CAFFEINE-HALOTHANE contracture testing (CHCT) of fresh, surgically removed skeletal muscle has been the basis for identifying individuals who are susceptible to malignant hyperthermia (MH). CHCT is invasive, expensive, and currently performed at five specialized centers in the United States, two in Canada, and one in Brazil, and has a sensitivity and specificity of 97% and 78%, respectively. 1Similar in vitro contracture testing has been used across Europe to identify MH-susceptible individuals and has sensitivity and specificity values of 99% and 94%, respectively. 2A less invasive, highly sensitive, and specific diagnostic test for MH has been actively sought for many years. Recent studies, coupling functional and genetic causes for MH, have brought genetic testing for this anesthetic-induced, life-threatening disease to the forefront. 3In this issue of the Journal, the report of a September 2002 meeting sponsored by the Malignant Hyperthermia Association of the United States represents an important first step toward a better diagnostic test for MH in North America. 4
Volatile anesthetics are the primary trigger of MH, causing an abnormally increased release of calcium within skeletal muscle cells. 3Mutations in the gene (RYR1) encoding the skeletal muscle calcium release channel (ryanodine receptor protein RyR1), are linked to MH susceptibility in humans, 5,6pigs, 7and dogs. 8In each species, skeletal muscle is characterized by abnormal in vitro contracture responses to caffeine and halothane. The MH syndrome is effectively prevented and treated by dantrolene, which inhibits intracellular Ca2+release from the sarcoplasmic reticulum Ca2+stores by binding to, and thus decreasing, the RyR1 channel open-state probability. 9,10Many RYR1 mutations have been expressed in heterologous systems (myotubes, COS-1, or human embryonic kidney cells) that show enhanced calcium fluxes when treated with RyR1 agonists. 11–13Collectively, this might seem to encompass and resolve the genetic basis for MH, but unfortunately, complicating issues exist. RyR1 is a homotetrameric protein. Each subunit has a molecular weight of 560 kDa (5,038 amino acids), making it one of the largest proteins known. Located on chromosome 19, the RYR1 gene spans 160,000 nucleotide bases, consists of 106 exons; as such, it is one of the most complex human genes. Consequently, most laboratories can only look at small pieces of the RYR1 gene when searching for mutations that might link to MH. Despite these technologic barriers, over 40 different MH-associated RYR1 mutations have been found in three different regions of the gene. Another complicating factor is that MH is genetically heterogenous; i.e. , mutations in RYR1 have not been identified in all MH families. Nevertheless, it is expected that once all mutations in RYR1 are identified, they may account for up to 70% of MH among all susceptible families. 14As for the other non-RYR1 genes associated with MH, mutations in the gene encoding the alpha subunit of the dihydropyridine receptor have been reported, but these seem to be very rare. 15,16Five other chromosomal loci (17q21–24, 1q32, 3q13, 7q21–24, and 5p) have linkage to MH, but the genes are not yet identified.
This Editorial View accompanies the following report of a scientific meeting: Sei Y, Sambuughin N, Muldoon S: Malignant Hyperthermia Genetic Testing in North America Working Group Meeting. Anesthesiology 2004; 100:464–5.
Diagnostic genetic screening for MH was initiated in Europe over 2 yr ago. The European Malignant Hyperthermia Group established guidelines for RYR1 mutation screening with 15 causative RYR1 mutations selected for initial testing. 17,18The first step is identification of an MH-susceptible individual using the validated in vitro contracture test. Screening with a panel of 15 different RYR1 mutations follows. If an RYR1 mutation is detected, then other first-degree relatives of that individual can be tested; those in whom the mutation is found are diagnosed as having MH without undergoing in vitro contracture testing. However, if the particular familial mutation is not found, the muscle (in vitro ) contracture test is required for MH diagnosis. This policy avoids false-negative diagnoses. In one European center, introduction of genetic testing allowed the diagnosis of MH susceptibility to be confirmed in approximately 50% of the proband’s relatives. 14
The search for a less invasive method than CHCT to diagnose MH has been ongoing for many years. Many approaches have been tried, but none have supplanted the muscle contracture test. 19Newer tests based on advances in molecular genetics and cellular physiology have the potential to be effective. The newer tests include measurements in Ca2+fluxes studied either in cultured skeletal muscle cells or in lymphoblastoid cells naturally expressing RYR1. Censier et al. 20reported enhanced intracellular calcium release from muscle cultured from MH-susceptible patients. Sei et al. 21,22identified and characterized the RYR1 in human B-lymphocytes and reported that Ca2+release induced by caffeine and 4-chloro-m-cresol was greater in cells from individuals susceptible to MH than in normal individuals or patients testing negative for CHCT. Also, Loke et al. 23recently demonstrated that direct sequencing of RYR1 transcripts from viable leukocytes could be used to analyze the complete RYR1 in blood samples. Further studies are required to determine the diagnostic potential of these tests.
