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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 (±)-[ 3 H]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.

Nocturnal frontal lobe epilepsy has been historically considered a channelopathy caused by mutations in subunits of the neuronal nicotinic acetylcholine receptor or in a recently reported potassium channel. However, these mutations account for only a minority of patients, and the existence of at least a new locus for the disease has been demonstrated. In 2005, we detected two nucleotide variations in the promoter of the CRH gene coding for the corticotropin releasing hormone in 7 patients. These variations cosegregated with the disease and were demonstrated to alter the cellular levels of this hormone. Here, we report the identification in an Italian affected family of a novel missense mutation (hpreproCRH p.Pro30Arg) located in the region of the CRH coding for the protein pro-sequence. The mutation was detected in heterozygosity in the two affected individuals. In vitro assays demonstrated that this mutation results in reduced levels of protein secretion in the short time thus suggesting that mutated people could present an altered capability to respond immediately to stress agents.

Ehlers-Danlos syndrome (EDS) is a clinically and genetically heterogeneous group of heritable connective tissue disorders (HCTDs) defined by joint laxity, skin alterations, and joint hypermobility. The latest EDS classification recognized 13 subtypes in which the clinical and genetic phenotypes are often overlapping, making the diagnosis rather difficult and strengthening the importance of the molecular diagnostic confirmation. New genetic techniques such as next-generation sequencing (NGS) gave the opportunity to identify the genetic bases of unresolved EDS types and support clinical counseling. To date, the molecular defects have been identified in 19 genes, mainly in those encoding collagen, its modifying enzymes or other constituents of the extracellular matrix (ECM). In this review we summarize the contribution of NGS technologies to the current knowledge of the genetic background in different EDS subtypes.

Inwardly rectifying potassium channels have been historically associated to several cardiovascular disorders. In particular, loss-of-function mutations in the Kir2.1 channel have been reported in cases affected by Andersen-Tawil syndrome while gain-of-function mutations in the same channel cause the short QT3 syndrome. Recently, a missense mutation in Kir2.1, as well as mutations in the Kir4.1, were reported to be involved in autism spectrum disorders suggesting a role of potassium channels in these diseases and introducing the idea of the existence of K+ channelautism spectrum disorders. Here, we report the identification in an Italian affected family of a novel missense mutation (p.Phe58Ser) in the KCNJ2 gene detected in heterozygosity in a proband affected by autism and borderline for short QT syndrome type 3. The mutation is located in the N-terminal region of the gene coding for the Kir2.1 channel and in particular in a very conserved domain. In vitro assays demonstrated that this mutation results in an increase of the channel conductance and in its open probability. This gain-of-function of the protein is consistent with the autistic phenotype, which is normally associated to an altered neuronal excitability.

Objective: To study the prevalence of DEPDC5 mutations in a series of 30 small European families with a phenotype compatible with autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). Methods: Thirty unrelated families referred with ADNFLE were recruited in France, Italy, Germany, Belgium, and Norway. Whole-exome sequencing was performed in 10 probands and direct sequencing of the DEPDC5 coding sequence in 20 probands. Testing for nonsense-mediated messenger RNA decay (NMD) was performed in lymphoblastic cells. Results: Exome sequencing revealed a splice acceptor mutation (c.2355-2A>G) in DEPDC5 in the proband of aGerman family. In addition, 3 nonsense DEPDC5 mutations (p.Arg487*, p.Arg1087*, and p.Trp1369*) were detected in the probands of 2 French and one Belgian family. The nonsense mutations p.Arg487* and p.Arg1087* were targeted by NMD, leading to the degradation of the mutated transcripts. At the clinical level, 78% of the patients with DEPDC5 mutations were drug resistant. Conclusions: DEPDC5 loss-of-function mutations were found in 13% of the families with a presentation of ADNFLE. The rate of drug resistance was high in patients with DEPDC5 mutations. Small ADNFLE pedigrees with DEPDC5 mutations might actually represent a part of the broader familial focal epilepsy with variable foci phenotype. © 2014 American Academy of Neurology.

Mutations in the SCN1A gene causing either loss or gain of function have been frequently found in patients affected by genetic epilepsy with febrile seizures plus (GEFS+) or Dravet syndrome (also named severe myoclonic epilepsy in infancy SMEI). By mutation screening of the SCN1A gene, we identified for the first time a case of two missense mutations in cis (p.[Arg1525Gln;Thr297Ile]) in all affected individuals of an Italian family showing GEFS+ and idiopathic generalized epilepsy (IGE). The p.Arg1525Gln mutation was not previously reported yet and was predicted to be pathological by prediction tools, whereas the p.Thr297Ile was already identified in patients showing SMEI. Functional studies revealed that the Nav1.1 channels harboring both mutations were characterized by a significant shift in the activation curve towards more positive potentials. Our data demonstrate that the p.Arg1525Gln represents a novel mutation in the SCN1A gene altering the channel properties in the co-presence of the p.Thr297Ile.

