Functional Characterization of 2 Known Ryanodine Receptor Mutations Causing Malignant Hyperthermia Anja H. Schiemann, PhD,* Neeti Paul,* Remai Parker,* Neil Pollock, MB ChB, FRCA, FANZCA, MD,† Terasa F. Bulger, MB ChB, FRCA, FANZCA,† and Kathryn M. Stowell, PhD* BACKGROUND: Malignant hyperthermia (MH) is a potentially lethal pharmacogenetic disorder. More than 300 variants in the ryanodine receptor 1 (RYR1) have been associated with MH; however, only 31 have been identified as causative. To confirm a mutation in RYR1 as being causative for MH, segregation of the potential mutation in at least 2 unrelated families with MH susceptibility must be demonstrated and functional assays must show abnormal calcium release compared with wild-type RYR1. METHODS: We used “Hot-spot” DNA screening to identify mutations in RYR1 in 3 New Zealand families. B-lymphoblastoid cells were used to compare the amount of calcium released on stimulation with 4-chloro-m-cresol between wild-type RYR1 cells and cells carrying the new variants in RYR1. RESULTS: We identified a known RYR1 mutation (R2355W) in 2 families and another more recently identified (V2354M) mutation in another family. Both mutations segregated with MH susceptibility in the respective families. Cell lines carrying a mutation in RYR1 showed increased sensitivity to 4-chloro-m-cresol. CONCLUSIONS: We propose that R2355W is confirmed as being an MH-causative mutation and suggest that V2354M is a RYR1 mutation likely to cause MH.  (Anesth Analg 2014;118:375–80)

M

alignant hyperthermia (MH; OMIM# 145600) is an autosomal dominant pharmacogenetic disorder triggered in MH-susceptible (MHS) individuals on exposure to volatile halogenated anesthetics. The symptoms of an MH episode include hyperthermia, muscle rigidity, tachycardia, hypoxemia, and metabolic acidosis and are due to rapidly rising myoplasmic calcium levels caused by an increased flux of calcium from the sarcoplasmic reticulum (SR) to the cytosol.1 In 50% to 70% of MH families,2 amino acid changes have been identified in the ryanodine receptor 1 (RYR1), which is the major skeletal muscle calcium-release channel and plays an important role in excitation–contraction coupling. In approximately 1% of cases, mutations are found in the α1S subunit of the dihydropyridine receptor.3,4 The RYR1 (located in the SR membrane) physically interacts with the voltage-dependent Ca2+ channel (dihydropyridine receptor) that is located in the T-tubule membrane. On depolarization of the plasma membrane in skeletal muscle, the dihydropyridine receptor undergoes conformational changes and consequently activates RYR1 that releases calcium from the SR into the sarcoplasm. This leads to muscle contraction as well as a range of other functional consequences.5 The RYR1 gene contains 106 exons and encodes approximately 15-kb-long cDNA.6 More than 300 variants From the *Institute of Fundamental Sciences, Massey University; and †Department of Anaesthesia and Intensive Care, MidCentral Health, Palmerston North Hospital, Palmerston North, New Zealand. Accepted for publication June 5, 2013. Funding: Funded by Massey University Research Fund and the Australian and New Zealand College of Anaesthetists (ANZCA, 12/022 and 09/13). The authors declare no conflicts of interest. Reprints will not be available from the authors. Address correspondence to Anja H. Schiemann, PhD, Institute of Fundamental Sciences, Massey University, Private Bag 11 222, Palmerston North 4442, New Zealand. Address e-mail to [email protected]. Copyright © 2013 International Anesthesia Research Society DOI: 10.1213/ANE.0b013e3182a273ea

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in RYR1 have been linked to MH, and 31 of these have been identified as being causative (European Malignant Hyperthermia Group, EMHG). Patients are diagnosed as MHS, MH-negative (MHN), or MH-equivocal according to the in vitro contracture test (IVCT),7 which requires invasive surgery, whereas a patient carrying a causative mutation (established by DNA testing) will be diagnosed as MHS and does not require IVCT. To classify a mutation as causative, segregation of MHS has to be demonstrated in at least 2 families, and functional assays have to show that a mutation in RYR1 leads to abnormal calcium release compared with wild-type RYR1.8 The aim of our study was to identify new variants or mutations associated with MH in New Zealand families and to increase the number of causative mutations recognized by the EMHG.

