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REVIEWS GENETIC MECHANISMS THAT UNDERLIE EPILEPSY Ortrud K. Steinlein Genetic factors can cause recurrent abnormal synchronization and episodic hyperexcitability of neuronal networks through various mechanisms. Many of the genes that have been implicated in idiopathic epilepsies code for ion channels, whereas syndromes with epilepsy as a main feature are caused by genes that are involved in functions as diverse as cortical development, mitochondrial function and cell metabolism. Each ‘epilepsy gene’ that is identified provides new and fascinating insights into the molecular basis of neuronal excitability and brain function. ABSENCE EPILEPSY A non-convulsive form of epilepsy that is characterized by a sudden, brief impairment of consciousness. MYOCLONUS Brief, involuntary twitching of a muscle or a group of muscles. Familiar examples of normal myoclonus include hiccups and jerks experienced when drifting off to sleep. FRAGILE X SYNDROME A genetic condition, commonly transmitted from mother to son, that is associated with mental retardation, abnormal facial features and enlarged testicles. Institute of Human Genetics, Friedrich-WilhelmsUniversity Bonn, School of Medicine, Wilhelmstrasse 31, 53111 Bonn, Germany. e-mail: [email protected] doi:10.1038/nrn1388 400 Epilepsies are characterized by recurrent seizures, which can cause motor, sensory, cognitive, psychic or autonomic disturbances. The seizures themselves are the clinical manifestation of an underlying transient abnormality of cortical neuronal activity, and the phenotypic expression of each seizure is determined by the point of origin of the hyperexcitability and its degree of spread in the brain. By convention, the diagnosis of epilepsy requires that the patient has had at least two unprovoked seizures. The seizures, which can last between a few seconds and a few minutes, can either be isolated or occur in series. Although its most basic manifestation starts at the single neuron level, epilepsy is fundamentally a circuit phenomenon, and seizures are only possible because the brain is organized in a series of interconnected neuronal networks. The initially localized hyperexcitability spreads into surrounding neuronal networks, where it might be either counterbalanced by inhibitory mechanisms, or, after involving more and more neurons, cause a clinically visible seizure. A related but different mechanism is found in absence seizures, where abnormal synchronization of the thalamocortical network, rather than an excessive recruitment of neurons, causes the seizures. Recurrent seizures can lead to permanent alterations of neuronal networks, paving the way for future seizures. The causes of sporadic or recurrent seizures are numerous, and they include acquired structural brain damage, altered metabolic states and inborn brain malformations. However, about 1% of all people develop | MAY 2004 | VOLUME 5 recurrent unprovoked seizures for no obvious reason and without any other neurological abnormalities. These are named ‘idiopathic epilepsies’, and they are assumed to be mainly genetic in origin. In some cases, the genetic point of origin has already been shown by successful cloning of the mutated gene1–15, but for most idiopathic epilepsies, especially the common forms like juvenile myoclonic epilepsy, or childhood and juvenile ABSENCE EPILEPSY, the genes that are involved in epileptogenesis remain unknown. In addition to the large group of idiopathic epilepsies, more than 200 single gene disorders are known in which epilepsy is a more or less important part of the phenotype. These include syndromes as diverse as neurodegenerative disorders from the group of progressive MYOCLONUS epilepsies, mental retardation syndromes like FRAGILE X SYNDROME or ANGELMAN’S SYNDROME, neuronal migration disorders or mitochondrial encephalomyopathies. Modes of inheritance for epilepsies All possible modes of inheritance are found in epilepsies, including AUTOSOMAL, X-chromosomal, mitochondrial and complex inheritance. Autosomal recessive inheritance is a common finding in epilepsies with an early age of onset and a progressive course. A recessive allele that causes early disablement or death in a homozygous state can be passed through the generations by healthy carriers. Monogenic idiopathic epilepsies, with their mostly benign course, are usually of autosomal dominant inheritance. Under a strict model of autosomal dominance, the mutant alleles of these genes should be expected to www.nature.com/reviews/neuro ©2004 Nature Publishing Group REVIEWS Box 1 | Modes of inheritance Monogenic: a single mutated gene is sufficient to cause the phenotype. Major gene effect: a highly penetrant mutation, which becames phenotypically manifest only if the genetic background of the patient provides additional minor mutations in other genes. Oligogenic: the phenotype is caused by mutations in a few genes. Polygenic: mutations in several genes are needed to generate the phenotype. The number of genes involved in different idiopathic epilepsies determines the mode of inheritance that underlies each syndrome. Epilepsies with oligogenic or polygenic inheritance are much more common than monogenic forms. The impact of any given mutation on the phenotype is determined by the effect the mutation has on the function of the gene, the importance of the gene for normal brain function and the existence of parallel pathways that might be able to compensate the defect. The impact of mutations is further modified by environmental factors, epigenetic effects and the genetic background of the patient. cause epilepsy in each of their carriers. However, family studies show that the penetrance of these mutations is often less than 100%, and that the age of onset, as well as the severity of the phenotype, varies within families16,17. This indicates that the clinical expression of genes that have been implicated in epilepsy can be modified by additional genes that have yet to be identified, and possibly by environmental factors. Monogenic