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• W2037711258 abstract "Human malignant hyperthermia (MH) is a clinical syndrome, not a single disease, triggered by exposure to volatile anesthetics and depolarizing skeletal muscle relaxants that is often, but not always, inherited as an autosomal dominant trait with incomplete penetrance and variable expressivity. MH arises from mutations that exhibit both locus heterogeneity (i.e., MH from different genes) and allelic heterogeneity (i.e., MH from different mutations in a single gene). In the present issue of the journal, Vukcevic et al.1 report testing for MH susceptibility using Epstein-Barr virus (EBV)-transformed lymphoblasts cultured from lymphocytes of humans known to be predisposed to MH by prior clinical event and skeletal muscle contracture test. The authors demonstrate greater cytoplasmic calcium (Ca2+) responses to 4-chlorocresol (4-cmc), a skeletal muscle Ca2+ release channel/ryanodine receptor (RYR1) agonist, in lymphoblasts from 5 MH patients with different mutations when compared with normal controls. The report is commendable in a number of respects including provision of detailed clinical summaries and in vitro contracture test (IVCT) data, the addition of corresponding genotypic information, the comparison of Ca2+ transients over a range of 4-cmc concentrations, and determination of EC50 values. In particular, the method described is noteworthy for its potential to address some of the challenges confronting MH diagnostic assays based on conventional contracture testing and genotyping. Independent of a clinical event, the most widely accepted method for ascertaining MH susceptibility is the ex vivo muscle biopsy configured for physiological testing of changes in contractility in the presence of volatile anesthetics (i.e., halothane) and the RYR1 agonist, caffeine. The tests, developed with different protocols in North America and Europe, are known as the caffeine halothane contracture test (CHCT) and the IVCT, respectively.2,3 Contracture strength thresholds that segregate normal, susceptible, and equivocal (in Europe) responses have been arrived at by consensus, based on data from patients with clinically documented MH in the absence of coincident myopathy (which may itself introduce bias4), and compared with the responses of normal individuals with an intentional bias favoring false-positive designations to not miss potentially susceptible individuals if a higher cutoff is chosen. The utility of the contracture test varies with its intended application, i.e., for differential diagnosis of a confusing event in the operating room, for MH research, and as a guide to drug selection in clinical care and genetic counseling. In the first case, if positive results are found, thereby indicating that an untoward perioperative event was most probably MH, the contracture test holds substantial value and may be lifesaving to a patient and family members. Negative results on a test performed for differential diagnosis are a greater challenge to interpretation, but may shift the focus away from a diagnosis of MH. For MH research, the value of the IVCT is unquestioned. Present-day knowledge of the molecular pathogenesis of human MH would be inconceivable without contracture test data to correlate with candidate genes and mutations in patients and pedigrees. However, as an instrument for genetic counseling, and as an aid to the caregiver tasked with selecting a clinical anesthetic regimen in a relative of an MH patient, contracture testing has well-recognized shortcomings. Contracture testing is invasive, scarring, expensive (approximately $5000), and is now performed at only 5 centers in North America (www.mhaus.org). There is not, and cannot be, experimental validation of contracture test results on children younger than 10 years of age, a population at higher risk for MH than adults. The interlaboratory replicability of the European IVCT (i.e., samples from the same patient tested in different laboratories using the same protocol) is not high (i.e., 56% between laboratories in the only published study5). Comparable results using the North American CHCT protocol and thresholds have yet to appear. Nor has contracture test reproducibility (i.e., samples from the same patient tested in the same laboratory on different days) been reported for either the European or North American test. Whereas a positive test result (and most probably an equivocal result), using whatever thresholds have been selected, provides evidence against the further use of trigger agents in a given patient or family member, the meaning of negative contracture tests in patients with enough indication to have the test in the first instance (e.g., the individual has a family member with MH) is not known. To measure the true incidence of false-negative contracture test results requires sufficient numbers of IVCT- and CHCT-negative patients to undergo subsequent anesthetics with trigger agents. Because an MH-susceptible individual may have as many as 30 uneventful anesthetics before experiencing a trigger,6 the number of anesthetics needed on contracture test–negative individuals to determine the false-negative incidence with precision is unsettled. Moreover, the incidence of false-negative (and false-equivocal) results is likely to vary between distinct MH-predisposing mutations in distinct genetic backgrounds. As the present study confirms, different RYR1 mutations confer varying degrees