Progress in brain research, volume 213

Advisory Editors

Stephen G. Waxman

Bridget Marie Flaherty Professor of Neurology
Neurobiology, and Pharmacology;
Director, Center for Neuroscience &
Regeneration/Neurorehabilitation Research
Yale University School of Medicine
New Haven, Connecticut
USA

Donald G. Stein

Asa G. Candler Professor
Department of Emergency Medicine
Emory University
Atlanta, Georgia
USA

Dick F. Swaab

Professor of Neurobiology
Medical Faculty, University of Amsterdam;
Leader Research team Neuropsychiatric Disorders
Netherlands Institute for Neuroscience
Amsterdam
The Netherlands

Howard L. Fields

Professor of Neurology
Endowed Chair in Pharmacology of Addiction
Director, Wheeler Center for the Neurobiology of Addiction
University of California
San Francisco, California
USA


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Contributors
Ste´phanie Baulac
Sorbonne Universite´s, UPMC Univ Paris 06, UM 75; INSERM, U1127; CNRS,
UMR 7225, and Institut du Cerveau et de la Moelle e´pinie`re, ICM, Paris, France
Ingmar Blu¨mcke
Department of Neuropathology, University Hospital Erlangen, Schwabachanlage,
Erlangen, Germany
John K. Cowell
Georgia Regents University Cancer Center, Augusta, GA, USA
Laura Flores-Sarnat
Department of Paediatrics, and Department of Clinical Neurosciences, Faculty
of Medicine and Alberta Children’s Hospital Research Institute, University of
Calgary, Calgary, Alberta, Canada
Antonio Gambardella
Institute of Neurology, Department of Medical Sciences, University Magna
Graecia, Catanzaro, Italy
David A. Greenberg
Battelle Center for Mathematical Medicine, Nationwide Children’s Hospital and
Pediatrics Department, Wexner Medical Center, Ohio State University, Columbus,
OH, USA

Ingo Helbig
Division of Neurology, The Children’s Hospital of Philadelphia, Philadelphia, USA
Shinichi Hirose
Department of Pediatrics, School of Medicine, and Central Research Institute for
the Molecular Pathomechanisms of Epilepsy, Fukuoka University, Fukuoka,
Japan
Katja Kobow
Department of Neuropathology, University Hospital Erlangen, Schwabachanlage,
Erlangen, Germany
Angelo Labate
Institute of Neurology, Department of Medical Sciences, University Magna
Graecia, Catanzaro, Italy
Holger Lerche
Department of Neurology and Epileptology, Hertie Institute for Clinical Brain
Research, University of Tu¨bingen, Tu¨bingen, Germany
Atul Maheshwari
Department of Neurology, Developmental Neurogenetics Laboratory, Baylor
College of Medicine Houston, TX, USA

v


vi

Contributors

Snezana Maljevic
Department of Neurology and Epileptology, Hertie Institute for Clinical Brain
Research, University of Tu¨bingen, Tu¨bingen, Germany
Berge A. Minassian

Division of Neurology, Department of Paediatrics; Program in Genetics and
Genome Biology, The Hospital for Sick Children, and Institute of Medical
Sciences, University of Toronto, Toronto, Ontario, Canada
Carlo Nobile
CNR-Neuroscience Institute, Section of Padua, Viale G, Colombo, Padova, Italy
Jeffrey L. Noebels
Department of Neurology, Developmental Neurogenetics Laboratory; Department
of Neuroscience, and Department of Molecular and Human Genetics, Baylor
College of Medicine, Houston, TX, USA
Harvey B. Sarnat
Department of Paediatrics; Department of Pathology (Neuropathology), and
Department of Clinical Neurosciences, Faculty of Medicine and Alberta Children’s
Hospital Research Institute, University of Calgary, Calgary, Alberta, Canada
Ortrud K. Steinlein
Institute of Human Genetics, University Hospital, Ludwig-Maximilians-University,
Munich, Germany
William L. Stewart
Battelle Center for Mathematical Medicine, Nationwide Children’s Hospital and
Pediatrics Department, Wexner Medical Center, Ohio State University, Columbus,
OH, USA
Pasquale Striano
Department of Neurosciences, Rehabilitation, Ophthalmology, Genetics,
Maternal and Child Health, University of Genoa, “G. Gaslini” Institute, Genova,
Italy


Preface
There has never been a time before in the history of epilepsy research when scientists
reached such a high level of knowledge. The amazing amount of data collected
within the last two decades greatly facilitated our understanding of basic concepts

of epileptogenesis. On the other hand, this progress came with the insight that the
mechanisms underlying seizure generation are far more complex than previously
thought. Twenty years ago, the introduction of the concept of epilepsies as channelopathies seemed to offer a plausible pathogenetic concept. Since then, it has become
obvious that ion channels are only part of the story, and that even a bona fide mutation within an ion channel cannot be taken as a proof that disturbed channel function directly translates into neuronal hyperexcitability. More complex mechanisms
have to be considered, and some of them might even precede the first clinically visible seizure by many years or even decades. It also has become obvious that a, most
likely large, number of genes exist that are directly associated with symptomatic or
genetic epilepsies but are neither coding for an ion channel subunit nor for a protein
that has any detectable interactions with such an ion channel. Apparently, new pathogenetic concepts are needed to guide researchers through the ever-increasing complexity of a field that less than half a century ago had still been dominated by the
hypothesis that a single “epilepsy gene” exists. Nowadays, it is clear that a large
number of epilepsy genes hide in our genome, and that these genes are able to cause
seizures by many different mechanisms, both directly and indirectly. The selection of
topics presented by the chapters in this book reflects this pathogenetic heterogeneity
as far as this is even possible in a single volume. These chapters are not aiming to
simply present a summary of facts but rather try to offer the reader a broad view of the
scientific concepts, theories, and approaches that presently dominate the different
fields in epilepsy research. The group of authors that contributed to this book is
as heterogeneous as the epilepsies themselves, including geneticists, electrophysiologists, and clinical researchers. This makes for a lively and sometimes refreshingly
controversial discussion, providing the readers with a wealth of different views, hypotheses, and ideas that hopefully create a fertile ground for the development of successful future research strategies.
Ortrud K. Steinlein

vii


CHAPTER

Genetic heterogeneity in
familial nocturnal frontal lobe
epilepsy

1

Ortrud K. Steinlein1
Institute of Human Genetics, University Hospital, Ludwig-Maximilians-University,
Munich, Germany
1
Corresponding author: Tel.: (+49)89-5160-4468; Fax: (+49)89-5160-4470,
e-mail address:

