This report describes two cases in which novel RYR1
variants are associated with fatal nonanesthetic awake episodes in children. Like other confirmed MHS mutations in RYR1,16,17
the D4505H variant resulted in a ~2-fold increase in the sensitivity to activation by caffeine. Importantly, this increase in caffeine sensitivity occurred when the D4505H variant was coexpressed with WT RYR1, demonstrating a “gain-of-function” effect of D4505H subunits on RYR1 release channel sensitivity, consistent with the known autosomal dominant pattern of inheritance of MH. Coexpression of the R3983C variant with WT RYR1 produced a more modest enhancement in RYR1 caffeine sensitivity. Remarkably, caffeine sensitivity was greatly potentiated when the two mutations were incorporated into the same subunit, but not on separate subunits. Specifically, the EC50
for caffeine activation was essentially the same for the D4505H variant when co-expressed with either WT RYR1 (EC50
= 1.64 ± 0.24 mM) or R3983C (EC50
= 1.53 ± 0.18 mM), while the R3983C-D4505H double mutant coexpressed with WT RYR1 (EC50
= 0.64 ± 0.17 mM) resulted in a further 2.5-fold increase in caffeine sensitivity and a nearly 5-fold increase in sensitivity compared to WT RYR1 alone (EC50
= 2.85 ± 0.49 mM). Thus, while the R3983C mutation only modestly altered RYR1 sensitivity by itself, it is a potent enhancer of D4505H-induced sensitization when present on the same subunit. On the other hand, peak caffeine-induced responses were not significantly different between WT RYR1 (ΔRatio= 0.52 ± 0.02) and any of the different coexpression conditions (ΔRatio was 0.54 ± 0.03, 0.55 ± 0.02, 0.52 ± 0.03, and 0.54 ± 0.03 for WT+R39833C, WT+D4505H, R3983C+D4505H, and WT+R3983C-D4505H, respectively). Nevertheless, we cannot exclude the possibility that minor changes in the levels of RYR1 expression in our experiments might alter release channel caffeine release sensitivity, but not efficacy.
Although the R3983C variant was the only RYR1 alteration identified in Case 1, susceptibility to nonanesthetic, stress-induced hyperthermic reactions appeared to be more pronounced in this patient compared to that of the child in Case 2 as evident from his clinical history. Discordance between susceptibility of the child in family 1 and the lower caffeine sensitivity of the R3893C variant following coexpression with RYR1 in RYR1-null myotubes could reflect differences between effects of the mutation in human versus
rabbit RyR1, effects in mature muscle fibers versus
developing myotubes, or the influence of a modifying variable present only in the patient (e.g.
, a mutation in regulatory or intronic regions of the RYR1
gene, WT allele silencing, or a second mutation at another MH gene locus). However, extended genetic analyses for additional mutations including three MH-associated mutations in the CACNA1S
gene and the most common mutations in the CPTII
gene were negative. An RYR1
mutation previously associated with King Denborough Syndrome was also not found.18
Nevertheless, it remains possible that the nonanesthetic events observed in this individual could involve the R3983C variant potentiating effects of a second, yet unidentified mutation in either a noncoding region of RYR1
or another MH susceptible loci.1
Intronic mutations resulting in altered splicing of RYR1
exons in core myopathies have been reported19,20
and epigenetic gene silencing of the normal RYR1
allele in skeletal muscle has also been demonstrated in families presenting with apparent recessively-inherited core myopathies.21,22
However, the only study of epigenetic RYR1
allelic silencing in MH found no evidence in 14 discordant cases from 11 independent families.23
Similar potentiating effects could explain why the proband in Case 2 possessing both the R3983C and D4505H RYR1
variants exhibited a more severe awake phenotype than either her mother or brother who carried only the D4505H variant.
The increase in internal nuclei observed in Case 1 is consistent with recent studies showing an increase in nuclear internalization in RyR1-related myopathies20,24
. Interestingly, Wilmshurst et al
identified a nonconservative substitution of a negatively charged RyR1 residue (E4502G) in an individual with centronuclear myopathy that is only three amino acids upstream of a similar nonconservative substitution of a negatively charged RyR1 residue (D4505H) identified in the proband in Case 2.24
Together, these findings are consistent with a histopathological continuum between MH- and myopathy-related RYR1 phenotypes.
