Caffeine and Halothane Sensitivity of Intracellular Ca2+ Release Is Altered by 15 Calcium Release Channel (Ryanodine Receptor) Mutations Associated with Malignant Hyperthermia and/or Central Core Disease

Abstract

Malignant hyperthermia (MH) and central core disease (CCD) are autosomal dominant disorders of skeletal muscle in which a potentially fatal hypermetabolic crisis can be triggered by commonly used anesthetic agents. To date, 17 mutations in the humanRYR1 gene encoding the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum (the ryanodine receptor) have been associated with MH and/or CCD. Although many of these mutations have been linked to MH and/or CCD, with high lod (log of the odds favoring linkage versus nonlinkage) scores, others have been found in single, small families. Independent biochemical evidence for a causal role for these mutations in MH is available for only two mutants. Mutations corresponding to the human MH mutations were made in a full-length rabbit RYR1 cDNA, and wild type and mutant cDNAs were transfected into HEK-293 cells. After about 48 h, intact cells were loaded with the fluorescent Ca2+indicator, fura-2, and intracellular Ca2+ release, induced by caffeine or halothane, was measured by photometry. Ca2+release in cells expressing MH or CCD mutant ryanodine receptors was invariably significantly more sensitive to low concentrations of caffeine and halothane than Ca2+ release in cells expressing wild type receptors or receptors mutated in other regions of the molecule. Linear regression analysis showed that there is a strong correlation (r = 0.95, p < 0.001) between caffeine sensitivity of different RYR1 mutants measured by the cellular Ca2+ photometry assay and by the clinical in vitro caffeine halothane contracture test (IVCT). The correlation was weaker, however, for halothane (r = 0.49, p > 0.05). Abnormal sensitivity in the Ca2+ photometry assay provides supporting evidence for a causal role in MH for each of 15 single amino acid mutations in the ryanodine receptor. The study demonstrates the usefulness of the cellular Ca2+ photometry assay in the assessment of the sensitivity to caffeine and halothane of specific ryanodine receptor mutants. Malignant hyperthermia (MH) and central core disease (CCD) are autosomal dominant disorders of skeletal muscle in which a potentially fatal hypermetabolic crisis can be triggered by commonly used anesthetic agents. To date, 17 mutations in the humanRYR1 gene encoding the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum (the ryanodine receptor) have been associated with MH and/or CCD. Although many of these mutations have been linked to MH and/or CCD, with high lod (log of the odds favoring linkage versus nonlinkage) scores, others have been found in single, small families. Independent biochemical evidence for a causal role for these mutations in MH is available for only two mutants. Mutations corresponding to the human MH mutations were made in a full-length rabbit RYR1 cDNA, and wild type and mutant cDNAs were transfected into HEK-293 cells. After about 48 h, intact cells were loaded with the fluorescent Ca2+indicator, fura-2, and intracellular Ca2+ release, induced by caffeine or halothane, was measured by photometry. Ca2+release in cells expressing MH or CCD mutant ryanodine receptors was invariably significantly more sensitive to low concentrations of caffeine and halothane than Ca2+ release in cells expressing wild type receptors or receptors mutated in other regions of the molecule. Linear regression analysis showed that there is a strong correlation (r = 0.95, p < 0.001) between caffeine sensitivity of different RYR1 mutants measured by the cellular Ca2+ photometry assay and by the clinical in vitro caffeine halothane contracture test (IVCT). The correlation was weaker, however, for halothane (r = 0.49, p > 0.05). Abnormal sensitivity in the Ca2+ photometry assay provides supporting evidence for a causal role in MH for each of 15 single amino acid mutations in the ryanodine receptor. The study demonstrates the usefulness of the cellular Ca2+ photometry assay in the assessment of the sensitivity to caffeine and halothane of specific ryanodine receptor mutants. Malignant hyperthermia (MH) 1The abbreviations used are: MH, malignant hyperthermia; CCD, central core disease; IVCT, in vitrocaffeine halothane contracture test; CHCT, caffeine halothane contracture test. is an autosomal dominant muscle disorder in which genetically susceptible individuals among populations of humans and domestic animals respond to the administration of potent inhalational anesthetics and depolarizing skeletal muscle relaxants with high fever and skeletal muscle rigidity (1Britt B.A. Schonbaum E. Lomax P. Thermoregulation: Pathology, Pharmacology and Therapy. Pergamon Press, New York1991: 179-292Google Scholar, 2MacLennan D.H. Phillips M.S. Science. 