Mutations in RYR1 in malignant hyperthermia and central core disease

The RYR1 gene encodes the skeletal muscle isoform ryanodine receptor and is fundamental to the process of excitation-contraction coupling and skeletal muscle calcium homeostasis. Mapping to chromosome 19q13.2, the gene comprises 106 exons and encodes a protein of 5,038 amino acids. Mutations in the gene have been found in association with several diseases: the pharmacogenetic disorder, malignant hyperthermia (MH); and three congenital myopathies, including central core disease (CCD), multiminicore disease (MmD), and in an isolated case of a congenital myopathy characterized on histology by cores and rods. The majority of gene mutations reported are missense changes identified in cases of MH and CCD. In vitro analysis has confirmed that alteration of normal calcium homeostasis is a functional consequence of some of these changes. Genotype-phenotype correlation studies performed using data from MH and CCD patients have also suggested that mutations may be associated with a range of disease severity phenotypes. This review aims to summarize the current understanding of RYR1 mutations reported in association with MH and CCD and the present viewpoint on the use of mutation data to aid clinical diagnosis of these conditions.     

Analysis of RYR1 haplotype profile in patients with malignant hyperthermia

This study represents a new approach to characterising patients at risk of malignant hyperthermia (MH) through the use of a recently published method for identifying high-risk haplotypes in candidate genes. We present analysis based upon the largest standardised and genotyped database of MH patients worldwide. We used unphased RYR1 SNP data directly to (1) assess RYR1 haplotype frequency differences between susceptible cases and control groups and (2) analyse population-based association via clustering of RYR1 haplotypes based on disease risk. Our results show a significant difference in RYR1 haplotype frequency between susceptible cases and UK Caucasian population controls. Furthermore we identify a high-risk cluster of haplotypes that is associated with the commonest UK MH mutation p.G2434R/c.7300G>A. These results demonstrate the applicability of this new and practical method for population based association analysis.     

Detection of a novel RYR1 mutation in four malignant hyperthermia pedigrees

Malignant hyperthermia (MH) is a potentially fatal autosomaldominant disorder of skeletal muscle and is triggered in susceptiblepeople by all commonly used inhalational anaesthetics and depolarizingmuscle relaxants. To date, six mutations in the skeletal muscleryanodine receptor gene (RYR1) have been identified in malignanthyperthermia susceptible (MHS) and central core disease (CCD)cases. Using SSCP analysis, we have screened the RYR1 gene inaffected individuals for novel MHS mutations and have identifieda G to A transition mutation which results in the replacementof a conserved Gly at position 2433 with an Arg. The Gly2433Argmutation was present in four of 104 unrelated MHS individualsinvestigated and was not detected in a normal population sample.This mutation is adjacent to the previously identified Arg2434Hismutation reported in a CCD/MH family and indicates that theremay be a second region in the RYR1 gene where MHS/CCD mutationscluster.     

Discordance between malignant hyperthermia susceptibility and RYR1 mutation C1840T in two Scandinavian MH families exhibiting this mutation

The ryanodine receptor 1 (RYR1) mutation C1840T has been reported to segregate with malignant hyperthermia (MH) susceptibility in several families. We have investigated several Scandinavian MH families with respect to five different RYR1 mutations reported to cause predisposition to MH, and we here report on two of the families in which the C1840T mutation was detected. In these two families there was recombination between MH susceptibility and this mutation in one and three individuals, respectively. These findings may suggest that it is necessary to reconsider the specificity of the IVCT and the role of C1840T as a cause of MH susceptibility in some families exhibiting this mutation.     

A simple method to detect the RYR1 mutation G1021A, a cause of malignant hyperthermia susceptibility

To search for the mutations RYR1 G1021A in families where malignant hyperthermia (MH) episodes have occurred, we have used an amplificationcreated restriction sites (ACRS) technique to detect the mutation. The previously described single-stranded conformation polymorphism (SSCP) technique was laborious and time consuming, but necessary to detect the mutation, whereas the method described here discriminates quickly and efficiently between homozygotes with the mutation, heterozygotes and homozygotes without the mutation.     

Search for three known mutations in the RYR1 gene in 48 Danish families with malignant hyperthermia

We have examined 48 Danish families in which malignant hyperthermia reactions have occurred, with respect to three of six published mutations in the gene for the calcium release channel of sarcoplasmic reticulum (the RYR1 gene) believed to cause malignant hyperthermia susceptibility in man. The mutations are Arg614Cys, also known as the "pig mutation"; Arg163Cys; and Ile403Met. The only mutation found was Arg163Cys, which was detected in only one family. The results of this study indicate that other mutations must underlie the disorder in most Danish malignant hyperthermia-susceptible families, and that the "pig mutation" is not a frequent cause of malignant hyperthermia susceptibility in Denmark.     

