Epilepsia, 50(7):1689–1696, 2009 doi: 10.1111/j.1528-1167.2009.02059.x
FULL-LENGTH ORIGINAL RESEARCH
Multidrug-resistant genotype (ABCB1) and seizure recurrence in newly treated epilepsy: Data from international pharmacogenetic cohorts *Cassandra Szoeke, yGraeme J. Sills, zPatrick Kwan, *Slave Petrovski, xMark Newton, yNikolas Hitiris, {Larry Baum, xSamuel F. Berkovic, yMartin J. Brodie, #Leslie J. Sheffield, and *Terence J. O’Brien *The Departments of Neurology, Medicine and Surgery, The Royal Melbourne Hospital, The University of Melbourne, Parkville, Victoria, Australia; yEpilepsy Unit, University Division of Cardiovascular & Medical Sciences, Western Infirmary, Glasgow, Scotland; zDepartment of Medicine and Therapeutics, The Chinese University of Hong Kong, Prince of Wales Hospital, Hong Kong, China; xDepartments of Medicine and Neurology, Austin Health, Melbourne, Australia; {School of Pharmacy, The Chinese University of Hong Kong, Shatin, Hong Kong, China; and #Department of Paediatrics, Murdoch Childrens Research Institute, The University of Melbourne, Parkville, Victoria, Australia
SUMMARY Purpose: The association between a specific polymorphism (3435C>T) in the ABCB1 gene, coding for the membrane drug transporter P-glycoprotein (PgP), and pharmacoresistance to seizure control is controversial. Studies have been limited by multiple drug use, chronic cohorts with varying definitions, and retrospective clinical data. Herein we examine the relationship of this polymorphism with seizure recurrence in three independent international cohorts of patients newly treated for epilepsy. Methods: Data were collected on demographics, medication details, and seizure control after 12 months of treatment. The distribution of ABCB1 3435C>T genotypes was compared between patients with and without recurrent unprovoked seizures. Results: Five hundred forty-two newly treated patients were enrolled (212 from Australia, 285 from Scotland, and 45 from Hong Kong). A total of
Despite the introduction of numerous new antiepileptic medications, the prevalence of seizure recurrence in patients with epilepsy remains unacceptably high and Accepted January 5, 2009; Early View publication May 11, 2009. Address correspondence to Professor Terence J. O’Brien, Department of Medicine, The Royal Melbourne Hospital, The University of Melbourne, Royal Parade, Parkville 305, Victoria, Australia. E-mail:
[email protected] Wiley Periodicals, Inc. ª 2009 International League Against Epilepsy
% had recurrent unprovoked seizures after 38.4% starting antiepileptic drug (AED) treatment. Genotype frequencies and ethnicity did not differ between the Scottish and Australian cohorts, but both were significantly different in the Hong Kong cohort. There was no significant relationship between the ABCB1 3435C>T genotype and the rate of recurrence of unprovoked seizures in the three cohorts individually or combined; however the epilepsy syndrome and a greater number of seizures pretreatment was associated with an increased risk of seizure recurrence. Conclusions: The ABCB1 3435C>T genotype does not have a major role in determining the efficacy of seizure control with initial AED therapy. The study highlights issues that arise in combining pharmacogenetic datasets from different ethnic regions and health systems, an approach that is essential to advance this field. KEY WORDS: Antiepileptic drugs, Pharmacogenomics, ABCB1, Pharmacoresistance.
