A novel SCN1A mutation associated with generalized epilepsy with febrile seizures plus--and prevalence of variants in patients with epilepsy

Abstract

We recently described mutations of the neuronal sodium-channel alpha-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.

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.

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.

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.

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.