Abstract
Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) was the first epilepsy in
humans that could be linked to specific mutations. It had been initially described as a channelopathy due to the fact that for nearly two decades mutations were exclusively found in subunits of the nicotinic acetylcholine receptor. However, newer findings demonstrate that the
molecular pathology of ADNFLE is much more complex insofar as this rare epilepsy can also
be caused by genes coding for non-ion channel proteins. It is becoming obvious that the different subtypes of focal epilepsies are not strictly genetically separate entities but that mutations within the same gene might cause a clinical spectrum of different types of focal
epilepsies.

Keywords
ADNFLE, nocturnal frontal lobe epilepsy, epileptic encephalopathy, acetylcholine receptor,
KCNT1, DEPDC5

1 INTRODUCTION
Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) was first described
as a distinct familial partial epilepsy in 1994 (Scheffer et al., 1995). Although rare, it
is often referred to not least because of its status as the very first idiopathic epilepsy in
humans for which the underlying genetic cause had been identified (Steinlein et al.,
1995). This was achieved at a time when molecular genetics was still a rather new
field, 300,000-marker genome-wide association studies unheard of, and highthroughput sequencing a vision rather than daily routine. Genotyping of only about
200 polymorphic markers led to the identification of a strong candidate locus for
ADNFLE on the tip of the long arm of chromosome 20 in a large Australian family
Progress in Brain Research, Volume 213, ISSN 0079-6123, />© 2014 Elsevier B.V. All rights reserved.

1


CHAPTER 1 Genetic heterogeneity

that included more than 25 affected individuals (Phillips et al., 1995). At that time,
this chromosomal region was already in the process of being characterized due to the
fact that some years previously it had been identified as a candidate region for another type of rare monogenic idiopathic epilepsy, named benign familial neonatal
convulsions (BFNCs) (Leppert et al., 1989). It turned out that the region on chromosome 20q contains two different ion channel subunit genes, CHRNA4 encoding the
a4-subunit of the neuronal nicotinic acetylcholine receptor and the voltage-gated potassium channel gene KCNQ2 (Steinlein et al., 1994). The latter one was proven to be
the major gene for BFNC, while CHRNA4 (and some years later CHRNB2) was identified as one of the main genes that cause ADNFLE (Biervert et al., 1998; De Fusco
et al., 2000; Singh et al., 1998; Steinlein et al., 1995). The identification of these first
two seizure-related genes introduced the concept of epilepsies as channelopathies, a
concept that has by now gotten firmly established by the discovery of several additional epilepsy-causing ion channel genes. Today, nearly 20 years later, ADNFLE is
again attracting attention by teaching us that one and the same disorder can be both a
channelopathy and a non-ion channel disorder (Dibbens et al., 2013; Ishida et al.,
2013; Ishii et al., 2013; Martin et al., 2013) (Fig. 1; Table 1).

2 CHRNA4 AND CHRNB2: THE “CLASSICAL” ADNFLE GENES
The nAChR subunit genes CHRNA4 and CHRNB2 are responsible for the clinical
phenotype in about 12–15% of ADNFLE patients with a strong family history
(Steinlein et al., 2012). Both genes are expressed throughout the brain and the proteins they encode ensemble to build one of the most widely expressed nAChRs
(3a4/2b2 or 2a4/3b2) in mammalian brain. The ubiquitous expression pattern of this
nAChR subtype is surprising given that mutations in these genes cause a seizure

KCNT1

CHRNA4

E

L
F
N
D
A

2

CHRNB2

DEPDC5

CHRNA2

DEPDC5
KCNT1

Frontal lobe

Parietal lobe

Temporal lobe

Occipital
lobe

DEPDC5

DEPDC5

FIGURE 1
Schematic overview summarizing the seizure origin of the known ADNFLE genes. Arrows
indicate the migrating seizures reported for several patients with KCNT1 mutations.


2 CHRNA4 and CHRNB2: The “classical” ADNFLE genes

Table 1 Clinical phenotypes associated with ADNFLE genes
Genes

Function

Clinical phenotypes

CHRNA4/
CHRNB2
CHRNA2
KCNT1

Ion channel

ADNFLE

Ion channel
Ion channel (signaling
function?)

DEPDC5

Non-ion channel

NFLE (ADNFLE?)
Malignant migrating partial
seizures
Early infantile epileptic
encephalopathy
Severe ADNFLE
Focal epilepsy with variable foci
ADNFLE

The question mark indicates that the clinical phenotype overlaps with that previously described in other
ADNFLE families but might not be identical

phenotype that originates from the frontal lobe and rarely shows secondary generalization. So far, it can only be speculated about the pathomechanisms that prevent
CHRNA4 and CHRNB2 mutations from having a more widespread effect.
A possible explanation for this phenomenon could be that in most parts of the brain
the effect the mutations have on neuronal excitability can be compensated by other
nAChR subunits. Another possibility would be that genes from other ion channel
families or even non-ion channel genes are involved in this restricted seizure activity.
So far, nearly all of the ADNFLE mutations identified within CHRNA4 or
CHRNB2 are missense mutations that cause amino acid exchanges within the second,
less often the first, transmembrane domain (Bertrand et al., 2005; Cho et al., 2003; De
Fusco et al., 2000; Hirose et al., 1999; Magnusson et al., 2003; Phillips et al., 1995;
Steinlein et al., 1995). The nAChR genes encode receptor subunits with four transmembrane domains. These are either directly or indirectly contributing to the structure that forms the walls of the ion channel and to the governing of the channels
opening and closing mechanism. The second transmembrane domain, consisting
of helical segments forming an inner ring (TM2) that shapes the pore, can be
regarded as a hot spot for ADNFLE mutations. Several of these mutations have been
identified more than once in unrelated families from different countries or even continents. This includes the neighboring mutations CHRNA4-Ser280Phe and CHRNA4Ser284Leu that are so far the most commonly detected ADNFLE mutations (Cho
et al., 2003; Hirose et al., 1999; Ito et al., 2000; McLellan et al., 2003; Phillips
et al., 2000; Rozycka et al., 2003; Steinlein et al., 1995, 2000). These two mutations

are only separated by a few amino acids, but nevertheless differ markedly with
respect to both their biopharmacological characteristics and the severity of the clinical phenotype they are associated with. Most of the patients carrying CHRNA4Ser280Phe present with an “epilepsy-only” phenotype, while many of those
with CHRNA4-Ser284Leu have additional neurological symptoms such as mildto-moderate mental retardation. Furthermore, the latter group of patients tend to have
an unusually early age of onset, while carriers of the CHRNA4-Ser280Phe mutation

