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RYANODINE receptors (RYRs) belong to the superfamily of ion-gated channels, having three different isoforms. Functional units are homotetramers of approximately 5,000 amino acids per subunit coded by 150-Kbp-long genes. The RYR type 1 is the main isoform expressed in skeletal muscle, forming the sarcoplasmic reticulum calcium release channel.1To date, more than 100 mutations in the huge gene that encodes the RYR type 1 (RYR1 ) have been associated with malignant hyperthermia (MH), central core disease (CCD), or both, but definitive evidence of a causative role is still lacking for most of them.2,3,4
Malignant hyperthermia is a pharmacogenetic disorder in which susceptible individuals develop a hypermetabolic state with generalized muscle contracture in response to a massive calcium release from the skeletal muscle sarcoplasmic reticulum after the administration of volatile general anesthetics or the depolarizing muscle relaxant succinilcholine.5,6The diagnosis of susceptibility to MH is achieved by the whole-muscle caffeine–halothane contracture test in North America7and by thein vitro contracture test (IVCT) in Europe,8whereas in Japan, it is determined by detecting an enhancement of the rate of calcium-induced calcium release (CICR) from the sarcoplasmic reticulum in comparison to reference values previously measured in healthy controls.9–11The CICR test is performed on skinned fibers prepared from patients’ skeletal muscle.
On the other hand,CCD is a congenital myopathy characterized by hypotonia in infancy, different degrees of proximal dominant muscle weakness, delayed motor development, and reduced muscle bulk. CCD derives its name from the “core” structures seen in type 1 skeletal muscle fibers, which consist of amorphous central areas that lack enzyme activity on nicotinamide adenine dinucleotide-tetrazolium reductase staining.12,13MH susceptibility and CCD are inherited mostly as autosomal dominant traits with variable penetrance. It seems clear by now that the main underlying pathomechanism in MH susceptibility is a dysregulation of calcium homeostasis in skeletal muscle determined by multiple interacting genetic factors, with approximately 50% of cases linked to theRYR1 and at least five other loci being apparently involved.14In contrast, the majority—if not all—of CCD cases are due toRYR1 mutations.15
Most of MH/CCD-related mutations have been found mainly in three regions or “hot spots”: domain 1, in the cytoplasmic aspect of the protein near the N-terminus between residues p.M1 and p.R614; domain 2, in the central region (p.R2163–p.R2458); and domain 3, in the C-terminal region (p.R4136–p.P4973) that encodes the transmembrane pore-forming segment.2However, it is possible that the so-called hot spots might be a result of experimental bias due to insufficient investigation of other gene regions.
In an effort to develop a less invasive diagnostic test for MH susceptibility, special attention has been given toRYR1 mutation screening. In a recent study, the detection rate of potentially causative mutations in North American MH-susceptible population increased from 25% to 70% through a systematic investigation of theRYR1 entire coding region in lieu of a screening limited to the hot spots.16
However, the vast size of theRYR1 gene and the genetic heterogeneity of the MH trait make of molecular diagnosis of MH susceptibility a painstaking task. Moreover, the incidence and distribution of mutations along theRYR1 gene in MH and in CCD patients is highly variable among populations.17Nevertheless, molecular genetic analysis represents a useful tool in the struggle for unraveling the pathogenic background of MH and CCD. When mutation frequencies in the correspondent geographic region are determined and integrated with pedigree information,18and the causative role of candidate mutations is functionally demonstratedin vitro ,19genetic analysis constitutes a supplement for the standard diagnostic tests inex vivo muscles. Considering these facts, we aimed to determine the frequency and distribution ofRYR1 mutations in the Japanese MH-susceptible population.
Materials and Methods
Patients
This study was approved by the institutional review board of the National Institute of Neuroscience, National Institute of Neurology and Psychiatry, Tokyo, Japan. We selected 58 unrelated Japanese patients diagnosed as MH susceptible by the CICR test (i.e. , enhanced CICR rate, see section titled MH Susceptibility Diagnosis: The CICR Test) from the database of our institution. The sample consisted of 33 males and 25 females ranging in age from 5 to 77 yr with a mean age of 33.5 ± 18.5 yr. Informed consent for genetic analysis was obtained from each patient or from their parents. The correspondent clinical records and biopsy specimens were reviewed—when available—and patients were classified according to the type of CICR enhancement (clear or abnormal) and according to the presence or absence of cores on muscle biopsy’s nicotinamide adenine dinucleotide-tetrazolium reductase staining.
