Central Core Disease Research Articles

Clinical and skeletal muscle biopsy characteristics of 25 patients with floppy infant syndrome

Floppy infant syndrome (FIS) comprises of a group of disorders with a common symptom of generalized hypotonia at birth or in early life, which causes diagnostic challenge. In the current work, we aimed to describe the clinical and histopathological characteristics of FIS to help improve the diagnosis of this condition. We collected information on the clinical characteristics, laboratory data, electrophysiological test results and detailed skeletal muscle biopsy histopathological features of 25 infants with FIS. We then discussed the final diagnoses and analyzed the neuromuscular features. Among the 25 infants with FIS, there were 7 cases of spinal muscular atrophy (SMA), 4 cases of congenital muscular dystrophy (CMD) (3 cases of merosin-deficient CMD and 1 case of Ullrich CMD), 8 cases of congenital myopathy (2 cases of central core disease, 1 case each of nemaline myopathy and centronuclear myopathy, and 4 cases of congenital fiber-type disproportion), and 6 cases of metabolic myopathy (3 cases of lipid storage myopathy, 2 cases of Pompe's disease, and 1 case of Leigh's syndrome). The histopathological characteristics of muscle biopsy were found to be distinct for these subgroups. FIS is caused by a variety of neuromuscular disorders that have common clinical manifestations, including SMA, CMD, congenital myopathies and metabolic myopathies. Skeletal muscle biopsy is an essential tool for the definite and differential diagnoses of FIS, especially of neuromuscular origin.

The dangers of MH

Q: I've heard a lot about malignant hyperthermia (MH). What is it and what warning signs should I be alert to? A: MH is a rare, genetic, life-threatening response to anesthesia characterized by skeletal muscle contractions and excessively high body temperatures that can reach 105 to 108° F (40.6 to 42.2° C). Metabolic acidosis—pH below 7.35 and a bicarbonate level below 22 mEq/L—can also occur, resulting in mental status changes and, potentially, coma. Dysrhythmias and cardiac arrest may also ensue if MH is left untreated. An autosomal dominant gene (RYR1) causes calcium abnormalities when the patient is exposed to succinylcholine, a powerful muscle relaxant used to temporarily paralyze the respiratory muscles during emergent or operative procedures. Although succinylcholine is the most common offender, MH may be caused by other anesthetics. MH has also been linked to patients with multiminicore myopathy—an inherited skeletal muscle disorder characterized by hypertonia—and central core disease. The patient with MH presents with a multitude of symptoms. Muscle rigidity and fasciculation are most commonly observed; however, be alert for hyperthermia, tachycardia, tachypnea, diaphoresis, arrhythmias, and cyanosis. Left untreated, secondary signs of MH include cardiac arrest, disseminated intravascular coagulation (DIC), acute renal failure, myoglobinuria, and rhabdomyolysis. Acid-base and electrolyte imbalances, such as hyperkalemia and hypercalcemia, may also occur. Various blood tests, diagnostic procedures, and clinical presentations can detect the severity of MH. Vital signs will reveal tachycardia, tachypnea, hyperthermia, and fluctuating BP. Respiratory and/or metabolic acidosis can be seen through arterial blood gases. A comprehensive metabolic panel will show electrolyte and kidney function abnormalities, such as an increase in blood urea nitrogen and creatinine, hyperkalemia, and hypercalcemia. Coagulation studies aid in detecting DIC, and urinalysis aids in detecting myoglobinuria. Dysrhythmias can be seen with continuous cardiac monitoring. Upon detection of MH, anesthesia and the concomitant surgery must be stopped. The surgical procedure is postponed and the trigger agent removed. Immediately administer dantroline sodium—a skeletal muscle relaxant—as a reversal agent. Hyperventilate the patient with 100% oxygen and administer I.V. fluids for hydration and dilution of the trigger agent/succinylcholine.Figure: A thorough pre-op history may prevent MH due to its genetic component.When hyperthermia occurs, apply cooling blankets; ice to the groin, axilla, and neck areas; or chilled I.V. fluids. During the use of cooling agents, frequently monitor the patient's temperature and perform frequent skin assessments. Continuous telemetry is essential for monitoring potential arrhythmias and tachycardia. A thorough preoperative history may prevent MH. It's imperative to inquire about a history of reactions to or complications from anesthesia in the patient or family. Genetic testing for RYR1 defects is available to pinpoint familial tendencies of MH. Instruct the patient with a known history of or potential for MH to wear a medical-alert bracelet.