epilepsies, and those with a more complex mode of inheritance, cannot be distinguished on the basis of their clinical features. One example of an idiopathic epilepsy with more than one possible form of inheritance is the syndrome of generalized epilepsy with febrile seizures plus (GEFS+), which is discussed below18. The spectrum of possible inheritance models in the common forms of idiopathic epilepsy extends from major gene effects, through oligogenic inheritance, to polygenic models (BOX 1). In the common forms of idiopathic epilepsies, the involvement of different genes probably explains why several epilepsy subtypes can occur within the same family or even the same patient. the coming years. The channels that are involved in idiopathic epilepsy belong to either the class of voltage-gated ion channels, which are important for action potential generation and control, or the class of ligand-gated ion channels, which are mainly involved in synaptic transmission. Mutations in voltage-gated potassium, sodium and chloride channels, as well as in ligand-gated acetylcholine and GABAA (γ-aminobutyric acid, subunit A) receptors, are known to cause different forms of idiopathic epilepsy1–15 (FIG. 2, TABLE 1). So far, most of the mutations have been found in families with rare epilepsies that follow monogenetic inheritance patterns, but ion channels are probably involved in the more common forms of idiopathic epilepsy too. Ion channels are directly linked to membrane excitability and to neurotransmitter release, and have been found to be responsible for various other inherited paroxysmal disorders, like hyper- and hypokalaemic periodic paralysis, PARAMYOTONIA CONGENITA, episodic ataxia and familial hemiplegic migraine19. The normal functioning of the cortex relies on a finely tuned balance between excitatory and inhibitory input, so any disturbance of this balance carries with it the possibility of uncontrolled hyperexcitability. Seizures can be induced either by the enhancement of excitatory stimuli or by impairment of inhibitory mechanisms. For example, inhibitory input can be disturbed directly by a mutated GABAA receptor, or indirectly by a mutation in a voltage-gated chloride channel that normally provides the chloride-efflux pathway, which is important for the inhibitory GABAA response9,10,15. A loss-of-function mutation in a voltage-gated sodium channel accessory β-subunit decreases the channel’s rate of inactivation Normal nAChR GABAA SCN K+ Ca2+ Na+ Cl– Na+ Neuronal excitability and communication ANGELMAN’S SYNDROME A genetic disorder that is caused by deletion or disruption of UBE3A (E6-AP). The symptoms of Angelman’s syndrome include hyperactivity, ataxia, problems with speech and language, and an unusually happy demeanour. AUTOSOMAL A term that refers to any chromosome in a cell that is not a sex chromosome. PARAMYOTONIA CONGENITA A rare autosomal dominant disorder in which muscle fibres are slow to relax after contraction. The excitability of neurons and their communications with each other depend on the action of various ion channels. The main structural feature that all ion channels share is the transmembrane pore, which shows distinct ion selectivity in different channel subtypes. The channel pore not only controls the type of ion that passes through it, but also the direction of ion flow (FIG. 1). Ion channels can rapidly alter the permeability of the membrane for certain ions, and this might, for example, change the resting potential to an action potential, or vice versa. It is therefore not surprising that ion channels have a key role in various disorders that are associated with hyper- or hypoexcitability of the affected tissue. Idiopathic epilepsies as channelopathies Idiopathic epilepsies are caused predominantly by mutations in genes that code for ion channels or their accessory subunits. So far, mutations in ten different ion channel subunit genes have been found in human idiopathic epilepsies, and this number is set to increase in NATURE REVIEWS | NEUROSCIENCE K+ Ca2+ Na+ Cl– KCNQ CLC K+ Cl– K+ Cl– LOF GOF Na+ Mutant Type of mutation: GOF LOF LOF Figure 1 | Gain- and loss-of-function in mutated ion channels. Simplified model demonstrating gain-of-function (GOF) and loss-of-function (LOF) effects caused by mutations in various channels that are associated with idiopathic epilepsy. From left to right: neuronal nicotinic acetylcholine receptor (nAChR) (genes: CHRNA4 and CHRNB2), GABAA (γ-aminobutyric acid, subtype A) receptor (genes: GABRG2 and GABRA1), voltage-gated sodium channel (SCN) (genes: SCN1A, SCN2A and SCN1B), voltage-gated potassium channel (KCNQ) (genes: KCNQ2 and KCNQ3) and voltagegated chloride channel (CLC) (gene: CLCN2). For each channel, the model presents only one of several possible pathogenetic mechanisms. The main ion currents are indicated by arrows. VOLUME 5 | MAY 2004 | 4 0 1 ©2004 Nature Publishing Group REVIEWS and causes hyperexcitability by increasing the transmembrane sodium influx6. Mutations in specific subtypes of potassium channels that are responsible for membrane repolarization lead to prolonged neuronal depolarization and therefore to spontaneous series of action potentials3–5 (FIG. 1). a CHRNA4/CHRNB2 b GABRG2/GABRA1 N N C C 1 2 3 1 4 2 3 4 c SCN1A/SCN2A 1 2 3 4 5 P 6 1 2 3 4 5 P 6 1 2 3 4 5 P 6 1 2 3 4 5 P 6 N C d KCNQ2/KCNQ3 e CLCN2 4 1 2 3 4 5 6 1 2 3 5 6 7 8 9–12 N N 13 C C Figure 2 | Subunit structures of ion channels involved in epileptogenesis. a | The neuronal nicotinic acetylcholine receptors (nAChRs) (CHRNA4/CHRNB2) are pentamers, with each subunit containing four transmembrane domains. At least ten nAChR subunits, which can assemble into heteromeric (α2–α6, β2–β4) or homomeric receptors (α7, α9), are expressed in the human brain. The binding of two agonist molecules is required for channel opening. The channel itself shows little selectivity among monovalent cations. b | The GABAA (γ-aminobutyric acid, subtype A) receptors (GABRG2/GABRA1) are ligand-gated ion channels that probably evolved from the same ancient genes