1Department of Anaesthesiology and Intensive Care Medicine, University of Leipzig, Liebigstr. 20a, D-04103 Leipzig, Germany. 2Institute for Human Genetics, University of Leipzig, Philipp-Rosenthal-Str. 55, D-04103 Leipzig, Germany*Corresponding author
Accepted for publication: March 6, 2001
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Abstract |
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Br J Anaesth 2001; 87: 2405
Keywords: complications, malignant hyperthermia; genetic factors, malignant hyperthermia; muscle skeletal, in vitro contracture test
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Introduction |
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The MH disposition in suspected individuals or families may be determined by in vitro contracture testing (IVCT) of a skeletal muscle biopsy; this is at present felt to be the most reliable diagnostic method. The procedures for IVCT were developed by the European and North American malignant hyperthermia groups (EMHG, NAMHG). They show a high sensitivity (99% for EMHG, 9297% for NAMHG) and specificity (94% for EMHG, 5378% for NAMHG).57 Besides determining MH disposition, the IVCT is the basic requirement for interpreting genetic MH data.
An important step towards understanding the pathogenesis of the disease was the identification of MH-associated genetic mutations. The ryanodine receptor (RYR1), which represents the calcium release channel of skeletal muscle sarcoplasmic reticulum, was considered to be the primary locus for MH susceptibility.810 In approximately 50% of MH families, a link with the coding region of the RYR1 gene (MHS1 locus on chromosome 19q1213.1) can be detected.11 Over 20 point mutations in the RYR1 gene have so far been found to segregate with the MHS phenotype.12 For most of these missense mutations it has been possible to identify functional effects on the calcium channel reflecting an increased sensitivity of calcium release to triggering substances.13 Because of this established genotype/phenotype correlation, carriers of a known MH-related mutation should be regarded as having a high risk of MH.1315 A genetic MH analysis may, therefore, be useful for family screening. Guidelines for molecular genetic work have recently been compiled by the EMHG.16 Using a family with a predisposition to MH as an example, we shall now discuss the diagnostic approaches currently available for determining the MH disposition.
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Patients and methods |
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Three months after this incident the patient underwent the IVCT.
In vitro contracture test
The IVCT was performed according to the criteria of the standard procedure of the European MH group.5 The test determines the threshold concentrations of halothane and caffeine that produce a contracture force 2 mN in an isolated muscle specimen. Depending on the results, the tested individual is classified as MH susceptible (MHS). MHS: contracture force
2 mN at a caffeine concentration of 2.0 mmol litre1 or less and a halothane concentration of 0.44 mmol litre1 or less; MH negative (MHN): contracture force
2 mN at a caffeine concentration of 3.0 mmol litre1 or more and a halothane concentration greater than 0.44 mmol litre1; or MH equivocal (MHE): contracture force
2 mN at a caffeine concentration of 2.0 mmol litre1 or less (MHEc) or a halothane concentration of 0.44 mmol litre1 or less (MHEh).
Molecular genetic investigation
Direct sequencing
Genomic DNA was prepared from whole blood. The index patient was screened for published MH-related mutations in the amino terminal and central region of the RYR1 gene by PCR amplification and direct sequencing. Purified PCR products were directly sequenced by the ABI Prism DNA Sequencing Kit (Perkin Elmer, USA). The samples were then loaded on 5.25% PAGE-PLUS gel (Amresco), electrophoresed in the ABI PRISM 377 Sequencer (Perkin Elmer) and aligned with Sequence Navigator Software (Perkin Elmer). Both strands were sequenced and compared to permit identification of ambiguities.
