Ryanodine Receptor Type 1 Mutations Research Articles

Malignant hyperthermia‐associated mutation of leaky RyR1 induces mitochondrial Ca2+ overload in the heart

Malignant hyperthermia (MH) is known as a pharmacogenetic disorder of skeletal muscle that shows hypermetabolic responses to anesthetic gases. It is reported that this syndrome is associated with the ryanodine receptor type 1 (RyR1) mutations. Although clinical manifestation of MH is mainly skeletal muscle disorder under anesthesia, sudden cardiac death (SCD) in the MH families in the conscious condition without anesthesia are also reported in the clinical case studies. However, the molecular mechanism underlying SCD in MH is completely unknown. We previously reported that a low level of RyR1 is expressed in the cardiac mitochondria, but not in the sarcoplasmic reticulum (SR) in the hearts, which may serves as an additional mitochondrial Ca2+ influx mechanism in cardiomyocytes distinct from the main pathway by the mitochondrial Ca2+ uniporter. Using knock‐in mice carrying a MH‐related RyR1 mutation Y522S (YS), we showed that YS hearts exhibit disrupted mitochondrial morphology. Moreover, ex‐vivo YS heart developed significantly higher number of multiple ventricular extrasystoles by β‐adrenergic stimulation compared to those observed in the wild‐type (WT) hearts. Therefore, we hypothesize that YS‐RyR1s form “leaky channel” at cardiac mitochondria and induce mitochondrial Ca2+ overload, followed by an increase in the mitochondrial reactive oxygen species (ROS) generation, which alters the cellular Ca2+ handing in cardiomyocytes. In this study, we used isolated cardiac mitochondria and cardiomyocytes from the YS mice to investigate the role of YS‐RyR1 in mitochondrial and cellular Ca2+ handling as well as ROS generation. We loaded the isolated mitochondria with Ca2+ or membrane‐potential sensitive dyes, plated them on the glass‐bottom dishes and assessed mitochondrial Ca2+ concentration ([Ca2+]m) and mitochondrial membrane potential (ψm) under the confocal microscopy. We found that YS mitochondria possess higher basal [Ca2+]m and depolarized ψm. Moreover, pretreatment with RyR1 blocker dantrolene decreased basal [Ca2+]m and hyperpolarized ψm in YS mitochondria. We next loaded cardiomyocytes with Ca2+ sensitive dyes and measured cellular Ca2+ handling. We found that YS myocytes had significantly higher basal cytosolic Ca2+ concentration as well as slower cytosolic Ca2+ clearance compared to WT. Pretreatment of dantrolene normalized the Ca2+ handing profiles in YS cardiomyocytes similar to those in WT. Finally, we found that YS cardiomyocytes exhibited higher mitochondrial superoxide (mSO) level and excessive cellular oxidation assessed by the staining with mSO‐sensitive dye, MitoSox Red and immunoblotting of carbonyl groups by OxyBlot, respectively. In summary, these results suggest that chronic mitochondrial Ca2+ overload via leaky mutant mRyR1 increases mSO production and cellular oxidation, which may alter cytosolic Ca2+ handling and increase the arrhythmogenic events in MH.Support or Funding InformationJ.O.‐U. was supported by NIH/NHLBI R01HL136757, NIH/NIGMS P30GM1114750, American Heart Association (AHA) 4BGIA18830032, AHA 16SDG27260248, Rhode Island Foundation #20164376 Medical Research Grant, American Physiological Society (APS), and 2017 Shih‐Chun Wang Young Investigator Award. B.S.J was supported by NIH/NIGMS U54GM115677 and P30GM1114750. S.S.S was supported by NIH/NHLBI R01HL093671 and R01HL122124.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Malignant hyperthermia‐associated mutation of RyR1 induces mitochondrial Ca2+ overload in the cardiomyocytes

