Mechanisms of human inherited epilepsies

Progress in Neurobiology 87 (2009) 41–57

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Progress in Neurobiology journal homepage: www.elsevier.com/locate/pneurobio

Mechanisms of human inherited epilepsies Christopher A. Reid a,b, Samuel F. Berkovic b,*, Steven Petrou a a b

Howard Florey Institute, The University of Melbourne, Parkville, Melbourne, Australia Epilepsy Research Centre, Department of Medicine, University of Melbourne, West Heidelberg, Vic. 3081, Australia

A R T I C L E I N F O

A B S T R A C T

Article history: Received 19 May 2008 Received in revised form 25 August 2008 Accepted 29 September 2008

It is just over a decade since the discovery of the first human epilepsy associated ion channel gene mutation. Since then mutations in at least 25 different genes have been described, although the strength of the evidence for these genes having a pathogenic role in epilepsy varies. These discoveries are allowing us to gradually begin to unravel the molecular basis of this complex disease. In the epilepsies, virtually all the established genes code for ion channel subunits. This has led to the concept that the idiopathic epilepsies are a family of channelopathies. This review first introduces the epilepsy syndromes linked to mutations in the various genes. Next it collates the genetic and functional analysis of these genes. This part of the review is divided into voltage-gated channels (Na+, K+, Ca2+, Cl and HCN), ligand-gated channels (nicotinic acetylcholine and GABAA receptors) and miscellaneous proteins. In some cases significant advances have been made in our understanding of the molecular and cellular deficits caused by mutations. However, the link between molecular deficit and clinical phenotype is still unknown. Piecing together this puzzle should allow us to understand the underlying pathology of epilepsy ultimately providing novel therapeutic strategies to complete the clinic-bench-clinic cycle. ß 2008 Elsevier Ltd. All rights reserved.

Keywords: Familial epilepsy Ion channels Function Genotype Phenotype

Contents 1. 2. 3. 4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Classification of human epilepsies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetic architecture of epilepsies and the dual role of physiological investigations . Voltage-gated channelopathies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Voltage-dependent Na+ channels. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.1. SCN1A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.2. SCN2A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.3. SCN1B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Voltage-dependent K+ channels. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.1. KCNQ2 and KCNQ3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.2. KCNA1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.3. Other mutations in K+ channels linked to epilepsy . . . . . . . . . . . . . . 4.3. Voltage-dependent Ca2+ channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.1. CACNA1H . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Voltage-dependent Cl channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4.1. CLCN2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. HCN. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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* Corresponding author at: Epilepsy Research Centre, Department of Medicine, University of Melbourne, Level 1, Neurosciences Building, Heidelberg Repatriation Hospital, Austin Health, 300 Waterdale Road, West Heidelberg, Vic. 3081, Australia. Tel.: +61 3 9496 2330; fax: +61 3 9496 2291. E-mail address: [email protected] (S.F. Berkovic). Abbreviations: ADNFLE, autosomal dominant nocturnal frontal lobe epilepsy; ADPEAF, autosomal dominant partial epilepsy with auditory features; AIS, axon initial segment; BFNIS, benign familial neonatal-infantile seizures; BFNS, benign familial neonatal seizures; CAE, childhood absence epilepsy; EA1, episodic ataxia type 1; EEG, electroencephalographic; EAAT1, excitatory amino acid transporter type 1; EEG, electroencephalography; FS, febrile seizures; GEFS+, generalized epilepsy with febrile seizures plus; HCN, hyperpolarization-activated cyclic nucleotide-gated channels; IGE, idiopathic generalised epilepsies; JAE, juvenile absence epilepsy; JME, juvenile myoclonic epilepsy; LGI1, leucine-rich glioma inactivated gene 1; MASS, monogenic audiogenic seizure-susceptible gene; ME2, malic enzyme 2; nAChR, nicotinic acetylcholine receptor; SMEI, severe myoclonic epilepsy in infancy; WT, wild type protein. 0301-0082/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.pneurobio.2008.09.016

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5.

6.

7. 8.

C.A. Reid et al. / Progress in Neurobiology 87 (2009) 41–57

Ligand-gated channelopathies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Nicotinic acetylcholine receptors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1.1. CHRNA4, CHRNB2 and CHRNA2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. GABA receptors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.1. GABRG2 and GABRA1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.2. GABRD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other genes linked to epilepsy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. LGI1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.1. Clinical syndrome and molecular findings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.2. Functional impact of mutations and potential neuronal mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. ATP1A2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. ME2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4. SLC1A3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5. EFHC1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.6. MASS1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.7. SLC2A1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Modelling the complex genetics of generalised epilepsy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Since the time of Hippocrates, epilepsy was recognized to have a familial component. In 1995, the first gene for idiopathic epilepsy was identified (Steinlein et al., 1995) and since then a series of discoveries has begun to gradually unravel the molecular basis of epilepsies. This has allowed the mechanistic basis of human epilepsy to be probed in a manner hitherto impossible. In the epilepsies, virtually all the established mutations are in genes that encode ion channel subunits. This has led to the concept that the idiopathic epilepsies are a family of channelopathies. Whether the ‘channelopathy’ concept will be relevant for most common epilepsies with complex inheritance remains an open question. Here we review genetic and physiological evidence that is aimed at bridging the genotype to phenotype gap. 2. Classification of human epilepsies This section provides a basic overview of epilepsy syndromes, specifically highlighting epilepsy phenotypes, the terms used to describe them and their acronyms. There are numerous forms of epilepsy, subdivided into clinical syndromes on the basis of age of onset, seizure patterns and other clinical, electroencephalographic (EEG) and imaging features. These are determined by genetic or acquired factors and, in many cases, there is evidence for a gene/ environmental interaction (Berkovic et al., 2006b). The clinical classification of the epilepsies is complex and evolving (Commission, 1989; Engel, 2001). Attempts are now being made to integrate more fundamental biological information, compared to the earlier approach based largely on clinical and electroencephalographic features. Over 50 epilepsy syndromes are described and they are broadly divided into generalized and focal (partial) syndromes reflecting the evidence that some seizure syndromes are generated bilaterally in the brain whereas others have a clear focal onset. This distinction remains clinically practical and helps guide treatment. The commonest epilepsy syndrome is febrile seizures (FS), which affect approximately 3% of all children. It is typically a selflimited disorder where a child under the age of 5 has seizures only in response to fever (Verity et al., 1985). Largely for reasons of inappropriate discrimination, FSs are not always regarded as ‘‘epilepsy’’ but there is no good reason to believe in a biological distinction. Sometimes the seizure syndrome associated with febrile seizures is more complicated and seizures without fever

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occur. This can be due to a variety of reasons but a common, relatively recent recognized condition is known as generalized epilepsy with febrile seizures plus (GEFS+) (Scheffer and Berkovic, 1997). In this familial epilepsy syndrome, seizures typically continue on to later childhood or adolescence when they usually remit but some individuals may have more severe phenotypes. The broad group of generalized epilepsies (comprising about 30% of cases, excluding febrile seizures) largely comprises the idiopathic generalized epilepsies which are a family of related syndromes, typically beginning in childhood or adolescence (but can begin at any time in life) and are associated with a characteristic EEG pattern of generalized spike-and-wave discharge (Loiseau et al., 1990). Patients have combinations of absence (loss of consciousness), myoclonic (sustained muscle contraction) and tonic–clonic (rhythmic jerking) seizures and the common associated syndromes include childhood absence epilepsy (CAE), juvenile absence epilepsy (JAE) and juvenile myoclonic epilepsy (JME) indicating the ages of onset and seizure types in the individual patients. More severe forms of generalized epilepsy such as myoclonic astatic epilepsy and Lennox–Gastaut syndrome are rare but provide great challenges in terms of effective therapy. The partial epilepsies (comprising about 60% of all patients with epilepsy) are generally subdivided by the lobe of electro-clinical onset, most commonly temporal or frontal—thus the terms temporal lobe epilepsy and frontal lobe epilepsy. These localization described syndromes can have heterogeneous aetiologies including genetic factors, major acquired lesions (head injury, stroke, tumour, etc.) or perhaps most commonly, a mixture of factors. A sub-set of the focal epilepsies demonstrates autosomal dominant inheritance and considerable progress has been made in the genetics of these (see below). These include autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) where the pattern of the frontal lobe seizures, generally beginning in the first two decades of life, are inherited as an autosomal dominant trait with approximately 70% penetrance (Scheffer et al., 1995). Inherited temporal lobe epilepsy syndromes include autosomal dominant partial epilepsy with auditory features (ADPEAF) where clinical onset is characteristically with auditory hallucinations, reflecting seizure onset in the lateral temporal region (Ottman et al., 1995). Syndromes that begin in the first year of life are more difficult to subdivide into focal or generalized types, probably reflecting the

