SLC6A1 variant pathogenicity, molecular function and phenotype: a genetic and clinical analysis

doi:10.1093/brain/awad292

. 2023 Dec 1;146(12):5198-5208.

doi: 10.1093/brain/awad292.

SLC6A1 variant pathogenicity, molecular function and phenotype: a genetic and clinical analysis

Arthur Stefanski¹, Eduardo Pérez-Palma², Tobias Brünger³, Ludovica Montanucci¹, Cornelius Gati⁴, Chiara Klöckner⁵, Katrine M Johannesen^{6

7}, Kimberly Goodspeed^{8

9}, Marie Macnee³, Alexander T Deng¹⁰, Ángel Aledo-Serrano¹¹, Artem Borovikov¹², Maina Kava^{13

14}, Arjan M Bouman¹⁵, M J Hajianpour¹⁶, Deb K Pal^{17

18}, Marc Engelen¹⁹, Eveline E O Hagebeuk²⁰, Marwan Shinawi²¹, Alexis R Heidlebaugh²², Kathryn Oetjens²², Trevor L Hoffman²³, Pasquale Striano^{24

25}, Amanda S Freed²⁶, Line Futtrup²⁷, Thomas Balslev^{27

28}, Anna Abulí²⁹, Leslie Danvoye³⁰, Damien Lederer³¹, Tugce Balci^{32

33}, Maryam Nabavi Nouri³⁴, Elizabeth Butler³⁵, Sarah Drewes³⁶, Kalene van Engelen³⁷, Katherine B Howell^{38

39

40}, Jean Khoury¹, Patrick May⁴¹, Marena Trinidad⁴², Steven Froelich⁴², Johannes R Lemke^{5

