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High-throughput sequencing (HTS) improved the molecular diagnosis in individuals with intellectual deficiency (ID) and helped to broaden the phenotype of previously known disease-causing genes. We report herein four unrelated patients with isolated ID, carriers of a likely pathogenic variant in KCNQ2, a gene usually implicated in benign familial neonatal seizures (BFNS) or early onset epileptic encephalopathy (EOEE). Patients were diagnosed by targeted HTS or exome sequencing. Pathogenicity of the variants was assessed by multiple in silico tools. Patients' ID ranged from mild to severe with predominance of speech disturbance and autistic features. Three of the four variants disrupted the same amino acid. Compiling all the pathogenic variants previously reported, we observed a strong overlap between variants causing EOEE, isolated ID, and BFNS and an important intra-familial phenotypic variability, although missense variants in the voltage-sensing domain and the pore are significantly associated to EOEE (p < 0.01, Fisher test). Thus, pathogenic variants in KCNQ2 can be associated with isolated ID. We did not highlight strong related genotype-phenotype correlations in KCNQ2-related disorders. A second genetic hit, a burden of rare variants, or other extrinsic factors may explain such a phenotypic variability. However, it is of interest to study encephalopathy genes in non-epileptic ID patients.

KCNQ2 and KCNQ3 pathogenic channel variants have been associated with a spectrum of developmentally regulated diseases that vary in age of onset, severity, and whether it is transient (i.e., benign familial neonatal seizures) or long-lasting (i.e., developmental and epileptic encephalopathy). KCNQ2 and KCNQ3 channels have also emerged as a target for novel antiepileptic drugs as their activation could reduce epileptic activity. Consequently, a great effort has taken place over the last 2 decades to understand the mechanisms that control the assembly, gating, and modulation of KCNQ2 and KCNQ3 channels. The current view that KCNQ2 and KCNQ3 channels assemble as heteromeric channels (KCNQ2/3) forms the basis of our understanding of KCNQ2 and KCNQ3 channelopathies and drug design. Here, we review the evidence that supports the formation of KCNQ2/3 heteromers in neurons. We also highlight functional and transcriptomic studies that suggest channel composition might not be necessarily fixed in the nervous system, but rather is dynamic and flexible, allowing some neurons to express KCNQ2 and KCNQ3 homomers. We propose that to fully understand KCNQ2 and KCNQ3 channelopathies, we need to adopt a more flexible view of KCNQ2 and KCNQ3 channel stoichiometry, which might differ across development, brain regions, cell types, and disease states.

Background The clinical features of KCNQ2-related disorders range from benign familial neonatal seizures 1 to early infantile epileptic encephalopathy 7. The genotype-phenotypic association is difficult to establish. Objective To explore potential factors in neonatal period that can predict the prognosis of neonates with KCNQ2-related disorder. Methods Infants with KCNQ2-related disorder were retrospectively enrolled in our study in Children’s Hospital of Fudan University in China from Jan 2015 to Mar 2020. All infants were older than age of 12 months at time of follow-up, and assessed by Bayley Scales of Infant and Toddler Development-Third Edition (BSID-III) or Wechsler preschool and primary scale of intelligence-fourth edition (WPPSI-IV), then divided into three groups based on scores of BSID-III or WPPSI-IV: normal group, mild impairment group, encephalopathy group. We collected demographic variables, clinical characteristics, neuroimaging data. Considered variables include gender, gestational age, birth weight, age of the initial seizures, early interictal VEEG, variant location, delivery type. Variables predicting prognosis were identified using multivariate ordinal logistic regression analysis. Results A total of 52 infants were selected in this study. Early interictal video-electro-encephalography (VEEG) (β = 2.77, 1.20 to 4.34, P = 0.001), and variant location (β = 2.77, 0.03 to 5.5, P = 0.048) were independent risk factors for prognosis. The worse the early interictal VEEG, the worse the prognosis. Patients with variants located in the pore-lining domain or S4 segment are more likely to have a poor prognosis. Conclusions The integration of early initial VEEG and variant location can predict prognosis. An individual whose KCNQ2 variant located in voltage sensor, the pore domain, with worse early initial VEEG background, often had an adverse outcome.

Peripheral nerve hyperexcitability syndrome comprises a heterogeneous group of diseases, clinically characterised by myokymia, fasciculation, muscle cramps and stiffness. The causes are either immune mediated or non-immune mediated. Non-immune-mediated forms are mostly genetic, relating to two main genes: KCNQ2 and KCNA1. Patients with KCNQ2 gene mutations typically present with epileptic encephalopathy, benign familial neonatal seizures and myokymia, though occasionally with purely peripheral nerve hyperexcitability. We report a woman with marked facial myokymia and distal upper limb contractures whose mother also had subtle facial myokymia; both had the c.G620A (p.R207Q) variant in the KCNQ2 gene. Patients with familial myokymia and peripheral nerve hyperexcitability syndrome should be investigated for KCNQ2 variants. This autosomal dominant condition may respond to antiepileptic medications acting at potassium channels.

KCNQ2 has been reported as a frequent cause of autosomal dominant benign familial neonatal seizures. De novo likely pathogenic variants in KCNQ2 have been described in neonatal or early infantile onset epileptic encephalopathy patients. Here, we report a three-generation family with six affected patients with a novel likely pathogenic variant (c.628C>T; p.Arg210Cys) in KCNQ2. Four family members, three adults and a child, presented with a childhood seizure onset with variability in the severity of seizures and response to treatment, intellectual disability (ID) as well as behavioral problems. The two youngest affected patients had a variable degree of global developmental delay with no seizures at their current age. This three-generation family with six affected members expands the phenotypic spectrum of KCNQ2 associated encephalopathy to KCNQ2 associated ID and or childhood onset epileptic encephalopathy. We think that KCNQ2 associated epileptic encephalopathy should be included in the differential diagnosis of childhood onset epilepsy and early onset global developmental delay, cognitive dysfunction, or ID. Furthermore, whole exome sequencing in families with ID and history of autosomal dominant inheritance pattern with or without seizures, may further broaden the phenotypic spectrum of KCNQ2 associated epileptic encephalopathy or encephalopathy.

Among neonatal epileptic syndromes, benign familial neonatal seizures (BFNS) are often due to autosomal-dominant mutations of the KCNQ2 gene. Seizures are usually characterized by asymmetric tonic posturing with apnea with onset in the first 7 days of life; they may even occur more than 10 times per day or evolve into status epilepticus. The delivery course of our patient was uneventful and family history was negative; on the second day of life the baby became pale, rigid, and apnoic during breastfeeding and appeared jittery and irritable when stimulated or examined. At age 3 days, she experienced clusters of generalized tonic seizures with pallor, desaturation, bradycardia, and partial response to intravenous phenobarbital; during her 4th and 5th days of life, three episodes of tonic seizures were noticed. At age 6 days, the patient experienced about 10 episodes of tonic seizures involving both sides of the body, which gradually responded to intravenous phenytoin. Electroencephalograms revealed abnormalities but brain MRI was normal. The patient is seizure-free since postnatal day 21; she is now 12 months old with cognitive development within normal limits at Bayley III Scale and mild motor delay. The patient is on maintenance therapy with phenobarbital since she was 7 months old. A de novo heterozygous mutation (c.853C>T/p.P285S) in the KCNQ2 gene was identified. We therefore describe a case of de novo KCNQ2-related neonatal convulsions with necessity of multiple anticonvulsants for the control of seizures, mutation occurring in the pore channel of the voltage-gated potassium channel subfamily Q member 2 associated with a likely benign course; furthermore, the same mutation of the KCNQ2 gene and a similar one (c.854C>A/p.P285H) have already been described in association with Ohtahara syndrome. Probably acquired environmental, perinatal and genetic risk factors are very important in determining the different phenotype; we hope that the rapid progress of analysis tools in molecular diagnosis can also be used in the search of an individualized therapeutic approach for these patients.