C.A. Reid et al. / Progress in Neurobiology 87 (2009) 41–57

immaturity of the nervous system where myelination is gradually occurring. A group of autosomal dominant epilepsy syndromes including benign familial neonatal seizures (BFNS), benign familial neonatal-infantile seizures (BFNIS) and benign familial infantile seizures begin in the first year of life with their names indicating the typical ages of onset (Berkovic et al., 2004). A much more severe infantile syndrome beginning around the age of 6 months is Dravet syndrome (also known as severe myoclonic epilepsy in infancy—SMEI) that can be regarded as the most severe phenotype of GEFS+. Here the disorder typically begins with prolonged seizures with fever, often down one side of the body, and thereafter follow heterogeneous patterns of seizures associated with developmental regression (Dravet et al., 2005). Classifications of epilepsies have attempted to encompass etiology and the terms ‘‘idiopathic’’ and ‘‘symptomatic’’ are often used. Idiopathic generalised epilepsies (IGE) have no evidence of an underlying cause. Most of the idiopathic epilepsies are benign, selflimited or easily treatable disorders and are believed to be largely genetic in origin. Conversely, the term symptomatic epilepsies is used when cases have a known or suspected cause and/or when there are associated neurological deficits such as intellectual disability or motor weakness. The boundaries between these two groups is blurred, and is increasingly blurring as knowledge increases, but the terms remain useful pragmatic designators in the clinic, providing the limitations are recognized. This review focuses on those epilepsies with a largely genetic cause, where the causative genes are known, although these presently account for a small minority of all human cases. We do not discuss the large number of rare ‘‘symptomatic’’ genetic epilepsies (e.g., Lafora disease, Rett syndrome, etc.) where seizure disorders are but one part of a complex phenotype, often including mental retardation, motor deficits and multi-system involvement. 3. Genetic architecture of epilepsies and the dual role of physiological investigations The initial successes in identifying epilepsy genes came from analysis of large pedigrees, where there was clinical genetic evidence of segregation of a major autosomal dominant gene. Study of these families was and remains important, as such families are a ‘‘fast track’’ to identifying causative genes of large effect. Such families are not, however, representative of the majority of human subjects with inherited epilepsies. As in all common diseases with an inherited component, epilepsies have complex genetics—that is they are determined by multiple genes with or without an environmental component (see Helbig et al., 2008 for a detailed discussion of the genetics of human epilepsy). However, the rare families with single genes of major effect are an extreme that we can take advantage of to determine critical pathways and set new principles regarding the causation of disease. It is worth noting that all epilepsy mutations discussed in this review express in a heterozygous manner (i.e., evident only in one allele). The pathological impact of each mutation is therefore either due to a dominant effect of the mutant protein within neurons (or glia) or a loss-of-function due to haploinsufficiency (i.e., a loss of ability to generate sufficient protein from the one ‘working’ allele). Physiological investigations have two major and at times subtly contradictory roles in this field. Fundamentally, physiological studies enable a logical series of experiments to bridge the gap between genotype and phenotype and identify targets for potential therapeutic intervention. Secondly, but often initially, the observation of a putative physiological change associated with a gene variant is used to validate that variant as contributing to disease. This can result in an element of circularity. The use of physiological

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tests to validate mutations is less important for major genes segregating in large pedigrees, particularly where there is independent confirmation of that gene in different pedigrees, ideally with different mutations. Here the genetic data alone speak clearly as to the relevance of the gene and physiology can be used for the important task of unravelling pathogenesis. In the case of genes contributing to a complex phenotype, however, the role of physiological testing can be more double handed, and the circularity problem can arise. Gene variants associated with complex phenotypes are much more difficult to identify by conventional genetic analysis and, moreover, because such variants might reasonably be expected to have a small physiological effect it may be difficult to identify such an effect in vitro. Indeed, identification of an in vitro effect might not necessarily be relevant to pathogenesis and, if used to validate a mutation where the genetic data is weak, it may lead to wrong and circular logic of causation. The review next discusses the genetic and physiological evidence linking mutations to epileptic phenotype. It is divided into voltage-gated channels (Na+, K+, Ca2+, and Cl), ligand-gated channels (nicotinic acetylcholine and GABAA receptors) and miscellaneous channels. Table 1 (voltage-dependent channels), Table 2 (ligand-gated) and Table 3 (miscellaneous genes) lists the genes reported in the idiopathic epilepsies and we tabulate these indicating our judgement as to the strength of the data, based on the clinical and molecular genetics, identification in multiple labs and extent of physiological validation. Genes that have been reported based solely on genetic association studies, which are prone to a variety of methodological problems, are not discussed (Cavalleri et al., 2005; Tan et al., 2004). Genome wide association studies have just begun to bear fruit in certain complex diseases Table 1 Voltage-gated ion channel genes associated with epilepsy. The table describes the gene and which epilepsy syndrome it is associated with. We attempt to put semiquantitative estimates as to the validity of associating the epileptic phenotype to each gene by scoring three criteria: genetic confirmation, in vitro functional assays and in vitro functional models as described in the key. Gene

SCN1A SCN2A SCN1B KCNQ2 KCNQ3 KCNA1 KCNMA1 KCNJ11 CACNA1H CACNA1A CACNB4 CLCN2 HCN1 &HCN2

Syndrome

Dravet/SMEI GEFS+ BFNIS GEFS+ BFNS BFNS Partial epilepsy and episodic ataxia Epilepsy and paroxysmal dyskinesia Epilepsy, neonatal diabetes IGE Epilepsy, migraine, episodic ataxia JME IGE IGE

Genetic confirmation

Functional confirmation In vitro

In vivo

+++ +++ ++ ++ +++ ++ ++

+++ ++ +++ +++ +++ +++ ++

+++

+

++

++

+++

++

++

++ +

++

+ +++

++ +++ ++

+ +

KEY—Genetic confirmation: +++, confirmed in at least 3 independent labs; ++, reported by 2 independent labs; +, mutation segregates in a large family. In vitro confirmation: +++, functional impact confirmed in more than 1 lab and in more than 1 in vitro expression system; ++, functional impact confirmed in more than 1 lab or in more than 1 in vitro expression system; +, functional impact only confirmed in a single lab or a single in vitro expression system; no functional deficit observed. In vivo confirmation: +++, validation based on known human mutation with seizure phenotype; ++, knock-out of gene is predictive of known channel dysfunction and results in seizure phenotype; +, available genetic models based on mutations not found in humans; no known animal model.

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Table 2 Ligand-gated ion channel associated with epilepsy. The table describes the gene and which epilepsy syndrome it is associated with. We attempt to put quantitative limits as to the validity of associating the epileptic phenotype to each gene by scoring three criteria: genetic confirmation, in vitro functional assays and in vitro functional models as described in the key. Gene

CHRNA4 CHRNB2 CHRNA2 GABRG2 GABRA1 GABRD

Syndrome

ADNFLE ADNFLE ADNFLE FS/GEFS+ IGE IGE/GEFS+

Genetic confirmation

Functional confirmation In vitro

In vivo

+++ ++ + +++ ++

++ ++ + +++ ++ +

+++

+++

KEY—Genetic confirmation: +++, confirmed in at least 3 independent labs; ++, reported by 2 independent labs; +, mutation segregates in a large family. In vitro confirmation: +++, functional impact confirmed in more than 1 lab and in more than 1 in vitro expression system; ++, functional impact confirmed in more than 1 lab or in more than 1 in vitro expression system; +, functional impact only confirmed in a single lab or a single in vitro expression system; no functional deficit observed. In vivo confirmation: +++, validation based on known human mutation with seizure phenotype; ++, knock-out of gene is predictive of known channel dysfunction and results in seizure phenotype; +, available genetic models based on mutations not found in humans; no known animal model. Table 3 Miscellaneous genes associated with epilepsy. The table describes the gene and which epilepsy syndrome it is associated with. We attempt to put quantitative limits as to the validity of associating the epileptic phenotype to each gene by scoring three criteria: genetic confirmation, in vitro functional assays and in vitro functional models as described in the key. Gene

Syndrome

Genetic confirmation

Functional confirmation

LGI1 ATP1A2

ADPEAF Infantile convulsions, migraine epilepsy syndromes IGE IGE Epilepsy, Migraine, episodic ataxia FS IGE and dyskinesia

+++ ++

++ +

++

++ +

++

++

In vitro

ME2 EFHC1 SLC1A3 MASS1 GLUT1

In vivo

KEY—Genetic confirmation: +++, confirmed in at least 3 independent labs; ++, reported by 2 independent labs; +, mutation segregates in a large family. In vitro confirmation: +++, functional impact confirmed in more than 1 lab and in more than 1 in vitro expression system; ++, functional impact confirmed in more than 1 lab or in more than 1 in vitro expression system; +, functional impact only confirmed in a single lab or a single in vitro expression system; no functional deficit observed. In vivo confirmation: +++, validation based on known human mutation with seizure phenotype; ++, knock-out of gene is predictive of known channel dysfunction and results in seizure phenotype; +, available genetic models based on mutations not found in humans; no known animal model.

(Kruglyak, 2008); at the time of writing no reports in epilepsy have been published. 4. Voltage-gated channelopathies 4.1. Voltage-dependent Na+ channels The primary role of voltage-dependent Na+ channels is for the initiation and propagation of action potentials, making them critical determinants of neuronal excitability. Nine genes encode the pore-forming a subunit, with four genes encoding the ancillary b subunits (Catterall et al., 2005). Unlike the different classes of K+ and Ca2+ channels, the functional properties of Na+ channels are very similar (Catterall et al., 2005). Mutations in both a and b subunits have been linked to epilepsy.

