Genetics of drug resistance

Epilepsia, 46(Suppl. 10):33–38, 2005 Blackwell Publishing, Inc.

C International League Against Epilepsy

Genetics of Drug Resistance Sanjay M. Sisodiya Department of Clinical and Experimental Epilepsy, Institute of Neurology, UCL, Queen Square, London, United Kingdom

Summary: Drug resistance in epilepsy affects about a third of patients and is an important clinical problem, associated with increased morbidity and mortality. It is important to consider carefully the definition of drug resistance. Recent interest in the field has focused on the potential molecular mechanisms underlying drug resistance. Environmental and seizure-related acquired causes are likely to contribute to the multifaceted basis of resistance in most cases. Genetic causes have attracted particular attention, partly because they may allow prediction of drug resistance and, potentially, rational treatment strategies.

Gene mutations, however, are unlikely to cause many cases of drug resistance. However, common variation in genes probably

will turn out to generate an important contribution to drug resistance phenomena. Associations between common variations in a number of genes and clinical drug resistance have now been published. However, to date, none of these associations has been unequivocally replicated by others to the extent that the original association has been accepted. Some of these associations are considered. Despite this apparently uninspiring record, the genetics of drug resistance are likely to prove productive in the near future, but their pursuit will require painstaking studies and multicenter collaboration. Key Words: Epilepsy—Drug resistance—Antiepileptic drugs—Genetics—Association studies.

The recent focus in epilepsy on resistance to treatment with antiepileptic drugs (AEDs) has demonstrated the pathophysiological complexity underlying this common and clinically important phenomenon. A number of articles have considered in some detail postulated relevant underlying mechanisms (1,2). This article focuses on the genetics of drug resistance, and considers some of the important principles: it is not intended to be a comprehensive survey of the literature.

previously unappreciated underlying pathophysiological mechanisms. If a patient with temporal lobe epilepsy due to hippocampal sclerosis fails to respond to carbamazepine, phenytoin, lamotrigine, and benzodiazepines but becomes seizure free on levetiracetam or pregabalin, is that patient drug resistant or not? Would this patient be or have been considered drug resistant in 1995, 2005, or 2015? There may currently be individuals whom we define as being drug resistant, whose epilepsy is drug resistant simply because we do not yet have drugs that are appropriate for the treatment of that individual’s epilepsy. For example, there may be individuals whose epilepsy is caused by mutation or variation in neuropeptide Y receptor genes, and as no current AEDs are thought to target these receptors, these patients would currently very likely be drug resistant. An operative and adaptive definition of drug resistance might be epilepsy in which seizures continue to occur in an individual despite the use of two, three, or more antiepileptic drugs appropriate for that individual’s epilepsy: this definition, it should be noted, may lead to different or changing phenotypic classification at different time points in the history of an individual’s epilepsy as newer, possibly more appropriate, drugs are tried. In addition, drug resistance in epilepsy can only be evaluated in treated patients. The untreated natural history of epilepsy cannot directly tell us about drug resistance.

Drug resistance in epilepsy is a major clinical problem. Pragmatic studies suggest that with the currently available AED armamentarium, about 30% of patients, in

DEFINITION OF DRUG RESISTANCE In clinical practice, most patients with drug resistance are easily recognized, but the definition of drug resistance in scientific studies must always be explicitly made: in any genetic study, a precise and consistent— but not necessarily a narrow—definition of the phenotype is paramount. The definition of drug resistance is not straightforward. Additionally, it will remain important to continually reevaluate existing definitions of “drug resistance” as advances take place both in our understanding of the pathobiology of epilepsy and in the availability of newer AEDs. Patients who were considered drug resistant under a given definition may not remain so as newer antiepileptic drugs are developed, or designed, to target Address correspondence and reprint requests to S.M. Sisodiya at Department of Clinical and Experimental Epilepsy, Institute of Neurology, UCL, Queen Square, London WC1N 3BG, U.K. E-mail: sisodiya@ion.ucl.ac.uk

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specialized epilepsy practices, have epilepsy that is drug resistant (3). Globally this represents a large number of patients with epilepsy, and these patients suffer disproportionately the burden of the mortality and morbidity associated with epilepsy. The point is made in most studies of drug-resistant epilepsy that it is always important to exclude other causes of apparent drug resistance, such as misdiagnosis, the use of inappropriate AEDs for a given syndrome, or dissociative seizures.

