Abstracts

Rationale: WERN, a co-operation of patients, clinicians and scientists, facilitated the study; The genetic basis of familial epilepsy in Wales, which provided a platform for novel gene-discovery and candidate gene analysis, and also has generated a translational multidisciplinary communication network which facilitates the dissemination of appropriate and accurate genetic outcomes from epilepsy research. We previously described a novel GABAA receptor subunit mutation, R97X in the gene encoding the ?2subunit of the GABAA receptor (GABRG2) in a family with a borderline GEFS phenotype. The mutation found to segregate with febrile seizures was not detected in 190 healthy controls. Structural modelling suggested the mutant polypeptide may traffick to the endoplasmic reticulum (ER), accumulate and be degraded. We set out to confirm this model using functional cell biology to best inform clinicians when counselling and providing information to the nuclear family harbouring the mutation. Methods: Polymerase chain reaction (PCR) was used to amplify GABRG2 coding regions; followed by DNA sequencing reactions we performed GABRG2 and SCN1A mutational analysis on the nuclear family. Following mutation discovery, GABAA receptor subunits were cloned and PCR directed mutagenesis performed. A neuronal phenotype cell line was cultured and magnetofected with GABR?1, GABR?2, and GABR?2 wild type(WT) or mutant receptor subunits. Surface and intracellular distribution of mutant and WT receptors were determined by co-labelling with anti-GABR?2 and anti-GABR?2,3 subunit antibodies and confocal analysis. Following cellular outcomes the mutation was confirmed in a diagnostic laboratory prior to communicating the result to the referring clinician and the affected family. Results: GABRG2 R97X introduces a premature stop codon, substitutes a highly conserved arginine and truncates GABRG2 with loss of all four transmembrane domains. WT GABR?2 immunoreactivity had a smooth distribution with clusters mainly detected on the cell surface and colocalised with GABR?2 immunoreactivity. Expression of GABR?2 R97X containing receptors resulted in less cell surface cluster immunoreactivity and more diffuse intracellular labelling. GABR?2 R97X immunoreactivity also clumped intracellularly around the nucleus in close proximity to the ER; validating the structural model. Conclusions: In this family GABAA receptor dysfunction represents a putative mechanism for GABRG2 R97X; which alters receptor composition and distribution by reducing expression and potentially compromising trafficking. Following these results, WERN collaborators including a clinical geneticist, genetic counsellor, molecular geneticist and a neurology registrar formulated a molecular report and facilitated the communication of this novel result to the original referring clinician and to the affected epilepsy family. This discovery apart from representing a molecular epilepsy outcome for the family and WERN demonstrates multidisciplinary translational research involving teams, networks and communication structures of epilepsy interested clinicians and scientists.