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Epilepsia, 47(10):1682–1690, 2006 Blackwell Publishing, Inc. C 2006 International League Against Epilepsy

Exploration of the Genetic Architecture of Idiopathic Generalized Epilepsies ∗ †Anne Hempelmann, ∗ †Kirsten P. Taylor, ‡Armin Heils, ∗ †Susanne Lorenz,

§Jean-Francois Prud’Homme, Rima Nabbout, Olivier Dulac, ¶Gabrielle Rudolf, ∗∗ Federico Zara, ††Amedeo Bianchi, ‡‡Robert Robinson, ‡‡R. Mark Gardiner, §§Athanasios Covanis, Dick Lindhout, ¶¶Ulrich Stephani, ‡Christian E. Elger, ∗∗∗ Yvonne G. Weber, ∗∗∗ Holger Lerche, †††Peter Nürnberg, ‡‡‡Katherine L. Kron, ‡‡‡Ingrid E. Scheffer, §§§John C. Mulley, ‡‡‡Samuel F. Berkovic, and ∗ †Thomas Sander ∗ Gene Mapping Center, Max-Delbrück Center, and †Department of Neurology, Charité University Medicine, Berlin, and ‡Clinic of Epileptology and Institute of Human Genetics, Rheinische Friedrich-Wilhelms-University, Bonn, Germany; §Généthon, Evry, Département de Neurologie Pédiatrique et de Maladies Métaboliques, Hopital Necker-Enfants Malades, APHP, Paris, and ¶ Clinique Neurologique, Hôpitaux Universitaires de Strasbourg, Strasbourg, France; ∗∗ Laboratory of Neurogenetics, U.O. Neuromuscular and Neurodegenerative Disease, Istituto Gaslini, University of Genova, Genova, and ††Epilepsy Centre, Department of Neurology, San Donato Hospital, Arezzo, Italy; ‡‡Department of Pediatrics and Child Health, The Rayne Institute, Royal Free and University College Medical School, London, United Kingdom; §§Department of Neurology, The Children’s Hospital “Agia Spohia,” Athens, Greece; Complex Genetics Section, Department of Medical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands; ¶ ¶ Clinic for Neuropaediatrics, University Clinics Schleswig Holstein, Kiel; ∗∗∗ Departments of Neurology and Applied Physiology, University of Ulm, Ulm, and †††Cologne Center for Genomics and Institute for Genetics, University of Cologne, Cologne, Germany; and ‡‡‡Epilepsy Research Centre and Department of Medicine (Neurology), University of Melbourne and Austin Health and Repatriation Medical Centre, Heidelberg, Victoria, and §§§Department of Genetic Medicine, Women’s and Children’s Hospital and School of Molecular and Biomedical Sciences, University of Adelaide, South Australia, Australia

Summary: Purpose: Idiopathic generalized epilepsy (IGE) accounts for ∼20% of all epilepsies and affects about 0.2% of the general population. The etiology of IGE is genetically determined, but the complex pattern of inheritance suggests an involvement of a large number of susceptibility genes. The objective of the present study was to explore the genetic architecture of common IGE syndromes and to dissect out susceptibility loci predisposing to absence or myoclonic seizures. Methods: Genome-wide linkage scans were performed in 126 IGE-multiplex families of European origin ascertained through a proband with idiopathic absence epilepsy or juvenile myoclonic epilepsy. Each family had at least two siblings affected by IGE. To search for seizure type–related susceptibility loci, linkage analyses were carried out in family subgroups segregating either

typical absence seizures or myoclonic and generalized tonic– clonic seizures on awakening. Results: Nonparametric linkage scans revealed evidence for complex and heterogeneous genetic architectures involving linkage signals at 5q34, 6p12, 11q13, 13q22-q31, and 19q13. The signal patterns differed in their composition, depending on the predominant seizure type in the families. Conclusions: Our results are consistent with heterogeneous configurations of susceptibility loci associated with different IGE subtypes. Genetic determinants on 11q13 and 13q22q31 seem to predispose preferentially to absence seizures, whereas loci on 5q34, 6p12, and 19q13 confer susceptibility to myoclonic and generalized tonic–clonic seizures on awakening. Key Words: Idiopathic generalized epilepsy—Complex inheritance—Absence seizure—Myoclonic seizure—Linkage.

