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.     

A novel RYR1 variant in an infant with a unique fetal presentation of central core disease

Ryanodine receptor type 1-related disorder (RYR1-RD) is the most common subgroup of congenital myopathies with a wide phenotypic spectrum ranging from mild hypotonia to lethal fetal akinesia. Genetic testing for myopathies is imperative as the diagnosis informs counseling regarding prognosis and recurrence risk, treatment options, monitoring, and clinical management. However, diagnostic challenges exist as current options are limited to clinical suspicion prompting testing including: single gene sequencing or familial variant testing, multi-gene panels, exome, genome sequencing, and invasive testing including muscle biopsy. The timing of diagnosis is of great importance due to the association of RYR1-RD with malignant hyperthermia (MH). MH is a hypermetabolic crisis that occurs secondary to excessive calcium release in muscles, leading to systemic effects that can progress to shock and death if unrecognized. Given the association of MH with pathogenic variants in RYR1, a diagnosis of RYR1-RD necessitates an awareness of medical team to avoid potentially triggering agents. We describe a case of a unique fetal presentation with bilateral diaphragmatic eventrations who had respiratory failure, dysmorphic facial features, and profound global hypotonia in the neonatal period. The diagnosis was made at several months of age, had direct implications on her clinical care related to anticipated need to long-term ventilator support, and ultimately death secondary an arrhythmia as a result of suspected MH. Our report reinforces the importance of having high suspicion for a genetic syndrome and pursuing early, rapid exome or genome sequencing as first line testing in critically ill neonatal intensive care unit patients and further evaluating the pathogenicity of a variant of uncertain significance in the setting of a myopathic phenotype.     

Ryanodine receptor mutations in malignant hyperthermia and central core disease

Malignant hyperthermia (MH) is a pharmacogenetic disorder of skeletal muscle that manifests in response to anesthetic triggering agents. Central core disease (CCD) is a myopathy closely associated with MH. Both MH and CCD are primarily disorders of calcium regulation in skeletal muscle. The ryanodine receptor (RYR1) gene encodes the key channel which mediates calcium release in skeletal muscle during excitation-contraction coupling, and mutations in this gene are considered to account for susceptibility to MH (MHS) in more than 50% of cases and in the majority of CCD cases. To date, 22 missense mutations in the 15,117 bp coding region of the RYR1 cDNA have been found to segregate with the MHS trait, while a much smaller number of these mutations is associated with CCD. The majority of RYR1 mutations appear to be clustered in the N-terminal amino acid residues 35-614 (MH/CCD region 1) and the centrally located residues 2163-2458 (MH/CCD region 2). The only mutation identified outside of these regions to date is a single mutation associated with a severe form of CCD in the highly conserved C-terminus of the gene. All of the RYR1 mutations result in amino acid substitutions in the myoplasmic portion of the protein, with the exception of the mutation in the C-terminus, which resides in the lumenal/transmembrane region. Functional analysis shows that MHS and CCD mutations produce RYR1 abnormalities that alter the channel kinetics for calcium inactivation and make the channel hyper- and hyposensitive to activating and inactivating ligands, respectively. The likely deciding factors in determining whether a particular RYR1 mutation results in MHS alone or MHS and CCD are: sensitivity of the RYR1 mutant proteins to agonists; the level of abnormal channel-gating caused by the mutation; the consequential decrease in the size of the releasable calcium store and increase in resting concentration of calcium; and the level of compensation achieved by the muscle with respect to maintaining calcium homeostasis. From a diagnostic point of view, the ultimate goal of development of a simple non-invasive test for routine diagnosis of MHS remains elusive. Attainment of this goal will require further detailed molecular genetic investigations aimed at solving heterogeneity and discordance issues in MHS; new initiatives aimed at identifying modulating factors that influence the penetrance of clinical MH in MHS individuals; and detailed studies aimed at describing the full epidemiological picture of in vitro responses of muscle to agents used in diagnosis of MH susceptibility.     

Correlations between genotype and pharmacological, histological, functional, and clinical phenotypes in malignant hyperthermia susceptibility

