GABRG2 mutations in genetic epilepsy with febrile seizures plus: structure, roles, and molecular genetics

Abstract

Genetic epilepsy with febrile seizures plus (GEFS+) is a genetic epilepsy syndrome characterized by a marked hereditary tendency inherited as an autosomal dominant trait. Patients with GEFS+ may develop typical febrile seizures (FS), while generalized tonic–clonic seizures (GTCSs) with fever commonly occur between 3 months and 6 years of age, which is generally followed by febrile seizure plus (FS+), with or without absence seizures, focal seizures, or GTCSs. GEFS+ exhibits significant genetic heterogeneity, with polymerase chain reaction, exon sequencing, and single nucleotide polymorphism analyses all showing that the occurrence of GEFS+ is mainly related to mutations in the gamma-aminobutyric acid type A receptor gamma 2 subunit (GABRG2) gene. The most common mutations in GABRG2 are separated in large autosomal dominant families, but their pathogenesis remains unclear. The predominant types of GABRG2 mutations include missense (c.983A → T, c.245G → A, p.Met199Val), nonsense (R136*, Q390*, W429*), frameshift (c.1329delC, p.Val462fs*33, p.Pro59fs*12), point (P83S), and splice site (IVS6+2T → G) mutations. All of these mutations types can reduce the function of ion channels on the cell membrane; however, the degree and mechanism underlying these dysfunctions are different and could be linked to the main mechanism of epilepsy. The γ2 subunit plays a special role in receptor trafficking and is closely related to its structural specificity. This review focused on investigating the relationship between GEFS+ and GABRG2 mutation types in recent years, discussing novel aspects deemed to be great significance for clinically accurate diagnosis, anti-epileptic treatment strategies, and new drug development.

Introduction

Genetic epilepsy with febrile seizures plus (GEFS+) is a common epileptic syndrome in children, which shows an autosomal dominant inheritance pattern and does not affect patient development. Scheffer et al. was the first to observed febrile seizures (FS, defined as convulsions and loss of consciousness within 24 h of the onset of febrile symptoms) which were either associated with fever in one family, among whom the majority of patients showed seizure termination in the middle of childhood (average age, 11 years). Members of this family were diagnosed with generalized epilepsy with febrile seizure plus [1]. It was subsequently suggested that generalized epilepsy with febrile seizure plus be renamed GEFS+, and further research on the electroclinical characteristics and genetics of GEFS+ family members was conducted [2]. The change in the the syndrome name was brought on by the recognition that many patients experience focal seizures, making the term “generalized epilepsy” inadequate. In 2001, the International League Against Epilepsy (ILAE) included GEFS+ as a new syndrome in the classification of epilepsy syndromes [3]. Following further research, the concept of generalized epilepsy with febrile seizure plus and name GEFS+ became accepted [4–8]. Mutations in the ligand-gated chloride channel gene (e.g., GABRG2) and neuronal voltage-gated sodium channel gene (e.g., SCN1A) are closely interrelated with GEFS+. Advances in molecular genetics in epilepsy have made rapid progress in recent years, revealing various mutant genes related to genetic epilepsy, laying the foundation for researchers to further study disease pathogenesis and gene function, and to promote the level of diagnosis and treatment of epilepsy.

There is significant evidence showing that GABA_A receptors (GABA_ARs) are the primary mediators of fast inhibitory synaptic transmission in the CNS, as thes have been repeatedly documented to play a vital role in animal models of seizures. Some GABRG2 gene mutations, such as missense [9–13], nonsense [14–16], splice site [17, 18], and frameshift mutations [19, 20], may cause GEFS+ (Fig. 1). These mutations may further alter GABA_AR expression, gating, and/or trafficking on the cell surface. Reduced GABA-evoked currents in neurons could also potentially cause neuronal disinhibition, while a finding of functional channel impairment in and of itself is not sufficient proof of pathogenicity. All pathophysiological mechanisms that lead to reduced GABA‐evoked currents in neurons can cause neuronal disinhibition and predispose affected patients to manifest seizures.

Fig. 1.

The schematic representation of the γ2 subunit topology, showing the position of different mutation types of GABRG2 gene. The pathogenic types of GABRG2 mutations was mainly divided into four types as follows: missense mutation, nonsense mutation, splice site mutation and frameshift mutation. Different types of GABRG2 mutations are not completely related to the disease, and the etiopathogenesis is varies. The red dots indicate missense mutation; the bule dots indicate nonsense mutation; the dark bule dots indicate splice-site mutation and the rose red dots indicate frameshift mutation

GABA_ARs form chloride ion channels, the currents of which can be modulated by several positive and negative allosteric regulators, including barbiturates, benzodiazepines, neurosteroids, bicuculline, picrotoxin, and zinc. In addition, GABA_ARs also affect neuronal excitability by mediating both phasic and inhibitory synaptic transmissions,as well as tonic and perisynaptic inhibition. Mutations in GABA_AR genes can cause epilepsy. Although some studies have presented hypothesesregarding the etiology of GEFS+, such as clathrin-independent endocytosis [21, 22], phosphorylation [23, 24], endoplasmic reticulum stress [25], and membrane dynamics [26], the specific underlying molecular mechanism still remains to be determined. In this updated and comprehensive review, we focused on the molecular genetic mechanisms of different clinical symptoms of epilepsy caused by various GABRG2 mutations, discussed the types of GABRG2 mutations, and highlighted significant advances in the mechanism of GABRG2 mutations in GEFS+, showing how new avenues are opening for the generation of new ideas and strategies for GEFS+ treatment.

