AU2010246325A1 - The SCN2A and SCN3A Loci for Idiopathic Generalized Epilepsy, Mutations thereof and Method using same to Assess, Diagnose, Prognose or treat Epilepsy - Google Patents

Description

1 Australian Patents Act 1990 - Regulation 2.3 Original Complete Specification, Standard Patent Invention Title: The SCN2A and SCN3A Loci for Idiopathic Generalized Epilepsy, Mutations thereof and Method using same to Assess, Diagnose, Prognose or Treat Epilepsy The following statement is a full description of this invention, including the best method of performing it known to the applicant: FIELD OF THE INVENTION The present invention relates to epilepsy. More particularly, the present invention relates to idiopathic generalized epilepsy (IGE) and to the identification of two loci 5 mapping to chromosome 2, which show a linkage with epilepsy in patients. The invention further relates to nucleic acid sequences, and protein sequences of these loci (SCNA), to variations and mutations in these sequences and to the use thereof to assess, diagnose, prognose or treat epilepsy. The invention also provides screening assays using SCN2A and/or SCN3A which can identify compounds which have 0 therapeutic benefit for epilepsy and related neurological disorders. BACKGROUND OF THE INVENTION Epilepsy is one of the most common neurological conditions, occurring in about 1.0% of the general population. The disease is characterised by paroxysmal abnormal 15 electrical discharges in the brain, which lead to transient cerebral dysfunction in the form of a seizure. A seizure is considered partial when the epileptic discharge is limited to part of one brain hemisphere, or generalised when it involves both cerebral hemispheres at the onset. The current classification of the epileptic syndromes rests on two criteria: 1) seizure type which may be generalised or partial at the onset, 20 according to clinical and EEG features; and 2) etiology, which may be idiopathic, cryptogenic and symptomatic. Symptomatic epilepsies have multiple and heterogeneous causes including brain injury, CNS infection, migrational and metabolic disorders. In the majority (65%) of the patients with either generalised or partial epilepsy, there is no underlying cause (idiopathic) or the cause is thought to be hidden or occult (cryptogenic). 25 Also, in the idiopathic epileptic syndromes, there is no evidence of cerebral dysfunction 2 other than the seizure, and the neurological examination is normal. There is now increasing evidence that in this latter group, genetic factors are important, especially for the idiopathic generalised epilepsy (IGE). In a recent study, Berkovic et al., (1998) showed a 62% concordance rate in monozygotic twins overall for epilepsy. In this study, a 5 higher concordance rate has been found in the generalised compared to the partial epilepsies, with 76% concordance rate for IGE. Recent studies using molecular genetic approaches have shown that many susceptibility genes for the epilepsies in human involve membrane ion channel and related proteins. These studies include the syndrome of benign familial neonatal convulsions where two loci have been identified [EBN1 on 10 chromosome 20, the KCNQ2 gene (a potassium channel); and EBN2 on chromosome 8, the KCNQ3 gene (also a potassium channel)] (Bievert et al., 1998; Charlier et al., 1998; Singh et al., 1998), as well as autosomal dominant nocturnal frontal lobe epilepsy [ADNFLE - chromosome 20, and the CHRNA4 gene (the neuronal nicotinic acetylcholine receptor alpha 4 subunit)] (Steinlein et al., 1995). More recently, there was a clinical 15 description of a new syndrome (GEFS), which consisted of generalised epilepsy with febrile seizures. According to the current classification of epileptic syndrome, this syndrome would fall in the category of IGE, based on the seizure and electroencaphalographic features. However, febrile seizures were present in all probands with GEFS, and the pattern of inheritance was clearly autosomal dominant, 20 which are not part of the usual IGE phenotype. This unique GEFS syndrome has been shown to be associated with a mutation on the beta-1 subunit of brain voltage-gated sodium channel (SCN1B) gene (Wallace et al., 1998). In addition, three different groups, including the group of the present inventors, have identified another locus on chromosome 2 in large kindred with this specific syndrome (GEFS). This region 25 contains many candidate genes, including a cluster of alpha subunits of sodium channels (SCNA). Voltage-gated sodium channels play an important role in the generation of action potential in nerve cells and muscle. The alpha subunit (SCNA) is the main component of the channel, and would be sufficient to generate an efficient channel when expressed in cells in vitro. In turn, the beta-1 and 2 subunits need an 30 alpha subunit to give an effective channel. The role of these subunits would be to modifiy the kinetic properties of the channel, mainly by fast inactivation of the sodium currents. The mutation found in the GEFS syndrome on the SCN1B gene was shown to reduce the fast inactivation of the sodium channels as compared to a normal SCNB1, when co-expressed with an alpha subunit. It is probable that this could be the 3 mechanism by which the mutation induce an hyperexcitability state in the brain, leading to seizure in humans. Interestingly, the mechanism of action of most of the anticonvulsant drugs is through a reduction of the repetitive firing of neurons, which is also known to be dependent on fast inactivation. These finding make it likely that 5 additional epilepsy genes will be identified by mutations in ion channels. There thus remains a need to identify whether IGE is caused by a mutation in a sodium channel (SCNA). There also remains a need to assess whether a mutation(s) in SCNA is associated with GEFS. There also remains a need to determine whether a 10 mutation that affects the fast inactivation of a sodium channel, given the particular phenotype of GEFS or IGE, could be linked to a region which includes SCNA genes. The present invention seeks to meet these and other needs. 15 The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety. SUMMARY OF THE INVENTION In one embodiment, the present invention relates to a genetic assay for determining 20 predisposition to epilepsy. In another embodiment, the present invention relates to a use of at least one of the loci of the present invention or an equivalent thereof (e.g., a loci in linkage disequilibrium therewith) as a marker for epilepsy and to determine the optimal treatment thereof 25 (e.g., to guide the treatment modalities, thereby optimizing treatment to a particular clinical situation). Yet in another embodiment, the present invention relates to an assay to screen for drugs for the treatment and/or prevention of epilepsy. In a particular embodiment, 30 such assays can be designed using cells from patients having a known genotype at one of the loci of the present invention. These cells harboring recombinant vectors can enable an assessment of the functionality of the SCN2A and/or SCN3A. Non-limiting examples of assays that could be used in accordance with the present invention include cis-trans assays similar to those described in U.S.P. 4,981,784.

4 It shall be understood that the determination of allelic variations in at least one of the loci of the present invention can be combined to the determination of allelic variation in other gene/markers linked to a predisposition to epilepsy. This combination of 5 genotype analyses could lead to better diagnosis programs and/or treatment of epilepsy. Non-limiting examples of such markers include SCN1B, EBN1, KCNQ2, EBN2, KCNQ3, ADNFLE, SCN1A and CHRNA4. In accordance with the present invention, there is therefore provided a method of 10 determining an individual's predisposition to epilepsy, which comprises determining the genotype of at least one locus selected from the group consisting of SCN2A and SCN3A. In one particular embodiment, the present invention provides a method of determining an individual's predisposition to epilepsy, which comprises determining a polymorphism (directly or indirectly by linkage disequilibrium) in a biological sample of 15 an individual and analyzing the allelic variation in at least one of the loci selected from SCN2A and SCN3A, thereby determining an individual's predisposition to epilepsy. In accordance with the present invention, there is also provided a method for identifying, from a library of compounds, a compound with therapeutic effect on ?0 epilepsy or other neurological disorders comprising providing a screening assay comprising a measurable biological activity of SCN2A or SCN3A protein or gene; contacting the screening assay with a test compound; and detecting if the test compound modulates the biological activity of SCN2A or SCN3A protein or gene; wherein a test compound which modulates the biological activity is a compound with 25 this therapeutic effect. Also provided within the present invention is a compound having therapeutic effect on epilepsy or other neurological disorders, identified by a method comprising: providing a screening assay comprising a measurable biological activity of SCN2A or SCN3A 30 protein or gene; contacting the screening assay with a test compound; and detecting if the test compound modulates the biological activity of SCN2A or SCN3A protein or gene, wherein a test compound which modulates the biological activity is a compound with this therapeutic effect.

5 SCN2A and SCN3A refer to genes and proteins for Sodium Channel, Neuronal Type 1, Alpha Subunit isoforms, and are described at OMIM # 182389 (Online Mendelian Inheritance in Man). These genes are structurally distinct sodium channel alpha subunit isoforms in brain, also known as brain types 11 and 111, respectively. Gene, 5 cDNA and protein sequences for the various isoforms are shown in SEQ ID NOs:1-66. Numerous methods for determining a genotype are known and available to the skilled artisan. All these genotype determination methods are within the scope of the present invention. In a particular embodiment of a method of the present invention, the 10 determination of the genotype comprises an amplification of a segment of one of the loci selected from the group consisting of SCN2A and SCN3A and in a particularly preferred embodiment, the amplification is carried out using polymerase chain reaction. 15 In a particular embodiment, a pair of primers is designed to specifically amplify a segment of one of the markers of the present invention. This pair of primers is preferably derived from a nucleic acid sequence of SCN2A or SCN3A or from sequences flanking these genes, to amplify a segment of SCN2A or SCN3A (or to amplify a segment of a loci in linkage disequilibrium with at least one of the loci of the 20 present invention). While a number of primers are exemplified herein, other primer pairs can be designed, using the sequences of the SCN2A and SCN3A nucleic acids molecules described hereinbelow. The same would apply to primer pairs from loci in linkage disequilibrium with the markers of the present invention. 25 Restriction fragment length polymorphisms can be used to determine polymorphisms at the SCN2A and SCN3A loci (and equivalent loci). While human SCN2A and SCN3A are preferred sequences (nucleic acid and proteins) in accordance with the present invention, the invention should not be so limited. 30 Indeed, in view of the significant conservation of these genes throughout evolution, sequences from different species, and preferably mammalian species, could be used in the assays of the present invention. Non-limiting examples include orthologs from rat and mice, which show significant conservation with their corresponding human genes.

