An Association between OXPHOS-Related Gene Expression and Malignant Hyperthermia Susceptibility in Human Skeletal Muscle Biopsies.

Malignant hyperthermia (MH) is a pharmacogenetic condition of skeletal muscle that manifests in hypermetabolic responses upon exposure to volatile anaesthetics. This condition is caused primarily by pathogenic variants in the calcium-release channel RYR1, which disrupts calcium signalling in skeletal muscle. However, our understanding of MH genetics is incomplete, with no variant identified in a significant number of cases and considerable phenotype diversity. In this study, we applied a transcriptomic approach to investigate the genome-wide gene expression in MH-susceptible cases using muscle biopsies taken for diagnostic testing. Baseline comparisons between muscle from MH-susceptible individuals (MHS, n = 8) and non-susceptible controls (MHN, n = 4) identified 822 differentially expressed genes (203 upregulated and 619 downregulated) with significant enrichment in genes associated with oxidative phosphorylation (OXPHOS) and fatty acid metabolism. Investigations of 10 OXPHOS target genes in a larger cohort (MHN: n = 36; MHS: n = 36) validated the reduced expression of ATP5MD and COQ6 in MHS samples, but the remaining 8 selected were not statistically significant. Further analysis also identified evidence of a sex-linked effect in SDHB and UQCC3 expression, and a difference in ATP5MD expression across individuals with MH sub-phenotypes (trigger from in vitro halothane exposure only, MHSh (n = 4); trigger to both in vitro halothane and caffeine exposure, MHShc (n = 4)). Our data support a link between MH-susceptibility and dysregulated gene expression associated with mitochondrial bioenergetics, which we speculate plays a role in the phenotypic variability observed within MH.

Epidemiological and clinical features of malignant hyperthermia: A scoping review.

Malignant hyperthermia (MH) is a potentially fatal inherited pharmacogenetic disorder related to pathogenic variants in the RYR1, CACNA1S, or STAC3 genes. Early recognition of the occurrence of MH and prompt medical treatment are indispensable to ensure a positive outcome. The purpose of this study was to provide valuable information for the early identification of MH by summarizing epidemiological and clinical features of MH. This scoping review followed the methodological framework recommended by Arksey and O'Malley. PubMed, Embase, and Web of science databases were searched for studies that evaluated the epidemical and clinical characteristics of MH. A total of 37 studies were included in this review, of which 26 were related to epidemiology and 24 were associated with clinical characteristics. The morbidity of MH varied from 0.18 per 100 000 to 3.9 per 100 000. The mortality was within the range of 0%-18.2%. Identified risk factors included sex, age, disorders associated with MH, and others. The most frequent initial clinical signs included hyperthermia, sinus tachycardia, and hypercarbia. The occurrence of certain signs, such as hypercapnia, delayed first temperature measurement, and peak temperature were associated with poor outcomes. The epidemiological and clinical features of MH varied considerably and some risk factors and typical clinical signs were identified. The main limitation of this review is that the treatment and management strategies were not assessed sufficiently due to limited information.

Evaluation of Malignant Hyperthermia Features in Patients with Pathogenic or Likely Pathogenic RYR1 Variants Disclosed through a Population Genomic Screening Program.

