Brain, Vol. 125, No. 12, 2668-2680,
December 2002
© 2002 Oxford University Press
A locus for simple pure febrile seizures maps to chromosome 6q22–q24
1 INSERM U289, 2 Département de Génétique, Cytogénétique et Embryologie, 3 Fédération de Neurologie, Hôpital Pitié-Salpêtrière, 4 Service de Neuro-Pédiatrie, Hôpital Saint Vincent de Paul, Paris and 5 Généthon, Evry, France
Correspondence to: Rima Nabbout, MD, INSERM U289, Hôpital Pitié-Salpêtrière, 47 boulevard de l’Hôpital, 75013 Paris, France E-mail: rimanabbout{at}yahoo.com
Received January 14, 2002. Revised June 12, 2002. Accepted June 15, 2002.
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Summary |
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Febrile seizures (FS) syndromes exhibit major clinical and genetic heterogeneity. We report a clinical and genetic study of three families with simple FS segregating as an autosomal dominant (AD) trait with high penetrance. All affected members presented a homogeneous phenotype of simple FS. The FS ceased before the age of 5 years. Among the 29 affected family members, only one patient presented two afebrile seizures, and none of the others developed concomitant or subsequent epilepsy. The phenotype differs from that previously reported in families presenting FS or generalized epilepsy with febrile seizures plus (GEFS+). After exclusion of already known loci for FS and GEFS+, we performed a genome-wide scan in the largest family. It led to the identification of a new locus on chromosome 6q22–q24 spanning 6.4 cM between D6S1620 and D6S975. For one of the other two families, the trait also segregated with this locus, but linkage studies could not restrict the candidate region further. The absence of linkage in the third family supports genetic heterogeneity of the AD form of pure simple FS. Sequence analysis excluded the implication of five candidate genes [A kinase anchoring protein 18 (AKAP18), syntaxin 7, putative neurotransmitter receptor (PNR), G protein receptor 57 (GPR57) and G protein receptor 58 (GPR58)] in the interval based on function. The locus mapping to 6q22–q24 seems to be the first identified locus responsible for pure simple FS, the most frequent form of FS. Studies are ongoing to identify the gene.
Keywords: simple febrile seizures; genetic heterogeneity; mapping; chromosome 6
Abbreviations: AD= autosomal dominant; AKAP18 = A kinase anchoring protein 18; FS = febrile seizures; FS+ = febrile seizures plus; GEFS+ = generalized epilepsy with febrile seizures plus; GPR57 = G protein receptor 57; GPR58 = G protein receptor 58; PNR = putative neurotransmitter receptor; SNP = single nucleotide polymorphism
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Introduction |
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TopSummaryIntroductionSubjects and methodsResultsDiscussionReferences |
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Febrile seizures (FS) are the most frequent type of seizures in children. They occur in neurologically healthy infants and children, between 6 months and 6 years of age. They are associated with fever but with no evidence of intracranial infection or a defined pathological or traumatic cause, and with no history of prior afebrile seizures (Consensus statement, 1980
; Commission on Classification and Terminology of the International League Against Epilepsy, 1989). They are usually benign, resolve spontaneously, do not require antiepileptic drug therapy and are not followed by permanent neurological sequelae. The incidence of FS in the North American population is 
2–5% in children under the age of 5 (Hauser, 1994; Hauser et al., 1996), but is as high as 7% in Japan (Tsuboi, 1984) and in Pacific island populations (Stanhope et al., 1972).
FS constitute a heterogeneous group of syndromes. The main risk is recurrence of FS, observed in 30% of patients (Berg et al., 1990). In addition, the risk of developing epilepsy later in life was found to be increased, although only moderately in population-based studies (Annegers et al., 1979, 1987; Ellenberg and Nelson, 1980; Berg, 1992), and antecedents of FS are reported by 10–15% of patients presenting epilepsy (Camfield et al., 1994; Hamati-Haddad and Abou-Khalil, 1998). The risk for later epilepsy is even greater in clinical and hospital-based studies which usually include more patients with complex diseases (Wallace, 1977), especially intractable temporal lobe epilepsy (Abou-Khalil et al., 1993; Guerreiro et al., 1999).
