Incidence of malignant hyperthermia reactions in 2,214 patients undergoing muscle biopsy.

To determine the incidence of malignant hyperthermia (MH) reactions after trigger-free anaesthesia in a large population of MH-susceptible (MHS) patients, the charts of 2,214 patients who underwent elective muscle biopsy for malignant hyperthermia were reviewed. Either general or regional anaesthesia with non-triggering drugs was used. For general anaesthesia, the trachea was intubated in the absence of muscle relaxants. The halothane/caffeine contracture test was performed on the biopsied muscle. Suspected MH reactions were identified according to their site of occurrence (in the operating room, recovery room or ward). Ninety-seven percent of patients received a general anaesthetic; 3% received a regional anaesthetic or field block. Of the 2214 patients whose muscles were biopsied, 1082 patients were biopsy-positive for MH. Five patients in whom MH reactions were diagnosed in the recovery room were all subsequently proved to be biopsy-positive for MH. Four of the five received intravenous dantrolene as part of their therapy; the fifth received only symptomatic therapy as parenteral dantrolene was not commercially available. All patients recovered completely from their reactions. We conclude that the incidence of MH reactions in biopsy-positive patients who receive a trigger-free anaesthetic for minor surgery is small (0.46%, (0.15-1.07%, 95% CI)). These reactions occur in the immediate postoperative period.

The substitution of Arg for Gly2433 in the human skeletal muscle ryanodine receptor is associated with malignant hyperthermia.

Single strand conformational polymorphism analysis was used to screen exons 43 and 44 in the skeletal muscle ryanodine receptor gene from 17 positively diagnosed members of families in which chromosome 19-linked malignant hyperthermia (MH) was segregating. A polymorphism in two unrelated individuals was found to result from the substitution of A for G7297, leading to the substitution of Arg for Gly2433. This mutation is adjacent to a mutation (Arg2434 to His) previously linked to MH and central core disease (Y. Zhang et al., Nature Genet. 1993, 5, 46-50). Subsequent screening showed the presence of the mutation in four of 106 MH families tested and its absence from about 1000 other chromosomes. The mutation was present in all six individuals in four families who had had an MH reaction, in two obligate carriers and in 10 individuals diagnosed as MH susceptible by the caffeine/halothane contracture test (CHCT). The mutation was present in an individual with a normal response to the CHCT and was absent in three individuals with a positive CHCT response. These discrepancies would be consistent with inaccuracies in the CHCT and/or with segregation of a second MH allele within two of the four affected families.

A mutation in the human ryanodine receptor gene associated with central core disease.

Central core disease (CCD) is a morphologically distinct, autosomal dominant myopathy with variable clinical features. A close association with malignant hyperthermia (MH) has been identified. Since MH and CCD genes have been linked to the skeletal muscle ryanodine receptor (RYR1) gene, cDNA sequence analysis was used to search for a causal RYR1 mutation in a CCD individual. The only amino acid substitution found was an Arg2434His mutation, resulting from the substitution of A for G7301. This mutation was linked to CCD with a lod score of 4.8 at a recombinant fraction of 0.0 in 16 informative meioses in a 130 member family, suggesting a causal relationship to CCD.

Polymorphisms and deduced amino acid substitutions in the coding sequence of the ryanodine receptor (RYR1) gene in individuals with malignant hyperthermia.

Twenty-one polymorphic sequence variants of the RYR1 gene, including 13 restriction fragment length polymorphisms (RFLPs), were identified by sequence analysis of human ryanodine receptor (RYR1) cDNAs from three individuals predisposed to malignant hyperthermia (MH). All RFLPs were detectable in PCR-amplified products, and their segregation was consistent with our initial finding of linkage to MH in the nine families previously informative for one or more intragenic markers (MacLennan et al., 1990, Nature 343:559-561). Four amino acid substitutions were identified in the study: Arg for Gly248, Cys for Arg470, Leu for Pro1785, and Cys for Gly2059. Of 45 families tested, a single family presented the Arg for Gly248 substitution where it segregated with malignant hyperthermia, making it a candidate mutation for predisposition to MH in man. The other three polymorphic substitutions failed to segregate with malignant hyperthermia in those families in which they occurred, implying that they represent polymorphisms with little or no effect on the function of the RYR1 gene.

