No evidence of mutations in the CACNA1S gene in the UK malignant hyperthermia population{dagger}

C. Brooks1, R. L. Robinson*,1, P. J. Halsall1 and P. M. Hopkins1

1MH Investigation Unit, Clinical Sciences Building, St James’ University Hospital, Leeds LS9 7TF, UK*Corresponding author

{dagger}Declaration of interest. This work was supported by donations from the British Malignant Hyperthermia Association.

Accepted for publication: December 4, 2001


    Abstract
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 Abstract
 Introduction
 Methods and results
 Comment
 References
 
Background. Malignant hyperthermia (MH) is an inherited, potentially fatal, pharmocogenetic disorder triggered by certain anaesthetic agents. In light of the reported genetic heterogeneity for the disorder and the recent introduction of DNA testing guidelines for the trait, we have assessed the role of the CACNA1S gene in MH susceptibility in UK patients. Linkage to this locus has previously been demonstrated in several European MH families.

Methods and results. We screened 200 unrelated MH-susceptible individuals for known CACNA1S mutations. With the aim to characterize further novel mutations at this locus, functionally relevant regions of the gene were also sequenced in 10 unrelated individuals from families where the involvement of other MH susceptibility loci was unlikely. No sequence variations were detected in any of the patients investigated.

Conclusions. Defects in CACNA1S are not a major cause of MH in the UK population. Diagnostic screening of this gene is unlikely to be of value to UK MH patients in the near future.

Br J Anaesth 2002; 88: 587–9

Keywords: complications, malignant hyperthermia; genetic factors, hyperthermia


    Introduction
 Top
 Abstract
 Introduction
 Methods and results
 Comment
 References
 
Malignant hyperthermia (MH) is an autosomal dominant inherited disorder in which a serious disturbance in skeletal muscle calcium regulation occurs when an individual is exposed to previously demonstrated potent inhalational anaesthetics or depolarizing neuromuscular blocking drugs. Without rapid intervention, a patient suffering an MH crisis may die. For the last 30 years, the only reliable method to determine an individual’s MH status has been by the in vitro contracture (IVC) test, whereby a muscle sample is biopsied and subsequently exposed to incremental doses of either caffeine or halothane and the relative contracture responses determined.1 In 2001, the European MH group published guidelines for the introduction of genetic testing for the condition.2

Genetic analyses mapped the MH susceptibility trait to the ryanodine receptor locus (RYR1) on chromosome 19q12–13.2. This gene encodes the skeletal muscle sarcoplasmic reticulum calcium release channel, a key protein involved in the process of excitation–contraction coupling. To date, over 30 different mutations have been detected in the gene,3 and 15 of these have been functionally characterized using in vitro cellular assays and are considered causative of MH.2 4 It is important to demonstrate that these mutations have a ‘pathogenic’ effect in a functional assay, as the majority of mutations identified in MH patients are missense mutations (amino acid substitutions), which may have a minimal effect on normal calcium channel function. Approximately 25% of UK MH pedigrees carry one of the 15 causative MH mutations. Whilst other, currently undetected, mutations in RYR1 may be responsible for a large percentage of MH cases (up to 50% show linkage), exclusion of linkage between MH susceptibility and RYR1 has been reported on numerous occasions.5 Linkage studies have implicated other candidate loci on chromosomes 1q, 3q, 5p, 7q and 17q. Therefore MH can be said to be a condition showing considerable allelic and locus heterogeneity.

Monnier and colleagues6 determined linkage to the CACNA1S gene on chromosome 1q with a high degree of probability (two-point LOD score of +4.38) in a large French MH pedigree. CACNA1S encodes the {alpha}1 subunit of the dihydropyridine receptor (DHPR), a transverse tubule calcium channel that is tightly coupled to the ryanodine receptor.7 The DHPR functions as the voltage sensor in excitation–contraction coupling. Upon further sequence analysis of MH-susceptible individuals within the pedigree, a G-to-A change at nucleotide 3333 in exon 25 of the CACNA1S gene, substituting arginine for histidine at amino acid 1086, was discovered, which segregated completely with the MH phenotype. This represented the first direct molecular evidence of MH locus heterogeneity. Subsequently Jurkatt-Rott and colleagues8 have found a similar amino acid substitution (Arg1086Cys) in exon 25 that displays partial segregation with the MH phenotype in a German pedigree. Both exons 25 and 26 of CACNA1S encode the intracellular loop that links domains III and IV within the DHPR {alpha}1 subunit. Although the functional significance of this region remains unclear, one model suggests that it in some way participates in signalling with the ryanodine receptor.

The aim of this study was to determine whether mutations in the CACNA1S gene predispose the UK population to MH susceptibility. Results of such analyses could have implications for the genetic diagnosis of MH in the future.


