Epilepsia, 45(11):1429–1442, 2004 Blackwell Publishing, Inc.  C 2004 International League Against Epilepsy

Invited Review

Genetic Association Studies in Epilepsy: “The Truth Is Out There” ∗ Nigel C. K. Tan, †John C. Mulley, and ∗ Samuel F. Berkovic ∗ Epilepsy Research Centre and Department of Medicine (Neurology), University of Melbourne, and †Department of Genetic Medicine, Women’s and Children’s Hospital and Department of Molecular Biosciences, University of Adelaide, Australia

Summary: Success has been achieved in identifying many mutations in rare monogenic epilepsy syndromes by using linkage analysis, but dissecting the genetic basis of common epilepsy syndromes has proven more difficult. Common epilepsies are genetically complex disorders believed to be influenced by variation in several susceptibility genes. Association studies can theoretically identify these genes, but despite more than 50 association studies in epilepsy, no consistent or convincing susceptibility genes have emerged, leading to scepticism about the association-study approach. We review the results of existing association studies in focal epilepsies, generalized epilepsies, febrile seizures, and epilepsy pharmacogenetics. By using an illustrative example, we discuss how methodologic issues of

sample size, selection of appropriate controls, population stratification, and significance thresholds can lead to bias and falsepositive associations; the importance of biologic plausibility also is emphasized. Newer methodologic refinements for association studies, such as use of two control groups, genomic control, haplotyping, and use of two independent datasets, are discussed. A summary of existing guidelines and a checklist for planning and appraising such association studies in epilepsy is presented. We remain cautiously optimistic that with methodologic refinements and multicenter collaborations with large sample sizes, association studies will ultimately be useful in dissecting the genetic basis of common epilepsy syndromes. Key Words: Complex disease—Spurious association—Checklist—Multicenter study.

Epilepsy is heterogeneous, incorporating numerous epilepsy syndromes with different etiologies. When a genetic basis exists for epilepsy, defining the genetic contribution has proven to be a formidable task (1). Success has been achieved in some families with rare monogenic epilepsy syndromes (2), with mutations of large effect where concordance between genotype and phenotype is reasonably strong. In contrast, progress has been slow for most of the common epilepsy syndromes encountered in daily clinical practice. It is posited that most epilepsies, much like other common diseases such as diabetes or asthma, are influenced by the effect of variation at several or multiple genes. Where this variation is in the form of common single nucleotide polymorphisms (SNPs), the model is termed the commondisease/common-variant hypothesis (3); some evidence exists to support this hypothesis (4). However, where the underlying genetic basis is a rare variation (present at a frequency of less than 1%), then the model is termed the common-disease/rare-variant hypothesis (5). Irrespective

of which model, or likely mixture of both models, each gene contributes a small or modest effect to the epilepsy phenotype, and by itself is insufficient to cause epilepsy. These are “susceptibility genes.” In addition, environmental factors may play a part (6). Concordance between genotype and phenotype is therefore relatively weak compared with monogenic disorders. Common epilepsy syndromes are thus polygenic or complex disorders, with the latter term preferred, as it also includes environmental influences. Approaches for genetic dissection of complex disorders have been reviewed in detail (6,7), and a summary of the two commonly used methods follows. LINKAGE ANALYSIS Linkage analysis has been the traditional tool used in monogenic diseases to narrow the number of candidate genes, one of which will have the underlying molecular defect (7). Classic linkage analysis usually requires large families with many affected members (multiplex families), or multiple families in which the same mutant gene is responsible. This procedure is efficient for monogenic disorders, in which the pathogenic effect is strong enough to define the affected status on clinical grounds, without too much confusion from incomplete penetrance (mutation

Accepted July 4, 2004. Address corresponding Author: Professor Samuel F Berkovic, Epilepsy Research Centre, University of Melbourne, 1st Floor Neurosciences Building, Austin Health, Banksia Street, Heidelberg West, VIC 3081, Australia. E-mail: [email protected]

