The structural organization of the human skeletal muscle ryanodine receptor gene (RYR1) and its involvement in malignant hyperthermia

grantor: University of TorontoThe ryanodine receptor ('RYR1') gene encoding the Ca2+ release channel of human skeletal muscle sarcoplasmic reticulum. has been cloned and the exon/intron boundaries have been determined. The gene contains 106 exons, of which two are alternatively spliced. The length of the gene, determined by the alignment of 16 genomic phage clones, a cosmid done and several long PCR products, was found to be approximately 158 kbp. Previously published errors in the cDNA sequence of the 'RYR1' gene were confirmed and corrected. In the 5' region of the 'RYR1' gene, a proposed CCAAT box and several Sp1 binding sites were identified within the proposed promoter sequence. SSCP analysis was used to screen 'RYR1' exons 44 and 45 in malignant hyperthermia (MH) patients. A polymorphism in two unrelated individuals was found to result from the substitution of A for G7300, leading to the substitution of Arg for Gly2434. Subsequent screening of MH probands identified the mutation in 4 of 106 families tested and its absence from about 1000 other chromosomes. The mutation was present in all individuals in 4 families who had a MH reaction. Discordance was found in 2 of the 4 families. The discrepancies between the clinical assessment and the genetic results could best be explained by inaccuracies in the caffeine/halothane contracture test (CHCT), leading to apparent false positive and negative test results. Knowledge of the structure of the 'RYR1' gene has also been utilized in studies. The first study involved the refinement of a diagnostic assay for the analysis of the Arg614 to Cys mutation in the human 'RYR1' gene. The second study involved the identification of a His for Arg2435 mutation in the 'RYR1' gene that was associated with central core disease in a large Canadian family. Genomic information was utilized to design a genomic based restriction endonuclease digestion based diagnostic test to screen patients with CCD. The determination of the 'RYR1' genomic structure now provides the basis for the analysis of 'RYR1' exons in MH or CCD probands from genomic DNA.Ph.D