Abstract
We recently described mutations of the neuronal sodium-channel α-subunit gene, SCN1A, on chromosome 2q24 in two families with generalized epilepsy with febrile seizures plus (GEFS+) type 2. To assess the contribution that SCN1A makes to other types of epilepsy, 226 patients with either juvenile myoclonic epilepsy, absence epilepsy, or febrile convulsions were screened by conformation-sensitive gel electrophoresis and manual sequencing of variants; the sample included 165 probands from multiplex families and 61 sporadic cases. The novel mutation W1204R was identified in a family with GEFS+. Seven other coding changes were observed; three of these are potential disease-causing mutations. Two common haplotypes, with frequencies of .67 and .33, were defined by five single-nucleotide polymorphisms (SNPs) spanning a 14-kb region of linkage disequilibrium. An SNP located 18 bp upstream of the splice-acceptor site for exon 3 was observed in 7 of the 226 patients but was not present in 185 controls, suggesting possible association with a disease mutation. This work has confirmed the role of SCN1A in GEFS+, by identification of a novel mutation in a previously undescribed family. Although a few candidate disease alleles were identified, the patient survey suggests that SCN1A is not a major contributor to idiopathic generalized epilepsy. The SCN1A haplotypes and SNPs identified here will be useful in future association and linkage studies.
Introduction
Recent progress in the positional cloning of genes responsible for monogenic epilepsies has demonstrated the involvement of ion-channel genes in several rare disorders (McNamara 1999). Sodium-channel mutations have been demonstrated for the epilepsy syndrome GEFS+ (generalized epilepsy with febrile seizures plus) (MIM 604236 and MIM 604233). Individuals with GEFS+ are characterized by febrile seizures that persist beyond age 6 years and by heterogeneous afebrile seizures that may include tonic-clonic, myoclonic, atonic, and absence seizures (Scheffer and Berkovic 1997). A mutation in the voltage-gated sodium-channel β-subunit SCN1B was identified in 1998 in a family with GEFS+ type 1 linked to chromosome 19p13 (Wallace et al. 1998). We described mutations of the neuronal sodium-channel α-subunit SCN1A in two families with GEFS+ type 2 linked to chromosome 2q24 (Escayg et al. 2000b). In the mouse, mutation of the related sodium-channel SCN2A also results in a dominantly inherited, progressive seizure disorder (Kearney et al. 2001).
SCN1A is one of the four voltage-gated sodium channels that are widely expressed in neurons of the CNS and peripheral nervous system (PNS) (Plummer and Meisler 1999). The differences in firing patterns and excitability of different classes of neurons are influenced by their specific complement of voltage-gated sodium, potassium, and calcium channels, as well as by the extent of posttranslational modifications of the channel proteins. Subtle differences in channel properties can have significant physiological effects and may underlie individual variation in neurological function (Kohrman et al. 1996; Bulman 1997).
The observation that patients with GEFS+ experience several types of seizures has suggested that genetic variation in SCN1A could contribute to other common forms of idiopathic generalized epilepsy. To test this possibility, we screened 226 patients with either juvenile myoclonic epilepsy (JME), childhood or juvenile absence epilepsy (CAE and JAE, respectively), febrile convulsions (FC), or generalized tonic-clonic seizures (GTCS). Twenty SCN1A variants were identified, including 8 amino acid substitutions and 12 noncoding variants. One amino acid substitution, W1204R, cosegregated with GEFS+ in a previously undescribed family, making this the third SCN1A mutation associated with GEFS+. Several variants identified in sporadic cases require further testing as possible susceptibility factors.
