WO2004039979A1

WO2004039979A1 - Means and methods for diagnosing and treating idiopathic generalized epilepsy (ige)

Info

Publication number: WO2004039979A1
Application number: PCT/EP2003/012086
Authority: WO
Inventors: Armin Heils; Karsten Haug
Original assignee: Rheinische Friedrich-Wilhelms-Uni Versität Bonn
Priority date: 2002-10-30
Filing date: 2003-10-30
Publication date: 2004-05-13
Also published as: AU2003276217A1

Abstract

The present invention relates to nucleic acid molecules encoding polypeptides which have amino acid sequences of the voltage-gated chloride channel CIC-2, wherein the glycine (Gly) residue corresponding to position (715) of the wild-type voltage-gated chloride channel CIC-2 is replaced by another amino acid residue or wherein amino acids corresponding to positions (74 to 117) of the wild-type voltage-gated chloride channel CIC-2 are deleted or wherein the wild-type translational reading frame of the voltage-gated chloride channel CIC-2 is altered due to an insertion of a nucleotide residue between position (596 and 597) of the corresponding wild-type nucleotide sequence. The invention further relates to polypeptides encoded by said nucleic acids, vectors and hosts comprising said nucleic acid molecules as well as to methods for producing polypeptides encoded by said nucleic acid molecules. The present invention also provides antibodies specifically directed to polypeptides encoded by said nucleic acid molecules. Additionally, primers for selectively amplifying said nucleic acid molecules are provided in the present invention as well as kits, compositions, particularly diagnostic compositions comprising said nucleic acids, vectors, polypeptides, antibodies and/or primers are provided. Also pharmaceutical compositions comprising nucleic acids encoding a functional voltage-gated chloride channel are provided. Moreover, the present invention relates to methods of diagnosing neurological diseases associated with the presence of any one of the aforementioned nucleic acids or polypeptides encoded therefrom as well as to uses and methods for treating neurological disorders/diseases employing a functional voltage-gated chloride channel CIC-2. Furthermore, the present invention also relates to methods for identifying molecules which are capable of specifically interacting with or altering the characteristics of the polypeptides of the invention as well as to methods for the production of pharmaceutical compositions.

Description

Means and methods for diagnosing and treating idiopathic generalized epilepsy (IGE)

The present invention relates to nucleic acid molecules encoding polypeptides which have amino acid sequences of the voltage-gated chloride channel CIC-2, wherein the glycine (Gly) residue corresponding to position 715 of the wild-type voltage-gated chloride channel CIC-2 is replaced by another amino acid residue or wherein amino acids corresponding to positions 74 to 117 of the wild-type voltage- gated chloride channel CIC-2 are deleted or wherein the wild-type translational reading frame of the voltage-gated chloride channel CIC-2 is altered due to an insertion of a nucleotide residue between position 596 and 597 of the corresponding wild-type nucleotide sequence. The invention further relates to polypeptides encoded by said nucleic acids, vectors and hosts comprising said nucleic acid molecules as well as to methods for producing polypeptides encoded by said nucleic acid molecules. The present invention also provides antibodies specifically directed to polypeptides encoded by said nucleic acid molecules. Additionally, primers for selectively amplifying said nucleic acid molecules are provided in the present invention as well as kits, compositions, particularly diagnostic compositions comprising said nucleic acids, vectors, polypeptides, antibodies and/or primers are provided. Also pharmaceutical compositions comprising nucleic acids encoding a functional voltage-gated chloride channel are provided. Moreover, the present invention relates to methods of diagnosing neurological diseases associated with the presence of any one of the aforementioned nucleic acids or polypeptides encoded therefrom as well as to uses and methods for treating neurological disorders/diseases employing a functional voltage-gated chloride channel CIC-2. Furthermore, the present invention also relates to methods for identifying molecules which are capable of specifically interacting with or altering the characteristics of the polypeptides of the invention as well as to methods for the production of pharmaceutical compositions. Epilepsy is a condition that has many forms and causes, but always features recurring seizures. An epileptic seizure is a convulsion or transient abnormal event experienced by the subject due to a paroxysmal discharge of cerebral neurones. Epilepsy, by definition, is the continuing tendency to have such seizures, even if a long interval separates attacks. A generalized convulsion or grand mal fit is the commonest recognized event.

It is known that epilepsy is one of the most frequent neurological diseases affecting about 3% of the population worldwide (Hauser (1996), Mayo Clin. Proc. 71 , 576- 586). Some of 3% of the population have two or more seizures during their lives. Around one-quarter of a million people in Britain take anticonvulsants. In Asia the prevalence is similar to that in Western nations; the condition is said to be over twice as common in Africa.

Clinical symptoms range from mild drowsiness to severe generalized convulsions and are induced by synchronous neuronal discharges due to an imbalance of inhibitory and excitatory activity in the brain. Idiopathic forms are genetically determined and account for about 40% of all epilepsies (Greenberg (1992), Neurology 42, 56-62; Berkovic (1998), Ann. Neurol. 43, 435-445). They are defined by recurrent seizures with characteristic clinical and electroencephalographic features in the absence of any detectable brain lesion. The most frequent idiopathic form of epilepsy is idiopathic generalized epilepsy (IGE). IGE comprises seven clinically delineated syndromes with age-related onset (Commission on Classification and Terminology of the International League Against Epilepsy (1989), Epilepsia 30, 389-399). The most common IGE subtypes are childhood and juvenile absence epilepsy (CAE, JAE), juvenile myoclonic epilepsy (JME) and epilepsy with grand mal seizures on awakening (EGMA) (Commission on Classification and Terminology of the International League Against Epilepsy, 1989, loc. cit.). Absence seizures are the leading symptom of CAE and JAE. They are characterized by a brief loss of consciousness (usually 10-20 s) and either manifest during childhood (CAE) or adolescence (JAE). JME manifests in adolescence with bilateral myoclonic jerks of arms and shoulders (myoclonic seizures) usually occurring in the early morning without a loss of consciousness. All types of IGE can be associated with generalized tonic-clonic seizures which typically occur on awakening, often provoked by sleep deprivation. When this is the only seizure type, patients are diagnosed with EGMA. In all subtypes of IGE, typical electroencephalographic features are generalized spike-wave (GSW-EEG) or poly-spike-wave (PSW-EEG) discharges reflecting a state of synchronized neuronal hyperexcitability. Several genes have already been identified to cause monogenic forms of idiopathic epilepsy. Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) has been shown to arise from mutations identified in genes encoding the 0₄- or β₂- subunit of neuronal nicotinic acetylcholine receptors (CHRNA4, CHRNB2). Mutations in two voltage-gated potassium channel genes (KCNQ2, KCNQ3) are associated with benign familial neonatal convulsions (BFNC). Mutations in three different sodium channel subunits (SCN1 B, SCN1A, SCN2A) _.as well as in the γ₂- subunit of the GABAA receptor (GABRG2) have been shown to cause generalized epilepsy with febrile seizures plus (GEFS⁺) (Steinlein (2000), Curr. Opin. Genet. Dev. 10, 286- 291; Berkovic (2001), Epilepsia 42, Suppl. 5: 16-23); Lerche (2001), Am. J. Med. Genet. 106, 146-159). Thus, all genes identified in idiopathic epilepsies so far encode ion channels. Recently, autosomal dominant partial epilepsy with auditory features, an idiopathic form of temporal lobe epilepsy, has been shown to be caused by mutations in the leucine-rich, glioma-inactivated 1 gene (LGI1), not encoding an ion channel (Kalachikov (2002), Nat. Genet. 30, 335-341). Regarding the genetic basis of pure forms of the common IGE subtypes, so far only one mutation in another GABAA receptor subunit gene (GABRA1) has been reported in a family with JME (Cossette (2002), Nat. Genet. 31 , 184-189). A single epilepsy gene whose mutations can cause the whole spectrum of common IGE subtypes has not been identified to date.

Recently, a genome wide search for chromosomal susceptibility loci of common IGE subtypes including _.130 IGE-multiplex families identified a novel IGE susceptibility locus on chromosome 3q26 (Z_NPL = 4.19 at D3S3725; P = 0.000017) (Sander (2000), Hum. Mol. Genet. 9, 1465-1472). One among many others of the candidate genes located in this chromosomal region is CLCN2 which encodes the voltage- gated chloride channel CIC-2 (Thiemann (1992), Nature 365, 57-60; Cid (1995), Hum. Mol. Genet. 4, 407-413). CIC-2 is strongly expressed in brain, in particular in γ-aminobutyric acid (GABA)-inhibited neurons (Smith (1995), J. Neurosci. 15, 4057- 4067; Sik (2000), Neuroscience 101 , 51-65). Several experimental results suggest an important role of this channel in establishing and maintaining a low intracellular chloride concentration ([Cl^"]i) which is necessary for an inhibitory GABA response. When hippocampal pyramidal neurons - the best studied model for GABA-ergic synaptic inhibition in the brain (Misgeld (1986), Science 232, 1413-1415; Thompson (1989a), J. Neurophysiol. 61 , 501-511, Thompson (1989b), J. Neurophysiol. 61, 512-523) - are loaded with chloride and when K-CI cotransport is simultaneously blocked by furosemide, a low [Cl^"]j is readjusted by activation of a chloride conductance with the physiological and pharmacological properties of CIC-2 (Staley (1994), J. Neurophysiol. 72, 273-284). In hippocampal neurons the expression of CIC-2 is correlated with the existence of a low [Cl^"]j and a hyperpolarizing GABA-ergic response. For example, CA1 and CA3 pyramidal neurons, which exhibit a hyperpolarizing inhibitory postsynaptic potential in response to activation of GABAA receptors, express high levels of CIC-2. In contrast, granule cells in the dentate gyms that are depolarized by GABA-ergic stimulation do not express this channel (Misgeld (1986), loc. cit; Staley (1994), loc.cit.; Smith (1995), loc. cit.; Sik (2000), loc. cit.). Furthermore, CIC-2 mRNA is upregulated postnatally in the rat hippocampus in parallel with the developmental switch of the GABA response from excitatory to inhibitory (Mladinic (1999), Proc. R. Soc. Lond. B Biol. Sci. 266, 1207-1213). Finally, experimental gene transfer of CIC- 2 into dorsal root ganglion neurons that do not express this channel clamps Eci to the membrane potential and changes the GABA response of these cells from excitatory to inhibitory (Staley (1996), Neuron 17, 543-551). Human CIC-2 channels exhibit unique gating features that allow them to act as a chloride efflux pathway well suited to establishing and maintaining a high transmembrane chloride gradient necessary for an inhibitory GABA response. Due to the coupling of channel activation to [Cl^"]i and the slow gating, they are closed under resting conditions as well as during action potentials or isolated excitatory postsynaptic potentials. Human CIC-2 channels open only with long-lasting changes of the transmembrane CI^" gradient when Eci becomes more positive than the membrane potential, for example when [Cl^"]j is increased after intense GABA-ergic inhibition (Staley (1994), loc. cit.; Thompson (1989a), loc. cit.; Thompson (1989b), loc. cit). As there is no voltage- and time-dependent inactivation, hCIC-2 channels remain open and extrude chloride until Eci approaches the resting membrane potential.

A passive transport mechanism such as a channel-mediated chloride flux is, however, by itself unable to account for the large transmembrane chloride gradient of many neurons. Primary or secondary active processes are necessary to generate an Eci more negative than the resting membrane potential. It is well established that an outwardly directed coupled transport of K⁺ and CI^" by the neuron-specific KCC2 transporter plays a key role in generating the low [Cl^"]j which is essential for GABA- ergic synaptic inhibition (Misgeld (1986), loc. cit.; Thompson (1989b), loc. cit.; Rivera (1999), Nature 397, 251-255; Hϋbner (2001), Neuron 30, 515-524; Woo (2002), Hippocampus 12, 258-268). Under physiological ionic conditions, K-CI cotransport by KCC2 is driven by the transmembrane K⁺ gradient and causes CI^" extrusion near the resting potential (Misgeld (1986), loc. cit.; Thompson (1989b), loc. cit.). However, increases of [K⁺]₀ affect the rate and the direction of this transport, i.e. at a low [Cl^"]i and a high [K⁺]₀ KCC2 may operate in reverse and accumulate internal chloride (Thompson (1989b), loc. cit.; Payne (1997), Am. J. Physiol. 273, C1516-1525; DeFazio (2000), J. Neurosci. 20, 8069-8076). High- frequency activity of GABA-ergic interneurons increases [K⁺]₀ in the vicinity of hippocampal pyramidal neurons (Kaila (1997), J. Neurosci. 17, 7662-7672). This will cause impaired CI^" extrusion or even inward K-CI cotransport by KCC2 and aggravate the intracellular chloride accumulation in dendrites and somata of pyramidal neurons produced by CI^" influx through GABAA receptors (Misgeld (1986), loc. cit; Thompson (1989a), loc. cit.; Thompson (1989b), loc. cit.; Payne (1997), loc. cit.; Kaila (1997), loc. cit.). Under these conditions, CIC-2 appears to be the primary chloride extrusion pathway to reestablish a negative Eci crucial to the preservation of an inhibitory GABA response.

The spread of electrical activity between cortical neurones is normally restricted. Synchronous discharge of neurones in normal brain takes place in small groups only; these limited discharges are responsible for the normal rhythms of the electro encephalo gram (EEG). During a seizure, large groups of neurones are activated repetitively and "hypersynchronously". There is a failure of inhibitory synaptic contact between neurones. This causes high-voltage spike-and-wave activity on the EEG. Epileptic activity confined to one area of the cortex is associated with specific symptoms and signs (partial seizures). This activity may remain focal or may spread to cause paroxysmal activity in both hemispheres and a generalized convulsion.

This spread is called secondary generalization of a partial seizure.

The main treatment options for people with epilepsy are medications, surgery, vagus nerve stimulation and a ketogenic diet. It is important to know that the same treatment does not work for every patient because the severity of epilepsy varies from patient to patient. Some patients will manage their epilepsy very well with medication while others will be better served by having surgery or using vagus nerve stimulation.

A few medications are currently approved for the treatment of epilepsy. Each of these medications has a unique list of benefits and side effects, and different medications are appropriate for different types of epilepsy. No one medication is proven to be the best treatment for epilepsy. Only a complete evaluation can determine what medication will work best for each individual patient.

Patients who do not respond well to medication may be candidates for surgery.

Some clinics offer different types of surgery for different types of epilepsy. A variety of sophisticated diagnostic tests will be used to determine if surgery is the best option.

Patients who have partial seizures that originate in one part of the brain may be candidates for a type of surgery in which that part of the brain is removed. This type of surgery is done only if it does not jeopardize normal function, and the part of the brain from which the seizure originates can be precisely pinpointed.

Patients who have generalized seizures are not usually candidates for surgery.

However, if the seizures are resulting in falls and injuries, a procedure called

"corpus callosotomy" may be considered. This procedure involves separating the nerve fibers that connect the two halves of the brain. While this surgery does not cure epilepsy or completely stop seizures, it can reduce the number and severity of seizures and the related falls and injuries.

Vagus nerve stimulation (VNS) is approved for the treatment of partial seizures in patients 12 years of age or older. VNS involves implantation of an electronic device under the skin in the chest. The device automatically delivers a stimulus as often as needed, from every few seconds to every few minutes. Patients usually do not become seizure-free with VNS; approximately 30 to 50 percent of patients can be expected to have a reduction in seizure activity.

The ketogenic diet has been used at some clinics. It is primarily used in childhood epilepsy. The mechanism by which the ketogenic diet works is unknown. The high fat, low-protein, no carbohydrate diet mimics some effects of starvation that seem to inhibit seizures. The diet is very rigid and carefully controlled, and must be supervised by a physician - sometimes in a hospital setting. Ketogenic diets have been used for epileptic children for many years with a success rate of approximately 50 percent. Close collaboration with an experienced dietitian knowledgeable in the implementation of the ketogenic diet, and dedication of the patient and his or her family is essential in order for this form of treatment to work.

So far it is known that CLCN2 encodes the voltage-gated chloride channel CIC-2 which is expressed in the brain, in particular in inhibitory neurons where it prevents chloride accumulation and ensures an inhibitory response to GABA. The human CLCN2 gene has been cloned and mapped to chromosome 3q26 (Cid (1995), loc. cit.), however, the gene has never been regarded as a potential candidate for epilepsy. Interestingly, even a targeted disruption of CLCN2 in a mouse does not exhibit neuronal hyperexcitability or lead to seizures (Bδsl (2001), EMBO J. 20, 1289-1299) suggesting a species-specific difference in the (patho-) physiological role of this channel. Mouse models often differ from human diseases. For example, transgenic mice with either a knock-out or knock-in of the gene CHRNA4, expected to be an animal model for the human disease of autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) (Berkovic (2001), loc. cit.) were not reported to develop seizures (Ross (2000), J. Neurosci. 20, 6431-6441 ; Labarca (2001), Proc. Natl. Acad. Sci. U. S. A. 98, 2786-2791). Compensatory mechanisms that are distinct in humans and mice as well as characteristic anatomical and physiological properties may account for these phenotypical differences.

Recently, mutations within two different genes encoding GABA_A receptor subunits have been identified in three families with GEFS⁺ (in one family associated with frequent absence seizures) or JME (Baulac (2001), Nat. Genet. 28, 46-48; Wallace (2001), Nat. Genet. 28, 49-52; Cossette (2002), loc. cit.). Attenuation of GABA-ergic synaptic inhibition might therefore evolve as a pathophysiological concept for a significant proportion of idiopathic epilepsies. Further genetic and pathophysiological studies of the inherited human epilepsies may open novel research directions, broaden our knowledge of the molecular mechanisms underlying epilepsy and finally contribute to the discovery of better pharmacological therapies.

Idiopathic generalized epilepsy (IGE) is an inherited neurological disorder affecting about 1 % of the world's population. So far, only several genes encoding neuronal ion channels have been identified in monogenic subtypes of idiopathic epilepsy (Steinlein (2000), loc. cit.; Berkovic (2001), loc. cit.; Lerche (2001), loc. cit.). However, no single epilepsy gene whose mutations can cause the whole spectrum of common idiopathic generalized epilepsy (IGE) subtypes has been identified until to date.

Since no well-suited medication nor diagnosis on a molecular level for idiopathic generalized epilepsy (IGE) is available, there is a need for identifying a gene whose mutations cause the whole spectrum of common IGE as well as for providing medicaments and methods for diagnosis and treatment of idiopathic generalized epilepsy (IGE).

Thus, the technical problem underlying the present invention is to provide means and methods for diagnosis and treating idiopathic generalized epilepsy (IGE).

The solution to said technical problem is achieved by providing the embodiments characterized in the claims.