Increased risk for later epilepsy is related to so-called ‘complex’ or ‘complicated’ FS as opposed to ‘simple’ FS, for which the risk is not greater than for the general population (Nelson and Ellenberg, 1976, 1978; Annegers et al., 1987).
The great majority of FS are ‘simple’. These are defined as generalized seizures that last <15 min, do not recur within 24 h, and have no post-ictal neurological anomalies (Nelson and Ellenberg, 1981). In contrast, complex FS or complicated FS are focal, prolonged, recur within 24 h or are associated with post-ictal neurological abnormalities, including Todd’s paresis (Nelson and Ellenberg, 1976, 1978). Long-lasting complex FS may be followed by mesial temporal sclerosis with epilepsy (Abou-Khalil et al., 1993; Guerreiro et al., 1999). A relationship between FS and epilepsy recently received further clarification with the description of the syndrome of febrile seizures plus (FS+) (Scheffer and Berkovic, 1997). The affected individuals experience numerous FS that persist after the age of 6 years and/or are associated with afebrile seizures, two characteristics that differ from simple FS. This syndrome recurs in families, exhibits autosomal dominant (AD) transmission and is known as ‘generalized epilepsy with febrile seizures plus (GEFS+)’ (Singh et al., 1999).
Twin and family studies have shown that there is an important genetic component in the aetiology of FS (Hauser et al., 1985; Tsuboi, 1987; Corey et al., 1991; Tsuboi and Endo, 1991). All modes of inheritance, AD, autosomal recessive and polygenic, have been described (Rich et al., 1987; Tsuboi, 1987; Johnson et al., 1996). Four genes underlying GEFS+ have been identified in recent years: SCN1B on chromosome 19q13.1 (Wallace et al., 1998), SCN1A (Escayg et al., 2000) together with SCN2A (Sugawara et al., 2001) on chromosome 2q24, and GABRG2 on chromosome 5q31.1–q33.1 (Baulac et al., 2001; Wallace et al, 2001a). No genes have yet been identified for FS, but four loci have been reported. Three were identified by parametric linkage analyses of large families: FEB1 on chromosome 8q13–q21 (Wallace et al., 1996), FEB2 on chromosome 19p13.3 in two families (Johnson et al., 1998; Kugler et al., 1998) and FEB3 on chromosome 2q23–q24 (Peiffer et al., 1999). A fourth locus, FEB4, was identified by a non-parametric analysis, on 5q14–q15 (Nakayama et al., 2000) in a series of 47 small families.
We report three families in which affected members express a homogeneous phenotype of simple FS. This trait segregated with an AD mode of inheritance. After exclusion of already known loci for FS and GEFS+, a genome-wide scan in the largest family spanning five generations led to the identification of a new locus for simple FS on chromosome 6q22–q24.
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Subjects and methods |
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Family collection and phenotyping
The study was performed with the approval of the Cochin-Port-Royal Hospital Ethics Committee. The families were selected after a public campaign to identify and collect families affected by epilepsy, organized by a network of clinicians and geneticists, the Association pour la Recherche sur la Génétique des Epilepsies, and Généthon, Evry, France. We selected for this study the three largest non-consanguinous families presenting simple FS. All families were of French origin. All adult members were interviewed by telephone with a detailed questionnaire. When there was a history of seizures, information on perinatal events, head trauma, age at seizure onset, duration and type of seizures, number of seizures, association with fever, intellectual outcome and antiepileptic drug therapy was obtained from the members experiencing seizures and from their parents. Parental information was available for all affected members, provided the parents were still alive. The parents of unaffected family members, especially spouses, were also interviewed in order to identify a possible bilinear inheritance of the trait. Additional data were obtained from the patients’ physicians and from medical records.
All family members or their legal representatives gave their informed consent to publish the pedigree, to contact their physicians and to request medical records.