6 In order to provide a clear and consistent understanding of terms used in the present description, a number of definitions are provided hereinbelow. 5 As used herein the term "RFLP" refers to restriction fragment length polymorphism. The terms "polymorphism", "DNA polymorphism" and the like, refer to any sequence in the human genome which exists in more than one version or variant in the population. 10 The term "linkage disequilibrium" refers to any degree of non-random genetic association between one or more allele(s) of two different polymorphic DNA sequences, that is due to the physical proximity of the two loci. Linkage disequilibrium is present when two DNA segments that are very close to each other on a given chromosome will tend to remain unseparated for several generations with the 15 consequence that alleles of a DNA polymorphism (or marker) in one segment will show a non-random association with the alleles of a different DNA polymorphism (or marker) located in the other DNA segment nearby. Hence, testing of a marker in linkage disequilibrium with the polymorphisms of the present invention at the SCN2A and/or SCN3A genes (indirect testing), will give almost the same information as testing for the 20 SCN2A and SCN3A polymorphisms directly. This situation is encountered throughout the human genome when two DNA polymorphisms that are very close to each other are studied. Linkage disequilibriums are well known in the art and various degrees of linkage disequilibrium can be encountered between two genetic markers so that some are more closely associated than others. 25 It shall be recognized by the person skilled in the art to which the present invention pertains, that since some of the polymorphisms or mutations herein identified in the SCN2A and/or SCN3A genes can be within the coding region of the genes and therefore expressed, that the present invention should not be limited to the 30 identification of the polymorphisms/mutations at the DNA level (whether on genomic DNA, amplified DNA, cDNA, or the like). Indeed, the herein-identified polymorphisms and/or mutations could be detected at the mRNA or protein level. Such detections of polymorphism identification on mRNA or protein are known in the art. Non-limiting examples include detection based on oligos designed to hybridize to mRNA or ligands 7 such as antibodies which are specific to the encoded polymorphism (i.e. specific to the protein fragment encoded by the distinct polymorphisms). Nucleotide sequences are presented herein by single strand, in the 5' to 3' direction, 5 from left to right, using the one letter nucleotide symbols as commonly used in the art and in accordance with the recommendations of the IUPAC-IUB Biochemical Nomenclature Commission. Unless defined otherwise, the scientific and technological terms and nomenclature 10 used herein have the same meaning as commonly understood by a person of ordinary skill to which this invention pertains. Generally, the procedures for cell cultures, infection, molecular biology methods and the like are common methods used in the art. Such standard techniques can be found in reference manuals such as for example Sambrook et al., (1989, Molecular Cloning - A Laboratory Manual, Cold Spring Harbor 15 Laboratories) and Ausubel et al., (1994, Current Protocols in Molecular Biology, Wiley, New York). The present description refers to a number of routinely used recombinant DNA (rDNA) technology terms. Nevertheless, definitions of selected examples of such rDNA terms 20 are provided for clarity and consistency. As used herein, "nucleic acid molecule", refers to a polymer of nucleotides. Non-limiting examples thereof include DNA (i.e. genomic DNA, cDNA, RNA molecules (i.e. mRNA) and chimeras of DNA and RNA. The nucleic acid molecule can be 25 obtained by cloning techniques or synthesized. DNA can be double-stranded or single-stranded (coding strand or non-coding strand [antisense]). The term "recombinant DNA" as known in the art refers to a DNA molecule resulting from the joining of DNA segments. This is often referred to as genetic engineering. 30 The term "DNA segment", is used herein, to refer to a DNA molecule comprising a linear stretch or sequence of nucleotides. This sequence when read in accordance with the genetic code, can encode a linear stretch or sequence of amino acids which can be referred to as a polypeptide, protein, protein fragment and the like.

8 The terminology "amplification pair" refers herein to a pair of oligonucleotides (oligos) of the present invention, which are selected to be used together in amplifying a selected nucleic acid sequence by one of a number of types of amplification 5 processes, preferably a polymerase chain reaction. Other types of amplification processes include ligase chain reaction, strand displacement amplification, or nucleic acid sequence-based amplification, as explained in greater detail below. As commonly known in the art, the oligos are designed to bind to a complementary sequence under selected conditions. 10 The nucleic acid (i.e. DNA, RNA or chimeras thereof) for practicing the present invention may be obtained according to well known methods. Oligonucleotide probes or primers of the present invention may be of any suitable 15 length, depending on the particular assay format and the particular needs and targeted genomes employed. In general, the oligonucleotide probes or primers are at least 12 nucleotides in length, preferably between 15 and 24 molecules, and they may be adapted to be especially suited to a chosen nucleic acid amplification system. As commonly known in the art, the oligonucleotide probes and primers can be designed 20 by taking into consideration the melting point of hydrizidation thereof with its targeted sequence (see below and in Sambrook et al., 1989, Molecular Cloning -A Laboratory Manual, 2nd Edition, CSH Laboratories; Ausubel et al., 1989, in Current Protocols in Molecular Biology, John Wiley & Sons Inc., N.Y.). 25 The term "DNA" molecule or sequence (as well as sometimes the term "oligonucleotide") refers to a molecule comprised of the deoxyribonucleotides adenine (A), guanine (G), thymine (T) and/or cytosine (C). Sometimes, in a double-stranded form, it can comprise or include a "regulatory element" according to the present invention, as the term is defined herein. The term "oligonucleotide" or "DNA" can be 30 found in linear DNA molecules or fragments, viruses, plasmids, vectors, chromosomes or synthetically derived DNA. As used herein, particular double-stranded DNA sequences may be described according to the normal convention of giving only the sequence in the 5' to 3' direction. Of course, as very well-known, DNA molecules or sequences are often in single stranded form.

9 "Nucleic acid hybridization" refers generally to the hybridization of two single-stranded nucleic acid molecules having complementary base sequences, which under appropriate conditions will form a thermodynamically favored double-stranded 5 structure. Examples of hybridization conditions can be found in the two laboratory manuals referred to above (Sambrook et al., 1989, supra and Ausubel et al., 1989, supra) and are commonly known in the art. In the case of a hybridization to a nitrocellulose filter, as for example in the well known Southern blotting procedure, a nitrocellulose filter can be incubated overnight at 65*C with a labeled probe in a 10 solution containing 50% formamide, high salt (5 x SSC or 5 x SSPE), 5 x Denhardt's solution, 1% SDS, and 100 pg/ml denatured carrier DNA (i.e. salmon sperm DNA). The non-specifically binding probe can then be washed off the filter by several washes in 0.2 x SSC/0.1% SDS at a temperature which is selected in view of the desired stringency: room temperature (low stringency), 420C (moderate stringency) or 65 0 C 15 (high stringency). The selected temperature is based on the melting temperature (Tm) of the DNA hybrid. Of course, RNA-DNA hybrids can also be formed and detected. In such cases, the conditions of hybridization and washing can be adapted according to well known methods by the person of ordinary skill. Stringent conditions will be preferably used (Sambrook et al.,1989, supra). Probes of the invention can be utilized with naturally occurring sugar-phosphate backbones as well as modified backbones including phosphorothioates, dithionates, alkyl phosphonates and a-nucleotides and the like. Modified sugar-phosphate backbones are generally taught by Miller, 1988, Ann. Reports Med. Chem. 23:295 and 25 Moran et al., 1987, Nucleic Acids Res., 14:5019. Probes of the invention can be constructed of either ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), and preferably of DNA. The types of detection methods in which probes can be used include Southern blots 30 (DNA detection), dot or slot blots (DNA, RNA), and Northern blots (RNA detection). Although less preferred, labeled proteins could also be used to detect a particular nucleic acid sequence to which it binds. More recently, PNAs have been described (Nielsen et al., 1999, Current Opin. Biotechnol., 10:71-75). PNAs could also be used to detect the polymorphisms of the present invention. Other detection methods include 10 kits containing probes on a dipstick setup and the like. Although the present invention is not specifically dependent on the use of a label for the detection of a particular nucleic acid sequence, such a label might be beneficial, by 5 increasing the sensitivity of the detection. Furthermore, it enables automation. Probes can be labeled according to numerous well known methods (Sambrook et al., 1989, supra). Non-limiting examples of labels include 3 H, 14 C, 32 P, and 35 S. Non-limiting examples of detectable markers include ligands, fluorophores, chemiluminescent agents, enzymes, and antibodies. Other detectable markers for use with probes, which 10 can enable an increase in sensitivity of the method of the invention, include biotin and radionucleotides. It will become evident to the person of ordinary skill that the choice of a particular label dictates the manner in which it is bound to the probe. As commonly known, radioactive nucleotides can be incorporated into probes of the 15 invention by several methods. Non-limiting examples thereof include kinasing the 5' ends of the probes using gamma 32 P ATP and polynucleotide kinase, using the Klenow fragment of Pol I of E. coli in the presence of radioactive dNTP (i.e. uniformly labeled DNA probe using random oligonucleotide primers in low-melt gels), using the SP6/T7 system to transcribe a DNA segment in the presence of one or more radioactive NTP, 20 and the like. As used herein, "oligonucleotides" or "oligos" define a molecule having two or more nucleotides (ribo or deoxyribonucleotides). The size of the oligo will be dictated by the particular situation and ultimately on the particular use thereof and adapted accordingly 25 by the person of ordinary skill. An oligonucleotide can be synthesized chemically or derived by cloning according to well known methods. As used herein, a "primer" defines an oligonucleotide which is capable of annealing to a target sequence, thereby creating a double stranded region which can serve as an 30 initiation point for nucleic acid synthesis under suitable conditions. Amplification of a selected, or target, nucleic acid sequence may be carried out by a number of suitable methods. See generally Kwoh et al., 1990, Am. Biotechnol. Lab. 8:14-25. Numerous amplification techniques have been described and can be readily adapted to suit particular needs of a person of ordinary skill. Non-limiting examples of 11 amplification techniques include polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), transcription-based amplification, the Q-beta replicase system and NASBA (Kwoh et al., 1989, Proc. NatI. Acad. Sci. USA 86, 1173-1177; Lizardi et al., 1988, BioTechnology 6:1197-1202; 5 Malek et al., 1994, Methods Mol. Biol., 28:253-260; and Sambrook et al., 1989, supra). Preferably, amplification will be carried out using PCR. Polymerase chain reaction (PCR) is carried out in accordance with known techniques. See, e.g., U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159; and 4,965,188 (the 10 disclosures of all four U.S. Patents are incorporated herein by reference). In general, PCR involves, a treatment of a nucleic acid sample (e.g., in the presence of a heat stable DNA polymerase) under hybridizing conditions, with one oligonucleotide primer for each strand of the specific sequence to be detected. An extension product of each primer which is synthesized is complementary to each of the two nucleic acid strands, 15 with the primers sufficiently complementary to each strand of the specific sequence to hybridize therewith. The extension product synthesized from each primer can also serve as a template for further synthesis of extension products using the same primers. Following a sufficient number of rounds of synthesis of extension products, the sample is analysed to assess whether the sequence or sequences to be detected 20 are present. Detection of the amplified sequence may be carried out by visualization following EtBr staining of the DNA following gel electrophores, or using a detectable label in accordance with known techniques, and the like. For a review on PCR techniques (see PCR Protocols, A Guide to Methods and Amplifications, Michael et al., Eds, Acad. Press, 1990). 25 Ligase chain reaction (LCR) is carried out in accordance with known techniques (Weiss, 1991, Science 254:1292). Adaptation of the protocol to meet the desired needs can be carried out by a person of ordinary skill. Strand displacement amplification (SDA) is also carried out in accordance with known techniques or 30 adaptations thereof to meet the particular needs (Walker et al., 1992, Proc. NatI. Acad. Sci. USA 89:392-396; and ibid., 1992, Nucleic Acids Res. 20:1691-1696). As used herein, the term "gene" is well known in the art and relates to a nucleic acid sequence defining a single protein or polypeptide. A "structural gene" defines a DNA 12 sequence which is transcribed into RNA and translated into a protein having a specific amino acid sequence thereby giving rise to a specific polypeptide or protein. It will be readily recognized by the person of ordinary skill, that the nucleic acid sequence of the present invention can be incorporated into anyone of numerous established kit formats 5 which are well known in the art. A "heterologous" (i.e. a heterologous gene) region of a DNA molecule is a subsegment of DNA within a larger segment that is not found in association therewith in nature. The term "heterologous" can be similarly used to define two polypeptidic segments not 10 joined together in nature. Non-limiting examples of heterologous genes include reporter genes such as luciferase, chloramphenicol acetyl transferase, beta galactosidase, and the like which can be juxtaposed or joined to heterologous control regions or to heterologous polypeptides. 15 The term "vector" is commonly known in the art and defines a plasmid DNA, phage DNA, viral DNA and the like, which can serve as a DNA vehicle into which DNA of the present invention can be cloned. Numerous types of vectors exist and are well known in the art. 20 The term "expression" defines the process by which a gene is transcribed into mRNA (transcription), the mRNA is then being translated (translation) into one polypeptide (or protein) or more. The terminology "expression vector" defines a vector or vehicle as described above but 25 designed to enable the expression of an inserted sequence following transformation into a host. The cloned gene (inserted sequence) is usually placed under the control of control element sequences such as promoter sequences. The placing of a cloned gene under such control sequences is often referred to as being operably linked to control elements or sequences. 30 Operably linked sequences may also include two segments that are transcribed onto the same RNA transcript. Thus, two sequences, such as a promoter and a "reporter sequence" are operably linked if transcription commencing in the promoter will produce an RNA transcript of the reporter sequence. In order to be "operably linked" it is not necessary that two sequences be immediately adjacent to one another.