43}, Jacob Tiller⁴⁴, Amber N Freed⁴⁴, Jing-Qiong Kang^{45

46

47

48

49}, Arthur Wuster⁴², Rikke S Møller^{6

50}, Dennis Lal^{1

51

52}

Affiliations

¹ Genomic Medicine Institute and Epilepsy Center, Cleveland Clinic, Cleveland, OH 44195, USA.
² Universidad del Desarrollo, Centro de Genética y Genómica, Facultad de Medicina Clínica Alemana, Santiago de Chile 7610658, Chile.
³ Cologne Center for Genomics (CCG), Medical Faculty of the University of Cologne, University Hospital of Cologne, Cologne 50931, Germany.
⁴ Department of Biological Sciences, Bridge Institute, USC Michelson Center for Convergent Bioscience, University of Southern California, Los Angeles, CA 90089, USA.
⁵ Institute of Human Genetics, University of Leipzig Medical Center, Leipzig 04103, Germany.
⁶ Department of Epilepsy Genetics and Personalized Medicine, The Danish Epilepsy Centre, Dianalund 4293, Denmark.
⁷ Department of Genetics, University Hospital of Copenhagen, Rigshispitalet, Copenhagen 2100, Denmark.
⁸ Children's Health, Medical Center, Dallas, TX 75235, USA.
⁹ Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
¹⁰ Clinical Genetics, Guys and St Thomas NHS Trust, London SE19RT, UK.
¹¹ Epilepsy Program, Neurology Department, Hospital Ruber Internacional, Madrid 28034, Spain.
¹² Research and Counseling Department, Research Centre for Medical Genetics, Moscow 115478, Russia.
¹³ Department of Neurology and Metabolic Medicine, Perth Children's Hospital, Perth 6009, Australia.
¹⁴ School of Paediatrics and Child Health, UWA Medical School, University of Western Australia, Perth 6009, Australia.
¹⁵ Department of Clinical Genetics, Erasmus MC, University Medical Center, Rotterdam 3015GD, The Netherlands.
¹⁶ Department of Pediatrics, Division of Medical Genetics and Genomics, Albany Medical College, Albany Med Health System, Albany, NY 12208, USA.
¹⁷ Department of Basic and Clinical Neurosciences, Institute of Psychiatry, Psychology and Neuroscience, King's College, London SE58AF, UK.
¹⁸ Department of Basic and Clinical Neurosciences, King's College Hospital, London SE59RS, UK.
¹⁹ Department of Pediatric Neurology, Amsterdam Public Health, Amsterdam University Medical Center, Amsterdam 1081HV, The Netherlands.
²⁰ Department of Pediatric Neurology, Stichting Epilepsie Instellingen Nederland (SEIN), Heemstede and Zwolle 2103SW, The Netherlands.
²¹ Division of Genetics and Genomic Medicine, Department of Pediatrics, St.Louis Children's Hospital, Washington University School of Medicine, St. Louis, MO 63110, USA.
²² Autism and Developmental Medicine Institute, Geisinger, Danville, PA 17837, USA.
²³ Department of Regional Genetics, Anaheim, Southern California Kaiser Permanente Medical Group, CA 92806, USA.
²⁴ Pediatric Neurology and Muscular Diseases Unit, IRCCS Istituto Giannina Gaslini, Genoa 16147, Italy.
²⁵ Department of Neurosciences, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health, University of Genoa, Genoa 16132, Italy.
²⁶ Department of Clinical Science, Kaiser Permanente Bernard J. Tyson School of Medicine, Pasadena, CA 91101, USA.
²⁷ Department of Paediatrics, Regional Hospital of Central Jutland, Viborg 8800, Denmark.
²⁸ Centre for Educational Development, Aarhus University, Aarhus 8200, Denmark.
²⁹ Department of Clinical and Molecular Genetics and Medicine Genetics Group, VHIR, University Hospital Vall d'Hebron, Barcelona 08035, Spain.
³⁰ Department of Neurology, Université catholique de Louvain, Cliniques universitaires Saint-Luc, Brussels 1200, Belgium.
³¹ Centre for Human Genetics, Institute for Pathology and Genetics, Gosselies 6041, Belgium.
³² Department of Pediatrics, Division of Medical Genetics, Western University, London, ON N6A3K7, Canada.
³³ Medical Genetics Program of Southwestern Ontario, London Health Sciences Centre and Children's Health Research Institute, London, ON N6A5A5, Canada.
³⁴ Department of Paediatrics, Division of Pediatric Neurology, London Health Sciences Centre, London, ON N6A5W9, Canada.
³⁵ GeneDx, Gaithersburg, MD 20877, USA.
³⁶ Department of Medical Genetics, UPMC Children's Hospital of Pittsburgh, Pittsburgh, PA 15224, USA.
³⁷ Medical Genetics Program of Southwestern Ontario, London Health Sciences Centre, London, ON N6A5W9, Canada.
³⁸ Department of Neurology, Royal Children's Hospital, Melbourne, VIC 3052, Australia.
³⁹ Department of Pediatrics, University of Melbourne, Melbourne, VIC 3052, Australia.
⁴⁰ Murdoch Children's Research Institute, Melbourne, VIC 3052, Australia.
⁴¹ Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Esch-sur-Alzette 4362, Luxembourg.
⁴² Translational Genomics, BioMarin Pharmaceutical Inc., Novato, CA 94949, USA.
⁴³ Center for Rare Diseases, University of Leipzig Medical Center, Leipzig 04103, Germany.
⁴⁴ SLC6A1 Connect, Frisco, TX 75034, USA.
⁴⁵ Department of Neurology, Vanderbilt University Medical Center, Nashville, TN 37240, USA.
⁴⁶ Neuroscience Graduate Program, Vanderbilt University, Nashville, TN 37235, USA.
⁴⁷ Department of Neurology, Vanderbilt Brain Institute, Nashville, TN 37235, USA.
⁴⁸ Department of Pharmacology, Vanderbilt University, Nashville, TN 37232, USA.
⁴⁹ Vanderbilt Kennedy Center of Human Development, Nashville, TN 37203, USA.
⁵⁰ Department of Regional Health Research, University of Southern Denmark, Odense 5000, Denmark.
⁵¹ Stanley Center of Psychiatric Research, Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA.
⁵² Department of Neurology, University of Texas Health Sciences Center at Houston, Houston, TX 77030, USA.

PMID: 37647852
PMCID: PMC10689929
DOI: 10.1093/brain/awad292

SLC6A1 variant pathogenicity, molecular function and phenotype: a genetic and clinical analysis

Arthur Stefanski et al. Brain. 2023.

. 2023 Dec 1;146(12):5198-5208.

doi: 10.1093/brain/awad292.

