GABRG2 Variants Associated with Febrile Seizures

Abstract

Febrile seizures (FS) are the most common form of epilepsy in children between six months and five years of age. FS is a self-limited type of fever-related seizure. However, complicated prolonged FS can lead to complex partial epilepsy. We found that among the GABA_A receptor subunit (GABR) genes, most variants associated with FS are harbored in the γ2 subunit (GABRG2). Here, we characterized the effects of eight variants in the GABA_A receptor γ2 subunit on receptor biogenesis and channel function. Two-thirds of the GABRG2 variants followed the expected autosomal dominant inheritance in FS and occurred as missense and nonsense variants. The remaining one-third appeared as de novo in the affected probands and occurred only as missense variants. The loss of GABA_A receptor function and dominant negative effect on GABA_A receptor biogenesis likely caused the FS phenotype. In general, variants in the GABRG2 result in a broad spectrum of phenotypic severity, ranging from asymptomatic, FS, genetic epilepsy with febrile seizures plus (GEFS+), and Dravet syndrome individuals. The data presented here support the link between FS, epilepsy, and GABRG2 variants, shedding light on the relationship between the variant topological occurrence and disease severity.

Keywords: GABAARs; febrile seizures; mutations.

Figure 1

Structural mapping of GABRG2 variants (A) Cryo-EM (PDB#: 6HUO) structure of the GABA_A receptor with the β3 subunits in blue, α1 subunits in red, and the γ2 subunit in yellow. γ2 variants were mapped onto the structure and represented as orange space-filled atoms. Binding sites for GABA, alprazolam (ALP), N-linked glycans (NAG), and PIP₂ are also shown in CPK space-filling atoms. (B,C) Top-side and bottom-side views of the extracellular (ECD in (B)) and transmembrane (TMD in (C)) domains of the 6HUO structure represented by ribbons with GABA, ALP, and NAG shown as sticks, and γ2 variants as space-filled representation. Subunit coloring is the same as in (A). Sequence alignments of human γ3, γ2 and γ1 GABA_AR subunits show the position of the variants in the γ2 subunit highlighted in yellow (black box). Residues in the alignments are colored according to their chemical properties. Structural domains related to the secondary structure are represented across subunits above the alignments as β-strands (β5, β5′, β8), loops (8 and 9), and transmembrane domains (M2, M3 and M4). Conserved sites (*) and sites with conservative replacements (:) are indicated below each position of the sequence alignment.

Figure 2

Mutant γ2 subunits induced structural rearrangements in structural domains important for GABA_A receptor gating. (A) GABA_AR γ2 tolerance landscape schematic representation of γ2 protein domains that did not tolerate missense variants. As shown, non-tolerated highly pathogenic variations shift the color gradient towards red, whereas tolerated variations with low pathogenicity shift the color gradient towards blue. Elements of the secondary structure of the GABA_AR γ2 subunit are shown in yellow (β-strands) and red (α-helices) rectangles. Bracket symbols delimit the extracellular (ECD), transmembrane (TDM), and cytoplasmic domains (CD). All GABA_AR γ2 subunit variants considered in this study were mapped to intolerant landscape regions. (B) Global structural perturbations shown on the Cryo-EM 6HUO structure after introduction of single point mutations in the γ2 subunit at positions 215, 325, 468, 464, and 457. The structural rearrangements in the secondary structure and side chain residues that differ among the WT (in gray) and the mutant simulation (RMS deviation ≥ 0.5 Å) are indicated in a different color from the WT simulation. (C) Enlarged views of structural domains showing structural rearrangements produced by the γ2R215H, γ2S325L, γ2Y468C, γ2F464S, and γ2F457S mutant subunits. Structural perturbations with RMS deviation ≥0.5 Å are shown and the conformational impact of the γ2 mutations to the α1β3γ2 GABA_AR are indicated. WT, wild type; RMS, root mean square.

Figure 3

Mutant γ2 subunits altered the kinetic properties of GABA_A receptors. (A) Close-up views of neighboring residues that were within a 6 Å radius at the site of the mutations corresponding to R215H, S325L, and Y468C in the γ2 subunit structures. The hydrogen bond network of the WT (left panels) and mutant structures are shown (right panels). Residues corresponding to the location of the substitution are colored orange, and the neighboring residues are colored as follows: carbon, white; oxygen, red; nitrogen, blue. Dashed lines indicate hydrogen bonds. (B,C) Superimposed representative current traces show deactivation ((B), top panel) and activation ((C), top panel) produced by 5 ms of GABA (1 mM) applications to WT (gray) and mutant receptors containing the γ2R215H (orange), γ2S325S (blue), and γ2Y468C (green) subunits. Traces were normalized to WT currents for clarity. Dashed lines represent zero currents. Bar graphs show average deactivation time constant ((B), bottom panel), and rise time ((C), bottom panel) from cells co-expressing α1β3 subunits with mutant or WT γ2 subunits. Values are expressed as mean ± S.D (see Table 2). Data points (circles) represent the number of patched wells per experimental condition acquired in three independent experiments. One-way ANOVA with Dunnett’s post-test were used to determine significance. **** p < 0.0001, *** p < 0.001, and * p < 0.05, respectively, relative to WT condition.