Structures and biological function of GABRG2 receptor

GABA_ARs constitute the major fast-inhibitory neurotransmitter system in the central nervous system. These receptors composed of five transmembrane-spanning subunits that assemble to form a pentameric ligand-gated chloride channel with various subunits (α1-6, β1-3, γ1-3, δ, ε, π, θ, and ρ1-3) [27, 28], resulting in significant heterogeneity between different GABA_AR. Based on Bayesian inference (BI) and maximum likelihood (ML) methods, a phylogenetic tree for GABA_ARs was constructed. According to the known GABA_ARs classification, the GABA_ARs protein was divided into 8 subgroups (groups I–VIII) (Fig. 2), while the γ genes from humans, rats and mice has been divided into five classes. Circular phylogenetic tree analysis of the amino acid sequences of γ subunits showed the highest similarity to β subunits. Further, the genomic location of 19 GABA_AR_S has been identified [29–31]. The genes encoding subunit α1, α6, β2, γ2 and π are all clustered on chromosome 5q; those encoding subunit α2, α4, β1, and γ1 genes are clustered on chromosome 4p12; subunit α3, ε, and θ genes are found on chromosome Xq28; subunit α5, β3, and γ3 are located on chromosome 15q12; subunit ρ1 and ρ2 genes are identified on chromosome 6q15; and subunit ρ3 and δ genes are mapped on chromosome 3q11.2 and chromosome 1p36.3, respectively. A summary of the genomic locations of GABA_AR subunit genes is provided in Table 1. Each receptor subunit has an intracellular domain (ICD), a transmembrane domain (TMD), and an extracellular domain (ECD). ECDs are primarily composed of β-sheets and comprise the agonist-binding sites, whereas TMDs consist of pore-forming α-helices, while structurally variable ICDs are involved in receptor assembly, trafficking, and clustering. Mutations in the genes encoded by these subunits can lead to abnormalities in the conformation or expression of the receptor, and contribute to neuronal hyperexcitation and seizures. The similarity of amino acid sequences among GABA_ARs subunits is approximately 15%–40%, while the similarity between different members of the same subunit family is 60%–80%. GABRG2 is located in the 5q31.1-33.1 region, which is composed of nine exon coding regions, eight introns, and a manipulated structure. This gene is transcribed to form mRNA, which is then translated into peptide chains in ribosomes, processed, and folded in the Golgi apparatus and the endoplasmic reticulum to form the correct conformation. GABRG2 mainly includes a large N-terminal ECD involved in endogenous ligand binding, a hydrophobic TMD (M1–4) composed of four α-helix regions, and an extracellular C-terminal. The M2 region of GABRG2 forms ion channel pores, and the hydrophobic M region is connected through a smaller intracellular ring between M1 and M2 and a larger intracellular ring between M3 and M4 (Fig. 1). This structure plays a crucial role in the connection, assembly, and transportation of other receptor subunits, mediating the rapid influx of chloride ions into the postsynaptic membrane, as well as the interaction of intracellular proteins [54]. Each TMD is connected by hydrophilic amino acids, while M2 is a domain formed by chloride ion channels that can selectively pass through negatively charged chloride ions.

Fig. 2.

Phylogenetic analysis of GABA_ARs genes constructed by MEGA (v 10). Phylogenetic tree of GABA_ARs gene nucleotide sequences constructed with MEGA X software in which the analyzed sequences are clustered into eight genotyping clusters (genotypic α, β, γ, δ, θ, π, ρ and ε) according to the amino acid sequences of GABA_ARs from humans, rats and mice. H: human; R: rat; M: mouse

Table 1.

Summary of the genomic location of 19 GABA-A receptor subunits

Subunit	Gene	Gene ID	OMIM ID	Chromosome	Locus	References
α1	GABRA1	2554	137,160	5	5q34	[32–34]
α2	GABRA2	2555	137,140	4	4p12	[32, 35]
α3	GABRA3	2556	305,660	X	Xq28	[36–38]
α4	GABRA4	2557	137,141	4	4p12	[32, 39]
α5	GABRA5	2558	137,142	15	15q12	[32, 40]
α6	GABRA6	2559	137,143	5	5q34	[32, 34]
β1	GABRB1	2560	137,190	4	4p12	[32]
β2	GABRB2	2561	600,232	5	5q34	[41–43]
β3	GABRB3	2562	137,192	15	15q12	[44]
γ1	GABRG1	2565	137,166	4	4p12	[32, 45]
γ2	GABRG2	2566	137,164	5	5q34	[32]
γ3	GABRG3	2567	600,233	15	15q12	[40, 46]
δ	GABRD	2563	137,163	1	1p36.3	[47]
ε	GABRE	2564	300,093	X	Xq28	[48]
π	GABRP	2568	602,729	5	5q34	[32, 49]
θ	GABRQ	55,879	300,349	X	Xq28	[50]
ρ1	GABRR1	2569	137,161	6	6q15	[32, 51, 52]
ρ2	GABRR2	2570	137,162	6	6q15	[32, 51, 52]
ρ3	GABRR3	200,959	618,668	3	3q11.2	[53]

The γ2 subunit participates in the complex ligand gate channel encoding the GABA_AR and is its main component. The most common type of GABA_AR in mammals comprises two α, two β, and one γ subunits (α1β2γ2, α1β3γ2, and α2β3γ2); α1β2γ2 is most distributed in the hippocampus and cortex. Approximately 60% of the GABA_AR subunits in the human brain have the α1β2γ2 combination [55], whereas α2β2γ2 and α3β2γ2 are most common in the amygdala, striatum, and spinal motoneurons. The α1β2γ2 has the pharmacological characteristics of type I benzodiazepines, whereas receptors containing the α2βγ2, α3βγ2, or α5βγ2 subtypes form type II benzodiazepines [56]. In 2018, Hibbs et al. obtained the first cryo-electron microscopy (Cryo-EM) structure of a heteromeric GABA_AR that composed of the human α1β2γ2 subunits [57]. Subsequently, the Cryo-EM structure of the rat α1β2γ2 heteropentamer was solved [58]. These Cryo-EM structures provided unprecedented insights not only into the overall architecture of heteropentameric GABA_ARs but also into the binding of diverse ligands including the agonist GABA (Fig. 3A–I). Finally, these Cryo-EM structures further demonstrated how membrane lipids interact with the transmembrane domain.

Fig. 3.