as the nAChRs, with whom they share several features, such as four transmembrane domains per subunit and a pentameric structure. GABAA receptors are selective for small anions and allow both chloride and bicarbonate to permeate. c | Voltage-gated sodium channels (SCN1A/SCN2A) are built from one α-subunit, which contains four tandem domains, each resembling the structure of a voltage-gated potassium channel subunit. Sodium channels are associated with two accessory β-subunits, which accelerate the gating kinetics of the channel. d | Voltage-gated potassium channels (KCNQ2/KCNQ3) are tetramers made up from homologous subunits. Each subunit contains six transmembrane domains. The fourth transmembrane domain carries several positively charged amino acids, which cause a conformational change on membrane depolarization. The linker between transmembrane domains 5 and 6 contains the selectivity filter that lines the ion pore. e | Voltage-gated chloride channels of the CLCN type comprise a gene family with nine mammalian members. They build homodimeric proteins, which probably contain two separate pores. CLCN channels conduct chloride ions across cell membranes, governing the electrical activity of cells. 402 | MAY 2004 | VOLUME 5 These examples demonstrate that mutated channels can cause neuronal hyperexcitability through numerous pathogenic mechanisms. In the following sections, some of the idiopathic epilepsies that are caused by mutated ion channels will be discussed in more detail. Familial nocturnal frontal lobe epilepsy. Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is a rare partial epilepsy that is characterized by clusters of brief motor seizures, which occur mostly during non-REM (rapid eye movement) sleep. ADNFLE shows considerable intrafamilial variation in terms of age of onset and severity. The motor features consist of thrashing hyperkinetic activity or tonic stiffening with superimposed clonic jerking. Secondary generalization with loss of consciousness can occur, but most patients remain conscious throughout their seizures20. In most ADNFLE families, the pathway that finally leads to partial seizures starts with a mutation in either the α4-subunit (CHRNA4) or the β2-subunit (CHRNB2) gene of the neuronal nicotinic acetylcholine receptor (nAChR)1,2. The nAChRs, like glycine, GABAA and serotonin receptors, are part of the superfamily of homo- and heteropentameric ligand-gated ion channels. One of the most common nAChRs in the mammalian brain assembles from α4- and β2-subunits, so it was not surprising to discover that both subunits are associated with the same type of epilepsy. However, it was surprising to find that a nAChR subtype that is ubiquitously present in the brain causes a partial type of epilepsy instead of a generalized one, and the reasons for this phenomenon are poorly understood. The first CHRNA4 mutation — a S248F amino-acid exchange within the second transmembrane region — was identified in 1995 (REF. 1). This was followed by several other descriptions of new mutations, either in CHRNA4 or in CHRNB2 (REFS 2,21–24). All of the known ADNFLE mutations are located within or close to the second or third transmembrane domain. Both transmembrane domains contribute to the walls of the ion channel, so it seems that only mutations that have a direct effect on the ion pore can cause ADNFLE. Reconstitution experiments in Xenopus laevis oocytes or human embryonic kidney (HEK) cells and PATCH CLAMP characterization of the known ADNFLE mutations revealed different electrophysiological and pharmacological profiles. Some mutations (α4S248F, α4776ins3) led to a reduction in calcium permeability, whereas other mutations (α4S252L) did not change the ionic selectivity of the channel. An increase in receptor desensitization was seen for only one of the known ADNFLE mutations (α4S248F). Furthermore, mutations α4S248F, α4776ins3 and β2V287M were effectively blocked by the anti-epileptic drug carbamazepine, whereas the drug response that was observed for some of the other mutants did not differ from that seen for the wild-type receptor25. These different functional properties might explain why some of the nAChR mutations seem to be associated with specific clinical features. For example, carriers of the α4776ins3 mutation are often affected by psychiatric disorders, whereas families with the www.nature.com/reviews/neuro ©2004 Nature Publishing Group REVIEWS Table 1 | Some of the genes that are involved in epilepsy Subtypes Gene symbol Phenotype Ion channel genes in idiopathic epilepsy Nicotinic acetylcholine receptors CHRNA4/CHRNB2 ADNFLE Potassium channels KCNQ2/KCNQ3 BFNC Sodium channels SCN1A/SCN2A/SCN1B GEFS+ Chloride channels CLCN2 IGE GABAA receptors GABRG2/GABRA1 GEFS+/IGE Non-ion channel genes in idiopathic epilepsy Function unknown LGI1 ADLTE G-protein coupled receptors MASS1/VLGR1 FS Polyglucosan metabolism EPM2A/EPM2B(NHLRC1) Lafora disease Cysteine protease inhibition CSTB Unverricht– Lundborg disease Respiratory chain MTTK/MTTL1 MERRF Lipidoses PPT Infantile NCL CLN2 Late infantile NCL CLN3 Juvenile NCL CLN5 Late infantile NCL, Finnish variant CLN6 Late infantile NCL, Indian variant CLN8 Northern epilepsy NEU1 Sialidosis metabolism Progressive myoclonus epilepsies Glycopeptide/oligosaccharide ADLTE, autosomal dominant lateral temporal lobe epilepsy; ADNFLE, autosomal dominant nocturnal frontal lobe epilepsy; BFNC, benign familial neonatal convulsions; FS, febrile seizures; GEFS+, generalized epilepsy with febrile seizures plus; IGE, idiopathic generalized epilepsies; MERRF, myoclonic epilepsy with ragged red fibres; NCL, neuronal ceroid lipofuscinosis. α4Ser252Leu mutation might have an increased risk for mental retardation26,27. The one characteristic that all mutations have in common is that they increase the acetylcholine sensitivity of nAChRs, indicating that a gain-of-function effect underlies this type of epilepsy. The mechanism by which the increased ACh sensitivity results in seizures is still speculative, but one attractive hypothesis is that the mutated