CAUSES OF DRUG RESISTANCE When true drug resistance is seen, causes may broadly be categorized into environmental or genetic. Environmental causes will not be considered further here, but might include alteration in drug receptors or targets, possibly associated with epileptogenesis or the consequences of seizures (4): it is, of course, possible that variable response to environmental insults—including seizures— may to some extent also be genetically determined. Genetic causes may be monogenic, oligogenic, or polygenic.

It is notable that even in most well-documented kindreds with Mendelian monogenic epilepsy reported in the literature, resistance to AED treatment is rarely reported to segregate faithfully with the epilepsy phenotype. Thus, as with etiology for most common epilepsies, in most cases drug resistance is also likely to be the eventual result of a combination of environmental and multiple, subtle, genetic factors: drug resistance thus counts as a “complex” trait.

Genetic variation that influences drug resistance in epilepsy may itself be rare, i.e., mutation: mutation is a form of genetic variation occurring in less than 1%, and often a much smaller proportion, of the population. On the other hand, genetic variation responsible for drug resistance may be more common: the most common type of genetic variation reportedly associated with drug-resistant epilepsy is single nucleotide polymorphism (SNP), variation at the level of a single nucleotide occurring in a larger percentage of the entire population, e.g., more than 5% or more than 8% of the population. SNPs are in fact probably the most common consequential form of genetic variation in the human genome, there being about 10 million SNPs in total across the human genome’s 3 billion base pairs.

GENE MUTATION AS A CAUSE OF DRUG RESISTANCE There are few examples in which a gene mutation appears to underlie drug resistance in epilepsy. The rare and severe epilepsy syndrome known as severe myoclonic epilepsy of infancy (SMEI) is now known in many cases to be caused by mutation in one of the genes encoding a subunit of the cerebrally-expressed sodium channel (SCN1A), though not all cases of SMEI are due to SCN1A mutation.

A large number of such mutations are now known, and Epilepsia, Vol. 46, Suppl. 10, 2005

all are de novo or seen at very low frequencies (5,6). It might be thought on this basis that perhaps common genetic variation, for example polymorphism, in the SCN1A gene might contribute at least partly to drug resistance in epilepsy in general. However, a detailed population genetic study in a large cohort of patients with drug-resistant epilepsy has shown that common variation in this gene, in fact, does not seem to associate with the broad phenotype of drug resistance across a wide range of epilepsy types and syndromes (C. Depondt, personal communication).

Another example of a mutation causing resistance to currently available antiepileptic drugs is refractory epilepsy associated with mutation in the SCN2A gene. A single example of this has been reported (7), the mutation causing epilepsy that is resistant to treatment with antiepileptic drugs. Although it may transpire that there are a large number of individual mutations in different genes that might cause individual cases of drug-resistant epilepsy, because by definition mutations are rare, and in practice often very rare, it is unlikely that gene mutation will emerge as a common genetic explanation for drug resistance overall.

COMMON GENETIC VARIATION AS A CONTRIBUTOR TO DRUG RESISTANCE In epilepsy, it is most likely that drug resistance, which is a common phenomenon, will be due to common genetic variation, when it has a genetic or partly genetic basis.

It is important when initially considering the entire set of genetic variations in any way associated with any epilepsy phenotype to draw a mental distinction between common genetic variation which contributes to the genetics of drug resistance, and common genetic variation which contributes to the pharmacogenetics of epilepsy.

While these two sets of variations will to some extent overlap, there will undoubtedly remain a set of variations that influences the way a drug is handled and specifically does not influence true resistance to antiepileptic drugs.

