Results of in vitro contracture testing of both parents of malignant hyperthermia susceptible probands

Background: Malignant Hyperthermia (MH) is regarded as a dominantly inherited condition. Therefore, most investigators do not test the second parent if the first parent is found to be MH susceptible (MHS). The purpose of this study was to validate this policy.
Methods: In vitro contracture tests (IVCT) have been performed in both parents of 101 MH susceptible probands. Diagnoses were made according to the European MH group protocol and include MH susceptible (MHS), MH equivocal (MHE) and MH negative (MHN). Our control material consists of 60 patients without any personal or family history of MH.
Results: Thirty-seven pairs of parents were MHS-MHN, 20 were MHE-MHN, 6 were MHS-MHS, 20 were MHS-MHE, 6 were MHE-MHE, and 12 were MHN-MHN. The frequency of MHE was greater in the parents than in the controls (26% versus 8%. P < 0.05). Several possible explanations exist: the IVCT produces false positive and/or false negative results; the MH genes may be more frequent in the population than previously expected; MH susceptibility may have more than one mode of inheritance; the mutation rate may not be negligible. Our test results in controls and fulminants point at a combination of these explanations.
Conclusions: We conclude that both parents should be tested whenever possible. For genetic research it is important that labelling any parent "presumed normal" may give misleading results.     

Multicentre evaluation of ryanodine contracture testing in malignant hyperthermia

A common protocol for in vitro contracture testing using the plant alkaloid ryanodine has been used by the European Malignant Hyperthermia Group since 1993. This protocol describes a test using I mumol/litre of high purity ryanodine (98%) added as a single bolus dose. The main aim of this study was to compare the results obtained with this test between laboratories with a view to assessing the validity of adopting common diagnostic end-points for use in future collaborative studies. In order to do this it was first necessary to determine the optimum cut- off values of the end-points for discriminating between patients diagnosed as susceptible or not to malignant hyperthermia. The end- points under evaluation were expressed in terms of time after application of ryanodine at which a certain degree of contracture develops. In this study, four end-points were investigated: time to initial contracture development (Ot); time to development of a 10-mN contracture (10t); time from addition of ryanodine to when baseline tension exceeds pre-drug tension (0tp); and time for contracture to reach 10 mN above pre-drug baseline tension (10tp). This protocol was developed initially and used by three investigating centres and the initial assessment of the end-points and their discriminatory ability was made using the first 100 patients from each of centres 1 and 2, and the first 90 patients from centre No. 3. Optimal cut-off values for each of the end-points were determined using logistic regression analysis. Discriminatory ability was improved by combining the Ot and 10t end-points (P < 0.05) but not significantly by combining the 0tp and 10tp end-points. Both methods of categorization were highly sensitive and specific compared with the current standard diagnostic tests. Results from eight additional diagnostic centres which have used the ryanodine contracture test more recently, while indicating that susceptible and normal individuals can be distinguished within a single laboratory, produced a level of variability between testing centres for the ryanodine contracture test that is incompatible with the use of common cut-off values. Possible causes for this variability between laboratories are discussed.      

Characterization of ryanodine receptor-mediated calcium release in human B cells: relevance to diagnostic testing for malignant hyperthermia

BACKGROUND: Mutations in the ryanodine type 1 receptor (RyR1) are causative for malignant hyperthermia. Studies in human B lymphocytes have shown that measurement of RyR1-mediated intracellular Ca(2+) (Ca(2+)(i)) release can differentiate between normal and malignant hyperthermia-susceptible individuals. The authors have further developed the B-cell assay by pharmacologically characterizing RyR1-mediated Ca release in two normal human B-cell lines and demonstrating increased sensitivity of lymphocytes to the RyR1 agonist 4-chloro-m-cresol (4-CmC) in the porcine model of MH. METHODS: Ca(2+)(i) was measured fluorometrically using fura-2 in populations of cells in suspension or with fluo-4 in single cells using confocal microscopy. The Dakiki and PP normal human B cell lines were used, as well as lymphocytes obtained from normal and malignant hyperthermia-susceptible pigs. 4-CmC was used to elicit RyR1-mediated Ca release; all experiments were performed in the absence of external Ca(2+). RESULTS: EC(50) values for 4-CmC were 0.98 and 1.04 mm for Dakiki and PP cells, respectively, demonstrating reproducibility. The 4-CmC-induced increase in Ca(2+)(i) was eliminated by thapsigargin and was unaffected by xestospongin C. The Ca(2+)(i) increase was separable from mitochondrial stores and was inhibited by azumolene. Caffeine did not induce Ca(2+)(i) release, but ryanodine depleted intracellular stores by 50%. Lymphocytes from pigs carrying the Arg614Cys mutation in RyR1 showed increased sensitivity to 4-CmC (EC(50) = 0.47 vs. 0.81 mm for cells derived from normal animals). CONCLUSIONS: RyR1-mediated Ca(2+) signals can be pharmacologically distinguished from other intracellular sources in human B cells, and alterations of RyR1 function can be successfully detected using Ca(2+) release from intracellular stores as an end point.     