Although human pedigrees carrying the ryanodine receptor type 1 (RyR1) mutations mainly show skeletal muscle disorders including malignant hyperthermia (MH), sudden cardiac death (SCD) in these MH families in the conscious condition without anesthesia are also frequently found in the clinical case studies. However, the molecular mechanism underlying cardiac phenotypes in human MH family is completely unknown. Our group previously reported that a low level of RyR1 is expressed in the mitochondria, but not in the sarcoplasmic reticulum (SR) in the rat and mouse hearts, which serves as an important mitochondrial Ca2+ influx pathway in addition to the mitochondrial Ca2+ uniporter in cardiomyocytes. We also showed, using knock‐in mice carrying a MH‐related RyR1 mutation Y522S (YS) that YS hearts exhibit disrupted mitochondrial morphology as well as compromised mitochondrial functions with a high cellular oxidative state. Moreover, ex‐vivo YS heart developed significantly higher number of multiple ventricular extrasystoles by β‐adrenergic stimulation compared those observed in the wild‐type (WT) hearts. Therefore, our main hypothesis is that YS‐RyR1s form “leaky channel” at mitochondria and induce mitochondrial Ca2+ overload, which alters the cellular Ca2+ handing in cardiomyocyte. Using isolated mitochondria loaded with Ca2+ or membrane‐potential sensitive dyes under the confocal microscope, we found that YS mitochondria have higher basal mitochondrial Ca2+ concentration, depolarized mitochondrial membrane potential. In addition, YS cardiomyocytes exhibit higher basal cytosolic Ca2+ concentration as well as slower cytosolic Ca2+ clearance compared to WT. Finally, pretreatment of RyR1 blocker dantrolene cancelled these changes in YS mitochondria and cardiomyocyes and normalized their Ca2+ handing profiles similar to those in WT. In summary, these results indicate that chronic mitochondrial Ca2+ overload via leaky mutant mRyR1 damages cardiac mitochondrial functions/structures, which may alter cytosolic Ca2+ handling, induce cellular oxidation, and increase the arrhythmogenic events in MH.Support or Funding Information16SDG27260248 AHA Scientist Development Grant

Gain-of-Function Mutation in Ryanodine Receptor Type 1 Modulates Murine Thymocyte Calcium Signaling and Autoimmune Response in Mice

Gain-of-function mutations in gene encoding ryanodine receptor type 1 (RyR1), an endo/sarcoplasmic reticulum Ca2+ release channel, are linked to a variety of skeletal muscular disorders. Other tissues, including cells of the immune system, have been shown to express RyR1. A previous study demonstrated enhanced immune responses in mice with gain-of-function RyR1 mutation (Vukcevic et al., 2013, J Cell Sci, 126: 3485-92). Here we report that after induction of an experimental autoimmune encephalomyelitis (EAE), a T cell-mediated autoimmune disease, the mice carrying gain-of-function RyR1 R163C mutation (R163C mice) displayed an earlier disease onset and augmentation of neurological symptoms compared with wild-type (WT) mice. In vitro studies revealed that splenocytes from the R163C mice have altered Ca2+ signaling compared with those from WT mice, which was revealed by challenging the cells with different concentrations of extracellular Ca2+ and Ca2+ mobilizing agent thapsigargin. No differences were found in the relative abundances of immune cell subtypes in spleens and lymph nodes from R163C and WT mice prior to the onset of the EAE. Nevertheless, immunostaining of the lumbar spinal cord sections after the onset of the clinical symptoms, revealed the larger pro-inflammatory infiltrate and axonal damage in the R163C mice compared with the WT mice, indicating that R163C mutation may affect immune cell trafficking to the CNS. These data indicate that altered Ca2+ handling in R163C mice immune cells may enhance these cells autoimmune responses and that gain-of-function mutations in RYR1 may represent a previously unknown risk factor for autoimmune diseases.