13 Expression control sequences will vary depending on whether the vector is designed to express the operably linked gene in a prokaryotic or eukaryotic host or both (shuttle vectors) and can additionally contain transcriptional elements such as enhancer elements, termination sequences, tissue-specificity elements, and/or translational initiation and termination sites. Prokaryotic expressions are useful for the preparation of large quantities of the protein encoded by the DNA sequence of interest. This protein can be purified according to ) standard protocols that take advantage of the intrinsic properties thereof, such as size and charge (i.e. SDS gel electrophoresis, gel filtration, centrifugation, ion exchange chromatography). In addition, the protein of interest can be purified via affinity chromatography using polyclonal or monoclonal antibodies. The purified protein can be used for therapeutic applications. 5 The DNA construct can be a vector comprising a promoter that is operably linked to an oligonucleotide sequence of the present invention, which is in turn, operably linked to a heterologous gene, such as the gene for the luciferase reporter molecule. "Promoter" refers to a DNA regulatory region capable of binding directly or indirectly to RNA .0 polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. For purposes of the present invention, the promoter is bound at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter will be found a transcription 25 initiation site (conveniently defined by mapping with S1 nuclease), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CCAT" boxes. Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the -10 and -35 consensus sequences. 30 In accordance with one embodiment of the present invention, an expression vector can be constructed to assess the functionality of specific alleles of the SCN2A and SCN3A sodium channels. Non-limiting examples of such expression vectors include a vector comprising the nucleic acid sequence encoding one of the sodium channels (or part thereof) according to the present invention. These vectors can be transfected in cells.

14 The sequences of the alpha subunit of the sodium channels in accordance with the present invention and their structure-function relationship could be assessed by a number of methods known to the skilled artisan. One non-limiting example includes the use of cells expressing the P-1 and p-2 subunits and the sequence of an alpha 5 subunit in accordance with the present invention. For example, an alpha subunit having a mutation, which is linked to epilepsy, could be compared to a sequence devoid of that mutation, as a control. In such cells, the functionality of the sodium channel could be tested as known to the skilled artisan and these cells could be used to screen for agents which could modulate the activity of the sodium channel. For 10 example, agents could be tested and selected, which would reduce the hyperexcitability state of the sodium channel (e.g., their reduction in fast inactivation). Agents known to the person of ordinary skill as affecting other sodium channels could be tested, for example, separately or in batches. Of course, it will be understood that the SCN2A and/or SCN3A genes expressed by these cells can be modified at will 15 (e.g., by in vitro mutagenesis or the like). As used herein, the designation "functional derivative" denotes, in the context of a functional derivative of a sequence whether a nucleic acid or amino acid sequence, a molecule that retains a biological activity (either function or structural; e.g., sodium 20 channel function or structure) that is substantially similar to that of the original sequence. This functional derivative or equivalent may be a natural derivative or may be prepared synthetically. Such derivatives include amino acid sequences having substitutions, deletions, or additions of one or more amino acids, provided that the biological activity of the protein is conserved. The same applies to derivatives of 25 nucleic acid sequences which can have substitutions, deletions, or additions of one or more nucleotides, provided that the biological activity of the sequence is generally maintained. When relating to a protein sequence, the substituting amino acid generally has chemico-physical properties which are similar to that of the substituted amino acid. The similar chemico-physical properties include, similarities in charge, bulkiness, 30 hydrophobicity, hydrophylicity and the like. The term "functional derivatives" is intended to include "fragments", "segments", "variants", "analogs" or "chemical derivatives" of the subject matter of the present invention. The genetic code, the chemico-physical characteristics of amino acids and teachings relating to conservative vs. non-conservative mutations are well-known in the art. Non-limiting examples of 15 textbooks teaching such information are Stryer, Biochemistry, 3rd ed.; and Lehninger, Biochemistry, 3rd ed. The functional derivatives of the present invention can be synthesized chemically or produced through recombinant DNA technology. All these methods are well known in the art. 5 The term "variant" refers herein to a protein or nucleic acid molecule which is substantially similar in structure and biological activity to the protein or nucleic acid of the present invention. 10 As used herein, "chemical derivatives" is meant to cover additional chemical moieties not normally part of the subject matter of the invention. Such moieties could affect the physico-chemical characteristic of the derivative (i.e. solubility, absorption, half life, decrease of toxicity and the like). Such moieties are exemplified in Remington's Pharmaceutical Sciences (1980). Methods of coupling these chemical-physical 15 moieties to a polypeptide or nucleic acid sequence are well known in the art. The term "allele" defines an alternative form of a gene which occupies a given locus on a chromosome. ?0 As commonly known, a "mutation" is a detectable change in the genetic material which can be transmitted to a daughter cell. As well known, a mutation can be, for example, a detectable change in one or more deoxyribonucleotide. For example, nucleotides can be added, deleted, substituted for, inverted, or transposed to a new position. Spontaneous mutations and experimentally induced mutations exist. The result of 25 mutations of a nucleic acid molecule is a mutant nucleic acid molecule. A mutant polypeptide can be encoded from this mutant nucleic acid molecule. As used herein, the term "purified" refers to a molecule having been separated from a cellular component. Thus, for example, a "purified protein" has been purified to a level 30 not found in nature. A "substantially pure" molecule is a molecule that is lacking in all other cellular components. As used herein, "SCNA biological activity" refers to any detectable biological activity of SCN2A or SCN3A gene or protein (herein sometimes collectively called SCNA genes 16 or SCNA proteins). This includes any physiological function attributable to an SCNA gene or protein. It can include the specific biological activity of SCNA proteins which is efflux of sodium or related ions. This includes measurement of channel properties such as, but not limited to: 1) the voltage-dependence of activation, a measure of the 5 strength of membrane depolarization necessary to open the channels, 2) the voltage dependence of steady state inactivation, a measure of the fraction of channels available to open at the resting membrane potential; and 3) the time course of inactivation. At a larger scale, SCNA biological activity includes transmission of impulses through cells, wherein changes in transmission characteristics caused by 10 modulators of SCNA proteins can be identified. Non-limiting examples of such measurements of these biological activities may be made directly or indirectly, such as through the transient accumulation of ions in a cell, dynamics of membrane depolarization, etc. SCNA biological activity is not limited, however, to these most important biological activities herein identified. Biological activities may also include 15 simple binding or pKa analysis of SCNA with compounds, substrates, interacting proteins, and the like. For example, by measuring the effect of a test compound on its ability to increase or inhibit such SCNA binding or interaction is measuring a biological activity of SCNA according to this invention. SCNA biological activity includes any standard biochemical measurement of SCNA such as conformational changes, ?0 phosphorylation status or any other feature of the protein that can be measured with techniques known in the art. Finally, SCNA biological activity also includes activities related to SCNA gene transcription or translation, or any biological activities of such transcripts or translation products. 25 As used herein, the terms "molecule", "compound", "agent" or "ligand" are used interchangeably and broadly to refer to natural, synthetic or semi-synthetic molecules or compounds. The term "molecule" therefore denotes for example chemicals, macromolecules, cell or tissue extracts (from plants or animals) and the like. Non limiting examples of molecules include nucleic acid molecules, peptides, ligands 30 (including, for example, antibodies and carbohydrates) and pharmaceutical agents. The agents can be selected and screened by a variety of means including random screening, rational selection and by rational design using for example protein or ligand modelling methods such as computer modelling. The terms "rationally selected" or "rationally designed" are meant to define compounds which have been chosen based 17 on the configuration of the interacting domains of the present invention. As will be understood by the person of ordinary skill, macromolecules having non-naturally occurring modifications are also within the scope of the term "molecule". For example, peptidomimetics, well known in the pharmaceutical industry and generally referred to 5 as peptide analogs can be generated by modelling as mentioned above. Similarly, in a preferred embodiment, the polypeptides of the present invention are modified to enhance their stability. It should be understood that in most cases this modification should not alter the biological activity of the protein. The molecules identified in accordance with the teachings of the present invention have a therapeutic value in 10 diseases or conditions in which sodium transport through the sodium channels is compromised by a mutation (or combination thereof) in one of the genes identified in accordance with the present invention. Alternatively, the molecules identified in accordance with the teachings of the present invention find utility in the development of compounds which can modulate the activity of the alpha subunit sodium channels 15 and/or the action potential in nerve cells and muscles cells (e.g., restore the fast inactivation of the sodium channel to normal levels). As used herein, agonists and antagonists also include potentiators of known compounds with such agonist or antagonist properties. In one embodiment, 20 modulators of the fast inactivation of the sodium channel in accordance with the present invention can be identified and selected by contacting the indicator cell with a compound or mixture or library of molecules for a fixed period of time. As used herein the recitation "indicator cells" refers to cells that express at least one 25 sodium channel a subunit (SCNA) according to the present invention. As alluded to above, such indicator cells can be used in the screening assays of the present invention. In certain embodiments, the indicator cells have been engineered so as to express a chosen derivative, fragment, homolog, or mutant of the combination of genotypes of the present invention. The cells can be yeast cells or higher eukaryotic 30 cells such as mammalian cells. In one particular embodiment, the indicator cell would be a yeast cell harboring vectors enabling the use of the two hybrid system technology, as well known in the art (Ausubel et al., 1994, supra) and can be used to test a compound or a library thereof. In another embodiment, the cis-trans assay as described in USP 4,981,784, can be adapted and used in accordance with the present 18 invention. Such an indicator cell could be used to rapidly screen at high-throughput a vast array of test molecules. In a particular embodiment, the reporter gene is luciferase or beta-Gal. 5 It shall be understood that the "in vivo" experimental model can also be used to carry out an "in vitro" assay. For example, cellular extracts from the indicator cells can be prepared and used in an "in vitro"test. A non-limiting example thereof includes binding assays. 10 In some embodiments, it might be beneficial to express a fusion protein. The design of constructs therefor and the expression and production of fusion proteins and are well known in the art (Sambrook et al., 1989, supra; and Ausubel et al., 1994, supra). Non-limiting examples of such fusion proteins include hemaglutinin fusions and 15 Gluthione-S-transferase (GST) fusions and Maltose binding protein (MBP) fusions. In certain embodiments, it might be beneficial to introduce a protease cleavage site between the two polypeptide sequences which have been fused. Such protease cleavage sites between two heterologously fused polypeptides are well known in the art. 20 In certain embodiments, it might also be beneficial to fuse the protein of the present invention to signal peptide sequences enabling a secretion of the fusion protein from the host cell. Signal peptides from diverse organisms are well known in the art. Bacterial OmpA and yeast Suc2 are two non-limiting examples of proteins containing 25 signal sequences. In certain embodiments, it might also be beneficial to introduce a linker (commonly known) between the interaction domain and the heterologous polypeptide portion. Such fusion protein find utility in the assays of the present invention as well as for purification purposes, detection purposes and the like. For certainty, the sequences and polypeptides useful to practice the invention include 30 without being limited thereto mutants, homologs, subtypes, alleles and the like. It shall be understood that generally, the sequences of the present invention should encode a functional (albeit defective) alpha subunit of sodium channels (SCNA). It will be clear to the person of ordinary skill that whether the SCNA sequence of the present invention, variant, derivative, or fragment thereof retains its function, can be 19 determined by using the teachings and assays of the present invention and the general teachings of the art. It should be understood that the SCNA protein of the present invention can be 5 modified, for example by in vitro mutagenesis, to dissect the structure-function relationship thereof and permit a better design and identification of modulating compounds. However, some derivative or analogs having lost their biological function may still find utility, for example for raising antibodies. These antibodies could be used for detection or purification purposes. In addition, these antibodies could also act as 10 competitive or non-competitive inhibitor and be found to be modulators of the activity of the SCNA proteins of the present invention. A host cell or indicator cell has been "transfected" by exogenous or heterologous DNA (e.g., a DNA construct) when such DNA has been introduced inside the cell. The 15 transfecting DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell. In prokaryotes, yeast, and mammalian cells for example, the transfecting DNA may be maintained on a episomal element such as a plasmid. With respect to eukaryotic cells, a stably transfected cell is one in which the transfecting DNA has become integrated into a chromosome so that it is inherited by 20 daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transfecting DNA. Transfection methods are well known in the art (Sambrook et al., 1989, supra; Ausubel et al., 1994 supra). The use of a mammalian cell as indicator can provide the advantage of furnishing an 25 intermediate factor, which permits for example the interaction of two polypeptides which are tested, that might not be present in lower eukaryotes or prokaryotes. It will be understood that extracts from mammalian cells for example could be used in certain embodiments, to compensate for the lack of certain factors. In general, techniques for preparing antibodies (including monoclonal antibodies and 30 hybridomas) and for detecting antigens using antibodies are well known in the art (Campbell, 1984, in "Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry and Molecular Biology", Elsevier Science Publisher, Amsterdam, The Netherlands) and in Harlow et al., 1988 (in: Antibody- A Laboratory Manual, CSH Laboratories). The present invention also provides polyclonal, monoclonal antibodies, 20 or humanized versions thereof, chimeric antibodies and the like which inhibit or neutralize their respective interaction domains and/or are specific thereto. From the specification and appended claims, the term therapeutic agent should be 5 taken in a broad sense so as to also include a combination of at least two such therapeutic agents. Further, the DNA segments or proteins according to the present invention could be introduced into individuals in a number of ways. For example, cells can be isolated from the afflicted individual, transformed with a DNA construct according to the invention and reintroduced to the afflicted individual in a number of 10 ways. Alternatively, the DNA construct can be administered directly to the afflicted individual. The DNA construct can also be delivered through a vehicle such as a liposome, which can be designed to be targeted to a specific cell type, and engineered to be administered through different routes. 15 For administration to humans, the prescribing medical professional will ultimately determine the appropriate form and dosage for a given patient, and this can be expected to vary according to the chosen therapeutic regimen (i.e. DNA construct, protein, cells), the response and condition of the patient as well as the severity of the disease. 20 Compositions within the scope of the present invention should contain the active agent (i.e. molecule, hormone) in an amount effective to achieve the desired therapeutic effect while avoiding adverse side effects. Typically, the nucleic acids in accordance with the present invention can be administered to mammals (i.e. humans) in doses 25 ranging from 0.005 to 1 mg per kg of body weight per day of the mammal which is treated. Pharmaceutically acceptable preparations and salts of the active agent are within the scope of the present invention and are well known in the art (Remington's Pharmaceutical Science, 16th Ed., Mack Ed.). For the administration of polypeptides, antagonists, agonists and the like, the amount administered should be chosen so as to 30 avoid adverse side effects. The dosage will be adapted by the clinician in accordance with conventional factors such as the extent of the disease and different parameters from the patient. Typically, 0.001 to 50 mg/kg/day will be administered to the mammal. The present invention also relates to a kit for diagnosing and/or prognosing epilepsy, 21 and/or predicting response to a medication comprising an assessment of a genotype at SCNA loci of the present invention (or loci in linkage disequilibrium therewith) using a nucleic acid fragment, a protein or a ligand, a restriction enzyme or the like, in accordance with the present invention. For example, a compartmentalized kit in 5 accordance with the present invention includes any kit in which reagents are contained in separate containers. Such containers include small glass containers, plastic containers or strips of plastic or paper. Such containers allow the efficient transfer of reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated and the agents or solutions of each container can 10 be added in a quantitative fashion from one compartment to another. Such containers will include in one particular embodiment a container which will accept the test sample (DNA protein or cells), a container which contains the primers used in the assay, containers which contain enzymes, containers which contain wash reagents, and containers which contain the reagents used to detect the extension products. 15 BRIEF DESCRIPTION OF THE DRAWINGS Having thus generally described the invention, reference will now be made to the accompanying drawings, showing by way of illustration a preferred embodiment thereof, and in which: ?0 Figure 1 shows the IGE candidate region on ch 2q23-q31. Order and distance between markers are according to Gyapay et al., 1994. Figure 2 shows the PCR primers used for genomic PCR-SSCP of SCN2A (SEQ ID 25 NO: 67-180); Figure 3 shows the mutation found in epilepsy patients in SCN2A (SEQ ID NO: 181 and 182); 30 Figure 4 shows the PCR primers used for genomic PCR-SSCP of SCN3A (SEQ ID NO: 183-272); and Figure 5 shows the mutation found in epilepsy patients in SCN3A (SEQ ID NO: 273 281).

