Abstracts

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. The availability of functional evidence of pathogenicity for human variants (particularly those with unknown clinical significance) is a key limiting factor in the clinical interpretation of genetic findings by ACMG guidelines. Although methods based on radioactive 3H-GABA uptake have been instrumental in understanding GAT-1 function and assessing the functional impact of human variants in SLC6A1, the use of radioactivity adds significant regulatory concerns, cost, and workflow complexity, which could be resolved by a fluorescence-based assay.

We developed a cell-based fluorescence assay of human GAT1 function based on iGABA-Snfr fluorescence using both transient transfection and stable HeLa cell lines.  We benchmarked our assay using domain deletions (ECD, CPD, and TM6-7), and several previously reported human variants (pathogenic, benign, and uncertain significance) in GAT1 using the ImageXpress Micro automated microscope in 96-well and 384-well format.

We show that iGABA-Snfr fluorescence faithfully reports the effect of GAT1-mediated GABA uptake when applied extracellularly (1-100uM). Domain deletions result in abrogration of uptake, while variants reduce uptake, as expressed by fractional change in iGABA fluorescence relative to WT control. High correspondence with the radioactive 3H-GABA assay was seen. Drug screening demonstrated a positive effect of the compound, 4-phenylbutyrate (4PB), as previously reported.

Our fluorescence-based assay may be useful for functional validation of human GAT1 variants and for drug screening to identify positive modulators of GAT1 without requiring radioactivity.