4.1.1. SCN1A SCN1A encodes the a1 (NaV1.1) subunit that forms a fast inactivating voltage-dependent Na+ channel. Historically this subunit has been thought to reside predominantly within the soma (Westenbroek et al., 1989). However, recent evidence suggests an axon initial segment (AIS) and nodes of Ranvier distribution potentially making it a critical subunit in the control of action potential generation and propagation (Duflocq et al., 2008). The a1 subunit is usually associated with one or two b subunits. 4.1.1.1. Clinical syndrome and molecular findings. Since first discovered in 2000 (Escayg et al., 2000b) over 100 mutations in SCN1A have been described in epilepsy (Mulley et al., 2005). The genotype–phenotype relationships are complex. Missense mutations at various sites in SCN1A protein are associated with GEFS+. While most individuals have mild self-limited phenotypes, the devastating disorder of Dravet syndrome can also be seen in patients with SCN1A mutations. More often, Dravet syndrome occurs as a sporadic disorder with de novo mutations (absent in both parents). Both missense mutations and nonsense mutations (those predicted to truncate the protein) are seen (Claes et al., 2001; Fujiwara et al., 2003; Kearney et al., 2006a; Nabbout et al., 2003; Ohmori et al., 2006, 2002). Recently, deletions of whole exons, multiple exons or the whole gene have been found in Dravet syndrome (Madia et al., 2006; Mulley et al., 2006; Suls et al., 2006). Vaccination, particularly with pertussis vaccine, was previously alleged to cause a severe epileptic encephalopathy. Such cases have recently been shown to usually have the clinical phenotype of Dravet syndrome and to have de novo SCN1A mutations (Berkovic et al., 2006a). Families with dominant disease inheritance, where the genetic evidence points to a single gene of large effect, make up only a small proportion of GEFS+ cases. The presence of marked phenotypic variation in some of these dominant families suggests that modifier genes, which are yet to be identified, also play a significant role. Most cases of GEFS+ appear to have complex inheritance, likely due to a number of genes. Even in the numerous cases with de novo mutations there is an expanding range of phenotypes, sometimes with identical mutations, again suggesting a role for modifier genes (Harkin et al., 2007). 4.1.1.2. Functional impact of mutations and potential neuronal mechanisms. Functional analysis of these mutations highlights the difficulty in correlating biophysical properties to the clinical phenotype. It also highlights the fact that we are still some way from truly defining the neuronal mechanisms that underlie these syndromes. Early electrophysiology and computer modelling studies attempted to provide a unifying concept for GEFS+ Na+ channel mutations, in which gain-of-function mutations drive hyperexcitability (Lossin et al., 2002). Several biophysical mechanisms have been proposed. For example; R1648H accelerated recovery from inactivation (Spampanato et al., 2001), possibly due to an impairment in a secondary ‘latch’ that stabilizes inactivation (Kahlig et al., 2006); W1204R showed a negative shift in the voltage-dependence of activation and steady-state inactivation (Spampanato et al., 2004a); and D188Y showed less usedependent decline in Na+ channel amplitude (Cossette et al., 2003). The D1866Y mutation also decreased the modulation by b1 subunit of the SCN1A protein resulting in an increased persistent current (Spampanato et al., 2004b). Increases in persistent Na+ current will contribute to depolarisation, thereby reducing the voltage required to fire action potentials. Persistent Na+ current can also contribute to the shaping of repetitive firing, the generation of rhythmicity and the amplification of both excitatory and inhibitory currents. Similarly, a mutation specific reduction in

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Na+ channel use-dependence will allow cells to fire at higher rates than normal increasing excitability. More recent studies have not supported the simple view that gain-of-function mutations in Na+ current should be responsible for hyperexcitability. A number of SCN1A mutations (e.g., V1353L, I1657C, R859C) linked to GEFS+ exhibit loss-of-function (Barela et al., 2006; Lossin et al., 2003). A trafficking deficit has also been proposed, which interestingly can be rescued with reduced temperature and specific anti-epileptic drugs (Rusconi et al., 2007). Further, as noted, the very severe disease Dravet syndrome is frequently linked to non-functional channels (Madia et al., 2006; Mulley et al., 2006; Ohmori et al., 2006; Suls et al., 2008). This argues that both increases and decreases in channel activity can underlie neuronal hyperexcitability that causes GEFS+. How can a gain-of-function and a loss-of-function result in a similar phenotype? Animal models have gone some way in explaining how a loss of SCN1A protein could enhance network excitability in Dravet syndrome. The Scn1a knock-out mouse models mimic the lossof-function mutations found in most cases of this disease (Yu et al., 2006). In some genetic backgrounds, heterozygous mice developed spontaneous seizures and sporadic death reflecting the severity of the disease in humans. Interestingly, although Na+ currents were essentially unchanged in hippocampal excitatory pyramidal neurons, the current was substantially reduced in inhibitory interneurons. This was due to differential compensation by other Na+ channel subunits within the different cell types. A significant reduction in interneuron function was postulated to underlie hyperexcitability in these animals. Similarly, a heterozygous knock-in mouse carrying the R1407X truncation mutation found in humans also displayed epileptic seizures early in life (Ogiwara et al., 2007). This study structurally isolates a SCN1A protein deficit to the AIS of a subpopulation of inhibitory interneurons in the developing neocortex. Animals heterozygous for the mutation show pronounced action potential attenuation during continuous firing in fast-spiking parvalbumin-positive interneurons. No deficit was evident in pyramidal neurons. Therefore, a selective loss of inhibitory neuron contribution during network activity may explain the severe excitable phenotype noted in Dravet syndrome. It is therefore possible that a loss- and gain-offunction can result in an excitable phenotype, via impact on different neuron types. It remains to be seen if this is true for more subtle loss- and gain-of-function mutations that result in the less severe GEFS+ phenotype. As an aside, analysis of the literature on Na+ channel mutant analysis raises the question of the real benefit of classifying channel mutations as loss- or gain-of-function. In some mutations, such as those that result in the absence of current, a loss-offunction is obvious. However, for others, where subtle, yet simultaneous, changes in voltage sensitivity and/or kinetics of activation, inactivation and recovery are seen, this simple classification scheme cannot fully capture the impact on channel function. Beyond this classification of a mutations impact at the molecular level, genetic compensation, cell biological effects and others all contribute to the overall consequence on networks and seizure genesis. 4.1.2. SCN2A Mutations in the SCN2A encoding the a2 (NaV1.2) subunit have also been linked to epilepsy. These subunits are found predominantly on unmyelinated axons (Westenbroek et al., 1989) including the AIS (Osorio et al., 2005). They are important for action potential initiation, propagation and the generation of repetitive firing.

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4.1.2.1. Clinical syndrome and molecular findings. Missense mutations in this gene have been most frequently associated with BFNIS, a mild, self-limited epilepsy syndrome of the first year of life (Berkovic et al., 2004; Heron et al., 2002; Striano et al., 2006; Herlenius et al., 2007). Earlier, a small Japanese family with complex inheritance contributing to febrile and afebrile seizures was observed with a missense R188W mutation (Sugawara et al., 2001). Similarly to that observed with the SCN1A mutations, where missense mutations often have mild phenotypes and truncations more severe ones. A de novo nonsense mutation in SCN2A (R102X) has been found in a single subject with more a severe epileptic phenotype that has similarities with Dravet syndrome (Kamiya et al., 2004). 4.1.2.2. Functional impact of mutations and potential neuronal mechanisms. The R188W mutation described in the Japanese family was reported to slow the inactivation of the channel and an augmentation of the Na+ current was predicted to underlie neuronal excitability (Sugawara et al., 2001). In vitro analysis of the L1563V mutation associated with BFNIS revealed an increase in excitability due to a depolarised shift in fast inactivation (Xu et al., 2007b). The mutation increased excitability of the neonatal isoform of the SCN2A channel but not the adult isoform (Xu et al., 2007b). The greater excitability of the adult relative to the neonatal isoform suggests a role for developmentally regulated splicing in controlling neuronal excitability (Xu et al., 2007b). Analysis of the R102X mutant protein, linked to a more severe epileptic phenotype, co-expressed with the wild-type (WT) channel revealed that mutated protein shifted the voltagedependence of inactivation of the WT protein in a hyperpolarizing direction (Kamiya et al., 2004). Subcellular localisation studies suggested that the dominant-negative regulation of the WT protein may be due to an effect on cytoskeletal interactions of the mutant protein (Kamiya et al., 2004). SCN2A channels harbouring the L1330F, L1563V, R223Q, and R1319Q mutations have also been transfected into pyramidal and bipolar neocortical neuronal cultures (Scalmani et al., 2006). Scalmani et al. conclude that mutation-mediated increases in Na+ current are responsible for neuronal hyperexcitability. 4.1.3. SCN1B SCN1B encodes the b1 ancillary subunit. Na+ channel b subunits are multifunctional, modulating channel gating, regulating the level of channel expression and potentially acting as a cell adhesion molecule (Isom, 2002). These channels are found in high abundance in several brain regions, although their precise subcellular location is unclear. 4.1.3.1. Clinical syndrome and molecular findings. The first mutation in SCN1B was described in an Australian family with GEFS+ (Wallace et al., 1998). A missense mutation was found at a highly conserved cysteine residue (C121W) in an extracellular domain. It is postulated that this mutation disrupts a disulfide bridge involved in the stabilisation of the immunoglobulin-like domain. A later study, adding credence to the importance of this channel region, described an epilepsy family with FS+ and early-onset absence epilepsy that was predicted to result from a 5 amino acid deletion in the same domain (Audenaert et al., 2003). Further families with SCN1B mutations have been recently described including two novel mutations (R85C and R85H) (Scheffer et al., 2007). Interestingly, five patients with the C121W mutation had confirmed temporal lobe epilepsy (Scheffer et al., 2007). 4.1.3.2. Functional impact of mutations and potential cellular mechanisms. Electrophysiological studies of co-expressed mutant