Mutations in KCNQ2 are associated with a range of electroclinical syndromes with dominant inheritance that are differentiated by the age at onset of the seizures and are associated with good prognosis. These are benign familial neonatal seizures (BFNS), benign familial neonatal-infantile seizures (BFNIS) and benign familial infantile seizures. Herein, we report the results of a systematic screening of KCNQ2 in 27 unrelated Bulgarian patients with compatible clinical diagnoses. Two pathogenic point mutations were identified: a novel splice-site c. 1526-2A > G variation causing BFNS and a missense c. 998G > A alteration in a patient with BFNIS, who subsequently developed benign epilepsy with centro-temporal spikes. Additionally, multiplex ligation-dependent probe amplification analysis and array comparative genomic hybridization assay detected a de novo deletion on 20q13.3 encompassing 0.41 Mb genomic region and covering 11 genes, including KCNQ2 and CHRNA4. This large-scale rearrangement was found in a patient with typical BFNS and no additional developmental abnormalities. Overall, KCNQ2 genetic defects were found in 11% of the patients in our cohort. These findings enrich the genetic epidemiology and mutation spectrum of KCNQ2 and allow adequate genetic counselling in the affected families.

SummaryObjective To evaluate treatment responses in benign familial neonatal epilepsy (BFNE). Methods We recruited patients with BFNE through a multicenter international collaboration and reviewed electroclinical and genetic details, and treatment response. All patients were tested at minimum for mutations/deletions in the KCNQ2, KCNQ3, and SCN2A genes. Results Nineteen patients were included in this study. A family history of neonatal seizures was positive in 16 patients, and one additional patient had a family history of infantile seizures. Mutations or deletions of KCNQ2 were found in 14, and of KCNQ3 in 2, of the 19 patients. In all patients, seizures began at 2–5 days of life and occurred multiple times per day. Four patients developed status epilepticus. Seizures were focal, alternating between hemispheres, and characterized by asymmetric tonic posturing associated with apnea and desaturation, followed by unilateral or bilateral asynchronous clonic jerking. Twelve of 19 patients were treated with multiple medications prior to seizure cessation. Seventeen of (88%) 19 patients were seizure-free within hours of receiving oral carbamazepine (CBZ) or oxcarbazepine (OXC). Earlier initiation of CBZ was associated with shorter hospitalization (p < 0.01). No side effects of CBZ were reported. All patients had normal development and remain seizure-free at a mean follow-up period of 7.8 years (6 months–16 years). Significance This study provides evidence that CBZ is safe and rapidly effective in neonates with BFNE, even in status epilepticus. We propose that CBZ should be the drug of choice in benign familial neonatal seizures.

The KCNQ2 gene codifies a subunit of the voltage-gated potassium M channel underlying the neuronal M-current. Classically, mutations in this gene have been associated with benign familial neonatal seizures, however, in recent years KCNQ2 mutations have been reported associated to early-onset epileptic encephalopathy. In this work, detailed familiar, clinical and genetic data were collected for 13 KCNQ2-positive patients revealed among a cohort of 80 epileptic pediatric probands from Spain who were analyzed through a targeted next-generation sequencing assay for 155 epilepsy-associated genes. This work shows for the first time the association between KCNQ2 mutations and startle attacks in 38% of patients, which opens the possibility to define electroclinical phenotypes associated to KCNQ2 mutations. It also demonstrates that KCNQ2 mutations contribute to an important percentage of Spanish patients with epilepsy. The study confirm the high genetic heterogeneity of this gene with 13 different mutations found, 10 of them novel and the better outcome of patients treated with sodium channel blockers.

Kv7.2 subunits encoded by the KCNQ2 gene provide a major contribution to the M-current (IKM), a voltage-gated K+ current crucially involved in the regulation of neuronal excitability. Heterozygous missense variants in Kv7.2 are responsible for epileptic diseases characterized by highly heterogeneous genetic transmission and clinical severity, ranging from autosomal-dominant Benign Familial Neonatal Seizures (BFNS) to sporadic cases of severe epileptic and developmental encephalopathy (DEE). Here, we describe a patient with neonatal onset DEE, carrying a previously undescribed heterozygous KCNQ2 c.418G &gt, C, p.Glu140Gln (E140Q) variant. Patch-clamp recordings in CHO cells expressing the E140Q mutation reveal dramatic loss of function (LoF) effects. Multistate structural modelling suggested that the E140Q substitution impeded an intrasubunit electrostatic interaction occurring between the E140 side chain in S2 and the arginine at position 210 in S4 (R210), this interaction is critically involved in stabilizing the activated configuration of the voltage-sensing domain (VSD) of Kv7.2. Functional results from coupled charge reversal or disulfide trapping experiments supported such a hypothesis. Finally, retigabine restored mutation-induced functional changes, reinforcing the rationale for the clinical use of Kv7 activators as personalized therapy for DEE-affected patients carrying Kv7.2 LoF mutations.

Neonatal epilepsy genetics is a rapidly expanding field with recent technological advances in genomics leading to an expanding list of genetic disorders associated with neonatal-onset epilepsy. The genetic causes of neonatal epilepsy can be grouped into the following categories: (i) malformations of cortical development, (ii) genetic-metabolic, (iii) genetic-vascular, (iv) genetic-syndromic, and (v) genetic-cellular. Clinically, epilepsy in the neonate shows phenotypic overlap with pathogenic variants in unrelated genes causing similar clinical presentation (locus heterogeneity) and variants in the same gene leading to a wide clinical spectrum ranging from benign familial neonatal seizures to more severe epileptic encephalopathies (variable expressivity). We suggest a diagnostic approach to obtaining a genetic diagnosis with emphasis on clinical features such as electro-clinical phenotype and magnetic resonance imaging findings. Rapid identification of genetic disorders with targeted treatments should be a clinical priority. Achieving a genetic diagnosis can be challenging in a rapidly changing genetic landscape, but is increasingly possible.

Benign familial neonatal seizures (BFNS) present a rare familial epilepsy syndrome caused by genetic alterations in the voltage-gated potassium channels Kv7.2 and Kv7.3, encoded by KCNQ2 and KCNQ3. While most BFNS families carry alterations in KCNQ2, mutations in KCNQ3 appear to be less common. Here, we describe a family with 6 individuals presenting with neonatal focal and generalized seizures. Genetic testing revealed a novel KCNQ3 variant, c.835G>T, cosegregating with seizures in 4 tested individuals. This variant results in a substitution of the highly conserved amino acid valine localized within the pore-forming transmembrane segment S5 (p.V279F). Functional investigations in Xenopus laevis oocytes revealed a loss of function, which supports p.V279F as a pathogenic mutation. When p.V279F was coexpressed with the wild-type (WT) Kv7.2 subunits, the resulting potassium currents were about 10-fold reduced compared to the WT Kv7.3 and Kv7.2 coexpression. Genotype-phenotype correlation shows an incomplete penetrance of p.V279F. Response to antiepileptic treatment was variable, but evaluation of treatment response remained challenging due to the self-limiting character of the disease. The identification of the pathogenic variant helped to avoid unnecessary investigations in affected family members and allowed guided therapy.

