The potential implication of MDR1 and NAC1 genetic polymorphisms on resistance to antiepileptic drugs among a Jordanian epileptic population: a cross-sectional study

Abstract Background Resistance to antiepileptic drugs (AEDs) remains one of the main challenges to neurologists. Polymorphisms of drug efflux transporters such as multidrug resistance (MDR1) gene and target sites such as the nucleus accumbens–associated 1 (NAC1) gene have been suggested to influence the responsiveness to treatment. Aim Evaluation of the association of MDR1 and NAC1 polymorphisms with AEDs resistance among Jordanian epileptic patients. Subjects and methods 86 Jordanian epileptics were included in the study. DNA was extracted and genotyping was conducted by polymerase chain reaction followed by sequencing. Nine single nucleotide polymorphisms (SNPs) on the MDR1 gene and six SNPs on the NAC1 gene were investigated. Results MDR1 and NAC1 polymorphisms don’t seem to influence the resistance to AEDs at the genotype or allele level. However, a strong association was found between MDR1 rs2032588 (OR = 5; 95%CI = [1.3–18.8], p = 0.01) and AEDs resistance among males at the allele level. Also, data revealed an association between MDR1 rs1128503 and AEDs resistance among females at the allele level. Conclusion The data suggest that MDR1 and NAC1 polymorphisms do not influence the AEDs resistance among Jordanian epileptics. However, there is a gender-dependent association between MDR1 polymorphisms and resistance to AEDs at two SNPs (rs2032588 and rs1128503).


Introduction
Epilepsy is a serious, chronic, and episodic neurological disease characterised by recurrent unprovoked epileptic seizures as a result of an abnormality in the neuronal activity in the brain (Falco-Walter et al. 2018) and affects over 70 million people worldwide (L€ oscher et al. 2020).According to the statistics of 2016, approximately 28,226 individuals suffered from epilepsy in Jordan (Beghi et al. 2019).Despite significant efforts in the treatment of epilepsy, about one-third of epileptic patients have refractory to pharmacotherapy (Kwan and Brodie 2000), impacting daily activity, social well-being, economic output, and quality of life for patients and their families (S anchez et al. 2010).The underlying mechanisms of resistance to antiepileptic drugs (AEDs) in drug-resistant epilepsy are still not fully understood (Potschka et al. 2001).The treatment failure depends on many elements including age; seizure type; type of epileptic syndrome; epilepsy aetiology; medical history; genetic factors influencing antiepileptic drugs pharmacokinetics or pharmacodynamics, or both (Fisher et al. 2014).
Two main hypotheses are potentially involved in the biological mechanism of AEDs resistance: target and transporter hypotheses.The transporter hypothesis, a potential mechanism for resistance, supports the over-expression of multidrug resistance (MDR1), recently named ATP-binding cassette subfamily B member1 (ABCB1), in blood-brain barrier endothelial cells in patients with epilepsy (Sisodiya 2003).The MDR1 gene is found in the human genome's chromosome 7p21 and consists of 29 exons (Sauna et al. 2007).The target hypothesis contends that there are alterations in drug intracellular target sites (receptors) such as nucleus accumbens-associated 1 (NAC1), also named voltage-gated sodium channel alpha subunit 1 (SCN1A) gene, resulting in decreased response to sodium channel blockers-Antiepileptic drugs (SCB-AEDs) (Sisodiya 2003).The NAC1 gene is located on chromosome 2q24 and contains 27 exons (Wallace et al. 2001).NAC1 has not only a possible causal role in epilepsy but also a potential influence on the efficacy of AEDs (Weale et al. 2003).The sodium channels reveal a large pore-forming alpha subunit associated with two smaller beta subunits (Baum et al. 2014;Catterall 2014).These ion channels are molecular targets for SCB-AEDs (Parker et al. 2016) that have been a cornerstone in the treatment of focal and generalised tonic-clonic seizures for more than 70 years.SCB-AEDs bind the alpha subunit (Kwan et al. 2008) which blocks ionic conductance through these channels (Kwan et al. 2008;Kumari et al. 2011).
Many previous studies have found that genetic polymorphism in the MDR1 and NAC1 genes is associated with drug-resistant epilepsy at different nucleotide positions.However, the results have not been consistent enough to confirm this association.The relationship between SNPs and refractory epilepsy in the two genes needs to be studied in different ethnicities, as there may be important differences in gene frequencies that influence ethnic populations differently.