Subjects and Methods
Subjects
DNA was obtained from index patients from 165 unrelated families with idiopathic generalized epilepsy (Haug et al. 2000; Sander et al. 2000). These patients included 72 with JME, 83 patients with either CAE or JAE, 4 with random GTCS on awakening, and 6 with a history of FC, who had at least one first-degree relative who was affected. Sixty-one sporadic cases of idiopathic generalized epilepsy (IGE) were also included in the study. Diagnostic classification followed the proposal of the Commission on Classification and Terminology of the International League Against Epilepsy (1989). The diagnosis of JME was based on repetitive myoclonic jerks, in arms and shoulders, on awakening. Diagnostic criteria for idiopathic absence epilepsy were a brief loss of consciousness with pyknoleptic daily clusters of absence seizures. The diagnosis for FC followed the proposal of the National Institutes of Health Consensus Development Panel (Consensus Statement 1980). Electroencephalography (EEG) recordings were obtained from most patients and demonstrated a normal background activity. Paroxysmal generalized spike-and-wave discharges were present in the patients with IGE. Each SCN1A variant observed in patients was assayed in 96–268 ethnically matched, unrelated controls who had been described elsewhere (Haug et al. 2000; Sander et al. 2000). Written consent was obtained from all participants, and the study was approved by the institutional ethics committees.
Mutation Detection
The 26 exons of SCN1A were individually amplified from genomic DNA in 28 PCR reactions using primers based on intronic sequence (Escayg et al. 2000b). PCR amplification was performed at 32 cycles of 1 min at 94°C, 45 s at 60°C, and 1 min at 72°C. PCR products were incubated at 99°C for 10 min, followed by 35 min at 68°C to facilitate heteroduplex formation, and were analyzed by conformation-sensitive gel electrophoresis (CSGE) with visualization by ethidium bromide staining as described elsewhere (Escayg et al. 2000a). PCR products with mobility variants were gel purified by means of the Qiaex II gel extraction kit (Qiagen) and were manually sequenced by means of the ThermoSequenase sequencing kit (USB).
Statistical Analysis
Allele and genotype frequencies in the patients and in the control groups were compared by either χ2 analysis or Fisher's exact test. A two-tailed type I error rate of 5% was chosen for the analyses. A correction for multiple testing was performed according to the Bonferroni procedure.
Results
W1204R: Cosegregation with GEFS+
Multiple members of a previously undescribed family from Germany had a history of febrile and afebrile seizures consistent with the classification of GEFS+ (Scheffer and Berkovic 1997). The four-generation pedigree is presented in figure 1. Six individuals in this family were classified as affected (table 1). Individuals III-1, IV-1, and IV-4 reported a history of childhood febrile and afebrile seizures. Individual II-3 had febrile seizures until age 5 years but did not have afebrile seizures. The proband (IV-3) was diagnosed with severe myoclonic seizures and did not have a history of febrile seizures. A detailed family history indicated that individual II-2 had epilepsy as an adult. The disease status of individual I-2 is unclear. EEG recordings and results of magnetic-resonance imaging (MRI) were obtained for selected individuals (table 1). Normal EEG patterns were recorded from the unaffected individuals: III-2, IV-2, and IV-5. Results of neurological examination were normal in all individuals except IV-1, who had mild intellectual disability and severe seizures that resulted in permanent institutional care. The clinical features of the affected members of this family are summarized in table 1.
Figure 1.
Mutation W1204R, cosegregating with disease in a family with GEFS+. A, Family with GEFS+, with 13 individuals from four generations. Six individuals were classified as affected. DNA was obtained from five affected and three unaffected family members. CSGE analysis of exon 18 of SCN1A identified a unique pattern in the proband (arrow). The same pattern was observed in the other affected individuals. B, Sequence analysis revealing a T→C transition (arrow) resulting in amino acid substitution W1204R.
Table 1.
Clinical Features of Affected Individuals from the Family with GEFS+[Note]
|
Age(years) |
Clinical Results |
||||
| Affected Individual | At Onset of FC | At Last FC | Type of Afebrile Seizure | EEG | MRI |
| II-2 | None | … | Unknown | Not available | Not available |
| II-3 | .5 | 5 | None | Not available | Not available |
| III-1 | 1 | 8 | GTCS | Normal | Not available |
| IV-1 | 2 | 7 | Myoclonic-astatic | Sharp slow waves, spike waves, polyspike wave | Normal |
| IV-3 | None | … | Myoclonic | Polyspike wave | Normal |
| IV-4 | 1 | 6 | GTCS | Irregular spike waves | Normal |
Note.— The family pedigree is shown in figure 1.