The role of the skeletal muscle ryanodine receptor gene in malignant hyperthermia.

Malignant hyperthermia (MH) is an inherited, potentially lethal condition in which sustained muscle contracture with attendant hypermetabolism and hyperthermia is triggered in humans, heterozygous for the gene defect, by inhalational anaesthetics and skeletal muscle relaxants, and in pigs, homozygous for the defect, by stress. Because muscle contracture could result from a defective Ca2+ release channel, we have focussed our attention on the linkage of MH to defects in the gene (RYR1) encoding the skeletal muscle Ca2+ release channel. We have cloned and sequenced human RYR1 cDNA and found restriction fragment length polymorphisms (RFLPs) in the human gene. We also localized RYR1 to human chromosome 19q13.1. Studies of the cosegregation of MH with these RFLPs established RYR1/MH linkage on human chromosome 19q13.1 (lod score of 4.2; recombinant fraction 0.0). We then sequenced MH and normal porcine RYR1 cDNAs. Mutation of C1843 to T, leading to substitution of Cys for Arg615, was the sole amino acid change noted between MH and normal animals. Linkage of this mutation to MH was established in a study of 338 informative meioses (lod score of 102; recombinant fraction 0.0). We identified the corresponding mutation in 1 of 35 human MH families studied and found cosegregation of the mutation and MH. The combination of a high lod score with crossing of a species barrier supports the causal nature of this mutation. Future studies are aimed at finding the major human MH mutations and establishing assays for their accurate diagnosis.

A substitution of cysteine for arginine 614 in the ryanodine receptor is potentially causative of human malignant hyperthermia.

Malignant hyperthermia (MH) is a devastating, potentially lethal response to anesthetics that occurs in genetically predisposed individuals. The skeletal muscle ryanodine receptor (RYR1) gene has been linked to porcine and human MH. Furthermore, a Cys for Arg substitution tightly linked to, and potentially causative of, porcine MH has been identified in the ryanodine receptor. Analysis of 35 human families predisposed to malignant hyperthermia has revealed the presence, and cosegregation with phenotype, of the corresponding substitution in a single family. This substitution, by analogy to the findings in pig, may be causal for predisposition to MH in this family.

Halothane-dependent release of intracellular Ca2+ in blood cells in malignant hyperthermia.

The concentration of ionized cytosolic calcium [( Ca2+]i) was determined in peripheral blood mononuclear cells from normal and malignant hyperthermia (MH)-susceptible humans and pigs, using the fluorescent Ca2+ indicator indo-1. [Ca2+]i was slightly but significantly elevated in cells from MH human cells relative to normal cells (198 +/- 18 nM, n = 15, and 146 +/- 14 nM, n = 11, respectively, P less than 0.05). Anesthetic concentrations of halothane in the cell suspension resulted in a rapid increase in [Ca2+]i in cells from both normal and MH humans or pigs. The increases (delta) were more pronounced in cells from MH subjects than from normal individuals (delta at 5.7 mM halothane: 245 +/- 53 vs. 57 +/- 11 nM, respectively) and from MH than from normal pigs (delta of 241 +/- 63 vs. 53 +/- 27 nM, respectively). Removal of extracellular Ca2+ obliterated the delta[Ca2+]i caused by halothane in cells from normal humans or pigs but only decreased by about half the delta[Ca2+]i in cells from MH humans or pigs. In 1,2-bis-(aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-loaded cells, in the absence of extracellular Ca2+, halothane failed to increase [Ca2+]i. This suggests that buffering Cai2+ with BAPTA precludes detection of release of Ca2+ from intracellular stores, explaining the previous observations made with quin2, a highly chelating Ca2+ indicator. It is concluded that clinical concentrations of halothane allow influx of Ca2+ in cells from both normal and MH-susceptible individuals but release Ca2+ from intracellular stores selectively in cells from the latter group.(ABSTRACT TRUNCATED AT 250 WORDS)

Ryanodine receptor gene is a candidate for predisposition to malignant hyperthermia.