largely unpredictable in the individual patient (Kwan & Brodie, 2000; Duncan et al., 2006). Therefore, examining the role of genetic contributors to the development of drug resistance has become an area of increasingly active research. Pharmacogenetics examines the effect on drug treatment outcomes of heterogeneity in genes for protein products involved in the pharmacokinetics or pharmacodynamics of drugs in vivo. The most closely examined genetic polymorphism that potentially may
1689
1690 C. Szoeke et al. predict resistance to antiepileptic drugs (AEDs) has been the ABCB1 (ATP Binding Cassette B1, also called multidrug resistance 1 [MDR1]) 3435C>T polymorphism. The ABCB1 gene attracted initial attention because of the proven influence of its gene product, P-glycoprotein (PgP), on the efficacy of oncology drugs (Shoemaker et al., 1983). This gene belongs to the ABC superfamily of transporter genes. These genes encode transmembrane proteins that transport substrates across cellular membranes and play an important role in maintaining the blood–brain barrier by protecting the brain from the accumulation of toxic compounds that have entered the bloodstream. Many hydrophobic drugs, like AEDs, are susceptible to the active efflux properties that are present in the blood–brain barrier. The proposed mechanism of these transporters is to remove the drugs from the cells, thereby limiting brain uptake of these medications. All eukaryotic genomes contain several families of genes capable of encoding efflux transporters, among which the ABC transporters are the largest. PgP is found to be overexpressed in epileptogenic foci (Tishler et al., 1995; Aronica et al., 2004; Lazarowski et al., 2004), and thus the possibility that common AEDs are a substrate of this efflux transporter has been suggested (Potschka et al., 2001; Cox et al., 2002; Weiss et al., 2003; Giessmann et al., 2004; Kwan & Brodie, 2005). Of the common AEDs prescribed—felbamate, gabapentin, lamotrigine, phenytoin, and topiramate—all have evidence suggesting that they are substrates for PgP, whereas levetiracetam is not, and the evidence for the two more common drugs, carbamazepine and valproate, remains equivocal (Owen et al., 2001; Potschka et al., 2001; Maines et al., 2005; Rivers et al., 2008). With respect to the 3435C>T polymorphism of the ABCB1 gene, the TT genotype has been found to
correlate with decreased PgP expression (Abbott et al., 2002); this is expected to lead to higher intracellular drug concentrations, which may be reflected in better seizure control. In contrast, the CC genotype is associated with increased gene product and presumably lower intracellular concentrations of antiepileptic medication, leading to reduced antiseizure efficacy. Recently the ABCB1 3435C>T genotypes were shown to be associated with different conformations of PgP, suggesting an effect on the protein folding (Kimchi-Sarfaty et al., 2007). Although there is a plausible biologic basis for polymorphisms in this gene relating to epilepsy outcome, investigations in several chronic epilepsy populations (Siddiqui et al., 2003; Aronica et al., 2004; Zimprich et al., 2004; Hung et al., 2005; Sills et al., 2005; Seo et al., 2006; Hung et al., 2007) have yielded inconsistent results (Table 1). Drug-resistant epilepsy has a multifactorial etiology affecting heterogeneous patient populations, with many disease- and patient-related factors that may affect treatment outcomes. The limitations in these studies have been outlined in subsequent publications (Szoeke et al., 2006). The importance of clinical phenotyping and difficulties with definitions, particularly in a chronic epilepsy population, has been an issue in these studies. In addition, the lack of replication between different centers may be attributed to site- or cohort-specific confounders. Only one previous study has examined the effect of this genetic polymorphism in newly treated patients, a population with less selection bias for medically refractory epilepsy than is inherent in the chronic epilepsy cohorts, and this revealed no association with a set of ABCB1 polymorphisms (Leschziner et al., 2006). However, this study did not account for ethnicity, epilepsy syndrome, or medication type.
Table 1. Previously published pharmacogenetics studies examining the association of the ABCB1 3435C>T polymorphism with AED efficacy, in order of publication Citation
Study size
Results
Cohort
Design
Siddiqui et al., 2003 Tan et al., 2004 Zimprich et al., 2004 Hung et al., 2005a Sills et al., 2005 Kim et al., 2006b Seo et al., 2006 Leschziner et al., 2006
n n n n n n n n
= = = = = = = =
315 609 210 331 400 171 210 503
CC more likely PR No association CC more likely PR CC more likely PR No association No association TT more likely PR No association
Kwan et al., 2007 Shahwan et al., 2007 Hung et al., 2007a Dericioglu et al., 2008 Lakhan et al., 2008
n n n n n
= = = = =
746 440 327 89 325
TT more likely PR No association CC more likely PR No association No association
Chronic epilepsy Chronic epilepsy Chronic epilepsy Chronic epilepsy Chronic epilepsy Chronic epilepsy Chronic epilepsy Newly treated epilepsy Chronic epilepsy Chronic epilepsy Chronic epilepsy Chronic epilepsy Chronic epilepsy
Retrospective Retrospective Retrospective Retrospective Retrospective Retrospective Retrospective Retrospective analysis of prospective trial Retrospective Retrospective Retrospective Retrospective Retrospective
AED, antiepileptic drug; PR, pharmacoresistant. a Publications from the same cohort. Epilepsia, 50(7):1689–1696, 2009 doi: 10.1111/j.1528-1167.2009.02059.x
1691 Outcome of Newly Treated Epilepsy In the current study we have analyzed, in newly treated epilepsy patients, the association of the ABCB1 3435C>T polymorphism with seizure recurrence, accounting for confounders including age, gender, ethnicity, epilepsy syndrome, and medication type. The newly treated design of this study corrects for the multiple drug and patient selection confounders experienced in previous chronic epilepsy studies. Importantly, three independently recruited international cohorts were investigated, allowing for assessment, within one study, of the generalizability of the findings across different patient populations which vary in their ethnic backgrounds and medical systems. This is important to examine in light of the need for future large multicenter international pharmacogenetic collaborations in order to have the power to allow the discovery of genes influencing AED response.
