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DNA Testing for Malignant Hyperthermia: The Reality and the Dream

Stowell, Kathryn M. PhD

doi: 10.1213/ANE.0000000000000063
Pediatric Anesthesiology: Review Article
Continuing Medical Education

The advent of the polymerase chain reaction and the availability of data from various global human genome projects should make it possible, using a DNA sample isolated from white blood cells, to diagnose rapidly and accurately almost any monogenic condition resulting from single nucleotide changes. DNA-based diagnosis for malignant hyperthermia (MH) is an attractive proposition, because it could replace the invasive and morbid caffeine-halothane/in vitro contracture tests of skeletal muscle biopsy tissue. Moreover, MH is preventable if an accurate diagnosis of susceptibility can be made before general anesthesia, the most common trigger of an MH episode. Diagnosis of MH using DNA was suggested as early as 1990 when the skeletal muscle ryanodine receptor gene (RYR1), and a single point mutation therein, was linked to MH susceptibility. In 1994, a single point mutation in the α 1 subunit of the dihydropyridine receptor gene (CACNA1S) was identified and also subsequently shown to be causative of MH. In the succeeding years, the number of identified mutations in RYR1 has grown, as has the number of potential susceptibility loci, although no other gene has yet been definitively associated with MH. In addition, it has become clear that MH is associated with either of these 2 genes (RYR1 and CACNA1S) in only 50% to 70% of affected families. While DNA testing for MH susceptibility has now become widespread, it still does not replace the in vitro contracture tests. Whole exome sequence analysis makes it potentially possible to identify all variants within human coding regions, but the complexity of the genome, the heterogeneity of MH, the limitations of bioinformatic tools, and the lack of precise genotype/phenotype correlations are all confounding factors. In addition, the requirement for demonstration of causality, by in vitro functional analysis, of any familial mutation currently precludes DNA-based diagnosis as the sole test for MH susceptibility. Nevertheless, familial DNA testing for MH susceptibility is now widespread although limited to a positive diagnosis and to those few mutations that have been functionally characterized. Identification of new susceptibility genes remains elusive. When new genes are identified, it will be the role of the biochemists, physiologists, and biophysicists to devise functional assays in appropriate systems. This will remain the bottleneck unless high throughput platforms can be designed for functional work. Analysis of entire genomes from several individuals simultaneously is a reality. DNA testing for MH, based on current criteria, remains the dream.

From the Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand.

Accepted for publication November 1, 2013.

Funding: Not applicable.

The author declares no conflicts of interest.

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Web site.

Reprints will not be available from the author.

Address correspondence to Kathryn M. Stowell, PhD, Institute of Fundamental Science, Massey University, Private Bag 11–222, Palmerston North, New Zealand. Address e-mail to k.m.stowell@massey.ac.nz.

The autosomal dominant pharmacogenetic disorder malignant hyperthermia (MH), while exhibiting a complex phenotype in clinical situations,1 is ultimately preventable simply by avoiding triggering agents. Because most MH-like symptoms occur during general anesthesia using volatile anesthetics or depolarizing muscle relaxants, knowledge of susceptibility to MH can lead clinicians to use alternative safe anesthetic procedures.2 Therefore, predictive diagnostic testing has been a primary goal since MH was first recognized in 1960.3 The in vitro contracture test (IVCT)4 and caffeine-halothane contracture test (CHCT)5 provide useful diagnostic tools although both tests have limitations in sensitivity and specificity.6,7 They are, however, extremely invasive and are subject to between-center variation.8 The ability to replace these physiological tests with a simple DNA test has been a major goal since 1990, when the first gene associated with MH susceptibility was identified.9,10 Once specific point mutations associated with MH susceptibility were identified, this goal became a reality but only for a very limited number of affected families. Highly specific, sensitive, and moreover noninvasive diagnostic tests using patient DNA were first introduced in New Zealand in 1998,11 to some extent realizing the dream of obviating the need for the IVCT. More than 15 years later, universal DNA testing for MH steadfastly remains a dream. This review is a summary of the background to MH diagnosis, the current situation as regards DNA testing, confounding factors, and potential for the future. Disease characteristics, pharmacology of triggering, clinical diagnosis, management, and related disorders are the subject of recent reviews12,13 and will not be covered in any detail here.

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BIOCHEMISTRY AND PHYSIOLOGY

At the physiological and biochemical level, MH predisposes susceptible individuals to an acute increase in cytosolic [Ca2+] during general anesthesia with volatile anesthetics or depolarizing muscle relaxants.13,14 In addition, several studies suggest that MH susceptibility is commonly associated with increased resting cytosolic [Ca2+].15–17 While this increased cytosolic [Ca2+] was originally suggested to result from a sustained increase in Ca2+ release from the sarcoplasmic reticulum (SR), via the ryanodine receptor (RYR1) calcium release channel, more recent evidence suggests that Ca2+ entry through nonselective cation channels in the sarcolemma may have a major role in elevating cytosolic [Ca2+] in both fulminant MH and in the resting state.18,19 This is known as store-operated calcium entry and is a response to depletion of the SR calcium store.20 Acutely increased cytosolic [Ca2+] results in a hypermetabolic state and can cause death if not treated with the drug dantrolene.21 While dantrolene binding sites have been identified on the surface of RYR1,22 the exact mechanism underlying the effects of this drug, which acts to decrease cytosolic [Ca2+], is currently unclear. Some evidence suggests that the cellular target of dantrolene is a cation channel residing in the sarcolemma.18

Calcium release from intracellular stores in skeletal muscle is regulated by close physical communication between 2 channels: the voltage-dependent Ca2+ channel, dihydropyridine receptor (DHPR), in the T-tubule membrane and the Ca2+ release channel RYR1 located in the SR membrane. The DHPR responds to depolarization of the T-tubule membrane in response to depolarization of the sarcolemma (contiguous with the T-tubule membrane), resulting in opening of the RYR1. This response to electrical stimulation17,23–28 results in a release of Ca2+ into the cytoplasm from stores in the SR, which triggers muscle contraction and has a range of other metabolic consequences. This process is known as excitation–contraction coupling. Ca2+ is returned to the SR by a Ca2+-ATPase (SERCA).29

RYR channel activity is modulated by many physiological and pharmacological agents, which have been used in many experimental systems. These include isolated muscle cell and membrane systems,30,31 cells in culture expressing mutated forms of rabbit or human RYR,32–37 B lymphocytes38–40 and myotubes,41–43 isolated from patients with RYR1 mutations, to study both Ca2+ release and protein–protein interactions. Studies using knock-in mice expressing mutated RYR1 provide the most physiologically relevant systems.44–50 Structural studies provide further insights into potential function of RYR1 mutations.51–61 The results of such studies have identified critical regions of both the RYR and DHPR essential for function; however, the mechanisms by which these mutations actually affect Ca2+ flux at the molecular level remain speculative. While a number of other proteins associated with the skeletal muscle Ca2+ channel have been identified,62–67 the complete protein complement of the channel is not currently known. Mutations in JSPR1, encoding the protein JP45 which is involved in excitation–contraction coupling and may regulate the DHPR, have also been implicated as factors that may modulate the effect of MH-associated RyR1 mutations.68 This complexity of the skeletal muscle calcium channel is a confounding factor in the search for new genes harboring MH-causative mutations, the interpretation of data in functional analyses, and hence also in implementing widespread DNA testing for MH susceptibility.

