KCNA2 Research Articles

P46 Functional investigation of a novel mutation causing a new phenotype for the KCNA1 gene

P46 Functional investigation of a novel mutation causing a new phenotype for the KCNA1 gene

Genetics of dizziness

Recent advances in next generation sequencing techniques (NGS) are increasing the number of novel genes associated with cerebellar and vestibular disorders. We have summarized clinical and molecular genetics findings in neuro-otolology during the last 2 years. Whole-exome and targeted sequencing have defined the genetic basis of dizziness including new genes causing ataxia: GBA2, TGM6, ANO10 and SYT14. Novel mutations in KCNA1 and CACNA1A genes are associated with episodic ataxia type 1 and type 2, respectively. Moreover, new variants in genes such as COCH, MYO7A and POU4F3 are associated with nonsyndromic deafness and vestibular dysfunction. Several susceptibility loci have been linked to familial vestibular migraine, suggesting genetic heterogeneity, but no specific gene has been identified. Finally, loci for complex and heterogeneous diseases such as bilateral vestibular hypofunction or familial Ménière disease have not been identified yet, despite their strong familial aggregation. Cerebellar and vestibular disorders leading to dizziness or episodic vertigo may show overlapping clinical features. A deep phenotyping including a complete familial history is a key step in performing a reliable molecular genetic diagnosis using NGS. Personalized molecular medicine will be essential to understand disease mechanisms as well as to improve their diagnosis and treatment.

High‐resolution molecular genomic autopsy reveals complex sudden unexpected death in epilepsy risk profile

Advanced variant detection in genes underlying risk of sudden unexpected death in epilepsy (SUDEP) can uncover extensive epistatic complexity and improve diagnostic accuracy of epilepsy-related mortality. However, the sensitivity and clinical utility of diagnostic panels based solely on established cardiac arrhythmia genes in the molecular autopsy of SUDEP is unknown. We applied the established clinical diagnostic panels, followed by sequencing and a high density copy number variant (CNV) detection array of an additional 253 related ion channel subunit genes to analyze the overall genomic variation in a SUDEP of the 3-year-old proband with severe myoclonic epilepsy of infancy (SMEI). We uncovered complex combinations of single nucleotide polymorphisms and CNVs in genes expressed in both neurocardiac and respiratory control pathways, including SCN1A, KCNA1, RYR3, and HTR2C. Our findings demonstrate the importance of comprehensive high-resolution variant analysis in the assessment of personally relevant SUDEP risk. In this case, the combination of de novo single nucleotide polymorphisms (SNPs) and CNVs in the SCN1A and KCNA1 genes, respectively, is suspected to be the principal risk factor for both epilepsy and premature death. However, consideration of the overall biologically relevant variant complexity with its extensive functional epistatic interactions reveals potential personal risk more accurately.

Episodic ataxia type 1 without episodic ataxia: the diagnostic utility of nerve excitability studies in individuals with KCNA1 mutations

Episodic ataxia type 1 (EA1) is caused by mutations in the KCNA1 gene encoding the fast potassium channel Kv1.1 and is characterized clinically by brief episodes of ataxia and continuous and spontaneous motor unit activity. Atypical presentations, in which the predominant manifestation is related to the peripheral nervous system, may lead to the diagnosis being missed or delayed, with the potential risk of individuals receiving inappropriate or unnecessary investigations and treatment. We present a case of a 15-year-old female with EA1 who had never had episodes of ataxia, and whose hand movements were initially thought to represent a tremor. Genetic screening for KCNA1 mutations was precipitated by the results of the nerve excitability studies (TROND protocol), which showed changes typical of reduced fast potassium channel conductance. This case highlights the utility of nerve excitability studies in identifying individuals with KCNA1 mutations.

