WO2009006793A1

WO2009006793A1 - Polymorphisms of scn2a associated with resistance to antiepileptic drugs and use thereof

Info

Publication number: WO2009006793A1
Application number: PCT/CN2008/001278
Authority: WO
Inventors: Larry Baum; Patrick Kwan
Original assignee: The Chinese University Of Hong Kong
Priority date: 2007-07-06
Filing date: 2008-07-07
Publication date: 2009-01-15

Abstract

Disclosed are polymorphisms of brain voltage-gated sodium channel type II alpha-subunit (SCN2A) which are associated with resistance to all mechanistic classes of anti-epileptic drugs (AEDs), or with resistance to the class of AEDs that block sodium channels, methods for effectively treating individual epilepsy patients, and uses of said polymorphisms. Particularly, the IVS7-31A>G (rs2304016) polymorphism of the SCN2A gene is associated with resistance to all classes of AEDs, and the R19K (rs17183814) polymorphism of the SCN2A gene is associated with resistance to the class of AEDs that block sodium channels.

Description

POLYMORPHISMS OF SCN2A ASSOCIATED WITH RESISTANCE TO ANTIEPILEPTIC

DRUGS AND USE THEREOF

RELATED APPLICATIONS This application claims priority to the U.S. Provisional Patent Application No.

60/958,653, entitled POLYMORPHISMS OF SCN2A ASSOCIATED WITH RESISTANCE TO ANTIEPILEPTIC DRUGS AND USE THEREOF, filed on July 6, 2007, the disclosure of which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The present invention generally relates to polymorphisms of brain voltage-gated sodium channel type II alpha-subunit (SCN2A) which are associated with resistance to anti-epileptic drugs (AED) in general, and specifically to the class of AEDs that block sodium channels, methods for effectively treating individual epilepsy patients, and uses of said polymorphisms.

BACKGROUND

Affecting up to 1% of the population, epilepsy is among the most common serious neurological disorders. Patients with epilepsy experience recurrent seizures, which are clinical manifestations of abnormal, sychronised, excessive discharge of cortical neurons. A seizure typically manifests as sudden, involuntary, disruptive, and often destructive sensory, motor, and cognitive phenomena. Seizures are frequently associated with physical harm to the body (e.g., tongue biting, limb breakage, and burns), a complete loss of consciousness, and incontinence. A typical seizure, for example, might begin as spontaneous shaking of an arm or leg and progress over seconds or minutes to rhythmic movement of the entire body, loss of consciousness, and voiding of urine or stool.

A single seizure most often does not cause significant morbidity or mortality, but severe or recurring seizures (epilepsy) result in major medical, social, and economic consequences. Epilepsy is most often diagnosed in children and young adults, making the long-term medical and societal burden severe for this population of patients. People with uncontrolled epilepsy are often significantly limited in their ability to work in many industries and cannot legally drive an automobile. An uncommon, but potentially lethal form of seizure is called status epilepticus, in which a seizure continues for more than 30 minutes. This continuous seizure activity may lead to permanent brain damage, and can be lethal if untreated. Epilepsy can result from a wide variety of causes, including head trauma (such as from a car accident or a fall), infection (such as meningitis), or from neoplastic, vascular or developmental abnormalities of the brain. In many cases, the cause is unknown and a genetic predisposition is suspected. While there is no known cure for epilepsy, chronic usage of anticonvulsant and antiepileptic medications can be effective in controlling seizures. The anticonvulsant and antiepileptic medications do not actually correct the underlying neurobiological alterations that cause seizures. Instead, the anticonvulsant and antiepileptic medications manage the patient's epilepsy by preventing or reducing the frequency of seizures. Anti-epileptic drugs (AEDs) are the mainstay of treatment.

There are a variety of AEDs acting via different mechanisms of action to enhance attenuation of excitation and/or facilitation of inhibition of neurotransmission. One class of AEDs is believed to act by blocking the repetitive firing of neuronal voltage-gated sodium channels, which are responsible for the upstroke of the neuronal action potential and ultimately control the intrinsic excitability of the nervous system. The main structural component of the neuronal sodium channel is the alpha-subunit (SCNA), which forms the ion conducting pore and confers voltage-dependency. Several isoforms of alpha-subunits are expressed in the brain, and sodium channel protein, brain (type) II alpha subunit (SCN2A) is the gene that encodes one such channel in the brain. AEDs can often very effectively prevent seizures. However, many patients only respond to some AEDs but not to others, and epilepsy is inadequately controlled by medication in up to one third of patients. Since it is difficult to predict which (or which mechanistic class of) AEDs will work for which patients on an individual level, drugs must often be tested one-after-another in each patient until an effective AED is found. This trial-and-error process exposes patients to the risk of side effects that each drug possesses, and extends the period during which patients risk suffering seizures, each of which carries a danger of injury or even death.

Therefore, to avoid these shortcomings in treatment approach, there is a long standing need to discover ways that may help to predict patients' response to AED therapy. One possible avenue is to identify genotypes associated with resistance to AEDs. To address this need, we have discovered associations of SCN2A polymorphisms with effectiveness of all classes of AEDs in general, as well as of the class of AEDs that act via blockade of sodium channels in epilepsy patients. A test which genotypes these or associated polymorphisms in epilepsy patients could help doctors decide whether or not to prescribe this class of AEDs in each patient. This is an example of a pharmacogenetic test.

SUMMARY OF INVENTION According to one aspect of the present invention, there are provided polymorphisms, polymorphic alleles, or heplotypes of the brain voltage-gated sodium channel type II alpha subunit (SCN2A) in a patient which are associated with resistance to all mechanistic classes of anti-epileptic drugs (AEDs) in general or with resistance specifically to AEDs that block sodium channels. In an embodiment of the invention, alleles of said polymorphisms associated with resistance to all mechanistic classes of AEDs in general are preferably selected from the group consisting of A alleles of SCN2A polymorphisms rsl965757, rs2304016 (splicing branch site polymorphism IVS7-31A>G), rs935403, the haplotype including the A alleles of rsl965757 and rs2304016, and the haplotype including the A alleles of rs2304016 and rs935403. Alleles associated with decreased resistance to all mechanistic classes of AEDs in general are preferably selected from the group consisting of the haplotype including the G alleles of rsl965757 and rs2304016, and the haplotype including the G alleles of rs2304016 and rs935403.