Authors

Affiliations

¹ Genomic Medicine Institute and Epilepsy Center, Cleveland Clinic, Cleveland, OH 44195, USA.
² Universidad del Desarrollo, Centro de Genética y Genómica, Facultad de Medicina Clínica Alemana, Santiago de Chile 7610658, Chile.
³ Cologne Center for Genomics (CCG), Medical Faculty of the University of Cologne, University Hospital of Cologne, Cologne 50931, Germany.
⁴ Department of Biological Sciences, Bridge Institute, USC Michelson Center for Convergent Bioscience, University of Southern California, Los Angeles, CA 90089, USA.
⁵ Institute of Human Genetics, University of Leipzig Medical Center, Leipzig 04103, Germany.
⁶ Department of Epilepsy Genetics and Personalized Medicine, The Danish Epilepsy Centre, Dianalund 4293, Denmark.
⁷ Department of Genetics, University Hospital of Copenhagen, Rigshispitalet, Copenhagen 2100, Denmark.
⁸ Children's Health, Medical Center, Dallas, TX 75235, USA.
⁹ Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
¹⁰ Clinical Genetics, Guys and St Thomas NHS Trust, London SE19RT, UK.
¹¹ Epilepsy Program, Neurology Department, Hospital Ruber Internacional, Madrid 28034, Spain.
¹² Research and Counseling Department, Research Centre for Medical Genetics, Moscow 115478, Russia.
¹³ Department of Neurology and Metabolic Medicine, Perth Children's Hospital, Perth 6009, Australia.
¹⁴ School of Paediatrics and Child Health, UWA Medical School, University of Western Australia, Perth 6009, Australia.
¹⁵ Department of Clinical Genetics, Erasmus MC, University Medical Center, Rotterdam 3015GD, The Netherlands.
¹⁶ Department of Pediatrics, Division of Medical Genetics and Genomics, Albany Medical College, Albany Med Health System, Albany, NY 12208, USA.
¹⁷ Department of Basic and Clinical Neurosciences, Institute of Psychiatry, Psychology and Neuroscience, King's College, London SE58AF, UK.
¹⁸ Department of Basic and Clinical Neurosciences, King's College Hospital, London SE59RS, UK.
¹⁹ Department of Pediatric Neurology, Amsterdam Public Health, Amsterdam University Medical Center, Amsterdam 1081HV, The Netherlands.
²⁰ Department of Pediatric Neurology, Stichting Epilepsie Instellingen Nederland (SEIN), Heemstede and Zwolle 2103SW, The Netherlands.
²¹ Division of Genetics and Genomic Medicine, Department of Pediatrics, St.Louis Children's Hospital, Washington University School of Medicine, St. Louis, MO 63110, USA.
²² Autism and Developmental Medicine Institute, Geisinger, Danville, PA 17837, USA.
²³ Department of Regional Genetics, Anaheim, Southern California Kaiser Permanente Medical Group, CA 92806, USA.
²⁴ Pediatric Neurology and Muscular Diseases Unit, IRCCS Istituto Giannina Gaslini, Genoa 16147, Italy.
²⁵ Department of Neurosciences, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health, University of Genoa, Genoa 16132, Italy.
²⁶ Department of Clinical Science, Kaiser Permanente Bernard J. Tyson School of Medicine, Pasadena, CA 91101, USA.
²⁷ Department of Paediatrics, Regional Hospital of Central Jutland, Viborg 8800, Denmark.
²⁸ Centre for Educational Development, Aarhus University, Aarhus 8200, Denmark.
²⁹ Department of Clinical and Molecular Genetics and Medicine Genetics Group, VHIR, University Hospital Vall d'Hebron, Barcelona 08035, Spain.
³⁰ Department of Neurology, Université catholique de Louvain, Cliniques universitaires Saint-Luc, Brussels 1200, Belgium.
³¹ Centre for Human Genetics, Institute for Pathology and Genetics, Gosselies 6041, Belgium.
³² Department of Pediatrics, Division of Medical Genetics, Western University, London, ON N6A3K7, Canada.
³³ Medical Genetics Program of Southwestern Ontario, London Health Sciences Centre and Children's Health Research Institute, London, ON N6A5A5, Canada.
³⁴ Department of Paediatrics, Division of Pediatric Neurology, London Health Sciences Centre, London, ON N6A5W9, Canada.
³⁵ GeneDx, Gaithersburg, MD 20877, USA.
³⁶ Department of Medical Genetics, UPMC Children's Hospital of Pittsburgh, Pittsburgh, PA 15224, USA.
³⁷ Medical Genetics Program of Southwestern Ontario, London Health Sciences Centre, London, ON N6A5W9, Canada.
³⁸ Department of Neurology, Royal Children's Hospital, Melbourne, VIC 3052, Australia.
³⁹ Department of Pediatrics, University of Melbourne, Melbourne, VIC 3052, Australia.
⁴⁰ Murdoch Children's Research Institute, Melbourne, VIC 3052, Australia.
⁴¹ Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Esch-sur-Alzette 4362, Luxembourg.
⁴² Translational Genomics, BioMarin Pharmaceutical Inc., Novato, CA 94949, USA.
⁴³ Center for Rare Diseases, University of Leipzig Medical Center, Leipzig 04103, Germany.
⁴⁴ SLC6A1 Connect, Frisco, TX 75034, USA.
⁴⁵ Department of Neurology, Vanderbilt University Medical Center, Nashville, TN 37240, USA.
⁴⁶ Neuroscience Graduate Program, Vanderbilt University, Nashville, TN 37235, USA.
⁴⁷ Department of Neurology, Vanderbilt Brain Institute, Nashville, TN 37235, USA.
⁴⁸ Department of Pharmacology, Vanderbilt University, Nashville, TN 37232, USA.
⁴⁹ Vanderbilt Kennedy Center of Human Development, Nashville, TN 37203, USA.
⁵⁰ Department of Regional Health Research, University of Southern Denmark, Odense 5000, Denmark.
⁵¹ Stanley Center of Psychiatric Research, Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA.
⁵² Department of Neurology, University of Texas Health Sciences Center at Houston, Houston, TX 77030, USA.

