Malignant Hyperthermia: Title and subTitle BreakUpdate on Susceptibility Testing

In 1962, Drs Denborough and Lovell and colleagues¹ from Melbourne, Australia, described a young man with a fractured tibia who was more concerned about receiving general anesthesia than about his leg. The patient had good reason: 10 of his relatives had died without explanation during or following general anesthesia. During his surgery (performed with halothane anesthesia), the patient developed severe hyperthermia, tachycardia, and tachypnea, and was ultimately rescued by aggressive cooling. When Denborough, a geneticist, investigated the patient’s family, he noted that the deaths followed an autosomal dominant pattern and concluded that there existed an inheritable disorder that caused susceptibility to life-threatening hyperthermia and hypermetabolism when a susceptible individual is exposed to general anesthetic agents.

Subsequent reported cases confirmed this association and suggested that an underlying skeletal muscle disorder may confer susceptibility to this disorder. Additional reports further defined the triggering agents, which include the volatile inhalational anesthetic gases (eg, ether, halothane, isoflurane, etc) and succinylcholine, a depolarizing neuromuscular blocker. Because of the high mortality (>70%) associated with this clinical syndrome in that era and the consistent presence of extreme hyperthermia, this syndrome was named malignant hyperthermia. A similar condition in certain breeds of swine (porcine stress syndrome) was described at about the same time and has served as a useful investigational model for the syndrome.

The incidence of acute MH susceptibility has been estimated to be as low as 1 in 250 000² and as high as 1 in 200,³ depending on the geographical region. The true prevalence is difficult to define because of unrecognized mild or aborted reactions, and the variable penetrance of the inherited trait. Furthermore, many MH-susceptible patients may never be exposed to anesthetic triggering agents. Almost all MH-susceptible patients are phenotypically normal and will only manifest the clinical characteristics of MH when exposed to anesthetic triggering agents. Because of improved monitoring standards that allow early detection of hypercarbia during general anesthesia and the availability of dantrolene, the mortality from acute MH is now estimated to be less than 5%.

The onset of acute MH is heralded by one or more signs of systemic hypermetabolism during or immediately after administration of a general anesthetic, during which a triggering agent is being or has been administered ( Article ). An overwhelming increase in oxygen consumption via uncontrolled glycolysis and aerobic metabolism leads to cellular hypoxia, progressive lactic acidosis, and hypercarbia from generation of excess carbon dioxide. Thus, the most common initial sign of acute MH is an unexplained rise in end-tidal carbon dioxide that does not easily abate with increased minute ventilation. Muscle rigidity is common and results from the abnormal elevation of intracellular calcium. Hyperthermia during MH is likely caused by continuous muscle activation with adenosine triphosphate breakdown that generates more heat than the body can dissipate to the environment. If untreated, continuing myocyte death and rhabdomyolysis result in life-threatening hyperkalemia and myoglobinuria. Additional life-threatening complications include disseminated intravascular coagulation, congestive heart failure, bowel ischemia, and compartment syndrome of the limbs secondary to profound muscle swelling.

Clinical Signs

Laboratory Findings

*In known cases of malignant hyperthermia (MH), there have been large variations in the intervals between exposure to the triggering agent and development of symptoms. Patients with MH have also demonstrated varying and unpredictable times for the initial signs to develop into a fulminant life-threatening crisis.
†Localized or generalized muscle rigidity, despite the presence of neuromuscular blockade, is a strongly specific indicator of MH when other signs are also present and is almost considered pathognomonic for the disease. The presence of masseter muscle spasm following administration of anesthetic triggering agents may herald the onset of MH in certain individuals.
‡Elevation of body temperature is a late sign of MH and may not be present at the time of the diagnosis. During fulminant acute MH, body temperature may increase at a rate of 1° to 2° C every 5 minutes.

Once recognized, treatment of MH includes immediate discontinuation of the anesthetic triggering agent and administration of dantrolene sodium, a specific antagonist of the pathophysiologic changes. Supportive measures are instituted to reverse the associated hyperthermia, acidosis, and hyperkalemia, and to prevent myoglobinuria-induced renal failure.