Potassium (K(+)) channels are expressed in almost every cells and are ubiquitous in neuronal and glial cell membranes. These channels have been implicated in different disorders, in particular in epilepsy. K(+) channel diversity depends on the presence in the human genome of a large number of genes either encoding pore-forming or accessory subunits. More than 80 genes encoding the K(+) channels were cloned and they represent the largest group of ion channels regulating the electrical activity of cells in different tissues, including the brain. It is therefore not surprising that mutations in these genes lead to K(+) channels dysfunctions linked to inherited epilepsy in humans and non-human model animals. This article reviews genetic and molecular progresses in exploring the pathogenesis of different human epilepsies, with special emphasis on the role of K(+) channels in monogenic forms.

Members of the ligand-gated neuronal nicotinic acetylcholine receptor (nAChR) gene family ( CHRNA4 and CHRNB2, coding for the alpha4 and beta2 subunits, respectively) are involved in autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). However, ADNFLE is genetically heterogeneous and mutations in CHRNA4 and CHRNB2 account for only a minority of ADNFLE cases. Additional nAChR subunits expressed in the brain are candidates for this epilepsy. The involvement of all genes coding for brain-expressed nAChR subunits, with known chromosome localization ( CHRNB2, 1q21; CHRNA2, 8p21; CHRNA6, CHRNB3, 8p11.2; CHRNA7, 15q14; CHRNA5/A3/B4, 15q24 and CHRNA4, 20q13.2) was investigated in four unrelated ADNFLE Italian families for at least three generations. Families were selected on the basis of anamnestic and videopolysomnographic analyses. Individuals were typed for polymorphic markers located in the above mentioned chromosome regions. Linkage and mutation analyses were performed. In none of the families was linkage between ADNFLE and the analysed chromosome regions detected. These findings support the hypothesis that genes different from those coding for alpha2-7 and beta2-4 neuronal nAChR subunits could be responsible for ADNFLE.

BACKGROUND:: Nocturnal frontal lobe epilepsy (NFLE) is an idiopathic partial epilepsy characterized by a wide spectrum of stereotyped motor manifestations, mostly occurring during non rapid eye movements sleep. NFLE is underdiagnosed since semiological similarities make it difficult to distinguish NFLE from parasomnias. In 1994, authors reported families with NFLE inherited as an autosomal dominant trait and they introduced the term of autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). A family history of possible NFLE is found in about 25% of cases. The genetic bases of the disease have been detected in a minority of cases. Mutations causing a gain of function of the neuronal nicotinic acetylcholine receptors were reported in 3 different subunits. REVIEW SUMMARY:: This review discusses the clinical aspects of NFLE and the diagnostic procedures. Furthermore, the genetic aspects are outlined. The main differentiating features characterizing NFLE are: (a) several attacks per night at any time during the night; (b) brief duration of the attacks; (c) stereotyped motor pattern. Nocturnal video-polysomnography is crucial for the diagnosis. Neurological examination in NFLE/ADNFLE is normal. About 30% of NFLE cases are resistant to antiepileptic drugs. Concerning the genetics, putative susceptibility nucleotide variations affecting the promoter of the CRH gene and altering the corticotrophin-releasing hormone levels have been reported in some NFLE patients. CONCLUSIONS:: Distinguishing NFLE seizures from paroxysmal nonepileptic sleep disorders is often difficult and sometimes impossible on clinical grounds alone. Nocturnal video-polysomnography is mandatory. Further genetic studies could help the diagnosis and treatment in NFLE patients.