Methods This international collaborative study consisted of three independent cohorts of patients; from Australia, Scotland, and Hong Kong, with newly diagnosed epilepsy, who had commenced an AED for the first time. The Australian cohort was prospectively recruited from patients presenting to first seizure clinics at the Royal Melbourne, Austin and Royal Perth Hospitals; the Scottish cohort comprised new-onset epilepsy patients entering monotherapy trials at the Epilepsy Unit, Western Infirmary, Glasgow; and the Hong Kong cohort was recruited from patients newly commencing therapy from inpatient wards and outpatient neurology clinics at the Prince of Wales Hospital. Patients who had commenced an AED for newly diagnosed epilepsy were eligible for inclusion in this study if they had experienced at least two unprovoked seizures or had a potentially epileptogenic lesion on their magnetic resonance imaging (MRI) scan and/or had epileptiform changes on their electroencephalography (EEG) recording. Patients who had experienced a single seizure without any of these MRI or EEG abnormalities were excluded from the analysis. Written informed consent was obtained from all patients, and the studies were approved by the respective local ethics committees. A neurologist with subspecialty expertise in epileptology assessed the patients at baseline, and data were collected on demographics, age, ethnicity, their initial medication details, and seizure and syndrome type. Seizure control after 12 months of AED treatment was assessed. For the Australian and Hong Kong cohorts, these data were obtained prospectively, whereas for the Scottish cohort these data were collated from prospective clinical trial notes. All patients who had commenced an antiepileptic medication for newly diagnosed epilepsy were eligible for inclusion in this study. Patients were classified as being AED ‘‘responsive’’ if they experienced no further unprovoked seizures over the
first 12 months of treatment, and as being ‘‘nonresponsive’’ if further seizures occurred that were not explained by medication noncompliance, inadequate dosing, or a major precipitating event. Precipitated seizures, defined as those seizures occurring due to known noncompliance, recreational drug use, or during initial drug titration phase (first month), were ignored for the purposes of this analysis. Differences in phenotype definition may be one of the reasons for the conflicting results reported by previous pharmacogenetic association studies in epilepsy. For this reason, rather than use the term ‘‘pharmacoresistant,’’ which arguably may apply only to patients who have failed multiple drug regimens, we focused on a clear clinical outcome of recurrent unprovoked seizures after 12 months on initial antiepileptic therapy. This is a clinically relevant endpoint, as the nonresponsive patients, while they may eventually achieve seizure control with subsequent medication changes, suffer the physical and psychosocial morbidity of having experienced further seizures despite treatment. In addition, previous work examining 20 years of follow-up has shown that only 5% of patients switch between responder and nonresponder groups initially classified from the first 12 months of follow-up (Mohanraj & Brodie, 2006). The pretreatment seizure frequency was separated into three categories: the first consisted of patients who experienced 1–2 seizures before starting antiepileptic medication, the second of patients experiencing 3–5 seizures, and the third patients who had >5 seizures pretreatment. Moreover, the epilepsy syndromes of the patients were collapsed into idiopathic generalized epilepsy (IGE) or localization related epilepsy (LRE), in accordance with the ILAE classification (Commission on Classification and Terminology of the International League Against Epilepsy, 1989). DNA was extracted from blood samples and genotyped for the ABCB1 3435C>T polymorphism. The genotyping methodology for each of the Australian (Petrovski et al., 2009), Scottish (Sills et al., 2005), and Hong Kong (Kwan et al., 2007) cohorts has been described in previous publications. Statistical analysis examined demographics and outcomes of interest among all three cohorts. For those factors that had a small number of patients (T genotypes were compared between patients who were responsive and those who were unresponsive to their initial AED treatment, and odds ratios were calculated between CC and TT genotypes. Finally, a multivariate logistical regression analysis examined the likelihood of genotype being associated with seizure outcome, while Epilepsia, 50(7):1689–1696, 2009 doi: 10.1111/j.1528-1167.2009.02059.x