nAChR alters the activity level of the thalamocortical loop in favour of oscillation by reinforcing the input from the thalamus25 (FIG. 3). PATCH CLAMP Technique whereby a very small electrode tip is sealed onto a patch of cell membrane, making it possible to record the flow of current through individual ion channels or pores within the patch. MISSENSE MUTATION A mutation that results in the substitution of an amino acid in a protein. Benign familial neonatal convulsions. The syndrome of benign familial neonatal convulsions (BFNC) — a rare autosomal, dominantly inherited epilepsy of the newborn — is characterized by unprovoked, generalized or multifocal seizures, typically starting within the first few days of life. The course of the disorder is usually benign and selflimiting, and, with or without pharmacotherapy, the seizures remit spontaneously within a few weeks or months28,29. Later seizures might occur in up to 15% of the patients, but they tend to occur infrequently. Recently, several patients with BFNC have been described in which the seizures did not respond well to anti-epileptic drugs and resulted in delayed development or psychomotor retardation30. It remains unclear whether these patients have a second unrecognized condition, or whether certain risk factors in combination with the BFNC mutation are responsible for the unfavourable outcome. NATURE REVIEWS | NEUROSCIENCE BFNC can be caused by mutations in the voltagegated potassium channel genes KCNQ2 or KCNQ3 (located on chromosome 20q13.3 and 8q24, respectively)3–5. The KCNQ2 and -3 genes encode subunits of the M-channel, a very slowly opening and closing potassium channel that is found ubiquitously in the brain31. The M-channel, which was first discovered some 20 years ago32, is a powerful controller of neuronal repetitive firing. It regulates the number of action potentials of individual neurons by opposing sustained membrane depolarization. All but two of the known BFNC mutations have been found in KCNQ2. Most of these mutations are truncating and are located in the large carboxy (C)-terminal portion that lies downstream of the six transmembrane domains. However, several MISSENSE MUTATIONS within the transmembrane domains have also been described. Most BFNC mutations cause only modest reductions (20–30%) in potassium currents in reconstitution experiments, indicating that even a slight alteration of M-channel activity is sufficient to cause epilepsy3. An exception to this haploinsufficiency concept is the KCNQ2/R207W mutation, which neutralizes a charged amino-acid residue within the channel’s voltage sensor in transmembrane domain 4. This mutation causes a more severe change in potassium channel properties by drastically slowing the voltage-dependent activation. At the clinical level, this results not only in BFNC, but also in myokymia — a spontaneous and repetitive involuntary contraction of muscle fibre groups33. In mice, Kcnq2 knockout experiments resulted in early post-natal lethality in homozygous animals, whereas heterozygous mice developed and behaved normally but showed an increased sensitivity to the chemoconvulsant pentylenetetrazole34. Nevertheless, they did not develop spontaneous seizures, which might indicate that Kcnq2 is not as important for M-channel function in the mouse brain as it is in humans. Recent studies have shown that dominantly inherited idiopathic early life seizures can also be caused by mutations in a gene that does not code for the M-channel. Two families with seizures starting within the first 3.5 months of life were found to have mutations in the voltage-gated sodium channel subunit SCN2A, which has also been implicated in GEFS+ (REF. 12). These findings once more highlight the complex genotype–phenotype relationships in idiopathic epilepsies. Generalized epilepsy with febrile seizures plus. A wide spectrum of epilepsy phenotypes can be present in families with GEFS+. Some of the more common phenotypes are febrile seizures, febrile seizures plus (FS+ — attacks with fever that continue beyond 6 years of age, sometimes interspersed with afebrile seizures), FS+ and absence seizures, FS+ and myoclonic seizures, and FS+ and atonic seizures or myoclonic-astatic epilepsy18. The mode of inheritance that underlies GEFS+ is still a matter for debate. In some families, the trait seems to be autosomal dominant, but in others it is probably better described as oligogenic or as a major gene effect.A genetic concept involving more than one gene would also be more consistent with the clinical variability that is VOLUME 5 | MAY 2004 | 4 0 3 ©2004 Nature Publishing Group REVIEWS a b Cortex Cortex Cholinergic fibres Excitatory projections Non-ion channel genes in idiopathic epilepsy Thalamus GABA releasing neurons Efference/afference Efference/afference Figure 3 | Model of interactions between the thalamus and cortical pyramidal cells. a | The thalamus and the layers of pyramidal cells in the cortex are interconnected by excitatory (predominantly glutamatergic) projections (thalamocortical and corticothalamic axons). The repetitive firing in the thalamocortical loop is inhibited by GABA (γ-aminobutyric acid)-releasing neurons, which are densely spaced within the thalamic reticular nucleus (RN). The thalamocortical and corticothalamic axons are connected to these GABA-releasing cells by collaterals and vice versa. The collaterals from the thalomocortical loop excite the GABA-releasing cells, which subsequently increase their inhibitory input in other parts of the thalamus but also prevent excessive inhibitory stimulus by connecting to other GABA-releasing neurons within the RN. Another regulating mechanism is provided by cholinergic fibres that release acetylcholine in the vicinity of the main dendrite of pyramidal cells, controlling input from external cell layers. b | A gainof-function mutation in the nicotinic acetylcholine receptor would probably decrease the input from external cortical layers. The reduced feedback from the external layers could reinforce the backpropagation from the thalamus