A Mutational Hotspot in TUBB2A Associated with impaired Heterodimer Formation and severe Brain Developmental Disorders

Gabriele Di Pasquale¹Jacopo Colella¹Carola Pia Di Cataldo¹MIGUEL A. SOLER²Sara Fortuna³Emma Mizrahi-Powell⁴Mathilde Nizon⁵Benjamin COGNE⁵Valentina Turchetti⁶Giuseppe Mangano⁷Francesco Fabrizio Comisi⁸Corrado Cecchetti⁹Alessandra Giliberti¹⁰Rosaria Nardello¹⁰Piero Pavone¹¹Raffaele Falsaperla¹²Gabriella Di Rosa¹³Gilad D. Evrony⁵Maurizio Del Vecchio¹Mariasavina Severino¹⁴Andrea Accogli¹⁵Alessandro Vittori^9*Vincenzo Salpietro^1*

• ¹Universita degli Studi dell'Aquila - Polo Coppito, L'Aquila, Italy
• ²Universita degli Studi di Udine, Udine, Italy
• ³Istituto Italiano di Tecnologia, Genoa, Italy
• ⁴New York University Grossman School of Medicine, New York, United States
• ⁵Nantes Universite, Nantes, France
• ⁶University College London Queen Square Institute of Neurology, London, United Kingdom
• ⁷Universita degli Studi di Enna 'Kore', Enna, Italy
• ⁸Universita degli Studi di Cagliari, Cagliari, Italy
• ⁹Department of Anesthesia, Intensive Care and Operating Sections, Bambino Gesù Children's Hospital (IRCCS), Rome, Italy
• ¹⁰Universita degli Studi di Palermo, Palermo, Italy
• ¹¹Universita degli Studi di Catania, Catania, Italy
• ¹²Universita degli Studi di Ferrara, Ferrara, Italy
• ¹³Universita degli Studi di Messina, Messina, Italy
• ¹⁴Istituto Giannina Gaslini, Genoa, Italy
• ¹⁵Universite de Montreal, Montreal, Canada

Abstract Introduction: Microtubules are essential components of the neuronal cytoskeleton. The α-and β-tubulins, variably expressed in the central nervous system, play key roles in neurogenesis and brain development. Pathogenic variants in TUBB2A have recently been identified as an ultra-rare cause of pediatric neurodevelopmental disorders (NDDs). However, the neurological and behavioral manifestations, genotype-phenotype correlations, and underlying disease mechanisms remain poorly understood due to the limited number of reported families. Methods: We describe a cohort of families presenting with microcephaly, global developmental delay, speech impairment, seizures and/or EEG abnormalities, movement disorders and severe behavioral disorders. Clinical assessments and brain imaging studies were conducted over a 10-year follow-up period. Genetic analysis was performed via whole-exome sequencing (WES), and structural modeling was used to investigate the functional impact of the identified variants. Results: WES revealed a novel recurrent heterozygous pathogenic variant in TUBB2A (NM_001069.3:c.1172G>A; NP_001060.1:p.Arg391His), identified as the cause of disease in multiple affected individuals from unrelated families. Comparative analysis with previously reported TUBB2A de novo variants confirmed that this novel recurrent mutation affects a highly conserved Arg391 residue within the longitudinal E-site heterodimer interface. Computational modeling demonstrated that the variant disrupts α/β-tubulin heterodimer formation, impairing binding stability at this critical interaction site. Discussion: Our findings expand the phenotypic and genotypic spectrum of TUBB2A-related disorders and identify Arg391 as a mutational hotspot linked to severe brain developmental disorders due to aberrant tubulin dynamics, highlighting the disruption of the α/β-tubulin heterodimer formation as the disease mechanism associated to this novel hotspot variant. These results provide new insights into