- Email: [email protected]
Multidrug resistance in patients undergoing resective epilepsy surgery is not associated with C3435T polymorphism in the ABCB1 (MDR1) gene
Epilepsy Research (2008) 80, 42—46
journal homepage: www.elsevier.com/locate/epilepsyres
Multidrug resistance in patients undergoing resective epilepsy surgery is not associated with C3435T polymorphism in the ABCB1 (MDR1) gene Nese Dericioglu a,∗, Melih O. Babaoglu b,1, Umit Yasar b,1, I. Burak Bal b,1, Atila Bozkurt b,1, Serap Saygi c,2 a
Hacettepe University, Institute of Neurological Sciences and Psychiatry, Sihhiye 06100, Ankara, Turkey Hacettepe University, School of Medicine, Department of Pharmacology, Sihhiye 06100, Ankara, Turkey c Hacettepe University, School of Medicine, Department of Neurology, Sihhiye 06100, Ankara, Turkey b
Received 30 July 2007; received in revised form 25 January 2008; accepted 5 March 2008 Available online 23 April 2008
KEYWORDS Multidrug resistance; Refractory epilepsy; Epilepsy surgery; ABCB1 (MDR1) gene; C3435T polymorphism
Summary Purpose: The C3435T polymorphism in the gene coding for P-glycoprotein (ABCB1) has been correlated with drug resistance in patients with epilepsy. However, replication studies have revealed conflicting results and the reason for this is not clear. We investigated the frequency of C3435T polymorphism in epileptic Turkish patients who underwent resective epilepsy surgery and compared our results with healthy controls. Methods: DNA samples were obtained from 100 healthy controls and 89 consecutive adult patients who underwent resective brain surgery due to refractory seizures at our epilepsy center. Genotypes for the C3435T polymorphism were determined by PCR and restriction analysis. Results: Comparison of drug-resistant patients and healthy controls revealed no significant difference in allele frequency (C vs. T; 2 = 0.015, p = 0.90) and genotype frequency (2 = 2.05, p = 0.36). The findings in the pure hippocampal sclerosis (HS) group (n = 73) were not significantly different from control subjects, either (allele frequency: 2 = 0.29, p = 0.59; genotype frequency: 2 = 2.14, p = 0.34). Conclusions: Our findings failed to prove an association between C3435T polymorphism and drug resistance in a sample of Turkish patients with refractory epilepsy who underwent resective brain surgery. © 2008 Elsevier B.V. All rights reserved.
∗
Corresponding author. Fax: +90 312 309 3451. E-mail addresses: [email protected] (N. Dericioglu), [email protected] (M.O. Babaoglu), [email protected] (U. Yasar), [email protected] (I.B. Bal), [email protected] (A. Bozkurt), [email protected] (S. Saygi). 1 Fax: +90 312 310 5312. 2 Fax: +90 312 309 3451. 0920-1211/$ — see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.eplepsyres.2008.03.004
C3435T polymorphism in the ABCB1 gene in drug resistant epilepsy
Introduction The pathogenesis underlying pharmacoresistance in epilepsy is unclear. One of the candidate mechanisms that has attracted considerable attention recently is the limitation of anticonvulsant medication access to the seizure focus by a number of efflux transporters, the most well known of which is P-glycoprotein (P-gp), encoded by the multidrug resistance ABCB1 (or MDR1) gene. P-glycoprotein is expressed at blood—tissue barriers and in organs with excretory functions. Therefore, it is thought to act as a physiologic defense by extruding xenobiotics from mammalian cells and affording protection of sensitive organs. The high level of P-gp in the cerebrovascular endothelium is believed to confer protection to the brain. It has been hypothesized that overexpression of P-gp and other efflux transporters in the cerebrovascular endothelium, in the region of the epileptic focus, may lead to drug resistance in epilepsy (Dombrowski et al., 2001). It has been claimed that a functional polymorphism of the ABCB1 gene may affect the absorption and tissue concentrations of numerous P-gp substrates (Hoffmeyer et al., 2000). Allelic differences in individual ABCB1 gene sequences may be associated with different expression levels of the gene. Several recent publications have demonstrated that the frequency of the C3435T mutation in exon 26 of the ABCB1 gene is significantly influenced by ethnicity (Ameyaw et al., 2001). Recently, Siddiqui et al. have reported that multidrugresistance in epilepsy is associated with a polymorphism in the drug transporter gene ABCB1 (Siddiqui et al., 2003). Patients with drug-resistant epilepsy were more likely to have the CC genotype at 3435 than the TT genotype. Nevertheless, replication studies afterwards have provided conflicting results (Tan et al., 2004; Zimprich et al., 2004; Sills et al., 2005; Kim et al., 2006a,b; Shahwan et al., 2007; Leschziner et al., 2007). The discrepancy between these findings has not been clarified yet. It is not known whether this is due to the different ethnic populations studied; the use of nonstandardized definitions of pharmacoresistance or due to other methodological issues. In this study, we aimed to investigate the frequency of the C3435T polymorphism in drug-resistant epileptic Turkish patients, where we defined multidrug resistance as ‘‘the patient’s absolute need for resective brain surgery due to uncontrolled seizures’’.
Patients and methods Patients Blood samples were collected prospectively from patients who underwent resective brain surgery for drug-resistant symptomatic or cryptogenic partial epilepsy (temporal: n = 86; extratemporal: n = 3) between July 2003 and May 2006 at our epilepsy center. All the patients had been followed for sufficiently long periods. They had at least 1 seizure/month, had failed at least two conventional antiepileptic drugs, and most, if not all, were on polytherapy. Pgp substrates such as carbamazepine, phenytoin or phenobarbital had been administered to all of the patients. Prior to surgery all the patients were investigated with long-term video-EEG monitoring and high resolution (1.5—3 T) cranial MRI. Interictal/ictal SPECT and PET were performed in selected cases. Patients were discussed
43
at a weekly multidisciplinary conference with all the available data (clinical history, interictal/ictal EEG, seizure semiology, structural ± functional neuroimaging studies), where a final consensus was reached. Patients who were offered surgery, but somehow were not operated during that time period were excluded. We compared our results with age-matched healthy volunteers whose demographic and genotypic findings have been reported recently (Tufan et al., 2007). The study was approved by the research ethics committee of Hacettepe University. All patients gave written informed consent.