In a preferred embodiment of the invention, alleles of said polymorphisms associated with resistance to the class of AEDs that block sodium channels are preferably selected from the group consisting of the following alleles of SCN2A polymorphisms: the A allele of the rsl7183814 (R 19K) polymorphism, the T allele of the rs2116658 polymorphism, and the haplotype including the A allele of R19K and T allele of rs2116658. Alleles associated with decreased resistance to the class of AEDs that block sodium channels are preferably selected from the group consisting of the haplotype including the G allele of Rl 9K and C allele of rs2116658.

According to another aspect of the present invention there is provided a method or a pharmacogenetic test of detecting polymorphic alleles or haplotypes of brain voltage-gated sodium channel alpha subunit SCN2A in an epilepsy patient, wherein said alleles or haplotypes are associated with either increased or decreased likelihood of resistance to either all classes of AEDs in general, or to the class of AEDs that blocks sodium channels, and wherein said method or test comprises the steps of providing samples containing DNA or RNA from the patient, and contacting the samples with agents for detecting existence of the polymorphisms. According to another aspect of the invention there is provided a method of identifying an epilepsy patient possessing either increased or decreased likelihood of resistance to anti-epileptic drugs (AEDs) that block sodium channels, comprising providing samples containing DNA or RNA from the patient, and contacting the samples with agents for detecting existence of polymorphic alleles or haplotypes of brain voltage-gated sodium channel alpha subunit which are associated with either increased or decreased likelihood of resistance to the AEDs or the class of AEDs that block sodium channels, wherein the presence of said polymorphic alleles or haplotypes indicates that the patient possesses either increased or decreased likelihood of resistance to anti-epileptic drugs (AEDs) that block sodium channels.

According to another aspect of the invention there is provided a method of identifying an epilepsy patient possessing either increased or decreased likelihood of resistance to all mechanistic classes of anti-epileptic drugs (AEDs) in general, comprising providing samples containing DNA or RNA from the patient; and contacting the samples with agents for detecting existence of polymorphic alleles or haplotypes of brain voltage- gated sodium channel alpha subunit which are associated with either increased or decreased likelihood of resistance to the AEDs or the all mechanistic classes of anti-epileptic drugs (AEDs) in general, wherein the presence of said polymorphic alleles or haplotypes indicates that the patient possesses either increased or decreased likelihood of resistance to the AEDs.

According to another aspect of the present invention there is provided a method for effectively treating an epilepsy patient, wherein said method comprises determining whether the patient has polymorphic alleles or haplotypes of brain voltage-gated sodium channel alpha subunit which are are associated with either increased or decreased likelihood of resistance to either all mechanistic classes of AEDs in general or to the class of AEDs that blocks sodium channels, and administering a class of AEDs or other treatment to the patient in order to increase the likelihood of successful treatment based on the results of the detection of the polymorphic alleles or haplotypes. According to a further aspect of the present invention there is provided a kit for determining polymorphic alleles or haplotypes of brain voltage-gated sodium channel alpha subunit SCN2A associated with either increased or decreased likelihood of resistance to either all classes of anti-epileptic drugs (AEDs) in general or to the class of AEDs that blocks sodium channels in a patient, comprising agents for detecting said alleles or haplotypes, standards of said alleles or haplotypes, and optionally inserts indicating that the presence of said alleles or haplotypes is a marker of an epilepsy patient being either more or less likely to resist all classes of anti-epileptic drugs in general or the class of AEDs that blocks sodium channels.

According to a further aspect of the present invention there is provided a kit for effectively treating an epilepsy patient, comprising agents for detecting polymorphic alleles or haplotypes of brain voltage-gated sodium channel alpha subunit SCN2A which are associated with resistance to either all classes of AEDs in general or to the class of AEDs that blocks sodium channels, standards of said polymorphisms,

AEDs, and optionally inserts indicating that whether the patient can be effectively treated or administered with said AEDs according to the results of the detection of polymorphic alleles or haplotypes. According to a further aspect of the present invention there is provided use of polymorphic alleles or haplotypes of SCN2A in preparing a kit for determining, a patient's polymorphisms of brain voltage-gated sodium channel alpha subunit SCN2A, which are associated with either increased or decreased likelihood of resistance to either all classes of anti-epileptic drugs (AEDs) in general or to the class of AEDs that blocks sodium channels. According to a further aspect of the present invention there is provided use of polymorphic alleles or haplotypes of SCNA which are associated with to either all classes of anti-epileptic drugs (AEDs) in general or to the class of AEDs that blocks sodium channels in preparing a kit for effectively treating an individual epilepsy patient.

In an embodiment, the kit of the invention may optionally comprise additional components useful for performing the methods of the invention. By way of example, the kit may comprise fluids (e.g. PBS buffer, SSC buffer) suitable for diluting samples or annealing complementary nucleic acids, one of more sample compartments, an instructional material which describes performance of a method of the invention, a sample of normal control, and the like. According to still another aspect of the present invention there is provided use of polymorphic alleles or haplotypes of SCN2A which are associated with either increased or decreased likelihood of resistance to either all classes of anti-epileptic drugs (AEDs) in general or to the class of AEDs that blocks sodium channels as a marker for consideration of non-AED therapy or for not prescribing certain AEDs to an epilepsy patient. BRIEF DESCRIPTION OF DRAWINGS

Figure 1 shows the results of Electrophysiology, panel A presents the result of the wild type of Rl 9K, panel B presents the result of 19K in which CTRL means control; and PHT represents the AED phenytoin.