Patients with known or suspected MH susceptibility who require surgery can be safely administered regional anesthesia (eg, spinal, epidural, or peripheral nerve block) with local anesthetics, or general anesthesia with nontriggering agents. These include barbiturates, benzodiazepines, opioids, propofol, etomidate, ketamine, nitrous oxide, and nondepolarizing neuromuscular blockers. Disadvantages to using these nontriggering agents include an increased time required in the preoperative period to prepare the continuous infusion devices and possibly increased cost, depending on the agent used.

The hypermetabolic syndrome that characterizes MH is caused by an altered regulation and an abnormally increased release of calcium from the sarcoplasmic reticulum into the sarcoplasm (Figure 1). The associated increased amounts of intracellular calcium result in an increase in oxygen consumption and anaerobic metabolism and cause the muscle rigidity and the clinical scenario described above.^{4 - 5} Calcium transport from the sarcoplasmic reticulum into the sarcoplasm is an integral component of the normal excitation-contraction process and is mediated by the ryanodine receptor, isoform 1 (RYR1).⁶ The calcium-regulating channel of RYR1 is located in the membrane of the sarcoplasmic reticulum and is directly influenced by depolarization via the transverse tubule system, which causes a structural change of the voltage-sensitive L-type (dihydropyridine) calcium channel.⁷ RYR1 is closely associated with other proteins that may influence calcium regulation, and include FK-506 binding protein and triadin. However, only RYR1 and dihydropyridine have been directly implicated in the pathophysiology of the MH process.

Inherited susceptibility to MH is conferred by an abnormal skeletal muscle RYR1 or associated structure that allows abnormal calcium release whenexposed to an anesthetic triggering agent.⁸ The mechanism by which an anesthetic triggering agent causes the abnormal calcium release that initiates the acute MH crisis is unknown, but prolonged RYR1 channel opening has been demonstrated in an experimental model.⁹ Dantrolene sodium, the treatment for MH, is known to inhibit calcium release via RYR1 antagonism.¹⁰

The most widely used and most sensitive method for determining whether an individual is susceptible to MH is the caffeine-halothane contracture test (CHCT).¹¹ The sensitivity of the CHCT is at least 97%, but the specificity is lower—up to 22% of patients may have a false-positive test.¹² The CHCT consists of excising a piece of skeletal muscle from the thigh and determining its contractile properties when exposed to the ryanodine receptor agonists halothane and caffeine. Abnormally high levels of contractile force (ie, contracture) are indicative of MH susceptibility. The CHCT is reserved for patients with a suspected MH event and for family members of known MH patients. However, the test is only available at 6 medical centers in the United States and 2 in Canada¹³ and, therefore, is unavailable to many patients. Furthermore, the cost of the CHCT is approximately $6000, which may or may not be reimbursed by third-party payers.

A slightly different protocol for testing MH susceptibility evolved in Europe and is referred to as the in vitro contracture test.¹⁴ The in vitro contracture test protocol is associated with a higher specificity (94%) and sensitivity (99%)¹¹ than the North American protocol. These reported differences are thought to be the result of greater methodological consistency between participating European centers than was in place among North American centers at the time that the published data were collected. Updated data for the aggregate of the North American centers have not been since obtained.

The decision to proceed with muscle contracture testing depends on a variety of factors including the features of the suspected MH event, family history, patients’ willingness to subject themselves to an additional surgical procedure, as well as the distance from the testing center. Some patients with suspected MH susceptibility will decline CHCT and consider themselves and their family members MH susceptible for all future anesthetics. However, many others will choose to undergo the muscle biopsy for a variety of personal reasons, including the establishment of a definitive diagnosis (especially if a myopathy is being considered), future possibility of serving in the military, unwillingness to limit their potential future options for anesthetic management, or for limiting their exposure to heat stress or vigorous exercise, which have been associated with acute MH symptoms in some susceptible individuals. Additional patients may want a definitive diagnosis for the benefit of their offspring.

After the discovery of MH-associated mutations in certain breeds of swine, researchers performed genetic linkage studies on human families with known MH susceptibility to discover analogous mutations.¹⁵ To date, 2 MH susceptibility genes have been identified and 4 have been mapped to specific chromosomes ( Article ) but have not been definitively identified. Depending on the population studied, up to 50% of these mutations can be mapped to the gene on chromosome 19q13.1 that encodes for RYR1.²⁵ Dihydropyridine receptor mutations have also been implicated in the pathophysiology of MH.^{23 ,26}

MHS1
MHS1 mutations are associated with the RYR1 gene on chromosome 19q13.1.^{16 - 17} These account for the majority of currently described MH-associated mutations.