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carriers who are not clinically affected), or phenocopies [such as relatives with epilepsy but different biologic disease mechanisms, which may or may not be genetic (e.g., poststroke seizures)]. Complex disorders such as the common epilepsy syndromes seen in daily clinical practice are theoretically amenable to linkage analyses, particularly nonparametric approaches (8); however, these have less power compared with linkage analyses carried out for monogenic disorders, and success with these approaches has been limited. The pathogenic effect of each putative “susceptibility gene” is small or modest, making detection difficult. To complicate matters, phenocopies and nonmendelian modes of genetic inheritance (8) further muddy the waters. Therefore those who are clinically affected may not all have the same susceptibility genes, whereas a particular susceptibility gene may be found in both affected and unaffected persons, frustrating our attempts at dissecting common epilepsies. Use of linkage methods for common epilepsy syndromes also is limited by availability of multiplex families. Although such families are not uncommon in the idiopathic generalized epilepsies or febrile seizures, they are less common in focal epilepsies. Two genome-wide linkage studies have thus been performed in idiopathic generalized epilepsies (9,10), resulting in several possible susceptibility loci; a novel epilepsy gene has been identified after investigation of one such locus (11). Overall success, although encouraging, has been limited, partly because of the practical difficulties and also because of relatively small sample sizes used so far. Attention has thus turned to association studies for dissecting complex diseases.

and consequent bias (12,13). Proof of an association ultimately lies in replication of the initial findings in several different populations and demonstrating that the associated allele plausibly alters biologic function, leading to disease (12,14).

NOT QUITE READY FOR PRIME TIME? Association studies may provide greater statistical power than linkage analysis in dissection of complex diseases under certain circumstances (15,16). Case collection also is easier. However, association studies have performed poorly thus far. A cross-disciplinary review showed that of more than 600 associations, only 166 were studied 3 times or more, and of these, only six were consistently replicated (17). Indeed, replication failure was the norm, leading one journal openly to discourage submission of association studies in complex disorders unless major biologic insights are found (18). This has led to scepticism about each new published association, and journals have therefore published guidelines for genetic association studies in an attempt to stem the tide of nonreplicable studies (19–22). However, methodology in association studies is evolving, and nonreplicable studies from even as recent as 5 to 6 years ago may reflect the lower stringency of the available methods of that time. New methods for correction of population stratification (13), increasing use of family-based controls (as opposed to population-based controls), use of two independent datasets of cases and controls within one study, and increasing rigor in defining statistical significance have gradually become more common in published studies (14).

ASSOCIATION STUDIES Association studies compare the frequency of specific alleles in affected cases against those in unaffected controls. Instead of tracking the co-inheritance or otherwise of marker and disease alleles within families, association studies generally involve populations (and usually larger numbers of individuals). An allele is said to be associated with the disease if its frequency differs between cases and controls more than would be predicted by chance (8), provided the controls are representative of the test population in all aspects other than disease affection status. Guilt by association, however, is not sufficient proof of causation (12). Linkage disequilibrium (LD) occurs when alleles are found together more than would be predicted by chance; an associated allele may therefore not be causative but instead in LD with the actual pathogenic allele. Alternatively, the association may have arisen by chance, or it may be artifactual because of methodologic weaknesses

Epilepsia, Vol. 45, No. 11, 2004

EPILEPSY: THE SCORE SO FAR The situation in neurology and epilepsy is not dissimilar, with many reports of a novel association cast into doubt by subsequent replication failure. Multiple conflicting and nonreplicable association studies have been done in the past 7 years for both focal and generalized epilepsy syndromes. Studies on febrile seizures and the pharmacogenetics of medically refractory epilepsy also have been performed. We summarize the results of the existing studies, with a brief analysis of their strengths and weaknesses, and endeavor to identify common limitations underlying these studies. Association studies of interleukin genes and temporal lobe epilepsy (TLE) provide a good overview of the problems of association studies and will be used as an example. Analysis of the other association studies is detailed in Tables 1 to 3. Finally, we also attempt to synthesize the existing sets of guidelines for association studies

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TABLE 1. Summary of association studies in focal epilepsy Gene and epilepsy syndrome

No. of cases

Findings

Notes

Overall interpretation

Interleukin 1β and temporal lobe epilepsy (TLE) See example Prodynorphin gene and TLE Stogmann 155 (43 with L-allele associated with increased et al. (39) family risk of TLE in subgroup with history of family history of seizures seizures)

Unclear if subgroup analysis was 1. If positive results were derived through prespecified before analysis or analysis of multiple subgroups defined not Corrected p value = 0.002 after primary analysis (as opposed to preplanned analyses), this may lead to false positives (22,42) a Tilgen 182 (46 with L-allele not associated with Both negative replication studies 2. Negative replication studies had low number of TLE with family history of et al. (40) family increased risk of TLE as a whole have only slightly more cases seizures; all three prodynorphin study history of or in subgroup with family with family history than initial findings pertain to small subgroups of seizures) history of seizures in both study. Initial association