DETAILED DESCRIPTIONS OF THE INVENTION

I. DEFINITIONS The term "allele" as used herein is any of one or more alternative forms of a gene, all of which alleles relates to one trait or characteristic. In a diploid cell or organism, the two alleles of a given gene occupy corresponding loci on a pair of homologous chromosomes.

The term "polymorphism" as used herein refers to the occurrence of two or more genetically determined alternative sequences or alleles in a population. A polymorphic marker or site is the locus at which divergence occurs. A polymorphism may comprise one or more base changes, an insertion, a repeat, or a deletion. A polymorphic locus may be as small as one base pair. The first identified allelic form is arbitrarily designated as the reference form and other allelic forms are designated as alternative or variant alleles. The allelic form occurring most frequently in a selected population is sometimes referred to as the wild type form. Diploid organisms may be homozygous or heterozygous for allelic forms. Single nucleotide polymorphisms (SNPs) are included in polymorphisms. The term ""polymorphism" as used herein also refers to "polymorphic allele".

As used herein, the term "anti-epileptic drug(s)" or "AED(s)" generally encompasses pharmacological agents that reduce the frequency or likelihood of a seizure. There are many drug classes that comprise the set of antiepileptic drugs (AEDs), and many different mechanisms of action are represented. For example, some medications are believed to increase the seizure threshold, thereby making the brain less likely to initiate a seizure. Other medications retard the spread of neural bursting activity and tend to prevent the propagation or spread of seizure activity. Some AEDs, such as the Benzodiazepines, act via the GABA receptor and globally suppress neural activity. However, other AEDs may act by modulating a neuronal calcium channel, a neuronal potassium channel, a neuronal NMDA channel, a neuronal AMPA channel, a neuronal metabotropic type channel, a neuronal sodium channel, and/or a neuronal kainite channel. The phrase "Anti-epileptic drugs that block sodium channels", "sodium-channel-blocking AEDs" used herein refers to anti-epileptic drugs that block sodium channels. The sodium-channel-blocking AEDs can be selected from the group consisting of topiramate, carbamazepine, oxcarbazepine, phenytoin, lamotrigine, zonisamide, felbamate, ethosuximide, and valproate (valproic acid), as well as other existing or new AEDs which may be identified to block sodium channels in the future.

The term "SCN2A" is used herein to refer to nucleic acids that encode a sodium channel type II alpha subunit polypeptide. The term "SCN2A" also refers to nucleic acids encoding a voltage-gated brain-specific sodium channel. The term "SCN2A" also refers generally to polypeptides encoded by SCN2A.

The polymorphisms of SCN2A comprise at least 309 SNPs, some of which are shown in the table below. We have discovered associations of SCN2A polymorphisms with response or resistance to all classes of AEDs in general or to the class of AEDs which block sodium channels in epilepsy patients.

Table A

The term "primer" as used herein refers to a single-stranded oligonucleotide capable of acting as a point of initiation for template-directed DNA synthesis under suitable conditions for example, buffer and temperature, in the presence of four different nucleoside triphosphates and an agent for polymerization, such as, for example, DNA or RNA polymerase or reverse transcriptase. The length of the primer, in any given case, depends on, for example, the intended use of the primer, and generally ranges from 15 to 30 nucleotides. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. A primer need not reflect the exact sequence of the template but must be sufficiently complementary to hybridize with such template. The primer site is the area of the template to which a primer hybridizes. The primer pair is a set of primers including a 5' upstream primer that hybridizes with the 5' end of the sequence to be amplified and a 3' downstream primer that hybridizes with the complement of the 3' end of the sequence to be amplified.

The term "probe" as used herein refers to sequences that are complementary to or mimic the polymorphisms of the present invention to hybridize specifically to the polymorphisms. Probes can comprise 10 to 20 nucleotides, or even longer where desired, such as 30, 40, 50, 60, 100, 200, 300, or 500 nucleotides or up to the full length of any one of SCNA. Such fragments can be readily prepared by, for example, chemical synthesis of the fragment, by application of nucleic acid amplification technology, or by introducing selected sequences into recombinant vectors for recombinant production. Probes can be immobilized on a surface and can be recognized by a particular target. See U.S. Pat. No. 6,582,908 as an example of arrays having all possible combinations of probes with 10, 12, and more bases.

The phrase "hybridizing specifically to" refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex nucleic acid mixture (e.g., total cellular DNA or RNA).

"Stringent hybridization conditions" and "stringent hybridization wash conditions" in the context of nucleic acid hybridization experiments such as Southern and Northern blot analysis are both sequence- and environment-dependent. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, part I chapter 2, Elsevier, New York, N. Y. Generally, highly stringent hybridization and wash conditions are selected to be about 5. degree. C. lower than the thermal melting point (T.sub.m) for the specific sequence at a defined ionic strength and pH. Typically, under "stringent conditions" a probe will hybridize specifically to its target subsequence, but to no other sequences.

The following are examples of hybridization and wash conditions that can be used to identify nucleotide sequences that are substantially identical to reference nucleotide sequences of the present invention: a probe nucleotide sequence preferably hybridizes to a target nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO.sub.4, 1 mM EDTA at 5O.degree. C. followed by washing in 2.times.SSC, 0.1% SDS at 50.degree. C; more preferably, a probe and target sequence hybridize in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO.sub.4, 1 mM EDTA at 50. degree. C. followed by washing in l.times.SSC, 0.1% SDS at 50.degree. C; more preferably, a probe and target sequence hybridize in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO.sub.4, 1 mM EDTA at 50.degree. C. followed by washing in 0.5.times.SSC, 0.1% SDS at 50. degree. C; more preferably, a probe and target sequence hybridize in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO.sub.4, 1 mM EDTA at 50.degree. C. followed by washing in 0.1. times. SSC, 0.1% SDS at 50. degree. C; more preferably, a probe and target sequence hybridize in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO.sub.4, 1 mM EDTA at 5O.degree. C. followed by washing in O.l.times.SSC, 0.1% SDS at 65.degree. C.

