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Pediatric Anesthesiology: Research Report

Ryanodine Receptor Type 1 Gene Variants in the Malignant Hyperthermia-Susceptible Population of the United States

Brandom, Barbara W., MD*; Bina, Saiid, PhD; Wong, Cynthia A., MD; Wallace, Tarina, MS; Visoiu, Mihaela, MD§; Isackson, Paul J., PhD; Vladutiu, Georgirene D., PhD; Sambuughin, Nyamkhishig, PhD; Muldoon, Sheila M., MD

Author Information
doi: 10.1213/ANE.0b013e31828a71ff

Dominant mutations in the ryanodine receptor type 1 gene (RYR1) encoding the skeletal muscle–specific intracellular calcium (Ca2+) release channel are a cause of malignant hyperthermia (MH) and central core disease (CCD). RYR1 variants associated with MH have been found in 50% to 86% of MH-susceptible (MHS) families depending on population of origin and study design.1–7 Recessive mutations in RYR1 are found in patients with congenital myopathies including nemaline myopathy, centronuclear myopathy, and congenital fiber type disproportion.8 Most of the subjects of these reports live in Europe or Japan. Differences in the frequency of some RYR1 mutations causative for MH1–4 and different variants of uncertain significance (VUS)1,3–5,7 have been observed in different populations. The aim of the present study was to analyze RYR1 in a large number of MHS subjects from the United States without prior genetic diagnosis. The overall goal of this project was to determine the heterogeneity of variants and mutations in RYR1 in the MHS population of the United States. Such a compendium of variants is important for the genetic diagnosis of MHS and will help in differentiating RYR1 variants that could be causative for MH from rare nonsynonymous polymorphisms.

METHODS

Population

Subject entry criteria included personal history of positive caffeine–halothane contracture test (CHCT), or in cases without CHCT results, personal anesthetic history of an MH event as judged by the MH Diagnostic Center director at the Uniformed Services University of the Health Sciences (USUHS, Bethesda, Maryland; SMM) or the director of the North American MH Registry (BWB), or a family history that included presumed death from MH and/or a positive CHCT in a family member. Subjects with histologic diagnosis of CCD were accepted into this study without CHCT results. Subjects were actively consented through the North American Malignant Hyperthermia Registry and the MH Diagnostic Center at the USUHS. Subjects in whom no genetic variants had been found in previous studies at USUHS3,9,10 were included in the present study. These subjects had previously been recruited from several MH diagnostic centers in the United States. In addition, 12 anonymized specimens were obtained from 1 MH diagnostic center that was no longer active (Northwestern University, Chicago, IL). This study was approved by the IRB at the University of Pittsburgh (Pittsburgh, PA), Northwestern University, and USUHS.

Clinical information, including the personal and family histories of the subjects, was variably obtained from the subjects themselves, data from the North American Malignant Hyperthermia Registry, and records from the MH biopsy center directors. Acute MH episodes were described by the raw score of the Clinical Grading Scale (CGS)11 when clinical details were available. CHCTs were performed and interpreted according to the criteria established by the North American Malignant Hyperthermia Group.12 Individuals were diagnosed MHS if any 1 or more of the 3 muscle exposures produced a contracture exceeding the diagnostic threshold. The threshold values for a positive test were ≥0.7 g contracture in the presence of 3% halothane and/or ≥0.3 g contracture in the presence of 2 mM caffeine. The contracture with the greatest tension in each subject is reported in this study. Only 1 muscle strip per subject was reported in this study. CCD was diagnosed by muscle histology performed by a staff neuropathologist at the Armed Forces Institute of Pathology (Washington, DC). The diagnosis of CCD was based on histochemical identification of amorphous areas (cores) that lack mitochondria and oxidative enzyme activity in type 1 muscle fibers.13