Malignant Hyperthermia‐associated Mutation of RyR1 Induces Mitochondrial Damages and Cellular Oxidation in the Heart

Human pedigrees carrying the ryanodine receptor type 1 (RyR1) mutations frequently show skeletal muscle (SM) disorders including malignant hyperthermia (MH). There are also case reports describing sudden cardiac death (SCD) in MH patients in the conscious condition without anesthesia. These observations cannot be explained as a secondary effect of their SM dysfunction; however, the molecular mechanism underlying cardiac phenotypes in MH is still unknown. We previously reported that a low level of RyR1 is expressed in the mitochondria (termed as mitochondrial RyR1: mRyR1), but not in the sarcoplasmic reticulum (SR) in the cardiomyocytes and serves as an important mitochondrial Ca2+ influx pathway in addition to the mitochondrial Ca2+ uniporter in cardiomyocytes. We also reported, using knock‐in mice carrying a MH‐related RyR1 mutation Y522S (YS) that YS hearts exhibit disrupted mitochondrial morphology as well as compromised mitochondrial functions with a high cellular oxidative state, which can account for cardiac phenotypes in MH. In addition, YS heart developed multiple ventricular extrasystoles by β‐adrenergic stimulation. Therefore, we hypothesize that YS‐RyR1s form “leaky channel” at mitochondria and induce mitochondrial Ca2+ overload, which alters the cellular Ca2+ handling in cardiomyocyte. Using isolated mitochondria or isolated cardiomyocytes under confocal microscope, we found that YS cardiomyocytes have higher basal mitochondrial Ca2+ concentration, depolarized mitochondrial membrane potential and slower cytosolic Ca2+ clearance as compared to WT. Moreover, pretreatment of RyR1 blocker dantrolene prevented these changes in YS cardiomyocytes and normalized their Ca2+ handling profiles similar to those in WT. In conclusion, these results indicate that chronic mitochondrial Ca2+ overload via leaky mutant mRyR1 damages cardiac mitochondrial functions/structures, reduces cytosolic Ca2+ buffering capacity and induces cellular oxidation, which may increase arrhythmogenic events in MH.Support or Funding InformationThis work was partly supported by American Heart Association (AHA) grant (14BGIA18830032 to J.O.‐U.), Medical Research Grant from W.W. Smith Charitable Trust (H1403 to J.O.‐U.) and NIH grants (2R01HL093671 and 1R01HL122124 to SSS).

Functional and genetic characterization of clinical malignant hyperthermia crises: a multi-centre study.

BackgroundMalignant hyperthermia (MH) is a rare pharmacogenetic disorder which is characterized by life-threatening metabolic crises during general anesthesia. Classical triggering substances are volatile anesthetics and succinylcholine (SCh). The molecular basis of MH is excessive release of Ca2+ in skeletal muscle principally by a mutated ryanodine receptor type 1 (RyR1). To identify factors explaining the variable phenotypic presentation and complex pathomechanism, we analyzed proven MH events in terms of clinical course, muscle contracture, genetic factors and pharmocological triggers.MethodsIn a multi-centre study including seven European MH units, patients with a history of a clinical MH episode confirmed by susceptible (MHS) or equivocal (MHE) in vitro contracture tests (IVCT) were investigated. A test result is considered to be MHE if the muscle specimens develop pathological contractures in response to only one of the two test substances, halothane or caffeine. Crises were evaluated using a clinical grading scale (CGS), results of IVCT and genetic screening. The effects of SCh and volatile anesthetics on Ca2+ release from sarcoplasmic reticulum (SR) were studied in vitro.ResultsA total of 200 patients met the inclusion criteria. Two MH crises (1%) were triggered by SCh (1 MHS, 1 MHE), 18% by volatile anesthetics and 81% by a combination of both. Patients were 70% male and 50% were younger than 12 years old. Overall, CGS was in accord with IVCT results. Crises triggered by enflurane had a significantly higher CGS compared to halothane, isoflurane and sevoflurane. Of the 200 patients, 103 carried RyR1 variants, of which 14 were novel. CGS varied depending on the location of the mutation within the RyR1 gene. In contrast to volatile anesthetics, SCh did not evoke Ca2+ release from isolated rat SR vesicles.ConclusionsAn MH event could depend on patient-related risk factors such as male gender, young age and causative RyR1 mutations as well as on the use of drugs lowering the threshold of myoplasmic Ca2+ release. SCh might act as an accelerant by promoting unspecific Ca2+ influx via the sarcolemma and indirect RyR1 activation. Most MH crises develop in response to the combined administration of SCh and volatile anesthetics.