Figure 4

Mutant γ2 subunits decreased GABA-evoked whole-cell currents. (A) Representative GABA current traces obtained following rapid application of 1 mM of GABA for 5 ms (arrow). The current traces from GABA_ARs containing mutant (in orange red, blue, and green) γ2 subunits were compared to WT (black) current traces. Dashed lines represent zero currents. Activation of GABA_A (α1β3γ2) receptors was measured on the SyncroPatch 384PE, and the whole-cell patch methodology and multi-hole NPC chips were used as described in Section 2. (B) Bar graphs show the average maximal GABA-evoked peak currents (I_MAX) for 1 mM of GABA in HEK293T cells co-expressing α1β3 subunits with wild-type (WT) or mutant γ2 subunits. γ2 variant subunit coloring is the same as in (A). Numerical data are reported as mean ± S.E.M. Data points represent the number of patched wells per experimental condition acquired in three independent experiments. One-way ANOVA with Dunnett’s post-test was used to determine significance. **** p < 0.0001, *** p < 0.001, and ** p < 0.01, respectively, relative to WT condition. (C) I_MAX variant over I_MAX WT ratios were calculated as estimates of the magnitude of the change in receptor activation by GABA. Panels represent the structural domain location of the γ2 variants (ECD, in gray; TMD, in blue; CD, in orange). Numerical data are reported as mean ± S.D. Some error bars cannot be displayed as they are smaller than the symbol size. A descriptive summary of the data is shown in Table 3.

Figure 5

Mutant γ2 subunits altered the potency of GABA_A receptors. (A) Activation concentration–response curves of GABA_A (α1β3γ2) receptors expressed in HEK293T cells on the SyncroPatch 384PE by GABA. A single concentration of GABA was applied to each well. Currents from each well were normalized to the maximal response within the well in the presence of 1 mM of GABA. The data were fitted using a three-parameters sigmoid model, as described in Section 2. The black line represents the fit for the WT condition. The colored lines represent the fit of each experimental condition that corresponds to the data in panel B. Numerical data are reported as mean ± S.E.M. Data points represent the number of successfully patched wells per experimental condition from three independent experiments. (B) pEC₅₀ (negative Log of the EC₅₀) values between WT and mutant γ2 subunits are plotted as the relative change in potency caused by the mutation. Numerical data are reported as mean ± S.D. A summary of the data is shown in Table 4.

Figure 6

Mutant γ2 subunits changed the composition of GABA_A receptors. (A) Wild-type or mutant γ2 subunits were co-transfected with α1β3 in HEK293T cells. Surface proteins were biotinylated and pulled down with streptavidin beads. Pulled-down proteins were separated by SDS-PAGE and probed with anti-α1, anti-β3, anti-γ2, or anti-ATPase antibodies. Total cell lysates (pull-down input) were collected, analyzed by SDS-PAGE, and equally blotted by anti-α1, anti-β3, anti-γ2, and anti-ATPase antibodies. Surface and total expression of GABA_ARs containing mutant γ2 subunits were normalized to WT α1β3γ2 subunits. Numerical data are reported as mean ± S.E.M. A summary of the data is shown in Table 5. (B) Surface/total expression ratios were determined as the fraction of surface to total altered GABA_AR subunit, as an indicator of the magnitude of the negative or positive effect of the presence of mutant γ2 subunits. Numerical data are reported as mean ± S.E.M. Some error bars cannot be displayed as they are smaller than the symbol size. The data were summarized in Table 6.

Figure 7

GABA_A receptor impairment profile caused by mutant γ2 subunits. Spider plots of the GABA_AR expression and function compare the impairment magnitude caused by mutant γ2 subunits. (A) The EC₅₀ and I_MAX of GABA_AR containing mutant γ2 subunits were normalized against WT. The data are related to the assays shown in Figure 4 and Figure 5. (B) Surface and total expression of GABA_AR co-expressing WT α1, β3, γ2, and mutant γ2 subunits were normalized against WT. These data are related to the assays shown in Figure 6. Values other than 1 indicate a loss (<1) or gain (>1) effect.

Figure 8

GABRG2 variants contributed to the pathogenesis of FS. (A) Comparison of the phenotypic profile of thirty GABRG2 variants reported in this and previous studies (Table 7). GABRG2 variants are grouped by structural domain location (ECD, extracellular domain; TMD, transmembrane domain; and CD, cytoplasmic domain), epilepsy phenotype (FS, febrile seizure; DEE, developmental epileptic encephalopathy; CAE, childhood absence epilepsy; GEFS+, genetic epilepsy with febrile seizures plus; GTCS, generalized tonic-clonic seizure; and MAE, myoclonic-astatic epilepsy), and pathogenicity (LP/P, likely pathogenic/pathogenic; and LB/B, likely benign/benign). The GABRG2 variants’ distribution in each category was assigned a number ranging from 1 to 28. Note that a specific variant may be represented in more than one category. (B) Occurrence of thirty GABRG2 variants by structural location (ECD, TMD, and CD) and epileptic phenotype. The number inside the bars corresponds to the variants included in that category. Variants that cause the most complex epileptic phenotypes occur in more than one GABA_AR structural domain. (C) Side view of the extracellular and transmembrane domains of two opposing γ2 subunits represented as rainbow ribbons. γ2 subunit variants are displayed in dark (the present study) and light gray (previously reported). In addition, key structural domains are indicated. (D,E) Doughnut plots classify GABRG2 variants by pathogenicity (LP/P, n = 28, and LB/B, n = 2) and structural location (NT, n = 13, and TM, n = 15, LP/P only), respectively. The plots show most variants are pathogenic, localizing preferentially to two key receptor activation domains. (F) Spider diagram comparing the pathogenicity and type of epilepsy profile of GABRG2 variants. The features of the thirty GABRG2 variants are summarized in Table 7.