Advertisement

Risk of malignant hyperthermia in patients carrying a variant in the skeletal muscle ryanodine receptor 1 gene

Open AccessPublished:October 18, 2022DOI:https://doi.org/10.1016/j.nmd.2022.10.003

      Highlights

      • Individuals with a variant in the RYR1-gene should be tested for MHS with IVCT.
      • Patients with MHS can have few to no abnormalities on ancillary testing.
      • Sometimes the combination of 2 VUS are necessary to express the MHS-trait.

      Abstract

      Malignant hyperthermia is a life-threatening disorder, which can be prevented by avoiding certain anesthetic agents. Pathogenic variants in the skeletal muscle ryanodine receptor 1-gene are linked to malignant hyperthermia. We retrospectively studied 15 patients who presented to our clinic with symptoms of muscle dysfunction (weakness, myalgia or cramps) and were later found to have a variant in the skeletal muscle ryanodine receptor 1-gene. Symptoms, creatine kinase levels, electromyography, muscle biopsy and in vitro contracture test results were reviewed. Six out of the eleven patients, with a variant of unknown significance in the skeletal muscle ryanodine receptor 1-gene, had a positive in vitro contracture test, indicating malignant hyperthermia susceptibility. In one patient, with two variants of unknown significance, both variants were required to express the malignant hyperthermia-susceptibility trait. Neurologists should consider screening the skeletal muscle ryanodine receptor 1-gene in patients with myalgia or cramps, even when few to no abnormalities on ancillary testing.

