Antonella Carsana

Recent advances in the diagnosis of malignant hyperthermia susceptibility: How confident can we be of genetic testing?

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 ∼30% of families investigated. Phenotype/genotype discordance in a single individual was observed in 10 out of 196 mutation-positive families. In five families a mutation-positive/IVCT-negative individual was observed, and in the other five families a mutation-negative/IVCT-positive individual was observed. These data represent the most comprehensive assessment of RYR1 mutation prevalence and genotype/phenotype correlation analysis and highlight the possible limitations of MH screening methods. The implications for genetic diagnosis are discussed.

Evolution of Vertebrate Ribonucleases: Ribonuclease A Superfamily

Publisher Summary This chapter covers evolution of ribonuclease A superfamily. The pyrimidine-specific ribonuclease A superfamily constitutes a group of homologous proteins with well-characterized and sequenced members isolated from many mammalian, avian, reptilian, and amphibian sources. No representatives have been found in fishes or nonvertebrate taxa. It is speculated that there exists a protein in these more distantly related taxa that has some sequence similarity with members of the ribonuclease A superfamily or has a similar fold of the polypeptide chain without demonstrable ribonuclease activity. Distantly related members of the ribonuclease A superfamily differ at more than 50% of the amino acid positions. The nomenclature of members of the ribonuclease A superfamily is rather chaotic, especially that of paralogous ribonucleases identified in the same species. This chapter discusses about ribonuclease superfamily. It explains concepts related to rodent ribonucleases. It also describes structure, evolution, and enzymatic properties of ribonuclease superfamily. An overview of mammalian ribonucleases 2 is also presented in this chapter. The chapter also elaborates in detail about primate ribonucleases and artiodactyl ribonucleases.

Double-stranded RNA

SummaryHigh molecular weight, fully double-stranded RNA (dsRNA) has been recognized as the genetic material of many plant, animal, fungal, and bacterial viruses (Diplomaviruses); virus-specific dsRNA is also found in cells infected with single-stranded RNA viruses.DsRNA has been identified in a variety of apparently normal eucaryotic cells and is associated with the ‘killer’ character of certain strains of Saccaromyces cerevisiae.The properties and significance of these various dsRNA species are described and discussed, as well as the available information concerning the biosynthesis of such RNA in virus-infected cells, its degradation by a variety of enzymes, and some problems concerning the variables which may control this process.Finally, the biological functions of dsRNA are briefly considered, as well as the structural properties important for its activity as an inducer of interferon and an inhibitor of protein synthesis.

Several interacting genes influence the malignant hyperthermia phenotype

Abstract. Malignant hyperthermia (MH), a potentially lethal disorder of skeletal muscle calcium homeostasis, manifests only on exposure to certain anaesthetic drugs. The mode of inheritance appears to be autosomal dominant with both locus and allelic heterogeneity having been reported. Association analysis of eight MH candidate loci in UK families has indicated that several genes influence susceptibility in individual families, rather than MH simply being a major gene defect. In support of this hypothesis, we present data on a replica analysis of an independent sample of European MH families.

Identification and functional characterization of malignant hyperthermia mutation T1354S in the outer pore of the Cavα1S-subunit

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(2+) channel (Ca(V)) α(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 >178 different positions on the ryanodine receptor (RYR1) gene but only to two on CACNA1S. Sequence analysis of CACNA1S revealed a c.4060A>T transversion resulting in amino acid exchange T1354S in the IVS5-S6 extracellular pore-loop region of Ca(V)α(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 α(1S)T1354S in dysgenic (α(1S)-null) myotubes. Whole cell patch-clamp analysis revealed that α(1S)T1354S produced significantly faster activation of L-type Ca(2+) currents upon 200-ms depolarizing test pulses compared with wild-type GFP-α(1S) (α(1S)WT). In addition, α(1S)T1354S-expressing myotubes showed a tendency to increased sensitivity for caffeine-induced Ca(2+) release and to larger action-potential-induced intracellular Ca(2+) transients under low (≤ 2 mM) caffeine concentrations compared with α(1S)WT. Thus our data suggest that an additional influx of Ca(2+) due to faster activation of the α(1S)T1354S L-type Ca(2+) current, in concert with higher caffeine sensitivity of Ca(2+) release, leads to elevated muscle contraction under pharmacological trigger, which might be sufficient to explain the MHS phenotype.

MOLECULAR EVOLUTION OF GENES ENCODING RIBONUCLEASES IN RUMINANT SPECIES

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.

