Malignant Hyperthermia—Molecular Testing Thierry GirardDepartments of Anesthesia and Research, University hospital, Basel, Switzerland Henrik RueffertDepartment of Anesthesiology and Intensive Care Medicine, University hospital,Leipzig, Germany Perioperative deaths associated with hyperthermia have been reported since introduction of general Malignant hyperthermia (MH) is triggered by all anesthesia in the 19th century. Only in 1960 did inhalative anesthetics as well as depolarizing muscle Denborough and Lovell describe the autosomal domi- relaxants in genetically predisposed individuals. Pre- nant mode of inheritance of this potentially fatal disease, symptomatic testing is important in this potentially which was then named ‘‘malignant hyperthermia.’’[1] fatal pharmacogenetic disease. In vitro challenging Malignant hyperthermia (OMIM 145600) is a classic of muscle samples with halothane and caffeine is the pharmacogenetic disorder. Affected individuals are basis of contracture testing, which until recently was free of any symptoms in daily life, but in geneti- the only accepted diagnostic procedure. Contracture cally predisposed individuals exposure to triggering testing is invasive and needs an open skeletal muscle agents causes a dramatic life-threatening increase in biopsy. Therefore, research in MH focuses on the development of less invasive procedures. In approxi-mately 85% of MH families, segregation analyses withmicrosatellite markers showed positive linkage of theMH susceptible (MHS) phenotype to the genetic locus of the skeletal muscle endoplasmatic calcium channel(ryanodine receptor, RYR1). Guidelines for molecular The clinical symptoms of MH reflect a significantly genetic diagnosis of MH were recently published and accelerated cell metabolism in the skeletal muscles, allow for molecular genetic diagnosis of MH suscep- which is initiated by certain triggering agents, i.e., all tibility in case of identification of a MH causative halogenated inhalative anesthetics and the depolariz- mutation. Because of the locus and allelic heterogene- ing muscle relaxant succinylcholine. The elevated mus- ity of MH, absence of MH associated mutations do cle activity leads to increases in skeletal muscle tone not allow for negative MH diagnosis.
(masseter spasm, general rigidity) and later to signsof muscular damage such as elevated creatine kinase,hyperkaliemia, myoglobinemia, and myoglobinuria.
Cellular and systemic hypermetabolism present with combined metabolic and respiratory acidosis as wellas an elevation in body temperature and finally result Malignant hyperthermia is a classic pharmacogenetic in multiorgan failure. During MH episodes, not all of disease. Apparently, normal individuals with a genetic the classic symptoms have to be present. Malignant predisposition to MH exhibit a potentially lethal hyperthermia might also present with a single symp- increase in metabolism following contact with trigger- tom only or symptoms only marginally outside a ing agents, i.e., all halogenated inhalative anesthetics normal range. These abortive cases make the diagnosis and the depolarizing muscle relaxant succinylcholine.
of MH on the basis of clinical presentation difficult This article briefly describes the clinical symptoms and only full-blown MH episodes can be accurately and therapy of MH before giving insight into the classified as true MH events. Early recognition and pathophysiology and related genetics. Traditional treatment in case of suspicion of a MH reaction influ- phenotyping methods that use contracture testing as ences the outcome. Monitoring during anesthesia has well as the recently introduced guidelines for molecular substantially changed in the last 20 years. Continuous genetic testing are discussed. The goal of this article measurement of carbon dioxide production, as well as is to elucidate the importance of presymptomatic test- arterial oxygen saturation together with improved ing for MH and the impact of molecular genetic alertness of the anesthesiologist towards MH, all considerably reduced the likelihood of full-blown Encyclopedia of Medical Genomics and Proteomics DOI: 10.1081/E-EDGP-120040294Copyright # 2005 by Taylor & Francis. All rights reserved.
Malignant Hyperthermia—Molecular Testing MH episodes. Supportive measures and specific weakness of the proximal limb.[11] Skeletal defects therapy are essential in case of a MH episode. Trigger including congential hip dislocation, thoracic defor- agents have to be discontinued followed by ventilation mities, pes cavus, clubfoot, and kyphoscoliosis are of the patient with pure oxygen and physical body associated with CCD. Like MH, CCD has been cooling. Specific therapy consists of the calcium chan- linked to the locus of RYR1. Central core disease is nel blocker dantrolene, which has to be immediately the only myopathy being closely associated with MH administered. With these therapeutic interventions, the mortality of MH has decreased dramatically from Because of its multitude of associated RYR muta- 70% to less than 10%.[2] The importance of dantrolene tions, MH shows considerable allelic heterogeneity; in the therapy of MH episodes makes it mandatory to locus heterogeneity has also been described. Besides have this drug available in every anesthetic institution chromosome 19, additional genetic loci on chromo- somes 1q32, 3q13.1, 5q, 17q11.2, 7q.23–q21.1 have The incidence of MH is difficult to determine.
