Clinical Cases and the Molecular Profiling of a Novel Childhood Encephalopathy-Causing GNAO1 Mutation P170R.

De novo mutations in GNAO1, the gene encoding the major neuronal G protein Gαo, cause a spectrum of pediatric encephalopathies with seizures, motor dysfunction, and developmental delay. Of the >80 distinct missense pathogenic variants, many appear to uniformly destabilize the guanine nucleotide handling of the mutant protein, speeding up GTP uptake and deactivating GTP hydrolysis. Zinc supplementation emerges as a promising treatment option for this disease, as Zn2+ ions reactivate the GTP hydrolysis on the mutant Gαo and restore cellular interactions for some of the mutants studied earlier. The molecular etiology of GNAO1 encephalopathies needs further elucidation as a prerequisite for the development of efficient therapeutic approaches. In this work, we combine clinical and medical genetics analysis of a novel GNAO1 mutation with an in-depth molecular dissection of the resultant protein variant. We identify two unrelated patients from Norway and France with a previously unknown mutation in GNAO1, c.509C>G that results in the production of the Pro170Arg mutant Gαo, leading to severe developmental and epileptic encephalopathy. Molecular investigations of Pro170Arg identify this mutant as a unique representative of the pathogenic variants. Its 100-fold-accelerated GTP uptake is not accompanied by a loss in GTP hydrolysis; Zn2+ ions induce a previously unseen effect on the mutant, forcing it to lose the bound GTP. Our work combining clinical and molecular analyses discovers a novel, biochemically distinct pathogenic missense variant of GNAO1 laying the ground for personalized treatment development.

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 protein Gαo, 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 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, Gßγ interactions, and G protein-coupled receptor (GPCR) coupling of the Gαo[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 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. FUNDING: This work was supported by the Joint Seed Money Funding scheme between the University of Geneva and the Hebrew University of Jerusalem.

Restoration of the GTPase activity and cellular interactions of Gαo mutants by Zn2+ in GNAO1 encephalopathy models.

De novo point mutations in GNAO1, gene encoding the major neuronal G protein Gαo, have recently emerged in patients with pediatric encephalopathy having motor, developmental, and epileptic dysfunctions. Half of clinical cases affect codons Gly203, Arg209, or Glu246; we show that these mutations accelerate GTP uptake and inactivate GTP hydrolysis through displacement Gln205 critical for GTP hydrolysis, resulting in constitutive GTP binding by Gαo. However, the mutants fail to adopt the activated conformation and display aberrant interactions with signaling partners. Through high-throughput screening of approved drugs, we identify zinc pyrithione and Zn2+ as agents restoring active conformation, GTPase activity, and cellular interactions of the encephalopathy mutants, with negligible effects on wild-type Gαo. We describe a Drosophila model of GNAO1 encephalopathy where dietary zinc restores the motor function and longevity of the mutant flies. Zinc supplements are approved for diverse human neurological conditions. Our work provides insights into the molecular etiology of GNAO1 encephalopathy and defines a potential therapy for the patients.

Longitudinal Study Depicting Differences in Complementary Feeding and Anthropometric Parameters in Late Preterm Infants up to 2 Years of Age.

Ensuring the nutritional demands of preterm (PT) infants during complementary feeding could contribute significantly to the infants' long-term health and development. However, the dietary guidelines for complementary feeding in PT are scarce. Thus, describing dietary intake and identifying nutritional targets for these infants could be of great interest. The aim of this study is to assess the food intake and anthropometric parameters in a Mediterranean infant cohort from 6 to 24 months and to identify nutritional targets especially focused on late preterm infants. This is a longitudinal prospective study analyzing information from administered questionnaires about general characteristics and food frequency consumption in 115 infants (20 PT (32 to 36 gestational weeks), 95 full-term (FT)) at 6, 12 and 24 months of age. Results show that the differences in the prevalence of underweight observed in PT infants vs. FT infants are maintained for up to 6 months of age but disappear at 12 and 24 months. The age of inclusion of new foods and the average intake of the main food groups was not different from that of FTs. Although protein intake at 6 months was directly correlated with weight gain and growth in FT, these associations were not observed in PT. At the nutritional level, the low intake of vitamin D in preterm infants is noteworthy. These findings may be useful when designing new intervention strategies for this population group.