Twenty-nine patients were recruited after experiencing an MH episode (table 1), 17 for having an MH relative, whereas the remaining 12 subjects had the CICR test under suspicion of MH susceptibility for different reasons: Three of them had been diagnosed with CCD, whereas the only remarkable finding among the others was an increase of serum creatine kinase in the absence of remarkable muscle symptoms.
Table 1:Clinicopathologic Diagnosis and RYR1 Phenotype of Japanese Malignant Hyperthermia–susceptible Individuals
Statistical Analysis
Association of the former categories with the presence or absence ofRYR1 mutations was statistically evaluated using chi-square test. Differences between proportions were analyzed with two-tailedz test. Statistical significance was defined asP < 0.05 orP < 0.01, when specified.
MH Susceptibility Diagnosis: The CICR Test
In this test, the sarcolemma of skeletal muscle fibers is chemically destroyed, thus allowing the stimulation of calcium release from sarcoplasmic reticulum using external calcium solutions at five different concentrations. If the patient’s CICR values are 1.5 SD above the normal average values at two or more calcium concentrations in two or more skinned fibers, the CICR rate is defined as being “clearly enhanced.” Otherwise, CICR test results are reported as “abnormally enhanced” or “not enhanced” (fig. 1).
Fig. 1:Representative results of the Ca2+-induced Ca2+release (CICR) test. Patients with a previous malignant hyperthermia episode typically have clear enhancement of the rate of CICR from sarcoplasmic reticulum (♦) in comparison with normal non–malignant hyperthermia controls (○). However, some malignant hyperthermia individuals have abnormal CICR results only at certain calcium concentrations (▴). Data are expressed as mean ± SD of the reference values for malignant hyperthermia susceptibility diagnosis in Japan.
Mutation Screening
Genomic DNA was extracted from either muscle biopsy samples or peripheral blood lymphocytes according to previously established protocols.20Using Web-based software,‡‡intronic primers approximately 25 bp long were designed to amplify all 106RYR1 exons with their flanking intron boundaries by polymerase chain reaction (PCR). The best condition for each set of primers was chosen after performing gradient temperature PCR (approximately 58°–70°C) using ExTaq polymerase with 10x ExTaq Buffer. LATaq polymerase with 2x GC Buffer-I were used for guanine/cytosine-rich exons. The PCR amplification mix contained a total volume of 25 μl composed of 20–100 ng genomic DNA, 0.4 μm of each primer, 250 μm of each deoxynucleotide triphosphate, and 1 U enzyme diluted in an appropriate buffer volume. Dimethyl sulfoxide, 0.5%, was added to the PCR mix to improve specific annealing when needed. Additional information on PCR conditions and primer sequences is available on the Anesthesiology Web site athttp://www.anesthesiology.org. Enzyme/buffer kits and deoxynucleotide triphosphate for PCR reactions were purchased from Takara (Tokyo, Japan). Enzyme and primers were removed after amplification by vacuum-filtration in Montage PCR96plates (Millipore; Bedford, MA). Purified PCR products ranging from approximately 250 to 1,000 bp were directly sequenced in both directions following the BigDye Terminator version 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) protocol on an automated ABI 3130xl Genetic Analyzer (Applied Biosystems-Hitachi, Foster City, CA). Before sequencing, all samples were further purified by centrifugation through Sephadex G-50 columns (Sigma-Aldrich, St. Louis, MO) in Multiscreen 96-Well HV plates (model MAHV N45; Millipore, Molsheim, France) to eliminate nonincorporated fluorescent dyes. DNA sequences were analyzed and compared with the reference sequence ofRYR1 21provided by the National Center for Biotechnology Information database on the Internet (gene: NM_000540; protein: NP_000531) using SeqScape version 2.5 software (Applied Biosystems). Mutations were detected by detailed eye-inspection of the sequencing data.
