Candidate loci in a threshold model of malignant hyperthermia.

Malignant Hyperthermia is an inherited pharmacogenetic skeletal muscle disorder, resulting in a hypermetabolic reaction under exposure to volatile anaesthetics or succinylcholine. The most common diagnostic variant for the UK MH cohort is the RYR1 p.(G2434R) mutation. Previous studies have indicated more than one gene may be involved in susceptibility, suggesting the disorder may adhere to a threshold model of inheritance. Muscle samples from MH patients have shown mitochondrial deficits, and downregulation of metabolic genes has been observed in a mouse model. Additionally, pathogenic variants in CPT2 and PYGM have been identified in susceptible individuals.
CRISPR/Cas gene editing was used to knock-out Cpt2 and Pygm in Ryr1 heterozygous p.(G2435R mouse myoblasts. Lentivirus transduction was used to introduce pathogenic CPT2 (p.(S113L), p.(P50H)) and PYGM variants (p.(R50X), p.(A193S)) into the knock-out clones. The sensitivity to triggering agents in the genetically modified cells was assessed via live cell calcium imaging. A case-control study was also conducted to identify regions of interest and incidence of pathogenic variants in 10 candidate genes (ACADVL, AMPD1, ATP2A1, CACNA1S, CACNA2D1, CASQ1, CPT2, PYGM, STAC3, TRPV1).
The Pygm knock-out clone exhibited increased sensitivity to triggering excitation contraction coupling (ECC) via KCl. The introduction of the p.(R50X) variant led to increased sensitivity to KCl and caffeine, indicating increased RYR1 sensitivity. The introduction of CPT2 variant p.(S113L) led to increased KCl sensitivity. The introduction of p.(P50H) led to increased caffeine sensitivity. The case-control study revealed several regions of interest across genes involved in ECC, calcium homeostasis and metabolism. Pathogenic variants in ACADVL and PYGM were also significantly over-represented in MH susceptible individuals.
These results suggest the possible contribution of candidate variants to a threshold model of MH, using a gene editing pipeline novel to MH research, alongside the identification of new candidates for investigation.