P.4.13 Central core disease (CCD): Improving the screening for mutations in RYR1 gene

Central core disease (CCD) is a congenital myopathy, characterized by the presence of central core-like areas in muscle fibers. Patients with RYR-related CCD usually have mild or moderate axial and proximal weakness, hypotonia and motor developmental delay. CCD is associated with susceptibility to malignant hyperthermia (MH), and both conditions have been linked to mutations in human RYR1 gene, which encodes a calcium release channel known as ryanodine receptor (RyR1). RYR1 mutational spectrum linked with CCD includes more than 200 described mutations mostly heterozygous dominant missense mutations and minor deletions or duplications. Definite diagnosis of CCD is done by clinical history and evaluation, histopathological analyses of muscle biopsy and, recently, genetic testing of gene’s “hot spots”, because RYR1 is a large gene and direct sequencing of each exon is laborious. Here, we tested Multiplex Ligation-dependent Probe Amplification (MLPA) as an alternative and simple method for screening patients with histological diagnosis or family history of CCD. This technique uses high-performance PCR to quantify up to 45 transcripts in small human DNA samples. It is based on annealing of two hemi-probes into its complementary sequence and was designed to detect the 33 most common mutations in RYR1 gene. From 41 patients diagnosed with CCD in three different institutions, 24 (58,5%) had mutations found in direct sequencing and 11 (26,8%) in MLPA. Seventeen patients (41,5%) had consistent results: 6 (14,6%) had their mutation diagnosed with sequencing and MLPA and neither method was able to find mutations in 11 patients (26,8%). Mutations in exon 101 were the most prevalent in sequencing (11 patients) and MLPA (4 patients). MLPA had shown to be a relevant tool to help diagnosing CCD; moreover, it is especially useful in diagnosing family members of patients with known mutations detectable by MLPA. Financial support: FAPESP-CEPID, CNPQ-INCT, FINEP.

P.4.12 Association of two mutations in the RYR1 gene in central core disease: Recessive or modifying effect?

Central core disease (CCD) is a congenital myopathy, characterized by the presence of central core-like areas in muscle fibers. Patients usually have mild or moderate axial and proximal weakness, hypotonia and motor developmental delay. CCD is associated with mutations in human RYR1 gene, which encodes a calcium release channel known as ryanodine receptor (RyR1). Here we present a family diagnosed with CCD with distinct phenotypes. A 48-year old female was referred to our service with clinical features suggesting a congenital myopathy. Histological analysis confirmed the diagnosis of CCD. Nine exons from the C-terminal region of RYR1 were screened for mutations in the index case. Two different missense mutations were identified: c.14256 A>C in exon 98 and c.14693 T>C in exon 102. Trans position of the 2 mutations was confirmed because patient's daughter, mother and sister carried the exon 98 mutation, but not the one in exon 102. The mutation in exon 102 have already been described as pathogenic, and is compatible with both severe and mild phenotypes, observed as intra and inter family variability. On the other hand, the c.14256 A>C mutation in exon 98 is a single nucleotide variation and its clinical significance is still untested. Considering the fact that patient's relatives showed no CCD phenotype, it could be inferred that this polymorphism alone is not responsible for the disease. Moreover, the effect of association of this polymorphism to exon 102's pathogenic mutation is not clear. Further studies about patients with both exon 98 polymorphism and exon 102 mutation could help establishing a better relation between these two findings. The case described above illustrates the present reality where new methods for wide genome screening are becoming more accessible and able to identify a great variety of mutations and polymorphisms in patients and their families. FAPESP-CEPID, CNPq-INCT, FINEP.