Genotyping procedure for ABCB1 3435C>T DNA was extracted from blood samples using QIAamp DNA blood kit (Qiagen, Hilden, Germany). The analysis of ABCB1 3435C>T was performed with a PCR-based endonuclease digestion method (Babaoglu et al., 2005; Drozdzik et al., 2003). The forward and reverse primers were 5 -TGTTTTCAGCTGCTTGATGG-3 and 5 -AAGGCATGTATGTTGGCCTC-3 , respectively (Quiagen, Hilden, Germany). The genomic fragment encompassing the point mutation of interest were amplified by using 100—200 ng DNA in a total volume of 50 L containing 100 M of each deoxynucleotides, 2 mM MgSO4 , 0.5 M of each primers and 2.5 units (U) Taq polymerase (New England Biolabs GmbH, Frankfurt, Germany). Amplification was performed using a Px2 Thermal Cycler (Thermo Electron Co., MA, USA) for 35 cycles, consisting of denaturation at 94 ◦ C for 30 s, annealing at 60 ◦ C for 30 s and extension at 72 ◦ C for 30 s between the initial denaturation at 94 ◦ C for 2 min and a final extension 72 ◦ C for 7 min. The amplified 197 bp fragment was then analyzed by endonuclease restriction in which 5 U of MboI (New England Biolabs GmbH, Frankfurt, Germany) was used for the detection of ABCB1 3435C>T substitution. 3435C allele was detected by the presence of two restriction fragments, which were 158 bp and 39 bp long. The 3435C>T substitution destroyed the cleavage site for the restriction enzyme. Restriction fragments were separated by electrophoresis in a 3% agarose gel and after ethidium bromide staining visualized using an ultraviolet transiluminator. Differences in genotype and allele frequencies between the groups were compared using the 2 -test. Significance was accepted at p < 0.05.
Results Demographic characteristics of the patients and healthy control subjects are shown in Table 1. Three different genotypes, CC, CT and TT were identified at position 3435 in exon 26 and their frequencies in the study group were 29.3% CC, 38.2% CT, 32.6% TT (Table 2). These values did not deviate significantly from the control group (2 = 2.05, p = 0.36). Since most of the patients in the study group had pure hippocampal sclerosis (HS) (82%), we also analyzed this subgroup separately. Genotype frequencies in this subgroup were as follows: 27.4% CC, 38.4% CT, 34.2% TT and were not significantly different from the control sample (2 = 2.14, p = 0.34) (Table 2). Similarly, no significant differences were noted in allele frequency between patients in the study group (p = 0.90; odds ratio = 0.97; 95% CI, 0.65—1.46) or patients with HS only (p = 0.59; odds ratio = 0.84; 95% CI, 0.55—1.27) and controls (Table 2).
Discussion Here, we report for the first time that a particular group of drug-resistant epilepsy patients who needed resective brain
44
N. Dericioglu et al.
Table 1
Characteristics of patients and healthy controls
Variable
Patients (n = 89)
Healthy controls (n = 100)a
Sex Male Female
42 47
46 54
Age Mean ± S.D.
28.7 ± 7.5
29.3 ± 14.5
Pathological findings HS only HS + CD CD DNET Tumor Non-specific findings
73 2 10 1 2 1
— — — — — —
HS: hippocampal sclerosis; CD: cortical dysplasia; DNET: dysembryoplastic neuroepithelial tumor. a Tufan et al. (2007).
surgery revealed no significant difference in genotype (CC vs. CT or TT) or allele (C vs. T) frequencies for the drug transporter ABCB1 C3435T polymorphism as compared to healthy control subjects in the Turkish population. The genotype frequencies in the healthy control group (Tufan et al., 2007) were similar to those reported in a previous study in healthy Turkish volunteers (Kerb et al., 2001) and in various Caucasian populations (Hoffmeyer et al., 2000; Ameyaw et al., 2001). The majority of patients with refractory epileptic seizures are resistant to many anticonvulsants, although the drugs do not all have the same mechanism of action. This suggests that a non-specific mechanism, such as seizureinduced overexpression of multidrug-resistance proteins, might be one of the causes of pharmacoresistant epilepsy (Sisodiya et al., 2002). The role of transporter proteins in drug-resistant epilepsy is an area of growing interest, since the first publication (Tishler et al., 1995). The most frequently studied protein in this regard is the P-gp which consists of 28 exons and is encoded by the ABCB1 gene (Callen et al., 1987). P-glycoprotein has wide substrate
specificity for lipophilic molecules including most of the major antiepileptic drugs (Loscher and Potschka, 2002). Genetic variations of ABCB1 have been studied extensively, reporting over 50 single nucleotide polymorphisms (SNP) (Ishikawa et al., 2004). Allelic variations in ABCB1 have attracted attention as a possible explanation for interindividual differences in drug response. In particular, a SNP in exon 26 (C—T transformation at position 3435) has been associated with altered expression levels and transport activity of P-gp. Increased levels and increased activity of P-gp are conferred by the CC genotype, whereas individuals with the TT genotype are reported to have reduced P-gp activity and higher plasma concentrations of substrate drugs after oral administration (Hoffmeyer et al., 2000). A recent study demonstrated that C3435T polymorphism is possibly the key genetic variation that determines the transporter—drug interaction possibly by altering the transporter’s substrate specificity (Kimchi-Sarfaty et al., 2007). In 2003, Siddiqui et al. reported for the first time that multidrug-resistance in epilepsy was associated with a polymorphism in the drug transporter gene ABCB1 (Siddiqui et al., 2003). As compared with drug-responsive epilepsy patients, subjects with drug-resistant epilepsy were more likely to have the CC genotype at ABCB1 3435 than the TT genotype. Comparison with control subjects without epilepsy also revealed similar findings. All the subsequent studies, except one (Hung et al., 2005), reported that there were no significant differences in genotype and/or haplotype frequencies between drug-resistant and drugresponsive patients or healthy controls (Tan et al., 2004; Zimprich et al., 2004; Sills et al., 2005; Kim et al., 2006a,b; Shahwan et al., 2007; Leschziner et al., 2007). The studies were usually conducted in heterogeneous patient groups with different phenotypic characteristics and in various ethnic groups such as Taiwanese (Hung et al., 2005), Korean (Kim et al., 2006a,b), Scottish (Sills et al., 2005), Irish (Shahwan et al., 2007) and British (Siddiqui et al., 2003; Leschziner et al., 2007) populations. The only study that supports the findings of the original study by Siddiqui et al. reported the genotypes of 10 different polymorphisms in drug-resistant epileptic patients (Hung et al., 2005). It was indicated that patients with certain haplotypes were more likely to be drug resistant and that there is significant link-
Table 2 Genotype and allele distribution of the C3435T polymorphism of the ABCB1 gene in the patients (all and hippocampal sclerosis (HS) only) and healthy control subjects Genotypes/alleles Genotypes CC CT TT Alleles C allele T allele a b
All patients (n, %)
95% CI
HS only (n, %)
95% CI
Healthy controlsa (n, %)
95% CI
26 (29.3%) 34 (38.2%) 29 (32.6 %) p = 0.36b
19.8—38.8 28.1—48.3 21.8—41.2
20 (27.4%) 28 (38.4%) 25 (34.2%) p = 0.34b
17.2—37.6 27.2—49.6 23.3—45.1
25 (25%) 49 (49%) 26 (26%)
16.5—33.5 39.2—58.8 17.4—34.6
86 (48.3%) 92 (51.7%) p = 0.90b
41.6—56.2 43.8—58.4
68 (46.6%) 78 (53.4%) p = 0.59b
38.5—54.7 45.3—61.5
99 (49.5 %) 101 (50.5%)
42.6—56.4 43.6—57.4
Tufan et al. (2007). As compared to the control group.