DNA samples from the eight living family members were screened for SCN1A mutations. The abnormal CSGE conformer of exon 18 was observed in the proband and in the other four affected individuals (fig. 1A). Sequence analysis of the PCR product revealed a T→C transition that was responsible for the amino acid substitution W1204R (fig. 1B). The three unaffected family members did not carry the mutation. This nonconservative amino acid substitution was not observed in 268 unaffected controls (536 chromosomes). W1204 is located in cytoplasmic loop 2, within 10 amino acids of the transmembrane segment D3S1 (fig. 2). The functional importance of this residue is demonstrated by evolutionary conservation in 12 of 13 mammalian, vertebrate, and invertebrate sodium channels (fig. 3).
Figure 2.
Amino acid substitutions in SCN1A that are identified in the present study. The sodium channel is composed of four internally homologous domains, D1–D4, each containing six transmembrane segments. The eight amino acid substitutions identified in the patient population are located in cytoplasmic loops 1 and 2. The only change that affects an evolutionarily conserved residue is W1204, located close to the cytoplasmic surface of transmembrane domain D3. Exon/intron boundaries are marked by horizontal bars; exon designations are in italics.
Figure 3.
Evolutionary conservation of tryptophan residue 1204. W1204 is located 10 residues upstream of transmembrane segment D3S1. This residue is highly conserved in vertebrate and invertebrate channels, including all of the other members of the mammalian gene family. The SCN1A sequence is that reported by Escayg et al. (2000b); GenBank accession numbers, from top to bottom, are M94055, AJ251507, M81758, M77235, AB027567.1, X82835, AF117907.1, AF188679, D37977, M22252, M32078, and AF047380. Electric eel = Electrophorus electricus; Drosophila = D. melanogaster; jellyfish = Polyorchis penicillatus.
Mutation Screening
Two hundred twenty-five additional patients were screened by exon amplification and CSGE. Nineteen additional variants were detected, including 18 single-nucleotide polymorphisms (SNPs) and 1 dinucleotide deletion (table 2); 7 of these SNPs encoded novel amino acid substitutions. Allele frequencies for six coding SNPs (cSNPs) were <1% and did not differ significantly between patient and control populations (P>.05, by Fisher's exact test). One coding variant, T1067A, was polymorphic in patients and controls, with an allele frequency of .33 in both groups.
Table 2.
Single-Nucleotide Substitutions in Human SCN1A
|
No. (Frequency) of Minor Allele in |
|||||
| Location | cDNA Nucleotidea(Amino Acid Change) | Major/Minor SNP Alleles | 226 Patients | 96 Controlsb | Haplotype of Minor Allelec |
| Intron 2 | 384−20 | G/C | 7 (.02) | 0 [185] | 2 |
| Intron 6 | 965−21 | T/C | 151 (.33) | 64 (.33) | 2 |
| Intron 7 | 1028+21 | C/T | …d | … | … |
| Exon 8 | 1131 | A/C | 2 (.004) | 4 (.02) [194] | 1 |
| Intron 8 | 1171−9,10 | TT/ΔTT | 16 (.04) | 7 (.04) | 1 |
| Exon 9 | 1212 | G/A | 151 (.33) | 64 (.33) | 2 |
| Exon 10 | 1625 (R542Q) | G/A | 1 (.002) | 0 | 1 |
| Exon 11 | 1811 (R604H) | G/A | 2 (.004) | 0 | 1 |
| Exon 12 | 2095 (V699I) | G/A | 1 (.002) | 0 | 1 |
| Exon 13 | 2292 | C/T | 151 (.33) | 64 (.33) | 2 |
| Intron 13 | 2416−37 | C/A | 151 (.33) | 64 (.33) | 2 |
| Intron 13 | 2416−72 | A/G | …d | … | … |
| Exon 16 | 3199 (T1067A) | A/G | 151 (.33) | 64 (.33) | 2 |
| Exon 16 | 3241 (G1081R) | G/A | 1 (.002) | 0 | … |
| Exon 17 | 3481 (A1161T) | G/A | 1 (.002) | 0 | 1 |
| Exon 17 | 3521 (T1174S) | C/G | 1 (.002) | 1 (.005) | … |
| Exon 18 | 3610 (W1204R) | T/C | 1 (.002) | 0 [268] | 1 |
| Intron 23 | 4476+33 | G/A | 4 (.009) | 2 (.01) | 2 |
| Exon 25 | 4731 | T/C | 1 (.002) | 1 (.005) | … |
| Exon 26 | 5418 | G/A | 8 (.02) | 3 (.005) [183] | 2 |
Three coding variants identified in familial cases did not cosegregate with the disease. V699I in exon 12 was identified in the proband of a family with JAE and FC. Other affected family members did not carry the mutation. R604H in exon 11 was identified in two probands from unrelated families with JME. The mutant allele in one of the two families carries the substitution A1161T, in exon 17, which cosegregates with R604H (table 2). R604H disrupts a protein kinase A (PKA) consensus site that is conserved in the four major sodium channels of the CNS (fig. 4A). Analysis of DNA from additional family members demonstrated multiple recombinations with the disease, including affected individuals in both families who did not carry the mutation (data not shown).