Idiopathic generalized epilepsy (IGE) accounts for ≤20% of all epilepsies and affects ∼0.2% of the gen-

eral population (Jallon and Latour, 2005). The IGE syndromes are characterized by the age-related occurrence of recurrent unprovoked generalized seizures in the absence of detectable brain lesions or metabolic abnormalities (ILAE, 1989). The most common subtypes are childhood absence epilepsy (CAE), juvenile absence epilepsy (JAE), juvenile myoclonic epilepsy (JME), and epilepsy with generalized tonic–clonic seizures (EGTCS) (Janz,

Accepted June 20, 2006. Address correspondence and reprint requests to Dr. T. Sander at Gene Mapping Center, Max-Delbrück-Center, Robert-Rössle-Str. 10, 13125 Berlin, Germany. E-mail: [email protected] The first two authors contributed equally to the present work. doi: 10.1111/j.1528-1167.2006.00677.x

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GENETIC ARCHITECTURE OF IDIOPATHIC GENERALIZED EPILEPSY 1997; Nordli, 2005). The key seizure types of these IGE syndromes are absence seizures (CAE, JAE), myoclonic seizures on awakening (JME), and generalized tonic–clonic seizures (EGTCS). The electroencephalographic signature of IGE seizures is marked by generalized spike–wave discharges (GSW-EEG), which reflect a synchronized hyperexcitability state of thalamocortical circuits (Blumenfeld, 2005). The etiology of IGE is genetically determined (Greenberg et al., 1992; Ottman, 2005), but the underlying genetic architecture of these common IGE syndromes is virtually unknown. The recurrence risk of IGE for first-degree relatives (5–8%) is 10- to 15-fold greater than the lifetime risk for IGE in the general population. Twin and family studies indicate an overlapping genetic component that is shared across the common IGE syndromes, but also provide evidence that different genetic configurations determine the phenotypic expression of certain seizure types, such as absence and myoclonic seizures (Berkovic et al., 1987; Beck-Mannagetta and Janz, 1991; Janz et al., 1992; Wirrell et al., 1996; Berkovic et al., 1998; Durner et al., 2001; Marini et al., 2004; Winawer et al., 2003, 2005). Molecular genetic approaches have identified causative gene mutations in rare monogenic forms of idiopathic epilepsies (ADLTE, ADNFLE, BFNC, GEFS+) (Gardiner, 2005; Mulley et al., 2005; Turnbull et al., 2005). Most of the currently known genes for human idiopathic epilepsies encode voltage-gated or ligand-gated ion channels (Mulley et al., 2005; Turnbull et al., 2005). These findings suggest that the monogenic epilepsies are primarily a family of channelopathies. No common gene variant has been identified that confers any substantial effect on the etiology of common IGE syndromes displaying complex inheritance. Current attempts to map susceptibility loci for genetically complex IGE syndromes have revealed several tentative susceptibility loci in the chromosomal regions 2q36 (Sander et al., 2000), 3q26 (Sander et al., 2000), 5p15 (Durner et al., 2001), 5q22 (Durner et al., 2001), 6p12 (Liu et al., 1996; Serratosa et al., 1996; Pinto et al., 2004), 6p23.1 (Greenberg et al., 1988, 1995, 2000; Durner et al., 1991; Sander et al., 1995, 1997), 8p12 (Durner et al., 1999), 8q24 (Zara et al., 1995; Fong et al., 1998), 10q25-q26 (Puranam et al., 2005), 14q23 (Sander et al., 2000), 15q14 (Elmslie et al., 1997), and 18q21 (Durner et al., 2001; Greenberg et al., 2005). For most implicated regions, replication studies have failed to establish unequivocal linkage relations, probably because of the confounding effects of phenotypic variability, complex inheritance, and genetic heterogeneity (Glazier et al., 2002; Ottman, 2005). To date, two genome scans of common IGE syndromes have been reported (Sander et al., 2000; Durner et al., 2001). Our previous genome scan of 130 IGE-multiplex families of European descent revealed linkage evidence for three IGE loci on chromo-