METHODS Blood and Tissue Samples

The study was approved by the Central Regional (Wellington, New Zealand) Human Ethics Committee. Blood and tissue samples were obtained with written informed consent at the time of collection.

Patient Presentation Patient AII:1 A 15-year-old boy underwent insertion of pins for a slipped right femoral epiphysis. He received an omnopon (papaveretum, contains a mixture of purified opium alkaloids including morphine, codeine, narcotine, and papaverine) premedication, and anesthesia was induced with thiopental 400 mg. Tracheal intubation was facilitated with 100 mg succinylcholine, and anesthesia was maintained with oxygen, nitrous oxide, and halothane. The procedure lasted 1 hour 25 minutes. The patient was stable throughout and after tracheal extubation; his heart rate (HR) was 80/min and arterial blood pressure 130/90 mm Hg with spontaneous respiration. Soon after transfer to the postanesthesia www.anesthesia-analgesia.org

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care unit, his condition quickly deteriorated; he became cyanosed, tachycardic, and tachypneic. His rectal temperature was measured at 39.5°C, quickly increasing to 40.5°C. His initial arterial blood gas (ABG) showed a Pco2 of 70 mm Hg and pH 7.15. He was given 200 mmol bicarbonate, 500 mg procainamide repeated within 30 minutes, and 16 mg decadron. He was quickly placed in a cold bath and subsequently given 25 mg chlorpromazine for shivering. He settled quickly with a pH of 7.35 and a Pco2 of 43.5 mm Hg. His potassium peaked at 4.8 mmol/L and creatine kinase (CK) was measured at >1000 units (n = 0.5–4.0) at 24 hours postoperatively and myoglobinuria was present. He was maintained on procainamide (250 mg 6 hourly) and made a full recovery. The reaction in retrospect ranked 6 on the Malignant Hyperthermia Clinical Grading Scale (MHCGS),9 indicating an almost certain likelihood of an MH reaction. This reaction occurred in 1973. Patient BI A 6-year-old girl had anesthesia for insertion of a grommet in her right ear. She was given nitrous oxide, oxygen, and halothane induction, and attempted tracheal intubation was facilitated with 25 mg succinylcholine with 1 repeat. The patient developed significant masseter spasm with some body rigidity, and her skin felt hot to the touch. The patient had an HR of 140/min, temperature of 37.8°C, and a respiratory rate of 44 breaths/min. The procedure was abandoned, and cooling fluids and body fanning administered. Dantrolene (1 mg/kg) was administered. The child made a full recovery, and surgery was performed at a later date. Her initial ABGs showed a pH of 7.31, Pco2 of 42 mm Hg, base deficit 5 mmol/L, and later, CK was measured at >3000 units (n < 200), and myoglobinuria was identified. Using the MHCGS, this reaction ranked 5, indicating a very likely MH reaction. Patient CII:3 A 30-year-old woman required anesthesia for removal of a retained placenta after normal vaginal delivery. She had a history of a tonsillectomy and no significant family history. She was premedicated with ranitidine and sodium citrate and was administered oxygen for 4 minutes. Anesthetic monitoring included arterial blood pressure, pulse oximeter saturation (Spo2), electrocardiogram, and capnography. Anesthesia was induced with 2 mg alfentanil, thiopental 350 mg, succinylcholine 120 mg, and atropine 0.6 mg. Tracheal intubation was attempted, but her mouth could not be opened because of masseter spasm. Marked generalized muscle spasm was noted. The spasm relaxed after 4 minutes, and no inhaled drug was administered. She developed an HR of 110/min, a maximum end tidal CO2 of 45 mm Hg, and CK postoperatively was 1374 units (n < 245). There was no record of ABG measurement. The patient was allowed to regain consciousness, and the procedure was performed under spinal anesthesia. This reaction ranked 3 on the MHCGS, indicating a somewhat less than likely MH reaction.

In Vitro Contracture Testing

Three patients from different families had experienced MH reactions during general anesthesia, and informed consent was obtained for an IVCT. IVCTs were performed at

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Palmerston North Hospital as part of the normal diagnostic procedures for MHS according to the EMHG protocol.7

Genetic Analysis

Genomic DNA from blood and lymphoblastoid cells was extracted using the Wizard™ DNA extraction kit according to the manufacturer’s instructions (Promega, Madison, WI). “Hot-Spot” regions in RYR1 (exons 6, 8–12, 14, 15, 17, 39–41, 43–47, 95, 98–104) were amplified by polymerase chain reaction (PCR) (primer sequences are available on request); the PCR products were purified using the Zymo OneStep™ PCR inhibitor removal kit (Zymo Research, Irvine, CA), and subsequently, dideoxysequencing was performed using the BigDye™ Terminator Version 3.1 kit on an ABI 3730 (Applied Biosystems, Foster City, CA) sequencer.

