Abstract
Epilepsy is a complex disease characterized by a predisposition to recurrent unprovoked seizures.
Abstract
1 Despite treatment with antiepileptic drugs (AEDs), about one-third of newly treated patients do not respond adequately to medications, making pharmacoresistance a major problem in the control of this condition. 2 Recent studies have investigated the association between over-expression of efflux transporters and excess efflux of AEDs across the blood-brain barrier (BBB) thereby leading to drug-resistant epilepsy. 3 P-glycoprotein (P-gp) was the first discovered human ABC transporter more than 30 years ago in drug-resistance ovarian cells obtained from Chinese hamsters. Keywords: Meta-analysis Epilepsy ABCB1 Polymorphism P-glycoprotein Antiepileptic drugs Pharmacoresistance
Abstract
Objective: The C3435T, a major allelic variant of the ABCB1 gene, is proposed to play a crucial role in drugresistance in epilepsy. The C/C genotype carriers reportedly are at higher risk of pharmacoresistance to AEDs, but only in some studies. The hypothesis of the C-variant associated risk and resistance to antiepileptic drugs (AEDs) has been hampered by conflicting results from inadequate power in casecontrol studies. To assess the role of C3435T polymorphism in drug-resistance in epilepsy, a systematic review and meta-analysis was conducted. Methods: Databases were obtained from the Cochrane Library, MEDLINE, EMBASE, major American and European conference abstracts, and www.google.my for genetic association studies up to February 2010. All the case-control association studies evaluating the role of ABCB1 C3435T in pharmacoresistance to AEDs were identified. The new definition of treatment outcome from International League Against Epilepsy (ILAE) was used for including studies for sub-analysis. To measure the strength of genetic association for the gene variant, the odds ratios (ORs) with 95% confidence intervals (CIs) were calculated using models of both fixed-and random-effects for comparisons of the alleles and genotypes with codominant (C/C vs. T/T, C/T vs. T/T), dominant (C/C + C/T vs. T/T), and recessive (C/C vs. C/T + T/T) models in overall and in ethnicity subgroups. The 19 studies were selected for the next sub-analysis based on the new definition of drug-responsiveness and drug-resistance from ILAE. The same analysis was also performed for treatment outcome and ethnicity subgroups.
Abstract
Results: A total of 22 association studies including 3231 (47.8%) drug-resistant patients and 3524 (52.2%) drug-responsive patients or healthy controls (genotyped for C3435T) were pooled in this meta-analysis. The allelic association of ABCB1 C3435T with risk of drug-resistance was not significant under fixedeffects model, 1.06 (95% CI 0.98-1.14, p = 0.12) and random-effects model, 1.10 (0.93-1.30, p = 0.28) in overall and in the subgroup analysis by ethnicity. Similar results were also obtained for all genetic models in the stratified analyses by new definition of drug-resistance by ILAE and ethnicity subgroups. There was no publication bias. Conclusion: We failed to show an association between the ABCB1 C3435T polymorphism and the risk of drug-resistance suggesting a revision in contribution of this polymorphism in the multi-drug transporters hypothesis of pharmacoresistance to AEDs in epilepsy. ß

gp expression is highly variable between different individuals. Inter-individual variability of P-gp activity may affect blood levels and drug distribution to the specific target compartment. 6 The ABCB1 gene is highly polymorphic and more than 50 variants reside in the coding region which can possibly cause altered function. The C3435T polymorphism is one of the most common polymorphisms in the ABCB1 gene. 7 There appears to be a possible link between the ABCB1 C3435T polymorphism and drug-resistance epilepsy but results from various studies indicate that this is controversial. The first pharmacogenetic study on this matter suggested a strong and significant association (P = 0.006) between the C/C genotype in the ABCB1 C3435T polymorphism and drug-resistant epilepsy. 8 Following that study, 21 replication studies were conducted to evaluate this hypothesis, but only nine confirmed the results of the first report (Table 2) . It is unclear why these reports have found such contradictory results. How can such conflicting results be interpreted? Is there any obvious effect of C3435T polymorphism on response to AEDs even if the phenotypes are almost the same? Two meta-analyses of three and eleven association studies published in 2007 and 2008, respectively did not confirm whether the C3435T in the ABCB1 gene contributes to the risk of drugresistance in epilepsy patients and certain ethnic subgroups. 9, 10 Therefore, to overcome the limitations of the individual studies and reliably assess the hypothesized ABCB1 C3435T polymorphism relationship with the risk of drug-resistance in epilepsy on the basis of the existing data, we provided pooled estimates using both fixed-and random-effects models in overall, the Asian and Caucasian populations and definition of treatment outcome using all genetic model analysis.