3


4

CHAPTER 1 Genetic heterogeneity

develop their seizures at an average age that is typical for most nAChR-caused nocturnal frontal lobe epilepsies (Bertrand et al., 2002; Cho et al., 2003; Hirose et al.,
1999; Ito et al., 2000; McLellan et al., 2003; Phillips et al., 2000; Rozycka et al.,
2003; Steinlein et al., 1995, 2000). On a molecular level, the two mutations differed
significantly with respect to their carbamazepine sensitivity, an antiepileptic drug
that in vivo was shown to be highly effective on CHRNA4-Ser280Phe carrying
nAChRs but not on those with the mutation CHRNA4-Ser284Leu (Bertrand et al.,
2002). These results, gained from the analysis of nAChRs expressed in Xenopus
oocytes, fit in with the observation that patients with the mutation CHRNA4Ser280Phe usually benefit from carbamazepine treatment, while sufficient seizure
reduction is rarely achieved by carbamazepine monotherapy in patients carrying
CHRNA4-Ser284Leu (Bertrand et al., 2002; Cho et al., 2003; Hirose et al., 1999;
Ito et al., 2000; McLellan et al., 2003; Phillips et al., 2000; Rozycka et al., 2003;
Steinlein et al., 1995, 2000).

3 THE CLINICAL SPECTRUM OF nAChR-CAUSED ADNFLE
The term nocturnal frontal lobe epilepsy describes a large group of partial epilepsies
that are heterogeneous in origin. ADNFLE as a rare monogenic disorder only accounts for a small proportion of these epilepsies that are mostly either symptomatic
or multifactorial. Patients with sporadic as well as familial nocturnal frontal lobe epilepsy mostly show hypermotoric seizures with movements and vocalizations. Due to
their often bizarre nature, the seizures might be misdiagnosed, for example, as a kind

of nonepileptic movement disorder, night terrors, or pseudoseizures. Electroencephalograms (EEGs) are not always helpful to establish the diagnosis because, as commonly found in frontal lobe epilepsies, they tend to be normal both interictally and
ictal. Consciousness is usually not impaired during seizures and postictal confusion
is not observed. Diurnal seizures might happen; however, most of these seizures occur during daytime naps, while seizures during wakefulness are a rare and infrequent
event (Scheffer et al., 1995; Vigevano and Fusco, 1993). Seizure onset most often
happens during the second decade of life; however, it can vary considerably even
within the same family (mean age of onset is 14 years (14 Æ 10 years)). In many individuals, seizures become milder and less frequent once they reach middle age.
A possible explanation for this phenomenon could be the normally occurring subtle
decline in the number of expressed nAChRs with age.
The very first reports described ADNFLE as a rather benign type of epilepsy that
affects otherwise healthy individuals and is readily controlled by carbamazepine.
However, follow-up reports put a question mark behind this initial assessment. This
was mainly due to the frequency with which additional major neurological symptoms
were found in patients affected by this “benign” epilepsy. A considerable degree of
interindividual variation is observed with respect to the neuropsychological development of the patients. It can range from normal intelligence to selected cognitive deficits to different degrees of mental retardation. Cognitive impairment seems to be


4 CHRNA2: A rare cause of familial NFLE

frequently associated with certain ADNFLE mutations while being rather rare with
other mutations (Bertrand et al., 2005; Hirose et al., 1999; Steinlein et al., 2012). The
same applies to psychiatric symptoms such as schizophrenia-like psychosis that was
present in most patients from a Norwegian ADNFLE family but is not usually seen in
other patients with the same disorder (Magnusson et al., 2003). The results of a metaanalysis including 19 families with 10 different mutations in either CHRNA4 or
CHRNB2 suggest that some of these mutations are frequently associated not only
with epilepsy but also with additional major cognitive or psychiatric symptoms,
while other ADNFLE mutations are preferentially found in “epilepsy-only” families.
Another feature in which patients with ADNFLE demonstrate considerable interindividual variability is the sensitivity with which their seizures respond to antiepileptic drug treatment. In many families, especially those with mutations such as
CHRNA4-Ser280Phe (previous name Ser248Phe), seizures are sufficiently controlled by the antiepileptic drug carbamazepine. Seizures in patients with other
ADNFLE mutations (for example, CHRNA4-Ser284Leu or CHRNA4-Thr293Ile
(previously named Ser252Leu or Thr265Ile)) do not respond easily to carbamazepine or other antiepileptic drugs and might require a multidrug treatment strategy.

Quite often, the latter type of ADNFLE mutation is associated with an increased risk
for major comorbidities such as mental retardation or psychiatric symptoms (Cho
et al., 2003; Hirose et al., 1999; Steinlein et al., 1995). The reservation must be made,
however, that for most nAChR mutations the number of known ADNFLE families is
still too small to derive reliable genotype–phenotype relations from them.