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CURRENT DIAGNOSIS

The “gold standard” for diagnosis of MH is the IVCT using skeletal muscle biopsy tissue after the European protocol4 or the CHCT after the North American protocol.5 DNA-based diagnostic tests were introduced in New Zealand in 199811 to augment the IVCT, and, while these are now becoming more accepted,69,70 they cannot completely replace the IVCT because of the heterogeneity of MH in both the number of RYR1 variants and the potential involvement of other genes.71–73 In addition, DNA-based diagnosis for MH susceptibility can be performed only in families with mutations that have been functionally characterized.72 This constraint, while appropriate, is a major hurdle to being able to introduce such tests more widely. In addition, an MH negative diagnosis cannot be made on a DNA test alone; rather, an IVCT is recommended where a DNA test is negative for a familial mutation.74

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GENETICS OF MALIGNANT HYPERTHERMIA

MH in humans is an autosomal dominant trait, but the penetrance of clinical MH varies among families while the factors that affect penetrance are unclear.75 The disorder also exhibits variation in expressivity, both in clinical presentation and in the IVCT, which adds further complexity to diagnosis. The first MH susceptibility locus was mapped to chromosome 19q13.1-13.2,9,10 the position of RYR1 encoding the skeletal muscle RyR1, with the first causative mutation being identified in 1992.76 Since then, the number of single nucleotide variants in RYR1 has grown to 397 (www.ncbi.nlm.nih.gov/SNP, accessed October 2013); however, mutations in RYR1 account for only approximately 50% to 70 % of families affected with MH.77 Five other loci have been identified by linkage analysis.78 Mutations, shown to be causative of MH in the CACNA1S gene, encoding the main subunit of the voltage-gated DHPR that interacts with the RYR1 channel, have been confirmed for an MH locus on chromosome 1q.79–81 Mutations in CASQ1, encoding the SR calcium buffer calsequestrin, have recently been identified.82 A mouse CASQ1 knock out demonstrates an MH-like phenotype;83 however, there is some doubt whether mutations in this gene are associated with MH in humans.84 Discordance between genotype and phenotype is common.71,75,85,86 In addition, some MH-susceptible (MHS) families present with >1 mutation.87–89 In short, despite the early promise of other genes being linked to MH susceptibility, RYR1 and CACNA1S are currently the only 2 genes where causative mutations have been identified. The frequency of MH has been estimated to be between 1 in 15,000 anesthetics in children and 1 in 50,000 in adults,7 but the actual prevalence of MH-associated mutations has been estimated to be as high as 1 in 2000.90,91 Because MH is both life threatening and preventable, it is of paramount importance to be able to both accurately and efficiently diagnose susceptibility to MH and thus effectively manage the MH patient during procedures requiring general anesthesia. Knowledge of the genetic defects resulting in MH is the starting point for introduction of widespread DNA testing for MH susceptibility. Candidate gene selection (among the approximately 20,000 in the human genome) for further screening, however, remains a major obstacle in the search for a new MH-associated gene.

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IDENTIFICATION OF GENETIC VARIANTS

The types of MH-associated genetic variants with potential to affect skeletal muscle calcium channel function include single nucleotide changes, codon deletion, frameshifts, and splice-site variations as well as potential epigenetic changes (modifications to the DNA that affect gene expression while not changing the actual DNA sequence).70,92,93 Single nucleotide changes include common variants that occur in the general population at a frequency >1% (generally known as polymorphisms). These are unlikely to be pathogenic, while those that occur less frequently have the potential to be pathogenic. It is therefore important to differentiate between a common benign polymorphism and a rare variant with potential pathogenicity. In the case of MH, a single nucleotide change causing a potential mutation would need to be proven causative by functional assay to be considered pathogenic. Most genetic variants associated with MH susceptibility are in fact single nucleotide changes.

There are many reports of screening strategies for mutation detection in RYR1 and CACNA1S, as well as other genes potentially associated with MH.77,81,82,90,94–99 For genes, the size of RYR1 (160,000 bp and 106 exons) and CACNA1S (93,500 bp and 44 exons) mutation screening is not trivial. Earlier gel-based methods for discovery of genetic variants are reviewed by Kwok and Chen,100,101 but these have now been superseded by DNA sequencing and related methods. The method of dideoxynucleotide chain termination for DNA sequencing, developed by Sanger,102 is the most commonly used method for mutation discovery.103,104 This method uses individual polymerase chain reaction (PCR) products (known as amplicons), amplified from genomic DNA, or cDNA prepared from muscle mRNA, as the template for DNA sequence analysis. As an example, approximately 20 separate amplicons need to be prepared by PCR, purified and subsequently sequenced to achieve complete coverage of the RYR1 coding region if cDNA is used as the starting template for PCR. Using genomic DNA as a starting template would require the preparation of considerably more amplicons. With the advent of next generation sequencing methods, it is now possible to interrogate the whole genome for the presence of rare variants. While a very powerful and attractive approach, it brings with it a new set of problems (see later). Once a familial mutation has been identified, shown to segregate with MH susceptibility and be functionally causative, a DNA-based diagnostic test can be designed and implemented. Thus, the process from mutation discovery to application of a DNA test for that mutation can take many years, yet another confounding factor in the quest for DNA-based diagnosis of MH.

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REQUIREMENTS FOR DNA TESTING

For clinical diagnosis, an assay must be reproducible, specific, sensitive, rapid, and relatively inexpensive. MH presents an immediate problem as many pathological single nucleotide changes are present in only 1 family; the disorder itself is relatively rare, and the number of potentially pathological rare variants associated with MH continues to grow. Nevertheless, DNA testing presents a significant advantage over the IVCTs; in that, only a small (approximately 1–3 mL) blood sample is required. DNA can also be extracted from buccal cells if any difficulty in obtaining a blood sample arises. The quality of DNA is important, but for routine applications (as described in the Supplemental Digital Content, http://links.lww.com/AA/A695), simple spectrophotometric measurements at 230, 260, and 280 nm are adequate to assess DNA concentration and purity.105 DNA testing can be performed on an individual of any age including the newborn by using cord blood for DNA extraction. The advent of PCR has revolutionized DNA testing for many genetic disorders, and for MH, it has the potential to eliminate (or significantly reduce) the need for the IVCT, at least in well-characterized families. Given our current lack of understanding of the molecular mechanisms leading to MH susceptibility, and the heterogeneity inherent in the disorder, universal application of DNA testing is not currently possible. Nevertheless, diagnosis of MH susceptibility by a DNA test can be used routinely in families where a causative familial mutation has been identified. A brief and by no means exhaustive summary including advantages and disadvantages of the types of assays available for the detection of single nucleotide changes, the most prevalent variant that can give rise to MH, is included as Supplemental Digital Content (http://links.lww.com/AA/A695).

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LIMITATIONS OF DNA TESTING FOR MH

A set of guidelines has been established for DNA testing for MH susceptibility.72 These present several limitations associated with being able to offer DNA testing for MH as an alternative to the IVCT or CHCT. The major confounding factors are summarized below.

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Heterogeneity

While most MHS families have genetic variants in RYR1, these account for only 50% to 70% of affected families.77 This range has been suggested to be a result of the type of screening method used to identify mutations. Lower values could be expected if only selected “hots pot”106 regions of the RYR1 gene were screened, while higher values could be expected if the entire RYR1 gene was screened.90,96 The number of single nucleotide changes identified in RYR1 is close to 400, while causative mutations in CACNA1S are relatively few and account for only approximately 1% of affected families.81,98 Complete screening of both RYR1 and CACNA1S in a number of centers71,77,81,85,104,107,108 clearly indicates that other genes are involved. Some families present with >1 RYR1 mutation,88,93,109,110 and others show discordance between the familial mutation and IVCT results.77,86 For these reasons, a diagnosis of MH negative cannot be established using a DNA test.72 Nevertheless, being able to make an MHS diagnosis using a simple DNA test does have value for specific families with 1 or more of the limited number of variants that have been classed as causative.

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Functional Analysis

A genetic variant must be classified as functionally causative before it can be used definitively in a DNA diagnosis of MH susceptibility.72 While a number of different methods can be adopted for this, none are trivial, and all require specialized equipment and a high level of technical skill, making functional characterization a major stumbling block in establishing DNA-based diagnostic assays for routine use. The simplest systems use ex vivo tissue including myotubes and lymphoblastoid cell lines isolated from patient samples.40–42,111–114 To take into account contribution from genetic background, samples from at least 2 unrelated families must be used to show causation.72 A more tractable system is a recombinant one, whereby individual variants can be introduced by site-directed mutagenesis and subsequently expressed in HEK293 cells as a heterologous system. Both rabbit and human RYR1 cDNA have been cloned32,36 and used in such studies.32,33,36,37,115–117 The sheer size of RYR1 cDNA (approximately 15,000 bp) is a logistical problem in both cloning and expression, but nevertheless, recent work36,37 has added several additional mutations that could be added to the list classed as causative by the European MH Group. The generation of dyspedic (RYR1 knock-out) and dysgenic (CACNA1S knock-out) mice has allowed development of more complex physiologically relevant, expensive, and technically demanding systems making use of knock-in mice.27,43–45,48–50,118–123 These systems have been used for functional characterization of variants in RYR1 and CACNA1S as well as interactions between the 2 channels. Caution does need to be exercised in interpreting the data obtained from such studies, because the phenotypes of some knock-in mice have been somewhat different to those of MH in humans.45,49 One of the criteria for inclusion is that the work be published. It is therefore possible that the number of causative mutations is in fact more extensive than that listed on the European MH Group website (http://www.emhg.org/).