Method

Search strategy and selection
All articles that examined the ABCB1 C3435T association with drug-resistance in epilepsy were identified. Databases were obtained from MEDLINE, EMBASE, as well as the Cochrane Database of Systematic Reviews, major American and European conference abstracts, and www.google.com and all relevant studies were compiled up to February 2010. Non-English language publications were excluded. Additionally, we hand searched the reference lists of retrieved full-text articles. MESH terms used included ''EPILEPSY'', ''POLYMORPHISM'', ''ABCB1'', ''C3435T'', ''DRUG-RESISTANT'', ''DRUG-RESPONSIVENESS'', ''ANTIEPILEPTIC DRUGS'', and ''ANTICONVULSANT DRUGS'' (including ''MONO-THERAPY'' and ''POLYTHERAPY''). Two reviewer independently assessed titles and abstracts of electronic searches, obtaining the full articles to assess for relevance where necessary.
Data extraction
For primary selection of the studies, all articles published till February 2010, were considered. All selected articles were examined for their appropriateness by two independent reviewers using an extraction template. Disagreements were documented and resolved by discussion with a third author. The case-control genetic association studies included in this meta-analysis had to meet the following criteria: (a) AED treatment of patients and compliance; (b) data on genotype distributions were available for both case and control groups; (c) genotype distribution of the control subjects conformed to the Hardy-Weinberg equilibrium; and (d) clear treatment outcome of either resistant or responsive to AEDs. Information on name of the first author, year of publication, country, journal, ethnic origin of the studied population, sample size, definition of drug-resistance and drug responsiveness, the types of epilepsy syndromes and AED treatment, genotyping methods, genotype and allele distributions, and confirmation of the diagnosis were abstracted. Genotype distributions reported in percentages were converted to actual numbers. If allele frequencies were not given, they were calculated from the corresponding genotype distributions. The control group was either drugresponsive epilepsy patients receiving AEDs or healthy people. In each study, if both drug-responsive patients and control data were available, we used drug-responsive patients' data for analyses. The phenotype definition of treatment outcome in each study was assessed according to the new three categories from the International League Against Epilepsy (ILAE): (1) drug-responsiveness as complete seizure freedom for at least one year; (2) drugresistant as failure of two tolerated and appropriately chosen and used AED schedules (whether as monotherapies or in combination) to achieve sustained seizure freedom; and (3) undetermined. 11 The consistent studies with outcome categories either 1 and 2 or 3 were classified as group one and two, respectively.