A search for three known RYR1 gene mutations in 41 Swedish families with predisposition to malignant hyperthermia

Eight mutations in the gene (the RYR1 gene) encoding the calcium release channel of sarcoplasmic reticulum (SR) in skeletal muscle are so far known to be very closely linked to malignant hyperthermia susceptibility in man and are regarded to be causative. We have examined 41 Swedish families where malignant hyperthermia had occurred in at least one member during anaesthesia, with respect to three of the known mutations. The mutations were Arg163Cys; Ile403Met and Arg614Cys (also known as the "pig mutation"). In three (i.e. 7%) of the families we detected the Arg614Cys mutation, and this was the only one of the mutations searched for that was observed. This indicates that other mutations than those searched for in this study must cause malignant hyperthermia susceptibility in most Swedish malignant hyperthermia susceptible families.     

Novel missense mutations and unexpected multiple changes of RYR1 gene in 75 malignant hyperthermia families

Tammaro A, Di Martino A, Bracco A, Cozzolino S, Savoia G, Andria B, Cannavo A, Spagnuolo M, Piluso G, Aurino S, Nigro V. Novel missense mutations and unexpected multiple changes of RYR1 gene in 75 malignant hyperthermia families. Malignant hyperthermia (MH) is an autosomal dominant pharmacogenetic disorder of skeletal muscle characterized by disturbance of intracellular calcium homeostasis in the sarcoplasmic reticulum. Mutations of the ryanodine receptor 1 (RYR1) gene account for most cases, with some studies claiming up to 86% of mutations in this locus. However, RYR1 gene is large and variants are common even in the normal population. We examined 54 families with MH susceptibility and 21 diagnosed with equivocal MH. Thirty‐five were selected for an anesthetic reaction, whereas the remainder for hyperCKemia. In these, we studied all 106 exons of the RYR1 gene. When no mutation was found, we also screened: sodium channel voltage‐gated, type IV alpha subunit (SCN4A), calcium channel voltage‐dependent, L type, alpha 1S subunit (CACNA1S), and L‐type voltage‐gated calcium channel alpha 2/delta‐subunit (CACNL2A). Twenty‐nine different RYR1 mutations were discovered in 40 families. Three other MH genes were tested in negative cases. Fourteen RYR1 amino acid changes were novel, of which 12 were located outside the mutational ‘hot spots'. In two families, the known mutation p.R3903Q was also observed in malignant hyperthermia‐nonsusceptible (MHN) individuals. Unexpectedly, four changes were also found in the same family and two in another. Our study confirms that MH is genetically heterogeneous and that a consistent number of cases are not due to RYR1 mutations. The discordance between in vitro contracture test status and the presence of a proven causative RYR1 mutation suggests that the penetrance may vary due to as yet unknown factors.     

Mutation screening of the RYR1 gene and identification of two novel mutations in Italian malignant hyperthermia families

Point mutations in the ryanodine receptor (RYR1) gene are associated with malignant hyperthermia, an autosomal dominant disorder triggered in susceptible people (MHS) by volatile anaesthetics and depolarising skeletal muscle relaxants. To date, 17 missense point mutations have been identified in the human RYR1 gene by screening of the cDNA obtained from muscle biopsies. Here we report single strand conformation polymorphism (SSCP) screening for nine of the most frequent RYR1 mutations using genomic DNA isolated from MHS patients. In addition, the Argl63Cys mutation was analysed by restriction enzyme digestion. We analysed 57 unrelated patients and detected seven of the known RYR1 point mutations. Furthermore, we found a new mutation, Arg2454His, segregating with the MHS phenotype in a large pedigree and a novel amino acid substitution at position 2436 in another patient, indicating a 15.8% frequency of these mutations in Italian patients. A new polymorphic site in intron 16 that causes the substitution of a G at position -7 with a C residue was identified.     

RYR mutation G1021A (Gly341Arg) is not frequent in Danish and Swedish families with malignant hyperthermia susceptibility

Malignant hyperthermia (MH) is a pharmacogenetic disorder. Susceptibility to MH (MHS) is presumed to be inherited in an autosomal dominant way. MH crises are triggered by halogenated inhalational anaesthetics and suxamethonium, and may be lethal if not treated early and adequately. Until now, eight mutations in the RYR1 gene have been described as causes of MHS phenotype in various MH families The mutation RYR1 G1021A (Gly341Arg) has been reported to account for approximately 10% of Caucasian MHS cases. However, in our study this mutation was discovered in only 1 out of 89 Scandinavian families, indicating that this mutation may be the cause of MHS in only about 1% of MHS families in those populations. The mutation may have been brought to Scandinavia by an immigrant.     