C3435T polymorphism in the ABCB1 gene in drug resistant epilepsy age disequilibrium among the SNPs C1236T, G2677T/A and C3435T in exons 12, 21 and 26, respectively. The haplotypes CGC, TGC and TTT were more likely to be associated with drug resistance in epilepsy (Hung et al., 2005). It was concluded that the three loci jointly influenced the treatment response for epileptic patients. Although most of these studies failed to demonstrate a significant association between ABCB1 genotype and epilepsy phenotype, the findings in patients with temporal lobe epilepsy (TLE) are worth mentioning. In their study, Tan et al. reported that in the TLE subgroup, genotype frequencies differed according to the presence of HS; TT genotype (contrary to the a priori hypothesis) was more common in TLE with HS (40.5%) than TLE without HS (24.5%) (Tan et al., 2004). In contrast to the above study, we failed to demonstrate any differences in the genotypic frequency in our patients with HS (frequency of the TT genotype = 34.2%). This might be due to the smaller number of patients in our study group; methodological differences (since we compared our study group with normal controls) or the finding in the previous study might be a false positive association due to chance, as the authors themselves have stated. The study by Zimprich et al. also failed to detect any difference between patients with TLE and non-epileptic controls (Zimprich et al., 2004). Contrary to the study by Tan et al. there were no differences between patients with mesial TLE and cryptogenic TLE. Whether an association exists or is dependent on the degree of drug-resistance awaits further investigations in this group. The precise definition of drug resistance may vary according to syndrome and subject, and awaits better understanding of the underlying mechanisms. Not surprisingly, in the studies mentioned above, drug resistance (e.g. <50% reduction in seizure frequency in the preceding year; >4—10 seizures/year; <2 to >6 seizures/month) and responsiveness (e.g. >50% reduction in seizure frequency in preceding year; complete seizure freedom >1—2 years) were defined in different ways. In our study, we preferred to define drug resistance as ‘‘the patient’s absolute need for resective brain surgery due to uncontrolled seizures after treatment with various drugs’’, thus minimizing any confusions with regard to the degree of drug resistance in a patient. After thorough investigation of the patients, we also excluded the possibility of including patients with pure psychogenic non-epileptic seizures that are known to constitute 10—20% of the patients referred to epilepsy centers (Benbadis and Allen Hauser, 2000). Surgical resection gave us the opportunity to have the pathological diagnosis. It is possible that different drug-resistance mechanisms are involved in localization-related epilepsies with various pathological substrates. Therefore, it seems plausible to perform the aforementioned association studies in phenotypically and perhaps pathologically homogenous groups of patients. A limitation of our study is the small sample size. In order to have a power of 80%, at least 318 patients were needed (Dupont and Plummer, 1998). However, considering that the study population consists of drug refractory epilepsy patients who underwent resective brain surgery, it is very unlikely to collect so many samples at a single center. To conclude, our study failed to demonstrate an association between ABCB1 C3435T polymorphism at exon 26 and antiepileptic drug resistance in a particular sample of Turk-
45
ish patients who needed brain resection for the treatment of multidrug-resistant epilepsy.
Acknowledgements This study was supported by a grant from the Scientific and Technical Research Council of Turkey (SBAGCOST B25-105S027). Drs. Babaoglu and Yasar are the recipients of the Turkish Academy of Sciences, Young Scientist Award (MOB/TUBA-GEBIP/2007—06 and UY/TUBAGEBIP/2005—17). The authors thank Dr. Y. Yardimci for technical assistance in genotyping.
