Functional and genetic characterization of clinical malignant hyperthermia crises: a multi-centre study

Abstract

Background: Malignant hyperthermia (MH) is a rare pharmacogenetic disorder which is characterized by life-threatening metabolic crises during general anesthesia. Classical triggering substances are volatile anesthetics and succinylcholine (SCh). The molecular basis of MH is excessive release of Ca2+ in skeletal muscle principally by a mutated ryanodine receptor type 1 (RyR1). To identify factors explaining the variable phenotypic presentation and complex pathomechanism, we analyzed proven MH events in terms of clinical course, muscle contracture, genetic factors and pharmocological triggers.

Methods: In a multi-centre study including seven European MH units, patients with a history of a clinical MH episode confirmed by susceptible (MHS) or equivocal (MHE) in vitro contracture tests (IVCT) were investigated. A test result is considered to be MHE if the muscle specimens develop pathological contractures in response to only one of the two test substances, halothane or caffeine. Crises were evaluated using a clinical grading scale (CGS), results of IVCT and genetic screening. The effects of SCh and volatile anesthetics on Ca2+ release from sarcoplasmic reticulum (SR) were studied in vitro.

Results: A total of 200 patients met the inclusion criteria. Two MH crises (1%) were triggered by SCh (1 MHS, 1 MHE), 18% by volatile anesthetics and 81% by a combination of both. Patients were 70% male and 50% were younger than 12 years old. Overall, CGS was in accord with IVCT results. Crises triggered by enflurane had a significantly higher CGS compared to halothane, isoflurane and sevoflurane. Of the 200 patients, 103 carried RyR1 variants, of which 14 were novel. CGS varied depending on the location of the mutation within the RyR1 gene. In contrast to volatile anesthetics, SCh did not evoke Ca2+ release from isolated rat SR vesicles.

Conclusions: An MH event could depend on patient-related risk factors such as male gender, young age and causative RyR1 mutations as well as on the use of drugs lowering the threshold of myoplasmic Ca2+ release. SCh might act as an accelerant by promoting unspecific Ca2+ influx via the sarcolemma and indirect RyR1 activation. Most MH crises develop in response to the combined administration of SCh and volatile anesthetics.

Figure 1

Effects of MH triggers on Ca²⁺ release. A: Uncontrolled myoplasmic Ca²⁺ release is the key to malignant hyperthermia. The most prominent cytosolic Ca²⁺ elevation results from the freeing of stored sarcoplasmic Ca²⁺ mediated by ryanodine receptor type 1 (RyR1). While volatile anesthetics stimulate Ca²⁺ release via RyR1, succinylcholine acts indirectly by activating the nicotinergic acetylcholine receptor (nAChR), a nonspecific cation channel, resulting in continuous local depolarisation. The depolarization can trigger propagated action potentials and will further activate the dihydropyridine receptors (DHPR, Ca_V1.1) leading to the gating of both Ca²⁺ release from the SR via RyR1 and L-type Ca²⁺ current from the extracellular space. B: Heavy SR from rat muscle was maximally preloaded with Ca²⁺ before testing the potential Ca²⁺ releasing agonists halothane, isoflurane, enflurane and succinylcholine. The resulting Ca²⁺ release is via the RyR1 channel. Halothane, isoflurane and enflurane induced Ca²⁺ release from the SR vesicles but succinylcholine had no detectable effect. Results are expressed as mean ± standard error from six separate SR specimens. Of the three anesthetics tested, halothane showed the greatest potency and efficacy.* C: Succinylcholine (SCh) significantly increases halothane induced contractures in malignant hyperthermia susceptible individuals. However, SCh alone does not lead to the development of pathological contractures in MHN or MHS individuals*. *Part of the data from Figure 1B and C was published in Klingler et al. in 2005 [25].

Figure 2

Clinical effects of volatile anesthetics. A: Box and whisker plots showing clinical grading scales (CGS) of 200 malignant hyperthermia susceptible (MHS, n = 165) or equivocal (MHE, n = 35) patients depending on the anesthetic agent used. Enflurane developed a significantly higher CGS compared to halothane, isoflurane and sevoflurane. B: CGS depending on the in vitro contracture test results: malignant hyperthermia susceptible (MHS), malignant hyperthermia equivocal halothane positive (MHEh) and caffeine positive (MHEc). A Mann–Whitney U-test was performed and yielded significant differences between MHS vs. MHEh, i.e. MHS vs. (MHEh + MHEc). C: Patients in this study with clinical crises that resulted in high MH Ranks (5 and 6) developed greater halothane and caffeine contractures than patients with lower MH Ranks (3 and 4). Asterisks (*, **) indicate significant differences. Columns represent mean ± standard error of the mean and black horizontal lines within the columns show median values.

Figure 3

Age and gender preponderance. Age and gender of 200 MH patients at the time of the clinical MH-episode.

Figure 4

Locations and effects of ryanodine receptor type 1 mutations. A: Amino acid (AS) sequence of the ryanodine receptor type 1 (RyR1) from the n-terminal end to the c-terminal end. Most of the mutations found in this study are located in one of the three hot spots: MH/CCD region 1: AS 35 to 614; MH/CCD region 2: AS 2163 to 2458; MH/CCD region 3: AS 4664 to 5020. B: Clinical grading scale (mean) for each RyR1 mutation in regard of the location of the patients mutation within the gene. C: Box plot showing clinical grading scales (CGS) depending on the location of the ryanodine receptor type 1 mutation. Boxes delineate the inter-quartile range (25% to 75%), black horizontal lines within the boxes show median values, whiskers indicate ranges and white squares represent mean values. Mann–Whitney U-test reveals significantly higher CGS of MH/CCD region 1, 2 and 3 compared to other regions of the protein.