Abstracts

Rationale: The heterotrimeric protein Go, whose a subunit is encoded by GNAO1, regulates ion channel function, neurotransmitter release, and neurite outgrowth. Mutations in GNAO1 have been identified in children with either epileptic encephalopathy (EIEE17) or neurodevelopmental delay with involuntary movements (NEDIM). The mechanism underlying this complex clinical spectrum is poorly understood. A mouse with a Gnao1 gain-of-function (GOF) knock-in mutation (Gnao1G184S/+) exhibits a mild seizure phenotype in C57Bl/6J mice. We have extended our previous study (Feng et al, Neurology, 2017) by analyzing the in vitro functions of 16 additional human GNAO1 mutations to validate our previously reported genotype-phenotype correlation. Moreover, electrophysiological and gait studies in mutant mice with both GOF and loss-of-function (LOF) Gnao1 mutations probe their potential with as models of the GNAO1 encephalopathies (EIEE17 and NEDIM). Methods: Wild-type and mutant Gαo were expressed in GNAS KO HEK293T cells; Gαo protein were levels tested by western blot. The ability of mutant Gαo to inhibit cAMP lev­els was assess­ed by co-expression with the a2A adrenergic receptor by determining concentration response curves for the a2 receptor agonist UK14,304. Also, Gnao1G184S/+ (GOF) and Gnao1-/+ (hetKO) animals of both sexes (age - 8 to 14 weeks) were analyzed by slice electrophysiology for IPSCs and EPSCs in hippocampus and cortex and gait was assessed using a DigiGait Imaging System.  Results: Previously, pathogenic human GNAO1 mutations showed both GOF and LOF behavior for cAMP regulation. Three pathogenic Arg209 mutations had relatively normal function. GNAO1 GOF mutations are associated with movement disorder with or without mild seizures (NEDIM) while the LOF mutations are associated with severe epilepsy. New mutations largely confirm that pattern.Moreover, a Gnao1G184S/+ GOF mutant mouse largely phenocopies the human R209C and G203R mutations with a mild seizure phenotype (sensitization to pentylenetetrazole kindling but few spontaneous seizures) plus showing evidence of a movement disorder. Hippocampal and cortical slices Gnao1 GOF mutant mice showed reduced spontaneous IPSCs but normal EPSCs. Digigait® analysis showed a striking reduction in stride length and increased paw angle variability in GOF (Gnao1G184S/+) mutant mice but not in Gnao1 hetKO mice.  Conclusions: De novo GNAO1 mutations have both GOF and LOF biochemical function with the former associated with seizures and the latter with movement disorder. Our GOF mutant mouse phenocopies this pattern. Further studies of human GNAO1 mutant alleles in mice are warranted. Funding: Michigan State University