Thus, the present invention relates to a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of:

(a) a nucleic acid sequence encoding a polypeptide which has an amino acid sequence of a voltage-gated chloride channel CIC-2, wherein the glycine (Gly) residue corresponding to position 715 of the wild-type voltage-gated chloride channel CIC-2 as depicted in SEQ ID NO: 2 is replaced by another amino acid; (b) a nucleic acid sequence encoding a polypeptide which has an amino acid sequence of a voltage-gated chloride channel CIC-2, wherein amino acids corresponding to positions 74 to 117 of the wild-type voltage-gated chloride channel CIC-2 as depicted in SEQ ID NO: 2 are deleted;

(c) a genomic nucleotide sequence encoding a voltage-gated chloride channel CIC-2 and which contains a mutation in intron 2 which leads to an aberrant splicing of the mRNA transcribed by said genomic nucleotide sequence resulting in a fusion of exons 2 and 4 thereby leading to the production of an mRNA lacking exon 3;

(d) a nucleic acid sequence encoding a polypeptide which has an amino acid sequence of a voltage-gated chloride channel CIC-2, wherein the wild-type translational reading frame of the voltage-gated chloride channel CIC-2 is altered due to an insertion between the nucleotides corresponding to position 596 and position 597 of the corresponding wild-type nucleotide sequence as depicted in SEQ ID NO: 1;

(e) the nucleotide sequence of SEQ ID NOs: 3, 5 or 7;

(f) a nucleic acid sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NOs: 4, 6 or 8;

(g) a nucleotide sequence which hybridizes to a nucleotide sequence defined in (a) or to the nucleotide sequence depicted in SEQ ID NO: 3 and which encodes a voltage-gated chloride channel CIC-2, in which the glycine (Gly) residue corresponding to position 715 of the wild-type voltage-gated chloride channel CIC-2 as depicted in SEQ ID NO: 2 is replaced by another amino acid residue;

(h) a nucleotide sequence which hybridizes to a nucleotide sequence defined in (b) or to the nucleotide sequence depicted in SEQ ID NO: 5 and which encodes a voltage-gated chloride channel CIC-2, wherein amino acids corresponding to positions 74 to 117 of the wild-type voltage-gated chloride channel CIC-2 as depicted in SEQ ID NO: 2 are deleted;

(i) a nucleotide sequence which hybridizes to a nucleotide sequence defined in (d) or to the nucleotide sequence depicted in SEQ ID NO: 7 and which encodes a voltage-gated chloride channel CIC-2, wherein the wild-type translational reading frame of the voltage-gated chloride channel CIC-2 is altered due to an insertion between the nucleotides corresponding to position 596 and position 597 of the corresponding wild type nucleotide sequence as depicted in SEQ ID NO: 1; and (j) a nucleic acid sequence being degenerate as a result of the genetic code to the nucleic acid sequence as defined in any one of (g) to (i).

It was surprisingly found that mutations in the CLCN2 gene which encodes the voltage-gated chloride channel CIC-2 can cause the whole spectrum of common subtypes of idiopathic generalized epilepsy. Three different CLCN2 mutations that co-segregate with the affection status in three unrelated IGE families are identified. Each mutation causes functional alterations that can explain neuronal hyperexcitability and the occurrence of epileptic seizures.

The three mutations described herein above are (i) a single amino acid substitution (G715E) caused by a point mutation in the respective wild-type codon, (ii) an atypical splicing (del74-117) caused by the deletion of an 11-bp fragment within the intron between exons 2 and 3, wherein said deleted 11-bp fragment is located in close proximity to a splice acceptor site and, thus, leads to aberrant splicing leading to skipping of exon 3 which results in an in-frame deletion of 44 amino acids corresponding to amino acids 74 to 117 of SEQ ID NO:2 , and (iii) a premature stop codon (M200fsX231) resulting from the insertion of a nucleotide residue between position 596 and 597 of the corresponding wild-type nucleotide sequence. All mutations produce functional alterations which provide distinct explanations for their pathogenic phenotypes. M200fsX231 and del74-117 cause a loss-of-function of CIC-2 channels, and are expected to decrease the transmembrane chloride gradient essential for GABA-ergic inhibition. Moreover, as demonstrated in the Examples herein below M200fsX231 and del74-117 are dominant-negative, i.e. they cause non-functionality of wild-type voltage-gated chloride channels when being co- expressed with said wild-type chloride channel. G715E causes a gain-of-function of CIC-2 channels, i.e. said G715E mutation results in an alteration of voltage- dependent gating that can cause membrane depolarization and hyperexcitability. These results establish that mutations in the CLCN2 gene encoding the voltage- gated chloride channel CIC-2 cause idiopathic generalized epilepsy. The identification of these mutations now allows the effective diagnosis of IGE, the development of methods for identifying compounds which can be used as therapeutics in IGE and the development of method of treatments for IGE.

Unless otherwise stated, the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, H.G.W, Nagel, B. and Kolbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland)

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step.

In accordance with the present invention, the term "nucleic acid sequence" means the sequence of bases comprising purine- and pyrimidine bases which are comprised by nucleic acid molecules, whereby said bases represent the primary structure of a nucleic acid molecule. Nucleic acid sequences include DNA, cDNA, genomic DNA, RNA, synthetic forms and mixed polymers, both sense and antisense strands, or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art.

When used herein, the term "polypeptide" means a peptide, a protein, or a polypeptide which encompasses amino acid chains of a given length, wherein the amino acid residues are linked by covalent peptide bonds. However, peptidomimetics of such proteins/polypeptides wherein amino acid(s) and/or peptide bond(s) have been, replaced by functional analogs are also encompassed by the invention as well as other than the 20 gene-encoded amino acids, such as selenocysteine. Peptides, oligopeptides and proteins may be termed polypeptides. The terms polypeptide and protein are often used interchangeably herein. The term polypeptide also refers to, and does not exclude, modifications of the polypeptide, e.g., glycosylation, acetylation, phosphorylation and the like. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. The term "voltage-gated chloride channel CIC-2", in accordance with this invention, denotes a polypeptide which has the characteristics of a voltage-gated ion channel CIC-2. Such characteristics include structural and/or functional characteristics. Structural characteristics refer to certain structural features which allow to classify a polypeptide as being a CIC-2 protein. One such feature is the amino acid sequence. In the context of the present invention a polypeptide is classified as a voltage-gated chloride channel CICI-2 if it shows a certain degree of sequence identity over its own length to the amino acid sequence of the human CIG-2 protein depicted in SEQ ID NO:2. This degree of sequence identity is at least 40%, more preferably at least 50%, even more preferably 60%, at least 70%, at least 80% at least 90% or at least 95%. It is particularly preferred that the degree of sequence identity is at least 65%. Moreover, structural characteristics of CIC-2 proteins are 10 to 12 transmembrane domains which could be analysed by using the program TMPRED (Hofmann (1993), Biol. Chem. 347, 166) or TMHMM (Krogh (2001), J. Mol. Bio. 305, 567-580) and 18 helical segments (Dutzler (2002), loc. cit.). Additionally, CIC-2 proteins dimerize and contain conserved domains designated "voltage_CLC" (PFAM Accession number: PF00654) and "CBS" (PFAM Accession number: PF00571), respectively, which can be identified by using the program PFAM (Bateman (2002), Nucl. Acids Res. 30, 276-280). In the context of the present invention a protein is classified as a CIC-2 protein if it displays at least one of the above-mentioned structural characteristics. Functional characteristics refer to properties related to the biological activity of the CIC-2 protein. In particular, CIC-2 is a chloride ion channel which allows chloride ions to pass from intracellular solution to extracellular solution, i.e. the efflux of chloride upon electrophysiological stimulation to establish and maintain a high transmembrane chloride gradient which is necessary for an inhibitory GABA response. Moreover, the voltage-gated chloride channel CIC-2 and forms naturally a dimer. Due to the coupling of channel activation to [Cl^"]j and the slow gating, they are closed under resting conditions as well as during action potentials or isolated excitatory postsynaptic potentials. CIC-2 channels open only with long-lasting changes of the transmembrane CI^" gradient when E_Cι becomes more positive than the membrane potential, for example when [CP]i is increased after intense GABA- ergic inhibition (Staley (1994), loc. cit.; Thompson (1989a), loc. cit.; Thompson (1989b), loc. cit.). As there is no voltage- and time-dependent inactivation, CIC-2 channels remain open and extrude chloride until Eci approaches the resting membrane potential. CIC-2 chloride channels are voltage-dependent and after hyperpolarisation of the membrane they permit efflux of intracellular chloride ions. Additionally, CIC-2 chloride channels permit efflux of chloride ions independent of voltage if the intracellular concentration of chloride ions is physiologically too high. The characteristics of CIC-2 channel proteins can be determined as mentioned, for example, in Jentsch (2002), Physiol Rev 82, 503-568. The term "voltage-gated chloride channels CIC-2" comprises functional and non-functional forms of the voltage-gated chloride channels CIC-2. A functional voltage-gated chloride channel CIC2 is understood to be a CIC-2 protein which has at least one of the above- mentioned functional characteristics which can be measured by methods known in the art and exemplified in the Examples herein. A non-functional voltage-gated chloride channel CIC-2 is a protein which can be classified as a CIC-2 protein due to structural characteristics as described above but which has lost at least one, preferably all, functional characteristics of a CIC-2 protein as described above. Non- functionality of the CIC-2 protein can, e.g., be determined by incubating erythrocytes having a CIC-2 protein in low osmotic solutions. Erythrocytes having a nonfunctional CIC-2 protein will display under these conditions a significant increase in swelling in comparison to erythrocytes expressing a functional CIC-2 protein, e.g., a wild-type CIC-2 protein, since it is assumed that the CIC-2 protein is involved in osmoregulation (Jentsch (2002), loc. cit). Thus, it is possible to determine the occurrence of a mutation in the voltage-gated chloride channel CIC-2 by measuring the chloride efflux of cells in low osmotic solutions. Cells harbouring a mutation in the CLCN2 gene encoding the CIC-2 protein show an altered chlorid efflux in comparison to cells harbouring a wild-type CIC-2 protein.

The three mutations described herein are characterized in that they have an amino acid replacement at a certain position, a deletion of a part of the amino acid sequence due to aberrant splicing or a nucleotide insertion at a certain position when compared to the corresponding wild-type human CIC-2 amino acid/nucleotide sequence as shown in SEQ ID NO:2/SEQ ID NO:1. The term "position" used in accordance with the present invention means the position of either an amino acid within an amino acid sequence depicted herein or the position of a nucleotide within a nucleic acid sequence depicted herein.

The position with respect to nucleotide sequences mentioned herein refer to the sequence shown in SEQ ID NO:1. This sequence represents the open reading frame of the assembled exons of the human CLCN2 gene encoding CIC-2. The corresponding genomic sequence including the introns is shown in SEQ ID NO:9. It is possible for the skilled person to identify the position in the genomic sequence corresponding to a position in SEQ ID NO:1 by aligning the sequences. Moreover, the exact locations of the exons and introns in SEQ ID NO:9 are described herein further below.

The term "an amino acid residue corresponding to position X of the wild-type voltage-gated chloride channel CIC-2 as depicted in SEQ ID NO:2" has the following meaning: The amino acid residue in question would be located at position X in the sequence of SEQ ID NO:2 if the sequence in which said amino acid residue occurs is compared and aligned with the amino acid sequence of SEQ ID NO:2. The amino acid sequence shown in SEQ ID NO:2 is the sequence of the human CIC-2 gene and is used as a reference sequence in the present invention. Accordingly, the term "a nucleotide residue corresponding to position Y of the wild- type nucleotide sequence as depicted in SEQ ID NO:1" means that a nucleotide residue in a CIC-2 encoding sequence would be located at position Y in SEQ ID NO:1 when the CIC-2 encoding sequence is compared and aligned with the sequence of SEQ ID NO:1. Amino acid and nucleotide sequences of other CIC-2 voltage-gated chloride channels from other organisms are known, e.g. from Cid (1995, loc.cit.) or Thiemann (1992, loc.cit.) and can also be retrieved from electronic data bases, such as GenBank or GenEMBL.

In order to determine whether an amino acid residue or nucleotide residue in a given CIC-2 sequence corresponds to a certain position in the amino acid sequence or nucleotide sequence of SEQ ID NO: 1/2, the skilled person can use means and methods well-known in the art, e.g. alignments, either manually or by using computer programs such as those mentioned further down below in connection with the definition of the term "hybridization" and degrees of homology. For example, BLAST2.0, which stands for Basic Local Alignment Search Tool (Altschul, Nucl. Acids Res. 25 (1997), 3389-3402; Altschul, J. Mol. Evol. 36 (1993), 290-300; Altschul, J. Mol. Biol. 215 (1990), 403-410), can be used to search for local sequence alignments. BLAST produces alignments of both nucleotide and amino acid sequences to determine sequence similarity. Because of the local nature of the alignments, BLAST is especially useful in determining exact matches or in identifying similar sequences. The fundamental unit of BLAST algorithm output is the High-scoring Segment Pair (HSP). An HSP consists of two sequence fragments of arbitrary but equal lengths whose alignment is locally maximal and for which the alignment score meets or exceeds a threshold or cutoff score set by the user. The BLAST approach is to look for HSPs between a query sequence and a database sequence, to evaluate the statistical significance of any matches found, and to report only those matches which satisfy the user-selected threshold of significance. The parameter E establishes the statistically significant threshold for reporting database sequence matches. E is interpreted as the upper bound of the expected frequency of chance occurrence of an HSP (or set of HSPs) within the context of the entire database search. Any database sequence whose match satisfies E is reported in the program output.

Analogous computer techniques using BLAST (Altschul (1997), loc. cit.; Altschul (1993), loc. cit; Altschul (1990), loc. cit.) are used to search for identical or related molecules in nucleotide databases such as GenBank or EMBL. This analysis is much faster than multiple membrane-based hybridizations. In addition, the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or similar. The basis of the search is the product score which is defined as:

%sequence identity x % maximum BLAST score . 100 and it takes into account both the degree of similarity between two sequences and the length of the sequence match. For example, with a product score of 40, the match will be exact within a 1-2% error; and at 70, the match will be exact. Similar molecules are usually identified by selecting those which show product scores between 15 and 40, although lower scores may identify related molecules. The first mutant described in the present invention is a nucleic acid sequence which encodes a voltage-gated chloride channel CIC-2 where the glycine (Gly) corresponding to position 715 of the wild-type voltage-gated chloride channel CIC- 2 as depicted in SEQ ID NO:2 is replaced by another aminό acid residue. This specific glycine residue lies within the intracellularly located C-terminus of the voltage-gated chloride channel CIC-2, and more particularly, it lies between T714 and S716 (as depicted in SEQ ID NO:2). The nucleotide sequence of the wild-type voltage-gated chloride channel CIC-2 is well known in the art and, inter alia, shown in SEQ ID NO. 1 (see also GenBank accession numbers S77770 and NM004366 as well as Cid (1995), loc. cit.). According to the standard genetic code (as illustrated, inter alia, in Stryer (1995), "Biochemistry", Freemann and Compagny, ISBN 0-7167-2009-4), the codons GGU, GGC, GGG, GGA code for glycine (Gly). Due to (a) point mutation(s) caused by, e.g., chemical and/or physical means or inaccuracy of the replication complex followed by a failure of the reparation machinery of a cell, a change of a single codon occur can be achieved. Possible types of point mutations are transitions, i.e. change of a purine or pyrimidine base for another purine or pyrimidine base, e.g. adenine to guanine or thymidine to cytosine or transversions, i.e. change of a purine or pyrimidine base for another pyrimide or purine base, e.g., adenine to thymidine or guanine to cytosine. Additionally a point mutation can also be caused by insertion or deletion of one or more nucleotides. The amino acid residue replacing the glycine at position 715 can in principle be any other amino acid residue, in particular a residue which naturally occurs in proteins. It can, e.g. be an aliphatic, aromatic, basic or acidic amino acid residue. Preferably it is an acidic amino acid residue, such as aspartate or glutamate. Most preferably, it is glutamate. In the mutant described in the Examples a transition from guanine to adenine at position 2144 of the wild-type nucleotide sequence depicted in SEQ ID NO: 1 took place which results in a change of the wild-type codon "GGG" encoding glycine (Gly) to "GAG" encoding glutamate (Glu).

Thus, an example and preferred embodiment for a mutation of the CIC-2 sequence in which GIy715 is replaced by glutamate is depicted in SEQ ID NO:3 (nucleotide sequence) and SEQ ID NO:4 (amino acid sequence). The replacement at position 715 preferably leads to a mutant CIC-2 protein which causes, when expressed in cells, membrane depolarization and hyperexcitability. More preferably the mutant displays the properties as described in Example 10 herein. These properties can be determined as described in Example 5.

The second mutation of the CIC-2 polypeptide described herein is a deletion of 44 amino acids corresponding to positions 74 to 117 of the wild-type CIC-2 sequence as depicted in SEQ ID NO: 2. Thus, the present invention also relates to nucleic acid sequences encoding a CIC-2 protein in which amino acids corresponding to positions 74 to 117 of the wild-type voltage-gated chloride channel CIC-2 as depicted in SEQ ID NO: 2 are deleted. This means, according to the present invention, that a fragment encompassing amino acid positions 74 to 117 of the corresponding wild-type amino acid sequence depicted in SEQ ID NO: 2 is deleted which results in a shortened polypeptide. An example for such a shortened polypeptide is depicted in SEQ ID NO: 6. Said shortened polypeptide is encoded by SEQ ID NO: 5. This type of second mutation as described herein preferably encodes a non-functional voltage-gated chloride channel CIC-2. In the present invention the deletion of a fragment encompassing amino acids 74 to 117 of the wild-type amino acid sequence depicted in SEQ ID NO: 2 is the result of an atypical splicing event during the maturation of the mRNA of the CLCN2 gene. Due to an 11- bp deletion in intron 2 (IVS2-14del11) in close proximity to the splice acceptor site of the wild-type nucleotide sequence depicted in SEQ ID NO: 9 atypical splicing takes place which leads to fusion of exons 2 and 4 of the wild-type mRNA, whereby exon 3 encoding amino acids 74 to 117 is skipped. The resulting protein (del74-117) depicted in SEQ ID NO: 6 lacks amino acids 74 to 117 of the corresponding wild type amino acid sequence depicted in SEQ ID NO: 2 such that amino acid position 74 of the deleted polypeptide depicted in SEQ ID NO: 5 corresponds to amino acid position 118 of the wild-type amino acid sequence depicted in SEQ ID NO: 2. Preferably, the nucleic acid sequence of the invention encodes a CIC-2 polypeptide in which exactly amino acids corresponding to positions 74 to 117 of SEQ ID NO:2 are deleted. However, also mutants are comprised in which either more or less amino acids within the CIC-2 amino acid sequence set forth in SEQ ID NO: 2 may be deleted due to, for example, atypical splicing or deletion of nucleotides of the nucleic acid molecule encoding CIC-2 or wrong posttranslational processes, as long as the CIC-2 voltage-gated chloride channel is non-functional. For example, it is also possible that further amino acids preceding amino acid position 74 or amino acids succeeding amino acid position 117 may be deleted or that less amino acids are deleted.

Preferably at least one, more preferably at least two, even more preferably at least three and most preferably at least 5 amino acid residues are further deleted upstream from the position corresponding to amino acid residue 74 and/or downstream of the position corresponding to amino acid residue 117 of SEQ ID NO:2.

However, it is preferred that not more than 20, preferably not more than 15, even more preferably not more than 10 and most preferably not more than 7 amino acid residues are further deleted upstream of the position corresponding to amino acid residue 74 of SEQ ID NO:2 or downstream of the position corresponding to amino acid residue 117 of SEQ ID NO:2.

