scholarly journals Scanning for Mutations of the Ryanodine Receptor (RYR1) Gene by Denaturing HPLC: Detection of Three Novel Malignant Hyperthermia Alleles

2003 ◽  
Vol 49 (5) ◽  
pp. 761-768 ◽  
Author(s):  
Angela Tammaro ◽  
Adele Bracco ◽  
Santolo Cozzolino ◽  
Maria Esposito ◽  
Antonietta Di Martino ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Malignant Hyperthermia ◽  
Ryanodine Receptor ◽  
Sudden Increase ◽  
Gene Encoding ◽  
In Vitro Contracture Test ◽  
Ryr1 Gene ◽  
Molecular Tests ◽  
Anesthetic Agents

Abstract Background: Malignant hyperthermia (MH) is a fatal autosomal dominant pharmacogenetic disorder characterized by skeletal muscle hypertonicity that causes a sudden increase in body temperature after exposure to common anesthetic agents. The disease is genetically heterogeneous, with mutations in the gene encoding the skeletal muscle ryanodine receptor (RYR1) at 19q13.1 accounting for up to 80% of the cases. To date, at least 42 RYR1 mutations have been described that cause MH and/or central core disease. Because the RYR1 gene is huge, containing 106 exons, molecular tests have focused on the regions that are more frequently mutated. Thus the causative defect has been identified in only a fraction of families as linked to chromosome 19q, whereas in others it remains undetected. Methods: We used denaturing HPLC (DHPLC) to analyze the RYR1 gene. We set up conditions to scan the 27 exons to identify both known and unknown mutations in critical regions of the protein. For each exon, we analyzed members from 52 families with positive in vitro contracture test results, but without preliminary selection by linkage analysis. Results: We identified seven different mutations in 11 MH families. Among them, three were novel MH alleles: Arg44Cys, Arg533Cys, and Val2117Leu. Conclusion: Because of its sensitivity and speed, DHPLC could be the method of choice for the detection of unknown mutations in the RYR1 gene.

Results of Contracture Tests with Halothane, Caffeine, and Ryanodine Depend on Different Malignant Hyperthermia-associated Ryanodine Receptor Gene Mutations

2002 ◽  
Vol 97 (2) ◽  
pp. 345-350 ◽  
Author(s):  
Marko Fiege ◽  
Frank Wappler ◽  
Ralf Weisshorn ◽  
Mark Ulrich Gerbershagen ◽  
Markus Steinfath ◽  
...  
Keyword(s):  
Malignant Hyperthermia ◽  
Ryanodine Receptor ◽  
Time Course ◽  
Clinical Symptoms ◽  
Receptor Gene ◽  
Gene Mutations ◽  
Onset Time ◽  
Release Channel ◽  
Ryr1 Gene

Background More than 20 mutations in the gene encoding for the ryanodine receptor (RYR1), a Ca2+ release channel of the skeletal muscle sarcoplasmic reticulum, have been found to be associated with malignant hyperthermia (MH). This study was designed to investigate the effects of different mutations in the RYR1 gene on contracture development in in vitro contracture tests (IVCT) with halothane, caffeine, and ryanodine. Methods Ninety-three MH-susceptible (MHS) patients, diagnosed by the standard IVCT with halothane and caffeine, were included in this prospective study. Surplus muscle specimens were used for an IVCT with 1 microm ryanodine. The contracture course during the ryanodine IVCT was described by the attainment of different time points: onset time of contracture and times when contracture reached 2 mN or 10 mN. In addition, all patients were screened for mutations of the RYR1 gene. Results In 36 patients, four different mutations of the RYR1 gene (C487-T, G1021-A, C1840-T, G7300-A) were found. The IVCT threshold concentrations of halothane and caffeine were lower in patients with the C487-T mutation compared with patients without a detected mutation in the RYR1 gene. In the IVCT with ryanodine, contracture levels of 2 mN and 10 mN were reached earlier in muscle specimens from patients with C487-T, C1840-T, and G7300-A mutations compared with specimens from patients with the G1021-A mutation and patients without detected mutation in the RYR1 gene. Conclusions The differences between the groups in the halothane and caffeine IVCT threshold concentrations and in the time course of contracture development in the ryanodine IVCT underline the hypothesis that certain mutations in the RYR1 gene could make the ryanodine receptor more sensitive to specific ligands. This may be an explanation for varying clinical symptoms of MH crisis in humans.

