Haploinsufficiency underlies the neurodevelopmental consequences of SLC6A1 variants

Abstract

Heterozygous variants in SLC6A1, encoding the GAT-1 GABA transporter, are associated with seizures, developmental delay, and autism. The majority of affected individuals carry missense variants, many of which are recurrent germline de novo mutations, raising the possibility of gain-of-function or dominant-negative effects. To understand the functional consequences, we performed an in vitro GABA uptake assay for 213 unique variants, including 24 control variants. De novo variants consistently resulted in a decrease in GABA uptake, in keeping with haploinsufficiency underlying all neurodevelopmental phenotypes. Where present, ClinVar pathogenicity reports correlated well with GABA uptake data; the functional data can inform future reports for the remaining 72% of unscored variants. Surface localization was assessed for 86 variants; two-thirds of loss-of-function missense variants prevented GAT-1 from being present on the membrane while GAT-1 was on the surface but with reduced activity for the remaining third. Surprisingly, recurrent de novo missense variants showed moderate loss-of-function effects that reduced GABA uptake with no evidence for dominant-negative or gain-of-function effects. Using linear regression across multiple missense severity scores to extrapolate the functional data to all potential SLC6A1 missense variants, we observe an abundance of GAT-1 residues that are sensitive to substitution. The extent of this missense vulnerability accounts for the clinically observed missense enrichment; overlap with hypermutable CpG sites accounts for the recurrent missense variants. Strategies to increase the expression of the wild-type SLC6A1 allele are likely to be beneficial across neurodevelopmental disorders, though the developmental stage and extent of required rescue remain unknown.

Keywords: GABA uptake; GAT-1; GAT1; SLC6A1; autism spectrum disorders; epilepsy with myoclonic-atonic seizures; missense vulnerability; neurodevelopmental delay.

Graphical abstract

Figure 1

Topology of GAT-1 and GABA uptake values by functional type (A) 2D representation of GAT-1 organized by 12 TM domains and linking or terminal chains. Only missense and in-frame variants are highlighted. (B) GABA uptake functional data as a percentage of WT and highlighted by activity type for 213 variants tested across the 599 amino acids of GAT-1. Individual data bars represent mean ± SEM of three biological replicates performed in triplicates. GoF, gain-of-function; LoF, loss-of-function; WT, wildtype.

Figure 2

GABA uptake of variants by phenotype and recurrence (A) GABA uptake data for 213 variants by predicted impact on GAT-1. PTVs and missense/in-frame variants are ascertained from clinical populations (left two categories) while 24 variants selected as controls were split between missense/in-frame and synonymous variants (right two categories) and used to set thresholds for loss of function and gain of function (dashed lines). (B and C) GABA uptake values by population allele count in 114,704 non-neuropsychiatric samples in gnomAD and by ClinVar clinical significance. (D) A Sankey plot summarizing the proportion of variants where ClinVar category could be reclassified. (E) GABA uptake values for de novo variants by the presence of seizures, DD, autism spectrum disorder (ASD), or schizophrenia. If phenotypes are comorbid (e.g., seizures and DD) the variant is shown for all phenotypes for which it is reported. All variants are shown in Figure S8B. (F) GABA uptake values of de novo variants observed in single individuals (1) or multiple individuals (2–11). Each variant is shown once. The four most recurrent variants (5, 6, 10, and 11) are labeled. Abbreviations: GoF, gain-of-function; LoF, loss-of-function; A93T, p.Ala93Thr; R211C, p.Arg211Cys; W495L, p.Trp495Leu. Statistical tests: E, two-sided Wilcoxon test. Colors: red, severe LoF; orange, LoF; blue, typical activity; pink, GoF. Boxplot whiskers are based on maximum and mimimum values within 1.5 times the interquartile range.

Figure 3

Clustering of surface localization and GABA uptake results of 86 missense variants and highlighted variants in GAT-1 structure and binding site (A) Individual GABA uptake values (y axis) correlated to surface localization results (x axis) and classified by K-means clustering as either present at the surface and low uptake (magenta), absent at the surface and low uptake (purple), or present at the surface and typical uptake (green). Variants with names in blue denote outliers. All other called variants are further illustrated in (B) and (E). (B) GAT-1 3D structure in its inward-open conformation with highlighted variants by cluster. (C) GAT-1 3D structure repeated and highlighted by TM domains. The red square indicates the binding site. (D) 2D topology of GAT-1 structure showing individual variants colored by cluster and corresponding TM domains highlighted from (C). (E) The GAT-1 binding site inset from (C) (red square) and relevant amino acids is shown with GABA bound. The WT and variant 3D structures of four variants are shown, and the variant insets are boxed in colors relevant to their clustering from (A) (magenta or purple). Two variants form part of the binding site for GABA (p.Gly63Ser and p.Ser295Leu), one variant is part of TM6 (p.Gly307Arg), and the variant in TM11 is the top recurrent variant (p.Val511Met, n = 6) with available surface localization data. Abbreviations: GoF, gain-of-function; LoF, loss-of-function; TM1, TM domain 1; TM3, TM domain 3; TM6, TM domain 6; TM8, TM domain 8; TM11, TM domain 11.

Figure 4

GAT-1 vulnerability underlies the enrichment of SLC6A1 missense variants (A) Correlation of individual GABA uptake data and ClinPred rankscore for 180 SCL6A1/GAT-1 missense variants. The red line shows the linear regression model with 95% confidence intervals represented by the red shading. The ClinPred value at the estimated loss-of-function threshold is indicated by the purple line (“LoF thres”). (B) Violin plots represent the ClinPred rankscore distribution for all possible missense variants in SLC6A1 and nine equivalent PTV-enriched ASD- and NDD-associated genes. The LoF thres line from (A) is also shown. (C) All 599 amino acids of GAT-1 are colored by the mean predicted GABA uptake of all possible missense variants using the linear regression model from (A) from the annotated ClinPred rankscores. Abbreviations: GoF, gain-of-function; LoF, loss-of-function. Statistical tests: A, linear regression; B, two-sided Wilcoxon test.

Figure 5

Recurrent missense variants in SLC6A1 are at hypermutable loci Estimated mutability based on three base-pair DNA sequences is shown for all 3,952 possible missense variants in SLC6A1 (x axis). A small number of variants have a ∼10-fold higher mutation rate due to the presence of CpG, as shown by the histogram (top). Predicted GABA uptake is predicted for each of these missense variants based on ClinPred (Figure 4). The number of unique individuals is indicated by size and color for each variant; the four most frequent are labeled (Figure 2; Table 1). LoF, loss-of-function.