Voltage-gated potassium channel gene mutations in KCNQ2 and KCNQ3 α subunits may result in benign familial neonatal seizure (BFNS). Previous reports have implicated KCNQ2 mutations in patients with BFNS who later developed rolandic epilepsy (RE). We describe an 8-year-old boy with BFNS who developed RE. A three-generation pedigree showed multiple individuals presenting with seizures within the first days of life. Whole exome sequencing in this patient revealed a novel mutation in the KCNQ2 pore region. To our knowledge, this is the first report of BFNS with RE described with a KCNQ2 mutation localizing to the pore domain.

Despite the evolution of new technologies for assessing neonatal brain function, electroencephalography (EEG) remains a powerful tool for neurological diagnosis and prognosis in neonates. It is considered the gold standard for distinguishing epileptic seizures from non-epileptic paroxysmal events and for detecting subclinical seizure activity in high-risk babies. In those babies severely ill, EEG is even more efficient as predictive test than the neurological examination. The prognostic value of neonatal EEG has been long recognized in term as well as in preterm infants. Background patterns, more that patterns of ictal discharges, correlate significantly with the long-term outcome. Although most abnormalities on the neonatal EEG are nonspecific, certain patterns can be highly suggestive for diagnosis. Prognostic value can be increased by obtaining early recordings, possibly within the first 48 hr of life, prolonged recordings to include samples of different activity states, and serial EEGs at short intervals to assess rapid changes that are likely to occur in high-risk infants. It is important to distinguish neonatal seizures from neonatal-onset epilepsies and epilepsy syndromes. Both benign and malignant neonatal epilepsy syndromes exist. While benign familial neonatal seizures represents a neonatal syndrome with benign outcome, early myoclonic encephalopathy and Ohtahara syndrome are the earliest form of age-dependent severe epilepsy syndromes. Early myoclonic encephalopathy and Ohtahara syndrome are both characterized by the presence of a burst-suppression EEG pattern. This pattern is usually associated with a poor prognosis and is considered the electroencephalographic correlate of a complete disconnection within the thalamocortical systems. Migrating partial seizures in infancy is a newly recognized epilepsy syndrome whose onset can be in the neonatal period. It is characterized by multifocal intractable seizures associated with psychomotor impairment. No etiology has been found so far. Different drugs currently used in neonatal intensive care unit, especially if in the toxic range, can alter the EEG background. Those effects need to be taken in account in the interpretation of neonatal EEG.

Benign familial neonatal epilepsy (BFNE), previously benign familial neonatal seizures, is a relatively rare epilepsy syndrome arising in newborns. The genetic basis for this syndrome has recently been defined. This review uses a case to illus- trate the challenges in the clinical approach to diagnosis in neonatal epilepsy, and gives the clinician a summary of the genetic basis for BFNE. Finally, an overview of counseling and clinical treatment of BFNE is discussed.

In order to clarify the phenotypes of 20q13.33 microdeletion, clinical manifestations and genetic findings from four patients are discussed in relation to chromosomal microdeletions at 20q13.33. All patients had epileptic seizures mostly beginning within the neonatal period and disappearing by 4 months of age, similar to epilepsy phenotypes of benign familial neonatal seizures. We performed array comparative, genomic hybridization analysis in order to investigate the chromosomal aberration. Developmental outcome was good in two patients with deletion restricted to three genes (CHRNA4, KCNQ2, and COL20A1), whereas delay in developmental milestones was observed in the other two with a wider range of deletion. Information obtained from array comparative genomic hybridization may be useful to predict seizure and developmental outcome, however, there is no distinctive pattern of abnormalities that would arouse clinical suspicion of a 20q13.33 microdeletion. Deletion of KCNQ2 and CHRNA4 does not appear to affect seizure phenotype. Molecular cytogenetic techniques, such as array comparative genomic hybridization, will be necessary to clarify the relationship between phenotypes and individual genes within this region.

Over one hundred mutations in the Kv7.2 (KCNQ2) gene encoding for phosphatidylinositol 4,5-bisphosphate (PIP2)-sensitive voltage-gated K+ channel subunits have been identified in early-onset epilepsies with wide phenotypic variability. By contrast, only few mutations in the closely related Kv7.3 (KCNQ3) gene have been reported, mostly associated with typical benign familial neonatal seizures (BFNS). We herein describe a patient affected by early onset epileptic encephalopathy (EOEE) carrying two Kv7.3 missense mutations (p.Val359Leu/V359L and p.Asp542Asn/D542N) in compound heterozygosis, each inherited from an asymptomatic parent. Patch-clamp recordings from transiently transfected CHO cells showed that, when incorporated in physiologically relevant Kv7.2 + Kv7.3 heteromeric channels, expression of Kv7.3 V359L or Kv7.3 D542N subunits failed to affect current density, whereas a significant decrease was instead observed when these mutant subunits were both simultaneously present. Modeling and functional experiments revealed that each variant decreased PIP2-dependent current regulation, with additive effects when the two were co-expressed. Moreover, expression of Kv7.2 subunits carrying the D535N variant previously described in three sporadic EOEE cases prompted functional changes more dramatic when compared to those of the corresponding D542N variant in Kv7.3, but similar to those observed when both Kv7.3 V359L and Kv7.3 D542N subunits were expressed together. Finally, the Kv7 activator retigabine restored channel dysfunction induced by each Kv7.2 or Kv7.3 variant(s). These results provide a plausible molecular explanation for the apparent recessive inheritance of the phenotype in the family investigated, and a rational basis for personalized therapy with Kv7 channel activators in EOEE patients carrying loss-of-function mutations in Kv7.2 or Kv7.3.

