Skeletal Muscle Ryanodine Receptor Gene Research Articles

VP417 Obstetric and gynaecological features in females carrying mutations in the skeletal muscle ryanodine receptor (RYR1) gene: a questionnaire study

VP417 Obstetric and gynaecological features in females carrying mutations in the skeletal muscle ryanodine receptor (RYR1) gene: a questionnaire study

Pre-operative exercise and pyrexia as modifying factors in malignant hyperthermia (MH)

Malignant hyperthermia (MH) is a life-threatening reaction triggered by volatile anesthetics and succinylcholine. MH is caused by mutations in the skeletal muscle ryanodine receptor (RYR1) gene, as is rhabdomyolysis triggered by exertion and/or pyrexia. The discrepancy between the prevalence of risk genotypes and actual MH incidence remains unexplained. We investigated the role of pre-operative exercise and pyrexia as potential MH modifying factors. We included cases from 5 MH referral centers with 1) clinical features suggestive of MH, 2) confirmation of MH susceptibility on Contracture Testing (IVCT or CHCT) and/or RYR1 genetic testing, and a history of 3) strenuous exercise within 72 h and/or pyrexia >37.5 °C prior to the triggering anesthetic. Characteristics of MH-triggering agents, surgery and succinylcholine use were collected. We identified 41 cases with general anesthesias resulting in an MH event (GA+MH, n = 41) within 72 h of strenuous exercise and/or pyrexia. We also identified previous general anesthesias without MH events (GA-MH, n = 51) in the index cases and their MH susceptible relatives. Apart from pre-operative exercise and/or pyrexia, trauma and acute abdomen as surgery indications, emergency surgery and succinylcholine use were also more common with GA+MH events. These observations suggest a link between pre-operative exercise, pyrexia and MH.

Pre-Operative Exercise and Pyrexia as Modifying Factors in Malignant Hyperthermia

Background: Malignant Hyperthermia (MH) is a life-threatening reaction triggered by volatile anaesthetics and succinylcholine characterized by hypercapnia, hypermetabolism and muscle breakdown. MH is most commonly caused by mutations in the skeletal muscle ryanodine receptor (RYR1) gene, as is rhabdomyolysis triggered by exertion and/or pyrexia. The discrepancy between risk genotypes and actual MH incidence remains unexplained. We hypothesized that MH is a compound event, reflecting a synergistic effect of predisposing genotype, triggering anaesthetic, and exercise and/or pyrexia as modifying factors. Methods: We performed a retrospective study and identified 41 patients with a history of exercise and/or pyrexia within 72 hours prior to an MH event. We compared the frequency of a history of exercise and/or pyrexia within 72 hours of the triggering agent in “eventful anaesthesias” (n=41) and in “uneventful anaesthesias” (n=51) in the index cases and/or their MH susceptible relatives. Indications for and urgency of surgery, and use of succinylcholine were also recorded. Findings: In eventful anaesthesias, there was a history of exercise and/or pyrexia within 72 hours prior to the anaesthetic in 24(0) and 21(1) patients, compared to 0 and 1 in uneventful anesthesias. Trauma and acute abdomen as surgery indications, emergency surgery and succinylcholine use were also more common with eventful anaesthesias. Interpretation: Exercise and/or pyrexia prior to general anaesthesia are risk factors for MH. Rhabdomyolysis may mimic an acute abdomen, whilst trauma surgery may carry a specific risk due to muscle damage. These observations should inform perioperative guidelines with the potential to reduce fatal anaesthesia complications. Funding Statement: This work is not funded. Declaration of Interests: Sheila Riazi, Luuk van den Bersselaar, Gunilla Islander, Luc Heytens, Marc M. J. Snoeck, Andrew Bjorksten, Robyn Gillies, George Dranitsaris, Anna Hellblom, Susan Treves, Nicol C. Voermans, Heinz Jungbluth: These authors declare no financial interests. Ethics Approval Statement: The retrospective case study was performed with Research Ethics Board approval from the University of Toronto (REB Nr 19-5365), and in accordance with the ethics guidelines issued by other participating centres.