Gain-of-function mutations in genes coding for subunits of the neuronal nicotinic acetylcholine receptor have been identified in 15% of families with autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE),1 and in the promoter of the corticotropin-releasing hormone (CRH) gene.1 In remaining families, the responsible genes are unknown. Patients with ADNFLE typically present many seizures per night involving intense motor agitation (frenetic movements of the trunk and limbs or sequence of various dystonic postures) lasting less than 1 minute. There are no clinical differences between families with and without identified mutations. The seizures seem to occur at the time of arousal from nonrapid eye movement (non-REM) sleep stage 2.2 Clinical and EEG observations and the identification by in vivo nuclear imaging in patients with ADNFLE carrying nicotinic receptor mutations of an increased nicotinic receptor density in the brainstem, suggesting a hyperactivity of the brainstem-thalamic cholinergic ascending arousal pathway, led to ADNFLE being considered as an arousal disorder.3 In parallel to the cholinergic system, the orexin/hypocretin system is also involved in inducing arousal from sleep. Orexin neurons, like cholinergic neurons, discharge …

Mutations of voltage-gated Na+ channels are the most common known cause of genetically determined epilepsy; Nav1.1 (SCN1A) is the most frequent target. They can cause both mild and severe forms, also in patients harboring the same mutation. We have recently characterized in a family with extreme phenotypes the first epileptogenic folding-defective Na+ channel mutant (Nav1.1-M1841T), whose loss of function is attenuated by interactions with associated proteins and drugs. We hypothesized that in vivo variability of the interactions may modulate the functional effect and thus the phenotype (Rusconi et al., 2007). Here we characterize another Nav1.1 folding-defective mutant (Nav1.1-R1916G) that, however, has been identified in a GEFS+ family with relatively mild phenotypes. This novel mutant shows a number of specific characteristics, but, similarly to Nav1.1-M1841T, it can be rescued by interactions with associated proteins and drugs. Thus, loss of function caused by folding defects that can be attenuated by molecular interactions may be a common pathogenic mechanism for Nav1.1 epileptogenic mutants. Folding defects can be present also in families showing only mild phenotypes in which, however, severe phenotypes could emerge within a permissive genetic background. © 2009 Wiley-Liss, Inc.

Objective Forty-six nocturnal frontal lobe epilepsy (NFLE) patients (in which the involvement of the CHRNA4 and CHRNB2 genes coding for neuronal nicotinic acetylcholine receptor (nAChRs) subunits associated to the disease were previously excluded) were analyzed for the presence of mutations in the CHRNA2 gene coding for the alpha2 subunit of the same receptor, which has been recently associated with the disease. Methods Mutational screening was performed by sequencing two polymerase chain reaction-amplified CHRNA2 DNA fragments, spanning the whole exon 6 and exon 7, respectively, which code for ∼75% of the mature protein and contain all four transmembrane domains contributing to the ion pore. Results No mutations were identified in the analyzed region of CHRNA2 . Conclusions These data demonstrate the rarity of the identified CHRNA2 mutations in NFLE patients, supporting the recently reported hypothesis of a restricted role for this gene in the disease.

Mutations responsible for autosomal dominant nocturnal frontal lobe epilepsy have been identified in two members of the neuronal nicotinic acetylcholine receptor gene family: CHRNA4 (ENFL1 locus) and CHRNB2 (ENFL3 locus) coding for α4 and β2 subunit, respectively. However, mutations in these genes account for only a minority (less than 10%) of cases. For a third ADNFLE locus (ENFL2) on chromosome 15q24 the gene was not identified. The involvement of the three loci in the pathogenesis of ADNFLE was investigated in 12 unrelated Italian families, selected on the basis of anamnestic and video-polysomnographic data. Compliant family members were typed for polymorphic markers spanning the analyzed chromosome regions. Linkage analyses excluded association of all chromosome regions with ADNFLE in 72% of cases. In two, four and one families it was impossible to ascertain or exclude association with ENFL1, ENFL2, or ENFL3, respectively, however, no mutations have been detected in the nicotinic receptor genes located in these regions. These data strongly suggest that ENFL1, ENFL2 and ENFL3 are minor loci for the disease and point to the existence of at least a fourth locus for ADNFLE.

Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is an idiopathic epilepsy, with a spectrum of clinical manifestations, ranging from brief, stereotyped, sudden arousals to more complex dystonic-dyskinetic seizures. Video-polysomnography allows a correct differential diagnosis. There is no difference between sporadic nocturnal frontal lobe epilepsy (NFLE) and ADNFLE in the clinical and neurophysiological findings. ADNFLE is the first idiopathic epilepsy for which a genetic basis has been identified. Mutations have been found in two genes (CHRNA4 and CHRNB2) coding for neuronal nicotinic receptor subunits (alpha4 and beta2, respectively). Contrasting data have been reported on the effect of these mutations on the functionality of the receptor. Moreover, the incomplete data on the neuronal network/s in which this receptor is involved, make difficult the understanding of the genotype-phenotype correlation. This is an overview on the clinical and genetic aspects of ADNFLE including a discussion of some open questions on the role of the neuronal nicotinic receptor subunit mutations in the pathogenesis of this form of epilepsy.