Assessment of Gene Polymorphism in GABAA1 Receptor among Sudanese Patients with Juvenile Myoclonic Epilepsy (JME)

Introduction: Juvenile myoclonic epilepsy (JME) is an idiopathic generalized epilepsy syndrome (IGE) with a strong genetic contribution. The main characters of JME are generalized convulsive or an absences seizures proceeded by myoclonic jerks. Gene variant of gamma-aminobutyric acid type A inhibitory receptor speculated to underlay JME etiology. Objective: This study aimed to screen for JME based on the International League against Epilepsy Commission on Classification and Terminology diagnostic criteria and to assess the link of polymorphism in the GABAA receptor gene, GABRA1 to the development of JME in Sudanese patients. Methods: Our epidemiological study enrolled 44 JME patients, only 23 participated in the genetic part and 35 matched healthy controls were also included. Blood genomic DNA was isolated and PCR basedrestriction fragment length polymorphism (RFLP) analysis was done. The data obtained were analyzed using computer software SPSS 21rt edition. Original Research Article Abdalla et al.; JPRI, 33(33B): 13-19, 2021; Article no.JPRI.70076 14 Results: The frequency of the mutant G allele was found to be 41.5% in patients and 45.5% in the controls. The genotype distribution of A and G alleles among patients were found to be (AA= 39%), (AG=39%) and (GG= 22%) and that of the controls were (AA=40%), (AG=30%) and (GG= 30%). Conclusion: The mutant G allele of the GABAA1 receptor does not affect the development of JME in Sudanese patients but the AG genotype may be a risk factor.


INTRODUCTION
It is reported that Juvenile myoclonic epilepsy represents 10% of all idiopathic generalized epilepsies, the disease is characterize by jerks and generalized tonic-clonic seizures (GTCS). The instability of neural networks caused by ion channel abnormalities is generally assumed to represent the basis of the pathophysiology of JME, abnormal cerebral structure and function mainly involving the frontal lobes have been reported [1]. Various heritability models have been used to clarify the genetic basis of JME, those models include major Mendelian inheritance or concurrent contribution of multiple genes with minor effects in non-Mendelian fashion [2]. Based on genetic epidemiological studies the classical JME appears to has complex genetic traits caused by interaction of several genetic and environmental risk factors [3]. A range of (17-49)% of patients with JME have relatives with different epileptic seizures [4]. The risk of having clinically evident JME is small and the disease burden varies across spectrum of individuals. Many studies have presumed that JME is an autosomal dominant disease of incomplete penetrance which means that some individuals who inherit JME genes do not express clinical JME. However, their children may inherit the clinically phenotyped syndrome [5,6,7]. It is reported that monogenic forms of IGEs are associate with various mutations in genes encoding for voltage and ligand-gated ion channels including a mutation in type A gammaaminobutyric acid (GABAA) receptor genes [8,9]. The instability of neural networks caused by ion channel abnormalities is assumed to represent the basis of the pathophysiology of JME [9,10]. Among JME patients-based studies, the extensive existent of central nervous system GABA inhibitory genetic abnormalities acquired an enormous concern [11]. GABAA receptors are ligand-gated chloride channels and their molecular structure comprises a heteropentameric protein complex assembled from 19 different classes of subunits. Epilepsycausing mutations have been identified in GABRA1 [8,12] and GABRG2 [13,14,15].
Several single nucleotide polymorphisms (SNPs) in the GABAA receptor have been described but only the intronic GABRA1 IVS11 + 15 A > G and the exonic GABRG2 588C > T gene polymorphisms are found to have functional significance in different neurological disorders. These gene variants have been attributed to several susceptibility factors for febrile seizures [16,17]. The mutant receptors have low affinity for GABA and they showed reduced chloride current amplitude when they fully saturated compared to wild-type receptors. Both inactivation and re-activation kinetics were significantly enhanced in mutant channels. In at least two mutations in GABRG2 [13,15] and two mutations in GABRA1 [12,18] has shown the reduction in the amplitude of GABA-evoked current due to reduced surface expression of the receptor protein caused by retention of the mutant receptors in the endoplasmic reticulum [18]. This is consistent with loss of function mutations found in at least four different subunits of the GABAA receptor for related epileptic syndromes. These suggest impairment in the degree of inhibition mediated by GABAA receptors in the pathophysiology of the classical IGE with autosomal dominant inheritance. High genetic heterogeneity has been observed in early linkage studies on JME, it is likely that additional ion channel genes will be involved in JME. Genes encoding GABAA receptor subunits come at the top of the list of candidates for epilepsy susceptibility [19]. Based on functional significance, previous observations and current knowledge, in our genetic study we investigated the possible link of the GABRA1IVS11 + 15 A > G (rs2279020) polymorphism with susceptibility to juvenile myoclonic epilepsy among Sudanese patients.

Patients and Controls
This research study consisted of two parts, an epidemiological and a genetic study. The proposal of the study was approved by the Ethical Research Committee of the National .The diagnosis of JME is based on the International League against Epilepsy Commission on Classification and Terminology diagnostic criteria. Forty-four patients were diagnosed as JME patients based on their EEGs characteristic features (consisting of generalized 3.5-6 Hz single, bifid and polyspikes slowwaves complexes on normal brain background activity) and only twenty-three patients agreed to participate in the genetic study. The study also included 35 healthy controls that were matched with patients by age, gender, ethnicity and residence. The demographic data and subjects' clinical history were obtained using a predesigned interviewing questionnaire. None of the controls experienced epileptic seizures or had a neuropsychiatric disease. Three ml of venous blood samples were collected from patients and controls and were preserved at -20°C for the genetic analysis.

