The drug-transporter gene MDR1 C3435T and G2677T/A polymorphisms and the risk of multidrug-resistant epilepsy in Turkish children

One-third of all individuals with epilepsy are resistant to antiepileptic drug (AED) treatment. Antiepileptic treatment response has been suggested to be modulated by genetic polymorphisms of drug efflux transporters. Several polymorphic variants within the multidrug resistance 1 (MDR1) gene, which encodes the major transmembrane efflux transporter P-glycoprotein, have been proposed to be associated with AED resistance in epilepsy patients. The aim of this study was to evaluate the effect of C3435T and G2677T/A polymorphisms of MDR1 on AED resistance in Turkish children with epilepsy. MDR1 C3435T and G2677T/A were genotyped in 152 patients with epilepsy, classified as drug-resistant in 69 and drug-responsive in 83. Genotypes of the C3435T and G2677T/A polymorphisms were determined by polymerase chain reaction followed by restriction fragment length polymorphism. Genotype and allele frequencies of C3435T and G2677T/A polymorphisms of the MDR1 gene did not differ between drug-resistant and drug-responsive epilepsy patients. Our results suggest that MDR1 C3435T and G2677T/A polymorphisms are not associated with AED resistance in Turkish epileptic patients. To clarify the exact clinical implication of the MDR1 polymorphisms on the multidrug resistance in epilepsy, further investigations in various ethnic populations would be necessary.


Introduction
Epilepsy is one of the most prevalent serious chronic neurological disorders, effecting approximately 1-2 % of the population [1]. About 30 % of patients with epilepsy do not respond to any of two to three first line antiepileptic drugs (AEDs) despite considerable advances in the therapy of epilepsy and these patients will develop refractory epilepsy [2]. Although efforts to predict pharmacoresistance have revealed several risk factors (an early onset of epilepsy, symptomatic or cryptogenic epilepsy, and generalized seizure) [3,4], the mechanisms underlying the resistance to AEDs in the epilepsy treatment are still not well-understood. Some potential mechanisms have been suggested regarding the transporters of the AEDs. There is accumulating evidence to suggest that an increase of functional expression of multidrug transporters produces pharmacokinetic changes that modify the access of AEDs to central nervous system targets [5]. Genetic polymorphisms in these transporters could account for their increased expression and/or functional activity. These genetic variations could be linked to changes on pharmacokinetic or pharmacodynamic of AEDs and effective responsiveness to AEDs [6].
The ATP-binding cassette (ABC) transporters, and especially ABCB1, are the focus of an international effort to establish their role in mediating drug resistance in a variety of human diseases including epilepsy [7]. Human multidrug resistant transporter gene (MDR1, also known as ABCB1), which is a member of the ABC superfamily, encodes a 170-kDa P-glycoprotein (P-gp) [8]. The P-gp is of particular interest, and was first identified in cancer cells, as a protein responsible for resistance to a wide variety of AEDs [9]. The transmembrane P-gp that functions as a multispesific drug-efflux transporter is considered to be associated with drug absortion and elimination. It is hypothesized that overexpression of an efflux transporter such as P-gp around an epiloptegenic focus, via reducing AED accumulation, may prevent AEDs from entering sites of action [10].
The expression, efflux, substrate spesificity, and mRNA stability of P-gp are influenced by various single nucleotide polymorphisms (SNPs) in the encoding gene, MDR1 [11]. Evidence is present for supporting the important role played by more gene polymorphisms on the function of P-gp via effecting its activity [12,13]. The MDR1 gene is highly polymorphic with more than 50 variants residing in the coding region and, which can possibly cause altered function [14].
A great number of studies were focused on two most common SNPs, C3435T (Exon 26, rs1045642) and G2677T/A (Exon 21, rs2032582) SNPs, to explore the associations with disease development and drug response in different populations [10]. The synonymous C3435T polymorphism was the first SNP, which has been shown to correlate with the expression levels and function of P-gp [15], although conflicting reports have subsequently appeared showing no correlation with the expression levels and function of P-gp [12]. The C3435T polymorphism is in linkage disequilibrium with nonsynonymous G2677T/A polymorphism (Ala893Ser/Thr), which may also contribute to the observed functional effect of variation at the C3435T polymorphism or at associated haplotypes [16]. Furthermore, the G2677T/A SNP has been shown to be associated with drug resistant epilepsy [17,18]. Thus, the MDR1 has been regarded as an interesting candidate gene potentially influencing the response to AED treatment.
Based on the hypothesis that MDR1 genetic polymorphisms can modulate drug response phenotype in epilepsy, the present study was carried out to evaluate the association of two SNPs [3435C [ T (rs1045642) and 2677G [ T/A (rs2032582)] of the MDR1 gene in drug-responsive and drug-resistance patients in a Turkish population.