Different views of a non-homology model of the heteropentameric α1β2γ2 GABA_AR as determined by Cryo-EM. A Illustration of GABA_AR structure illustrating the extracellular, transmembrane and intracellular portions in rat model. B Top view of α1β2γ2 GABA_AR ECD. C View of the receptor from the ICD (bottom view). D The side view of the receptor exhibits the different domains common to all Cys-loop receptors: the ECD, the TMD and the ICD in mice model. E Top view of the receptor. F Bottom view of the receptor. G Cartoon representation of the receptor viewed parallel to the membrane plane in human model. H and I show the receptor from the perspective of the synapse of the ECD and ICD, respectively. The γ2 subunit is colored light bule, β2 subunit light yellow and α1 gray. ECD: extracellular domain; TMD: transmembrane domain; ICD: intracellular domain

GABA_AR is an important chloride channel receptor on the cell membrane that plays a vital role in maintaining the internal and external electrical balance of cells. These receptors can exist in three open states on the cell membrane, with average durations of approximately 0.5, 2.5, and 8 ms. The single-channel current formed by the α1β1γ2 receptor has multiple open and closed states, whereas the current formed by the α1β1δ receptor is small, and the channel is closed under unpredictable conditions [59]. When the expression of the α1 or γ2 subunit is inhibited by gene knockout, the number of GABA_ARs and affinity for GABA are significantly decreased, resulting in an increased susceptibility to epileptic seizures. Indeed, GEFS+ is the most common disease caused by γ2 mutation. When any of the subunits involved in GABA_AR formation are mutated, their activity becomes impaired, resulting in reduced GABA-mediated synaptic inhibition, leading to neuronal hyperexcitability. Although we have some understanding of the GABA_AR and its constituent subunit structure, several variations in the recombination order and combinations of the receptor subunits in different species and cell types exist. Further, the mechanism of phenotypic variation caused by GABA_AR family genes remains unclear, requiring further investigation.

Recent advances in clinical research of GEFS+

Clinical features

Exome sequencing of patients from families with a high incidence of GEFS+ showed that mutations in GABRG2 was the primary cause of GEFS+, an autosomal dominant inherited disease with high genetic and phenotypic heterogeneities. Incomplete penetrance and different degrees of gene expression are theprimary reasons underlying these different clinical manifestations. According to the classification criteria for seizures and epileptic syndromes of the ILAE [60–63], the most common clinical manifestation of GEFS+ is FS, although generalized tonic–clonic seizures (GTCSs, involving loss of consciousness, generalized muscle tonic and convulsions) can occur in children between 3 months and 6 years of age, often occurring concourrent with fever (≥ 38 ℃) [64]. The second most common manifestation is febrile seizure plus (FS+), with thses patients generally experiencing febrile tonic–clonic seizures before 3 months and/or after 6 years of age, with some further presenting with both febrile and afebrile GTCS. FS+ has features that are significantly different from FS syndrome.

In addition to the two common clinical symptoms described above, GEFS+ may also present as FS/FS+ with absence, myoclonic, focal, or dystonic seizures, for which the most severe symptoms are myoclonic sudden epilepsy and severe myoclonic epilepsy in infants [1, 2, 26, 64–66]. One study of 201 individuals from 31 GEFS+ families showed that FS and FS+ occurred in 41% and 20%, respectively [2], amomg whom the median age at first onset was 12 months and 14 months, respectively, and the age of remission was 2 years and 6–34 years, respectively. There were three types of FS+ in this study: (1) FS, which lasted for more than 6 years; (2) GTCS beyond FS; (3) only FS after the age of 6. Another study of eight GEFS+ families (58 individuals) showed that the heterogeneity of patients' clinical presentation was influenced by genetic modifications and environmental factors, which affected the clinical symptoms and natural history of the disease [66].

Diagnosis principles

The diagnosis of GEFS+ is difficult in clinical practice, particularly in patients with familial febrile seiures plus. Detailed electroclinical evaluations including scalp and video electroencephalograms and brain magnetic resonance imaging were performed. Clinical seizure data were obtained from parents or witnesses, and an epilepsy questionnaire was administered. Blood samples from probands and family members with the disease were collected and analyzed by whole-exome sequencing, targeted next-generation sequencing of epilepsy, or other techniques [2, 66, 67]. The inclusion criteria were as follows: two or more family members with GEFS+, and at least one with FS+; or there was no FS+ in the family, but three or more patients presented with symptoms of GEFS+, such as FS or myoclonic absence epilepsy (MAE). The exclusion criteria were FS only and Dravet syndrome in the proband’s family.

Analysis of GABRG2 mutation types and potential pathogenesis in genetic epilepsy

GEFS+ occurs as a result of multiple gene expression levels and environmental factors. The pathological mechanism is complex, including fever, inflammatory mediators, and genetic susceptibility, among which changes in genetic susceptibility factors and corresponding molecules are the most important causes of the disease. In addition to coding ligand-gated chloride channels, molecular genetic mechanisms also include mutations in genes encoding voltage-gated sodium channels (SCN1A/SCN2A/SCN1B) [68–71], as well as pro- and anti-inflammatory factor genes [72]. Theese gene mutations, as well as sporadic forms of other phenotypes, have been found in both large and small families, and many of them do not follow Mendelian inheritance. Furthermore, the incidence of GEFS+ in small families in the population is higher than that reported in the literature[6], and there are no particularly effective antiepileptic drugs for the minority of patients with the phenotypes. Although mutations or polymorphisms of these genes are related to the pathogenesis of GEFS+, the pathogenesis of GEFS+ has not yet been elucidated. Each mutation is linked to a different GABRG2 protein synthesis disorders through different molecular mechanisms as detailed below in Table 2. Herein, we review the research progress in diseases caused by GABRG2 gene mutations, providing a reference for clinical treatment decisions, as well as a baseline for the development of new anti-epileptic drugs.

Table 2.

GABRG2 gene mutations and variants associated with genetic epilepsies and their underlying molecular and biochemical defects mechanisms

