Association between the α_1a calcium channel gene CACNA1A and idiopathic generalized epilepsy

Idiopathic generalized epilepsy (IGE) is a common disorder with a strong genetic component. For common forms of IGE, the pattern of inheritance is complex, probably resulting from the action of a few or many genes of small to moderate effect. In this study we used an association strategy to investigate whether genetic variation of the α_1A subunit of the voltage-gated calcium channel gene (CACNA1A) influences risk for IGE. The α_1A subunit is the pore-forming channel in P/Q-type channels that plays an important role in neurotransmitter release.1 Mutations in the mouse homologue cause seizures and ataxia in tottering and leaner mice.2 In humans, mutations in CACNA1A result in episodic ataxia type 2, spinocerebellar ataxia type 6, and familial hemiplegic migraine.3 In an earlier study of only 55 probands, no association was reported between a CAG repeat polymorphism in exon 47 and the common subtypes of IGE.4

Although IGE includes many syndromes, their phenotypes overlap and more than one subtype is often observed within families, suggesting genes common to more than one subtype. We collected DNA from a cohort of Caucasian patients with IGE, unselected for subtype, at Kent and Canterbury Hospital. All probands had generalized spike and wave on EEG, normal background rhythm, and a clinical history consistent with IGE. Control subjects consisted of patients at the same hospital and the same ethnic group with no history of epilepsy or blackouts. We also collected DNA from both parents, when available, for within-family tests of association.

Results of this study are shown in the table. We analyzed four single nucleotide polymorphisms (SNP) in exons 6, 8, 16, and 20 (SNP6, -8, -16 and -20) and one simple sequence repeat polymorphism in intron 8 (D19S1150). Of these, SNP8 (OR, 1.8; 95% CI, 1.3 to 2.4; p = 0.00033) and D19S1150 (p = 0.032) showed evidence of allelic association. When corrected for multiple testing the association of SNP8 was still significant (Bonferroni-adjusted p = 0.0016). The D19S1150 association derives mainly from an excess of allele 6 in the probands compared with the control subjects (16.9% versus 9.6%, χ² = 8.28; df = 1; p = 0.004; OR, 1.9; 95% CI, 1.2 to 3.0). All genotype frequencies were within Hardy–Weinberg equilibrium. Power calculations showed that the sample size had 80% power at p = 0.05 to detect associations with SNP6 at an OR of 4.2, with SNP16 at an OR of 1.9, and with SNP20 at an OR of 2.1. Although SNP6 is too infrequent to be informative with our sample size, smaller gene effects would be detected in SNP16 and SNP20, but not as small as the gene effect associated with SNP8.

For the two associated markers, we controlled for the possible effects of population stratification by performing within-family analyses in the subset of probands with parental DNA. For SNP8, haplotype-based relative risk analysis demonstrated a significant difference between transmitted and untransmitted alleles (p = 0.044). Moreover, there was no difference between untransmitted and control allele frequencies (36.1% versus 36.8%) suggesting similar genetic background between patients and control subjects (see table). The transmission disequilibrium test showed that of 65 informative transmissions, 42 A-alleles were transmitted versus 23 untransmitted (p = 0.019; OR, 1.8). For D19S1150, neither haplotype relative risk nor transmission disequilibrium test analyses yielded significant results, although similar control and untransmitted allele frequencies again suggest matching genetic backgrounds between patients and control subjects. Because of lower sample size, the within-family studies had much less power than the case-control studies.

These results provide the first direct evidence that CACNA1A is involved in the etiology of IGE. SNP8 is a silent polymorphism and is unlikely to have a functional effect itself. The association with IGE is therefore likely to result from linkage disequilibrium between SNP8 and a nearby functional polymorphism. For most outbred populations, linkage disequilibrium between SNP is estimated to extend between 3 and 50 kb5,6⇓ and up to 1000 kb for simple sequence repeat polymorphisms.7 CACNA1A has not been completely sequenced, but the size of genomic region spanning the coding region has been estimated at 300 kb.3 Thus, the putative IGE susceptibility locus is likely to lie within 50 kb of exon 8. The CAG repeat polymorphism at the 3′ end of the gene that failed to show association4 is well outside this range.

Further work is now required to delineate the region most likely to contain the functional variant giving rise to this association. We are presently screening CACNA1A for mutations around SNP8 to define the region of interest more precisely. We are also engaged in detailed clinical characterization of patients in our IGE sample, which will enable us to ascertain whether these results are due to a small gene effect throughout the sample or a larger gene effect in a subset.