The present invention also provides a mutation in the gene encoding CIC-2 which is related to IGE and which is characterized in that it is a mutation which occurs in intron 2 of the genomic sequence encoding CIC-2 and leads to an aberrant splicing of the mRNA transcribed from said gene insofar as exons 2 and 4 are fused and exon 3 is skipped. Since exon 3 encodes amino acid residues corresponding to residues 74 to 117 of SEQ ID NO:2, the result is a shortened polypeptide lacking 44 amino acids compared to the wild-type CIC-2 protein.

The exon/intron structure of the gene encoding the CIC-2 protein is known to the person skilled in the art. The genomic sequences of CIC-2 encoding genes of human, rat, mouse, guinea pig and rabbit are, e.g. published or available in - Cid (1995), loc. cit.; Chu (1996), Nucl. Acids Res. 24, 3453-3457); Hathaway (1999), direct submission to GenBank at NCBI/NIH; Cid (1998) direct submission to GenBank at NCBI/NIH; Furukawa (1995), FEBS Lett. 375, 56-62 - and available in databases under accession numbers NP004357, NP058833, AAD26466, AAD37113 and S68210. The genomic sequence and exon/intron structure of the human gene encoding CIC-2 is, e.g., evident from data base entry NM004357 at the Human Genome Browser Gateway. There, exons are indicated in upper cases and introns are indicated in lower cases. The genomic sequence of the human gene encoding CIC-2 is also evident from SEQ ID NO:9. The exons and introns are indicated. In particular, exons 2 and 3 and intron 2 are located at the following positions.

Table 1 : Positions of exons and introns in the wild-type CLCN2 gene as shown in SEQ ID NO: 9

A mutation according to the invention which leads to a fusion of exons 2 and 4 of a CIC-2 encoding gene may, e.g., be a mutation which prevents interaction of the splice donor and splice acceptor sites necessary to fuse exons 2 and 3 but which does not prevent fusion of exons 2 and 4. Preferably, such a mutation abolishes the function of the splice acceptor site necessary for the fusion of exons 2 and 3. Even more preferably such a mutation is a sequence alteration in intron 2 close to or in the splice acceptor site. More preferably it is a deletion close to or overlapping the splice acceptor site. One example of a mutation in intron 2 of a CIC-2 encoding gene is the deletion of 11 bp corresponding to nucleotides 2653 to 2663 in SEQ ID NO:9.

The mutation in intron 2 as described above preferably leads to an ORF which, when expressed, leads to the synthesis of a non-functional CIC-2 protein. A further mutation in a CIC-2 encoding polynucleotide found to be correlated with IGE is an insertion between nucleotides corresponding to positions 596 and 597 of the corresponding wild-type sequence shown in SEQ ID NO:1 which leads to a premature stop codon due to a shift in the wild-type translational reading frame. The term "wild-type translational reading frame" when used in accordance with the present invention means that only this possibility out of three possibilities to read the nucleotide sequence of the CIC-2 gene beginning with a start codon (ATG) in a triplett pattern results in the amino acid sequence depicted in SEQ ID NO: 2. Accordingly, an alteration of the wild-type translational reading frame changes the reading frame and, thus, the amino acid sequence following the frame shift. These mutations are called frame-shift mutations.

Preferably, the insertion between the nucleotides corresponding to position 596 and 597 of SEQ ID NO: 1 results in a frameshift which has the effect that the nucleotide triplet corresponding to nucleotides 690 to 692 of SEQ ID NO:1 , which constitutes a stop codon, lies in frame, thereby leading to a premature termination of translation. Such an insertion may be an insertion of 1 nucleotide or of (1 + 3 x X) nucleotides with X being an integer >1.

Preferably the insertion is only one nucleotide so that position 597 of the wild-type nucleotide sequence depicted in SEQ ID NO:1 is then residue 598 in a correspondingly mutated sequence. The inserted nucleotide(s) may be any type of nucleotide, e.g. adenine, guanine, cytidine or thymidine, or analogs or derivatives thereof which can be incorporated into nucleic acid molecules. In case the insertion is just one nucleotide, the nucleotide is preferably a guanine. The nucleotide sequence of an example of such a mutation is set forth in SEQ ID NO:7. This mutation is designated M200fsX231. In this mutation, the insertion of a nucleotide residue between positions 596 and 597 of the wild-type nucleotide sequence depicted in SEQ ID NO: 1 leads to a +1 frame shift mutation. Said mutation results due to the generation a premature stop codon present in the +1 reading frame in comparison to the wild-type amino acid sequence depicted in SEQ ID NO: 2 in a shortened polypeptide depicted in SEQ ID NO: 8. Said shortened polypeptide is non-functional due to the generation of a premature stop codon in the +1 reading frame.

The insertion may alternatively also be an insertion of 2 nucleotides or of (2 + 3 x X) nucleotides with X being an integer >1. Such a mutation also leads to a premature termination and, thus, a shorter polypeptide due to the fact that the nucleotide triplet at positions 1022 to 1024 of SEQ ID NO:1 , which constitutes a stop codon, comes into frame.

The insertion as described herein-above preferably leads to a mutation in the CIC-2 encoding sequence which, when expressed, leads to the synthesis of a nonfunctional CIC-2 protein.

The present invention also relates to nucleic acid molecules which hybridize to one of the above described nucleic acid molecules and which encode a CIC-2 protein with one of the above described mutations.

The term "hybridizes" as used in accordance with the present invention may relate to hybridizations under stringent or non-stringent conditions. If not further specified, the conditions are preferably non-stringent. Said hybridization conditions may be established according to conventional protocols described, for example, in Sambrook, Russell "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory, N.Y. (2001); Ausubel, "Current Protocols in Molecular Biology", Green Publishing Associates and Wiley Interscience, N.Y. (1989), or Higgins and Hames (Eds) "Nucleic acid hybridization, a practical approach" , IRL Press Oxford, Washington DC, (1985). The setting of conditions is well within the skill of the artisan and can be determined according to protocols described in the art. Thus, the detection of only specifically hybridizing sequences will usually require stringent hybridization and washing conditions such as O.lxSSC, 0.1% SDS at 65°. Non- stringent hybridization conditions for the detection of homologous or not exactly complementary sequences may be set at 6xSSC, 1% SDS at 65°C. As is well known, the length of the probe and the composition of the nucleic acid to be determined constitute further parameters of the hybridization conditions. Note that variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hydridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility. Hybridizing nucleic acid molecules also comprise fragments of the above described molecules. Such fragments may represent nucleic acid sequences which code for a non-functional voltage-gated chloride channel CIC-2 or a nonfunctional fragment thereof, and which have a length of at least 12 nucleotides, preferably at least 15, more preferably at least 18, more preferably of at least 21 nucleotides, more preferably at least 30 nucleotides, even more preferably at least 40 nucleotides and most preferably at least 60 nucleotides. Furthermore, nucleic acid molecules which hybridize with any of the aforementioned nucleic acid molecules also include complementary fragments, derivatives and allelic variants of these molecules. Non-functional fragments of a voltage-gated chloride channel CIC- 2 may be comprised in a fusion and/or chimeric protein. Additionally, a hybridization complex refers to a complex between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary G and C bases and between complementary A and T bases; these hydrogen bonds may be further stabilized by base stacking interactions. The two complementary nucleic acid sequences hydrogen bond in an antiparallel configuration. A hybridization complex may be formed in solution (e.g. C₀t or R₀t analysis) or between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., membranes, filters, chips, pins or glass slides to which, e.g., cells have been fixed). The terms complementary or complementarity refer to the natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing. For example, the sequence "A-G-T" binds to the complementary sequence "T-C-A". Complementarity between two single-stranded molecules may be "partial", in which only some of the nucleic acids bind, or it may be complete when total complementarity exists between single-stranded molecules. The degree of complementartity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, which depend upon binding between nucleic acids strands. The term "hybridizing sequences" preferably refers to sequences which display a sequence identity of at least 40%, preferably at least 50%, more preferably at least 60%, even more preferably at least 70%, particularly preferred at least 80%, more particularly preferred at least 90%, even more particularly preferred at least 95% and most preferably at least 97% identity with a nucleic acid sequence as described above encoding a CIC-2 protein having a described mutation. Moreover, the term "hybridizing sequences" preferably refers to sequences encoding a CIC-2 protein having a sequence identity of at least 40%, preferably at least 50%, more preferably at least 60%, even more preferably at least 70%, particularly preferred at least 80%, more particularly preferred at least 90%, even more particularly preferred at least 95% and most preferably at least 97% identity with an amino acid sequence of a CIC-2 mutant as described herein above.

In accordance with the present invention, the term "identical" or "percent identity" in the context of two or more nucleic acid or amino acid sequences, refers to two or more sequences or subsequences that are the same, or that have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 60% or 65% identity, preferably, 70-95% identity, more preferably at least 95% identity), when compared and aligned for maximum correspondence over a window of comparison, or over a designated region as measured using a sequence comparison algorithm as known in the art, or by manual alignment and visual inspection. Sequences having, for example, 60% to 95% or greater sequence identity are considered to be substantially identical. Such a definition also applies to the complement of a test sequence. Preferably the described identity exists over a region that is at least about 15 to 25 amino acids or nucleotides in length, more preferably, over a region that is about 50 to 100 amino acids or nucleotides in length. Those having skill in the art will know how to determine percent identity between/among sequences using, for example, algorithms such as those based on CLUSTALW computer program (Thompson (1994), Nucl. Acids Res. 2, 4673-4680) or FASTDB (Brutlag (1990), Comp. App. Biosci. 6, 237-245), as known in the art. Although the FASTDB algorithm typically does not consider internal non-matching deletions or additions in sequences, i.e., gaps, in its calculation, this can be corrected manually to avoid an overestimation of the % identity. CLUSTALW, however, does take sequence gaps into account in its identity calculations. Also available to those having skill in this art are the BLAST and BLAST 2.0 algorithms (Altschul (1977), Nucl. Acids Res., 25: 33893402). The BLASTN program for nucleic acid sequences uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, and an expectation (E) of 10. The BLOSUM62 scoring matrix (Henikoff (1989), Proc. Natl. Acad. Sci., USA, 89, 10915) uses alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.

Moreover, the present invention also relates to nucleic acid molecules the sequence of which is degenerate in comparison with the sequence of an above-described hybridzing molecule. When used in accordance with the present invention the term "being degenerate as a result of the genetic code" means that due to the redundancy of the genetic code different nucleotide sequences code for the same amino acid.

The nucleic acid molecules according to the invention may be derived from any organism encoding corresponding CIC-2 voltage-gated chloride channels. For example, CIC-2 voltage-gated chloride channels have been reported in various organisms, like in rabbit or guinea pig (see, inter alia, Lam, Nature 396 (1998), 125- 126; Chiu, Molecular Biology and Evolution 16 (1999), 826-838). In a preferred embodiment the nucleic acid molecule of the invention is derived from a vertebrate, preferably from a mammal, even more preferably the nucleic acid molecule is derived from rabbit or guinea pig, and most preferably the nucleic acid is derived from mouse, rat or human.

The nucleic acid molecule according to the invention may be any type of nucleic acid, e.g. DNA, RNA or PNA (peptide nucleic acid). The DNA may, for example, be cDNA. In a preferred embodiment it is a genomic DNA. The RNA may be, e.g., mRNA. The nucleic acid molecule may be natural, synthetic or semisynthetic or it may be a derivative, such as peptide nucleic acid (Nielsen, Science 254 (1991), 1497-1500) or phosphorothioates. Furthermore, the nucleic acid molecule may be a recombinantly produced chimeric nucleic acid molecule comprising any of the aforementioned nucleic acid molecules either alone or in combination.

Preferably, the nucleic acid molecule of the present invention is part of a vector. Therefore, the present invention relates in another embodiment to a vector comprising the nucleic acid molecule of this invention. Such a vector may be, e.g., a plasmid, cosmid, virus, bacteriophage or another vector used e.g. conventionally in genetic engineering, and may comprise further genes such as marker genes which allow for the selection of said vector in a suitable host cell and under suitable conditions.

The nucleic acid molecules of the present invention may be inserted into several commercially available vectors. Nonlimiting examples include plasmid vectors compatible with mammalian cells, such as pUC, pBluescript (Stratagene), pET (Novagen), pREP (Invitrogen), pCRTopo (Invitrogen), pcDNA3 (Invitrogen), pCEP4 (Invitrogen), pMC1 neo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO- pSV2neo, pBPV-1 , pdBPVMMTneo, pRSVgpt, pRSVneo, pSV2-dhfr, pUCTag , plZD35, pLXIN and pSIR (Clontech) and plRES-EGFP (Clontech). Baculovirus vectors such as pBlueBac, BacPacz Baculovirus Expression System (CLONTECH), and MaxBacTM Baculovirus Expression System, insect cells and protocols (Invitrogen) are available commercially and may also be used to produce high yields of biologically active protein, (see also, Miller (1993), Curr. Op. Genet. Dev., 3, 9; O'Reilly, Baculovirus Expression Vectors: A Laboratory Manual, p. 127). In addition, prokaryotic vectors such as pcDNA2; and yeast vectors such as pYes2 are nonlimiting examples of other vectors suitable for use with the present invention. For vector modification techniques, see Sambrook and Russel (2001), loc. cit. Vectors can contain one or more replication and inheritance systems for cloning or expression, one or more markers for selection in the host, e. g. antibiotic resistance, and one or more expression cassettes.

The coding sequences inserted in the vector can be synthesized by standard methods, isolated from natural sources, or prepared as hybrids. Ligation of the coding sequences to transcriptional regulatory elements (e. g., promoters, enhancers, and/or insulators) and/or to other amino acid encoding sequences can be carried out using established methods.

Furthermore, the vectors may, in addition to the nucleic acid sequences of the invention, comprise expression control elements, allowing proper expression of the coding regions in suitable hosts. Such control elements are known to the artisan and may include a promoter, translation initiation codon, translation and insertion site or internal ribosomal entry sites (IRES) (Owens (2001), Proc Natl Acad Sci USA 98,1471-1476) for introducing an insert into the vector. Preferably, the nucleic acid molecule of the invention is operatively linked to said expression control sequences allowing expression in eukarvotic or prokaryotic cells. Particularly preferred are in this context control sequences which allow for correct expression in neuronal cells and/or cells derived from nervous tissue.

Control elements ensuring expression in eukaryotic and prokaryotic cells are well known to those skilled in the art. As mentioned above, they usually comprise regulatory sequences ensuring initiation of transcription and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptional as well as translational enhancers, and/or naturally-associated or heterologous promoter regions. Possible regulatory elements permitting expression in for example mammalian host cells comprise the CMV- HSV thymidine kinase promoter, SV40, RSV-promoter (Rous sarcome virus), human elongation factor 1 -promoter, CMV enhancer, CaM-kinase promoter or SV40-enhancer.

For the expression for example in nervous tissue and/or cells derived therefrom, several regulatory sequences are well known in the art, like the minimal promoter sequence of human neurofilament L (Charron, J. Biol. Chem 270 (1995), 25739- 25745). For the expression in prokaryotic cells, a multitude of promoters including, for example, the tac-lac-promoter, the lacUVδ or the trp promoter, has been described. Beside elements which are responsible for the initiation of transcription such regulatory elements may also comprise transcription termination signals, such as SV40-poIy-A site or the tk-poly-A site, downstream of the polynucleotide. In this context, suitable expression vectors are known in the art such as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pRc/CMV, pcDNAI , pcDNA3 (In- Vitrogene, as used, inter alia in the appended examples), pSPORTI (GIBCO BRL) or pGEMHE (Promega), or prokaryotic expression vectors, such as lambda gt11. An expression vector according to this invention is at least capable of directing the replication, and preferably the expression, of the nucleic acids and protein of this invention. Suitable origins of replication include, for example, the Col E1 , the SV40 viral and the M 13 origins of replication. Suitable promoters include, for example, the cytomegalovirus (CMV) promoter, the lacZ promoter, the gal 10 promoter and the Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) polyhedral promoter. Suitable termination sequences include, for example, the bovine growth hormone, SV40, lacZ and AcMNPV polyhedral polyadenylation signals. Examples of selectable markers include neomycin, ampicillin, and hygromycin resistance and the like. Specifically-designed vectors allow the shuttling of DNA between different host cells, such as bacteria-yeast, or bacteria-animal cells, or bacteria-fungal cells, or bacteria invertebrate cells.

Beside the nucleic acid molecules of the present invention, the vector may further comprise nucleic acid sequences encoding for secretion signals. Such sequences are well known to the person skilled in the art. Furthermore, depending on the expression system used leader sequences capable of directing the expressed polypeptide to a cellular compartment may be added to the coding sequence of the nucleic acid molecules of the invention and are well known in the art. The leader sequence(s) is (are) assembled in appropriate phase with translation, initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein, or a part thereof, into, inter alia, the extracellular membrane. Optionally, the heterologous sequence can encode a fusion protein including an C- or N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and, as desired, the collection and purification of the proteins, antigenic fragments or fusion proteins of the invention may follow. Of course, the vector can also comprise regulatory regions from pathogenic organisms. Furthermore, said vector may also be, besides an expression vector, a gene transfer and/or gene targeting vector. Gene therapy, which is based on introducing therapeutic genes (for example for vaccination) into cells by ex-vivo or in-vivo techniques is one of the most important applications of gene transfer. Suitable vectors, vector systems and methods for in-vitro or in-vivo gene therapy are described in the literature and are known to the person skilled in the art; see, e.g., Giordano, Nature Medicine 2 (1996), 534-539; Schaper, Circ. Res. 79 (1996), 911- 919; Anderson, Science 256 (1992), 808-813, Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ. Res. 77 (1995), 1077-1086; Wang, Nature Medicine 2 (1996), 714-716; WO 94/29469; WO 97/00957, Schaper, Current Opinion in Biotechnology 7 (1996), 635-640 or Verma, Nature 389 (1997), 239-242 and references cited therein.

The nucleic acid molecules of the invention and vectors as described herein above may be designed for direct introduction or for introduction via liposomes, or viral vectors (e.g. adenoviral, retroviral) into the cell. Additionally, baculoviral systems or systems based on vaccinia virus or Semliki Forest Virus can be used as eukaryotic expression system for the nucleic acid molecules of the invention. In addition to recombinant production, fragments of the protein, the fusion protein or antigenic fragments of the invention may be produced by direct peptide synthesis using solid- phase techniques (cf Stewart et al. (1969) Solid Phase Peptide Synthesis, WH Freeman Co, San Francisco; Merrifield, J. Am. Chem. Soc. 85 (1963), 2149-2154). In vitro protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved, for example, using Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer, Foster City CA) in accordance with the instructions provided by the manufacturer. Various fragments may be chemically synthesized separately and combined using chemical methods to produce the full length molecule.

The present invention in addition relates to a host transformed with a vector of the present invention or to a host comprising the nucleic acid molecule of the invention. Said host may be produced by introducing said vector or nucleotide sequence into a host cell which upon its presence in the cell mediates the expression of a protein encoded by the nucleotide sequence of the invention or comprising a nucleotide sequence or a vector according to the invention wherein the nucleotide sequence and/or the encoded polypeptide is foreign to the host cell.

