Loss of heterozygosity of the H4833Y mutation on RYR1 gene causing malignant hyperthermia : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University, Palmerston North

Abstract

Malignant hyperthermia is a potentially fatal pharmacological disorder and is triggered by volatile anaesthetics in predisposed individuals. Mutations in the RYR1 gene, encoding the skeletal muscle calcium receptor channel have been linked to MH susceptibility. Over 200 point mutations have been have been found to date in the RYR1 gene linked to MHS worldwide. EBV-immortalization is regularly used worldwide as an effective procedure for inducing long-term growth of human B lymphocytes. In the current study, it was observed that immortalized lymphocytes from MHS patients heterozygous for the missense mutation H4833Y when initially cultured expressed both wild type and mutant allele but after a few weeks of culture they seemed to lose the mutant allele. High resolution melting assays and hybridization probe assays showed the loss of heterozygosity and this was confirmed using DNA sequencing. Genotyping and haplotype analysis using three intragenic RFLPs and two (CA)n repeat microsatellite markers tightly linked to the RYR1 gene showed a definite change in the haplotype, suggesting more widespread changes in the genome upon short-term culture of EBV-immortalized B-lymphocytes