Melody Li

KCNT1 gain of function in 2 epilepsy phenotypes is reversed by quinidine

Mutations in KCNT1 have been implicated in autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) and epilepsy of infancy with migrating focal seizures (EIMFS). More recently, a whole exome sequencing study of epileptic encephalopathies identified an additional de novo mutation in 1 proband with EIMFS. We aim to investigate the electrophysiological and pharmacological characteristics of hKCNT1 mutations and examine developmental expression levels.

Quinidine in the treatment of KCNT1-positive epilepsies

We report 2 patients with drug‐resistant epilepsy caused by KCNT1 mutations who were treated with quinidine. Both mutations manifested gain of function in vitro, showing increased current that was reduced by quinidine. One, who had epilepsy of infancy with migrating focal seizures, had 80% reduction in seizure frequency as recorded in seizure diaries, and partially validated by objective seizure evaluation on EEG. The other, who had a novel phenotype, with severe nocturnal focal and secondary generalized seizures starting in early childhood with developmental regression, did not improve. Although quinidine represents an encouraging opportunity for therapeutic benefits, our experience suggests caution in its application and supports the need to identify more targeted drugs for KCNT1 epilepsies. Ann Neurol 2015;78:995–999

A functional correlate of severity in alternating hemiplegia of childhood

OBJECTIVE Mutations in ATP1A3, the gene that encodes the α3 subunit of the Na(+)/K(+) ATPase, are the primary cause of alternating hemiplegia of childhood (AHC). Correlations between different mutations and AHC severity were recently reported, with E815K identified in severe and D801N and G947R in milder cases. This study aims to explore the molecular pathological mechanisms in AHC and to identify functional correlates for mutations associated with different levels of disease severity. METHODS Human wild type ATP1A3, and E815K, D801N and G947R mutants were expressed in Xenopus laevis oocytes and Na(+)/K(+) ATPase function measured. Structural homology models of the human α3 subunit containing AHC mutations were created. RESULTS The AHC mutations examined all showed similar levels of reduction in forward cycling. Wild type forward cycling was reduced by coexpression with any mutant, indicating dominant negative interactions. Proton transport was measured and found to be selectively impaired only in E815K. Homology modeling showed that D801 and G947 lie within or near known cation binding sites while E815 is more distal. Despite its effect on proton transport, E815K was also distant from the proposed proton transport route. INTERPRETATION Loss of forward cycling and dominant negativity are common and likely necessary pathomechanisms for AHC. In addition, loss of proton transport correlated with severity of AHC. D801N and G947R are likely to directly disrupt normal Na(+)/K(+) binding while E815K may disrupt forward cycling and proton transport via allosteric mechanisms yet to be elucidated.

Dominant KCNA2 mutation causes episodic ataxia and pharmacoresponsive epilepsy.

Objective: To identify the genetic basis of a family segregating episodic ataxia, infantile seizures, and heterogeneous epilepsies and to study the phenotypic spectrum of KCNA2 mutations. Methods: A family with 7 affected individuals over 3 generations underwent detailed phenotyping. Whole genome sequencing was performed on a mildly affected grandmother and her grandson with epileptic encephalopathy (EE). Segregating variants were filtered and prioritized based on functional annotations. The effects of the mutation on channel function were analyzed in vitro by voltage clamp assay and in silico by molecular modeling. KCNA2 was sequenced in 35 probands with heterogeneous phenotypes. Results: The 7 family members had episodic ataxia (5), self-limited infantile seizures (5), evolving to genetic generalized epilepsy (4), focal seizures (2), and EE (1). They had a segregating novel mutation in the shaker type voltage-gated potassium channel KCNA2 (CCDS_827.1: c.765_773del; p.255_257del). A rare missense SCN2A (rs200884216) variant was also found in 2 affected siblings and their unaffected mother. The p.255_257del mutation caused dominant negative loss of channel function. Molecular modeling predicted repositioning of critical arginine residues in the voltage-sensing domain. KCNA2 sequencing revealed 1 de novo mutation (CCDS_827.1: c.890G>A; p.Arg297Gln) in a girl with EE, ataxia, and tremor. Conclusions: A KCNA2 mutation caused dominantly inherited episodic ataxia, mild infantile-onset seizures, and later generalized and focal epilepsies in the setting of normal intellect. This observation expands the KCNA2 phenotypic spectrum from EE often associated with chronic ataxia, reflecting the marked variation in severity observed in many ion channel disorders.