MHS2
MHS2 mutations are found in North American and southern African families and have been linked to chromosomal locus 17q11.2-q24, which is associated with the voltage-dependent sodium channel of the skeletal muscle membrane.^{18 - 19}

MHS3
MHS3 mutations have been linked to chromosomal locus 7q21-q22, the site of coding for the α₂/δ subunit of the dihydropyridine receptor, the T-tubule bound voltage sensor for RYR1.²⁰ However, causative genes have yet to be located.

MHS4
MHS4 mutations have been linked to chromosomal locus 3q13.1.²¹ Like MHS3, the causative genes have yet to be located.

MHS5
MHS5 mutations are associated with the gene encoding the α₁ subunit of the dihydropyridine receptor at chromosomal locus 1q32,²² and they account for 1% of all MHS cases.²³

MHS6
MHS6 mutations are linked to chromosomal locus 5p²⁴ without a known causative gene. Evidence for the MHS6 locus is weak, and its validity remains to be confirmed.

As MH-susceptible families have been studied, additional mutation loci have been found and classified by a numbering system according to their general location using the prefix “MHS” (Box 2).

MH-associated mutations are frequently unique and confined to single families or kindred. To date, more than 60 mutations in RYR1 have been associated with an abnormal contracture test or an irrefutable clinical episode of MH.²⁷ Additional mutations in less investigated regions of the RYR1 or in other genes must exist to account for the remainder of MH-susceptible patients. Although MH-susceptible mutations tend to cluster in certain areas of the gene, the search for additional mutations by linkage analysis is made difficult by the extremely large size of the RYR1 gene, which contains 106 exons consisting of 159 000 base pairs and 5038 amino acids.²⁸ Furthermore, the RYR1 gene has at least 16 polymorphisms in the coding region¹⁵ and more than half of the RYR1 mutations are private.

The majority of MH-related mutations in the RYR1 gene are located in 1 of 3 hot spots: a region close to the N-terminus clustered between amino acid residues 35 and 614 (MH region 1); a central region (within the sarcoplasmic reticulum membrane) between amino acid residues 2163 and 2458 (MH region 2); and a region in the C-terminal transmembrane region, between amino acid residues 4643 and 4898. The N-terminus and central regions of the RYR1 gene appear to be involved in interdomain interactions responsible for the closed state of the receptor (ie, inhibiting calcium flow). Mutations in this area that unzip the interacting domains may be responsible for abnormally elevated calcium flow through the receptor channel when appropriately activated by triggering agents.²⁹ Nevertheless, altered calcium regulation will result from any MH-associated RYR1 mutation regardless of its location within the different hot spots.³⁰ Malignant hyperthermia–susceptible individuals without an overt myopathy are likely to have mutations at the N-terminus, whereas those patients with a clinical myopathy (ie, central core disease) are likely to have mutations at the C-terminus.^{31 - 32}

Mutation analysis is now available on a clinical basis in the United States. A DNA sample for this test can be obtained from buccal cells, white blood cells, muscle cells, or other tissue. The North American mutation analysis protocol currently screens for 17 of the most common RYR1 mutations, and, at the present time, it is expected to detect up to 25% of patients with MH susceptibility, depending on the population studied.²⁷

RYR1 analysis will likely be recommended for (1) patients with a positive CHCT or in vitro contracture test result; (2) relatives of patients with known MH susceptibility by contracture test or RYR1 mutation; and (3) patients or their relatives without CHCT who experienced a highly suspicious clinical episode of MH. Patients with evidence of possible MH, such as masseter muscle rigidity, high fever during or immediately following a general anesthetic, or unexpected rhabdomyolysis during or following a general anesthetic, and surviving heat stroke victims with a family history of MH susceptibility are potential candidates for genetic testing, which will be made available to patients by referral from a physician or genetic counselor. At the present time, the cost of obtaining the RYR1 screening is approximately $800. However, in family member of probands in whom a mutation is already known, the cost to screen fora particular mutation is approximately $200. But, CHCT is still recommended for family members of a proband with a suspicious MH episode but without a known causative mutation.