P.4.14 A novel mutation in RYR1 gene for central core disease in a Chinese family

Central core disease (CCD) is a mild, non-progressive or slowly progressive congenital myopathy with proximal symmetrical muscle weakness. CCD is transmitted as an autosomal dominant or recessive trait and at least one gene responsible is the ryanodine receptor gene at chromosome 19q13.1. Here we report the clinical and myopathological changes in a Chinese family with central core disease in an autosomal dominant pattern. The proband showed early onset congenital myopathy with severe contractures, delayed motor milestones, both proximal and distal muscle weakness, normal serum creatine kinase (CK), a non-progressive course, with muscle biopsy findings of central cores and in addition, marked proliferation of connective and adipose tissue, and variation in fibre size. The mother and the younger sister of the proband had similar but mild symptoms with the proband. Molecular genetic analysis showed a heterozygous sequence change in exon 102 (c.14692G > A; p.Gly4897Asp) in RYR1 gene not previously reported. The uncommon clinical distribution of weakness and the severe morphological abnormalities on muscle histology, which had some features of a muscular dystrophy had never been reported in an autosomal dominant CCD family.

O.22 Autosomal dominant Core Congenital Myopathy caused by a mutation in the MYH7 gene

Autosomal dominant (AD) Central core disease (CCD) named currently congenital myopathy with core is an inherited disorder characterised by the presence of cores which are well-limited rounded areas devoid of any oxidative staining. The cores extend almost along the full length of the fibres; these areas correspond to sarcomeric disorganisation, Z line streaming and absence of mitochondria. Genetic studies of AD-CCD families demonstrated the presence of heterozygous mutations in the RYR1 gene in the large majority of families. However, this gene was excluded in some families, suggesting a genetic heterogeneity of AD-Core myopathies (Romero et al., 2005). To enlarge the genetic spectrum of autosomal dominant congenital myopathies with cores demonstrating mutations in a second gene. AD-CCD family with three affected members: the mother and two of three siblings. The symptoms began during the early childhood with delayed motor development. Later they develop proximal weakness, hypertrophy of calves, scapular winging and significant weakness (amyotrophic) of quadriceps and tibialis anterior. No cardiac or ocular involvement was noted. The muscle biopsies sections showed a particular pattern: relatively large and eccentric cores (placed near the subsarcolemmal regions) associated with type 1 predominance and fibre type disproportion in a patient. The large majority of the cores have abrupt borders. Electron microscopy confirmed the presence of multiple large disorganised sarcomeric areas as characteristic unstructured and structured cores. Moreover, some fibres contain focal disorganised areas. Exome sequencing analysis identified a heterozygous missense mutation L1723P in MYH7 segregating with the disease and affecting a conserved residue in the tail domain. With this study we describe MYH7 as a second causative gene for AD-CCD in addition to RYR1 . Our study enlarges the genetic spectrum of AD-CCD myopathies.

P.4.9 Variable clinical and histological features in severe congenital RYR1 associated myopathy

<i>RYR1</i> associated myopathy classically manifests as central core disease with a static to slowly progressive course beginning with congenital hypotonia. Recently the genetic, clinical and histological spectrum has expanded considerably. We report on a series of 11 patients with early neonatal onset <i>RYR1</i> associated myopathy due to recessive and dominant mutations representing the severe end of the clinical spectrum. Eleven patients with severe neonatal onset <i>RYR1</i> associated myopathy confirmed by genetic testing were ascertained. Clinical features associated with the severe neonatal presentation of <i>RYR1</i> associated myopathy included decreased fetal movement, hypotonia, poor feeding, respiratory involvement, arthrogryposis, ophthalmoplegia in three patients, and femur fractures or hip dislocation at birth. Four patients had dominant <i>RYR1</i> mutations, and seven had recessive <i>RYR1</i> mutations. One patient had a cleft palate, and another a congenital rigid spine phenotype – findings not previously described in the literature in patients with early onset <i>RYR1</i> mutations. Muscle biopsy was performed in all patients at ages ranging from 3weeks of life to 4years old. Marked type I fiber predominance approaching type I uniformity was common. All dominant cases had cores while recessive cases had extremely diverse pathology including central cores, multi minicores, more subtle unevenness or normal staining on NADH. Centralized nuclei, extensive fatty and connective tissue infiltration, extremely small fibers in the absence of active degeneration and regeneration were also seen as part of this spectrum. This series confirms and expands the clinical and histological variability associated with severe congenital <i>RYR1</i> associated myopathy. Both dominant and recessive mutations of the <i>RYR1</i> gene can result in a severe neonatal onset phenotype, but more clinical and histological heterogeneity has been seen in those with recessive <i>RYR1</i> gene mutations. The absence of cores should not preclude the consideration of <i>RYR1</i> associated myopathy.

