Pharmacogenomic association study on the role of drug metabolizing, drug transporters and drug target gene polymorphisms in drug-resistant epilepsy in a north Indian population

BACKGROUND: In epilepsy, in spite of the best possible medications and treatment protocols, approximately one-third of the patients do not respond adequately to anti-epileptic drugs. Such interindividual variations in drug response are believed to result from genetic variations in candidate genes belonging to multiple pathways. MATERIALS AND METHODS: In the present pharmacogenetic analysis, a total of 402 epilepsy patients were enrolled. Of them, 128 were diagnosed as multiple drug-resistant epilepsy and 274 patients were diagnosed as having drug-responsive epilepsy. We selected a total of 10 candidate gene polymorphisms belonging to three major classes, namely drug transporters, drug metabolizers and drug targets. These genetic polymorphism included CYP2C9 c.430C>T (*2 variant), CYP2C9 c.1075 A>C (*3 variant), ABCB1 c.3435C>T, ABCB1c.1236C>T, ABCB1c.2677G>T/A, SCN1A c.3184 A> G, SCN2A c.56G>A (p.R19K), GABRA1c.IVS11 + 15 A>G and GABRG2 c.588C>T. Genotyping was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods, and each genotype was confirmed via direct DNA sequencing. The relationship between various genetic polymorphisms and responsiveness was examined using binary logistic regression by SPSS statistical analysis software. RESULTS: CYP2C9 c.1075 A>C polymorphism showed a marginal signifi cant difference between drug resistance and drug-responsive patients for the AC genotype (Odds ratio [OR] = 0.57, 95% confi dence interval [CI] = 0.32–1.00; P = 0.05). In drug transporter, ABCB1c.2677G>T/A polymorphism, allele “A” was associated with drug-resistant phenotype in epilepsy patients (P = 0.03, OR = 0.31, 95% CI = 0.10–0.93). Similarly, the variant allele frequency of SCN2A c.56 G>A single nucleotide polymorphism was signifi cantly higher in drug-resistant patients (P = 0.03; OR = 1.62, 95% CI = 1.03, 2.56). We also observed a signifi cant difference at the genotype as well as allele frequencies of GABRA1c.IVS11 + 15 A > G polymorphism in drug-resistant patients for homozygous GG genotype (P = 0.03, OR = 1.84, 95% CI = 1.05–3.23) and G allele (P = 0.02, OR = 1.43, 95% CI = 1.05–1.95). CONCLUSIONS: Our results showed that pharmacogenetic variants have important roles in epilepsy at different levels. It may be noted that multi-factorial diseases like epilepsy are also regulated by various other factors that may also be considered in the future.


