Malignant hyperthermia (MH) is an autosomal dominant pharmacogenetic disorder triggered by volatile halogenated anesthetics including halothane, isoflurane, enflurane, desflurane, and sevoflurane, and the depolarizing neuromuscular blocking drug, succinylcholine.1 2 2+ release channel of the sarcoplasmatic reticulum, the ryanodine receptor 1 (RYR1), plays a central role in the excitation–contraction coupling. Each subunit of the homotetrameric RYR1 comprises 5038 amino acid residues, making the receptor the largest known ion channel. Mutations within the RYR1 protein are the major cause of MH and central core disease. Mutations in the RYR1 protein are considered to account for susceptibility to MH in >70% of all cases.3
Patients harboring one of the known causative mutations in the RYR1 gene can be diagnosed as MH susceptible (MHS) by a less invasive genetic blood test without the need for a muscle biopsy. However, >170 genetic variants have been detected in RYR1 and linked to MH until now. More and more variants outside the “hot spots” have been identified in the last few years.3 – 5 a
METHODS
IVCT
The study was approved by the Institutional Ethics Committee of the Medical University of Vienna (Vienna, Austria). Written informed consent was obtained from all patients scheduled for an IVCT because of suspected MH susceptibility. The IVCT is performed when there is a family history of MH or after a possible MH episode during general anesthesia. Individuals with an ASA grading scale of 4 and 5 and children younger than 16 years are excluded from the test in our center. The IVCT was performed according to the protocol of the EMHG, in which muscle specimens are exposed to incremental doses of halothane and caffeine.6
For this study, sequencing of the entire coding region of the RYR1 gene was performed only from individuals with a clear MH disposition (contracture of at least 0.2 g to a halothane concentration of 0.11 mM (0.5%), and a caffeine concentration of 1 mM).
Cell Culture
Sera and media for cell culture were obtained from PAA (Linz, Austria). Trypsin-EDTA, glutamax, penicillin, streptomycin, gentamicin, and amphotericin B were from GIBCO (Vienna, Austria), trypsin from Roche (Vienna, Austria), and fura2-AM from Molecular Probes (Invitrogen, Lofer, Austria). Fibronectin and matrigel were purchased from BD Biosciences (Bedford, MA). All other chemicals were obtained from Sigma Aldrich GmbH (Vienna, Austria).
Muscle obtained at the time of biopsy for the IVCT was cut and digested to grow cultured human skeletal muscle cells as described previously.7
Cells were kept at 37°C under 2.5% CO2 in an incubator, grown close to confluence, and reseeded on 25-mm glass cover slips coated with fibronectin or matrigel. Adherent cells were exposed to differentiation medium to promote fusion of myoblasts to multinucleated myotubes in an incubator with 5% CO2 .
Genetic Analysis
Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the Genebank reference sequence NM_000540.2 for the human RYR1.
Amino acid substitutions for the human RYR1 (Genebank accession no. NP_000531.2) are named according to their position with the first methionine encoded by the ATG start codon designated as amino acid number “1.”