RYR1 Screening

Genetic analysis was performed in a tiered manner. Initially, 30 exons in the 3 mutational hotspots of RYR1 were screened for variants. Subsequently 50, 70, 100 exons, or the entire RYR1 coding region were examined as material resources allowed. In 17 of the subjects, ≥100 exons of RYR1 were examined. Sequencing of RYR1 was performed as described previously.3 In brief, complementary DNA was synthesized using RNA extracted from the frozen muscle biopsy samples, and then amplified in 26 overlapping fragments. In the absence of muscle samples, exons were amplified using genomic DNA extracted from peripheral blood and intronic primers designed for each exon. The RYR1 variants were determined by direct sequencing using an ABI 3100 DNA analyzer (Applied Biosystems, Foster City, CA). The newly identified RYR1 variants were compared with the single nucleotide polymorphism database at the National Center for Biotechnology Information. a The variants of 2 subjects were identified by microarray technology using a Goldengate platform with VeraCode technology (Illumina, Inc., San Diego, CA) as previously described.14 The frequency of novel RYR1 variants identified in this study was determined in healthy unrelated population controls using restriction enzyme or DNA sequencing analysis as described previously.3 Controls were 100 Caucasian individuals, 50 of whom were MH negative by CHCT. The others were healthy individuals from unrelated families enrolled into a genetic study for screening of a familial MH mutation in RYR1.

Screening of Other Genes

In subjects in whom no abnormalities were found in ≥100 exons of RYR1, the α-1 subunit of the dihydropyridine receptor gene (CACNA1S) was screened for 4 variants, Arg174Trp in exon 4, Arg1086His and Arg1086Ser in exon 26, and Thr1354Ser in exon 44 using genomic DNA extracted from peripheral blood. Primers specific to these exons were designed; primer sequences are available on request. Restriction enzyme MspI was used to screen Arg174Trp, and enzyme HhaI was used to screen Arg1086 and Arg1086Ser mutations. The Thr1354Ser was screened using direct sequencing of exon 44 polymerase chain reaction product. In 1 case of fatal MH, the entire CACNA1S was examined. In 1 case with histologic diagnosis of McArdle disease (glycogen storage disease type 5) and no findings in RYR1, the most common mutation, Arg50X, in the myophosphorylase gene (PYGM) was screened using NIaIII restriction enzyme analysis followed by sequencing.

A variant was defined as any change to the wild-type sequence, other than common polymorphisms previously found in 1 in 100 control subjects, whether it was found in MHS or control subjects. To be considered a candidate MH-causative mutation, the variant must be nonsynonymous (alter the amino acid), be present in MHS subjects only, be absent in at least 100 control subjects, and be absent in all published databases listing RYR1 polymorphisms. The pathogenic MHS mutation should segregate with MHS in 2 unrelated families, and be shown to induce biophysical changes consistent with MHS.b The concomitant presence of nonsynonymous polymorphisms was not counted as a second or multiple variant in MHS subjects in this study. Mutations and variants identified in the RYR1 were denoted according to Human Genome Variation guidelines for nomenclature.c Variants are numbered by amino acid position according to the RYR1 protein sequence NP_000531.2.

Statistical Analysis

Data are summarized using means and SD and point estimates with 95% confidence intervals. Mean values of contractures in groups of subjects with different genetic findings were compared by analysis of variance followed by Student-Newman-Keuls tests using PASW Statistics 18.0.0 (SPSS Inc., Chicago, IL).

RESULTS

DNA analysis was performed on 120 unrelated subjects (Fig. 1; individual numbered subjects are described in Supplemental Digital Content 1, http://links.lww.com/AA/A524). CHCT were reported for 108 subjects, 1 of whom was found to have CCD diagnosed by histopathology (subject 52). In subjects who did not undergo CHCT, CGS scores of their suspected MH episodes, when calculable, ranged from 23 to 76. These 7 subjects were numbered 6, 15, 25, 31, 51, 53, and 63 in Supplemental Digital Content 1 (http://links.lww.com/AA/A524). Subject 53 died of MH (CGS 76). Subject 51 had progressive CCD, diagnosed by histopathology, after a severe MH episode during general anesthesia (CGS 53). Subjects 17 and 27 had CCD diagnosed after a suspected MH episode. Two subjects, 8 and 49, had no CHCT results because they underwent muscle contracture tests before standardization of the CHCT, due to their history suggestive of MH susceptibility (Supplemental Digital Content 1, http://links.lww.com/AA/A524), with abnormal results. One subject, 50, had a very strong family history of MH, but was unable to undergo CHCT.