Introduction
Epilepsy is a common, serious, but treatable neurological disorder, affecting at least 60 million people worldwide. [1] Several pharmacological agents are available in the market for the treatment of epilepsy.
These anti-epileptic drugs (AEDs) increase inhibition, decrease excitation or prevent aberrant burst-fi ring of the neurons. [1] However, 20-30% of the epilepsy patients do not respond adequately to the currently available AEDs, and there is high incidence of adverse drug reactions. [2] Therefore, drug resistance and adverse reactions are important clinical problems in the treatment of epilepsy. Large interindividual variation in effi cacy and adverse effects of anti-epileptic therapy presents opportunities and challenges in newly emerging areas of pharmacogenomics. In the post genomic era, the interindividual variations in drug response have been attributed to allelic variations in the genes. [3][4][5] These genetic variations can potentially affect the individual responsiveness to the drug at several steps, which include drug absorption, drug distribution, drug metabolism, drug elimination and drug concentration at target sites. [6] There is a growing list of polymorphisms found in different classes of genes encoding drugmetabolizing enzymes (DMEs), drug transporters, receptors and drug targets, which have been linked to drug effects in humans. In pharmacotherapy, drug metabolism represents a prominent pathway both in qualitative and quantitative elimination of drugs, including AEDs. It is accomplished by the hepatic (metabolism) and/or renal (excretion) route, which comprises the socalled phase I [(e.g., oxidative reactions catalyzed by various cytochrome P-450 enzymes (CYPs)] and phase II (e.g., conjugations like glucuronidation) reactions. The CYP family comprises major phase-I DMEs, and is responsible for the metabolism of many commonly prescribed AEDs. Variants with very high enzymatic activity may be associated with a need for higher drug dosages than usually prescribed, but low or absence of biotransformation capacity may result in treatment failure due to inadequate drug levels. [7] Thus, there are a variety of potential mechanisms by which polymorphic drug metabolism can affect AED responsiveness. [8] Several classes of drug transport proteins are also shown to have a pharmacogenetic relevance. The ABC proteins are products of multidrug resistance (MDR1/ABCB1) genes that are expressed widely, including capillary endothelial cells that constitute the blood-brain barrier responsible for ATP-mediated effl ux of different compounds outside the brain, serving as a defense mechanism. [9,10] The ABCB1 (MDR1) gene codes for prototype molecule P-glycoprotein (Pgp) that is recognized to be a key element in regulating access of a diverse array of therapeutic agents to the brain and other tissues. [11,12] Several commonly used AEDs have been proposed to be substrates for P-gp-mediated transport. [13] A large number of genetic variations have been documented in human ABCB1, and some of them have been shown to the infl uence dosage of AEDs. The role of voltage-gated and ligand-gated ion channels in epileptogenesis of both genetic and acquired epilepsies, and as targets in the development of new AEDs, is very important. [14] Ionic currents generated through sodium channels are inhibited by a number of different types of therapeutically important AEDs. Several single nucleotide polymorphisms (SNPs) in the sodium channel genes have been described so far, but only a few, including SCN1A p. Thr1067Ala or c.3184 A>G (rs2298771) and SCN2A p.Arg19Lys or c.56 G>A (rs17183814) gene polymorphisms have been found to have a functional signifi cance in the different neurological disorders. [15] In the central nervous system, γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter that controls neuronal excitability and network interactions in the cerebral cortex of the brain. AEDs such as benzodiazepines, phenobarbital, gabapentin and topiramate are important targets of the GABAA receptor. [16] Recently, it has been reported that AED-resistant rats differ from drug-responsive rats in GABAA receptor subunit expression in a rat model of temporal lobe epilepsy. Therefore, genetic variations in GABAA receptor subunits may also be involved in resistance to AEDs. [17] In this way, individual genotype influences almost all stages of pharmacokinetics (hepatic drug-metabolizing enzymes, membrane drug transporters) and pharmacodynamics (sodium channels, GABA receptors, etc.). [18] Therefore, the present study was planned to study the genetic polymorphisms in genes for cytochrome P450, multidrug transporter, voltage-gated sodium ion channels and ligand-gated GABAA receptor genes in drug responsiveness in patients undergoing treatment for epilepsy.

Materials and Methods
The study was retrospective and consisted of patients

Defi nition of drug resistance and responsiveness
The main criterion for drug resistance was the occurrence of at least four seizures over a period of 1 year, with three appropriate AEDs at maximum tolerated doses. [19,20] Patients who had undergone surgery for seizure control were considered refractory, irrespective of their outcome after surgery. The epilepsy patients who had complete freedom from seizures for at least 1 year from the last follow-up visit were considered drug responsive.

Sample DNA extraction
A venous blood sample (5 ml) was collected from each subject and was kept frozen till DNA extraction.
Genomic DNA was isolated from peripheral blood leukocytes by the salting-out protocol. [21] Extracted DNA was quantifi ed using a Nanodrop Analyzer (ND-1000) spectrophotometer (Nano Drop Technologies Inc., Wilmington, DE, USA). The plasma was separated and stored at -20°C for drug level assay.

Selection of candidate genes
The candidate genes were selected on the basis of their functional role, current biological knowledge of epilepsy, a reported prevalence of at least 5% for the variant allele and published evidence of an association with epilepsy and other neurological disorders. We aimed to select, as candidate genes, major drug targets (voltage and ligand gated), multidrug transporters (ABCB1) and metabolizers (CYP2C9*2 and CYP2C9*3) of the principal AEDs for use in future pharmacogenetic studies. Details of candidate genes selected for the study are given in Table 1.

Genotyping of genetic markers used in the study
The genotypes were determined by the PCR-RFLP method. Primers, annealing temperature, amplified fragment size, restriction pattern and restriction enzymes used are listed in Table 2.

Statistical analysis
The sample size was calculated using the QUANTO 1.1 program (hydra.usc.edu/gxe). The desired power of our study was set at 80%. Relative risks for power calculation were set at 2. Descriptive statistics of patients and controls were presented as the mean and standard deviations (SDs) for continuous measures, while frequencies and percentages were used for categorical measures. The relationship between various genotypes and responsiveness was examined using the binary logistic regression. Association was expressed as odds ratios (OR) or risk estimates with 95% confi dence intervals (CI). The association was considered to be signifi cant when the P-value was <0.05.