      Keywords

      1. Introduction

      Malignant hyperthermia (MH) is a rare, but life-threatening condition [
      • Rosenberg H.
      • Davis M.
      • James D.
      • Pollock N.
      • Stowell K.
      Malignant hyperthermia.
      ]. This pharmacogenetic disorder of skeletal muscle presents as a hypermetabolic response to potent volatile anesthetic gasses such as halothane and isoflurane and the depolarizing muscle relaxant succinylcholine [
      • Denborough M.
      Malignant hyperthermia.
      ,
      • Hopkins P.M.
      Malignant hyperthermia: pharmacology of triggering.
      ]. The clinical signs of MH include hyperthermia, tachycardia, tachypnea, increased carbon dioxide production, increased oxygen consumption, acidosis, muscle rigidity and rhabdomyolysis [
      • Rosenberg H.
      • Pollock N.
      • Schiemann A.
      • Bulger T.
      • Stowell K.
      Malignant hyperthermia: a review.
      ,
      • Halliday N.J.
      Malignant hyperthermia.
      ,
      • Ellis F.R.
      • Halsall P.J.
      • Christian A.S.
      Clinical presentation of suspected malignant hyperthermia during anaesthesia in 402 probands.
      ]. The pathophysiologic changes of MH are due to an uncontrolled increase in myoplasmic calcium, leading to increased muscle activation [
      • Hopkins P.M.
      • Gupta P.K.
      • Bilmen J.G.
      Malignant hyperthermia.
      ]. As a result, the muscle membrane integrity becomes compromised due to ATP depletion, which eventually leads to hyperkalemia and rhabdomyolysis [
      • Rosenberg H.
      • Davis M.
      • James D.
      • Pollock N.
      • Stowell K.
      Malignant hyperthermia.
      ,
      • Maclennan D.H.
      • Zvaritch E.
      Mechanistic models for muscle diseases and disorders originating in the sarcoplasmic reticulum.
      ,
      • MacLennan D.H.
      • Phillips M.S.
      Malignant hyperthermia.
      ]. By identifying those at risk of this potentially fatal condition, it can be avoided. Avoidance of anesthetic triggers and monitoring of patients at risk reduces the risk of a fatal outcome [
      • Rüffert H.
      • Bastian B.
      • Bendixen D.
      • Girard T.
      • Heiderich S.
      • Hellblom A.
      • et al.
      Consensus guidelines on perioperative management of malignant hyperthermia suspected or susceptible patients from the European Malignant Hyperthermia Group.
      ]. Treatment of a MH-crisis should include immediate cessation of triggering agents and administration of the muscle relaxant dantrolene, which will counteract the uncontrolled rise in calcium and its potentially fatal consequences [
      • Hopkins P.M.
      • Gupta P.K.
      • Bilmen J.G.
      Malignant hyperthermia.
      ,
      • Glahn K.P.
      • Ellis F.R.
      • Halsall P.J.
      • Müller C.R.
      • Snoeck M.M.
      • Urwyler A.
      • et al.
      Recognizing and managing a malignant hyperthermia crisis: guidelines from the European Malignant Hyperthermia Group.
      ].
      Central in the pathogenesis of MH is the ryanodine receptor of skeletal muscle, further referred to as RyR1 [
      • Rueffert H.
      • Olthoff D.
      • Deutrich C.
      • Meinecke C.D.
      • Froster U.G.
      Mutation screening in the ryanodine receptor 1 gene (RYR1) in patients susceptible to malignant hyperthermia who show definite IVCT results: identification of three novel mutations.
      ,
      • Robinson R.
      • Carpenter D.
      • Shaw M.A.
      • Halsall J.
      • Hopkins P.
      Mutations in RYR1 in malignant hyperthermia and central core disease.
      ,
      • McCarthy T.V.
      • Quane K.A.
      • Lynch P.J.
      Ryanodine receptor mutations in malignant hyperthermia and central core disease.
      ]. The ryanodine receptors (RyR) form a group of intracellular calcium release channels on the sarcoplasmic reticulum (SR) or endoplasmic reticulum (ER) in muscles and nerves [
      • Zalk R.
      • Marks A.R.
      Ca(2+) Release channels join the 'resolution revolution'.
      ,
      • Kushnir A.
      • Wajsberg B.
      • Marks A.R.
      Ryanodine receptor dysfunction in human disorders.
      ]. Alongside MH, pathogenic variants in the RYR1-gene are linked to exertional rhabdomyolysis, King-Denborough syndrome, late-onset axial myopathy and congenital myopathies such as central core disease and minicore myopathy with external ophtalmoplegia [
      • Lawal T.A.
      • Todd J.J.
      • Witherspoon J.W.
      • Bönnemann C.G.
      • Dowling J.J.
      • Hamilton S.L.
      • et al.
      Ryanodine receptor 1-related disorders: an historical perspective and proposal for a unified nomenclature.
      ,
      • Jungbluth H.
      • Treves S.
      • Zorzato F.
      • Sarkozy A.
      • Ochala J.
      • Sewry C.
      • et al.
      Congenital myopathies: disorders of excitation-contraction coupling and muscle contraction.
      ]. Malignant hyperthermia has an autosomal dominant inheritance, congenital myopathies an autosomal recessive or dominant inheritance. There is growing evidence for a clinical and histopathological continuum between patients with MH-susceptibility (MHS) and congenital myopathies [
      • van den Bersselaar L.R.
      • Kruijt N.
      • Scheffer G.J.
      • van Eijk L.
      • Malagon I.
      • Buckens S.
      • et al.
      The neuromuscular and multisystem features of RYR1-related malignant hyperthermia and rhabdomyolysis: a study protocol.
      ]. The phenotype is dependent on the pathogenic variant, but some patients with central core or multiminicore disease will also suffer from MH [
      • Hopkins P.M.
      • Gupta P.K.
      • Bilmen J.G.
      Malignant hyperthermia.
      ]. Currently more than 400 variants of unknown significance in the RYR1-gene have been identified, but the specific MH risk for each of these variants is uncertain [
      • Litman R.S.
      • Griggs S.M.
      • Dowling J.J.
      • Riazi S.
      Malignant hyperthermia susceptibility and related diseases.
      ]. Until now, 48 variants have been identified as pathogenic for MHS according to the European Malignant Hyperthermia Group (EMHG) (www.emhg.org). The criteria of the EMHG recommend that, when a variant of unknown significance is detected, a functional assay with a muscle biopsy and in vitro contracture test (IVCT) is required [
      • Hopkins P.M.
      • Rüffert H.
      • Snoeck M.M.
      • Girard T.
      • Glahn K.P.
      • Ellis F.R.
      • et al.
      European Malignant Hyperthermia Group guidelines for investigation of malignant hyperthermia susceptibility.
      ]. Thus, a variant must be both genetically and functionally characterized before being reclassified [
      • Stowell K.M.
      Malignant hyperthermia: a pharmacogenetic disorder.
      ].
      The IVCT is currently considered the gold standard for diagnosis of an individual's MHS [
      • Hopkins P.M.
      • Rüffert H.
      • Snoeck M.M.
      • Girard T.
      • Glahn K.P.
      • Ellis F.R.
      • et al.
      European Malignant Hyperthermia Group guidelines for investigation of malignant hyperthermia susceptibility.
      ]. The IVCT is based on the contractile response of the individual's fresh muscle tissue in the presence of caffeine or halothane. The sensitivity of the IVCT conducted according to the EMHG protocol has been evaluated as 99% with a specificity of 94% [
      • Hopkins P.M.
      • Gupta P.K.
      • Bilmen J.G.
      Malignant hyperthermia.
      ,
      • Hopkins P.M.
      • Rüffert H.
      • Snoeck M.M.
      • Girard T.
      • Glahn K.P.
      • Ellis F.R.
      • et al.
      European Malignant Hyperthermia Group guidelines for investigation of malignant hyperthermia susceptibility.
      ,
      • Ording H.
      • Brancadoro V.
      • Cozzolino S.
      • Ellis F.R.
      • Glauber V.
      • Gonano E.F.
      • et al.
      In vitro contracture test for diagnosis of malignant hyperthermia following the protocol of the European MH Group: results of testing patients surviving fulminant MH and unrelated low-risk subjects. The European Malignant Hyperthermia Group.
      ]. Therefore, in patients with a negative IVCT, MHS is reasonably ruled out [
      • Hopkins P.M.
      • Rüffert H.
      • Snoeck M.M.
      • Girard T.
      • Glahn K.P.
      • Ellis F.R.
      • et al.
      European Malignant Hyperthermia Group guidelines for investigation of malignant hyperthermia susceptibility.
      ]. Several in silico prediction methods have been developed to reveal the pathogenicity of a detected variant, but none can replace the IVCT so far [
      • Hoppe K.
      • Jurkat-Rott K.
      • Kranepuhl S.
      • Wearing S.
      • Heiderich S.
      • Merlak S.
      • et al.
      Relevance of pathogenicity prediction tools in human RYR1 variants of unknown significance.
      ,
      • Matthijs G.
      • Souche E.
      • Alders M.
      • Corveleyn A.
      • Eck S.
      • Feenstra I.
      • et al.
      Guidelines for diagnostic next-generation sequencing.
      ].
      The aim of this study is to describe the findings on clinical assessment and ancillary testing (creatine kinase (CK) levels, EMG, muscle biopsy, IVCT) in patients with neuromuscular complaints that turned out to be secondary to MHS, as determined by IVCT. Additionally, we want to stress the importance of considering a RYR1-variant in patients with muscle complaints, given the potential medico-legal issues when MHS is missed.