22 Sequences are also shown in the Sequence Listing. For example, SEQ ID NO: 1 shows the cDNA sequence of the adult form of SCN2A; SEQ ID NO: 2 shows the cDNA sequence of the neonatal form of SCN2A; SEQ ID NO: 3 shows the protein 5 sequence of the adult form of SCN2A; SEQ ID NO: 4 shows the protein sequence of the neonatal form of SCN2A; SEQ ID NOs:5-32 show the genomic sequence of SCN2A; SEQ ID NO: 33 shows the cDNA sequence of the adult form of SCN3A; SEQ ID NO: 34 shows the cDNA sequence of the neonatal form of SCN3A; SEQ ID NO: 35 shows the protein sequence of the adult form of SCN3A; SEQ ID NO: 36 shows the 10 protein sequence of the neonatal form of SCN3A; and SEQ ID NOs:37-66 show the genomic sequence of SCN3A. Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of preferred 15 embodiments with reference to the accompanying drawing which is exemplary and should not be interpreted as limiting the scope of the present invention. DESCRIPTION OF THE PREFERRED EMBODIMENT Epilepsy is one of the most common neurological conditions, affecting 1-2% of the 20 general population. Familial aggregation studies have shown an increased risk for epilepsy in relatives of probands with different types of epilepsy, and especially for the idiopathic generalized epilepsies (IGEs). The epilepsy genes identified to date account for a very small proportion of all the epilepsies. In addition, they have been identified in rare syndromes where the pattern of inheritance was clearly Mendelian. This is not the 25 case for the vast majority of epileptic patients, however, where the pattern of inheritance is not compatible with a simple Mendelian model. In fact, most authors consider epilepsy to be the result of a combination of many different genetic and environmental factors, features of a complex trait. While the pattern of inheritance is not Mendelian, sporadic IGE cases may be caused by specific mutations in the same 30 genes. Based on this assumption, a large cohort of IGE patients was tested for mutation in the SCNA genes. In order to localize the gene causing epilepsy in a large family segregating an autosomal dominant form of IGE, 41 family members, including 21 affected individuals, 23 were genotyped. A detailed clinical description of this family has been reported elsewhere (Scheffer and Berkovic, 1997). The majority of patients in this family present a benign epilepsy syndrome occurring in childhood and characterized by frequent generalized tonic-clonic seizures not always associated with fever: a syndrome called 5 febrile seizures plus (FS+). However, several patients presented other types of generalized seizures (GTCS) as well, such as myoclonic seizures and absences (Scheffer and Berkovic, 1997). Mean age at onset was 2.2 years and offset was 11.7 years. Neurological examination and intellect were normal in all individuals except one, who had moderate intellectual disability. EEG recordings were normal in most patients. 10 However, in three individuals generalized epileptiform activity was found and four patients had mild or moderate diffuse background slowing. Table 1 shows the different types of seizures found in the 21 patients included in this study. Table 1. Different types of generalized seizures found in the 21 15 patients included in the linkage analysis. Type of seizures n Febrile convulsions alone 9 GTCSsa + absence seizures 4 GTCSs + myoclonic seizures 1 GTCSs + atonic seizures 1 Solitary afebril GTCS 1 Secondary epilepsy + mental retardation 1 Unwitnessed events 4 _a GTCS: generalized tonic clonic seizure A genome wide search examining 190 markers identified linkage of IGE to chromosome (ch) 2 based on an initial positive lod score for marker D2S294 (Z=4.4); 20 (=0). A total of 24 markers were tested on ch 2q in order to define the smallest IGE candidate region. Table 2 shows the two-point lod scores for 17 markers spanning the IGE candidate region. The highest lod score (Zmax=5.29; (=0) was obtained with marker D2S324. Critical recombination events mapped the IGE gene to a 29cM region flanked by markers D2S156 and D2S311, assigning the IGE locus to ch 2q23-q31 25 (Figure 1). Although the relationship of FS+ with other IGE phenotypes remains unclear, the observation that in this family, several affected individuals have different 24 types of generalized seizures, suggests that seizure predisposition determined by the ch 2q-IGE gene could be modified by other genes and/or environmental factors, to produce different seizure types.

