Abstracts

Rationale: The alpha subunit of Go, Gao (encoded by GNAO1) regulates neurotransmitter release and ion channel opening, which are important in the development and prevention of epileptic seizures. Although a link between mutations in Gao protein and epilepsy has been identified, the underlying mechanisms are not clear. In the current study, we assessed four de novo mutations in GNAO1 (G203R, I279N, D174G, ?"T191-F197) previously reported in children with epileptic encephalopathy (EIEE17) by testing their expression level and function to better understand the mechanisms of GNAO1-associated encephalopathy. Methods: Wild type and mutant Gao were expressed in HEK293T cells using the pCI plasmid and their expression levels were tested by western blot/IR fluorescence detection. Function was tested by co-expression with the a2a adrenergic receptor and concentration response curves for inhibition of cAMP levels by the a2 receptor agonist UK14,304 were assessed using the LANCE Ultra cAMP assay (Perkin Elmer, Waltham, MA). Results were compared to those for a known gain-of-function mutant (G184S) that we have shown causes an epilepsy phenotype in C57Bl/6J mice. Results: The Gao G203R mutant expressed normally in HEK293T cells, but the I279N, D174G, and ?"T191-F197 mutants all had decreased protein expression. I279N, D174G and ?"T191-F197 exhibit different degrees of reduction in the maximal inhibition of cAMP levels (i.e. loss of function). Unexpectedly, for the G203R mutation, the EC50 for UK14,304 was markedly reduced (EC50 12.1nM vs 31.5nM for WT Gao) while maximum inhibition of cAMP production was unchanged from WT Gao(102.11.0%). This gain-of-function behavior was similar to that of the G184S mutant (UK14,304 EC50 11.7nM with a 107.70.9% maximal response comparing to WT Gao). Furthermore, the two complete loss-of-function mutants D174G and ?"T191-F197 did not exhibit dominant negative effects. A number of new de novo mutations have been published since and analysis of these mutant alleles is underway. Conclusions: EIEE17 presents in infancy with early onset of intractable seizure, poor psychomotor development and possible brain abnormalities. Some patients may show involuntary movements. All GNAO1 mutants described have arisen de novo. Functional studies demonstrate that GNAO1 mutation associated epileptic encephalopathy result in both loss and gain of Gao function, suggesting a unique role of Gao modulation in the genesis of epileptic encephalopathy. Funding: This work was supported by the National Institutes of Health [R01 GM039561].