Malignant hyperthermia susceptibility (MHS) is a subclinical pharmacogenetic disorder caused by an impairment of skeletal muscle calcium homeostasis in response to triggering agents. While in vitro contracture testing (IVCT) is the gold standard for defining MHS, molecular analysis is increasingly used to diagnosis MHS. Mutations associated with MHS have been reported in two genes: RYR1 and CACNA1S. Mutations in RYR1 are also responsible for central core disease (CCD), a myopathy that can be associated with a positive IVCT response. We report here the results of correlation studies performed with molecular, pharmacological, histological, and functional data obtained in 175 families (referred to as confirmed (129) or potential (46) MHS families). Extensive molecular analysis allowed us to identify a variant in 60% of the confirmed MHS families, and resulted in the characterization of 11 new variants in the RYR1 gene. Most mutations clustered to MH1 and MH2 domains of RYR1. Functional analysis allowed us to assign a causative role for seven MHS mutations that we propose to add to the panel of MHS mutations used for genetic testing. The use of genetic data to determine MHS status led to a 99.5% sensitivity for IVCT. IVCT-positive/mutation-negative diagnoses were analyzed not only in terms of specificity for IVCT, but also to assess the presence of a second MHS trait in families, and the genetic heterogeneity of the disease. Histological analyses revealed the presence of cores in more than 20% of muscle biopsies originating from 242 genotyped and tested MHS patients who did not present with clinical symptoms. This indicates that these patients must be considered as MHS patients with cores, and are clearly differentiated from CCD patients who have been tested positive for MHS.     

Next‐generation DNA sequencing of a Swedish malignant hyperthermia cohort

Malignant hyperthermia (MH)‐related mutations have been identified in the ryanodine receptor type 1 gene (RYR1) and in the dihydropyridine gene (CACNA1S), but about half of the patients do not have causative mutations in these genes. We wanted to study the contribution of other muscle genes to the RYR1 phenotypes. We designed a gene panel for sequence enrichment targeting 64 genes of proteins involved in the homeostasis of the striated muscle cell. Next‐generation sequencing (NGS) resulted in >50,000 sequence variants which were further analyzed by software filtering criteria to identify causative variants. In four of five patients we identified previously reported RYR1 mutations while the fifth patient did not show any candidate variant in any of the genes investigated. In two patients pathogenic variants were found in other genes known to cause a muscle disorders. All but one patient carried likely benign rare polymorphisms. The NGS technique proved convenient in identifying variants in the RYR1. However, with a clinically variable phenotype‐like MH, the pre‐selection of genes poses problems in variant interpretation.     

Recessive RYR1 mutations in a patient with severe congenital nemaline myopathy with ophthalomoplegia identified through massively parallel sequencing

Nemaline myopathy (NM) is a group of congenital myopathies, characterized by the presence of distinct rod-like inclusions "nemaline bodies" in the sarcoplasm of skeletal muscle fibers. To date, ACTA1, NEB, TPM3, TPM2, TNNT1, and CFL2 have been found to cause NM. We have identified recessive RYR1 mutations in a patient with severe congenital NM, through high-throughput screening of congenital myopathy/muscular dystrophy-related genes using massively parallel sequencing with target gene capture. The patient manifested fetal akinesia, neonatal severe hypotonia with muscle weakness, respiratory insufficiency, swallowing disturbance, and ophthalomoplegia. Skeletal muscle histology demonstrated nemaline bodies and small type 1 fibers, but without central cores or minicores. Congenital myopathies, a molecularly, histopathologically, and clinically heterogeneous group of disorders are considered to be a good candidate for massively parallel sequencing.     

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.     

A new mutation in the skeletal ryanodine receptor gene (RYR1) is potentially causative of malignant hyperthermia, central core disease, and severe skeletal malformation

Malignant hyperthermia susceptibility (MHS) and central core disease (CCD) have been shown to result from missense mutations in the ryanodine receptor gene of the skeletal muscle (RYR1). A 15-year-old patient who had spondylocostal dysostosis (SCD) developed an MH crisis during general anesthesia. The patient was characterized phenotypically by block vertebrae, vertebral fusion, short neck and thorax, fused ribs, craniofacial abnormalities, spina bifida occulta, and a diaphragmatic defect closed surgically in early infancy. The diagnosis MH susceptible (MHS) was confirmed by the in vitro contracture test (IVCT) on a muscle biopsy. Surprisingly, the histopathological investigation revealed the presence of CCD too. Molecular genetic investigation of the RYR1 gene was performed to search for known MH-related mutations. Cluster regions of the RYR1 gene, in which mutations have already been found, were examined by direct automated sequencing. In addition to the diagnosis MHS and CCD we were able to identify a novel RYR1 mutation in exon 46: 7358ATC > ACC, resulting in an Ile2453Thr substitution. This mutation was also present in the mother, in whom MH disposition and CCD were determined by muscle investigations. We suggest that the newly identified RYR1 mutation is closely associated with MH and CCD. A probable causative role of the RYR1 gene in SCD patients should be assessed by further genetic investigations.     

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.     