Figure 1
Figure 1:
This flow chart illustrates the outcomes of DNA sequencing for subjects with and without prior caffeine–halothane contracture test (CHCT). A variant of uncertain significance (VUS) is a variant that has been previously reported, but it is not yet proven to be malignant hyperthermia (MH)-causative. A novel variant is a variant that has not been previously reported, and it has not been proven to be MH-causative. A polymorphism is a change in the amino acid sequence of the gene that has been found at least once in 100 normal subjects and is not expected to be pathogenic. The numbers in parentheses are the numbers of subjects found to have this type of change inRYR1. Two novel variants in the ryanodine receptor type 1 gene (RYR1) were found in 1 subject. A novel variant and a VUS in RYR1 were found in 1 subject. Two different VUS were found in 2 subjects. A VUS and a polymorphism were found in 1 subject. A novel variant and a polymorphism in RYR1 were found in another subject. One subject had 2 polymorphisms in RYR1. In summary, there were 7 subjects who had 2 different variants when polymorphisms in RYR1 are included in this count. Thus, the sum of the numbers in the subboxes is 7 more than the number of subjects in this study reported to have mutations, VUS, novel variants and/or polymorphisms. (see Table 4 and Supplemental Digital Content 1, http://links.lww.com/AA/A524)

Ryanodine Receptor Gene Findings

Ten known RYR1 mutations causative for MH were identified in 26 subjects (Table 1). A previously reported variant of unknown significance or a novel variant in RYR1 was identified in an additional 36 subjects (Table 2).3–7,15–35 Thus, 62 of 120 or 52% (95% confidence interval, 43%–61%) of all the subjects had a causative mutation or other variant in RYR1.

Table 1
Table 1:
Known Malignant Hyperthermia (MH)–Causative Mutations
Table 2
Table 2:
RYR1 Variants of Uncertain Significance in Malignant Hyperthermia-Susceptible Subjects

Eleven of the 16 novel variants reported in this study were found in 1 of the 3 previously defined hotspots of RYR127 in exons 2, 14, 41, 44, 90, 91, 100, and 102, and 5 were found outside of these hotspots, in exons 24, 66, and 76. Four novel variants were at amino acid residues, Gly40 in exon 2, Arg1043 in exon 24, Arg2248 in exon 41, and Ala4906 in exon 102, where different variants had been previously reported.5,7,15,16,30 These novel variants were not present in any of the 100 control subjects.

Contracture test results are described in Table 3. There were no significant differences between the maximum contractures in subjects with MH-causative mutations and subjects with VUS in RYR1. The maximum number of contractures in subjects with MH mutations or VUS in RYR1 were significantly more than the maximum number of contractures in those with no abnormality, which includes polymorphisms, found in RYR1.

Table 3
Table 3:
Caffeine–Halothane Contractures Are Greater in Malignant Hyperthermia-Susceptible Subjects with RYR1 MH Mutations and Variants Than in Those with No Abnormalities Found in RYR1

Known MH-causative RYR1 mutations were found in 7 of the 12 subjects (6, 8, 15, 27, 31, 49, and 50 in Supplemental Digital Content 1, http://links.lww.com/AA/A524) who had not undergone CHCT. Previously reported VUS in RYR1 were found in 2 other subjects in this group (51 and 63), 1 of whom also had histologically diagnosed CCD. Novel variants in RYR1 were found in 2 subjects (17 and 53), 1 of whom was the anesthetic-induced fatality and the other had CCD with no history of MH. Only 1 of these 12 subjects (25) who had not undergone CHCT had no findings in the 106 exons of RYR1 other than a known polymorphism. This subject experienced an MH event with CGS 38 (very likely MH). Clinical details of these cases are described further in Supplemental Digital Content 1 (http://links.lww.com/AA/A524).