Subjects
One hundred patients were invited to take part in our crosssectional study if they were diagnosed as epileptic patients by neurologists; treated with antiseizure for at least twelve months; and attending a neurology clinic at Al-Basheer Hospital, Amman, Jordan.The patients were recruited over a period of 3 months (June-September 2018).Eighty-six patients were accepted and fulfilled the recruitment criteria.We excluded from the study patients with known epilepsyrelated genetic abnormality and those who were non-adherent to AEDs.Adherence to medications was assessed utilising the Morisky scale (Moon et al. 2017).The confidentiality and rights for all participants in our study were maintained and written informed consent was provided to all of the subjects.The approval of the study was done by the School of Pharmacy Scientific Research committee, Deanship of Academic Research, and the Institutional Review Board (IRB) of Al-Basheer Hospital and the Ministry of Health in Jordan (MBA/IRB/8147 on May 27th, 2018).
Clinical information including age, gender, clinical history of the patient and his or her family, lifestyle information, date of starting treatment with antiepileptic drugs and other relevant information was gathered for each patient.We collected all these data from patient interviews or medical records.Positive family history was defined as the incidence of epilepsy in first-degree relatives.The resistance to AEDs was defined by the International League Against Epilepsy (ILAE) (Kwan et al. 2010).The participants were considered as drug-resistant if the treatment with monotherapy or in combination was accurately administered for at least one year at full adequate doses and epileptic seizures persisted (Ajmi et al. 2018).While the patients were presumed as drugresponsive if they were free from seizures for at least 12 months during treatment with monotherapy or in combination (Ajmi et al. 2018).
Appropriate reverse and forward primer sets were prepared (Table 1).The conditions of PCR were as follows: 2 ml (50-100 ng) of DNA was amplified by adding 10 ml of OneTaqV R Quick-LoadV R master mix, the forward and reverse primers, 1.5 ml (10 mM) and 3.5 ml of water for injection.Based on master mix protocol and several optimisations, the thermal cycler program was as follows: an initial denaturation step at 94 C for 3 min followed by 39 cycles of denaturation at 94 C for 30 s, annealing for 15 s at 62 C, an extension step at 68 C for 60 s, and a final extension step at 68 C for 5 min.PCR products were visualised on agarose gel (2%) stained using RedSafeV R .Sharp PCR products were sequenced using the Sanger sequencing technique by GENEWIZ Technical Support Group, USA (http://www.genewiz.com).

Data management and statistical analysis
The statistical analysis for data was realised using Statistical Package for the Social Sciences (SPSS) software version 22 (SPSSV R Inc., Chicago, USA).We used Chi-square or Fisher's exact test, as appropriate, to compare discrete variables such as clinical factors, and allelic and genotypic distribution between the responsive and non-responsive groups.Results were expressed as counts and frequencies.The allelic and genotypic frequencies for different alleles among participants were estimated from the results of the sequencing and read using Chromas Lite software version 2.1.1 (Skalska et al. 2015).Genotypic and allelic frequencies were analysed in concordance with the Hardy-Weinberg equilibrium (Chen et al. 2017).We evaluated the strength of associations between genotype/haplotype, and drug-resistance by odds ratios (OR) and 95% confidence intervals (95% CI).Results were considered to be statistically significant if the p-value was less than 0.05.

Subjects' description and clinical characteristics
In this study, a total of 86 epileptic patients were enrolled; 56 (65.1%) were classified as drug-resistant and 30 (34.9%) as drug-responsive.Only 72 out of the 86 patients were taking SCB-AEDs and 21(29.2%) of these patients were classified as drug-responsive, while 51 (70.8%) were drug-nonresponsive.
At the patients' interviews, the mean (SD) for the ages was 30.4 (±12.6)years, whereas the mean age at the seizure onset was 19.9 (±12.9)years.There was no significant difference in gender: 46 males (53.5%); and 40 females (46.5%).
Comparisons of the demographic and clinical characteristics between drug-responsive and drug-nonresponsive epileptics are shown in Table 2.There were no differences between the two groups according to gender, age, or family history.Resistance to AEDs was 3.7 times more likely to be observed in patients treated with more than one AED (OR ¼ 0.27; 95%CI ¼ [0.11 À 0.7], p ¼ 0.006).Patients taking carbamazepine were 5 times more likely to be observed in the resistant group than those not taking carbamazepine (OR ¼ 5; 95%CI ¼ [1.4-10], p ¼ 0.01).This is because carbamazepine is more frequently prescribed in bitherapy and more (Table 2).