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

Malignant hyperthermia (MH) in man is an autosomal dominant disorder of skeletal muscle Ca(2+)-regulation. During anesthesia in predisposed individuals, it is triggered by volatile anesthetics and depolarizing muscle relaxants. In >50% of the families, MH susceptibility is linked to the gene encoding the skeletal muscle ryanodine receptor (RYR1), the calcium release channel of the sarcoplasmic reticulum, on chromosome 19q12-13.2. To date, 21 RYR1 mutations have been identified in a number of pedigrees. Four of them are also associated with central core disease (CCD), a congenital myopathy. Screening for these 21 mutations in 105 MH families including 10 CCD families phenotyped by the in vitro contracture test (IVCT) according to the European protocol revealed the following approximate distribution: 9% Arg-614-Cys, 1% Arg-614-Leu, 1% Arg-2163-Cys, 1% Val-2168-Met, 3% Thr-2206-Met and 7% Gly-2434-Arg. In one CCD family, the disease was caused by a recently reported MH mutation, Arg-2454-His. Two novel mutations, Thr-2206-Arg and Arg-2454-Cys were detected, each in a single pedigree. In the 109 individuals of the 25 families with RYR1 mutations cosegregation between genetic result and IVCT was almost perfect, only three genotypes were discordant with the IVCT phenotypes, suggesting a true sensitivity of 98.5% and a specificity of minimally 81.8% for this test. Screening of the transmembraneous region of RYR1 did not yield a new mutation confirming the cytosolic portion of the protein to be of main functional importance for disease pathogenesis.     

An autosomal dominant congenital myopathy with cores and rods is associated with a neomutation in the RYR1 gene encoding the skeletal muscle ryanodine receptor

Central core disease (CCD) and nemaline myopathy (NM) are congenital myopathies for which differential diagnosis is often based on the presence either of cores or rods. Missense mutations in the skeletal muscle ryanodine receptor gene (RYR1) have been identified in some families with CCD. Mutations in the alpha-tropomyosin and alpha-actin genes have been associated with most dominant forms of NM. Analysis of the RYR1 cDNA in a French family identified a novel Y4796C mutation that lies in the C-terminal channel-forming domain of the RyR1 protein. This mutation was linked not only to a severe and penetrant form of CCD, but also to the presence of rods in the muscle fibres and to the malignant hyperthermia susceptibility (MHS) phenotype. The Y4796C mutation was introduced into a rabbit RYR1 cDNA and expressed in HEK-293 cells. Expression of the mutant RYR1 cDNA produced channels with increased caffeine sensitivity and a significantly reduced maximal level of Ca(2+) release. Single-cell Ca(2+) analysis showed that the resting cytoplasmic level was increased by 60% in cells expressing the mutant channel. These data support the view that the rate of Ca(2+) leakage is increased in the mutant channel. The resulting chronic elevation in myoplasmic concentration is likely to be responsible for the severe expression of the disease. Haplotyping analysis indicated that the mutation arose as a neomutation in the proband. This first report of a neomutation in the RYR1 gene has strong implications for genetic linkage studies of MHS or CCD, two diseases characterized by a genetic heterogeneity.     

Diagnosis of malignant hyperthermia: a comparison of the in vitro contracture test with the molecular genetic diagnosis in a large pedigree.

Malignant hyperthermia (MH) is an inherited skeletal muscle disorder and is one of the major causes of death resulting from anaesthesia. MH is currently diagnosed by the in vitro contracture test performed on a muscle biopsy. Genetic linkage analysis on an Irish MH pedigree showed that when the thresholds for the standardised European protocol for MHS diagnosis was applied, linkage between the MHS phenotype and the RYR1 locus was excluded. When we raised the threshold values for assignment of MHS status and assumed MHN diagnosis in subjects where this threshold was not attained, tight linkage between MHS and RYR1 markers was observed, suggesting that MHS is linked to the RYR1 locus in this pedigree. Confirmation of these results was borne out by the fact that all of the MHS patients in the pedigree exceeding the raised threshold carried the known MHS Gly341Arg RYR1 mutation. The results obtained could be explained (1) by false positive diagnosis of MHS in the recombinant subjects, (2) by the presence of a mutation in a predisposing gene other than RYR1, or (3) by the presence of mild subclinical myopathies. The implications of these results for heterogeneity studies is discussed.     

Exclusion of defects in the skeletal muscle specific regions of the DHPR alpha 1 subunit as frequent causes of malignant hyperthermia.

The molecular defect predisposing to the majority of malignant hyperthermia (MH) cases is unknown, although various point mutations in the ryanodine receptor gene (RYR1) have been associated with susceptibility in a small proportion of cases. We report here that one of these, the Arg163Cys substitution, does not cosegregate with MH susceptibility. Comparison of cDNA sequences encoding the skeletal muscle specific components of the dihydropyridine receptor alpha 1 subunit between MH susceptible (MHS) and MH non-susceptible (MHN) patients was made in subjects without the reported MH linked RYR1 mutations. There were no differences within the sequence encoding the II-III loop or the IS3/IS3-IS4 segment, excluding defects in these functional segments of the alpha 1 subunit as frequent causes of MH.     

