Professional Documents
Culture Documents
doi: 10.1093/bja/aev225
Review
REVIEW
Abstract
It is 30 yr since the British Journal of Anaesthesia published the first consensus protocol for the laboratory diagnosis of malignant
hyperthermia susceptibility from the European Malignant Hyperthermia Group. This has subsequently been used in more than
10 000 individuals worldwide to inform use of anaesthetic drugs in these patients with increased risk of developing malignant
hyperthermia during general anaesthesia, representing an early and successful example of stratified medicine. In 2001, our
group also published a guideline for the use of DNA-based screening of malignant hyperthermia susceptibility. We now present
an updated and complete guideline for the diagnostic pathway for patients potentially at increased risk of developing malignant
hyperthermia. We introduce the new guideline with a narrative commentary that describes its development, the changes to
previously published protocols and guidelines, and new sections, including recommendations for patient referral criteria and
clinical interpretation of laboratory findings.
Key words: CACNA1S; excitation–contraction coupling; malignant hyperthermia; malignant hyperthermia susceptibility,
diagnosis; RYR1; skeletal muscle
The European Malignant Hyperthermia Group (EMHG) was muscle samples were more sensitive to the contracture-inducing
formed in 19831 with the principal objective of standardizing properties of caffeine, or caffeine with halothane,2 or halothane
the laboratory diagnosis of malignant hyperthermia (MH) sus- alone3 than normal muscle, but with such interlaboratory vari-
ceptibility. At that time, pharmacological challenge tests carried ation that comparisons of results were impossible. One particular
out on excised skeletal muscle biopsies were being used for this area of contention was the combined caffeine–halothane test,
purpose by several groups in North America, Europe, South Africa, which had proponents in North America but not in Europe. In
and Australia. These tests were based on the findings that MH 1984, the EMHG published its protocol for an in vitro contracture
† Contributors to the guidelines from member laboratories of the European Malignant Hyperthermia Group are listed in Appendix 3.
© The Author 2015. Published by Oxford University Press on behalf of the British Journal of Anaesthesia. All rights reserved.
For Permissions, please email: journals.permissions@oup.com
1
Hopkins et al. | 2
test (IVCT),4 and this has formed the bedrock of clinical diagnosis writing committee, with two rounds of consultation with repre-
and phenotyping for research in Europe throughout the past sentatives from each member laboratory of the EMHG. The final
30 yr. The protocol has been the subject of annual review by version of the guideline (Appendix 1) was agreed by the EMHG
the EMHG and, since the launch of the EMHG website (www. in May 2014. We will now discuss the rationale for elements of
emhg.org), the latest version of the protocol has been available the guideline that were not included in either the IVCT protocol
on-line. Member laboratories of the EMHG participate in a quality or the previous genetic diagnosis guideline, and the major
assurance-programme, with a principal focus on compliance changes to these previous documents.
with the protocol.
Although the protocol was developed through an informal
consensus approach, it was based on the collective experience
Patient referral criteria
of the EMHG founding members, which in 1984 already encom- This is a new addition to previous guidelines. For possible malig-
passed investigation by muscle biopsy of more than 1500 indivi- nant hyperthermia reactions during general anaesthesia, we
duals at risk of MH susceptibility either through a history of a considered using the score provided by the Clinical Grading
suspected MH reaction or a family history. In 1984, there was no Scale.5 This can provide an estimate of the likelihood of a malig-
obvious means of validating the test because there were insuffi- nant hyperthermia reaction, but the decision required in the
cient data on the outcome of the test in low-risk individuals and diagnostic setting is whether MH can be excluded or not, for
no consensus on the clinical criteria for assigning ‘true-positive’ which the Clinical Grading Scale is generally not helpful. When
that seems likely in perhaps a minority of patients with exer- or both caffeine and halothane tests is MHS. A suffix is then
tional heat illness. There are indeed well-recognized non-genetic added to indicate an abnormal response to halothane (h) or caf-
factors that predispose to exertional heat illness,32 and we rec- feine (c), so that there are now four laboratory diagnostic groups:
ommend that these are excluded before further investigation, be- MHShc (formerly MHS); MHSh (formerly MHE); MHSc (formerly
cause the purpose of further tests is to identify individuals (and MHE); and MHN (unchanged).