Results
Thirty-six distinct DNA sequence variations inducing predicted amino acid changes were found in 41 of 58 patients, consisting of 34 single nucleotide substitutions (all in heterozygous state except in three cases), one 9-bp duplication, and one 3-bp deletion (table 1). The latter was detected as a downstream sequence overlapping that was further confirmed by cloning and sequencing both alleles separately. The affected residues were not limited to the hot spots but rather distributed along the wholeRYR1 gene (fig. 2). To rule out the presence of polymorphisms, we sequenced all of the exons carrying novel sequence variations in 60 control DNA samples available from previous investigations and, in addition, checked a database of common gene variations in the Japanese population.22The control subjects neither belonged to MH pedigrees nor had any known neuromuscular disorder. We also checked the evolutionary conservation of the affected residues amongRYR1 from different species (fig. 3). Two substitutions in exon 34 (c.4999C>T, p.R1667C present in five patients and c.5287C>T, p.P1773S in one patient) were found in two control samples each, upon which we presumed they were nonsynonymous single nucleotide polymorphisms. The substitution c.11266C>G (p.Q3756E) previously reported as nonpathogenic16was found in seven patients. In addition, another nonsynonymous polymorphism (c.5287C>T, p.P1763S) was present in two control samples but not among MH-susceptible individuals. Twenty-nine silent single nucleotide polymorphisms were in addition found (data not shown).
Fig. 2:Distribution of sequence variations along the ryanodine receptor type 1 gene. Exons harboring sequence variations that were detected in this study are represented as solid boxes , in light or dark gray , depending on their location within or outside the hot spots, respectively. Predicted amino acid substitutions written above the exon boxes have been detected in Japan and mainland Asia ( underlined if previously reported in other populations), whereas those below the boxes have been reported in Western countries. Nonsynonymous single nucleotide polymorphisms are indicated in light gray .
A list of references corresponding to each sequence variation is available on the Anesthesiology Web site at http://www.anesthesiology.org. †= Detected in this study; ⊙= associated with cores. The occurrence of recurrent substitutions is written between parentheses. Fig. 3:Evolutionary conservation among species of the ryanodine receptor type 1 gene: residues affected by sequence variations in malignant hyperthermia–susceptible Japanese. Sequence variations in boldface have not been previously reported. Symbols represent the phenotype of the mutation carriers. CICR = Ca2+-induced Ca2+release test: ♦= clearly enhanced; UNHANDLED_ENTITY=abnormally enhanced; ⊙= cores in muscle biopsy. Nonsynonymous single nucleotide polymorphisms detected in control DNA samples are written in light gray . Nonconserved residues are highlighted with a gray background .
Of 33 potentially pathogenic sequence variations, 28 (84.8%) were private (i.e. , present only in one pedigree). Two homozygous nucleotide changes were found in the same patient, the child of a consanguineous marriage. Two different heterozygous substitutions were identified in six patients as well. Interestingly, the proportion of patients with clearly enhanced CICR was significantly greater in this group than among those who carried only one plausibly pathogenic substitution (0.83vs. 0.41, respectively;z = 2.41;P = 0.016, two tailed).
TheRYR1 genotype was associated with the type of CICR enhancement and with the presence of cores on nicotinamide adenine dinucleotide-tetrazolium reductase staining in the correspondent muscle biopsies. Pathology specimens were available in 51 patients, and cores were identified in 15 of them. Patients with clearly enhanced CICR, as well as those with cores, were more likely to bearRYR1 mutations (tables 2 and 3). Besides, 13 of 27 MH-susceptible patients (48.1%) withRYR1 mutations had cores, whereas such structures were seen only in 2 patients (J-38 and J-56) among those without mutation. There was no significant difference in the incidence of mutations between patients with clinical MH events (62.1%)versus those who underwent CICR testing by virtue of familial risk only (58.8%).