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b (C121W) subunit and pore-forming a subunit, in both mammalian and Xenopus oocycte expression systems, are consistent with loss of b subunit function. The result is a potential increase in Na+ current due to slowing of inactivation, increased availability of channels at hyperpolarized potentials, and reduction in the channel rundown during high-frequency activation (Meadows et al., 2002; Wallace et al., 1998). A similar loss of modulatory effects of the b subunit on SCN2A channels was noted for the R85C and R85H mutations (Xu et al., 2007a). A net increase in Na+ current is predicted to increase neuron excitability. A computer modelling study based on the R85C and R85H mutations suggests that excitability was most sensitive to shifts in voltage-dependence of activation, a finding likely to be translatable to other Na+ channel mutations (Thomas et al., 2007). The impact of the C121W (or other) mutation may not be limited to its effect on Na+ channel kinetics. A cell adhesion assay demonstrated a disruption in the ability of the mutated b subunit to mediate protein–protein interactions (Meadows et al., 2002). Interestingly, the homozygous Scn1b knock-out mouse displays spontaneous generalised seizures (Chen et al., 2004). An abnormal expression pattern for the SCN1A and SCN3A protein in hippocampal neurons, a disruption in axoglial communication and/or a developmental disruption in neurite growth are present in the knock-out animal (Chen et al., 2004; Davis et al., 2004). Dysfunction of any of the adhesion roles played by the b1 subunit could therefore be equally responsible for an excitable phenotype. 4.2. Voltage-dependent K+ channels K+ channels are the most diverse group of ion channels with over 70 subunits discovered to date. These channels can be classified into three structural families depending on the number of transmembrane domains in each subunit. Voltage-dependent K+ channels contain six transmembrane domains and include several subfamilies such as KCNQ, KV1 and KCa1 channels (Alexander et al., 2008) in which epilepsy-causing mutations have been described. The inward-rectifier K+ channels contain two transmembrane domains. Again, there are several subclasses, with mutations in the Kir6 and Kir4 subclasses potentially implicated in epilepsy. Collectively these channels play a wide variety of roles in defining neuronal excitability including the modulation of neuronal firing patterns, defining resting membrane potential and the modulation of neurotransmitter release. The four transmembrane domains (or two pore domain) family is an additional family that is responsible for background or leak K+ currents (Patel and Honore, 2001). There are no documented epilepsy mutations in this family. 4.2.1. KCNQ2 and KCNQ3 Mutations in a voltage-activated K+ channel, specifically those encoded by KCNQ2 and KCNQ3, provide us with a relatively complete picture linking mutation to cellular mechanisms underlying an epileptic phenotype. Heteromeric and homomeric channels encoded by KCNQ2 (KV7.2) and KCNQ3 (KV7.3) have the electrophysiological and pharmacological properties characteristic of M channels (Selyanko et al., 2001; Wang et al., 1998). The Mcurrent is a non-inactivating voltage-gated K+ current that is open during prolonged depolarisation and mediates the medium afterhyperpolarisation conductance. 4.2.1.1. Clinical syndrome and molecular findings. BFNS is an autosomal-dominant epilepsy in which the vast majority of families have mutations in KCNQ2, with a few in KCNQ3. Over 50 mutations in KCNQ2 and three in KCNQ3 have been identified in families affected by this form of epilepsy (Bassi et al., 2005; Biervert et al., 1998; Borgatti et al., 2004; Coppola et al., 2003;

Dedek et al., 2001; Hirose et al., 2000; Hunter et al., 2006; Richards et al., 2004; Lee et al., 2000; Lerche et al., 1999; Miraglia del Giudice et al., 2000; Singh et al., 1998, 2003; Tang et al., 2004; Zhou et al., 2006). De novo mutations in KCNQ2 have also been found in patients with benign neonatal seizures without family history (Claes et al., 2004). Approximately 50% of mutations are predicated to truncate the protein and the remainder are missense mutations. 4.2.1.2. Functional impact of mutations and potential neuronal mechanisms. The majority of mutations occurring in the KCNQ2 protein are in the C-terminus, a region likely to be important in subunit co-assembly (Schwake et al., 2003). The C-terminus also contains domains that interact with calmodulin (Wen and Levitan, 2002) and a dysfunction in the KCNQ2-calmodulin interaction has been noted for mutations linked to epilepsy (Richards et al., 2004). However, the most consistent finding is loss-of-function for channels harbouring mutations linked to BFNS via a variety of biophysical and/or trafficking mechanisms (Bassi et al., 2005; Borgatti et al., 2004; Dedek et al., 2001; Hunter et al., 2006; Lerche et al., 1999; Schroeder et al., 1998; Schwake et al., 2000; Singh et al., 2003; Soldovieri et al., 2006, 2007; Wuttke et al., 2008). Activation of M-channels tends to stabilise membrane potential thereby limiting action potential firing. A loss of this ability, due to mutations, would therefore be expected to increase neuronal excitability. Animal studies go some way in providing a more complete understanding of this mechanism. Importantly, a conditional transgenic mouse harbouring a dominant-negative pore mutation (G279S) in KCNQ2, found in humans, developed spontaneous seizures (Peters et al., 2005). Electrophysiological recordings from CA1 pyramidal neurons showed a reduced medium after-polarisation in the mutant mice, and as a consequence a reduced spike-frequency adaptation potentially explaining the excitable phenotype (Peters et al., 2005). A similar finding was observed in CA1 pyramidal neurons of a spontaneous mutation in Kcnq2 in mice that have an altered seizure threshold (Otto et al., 2006). Both KCNQ2 and KCNQ3 protein are also found in parvalbumin-positive hippocampal interneuron, but what impact a loss-of-function of these channels in these cells has on neuronal networks is unclear. KCNQ2 and KCNQ3 protein co-localise at the AIS and/or nodes of Ranvier in some neurons (Devaux et al., 2004; Schwarz et al., 2006). Here they play an integral role in defining nodal excitability including spike-frequency adaptation and thus conductance along myelinated axons (Schwarz et al., 2006). A hypo-functioning mutated channel at nodes could also drive hyperexcitable neuronal circuits (Devaux et al., 2004). Therefore, a loss-offunction either preventing somatic or axonal spike-frequency adaptation is likely to drive hyperexcitability in patients harbouring mutations in KCNQ2 and KCNQ3. Within a few weeks or months of birth, BFNS spontaneously remits in the vast majority of patients, the basis of which is unresolved. Recent immunohistochemical analysis of human tissue suggests that simultaneous high expression of both subunits occurs at times that coincides with the onset of BFNS (Kanaumi et al., 2008). However, the developmental pattern of KCNQ2 and KCNQ3 protein expression seems unlikely to explain remission since the expression of both subunits increases during maturation (Tinel et al., 1998; Geiger et al., 2006; Weber et al., 2006). A further plausible hypothesis is that remission coincides with a switch of the transmitter GABA as a predominantly excitatory transmitter in early development to its more traditional inhibitory role (Rivera et al., 1999). During early development periods, inhibition based on K+ flux may be more critical and a loss-of-function due to mutations in KCNQ2/3 could result in hyperexcitability sufficient to generate seizures (Okada et al., 2003). It must be noted that

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approximately 10% of patients suffering BFNS do go on to have further seizures suggesting an ongoing fundamental physiological alteration that is prone to have a clinical effect in certain circumstances. 4.2.2. KCNA1 KCNA1 encodes the KV1.1 channel that is widely expressed throughout the nervous system. The channel concentrates on the axonal membrane and presynaptic nerve terminals where it helps to repolarise and shape action potentials. Blocking K+ channels broadens the action potential increasing the flux of Ca2+ into the presynaptic terminal, in turn increasing neurotransmitter release. 4.2.2.1. Clinical syndrome and molecular findings. Mutations in KCNA1 cause the dominant disorder Episodic Ataxia Type 1 (EA1, (Browne et al., 1994)). Although not present in every patient with EA1, epilepsy is over represented in some EA1 families. Thus KCN1A cannot be regarded as a gene of major effect in causing epilepsy, but appears to be a risk factor for seizures, presumably interacting with the genetic background in these families (Eunson et al., 2000; Zuberi et al., 1999). 4.2.2.2. Functional impact of mutations and potential neuronal mechanisms. Analysis of mutant channels suggests impaired function through a variety of mechanisms including channel assembly, trafficking and kinetics (Cusimano et al., 2004; Eunson et al., 2000; Rea et al., 2002; Zuberi et al., 1999). The severity of loss-of-function may loosely correlate to behavioural phenotype (Rea et al., 2002). A mouse model in which Kcn1a is entirely knocked out displays spontaneous seizures (Smart et al., 1998). The passive neuronal properties of the knock-out mouse were normal. However, an increase in the frequency and amplitude of spontaneous and miniature inhibitory postsynaptic currents (mIPSC), but not excitatory postsynaptic currents (EPSC) was observed in the layer V pyramidal neurons (van Brederode et al., 2001). Similarly, mice harbouring the loss-of-function V408A mutation linked to EA1 showed an increase in spontaneous IPSCs (but not miniature IPSC) onto Purkinje cells (Herson et al., 2003). The increase in GABA release presumably reflects, at least in part, the broadening of action potentials in the terminal, but the basis of the relative selectivity for GABAergic terminals is not clear. Also, the mechanism via which enhanced GABA release mediates the behavioural changes noted in these animals and the relevance to epilepsy remains to be determined. 4.2.3. Other mutations in K+ channels linked to epilepsy Mutations in three other K+ channel genes have been reported in human epilepsy. A mutation in KCNMA1 encoding the poreforming subunit of the BK channel (KCa1.1) has been found in a family suffering coexistent generalised epilepsy and paroxysmal dyskinesia (Du et al., 2005). The BK channel is a large conductance K+ channel that is gated by depolarisation and increased intracellular Ca2+. Functional studies indicate a fivefold increase in sensitivity to Ca2+ of the mutant channel (Du et al., 2005). The authors propose that the ability of the enhanced BK channel to repolarise neurons following an action potential allows them to fire at higher frequencies. This hypothesis is supported by studies on b4 accessory subunit knock-out mice that displays spontaneous nonconvulsive seizures (Brenner et al., 2005). An important function of b4 subunits is to reduce BK channel function. In the knock-out mouse, the consequential gain-of-function of the BK channel underlies a ‘sharpening’ of action potentials allowing the cell to support higher firing frequency. Heterozygous mutations found in KCNJ11 (Kir6.2) can cause permanent neonatal diabetes mellitus that in severe cases can be

