CHRNA2 and Nocturnal Frontal Lobe Epilepsy: Identification and Characterization of a Novel Loss of Function Mutation

Abstract

Mutations in genes coding for subunits of the neuronal nicotinic acetylcholine receptor (nAChR) have been involved in familial sleep-related hypermotor epilepsy (also named autosomal dominant nocturnal frontal lobe epilepsy, ADNFLE). Most of these mutations reside in CHRNA4 and CHRNB2 genes, coding for the α4 and β2 nAChR subunits, respectively. Two mutations with contrasting functional effects were also identified in the CHRNA2 gene coding for the α2 subunit. Here, we report the third mutation in the CHRNA2, found in a patient showing ADNFLE. The patient was examined by scalp EEG, contrast-enhanced brain magnetic resonance imaging (MRI), and nocturnal video-polysomnographic recording. All exons and the exon-intron boundaries of CHRNA2, CHRNA4, CHRNB2, CRH, KCNT1 were amplified and Sanger sequenced. In the proband, we found a c.754T>C (p.Tyr252His) missense mutation located in the N-terminal ligand-binding domain and inherited from the mother. Functional studies were performed by transient co-expression of α2 and α2 ^Tyr252His , with either β2 or β4, in human embryonic kidney (HEK293) cells. Equimolar amounts of subunits expression were obtained by using F2A-based multi-cistronic constructs encoding for the genes relative to the nAChR subunits of interest and for the enhanced green fluorescent protein. The mutation reduced the maximal currents by approximately 80% in response to saturating concentrations of nicotine in homo- and heterozygous form, in both the α2β4 and α2β2 nAChR subtypes. The effect was accompanied by a strong right-shift of the concentration-response to nicotine. Similar effects were observed using ACh. Negligible effects were produced by α2^Tyr252His on the current reversal potential. Moreover, binding of (±)-[³H]Epibatidine revealed an approximately 10-fold decrease of both K_d and B_max (bound ligand in saturating conditions), in cells expressing α2^Tyr252His. The reduced B_max and whole-cell currents were not caused by a decrease in mutant receptor expression, as minor effects were produced by α2^Tyr252His on the level of transcripts and the membrane expression of α2β4 nAChR. Overall, these results suggest that α2^Tyr252His strongly reduced the number of channels bound to the agonist, without significantly altering the overall channel expression. We conclude that mutations in CHRNA2 are more commonly linked to ADNFLE than previously thought, and may cause a loss-of-function phenotype.

Keywords: ADNFLE; ADSHE; frontal lobe epilepsy; genetics; nicotinic receptor; patch-clamp.

Figure 1

Hypnogram of the patient with the distribution of nocturnal episodes.

Figure 2

Genetics. (A) Pedigree of the family in which the mutation has been identified. The arrow points to the proband. Genotypes are shown. T/T: wild-type (WT) genotype; T/C: heterozygous genotype. Squares indicate males while circles indicate females. The legend of each kind of symbol filling is reported. (B) Electropherogram from the proband heterozygous for the transition c.754T>C (RefSeq NM_000742.3) that corresponds to the missense mutation p.Tyr252His. (C) Amino acid multiple alignment of the α2 subunit of the nAChR sequence displaying evolutionary conservation of Tyrosine Y residue across species.

Figure 3

CHRNA2 mRNA levels detected by real-time quantitative PCR in HEK293 cells transfected with tricistronic vectors containing either the wild-type (WT) or the mutant CHRNA2 (p.Tyr252His) in combination with CHRNB4 (A) or CHRNB2 (B) cDNAs and the eGFP reporter. Data represent the mean ± SEM (n = 3) and are expressed as fold increase of mRNA levels normalized to eGFP transcript levels and to non-transfected HEK293 cells (NT). (C) Equal amount of membranes proteins from HEK293 cells transfected with either α2β4 (lanes 1 and 2), or α2^Tyr252Hisβ4 (α2^∗β4; lanes 3 and 4) or untransfected (lanes 5 and 6) were separated on 9% acrylamide SDS gels, electrotransferred to nitrocellulose, probed with 5 μg/ml of the anti-α2 or anti-β4 primary Ab (as indicated), and then incubated with the secondary Ab (anti-rabbit Ly-Cor IRDye800RD, dilution 1:20000). The IR signal was measured using an Odyssey CLx – Infrared Imaging System and the signal intensity of the WB bands of the α2 and β4 subunits was quantified using iStudio software. The arrows indicate the α2 or β4 subunits.

