Epilepsy Research (2009) 84, 86—90

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SHORT COMMUNICATION

Lack of association between ABCB1, ABCG2, andABCC2 genetic polymorphisms and multidrugresistance in partial epilepsy

Dong Wook Kima, Sang Kun Leeb,∗, Kon Chub, In-Jin Jangc, Kyung-Sang Yuc,Joo-Youn Choc, Seon-Jeong Kimc

a Department of Neurology, Konkuk University Medical Center, Seoul, Republic of Koreab Comprehensive Epilepsy Center, Department of Neurology, Seoul National University Hospital, Seoul, Republic of Koreac Department of Pharmacology and Clinical Pharmacology, Seoul National University Hospital, Seoul, Republic of Korea

Received 10 September 2008; received in revised form 23 November 2008; accepted 13 December 2008Available online 22 January 2009

KEYWORDSATP-binding cassette(ABC) transporters;Multidrug resistance;Antiepileptic drugs;Epilepsy

Summary ATP-binding cassette (ABC) transporters participate in drug disposition andresponse in various conditions, and many polymorphisms in ABC transporter genes have beenrecognized in association with altered transporter functions of various drugs. Studies on epilepsyhave focused on the C3435T polymorphism of the ABCB1 gene, but other ABC transporters arealso thought to be involved in the transport of antiepileptic drugs. We have evaluated the func-tional polymorphisms of ABCB1, ABCG2, and ABCC2 genes with regard to epilepsy drug responsein partial epilepsy, and have investigated the potential of combined effects of polymorphismsin more than one transporter gene. We studied 6 genetic polymorphisms in 3 transporter genesin 193 drug responders and 198 nonresponders. There was no significant difference betweenthe two groups, and we did not observe any combined effects of ABCB1 and ABCG2 genetic

polymorphisms. Our study suggests that genetic polymorphisms in ABC transporters may not besignificant predictors of drug re© 2008 Elsevier B.V. All rights re

∗ Corresponding author at: Department of Neurology, SeoulNational University Hospital, 28, Yongkeun dong, Chongno Ku, Seoul,110-744, Republic of Korea. Tel.: +82 2 20722923;fax: +82 2 36727553.

E-mail address: sangunlee@dreamwiz.com (S.K. Lee).

I

Eadinett

0920-1211/$ — see front matter © 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.eplepsyres.2008.12.001

sponse in epilepsy patients.served.

ntroduction

pilepsy is a common neurological disorder that affectspproximately 1% of the population. Although antiepilepticrugs (AEDs) effectively control seizures in most patients,

t is estimated that a third of the affected population doesot have a successful seizure control, known as refractorypilepsy. Most patients with refractory epilepsy are resistanto several AEDs having different mechanisms. This supportshe pharmacokinetic mechanism of drug resistance, which

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ABC genetic polymorphism and MDR in partial epilepsy

says that AEDs do not reach the epileptogenic focus by activeefflux mediated by locally overexpressed drug transporters(Sisodiya, 2003).

ATP-binding cassette (ABC) transporters are a superfam-ily of efflux proteins that use ATP hydrolysis to extractdrugs from a eukaryotic cell, lowering the intracellularconcentration of the drugs. The major ABC transportersinclude P-glycoprotein (PGP, ABCB1), members of the mul-tidrug resistance protein (MRP, ABCC) family (MRP1-9), andbreast cancer resistance protein (BCRP, ABCG2). The pres-ence of these transporters in the liver, bowel, kidney, andblood—brain barrier (BBB) suggests that they are key playersin physiological cellular defense system. ABC transportersare increasingly recognized to be important for drug dispo-sition and response in various conditions, including cancer,infection, and epilepsy, and a number of genetic poly-morphisms in ABC transporters have been associated withaltered transporter functions of various drugs (Lazarowskiet al., 2007; Szakacs et al., 2008). For epilepsy, PGP, theproduct of the ABCB1 gene, has been extensively studied asa candidate protein responsible for resistance to many AEDs.The C3435T polymorphism of the ABCB1 gene or haplotypeincluding this polymorphism has been associated with mul-tidrug resistance in several studies with epilepsy patients(Siddiqui et al., 2003; Zimprich et al., 2004; Hung et al.,2005), but many other studies failed to replicate any associ-ation with the C3435T polymorphism (Tan et al., 2004; Sillset al., 2005; Kim et al., 2006a,b; Leschziner et al., 2006;Kwan et al., 2007; Dericioglu et al., 2008).