References Ameyaw, M.M., Regateiro, F., Li, T., Liu, X., Tariq, M., Mobarek, A., Thornton, N., Folayan, G.O., Githang’a, J., Indalo, A., Ofori-Adjei, D., Price-Evans, D.A., McLeod, H.L., 2001. MDR1 pharmacogenetics: frequency of the C3435T mutation in exon 26 is significantly influenced by ethnicity. Pharmacogenetics 11, 217—221. Babaoglu, M.O., Bayar, B., Aynacioglu, A.S., Kerb, R., Abali, H., Celik, I., Bozkurt, A., 2005. Association of the ABCB1 3435C>T polymorphism with antiemetic efficacy of 5-hydroxytryptamine type 3 antagonists. Clin. Pharmacol. Ther. 78, 619—626. Benbadis, S.R., Allen Hauser, W., 2000. An estimate of the prevalence of psychogenic non-epileptic seizures. Seizure 9 (4), 280—281. Callen, D.F., Baker, E., Simmers, R.N., Seshadri, R., Roninson, I.B., 1987. Localization of the human multidrug resistance gene, MDR1, to 7q21.1. Hum. Genet. 77, 142—144. Dombrowski, S.M., Desai, S.Y., Marroni, M., Cucullo, L., Goodrich, K., Bingaman, W., Matberg, M.R., Bengez, L., Janigro, D., 2001. Overexpression of multiple drug resistance genes in endothelial cells from patients with refractory epilepsy. Epilepsia 42 (12), 1501—1506. Drozdzik, M., Bialecka, M., Mysliwiec, K., Honczarenko, K., Stankiewicz, J., Sych, Z., 2003. Polymorphism in the Pglycoprotein drug transporter MDR1 gene: a possible link between environmental and genetic factors in Parkinson’s disease. Pharmacogenetics 13, 259—263. Dupont, W.D., Plummer, W.D., 1998. Power and sample size calculations for studies involving linear regression. Control Clin. Trials 19, 589—601. Hoffmeyer, S., Burk, O., von Richter, O., Arnold, H.P., Brockm¨ oller, J., Johne, A., Cascorbi, I., Gerloff, T., Roots, I., Eichelbaum, M., Brinkmann, U., 2000. Functional polymorphisms of the human multidrug-resistance gene: multiple sequence variations and correlation of one allele with P-glycoprotein expression and activity in vivo. Proc. Natl. Acad. Sci. U.S.A. 97, 3473— 3478. Hung, C.C., Tai, J.J., Lin, C.J., Lee, M.J., Liou, H.H., 2005. Complex haplotypic effects of the ABCB1 gene on epilepsy treatment response. Pharmacogenomics 6 (4), 411—417. Ishikawa, T., Onishi, Y., Hirano, H., Oosumi, K., Nagakura, M., Tarui, S., 2004. Pharmacogenomics of drug transporters: a new approach to functional analysis of the genetic polymorphisms of ABCB1 (P-glycoprotein/MDR1). Biol. Pharm. Bull. 27, 939— 948. Kerb, R., Aynacioglu, A.S., Brockm¨ oller, J., Schlagenhaufer, R., Bauer, S., Szekeres, T., Hamwi, A., Fritzer-Szekeres, M., Baum¨ gartner, C., Ongen, H.Z., G¨ uzelbey, P., Roots, I., Brinkmann, U., 2001. The predictive value of MDR1 CYP2C9 and CYP2C19 polymorphisms for phenytoin plasma levels. Pharmacogenom. J. 1, 204—210.
46 Kim, D.W., Kim, M., Lee, S.K., Kang, R., Lee, S.Y., 2006a. Lack of association between C3435T nucleotide MDR1 polymorphism and multidrug-resistant epilepsy. Seizure 15 (5), 344—347. Kim, Y.O., Kim, M.K., Woo, Y.J., Lee, M.C., Kim, J.H., Park, K.W., Kim, E.Y., Roh, Y.I., Kim, C.J., 2006b. Single nucleotide polymorphisms in the multidrug resistance 1 gene in Korean epileptics. Seizure 15, 67—72. Kimchi-Sarfaty, C., Oh, J.M., Kim, I., Sauna, Z.E., Calcagno, A.M., Ambudkar, S.V., Gottesman, M.M., 2007. A silent polymorphism in the MDR1 gene changes substrate specificity. Science 315, 525—528. Leschziner, G.D., Andrew, T., Leach, J.P., Chadwick, D., Coffey, A.J., Balding, D.J., Bentley, D.R., Pirmohamed, M., Johnson, M.R., 2007. Common ABCB1 polymorphisms are not associated with multidrug resistance in epilepsy using a gene-wide tagging approach. Pharmacogenet. Genom. 17, 217—220. Loscher, W., Potschka, H., 2002. Role of multidrug transporters in pharmacoresistance to antiepileptic drugs. J. Pharmacol. Exp. Ther. 301, 7—14. Shahwan, A., Murphy, K., Doherty, C., Cavalleri, G.L., Muckian, C., Dicker, P., McCarthy, M., Kinirons, P., Goldstein, D., Delanty, N., 2007. The controversial association of ABCB1 polymorphisms in refractory epilepsy: an analysis of multiple SNPs in an Irish population. Epilepsy Res. 73 (2), 192—198. Siddiqui, A., Kerb, R., Weale, M.E., Brinkmann, U., Smith, A., Goldstein, D.B., Wood, N.W., Sisodiya, S.M., 2003. Association of
N. Dericioglu et al. multidrug resistance in epilepsy with a polymorphism in the drug-transporter gene ABCB1. N. Engl. J. Med. 348, 1442—1448. Sills, G.J., Mohanraj, R., Butler, E., McCrindle, S., Collier, L., Wilson, E.A., Brodie, M.J., 2005. Lack of association between C3435T polymorphism in the human multidrug resistance (MDR1) gene and response to antiepileptic drug treatment. Epilepsia 46 (5), 643—647. Sisodiya, S.M., Lin, W.R., Harding, B.N., Sguier, M.V., Thom, M., 2002. Drug resistance in epilepsy: human epilepsy. Novart. Found. Symp. 243, 167—174. Tan, N.C.K., Heron, S.E., Scheffer, I.E., Pelekanos, J.T., McMahon, J.M., Vears, D.F., Mulley, J.C., Berkovic, S.F., 2004. Failure to confirm association of a polymorphism in ABCB1 with multidrugresistant epilepsy. Neurology 63, 1090—1092. Tishler, D.M., Weinberg, K.I., Hinton, D.R., Barbaro, N., Annett, G.M., Raffel, C., 1995. MDR1 gene expression in brain of patients with medically intractable epilepsy. Epilepsia 36, 1—6. Tufan, A., Babaoglu, M.O., Akdogan, A., Yasar, U., Calguneri, M., Kalyoncu, U., Karadag, O., Hayran, M., Ertenli, A.I., Bozkurt, A., Kiraz, S., 2007. Association of drug transporter gene ABCB1 (MDR1) 3435C to T polymorphism with colchicine response in familial Mediterranean fever. J. Rheumatol. 34 (7), 1540—1544. Zimprich, F., Sunder-Plassmann, R., Stogmann, E., Gleiss, A., Dal-Bianco, A., Zimprich, A., Plumer, S., Baumgartner, C., Mannhalter, C., 2004. Association of an ABCB1 gene haplotype with pharmacoresistance in temporal lobe epilepsy. Neurology 63, 1087—1089.