By "foreign" it is meant that the nucleotide sequence and/or the encoded polypeptide is either heterologous with respect to the host, this means derived from a cell or organism with a different genomic background, or is homologous with respect to the host but located in a different genomic environment than the naturally occurring counterpart of said nucleotide sequence. This means that, if the nucleotide sequence is homologous with respect to the host, it is not located in its natural location in the genome of said host, in particular it is surrounded by different genes. In this case the nucleotide sequence may be either under the control of its own promoter or under the control of a heterologous promoter. The location of the introduced nucleic acid molecule or the vector can be determined by the skilled person by using methods well-known to the person skilled in the art, e.g., Southern Blotting. The vector or nucleotide sequence according to the invention which is present in the host may either be integrated into the genome of the host or it may be maintained in some form extrachromosomally. In this respect, it is also to be understood that the nucleotide sequence of the invention can be used to restore or create a mutant gene via homologous recombination.

Said host may be any prokaryotic or eukaryotic cell. Suitable prokaryotic/bacterial cells are those generally used for cloning like E. coli, Salmonella typhimurium, Serratia marcescens or Bacillus subtilis. Said eukaryotic host may be a mammalian cell, an amphibian cell, a fish cell, an insect cell, a fungal cell, a plant cell or a bacterial cell (e.g., E coli strains HB101, DH5a, XL1 Blue, Y1090 and JM101), Eukaryotic recombinant host cells are preferred. Examples of eukaryotic host cells include, but are not limited to, yeast, e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis or Pichia pastoris cells, cell lines of human, bovine, porcine, monkey, and rodent origin, as well as insect cells, including but not limited to, Spodoptera frugiperda insect cells and Drosophila- derived insect cells as well as zebra fish cells. Mammalian species-derived cell lines suitable for use and commercially available include, but are not limited to, L cells, CV-1 cells, COS-1 cells (ATCC CRL 1650), COS-7 cells (ATCC CRL 1651), HeLa cells (ATCC CCL 2), C1271 (ATCC CRL 1616), BS-C-1 (ATCC CCL 26) and MRC- 5 (ATCC CCL 171).

In a particularly preferred embodiment said mammalian cell is a neuronal cell and/or a cultured cell like, inter alia, a HEK 293 (human embryonic kidney) cell, a CHO, HeLa, NIH3T3, BHK, PC12 cell or a neuronal stem cell preferably derived from a mammal and more preferably from a human.

In another more preferred embodiment said amphibian cell is an oocyte. In an even more preferred embodiment said oocyte is a frog oocyte, particularly preferred a Xenopus laevis oocyte.

In a more preferred embodiment, the host according to the invention is a non- human transgenic organism. Said non-human organism may be a mammal, amphibian, a fish, an insect, a fungus or a plant. Particularly preferred non-human transgenic animals are Drosophila species, Caenorhabditis elegans, Xenopus species, zebra fish, Spodoptera frugiperda, Autographa califomica, mice and rats. Transgenic plants comprise, but are not limited to, wheat, tobacco, parsley and Arabidopsis. Transgenic fungi are also well known in the art and comprise, inter alia, yeasts, like S. pombe or S. cerevisae, or Aspergillus, Neurospora or Ustilago species.

In another embodiment, the present invention relates to a method for producing the polypeptide encoded by a nucleic acid molecule of the invention comprising culturing/raising the host of the invention and isolating the produced polypeptide.

A large number of suitable methods exist in the art to produce polypeptides in appropriate hosts. If the host is a unicellular organism or a mammalian or insect cell, the person skilled in the art can revert to a variety of culture conditions that can be further optimized without an undue burden of work. Conveniently, the produced protein is harvested from the culture medium or from isolated (biological) membranes by established techniques. Furthermore, the produced polypeptide may be directly isolated from the host cell. Said host cell may be part of or derived from a part of a host organism, for example said host cell may be part of the CNS of an animal or the harvestable part of a plant. Additionally, the produced polypeptide may be isolated from fluids derived from said host, like blood, milk or cerebrospinal fluid.

Additionally the present invention relates to a polypeptide that is encoded by a nucleic acid molecule of the invention or produced by the method of the invention. The polypeptide of the invention may accordingly be produced by microbiological methods or by transgenic mammals. It is also envisaged that the polypeptide of the invention is recovered from transgenic plants. Alternatively, the polypeptide of the invention may be produced synthetically or semi-synthetically.

For example, chemical synthesis, such as the solid phase procedure described by Houghton (1985), Proc. Natl. Acad. Sci. USA (82), 5131-5135, can be used. Another method is in vitro translation of mRNA. A preferred method involves the recombinant production of protein in host cells as described above. For example, nucleotide acid sequences comprising all or a portion of any one of the nucleotide sequences according to the invention can be synthesized by PCR, inserted into an expression vector, and a host cell transformed with the expression vector. Thereafter, the host cell is cultured to produce the desired polypeptide, which is isolated and purified. Protein isolation and purification can be achieved by any one of several known techniques; for example and without limitation, ion exchange chromatography, gel filtration chromatography and affinity chromatography, high pressure liquid chromatography (HPLC), reversed phase HPLC, preparative disc gel electrophoresis. In addition, cell-free translation systems can be used to produce the polypeptides of the present invention. Suitable cell-free expression systems for use in accordance with the present invention include rabbit reticulocyte lysate, wheat germ extract, canine pancreatic microsomal membranes, E. coli S30 extract, and coupled transcription/translation systems such as the TNT-system (Promega). These systems allow the expression of recombinant polypeptides or peptides upon the addition of cloning vectors, DNA fragments, or RNA sequences containing coding regions and appropriate promoter elements. As mentioned supra, protein isolation/purification techniques may require modification of the proteins of the present invention using conventional methods. For example, a histidine tag can be added to the protein to allow purification on a nickel column. Other modifications may cause higher or lower activity, permit higher levels of protein production, or simplify purification of the protein.

In a further embodiment, the present invention relates to an antibody specifically directed to a polypeptide of the invention, wherein said antibody specifically reacts with an epitope generated and/or formed by the mutation in the voltage-gated chloride channel CIC-2 selected from the group consisting of:

(i) an epitope presented by a polypeptide which has an amino acid sequence of a voltage-gated chloride channel CIC-2, wherein the glycine (Gly) residue corresponding to position 715 of the wild-type voltage-gated chloride channel

CIC-2 as depicted in SEQ ID NO: 2 is replaced by another amino acid residue;

(ii) an epitope presented by a polypeptide which has an amino acid sequence of a voltage-gated chloride channel CIC-2, wherein amino acids corresponding to positions 74 to 117 of the wild-type voltage-gated chloride channel CIC-2 as depicted in SEQ ID NO: 2 are deleted; and

(iii) an epitope presented by a polypeptide which has an amino acid sequence of a voltage-gated chloride channel CIC-2, wherein the wild-type translational reading frame of the voltage-gated chloride channel CIC-2 is altered due to an insertion between the nucleotides corresponding to position 596 and position 597 of the corresponding wild type nucleotide sequence as depicted in SEQ ID NO: 1.

With respect to preferred embodiments of (i) to (iii) the same applies as described above in connection with the nucleic acid molecules. The term "specifically" in this context means that the antibody reacts with the mutant CIC-2 protein but not with a wild-type CIC-2 protein. Preferably this term also means that such an antibody does not bind to other mutant forms of the CIC-2 protein, in particular those described herein. Whether the antibody specifically reacts as defined herein above can easily be tested, inter alia, by comparing the reaction of said antibody with a wild-type voltage-gated chloride channel CIC-2 (or a subunit or a fragment thereof) with the reaction of said antibody with a mutant CIC-2 polypeptide of the invention. The antibody of the present invention can be, for example, polyclonal or monoclonal. The term "antibody" also comprises derivatives or fragments thereof which still retain the binding specificity. Techniques for the production of antibodies are well known in the art and described, e.g. in Harlow and Lane "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988. These antibodies can be used, for example, for the immunoprecipitation and immunolocalization of the polypeptides of the invention as well as for the monitoring of the presence of such polypeptides, for example, in recombinant organisms or in diagnosis. They can also be used for the identification of compounds interacting with the proteins according to the invention (as mentioned herein below). For example, surface plasmon resonance as employed in the BIAcore system can be used to increase the efficiency of phage antibodies which bind to an epitope of the polypeptide of the invention (Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13).

The present invention furthermore includes chimeric, single chain and humanized antibodies, as well as antibody fragments, like, inter alia, Fab fragments. Antibody fragments or derivatives further comprise F(ab')₂, Fv or scFv fragments; see, for example, Harlow and Lane, loc. cit.. Various procedures are known in the art and may be used for the production of such antibodies and/or fragments. Thus, the (antibody) derivatives can be produced by peptidomimetics. Further, techniques described for the production of single chain antibodies (see, inter alia, US Patent 4,946,778) can be adapted to produce single chain antibodies to polypeptide(s) of this invention. Also, transgenic animals may be used to express humanized antibodies to polypeptides of this invention. Most preferably, the antibody of this invention is a monoclonal antibody. For the preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples for such techniques include the hybridoma technique (Kδhler and Milstein (1975), Nature 256, 495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor, Immunology Today 4 (1983), 72) and the EBV- hybridoma technique to produce human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985), 77-96). Techniques describing the production of single chain antibodies (e.g., U.S. Patent 4,946,778) can be adapted to produce single chain antibodies to immunogenic polypeptides as described above. Furthermore, transgenic mice may be used to express humanized antibodies directed against said immunogenic polypeptides. It is in particular preferred that the antibodies/antibody constructs as well as antibody fragments or derivatives to be employed in accordance with this invention or capable to be expressed in a cell. This may, inter alia, be achieved by direct injection of the corresponding proteineous molecules or by injection of nucleic acid molecules encoding the same. Furthermore, gene therapy approaches are envisaged. Accordingly, in context of the present invention, the term "antibody molecule" relates to full immunoglobulin molecules as well as to parts of such immunoglobulin molecules. Furthermore, the term relates, as discussed above, to modified and/or altered antibody molecules, like chimeric and humanized antibodies. The term also relates to monoclonal or polyclonal antibodies as well as to recombinantly or synthetically generated/synthesized antibodies. The term also relates to intact antibodies as well as to antibody fragments thereof, like, separated light and heavy chains, Fab, Fab/c, Fv, Fab', F(ab')₂. The term "antibody molecule" also comprises bifunctional antibodies and antibody constructs, like single chain Fvs (scFv) or antibody-fusion proteins. It is also envisaged in context of this invention that the term "antibody" comprises antibody constructs which may be expressed in cells, e.g. antibody constructs which may be transfected and/or transduced via, inter alia, viruses or vectors. It is in particular envisaged that such antibody constructs specifically recognize the polypeptides of the present invention. It is, furthermore, envisaged that said antibody construct is employed in gene therapy approaches.

The present invention relates also to an aptamer specifically binding to a polypeptide according to the invention wherein said aptamer reacts with an epitope of a polypeptide of the present invention as well as to an aptamer specifically directed to a corresponding nucleic acid molecule according to the invention. In accordance with the present invention, the term "aptamer" means nucleic acid molecules that can bind to target molecules. Aptamers commonly comprise RNA, single stranded DNA, modified RNA or modified DNA molecules. The preparation of aptamers is well known in the art and may involve, inter alia, the use of combinatorial RNA libraries to identify binding sides (Gold, Ann. Rev. Biochem 64 (1995), 763-797). Furthermore, the present invention relates to a primer or pair of primers capable of specifically amplifying the nucleic acid molecules of the present invention. The term "primer" when used in the present invention means a single-stranded nucleic acid molecule capable of annealing the nucleic acid molecule of the present application and thereby being capable of serving as a starting point for amplification. Said term also comprises oligoribo- or desoxyribonucleotides which are complementary to a region of one of the strands of a nucleic acid molecule of the present invention. According to the present invention the term "pair of primers" means a pair of primers that are with respect to a complementary region of a nucleic acid molecule directed in the opposite direction towards each other to enable, for example, amplification by polymerase chain reaction (PCR).

The term "amplifying" refers to repeated copying of a specified sequence of nucleotides resulting in an increase in the amount of said specified sequence of nucleotides.

When used in the present invention the term "specifically" means that only the nucleic acid molecules as described herein above are amplified and nucleic acid molecules encoding the wild-type CIC-2 voltage-gated receptor as depicted in SEQ ID NO: 1 are not amplified. Thus, a primer according to the invention is preferably a primer which binds to a region of a nucleic acid molecule of the invention which is unique for this molecule and which is not present in the wild-type CIC-2 encoding sequence, i.e. the primer binds in a region in which one of the above described mutations occur. In connection with a pair of primers according to the invention it is possible that one of the primers of the pair is specific in the above described meaning or both of the primers of the pair are specific. In both cases, the use of such a pair of primers would allow to specifically amplify a mutant of the invention as described herein-above but not the wild-type CIC-2 encoding sequence. The 3'-OH end of a primer is used by a polymerase to be extended by successive incorporation of nucleotides. The primer or pair of primers of the present invention can be used, for example, in primer extension experiments on template RNA according to methods known by the person skilled in the art. Preferably, the primer or pair of primers of the present invention are used for amplification reactions on template RNA or template DNA, preferably cDNA or genomic DNA. The terms "template DNA" or "template RNA" refers to DNA or RNA molecules or fragments thereof of any source or nucleotide composition, that comprise a target nucleotide sequence as defined above. The primer or pair of primers can also be used for hybridization experiments as known in the art. Preferably, the primer or pair of primers are used in polymerase chain reactions to amplify sequences corresponding to a sequence of the nucleic acid molecule of the present invention. It is known that the length of a primer results from different parameters (Gillam (1979), Gene 8, 81-97; Innis (1990), PCR Protocols: A guide to methods and applications, Academic Press, San Diego, USA). Preferably, the primer should only hybridize or bind to a specific region of a target nucleotide sequence. The length of a primer that statistically hybridizes only to one region of a target nucleotide sequence can be calculated by the following formula: (%) ^x (whereby x is the length of the primer). For example a hepta- or octanucleotide would be sufficient to bind statistically only once on a sequence of 37 kb. However, it is known that a primer exactly matching to a complementary template strand must be at least 9 base pairs in length, otherwise no stable-double strand can be generated (Goulian (1973), Biochemistry 12, 2893-2901). It is also envisaged that computer-based algorithms can be used to design primers capable of amplifying the nucleic acid molecules of the invention. Preferably, the primers of the invention are at least 10 nucleotides in length, more preferred at least 12 nucleotides in length, even more preferred at least 15 nucleotides in length, particularly preferred at least 18 nucleotides in length, even more particularly preferred at least 20 nucleotides in length and most preferably at least 25 nucleotides in length. The invention, however, can also be carried out with primers which are shorter or longer.

It is also envisaged that the primer or pair of primers is labeled. The label may, for example, be a radioactive label, such as ³²P, ³³P or ³⁵S. In a preferred embodiment of the invention, the label is a non-radioactive label, for example, digoxigenin, biotin and fluorescence dye or a dye.

In another preferred embodiment said primers are selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21 In yet another embodiment, the present invention relates to a composition comprising a nucleic acid molecule, a vector, a polypeptide, an antibody, an aptamer and/or a primer or pair of primers of the invention.

The term "composition", as used in accordance with the present invention, comprises at least one nucleic acid molecule, vector, polypeptide, an antibody and/or primer or pair of primers of this invention. It may, optionally, further molecules capable of altering the characteristics of the polypeptides of the invention or specifically interacting with the polypeptides of the invention thereby, for example, suppressing, blocking, modulating and/or activating their function which have neuroprotective, nootropic and/or antiepileptic properties. The composition may be in solid, liquid or gaseous form and may be, inter alia, in the form of (a) powder(s), (a) tablet(s), (a) solution(s) or (an) aerosol(s).

In a preferred embodiment the composition according to the invention is a diagnostic composition, optionally further comprising suitable means for detection. As described above, the present invention is based on the surprising finding that certain types of mutations in the CIC-2 protein are connected with IGE. Thus, the knowledge of these mutations now allows to diagnose IGE in an easy way. The diagnostic composition comprises at least one of the aforementioned compounds of the invention. The diagnostic composition may be used, inter alia, for methods for determining the presence and/or expression of the nucleic acids and/or polypeptides of the invention. This may be effected by detecting, e.g., the presence of a corresponding gene in the genetic material of an individual or the presence of the corresponding mRNA which comprises isolation of DNA or RNA from a cell derived from said individual, contacting the DNA or RNA so obtained with a nucleic acid probe as described above under hybridizing conditions, and detecting the presence of .mRNAs hybridized to the probe. Alternatively, the diagnostic composition may also be used for detecting the presence of a nucleic acid molecule of the invention by PCR. Furthermore, polypeptides of the invention can be detected with methods known in the art, which comprise, inter alia, immunological methods, like, RIA, FIA, ELISA, FACS or Western blotting.

Furthermore, the diagnostic composition of the invention may be useful, inter alia, in detecting the prevalence, the onset or the progress of a disease related to the expression of a polypeptide of the invention. Accordingly, the diagnostic composition of the invention may be used, inter alia, for assessing the prevalence, the onset and/or the disease status of neurological, neurodegenerative and/or neuro- psychiatric disorders, as defined herein above. It is also contemplated that the diagnostic composition of the invention may be useful in discriminating (the) stage(s) of a disease.

The diagnostic composition optionally comprises suitable means for detection. The nucleic acid molecule(s), vector(s), host(s), antibody(ies), aptamer(s), polypeptide(s) described above are, for example, suitable for use in immunoassays in which they can be utilized in liquid phase or bound to a solid phase carrier. Examples of well- known carriers include glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amyloses, natural and modified celluloses, polyacrylamides, agaroses, and magnetite. The nature of the carrier can be either soluble or insoluble for the purposes of the invention. Solid phase carriers are known to those in the art and may comprise polystyrene beads, latex beads, magnetic beads, colloid metal particles, glass and/or silicon chips and surfaces, nitrocellulose strips, membranes, sheets, duracytes and the walls of wells of a reaction tray, plastic tubes or other test tubes. Suitable methods of immobilizing nucleic acid molecule(s), vector(s), host(s), antibody(ies), aptamer(s), polypeptide(s), etc. on solid phases include but are not limited to ionic, hydrophobic, covalent interactions or (chemical) crosslinking and the like. Examples of immunoassays which can utilize said compounds of the invention are competitive and non-competitive immunoassays in either a direct or indirect format. Commonly used detection assays can comprise radioisotopic or non-radioisotopic methods. Examples of such immunoassays are the radioimmunoassay (RIA), the sandwich (immunometric assay) and the Northern or Southern blot assay. Furthermore, these detection methods comprise, inter alia, IRMA (Immune Radioimmunometric Assay), EIA (Enzyme Immuno Assay), ELISA (Enzyme Linked Immuno Assay), FIA (Fluorescent Immuno Assay), and CLIA (Chemioluminescent Immune Assay). Furthermore, the diagnostic compounds of the present invention may be are employed in techniques like FRET (Fluorescence Resonance Energy Transfer) assays. Appropriate labels and methods for labeling are known to those of ordinary skill in the art. Examples of the types of labels which can be used in the present invention include inter alia, fluorochromes (like fluorescein, rhodamine, Texas Red, etc.), enzymes (like horse radish peroxidase, β-galactosidase, alkaline phosphatase), radioactive isotopes (like ³²P, ³³P, ³⁵S or ¹²⁵l), biotin, digoxygenin, colloidal metals, chemi- or bioluminescent compounds (like dioxetanes, luminol or acridiniums). A variety of techniques are available for labeling biomolecules, are well known to the person skilled in the art and are considered to be within the scope of the present invention and comprise, inter alia, covalent coupling of enzymes or biotinyl groups, phosphorylations, biotinylations, random priming, nick-translations, tailing (using terminal transferases). Such techniques are, e.g., described in Tijssen, "Practice and theory of enzyme immuno assays", Burden and von Knippenburg (Eds), Volume 15 (1985); "Basic methods in molecular biology", Davis LG, Dibmer MD, Battey Elsevier (1990); Mayer, (Eds) "Immunochemical methods in cell and molecular biology" Academic Press, London (1987); or in the series "Methods in Enzymology", Academic Press, Inc.