their relatives) at risk of recurrent episodes, rather than to con- The second limitation of the old (and indeed new) laboratory
firm the clinical diagnosis of heat illness per se. classification system is that it is crude, not using a wealth of data
that can be derived from the contracture studies. This was best
appreciated at the outset of molecular genetic investigations of
The in vitro contracture test MH susceptibility, because accurate phenotyping is essential
Only very minor adjustments have been made to the technical for robust genetics. The EMHG adopted, although never pub-
conduct of the IVCT compared with the last published version6 lished, a recommendation that the minimal data set for the
because the validation study revealed a clinically useful and ro- IVCT phenotype should be the threshold concentration (concen-
bust test.6 One of these amendments is to stipulate an increased tration producing a contracture of 2 mN or 0.2 g force) of halo-
minimal length of muscle specimen for use in the tests, because thane and caffeine, along with the tension produced at 2%
muscle length has emerged as the major determinant of speci- halothane and at 2 mM caffeine. Even more sophisticated ap-
men viability (EMHG, unpublished data). The EMHG quality-as- proaches have been proposed,34 35 and the genetic basis for the
Recent evidence has also confirmed the foresight of the previ- be easy to publish, and we have therefore removed the need for
ous guidelines in requiring functional analysis of missense functional analyses using previously described methods to be
variants before their adoption for diagnostic use. Kim and collea- published before accepting the relevant variant for diagnostic
gues18 reported the prevalence of rare RYR1 variants in a control use. Instead, reports of functional analyses will be peer reviewed
sample to be 6%. It is interesting that generic guidelines for in- by members of the EMHG with the relevant expertise.
terpretation of findings of variants in other genes adopt a similar Advances in genetic technology have prompted a change in
approach.38 These generic guidelines also propose using segrega- our recommendations for the diagnostic pathway of patients re-
tion analyses39 to indicate likely pathogenicity, but in our experi- ferred for investigation of MH susceptibility (Fig. 1). Our previous
ence it is rare to generate sufficient statistical power to enable genetic testing guideline19 recommended muscle biopsy and
this, even when combining data from several families carrying IVCT as the primary investigation for the index case, with subse-
the same variant. quent mutation screening when the IVCT confirmed MH suscep-
Unfortunately, the costs and technical difficulties associated tibility. We now consider DNA screening to be a viable alternative
with conducting rigorous functional analyses are rate limiting. Al- primary diagnostic approach to the IVCT.45 The more than doub-
though more than 180 RYR1 variants have been associated with ling in number of functionally characterized variants associated
MH susceptibility, only 33 have been shown to have functional ef- with MH susceptibility has increased the proportion of MH fam-
fects consistent with MH pathogenicity (www.emhg.org). Two ilies able to benefit from DNA diagnostics from ∼25% in 2001 to
variants in CACNA1S have also been shown to be functionally more than 40% now (data extrapolated from Carpenter and col-
MH susceptible
References
MH susceptible* MH negative
1. Ellis FR. European Malignant Hyperpyrexia Group. Br J Anaesth
1984; 56: 1181–2
Fig 1 Diagnostic pathway for investigation of MH susceptibility. The
2. Kalow W, Britt BA, Terreau ME, Haist C. Metabolic error of
decision to pursue either DNA screening or muscle biopsy and IVCT in
muscle metabolism after recovery from malignant hyper-
the first instance will be made on a patient-by-patient basis by the MH
diagnostic centre in consultation with the patient and their health-care
thermia. Lancet 1970; 2: 895–8
funder. Factors that may be taken into consideration will include the 3. Ellis FR, Keaney NP, Harriman DG, et al. Screening for malig-
availability of the respective tests (including turnaround time), the nant hyperpyrexia. Br Med J 1972; 3: 559–61
urgency of the test (for example, if the patient is awaiting surgery), 4. The European Malignant Hyperpyrexia Group. A protocol for
the prior probability of a positive diagnosis, and the costs of the tests in
the investigation of malignant hyperpyrexia (MH) suscepti-
the relevant laboratory. The prior probability of a positive diagnosis will
bility. Br J Anaesth 1984; 56: 1267–9
be estimated by the clinical director of the MH centre based on clinical
judgement, published data,5 44 or a combination of these. *These patients
5. Larach MG, Localio AR, Allen GC, et al. A clinical grading scale to
should be invited to take part in research studies of the genetic basis of predict malignant hyperthermia susceptibility. Anesthesiology