Bioenergetic defects in muscle fibers of RYR1 mutant knock-in mice associated with malignant hyperthermia

Mutations in the skeletal muscle ryanodine receptor gene (RYR1) can cause susceptibility to malignant hyperthermia (MH), a potentially lethal genetic condition triggered by volatile anesthetics. MH is associated with hypermetabolism, which has directed research interest into oxidative phosphorylation and muscle bioenergetics. The most common cause of MH in the United Kingdom is the c.7300G>A RYR1 variant, which is present in ∼16% of MH families. Our study focuses on the MH susceptible G2435R-RYR1 knock-in mouse model, which is the murine equivalent of the human c.7300G>A genotype. Using a combination of transcriptomics, protein expression, and functional analysis, we investigated adult muscle fiber bioenergetics in this mouse model. RNA-Seq data showed reduced expression of genes associated with mitochondria and fatty acid oxidation in RYR1 mutants when compared with WT controls. Mitochondrial function was assessed by measuring oxygen consumption rates in permeabilized muscle fibers. Comparisons between WT and homozygous G2435R-RYR1 mitochondria showed a significant increase in complex I-facilitated oxidative phosphorylation in mutant muscle. Furthermore, we observed a gene-dose-specific increase in reactive oxygen species production in G2435R-RYR1 muscle fibers. Collectively, these findings provide evidence of metabolic defects in G2435R-RYR1 knock-in mouse muscle under basal conditions. Differences in metabolic profile could be the result of differential gene expression in metabolic pathways, in conjunction with mitochondrial damage accumulated from chronic exposure to increased oxidative stress.

New Compound Heterozygous Splice Site Mutations of the Skeletal Muscle Ryanodine Receptor (RYR1) Gene Manifest Fetal Akinesia: A Linkage with Congenital Myopathies

Mutations in the skeletal muscle ryanodine receptor (RYR1) gene have been linked to malignant hyperthermia susceptibility, central core disease, and minicore myopathy with external ophthalmoplegia. RYR1 is an intracellular calcium release channel and plays a crucial role in the sarcoplasmic reticulum and transverse tubule connection. Here, we report 2 fetuses from the same parents with compound heterozygous mutations in the RYR1 gene (c.10347+1G>A and c.10456-2Α>G) who presented with fetal akinesia and polyhydramnios at 27 and 19 weeks of gestation with intrauterine growth restriction in the third pregnancy. The prospective parents of the fetuses were heterozygous carriers for c.10456-2Α>G (mother) and c.10347+1G>A (father). Both mutations affect splice sites resulting in dysfunctional protein forms probably missing crucial domains of the C-terminus. Our findings reveal a new RYR1 splice site mutation (c.10456-2Α>G) that may be associated with the clinical features of myopathies, expanding the RYR1 spectrum related to these pathologies.

DNA Testing for Malignant Hyperthermia

The advent of the polymerase chain reaction and the availability of data from various global human genome projects should make it possible, using a DNA sample isolated from white blood cells, to diagnose rapidly and accurately almost any monogenic condition resulting from single nucleotide changes. DNA-based diagnosis for malignant hyperthermia (MH) is an attractive proposition, because it could replace the invasive and morbid caffeine-halothane/in vitro contracture tests of skeletal muscle biopsy tissue. Moreover, MH is preventable if an accurate diagnosis of susceptibility can be made before general anesthesia, the most common trigger of an MH episode. Diagnosis of MH using DNA was suggested as early as 1990 when the skeletal muscle ryanodine receptor gene (RYR1), and a single point mutation therein, was linked to MH susceptibility. In 1994, a single point mutation in the α 1 subunit of the dihydropyridine receptor gene (CACNA1S) was identified and also subsequently shown to be causative of MH. In the succeeding years, the number of identified mutations in RYR1 has grown, as has the number of potential susceptibility loci, although no other gene has yet been definitively associated with MH. In addition, it has become clear that MH is associated with either of these 2 genes (RYR1 and CACNA1S) in only 50% to 70% of affected families. While DNA testing for MH susceptibility has now become widespread, it still does not replace the in vitro contracture tests. Whole exome sequence analysis makes it potentially possible to identify all variants within human coding regions, but the complexity of the genome, the heterogeneity of MH, the limitations of bioinformatic tools, and the lack of precise genotype/phenotype correlations are all confounding factors. In addition, the requirement for demonstration of causality, by in vitro functional analysis, of any familial mutation currently precludes DNA-based diagnosis as the sole test for MH susceptibility. Nevertheless, familial DNA testing for MH susceptibility is now widespread although limited to a positive diagnosis and to those few mutations that have been functionally characterized. Identification of new susceptibility genes remains elusive. When new genes are identified, it will be the role of the biochemists, physiologists, and biophysicists to devise functional assays in appropriate systems. This will remain the bottleneck unless high throughput platforms can be designed for functional work. Analysis of entire genomes from several individuals simultaneously is a reality. DNA testing for MH, based on current criteria, remains the dream.

