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

• Published in: Med (New York, N.Y. : Online) Vol. 4; no. 5; pp. 311 - 325.e7
• Main Authors: Koval, Alexey, Larasati, Yonika A., Savitsky, Mikhail, Solis, Gonzalo P., Good, Jean-Marc, Quinodoz, Mathieu, Rivolta, Carlo, Superti-Furga, Andrea, Katanaev, Vladimir L.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 12.05.2023
• Subjects: Child; drug discovery; Drug Evaluation, Preclinical; G protein; GNAO1; GTP-Binding Protein alpha Subunits, Gi-Go - chemistry; GTP-Binding Protein alpha Subunits, Gi-Go - genetics; GTP-Binding Protein alpha Subunits, Gi-Go - metabolism; GTP-Binding Proteins - genetics; GTP-Binding Proteins - metabolism; Guanosine Triphosphate; High-Throughput Screening Assays; Humans; Mutation - genetics; pediatric encephalopathy; personalized medicine; signaling; GNAO1; pediatric encephalopathy; signaling; drug discovery; Translation to patients; personalized medicine; G protein
• ISSN: 2666-6340; 2666-6340
• DOI: 10.1016/j.medj.2023.03.001

The GNAO1 gene, encoding the major neuronal G protein Gαo, is mutated in a subset of pediatric encephalopathies. Most such mutations consist of missense variants. In this study, we present a precision medicine workflow combining next-generation sequencing (NGS) diagnostics, molecular etiology analysis, and personalized drug discovery. We describe a patient carrying a de novo intronic mutation (NM_020988.3:c.724-8G>A), leading to epilepsy-negative encephalopathy 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 Gαo. 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, intracellular localization, Gβγ interactions, and G protein-coupled receptor (GPCR) coupling of the Gαo[insPQ] mutant protein remain unchanged. 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 analysis 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 Gαo. This work was supported by the Joint Seed Money Funding scheme between the University of Geneva and the Hebrew University of Jerusalem. [Display omitted] •Splice-site mutation in GNAO1 produces aberrant Gαo with a two-amino acid insertion•Insertion changes switch III dynamics and interactions within Gαo and with effectors•Pathogenic Gαo[insPQ] displays aberrant GTP uptake and hydrolysis•High-throughput screening platform is established for personalized drug discovery Mutations in the gene GNAO1 lead to severe pediatric brain disorders with epilepsy, motor dysfunction, and developmental and intellectual delay. No curative therapy currently exists for this devastating disease. A team of neurologists, clinical geneticists, and biologists focus here on a particular mutation in the GNAO1 gene. Through an exhaustive clinical, genetic, cellular, and molecular characterization, they identify the core mechanisms of the disease caused by this specific mutation. This further allows us to establish a high-throughput screening platform to look for molecules that would correct the abnormal functioning of the mutant protein. This multidisciplinary work validates a personalized medicine research and drug discovery pipeline that can be expanded to other genetic disorders. A splice-site mutation in GNAO1 encoding the major neuronal G protein Gαo leads to severe pediatric encephalopathy. Koval et al. discover that this mutation leads to an in-frame insertion of two extra amino acids, a novel pairing between the regulatory switch III and switch II loops, and aberrant GTP uptake and hydrolysis.