4 CHRNA2: A RARE CAUSE OF FAMILIAL NFLE
So far, only a single mutation (Ile279Asn) has been described in CHRNA2, a gene
that encodes one of the major a-subunits of the nAChR (Aridon et al., 2006; Combi
et al., 2009; Gu et al., 2007). The mutation was found in a family of Italian origin in
which 10 members were affected by nocturnal epilepsy. The seizure phenotype was
characterized by arousal from sleep, followed by prominent fear sensation and tongue movements. Compared to other ADNFLE families, a rather high rate of nocturnal wanderings was reported. It is therefore not entirely clear yet if the phenotype in
this family is indeed ADNFLE or if is better classified as a separate entity of nocturnal frontal lobe epilepsy (Hoda et al., 2009). Analyses of CHRNA2-Ile279Asn
on a molecular basis showed that expression of nAChRs carrying this mutation in
Xenopus oocytes significantly increases the number of receptors expressed at the
membrane surface. The mutated receptors also yielded higher ACh-evoked currents
and showed a markedly increased sensitivity toward their natural agonist acetylcholine. Taken together, it can be concluded that, comparable to the impact ADNFLE
mutations within the CHRNA4 and CHRNB2 genes have, the CHRNA2 mutation
results in a gain-of-function effect, at least in the Xenopus oocyte model system. This
effect was even stronger when CHRNA2 was coexpressed with CHRNB2 instead of
CHRNB4 (Aridon et al., 2006; Hoda et al., 2009).

5


6

CHAPTER 1 Genetic heterogeneity

5 BIOPHARMACOLOGICAL PROFILES OF nAChR MUTATIONS

The CHRNA2 mutation Ile279Asn displayed, similar to previously described
epilepsy-causing mutations in nAChRs, its own distinct biopharmacological profile.
Unlike the wild-type receptor, which already responds to the open-channel blocker
carbamazepine at low doses, blocking of the CHRNA2-Ile279Asn-carrying nAChRs
was only achieved at carbamazepine doses above 40 mM (Hoda et al., 2009). It is
therefore possible that carbamazepine would be ineffective if used as an antiepileptic
drug in patients with CHRNA2-Ile279Asn-caused nocturnal seizures. It has not been
entirely understood yet why some ADNFLE mutations are good responders with regard to carbamazepine and some are not. It had been speculated that these differences
might be due to the specific position of the mutated amino acids in relation to the ion
channel lumen (Hoda et al., 2009). However, there are no clear differences between
the mutations of carbamazepine responders versus nonresponders with respect to
their localization within the protein.
It is also not understood yet why only some nAChR mutations exhibit the same
gain-of-function effect after application of either nicotine or acetylcholine, while
other mutations differ with respect to these two agonists. For reasons unknown, some
of the nAChR mutations are less, while others are more sensitive toward nicotine
when compared to the wild type (Hoda et al., 2008). It is therefore possible that
smoking or other means of nicotine consumption might exacerbate seizure activity
in carriers of certain mutations, while others might benefit from it. The latter effect
has already been shown for patients from two Norwegian families with ADNFLE
mutations CHRNA4-776ins3 and CHRNA4-Ser248Phe (Brodtkorb and Picard,
2006). However, it is possible that experiments involving nicotine application are
not safe enough to be conducted with carriers of mutations showing high affinity toward nicotine. For example, the CHRNA2 mutation Ile279Asn, which belongs to the
latter group, displays a rather pronounced gain-of-function effect after nicotine application in the Xenopus model (Hoda et al., 2009). It is therefore possible that nicotine consumption would have a marked negative effect on seizure control in patients
with this mutation.

6 SEVERE ADNFLE CAUSED BY KCNT1 MUTATIONS
Recently, genomic mapping in a family with genetic epilepsy led to the discovery of
KCNT1 as a new causative gene for both ADNFLE and epileptic encephalopathy.
Extension of the study subsequently identified several additional familial and sporadic cases of KCNT1-caused ADNFLE (Heron et al., 2012). The clinical phenotype

differed from the above described nAChR-caused ADNFLE insofar as the nocturnal
seizures were frequently associated with major comorbidities. Intellectual disability
was more common in KCNT1 mutation carriers than in those with CHRNA4 or
CHRNB2 mutations, and the same was true for psychiatric symptoms or behavioral
problems. There was also a significantly lower age of onset (mean 6 years, 4 years


6 Severe ADNFLE caused by KCNT1 mutations

lower than in nAChR-caused ADNFLE), together with complete penetrance (compared to an average 60–80% in nAChR-caused ADNFLE). Interestingly, another
report was published in the same journal issue also describing patients with mutations in KCNT1, but those patients did not present with ADNFLE but with the much
more severe phenotype of malignant migrating partial seizures of infancy (Barcia
et al., 2012). This disorder is part of the group of early onset epileptic encephalopathies that includes different catastrophic childhood epilepsies with poor prognosis.
In most of these epilepsies, the outcome is characterized by psychomotoric disability
that can be severe. Patients with malignant migrating partial seizures of infancy
experience pharmacoresistant polymorphic focal seizures and psychomotor development arrests after the first months of life. EEGs demonstrate that the seizures are not
always concentrating on the same region (as they do from the frontal lobe in ADNFLE) but can arise from various areas of the brain. Furthermore, during a seizure,
epileptic activity can be seen to migrate from one part of the brain to another one
(hence the name of the syndrome).
The KCNT1 gene (also known as SLACK or ENFL5) encodes an outwardly rectifying sodium-activated potassium channel (KCa4.1) that (although calcium does not
act as its main activator) due to sequence homologies belongs to the subfamily of
calcium-activated potassium channels. KCNT1 is expressed in two alternatively
spliced isoforms of different length. Both isoforms contain six putative
membrane-spanning regions as well as an extended COOH terminus. The open probability of KCa4.1 channels increases with depolarization, implying intrinsic voltage
dependence. In brain, the KCa4.1 potassium channel has been shown to be widely
expressed (Joiner et al., 1998). Furthermore, the KCNT1 gene shows a high level
of sequence conservation, a fact that strongly suggests an important functional role,
most likely in neuronal excitability. Interestingly, the major areas of KCNT1 expression include the substantia nigra, frontal cortex, deep cerebellar nuclei, trigeminal
system, subthalamic nuclei, rubrospinal tract, reticular formation, and vestibuloocular tract. Several of these brain structures participate in the regulation of movement and posture, and it has therefore been postulated that KCa4.1 might be important
for motor control (Bhattacharjee et al., 2002). Such a role would fit well with the

finding that mutations in KCNT1 are one of the causes of ADNFLE, a disorder characterized mainly by motor seizures.
So far, it is not entirely clear how mutations in the KCNT1 gene are able to cause
two different seizure phenotypes. Obviously, the mutations found in malignant migrating partial seizures of infancy all occurred de novo, as it is mostly the case in
intellectually debilitating genetic conditions with an age of onset before adulthood.
However, de novo mutations are also found in some ADNFLE patients. Both the
KCNT1 mutations causing severe ADNFLE and the ones found in malignant migrating partial seizures of infancy are missense mutations (Barcia et al., 2012; Heron
et al., 2012). In the latter condition, there seem to be hotspots within KCNT1 for this
mutational mechanism because both the mutation Gly288Ser and Arg428Gln occurred more than once in unrelated patients (Barcia et al., 2012; Ishii et al.,
2013). Several of the so far published mutations occurred at different CpG sites