The entire cDNA of the RYR1 of family member A-III:2 (Fig. 1 , family A) and B-II:3 (Fig. 1 , family B) was amplified and sequenced. RNA was isolated from differentiated muscle cells on day 4 after changing from the growth medium to the differentiation medium with the peqgold total RNA kit from peqlab (Erlangen, Germany). Subsequently, the RNA was reverse transcribed into cDNA using the SuperScript First-Strand cDNA Synthesis System (Invitrogen, Lofer, Austria). The cDNAs were used as templates for amplification of parts of the RYR1 sequence with the polymerase chain reaction (PCR) technique. Oligonucleotide primers were used to amplify 23 overlapping sequences of the RYR1 gene.8
Figure 1: Pedigrees and results of the in vitro contracture tests (IVCT). The diagnoses of the IVCT are given as MHS (black symbols = malignant hyperthermia susceptible), MHEH (checkered = malignant hyperthermia equivocal to halothane), or MHN (white = malignant hyperthermia normal), and ? not tested in the IVCT. The results of the genetic tests are given as the position of an amino acid exchange or in case of no exchange, the position is shown in brackets. Arrows denote the index patients; + = heterozygous for the mutant allele; − = homozygous for the wild type allele. A, Family A: the entire cDNA of the ryanodine receptor 1 (RYR1) of family member A-III:2 was sequenced and a heterozygous transversion from guanine to cytosine at position 1834 of the cDNA implies an exchange of alanine 612 to proline. All family members of the third generation are heterozygous carriers of this p.A612P variant. The index patient (A-III:4) was not tested by the IVCT but was diagnosed MHS because of the symptoms during an anesthesia. In addition the p.A612P variant was found in his genomic DNA. B, Muscle contractures induced by increasing concentrations of halothane and caffeine in the IVCT from the individual A-III:2. C, Pedigree of family B. Family member B-I:2 was diagnosed MHEH; B-II:3 and B-II:4 are MHS. While the index patient (arrow) is heterozygous for the causative MH mutation p.R2458H Figure 1. (Continued) (an exchange of arginine to histidine) and the new RYR1 variant p.R3348C (a change of arginine to cysteine), his mother, his sister, and his half-sister are heterozygous carriers of only 1 of these variants. D, Muscle strips of the individual with the new p.R3348C (MHEH) variant reacted to halothane but not to caffeine in the IVCT (top trace). The muscle strip with the double mutation from the index patient (bottom trace) showed the maximum contracture already at the lowest halothane concentration used.
To check whether one of the newly found variants was present in other family members or control individuals, we extracted genomic DNA with the “DNA isolation kit for mammalian blood” (Roche Applied Science, Austria) from peripheral blood according to the manufacturer's protocol. Intronic primers to amplify RYR1 exons 17, 46, and 67 with their flanking intron boundaries were designed and used with AmpliTaq polymerase (Invitrogen, Lofer, Austria). To exclude a possible involvement of a known MH causative DHP-receptor mutation, the primers 25i5 and 25i3 were used for detection of the R1086H substitution in the α1 subunit of the L-type Ca2+ channel.9
PCR products were then purified with the Spin PCRapid purification kit (Invitek, Berlin, Germany) and sequenced by a commercial sequencing laboratory (DI Martin Ibl, Vienna, Austria). Both strands of all PCR products were sequenced.
Determination of Ca2+ Concentration
Cells on coverslips were incubated with the Ca2+ -sensitive fluorescent dye fura-2/AM. The loading buffer was Tyrode's solution (137 mM NaCl, 5.6 mM glucose, 5.4m KCl, 2.2 mM NaHCO3 , 1.1 mM MgCl2 , 0.4 mM NaH2 PO4 , 10 mM HEPES/Na, 1.8 mM CaCl2 , pH 7.4) supplemented with 5 to 10 μM fura-2/AM and 0.025% F-127 pluronic. After incubation in loading buffer for 45 to 60 minutes at 37°C, unloaded dye was washed out and coverslips were placed into a perfusion chamber of a Nikon Diaphot-300 fluorescence microscope at 400× magnification (Nikon, Vienna, Austria). Only cells that reacted to depolarization solution HK (HK: Tyrode's solution with 60 mM KCl and 80 mM NaCl, no Ca2+ added) with an increase in intracellular Ca2+ concentration ([Ca2+ ]i ) were used for experiments, assuming a skeletal muscle specific excitation–contraction coupling. Fluorescence intensity was monitored at an emission wavelength of 510 nm by altering excitation wavelengths between 340 and 380 nm using a monochromator (VisiTech, Sunderland, United Kingdom).
For measurement of [Ca2+ ]i , images were recorded with a sample interval of 0.5 seconds to 2 seconds. Stored images were analyzed using the QC2000 software package from VisiTech. Background subtraction, rationing, and calculation of [Ca2+ ]i were done offline using the SigmaPlot 11.0 program (Systat Software Inc., Chicago, IL). Resting [Ca2+ ]i was defined by the average of the first 20 data points before application of any substances. The value of a [Ca2+ ]i transient was determined by the peak value reached within the time of substance application.
Calibration of fura-2 fluorescence signals to calculate [Ca2+ ]i values were done according to Grynkiewicz et al.10 11
Concentration response curves were assessed by application of increasing concentrations of substances to a cell and measurement of [Ca2+ ]i . After washout of the substance and decline of [Ca2+ ]i to the resting level, the next higher concentration was applied. Least squares fitting of concentration response curves to the Hill equation was performed with SigmaPlot. Because of the high volatility of halothane and evaporation from the solution, the real concentration of halothane in the applied solutions was determined by gas chromatography for each concentration (Fig. 2 ). The halothane concentrations in Figure 3 are depicted as means ± SE.
