Abstract
Actin-like 6a (ACTL6A) encodes an Actin-Related Protein (ARP) primarily known for its role in the BRG1-associated factor (BAF) chromatin remodeling complex. While the BAF complex has been extensively studied, the specific function of ACTL6A within this complex remains relatively unexplored. Recently, the Bicknell lab identified a missense variant (p.Pro305Ser) in a New Zealand father-daughter duo, raising the possibility of ACTL6A's involvement in neurodevelopmental disorders (NDDs). Although a few ACTL6A variants have been reported in NDDs, functional evidence linking them to disease mechanisms is still lacking. Missense variants in other BAF complex subunits have been implicated in disorders such as Coffin-Siris syndrome and Nicolaides–Baraitser syndrome, both of which share clinical features—such as neurodevelopmental delay and craniofacial dysmorphisms—with patients harboring ACTL6A variants, including the New Zealand duo. Therefore, we hypothesised that ACTL6A variants could impair cellular function by destabilizing the protein, disrupting its localization, or altering its protein interactions. To investigate this, we cloned the ACTL6A sequence into a plasmid and introduced patient variants via site-directed mutagenesis. These plasmids were transfected into HEK293FT cells, and protein samples were optimised for Western Blotting. To assess protein stability, cycloheximide assays were performed, which showed the variants didn’t induce significant instability of ACTL6A 12 hours after protein synthesis inhibition. As the WT ACTL6A protein was not showing any visible reduction in protein levels, the assay may need to be tracked up to 24 hours post protein synthesis-inhibition before conclusions can be drawn. To examine the impact of variants on protein localisation, we transfected U2OS cells with the plasmids and visualized ACTL6A protein via immunofluorescence. Notably, the p.Pro126Leu variant displayed an aggregate-like phenotype, with significantly reduced ACTL6A levels in the nucleus. This suggests that the variant may cause protein misfolding, leading to cytoplasmic aggregation and impaired nuclear localisation. While these findings indicate a potential disruption in ACTL6A protein localisation for the p.Pro126Leu variant, further studies are needed to confirm this and the effects each variant has on ACTL6A’s stability and protein interactions. This work presents initial evidence suggesting that ACTL6A variants may play functional roles in neurodevelopmental disorders, contributing to their classification using ACMG guidelines. The conclusions drawn from this project will establish a foundation for future research aimed at developing diagnostic tools and enhancing our understanding of ACTL6A's role within the BAF complex and its impact on brain development.