1992; 256: 789-794Crossref PubMed Scopus (417) Google Scholar, 3MacLennan D.H. Phillips M.S. Dawson D.C. Frizzell R.A. Ion Channels and Genetic Diseases. The Rockefeller University Press, New York1995: 89-100Google Scholar). Central core disease (CCD) is a rare, non-progressive myopathy, presenting in infancy and characterized by hypotonia and proximal muscle weakness (4Shy G.M. Magee K.R. Brain. 1956; 79: 610-621Crossref PubMed Scopus (302) Google Scholar). An important feature of CCD is its close association with MH susceptibility (5Denborough M.A. Dennett X. Anderson R.M. Br. Med. J. 1973; 1: 272-273Crossref PubMed Scopus (121) Google Scholar). Although diagnosis of CCD is made on the basis of the lack of oxidative enzymatic activity in central regions of skeletal muscle fibers (6Dubowitz V. Pearse A.G.E. Lancet. 1960; : 23-24Abstract PubMed Scopus (78) Google Scholar), the only accepted diagnostic test for MH susceptibility in humans is the North American caffeine halothane contracture test (CHCT) (7Larach M.G. Anesth. Analg. 1989; 69: 511-515Crossref PubMed Google Scholar) or its European counterpart, the in vitro contracture test (IVCT) (8European Malignant Hyperthermia Group Br. J. Anaesth. 1984; 56: 1267-1271Abstract Full Text PDF PubMed Scopus (481) Google Scholar). These tests are based on the hypersensitivity of contracture, induced in muscle strips obtained by biopsy, in the presence of caffeine or halothane. Early genetic linkage studies showed that a single amino acid mutation, Arg615 → Cys (R615C), in the skeletal muscle ryanodine receptor gene (RYR1) is tightly linked to MH in swine (8European Malignant Hyperthermia Group Br. J. Anaesth. 1984; 56: 1267-1271Abstract Full Text PDF PubMed Scopus (481) Google Scholar,9Fujii J. Otsu K. Zorzato F. de Leon S. Khanna V.K. Weiler J.E. O'Brien P.J. MacLennan D.H. Science. 1991; 253: 448-451Crossref PubMed Scopus (1257) Google Scholar). Further genetic and biochemical data have supported the view that this mutation is causal of MH in swine and humans (2MacLennan D.H. Phillips M.S. Science. 1992; 256: 789-794Crossref PubMed Scopus (417) Google Scholar). The corresponding mutation (R614C) has been linked to MH in some human families (11Gillard E.F. Otsu K. Fujii J. Khanna V.K. De Leon S. Derdemezi J. Britt B.A. Duff C.L. Worton R.G. MacLennan D.H. Genomics. 1991; 11: 751-755Crossref PubMed Scopus (278) Google Scholar, 12Hogan K. Couch F. Powers P.A. Gregg R.G. Anesth. Analg. 1992; 75: 441-448Crossref PubMed Scopus (35) Google Scholar), but in other families, discordance has been reported (13Deufel T. Sudbrak R. Feist Y. Rubsam B. Du Chesne I. Schafer K.L. Roewer N. Grimm T. Lehmann-Horn F. Hartung E.J. Müller C.R. Am. J. Hum. Genet. 1995; 56: 1334-1342PubMed Google Scholar, 14Serfas K.D. Bose D. Patel L. Wrogemann K. Phillips M.S. MacLennan D.H. Greenberg C.R. Anesthesiology. 1996; 84: 322-329Crossref PubMed Scopus (34) Google Scholar). Discordance raises questions concerning the causal nature of such mutations, but other reasons for discordance include the possibility that two MH mutations might be segregating in one family or that the in vitro contracture test for MH susceptibility fails to provide a phenotypic diagnosis of MH with sufficient accuracy for genetic analysis (15MacLennan D.H. Curr. Opin. Neurol. 1995; 8: 397-401Crossref PubMed Scopus (40) Google Scholar). Analysis of the CHCT as a diagnostic test for MH indicates that it can achieve 97% sensitivity and 79% specificity, 2Allen, G. C., Larach, M. G., and Kunselman, A. R., (1997) Anesthesiology, in press. whereas the IVCT can achieve 99% sensitivity and 93.6% specificity (17Ording H. for the European Malignant Hyperthermia Group Acta Anesthesiol. Scand. 