The FS trait was AD in all families. The first family (family 1) had 162 members over five generations (Fig. 1). Fourteen family members had died before the study began. FS was reported in 15 family members. Thirty-nine consented to participate in the genetic study, and samples of their DNA were obtained.
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) indicate the members of generation II who did not have offspring with FS. An asterisk indicates branches of the family who declined to participate in the genetic study.The other two families (family 2 and family 3) spanned three generations. They consisted of 20 and 27 members, respectively, with seven affected members in each family. Twelve DNA samples were available for family 2 and 14 for family 3.
Genotyping
DNA was extracted at the Généthon DNA bank by standard procedures. Family members were genotyped with microsatellites in order to exclude reported loci for FS and GEFS+: D8S553, D8S1840, D8S530 for FEB1 (Wallace et al., 1996), D19S565, D19S591, D19S209, D19S216 for FEB2 (Johnson et al., 1998; Kugler et al., 1998), D5S644, D5S652, D5S2079 for FEB4 (Nakayama et al., 2000), D19S414, D19S868, D19S425 for GEFS+1 (Wallace et al., 1998), D2S156, D2S382, D2S2330, D2S2345, D2S326 for GEFS+2 and FEB3 (Baulac et al., 1999; Moulard et al., 1999; Peiffer et al., 1999; Lopes-Cendes et al., 2000) and D5S422, D5S2093, D5S621 for GEFS+3 (Baulac et al., 2001; Wallace et al., 2001a).
We performed a genome-wide scan in the largest family (family 1) with 380 fluorescent microsatellites from the ABI-PRISM linkage mapping set, version 2 (Perkin Elmer) selected from the Généthon map (Dib et al. 1996). The 20 markers on chromosome X were not used because of a male to male transmission. The PCRs were prepared as follows: 50 ng of DNA, 5 pmol of each primer, 2.5 mM of each dNTP, 1.5 µl of 10x PCR buffer and 0.6 U of Ampli Taq Gold DNA polymerase in a final volume of 15 µl. Samples were incubated for 12 min at 95°C in order to activate the Taq polymerase, then for 15 s at 89°C, 15 s at 55°C and 30 s at 72°C for 35 cycles, followed by an elongation for 10 min at 72°C at the end of each PCR. The PCR products were pooled with the Genescan 400HD size standard and were loaded on a 4% acrylamide gel for electrophoresis (ABI PRISMR 377 DNA sequencer, PE Biosynthesis).
Linkage analyses
Affected status was assigned when the occurrence of one or more FS was documented. For generation II in family 1, since the parents of the affected members could no longer be interviewed, we considered the status of this generation as unknown in order to increase the stringency of genetic analysis.
Segregation of the trait was considered AD with a 75% penetrance estimated by the method of Johnson et al. (1996). Pairwise and multipoint LOD scores were calculated with the MLINK and LINKMAP programs of the NT link package, version 3.0 (Schaffer et al., 1994). The disease gene frequency was estimated at 0.0001. The phenocopy rate was defined as 3%, since the frequency of FS in the general population ranges from 2 to 5%. The recombination fraction 
was considered to be equal in males and females. For the genome scan, all markers were analysed assuming equal allele frequencies. For multipoint analysis, the order of the markers was that of the Généthon linkage map. Calculations were performed at Infobiogen (Villejuif, France).
Mutation detection
We sequenced five candidate genes in the 6q22–q24 region encoding the A kinase anchoring protein 18 (AKAP18), syntaxin 7, a putative neurotransmitter receptor (PNR), G protein receptor 58 (GPR58) and G protein receptor 57 (GPR57). The PCR products covering the coding exons and exon–intron boundaries were sequenced using the BigDye Deoxy Terminator Cycle sequencing kit (Applied Biosystems) on an Applied Biosystems Sequencer 377. The primers designed for all genes with their respective annealing temperature are shown in Table 1. We analysed both strands using Sequencing Analysis and AutoAssembler software. For each gene, we tested three affected members, two from family 1 (III.3 and III.11) and one from family 2 (II.4), and two controls.