Figure 2: Time course of [Ca2+ ]i in myotubes for the determination of halothane concentration response curves. A, [Ca2+ ]i in a cell from an individual diagnosed as malignant hyperthermia (MH) nonsusceptible (MHN; double peaks during application of activators of the RYR1 can be seen occasionally and are also observed with other substances). B, Susceptible to MH with an exchange of arginine to histidine at position 2458 (p.R2458H, MHS). C, Susceptible to MH with p.R2458H and the additional exchange of an arginine to cysteine at position 3348 (p.R3348C). D, Equivocal to halothane (MHEH) with the p.R3348C variant. HK Ca2+ = 0 depicts application of a Ca2+ -free solution with increased potassium to depolarize the cell, demonstrating skeletal muscle type excitation contraction coupling. Increasing concentrations of halothane resulted in increasing Ca2+ transients. Numbers above the application bars give the concentration of halothane in millimolar.
Figure 3: Halothane concentration–response curves. Cells with mutated RYR1 showed a shift of the EC50 values to lower concentrations. These cells were clearly more sensitive to halothane than malignant hyperthermia nonsusceptible (MHN) control cells. Lines are least squares fits through the symbols according to the Hill equation; data are given as mean ± SE with the number of single-cell experiments ranging from 9 to 15.
Cells from the MH negative (MHN) control group were derived from 6 nonrelated individuals and from 2 relatives of family A; the cells in the p.A612P group stem from 3 members of 1 family; and cells with the p.R2458H, the p.R3348C, and the p.R2458H/p.R3348C variants were from 1 person each from the same family.
Determination of Halothane Concentrations
Halothane-containing solutions were prepared by dilution of a saturated solution of halothane in Tyrode's solution for the concentrations 0.1 mM, 0.3 mM, 0.5 mM, and 1 mM; for higher concentration (3 mM, 5 mM, and 10 mM) a 20% halothane solution in dimethylsulfoxide was prepared and diluted. The real concentration of halothane in the solution used for experiments was determined by gas chromatography and is indicated in Figures 2 and 3 . Gas chromatography was performed by analyzing the equilibrated gas phase of the sample with a head space sampler (Model 7694, Agilent Technologies, Vienna, Austria) and an HP 5890 gas chromatograph (Agilent Technologies, Austria). A DB624, 3-μm (30 m × 0.53 mm) separation column (J&W Scientific, Agilent Technologies, Austria), and nitrogen as a carrier gas at 60°C were used in combination with a flame ionization detector for the quantification of halothane.
Statistical Analysis
Data of cells from MHS and MH equivocal to halothane (MHEH) individuals with the new variants were compared with data of cells from MHN individuals without the variants. Results of single cell experiments were treated as independent for statistical analysis even when the cells were derived from the same individual. The EC50 and maximum Ca2+ release values were determined by fitting each experiment to the Hill equation. The calculated maximum Ca2+ release value was used for normalization of the concentration response curves. Data were tested for normality with the Shapiro Wilk test, and parametric data were compared with the 2-sample t test, whereas nonparametric data were compared with the Mann–Whitney rank sum test. P values smaller than 0.05 were considered to indicate a statistical significant difference. All data are shown as mean ± SE. Statistical analysis and curve fitting was performed using SigmaPlot 11.0.
RESULTS
Genetic Analysis
For this study, we sequenced the RYR1 gene only from individuals with a clear positive (MHS) test result but without a previously identified MH mutation. From representatives of 4 Austrian families the cDNA of the RYR1 was analyzed; in 3 of them new variants were found and characterized. One of these new variants was described elsewhere.12
Family A.