P.4.10 Exon skipping as a therapeutic strategy applied to a RyR1 mutation causing severe core myopathy

Central Core Disease is a myopathy resulting generally from a mutation in the RYR1 gene, encoding the skeletal muscle calcium release channel RyR1. No treatment is currently available for this disease. We studied a pathological situation in which an affected child harbors two recessive mutations, resulting in a massive reduction in the RyR1 amount. The paternal mutation inducing the inclusion of a new in frame exon in the mRNA of RyR1, resulted in the insertion of additional amino-acids and destabilization of the protein. We hypothesized that inducing the skipping of this exon would be sufficient to restore RyR1 expression and normalization of calcium releases. We developed U7-AON lentiviral vectors to induce exon-skipping on affected primary muscle cells. The efficiency of the exon skipping at the mRNA level, at the protein level and at the functional level using calcium imaging were evaluated. We observed in these affected primary muscle culture a reduction in the inclusion of the additional exon, an increase in the RyR1 protein expression, and a restoration of normal calcium releases. This study is the first demonstration of the potential of exon skipping for the therapy of Central Core Disease, from the molecular to the functional level.

Type 2 Ryanodine Receptor Domain A Contains a Unique and Dynamic α-Helix That Transitions to a β-Strand in a Mutant Linked with a Heritable Cardiomyopathy

Ryanodine receptors (RyRs) are large tetrameric calcium (Ca2+) release channels found on the sarcoplasmic reticulum that respond to dihydropyridine receptor activity through a direct conformational interaction and/or indirect Ca2+ sensitivity, propagating sarcoplasmic reticulum luminal Ca2+ release in the process of excitation–contraction coupling. There are three human RyR subtypes, and several debilitating diseases are linked to heritable mutations in RyR1 and RyR2 including malignant hypothermia, central core disease, catecholaminergic polymorphic ventricular tachycardia (CPVT) and arrhythmogenic right ventricular dysplasia type 2 (ARVD2). Despite the recent appreciation that many disease-associated mutations within the N-terminal RyRABC domains (i.e., residues 1–559) are located in the putative interfaces mediating tetrameric channel assembly, the precise structural and dynamical consequences of the mutations are not well understood. We used solution nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography to examine the effect of ARVD2-associated (i.e., R176Q) and CPVT-associated [i.e., P164S, R169Q and delta exon 3 (Δ3)] mutations on the structure and dynamics of RyR2A. Our solution NMR data exposed a mobile α-helix, unique to type 2; further, this α2 helix rescues the β-strand lost in RyR2A Δ3 but remains dynamic in the hot-spot loop (HS-loop) P164S, R169Q and R176Q mutant proteins. Docking of our X-ray crystal/NMR hybrid structure into the RyR1 cryo-electron microscopy map revealed that this RyR2A α2 helix is in close proximity to dense “columns” projecting toward the channel pore. This is in contrast to the HS-loop mutations that cause structural changes largely localized to the intersubunit interface between adjacent ABC domains. Taken together, our data suggest that ARVD2 and CPVT mutations have at least two distinct structural consequences linked to channel dysfunction: perturbation of the HS-loop (i.e., domain A):domain B intersubunit interface and disruption of the communication between the N-terminal region and the channel domain.