GABRG2 mutation or variant	GABAARs structural domain	GABAARs functional domain	Potential mechanisms	Seizure types	Family history: Y or N	Seizures fever sensitivity: Y or N	Cluster seizures: Y or N	References
Missense mutations
R43Q (R82Q)	NT	Binding	Impaired oligomerization, impaired surface expression and ER retention	FS, CAE	Y	Y	N	[9, 22, 73–79]
R138G (R177G)	NT	Binding	Incorporated into GABAARs and conferred altered current desensitization; impaired surface expression and ER retention	FS	Y	Y	N	[11–13]
K289M (K328M)	M2-M3 loop	Coupling	Gating defect	FS, GEFS+	Y	Y	N	[10, 26, 84–87]
G257R	NT	Binding	Impaired surface expression and ER retention	RE	N	Y	N	[18]
I389V	M3-M4 loop	Intracellular	Unclear	RE	N	N	N	[18]
P83S	NT	Binding	Impaired surface expression and ER retention	IGE, FS, GEFS+	Y	Y	N	[89, 107]
N40S (N79S)	NT	Binding	Impaired receptor trafficking and increased retention of the receptor in intracellular compartments	GTCS	N	N	N	[89, 91, 92]
R323Q	M2-M3 loop	Coupling	Impaired surface expression; decreased GABA-evoked currents, increased by 25% the fractional Zn2+ inhibition of currents	GEFS+, FS, myoclonic	Y	Y	N	[18, 25]
A106T	NT	Binding	Impaired surface expression; decreased GABA-evoked currents	GTCS, DEE	Y	N	N	[25, 65]
I107T	NT	Binding	Impaired surface expression; decreased GABA-evoked currents, increased by 25% the fractional Zn2+ inhibition of currents; more stable than wild-type subunits and/or were retained in the endoplasmic reticulum	Tonic	Y	N	N	[25]
P282S	M1	Pore	Impaired surface expression; decreased GABA-evoked currents, increased by 25% the fractional Zn3+ inhibition of currents; more stable than wild-type subunits and/or were retained in the endoplasmic reticulum	Atypical absences	Y	N	N	[25]
R323W	M2-M3 loop	Coupling	Impaired surface expression; decreased GABA-evoked currents, increased by 25% the fractional Zn4+ inhibition of currents; more stable than wild-type subunits and/or were retained in the endoplasmic reticulum	GTCS	Y	N	N	[25]
F343L	M3	Pore	Impaired surface expression; decreased GABA-evoked currents	Tonic, epileptic encephalopathy	Y	N	N	[25, 93–95]
P282T	M1	Pore	Causing accumulation of the subunit in the endoplasmic reticulum (ER) and impairing the surface trafficking	GTCS, atonic	Y	N	N	[96]
P302L	M2	Pore	Impaired surface expression; decreased GABA-evoked currents	DS	Y	Y	N	[97]
S306F	M2	Pore	Impaired surface expression; decreased GABA-evoked currents	Clonic	Y	N	N	[96]
c.595A>G (p.Met199Val)	NT	Binding	Impaired surface expression; decreased GABA-evoked currents	FS, GTCS, absences	Y	Y	N	[19]
Nonsense mutation
R136*	NT	Binding	Impaired surface expression and total expression; compromised receptor trafficking	GEFS+, GGE	Y	Y	N	[15, 98]
Q1X (Q40X)	NT	Binding	Impaired axonal transport; decreased GABA-evoked currents	SMEI, DS	Y	Y	N	[16, 54]
W390X (W429X)	M3-M4 loop	Coupling	Impaired surface expression; decreased GABA-evoked currents	Focal seizure, GEFS+, DS	Y	Y	Y	[54, 67, 98]
Q351X (Q390X)	M3-M4 loop	Coupling	Impaired surface expression; ER Retention, ERAD, dominant negative effect; decreased GABA-evoked currents;	GEFS+, FS, FS+, SMEI, DS	Y	Y	N	[54, 98]
T90M	NT	Binding	Impaired surface expression, ER retention, and channel gating defects	SHE	N	N	Y	[99]
Q217X	NT	Binding	Reduced synaptic clustering and distribution of GABAAR	SHE	N	N	Y	[99]
T317N	M2	Pore	Impaired surface expression, ER retention, and channel gating defects	SHE	N	N	Y	[99]
Splice-site mutation
c.588+2T>G (IVS6+2T → G)	M1-M2 loop (intron 6)	Coupling	Impaired surface expression; ER retention	FS, CAE	Y	Y	N	[17, 20]
c.549-3T>G (IVS4-3T → G)	NT (intron 4)	Binding	Not reported	RE	Y	N	N	[18]
c.631+4A>G (IVS5+4A → G)	NT (intron 5)	Binding	Not reported	GTCS, FS+	Y	Y	N	[67]
c.631+5G>T (IVS5+5G → T)	NT (intron 5)	Binding	Not reported	GTCS, focal seizure	Y	N	Y	[67]
c.922+1G>T (IVS7+1G → T)	M2 (intron 7)	Pore	Not reported	Focal seizure	Y	Y	N	[67]
c.1128+5G>A (IVS8+5G → A)	M3-M4 loop (intron 8)	Coupling	Not reported	MS, GTCS	N	Y	Y	[67]
c.1249-7C>T (IVS9-7C → T)	M3-M4 loop (intron 9)	Coupling	Not reported	Focal seizure	Y	Y	Y	[67]
Frameshift mutation
p.Ser443delC (c.1329delC)	M3–M4 loop (exon 9)	Binding	Impaired surface expression, ER retention, and channel gating defects; decreased GABA-evoked currents;	GEFS+	Y	Y	Y	[100]
p.Val462fs*33 (c.1382delG)	M4 (exon 9)	Pore	Not reported	FS	Y	Y	N	[19]
p.Pro59fs*12 (c.174_175delinsA)	NT (exon 2)	Binding	Not reported	FS	Y	Y	N	[19]
p.Glu402fs*3 (c.1206delinsTTCAT)	M3–M4 loop (exon 9)	Coupling	Not reported	FS	Y	Y	N	[19]

FS febrile seizures, CAE childhood absence epilepsy, GEFS+ genetic epilepsy and febrile seizures plus, RE rolandic epilepsy, IGE idiopathic generalized epilepsy, GTCS generalized tonic–clonic seizure, DEE developmental and epileptic encephalopathy, DS Dravet syndrome, GGE genetic general epilepsy, SMEI severe myoclonic epilepsy in infancy, FS+ febrile seizures plus, SHE sleep-related hypermotor epilepsy, MS myoclonic seizure