and deeper cortical layers. Such an imbalance could lead to seizures by enhancing oscillation in the thalamocortical loop. observed within and between GEFS+ families. Nevertheless, only one mutation has so far been identified in each GEFS+ family. Mutations have been identified in genes for three voltage-gated sodium channel subunits (SCN1A, SCN2A, SCN1B)6–8,35, as well as in the γ2 subunit gene (GABRG2) of the GABAA receptor9,10,36–38. SCN1A, the most frequently involved gene, codes for a large receptor protein comprised of four domains, each of which contains six transmembrane domains. Heterologous expression of SCN1A with the accessory subunits SCN1B and SCN2B in mammalian cells showed that certain GEFS+ mutations cause a subtle gating defect. At depolarized potentials, the fast inactivation was less complete than normal, leading to a persistent inward sodium current39. This gain-of-function effect probably enhances neuronal excitability by prolonged membrane depolarization. Other GEFS+ mutations showed a reduction in current density and accelerated recovery from slow inactivation and did not cause a persistent sodium current. These findings indicate that either an increase or a decrease in sodium channel activity can result in seizures40,41. 404 Mutations in the SCN1A gene have also been found in severe myoclonic epilepsy of infancy (SMEI or Dravet’s syndrome), an intractable epilepsy of early childhood that sometimes co-occurs in GEFS+ families42. So far, the genetic relationship between GEFS+ and SMEI remains unclear. The most likely explanation is that SMEI has a complex mode of inheritance, and that some of the genes that are involved in this disorder are also responsible for the predisposition to seizure phenotypes from the GEFS+ spectrum. Mutations within the γ2-subunit of the GABAA receptor were described as another cause of GEFS+ (REFS 9,10,36–38). However, the most common afebrile seizure type in families with GABRG2 mutations is absence seizures. Therefore, it remains a matter for debate whether GABRG2 mutations indeed cause a subtype of GEFS+, or whether they predispose for childhood absence epilepsy. | MAY 2004 | VOLUME 5 Although ion channels undoubtedly have an important role in idiopathic epilepsies, we should not overlook the fact that other pathways can lead to neuronal hyperexcitability. Mutant forms of any gene that is involved in neuronal plasticity, development of neuronal networks or neuronal metabolism are potential candidates for causing epileptogenesis. Furthermore, even genes that encode molecules that control more upstream events, such as transcription factors or vesicle proteins, have to be regarded as having a potential involvement in epilepsy. Familial lateral temporal lobe epilepsy. Autosomal dominant lateral temporal lobe epilepsy (ADLTE, also known as autosomal dominant partial epilepsy with auditory features) is an idiopathic syndrome that is characterized by simple partial seizures with mainly acoustic and sometimes even visual hallucinations43. In some families, the seizures can start with a brief sensory aphasia without reduced consciousness44. The identification of LGI1 (leucine-rich glioma inactivated gene 1, located on chromosome 10q24) as the gene that is responsible for ADLTE came as a surprise, because all the genes that were previously implicated in idiopathic epilepsy coded for ion channel subunits. The LGI1 mutations that have been found in ADLTE families so far are mostly truncating mutations, but several missense mutations have also been described11,45–48. The function of the LGI1 protein is still unknown, but sequence analysis showed that it is not an ion channel subunit, and probably not even a membrane-bound protein. The main sequence characteristics of LGI1, and its three homologous genes LGI2, LGI3 and LGI4 (REF. 49), are a conserved leucine-rich repeat (LRR) domain, followed by seven copies of an epitempin repeat50,51. The LRR consists of repeated β-strands and α-helices that are connected by loops. The LRR domain usually acts as a framework for protein–protein interactions, and it is present in numerous proteins with diverse functions. The nature of possible binding partners for LGI proteins remains elusive. www.nature.com/reviews/neuro ©2004 Nature Publishing Group REVIEWS Common forms of idiopathic epilepsy LGI1–LG14 LRR Epitempin repeat 100 aa MASS1/VLGR1 Calx-β domain Epitempin repeat TM 1000 aa Figure 4 | Epitempin repeat in epilepsy-associated genes. The proteins encoded by both LGI1, the gene involved in autosomal dominant lateral temporal lobe epilepsy, and the MASS1/VLGR1 gene, which is mutated in the Frings mouse model for audiogenic seizures and in one family with febrile seizures, share the epitempin repeat13,49. The MASS1/VLGR1 protein — one of the largest membrane proteins and a member of the superfamily of seven-helix G-protein coupled receptors — contains six complete and one degenerate copy of the epitempin repeat. MASS1/VLGR1 is expressed in the developing nervous system rather than in the adult brain. Its overall structure shows homologies to large G-protein coupled receptors from the flamingo family, the latrophilins and the brain angiogenesis inhibitors. These receptors are mainly involved in neuronal development. Furthermore, the leucine-rich repeat (LRR) domain subtype present in the LGI1 protein is also found in Slit, a neurogenic protein involved in axonal guidance47. It is therefore tempting to speculate that both MASS1/VLGR1 and LGI1 cause epilepsy by interference with normal brain development. aa, amino acid residues; TM, transmembrane domain. β-PROPELLER A protein domain that consists of an array of β-sheet motifs, which are configured in a ring to resemble the blades of a propeller. G PROTEIN A heterotrimeric GTP-binding and -hydrolysing protein that interacts with cell-surface receptors, often stimulating or inhibiting the activity of a downstream enzyme. G proteins consist of three subunits: the α-subunit, which contains the guanine-nucleotide-binding site; and the β- and γ-subunits, which