journal homepage: www.elsevier.com/locate/epilepsyres
Multidrug resistance in patients undergoing resective epilepsy surgery is not associated with C3435T polymorphism in the ABCB1 (MDR1) gene Nese Dericioglu a,∗, Melih O. Babaoglu b,1, Umit Yasar b,1, I. Burak Bal b,1, Atila Bozkurt b,1, Serap Saygi c,2 a
Hacettepe University, Institute of Neurological Sciences and Psychiatry, Sihhiye 06100, Ankara, Turkey Hacettepe University, School of Medicine, Department of Pharmacology, Sihhiye 06100, Ankara, Turkey c Hacettepe University, School of Medicine, Department of Neurology, Sihhiye 06100, Ankara, Turkey b
Received 30 July 2007; received in revised form 25 January 2008; accepted 5 March 2008 Available online 23 April 2008
KEYWORDS Multidrug resistance; Refractory epilepsy; Epilepsy surgery; ABCB1 (MDR1) gene; C3435T polymorphism
Summary Purpose: The C3435T polymorphism in the gene coding for P-glycoprotein (ABCB1) has been correlated with drug resistance in patients with epilepsy. However, replication studies have revealed conflicting results and the reason for this is not clear. We investigated the frequency of C3435T polymorphism in epileptic Turkish patients who underwent resective epilepsy surgery and compared our results with healthy controls. Methods: DNA samples were obtained from 100 healthy controls and 89 consecutive adult patients who underwent resective brain surgery due to refractory seizures at our epilepsy center. Genotypes for the C3435T polymorphism were determined by PCR and restriction analysis. Results: Comparison of drug-resistant patients and healthy controls revealed no significant difference in allele frequency (C vs. T; 2 = 0.015, p = 0.90) and genotype frequency (2 = 2.05, p = 0.36). The findings in the pure hippocampal sclerosis (HS) group (n = 73) were not significantly different from control subjects, either (allele frequency: 2 = 0.29, p = 0.59; genotype frequency: 2 = 2.14, p = 0.34). Conclusions: Our findings failed to prove an association between C3435T polymorphism and drug resistance in a sample of Turkish patients with refractory epilepsy who underwent resective brain surgery. © 2008 Elsevier B.V. All rights reserved.
∗
Corresponding author. Fax: +90 312 309 3451. E-mail addresses: [email protected] (N. Dericioglu), [email protected] (M.O. Babaoglu), [email protected] (U. Yasar), [email protected] (I.B. Bal), [email protected] (A. Bozkurt), [email protected] (S. Saygi). 1 Fax: +90 312 310 5312. 2 Fax: +90 312 309 3451. 0920-1211/$ — see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.eplepsyres.2008.03.004
C3435T polymorphism in the ABCB1 gene in drug resistant epilepsy
Introduction The pathogenesis underlying pharmacoresistance in epilepsy is unclear. One of the candidate mechanisms that has attracted considerable attention recently is the limitation of anticonvulsant medication access to the seizure focus by a number of efflux transporters, the most well known of which is P-glycoprotein (P-gp), encoded by the multidrug resistance ABCB1 (or MDR1) gene. P-glycoprotein is expressed at blood—tissue barriers and in organs with excretory functions. Therefore, it is thought to act as a physiologic defense by extruding xenobiotics from mammalian cells and affording protection of sensitive organs. The high level of P-gp in the cerebrovascular endothelium is believed to confer protection to the brain. It has been hypothesized that overexpression of P-gp and other efflux transporters in the cerebrovascular endothelium, in the region of the epileptic focus, may lead to drug resistance in epilepsy (Dombrowski et al., 2001). It has been claimed that a functional polymorphism of the ABCB1 gene may affect the absorption and tissue concentrations of numerous P-gp substrates (Hoffmeyer et al., 2000). Allelic differences in individual ABCB1 gene sequences may be associated with different expression levels of the gene. Several recent publications have demonstrated that the frequency of the C3435T mutation in exon 26 of the ABCB1 gene is significantly influenced by ethnicity (Ameyaw et al., 2001). Recently, Siddiqui et al. have reported that multidrugresistance in epilepsy is associated with a polymorphism in the drug transporter gene ABCB1 (Siddiqui et al., 2003). Patients with drug-resistant epilepsy were more likely to have the CC genotype at 3435 than the TT genotype. Nevertheless, replication studies afterwards have provided conflicting results (Tan et al., 2004; Zimprich et al., 2004; Sills et al., 2005; Kim et al., 2006a,b; Shahwan et al., 2007; Leschziner et al., 2007). The discrepancy between these findings has not been clarified yet. It is not known whether this is due to the different ethnic populations studied; the use of nonstandardized definitions of pharmacoresistance or due to other methodological issues. In this study, we aimed to investigate the frequency of the C3435T polymorphism in drug-resistant epileptic Turkish patients, where we defined multidrug resistance as ‘‘the patient’s absolute need for resective brain surgery due to uncontrolled seizures’’.
Patients and methods Patients Blood samples were collected prospectively from patients who underwent resective brain surgery for drug-resistant symptomatic or cryptogenic partial epilepsy (temporal: n = 86; extratemporal: n = 3) between July 2003 and May 2006 at our epilepsy center. All the patients had been followed for sufficiently long periods. They had at least 1 seizure/month, had failed at least two conventional antiepileptic drugs, and most, if not all, were on polytherapy. Pgp substrates such as carbamazepine, phenytoin or phenobarbital had been administered to all of the patients. Prior to surgery all the patients were investigated with long-term video-EEG monitoring and high resolution (1.5—3 T) cranial MRI. Interictal/ictal SPECT and PET were performed in selected cases. Patients were discussed
43
at a weekly multidisciplinary conference with all the available data (clinical history, interictal/ictal EEG, seizure semiology, structural ± functional neuroimaging studies), where a final consensus was reached. Patients who were offered surgery, but somehow were not operated during that time period were excluded. We compared our results with age-matched healthy volunteers whose demographic and genotypic findings have been reported recently (Tufan et al., 2007). The study was approved by the research ethics committee of Hacettepe University. All patients gave written informed consent.