Figure 4.
SCN1A variants R604H and R542Q, which disrupt potential sites of protein phosphorylation. A, Consensus protein kinase A site in exon 11 (underlined), which is conserved in several sodium channels and is disrupted by the substitution R604H. B, Consensus tyrosine kinase phosphorylation site in exon 10 (underlined), which is present only in SCN1A and is disrupted by the substitution R542Q.
Three cSNPs were identified in sporadic cases of epilepsy for which family records were not available. R542Q was found in a patient with JME and is located in a consensus tyrosine kinase phosphorylation site (fig. 4B). G1081R and T1174S were identified in patients with JAE and JME, respectively, and T1174S was also observed in one control (table 2). Since family members were not available for analysis, the role that these amino acid substitutions play in seizure susceptibility could not be evaluated and warrants further analysis. The relationship between the eight coding variants and disease status is summarized in table 3.
Table 3.
Coding Variation in SCN1A, and Relationship to Disease
| Substitution | Domain | Type of Epilepsy | Genetic Observation(s) |
| W1204R | Loop 2 | GEFS+ type 2 | Cosegregates with disease |
| R604H | Loop 1 | Familial JME (two families) | Discordant; absent in several affected family members |
| V699I | Loop 1 | Familial JAE with FC | Discordant; absent in affected parent and affected sib |
| A1161T | Loop 2 | Familial JME | Discordant; absent in several affected family members |
| R542Q | Loop 1 | Sporadic JME | Family not available |
| G1081R | Loop 2 | Sporadic JAE | Family not available |
| T1174S | Loop 2 | Sporadic JME | Family not available |
| T1067A | Loop 2 | All | Polymorphic in patients and controls |
Noncoding SNPs
Seven noncoding SNPs were present at polymorphic frequencies (i.e., >1% ) (table 2), and five were present at lower frequencies. Allele frequencies did not differ significantly between the 226 patients and 96–194 ethnically matched controls, with one exception: the SNP in intron 2 appeared to be more frequent in patients (7/226) than in controls (0/185) (P=.019, by Fisher's exact test [two tailed]), but the difference between the patients and controls was not significant after correction for multiple testing. The patients with this variant were diagnosed as having JAE (4 cases), JME (2 cases), or CAE (1 case). Additional studies, in independent samples, will be required in order to determine whether this SNP is associated with predisposition to epilepsy.
SCN1A Haplotypes
Two major haplotypes are defined by the consistent association of five SNPs in SCN1A in 322 unrelated individuals, including patients and controls (table 4). Haplotypes 1 and 2 exhibit frequencies of .67 and .33 in this northern-European population. The genotype frequencies are in agreement with Hardy-Weinberg expectations. The haplotypes were confirmed by cosegregation in meiosis (data not shown). Minor haplotypes are defined by the rare SNPs, which are all restricted to either haplotype 1 or haplotype 2 (table 2).
Table 4.