Genome-Wide Identification and Validation of a Novel Methylation Biomarker, SDC2, for Blood-Based Detection of Colorectal Cancer

Aberrant DNA methylation has shown promise as a biomarker for the early detection of cancer. To discover novel genes frequently methylated at an early stage in colorectal cancer (CRC), DNA microarray analysis coupled with enriched methylated DNA was performed in primary tumors and compared with adjacent nontumor tissues of 12 patients with CRC at stages I to IV. Stepwise filtering for candidate selection in microarray data analysis yielded a set of genes that are highly methylated across all CRC tumors and that can be used as a composite biomarker for CRC detection. Verification assay identified the SDC2 gene as a potential methylation biomarker for early CRC detection. In clinical validation in tissues from 139 CRC patients, a much higher level of aberrant SDC2 methylation was measured in most primary tumors (97.8%), compared with corresponding nontumor tissue of CRC patients, irrespective of clinical stage. Clinical validation of SDC2 methylation in serum DNA from CRC patients (n = 131) at stages I to IV and from healthy individuals (n = 125) by quantitative methylation-specific PCR demonstrated a high sensitivity of 87.0% (95% CI, 80.0% to 92.3%) in detecting cancers, with a specificity of 95.2% (95% CI, 89.8% to 98.2%). Importantly, sensitivity at stage I was 92.3%, indicating the potential of SDC2 methylation as a blood-based DNA test for early detection of CRC.

Clinical, genetic, neurophysiological and functional study of new mutations in episodic ataxia type 1

Background and objectiveHeterozygous mutations in KCNA1 cause episodic ataxia type 1 (EA1), an ion channel disorder characterised by brief paroxysms of cerebellar dysfunction and persistent neuromyotonia. This paper describes four...

Ten Years’ Follow-Up of a Family With Myokymia and Muscle Cramps Without Ataxia

We report 10 years' follow-up of the previously described family with a novel mutation of the KCNA1 gene. The family consisted of 3 affected boys (first seen at ages 3, 11, and 12) and their affected mother and asymptomatic father and sister. They clinically presented with diffuse myokymia, muscle cramps, and lower limb spasticity without ataxia, but episodic ataxia developed later during adolescence and early adulthood. Long-term follow-ups of families with known KCNA1 gene mutation are rarely mentioned in the literature. Treatment with carbamazepine, 600 to 800mg daily resulted in cessation of muscle cramps and marked improvement of lower leg symptoms. In the youngest child, after 2 years carbamazepine had to be changed to oxcarbazepine because of side effects. Carbamazepine and oxcarbazepine are both effective in treatment of symptoms related to KCNA1 gene mutation. Symptoms will reoccur if treatment is stopped and there is variability of symptom severity between family members.

Voltage‐gated potassium channels and the diversity of electrical signalling

Since Hodgkin and Huxley discovered the potassium current that underlies the falling phase of action potentials in the squid giant axon, the diversity of voltage-gated potassium (Kv) channels has been manifested in multiple ways. The large and extended potassium channel family is evolutionarily conserved molecularly and functionally. Alternative splicing and RNA editing of Kv channel genes diversify the channel property and expression level. The mix-and-match of subunits in a Kv channel that contains four similar or identical pore-forming subunits and additional auxiliary subunits further diversify Kv channels. Moreover, targeting of different Kv channels to specific subcellular compartments and local translation of Kv channel mRNA in neuronal processes diversify axonal and dendritic action potentials and influence how synaptic plasticity may be modulated. As one indication of the evolutionary conservation of Kv1 channel functions, mutations of the Shaker potassium channel gene in Drosophila and the KCNA1 gene for its mammalian orthologue, Kv1.1, cause hyperexcitability near axon branch points and nerve terminals, thereby leading to uncontrolled movements and recapitulating the episodic ataxia-1 (EA1) symptoms in human patients.