      2. Methods

      This study is a retrospective analysis of data from 15 individuals referred to the adult neuromuscular outpatient clinic of Ghent University Hospital and Sint Lucas General Hospital Ghent. The patients presented to one of these hospitals between 1/1/2016 and 30/06/2021 and were included if a variant of unknown significance or (likely) pathogenic variant in RYR1-gene was found in the work-up for their neuromuscular complaints (or when performing segregation analysis). Information for this study was gathered from electronic medical records of the included individuals. We retrospectively reviewed the initial symptoms at presentation, serum CK, family history, results of the muscle biopsy, EMG, genetic testing, IVCT and the pathogenicity prediction. When patients presented to us with unexplained muscle weakness and/or myalgia/cramps, which were considered to be neuromuscular in origin by the clinician, CK levels and EMG were usually performed first. If no cause was identified, we proceeded to muscle biopsy +/- genetic testing, depending on the urgency (for example, clinical deterioration, wish for children, impeding anesthesia and other factors). The indications for performing a 216-gene panel, including well-known neuromuscular diseases were: (1) various unexplained complaints, such as muscle weakness, cramps, non-specific myalgia, with or without increased serum CK or (2) an adverse anesthetic event of the individual.
      Thirteen of the 15 individuals underwent this panel. Some underwent additional testing for other genes (e.g. facioscapulohumeral dystrophy or myotonic dystrophies) depending on their clinical presentation. Two of the 15 individuals (patient 7 and 8) underwent targeted mutation analysis because of a MH-susceptible child (patient 6) who carried two variants in the RYR1-gene. In addition to genetic screening, an IVCT was performed at the University of Antwerp (J. De Puydt). Genetic testing for MHS and IVCT were carried out in accordance with the guidelines of the EMHG (https://www.emhg.org/testing-for-mh) [
      • Hopkins P.M.
      • Rüffert H.
      • Snoeck M.M.
      • Girard T.
      • Glahn K.P.
      • Ellis F.R.
      • et al.
      European Malignant Hyperthermia Group guidelines for investigation of malignant hyperthermia susceptibility.
      ]. Written informed consent was obtained from individuals prior to revision of their electronic records and the study design was approved by the Ethics Committee of the University Hospital of Ghent, Belgium.
      Genetic screening – Gene panel analysis for neuromuscular diseases (including the RYR1-gene) was performed by applying whole-exome sequencing (WES) on genomic DNA extracted from blood samples of the 13 individuals using the SureSelect XT Human All Exon V6 enrichment kit (Agilent Technologies), followed by sequencing on the HiSeq3000 sequencer (Illumina). Variants with a low-quality score were confirmed with an independent test (polymerase chain reaction (PCR) amplification followed by Sanger sequencing). Nucleotide numbering is according to the Human Genome Variation Society guidelines (HGVS). Variant classification for MHS is based on the recently published guidelines by the ClinGen expert panel [
      • Johnston J.J.
      • Dirksen R.T.
      • Girard T.
      • Gonsalves S.G.
      • Hopkins P.M.
      • Riazi S.
      • et al.
      Variant curation expert panel recommendations for RYR1 pathogenicity classifications in malignant hyperthermia susceptibility.
      ]. Since MHS primarily results from missense alterations in RYR1, this classification system is developed for missense variants. Other alterations (e.g. nonsense, splice site) rather result in a loss of function and are thus more likely to potentially cause a RYR1-related myopathy. Variant classification for RYR1-related myopathies is performed using an in house developed tool based on the ACMG [
      • Richards S.
      • Aziz N.
      • Bale S.
      • Bick D.
      • Das S.
      • Gastier-Foster J.
      • et al.
      Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.
      ] and ACGS (www.acgs.uk.com/quality/best-practice-guidelines/) guidelines in the following classes: (1) benign, (2) likely benign (>95% certainty that the variant is benign), (3) variant of unknown significance, (4) likely pathogenic (>95% certainty that the variant is pathogenic), (5) pathogenic. The classification system cannot distinguish between RYR1-variants causing MHS and variants causing a RYR1-relateded myopathy [
      • Hopkins P.M.
      • Gupta P.K.
      • Bilmen J.G.
      Malignant hyperthermia.
      ].
      IVCT – MHS was diagnosed using the IVCT in accordance with the recommendations of the EMHG [
      • Hopkins P.M.
      • Rüffert H.
      • Snoeck M.M.
      • Girard T.
      • Glahn K.P.
      • Ellis F.R.
      • et al.
      European Malignant Hyperthermia Group guidelines for investigation of malignant hyperthermia susceptibility.
      ]. The IVCT determines the contracture susceptibility of surgically excised muscle specimens to halothane and caffeine. Muscle samples from all 15 individuals were tested, irrespective of whether a pathogenic mutation or a variant of unknown significance (VUS) was detected. Biopsies were taken from the vastus lateralis muscle under locoregional anesthesia. Muscle bundles from individuals who are MHS have lower contracture thresholds to halothane and caffeine relative to MH-negative (MHN) individuals. A contracture force of ≥0,2 gr at a halothane concentration ≤2% and a caffeine concentration ≤2 mM defines a positive result for MHS [
      • Hopkins P.M.
      • Rüffert H.
      • Snoeck M.M.
      • Girard T.
      • Glahn K.P.
      • Ellis F.R.
      • et al.
      European Malignant Hyperthermia Group guidelines for investigation of malignant hyperthermia susceptibility.
      ].