Malignant hyperthermia (MH) is a potentially lethal condition in which sustained muscle contracture, with attendant hypercatabolic reactions and elevation in body temperature, are triggered by commonly used inhalational anaesthetics and skeletal muscle relaxants. In humans, the trait is usually inherited in an autosomal dominant fashion, but in halothane-sensitive pigs with a similar phenotype, inheritance of the disease is autosomal recessive or co-dominant. A simple and accurate non-invasive test for the gene is not available and predisposition to the disease is currently determined through a halothane- and/or caffeine-induced contracture test on a skeletal muscle biopsy. Because Ca2+ is the chief regulator of muscle contraction and metabolism, the primary defect in MH is believed to lie in Ca2+ regulation. Indeed, several studies indicate a defect in the Ca2+ release channel of the sarcoplasmic reticulum, making it a prime candidate for the altered gene product in predisposed individuals. We have recently cloned complementary DNA and genomic DNA encoding the human ryanodine receptor (the Ca2(+)-release channel of the sarcoplasmic reticulum) and mapped the ryanodine receptor gene (RYR) to region q13.1 of human chromosome 19 (ref. 14), in close proximity to genetic markers that have been shown to map near the MH susceptibility locus in humans and the halothane-sensitive gene in pigs. As a more definitive test of whether the RYR gene is a candidate gene for the human MH phenotype, we have carried out a linkage study with MH families to determine whether the MH phenotype segregates with chromosome 19q markers, including markers in the RYR gene. Co-segregation of MH with RYR markers, resulting in a lod score of 4.20 at a linkage distance of zero centimorgans, indicates that MH is likely to be caused by mutations in the RYR gene.

Canine stress syndrome/malignant hyperthermia susceptibility: calcium-homeostasis defect in muscle and lymphocytes.

This study provides the first comprehensive characterisation of the calcium (Ca) homeostasis defects found in muscle and lymphocytes of a malignant hyperthermia (MH)-susceptible dog. Novel findings regarding this dog are reported, compared to controls. First, a canine stress syndrome occurs, analogous to the porcine stress syndrome; susceptibility can be identified by exercise challenge testing. Secondly, caffeine causes Ca release from muscle sarcoplasmic reticulum in a greater amount and at a greater rate. Thirdly, there is a compensatory increase in Ca sequestration by sarcoplasmic reticulum. Fourthly, lymphocytes have lower cytosolic-free Ca and a greater ability to prevent Ca increase. Halothane increases Ca by a greater amount and rate. Fifthly, muscle is more resistant to the contracture-producing effects of caffeine, as occurs in the non-rigid variant of MH susceptibility in man. This resistance, despite increased caffeine-induced release through the Ca channel, may be attributable to increased Ca sequestration by sarcoplasmic reticulum. Finally, erythrocyte osmotic fragility and creatine kinase tests fail to distinguish between the MH-susceptible dog and controls.

Malignant hyperthermia susceptibility: biochemical basis for pathogenesis and diagnosis.

Malignant hyperthermia (MH) is a hypermetabolic and hypercontractile syndrome triggered by anesthesia or various stressors that cause a sustained increase in sarcoplasmic ionized Ca. Susceptibility is apparently inherited in an autosomal dominant pattern. The primary molecular defect results in hypersensitive ligand-gating of the Ca-release channel of sarcoplasmic reticulum (SR) in skeletal muscle: channel opening is stimulated by abnormally low concentrations of agonist. We attribute MH to a mutation in the gene for the fast twitch muscle isoform of the Ca-channel, resulting in the expression of a cardiac-like isoform in fast muscle. Syndromes with some resemblance to MH can occur due to other genetic or acquired imbalances in Ca-flux across SR that favor net release of Ca. Either defective uptake or release can be detected as increased sensitivity of muscle to the contracture-producing effects of caffeine and halothane. Thus, caffeine and/or halothane contracture tests for MH-susceptibility may give false positives when there is decreased Ca-uptake, such as in muscular dystrophies. Ca-channel hypersensitivity and decreased Ca-uptake activity can be detected by assays using isolated SR. Functional assays using lymphocytes are being assessed as potential replacements for muscle contracture tests. Polymorphism analysis of proteins or nucleic acids for the MH or closely-linked genes has been used to trace the inheritance of MH-susceptibility.

