the ATP2A1 gene. Almost 50 years after the initial case presentation, only 18 patients have been reported and many questions regarding the clinical phenotype and results of ancillary investigations remain unanswered, likely leading to incomplete recognition and consequently under-diagnosis. Additionally, little is known about the natural history of the disorder, genotype-phenotype correlations, and the effects of symptomatic treatment. We studied the largest cohort of Brody disease patients to date (n = 40), consisting of 22 new patients (19 novel mutations) and all 18 previously published patients. This observational study shows that the main feature of Brody disease is an exercise-induced muscle stiffness of the limbs, and often of the eyelids. Onset begins in childhood and there was no or only mild progression of symptoms over time. Four patients had episodes resembling malignant hyperthermia. The key finding at physical examination was delayed relaxation after repetitive contractions. Additionally, no atrophy was seen, muscle strength was generally preserved, and some patients had a remarkable athletic build. Symptomatic treatment was mostly ineffective or produced unacceptable side effects. EMG showed silent contractures in approximately half of the patients and no myotonia. Creatine kinase was normal or mildly elevated, and muscle biopsy showed mild myopathic changes with selective type II atrophy. Sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) activity was reduced and western blot analysis showed decreased or absent SERCA1 protein. Based on this cohort, we conclude that Brody disease should be considered in cases of exercise-induced muscle stiffness. When physical examination shows delayed relaxation, and there are no myotonic discharges at electromyography, we recommend direct sequencing of the ATP2A1 gene or next generation sequencing with a myopathy panel. Aside from clinical features, SERCA activity measurement and SERCA1 western blot can assist in proving the pathogenicity of novel ATP2A1 mutations. Finally, patients with Brody disease may be at risk for malignant hyperthermia-like episodes, and therefore appropriate perioperative measures are recommended. This study will help improve understanding and recognition of Brody disease as a distinct myopathy in the broader field of calcium-related myopathies

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-offunction 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.</p