It has been reported that coding mutations in the EFHC1 (for EF-hand containing) gene are the cause of perhaps up to 9% of all cases of juvenile myoclonic epilepsy (JME).1 The major evidence supporting this conclusion includes the following: (1) Hispanic and Dutch families with epilepsy show linkage to a locus that includes EFHC1,2–6 (2) highly penetrant EFHC1 coding mutations segregate with JME in families reporting Hispanic and Central American ancestry,7 (3) rare EFHC1 mutations have been reported to be both inherited and de novo in sporadic JME cases,8 (4) Efhc1-deficent mice exhibit spontaneous seizures,9 (5) EFHC1 regulates important neurodevelopmental events in cell culture.10,11 However, despite this evidence, notable contradictions still remain. For instance, in the study that initially proposed mutations in EFHC1 as a cause of JME,7 four separate coding mutations were found to segregate in 6 of the 30 JME families that showed linkage to the EFHC1-containing 6p12-p11 locus. Although these four mutations were not found in 382 Hispanic controls, neither they nor any other EFCH1 coding mutations were found segregating in the other 24 families in the study. Therefore, the six families were the only families in which these four EFHC1 mutations were found to segregate with JME. It is notable that when these six families were excluded from the analysis, evidence in favor of linkage remained.7 Although these observations support the hypothesis of a JME gene existing in the region, they cast some doubt on the importance of EFHC1 coding mutations for disease and/or on the role of EFHC1 in JME susceptibility. A subsequent study of Dutch JME patients also found linkage to the same region5 but failed to find any exonic mutations in EFHC1 associated with JME.12 Thus, in the majority of 6p12-p11-linked JME families from both Hispanic and Dutch populations, EFHC1 coding mutations do not segregate with disease. This means that other elements in the region, either within EFHC1 or in some other gene, must be responsible for the genetic signal. These elements might include regulatory factors affecting EFHC1. However, an additional possibility that cannot be ruled out is that EFHC1 is not the actual causative gene and that mutations in some unexamined nearby element functionally unrelated to EHFC1 are the cause of 6p12-p11-linked JME. One example that illustrates how causal mutations can be in a gene nearby a gene initially thought to be causal is in benign familial neonatal seizures (BFNS). Initially, given the observation of a nonsense mutation in CHRNA4, the gene encoding the neuronal nicotinic cholinergic receptor α4 subunit, segregating13 with BFNS in a linked region of chromosome 20q,14 this mutation was thought to be causal. At the time of its discovery, the evidence seemed convincing. However, it was ultimately shown that mutations in a nearby gene KCNQ2 were in fact the cause of 20q-linked BFNS.15,16 Thus, whether one considers CHRNA4 and BFNS or EFHC1 and JME, the existence of segregating mutations is not proof of causality, especially in the case of EFHC1, which also happens to have several coding mutations that are unrelated to epilepsy.7,17,18 (A fact often ignored in a cosegregation argument is that, in a linked region, all variants within the region, including those not in the disease gene itself, are more likely to cosegregate with the disease, by the definition of linkage analysis. This means that cosegregation alone cannot prove that a variant is the cause of the disease.) Importantly, if it is the case that another gene at 6p11-12 is causal for JME, the mutations are unlikely to be coding mutations, as mutation analysis of several genes in the region seems to rule out this possibility.18 More recently, exome-wide sequencing efforts have raised another issue regarding the role of EFHC1 in JME. Specifically, two of the four mutations initially reported to cause JME with high penetrance, and reported to co-segregate with JME,7 are variants found in individuals in the National Heart, Blood and Lung Institute Exome Variant Server (EVS: http://evs.gs.washington.edu/EVS) database.19 The EFHC1 F229L variant (rs137852776) is relatively rare (0.4%) and carriers in the EVS database could possibly be JME patients, since the sample is unphenotyped. However, the frequency of the other variant, the EFHC1 P77T-R221H haplotype (rs149055334-rs79761183), is found in >2% in the EVS data base among African Americans, a surprisingly high frequency for a haplotype reported to cause JME with roughly 80% penetrance. Without phenotypic data on these individuals, we cannot rule out the possibility that the EVS individuals with this haplotype are also JME patients, although a frequency of >2% significantly exceeds the prevalence of all forms of JME in any population. Therefore, an important question becomes, for example, “What does it mean to be an African American carrying the EFHC1 P77T-R221H allele and how does this impact individual susceptibility to epilepsy?” The reported role of pathogenic EFHC1 variants in JME7,8 has been mentioned in reports weighing the benefits and harm of genetic testing in the epilepsies (e.g., Ottman et al.20), with the causative role of EFHC1 in JME listed as one of the most well-accepted findings. Ottman et al. note that being told that one carries a risk allele for epilepsy can lead to anxiety, stigmatization, and discrimination. Therefore, it is important to distinguish between “susceptibility” alleles and “causal” alleles. “Susceptibility” alleles interact with other genetic or environmental factors to increase the chances of developing disease, but they need not have any direct effect on the underlying disease mechanism and are not necessary for the disease to manifest. In contrast, the defining characteristic of “causal” alleles is their clear direct involvement in the disease mechanism with interactions between other genetic or environmental factors being of secondary importance in disease expression. If the putative EFHC1 disease alleles are in fact causative, as is the case for genes involved in several severe, monogenic forms of epilepsy (e.g., SCN1A in Dravet syndrome21), their interpretation in the clinic will be substantially different than if they are merely susceptibility alleles. Therefore, it is essential that the risk attributed to genetic variants not be overstated. Furthermore, understanding which category EFHC1 fits into is of importance in understanding the mechanisms underlying epilepsy susceptibility. This is especially important because, unlike the ion channel–encoding candidate genes, which have been studied extensively in epileptogenesis, EFHC1 appears to function in neural development and differentiation,22,23 a mechanism for epileptogenesis only recently being widely acknowledged.1,24 Because of the need to properly assess the effect of EFHC1 disease alleles in JME, we investigated the reportedly strong effect of EFHC1 coding mutations in Hispanic patients with epilepsy, where the effect is presumed to be particularly large. Our controls were carefully selected so as to have no family history of epilepsy (as opposed to the unphenotyped individuals in the EVS [http://evs.gs.washington.edu/EVS] and 1,000 Genomes databases [http://www.1000genomes.org]. We measured the frequency of the reportedly pathogenic EFHC1 P77T-R221H and F229L alleles in specific populations and used 1,000 Genomes data to supplement our findings. Our study does not find an excess of EFHC1 mutations in JME patients, but may suggest a possible ancestry-specific component governing susceptibility to EFHC1-based JME. Such possible interactions must be considered when assigning risk values to susceptibility alleles in JME and in all complex disorders, especially when considering such variants in applications such as designing genetic testing panels for assessing risk for a particular disease.

Summary Mutations in KCNQ2 and KCNQ3 were originally described in infants with benign familial neonatal seizures (BFNS). Recently, KCNQ2 mutations have also been shown to cause epileptic encephalopathy. This report describes three infants carrying abnormalities of KCNQ2 and one infant with a KCNQ3 mutation. The different KCNQ2 abnormalities led to different phenotypes and included a novel intragenic duplication, c.419_430dup, in an infant with BFNS, a 0.761Mb 20q13.3 contiguous gene deletion in an infant with seizures at 3 months, and a recurrent de novo missense mutation c.881C>T in a neonate with “KCNQ2-encephalopathy.” The mutation in KCNQ3, c.989G>A, was novel and occurred in an infant with BFNS. KCNQ-related seizures often present with tonic/clonic manifestations, cyanosis, or apnea. Certain genotype–phenotype correlations help predict outcome. Similarly affected family members suggests benign familial “KCNQ-related” epilepsy, whereas neonatal seizures with unexplained multifocal epileptiform discharges or burst suppression on electroencephalography, and acute abnormalities of the basal ganglia/thalami are suggestive of KCNQ2-encephalopathy, which is often sporadic. 20q13.33 contiguous gene deletion encompassing KCNQ2 may harbor atypical features depending on deletion size. Although the phenotype often guides direct targeted gene testing in these conditions, array CGH should also be considered in suspected sporadic or atypical familial cases to diagnose 20q13.33 deletion.