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somes 2q36, 3q26, and 14q23 (Sander et al., 2000). The second IGE genome scan included 91 European families ascertained through probands with adolescent-onset IGE, of which 38 families were multiplex or multigenerational (Durner et al., 2001). Parametric linkage analyses yielded significant evidence for a locus common to most IGEs on chromosome 18q21 and syndrome- and seizure type–related susceptibility loci on chromosome 6p21 for JME-related IGEs, 8p12 for non-JME forms of IGE, and two loci for absence seizures in the chromosomal regions 5p15 and 5q22. Despite the discordant and inconsistent linkage findings, positional candidate gene analyses have identified functional mutations with functional effects in two susceptibility genes (CLCN2 on 3q27, EFHC1 on 6p12) in a few families with common IGE syndromes (Haug et al., 2003; D’Agostino et al., 2004; Suzuki et al., 2004). In addition, linkage disequilibrium mapping at two linkage regions on 6p21.3 and 18q21.1 has suggested two potential susceptibility genes (BRD2, ME2) predisposing to common IGE subtypes (Pal et al., 2003; Greenberg et al., 2005). The current linkage findings for IGE in general, as well as for certain IGE subtypes, reflect the likely oligogenic architecture of common familial IGE syndromes. Accelerating progress in gene discoveries for mono- and oligogenic IGE subtypes emphasizes the great potential of molecular genetic approaches for dissecting the complex genetic basis of common IGE syndromes, but large multicenter collaborations are necessary to achieve sufficient statistical power to clarify the underlying phenotype–genotype relations. The present genome-wide linkage scan of 126 IGE-multiplex families of European origin was designed to explore the genetic architecture of common IGE syndromes and to dissect out seizure type–related susceptibility genes predisposing to absence or myoclonic seizures. METHODS Family ascertainment The study included 126 IGE-multiplex families of European descent, which were collected from one Australian and six European research groups. None of the present families was part of our previous genome scan (Sander et al., 2000). The families were derived from: Germany (n = 73), France (n = 12), Greece (n = 7), Italy (n = 5), United Kingdom (n = 4), The Netherlands (n = 1), Serbia/Montenegro (n = 1), Norway (n = 1), and Australia and New Zealand (n = 22, mixed European origin). The study protocol was approved by the institutional review boards of the participating centers. Written informed consent was obtained from all participants and parents/guardians in the case of minors. Inclusion criteria were (a) proband affected by either JME, JAE, or CAE; (b) one or more siblings with either unprovoked generalized seizures or GSW discharges on the routine EEG; Epilepsia, Vol. 47, No. 10, 2006

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and (c) both parents, if available, or sufficient siblings to reconstruct the missing parental genotypes. Exclusion criteria for family members were the presence of any abnormal neurologic finding, mental retardation, and morphologic or metabolic brain disorders, or stimulus-induced seizures only. Clinical evaluation The diagnoses of IGE syndromes were performed according to the current classification of the International League Against Epilepsy (ILAE, 1989). Idiopathic absence epilepsies (IAEs) begin with typical absence seizures, starting at the age of 4–20 years. JME has an adolescent onset between the ages of 8 and 20 years and is characterized clinically by bilateral synchronous irregular myoclonic jerks of the upper arms and shoulders without loss of consciousness, typically on awakening. Epilepsy with generalized tonic–clonic seizures on awakening (EGMA) usually starts at the age of 8–25 years, and at least two thirds of all seizures (n > 2) occur within the first hour after awakening or during phases of decreased vigilance. The entire sample of 126 IGE-multiplex families included 675 family members, of which 579 individual members were genotyped. In total, 97 (77%) were nuclear, and 29 (23%) were extended families. Epilepsy syndromes and epileptic seizures in the entire family sample and both subgroups are shown in Table 1. Detailed information regarding the pedigrees and syndromic classification of the affected members is available at http://www.unikiel.de/pediatrics/FrameNeuro.html. Family groups and trait definitions To search for susceptibility loci common to a broad spectrum of IGE syndromes, family members with either unprovoked generalized seizures or unprovoked GSWEEG were classified as “affected” (n = 356; IGE-trait definition) in the entire sample of 126 IGE-multiplex families. Among the affected individuals, 13 clinically unaffected family members exhibited unprovoked GSW discharges during their routine EEGs. In the context of at least one first-degree relative with IGE, the GSW-EEG was considered as subclinical expression of the IGE trait (Blumenfeld, 2005). The same IGE-trait definition was used in our previous genome scan (Sander et al., 2000) and therefore allows comparisons with the original linkage findings. To dissect out susceptibility genes for absence or myoclonic seizures, we narrowed the seizure type–related spectrum in two stages. First (stage I), we subdivided two distinct family subgroups by the presence or absence of JME, comprising 53 JME families and 73 IAE families without JME members (Table 1). Genome-wide linkage scans were then carried out under the IGE-trait definition. Further to maximize genetic homogeneity by narrowing familial phenotypic variance (stage II), we distinguished two subgroups of multiplex families, which contained Epilepsia, Vol. 47, No. 10, 2006