Malignant hyperthermia: excitation-contraction coupling, Ca2+ release channel, and cell Ca2+ regulation defects

1996 ◽  
Vol 76 (2) ◽  
pp. 537-592 ◽  
Author(s):  
J. R. Mickelson ◽  
C. F. Louis
Keyword(s):  
Skeletal Muscle ◽  
Malignant Hyperthermia ◽  
Contractile Activity ◽  
Underlying Mechanism ◽  
Clinical Test ◽  
Release Channel ◽  
Excitation Contraction Coupling ◽  
Contraction Coupling ◽  
Anesthetic Agents

Malignant hyperthermia (MH) is a disorder of skeletal muscle in which certain anesthetic agents trigger a sustained elevation in myoplasmic Ca2+ concentration that activates metabolic and contractile activity. This review focuses on the biochemical and physiological alterations in the skeletal muscle of MH-susceptible (MHS) pigs and humans that appear responsible for this inherited disorder. In porcine MH, these studies identified the skeletal muscle sarcoplasmic reticulum Ca2+ release channel gene (RYR1) as the site of the defect. A mutation in this protein results in altered excitation-contraction coupling and secondary changes in porcine muscle structure and function. Although RYR1 mutations have been reported in many MHS human families, there is also significant genetic heterogeneity, and much less is known as to the underlying mechanism responsible for altered human myoplasmic Ca2+ regulation. The effects of caffeine and anesthetic agents on MHS and normal muscle are also discussed to better understand the basis for the in vitro clinical test for this disorder and mechanisms responsible for the initiation and maintenance of MH episodes in susceptible individuals. Finally, we examine the possiblity of a defect in Ca2+ regulation in tissues other than skeletal muscle. Current understanding of the molecular basis of MH elegantly illustrates the successful integration of knowledge obtained from all fields of biological and clinical science.

Detection of Proton Release from Cultured Human Myotubes to Identify Malignant Hyperthermia Susceptibility

2002 ◽  
Vol 97 (5) ◽  
pp. 1059-1066 ◽  
Author(s):  
Werner Klingler ◽  
Christoph Baur ◽  
Michael Georgieff ◽  
Frank Lehmann-Horn ◽  
Werner Melzer
Keyword(s):  
Skeletal Muscle ◽  
Malignant Hyperthermia ◽  
Metabolic Activation ◽  
Malignant Hyperthermia Susceptibility ◽  
Proton Release ◽  
Release Channel ◽  
Proton Secretion ◽  
In Vitro Contracture Test ◽  
Human Myotubes

Background Malignant hyperthermia (MH) is a pharmacogenetic disorder of skeletal muscle. During general anesthesia, a life-threatening hypermetabolic state may occur resulting from increased release of Ca2+ from the sarcoplasmic reticulum in skeletal muscle. Diagnosis of MH susceptibility requires surgical muscle biopsies to measure force in response to chemical stimulation (in vitro contracture test, IVCT). Here, the authors investigated an alternative way of discriminating MH-susceptible (MHS) from normal (MHN) subjects by using cultured human myotubes and measuring proton release as an indicator of cellular metabolism. Methods Myotubes were stimulated with the Ca2+ release channel agonist 4-chloro-m-cresol (4-CmC), leading to metabolic activation and proton secretion. The rate of extracellular acidification was recorded with a silicon sensor chip. Results A stepwise increase in 4-CmC concentration led to a phasic-tonic increase in the acidification rate. The response, measured at different concentrations of 4-CmC, was considerably larger in cultures from MHS compared with MHN subjects and correlated well with the force response in the IVCT. Conclusions The enhanced metabolism of cultured skeletal myotubes, likely originating from an increased myoplasmic Ca2+ concentration, can be monitored by studying the proton secretion rate. Because the method seems to be able to distinguish normal from pathologic phenotypes, it is a promising technique for possible future use in less invasive MH testing.

