Mutation selection

There are a large number of RYR1 mutations reported that are linked to MH and/or CCD although functional studies have been performed for only a limited number of mutations. A major objective in this study was to construct the human RYR1 cDNA clone and perform functional analyses using this cDNA. Functional studies using human cDNA had not been reported at the outset of this study or when this thesis was submitted. Although it was uncertain if cloning of RYR1 cDNA was feasible, western blotting clearly showed the expressed RYR1 protein. As the cloning and expression was successful, it was then necessary to select RYR1 mutations for mutant construction and subsequent functional studies.

Six RYR1 mutations were selected and mutants were constructed for these mutations: two N-terminal mutations linked to MH, R163C and G248R, two C-terminal mutations linked to MH, T4826I and H4833Y, and two C-terminal mutations linked to CCD, I4898T and G4899R. As human RYR1 cDNA had not been used previously in functional studies, mutations that had been used in previous work using rabbit RYR1 cDNA were selected in order to test the use of human RYR1 cDNA in functional analyses. The R163C and G248R mutations are two of the most common RYR1 mutations identified widely throughout the world. The rabbit RYR1 cDNA mutant clones had been constructed for these mutations and functional analyses had been performed showing their hypersensitivity against agonists such as halothane and

caffeine using HEK-293 cells in transient transfections [Tong, et al., 1997; Yang, et al., 2003]. This work suggested that they should make sound positive controls for functional studies using human RYR1 cDNA. They should also be useful in establishing the experimental procedures for other RYR1 mutations that are either currently unknown or uncommon with unpredictable responses against drugs.

Two C-terminal RYR1 mutations linked to MH were also selected: T4826I and H4833Y. Both have been identified in New Zealand Maori MH families and clearly co-segregate in MHS patients who showed strong positive reactions during the IVCT [Anderson, et al., 2008; Brown, et al., 2000]. Functional studies have also been performed for the T4826I mutation using rabbit RYR1 cDNA and for the H4833Y mutation using human B lymphocytes showing their hypersensitivity to agonists [Anderson, et al., 2008; Yang, et al., 2003]. At the time the H4833Y mutation was selected for the current work, the functional study using B lymphocytes had not been started. These four mutations are located in the N-terminal hotspot region 1 or C-terminal region 3, respectively. Comparison of several RYR1 mutations from each hotspot region using the same experimental conditions may be of use in showing that RYR1 mutations linked to MH have similar hypersensitivity against drugs regardless of the location of the mutations. Mutants with RYR1 mutations from hotspot region 2, however, were not constructed because of the difficulties associated with cloning after mutagenesis. Site-directed mutagenesis using the MEGAWHOP technique [Miyazaki and Takenouchi, 2002] requires PCR amplification using a plasmid as a template. An RYR1 fragment insert in the template plasmid should not be larger than 3 kb for successful and correct elongation. The pBSXC+ (2.7 kb insert) and pBSH+ (2.2 kb) subclones were used as templates for N-terminal (hotspot region 1) or C-terminal (hotspot region 3) mutations, respectively. It was not feasible, however, to design a subclone which contained a ~3 kb fragment covering the central hotspot region 2 because of unavailability of unique restriction enzyme sites. The pBSHK+ (4.5 kb) and pBSKO+ (5.8 kb) subclones cover region 2, but each is too large for use as a PCR template. The pBSKS+1177 (1.2 kb) or pBSKCII+ (2.7 kb) may be suitable to be used for mutagenesis of mutations located on region 2 although there are many subsequent steps of subcloning to construct pcRYR1+mutation after mutagenesis. Hence, MH mutations located only in either

region 1 or region 3 were selected in this study.

Functional studies for CCD mutations have recently been reported using the rabbit cDNA or transgenic mice [Durham, et al., 2008; Lynch, et al., 1999]. Two C-terminal CCD mutations I4898T and G4898R were selected in this study to compare with MH mutations under the same experimental conditions. Both mutations are common CCD mutations which have been identified only in CCD patients and not in MH families. All mutations selected in this study have been used for previous functional studies. While the work carried out may not have produced new data, this is the first study using human RYR1 cDNA. Nevertheless it is important to establish experimental procedures for cloning, mutagenesis, and protein expression for future work using other novel or uncharacterised RYR1 mutations. Some other RYR1 mutations including R44C, R401C and R533C were also selected but were not used for functional analyses in this study because of lack of time. Only mutagenesis was completed for some mutations such as G341R and mutagenesis and cloning was completed but [3H]ryanodine binding assays were not performed for some mutations such as R44C. These mutations have been identified previously, but functional analyses have not yet been reported. The common mutations used in previous studies such as R163C were intended as positive controls to validate the human RYR1 cDNA clone and establish its utility prior to analysing uncharacterised mutations. Further analyses of the other mutations already prepared by mutagenesis and cloned into the full-length human

RYR1 cDNA may be of interest for future work. This study provides a positive control for future studies using human RYR1 cDNA.