Mild clinical and histopathological features in patients who carry the frequent and causative malignant hyperthermia RyR1 mutation p.Thr2206Met.

OBJECTIVE: Malignant hyperthermia (MH) is a classically unapparent pharmacogenetic disorder of the skeletal muscles triggered by inhalational anesthetics or depolarizing muscle relaxants. The disposition to MH is inherited in an autosomal-dominant manner and is primarily due to mutations in the gene for the ryanodine receptor type 1 (RyR1). The present study intended to analyze whether mild muscular symptoms (elevation of the resting CK, cramps in the calves, slight calf hypertrophy) may be associated with susceptibility to MH and/or with histopathological changes. METHODS: A muscle biopsy was taken from 12 out of 44 blood relatives (three generations) of a large family and was investigated with the halothane/caffeine in vitro contracture test (IVCT). Afterwards a histological, histochemical and immunhistological examination was performed. Altogether in 29 persons the DNA was analyzed for mutations in the RyR1-gene. RESULTS: Eight persons were diagnosed as susceptible to MH (MHS) by the IVCT, 4 were MH negative. All MHS persons carried the MH causative c.6617C > T (Thr2206Met) mutation and showed slight clinical signs of a myopathy as well as mild biopsy changes with isolated hypotrophic fibers and disseminated small areas with reduction of oxidative staining (multi-minicore like lesions). The Thr2206Met mutation was identified in another further 9 relatives who also experienced mild myopathological features. Clinical MH incidents were not reported in this large family. CONCLUSION: The RyR1 Thr2206Met mutation is one of the most frequent mutations in the European MH population but carriers are normally healthy. In this study we could demonstrate that the MH causative Thr2206Met mutation may also be associated both with clinical symptoms of a mild myopathy and histopathological changes in the oxidative inter myofibrillar network.

Calcium release from sarcoplasmic reticulum is facilitated in human myotubes derived from carriers of the ryanodine receptor type 1 mutations Ile2182Phe and Gly2375Ala.

Malignant hyperthermia (MH) is caused by increased calcium release from sarcoplasmic reticulum, triggered by volatile anesthetics or depolarizing muscle relaxants. Numerous mutations associated with MH have been detected in the skeletal muscle type ryanodine receptor gene (RyR1), but so far facilitated calcium release has only been demonstrated for a few of them. This is a prerequisite for confirming the causative role of an RyR1 mutation for MH. Calcium release from sarcoplasmic reticulum induced by 4-chloro-m-cresol (4CmC), caffeine, and halothane was determined in human myotubes by calcium imaging. The RyR1 Ile2182Phe mutation and the RyR1 Gly2375Ala mutation have been identified in individuals susceptible to MH. In myotubes of individuals carrying the RyR1 Ile2182Phe or the RyR1 Gly2375Ala mutation, the EC(50) for caffeine and halothane was reduced; in the Ile2182Phe myotubes, the EC(50) for 4CmC was also reduced, all consistent with facilitated calcium release from the sarcoplasmic reticulum. From these data we conclude that both mutations are pathogenic for MH.

Recent advances in the diagnosis of malignant hyperthermia susceptibility: how confident can we be of genetic testing?

Malignant hyperthermia (MH) is a condition that manifests in susceptible individuals only on exposure to certain anaesthetic agents. Although genetically heterogeneous, mutations in the RYR1 gene (19q13.1) are associated with the majority of reported MH cases. Guidelines for the genetic diagnosis for MH susceptibility have recently been introduced by the European MH Group (EMHG). These are designed to supplement the muscle biopsy testing procedure, the in vitro contracture test (IVCT), which has been the only means of patient screening for the last 30 years and which remains the method for definitive diagnosis in suspected probands. Discordance observed in some families between IVCT phenotype and susceptibility locus genotype could limit the confidence in genetic diagnosis. We have therefore assessed the prevalence of 15 RYR1 mutations currently used in the genetic diagnosis of MH in a sample of over 500 unrelated European MH susceptible individuals and have recorded the frequency of RYR1 genotype/IVCT phenotype discordance. RYR1 mutations were detected in up to approximately 30% of families investigated. Phenotype/genotype discordance in a single individual was observed in 10 out of 196 mutation-positive families. In five families a mutation-positive/IVCT-negative individual was observed, and in the other five families a mutation-negative/IVCT-positive individual was observed. These data represent the most comprehensive assessment of RYR1 mutation prevalence and genotype/phenotype correlation analysis and highlight the possible limitations of MH screening methods. The implications for genetic diagnosis are discussed.