1997; 41: 955-966Crossref PubMed Scopus (280) Google Scholar). The IVCT protocol permits an equivocal diagnosis (MHE) if the muscle biopsy responds to either caffeine or halothane, but not both. To date, 17 point mutations have been described in humanRYR1. Amino acid residue 4 (Gly4) in the rabbitRYR1 sequence, expressed in this study, is deleted in the human RYR1 sequence, accounting for discrepancies in numbering of these mutations in the two species. Twelve mutations have been linked to human MH only (C35R, G248R, G341R, R552W, R614C, R614L, R2163C, V2168M, T2206M, G2435R, R2458C and R2458H), and five have been linked to human CCD plus MH (R163C, I403M, Y522S, R2163H, and R2436H) in several human families (11Gillard E.F. Otsu K. Fujii J. Khanna V.K. De Leon S. Derdemezi J. Britt B.A. Duff C.L. Worton R.G. MacLennan D.H. Genomics. 1991; 11: 751-755Crossref PubMed Scopus (278) Google Scholar, 18Zhang Y. Chen H.S. Khanna V.K. De Leon S. Phillips M.S. Schappert K. Britt B.A. Brownell A.K.W. MacLennan D.H. Nat. Genet. 1993; 5: 46-50Crossref PubMed Scopus (291) Google Scholar, 19Phillips M.S. Khanna V.K. De Leon S. Frodis W. Britt B.A. MacLennan D.H. Hum. Mol. Genet. 1994; 3: 2181-2186Crossref PubMed Scopus (51) Google Scholar, 20Quane K.A. Healy J.M.S. Keating K.E. Manning B.M. Couch F.J. Palmucci L.M. Doriguzzi K. H. D. W. Müller C.R. Nat. Genet. 1993; 5: PubMed Scopus Google Scholar, K.A. Keating K.E. Healy J.M.S. Manning B.M. R. I. N. J. Genomics. 1994; PubMed Scopus Google Scholar, K.A. Keating K.E. Manning B.M. Healy J.M.S. K. M. L. R. P. N. J. Hum. Mol. Genet. 1994; 3: PubMed Scopus Google Scholar, K.E. K.A. Manning B.M. M. Hartung E. K. A. M. Müller C.R. Hum. Mol. Genet. 1994; 3: PubMed Scopus Google Scholar, K.E. L. P.J. K.A. M. J. Med. Genet. 1997; PubMed Scopus Google Scholar, P.J. R. H. N. K.A. P. G. I. J. Anesthesiology. 1997; PubMed Scopus Google K.A. H. Keating K.E. Manning B.M. R. D. K. Lehmann-Horn F. T. Br. J. 1997; 79: Full Text PDF PubMed Scopus Google Scholar). M. K. A. H. A. V. M. J. E. L. M. P. J. P. K. D. G. L. K. T. W. J. J. A. R. and T. V. for These mutations in two in the of RYR1 between and and between and these mutations, only the human mutation has been characterized in the of the corresponding mutation in this has been to be more sensitive to caffeine and halothane than the Ca2+release channel in tests S. PubMed Scopus Google Scholar), single channel M. R. J. J. B.A. J. Full Text PDF Scopus Google Scholar, M. Am. J. 1993; PubMed Google Scholar), and cellular Ca2+ photometry K. K. Y. T. M. T. M. J. 1994; Full Text PDF PubMed Google Scholar, S. F. P. M. B. Zorzato F. J. 1994; PubMed Scopus Google Scholar). The mutation has been to have than in human an of its M. L. T. Lehmann-Horn F. A. J. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). the of these mutations, there is biochemical evidence to causal role in MH linkage data are available for several of the mutations, but such as the mutation, found in the RYR1 gene in a single MH family of five (11Gillard E.F. Otsu K. Fujii J. Khanna V.K. De Leon S. Derdemezi J. Britt B.A. Duff C.L. Worton R.G. MacLennan D.H. Genomics. 1991; 11: 751-755Crossref PubMed Scopus (278) Google Scholar), or the mutation, found in two of a single, small CCD K.A. Healy J.M.S. Keating K.E. Manning B.M. Couch F.J. Palmucci L.M. Doriguzzi K. H. D. W. Müller C.R. Nat. Genet. 1993; 5: PubMed Scopus Google Scholar), than causal of in these and other mutations that in a with MH and/or CCD is an important this study, have each of 15 MH mutations into a rabbit RYR1 cDNA, expressed each mutation in HEK-293 and measured the intracellular of wild type and mutated to caffeine and halothane. The caffeine and halothane of RYR1 measured in the Ca2+ photometry assay and by the clinical IVCT, were The study that each mutant is more sensitive to the of caffeine and halothane than the wild type strong correlation was between the caffeine measured in the two but a correlation was for halothane for were obtained New and were K. J. Full Text PDF PubMed Google Scholar) was a University of and were was and ryanodine was obtained was other were of The and of the full-length rabbit skeletal muscle ryanodine receptor (RYR1) has