Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Aug:342:113723.
doi: 10.1016/j.expneurol.2021.113723. Epub 2021 May 5.

Genetic mosaicism, intrafamilial phenotypic heterogeneity, and molecular defects of a novel missense SLC6A1 mutation associated with epilepsy and ADHD

Sarah Poliquin  1 , Inna Hughes  2 , Wangzhen Shen  3 , Felicia Mermer  3 , Juexin Wang  4 , Taralynn Mack  1 , Dong Xu  4 , Jing-Qiong Kang  5
Affiliations

Genetic mosaicism, intrafamilial phenotypic heterogeneity, and molecular defects of a novel missense SLC6A1 mutation associated with epilepsy and ADHD

Sarah Poliquin et al. Exp Neurol. 2021 Aug.

Abstract

Background: Mutations in SLC6A1, encoding γ-aminobutyric acid (GABA) transporter 1 (GAT-1), have been recently associated with a spectrum of neurodevelopmental disorders ranging from variable epilepsy syndromes, intellectual disability (ID), autism and others. To date, most identified mutations are de novo. We here report a pedigree of two siblings associated with myoclonic astatic epilepsy, attention deficit hyperactivity disorder (ADHD), and ID.

Methods: Next-generation sequencing identified a missense mutation in the SLC6A1 gene (c.373G > A(p.Val125Met)) in the sisters but not in their shared mother who is also asymptomatic, suggesting gonadal mosaicism. We have thoroughly characterized the clinical phenotypes: EEG recordings identified features for absence seizures and prominent bursts of occipital intermittent rhythmic delta activity (OIRDA). The molecular pathophysiology underlying the clinical phenotypes was assessed using a multidisciplinary approach including machine learning, confocal microscopy, and high-throughput 3H radio-labeled GABA uptake assays in mouse astrocytes and neurons.

Results: The GAT-1(Val125Met) mutation destabilizes the global protein conformation and reduces transporter protein expression at total and cell surface. The mutant transporter protein was localized intracellularly inside the endoplasmic reticulum (ER) in both HEK293T cells and astrocytes which may directly contribute to seizures in patients. Radioactive 3H-labeled GABA uptake assay indicated the mutation reduced the function of the mutant GAT-1(Val125Met) to ~30% of the wildtype.

Conclusions: The seizure phenotypes, ADHD, and impaired cognition are likely caused by a partial loss-of-function of GAT-1 due to protein destabilization resulting from the mutation. Reduced GAT-1 function in astrocytes and neurons may consequently alter brain network activities such as increased seizures and reduced attention.

Keywords: ADHD; GABA transporter 1; Mosaicism; Myoclonic atonic epilepsy (MAE); Phenotypic heterogeneity; Protein stability.

PubMed Disclaimer

Conflict of interest statement

Competing interests

The authors declare that they are no competing interests.