malignant hyperthermia. IVCT, in vitro contracture test; MH, malignant 1994; 80: 771–9
hyperthermia; MHN, in vitro contracture test laboratory classification 6. Ording H, Brancadoro V, Cozzolino S, et al. In vitro contracture
applied when all contracture tests are negative; MHShc, MHSh, and MHSc,
test for diagnosis of malignant hyperthermia following the
IVCT laboratory classifications applied when contracture responses to
protocol of the European MH Group: results of testing patients
both halothane and caffeine are abnormal, response to halothane alone
is abnormal, or response to caffeine alone is abnormal, respectively. surviving fulminant MH and unrelated low-risk subjects. Acta
Anaesthesiol Scand 1997; 41: 955–66
5 | Malignant hyperthermia diagnostic guidelines
7. McCarthy TV, Healy JM, Heffron JJ, et al. Localization of the 25. Isaacs H, Barlow MB. Malignant hyperpyrexia during anaes-
malignant hyperthermia susceptibility locus to human thesia: possible association with subclinical myopathy. Br
chromosome 19q12–13.2. Nature 1990; 342: 562–4 Med J 1970; 1: 275–7
8. MacLennan DH, Duff C, Zorzato F, et al. Ryanodine receptor 26. Ellis FR, Clarke IM, Modgill M, Currie S, Harriman DG. Evalu-
gene is a candidate for predisposition to malignant hyper- ation of creatine phosphokinase in screening patients for
thermia. Nature 1990; 343: 559–61 malignant hyperpyrexia. Br Med J 1975; 3: 511–3
9. Levitt RC, Olckers A, Meyers S, et al. Evidence for the localiza- 27. Weglinski MR, Wedel DJ, Engel AG. Malignant hyperthermia
tion of a malignant hyperthermia susceptibility locus (MHS2) testing in patients with persistently increased serum creatine
to human chromosome 17q. Genomics 1992; 14: 562–6 kinase levels. Anesth Analg 1997; 84: 1038–41
10. Iles DE, Lehmann-Horn F, Scherer SW, et al. Localization of 28. Hopkins PM, Ellis FR, Halsall PJ. Evidence for related myop-
the gene encoding the α2/δ-subunits of the L-type voltage- athies in exertional heat stroke and malignant hyperthermia.
dependent calcium channel to chromosome 7q and ana- Lancet 1991; 338: 1491–2
lysis of the segregation of flanking markers in malignant 29. Bendahan D, Kozak-Ribbens G, Confort-Gouny S, et al. A
hyperthermia susceptible families. Hum Mol Genet 1994; 3: noninvasive investigation of muscle energetics supports
969–75 similarities between exertional heat stroke and malignant
11. Robinson R, Monnier N, Wolz W, et al. A genome-wide search hyperthermia. Anesth Analg 2001; 93: 683–9
for susceptibility loci in two European malignant hyperther- 30. Tobin JR, Jason DR, Nelson TE, Sambuughin N. Malignant
cells transfected with malignant hyperthermia or central Appendix 1: European Malignant Hyperthermia
core disease mutant Ca2+ release channels. J Biol Chem 1999; Group guideline for the investigation of
274: 693–702
malignant hyperthermia susceptibility
43. Yang T, Ta TA, Pessah IN, Allen PD. Functional defects in six
ryanodine receptor isoform-1 (RyR1) mutations associated Investigation of malignant hyperthermia (MH) susceptibility ini-
with malignant hyperthermia and their impact on skeletal ex- tially involves clinical evaluation of a patient’s risk based on their
citation-contraction coupling. J Biol Chem 2003; 278: 25722–30 anaesthetic and medical history, and relevant family history.
44. Ellis FR, Halsall PJ, Christian AS. Clinical presentation of sus- Further investigation is indicated when increased risk of suscep-
pected malignant hyperthermia during anaesthesia in 402 tibility to MH cannot be excluded. The highest sensitivity for de-
probands. Anaesthesia 1990; 45: 838–41 tecting susceptibility to MH is provided by pharmacological
45. Forrest KM, Foulds N, Millar JS, et al. RYR1-related malignant challenge tests carried out on freshly excised skeletal muscle in
hyperthermia with marked cerebellar involvement – a para- controlled laboratory conditions. These tests, when carried out
digm of heat-induced CNS injury? Neuromuscul Disord 2015; according to the following protocol, are collectively referred to
25: 138–40 as the in vitro contracture test, or IVCT. The IVCT is recommended
46. Tong J, Oyamada H, Demaurex N, Grinstein S, McCarthy TV, for individuals considered to be at increased risk of MH either as a
MacLennan DH. Caffeine and halothane sensitivity of intra- first-line test or when DNA analyses have failed to confirm the
cellular Ca2+ release is altered by 15 calcium release channel high-risk status. DNA analyses are less invasive than the IVCT
stated with a maximum deviation of (10%), and its pH should a sustained increase of at least 2 mN (0.2 g) in baseline force
be in the range 7.35–7.45 at 37°C. from the lowest force reached. In addition, the maximal con-
5. The muscle should be transported to the laboratory in Krebs– tracture achieved at a caffeine concentration of 2 mmol litre−1
Ringer solution at ambient temperature. In the laboratory, it should be reported. Please note that the lowest force is not ne-
should be kept at room temperature and carboxygenated. cessarily the same as the predrug force.