1692 C. Szoeke et al. accounting for age, gender, ethnicity, country, medication type, and syndrome type.
Results Five hundred forty-two newly treated patients were enrolled in this collaboration (212 from Australia, 285 from Scotland, and 45 from Hong Kong). ABCB1 3435C>T genotype was available for all patients. The distributions of 3435C>T genotypes were consistent with the Hardy-Weinberg equilibrium (p > 0.05). A total of 208 (38.4%) of the patients had one or more recurrent unprovoked seizures within 1 year of initial treatment, excluding any that occurred during the first month in the period of drug titration, and were, therefore, classified as being nonresponsive to the initial AED therapy. Table 2 shows the basic demographic, initial treatment, pretreatment seizure frequency, syndrome type, and ABCB1 genotype distribution characteristics of the three cohorts. There was no significant difference in age or gender between the cohorts. There was a significant difference in both ethnicity and genotype frequencies when the Hong Kong cohort was compared with the Australian and Scottish cohorts, but no differences between the latter two cohorts. Moreover, pretreatment seizure frequency was
markedly different between the cohorts (p < 0.001). All three cohorts differed in the rates of seizure recurrence (p < 0.001) and the distribution of the types of AED taken (p < 0.001). The three cohorts were not significantly different in their distribution of epilepsy syndrome type, with a predominance of localization-related epilepsies. The distribution of the genotypes for the ABCB1 3435C>T single nucleotide polymorphism (SNP) in pharmacoresponsive and nonresponder patients for the combined cohorts is given in Table 3. No significant associations between the genotype and responsiveness to initial AED treatment were found in any of the individual cohorts, nor when combined [odds ratio (OR) = 0.79, 95% confidence interval (CI) 0.48–1.28, p = 0.55]. The odds ratio estimate was >1.0 for CC versus TT genotype in the Australian cohort and T polymorphism
Female Male Carbamazepine Valproate Lamotrigine Phenytoin Levetiracetam Topiramate Other AED Nonresponder Responder Caucasian Asian Other IGE LRE Unclassified 1–2 seizures 3–5 seizures >5 seizures unknown CC CT TT
Scotland (n = 285)
Combined (n = 542)
Hong Kong (n = 45)
n mean
% or (SD)
n mean
% or (SD)
n mean
% or (SD)
p-value
n mean
% or (SD)
39 102 110 112 81 4 9 6 0 0 64 148 196 2 14 57 144 11 80 45 87 0 55 94 63
(19.7) 48.1% 51.9% 52.8% 38.2% 1.9% 4.3% 2.8% 0.0% 0.0% 30.2% 69.8% 92.5% 0.9% 6.6% 26.9% 67.9% 5.2% 37.74% 21.23% 41.03% N/A 26.0% 44.3% 29.7%
40 128 157 19 92 115 1 22 16 20 133 152 281 4 0 92 185 8 71 81 130 3 54 141 90
(17.6) 44.9% 55.1% 6.7% 32.3% 40.3% 0.4% 7.7% 5.6% 7.0% 46.7% 53.3% 98.6% 1.4% 0.0% 32.3% 64.9% 2.8% 25.2% 28.7% 46.1% N/A 18.9% 49.5% 31.6%
48 18 27 1 21 8 15 0 0 0 11 34 1 44 0 19 25 1 27 7 10 1 14 28 3
(25.1) 40.0% 60.0% 2.2% 46.7% 17.8% 33.3% 0.0% 0.0% 0.0% 24.4% 75.6% 2.2% 97.8% 0.0% 42.2% 55.6% 2.2% 61.3% 16.0% 22.7% N/A 31.1% 62.2% 6.7%
>0.05 >0.05
40.2 248 294 132 194 127 25 28 16 20 208 334 478 50 14 168 354 20 178 133 227 4 123 263 156
(19.3) 45.8% 54.2% 24.3% 35.8% 23.4% 4.6% 5.2% 3.0% 3.7% 38.4% 61.6% 88.2% 9.2% 2.6% 31.0% 65.3% 3.7% 33.1% 24.7% 42.2% N/A 22.7% 48.5% 28.8%
5 seizures was predictive of drug responsiveness.
Discussion 95.0% CI
Age Antiepileptic drug (AED) Gender Syndrome CC vs. TT CT vs. TT Ethnicity >5 vs. 1–2 pretreatment seizures >5 vs. 3–5 pretreatment seizures
Sig.
