The Functional Impact of Cis Acting Ryanodine Receptor Type 1 Mutations in a Child with a Fatal Spontaneous MH Event

Mutations in the RYR1 gene result in MH, a pharmacogenetic disorder of skeletal muscle typically triggered by administration of anesthetics. However, cases of sudden death during exertion, heat challenge, and febrile illness in the absence of triggering drugs have been reported in individuals with RYR1 gene variants. We describe the clinical history and genetic analysis of a child that suffered a fatal, non-anesthetic MH episode associated with febrile illness who was heterozygous for two novel RYR1 variants where one variant, R3983C, occurred de-novo and another, D4505H, was inherited. Effects of the two variants on RYR1 sensitivity to activation by caffeine were assessed following expression in RYR1-null myotubes. The single (R3983C and D4505H) and double (R3983C-D4505H) variants were engineered into a full-length rabbit RYR1 cDNA and introduced into RyR1-null myotubes via nuclear microinjection. Effects of the different heterotypic expression conditions (WT+R3983C, WT+D4505H, R3983C+D4505H, and WT+R3983C-D4505H) on RYR1 activation by caffeine were determined in indo-1-loaded myotubes. Compared to WT RYR1 alone (EC50 = 1.5 mM), the caffeine sensitivity of RYR1 Ca2+ release was only modestly increased following co-expression of either R3983C (EC50 = 1.3 mM) or D4505H (EC50 = 0.9 mM). Remarkably, co-expression of WT RYR1 with the double mutant in cis (R3983C-D4505H) produced a much stronger sensitization of caffeine-induced release (EC50 = 0.3) than that observed following co-expression of the two variants on separate subunits (EC50 = 0.9). Thus, the R3893C mutation potentiates D4505H-mediated sensitization of caffeine-induced RYR1 Ca2+ release when these mutations are in cis or present on the same subunit, but not when present on separate subunits. These results indicate that allelic segregation can be a critical, and heretofore unappreciated, pathogenic factor in compound heterozygous MH individuals.