Voltage-gated Potassium Channels and the Diversity of Electrical Signaling

Abstract Since Hodgkin and Huxley discovered the potassium current that underlies the falling phase of action potentials in the squid giant axon, the diversity of voltage-gated potassium (Kv) channels has been manifested in multiple ways. The large and extended potassium channel family is evolutionarily conserved molecularly and functionally. Alternative splicing and RNA editing of Kv channel genes diversify the channel property and expression level. The mix-and-match of subunits in a Kv channel that contains four similar or identical pore-forming subunits and additional auxiliary subunits further diversify Kv channels. Moreover, targeting of different Kv channels to specific subcellular compartments and local translation of Kv channel mRNA in neuronal processes diversify axonal and dendritic action potentials and influence how synaptic plasticity may be modulated. As one indication of the evolutionary conservation of Kv1 channel functions, mutations of the Shaker potassium channel gene in Drosophila and the KCNA1 gene for its mammalian orthologue, Kv1.1, cause hyperexcitability near axon branch points and nerve terminals, thereby leading to uncontrolled movements and recapitulating the episodic ataxia-1 (EA1) symptoms in human patients.

Experimental validation of in silico predicted KCNA1, KCNA2, KCNA6 and KCNQ2 genes for association studies of peripheral nerve hyperexcitability syndrome in Jack Russell Terriers

KCNA1, KCNA2, KCNA6 and KCNQ2 are associated with peripheral nerve hyperexcitability in humans. In order to determine if these genes are also involved in Jack Russell Terriers with a similar syndrome characterized by myokymia and neuromyotonia, their predicted canine orthologs were first validated experimentally. They were found either incompletely or even incorrectly annotated, mainly due to gaps in the canine genomic sequence and insufficient transcript data. Canine KCNQ2 was found to contain 20 coding exons, of which three are not described in humans. It encodes for at least 14 different transcript variants in the frontal cortex of a single dog, of which only four are also described in humans. Mutation detection in Jack Russell Terriers diagnosed with peripheral nerve hyperexcitability revealed no pathogenetic relevant structural mutations. However, the four missense sequence variations and the 14 transcript variants of KCNQ2 will contribute to the study of the functional diversity of voltage-gated potassium channels.

Paroxysmal neuromyotonia: A new sporadic channelopathy

Neuromyotonia is a heterogeneous group of genetic and autoimmune channelopathies resulting in hyperexcitability of peripheral nerves. We report an unusual case of neuromyotonia, which to our knowledge has not been previously described. The patient developed intermittent attacks of severe painful muscle stiffness accompanied by sweating, myokymia and raised serum creatine kinase. Genetic analysis of KCNA1, KCNQ2 and SCN4A genes did not identify pathogenic mutation. Serum voltage-gated potassium channel antibody was also negative. He was successfully treated with acetazolamide and carbamazepine. This appears to be a new neuromuscular disease, “paroxysmal neuromyotonia”, the etiology of which is still unknown.

Deficits in Responding to Brief Noise Offsets in Kcna1 −/− Mice Reveal a Contribution of This Gene to Precise Temporal Processing Seen Previously Only for Stimulus Onsets

The voltage-gated potassium channel subunit Kv1.1 encoded by the Kcna1 gene is expressed in many brainstem nuclei, and electrophysiological studies of Kcna1-null mutant (-/-) single neurons suggest that channels containing this subunit are critical for precise processing of rapid acoustic perturbations. We tested the hypothesis that brief offsets of a background noise are behaviorally less salient for Kcna1 -/- mice, measured by changes in noise offset inhibition of acoustic startle reflexes (ASR). In experiment 1, noise offset was followed by ASR-eliciting sound bursts either after 1-10 ms quiet intervals or after the return of noise for 10-290 ms following 10-ms quiet gaps. ASR inhibition to offset and gaps was initially higher in +/+ mice but persisted longer in -/- mice. Experiment 2 contrasted brief abrupt offsets with ramped offsets of the same duration up to 10 ms, the ramps intended to simulate progressively slower internal decays of afferent processing. Both groups had greater inhibition for abrupt offsets at asymptote, and this difference was evident at the 1-ms interval in +/+ but not -/- mice. Further, the asymptotic effect of ramped offsets in +/+ mice was equal to that produced by abrupt offsets in null mutants, suggesting more perseveration of internal afferent activity following noise offset in -/- mice. Overall, these data are consistent with prior electrophysiological studies showing that the neural mechanisms for processing acoustic transients are less effective in Kcna1 -/- mice and support previous proposals that Kv1.1 contributes to the perception of animal vocalizations and human speech.