Male preponderance of patients testing positive for malignant hyperthermia susceptibility

BACKGROUND: Malignant hyperthermia susceptibility is diagnosed using an in vitro contracture test (IVCT). In families in which the mutation is known, genetic tests are also available. The inheritance pattern is regarded as autosomal dominant, which predicts equal proportions of men and women affected. The aim of this study was to investigate whether there were sex differences in the diagnostic outcome of the 1407 patients tested for malignant hyperthermia in Sweden between 1985 and 2005. METHODS: Information about sex, diagnosis, IVCT result and kinship was analysed. Comparisons were made between the two sexes. Probands and relatives were analysed separately in order to eliminate bias caused by the type of surgery performed in the two sexes. RESULTS: Males, more than females, revealed a pathological outcome in IVCT. Amongst male relatives, the fraction of pathological outcome in IVCT was 0.70 [95% confidence interval (CI), 0.66-0.74]; the corresponding value for females was 0.40 (95% CI, 0.36-0.44). CONCLUSION: A significant difference was observed in the sex distribution of outcome of IVCT, with significantly more males revealing a pathological IVCT. This indicates the influence of one or several factors related to sex in the outcome of IVCT, for example different expression of calcium handling proteins in the sexes, a complex pattern of inheritance or unknown environmental factors.     

The genetics of malignant hyperthermia

The genetic evaluation of the ryanodine type one receptor (RYR1) gene is unlikely to be a useful screening test of malignant hyperthermia susceptibility. But when significant suspicion of malignant hyperthermia has been raised by well-documented clinical events or strong family history, the genetic evaluation of RYR1 could secure a diagnosis and indicate appropriate treatment for both the index patient and many relatives of all ages, including the youngest.     

The malignant neuroleptic syndrome and malignant hyperthermia

We report on a patient with neuroleptic malignant syndrome (NMS) caused by a therapy for endogenous depression. The symptoms were hyperpyrexia (39.2 degrees C), rigidity, elevated creatine kinase (CK: 594 U/l) and coma. After transfer from an outside hospital, he was treated, at first without effect with dantrolene p.o. (80 mg q.i.d.) and i.v. (1 mg/kg-1/h-1). Clinical improvement and temperature reduction were noted when the levels of neuroleptic drugs fell during unspecific intensive care with mechanical ventilation, sedation (flunitrazepam, barbiturates), relaxation (pancuronium), and hydration. After uncomplicated weaning from the ventilator the patient became more cooperative and was returned to the psychiatric ward. Further treatment took the form of combined drug therapy with biperiden and flunitrazepam and in addition a series of 12 electroconvulsive therapies (ECT). The elevated CK levels initially decreased, serum potassium levels were found to be within normal limits, and myoglobinuria was not detected during the further course. Trigger agents for NMS are antipsychotic drugs such as thioxanthenes, phenothiazines and butyrophenones. Because the signs and symptoms are so similar to those of malignant hyperthermia (MH), it has been suggested that NMS and MH are related diseases. The postulated mechanisms of NMS become apparent in the CNS, whereas those of MH affect the muscle cell itself. An abnormal in vitro contraction test after NMS should suggest to triggering of MH crisis after succinylcholine administration in anaesthesia for ECT.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prolonging viability of swine muscle biopsy specimens in malignant hyperthermia testing

In North America, the caffeine halothane contracture test (CHCT) is the standard test for the diagnosis of malignant hyperthermia (MH). Current CHCT protocol recommends that the test be completed within 5 h of muscle excision. The purpose of this study was to investigate whether the period of skeletal muscle viability could be extended to 24 h. We tested the gracilis muscle from normal (n = 8) and MH-susceptible swine (n = 8). After baseline (1-2 h after excision) CHCT, the remaining muscles were placed into one of the following four treatment groups. In Groups 1 and 2, the muscles remained under tension and were stored in Krebs buffer (pH 7.4) at 23 degrees C-25 degrees C (clamped-warm) and 4 degrees C (clamped-cold), respectively. In Groups 3 and 4, the muscle strips were dissected, and the ends were tied with silk sutures, cut from the clamp, and placed in Krebs buffer at 23 degrees C-25 degrees C (free-warm) and 4 degrees C (free-cold), respectively. The responses of the treatment groups to halothane (3%) and caffeine (0.5-32 mM) were tested 22-26 h after excision. The clamped-warm storage was the only storage method to correctly diagnose MH susceptibility in all muscle strips tested. This finding was also confirmed in muscle stored under clamped-warm conditions and shipped overnight to another testing center for a parallel CHCT.     