Missense mutations

In 2001, Wallace et al. conducted exome sequencing research on children in families with absence epilepsy and FS, identifying a missense mutation (c.245G → A; R43Q) in the second exon region of GABRG2 (c.245G → A; R43Q), which transformed the highly conserved arginine residue into glutamine at residue 43 of the mature Gabrg2 protein [9]. This R43Q mutation is located in the first two high-affinity benzodiazepine binding domains of the GABA_AR receptors, which does not change the reactivity of the Cl⁻ current to GABA, but caused a reduction in the sensitivity to diazepam and flunitrazepam. Further, sensitivity to imidazolidine drugs such as zolpidem is increased, which provides a physiological basis for the prevention of epilepsy [9, 73, 74]. In vivo and in vitro studies have shown that the R43Q mutation reduces the expression of GABA_AR on the cell surface, significantly reduces the synaptic current mediated by it, reduces synaptic inhibition, and triggers increased neuronal excitability [75–78]. Witsch et al. found that the spontaneous firing activity of pyramidal neurons in layers 2/3 and 5/6 of the cerebral cortex of R43Q mutant mice was significantly increased compared to that of wild-type mice [79]. R43Q mutations can also attenuate endocytosis by GABA_AR antagonists, affecting the transport of receptors in cells [22]. In addition, the R43Q mutation affects neuron development, thereby affecting the stability of the entire brain network. As such, reversing brain developmental dysfunction is not possible by simply targeting a receptor to compensates for defects [80]. This mutation can also change the density of neurons in the dentate gyrus of the hippocampus, resulting in changes in the hippocampal structure and volume reduction [81], changes in the cortical microcircuits [82], and a decreases in the connections between neurons in the cerebral cortex [83], leading to the weakening of the inhibitory activity of neurons and the occurrence of epilepsy. One study found that a γ2 mutation (R139G) at one of the two benzodiazepine binding sites located at the N-terminal of the γ2 subunit reduced the sensitivity to benzodiazepine, and that the only clinical symptom caused by this mutation was FS [11]. Residue R139G is a conserved amino acid residue in the γ2 subunit. In the cysteine ring receptor family, polar and charged amino acid residues are present at this site.

The mutation (K289M) of the γ2 subunit, which is located in the short extracellular loop between the second and third transmembrane domains, was first identified in studies of GEFS+ families [10]. In exon 8, the base A → T is switched, resulting in the replacement of the positively charged lysine with neutral methionine. The mutation region is related to the gating of ligand-gated ion channels and is also an important region linked to autosomal dominant inheritance in generalized epilepsy. In proteins with the K289M mutation, the average time of its channel opening is reduced, and the duration of the rapid GABA-evoked current is shortened, leading to a reduction in the duration of the inhibitory postsynaptic current, reducing the inhibitory effect of neurons and subsequently inducing epilepsy [84–86]. The K289M mutation had no significant effect on cell membrane transport and recombinant γ2 synapse aggregation, but did accelerate the attenuation of the synaptic current, which may have different effects on the GABAergic signaling pathway [87]. With an increase in temperature, the number of GABA_AR clusters and the frequency of micro-inhibitory postsynaptic currents (mIPSCs) in neurons expressing the K289M mutation significantly decreased, triggering the escape of receptors from postsynaptic regions and further reducing the inhibitory effect of GABAergic transmission [26]. However, another study showed that the K289M mutation is rare in children with FS and GEFS+ [88]. In 2015, p.G257R and p.I389V GABRG2-missense mutations were identified in patients with Rolandic epilepsy (RE) [18].

The missense mutations N79S, R82Q, P83S, and R177G are all located in the N-terminal loop of the extracellular region of the γ2 subunit [13, 88, 90], and all result in some degree of impairment in the normal assembly function of the GABA_AR. Studies have shown that the γ2 (N79S) subunit can be efficiently assembled into GABA_AR, and that its normal receptor transport changes only slightly. Research has also shown that the N79S mutation is a rare or susceptible variant. However, R82Q and P83S mutations can impair the normal assembly of pentamers, resulting in the retention and degradation of synthetic receptors in the endoplasmic reticulum, as well as the reduction of normal GABA_AR synthesis. The co-expression assay of wild-type or mutant γ2 subunits with α1 and β2 showed that the cell membrane expression and overall expression level of wild-type and mutant γ2 subunit proteins increased after 24 h incubation at 30℃, indicating that lower temperatures can increase the stability of GABA_AR. A novel γ2 mutation (R177G) was also detected in a family with complex FS [13]. This R177G mutation resulted in the retention of the γ2 subunit in the endoplasmic reticulum and its degradation by the ubiquitin–proteasome system, which led to the dysfunction of GABA_ARs surface synthesis, thereby reducing the inhibitory function of neurons and increasing the excitability of neurons is increased, thus triggering seizures.

In a prior study of 140 children with epilepsy who underwent genetic screening, one patient with GTCS had a γ2 (c.236A>G: p.N40S) mutation [91]. This N40S mutation affects asparagine at amino acid residue 40 of the mature γ2 subunit, resulting in its replacement with serine. This mutant region is adjacent to the first of the two high-affinity benzodiazepine-binding domains of the γ2 subunit, resulting in ion channel dysfunction and promotion of GTCS. Migita et al. found that the N40S mutation led to a significant increase in the Hill coefficient of the N40S receptor, which caused a change in the GABA concentration effect, triggering epilepsy [92]. In 2017, Shen et al. conducted next-generation sequencing to investigate patients diagnosed with epileptic encephalopathy, identifying six missense mutations in GABRG2 (A106T, I107T, P282S, R323W, F343L, and R323Q) [25]. In vitro, the γ2 (R323Q) mutation reduced the expression of GABA_AR on the cell surface and the amplitude of the GABA-induced whole-cell current. However, different mutations in γ2 lead to different degrees of current amplitude reduction, indicating that GABAergic neurotransmission dysfunction is involved in the pathogenesis of genetic epilepsy [18, 25, 101]. In addition, Zou et al. showed that the γ2 (A106T) mutation is accompanied by other severe phenotypes besides the common FS symptoms, such as the early onset of the first seizure, significant motor and language retardation, intellectual disorder, hypotonia, dyskinesia, malformation, and visual impairment [102]. Similar to the P282S mutation, the γ2 (P282T) mutation is located in the M1 transmembrane domain of GABRG2 (c.844C>A; p.P282T) [96, 103]. This P282S mutation causes threonine 282 to be replaced by serine. Threonine and serine are polar amino acids, and their effects on polyprotein structures are very similar. The effect of γ2 (P282T) on the protein structure and function was similar to that of γ2 (P282S). Both mutations lead to similar clinical manifestations, except that patients with γ2 (P282T) mutations display more clinical features of neurodegeneration. Next-generation sequencing was performed on a patient with Dravet syndrome showed that the base encoding leucine in the cytoplasm was located in the M2 segment of the transmembrane domain of the γ2 subunit (c.905C>T; P302L), which influences the high conservation of the transmembrane segment M2 located in the pore domain of the GABA_AR γ2 subunit. The γ2 (P302L) mutation causes GABA_AR to lose approximately 90% of its function by affecting the closing, opening, and current desensitization of ion channels in the receptor conduction pathway and finally increasing the excitability of neurons.