Highlights in basic autonomic neurosciences: Epileptic seizure and sudden death

People with epilepsy die unexpectedly for unknown pathological reasons at a rate 24 times greater than the general population. Such deaths are classified as sudden unexplained death in epilepsy (SUDEP). The etiology of the SUDEP is frequently unclear which prevents the development of a rational treatment of this problem. Mice lacking Kv1.1 Shaker-like potassium channels encoded by the Kcna1 gene exhibit severe seizures and die prematurely. In this article the authors postulated that Kv1.1-potassium deficiency may underlie primary neurogenic cardiac dysfunction and cause SUDEP. EEG–ECG recordings showed that Kcna1-null mice display atrioventricular (AV) conduction blocks, as well as bradycardia and premature ventricular contractions. During seizures the occurrence of AV conduction blocks increased, which might cause SUDEP. Then the authors used atropine and propranolol to block the autonomic nervous system and found that atropine ameliorated AV conduction block indicating excessive parasympathetic tone may cause a neurocardiac defect. There were no changes in Kv1.1-deficient cardiac structure, but extensive Kv1.1 expression in juxtaparanodes of the wild-type vagus nerve, the primary source of parasympathetic input to the heart. The authors concluded that Kv1.1 deficiency leads to impaired neural control of cardiac rhythmicity due in part to aberrant parasympathetic neurotransmission, making Kcna1 a strong candidate gene for causing human SUDEP.

Kcna1-mutant rats dominantly display myokymia, neuromyotonia and spontaneous epileptic seizures

Mutations in the KCNA1 gene, which encodes for the α subunit of the voltage-gated potassium channel Kv1.1, cause episodic ataxia type 1 (EA1). EA1 is a dominant human neurological disorder characterized by variable phenotypes of brief episodes of ataxia, myokymia, neuromyotonia, and associated epilepsy. Animal models for EA1 include Kcna1-deficient mice, which recessively display severe seizures and die prematurely, and V408A-knock-in mice, which dominantly exhibit stress-induced loss of motor coordination. In the present study, we have identified an N-ethyl-N-nitrosourea-mutagenized rat, named autosomal dominant myokymia and seizures (ADMS), with a missense mutation (S309T) in the voltage-sensor domain, S4, of the Kcna1 gene. ADMS rats dominantly exhibited myokymia, neuromyotonia and generalized tonic–clonic seizures. They also showed cold stress-induced tremor, neuromyotonia, and motor incoordination. Expression studies of homomeric and heteromeric Kv1.1 channels in HEK cells and Xenopus oocytes, showed that, although S309T channels are transferred to the cell membrane surface, they remained non-functional in terms of their biophysical properties, suggesting a dominant-negative effect of the S309T mutation on potassium channel function. ADMS rats provide a new model, distinct from previously reported mouse models, for studying the diverse functions of Kv1.1 in vivo, as well as for understanding the pathology of EA1.

Low‐voltage activated Kv1.1 subunits are crucial for the processing of sound source location in the lateral superior olive in mice

Voltage-gated potassium (Kv) channels containing Kv1.1 subunits are strongly expressed in neurons that fire temporally precise action potentials (APs). In the auditory system, AP timing is used to localize sound sources by integrating interaural differences in time (ITD) and intensity (IID) using sound arriving at both cochleae. In mammals, the first nucleus to encode IIDs is the lateral superior olive (LSO), which integrates excitation from the ipsilateral ventral cochlear nucleus and contralateral inhibition mediated via the medial nucleus of the trapezoid body. Previously we reported that neurons in this pathway show reduced firing rates, longer latencies and increased jitter in Kv1.1 knockout (Kcna1−/−) mice. Here, we investigate whether these differences have direct impact on IID processing by LSO neurons. Single-unit recordings were made from LSO neurons of wild-type (Kcna1+/+) and from Kcna1−/− mice. IID functions were measured to evaluate genotype-specific changes in integrating excitatory and inhibitory inputs. In Kcna1+/+ mice, IID sensitivity ranged from +27 dB (excitatory ear more intense) to −20 dB (inhibitory ear more intense), thus covering the physiologically relevant range of IIDs. However, the distribution of IID functions in Kcna1−/− mice was skewed towards positive IIDs, favouring ipsilateral sound positions. Our computational model revealed that the reduced performance of IID encoding in the LSO of Kcna1−/− mice is mainly caused by a decrease in temporal fidelity along the inhibitory pathway. These results imply a fundamental role for Kv1.1 in temporal integration of excitation and inhibition during sound source localization.