BACKGROUND: Malignant hyperthermia (MH) susceptibility is a heritable musculoskeletal disorder that can present as a potentially fatal hypermetabolic response to triggering anesthesia agents. Genomic screening for variants in MH-associated genes RYR1 and CACNA1S provides an opportunity to prevent morbidity and mortality. There are limited outcomes data from disclosing variants in RYR1, the most common MH susceptibility gene, in unselected populations. The authors sought to identify the rate of MH features or fulminant episodes after triggering agent exposure in an unselected population undergoing genomic screening including actionable RYR1 variants. METHODS: The MyCode Community Health Initiative by Geisinger (USA) is an electronic health record-linked biobank that discloses pathogenic and likely pathogenic variants in clinically actionable genes to patient-participants. Available electronic anesthesia and ambulatory records for participants with actionable RYR1 results returned through December 2020 were evaluated for pertinent findings via double-coded chart reviews and reconciliation. Descriptive statistics for observed phenotypes were calculated. RESULTS: One hundred fifty-two participants had an actionable RYR1 variant disclosed during the study period. None had previous documented genetic testing for MH susceptibility; one had previous contracture testing diagnosing MH susceptibility. Sixty-eight participants (44.7%) had anesthesia records documenting triggering agent exposure during at least one procedure. None received dantrolene treatment or had documented muscle rigidity, myoglobinuria, hyperkalemia, elevated creatine kinase, severe myalgia, or tea-colored urine. Of 120 possibly MH-related findings (postoperative intensive care unit admissions, hyperthermia, arterial blood gas evaluation, hypercapnia, or tachycardia), 112 (93.3%) were deemed unlikely to be MH events; 8 (6.7%) had insufficient records to determine etiology. CONCLUSIONS: Results demonstrate a low frequency of classic intraanesthetic hypermetabolic phenotypes in an unselected population with actionable RYR1 variants. Further research on the actionability of screening for MH susceptibility in unselected populations, including economic impact, predictors of MH episodes, and expanded clinical phenotypes, is necessary.

Effects of Remimazolam on Intracellular Calcium Dynamics in Myotubes Derived from Patients with Malignant Hyperthermia and Functional Analysis of Type 1 Ryanodine Receptor Gene Variants.

Remimazolam is a novel general anesthetic and its safety in patients with malignant hyperthermia (MH) is unknown. We used myotubes derived from the skeletal muscle of patients with MH to examine the response to ryanodine receptor 1 (RYR1) agonist and remimazolam in MH-susceptible patients. Patients underwent muscle biopsy for the Ca2+-induced Ca2+ release (CICR) rate test, a diagnostic tool for MH in Japan. Ten patients had myotubes obtained from skeletal muscle cultures, and the genes associated with malignant hyperthermia in these patients were analyzed. The EC50 of caffeine, cresol, and remimazolam to induce intracellular calcium concentration change were compared between myotubes from CICR-negative genetic test patients and myotubes from other patients. Eight of the ten were CICR-positive, five of whom had RYR1 causative gene mutations or variants. Two patients had CICR-negative genetic tests, and as expected had the highest EC50 (the concentration of a drug that gives a half-maximal response) in response to caffeine, 4CmC and remimazolam. Three patients had a positive CICR but no known variants in RYR1 or CACNA1S (voltage-gated calcium channel subunit alpha1S). Myotubes in these patients had significantly lower EC50s for all agents than myotubes in CICR-negative patients. When myotubes from a patient who was CICR-negative and had no gene variant were used as a control, myotubes from CICR-positive patients were more hyper-responsive than controls to all stimulants used. The EC50 for remimazolam was lowest for myotubes from CICR-positive, RYR1-mutant patients, at 206 µM (corresponding to 123 µg/mL). The concentration was more than 80-times higher than the clinical concentration. RYR1 gene variants in R4645Q and W5020G were shown to be causative gene mutations for MH. Intracellular calcium in myotubes from MH patients are elevated at high concentrations of remimazolam but not at clinically used concentrations of remimazolam. Remimazolam appears to be safe to use in patients with MH.

Functional characterization of RYR1 variants identified in malignant hyperthermia susceptible individuals.

Malignant hyperthermia is a pharmacogenetic disorder triggered by halogenated anesthetic agents in genetically predisposed individuals. Approximately 70 % of these individuals carry mutations in RYR1, the gene encoding the ryanodine receptor calcium channel of skeletal muscle. In this study, we performed functional analysis of 5 RYR1 variants identified in members from 8 families who had been diagnosed by the IVCT. Of the 68 individuals enrolled in the study, 43 were diagnosed as MHS, 23 as MHN, and 2 individuals were not tested. Here we demonstrate that the 5 RyR1 variants cause hypersensitivity to RyR1 agonist-mediated calcium release. According to the EMHG scoring matrix these five genetic variants can be classified as follows: c.8638G>A (p.E2880K) and c.11314C>T (p.R3772W) likely pathogenic, c.11416G>A (p.G3806R), c.14627A>G (p.K4876R) and c.14813T>C (p.I4938T), pathogenic (RefSeq NM_000540.3). We propose that the newly functionally characterized RYR1 variants, be included in the panel of variants to be used for the molecular diagnosis of MHS.