For example, variations in genes encoding cytochrome P-450 drug metabolizing enzymes undoubtedly influence the pharmacokinetics of a number of antiepileptic drugs such as phenytoin (8); however, these genetic variants have effects on serum drug levels, which can in general be overcome, to achieve control of seizures, by changes in drug dosing. It is thus unlikely that such variation contributes commonly to drug resistance. On the other hand, variation in genes encoding neuronal glutamate receptors may contribute to drug resistance, but is unlikely to contribute to pharmacokinetics.

The contribution of common genetic variation to any given trait of interest is not in general a Mendelian characteristic: the common variants rarely have a major effect individually. Currently, case-control genetic association studies dominate the field in human genetics in general,

GENETICS OF DRUG RESISTANCE partly because of discoveries regarding the architecture of the human genome and the development of appropriate investigational strategies and tools. Case-control association studies examine the difference in distribution of alleles and genotypes at chosen polymorphic loci in cohorts of moreor-less defined disease phenotypes compared with other phenotypes, ideally differing only at the trait of interest.

Such genetic association studies are frequently reported in the literature for all manner of human diseases: it is likely that common genetic variation does contribute to susceptibility to common diseases (9). However, across the spectrum of human diseases, only a handful of such studies have actually been reliably replicated (10). The repetition of a reported experimental result (in this case, an association) forms a fundamental part of the process of acceptance of that result as part of the body of human knowledge. Numerous excellent articles have considered the reasons for the failure of most replication studies in human genetics (11).

The selection of cohorts for study, the power of cohorts to identify a reported association (itself partly related to the magnitude of the effect of the variation under consideration), adequate coverage of all common genetic variation within the studied gene, the lack of knowledge of the actual causal variation driving the association, occult genetic stratification between cases and controls, and poor statistical methodology are among the reasons for replication failure. However, the most common reason for failure is probably that the initially reported positive result is, in fact, a false positive. The prevalence of such complicating factors in human association studies is apparent from the literature on the genetics of drug resistance in epilepsy.

The problems besetting replication in epilepsy genetic association studies have recently been thoroughly reviewed in an excellent paper from Berkovic and colleagues (12).

The points raised in that paper undoubtedly apply to the studies considered in the rest of this report, as considered explicitly in Tan et al.’s review (12).

REPORTED COMMON GENETIC CAUSES OF DRUG RESISTANCE Notwithstanding the above caveats, a number of genetic association studies have been published addressing drug resistance in epilepsy: some of these will now be considered.

Interleukin IL-1β gene Interleukin IL-1β is a proinflammatory cytokine, receptors for which have been found in the hippocampus. In 2000, an association was reported in a Japanese study between a single nucleotide polymorphism in the promoter of the interleukin IL-1 β gene and the phenotype of temporal lobe epilepsy with hippocampal sclerosis, compared to those with temporal lobe epilepsy without hippocampal sclerosis or nontemporal lobe epilepsy (13). A num-

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ber of studies have since attempted to replicate this initial finding, largely without success except in one cohort of patients with partial epilepsy (14–18). Although there are biological grounds to consider the relevance of this particular gene in refractory epilepsy, currently a major effect for this particular polymorphism is difficult to sustain.

Prodynorphin gene Prodynorphin is a seizure-suppressing peptide of considerable potency. Functional polymorphism is known to exist in the gene promoter, in this case taking the form of the length of a variable tandem repeat. In a study of European patients, a significant association was noted between this variant in the prodynorphin gene and the phenotype of “familial risk” temporal lobe epilepsy, compared with “nonfamilial” temporal lobe epilepsy (19). Patients in both of these studied groups were presurgical candidates, and therefore presumably had drug-resistant epilepsy. Three replication studies have not supported the original finding, but power considerations mean that a role for this variant of this gene cannot conclusively be excluded at this time (18,20,21).