Figures

Figure 1.
Figure 1.. A novel SLC6A1 missense mutation GAT-1(Val125Met) in two sisters associated with myoclonic astatic epilepsy.
(A). Schematic representation of GAT-1 protein topology and locations of GAT-1 variants previously identified in patients associated with a spectrum of epilepsy syndromes. It is predicted that GAT-1 contains 12 transmembrane domains. Val125Met (V125M) is located at the 3rd transmembrane helix of the GAT-1 protein. The positions of variants are based on the published LeuT crystal structure. (B) Pedigree and the genotype. A missense mutation was found in the proband and the half-sister but not in the rest of the family members. (C). Amino acid sequence homology shows that valine (V) at residue 125 is highly conserved in SCL6A1 in humans (Accession NO.NP_003033.3) and across species as shown in boxed region.
Figure 2.
Figure 2.. Electroencephalogram (EEG) of two sisters carrying the GAT-1(Val125Met) encoding mutation showing high amplitude burst of 3 Hz spike wave discharges and prominent occipital intermittent rhythmic delta activity (OIRDA).
(A, B) Prominent bursts of Occipital Intermittent Rhythmic Delta Activity in the Proband (A, age 8) and Patient 2 (B, age 4) occurred throughout the waking state. (C, D). Video EEG recordings showed sudden burst of generalized high amplitude bursts of 3.0 Hz spike and slow waves in proband (C) and the half-sister (D). (E, F), Typical absence for proband was associated with a myoclonic jerk and loss of tone—noted by muscle artifact (red arrows), sensitivity 15 microvolts (E) and a typical abnormal spike wave discharges for patient 2 (D) arising out of sleep, which was asymptomatic. Sensitivity 15 microvolts.
Figure 3.
Figure 3.. Modeling of the mutant GAT-1(Val125Met) protein with machine learning tools.
(A-B). Tertiary structures of both the wildtype (A) and Val125Met mutant (B) GAT-1 protein are predicted by I-TASSER and DynaMut. The valine at residue 125 is mutated to methionine, both highlighted in light green, alongside with the surrounding residues. The interatomic interactions were predicted by DynaMut, where halogen bonds are depicted in blue and hydrogen bonds are colored in red. The Val125Met mutation results in the addition of sulfur into the sidechain, not causing drastic changes in polarity and charge, but making the protein less hydrophobic. (C). Machine learning tools predicted ΔΔG (Kcal/mol) of the mutant GAT-1(Val125Met) protein. Bars in the positive direction are predicted as stabilizing while bars in the negative direction are predicted as destabilizing.
Figure 4.
Figure 4.. The GABA reuptake function of the mutant GAT-1(Val125Met) transporter was reduced in non-neuronal cells and neurons.
A-B. HEK293T cells or mouse cortical astrocytes were transfected with wildtype GAT-1YFP (wt), or the mutant GAT-1(Val125Met, V125M)YFP cDNAs (1μg per a 35mm2 dish) for 48 hrs before 3H radioactive GABA uptake assay. C. Mouse cortical neurons were transfected with the wildtype or the mutant GAT-1(Val125Met) cDNAs at day 7 in culture. 3H radioactive GABA uptake assay was performed after 8 days of transfection. GABA flux was measured after 30 min transport at room temperature. The influx of GABA, expressed in pmol/μg protein/min, was averaged from duplicates for each condition and for each transfection. The average counting was taken as n = 1. The untransfected condition was taken as baseline flux, which was subtracted from both the wildtype and the mutant conditions in HEK293T cells (A). The pmol/μg protein/min in the mutant was then normalized to the wildtype from each experiment, which was arbitrarily set as 100%. (***p < 0.01 vs. wt, n=4-7 different transfections). Cl-966 (100μm) was applied 30 min before preincubation and removed during preincubation. (One-sample t test. Values were expressed as mean ± S.E.M).
Figure 5.
Figure 5.. Both the total and cell surface expression of the mutant GAT-1(Val125Met) transporter protein was reduced.