Genotype-phenotype correlations in recessive RYR1-related myopathies

BackgroundRYR1 mutations are typically associated with core myopathies and are the most common overall cause of congenital myopathy. Dominant mutations are most often associated with central core disease and malignant hyperthermia, and genotype-phenotype patterns have emerged from the study of these mutations that have contributed to the understanding of disease pathogenesis. The recent availability of genetic testing for the entire RYR1 coding sequence has led to a dramatic expansion in the identification of recessive mutations in core myopathies and other congenital myopathies. To date, no clear patterns have been identified in these recessive mutations, though no systematic examination has yet been performed.MethodsIn this study, we investigated genotype-phenotype correlations in a large combined cohort of unpublished (n = 14) and previously reported (n = 92) recessive RYR1 cases.ResultsOverall examination of this cohort revealed nearly 50% of cases to be non-core myopathy related. Our most significant finding was that hypomorphic mutations (mutations expected to diminish RyR1 expression) were enriched in patients with severe clinical phenotypes. We also determined that hypomorphic mutations were more likely to be encountered in non-central core myopathies. With analysis of the location of non-hypomorphic mutations, we found that missense mutations were generally enriched in the MH/CCD hotspots and specifically enriched in the selectivity filter of the channel pore.ConclusionsThese results support a hypothesis that loss of protein function is a key predictive disease parameter. In addition, they suggest that decreased RyR1 expression may dictate non-core related pathology though, data on protein expression was limited and should be confirmed in a larger cohort. Lastly, the results implicate abnormal ion conductance through the channel pore in the pathogenesis in recessive core myopathies. Overall, our findings represent a comprehensive analysis of genotype-phenotype associations in recessive RYR1-myopathies.

Case Scenario: Hypotonia in Infancy

NFANTS with hypotonia of unknown etiology pose a unique challenge as many of the potential diagnoses have major, often conflicting, anesthetic management implications. The differential diagnosis of hypotonia is long and includes possibilities such as Duchenne muscular dystrophy, central core disease, and multiminicore disease. Intravenous anesthetic techniques are recommended because hyperkalemia or malignant hyperthermia is associated with the use of volatile anesthetics. However, the differential diagnosis of infants with hypotonia also includes mitochondrial disorders. In children with mitochondrial disorders, an intravenous anesthetic technique that includes propofol could lead to metabolic decompensation because propofol alters mitochondrial electron transfer. This dilemma is often encountered when hypotonic infants require anesthesia for diagnostic tests such as magnetic resonance imaging and muscle biopsies for definitive diagnosis.

Oxygen-coupled Redox Regulation of the Skeletal Muscle Ryanodine Receptor/Ca2+ Release Channel (RyR1): SITES AND NATURE OF OXIDATIVE MODIFICATION

In mammalian skeletal muscle, Ca(2+) release from the sarcoplasmic reticulum (SR) through the ryanodine receptor/Ca(2+)-release channel RyR1 can be enhanced by S-oxidation or S-nitrosylation of separate Cys residues, which are allosterically linked. S-Oxidation of RyR1 is coupled to muscle oxygen tension (pO2) through O2-dependent production of hydrogen peroxide by SR-resident NADPH oxidase 4. In isolated SR (SR vesicles), an average of six to eight Cys thiols/RyR1 monomer are reversibly oxidized at high (21% O2) versus low pO2 (1% O2), but their identity among the 100 Cys residues/RyR1 monomer is unknown. Here we use isotope-coded affinity tag labeling and mass spectrometry (yielding 93% coverage of RyR1 Cys residues) to identify 13 Cys residues subject to pO2-coupled S-oxidation in SR vesicles. Eight additional Cys residues are oxidized at high versus low pO2 only when NADPH levels are supplemented to enhance NADPH oxidase 4 activity. pO2-sensitive Cys residues were largely non-overlapping with those identified previously as hyperreactive by administration of exogenous reagents (three of 21) or as S-nitrosylated. Cys residues subject to pO2-coupled oxidation are distributed widely within the cytoplasmic domain of RyR1 in multiple functional domains implicated in RyR1 activity-regulating interactions with the L-type Ca(2+) channel (dihydropyridine receptor) and FK506-binding protein 12 as well as in "hot spot" regions containing sites of mutation implicated in malignant hyperthermia and central core disease. pO2-coupled disulfide formation was identified, whereas neither S-glutathionylated nor sulfenamide-modified Cys residues were observed. Thus, physiological redox regulation of RyR1 by endogenously generated hydrogen peroxide is exerted through dynamic disulfide formation involving multiple Cys residues.