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Polymorphisms which did not affect a restriction site were analysed in relatives and controls by sequencing.
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Results |
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Phenotype study
The clinical profile of affected members is summarized in Table 2.
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In family 1, the age at onset of FS ranged from 10 months to 2.5 years, with a mean age of 17.5 months. The age at examination of the family members ranged from 19 to 78 years (mean 48 years), except for subjects of generation V who were aged 4, 8, 9 and 10 years and were not affected (Fig. 1). The seizures were described as generalized in all cases, tonic–clonic in nine, hypertonic in six and hypotonic in one (IV.8). Most seizures were brief, ranging from 1 to 5 min, but lasted up to 10 min in two patients (III.3 and III.4). The mean number of FS was three (ranging from two to five), except in patient IV.8 who presented a single hypotonic FS. Interictal EEGs were performed in five patients (III.8, IV.1, IV.6, IV.8 and IV.10) and were normal. Only one patient (IV.1) underwent brain imaging, and the CT scan was normal. One patient (IV.1) received phenobarbital for a period of 3 years. Patient IV.6 had two brief generalized convulsions during the eruptive phase of measles. He had slight concomitant alteration of consciousness, and lumbar puncture revealed 42 white blood cells/mm3, mainly lymphocytes. He was therefore considered to be unaffected since the seizures were associated with meningitis of viral origin, excluding the diagnosis of FS (Joint Working Group of the Research Unit of the Royal College of Physicians and the British Paediatric Association, 1991
). In family 2, the age at onset of FS ranged from 9 months to 2.5 years, with a mean age of 15.5 months, and the age at examination of the family members ranged from 15 to 76 years (mean 33 years). Seizures were described as generalized tonic–clonic in six affected members, hypertonic in one and hypotonic in one (III.6). Most seizures were brief, ranging from 1 to 3 min, but lasted up to 10 min in one patient (III.13). In the oldest patient (I.2), information was available from her medical file. The mean number of FS was three (ranging from two to five), except in patient III.6 who presented a single FS. One patient (II.5) presented two afebrile seizures at the ages of 17 and 25 years. These seizures occurred while the patient was awake and lasted <2 min. His EEG was normal and he received no antiepileptic drugs. He was 45 years old at the time of this study and convulsions had not recurred. Interictal EEGs were performed in five patients (II.2, II.4, III.3, III.5 and III.7) and were normal. Two patients had a brain MRI (III.3 and III.5) which was also normal. These two patients received valproate for a period of 3 years, although their clinical pattern was not different from that of the other family members.
In family 3, the age at onset of FS ranged from 9 months to 4 years, with a mean age of 22 months, and the age at examination of the family members ranged from 10 to 60 years (mean 28 years). Seizures were generalized tonic–clonic in five and hypertonic in two. They were brief and lasted <5 min in all patients. The mean number of FS was two (ranging from two to four). Interictal EEGs performed in five patients (III.5, IV.3, IV.4, IV.5 and IV.6) were normal. Brain imaging was done for IV.3, and his CT scan was normal. Two patients (III.5 and IV.3) received antiepileptic drugs until the age of 4 years (Table 2).
In these three families, all affected members presented FS that fulfil the criteria for simple FS. Cessation of FS was reported before or at the age of 5 years. Of the 29 affected members, only one presented later afebrile seizures (II.5, family 2). All affected members had normal psychomotor development.
Linkage analyses
In the three families, pairwise analyses were performed for markers of the loci already identified for FS (8q, 19p and 5q) and GEFS+ (19q, 2q and 5q). LOD scores were negative for all markers at = 0.00. The exclusion of these loci was confirmed by multipoint analyses (data not shown).