The term "array" or "microarray" as used herein refers to an intentionally created collection of molecules which can be prepared either synthetically or biosynthetically. The molecules in the array can be identical or different from each other. The array can assume a variety of formats, for example, libraries of soluble molecules comprising the polymorphisms of the invention tethered to resin beads, silica chips, or other solid supports.

The microarray can be used to monitor the expression level of large numbers of genes simultaneously and to identify polymorphisms. Microarrays may be prepared, used, and analyzed using methods known in the art. (For example, see Schena et al., 1996; Heller et al., 1997).

II. Determining Polymorphisms Resistant to AEDs

Epilepsy patients who take AEDs for >1 year are selected in a study of the invention.

Patients who have not experienced any seizure for at least a year up to the date of recruitment, and receiving a stable dose of AED treatment, are considered to have drug responsive epilepsy.

Patients who have had an average of one seizure or more per month over the previous year despite treatment with two or more AEDs at therapeutic dosages and/or serum drug concentrations are considered to have drug resistant epilepsy. In this context, AEDs must have failed primarily due to inadequate efficacy instead of adverse effects. Samples, such as blood samples, are collected from the patients. DNA and/or RNA are extracted from samples of the two sorts of patients. Methods for extracting DNA and RNA are well known in the art.

We use a haplotype-tagging strategy for SCNA, particularly SCNlA, SCN2A, and SCN3A, which are located in a cluster on chromosome 2.

In a preferred embodiment, on the HapMap.org website, SCN2A is selected and zoomed out 10% to include flanking regions, showing 105.5 kbp from positions 165,971,301 to 166,076,842 on chromosome 2. Similarly, SCN3A is selected and zoomed out 10%, showing 128.2 kbp from positions 165,763,721 to 165,891,884 on chromosome 2. CHB (Chinese Beijing) SNP genotype data from HapMap Data Rel#21/phaseII JulO6 are downloaded from the 313.1 kbp region from 165,763,721 to 166,076,842, including the two genes (SCN2A and SCN3A) and flanking regions.

The Tagger function of the Haploview program

(http://www.broad.mit.edu/mpg/haploview) is used with default settings, except that the minimum minor allele frequency is set at 5%. Aggressive tagging is selected, the r² threshold is 0.8, and the LOD threshold for multi-marker tests is 3. One of ordinary skill in the art will understand that other r² threshold or LOD threshold can be selected as desired.

Markers between the two genes, in the region from 165,891,884 to 165,971,301, are deselected. We force the inclusion of a coding SNP: rsl7183814 (SCN2A R19K). Some markers are incompatible with others in our genotyping assay design, and

Tagger is run again with such markers forced to be excluded. This process is repeated several times until all markers generated are compatible. We chose 19 tagging SNPs: 14 in or flanking SCN2A and 5 in or flanking SCN3A, capturing 172 of 200 alleles with r²>0.8 (mean=0.96). The smaller number of tagging SNPs for SCN3A was due to its relatively simpler haplotype structure.

In another preferred embodiment, on the HapMap.org website, SCNlA is selected and zoomed out 10% to include flanking regions, showing 90.6 kbp from positions 166,669,112 to 166,759,757 on chromosome 2. We force inclusion of a coding SNP, rs2298771 (AlalO56Thr), and a SNP reportedly associates with AED dose, rs3812718 (TVS5-91G>A), and chose four more tagging SNPs, capturing 85 of 99 alleles with mean r²>0.8 (mean=0.98).

III. Polymorphisms Associated with Resistance or Response to AEDs According to the invention, polymorphisms in moderate or strong linkage disequilibrium with the polymorphisms we identified and specified may also be associated with resistance or response to AEDs and will be included in the invention..

In the present invention, the inventors find polymorphisms of brain voltage-gated sodium channel type II alpha subunit (SCN2 A) associated with resistance to all classes of AEDs in general or to the class of AEDs that block sodium channels. In one preferable embodiment of the invention, alleles of said polymorphisms associated with resistance to all classes of AEDs in general are preferably selected from the group consisting of A alleles of SCN2 A polymorphisms rsl965757, rs2304016 (splicing branch site polymorphism IVS7-31A>G), rs935403, the haplotype including the A alleles of rsl965757 and rs2304016, and the haplotype including the A alleles of rs2304016 and rs935403. Alleles associated with decreased resistance to all classes of AEDs in general are preferably selected from the group consisting of the haplotype including the G alleles of rsl965757 and rs2304016, and the haplotype including the G alleles of rs2304016 and rs935403.

Alleles of said polymorphisms associated with resistance to the class of AEDs that block sodium channels are preferably selected from the group consisting of the following alleles of SCN2A polymorphisms: the A allele of the Rl 9K polymorphism, the T allele of the rs2116658 polymorphism, and the haplotype including the A allele of R19K and T allele of rs2116658. Alleles associated with decreased resistance to the class of AEDs that block sodium channels are preferably selected from the group consisting of the haplotype including the the G allele of R19K and C allele of rs2116658. In a more preferred embodiment of the invention, said polymorphisms include the

Rl 9K polymorphism or IVS7-31A>G polymorphism, hi one embodiment of the invention, said polymorphisms also comprise polymorphisms near SCN2A and in moderate or strong linkage disequilibrium with any of the above polymorphisms or haplotypes.

