Molecular mechanisms of hotspot variants in cytoskeletal β-actin associated with Baraitser-Winter syndrome.

Baraitser-Winter cerebrofrontofacial syndrome (BWCFF) is the most common and best-defined clinical entity associated with heterozygous single-point missense mutations in cytoskeletal β-actin. Patients present with distinct craniofacial anomalies and neurodevelopmental disabilities of variable severity. To date, the most frequently observed variants affect residue R196 of cytoskeletal β-actin. Patients carrying the p.R196 variants are likely to suffer from pachygyria, probably due to neuronal migration defects contributing to the development of abnormal convolutions of the cerebral cortex. Here, we report on the recombinant production, purification and characterization of the BWCFF hotspot variants p.R196H, p.R196C and p.R196S. Our findings reveal that the stability of the monomeric variants remains unaffected, suggesting that the disease mechanism involves the incorporation of these variants into actin filaments. This incorporation alters F-actin stability and polymerization dynamics to varying degrees, depending on the specific variant. These effects are consistent with the positioning of residue R196 near the helical filament axis. Observed changes include an increased critical concentration for polymerization, reduced elongation rates and accelerated filament depolymerization. Within the actin-related protein 2/3 (Arp2/3)-generated branch junction complex, which is critical for processes such as cell migration and endocytosis, residue R196 is located at the interface between the first protomer of the nucleated filament and the Arp2 subunit. Variant p.R196H specifically results in reduced branching efficiency and impaired branch stability. Future research will seek to elucidate the impact of these actin filament defects on cellular processes and their contribution to the multifaceted pathophysiology of BWCFF, with a particular emphasis on cortical development.