Malignant hyperthermia.

Malignant hyperthermia is a hereditary trait characterized by hypercatabolic reactions induced by anesthetic drugs, or physical or emotional stress. Patients must be treated quickly and efficiently in order to prevent irreversible organ damage and death.

Selective increase in cytoplasmic calcium by anesthetic in lymphocytes from malignant hyperthermia-susceptible pigs.

Anesthetic-induced malignant hyperthermia in pigs and humans is characterized by muscle rigidity and rapid, often fatal, increases in body temperature. A defect in Ca2+ homeostasis has been suspected as underlying the disease, based on the preventive effect of dantrolene sodium, an agent thought to reduce Ca2+ levels in the cytoplasm. We describe here direct measurements of cytoplasmic ionized Ca2+ levels in lymphocytes from seven normal and 12 malignant hyperthermia-susceptible pigs, using the fluorescent indicator quin2. No differences in the concentration of cytoplasmic ionized Ca2+ were found in cells from malignant hyperthermia-susceptible pigs (160 +/- 10 nM) relative to the controls (150 +/- 10 nM). However, addition of halothane in vitro caused a significant increase (to 270 +/- 30 nM) in lymphocytes from malignant hyperthermia-susceptible pigs, but not from normal pigs (180 +/- 10 nM). The halothane-mediated increase in cytoplasmic ionized Ca2+ required extracellular Ca2+. It is suggested that general anesthetics such as halothane increase the permeability of the cell surface to Ca2+, and that this increase may, on its own or indirectly, increase the cytoplasmic level of ionized Ca2+ during a malignant hyperthermia crisis. The detection of a halothane-dependent increase in cytoplasmic ionized Ca2+ selectively in malignant hyperthermia-susceptible pigs could be the basis for a noninvasive test for malignant hyperthermia.

Anaesthetic-induced increase in ionised calcium in blood mononuclear cells from malignant hyperthermia patients.

The cytoplasmic concentration of ionised calcium, [Ca2+]i, is believed to be altered by agents that induce a malignant hyperthermia (MH) crisis in susceptible individuals. MH patients were identified by the halothane and halothane/caffeine contracture tests done in isolated muscle biopsy specimens. [Ca2+]i was measured in isolated peripheral blood mononuclear cells from MH patients and controls by means of the fluorescent calcium ion indicator quin2. In the absence of halothane there was no significant difference in [Ca2+]i in cells from normal and MH patients. Addition of halothane (4 microliter/ml) significantly increased [Ca2+]i in cells from MH patients but not in controls. The halothane-induced increase in [Ca2+]i required extracellular calcium ions. This is the first evidence of the mechanism of action of halothane in cells of MH patients; the differential effect of halothane on [Ca2+]i might constitute the basis for a non-invasive screening test for MH.

Comparison of metabolites in skeletal muscle biopsies from normal humans and those susceptible to malignant hyperthermia.

In earlier work on malignant hyperthermia (MH) susceptible pigs the concentration of muscle metabolites differed from that found in normal control pigs. Therefore, in the present study these metabolites were measured in human muscle biopsies to find out whether normal individuals could be discriminated from MH-susceptible persons. Analysis of skeletal muscle metabolites was performed on skeletal muscle obtained from humans (n = 68) being screened to exclude or confirm susceptibility to MH. Three groups were identified based on the reaction pattern of a skeletal muscle sample exposed in vitro to caffeine or halothane 1% plus caffeine: 1) MH susceptible (MHS; n = 19); 2) normal humans, (controls; n = 31); and 3) intermediate-reaction type (K-type:n = 18). No significant differences were found in metabolite levels of phosphocreatine (normal, MHS, and K-type: 13.20 vs. 13.74 vs. 14.42 nmol/mg wet weight, respectively), creatine (16.30 vs. 16.94 vs. 15.06 nmol/mg wet weight, respectively), adenosine triphospate (3.75 vs. 3.98 vs. 3.89 nmol/mg wet weight, respectively) and lactate (3.73 vs. 3.65 vs. 3.79 nmol/mg wet weight, respectively). It is concluded that analysis of skeletal muscle metabolites cannot be used as a screening test to confirm or exclude MH susceptibility in humans.

