Abstract
We recently identified de novo missense variants affecting the small GTPase ARF3 as the cause of a disorder characterized by developmental delay/intellectual disability, microcephaly, brain atrophy, epilepsy and minor skeletal defects. In vitro and in vivo analyses documented impaired Golgi integrity, vesicle trafficking, and brain and body axes development. Here, we report clinical features of five additional patients and the functional characterization of three novel ARF3 variants. Cell-based assays corroborate a deleterious, variant-specific effect on protein stability, GTP binding and Golgi morphology. Zebrafish models confirm the dominant behavior of the tested variants and their variable impact on development. ARF3 mutants significantly affected Golgi integrity in vivo as well as brain size, recapitulating patients’ microcephaly. These findings expand the ARF3-related Golgipathy mutational spectrum, strengthen previous observations linking variants with dominant negative behavior to a markedly severe phenotype, and underscore the specific vulnerability of the nervous system to ARF and Golgi dysfunction.
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Data availability
The functional data generated in this study can be found in the supplementary material. The sequencing data are not publicly available due to due to privacy/ethical restrictions. The new variants identified in this work and their clinical association have been submitted to ClinVar (SCV006550956-SCV006550959).
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Acknowledgements
We thank the families participating in the study. One patient included in this study was diagnosed thanks to Defidiag (NCT04154891), a project supported by The French Ministry of Health in the framework of the French initiative for genomic medicine (https://pfmg2025.fr/) and by French government funding from the Agence Nationale de la Recherche under the “Investissements d’avenir” program (ANR- 10-IAHU-01).
Funding
The work was supported by “Fondazione Telethon ETS” (grant number GMR23T1168, to AL), Italian Ministry of Health (5x1000, to AL; Current Research Funds, PNRR-MR1-2022-12376811 and RF-2021-12374963, to MT) and Istituto Superiore di Sanità (Bando Ricerca Indipendente ISS20-2e15b898baf0, to SC).
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VM, SC, EZ: functional analyses (in vitro); GF, CP: functional analyses (in vivo); FCR: clinical and molecular data assessment and data curation; MC: structural analyses; CS-B, CB, SEC, BG, AdSM, DK, ES, CC, TH, A-SH, LvE, EEB, UH, AS: clinical and molecular data collection; MT, AL: study coordination, funding acquisition, writing.
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All subjects were referred for diagnostic genetic testing. Clinical data and DNA samples were collected, stored, and used in accordance with the ethical standards of the Declaration of Helsinki, following written informed consent from participating families. The study was approved by the local Institutional Ethical Committee of the Ospedale Pediatrico Bambino Gesù, Rome (ref. RF-2021-12374963 and PNRR-MR1-2022-12376811). Written informed consents were obtained for publication of individual pictures and clinical details. All animal experiments were conducted according to ARRIVE guidelines (https://arriveguidelines.org/) upon approval of the Italian Ministry of Health (Direzione Generale della Salute Animale - Ufficio 6 EX DGSAF, ref.n° 652/2024-PR).
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Muto, V., Fasano, G., Radio, F.C. et al. Newly identified ARF3 variants strengthen the causal link between Golgi fragmentation and brain malformations. Eur J Hum Genet 34, 438–443 (2026). https://doi.org/10.1038/s41431-025-02001-w
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DOI: https://doi.org/10.1038/s41431-025-02001-w