Analysis of histomorphology in malignant hyperthermia-susceptible patients

Malignant hyperthermia (MH) is a potentially lethal disorder of skeletal muscle triggered by anesthetic agents. A histomorphological examination of diseased muscle may provide insight into MH pathophysiology, but it is not a routine part of standard-of-care practice for the identification of MH-susceptibility. In this study, we investigated muscle histomorphology in a large cohort of MH-susceptible (MHS) patients and examined its relationship to genotype and phenotype. All consenting patients who were identified as MHS based on a caffeine-halothane contracture test (CHCT) performed during 1992-2011 were retrospectively identified and recruited for this study. Results of the histomorphological examination, which is a routine part of our centre-specific practice, were reviewed. Patient demographics, MH proband status, histological features, CHCTs, and genetic results for MH-causative mutations were summarized. Seven of the 399 patients classified as MHS had histological characteristics consistent with central core disease, and one patient was a carrier of Duchenne's muscular dystrophy. Eighty-six (22%) patients had histological abnormalities, and five (6%) of these had evidence of "frank" myopathy. No histologic abnormalities were consistent among the MHS patients; however, a higher proportion of MH probands had abnormal histomorphology compared with the general MHS population, and patients with evidence of "frank" myopathy showed similarities in clinical history, biochemistry, CHCT, and genetic testing. Despite the inability of the histomorphological examination to identify consistent features in MHS patients, histology may serve as a potential adjunct to CHCT and aid in the identification of other myopathies. Nevertheless, the specifics of its utility ought to be assessed in other studies and by way of formal cost-effectiveness analysis.

Exon Skipping as a Therapeutic Strategy Applied to anRYR1Mutation with Pseudo-Exon Inclusion Causing a Severe Core Myopathy

Central core disease is a myopathy often arising from mutations in the type 1 ryanodine receptor (RYR1) gene, encoding the sarcoplasmic reticulum calcium release channel RyR1. No treatment is currently available for this disease. We studied the pathological situation of a severely affected child with two recessive mutations, which resulted in a massive reduction in the amount of RyR1. The paternal mutation induced the inclusion of a new in-frame pseudo-exon in RyR1 mRNA that resulted in the insertion of additional amino acids leading to the instability of the protein. We hypothesized that skipping this additional exon would be sufficient to restore RyR1 expression and to normalize calcium releases. We therefore developed U7-AON lentiviral vectors to force exon skipping on affected primary muscle cells. The efficiency of the exon skipping was evaluated at the mRNA level, at the protein level, and at the functional level using calcium imaging. In these affected cells, we observed a decreased inclusion of the pseudo-exon, an increased RyR1 protein expression, and a restoration of calcium releases of normal amplitude either upon direct RyR1 stimulation or in response to membrane depolarization. This study is the first demonstration of the potential of exon-skipping strategy for the therapy of central core disease, from the molecular to the functional level.

