High-throughput imaging of GABA fluorescence as a functional assay for variants in the neurodevelopmental gene, SLC6A1

Abstract Pathogenic variants in SLC6A1, which encodes GABA Transporter 1 (GAT-1), are associated with developmental delay, autism, epilepsy (e.g., epilepsy with myoclonic astatic seizures [EMAS]), and possibly schizophrenia. Functional assays to establish pathogenicity of human variants is a key limiting factor in the clinical interpretation of genetic findings. Methods based on radioactive [3H]-GABA uptake come with significant regulatory concerns, cost, and workflow complexity, which could be resolved by an alternate assay. To address this issue, we developed a high-content fluorescence imaging assay of GAT-1-mediated GABA uptake using a genetically encoded GABA sensor, iGABA-Snfr. We demonstrated that pathogenic variants strongly reduced uptake (mean, –89.4% [95% CI, –71.5% to –107.3%]). Some variants of uncertain significance (VUS) were associated with reduced GABA uptake (G111R, S459R, V511M; mean, –101.2% [95% CI –81.1% to –121.3%]), whereas others showed only mild reduction (R211C, R566H, F242V, R419C; mean, –33.6% [95% CI –17.2% to –50.1%]), supporting variant reclassification. Variant-specific effects on iGABA were highly correlated with the results of the radioactive [3H]-GABA assay (R2=0.8095, p<0.0001). The molecular chaperone 4-phenylbutyric acid (4PBA) was associated with ∼35% increase in iGABA. This non-radioactive assay is suitable for functional validation and high-throughput screening to identify positive modulators of GAT-1. Competing Interest Statement The authors have declared no competing interest. Footnotes The order of the author list was corrected.