Absence of the causative mutation in a family member of a proband is not sufficient to rule out MH susceptibility. However, once a mutation is detected in a family, those with the mutation should be considered MH susceptible.

Limitations of genetic testing include the relatively low sensitivity (25%) and the considerable amount of interindividual and intraindividual variability in phenotypic expression of the MH syndrome in individuals known to be susceptible.³³ Some susceptible individuals may not manifest MH-like signs when they are exposed to triggering agents. Furthermore, some individuals known to be MH susceptible based on genetic screening may not demonstrate a positive contracture test.^{34 - 36} This phenomenon, which is known as discordance, indicates the disagreement between the genotypic and phenotypic test results and has been documented by European MH researchers. A multicenter study that included 500 unrelated patients found an overall discordance in approximately 10% of individuals or 2.6% of families referred for genetic diagnostic studies.³³ Similar studies have not been performed in MH-susceptible families in North America; therefore, the incidence of discordance in this population is unknown.

The reasons for this discordance are unknown but may be related to the presence of a cosegregating causal mutation, variability of test results between testing laboratories, and certain clinical modulators, such as the fatty acid component of the muscle³⁷ or sodium channel composition.³⁸ Furthermore, the MH mutation may require linkage with additional mutations associated with myopathies, such as central core disease.³⁹

Another major limitation of RYR1 screening analysis is that there are many additional causal mutations (23 identified to date), many of them unique, appearing in only one family. Current techniques of analysis are limited—testing for additional mutations is quite costly with regard to cost and time.

Reports of MH in patients with underlying muscle diseases have led to a greater understanding of the genetic association between MH susceptibility and these entities. Malignant hyperthermia susceptibility is associated with central core myopathy^{25 ,40} and has been documented in patients with multiminicore disease,⁴¹ hypokalemic periodic paralysis,^{42 - 43} heat stroke,^{44 - 45} and exercise-induced rhabdomyolysis.⁴⁶ Neuroleptic malignant syndrome, which may occur when an individual is exposed to an antipsychotic medication, shares many similarities with MH, including the clinical presentation and treatment (ie, dantrolene). However, no known genetic or causal association exists between MH susceptibility and neuroleptic malignant syndrome.

Patients with a dystrophinopathy (eg, Duchenne muscular dystrophy, Becker muscular dystrophy) may often develop life-threatening hyperkalemia and rhabdomyolysis when exposed to anesthetic triggering agents.^{47 - 48} This reaction shares many clinical features with acute MH but the etiology is likely to be different because the gene for the dystrophin protein is located on the X chromosome.

The possibility of an MH-susceptible human developing a fatal MH crisis in the absence of an anesthetic trigger, as occurs in susceptible swine, has been debated for many years. Such deaths have occurred in individuals who harbor a causative mutation^{44 ,49} but are exceedingly rare and are often associated with severe heat stress or exercise intolerance.

A proposed sequence of testing for patients thought to be MH susceptible is presented in Figure 2. At present, the invasive muscle contracture test remains the gold standard for determining MH susceptibility. Molecular genetic testing via RYR1 screening analysis is in its infancy and will become more useful as additional causative mutations are identified and additional exons are included in screening analysis to detect novel mutations. Because the RYR1 gene is extraordinarily large, complete testing of all possible mutation locations is expensive and time consuming. Therefore, additional methods for determining MH susceptibility are being investigated. One method uses nuclear magnetic resonance spectroscopy to evaluate adenosine triphosphate depletion during graded exercise in vivo because MH-susceptible individuals have a greater breakdown of adenosine triphosphate and creatine phosphate as well as an increase in intracellular acid content compared with those who are not susceptible.⁵⁰

Another avenue of investigation involves the insertion of a microdialysis catheter directly into muscle. Injection of caffeine will elicit an enhanced release of carbon dioxide in MH-susceptible individuals in vivo.⁵¹

A third possible method of MH susceptibility testing uses B-lymphocytes, which harbor the RYR1 protein and release calcium when stimulated with caffeine. The B-lymphocytes of MH susceptible patients demonstrate an abnormally enhanced calcium release.⁵² Presently, however, these aforementioned alternative susceptibility tests have not been sufficiently validated to be clinically useful.