of cellular dysfunction, as measured by agonist-induced Ca2+ release, in samples from patients with distinct genetic backgrounds, thereby undermining confidence that 1 or 2 threshold contracture test values are sufficient to segregate all susceptibility to the MH syndrome into 2 or 3 categories (i.e., negative, positive, and/or equivocal) for the purposes of genetic counseling. Accordingly, no such investigation of the incidence of false-negative contracture test responses has been, or is likely to be conducted, and no peer-reviewed scientific data are available to the concerned patient, family member, or caregiver regarding the safety of administering drugs that trigger MH to patients who are contracture test negative (or equivocal). Although genetic testing for MH differential diagnosis and counseling is minimally invasive, requiring only a few drops of blood or a cheek swab for DNA isolation, is easily replicated and reproduced, and is less costly than contracture testing depending on the number of mutations sought, inherent deficiencies also constrain widespread MH genotyping. Only about 60% of human MH susceptibility may be correlated with DNA sequence variations in the RYR1 gene.7 Although >200 amino acid substitutions in RYR1 have been identified, only 26 have been experimentally classified as causal mutations (see list maintained at www.emhg.org). Even with full-length RYR1 sequencing, a number of mechanisms for MH genetic pathology may be missed, for example, copy number variations, epigenomic modifications, and polymorphisms and alternate splice sites in unsequenced introns and promoter regions. Most importantly, a full-length genomic characterization of RYR1 leaves the contribution of other known and suspected loci unscreened. At least 4 additional genetic loci have been associated with MH predisposition. With the exception of the skeletal muscle isoform of the α subunit of the voltage-dependent calcium channel (CACNA1S),8 also known as the dihydropyridine receptor (DHPR), other MH genes remain unknown. Polymorphisms at these loci may themselves act as Mendelian determinants of MH susceptibility, or they may serve in concert with known loci to amplify MH susceptibility. Thus, in the setting of both locus and allelic genetic heterogeneity, interpretation of MH genetic test data is not straightforward. Whereas the presence of a shared MH mutation in family members of a proband who has had a clear-cut clinical MH trigger permits identification of these individuals as MH susceptible, the absence of 1 or more genotypes in question from a partial panel (i.e., a negative genetic test result) is not interpretable. In most, if not all cases, a patient will not have been harmed by the genetic testing, and his or her a priori risk of MH will be somewhat less than that in the untested population. Indeed, it has been estimated that up to 1 of 2000 patients carry alleles that may predispose to MH upon exposure to trigger agents.9 Unfortunately, in view of this figure, genotyping for MH susceptibility will remain incomplete for the foreseeable future. Relying on earlier work,10 Vukcevic et al. reasoned that white cells from patients with previously uncharacterized RYR1 mutations and a history of MH susceptibility by clinical event and IVCT abnormalities would also respond to lower concentrations of 4-cmc than cells from normal individuals. The authors asked study participants' centers to have anticoagulated whole blood samples drawn by their primary care physicians and forwarded by mail to a testing site in Würzberg, Germany for white blood cell isolation. There, B-cell lymphocytes were purified, cultured, and transformed into patient-specific, lymphoblastoid (i.e., dedifferentiated) cell lines via EBV infection.11 The EBV-transformed lymphoblasts, which express the skeletal muscle RYR1 isoform, were then tested for intracellular Ca2+ release from endoplasmic reticulum stores using changes in Ca2+-sensitive fluorescent dye intensity in response to increasing concentrations of 4-cmc, and the responses were compared with those in cells from control participants. The authors report that cell lines from each MH-susceptible individual with a distinct mutation responded to 4-cmc with statistically significant lower EC50 values than cells from normal individuals, and that the magnitude of the responses differed between individuals with different mutations. If confirmed, it is possible to envision a test devised for both research and clinical applications based on the present data that may complement contracture testing and genotypic data for the diagnosis of MH susceptibility and counseling. Such a test holds promise to be noninvasive, inexpensive, patient- and family-specific, and amenable to a formal characterization for analytical and clinical validity including measures of both replicability and reproducibility. For this to occur, however, a number of questions must first be answered. The size of the present study is small, with just 3 of 5 MH patients lacking a coexisting myopathy, and the remaining 2 with central core disease and an undiagnosed myopathy, respectively. Will similar results be observed when more patients, some with the same mutation from the same family and some with the same mutation from different families, are tested? Additionally, the magnitudes of response differences between patients and normal controls are small in the reported Ca2+ release assays, but the variances are relatively large, particularly