J.J.A. Heffron

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

Background: Determination of sensitivity and specificity of the in vitro contracture test (IVCT) for malignant hyperthermia (MH) susceptibility using the European MH Group (EMHG) protocol has been performed in some laboratories but only on a small sample from the combined EMHG. Thus, the purpose of the present study was to determine combined EMHG sensitivity and specificity of the test.

Porcine Stress Syndromes

Publisher Summary This chapter discusses that porcine stress syndrome (PSS) is characterized by acute death induced by natural stressors—such as—transport, high ambient temperature, exercise, fighting, service, and parturition. Malignant hyperthermia (MH) is a pharmacogenetic disease. Identification of pigs susceptible to any of the stress syndromes is of great importance. The chapter discusses that serological tests have focused on the measurement of serum enzymes. Hematological tests have analyzed platelet function, blood groups, and erythrocyte fragility in attempts to find a predictive test. In general, the rationale has promised much, but the results have been disappointing. In man, susceptibility to MH is well correlated with clinical myopathies. There is no doubt that sympathetic nerve activity associated with stress causes the release of adrenaline and nonadrenaline. It is well recognized that thyroid hormones regulate tissue metabolic rates. Hypermetabolism of tissues and hyperthermia are characteristic of PSS and MH, and a priori link between thyroid hormone activity and stress seems likely. The chapter discusses that although it is possible for clinically normal pigs to develop stress syndromes, the data presented here argue that pigs most likely to develop MH and PSS have a “homozygote” genetic defect involving a single gene. A whole range of responses to stress or drugs can, therefore, be envisaged and breed variations and the difficulty of finding unequivocal predictive tests all can be accounted for by variation in the severity of the inherited defect.

Identification of Novel Mutations in the Ryanodine-Receptor Gene (RYR1) in Malignant Hyperthermia: Genotype-Phenotype Correlation

Malignant hyperthermia (MH) is a pharmacogenetic disorder of skeletal muscle that is triggered in genetically predisposed individuals by common anesthetics and muscle relaxants. The ryanodine receptor (RYR1) is mutated in a number of MH pedigrees, some members of which also have central core disease (CCD), an inherited myopathy closely associated with MH. Mutation screening of 6 kb of the RYR1 gene has identified four adjacent novel mutations, C6487T, G6488A, G6502A, and C6617T, which result in the amino acid alterations Arg2163Cys, Arg2163His, Val2168Met, and Thr2206Met, respectively. Collectively, these mutations account for 11% of MH cases and identify the gene segment 6400-6700 as a mutation hot spot. Correlation analysis of the in vitro contracture-test data available for pedigrees bearing these and other RYR1 mutations showed an exceptionally good correlation between caffeine threshold and tension values, whereas no correlation was observed between halothane threshold and tension values. This finding has important ramifications for assignment of the MH-susceptible phenotype, in genotyping studies, and indicates that assessment of recombinant individuals on the basis of caffeine response is justified, whereas assessment on the basis of halothane response may be problematic. Interestingly, the data suggest a link between the caffeine threshold and tension values and the MH/CCD phenotype.

Central core disease. A correlated genetic, histochemical, ultramicroscopic, and biochemical study.

Two patients suffering from central core disease are presented. The condition is associated with musculoskeletal abnormalities which have been traced back over five generations. In addition to the typical histochemical findings, electronmicroscopic study has revealed the presence of both structured and non-structured cores in adjacent areas. The calcium uptake by the sarcoplasmic reticulum was reduced to one-third of normal. Phosphorylase activity was normal in the one case and reduced to 63% in the other. Actomyosin Mg2+-activated ATPase activity was decreased, as was the Ca2+-dependent ATPase of the sarcoplasmic reticulum.

Evidence for a relationship between ATP hydrolysis and changes in extracellular space and fibre diameter during rigor development in skeletal muscle.

Abstract 1. 1. The postmortem time course of rigor mortis was determined by pH, phosphocreatine and ATP changes in skeletal muscles from adult mice. Fibre diameter was measured on isolated unfixed fibres from pre-rigor and rigor muscles. 2. 2. A significant decrease (14–16 per cent; P 3. 3. Measurement of the ECS showed that it increased after 1 hr postmortem (P 4. 4. An almost complete incision in a rigor muscle (4 hr postmortem) gave an ECS value of 66.4 ml/100 g compared to 36.4 ml/100 g in the undamaged rigor control. A value similar to that of the damaged muscle 4 hr postmortem was obtained for undamaged 24 hr postmortem muscles (74.6 ml/100 g), indicating complete sarcolemmal permeability to inulin. 5. 5. In addition to the effect of rigor on skeletal muscle fibre diameter, this study illustrates the importance of standardizing postmortem time for ECS and muscle fibre size measurements.

A multicenter study of 4-chloro-m-cresol for diagnosing malignant hyperthermia susceptibility.

