Association between GABRG2 rs211037 polymorphism and idiopathic generalized epilepsies: a meta-analysis

We performed this meta-analysis to investigate the association between GABRG2 rs211037polymorphism and the risk for idiopathic generalized epilepsies (IGEs). Medline, Embase, Cochrane Library and Chinese National Knowledge Infrastructure (CNKI) databases were searched for eligible studies (until May 5, 2020) on the association between GABRG2 rs211037 polymorphism and IGE. The odds ratios were calculated using a fixed or random model in STATA 15.0 software. Subgroup analyses for ethnicity, age, source of controls, type of seizure syndrome and therapeutic responses were conducted. We found no significant associations between GABRG2 rs211037 polymorphism and the susceptibility to IGEs. In addition, no significant association was detected between GABRG2 rs211037 polymorphism and drug resistance in IGE patients. The results did not change after stratification by Asian population, healthy controls, children, juvenile myoclonic epilepsy, and childhood absence epilepsy. The current studies indicated that the GABRG2 rs211037 polymorphism was not related to susceptibility or drug resistance of IGE. Further well-designed studies are needed to verify the results.


Background
Idiopathic generalized epilepsies (IGEs) are featured by recurring generalized seizures without intracranial lesions [1], affecting 0.3% of the general population and accounting for 30% of all epilepsy disorders [1]. Epilepsy with generalized tonic clonic seizures (EGTCS), childhood absence epilepsy (CAE), juvenile myoclonic epilepsy (JME) and juvenile absence epilepsy (JAE) and were common syndromes of IGEs [2]. The causes of IGE are highly heterogeneous and genetic factors seem to be an important one.
Previous studies indicated that disruption of the gamma-amino butyric acid (GABA)-mediated inhibitory neurotransmission results in neuronal hyperexcitability, which is proved to participate in epileptogenesis [3][4][5]. Genetic alterations can influence channel function, increase neuronal excitability and induce neuronal networks into synchronous activity, and lead to in epileptic seizures [6]. Recent studies have identified GABRG2 genetic variants in relation with increased risk of epilepsy, of which rs211037 (C588T) is receiving much attention [7]. The GABRG2 rs211037 variant may alter the transcription and translation efficiency of GABAA receptor subunits, modify the composition of receptor, and influence the sensitivity to extrinsic environmental signals [8].
The association between GABRG2 rs211037 polymorphism and the risk for IGE have been investigated in several studies. Nevertheless, the results are inconsistent. Haerian BS et.al. conducted a meta-analysis with 4 studies included and found no significant association between GABRG2 rs211037 and the susceptibility to IGEs [7]. However, several studies investigating the association between GABRG2 rs211037 and IGEs since 2003. In the study, we performed an updated meta-analysis to assess the relationship between GABRG2 rs211037 and IGEs.

Literature search strategy
Eligible studies were searched in the databases of Medline, Embase, Wanfang, Cochrane Library and Chinese National Knowledge Infrastructure (CNKI) (until May 2020) using the following search keywords: 'epilepsy' OR 'seizure', 'polymorphism' OR 'variant' OR 'mutation', and 'GABARG2'. In addition, we searched the references of reviews and relevant studies. This meta-analysis was conducted according to Meta-analysis for Observational Studies in Epidemiology guidelines.

Inclusion and exclusion criteria
Two authors (Xiaohui Yang and Hongyun Wei) screened each eligible article independently, and a third author (Xiaosa Chi) would rejudge the article in case of any disagreement. The study inclusion criteria were: (1) assessing the relation between GABRG2 rs211037 polymorphism and IGEs; (2) case-control or cohort study; (3) providing sufficient data on genotype distributions; and (4) in English or Chinese language. Studies were excluded if they: (1) are reviews and meeting abstracts; (2) did not report sufficient data; and (3) are animal studies or in vitro experiments.

Data extraction
Baseline information were extracted in duplicates from all of the eligible studies by Xiaohui Yang and Hongyun Wei independently, and any dispute was figured out by discussion. The extracted data included: name of the Fig. 1 Flow chart of study selection. Ultimately, 11 articles were identified first author, publication year, country of origin, ethnicity, source of cases and controls, study period, genotyping methods, quality control, the numbers of cases and controls, and frequencies of genotype.