Detection methods comprise, but are not limited to, autoradiography, fluorescence microscopy, direct and indirect enzymatic reactions, etc.

Said diagnostic composition may be used for methods for detecting the presence and/or abundance of a nucleic acid molecule of the invention in a biological and/or medical sample and/or for detecting expression of such a nucleic acid molecule (e.g. by determining the mRNA or the expressed polypeptide). Furthermore, said diagnostic composition may also be used in methods of the present invention, inter alia, for the detection of specific antagonists or agonists for CIC-2 voltage-gated chloride channels (see herein below).

In a preferred embodiment the present invention relates to diagnostic composition designed for use in a method in which the occurrence of the mutation in the voltage- gated chloride channel CIC-2 gene is determined by PCR, immunological methods and/or electrophysiological methods as described herein below and in the Examples. Additionally, it is possible to determine the occurrence of a mutation in the voltage-gated chloride channel CIC-2 by measuring the chloride efflux of cells in low osmotic solutions. Cells harbouring a mutation in the CLCN2 gene encoding the CIC-2 protein show an altered chlorid efflux in comparison to cells harbouring a wild-type CIC-2 protein (Jentsch (2002), loc. cit.).

In yet another aspect the present invention relates to the use of a nucleic acid molecule, a vector, a polypeptide, an antibody, aptamer and/or a primer or pair of primers of the present invention for the preparation of a diagnostic composition for the detection of a neurological disease/disorder.

In another embodiment the present invention relates to a method of diagnosing a neurological disease or a susceptibility to a neurological disease comprising the step of determining in a sample obtained from an individual whether the CIC-2 protein expressed in the cells of said individual is non-functional or shows an altered voltage-dependent gating in comparison to the wild-type CIC-2 protein. "Nonfunctional" means that the CIC-2 protein has lost at least one functional property displayed by the wild-type CIC-2 protein as described herein above. Preferably, "non-functional" means that the CIC-2 protein does no longer function as a channel. Non-functionality may, e.g., be caused by the fact that one allele occurring in an individual codes for a CIC-2 protein which leads to non-functional dimers (dominant negative mutation). Whether a CIC-2 protein in an individual is functional or nonfunctional can be determined as described herein above and in the examples. The term "altered voltage-dependent gating" means that the respective CIC-2 protein reacts in a different way to voltage than the wild-type CIC-2 protein. This can be determined as described in the examples. Preferably, the CIC-2 protein showing an altered voltage-dependent gating shows properties which result in membrane depolarization and/or hyperexcitability. This can be determined as described in Example 5.

The present invention also relates to a method of diagnosing a neurological disease or susceptibility to a neurological disease comprising the step of determining in a sample obtained from an individual whether the voltage-gated chloride channel CIC- 2 protein or gene shows a mutation selected from the group consisting of: (a) a replacement of the glycine (Gly) residue corresponding to position 715 of the wild-type voltage-gated chloride channel CIC-2 as depicted in SEQ ID NO: 2 by another residue;

(b) a deletion of at least amino acids corresponding to positions 74 to 117 of the wild-type voltage-gated chloride channel CIC-2 as depicted in SEQ ID NO: 2;

(c) a genomic nucleotide sequence encoding a voltage-gated chloride channel CIC-2 and which contains a mutation in intron 2 which leads to an aberrant splicing of the mRNA transcribed by said genomic nucleotide sequence resulting in a fusion of exons 2 and 4 thereby leading to the production of an mRNA lacking exon 3; and

(d) an insertion between the nucleotides corresponding to position 596 and position 597 of the corresponding wild-type nucleotide sequence as depicted in SEQ ID NO: 1 leading to an alteration of the wild-type translational reading frame of the voltage-gated chloride channel CIC-2.

With respect to the preferred embodiments of the mutations (a) to (d) the same applies as already described above in connection with the nucleic acid molecules according to the invention.

In accordance with the present invention by the term "sample" is intended any biological sample obtained from an individual, cell line, tissue culture, or other source containing polynucleotides or polypeptides or portions thereof. As indicated, biological samples include body fluids (such as blood, sera, plasma, urine, synovial fluid and spinal fluid) and tissue sources found to express the polynucleotides of the present invention. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. A biological sample which includes genomic DNA, mRNA or proteins is preferred as a source.

As described herein above, mutations of the CIC-2 encoding gene CLCN2 can occur on DNA level or on mRNA level and result in CIC-2 voltage-gated chloride channels which show either an altered function or no function when compared to the wild-type CIC-2 voltage-gated chloride channel as described herein. Thus, various methods on DNA level, RNA level or protein level exist for determining whether the voltage-gated chloride channel CIC-2 shows a mutation as described herein above. Consequently, mRNA, cDNA, DNA and genomic DNA are the preferred nucleic acid molecules to be used in the below mentioned methods. Also polypeptides or fragments thereof are preferred if a mutation in the CIC-2 voltage- gated chloride channel as described herein is to be determined.

Preferably, a point mutation leading to the replacement of the glycine (Gly) residue at position 715 of the corresponding wild-type CIC-2 amino acid sequence depicted in SEQ ID NO: 2 by another amino acid can be determined by PCR. Said PCR is followed by a restriction fragment length polymorphism (RFLP) analysis if due to the point mutation a recognition site for a restriction endonuclease is generated which is not present in the wild-type nucleotide sequence or a recognition site for a restriction enzyme is created which does not occur in the wild-type CLCN2. It is also preferred that a recognition site for a restriction endonuclease is lost due to a point mutation in the wild-type CLCN-2 nucleic acid sequence depicted in SEQ ID NO: 1. Accordingly, the primers depicted in SEQ ID NOs: 16 and 17, respectively are used to amplify a fragment comprising at least the nucleotide residues encoding the amino acid residue corresponding to position 715 of SEQ ID NO: 1. The temperature for annealing the primers to the template nucleotide sequence is preferably at least 62°C for preferably 30 sec, the temperature for denaturation is preferably at least 95°C for preferably 30 sec and the temperature for extension of the annealed primers is preferably at least 72°C for preferably 60 sec. The cycle of denaturation, annealing and extension is preferably carried out for at least 35 times. As is shown in the appended Example infra, the amplification results in a 222 bp fragment irrespective whether the point mutation is present or not. Said 222 bp fragment is preferably subject to treatment with preferably the restriction endonuclease Ital which recognizes the nucleotide sequence GCNGC, wherein N is any nucleotide (G, A, T or C). However, any restriction endonuclease which recognizes said sequence may be used. Thereby, the 222 bp fragment comprising the wild-type CLCN-2 nucleotide sequence is cleaved in a 117 bp, 88 bp and 17 bp fragment, whereas the 222 bp fragment comprising at least the nucleotide residues encoding the amino acid residue corresponding to position 715 of SEQ ID NO: 1 is cleaved in 117 bp and 105 bp fragment. To visualize the restriction fragments, preferably gel electrophoresis on a 10% polyacrylamidegel is performed. More preferably said mutation can be determined by PCR using primers and conditions that allow only an amplification of the wild-type nucleotide sequence encoding a glycine at position 715, but not of the nucleotide sequence of a nucleic acid molecule encoding a different amino acid residue at the corresponding position. Such a method has been used, for example, in Loubradou (2001), Mol. Microbiol. 40, 719-730, to demonstrate a point mutation in a fungal resistance gene. It is even more preferred that PCR is performed to determine a mutation using primers and conditions that allow no amplification if the wild-type nucleotide sequence encoding Gly 715 is present, but only if another amino acid residue is encoded at position 715. Particularly preferred is a method using PCR and primers under conditions that allow amplification of a fragment comprising at least the nucleotide residues encoding the amino acid residue corresponding to position 715 of SEQ ID NO:1. Said PCR is followed by e.g., sequencing and/or single strand conformation analysis (SSCA). Said fragment is preferably of at least 25 nucleotides in length, more preferred of at least 50 nucleotide in length, even more preferred of at least 75 nucleotides in length, particularly preferred of at least 100 nucleotides in length and more particularly preferred of at least 200 nucleotides in length and most preferred of at least 250 nucleotides in length. Said primers are preferably of at least 12 nucleotides in length, more preferred of at least 15 nucleotides in length, even more preferred of at least 18 nucleotides in length and most preferred of at least 21 nucleotides in length as depicted in SEQ ID NO: 10 and SEQ ID NO: 11. The temperature for annealing said primers is preferably at least 50°C, more preferred at least 55°C and most preferred at least 58°C. The temperature for denaturation is preferably at least 95°C for preferably at least 10 sec, more preferably at least 20 sec, even more preferred at least 30 sec and most preferred at least 60 sec. However, depending on the length and the G-C content of the nucleic acid sequence to be amplified the temperature for denaturation may be shorter or longer. The temperature for extension of the annealed primers is preferably at least 10 sec, more preferably at least 20 sec, even more preferred at least 30 sec and most preferred at least 60 sec. A PCR reaction comprising the aforementioned conditions is exemplified in the Examples herein below. The subsequent sequencing and/or SSCA is carried out as known in the art. Preferably, the PCR fragments are separated on a 10% polyacrylamide gel at 4°C or also preferred at room temperature. PCR fragments showing a SSCA band shift are amplified with the primers under conditions as mentioned above and are subsequently sequenced. Alternatively, it is also possible to directly sequence genomic DNA in order to determine whether a mutation in the CLCN2 gene has occurred. A direct genomic sequencing approach is, for example, demonstrated for baker's yeast in Horecka (2000), Yeast 16, 967-970.

Preferably, a deletion is determined by using hybridization techniques as known in the art. In particular, a primer is designed as mentioned herein above that is capable to only hybridize to wild-type genomic DNA as depicted in SEQ ID NO: 9 but not to a nucleotide sequence comprising a deletion of a fragment between nucleotides 2653 and 2663 of SEQ ID NO:9. Also preferred is the method of fluorescent in situ hybridization (FISH) for determining on whole chromosomes, in particular on chromosome 3q26 that said chromosome has the above mentioned deletion. Even more preferred is that a deletion of nucleotide residues as described herein may be determined by using PCR, wherein one primer of a pair of primers is located within the region of genomic DNA comprising said deletion. Preferably, said deletion is between nucleotide positions 2653 and 2663 as depicted in SEQ ID NO: 9. Thus, under the appropriate conditions no PCR fragment will result if the genomic DNA comprises said deletion. It is particularly preferred that PCR using primers which are located upstream or downstream of the deletion is performed to determine said deletion. Under appropriate conditions as mentioned herein above, both a fragment of genomic DNA of the wild-type nucleotide sequence as set forth in SEQ ID NO: 1 and a fragment of the nucleotide sequence comprising a deletion of preferably the nucleotides between positions 2653 and 2663 as depicted in SEQ ID NO: 9 will be amplified. However, by performing gel electrophoresis it can be evaluated that the fragment comprising the deletion will be shorter than the corresponding fragment of the wild-type sequence. As is shown in the appended Examples infra, the primers depicted in SEQ ID NO: 18 and 19, respectively, are used for the aforementioned method. In particular, using the primers depicted in SEQ ID NO: 18 and 19, respectively, results in a 250 bp fragment if genomic DNA of the wild-type nucleotide sequence as set forth in SEQ ID NO: 1is the template. On the other hand, if the nucleotide sequence comprising a deletion of preferably the nucleotides between positions 2653 and 2663 as depicted in SEQ ID NO: 9 is used as template, said primers amplify a fragment of 239 bp. The PCR is carried out by preferably at least 35 times denaturing the template nucleic acid as described above at a temperature of preferably at least 95°C for preferably at least 30 sec, annealing the primers at a temperature of preferably at least 62°C for preferably 30 sec and extending said primers at a temperature of preferably at least 72°C for preferably at least 60 sec. Said PCR is followed by a treatment preferably with the restriction endonuclease Bsgl which recognizes the nucleotide sequences

GTGCAGNNNNNNNNNNNNNNNN. However, any restriction endonuclease which recognizes said sequence may be used. Due to the deletion of preferably the nucleotides between positions 2653 and 2663 as depicted in SEQ ID NO: 9 the muatant is not cleaved since the nucleotide sequence comprising the Bsgl recognition site is absent. The wild-type sequence, however, is cleaved which results in a 212 bp and a 38 bp restriction fragment. The resulting restriction fragments are preferably separated on a 3% agarose gel. Although said deletion was shown to occur in intron 2 of the nucleotide sequence depicted in SEQ ID NO: 9 it may also be possible to determine said deletion on unspliced mRNA or its corresponding cDNA. In an alternative approach a PCR method can be applied using spliced • mRNA or the corresponding cDNA as template. In particular, as described herein and shown in the Examples herein below said deletion occurs in intron 2 of the nucleotide sequence as depicted in SEQ ID NO: 9 and leads to aberrant splicing. As a consequence exons 2 and 4 are fused whereby exon 3 is skipped. Thus, it is possible to determine on the level of spliced mRNA or the corresponding cDNA whether exon 3 is present or missing. To this end, it is e.g. possible to design a first primer which is capable of binding to exon 2 preferably in close proximity to the 3'-end of exon 2 and a second primer which is capable of binding to exon 4 preferably in close proximity to the 5'-end of said exon 4. Under appropriate conditions a larger PCR fragment will result if spliced mRNA or cDNA (comprising a wild-type exon1-exon2-exon3-exon4-exon5-24 arranged structure as depicted in SEQ ID NO: 1) not resulting from an aberrant splicing event is used as a template since the mRNA or cDNA (comprising an exon1-exon2-exon4-exon5-24 arranged structure as depicted in SEQ ID NO: 5) resulting from an aberrant splicing event does not comprise exon 3. It is also possible that an oligonucleotide is designed which is only capable to hybridize to mRNA or corresponding cDNA resulting from an aberrant splicing. Such an oligonucleotide may be designed so as to be able to bind to a short region in the 3' end of exon 2 and to a short region in the 5' region of exon 4 and will only bind if exon 2 and 4 are directly fused together. Most preferred is a PCR-based approach using genomic DNA as template and the above mentioned conditions and primers that are preferably of at least 12 nucleotides in length, more preferred of at least 15 nucleotides in length, even more preferred of at least 18 nucleotides in length and most preferred of at least 21 nucleotides in length as depicted in SEQ ID NO: 12 and SEQ ID NO: 13. Said PCR- based approach is e.g. followed by sequencing and/or SSCA as described herein above. Bands showing an alteration in comparison to "wild-type" bands may be reamplified and sequenced to determine whether the amplified nucleic acid sequence has said deletion.

Also an insertion in a nucleic acid sequence as described herein is preferably determined by PCR-based approaches. In particular, as already mentioned herein above, one of the two primers used in a PCR is designed in a manner that it is either capable to bind only to the wild-type nucleic acid sequence not comprising an insertion between nucleotide position 596 and 597 as depicted in SEQ ID NO: 1 or capable to bind only to a nucleotide sequence comprising an insertion at said positions. Thus, in the first case no PCR fragment will result if the nucleotide sequence comprises an insertion and in the second case a PCR fragment will result from said nucleotide sequence comprising said insertion. As a template nucleic acid molecule either cDNA or genomic DNA is preferred to be used. More preferably, PCR with appropriate primers located upstream and downstream of positions 596 and 597 as depicted in SEQ ID NO: 1 is followed by RFLP analysis to determine whether an insertion has occurred. Said RFLP analysis is possible if due to the insertion an endonuclease restriction site is generated that is not present in the wild- type nucleic acid sequence depicted in SEQ ID NO: 1 or a restriction site is destroyed which occurs in the wild-type sequence. Namely, the primers depicted in SEQ ID NO: 20 and 21 , respectively, are used to amplify a template nucleic acid molecule as described above. The following PCR conditions are applied: preferably at least 35 cycles of denaturation at a temperature preferably of at least 95°C for preferably at least 30 sec, annealing at a temperature of preferably at least 64°C for preferably at least 30 sec and extension at a temperature of preferably at least 72°C for preferably at lest 60 sec. Said PCR results in the generation of a 255 bp fragment in case of a wild-type nucleic acid template not comprising an insertion between nucleotide position 596 and 597 as depicted in SEQ ID NO: 1 or in a 256 bp fragment in case of a nucleic acid template comprising an insertion between nucleotide position 596 and 597 as depicted in SEQ ID NO: 1. Afterwards, the fragments are treated preferably with the restriction endonuclease Mwol which recognizes the nucleotide sequence GCNNNNNNNGC. However, any restriction endonuclease which recognizes said sequence may be used. As described above, an insertion between nucleotide position 596 and 597 as depicted in SEQ ID NO: 1 results in the generation of a recognition site for Mwol and, accordingly, the 256 bp fragment comprising said insertion will be cleaved in a 115 bp, a 112 bp, a 17 bp and a 12 bp fragment, whereas the 255 bp wild-type fragment will be cleaved in a 115 bp, a 112 bp and 28 bp fragment which are visualized on a 10% polyacrylamidegel by methods known in the art. More preferably PCR using genomic DNA as template and the above mentioned conditions followed by SSCA as described herein above is performed to determine whether an insertion between positions 596 and 597 as depicted in SEQ ID NO: 1 has taken place. The primers used for this purpose are preferably of at least 12 nucleotides in length, more preferred of at least 15 nucleotides in length, even more preferred of at least 18 nucleotides in length and most preferred of at least 21 nucleotides in length as depicted in SEQ ID NO: 14 and SEQ ID NO: 15.

It is also possible to determine the above-described CIC-2 mutations on the protein level. Some of the mutations described above lead to shortened versions of the CIC-2 protein. Thus, it is conceivable to determine the occurrence of these mutations by determining the length or molecular weight of the CIC-2 protein expressed in an individual, e.g. by SDS PAGE.