at least two siblings affected by either typical absence seizures without JME-related myoclonic seizures or JMErelated myoclonic seizures and generalized tonic–clonic seizures on awakening. Linkage analyses were performed in the family subgroups by using the corresponding seizure type–related trait definition. To search for susceptibility loci predisposing to typical absence seizures, we formed a subgroup of 52 IAE-multiplex families without JME members that included at least two siblings with either CAE or JAE. For linkage analysis, only family members with IAE were considered as “affected” (n = 118; IAEtrait definition). To dissect out genes conferring susceptibility to myoclonic and generalized tonic–clonic seizures on awakening, we defined a subgroup of 28 families of JME patients in which at least one other sibling was affected by either JME or EGMA. For linkage analysis, only family members with either myoclonic (JME) and generalized tonic–clonic seizures on awakening (EGMA) were classified as “affected” (n = 70; JME-trait definition), irrespective of the presence of absence seizures. Eight family members with IAE but without JME or EGMA were classified as “unknown.” The rationale for combining myoclonic and generalized tonic–clonic seizures on awakening reflects the remarkable syndromic overlap of JME and EGMA, with their adolescent onset and predominant occurrence on awakening (Janz, 1997; Andermann and Berkovic, 2001). Moreover, it has been shown that generalized tonic–clonic seizures on awakening are frequently preceded by series of myoclonic jerks (Delgado-Escueta et al., 1999). With regard to the frequent age-related co-occurrence of myoclonic seizures, absence seizures, and GTCS on awakening in IGE patients and the common familial clustering of JME, IAE, and EGMA (Berkovic et al., 1987; Beck-Mannagetta and Janz, 1991; Janz et al., 1992; Wirrell et al., 1996; Janz, 1997; Berkovic et al., 1998; Andermann and Berkovic, 2001; Marini et al., 2004; Winawer et al., 2003, 2005b57; Martinez-Juarez et al., 2006), we did not exclude JME/EGMA-multiplex families, in which relatives exhibited absence seizures. Nine of the 28 JME/EGMA-multiplex families included family members with absence seizures, and seven of 70 family members affected by JME or EGMA also exhibited absence seizures during the course of their epilepsy. Genome scan panel Genotyping of short tandem repeat (STR) polymorphisms and quality checks of the genotype data were carried out as described previously (Tauer et al., 2005). In total, 454 STR markers (http://www.msz.mdcberlin.de/marker.html) were preferentially selected from the Marshfield marker set (Broman et al., 1998), covering all 22 autosomes and the X chromosome. In addition, 185 STR polymorphisms were genotyped for enhancing the information content (>85%) in regions of interest, including

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TABLE 1. Clinical characterization of epilepsies in 126 IGE-multiplex families Family samplea

Fam. N

Ind. N

JME

CAE

JAE

EGMA

EGTCS

SS

GSW

Uncl. Epil.