Autosomal Dominant Canine Malignant Hyperthermia Is Caused by a Mutation in the Gene Encoding the Skeletal Muscle Calcium Release Channel (RYR1 )

2001 ◽  
Vol 95 (3) ◽  
pp. 716-725 ◽  
Author(s):  
Monica C. Roberts ◽  
James R. Mickelson ◽  
Edward E. Patterson ◽  
Thomas E. Nelson ◽  
P. Jane Armstrong ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Calcium Release ◽  
Autosomal Dominant ◽  
Calcium Release Channel ◽  
Gene Encoding ◽  
Release Channel ◽  
In Vitro Contracture Test ◽  
Mixed Breed ◽  
Muscle Calcium

Background Malignant hyperthermia (MH) is an inherited disorder of skeletal muscle characterized by hypercarbia, rhabdomyolysis, generalized skeletal muscle contracture, cardiac dysrhythmia, and renal failure, that develops on exposure to succinylcholine or volatile anesthetic agents. All swine and up to 50% of human MH events are thought to be associated with mutations in the calcium release channel of the sarcoplasmic reticulum, also known as the ryanodine receptor (RYR1). Events resembling MH have been reported in other species, but none have undergone genetic investigation to date. Methods To determine the molecular basis of canine MH, a breeding colony was established with a male, mixed-breed, MH-susceptible (MHS) dog that survived an in vivo halothane-succinylcholine challenge. He was mated to three unaffected females to produce four litters and back-crossed to an affected daughter to produce one litter. One of his MHS sons was mated to an unaffected female to produce an additional litter. Forty-seven dogs were phenotyped with an in vitro contracture test and diagnosed as MHS or MH normal based on the North American in vitro contracture test protocol. Nine microsatellite markers in the vicinity of RYR1 on canine chromosome 1 (CFA01) were tested for linkage to the MHS phenotype. Mutational analysis in two MHS and two MH-normal dogs was performed with direct sequencing of polymerase chain reaction products and of cloned fragments that represent frequently mutated human RYR1 regions. A restriction fragment length polymorphism was chosen to detect the candidate mutation in the pedigree at large. Results Pedigree inspection revealed that MHS in this colony is transmitted as an autosomal dominant trait. FH2294, the marker closest to RYR1, is linked to MHS at a theta = 0.03 with a LOD score of 9.24. A T1640C mutation gives rise to an alanine for valine substitution of amino acid 547 in the RYR1 protein, generating a maximum LOD score of 12.29 at theta = 0.00. All dogs diagnosed as MHS by in vitro contracture test were heterozygous for the mutation, and all MH-normal dogs were homozygous for the T1640 allele. Conclusions These results indicate that autosomal dominant canine MH is caused by a mutation in the gene encoding the skeletal muscle calcium release channel and that the MHS trait in this pedigree of mixed-breed dogs is in perfect cosegregation with the RYR1 V547A mutation.

scholarly journals Identification and Biochemical Characterization of a Novel Ryanodine Receptor Gene Mutation Associated with Malignant Hyperthermia

2008 ◽  
Vol 108 (2) ◽  
pp. 208-215 ◽  
Author(s):  
Ayuk A. Anderson ◽  
Rosemary L. Brown ◽  
Brenda Polster ◽  
Neil Pollock ◽  
Kathryn M. Stowell
Keyword(s):  
Skeletal Muscle ◽  
Malignant Hyperthermia ◽  
B Lymphocytes ◽  
Ryanodine Receptor ◽  
Calcium Release ◽  
Dna Analysis ◽  
Biochemical Characterization ◽  
Receptor Gene ◽  
Diagnostic Tools

