{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,5,29]],"date-time":"2026-05-29T09:26:09Z","timestamp":1780046769629,"version":"3.53.1"},"posted":{"date-parts":[[2025,11,7]]},"group-title":"Neuroscience","reference-count":15,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,11,7]],"date-time":"2025-11-07T00:00:00Z","timestamp":1762473600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000065","name":"NINDS","doi-asserted-by":"crossref","award":["K08NS118107 (CMM)"],"award-info":[{"award-number":["K08NS118107 (CMM)"]}],"id":[{"id":"10.13039\/100000065","id-type":"DOI","asserted-by":"crossref"}]},{"name":"Diamond Blackfan Chair in Neuroscience Research"},{"name":"Robinson Fund for Transformative Research in Epilepsy"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,11,10]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Pathogenic variants in\n                  <jats:italic>SLC6A1<\/jats:italic>\n                  , 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, \u201389.4% [95% CI, \u201371.5% to \u2013107.3%]). Some variants of uncertain significance (VUS) were associated with reduced GABA uptake (G111R, S459R, V511M; mean, \u2013101.2% [95% CI \u201381.1% to \u2013121.3%]), whereas others showed only mild reduction (R211C, R566H, F242V, R419C; mean, \u201333.6% [95% CI \u201317.2% to \u201350.1%]), supporting variant reclassification. Variant-specific effects on iGABA were highly correlated with the results of the radioactive [3H]-GABA assay (R\n                  <jats:sup>2<\/jats:sup>\n                  =0.8095, p&lt;0.0001). The molecular chaperone 4-phenylbutyric acid (4PBA) was associated with \u223c35% increase in iGABA. This non-radioactive assay is suitable for functional validation and high-throughput screening to identify positive modulators of GAT-1.\n                <\/jats:p>","DOI":"10.1101\/2025.11.06.687003","type":"posted-content","created":{"date-parts":[[2025,11,8]],"date-time":"2025-11-08T03:05:16Z","timestamp":1762571116000},"source":"Crossref","is-referenced-by-count":0,"title":["High-throughput imaging of GABA fluorescence as a functional assay for variants in the neurodevelopmental gene,\n                  <i>SLC6A1<\/i>"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5460-4550","authenticated-orcid":false,"given":"Christopher Michael","family":"McGraw","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Guoqi","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gillian","family":"Prinzing","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8123-4092","authenticated-orcid":false,"given":"Kimberly","family":"Wiltrout","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7350-5136","authenticated-orcid":false,"given":"Annapurna","family":"Poduri","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2025111215153972000_2025.11.06.687003v2.1","doi-asserted-by":"publisher","DOI":"10.1016\/j.expneurol.2019.112973"},{"key":"2025111215153972000_2025.11.06.687003v2.2","doi-asserted-by":"publisher","DOI":"10.1016\/j.ajhg.2015.02.016"},{"key":"2025111215153972000_2025.11.06.687003v2.3","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-60327-317-6_1"},{"key":"2025111215153972000_2025.11.06.687003v2.4","doi-asserted-by":"publisher","DOI":"10.1111\/epi.13986"},{"key":"2025111215153972000_2025.11.06.687003v2.5","doi-asserted-by":"publisher","DOI":"10.1111\/epi.17731"},{"key":"2025111215153972000_2025.11.06.687003v2.6","doi-asserted-by":"publisher","DOI":"10.1038\/s41592-019-0471-2"},{"key":"2025111215153972000_2025.11.06.687003v2.7","doi-asserted-by":"publisher","DOI":"10.1111\/epi.14531"},{"key":"2025111215153972000_2025.11.06.687003v2.8","doi-asserted-by":"publisher","DOI":"10.1093\/braincomms\/fcac144"},{"key":"2025111215153972000_2025.11.06.687003v2.9","doi-asserted-by":"publisher","DOI":"10.1038\/s41593-019-0565-2"},{"key":"2025111215153972000_2025.11.06.687003v2.10","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-4757-6376-8_6"},{"key":"2025111215153972000_2025.11.06.687003v2.11","doi-asserted-by":"publisher","DOI":"10.1093\/braincomms\/fcae110"},{"key":"2025111215153972000_2025.11.06.687003v2.12","doi-asserted-by":"publisher","DOI":"10.1016\/j.ajhg.2024.04.021"},{"key":"2025111215153972000_2025.11.06.687003v2.13","doi-asserted-by":"publisher","DOI":"10.1016\/j.ajhg.2021.07.007"},{"key":"2025111215153972000_2025.11.06.687003v2.14","doi-asserted-by":"publisher","DOI":"10.1016\/j.neuron.2007.10.021"},{"key":"2025111215153972000_2025.11.06.687003v2.15","doi-asserted-by":"publisher","DOI":"10.1177\/1087057111405851"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.1101\/2025.11.06.687003","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T09:58:06Z","timestamp":1768471086000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.1101\/2025.11.06.687003"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,11,7]]},"references-count":15,"URL":"https:\/\/doi.org\/10.1101\/2025.11.06.687003","relation":{},"subject":[],"published":{"date-parts":[[2025,11,7]]},"subtype":"preprint"}}