Cortical microarchitecture changes in genetic epilepsy

Objective: The human GABAAγ2(R43Q) mutation is associated with genetic epilepsy. Because of the role of γ-aminobutyric acid (GABA) in brain development, we asked whether this epilepsy mutation might affect excitability by changing cortical cytoarchitecture. Methods: We used a mouse model harboring a heterozygous R43Q missense mutation in the GABAA receptor subunit γ2, as identified in a family with absence epilepsy and febrile seizures. Three-dimensional quantification of immunostained neurons (NeuN), inhibitory neurons (GABA), and inhibitory neuron subpopulations (calretinin, parvalbumin, and calbindin) was performed in fiducial somatosensory cortical columns of seizure-naive GABAAγ2(R43Q) and control mice. Results: Of note, the densities of GABA-, calretinin-, parvalbumin-, and calbindin-containing neurons were increased, and somewhat perplexing, the ratio between putative excitatory and inhibitory neurons was decreased in GABAAγ2(R43Q) mice. Differences were detected in a layer-specific manner with greater overall effects in layers 2/3, 5, and 6, as compared with layers 1 and 4. Conclusions: Our results suggest that the γ2(R43Q) mutation significantly affects cortical microcircuitry in the cortex of this model of human genetic epilepsy.

Precision therapy for epilepsy due to KCNT1 mutations: A randomized trial of oral quinidine

Objective To evaluate quinidine as a precision therapy for severe epilepsy due to gain of function mutations in the potassium channel gene KCNT1. Methods A single-center, inpatient, order-randomized, blinded, placebo-controlled, crossover trial of oral quinidine included 6 patients with severe autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) due to KCNT1 mutation. Order was block randomized and blinded. Four-day treatment blocks were used with a 2-day washout between. Dose started at 900 mg over 3 divided doses then, in subsequent participants, was reduced to 600 mg, then 300 mg. Primary outcome was seizure frequency measured on continuous video-EEG in those completing the trial. Results Prolonged QT interval occurred in the first 2 patients at doses of 900 and 600 mg quinidine per day, respectively, despite serum quinidine levels well below the therapeutic range (0.61 and 0.51 μg/mL, reference range 1.3–5.0 μg/mL). Four patients completed treatment with 300 mg/d without adverse events. Patients completing the trial had very frequent seizures (mean 14 per day, SD 7, median 13, interquartile range 10–18). Seizures per day were nonsignificantly increased by quinidine (median 2, 95% confidence interval −1.5 to +5, p = 0.15) and no patient had a 50% seizure reduction. Conclusion Quinidine did not show efficacy in adults and teenagers with ADNFLE. Dose-limiting cardiac side effects were observed even in the presence of low measured serum quinidine levels. Although small, this trial suggests use of quinidine in ADNFLE is likely to be ineffective coupled with considerable cardiac risks. Clinical trials registration Australian Therapeutic Goods Administration Clinical Trial Registry (trial number 2015/0151). Classification of evidence This study provides Class II evidence that for persons with severe epilepsy due to gain of function mutations in the potassium channel gene KCNT1, quinidine does not significantly reduce seizure frequency.