198th ENMC International Workshop: 7th Workshop on Centronuclear (Myotubular) myopathies, 31st May – 2nd June 2013, Naarden, The Netherlands

1. Introduction and overviewSixteen clinicians and basic scientists from 4 countriesconvened from the 31st of May to the 2nd of June 2013 inNaarden, The Netherlands, for the 198th ENMC sponsoredWorkshop on Centronuclear/Myotubular myopathies(CNM/MTM). The workshop was also attended by AnneLennox and Melanie Spring as representatives of theMyotubular Trust, a European patient support group forpatients affected by myotubular (centronuclear) myopathies,and by Hal Landy, Deborah Ramsdell and MatthewPatterson, representatives of 2 industry partners, Valerionand Audentes Therapeutics, with an interest in developingspecific therapies for CNM/MTM.Following a welcome from Daniel Zollinger, ENMCrepresentative, and the chairpersons of the workshop,Carina Wallgren-Pettersson (Helsinki, Finland) andJocelyn Laporte (Illkirch, France) gave an overview ofnew developments in the field: Since the most recent, 6thENMC MTM/CNM workshop held in January 2009 [1],further in-depth phenotypical characterization has lead tothe proposal of histopathological subclasses [2], wideningof the clinical spectrum for genetically already resolvedforms [3–5] and refinement of the molecular diagnosis[6,7]. Mutations in the skeletal muscle ryanodine receptor(RYR1) gene [8–10] and the CCDC78 gene [11] have nowbeen associated with congenital myopathies withprominent internal nuclei and the arrival of nextgeneration sequencing for genetic diagnosis has lead tothe identification of further novel genes [11]. Models tostudy and/or to rescue the affected cellular pathways arenow available in yeast [12,13], Caenorhabditis elegans [14–16], drosophila [17], zebrafish [18,19], mouse [20–23] anddog [24]. Those models have suggested several potentialpathogenic mechanisms, including alteration of triads andcalcium handling [18,25], defects of the neuromuscularjunction [19,26,27], satellite cells [28], mitochondria andthe desmin cytoskeleton [29], as well as the autophagypathway [23,30]. Some of those pathogenic mechanismsare common to different forms of CNM [31,32]. Severalproof-of-concept studies aimed at amelioration of theCNM phenotypes have recently been reported, includingreplacement of the myotubularin protein through either

Mutations in RYR1 are a common cause of exertional myalgia and rhabdomyolysis

Mutations in the skeletal muscle ryanodine receptor (RYR1) gene are a common cause of neuromuscular disease, ranging from various congenital myopathies to the malignant hyperthermia (MH) susceptibility trait without associated weakness. We sequenced RYR1 in 39 unrelated families with rhabdomyolysis and/or exertional myalgia, frequent presentations in the neuromuscular clinic that often remain unexplained despite extensive investigations. We identified 9 heterozygous RYR1 mutations/variants in 14 families, 5 of them (p.Lys1393Arg; p.Gly2434Arg; p.Thr4288_Ala4290dup; p.Ala4295Val; and p.Arg4737Gln) previously associated with MH. Index cases presented from 3 to 45 years with rhabdomyolysis, with or without exertional myalgia (n=12), or isolated exertional myalgia (n=2). Rhabdomyolysis was commonly triggered by exercise and heat and, less frequently, viral infections, alcohol and drugs. Most cases were normally strong and had no personal MH history. Inconsistent additional features included heat intolerance, and cold-induced muscle stiffness. Muscle biopsies showed mainly subtle changes. Familial RYR1 mutations were confirmed in relatives with similar or no symptoms. These findings suggest that RYR1 mutations may account for a substantial proportion of patients presenting with unexplained rhabdomyolysis and/or exertional myalgia. Associated clinico-pathological features may be subtle and require a high degree of suspicion. Additional family studies are paramount in order to identify potentially MH susceptible relatives.

A novel late-onset axial myopathy associated with mutations in the skeletal muscle ryanodine receptor (RYR1) gene

Mutations in the skeletal muscle ryanodine receptor (RYR1) gene are a common cause of inherited neuromuscular disorders and have been associated with a wide clinical spectrum, ranging from various congenital myopathies to the malignant hyperthermia susceptibility (MHS) trait without any associated weakness. RYR1-related myopathies are usually of early-childhood onset. Here we present 11 patients from 8 families with a late-onset axial myopathy associated with RYR1 variants. Patients presented between the third and seventh decade of life to neuromuscular centres in Norway, the Netherlands and the United Kingdom with predominant axial muscle involvement, comprising variable degrees of lumbar hyperlordosis, scapular winging and/or camptocormia. Marked myalgia was commonly associated. Serum creatine kinase levels were normal or moderately elevated. Muscle imaging showed consistent involvement of the lower paravertebral muscles and the posterior thigh. Muscle biopsy findings were often discrete, featuring variability in fibre size, increased internal nuclei and unevenness of oxidative enzyme staining, but only rarely overt cores. RYR1 sequencing revealed heterozygous missense variants, either previously associated with the MHS trait or localizing to known MHS mutational hotspots. These findings indicate that MHS-related RYR1 mutations may present later in life with prominent axial weakness but not always typical histopathological features. We propose a combined effect of RyR1 dysfunction, aging and particular vulnerability of axial muscle groups as a possible pathogenic mechanism. RYR1 is a candidate for cases with "idiopathic" camptocormia or bent spine syndrome (BSS).