function as a heterodimer. FRINGS MOUSE MODEL An inbred mouse strain with a seizure phenotype that is characterized by wild running, loss of righting reflex, tonic flexion and tonic extension in response to high-intensity sound stimulation. AUDIOGENIC EPILEPSY A form of epilepsy in which the seizures are provoked by auditory stimuli. The second hallmark of LGI1 — the epitempin repeat — is located in the C-terminus and consists of a tandem repeat with a core of about 50 residues, which probably fold into a β-PROPELLER structure51. The function of the epitempin repeat is unknown, but it was also found in another gene that has been associated with epilepsy — the MASS1/VLGR1 gene (FIG. 4). MASS1/ VLGR1 codes for the very large G-PROTEIN coupled receptor, and mutations in this gene are responsible for the 52 FRINGS MOUSE MODEL of AUDIOGENIC EPILEPSY . Mutations in the MASS1/VLGR1 gene also seem to be a rare cause of febrile seizures in humans13. In the MASS1/VLGR1 protein, the epitempin repeat is part of the ligand-binding ectodomain, so, like the LRR domain, this repeat might be involved in protein–protein interactions. This raises the question of whether this formerly unknown sequence signature might be involved in a new mechanism of epileptogenesis. One possibility is that the epitempin repeat might be important for mechanisms like synaptogenesis or axon guidance that depend on a communication between cells, or between cells and extracellular matrix proteins. In such a model, the epilepsy that is caused by mutations in the LGI1 gene could be the result of abnormalities in synapse formation or neuronal migration. LGI1 was first cloned from the translocation breakpoint of a glioma cell line, and was subsequently shown to be downregulated in many, but not all, glioblastoma cell lines that were tested53,54. As no mutations or deletions of LGI1 were found in these cell lines, it remains unclear whether the loss of LGI1 expression is indeed a causative event in tumorigenesis. However, re-expression of LGI1 in cell lines that lack LGI1 mRNA significantly reduced cell proliferation, supporting the hypothesis that LGI1 is a tumour suppressor gene54. No evidence has been found that the LGI1 mutations in families with ADLTE increase the rate of brain tumours or other malignancies, excluding a role for LGI1 as a first-step or high-penetrance tumour suppressor gene55. NATURE REVIEWS | NEUROSCIENCE In childhood and adolescence, about 30–40% of all epilepsies belong to the group of idiopathic generalized epilepsies (IGE). These include age-related subtypes like juvenile myoclonic epilepsy, childhood absence epilepsy, juvenile absence epilepsy and grand mal epilepsy on awakening. Depending on the age of onset, individuals with IGE might present with different subtypes, and they often have a family history for epilepsy. The concordance rate in monozygotic twins is up to 95%, supporting an almost complete genetic aetiology of IGE56. First-degree relatives of patients with IGE have a risk of about 8–12% to be affected by IGE, whereas the recurrence risk for second-degree relatives is only about 1–2% (equal to the epilepsy risk in the general population). These empirical risk numbers are more compatible with an oligogenic than a monogenic mode of inheritance in IGE. Several genes are probably involved in each patient, and the rapid decrease of the recurrence risk implies that the interaction of these gene loci is multiplicative rather than additive. One attractive theory is that some IGE genes determine the seizure threshold by influencing neuronal excitability, whereas other genes are responsible for the age of onset, and therefore the seizure subtype57. However, this theory has not yet been supported by experimental data. So far, little is known about the genes that underlie epileptogenesis in IGE. Linkage studies and association approaches have highlighted numerous candidate regions within the genome, but replication studies usually failed to confirm the initial observations. One example is the association with IGE that was found for the α1A-calcium channel subunit gene (CACNA1A) on chromosome 19 in one study58, but could not be replicated using an independent approach59. Missense mutations in another calcium channel gene — CACNA1H on chromosome 16p13.3 — were found in several patients with childhood absence epilepsy60. These results indicate that CACNA1H might be a susceptibility gene that is involved in the pathogenesis of IGE. Of interest is the chromosomal region 3q26, which was marked as a susceptibility region for common IGE subtypes in a genome-wide search61. Follow-up studies in this region identified mutations within the voltagegated chloride channel gene CLCN2 in 3 of 46 unrelated IGE families, indicating a role for CLCN2 as a minor locus for IGE. The µ-opioid receptor subunit gene (OPRM1) was found to be associated with IGE in two independent studies, but no mutations have been identified so far62,63. In the future, even larger samples of patients with IGE will be needed to overcome the obstacles that come with the task of identifying genes in disorders with complex inheritance. Syndromes with epilepsy as a key feature More than 200 inherited syndromes are known in which epileptic seizures are an important, but not the only, clinically prominent feature. In these syndromes, the epilepsy is often accompanied by other neurological symptoms, such as mental retardation, dementia or ataxia. The genes that underlie these syndromes are VOLUME 5 | MAY 2004 | 4 0 5 ©2004 Nature Publishing Group REVIEWS INTENTION TREMOR A tremor that is exacerbated by voluntary goal-directed movements; for example, trying to put a key in a lock. DYSARTHRIA A speech impairment that is caused by damage to the nerves or muscles that control speech articulation. Although the speech is difficult to understand, it is usually linguistically normal, thereby distinguishing this condition from language disorders. RNA INTERFERENCE (RNAi). A method by which double-stranded RNA that is encoded on an exogenous vector can be used