7


8

CHAPTER 1 Genetic heterogeneity

within the KCNT1 gene. These sites are known to be prone to mutational events, most
likely because the methylated cytosines are vulnerable to spontaneous deamination
into thymine.
Most of the mutations in both severe ADNFLE and malignant migrating partial
seizures of infancy are affecting conserved amino acid residues that are located
within the large COOH-terminal region of KCNT1, a region of so far unknown function. It is therefore not clearly evident if KCNT1-caused epilepsy belongs to the
group of channelopathies, or if it is caused by different mechanisms. The COOHterminal region is located within the cytoplasma and contains several conserved
sequence motifs. These are believed to facilitate the interaction with a network of
proteins that regulate channel activity. One of the genes involved in this network
is FMR1, encoding the FMRP protein that is involved in rapid, activity-regulated
transport of mRNAs and has an important role in synaptogenesis and neuronal plasticity (Deng et al., 2013). Loss of FMRP is known to cause one of the most common
inherited mental retardation disorders, the fragile X syndrome (Kremer et al., 1991).
Under physiological conditions, FMRP binds selectively to sequences at the KCa4.1

COOH terminus and subsequently activates the potassium channel (Brown et al.,
2010). Mutations within the COOH terminus might introduce conformational alterations that could interfere with the binding of FMRP, causing changes in the firing
pattern of neurons expressing the KCa4.1 potassium channel. It has also been speculated that the binding to KCa4.1 might in reverse modulate functions of FMRP such
as the regulation of the transport of its cargo mRNAs or the activity-dependent increases in the translation of these mRNAs (Li et al., 2009; Schutt et al., 2009).
The direct functional association between the potassium channel KCa4.1 and
FMRP, a protein whose loss of function is known to inflict profound intellectual disability, might explain the observation that KCNT1 mutations not only cause epilepsy
but are responsible for a much broader neurological phenotype that can include both
cognitive and psychiatric features (Barcia et al., 2012). However, it cannot be excluded that other, so far unknown, mechanisms are responsible for the clinical spectrum associated with KCNT1 mutations. The COOH-terminal region of KCa4.1
contains sequence motifs such as phosphorylation sites, tandem regulators of potassium conductance domains, and nicotinamide adenine dinucleotide binding sites.
One of the mutations discovered in ADNFLE patients affects an amino acid within
such a nicotinamide adenine dinucleotide binding site, while another one targets an
amino acid directly adjacent to this site. Both mutations were associated with an
ADNFLE phenotype that was more pronounced than that observed with mutations
in other parts of the C terminus (Heron et al., 2012). It is therefore possible that a
close relationship exists between the position of the KCNT1 mutation and the clinical
phenotype.
So far, the number of known families with KCNT1 mutations is too small to deduce reliable genotype–phenotype correlations that could be useful for the genetic
counseling of affected families. However, strong genotype–phenotype relationships
seem to exist, a hypothesis that is supported by the fact that the few recurrent KCNT1
mutations that have been reported are causing a roughly uniform phenotype (Barcia


7 DEPDC5 as a cause of familial focal epilepsy

et al., 2012; Heron et al., 2012; Ishii et al., 2013). So far, there is no evidence that a
mutation might be able to cause both severe ADNFLE and malignant migrating partial seizures of infancy. There is, however, some indication that the phenotypic spectrum associated with KCNT1 mutations might not be restricted to either of these
clinical phenotypes. A case report described a single patient with a de novo KCNT1
mutation and a severe clinical phenotype that includes profound psychomotor retardation, microcephaly, deficient neuronal myelination, and therapy-resistant myoclonic seizures (Vanderver et al., 2013). The EEG did not show any signs
compatible with the migrating partial seizures seen in the above described patients,
and it is therefore possible that KCNT1 mutations are associated with a broader spectrum on early infantile epileptic encephalopathies.

7 DEPDC5 AS A CAUSE OF FAMILIAL FOCAL EPILEPSY
Mutations within the DEPDC5 gene (alternative name KIAA0645) have been recently found in patients with familial focal epilepsies (Dibbens et al., 2013; Ishida
et al., 2013; Martin et al., 2013). The phenotypic spectrum in these families included
the subtypes ADNFLE, familial temporal lobe epilepsy, and familial focal epilepsy
with variable foci. The majority of these mutations are nonsense mutations that can
be expected to introduce premature stop codons resulting in nonsense-mediated
mRNA degradation, thus causing a loss-of-function effect. The frequency of
DEPDC5 mutations in patients with familial focal epilepsy was estimated to be about
12–27%, rendering DEPDC5 one of the most frequent causes detected so far in genetic epilepsy. The penetrance seems to be lower when compared to ADNFLE
caused by nAChR mutations; however, this might be a bias due to the so far low number of known families (Ishida et al., 2013). The clinical phenotype in patients with
DEPDC5 mutations is rather benign insofar as most of them are of normal intellect
without any detectable structural brain lesions. However, autism spectrum disorder
or intellectual disability has been described in some affected individuals (Dibbens
et al., 2013; Ishida et al., 2013; Martin et al., 2013).
DEPDC5 encodes the DEP domain-containing protein 5 that is ubiquitously
expressed in human tissues. The DEP domain was named from the initials of three
proteins, disheveled (Dsh), Egl-10, and pleckstrin (Klingensmith et al., 1994; Koelle
and Horvitz, 1996). So far, not much is known about the structure and function of
either DEPDC5 or its DEP domain. There are a few reports that discuss a possible
role of DEPDC5 in the pathogenesis of different malignancies; however, the evidence for a role of DEPDC5 in carcinogenesis is far from conclusive. The gene is
located on chromosome 22 in a region that was found to harbor a homozygous deletion common to two cases of glioblastoma (Seng et al., 2005). However, structural
aberrations of chromosome 22 are a frequent occurrence in astrocytic tumors, and
this observation could have therefore been caused by coincidental occurrence.
In another report, an intronic single nucleotide polymorphism within the
DEPDC5 gene was described as a risk factor significantly associated with the