Figure 4

Whole-cell currents from nAChR receptors containing or not α2^Tyr252His. (A) Representative whole-cell current traces elicited at –60 mV by the indicated concentration of nicotine, in cells expressing α2/β4 (wild type), α2^Tyr252His/β4 (homozygote), or α2/ α2^Tyr252His/β4 (heterozygote) receptors, as indicated. The bars above the current traces mark the time of nicotine application. The time gaps between consecutive traces represents about 2 min in the absence of agonist. (B) Same as (A), except that ACh was used instead of nicotine, at the indicated concentrations. Cells expressed either α2/β4 (wild type), or α2^Tyr252His/β4 (homozygote) receptors.

Figure 5

α2^Tyr252His decreases the maximal current density, without altering V_rev. (A) Bars represent average peak whole-cell current densities measured at the indicated concentrations of nicotine or ACh, in cells expressing α2β4, α2^Tyr252Hisβ4, or α2^Tyr252His/α2β4. The results of representative measurements are shown for 1 μM nicotine (p = 0.00005 between WT, n = 17, and homozygotes, n = 12; p = 0.0002 between WT and heterozygotes, n = 8), 30 μM nicotine (p = 0.0075; n = 9 for WT and n = 20 for homozygotes), 100 μM nicotine (p = 0.02 between WT, n = 9, and homozygotes, n = 22; p = 0.007 between WT and heterozygotes, n = 21), 300 μM nicotine (p = 0.02 between WT, n = 8, and homozygotes, n = 22; p = 0.01 between WT and heterozygotes, n = 21), 10 μM ACh (p = 0.0023; n = 17 for WT and 15 for homozygotes), 100 μM ACh (p = 0.010; n = 22 for WT and n = 19 for homozygotes), ^∗p < 0.05; ^∗∗p < 0.01. (B) Same as (A), but for α2β2 and α2^Tyr252Hisβ4 receptors. For 1 μM nicotine (p = 0.0004; n = 10 for WT and n = 7 for homozygotes), 10 μM nicotine (p = 0.0008; n = 11 for WT and n = 7 for homozygotes), 300 μM nicotine (p = 0.0006; n = 12 for WT and n = 11 for homozygotes). (B) Same as (A), but for α2β2 and α2^Tyr252Hisβ4 receptors, tested with 300 μM nicotine (p = 0.0006; n = 12 for WT and n = 11 for homozygotes), ^∗∗p < 0.01. (C) Representative current traces for the indicated receptor type, obtained by stimulating the cell with 1 s voltage ramps (–60 to +20 mV), in the presence or absence of 600 μM nicotine. The background current was subtracted to the one obtained in the presence of nicotine. V_rev was estimated by fitting the currents with a polynomial function. (D) Average V_rev values measured in WT (n = 11) and mutant (n = 11) receptors. The reported values were not significantly different between WT and mutant (with unpaired t-test).

Figure 6

Concentration-response analysis. (A) Concentration-response relation derived from patch-clamp results for α2β4 receptors. Data points are average peak whole-cell currents, normalized to the current elicited by 300 μM nicotine in WT receptors. Continuous line is fit to equation (1). The relative estimated parameters were: EC_50high: 0.08 ± 0.027 μM; EC_50low: 24.7 ± 2.76 μM; nH1: 2.41 ± 2.6; nH2: 1.24 ± 0.15. (B) Same as (A), for α2^Tyr252Hisβ4. In this case, peak currents are normalized to the current elicited by 800 μM nicotine. Representative currents are shown in the inset. Continuous line is fit to equation (1). The relative estimated parameters were: EC_50high: 23.4 ± 23 μM; EC_50low: 275.7 ± 12.5 μM; nH1: 0.87 ± 0.27; nH2: 3.44 ± 0.81. (C) Saturation binding experiments aimed to determine K_d and B_max of [³H]Epibatidine in cells transfected with α2β4, or α2^Tyr252Hisβ4 (α2^∗β4), or non-transfected. Curves were obtained from two independent saturation experiments using a non-linear least squares analysis program using GraphPad Prism version 6.