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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 ?4 and ?2 nAChR subunits, respectively. Two mutations with contrasting functional effects were also identified in the CHRNA2 gene coding for the ?2 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 ?2 and ?2 Tyr252His , with either ?2 or ?4, 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 ?2?4 and ?2?2 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 ?2Tyr252His on the current reversal potential. Moreover, binding of (±)-[3H]Epibatidine revealed an approximately 10-fold decrease of both Kd and Bmax (bound ligand in saturating conditions), in cells expressing ?2Tyr252His. The reduced Bmax and whole-cell currents were not caused by a decrease in mutant receptor expression, as minor effects were produced by ?2Tyr252His on the level of transcripts and the membrane expression of ?2?4 nAChR. Overall, these results suggest that ?2Tyr252His 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.

SUBMITTER: Villa C

PROVIDER: S-EPMC6379349 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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

Villa Chiara C Colombo Giulia G Meneghini Simone S Gotti Cecilia C Moretti Milena M Ferini-Strambi Luigi L Chisci Elisa E Giovannoni Roberto R Becchetti Andrea A Combi Romina R

Frontiers in molecular neuroscience 20190212

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 repor ...[more]

PMID: 30809122

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Project description:Previously characterized nicotinic acetylcholine receptor (nAChR) autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE)-associated mutations are found in ?2, ?4 and ?2 subunit transmembrane (TM) domains. They predominantly increase ACh potency and, for ?2-subunit mutants, increase macroscopic currents. Two recently-identified mutations, ?4(R336H) and ?2(V337G), located in the intracellular cytoplasmic loop (C2) have been associated with non-familial NFLE. Effects of these mutations on ?4?2-nAChR function and expression were studied for the first time, using two-electrode voltage clamp recordings in Xenopus laevis oocytes. Biased-ratio preparations elucidated the mutations' effects at alternate isoforms: high-sensitivity [HS; (?4)2(?2)3] or low-sensitivity [LS; (?4)3(?2)2] via 1:10 or 30:1 [?4:?2] cRNA injection ratios, respectively. An unbiased (1:1 [?4:?2] cRNA) injection ratio was also used to study potential shifts in isoform expression. ?4(R336H)-containing receptors showed significant increases in maximal ACh-induced currents (Imax) in all preparations (140% increase compared to wild type control). ?2(V337G)-containing receptors significantly increased Imax in the LS-favoring preparation (20% increase compared to control). Expression of either mutation consistently produced enrichment of HS-isoform expression in all preparations. ?4?2-nAChR harboring either NFLE mutant subunit showed unchanged ACh, sazetidine-A, nicotine, cytisine and mecamylamine potency. However, both mutant subunits enhanced partial agonist efficacies in the LS-biased preparation. Using ?2-subunit-specific [(125)I]mAb 295 immunolabeling, nAChR cell-surface expression was determined. Antibody binding studies revealed that the ?2(V337G) mutation tended to reduce cell-surface expression, and function per receptor was significantly increased by either NFLE mutant subunit in HS-favoring preparations. These findings identify both common and differing features between TM- and C2-domain AD/NFLE-associated mutations. As we discuss, the shared features may be particularly salient to AD/NFLE etiology.

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

Publications

<i>CHRNA2</i> and Nocturnal Frontal Lobe Epilepsy: Identification and Characterization of a Novel Loss of Function Mutation.

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