been linked to MH.[12] However, for most of these loci, Clinical data probably underestimate the true genetic no candidate gene has yet been identified, except for predisposition, because the phenotypic presentation chromosomes 1q23 and 7q23–q21.1. Here, the genes after contact with triggering agents is highly variable encoding for the alpha1- and alpha2=delta subunit and many full-blown episodes occurred in patients of the DHPR could be detected. In MHS patients, who had previously undergone several uneventful Monnier et al.[13] found a mutation in the gene encod- anesthesias despite having contact with trigger agents.
ing for the alpha1-subunit of DHPR (CACNA1S).
Former estimates of about 1 : 15,000 anesthetics in Furthermore, results of an extended transmission dis- children and about 1 : 50,000 in adults might well equilibrium test (ETDT) on European MH families suggested that multiple interacting genes influence theMH phenotype while the RYR1 gene on chromosome19 represents the major locus for MH susceptibility.[14] The animal model of MH, the porcine stress syndrome, allowed for a deeper insight into the pathophysiologyof MH. Compared with MH negative (MHN) pigs, Specific testing must be used to diagnose MH suscep- skeletal muscle strips from MHS pigs showed an tibility, as MH is a subclinical myopathy. The aim of increase in myoplasmic calcium concentration if chal- testing for MH is to establish the MH status of lenged with triggering agents.[3] Myoplasmic calcium 1) patients who had a possible MH episode and concentration is essentially regulated by the calcium 2) relatives of patients already tested and found to be release channel of the sarcoplasmic reticulum: the MHS. Presymptomatic testing for MH confirms or skeletal muscle isoform of the RYR1. Moreover, the excludes MH susceptibility before the administration intracellular Ca2þ release is finely co-ordinated with of trigger agents. This is important, because safe alter- the voltage dependent dihydropyridine (DHPR) recep- natives exist for anesthesia in patients known to be tor of the transverse tubule interacting with RYR1.
MHS. Regional anesthesia with any local anesthetic Linkage analyses revealed RYR1 as the primary locus and total intravenous anesthesia without the use of for MH susceptibility.[4,5] The gene encoding for depolarizing neuromuscular blocking drugs is safe for RYR1 is located on chromosome 19q13.1. Ryanodine MHS patients.[2] The gold standard for determining a receptor channel is a homotetramer and represents one predisposition for MH is an in vitro muscle contrac- of the largest proteins in the human body. Each sub- ture test. After an open muscle biopsy, fresh human unit consists of 5038 amino acids, encoded by a genomic skeletal muscle is challenged in vitro with caffeine DNA of 160 kb and 106 exons.[6] In pigs, a mutation at and halothane. Contractile thresholds to both drugs nucleotide position 1843, substituting a thymine for are determined in several muscle strips. Standardized a cytosine is responsible for MH in an autosomal protocols for the European in vitro contracture test recessive mode of inheritance.[7] The same mutation (IVCT) and the North American caffeine–halothane was found in humans at position 1840, but in contrast contracture test (CHCT) have been published.[15,16] to pigs the MH disposition is dominantly inherited.
Although the two protocols slightly differ, they both Up to 85% of MH families show linkage to the locus yield reproducible and comparable results.
of RYR1.[8] Today more than 40 mutations in the Because MH is a potentially fatal disease, any RYR1 gene are associated with MH and=or central diagnostic test requires a high sensitivity in order to core disease (CCD).[9,10] Central core disease is an keep the risk of false-negative MH diagnoses as low autosomal dominant congential myopathy, clinically as possible.[17] In case of a wrong MHN diagnosis, characterized by a slow or nonprogressive muscle the application of MH triggering agents could be Malignant Hyperthermia—Molecular Testing detrimental. The sensitivity and specificity of the IVCT Mutations of the gene encoding the ryanodine receptor type 1 gene included in guidelines for molecular Since an invasive open muscle biopsy is required for genetic diagnosis of malignant hyperthermia (MH) contracture testing, MH research has focused on the establishment of noninvasive or less invasive testing for MH susceptibility. With the introduction of mole- cular genetic methods and the identification of a single mutation responsible for the porcine stress syndrome, many anesthesiologists hoped that finally noninvasive testing for MH would soon be available. However,allelic and locus heterogeneity of MH has diminished this expectation. Recently, an important step towards less invasive MH testing has been made by the European Malignant Hyperthermia Group (EMHG).
