C1840-T mutation in the human skeletal muscle ryanodine receptor gene: frequency in northern German families susceptible to malignant hyperthermia and the relationship to in vitro contracture response

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.

Download Full-text

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

Anesthesiology ◽

10.1097/00000542-200208000-00010 ◽

2002 ◽

Vol 97 (2) ◽

pp. 345-350 ◽

Cited By ~ 16

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.

Download Full-text

Functional Studies of RYR1 Mutations in the Skeletal Muscle Ryanodine Receptor Using Human RYR1 Complementary DNA

Anesthesiology ◽

10.1097/aln.0b013e3181d69283 ◽

2010 ◽

Vol 112 (6) ◽

pp. 1350-1354 ◽

Cited By ~ 20

Author(s):

Keisaku Sato ◽

Neil Pollock ◽

Kathryn M. Stowell

Keyword(s):

Skeletal Muscle ◽

Malignant Hyperthermia ◽

Calcium Channel ◽

Ryanodine Receptor ◽

Human Skeletal Muscle ◽

Binding Assays ◽

Complementary Dna ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells

Background Malignant hyperthermia is associated with mutations within the gene encoding the skeletal muscle ryanodine receptor, the calcium channel that releases Ca from sarcoplasmic reticulum stores triggering muscle contraction, and other metabolic activities. More than 200 variants have been identified in the ryanodine receptor, but only some of these have been shown to functionally affect the calcium channel. To implement genetic testing for malignant hyperthermia, variants must be shown to alter the function of the channel. A number of different ex vivo methods can be used to demonstrate functionality, as long as cells from human patients can be obtained and cultured from at least two unrelated families. Because malignant hyperthermia is an uncommon disorder and many variants seem to be private, including the newly identified H4833Y mutation, these approaches are limited. Methods The authors cloned the human skeletal muscle ryanodine receptor complementary DNA and expressed both normal and mutated forms in HEK-293 cells and carried out functional analysis using ryanodine binding assays in the presence of a specific agonist, 4-chloro-m-cresol, and the antagonist Mg. Results Transiently expressed human ryanodine receptor proteins colocalized with an endoplasmic reticulum marker in HEK-293 cells. Ryanodine binding assays confirmed that mutations causing malignant hyperthermia resulted in a hypersensitive channel, while those causing central core disease resulted in a hyposensitive channel. Conclusions The functional assays validate recombinant human skeletal muscle ryanodine receptor for analysis of variants and add an additional mutation (H4833Y) to the repertoire of mutations that can be used for the genetic diagnosis of malignant hyperthermia.

Download Full-text

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

Clinical Chemistry ◽

10.1373/49.5.761 ◽

2003 ◽

Vol 49 (5) ◽

pp. 761-768 ◽

Cited By ~ 31

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.

Download Full-text

Treatment of Normal Skeletal Muscle with FK506 or Rapamycin Results in Halothane-induced Muscle Contracture

Anesthesiology ◽

10.1097/00000542-199809000-00020 ◽

1998 ◽

Vol 89 (3) ◽

pp. 693-698. ◽

Cited By ~ 8

Author(s):

Richard L. Brooksbank ◽

Margaret E. Badenhorts ◽

Hyam Isaacs ◽

Nerina Savage

Keyword(s):

Carbon Dioxide ◽

Skeletal Muscle ◽

Malignant Hyperthermia ◽

Ryanodine Receptor ◽

Human Skeletal Muscle ◽

Receptor Type ◽

Muscle Strip ◽

Healthy Human ◽

Healthy Persons

Background FKBP12 is a protein that is closely associated with the ryanodine receptor type 1 of skeletal muscle and modulates Ca2+ release by the channel. The immunosuppressants FK506 and rapamycin both bind to FKBP12 and in turn dissociate the protein from the ryanodine receptor. By treating healthy human skeletal muscle strips with FK506 or rapamycin and then subjecting the strips to the caffeine-halothane contracture test, this study determined that FK506 and rapamycin alter the sensitivity of the muscle strip to halothane, caffeine, or both. Methods Skeletal muscle strips from 10 healthy persons were incubated in Krebs medium equilibrated with a 95% oxygen and 5% carbon dioxide mixture, which contained either 12 microM FK506 (n = 8) or 12 microM rapamycin (n = 6), for 15 min at 37 degrees C. The strips were subjected to the caffeine-halothane contracture test for malignant hyperthermia according to the European Malignant Hyperthermia Group protocol. Results Treatment of normal skeletal muscle strips with FK506 and rapamycin resulted in halothane-induced contractures of 0.44+/-0.16 g and 0.6+/-0.49 g, respectively, at 2% halothane. Conclusions The results obtained show that pre-exposure of healthy skeletal muscle strips to either FK506 or rapamycin is sufficient to give rise to halothane-induced contractures. This is most likely caused by destabilization of Ca2+ release by the ryanodine receptor as a result of the dissociation of FKBP12. This finding suggests that a mutation in FKBP12 or changes in its capacity to bind to the ryanodine receptor could alter the halothane sensitivity of the skeletal muscle ryanodine receptor and thereby predispose the person to malignant hyperthermia.