Association of MDR1 polymorphisms with resistance to AEDs
The distribution of MDR1 genotypes and alleles is listed in Table 3.The results of genotyping for all polymorphisms were consistent with the Hardy-Weinberg equilibrium (p > 0.05).The analysis showed none of the studied genotypes or alleles were associated with resistance to AEDs.However, we re-analysed associations within blocks containing either males or females to overcome the unequal distribution of gender between the two groups.The data based on patients' gender (Table 4), revealed a statistically significant association among males in rs2032588 at the allele level; males with T alleles were 5 times more likely to be observed in the non-responsive group compared to those with C alleles (OR ¼ 5; 95%CI ¼ [1.3-18.8],p ¼ 0.01).With regards to C1236T (rs1128503), the analysis revealed that females with C alleles were 2.5 times more likely to be in the non-responsive group compared to those with T alleles (OR ¼ 2.5; 95%CI ¼ [1.02-6.5], p ¼ 0.04).

Association of NAC1 polymorphisms with resistance to AEDs
The frequencies of genotypes and alleles of the aforementioned NAC1 SNPs are shown in Table 5. Hardy-Weinberg equilibrium for all polymorphisms was observed (p > 0.05).None of the study's SNPs were significantly associated with SCB-AEDs resistance.However, to overcome the unequal distribution of gender, researchers re-analysed associations within blocks containing either males or females.Stratifying data based on patients' gender (Table 6), revealed none of the studied genotypes or alleles were associated with resistance to AEDs.

