No abstract

No abstract

Malignant hyperthermia (MH) is a condition that manifests in susceptible individuals only on exposure to certain anaesthetic agents. Although genetically heterogeneous, mutations in the RYR1 gene (19q13.1) are associated with the majority of reported MH cases. Guidelines for the genetic diagnosis for MH susceptibility have recently been introduced by the European MH Group (EMHG). These are designed to supplement the muscle biopsy testing procedure, the in vitro contracture test (IVCT), which has been the only means of patient screening for the last 30 years and which remains the method for definitive diagnosis in suspected probands. Discordance observed in some families between IVCT phenotype and susceptibility locus genotype could limit the confidence in genetic diagnosis. We have therefore assessed the prevalence of 15 RYR1 mutations currently used in the genetic diagnosis of MH in a sample of over 500 unrelated European MH susceptible individuals and have recorded the frequency of RYR1 genotype/IVCT phenotype discordance. RYR1 mutations were detected in up to B30% of families investigated. Phenotype/genotype discordance in a single individual was observed in 10 out of 196 mutation-positive

Pirone A, Schredelseker J, Tuluc P, Gravino E, Fortunato G, Flucher BE, Carsana A, Salvatore F, Grabner M. Identification and functional characterization of malignant hyperthermia mutation T1354S in the outer pore of the Cav␣1S-subunit. Am J Physiol Cell Physiol 299: C1345-C1354, 2010. First published September 22, 2010; doi:10.1152/ajpcell.00008.2010.-To identify the genetic locus responsible for malignant hyperthermia susceptibility (MHS) in an Italian family, we performed linkage analysis to recognized MHS loci. All MHS individuals showed cosegregation of informative markers close to the voltage-dependent Ca 2ϩ channel (CaV) ␣1S-subunit gene (CACNA1S) with logarithm of odds (LOD)-score values that matched or approached the maximal possible value for this family. This is particularly interesting, because so far MHS was mapped to Ͼ178 different positions on the ryanodine receptor (RYR1) gene but only to two on CACNA1S. Sequence analysis of CACNA1S revealed a c.4060AϾT transversion resulting in amino acid exchange T1354S in the IVS5-S6 extracellular pore-loop region of CaV␣1S in all MHS subjects of the family but not in 268 control subjects. To investigate the impact of mutation T1354S on the assembly and function of the excitation-contraction coupling apparatus, we expressed GFP-tagged ␣1ST1354S in dysgenic (␣1S-null) myotubes. Whole cell patch-clamp analysis revealed that ␣1ST1354S produced significantly faster activation of L-type Ca 2ϩ currents upon 200-ms depolarizing test pulses compared with wild-type GFP-␣1S (␣1SWT). In addition, ␣1ST1354S-expressing myotubes showed a tendency to increased sensitivity for caffeine-induced Ca 2ϩ release and to larger action-potential-induced intracellular Ca 2ϩ transients under low (Յ2 mM) caffeine concentrations compared with ␣1SWT. Thus our data suggest that an additional influx of Ca 2ϩ due to faster activation of the ␣1ST1354S L-type Ca 2ϩ current, in concert with higher caffeine sensitivity of Ca 2ϩ release, leads to elevated muscle contraction under pharmacological trigger, which might be sufficient to explain the MHS phenotype. Skeletal muscle EC coupling is known as a signaling mechanism (10) between the sarcolemmal voltage-gated L-type Ca 2ϩ channel and the SR Ca 2ϩ release channel or ryanodine receptor type-1 (RyR1). Membrane depolarization induces conformational changes in the voltage sensing and pore forming Ca V ␣ 1S -subunit of the L-type Ca 2ϩ channel, which opens the RyR1 via protein-protein interaction. Consequently, Ca 2ϩ released from the SR stores activates the contractile apparatus of the muscle fibers. Genes encoding the key proteins of the EC coupling machinery are the molecular suspects for MH susceptibility. Linkage studies established the RyR1 gene (RYR1) as the main relevant gene (23, 24) that accounts for MHS in Ͼ50% of MH families. So far, a genome-wide search has identified additional MHS loci on chromosomes 1q32 (26) (MHS5, OMIM 601887) where the gene coding for the Ca V ␣ 1S -subunit is located, 7q21-22 (18) (MHS3, OMIM 154276) the gene lo...

Phylogenetic analysis, based on the primary structures of mammalian pancreatic-type ribonucleases, indicated that gene duplication events, which occurred during the evolution of ancestral ruminants, gave rise to the three paralogous enzymes present in the bovine species. Herein we report data that demonstrate the existence of the orthologues of the bovine pancreatic, seminal, and cerebral ribonucleases coding sequences in the genomes of giraffe and sheep. The "seminal" sequence is a pseudogene in both species. We also report an analysis of the transcriptional expression of ribonuclease genes in sheep tissues. The data presented support a model for positive selection acting on the molecular evolution of ruminant ribonuclease genes.