9

10

CHAPTER 1 Genetic heterogeneity

likelihood of progression to hepatocellular carcinoma in patients with chronic viral
hepatitis (Miki et al., 2011). The polymorphism rs1012068 was detected in a genomewide association study, remained significant after Bonferroni correction for multiple
testing, and was confirmed in a follow-up replication study (Miki et al., 2011). The
association between rs1012068 and the risk for hepatocellular carcinoma can therefore
be regarded as robust; however, with an odds ratio of about 2 in males (lower in females), the risk conferred by this polymorphism is rather small. Furthermore, it is unknown if rs1012068 itself is functional or if it only acts as a placeholder for another
linked polymorphism within or outside DEPDC5. Additional albeit indirect evidence
for a role of DEPDC5 in tumorigenesis is provided by the observation that DEPDC1, a
gene containing a DEP domain similar to that present in DEPDC5, has been linked to
bladder carcinogenesis (Kanehira et al., 2007). Further studies are needed to clarify if
DEPDC5 indeed participates in the molecular pathology of malignancies and, if so,
how this relates to its established role in epileptogenesis.
It is nevertheless interesting that with DEPDC5 yet another epilepsy gene has
been identified that was discussed as a possible cancer gene before being discovered
to cause a monogenic type of epilepsy. The same happened several years previously
with a different gene, LGI1 on chromosome 10q, which is responsible for autosomal
dominant temporal lobe epilepsy (also named autosomal dominant partial epilepsy
with auditory features) (Chernova et al., 1998; Gu et al., 2002; Kalachikov et al.,
2002). It is therefore tempting to speculate that at least some of the pathways leading
to cancer might have parts in common with those that cause epilepsy. One such connection could be provided by the putative role of DEPDC5 within the mTOR
pathway.
The mTOR complex 1 is known as one of the most important regulators of cell
growth and has been frequently found to be deregulated in different common multifactorial disorders including malignancies and diabetes mellitus (Laplante and
Sabatini, 2012). The mTOR complex 1 is able to sense amino acid levels by interacting with a complex signaling machine (Zoncu et al., 2011). Part of this machine is
the GATOR complex, a multiprotein Rag-interacting complex that contains the
DEPDC5 protein as one of its components (Bar-Peled et al., 2013). Experimentally
induced loss of function in GATOR resulted in hyperactive mTOR complex 1 signaling (Bar-Peled et al., 2013). This observation is of interest with respect to epileptogenesis because aberrant mTOR complex 1 signaling is known to cause

disturbances in neuronal migration and cortical lamination. This has been demonstrated in different neuronal migration disorders, including tuberous sclerosis. The
molecular mechanisms leading to tuber formation during brain development in patients with tuberous sclerosis include loss-of-function mutations in either the TSC1 or
the TSC2 gene that are part of the mTOR signaling cascade. This results in constitutive mTOR activation which in turn interferes with the development of the cerebral
cortex (Prabowo et al., 2013; Tsai et al., 2012). A possibility would be that mutations
in DEPDC5 have a less dramatic effect on mTOR complex 1 signaling but disturb it
enough to introduce microscopic changes in brain cytoarchitecture or synaptic


References

connectivity that are able to promote focal epilepsy without causing macroscopically
visible structural malformations.

8 CONCLUSIONS
ADNFLE was the first epilepsy in humans for which mutations have been identified.
By now, it has also become the prototype of a neurological disorder that can be
caused by genes coding for either ion channels or non-ion channels. The ADNFLE
patients belonging to either one of these genetic subgroups are not easily distinguishable from each other on the basis of their clinical characteristics alone. One possible
indication pointing toward a non-ion channel origin can be the observation of family
members with other types of focal epilepsies, but this does not apply to all families
concerned. Non-ion channel ADNFLE patients tend to have a more severe phenotype, both with respect to an earlier age of seizure onset and a higher frequency
of additional major symptoms such as mental retardation. However, seriously affected individuals or even whole families with a severe course of the disorder are
also found in the group of ion channel ADNFLE patients, rendering the clinical
course a not very reliable criterion to differentiate between both groups. As in many
other rare types of epilepsy, genetic testing has therefore become a routine instrument in the classification of ADNFLE patients. Nevertheless, mutations are still only
detectable in far less than half of the ADNFLE families. This implicates that additional genes exist that are able to cause this clinical phenotype. Given the rapid progress in sequencing technologies, it can be expected that at least some of these genes
will be identified within the next few years. It will be most interesting to see to which
functional classes these genes belong, and if these new genes are able to further shed
light on the obviously complex pathomechanisms that underlie nocturnal frontal lobe
epilepsy. Already, the clinical similarities between the two groups of patients pose

the question whether the proteins encoded by the ion channel and non-ion channel
ADNFLE genes are involved in some of the same, so far unknown functional
pathway(s). Uncovering such common pathways not only will greatly facilitate
our understanding of the molecular basis of epileptogenesis but hopefully will also
be able to reveal new therapeutic targets.

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15


CHAPTER

Potassium channel genes
and benign familial neonatal
epilepsy

2

Snezana Maljevic1, Holger Lerche
Department of Neurology and Epileptology, Hertie Institute for Clinical Brain Research, University
of Tu¨bingen, Tu¨bingen, Germany
1
Corresponding author: Tel.: +49-7071-29-81922; Fax: +49-7071-29-4698,
e-mail address:

Abstract
Several potassium channel genes have been implicated in different neurological disorders including genetic and acquired epilepsy. Among them, KCNQ2 and KCNQ3, coding for KV7.2
and KV7.3 voltage-gated potassium channels, present an example how genetic dissection of an
epileptic disorder can lead not only to a better understanding of disease mechanisms but also
broaden our knowledge about the physiological function of the affected proteins and enable
novel approaches in the antiepileptic therapy design. In this chapter, we focus on the neuronal

KV7 channels and associated genetic disorders—channelopathies, in particular benign familial
neonatal seizures, epileptic encephalopathy, and peripheral nerve hyperexcitability (neuromyotonia, myokymia) caused by KCNQ2 or KCNQ3 mutations. Furthermore, strategies using
KV7 channels as targets or tools for the treatment of epileptic diseases caused by neuronal
hyperexcitability are being addressed.