Genotyping procedure for ABCB1 3435C>T DNA was extracted from blood samples using QIAamp DNA blood kit (Qiagen, Hilden, Germany). The analysis of ABCB1 3435C>T was performed with a PCR-based endonuclease digestion method (Babaoglu et al., 2005; Drozdzik et al., 2003). The forward and reverse primers were 5 -TGTTTTCAGCTGCTTGATGG-3 and 5 -AAGGCATGTATGTTGGCCTC-3 , respectively (Quiagen, Hilden, Germany). The genomic fragment encompassing the point mutation of interest were amplified by using 100—200 ng DNA in a total volume of 50 L containing 100 M of each deoxynucleotides, 2 mM MgSO4 , 0.5 M of each primers and 2.5 units (U) Taq polymerase (New England Biolabs GmbH, Frankfurt, Germany). Amplification was performed using a Px2 Thermal Cycler (Thermo Electron Co., MA, USA) for 35 cycles, consisting of denaturation at 94 ◦ C for 30 s, annealing at 60 ◦ C for 30 s and extension at 72 ◦ C for 30 s between the initial denaturation at 94 ◦ C for 2 min and a final extension 72 ◦ C for 7 min. The amplified 197 bp fragment was then analyzed by endonuclease restriction in which 5 U of MboI (New England Biolabs GmbH, Frankfurt, Germany) was used for the detection of ABCB1 3435C>T substitution. 3435C allele was detected by the presence of two restriction fragments, which were 158 bp and 39 bp long. The 3435C>T substitution destroyed the cleavage site for the restriction enzyme. Restriction fragments were separated by electrophoresis in a 3% agarose gel and after ethidium bromide staining visualized using an ultraviolet transiluminator. Differences in genotype and allele frequencies between the groups were compared using the 2 -test. Significance was accepted at p < 0.05.
Results Demographic characteristics of the patients and healthy control subjects are shown in Table 1. Three different genotypes, CC, CT and TT were identified at position 3435 in exon 26 and their frequencies in the study group were 29.3% CC, 38.2% CT, 32.6% TT (Table 2). These values did not deviate significantly from the control group (2 = 2.05, p = 0.36). Since most of the patients in the study group had pure hippocampal sclerosis (HS) (82%), we also analyzed this subgroup separately. Genotype frequencies in this subgroup were as follows: 27.4% CC, 38.4% CT, 34.2% TT and were not significantly different from the control sample (2 = 2.14, p = 0.34) (Table 2). Similarly, no significant differences were noted in allele frequency between patients in the study group (p = 0.90; odds ratio = 0.97; 95% CI, 0.65—1.46) or patients with HS only (p = 0.59; odds ratio = 0.84; 95% CI, 0.55—1.27) and controls (Table 2).
Discussion Here, we report for the first time that a particular group of drug-resistant epilepsy patients who needed resective brain
44
N. Dericioglu et al.
Table 1
Characteristics of patients and healthy controls
Variable
Patients (n = 89)
Healthy controls (n = 100)a
Sex Male Female
42 47
46 54
Age Mean ± S.D.
28.7 ± 7.5
29.3 ± 14.5
Pathological findings HS only HS + CD CD DNET Tumor Non-specific findings
73 2 10 1 2 1
— — — — — —
HS: hippocampal sclerosis; CD: cortical dysplasia; DNET: dysembryoplastic neuroepithelial tumor. a Tufan et al. (2007).
surgery revealed no significant difference in genotype (CC vs. CT or TT) or allele (C vs. T) frequencies for the drug transporter ABCB1 C3435T polymorphism as compared to healthy control subjects in the Turkish population. The genotype frequencies in the healthy control group (Tufan et al., 2007) were similar to those reported in a previous study in healthy Turkish volunteers (Kerb et al., 2001) and in various Caucasian populations (Hoffmeyer et al., 2000; Ameyaw et al., 2001). The majority of patients with refractory epileptic seizures are resistant to many anticonvulsants, although the drugs do not all have the same mechanism of action. This suggests that a non-specific mechanism, such as seizureinduced overexpression of multidrug-resistance proteins, might be one of the causes of pharmacoresistant epilepsy (Sisodiya et al., 2002). The role of transporter proteins in drug-resistant epilepsy is an area of growing interest, since the first publication (Tishler et al., 1995). The most frequently studied protein in this regard is the P-gp which consists of 28 exons and is encoded by the ABCB1 gene (Callen et al., 1987). P-glycoprotein has wide substrate
specificity for lipophilic molecules including most of the major antiepileptic drugs (Loscher and Potschka, 2002). Genetic variations of ABCB1 have been studied extensively, reporting over 50 single nucleotide polymorphisms (SNP) (Ishikawa et al., 2004). Allelic variations in ABCB1 have attracted attention as a possible explanation for interindividual differences in drug response. In particular, a SNP in exon 26 (C—T transformation at position 3435) has been associated with altered expression levels and transport activity of P-gp. Increased levels and increased activity of P-gp are conferred by the CC genotype, whereas individuals with the TT genotype are reported to have reduced P-gp activity and higher plasma concentrations of substrate drugs after oral administration (Hoffmeyer et al., 2000). A recent study demonstrated that C3435T polymorphism is possibly the key genetic variation that determines the transporter—drug interaction possibly by altering the transporter’s substrate specificity (Kimchi-Sarfaty et al., 2007). In 2003, Siddiqui et al. reported for the first time that multidrug-resistance in epilepsy was associated with a polymorphism in the drug transporter gene ABCB1 (Siddiqui et al., 2003). As compared with drug-responsive epilepsy patients, subjects with drug-resistant epilepsy were more likely to have the CC genotype at ABCB1 3435 than the TT genotype. Comparison with control subjects without epilepsy also revealed similar findings. All the subsequent studies, except one (Hung et al., 2005), reported that there were no significant differences in genotype and/or haplotype frequencies between drug-resistant and drugresponsive patients or healthy controls (Tan et al., 2004; Zimprich et al., 2004; Sills et al., 2005; Kim et al., 2006a,b; Shahwan et al., 2007; Leschziner et al., 2007). The studies were usually conducted in heterogeneous patient groups with different phenotypic characteristics and in various ethnic groups such as Taiwanese (Hung et al., 2005), Korean (Kim et al., 2006a,b), Scottish (Sills et al., 2005), Irish (Shahwan et al., 2007) and British (Siddiqui et al., 2003; Leschziner et al., 2007) populations. The only study that supports the findings of the original study by Siddiqui et al. reported the genotypes of 10 different polymorphisms in drug-resistant epileptic patients (Hung et al., 2005). It was indicated that patients with certain haplotypes were more likely to be drug resistant and that there is significant link-
Table 2 Genotype and allele distribution of the C3435T polymorphism of the ABCB1 gene in the patients (all and hippocampal sclerosis (HS) only) and healthy control subjects Genotypes/alleles Genotypes CC CT TT Alleles C allele T allele a b
All patients (n, %)
95% CI
HS only (n, %)
95% CI
Healthy controlsa (n, %)
95% CI
26 (29.3%) 34 (38.2%) 29 (32.6 %) p = 0.36b
19.8—38.8 28.1—48.3 21.8—41.2
20 (27.4%) 28 (38.4%) 25 (34.2%) p = 0.34b
17.2—37.6 27.2—49.6 23.3—45.1
25 (25%) 49 (49%) 26 (26%)
16.5—33.5 39.2—58.8 17.4—34.6
86 (48.3%) 92 (51.7%) p = 0.90b
41.6—56.2 43.8—58.4
68 (46.6%) 78 (53.4%) p = 0.59b
38.5—54.7 45.3—61.5
99 (49.5 %) 101 (50.5%)
42.6—56.4 43.6—57.4
Tufan et al. (2007). As compared to the control group.