It is also possible to determine the mutations of the CIC-2 voltage-gated channel as described herein by using the antibodies of the present invention. Said antibodies specific for said mutations of CIC-2 proteins will be determined by assay techniques such as radioimmunoassays, competitive-binding assays, Western blot analysis and ELISA assay. Also preferred are classical immunohistological methods. The finding, described in the present invention, that certain mutations in the CIC-2 encoding gene and/or the corresponding protein are connected with IGE is indicative that the non- or dysfunction of the CIC-2 protein is responsible for various forms of IGE. Thus, the finding of these mutations not only allows the diagnosis of IGE by determining whether the above-described mutations occur in an individual. It also allows to develop a treatment for IGE which has been diagnosed to be the result of a mutation in the CIC-2 encoding gene. Such a treatment can, e.g., comprise the introduction of a nucleic acid molecule encoding a functional wild-type CIC-2 protein thereby restoring in said individual the CIC-2 activity.

Thus, in another embodiment the present invention also relates to a pharmaceutical composition. In accordance with the present invention the term "pharmaceutical composition" relates to a composition comprising a nucleic acid molecule comprising a nucleotide sequence which encodes a functional voltage-gated chloride channel CIC-2 and which is selected from the group consisting of:

(a) a nucleotide sequence encoding a polypeptide comprising the amino acid sequence as depicted in SEQ ID NO: 2;

(b) a nucleotide sequence comprising the nucleotide sequence as depicted in SEQ ID NO: 1 ;

(c) a nucleotide sequence which hybridizes to the nucleotide sequence of (a) or (b); and

(d) a nucleotide sequence which is degenerated as a result of the genetic code to the nucleotide sequence of (c).

Such pharmaceutical compositions comprise a therapeutically effective amount of a nucleic acid molecule encoding a functional CIC-2 protein and, optionally, a pharmaceutically acceptable carrier. The pharmaceutical composition may be administered with a physiologically acceptable carrier to a patient, as described herein. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency or other generally recognized pharmacopoeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Such compositions will contain a therapeutically effective amount of the aforementioned compounds, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

In another preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilised powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration. The pharmaceutical composition of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

In vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. Preferably, the pharmaceutical composition is administered directly or in combination with an adjuvant. The pharmaceutical composition is preferably designed for the application in gene therapy. The technique of gene therapy has already been described above in connection with the nucleic acid molecules of the invention and all what has been said there also applies in connection with the pharmaceutical composition. For example, the nucleic acid molecule in the pharmaceutical composition is preferably in a form which allows its introduction, expression and/or stable integration into cells of an individual to be treated.

In another aspect the present invention relates to a method of treating a neurological disease comprising administering a therapeutically effective amount of the pharmaceutical composition described herein above to a subject suffering from said disease.

In the context of the present invention the term "subject" means an individual in need of a treatment of a neurological disease. Preferably, the subject is a vertebrate, even more preferred a mammal, particularly preferred a human. The term "administered" means administration of a therapeutically effective dose of the aforementioned nucleic acid molecule encoding a functional CIC-2 protein to an individual. By "therapeutically effective amount" is meant a dose that produces the effects for which it is administered. The exact dose will depend on the purpose of the treatment and will be ascertainable by one skilled in the art using known techniques. As is known in the art and described above, adjustments for systemic versus localized delivery, age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art. The methods are applicable to both human therapy and veterinary applications. The compounds described herein having the desired therapeutic activity may be administered in a physiologically acceptable carrier to a patient, as described herein. Depending upon the manner of introduction, the compounds may be formulated in a variety of ways as discussed below. The concentration of therapeutically active compound in the formulation may vary from about 0.1-100 wt %. The agents maybe administered alone or in combination with other treatments. The administration of the pharmaceutical composition can be done in a variety of ways as discussed above, including, but not limited to, orally, subcutaneously, intravenously, intra-arterial, intranodal, intramedullary, intrathecal, intraventricular, intranasally, intrabronchial, transdermally, intranodally, intrarectally, intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally, or intraocularly. In some instances, for example, in the treatment of wounds and inflammation, the candidate agents may be directly applied as a solution dry spray. The attending physician and clinical factors will determine the dosage regimen. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. A typical dose can be, for example, in the range of 0.001 to 1000 μg; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. The dosages are preferably given once a week, however, during progression of the treatment the dosages can be given in much longer time intervals and in need can be given in much shorter time intervals, e.g., daily. In a preferred case the immune response is monitored using herein described methods and further methods known to those skilled in the art and dosages are optimized, e.g., in time, amount and/or composition. Dosages will vary but a preferred dosage for intravenous administration of DNA is from approximately 10⁶ to 10¹² copies of the DNA molecule. If the regimen is a continuous infusion, it should also be in the range of 1 μg to 10 mg units per kilogram of body weight per minute, respectively. Progress can be monitored by periodic assessment. The pharmaceutical composition of the invention may be administered locally or systemically. Administration will preferably be parenterally, e.g., intravenously. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. It is also envisaged that the pharmaceutical compositions are employed in co- therapy approaches, i.e. in co-administration with other medicaments or drugs, for example other drugs for preventing, treating or amelioration epilepsy, in particular IGE.

The invention also relates to the use of a nucleic acid molecule encoding a functional CIC-2 protein as described herein above in connection with the pharmaceutical composition for the preparation of a pharmaceutical composition for treating a neurological disease.

In a more preferred embodiment said neurological disease to be treated with the aforementioned pharmaceutical composition is an idiopathic generalized epilepsy (IGE). Particularly preferred, said idiopathic generalized epilepsy is selected from the group consisting of childhood absence epilepsy (CAE), juvenile absence epilepsy (JAE), juvenile myoclonic epilepsy (JME) and epilepsy with grand mal seizures on awakening (EGMA).

Additionally, the present invention relates to a kit comprising the nucleic acid molecule, the vector, the host, the polypeptid, the antibody or the aptamer, the primer or pair of primers of the invention or the molecule as identified or characterized in a method herein below of the present invention. Advantageously, the kit of the present invention further comprises, optionally (a) reaction buffer(s), storage solutions and/or remaining reagents or materials required for the conduct of scientific or diagnostic assays or the like. Furthermore, parts of the kit of the invention can be packaged individually in vials or bottles or in combination in containers or multicontainer units.

The kit of the present invention may be advantageously used, inter alia, for carrying out the method of producing a polypeptide of the invention, the method(s) of identification and/or characterization of molecules specifically interacting with CIC-2 voltage-gated chloride channels as described herein below and/or it could be employed in a variety of applications referred herein, e.g., as diagnostic kits, as research tools or therapeutic tools. Additionally, the kit of the invention may contain means for detection suitable for scientific, medical and/or diagnostic purposes. The manufacture of the kits follows preferably standard procedures which are known to the person skilled in the art.

Furthermore, the present invention relates to a method for identifying molecules which are capable of specifically interacting with a polypeptide of the present invention, comprising the steps of (a) contacting a polypeptide of the present invention with a molecule to be tested; and (b) determining whether said molecule is capable of specifically interacting with said polypeptide. The polypeptide may be provided directly or by expression of a corresponding nucleic acid molecule or vector of the invention, e.g., in vitro or in a suitable host cell.

Additionally, the present invention relates to a method for the characterization of molecules which are capable of altering characteristics of the polypeptides of the present invention, comprising the steps of (a) contacting a polypeptide of the invention with said molecule; and (b) determining whether the molecule alters a characteristic of said polypeptide.

Said identification and/or characterization of molecules which are capable of interacting with or altering characteristics of the polypeptide of this invention, may be, inter alia, achieved by transfecting an appropriate host with a nucleic acid molecule of invention. Said hosts comprise, but are not limited to, HEK 293 cells or are injected into frog oocytes, preferably a Xenopus oocyte for functional expression (Goldin (1992), Methods Enzymol. 207, 266). Expressed CIC-2 voltage-gated channels con be examined using standard two-electrode voltage clamp techniques (see Stuhmer (1992), Methods Enzymol. 207, 319; Kohler (1996), Science 273, 1709). After expression of a CIC-2 voltage-gated chloride channel as defined herein, membrane currents may be deduced in the absence and/or presence of the molecule to be identified and/or characterized. Methods for the deduction of membrane currents are well known in the art and comprise, e.g., patch clamp methods as described in Hamill, Pfluger's Arch. 391 (1981), 85-100 or two-electrode voltage clamp in oocytes, as described in Methfessel, Pflϋgers Archive 407 (1986) 577-588.

Furthermore, the present invention relates to a method of screening for molecules which are capable of interacting with the polypeptide of this invention, comprising the steps of (a) contacting a polypeptide of the invention with a molecule; and (b) measuring and/or detecting a response; and (c) comparing said response to a standard response as measured in the absence of said candidate molecule.

Potential candidate molecules or candidate mixtures of molecules may be, inter alia, substances, compounds or compositions which are of chemical or biological origin, which are naturally occurring and/or which are synthetically, recombinantly and/or chemically produced. Thus, candidate molecules may be proteins, protein- fragments, peptides, amino acids and/or derivatives thereof or other compounds, such as ions, which bind to and/or interact with wild-type CIC-2 voltage-gated chloride channels. Such binding and/or interacting candidate compounds may be found employing, inter alia, yeast two-hybrid systems or modified yeast two-hybrid systems as described, for example, in Fields, Nature 340 (1989), 245-246; Gyuris, Cell 75 (1993), 791-801 ; or Zervos, Cell 72 (1993), 223-232.

Furthermore, potential candidate molecules may be contacted with a cell, such as an oocyte or a HEK 293 cell, which expresses a polypeptide of the invention or with a membrane patch comprising a polypeptide of the invention and a corresponding response (inter alia, a dose-response response, a current- response, or single current channel response) may be measured in order to elucidate any effect said candidate molecule causes.

Within the scope of the present invention are also methods for identifying, characterizing and for screening of molecules which are capable of interacting with CIC-2 voltage-gated chloride channels which comprise so-called high- throughput screening methods and similar approaches which are known in the art (Spencer, Biotechnol. Bioeng. 61 (1998), 61-67; Oldenburg, Annu. Rev. Med. Chem. 33 (1998), 301-311) carried out using 96-well, 384-well, 1536-well (and other) commercially available plates. Further methods to be employed in accordance with the present invention comprise, but are not limited to, homogenous fluorescence readouts in high-throughput screenings (as described, inter alia, in Pope, Drug Discovery Today 4 (1999), 350-362). The method of the present invention for identification, characterization and/or screening of molecules capable of interacting with CIC-2 voltage-gated chloride channels can, inter alia, employ hosts as defined herein which express the polypeptide of the present invention. Cell-based assays, instrumentation for said assays and/or measurements are well-known in the art and described, inter alia, in Gonzalez, Drug Discovery Today 4 (1999), 431-439 or Ramm, Drug Discovery Today 4 (1999), 401-410. It is also envisaged that the high through put screens described herein are conducted by using, for example cRNA, i.e. synthetic RNA from a cDNA construct) that can be introduced in host cells, such as Xenopus oocytes using routine methods in the art. As an example, direct nucleic acid injection can be employed, such as the Eppendorf microinjection system (Micromnipulator 5171 and Transjector 5242). The injected/transformed cells can be analyzed for chloride currents about 4 hours later using patch-clamp techniques which are commonly practiced in the art.

Additionally, the present invention relates to a method for the production of a pharmaceutical composition comprising the steps of a method of the invention for identifying, characterizing and/or screening of molecules which are capable of interacting with CIC-2 voltage-gated chloride channels and further comprising a step, wherein a derivative of said identified, characterized and/or screened molecule is generated. Such a derivative may be generated by, inter alia, peptidomimetics.

The invention furthermore relates to a method for the production of a pharmaceutical composition comprising the steps of a method of the invention for identifying, characterizing, screening and/or derivatizing of molecules which are capable of interacting with CIC-2 voltage-gated chloride channels and formulating the molecules identified, characterized, screened and/or derivatized in pharmaceutically acceptable form.

In a more preferred embodiment the present invention relates to a method wherein said molecule(s) comprise(s) (a) neuroprotective, (a) nootropic and/or (a) antiepileptic molecule(s).

In a yet more preferred embodiment the present invention relates to a method wherein said molecule(s) are antagonist(s), partial antagonist(s), partial agonist(s) and/or agonist(s) for a voltage-gated chloride channel CIC-2.

In accordance with the present invention, the term "antagonist" denotes molecules/substances, which are capable of inhibiting and/or reducing an agonistic effect. The term "antagonist" comprises competitive, non-competitive , functional and chemical antagonists as described, inter alia, in Mutschler, "Arzneimittelwirkungen" (1986), Wissenschaftliche Verlagsgesellschaft mbH, Stuttgart, Germany. The term "partial antagonist" in accordance with the present invention means a molecule/substance that is capable of incompletely blocking the action of agonists through, inter alia, a non-competitive mechanism. As "agonist", in accordance with this invention, molecules/substances are denoted which have an affinity as well as an intrinsic activity. Mostly, said intrinsic activity (oc) is defined as being proportional to the quotient of the effect, triggered by said agonist (E_A) and the effect which can be maximally obtained in a given biological system (E_max): therefore, the intrinsic activity can be defined as

The highest relative intrinsic activity results from E_A/Em_ax=1. Agonists with an intrinsic activity of 1 are full agonists, whereas substances/molecules with an intrinsic activity of >0 and <1 are partial agonists. Partial agonists show a dualistic effect, i.e. they comprise agonistic as well as antagonistic effects.

The person skilled in the art can, therefore, easily employ the compounds and the methods of this invention in order to elucidate the agonistic and/or antagonistic effects and/or characteristics of a compound/molecule/substance to be identified and/or characterized in accordance with any of the above described methods. Preferably, an identified antagonist of the voltage-gated chloride channel CIC-2 comprising the G715E mutation may be useful to reestablish the electrophysiological properties normally shown by wild-type CIC-2 voltage-gated chloride channels. In particular the altered chloride-dependent gating of the G715E mutation may be reversed.

It is also preferred that an identified agonist of the voltage-gated chloride channel CIC-2 resulting from either the deletion of amino acids 74 to 117 or the insertion of nucleotides between position 596 and 597 of the corresponding wild-type nucleotide sequence may be useful to reestablish the lost functionality of the CIC- 2 voltage-gated chloride channel.

The anagonist(s), partial anatagonist(s), partial agonist(s) and /or agonist(s) for the voltage-gated chlorid channel CIC-2 is preferably selected from aptamers, aptazymes, RNAzymes, antibodies, affybodies, trinectins, anticalins, or the like compounds. The effect of the compounds on the activity of the voltage-gated chlorid channel CIC-2 may be assayed by testing the effect of the compound in an electrophsyiological recording to obtain the voltage dependence of channel activation. A suitable assay is described, e.g., in Example 6. Techniques for the production of suitable compounds are well known in the art. Suitable compounds are e.g., antibodies, described in Harlow and Lane " Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988. Suitable compounds are also aptamers. Their preparation is well known in the art, e.g. Gold (1995), Ann. Rev. Biochem. 64: 763-797.

Further suitable compounds are e.g., anticalins, described in EP 1 017 814. Said European patent also describes the process of preparing such anticalins with the ability to bind a specific target. Also suitable molecules are Trinectins (Phylos Inc., Lexington, Massachusetts, USA, or Xu (2002), Chem. Biol. 9:933). Another kind of suitable molecules are affybodies (see Hansson (1999), Immunotechnology 4:237- 252, or Henning (2002), Hum Gene Ther. 13:1427-1439, and references therein).

The Figures show:

Figure 1 Segregation analysis of three different CLCN2 mutations in families with common IGE subtypes.

Individuals carrying one of the mutations are denoted by +/m, whereas the absence of a mutation is denoted by +/+. Filled symbols represent individuals diagnosed with either juvenile myoclonic epilepsy (JME), juvenile absence epilepsy (JAE), childhood absence epilepsy (CAE), or epilepsy with grand mal seizures on awakening (EGMA). Three individuals were regarded as affected based on their pathological electroencephalogram showing spontaneous generalized spike-wave (sw-EEG) and/or poly-spike-wave (psw-EEG) discharges.

(A) Family 1 was ascertained through individual IV: 1 who was diagnosed with JME. Segregation analyses showed perfect co-segregation of the mutation and the affection status in this family.

(B) The index patient in family 2 (IV:4) experienced daily clusters of absence seizures during childhood (CAE). All affected family members carry the mutation, whereas the occurrence of the mutation in the unaffected individual 111:11 indicates incomplete penetrance of this mutation.

(C) Family 3 was ascertained through the index patient ll:2 whose epilepsy syndrome was classified as JAE. All affected family members were found to be heterozygous for the mutation.

Detailed clinical information is given in Experimental Procedures.

Figure 2 Detection of three different CLCN2 mutations in families with common IGE subtypes.

(A) In family 1 , a G-insertion was found in bp-position 597 (597insG) (arrow) resulting in a translational reading frameshift and a premature stop codon in position 231 (M200fsX231).

(B) An 11-bp deletion (underlined) in intron 2 (IVS2-14del11) close to the splice acceptor site was found in family 2 (left panel). Results of a RT- PCR using primers binding to exon 1 and exon 5 showed the wild-type (540 bp) and a novel splice variant (408 bp). Sequencing of the alternative splice product (right panel) demonstrated the loss of exon 3 leading to the loss of 44 amino acid residues (del74-117).

(C) A G→A transition (G2144A) (arrow) identified in family 3 results in a non-conservative amino acid exchange (G715E) in the C-terminus of the protein.

Figure 3 Functional Characterization of wild-type hCIC-2 Channels.

(A) Membrane topology model of CIC-2 (Thiemann et al., 1992; Cid et al., 1995), based on the high-resolution structure of a CIC channel from S. typhimurium with 18 helical segments (Dutzler et al., 2002). CIC-2 is a 898 amino acid polypeptide which differs from the prokaryotic isoforms in the existence of a 333 amino acid cytoplasmic C-terminus that exhibits two CBS domains (Ponting (1997), J. Mol. Med. 75, 160-163).

(B) Voltage-clamp protocol and a typical whole-cell recording from a tsA201 cell expressing wild-type hCIC-2 channels.

(C) Voltage dependence of the relative open probability (P_open) of wild- type hCIC-2 channels for different internal chloride concentrations ([Cl^"]j). To enable measurements at low [Cl^"]j, the construct wild-type-FCYENE yielding enhanced expression levels was used (Ma (2001), Science 291, 316-319). Lines represent fits of a standard Boltzmann function to the data points. Midpoints of activation (V₀.s) at different [Cl^"]ι were as follows: 124 mM: -77 ± 2 mV (filled circles, n = 10); 34 mM: -128 + 7 mV (open squares, n = 9); 15 mM: -131 + 4 mV (filled triangles , n = 7); 4 mM: -159 ± 7 mV (open diamonds, n = 5). The vertical dashed lines represent the calculated chloride equilibrium potentials (Eci) at 4 ( — ), 15 ( ), 34 (-.-.-) and 124 (-..-..-) mM [Cl^"]i.

(D) Midpoint of activation (V₀.s) as a function of [Cl^"]ι shown as a semi- logarithmic plot (n = 5-10, as in (C)). The line represents linear regressions (r² = 0.92). Figure 4 Functional Characterization of Two Loss-of-Function hCIC-2 Mutations.