Statistical analysis
We calculate the pooled odds ratios (ORs) and corresponding 95%CI to estimate the strength of association between GABRG2 rs211037 polymorphism and the risk for IGE. The dominant model, recessive model, co-dominant model and allele model were conducted to evaluate the association. In addition, subgroup analyses were conducted for ethnicity, age, source of control (non-IGE or healthy control), JME, CAE, and drug resistance. We calculated Z test to evaluate the significance of pooled ORs, and regarded P < 0.05 as statistically significant.
We evaluated the inter-study heterogeneity by Cochran's Q test and I 2 statistic. I 2 ≥ 50% was considered as statistically significant. In this condition, the randomeffects model was applied to count pooled OR; otherwise, the fixed-effects model was applied. Moreover, publication bias was evaluated using visual inspection of Funnel plot which was obtained from Begg's test. All data was analyzed with STATA (version 15.0; Stata Corp, College Station, TX).  AS-PCR Allele-specific polymerase chain reaction, CAE childhood absence epilepsy, IGE childhood idiopathic generalized epilepsy, JME Juvenile myoclonic epilepsy, MassARRAY matrixassisted laser desorption/ionization time of flight mass spectrometry, NA not available, PCR-RFLP polymerase chain reaction-restriction fragment length polymorphism, RT-PCR reverse transcription-polymerase chain reaction, tSNP a tagging single nucleotide polymorphism
When stratified by seizure syndrome, we found no significant relationship in patients with CAE. Besides, the GABRG2 rs211037 polymorphism was not significantly related to the risk of JME.
We further performed a subgroup meta-analysis to explore the relationship between GABRG2 rs211037 polymorphism and drug resistance in patients with IGE, and no significant association was detected (TT + CT vs CC: OR = 1.64, 95%CI = 0.87-3.07, P = 0.002, Fig. 4).

Discussion
Previous studies concerning the role of GABRG2 in IGEs have come to controversial results, so in this study, we performed the meta-analysis to investigate the association of GABRG2 rs211037 polymorphism with the risk for IGE. The results showed no relationship between GABRG2 rs211037 polymorphism and IGE. Also no significant associations were found in subgroups of healthy  control, Asian ethnicity, JME, CAE or children. Furthermore, the GABRG2 rs211037 polymorphism was not significantly related to drug resistance. As a major inhibitory neurotransmitter, GABA affects neuronal excitability and network interactions in the brain [20]. Its binding to receptors leads to the influx of chloride ions through ion channels, resulting in fast postsynaptic inhibition in the brain [21]. Expression and conformation modification of GABAA receptors may be involved in epileptogenesis, such as mRNA lability, irregular subunit folding, distortions of the GABA channel, and glycosylation defects [22,23]. Mutations in GABRG2 affect the expression and trafficking of GABAA receptors to the cell membrane [4,24], and are reported to be associated with IGEs [25]. In addition, the γ2 subunit has been proved to influence the kinetics, synaptic and postsynaptic clustering and maintenance of GABAA-associated channels [26][27][28]. All these may contribute to epilepsy development. Moreover, lower amplitude of GABA-activated currents was detected in HEK293 cells which expresses the mutant GABAA receptor, suggesting that dysfunction of this receptor may lead to seizures [29]. Further studies are needed to explore the effect of GABAA in idiopathic epilepsy. In addition, we performed a subgroup analysis in Asian people, but failed to detect any association, which is inconsistent with previous studies showing that GABRG2 rs211037 polymorphism is associated with susceptibility to IGE in Asian people. More studies are needed to clarify this discrepancy. In addition, there are relevant few studies performed in Caucasian populations. Therefore, future studies should be performed with more ethnicities.
Moreover, no association was detected when stratified by subtype of epilepsy syndrome including CAE and JME. Seizure syndromes in IGE have common genetic predisposition but various clinical manifestations. The age at onset of the various subgroups included in IGEs differed. The reasons for the discrepancy between previous studies and ours in the associations of GABRG2 rs211037 polymorphism with IGE may be the lack of specific subtype analysis in previous studies. Meanwhile, a great many genes participated in the mechanisms of IGEs, and the effect of GABRG2 on IGEs may be little [13]. Therefore, genome-wide association studies are essential to explore the association between GABRG2 and IGEs.
In this meta-analysis, we also found no relation between GABRG2 rs211037 polymorphism and drug resistance. It has been reported that the expression of GABAA receptor subunit in drug-resistant rats differs from that in the drug-responsive rats of temporal lobe epilepsy [30]. Abou et al. showed that the GABRG2 rs211037 polymorphism may contribute to the resistance to antiepileptic drugs (AEDs) among Egyptian children [9]. Several AEDs such as topiramate, gabapentin, and phenobarbital target the GABAA receptor and alter the concentrations of GABA [31]. Nevertheless, relevant studies are still limited, and further research is needed to clarify the relationship and the underlying pathogenesis, which may contribute to identifying therapeutic targets for the development of novel AEDs.
Our study has some limitations. First, our searches were made in limited databases, thus other relevant paper might be missed out. Second, relatively few cases and controls were available in some subgroups, which may influence the power of the test. Third, in most studies Asian participants were employed, so our results may not be suitable for other ethnicities. Finally, further analysis of gene-gene and loci-loci interactions was not conducted on account of insufficient data.
In conclusion, we found no associations between the GABRG2 rs211037 polymorphism and the risk for IGE. Subgroup analysis stratified by ethnicity, age, healthy control and drug resistance provided similar results. Further well-designed case-control studies with larger sample sizes are needed.