The analyses of the genome-wide scan in the largest family (family 1) showed positive pairwise LOD scores at = 0.00 for 28 out of 380 markers. For every positive locus, two additional markers were genotyped. These regions were then excluded by haplotype reconstruction and multipoint analyses, except for three adjacent markers on chromosome 6 including D6S262. The highest LOD score was obtained for the marker D6S1572 (Zmax = 3.54 at = 0.00). Additional markers in the region D6S407, D6S1620, D6S1572, D6S435, D6S457, D6S1656 and D6S472 were then tested, the majority of which yielded positive LOD scores >2.5 at = 0.00 (Table 3). Multipoint analysis with markers D6S407, D6S1620, D6S1572, D6S262 and D6S472 confirmed the linkage, with a maximum LOD score of 4.82 between markers D6S1572 and D6S472.
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Haplotype reconstruction and analysis of recombination events were performed for markers D6S407, D6S1620, D6S1572, D6S262, D6S1656, D6S472 and D6S975. The centromeric boundary of the candidate interval was defined by a recombination between D6S1620 and D6S1572 in patient IV.24 and the telomeric boundary by a recombination in patient IV.1 between D6S472 and D6S975. The size of the interval was
6.4 cM. All affected members shared the same haplotype (3-2-2-3-9-4) (Fig. 2, grey bars), except patient IV.8 who could be considered as a phenocopy. He presented a single FS of brief duration <1 min, described as hypotonic, that differed from the tonic–clonic or the hypertonic seizures of other affected family members. The unaffected individuals II.1, II.6, II.8, II.11 and II.13, who did not share the haplotype associated with the disease, had no affected children or grandchildren (arrows on Fig. 1), supporting the hypothesis that they are not carriers of the mutation.
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In the other two families with the same phenotype, we tested seven markers in the candidate region: D6S407, D6S1620, D6S1572, D6S262, D6S1656, D6S472 and D6S975. Positive LOD scores were obtained by pairwise analyses in family 2. The maximum LOD score value reached 1.91 (Table 4) for D6S262, which was higher than the threshold value of 1.7 for a linkage to a known locus (Ott, 1991
). This linkage was reinforced by multipoint analysis, which yielded a maximum value of 1.93 between D6S262 and D6S472. All affected individuals shared the same haplotype (2-3-1-1-5-1-5), except for patient III.6 who presented, like individual IV.8 in family 1, a single brief hypotonic FS and was also considered to be a phenocopy (Fig. 3, grey bars). The boundaries of the candidate interval were defined by a recombination between the markers D6S1702 and D6S407 at the centromeric boundary (II.4) and between the markers D6S975 and D6S270 at the telomeric boundary (III.7), spanning an interval of 11.5 cM. However, haplotype reconstruction in family 2 did not help to restrict the candidate region since the candidate interval for family 1 (D6S1620–D6S472) was included within that for family 2 (D6S1702–D6S270) (Fig. 3). The difference between the two haplotypes for D6S1572, D6S262, D6S1656 and D6S472, which was (3-2-9-4) for family 1 and (1-1-5-1) for family 2, suggests the likelihood of two different mutational events in these families.
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In family 3, negative pairwise LOD scores were obtained for the same markers (Table 5). Multipoint LOD score values were less than –2.00 between D6S1620 and D6S1656. The haplotype reconstruction is shown in Fig. 4. These results support the absence of linkage for family 3 to the 6q22–q24 locus.
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Sequencing of candidate genes
The candidate interval spans 6.4 cM and contains 22 genes and many expressed sequence tags (Genemap 99, NCBI Sequence Viewer, Sanger Center, HGREP). Five of these genes could be considered candidates due to their functions: AKAP18, syntaxin 7, PNR, GPR58 and GPR57. We tested these genes by direct sequencing of three affected members, two from family 1 (III.3 and III.11) and one from family 2 (II.4), and two controls.