Discussion
One of the main challenges for neurologists in epilepsy care is the resistance to AEDs (Mosh e et al. 2015).The variation in response to AEDs among epileptic patients receiving the same therapy can be directly related to the genetic profile.The resistance to AEDs has been hypothesised to involve the over-expression of drug efflux transporters, such as MDR1, at the blood-brain barrier reducing antiseizure drug concentration that affects treatment response (L€ oscher et al. 2020).Several previous studies indicate that antiseizure drugs are MDR1 substrates, including carbamazepine (Potschka et al. 2001), phenytoin (Potschka and L€ oscher 2001), lamotrigine, and others (Potschka et al. 2002).The variability in AEDs response may be associated with genetic polymorphisms of the MDR1 gene.Furthermore, the effectiveness of SCB-AEDs can be impacted by genetic polymorphisms of the NAC1 gene altering the pharmacological goal sensitivity (Weale et al. 2003).As a consequence, the detection of MDR1 and NAC1 genetic polymorphisms may lead to early recognition of patients at high risk of AEDs resistance and the initiation of individualised pharmacotherapy for epileptic patients (Bao et al. 2018).
There are previous studies conducted with genes other than MDR1 and NAC1.Eltalal et al. revealed Egyptian epileptic patients with CYP2C9 Ã 2(TT) genotype were more likely to develop resistance to AEDs (Eltalal et al. 2021).Another study suggested that ABCC2 rs717620 was associated with the poor response to AEDs, and none of the ABCC2 haplotypes The prevalence of refractory epilepsy is affected by definition of resistance to antiepileptics.In the current epileptics' cohort, the resistance varied dramatically from 11.6% to 65.1% based on the used definitions.If utilising a definition which describes the resistance as "the occurrence of at least three seizures at maximum tolerated doses over the year prior to recruitment with trials of more than three effective antiepileptic drugs", the non-responders would be only 11.6% (Kim et al. 2009).Whereas if utilising a definition used in Eltalal et al. which defines AEDs resistance as "the occurrence of at least four seizures over one year with three appropriate AEDs", the resistant patients would be 53% (Eltalal et al. 2021).However, the figure rises to 65.1% of the samples when using the ILAE definition which states that patients are considered to be drug-resistant if the treatment with monotherapy or in combination accurately administered for at least one year at full adequate doses and epileptic seizures persist (Scheffer et al. 2018;Ajmi et al. 2018).
The aim of our study was to evaluate the association of genetic polymorphisms of MDR1 at nine specific positions, and NAC1 at six positions, with resistance to AEDs and we found no association between these polymorphisms and refractory to AEDs.Researchers succeeded in overcoming imbalanced gender distribution between the two groups by re-analysing associations within blocks containing either males or females.Gender-based re-analysis revealed significant findings only at two SNPs of MDR1, rs1128503 and rs2032588.Regarding C1236T(rs1128503), the well-known MDR1 SNP, it is associated with resistance among females but not among males or the whole sample irrespective of gender.Resistant epileptic patients were more likely to have the C alleles than T alleles compared with responsive patients.The lack of association in the whole sample has been confirmed in previously conducted research.Twenty studies in the meta-analysis by Li et al. examined the association between MDR1 C1236T and resistance to AEDs.The findings revealed that there was no evident interaction between the variant C1236T and resistance to AEDs irrespective of studied ethnicities (Li et al. 2015).However, the hypothesis that MDR1 C1236T polymorphisms affected the efficacy of therapy in epileptic patients was supported by a few studies (Sun et al. 2016;Chouchi et al. 2019).
Regarding genetic polymorphism of MDR1 at rs2032588 position, the results of the current study showed resistance among males but not among females or the whole sample irrespective of gender.No previous studies reported its impact on the responsiveness to AEDs and our study is the first study to establish such an association among males with relatively good strength (OR ¼ 5).The gender-dependent results of the current study may be related to gender differences in response to pharmacotherapy which has been reported with many proteins (Anderson 2008).An Iranian study reported a lack of association between C1236T(rs1128503) and AEDs responsiveness.However, gender-based analysis revealed significant associations among females but not males.Females with 1236CC/CT had an increased risk for resistance to drugs (Maleki et al. 2010).
There are limited previous studies regarding the rs2235048 position (Escalante-Santiago et al. 2014;Gao et al. 2019); these two studies did not reveal any association between the SNP and resistance to AEDs, and the results are consistent with those from the current study.With regards to the relationship between MDR1 C3435T(rs1045642) polymorphisms and responsiveness to AEDs, the literature reveals indecisive results.Many studies have confirmed no association between drug-refractory epilepsy and the genetic polymorphisms of MDR1 C3435T.Two Turkish studies (Ozgon et al. 2008;Alpman et al. 2010), and one study from Korea (Kim et al. 2006) did not find any correlations.Alternatively, this association has been supported by other studies (Siddiqui et al. 2003;Hung et al. 2007;Ajmi et al. 2018).A comprehensive meta-analysis that included 57 studies, suggested that patients with a C allele in C3435T position had a significantly higher risk of resistance to AEDs among Caucasians, but not among Indians or Asians (Li et al. 2015), a result that is not consistent with the results from our study.This finding is supported by the study by Hoffmeyer et al. which hypothesised that the C allele is correlated with increased activity of the MDR1 protein (Hoffmeyer et al. 2000).However, a study from Tunisia published in 2018, proposed the T allele as the allele responsible for AEDs resistance among Tunisian epileptic patients (Ajmi et al. 2018).
The association of genetic polymorphism of MDR1 G2677T/A (rs2032582) and resistance to AEDs has been supported by some studies (Subenthiran et al. 2013;Yu et al. 2015;Ajmi et al. 2018).A meta-analysis by Yu et al. reported that ethnicity has an effect on MDR1 at G2677T/A position and the response to AEDs; it revealed that Asians have a significantly increased risk of drug-resistance epilepsy (Yu et al. 2015).The conflicting findings of the association between studied MDR1 SNPs and resistance to AEDs, as confirmed from published studies, may be attributed to differences in Fisher's exact test or chi-square test (as appropriate) were used to estimate p-value; p: probability.p-value of <0.05 is considered significant.Values expressed as N and %; N: number; % was calculated out of available data.OR: odds ratio; CI: confidence interval.Odds ratio and its 95% CI was not calculated for p > 0.05.
frequencies of the genotypes/alleles among different ethnicities.Many previous types of research have indicated that the frequencies of the genotypes of different genes that have been studied among the Jordanian population are similar to those reported among Caucasians (Yousef et al. 2012;Al-Motassem et al. 2015;Abuhaliema et al. 2016;Amrani et al. 2016;Shafagoj et al. 2018).Regarding NAC1, our observations suggest none of the examined SNPs modulate the response to AEDs.Interestingly, to date, there have been no previously published studies that investigated the effect of NAC1 SNPs (rs3812719, rs2195144, and rs2217199) on the SCB-AEDs resistance.Further investigations among different ethnicities are needed for the confirmation of any association.Regarding the rs2298771 position, several studies investigated the impact of this SNP on response to antiseizure drugs and have yielded indecisive results.A meta-analysis by Haerian et al. of four Asian studies, three from Malaysia and one from Hong Kong, revealed there was no significant association at the level of alleles, or genotypes, with drug  Indian; 2 Han Chinese; 1Italian) in which A allele was significantly associated with resistance to SCB-AEDs, while carriers of AA genotype had significantly increased risk of drug-resistance epilepsy (Bao et al. 2018).An Egyptian study reported that the frequency of G allele and AG genotype was significantly higher in the resistant cohort than that in responsive one (Abo El Fotoh et al. 2016).The conflicting results could be attributed to the differences in ethnicity (Manna et al. 2011;Wang et al. 2014;Bertok et al. 2017), and anti-epileptic regimens (Hung et al. 2012;Shi et al. 2019).
Regarding NAC1 rs3812718, our study showed that there is no association with resistance to AEDs.Previous studies regarding this SNP revealed conflicting results.The Haerian et al. meta-analysis reported that none of the examined studies (1 Japanese; 1 Spanish; 1Italian; 1 Indian; 3 Malaysian and 1 Hong Kong) revealed a significant association with resistance to SCB-AEDs in the context of NAC1 rs3812718 polymorphism (Haerian et al. 2013).Alternatively, T. Abe et al. demonstrated the association between the AA genotype and lack of response to Carbamazepine among Japanese epileptic patients (Abe et al. 2008).On the other hand, three studies (British, Chinese, and Greek) found the carriers of AA and AG genotypes in rs3812718 have the maximum maintenance doses of carbamazepine and phenytoin (Tate et al. 2005(Tate et al. , 2006;;Angelopoulou et al. 2017).The discrepancy between the present results and those of previous studies may be due to the use of different medications and dosing strategies.Regarding rs1972445, only one previous study (Yip et al. 2014) reported the impact of NAC1 at this position on responsiveness to multiple AEDs and the result were not consistent with those from our study.
The discrepancy between the two genes studies may be because of differences in sample size.Another cause of inconsistency in the results may be the use of variant definitions of "resistance to AEDs" by the different studies (Ebid et al. 2007;Ozgon et al. 2008;Alpman et al. 2010;Sayyah et al. 2011;Li et al. 2015;Chouchi et al. 2019).Other factors may also have an effect, such as differences in inclusion/exclusion criteria, the ethnicities of the study populations, the heterogeneity of epilepsy, interactions between unstudied genes, or interactions of the genes with environmental factors.Our study has the strength of considering new polymorphisms in the NAC1 gene, however, it also has some limitations: (a) a small sample size; and (b) the study was performed among only Jordanians which may explain the inconsistent results.In conclusion, the results of the current study suggest a gender-dependent correlation between MDR1 genetic polymorphisms and resistance to AEDs.Additionally, our study reported valuable information that suggests no association of NAC1 polymorphisms with response to SCB-AEDs.Further prospective cohort studies with larger samples from different ethnicities are required to confirm our results and to draw findings from different comparisons.
were associated with resistance to AEDs(Al-Eitan et al. 2019b).Additionally, Al-Eitan et al. found that the GRM4 rs2451334 GG genotype was distributed more among good responsive epileptic patients (Al-Eitan, Al-Dalalah, and Aljamal 2019c).On the other hand, Al-Eitan et al. confirmed no association between eight SNPs within CYP3A5, CHRM2, and ZNF498 genes and responsiveness to AEDs treatment (Al-Eitan et al. 2019a).Moreover, Al-Eitan et al. suggested that KCNA1 and KCNV2 SNPs do not influence drug responsiveness in epileptic patients (Al-Eitan et al. 2018).
Tm: melting temperature as calculated by primer-BLAST software program.