RYR1 was not examined to the same extent in all 58 subjects (1 of whom did not undergo CHCT) in whom no variants were found because of the limited availability of DNA. However, there was no significant difference in maximum contractures between groups with more or fewer exons examined (Table 3).

Previously described polymorphisms (variants that are not expected to be pathologic that are found in at least 1 in 100 normal subjects) in RYR1 were found in 9 of these 58 subjects and in 2 subjects who had RYR1 VUS and in 1 who had a novel RYR1 variant. Contracture test results for 8 of these 9 subjects are summarized in Table 3. The observed polymorphisms included Val974Met, Arg1109Leu, and Ile2321Val (each in 1 subject), Lys1393Arg, Pro1787Leu and Gly2060Cys (each in 3 subjects). In 1 subject, both Pro1787Leu and Gly2060Cys were found. The clinical details of these subjects are presented in Supplemental Digital Content 1 (http://links.lww.com/AA/A524).

RYR1 Compound Heterozygotes and Variants in CACNA1S and PYGM

Two variants, novel or previously observed VUS in RYR1, were found in 4 of the 120 subjects (Table 4 and Supplemental Digital Content 1, http://links.lww.com/AA/A524). This count does not include polymorphisms. One subject listed in Table 4 was not included in Table 2, or in the total count of subjects in this study, because this subject had an MH episode after which a sibling underwent CHCT. Both siblings were entered into this study before it was recognized that they were related. The proband in this family had a known RYR1 mutation causative for MH and a novel variant. The sibling of this proband, who had strongly positive results on the contracture test (Table 2), had only the novel variant in RYR1. In the subject who died of MH, a novel variant, Val875Met, was found in exon 20 of CACNA1S in addition to a novel RYR1 variant, Arg3283Gln.

Table 4
Table 4:
Individuals with 2 Genetic Variants

In the 17 subjects in whom no variants were found in >100 exons of RYR1, the CACNA1S was examined for 4 variants. None of the 4 CACNA1S variants associated with MHS in previous studies was found. Two of the 17 subjects reported that a first-degree relative died during anesthesia, and 1 reported that a more distant relative died during anesthesia. In contrast, in the families of 18 different subjects in whom RYR1 variants were found (8 causative MH mutations and 10 VUS), 13 subjects reported perioperative deaths in first-degree relatives, and 5 reported perioperative deaths in more distant relatives. Unfortunately, the records documenting the cause of death were not available in most of these cases. It is certainly possible that the cause of death was not MH. Nevertheless, these unexpected deaths motivated family members to undergo CHCT. The clinical data available for these cases are presented in Supplemental Digital Content 1 (http://links.lww.com/AA/A524).

A homozygous Arg50X mutation was found in PYGM in 1 subject who had repeated episodes of muscle weakness and pain after exercise. His basal creatine kinase was 3000 to 6000 IU. After moderate exercise, creatine kinase increased to >100,000 IU. He had never had anesthesia, but a second-degree relative was reported to have had anesthetic complications; therefore, CHCT was performed. The maximum contracture was 0.28 g in the presence of 2 mM caffeine and 2.68 g in the presence of 3% halothane. Examination of the entire RYR1 gene did not identify a variant. An additional muscle biopsy was performed, and McArdle disease was diagnosed histologically.