GABRA1 (rs2279020) and GABRG2 (rs211037) polymorphism in drug-resistant epilepsy
We observed a signifi cant difference at the genotype as  Table 7]. However, in GABRG2, 588C>T did not show any significant differences in drug-resistant versus drug-responsive epilepsy patients either at the genotype or the allele levels [ Table 7]. This suggests that GABRA1 IVS11 + 15 A>G intronic polymorphism may be involved in the drug response in epilepsy patients.

Discussion
Multiple genes are known to be responsible for drug responsiveness. For example, DMEs [29] include mainly cytochrome P450, drug transporters (MDR1) and drug targets, which include sodium channels, potassium channels and GABA receptors. In observations that identifi ed CYP2C9 as a major drug metabolizer for commonly prescribed AEDs. [22] CYP2C9 is responsible for the hydroxylation of up to 90% of serum phenytoin. Among AEDs, phenytoin, carbamazepine and phenobarbital mainly undergo signifi cant metabolism by cytochrome P450 isozymes. Earlier studies have also observed a strong association between

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CYP2C9 genotype and phenytoin maintenance dose requirement. [30][31] Weide et al. [30] found that CYP2C9 allelic variants affect phenytoin dose requirement; for patients carrying at least one mutant CYP2C9 allele, the mean phenytoin dose required to achieve a therapeutic serum concentration was about 37% lower than the mean dose required by wild-type individuals.
Another similar study from Taiwan revealed that the CYP2C9 and CYP2C19 polymorphisms have dramatic effects on the population pharmacokinetic parameters of phenytoin. [32] On the basis of these observations,  [19] Although all defi nitions are signifi cantly associated with longer-term outcome, no single preferred defi nition of intractable epilepsy exists. [34] There needs to be consensus for a single defi nition of drug resistance in epilepsy so that results from different studies can be easily compared.
Until now, very few studies have correlated drug targets like sodium channels in drug resistance and therapeutic dosage in patients with epilepsy. Therefore, we looked for an association of the two polymorphisms with drugresistance phenotype in our patient groups. Even though SCN1A c.3184 A>G polymorphism was associated with generalized epilepsy, [35] it showed no infl uence on the multidrug resistance phenotype in patients with epilepsy.
Similarly, Kwan et al. [36] also found no involvement of this polymorphism in drug-resistant epilepsy. However, SCN1A IVS5-91 G>A intronic polymorphism of the same gene has been reported to show a populationspecifi c association with carbamazepine and multiple drug-resistance epilepsy. [37][38][39] It is now well established that various AEDs mediate their action through GABA binding. [40] It is also hypothesized that target receptor sites are somehow altered in the epileptic brain so that they are much less sensitive to the administered AEDs. In the present study, we found the involvement of GABRA1 IVS11 + 15 A>G polymorphism in modulating drug response in pharmacotherapy, while GABRG2 588C>T was not found to be associated with drug resistance in north Indian epilepsy subjects. Several mutations in this gene have also been reported to be involved in epilepsy causation that result in loss of function of GABAA receptors via a reduction in GABA expression and accelerated deactivation. [41] It is possible that the association of GABRA1 IVS11 + 15 A>G polymorphism with refractory phenotype in our study may occur due to changes in the structure and function of inhibitory GABAA receptors. [42] Excessive glutamate excitation and activation of drug resistance genes may also contribute to changes in the GABA receptor conformation and loss of drug effi cacy. In the above-mentioned facts, we have provided evidence that genetic variations in all three classes of genes have some role in multiple drug resistance; but, it would be desirable to replicate them in larger cohorts. It should also be kept in mind that the overall genetic effect of these genes may have a greater role in determining the drug responsiveness rather than a single gene and its genetic polymorphisms alone. Response to newly administered AED treatments is highly dependent on past treatment history. [43] Other factors such as type of epilepsy, duration of seizure and no seizure prior to initiation of drug therapy [43] may also be responsible for differences in drug responsiveness.
Therefore, the current studies associating particular genes and their variants with seizure control or adverse events have inherent weaknesses, yet it is believed that future pharmacogenomic studies with clearly defi ned phenotypes involving a multicenter approach will result in therapeutic application.