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Case Reports
. 2023 Jun;10(6):1046-1053.
doi: 10.1002/acn3.51786. Epub 2023 May 16.

Dominant-negative variant in SLC1A4 causes an autosomal dominant epilepsy syndrome

Jonai Pujol-Giménez  1   2 , Ghayda Mirzaa  3   4   5 , Elizabeth E Blue  5   6   7 , Giuseppe Albano  1   2 , Danny E Miller  4   5   8 , Aimee Allworth  6 , James T Bennett  3   4   5   9 , Peter H Byers  6   8 , Sirisak Chanprasert  6 , Jingheng Chen  7 , Daniel Doherty  4   5 , Andrew B Folta  6 , Madelyn A Gillentine  10 , Ian Glass  4   5 , Anne Hing  4 , Martha Horike-Pyne  6 , Kathleen A Leppig  11 , Azma Parhin  6 , Jane Ranchalis  6 , Wendy H Raskind  6 , Elisabeth A Rosenthal  6 , Ulrike Schwarze  8 , Sam Sheppeard  6 , Samuel Strohbehn  6 , Virginia P Sybert  6 , Andrew Timms  9 , Mark Wener  8 ; University of Washington Center for Mendelian Genomics (UW-CMG)a, Undiagnosed Diseases Network (UDN); Michael J Bamshad  4   5 , Fuki M Hisama  5   6 , Gail P Jarvik  5   6   12 , Katrina M Dipple  4   5 , Matthias A Hediger  1   2 , Andrew B Stergachis  5   6   12
Collaborators, Affiliations
Case Reports

Dominant-negative variant in SLC1A4 causes an autosomal dominant epilepsy syndrome

Jonai Pujol-Giménez et al. Ann Clin Transl Neurol. 2023 Jun.

Abstract

SLC1A4 is a trimeric neutral amino acid transporter essential for shuttling L-serine from astrocytes into neurons. Individuals with biallelic variants in SLC1A4 are known to have spastic tetraplegia, thin corpus callosum, and progressive microcephaly (SPATCCM) syndrome, but individuals with heterozygous variants are not thought to have disease. We identify an 8-year-old patient with global developmental delay, spasticity, epilepsy, and microcephaly who has a de novo heterozygous three amino acid duplication in SLC1A4 (L86_M88dup). We demonstrate that L86_M88dup causes a dominant-negative N-glycosylation defect of SLC1A4, which in turn reduces the plasma membrane localization of SLC1A4 and the transport rate of SLC1A4 for L-serine.

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Conflict of interest statement

DEM is on a scientific advisory board at Oxford Nanopore Technologies (ONT). DEM is engaged in a research agreement with ONT and they have paid for him to travel to speak on their behalf.

Figures

Figure 1
Figure 1
Structural relationship between pathogenic variants within SLC1A4. Upper and side view of the cryo‐EM structure of human SLC1A4. Transport and scaffold domains for each subunit of the trimer are highlighted as indicated. Previously reported disease‐linked SLC1A4 variants (E256, R457, G374, and G381) are colored (pink, green, and yellow). Residues duplicated in the mutation under study L86, R87, and M88 are colored in red.
Figure 2
Figure 2
SLC1A4L86_M88dup has a dominant‐negative impact on SLC1A4 function. (A) Uptake of 25 μmol/L [3H]‐L‐serine (left) and 25 μμmol/L [3H]‐L‐alanine (right) by HEK293T cells transfected with the indicated constructs. (B, C) [3H]‐L‐serine kinetics by HEK293T cells transfected with (B) SLC1A4WT (WT) or SLC1A4L86_M88dup (L86_M88dup), (C) or co‐transfected with both empty vector and SLC1A4WT (EV + WT), or SLC1A4WT and SLC1A4L86_M88dup (WT + L86_M88dup). (below) Kinetic parameters obtained by fitting the results to the Hill equation. Data represented as mean ± SD and obtained from two independent experiments with 2–16 technical replicates each. *P < 0.001.
Figure 3
Figure 3
SLC1A4L86_M88dup has a dominant‐negative impact on SLC1A4 N‐glycosylation. (A) Immunoblots showing the localization of overexpressed HA‐tagged SLC1A4WT (WT) or SLC1A4L86_M88dup (dLRM) within the plasma membrane and non‐membrane fractions. (below) Actin control. (right) Quantification of the optical density (O.D.) for the ~60 and ~80 kDa bands in the membrane and non‐membrane fractions (mean ± SD). ns, non‐significant P > 0.05; *P < 0.01. (B) Immunoblot showing the molecular weight of overexpressed HA‐tagged SLC1A4WT (WT) in untreated cell lysates, as well as cell lysates treated with the N‐glycosidase PNGase F prior to the immunoblotting. (C) Same as (A), but using cells co‐transfected with both empty vector and SLC1A4WT (EV + WT), or SLC1A4WT and SLC1A4L86_M88dup (WT + L86_M88dup). (D) Cryo‐EM structure of human SLC1A4 with residues Leu88, Arg87, and Met88 colored in red, blue, and white respectively. Putative N‐linked glycosylation sites, located in an extracellular loop present at the end of TM4 are colored in yellow.
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