Core myopathies and malignant hyperthermia susceptibility: a review

The core myopathies are a subset of myopathies that present in infancy with hypotonia and muscle weakness. They were formerly considered a rare type of congenital myopathy but are now recognized as being more prevalent. Due to their genetic linkage to mutations in the ryanodine receptor gene (RYR1), core myopathies (in particular, central core disease) carry a high risk of malignant hyperthermia susceptibility. In this review article, we describe the phenotypical, genetic, and histopathological characteristics of core myopathies and further describe the currently understood nature of their risk of malignant hyperthermia. We also review the level of suspicion a clinician should exhibit with a child who has a possible core myopathy or other congenital myopathy presenting for an anesthetic prior to a definitive genetic analysis. For this review article, we performed literature searches using the key words anesthesiology, core myopathies, pediatric neurology, malignant hyperthermia, genetics, ryanodine receptor, and molecular biology. We also relied on literature accumulated by the two authors, who served as hotline consultants for the Malignant Hyperthermia Hotline of the Malignant Hyperthermia Association of the United States (MHAUS) for the past 12years.

Congenital myopathies--clinical features and frequency of individual subtypes diagnosed over a 5-year period in the United Kingdom.

The congenital myopathies are a group of inherited neuromuscular disorders mainly defined on the basis of characteristic histopathological features. We analysed 66 patients assessed at a single centre over a 5 year period. Of the 54 patients where muscle biopsy was available, 29 (54%) had a core myopathy (central core disease, multi-minicore disease), 9 (17%) had nemaline myopathy, 7 (13%) had myotubular/centronuclear myopathy, 2 (4%) had congenital fibre type disproportion, 6 (11%) had isolated type 1 predominance and 1 (2%) had a mixed core-rod myopathy. Of the 44 patients with a genetic diagnosis, RYR1 was mutated in 26 (59%), ACTA1 in 7 (16%), SEPN1 in 7 (16%), MTM1 in 2 (5%), NEB in 1 (2%) and TPM3 in 1 (2%). Clinically, 77% of patients older than 18 months could walk independently. 35% of all patients required ventilatory support and/or enteral feeding. Clinical course was stable or improved in 57/66 (86%) patients, whilst 4 (6%) got worse and 5 (8%) died. These findings indicate that core myopathies are the most common form of congenital myopathies and that more than half can be attributed to RYR1 mutations. The underlying genetic defect remains to be identified in 1/3 of congenital myopathies cases.

Ca2+ Release in Muscle Fibers Expressing R4892W and G4896V Type 1 Ryanodine Receptor Disease Mutants

The large and rapidly increasing number of potentially pathological mutants in the type 1 ryanodine receptor (RyR1) prompts the need to characterize their effects on voltage-activated sarcoplasmic reticulum (SR) Ca2+ release in skeletal muscle. Here we evaluated the function of the R4892W and G4896V RyR1 mutants, both associated with central core disease (CCD) in humans, in myotubes and in adult muscle fibers. For both mutants expressed in RyR1-null (dyspedic) myotubes, voltage-gated Ca2+ release was absent following homotypic expression and only partially restored following heterotypic expression with wild-type (WT) RyR1. In muscle fibers from adult WT mice, both mutants were expressed in restricted regions of the fibers with a pattern consistent with triadic localization. Voltage-clamp-activated confocal Ca2+ signals showed that fiber regions endowed with G4896V-RyR1s exhibited an ∼30% reduction in the peak rate of SR Ca2+ release, with no significant change in SR Ca2+ content. Immunostaining revealed no associated change in the expression of either α1S subunit (Cav1.1) of the dihydropyridine receptor (DHPR) or type 1 sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA1), indicating that the reduced Ca2+ release resulted from defective RyR1 function. Interestingly, in spite of robust localized junctional expression, the R4892W mutant did not affect SR Ca2+ release in adult muscle fibers, consistent with a low functional penetrance of this particular CCD-associated mutant.