IV. Pharmacogenetic Tests or Methods

There is currently no pharmacogenetic test to guide doctors in prescribing AEDs. Since many patients only respond to some AEDs but not to others, and epilepsy is inadequately controlled by medication in up to one third of patients, a method or a pharmacogenetic test which scans associated polymorphisms in epilepsy patients could help doctors decide whether or not to prescribe certain AEDs in a patient. That means the tests or methods assist doctors to classify epilepsy patients into two classes, i.e. patients whose epilepsy is likely to be resistant to AEDs and patients whose epilepsy is likely to be responsive to AEDs. In a preferred embodiment, said AEDs are the class of AEDs which block sodium channels.

This test or method would allow such guidance, reducing the time to find effective AEDs and reducing the number of ineffective AEDs tested in each patient, thus decreasing the risk of side effects from ineffective AEDs and decreasing the danger of seizures during the period before an effective AED is identified.

In one embodiment of the method or test, samples are collected from epilepsy patients and subjected to the method or the pharmacogenetic test. The samples used in the test will be any samples containing nucleic acids (including DNA or RNA), such as blood samples, body fluids, secretions, etc., however, blood samples are preferred. The methods for extracting DNA or RNA are well known in the art and as described in the Sambrook and Russell, Molecular Cloning: A Laboratory Manual 3^rd Edition, New York: Cold Spring Harbor Laboratory Press, 2001. The method or the pharmacogenetic test of the invention will involve detection methods of single nucleotide polymorphisms to determining the presence of the polymorphisms associated with resistance to AEDs in a patient.

Detection methods of single nucleotide polymorphisms are well known in the art such as the methods described in Zhao, et al., Progress in Detection Methods of Single Nucleotide Polymorphisms. Hereditas, 2005;27(l): 123-129, and may comprise a method based on a gene chip, HyBeacon™ probes, fluorescence resonance such as molecular beacon, alphascreen and reversed enzyme activity DNA interrogation test (READIT), fluorescence polarization (FP), TaqMan, mass spectrometry including solid phase capture able-single base extension (SPC-SBE), GOOD assay and electrospray ionization mass spectrometry (ESI-MS), bacterial magnetic particle (BMP), or other existing or new methods.

If an epilepsy patient possess the polymorphisms associated with resistance either to all classes of AEDs in general or to the class of AEDs that block sodium channels, the patient will be identified as an epilepsy patient of the AEDs resistant. Accordingly, the pharmacogenetic tests or the methods of the invention for detecting the polymorphisms associated with resistance either to all classes of AEDs in general or to the class of AEDs that block sodium channels can be used in a method for identifying a an epilepsy patient being resistant to either to all classes of AEDs in general or to the class of AEDs that block sodium channels. In an embodiment of the invention, there is provided a method for identifying possessing either increased or decreased likelihood of resistance to anti-epileptic drugs (AEDs) that block sodium channels, comprising providing samples containing DNA or RNA from the patient, and contacting the samples with agents for detecting existence of polymorphic alleles or haplotypes of brain voltage-gated sodium channel alpha subunit which are associated with either increased or decreased likelihood of resistance to the AEDs or the class of AEDs that block sodium channels, wherein the presence of said polymorphic alleles or haplotypes indicates that the patient possesses either increased or decreased likelihood of resistance to anti-epileptic drugs (AEDs) that block sodium channels. hi another embodiment of the invention, there provided a method of identifying an epilepsy patient possessing either increased or decreased likelihood of resistance to all mechanistic classes of anti-epileptic drugs (AEDs) in general, comprising providing samples containing DNA or RNA from the patient, and contacting the samples with agents for detecting existence of polymorphic alleles or haplotypes of brain voltage-gated sodium channel alpha subunit which are associated with either increased or decreased likelihood of resistance to the AEDs or the all mechanistic classes of anti-epileptic drugs (AEDs) in general, wherein the presence of said polymorphic alleles or haplotypes indicates that the patient possesses either increased or decreased likelihood of resistance to the AEDs.

V. Methods for Effectively Treating Individual Epilepsy Patients The invention also relates to a method for effectively treating individual epilepsy patients comprising detecting polymorphisms associated with said resistance, followed by administering an AED to a patient who does not have the alleles or haplotypes associated with said resistance.

In one embodiment of the invention, detecting polymorphisms is performed through a pharmacogenetic test of the invention. For example, if the alleles or haplotypes of a patient comprise those selected from the group consisting of the A allele of the Rl 9K polymorphism, the T allele of the rs2116658 polymorphism, the haplotype including the A allele of R19K and T allele of rs2116658, or alleles or haplotypes in moderate or strong linkage disequilibrium with those polymorphisms, a doctor or a physician will not prescribe the class of AEDs that block sodium channels but AEDs that act by other mechanisms of action instead. As another example, if the alleles or haplotypes of a patient comprise those selected from the group consisting of the A alleles of SCN2A polymorphisms rsl965757, rs2304016 (splicing branch site polymorphism rVS7-31A>G), rs935403, the haplotype including the A alleles of rsl965757 and rs2304016, and the haplotype including the A alleles of rs2304016 and rs935403, a doctor or a physician may more readily consider non-drug treatment such as surgery.

VI. Kits

In one aspect, the invention provides a kit for detecting alleles or haplotypes of polymorphisms of SCN2A associated with resistance to all classes of AEDs or specifically to the class of AEDs that block sodium channels, comprising agents for detecting said polymorphisms, standards of said polymorphisms, and inserts indicating that the presence of said alleles or haplotypes is a marker of an epilepsy patient being resistant to anti-epileptic drugs. In one embodiment of the invention, the agents comprise a probe, a primer, or a microarray for detecting said polymorphisms. Optionally, the kit can comprise other agents or materials commonly used in assays such as Southern blotting, Northern blotting, or PCR.

In another aspect, the invention also provides a kit for effectively treating individual epilepsy patients. In one embodiment of the kit for treating epilepsy, the kit can comprise the agents for detecting polymorphisms of SCN2A associated with resistance to all classes of anti-epileptic drugs (AED) in general, as well as the class of anti-epileptic drugs (AED) that block sodium channels.