      3. Results

      Results on individual patients are summarized in Table A.
      Table ASchematic overview of the key patient data.
      PatientClassification (for MHS, prior to IVCT)RegionIVCTSymptoms at presentationSerum CK (U/L)EMGMuscle biopsyGeneral anesthesia in medical history
      1Variant of unknown significancec.14474G>A, p.(Arg4825His), exon 100+Myalgia mainly post exercise286 - 81 000Non-irritable myopathyMild myopathic changes
      2Variant of unknown significancec.3379C>T, p.(Arg1127Cys), exon 25+Myalgia and spasms in proximal muscles70NormalNormal+
      3Variant of unknown significancec.3877C>A, p.(Pro1293Thr), exon 28+Myalgia, proximal and facial weakness and contractures118Minimal myopathic changes in biceps muscleNormal+
      4Variant of unknown significancec.9796A>C, p.(Met3266Leu), exon 66+Proximal muscle weakness, mild distal weakness221 - 450Non-irritable myopathyMyopathy with central cores
      5Variant of unknown significancec.6486C>G, p.(Ile2162Met), exon 39+Muscle atrophy, proximal muscle weakness, cramps990NormalMild muscular dystrophy
      6Two variants of unknown significancec.13760C>T, p.(Pro4587Leu), exon 95 and c.13918A>G, p. (Met4640Val), exon 95+Proximal muscle weakness and fatigue159Mild non- irritable myopathyMyopathy with core formations
      7Variant of unknown significancec.13918A>G, p.(Met 4640Val), exon 95 (father of patient 6)No symptoms155NDMyopathy with core formations+
      8Variant of unknown significancec.13760C>T, p. (Pro4587Leu), exon 95 (mother of patient 6)No symptoms71NDNormal+
      9Variant of unknown significancec.3953C>T, p.(Ala1318Val), exon 28Myalgia, exercise intolerance51 - 32,000NormalNormal+
      10NA, (variant of unknown significance)c.8401–8C>G, p. (?), intron 53Diffuse muscle weakness and myalgia92 - 596NormalNormal+
      11Variant of unknown significance and NA, (pathogenic variant)(3) c.13664A>G, p. (Tyr4555Cys), exon 49 (5) c.10347+1G>A, p.(?), intron 68Proximal muscle weakness and myalgia572 - 3400NDUnspecific myopathy, mild increased variation in muscle fiber size
      12NA, (pathogenic variant)c.10859G>A, p.(Trp3620Ter), exon 74Myalgia after exercise743 - 1200NormalMultiple fibers with central attenuation of the intermyofibrillar network, sometimes with the morphology of classic cores
      13Pathogenic variant)c.14545G>A, p.(Val4849Ile), exon 101+Muscle cramps and myalgia306 - 700NDMultiple fibers with central attenuation of the intermyofibrillar network, without the morphology of classic cores+
      14Pathogenic variantc.7042_7044del, p.(Glu2348de), exon 44+Myalgia in rest and during exercise406NormalMyopathic changes with mild changes of the intermyofibrillar network without cores
      15Pathogenic variantc. 7523G>A, p.(Arg2508His), exon 47+Muscle cramps, mild proximal muscle weakness252NDMyopathy with central cores+, resulting in MH
      Schematic overview of the relevant patient data i.e. classification of the variant based on the criteria for MHS by the ClinGen expert panel
      • Johnston J.J.
      • Dirksen R.T.
      • Girard T.
      • Gonsalves S.G.
      • Hopkins P.M.
      • Riazi S.
      • et al.
      Variant curation expert panel recommendations for RYR1 pathogenicity classifications in malignant hyperthermia susceptibility.
      , location of the variant in the RYR1-gene, result of the IVCT, symptoms, CK levels (normal values are 10–195 U/L for men and 10–170 U/L for women), EMG and muscle biopsy results, previous exposure to general anesthesia. The ClinGen classification in not useful for splice variants (patients 10 and 11) and nonsense variants (patient 12) since these are considered to possibly cause RYR1-related myopathies instead of MHS. The genetic classification following the ACMG/ACGS-criteria for these 3 patients is noted between brackets. The variants in patient 13, 14 and 15 are EMHG diagnostic mutations. Therefore, the variants in patient 14 and 15, who were identified as likely pathogenic by ClinGen were changed to pathogenic. ND =not done. NA = no information available.

      3.1 Genetic data

      Based on the ClinGen criteria for MHS, the variants were classified as follows: of the 15 individuals with a variant in the RYR1-gene, 9 individuals (patient 1, 2, 3, 4, 5, 7, 8, 9, 11) harbored one variant of unknown significance (VUS, class 3) and one individual (patient 6) was compound heterozygous for two VUS in trans. Patient 11 also carried an intronic variant in cis, that was classified as a pathogenic variant (class 5) using the ACMG/ACGS-criteria for RYR1-related myopathies. Patient 14 and 15 had a likely pathogenic variant according to the ClinGen classification, but since these variants were known to be an EMHG-mutation, they were upgraded to pathogenic. Also patient 13 had a variant that was known to be an EMHG-mutation. The variant classification was mostly the same when the variant was classified using the ClinGen classification (for MHS) or the ACMG/ACGS-criteria for RYR1-related myopathies. An exception were the nonsense (patient 12) and splice site variants (patient 10, 11), for which the ClinGen classification is not useful, since these type of genetic defects are rather known to result in a loss of function and thus a possible RYR1-related myopathy (instead of MHS). In total, forteen different variants in the RYR1-gene were detected. Ten of these variants were absent in the gnomAD v2.1.1 population databases: p.(Arg4825His) (c.14474G>A in exon 100), p.(Met3266Leu) (c.9796A>C in exon 66), p.(Ile2162Met) (c.6486C>G in exon 39), p.(Pro4587Leu) (c.13760C>T in exon 95), p.(Met4640Val) (c.13918A>G in exon 95), (c.8401–8C>G in intron 53), p.(Trp3620Ter) (c.10859G>A in exon 74), p.(Tyr4555Cys) (c.13664A>G in exon 49), splice site variant c.10347+1G>A in intron 68 and p.(Ala1318Val) (c.3953C>T in exon 28).

      3.2 IVCT data

      In vitro contracture testing classified 9 of all 15 individuals with a variant in the RYR1-gene as MHS, whilst six were classified as MHN. Of the 9 individuals identified with one VUS, 5 were classified as MHS (patient 1, 2, 3, 4, 5). The one individual identified with two variants of unknown significance (patient 6) was classified as MHS. The individual identified with both a VUS (for MHS) and a pathogenic variant (for RYR1-myopathies) (patient 11) was classified as MHN. The three individuals with a pathogenic variant (patient 13, 14, 15) were classified as MHS. One positive IVCT result does not change the ClinGen result, since one IVCT result is not considered to be a strong enough argument to change the classification. However, one negative IVCT result in combination with other benign criteria or arguments allows downgrading a class 3 into a class 2 variant for MHS, as could be applied to patient 9.

      3.3 Clinical features

      Eleven patients, that presented to our neuromuscular clinic, suffered from non-specific myalgia, muscle fatigue and/or cramps. Seven had muscle weakness, especially proximal. Of the 9 patients that turned out to be MHS, 4 only had myalgia or cramps, 1 only weakness and 4 both.