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accompanied with developmental delay and seizures that may be treatable with sulphonylurea drugs (Proks et al., 2004; Shimomura et al., 2007). KCNJ11 encodes the pore-forming subunit of the ATPsensitive K+ channel (Kir6.2). All mutations increased resting current by reducing the inhibition by ATP. KCNJ10 encodes Kir4.1, an inwardly rectifying K+ channel that regulates extracellular K+ concentration. A common missense mutation in KCNJ10 is postulated to confer seizure resistance to individuals (Buono et al., 2004; Lenzen et al., 2005a). However, functional analysis did not produce any observable changes in channel activity raising questions as to the relevance of this gene to seizure susceptibility phenotypes (Shang et al., 2005). 4.3. Voltage-dependent Ca2+ channels Voltage-activated Ca2+ channels support a number of dynamic processes in neurons with both presynaptic and postsynaptic Ca2+ channels being critical determinants of neuronal excitability. Mutations in genes encoding these channels are therefore logical candidates in the context of epilepsy phenotypes. Electrophysiological studies indicate there are at least six Ca2+ channel types: L-, N-, P-, Q-, R- and T-type (Catterall, 2000). Voltage-activated Ca2+ channels can be divided into high voltage-activated or low voltageactivated classes based on the membrane potential range over which the channel is activated. The key determinant of Ca2+ channel subtypes character is their a1 pore-forming subunit. However, additional ancillary subunits significantly influence the kinetics of Ca2+ channels. Absence epilepsy in a number of mice models is caused by recessive defects in genes encoding a variety of high voltage-activated Ca2+ channels. For example, Rolling-nagoya (Mori et al., 2000) and rocker (Zwingman et al., 2001) mice pathologies are due to defects in the P/Q-type Ca2+ channel a subunit gene while lethargic (Burgess et al., 1997), ducky (Barclay et al., 2001), and stargazer (Letts et al., 1998) have defects in genes for the ancillary b4–, a2d, and g2 subunits respectively. Mutations in these channels are rarely observed in humans. A heterozygous mutation in CACNA1A (P/Q-type Ca2+ channel) has been described in one individual with a complex phenotype that included generalised epilepsy (Jouvenceau et al., 2001). Further, a small family exhibiting absence epilepsy combined with ataxia has a mutation in CACNA1A (Imbrici et al., 2004). Functional studies implicate a loss of P/Q-type Ca2+ channel function in both cases (Imbrici et al., 2004; Jouvenceau et al., 2001). Similarly, a mutation in CACNB4 (b4 subunit) described in one patient with JME resulted in a loss of protein function (Escayg et al., 2000a). However, to date, the most compelling evidence of epilepsy implicated Ca2+ channel is for CACN1AH that encodes a low-threshold T-type Ca2+ channel. 4.3.1. CACNA1H Low voltage-activated Ca2+ channels belonging to the CaV3 family contain three members (CaV3.1 (a1G), CaV3.2 (a1H), CaV3.3 (a1I)) (Catterall, 2000). They are preferentially located in neuronal dendrites where they define neuronal excitability (Perez-Reyes, 2003). This includes mediating low-threshold action potentials that support burst firing (Destexhe et al., 1996; Huguenard and McCormick, 1992) and boosting of synaptic inputs (Cook and Johnston, 1997; Migliore and Shepherd, 2002). Within the thalamocortical circuitry CACN1AH protein is found predominantly in layer V of the cortex and in the reticular thalamic nuclei of the thalamus (Talley et al., 1999). 4.3.1.1. Clinical syndrome and molecular findings. Over 30 mutations located in CACNA1H that encodes the CaV3.2 subunit have been found in IGE cases (Chen et al., 2003; Heron et al., 2007, 2004; Khosravani et al., 2004, 2005; Vitko et al., 2005). Initial studies by

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Chen et al. (2003) found 12 rare missense mutations in 14 of 118 patients suffering CAE suggesting that it may be a susceptibility gene involved in the pathogenesis of this disease. Heron et al. (2004) discovered four missense mutations in patients with IGE but none of the mutations were found in patients with JAE or CAE. Interestingly, evaluation of 44 pedigrees and 220 unrelated patients with CAE of Caucasian European origin also failed to find evidence for mutations in CACNA1H (Chioza et al., 2006) suggesting a population bias, although the gene was not systematically sequenced. 4.3.1.2. Functional impact of mutations and potential neuronal mechanisms. Classical 3 Hz spike-wave discharges associated with absence epilepsy involve inappropriate oscillations of the thalamocortical network (Crunelli and Leresche, 2002). Low threshold spikes, which trigger bursting, are mediated by T-type Ca2+ channels and play an important role in regulating neuronal excitability in thalamic neurons where they are highly expressed (Perez-Reyes, 2003). Several lines of evidence support the hypothesis that changes in T-type Ca2+ channel function are a significant factor in the pathogenesis of absence epilepsy. Firstly, the anti-absence drug ethosuximide blocks T-type Ca2+ channels at physiologically relevant concentrations (Coulter et al., 1989). Secondly, quantitative increases in expression and function of Ttype Ca2+ channels occur in mouse and rat models of absence epilepsy (Talley et al., 2000; Tsakiridou et al., 1995; Zhang et al., 2004). Finally, mice that lack Cacna1g (CaV3.1) are resistant to spontaneous absence seizures exhibited in the P/Q Ca2+ channel knock-out mouse (Kim et al., 2001). Together this provides a framework in which a gain-of-function in the T-type Ca2+ channel is predicted to increase thalamo-cortical hyperexcitability. Functional studies were undertaken on five mutations found in the study of Chen et al. (2003) in Chinese subjects. Whole-cell voltage clamp recordings from HEK293 cells transfected with the mutated rat CaV3.2 cDNA showed a hyperpolarisation shift in activation in F161L and E2832K mutants and a slowing of inactivation for V831M (Khosravani et al., 2004) representing a gain-of-function. There were no changes with two other mutations. Vitko et al. (2005) extended these studies by specifically examining the functional effects of 12 CAE-specific mutations in human CaV3.2 cDNA. Interestingly, they found both quantitative and qualitative differences of the same mutation between rat and human cDNA highlighting a need for caution when using rodent cDNA. Eleven of twelve mutations altered channel function, including altered voltage-dependence of activation, altered voltage-dependence of inactivation and altered rate of recovery from the inactivated state. Incorporation of these findings into a computer simulation of the thalamic region, based on Hodgkin– Huxley-type equations, predicted that seven SNPs increased firing of neurons, with three inducing oscillations at frequencies expected during absence seizures (Vitko et al., 2005). Mutations for which the model predicted this behaviour increased persistent T-type current that is expected to lead to prolonged membrane depolarisation. However, three mutations predicted a reduced firing rate. The basis for this discrepancy is yet to be determined. In a further study two of three missense mutations identified in patients with IGE exhibited small functional changes in the activation and inactivation kinetics representing a gain-of-function that is likely to contribute to enhanced neuronal firing (Khosravani et al., 2005). More recently, analysis of 11 new variants showed that 9 had altered gating with most predicting increased Ca2+ current (Heron et al., 2007). Changes in trafficking have also been postulated with mutations clustered around the intracellular I-II loop of the CaV3.2 showing increased cell surface

expression of the channel potentially increasing channel activity (Vitko et al., 2007). Variations in CACNA1H are commonly found in the general population. One such mutation, R788C, occurs in about 20% of Chinese CAE patients (Chen et al., 2003) raising the possibility that two mutations in the gene may have a unique impact on channel function. To test this, both the R788C and G773D mutation were introduced into the channel producing significantly different activation and inactivation kinetics to channels harbouring either mutation alone, arguing that a ‘gene dosing’ may occur (Vitko et al., 2005). The large potential for alternative splicing of the CaV3.2 channel adds an additional twist to an already complex story. Although most studies that have investigated CaV3.2 function have done so in only one splice variants, it can be alternatively spliced at between 12 and 14 sites with the potential to generate over 4000 alternative mRNA sequences (Zhong et al., 2006). Zhong et al. confirmed that a number of these variants can form functional CaV3.2 channels, and that these channels do differ in their gating properties. This raises the question—do various mutants linked to epilepsy impact differently on the alternative spliced variants? In most cases the impact of mutations were comparable or larger than those noted in previous studies. However, a number of mutations linked to IGE and CAE are clustered in or near segments associated with anomalous splicing. In three examples with missense or silent mutations associated with CAE/IGE that did little to change gating kinetics, significant alterations occurred in regions that are predicted to regulate splicing bias. In this scheme inappropriate over/under expression of a variant (with distinct kinetic properties) could drive hyperexcitability. 4.4. Voltage-dependent Cl channels Cl channels are a functionally distinct group of anion selective channels that play a role in regulating the excitability of neurons. In particular, the CLC family that is activated by voltage may be involved in the pathogenesis of certain epilepsies. 4.4.1. CLCN2 CLCN2 encodes a chloride channel (ClC-2) gated by hyperpolarizing potentials. These currents may have a role in K+ buffering, pH regulation and volume regulation (Walz, 2002). However, the role this channel plays in the central nervous system is still unclear. 4.4.1.1. Clinical syndrome and molecular findings. Mutations in CLCN2 have been rarely associated with the common forms of idiopathic generalised epilepsy, including CAE, JAE, JME and grand mal seizures on awakening (D’Agostino et al., 2004; Everett et al., 2007; Haug et al., 2003). 4.4.1.2. Functional impact of mutations and potential neuronal mechanisms. Three primary mutations in CLCN2 have been analysed at the functional level. Two impediments limit our ability to predict the cellular mechanisms that potentially drive neuronal hyperexcitability in patients harbouring these mutations. Firstly, the role the CIC-2 plays in the central nervous system is unclear. Secondly, there is conflicting literature on the basic impact of these mutations. Haug et al. (2003) described a mutation predicted to result in a premature stop codon (M200fsX231) that was shown to exert a dominant-negative effect on WT protein that leads to significant loss-of-function of the protein. In contrast, others find that although the mutant protein is non-functional, it does not exert a dominant negative effect of on the WT protein (Niemeyer et al., 2004). Niemeyer et al. (2004) postulate that haploinsufficiency, at least for the M200fsX231 mutation, results