Study population
A total of 152 Turkish children with epilepsy treated with AEDs were included in this study. The patients were diagnosed and classified according to the guidelines of the International League against Epilepsy. Patients, who had an established clinical diagnosis of epilepsy (defined by the occurrence of two or more unprovoked seizures) confirmed by an electroencephalogram examination by an expert were included in the study. Exclusion criteria consisted of severe adverse drug reactions, poor compliance with AEDs, history of pseudo-seizures, alcohol or drug abuse, or any other malignant diseases such as brain tumor, secondary metastasis, hepatic failure, or renal failure.
Patients were included if they had either drug-resistant or drug-responsive epilepsy according to the following definitions and criteria. The main criterion for drug resistance was the occurrence of at least four seizures over a period of 1 year with three AEDs, such as carbamazepine ? valproate ? levetiracetam, or carbamazepine ? valproate ? diphenyl hydantoin, according to the epileptic seizure type at maximum tolerated doses. Of the 152 patients, 69 were classified as drug resistant (40 males, 29 females, mean age: 9.2 ± 3.6 years) and 83 were drug responsive (51 males, 32 females, mean age: 9.3 ± 3.8 years. The protocol for this study was approved by the Regional Ethics Committee and was carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans. An informed consent was signed by each participant or responsible adult.

Blood samples and DNA isolation
Venous blood samples (3 ml) were collected in EDTA vacutainer tubes for DNA extraction. Immediately after collection, whole blood was stored in aliquots at -20°C until use. Genomic DNA was extracted from leukocytes using Roche DNA purification kit (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer's instructions. (Applied Biosystems, California, USA) under the following conditions: For MDR1 C3435T polymorphism, initial denaturation at 94°C for 4 min, followed by 35 cycles each of 94°C for 30 s, 56°C for 30 s, 72°C for 30 s; and a final extension at 72°C for 5 min; for MDR1 G2677T/A polymorphism, initial denaturation at 94°C for 3 min, followed by 40 cycles each of 94°C for 20 s, 55°C for 20 s, 72°C for 30 s; and a final extension at 72°C for 5 min. The PCR products were digested by restriction endonucleases of MboI at 37°C (for C3435T), BanI at 37°C (for G2677T) and BsrI at 65°C (for G2677A) for 3 h and analyzed with 3 % agarose gel electrophoresis.

Statistical analysis
Mean and standard deviations (SDs) are presented in case of continuous variables. Differences between the means of the two continuous variables were evaluated by the Student's t test. Chi square (v 2 ) or Fischer's exact test (twosided) was used to compare the association between the genotypes and alleles in relation to the cases, and test for deviation of genotype distribution from Hardy-Weinberg equilibrium. P values of \0.05 were considered statistically significant. The odds ratio (OR) and their 95 % confidence intervials (CIs) were calculated to estimate the strength of the association. Statistical analysis was performed using a statistical software package, SPSS 16.0 (SPSS Inc, USA). Haplotype frequency analysis and linkage disequilibrium for the two MDR1 polymorphisms were performed by using the Haploview 4.2 program.

Results
Basic demographic and clinical charecteristics of epilepsy patients with drug-resistant and drug-responsive are presented Table 1. The groups were not significantly different with respect to the age and gender (all values of P [ 0.05).
No significant deviations from Hardy-Weinberg equilibrium were observed in epilepsy patients (P = 0.68 for MDR1 C3435T and P = 1.00 for MDR1 G2677T/A). Table 2 shows the genotype and allele frequencies of the polymorphisms in MDR1 gene for both drug-resistant and drug-responsive epilepsy patients. The distribution of MDR1 C3435T and G2677T/A genotypes within drugresistant (P = 0.76 and P = 1.00, respectively) and drugresponsive (P = 0.88 and P = 0.98, respectively) groups were not significantly different from that under Hary-Weinberg equilibrium. No statistically significant differences were detected for the genotype and allele frequencies of the polymorphisms in the MDR1 gene between the drugresistant and drug-responsive groups. Table 3 lists the haplotypes of MDR1 polymorphisms in the drug-resistant and drug-responsive epilepsy patients. Haplotype frequencies of these polymorphisms in the MDR1 gene did not differ significantly between drugresistant and drug-responsive epilepsy patients. The linkage disequilibrium test of two SNPs in MDR1 (Rs1045642 and rs2032582) were in strong linkage disequilibrium (D 0 = 0.991).