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Discordance

While discordance has been mentioned above, it deserves a separate discussion. The IVCT and CHCT are neither 100% specific nor sensitive6,124; nevertheless, the 2 tests are the gold standard reference point in establishing genotype/phenotype correlations for MH susceptibility. As physiological tests, they are both subject to many external factors that are not present in a DNA-based test. These include interlaboratory variability,8 accuracy of measurement of the concentrations of both caffeine and halothane, as well as the accepted practice of recording maximum values rather than averages as would be the norm in a standard analytical procedure. Hence, establishing 100% concordance between CHCT/IVCT and DNA data will remain problematic. In addition, there is clear evidence that >1 gene is associated with MH susceptibility in some families.71,73,77,85,86,125 Until such time as discordance can be eliminated, either by a more definitive physiological test or by the identification of all genes and associated causative mutations, MH negative will not be able to be diagnosed by a DNA test. This remains a huge obstacle in the quest for universal application of DNA testing for MH susceptibility.

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NEXT GENERATION SEQUENCING

The development of next generation sequencing (NGS) technologies provides another set of tools with which to interrogate the human genome. The ability to sequence the entire genome, however, should be approached with caution. The sheer amount of information that can be provided may be overwhelming, and the tools and skills required to actually analyze the data in a meaningful manner are far from trivial.126–128 There are several approaches that can be taken with this technology using a range of different instruments and associated chemistries.129–131 Some laboratories may have a personal instrument for in-house use, while others may choose to outsource preparation of samples, instrumentation, and analysis. Template preparation, DNA sequencing chemistries, data collection, genome sequence alignment, and assembly as well as potential applications of NGS have been very well described and illustrated in a recent technical review.132

While each commercial instrument and associated chemistry is very different, the principles are in common. In short, a genome is fragmented or sectioned to produce manageable-sized pieces of DNA. The resultant DNA fragments are immobilized on a solid surface and then simultaneously used as templates in thousands to billions of spatially separated sequencing reactions in relatively short read lengths. The resultant sequencing information yields multiple sets of overlapping sequences, which can then be aligned to the reference human genome. As each individual fragment of DNA from the genome is represented many times in the sequencing data, it is possible to accurately determine the presence of a variant. The sheer volume of data produced calls for the development of sophisticated software and processes to read and align the DNA sequence, determine the quality of the data, identify sequence variants, and assess true variation compared with machine artifacts.133–135 Following is a brief summary of potential applications of NGS for MH mutation screening and discovery.

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Amplicon Sequencing for Selected Candidate Genes

Anyone who has undertaken the laborious task of amplifying the cDNA or genomic DNA representing RYR1 in small sections, purified the resultant amplicons and then submitted them individually for dideoxy chain termination (Sanger) sequencing will realize the advantages in both cost and time saving of using NGS for amplicon sequencing.136–138 The template can be either cDNA or genomic DNA, which extends the accessibility of the methodology, because genomic DNA can be isolated very easily from a blood sample. A set of PCR primers to amplify specific exons or the entire length of the gene, in a series of segments, can be easily designed and tested for specificity. The instrument and chemistry used for sequencing will determine the average length of amplicon produced. PCR products (amplicons) are pooled and a library generated, again according to appropriate chemistry and the instrument to be used. As a first screen, RYR1 and CACNA1S would be suitable and reasonable genes to interrogate. Because of the technology, samples from many individuals can be analyzed simultaneously by the use of “barcode” sequencing tags.139 Dedicated software divides the data obtained into bins according to the barcode representing each individual and then removes the barcoding tags before sequence analysis. The sequence obtained can then be aligned, with appropriate software, to the reference sequence for the genes under study and genetic variants identified. Interrogation of these variants, against the several accessible databases of human sequence information, can then begin with the aim of identifying novel variants that alter amino acid sequence and hence potentially protein function. This type of framework for analysis can be implemented easily as a diagnostic tool for families newly identified as being susceptible to MH. Of course, any putative novel mutation would need to be functionally characterized before it could be deemed causative and subsequently used as the basis of a diagnostic test. The major disadvantage of this method is that not all amplicons will be sequenced with the same efficiency, so some may be underrepresented or missing completely in the analysis. Individual Sanger sequencing reactions for any missed amplicons may need to be performed separately for 100% coverage. Once a familial mutation has been identified, then a simple PCR-based assay can be designed, validated, and implemented. The method of choice for simplicity, speed, and cost is high resolution amplicon melting (see Supplemental Digital Content, http://links.lww.com/AA/A695).

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Sequence Capture for Selected Candidate Genes

The net can be spread more widely for families where no mutations in either RYR1 or CACNA1S have been identified. Targeted sequence capture, whereby a set of probes either on an array140 or in solution,141 can be used to simultaneously capture many specified genes with potential roles in MH. DNA samples from several individuals can be analyzed simultaneously by barcoding as for amplicon sequencing. An array-based targeted sequence capture method has recently been used for 2 families with apparently no mutations in RYR1 as detected by Sanger sequencing.142 In general terms, genomic DNA is fragmented, size selected, and hybridized to the probes. The hybridized probes are then washed free of unbound DNA. The captured DNA fragments are separated from the probes by denaturing the probe/DNA hybrid and subjected to next generation sequencing, together with an appropriate bioinformatic pipeline for alignment, and variant detection. Targeted capture has an advantage over whole exome sequencing in that the number of genes interrogated is limited; therefore, the relative number of candidate variants that would progress through to segregation analysis will be small. The major disadvantages with this method are that all the genes likely to be associated with MH are not currently known, and not all exons for every gene may be represented in the sequence analysis. As above, alternative strategies would need to be applied to achieve 100% coverage.

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Whole Exome Capture

In terms of cost effectiveness, the method of choice for variant discovery would be whole exome capture, where probes representing all of the known expressed genomes are used in solution to capture exons from a DNA sample.143 This method generally results in sequencing all exon-intron boundaries as well because the genomic DNA to be analyzed is fragmented randomly. A variety of commercial library (randomly fragmented genomic DNA representing the whole genome) preparation kits are available for different total numbers of genes and a wide range of service providers. While it is possible to identify a potential variant in a novel gene, the amount of natural variability among individuals is likely to be a bottleneck in whole exome sequencing. The researcher must have access to sufficiently large family trees so that samples for analysis can be appropriately selected. This can be assisted initially by performing a linkage analysis (with microsatellite markers or a single nucleotide polymorphism array) to identify potentially linked genes.144 Even then, the number of variants obtained may still preclude establishing a workable set of candidate variants to check for segregation. Variants for further study can be limited by the judicious use of software that screen out, or select for, particular gene sets. For example, one may look for variants, in the first instance, that occur in genes expressed only in skeletal muscle. Assistance from a dedicated bioinformatician is an essential requirement for the success of this type of analysis. The simplest and most inexpensive method for subsequent segregation studies, for candidate variants, is high resolution melt analysis. That is, all MHS family members, who have been phenotyped by a clinical MH reaction or positive IVCT/CHCT, must carry the variant, and the variant must be absent from at least 100 individuals who have been phenotyped MH negative. Exome sequencing has been used recently to analyze genomic DNA, from individuals in 4 MHS families, where mutations in RYR1 and CACNA1S had been previously excluded by Sanger sequencing using cDNA prepared from probands’ muscle mRNA.145 It is interesting to note that this study revealed 3 novel rare heterozygous variants in RYR1 and 1 in CACNA1S. Review of original Sanger sequencing data did indeed reveal the presence of these variants, but the original software used had miscalled them as homozygous. This exercise highlights the additional sensitivity that can be gained from NGS methods and provides a great deal of promise in the quest for the identification of new potentially pathogenic variants associated with MH susceptibility. A potential systematic approach from variant detection to implementation of a familial DNA-based diagnostic test for MH is represented in Figure 1.

