Functional characterization of a de novo ACTB variant and its impact on β-actin cytoskeletal organization and cell motility

Background: Actin is a major component of the cytoskeleton and plays a key role in maintaining cell shape, motility, and intracellular organization, ACTB encoding cytoplasmic β-actin, is associated with several human disorders, including Baraitser-Winter syndrome and ACTB-associated syndromic thrombocytopenia. Although variants in exons 5 and 6 are mainly associated with the latter, their contribution to the broader ACTB-related disease spectrum remains unclear. Here, we describe a novel de novo ACTB missense variant in a pediatric patient with a Baraitser-Winter phenotype and investigate its functional consequences. Methods: Whole-exome sequencing identified a de novo missense variant in ACTB, c.959T>C (p.Leu320Pro), in a pediatric patient. Structural analysis predicted that the amino acid substitution introduces steric clashes, potentially leading to local conformational changes in the β-actin protein. Functional characterization was performed using HEK293T cells transiently transfected with plasmid expressing the mutant ACTB. Cell morphology and cytoskeletal organization were evaluated by immunofluorescence and confocal microscopy. β-actin and phalloidin staining were used to assess the levels of globular (G-) actin and polymerized filamentous (F-) actin, respectively. Results: Evolutionary analysis showed that Leu320 is predominantly conserved as a hydrophobic residue, whereas proline was observed in less than 1% of aligned sequences. Structural modelling indicated that the p.Leu320Pro substitution results in the loss of two backbone hydrogen bonds involving residue 320 and introduces steric clashes with the highly conserved Glu316 residue. Microscope analysis revealed increased β-actin staining together with reduced phalloidin fluorescence in cells expressing the mutant protein, indicating impaired actin polymerization. These alterations were associated with changes in cell morphology and reduced cell viability, supporting a functional impact of the identified variant on cytoskeletal organization. Conclusions: Our findings demonstrate that the de novo ACTB c.959T>C (p.Leu320Pro) variant induces a local structural alteration of β-actin and is associated with altered cytoskeletal organization in cells. These results expand the spectrum of pathogenic ACTB variants and provide further insight into the molecular mechanisms underlying ACTB-related disorders. Further studies will be required to fully elucidate the pathogenic mechanisms of this variant and its effects in disease-relevant cellular models.