      3.4 Ancillary investigations

      Blood tests were performed on all individuals following symptom presentation looking primarily at the CK levels. In the table, CK levels at rest are included for all patients (in U/L). Two patients were referred to us after an episode of rhabdomyolysis (patient 1 and patient 9). Patient 1 had several episodes of exertional myalgia, sometimes with myoglobinuria and had elevated CK values at rest. Patient 9 only had rhabdomyolysis once, without a clear trigger, but had normal CK at rest. In 9 out of 15 individuals with a variant in the RYR1-gene, CK levels at rest were elevated. Out of the 9 patients who turned out to have a positive IVCT, 6 had elevated CK levels at baseline.
      An EMG was performed on 10 of the 15 individuals. Four individuals out of the 10 showed an abnormal EMG with chronic myopathic findings: these patients were diagnosed with a VUS and were MHS.
      Light microscopy studies of muscle biopsy were, in addition to the IVCT, performed on all individuals included in this study. Several abnormal muscle biopsies were found in patients diagnosed with a variant in the RYR1-gene. Individuals with a pathogenic variant all showed changes in the intermyofibrillar pattern with or without central cores. In addition, five patients with a VUS showed abnormal muscle biopsies and four of them were proven MHS.

      3.5 Special findings

      One individual (patient 6) was diagnosed with two variants of unknown significance in the RYR1-gene: p.(Met4640Val) (c.13918A>G) and p.(Pro4587Leu) (c.13760C>T). Since in vitro contracture testing showed this individual to be MHS, further investigations were conducted in both parents (patient 7 and 8). Genetic testing identified the p.(Met4640Val) (c.13918A>G) variant in one parent (patient 7) and the p.(Pro4587Leu) (c.13760C>T) variant in the other parent (patient 8). Remarkably, IVCT showed both these variants to be MHN, which indicates that probably only the combination of the two variants will pose a risk for MH. The individual with two variants initially presented with motor development delay in childhood and complaints of proximal muscle weakness and fatigue. CK levels were normal, but an EMG showed a mild non-irritable myopathy. The muscle biopsy showed a myopathy with core formations. Both parents, however, did not have muscle complaints and had normal CK levels.