Keywords
KCNQ2, KCNQ3, M-current, retigabine, heterologous expression, dominant-negative effect,
haploinsufficiency, developmental expression

1 INTRODUCTION
Each of approximately 85 billion neurons in the human brain greatly relies in its
function on the specific expression of relatively small proteins—ion channels—in
its membrane. These proteins provide a unique milieu in which information can
be generated and transmitted to control both movement of the little toe and creation
of a space shuttle or The Fifth Symphony. In other words, ion channels form selective
pores for different ions, which can open and close in a regulated manner and thus
determine the ion flux over membrane, presenting the basis of the electrical excitability. Essentially, changes in membrane potential allow opening of voltage-gated
Progress in Brain Research, Volume 213, ISSN 0079-6123, />© 2014 Elsevier B.V. All rights reserved.

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18

CHAPTER 2 K+ channel genes and benign familial neonatal epilepsy

ion channels, whereas binding of specific chemical messengers (neurotransmitters)
evokes ion passage through ligand-gated ion channels (Lerche et al., 2005).
Voltage-gated ion channels are responsible for the generation of action potentials
and their conduction along the axons, as well as for establishing and revoking

membrane potential at rest. When action potentials arrive on the presynaptic
membrane, they induce Ca2+ influx and the release of neurotransmitters, which bind
to the ligand-gated postsynaptic channels and provide the information transmission
between cells. Neurons can be distinguished by the chemical messengers they
release: excitatory neurons communicate via glutamate or acetylcholine, whereas
neurotransmitters produced by inhibitory neurons are g-aminobutyric acid (GABA)
and glycine. Ion channels are further characterized by specific temporal and spatial
distribution and can inhabit different neuronal compartments. In excitatory pyramidal cells, the specific voltage-gated sodium channels, such as NaV1.2 and NaV1.6
(Liao et al., 2010), or potassium channels, such as KV7.2 and KV7.3 (Maljevic
et al., 2008), are expressed in the axon initial segments (AISs), the origin site of
action potentials. In contrast, the NaV1.1 sodium channel is found at the AISs
of the inhibitory neurons (Ogiwara et al., 2007). Ligand-gated channels occupy
postsynaptic membranes in dendrites, but some voltage-gated ion channels are also
found at these sites (Vacher et al., 2008).
The majority of genetic defects detected thus far in idiopathic epilepsies affect
ion channels. Genetic alterations can affect channel function and thereby alter
the electrical impulse, modifying neuronal excitability and driving networks of
neurons into synchronous activity, which can finally lead to an epileptic seizure.
Moreover, mutations within the postsynaptic receptors can affect the conduction
between cells and thus present an epilepsy-causing defect.
Within a healthy brain, ion channels, ingrained in membranes of excitatory and
inhibitory neurons, are providing a neuronal balance. Epileptic seizures can be
elicited by disruption of this balance caused by ion channel defects and treated by
anticonvulsants that are mainly affecting ion channels. It is a challenge to our
understanding how and why genetic alterations resulting in epileptic seizures do
not cause disease phenotype interictally. Furthermore, genetic epilepsy disorders
occur at certain age and can in some cases remit spontaneously, indicating that specific patterns of ion channel function or expression may be responsible for the seizure
precipitation.
Potassium channel genes cover a number of important physiological functions
and have, therefore, been under a detailed investigation in relation to genetic epilepsies. Indeed, in the past 20 years, several potassium channels have been associated

with epilepsy. Especially one potassium channel family, the KCNQ channels,
drew much attention since mutations in the KCNQ genes have been linked to
different human inherited diseases. Mutations in KCNQ2 and KCNQ3 genes were
the first potassium channel mutations associated with an epileptic phenotype in
benign familial neonatal seizures (BFNS) (Biervert et al., 1998; Charlier et al.,
1998; Schroeder et al., 1998). In the meantime, the phenotype spectrum related to
these channels extended, including among others severe epileptic encephalopathy


2 Potassium channels

(EE) (Weckhuysen et al., 2012). In parallel, development of the newly approved drug
retigabine, which is acting as an opener of these channels, has started a new era in the
development of antiepileptic drugs.

2 POTASSIUM CHANNELS
Subunits of potassium (K+) channels are encoded by approximately 80 genes (KCN)
in mammals, and present the most divergent of all ion channel families. They are
widely expressed throughout the body having various physiological functions
(Coetzee et al., 1999). The specificity of these channels for K+ over other cations
is defined by a highly conserved amino acid sequence, the so-called GYG signature
sequence, which enables selective transmission of K+ by replacing the six water
molecules that surround these ions. The K+ channel from a Streptomyces lividans
bacterium KscA was the first crystallized ion channel (Doyle et al., 1998). Subsequently determined crystal structures of mammalian channels revealed that conformational changes, which open and close the pore, take place within its inner part
in response to membrane depolarization, binding of Ca2+ or other regulatory
mechanisms (Long et al., 2005).
Based on the number of transmembrane (TM)-spanning regions in each subunit
and their physiological and pharmacological characteristics, K+ channels are
grouped into 2TM, 4TM, and 6TM or 7TM families (Gutman et al., 2005). All
potassium channel genes are thought to emerge by gene duplication from a single