Clinical History of Subjects in Whom No RYR1 Variants or Polymorphisms and None of 4 CACNA1S Variants Were Found

All 49 of these subjects had CHCT results (Supplemental Digital Content 1, http://links.lww.com/AA/A524). In this group, 17 underwent CHCT because of a family history of an MH event. In 3 families, between 3 and 5 positive CHCTs per family unit were documented. In 1 of these 3 families, there was a perioperative death. In another family, there was a perioperative cardiac arrest with death and subsequently positive CHCT in a first-degree relative who was a subject in this study. The clinical presentation of the 7 subjects who were reported to have had an MH event, with no family history of MH, in whom no RYR1 or CACNA1S variants were observed, included masseter muscle rigidity in 2 subjects, and 1 had CGS of 38 (very likely MH).11 Another had a CGS of 18 and the rest had CGS of ≤15. Rhabdomyolysis without more evidence for MH was the indication for CHCT in 3 subjects. There was no documented indication for CHCT in 22 of the subjects in whom there were no genetic findings. There were no deaths attributed to MH in the 49 subjects in whom no genetic variants were found in RYR1 or CACNA1S or in the 9 subjects in whom only a polymorphism in RYR1 was observed.

DISCUSSION

In this sample of 120 subjects with the diagnosis of MHS, we found 10 MH-causative mutations, 16 novel RYR1 variants, 1 novel CACNA1S variant, and 18 previously reported VUS in RYR1. A 2005 study of 30 subjects from North America, using direct sequencing of the entire coding region of RYR1,3 found 7 previously reported known MH mutations in 10 subjects, 2 VUS in 2 subjects, and 9 novel RYR1 variants in 9 other subjects. Seven of these previously observed mutations and variants were also found in this study of 120 subjects, but none of the 9 novel variants reported in 2005 was observed again. Gly2434Arg was the most frequent MH mutation reported in 2001, 2005, and in the current study. This mutation was also the most frequently observed MH mutation in Great Britain37 and Canada,6 but not in France,2 Japan,4 Australia,27 Switzerland,5 or Italy.7 Numerous cohort and case studies have reported RYR1 variants in subjects with clinical evidence of MH, including positive contracture tests. When these reports were comprehensively reviewed by Robinson et al.16 in 2006, 178 missense RYR1 variants were identified. As of November 2012, 414 unique variants in RYR1 had been reported in the Leiden Open Variation Database.d This report adds more RYR1 variants to this list. Thus, the number of described RYR1 variants is increasing rapidly, but the number proven to meet the strict criteria of a causative mutation is an order of magnitude lower.

Previous studies reported a high percentage of RYR1 variants thought to be unique or private to 1 family. This report supports the reassignment of variants from the private to the recurrent category. In 8 subjects in this study, a variant of uncertain significance was found that had been published only once before.6,16,17,28,31 Other VUS observed in this study have been reported at least twice previously, but not yet widely accepted as MH-causative mutations.4,5,7,16,18–22,24–27,30,32–35 Thus, this article provides data to support the potential MH-causative nature of many VUS in RYR1. Many variants listed in Table 2 have been observed previously in association with MHS, but the functional tests needed to prove that these variants cause the MH syndrome have not yet been performed.

The novel variants and VUS in RYR1 documented in this study support the need for examination of the entire RYR1 gene in MHS patients when searching for the genetic marker of this condition. Nine of the VUS observed in this study were outside of the RYR1 hotspots as these were defined.27 Fatal episodes of MH, MH death in a sibling, CCD, positive CHCT results, and observation of the same variant in other studies4 were reported in these 9 subjects.

In contrast to earlier claims that RYR1 variants associated with CCD are found in the C terminal exons 85 to 103,16 we observed RYR1 variants in exons 24, 47, and 66 in subjects with CCD. The same variant in exon 47 was observed in Japanese subjects with MH and CCD.4 The other 2 are novel RYR1 variants.