Table 2:Association between CICR Status and RYR1 Mutation Occurrence
Table 3:Association between the Occurrence of Cores versus RYR1 Mutations
Discussion
Previous genetic investigations in the Japanese MH population either were limited to certain mutational spots or evaluated just a few pedigrees.23–25To the best of our knowledge, this is the first report for most of the potentially pathogenic sequence variations found in our sample (26 of 33), with exception of 6 mutations formerly found in MH population of Western origin (i.e. , c.487C>T, p.R163C26; c.1022G>C, p.G341R27; c.1598G>A, p.R533H28; c. 6488G>A, p.R2163H29; c.7304G>A, p.R2435H30; and c.7373G>A, p.R2458H31) and 1 mutation reported in a Japanese MH-susceptible pedigree (c.14512C>G, p.L4838V25). In addition, the residues p.D16632and p.R245233were also affected as in other studies, but with different predicted amino acid substitutions. The location and distribution of mutations in our Japanese cohort differed markedly from that of MH population in Western countries. For example, in some European cohorts, p.G341R has been found in up to 10% of MH-susceptible individuals,27whereas it was present only in one Japanese patient. Similarly, p.R613C—common in North America34and Germany28with incidences of 9% and 4%, respectively—was not found in our cohort. It is worth noting that the distribution of mutations along theRYR1 gene is not limited to the widely accepted hot spots, even though they occur more frequently in some regions of the gene. In fact, more than one third of the candidate mutations in this cohort were found outside the hot spots.
Mutation Detection Rate
The first genetic study of the entireRYR1 coding region in the MH-susceptible North American population was recently published, yielding a mutation detection rate of 70%.16The authors performed an initial screening using denaturing high-performance liquid chromatography on reverse-transcriptase PCR–amplified complementary-DNA strands, which was followed—to confirm the presence of mutation—by direct sequencing of genomic DNA in those patients whose samples had abnormal chromatography elution patterns. In contrast, we obtained a significantly smaller detection rate of potentially pathogenic sequence variations (56.9%;z = 2.12,P = 0.017). This divergence may be due in part to differences in the selection criteria. We included all available samples from unrelated patients clinically regarded as MH susceptible based on the enhancement of CICR, whereas the North American survey considered caffeine–halothane contracture test results for the recruitment of patients. The concordance among the CICR test, the caffeine–halothane contracture test, and the IVCT test has been evaluated in a previous investigation,35reporting a complete accordance only in 13 of 21 cases. Nevertheless, patients who have had an MH episode were recognized as MH susceptible by all tests. In our study, the incidence ofRYR1 mutations was higher among patients with a clear CICR enhancement (72.4%;table 1). Therefore, we believe that the higher mutation detection rate yielded by the cited North American survey resulted from the exclusion of equivocal results on the caffeine–halothane contracture test.36
MH Susceptibility with Cores
We evaluated muscle pathology whenever available (51 of 58 cases), identifying cores—or core-like structures—in almost one third of patients. Cores were more frequently seen among individuals withRYR1 mutations in comparison to those without mutation (48.1%vs. 8.3%, respectively). From another viewpoint,RYR1 mutations were present in most of our patients with cores (86.7%), indicating that the presence of cores may be a good predictor of MH susceptibility due toRYR1 mutations. Eight patients with cores were not categorized as having CCD because their pathologic findings (e.g. , just a few, rather eccentric, core-like structures with poorly defined borders) differed greatly from those seen in canonical CCD.13In addition, they did not present any clinical signs or symptoms of myopathy.
Interestingly, we did not find any patient with the typical pathologic characteristics seen in CCD cases with mutations in domain 3 (i.e. , type 2 fiber deficiency and central cores with sharp borders in most of type 1 fibers).15Instead, we recognized just a few core-like structures in only two of the C-terminal mutation carriers (J-14 and J-51). All CCD patients included in our survey have an enhanced CICR from the sarcoplasmic reticulum. Contrastingly, CCD patients with mutations in domain 3 may exhibit normal functional results inex vivo muscle tests.37This suggests that the domain 3 mutations associated with MH susceptibility may be functionally different from those located in the same domain and associated with typical CCD. Furthermore, in accordance with our results, CCD-related mutations situated outside theRYR1 pore-forming region are more likely to produce hyperresponsive channels rather than excitation–contraction uncoupling mutants.3,38
MH Susceptibility withoutRYR1 Mutations
We did not find anyRYR1 mutation in 25 patients, of whom 11 had had a clinical episode of MH, 7 had a relative who experienced an MH crisis, 4 had increased creatine kinase with no other symptoms, 1 had multiminicore disease, and the remaining 2 were finally diagnosed with limb-girdle muscular dystrophy 2A. Despite that we may overlook otherRYR1 defects by doing only direct sequencing (e.g. , splicing abnormalities,39epigenetic modifications, large deletions38or duplications), it seems reasonable to consider the influence of another causative MH susceptibility locus in these patients.