In addition to PGP, other ABC transporters can be involvedin drug resistance in epilepsy patients. ABC transporters ofthe ABCC family and ABCG2 are also normally expressed inthe (BBB), and have been suggested to reduce the brain pen-etration of many AEDs. Overexpression of these transportershas been reported in a human epileptogenic brain (Sisodiyaet al., 2006) and in experimental models (van Vliet et al.,2005), and there was a compensatory change with upregu-lation of one transporter in response to the lack of anothertransporter (Hoffmann and Loscher, 2007). However, therehave been few studies on epilepsy patients that have investi-gated the contribution of functional polymorphisms of otherABC transporter genes to the AED response, and the com-bined effect of polymorphisms in multiple transporter geneshas not been evaluated.

In this study, we determined the effect of functionalpolymorphisms of ABCB1, ABCG2, and ABCC2 genes on theepilepsy drug response, and investigated potential combinedeffects of polymorphisms in multiple transporter genes inindividual patients.

Methods

Subjects

Patients were consecutively recruited through the comprehensiveepilepsy center at Seoul National University Hospital, South Korea.We tried to include patients with partial onset seizures only, there-

fore excluded patients with clinical or electroencephalographicevidences of generalized onset seizures. Drug-resistant or drug-responsive epilepsy was determined as follows. Drug resistancewas defined as having at least four seizures during the previousyear while trying at least three antiepileptic medications at maxi-

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87

al tolerated doses. The maximum tolerated dose was the highestose that did not lead to clinical side effects. Drug response wasefined as a complete freedom from seizure while using one or twontiepileptic medications for at least a year before the last follow-p visit. Seizures were not considered for the purpose of this study ifhey are associated with provocation events such as alcohol intake,leep deprivation and irregular medication. Patients who did nott into the drug-resistant or drug-responsive epilepsy categoriesere excluded from this study. All epilepsy patients were ethnicallyomogeneous Koreans. All subjects gave written informed consento participate in this study, which was approved by the institutionalthics committee.

enotyping of MDR1, ABCG2, and ABCC2 polymorphisms

enomic DNA was extracted from the peripheral whole blood ofach volunteer using a QIAamp DNA Blood Mini Kit (QIAGEN GmbH,ermany). Genotyping used TaqMan allelic discrimination assaysn an AB 7500 Real time PCR System (Applied Biosystems, Fosterity, CA, USA). A volume of 10 �L of PCR reaction mixture was pre-ared with 5 �L of 2× TaqMan Genotyping Master Mix, 0.5 �L of 20×rug Metabolism Genotyping Assay Mix, 3.5 �L of DNase-free water,nd 1 �L of genomic DNA. Genotyping for ABCB1 1236C>T (assayD = C 7586662 10), ABCG2 421C>A (assay ID: C 15854163 70),nd ABCC2 1249G>A (assay ID = C 22272980 20) single nucleotideolymorphisms (SNPs) were performed using Validated TaqManenotyping Assays purchased from Applied Biosystems. The primernd probe sequences are listed in Table 1. PCR reactions were car-ied out as follows: initial denaturation at 95 ◦C for 10 min, 50 cyclesf denaturation at 92 ◦C for 15 s, and then anneal/extension at 60 ◦Cor 1 min. The allelic discrimination results were determined aftermplification by performing an end-point read. The AB Sequenceetection System 7500 v1.4 software package (Applied Biosystems)as used in the analyses. We repeated the TaqMan assay on 10% of

he samples.

tatistical tests

e compared the clinical characteristics of the drug-responsiveroup and the drug-resistant group with Student’s t-test or theann—Whitney U-test for continuous variables and with a �2 foratergorical variables. Differences in genotype and allele frequen-ies between the groups were compared using the �2-test or Fishersxact test. Because there is a recent study that has reported theresence of a C3435T polymorphism in combination with one orwo other polymorphisms (C1236T and G2677T/A) in the ABCB1ene may be associated with a reduced function of PGP (Kimchi-arfaty et al., 2007), we investigated whether patients that areomozygous for reduced PGP activity (patients with 3435TT in com-ination with 1236TT or 2677 T/AT/A genotype) were more likelyo be drug responders. We also investigated whether patients witht least one homozygote for altered BCRP activity (patients with4AA or 421AA genotypes) would have different chance to be drugesponders. Finally, we investigated potential combined effects ofolymorphisms in multiple transporter genes in individual patients.ll analyses were conducted using SPSS version 14.0 (SPSS Inc.,hicago, IL). P < 0.05 was considered significant.