when the standard errors of the mean are corrected to standard deviations. Will statistical significance and clinical significance be maintained when variances between different patients with the same mutation are tested against normal controls, rather than variances between different samples of cells cultured from the same patient? Although EBV-transformed cells have many attractive properties as test vehicles, it is not known whether the RYR1 channel expressed in lymphoblastoid cells is identically regulated in comparison to that of skeletal muscle. Is the response of RYR1 in circulating white cells, therefore, a true surrogate for the MH responsiveness of skeletal myocytes? Of note, the authors have not provided positive control data taken, for example, from patients expressing mutations with clear-cut MH causality, nor have they provided the reader with information regarding selection of the normal control participants. How many individuals comprised the normal control group? Were the normal participants previously anesthetized without event? How often? Did they have contracture tests? What were the results? Did they have a relative with MH? Were they genotyped? If they were genotyped, how extensive was this evaluation? Nor do the authors justify selection of 4-cmc. Were other agents tried (e.g., halothane or caffeine) and failed? Was effort given to testing agents in combination to magnify differences between patient and normal responses, and to control the magnitude of variances? Answers to these and other methodological questions are critical to more widespread development of the present and similar assays that aim to fill in gaps in contracture testing and genotyping outlined above. The present investigation is not the first to correlate an MH genotype with an altered physiological phenotype in lymphoblastoid cells exposed to 4-cmc. Zullo et al.12 recently immortalized lymphoblastoid cell lines from normal and MH-susceptible patients with diverse RYR1 mutations, and reported enhanced 4-cmc–induced acidification of the cell medium in samples from patients with a history of MH when compared with normal controls. Ca2+ fluxes were not directly measured. Whereas these authors demonstrated that a subset of cell lines with specific mutations exhibit increments in extracellular acidification, other cell lines from MH-susceptible individuals, for example, those expressing the Cys4664Arg mutation, did not acidify the medium even to the same degree as observed in control cells, thereby suggesting that MH may not be the consequence of a single shared pathway and that not all steps in MH pathogenesis are modeled by tissues other than muscle. Although Ca2+ fluxes may be an appropriate index for MH susceptibility in patients with RYR1 mutations, it is not yet known whether Ca2+ fluxes play a role in MH in individuals who have mutations at other known or as yet unidentified genetic loci. Thus, the general applicability of the methods proposed by Zullo et al. and those of the present article awaits acquisition of further data. The future of phenotypic testing for uncommon genetic disorders, and in particular pharmacogenetic disorders such as MH, however, seems brighter to us than might be apparent from the above discussion. Notably, stem cell research and somatic nuclear transfer offer possibilities not dreamed of when contracture testing for MH was initially developed. It is now possible to isolate patient-specific nuclei carrying mutations of interest from such sources as white blood cells or skin fibroblasts, and transfer them into pluripotential cells. In turn, the modified cells may then be induced to form muscle cells upon which exposure to trigger agents and physiological testing (for example, by measuring Ca2+ transients and acidification in response to various MH triggers) can proceed.13,14 In addition, the ability to generate patient-specific induced pluripotent stem cells from readily available tissues and to transform them into differentiated cell lines for further investigation has recently been described.15–17 Thus, the panoply of human genetic heterogeneity is now amenable to quantification with rigorous control of the polymorphism of interest, the genetic background of the sample, and the cellular apparatus of the relevant differentiated cell. Correlation of data from these methods with detailed clinical information, genotyping, and with quantitative rather than categorical data derived from contracture testing and the cellular assays reported herein will be critical to their validation and refinement. Because the mutation itself need not be known to discriminate modified cells from MH and non-MH sources, phenotypes resolved in this fashion may also be fitting substrates for pedigree and genome-wide association studies aimed at discerning novel genetic loci capable of imparting MH susceptibility. Accordingly, we believe that comprehensive and well-validated preoperative testing for MH susceptibility is not an insurmountable aim, but rather one that will require the acquisition of an expanded and integrated test repertoire. AUTHOR CONTRIBUTIONS Both authors helped write the manuscript, and approved the final manuscript." @default.
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• W2037711258 date "2010-07-01" @default.
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• W2037711258 title "Out of a Cell into This Darkened Space" @default.
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