Objective Mutations in KCNQ2 and KCNQ3, encoding the voltage-gated potassium channels KV7.2 and KV7.3, are known to cause benign familial neonatal seizures mainly by haploinsufficiency. Here, we set out to determine the disease mechanism of 7 de novo missense KCNQ2 mutations that were recently described in patients with a severe epileptic encephalopathy including pharmacoresistant seizures and pronounced intellectual disability. Methods Mutations were inserted into the KCNQ2 cDNA. Potassium currents were recorded using 2-microelectrode voltage clamping, and surface expression was analyzed by a biotinylation assay in cRNA-injected Xenopus laevis oocytes. Results We observed a clear loss of function for all mutations. Strikingly, 5 of 7 mutations exhibited a drastic dominant-negative effect on wild-type KV7.2 or KV7.3 subunits, either by globally reducing current amplitudes (3 pore mutations) or by a depolarizing shift of the activation curve (2 voltage sensor mutations) decreasing potassium currents at the subthreshold level at which these channels are known to critically influence neuronal firing. One mutation significantly reduced surface expression. Application of retigabine, a recently marketed KV7 channel opener, partially reversed these effects for the majority of analyzed mutations. Interpretation The development of severe epilepsy and cognitive decline in children carrying 5 of the 7 studied KCNQ2 mutations can be related to a dominant-negative reduction of the resulting potassium current at subthreshold membrane potentials. Other factors such as genetic modifiers have to be postulated for the remaining 2 mutations. Retigabine or similar drugs may be used as a personalized therapy for this severe disease. Ann Neurol 2014;75:382–394

International audience; Mutations in KCNQ2 and KCNQ3 genes are responsible for benign familial neonatal seizures and epileptic encephalopathies. Some of these mutations have been shown to alter the binding of calmodulin (CaM) to specific C-terminal motifs of KCNQ subunits, known as the A and B helices. Here, we show that the mutation I342A in the A helix of KCNQ3 abolishes CaM interaction and strongly decreases the heteromeric association with KCNQ2. The assembly of KCNQ2 with KCNQ3 is essential for their expression at the axon initial segment (AIS). We find that the I342A mutation alters the targeting of KCNQ2/3 subunits at the AIS. However, the traffic of the mutant channels was rescued by provision of exogenous CaM. We show that CaM enhances the heteromeric association of KCNQ2/KCNQ3-I342A subunits by binding to their B helices in a calcium-dependent manner. To further assert the implication of CaM in channel assembly, we inserted a mutation in the second coil-coil domain of KCNQ2 (KCNQ2-L638P) to prevent its heteromerization with KCNQ3. We observe that the expression of a Ca(2+)-insensitive form of CaM favours the assembly of KCNQ3 with KCNQ2-L638P. Our data thus indicate that both apoCaM and Ca(2+)/CaM bind to the C-terminal domains of KCNQ2 and KCNQ3 subunits, and regulate their heteromeric assembly. Hence, CaM may control the composition and distribution of KCNQ channels in neurons.

Summary Purpose KCNQ2 mutations have been found in patients with benign familial neonatal seizures, myokymia, or early onset epileptic encephalopathy (EOEE). In this study, we aimed to delineate the clinical spectrum of EOEE associated with KCNQ2 mutation. Methods A total of 239 patients with EOEE, including 51 cases with Ohtahara syndrome and 104 cases with West syndrome, were analyzed by high-resolution melting (HRM) analysis or whole-exome sequencing. Detailed clinical information including electroencephalography (EEG) and brain magnetic resonance imaging (MRI) were collected from patients with KCNQ2 mutation. Key Findings A total of nine de novo and one inherited mutations were identified (two mutations occurred recurrently). The initial seizures, which were mainly tonic seizures, occurred in the early neonatal period in all 12 patients. A suppression-burst pattern on EEG was found in most. Only three patients showed hypsarrhythmia on EEG; eight patients became seizure free when treated with carbamazepine, zonisamide, phenytoin, topiramate, or valproic acid. Although the seizures were relatively well controlled, moderate-to-profound intellectual disability was found in all except one patient who died at 3 months. Significance De novo KCNQ2 mutations are involved in EOEE, most of which cases were diagnosed as Ohtahara syndrome. These cases showed distinct features with early neonatal onset, tonic seizures, a suppression-burst EEG pattern, infrequent evolution to West syndrome, and good response to sodium channel blockers, but poor developmental prognosis. Genetic testing for KCNQ2 should be considered for patients with EOEE.

Background/Purpose Pediatric epilepsy caused by a KCNQ2 gene mutation usually manifests as benign familial neonatal seizures (BFNS) during the 1 st week of life. However, the exact mechanism, phenotype, and genotype of the KCNQ2 mutation are unclear. Methods We studied the KCNQ2 genotype from 75 nonconsanguineous patients with childhood epilepsy without an identified cause (age range: from 2 days to 18 years) and from 55 healthy adult controls without epilepsy. KCNQ2 mutation variants were transfected into HEK293 cells to investigate what functional changes they induced. Results Four (5%) of the patients had the E515D KCNQ2 mutation, which the computer-based PolyPhen algorithm predicted to be deleterious. Their seizure outcomes were favorable, but three had an intellectual disability. Two patients with E515D presented with continuous spikes and waves during slow-wave sleep (CSWS), and the other two presented with BFNS. We also analyzed 10 affected family members with the same KCNQ2 mutation: all had epilepsy (8 had BFNS and 2 had CSWS). A functional analysis showed that the recordings of the E515D currents were significantly different ( p KCNQ2 E515D variants are less sensitive to voltage and require stronger depolarization to reach opening probabilities than those with the wild type or N780T (a benign polymorphism). Conclusion KCNQ2 mutations can cause various phenotypes in children: they lead to BFNS and CSWS. We hypothesize that patients with the KCNQ2 E515D mutation are susceptible to seizures.

Benign neonatal sleep myoclonus is an uncommon, nonepileptic disorder characterized by myoclonic jerks appearing in the neonatal period that occur predominantly during sleep. Although self-limiting, the disorder is frequently confused with epileptic neonatal seizures. A few familial cases have been reported; however the genetics has not been studied. We ascertained 3 families with 2 or more affected individuals and analyzed the pedigrees. We used microsatellite markers to determine if the disorder was possibly linked to KCNQ2 or KCNQ3, the 2 genes that cause most cases of benign familial neonatal seizures, a disorder that it could be easily confused with. The 3 pedigrees, including one with 4 affected individuals, were suggestive of autosomal dominant inheritance. The loci for KCNQ2 and KCNQ3 were excluded in the 2 larger families. We conclude that benign neonatal sleep myoclonus can show autosomal dominant inheritance and is not allelic with benign familial neonatal seizures. Refereed/Peer-reviewed

Changes in voltage-dependent gating represent a common pathogenetic mechanism for genetically inherited channelopathies, such as benign familial neonatal seizures or peripheral nerve hyperexcitability caused by mutations in neuronal K(v)7.2 channels. Mutation-induced changes in channel voltage dependence are most often inferred from macroscopic current measurements, a technique unable to provide a detailed assessment of the structural rearrangements underlying channel gating behavior; by contrast, gating currents directly measure voltage-sensor displacement during voltage-dependent gating. In this work, we describe macroscopic and gating current measurements, together with molecular modeling and molecular-dynamics simulations, from channels carrying mutations responsible for benign familial neonatal seizures and/or peripheral nerve hyperexcitability; K(v)7.4 channels, highly related to K(v)7.2 channels both functionally and structurally, were used for these experiments. The data obtained showed that mutations affecting charged residues located in the more distal portion of S(4) decrease the stability of the open state and the active voltage-sensing domain configuration but do not directly participate in voltage sensing, whereas mutations affecting a residue (R4) located more proximally in S(4) caused activation of gating-pore currents at depolarized potentials. These results reveal that distinct molecular mechanisms underlie the altered gating behavior of channels carrying disease-causing mutations at different voltage-sensing domain locations, thereby expanding our current view of the pathogenesis of neuronal hyperexcitability diseases.