esults

n total, 193 patients (49.4%) were classified as drug

esponders and 198 as nonresponders (50.6%). In the drug-esponsive group, the subjects consisted of 101 men and 92omen, and the mean age was 34.7 ± 8.2 years. In the drug-

esistant group, the subjects consisted of 118 men and 80omen, and the mean age was 33.4 ± 9.3 years. There was

88 D.W. Kim et al.

Table 1 Primer and probe sequences.

SNP Primers Probes

ABCB1 2677G>T 5′-GAAATGAAAATGTTGTCTGGACAAGCA-3′ VIC-TTCCCAGCACCTTC5′-CTTAGAGCATAGTAAGCAGTAGGGAGT-3′ FAM-TTCCCAGAACCTTC

ABCB1 2677G>A 5′-GAAATGAAAATGTTGTCTGGACAAGCA-3′ VIC-TTCCCAGCACCTTC5′-CTTAGAGCATAGTAAGCAGTAGGGAGT-3′ FAM-CTTCCCAGTACCTTC

ABCB1 3435C>T 5′-CTGTTTGACTGCAGCATTGCT-3′ VIC-CCCTCACGATCTCTT5′-ATGTATGTTGGCCTCCTTTGCT-3′ FAM-CCCTCACAATCTCTT

′ AGTA ′

CAGG

ntwnptwbtrwflgpi

D

AistcmM(W

ABCG2 34G>A 5 -CTCTCCAGATGTCTTCC5′-CCTTCAGTAAATGCCTT

SNP = single nucleotide polymorphism.

o difference in sex (P = 0.15) and age (P = 0.71) betweenhe two groups. Drug responders were using 1.40 ± 9.49 AEDshile nonresponders were using 3.89 ± 9.3 AEDs. There waso significant difference in the genotypes of six polymor-hisms in the three ABC transporter genes between thewo groups (Table 2). We investigated whether patientsith reduced PGP or BCRP activity were more likely toe drug responders, but we found no difference betweenhe groups. Finally, we investigated whether patients witheduced transporter function in more than one transporterere more likely to be drug responders. Because the allelic

requency of 1249G>A polymorphism in the ABCC2 gene was

ow, we studied the combined effects of ABCB1 and ABCG2enetic polymorphisms on the drug response in epilepsyatients, but we found no reduced risk of drug resistancen patients with reduced activity of PGP and BCRP (Table 3).

Table 2 Lack of association of polymorphisms in ABC trans-porter genes and drug response in epilepsy patients.

Genotype frequency P-value*

ABCB11236C>T w/w w/m m/mDrug-responsive 31 100 62Drug-resistant 25 104 69 0.5972677G>T/A w/w w/m m/mDrug-responsive 37 93 63Drug-resistant 30 98 70 0.5583435C>T w/w w/m m/mDrug-responsive 81 90 22Drug-resistant 73 97 28 0.513

ABCG234G>A w/w w/m m/mDrug-responsive 105 69 19Drug-resistant 104 79 15 0.581421C>A w/w w/m m/mDrug-responsive 98 74 21Drug-resistant 103 77 18 0.303

ABCC21249G>A w/w w/m m/mDrug-responsive 165 27 1Drug-resistant 162 35 1 0.326**

w = wild type allele; m = mutant type allele; ABC = ATP-bindingcassette; * = from a �2-test; ** = compared using a 2 × 2 table;w/w genotype versus w/m + m/m genotype.