      4. Discussion

      In this study, eleven individuals were characterized with one or two VUS. The patients included were seen and referred for IVCT, prior to the publication of the new classification system for RYR1/MHS. In retrospect, patient 10 and 12 should not have been referred for IVCT to rule out MHS, since these splice site and nonsense variants are unlikely to result in a gain of function. Out of these 11 patients, six individuals were eventually proven MHS by IVCT. This illustrates that it is important to further investigate individuals with a VUS in the RYR1-gene [
      • Hopkins P.M.
      • Rüffert H.
      • Snoeck M.M.
      • Girard T.
      • Glahn K.P.
      • Ellis F.R.
      • et al.
      European Malignant Hyperthermia Group guidelines for investigation of malignant hyperthermia susceptibility.
      ,
      • Stowell K.M.
      Malignant hyperthermia: a pharmacogenetic disorder.
      ]. Furthermore, there seems to be an overlap with some mutations that are associated with CCD, also increasing the risk of MH [
      • Hopkins P.M.
      • Gupta P.K.
      • Bilmen J.G.
      Malignant hyperthermia.
      ,
      • Robinson R.L.
      • Brooks C.
      • Brown S.L.
      • Ellis F.R.
      • Halsall P.J.
      • Quinnell R.J.
      • et al.
      RYR1 mutations causing central core disease are associated with more severe malignant hyperthermia in vitro contracture test phenotypes.
      ]. Thus, functional testing for MHS in patients with a congenital myopathy and a (likely) pathogenic variant in the RYR1-gene can also be informative. According to the EMHG, the IVCT remains the gold standard test for (re)classification of variants in the RYR1-gene as pathogenic for MHS [
      • Litman R.S.
      • Griggs S.M.
      • Dowling J.J.
      • Riazi S.
      Malignant hyperthermia susceptibility and related diseases.
      ]. Patients with a positive IVCT, should be considered at risk for MH and given a warning card. Family members also need gene testing and should avoid undergoing anesthesia with MH-triggering agents while awaiting results [
      • Litman R.S.
      • Griggs S.M.
      • Dowling J.J.
      • Riazi S.
      Malignant hyperthermia susceptibility and related diseases.
      ,
      • Riazi S.
      • Bersselaar L.
      • Islander G.
      • Heytens L.
      • Snoeck M.M.J.
      • Bjorksten A.
      • et al.
      Pre-operative exercise and pyrexia as modifying factors in malignant hyperthermia (MH).
      ]. Relatives identified with the same variant as their affected family member are assumed to be MHS. Some of the patients in this study had a positive family history of muscle complaints in first degree relatives, often previously diagnosed as fibromyalgia, but none had a family member with MH. Recently, a large multi-center study among four MH-units also reported positive IVCT's in patients without a personal/family history of an adverse anesthetic event [
      • van den Bersselaar L.R.
      • Hellblom A.
      • Gashi M.
      • Kamsteeg E.J.
      • Voermans N.C.
      • Jungbluth H.
      • et al.
      Referral indications for malignant hyperthermia susceptibility diagnostics in patients without adverse anesthetic events in the era of next-generation sequencing.
      ].
      In this study, a particularly interesting family was observed: one patient had two VUS in the RYR1-gene and a positive IVCT. After segregation analysis both parents were identified with one VUS, but had a negative IVCT, implicating that only when both of these variants are present, the MHS-trait is expressed.
      Of the patients with MHS, four had already undergone general anesthesia, only causing clinically obvious MH in patient 15. This patient, who has a pathogenic mutation in the RYR1-gene, presented with MH during general anesthesia for reconstruction of the middle ear. This discrepancy can be explained by the existence of incomplete penetrance, as well as the fact that some MH-crises are missed based on the variable manifestations [
      • Rueffert H.
      • Olthoff D.
      • Deutrich C.
      • Meinecke C.D.
      • Froster U.G.
      Mutation screening in the ryanodine receptor 1 gene (RYR1) in patients susceptible to malignant hyperthermia who show definite IVCT results: identification of three novel mutations.
      ,
      • Denborough M.A.
      • Forster J.F.
      • Lovell R.R.
      • Maplestone P.A.
      • Villiers J.D.
      Anaesthetic deaths in a family.
      ]. Incomplete penetrance of the MHS-trait implies that the genetic defect either requires additional factors for MH to occur, or that other factors can prevent the occurrence [
      • Riazi S.
      • Kraeva N.
      • Hopkins P.M.
      Malignant hyperthermia in the post-genomics era: new perspectives on an old concept.
      ,
      • Robinson R.L.
      • Carpenter D.
      • Halsall P.J.
      • Iles D.E.
      • Booms P.
      • Steele D.
      • et al.
      Epigenetic allele silencing and variable penetrance of malignant hyperthermia susceptibility.
      ]. This explains why up to 50% of the individuals with MHS described in literature have undergone anesthesia uneventfully despite the use of volatile anesthetics [
      • Rosenberg H.
      • Pollock N.
      • Schiemann A.
      • Bulger T.
      • Stowell K.
      Malignant hyperthermia: a review.
      ,
      • Riazi S.
      • Kraeva N.
      • Hopkins P.M.
      Malignant hyperthermia in the post-genomics era: new perspectives on an old concept.
      ,
      • Klingler W.
      • Heiderich S.
      • Girard T.
      • Gravino E.
      • Heffron J.J.
      • Johannsen S.
      • et al.
      Functional and genetic characterization of clinical malignant hyperthermia crises: a multi-centre study.
      ]. An additional explanation for the variable occurrence of MH-crises is that newer inhalational anesthetics are a less potent trigger for MH. Lastly, variable occurrence of MH-crises may also reflect the variable presence of additional modifying factors, such as intense exercise and/or pyrexia in genetically susceptible individuals [
      • Riazi S.
      • Bersselaar L.
      • Islander G.
      • Heytens L.
      • Snoeck M.M.J.
      • Bjorksten A.
      • et al.
      Pre-operative exercise and pyrexia as modifying factors in malignant hyperthermia (MH).
      ].
      Patients with muscular dystrophies (Becker, Duchenne), that are not MHS, can develop a MH-like reactions when exposed to succinylcholine [
      • Gurnaney H.
      • Brown A.
      • Litman R.S.
      Malignant hyperthermia and muscular dystrophies.
      ,
      • Heiman-Patterson T.D.
      • Rosenberg H.
      • Fletcher J.E.
      • Tahmoush A.J.
      Halothane-caffeine contracture testing in neuromuscular diseases.
      ]. The management of this condition with rhabdomyolysis and hyperkalemia is different from that of MH and consists of cardiac support and reduction of potassium levels. Such a MH-like reaction has also been described in patients with myotonic dystrophy type 1, certain metabolic and mitochondrial myopathies [
      • De Wel B.
      • Claeys K.G.
      Malignant hyperthermia: still an issue for neuromuscular diseases?.
      ]. The sensitivity and specificity of the IVCT has been validated in patients with suspected MH, but might be different in a less specific population.
      Of all 9 patients with MHS, 2 had a normal biopsy and 7 an abnormal one with findings ranging from mild changes to central cores. Previous studies already reported a broad spectrum of histopathologic abnormalities in patients with RYR1-related MH, with some also having normal biopsies [
      • Knuiman G.J.
      • Küsters B.
      • Eshuis L.
      • Snoeck M.
      • Lammens M.
      • Heytens L.
      • et al.
      The histopathological spectrum of malignant hyperthermia and rhabdomyolysis due to RYR1 mutations.
      ].
      Other aspects of RYR1-related MH described in our study have been confirmed previously. For instance, (non-specific) myalgia, muscle fatigue and/or cramps, present in all of our MHS-patients, have already been associated with RYR1-variants in several studies [
      • Dlamini N.
      • Voermans N.C.
      • Lillis S.
      • Stewart K.
      • Kamsteeg E.J.
      • Drost G.
      • et al.
      Mutations in RYR1 are a common cause of exertional myalgia and rhabdomyolysis.
      ,
      • Witting N.
      • Laforêt P.
      • Voermans N.C.
      • Roux-Buisson N.
      • Bompaire F.
      • Rendu J.
      • et al.
      Phenotype and genotype of muscle ryanodine receptor rhabdomyolysis-myalgia syndrome.
      ,
      • Snoeck M.
      • van Engelen B.G.
      • Küsters B.
      • Lammens M.
      • Meijer R.
      • Molenaar J.P.
      • et al.
      RYR1-related myopathies: a wide spectrum of phenotypes throughout life.
      ]. These symptoms have a significant effect on quality of life [
      • van Ruitenbeek E.
      • Custers J.A.E.
      • Verhaak C.
      • Snoeck M.
      • Erasmus C.E.
      • Kamsteeg E.J.
      • et al.
      Functional impairments, fatigue and quality of life in RYR1-related myopathies: a questionnaire study.
      ]. Five had muscle weakness, especially proximal, which has also been described in MH-related RYR1-variants [
      • Snoeck M.
      • van Engelen B.G.
      • Küsters B.
      • Lammens M.
      • Meijer R.
      • Molenaar J.P.
      • et al.
      RYR1-related myopathies: a wide spectrum of phenotypes throughout life.
      ,
      • de Souza A.
      Adult-onset selective quadriceps femoris weakness in RYR1-related myopathy.
      ]. This underscores the wide spectrum of RYR1-related neuromuscular disease.
      One of the six patients with a VUS didn't have any objective clinical finding or abnormality on ancillary testing (CK levels, EMG, biopsy). Although we are aware of the small numbers in this case series, requiring caution in drawing major conclusions, this is important to mention, especially given the prevalence of unexplained muscle complaints in a neuromuscular outpatient clinic.
      The most important limitation of our study is its low number of patients and its retrospective nature. Nevertheless, given there is a preventative treatment for this life-threatening condition and given the potential medicolegal issues when missed, we consider it important to raise awareness amongst neuromuscular neurologists about testing for a variant in the RYR1-gene in patients with non-specific muscle complaints, even in the absence of muscle weakness or CK elevation. We would recommend considering this diagnosis in (young) patients with invalidating muscle aches/cramps, especially if there are abnormalities on clinical exam or (at least) one ancillary test (CK's, EMG, biopsy) and/or if there is a positive family history of similar complaints. From a therapeutic standpoint, if MHS is proven, dantrolene orally might provide a relief from their symptoms of myalgia and cramps [
      • Butala B.N.
      • Kang A.
      • Guron J.
      • Brandom B.W.
      Long term oral dantrolene improved muscular symptoms in a malignant hyperthermia susceptible individual.
      ]. Neurologists should keep in mind that, if a variant is found, this will likely lead to an IVCT, requiring a muscle biopsy to determine the relevance of the variant.