Episodic ataxia type 1 mutations affect fast inactivation of K+ channels by a reduction in either subunit surface expression or affinity for inactivation domain

Episodic ataxia type 1 (EA1) is an autosomal dominant disorder characterized by continuous myokymia and episodic attacks of ataxia. Mutations in the gene KCNA1 that encodes the voltage-gated potassium channel Kv1.1 are responsible for EA1. In several brain areas, Kv1.1 coassembles with Kv1.4, which confers N-type inactivating properties to heteromeric channels. It is therefore likely that the rate of inactivation will be determined by the number of Kv1.4 inactivation particles, as set by the precise subunit stoichiometry. We propose that EA1 mutations affect the rate of N-type inactivation either by reduced subunit surface expression, giving rise to a reduced number of Kv1.1 subunits in heterotetramer Kv1.1-Kv1.4 channels, or by reduced affinity for the Kv1.4 inactivation domain. To test this hypothesis, quantified amounts of mRNA for Kv1.4 or Kv1.1 containing selected EA1 mutations either in the inner vestibule of Kv1.1 on S6 or in the transmembrane regions were injected into Xenopus laevis oocytes and the relative rates of inactivation and stoichiometry were determined. The S6 mutations, V404I and V408A, which had normal surface expression, reduced the rate of inactivation by a decreased affinity for the inactivation domain while the mutations I177N in S1 and E325D in S5, which had reduced subunit surface expression, increased the rate of N-type inactivation due to a stoichiometric increase in the number of Kv1.4 subunits.

Nerve excitability studies characterize KV1.1 fast potassium channel dysfunction in patients with episodic ataxia type 1

Episodic ataxia type 1 is a neuronal channelopathy caused by mutations in the KCNA1 gene encoding the fast K(+) channel subunit K(v)1.1. Episodic ataxia type 1 presents with brief episodes of cerebellar dysfunction and persistent neuromyotonia and is associated with an increased incidence of epilepsy. In myelinated peripheral nerve, K(v)1.1 is highly expressed in the juxtaparanodal axon, where potassium channels limit the depolarizing afterpotential and the effects of depolarizing currents. Axonal excitability studies were performed on patients with genetically confirmed episodic ataxia type 1 to characterize the effects of K(v)1.1 dysfunction on motor axons in vivo. The median nerve was stimulated at the wrist and compound muscle action potentials were recorded from abductor pollicis brevis. Threshold tracking techniques were used to record strength-duration time constant, threshold electrotonus, current/threshold relationship and the recovery cycle. Recordings from 20 patients from eight kindreds with different KCNA1 point mutations were compared with those from 30 normal controls. All 20 patients had a history of episodic ataxia and 19 had neuromyotonia. All patients had similar, distinctive abnormalities: superexcitability was on average 100% higher in the patients than in controls (P < 0.00001) and, in threshold electrotonus, the increase in excitability due to a depolarizing current (20% of threshold) was 31% higher (P < 0.00001). Using these two parameters, the patients with episodic ataxia type 1 and controls could be clearly separated into two non-overlapping groups. Differences between the different KCNA1 mutations were not statistically significant. Studies of nerve excitability can identify K(v)1.1 dysfunction in patients with episodic ataxia type 1. The simple 15 min test may be useful in diagnosis, since it can differentiate patients with episodic ataxia type 1 from normal controls with high sensitivity and specificity.