PMID: 37647852
PMCID: PMC10689929
DOI: 10.1093/brain/awad292

Abstract

Genetic variants in the SLC6A1 gene can cause a broad phenotypic disease spectrum by altering the protein function. Thus, systematically curated clinically relevant genotype-phenotype associations are needed to understand the disease mechanism and improve therapeutic decision-making. We aggregated genetic and clinical data from 172 individuals with likely pathogenic/pathogenic (lp/p) SLC6A1 variants and functional data for 184 variants (14.1% lp/p). Clinical and functional data were available for a subset of 126 individuals. We explored the potential associations of variant positions on the GAT1 3D structure with variant pathogenicity, altered molecular function and phenotype severity using bioinformatic approaches. The GAT1 transmembrane domains 1, 6 and extracellular loop 4 (EL4) were enriched for patient over population variants. Across functionally tested missense variants (n = 156), the spatial proximity from the ligand was associated with loss-of-function in the GAT1 transporter activity. For variants with complete loss of in vitro GABA uptake, we found a 4.6-fold enrichment in patients having severe disease versus non-severe disease (P = 2.9 × 10-3, 95% confidence interval: 1.5-15.3). In summary, we delineated associations between the 3D structure and variant pathogenicity, variant function and phenotype in SLC6A1-related disorders. This knowledge supports biology-informed variant interpretation and research on GAT1 function. All our data can be interactively explored in the SLC6A1 portal (https://slc6a1-portal.broadinstitute.org/).

Keywords: SLC6A1; autism; epilepsy; genetics; neurodevelopmental disorder.

PubMed Disclaimer

Conflict of interest statement

D.L. consults for Encoded Therapeutics Inc., San Francisco, USA. A.W. and S.F. are employees of BioMarin Pharmaceutical and hold common stock. M.T. is a former BioMarin employee. E.B. is an employee of GeneDx, LLC. All other authors report no competing interests.

Figures

**Figure 1**
SLC6A1 portal: user interface and functionality (https://slc6a1-portal.broadinstitute.org/). Four main menu items hold different functionalities of the SLC6A1 portal. Basic information: key milestones in *SLC6A1* research and summary statistics of the clinical information. Educational resources: resources, links to family advocacy groups and our in-house produced an animated whiteboard explainer video. Variant analysis: clinical significance according to ClinVar and comparative information on the selected variant with other similar variants. Research: visualizations of variant annotations, clinical phenotypes and functional data based on multiple filter options. Efforts to add more data to our online resource are motivated by an increased ability to understand the logic of structure to function to phenotype relations. Furthermore, easy access, the ability to explore the data and educational resources are additional features of our web portal. Given that very few clinicians and caregivers can collect data and perform bioinformatics analyses, the portal enables anyone with access to the internet to explore the data, understand and develop hypotheses.