Congenital myopathy with focal loss of cross-striations revisited

In 1977 Wijngaarden et al. reported a Dutch family with a congenital myopathy characterized by external ophthalmoplegia and a remarkable histological feature, focal loss of cross-striations. A small number of other families with similar clinical and pathological features led to the consideration of this congenital myopathy as a distinct entity. Here we present more than 30years of follow-up from the Dutch family and report recently identified compound heterozygous mutations in the skeletal muscle ryanodine receptor (RYR1) gene, c.10627-2A>G and p.Arg3539His (c.10616G>A). Focal loss of cross-striations on muscle biopsy is another histopathological feature that should raise the possibility of RYR1 involvement.

Association of halothane sensitivity with growth and meat quality in pigs

Previous reports have indicated that a proportion of pigs, homozygous normal for the skeletal muscle ryanodine receptor gene (RYR1), was halothane sensitive, and this was associated with poor meat quality when pigs were handled aggressively. This study was conducted to evaluate halothane sensitivity in RYR1-normal pigs, managed under simulated commercial conditions, to ascertain the association of halothane sensitivity with growth rate and meat quality. A total of 363 pigs across four farrowing groups, from seven Landrace sires and 38 Yorkshire–Landrace F1 dams, were tested at 8 weeks of age for halothane sensitivity using a closed system that delivered 5% halothane at 2 l/min for 3 (group 1) or 2 (groups 2 to 4) min. After 1 min, limb rigidity, limb tremors and abdominal discoloration were evaluated on a binomial scale with 0 indicating no reaction and 1 indicating reaction. Testing was repeated 2 days later. At 10 weeks of age, pigs were moved to finishing pens and not moved again until marketing. Within farrowing group, pigs were harvested in one of two groups, and at marketing were moved a distance of 91 m, weighed, tattooed, loaded and transported a distance of 550 km to a commercial harvest plant. After overnight rest, pigs were harvested and the pH of the loin muscle was measured at 45 min (pH45) after stunning. After an 18-h chill, loin muscle pH (pHu), International Commission on Illumination (CIE) L*, a*, b*, color (1 to 6) and marbling (1 to 10) scores and fluid loss percent were collected. Generalized linear mixed models were used to estimate repeatabilities for response to halothane challenge. Repeatabilities for limb rigidity for the front right and left legs were 0.24 and 0.31, respectively, whereas rear right and left leg repeatabilities were 0.19 and 0.17, respectively. Repeatabilities for front right and left leg tremors were 0.16 and 0.20, respectively. Growth rate was not influenced by any measure of halothane sensitivity. Carcasses from pigs exhibiting limb rigidity tended to have lower pH45 (5.88 v. 5.97; P = 0.06), similar pHu (5.47 v. 5.49; P = 0.32), less pH decline from 45 min to 18 h (−0.40 v. −0.50; P = 0.04) and a tendency for greater fluid loss percent (5.01 v. 4.55; P = 0.08) than carcasses from pigs that did not exhibit limb rigidity during halothane challenge. A proportion of pigs normal for RYR1 did exhibit limb rigidity during halothane gas challenge, and subsequently tended to have lower 45 min pH and greater longissimus muscle fluid loss post harvest.

A study of a family with the skeletal muscle RYR1 mutation (c.7354C>T) associated with central core myopathy and malignant hyperthermia susceptibility

Congenital myopathies are early onset hereditary muscle disorders. A sub-group of these is associated with malignant hyperthermia susceptibility. Mutations in the skeletal muscle ryanodine receptor ( RYR1) gene have been associated with various congenital myopathy phenotypes and may also cause malignant hyperthermia susceptibility. We describe nine affected members of an extended family presenting with a myopathy typically manifesting as upper eye lid ptosis, quadriceps atrophy and patellar dislocation. Three affected members underwent extensive genetic testing and have a RYR1 exon 46 c.7354C>T gene mutation; two of whom had muscle biopsies – both demonstrated central core myopathy. The only affected family member who underwent testing for malignant hyperthermia susceptibility was shown to be positive. The clinical phenotypes seen among affected family members varies widely in severity, and have features in common with those congenital myopathies associated with malignant hyperthermia susceptibility, raising the possibility that these conditions represent a spectrum of disease.

