Anaesthesia and Genetics of the Ryanodine 1 Receptor

Abstract: The only validated method to characterize the phenotype and to reach a definitive diagnosis of Malignant Hyperthermia Susceptibility (MHS; OMIM '145600), a pharmacogenetic disease linked to the Ryanodine 1 receptor gene (RYR1; OMIM '180901), is an invasive muscle contraction test requiring a muscle biopsy, according to the European (IVCT) protocol or to the North American (CHCT) protocol. The series of scientific papers in this thesis tries to reach as far as possible in diagnosing MHS from peripheral venous blood samples collected at the patients’ local primary care provider and sent to the laboratory over significant transportation time and distance. Study I uses a traditional method of direct sequencing of a limited number of central exons of the RYR1 gene in the Swedish population with an outcome of only one fifth of the tested probands positive for a known MH causative mutation. Study II tries to bypass the laborious direct sequencing method of the very large RYR1 gene by using a method of stabilizing RNA in test tubes for transportation and synthesizing of RYR1cDNA for direct sequencing. Study IV implies a recent method based on establishing and analysing High Resolution Melting (HRM) DNA curves of 131 amplicons of the RYR1 on stable genomic DNA with an encouraging finding of a sequence variant in 81 % of the tested patients and similarly reducing the sequencing work by 79 %. By analysing all the found sequence variants in studies I, II and IV leading to amino acid changes in the final RYR1 receptor, it seems that the formerly known hotspot (N-terminal, central and C-terminal) distribution pattern can be seen, but sequence variants appear also outside of these traditional hotspots. Therefore coverage of the total coding region of the gene remains necessary. Another fact is that about half of the found sequence variants leading to amino acid changes are previously unknown and therefore uncharacterized at functional level. Study III presents a method of measuring the [Ca2+]i resting level and the increase in cytoplasmic [Ca2+]i level in prepared B lymphoblastic cell clones from patients carrying the actual candidate mutations, after influence of a RYR1 agonist. And of combining the outcome data on cellular level with clinical data. The series of studies I-IV presented in this thesis cannot give a definitive method to reach the diagnosis MHS, or even more important; to reach the definitive diagnosis of MHN, without an IVCT. But it can present advanced techniques to describe the genetical status of the patients and the impact of new previously unknown candidate mutations at functional level, and to combine this data with clinical data, based on only a simple venous blood sample. Once a MH causative mutation is found in a pedigree this allows for predictive testing in the family members and the individuals positive for the actual mutation can be assigned MHS without an IVCT, whereas the individuals negative for the mutation must go through IVC testing in order to confirm the MHN status.