High Resolution Melting Analysis

Segregation of variants in families and the presence of variants in lymphoblastoid cells were established using high resolution melting (HRM) analysis as described previously.10 LightCycler Probe Design Software 2.0 (Roche, Mannheim, Germany) was used to design primers; real-time PCR, and HRM analysis were performed on the LightCycler 480 System (Roche) using the SsoFast™ EvaGreen® Supermix (Bio-Rad, Hercules, CA) according to the manufacturer’s instructions. HRM reaction mix contained 1× SsoFast™ EvaGreen® Supermix, 0.3 μM of each primer, and 10 to 60 ng genomic DNA. Assays were performed in 96-well plates in a 10 μL volume.

Immortalized B-Lymphoblastoid Cell Lines

B-lymphocytes were extracted from patients’ whole blood and transformed using the Epstein-Barr virus as previously described.11 HRM analysis was used to confirm the presence of the variants in DNA isolated from lymphoblastoid cell lines.

Calcium Release Assays

B-lymphoblastoid cells were loaded with fura-2/AM (4 μM final concentration, Invitrogen Life Technologies, Carlsbad, CA) and 0.05% pluronic F-127 in balanced salt solution (BSS) buffer (140 mM NaCl, 2.8 mM KCl, 1 mM MgCl2, 10 mM HEPES, pH 7.3, 2 mM CaCl2, and 10 mM glucose) for 1 hour at 37°C in the dark. Loaded cells were washed once in BSS buffer, then in calcium-free BSS buffer containing 2 mM EGTA. One × 106 cells/mL (final volume 2 mL) were used to measure changes in intracellular Ca2+ concentration for MHS patients and MHN controls after addition of increasing concentrations of 4-chloro-m-cresol (4-CmC) in a spectrofluorometer with a magnetic stirrer (LS-50, Perkin Elmer, Norwalk, CT). In addition, calcium release in fura-2/AM loaded B-lymphoblastoid cells was measured after addition of 400 nM thapsigargin. All experiments were performed in calcium-free BSS buffer containing 2 mM EGTA.

Statistical Analysis

Origin software (v. 8.5.1, Microcal Software, Northampton, MA) was used for statistical analysis and for generation of concentration–response curves. Results were calculated as mean values (±SEM) of n results, and 50% effective concentration (EC50) values were calculated from sigmoidal curve

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Figure 1. Segregation analysis of families A (R2355W), B (R2355W), and C (V2354M). Black symbols indicate MH-susceptible (MHS), checkered MH-equivocal (MHE), white symbols with N indicate MH-negative (MHN), white symbols with question marks indicate in vitro contracture test (IVCT) has not been performed. The ryanodine receptor 1 (RYR1) mutations are represented by filled bars under symbols. Arrows indicate the probands. MHN = MH-negative; MHE = MH-equivocal; MHS = MH-susceptible; RYR1 = ryanodine receptor 1; IVCT = In vitro contracture test.

fitting of the data points. Statistical analysis was performed using the Student t test for paired samples or analysis of variance (ANOVA) when >2 groups were compared.

Bioinformatic Tools

ClustalW2 (www.ebi.ac.uk/Tools/msa/clustalw2/) was used to generate protein sequence alignments. Five different programs were used for computational prediction of the severity of the amino acid change at the given location in RYR1. These include PolyPhen2 (http://genetics.bwh.harvard.edu/pph2/),12 Pmut (http://mmb2.pcb.ub.es:8080/ PMut/),13 SIFT (http://sift.jcvi.org/),14 MutPred (http:// mutpred.mutdb.org/),15 and SNPs&GO (http://snps-andgo.biocomp.unibo.it/snps-and-go/).16 These programs use evolutionary information or information derived from multiple sequence alignments and protein structural and functional parameters to predict the effect of an amino acid change on protein function.