The subjects in this study who underwent CHCT were evaluated by anesthesiologists at 1 of several MH diagnostic centers. Review of personal medical and anesthetic history and family history are part of these evaluations. These anesthesiologists may consult with a neurologist before proceeding with CHCT so that maximum diagnostic use can be made of the patient’s muscle biopsy. Not all patients who present for CHCT are accepted for this procedure. If there is not a high prior probability of MH being present, the CHCT is not warranted.38

Individuals who had not undergone CHCT were subject to similar scrutiny before entry into this study. The high yield of RYR1 findings in this subset without a personal history of CHCT is likely due to the rigor of that review. Review of medical records before testing an individual for MHS includes detailed review of the anesthetic record, including minute ventilation and exhaled gas concentrations. These details can be clearly provided by the electronic anesthetic record.39 Other clinical and laboratory data should also be part of this assessment.40 Often, however, medical records, especially paper records, are incomplete. Additionally, because anesthesia providers should intervene to treat MH before it becomes fulminant, severe hypercarbia and rhabdomyolysis are averted. Thus, in many cases a CGS associated with “very likely MH” (CGS of 35–49) or “almost certain MH,” (CGS of ≥50),11 will not be observed. This was the case in the cohort reported in this study.

The observation of subjects with 2 variants is consistent with the previous claim that the frequency of an MH-causative genetic variant may be as much as 1 in 2000 to 3000 people in the general population.4,35

The percentage of subjects in this study in whom variants in RYR1 were identified is less than in several previously published studies. This is likely due in part to our inability to examine the entire RYR1 gene in all subjects because of the limited amount of genetic material available from MH diagnostic centers that have closed. Nevertheless, it is of interest that in those subjects in whom ≥100 RYR1 exons were examined without identification of any variant, the average maximum contractures were less than in the groups in which either known MH-causative mutations or VUS were found in the RYR1 gene. Because of the high sensitivity and low specificity of the CHCT, there can be false-positive CHCT results in as many as 22% of those tested.41 Therefore, the personal anesthetic and medical histories and family histories have to be re-examined to diagnose the underlying condition. It may be that the low specificity of the CHCT produces clinically positive results in individuals who are not really at increased risk of experiencing MH. For example, the subject who was homozygous for a mutation causing McArdle disease had a maximum caffeine contracture <0.3 g, but was enlisted in this study because he had a maximum halothane contracture of >2 g and suspicion of an adverse anesthetic event in a relative. The medical history of the individual should guide testing so that the differential diagnosis of exercise-induced rhabdomyolysis, the chief complaint of this individual, includes more than RYR1 variants.

CHCT may have positive results, diagnostic of MHS, in myopathic conditions with elevated calcium in muscle that do not include genetic abnormalities of RYR1. Appropriately focused blood testing could provide a diagnosis without performing muscle biopsy. When muscle biopsy is performed, comprehensive pathologic examination should be carefully planned prospectively to maximize diagnostic yield. If muscle contracture testing is needed to evaluate MHS, the biopsy should be performed at an MH diagnostic center (see www.mhaus.org for a list of active MH diagnostic centers in North America). CHCT remains the only method available in North America to confirm that the diagnosis is not MHS.

Previous work has demonstrated that greater halothane contractures are more likely to be found in individuals with MH-causative mutations in RYR1.42 Yet our subjects without abnormalities in RYR1 had been diagnosed as MHS by the CHCT. They or their first-degree relatives experienced adverse anesthetic events. It is necessary to continue to collect detailed anesthetic records, family histories, and biologic specimens from such people to discover the genetic factors associated with these adverse events. It may be that a new genetic locus for MH can be identified in subjects with strong family histories of adverse anesthetic events without RYR1 variants. Variants in regulatory regions and deep intronic variants of RYR1, or in areas of CACNA1S not examined in this study, may be responsible for the MHS diagnosis in this group. Given the dispersion of the population and potential difficulty confirming anesthetic details, it is necessary to continue collection of data in a central repository such as the North American MH Registry, so that future studies of this potentially life-threatening syndrome can be supported.