Statistical analysis
The meta-analysis was performed to examine the overall association for allelic (C vs. T) and genotype genetic models (C/C vs. T/T and C/T vs. T/T, C/C + C/T vs. T/T, and C/C vs. C/T + T/T, assuming co-dominant, dominant, and recessive effects) of the 3435C allele and the risk of resistance to AEDs. Deviation of Hardy-Weinberg equilibrium (HWE) was examined by x 2 tests. The per-allele odds ratio (OR) of the rare allele (3435T) as well as the corresponding 95% confidence intervals (CI) and p value were calculated as using a comparative two group outcomes statistics to compare drugresistant and drug-responsive patients. Fixed-effects summary measures were calculated as inverse-variance-weighted average of the log OR if there was no heterogeneity (p > 0.10) and randomeffects where substantial heterogeneity (p < 0.10) exist. To measure the strength of genetic association, we used the I 2 test to assess the proportion of statistical heterogeneity and the Qstatistic test with a p < 0.10 to define a significant degree of heterogeneity. A sensitivity analysis was carried out by excluding each study in turn to investigate the extent to which they contributed to the heterogeneity. Subsidiary analyses including subgroup analyses were performed to evaluate the effects of either ethnicity in overall of studies for allelic model or definition of treatment outcome and ethnicity in overall and subgroups under all genetic models. Ethnic group was defined as Asian and Caucasians. The new definition of treatment outcome from ILAE was used for sub-analysis of both subgroups one and two and ethnicity in the first subgroup. All probability values are 2-sided, and values of P < 0.05 were considered statistically significant. The evidence of publication bias was assessed by visual funnel plot inspection and Egger's test. Statistical analyses were performed using validated Meta-analysis Made Easy (MIX) version 1.7. 
Results

Study characteristics
The initial search with the keywords and the subject terms identified 22 publications that met the inclusion criteria and were eligible for review. Out of the 22 studies, nine were positive studies among which the carriers homozygous for the 3435T allele in three studies were significantly higher in the drug-resistant than in the drug-responsive patients. Among the 22 included studies, there was considerable diversity between ethnic groups. Fourteen articles represented studies with the Asian populations, [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] while seven were carried out in European descent populations. 8, [28] [29] [30] [31] [32] [33] Patients were recruited from Egypt (one), 19 Australia (one), 30 China (five studies), 14, 15, 18, 21, 25 Croatia (one), 28 Germany (one), 33 India (two studies), 26, 27 Ireland (one), 15 Japan (one), 17 Korea (three studies), 15, 16, 24 Scotland (one), 31 Turkey (two studies), 22,23 and United Kingdom (two studies). 8, 29 In one multi-centre cooperation study, subjects were collected from Australia, Hong Kong and Scotland. 13 We divided this study into three sub-studies for metaanalysis. Hence, the total number of studies for meta-analysis could be considered to be 24 instead of 22 for analyses. Characteristics of the included studies are presented in Table 1 .
Study characteristics
The included studies provided a total of 6755 subjects, 3231 (47.8%) drug-resistant patients and 3524 (52.2%) drug-responsive epilepsy patients or healthy controls. The median number of sample size was 288 (range, 45-609). The genotype and allele distributions of ABCB1 C3435T in the studies are shown in Table 2 . The ABCB1 C3435T polymorphism was found to occur in frequencies consistent with HWE in the drug-resistant and drug-responsive epilepsy patients or healthy populations of the published studies. Although the basic study design was the same, a wide variety of epilepsy syndromes, AED types, and definition of treatment outcomes were used. From 24 studies, 19 studies were compatible with the new outcome categories from ILAE, 12 performed in the Asian populations [13] [14] [15] [16] 18, [20] [21] [23] [24] [25] [26] [27] and seven in the Caucasian populations 8, 13, [28] [29] [30] [31] (Table 4) , while five studies were incompatible 17, 19, 22, [32] [33] (Table 5 ).