GABA(B) receptor 1 gene GABA receptors have been widely implicated in a range of processes in seizures and epileptogenesis. A putatively functional, amino acid-changing single nucleotide polymorphism (G1465A) in the GABA(B) receptor 1 gene GABBR1 has been studied in a cohort of European patients. An association was noted with variation at this SNP in patients with “severe” temporal lobe epilepsy versus a cohort of patients with “mild” (i.e., nondrug resistant) temporal lobe epilepsy (22). Again, in a single replication study, this association has not been replicated (18).

Prion protein gene Mice lacking the prion protein gene are reportedly more sensitive to chemoconvulsants (23). The group originally reporting this finding went on to pursue a study of variation in the human prion protein gene PRNP and association with epilepsy (24). These workers adopted a candidate gene approach, and sought to establish common variation in this gene, and to study its association with drug resistance. In a cohort of 100 patients with drug-resistant surgically-operated temporal lobe epilepsy, in comparison to a control subjects, a strong association was noted between a particular polymorphism in the human PRNP gene and the phenotype of drug resistance in temporal lobe epilepsy, and separately for the same SNP with the phenotype of poor outcome following temporal lobe surgery.

However, this study has also failed to be replicated (18).

It is also notable that the reported single nucleotide polymorphism was not found in 180 control subjects, from the authors’ own paper, nor in other cohorts of normal individuals (25); population heterogeneity (ethnic admixture) was also a problem in this study.

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Apolipoprotein E gene Common variation in the apolipoprotein E gene has been associated with a range of human disease phenotypes, including for example age of onset of Alzheimer’s disease. A study in Australian patients reported an association between variation in this gene and the length of the latent interval in patients with typical, drug-resistant, temporal lobe epilepsy (26). This intriguing finding, which might have opened up the possibility of rational intervention during the latent period to prevent the development of refractory temporal lobe epilepsy, has also not been replicated (18), nor previously seen (27,28).

ABCB1, the P-glycoprotein-encoding gene Common variation in the gene ABCB1, encoding the broad-spectrum nonspecific drug transport protein, Pglycoprotein, has been associated with a wide range of phenotypes across the spectrum of human diseases. The most commonly studied variant in this gene is the polymorphism C3435T. In a study of patients with drugresistant epilepsy compared to those with drug-sensitive epilepsy, there was an association with this polymorphism (29). However a large, well-powered, exact replication study failed to replicate this association (30); a partial replication was achieved in a separate much smaller study (31).

ANY CAUSE FOR OPTIMISM? This brief survey of published associations between common genetic variation and the phenotype of drug resistance would appear to show that there are no currently accepted common genetic factors associated with drug resistance. It is worth considering this a little further to set these results in context, by considering the position of genetics, in particular population genetics, with respect to the biology of a disease overall. The P-glycoprotein association is illustrative (32). It is now well accepted that there is an excess of P-glycoprotein seen focally in human epileptogenic brain tissue, being found to be expressed, by immunohistochemistry, by reactive glia within epileptogenic regions and not by glia in adjacent, nonepileptogenic, regions. Some workers have also shown, again using immunohistochemistry in brain tissue, expression of P-glycoprotein by neurons within epileptogenic regions. Normal glia and normal neurons do not express P-glycoprotein that is detectable by immunohistochemistry. Other drug transporters, some of which in common with P-glycoprotein appear to be able to transport at least some AEDs at least under some circumstances, are also overexpressed in epileptogenic brain tissue. There is now a considerable body of evidence from in vitro studies and animal models to suggest that P-glycoprotein, and some related transporters, are indeed able to transport some AEDs (33). The current model for the role of these proteins in mediating drug resistance in epilepsy indepenEpilepsia, Vol. 46, Suppl. 10, 2005

dent of genetic factors is as follows: in epileptogenic regions of the brain, P-glycoprotein and other transporters are found not only in their normal situation, for example in the capillary endothelium, but also in reactive glia, and possibly also in neurons within epileptogenic regions; in concert with the demonstrated ability of some of these transporter proteins to export antiepileptic drugs, the net effect is postulated to be a reduction in the active local concentration of AEDs, particularly at their site of action at the surface of, or within, neurons. It is important to note that this overexpression is thought to occur focally in epileptogenic brain regions and not in adjacent or distant brain regions, which are not primarily involved in generating the epilepsy. The findings upon which this model is based have been corroborated by a number of studies, and are independent of the disputed genetic findings. The initial genetic findings suggested that in addition to local overexpression, genetic factors might also influence the expression or activity of the upregulated protein, thus further contributing to the phenomenon of drug resistance.