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in a reduced number of active channels. A loss-of-function, either through haploinsufficiency or dominant negative effects, may increase hyperexcitability by reducing the Cl gradient essential for GABAergic inhibition. However, absence of seizures or of a reduced seizure threshold in the ClC-2 knock-out mouse highlights the difficulty in directly correlating a loss-of-function with an excitable phenotype (Blanz et al., 2007; Nehrke et al., 2002). A missense mutation (G715E) was also found in an unrelated family (Haug et al., 2003). The G715E mutation alters the voltagedependence of gating making it less sensitive to internal chloride concentration. It was proposed that during intense neuronal activity a window occurs in which Cl may be above equilibrium and increased CLC-2 function of the mutant would increase excitability under these conditions. However, change in Cl sensitivity and gating properties were not observed by Niemeyer et al. (2004), so the relevance of this mechanism remains unclear. The affinity of the ATP binding to the cystathionine-b-synthase domain in ClC-2 channels is reduced by an order of magnitude in the G715E mutated protein (Scott et al., 2004). Replacement of ATP with AMP accelerates both opening and closing kinetics of the ClC2 channel, an effect significantly impaired in the heteromeric mutant/WT receptor. Again it is difficult to speculate on how this could result in hyperexcitability, although it was postulated that a depletion of ATP during intense neuronal activity might contribute to epilepsy noted in patients harbouring this mutation. It is important to note that mutations in CLCN2 are most likely to impart an increased susceptibility to develop IGE, and may not be pathogenic per se. Determining other mutations (or environmental factors) that work in concert with mutated ClC-2 will significantly improve our ability to narrow down potential cellular mechanisms involving dysfunction in this gene.

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distributed in the central nervous system. Genes encoding a total of 17 subunits (a1–10, b1–4, d, e and g) have been identified (Kalamida et al., 2007). Mutations in a and b subunit classes have been described in epilepsy. 5.1.1. CHRNA4, CHRNB2 and CHRNA2 CHRNA4 and CHRNB2 encode the a4 and b2 subunits, respectively, which combine to form the most abundant nAChR in the brain (Gotti et al., 1997). CHRNA2 encodes the a2 subunit that is found predominantly in GABAergic interneurons (Son and Winzer-Serhan, 2006). nAChRs are found both pre- and postsynaptically on pyramidal neurons and interneurons (Alkondon and Albuquerque, 2004). One of the major roles of nAChRs is to modulate neurotransmitter release, including the primary transmitters glutamate and GABA (Alkondon and Albuquerque, 2004). 5.1.1.1. Clinical syndrome and molecular findings. ADNFLE is an idiopathic epileptic syndrome with focal seizures arising from the frontal regions and occurs predominantly from stage 2 of sleep (Scheffer et al., 1994). Several mutations in both CHRNA4 and CHRNB2 have been found in families with ADNFLE together with a mutation in the CHRNA2 found in a single family (Aridon et al., 2006; Bertrand et al., 2005; De Fusco et al., 2000; Hirose et al., 1999; Phillips et al., 2001; Steinlein et al., 1997, 1995). These mutations were all initially found to cluster within or around M2, the pore-forming region of the nAChR. Recently, evidence for mutations in M3 of CHRNB2 have been found, one of which was associated with additional memory defects (Bertrand et al., 2005; Hoda et al., 2008). Not all patients suffering ADNFLE have mutations in nAChR. In a recent account, a mutation of the corticotropin-releasing hormone gene has been linked to a family with ADNFLE (Combi et al., 2005).

4.5. HCN Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are encoded by four genes (HCN1–4). HCN channels mediate hyperpolarization-activated currents (Ih) in the brain where they contribute to resting membrane potential (Lupica et al., 2001), to the shaping of synaptic inputs, and to the generation of rhythmic and synchronized neuronal events (Maccaferri and McBain, 1996; Magee, 1999). Several human and animal studies into temporal lobe epilepsy have reported alterations in HCN expression in the hippocampus and related limbic structures that could alter Ih and potentially contribute to the development of epilepsy (Santoro and Baram, 2003). More recently there are reports of alterations in HCN expression and Ih in genetic models of absence epilepsy (Budde et al., 2005; Kole et al., 2007; Kuisle et al., 2006). Together, these results implicate transcriptional changes in HCN and thus Ih in the pathogenesis of various forms of epilepsy. However, there is little evidence that mutations in HCN contribute to epilepsy. A mutation analysis of HCN1 and HCN2 in unrelated patients with IGE revealed several functional variants (Tang et al., 2008). However, in vitro analysis demonstrated only a trend for impact of some of these variants on channel function. Further studies are warranted to establish if mutations in HCN contribute to an epileptic phenotype in man. 5. Ligand-gated channelopathies 5.1. Nicotinic acetylcholine receptors Nicotinic acetylcholine receptors are members of the Cys-loop family of transmitter-gated ion channels and form as homo- or hetero-meric pentamers (Alexander et al., 2008). They are ligandgated ion channels permeable to Na+, K+ and Ca2+ and are widely

5.1.1.2. Functional impact of mutations and potential neuronal mechanisms. Common clinical phenotypes can be caused by a range of mutations in the different nAChR subunits (McLellan et al., 2003). In contrast, at the molecular level there seems to be a range of biophysical consequences of the various identified mutations (Sutor and Zolles, 2001). However, one common effect of mutations in the a4 and b2 subunits is an increase in the sensitivity of the receptor to acetylcholine (Bertrand et al., 2005, 2002; Hoda et al., 2008). Furthermore, recent evidence shows that this is also the case for the a2 subunit (Aridon et al., 2006) suggesting a convergent physiological pathway may underpin ADNFLE. One potential consequence of this heightened acetylcholine sensitivity is an increase in pre-synaptic Ca2+ influx that would increase transmitter release (Reid et al., 1998). Another mechanism has been proposed in which changes in Ca2+-mediated modulation of mutant nAChR’s is impaired with the impact most pronounced at excitatory synapses (Rodrigues-Pinguet et al., 2003, 2005). Knock-in mouse models based on human mutations provide support for a gain-of-function of the nAChR in ADNFLE (Klaassen et al., 2006; Teper et al., 2007). Mutant mice display abnormal EEG patterns consistent with seizure activity (Klaassen et al., 2006), as well as a dystonic phenotype that may form part of the spectrum observed in ADNFLE (Teper et al., 2007). It should be noted that Teper et al. did not observe spontaneous epileptic activity. The most striking cellular phenotype is a >20-fold increase in nicotineevoked synaptic release exclusively at inhibitory synapses on to layer II/III pyramidal neurons in the cortex (Klaassen et al., 2006). A model is proposed in which asynchronous firing layer II/III pyramidal cells are synchronized following recovery from significant GABAA-mediated inhibition triggered by cholinergic activation of mutant nAChRs (Klaassen et al., 2006). This is

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supported by the fact that seizures are blocked by sub-convulsive doses of a GABAA receptor antagonist. Mann and Mody (2008) further discuss at the circuit level how a seemingly paradoxical increase in neocortical GABAA receptor-mediated current could be epileptogenic. 5.2. GABA receptors Gamma-aminobutyric acid (GABA) is the predominant inhibitory transmitter within the central nervous system and acts through three receptor classes: the ionotropic GABAA and GABAC receptors and the metabotropic GABAB receptors. From a genetic perspective all mutations documented to date are localised to GABAA receptors. The GABAA receptor is a transmitter-gated ion channel of the Cys-loop family (Alexander et al., 2008). GABAA receptors are pentameric proteins with a central Cl permeant pore that is formed from various combinations of proteins encoded by the a, b, g, d, e, and u subunits gene families. A vast array of functional GABAA receptors can be formed from combinations of these subunits in a brain-region and development-specific manner. 5.2.1. GABRG2 and GABRA1 GABRG2 encodes the g2 subunit and GABRA1 encodes a1 subunit. The most common GABAA receptor in the brain is the a1b2g2 complex. Both GABRG2 and GABRA1 have been implicated in epilepsy. 5.2.1.1. Clinical syndrome and molecular findings. Two independent groups simultaneously identified mutations in GABRG2 found in families with GEFS+ and CAE (Baulac et al., 2001; Wallace et al., 2001). Two further mutations in GABRG2 have since been associated with GEFS+, Dravet syndrome, CAE and FS (Harkin et al., 2002; Kananura et al., 2002). Both mutations are predicted to lead to truncated protein (Harkin et al., 2002; Kananura et al., 2002). A GABRG2 mutation associated specifically to FS has also been described (Audenaert et al., 2006). Evidence for involvement of GABRA1 in epilepsy has been provided by a familial mutation associated with JME (Cossette et al., 2002) and a sporadic case of CAE (Maljevic et al., 2006). 5.2.1.2. Functional impact of mutations and potential neuronal mechanisms. Several in vitro studies have been performed to determine the functional consequence of the various mutations. Analysis of the R43Q mutation associated with FS and CAE alters benzodiazepine sensitivity, receptor kinetics, assembly, trafficking, and cell surface expression (Bianchi et al., 2002; Bowser et al., 2002; Kang et al., 2006; Sancar and Czajkowski, 2004). These in vitro findings are consistent with a reduction in GABAA receptor-mediated current due to the mutation under the various conditions. The residues surrounding R43 in the g2 subunit appear to be important for inter-subunit interactions and are impaired by the Q43 mutant, providing a potential molecular basis for the kinetic and trafficking changes noted (Hales et al., 2005). Functional analysis of K289M associated with childhood generalised tonic–clonic epilepsy similarly revealed a reduced GABA-mediated current (Baulac et al., 2001). Further kinetic analysis of this mutation demonstrated an acceleration of deactivation (Bianchi et al., 2002). An acceleration of synaptic event decay has also been observed in hippocampal neurons transfected with g2(K289M) (Eugene et al., 2007). R139G, linked exclusively to FS, subtly altered desensitisation kinetics and significantly reduces benzodiazepine sensitivity (Audenaert et al., 2006), qualitatively similar to that observed for R43Q (Wallace et al., 2001). The Q351X truncation mutation in a family