FC

IGE IAE IAE-MP JME JME-MP

126 73 52 53 28

675 357 253 318 171

90 — — 90 58

144 115 96 29 6

46 32 22 14 3b

24 12 5 12 11

35 22 12 13 4

4 2 1 2 —

13 11 3 2 1

4 1 — 3 1

5 2 2 3 2

IGE, idiopathic generalized epilepsy; IAE, idiopathic absence epilepsy; CAE, childhood absence epilepsy; JAE, juvenile absence epilepsy; JME, juvenile myoclonic epilepsy; EGMA, epilepsy with tonic–clonic seizures on awakening; EGTCS, epilepsy with generalized tonic–clonic seizure; SS, unprovoked single generalized tonic–clonic seizure; GSW, unprovoked generalized spike–wave discharges in the EEG; FC, febrile convulsion; Uncl. Epil., unclassified epilepsy; IGE, entire sample of families containing at least two siblings with IGE; IAE-MP, families containing at least two siblings with IAE; JME-MP, families of JME patients containing at least two siblings with either JME or EGMA. a One diagnostic category only was given for each subject, using a hierarchic order. b One JAE patient also exhibited EGMA.

candidate regions of current and previous linkage hints and known epilepsy genes. Marker locations were obtained from the sex-averaged Marshfield genetic map (Broman et al., 1998). The average interval between adjacent markers was 5.3 cM (all gaps, 3.58–3.60); genome-wide “suggestive” linkage evidence was established for an NPL score expected to occur once by chance in a genome-wide scan (ZNPL > 2.65–2.70) (Lander and Kruglyak, 1995). For confirmation of previous significant linkage findings, a nominal p value of 0.01 was required (Lander and Kruglyak, 1995). RESULTS To explore the complex pattern of susceptibility loci underlying familial IGE syndromes, we performed a genome-wide nonparametric linkage (NPL) scan in the entire sample of 126 IGE-multiplex families, assuming the IGE-trait definition (Fig. 1). Four NPL scores in the chromosomal regions 5q34 (ZNPL = 3.11 at GABGR2; Marshfield position, 163.3), 11q13 (ZNPL = 3.37 near D11S4095, pos. 67.4), 13q22-q31 (ZNPL = 3.01 at D13S800, pos. 55.3), and 19q13 (ZNPL = 3.49 at D19S900, pos. 67.4) exceeded the empiric significance threshold for “suggestive” linkage evidence (ZNPL > 2.66). Although none of the observed linkage signals reached evidence for “significant” linkage, the actual number of four NPL peaks >3.00 is highly unlikely to occur by chance (p < 0.001). To search for susceptibility genes predisposing to typical absence seizures, linkage analysis was initially carried out in 73 IAE families without JME members, assuming the IGE-trait definition. A maximum multipoint NPL score of 3.80 was found in the chromosomal region 11q13 at D11S1975 (pos. 72.4). Simulation analysis of the present data set indicated that a ZNPL = 3.80 occurs by chance 0.018 times in a genome-wide NPL scan, Epilepsia, Vol. 47, No. 10, 2006

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FIG. 1. Genome-wide nonparametric llinkage (NPL) results for the idiopathic generalized epilepsy (IGE)-trait definition in 126 IGE-multiplex families. The chromosomes are arranged in linear scale from pter to qter. Empirically derived genome-wide significance thresholds are indicated in the plots (horizontal lines).