25 Table 2. Two-point lod-scores for 17 markers localized on ch 2q23-q31. Recombination fractions Locus 0 0.05 0.1 0.15 0.2 0.3 0.4 Zmax max D2S142 0.99 1.94 1.97 1.85 1.68 1.22 0.66 1.98 0.078 D2S284 1.3 1.18 1.06 0.94 0.82 0.57 0.3 1.3 0 D2S306 1.9 2.82 2.74 2.52 2.25 1.6 0.85 2.82 0.057 D2S156 2.15 3.05 2.96 2.73 2.43 1.73 0.93 3.05 0.056 D2S354 4.72 4.26 3.82 3.4 2.97 2.1 1.13 4.72 0 D2Si1 5.15 4.71 4.26 3.78 3.29 2.26 1.17 5.15 0 D2S124 3.5 3.2 2.89 2.58 2.26 1.58 0.84 3.5 0 D2S382 4.31 3.93 3.54 3.14 2.74 1.91 1.02 4.31 0 D2S399 0.48 0.4 0.33 0.27 0.22 0.14 0.08 0.48 0 D2S294 4.4 4.04 3.65 3.25 2.84 2 1.07 4.4 0 D2S335 4.76 4.32 3.91 3.51 3.1 2.22 1.21 4.76 0 D2S333 1.42 1.23 1.04 0.87 0.72 0.45 0.22 1.4 0 D2S324 5.29 4.72 4.16 3.63 3.13 2.15 1.14 5.29 0 D2S384 3.85 3.52 3.17 2.82 2.45 1.69 0.89 3.85 0 D2S152 1.9 1.7 1.52 1.36 1.2 0.87 0.48 1.9 0 D2S311 -0.81 1.62 1.66 1.58 1.46 1.11 0.63 1.66 0.085 D2S155 -5.21 0.57 1.12 1.29 1.29 1.04 0.59 1.3 0.17 26 Haplotypes using 17 markers spanning the IGE candidate region were constructed (data not shown). The centromeric boundary was defined by a recombination event between the markers D2S156 and D2S354; whereas a recombination between the markers D2S1 52 and D2S31 1 set the telomeric boundary. These critical recombination 5 events localized the IGE gene to a 29cM region flanked by markers D2S156 and D2S311 (Figure 1). Over the last four decades, family studies provided two important pieces of evidence supporting the role of genetic factors in determining susceptibility to seizures: 1) 10 familial aggregation studies have shown evidence for an increased risk for epilepsy in relatives of probands with different types of epilepsy. In two studies standardized morbidity ratios for unprovoked seizures in relatives of individuals with idiopathic childhood-onset epilepsy varied from 2.5 to 3.4 in siblings and 6.7 in offspring (Anneger et al., 1982; Ottman et al., 1989); and 2) the presence of higher concordance 15 rates for epilepsy in monozygotic than in dizygotic twins. Different studies showed concordance rates varying from 54 to 11 % in monozygotic twins and 10 to 5% in dizygotic pairs (Inouye, 1960; Lennox, 1960; Harvald and Hauge, 1965; Corey et al., 1991; Silanpaa et al., 1991). ?0 It is now generally accepted that seizure susceptibility probably reflects complex interactions of multiple factors affecting neuronal excitability and that most common genetic epilepsies display familial aggregation patterns that are not explained by segregation of a single autosomal gene (Andermann, 1982; Ottman et al., 1995). This of course significantly makes more complex one's ability to isolate genes which 25 predispose or induce epilepsy. However, some specific epileptic syndromes, which aggregate in families, may result from definable monogenic abnormalities. These families present a unique opportunity to rapidly map genes that play a role in determining predisposition to seizures. 30 To date, there are a total of six loci (Greenberg et al., 1988; Leppert et al., 1989; Lewis et al., 1993; Elmslie et al., 1997; Guipponi et al., 1997; Wallace et al., 1998), for which three genes have been identified in specific IGE syndromes (Bievert et al., 1998; Singh et al., 1998; Wallace et al., 1998). Interestingly, all three genes are ion channels, including a mutation found in the Na+-channel (1 in a Tasmanian family with febrile 27 seizures and generalized epilepsy (Wallace et al., 1998). While the candidate interval identified in our kindred remains large, a number of interesting genes map to the region. These include a cluster of Na+ channel genes and K+ channel genes (electronic data base search), as well as the GAD1 gene, which encodes for glutamate 5 decarboxylase, an enzyme involved in the syntheses of y-aminobutyric acid (GABA) (Bu and Tobin, 1994). GABA is one of the major neurotransmitters involved in synaptic inhibition in the central nervous system (Barnard et al., 1987). However, the large size of the candidate interval will require further refinement of the locus prior to the identification of the gene responsible for IGE in the kindred studied herein. 10 Fifty-three % (9/17) of affected individuals in the large IGE family described herein, who had their seizures classified, had only febrile convulsions. However, 41 % of patients (7/17) presented with different types of generalized seizures. These findings may indicate that, although the predisposition to IGE in this family is determined by a 15 single gene localized on ch2q23-q31, the different types of generalized seizures occurring in the same family may have resulted from interactions among genetic and/or environmental modifiers. In conclusion, a locus for IGE was mapped on ch 2q23-q31. This locus seems to be ?0 associated with a specific IGE syndrome, FS +. However, the relationship of FS+ with other IGE phenotypes, and the role of the ch 2q locus in other FS+ families and in other forms of IGE are still undetermined. Having identified a locus for IGE on chromosome 2q23-q31, it was next verified 25 whether mutations and/or polymorphisms could be linked to epilepsy. Public databases were screened to identify potential genes in that chromosome region. The blasts of the databases were also oriented to identify more specifically, membrane channels since seizures in mice and human are known to be associated with membrane channels. Having identified membrane channel coding sequences or parts 30 thereof by the computer searches, the candidate genes, potentially involved in epilepsy, had to be validated as susceptibility genes for the disease. Two approaches were used. The first one was to test the candidate genes for mutations in a family comprising members having the disease (data not shown). The second approach was as follows. Since it is known that epilepsy results from a lower seizure threshold, and 28 that generalized epilepsy results, in many instances, from a generalized lowering of the seizure threshold, the following hypothesis was formulated. The gene which results in epilepsy in the large family (that enabled the focusing chromosome 2q23-q31) should have other, less severe, mutations that would cause epilepsy in people who have only 5 a weak family history of epilepsy. The sodium channel genes were chosen because they are involved in key electrical functions and could thus be good candidates. To formally test the hypothesis, many (60 to 70) unrelated cases of epilepsy were tested for mutations in these candidate genes. Surprisingly, mutations were found in two candidate genes. 10 In order to assess whether mutations/polymorphisms could be identified and correlated to epilepsy, a panel of 70 to 80 epileptic patients (IGE) were tested for mutations in SCN2A and SCN3A, using Single-strand conformation polymorphism (SSCP). SSCP analysis enables the detection of mutations as small as single-base substitutions. 15 Indeed, such substitutions, by altering the conformations of single-strand DNA molecules, affect the electrophoretic mobilities thereof in non-denaturing gels. Thus, one can distinguish among sequences by comparing the mobilities of wild type (wt), mutant DNA, or different alleles of a given locus. The identification of single base substitutions of genes using SSCP is well known in the art, and numerous protocols 20 are available therefor. A non-limiting example thereof includes fluorescence-based SSCP analysis, following PCR carried out using fluorescent-labeled primers specific for the DNA regions one wishes to amplify. Upon the identification of differences between normal and epileptic mobilities for one of 25 the SCNA loci of the present invention, the amplified fragments were sequenced and the nucleic acid sequences between a normal patient and an epileptic patient (IGE) compared. This comparison enabled the identification of mutations in SCN2A, and SCN3A. To assess, whether this difference in sequence or mutation was significantly associated with the disease, SSCP analysis was performed once again using a large 30 cohort of normal patients. This analysis enabled to show that the mutations identified by SSCP and confirmed by sequence analysis were not present in the large cohort of normal patients tested, thereby showing that the mutations identified correlated with IGE, for the population tested. Taken together, these results show that SCN2A and SCN3A are validated genes 29 associated with epilepsy and more specifically with IGE. This invention now establishes, for the first time, that SCN2A and SCN3A are directly responsible for idiopathic generalized epilepsy (IGE) in certain human populations. 5 Further, this discovery suggests that compounds which modulate the activity of SCN2A and SCN3A may have application far beyond the small groups of families with IGE, and may have applicability for treating many or all forms of epilepsy and related neurological disorders. It is therefore an object of this invention to provide screening assays using SCN2A and/or SCN3A which can identify compounds which have 10 therapeutic benefit for epilepsy and related neurological disorders. This invention also claims those compounds, the use of these compounds in treating epilepsy and related neurological disorders, and any use of any compounds identified using such a screening assay in treating epilepsy and related neurological disorders. 15 Generally, high throughput screens for one or more SCN2A or SCN3A (herein collectively called SCNA) sodium channels modulators i.e. candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) may be based on assays which measure biological activity of SCNA. The invention therefore provides a method (also referred to herein as a "screening assay") 20 for identifying modulators, which have a stimulatory or inhibitory effect on, for example, SCNA biological activity or expression, or which bind to or interact with SCNA proteins, or which have a stimulatory or inhibitory effect on, for example, the expression or activity of SCNA interacting proteins (targets) or substrates. 25 Examples of methods available for cell-based assays and instrumentation for screening ion-channel targets are described in the review by Gonzalez et al., (Drug Discov. Today 4:431-439, 1999), and high-throughput screens for ion-channel drugs are described in review by Denyer et al., (Drug Discov. Today 3:323-332, 1998). Such assays include efflux of sodium or related ions that can be measured in a cell line 30 (recombinant or non-recombinant) using fluorescence-based assays using both sodium indicator dyes and voltage sensing dyes. Preferred assays employ 14C guanidine flux and/or sodium indicator dyes such as SBFI and voltage sensing dyes such as DiBAC. Oxonal dyes such as DiBAC 4 are responsive to membrane depolarization. Hyper-polarization results in removal of the dye from the cell by passive 30 diffusion, while depolarization results in concentration of the dye within the cell. In one embodiment, the invention provides assays for screening candidate or test compounds which interact with substrates of a SCNA protein or biologically active 5 portion thereof. In another embodiment, the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of a SCNA protein or polypeptide or biologically active portion thereof. 10 In one embodiment, an assay is a cell-based assay in which a cell which expresses a SCNA protein or biologically active portion thereof, either natural or recombinant in origin, is contacted with a test compound and the ability of the test compound to modulate SCNA biological activity, e.g., modulation of sodium efflux activity, or binding 15 to a sodium channel or a portion thereof, or any other measurable biological activity of SCNA is determined. Determining the ability of the test compound to modulate SCNA activity can be accomplished by monitoring, for example, the release of a neurotransmitter or other compound, from a cell which expresses SCNA such as a neuronal cell, e.g., a substantia nigra neuronal cell, or a cardiac cell upon exposure of 20 the test compound to the cell. Furthermore, determining the ability of the test compound to modulate SCNA activity can be accomplished by monitoring, for example, the change in current or the change in release of a neurotransmitter from a cell which expresses SCNA upon exposure to a test compound. Currents in cells can be measured using the patch-clamp technique as described in the Examples below 25 using the techniques described in, for example, Hamill et al., 1981 Pfluegers Arch. 391:85-100. Alternatively, changes in current can be measured by dye based fluorescence assays described below. Determining the ability of the test compound to modulate binding of SCNA to a 30 substrate can be accomplished, for example, by coupling the SCNA agent or substrate with a radioisotope or enzymatic label such that binding of the SCNA substrate to SCNA can be determined by detecting the labeled SCNA substrate in a complex. For example, compounds (e.g., SCNA agents or substrates) can be labeled with 1251 35 s, 4C, or 3 H, either directly or indirectly, and the radioisotope detected by direct counting 31 radio-emission or by scintillation counting. Alternatively, compounds can be enzymatically labeled with, for example, horseradish peroxidase or alkaline phosphatase. In these assays, compounds which inhibit or increase substrate binding to SCNA are useful for the therapeutic objectives of the invention. 5 It is also within the scope of this invention to determine the ability of a compound (e.g., SCNA substrate) to interact with SCNA without the labeling of any of the interactants. For example, a microphysiometer can be used to detect the interaction of a compound with SCNA without the labeling of either the compound or the SCNA (McConnell H.M. 0 et al., (1992), Science 257:1906-1912). As used herein, a "microphysiometer" (e.g., Cytosensor) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between a compound and SCNA. 15 Modulators of SCNA can also be identified through the changes they induce in membrane potential. A suitable instrument for measuring such changes is the VIPR TM (voltage ion probe reader) from Aurora Biosciences. This instrument works together with a series of voltage-sensing ion probe assays. The probes sense changes in ?0 transmembrane electrical potential through a voltage-sensitive FRET mechanism for which the ratio donor fluorescence emission to acceptor fluorescence emission reveals the extent of cell depolarization for both sodium and potassium channels. Depolarization results from transport of a quencher across the membrane and far enough away from a membrane-bound fluorescence emitter to relieve the initial 25 quenching and produce light at the emission wavelength of the emitter. The system follows fluorescence at two wavelengths, both the intensities and ratios change during cell depolarization. The reader permits detection of sub-second, real-time optical signals from living cells in a microplate format. The system is amenable to manual operation for assay development or automation via robots for high-throughput 30 screening. In another embodiment, the assay is a cell-based assay comprising a contacting of a cell containing a target molecule (e.g., another molecule, substrate or protein that interacts with or binds to SCNA) with a test compound and determining the ability of 32 the test compound to indirectly modulate (e.g., stimulate or inhibit) the biological activity of SCNA by binding or interacting with the target molecule. Determining the ability of the test compound to indirectly modulate the activity of SCNA can be accomplished, for example, by determining the ability of the test compound to bind to 5 or interact with the target molecule and thereby to indirectly modulate SCNA , to modulate sodium efflux, or to modulate other biological activities of SCNA. Determining the ability of the SCNA protein or a biologically active fragment thereof, to bind to or interact with the target molecule can be accomplished by one of the methods described above or known in the art for determining direct binding. In a preferred 10 embodiment, determining the ability of the test compound's ability to bind to or interact with the target molecule and thereby to modulate the SCNA protein can be accomplished by determining a secondary activity of the target molecule. For example, the activity of the target molecule can be determined by detecting induction of a cellular second messenger of the target (e.g., intracellular Ca2+, diacylglycerol, IP3, 15 and the like), detecting catalytic/enzymatic activity of the target on an appropriate substrate, detecting the induction of a reporter gene (comprising a target-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, such as luciferase), or detecting a target-regulated cellular response such as the release of a neurotransmitter. Alternatively, recombinant cell lines may employ ?0 recombinant reporter proteins which respond, either directly or indirectly to sodium efflux or secondary messengers all as known in the art. In yet another embodiment, an assay of the present invention is a cell-free assay in which a SCNA protein or biologically active portion thereof, either naturally occurring or 25 recombinant in origin, is contacted with a test compound and the ability of the test compound to bind to, or otherwise modulate the biological activity of, the SCNA protein or biologically active portion thereof is determined. Preferred biologically active portions of the SCNA proteins to be used in assays of the present invention include fragments which participate in interactions with non-SCNA molecules, (e.g., other 30 channels for sodium, potassium or Ca+ or fragments thereof, or fragments with high surface probability scores for protein-protein or protein-substrate interactions). Binding of the test compound to the SCNA protein can be determined either directly or indirectly as described above. In a preferred embodiment, the assay includes contacting the SCNA protein or biologically active portion thereof with a known 33 compound which binds SCNA to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a SCNA protein, wherein determining the ability of the test compound to interact with a SCNA protein comprises determining the ability of the test compound to preferentially 5 bind to SCNA or biologically active portion thereof as compared to the known compound. In another embodiment, the assay is a cell-free assay in which a SCNA protein or biologically active portion thereof is contacted with a test compound and the ability of l0 the test compound to modulate (e.g., stimulate or inhibit) the activity of the SCNA protein or biologically active portion thereof is determined. Determining the ability of the test compound to modulate the activity of a SCNA protein can be accomplished, for example, by determining the ability of the SCNA protein to bind to a SCNA target molecule by one of the methods described above for determining direct binding. 15 Determining the ability of the SCNA protein to bind to a SCNA target molecule can also be accomplished using a technology such as real-time Biomolecular Interaction Analysis (BIA, Sjolander, S. and Urbaniczky, C., (1991) Anal. Chem. 63:2338-2345 and Szabo et al., (1995) Curr. Opin. Struct. Biol. 5:699-705). As used herein, "BIA" refers to a technology for studying biospecific interactions in real time, without labeling 20 any of the interactants (e.g., BIA core). Changes in the optical phenomenon of surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules. In an alternative embodiment, determining the ability of the test compound to modulate 25 the activity of a SCNA protein can be accomplished by determining the ability of the test compound to modulate the activity of an upstream or downstream effector of a SCNA target molecule. For example, the activity of the test compound on the effector molecule can be determined or the binding of the effector to SCNA can be determined as previously described. 30 The cell-free assays of the present invention are amenable to use of both soluble and/or membrane-bound forms of isolated proteins. In the case of cell-free assays in which a membrane-bound form of an isolated protein is used (e.g., a sodium channel) it may be desirable to utilize a solubilizing agent such that the membrane-bound form 34 of the isolated protein is maintained in solution. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton@ X-1OO, Triton@ X-1 14, Thesit®, isotridecylpoly(ethylene glycol ether)n, 3-[(3 5 cholamidopropyl)dimethy-amino]-1 -propane sulfonate (CHAPS), 3-3 cholamidopropyl)dimethylamino]-2-hydroxy-l-propane sulfonate (CHAPSO), or N dodecyl-N,N-dimethyl-3-ammnonio-1-propane sulfonate. In more than one embodiment of the above assay methods of the present invention, it 10 may be desirable to immobilize either SCNA or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to a SCNA protein or interaction of a SCNA protein with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for 15 containing the reactants. Examples of such vessels include microtitre plates, test tubes and micro-centrifuge tubes. In one embodiment a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase/SCNA fusion proteins or glutathione-S transferase/target fusion proteins can be adsorbed onto glutathione sepharose beads ?0 (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtitre plates, which are then combined with the test compound or the test compound and either the non adsorbed target protein or SCNA protein and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation the beads or microtitre plate wells are washed to remove any 25 unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of SCNA binding or activity determined using standard techniques. 30 Other techniques for immobilizing proteins on matrices (and well-known in the art) can also be used in the screening assays of the invention. For example, either a SCNA protein or a SCNA target molecule can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated SCNA protein or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., 35 biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with SCNA protein or target molecules but which do not interfere with binding of the SCNA protein to its target molecule can be derivatized to the wells of the plate, and 5 unbound target or SCNA protein trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST immobilized complexes, include immunodetection of complexes using antibodies reactive with the SCNA protein or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the SCNA protein or 10 target molecule. In a preferred embodiment, candidate or test compounds or agents are tested for their ability to inhibit or stimulate a SCNA molecule's ability to modulate vesicular traffic and protein transport in a cell, e.g., a neuronal or cardiac cell using the assays described in 15 for example Komada M. et al., (1999) Genes Dev.13(11):1475-85, and Roth M.G. et al., (1999) Chem. Phys. Lipids. 98(12):141-52. In another preferred embodiment candidate, or test compounds or agents are tested for their ability to inhibit or stimulate or regulate the phosphorylation state of a SCNA ?0 channel protein or portion thereof, or an upstream or downstream target protein, using for example an in vitro kinase assay. Briefly, a SCNA target molecule (e.g., an immunoprecipitated sodium channel from a cell line expressing such a molecule), can be incubated with radioactive ATP, e.g., [gamma-32P]-ATP, in a buffer containing MgCl 2 and MnC1 2 , e.g., 10 mM MgCl 2 and 5 mM MnC1 2 . Following the incubation, the 25 immunoprecipitated SCNA target molecule (e.g., the sodium channel), can be separated by SDS-polyacrylamide gel electrophoresis under reducing conditions, transferred to a membrane, e.g., a PVDF membrane, and autoradiographed. The appearance of detectable bands on the auto radiograph indicates that the SCNA substrate, e.g., the sodium channel, has been phosphorylated. Phosphoaminoacid 30 analysis of the phosphorylated substrate can also be performed in order to determine which residues on the SCNA substrate are phosphorylated. Briefly, the radiophosphorylated protein band can be excised from the SDS gel and subjected to partial acid hydrolysis. The products can then be separated by one-dimensional electrophoresis and analyzed on, for example, a phosphoimager and compared to 36 ninhydrin-stained phosphoaminoacid standards. Assays such as those described in, for example, Tamaskovic R. et al., (1999) Biol. Chem. 380(5):569-78. In another preferred embodiment, candidate or test compounds or agents -are tested 5 for their ability to inhibit or stimulate a SCNA molecule's ability to associate with (e.g., bind) calcium, using for example, the assays described in Liu L., (1999) Cell Signal. 11(5):317-24 and Kawai T. et al., (1999) Oncogene 18(23):3471-80. In another preferred embodiment, candidate or test compounds or agents are tested 10 for their ability to inhibit or stimulate a SCNA molecule's ability to modulate chromatin formation in a cell using for example the assays described in Okuwaki M. et al., (1998) J. Biol. Chem. 273(51):34511-8 and Miyaji-Yamaguchi M., (1999) J. Mol. Biol. 290(2): 547-557. 15 In yet another preferred embodiment candidate or test compounds or agents are tested for their ability to inhibit or stimulate a SCNA molecule's ability to modulate cellular proliferation, using for example, the assays described in Baker F.L. et al., (1995) Cell Prolif. 28(1):1-15, Cheviron N. et al., (1996) Cell Prolif. 29(8):437-46. Hu Z. W. et al., (1999) J. Pharmacol. Exp. Ther. 290(1):28-37 and Elliott K. et al., (1999) 20 Oncogene 18(24):3564-73. In a preferred embodiment, candidate or test compounds or agents are tested for their ability to inhibit or stimulate a SCNA molecule's ability to regulate it's association with the cellular cytoskeleton. Using for example, the assays similar to those described in 25 Gonzalez C. et al., (1998) Cell Mol. Biol. 44(7):1117-27 and Chia C.P. et al., (1998) Exp. Cell Res. 244(1):340-8. In another preferred embodiment, candidate or test compounds or agents are tested for their ability to inhibit or stimulate a SCNA molecule's ability to modulate membrane 30 excitability, using for example, the assays described in Bar-Sagi D. et al., (1985) J. Biol. Chem. 260(8):4740-4 and Barker J.L. et al., (1984) Neurosci. Lett. 47(3):313-8. In another preferred embodiment, candidate or test compounds or agents are tested for their ability to inhibit or stimulate a SCNA molecule's ability to modulate cytokine 37 signaling in a cell, (e.g., a neuronal or cardiac cell), the assays described in Nakashima Y. et al., (1999) J. Bone Joint Surg. Am. 81 (5):603-15. In another embodiment, modulators of SCNA expression are identified in a method 5 wherein a cell is contacted with a candidate compound and the expression of SCNA mRNA or protein in the cell is determined. The level of expression of SCNA mRNA or protein in the presence of the candidate compound is compared to the level of expression of SCNA mRNA or protein in the absence of the candidate compound. The candidate compound can then be identified as a modulator of SCNA expression based 10 on this comparison. For example, when expression of SCNA mRNA or protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of SCNA mRNA or protein expression. Alternatively, when expression of SCNA mRNA or protein is less (statistically significantly less) in the presence of the candidate 15 compound than in its absence, the candidate compound is identified as an inhibitor of SCNA mRNA or protein expression. The level of SCNA mRNA or protein expression in the cells can be determined by methods described herein or other methods known in the art for detecting SCNA mRNA or protein. 20 The assays described above may be used as initial or primary screens to detect promising lead compounds for further development. Often, lead compounds will be further assessed in additional, different screens. Therefore, this invention also includes secondary SCNA screens which may involve electrophysiological assays utilizing mammalian cell lines expressing the SCNA channels such as patch clamp 25 technology or two electrode voltage clamp and FRET-based voltage sensor. Standard patch clamp assays express wild type and mutant channels in Xenopus oocytes, and examine their properties using voltage-clamp electrophysiological recording. Wild type sodium channels are closed at hyperpolarized membrane potentials. In response to membrane depolarization the channels open within a few hundred microseconds, 30 resulting in an inward sodium flux, which is terminated within a few milliseconds by channel inactivation. In whole cell recordings, rapid activation and inactivation of thousands of sodium channels distributed throughout the cell membrane results in a transient inward sodium current that rises rapidly to peak amplitude and then decays to baseline within a few milliseconds.

