Functional characterisation of novel ‘AUSTRIAN’ putative MH variants of the RYR 1

Malignant hyperthermia (MH) is still a live threatening, anaesthesia related complication. In an estimated 80% of MH cases an underlying mutation in the ryanodine receptor 1 (RYR1) causes problems with increased Ca2+ release during anaesthesia. To diagnose this potentially lethal pharmacogenetic disorder, the in vitro contracture test (IVCT), which requires an expensive and invasive surgical intervention, is still the only mean when the underlying causative mutation is unknown. For members of a family with a known MH mutation, a less invasive genetic test can be offered. To expand the numbers of beneficiaries of this minimal invasive test, our clinical department has started to characterize Austrian MH individuals on a genetic basis to identify possible MH causative mutations and to offer a genetic test for those individuals with a known MH mutation. Up to now we were able to identify three new mutations but the benefit for the patients to undergo genetic testing of these mutations is only possible as soon as these mutations are approved as causative by the European MH group. Such an approval demands segregation of the mutation with the MH phenotype in at least two independent families with MH history. Since the frequency of the newly found mutations is extremely low and the number of family members is often insufficient, it is foreseeable that approval to these mutations will not be given on the basis of population genetics. An alternative to population genetics is the experimental approach. To prove the causality of a newly found variant, it is introduced into the coding sequence of the RYR1. This mutated form of the RYR1 is then expressed in a cell line. These cells with the mutated receptors -as compared to the cells with the wild-type receptors- are prone to increased Ca2+ release in the presence of specific pharmacologic activators of the RYR such as halothane, caffeine or 4-chloro-m-cresol (4-CmC). Mutated receptors, which are responsible for an MH phenotype, show increased sensitivities when compared to wild type channels. Since MH is a muscle related disorder in addition to HEK-293 cells, a muscle cell line from mice lacking a functional RyR1 will be used to express the RYR1 in its natural environment. The proof of causality for new mutations would enable us to perform the genetic test with members of families with these mutations. Since the IVCT is an invasive and expensive surgical intervention, the genetic test not only is a major relief for the patient but also saves money for the health insurance (by reducing the duration of employee`s illness) and the hospital, which currently has to take over the expenses of the IVCT.

The aim of the project was the determination of a causal role of three new variants of the ryanodine receptor 1 (RYR1) which were identified in Austrian individuals found to be susceptible to malignant hyperthermia (MH). MH is a pharmacogenetic disorder where affected individuals react to halogenated volatile anaesthetics such as halothane or sevoflurane among others with an increase in body temperature and muscle rigidity. If untreated MH episodes can lead to the death of affected individuals. Most cases of MH are caused by mutations in the RYR1 gene which codes for an intracellular Ca2+ release channel pivotal for the ability of skeletal muscle cells to contract upon neuronal stimulation. Therefore, we tried to express full length wildtype and mutated RyR1 in HEK-293 cells and to measure the sensitivity of Ca2+ release through the RyR1 against pharmacologic activators such as caffeine. Variants which show a higher sensitivity against this substance can therefore be classified as MH mutations. The identification of MH mutations enables us to perform a genetic test for MH instead of doing the invasive in vitro contracture testing.A specific problem of this task is the huge size of the coding sequence of the RYR1 gene with more than 15,000 base pairs which makes it prone to recombination when introduced into a plasmid vector. We investigated three mutations (1834G>C, 10042C>G and 11953C>T) two of which could be introduced into the full length RYR1 clone via shorter subclones. To verify the mutations we sequenced the whole length of the plasmid which showed repeatedly recombinations of the wildtype as well as the mutated forms. These recombinations resulted in an early stop of translation and therefore no functional channels were expressed. Although we tested various bacterial strains known to be deficient for recombinations, we were until now not able to solve this problem. Recurrently, mutations which looked correct in the sequencing gave no functional expression in HEK cells after a subsequent DNA isolation.Obviously the RYR1 DNA is very unstable and is expressed only weakly in HEK cells, thats why another expression system seems to be favourable for this task which uses bacterial artificial chromosomes (BAC expression vector). This vector system can accept DNA inserts of up to a size of 350 kb and yields high protein expression levels with good stability of the DNA for prolonged expression. We plan to change the expression system to a BAC expression vector and we will launch a cooperation with the Institute of Pharmacology of the Medical University of Vienna.