Figure 1

Figure 1

Apart from availability of suitable family trees with adequate CHCT/IVCT data, the major limitations of this technology include the sheer volume of data generated, the logistics and cost of data storage, and the laborious systematic exclusion of variants that do not segregate with disease.134,146 In addition, some coding sequences will not be represented at all, or the sequencing coverage (number of times a single sequence is represented in the dataset) will be too low for accuracy. Mutations in the noncoding region of genes, that may be associated with MH susceptibility, are also likely to be missing from the sequencing data. For completeness, whole exome sequencing could be teamed with analysis of the epigenome (all the chromatin and DNA modifications associated with the DNA itself), transcriptome (that part of the genome that is expressed in a specific cell type), and metabolome (all the metabolites that are produced in a particular tissue) in a systems biology approach to generate tangible linkages between disease-associated genetic variants and their phenotypic expression.147,148 This approach would, however, require the use of muscle samples, which make a retrospective study more challenging.

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ETHICAL CONSIDERATIONS

The advent of whole genome or even part genome sequencing presents some ethical dilemmas. While MH-associated genes are obviously the focus, the analysis will necessarily also identify variants associated with other disorders. Even judicious screening cannot eliminate all incidental findings. While some may be known risk factors, others will have no clearly defined disease association. Genome sequencing can provide both a diagnostic and a screening function, but the complexity of the data obtained and its true value in prognosis will necessarily be difficult, if not impossible, to convey to most patients. At present, whole genome sequencing rests largely in the research arena, but as costs decrease and more “user-friendly” bioinformatics pipelines are established, the technology will become accessible as a diagnostic tool. Carefully worded consent forms will therefore need to be developed and implemented to safeguard patients, their families, and the laboratories doing the analyses.149 A recent editorial summarizes very eloquently the potential ethical dilemmas associated with whole genome sequencing, especially that of the newborn.150

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CONCLUSIONS

While we are still some way from the dream of eliminating the CHCT/IVCT, tremendous progress has been made in variant screening with at least a small number of mutations being accepted as diagnostic. Even though currently limited to an MHS diagnosis, this is still very valuable in cost to health care providers as well as patient comfort and safety. There is much work to be done, however, first in the functional characterization of the myriad known RYR1 variants linked to MH but not yet shown to be causative. Completion of this task would significantly increase the number of families worldwide who could be offered DNA-based testing. The quest for new “hot” genes must continue, and fortunately, NGS provides the technology and instrumentation to achieve this knowledge. This will bring new challenges in developing appropriate experimental systems with which to prove causality. A difficulty immediately arises in securing sufficient research funding for what is still considered a rare disorder. It will be our tasks as research scientists to convince the funding bodies to invest in understanding the molecular pathology associated with genetic variants that perturb the structure and function of this giant channel that has so many critically important cellular roles, including susceptibility to MH. This is the reality.

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DISCLOSURES

Name: Kathryn M. Stowell, PhD.

Contribution: This author designed the strategy for the review, sourced appropriate literature, and prepared the manuscript.

Attestation: Kathryn Stowell approved the final manuscript and is the archival author.

This manuscript was handled by: Peter J. Davis, MD.