Benign familial neonatal seizures (BFNS) are a dominant epilepsy syndrome caused by mutations in the voltage-gated potassium channels K(V) 7.2 and K(V) 7.3. We examined the molecular pathomechanism of a BFNS-causing mutation (p.N258S) in the extracellular S5-H5 loop of K(V) 7.2. Wild type (WT) and mutant channels, expressed in both Xenopus laevis oocytes and CHO cells, were studied using electrophysiological techniques. The results revealed a pronounced loss-of-function with a dominant-negative effect of the mutant on WT K(V) 7.2 and K(V) 7.3 channels. Since single-channel recordings of K(V) 7.3-K(V) 7.2 and K(V) 7.3-N285S concatemers showed similar properties for both constructs, we hypothesized that the observed reduction in current amplitude was due to a folding and trafficking defect, which was confirmed by biochemical and immunocytochemical experiments revealing a reduced number of mutant channels in the surface membrane. Furthermore, rescuing experiments revealed that upon specific incubation of transfected CHO cells-either at lower temperatures of

Genetic factors play an increasingly recognized role in idiopathic epilepsies. Positional cloning strategies in multigenerational families with autosomal dominant transmission have revealed several genes. Most of epilepsy-genes encode ion channels subunits or receptors for neurotransmitters: voltage-gated potassium channels (KCNQ2, KCNQ3) for benign familial neonatal seizures; voltage-gated sodium channel subunits (SCN1B, SCN1A, SCN2A) in generalized epilepsy with febrile seizures plus (GEFS +), severe myoclonic epilepsy of infancy or Dravet syndrome and benign familial neonatal-infantile seizures; nicotinic acetylcholine receptor subunits (CHRNA4, CHRNA2, CHRNB2) in autosomal dominant nocturnal frontal lobe epilepsy, and GABA A receptor subunits for GEFS+ and autosomal dominant juvenile myoclonic epilepsy.

Interest in Morvan's disease or syndrome has grown, owing to its close links with various potassium channelopathies. Potassium is crucial for gating mechanisms (channel opening and closing), and especially for repolarization. Defective potassium regulation can lead to neuronal hyperexcitability. There are three families of potassium channels: voltage-gated potassium channels or VGKC (Kv1.1-Kv1.8), inward rectifier K+ channels (Kir), and two-pore channels (K2p). VGK channels are the commonest, and especially those belonging to the Shaker group (neuromyotonia and Morvan's syndrome, limbic encephalitis, and type 1 episodic ataxia). Brain and heart K+ channelopathies are a separate group due to KCNQ1 mutation (severe type 2 long QT syndrome). Kv7 channel mutations (in KNQ2 and KCNQ3) are responsible for benign familial neonatal seizures. Mutation of the Ca+ activated K+ channel gene causes epilepsy and paroxysmal dyskinesia. Inward rectifier K+ channels regulate intracellular potassium levels. The DEND syndrome, a treatable channelopathy of the brain and pancreas, is due to KCNJ1 mutation. Andersen's syndrome, due to KCNJ2 mutation, is characterized by periodic paralysis, cardiac arrythmia, and dysmorphia. Voltage-insensitive K2p channelopathies form a final group.

Objective: Mutations of the genes encoding subunits of potassium voltage-gated channel, KCNQ2 and KCNQ3 , have been identified in patients with benign familial neonatal seizures (BFNS). This study set out to determine the frequency of microchromosomal deletions of KCNQ2 or KCNQ3 associated with BFNS. Methods: The study subjects were patients with BFNS (n = 22). Microdeletions were sought by multiplex ligation-dependent probe amplification and then confirmed by fluorescence in situ hybridization and characterized by array-based comparative genomic hybridization. Results: Heterozygous multiple exonic deletions of KCNQ2 were identified in 4 of 22 patients with BFNS. Concomitant deletions of adjacent genes, including nicotinic cholinergic receptor α4 ( CHRNA4 ), were detected in 2 of the 4 cases. The clinical courses of patients with deletions of both KCNQ2 and CHRNA4 were those of typical BFNS, and none presented with the phenotype of autosomal dominant nocturnal frontal lobe epilepsy, some of which are caused by mutations of CHRNA4 . Conclusions: Our findings indicate that the clinical courses of patients with deletions of both KCNQ2 and CHRNA4 are indistinguishable from those of patients with deletions of KCNQ2 only.

This article aimed to review the latest genes associated with idiopathic focal and generalized epilepsies.PubMed and Entrez Gene searches pertaining to this work was conducted using specific keyword search terms related to genes and various listed subtopics related to idiopathic epilepsy syndromes.Mutations in the cholinergic receptor, neuronal nicotinic, alpha2, alpha4 and beta2 subunit genes have been found in autosomal dominant nocturnal frontal lobe epilepsy. Mutations of potassium voltage-gated channel, KQT-like subfamily, members 2 and 3 genes were identified to be responsible for benign familial neonatal seizures. The voltage-gated sodium channel genes and gamma-aminobutyric acid receptor alpha subunit genes may be involved in the pathogenesis of generalized epilepsy with febrile seizure plus. Mutations of gamma-aminobutyric acid receptor alpha1, gamma-aminobutyric acid receptor delta, calcium channel voltage-dependent beta4 subunit and chloride channel 2 gene are associated with juvenile myoclonic epilepsy. In addition, mutations of leucine-rich, glioma-inactivated 1 gene leads to genetic abnormalities of familial lateral temporal lobe epilepsy. EF-hand domain (C-terminal)-containing 1 gene can cause some patterns of juvenile myoclonic and juvenile absence epilepsies.Genetic factors play an important role in idiopathic epilepsy syndromes. Ion channel genes and some non-ion channel genes contribute to the pathogenesis of idiopathic epilepsies. Based on these findings, genetic diagnosis and new treatment strategies to part of idiopathic epilepsies become possible in the future.