to interfere with normal RNA processing, causing rapid degradation of the endogenous RNA and thereby precluding translation. This provides a simple way of studying the effects of the absence of a gene product in simple organisms and in cells. involved in tasks as different as glycogen metabolism, respiratory chain activity and brain development. In contrast to the idiopathic epilepsies, the course of these disorders is often not benign — they are not a result of an episodic and reversible disturbance of brain function, but rather a developmental abnormality or irreversible and progressive neuronal cell loss in the brain. Some of the mechanisms by which certain genes can cause syndromes with epilepsy are discussed in the context of two disorders from the group of progressive myoclonus epilepsies and a typical example of a neuronal migration disorder. Unverricht–Lundborg disease. Progressive myoclonus epilepsy type 1 (EPM1), or Unverricht–Lundborg disease (also known as Baltic or Mediterranean myoclonus epilepsy), is an autosomal recessive neurodegenerative disorder that is characterized by progressive, stimulisensitive myoclonic jerks and generalized tonic-clonic seizures. The age of onset is between 6 and 18 years of age, and the course of the disorder is usually about 10 to 20 years in duration. Mental deterioration, INTENTION TREMOR, DYSARTHRIA and mild ataxia can develop in later stages of the disorder64. Pathological findings demonstrate a marked loss of Purkinje cells in the cerebellum, neuronal loss in the spinal cord and the medial thalamus, and a proliferation of Bergmann glia65. In most patients, Unverricht–Lundborg disease is caused by a dodecamer repeat that is located upstream of the initiation codon of the CSTB (cystatin B, also stefin B) gene66. Expansion of the unstable dodecamer repeat probably prevents transcription of CSTB by increasing the distance between the transcription factor binding sites and the transcription initiation site. CSTB is a member of the family of type 1 cystatins, which are intracellular inhibitors of cysteine proteases, and are likely to protect the organism from proteolysis by controlling the activities of endogenous proteases. CSTBdeficient mice develop progressive ataxia and myoclonic seizures owing to extensive apoptotic cell death in the cerebellum, and they provide a good animal model for Unverricht–Lundborg disease67,68. CSTB is known to inhibit several different proteases in vitro, including cathepsins B, H, L and S. In vivo studies in knockout mice demonstrated that cathepsin S levels increase in the absence of CSTB, indicating a feedback mechanism between the two proteins. The imbalance between the protease inhibitor CSTB and proteases such as cathepsin S probably initiates the cascade of cellular events that lead to progressive cell death and finally to clinical symptoms in Unverricht–Lundborg disease68. Table 2 | Lissencephaly genes Gene Chromosome Phenotype RELN 7q22 Lissencephaly and cerebellar hypoplasia LIS1 (PAFAH1B1) 17p13.3 Lissencephaly type 1/Miller–Dieker Syndrome YWHAE (14-3-3ε) 17p13.3 Miller–Dieker Syndrome DCX (XLIS) Xq22.3-q23 Lissencephaly type 1/double cortex syndrome ARX Xp22.13 Lissencephaly and genital abnormalities 406 | MAY 2004 | VOLUME 5 Lafora disease. Another autosomal recessive neurodegenerative disorder in which seizures are an important part of the phenotype is Lafora progressive myoclonus epilepsy. Lafora disease is the most common form of adolescent-onset progressive epilepsy, and it leads to cognitive decline, dementia and finally death within 10 years of onset. The presence of intracellular polyglycosan inclusion bodies — so-called Lafora bodies — is pathognomonic for Lafora disease. In about 70–80% of the patients, homozygous mutations of the EPM2A gene on chromosome 6q24 are found69. A second gene that is associated with Lafora disease (EPM2B/NHLRC1) was recently identified on chromosome 6p22.3 (REF. 70). EPM2A and EPM2B encode the proteins laforin and malin, respectively. Laforin contains a dual-specifity phosphatase catalytic domain and a carbohydratebinding domain. Experiments with transgenic mice demonstrated that laforin interacts with itself and with the glycogen-targeting regulatory subunit R5 of protein phosphatase 1. Most EPM2A mutations that are found in patients with Lafora interfere with the phosphatase activity of laforin and disrupt its binding capacity to glycogen and polyglycosans, as well as the interaction with R5 (REF. 71). Malin is characterized by a zinc finger of the RING type and six NHL-repeat protein–protein interaction domains. The presence of a RING finger implies that malin is involved in the ubiquitin pathway, specifying substrates that have to be removed by the proteasome system. It has been suggested that mutations in either laforin or malin might lead to improper clearance and subsequent accumulation of polyglycosans in dendrites, and that the resulting disturbance of neuronal function is responsible for the clinical features of Lafora disease70. X-linked lissencephaly/double cortex syndrome. Several lissencephaly syndromes are known in which mutated genes cause a failure of neuronal migration during embryonic development, resulting in cross-neocortical disorganization (TABLE 2). The most common type of lissencephaly is classical lissencephaly, or LIS1, which is characterized by a smooth cerebral surface, a thick cortex and no other brain malformations. The X-linked lissencephaly/double cortex syndrome (XLIS) is an X-chromosomal, dominantly inherited neuronal migration disorder, which manifests in males as classical lissencephaly and in females as subcortical band heterotopia (double cortex) (FIG. 5). The affected individuals have epilepsy and mental retardation of variable severity, and the clinical phenotype is usually more pronounced in hemizygous males than in heterozygous females. The doublecortin (DCX) gene, which is located on chromosome Xq22.3-q23, was identified as the causative gene in XLIS. It encodes a microtubule-associated protein that is expressed