4-Phenylbutyrate Plus Wildtype GAT-1 Augmentation: A Dual Therapy to Rescue SLC6A1 Variant-Associated Developmental...

Pathogenic variants in SLC6A1, which encodes the γ-aminobutyric acid (GABA) transporter GAT-1, cause developmental and epileptic encephalopathies (DEEs) through reduced GABA uptake, impaired transporter trafficking, and functional haploinsufficiency. 4-phenylbutyrate (PBA) is a clinically available small molecule with chemical-chaperone and histone-deacetylase-inhibitor activities that can rescue misfolded GABAergic proteins, but variant-level rescue data are needed to guide precision treatment.

We report a novel de novo missense mutation p.Ala305Val in GAT-1 encoding SLC6A1, in a patient with myoclonic-atonic epilepsy and a developmental and epileptic encephalopathy phenotype. Ala305Val was compared with the residue-matched comparator p.Ala305Thr (Ala305Thr). Variant effects were evaluated by (i) protein-structure prediction across nine stability-prediction algorithms using the cryo-EM-derived human GAT-1 template (PDB 7Y7W); (ii) 3H-GABA uptake assays in HEK293T cells and in human iPSC-derived astrocytes and cortical neurons; (iii) live-cell confocal microscopy of ER colocalization; (iv) pharmacologic rescue with PBA, TUDCA and salubrinal (v) and GAT-1 cDNA gene-augmentation, alone and in combination with PBA.

AI-based stability predictors uniformly indicated destabilization of GAT-1 p.Ala305Val and GAT-1 p.Ala305Thr. GAT-1 p.Ala305Val reduced 3H GABA uptake across HEK293Ts, astrocytes, and neurons. The mutant transporter accumulated within the endoplasmic reticulum (ER), with ER colocalization rising from approximately 30% in wildtype to ~80% in GAT-1 p.Ala305Val; PBA reduced ER retention to approximately ~40% and restored total GAT-1 fluorescence toward wildtype levels. Pharmacochaperones (PBA, TUDCA) restored GABA uptake for the mutant transporters. Wildtype GAT-1 gene augmentation improved GABA uptake in the heterozygous condition but combined PBA plus wildtype allele augmentation produced rescue greater than either intervention alone in the available dose-response ranges.

GAT-1 p.Ala305Val is a trafficking-impaired, loss-of-function variant whose dysfunction is amenable to two convergent therapeutic axes: pharmacologic correction of folding and trafficking, and augmentation of functional transporter expression. These findings support a two-pronged precision-medicine framework for SLC6A1-related DEEs in which PBA increased the transporter function augmented by genetic approaches.