During general anesthesia for tonsillectomy a 6-year-old boy developed signs of MH. A first sign after induction with nitrous oxide and halothane were livid spots on both upper limbs. After the application of succinylcholine (1 mg/kg), minor tachycardia, tachypnea, and spasms of the masseter and upper limbs were observed but no hyperthermia. The day after the episode, GOT (654 U/l) and GPT (163 U/l) were elevated as well as other variables (total bilirubin 41 μM; LDH: 1560 U/L; alkaline phosphatase: 363 U/L; blood urea nitrogen: 11.2 mM; creatinine: 140 μM). The creatine phosphokinase value increased to 3777 U/l 2 days after the episode and myoglobinuria was diagnosed, whereas the other variables tested tended to normalize. Because of his age, the index patient did not undergo the IVCT. The mother and 3 siblings of the child were diagnosed MHS (Fig. 1 A), whereas the father and 2 cousins of the mother were diagnosed not MH susceptible (MHN) by the IVCT. Unfortunately, neither muscle biopsy samples nor blood samples from the mother and the father were available. Hence, the entire RYR1 cDNA of family member A-III:2 was sequenced. A transversion was identified in exon 17 at position c.1834G>C (rs118204423) and implies an amino acid exchange from alanine to proline at position 612 of the protein. This variant was also detected by analysis of the respective genomic DNAs in his 3 siblings (including the index patient) but not in their aunt and uncle, who were diagnosed MHN by the IVCT.
Furthermore, this variant was not found in 73 other MHS individuals of other families nor in 100 MHN individuals.
Family B.
In the context of a shoulder surgery, the patient had general anesthesia with fentanyl, propofol, and sevoflurane. After 50 minutes, he developed signs of a possible MH crisis but without muscle rigidity; the anesthesia was aborted, and therapy with dantrolene (8 mg/min for 30 minutes) was commenced. During the crisis the creatine phosphokinase value was 1891 U/L and increase to 2952 U/L after 24 hours. Histologically, a minimal unspecific myopathic syndrome was diagnosed with slightly decreased oxidative enzyme activity in the core regions of the muscle fibers. The index patient and his sister were diagnosed MHS by the IVCT, while their mother was diagnosed MHEH (family B in Fig. 1 C). The complete cDNA of the RYR1 gene of the index patient was sequenced and 2 compound heterozygous mutations were detected: a transition from guanine to adenine at position c.7373 (leading to amino acid exchange p.R2458H) and a transition from cytosine to guanine in exon 67 at position c.10,042 (rs118204421, amino acid exchange p.R3348C). While family member B-II:4 is heterozygous for the causative MH mutation c.7373,13 14
Ca2+ Imaging Experiments
For this study, differentiated skeletal muscle cells from MHS individuals A-III:1, A-III:2, A-III:3 (Fig. 1 , family A), B-I:2, B-II:3, and B-II:4 (Fig. 1 , family B) were used for Ca2+ -imaging experiments to determine their Ca2+ -release properties in the presence of caffeine, 4-chloro-m-cresole (4-CmC), and halothane. As controls cells from 6 nonrelated MHN individuals (control group) and from 2 related individuals from family A (A-II:3 and A-II:4, control Relative) without the variants were used. The Ca2+ -releasing agents specifically activate the RYR1 to cause Ca2+ release. The resulting changes in the intracellular Ca2+ concentration ([Ca2+ ]i ) and the sensitivities to these pharmacological agents were determined. To calculate the concentration– response curves according to the Hill equation, the specific substances were applied in increasing concentrations to differentiated muscle cells: 0.1 to 10 mM halothane (Fig. 2 A), 1 to 60 mM caffeine, and 10 to 1000 μM 4-CmC while the [Ca2+ ]i was monitored. MHS cells with the A612P and the double mutation displayed the lowest EC50 values for all substances tested (Table 1 ).
Table 1: Pharmacological Variables of Cells from Different Diagnostic Groups
The EC50 value for Ca2+ release by halothane in MHN control cells was 1.4 mM (14 cells from 2 individuals). In comparison, cells with either the R2458H (n = 15) or the p.R3348C (n = 13) mutation reacted about 3 times more sensitively. When both mutations were present in the Ca2+ -release channel (n = 12), the EC50 value was further decreased by about 50% (Table 1 and Fig. 3 ).
For caffeine the EC50 value of all MHS and also the MHEH cells was approximately 1 mM, whereas MHN cells (26 cells from 5 individuals) reached the half maximum Ca2+ increase at 6.5 mM caffeine (Table 1 , Fig. 4 ). The situation was less clear when cells were perfused with 4-CmC. Although double-mutated cells (n = 7) were about twice as sensitive as control cells (29 cells from 3 individuals), p.R2458H cells (n = 9) and p.R3348C cells (n = 18) did not show increased sensitivities (Fig. 5 ).