Meta-analysis
In a pooled analysis of 24 studies, no significant allelic association was recorded under either fixed-effects model 1.06 (95% CI 0.98-1.14, p = 0.12) or random-effects model, 1.10 (0.93-1.30, p = 0.28) indicating that the C allele is not associated with the risk of resistance to AED in epilepsy as compared to the T allele ( Fig. 1 , Table 3 ). The wide variation in the C allele frequency in drug-responsive and drug-resistant patients caused high and significant heterogeneity (I 2 = 80.7%, p < 0.0001). A sensitivity analysis which excluded each study in turn, demonstrated a decrease of the pooled OR from 1.06 to 0.93 but still non-significant (95% CI 0.86-1.01, p = 0.09). This heterogeneity was contributed mainly by the six positive studies 8, 14, 17, 19, 20, 28 among which the effect of two studies 14, 19 was higher. Removal of these six studies from meta-analysis gave 24.48% (p = 0.17) heterogeneity and showed that they have the highest effect on ABCB1 C3435T allelic association with the risk of resistance to AEDs. The ethnicity-based subgroup meta-analysis examining allelic model, also showed no significant association in the either Asian or Caucasian populations (Table 3) . In a comparable genotype data, the association of all genetic models with drug-resistance was not statistically significant. Subsidiary analyses of ethnicity in the 15 and nine studies from the Asians and Caucasians, respectively showed no significant associations in all genetic models. However, the risk of drugresistance in the Caucasian population for the allelic model (p = 0.10) and co-dominant (C/C vs. T/T: p = 0.10), dominant (p = 0.38), and recessive (p = 0.09) effects of the ABCB1 C3435T was greater than in Asians (p = 0.78, p = 0.91, p = 0.93, and p = 0.68, respectively). A wide range of heterogeneity (0-85.6%) was observed among the studies in both the Asian and Caucasian populations. Unlike the co-dominant (C/T vs. T/T) and dominant genetic models in the Caucasians (p = 0.50 and p = 0.12, respectively), the heterogeneity in the rest was significance ( Table 3 ).
Meta-analysis
The stratified analysis using the new definition of ILAE 11 for treatment outcomes was performed for the 19 compatible (Table 4) and five incompatible studies ( Table 5 ). The subsidiary analysis based on this new definition of treatment outcomes and ethnicity in the compatible and incompatible studies did not show any significant association under all genetic models ( Table 4 ). The range of heterogeneity in the compatible group was wide (14.3-83.3%) and significant, except of Caucasians under co-dominant (C/ T vs. T/T) (p = 0.32) and dominant (p = 0.05) models. Similarly, the incompatible studies showed a wide range of heterogeneity (58.8- 
Discussion
It was hypothesized that the ABCB1 C3435T polymorphism is related with the risk of resistance to AEDs in epilepsy. Siddiqui et al. (2003) for the first time examined whether C3435T polymorphism was associated with resistance to AEDs in the Caucasians with epilepsy. The frequency of the C/C genotype among drug-resistant patients was significantly higher than in responsive patients (P = 0.006). 8 Zimprich et al. (2004) confirmed the results of the original report (p = 0.035), 34 but the outcome of the study of with the exact replication of the first study and almost twice the sample size in the Caucasians was negative. 30 Similarly, a comprehensive genome wide approach 35 and also a combined cohort study in the Scottish, Australian, and Hong Kong populations, failed to confirm the original findings. 13 In total, 22 studies attempted to examine this hypothesis in epilepsy, but only nine found significant association in which three reported that the T/T genotype was more frequent in drug-resistant patients. There have been also two meta-analysis studies of the association of ABCB1 C3435T with drug-resistance. The first report included three studies performed in 1073 Caucasians patients with the same definition of drug-responsiveness and drug-resistance 9 and the second one included 11 studies involving 3371 patients with different ethnicities and definition of drug-responsiveness and drug-resistance. 10 Neither studies confirmed this association and stratification of ethnic subgroups in the second meta-analysis also provided no further evidence. The findings of the present meta-analysis indicate that neither the C allele nor the T allele carriers of the ABCB1 C3435T polymorphism confer significant risk to drug-resistance in epilepsy. Similar results were found for all genotype genetic models in overall of studies. In the subgroup analysis for the Asian and the Caucasian populations, none of the genetic comparisons showed a significant association. Hence, the substitution of C to T at position 3435 of the exon 26 of the ABCB1 gene does not effect on response to AEDs in the epilepsy patients with different ethnicities. As different definitions of drug-responsiveness and drug-resistance in the patients with various ethnicities were included in the studies, we therefore carried out subgroup metaanalyses based on the new definition drug-resistance by the ILAE 11 and ethnicity. Subsidiary analyses of the definition of treatment outcomes in the 19 included and five excluded studies as well as by ethnicity in the included reports did not show any association under all genetic models.