Pancreatitis in RYR1-related disorders.

Mutations in RYR1 encoding the ryanodine receptor (RyR) skeletal muscle isoform (RyR1) are a common cause of inherited neuromuscular disorders. Despite its expression in a wide range of tissues, non-skeletal muscle manifestations associated with RYR1 mutations have only been rarely reported. Here, we report three patients with a diagnosis of Central Core Disease (CCD), King-Denborough Syndrome (KDS) and Malignant Hyperthermia Susceptibility (MHS), respectively, who in addition to their (putative) RYR1-related disorder also developed symptoms and signs of acute pancreatitis. In two patients, episodes were recurrent, with severe multisystem involvement and sequelae. RyR1-mediated calcium signalling plays an important role in normal pancreatic function but has also been critically implicated in the pathophysiology of acute pancreatitis, particularly in bile acid- and ethanol-induced forms. Findings from relevant animal models indicate that pancreatic damage in these conditions may be ameliorated through administration of the specific RyR1 antagonist dantrolene and other compounds modifying pancreatic metabolism including calcium signalling. These observations suggest that patients with RYR1 gain-of-function variants may be at increased risk of developing acute pancreatitis, a condition which should therefore be considered in the health surveillance of such individuals.

Congenital Myasthenic Syndrome With Malignant Hyperthermia Susceptibility in a Child Undergoing Adenotonsillectomy for Obstructive Sleep Apnea: A Case Report.

Congenital myasthenic syndromes are rare genetic diseases involving pathologic proteins in the neuromuscular junction. Malignant hyperthermia susceptibility is a genetic disorder involving a hypermetabolic response to volatile anesthetics and depolarizing neuromuscular blocking agents. We present the first reported case of a 3-year-old boy with both congenital myasthenic syndrome and malignant hyperthermia susceptibility, resulting from a mutation in the ryanodine receptor type 1 gene, who underwent an adenotonsillectomy for severe obstructive sleep apnea. We discuss the anesthetic challenges in navigating these 3 comorbidities in the setting of airway surgery.

Isoflurane Alters Presynaptic Endoplasmic Reticulum Calcium Dynamics in Wild-Type and Malignant Hyperthermia-Susceptible Rodent Hippocampal Neurons.

Volatile anesthetics reduce excitatory synaptic transmission by both presynaptic and postsynaptic mechanisms which include inhibition of depolarization-evoked increases in presynaptic Ca2+ concentration and blockade of postsynaptic excitatory glutamate receptors. The presynaptic sites of action leading to reduced electrically evoked increases in presynaptic Ca2+ concentration and Ca2+-dependent exocytosis are unknown. Endoplasmic reticulum (ER) of Ca2+ release via ryanodine receptor 1 (RyR1) and uptake by SERCA are essential for regulation intracellular Ca2+ and are potential targets for anesthetic action. Mutations in sarcoplasmic reticulum (SR) release channels mediate volatile anesthetic-induced malignant hyperthermia (MH), a potentially fatal pharmacogenetic condition characterized by unregulated Ca2+ release and muscle hypermetabolism. However, the impact of MH mutations on neuronal function are unknown. We used primary cultures of postnatal hippocampal neurons to analyze volatile anesthetic-induced changes in ER Ca2+ dynamics using a genetically encoded ER-targeted fluorescent Ca2+ sensor in both rat and mouse wild-type (WT) neurons and in mouse mutant neurons harboring the RYR1 T4826I MH-susceptibility mutation. The volatile anesthetic isoflurane reduced both baseline and electrical stimulation-evoked increases in ER Ca2+ concentration in neurons independent of its depression of presynaptic cytoplasmic Ca2+ concentrations. Isoflurane and sevoflurane, but not propofol, depressed depolarization-evoked increases in ER Ca2+ concentration significantly more in mouse RYR1 T4826I mutant neurons than in wild-type neurons. The RYR1 T4826I mutant neurons also showed markedly greater isoflurane-induced reductions in presynaptic cytosolic Ca2+ concentration and synaptic vesicle (SV) exocytosis. These findings implicate RyR1 as a molecular target for the effects of isoflurane on presynaptic Ca2+ handling.