The group published guidelines for molecular genetic diagnosis of MH susceptibility in 2001.[18] Fifteen RYR1 mutations were included in these guidelines,all of which had been proven to be causative through functional analyses. This list of mutations approved for molecular MH diagnoses is constantly up- dated, and since publication of the guidelines, another As mentioned above, any diagnostic approach to MH has to accomplish thehighest possible sensitivity. Incorrect negative MH diagnoses have to be prevented. This is important not only for the individuals who are tested, but also for their offspring. Therefore, the EMHG guidelines for molecular genetic testing in MH do not allow for negative MH diagnosis exclusively on the basis of genetic investigations. Following the EMHG guide-lines, first-degree relatives of individuals carrying a MH causative mutation are investigated for this familial mutation. While mutation carriers can be diagnosed MHS, absence of the familial mutation does EMHG, included in the guidelines of the European MH Group; not allow for a MHN diagnosis (Fig. 1). The latter sub- NAMHG, included in the North American MH Group.
jects subsequently require an open muscle biopsyfollowed by contracture testing in order to have theirMH status established.[18] A similar proposal was is cost-effective if performed in selected individuals.
made by the North American MH Group, suggesting Total costs of contracture testing is estimated to be 17 MH associated mutations in the RYR1 gene at U.S.$ 5000–6000[9] and although charges for genetic (Table 1).[9] Knowledge of mutation frequency for investigations are not yet defined in every country, they centers performing molecular genetic investigations can be expected to be considerably less. Considering in MH is necessary in order to reduce the number of the fact that contracture testing involves an open mutations screened for. For several countries, such muscle biopsy and can only be performed at specific investigations were determined and found to be geogra- centers, the advantages of molecular testing are phically different.[19] Therefore, it appears to be useful obvious from the patients’ point of view.
to screen index patients of MH families for the most Molecular genetic diagnosis of MH is challenged by frequent MH mutations of their region. Once a muta- reports of discordances between the genotype and tion is identified in a family, then additional relatives phenotypes determined by IVCT,[20] as well as by can be investigated by molecular methods. If detailed spontaneous occurrences of MH.[21] We have so pedigree information is available and individuals are far performed genetic analyses in 67 individuals, 32 carefully selected, then a 50% rate of positive molecu- of them were found to be MHS. Of the remaining lar investigations is to be expected. Even though 35, 20 genetically negative persons underwent IVCT patients with negative genetic results have to undergo and all but one were MHN.[19] This calculates to both, genetic and contracture testing, molecular testing a negative predictive value of 0.95 (95% confidence Malignant Hyperthermia—Molecular Testing The heterogenetic nature of the disease leads to twoimportant drawbacks: 1) Patients not carrying thefamilial mutation must still undergo contracturetesting and 2) only 50% of MH families have knownmutations, some of which are not yet classified as beingcausative. Research in MH will continue to focus onthe aim to offer less invasive MH testing for as manypatients as possible. This involves screening for novelmutations followed by characterization using func-tional analyses. In addition, research also continuesto focus on alternative diagnostic procedures. Severalresearch groups have published preliminary resultsusing either Epstein-Barr Virus (EBV)-immortalizedb-lymphocytes, primary cultures of human skeletalmuscle cells or in vivo microinjections of trigger agents.
In the future, increased knowledge of causative MH mutations and the development of molecular genetictechniques and gene chip technology might lead to pre-operative genetic analysis and risk profiles. This wouldcertainly increase perioperative patient safety.
We would like to thank Mrs. Joan Etlinger, B.A.,Scientific 1. Denborough, M.; Lovell, R.R. Anaesthetic deaths 2. Gronert, G.; Antonigni, J.; Pessah, I. Malignant hyperthermia. In Anesthesia, 5th Ed.; Miller, R., Fig. 1 Diagram of the diagnostic procedures for malignant Ed.; Churchill Livingstone: New York, 2000; 3. Iaizzo, P.A.; Klein, W.; Lehmann-Horn, F.
interval 0.75–0.99) for genetic testing and thus, the Fura-2 detected myoplasmic calcium and its cor- conservative approach of the EMHG guidelines seems relation with contracture force in skeletal muscle to be justified. Today molecular testing for MH is an from normal and malignant hyperthermia suscep- important progress and clearly advantageous for the tible pigs. Pflugers Arch. 1988, 411 (6), 648–653.
patients. Contracture testing and molecular genetics, 4. McCarthy, T.V.; Healy, J.M.; Heffron, J.J.; the two pillars of MH diagnosis are complementary Lehane, M.; Deufel, T.; Lehmann-Horn, F.; Farrall, M.; Johnson, K. Localization of themalignant hyperthermia susceptibility locus tohuman chromosome 19q12–13.2. Nature 1990,343 (6258), 562–564.