achieving genome-wide “significance” for linkage (Pemp = 0.018). Two NPL peaks in the chromosomal regions 13q22-q31 (ZNPL = 3.19 at D13S800, pos. 55.3) and 19q13 (ZNPL = 2.76 at D19S900, pos. 67.4) exceeded the empiric threshold for “suggestive” linkage evidence (ZNPL > 2.66) (Fig. 2A). To increase genetic homogeneity by narrowing familial phenotypic variance, linkage analysis was then conducted in a subgroup of 52 IAE-multiplex families under the IAE trait. Likewise, the genome-wide scan yielded three NPL peaks in the chromosomal regions 11q13 (ZNPL = 2.82 at D11S1314, pos. 73.6), 13q22-q31 (ZNPL = 2.71 at D13S793, pos. 76.3), and 19q13 (ZNPL = 2.66 at D19S900, pos. 67.4), exceeding the empirical threshold for “suggestive” linkage evidence (ZNPL > 2.65) (Fig. 2B). To dissect susceptibility genes predisposing to JMErelated seizure types, NPL analysis was first (stage I) performed in 53 JME families under the IGE-trait definition. The genome-wide scan revealed two NPL peaks in the chromosomal regions 5q34 (ZNPL = 2.77 at GABGR2D5S423, pos. 163.3–169.4) and 19q13 (ZNPL = 3.08 near D19S246, pos. 78.1), achieving empiric evidence for “suggestive” linkage (ZNPL > 2.70) (Fig. 3A). When the phenotypic variance in the 28 JME/EGMA-multiplex families was further narrowed by use of the JME-trait definition, NPL peaks exceeding the empiric threshold of “suggestive” linkage (ZNPL > 2.70) were obtained in the chromosomal regions 6p12 (ZNPL = 3.05 at D6S2410, pos. 73.1) and 19q13 (ZNPL = 3.46 near D19S585, pos. 79.5) (Fig. 3B). Taking into account that linkage of JME to the EFHC1 locus (pos. 75.7) on 6p12 has been established (Liu et al., 1996; Serratosa et al., 1996; Pinto et al., 2004), the present NPL score of 3.05 near to EFHC1 locus provided “confirmatory” evidence for linkage (p < 0.01). Because one Dutch JME family (family 9831) was also part of a recent linkage study confirming evidence for Epilepsia, Vol. 47, No. 10, 2006

FIG. 2. A: Genome-wide nonparametric llinkage (NPL) results for the idiopathic generalized epilepsy (IGE) trait definition in 73 IAE-multiplex families. B: Genome-wide NPL results for the IAEtrait definition in 52 IAE-multiplex families. The chromosomes are arranged in linear scale from pter to qter. Empirically derived genome-wide significance thresholds are indicated in the plots (horizontal lines).

linkage of JME-related traits to the EFHC1 region (Pinto et al., 2004), we repeated linkage analyses of the 6p12 region without this family. Likewise, “confirmatory” evidence for linkage was obtained at D6S2410 (ZNPL = 2.95, nominal p = 0.0017; pos. 73.1). DISCUSSION The aim of this study was to explore the genetic architecture of common familial IGE syndromes and to dissect seizure type–related loci predisposing to either absence or JME-related myoclonic seizures. Our genome-wide NPL scan for susceptibility loci common to most IGEs yielded a complex pattern of NPL signals in the entire sample of 126 IGE-multiplex families (Fig. 1). Four NPL peaks in the chromosomal regions 5q34, 11q13, 13q22-q31, and 19q13 achieved empirically derived thresholds of “suggestive” evidence for linkage (ZNPL > 2.70). Taking into account that one NPL score ≥2.70 would be expected to occur by chance in the present genome scan, the actual number of four NPL peaks >3.00 is highly unlikely to occur by chance (p < 0.001) and reflects the complex and

GENETIC ARCHITECTURE OF IDIOPATHIC GENERALIZED EPILEPSY

FIG. 3. A: Genome-wide nonparametric llinkage (NPL) results for the idiopathic generalized epilepsy (IGE)-trait definition in 53 JME families. B: Genome-wide NPL results for the JME-trait definition in 28 JME/EGMA-multiplex families. The chromosomes are arranged in linear scale from pter to qter. Empirically derived genome-wide significance thresholds are indicated in the plots (horizontal lines).

heterogeneous genetic architecture of IGE in the present IGE-multiplex families. Twin and family studies suggest that shared and distinct genetic factors confer susceptibility to absence and myoclonic seizures (Janz et al., 1992; Wirrell et al., 1996; Berkovic et al., 1998; Durner et al., 2001; Winawer et al., 2003, 2005; Marini et al., 2004). To dissect out seizure type–related susceptibility loci for either typical absence or myoclonic seizures, we narrowed the familial phenotypic variance by subgrouping of families according to their predominant seizure type and by evaluating seizure type–related trait definitions. Assuming that some of the genetic effects on certain seizure types are essential, families containing several affected members will tend to be concordant for the seizure type of interest. By maximizing phenotypic and thereby genetic homogeneity, our present linkage results revealed two susceptibility loci for typical absence seizures in the chromosomal regions 11q13 and 13q22-q31. In contrast, we found evidence for two different susceptibility loci on chromosomes 6p12 and 19q13, predisposing to myoclonic or generalized tonic–clonic seizures on awakening. The susceptibility locus on 6p12