Two Major SCN1A Haplotypes[Note]
|
SNP at |
|||||
| Haplotype (No. [Frequency]) | Intron 6: 965-21 | Exon 9: 1212 | Exon 13: 2292 | Intron 13: 2416−37 | Exon 16: 3199 |
| 1 (429 [.67]) | T | G | C | C | A |
| 2 (215 [.33]) | C | A | T | A | G |
Note.— Data are for 322 unrelated individuals. Genotype frequencies were consistent with Hardy-Weinberg equilibrium.
The SNPs defining the haplotypes span a 14-kb region of linkage disequilibrium (LD) in SCN1A (fig. 5). The positions of related sodium-channel genes in the cluster on chromosome 2q24 are shown on the clone contig, which was generated by BLAST alignment of cDNA and genomic sequences in the GenBank high-throughput genomic-sequence database. In the cluster, SCN1A and SCN2A are orientated tail-to-tail, with their C-terminal coding exons separated by 600 kb. SCN2A and SCN3A are orientated head-to-head separated by 100 kb that could contain shared regulatory sequences. The PNS-channel SCN9A is located 120 kb distal to SCN1A. The microsatellite D2S2330, located between SCN1A and SCN2A, is closely linked to the inherited epilepsy locus FEB3 (Peiffer et al. 1999), indicating that SCN1A and SCN2A are strong candidates genes for this disorder.
Figure 5.
Physical map of sodium-channel gene cluster on chromosome 2q24. Arrows indicate direction of transcription of sodium-channel genes. The positions of polymorphic microsatellites are indicated. BLAST analysis of the GenBank high-throughput genomic-sequence database was used to construct the 1.6-Mb contig containing SCN1A, SCN2A, SCN3A, and SCN9A. The GenBank accession number of each bacterial artificial chromosome is indicated. LD = 14-kb region of LD in SCN1A.
Discussion
This is the first systematic study of population variation in a neuronal sodium-channel α-subunit gene. We have identified 20 variants of SCN1A, including 8 amino acid substitutions, in 226 patients with IGE subtypes and FC. The amino acid substitution W1204R has been found to cosegregate with GEFS+, making this the third SCN1A mutation associated with this disease (Escayg et al. 2000b). It is interesting that the proband in this family had JME, without any history of febrile seizures or other seizure types. Four other variants identified in this study are potentially associated with disease. The difference, in frequency of the SNP in intron 2, between patients (7/226) and controls (0/185) approached significance, and follow-up studies to detect possible disease association with this variant are warranted. The three amino acid substitutions identified in sporadic cases of epilepsy could also be predisposing mutations and will require further analysis.
The 226 patients were screened for all 26 exons of SCN1A, and variants were found in 15 exons (table 2). The 96 controls were then screened for these 15 exons only. No novel variants were found in the controls. Rare coding variants of SCN1A were present in 8/226 patients and 1/96 controls. As more data accumulate, it will be interesting to determine whether rare coding variants of sodium channels are consistently more prevalent in patient populations.
All of the coding variants identified in this study are located in the cytoplasmic loops of the sodium-channel protein (fig. 2). The voltage-gated sodium channels contain four domains (D1–D4), each with six transmembrane segments. The transmembrane segments are among the most highly conserved protein domains known. The large cytoplasmic loops are less well conserved among different members of the sodium-channel family, but they are well conserved among mammalian orthologues. The two cytoplasmic loops of human SCN1A exhibit amino acid sequence identity of 98% (330/337) and 96% (213/221) with SCN1A from rat or mouse, suggesting functional constraints throughout these domains. In the cytoplasmic loops, the only functional motifs that have been recognized thus far are phosphorylation sites (Smith and Goldin 1997; Ratcliffe et al. 2000). Phosphorylation of five protein kinase A sites in loop 1 of SCN2A can attenuate sodium current amplitude (Smith and Goldin 1997). The PKA site corresponding to R604H had a minor effect on current amplitude in a previous study (Smith and Goldin 1997) and did not cosegregate with the seizure disorder in two families with JME. Sodium channels may be modulated by tyrosine phosphorylation (Ratcliffe et al. 2000), and the variant R542Q disrupts a predicted tyrosine kinase site in exon 10 of SCN1A. Another possible function of the cytoplasmic loops is channel clustering and subcellular localization (Sampo et al. 2000), but specific residues involved in these processes have not been identified.