5. MacLennan, D.H.; Duff, C.; Zorzato, F.; Fujii, J.; Phillips, M.; Korneluk, R.G.; Frodis, W.; Britt, The introduction of the guidelines for molecular B.A.; Worton, R.G. Ryanodine receptor gene genetic diagnosis of MH susceptibility is an important is a candidate for predisposition to malignant hyperthermia. Nature 1990, 343 (6258), 559–561.
Malignant Hyperthermia—Molecular Testing 6. Phillips, M.S.; Fujii, J.; Khanna, V.K.; DeLeon, voltage-dependent calcium-channel receptor in S.; Yokobata, K.; de Jong, P.J.; MacLennan, skeletal muscle. Am. J. Hum. Genet. 1997, 60 (6), D.H. The structural organization of the human skeletal muscle ryanodine receptor (RYR1) gene.
14. Robinson, R.; Hopkins, P.; Carsana, A.; Gilly, 7. Fujii, J.; Otsu, K.; Zorzato, F.; de Leon, S.; Jurkat-Rott, K.; Muller, C.; Shaw, M.A. Several MacLennan, D.H. Identification of a mutation hyperthermia phenotype. Hum. Genet. 2003, in porcine ryanodine receptor associated with malignant hyperthermia. Science 1991, 253 (5018), 15. European Malignant Hyperpyrexia Group. A protocol for the investigation of malignant hyper- 8. Robinson, R.; Curran, J.L.; Hall, W.J.; Halsall, pyrexia (MH) susceptibility. Br. J. Anaesth. 1984, P.J.; Hopkins, P.M.; Markham, A.F.; Stewart, A.D.; West, S.P.; Ellis, F.R. Genetic heterogeneity 16. Larach, M.G. Standardization of the caffeine and HOMOG analysis in British malignant hyper- thermia families. J. Med. Genet. 1998, 35 (3), Anesth. Analg. 1989, 69 (4), 511–515.
9. Nelson, T.E.; Rosenberg, H.; Muldoon, S.M.
17. Allen, G.C.; Brubaker, C.L. Human malignant Genetic testing for malignant hyperthermia in hyperthermia associated with desflurane anesthe- North America. Anesthesiology 2004, 100 (2), sia. Anesth. Analg. 1998, 86 (6), 1328–1331.
18. Urwyler, A.; Deufel, T.; McCarthy, T.; West, S.
10. McCarthy, T.V.; Quane, K.A.; Lynch, P.J. Rya- Guidelines for molecular genetic detection of nodine receptor mutations in malignant hyper- susceptibility to malignant hyperthermia. Br. J.
thermia and central core disease. Hum. Mutat.
19. Girard, T.; Treves, S.; Voronkov, E.; Siegemund, 11. Tilgen, N.; Zorzato, F.; Halliger-Keller, B.; M.; Urwyler, A. Molecular genetic testing for malignant hyperthermia susceptibility. Anesthe- Schneider, C.; Hauser, E.; Lehmann-Horn, F.; Muller, C.R.; Treves, S. Identification of four 20. Robinson, R.L.; Anetseder, M.J.; Brancadoro, V.; novel mutations in the C-terminal membrane Van Broekhoven, C.; Carsana, A.; Censier, K.; spanning domain of the ryanodine receptor 1: Fortunato, G.; Girard, T.; Heytens, L.; Hopkins, association with central core disease and altera- P.M.; Jurkat-Rott, K.; Klinger, W.; Kozak- tion of calcium homeostasis. Hum. Mol. Genet.
Ribbens, G.; Krivosic, R.; Monnier, N.; Nivoche, Y.; Olthoff, D.; Rueffert, H.; Sorrentino, V.; 12. Jurkat-Rott, K.; McCarthy, T.; Lehmann-Horn, Tegazzin, V.; Mueller, C.R. Recent advances in F. Genetics and pathogenesis of malignant the diagnosis of malignant hyperthermia suscep- tibility: how confident can we be of genetic testing? Eur. J. Hum. Genet. 2003, 11 (4), 342–348.
13. Monnier, N.; Procaccio, V.; Stieglitz, P.; Lunardi, 21. Rueffert, H.; Olthoff, D.; Deutrich, C. Sponta- J. Malignant-hyperthermia susceptibility is asso- neous occurrence of the disposition to malignant ciated with a mutation of the alpha 1-subunit hyperthermia. Anesthesiology 2004, 100 (3), of the human dihydropyridine-sensitive L-type



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