Odds ratio
Lower
Upper
0.28 0.27 0.80 0.02 0.47 0.92 0.87 T genotype was not predictive of drug responsiveness (Table 5). However, logistic
In this study no significant association of the ABCB1 3435C>T genotype with spontaneous seizure recurrence over 12 months after initial AED treatment was found, either in the study population as a whole or in the individual international cohorts that comprised the study population. Neither was a significant association seen when the analysis was restricted to patients with localization-related (focal) epilepsies, the group to which the hypothesis is most applicable (Abbott et al., 2002). The strengths of this study are that the cohort consisted entirely of newly treated patients, thereby avoiding many of the selection biases inherent in chronic epilepsy cohorts, and that the analysis was performed on three independent international cohorts from different ethnic and genetic backgrounds treated in different medical health systems. It is important to point out that this study cannot definitively show that the ABCB1 gene is unrelated to pharmacoresistance, but rather that the specific ABCB1 3435C>T polymorphism does not have a major association with response to initial AED treatment in its own right. Epilepsia, 50(7):1689–1696, 2009 doi: 10.1111/j.1528-1167.2009.02059.x
1694 C. Szoeke et al. Although clinical and syndromic factors have been considered, other genetic factors may override or enhance the effect the 3435C>T polymorphism has on responsiveness. Therefore, a more complete genetic study to discover other genes that may be influencing pharmacoresistance is required. Identifying a single polymorphism of sufficient strength to entirely explain the observed pharmacoresistance in patients with epilepsy seems unlikely. There are a large number of genes that could theoretically contribute to the phenomenon of pharmacoresistance in epilepsy, and the study of resistance to AEDs in humans can be effectively carried out only if all such genes are considered, rather than a handful of selected candidates. Even among the ABC genes, there are several other classes of encoded proteins, such as the multidrug resistance– associated proteins (MRPs) and breast cancer resistance proteins (BCRPs), which are expressed in the blood–brain barrier and could potentially contribute to reducing the brain penetration of AEDs in patients with pharmacoresistant epilepsy (Begley, 2004). Previous discussions of multidrug resistance (MDR)– association studies have commented on the importance of linkage disequilibrium, as ABCB1 3435C>T is a synonymous polymorphism that does not result in alteration of the amino acid sequence in the transcribed protein. In addition, patterns of linkage disequilibrium differed across ethnic groups. In our collaboration, we noted different frequencies of ABCB1 3435CC, CT, and TT genotypes in patients of Caucasian and Asian ethnicity, and a different proportion of genotype frequencies in drug-responsive individuals—but not between individual cohorts when the confounding influence of ethnicity was removed. This highlights the importance of considering ethnicity and including this factor as a potential confounder in pharmacogenetic association analyses. An important observation of this study is the differences in the rates of seizure recurrence, the number of seizures prior to treatment, and patterns of AED use between countries, which are not accounted for by differences in epilepsy syndromes. The latter two most likely represent local differences in prescribing habits, but also in some cases (predominantly the Scottish cohort) the inclusion of patients undergoing randomized clinical trials in which the choice of medication is necessarily limited. With the importance of large-scale patient recruitment to achieve sufficient statistical power for genome-wide pharmacogenetic studies, international collaboration is likely to be essential. This study demonstrates important differences in country-specific factors that will need to be accounted for in these collaborative efforts. The importance of collecting data on type of medication, epilepsy syndrome, and ethnicity to allow for differences in these characteristics between different international sites needs to be accounted for in the overall analysis. Epilepsia, 50(7):1689–1696, 2009 doi: 10.1111/j.1528-1167.2009.02059.x
The number of seizures prior to treatment has been advocated as a method of assessing disease severity for pharmacogenetic studies of AED response (Rogawski & Johnson, 2008), and has consistently been shown to be predictive of the success of seizure control in newly treated epilepsy (Kim et al., 2006a; Leschziner et al., 2006; Mohanraj & Brodie, 2006; Schiller & Najjar, 2008). As Rogawski and Johnson note: ‘‘Indeed, the frequency of seizures in the early phase of epilepsy is the dominant risk factor influencing the chance of remission of seizures, outweighing the contribution from other factors associated with prognosis including etiology of epilepsy, seizure type or the results of EEG or imaging investigations.’’ Consistent with this, our multivariate logistic regression analysis showed that a higher number of seizures pretreatment (>5 vs. 1–2) was predictive of recurrent seizures posttreatment, but even when this was accounted for there was no significant effect of the ABCB1 3435C>T genotype (Table 5). One of the potential limitations of our study is the short (1 year) follow-up period; however, recurrence of seizures at 1 year has been shown to be highly predictive of longterm medical refractoriness (Hauser et al., 1998; Kalita et al., 2005), and this is an important clinical endpoint given that it is the length of seizure freedom that is commonly required for the patient to be allowed to resume driving. Another potential limitation is that AED levels were not collected in a sufficiently consistent manner, within or between cohorts, to be utilized in this study. In addition, although an important purpose of the study was to compare possible confounders to detecting pharmacogenetic predictors of seizure outcome across international cohorts, the size of the patient populations, individually and collectively, was almost certainly too small (particularly in the case of the Hong Kong cohort) to have sufficient power to detect anything other than a large effect of the polymorphism on seizure recurrence. Furthermore, larger trials could permit subgroup analysis by drug and syndrome with greater power than was possible in this study. In addition to the interest in AED response, this ABCB1 polymorphism has been implicated as contributing to drug resistance in cancer (Gottesman et al., 2002), rheumatoid arthritis (Llorente et al., 2000), and inflammatory bowel disease (Farrell et al., 2000). However, it remains controversial whether and which AEDs are significantly transported by PgP. Recent work utilizing highly sensitive in vitro whole-cell assays reported that carbamazepine, valproate, phenytoin, lamotrigine, and primidone do not interact with ABCB1 (Rivers et al., 2008). These results are consistent with recent findings from another group that has also specifically examined this with in vitro assays (Baltes et al., 2007a, 2007b). However, when the same group used the concentration equilibrium transport assay system, which evaluates active transport independently of the passive permeability component, several AEDs, but
1695 Outcome of Newly Treated Epilepsy not carbamazepine, were reported to be substrates (LunaTorts et al., 2008). The experience in epilepsy highlights that a clear biologic hypothesis and association of outcomes is not sufficient proof of causation (Page et al., 2003). The importance of stringent methodologies for future studies, incorporating collection of information on significant confounders is essential to provide meaningful results. The results of this and previous studies examining potential pharmacogenetic predictors of AED response support the complex nature of pharmacoresistance in epilepsy. Rather than being dependent on one gene polymorphism, pharmacoresistance in epilepsy is more likely to be determined by a combination of multiple polymorphisms and environmental factors, which collectively determine the outcome in any given individual. In addition, it is likely that different combinations of these polymorphisms will account for the differing degrees of pharmacoresistance observed in practice. Future studies should include the analysis of multiple markers, and the interdependencies between markers, to elucidate the impact of an individual’s genetic variability on the development of pharmacoresistance (Petrovski et al., 2009). To have sufficient power, this kind of study is likely to require the enrolment of large numbers of patients, requiring collaboration and data collection from multiple sites and countries. The present study begins this process and also illustrates the analytical complexity involved in such studies, particularly in terms of phenotypic definitions, ethnic variations in frequency of polymorphisms and patterns in linkage disequilibrium, and variations in prescribing practice. These issues must be addressed in future collaborative efforts.
Acknowledgments The study was supported in the past by a Pfizer Neuroscience Grant (C.S.) and a grant from the Victor Hurley Foundation of Melbourne Health (C.S.). A Bio21 Fellowship (C.S.), University of Melbourne fostered the international collaboration. The work was also partly supported by a grant from the Research Grants Council of the Hong Kong Special Administrative Region, China (Project no. CUHK4466/06M) (P.K., L.B., T.J.O.). BioGrid Australia was responsible for database management. Dr. Nick Lawn enrolled the patients from the Royal Perth Hospital, Western Australia. We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines. Disclosure: C.S. has received a Pfizer Neuroscience Grant. T.J.O. has received financial support for his research from the NH&MRC (Australia), NARSAD (U.S.A.), DEST (Australia), The Royal Melbourne Hospital Neuroscience Foundation, The Victorian Neurotrauma Initiative, and research and educational grants from the Pharmaceutical Industry (Pfizer, UCB, Jansen-Cilag, GSK, Abbott, Sanofi-Aventis). In addition he is on a Medical Advisory Board for Jansen-Cilag (Australia) and Pfizer (Australia), work for which he has received Honoraria. L.J.S. has received financial support from the Murdoch Childrens Research Institute for this research. He is a director of a pharmacogenetic testing company, which does not do genetic testing for ABCB1. Other authors report no conflict of interest.
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