The patients with high creatine kinase of unknown origin were originally referred by their physicians (or anesthesiologists) after preoperative routine workup to rule out the possibility of MH susceptibility. As stated previously, the CICR test is designed to detect specific abnormalities on calcium release from the sarcoplasmic reticulum, which is mainly governed by the RYR1 in skeletal muscle. Nevertheless, it may also be secondarily influenced by disturbances of calcium homeostasis in the myocyte from other sources, such as it occurs in individuals affected by neuromuscular syndromes who have positive IVCT results.40,41This might be the case of patients J-56, J-57, and J-58, who had mild clinical manifestations of myopathy. However, it is safer to consider such patients as MH susceptible at the moment of making clinical decisions, having no additional evidence to infer their actual likelihood to experience an MH episode. Actually, we found sequence variations potentially causative of MH in two patients with high creatine kinase (J-51 and J-52), no pertinent MH history, and no remarkable signs or symptoms.
Patients with Two Candidate Mutations
Several probands whose parents both had abnormal IVCT results have been found to harbor two different causative mutations, which is defined as a compound heterozygous state.42In previous reports, patients with compound heterozygous mutations could not be differentiated on the basis of their IVCT results.42However, in our study, five of six patients with two candidate mutations had clear CICR enhancement. In four of them, the causative role of at least one of the mutations was demonstrated previously.28–30,32There still remains the possibility that one of the substitutions is actually a polymorphism, as evidenced in patient J-12. Nevertheless, we could reasonably suggest that a more pronounced enhancement of CICR in these patients may be the result of having two mutations with an additive effect. In the case of patient J-51, who had cores in the muscle biopsy, we also speculate that the “core” and “MH susceptibility” traits might have resulted from one mutation each based on the location of the respective mutations within two different functional domains of the protein.15,43Unfortunately, we were not able to confirm the state of compound heterozygosity by haplotyping or segregation analysis as reported in other investigations44,45because no additional DNA samples from the affected families were available.
Prevalence of MH Trait in Japan
The prevalence of MH in the general population is presumed to be around 1:10,000, based on crisis reports and on its autosomal dominant pattern of inheritance.40,42However, considering the higher-than-expected occurrence of individuals with two possibly predisposing MH alleles, we may want to question this somewhat arbitrary assumption. Having documented a series of MH susceptible patients with a compound heterozygous state, Monnieret al. 42estimated that the incidence of the MH trait in the French population might be as high as 1 in 3,000 people. Based on the size of the Japanese population (1.3 × 108) and on the probability of finding a compound heterozygous MH-susceptible person according to the previously stated incidence (10−4× 10−8× 0.25), we should not have identified more than one patient with compound heterozygous mutations. Taking into account only those patients with two mutations affecting well-conserved residues, we still have four cases in which a compound heterozygous state is most likely. Therefore, we estimate that the prevalence of the MH-predisposing phenotype in Japan might be closer to 1 in 2,000 people. However, this hypothesis may be confirmed only if a noninvasive test suitable for massive screening of MH susceptibility becomes available in the future.
In summary, we determined the frequency and distribution ofRYR1 mutations in a large cohort, evidencing that they account for 56.9% of Japanese MH-susceptible patients. Albeit the causative role of many newly reported nucleotide substitutions remains to be proven, our study represents the cornerstone for establishing a protocol of molecular genetic screening as an aid for the diagnosis of MH susceptibility in Japan. The incidence ofRYR1 mutations is higher in MH-susceptible patients with clear CICR enhancement and in those with identifiable cores on their muscle biopsies. The results of this work raise additional indicia of the genetic heterogeneity underlying MH susceptibility. Screening the whole open reading frame of theRYR1 gene is crucial for genetic investigations in MH-susceptible individuals, because many mutations occur outside the previously known hot spots and some patients may carry two different MH-predisposing sequence variations. Finally, we think that the prevalence of MH susceptibility in the Japanese population might be significantly higher than it is believed.
The authors thank patients, their families, and physicians for their cooperation.
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