Restriction enzyme analysis
A 918-bp17 PCR product of RYR1 exon 17 was digested with the restriction enzyme RsaI. Standard PCR conditions were used for amplification: denaturation at 95°C for 1 min, annealing at 62°C for 1 min, extension at 72°C for 2 min, 30 cycles; forward primer 5'-TTG CCA CAT CTT ATC CCG ATG CGC; reverse primer 5'-GAA CCT GTC CAG AGA TGC AGT CCA TC. After purification, the PCR product was mixed with 10 U of the restriction enzyme RsaI and incubated for 8 h at 37°C. According to GenBank (Accession no.: U48557) the following fragments were to be expected (OMIGA 1.1, Oxford Molecular Ltd.): normal type 166, 553, 12, 185, 2 bp; heterozygous C1840T-mutation 166, 553, 719, 12, 185, 2 bp; homozygous C1840T-mutation 719, 185, 12, 2 bp. A 719-bp fragment occurred because of the loss of one restriction site (553+ 166 bp).
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Results |
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Discussion |
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The Arg614Cys mutation was the first human mutation found to be associated with MH.8 The majority of patients carrying this mutation were classified as MH-susceptible in the IVCT. However, some cases of discordance between the genotype and the phenotype were described for the Arg614Cys mutation, and this raised doubts as to whether the mutation is really the cause.20 21 Most doubts concerned discrepancies between MHS results in the IVCT and the absence of the mutation. Attempts were made to explain these findings by implicating further independent unknown mutations, which might cause MH; therefore, suggesting that the single-gene model underlying MH may be incorrect. This interpretation was reinforced by the identification of five additional MH loci on chromosomes 17q,7q, 3q, 1q, and 5p22-25.
On the other hand, an explanation for the occurrence of genotype/phenotype discordance may lie in the IVCT, which does not guarantee total phenotypic accuracy. According to the EMHG procedure the IVCT has a sensitivity of 99% and a specificity of 94%.6 Moreover, a recent study shows a considerable between-centre variability of the IVCT results for the same patient.26
The study described here is an excellent example of the effects of the Arg614Cys mutation on the MH phenotype.
A strong correlation between clinical, IVCT and molecular genetic findings was first established for the homozygous index patient who had developed an MH crisis. Further more, both the homozygous sister (II:9) and the heterozygous parents (I:1, I:2) were diagnosed as MHS in the IVCT. The two homozygous individuals showed much greater contracture responses to both halothane and caffeine than the heterozygous subjects. The absence of the Arg614Cys mutation in all MHN individuals also supports the strong association of the mutations with susceptibility. No genotype/phenotype discordance was observed for MHS or MHN individuals.
These results support previous assumptions that inheritance of homozygous-dominant alleles is associated with a more severe phenotype possibly arising from a more deleterious change to the channel structure. On the other hand, both the homozygous and the heterozygous individuals were phenotypically healthy and there were no clinical signs indicating the presence of the MH disposition.27 28
Our findings suggest that the Arg614Cys mutation is the cause of the MH phenotype in this family. The mutation was inherited over three generations and was only present in MHS individuals. Our results are supported by functional expression studies in which Arg614Cys mutant channels showed an increased sensitivity of intracellular calcium release to halothane and caffeine13 or in which the calcium release was activated at lower depolarizing potentials.29
A second objective of this study was to assign MH susceptibility from identification of the familial mutation.16 On the basis of the genotype/phenotype correlation described above we assigned the MHS diagnosis to eight further mutation carriers in this family without performing the invasive IVCT. These individuals, especially, profited from the identification of the familial RYR1 mutation and did not have to undergo the muscle biopsy; this was reflected in a much greater compliance on their part concerning the MH investigation. Individuals who do not carry the familial mutation should still undergo the IVCT because of the genetic complexity of MH.16
After establishing the MH disposition in the index patient, a commonly used testing strategy is to perform an IVCT on one parent in the first instance. If this produced a clear MHS diagnosis it would be possible to assume that the other parent is MHN and does not need to undergo the invasive IVCT because the likelihood of the other parent also being MH-susceptible is very small (1:10 000 to 1:60 000 where volatile anaesthetics are used30 31). With our family, this strategy could have had disastrous consequences for the second parent. Our results support the suggestion that both parents of a MHS subject should always be investigated.32
Our study has demonstrated that genetic data for MH status can in some cases provide additional diagnostic information and prove a valuable supplement to diagnostic testing for MH.
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References |
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