Figure 4: Caffeine concentration–response curves. Sensitivity of cells with the RYR1 variants to caffeine as a test substance. MHS (malignant hyperthermia susceptible) cells were 4 (p.A612P) to 8 times (double variant) more sensitive to caffeine than control MHN (malignant hyperthermia nonsusceptible) cells. MHN cells from relatives of the p.A612P carriers (MHN Relative) have the same caffeine sensitivity as normal cells from nonrelated persons. Although the p.R3348C variant was diagnosed MHEH (malignant hyperthermia equivocal to halothane) in the in vitro contracture test, on the cellular level the Ca2+ -release properties resemble those of MHS cells. Data are mean ± SE from 9 to 26 single-cell experiments. Curves were obtained by least square fitting of the symbols to the Hill equation.
Figure 5: The 4-chloro-m-cresol (4-CmC) concentration–response curves. Cells with the p.A612P and the double mutation in the RYR1 showed similar sensitivities for 4-CmC. In contrast, cells with the p.R2458H and the p.R3348C variant were unaffected. Malignant hyperthermia nonsusceptible (MHN) cells from relatives of the p.A612P carriers (MHN Relative) have the same 4-CmC sensitivity as cells from nonrelated normal individuals. Data are mean ± SE from 7 to 29 single-cell experiments. Curves were obtained by least square fitting of the symbols to the Hill equation.
The analysis of the maximum Ca2+ increase revealed that cells with the double mutation/variant p.R2458H/p.R3348C in RYR1 showed a significantly reduced maximum Ca2+ increase by caffeine, 4-CmC and halothane (Table 1 ) in comparison with MHN cells. Cells with the p.R2458H and the p.R3348C variants showed a small but not significant reduction in maximum Ca2+ increase. Cells with the A612P variant did not show a significant change in the maximum Ca2+ increase with caffeine or 4-CmC, but it was slightly stronger with halothane.
The mean cytoplasmic resting [Ca2+ ]i in MHN cells was determined to be 75 ± 5 nM SEM, (n = 69 cells from 6 nonrelated individuals) but only 37 ± 5 nM in MHN cells of family A, whereas cells bearing the A612P variant showed a resting [Ca2+ ]i of 96 ± 4 nM (n = 59, P < 0.001). Significantly increased resting [Ca2+ ]i was also found in cells harboring the p.R2458H variant (91 ± 4 nM; n = 33, P = 0.007) and the p.R3348C variant (89 ± 4 nM; n = 49, P = 0.033) but not in cells with the double amino acid exchange p.R3348C/p.R2458H (78 ± 5 nM; n = 32, P = 0.313).
DISCUSSION
We found 2 new genetic variants in individuals with a clear MH disposition. For the following reasons we assign these variants a causative role in the etiology of MH:
The p.A612P as well as p.R3348C are located in evolutionary highly conserved regions.
The p.A612P is in close proximity to one of the most common causative MH mutations (p.R614C).
Ca2+ -release experiments showed increased sensitivity of cells with the new variants against caffeine and halothane, specific activators of the RYR1.
A missense mutation at the p.R3348 site has already been found in another nonrelated MHS subject.14
In >100 MH nonsusceptible individuals these variants were absent.
The substitution of an alanine by a proline in the newly found variant p.A612P could easily affect the function of the RYR1 because unlike alanine, proline is hydrophilic and acts as a disruptor of secondary structure elements such as α helices and β sheets. The first RYR1 mutation linked to MH was p.R614C,15 – 17 3 18 19 2+ channel and render it more sensitive to RYR1 activators. The region from amino acid 609 to 615—NGVA VRS— is highly conserved among various species. It is identical in rabbits (Oryctolagus cuniculus ), zebrafish (Danio rerio ), fruit flies (Drosophila melanogaster ), and roundworms (Caenorhabditis elegans ). In addition, the conserved character of this region within the RYR1, RYR2, and RYR3 isoforms implies an important functional role of this domain.