Discussion
Drug-resistant epilepsy patients tend to be resistant to a broad range of AEDs with a varied mode of action [5,7]. The functional characteristics of non-specific drug transporter proteins could explain broader resistance across types of epilepsy and to a range of AEDs [21]. Among these transporter proteins, P-gp is considered as one of the most potent contributer to the phenomenon of clinical drug resistance to AEDs.
Various genetic polymorphisms in the MDR1 gene could be linked with altered in vivo transport activity of P-gp which may play a facilitatory role in drug-resistant epilepsy [6]. These genetic polymorphisms among epilepsy patients are likely to contribute to different responses in patients, who have similar epilepsy syndromes and are taking similar, or even the same, doses of medication.
So in the present study, we investigated two SNPs in MDR1 gene in drug-resistant and drug-responsive epilepsy patients. Results of the present study demonstrated that genotype and allele frequencies of C3435T and G2677T/A polymorphisms of the MDR1 gene did not differ between drug-responsive and drug resistant epilepsy patients. Furthermore, the results of this study also showed no haplotype association of these MDR1 polymorphisms with drug-resistant epilepsy.
The results from various studies focusing on the role of MDR1 C3435T and G2677T/A polymorphisms in AED responsiveness are conflicting. Consistent with the present study, several studies have found no significant associations between drug-resistance epilepsy and the MDR1 C3435T and G2677T/A polymorphisms [22,23]. Two studies from Turkey also reported that there were no associations between the polymorphisms of the MDR1 gene and drugresistant epilepsy [24,25]. Similarly, Kim et al. [26] observed no association between the polymorphisms of G2677T/A and 3435C [T and drug resistance in Korean patients. However, contrary to the above studies' results, Hung et al. [17] found that 3435C[T and 2677G[T in the MDR1 gene contributed to drug-resistant epilepsy. Siddiqui et al. [27] demonstrated that the frequency of the 3435CC genotype among drug-resistant patients (27.5 %) was significantly higher than in drug-responsive patients or nonepileptic controls (15.7 or 18.5 %, respectively) among Caucasian subjects. In contrast to these results, Kwan et al. [28] reported that patients with drug-resistant epilepsy had a higher frequency of the 3435TT genotype (16.7 %) in comparison to drug-responsive epileptics or nonepileptic controls (10.6 or 7.4 %, respectively). Furthermore, Seo et al. also demonstrated that drug-resistant epilepsy patients were significantly more likely to have the T allele and the TT genotype at C3435T when compared to drug-responsive patients (50 vs 27; 36.9 vs 16.7 %, respectively). In addition, Seo et al. [29] found that patients with drug-resistant epilepsy had higher frequencies of the TT genotypes at G2677T/A in comparison to the drug-responsive patients (26.2, 23.8 %, respectively).
The differences observed in these reports may be due to the ethnic differences in genotype/allele frequencies. Genetic polymorphisms often vary between ethnic groups. Ethnic differences in genotype frequency for the investigated polymorphisms might effect the results in genetic studies. Distribution of MDR1 C3435T polymorphism also displays an ethnic difference. For example, MDR1 3435CC was less common than 3435TT in Caucasian patients (16-19 vs 27-33 % in drug-responsive patients) [22,27], while the opposite of this was observed in Han Chinese cohort (38 vs 7 % in drug responsive patients) [28], similar to other Asian populations [30]. Furthermore, contradictory data reported in these epidemiologic studies may be due to other several factors such as number of patients, combinations of susceptibility variants, the isolated nature of the study populations, heterogeneity of epilepsy and geneenvironment interactions.  The authors assessed the association of MDR1 haplotypes derived from C3435T, G2677T/A, and C1236T loci in response to AEDs in different epilepsy populations [31]. In consistency with our results, Alpman et al. [25] found that the haplotypes of C3435T and G2677T/A were not associated with drug resistant epilepsy in Turkish epileptics. Furthermore, Meng et al. [32] failed to detect any associations between drug-resistance and haplotypes of C3435T, G2677T/A, and C1236T polymorphisms. However, Siddiqui et al. [27] reported that the C-G-C haplotype of C3435T, G2677T/A, and C1236T was associated with the AED-resistance. Zimprich et al. [18] also identified that C-G-C haplotype significantly increased the risk for pharmacoresistance. Contrary to this, Seo et al. [29] found that patients with drug-resistant epilepsy had higher frequencies of the T-T-T haplotype at C3435T, G2677T/A, and C1236T than patients with drug-responsive epilepsy (42.1, 30.4 %, respectively).
In conclusion, our results indicate that MDR1 C3435T and G2677T/A polymorphisms are not associated with drug-resistant epilepsy in the study population. The limitation of the current study is the small sample size. To clarify the exact clinical implication of the MDR1 polymorphisms on the multidrug resistance in epilepsy, further investigations in various ethnic populations would be necessary.