Null mutations causing depletion of the type 1 ryanodine receptor (RYR1) are commonly associated with recessive structural congenital myopathies with cores

Mutations of the ryanodine receptor cause dominant and recessive forms of congenital myopathies with cores. Quantitative defects of RYR1 have been reported in families presenting with recessive forms of the disease and epigenic regulation has been recently proposed to explain potential maternal monoallelic silencing of the RYR1 gene. We investigated nine families presenting with a recessive form of the disease and showing a quantitative defect of RYR1 expression. Genetic analysis allowed the identification of a mutation on both alleles of the RYR1 gene for all patients, 15 being novel variants. We evidenced for all patients an alteration of the expression of the RYR1 gene caused by amorphic mutations responsible either for mRNA or protein instability. In seven families the variant present on the second allele was a missense mutation. In the remaining two families the second variant led to a hypomorphic expression of the RYR1 gene and was associated with a severe neonatal phenotype, pointing out the minimal amount of RYR1 needed for skeletal muscle function. Noticeably, a novel additional exon 3b was characterized in the most severely affected cases. This study showed that all cases presenting with a quantitative defect of RYR1 expression in our panel of patients affected by recessive core myopathies were caused by the presence of one recessive null allele and that variability of the phenotype depended on the nature of the mutation present on the second allele. Our study also indicated that presence of a second mutation must be investigated in sporadic cases or in dominant cases presenting with a familial clinical variability.     

Abnormal myosin post-translational modifications and ATP turnover time associated with human congenital myopathy-related RYR1 mutations

Aim

Conditions related to mutations in the gene encoding the skeletal muscle ryanodine receptor 1 (RYR1) are genetic muscle disorders and include congenital myopathies with permanent weakness, as well as episodic phenotypes such as rhabdomyolysis/myalgia. Although RYR1 dysfunction is the primary mechanism in RYR1-related disorders, other downstream pathogenic events are less well understood and may include a secondary remodeling of major contractile proteins. Hence, in the present study, we aimed to investigate whether congenital myopathy-related RYR1 mutations alter the regulation of the most abundant contractile protein, myosin.

Methods

We used skeletal muscle tissues from five patients with RYR1-related congenital myopathy and compared those with five controls and five patients with RYR1-related rhabdomyolysis/myalgia. We then defined post-translational modifications on myosin heavy chains (MyHCs) using LC/MS. In parallel, we determined myosin relaxed states using Mant-ATP chase experiments and performed molecular dynamics (MD) simulations.

Results

LC/MS revealed two additional phosphorylations (Thr1309-P and Ser1362-P) and one acetylation (Lys1410-Ac) on the β/slow MyHC of patients with congenital myopathy. This method also identified six acetylations that were lacking on MyHC type IIa of these patients (Lys35-Ac, Lys663-Ac, Lys763-Ac, Lys1171-Ac, Lys1360-Ac, and Lys1733-Ac). MD simulations suggest that modifying myosin Ser1362 impacts the protein structure and dynamics. Finally, Mant-ATP chase experiments showed a faster ATP turnover time of myosin heads in the disordered–relaxed conformation.

Conclusions

Altogether, our results suggest that RYR1 mutations have secondary negative consequences on myosin structure and function, likely contributing to the congenital myopathic phenotype.     

Familial and sporadic forms of central core disease are associated with mutations in the C-terminal domain of the skeletal muscle ryanodine receptor.

Central core disease (CCD) is an autosomal dominant congenital myopathy. Diagnosis is based on the presence of cores in skeletal muscles. CCD has been linked to the gene encoding the ryanodine receptor (RYR1) and is considered to be an allelic disease of malignant hyperthermia susceptibility. However, the report of a recessive form of transmission together with a variable clinical presentation has raised the question of the genetic heterogeneity of the disease. Analyzing a panel of 34 families exclusively recruited on the basis of both clinically and morphologically expressed CCD, 12 different mutations of the C-terminal domain of RYR1 have been identified in 16 unrelated families. Morphological analysis of the patients' muscles showed different aspects of cores, all of them associated with mutations in the C-terminal region of RYR1. Furthermore, we characterized the presence of neomutations in the RyR1 gene in four families. This indicates that neomutations into the RyR1 gene are not a rare event and must be taken into account for genetic studies of families that present with congenital myopathies type 'central core disease'. Three mutations led to the deletion in frame of amino acids. This is the first report of amino acid deletions in RYR1 associated with CCD. According to a four-transmembrane domain model, the mutations concentrated mostly in the myoplasmic and luminal loops linking, respectively, transmembrane domains T1 and T2 or T3 and T4 of RYR1.     

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 approximately 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.     

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 RYR1^Cys4664Arg 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. © 2009 Wiley‐Liss, Inc.     

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.