Changes in cytoplasmic free calcium caused by halothane. Role of the plasma membrane and intracellular Ca2+ stores.

Malignant hyperthermia is a muscle disease characterized by an abnormal response to anaesthetics, stress, and exercise. It is typified by muscle contracture and a dramatic elevation in body temperature. A defect in the regulation of the concentration of cytoplasmic free calcium, [Ca2]i, is thought to underlie this disease, but the actual [Ca2+]i was not measurable until recently. We have shown that the anaesthetic halothane increases [Ca2+]i in isolated lymphocytes from malignant hyperthermia-susceptible humans and pigs but not in the normal counterparts. In this report we extend these observations to a larger number of cases and analyze the molecular mechanisms responsible for the increase. The halothane-mediated rise in [Ca2+]i required external Ca2+ and was prevented by nifedipine, an inhibitor of the voltage-sensitive Ca2+ channels of the cell membrane. In addition, the effect of halothane on the releasable Ca2+ from intracellular stores was determined by measuring the size of the releasable pool before and after addition of the anaesthetic. After addition of halothane, about 73% of this Ca2+ pool was still available for release by the Ca2+ ionophore ionomycin in cells from normal humans and pigs. In contrast, only about 45% of the free Ca2+ in intracellular stores was left after treatment with halothane in cells from malignant hyperthermia-susceptible humans and swine. These results indicate that halothane acts both at the cell membrane and at intracellular organelles, and that this action results in a net increase in [Ca2+]i in malignant hyperthermia, but not in normal cells. The action at the cell membrane appears to be on the voltage-sensitive Ca2+ channels.(ABSTRACT TRUNCATED AT 250 WORDS)

Failure of the tourniquet-twitch test as a diagnostic or screening test for malignant hyperthermia.

We have performed the tourniquet-twitch test of Roberts and Ryan in normal and in malignant hyperthermia (MH) patients and relatives. This test measures the ratio of electrically induced thumb twitches noted after 10 min of ischemia with those noted immediately prior to the ischemia. We found no significant differences in this ratio between normal subjects and those who have had MH reactions, or relatives of such individuals. Furthermore, we have observed no significant differences in tourniquet-twitch ratios between those with normal caffeine-halothane contractures and persons with caffeine-halothane contractures tests positive for MH. These findings do not agree with those of Roberts and Ryan, who reported that tourniquet-twitch ratios were higher in MH patients than in normal patients. We have, however, determined that subjects with tourniquet-twitch ratios greater than or equal to 1.8 are substantially younger than those with tourniquet-twitch ratios less than or equal to 1.0. Therefore we do not believe that the tourniquet-twitch test is useful as a diagnostic, or even as a screening test for MH.

Failure of the platelet-halothane nucleotide depletion test as a diagnostic or screening test for malignant hyperthermia.

A platelet-halothane nucleotide or bioassay test has been suggested as an accurate means for diagnosis of malignant hyperthermia (MH). For the last three years we performed this test in all patients at risk for MH and in all normal control patients undergoing skeletal muscle biopsy in our diagnostic unit. The reliability of the platelet-halothane nucleotide test has been assessed on the basis of the presence or absence of a previous MH reaction and degree of relationship of relatives to those who have had reactions, and on the basis of caffeine-halothane contracture test values (positive or negative for MH). We have found no significant difference in platelet-halothane nucleotide test values in malignant hyperthermia susceptible (MHS) patients or relatives of MHS patients and normal controls, and no relationship between the nucleotide test and caffeine-halothane contracture test results. We conclude that the platelet-halothane nucleotide test is not of assistance in the diagnosis of or in the screening for MH.