with GEFS+ traps GABAA receptors in the lumen of the endoplasmic reticulum (Harkin et al., 2002). Finally, the A322D mutation in the a1 subunit results in a loss-of-function of GABAA receptors via a reduction in GABA sensitivity, accelerated deactivation and a reduction in cell surface expression of GABAA receptors (Krampfl et al., 2005), a process likely to involve rapid endoplasmic reticulum-associated degradation (Gallagher et al., 2007). Collectively, these data suggest a reduction in GABAA receptor-mediated inhibition may result in neuronal hyperexcitability. This view is supported, at least in part, by a knock-in mouse model carrying the R43Q mutation that recapitulates the absence-seizure phenotype seen in patients (Tan et al., 2007). Synpatically, a small yet reproducible change in inhibitory currents was seen in layer 2/3 cortical pyramidal neurons of these mice (Tan et al., 2007). Additionally, recent work shows that the R43Q mutation can also have a developmental impact on neuronal network excitability (Chiu et al., 2008). This suggests that mutation-mediated dysfunction can trigger a cascade of events (e.g., morphological and/or transcriptional changes) that define long-term network stability. These complex series of events can only be fully expressed and investigated in animal models. Reduced cell surface expression of mutant receptors via a ‘dominant negative’ effect appears to be a common in vitro finding for mutations in GABRG2 and GABRA1 including the g2(R43Q) (Bianchi et al., 2002; Kang and Macdonald, 2004; Sancar and Czajkowski, 2004). However, although the knock-in mouse model carrying the R43Q mutation showed reduced g2 subunit expression, a1 subunit expression was normal, only partially supporting previous in vitro findings and ruling out a ‘classical’ dominantnegative effect (Tan et al., 2007). This was also seen in experiments using recombinant expression of R43 and Q43 GFP tagged g2 subunits in hippocampal cultures (Frugier et al., 2007). These results again highlight the caution required when interpreting data from simple expression systems that lack the complexity of neurons. Tonic inhibition, which carries far more current that classic phasic inhibition (Glykys and Mody, 2007), has been implicated in catamenial epilepsy (Maguire et al., 2005) and may play a role in the pathology of seizure genesis in patients with g2(R43Q). Recombinant expression of R43 or Q43 g2 subunits in cultured hippocampal neurons revealed little difference in phasic inhibitory synaptic currents (Eugene et al., 2007). However, examination of tonic GABA currents in the same preparation showed a significant decrease that correlated to a reduction in the surface expression of the GABAA a5 subunit (Eugene et al., 2007). Patients harbouring mutations in the g2 subunit have epilepsy that is frequently triggered by fever. Motivated by the knowledge that the g2 subunit is critical to receptor trafficking (Alldred et al., 2005; Keller et al., 2004), investigators studied the temperaturesensitivity of the a1b2g2 GABAA receptor containing g2 mutations R43Q, K289M and Q351X (Kang et al., 2006). Increasing incubation temperature selectively decreases mutant receptor expression suggesting that real time reductions in GABAA receptor expression contribute to the genesis of seizures triggered by fever. It is debatable as to whether this represents a general mechanism for FS (Berkovic and Petrou, 2006). 5.2.2. GABRD GABRD encodes the d subunit that forms heteromeric extra- or peri-synaptic channels that differ from the synaptic GABAA channels. As a consequence of this extra-synaptic localization, high GABA affinity, and minimal desensitization ideally position d subunit containing GABAA receptors to respond to ambient GABA and contribute to tonic inhibitory current (Mody, 2001).

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5.2.2.1. Clinical syndrome and molecular findings. Recently, two mutations in GABRD have been proposed as susceptibility alleles for GEFS+ and JME (Dibbens et al., 2004). Confirmation from other patient cohorts will be required to verify GABRD as a true susceptibility epilepsy gene. 5.2.2.2. Functional impact of mutations and neuronal cellular mechanisms. Functional comparison of a1b2d and a1b2d (E177A or R220H) containing receptors revealed a mutationspecific reduction in GABA-mediated current suggesting that a reduction in tonic inhibition may be a risk factor for epilepsy (Dibbens et al., 2004). Further functional studies, but based on the a4b2d subunit combination that is thought to be a more physiologically relevant isoform, showed a reduction in surface expression only for the homozygous mutant channel (Feng et al., 2006). 6. Other genes linked to epilepsy A number of other epilepsies have been linked to mutations in a variety of other proteins. We will briefly discuss these next. 6.1. LGI1 The leucine-rich glioma inactivated gene 1 (LGI1) encodes a protein that is characterised by leucine-rich repeats and that has been proposed to play a role in protein–protein interactions (Kobe and Deisenhofer, 1995). The biological function of this protein in the central nervous system is largely unknown. Many believe that the LGI1 protein is a secreted neuronal protein (Fukata et al., 2006; Sirerol-Piquer et al., 2006). Recent evidence suggests it may also play a role as an ancillary subunit of a channel that underlies the Atype K+ current present in presynaptic terminals (Schulte et al., 2006). 6.1.1. Clinical syndrome and molecular findings Mutations in LGI1 have been identified in patients suffering autosomal dominant partial epilepsy with auditory features (ADPEAF) that is characterised by partial seizures with auditory or other sensory hallucinations (Kalachikov et al., 2002; MoranteRedolat et al., 2002; Ottman et al., 2004). 6.1.2. Functional impact of mutations and potential neuronal mechanisms Several mechanisms explaining how a mutated LGI1 protein may result in epilepsy have been proposed recently. The first relates to the LGI1 protein as an ancillary subunit to the A-type of K+ channel. The co-expression of KV1.1, KV1.4 and KVb1 are thought to closely resemble the A-type potassium current that is present in presynaptic terminals responsible for repolarisation (Monaghan et al., 2001). The KVb1 subunit contributes significantly to the rapid inactivation of this heteromeric channel. Functional LGI1 protein inhibits the interaction between KV1.1 and KVb1 resulting in a significant slowing of inactivation (Schulte et al., 2006). However, LGI1 protein harbouring mutations linked to ADPEAF did not inhibit the interaction, resulting in a channel that inactivated significantly faster than its predicted WT counterpart. The faster inactivation of A-type channels (containing mutant LGI1 protein) may result in a broadening of action potentials causing increased transmitter release in ADPEAF patients. Schulte et al. (2006) provide evidence that LGI1 and KV1.1 are co-localised to the termination zone of the excitatory entorhinal perforant path. A model is proposed in which an enhanced excitation, specifically during high frequency input, triggers focal epileptic activity in the hippocampus in patients harbouring mutations in LGI1. A second

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mechanism relating to LGI1 putative secretory role has also been proposed. Mutated LGI1 protein is unable to bind to the transmembrane protein, ADAM22 that is associated with the postsynaptic density (Fukata et al., 2006). A reduction in excretion of the mutated LGI1 protein has also been described (Sirerol-Piquer et al., 2006). Normally the interaction between the LGI1 and ADAM22 protein specifically increases AMPA receptor-mediated synaptic currents (Fukata et al., 2006). How a loss of this function may cause epilepsy remains unclear. 6.2. ATP1A2 ATP1A2 encodes the a2 subunit of the Na+/K+-ATPase pump. The Na+/K+-ATPase is ubiquitously found on cell plasma membranes where it is critical in maintaining the electrochemical gradient. Mutations in this gene have been associated with familial hemiplegic migraine (Vanmolkot et al., 2003). A family harbouring a missense mutation (R689Q) also had a history of BFIS (Vanmolkot et al., 2003). Functional analysis of this mutation reveals loss-of-function due to a reduction in catalytic turnover (Segall et al., 2005). The impact of this on cellular excitability is unknown but it may delay extracellular clearance of K+ or alter Ca2+ handling of neurons. Further studies have suggested that ATP1A2 mutations should be considered in a wider spectrum of phenotypes where epilepsy and migraine coexist (Deprez et al., 2008). 6.3. ME2 Malic enzyme 2 (ME2) is a mitochondrial enzyme that converts malate to pyruvate essential for the synthesis of GABA in neurons. Linkage and association analysis revealed a nine-SNP haplotype that was observed with increased frequency in a population of patients with IGE (Greenberg et al., 2005). However, a similar analysis did not reveal such an association in a German IGE population suggesting that mutations in this gene may not be a critical determinant of this phenotype (Lenzen et al., 2005b). 6.4. SLC1A3 SLC1A3 encodes the excitatory amino acid transporter 1 (EAAT1) that is expressed on glial. These proteins are partly responsible for regulating extracellular glutamate concentration. A single patient with episodic ataxia, seizures, migraine and hemiplegia was noted to have a mutation in SLC1A (Jen et al., 2005). Functional analysis reveals a significant reduction in the expression of the protein. This resulted in a significant reduction in glutamate uptake providing a possible mechanism for enhanced neuronal excitability. 6.5. EFHC1 EFHC1 encodes a protein with a Ca2+-binding EF-hand motif, but for which no function is known. EFHC1 has been described as a susceptibility gene for JME (Suzuki et al., 2004) and related generalized epilepsy phenotypes (Annesi et al., 2007; Stogmann et al., 2006). Functional analysis reveals that the EFHC1 protein increases R-type voltage-dependent Ca2+ current, and that this increase is not seen with the mutated protein (Suzuki et al., 2004). Other studies have suggested that the EFHC1 protein plays a role in cell division through association with the mitotic spindle (de Nijs et al., 2006) with further evidence suggesting a ciliary role (King, 2006). How dysfunction in these may form the cellular basis of excitability is unclear.