In one case of epilepsy in an infant with migrating focal seizures [96, 103], the M2 segment of the transmembrane domain of the γ2 subunit was mutated (c.917C>T; S306F). This mutation was close to γ2 (P302L), a common pore-lining residue that forms a part of the inner surface of the γ2 subunit pores. However, the underlying pathogenic mechanism requires further study. A whole-exon sequencing study of 14 patients diagnosed with FS revealed that a base in exon 4 of GABRG2 was replaced (c.529C>G; R139G), resulting in the substitution of a highly conserved arginine with glycine in the mature peptide located at position 139 of the second benzodiazepine binding site of the γ2 subunit. Electrophysiological studies showed that this R139G mutation altered the current desensitization and reduced the enhancement of benzodiazepine. The rapid desensitization stage is the main factor in the formation of an inhibitory postsynaptic current. Increasing the rapid desensitization stage reduces the amplitude of the inhibitory postsynaptic current, leading to epilepsy. In 2015, Boillot et al. conducted an exon sequencing study on an FS family with early absence seizures and patients with GTCS, identifying the missense mutation c.595A>G (c.595A>G; p.Met199Val) located in exon 5 of GABRG2 [19]. The discovery of the p.Met199Val mutation in the N-terminal of the extracellular loop of γ2 protein further expands the genetic spectrum of GABRG2 mutations. The mutation c.968G>A; p.Arg323Gln of GABRG2 was first identified in patients with GEFS+ with mild cognitive impairment [55] and is characterized by high levels of repetition.

Nonsense mutation

Gene sequencing was conducted on 80 families with confirmed epilepsy identifying a base switch from C to T at position 406 of GABRG2 (c.406 C>T; p.Arg136*) [15]. A premature stop codon (TGA) was also introduced at the highly conserved arginine residue at position 136 (p.R136*) in the immature GABRG2 polypeptide sequence was also identified. This mutation resulted in the shortening of the GABRG2 protein chain, loss of all four transmembrane domains and the C-terminal, and partial retention of the N-terminal [11, 19]. In vitro tests showed that this mutation of γ2 (p.R136*) resulted in the dysfunction of its receptor transport, decreased expression of γ2 protein on the cell surface, and aggregations in the nucleus and endoplasmic reticulum, which may be the cause of epilepsy [15, 98]. In some patients with severe epileptic syndrome, a translational premature stop codon (PTC) has been found at the position encoding the first amino acid in the γ2 subunit mRNA, resulting in a γ2 mutation (c.118C>T; p.Q40X) [16, 104, 105]. The γ2 (Q40X) mutant mRNA can be degraded by nonsense-mediated mRNA; however, the undegraded mutant mRNA can be translated into a shortened peptide, similar to a signal peptide. The γ2 (Q40X) mutant subunit fails to assemble into a functional GABA_AR, which reduces the amplitude of the GABA-evoked currents. In addition, the Q40X mutation impaired the axonal transport of α1 and β2 subunits. In 2008, Sun et al. found a heterozygous mutation in exon 9 of the GABRG2 gene in several Chinese patients with GEFS+ (c.1287G>A; p.W390X), the genetic codon changes from TGG to TGA, resulting in the replacement of tryptophan at position 390 by the stop code, shortening the intracellular loop channel protein between the third and fourth transmembrane domains [106]. Another study also identified a γ2 (W390X) mutation in Chinese patients with GEFS+; the family conforms to autosomal dominant inheritance with incomplete penetrance, and the W390X mutation may be a pathogenic gene in Chinese patients with GEFS+ [107].

In a GEFS+ family, DNA sequencing revealed for the first time that a base C>T transition (c.1168C>T; Q351X) occurred at nucleotide position 1168 of the intracellular loop between the third and fourth transmembrane domains of the GABRG2 gene by DNA sequencing [14]. This single-base transition resulted in the introduction of a premature stop codon at Q351X in the mature GABRG2 protein, resulting in the complete loss of the fourth transmembrane domain of GABRG2, and leading to the shortening of the synthesized GABRG2 protein. In vitro studies have further shown that sensitivity to GABA transmitters is lost after γ2 (Q351X) mutation, and its expressed protein is retained in the endoplasmic reticulum, causing a dysfunction of receptor transport, aggregation, assembly, and synaptic maintenance, leading to impaired GABA_AR channel function and lowering of the seizure threshold [14, 108, 109]. Using rat cortical neurons with the Q351X mutation, researchers have shown that the mutation can form a high molecular weight protein, which is a common phenomenon of shortening or misfolding of the GABA_AR subunit protein, causing dysfunction of GABA_A receptor transport, resulting in abnormal GABA_AR channel function [110]. This is similar to the mechanism of protein chain shortening caused by many other mutations, including the 39 amino acid residue signal peptide sequence in the immature γ2 (Q390X) mutation. The Q390X mutation is also called GABRG2 (Q351X), which does not include the 39 amino acid residue signal peptide sequence [111].

In the Gabrg2^+/Q390X knock-in (KI) mouse model, it was found that Gabrg2^+/Q390X could not only damage the transmission of inhibitory neurotransmitters but also caused the accumulation and aggregation of γ2 (Q390X) subunits in the cell, activating caspase 3 and consequently causing extensive, age-dependent neurodegeneration [111–113]. Another study indicated that γ2 (Q390X) KI mice showed myoclonic convulsions, GTCS, and anxiety symptoms with an increase in temperature, suggesting that GABRG2 (Q390X) mutations may alter the temperature regulation of the brain and induce seizures in the process of temperature increase [114]. The electroencephalogram of the γ2 (Q390X) KI mouse model showed that the inhibition of cortical neurons was reduced, and spike discharges occurred. The threshold of epileptic seizures induced by pentylenetetrazol was decreased, and the overexpression of the wild-type γ2 subunit increased mIPSCs and diminished the symptoms of epilepsy attack in γ2 (Q390X) KI mice [115]. Further, one a study showed that a combination of staventanol and diazepam can significantly improve seizure symptoms and increase the survival rate of Gabrg2^+/Q390X KI mice [114]. However, the use of staventanol alone does not affect most epileptic symptoms, and it can only be used as an adjunctive drug for GABA_AR-deficient diseases. In a recent study, three rare GABRG2 variants (T90M, Q217X and T317N) associated with sleep‑related hypermotor epilepsy were detected [99]. Functional analysis showed that the Q217X variant reduced synaptic clustering and distribution of GABA_AR, while the T90M and T317N variants decreased GABA-evoked current amplitudes by diverse mechanisms including endoplasmic reticulum retention, gating defects and impaired surface expression.