6. The time from biopsy to completion of the tests should not 12. The static halothane test and measurement of static halo-
exceed 5 h. thane threshold. The halothane threshold is obtained using
7. The tests should be performed at 37°C in a tissue bath the halothane concentrations 0.11, 0.22, 0.44, and an optional
perfused either intermittently or continuously with Krebs– concentration of 0.66 mmol litre−1 as equivalent to 0.5, 1.0, 2.0,
Ringer solution and carboxygenated continuously. At least and 3.0 Vol%, respectively, from a serviced and calibrated
four tests should be performed, each one using a fresh speci- vaporizer. It is recommended that the halothane concentra-
men. These include two static caffeine tests (see 11 below) tion in the gas phase should be measured close to the inlet
and two halothane tests. The halothane test could consist port of the tissue bath or the tissue bath concentration should
of either one static (see 12 below) and one dynamic test (see be measured regularly using gas chromatography, or both
13 below) or two static tests. Each laboratory should be con- (see under Quality Control, below). The specimen should be
sistent in the method used. Separate tissue baths should be exposed to each halothane concentration for at least 3 min
used for different agents. or until maximal contracture is reached. The result of this
• MHSc: a caffeine threshold at a caffeine concentration of 2.0 hyperthermia. An MHN-tested individual cannot transmit
mmol litre−1 or less and a halothane threshold concentra- MH risk to their offspring.
tion above 0.44 mmol litre−1 in all halothane tests.
• MHN: a caffeine threshold at a caffeine concentration of
3 mmol litre−1 or more in all caffeine tests and a halothane D. Molecular genetic detection of susceptibility
threshold concentration above 0.44 mmol litre−1 in all halo- to malignant hyperthermia
thane tests. Although an MH episode must be considered a multifactorial se-
15. Quality control. Viability in any specimen used should be de- quence of events, the genetic basis for MH susceptibility is largely
monstrated by twitches ≥10 mN (1 g) at the beginning of a test attributable to mutations in the RYR1 gene. Despite several link-
and, for the caffeine test, a response to 32 mmol litre−1 ≥50 age and screening studies, mutations associated with MHS have
mN (5 g) at the end. The concentrations of halothane and caf- been found only in RYR1 and, more rarely, in CACNA1S, the gene
feine in the tissue bath should be checked at least every 6 for the skeletal muscle L-type voltage-dependent Ca2+ channel
months. The samples should be obtained directly from the (Cav1.1 or DHPR).
tissue bath in the same dynamic conditions as when testing. The great majority of mutations reported in RYR1 result in the
Samples for determination of halothane concentrations replacement of an individual amino acid. With the currently
should be obtained immediately after the gas flow has been available algorithms, it is challenging to predict the functional
stopped to avoid sampling from the gas phase. Halothane consequence of a given amino acid substitution within a large
Appendix 2: Characterization of RYR1 functionally characterized using any of the previously de-
sequence variants scribed methods (section 2 above), data can be submitted
directly to the EMHG through its website. Functional data
1. Genetic characterization. Each variant should be fully charac- acquired using novel methods will require validation through
terized at the genetic level, including: publication in a peer-reviewed journal.
• A full description at the DNA and protein level, considering
aspects of evolutionary conservation and change in charge,
polarity, or structure introduced by the amino acid Disclaimer
replacement;
• Co-segregation of the variant with the disease in the family These guidelines represent the views of the European Malignant
or families affected; and Hyperthermia Group. They are based on careful consideration
• Assessment of the prevalence of the variant in a relevant and interpretation of the available evidence at the time that
population by means of database searches [e.g. dbSNP (http:// they were agreed. They are intended principally for clinical scien-
www.ncbi.nlm.nih.gov/SNP), 1000 Genomes (http://browser. tists and clinicians involved in the laboratory diagnosis of malig-
1000genomes.org), and exome variant server (http://evs.gs. nant hyperthermia, who are encouraged to take them fully into
washington.edu/EVS/)]. It is anticipated that pathogenic account when exercising their diagnostic judgement. The guide-
variants will have a minor allele frequency <1%. The lines do not override the individual responsibility for laboratory
directors and diagnostic clinicians to make appropriate decisions