The situation is less clear with the second new variant, R3348C, because the family affected is very small and the interpretation of the heredity within the pedigree is also complicated by the occurrence of another known causative MH mutation (p.R2458H20 14 21 Sus scrofa ), mice (Mus musculus ), rat (Rattus norvegicus ), and zebrafish (Danio rerio ) shows that the amino acids near arginine (R) p.3348—QPIVSR A— are totally conserved. The RYR2 and RYR3 isoforms in mammals have a lysine at this position, which is chemically very similar, so the amino acid in position p.3348 of RYR1 is highly conserved among the species.
At the cellular level the phenotype of p.R3348C cells showed sensitivities for halothane and caffeine, which are more consistent with an MHS diagnosis than with an MHEH diagnosis. The EC50 values for caffeine (1.2 mM), and halothane (0.33 mM) in the cellular test were significantly lower than in MHN control cells (6.5 mM caffeine and 1.4 mM halothane, respectively). Interestingly, the EC50 for 4-CmC was unchanged in p.R3348C cells (103 μM vs 100 μM in MHN control cells; Table 1 ) as well as in cells with the causative p.R2458H mutation. 4-CmC is often not able to produce significant differences between MHEH and MHN cells,22 23 24
The maximum Ca2+ increase reached during the application of caffeine, 4-CmC, and halothane was significantly reduced in cells with the double mutation/variant p.R2458H/p.R3348C (Table 1 ). Cells with the p.A612P variant showed an increased maximum Ca2+ release for halothane, whereas cells harboring the p.R2458H or the p.R3348C mutations did not differ significantly from the control group (Table 1 ). The decrease in the maximum Ca2+ increase without increased resting [Ca2+ ]i for the double-mutated cells could be the result of an at least partially defective sarcoplasmic reticulum Ca2+ -release channel. In the histology a minor unspecific myopathy was diagnosed, as can be found often in MHS subjects. How these alterations are related to a functional change in the RYR1 due to the found variants remains elusive.
To confirm these new genetic variants as causative MH mutations, further cases in other nonrelated MH families have to be found. However, the absence of these new mutations in >100 MHN control individuals supports their possible causative role for MH. Alternatively, functional in vitro expression of mutated RyR1 in dyspedic muscle cells could prove their causative role.
The presence of 2 possible MH mutations/variants in the RYR1 of the index patient of family B shows the difficulty of performing valid segregation analyses without sequencing the entire coding region of RYR1. From the clinical point of view the occurrence of double mutations could exacerbate the MH syndrome in affected individuals. In the IVCT the index patient of family B carrying the double variant in the RYR1 showed maximum tension development already at the lowest halothane concentration. Although sevoflurane is considered the least potent trigger substance since it is the least potent volatile anesthetic to induce muscle contractures in the IVCT,25 26
The possibility of double mutations led to the guidelines of the EMHG for genetic testing27
CONCLUSIONS
This study describes and characterizes novel RYR1 variants not only in, but also outside, the MH “hot spot” regions of RYR1, which seem to have causative effects. Finally, the identification and characterization of such novel, putative causative, MH mutations is important to refine genetic testing and our understanding of the molecular mechanisms leading to MH.
DISCLOSURES
Name: Alexius Kaufmann, PhD.
Contribution: This author helped conduct the study, analyze the data, and write the manuscript.
Attestation: Alexius Kaufmann has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Birgit Kraft, MD.
Contribution: This author helped conduct the study.
Attestation: Birgit Kraft has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Andrea Michalek-Sauberer, MD.
Contribution: This author helped conduct the study and write the manuscript.
Attestation: Andrea Michalek-Sauberer has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Marta Weindlmayr, PhD.
Contribution: This author helped conduct the study and analyze the data.
Attestation: Marta Weindlmayr has seen the original study data and approved the final manuscript.
Name: Hans G. Kress, MD.
Contribution: This author helped design the study and write the manuscript.
Attestation: Hans G. Kress approved the final manuscript.
Name: Ferdinand Steinboeck, PhD.
Contribution: This author helped analyze the data and write the manuscript.
Attestation: Ferdinand Steinboeck has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Lukas G. Weigl, PhD.
Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.
Attestation: Lukas G. Weigl has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
This manuscript was handled by: Peter J. Davis, MD.
a Available at: http://www.emhg.org/genetics/mutations-in-ryr1/ Cited Here
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