Putative malignant hyperthermia mutation CaV1.1-R174W is insufficient to trigger a fulminant response to halothane or confer heat stress intolerance.

Malignant hyperthermia susceptibility (MHS) is an autosomal dominant pharmacogenetic disorder that manifests as a hypermetabolic state when carriers are exposed to halogenated volatile anesthetics or depolarizing muscle relaxants. In animals, heat stress intolerance is also observed. MHS is linked to over 40 variants in RYR1 that are classified as pathogenic for diagnostic purposes. More recently, a few rare variants linked to the MHS phenotype have been reported in CACNA1S, which encodes the voltage-activated Ca2+ channel CaV1.1 that conformationally couples to RyR1 in skeletal muscle. Here, we describe a knock-in mouse line that expresses one of these putative variants, CaV1.1-R174W. Heterozygous (HET) and homozygous (HOM) CaV1.1-R174W mice survive to adulthood without overt phenotype but fail to trigger with fulminant malignant hyperthermia when exposed to halothane or moderate heat stress. All three genotypes (WT, HET, and HOM) express similar levels of CaV1.1 by quantitative PCR, Western blot, [3H]PN200-110 receptor binding and immobilization-resistant charge movement densities in flexor digitorum brevis fibers. Although HOM fibers have negligible CaV1.1 current amplitudes, HET fibers have similar amplitudes to WT, suggesting a preferential accumulation of the CaV1.1-WT protein at triad junctions in HET animals. Never-the-less both HET and HOM have slightly elevated resting free Ca2+ and Na+ measured with double barreled microelectrode in vastus lateralis that is disproportional to upregulation of transient receptor potential canonical (TRPC) 3 and TRPC6 in skeletal muscle. CaV1.1-R174W and upregulation of TRPC3/6 alone are insufficient to trigger fulminant malignant hyperthermia response to halothane and/or heat stress in HET and HOM mice.

Rhabdomyosarcoma Associated with Core Myopathy/Malignant Hyperthermia: Combined Effect of Germline Variants in RYR1 and ASPSCR1 May Play a Role.

Rhabdomyosarcomas have been described in association with thyroid disease, dermatomyositis, Duchenne muscular dystrophy, and in muscular dystrophy models but not in patients with ryanodine receptor-1 gene (RYR1) pathogenic variants. We described here an 18-year-old male who reported a cervical nodule. Magnetic resonance images revealed a mass in the ethmoidal sinus corresponding to rhabdomyosarcoma. As his father died from malignant hyperthermia (MH), an in vitro contracture test was conducted and was positive for MH susceptibility. Muscle histopathological analysis in the biopsy showed the presence of cores. Molecular analysis using NGS sequencing identified germline variants in the RYR1 and ASPSCR1 (alveolar soft part sarcoma) genes. This report expands the spectrum of diseases associated with rhabdomyosarcomas and a possible differential diagnosis of soft tissue tumors in patients with RYR1 variants.

Muscle Ultrasound Abnormalities in Individuals with RYR1-Related Malignant Hyperthermia Susceptibility.

BACKGROUND: Variants in RYR1, the gene encoding the ryanodine receptor-1, can give rise to a wide spectrum of neuromuscular conditions. Muscle imaging abnormalities have been demonstrated in isolated cases of patients with a history of RYR1-related malignant hyperthermia (MH) susceptibility. OBJECTIVE: To provide insights into the type and prevalence of muscle ultrasound abnormalities and muscle hypertrophy in patients carrying gain-of-function RYR1 variants associated with MH susceptibility and to contribute to delineating the wider phenotype, optimizing the diagnostic work-up and care for MH susceptible patients. METHODS: We performed a prospective cross-sectional observational muscle ultrasound study in patients with a history of RYR1-related MH susceptibility (n = 40). Study procedures included a standardized history of neuromuscular symptoms and a muscle ultrasound assessment. Muscle ultrasound images were analyzed using a quantitative and qualitative approach and compared to reference values and subsequently subjected to a screening protocol for neuromuscular disorders. RESULTS: A total of 15 (38%) patients had an abnormal muscle ultrasound result, 4 (10%) had a borderline muscle ultrasound screening result, and 21 (53%) had a normal muscle ultrasound screening result. The proportion of symptomatic patients with an abnormal result (11 of 24; 46%) was not significantly higher compared to the proportion of asymptomatic patients with an abnormal ultrasound result (4 of 16; 25%) (P = 0.182). The mean z-scores of the biceps brachii (z = 1.45; P < 0.001), biceps femoris (z = 0.43; P = 0.002), deltoid (z = 0.31; P = 0.009), trapezius (z = 0.38; P = 0.010) and the sum of all muscles (z = 0.40; P < 0.001) were significantly higher compared to 0, indicating hypertrophy. CONCLUSIONS: Patients with RYR1 variants resulting in MH susceptibility often have muscle ultrasound abnormalities. Frequently observed muscle ultrasound abnormalities include muscle hypertrophy and increased echogenicity.