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REFERENCES

1. Rüffert H, Olthoff D, Deutrich C, Froster UG. [Current aspects of the diagnosis of malignant hyperthermia]. Anaesthesist. 2002;51:904–13
2. Denborough MA. Malignant hyperpyrexia. Compr Ther. 1975;1:51–6
3. Denborough M, Lovell R. Anaesthetic deaths in a family. Lancet. 1960;2:45
4. European, Malignant, Hyperpyrexia, Group. . A protocol for the investigation of malignant hyperpyrexia (MH) susceptibility. Br J Anaesth. 1984;56:1267–9
5. Larach MG. Standardization of the caffeine halothane muscle contracture test. North American Malignant Hyperthermia Group. Anesth Analg. 1989;69:511–5
6. Allen GC, Larach MG, Kunselman AR. The sensitivity and specificity of the caffeine-halothane contracture test: a report from the North American Malignant Hyperthermia Registry. The North American Malignant Hyperthermia Registry of MHAUS. Anesthesiology. 1998;88:579–88
7. Ording H. Current diagnostic methods of malignant hyperthermia: an evaluation. Acta Anaesthesiol Belg. 1990;41:103–6
8. Ording H, Islander G, Bendixen D, Ranklev-Twetman E. Between-center variability of results of the in vitro contracture test for malignant hyperthermia susceptibility. Anesth Analg. 2000;91:452–7
9. MacLennan DH, Duff C, Zorzato F, Fujii J, Phillips M, Korneluk RG, Frodis W, Britt BA, Worton RG. Ryanodine receptor gene is a candidate for predisposition to malignant hyperthermia. Nature. 1990;343:559–61
10. McCarthy TV, Healy JM, Heffron JJ, Lehane M, Deufel T, Lehmann-Horn F, Farrall M, Johnson K. Localization of the malignant hyperthermia susceptibility locus to human chromosome 19q12-13.2. Nature. 1990;343:562–4
11. Stowell KM, Brown R, James D, Couchman K, Hodges M, Pollock N. Malignant hyperthermia in New Zealand. NZ BioScience. 1999;7:12–7
12. Rosenberg H, Sambuughin N, Riazi S, Dirksen R. Malignant Hyperthermia Susceptibility. GeneReviews (Internet). 2010 Available at: www.ncbi.nlm.nih.gov/books/NBK1146. Accessed October 2013.
13. Hopkins PM. Malignant hyperthermia: pharmacology of triggering. Br J Anaesth. 2011;107:48–56
14. MacLennan DH, Chen SR. The role of the calcium release channel of skeletal muscle sarcoplasmic reticulum in malignant hyperthermia. Ann N Y Acad Sci. 1993;707:294–304
15. Vukcevic M, Broman M, Islander G, Bodelsson M, Ranklev-Twetman E, Müller CR, Treves S. Functional properties of RYR1 mutations identified in Swedish patients with malignant hyperthermia and central core disease. Anesth Analg. 2010;111:185–90
16. Yang T, Esteve E, Pessah IN, Molinski TF, Allen PD, López JR. Elevated resting [Ca(2+)](i) in myotubes expressing malignant hyperthermia RyR1 cDNAs is partially restored by modulation of passive calcium leak from the SR. Am J Physiol Cell Physiol. 2007;292:C1591–8
17. Eltit JM, Bannister RA, Moua O, Altamirano F, Hopkins PM, Pessah IN, Molinski TF, López JR, Beam KG, Allen PD. Malignant hyperthermia susceptibility arising from altered resting coupling between the skeletal muscle L-type Ca2+ channel and the type 1 ryanodine receptor. Proc Natl Acad Sci U S A. 2012;109:7923–8
18. Eltit JM, Ding X, Pessah IN, Allen PD, Lopez JR. Nonspecific sarcolemmal cation channels are critical for the pathogenesis of malignant hyperthermia. FASEB J. 2013;27:991–1000
19. Duke AM, Hopkins PM, Calaghan SC, Halsall JP, Steele DS. Store-operated Ca2+ entry in malignant hyperthermia-susceptible human skeletal muscle. J Biol Chem. 2010;285:25645–53
20. Dirksen RT. Checking your SOCCs and feet: the molecular mechanisms of Ca2+ entry in skeletal muscle. J Physiol. 2009;587:3139–47
21. Austin KL, Denborough MA. Drug treatment of malignant hyperpyrexia. Anaesth Intensive Care. 1977;5:207–13
22. Paul-Pletzer K, Yamamoto T, Bhat MB, Ma J, Ikemoto N, Jimenez LS, Morimoto H, Williams PG, Parness J. Identification of a dantrolene-binding sequence on the skeletal muscle ryanodine receptor. J Biol Chem. 2002;277:34918–23
23. Ikemoto N, Yamamoto T. Regulation of calcium release by interdomain interaction within ryanodine receptors. Front Biosci. 2002;7:d671–83
24. Meissner G, Lu X. Dihydropyridine receptor-ryanodine receptor interactions in skeletal muscle excitation-contraction coupling. Biosci Rep. 1995;15:399–408
25. Protasi F, Paolini C, Nakai J, Beam KG, Franzini-Armstrong C, Allen PD. Multiple regions of RyR1 mediate functional and structural interactions with alpha(1S)-dihydropyridine receptors in skeletal muscle. Biophys J. 2002;83:3230–44
26. Avila G. Intracellular Ca2+ dynamics in malignant hyperthermia and central core disease: established concepts, new cellular mechanisms involved. Cell Calcium. 2005;37:121–7
27. Bannister RA, Grabner M, Beam KG. The alpha(1S) III-IV loop influences 1,4-dihydropyridine receptor gating but is not directly involved in excitation-contraction coupling interactions with the type 1 ryanodine receptor. J Biol Chem. 2008;283:23217–23
28. Dayal A, Schredelseker J, Franzini-Armstrong C, Grabner M. Skeletal muscle excitation-contraction coupling is independent of a conserved heptad repeat motif in the C-terminus of the DHPRbeta(1a) subunit. Cell Calcium. 2010;47:500–6
29. Gommans IM, Vlak MH, de Haan A, van Engelen BG. Calcium regulation and muscle disease. J Muscle Res Cell Motil. 2002;23:59–63
30. O’Sullivan GH, McIntosh JM, Heffron JJ. Abnormal uptake and release of Ca2+ ions from human malignant hyperthermia-susceptible sarcoplasmic reticulum. Biochem Pharmacol. 2001;61:1479–85
31. Shtifman A, Ward CW, Yamamoto T, Wang J, Olbinski B, Valdivia HH, Ikemoto N, Schneider MF. Interdomain interactions within ryanodine receptors regulate Ca2+ spark frequency in skeletal muscle. J Gen Physiol. 2002;119:15–32
32. Tong J, Oyamada H, Demaurex N, Grinstein S, McCarthy TV, MacLennan DH. Caffeine and halothane sensitivity of intracellular Ca2+ release is altered by 15 calcium release channel (ryanodine receptor) mutations associated with malignant hyperthermia and/or central core disease. J Biol Chem. 1997;272:26332–9
33. Oyamada H, Oguchi K, Saitoh N, Yamazawa T, Hirose K, Kawana Y, Wakatsuki K, Oguchi K, Tagami M, Hanaoka K, Endo M, Iino M. Novel mutations in C-terminal channel region of the ryanodine receptor in malignant hyperthermia patients. Jpn J Pharmacol. 2002;88:159–66
34. Loke JC, Kraev N, Sharma P, Du G, Patel L, Kraev A, MacLennan DH. Detection of a novel ryanodine receptor subtype 1 mutation (R328W) in a malignant hyperthermia family by sequencing of a leukocyte transcript. Anesthesiology. 2003;99:297–302
35. Querfurth HW, Haughey NJ, Greenway SC, Yacono PW, Golan DE, Geiger JD. Expression of ryanodine receptors in human embryonic kidney (HEK293) cells. Biochem J. 1998;334(Pt 1):79–86
36. Sato K, Pollock N, Stowell KM. Functional studies of RYR1 mutations in the skeletal muscle ryanodine receptor using human RYR1 complementary DNA. Anesthesiology. 2010;112:1350–4
37. Sato K, Roesl C, Pollock N, Stowell KM. Skeletal muscle ryanodine receptor mutations associated with malignant hyperthermia showed enhanced intensity and sensitivity to triggering drugs when expressed in human embryonic kidney cells. Anesthesiology. 2013;119:111–8
38. Sei Y, Brandom BW, Bina S, Hosoi E, Gallagher KL, Wyre HW, Pudimat PA, Holman SJ, Venzon DJ, Daly JW, Muldoon S. Patients with malignant hyperthermia demonstrate an altered calcium control mechanism in B lymphocytes. Anesthesiology. 2002;97:1052–8
39. Tilgen N, Zorzato F, Halliger-Keller B, Muntoni F, Sewry C, Palmucci LM, Schneider C, Hauser E, Lehmann-Horn F, Müller CR, Treves S. Identification of four novel mutations in the C-terminal membrane spanning domain of the ryanodine receptor 1: association with central core disease and alteration of calcium homeostasis. Hum Mol Genet. 2001;10:2879–87
40. Anderson AA, Brown RL, Polster B, Pollock N, Stowell KM. Identification and biochemical characterization of a novel ryanodine receptor gene mutation associated with malignant hyperthermia. Anesthesiology. 2008;108:208–15