Epileptic seizures occur commonly in the first days after birth.1 Such neonatal seizures are usually symptomatic, arising as a result of developmental abnormalities, in utero injuries, perinatal hypoxia-ischemia, infection, and other causes. Patients experiencing neonatal seizures are at substantial risk of mortality and long-term morbidity, including static encephalopathy, cerebral palsy, and chronic epilepsy.2, 3 The first-line drugs given to neonates, phenobarbital and phenytoin, are effective in less than 50% of cases4. Moreover, phenobarbital, benzodiazepines and phenytoin have been shown to cause widespread neuronal apoptosis when given to young rodents, raising concerns about administration of these drugs in human infants.5-7 The need for new treatments for neonatal seizures that are both safer and more effective has been widely acknowledged.1, 7, 8 Genetic, physiological, and pharmacological evidence calls attention to KCNQ potassium channels (also termed Kv7 and M-channels) as potential molecular targets for treatment of neonatal seizures.9 Loss-of-function mutations in the KCNQ2 and KCNQ3 genes cause benign familial neonatal seizures (BFNS), an uncommon but highly penetrant, dominantly-inherited syndrome characterized by seizures that recur frequently over the first weeks of life.10-12 Experimental inhibition of KCNQ channels in rodents, by pharmacological and genetic methods, dramatically promotes neuronal and network hyperexcitability and seizures in neonatal animals and in slices from neonatal brain; these effects diminish or disappear progressively with maturation. KCNQ2 and KCNQ3 are highly expressed on both myelinated and unmyelinated axons, and are strictly colocalized with clusters of voltage-gated sodium channels in the proximal axon (where action potentials initiate) and at the nodes of Ranvier.14, 17-19 It has been proposed that this strategic localization at the exact sites where action potentials arise and are conducted allows KCNQ channels to exert strong control over neuronal firing.17, 18 Selective neuronal KCNQ channel openers have been identified among drugs already in human use, including diclofenac (a widely used anti-inflammatory agent), flupirtine (a non-opioid analgesic in approved use in Europe) and retigabine (which is currently in phase 3 clinical trials for adult partial epilepsy in the U.S. and Europe).20-22 Additional potent and selective KCNQ channel openers have recently been discovered through directed high-throughput screening and have begun to be subjected to preclinical testing for adult epilepsy and pain.23-25 KCNQ openers increase channel activity significantly above normal levels by speeding opening rates, slowing closing rates, and reducing the membrane depolarization required for activation. Flupirtine and retigabine are close structural analogues (Figure 1). Flupirtine and retigabine have been shown to exhibit neuroprotective activity in several experimental systems, including models of infantile neuronal ceroid lipofuscinosis.26-28 Remarkably, KCNQ channel opener compounds have not been evaluated as a potential treatments for neonatal seizures or status epilepticus (SE). The human KCNQ2 and KCNQ3 channel loss-of-function phenotype, BFNS, suggests the channels are present, active and important during the neonatal period. Thus, augmenting KCNQ channel activity is a rational strategy for safe suppression of hyperexcitability during the neonatal period. Figure 1 Flupirtine and retigabine are close analogues In early trials using adult rodent models and adult human patients, flupirtine showed evidence of anti-epileptic efficacy.29 Here, we have tested flupirtine's utility for treatment of neonatal seizures and SE. We have compared flupirtine to two drugs widely used for neonatal seizures, phenobarbital and diazepam. In two well-established models, using the glutamate receptor agonist kainic acid or the convulsant gas flurothyl, flupirtine appeared more effective than either standard agent in preventing the onset of seizures in rat pups. In the kainic acid model, which produces seizures that evolve into SE lasting hours, flupirtine was more effective than either standard agent in diminishing the severity of electrographic and behavioral seizures. Indeed, flupirtine was effective even when its administration was withheld until after kainate-induced seizure activity had progressed to continuous SE. These findings indicate that KCNQ channel opener therapy for neonatal seizures deserves further investigation.

The K(v)7.2 subunits are the main molecular determinants of the M-current, a widespread K(+) current regulating neuronal excitability. Mutations in the K(v)7.2 gene cause benign familial neonatal seizures, an autosomally inherited human epilepsy. The benign familial neonatal seizure-causing mutations include those at arginine residues at positions 207 and 214 in the S(4) segment of K(v)7.2. In this study, each of the six S(4) arginines was individually replaced with neutral glutamines, and the functional properties of mutant channels were studied by whole-cell and single-channel voltage-clamp measurements. The results obtained suggest that each S(4) arginine residue plays a relevant role in the voltage-dependent gating of K(v)7.2 channels. In particular, a decreased positive charge at the N-terminal end of S(4) stabilized the activated state of the voltage-sensor, whereas positive-charge neutralization at the C-terminal end of S(4) favored the resting conformation. Strikingly, neutralization of a single arginine at position 201 was sufficient to cause a significant loss of voltage dependence in channel activation. Moreover, by comparing the functional properties of glutamine versus tryptophan substitution, we found steric bulk to play a relevant role at position 207, but not at position 214, in which the main functional effect of this disease-causing mutation seems to be a consequence of the loss of the positive charge.

Voltage-gated K+ channels of the KV7 (KCNQ) family have been identified in the last 10–15 years by discovering the causative genes for three autosomal dominant diseases: cardiac arrhythmia (long QT syndrome) with or without congenital deafness (KCNQ1), a neonatal epilepsy (KCNQ2 and KCNQ3) and progressive deafness alone (KCNQ4). A fifth member of this gene family (KCNQ5) is not affected in a disease so far. Four genes (KCNQ2–5) are expressed in the nervous system. This review is focused on recent findings on the neuronal KV7 channelopathies, in particular on benign familial neonatal seizures (BFNS) and peripheral nerve hyperexcitability (PNH, neuromyotonia, myokymia) caused by KCNQ2 mutations. The phenotypic spectrum associated with KCNQ2 mutations is probably broader than initially thought, as patients with severe epilepsies and developmental delay, or with Rolando epilepsy have been described. With regard to the underlying molecular pathophysiology, it has been shown that mutations with very subtle changes restricted to subthreshold voltages can cause BFNS thereby proving in a human disease model that this is the relevant voltage range for these channels to modulate neuronal firing. The two mutations associated with PNH induce much more severe channel dysfunction with a dominant negative effect on wild type (WT) channels. Finally, KV7 channels present interesting targets for new therapeutic approaches to diseases caused by neuronal hyperexcitability, such as epilepsy, neuropathic pain, and migraine. The molecular mechanism of KV7 activation by retigabine, which is in phase III clinical testing to treat pharmacoresistant focal epilepsies, has been recently elucidated as a stabilization of the open conformation by binding to the pore region.

Benign familial neonatal convulsions (BFNC, also named benign familial neonatal seizures, BFNS) is a rare autosomal dominant inherited epilepsy syndrome with clinical and genetic heterogeneity. Two voltage-gated potassium channel subunit genes, KCNQ2 and KCNQ3, have been identified to cause BFNC1 and BFNC2, respectively. To date, only three mutations of KCNQ3, all located within exon 5, have been reported. By limited linkage analysis and mutation analysis of KCNQ3 in a Chinese family with BFNC, we identified a novel missense mutation of KCNQ3, c.988C>T located within exon 6. c.988C>T led to the substitution Cys for Arg in amino acid position 330 (p.R330C) in KCNQ3 potassium channel, which possibly impaired the neuronal M-current and altered neuronal excitability. Seizures of all BFNC patients started from day 2 to 3 after birth and remitted during 1 month, and no recurrence was found. One family member who displayed fever-associated seizures for two times at age 5 years and was diagnosed as febrile seizures, however, did not carry this mutation, which suggests that febrile seizures and BFNC have different pathogenesis. To our knowledge, this is the first report of KCNQ3 mutation in Chinese family with BFNC.

We first review the clinical presentation and current therapeutic approaches available for treating Unverricht-Lundborg disease (ULD), a progressive myoclonus epilepsy. Next, we describe the identification of disease causing mutations in the gene encoding cystatin B (CSTB). A Cstb-deficient mouse model, which recapitulates the key features of ULD including myoclonic seizures, ataxia, and neuronal loss, was generated to shed light on the mechanisms contributing to disease pathophysiology. Studies with this model have elucidated the diverse biological roles for Cstb from functioning as a protease inhibitor, to regulating glial activation, oxidative stress, serotonergic neurotransmission, and hyperexcitability. These findings set the stage for future studies that may open avenues to improved therapeutic approaches.