Mice with RyR1 mutation (Y524S) undergo hypermetabolic response to simvastatin.

BackgroundStatins are widely used drugs for the treatment of hyperlipidemia. Though relatively safe, some individuals taking statins experience rhabdymyolysis, muscle pain, and cramping, a condition termed statin-induced myopathy (SIM). To determine if mutations in the skeletal muscle calcium (Ca2+) release channel, ryanodine receptor type 1 (RyR1), enhance the sensitivity to SIM we tested the effects of simvastatin, the statin that produces the highest incidence of SIM in humans, in mice with a mutation (Y524S, ‘YS’) in RyR1. This mutation is associated with malignant hyperthermia in humans. Exposure of mice with the YS mutation to mild elevations in environmental temperature produces a life-threatening hypermetabolic response (HMR) that is characterized by increased oxygen consumption (VO2), sustained muscle contractures, rhabdymyolysis, and elevated core body temperature.MethodsWe assessed the ability of simvastatin to induce a hypermetabolic response in the YS mice using indirect calorimetry and to alter Ca2+ release via RyR1 in isolated flexor digitorum brevis (FDB) fibers from WT and YS mice using fluorescent Ca2+ indicators. We also tested the ability of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) to protect against the simvastatin effects.ResultsAn acute dose of simvastatin triggers a hypermetabolic response in YS mice. In isolated YS muscle fibers, simvastatin triggers an increase in cytosolic Ca2+ levels by increasing Ca2+ leak from the sarcoplasmic reticulum (SR). With higher simvastatin doses, a similar cytosolic Ca2+ increase occurs in wild type (WT) muscle fibers. Pre-treatment of YS and WT mice with AICAR prevents the response to simvastatin.ConclusionsA mutation in RyR1 associated with malignant hyperthermia increases susceptibility to an adverse response to simvastatin due to enhanced Ca2+ release from the sarcoplasmic reticulum, suggesting that RyR1 mutations may underlie enhanced susceptibility to statin-induced myopathies. Our data suggest that AICAR may be useful for treating statin myopathies.

Sevoflurane is less sensitive than halothane for in vitro detection of malignant hyperthermia susceptibility

Sevoflurane is a known triggering agent of malignant hyperthermia (MH). The present study analyzed different effects of sevoflurane on skeletal muscle of MH susceptible and nonsusceptible individuals in vitro and compared the results to the standardized test protocol with halothane and caffeine. A potential influence of a present ryanodine receptor type 1 (RyR1) mutation was investigated. Muscle bundles of 24 MH-susceptible patients with or without an RyR1 mutation, 35 MH-nonsusceptible and 10 MH-equivocal patients were exposed either to sevoflurane 8 vol% bolus or increasing doses of 2, 4, 6, and 8 vol%. In MH-positive patients, a screening for mutations in the RyR1 gene was performed. The in vitro parameters initial length, weight, predrug resting tension, and predrug twitch height did not differ between the groups. Sevoflurane caused significant contractures in MH-susceptible but not in MH-nonsusceptible muscle after increasing doses [1.4 (0.3-6.0) vs. 0 (0-0) mN] and after bolus application [6.9 (2.4-21.4) vs. 0 (0-0) mN]. However, only 50% of the susceptible patients developed contractures ≥ 2 mN after increasing concentrations while 83% did so after rapid bolus administration. Presence of an RyR1 mutation was detected in 36% of the examined MH-positive patients but had no influence on developing contractures. Sevoflurane-induced contractures do not reliably detect MH susceptibility on an individual level. Therefore, sevoflurane is no suitable alternative for diagnostic use. Mutation-specific effects regarding contracture sizes after incubation with sevoflurane, halothane, or caffeine were not found.

