Abstracts

Rationale: Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is an epilepsy syndrome characterized by clusters of motor seizures arising from sleep, usually occur­ring in individuals of normal intellect (Scheffer et al., 1995 Brain 118:61-73). ADNFLE has been causally associated with neuronal acetylcholine receptor (nAChR) subunit alpha-2 (CHRNA2) dysfunction in only one family, in which a gain of function mutation was demonstrated (Aridon et al., 2006, Am J Hum Genet 79:342-350). We assessed the mutation frequency in CHRNA2 in a large cohort of patients with NFLE. Upon finding a novel mutation in CHRNA2 in a large Italian family, we tested in vitro its functional effects. Methods: We sequenced the exons covering all the coding regions of CHRNA2 (Entrez Gene ID: 1135, accession number: NM_000742.3) and their flanking intronic regions in 150 patients with NFLE (73 sporadic; 77 familial) in most of whom diagnosis was validated by EEG recording of seizures. In all patients, mutation screening of CHRNA4 and CHRNB2 was negative. We measured whole-cell currents in HEK cells in both wild type and mutant α2β4 and α2β2 nAChR subtypes upon stimulation with nicotine at different concentrations. Results: We found a c.889A >T (p.Ile297Phe) mutation in 1 out of 150 (≈ 0.6%) patients (1.2% of familial cases). Genetic segregation and video-EEG studies confirmed that all affected family members, exhibiting epilepsy with paroxysmal arousals and nocturnal paroxysmal dystonia-like attacks, carried the p.Ile297Phe mutation. Oxcarbazepine treatment was effective in all. Functional studies demonstrated that the whole-cell current density was reduced to about 40% in heterozygosity (α2β4: n=27; p<0.01; α2β2: n=5; p<0.05) and to 0% in homozygosity (α2β4: n=30) (Figure 1), with minor effects on channel permeability and sensitivity to the agonist (average V_rev: -1.1 ± 3 mV for the WT (n = 9); -4.2 ± 2.1 for the heterozygote (n = 5); not statistically significant differences). These results indicate that the p.Ile297Phe mutation decreases the functional expression of the heteromeric nAChR isoforms containing the mutant α2 subunit. Conclusions: Although infrequent, CHRNA2 mutations have a causative role in ADNFLE. Loss of function, not just gain of function, of the channel has pathogenic effects. CHRNA2 mutation screening should be considered in patients with ADNFLE.