A, B. HEK293T cells were transfected with wildtype GAT-1YFP (wt), or the mutant GAT-1(Val125Met, V125M)YFP cDNAs (3μg/60 mm2) for 48 hrs. The cells (A) were either harvested directly after wash with PBS for total lysates or followed by cell surface biotinylation to isolate the cell surface bound proteins (B). The total lysates (A) or isolated surface protein (B) were then analyzed by SDS-PAGE. Membranes were immunoblotted with rabbit anti-GAT-1 (1:300). (C). The total protein integrated density values (IDVs) from the total lysates (C) or isolated cell surface protein (D) were measured. The abundance of the mutant GAT-1(Val125Met) transporter was normalized to the wildtype cells expressing GAT-1YFP. In C, the total protein abundance was measured by adding up all the bands between 90-110 KDa. The total protein IDVs of either the wildtype or the mutant was normalized to its loading control. The abundance of the mutant transporter was then normalized to the wildtype. (**p < 0.01; ***p < 0.001 vs. wt, n=4 different transfections, One-sample t test, Values were expressed as mean ± S.E.M).
Figure 6.
Figure 6.. The mutant GAT-1(V125M) transporters were retained inside the endoplasmic reticulum in HEK 293T cells
(A) HEK293T cells were transfected with wildtype GAT-1YFP or the mutant GAT-1(Val125Met, V125M)YFP with the pECFP-ER marker (ERCFP) at 2:1 ratio (2 μg:1μg cDNAs) for 48 hrs. Live cells were examined under a confocal microscopy with excitation at 458 nm for CFP, 514 nm for YFP. All images were single confocal sections averaged from 8 times to reduce noise, except when otherwise specified. (B) The GAT-1YFP fluorescence overlapping with ERCFP fluorescence was quantified by Metamorph with colocalization percentage. Tu stands for Tunicamycin (10 μg/ml) treated for 16 hrs. (***p < 0.001 V125M vs. wt, V125M §§ vs wt+Tun. N=7-8 representative fields from 4 different transfections. One-way analysis of variance (ANOVA) and Newman-Keuls test was used to determine significance compared to the wt condition and between mutation and wt+Tu. Values were expressed as mean ± S.E.M).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Roth FC, Draguhn A. GABA metabolism and transport: effects on synaptic efficacy. Neural Plast 2012;2012:805830. - PMC - PubMed
    1. Carvill GL, McMahon JM, Schneider A, Zemel M, Myers CT, Saykally J, Nguyen J, Robbiano A, Zara F, Specchio N, Mecarelli O, Smith RL, Leventer RJ, Moller RS, Nikanorova M, Dimova P, Jordanova A, Petrou S, Helbig I, Striano P, Weckhuysen S, Berkovic SF, Scheffer IE, Mefford HC. Mutations in the GABA Transporter SLC6A1 Cause Epilepsy with Myoclonic-Atonic Seizures. Am J Hum Genet 2015. May 7;96(5):808–15. - PMC - PubMed
    1. Johannesen KM, Gardella E, Linnankivi T, Courage C, de Saint MA, Lehesjoki AE, Mignot C, Afenjar A, Lesca G, Abi-Warde MT, Chelly J, Piton A, Merritt JL, Rodan LH, Tan WH, Bird LM, Nespeca M, Gleeson JG, Yoo Y, Choi M, Chae JH, Czapansky-Beilman D, Reichert SC, Pendziwiat M, Verhoeven JS, Schelhaas HJ, Devinsky O, Christensen J, Specchio N, Trivisano M, Weber YG, Nava C, Keren B, Doummar D, Schaefer E, Hopkins S, Dubbs H, Shaw JE, Pisani L, Myers CT, Tang S, Tang S, Pal DK, Millichap JJ, Carvill GL, Helbig KL, Mecarelli O, Striano P, Helbig I, Rubboli G, Mefford HC, Moller RS. Defining the phenotypic spectrum of SLC6A1 mutations. Epilepsia 2018. Feb;59(2):389–402. - PMC - PubMed
    1. Mattison KA, Butler KM, Inglis GAS, Dayan O, Boussidan H, Bhambhani V, Philbrook B, da SC, Alexander JJ, Kanner BI, Escayg A. SLC6A1 variants identified in epilepsy patients reduce gamma-aminobutyric acid transport. Epilepsia 2018. Sep;59(9):e135–e141. - PubMed
    1. Cai K, Wang J, Eissman J, Wang J, Nwosu G, Shen W, Liang HC, Li XJ, Zhu HX, Yi YH, Song J, Xu D, Delpire E, Liao WP, Shi YW, Kang JQ. A missense mutation in SLC6A1 associated with Lennox-Gastaut syndrome impairs GABA transporter 1 protein trafficking and function. Exp Neurol 2019. Jun 6;320:112973. - PMC - PubMed

Publication types

MeSH terms

Substances