In an embodiment of the invention, the agents for detecting said polymorphisms of the invention can be the agents used in any assay for detecting single nucleotide polymorphisms, such as a probe, a primer or a microarray designed according to the polymorphisms to be tested, hi preferred embodiments of the invention, the assay for detecting single nucleotide polymorphisms can a method based on gene chip, HyBeacon™ probes, fluorescence resonance such as molecular beacon, alphascreen and reversed enzyme activity DNA interrogation test (READIT), fluorescence polarization (FP), TaqMan, mass spectrometry including solid phase capture able-single base extension (SPC-SBE), GOOD assay and electrospray ionization mass spectrometry (ESI-MS), or bacterial magnetic particles (BMP) and other methods as described in Zhao, et al., Progress in Detection Methods of Single Nucleotide Polymorphisms. Hereditas, 2005 ;27(1): 123-129, and references as cited therein.

VII. Use of Polymorphisms of SCN2A

The present invention also relates to use of polymorphisms of SCN2 A associated with resistance to anti-epileptic drugs (AEDs) in treating epilepsy patients.

The associated polymorphisms may be used to design probes or primers that could, for example, hybridize to the DNA in samples, or could be used to amplify the DNA samples using, for example, polymerase chain reaction techniques. Such probes or primers may be detectably labeled. By "detectably labeled" is meant any means for marking and identifying the presence of a molecule, e. g., an oligonucleotide probe or primer, a gene or fragment thereof, or a molecule. Said polymorphisms can also be used in preparing an array or a microarray to detect polymorphisms of epilepsy patients. Accordingly, in embodiments of the invention, the polymorphisms of the invention can be used in manufacture of kits of the invention as described herein. In other embodiment of the invention, said polymorphisms can also be used as a marker for not prescribing the AEDs to an epilepsy patient who possesses the polymorphisms associate with resistance to the AEDs. One of ordinary skill in the art will understand other use of the polymorphisms of the invention in view of the disclosure herein.

EXAMPLES

EXAMPLE 1 Scanning Polymorphisms in Patients Resistant or Responding to AEDs Methods and Materials

Chinese epilepsy patients administered with AEDs for >1 year were studied. Patients who had not experienced any seizure for at least a year up to the date of recruitment, and received a stable dose of AED treatment, were considered to have drug responsive epilepsy. Patients who had had an average of one seizure or more per month over the previous year despite treatment with two or more AEDs at therapeutic dosages and/or serum drug concentrations were considered to have drug resistant epilepsy. In this context, AEDs must have failed primarily due to inadequate efficacy instead of adverse effects. 282 patients with drug responsive epilepsy and 216 patients with drug resistant epilepsy were included. We used a haplotype-tagging strategy for SCNlA, SCN2A, and SCN3A, which are located in a cluster on chromosome 2. On the HapMap.org website, SCN2A was selected and zoomed out 10% to include flanking regions, showing 105.5 kbp from positions 165,971,301 to 166,076,842 on chromosome 2. Similarly, SCN3A was selected and zoomed out 10%, showing 128.2 kbp from positions 165,763,721 to 165,891,884 on chromosome 2. CHB (Chinese Beijing) SNP genotype data from HapMap Data Rel#21/phaseII JulO6 were downloaded from the 313.1 kbp region from 165,763,721 to 166,076,842, including the two genes (SCN2A and SCN3A) and flanking regions. The Tagger function of the Haploview program (http://www.broad.mit.edu/mpg/haploview) was used with default settings, except that the minimum minor allele frequency was set at 5%. Aggressive tagging was selected, the r² threshold was 0.8, and the LOD threshold for multi-marker tests was 3. Markers between the two genes, in the region from 165,891,884 to 165,971,301, were deselected. We forced the inclusion of a coding SNP: rsl7183814 (SCN2A R19K). Some markers were incompatible with others in our genotyping assay design, and Tagger was run again with such markers forced to be excluded; this process was repeated several times until all markers generated were compatible. We chose 19 tagging SNPs: 14 in or flanking SCN2A and 5 in or flanking SCN3A, capturing 172 of 200 alleles with r²>0.8 (mean=0.96). The smaller number of tagging SNPs for SCN3A was due to its relatively simpler haplotype structure. On the HapMap.org website, SCNlA was selected and zoomed out 10% to include flanking regions, showing 90.6 kbp from positions 166,669,112 to 166,759,757 on chromosome 2. We forced inclusion of a coding SNP, rs2298771 (AlalO56Thr), and a SNP reportedly associated with AED dose, rs3812718 (IVS5-91G>A), and chose four more tagging SNPs, capturing 85 of 99 alleles with mean r²>0.8 (mean=0.98).

DNA and RNA were extracted from blood samples. SNPs were genotyped using a Sequenom Mass Array System. Statistics

Haploview 3 and Epi 6 software were used to analyze association of alleles and haplotypes with AED resistance. For each polymorphism, the data were analyzed by chi square test for the two classes of patients and either the three genotypes or the two alleles. Haploview estimated haplotypes using an accelerated EM algorithm. Results

A alleles of SCN2A polymorphisms rsl965757, rs2304016 (splicing branch site polymorphism IVS7-31A>G), rs935403, the haplotype including the A alleles of rsl965757 and rs2304016, and the haplotype including the A alleles of rs2304016 and rs935403 were associated with drug resistance to all classes of AEDs in general: OR=I.4, p=0.02; OR=2.0, p=0.007; OR=I.5, p=0.03; OR=I.4, p=0.02; and OR=I.4, p=0.02, respectively. The haplotype including the G alleles of rsl965757 and rs2304016, and the haplotype including the G alleles of rs2304016 and rs935403 were associated with decreased drug resistance: OR=0.5, p=0.007; and OR=0.5, p=0.02. In 92 patients responsive to AEDs that block sodium channels and 193 patients resistant to such drugs, the T allele of rs211658, the A allele of rsl7183814 (R19K), and the haplotype including the A allele of R19K and T allele or rs2116658 were associated with resistance: OR=2.0, ρ=0.02; OR=I .9, p=0.03; and OR=I.9, p=0.03; respectively. In the same patients, the haplotype including the G allele of R19K and C allele or rs2116658 were associated with decreased resistance: OR=O.6, p=0.03.