(A), (D) Proposed topology models of CIC-2 as shown in Figure 3A for the M200fsX231 mutation (A) and the del74-117 splice variant (D). (B), (E) Voltage-clamp protocol and typical whole-cell recordings from tsA201 cells expressing mutant hCIC-2 channels (B: M200fsX231, E: del74-117). There was no detectable current in transfected cells. (C) Voltage dependence of averaged peak current amplitudes from cells transfected with wild-type (filled circles, n = 17), M200fsX231 (open circles, n = 12), M200fsX231-FCYENE (open diamonds, n = 4), wild-type- M200fsX231-FCYENE concatamer (open triangles, n = 13), or from cells co-transfected with wild-type and M200fsX231 (open squares, n = 14). Cells expressing wild-type alone or the same amount of wild-type plus the same amount of M200fsX231 (co-transfection) were transfected and measured on the same days in parallel.

(F) Voltage dependence of averaged peak current amplitudes from cells transfected with wild-type (filled circles, n = 8), del74-117 (open circles, n = 6), del74-117-FCYENE (open diamonds, n = 9), wild-type-del74-117- FCYENE (open triangles down, n = 7) and del74-117-wild-type-FCYENE (open triangles up, n = 7) concatamers, and from cells co-transfected with wild-type and del74-117 (open squares, n = 8). Cells expressing wild-type alone or the same amount of wild-type plus the same amount of del74-117 (co-transfection) were transfected and measured on the same days in parallel.

Figure 5 Functional Characterization of the Point Mutation G715E.

(A) Proposed topology model of CIC-2 as shown in Figure 3A showing the position of the point mutation G715E within the intracellularly located C-terminus of the channel.

(B) Voltage-clamp protocol and a typical whole-cell recording from a tsA201 cell expressing G715E hCIC-2 channels.

(C) Voltage dependence of P_open of G715E-hCIC-2-FCYENE channels (compare Figure 3C, Ma et al., 2001). V₀.₅ at different [Cl^"]ι were as follows: 124 mM: -87 ± 5 mV (filled circles, n = 7); 34 mM: -100 ± 5 mV (open squares, n = 4, p < 0.05 compared to wild-type); 15 mM: -113 + 6 mV (filled triangle, n = 8, p < 0.05 compared to wild-type); 4 mM: -136 ± 7 mV (open diamonds, n = 8, p < 0.05 compared to wild-type). Vertical lines represent Eci as in Figure 3C.

(D) Midpoint of activation (V₀.₅) as a function of [Cl^"]ι shown as a semi- logarithmic plot for wild-type (filled circles) and G715E (open circles) (n = 4-10, as in (C) and Figure 3C). Lines represent linear regressions (wild- type: r² = 0.92; G715E: r² = 0.98).

The examples illustrate the invention.

Example 1: Mutation Screening

For mutation screening single strand conformation analysis (SSCA) was carried out using primers which allowed amplification of the entire CLCN2 coding region and adjacent intronic sequences. ^' PCR fragments were separated on a 10% polyacrylamide gel at 4°C and at room temperature. Aberrant bands were sequenced (ABI 377). In case of a sequence variation the entire CLCN2 coding region in the index patients was sequenced and all available family members were genotyped.

Example 2: Analysis of mutations in the CIC-2 coding sequence by PCR and

SSCA

To analyse the herein identified mutations of the CLCN2 gene encoding the voltage- gated chloride channel CIC-2, PCR is employed. Genomic DNA was extracted from 10 ml aliquots of EDTA-anticoagulated blood samples, using a salting-out method. PCR cycles were performed in a MJ Research thermocycler with the following conditions: 35 cycles of denaturation at 95°C,for 30 sec, annealing at 58°C for 30 sec, and extension at 72°C for 60 sec. Each PCR was done in a final volume of 25 μl containing 100 ng of genomic DNA, 10 pmol of each the forward and reverse primer as depicted herein below, 200 μM of each dNTP, 15 mM MgCI₂, 50 mM KCI, 10 mM Tris-HCI (pH 8.3), and 0.5 U Taq DNA Polymerase.

The following primers were used to identify the point mutation in the mutant CLCN2 gene leading to the corresponding G715E mutation in the voltage-gated chloride channel CIC-2 as depicted in SEQ ID NO: 2: 5'-TGTCTTCCTTACCTTTCCTGG-3' (forward primer) (depicted in SEQ ID NO: 10) and 5'- ACTGCAGGGTTAATGACGTGG-3' (reverse primer) (depicted in SEQ ID NO: 11).

The following primers were used to identify the mutation leading to an atypical splicing of the CLCN2 mRNA (del74-117) which results in the deletion of the corresponding amino acids 74 to 117 of the wild-type voltage-gated chloride channel CIC-2 as depicted in SEQ ID NO: 2: 5'-AATATGGACGGAGCCGTTGCG-3' (forward primer) (depicted in SEQ ID NO: 12) and 5'-AGCTGACCAATGCCATGAGAAG-3' (depicted in SEQ ID NO: 13).

The following primers were used to identify an insertion of a nucleotide between positions 596 and 597 of the corresponding wild-type CLCN2 sequence as depicted in SEQ ID NO: 1 leading to a premature stop codon (M200fsX231) within the CLCN2 sequence as depicted in SEQ ID NO: 1 which results in a truncated CIC-2 protein: 5'-TGCATCGAATGCCTCTCCTG-3' (forward primer) (depicted in SEQ ID NO: 14) and 5'-CCACCAGGAGGGACTCCTTC-3' (reverse primer) (depicted in SEQ ID NO: 15).

PCR fragments were separated on a 10% polyacrylamide gel at 4°C and at room temperature, respectively. PCR fragments showing a SSCA band shift were amplified again prior to direct sequence analysis, which was carried out on an automated sequence analyser (ABI 377).

Example 3: Analysis of mutations in the CIC-2 coding sequence by PCR and restriction endonuclease digestion

Genomic DNA was obtained as described in Example 2, supra. The PCR machine used is also described in Example 2, supra.

The following primers were used to identify the point mutation in the mutant CLCN2 gene leading to the corresponding G715E mutation in the voltage-gated chloride channel CIC-2 as depicted in SEQ ID NO: 2: 5'- GCACATGCAGGAGCGCAGA -3' (forward primer) (depicted in SEQ ID NO: 16) and 5'- CCTGCCGACTCTGCGCTG - 3' (reverse primer) (depicted in SEQ ID NO: 17).

PCR conditions were as follows: 35 cycles of denaturation at 95°C for 30 sec, annealing at 62°C for 30 sec, and extension at 72°C for 60 sec. Each PCR is done in a final volume of 25 μl containing 100 ng of genomic DNA, 10 pmol of each the forward and reverse primers, 200 μM of each dNTP, 1.5 mM MgCI₂, 50 mM KCI, 10 mM Tris-HCI (pH 8.3), and 0.5 U Taq DNA polymerase. PCR product sizes: 222 bp (wild type) 222 bp (mutant)

PCR is followed by restriction enzyme digest with lta\. Fragments can be separated on a 10% polyacrylamide gel and visualized by standard silver staining procedure.

Bands: 117 bp, 88 bp and 17 bp (wild type)

117 bp and 105 bp (mutant)

The following primers were used to identify the mutation leading to an atypical splicing of the CLCN2 mRNA (del74-117) which results in the deletion of the corresponding amino acids 74 to 117 of the wild-type voltage-gated chloride channel CIC-2 as depicted in SEQ ID NO: 2: 5'- CGGGCTGCCCCAGAGCTC -3' (forward primer) (depicted in SEQ ID NO: 18) and 5'- GATACTAGGAACTTGTGGCAG -3' (depicted in SEQ ID NO: 19).

PCR-conditions: 35 cycles of denaturation at 95°C for 30 sec, annealing at 62°C for 30 sec, and extension at 72°C for 60 sec. Each PCR is done in a final volume of 25 μl containing 100 ng of genomic DNA, 10 pmol of each the forward and reverse primers, 200 μM of each dNTP, 1.5 mM MgCI₂, 50 mM KCI, 10 mM Tris-HCI (pH 8.3), and 0.5 U Taq DNA polymerase.

^JCR product sizes: 250 bp (wild type) 239 bp (mutant)

³CR is followed by restriction enzyme digest with Bsgl. Fragments can be separated and visualized on a 3% agarose gel

3ands: 212 bp and 38 bp (wild type)

239 bp (mutant)

The following primers were used to identify an insertion of a nucleotide between )ositions 596 and 597 of the corresponding wild-type CLCN2 sequence as depicted n SEQ ID NO: 1 leading to a premature stop codon (M200fsX231) within the CLCN2 sequence as depicted in SEQ ID NO: 1 which results in a truncated CIC-2 protein: 5'- TGGATGTCCCGGGGCTTGAAC -3' (forward primer) (depicted in SEQ ID NO: 20) and 5'- TCTTTGCCAAGCGGCAATCCC -3' (reverse primer) (depicted in SEQ ID NO: 21).

PCR-conditions: 35 cycles of denaturation at 95°C for 30 sec, annealing at 64°C for 30 sec, and extension at 72°C for 60 sec. Each PCR is done in a final volume of 25 μl containing 100 ng of genomic DNA, 10 pmol of each the forward and reverse primers, 200 μM of each dNTP, 1.5 mM MgCI₂, 50 mM KCI, 10 mM Tris-HCI (pH 8.3), and 0.5 U Taq DNA polymerase.

PCR product sizes: 255 bp (wild type) 256 bp (mutant)

PCR is followed by restriction enzyme digest with Mwol. Fragments can be separated on a 10% polyacrylamide gel and visualized by standard silver staining procedure.

Bands: 115 bp, 112 bp and 28 bp (wild type)

115 bp, 112 bp, 17bp and 12 bp (mutant)

Example 4: Quantitative Competitive RT-PCR

To determine the relative amount of mutant and wild-type CIC-2 mRNA in peripheral blood cells from two individuals carrying the IVS2-14del11 mutation (lll:7, IV:4), one family member carrying two wild-type alleles (lll:5) (Figure 1 B) and one independent control subject, a quantitative competitive (QC) RT-PCR assay was established. Total RNA was extracted from EDTA-anticoagulated blood samples using the Qiamp^® RNA Blood Mini Kit (Qiagen) according to the manufacturers instructions. The concentrations of total RNA prepared were determined by OD₂₆o and adjusted by semiquantitative RT-PCR using the human hypoxanthine phosphoribosyltransferase 1 transcript as an internal standard. Competitors for mutant and wild-type CIC-2 were generated in the pCR2.1-TOPO vector (Invitrogen). A 91-bp internal deletion was introduced in both competitors by standard recombinant PCR-technologies to produce PCR products of different lengths. For QC-RT-PCR target-specific primer sets binding to either the wild-type cDNA or the mutant CIC-2 cDNA were generated. Serially ten-fold diluted competitor DNAs were added to RT-PCR tubes. One-tube QC-RT-PCR was performed under the following conditions: Reverse transcription at 50°C for 30 minutes, 95°C for 15 minutes, and 23 identical cycles of denaturation (94°C for 30 seconds), annealing (68°C for 20 seconds) and extension (72°C for 1 minute). The last cycle was followed by an extra 5 minutes at 72°C. QC-RT-PCR products were run on a 2% agarose gel. Band intensities were analyzed and quantified optically using an image processing system (Gel-doc 1000 camera system and image analysis software, molecular analysis software, Bio-Rad Lab Inc.). The relative amounts of wild-type and mutant CIC-2 cDNA obtained in two patients carrying the IVS2-14del11 mutation and two controls were calculated by determining the equivalent point in a logarithmic linear regression plot using mean values of five independent experiments in each individual.

Example 5: Plasmid Construction, Mutagenesis and Transfection

The full-length human CIC-2 cDNA in plasmid pBK-RSV (Invitrogen) (Cid (1995), loc. cit.) was subcloned into pcDNA3.1 (Invitrogen) after modifying the original construct by PCR-techniques to produce a N-terminal Not l restriction site (pcDNA3.1-hCIC-2). To increase expression levels, an endoplasmatic reticulum (ER) export signal (FCYENE) (Ma (2001), loc. cit.) was introduced at the C-terminus of hCIC-2 and an additional C-terminal Xba I restriction site was created to subclone this construct into pRcCMV (Invitrogen) (pRcCMV-hCIC-2-FCYENE). Mutations were introduced into both constructs by PCR-based techniques and verified by sequencing. For wild-type and all mutations, constructs with and without FCYENE exhibited indistinguishable biophysical properties. Transient transfection of tsA201 cells using the Ca₃(P04)2 technique was performed as previously described (Fahlke (1997b), J. Gen. Physiol. 109, 93-104). For each mutation, at least two independent recombinants were examined. Concatameric constructs linking two wild-type or one mutant and one wild-type hCIC-2 sequence in a single open reading frame were designed as described previously for CIC-1 (Fahlke (1997b), loc. cit.). Transfection of concatameric wild- type-wild-type constructs in tsA201 cells resulted in peak current amplitudes comparable with monomeric wild-type constructs. Co-transfections of wild-type and mutant CIC-2 were performed in a 1 :1 ratio of transfected cDNA. To examine a possible dominant negative effect of M200fsX231 and del74-117 on the wild-type, cells were transfected on the same day with either wild-type alone or the same amount of wild-type plus the same amount of mutant cDNA.

Example 6: Electrophysiological Recordings

Standard whole-cell patch clamp recordings were performed as described (Fahlke (1997b), loc. cit.). The standard extracellular (bath) solution contained (in mM): 140 NaCI, 4 KCI, 2 CaCI₂, 1 MgCI₂₎ 5 HEPES, pH 7.4. Four intracellular (pipette) solutions with different [Cl^"]ι were composed as follows (in mM): (i) 120 NaCI, 2 MgCI₂, 5 EGTA, 10 HEPES, pH 7.4 ([Cl^"]ι = 124 mM); (ii) 30 NaCI, 90 Na-Glutamate, 2 MgCI₂, 5 EGTA, 10 HEPES, pH 7.4 ([Cl^"]j = 34 mM); (iii) 11 NaCI, 109 Na- Glutamate, 2 MgCI₂, 5 EGTA, 10 HEPES, pH 7.4 ([CI^"]ι = 15 mM); (iv) 120 Na- Glutamate, 2 MgCI₂, 5 EGTA, 10 HEPES, pH 7.4 ([Cl^"]ι = 4 mM). Agar bridges were used to connect the bath solution and, when the intracellular solution contained glutamate, also the pipette solution to the amplifier. Between voltage steps, cells were held to potentials close to the calculated chloride equilibrium potential. Junction potentials calculated using the JPCalc software were used to correct results (Barry (1994), J. Neurosci. Methods 51 , 107-116).

To obtain the voltage dependence of activation, the instantaneous current amplitude determined 200 μs after a voltage step to 75 mV was measured after test pulses to different voltages (V), normalized to its maximum value and plotted versus the test potential. To reach steady-state conditions, the test pulse duration was adjusted to 2.5 s. Steady-state activation curves obtained in this manner were fit using a single Boltzmann function plus a constant term: l(V) = A/(1 + exp(V - V₀.s)/kv) + C, with l(V) representing the voltage-dependent current amplitude, A an amplitude factor, V₀.5 the voltage of half-maximal activation, kv a slope factor and C a constant term. After subtraction of the leak current component C, current amplitudes were divided by A to obtain the relative steady state open probability of hCIC-2 (Figure 4). Data were analyzed by a combination of pClamp (Axon Instruments, Foster City, CA, USA) and SigmaPlot (Jandel Scientific, San Rafael, CA, USA) programs. For statistic evaluation Student's T-Test was used. All data are shown as means ± SEM.

Example 7: Study Sample and Family Histories

All 46 families investigated were recruited at the Klinik fur Epileptologie and Klinik fur Padiatrie, Universitat Bonn, and the Neurologische Klinik, Universitatsklinikum Charite, Berlin, Germany. Diagnostic classification was performed by experienced neurologists following standard criteria (Commission on Classification and Terminology of the International League Against Epilepsy (1989), loc. cit.). EEG recordings were obtained from all patients and clinically unaffected family members. Individuals were regarded as affected, when they presented either with a history of primary generalized seizures or an EEG pattern typical for IGE such as generalized spike and wave or polyspike-wave discharges. The study was approved by the local Ethics Committees of the participating centres and written informed consent was obtained from all participants. In family 1 (Figure 1A), the leading IGE syndrome was JME, except for individual IV:3 who was diagnosed with EGMA. All clinically affected family members experienced frequent generalized tonic-clonic seizures. Myoclonic jerks often occurred in a series finally followed by generalized tonic-clonic seizures. Affected individuals only became seizure-free when treated with high dosages of valproate in combination with other antiepileptic drugs. In familiy 2 (Figure 1 B), all affected family members except for individual IV:4 exclusively experienced no more than one or two generalized tonic-clonic seizures on awakening, and these patients became seizure-free without requiring antiepileptic medication. One child (1V:4) presented with CAE with typical pyknoleptic daily clusters of absence seizures at the age of four years. The child became seizure-free when treated with valproate. In family 3 (Figure 1C), two affected siblings with JAE wee diagnosed. The disease status of the father remained unclear. In all three families, one individual was regarded as affected based on unprovoked generalized epileptiform discharges in the EEG. Example 8: Mutation Screening in 46 IGE Families with Linkage Evidence for the Chromosome 3q26 Susceptibility Locus

The genomic organization of the human CLCN2 gene was determined by comparing the published cDNA sequence (GenBank accession number NM_004366) with the genomic clone AC078797. 24 coding exons were identified, and a PCR-based strategy to amplify all coding exons and adjacent splice sites from genomic DNA was established. The CLCN2 gene was screened in index patients of 46 IGE families linked to chromosome 3q26 using single strand conformation analysis (SSCA). Direct sequencing of aberrant bands revealed three heterozygous mutations that co-segregate with the IGE trait in three unrelated families, presenting with the typical IGE subtypes of EGMA, CAE, JAE, or JME, respectively (Figure 1) (a detailed description of the clinical features of all three families is given in Example 2). None of these mutations was identified in a total of 360 control chromosomes. No further CLCN2 mutations were found in the index patients by sequencing all coding exons and adjacent splice sites.

The leading IGE syndrome in family 1 was JME presenting with frequent myoclonic and generalized tonic clonic seizures (Figure 1A). A single nucleotide insertion in bp-position 597 (597insG) (Figure 2A) was detected within exon 5 of individual IV: 1. The 597insG mutation alters the normal translational reading frame and predicts a premature stop codon (M200fsX231) that severely truncates the protein (Figure 4A). Affected individuals of family 2 experienced rare generalized tonic clonic seizures on awakening (EGMA), except individual IV:4 who exclusively suffered from absence seizures (CAE) (Figure 1 B). An 11-bp deletion in intron 2, IVS2-14del11 , was identified in this family, close to the splice acceptor site. This finding prompted to search for alternative CIC-2 splice products. RT-PCR using primers located in exon 1 and exon 5 demonstrated the presence of a novel CIC-2 splice variant lacking exon 3 (delexon3) (Figure 2B). Skipping of exon 3 leads to an in-frame deletion of 44 amino acid residues (del74-117) (Figure 4D). Since this splice variant was also found in healthy controls, a quantitative competitive RT-PCR assay was established. In both tested patients carrying the IVS2-14del11 mutation the number of wild-type mRNA copies was reduced (60 % and 66 % of the average number found in two controls), whereas the number of delexon3 mRNA copies was 8-fold increased. Accordingly, in the two control subjects a delexon3 was found: wild-type mRNA ratio of 5 : 95 and in patients carrying the intronic 11 bp deletion, the ratio was found to be 40 : 60.