been described F. Fujii J. Otsu K. Phillips M.S. G. MacLennan D.H. J. Full Text PDF PubMed Google Scholar, R. MacLennan D.H. 1993; PubMed Scopus Google Scholar, P. L. MacLennan D.H. J. 1997; Full Text PDF PubMed Scopus Google Scholar). as of mutations are by it was to a of that can be full-length RYR1 for and J. E.F. T. Press, Scholar). the into by of five and the of five of about as in B. The five are the in and the five into RYR1 by are the The and the were into the R. MacLennan D.H. 1993; PubMed Scopus Google Scholar) To with these two and were by the of into the between and in The in in the of the by the RYR1 was into to and were into to The was into to and were into to The was into to and the was into to was into to The and with five were into to in the The were into and to as The cDNA, with not be into the which has and was with a and with to a full-length was into the of R. the and the mutant were into the of an were or as by the small in and into the of or for by S. A. PubMed Scopus Google Scholar). The of the mutated of the was by the by the of F. S. S. A. PubMed Scopus Google Scholar). mutations were into in or for in HEK-293 cells. Mutations and were into the and mutations and were into the Mutations R2163C, R2163H, or were into The of wild type in these and HEK-293 cells were in with amino 4 of of of and was by the as described by Chen and H. Mol. PubMed Scopus Google Scholar). of were used to cells were in the but with or with was as described by Chen R. MacLennan D.H. 1993; PubMed Scopus Google Scholar). and were used to expressed ryanodine receptor in HEK-293 cells. and were used in the of the was on one of cells by PubMed Scopus Google Scholar), and to H. T. J. S. A. PubMed Scopus Google Scholar). The were with a of and with a of in a and the were with the and to a was used to the of caffeine on Ca2+ release by the different ryanodine receptor expressed in HEK-293 cells. and were as described was and Ca2+ photometry was on intact cells about 48 G. M. J. Full Text PDF PubMed Scopus Google Scholar). The cells were loaded with for and in a in A. D. J. T. J. PubMed Scopus Google Scholar). The were in a on the of an The were and a The was a into the of each of the two were used to the Ca2+ are as the of with to of caffeine or halothane were on one of the and on the was a of a of the of were and to the release caffeine for the caffeine and the halothane The European IVCT protocol (8European Malignant Hyperthermia Group Br. J. Anaesth. 1984; 56: 1267-1271Abstract Full Text PDF PubMed Scopus (481) Google Scholar) was used in The of of these tests and analysis is by Manning Linear regression analysis was An test was used for of between < was to The and of RYR1 have been reported F. Fujii J. Otsu K. Phillips M.S. G. MacLennan D.H. J. Full Text PDF PubMed Google Scholar, R. MacLennan D.H. 1993; PubMed Scopus Google Scholar, P. L. MacLennan D.H. J. 1997; Full Text PDF PubMed Scopus Google Scholar). the of 15 mutations in the human ryanodine receptor which have been linked to MH (11Gillard E.F. Otsu K. Fujii J. Khanna V.K. De Leon S. Derdemezi J. Britt B.A. Duff C.L. Worton R.G. MacLennan D.H. Genomics. 1991; 11: 751-755Crossref PubMed Scopus (278) Google Scholar, 18Zhang Y. Chen H.S. Khanna V.K. De Leon S. Phillips M.S. Schappert K. Britt B.A. Brownell A.K.W. MacLennan D.H. Nat. Genet. 1993; 5: 46-50Crossref PubMed Scopus (291) Google Scholar, 19Phillips M.S. Khanna V.K. De Leon S. Frodis W. Britt B.A. MacLennan D.H. Hum. Mol. Genet. 1994; 3: 2181-2186Crossref PubMed Scopus (51) Google Scholar, 20Quane K.A. Healy J.M.S. Keating K.E. Manning B.M. Couch F.J. Palmucci L.M. Doriguzzi K. H. D. W. Müller C.R. Nat. Genet. 1993; 5: PubMed Scopus Google Scholar, K.A. Keating K.E. Healy J.M.S. Manning B.M. R. I. N. J. Genomics. 1994; PubMed Scopus Google Scholar, K.A. Keating K.E. Manning B.M. Healy J.M.S. K. M. L. R. P. N. J. Hum. Mol. Genet. 1994; 3: PubMed Scopus Google Scholar, K.E. K.A. Manning B.M. M. Hartung E. K. A. M. Müller C.R. Hum. Mol. Genet. 1994; 3: PubMed Scopus Google Scholar, K.E. L. P.J. K.A. M. J. Med. Genet. 1997; PubMed Scopus Google Scholar, P.J. R. H. N. K.A. P. G. I. J. Anesthesiology. 