L Heytens Responsible Research Group Maligne Hyperthermia University of Antwerp
C Van Broeckhoven Scientific Director Department of Molecular Genetics VIB8 University of Antwerp)
Summary of research proposal
Over the last 14 years the Malignant Hyperthermia -laboratory of the University of Antwerp has performed over 400 in vitro contracture tests to establish the MH-phenotype in referred probands and their family members. This allowed us to establish a phenotype databank concerning 27 families with at least two generations investigated.
In the majority of families, MH results from a defect in the regulation of calcium release in skeletal muscle due to dominant mutations in the calcium channel of the sarcoplasmic reticulum, the so-called ryanodine receptor gene RYR1. Over the years a large number of mutations have been uncovered in this gene of which 15 are relatively prevalent and thought to be causative. This has resulted in recommendations for the use of genetic analysis in conjunction with the in vitro contracture test issued by the European Malignant Hyperthermia Group in 2001.
The goal of this project is to screen these 27 families for the 15 most prevalent causative RYR1 mutations and thus offer the possibility of non-invasive molecular genetic diagnosis to the non-biopsied members of these families.
Malignant Hyperthermia (MH) is an autosomal dominant, genetically heterogeneous trait, which manifests as an acute rhabdomolysis during general anaesthesia when susceptible individuals are exposed to certain triggering agents, mainly the volatile anaesthetics and succinylcholine. Classical fulminant MH presents with a combination of the following clinical and biochemical signs : tachycardia, arrhythmias, increasing haemodynamic instability, early tachy- and polypnea, pronounced hypercapnia, respiratory and mixed acidosis, generalized rigidity, hyperthermia, postoperative myalgia and myoglobunuria, grossly increased CPK and finally late systemic complications such as hyperkalemia, renal failure, disseminated intravascular coagulation and eventually cardiac arrest.
This potentially life-threatening anaesthesia-induced event reflects disturbed skeletal muscle calcium homeostasis as a result of defects in the genes coding for the proteins involved in skeletal muscle excitation-contraction coupling, mainly the RYR1 gene.
The skeletal muscle EC coupling mechanism is the process whereby the depolarisation of the muscle surface membrane and the transverse tubule netwrok leads to calcium release from the sarcoplasmic reticulum (SR). The transverse tubules contain calcium channels that are voltage activated, the so-called dihydropyridinereceptors (DHPR). Depolarisation of these receptors induces a tri-dimensional conformational change which in turn is mechanically coupled to the calcium-release channel of the SR, the ryanodine receptor (RYR). This homotetrameric structure is the main channel for the release of calcium from the SR into the sarcoplasm and thus to initiate contraction.
The 15.5 kb cDNA of the corresponding RYR1-gene on chromosome 19 encodes a 5035 amino acid protein. Four identical subunits of 565 kDa form the homotetramic RYR in the SR-membrane. The 20% COOH-terminal end of the molecule forms transmembrane channel domains whereas the NH2 terminal forms a large cytosolic protrusion which extends towards the transverse tubule and makes contact with the DHPR.
Mickelson (1) demonstrated that a significant difference in calcium releasing activity existed between MH-susceptible and normal SR-preparations, allowing longer open time probability and thus an increased rate of calcium release, which in turn induces several uncontrolled metabolic processes ultimately leading to severe hyperacute rhabdomyolysis.
Susceptibility to MH is normally diagnosed by in vitro contracture testing (IVCT) with caffeine and halothane. The MH-laboratory of the University of Antwerp is the national reference centre for MH-diagnosis and member of the European Group for Malignant Hyperthermia. The IVC-test requires a sample of skeletal muscle tissue, which is exposed in vitro to incremental doses of different specific testing agents and the contracture response measured. The test is considered positive (MHS) if a sustained contracture of at least 2 mN is obtained in two different muscle bundles at caffeine concentrations of 2 mM or less, and halothane concentrations of 2 Vol% or less. Normal individuals (MHN) do not react at the threshold concentrations of either agent. The result is called equivocal (MHE) when a significant contracture (2 mN or more) is obtained with only one test substance e.g. MHEh if reacting to halothane and MHEc when reacting to caffeine only. Patients classified as MHS and MHE are considered clinically at risk to MH.
This test has been standardised across Europe by the EMHG and shows a high degree of sensitivity (99%) and specificity (93.5) (2).
However, it is an invasive test, a fact which very much hampers its widespread use as a screening instrument. Especially in children, a minimally invasive diagnostic test would be very welcome. Therefore it has always been a long term goal of the EMHG to promote the development of a non-invasive test and mainly to assess whether DNA based diagnosis for MH is feasible.
Genetically, MH-susceptibility exhibits an autosomal dominant mode of inheritance with an estimated prevalence of 1 in 8,500. It has been shown to demonstrate both locus and allelic heterogeneity with 5 distinct susceptibility loci so far identified.
Fortunately, up to 80% of families demonstrate linkage to one locus on chromosome 19q13.1, now known to correspond with the ryanodine receptor gene (RYR1). The remaining loci have been identified only in isolated families.