RESULTS

Hot-Spot PCR screening in families A and B identified a c.7063 C > T change (rs193922803) in exon 44 in RYR1, resulting in an amino acid change from arginine to tryptophan at position 235517 and in family C a c.7060 G > A change,18 resulting in an amino acid change from valine to methionine at position 2354. Both changes segregated with MHS in the families (Fig. 1, Table 1). HRM screening did not detect either variant in >100 MHN individuals. Computational February 2014 • Volume 118 • Number 2

Table 1.  In Vitro Contracture Test Data of Families A, B, and C Sequence variant or mutation found in RYR1 Patient Caffeine (g) Halothane (g) Diagnosis AI 0.05 0.35 MHN — AII:1 0.6 1.8 MHS R2355W AII:2 0.6 1.5 MHS R2355W AII:3 2.2 3.5 MHS R2355W AII:4 0 0.1 MHN No DNA available AIII:1 2.2 2.2 MHS R2355W AIII:2 1 2 MHS R2355W AIII:5 1.6 1.5 MHS R2355W AIV:1 1.6 0.8 MHS R2355W BI 1.5 1.2 MHS R2355W CI:1 0.1 0.2 MHN — CI:2 2.2 2.5 MHS V2354M CI:3 1.2 2.8 MHS V2354M CI:4 0 0.1 MHN — CII:1 0.95 1.4 MHS No DNA available CII:2 2 5.2 MHS V2354M CII:3 3.5 3.9 MHS V2354M CII:4 0 0 MHN — CII:5 2.8 2.8 MHS V2354M CII:6 0 0.8 MHE — Control 1 0 0.1 MHN — Control 2 0.1 0.2 MHN — Maximal contractures in vitro contracture test (IVCT) at the threshold concentrations of caffeine (2 mmol/L) and halothane (2%). Caffeine (n ≤ 0.2 g tension), Halothane (n ≤ 0.4 g tension). MHN = MH-negative; MHE = MH-equivocal; MHS = MH-susceptible; RYR1 = ryanodine receptor 1.

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Table 2.  Bioinformatic Analysis of Ryanodine Receptor 1 (RYR1) Variants Change V2354M R2355W

PolyPhen2 Probably damaging Probably damaging

Pmut Neutral Pathological

SIFT Damaging Damaging

MutPred 0.755 0.951

SNPs&GO Disease Disease

Reference 18 and this study 17 and this study

The output of MutPred shows the probability that an amino acid substitution is disease associated.

analysis of the predicted effects of the amino acid substitutions on protein function is shown in Table 2. All programs predicted the amino acid changes to have a damaging impact on protein function except for Pmut in the case of the V2354M change, which was predicted not to have an effect. Lymphoblastoid cell lines were used to determine whether the mutations affected the sensitivity of RYR1 to stimulation by 4-CmC (Fig. 2). As a control, cell lines from 2 nonrelated MHN individuals were used. Concentration– response curves were calculated using increasing concentrations of 4-CmC, and the amount of Ca2+ released was calculated as a percentage relative to Ca2+ released by 1000 µM 4-CmC. In the presence of the mutations, the concentration–response curves showed a significant shift to the left compared with the healthy controls. Lymphoblastoid cell lines carrying mutations in RYR1 showed significantly lower EC50 values for 4-CmC–induced Ca2+ release compared with wild-type cell lines (Table 3).

DISCUSSION

Three patients who experienced an MH reaction were screened for potential mutations in the RYR1 gene with Hot-Spot PCR and direct sequencing analysis. In 2 families, we found a previously described R2355W17 mutation and in another family a V2354M variant.18 We cannot exclude the presence of further potential variants in RYR1

Figure 2. 4-Chloro-m-cresol (4-CmC) concentration–response curves of B lymphoblastoid cell lines carrying either the R2355W or V2354M mutation (black symbols, closed squares, AII:3; closed triangles, AIII:3; closed circles, AIII:4; closed inverted triangles, BII; and closed diamonds, CII:2) show an increased sensitivity to 4-CmC compared with MH-negative (MHN) control cell lines (white symbols, open circles, control 1; open inverted triangles, control 2). The increase of Ca2+ released on stimulation with 4-CmC is expressed as the percentage of maximal Ca2+ released by 1000 μM 4-CmC. Concentration–response curves were generated using Origin software; results are shown as mean ± SEM (n = 5–7).