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was observed only in the JME families but not in the entire sample of IGE-multiplex families. Thus this susceptibility locus appears to be a major genetic determinant for the phenotypic expression of bilateral myoclonic seizures on awakening or, more generally, generalized seizures triggered by an impaired control of the excitability state of thalamocortical circuits associated with awakening (Janz, 1997; Andermann and Berkovic, 2001). Overall, our results suggest that syndromes with absence seizures compared with those with myoclonic seizures on awakening differ in their genetic predisposition. The present linkage results in JME/EGMA-multiplex families confirmed previous evidence for linkage to the chromosomal region 6p12 in families from Belize, Mexico, and The Netherlands (Liu et al., 1996; Serratosa et al., 1996; Pinto et al., 2004). Recent mutation analysis of the positional candidate gene EFHC1 identified five missense mutations in affected family members of six unrelated Mexican families with JME, suggesting that the EFHC1 gene is a responsible disease gene (Suzuki et al., 2004). Among affected relatives of JME families showing linkage to 6p12, JME was most common (40%), followed by EGTCS/EGMA only (36%), but CAE was relatively rare (Martinez-Juarez et al., 2006). The present subgroup of JME/EGMA-multiplex families showed a similar phenotypic spectrum (classic JME). Here we demonstrate that it is possible to dissect IGE-loci reproducibly within oligogenic traits by narrowing the familial phenotypic variance and by applying defined phenotype–genotype relations. However, it remains to be determined whether genetic variation of the EFHC1 gene accounts for the observed linkage findings. Recently, Tauer et al. (2005) mapped a susceptibility locus for photosensitive IGE to the chromosomal region 13q31 in 25 families, in which photosensitivity was strongly associated with IGE. Twenty-three of these 25 photosensitive IGE-multiplex families were taken from the present study sample (without knowledge of the linkage results) to explore whether photoparoxysmal response could serve as an electroencephalographic endophenotype for cortical hyperexcitability (Parra et al., 2005). Because the age-related occurrence of PPR was not examined systematically in all of the investigated 126 IGE-multiplex families, further replication studies are necessary to validate this tentative phenotype–genotype relation. Furthermore, the present linkage hint of IGE to the chromosomal region 5q34 highlights an interesting candidate region harboring a cluster of four genes encoding GABAA -receptor subunits (GABRA1, GABRA6, GABRB2, GABRG2). Mutations in the GABRG2 gene have been identified in two families with generalized epilepsy and febrile seizures plus (GEFS++ ) and in two families with febrile seizures and CAE, respectively (Gardiner, 2005; Mulley et al., 2005; Turnbull et al., 2005). Moreover, two missense mutations of the GABRA1 gene have recently been described, Epilepsia, Vol. 47, No. 10, 2006