Muscle Magnetic Resonance Imaging in Congenital Myopathies Due to Ryanodine Receptor Type 1 Gene Mutations

To establish the consistency of the previously reported pattern of muscle involvement in a large cohort of patients with molecularly defined ryanodine receptor type 1 (RYR1)-related myopathies, to identify possible additional patterns, and to compare magnetic resonance imaging (MRI) findings with clinical and genetic findings. Blinded analysis of muscle MRI patterns of patients with congenital myopathies with dominant or recessive RYR1 mutations and control patients without RYR1 mutations. We compared MRI findings with the previously reported pattern of muscle involvement. Data from 3 tertiary referral centers. Thirty-seven patients with dominant or recessive RYR1 mutations and 23 controls with other myopathies. Each MRI was classified as typical if it was identical to the reported pattern, consistent if it was similar to the reported one but with some additional features, or different. Images with no or few changes were classified as uninformative. Twenty-one of 37 patients with RYR1 mutations had a typical pattern; 13 had a consistent pattern. Two patients had uninformative MRIs and only 1 had a different pattern. Compared with patients with dominant mutations, patients with recessive mutations and ophthalmoparesis had a more diffuse pattern, classified as consistent in 6 of 8. In contrast, 10 of 11 with recessive mutations but without ophthalmoparesis had a typical pattern. All MRIs of 23 control patients were classified as different. Our results suggest that muscle MRI is a powerful predictor of RYR1 involvement in patients with a congenital myopathy, especially if they carry a dominant mutation or recessive mutations without ophthalmoparesis.

Ryanodine receptor type 1 gene mutations found in the Canadian malignant hyperthermia population

Malignant hyperthermia (MH) is an autosomal dominant pharmacogenetic disorder that is manifested on exposure of susceptible individuals to halogenated anesthetics or succinylcholine. Since MH is associated primarily with mutations in the ryanodine receptor type 1 (RYR1) gene, the purpose of this study was to determine the distribution and frequency of MH causative RyR1 mutations in the Canadian MH susceptible (MHS) population. In this study, we screened a representative cohort of 36 unrelated Canadian MHS individuals for RYR1 mutations by sequencing complete RYR1 transcripts and selected regions of CACNA1S transcripts. We then analyzed the correlation between caffeine-halothane contracture test (CHCT) results and RYR1 genotypes within MH families. Eighty-six percent of patients had at least one RyR1 mutation (31 out of 36), five of which were unrelated individuals who were double-variant carriers. Fifteen of the 27 mutations identified in RYR1 were novel. Eight novel mutations, involving highly conserved amino acid residues, were predicted to be causal. Two of the mutations co-segregated with the MHS phenotype within two large independent families (a total of 79 individuals). Fourteen percent of MHS individuals (five out of 36) carried neither RYR1 nor known CACNA1S mutations. The distribution and frequency of MH causative RyR1 mutations in the Canadian MHS population are close to those of European MHS populations. Novel mutations described in this study will contribute to the worldwide pool of MH-associated mutations in the RYR1 gene, ultimately increasing the value of MH genetic diagnostic testing.

The Genetics of Malignant Hyperthermia and Related Muscular Syndromes

To the Editor: The review by Capacchione and Muldoon1 discusses relationships between malignant hyperthermia (MH) and other muscular diseases presenting with some of the clinical features of MH. The task is made more difficult because of the uncertain relationships between a positive in vitro contracture test for halothane and caffeine, specific expression of the triggering mechanism of MH, and the presence of a genetic defect in the muscular ryanodine receptor type 1 (RYR1) gene. Capacchione and Muldoon state that “a negative RYR1 screen with a positive contracture test does not necessarily mean that the patient is not MH susceptible as there are likely yet unidentified non-RYR1 mutations that confer MHS (MH susceptibility).” Indeed, in the 20% to 30% of individuals lacking an RYR1 mutation, genetic alterations of other proteins involved in calcium release or its regulation could be present.2 Monnier et al.3 described such a mutation in the α-1 subunit of the human dihydropyridine receptor responsible for MH in a French family. Protasi et al.2 and Dainese et al.,4 studying knockout mice lacking skeletal muscle calsequestrin, a calcium-binding and buffering protein involved in the modulation of calcium release from RYR1, suggested that this mutation could be involved in MH and environmental heat stroke. On the basis of analogies between MH and catecholaminergic polymorphic ventricular tachycardia, a cardiac disorder caused by mutation in the cardiac RYR2 gene, MacLennan and Chen5 suggested that both diseases may have a common mechanism. In fact, a mutation in RyR can decrease the threshold for calcium release from sarcoplasmic reticulum, and a mutation in calsequestrin can increase the free calcium concentration over the same threshold, giving rise, in the presence of halothane and catecholamines, to contracture (MH) or arrhythmias (catecholaminergic polymorphic ventricular tachycardia), respectively. It is also possible that a mutation in different genes involved in the production and regulation of different calcium handling proteins could explain not only MH and similar muscular diseases but also some cardiac-related diseases sharing the same genetic anomaly. Luca Siracusano, MD Viviana Girasole, MD Department of Neuroscience, Psychiatric and Anesthesiological Sciences University of Messina Messina, Italy [email protected] or [email protected]