DNA isolation and PCR-RFLP Genotyping of GABRA1 (rs2279020)
The gene variants were detected by methods described by Chou IC et al. [15] and Park Cs et al. [20]. Genomic DNA was isolated from peripheral blood leucocytes using the standard salting-out method with slight modifications [21]. GABRA1 (rs2279020) genotyping was performed using PCR-RFLP analysis (Table 1). PCR conditions were as follows: a denaturing step at 95°C for 5 min, then 30 cycles at 94°C for 30s, the annealing temperature is 60°C for 30s, then 72°C for 30 s, and a final extension at 72°C for 7 min. PCR products of 165 bp was observed for GABRA1 (rs2279020) which was then digested using the specific restriction endonuclease AvaII (Fermentas Inc., USA) RFLP assay in GABRA1IVS11 + 15 A > G polymorphism was used to distinguish the A/G substitution at nucleotide 15 of the last intron and the products were separated on 2% agarose gel Figure.

RESULTS
The study revealed that out of the 3523 patients attended to our clinics, 1460 (41.4%) had normal EEGs (Fig. 1) while 2063 (58.6%) patients had abnormal EEGs (Fig. 2), of those patients only 44(2.13%) their EEGs showed the characteristics electrical features of JME with moderate females' predominance (56.81%) females and (43.2%) males (Table 2). Absence attacks were confirmed in (77.27%) of JME patients and the generalized tonic-colonic convulsions GTCS reported in (84.1%) of patients, with a mean age of onset (13.92 ± 5.65) years (Table 3). Of the JME patients, 92% showed no aura symptoms and postictal sleepiness was confirmed in (75.7%) of them, but all patients lost their consciousness. The mean age of JME patients at diagnosis was (19.55 ± 8.98) years; (75%) of them were between 10 and 30 years. Myoclonic jerks were detected in (90.9%) of patients with a mean age of onset (10.48 ± 4.81) years. Sleep disturbance was found in (40.9%), memory capacities declined in (79.5%), and school or work performance deteriorated in (86.4%). Of JME patients (75%) had positive family history of epilepsy; of whom (66.3%) were first degree, while (33.7%) were second-degree relatives. The results of the genetic study showed that for the GABRA1IVS11 + 15 A > G (rs2279020) polymorphism, the frequency of the A allele was found to be 41% in patients and 45% in controls and that of G allele was 41% among patients and 45% among the control group. The genotype distribution of A and G alleles for patient were (AA= 39%), (AG=39%) and (GG= 22%) and that of the controls were (AA=40%), (AG=30%) and (GG= 30%). The male: female ratio of patients was 2:2.5 and that of controls was 3:3.5 Table  [2].

DISCUSSION
At the time of our epidemiological study 44 (2.13%) of the patients were found to be typical cases of JME. Of those patients 77.3% showed the classical symptoms triad (jerks, absences and GTCS). This finding is inconsistent with Murthy et al. observation, who found it in 17.5 % of patients [22]. The significant distressing event for 84.1% of our patients was the generalized tonic-colonic convulsions; Ali et al. documented the GTCS in 94.1% of patients with JME [23]. In all Sudanese JME patients, the GTCS followed the MJs by 4.4 years compared to 3.3 years indicated by Asconape and Penry (1984). It was evident that most of our patients didn't come for specialized medical advice until they developed GTCS. As far as we know, this is the first study that assessed genetic polymorphism of GABRA1 (rs2279020) in Sub-Saharan African patients with JME. Our findings heightened the assumption of no relationship between G allele and JME phenotype. Genotyping disclosed the frequency of the mutated G allele among patients and controls was so close, 41.5% and 45%, respectively. It is also similar to the results of the studies carried out by Kananura et al. [14] and Chou IC et al. [15]. This consensus has been scaffolded by the Bruna Priscila dos Santos et al. [3] meta-analysis outcomes. There is no significant association between IGEs and either A or G alleles by reviewing over 50 populationbased genetic association studies investigating polymorphism with JME. But the inconsistency appeared when our results likened to Ritu Kumari et al. [24], who found that the muted G allele was significantly high in Indian patients with IGEs. This discrepant outcome might be due to our small sample size and also the effect of other environmental and genetic factors that supporting the speculation of non-Mendelian heredity. Considering the general population, including both patient (P/C) control individuals, the prevalence of homozygous and heterozygous carriers was AA (39% / 40%), AG (39% /30%), and GG (22%/30%). Showed no significant difference except for the AG genotype, which is higher by 10% in patients. These findings agree with the results obtained in India and China by Kananura et al. and Chou IC et al, respectively [14][15].

CONCLUSION
The prevalence of JME among Sudanese epileptic patients is not different from international reports with clues of family's occurrence. The mutant G allele of the GABAA1 receptor gene do not affect the development of JME in Sudanese patients. The AG genotype of GABAA1 receptor may be a risk factor for JME in Sudan.

LIMITATIONS AND RECOMMENDA-TIONS
We recommend a study with a large sample size, including detailed clinical investigations and genetic analysis of other subunits of GABA receptor, to demonstrate the role of the family history in the disease's occurrence in Sudan and to confirm our results.
The culture of research participation among the Sudanese population is feeble, particularly regarding their genetic research. More than half of our initial recruited patients (44 patients) refused to participate in the genetic analysis.

CONSENT AND ETHICAL APPROVAL
The proposal of the study was approved by the Ethical Research Committee of the National Ribat University in Khartoum. All participants or their legal guardians signed a written informed consent disclosing their rights and confidentiality guarantees before starting the study procedures.