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ATGTC-3 VIC-TCCTTGTGACATTGGGATTCAT-3′ FAM-CCTTGTGACACTGGGAT

iscussion

lthough the role of PGP has been most extensively studiedn patients with refractory epilepsy, other ABC transportersuch as MRP2 and BCRP may have a role in modulatinghe brain penetration of many AEDs. ABC transporters haveonsiderable overlap of substrates. Phenytoin and carba-azepine are suggested as substrates of both PGP andRP2, and valproic acid is suggested as a substrate of MRP2

Potschka and Loscher, 2001; Potschka et al., 2003a,b).e studied G34A and C421A polymorphisms of ABCG2 geneecause they are the most common between differentthnic groups (Zamber et al., 2003), and the C421A poly-orphism has been associated with decreased expression

f the BCRP protein (Kondo et al., 2004), and the G34Aolymorphism has been associated with decreased BCRPransporter activity (Mizuarai et al., 2004). The effectf genetic polymorphism of ABCC2 gene is more complexecause the ABCC2 polymorphisms may be associated withoth increased and decreased expression and function. Wetudied G1249A polymorphism because this polymorphismas strongly associated with higher activity of transporter

unction (Haenisch et al., 2008).Our data did not identify any significant association

etween genetic polymorphisms in ABC transporters or anyombined effects in response to AED treatment. Severalxplanations are possible for this lack of association. Numer-us clinical and pathological factors can contribute to drugesistance. Symptomatic epilepsy is more drug resistant thandiopathic epilepsy and a history of multiple seizures beforereatment predicts AED resistance. Pathological character-stics, such as hippocampal sclerosis, cortical dysplasia,nd severe brain injury have also been associated with aigh rate of AED resistance. Furthermore, non-ABC trans-orters, such as major vault protein, can also participaten transporter of AEDs, which makes the pharmacokineticechanism of drug resistance more complicated (Sisodiya et

l., 2006). Therefore, genetic polymorphisms in ABC trans-orters may be only a weak contributor, and the effects ofhese polymorphisms could not be identified in our small,onstratified study. A study by Leschziner et al. (2006) failedo find an association between the C3435T polymorphism

n the ABCB1 gene with drug response, but it did identifyhat the number of seizures before treatment was the onlyignificant factor affecting treatment outcome.

This lack of an association may be related to the fact thathe function of ABC transporters depends on the differences

ABC genetic polymorphism and MDR in partial epilepsy 89

Table 3 Lack of association of combined genetic polymorphisms in ABC transporter genes and drug response in epilepsy patients.

Drug-responsive Drug-resistant P-value

ABCB1Patients with reduced PGP activity 20 27Other patients 173 171 0.320*

ABCG2Patients with reduced BCRP activity 40 33Other patients 153 165 0.303*

ABCB1 and ABCG24

89

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Patients with reduced PGP and BCRP activityOther patients 1

ABC = ATP-binding cassette; * = from a �2-test, ** = using Fishers ex

in gene expression. It has been suggested that the trans-porter function may be exaggerated when the transcriptionmachinery is stressed. Therefore, the genotype dependenceof the transporter function may be different between thepopulations studied, and the effects of the genotype maybe more evident in populations with greater drug resistance(Sisodiya and Goldstein, 2007).

Finally, some experimental studies have doubted thefundamental fact that most AEDs are substrates of ABCtransporters. Although a number of AEDs have been iden-tified as substrates of ABC transporters in rodent models,recent experimental models have demonstrated significantdifferences in the efflux transport of AEDs by ABC trans-porters between species, and have suggested that AEDs areeither not substrates or are very weak substrates of ABCtransporters in humans (Baltes et al., 2007). Unlike classicsubstrates of ABC transporters, most AEDs are lipophilic andhave high passive permeability across the BBB. If human ABCtransporters do not transport AEDs, it would be a reason-able explanation of our results. Recent studies have failedto show significant interactions between BCRP protein andseveral AEDs (Cerveny et al., 2006), and association betweenthe drug-resistant epilepsy and variations of ABCC2 geneincluding G1249A polymorphism (Seo et al., 2008).

In summary, we did not identify a significant associa-tion between the genetic polymorphisms in ABC transportersor their combined effects in response to AED treatment.Although we could not control all possible factors that mayhave influenced the results, our study suggests that geneticpolymorphisms in ABC transporters may not be significantpredictors of drug response in epilepsy patients.

Acknowledgment

This study was supported by a grant from the Korea Health 21R&D Project, Ministry for Health, Welfare and Family Affairs,ROK (A030001).

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