Enhanced RyR1 Channel Activity by the Knock-In Mouse that Expresses Human Malignant Hyperthermia Mutation T4826I

Malignant hyperthermia (MH) is a life-threatening pharmacogenetic syndrome of skeletal muscle associated with mutations in the Ca2+ release channel - ryanodine receptor type 1 (RyR1). A genetically engineered knock-in mouse that expresses the human C-terminal MH mutation T4826I RyR1 was created. Both RyR1-T4826I/WT (HET) and RyR1-T4826I/T4826I (HOM) mice survive and thrive if unchallenged with triggering agents. Sarcoplasmic reticulum membranes from HET, HOM, and wild type (WT) mice were prepared to study the biochemical and biophysical properties of RyR1complexes using [3H]ryanodine ([3H]Ry) binding analysis, western blotting, and single channels incorporated in bilayer lipid membranes. The results from this study reveal: (1) Significantly elevated [3H]Ry binding in the preparations with rank order HOM >>HET>WT), (2) significant diminution in the inhibitory potency of Ca2+ and Mg2+ for both HOM and HET, (3) initial rates of [3H]Ry binding with rank order HOM>>HET>WT when measured at either 25 °C or 37°C, (4) Significantly greater open probability, longer mean open dwelling time and shorter mean closed times with HOM and HET channels. These data indicate that the T4826I RyR1 mutation confers significantly destabilizes the closed channel conformation of RyR1, and the altered channel properties may be mostly responsible for the abnormal intracellular Ca2+ homeostasis and the MH susceptibility. (Words: 202).

Postmortem molecular screening for mutations in ryanodine receptor type 1 (RYR1) gene in psychiatric patients suspected of having died of neuroleptic malignant syndrome

Postmortem diagnosis of neuroleptic malignant syndrome (NMS) is difficult to perform, because the clinical symptoms just before death are not usually available. Malignant hyperthermia (MH) is a catastrophic, life-threatening hypermetabolic syndrome triggered by certain anesthetics. Ryanodine receptor type 1 (RYR1) gene mutations are known to be involved in susceptibility to MH. Similarities in clinical features, such as elevated body temperature, between NMS and MH have led to the suggestion that NMS is a neurogenic form of MH. In this study, we analyzed possible mutations of the RYR1 gene in 11 psychiatric patients suspected at autopsy to have died of NMS. All cases were suspected of having elevated body temperature at death, and their causes of death could not be determined by autopsy examinations. Two mutations (R4645Q and A612T) in the RYR1 gene were identified. The R4645Q mutation has previously been reported in MH patients, but five heterozygous mutations were also found in 400 Japanese control alleles. The other mutation was novel, and was not found in the same control alleles. The results of this study provide the first successful identification of RYR1 mutations in psychiatric patients suspected at autopsy of having died of NMS. However, the association between RYR1 gene mutations and cause of death in psychiatric patients suspected of dying of NMS remains unclear.