41. Wehner M, Rueffert H, Koenig F, Olthoff D. Calcium release from sarcoplasmic reticulum is facilitated in human myotubes derived from carriers of the ryanodine receptor type 1 mutations Ile2182Phe and Gly2375Ala. Genet Test. 2003;7:203–11
42. Wehner M, Rueffert H, Koenig F, Neuhaus J, Olthoff D. Increased sensitivity to 4-chloro-m-cresol and caffeine in primary myotubes from malignant hyperthermia susceptible individuals carrying the ryanodine receptor 1 Thr2206Met (C6617T) mutation. Clin Genet. 2002;62:135–46
43. Yang T, Ta TA, Pessah IN, Allen PD. Functional defects in six ryanodine receptor isoform-1 (RyR1) mutations associated with malignant hyperthermia and their impact on skeletal excitation-contraction coupling. J Biol Chem. 2003;278:25722–30
44. Andronache Z, Hamilton SL, Dirksen RT, Melzer W. A retrograde signal from RyR1 alters DHP receptor inactivation and limits window Ca2+ release in muscle fibers of Y522S RyR1 knock-in mice. Proc Natl Acad Sci U S A. 2009;106:4531–6
45. Barrientos GC, Feng W, Truong K, Matthaei KI, Yang T, Allen PD, Lopez JR, Pessah IN. Gene dose influences cellular and calcium channel dysregulation in heterozygous and homozygous T4826I-RYR1 malignant hyperthermia-susceptible muscle. J Biol Chem. 2012;287:2863–76
46. Chelu MG, Goonasekera SA, Durham WJ, Tang W, Lueck JD, Riehl J, Pessah IN, Zhang P, Bhattacharjee MB, Dirksen RT, Hamilton SL. Heat- and anesthesia-induced malignant hyperthermia in an RyR1 knock-in mouse. FASEB J. 2006;20:329–30
47. De Crescenzo V, Fogarty KE, Lefkowitz JJ, Bellve KD, Zvaritch E, MacLennan DH, Walsh JV Jr. Type 1 ryanodine receptor knock-in mutation causing central core disease of skeletal muscle also displays a neuronal phenotype. Proc Natl Acad Sci U S A. 2012;109:610–5
48. Estève E, Eltit JM, Bannister RA, Liu K, Pessah IN, Beam KG, Allen PD, López JR. A malignant hyperthermia-inducing mutation in RYR1 (R163C): alterations in Ca2+ entry, release, and retrograde signaling to the DHPR. J Gen Physiol. 2010;135:619–28
49. Loy RE, Orynbayev M, Xu L, Andronache Z, Apostol S, Zvaritch E, MacLennan DH, Meissner G, Melzer W, Dirksen RT. Muscle weakness in Ryr1I4895T/WT knock-in mice as a result of reduced ryanodine receptor Ca2+ ion permeation and release from the sarcoplasmic reticulum. J Gen Physiol. 2011;137:43–57
50. Yang T, Riehl J, Esteve E, Matthaei KI, Goth S, Allen PD, Pessah IN, Lopez JR. Pharmacologic and functional characterization of malignant hyperthermia in the R163C RyR1 knock-in mouse. Anesthesiology. 2006;105:1164–75
51. Amador FJ, Liu S, Ishiyama N, Plevin MJ, Wilson A, MacLennan DH, Ikura M. Crystal structure of type I ryanodine receptor amino-terminal beta-trefoil domain reveals a disease-associated mutation “hot spot” loop. Proc Natl Acad Sci U S A. 2009;106:11040–4
52. Kimlicka L, Van Petegem F. The structural biology of ryanodine receptors. Sci China Life Sci. 2011;54:712–24
53. Lobo PA, Van Petegem F. Crystal structures of the N-terminal domains of cardiac and skeletal muscle ryanodine receptors: insights into disease mutations. Structure. 2009;17:1505–14
54. Raina SA, Tsai J, Samsó M, Fessenden JD. FRET-based localization of fluorescent protein insertions within the ryanodine receptor type 1. PLoS One. 2012;7:e38594
55. Tung CC, Lobo PA, Kimlicka L, Van Petegem F. The amino-terminal disease hotspot of ryanodine receptors forms a cytoplasmic vestibule. Nature. 2010;468:585–8
56. Yuchi Z, Lau K, Van Petegem F. Disease mutations in the ryanodine receptor central region: crystal structures of a phosphorylation hot spot domain. Structure. 2012;20:1201–11
57. Samsó M, Feng W, Pessah IN, Allen PD. Coordinated movement of cytoplasmic and transmembrane domains of RyR1 upon gating. PLoS Biol. 2009;7:e85
58. Samsó M, Shen X, Allen PD. Structural characterization of the RyR1-FKBP12 interaction. J Mol Biol. 2006;356:917–27
59. Serysheva II, Ludtke SJ, Baker ML, Cong Y, Topf M, Eramian D, Sali A, Hamilton SL, Chiu W. Subnanometer-resolution electron cryomicroscopy-based domain models for the cytoplasmic region of skeletal muscle RyR channel. Proc Natl Acad Sci U S A. 2008;105:9610–5
60. Wagenknecht T, Radermacher M. Ryanodine receptors: structure and macromolecular interactions. Curr Opin Struct Biol. 1997;7:258–65
61. Wang R, Zhong X, Meng X, Koop A, Tian X, Jones PP, Fruen BR, Wagenknecht T, Liu Z, Chen SR. Localization of the dantrolene-binding sequence near the FK506-binding protein-binding site in the three-dimensional structure of the ryanodine receptor. J Biol Chem. 2011;286:12202–12
62. MacKrill JJ. Protein-protein interactions in intracellular Ca2+-release channel function. Biochem J. 1999;337(Pt 3):345–61
63. Anderson AA, Altafaj X, Zheng Z, Wang ZM, Delbono O, Ronjat M, Treves S, Zorzato F. The junctional SR protein JP-45 affects the functional expression of the voltage-dependent Ca2+ channel Cav1.1. J Cell Sci. 2006;119:2145–55
64. Bleunven C, Treves S, Jinyu X, Leo E, Ronjat M, De Waard M, Kern G, Flucher BE, Zorzato F. SRP-27 is a novel component of the supramolecular signalling complex involved in skeletal muscle excitation-contraction coupling. Biochem J. 2008;411:343–9
65. Treves S, Franzini-Armstrong C, Moccagatta L, Arnoult C, Grasso C, Schrum A, Ducreux S, Zhu MX, Mikoshiba K, Girard T, Smida-Rezgui S, Ronjat M, Zorzato F. Junctate is a key element in calcium entry induced by activation of InsP3 receptors and/or calcium store depletion. J Cell Biol. 2004;166:537–48
66. Treves S, Thurnheer R, Mosca B, Vukcevic M, Bergamelli L, Voltan R, Oberhauser V, Ronjat M, Csernoch L, Szentesi P, Zorzato F. SRP-35, a newly identified protein of the skeletal muscle sarcoplasmic reticulum, is a retinol dehydrogenase. Biochem J. 2012;441:731–41
67. Treves S, Vukcevic M, Maj M, Thurnheer R, Mosca B, Zorzato F. Minor sarcoplasmic reticulum membrane components that modulate excitation-contraction coupling in striated muscles. J Physiol. 2009;587:3071–9
68. Yasuda T, Delbono O, Wang ZM, Messi ML, Girard T, Urwyler A, Treves S, Zorzato F. JP-45/JSRP1 variants affect skeletal muscle excitation-contraction coupling by decreasing the sensitivity of the dihydropyridine receptor. Hum Mutat. 2013;34:184–90
69. Litman RS, Rosenberg H. Malignant hyperthermia: update on susceptibility testing. JAMA. 2005;293:2918–24
70. Rosenberg H, Rueffert H. Clinical utility gene card for: malignant hyperthermia. Eur J Hum Genet. 2011;19
71. Robinson RL, Anetseder MJ, Brancadoro V, van Broekhoven C, Carsana A, Censier K, Fortunato G, Girard T, Heytens L, Hopkins PM, Jurkat-Rott K, Klinger W, Kozak-Ribbens G, Krivosic R, Monnier N, Nivoche Y, Olthoff D, Rueffert H, Sorrentino V, Tegazzin V, Mueller CR. Recent advances in the diagnosis of malignant hyperthermia susceptibility: how confident can we be of genetic testing? Eur J Hum Genet. 2003;11:342–8
72. Urwyler A, Deufel T, McCarthy T, West SEuropean Malignant Hyperthermia Group. . Guidelines for molecular genetic detection of susceptibility to malignant hyperthermia. Br J Anaesth. 2001;86:283–7
73. Robinson R, Curran JL, Hall WJ, Halsall PJ, Hopkins PM, Markham AF, Stewart AD, West SP, Ellis FR. Genetic heterogeneity and HOMOG analysis in British malignant hyperthermia families. J Med Genet. 1998;35:196–201
74. Girard T, Treves S, Voronkov E, Siegemund M, Urwyler A. Molecular genetic testing for malignant hyperthermia susceptibility. Anesthesiology. 2004;100:1076–80
75. McCarthy TV, Quane KA, Lynch PJ. Ryanodine receptor mutations in malignant hyperthermia and central core disease. Hum Mutat. 2000;15:410–7
76. MacLennan DH, Otsu K, Fujii J, Zorzato F, Phillips MS, O’Brien PJ, Archibald AL, Britt BA, Gillard EF, Worton RG. The role of the skeletal muscle ryanodine receptor gene in malignant hyperthermia. Symp Soc Exp Biol. 1992;46:189–201
77. Robinson R, Carpenter D, Shaw MA, Halsall J, Hopkins P. Mutations in RYR1 in malignant hyperthermia and central core disease. Hum Mutat. 2006;27:977–89
78. Jurkat-Rott K, McCarthy T, Lehmann-Horn F. Genetics and pathogenesis of malignant hyperthermia. Muscle Nerve. 2000;23:4–17