1997; PubMed Scopus Google Scholar, K.A. H. Keating K.E. Manning B.M. R. D. K. Lehmann-Horn F. T. Br. J. 1997; 79: Full Text PDF PubMed Scopus Google Scholar, E.F. Otsu K. Fujii J. Duff De Leon S. Khanna V.K. Britt B.A. Worton R.G. MacLennan D.H. Genomics. 1992; PubMed Scopus Google the of these 15 mutations cDNA, to the that were in the to and the that were used for The the in which the was in for mutant and to full-length The of a RYR1 in HEK-293 cells and of the have been described P. L. MacLennan D.H. J. 1997; Full Text PDF PubMed Scopus Google Scholar). used this in this of regions of and RYR1 HEK-293 cells with a the ryanodine receptor. The for this between amino and the of the ryanodine receptor. was in between wild type and MH mutant RYR1 cells. was used to an of the regions about cells the of of = of the cells for ryanodine receptor regions were for cellular Ca2+ photometry. of of transfected and HEK-293 cells showed that there were in the of or of between wild type and mutant ryanodine HEK-293 cells were transfected with different of RYR1 plus of or 4 The by but was in by or in caffeine by the cellular Ca2+ that was not by and that the of expressed for a caffeine in an was low to of and to of halothane for HEK-293 cells and for cells transfected with the wild type Ca2+ release the MH mutant channel and the CCD mutant channel in which about HEK-293 cells were the of concentrations of caffeine are in A. there was in the measured in HEK-293 cells and in RYR1 HEK-293 cells. to a small in in HEK-293 but were in the Ca2+ release in HEK-293 cells transfected with the and mutant cDNAs that these mutants were sensitive to concentrations of caffeine and halothane than HEK-293 cells transfected with wild of caffeine by the Ca2+ to to a of about were measured for each in caffeine and halothane and to the for Ca2+ release in the of or to the obtained with caffeine in the of halothane. the in this for wild G2435R, and mutant are in were and The in that HEK-293 cells not respond to halothane concentrations but respond to halothane. HEK-293 cells transfected with wild type were and halothane, but cells transfected with MH mutant and CCD mutant to and as HEK-293 cells to halothane, it was not to a to halothane in cells. the of halothane were to the to which was The for halothane of wild G2435R, and in were and 4 for caffeine of Ca2+release are for wild type and 15 mutant Ca2+release channel expressed in HEK-293 cells. are for halothane of Ca2+ release the the 15 mutations linked to MH, Ca2+ release in was significantly more sensitive to concentrations of caffeine and halothane 4 than was the wild type by cellular Ca2+ photometry. The was the mutant IVCT data for different RYR1 mutations have been it to test with the IVCT data for the different mutations between caffeine and halothane measured by cellular and by IVCT were to was a correlation of correlation = between for caffeine and halothane, measured by cellular Ca2+ photometry 4 but there was correlation between the caffeine and halothane measured by IVCT 4 that a muscle biopsy a might have a strong to caffeine but not to halothane, be however, a single data the IVCT halothane to the R2458C mutation, is an mutation was to caffeine in and to halothane in the cellular Ca2+ photometry but was sensitive to halothane in the IVCT was a strong correlation (r = 0.95, p < 0.001) between the IVCT caffeine and the caffeine measured in the cellular Ca2+ photometry assay but not between the IVCT halothane and halothane 4 measured by cellular Ca2+ photometry. this study, the of HEK-293 cells for the and analysis of wild type and MH or CCD mutant Ca2+ release in the of the expressed Arg615 → Cys RYR1 mutant cellular Ca2+ of a high of Ca2+ release in several different was by Otsu K. K. Y. T. M. T. M. J. 1994; Full Text PDF PubMed Google Scholar), used cells as and that the mutant Ca2+ release channel is more sensitive than wild type to caffeine and halothane, and by S. F. P. M. B. Zorzato F. J. 1994; PubMed Scopus Google Scholar), used cells to that the mutant Ca2+ release channel is more sensitive to than wild The single channel of the Ca2+release expressed in HEK-293 cells the used in this study, have been in to be to the channel P. L. MacLennan D.H. J. 1997; Full Text PDF PubMed Scopus Google Scholar). Although of Ca2+ release sensitive to have been reported in HEK-293 cells J. P. S. 