      Study roles

      L.J. was responsible for collection and analysis of the data. J.D.B. and S.H were responsible for intellectual content and study design. S.S and M.M performed the molecular genetic testing and interpretation. L.J and S.H were involved in drafting the manuscript. J.D.B., J.D., S.S were responsible for evaluation of the manuscript.

      Declaration of Competing Interest

      J.D.B. and S.H. are a member of the European Reference Network for Neuromuscular Diseases. There is no conflict of interest. No targeted funding was received for this study.

      Acknowledgments

      We kindly want to thank dr David Bourke, neurologist at Capital and Coast District Health Board, New Zealand, for proofreading the manuscript.

      References

        • Rosenberg H.
        • Davis M.
        • James D.
        • Pollock N.
        • Stowell K.
        Malignant hyperthermia.
        Orphanet J Rare Dis. 2007; 2: 21
        • Denborough M.
        Malignant hyperthermia.
        Lancet. 1998; 352: 1131-1136
        • Hopkins P.M.
        Malignant hyperthermia: pharmacology of triggering.
        Br J Anaesth. 2011; 107: 48-56
        • Rosenberg H.
        • Pollock N.
        • Schiemann A.
        • Bulger T.
        • Stowell K.
        Malignant hyperthermia: a review.
        Orphanet J Rare Dis. 2015; 10: 93
        • Halliday N.J.
        Malignant hyperthermia.
        J Craniofac Surg. 2003; 14: 800-802
        • Ellis F.R.
        • Halsall P.J.
        • Christian A.S.
        Clinical presentation of suspected malignant hyperthermia during anaesthesia in 402 probands.
        Anaesthesia. 1990; 45: 838-841
        • Hopkins P.M.
        • Gupta P.K.
        • Bilmen J.G.
        Malignant hyperthermia.
        Handb Clin Neurol. 2018; 157: 645-661
        • Maclennan D.H.
        • Zvaritch E.
        Mechanistic models for muscle diseases and disorders originating in the sarcoplasmic reticulum.
        Biochim Biophys Acta. 2011; 1813: 948-964
        • MacLennan D.H.
        • Phillips M.S.
        Malignant hyperthermia.
        Science. 1992; 256: 789-794
        • Rüffert H.
        • Bastian B.
        • Bendixen D.
        • Girard T.
        • Heiderich S.
        • Hellblom A.
        • et al.
        Consensus guidelines on perioperative management of malignant hyperthermia suspected or susceptible patients from the European Malignant Hyperthermia Group.
        Br J Anaesth. 2021; 126: 120-130
        • Glahn K.P.
        • Ellis F.R.
        • Halsall P.J.
        • Müller C.R.
        • Snoeck M.M.
        • Urwyler A.
        • et al.
        Recognizing and managing a malignant hyperthermia crisis: guidelines from the European Malignant Hyperthermia Group.
        Br J Anaesth. 2010; 105: 417-420
        • Rueffert H.
        • Olthoff D.
        • Deutrich C.
        • Meinecke C.D.
        • Froster U.G.
        Mutation screening in the ryanodine receptor 1 gene (RYR1) in patients susceptible to malignant hyperthermia who show definite IVCT results: identification of three novel mutations.
        Acta Anaesthesiol Scand. 2002; 46: 692-698
        • Robinson R.
        • Carpenter D.
        • Shaw M.A.
        • Halsall J.
        • Hopkins P.
        Mutations in RYR1 in malignant hyperthermia and central core disease.
        Hum Mutat. 2006; 27: 977-989
        • McCarthy T.V.
        • Quane K.A.
        • Lynch P.J.
        Ryanodine receptor mutations in malignant hyperthermia and central core disease.
        Hum Mutat. 2000; 15: 410-417
        • Zalk R.
        • Marks A.R.
        Ca(2+) Release channels join the 'resolution revolution'.
        Trends Biochem Sci. 2017; 42: 543-555
        • Kushnir A.
        • Wajsberg B.
        • Marks A.R.
        Ryanodine receptor dysfunction in human disorders.
        Biochim Biophys Acta Mol Cell Res. 2018; 1865: 1687-1697
        • Lawal T.A.
        • Todd J.J.
        • Witherspoon J.W.
        • Bönnemann C.G.
        • Dowling J.J.
        • Hamilton S.L.
        • et al.
        Ryanodine receptor 1-related disorders: an historical perspective and proposal for a unified nomenclature.
        Skelet Muscle. 2020; 10: 32
        • Jungbluth H.
        • Treves S.
        • Zorzato F.
        • Sarkozy A.
        • Ochala J.
        • Sewry C.
        • et al.
        Congenital myopathies: disorders of excitation-contraction coupling and muscle contraction.
        Nat Rev Neurol. 2018; 14: 151-167
        • van den Bersselaar L.R.
        • Kruijt N.
        • Scheffer G.J.
        • van Eijk L.
        • Malagon I.
        • Buckens S.
        • et al.
        The neuromuscular and multisystem features of RYR1-related malignant hyperthermia and rhabdomyolysis: a study protocol.
        Medicine (Baltimore). 2021; 100: e26999
        • Litman R.S.
        • Griggs S.M.
        • Dowling J.J.
        • Riazi S.
        Malignant hyperthermia susceptibility and related diseases.
        Anesthesiology. 2018; 128: 159-167
        • Hopkins P.M.
        • Rüffert H.
        • Snoeck M.M.
        • Girard T.
        • Glahn K.P.
        • Ellis F.R.
        • et al.
        European Malignant Hyperthermia Group guidelines for investigation of malignant hyperthermia susceptibility.
        Br J Anaesth. 2015; 115: 531-539
        • Stowell K.M.
        Malignant hyperthermia: a pharmacogenetic disorder.
        Pharmacogenomics. 2008; 9: 1657-1672
        • Ording H.
        • Brancadoro V.
        • Cozzolino S.
        • Ellis F.R.
        • Glauber V.
        • Gonano E.F.
        • et al.