79. Monnier N, Procaccio V, Stieglitz P, Lunardi J. Malignant-hyperthermia susceptibility is associated with a mutation of the alpha 1-subunit of the human dihydropyridine-sensitive L-type voltage-dependent calcium-channel receptor in skeletal muscle. Am J Hum Genet. 1997;60:1316–25
80. Toppin PJ, Chandy TT, Ghanekar A, Kraeva N, Beattie WS, Riazi S. A report of fulminant malignant hyperthermia in a patient with a novel mutation of the CACNA1S gene. Can J Anaesth. 2010;57:689–93
81. Carpenter D, Ringrose C, Leo V, Morris A, Robinson RL, Halsall PJ, Hopkins PM, Shaw MA. The role of CACNA1S in predisposition to malignant hyperthermia. BMC Med Genet. 2009;10:104
82. Capacchione JF, Sambuughin N, Bina S, Mulligan LP, Lawson TD, Muldoon SM. Exertional rhabdomyolysis and malignant hyperthermia in a patient with ryanodine receptor type 1 gene, L-type calcium channel alpha-1 subunit gene, and calsequestrin-1 gene polymorphisms. Anesthesiology. 2010;112:239–44
83. Protasi F, Paolini C, Dainese M. Calsequestrin-1: a new candidate gene for malignant hyperthermia and exertional/environmental heat stroke. J Physiol. 2009;587:3095–100
84. Kraeva N, Zvaritch E, Frodis W, Sizova O, Kraev A, MacLennan DH, Riazi S. CASQ1 gene is an unlikely candidate for malignant hyperthermia susceptibility in the North American population. Anesthesiology. 2013;118:344–9
85. Robinson R, Hopkins P, Carsana A, Gilly H, Halsall J, Heytens L, Islander G, Jurkat-Rott K, Müller C, Shaw MA. Several interacting genes influence the malignant hyperthermia phenotype. Hum Genet. 2003;112:217–8
86. Brown RL, Pollock AN, Couchman KG, Hodges M, Hutchinson DO, Waaka R, Lynch P, McCarthy TV, Stowell KM. A novel ryanodine receptor mutation and genotype-phenotype correlation in a large malignant hyperthermia New Zealand Maori pedigree. Hum Mol Genet. 2000;9:1515–24
87. Monnier N, Krivosic-Horber R, Payen JF, Kozak-Ribbens G, Nivoche Y, Adnet P, Reyford H, Lunardi J. Presence of two different genetic traits in malignant hyperthermia families: implication for genetic analysis, diagnosis, and incidence of malignant hyperthermia susceptibility. Anesthesiology. 2002;97:1067–74
88. Zhou H, Yamaguchi N, Xu L, Wang Y, Sewry C, Jungbluth H, Zorzato F, Bertini E, Muntoni F, Meissner G, Treves S. Characterization of recessive RYR1 mutations in core myopathies. Hum Mol Genet. 2006;15:2791–803
89. Kaufmann A, Kraft B, Michalek-Sauberer A, Weindlmayr M, Kress HG, Steinboeck F, Weigl LG. Novel double and single ryanodine receptor 1 variants in two Austrian malignant hyperthermia families. Anesth Analg. 2012;114:1017–25
90. Ibarra M CA, Wu S, Murayama K, Minami N, Ichihara Y, Kikuchi H, Noguchi S, Hayashi YK, Ochiai R, Nishino I. Malignant hyperthermia in Japan: mutation screening of the entire ryanodine receptor type 1 gene coding region by direct sequencing. Anesthesiology. 2006;104:1146–54
91. Rosenberg H, Davis M, James D, Pollock N, Stowell K. Malignant hyperthermia. Orphanet J Rare Dis. 2007;2:21
92. Kimura T, Lueck JD, Harvey PJ, Pace SM, Ikemoto N, Casarotto MG, Dirksen RT, Dulhunty AF. Alternative splicing of RyR1 alters the efficacy of skeletal EC coupling. Cell Calcium. 2009;45:264–74
93. Zhou H, Brockington M, Jungbluth H, Monk D, Stanier P, Sewry CA, Moore GE, Muntoni F. Epigenetic allele silencing unveils recessive RYR1 mutations in core myopathies. Am J Hum Genet. 2006;79:859–68
94. Galli L, Orrico A, Lorenzini S, Censini S, Falciani M, Covacci A, Tegazzin V, Sorrentino V. Frequency and localization of mutations in the 106 exons of the RYR1 gene in 50 individuals with malignant hyperthermia. Hum Mutat. 2006;27:830
95. Gillies RL, Bjorksten AR, Davis M, Du Sart D. Identification of genetic mutations in Australian malignant hyperthermia families using sequencing of RYR1 hotspots. Anaesth Intensive Care. 2008;36:391–403
96. Sambuughin N, Holley H, Muldoon S, Brandom BW, de Bantel AM, Tobin JR, Nelson TE, Goldfarb LG. Screening of the entire ryanodine receptor type 1 coding region for sequence variants associated with malignant hyperthermia susceptibility in the north american population. Anesthesiology. 2005;102:515–21
97. Broman M, Heinecke K, Islander G, Schuster F, Glahn K, Bodelsson M, Treves S, Müller C. Screening of the ryanodine 1 gene for malignant hyperthermia causative mutations by high resolution melt curve analysis. Anesth Analg. 2011;113:1120–8
98. Brandom BW, Bina S, Wong CA, Wallace T, Visoiu M, Isackson PJ, Vladutiu GD, Sambuughin N, Muldoon SM. Ryanodine receptor type 1 gene variants in the malignant hyperthermia-susceptible population of the United States. Anesth Analg. 2013;116:1078–86
99. Sambuughin N, Capacchione J, Blokhin A, Bayarsaikhan M, Bina S, Muldoon S. The ryanodine receptor type 1 gene variants in African American men with exertional rhabdomyolysis and malignant hyperthermia susceptibility. Clin Genet. 2009;76:564–8
100. Kwok PY, Chen X. Detection of single nucleotide polymorphisms. Curr Issues Mol Biol. 2003;5:43–60
101. Gingeras TR, Higuchi R, Kricka LJ, Lo YM, Wittwer CT. Fifty years of molecular (DNA/RNA) diagnostics. Clin Chem. 2005;51:661–71
102. Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977;74:5463–7
103. Kwok PY, Duan S. SNP discovery by direct DNA sequencing. Methods Mol Biol. 2003;212:71–84
104. Levano S, Vukcevic M, Singer M, Matter A, Treves S, Urwyler A, Girard T. Increasing the number of diagnostic mutations in malignant hyperthermia. Hum Mutat. 2009;30:590–8
105. Wilfinger WW, Mackey K, Chomczynski P. Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity. Biotechniques. 1997;22:474–6 478–81
106. Sei Y, Sambuughin NN, Davis EJ, Sachs D, Cuenca PB, Brandom BW, Tautz T, Rosenberg H, Nelson TE, Muldoon SM. Malignant hyperthermia in North America: genetic screening of the three hot spots in the type I ryanodine receptor gene. Anesthesiology. 2004;101:824–30
107. Carpenter D, Robinson RL, Quinnell RJ, Ringrose C, Hogg M, Casson F, Booms P, Iles DE, Halsall PJ, Steele DS, Shaw MA, Hopkins PM. Genetic variation in RYR1 and malignant hyperthermia phenotypes. Br J Anaesth. 2009;103:538–48
108. Schiemann AH, Dürholt EM, Pollock N, Stowell KM. Sequence capture and massively parallel sequencing to detect mutations associated with malignant hyperthermia. Br J Anaesth. 2013;110:122–7
109. Kossugue PM, Paim JF, Navarro MM, Silva HC, Pavanello RC, Gurgel-Giannetti J, Zatz M, Vainzof M. Central core disease due to recessive mutations in RYR1 gene: is it more common than described? Muscle Nerve. 2007;35:670–4
110. Ducreux S, Zorzato F, Ferreiro A, Jungbluth H, Muntoni F, Monnier N, Müller CR, Treves S. Functional properties of ryanodine receptors carrying three amino acid substitutions identified in patients affected by multi-minicore disease and central core disease, expressed in immortalized lymphocytes. Biochem J. 2006;395:259–66
111. Ducreux S, Zorzato F, Müller C, Sewry C, Muntoni F, Quinlivan R, Restagno G, Girard T, Treves S. Effect of ryanodine receptor mutations on interleukin-6 release and intracellular calcium homeostasis in human myotubes from malignant hyperthermia-susceptible individuals and patients affected by central core disease. J Biol Chem. 2004;279:43838–46
112. Kraeva N, Zvaritch E, Rossi AE, Goonasekera SA, Zaid H, Frodis W, Kraev A, Dirksen RT, Maclennan DH, Riazi S. Novel excitation-contraction uncoupled RYR1 mutations in patients with central core disease. Neuromuscul Disord. 2013;23:120–32
    113. Wehner M, Rueffert H, Koenig F, Olthoff D. Functional characterization of malignant hyperthermia-associated RyR1 mutations in exon 44, using the human myotube model. Neuromuscul Disord. 2004;14:429–37
      114. Zorzato F, Yamaguchi N, Xu L, Meissner G, Müller CR, Pouliquin P, Muntoni F, Sewry C, Girard T, Treves S. Clinical and functional effects of a deletion in a COOH-terminal lumenal loop of the skeletal muscle ryanodine receptor. Hum Mol Genet. 2003;12:379–88
        115. Avila G, Dirksen RT. Functional effects of central core disease mutations in the cytoplasmic region of the skeletal muscle ryanodine receptor. J Gen Physiol. 2001;118:277–90