What is malignant hyperthermia susceptibility?

The molecular mechanisms of susceptibility to malignant hyperthermia are complex. The malignant hyperthermia susceptibility phenotype should be reserved for patients who have a personal or family history consistent with malignant hyperthermia under anaesthesia and are subsequently demonstrated through diagnostic testing to be at risk.

[Analysis of a case of Multiple pterygium syndrome due to a novel variant of CHRNG gene].

OBJECTIVE: To explore the clinical characteristics and genetic etiology of a child with multiple pterygium syndrome (MPS). METHODS: A child with MPS who was treated at the Orthopedics Department of Guangzhou Women and Children's Medical Center Affiliated to Guangzhou Medical University on August 19, 2020 was selected as the study subject. Clinical data of the child was collected. Peripheral blood samples of the child and her parents were also collected. Whole exome sequencing (WES) was carried out for the child. Candidate variant was validated by Sanger sequencing of her parents and bioinformatic analysis. RESULTS: The child, an 11-year-old female, had a complain of "scoliosis found 8 years before and aggravated with unequal shoulder height for 1 year". WES results revealed that she has carried a homozygous c.55+1G>C splice variant of the CHRNG gene, for which both of her parents were heterozygous carriers. By bioinformatic analysis, the c.55+1G>C variant has not been recorded by the CNKI, Wanfang data knowledge service platform and HGMG databases. Analysis with Multain online software suggested that the amino acid encoded by this site is highly conserved among various species. As predicted with the CRYP-SKIP online software, the probability of activation and skipping of the potential splice site in exon 1 caused by this variant is 0.30 and 0.70, respectively. The child was diagnosed with MPS. CONCLUSION: The CHRNG gene c.55+1G>C variant probably underlay the MPS in this patient.

Molecular Aspects Implicated in Dantrolene Selectivity with Respect to Ryanodine Receptor Isoforms.

Dantrolene is an intra-cellularly acting skeletal muscle relaxant used for the treatment of the rare genetic disorder, malignant hyperthermia (MH). In most cases, MH susceptibility is caused by dysfunction of the skeletal ryanodine receptor (RyR1) harboring one of nearly 230 single-point MH mutations. The therapeutic effect of dantrolene is the result of a direct inhibitory action on the RyR1 channel, thus suppressing aberrant Ca2+ release from the sarcoplasmic reticulum. Despite the almost identical dantrolene-binding sequence exits in all three mammalian RyR isoforms, dantrolene appears to be an isoform-selective inhibitor. Whereas RyR1 and RyR3 channels are competent to bind dantrolene, the RyR2 channel, predominantly expressed in the heart, is unresponsive. However, a large body of evidence suggests that the RyR2 channel becomes sensitive to dantrolene-mediated inhibition under certain pathological conditions. Although a consistent picture of the dantrolene effect emerges from in vivo studies, in vitro results are often contradictory. Hence, our goal in this perspective is to provide the best possible clues to the molecular mechanism of dantrolene's action on RyR isoforms by identifying and discussing potential sources of conflicting results, mainly coming from cell-free experiments. Moreover, we propose that, specifically in the case of the RyR2 channel, its phosphorylation could be implicated in acquiring the channel responsiveness to dantrolene inhibition, interpreting functional findings in the structural context.