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Background To determine whether malignant hyperthermia (MH) susceptibility in a Canadian pedigree is associated with a mutation in the ryanodine receptor subtype 1 (RYR1) gene, the complete RYR1 transcript obtained from the leukocytes of one MH-susceptible family member was sequenced, using a newly developed protocol. Methods RNA was extracted from leukocytes and converted into complementary DNA. Overlapping fragments of RYR1 complementary DNA were amplified by the polymerase chain reaction and used for double-strand sequencing to find a single mutation likely to be causal of MH susceptibility. Inheritance of the mutation in the family was studied by restriction endonuclease analysis and/or sequencing of genomic DNA and compared to available caffeine halothane contracture test data. The mutation was introduced into rabbit RYR1 complementary DNA, the complementary DNA was expressed in human embryonic kidney line 293 cells, and Ca2+ release by the mutant Ca2+ release channel was measured following the addition of caffeine and halothane. Results A novel arginine 328 to tryptophan mutation in RYR1 was detected by direct sequencing of the RYR1 transcript from leukocytes of one MH-susceptible individual. A causal role for this mutation in MH is indicated by cosegregation of the mutation with the MH-susceptible phenotype within the family and by the demonstration that the mutant channel has increased sensitivity to both caffeine and halothane. Conclusions The feasibility of using complete RYR1 transcripts from leukocytes for sequence analysis offers an efficient and noninvasive method for scanning RYR1 for novel mutations.

Background Malignant hyperthermia (MH) is an important cause of anesthesia-induced death. Malignant hyperthermia susceptibility is diagnosed using the in vitro caffeine/halothane contracture test (CHCT) in fresh muscle biopsy specimens. The CHCT test is highly invasive, expensive, and lacks 100% specificity. Genetic and biochemical evidence provide strong support for the view that the substitution of cysteine for arginine 614 (Arg614Cys) in the human ryanodine receptor gene is one of several mutations that are likely to cause human MH. DNA testing was compared with CHCT as a means of predicting MH susceptibility in a large MH family in which the Arg614Cys mutation was detected. Methods A comparison of CHCT and DNA-based diagnosis was conducted in a large Manitoba Mennonite MH kindred identified by an index patient who died at age 45 yr of an MH crisis after general anesthesia. The presence of the Arg614Cys mutation was detected through a combination of polymerase chain reaction and restriction endonuclease digestion. Blood samples for DNA analysis were obtained from 68 family members, including 19 who had undergone muscle biopsies and 1 who had a documented crisis but did not undergo biopsy. Family members were classified as MH-susceptible or MH-normal on the basis of the CHCT. Results Twenty-two persons were found to be heterozygous for the Arg614Cys mutation. Five of these persons had prior positive CHCT results and one had an MH crisis but did not undergo biopsy. On DNA testing, 44 persons were found to be homozygous for the normal allele. Of these, ten had been classified as MH-normal and five as MH-susceptible on the basis of the CHCT. On reevaluation of the data obtained in our earlier CHCT diagnoses, we found that the condition of the muscle was poor, with no twitch, for three of five individuals homozygous for the normal allele but originally classified as MH-susceptible and for one who was homozygous for the normal allele and originally classified as MH-normal. Caffeine/halothane contracture test results for these four persons were considered invalid. The twitch response was good for the two remaining persons who were homozygous for the normal allele but classified as MH- susceptible, because contracture was observed with appropriately low levels of both caffeine and halothane. Conclusions An absolute correlation between DNA test results and CHCT assignment could not be made in this kindred. Possible explanations for discordance are that the Arg614Cys mutation is not linked to MH, that a second MH mutation is segregating in the family, or that there are errors in the CHCT. Because there is strong evidence supporting the causal nature of the Arg614Cys mutation, the discordant persons are not closely related within the pedigree as they would be if a second MH mutation were segregating, and the CHCT is not 100% accurate, we propose that the observed discordance between DNA test results and CHCT assignment in this kindred results from two false-positive diagnoses by the CHCT.