Kv7.2 and Kv7.3 subunits underlie the M-current, a neuronal K+ current characterized by an absolute functional requirement for phosphatidylinositol 4,5-bisphosphate (PIP2). Kv7.2 gene mutations cause early-onset neonatal seizures with heterogeneous clinical outcomes, ranging from self-limiting benign familial neonatal seizures to severe early-onset epileptic encephalopathy (Kv7.2-EE). In this study, the biochemical and functional consequences prompted by a recurrent variant (R325G) found independently in four individuals with severe forms of neonatal-onset EE have been investigated. Upon heterologous expression, homomeric Kv7.2 R325G channels were non-functional, despite biotin-capture in Western blots revealed normal plasma membrane subunit expression. Mutant subunits exerted dominant-negative effects when incorporated into heteromeric channels with Kv7.2 and/or Kv7.3 subunits. Increasing cellular PIP2 levels by co-expression of type 1γ PI(4)P5-kinase (PIP5K) partially recovered homomeric Kv7.2 R325G channel function. Currents carried by heteromeric channels incorporating Kv7.2 R325G subunits were more readily inhibited than wild-type channels upon activation of a voltage-sensitive phosphatase (VSP), and recovered more slowly upon VSP switch-off. These results reveal for the first time that a mutation-induced decrease in current sensitivity to PIP2 is the primary molecular defect responsible for Kv7.2-EE in individuals carrying the R325G variant, further expanding the range of pathogenetic mechanisms exploitable for personalized treatment of Kv7.2-related epilepsies.

BACKGROUND: Benign familial neonatal seizures are most often caused by mutations in the voltage-gated potassium channel subunit gene KCNQ2. More than 60 mutations have been described in BFNS families, approximately half of which lead to protein truncation. The hypothesis of this study was that deletion or duplication of >or=1 exons of KCNQ2 could cause BFNS in cases without coding or splicing mutations. METHODS: Multiplex ligation-dependent probe amplification (MLPA) was used to test a group of 21 unrelated patients with clinical features consistent with either BFNS, benign familial neonatal-infantile seizures or sporadic neonatal seizures, for exonic deletions and duplications. RESULTS: Three deletions and one duplication mutation were identified in four familial cases and cascade testing of their available family members showed that the mutations segregated with the phenotype in each family. The junction fragment for one of the deletions was amplified by PCR and sequenced to characterise the breakpoint and verify that a deletion had occurred. CONCLUSIONS: Submicroscopic deletions or duplications of KCNQ2 are seen in a significant proportion of BFNS families: four of nine (44%) cases previously testing negative for coding or splice site mutation by sequencing KCNQ2 and KCNQ3. MLPA is an efficient second-tier testing strategy for KCNQ2 to identify pathogenic intragenic mutations not detectable by conventional DNA sequencing methods.

Background: Peripheral nerve hyperexcitability (PNH) is characterized by muscle overactivity due to spontaneous discharges of lower motor neurons usually associated with antibodies against voltage-gated potassium channels. PNH may also occur in combination with episodic ataxia or epilepsy caused by mutations in K V 1.1 or K V 7.2 channels. Only one PNH-associated mutation has been described so far in K V 7.2 (R207W), in a family with both PNH and neonatal seizures. Methods: PNH was characterized by video and electromyography. The KCNQ2 gene was sequenced and K V 7.2 channels were functionally characterized using two-microelectrode voltage-clamping in Xenopus oocytes. Results: In a patient with PNH without other neurologic symptoms, we identified a novel KCNQ2 mutation predicting loss of a charged residue within the voltage sensor of K V 7.2 (R207Q). Functional analysis of both PNH-associated mutants revealed large depolarizing shifts of the conductance-voltage relationships and marked slowing of the activation time course compared to wild type (WT) channels, less pronounced for R207Q than R207W. Co-expression of both mutant with WT channels revealed a dominant negative effect reducing the relative current amplitudes after short depolarizations by >70%. The anticonvulsant retigabine, an activator of neuronal K V 7 channels, reversed the depolarizing shift. Conclusions: Mutations in KCNQ2 can cause idiopathic PNH alone and should be considered in sporadic cases. Both K V 7.2 mutants produce PNH by changing voltage-dependent activation with a dominant negative effect on the WT channel. This distinguishes them from all hitherto examined Kv7.2 or K V 7.3 mutations which cause neonatal seizures by haploinsufficiency. Retigabine may be beneficial in treating PNH.

Summary Background Benign familial neonatal convulsions (BFNC) is a rare autosomal dominant seizure disorder usually described to be characterized by a benign course, spontaneous remission and normal psychomotor development. The latter statement had come under consideration when a few case reports of families with less than favorable outcomes were published. Methods Since 1998 a total of 112 families suspected to have BFNC have been referred to our lab for genetic testing. Within this sample we identified private KCNQ2 mutations in 17 BFNC families. For 10 of those 17 families follow up information about the psychomotor development and the outcome were available. Results In 4 (40%) of the 10 families at least 1 affected individual showed delayed psychomotor development or mental retardation. Three of the four mutations were familial, while the fourth mutation was de novo. Mutations associated with an unfavorable outcome tended to be located within the functionally critical S5/S6 regions of the KCNQ2 gene. Conclusions Our data raise the question if BFNC can indeed be described as a benign disorder, and which are the genetic and/or environmental factors that influence the outcome.

KCNQ2 and KCNQ3 K+ channel subunits underlie the muscarinic-regulated K+ current (I(KM)), a widespread regulator of neuronal excitability. Mutations in KCNQ2- or KCNQ3-encoding genes cause benign familiar neonatal convulsions (BFNCs), a rare autosomal-dominant idiopathic epilepsy of the newborn. In the present study, we have investigated, by means of electrophysiological, biochemical, and immunocytochemical techniques in transiently transfected cells, the consequences prompted by a BFNC-causing 1-bp deletion (2043deltaT) in the KCNQ2 gene; this frameshift mutation caused the substitution of the last 163 amino acids of the KCNQ2 C terminus and the extension of the subunit by additional 56 residues. The 2043deltaT mutation abolished voltage-gated K+ currents produced upon homomeric expression of KCNQ2 subunits, dramatically reduced the steady-state cellular levels of KCNQ2 subunits, and prevented their delivery to the plasma membrane. Metabolic labeling experiments revealed that mutant KCNQ2 subunits underwent faster degradation; 10-h treatment with the proteasomal inhibitor MG132 (20 microm) at least partially reversed such enhanced degradation. Co-expression with KCNQ3 subunits reduced the degradation rate of mutant KCNQ2 subunits and led to their expression on the plasma membrane. Finally, co-expression of KCNQ2 2043deltaT together with KCNQ3 subunits generated functional voltage-gated K+ currents having pharmacological and biophysical properties of heteromeric channels. Collectively, the present results suggest that mutation-induced reduced stability of KCNQ2 subunits may cause epilepsy in neonates.

Recent exciting developments in epilepsy genetics have led to significant insights into the mechanisms underlying seizure disorders. Success in epilepsy genetics research to date has resulted from identification of genes responsible for rare monogenic disorders, the majority encoding either voltage- or ligand-gated ion channels. For some conditions, such as benign familial neonatal seizures, an understanding of the underlying genetics is helpful in predicting prognosis. However, for other disorders, such as autosomal dominant nocturnal frontal lobe epilepsy, phenotypic severity is determined by factors other than the major dominant nicotinic subunit mutation found in some families. Further complexity arises when single-gene mutations give rise to heterogeneous phenotypes, as typically occur with generalized epilepsy with febrile seizures plus. Another area of increasing genetic endeavour, pharmacogenetics will allow tailoring of antiepileptic medication for each patient. Pharmacogenetics explores genetic polymorphisms in genes coding for drug-metabolizing enzymes, receptors and transporters. Polymorphisms have been identified that result in marked ethnic and interindividual differences in response to treatment. With further understanding of the impact of these differences, pharmacogenetic screening is likely to guide the management of epilepsy in the future.