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Table 3.  EC50 Values for 4-Chloro-m-Cresol Induced Calcium Release from B-Lymphoblastoid Cells Cell line AII:3 AIII:3 AIII:4 BII CII:2 Control 1 Control 2

Sequence variant or mutation found in RYR1 R2355W R2355W R2355W R2355W V2354M — —

EC50 4-chloro-m-cresol (µM) 558.8 ± 8.3* 561.9 ± 5.3* 558.8 ± 13.2* 568.5 ± 23.4* 594.6 ± 9.6* 692.9 ± 14.7 683.4 ± 17.5

Results are shown as mean values (±SEM) of n results. *P < 0.05.

in these patients because the complete RYR1 gene was not sequenced; however, both the mutations segregate with MHS in families A and C (the only other person in family B carrying the R2355W mutation has not had an IVCT because of her young age). According to the IVCT, patient CII:6 was classified as MH-equivocal; however, HRM analysis and Sanger sequencing showed that this patient lacks the familial V2354M mutation. This discrepancy could be due to the fact that the IVCT has a 99% sensitivity and a 94% specificity.7 The variants were not present in >100 MHN individuals, which makes them more likely to be potential mutations rather than polymorphisms. The amino acid changes are located in an evolutionarily highly conserved region (Fig. 3). Valine at position 2354 in RYR1 is conserved in all human RYR isoforms and also among different species (mouse, opossum, chicken, and tetraodon). Arginine at position 2355 in RYR1 is conserved in human RYR2, mouse, opossum, and tetraodon. At the same position in human RYR3 and chicken, the arginine is replaced by a lysine, which has similar chemical properties to arginine. Furthermore, V2354M and R2355W are very closely located to the causative A2350T mutation.17,19 Computational analysis of the amino acid changes suggested that the changes have a damaging impact on protein function. The V2354M change was

Figure 3. Multiple protein sequence alignments using ClustalW2. The arrows indicate the amino acid substitutions V2354M and R2355W. Sequence alignments were generated using ryanodine receptor 1 (RYR1) amino acid sequences of human, mouse (mammal), opossum (marsupial), chicken (bird) and Tetraodon (fish), human RYR2 (cardiac isoform) and human RYR3 (ubiquitously expressed).

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regarded as “neutral” by Pmut, which has a 83.5% overall success rate.20 Valine and methionine are both nonpolar, hydrophobic amino acids whereas an arginine (polar and hydrophilic) to tryptophan (nonpolar and hydrophobic) change would be more likely to have an effect on protein function. The mutation resulting in a V2168M change has however been proven to be causative.21–23 Sei et al.24 demonstrated that B-lymphocytes express a functional RYR1, and since then, we and other researchers have used B-lymphoblastoid cell lines to investigate the effect of certain RYR1 mutations on calcium release on stimulation with 4-CmC.25–28 Functional assays performed using human myotubes17 showed that the R2355W mutation did increase sensitivity to 4-CmC and reduced EC50, but the resting calcium concentration was not elevated. We have also shown using lymphoblastoid cell lines that both R2355W and V2354M cell lines showed increased sensitivity to 4-CmC compared with MHN cell lines and had significantly lower EC50 values. EMHG guidelines state that to classify a mutation as causative, segregation of the disease in at least 2 families has to be demonstrated, and functional studies must show that a certain mutation in RYR1 leads to abnormal calcium release compared with wild-type RYR1.8 These criteria have now been fulfilled for the R2355W mutation as Wehner et al.,17 and our group have demonstrated in 3 families that this mutation in RYR1 leads to increased calcium release on stimulation with 4-CmC compared with wild-type RYR1. In addition, this mutation has been identified in families in the United Kingdom2,29,30 and the United States.18 Furthermore, we have shown the absence of the mutation in >100 control samples. Therefore, we suggest that R2355W can be included in the list of causative mutations. To confirm V2354M as a causative mutation, more families with this mutation will need to be identified, and functional assays using HEK293 cells that express a mutant RYR1 could be used to demonstrate its association with MH. E DISCLOSURES

Name: Anja H. Schiemann, PhD. Contribution: This author helped conduct the study, analyze the data and prepare the manuscript. Attestation: Anja H. Schiemann approved the final manuscript and attests to the integrity of the original data and the analysis reported in this manuscript. Anja H. Schiemann is the archival author. Name: Neeti Paul. Contribution: This author helped conduct the study. Attestation: Neeti Paul approved the final manuscript. Name: Remai Parker. Contribution: This author helped conduct the study. Attestation: Remai Parker approved the final manuscript. Name: Neil Pollock, MB ChB, FRCA, FANZCA, MD. Contribution: This author helped conduct the study, analyze the data, and prepare the manuscript. Attestation: Neil Pollock approved the final manuscript. Name: Terasa F. Bulger, MB ChB, FRCA, FANZCA. Contribution: This author helped conduct the study. Attestation: Terasa Bulger approved the final manuscript. Name: Kathryn M. Stowell, PhD. Contribution: This author helped design and conduct the study, analyze the data, and prepare the manuscript.