The absence of proof by replication for the initial association does not exclude a role for P-glycoprotein in mediating or contributing to drug resistance, but instead mandates comprehensive multimodal assessment of its potential role, including further functional studies in vivo, ex vivo, or in vitro, or in terms of assessment of the effect of ABCB1 polymorphisms on mRNA and protein quantity.

In addition, the polymorphism that was initially studied may not itself be the causal variant, and strategies to identify other possible candidates for the causal variant are warranted: one such has been performed (34).

This example illustrates the idea that thorough evaluation of the role of an agent postulated to have an effect in drug resistance is warranted if there is good biological evidence to consider that that agent does have a role, and that this need for evaluation is independent of genetic findings, and indeed is an essential follow-up to positive genetic associations. Additionally, the stringency for the evaluation and acceptance of genetic findings can legitimately be less when there is good biological motivation for the study of a given agent (11).

CONCLUSIONS: THE PRESENT AND THE FUTURE The current state of play is that there are known a few gene mutations that cause Mendelian epilepsy, which is also drug resistant. There are, however, no common genetic variations that have been robustly proven to contribute to drug resistance in non-Mendelian epilepsies, nor any markers for such drug resistance. However, a role for the genes already studied, or the proteins encoded, cannot yet be completely excluded for the majority of the genes studied. Nevertheless, it remains the case that

GENETICS OF DRUG RESISTANCE currently there are no genetic predictors for drug resistance in epilepsy.

Any doubt about the value of further research in this area should be dispelled, however, by an example from cancer disease genetics. While analogy between cancer and epilepsy resistance genetics cannot be carried too far, there may be some parallels. Nonsmall cell lung carcinoma is a particularly difficult disease to treat, most patients being resistant to currently available anticancer drugs. The epidermal growth factor receptor plays an important role in the biology of nonsmall cell lung cancer. In the search for new therapeutic agents, gefitinib was synthesized: this is a tyrosine kinase inhibitor that targets the epidermal growth factor receptor (encoded by EGFR). In initial studies, gefitinib did not appear to have any useful effect in the majority of patients. It was, however, noted that a minority of patients responded extremely well. In a recently published study, it has been shown that eight out of nine responders to gefitinib had somatic mutations (i.e., in their cancer cells) in EGFR, while none of seven nonresponders had such mutations (35). This finding may radically alter the perception and future role of gefitinib: it should be possible to predict which patients will respond to the drug, and also those who will not respond, and might be spared any adverse effects (36). The importance of this finding for disease genetics more broadly, and for the implications for health care provision, has not gone unnoticed.

In the future, candidates for genes mediating or contributing to drug resistance in epilepsy may emerge from pharmacogenetics (in particular, from the study of genes whose products influence drug kinetics, drug transport, and drug targets; (37)), from evolving understanding of disease biology, and from animal models. In addition, genome-wide screens for the identification of candidate genes and polymorphisms are now becoming a reality, as for example shown in the search for genes underlying hepatic adverse effects in patients exposed to tranilast, an antirestenosis drug no longer under development (38).

Careful methodology in association studies is undoubtedly essential if such studies are to be robust and replicable. A practical framework for such robust studies in epilepsy is now in place (12), and continues to develop in general (39).

Collaboration between centers of expertise with access to large, well-phenotyped cohorts of patients with epilepsy and control subjects will also be essential, especially if specific phenotypes, such as drug resistance, are to be examined. Despite the difficulties and complexities, the study of the genetics of drug resistance promises not only to be exciting, but also to illuminate the pathophysiology of epilepsy and potentially to provide new treatment targets and treatment strategies for patients with drugresistant epilepsy.

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