The goal of a simple non-invasive DNA test for susceptibility to malignant hyperthermia (MH) has remained elusive. Given the complexity of the disorder at the genetic level, inherent with a genetically heterogeneous1 condition, the initial aim has been to introduce limited DNA testing for some families. This issue contains the first comprehensive description of the circumstances in which limited DNA testing may be introduced.2 This is in the form of guidelines produced by the European Malignant Hyperthermia Group (EMHG). It is, however, important to appreciate that these guidelines represent a consensus statement and not a validated process. In this editorial we examine the evidence underpinning the guidelines and the steps required for them to be validated.
For the last 30 yr the invasive in vitro muscle contracture test (IVCT) has been the only recognized diagnostic tool to assess MH status. A standardized European protocol was published in 1984.3 Using this assay patients are assigned MH susceptible (MHS), MH normal (MHN) or MH equivocal (MHE) according to the in vitro contracture response of viable muscle tissue following exposure to halothane and separately to caffeine. An increase in contracture of 0.2 g at a threshold dose of 2% halothane, caffeine 2 mM is considered a positive test result. The MHE class includes patients who react to only one of the triggering agents used in the test. Thresholds for this testing procedure have been set to avoid potentially dangerous false-negative diagnoses. Consequently, a false-positive rate of approximately 6% has been suggested from IVCT analysis of patients with a low-risk of MH, using the European IVCT protocol.4 This can create problems for genetic studies when trying to assess the degree of concordance between IVCT phenotype and MH genotype, an exercise essential in the validation of any genetic testing procedure.
A primary aim of the EMHG has been to standardize testing procedures for MH susceptibility to attain consistency between centres offering a screening service.3 It should be emphasized that the IVC-testing procedure will retain a central role for the diagnosis of MH in the proposed genetic testing strategy and that genetic testing will not be beneficial to all families. Only on confirmation of MH in a family by an IVCT positive result in a suspected index case would it be appropriate to refer a patient for molecular genetic analysis. Confirmation of MH status through genetic analysis could then lead to quicker results for the rest of the kinship, as opposed to the present situation where IVCT is offered to relatives.
In the EMHG guidelines, two types of analyses are proposed for the genetic investigations.2
(i) Mutation testing is proposed only for the RYR1 gene (encoding the sarcoplasmic reticulum calcium release channel) and then only for RYR1 mutations recognized as causative of MH. Genetic heterogeneity in MH is well documented with potentially five other susceptibility loci reported through linkage analyses of MH pedigrees.58 However, defects in the RYR1 gene account for a major proportion of MH cases (up to 80% of families investigated to date9). Restriction of genetic testing to the analysis of causative mutations has been primarily in this direction because all RYR1 mutations reported to date are missense.10 The pathogenicity of missense mutations must be carefully considered. Often the effect of such substitutions on protein function is minimal if the side chain of the replacing amino acid is similar to the original (conservative substitutions). Replacements where the side chains are different (nonconservative substitutions) are more likely to have some effect on protein function. Evidence to support the involvement of missense mutations in disease is gained: by considering the position of the affected amino acid in the protein e.g. at a functionally relevant site, or at a site which is conserved across species, or between members of a gene family; by the exclusive segregation of the mutation with the trait; by lack of segregation in a normal control population; and ideally by functional characterization in an appropriate assay.
The 15 mutations listed in the genetic testing guidelines occur in the myoplasmic RYR1 domain at sites shown to be conserved across the RYR genes characterized to date and in RYR functional homologues cloned from other species.10 Ten of the 15 mutations result in charge/polarity changes of the amino acid side chain. In addition, no reports have described any of the mutations occurring in patient control groups. All 15 have been investigated in functional assays using cultured cells transfected with mutant RYR channels.1115 Mutations that were shown to give a higher amplitude of calcium release in response to low concentrations of caffeine and halothane compared to wild-type RYR channel controls in these studies have been defined as causative mutations in the EMHG guidelines. While the data available support a potential pathological role for these mutations in predisposition to MH susceptibility, the non-physiological conditions and small numbers of cells used in these studies, along with the apparent increasing genetic complexity of the disorder favour a more cautious interpretation of their functional significance. On genetic investigation the guidelines dictate that if a causative mutation were identified in a family member, then the individual would be considered susceptible to MH. If no mutation was detected, even if a familial mutation had previously been identified, the patient must be referred for IVC-testing to confirm MH status.
(ii) Assuming an autosomal dominant mode of inheritance, segregation analysis of genetic markers close to the known MHS loci will have a role in genetic testing. This role will be limited compared to that of mutation screening because the approach is dependent upon the characterization of an extended MH pedigree by the IVCT in order to achieve a statistically significant threshold for linkage. The likelihood of genetic linkage is expressed as the log of the odds ratio, or lod score: a lod score of +3.0 (corresponding to odds of 1000:1) is generally considered as sufficient to establish linkage. This approach will have most relevance to families already extensively characterized by the IVCT and in whom no RYR1 mutation has been found. If a high-risk haplotype can be established within a family, an individual not previously investigated may be considered at risk of MH, on the basis of genetic data alone, if found to carry the high-risk haplotype identified in the other IVCT-MHS cases in the family. As with the application of mutation testing, the EMHG guidelines advise that an individual must be referred for IVC-testing if the high-risk haplotype is not found.
To the uninitiated the guidelines for testing may seem highly conservativebut for good reason. Genetic testing for MH susceptibility is certainly not at the stage where it may be used in replacement of the IVCT. Thus any genetic analyses must be conducted in co-operation with a recognized IVCT centre. The reason for caution is a consequence of reported discordance between IVCT and genetic data in MH families. Examples include IVC-tested MH normal patients carrying an RYR1 mutation/high-risk haplotype and/or IVC-tested MH susceptible patients who do not carry the familial RYR1 mutation/high-risk haplotype. There are several possible explanations for discordance: a false +ve/ve IVCT result (the way the thresholds were set for the assay suggests that some false positive diagnoses are inevitable); variable penetrance of the mutation detected; or additional genetic factors may be involved in determination of phenotype in a discordant individual. This raises the question as to the appropriateness of the assumed genetic model for the trait.16
Until these observations can be more fully explained, the guidelines advise that for predictive diagnosis in families, a conservative approach is appropriate i.e. either the MHS test result of the IVCT, or the presence of the high-risk haplotype/causative mutation will be used to infer an MH susceptible phenotype. An MH negative diagnosis must be confirmed by a normal IVCT result. In this way, false-negative diagnoses are minimized but an increase in false-positive diagnoses is possible. Therefore, a current objective for the EMHG must be to substantiate these guidelines for genetic testing. In a retrospective approach, this will involve extensive genetic characterization of families previously investigated by the IVCT. Data generated from such studies will be pivotal for the validation and appropriate revision of the guidelines if necessary, whereupon it may be necessary to revisit diagnoses based on genetic data alone in some patients.
In conclusion, the publication of a group consensus on guidelines for genetic testing for MH susceptibility is welcomed. The literature to date has not clarified directly, or considered how appropriate genetic testing is for this condition. Data from European studies suggests that such a service will benefit many families, reducing the number of individuals that have to undergo the current invasive testing procedure, and allowing those individuals who may not be eligible for IVC-testing (very young/old) to be investigated.
R. L. Robinson
P. M. Hopkins
Malignant Hyperthermia Investigation Unit
Academic Unit of Anaesthesia
St Jamess University Hospital
Leeds LS9 7TF
UK
References
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