38 Tertiary screens may involve the study of the identified modulators in rat and mouse models for epilepsy. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model. For example, a 5 test compound identified as described herein (e.g., a SCNA modulating agent, an antisense SCNA nucleic acid molecule, a SCNA-specific antibody, or a SCNA-binding partner) can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal model to determine the mechanism of action of such 10 an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatment (e.g., treatments of different types of epilepsy or CNS disorders), as described herein. The test compounds of the present invention can be obtained using any of the 15 numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one compound' library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other !0 four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, Anticancer Drug Des. 12: 145, 1997). Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al., (1993) Proc. Natl. Acad. Sci. USA. 90:6909; Erb et al., (1994) Proc. NatI. Acad. Sci. USA 91:11422; Zuckermann et al., (1994), J. Med. Chem. 37:2678; Cho et 25 al., (1993) Science 261 :1303; Carrell et al., (1994) Angew. Chem, Int. Ed Engl. 33:2059; Carell et al., (1994) Angew. Chem, Int. Ed. Engl. 33:2061; and in Gallop et al., (1994), Med Chem. 37:1233. Libraries of compounds may be presented in solution (e.g., Houghten, (1992) Biotechniques 13:412-421), or on beads (Lam, (1991) Nature 354:82-84), chips (Fodor, (1993) Nature 364:555-556), bacteria (Ladner, USP 30 5,223,409), spores (Ladner, USP '409), plasmids (Cull et al., (1992) Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage (Scott and Smith, (1990) Science 249:386-390). Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al., (1993), Proc. Natl. Acad. Sci. USA 90:6909; Erb et al., (1994), Proc. Nati. Acad. Sci. USA 91: 11422; Zuckermann et al., (1994), J. Med.