Most of the mutations were found as the result of a collaborative European search for new RYR1 mutations, initiated by Tommy Mc Carthy (Cork, Ireland). To this date about 70 RYR1 mutations have been reported to be linked to MH-susceptibility.
These mutations appear to cluster in 2 regions namely the N-terminal region bp 1 - 700 and a second central region bp 2300-2500. Both correspond to gene subunits coding for the cytoplasmic foot structure of RYR. A third region of the gene, the C-terminal region also harbours mutations found in central core disease, a structural myopathy that strongly predisposes to MH-susceptibility. This third region is believed to code for the transmembrane pore.
The pathogenicity of the majority of these mutations however is still under consideration. Fifteen of these mutations are listed in the genetic testing guidelines by the EMHG as they occur in the myoplasmic RYR1 domain at sites shown to be conserved across the RYR genes and have been investigated in functional assays using cultured cells transfected with mutant channels. On the basis of these findings the following mutations - given as RYR1 amino acid change - have been defined as 'causative' mutations in the EMHG guidelines : Cys35Arg, Arg163Cys, Gly248Arg, Gly341Arg, Ile403Met, Tyr522Ser, Arg552Trp, Arg614Cys, Arg614Leu, Arg2163Cys, Arg2163His, Gly2434Arg, Arg2435His, Arg2458Cys, Arg2458His.
A recent European survey including data from 10 MH units, including our own partial results, showed that the most frequent RY1 mutations in Europe are Gly341Arg, Arg614Cys and Gly2434Arg accounting respectively for 14.8, 28.6 and 34.2% of the mutation positive cases (3).
As a result of this the stage has now been reached where genetic screening for the condition can be offered, albeit in a limited capacity. The testing guidelines according to which genetic diagnosis has to be performed have been published in 2001 (4).
Since 1990 our centre has performed over 400 biopsies for IVC-testing of MH susceptibility to establish the MH-phenotype in a number of probands and family members. This database including information about 27 families in which at least 2 generations have been phenotyped, can now serve as a basis to carry out a comprehensive assessment of the prevalence of the different reported mutations in the Belgian population.
In a previous study by our centre limited mutaton analysis of RYR1 was already performed. At that time 11 families were sufficiently phenotyped by the IVCT - 82 individuals tested of which 42 were MHS, 31 MHN and 9 MHE - to allow the search for phenotype-genotype correlation and the following mutations : Arg146Cys, Gly248Arg, Arg2434His, Arg163Cys, Ile403Met and Gly341Arg.
This screening showed the Gly341Arg mutation to be present in 3 out of 11 families. The mutation clearly co segregated with the MHS phenotype in each family. Further analysis showed the mutation to have the same genetic background in all three families, suggesting the presence of a founder effect.
In a fourth family the Arg2163His mutation was found.
The main goals of this research proposal are :
- To obtain a comprehensive assessment of the prevalence of the different internationally reported mutations in the Belgian population and through this
- offer a non-invasive diagnostic test to children and family members of a proband in which one of these causative RYR1 mutations is found.
For this planned mutation analysis of RYR1, 27 Belgian families have been selected on the basis of the information available in our database. All families were referred to our MH laboratory after a suspected clinical MH episode in a relative. Informed consent was obtained from all individuals regarding blood sampling. The number of MH susceptible individuals per family ranges from xxxx to xxxx.
Over the previous years blood has been sampled from all individuals and DNA extracted and stored.
- Step 1. For the mutation screening one key individual will be selected in each family on the basis of a clearly positive IVCT result and the position of the subject within the pedigree. This individual will often, but not invariably, be the proband. The detection technique used i.e. allele-specific PCR, SSCP analysis and RFLP analysis will be performed as published in the literature.
- Step 2.In those families where one of these 15 mutations is identified in the key individual, all other family members will be screened for that particular mutation.
- Step 3. Study of genotype-phenotype correlation to assess concordance/discordance.
- Step 4. Offering of genetic counselling via a medical genetic lab to those families with an identified mutation and good concordance between genotype and phenotype.
- Mickelson JR. Malignant hyperthermia - Excitation-contraction coupling Ca2+ release channel, and cell Ca2+ regulation defects. Phys Rev 76, 537 - 592, 1996.
- 0rding H. In vitro contracture test for diagnosis of malignant hyperthermia following the protocol of the European MH Group : results fo testing patients surviving fulminant MH an dunrelated low-risk subjects. Acta Anaesthesiol Scand 41, 955-966,1997.
- RL Robinson, MJ Anetseder, V Brancadoro, C Van Broeckhoven, A Carsane, K Censier, Fortunato G, Girard T, Heytens L, Hopkins PM, Jurkat-Rott K, Klinler W, Kozak-Ribbens G, Krivosic R, Monnier N, Nivoche Y, Olthoff D, Rueffert H, Sorrentino V, Tegazzin, Muller CR. Recent advances in the diagnosis of malignant hyperthermia susceptibility : how confident can we be of genetic testing? Eur J Hum Genet, 342 - 348, 2003.
- Urwyler a, Deufel T, McCarthy t and West S. Guidelines for the molecular detection of susceptibility to malignant hyperthermia. Br J Anaesth 86, 283 - 287, 2001
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