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Attestation: Kathryn M. Stowell approved the final manuscript and attests to the integrity of the original data and the analysis reported in this manuscript. This manuscript was handled by: Peter J. Davis, MD. ACKNOWLEDGMENTS

We thank Trish McLenachan and Robyn Marston for technical assistance. REFERENCES 1. MacLennan DH, Phillips MS. Malignant hyperthermia. Science 1992;256:789–94 2. Robinson R, Carpenter D, Shaw MA, Halsall J, Hopkins P. Mutations in RYR1 in malignant hyperthermia and central core disease. Hum Mutat 2006;27:977–89 3. Monnier N, Procaccio V, Stieglitz P, Lunardi J. Malignanthyperthermia susceptibility is associated with a mutation of the alpha 1-subunit of the human dihydropyridine-sensitive L-type voltage-dependent calcium-channel receptor in skeletal muscle. Am J Hum Genet 1997;60:1316–25 4. Carpenter D, Ringrose C, Leo V, Morris A, Robinson RL, Halsall PJ, Hopkins PM, Shaw MA. The role of CACNA1S in predisposition to malignant hyperthermia. BMC Med Genet 2009;10:104 5. Maclennan DH, Zvaritch E. Mechanistic models for muscle diseases and disorders originating in the sarcoplasmic reticulum. Biochim Biophys Acta 2011;1813:948–64 6. Phillips MS, Fujii J, Khanna VK, DeLeon S, Yokobata K, de Jong PJ, MacLennan DH. The structural organization of the human skeletal muscle ryanodine receptor (RYR1) gene. Genomics 1996;34:24–41 7. Ording H, Brancadoro V, Cozzolino S, Ellis FR, Glauber V, Gonano EF, Halsall PJ, Hartung E, Heffron JJ, Heytens L, Kozak-Ribbens G, Kress H, Krivosic-Horber R, Lehmann-Horn F, Mortier W, Nivoche Y, Ranklev-Twetman E, Sigurdsson S, Snoeck M, Stieglitz P, Tegazzin V, Urwyler A, Wappler F. 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;41:955–66 8. Urwyler A, Deufel T, McCarthy T, West S; European Malignant Hyperthermia Group. Guidelines for molecular genetic detection of susceptibility to malignant hyperthermia. Br J Anaesth 2001;86:283–7 9. Larach MG, Localio AR, Allen GC, Denborough MA, Ellis FR, Gronert GA, Kaplan RF, Muldoon SM, Nelson TE, Ording H. A clinical grading scale to predict malignant hyperthermia susceptibility. Anesthesiology 1994;80:771–9 10. Grievink H, Stowell KM. Identification of ryanodine receptor 1 single-nucleotide polymorphisms by high-resolution melting using the LightCycler 480 System. Anal Biochem 2008;374:396–404 11. Anderson AA, Brown RL, Polster B, Pollock N, Stowell KM. Identification and biochemical characterization of a novel ryanodine receptor gene mutation associated with malignant hyperthermia. Anesthesiology 2008;108:208–15 12. Adzhubei IA, Schmidt S, Peshkin L, Ramensky VE, Gerasimova A, Bork P, Kondrashov AS, Sunyaev SR. A method and server for predicting damaging missense mutations. Nat Methods 2010;7:248–9 13. Ferrer-Costa C, Gelpí JL, Zamakola L, Parraga I, de la Cruz X, Orozco M. PMUT: a web-based tool for the annotation of pathological mutations on proteins. Bioinformatics 2005;21:3176–8 14. Ng PC, Henikoff S. Predicting deleterious amino acid substitutions. Genome Res 2001;11:863–74 15. Li B, Krishnan VG, Mort ME, Xin F, Kamati KK, Cooper DN, Mooney SD, Radivojac P. Automated inference of molecular mechanisms of disease from amino acid substitutions. Bioinformatics 2009;25:2744–50 16. Calabrese R, Capriotti E, Fariselli P, Martelli PL, Casadio R. Functional annotations improve the predictive score of human disease-related mutations in proteins. Hum Mutat 2009;30:1237–44