39 Chem. 37:2678; Cho et al., (1993), Science 261:1303; Carell et al., (1994) Angew. Chem Int. Ed. Engl. 33:2059, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product. 5 It is recognized by the inventors that certain therapeutic agents have been identified for cardiac, muscular, chronic pain, acute pain and other disorders, and analgesics and anesthetics that are modulators of sodium channels. Use of these sodium channel modulators for treating epilepsy and related neurological disorders also falls within the scope of this invention. In one embodiment of this invention, sodium channel blockers 10 are modified to achieve improved transport across the blood brain barrier in order to have direct effect on neuronal SCNA proteins and genes. Descriptions of such compounds are found at Hunter, JC et al., Current Opinion in CPNS Invest. Drugs. 1999 1(1):72-81; Muir KW et al., 2000, Cerebrovasc. Disc. 10(6):431-436; Winterer, G., 2000, Pharmacopsychiatry 33(5):182-8; Clare et al., 2000, Drug. Discov. Today 15 5(11):506-520; Taylor CP et al., 2000, Adv. Pharmacol. 39:47-98, and Pugsley MK et al., 1998, Eur. J. Pharmacol. 342(1)93-104. It is also recognized by the inventors that compounds which modulate (i.e. either upregulate or downregulate) transcription and translation of SCNA genes are useful for 20 treating epilepsy or related neurological disorders. According to this invention, test compounds which modulate the activity of promoter elements and regulatory elements of sodium channel genes are useful for treating these disorders. In summary, based on the disclosure herein, those skilled in the art can develop SCNA 25 screening assays which are useful for identifying compounds which are useful for treating epilepsy and other disorders which relate to potentiation of SCNA expressing cells. The assays of this invention may be developed for low-throughput, high throughput, or ultra-high throughput screening formats. 30 The assays of this invention employ either natural or recombinant SCNA protein. Cell fraction or cell free screening assays for modulators of SCNA biological activity can use in situ, purified, or purified recombinant SCNA proteins. Cell based assays can employ cells which express SCNA protein naturally, or which contain recombinant SCNA gene constructs, which constructs may optionally include inducible promoter 40 sequences. In all cases, the biological activity of SCNA can be directly or indirectly measured; thus modulators of SCNA biological activity can be identified. The modulators themselves may be further modified by standard combinatorial chemistry techniques to provide improved analogs of the originally identified compounds. 5 Finally, portions or fragments of the SCNA cDNA sequences identified herein (and the corresponding complete gene sequences) can be used in numerous ways as polynucleotide reagents. For example, these sequences can be used to: (i) map their respective genes on a chromosome and thus, locate gene regions associated with 10 genetic disease (mutations/polymorphisms) related to epilepsy or CNS disorders that involve SCNA directly or indirectly; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample. The present invention is illustrated in further detail by the following non-limiting 15 examples. EXAMPLE 1 Molecular analysis Genomic DNA was extracted from blood samples (Sambrook et al., 1989) or 20 lymphoblastoid cell lines (Anderson and Gusella, 1984) from each individual. A panel of 210 dinucleotide (CA)n repeat polymorphic markers with high heterozygosity (75%) were chosen from the 1993-94 Genethon map (Gyapay et al., 1994). Dinucleotide markers were spaced an average of 20 cM from each other throughout the 22 autosomes. 25 Genotyping of microsatellite markers was accomplished by polymerase chain reaction (PCR). The reaction mixture was prepared in a total volume of 13:1, using 80ng genomic DNA; 1.25:1 10x buffer with 1.5mM MgCl 2 ; 0.65:1 BSA (2.0mg/ml); 100ng of each oligonucleotide primer; 200mM dCTP, dGTP and dTTP; 25mM dATP; 1.5mCi 30 [35S] dATP; and 0.5units Taq DNA polymerase (Perkin-Elmer). Reaction samples were transferred to 96 well plates and were subjected to: 35 cycles of denaturation for 30 seconds at 94'C, annealing for 30 seconds at temperatures varying from 55*C to 57*C depending on the specificity of the oligonucleotide primers, and elongation for 30 seconds at 72*C. PCR reaction products were electrophoresed on 6% denaturing 41 polyacrylamide sequencing gels. EXAMPLE 2 Genetic analysis 5 Two-point linkage analysis was carried out using the MLINK program version 5.1 from the LINKAGE computer package (Lathrop et al., 1984). Precise values for Zmax were calculated with the ILINK program from the same computer package. Lod scores were generated based on an autosomal dominant mode of inheritance, 80% penetrance, disease gene frequency of 1:500 and allele frequencies for all allele markers 10 calculated from the pedigree using the computer program ILINK (Lathrop et al., 1984). EXAMPLE 3 Mutations in SCN2A in IGE patients Genomic DNA form IGE and normal patients was obtained by conventional methods. 15 Primers used to amplify the genomic DNA are shown in Figure 2. Following PCR, SSCP analysis was performed and mutations in SCN2A were identified as follows (Figure 3): (1) Lys908Arg: normal: TAC AAA GAA (SEQ ID NO: 181) for patient numbers always 20 preceded by R; R9782 (Patient with IGE): TAC AGA GAA (SEQ ID NO: 182). The mutation is thus a conservative aa change, located in an extracellular domain between TM domains llS5 and IIS6; in 1/70 IGE patients; 0/96 normal controls. The mutation involves an important component of the SCN gene, since the S5 and S6 segments are thought to form the wall of the transmembrane pore which allows the sodium to enter 25 the cell. This may have an influence on the gating control of the pore. (2) Leu768Val, in individuals R8197, R9062 and R9822 (all IGE patients) (found in 3/70 IGE patients and 0/65 control subjects). The mutations is in the IV-S6 component of the sodium channel, which is important in the inactivation of the channel (see above 30 for more detail). EXAMPLE 4 Mutations in SCN3A in IGE patients Genomic DNA from IGE and normal patients was obtained by conventional methods. Primers used to amplify the genomic DNA are shown in Figure 4. Following PCR, 42 SSCP analysis was performed and mutations in SCN3A were identified as follows (Figure 5): (1) Asn43DEL: allele 1: CAA GAT AAT GAT GAT GAG (SEQ ID NO: 273); allele 2: 5 CAA GAT --- GAT GAT GAG (SEQ ID NO: 274); in open reading frame deletes 1 aa: DNDDEN->QDDDEN, in the cytoplasmic N-terminal segment; in IGE patients, the frequency of allele 1 = 131/146 (0.90); allele 2= 15/146 (0.10); for IGE patients: homozygotes (22): R3958, R9632; heterozygotes (12): R9049, R9152, R9649, R9710, R9896, R10069, R10191, R10213, R9993, R10009, R10256 . Of note, 2 patients are .0 homozygous for the rare allele and all patients have IGE. In controls: allele 1 = 145/154 (0.94); allele 2 = 9/154 (0.06) and no 22 homozygotes were found. (2) normal: tggtgtaaggtag (SEQ ID NO: 275), R10670 (IGE patient): tggtataaggtag (SEQ ID NO: 276), in conserved intron between 5N & 5A exons, significance 15 uncertain. (3) normal: ccccttatatctccaac (SEQ ID NO: 277), R10250 (IGE patient): ccccttatayctccaac (SEQ ID NO: 278); in conserved intron between 5N & 5A exons, significance uncertain. ?0 (4) Vall03511e: normal: AAA TAC GTA ATC GAT (SEQ ID NO: 279), R9269 (IGE patient): AAA TAC RTA ATC GAT (SEQ ID NO: 281); (GTA>ATA = Val>lle). The mutation is thus a conservative aa change which destroys a SnaBI site (this could thus be used as a polymorphism identifiable by restriction enzyme digestion). In 25 cytoplasmic domain bw 1l-S6 & Ill-Si TMs; found in 1/70 IGE alleles; and 0/70 controls.