Splice site mutation

Kananura et al. reported GABRG2 (IVS6+2T → G) splice site mutations in children with absence seizures and FS. Direct sequencing showed that a pair of base transpositions occurred at the splice donor site of intron 6, with guanine replacing thymidine (IVS6+2T → G). In addition, this mutation disrupted the conserved splice site motif “gt” of intron 6, turning it into “gg” [17]. An intron splice site mutation is another type of mutation generated by PTC, including nonsense, deletion, and frameshift mutations [54]. In vitro studies have shown that the mutation of γ2 (IVS6+2T → G) produces a stable and non-functional shortened γ2 subunit by reducing the transcription level of GABRG2, thereby thus the assembly of GABA_ARs and reducing GABAergic inhibition [20]. Intriguingly, the GABRG2 (c.549-3T>G) mutation was also found in two patients with typical Rolandic epilepsy [18]. However, due to the shortage of GABRG2 in human blood, the possible pathogenic effect of the c.549-3T>G splicing site mutation on the RNA of patients could not be evaluated.Reasonably designed in vitro and in vivo experiments are needed to explore its pathogenic mechanisms. Finally, in one recent report, c.631+4A>G, c.631+5G>T, c.922+1G>T, c.1128+5G>A, and c.1249-7C>T variations in GABRG2 were identified in 35 patients with FS+ [67].

Frameshift mutation

The GABRG2 mutation (c.1329delC) in the last exon of GABRG2 in a GEFS+ family was identified in 2013, causing the loss of a cytosine nucleotide in the Ser 443 codon TCC, shifting the open reading frame, which resulted in the loss of the natural stop codon and the generation of a new stop codon (p.Tyr444Metfs*51) in the 3’ untranslated region [100, 116]. This mutation resulted in the loss of 24 amino acids from the C-terminal of the γ2 subunit, as well as the reacquisition of 50 amino acids which differed from the natural variation, thus reducing the hydrophobicity of the C-terminal. The γ2 (c.1329delC) mutant subunit is not expressed on the cell membrane surface, instead remaining in the endoplasmic reticulum, which reduces its total expression and affects the function of GABA_ARs on the cell surface. Boillot et al. carried out exon sequencing studies on 107 FS+ families, identifying two new GABRG2 frameshift mutations (p.Val462fs*33 and p.Pro59fs*12), which introduced an incorrect stop codon into the reading frame, resulting in shortening of the γ2 protein chain and causing GABRG2 dysfunction [19]. These studies further confirmed that the GABRG2 mutation is an important pathogenic factor in genetic epilepsy. In addition, other uncommon mutations, such as γ2 (p.Glu402fs*3), and mutation types, such as exon deletion, were also identified in this study.

Role of the GABRG2 mutation in clinical diagnosis and treatment of epilepsy

The GABRG2 gene mutation is an autosomal dominant inherited disease which is accompanied by incomplete penetrance. Sporadic focal epilepsy may also be diagnosed in some families with mutations. In recent years, many studies have suggested that there is significant heterogeneity in the clinical manifestations and gene mutations in GEFS+ families from different countries, races, and regions. GABRG2 mutations are not uncommon in the GEFS+ family population, but due to their complex and diverse clinical manifestations, more attention should be paid to clinical diagnosis and treatment. Based on human genome research achievements, the target genes of patients with a typical GEFS+ family genetic history should be tested first. If the target gene test result is negative, panel detection can subsequently be performed. For patients with atypical GEFS+, chromosomal microarray detection is first performed, followed by the target gene panel method. If the above two tests are negative, gene sequencing technology can be used to screen for mutation sites, such as whole genome and exome sequencing [117, 118].

Although the spectrum of GABRG2 mutations is diverse, different mutation types have many similar pathophysiological mechanisms, mainly leading to the impairment of normal assembly, transportation, and expression of GABA_ARs, resulting in the reduction of inhibitory synaptic receptors and an increase in neuronal excitability. The main pathogenic mechanisms include neuroinflammation, autophagy deficiency, and endoplasmic reticulum stress (Fig. 4). The proportion of drug-refractory epilepsy in patients with GABRG2 mutation-related epilepsy is relatively high, but there have been relatively few studies on this topic. The use of stilopenol combined with diazepam can effectively improve the clinical symptoms of patients [116]. Indeed, one study showed that vorinostat, which has been used in the treatment of epilepsy caused by GABRA1 mutations, can ameliorate the inhibitory synaptic damage caused by γ2 missense mutations and could be considered a treatment for epilepsy caused by GABRG2 mutations [119]. Overexpression of the γ2 wild-type subunit receptor has been shown to significantly reduce mortality in Dravet syndrome mice [115]. Some studies have further suggested that gene therapy using antisense oligonucleotide technology is also a new avenue for the treatment of diseases caused by GABRG2 mutations [120, 121]. Clinical data has further shown that valproate and levetiracetam may be suitable for patients with GABRG2 variants [67]. In another study, oxcarbazepine was used in patients with the GABRG2 variant, achieving a good prognosis [102]. In our previous studies, we successfully constructed a cell line and the mouse model of GABRG2 knockout, which will provide a good research vector for further revealing the pathogenic mechanism of GEFS+ and help provide a reference for the treatment of this disease [122, 123]. Our data further indicated that neuroinflammation may be involved in GEFS+ induced by GABRG2 mutations (unpublished), and anti-inflammatory strategies might be a potential treatment for GEFS+. Valproic acid, levetiracetam, zonisamide, topiramate, lamotrigine, and lacosamide are all effective treatments for myoclonic seizures (MS) and GTCS [124]. A recent study showed that the γ subunit impairs the glutamate allosteric potentiation of GABA_ARs by reducing the number of glutamate-binding pockets. A single mutation in the pocket-forming amino acid residues of either α1 or β2 in α1β2γ2 receptors is sufficient to eliminate glutamate modulation [125]. Valproic acid can increase GABA (by increasing synthesis and decreasing degradation), inhibit voltage-sensitive sodium and T-type calcium channels, and possibly reduce glutamate synthesis [126]. A disruption in the the dynamic balance between glutamate receptor-mediated neuronal excitation and GABA_AR_S-mediated inhibition can contribute to the occurrence of nervous system diseases, including neurological paroxysmal disorders and neurodegenerative diseases.