        116. Brini M, Manni S, Pierobon N, Du GG, Sharma P, MacLennan DH, Carafoli E. Ca2+ signaling in HEK-293 and skeletal muscle cells expressing recombinant ryanodine receptors harboring malignant hyperthermia and central core disease mutations. J Biol Chem. 2005;280:15380–9
        117. Migita T, Mukaida K, Hamada H, Yasuda T, Haraki T, Nishino I, Murakami N, Kawamoto M. Functional analysis of ryanodine receptor type 1 p.R2508C mutation in exon 47. J Anesth. 2009;23:341–6
        118. Adams BA, Tanabe T, Mikami A, Numa S, Beam KG. Intramembrane charge movement restored in dysgenic skeletal muscle by injection of dihydropyridine receptor cDNAs. Nature. 1990;346:569–72
        119. Bannister RA, Pessah IN, Beam KG. The skeletal L-type Ca(2+) current is a major contributor to excitation-coupled Ca(2+) entry. J Gen Physiol. 2009;133:79–91
        120. Weiss RG, O’Connell KM, Flucher BE, Allen PD, Grabner M, Dirksen RT. Functional analysis of the R1086H malignant hyperthermia mutation in the DHPR reveals an unexpected influence of the III-IV loop on skeletal muscle EC coupling. Am J Physiol Cell Physiol. 2004;287:C1094–102
        121. Dirksen RT, Avila G. Distinct effects on Ca2+ handling caused by malignant hyperthermia and central core disease mutations in RyR1. Biophys J. 2004;87:3193–204
        122. Lefebvre R, Legrand C, Groom L, Dirksen RT, Jacquemond V. Ca2+ release in muscle fibers expressing R4892W and G4896V type 1 ryanodine receptor disease mutants. PLoS One. 2013;8:e54042
        123. Lee EH, Lopez JR, Li J, Protasi F, Pessah IN, Kim DH, Allen PD. Conformational coupling of DHPR and RyR1 in skeletal myotubes is influenced by long-range allosterism: evidence for a negative regulatory module. Am J Physiol Cell Physiol. 2004;286:C179–89
        124. Ording H, Brancadoro V, Cozzolino S, Ellis FR, Glauber V, Gonano EF, Halsall PJ, Hartung E, Heffron JJ, Heytens L, Kozak-Ribbens G, Kress H, Krivosic-Horber R, Lehmann-Horn F, Mortier W, Nivoche Y, Ranklev-Twetman E, Sigurdsson S, Snoeck M, Stieglitz P, Tegazzin V, Urwyler A, Wappler F. In vitro contracture test for diagnosis of malignant hyperthermia following the protocol of the European MH Group: results of testing patients surviving fulminant MH and unrelated low-risk subjects. The European Malignant Hyperthermia Group. Acta Anaesthesiol Scand. 1997;41:955–66
        125. Robinson RL, Curran JL, Ellis FR, Halsall PJ, Hall WJ, Hopkins PM, Iles DE, West SP, Shaw MA. Multiple interacting gene products may influence susceptibility to malignant hyperthermia. Ann Hum Genet. 2000;64:307–20
        126. Pavlopoulos GA, Oulas A, Iacucci E, Sifrim A, Moreau Y, Schneider R, Aerts J, Iliopoulos I. Unraveling genomic variation from next generation sequencing data. BioData Min. 2013;6:13
        127. Nielsen R, Paul JS, Albrechtsen A, Song YS. Genotype and SNP calling from next-generation sequencing data. Nat Rev Genet. 2011;12:443–51
        128. Cooper GM, Shendure J. Needles in stacks of needles: finding disease-causal variants in a wealth of genomic data. Nat Rev Genet. 2011;12:628–40
        129. Xu Y, Jiang H, Tyler-Smith C, Xue Y, Jiang T, Wang J, Wu M, Liu X, Tian G, Wang J, Wang J, Yang H, Zhang XAsan. . Comprehensive comparison of three commercial human whole-exome capture platforms. Genome Biol. 2011;12:R95
        130. Clark MJ, Chen R, Lam HY, Karczewski KJ, Chen R, Euskirchen G, Butte AJ, Snyder M. Performance comparison of exome DNA sequencing technologies. Nat Biotechnol. 2011;29:908–14
        131. Lam HY, Clark MJ, Chen R, Chen R, Natsoulis G, O’Huallachain M, Dewey FE, Habegger L, Ashley EA, Gerstein MB, Butte AJ, Ji HP, Snyder M. Performance comparison of whole-genome sequencing platforms. Nat Biotechnol. 2012;30:78–82
        132. Metzker ML. Sequencing technologies-the next generation. Nat Rev Genet. 2010;11:31–46
        133. DePristo MA, Banks E, Poplin R, Garimella KV, Maguire JR, Hartl C, Philippakis AA, del Angel G, Rivas MA, Hanna M, McKenna A, Fennell TJ, Kernytsky AM, Sivachenko AY, Cibulskis K, Gabriel SB, Altshuler D, Daly MJ. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat Genet. 2011;43:491–8
        134. D’Antonio M, D’Onorio De Meo P, Paoletti D, Elmi B, Pallocca M, Sanna N, Picardi E, Pesole G, Castrignanò T. WEP: a high-performance analysis pipeline for whole-exome data. BMC Bioinformatics. 2013;14(Suppl 7):S11
        135. Berger B, Peng J, Singh M. Computational solutions for omics data. Nat Rev Genet. 2013;14:333–46
        136. De Leeneer K, Hellemans J, De Schrijver J, Baetens M, Poppe B, Van Criekinge W, De Paepe A, Coucke P, Claes K. Massive parallel amplicon sequencing of the breast cancer genes BRCA1 and BRCA2: opportunities, challenges, and limitations. Hum Mutat. 2011;32:335–44
        137. Moonsamy PV, Williams T, Bonella P, Holcomb CL, Höglund BN, Hillman G, Goodridge D, Turenchalk GS, Blake LA, Daigle DA, Simen BB, Hamilton A, May AP, Erlich HA. High throughput HLA genotyping using 454 sequencing and the Fluidigm Access Array™ System for simplified amplicon library preparation. Tissue Antigens. 2013;81:141–9
        138. Taudien S, Groth M, Huse K, Petzold A, Szafranski K, Hampe J, Rosenstiel P, Schreiber S, Platzer M. Haplotyping and copy number estimation of the highly polymorphic human beta-defensin locus on 8p23 by 454 amplicon sequencing. BMC Genomics. 2010;11:252
        139. Parameswaran P, Jalili R, Tao L, Shokralla S, Gharizadeh B, Ronaghi M, Fire AZ. A pyrosequencing-tailored nucleotide barcode design unveils opportunities for large-scale sample multiplexing. Nucleic Acids Res. 2007;35:e130
        140. Okou DT, Steinberg KM, Middle C, Cutler DJ, Albert TJ, Zwick ME. Microarray-based genomic selection for high-throughput resequencing. Nat Methods. 2007;4:907–9
        141. Sulonen AM, Ellonen P, Almusa H, Lepistö M, Eldfors S, Hannula S, Miettinen T, Tyynismaa H, Salo P, Heckman C, Joensuu H, Raivio T, Suomalainen A, Saarela J. Comparison of solution-based exome capture methods for next generation sequencing. Genome Biol. 2011;12:R94
        142. Scheimann A, Pollock N, Stowell KM. Sequence capture and massively parallel sequencing to detect mutations associated with malignant hyperthermia. Br J Anaesth. 2013;110:122–7
        143. Priya RR, Rajasimha HK, Brooks MJ, Swaroop A. Exome sequencing: capture and sequencing of all human coding regions for disease gene discovery. Methods Mol Biol. 2012;884:335–51
        144. Ehm MG, Karnoub MC, Sakul H, Gottschalk K, Holt DC, Weber JL, Vaske D, Briley D, Briley L, Kopf J, McMillen P, Nguyen Q, Reisman M, Lai EH, Joslyn G, Shepherd NS, Bell C, Wagner MJ, Burns DKAmerican Diabetes Association GENNID Study Group. Genetics of NIDDM. . Genomewide search for type 2 diabetes susceptibility genes in four American populations. Am J Hum Genet. 2000;66:1871–81
        145. Kim JH, Jarvik GP, Browning BL, Rajagopalan R, Gordon AS, Rieder MJ, Robertson PD, Nickerson DA, Fisher NA, Hopkins PM. Exome sequencing reveals novel rare variants in the ryanodine receptor and calcium channel genes in malignant hyperthermia families. Anesthesiology. 2013;119:1054–65
        146. Isakov O, Perrone M, Shomron N. Exome sequencing analysis: a guide to disease variant detection. Methods Mol Biol. 2013;1038:137–58
        147. Hawkins RD, Hon GC, Ren B. Next-generation genomics: an integrative approach. Nat Rev Genet. 2010;11:476–86
        148. Peterlin B, Maver A. Integrative ‘omic’ approach towards understanding the nature of human diseases. Balkan J Med Genet. 2012;15:45–50
        149. Burke W, Matheny Antommaria AH, Bennett R, Botkin J, Clayton EW, Henderson GE, Holm IA, Jarvik GP, Khoury MJ, Knoppers BM, Press NA, Ross LF, Rothstein MA, Saal H, Uhlmann WR, Wilfond B, Wolf SM, Zimmern R. Recommendations for returning genomic incidental findings? We need to talk! Genet Med. 2013;15:854–9
        150. . Sequenced from the start. Nature. 2013;501:135

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