RYR1 mutations causing central core disease are associated with more severe malignant hyperthermia in vitro contracture test phenotypes

Malignant hyperthermia (MH) and central core disease (CCD) are autosomal dominant disorders of skeletal muscle. Susceptibility to MH is only apparent after exposure to volatile anesthetics and/or depolarizing muscle relaxants. CCD patients present with diffuse muscular weakness but are also at risk of MH. Mutations in RYR1 (19q13.1), encoding a skeletal muscle calcium release channel (ryanodine receptor), account for the majority of MH and CCD cases. Fifteen RYR1 N-terminal mutations are considered causative of MH susceptibility, five of which are also associated with CCD. In the first extensive UK population survey, eight of 15 mutations were detected in 85 out of 297 (29%) unrelated MH susceptible cases, with G2434R detected in 53 cases (18%). Mutation type was shown to affect significantly MH phenotypes (in vitro contracture test (IVCT) response to caffeine, halothane, and ryanodine). RYR1 mutations associated with both CCD and MH (R163C, R2163H, R2435H) had more severe caffeine and halothane response phenotypes than those associated with MH alone. Mutations near the amino terminal (R163C, G341R) had a relatively greater effect on responses to caffeine than halothane, with a significantly increased caffeine:halothane tension ratio compared to G2434R of the central domain. All phenotypes were more severe in males than females, and were also affected by muscle specimen size and viability. Discordance between RYR1 genotype and IVCT phenotype was observed in seven families (nine individuals), with five false-positives and four false-negatives. This represents the most extensive study of MH patient clinical and genetic data to date and demonstrates that RYR1 mutations involved in CCD are those associated with one end of the spectrum of MH IVCT phenotypes.     

Identification of a novel mutation in the ryanodine receptor gene (RYR1) in a malignant hyperthermia Italian family

Malignant hyperthermia (MH) is an inherited autosomal dominant pharmacogenetic disorder and is one of the main causes of death subsequent to anaesthesia. Around 50% of affected families are linked to the ryanodine receptor (RYR1) gene. To date, 19 mutations have been identified in the coding region of this gene and appear to be associated with the MH-susceptible phenotype. Here we report the identification by two independent methods of a novel mutation associated with the MH-susceptible phenotype in the RYR1 gene: the 6488G-->C transversion, resulting in the replacement of the Arg2163 with a proline residue.     

Frequency and localization of mutations in the 106 exons of the RYR1 gene in 50 individuals with malignant hyperthermia

Malignant hyperthermia (MH) is a dominantly inherited pharmacogenetic condition that manifests as a life-threatening hypermetabolic reaction when a susceptible individual is exposed to common volatile anesthetics and depolarizing muscle relaxants. Although MH appears to be genetically heterogeneous, RYR1 is the main candidate for MH susceptibility. However, since molecular analysis is generally limited to exons where mutations are more frequently detected, these are routinely found only in 30-50% of susceptible subjects. In this study the entire RYR1 coding region was analyzed in a cohort of 50 Italian MH susceptible (MHS) subjects. Thirty-one mutations, 16 of which were novel, were found in 43 individuals with a mutation detection rate of 86%, the highest reported for RYR1 in MH so far. These data provide clear evidence that mutations in the RYR1 gene are the predominant cause of MH.     

TRPV1 variants impair intracellular Ca2+ signaling and may confer susceptibility to malignant hyperthermia

PurposeMalignant hyperthermia (MH) is a pharmacogenetic disorder arising from uncontrolled muscle calcium release due to an abnormality in the sarcoplasmic reticulum (SR) calcium-release mechanism triggered by halogenated inhalational anesthetics. However, the molecular mechanisms involved are still incomplete.MethodsWe aimed to identify transient receptor potential vanilloid 1(TRPV1) variants within the entire coding sequence in patients who developed sensitivity to MH of unknown etiology. In vitro and in vivo functional studies were performed in heterologous expression system, trpv1^−/− mice, and a murine model of human MH.ResultsWe identified TRPV1 variants in two patients and their heterologous expression in muscles of trpv1^−/− mice strongly enhanced calcium release from SR upon halogenated anesthetic stimulation, suggesting they could be responsible for the MH phenotype. We confirmed the in vivo significance by using mice with a knock-in mutation (Y524S) in the type I ryanodine receptor (Ryr1), a mutation analogous to the Y522S mutation associated with MH in humans. We showed that the TRPV1 antagonist capsazepine slows the heat-induced hypermetabolic response in this model.ConclusionWe propose that TRPV1 contributes to MH and could represent an actionable therapeutic target for prevention of the pathology and also be responsible for MH sensitivity when mutated.     

The ryanodine receptor type 1 gene variants in African American men with exertional rhabdomyolysis and malignant hyperthermia susceptibility

It has been suggested that exertional rhabdomyolysis (ER) and malignant hyperthermia (MH) are related syndromes. We hypothesize that patients with unexplained ER harbor mutations in the ryanodine receptor gene type 1 (RYR1), a primary gene implicated in MH, and therefore ER patients are at increased risk for MH. Although there are reported cases of MH in individuals of African descent, there are no data available on molecular characterization of these patients. We analyzed RYR1 in six, unrelated African American men with unexplained ER, who were subsequently diagnosed as MH susceptible (MHS) by the Caffeine Halothane Contracture Test. Three novel and two variants, previously reported in Caucasian MHS subjects, were found in five studied patients. The novel variants were highly conserved amino acids and were absent among 230 control subjects of various ethnic backgrounds. These results emphasize the importance of performing muscle contracture testing and RYR1 mutation screening in patients with unexplained ER. The MHS‐associated variant Ala1352Gly was identified as a polymorphism predominant in individuals of African descent. Our data underscore the need for investigating RYR1 across different ethnic groups and will contribute to interpretation of genetic screening results of individuals at risk for MH.