We determined the genomic structure of AKAP18 by aligning the cDNA sequence (GI: 14755533) with the working draft sequence of chromosome 6 (GI: 14755617) (NCBI, Map Viewer). The gene spans 138 kb and is composed of seven exons covering 2793 bp. We used intronic primers (Table 1) to amplify the seven coding exons and sequenced 80 bp in the introns at the 5' and 3' borders of each exon in order to examine the splice sites. We also analysed 300 bp of the 5' end upstream (exon 1) from the first coding ATG. We found a heterozygous single nucleotide polymorphism (SNP) G578A in exon 5 in the two patients of family 1, but not in the patient from family 2. This polymorphism changed the protein sequence, replacing a serine by an asparagine at residue 193 (S193N) of the AKAP18 protein. Direct DNA sequencing in all family members showed that this SNP did not co-segregate with the disease phenotype. Furthermore, after sequencing 96 chromosomes from European controls, the allele frequency was 47% for G and 53% for A. Genotypes of controls were in Hardy–Weinberg equilibrium.
Alignment of the syntaxin 7 cDNA sequence (GI: 15300438) and the sequence of yeast artificial chromosome (YAC) NT_25741 of chromosome 6 revealed a genomic organization of 10 exons covering 1586 bp with the coding region within exons 2–10. We designed a pair of intronic primers for each exon, except for exon 10 that was divided into three parts (A, B and C) since it spans 841 bp. Primers were intronic at an average of 80 bp from the 5' and 3' end of each exon. Primers for exon 10 were intronic at the 5' end of 10A and at the 3' end of 10C, and were exonic for the other fragments (Table 1). In addition to the SNPs already reported, we detected two new SNPs (T1221C and G1402A) in the 3'-non-coding terminal portion of the mRNA.
The genes for GPR58, GPR57 and PNR share some sequence identity and are located close to each other in the candidate region on 6q22–q24. Each of these three genes is encoded by a single exon. GPR58 spans 921 bp (GI: 13643426), GPR57 1030 bp (GI: 13643419) and PNR 1014 bp (GI: 13643411). We designed primers in order to obtain PCR fragments of 300 bp (Table 1). GPR58 and GPR57 were divided into five fragments and PNR into six fragments. Direct sequencing showed a C139T polymorphism in the 5'-untranslated region of GPR57. In PNR, we detected a G192A polymorphism that did not change the amino acid encoded (GCG/GCA = alanine), and did not segregate with the disease in either family 1 or family 2.
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Discussion |
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TopSummaryIntroductionSubjects and methodsResultsDiscussionReferences |
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After the exclusion of the known loci for FS and GEFS+ in three French families in which simple FS segregates as an AD trait, we identified a new locus for FS on chromosome 6q22–q24 in the largest family. The smallest genetic interval extends from D6S1620 to D6S472, spanning
6.4 cM on the sex-averaged linkage map. One of the two other families presented linkage to this locus, but haplotype reconstruction did not restrict the candidate interval further.
The methodology for phenotyping was as strict as possible. The data were collected by interviewing the parents, when alive, of each affected member, in order to obtain a reliable description of the ictal events. Parents are unlikely to forget details of such an impressive and unexpected event, even several decades later (van Stuijvenberg et al., 1999). Information about members of generation II in family 1 was difficult to obtain since the parents were deceased at the time of this study. Furthermore, no medical records were available, because the parents were convinced that their children’s condition, ‘having worms’ according to local terminology, was innocuous and transitory, and did not seek medical care. Although the members of generation II reported learning from their parents that they had manifested the same ictal events as their children or grandchildren, i.e. brief and generalized FS without afebrile seizures, we had no formal evidence of the occurrence of FS. The status of this generation was therefore considered as unknown.