Fig. 4.

Schematic representation of molecular biogenesis, assembly, and transport of GABA_A receptor subunits. Only those receptors that are properly assembled and reached the cell surface and synapses can perform the function of chloride ions, while subunits located in intercellular compartments have no function. Missense mutation or nonsense mutation can be affected by nonsense mediated mRNA degradation (NMD) system, or degraded in endoplasmic reticulum-associated degradation (ERAD). Therefore, the GABRG2 mutation site type generally does not exist on the cell surface or in synapses. The arrows show the targeted subcellular locations of GABRG2 mutations

A recent clinical trial showed that cannabidiol (CBD) exerts an effect on the treatment of clinically relevant febrile convulsions, reducing seizure frequency [127, 128]. Cannabidiol, derived from the cannabis plant, is a major non-psychoactive cannabinoid that reduces neuronal excitability and limits seizures through its action on multiple targets [129, 130]. CBD is a positive allosteric modulator of GABA_ARs, either enhancing inhibitory GABA_A receptor activation [131], potentiating GABA-mediated inhibitory currents [132], or enhancing the amplitude of the GABA-evoked currents [133], exerting anti-inflammatory, antioxidant and neuroprotective properties [134–137]. CBD also ameliorates epilepsy by acting on the metabolism, calcium signaling pathway, and tuberculosis pathways in the hippocampus [138]. Suberanilohydroxamic acid (SAHA) has been proved to be effective at treating spontaneous seizures in zebrafish caused by GABRG2 mutation [93]. Sandouka and coworkers showed that dimethyl fumarate (DMF) can significantly increase Nrf2 activity, reduce neuronal cell mortality, decrease seizure frequency, and reverse epilepsy-induced behavioral disorders [139]. Given the heterogeneity in GABRG2 mutation-related clinical features, early diagnosis is challenging. However, there is a lack of explanation to explain this heterogeneity. Therefore, epilepsy caused by mutations of GABRG2 needs to be treated after achieving definitive diagnosis.

In addition to the wealth of evidence showing that GABRG2 mutations can cause GEFS+, many studies have shown that SCN1A [1, 70, 140–146], SCN1B [147, 66, 106], SCN2A [148, 149] and SCN9A [150, 151] mutations can also cause GEFS+. Our previous study suggested that GABR2 mutations can affect the expression of SCN1A on the cell surface [122]. The co-expression of GABRG2 with SCN1A, SCN1B, SCN2A, and SCN9A was observed in the STRING database. The results of the protein–protein interaction network between GABRG2 and the above factors are shown in Fig. 5. These co-expressed factors are directly affected by the GABRG2 gene mutations. Until now, few reports have directly investigated the relationship between GABRG2 and these factors; as such, the specific mechanism remains unclear and further studies are needed. This suggests that in addition to GABRG2 mutations, we need to pay attention to the changes of these factors in the study of genetic epilepsy, which may provide help for the treatment of GEFS+. Potassium channel gene therpy has been successfully applied to rodent models of focal epilepsy[152–154].

Fig. 5.

Summary network showing protein–protein interactions (PPI) network as identified by the STRING database. Only essential interacting partners are shown. The PPI network of the eighteen hub genes associated with GABRG2

Conclusion and future perspectives

In this review, we highlighted the different clinical representations resulting from different mutation types of the GABRG2 gene. GABRG2 mutations are influenced by a variety of factors in the process of GEFS+, including genetic regulation, apoptosis, inflammatory response, cell signaling, transcription factors, and proteins with other functions. However, there are still many problems worth exploring for the clinical diagnosis and treatment of GEFS+ caused by GABRG2 mutation. As a disease caused by mutations in the genes encoding chloride channels, GEFS+ displays genetic characteristics and clinical manifestations distinct different from those of classic monogenic epilepsy. The pathophysiological mechanism and precise diagnosis and treatment of hereditary epilepsy still depend on the progress of gene and molecular diagnoses. The maturation of gene cloning methods and clinical application of CRISPR/Cas9 gene editing technology will also provide a new theoretical basis for the diagnosis, treatment, and prevention of GEFS+.

Abbreviations

GEFS+

Genetic epilepsy with febrile seizures plus

FS

Febrile seizures

GTCSs

Generalized tonic–clonic seizures

GABRG2

Gamma-aminobutyric acid type A receptor gamma 2 subunit

ICD

Intracellular domain

TMD

Transmembrane domain

ECD

Extracellular domain

MAE

Myoclonic absence epilepsy

mIPSCs

Micro-inhibitory postsynaptic currents

RE

Rolandic epilepsy

SHE

Sleep-related hypermotor epilepsy

BI

Bayesian inference

ML

Maximum likelihood

CBD

Cannabidiol

DMF

Dimethyl fumarate

SAHA

Suberanilohydroxamic acid

CAE

Childhood absence epilepsy

IGE

Idiopathic generalized epilepsy

DEE

Developmental and epileptic encephalopathy

DS

Dravet syndrome

GGE

Genetic general epilepsy

SMEI

Severe myoclonic epilepsy in infancy

MS

Myoclonic seizure

Author contributions

XXL and TS were responsible for conceiving the manuscript ideas, SNG and YYS were in charge of the literature search and participated in the design of the review. JWD, CC, YHW and PDL contributed to the initial draft. XJW initiated the study and revised the manuscript. The content of this article was made by consensus of all the authors. All the listed authors contributed substantially, directly, and intellectually to the work and approved it for publication.

Funding

The work described in this article was supported by the China Association Against Epilepsy (Grant No. CU-2022-002) and the National Natural Science Foundation of China (Grant No. 81971085).

Data availability

Not applicable.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.