Discussion
The current meta-analysis provides a comprehensive assessment of ABCB1 C3435T variant and drug-resistance risk. Meanwhile our meta-analysis does not support an association of C3435T polymorphism with risk of resistance to AEDs. Compared with the precious meta-analysis, the present study is much larger, with almost twice as many as the cases as the earlier meta-analysis. Furthermore, we assessed not only the association between the C3435T polymorphism and drug-resistance risk in the consistent and inconsistent studies with the new definition of ILAE for treatment outcome in epilepsy but also for the stratified subgroup by ethnicity in the consistent group with ILAE definition. The nonconcordance in the studies may be explained by five phenomena: (i) publication bias which can be caused by false positive results, found by chance as a result of insufficient sample size and low statistical power. 36, 37 The meta-analysis by Bournissen et al (2008) 10 and our results, however, did not indicate significant publication bias. Moreover, sample size of the included 22 genetic association studies in the current meta-analysis was quite small, the median being 288. In order to have a power of 80%, it needs a large sample size or collaboration between multi-centres and countries 38 (20) .
Discussion
(ii) Population diversity with different race and types of seizures and epilepsy syndromes may cause variety in AEDs type administration, dosage, and treatment duration. 39 However, the data of Bournissen et al (2008) and the current metaanalysis showed no evidence that the ABCB1 C3435T polymorphism is associated with the risk of resistance to AEDs in the Asians and Caucasians. Furthermore, the data were not sufficient enough to allow us to undertake further subgroup analyses on specific subsets of epilepsy types/syndromes and AEDs type. (iii) Variability in the definitions of treatment outcomes to distinguish drugresistance from drug-responsiveness in epilepsy may cause variations in the results. 40 Since, those patients who are classified as drug-resistant in some studies may be drug-responsiveness.
Discussion
41 Despite of the fact that we have excluded five studies, there is no effect on the overall results as well as the results in the Asians and Caucasian subgroups. Furthermore, a short-term follow-up of newly diagnosed patients in the cohort studies leads to misclassification. The shortest follow-up period used by new definition of drug-resistance and drug-responsiveness from ILAE was 12 months, 11 while the range of response to AEDs is 2-16 years (median, 5 years); 3 the shortest follow-up periods for both drugresistance and drug-responsiveness to AEDs in the 22 included studies in this meta-analysis was 3 months.(iv) It is not clear whether some AEDs are substrates of P-gp; [42] [43] [44] hence justification of the results is difficult. Moreover, the interaction between AEDs in poly-therapy regimens can influence the response to AEDs. 45 In this study, because of data limitation; we did not carry out the stratification analyses by AED monotherapy regimens. (v) The complex gene-gene (epistasis) and/or gene-environment interactions contribute to etiology and response to AEDs in epilepsy. The marginal effect of each susceptibility gene is small but the effect of these genes acting through a set of genes and their variants epistically in the same pathway may be large. 46 The discrepant results may be caused by cross-tabulation of the C3435T polymorphism with specific variants either in the ABCB1 gene or other genes. 39 In addition, C3435T might have only marginal functional significance as suggested by Cascorbi. 47 Such a variantvariant interaction may play an important role in gene-disease associations than individual polymorphism in epilepsy and cause variety in AED types administration, dosage, and treatment duration across and within the populations with different features. 39 Further studies in larger and different populations need to be conducted to examine this hypothesis.
Conclusions
The results of this meta-analysis do not provide support for the association of the ABCB1 C3435T polymorphism with risk of resistance to AEDs in different races and suggest a revision in contribution of this polymorphism in the multi-drug transporters hypothesis of resistance to AEDs in epilepsy.

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