Spectrum of congenital myopathies: A single centre experience

Congenital myopathies (CMs) are rare and they are clinically and genetically heterogeneous. Muscle biopsy is characterized by structural abnormality that is diagnostic. There are few studies from India. This is a retrospective study of 12 years. The demographic data, clinical features and laboratory data of patients diagnosed as CMs on muscle biopsy were retrieved from medical records. The slides were reviewed for morphological and structural abnormalities using the following stains hematoxylin and eosin, modified Gomori trichrome, masson trichrome, periodic acid schiff, adenosine triphosphatase preincubated at pH 9.4, 4.6 and 4.3, nicotinamide adenine dinucleotide tetrazolium reductase, succinic dehydrogenase and cytochrome c oxidase. Immunohistochemistry was performed with dystrophin, sarcoglycans and desmin wherever necessary. There were 50 patients with CMs: Centronuclear myopathy (23), myotubular myopathy (3) and central core disease (CCD) (8), nemaline myopathy (5), congenital fiber type proportion (10) and desmin related myopathy with arrythmogenic right ventricular cardiomyopathy (ARVD) (1). Of the 50 patients, 30 (60%) presented in the first decade of life. Proximal muscle weakness and hypotonia were the common presenting features. Type 1 atrophy and predominance were seen in most cases on muscle biopsy. CCD had one patient with high creatine phosphokinase levels, biopsy in one patient showed both rods and cores, in the other limb girdle muscular dystrophy like picture and one biopsy showed uniform type 1 fibers. There was one desmin related myopathy with ARVD, who had cardiac transplantation and both skeletal and cardiac muscle showed characteristic rimmed vacuoles and inclusions positive for desmin. CMs are rare and the diagnosis can only be established on muscle biopsy. Defining the specific CMs helps the clinician in counseling the patient and family.

In Situ Visualization of Mouse Skeletal Muscle Ryanodine Receptor by Cryo-Electron Tomography

Among three isoforms of mouse ryanodine receptors (RyRs), which share 70% similarities with their amino acid sequences, the type 1 skeletal muscle ryanodine receptor (RyR1) is the most extensively studied and known. Structurally, RyR1 is made up of four identical subunits. Each subunit has a molecular mass of approximately 500-600 kDa. This isoform contains at least two functional domains: a carboxyl-terminal hydrophobic domain or trans-membrane domain that contains the Ca2+ release channel pore, and a large amino-terminal domain or cytoplasmic domain that contains sites responsible for the channel regulation. Physiologically, RyR1 functions as a channel for calcium-induced calcium release (CICR) during excitation-contraction coupling (EC-coupling), in which Ca2+ release from the sarcoplasmic reticulum is followed by the subsequent muscle contraction. In central core disease, mutations in RyR1 may promote disruption of EC-coupling resulting in skeletal muscle weakness and lower limb deformities. The important role for RyR1 in this physiological function drives the need for high resolution and comprehensive structural information. Despite many structural studies on RyR1, however, the mechanism underlying this phenomenon remains enigmatic. Advances in techniques and approaches to observe a structure of RyR1 is important to address this question. In this study, we used immunogold for in situ visualization of mice RyR1 by electron tomography. To our knowledge, this is the first report on the application of electron tomography for in situ visualization of RyR1.

The General Anaesthetic Binding Site of Calmodulin Disrupts Ryanodine Peptide Binding

The skeletal muscle Ryanodine Receptor (RyR1) is a large calcium release channel involved in excitation-contraction coupling. It is also the target for hundreds of disease mutations that cause malignant hyperthermia (MH) or skeletal muscle disorders like central core disease (CCD). MH is typically triggered by volatile anesthetics, but their binding site on RyR1 has not been fully described. RyR1 is under the control of several auxiliary proteins. One of these is Calmodulin (CaM), a Ca2+-binding protein that can suppress RyR1 activity at elevated Ca2+ concentrations. Here we investigate how CaM can bind to RyR1, and how this may be affected by volatile anesthetics. We found that CaM can bind to at least three different RyR1 peptides, with the affinity and lobe specificity being altered substantially by the Ca2+ concentrations. In addition, we identified two binding sites for sevoflurane, a volatile anesthetic, on Ca2+/CaM. The anesthetic binds to a pocket that is involved in binding RyR1 peptides. In addition, it can alter the affinity of the N-terminal CaM lobe for Ca2+. These findings suggest that binding of anesthetics to CaM may be involved in the pathophysiology of malignant hyperthermia.