Standardization of the in vitro contracture test (IVCT) for malignant hyperthermia (MH) susceptibility has resulted in very rare false negative tests. However, false positive results stigmatizing the patient seem to be more frequent than false negative results and make supplementary tests desirable. This multicenter approach studied the usefulness of an IVCT with 4-chloro-m-cresol (4-CmC), a ryanodine receptor-specific agonist for a better definition of MH susceptibility. Diagnosis made by the standard IVCT was compared with the results of this 4-CmC test on muscle specimens of 202 individuals from 6 European MH centers. In the 4-CmC test, the results of the MH susceptible group differed significantly from both the MH normal and the MH equivocal group. 4-CmC revealed a qualitatively dose response-curve similar to caffeine. A correlation index of r = 0.79 for the concentration thresholds underlined the strong concordance of the caffeine and the 4-CmC effects. The optimal threshold concentration was determined to be 75 &mgr;M in the pooled data of all centers and is much lower than that of caffeine (2 mM), suggesting a more than 25-fold higher affinity of 4-CmC. The predictive value of 4-CmC is as high as that of caffeine and consequently higher than that of halothane. 4-CmC seems to be a suitable drug to refine diagnosis of MH susceptibility and could be used as an additional test substance. Implications Although in vitro contracture testing for malignant hyperthermia diagnosis is well standardized, with a relatively high sensitivity and specificity, false test results cannot be excluded and may be associated with serious disabilities for the concerned individuals. In this multicenter study, 4-chloro-m-cresol was evaluated as a new test substance for the in vitro contracture testing. Its use improves the accuracy of in vitro diagnosis of malignant hyperthermia susceptibility.

Detection of a novel mutation in the ryanodine receptor gene in an Irish malignant hyperthermia pedigree: correlation of the IVCT response with the affected and unaffected haplotypes.

Defects in the ryanodine receptor (RYR1) gene are associated with malignant hyperthermia (MH), an autosomal dominant disorder of skeletal muscle and one of the main causes of death resulting from anaesthesia. Susceptibility to MH (MHS) is determined by the level of tension generated in an in vitro muscle contracture test (IVCT) in response to caffeine and halothane. To date, mutation screening of the RYR1 gene in MH families has led to the identification of eight mutations. We describe here the identification of a novel mutation, Arg552Trp, in the RYR1 gene, which is clearly linked to the MHS phenotype in a large, well characterised Irish pedigree. Considering that the RYR1 protein functions as a tetramer, correlation of the IVCT with the affected and unaffected haplotypes was performed on the pedigree to investigate if the normal RYR1 allele in affected subjects contributes to the variation in the IVCT. The results show that the normal RYR1 allele is unlikely to play a role in IVCT variation.

Skeletal muscle sarcoplasmic reticulum in porcine malignant hyperthermia

To examine the function of sarcoplasmic reticulum (SR) in malignant hyperthermia, SR was isolated from semitendinosus muscle of normal and genetically susceptible Poland China swine. Determinations included rate of calcium binding (oxalate absent), rate and capacity of calcium uptake (oxalate present), and spontaneous calcium release (in the absence of ionic depolarization or calcium) with and without halothane, using the millipore filtration technique. Rate of calcium binding, and rate and capacity of calcium uptake were decreased, and spontaneous calcium release was greater in SR fragments from susceptible swine as compared to those from normal swine. Halothane 0.5% slightly increased the rate of calcium binding in susceptible and normal SR. Above 1%, halothane decreased calcium binding rate, and uptake rate and capacity, and increased calcium release similarly in susceptible and normal SR. These differences in SR function were insufficient to explain the etiology of malignant hyperthemia, nor did the effect of halothane account for its triggering action.

Weakness associated with the pathological presence of lipid in skeletal muscle: a detailed study of a patient with carnitine deficiencey.

A patient with muscular weakness demonstrating pathological lipid accumulation and abnormal mitochondria in skeletal muscle has been studied. The lipid accumulation and mitochondrial changes are thought to be related to the established deficiency of carnitine in this patients muscle. The symptoms of muscular weakness associated with lipid accumulation in the skeletal muscle in the absence of complaint of muscle cramps or myglobinuria are thought to be diagnostic of carnitine deficiency. The failure of the sarcoplasmic reticulum to accumulate Ca2+ is discussed. The patients strength responded dramatically when propranolol was added to his steroid therapy.

Abnormal uptake and release of Ca2+ ions from human malignant hyperthermia-susceptible sarcoplasmic reticulum

Malignant hyperthermia (MH) is a pharmacogenetic myopathy that occurs in humans and several other mammalian species. There has been limited investigation of Ca2+ transport by human heavy sarcoplasmic reticulum (HSR) vesicles despite the fact that mutations of the ryanodine receptor Ca2+ release channel have been linked to inheritance of MH. In this study, the Ca2+ release and uptake mechanisms in human MH-susceptible HSR (MHS) vesicles were investigated and the kinetics and sensitivity compared to normal vesicles. Alterations in Ca2+ regulation were thereby elucidated. HSR vesicles from 6 normal (MHN) and 5 MHS patients were compared using a dual-wavelength continuous Ca2+ flux assay in the presence of pyrophosphate. The loading capacity and loading rate of Ca2+ in MHS vesicles were reduced by almost 50%. These parameters were restored to normal when the Ca2+ channel blocker ruthenium red was added. Calcium-induced calcium release, halothane-induced calcium release, and trifluoperazine-induced calcium release were clearly elevated in MHS HSR vesicles compared to MHN vesicles. The results suggest that MH ryanodine receptors exist in a more open resting state than those in normal muscle.