The phenotypes of affected members of these three families are homogeneous and consisted of generalized FS of brief duration that recurred a few times. Onset was in most cases at 18 months of age. None of the 29 affected members suffered FS after 5 years of age, and no family member, whether or not he had FS, had presented epilepsy or mental retardation. Children who develop afebrile seizures after FS usually do so within 5 years of the initial convulsion (Wolf and Forsythe, 1989). As 65% of the studied population were older than 40 years and 95% were older than 15 years, it is highly unlikely that afebrile seizures or epilepsy related to FS would develop later. One family member also presented afebrile seizures (II.5, family 2). This patient had two brief generalized tonic–clonic seizures. They did not recur and did not necessitate any further treatment, and the subject remained with normal EEG and normal MRI. The incidence of afebrile seizures in affected patients was therefore 3% (one out of 29) (confidence interval 0.09–17.76). The incidence of generalized tonic–clonic seizures in the general population (0.5%) is within this interval (Hauser et al., 1996) and the difference is not significant. We decided therefore to assign to this subject the phenotype of simple FS. The characteristics of our population were consistent with the criteria of simple FS (Nelson and Ellenberg, 1981). One patient in each family was considered to be a phenocopy (IV.8 in family 1 and III.6 in family 2). They presented a single hypotonic FS. It is notoriously difficult to differentiate a brief hypotonic episode from non-convulsive events in infancy.
The phenotype of the present families differs from those reported in genetic studies on familial FS and GEFS+. In the family with linkage to FEB1 on 8q (Wallace et al., 1996), 10 affected individuals presented a single FS, and three of them (30%) later developed afebrile seizures. Later afebrile seizures were also reported by Nakayama et al. (2000) in one of the families with linkage to FEB4 on 5q14. This locus was identified in a non-parametric study of 47 small families, the largest of which comprised three out of seven (40%) affected members who developed afebrile seizures following FS. In the family with linkage to FEB2 on 19p13.3 (Johnson et al., 1998), none of the family members developed afebrile seizures but the FS were rather long (average duration 15 min), and probably correspond to complex FS (Nelson and Ellenberg, 1981). In another family with linkage to the same locus (Kugler et al., 1998), 43% of the affected members presented long-lasting complex FS with no afebrile seizures. The phenotype associated with 19p13.3 could therefore be considered as pure familial complex FS. In the family with linkage to FEB3 on 2q23–q24 (Peiffer et al., 1999), eight out of 21 members with FS also presented afebrile seizures before the age of 10 years. The mean number of FS in the subgroup who later developed afebrile seizures was 10, higher than the usual number of recurrences in simple FS since only 9% of affected children have three or more FS (Berg et al., 1990). This family resembled families with GEFS+, since FS were associated with afebrile generalized seizures. However, the FS stopped shortly before the age of 6 years, in contrast to the usual persistence of FS beyond the age of 6 years in GEFS+ (Scheffer and Berkovic, 1997). Interestingly, three families with GEFS+ (GEFS+2) were linked to the same locus on 2q23–q24 (Baulac et al., 1999; Moulard et al., 1999; Lopes-Cendes et al., 2000), and mutations were later shown to affect the same gene, SCN1A, in two of these three families (Escayg et al., 2000). Other mutations have been identified in the SCN1A gene (Escayg et al., 2001; Wallace et al., 2001b) and in the closely linked SCN2A gene (Sugawara et al., 2001). To date, no mutation in these genes has been reported in the family with linkage to FEB3 (Peiffer et al., 1999).
On the basis of the previously reported and the present data, some phenotype–genotype correlation could be made in patients with FS. The genes on chromosomes 2q, 19q and 5q31–q33 are responsible for GEFS+, and the loci on chromosome 8q (FEB1) and 5q14–q15 (FEB4) map for FS associated with afebrile seizures but without the other criteria for GEFS+. FEB3 on 2q23–q24 seems to be related to the GEFS+ group. The locus on 19p (FEB2) appears to be associated with pure, but complex FS, whereas the phenotype associated with the locus on 6q, that we describe here, seems to be that of pure and simple FS. In addition, the absence of linkage in the third family supports the existence of genetic heterogeneity in the AD form of pure simple FS.