Table 1. Association of polymorphisms with response to all classes of AEDs in general:

SNP rs# Genotype frequencies P

Remission Refractory Genotypes

1965757 0.470 AA 0.437 AG 0.093 GG 0.563 AA 0.386 AG 0.051 GG 0.06

2304016 0.806 AA 0.191 AG 0.004 GG 0.912 AA 0.074 AG 0.014 GG 0.001*

935403 0.503 AA 0.400 AG 0.097 GG 0.598 AA 0.358 AG 0.045 GG 0.07

Table 2. Association of polymorphisms with response to the class of AEDs that block sodium channels:

*Chi-square calculated for the more common homozygote vs. the other two genotypes combined.

EXAMPLE 2 Electrophysiology Electrophysiology was performed on HEK cells transfected with SCN2A expression plasmids. To study the effect of drugs on the sodium current, sodium current was evoked by holding the voltage at fixed values (-140 mV to -20 mV) and cycling through the following voltage pattern at 2 Hz: 0 mV for 10 milliseconds, back to the original holding voltage for 30 milliseconds, slight hyperpolarization (by 10 or 20 mV) for 20 milliseconds, and back to the original holding voltage. We first ran the protocols for 20 sweeps, waited a couple of minutes until the current was stable, then recorded the current (Control, 20 sweeps). We then added the drugs, waited for 5 minutes, then recorded the current again (Drug, 20 sweeps). We then compared the amplitude of sodium current before and after drugs to see the degree to which drugs blocked the sodium channel. The amplitude of sodium current ranged from 1 mA to 6 mA. All the experiments were performed at room-temperature (~20°C). Pipette resistance was 2-4 MΩ, Cm=I 0~40 pF. Cell and electrode capacitance and series resistance were compensated. Sampling time was 8 μs (125 kHZ), and the 4 pole lowpass Bessel filter was 10 kHZ. Residual linear leak and capacitance were subtracted using a P/4 protocol.

Electrophysiology evidence suggests that the Rl 9K polymorphism may have an effect on resistance to the class of AEDs that block sodium channels; for example, the AED phenytoin blocked the 19K form of the SCN2A protein to a lesser degree than the wild-type channel at holding potentials of -60 mV or higher, as shown in Figure 1.

EXAMPLE 3 Real Time PCR Real time PCR evidence suggests that IVS7-31A>G (rs2304016) is associated with an effect on SCN2A expression, perhaps by directly affecting mRNA splicing. RNA was purified from fresh blood samples of human subjects previously genotyped for SCN2A polymorphisms. Ficoll reagent was used to isolate white blood cells and Qiagen RNeasy kits were used to isolate RNA from white blood cells. From each sample, 500 ng RNA was reverse-transcribed to cDNA using random hexamers and Superscript II Reverse Transcriptase. Real-time PCR with primers in SCN2A, and in 18S rRNA as a control RNA, was performed on a real-time PCR machine (Applied Biosystems) using 10% of the cDNA for each sample. SCN2A mRNA levels increased with the number of IVS7-31G alleles in each subject (p=0.013, ANOVA).

While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention.

All patents, patent applications, and other publications cited in this application are incorporated by reference in their entirety.

Claims

WHAT IS CLAIMED IS:

1. A method of identifying an epilepsy patient possessing either increased or decreased likelihood of resistance to anti-epileptic drugs (AEDs) that block sodium channels, or all mechanistic classes of AEDs, comprising providing samples containing DNA or RNA from the patient; and contacting the samples with agents for detecting existence of polymorphic alleles or haplotypes of brain voltage-gated sodium channel alpha subunit which are associated with either increased or decreased likelihood of resistance to the class of AEDs that block sodium channels, or the all mechanistic classes of AEDs, wherein the presence of said polymorphic alleles or haplotypes indicates that the patient possesses either increased or decreased likelihood of resistance to the AEDs that block sodium channels or the all mechanistic classes of AEDs.

2. The method of claim 1, wherein the polymorphic alleles or haplotypes associated with increased resistance to the class of AEDs that blocks sodium channels are selected from the group consisting of the A allele of the rsl7183814 (R19K) polymorphism, the T allele of the rs2116658 polymorphism, and the haplotype including the A allele of Rl 9K and T allele of rs2116658.

3. The method of claim 1, wherein the haplotypes associated with decreased resistance to the class of AEDs that blocks sodium channels comprise the haplotype including the G allele of R19K and C allele of rs2116658.

4. The method of claim 1, wherein one of the polymorphic alleles is the K allele of the R19K (rsl7183814) polymorphism of the SCN2A gene, the patient possessing the K allele more commonly shows the resistance to the class of AEDs that blocks sodium channels.

5. The method of claim 1, wherein one of the polymorphic alleles is the T allele of the rs2116658 polymorphism of the SCN2A gene, the patient possessing the T allele more commonly shows the resistance to the class of AEDs that blocks sodium channels.

6. The method of claim 1, wherein one of the haplotypes includes the A allele of R19K and T allele of rs2116658 of the SCN2A gene, the patient possessing the above haplotype more commonly shows resistance to the class of AEDs that blocks sodium channels.

7. The method of claim 1, wherein one of the haplotypes includes the G allele of R19K and C allele of rs2116658 of the SCN2A gene, the patient possessing the above haplotype less commonly showing resistance to the class of AEDs that blocks sodium channels.

8. The method of any one of claims 1-7, wherein the class of AED that blocks sodium channels can be selected from the group consisting of topiramate, carbamazepine, oxcarbazepine, phenytoin, lamotrigine, zonisamide, felbamate, ethosuximide, valproate, and other existing or new AEDs that block sodium channels.