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6.6. MASS1 A naturally occurring mutation in the monogenic audiogenic seizure-susceptible (MASS) gene has been reported in Frings mouse that is prone to audiogenic seizures (Skradski et al., 2001). The MASS1 gene product is a fragment of the very large G-proteincoupled receptor (VLGR1) that may have an important developmental role. Linkage to febrile seizures has mapped to region 5q14 that contains MASS1. A rare nonsense mutation (S2652X) has been identified in one family with febrile and afebrile seizures (Nakayama et al., 2002) although this mutation does not explain febrile seizures in most families linked to this chromosome. Further studies confirm linkage of febrile seizures to chromosome 5q14 highlighting the importance of this region, but again does not support a role for MASS1 (Deprez et al., 2006). 6.7. SLC2A1 Mutations in SLC2A1 that encode the glucose transporter (GLUT1) are a well-established cause of a severe infantile encephalopathy with microcephaly, intellectual disability and seizures and are regarded as a ‘‘symptomatic’’ form of epilepsy (De Vivo et al., 1991). Recently a number of families have been described where subjects are intellectually normal but present with epilepsy closely resembling IGE as well as paroxysmal dyskinesias and, in one family, haemolytic anemia (Suls et al., 2008; Weber et al., 2008). Impaired glucose transport was confirmed in Xenopus oocytes. Importantly, this disorder may be treatable by ketogenic diet. 7. Modelling the complex genetics of generalised epilepsy It is becoming increasingly clear that genetic background is a significant contributor to clinical heterogeneity, with incomplete penetrance and phenotypic variability common features of familial epilepsy. Phenotypic heterogeneity can be modelled in rodents using mice strains with known seizure susceptibility differences. For instance, the seizure phenotype of hetrerozygous SCN1A mice depends heavily on genetic background (Yu et al., 2006). Similarly, the seizure-prone DBA/2J mice carrying the R43Q mutation exhibit a more severe seizure phenotype than C57B/6 mice (Tan et al., 2007). Investigating basal differences in neuronal function of different strains may lead to a better understanding of predisposing and protective neurobiological factors as determined by genetic background. For example, DBA/2J have increased GABAergic input into the thalamus when compared to the C57B/6 strain (Tan et al., 2008). The DBA/2J strain also had distinct sleep EEG frequency power, a trait that may be predictive for seizure outcome (Tan et al., 2008). Complex inheritance has also been modelled by breeding mice harbouring different mutations. Double mutant mice carrying Scn2a and Kcnq2 mutations exhibit severe epilepsy demonstrating that a combination of mild phenotype alleles can result in a more severe phenotype (Kearney et al., 2006b). A combination of mutants can also be protective against epilepsy (Glasscock et al., 2007; Kim et al., 2001; Martin et al., 2007). Study of the effect of multiple mutations in animal models may well give insights into approaching the complex genetic architecture of epilepsy in humans. 8. Future directions The last decade has revealed unanticipated complexity in the genetic architecture of human epilepsies. Rather than there being a handful of genes predisposing to epilepsy in man, current evidence suggests that dozens or perhaps hundreds of genes may play a role.

Of the many genes identified, the majority code for ion channel subunits. The understanding of genotype–phenotype relationships has also proved challenging and a genetic classification of clinical epilepsies is currently elusive. The large number of electrophysiological studies on ion channel genes of major effect has sometimes had apparently conflicting effects in vitro. The diversity of mutations that can cause a similar phenotype argue for points of physiological convergence in determining phenotypes; this is the case for Mendelian epilepsies where most progress has been made, and is likely to be even more relevant in epilepsies with complex inheritance where gene discovery is just beginning. Identifying such convergences, be they anatomical structures or distributed networks, is essential for deeper understanding of mechanisms and for designing therapeutic strategies; it is important to recognize that in vitro studies are opaque to such convergences. It has become increasingly clear that studies in knock-in mice that recapitulate the human genotype and phenotype are the most powerful tool for future investigation. Acknowledgements We would like to thank Prof. Fred Mendelsohn and Karen Oliver for critically reading this manuscript. This work was funded in part by the NHMRC through a program grant (SFB, SP) and project grant (CAR, SP). CAR was also supported by a RD Wright Fellowship, University of Melbourne. References Alexander, S.P., Mathie, A., Peters, J.A., 2008. Guide to receptors and channels (GRAC), 3rd edition. Br. J. Pharmacol. 153 (Suppl. 2), S1–209. Alkondon, M., Albuquerque, E.X., 2004. The nicotinic acetylcholine receptor subtypes and their function in the hippocampus and cerebral cortex. Prog. Brain Res. 145, 109–120. Alldred, M.J., Mulder-Rosi, J., Lingenfelter, S.E., Chen, G., Luscher, B., 2005. Distinct gamma2 subunit domains mediate clustering and synaptic function of postsynaptic GABAA receptors and gephyrin. J. Neurosci. 25, 594–603. Annesi, F., Gambardella, A., Michelucci, R., Bianchi, A., Marini, C., Canevini, M.P., Capovilla, G., Elia, M., Buti, D., Chifari, R., Striano, P., Rocca, F.E., Castellotti, B., Cali, F., Labate, A., Lepiane, E., Besana, D., Sofia, V., Tabiadon, G., Tortorella, G., Vigliano, P., Vignoli, A., Beccaria, F., Annesi, G., Striano, S., Aguglia, U., Guerrini, R., Quattrone, A., 2007. Mutational analysis of EFHC1 gene in Italian families with juvenile myoclonic epilepsy. Epilepsia 48, 1686–1690. Aridon, P., Marini, C., Di Resta, C., Brilli, E., De Fusco, M., Politi, F., Parrini, E., Manfredi, I., Pisano, T., Pruna, D., Curia, G., Cianchetti, C., Pasqualetti, M., Becchetti, A., Guerrini, R., Casari, G., 2006. Increased sensitivity of the neuronal nicotinic receptor alpha 2 subunit causes familial epilepsy with nocturnal wandering and ictal fear. Am. J. Hum. Genet. 79, 342–350. Audenaert, D., Claes, L., Ceulemans, B., Lofgren, A., Van Broeckhoven, C., De Jonghe, P., 2003. A deletion in SCN1B is associated with febrile seizures and early-onset absence epilepsy. Neurology 61, 854–856. Audenaert, D., Schwartz, E., Claeys, K.G., Claes, L., Deprez, L., Suls, A., Van Dyck, T., Lagae, L., Van Broeckhoven, C., Macdonald, R.L., De Jonghe, P., 2006. A novel GABRG2 mutation associated with febrile seizures. Neurology 67, 687–690. Barclay, J., Balaguero, N., Mione, M., Ackerman, S.L., Letts, V.A., Brodbeck, J., Canti, C., Meir, A., Page, K.M., Kusumi, K., Perez-Reyes, E., Lander, E.S., Frankel, W.N., Gardiner, R.M., Dolphin, A.C., Rees, M., 2001. Ducky mouse phenotype of epilepsy and ataxia is associated with mutations in the Cacna2d2 gene and decreased calcium channel current in cerebellar Purkinje cells. J. Neurosci. 21, 6095–6104. Barela, A.J., Waddy, S.P., Lickfett, J.G., Hunter, J., Anido, A., Helmers, S.L., Goldin, A.L., Escayg, A., 2006. An epilepsy mutation in the sodium channel SCN1A that decreases channel excitability. J. Neurosci. 26, 2714–2723. Bassi, M.T., Balottin, U., Panzeri, C., Piccinelli, P., Castaldo, P., Barrese, V., Soldovieri, M.V., Miceli, F., Colombo, M., Bresolin, N., Borgatti, R., Taglialatela, M., 2005. Functional analysis of novel KCNQ2 and KCNQ3 gene variants found in a large pedigree with benign familial neonatal convulsions (BFNC). Neurogenetics 6, 185–193. Baulac, S., Huberfeld, G., Gourfinkel-An, I., Mitropoulou, G., Beranger, A., Prud’homme, J.F., Baulac, M., Brice, A., Bruzzone, R., LeGuern, E., 2001. First genetic evidence of GABA(A) receptor dysfunction in epilepsy: a mutation in the gamma2-subunit gene. Nat. Genet. 28, 46–48. Berkovic, S.F., Harkin, L., McMahon, J.M., Pelekanos, J.T., Zuberi, S.M., Wirrell, E.C., Gill, D.S., Iona, X., Mulley, J.C., Scheffer, I.E., 2006a. De-novo mutations of the sodium channel gene SCN1A in alleged vaccine encephalopathy: a retrospective study. Lancet Neurol. 5, 488–492.

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