As more is being understood about the nature of susceptibility to MH it is becoming increasingly apparent that it is complex and cannot always be simply described as autosomal dominant. There is evidence for variation in clinical severity and IVCT phenotype, resulting from the same mis-sense change in RYR1 [19, 20]. There are also reports of compound heterozygotes in RYR1 [[12, 21], unpublished UK observations] and an individual with mutations in both RYR1 and CACNA1S . Furthermore, we have previously demonstrated, using transmission disequilibrium testing, that multiple interacting gene products affect susceptibility to MH [10, 22]. Even within families that showed linkage to RYR1 evidence has been provided for linkage to other loci elsewhere in the genome .
This study focused on CACNA1S encoding the α1 subunit of the DHPR. In the largest study to date, of 50 MH patients, we identified a single, potentially pathogenic, variant p.Arg174Trp. The p.Arg174Trp change is situated at a site that is conserved in rabbit, cat, mouse, and zebrafish and causes a change in the charge of the amino acid from basic to non-polar. The amino acid in question lies in the S4 segment domain of the DHPR thought to function as a voltage sensor, thus a change in charge may alter the voltage sensor mechanism and consequently disrupt the cellular calcium homoeostasis. Further functional work to support these observations would be valuable.
This work also identified two other variants (p.Gly258Asp, p.Ser606Asn) thought to be polymorphic as they do not show disease concordancy, but which were not present in control chromosomes. Whilst it is likely that they are indeed polymorphisms, the fact that they lie in conserved regions of the protein and cause a change in the polarity of the amino acid substituted suggests otherwise and there is the potential that they could play a minor role in, or have a modifier effect on, disease phenotype. Since there is evidence that MH may not necessarily be a simple single gene disorder, there is the possibility that both of these changes are present together with an additional major change and in some instances account for discordancy with disease; i.e. these mutations may be necessary, but not sufficient, to cause MH susceptibility in particular individuals.
Comparison of CACNA1S haplotype frequencies between susceptible cases and UK Caucasian population controls identified no significant haplotype frequency differences. Even given that this is the largest standardised and genotyped MH database worldwide, there are a limited number of MH patients, which could reduce the power to detect a significant association between CACNA1S haplotype with MH. However, the haplotype analysis does provide some evidence for an elevated haplotype diversity, potentially resulting from the high rate of recombination observed across the locus and a low level of linkage disequilibrium detected, as seen in the present study and consistent with that observed in the HapMap project. Coupling this observation with the now growing number of reported non-pathogenic non-synonymous changes described across CACNA1S, it is possible that this locus can tolerate a high degree of variability. This variability could affect the conformation of the DHPR protein, which has implications not only in the E-C coupling of skeletal muscle but also in that of cardiac muscle.
Our data suggest that, whilst CACNA1S may play a role in MH manifestation in the UK, it is not a major locus, thereby suggesting that there are other loci with importance in MH susceptibility. As well as the reported linkage to chromosome 1q and 19q there are alternative loci proposed on chromosomes 7q  and 17q , however no contributory mutations have been identified in these regions. It is highly probable that any novel genes for MH susceptibility will play a minor role. An alternative method to identify genes responsible for MH could be to take a candidate gene approach and focus on genes whose products are directly involved with E-C coupling and Ca2+ regulation, for example the other subunits of the DHPR, calmodulin and JP-45 .