Another approach has been the use of nuclear magnetic resonance spectroscopy to noninvasively measure adenosine triphosphate, pH, creatine phosphate, and other high-energy phosphates. 24With exercise, MH-susceptible individuals demonstrate greater depletion of high-energy phosphates and a decrease in pH compared to people without MH. Yet other investigators have shown that in vivo microinjection of caffeine in muscle produces an increase in carbon dioxide output and hydrogen ion production in MH-susceptible indiviuals. 25A multicenter study to evaluate this test in a larger number of patients is in the planning stages in European MH centers.
The disadvantages of contracture testing are that CHCT must be performed on fresh, surgically removed skeletal muscle (usually vastus lateralis), and total costs at one of the MH diagnostic centers (including testing, anesthesia, preoperative surgical assessment, and hospital charges) can range from $5,000 to $6,000. With the reduced number of MH testing centers, patients can incur losses in time, travel, and housing costs. In 2002, the North American MH Genetics Group 4developed guidelines for genetic MH diagnosis, taking advantage of the European Malignant Hyperthermia Group model. In addition, for the past 5 yr, the North American MH group’s active research program has screened patients diagnosed as having MH by CHCT for RYR1 mutations and has found most results to be consistent with the European data. 26However, some mutations appear to be specific to the North American population. 27,28On the basis of these results, the North American MH Genetics Group has identified the priorities for initial MH genetic screening. The panel of 17 RYR1 mutations proposed at the recent genetic workshop will be used. This panel will be updated as new causative mutations are discovered. Families to be tested must be identified by a CHCT-positive result or by a strong history for MH and will be referred from a MH Diagnostic Center. The North American MH Registry database can be used to identify potential families and to maintain the results of genetic testing. Samples of DNA (blood or buccal cells) will be obtained and sent to the genetic testing laboratory. The genetic MH testing laboratory must be a Clinical Laboratory Improvement Act–certified laboratory to receive Medicare and Medicaid payments. For each family, initial testing will involve screening for the 17 mutations recommended in individuals determined to have MH and will be the most costly (estimates are unavailable). Once a mutation is identified in the affected member, then family members will be offered testing for the presence of the family-specific mutation. Mutation-positive members would be regarded as MH-positive without further CHCT testing, and the cost will be considerably less. To avoid the danger of a false-negative diagnosis, it will remain necessary to continue performance of CHCT for diagnosis of those family members who do not carry the familial RYR1 mutation. The initial genetic screening for MH will be limited by low sensitivity because the recommended panel of mutations does not cover all potential mutations. At this time, it is not practical to screen the entire RYR1 gene or all RYR1 mutations in each individual with MH. However, the panel of 17 mutations having the highest frequency of occurrence among North American MH families is a starting point. This step introduces new diagnostic tools to the MH centers and, in those MH-positive families in which a causative RYR1 mutation is identified, many individuals will be spared the expensive and invasive CHCT test. As is standard practice in the diagnosis of other genetic diseases, genetic counseling will be necessary; initially, this may be performed through the MH Diagnostic Center from which the patient was referred. Future developments of MH genetic screening will be documented on the Web site of the Malignant Hyperthermia Association of the United States. §