Screening of the ryanodine receptor gene in 105 malignant hyperthermia families: novel mutations and concordance with the in vitro contracture test

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.

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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

Journal of Molecular Medicine ◽

10.1007/bf00203617 ◽

1995 ◽

Vol 73 (1) ◽

pp. 35-40 ◽

Cited By ~ 9

Author(s):

M. Steinfath ◽

S. Singh ◽

J. Scholz ◽

K. Becker ◽

C. Lenzen ◽

...

Keyword(s):

Skeletal Muscle ◽

Malignant Hyperthermia ◽

Ryanodine Receptor ◽

Gene Frequency ◽

Human Skeletal Muscle ◽

Receptor Gene ◽

The Relationship

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Evidence for Susceptibility to Malignant Hyperthermia in Patients with Exercise-induced Rhabdomyolysis

Anesthesiology ◽

10.1097/00000542-200101000-00019 ◽

2001 ◽

Vol 94 (1) ◽

pp. 95-100 ◽

Cited By ~ 146

Author(s):

Frank Wappler ◽

Marko Fiege ◽

Markus Steinfath ◽

Kamayni Agarwal ◽

Jens Scholz ◽

...

Keyword(s):

Malignant Hyperthermia ◽

Receptor Gene ◽

Heat Stroke ◽

Histologic Examination ◽

In Vitro Contracture Test ◽

Genetic Abnormalities ◽

History Of ◽

Exercise Induced ◽

Muscle Biopsies

Background Malignant hyperthermia (MH), heat stroke, and exercise-induced rhabdomyolysis (ER) were suspected to be related syndromes. However, it is not known whether individuals with history of ER have an increased incidence of susceptibility to MH. To establish an association between ER and susceptibility to MH, the authors determined the MH status in patients with a history of MH-like episodes induced by physical stress. Methods Twelve unrelated patients with ER, 18 patients with anesthesia-induced MH, and 28 controls were investigated with the in vitro contracture test (IVCT) according to the European MH Group protocol and the ryanodine contracture test. In addition, all patients were screened for genetic mutations, and histology was performed on muscle specimens. Results Ten ER patients had positive IVCT results, one patient had a negative test result, and one patient showed equivocal responses. Samples from patients with positive IVCT results showed pronounced contractures after exposition to ryanodine, as opposed to specimens from patients with negative IVCT results, which developed contractures slowly. Three ER patients had mutations at the ryanodine receptor gene. All anesthesia-induced MH patients had positive IVCT results, two of them presented the C1840T mutation. The control patients had normal contracture test results and no typical MH mutations. Histologic examination determined no specific myopathies in any patient. Conclusions Regarding these results, the authors recommend performing muscle biopsies for histologic examination and IVCT in patients with ER. In addition, the patient should be seen by a neurologist and screened for genetic abnormalities to shed light on the genetics of MH.

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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.

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Room E, 10/17/2000 2: 00 PM - 4: 00 PM (PS) Screening of the Ryanodine Receptor Gene and Identification of Novel Mutations in North American Malignant Hyperthermia Population

Anesthesiology ◽

10.1097/00000542-200009001-00111 ◽

2000 ◽

Vol 93 (Supplement) ◽

pp. A-111

Author(s):

N. Sambuughin ◽

Y. Sei ◽

T. Nelson ◽

H. Rosenberg ◽

S. Muldoon

Keyword(s):

Malignant Hyperthermia ◽

Ryanodine Receptor ◽

North American ◽

Receptor Gene ◽

Novel Mutations

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

Anesthesiology ◽

10.1097/01.anes.0000299431.81267.3e ◽

2008 ◽

Vol 108 (2) ◽

pp. 208-215 ◽

Cited By ~ 19

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.

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