In summary, these results contribute to increasing the usefulness of genetic testing of MHS by documenting the presence of RYR1 variants in independent families and by describing novel variants. These results are consistent with previous work that identified MH-causative mutations more often in those with greater muscle contractures on in vitro testing.42 Technologic improvements in electronic health records and genetic analysis should lead to a more comprehensive view of both the phenotype and the genetic basis of MHS. Continued detailed reporting of the phenotypes of MHS is necessary to support interpretation of genetic results.

RECUSE NOTE

Dr. Cynthia Wong is the Section Editor for Obstetric Anesthesiology for the Journal. This manuscript was handled by Dr. Steven L. Shafer, Editor-in-Chief, and Dr. Wong was not involved in any way with the editorial process or decision.

ACKNOWLEDGMENTS

We acknowledge the contributions of Drs. H. Rosenberg, T.E. Nelson, T. Tautz, J. Tobin, and all the other Directors of MH Diagnostic Testing Centers in North America who have contributed reports to the North American MH Registry and specimens to previous studies of MH genetics.

DISCLOSURES

Name: Barbara W. Brandom, MD.

Contribution: This author helped design and conduct the study, analyze the data, and write the manuscript.

Attestation: Barbara W. Brandom 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.

Conflicts of Interest: Barbara W. Brandom received research support from Malignant Hyperthermia Association of the United States which is a not-for-profit group. She is the current director of the North American MH Registry, which is a subsidiary of MHAUS.

Name: Saiid Bina, PhD.

Contribution: This author helped write the manuscript and with laboratory work.

Attestation: Saiid Bina has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Conflicts of Interest: The author has no conflicts of interest to declare.

Name: Cynthia A. Wong, MD.

Contribution: This author helped conduct the study, write the manuscript, and provided subject materials.

Attestation: Cynthia A. Wong has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Conflicts of Interest: The author has no conflicts of interest to declare.

Name: Tarina Wallace, MS.

Contribution: This author helped conduct the study and with laboratory work.

Attestation: Tarina Wallace has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Conflicts of Interest: The author has no conflicts of interest to declare.

Name: Mihaela Visoiu, MD.

Contribution: This author helped conduct the study and write the manuscript.

Attestation: Mihaela Visoiu has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Conflicts of Interest: The author has no conflicts of interest to declare.

Name: Paul J. Isackson, PhD.

Contribution: This author helped with laboratory work and provided important editorial comments.

Attestation: Paul J. Isackson has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Conflicts of Interest: The author has no conflicts of interest to declare.

Name: Georgirene D. Vladutiiu, PhD.

Contribution: This author helped with laboratory work and provided important editorial comments.

Attestation: Georgirene D. Vladutiiu has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Conflicts of Interest: The author has no conflicts of interest to declare.

Name: Nyamkhishig Sambuughin, PhD.

Contribution: This author helped conduct the study, write the manuscript, and helped with genetic analysis.

Attestation: Nyamkhishig Sambuughin has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Conflicts of Interest: The author has no conflicts of interest to declare.

Name: Sheila M. Muldoon, MD.

Contribution: This author helped design and conduct the study, and write the manuscript.

Attestation: Sheila M. Muldoon has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Conflicts of Interest: The author has no conflicts of interest to declare.

a NCBI. dbSNP short genetic variations. Available at: http://www.ncbi.nlm.nih.gov/projects/SNP. Accessed February 28, 2013.
Cited Here

b Universität Basel. European Malignant Hyperthermia Group (EMHG).Available at: http://www.emhg.org. Accessed February 28, 2013.
Cited Here

c Human Genome Variation Society.Guidelines & recommendations. Available at: http://www.hgvs.org/rec.html. Accessed February 28, 2013.
Cited Here

d dLeiden Open Variation Database. RYanodine Receptor 1 (skeletal) (RYR1). Available at: http://www.dmd.nl/nmdb2/home.php?select_db=RYR1. Accessed February 28, 2013.
Cited Here

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