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17. Wehner M, Rueffert H, Koenig F, Olthoff D. Functional characterization of malignant hyperthermia-associated RyR1 mutations in exon 44, using the human myotube model. Neuromuscul Disord 2004;14:429–37 18. Brandom BW, Bina S, Wong CA, Wallace T, Visoiu M, Isackson PJ, Vladutiu GD, Sambuughin N, Muldoon SM. Ryanodine receptor type 1 gene variants in the malignant hyperthermiasusceptible population of the United States. Anesth Analg 2013;116:1078–86 19. Sambuughin N, Nelson TE, Jankovic J, Xin C, Meissner G, Mullakandov M, Ji J, Rosenberg H, Sivakumar K, Goldfarb LG. Identification and functional characterization of a novel ryanodine receptor mutation causing malignant hyperthermia in North American and South American families. Neuromuscul Disord 2001;11:530–7 20. Ferrer-Costa C, Orozco M, de la Cruz X. Sequence-based prediction of pathological mutations. Proteins 2004;57:811–9 21. Girard T, Cavagna D, Padovan E, Spagnoli G, Urwyler A, Zorzato F, Treves S. B-lymphocytes from malignant hyperthermia-susceptible patients have an increased sensitivity to skeletal muscle ryanodine receptor activators. J Biol Chem 2001;276:48077–82 22. Yang T, Ta TA, Pessah IN, Allen PD. Functional defects in six ryanodine receptor isoform-1 (RyR1) mutations associated with malignant hyperthermia and their impact on skeletal excitation-contraction coupling. J Biol Chem 2003;278:25722–30 23. Ducreux S, Zorzato F, Müller C, Sewry C, Muntoni F, Quinlivan R, Restagno G, Girard T, Treves S. Effect of ryanodine receptor mutations on interleukin-6 release and intracellular calcium homeostasis in human myotubes from malignant hyperthermia-susceptible individuals and patients affected by central core disease. J Biol Chem 2004;279:43838–46

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24. Sei Y, Gallagher KL, Basile AS. Skeletal muscle type ryanodine receptor is involved in calcium signaling in human B lymphocytes. J Biol Chem 1999;274:5995–6002 25. Levano S, Vukcevic M, Singer M, Matter A, Treves S, Urwyler A, Girard T. Increasing the number of diagnostic mutations in malignant hyperthermia. Hum Mutat 2009;30:590–8 26. Vukcevic M, Broman M, Islander G, Bodelsson M, RanklevTwetman E, Müller CR, Treves S. Functional properties of RYR1 mutations identified in Swedish patients with malignant hyperthermia and central core disease. Anesth Analg 2010;111:185–90 27. Ducreux S, Zorzato F, Ferreiro A, Jungbluth H, Muntoni F, Monnier N, Müller CR, Treves S. Functional properties of ryanodine receptors carrying three amino acid substitutions identified in patients affected by multi-minicore disease and central core disease, expressed in immortalized lymphocytes. Biochem J 2006;395:259–66 28. Schiemann AH, Dürholt EM, Pollock N, Stowell KM. Sequence capture and massively parallel sequencing to detect mutations associated with malignant hyperthermia. Br J Anaesth 2013;110:122–7 29. Carpenter D, Robinson RL, Quinnell RJ, Ringrose C, Hogg M, Casson F, Booms P, Iles DE, Halsall PJ, Steele DS, Shaw MA, Hopkins PM. Genetic variation in RYR1 and malignant hyperthermia phenotypes. Br J Anaesth 2009;103:538–48 30. Klein A, Lillis S, Munteanu I, Scoto M, Zhou H, Quinlivan R, Straub V, Manzur AY, Roper H, Jeannet PY, Rakowicz W, Jones DH, Jensen UB, Wraige E, Trump N, Schara U, Lochmuller H, Sarkozy A, Kingston H, Norwood F, Damian M, Kirschner J, Longman C, Roberts M, Auer-Grumbach M, Hughes I, Bushby K, Sewry C, Robb S, Abbs S, Jungbluth H, Muntoni F. Clinical and genetic findings in a large cohort of patients with ryanodine receptor 1 gene-associated myopathies. Hum Mutat 2012;33:981–8

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Functional characterization of 2 known ryanodine receptor mutations causing malignant hyperthermia.

Malignant hyperthermia (MH) is a potentially lethal pharmacogenetic disorder. More than 300 variants in the ryanodine receptor 1 (RYR1) have been asso...
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