C3435T polymorphism in the ABCB1 gene in drug resistant epilepsy age disequilibrium among the SNPs C1236T, G2677T/A and C3435T in exons 12, 21 and 26, respectively. The haplotypes CGC, TGC and TTT were more likely to be associated with drug resistance in epilepsy (Hung et al., 2005). It was concluded that the three loci jointly influenced the treatment response for epileptic patients. Although most of these studies failed to demonstrate a significant association between ABCB1 genotype and epilepsy phenotype, the findings in patients with temporal lobe epilepsy (TLE) are worth mentioning. In their study, Tan et al. reported that in the TLE subgroup, genotype frequencies differed according to the presence of HS; TT genotype (contrary to the a priori hypothesis) was more common in TLE with HS (40.5%) than TLE without HS (24.5%) (Tan et al., 2004). In contrast to the above study, we failed to demonstrate any differences in the genotypic frequency in our patients with HS (frequency of the TT genotype = 34.2%). This might be due to the smaller number of patients in our study group; methodological differences (since we compared our study group with normal controls) or the finding in the previous study might be a false positive association due to chance, as the authors themselves have stated. The study by Zimprich et al. also failed to detect any difference between patients with TLE and non-epileptic controls (Zimprich et al., 2004). Contrary to the study by Tan et al. there were no differences between patients with mesial TLE and cryptogenic TLE. Whether an association exists or is dependent on the degree of drug-resistance awaits further investigations in this group. The precise definition of drug resistance may vary according to syndrome and subject, and awaits better understanding of the underlying mechanisms. Not surprisingly, in the studies mentioned above, drug resistance (e.g. <50% reduction in seizure frequency in the preceding year; >4—10 seizures/year; <2 to >6 seizures/month) and responsiveness (e.g. >50% reduction in seizure frequency in preceding year; complete seizure freedom >1—2 years) were defined in different ways. In our study, we preferred to define drug resistance as ‘‘the patient’s absolute need for resective brain surgery due to uncontrolled seizures after treatment with various drugs’’, thus minimizing any confusions with regard to the degree of drug resistance in a patient. After thorough investigation of the patients, we also excluded the possibility of including patients with pure psychogenic non-epileptic seizures that are known to constitute 10—20% of the patients referred to epilepsy centers (Benbadis and Allen Hauser, 2000). Surgical resection gave us the opportunity to have the pathological diagnosis. It is possible that different drug-resistance mechanisms are involved in localization-related epilepsies with various pathological substrates. Therefore, it seems plausible to perform the aforementioned association studies in phenotypically and perhaps pathologically homogenous groups of patients. A limitation of our study is the small sample size. In order to have a power of 80%, at least 318 patients were needed (Dupont and Plummer, 1998). However, considering that the study population consists of drug refractory epilepsy patients who underwent resective brain surgery, it is very unlikely to collect so many samples at a single center. To conclude, our study failed to demonstrate an association between ABCB1 C3435T polymorphism at exon 26 and antiepileptic drug resistance in a particular sample of Turk-
45
ish patients who needed brain resection for the treatment of multidrug-resistant epilepsy.
Acknowledgements This study was supported by a grant from the Scientific and Technical Research Council of Turkey (SBAGCOST B25-105S027). Drs. Babaoglu and Yasar are the recipients of the Turkish Academy of Sciences, Young Scientist Award (MOB/TUBA-GEBIP/2007—06 and UY/TUBAGEBIP/2005—17). The authors thank Dr. Y. Yardimci for technical assistance in genotyping.