In family 3, two affected individuals were diagnosed with JAE (Figure 1C). A single nucleotide exchange in bp-position 2144 was identified (G2144A) predicting the substitution of glutamate for glycine at position 715 (G715E) (Figure 2C) located C- terminal to the last transmembrane helix (Figure 5A).

The results of the present invention as illustrated in the Examples herein show that CLCN2 is the first epilepsy gene, mutations of which can cause the whole spectrum of common IGE subtypes. Therefore, these results support the neurobiological concept that the clinically different forms of this disease such as CAE, JAE, JME and EGMA share an overlapping genetic predisposition (Reutens (1995), Neurology 45, 1469-1476 OK). A different genetic background or unknown modifier genes might be responsible for the development of a predominant clinical phenotype, as was also observed in each of the families presented in this study. Thus, the results of the present invention provide further evidence for an important role of a genetically driven dysfunction of GABA-ergic synaptic inhibition in epileptogenesis which has often been discussed in the etiology of partial and generalized epilepsy.

Example 9: Wild-type Human CIC-2 Channels Constitute a Sole Chloride Efflux Pathway

To define the specific functional alterations caused by the two mutations and the splice variant, wild-type and mutant human CIC-2 (hCIC-2) channels were expressed in tsA201 cells and their functional properties were studied using the whole-cell patch clamp technique. Characteristic current recordings from a cell expressing wild-type hCIC-2 are shown in Figure 3B. The channels were closed at positive potentials and activated slowly upon membrane hyperpolarization. There was no indication for a voltage- and time-dependent inactivation (data not shown). The relative open probability depended not only on the membrane potential, but also on the intracellular chloride concentration ([C!^"]j) (Figure 3C). Decreasing [Cl^"]j resulted in a parallel shift of the activation curve to more hyperpolarized potentials. This particular dependence of gating on [Cl^"]ι ensures that wild-type hCIC-2 channels only open at potentials negative to the chloride reversal potential (Eci). wild-type hCIC-2 channels thus constitute an exclusive efflux pathway for chloride ions, quite similar to that shown for native CIC-2 channels in a rat hippocampal slice preparation (Staley (1994), loc. cit.). Figure 3D illustrates the relationship between [Cl^"]ι and the midpoint of activation (V₀.s) for wild-type hCIC-2 channels. The midpoint of activation changes in a logarithmic relationship with [Cl^"j|. This behavior is compatible with a model in which the voltage dependence of channel opening arises for the most part from voltage-dependent binding of intracellular CI^" (Cui (1997), J. Gen. Physiol. 109, 647-673).

It is known that coupling between ion permeation and gating is much tighter in CIC channels (Richard (1990), Science 247, 1208-1210; Pusch (1995), Nature 373, 527- 531) than for example in voltage-gated cation channels. The findings of the present invention show that the specific effects of permeating anions on CIC gating are more than a biophysical peculiarity. They are key to the physiological function of certain CIC channels. A comparison between the two CIC isoforms expressed in excitable membranes, CIC-1 and CIC-2, nicely illustrates the evolutionary optimisation of chloride-dependent gating in CIC channels. Activation gating of CIC-1 is almost independent of [Cl^"]j (Fahlke (1995), Neuron 15, 463-472), and this feature allows the muscle CIC isoform to provide the characteristic large resting conductance of the sarcolemma at a low [Cl^"]j. A naturally occurring mutation that couples CIC-1 gating to [CI-],, substantially reduces the resting chloride conductance and causes myotonia congenita, a genetic disease characterized by muscle hyperexcitability (Fahlke (1995, loc. cit.). In contrast, CIC-2 regulates internal anion composition and does not contribute to the resting conductance. Gating of wild-type CIC-2 critically depends on [Cl^"]i and a genetically induced alteration of the chloride dependence of gating causes neuronal hyperexcitability.

The above-mentioned results show that mutations in CLCN2, a gene encoding the neuronal chloride channel CIC-2, can cause several common IGE subtypes. Moreover, said results demonstrate an important role of CIC-2 in the regulation of neuronal excitability in humans and explain the physiological importance of the peculiar gating features of this CIC isoform. Example 10:The M200fsX231 and del74-117 Mutations Cause a Loss-of- Function of CIC-2 Channels

The M200fsX231 mutation predicts a truncated channel protein lacking major sequence determinants of the ionic pore (Fahlke (1997a), Nature 390, 529-532; Dutzler (2002), Nature 415, 287-294) (Figure 4A). Interestingly, heterologous expression of M200fsX231 mutant channels did not yield any detectable chloride current (Figure 4B, C). However, as patients carrying this mutation are heterozygous, only a minority of CIC-2 channels will be homomeric mutant channels. CIC channels are dimeric proteins (Miller (1982), Philos. Trans. R. Soc. Lond. B Biol. Sci. 299, 401-411 ; Dutzler (2002), loc. cit.) and therefore, channels consisting of one wild-type and one mutant subunit will represent the largest fraction of CIC-2 channels in heterozygous patients if the mutant is able to interact with the wild-type subunit. To study the functional properties of these heterodimeric channels, a concatameric construct that links one wild-type and one mutant allele in a single open reading frame was expressed, and additionally wild-type and mutant co-expression experiments were performed. The concatamer was non-functional, and co-transfection with wild-type and mutant cDNAs in a 1 :1 ratio resulted in a significantly smaller chloride current than that obtained from transfection of wild-type channels alone (peak current amplitude at -125 mV: 2.1 ± 0.4 nA, n = 17 (wild- type); 0.5 ± 0.3 nA, n =14 (Co-transfection of wild-type and M200fsX231), p<0.01) (Figure 4C). This dominant negative effect predicts a substantial reduction of functional CIC-2 channels in heterozygous patients.

The splice variant delexon3 predicts the deletion of helix B in the N-terminal part of the channel (del74-117) (Dutzler (2002), loc. cit.) (Figure 4D). Neither this mutant channel nor the concatameric proteins consisting of one wild-type and one del74- 117 subunit were functional (Figure 4E, F). Coexpression experiments revealed a dominant negative effect of del74-117 (peak current amplitude at -125 mV: 1.19 ± 0.46 nA, n = 8 (wild-type); 0.18 ± 0.05 nA, n = 8 (Co-transfection of wild-type and del74-117), p<0.05) (Figure 4F). Non-functional homodimeric and heterodimeric mutant channels will cause a reduced number of functional CIC-2 channels in heterozygous patients carrying the IV2-14del11 mutation.

The quantitative competitive RT-PCR assay predicted a lower expression of the del74-117 splice variant compared to that of wild-type. Therefore, a less pronounced effect of the IVS2-14del11 mutation on chloride current amplitudes is expected than for the 597insG mutation encoding the truncated mutant M200fsX231 (assuming a 50:50 expression of wild-type and mutant alleles in heterozygotes carrying the 597insG mutation). These findings are consistent with the less severe epileptic phenotype in family 2 compared to family 1.

The results obtained from the heterologous expression system predict a substantial reduction of CIC-2 channel function in patients carrying the 597insG or the IVS2- 14del11 mutation. This will impair chloride efflux and presumably result in an intracellular chloride accumulation. Consequently, the inhibitory GABA response will be reduced or might even become excitatory and thus cause epileptic seizures.

Example 11 :G715E Alters Chloride-Dependent Gating

In contrast to M200fsX231 and del74-117, G715E channels were functional but exhibited an altered voltage-dependent gating (Figure 5). Neither current amplitudes (at -125 mV: wild-type: -2.8 ± 0.6 nA, n = 7; G715E: -2.8 ± 0.9 nA, n = 6), nor the voltage dependence of the relative open probability at a high [Cl^"]j (Figure 5B, C) differed significantly from wild-type channels. However, G715E channels displayed a distinct CI^" dependence of channel opening. At [Cr]ι, of 4, 15 and 34 mM, the voltage dependence of activation of G715E channels was shifted to more positive potentials compared with wild-type channels (Figure 5C, D, p < 0.05). As observed for wild-type channels, the V₀.₅ of G715E channels changed in a logarithmic relationship with [Cl^"]ι. Assuming that the voltage dependence of channel opening arises for the most part from voltage-dependent binding of intracellular CI^" (Cui (1997), loc. cit), this dependence indicates that G715E channels exhibit a higher KD (at 0 mV) and a steeper voltage dependence of CI^" binding than wild-type channels. These alterations result in opening of mutant channels at less negative potentials than wild-type channels in a physiological chloride range.

The above-mentioned results demonstrate that the G715E mutation apparently causes neuronal hyperexcitability by a distinct mechanism, i.e. by conducting an enhanced chloride efflux. An efflux of negatively charged chloride ions can cause cell depolarization and thereby alter neuronal excitability. The particular chloride dependence of channel opening of wild-type hCIC-2 channels (Figures 3C, D) presumably prevents substantial membrane depolarization by keeping the CI^" efflux at a low level. In contrast, G715E channels open at less negative potentials due to their different chloride sensitivity (Figure 5C, D). After intense synaptic activation causing membrane depolarization by activation of glutamate receptors and GABAA receptor-mediated CI^" influx, mutant channels will conduct an increased CI^" outward current upon repolarization when Eci becomes more positive than the membrane potential. This abnormal conductance may thus induce recurrent membrane depolarization beyond action potential threshold, quite analogous to pacemaker channels in heart and brain. Such discharges can explain the initiation of epileptic seizures. The alteration of CIC-2 gating by G715E represents a gain-of-function and can explain why heterozygous patients are clinically affected. The findings of the present invention demonstrated, inter alia, in the above described Examples show that loss-of-function as well as gain-of-function CLCN2 mutations can cause epilepsy in humans.

Claims

1. A nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of:

(a) a nucleic acid sequence encoding a polypeptide which has an amino acid sequence of a voltage-gated chloride channel CIC-2, wherein the glycine (Gly) residue corresponding to position 715 of the wild- type voltage-gated chloride channel CIC-2 as depicted in SEQ ID NO: 2 is replaced by another amino acid residue;

(b) a nucleic acid sequence encoding a polypeptide which has an amino acid sequence of a voltage-gated chloride channel CIC-2, wherein amino acids corresponding to positions 74 to 117 of the wild-type voltage-gated chloride channel CIC-2 as depicted in SEQ ID NO: 2 are deleted;

(c) a genomic nucleotide sequence encoding a voltage-gated chloride channel CIC-2 and which contains a mutation in intron 2 which leads to an aberrant splicing of the mRNA transcribed by said genomic nucleotide sequence resulting in a fusion of exons 2 and 4 thereby leading to the production of an mRNA lacking exon;

(e) the nucleotide sequence of SEQ ID NOs: 3, 5 or 7;

(i) a nucleotide sequence which hybridizes to a nucleotide sequence defined in (d) or to the nucleotide sequence depicted in SEQ ID NO: 7 and which encodes a voltage-gated chloride channel CIC-2, wherein the wild-type translational reading frame of the voltage- gated chloride channel CIC-2 is altered due to an insertion between the nucleotides corresponding to position 596 and position 597 of the corresponding wild type nucleotide sequence as depicted in SEQ ID NO: 1 ; and

(j) a nucleic acid sequence being degenerate as a result of the genetic code to the nucleic acid sequence as defined in any one of (g) to (i).

2. The nucleic acid molecule of claim 1 derived from mouse, rat or human.

3. The nucleic acid molecule of claim 1 or 2 which is DNA, RNA, PNA or phosphorothioates.

4. A vector comprising the nucleic acid molecule of any one of claims 1 to 3.

5. The vector of claim 4 which is an expression vector, a gene targeting vector and/or a gene transfer vector.

6. A host transformed with a vector of claim 4 or 5 or transformed with the nucleic acid molecule of any one of claims 1 to 3.

7. The host of claim 6 which is a mammalian cell, an amphibian cell, a fish, an insect cell, a fungal cell, a plant cell or a bacterial cell.

8. The host of claim 7, wherein said mammalian cell is a CHO cell.

9. The host of claim 7, wherein said amphibian cell is an oocyte, preferably a Xenopus oocyte.

10. The host of claim 9, wherein said oocyte is a frog oocyte.

11. The host of claim 6 which is a non-human transgenic organism.

12. The host of claim 11 , wherein said non-human organism is a mammal, amphibian, a fish, an insect, a fungus or a plant.

13. A method for producing the polypeptide encoded by a nucleic acid molecule of any one of claims 1 to 3 comprising culturing/raising the host of any one of claims 6 to 11 and isolating the produced polypeptide.

14. A polypeptide encoded by the nucleic acid molecule of any one of claims 1 to 3 or produced by the method of claim 13.

15. An antibody specifically directed to the polypeptide of claim 14, wherein said antibody specifically reacts with an epitope generated and/or formed by the mutation in the voltage-gated chloride channel CIC-2 selected from the group consisting of:

(i) an epitope presented by a polypeptide which has an amino acid sequence of a voltage-gated chloride channel CIC-2, wherein the glycine (Gly) residue corresponding to position 715 of the wild-type voltage-gated chloride channel CIC-2 as depicted in SEQ ID NO: 2 is replaced by another amino acid residue;

(ii) an epitope presented by a polypeptide which has an. amino acid sequence of a voltage-gated chloride channel CIC-2, wherein amino acids corresponding to positions 74 to 117 of the wild-type voltage- gated chloride channel CIC-2 as depicted in SEQ ID NO: 2 are deleted; and (iii) an epitope presented a polypeptide which has an amino acid sequence of a voltage-gated chloride channel CIC-2, wherein the wild-type translational reading frame of the voltage-gated chloride channel CIC-2 is altered due to an insertion between the nucleotides corresponding to position 596 and position 597 of the corresponding wild type nucleotide sequence as depicted in SEQ ID NO: 1.

16. The antibody of claim 15 which is a monoclonal antibody.

17. An aptamer specifically binding to a nucleic acid molecule of any one of claims 1 to 3 or to the polypeptide of claim 14.

18. A primer or pair of primers capable of specifically amplifying a nucleic acid molecule as defined in any one of claims 1 to 3.

19. The primer of pair of primers of claim 18, which is selected from the group consisting of SEQ ID NOs.: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 and 21.

20. A composition comprising the nucleic acid molecule of any one of claims 1 to 3, the vector of claim 4 or 5, the polypeptide of claim 14, the antibody of claim 15 or 16, the aptamer of claim 17 and/or the primer or pair of primers of claim 18 or 19.

21. The composition of claim 20 which is a diagnostic composition.

22. The diagnostic composition of claim 21 , optionally further comprising suitable means for detection.

23. Use of the nucleic acid molecule of any one of claims 1 to 3, the vector of claim 4 or 5, the polypeptide of claim 14, the antibody of claim 15 or 16, the aptamer of claim 17 and/or the primer or pair of primers of claim 18 or 19 for the preparation of a diagnostic composition for the detection of a neurological disease/disorder.

24. A method of diagnosing a neurological disease or a susceptibility to a neurological disease comprising the step of determining in a sample obtained from an individual whether the CIC-2 protein expressed in the cells of said individual is non-functional or shows an altered voltage- dependent gating in comparison to the wild-type CIC-2 protein.

25. A method of diagnosing a neurological disease or a susceptibility to a neurological disease comprising the step of determining in a sample obtained from a patient whether the voltage-gated chloride channel CIC-2 gene shows a mutation selected from the group consisting of:

(a) a replacement of the glycine (Gly) residue corresponding to position 715 of the wild-type voltage-gated chloride channel CIC-2 as depicted in SEQ ID NO: 2 by another amino acid residue;

(b) a deletion of amino acids corresponding to positions 74 to 117 of the wild-type voltage-gated chloride channel CIC-2 as depicted in SEQ ID NO: 2; and

(c) an insertion between the nucleotides corresponding to position 596 and position 597 of the corresponding wild type nucleotide sequence as depicted in SEQ ID NO: 1 leading to an alteration of the wild-type translational reading frame of the voltage-gated chloride channel CIC-2.

26. The method of claim 25, wherein the occurrence of the mutation in the voltage-gated chloride channel CIC-2 gene is determined by PCR, immunoiogical methods and/or electrophysiological methods.

27. A pharmaceutical composition comprising a nucleic acid molecule comprising a nucleotide sequence which encodes a functional voltage- gated chloride channel CIC-2 and which is selected from the group consisting of:

28. A method of treating a neurological disease comprising administering a therapeutically effective amount of the nucleic acid molecule as defined in claim 27 to a subject suffering from said disease.

29. Use of the nucleic acid molecule as defined in claim 27 for the preparation of a pharmaceutical composition for treating a neurological disease.

30. The use of claim 23 or 29 or the method of claim 25, 26 or 28, wherein said neurological disease is an idiopathic generalized epilepsy (IGE).

31. The use or the method of claim 30, wherein said idiopathic generalized epilepsy (IGE) is selected from the group consisting of childhood absence epilepsy (CAE), juvenile absence epilepsy (JAE), juvenile myoclonic epilepsy (JME) and epilepsy with grand mal seizures on awakening (EGMA).

32. A kit comprising the nucleic acid molecule of any one of claims 1 to 3, a vector of claim 4 or 5, a host of any one of claims 6 to 11 , a polypeptide of claim 14, an antibody of claim 15 or 16, an aptamer of claim 17 and/or a primer or pair of primers of claim 18 or 19.

33. A method for identifying molecules which are capable of specifically interacting with the polypeptide of claim 14, comprising the steps of

(a) contacting a polypeptide of claim 14 with a molecule to be tested; and

(b) determining whether said molecule is capable of specifically interacting with said polypeptide.

34. A method for the characterization of molecules which are capable of altering characteristics of the polypeptide of claim 14, comprising the steps of

(a) contacting a polypeptide of claim 14 with said molecule; and

(b) determining whether the molecule alters a characteristic of the polypeptide of claim 14.

35. A method of screening for molecules which are capable of interacting with the polypeptide of claim 14, comprising the steps of

(a) contacting a polypeptide of claim 14 with said molecule; and

(b) measuring and/or detecting a response; and

(c) comparing said response to a standard response as measured in the absence of said candidate molecule.

36. A method for the production of a pharmaceutical composition comprising the steps of the method of any one of claims 33 to 35 and comprising a further step, wherein a derivative of said identified, characterized and/or screened molecule is generated.

37. A method for the production of a pharmaceutical composition comprising the steps of the method of any one of claims 33 to 36 and formulating the molecules identified, characterized, screened and/or derivatized in pharmaceutically acceptable form.

38. The method of any one of claims 33 to 37, wherein said molecule(s) comprise(s) (a) neuroprotective, (b) nootropic molecule(s) and (c) antiepilectic molecule(s).

39. The method of any one of claims 33 to 38, wherein said molecule(s) comprise(s) antagonist(s), partial antagonist(s), partial agonist(s) and/or agonist(s) for a voltage-gated chloride channel CIC-2 .