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Malignant hyperthermia (MH) in man is an autosomal dominant disorder of skeletal muscle Ca(2+)-regulation. During anesthesia in predisposed individuals, it is triggered by volatile anesthetics and depolarizing muscle relaxants. In >50% of the families, MH susceptibility is linked to the gene encoding the skeletal muscle ryanodine receptor (RYR1), the calcium release channel of the sarcoplasmic reticulum, on chromosome 19q12-13.2. To date, 21 RYR1 mutations have been identified in a number of pedigrees. Four of them are also associated with central core disease (CCD), a congenital myopathy. Screening for these 21 mutations in 105 MH families including 10 CCD families phenotyped by the in vitro contracture test (IVCT) according to the European protocol revealed the following approximate distribution: 9% Arg-614-Cys, 1% Arg-614-Leu, 1% Arg-2163-Cys, 1% Val-2168-Met, 3% Thr-2206-Met and 7% Gly-2434-Arg. In one CCD family, the disease was caused by a recently reported MH mutation, Arg-2454-His. Two novel mutations, Thr-2206-Arg and Arg-2454-Cys were detected, each in a single pedigree. In the 109 individuals of the 25 families with RYR1 mutations cosegregation between genetic result and IVCT was almost perfect, only three genotypes were discordant with the IVCT phenotypes, suggesting a true sensitivity of 98.5% and a specificity of minimally 81.8% for this test. Screening of the transmembraneous region of RYR1 did not yield a new mutation confirming the cytosolic portion of the protein to be of main functional importance for disease pathogenesis.

Malignant hyperthermia is an inherited autosomal disorder of skeletal muscle in which certain volatile anesthetics and depolarizing muscle relaxants trigger an abnormally high release of Ca 2؉ from the intracellular Ca 2؉ store, the sarcoplasmic reticulum. In about 50% of cases, malignant hyperthermia susceptibility is linked to the gene encoding the skeletal muscle ryanodine receptor/Ca 2؉ release channel (RYR1). To date, eight point mutations have been identified in human RYR1. Although these mutations are thought to lead to an increased caffeine and halothane sensitivity in the contractile response of skeletal muscle, their functional consequences have not been investigated on the molecular level. In the present study, we provide the first functional characterization of a point mutation located in the central part of RYR1, Gly 2434 3 Arg. Using high affinity [3 H]ryanodine binding as the experimental approach, we show that this mutation enhances the sensitivity of RYR1 to activating concentrations of Ca 2؉ and to the exogenous and diagnostically used ligands caffeine and 4-chloro-m-cresol. In parallel, the sensitivity to inhibiting concentrations of Ca 2؉ and calmodulin was reduced, transferring the mutant Ca 2؉ release channel into a hyperexcitable state. Malignant hyperthermia (MH)1 is a pharmacogenetic skeletal myopathy of humans and swine and is one of the main causes of death due to anesthesia. Predisposed patients are at high risk for undergoing a fulminant MH crisis when exposed to certain volatile anesthetics and depolarizing muscle relaxants commonly used in anesthesia. A point mutation (Arg 615 3 Cys) in the skeletal muscle ryanodine receptor (RYR1), which functions as the sarcoplasmic reticulum (SR) Ca 2ϩ release channel, has been linked to porcine stress syndrome, an equivalent to human MH (1). Contrary to porcine stress syndrome, human MH is a genetically heterogeneous skeletal muscle disorder. Based on genetic linkage studies, three MH loci are known. The first has been mapped to chromosome 19q12-13.2 encompassing the gene that in homology with the animal model encodes the RYR1 (2, 3), the second has been mapped to chromosome 7q including the gene for the ␣ 2 /␦ subunit of the skeletal muscle dihydropyridine receptor (4), and the third has been mapped to chromosome 3q13.1 (5). To date, mutations have only been identified in RYR1 that count for approximately 50% of human MH cases (reviewed in Ref. 6). MH mutations seem to cluster in two areas of the RYR1 sequence. Six mutations have been localized in the N-terminal sequence of RYR1 containing a homologous mutation to that identified in porcine MH-susceptible (MHS) muscle. Two further mutations have been found in the central amino acid sequence. In vitro, all these mutations induce a hypersensitivity of biopsied muscle to the contracture-triggering agents caffeine and halothane. This enhanced sensitivity is exploited in the diagnostic in vitro contracture test (IVCT). According to the European test protocol (7), dissected muscle fiber bundles are ...

Peripheral blood cell DNA from a patient with a chronic myeloproliferative disorder was tested in the tumorigenicity assay. Upon tumor induction in nude mice we isolated a human oncogene by means of genomic cloning, exon trap analysis and cDNA cloning. Sequence analysis revealed a fusion product of the p85b subunit of phosphatidylinositol (PI) 3-kinase and HUMORF8, a putative deubiquitinating enzyme, which has been generated during the DNA transfection process. Application of the tumorigenicity assay to various p85b and HUMORF8 cDNA constructs indicated that the recombination of both genes rather than the truncation of one of the fusion partners renders the chimeric protein tumorigenic. Moreover, sequence analysis of human wildtype p85b revealed an alanine for serine substitution at a site important for the regulation of the lipid kinase activity of PI 3-kinase in human p85a. This variation may relate to di erences in the mode of signal transduction from both p85 isoforms.