Increased sensitivity to 4-chloro-m-cresol and caffeine in primary myotubes from malignant hyperthermia susceptible individuals carrying the ryanodine receptor 1 Thr2206Met (C6617T) mutation.

Malignant hyperthermia (MH) is an autosomal-dominant disorder of skeletal muscle, triggered by volatile anaesthetics and depolarizing muscle relaxants. The causative defect lies in the control of Ca(2+) release from the sarcoplasmic reticulum in skeletal muscle. Numerous mutations have been detected in the ryanodine receptor 1 (RyR1) gene, but so far an MH-causative role has only been confirmed for 16 human RyR1 mutations. In this report we show that myotubes derived from individuals carrying the RyR1 Thr2206Met (C6617T) mutation have an abnormal response of the intracellular calcium concentration to 4-chloro-m-cresol and to caffeine. Satellite cells were obtained from muscle biopsies of patients referred for diagnosing MH. The intracellular calcium concentration in response to 4-chloro-m-cresol and to caffeine was investigated by fluorescence calcium imaging. In myotubes the half-maximal activation concentration (EC(50)) for 4-chloro-m-cresol was reduced from 203 micro m (wild type) to 98 micro m (Thr2206Met), and for caffeine from 3.8 mm to 1.8 mm. From the reduction of EC(50) we conclude that the RyR1 Thr2206Met mutation is pathogenic for MH.

Determination of a positive malignant hyperthermia (MH) disposition without the in vitro contracture test in families carrying the RYR1 Arg614Cys mutation.

Molecular genetic methods are used with caution for determining positive malignant hyperthermia (MH) disposition in clinical MH diagnosis because of the genetic variability of this disease. But under defined conditions, genotyping can have an advantage over the standardized in vitro contracture test (IVCT) in respect of invasive approach, specificity, patient compliance, and the work and expense involved in the method. We aim to demonstrate this using 10 families with the Arg614Cys mutation in the ryanodine receptor as an example. Fifty-one index patients who had been classified as MH-susceptible (MHS) in the IVCT (European MH protocol) after a clinical MH incident or suspected MH were screened for the Arg614Cys (C1840-->T) mutation in the RYR1 gene because this mutation is more common in German MH families (9%). The family members of those index patients, in whom a Arg614Cys mutation was detectable, were also screened for the presence of this mutation (n=136), and the results were subjected to a more detailed analysis including existing IVCT findings (n=71). The Arg614Cys mutation was identified in a total of 64 members of the 10 independent families. In 35 individuals in this group, there was a definite concordance between the MHS diagnosis in the IVCT and the presence of the Arg614Cys mutation. No individual phenotyped as MH-negative carried the mutation. On the basis of the guidelines of the EMHG for molecular genetic detection of MH susceptibility, 29 individuals who bore the Arg614Cys mutation were classified as MHS without the IVCT. If a causal mutation is detected in an MH family, the MHS diagnosis can be deduced without the invasive IVCT in all other mutation carriers. Despite inclusion of only one (Arg614Cys) of all known MH mutations, the study emphasizes the practical use of a genetic approach for determination of a positive MH diagnosis.

Homozygous and heterozygous Arg614Cys mutations (1840C-->T) in the ryanodine receptor gene co-segregate with malignant hyperthermia susceptibility in a German family.

The determination of susceptibility to malignant hyperthermia (MH) by genetic investigation is a controversial issue because of the genetic heterogeneity of this disorder. The requirement for such an approach in MH diagnosis is a strong correlation between MH-associated genetic abnormalities and phenotypic findings in the in vitro contracture test (IVCT). After a severe clinical MH crisis during general anaesthesia a patient was diagnosed by the IVCT in which susceptibility to MH was confirmed. Genetic screening for MH-related mutations in the RYR1 gene revealed the presence of a homozygous 1840C-->T base exchange (Arg614Cys substitution) in this patient. A specific search for this defect in 20 relatives led to the identification of a total of 11 Arg614Cys mutations. Of these, 10 were heterozygous (including both parents) and one was homozygous (sister). Further IVCTs were subsequently performed on the parents of the index patient, the homozygous sister and all relatives who did not carry the Arg614Cys in order to determine the genotype/phenotype correlation. After analysing these data, and because of the strong correlation between clinical, phenotypic, and genetic results in the index patient, we assigned the diagnosis 'MHS' to all the remaining Arg614Cys mutation carriers of that family without performing the IVCT.