1995; PubMed Scopus Google Scholar), have been to skeletal muscle ryanodine receptor in HEK-293 cells several ryanodine in and of HEK-293 P. L. MacLennan D.H. J. 1997; Full Text PDF PubMed Scopus Google Scholar). however, the of Ca2+ release single HEK-293 cells. it was important to photometry with a of HEK-293 cells that of Ca2+release were by with cells. The the of a to the of low concentrations of caffeine or halothane cells. were the the that have in the of this assay was the of in might were on the basis of of and were on different regions of the caffeine and halothane one of the to on to for caffeine and halothane not which were were to release obtained with in the The of the of halothane was halothane, to in was in assay to achieve the specific concentrations of halothane used in cellular Ca2+ photometry. The halothane was on HEK-293 cells transfected with RYR1 as were in the of the contracture, that of halothane is not a in cellular Ca2+ photometry mutations to MH or CCD, in other regions of have not sensitivity to caffeine and halothane. and D. H. have the of 15 mutations in rabbit skeletal muscle ryanodine which are associated with MH or CCD in the corresponding human Ca2+release data that of the mutant ryanodine receptor respond significantly to low concentrations of caffeine and halothane. The in transfected HEK-293 cells are to be to in the of the of the caffeine for the RYR1 mutations obtained by cellular Ca2+ photometry and by the muscle biopsy IVCT that there was an correlation between The correlation is the caffeine data were obtained different a of several The close correlation that demonstrates that the cellular Ca2+ photometry described be a of RYR1 MH the cellular Ca2+ photometry a correlation was between sensitivity to caffeine and sensitivity to halothane 4 the correlation between the halothane in the cellular assay and the halothane in the IVCT was not 4 is not as a correlation between the caffeine and halothane obtained for the IVCT 4 The correlation between the halothane in the cellular Ca2+ photometry assay and in the IVCT the different nature of the cellular and muscle biopsy such in halothane in the are that in of the correlation was between the halothane and caffeine in the cellular assay but not in the in the IVCT, there was a correlation between halothane and and the of to halothane, but not to in the IVCT different in it is that the halothane of mutations be more measured in the cellular assay than in the The IVCT were whereas the cellular assay be to a the for caffeine and halothane are the for caffeine and halothane in the the that the cellular assay a of the IVCT The of this study to a of biochemical and genetic evidence that mutations in the RYR1 gene are causal of malignant in the diagnosis of MH by in is that the tests can close to but with more than specificity (17Ording H. for the European Malignant Hyperthermia Group Acta Anesthesiol. Scand. 1997; 41: 955-966Crossref PubMed Scopus (280) Google and the that are with the test are not to be to is more that the contracture test the of gene in the muscle strips which are it is that muscle cells have the to for genetic that might be PubMed Scopus Google Scholar, A. Khanna V.K. G. MacLennan D.H. Nat. Genet. 1996; PubMed Scopus Google Scholar, Am. J. 1994; PubMed Google Scholar). CHCT in a family not only the of the RYR1 mutation MH, but the different genetic of each the of in that MH are in than in and susceptibility in the to (1Britt B.A. Schonbaum E. Lomax P. Thermoregulation: Pathology, Pharmacology and Therapy. Pergamon Press, New York1991: 179-292Google Scholar). of these diagnostic it is important to the possibility that the CHCT be by based tests for tests achieve however, has been to which RYR1 mutations in causal of MH and which are is that linkage data mutations and MH not be of the of the IVCT (15MacLennan D.H. Curr. Opin. Neurol. 1995; 8: 397-401Crossref PubMed Scopus (40) Google Scholar). P. and D. H. Am. J. in press. that an is to test each MH mutation, as are for sensitivity to caffeine and halothane and in the of the genetic by HEK-293 cells. mutation is to in sensitivity than wild type to the Ca2+ of caffeine and halothane, such a mutation be to be and administration of Chen and for and for and de Leon for the of