**Figure 2**
**Domain-wide analysis of patient and population variants.** (A) The TM1/6, scaffold and EL4 regions harboured mainly variants with low activity, whereas the N-/C-terminal domain contained mostly variants with wild-type (WT) activity. The dotted line represents the cut-off that separates wild-type activity variants (>42.8% of wild-type activity) from low activity variants (<42.8% of wild-type activity). (B) A domain-wide comparison of patient versus population variants shows enrichment of patient variants in TM1/6 and EL4. The N- and C-terminal regions are depleted for patient variants. The dotted line represents a balance between patient and population variants. TM1/6 = transmembrane helix 1/6; TMD-other = transmembrane domain other; EL2/3/4 = extracellular loops 2/3/4; OR = odds ratio.

**Figure 3**
**The spatial relation of *SLC6A1* variants is associated with function and position within the GAT1 protein structure 3D structure (PDB id: 7SK2).** (A) GAT1 3D structure colour-coded in red regions with nearly complete loss-of-function (LoF) variants (<10% of normalized wild-type activity) and in yellow regions with wild-type activity variants (>42.8% of normalized wild-type activity). (B) Side view of the GAT1 3D structure. *SLC6A1* variants were categorized in the same three activity groups [0–10% (*left*), 10–42.8% (*middle*), > 42.8% (*right*)] and mapped onto the GAT1 3D structure. The variants with the lowest and medium average functional activity tend to be closer to the ligand, whereas variants with wild-type activity tend to face outwards. (C) Box plot showing the quantification of each variant’s distance (Å) from the ligand by the three activity groups. **Significant after Bonferroni multiple test correction; *Nominally significant; n.s. = not significant; WT = wild-type.

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References

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[1] Scimemi A. Structure, function, and plasticity of GABA transporters. Front Cell Neurosci. 2014;8:161. - PMC - PubMed

[2] Scimemi A. Structure, function, and plasticity of GABA transporters. Front Cell Neurosci. 2014;8:161. - PMC - PubMed

[3] Goodspeed K, Pérez-Palma E, Iqbal S, et al. . Current knowledge of SLC6A1-related neurodevelopmental disorders. Brain Commun. 2020;2:fcaa170. - PMC - PubMed

[4] Goodspeed K, Pérez-Palma E, Iqbal S, et al. . Current knowledge of SLC6A1-related neurodevelopmental disorders. Brain Commun. 2020;2:fcaa170. - PMC - PubMed

[5] Johannesen KM, Gardella E, Linnankivi T, et al. . Defining the phenotypic spectrum of SLC6A1 mutations. Epilepsia. 2018;59:389–402. - PMC - PubMed

[6] Johannesen KM, Gardella E, Linnankivi T, et al. . Defining the phenotypic spectrum of SLC6A1 mutations. Epilepsia. 2018;59:389–402. - PMC - PubMed

[7] Carvill GL, McMahon JM, Schneider A, et al. . Mutations in the GABA transporter SLC6A1 cause epilepsy with myoclonic-atonic seizures. Am J Hum Genet. 2015;96:808–815. - PMC - PubMed

[8] Carvill GL, McMahon JM, Schneider A, et al. . Mutations in the GABA transporter SLC6A1 cause epilepsy with myoclonic-atonic seizures. Am J Hum Genet. 2015;96:808–815. - PMC - PubMed

[9] Mermer F, Poliquin S, Rigsby K, et al. . Common molecular mechanisms of SLC6A1 variant-mediated neurodevelopmental disorders in astrocytes and neurons. Brain. 2021;144:2499–2512. - PMC - PubMed

[10] Mermer F, Poliquin S, Rigsby K, et al. . Common molecular mechanisms of SLC6A1 variant-mediated neurodevelopmental disorders in astrocytes and neurons. Brain. 2021;144:2499–2512. - PMC - PubMed

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SLC6A1 variant pathogenicity, molecular function and phenotype: a genetic and clinical analysis

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SLC6A1 variant pathogenicity, molecular function and phenotype: a genetic and clinical analysis

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Conflict of interest statement

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