        In vitro contracture test for diagnosis of malignant hyperthermia following the protocol of the European MH Group: results of testing patients surviving fulminant MH and unrelated low-risk subjects. The European Malignant Hyperthermia Group.
        Acta Anaesthesiol Scand. 1997; 41: 955-966
        • Hoppe K.
        • Jurkat-Rott K.
        • Kranepuhl S.
        • Wearing S.
        • Heiderich S.
        • Merlak S.
        • et al.
        Relevance of pathogenicity prediction tools in human RYR1 variants of unknown significance.
        Sci Rep. 2021; 11: 3445
        • Matthijs G.
        • Souche E.
        • Alders M.
        • Corveleyn A.
        • Eck S.
        • Feenstra I.
        • et al.
        Guidelines for diagnostic next-generation sequencing.
        Eur J Hum Genet. 2016; 24: 1515
        • Johnston J.J.
        • Dirksen R.T.
        • Girard T.
        • Gonsalves S.G.
        • Hopkins P.M.
        • Riazi S.
        • et al.
        Variant curation expert panel recommendations for RYR1 pathogenicity classifications in malignant hyperthermia susceptibility.
        Genet Med. 2021; 23: 1288-1295
        • Richards S.
        • Aziz N.
        • Bale S.
        • Bick D.
        • Das S.
        • Gastier-Foster J.
        • et al.
        Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.
        Genet Med. 2015; 17: 405-424
        • Robinson R.L.
        • Brooks C.
        • Brown S.L.
        • Ellis F.R.
        • Halsall P.J.
        • Quinnell R.J.
        • et al.
        RYR1 mutations causing central core disease are associated with more severe malignant hyperthermia in vitro contracture test phenotypes.
        Hum Mutat. 2002; 20: 88-97
        • van den Bersselaar L.R.
        • Hellblom A.
        • Gashi M.
        • Kamsteeg E.J.
        • Voermans N.C.
        • Jungbluth H.
        • et al.
        Referral indications for malignant hyperthermia susceptibility diagnostics in patients without adverse anesthetic events in the era of next-generation sequencing.
        Anesthesiology. 2022; 136: 940-953
        • Denborough M.A.
        • Forster J.F.
        • Lovell R.R.
        • Maplestone P.A.
        • Villiers J.D.
        Anaesthetic deaths in a family.
        Br J Anaesth. 1962; 34: 395-396
        • Riazi S.
        • Kraeva N.
        • Hopkins P.M.
        Malignant hyperthermia in the post-genomics era: new perspectives on an old concept.
        Anesthesiology. 2018; 128: 168-180
        • Robinson R.L.
        • Carpenter D.
        • Halsall P.J.
        • Iles D.E.
        • Booms P.
        • Steele D.
        • et al.
        Epigenetic allele silencing and variable penetrance of malignant hyperthermia susceptibility.
        Br J Anaesth. 2009; 103: 220-225
        • Klingler W.
        • Heiderich S.
        • Girard T.
        • Gravino E.
        • Heffron J.J.
        • Johannsen S.
        • et al.
        Functional and genetic characterization of clinical malignant hyperthermia crises: a multi-centre study.
        Orphanet J Rare Dis. 2014; 9: 8
        • Riazi S.
        • Bersselaar L.
        • Islander G.
        • Heytens L.
        • Snoeck M.M.J.
        • Bjorksten A.
        • et al.
        Pre-operative exercise and pyrexia as modifying factors in malignant hyperthermia (MH).
        Neuromuscul Disord. 2022; 32: 628-634
        • Gurnaney H.
        • Brown A.
        • Litman R.S.
        Malignant hyperthermia and muscular dystrophies.
        Anesth Analg. 2009; 109: 1043-1048
        • Heiman-Patterson T.D.
        • Rosenberg H.
        • Fletcher J.E.
        • Tahmoush A.J.
        Halothane-caffeine contracture testing in neuromuscular diseases.
        Muscle Nerve. 1988; 11: 453-457
        • De Wel B.
        • Claeys K.G.
        Malignant hyperthermia: still an issue for neuromuscular diseases?.
        Curr Opin Neurol. 2018; 31: 628-634
        • Knuiman G.J.
        • Küsters B.
        • Eshuis L.
        • Snoeck M.
        • Lammens M.
        • Heytens L.
        • et al.
        The histopathological spectrum of malignant hyperthermia and rhabdomyolysis due to RYR1 mutations.
        J Neurol. 2019; 266: 876-887
        • Dlamini N.
        • Voermans N.C.
        • Lillis S.
        • Stewart K.
        • Kamsteeg E.J.
        • Drost G.
        • et al.
        Mutations in RYR1 are a common cause of exertional myalgia and rhabdomyolysis.
        Neuromuscul Disord. 2013; 23: 540-548
        • Witting N.
        • Laforêt P.
        • Voermans N.C.
        • Roux-Buisson N.
        • Bompaire F.
        • Rendu J.
        • et al.
        Phenotype and genotype of muscle ryanodine receptor rhabdomyolysis-myalgia syndrome.
        Acta Neurol Scand. 2018; 137: 452-461
        • Snoeck M.
        • van Engelen B.G.
        • Küsters B.
        • Lammens M.
        • Meijer R.
        • Molenaar J.P.
        • et al.
        RYR1-related myopathies: a wide spectrum of phenotypes throughout life.
        Eur J Neurol. 2015; 22: 1094-1112
        • van Ruitenbeek E.
        • Custers J.A.E.
        • Verhaak C.
        • Snoeck M.
        • Erasmus C.E.
        • Kamsteeg E.J.
        • et al.
        Functional impairments, fatigue and quality of life in RYR1-related myopathies: a questionnaire study.
        Neuromuscul Disord. 2019; 29: 30-38
        • de Souza A.
        Adult-onset selective quadriceps femoris weakness in RYR1-related myopathy.
        Neurol Sci. 2022; 43: 3453-3455
        • Butala B.N.
        • Kang A.
        • Guron J.
        • Brandom B.W.
        Long term oral dantrolene improved muscular symptoms in a malignant hyperthermia susceptible individual.
        J Neuromuscul Dis. 2016; 3: 115-119