{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,20]],"date-time":"2026-09-20T03:17:42Z","timestamp":1789874262463,"version":"4.0.1"},"reference-count":30,"publisher":"MDPI AG","issue":"19","license":[{"start":{"date-parts":[[2025,9,24]],"date-time":"2025-09-24T00:00:00Z","timestamp":1758672000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"JSPS KAKENHI","award":["JP22H03049"],"award-info":[{"award-number":["JP22H03049"]}]},{"name":"JSPS KAKENHI","award":["JP20K21589"],"award-info":[{"award-number":["JP20K21589"]}]}],"content-domain":{"domain":["www.mdpi.com"],"crossmark-restriction":true},"short-container-title":["Cells"],"abstract":"<jats:p>RAC3 encodes a small Rho-family GTPase essential for cytoskeletal regulation and neurodevelopment, and de novo RAC3 variants typically act as gain-of-function alleles that cause severe neurodevelopmental disorders. In this study, we analyzed a fetus with multisystem congenital anomalies and identified a de novo RAC3 p.(T17R) variant by genome sequencing. To elucidate the pathogenicity of this variant, we combined in silico variant prioritization, structural and energetic modeling, and pathogenicity prediction with in vitro biochemical assays, including GDP\/GTP exchange, GTP hydrolysis, effector pull-down, and luciferase reporter analyses in COS7 cells, as well as morphological analysis of primary hippocampal neurons. Furthermore, we performed in vivo analyses using a mouse in utero electroporation to assess cortical neuron migration, axon extension, and dendritic development. Our biochemical results suggest that RAC3-T17R exhibits markedly increased GDP\/GTP exchange, with a preference for GDP binding, and undetectable GTP hydrolysis. The mutant displayed minimal binding to canonical RAC effectors (PAK1, MLK2, and N-WASP) and failed to activate SRF-, NF\u03baB-, or AP1-dependent transcription. Neuronal overexpression of RAC3-T17R impaired axon formation in vitro, while in vivo expression delayed cortical neuron migration and axon extension and reduced dendritic arborization. Clinically, the fetus exhibited corpus callosum agenesis, microcephaly, organomegaly, and limb contractures. Collectively, these findings indicate that the RAC3 p.(T17R) variant may represent a signaling-deficient allele with pleiotropic, variant-specific mechanisms that disrupt corticogenesis and broader organogenesis. Our multi-tiered in silico\u2013in vitro\u2013in vivo approach demonstrates that noncanonical RAC3 variants can produce complex, multisystem developmental phenotypes beyond previously recognized RAC3-related neurodevelopmental disorders.<\/jats:p>","DOI":"10.3390\/cells14191499","type":"journal-article","created":{"date-parts":[[2025,9,25]],"date-time":"2025-09-25T07:46:49Z","timestamp":1758786409000},"page":"1499","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["A Pleiotropic and Functionally Divergent RAC3 Variant Disrupts Neurodevelopment and Impacts Organogenesis"],"prefix":"10.3390","volume":"14","author":[{"given":"Ryota","family":"Sugawara","sequence":"first","affiliation":[{"name":"Department of Molecular Neurobiology, Institute for Developmental Research, Aichi Developmental Disability Center, 713-8 Kamiya, Kasugai 480-0392, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2194-7239","authenticated-orcid":false,"given":"Marcello","family":"Scala","sequence":"additional","affiliation":[{"name":"Department of Neurosciences, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health, University of Genoa, 16132 Genoa, Italy"},{"name":"Unit of Medical Genetics, IRCCS Giannina Gaslini Institute, 16147 Genoa, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sara","family":"Cabet","sequence":"additional","affiliation":[{"name":"Pediatric and Fetal Imaging Department, Femme-M\u00e8re-Enfant Hospital, Hospices Civils de Lyon, Claude Bernard Lyon 1 University, 69500 Lyon, France"},{"name":"Pathology and Genetics of Neuron and Muscle, Neuromyogene Institute, CNRS UMR 5261 INSERM U1315, University of Lyon\u2013Universit\u00e9 Claude Bernard Lyon 1, 69500 Lyon, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carine","family":"Abel","sequence":"additional","affiliation":[{"name":"Department of Genetics, Groupement Hospitalier Est, Hospices Civils de Lyon, 69777 Bron, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Louis","family":"Januel","sequence":"additional","affiliation":[{"name":"Department of Genetics, Groupement Hospitalier Est, Hospices Civils de Lyon, 69777 Bron, France"},{"name":"Plan France Medecine G\u00e9nomique, Auragen Laboratory, 69003 Lyon, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7691-9492","authenticated-orcid":false,"given":"Gaetan","family":"Lesca","sequence":"additional","affiliation":[{"name":"Pathology and Genetics of Neuron and Muscle, Neuromyogene Institute, CNRS UMR 5261 INSERM U1315, University of Lyon\u2013Universit\u00e9 Claude Bernard Lyon 1, 69500 Lyon, France"},{"name":"Department of Genetics, Groupement Hospitalier Est, Hospices Civils de Lyon, 69777 Bron, France"},{"name":"Plan France Medecine G\u00e9nomique, Auragen Laboratory, 69003 Lyon, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Laurent","family":"Guibaud","sequence":"additional","affiliation":[{"name":"Pediatric and Fetal Imaging Department, Femme-M\u00e8re-Enfant Hospital, Hospices Civils de Lyon, Claude Bernard Lyon 1 University, 69500 Lyon, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fr\u00e9d\u00e9rique","family":"Le Breton","sequence":"additional","affiliation":[{"name":"Department of Pathology, Hospices Civils de Lyon, 69777 Bron, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hiroshi","family":"Ueda","sequence":"additional","affiliation":[{"name":"United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, Yanagido, Gifu 501-1193, Japan"},{"name":"Department of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu University, Yanagido, Gifu 501-1193, Japan"},{"name":"Center for One Medicine Innovative Translational Research (COMIT), Gifu University, Yanagido, Gifu 501-1193, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3324-5998","authenticated-orcid":false,"given":"Hidenori","family":"Tabata","sequence":"additional","affiliation":[{"name":"Department of Molecular Neurobiology, Institute for Developmental Research, Aichi Developmental Disability Center, 713-8 Kamiya, Kasugai 480-0392, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6527-477X","authenticated-orcid":false,"given":"Hidenori","family":"Ito","sequence":"additional","affiliation":[{"name":"Department of Molecular Neurobiology, Institute for Developmental Research, Aichi Developmental Disability Center, 713-8 Kamiya, Kasugai 480-0392, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6827-8434","authenticated-orcid":false,"given":"Koh-ichi","family":"Nagata","sequence":"additional","affiliation":[{"name":"Department of Molecular Neurobiology, Institute for Developmental Research, Aichi Developmental Disability Center, 713-8 Kamiya, Kasugai 480-0392, Japan"},{"name":"Department of Neurochemistry, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Nagoya 466-8550, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,9,24]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"20384","DOI":"10.1074\/jbc.272.33.20384","article-title":"Characterization of RAC3, a Novel Member of the Rho Family","volume":"272","author":"Haataja","year":"1997","journal-title":"J. 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