Table 2 .
Comparison of clinical and patient's characteristics according to response to AEDs.Values expressed as N and %; N; number; % was calculated out of available data.AEDs; antiepileptic drugs.Fisher's exact test or chi-square test (as appropriate) were used to estimate p-value p, Probability.p-value of <0.05 is considered significant.OR: odds ratio; CI: confidence interval.Odds ratio and its 95% CI was not calculated for p > 0.05.

Table 3 .
Genotype and allele frequencies of MDR1 polymorphisms.

Table 3 .
Continued.Fisher's exact test or chi-square test (as appropriate) were used to estimate p-value, p, Probability.p-value of <0.05 is considered significant.Values expressed as N and %; N: number; % was calculated out of available data.

Table 4 .
Comparison of response with MDR1 genotypes according to the patient's gender.

Table 5 .
Genotype and allele frequencies of NAC1 polymorphisms.Fisher's exact test or chi-square test (as appropriate) were used to estimate p-value; p: Probability.p-value of <0.05 is considered significant.Values expressed as N and %; N: number; % was calculated out of available data.
(Ghazala et al. 2022)leptic patients(Haerian et al. 2013).Moreover, in Arabic regions, a more recent publication, from Ghazala,et al. revealed no significant association between responsive and resistant groups at the level of genotypes and alleles among Egyptian children with epilepsy(Ghazala et al. 2022).On the other hand, a later meta-analysis conducted by Yi Bao et al. confirmed this association through four studies (1

Table 6 .
Comparison of response with NAC1 genotypes & alleles according to the patient's gender.
as N and %; N: number; % was calculated out of available data.