43 EXAMPLE 5 Further validation of the role of SCN2A and SCN3A, and specific mutations thereof in IGE and epilepsy in general 5 A number of methods could be used to further validate the role of SCN2A, SCN3A, and specific mutations thereof in IGE. For example, additional patients could be screened for mutations in SCN2A or SCN3A. Furthermore, additional normal patients could be screened in order to validate that the mutations identified significantly correlate with disease, as opposed to reflecting a polymorphism which is not linked to 10 IGE. Polymorphisms which are not directly linked to IGE, if in linkage disequilibrium with a functional mutation linked to IGE, could still be useful in diagnosis and/or prognosis assays. In addition, functional studies can be carried. Numerous methods are amenable to the skilled artisan. One particularly preferred functional assay involves the use of Xenopus oocytes and recombinant constructs harboring normal or 15 mutant sequence of SCN2A or SCN3A. Xenopus oocytes have been used in functional assays to dissect the structure-function relationship of the cyclic AMP modulated potassium channel using recombinant KCNQ2 and KCNQ3 (Schroeder et al., 1998). As well, it has been used to dissect the structure-function relationship of the beta subunit of the sodium channel (SCN1B gene; Wallace et al., 1998). 20 Other examples of informative structure-function studies include characterization of the functional consequence of mutations, such as naturally occurring mutations, in a sodium channel gene. In this regard, a mutation associated with a loss or reduction in function of the sodium channel could at least in part explain its role in epilepsy. Such a 25 functional consequence is may also be expected to be observed with other mutations identified in SCNA2 and SCNA3. Although the present invention has been described hereinabove by way of preferred embodiments thereof, it can be modified, without departing from the spirit and nature of 30 the subject invention as defined in the appended claims. The term "comprise" and variants of the term such as "comprises" or "comprising" are used herein to denote the inclusion of a stated integer or stated integers but not to exclude any other integer or any other integers, unless in the context or usage an 44 exclusive interpretation of the term is required. Any reference to publications cited in this specification is not an admission that the disclosures constitute common general knowledge in Australia. 5 45 REFERENCES: Andermann E (1982) Multifactorial inheritance of generalized and focal epilepsy. In: Anderson VE, Hauser WA, Penry JK, Sing CF (eds) Genetic Basis of the Epilepsies. New York, Raven Press, pp:355-374 5 Anderson MA and Gusella JF (1984) Use of cyclosporin A in establishing Epstein Barr virus-transformed human lymphoblastoid cell lines. In Vitro 20:856-858 Anneger JF, Hauser WA, Anderson VE (1982) Risk of seizures among relatives of patients with epilepsy: families in a defined population. In: Anderson VE, Hauser WA, Sing L, Porter R (eds) The Genetic Basis of the Epilepsies, Raven Press, New York, 0 pp 151-159 Barnard EA, Darlison MG, Seeburg P (1987) Molecular biology of the GABAA receptor: the receptor/channel superfamily. Trends Neurosci 10:502-509. Berkovic SF, et al. Epilepsies in twins: genetics of the major epileptic syndromes. Ann Neurol. 43:435-445 (1998). 5 Bievert C, Schoeder BC, Kubisch C, Berkovic SF, Propping P, Jentsch TJ, Steinlein OK (1998) A potassium channel mutation in neonatal human epilepsy. Science 279:403-406 Bu DF, Tobin AJ (1994) The exon-intron organization of the genes (GAD1 and GAD2) encoding two human glutamate decarboxylases (GAD67 and GAD65) suggests that .0 they derive from a common ancestral GAD. Genomics 1:222-228. Charlier C, et al. A pore mutation in a novel KGT-like potassium channel gene in an idiopathic epilepsy family. Nat. Genet. 18:53-55 (1998). Commission on Classification and Terminology of the International League against Epilepsy (1989) Proposal for revised clinical and eletroencephalographic classification 25 of epileptic seizures. Epilepsia 22:489-501 Corey LA, Berg K, Pellock JM, Solaas MH, Nance WE, DeLorenzo RJ (1991) The occurrence of epilepsy and febrile seizures in Virginian and Norwegian twins. Neurology 41:433-436 Elmslie FV, Rees M, Williamson MP, Kerr M, Kjeldsen MJ, Pang KA, Sundqvist A, et al 30 (1997) Genetic mapping of a major susceptibility locus for juvenile myoclonic epilepsy on chromosome 15q. Hum Mol Genet 6:1329-1334 Engel JJ, Pedley TA (1998) What is epilepsy? In: Engel JJ, Pedley TA (eds) Epilepsy a Comprehensive Textbook, Lippincott-Raven Publishers, Philadelphia, pp: 1-10. Greenberg DA, Delgado-Escueta AV, Widelitz H, Sparkes RS, Treiman L, Maldonado 46 HM, et al (1988) Juvenile myoclonic epilepsy (JME) may be linked to the BF and HLA loci on human chromosome 6. Am J Hum Genet 31:185-192 Guipponi M, Rivier F, Vigevano F, Beck C, Crespel A, Echenne B, Lucchini P, et al (1997) Linkage mapping of benign familial infantile seizures (BFIS) to chromosome 5 19q. Hum Mol Genet 6:473-477 Gyapay G, Morissette J, Vignal A, et al. (1994) The 1993-94 Genethon human genetic linkage map. Nat Genet 7:246-339 Harvald B and Hauge M (1965) Hereditary factors elucidated by twin studies. In: Neel JV, Shaw MW, Schull WJ (eds) Genetics and the Epidemiology of Chronic Diseases, 10 Washington Public Health Service Publications 1163:61-76 Inouye E (1960) Observations on forty twin index cases with chronic epilepsy and their co-twins. J Nerv Ment Dis 130: 401-416 Lathrop GM, Lalouel JM, (1984) Easy calculations of lod scores and genetic risks on small computers. Am J Hum Genet 36:460-465 15 Lennox WG, Lennox MA (1960) Epilepsy and related disorders. Boston, Little.Brown. Leppert M, Anderson VE, Quattlebaum T, Staufe D, O'Connell P, Nakamura Y, Lalouel JM, et al (1989) Benign familial neonatal convulsions linked to genetic markers on chromosome 20. Nature 337:647-648 Lewis TB, Leach RJ, Ward K, O'Connell P, Ryan SG (1993) Genetic Heterogeneity in ?0 benign familial neonatal convulsions: identification of a new locus on chromosome 8q. Am J Hum Genet 53:670-675 Ottman R, Annegers JF, Hauser WA, Kurland LT (1989) Seizure risk in offspring of parents with generalized versus partial epilepsy. Epilepsia 30:157-161 Ottman R, Hauser WA, Barker-Cummings C, Lee JH, Risch N (1997) Segregation 25 analysis of cryptogenic epilepsy and an empirical test of the validity of the results. Am J Hum Genet 60:667-675 Sambrook J, Fritsch EF, Maniatis T (eds) (1989) Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp E.3-E.4 Scheffer IE and Berkovic SF (1997) Generalised epilepsy with febrile seizures plus: a 30 genetic disorder with heterogeneous clinical phenotypes. Brain 120: 479-490. Schroeder et al.,1998, Nature 396:687-690. Silanpaa M, Koskenvuo M, Romanov K, Kaprio J (1991) Genetic factors in epileptic seizures: evidence from a large twin population. 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Claims (13)

1. A method of determining an individual's predisposition to epilepsy and/or development of epilepsy, as well as predicting this individual's response to medication, 5 said method comprising the step of determining the genotype of at least one gene selected from SCN2A and SCN3A of the individual, or of a DNA variant, equivalent, or mutation which shows a linkage disequilibrium therewith, thereby determining an individual's predisposition to epilepsy and/or development of epilepsy. 10

2. The method of claim 1, wherein the step of determining the SCN2A or SCN3A genotype comprises restriction endonuclease digestion.

3. The method of claim 1 or 2, wherein the step of determining the SCN2A or SCN3A genotype comprises hybridizing with allele specific oligonucleotides. 15

4. The method of claim 1, which further comprises a step, prior to determining the SCN2A or SCN3A genotype, of amplifying a segment of the SCN2A or SCN3A using polymerase chain reaction. 20

5. The method of claim 1, wherein the step of determining the SCN2A or SCN3A genotype comprises a sequencing of SCN2A or SCN3A , or parts thereof.

6. The method of claim 1, wherein the SCN2A or SCN3A genotype is determined using a polymorphic variant site in linkage disequilibrium with at least one allelic variant 25 or mutant identified in accordance with the present invention.

7. An assay for screening a test agent and selecting an agent which modulates inactivation of a sodium channel involved in epilepsy comprising: a) a recombinant SCN2A or SCN3A gene which encodes an alpha subunit of 30 said sodium channel or functional fragment thereof; and b) assaying a function of said sodium channel; wherein an agent can be selected when an observable difference is observed between the inactivation of said sodium channel in the presence of said test agent, as compared to in an absence thereof, and wherein a malfunction of said 49 sodium channel is associated with epilepsy.

8. An assay for screening a test agent and selecting an agent which modulates the activity of a sodium channel involved in epilepsy comprising: 5 a) a recombinant SCN2A or SCN3A gene which encodes an alpha subunit of said sodium channel or functional fragment thereof; and b) assaying the activity of said sodium channel; wherein an agent can be selected when an observable difference is observed between the activity of said sodium channel in the presence of said test agent, 0 as compared to in an absence thereof, and wherein a malfunction of said sodium channel is associated with epilepsy.

9. A method of using specific alleles of the SCN2A or SCN3A genes, or variant, equivalent, or mutation thereof which shows linkage disequilibrium therewith, to set-up 15 a screening assay for agents destined to modulate sodium channel function for the purpose of identifying agents having an application in epilepsy therapy.

10. A method for identifying, from a library of compounds, a compound with therapeutic effect on epilepsy or other neurological disorders comprising: 0 a) providing a screening assay comprising a measurable biological activity of SCN2A or SCN3A protein or gene; b) contacting said screening assay with a test compound; and c) detecting if said test compound modulates the biological activity of SCN2A or SCN3A protein or gene; 25 wherein a test compound which modulates said biological activity is a compound with said therapeutic effect.

11. The method of claim 10, wherein the test compound with said therapeutic effect is further modified by combinatorial or medicinal chemistry to provide further analogs of 30 said test compound also having said therapeutic effect.

12. A compound having therapeutic effect on epilepsy or other neurological disorders, identified by a method comprising, a) providing a screening assay comprising a measurable biological activity 50 of SCN2A or SCN3A protein or gene; b) contacting said screening assay with a test compound; and c) detecting if said test compound modulates the biological activity of SCN2A or SCN3A protein or gene; 5 wherein a test compound which modulates said biological activity is a compound with said therapeutic effect.

13. The compound of claim 12, wherein the compound with said therapeutic effect is further modified by combinatorial or medicinal chemistry to provide analogs of said 0 compound also having said therapeutic effect. Dated: 19 November 2010