Malignant 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 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.
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.
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 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.
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.
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 Palmerston North Hospital as part of the normal diagnostic procedures for MHS according to the EMHG protocol.7
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.
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 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.
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-and-go.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.
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 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).
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 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 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.
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.
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.
We thank Trish McLenachan and Robyn Marston for technical assistance.
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© 2014 International Anesthesia Research Society
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