Such genotype–phenotype correlation could have a major impact on the prognosis of the disease and will help to elucidate the underlying mechanism. The study of some GEFS+ families established a link between FS+ and severe myoclonic epilepsy in infancy (Dravet syndrome), one of the most severe epilepsy syndromes of early infancy (Singh et al., 2001). This intractable epilepsy associates onset in the first year of life with both febrile and afebrile, generalized or focal seizures, followed later by myoclonic jerks and cognitive and motor impairment (Dravet et al., 1992). The study of the SCN1A gene responsible for GEFS+ showed that truncating mutations in this gene cause Dravet syndrome (Claes et al., 2001; Sugawara et al., 2002). Recently, a new mutation in the GABRG2 gene, also reported in GEFS+, was identified in a family with GEFS+ in which one patient had a phenotype consistent with Dravet syndrome (Harkin et al., 2002). Similarly, studies have shown that the distinction between simple and complex FS has prognostic value since only the latter carries the risk of later onset of temporal lobe epilepsy. (Abou-Khalil et al., 1993). It should therefore be of major interest for the sake of early diagnosis and therapeutic decision in severe conditions such as Dravet syndrome to distinguish genes for GEFS+ from those for pure FS, and those for pure simple FS from those for pure complex FS.
To date, 22 genes and a number of expressed sequence tags have been mapped to the candidate region on 6q22–q24. Since FS are due to a discharge in cortical neurones and consist of an age-dependent disorder, the selection of candidate genes was based on their expression in neurones and their possible involvement in cell excitability and/or plasticity. We selected five potential candidate genes for sequencing because of their putative function, the genes coding for AKAP18, syntaxin 7, PNR, GPR58 and GPR57.
AKAP18 protein is expressed in many tissues including the cerebral cortex. It targets the cAMP-dependent protein kinase to the plasma membrane, and permits its functional coupling with L-type calcium channels (Fraser et al., 1998), which are voltage-gated channels controlling a variety of neuronal functions implicated in epileptogenesis (Empson and Jefferys, 2001). Since ion channels have already been implicated in several human idiopathic epilepsies and rodent models of epilepsy present mutations in calcium channel subunits (Burgess and Noebels, 2000), the AKAP18 protein was suspected to play a role in epileptogenesis. However, we did not find any causative mutation when the seven coding exons, the exon–intron boundaries and the 300 bp upstream from the first coding ATG were sequenced.
After the exclusion of this highly attractive candidate gene, we sequenced the syntaxin 7 gene. This protein is required for the docking of synaptic vesicles and may be involved in manifestations of synaptic plasticity (Kamphuis et al., 1985; Hinz et al., 2001). No mutation was found in this gene, but two SNPs were identified in the 5'-non-translated terminal portion of the mRNA.
The three genes PNR, GPR58 and GPR57, which encode homologous G protein receptors, were also sequenced. These proteins present a highly conserved structure, with significant homology to the 
2 adrenergic receptor and to the D2 dopamine receptor (Zeng et al., 1998; Lee et al., 2000). This homology with CNS receptors made them interesting candidates. No mutation causative of the disease was found, but we identified a new SNP in the 5'-untranslated region of the gene of GPR57, and an SNP in PNR that did not change the amino acid sequence.
In conclusion, we have mapped a new locus for FS on chromosome 6q22–q24 in two families presenting a phenotype of pure simple FS. This phenotype is markedly homogeneous and differs from those previously reported for FS or GEFS+ with known loci. In addition, the exclusion of the locus in a third family with the same phenotype strongly supports genetic heterogeneity of the AD form of pure simple FS. Although an extensive search for causative mutations was performed in the candidate genes, no mutation was identified. Better knowledge of the human genome and ongoing genetic study of the region should permit identification of a gene underlying simple FS.
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We wish to thank all members of the families for their participation in this study, Drs M. Ruberg and C. Chiron for critically reading the manuscript, and Dr G. Stevanin for his valuable advice. This study was supported by grants from the Association pour la Recherche sur la Génétique des Epilepsies (ARGE), funded by Sanofi-Synthelabo, Généthon, the Association pour le Développement de la Recherche sur les Maladies Neurologiques et Psychiatriques (ADRMGNP) and the Association Française contre les Myopathies (AFM). A.H. was supported by ARGE, and R.N. by the Ligue Française Contre les Epilepsies (LFCE).
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