    Methods and results
 Top
 Abstract
 Introduction
 Methods and results
 Comment
 References
 
A cohort of 200 unrelated IVC-tested MH-susceptible individuals representative of the UK MH population was tested to determine the presence or absence of the G3333A mutation (exon 25 of DHPR {alpha}1s) using a restriction enzyme digest assay.6 This sample size was sufficient to detect a mutation with <1% prevalence. Final analysis provided us with no evidence of this mutation existing within the UK MH population, giving an estimated frequency of <0.25% (upper 95% confidence interval: 0.75%) in this group. We concluded that alongside further investigation of this domain (III–IV linking region), other regions of the DHPR {alpha}1s that interact with the ryanodine receptor protein should be analysed for possible MH associated mutations. The II–III cytoplasmic loop, an important determinant in excitation–contraction coupling,9 is one such region encoded by the CACNA1S exons 14 to 18.10

A group of MH-susceptible individuals representing 10 UK MH pedigrees was selected. Three exhibited discordance between the RYR1 genotype and MH-susceptibility phenotype, implying the possible presence of a second/alternative causative mutation, or linkage to another susceptibility locus. One represented a pedigree in which a known RYR1 mutation (G7300A) completely segregated with the MH phenotype and therefore served as a CACNA1S mutation negative control. The remaining seven represented pedigrees in which known mutations were excluded and evidence of linkage to chromosome 19 was inconclusive. Genomic DNA samples from all representative MH-susceptible individuals were amplified by the polymerase chain reaction using exon-specific primers for exons 14–18 and 25–26 (Table 1) and sequenced.


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Table 1 Primer sequences used for CACNA1S sequence analysis
 
No sequence variations were identified within this cohort for the regions investigated. Therefore mutations in CACNA1S, or at least these specific regions, do not appear to be a significant cause of MH in the UK MH population. It would also suggest that exons 14–18 and 25–26 are highly conserved regions of the gene and are therefore crucial to the normal function of the DHPR.


    Comment
 Top
 Abstract
 Introduction
 Methods and results
 Comment
 References
 
As our investigation has provided no evidence of mutations in the CACNA1S gene in the UK MH population, there is currently no basis to propose adding mutation screening of CACNA1S to that of RYR1 in guidelines for the DNA-based diagnosis of MH for UK patients.2 Screening for mutations in this gene may, however, be beneficial in the assessment of MH status in other populations. Further studies examining physical or functional interactions between the DHPR and the ryanodine receptor may highlight other regions in the {alpha}1 subunit that may harbour mutations causative of MH. Alternatively, other genes of the DHPR heterotetramer complex – ß and {gamma} subunit genes on chromosome 17q, and the {alpha}2/{delta} subunit gene on chromosome 7q – may warrant investigation. Numerous potential candidate genes have been proposed for MH on the basis that they code for proteins involved in skeletal muscle calcium homeostasis, the disruption of which appears to be the primary physiological defect in patients suffering an MH reaction. Examples include genes coding for triadin, calsequestrin and the tacrolimus-binding protein, FKBP12. Genetic analysis to date would indicate that, if involved, such candidates are likely to be rare susceptibility loci. Evidence indicates that defects in the RYR1 gene account for the majority of MH cases. Future research may therefore be better prioritized towards determining the presence of novel mutations within this locus.


    References
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 Abstract
 Introduction
 Methods and results
 Comment
 References
 
1 European Malignant Hyperpyrexia Group. A protocol for investigation of malignant hyperthermia susceptibility. Br J Anaesth 1984; 56: 1267–9[Abstract]

2 Urwyler A, Deufel T, McCarthy T, West S. Guidelines for the molecular detection of susceptibility to malignant hyperthermia Br J Anaesth 2001; 86: 283–7[Abstract/Free Full Text]

3 McCarthy TV, Quane KA, Lynch PJ. Ryanodine receptor mutations in malignant hyperthermia and central core disease. Hum Mutat 2000; 15: 410–7[ISI][Medline]

4 Tong J, Oyamada H, Demaurex N, Grinstein S, McCarthy TV, MacLennan DH. Caffeine and halothane sensitivity of intracellular Ca2+ is altered by calcium release channel (ryanodine receptor) mutations associated with malignant hyperthermia and/or central core disease. J Biol Chem 1997; 272: 26332–9[Abstract/Free Full Text]

5 Robinson R, Curran J, Hall W, et al. Genetic heterogeneity and HOMOG analysis in British malignant hyperthermia families. J Med Genet 1998; 35: 196–201[Abstract]

6 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 calcium channel receptor in skeletal muscle. Am J Hum Genet 1997; 60: 1316–25[ISI][Medline]

7 Nakai J, Sekiguchi N, Rando TM, Allen PD, Beam KG. Two regions of the ryanodine receptor involved in coupling with L-type calcium channels. J Biol Chem 1998; 273: 13403–6[Abstract/Free Full Text]

8 Jurkatt-Rott K, Hang C, Sipos I, et al. III-IV loop of cardiac L-type calcium channel contributes to fast inactivation as implied by a naturally occurring disease-causing mutation. Pflügers Arch 2000; 439: R332

9 Tanabe T, Beam KG, Adams BA, Niidome T, Numa S. Regions of the skeletal muscle dihydropyridine receptor critical for excitation–contraction coupling. Nature 1990; 346: 567–9[ISI][Medline]

10 Hogan K, Gregg RG, Powers PA. The structure of the gene encoding the skeletal muscle {alpha}1-subunit of the dihydropyridine-sensitive L-type calcium channel (CACNL1A3). Genomics 1996; 31: 392–4[ISI][Medline]





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