in migrating neuroblasts72. Dcx-knockout mice did not show any neocortical malformations, so they did not provide a good model for studying the mechanisms that underlie DCX pathology73. However, using the RNA INTERFERENCE (RNAi) method, a technology that allows acute targeting and disruption of a specific RNA, Bai et al.74 showed that knocking down www.nature.com/reviews/neuro ©2004 Nature Publishing Group REVIEWS a Normal b DCX female c DCX male Figure 5 | Magnetic resonance imaging (MRI) scans of patients with X-linked lissencephaly/double cortex syndrome. a | MRI brain scan of an unaffected individual. b | Female individual with a DCX mutation. The white matter shows a heterotopic band of neurons (‘subcortical band heterotopia’, marked by an arrow), which underlies the normal cortex. c | Male individual with a DCX mutation. The cortex is abnormally thick and shows poor formation of gyri and sulci (‘smooth brain’). Reproduced, with permission, from REF. 77 (2001) Macmillan Magazines Ltd. DCX in utero impairs radial migration of neurons and causes some neurons to occupy inappropriate laminar positions. The phenotypic differences in males and females are explained by the random X-inactivation that causes a mosaic status for the remaining normal DCX allele in neuronal cells of hemizygous females, leading to a mixed pattern of normally migrated and migrationarrested cells. The abnormal microarchitecture of the cortex is likely to create aberrant neuronal networks, which can generate focal or secondarily generalized cortical hyperexcitability. Mitochondrial inheritance and MERRF Mitochondrial encephalomyopathies are a genetically and phenotypically heterogeneous group of disorders that are caused by either mutations in the maternally inherited mitochondrial genome or by mutations in the nuclear DNA. So far, more than 200 disease-causing mutations of mitochondrial DNA (mtDNA) are known. The variability of the clinical phenotypes in mitochondrial disorders is mostly due to heteroplasmy of mtDNA; that is, nonuniform distribution of mitochondria in different tissues and the coexistence of mutated and wild-type mtDNA within the same cell organelle. One of the best-known mitochondrial syndromes is MERRF (myoclonic epilepsy with ragged red fibres), which is characterized by myoclonus, epilepsy, ataxia, muscle weakness, hearing loss, and elevated serum lactate and pyruvate levels. Muscle biopsy typically shows ‘ragged red’-fibres and paracrystalline inclusions in 1. 2. 3. 4. Steinlein, O. et al. A missense mutation in the neuronal nicotinic acetylcholine receptor α4 subunit is associated with autosomal dominant nocturnal frontal lobe epilepsy. Nature Genet. 11, 201–203 (1995). This paper described the identification of the first gene that was found to be responsible for idiopathic epilepsy in humans. Fusco, M. D. et al. The nicotinic receptor β2 subunit is mutant in nocturnal frontal lobe epilepsy. Nature Genet. 26, 275–276 (2000). Biervert, C. et al. A potassium channel mutation in neonatal human epilepsy. Science 279, 403–406 (1998). Singh, N. A. et al. A novel potassium channel gene, KCNQ2, is mutated in an inherited epilepsy of newborns. Nature Genet. 18, 25–29 (1998). 5. 6. 7. 8. subsarcolemmal mitochondria. The MERRF syndrome can be caused by mutations in at least two different mitochondrial genes, MTTK and, less often, MTTL1 (REFS 75,76). MTTK and MTTL1 code for the transfer RNAs for lysine and leucine, respectively. Mutations in these genes result in a severe defect in the translation of mitochondrial genes, causing multiple deficiencies in the cell’s respiratory chain. In MERRF, the impairment of mitochondrial protein synthesis mainly affects the cytochrome c oxidase complex (COX). The broad spectrum of possible clinical features in MERRF is due to the wide range of cellular pathways and functions in which mitochondria are involved. The oxidative phosphorylation defect results in cell dysfunction and apoptosis in tissues that are sensitive to decreased ATP production. Mitochondrial defects therefore manifest primarily in organs with a high energy demand, such as brain and muscle. A possible cause of seizures in mitochondrial encephalomyopathies could be an imbalance of excitatory and inhibitory mechanisms within neuronal networks that is caused by functionally impaired neurons. The future of epilepsy genetics It is difficult to predict how many epilepsy-associated genes are still waiting to be discovered in the human genome. We have already learned that ion channel defects are one of the main causes of idiopathic epilepsies, and that syndromes in which epilepsy is an important feature can be caused by genes that are involved in functions as diverse as neuronal migration, glycogen metabolism and respiratory chain activity. There are many possibilities if one wants to speculate about the nature of genes that might cause epilepsy when mutated. Such genes might code for cell adhesion proteins and neurotrophic factors that are involved in establishing brain architecture during embryogenesis, synaptic signalling proteins that govern synapse formation and synaptic connectivity, or proteins that are involved in extracellular matrix composition. 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Online links DATABASES The following terms in this article are linked online to: Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene CACNA1A | CACNA1H | CHRNA4 | CHRNB2 | CLCN2 | CSTB | DCX | EPM2A | EPM2B/NHLRC1 | GABRG2 | KCNQ2 | KCNQ3 | LGI1 | LGI2 | LGI3 | LGI4 | MASS1/VLGR1 | MTTK | MTTL1 | OPRM1 | SCN1A | SCN1B |SCN2A OMIM: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM ADNFLE | BFNC | childhood absence epilepsy | EPM1 | GEFS+ | grand mal epilepsy on awakening | IGE | juvenile absence epilepsy | juvenile myoclonic epilepsy | Lafora progressive myoclonus epilepsy | LIS1 | MERRF | SMEI | XLIS FURTHER INFORMATION Encyclopedia of Life Sciences: http://www.els.net/ epilepsy Access to this interactive links box is free online. www.nature.com/reviews/neuro ©2004 Nature Publishing Group