Nongenetic factors influence severity of episodic ataxia type 1 in monozygotic twins

Episodic ataxia type 1 (EA1) is a monogenic channelopathy caused by mutations of the potassium channel gene KCNA1. Affected individuals carrying the same mutation can exhibit considerable variability in the severity of ataxia, neuromyotonia, and other associated features. We investigated the phenotypic heterogeneity of EA1 in 2 sets of identical twins to determine the contribution of environmental factors to disease severity. One of the mutations was also found in a distantly related family, providing evidence of the influence of genetic background on the EA1 phenotype. We evaluated 3 families with an EA1 phenotype, 2 of which included monozygotic twins. We sequenced the KCNA1 gene and studied the biophysical consequences of the mutations in HEK cells. We identified a new KCNA1 mutation in each pair of twins. Both pairs reported striking differences in the clinical severity of symptoms. The F414S mutation identified in one set of twins also occurred in a distantly related family in which seizures complicated the EA1 phenotype. The other twins had an R307C mutation, the first EA1 mutation to affect an arginine residue in the voltage-sensor domain. Both mutants when expressed exerted a dominant-negative effect on wild-type channels. These results broaden the range of KCNA1 mutations and reveal an unexpectedly large contribution of nongenetic factors to phenotypic variability in EA1. The occurrence of epilepsy in 1 of 2 families with the F414S mutation suggests an interplay of KCNA1 with other genetic factors.

The voltage‐operated Potassium channel Kv1.1 as new player in renal Magnesium handling

Genetic studies in families with hereditary forms of renal Mg2+ wasting revealed new genes involved in Mg2+ homeostasis such as Mg2+ channel TRPM6. Likewise, this study aims to find the affected gene in a Brazilian family with isolated dominant hypomagnesemia. SNP‐based linkage analysis was used to identify a 11.6 cM critical region on chromosome 12. We sequenced the KCNA1 gene and identified a heterozygous A763G mutation that co‐segregated with the disorder. KCNA1 encodes voltage‐gated K+ channel Kv.1.1 in which the mutation causes substitution of a conserved asparagine at position 255 for an aspartic acid (N255D). Kv.1.1 is expressed in kidney, where it co‐localizes with TRPM6 at the distal convoluted tubule (DCT) luminal membrane as shown by immunohistochemistry. Functional analysis showed that the N255D mutation results in a non‐functional channel with a dominant negative effect on the wild‐type channel. Next, we revealed that the N255D channel fails to hyperpolarize the cell membrane. The N255 residue was further characterized by substitution with six amino acids (E, Q, A, V, T, H). The Kv1.1 N255E and N255Q mutants were not functional, while the other mutants were similar to wild‐type. Yet, their channel kinetics significantly changed, suggesting a role for Kv1.1 N255 in channel gating. Kv1.1 likely establishes the membrane potential across the DCT luminal membrane to drive TRPM6‐mediated Mg2+ reabsorption.

A missense mutation in the Kv1.1 voltage-gated potassium channel–encoding gene KCNA1 is linked to human autosomal dominant hypomagnesemia

Primary hypomagnesemia is a heterogeneous group of disorders characterized by renal or intestinal magnesium (Mg2+) wasting, resulting in tetany, cardiac arrhythmias, and seizures. The kidney plays an essential role in maintaining blood Mg2+ levels, with a prominent function for the Mg2+-transporting channel transient receptor potential cation channel, subfamily M, member 6 (TRPM6) in the distal convoluted tubule (DCT). In the DCT, Mg2+ reabsorption is an active transport process primarily driven by the negative potential across the luminal membrane. Here, we studied a family with isolated autosomal dominant hypomagnesemia and used a positional cloning approach to identify an N255D mutation in KCNA1, a gene encoding the voltage-gated potassium (K+) channel Kv1.1. Kv1.1 was found to be expressed in the kidney, where it colocalized with TRPM6 along the luminal membrane of the DCT. Upon overexpression in a human kidney cell line, patch clamp analysis revealed that the KCNA1 N255D mutation resulted in a nonfunctional channel, with a dominant negative effect on wild-type Kv1.1 channel function. These data suggest that Kv1.1 is a renal K+ channel that establishes a favorable luminal membrane potential in DCT cells to control TRPM6-mediated Mg2+ reabsorption.