ancestor gene ( Jegla et al., 2009) having 2TM segments. This structure is characteristic for the inward-rectifier K+ channel family (KIR), including ATP-sensitive K+
channels which associate with sulfonylurea subunits to regulate cellular metabolism
and G-protein-coupled KIR channels. As in the majority of K+ channel families,
functional pore is formed by four subunits (Hibino et al., 2010). As a matter of fact,
the 4TM K+ channel family is the only one in which the functional pore is formed by
two subunits. These channels are unique because they contain two instead of one
pore-forming loop. The 4TM, responsible for the leak currents in neuronal cells,
are active at rest and have constitutively open channel gate (Plant et al., 2013).
K+ channels, which are voltage-insensitive and activated by low concentrations
of internal Ca2+, comprise the 6TM family of “small-conductance” (SK) and
“intermediate-conductance” (IK) KCa channels. Ca2+ does not bind directly on these
channels but is instead bound to calmodulin (CaM), which induces conformational
changes resulting in pore opening (Wei et al., 2005). In the 7TM KCa1.1, so-called
big-conductance (BK) channels, the N-terminus makes a seventh pass through
the membrane to the extracellular side. These channels are expressed in a broad
variety of cells and binding of Ca2+ is not dependent on its association with CaM
(Shieh et al., 2000).
The largest family of K+ channels is encoded by about 40 genes and encompasses
voltage-gated (KV) channels. KV channels consist of four a-subunits, each containing 6TM regions, which form a single pore (Fig. 1) (Gutman et al., 2005). A short

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CHAPTER 2 K+ channel genes and benign familial neonatal epilepsy

FIGURE 1
Structure and function of KV channels. Functional voltage-gated potassium channels (KV)
are made of four subunits. A typical structure of a single KV channel subunit is shown on the

left. Due to their different characteristics, KV channels play diverse physiological roles.
Whereas several KV channels are directly implicated in the membrane repolarization during
an action potential, KV7.2/KV7.3 are active in the subthreshold range and important for
the regulation of resting membrane potential and prevention of repetitive firing (middle).
Examples for specific somatodendritic and axonal localization of KV channels are presented
on the right.

amino acid sequence, containing positively charged arginine residues, forms the
fourth TM segment S4 responsible for the channel regulation by voltage and therefore named the voltage sensor. In response to changes in membrane potential, conformational changes within this region will affect the movement of the channel gate
in the intracellular side of the pore-forming S5–S6 loop. Amino and C-terminal
domain are located inside the cell and can vary in their length in different subunits.
The subunit assembly domain is usually found at the N-terminus, except in KCNQ
(KV7) and hERG (KV11) channels, where it is located in the C-terminus. These parts
of the channel also contain binding domains for auxiliary subunits or other regulatory
proteins (Gutman et al., 2005).
Functional diversity of potassium channels is further increased by their
heteromerization into dimers (KIR) or tetramers (KV) and interaction with a number
of auxiliary subunits. mRNA splicing and posttranslational modifications also
contribute to the K+ channel diversity (Gutman et al., 2005; Shieh et al., 2000).

2.1 HOW POTASSIUM CHANNELS REGULATE NEURONAL
EXCITABILITY
KV channels usually colocalize with voltage-dependent Na+(NaV) or Ca2+(CaV)
channels in excitable cells and are responsible for the cell membrane repolarization
or hyperpolarization. During an action potential, cell membrane is depolarizing
ought to the influx of Na+ ions through the NaV channels and the repolarization phase
is determined by the inactivation of NaV channels, as well as by efflux of K+ ions due
to the concentration gradient across the membrane upon the opening of KV channels

2 Potassium channels

(Lehmann-Horn and Jurkat-Rott, 1999). Slower than the NaV channels, some KV
channel subunits generate fast K+ currents across the membrane, which can also
inactivate and are recognized as A-type potassium currents (Shieh et al., 2000).
Inactivation is a state of the channel protein in which, although still in the open
conformation, the channel pore is not permeable due to occlusion by an amino
terminal sequence (fast, N-type or ball-and-chain sequence inactivation) or a conformational change within a pore (slow, P- or C-type inactivation). An important
potassium current in neurons is the so-called M-current, a noninactivating slow
current which is activated at subthreshold voltages and can be regulated by muscarinic agonists, which is where the name comes from (Brown and Adams, 1980).
Physiologically, the A-currents will have a larger impact on the initial action potentials within a spike train whereas M-current will determine the response to multiple
spikes, when A-current is inactivated (Bean, 2007; Brown and Adams, 1980).
Typical A-type KV channels are found in KV1–KV4 subfamilies, while KV7
(KCNQ) and KV11 (hERG) produce the M-currents (Fig. 1) (Shieh et al., 2000).
Within the central and peripheral nervous systems, the a subunits of KV channel
family are expressed in both neurons and glial cells and besides excitability also
affects Ca2+ signaling, secretion, volume regulation, proliferation, and migration.
Within a single neuron, they can occupy different subdomains indicating their specialized physiological roles ( Jensen et al., 2011). For instance, KV2 and KV4 present
somatodendritic channels, KV1 subunits are found on axons and nerve terminals,
KV7 reside mainly at AISs and nodes of Ranvier, and KV3 are expressed in dendritic
or axonal domains, depending on the neuronal cell type or a splice variant (Fig. 1)
(Vacher et al., 2008). A variety of molecular mechanisms, including interactions
with other neuronal proteins, determine specific distribution of KV channels in
neuronal membrane subdomains, which is also dependent on and regulated by
neuronal activity ( Jensen et al., 2011; Misonou and Trimmer, 2004).

2.2 POTASSIUM CHANNELS IN EPILEPSY AND RELATED DISORDERS
The major physiological roles that potassium channels play in the nervous system
indicate they may be involved in a number of neuronal disorders characterized by
increased excitability, such as epilepsy, migraine, naturopathic pain, ataxia, and others.

Diseases caused by dysfunction of ion channels are called “channelopathies.” Before
we concentrate on the neonatal seizures and the associated neuronal KCNQ2/3 channelopathies, we will shortly address the involvement of other potassium channels in
epilepsy and pertinent diseases.

2.2.1 Mutations in KV1.1 Cause Episodic Ataxia
KCNA1 gene encodes KV1.1 channel, which is the human homolog of the Shaker
potassium channel of the fruit fly Drosophila melanogaster. Mutations causing a loss
of function of the Shaker channel in fruit flies are related to the leg-shaking phenotype occurring episodically or upon ether anesthesia. As mentioned before, KV1.1
channels mediate the fast-inactivating A-currents known to regulate the repolarizing

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