Download Full-text

Cosegregation of porcine malignant hyperthermia and a probable causal mutation in the skeletal muscle ryanodine receptor gene in backcross families

Genomics ◽

10.1016/0888-7543(91)90083-q ◽

1991 ◽

Vol 11 (3) ◽

pp. 744-750 ◽

Cited By ~ 92

Author(s):

Kinya Otsu ◽

Vijay K. Khanna ◽

Alan L. Archibald ◽

David H. MacLennan

Keyword(s):

Skeletal Muscle ◽

Malignant Hyperthermia ◽

Ryanodine Receptor ◽

Receptor Gene ◽

Causal Mutation

Download Full-text

Increased sensitivity of the ryanodine receptor to halothane-induced oligomerization in malignant hyperthermia-susceptible human skeletal muscle

Journal of Applied Physiology ◽

10.1152/japplphysiol.00537.2003 ◽

2004 ◽

Vol 96 (1) ◽

pp. 11-18 ◽

Cited By ~ 5

Author(s):

Louise Glover ◽

James J. A. Heffron ◽

Kay Ohlendieck

Keyword(s):

Skeletal Muscle ◽

Sarcoplasmic Reticulum ◽

Malignant Hyperthermia ◽

Ryanodine Receptor ◽

Human Skeletal Muscle ◽

Dihydropyridine Receptor ◽

Threshold Concentration ◽

Release Channel ◽

Functional Dynamics ◽

Increased Sensitivity

Mutations in the skeletal muscle RyR1 isoform of the ryanodine receptor (RyR) Ca2+-release channel confer susceptibility to malignant hyperthermia, which may be triggered by inhalational anesthetics such as halothane. Using immunoblotting, we show here that the ryanodine receptor, calmodulin, junctin, calsequestrin, sarcalumenin, calreticulin, annexin-VI, sarco(endo)plasmic reticulum Ca2+-ATPase, and the dihydropyridine receptor exhibit no major changes in their expression level between normal human skeletal muscle and biopsies from individuals susceptible to malignant hyperthermia. In contrast, protein gel-shift studies with halothane-treated sarcoplasmic reticulum vesicles from normal and susceptible specimens showed a clear difference. Although the α2-dihydropyridine receptor and calsequestrin were not affected, clustering of the Ca2+-ATPase was induced at comparable halothane concentrations. In the concentration range of 0.014–0.35 mM halothane, anesthetic-induced oligomerization of the RyR1 complex was observed at a lower threshold concentration in the sarcoplasmic reticulum from patients with malignant hyperthermia. Thus the previously described decreased Ca2+-loading ability of the sarcoplasmic reticulum from susceptible muscle fibers is probably not due to a modified expression of Ca2+-handling elements, but more likely a feature of altered quaternary receptor structure or modified functional dynamics within the Ca2+-regulatory apparatus. Possibly increased RyR1 complex formation, in conjunction with decreased Ca2+ uptake, is of central importance to the development of a metabolic crisis in malignant hyperthermia.

Download Full-text

Analysis of expression of the human ryanodine receptor gene in malignant hyperthermia skeletal muscle tissue

Biochemical Society Transactions ◽

10.1042/bst019046s ◽

1991 ◽

Vol 19 (1) ◽

pp. 46S-46S ◽

Cited By ~ 3

Author(s):

C. M. Gillian ◽

J.J.A. Heffron ◽

M. Lehane ◽

A. Marks ◽

T.V. McCarthy

Keyword(s):

Skeletal Muscle ◽

Malignant Hyperthermia ◽

Ryanodine Receptor ◽

Muscle Tissue ◽

Receptor Gene ◽

Skeletal Muscle Tissue

Download Full-text

Altered binding site for Ca2+ in the ryanodine receptor of human malignant hyperthermia

AJP Cell Physiology ◽

10.1152/ajpcell.1991.261.2.c237 ◽

1991 ◽

Vol 261 (2) ◽

pp. C237-C245 ◽

Cited By ~ 30

Author(s):

H. H. Valdivia ◽

K. Hogan ◽

R. Coronado

Keyword(s):

Skeletal Muscle ◽

Malignant Hyperthermia ◽

Binding Site ◽

Ryanodine Receptor ◽

Human Skeletal Muscle ◽

Scatchard Analysis ◽

Dependent Manner ◽

Binding Properties ◽

Release Channel ◽

Planar Bilayers

The binding properties of [3H]ryanodine, a specific ligand of the receptor complex that forms the Ca2+ release channel of sarcoplasmic reticulum, were studied in normal (N) and malignant hyperthermia-susceptible (MH) human skeletal muscle. Integrity of the solubilized ryanodine receptor was demonstrated by single-channel recordings in planar bilayers and by the changes produced by activators and inhibitors of the Ca2+ release channel on the binding properties of [3H]ryanodine. N and MH receptors were capable of binding [3H]ryanodine in a Ca(2+)-dependent manner. Scatchard analysis showed that a single binding site for [3H]ryanodine was present in either N or MH muscle. Binding affinity was approximately the same in N and MH (Kd approximately 7 nM), when the Ca2+ concentration was greater than 30 microM. At 0.3 microM Ca2+, MH receptors displayed a higher affinity for [3H]ryanodine (Kd = 4.1 +/- 1.0 nM) than N receptors (Kd = 7.1 +/- 0.8 nM). The presence of a single Kd for [3H]ryanodine in MH muscle, distinct from that of N muscle, indicated that MH muscle does not have detectable levels of N receptors. Ca2+ dependence of [3H]ryanodine binding further suggested that MH receptors had a higher affinity for Ca2+ (Kd[Ca2+] = 120 +/- 50 nM) than N receptors (Kd[Ca2+] = 250 +/- 80 nM). Caffeine increased [3H]ryanodine binding at submicromolar [Ca2+], and the effect was larger in MH. Apparent affinity constants for caffeine were 13 +/- 1.8 mM in N and 6 +/- 0.8 mM in MH receptors. Evidently, the ryanodine receptor of MH-susceptible human skeletal muscle has an unusually high sensitivity to Ca2+ which is augmented by caffeine.(ABSTRACT TRUNCATED AT 250 WORDS)

Download Full-text