References Ameyaw, M.M., Regateiro, F., Li, T., Liu, X., Tariq, M., Mobarek, A., Thornton, N., Folayan, G.O., Githang’a, J., Indalo, A., Ofori-Adjei, D., Price-Evans, D.A., McLeod, H.L., 2001. MDR1 pharmacogenetics: frequency of the C3435T mutation in exon 26 is significantly influenced by ethnicity. Pharmacogenetics 11, 217—221. Babaoglu, M.O., Bayar, B., Aynacioglu, A.S., Kerb, R., Abali, H., Celik, I., Bozkurt, A., 2005. Association of the ABCB1 3435C>T polymorphism with antiemetic efficacy of 5-hydroxytryptamine type 3 antagonists. Clin. Pharmacol. Ther. 78, 619—626. Benbadis, S.R., Allen Hauser, W., 2000. An estimate of the prevalence of psychogenic non-epileptic seizures. Seizure 9 (4), 280—281. Callen, D.F., Baker, E., Simmers, R.N., Seshadri, R., Roninson, I.B., 1987. Localization of the human multidrug resistance gene, MDR1, to 7q21.1. Hum. Genet. 77, 142—144. Dombrowski, S.M., Desai, S.Y., Marroni, M., Cucullo, L., Goodrich, K., Bingaman, W., Matberg, M.R., Bengez, L., Janigro, D., 2001. Overexpression of multiple drug resistance genes in endothelial cells from patients with refractory epilepsy. Epilepsia 42 (12), 1501—1506. Drozdzik, M., Bialecka, M., Mysliwiec, K., Honczarenko, K., Stankiewicz, J., Sych, Z., 2003. Polymorphism in the Pglycoprotein drug transporter MDR1 gene: a possible link between environmental and genetic factors in Parkinson’s disease. Pharmacogenetics 13, 259—263. Dupont, W.D., Plummer, W.D., 1998. Power and sample size calculations for studies involving linear regression. Control Clin. Trials 19, 589—601. Hoffmeyer, S., Burk, O., von Richter, O., Arnold, H.P., Brockm¨ oller, J., Johne, A., Cascorbi, I., Gerloff, T., Roots, I., Eichelbaum, M., Brinkmann, U., 2000. Functional polymorphisms of the human multidrug-resistance gene: multiple sequence variations and correlation of one allele with P-glycoprotein expression and activity in vivo. Proc. Natl. Acad. Sci. U.S.A. 97, 3473— 3478. Hung, C.C., Tai, J.J., Lin, C.J., Lee, M.J., Liou, H.H., 2005. Complex haplotypic effects of the ABCB1 gene on epilepsy treatment response. Pharmacogenomics 6 (4), 411—417. Ishikawa, T., Onishi, Y., Hirano, H., Oosumi, K., Nagakura, M., Tarui, S., 2004. Pharmacogenomics of drug transporters: a new approach to functional analysis of the genetic polymorphisms of ABCB1 (P-glycoprotein/MDR1). Biol. Pharm. Bull. 27, 939— 948. Kerb, R., Aynacioglu, A.S., Brockm¨ oller, J., Schlagenhaufer, R., Bauer, S., Szekeres, T., Hamwi, A., Fritzer-Szekeres, M., Baum¨ gartner, C., Ongen, H.Z., G¨ uzelbey, P., Roots, I., Brinkmann, U., 2001. The predictive value of MDR1 CYP2C9 and CYP2C19 polymorphisms for phenytoin plasma levels. Pharmacogenom. J. 1, 204—210.
46 Kim, D.W., Kim, M., Lee, S.K., Kang, R., Lee, S.Y., 2006a. Lack of association between C3435T nucleotide MDR1 polymorphism and multidrug-resistant epilepsy. Seizure 15 (5), 344—347. Kim, Y.O., Kim, M.K., Woo, Y.J., Lee, M.C., Kim, J.H., Park, K.W., Kim, E.Y., Roh, Y.I., Kim, C.J., 2006b. Single nucleotide polymorphisms in the multidrug resistance 1 gene in Korean epileptics. Seizure 15, 67—72. Kimchi-Sarfaty, C., Oh, J.M., Kim, I., Sauna, Z.E., Calcagno, A.M., Ambudkar, S.V., Gottesman, M.M., 2007. A silent polymorphism in the MDR1 gene changes substrate specificity. Science 315, 525—528. Leschziner, G.D., Andrew, T., Leach, J.P., Chadwick, D., Coffey, A.J., Balding, D.J., Bentley, D.R., Pirmohamed, M., Johnson, M.R., 2007. Common ABCB1 polymorphisms are not associated with multidrug resistance in epilepsy using a gene-wide tagging approach. Pharmacogenet. Genom. 17, 217—220. Loscher, W., Potschka, H., 2002. Role of multidrug transporters in pharmacoresistance to antiepileptic drugs. J. Pharmacol. Exp. Ther. 301, 7—14. Shahwan, A., Murphy, K., Doherty, C., Cavalleri, G.L., Muckian, C., Dicker, P., McCarthy, M., Kinirons, P., Goldstein, D., Delanty, N., 2007. The controversial association of ABCB1 polymorphisms in refractory epilepsy: an analysis of multiple SNPs in an Irish population. Epilepsy Res. 73 (2), 192—198. Siddiqui, A., Kerb, R., Weale, M.E., Brinkmann, U., Smith, A., Goldstein, D.B., Wood, N.W., Sisodiya, S.M., 2003. Association of
N. Dericioglu et al. multidrug resistance in epilepsy with a polymorphism in the drug-transporter gene ABCB1. N. Engl. J. Med. 348, 1442—1448. Sills, G.J., Mohanraj, R., Butler, E., McCrindle, S., Collier, L., Wilson, E.A., Brodie, M.J., 2005. Lack of association between C3435T polymorphism in the human multidrug resistance (MDR1) gene and response to antiepileptic drug treatment. Epilepsia 46 (5), 643—647. Sisodiya, S.M., Lin, W.R., Harding, B.N., Sguier, M.V., Thom, M., 2002. Drug resistance in epilepsy: human epilepsy. Novart. Found. Symp. 243, 167—174. Tan, N.C.K., Heron, S.E., Scheffer, I.E., Pelekanos, J.T., McMahon, J.M., Vears, D.F., Mulley, J.C., Berkovic, S.F., 2004. Failure to confirm association of a polymorphism in ABCB1 with multidrugresistant epilepsy. Neurology 63, 1090—1092. Tishler, D.M., Weinberg, K.I., Hinton, D.R., Barbaro, N., Annett, G.M., Raffel, C., 1995. MDR1 gene expression in brain of patients with medically intractable epilepsy. Epilepsia 36, 1—6. Tufan, A., Babaoglu, M.O., Akdogan, A., Yasar, U., Calguneri, M., Kalyoncu, U., Karadag, O., Hayran, M., Ertenli, A.I., Bozkurt, A., Kiraz, S., 2007. Association of drug transporter gene ABCB1 (MDR1) 3435C to T polymorphism with colchicine response in familial Mediterranean fever. J. Rheumatol. 34 (7), 1540—1544. Zimprich, F., Sunder-Plassmann, R., Stogmann, E., Gleiss, A., Dal-Bianco, A., Zimprich, A., Plumer, S., Baumgartner, C., Mannhalter, C., 2004. Association of an ABCB1 gene haplotype with pharmacoresistance in temporal lobe epilepsy. Neurology 63, 1087—1089.