Characterization of variants in the RYR3 gene associated with epileptic encephalopathy

Ryanodine receptor type 3 (RyR3) is an intracellular calcium release channel localized in the endoplasmic reticulum. Although initially identified in the brain, RyR3 is ubiquitously expressed in multiple tissue. In the central nervous system, RyR3 is highly expressed in the hippocampus and striatum and represents the only isoform present in dendritic spines of primary hippocampal neurons, where it amplifies post synaptic Ca2+ signals and contributes to synaptic plasticity, neuronal development and memory process. To date, more than 1200 RYR3 sequence variants, found in patients with different disorders, have been reported in the ClinVar database and, recently, around 75% of RYR3 variants have been found in patients affected by epileptic encephalopathy. Thanks to the extensive use of whole genome and/or exome sequencing techniques in routine diagnostic procedures, the number of variants in RYR3 that may be potentially associated with epilepsy is expected to rise, extending the need for variant classification and validation to confirm their clinical significance and to improve early diagnosis, risk stratification and management of patients carrying RYR3 variants. To this aim, in collaboration with Dr. Van Petegem’s group at the University of British Columbia in Canada, we selected seven variants associated with epileptic encephalopathy, located in the chloride (RyR3-C64Y, RyR3-R287Q, RyR3-R287W and RyR3-R412W) or in the ATP binding sites (RyR3-R270Q, RyR3-R270W and RyR3-T313N) within the N-terminal region of RyR3. To investigate the biological role of each variant, stable cell clones were generated by transfecting HEK293 cells with expression vectors for wild type or mutant RyR3 channels. For each variant, stable clones were selected and each variant was characterized by calcium imaging studies to determine their effect and their possible correlation with epileptic encephalopathy. The results obtained indicate that alterations localized at the chloride or ATP binding sites within the N-terminal region of the channel can modulate receptor function through allosteric mechanism. From a functional perspective, the variants display heterogeneous effects, including features that mirror those of typical gain of function mutations. Variants R412W and T313N showed a possible hypersensitive activation threshold in the presence of extracellular Ca2+, while variant R287W showed increased sensitivity to caffeine; these features may result in an increase in Ca2+-induced Ca2+ release that may lower the threshold for firing action potentials, leading to repetitive, hypersynchronous bursting characteristic of seizure disorders.