Background Mutations in the skeletal muscle ryanodine receptor gene may result in altered calcium release from sarcoplasmic reticulum stores, giving rise to malignant hyperthermia (MH). MH is a pharmacogenetic skeletal muscle disorder triggered by volatile anesthetics and depolarizing muscle relaxants. Diagnosis of MH is by in vitro contracture testing of quadriceps muscle. DNA analysis of causative mutations is limited by the large number of mutations that cosegregate with MH and the relatively few that have been biochemically characterized. Methods DNA sequence analysis was used to screen the skeletal muscle ryanodine receptor gene in MH-susceptible individuals. A diagnostic test using real-time polymerase chain reaction was developed to detect the mutation in individuals diagnosed as MH susceptible by in vitro contracture testing. The functional relevance of this mutation was examined in Epstein-Barr virus-immortalized B-lymphoblastoid cells. Results A novel ryanodine receptor mutation (cytosine 14997 thymine resulting in a histidine 4833 tyrosine substitution) was identified in pathology specimens from two patients with fatal MH reactions. B lymphocytes from patients with this mutation were approximately twofold more sensitive than MH-negative cells to activation with 4-chloro-m-cresol. The amount of Ca released from B lymphocytes of MH-susceptible patients was significantly greater than that released from cells of family members without this mutation. Haplotype analysis suggests that both families had a common ancestor. Conclusions DNA analysis to detect mutations which cosegregate with MH as well as biochemical assays on cultured lymphocytes obtained from blood can serve as useful diagnostic tools for MH susceptibility and genotype-phenotype correlations.

scholarly journals Therapeutic effects of novel type 1 ryanodine receptor inhibitor on malignant hyperthermia

2021 ◽  
Vol 154 (9) ◽  
Author(s):  
Toshiko Yamazawa
Keyword(s):  
Skeletal Muscle ◽  
Malignant Hyperthermia ◽  
Ryanodine Receptor ◽  
Half Life ◽  
Water Solubility ◽  
Heat Stroke ◽  
Therapeutic Effects ◽  
Ryr1 Gene ◽  
Plasma Half Life

Ca2+-induced Ca2+ release (CICR) is mediated by ryanodine receptors, a Ca2+ release channel in the sarcoplasmic/endoplasmic reticulum (SR/ER), and plays an important role in various tissues. Type 1 ryanodine receptor (RYR1) plays a key role during excitation–contraction coupling of skeletal muscle. Mutations in RYR1 overactivate the channel to cause malignant hyperthermia (MH). MH is a serious complication characterized by skeletal muscle rigidity and elevated body temperature in response to commonly used inhalational anesthetics. Thus far, >300 mutations in the RYR1 gene have been reported in patients with MH. Some heat stroke triggered by exercise or environmental heat stress is also related to MH mutations in the RYR1 gene. The only drug approved for ameliorating the symptoms of MH is dantrolene, which has been first developed in the 1960s as a muscle relaxant. However, dantrolene has several disadvantages for clinical use: poor water solubility, which makes rapid preparation difficult in emergency situations, and long plasma half-life, which causes long-lasting side effects such as muscle weakness. Here, we show that a novel RYR1-selective inhibitor, 6,7-(methylenedioxy)-1-octyl-4-quinolone-3-carboxylic acid (compound 1 [Cpd1]), effectively rescues MH and heat stroke in new mouse model (RYR1-p.R2509C) relevant to MH. Cpd1 has great advantages of higher water solubility and shorter plasma half-life compared with dantrolene. Our data suggest that Cpd1 has the potential to be a promising new candidate for effective treatment of patients carrying RYR1 mutations. Finally, we have recently identified that heat directly activates RYR1, which induces Ca2+ release from intracellular stores. Our results provide direct evidence that heat induces Ca2+ release (HICR) from the SR through the mutants rather than wild type RYR1, causing an immediate rise in the cytosolic Ca2+ concentration.

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