Importance of fasting in the lymphocyte calcium test for malignant hyperthermia

Anaesthetic-induced increases in cytoplasmic free Ca2+ have been reported to be greater in lymphocytes from malignant hyperthermia (MH) susceptible patients than in those from controls, suggesting that this may be the basis for a less invasive test for MH susceptibility. In the present study the cytoplasmic Ca2+ concentrations of lymphocytes were monitored with indo-1 in 14 control subjects (nine fasted and five nonfasted) and five fasted MH susceptible and three fasted nonsusceptible patients, diagnosed by the halothane and caffeine contracture tests. No relationship was observed between MH susceptibility and Ca2+ concentrations in lymphocytes in the absence or presence of halothane. There was, however, a relationship in control subjects between fasting and the response of lymphocytes to halothane, with the halothane-induced Ca2+ increase being considerably larger in nonfasted subjects.     

Multicentre evaluation of in vitro contracture testing with bolus administration of 4-chloro-m-cresol for diagnosis of malignant hyperthermia susceptibility

BACKGROUND AND OBJECTIVE: The in vitro contracture test with halothane and caffeine is the gold standard for the diagnosis of susceptibility to malignant hyperthermia (MH). However, the sensitivity of the in vitro contracture test is between 97 and 99% and its specificity is 78-94% with the consequence that false-negative as well as false-positive test results are possible. 4-Chloro-m-cresol is potentially a more specific test drug for the in vitro contracture test than halothane or caffeine. This multicentre study was designed to investigate whether an in vitro contracture test with bolus administration of 4-chloro-m-cresol can improve the accuracy of the diagnosis of susceptibility to MH. METHODS: Three hundred and fifty-two patients from 11 European MH laboratories participated in the study. The patients were first classified as MH susceptible, MH normal or MH equivocal by the in vitro contracture test according to the European MH protocol. Muscle specimens surplus to diagnostic requirements were used in this study (MH susceptible = 103 viable samples; MH equivocal = 51; MH normal = 204). 4-Chloro-m-cresol was added to achieve a concentration of 75 micromol L(-1) in the tissue bath. The in vitro effects on contracture development and muscle twitch were observed for 60 min. RESULTS: After bolus administration of 4-chloro-m-cresol, 75 micromol L(-1), 99 of 103 MH-susceptible specimens developed marked muscle contractures. In contrast, only two of 204 MH-normal specimens showed an insignificant contracture development following 4-chloro-m-cresol. From these results, a sensitivity rate of 96.1% and a specificity rate of 99.0% can be calculated for the in vitro contracture test with bolus administration of 4-chloro-m-cresol 75 micromol L(-1). Forty-three patients were diagnosed as MH equivocal, but only specimens from 16 patients developed contractures in response to 4-chloro-m-cresol, indicating susceptibility to MH. CONCLUSIONS: The in vitro contracture test with halothane and caffeine is well standardized in the European and North American test protocols. However, this conventional test method is associated with the risk of false test results. Therefore, an improvement in the diagnosis of MH is needed. Regarding the results from this multicentre study, the use of 4-chloro-m-cresol could increase the reliability of in vitro contracture testing.     

Pathophysiology of malignant hyperthermia

Malignant hyperthermia (MH) is a pharmacogenetic disease in man and animals. It primarily involves skeletal muscle tissue, but other tissues might be affected to a lesser degree. Calcium homeostasis in muscle cells is upset in susceptible individuals, so that various agents and circumstances can increase the free, ionised intracellular calcium concentration to damaging levels. The primary defect is not known at present, but is believed to involve an abnormally sensitive calcium-induced calcium release mechanism. Thus small, localised increases in calcium concentration releases more calcium so that a vicious cycle is triggered. The increased calcium concentration causes multiple effects in the muscles by stimulating contraction and a hypermetabolic state, clinically observed as rigidity and fever. If demands on the homeostatic mechanisms to lower the calcium concentration become exhausted, and metabolism is insufficient to supply enough phosphocreatine and ATP, membrane potentials cannot be maintained, and permeability of the cell membranes increase. This causes loss of phosphate and H+ as well as K+ and Mg++, and later myoglobin and creatine kinase. Thereby oxidative metabolism is further impeded with formation of lactate as a result. The ensuing acidosis stimulates sympathetic innervation, resulting in tachycardia, high blood pressure, and vasoconstriction. Hyperkalemia causes arrhythmia. Dantrolene inhibits the release of calcium and can halt the process if given before depletion of the energy rich phosphates is too advanced.