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one in a large French-Canadian family with autosomal dominant inheritance of JME (Cossette et al., 2002), and another one in a “sporadic case” of CAE (Maljevic et al., 2006). Together, these findings suggest that these four GABAA -receptor genes are promising positional candidate genes for IGE. None of the linkage signals on chromosomes 11q13 and 19q13 implicated in the present study has been reported in previous linkage studies of human idiopathic epilepsy. These genomic regions contain several genes involved in neuronal signal transduction (e.g., ADRBK1, ATP1A3, CACNG6, CAGNG7, CACNG8, CLCNS1A, GNG3, GRIK5, GRIN2D, SLC1A5), neurotransmitter metabolism (ALDH3B1, ALDH3B2), synaptogenesis (SHANK1, SHANK2, SYT3, SYNGR4), and neurodevelopment (PLAUR), which we consider as plausible positional candidate genes. Our attempt to replicate previous linkage claims failed to confirm most of the reported IGE susceptibility loci, except for the JME locus on chromosome 6p12. Although we used a consistent research design across both of our genome scans, the present linkage results of the entire family sample do not confirm our previous linkage findings on chromosomes 2q36, 3q26, and 14q23 in 130 IGEmultiplex families (Sander et al., 2000). Likewise, our present NPL analyses do not provide supportive linkage evidence for any published linkage claim on 5p15 (Durner et al., 2001), 5q22 (Durner et al., 2001), 6p23.1 (Greenberg et al., 1988, 1995, 2000; Durner et al., 1991; Sander et al., 1995, 1997), 8p12 (Durner et al., 1999), 8q24 (Zara et al., 1995; Fong et al., 1998), 15q14 (Elmslie et al., 1997), and 18q21 (Durner et al., 2001). This failure can be attributed to a number of reasons, such as locus heterogeneity, variability in study designs, phenotype definitions, sampling variations, low statistical power, inappropriate statistical modeling, and putative false-positive linkage hints. Although the number of replications of current linkage claims is disappointingly low, the current data provide realistic insights into the complexity of the genetic architecture of the common IGE syndromes. The discordant linkage findings reported so far contradict the optimistic view that a few common major susceptibility genes account for a substantial fraction of the overall genetic variance of IGE syndromes (Durner et al., 2001). The genetic predisposition for IGE likely represents a biologic continuum, in which a small fraction follows monogenic inheritance, whereas the majority of common IGE syndromes presumably display an oligogenic or polygenic predisposition. Given that a considerable proportion of IGEmultiplex families follow polygenic inheritance (Ottman, 2005; Tan et al., 2006), not all of the families used are suitable for linkage mapping (Glazier et al., 2002). Moreover, the present linkage results demonstrate that the configuration of the underlying susceptibility loci may vary remarkably depending on the composition of IGE subtypes in the family sample. In the presence of extensive genetic Epilepsia, Vol. 47, No. 10, 2006

heterogeneity, initial linkage findings often reflect sampling variations, leading to a random overrepresentation of a few major genes. Subsequent genome scans are therefore more likely to map other susceptibility genes that are again accumulated by chance. Consequently, replication studies would require a large number of families (>1000) to detect a specific major gene identified in a previous study (Suarez et al., 1994; Altmuller et al., 2001; Tan et al., 2006). In summary, the present genome scans revealed a complex pattern of linkage signals (5q34, 6p12, 11q13, 13q22q31, and 19q13) that differed in their composition depending on the predominant seizure type in the multiplex families. Configurations of distinct and shared genetic effects seem to influence risk for different IGE subtypes. Genetic determinants on 11q13 and 13q22-q31 may confer susceptibility to absence seizures, whereas loci on 5q34, 6p12, and 19q13 preferentially predispose to myoclonic seizures and GTCS on awakening. The discordant linkage findings observed so far suggest that the genetic architecture of the majority of common IGE syndromes is more complex and heterogeneous than initially expected. In future, three linkage-related approaches will be essential to dissect the oligogenic predisposition to common IGE syndromes. First, large multicenter collaborations using standardized protocols for phenotyping of IGE traits will ensure relative phenotypic homogeneity and are imperative to achieve sufficient power to disentangle the complex genetic basis of IGE traits. Second, clinical genetics must delineate suitable endophenotypes (e.g., seizure type, photosensitivity, precipitating factors) and to specify phenotype–genotype relations that are closer to the molecular pathways of the underlying susceptibility genes. Finally, more-advanced linkage statistics will take into account gene interactions (Bell et al., 2006). However, even if genetic diversity will hinder the dissection of the complex genetic basis of common familial IGE syndromes, it can be expected that most molecular defects will functionally converge on a few common processes regulating cortical synchronization (Noebels, 2003). Thus the functional characterization of these key molecular pathways will provide important insights into epileptogenesis and may have equally significant therapeutic implications. Acknowledgment: We gratefully acknowledge all families participating in the present study and all the members of the clinical centers (Drs. Brodtkorb, Ernst, Janz, Kasper, Muhle, Neubauer, Schmitz, and Steinhoff) for their support. We thank Carolin Engel, Heike Fischer, Ilona Lichter, and Nadine Wittstruck for their excellent technical assistance in STR genotyping. This work was supported by grants of the Deutsche Forschungsgemeinschaft (Sa434/3–1), the German National Genome Research Network (BMBF-NGFN2: 01GS0474, 01GS0479, 01GR0416), the Stiftung Michael, and the German Volkswagen Foundation.

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