The amino acid substitution T1067A was observed at a frequency of .33 both in patients and in controls and is clearly a nonpathogenic polymorphism. The substitutions G1081R and T1174S were observed at very low frequency in sporadic cases, and T1174S was also present in one control individual. Understanding the significance of the coding variants in the cytoplasmic loops will depend on further elucidation of the functions of these domains.
The two previously identified SCN1A mutations in GEFS+ were located in the voltage-responsive S4 segments (Escayg et al. 2000b). The GEFS+ mutation described here, W1204R, is located in the second cytoplasmic loop, 10 residues distant from the cytoplasmic surface of the transmembrane segment D3S1. The evolutionary conservation of W1204 is strong evidence for an important function for this residue. Many disease mutations of the muscle and cardiac sodium channels SCN4A and SCN5A are located in similar sites near the cytoplasmic surfaces of transmembrane segments (Bulman 1997). There are evidently functional constraints on other residues, between W1204 and the start of D3S1, since they are highly conserved in the 11 vertebrate and 2 invertebrate sodium channels compared in figure 3. The number of channels with identical amino acids are as follows: W1204, 12/13; W1205, 11/13; R1208, 13/13;, R/K1209, 13/13; and T1210, 10/13. Functional analysis of the W1204R mutation in Xenopus oocytes and mammalian cells will be important for understanding its effect on channel activity.
Four GEFS+ mutations are now known—three in SCN1A and one in the β-subunit SCN1B. If the SCN1B mutation is a loss-of-function mutation, as has been reported elsewhere (Wallace et al. 1998), it would result in slower inactivation of the associated α-subunit, leading to increased persistent current and neuronal hyperexcitability. Persistent sodium currents are thought to underlie ictal epileptiform activity in the brain, and SCN1A is expressed in the cell bodies of many neurons that generate persistent currents. Thus, a common electrophysiological mechanism may underlie epilepsies caused by mutations in SCN1A and SCN1B. Structure/function analysis in heterologous expression systems and in transgenic mice will be required in order to elucidate the precise kinetic and physiological consequences of these mutations.
The present study confirms the role that SCN1A plays in the etiology of GEFS+. The phenotypic variability in families with GEFS+ raises the possibility that the incidence of GEFS+ has been underestimated, since, in the absence of a complete family medical history, individuals with GEFS+ may be misclassified as manifesting one of the more common syndromes of IGE. SCN1A should also be considered as a candidate for other inherited epilepsy syndromes associated with FC, which were not tested in the present study. When functional assays for functional domains in the cytoplasmic loops of the sodium channels are developed, it will be of great interest to determine whether the variants described here influence channel function. The SNPs and the physical map of the sodium-channel gene cluster will be useful for testing the contributions that these genes make to other inherited disorders. Studies of allelic mutations of SCN8A in the mouse have demonstrated that mutations of a single sodium channel can generate a variety of neurological symptoms (e.g., ataxia, tremor, a dystonic movement disorder, and paralysis) (Meisler et al. 1997, and in press). We anticipate that mutations in the human sodium channels will be found to contribute to a broad spectrum of inherited disease.
Electronic-Database Information
Accession numbers and URLs for data in this article are as follows:
- BLAST, http://www.ncbi.nlm.nih.gov/BLAST/ (for alignment of cDNA and genomic sequences)
- GenBank Overview, http://www.ncbi.nlm.nih.gov/Genbank/GenbankOverview.html (for 1204 residues in human SCN2A [accession number M94055], human SCN3A [AJ251507], SCN4A [accession number M81758], human SCN5A [accession number M77235], human SCN8A [accession number AB027567.1], human SCN9A [accession number X82835], human SCN10A [accession number AF117907.1], human SCN11A [accession number AF188679], Fugu rubripes [accession number D37977], Electrophorus electricus [accession number M22252], Drosophila melanogaster [accession number M32078], and Polyorchis penicillatus [accession number AF047380])
- Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/Omim (for GEFS+ [MIM 604236] and GEFS+ type 2 [MIM 604236 and MIM 604233])
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