Increased sensitivity of the neuronal nicotinic receptor alpha 2 subunit causes familial epilepsy with nocturnal wandering and ictal fear

Abstract

Sleep has traditionally been recognized as a precipitating factor for some forms of epilepsy, although differential diagnosis between some seizure types and parasomnias may be difficult. Autosomal dominant frontal lobe epilepsy is characterized by nocturnal seizures with hyperkinetic automatisms and poorly organized stereotyped movements and has been associated with mutations of the alpha 4 and beta 2 subunits of the neuronal nicotinic acetylcholine receptor. We performed a clinical and molecular genetic study of a large pedigree segregating sleep-related epilepsy in which seizures are associated with fear sensation, tongue movements, and nocturnal wandering, closely resembling nightmares and sleep walking. We identified a new genetic locus for familial sleep-related focal epilepsy on chromosome 8p12.3-8q12.3. By sequencing the positional candidate neuronal cholinergic receptor alpha 2 subunit gene (CHRNA2), we detected a heterozygous missense mutation, I279N, in the first transmembrane domain that is crucial for receptor function. Whole-cell recordings of transiently transfected HEK293 cells expressing either the mutant or the wild-type receptor showed that the new CHRNA2 mutation markedly increases the receptor sensitivity to acetylcholine, therefore indicating that the nicotinic alpha 2 subunit alteration is the underlying cause. CHRNA2 is the third neuronal cholinergic receptor gene to be associated with familial sleep-related epilepsies. Compared with the CHRNA4 and CHRNB2 mutations reported elsewhere, CHRNA2 mutations cause a more complex and finalized ictal behavior.

Figure 1.

Pedigree structure and disease-haplotype segregation. Individuals available for typing are indicated by the generated genotype data. The arrow indicates the proband. Blackened symbols denote individuals affected by epilepsy with nocturnal wandering and ictal fear; unblackened symbols denote unaffected individuals. Circles and squares indicate females and males, respectively. Marker order and distances in cM are given in table 2. The disease haplotype is boxed.

Figure 2.

CHRNA2 mutation in familial epilepsy with nocturnal wandering and ictal fear. A, Electropherograms of CHRNA2 gene sequence spanning the missense mutation in the proband (top) and a control (bottom). The arrow indicates the 836T→A transversion. B, DHPLC elution profile of the heterozygous proband (left) and a wild-type (wt) control (right). C, Evolutionary conservation of different α-subunit I279 residues among different species (UniProtKB/Swiss-Prot).

Figure 3.

Electrophysiological properties of wild-type and mutant α2/β4 nicotinic receptors. Representative current traces evoked by pulses of the indicated acetylcholine concentration for either α2/β4 (A) or α2^I279N/β4 (B) nAChRs. Note the larger response in α2^I279N/β4 receptors at nonsaturating doses of agonist. The maximal response was always tested at regular intervals during the experiment, to exclude artifacts due to channel rundown. Similar results were obtained with nicotine. No significant difference was observed in the current density of wild-type (α2/β4), mutant (α2^I279N/β4), and heterozygous (α2+α2^I279N/β4) receptors, tested in the presence of 100 μM agonist. C and D, Dose-response curves to acetylcholine (C) and nicotine (D). Data points are average peak currents obtained at the indicated ligand concentration and normalized to the current obtained in the presence of 100 μM agonist (n=6 for each concentration point). The experimental points were fitted with a single-term empirical Hill equation, providing the following estimates for EC₅₀: acetylcholine, 3.94 ± 0.31 μM (for α2/β4) and 0.64 ± 0.14 μM (for α2^I279N/β4); nicotine, 2.84 ± 0.16 μM (for α2/β4), 0.52 ± 0.07 μM (for α2^I279N/β4), and 1.66 ± 0.25 μM (for α2+α2^I279N/β4).

Figure 4.

Expression pattern of CHRNA2, CHRNA4, and CHRNB2 nicotinic subunits in human brain. For quantitative analysis of different α and β subunits' expression in the human brain, we optimized a semiquantitative RT-PCR method coupled with Southern blot analysis. With this analysis, a relatively stronger expression was observed in the thalamus for CHRNA2 mRNA, whereas mild expression was seen in the other brain regions analyzed for both CHRNA2 and CHRNA4. CHRNB2 showed a homogeneous level of expression throughout the different brain areas, with the exception of the temporal cortex. GAPDH was used as positive control. RT- = negative control.