In-depth molecular profiling of an intronic GNAO1 mutant as the basis for personalized high-throughput drug screening

Background: The GNAO1 gene, encoding the major neuronal G pro-tein Gao, is mutated in a subset of pediatric encephalopathies. Most such mutations consist of missense variants.Methods: In this study, we present a precision medicine workflow combining next-generation sequencing (NGS) diagnostics, molecular etiology analysis, and personalized drug discovery.Findings: We describe a patient carrying a de novo intronic mutation (NM_020988.3:c.724-8G>A), leading to epilepsy-negative encephalop-athy with motor dysfunction from the second decade. Our data show that this mutation creates a novel splice acceptor site that in turn causes an in-frame insertion of two amino acid residues, Pro-Gln, within the regulatory switch III region of Gao. This insertion misconfigures the switch III loop and creates novel interactions with the catalytic switch II region, resulting in increased GTP uptake, defective GTP hydrolysis, and aberrant interactions with effector proteins. In contrast, intracel-lular localization, Gbg interactions, and G protein-coupled receptor (GPCR) coupling of the Gao[insPQ] mutant protein remain unchanged.Conclusions: This in-depth analysis characterizes the heterozygous c.724-8G>A mutation as partially dominant negative, providing clues to the molecular etiology of this specific pathology. Further, this anal-ysis allows us to establish and validate a high-throughput screening platform aiming at identifying molecules that could correct the aberrant biochemical functions of the mutant Gao.Funding: This work was supported by the Joint Seed Money Funding scheme between the University of Geneva and the Hebrew University of Jerusalem.