O.15 Mutations in the skeletal muscle ryanodine receptor (RYR1) gene presenting with exertional myalgia and rhabdomyolysis

O.15 Mutations in the skeletal muscle ryanodine receptor (RYR1) gene presenting with exertional myalgia and rhabdomyolysis

P3.41. Clinical and genetic findings in a large cohort of patients with congenital myopathies due to mutations in the skeletal muscle ryanodine receptor (RYR1) gene

P3.41. Clinical and genetic findings in a large cohort of patients with congenital myopathies due to mutations in the skeletal muscle ryanodine receptor (RYR1) gene

P3.43 An adult-onset, slowly progressive axial myopathy with cataracts due to mutations in the skeletal muscle ryanodine receptor (RYR1) gene

P3.43 An adult-onset, slowly progressive axial myopathy with cataracts due to mutations in the skeletal muscle ryanodine receptor (RYR1) gene

1FC2.5 Recessive mutations in the skeletal muscle ryanodine receptor (RYR1) gene may mimick X-linked centronuclear (myotubular) myopathy (XLMTM)

of nighttime BiPAP use in 45 patients at an average age of 11.34±4.28 years. Patients at the mildest end of the phenotypic spectrum did not demonstrate a clear pattern of decline in respiratory function. Conclusions: Forced vital capacity is a viable outcome measure for patients at the moderate to severe end of the collagen VI myopathy phenotypic spectrum, a finding which should inform optimal surveillance and clinical trial planning.

King–Denborough syndrome with and without mutations in the skeletal muscle ryanodine receptor ( RYR1) gene

King–Denborough syndrome (KDS), first described in 1973, is a rare condition characterised by the triad of dysmorphic features, myopathy, and malignant hyperthermia susceptibility (MHS). Autosomal dominant inheritance with variable expressivity has been reported in several cases. Mutations in the skeletal muscle ryanodine receptor ( RYR1) gene have been implicated in a wide range of myopathies such as central core disease (CCD), the malignant hyperthermia (MH) susceptibility trait and one isolated patient with KDS. Here we report clinical, pathologic and genetic features of four unrelated patients with KDS. Patients had a relatively uniform clinical presentation but muscle biopsy findings were highly variable. Heterozygous missense mutations in RYR1 were uncovered in three out of four families, of which one mutation was novel and two have previously been reported in MH. Further RyR1 protein expression studies performed in two families showed marked reduction of the RyR1 protein, indicating the presence of allelic RYR1 mutations not detectable on routine sequencing and potentially explaining marked intrafamilial variability. Our findings support the hypothesis that RYR1 mutations are associated with King–Denborough syndrome but that further genetic heterogeneity is likely.

P68 The pathological spectrum associated with mutations in the skeletal muscle ryanodine receptor (RYR1) gene

P68 The pathological spectrum associated with mutations in the skeletal muscle ryanodine receptor (RYR1) gene

Multi-minicore disease and atypical periodic paralysis associated with novel mutations in the skeletal muscle ryanodine receptor (RYR1) gene

The skeletal muscle ryanodine receptor plays a crucial role in excitation–contraction (EC) coupling and is implicated in various congenital myopathies. The periodic paralyses are a heterogeneous, dominantly inherited group of conditions mainly associated with mutations in the SCN4A and the CACNA1S genes. The interaction between RyR1 and DHPR proteins underlies depolarization-induced Ca 2+ release during EC coupling in skeletal muscle. We report a 35-year-old woman presenting with signs and symptoms of a congenital myopathy at birth and repeated episodes of generalized, atypical normokalaemic paralysis in her late teens. Genetic studies of this patient revealed three heterozygous RYR1 substitutions (p.Arg2241X, p.Asp708Asn and p.Arg2939Lys) associated with marked reduction of the RyR1 protein and abnormal DHPR distribution. We conclude that RYR1 mutations may give rise to both myopathies and atypical periodic paralysis, and RYR1 mutations may underlie other unresolved cases of periodic paralysis with unusual features.

Multi-minicore disease and atypical periodic paralysis associated with novel mutations in the skeletal muscle ryanodine receptor (RYR1) gene

Multi-minicore disease and atypical periodic paralysis associated with novel mutations in the skeletal muscle ryanodine receptor (RYR1) gene