SEQUENCES RELATED TO THE OX PANCREATIC RIBONUCLEASE CODING REGION IN THE GENOMIC DNA OF MAMMALIAN-SPECIES

Mammalian pancreatic ribonucleases form a family of homologous proteins that has been extensively investigated. The primary structures of these enzymes were used to derive phylogenetic trees. These analyses indicate that the presence of three strictly homologous enzymes in the bovine species (the pancreatic, seminal, and cerebral ribonucleases) is due to gene duplication events which occurred during the evolution of ancestral ruminants.In this paper we present evidence that confirms this finding and that suggests an overall structural conservation of the putative ribonuclease genes in ruminant species.We could also demonstrate that the sequences related to ox ribonuclease coding regions present in genomic DNA of the giraffe species are the orthologues of the bovine genes encoding the three ribonucleases mentioned above.

Analysis of Dystrophin Gene Deletions Indicates that the Hinge III Region of the Protein Correlates with Disease Severity: Dystrophin Gene Deletions and Genotype-phenotype Correlation

We have investigated the frequency of deletions in the dystrophin gene in 108 unrelated Duchenne and Becker muscular dystrophy (DMD/BMD) patients from southern Italy (DMD, n. 47; BMD, n. 61) and identified 89 deletions. The de novo mutation rate (about 30%), and the preferentially maternal origin of deletional mutations, analysed in families in which the maternal grandparents were available or their haplotypes could be unequivocally reconstructed, are in agreement with data reported for other populations. The correlation between BMD phenotype and type of deletion suggests that, in the distal rod domain region, the deletion size may not be as crucial as the particular combination of missing exons. In fact, we provide immunohistochemical and clinical evidence that in‐frame deletion of the hinge III region in the distal rod domain results in a milder phenotype as compared with shorter deletions that do not include the hinge III region. Our data obtained in BMD patients, by confirming inferences arising from minigene transfection experiments in mdx mice, represent an important contribution to gene therapy approaches.

Ionic control of enzymic degradation of double-stranded RNA

The pattern of the degradation of various double-stranded polyribonucleotides by several ribonucleases (bovine RNAase A and its cross-linked dimer, bovine seminal RNAase, and pike-whale pancreatic RNAase) has been studied as a function of ionic strength and pH. It appears that (1) there is no direct correlation between the secondary structure of double-stranded RNA and its resistance against enzymatic breakdown, i.e., the stability of the secondary structure of double-helical RNA is not the main variable in the process. (2) The acstivity responses of the enzymes examined to changes of ionic strength and pH suggest that enzymic degradation of double-stranded RNA is mainly controlled by ion concentration, and that the process may fall within the phenomena interpreted by the theory of the ionic control of biochemical reactions advanced by Douzou and Maurel (Douzou, P. and Maurel, P. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 1013--1015). (3) The activity curves of the enzyme studied show, at a given pH, a shift toward higher ionic strengths as a function of the basicity of the enzyme protein. This finding explains the already observed correlation between number and/or density of positive charges of a ribonuclease molecule and its ability to attack double-stranded RNA in 0.15 M sodium chloride/0.015 M sodium citrate (SSC). (4) A careful analysis of the influence of ionic strength and pH on the reaction appears to be necessary in order to characterize a ribonuclease which shows activity towards double-stranded RNAs, and to allow a meaningful comparison between different enzymes capable of attacking these substrates.

Functional Characterization of Ryanodine Receptor (RYR1) Sequence Variants Using a Metabolic Assay in Immortalized B-Lymphocytes

Mutations in the RYR1 gene are linked to malignant hyperthermia (MH), central core disease and multi‐minicore disease. We screened by DHPLC the RYR1 gene in 24 subjects for mutations, and characterized functional alterations caused by some RYR1 variants. Three novel sequence variants and twenty novel polymorphisms were identified. Immortalized lymphoblastoid cell lines from patients with RYR1 variants and from controls were stimulated with 4‐chloro‐m‐cresol (4‐CmC) and the rate of extracellular acidification was recorded. We demonstrate that the increased acidification rate of lymphoblastoid cells in response to 4‐CmC is mainly due to RYR1 activation. Cells expressing RYR1 variants in the N‐terminal and in the central region of the protein (p.Arg530His, p.Arg2163Pro, p.Asn2342Ser, p.Glu2371Gly and p.Arg2454His) displayed higher activity compared with controls; this could account for the MH‐susceptible phenotype. Cell lines harboring RYR1Cys4664Arg were significantly less activated by 4‐CmC. This result indicates that the p.Cys4664Arg variant causes a leaky channel and depletion of intracellular stores. The functional changes detected corroborate the variants analyzed as disease‐causing alterations and the acidification rate measurements as a means to monitor Ca2+‐induced metabolic changes in cells harboring mutant RYR1 channels.