It is important to note that a different de novo
missense mutation of the same RYR1 residue R3983 (R3983H) was recently described in a case report of a child with previous MH history that experienced a fatal non-anesthetic episode following ondansetron administration.4
This child also presented with clinical and histopathologic signs consistent with multi-minicore disease. Although multi-minicore disease is typically inherited as a recessive myopathy, neither a second RYR1
variant nor monoallelic RYR1
expression were reported in this child. In addition, this report did not determine whether the identified R3893H variant was causative of increased MH susceptibility by assessing its impact on RYR1 function. Nevertheless, the identification of de novo
mutations to the identical RYR1 residue (R3893) in three independent families indicates that sporadic cases of MH due to de novo
mutations in the RYR1 gene are likely to be more common than previously appreciated.
The R3983 residue is located within a putative ryanodine receptor and inositol 1,4,5-triphosphate receptor homology associated domain that spans RYR1 residues 3870–3992. While the homology associated domain is specific to RYR and IP3R, the function of this domain for this superfamily of intracellular Ca2+
release channels is unknown. The R3983 residue is conserved across species in all three RYR isoforms, while the D4505 residue is conserved only in RYR1 (). In fact, the D4505 residue is located within RYR1 divergent region 1 (D1; residues 4254–4631); one of three evolutionarily divergent regions of RYR isoforms.25
The D1 region maps to part of the “handle” domain on the RYR cryoelectron microscopy three dimensional structure.26
According to current RYR topological models,27
the D1 region includes at least one transmembrane domain and adjacent cytoplasmic and intraluminal sequences. Interestingly, deletion of the majority of the RYR1 D1 region (Δ4274–4535) potentiates voltage-gated Ca2+
release and enhances release channel sensitivity to activation by the dihydropyridine receptor.28
Based on these results, the D1 region functions as a negative regulatory module that increases the energy barrier for Ca2+
release channel opening. Thus, the D4505H mutation may enhance RYR1 release channel sensitivity to activation by disrupting the integrity of the D1 negative regulatory module.
Our results demonstrate that the functional impact of the two variants expressed in RyR1-nullmyotubes depends on whether the two variants are located on common or separate subunits. Genetic analysis of the second family revealed that the two variants are localized to separate subunits in Case 2. Although the caffeine sensitivity with the variants on separate subunits is not as high as when they localize to the same subunit, the allelic relationship in Case 2 does not necessarily imply similar a expression level of the two proteins. Furthermore, coexpression of the two variants on separate cDNAs resulted in a 2-fold increase in caffeine sensitivity, consistent with the child’s awake episodes and heat sensitivity. The unusually high caffeine sensitivity when the two variants localize to the same subunit demonstrates for the first time an allele-dependent synergism between two novel RYR1 gene variants. Our results are consistent with the two residues contributing to a negative regulatory module within the D1 region of each monomer. As a result, variants of both residues within the same subunit may lead to a synergistic antagonism of D1 function that potentiates RYR1 release channel sensitivity to activation. Together, these findings indicate that allelic segregation can be a critical, and heretofore unappreciated, pathogenic factor in MH individuals.
R3983C and D4505H ryanodine receptor variants were identified in a child that experienced a fatal, nonanesthetic awake episode. The impact of the two variants depends on whether they are located on common or separate alleles.
What we already know about this topic
* A subset of malignant hyperthermia susceptible patients can develop malignant hyperthermia-like symptoms in response to nonanesthetic stimuli.
* The interactions between genetic risk factors and environmental triggers for such "awake episodes" are unclear.
What this article tells us that is new
* Examination of the type I ryanodine receptor gene in two unrelated children who experienced fatal, non-anesthetic awake episodes revealed the presence of an identical new variant in both and a second unique variant in one.
* Functional analyses of the two variants in myotubes demonstrate that allelic segregation and genetic background play a critical role in the expression of symptoms.