Increasing the number of diagnostic mutations in malignant hyperthermia

Malignant hyperthermia (MH) is an autosomal dominant disorder characterized by abnormal calcium homeostasis in skeletal muscle in response to triggering agents. Today, genetic investigations on ryanodine receptor type 1 (RYR1) gene and alpha1 subunit of the dihydropyridine receptor (DHPR) (CACNA1S) gene have improved the procedures associated with MH diagnosis. In approximately 50% of MH cases a causative RYR1 mutation was found. Molecular genetic testing based on RYR1 mutations for MH diagnosis is challenging, because the causative mutations, most of which are private, are distributed throughout the RYR1 gene. A more comprehensive genetic testing procedure is needed. Therefore, we aim to expand the genetic information related to MH and to evaluate the effect of mutations on the MH phenotype. Performing an in-depth mutation screening of the RYR1 transcript sequence in 36 unrelated MH susceptible (MHS) patients, we identified 17 novel, five rare, and eight non-disease-causing variants in 23 patients. The 13 remaining MHS patients presented no known variants, neither in RYR1 nor in the CACNA1S binding regions to RYR1. The 17 novel variants were found to affect highly conserved amino acids and were absent in 100 controls. Excellent genotype-phenotype correlations were found by investigating 21 MHS families-a total of 186 individuals. Epstein-Barr virus (EBV) lymphoblastoid cells carrying four of these novel mutations showed abnormal calcium homeostasis. The results of this study contribute to the establishment of a robust genetic testing procedure for MH diagnosis.

A Double Mutation of the Ryanodine Receptor Type 1 Gene in a Malignant Hyperthermia Family with Multiminicore Myopathy

Background and purposeAt least 100 Ryanodine receptor type 1 (RYR1) mutations associated with malignant hyperthermia (MH) and central core disease (CCD) have been identified, but 2 RYR1 mutations accompanying multiminicore myopathy in an MH and/or CCD family have been reported only rarely.MethodsFifty-three members of a large MH family were investigated with clinical, histopathologic, RYR1 mutation, and haplotyping studies. Blood creatine kinase (CK) and myoglobin levels were also measured where possible.ResultsSequencing of the entire RYR1 coding region identified a double RYR1 mutation (R2435H and A4295V) in MH/CCD regions 2 and 3. Haplotyping analysis revealed that the two missense heterozygous mutations (c.7304G>A and c.12891C>T) were always present on a common haplotype allele, and were closely cosegregated with histological multiminicores and elevated serum CK. All the subjects with the double mutation showed elevated serum CK and myoglobin, and the obtained muscle biopsy samples showed multiminicore lesions, but only two family members presented a late-onset, slowly progressive myopathy.ConclusionsWe found multiminicore myopathy with clinical and histological variability in a large MH family with an unusual double RYR1 mutation, including a typical CCD-causing known mutant. These results suggest that multiminicore lesions are associated with the presence of more than two mutations in the RYR1 gene.

Screening of the Entire Ryanodine Receptor Type 1 Coding Region for Sequence Variants Associated with Malignant Hyperthermia Susceptibility in the North American Population

Malignant hyperthermia (MH) is a life-threatening and frequently fatal disorder triggered by commonly used anesthetics. MH susceptibility is a genetically determined predisposition to the development of MH. Mutations in the ryanodine receptor type 1 (RYR1) gene are the major cause of MH susceptibility. The authors sought to develop a reliable genetic screening strategy based on efficient and relatively inexpensive mutation-detection procedures. A cohort (n = 30) of North American MH patients and MH-susceptible individuals was studied. RNA and DNA extracted from muscle tissue or blood lymphocytes were used for analysis. The entire RYR1 coding region was amplified in 57 overlapping fragments and subjected to denaturing high-performance liquid chromatography analysis followed by direct nucleotide sequencing to characterize RYR1 alterations. Nine previously reported and nine unknown RYR1 mutations were identified in 21 of 30 studied patients (70%). Some of the new mutations were located outside of known mutational "hot spots," suggesting that RYR1 contains previously unknown mutation-prone areas requiring analysis. The North American MH/MH-susceptible population is characterized by a high RYR1 allelic heterogeneity. Denaturing high-performance liquid chromatography analysis of RNA samples extracted from the biopsied skeletal muscle followed by DNA sequencing is a highly efficient methodology for RYR1 mutation detection. This approach allows increasing the rate of mutation detection to 70% and identifying mutations in the entire RYR1 coding region.