Gabapentin modulates HCN4 channel voltage-dependence

Gabapentin (GBP) is widely used to treat epilepsy and neuropathic pain. There is evidence that GBP can act on hyperpolarization-activated cation (HCN) channel-mediated Ih in brain slice experiments. However, evidence showing that GBP directly modulates HCN channels is lacking. The effect of GBP was tested using two-electrode voltage clamp recordings from human HCN1, HCN2, and HCN4 channels expressed in Xenopus oocytes. Whole-cell recordings were also made from mouse spinal cord slices targeting either parvalbumin positive (PV+) or calretinin positive (CR+) inhibitory neurons. The effect of GBP on Ih was measured in each inhibitory neuron population. HCN4 expression was assessed in the spinal cord using immunohistochemistry. When applied to HCN4 channels, GBP (100 μM) caused a hyperpolarizing shift in the voltage of half activation (V1/2) thereby reducing the currents. Gabapentin had no impact on the V1/2 of HCN1 or HCN2 channels. There was a robust increase in the time to half activation for HCN4 channels with only a small increase noted for HCN1 channels. Gabapentin also caused a hyperpolarizing shift in the V1/2 of Ih measured from HCN4-expressing PV+ inhibitory neurons in the spinal dorsal horn. Gabapentin had minimal effect on Ih recorded from CR+ neurons. Consistent with this, immunohistochemical analysis revealed that the majority of CR+ inhibitory neurons do not express somatic HCN4 channels. In conclusion, GBP reduces HCN4 channel-mediated currents through a hyperpolarized shift in the V1/2. The HCN channel subtype selectivity of GBP provides a unique tool for investigating HCN4 channel function in the central nervous system. The HCN4 channel is a candidate molecular target for the acute analgesic and anticonvulsant actions of GBP.

Gain-of-function HCN2 variants in genetic epilepsy.

Genetic generalized epilepsy (GGE) is a common epilepsy syndrome that encompasses seizure disorders characterized by spike‐and‐wave discharges (SWDs). Pacemaker hyperpolarization‐activated cyclic nucleotide‐gated channels (HCN) are considered integral to SWD genesis, making them an ideal gene candidate for GGE. We identified HCN2 missense variants from a large cohort of 585 GGE patients, recruited by the Epilepsy Phenome‐Genome Project (EPGP), and performed functional analysis using two‐electrode voltage clamp recordings from Xenopus oocytes. The p.S632W variant was identified in a patient with idiopathic photosensitive occipital epilepsy and segregated in the family. This variant was also independently identified in an unrelated patient with childhood absence seizures from a European cohort of 238 familial GGE cases. The p.V246M variant was identified in a patient with photo‐sensitive GGE and his father diagnosed with juvenile myoclonic epilepsy. Functional studies revealed that both p.S632W and p.V246M had an identical functional impact including a depolarizing shift in the voltage dependence of activation that is consistent with a gain‐of‐function. In contrast, no biophysical changes resulted from the introduction of common population variants, p.E280K and p.A705T, and the p.R756C variant from EPGP that did not segregate with disease. Our data suggest that HCN2 variants can confer susceptibility to GGE via a gain‐of‐function mechanism.

Lack of response to quinidine in KCNT1-related neonatal epilepsy

To evaluate the clinical efficacy and safety of quinidine in patients with KCNT1‐related epilepsy of infancy with migrating focal seizures (EIMFS) in the infantile period and to compare with the effect of quinidine on mutant channels in vitro.

KCTD12 modulation of GABA(B) receptor function

The molecular composition and functional diversity of native GABAB receptors (GABABR) are still poorly understood, thus hindering development of selective GABABR ligands. Potassium channel tetramerization domain‐containing protein (KCTD) 12 is a GABABR auxiliary subunit and mouse KCTD12 can alter GABABR function. In this study, we sought to characterize the effects of human KCTD12 on GABABR kinetics and pharmacology, using an automated electrophysiological assay. Seizure susceptibility and ethanol consumption were also investigated in a KCTD12 knockout mouse model. Human KCTD12 co‐expression altered the kinetics of GABABR‐mediated GIRK channels, speeding rates of both activation and desensitization. Analysis of concentration‐response curves showed that KCTD12 coexpression did not alter effects of the agonists GABA or baclofen on GABABR. KCTD12 coexpression enhanced the potentiating effects of the positive allosteric modulator CGP7930, and its effects on GABABR activation and desensitization. The function of KCTD12 in vivo was examined, using the KCTD12 knockout mouse model. The knockout mice were more resistant to a pentylenetetrazole proconvulsant challenge suggesting reduced seizure susceptibility. In the two bottle preference test, KCTD12 knockout mice demonstrated a reduced consumption at high ethanol concentrations. In summary, human KCTD12 accelerated the kinetics of GABABR in vitro, in a manner possibly sensitive to allosteric pharmacological modulation. This study also provides novel in vivo evidence that the interaction between KCTD12 and GABABR is of physiological significance, and may be a mechanism to more selectively modulate GABABR.