9. The method of claim 1, wherein the polymorphic alleles or haplotypes associated with increased resistance to all mechanistic classes of anti-epileptic drugs (AEDs) are selected from the group consisting of A alleles of SCN2A polymorphisms rs 1965757, rs2304016 (splicing branch site polymorphism IVS7-31A>G), rs935403, the haplotype including the A alleles of rsl965757 and rs2304016, and the haplotype including the A alleles of rs2304016 and rs935403.

10. The method according to claim 1, wherein the haplotypes associated with decreased resistance to all mechanistic classes of AEDs are selected from the group consisting of the G alleles of rsl965757 and rs2304016, and the haplotype including the G alleles of rs2304016 and rs935403.

11. The method according to claim 1, wherein one of the polymorphic alleles is the A allele of the rsl965757 polymorphism of the SCN2A gene, the patient possessing the A allele more commonly shows resistance to all mechanistic classes of AEDs.

12. The method according to claim 1, wherein one of the polymorphic alleles is the A allele of the rs2304016 polymorphism of the SCN2A gene, the patient possessing the A allele more commonly shows resistance to all mechanistic classes of AEDs.

13. The method according to claim 1, wherein one of the polymorphic alleles is the A allele of the rs935403 polymorphism of the SCN2A gene, the patient possessing the A allele more commonly shows resistance to all mechanistic classes of AEDs.

14. The method according to claim 1, wherein one of the haplotypes includes the A alleles of rsl965757 and rs2304016 of the SCN2A gene, the patient possessing the above haplotype more commonly shows resistance to all mechanistic classes of AEDs.

15. The method according to claim 1, wherein one of the haplotypes includes the A alleles of rs2304016 and rs935403 of the SCN2A gene, the patient possessing the above haplotype more commonly shows resistance to all mechanistic classes of AEDs.

16. The method according to claim 1, wherein one of the haplotypes includes the G alleles of rsl965757 and rs2304016 of the SCN2A gene, the patient possessing the above haplotype less commonly shows resistance to all mechanistic classes of AEDs.

17. The method according to claim 1, wherein one of the haplotypes includes the G alleles of rs2304016 and rs935403 of the SCN2A gene, the patient possessing the above haplotype less commonly shows resistance to all mechanistic classes of AEDs.

18. The method of any one of claims 1-17, wherein the class of AED that blocks sodium channels can be selected from the group consisting of topiramate, carbamazepine, oxcarbazepine, phenytoin, lamotrigine, zonisamide, felbamate, ethosuximide, valproate, and other AEDs that block sodium channels.

19. The method of any one of claims 1-18, wherein said AEDs (not limited to drugs that block sodium channels) can be selected from the above drugs as well as gabapentin, levetiracetam, phenobarbital, pregabalin, benzodiazepines, vigabatrin, tiagabine, and other drugs treating epilepsy.

20. The method of any one of claims 1-19, wherein said polymorphic alleles or haplotypes comprise other polymorphic alleles or haplotypes in or near the SCN2A gene and in moderate or strong linkage disequilibrium with the polymorphic alleles or haplotypes.

21. A method for effectively treating an epilepsy patient, wherein said method comprises providing samples containing DNA or RNA from the patient; contacting the samples with agents for detecting existence of polymorphic alleles or haplotypes of brain voltage-gated sodium channel alpha subunit SCN2A which are associated with either increased or decreased likelihood of resistance to either all mechanistic classes of

AEDs or to the class of AEDs that blocks sodium channels to suggest whether the patient can be effectively treated or administered with said AEDs; and administering a class of AEDs or other treatment to the patient in order to increase the likelihood of successful treatment based on the results of the detection of the polymorphic alleles or haplotypes .

22. The method of claim 21, wherein the polymorphic alleles or haplotypes are selected from the group consisting of the alleles or haplotyes used in the method according to any one of claims 1 -20.

23. A kit for determining polymorphic alleles or haplotypes of brain voltage-gated sodium channel alpha subunit SCN2A associated with either increased or decreased likelihood of resistance to either all classes of anti-epileptic drugs (AEDs) or to the class of AEDs that blocks sodium channels in a patient, comprising agents for detecting said alleles or haplotypes; standards of said alleles or haplotypes; and optionally inserts indicating that the presence of said alleles or haplotypes is a marker of an epilepsy patient being either more or less likely to resist all classes of anti-epileptic drugs or the class of AEDs that blocks sodium channels.

24. The kit of claim 23, wherein the polymorphic alleles or haplotypes are selected from the group consisting of the alleles or haplotyes used in the method according to any one of claims 1-20.

25. The kit of claim 23 or 24, wherein the agents for detecting said polymorphic alleles or haplotypes can be the agents used in any assay for detecting single nucleotide polymorphisms, such as a probe, a primer or a microarray designed according to the polymorphic alleles or haplotypes to be tested.

26. A kit for effectively treating an epilepsy patient, comprising agents for detecting polymorphic alleles or haplotypes of brain voltage-gated sodium channel alpha subunit SCN2A which are associated with resistance to either all classes of AEDs or to the class of AEDs that blocks sodium channels; standards of said alleles or haplotypes; AEDs; and optionally inserts indicating that whether the patient can be effectively treated or administered with said AEDs according to the results of the detection of polymorphic alleles or haplotypes.

27. The kit of claim 26, wherein the polymorphic alleles or haplotypes are selected from the group consisting of the alleles or haplotyes used in the method according to any one of claims 1-20.

28. The kit of claim 26 or 27,wherein the agents for detecting said polymorphic alleles or haplotypes can be the agents used in any assay for detecting single nucleotide polymorphisms, such as a probe, a primer or a microarray designed according to the polymorphic alleles or haplotypes to be tested.

29. Use of polymorphic alleles or haplotypes of SCN2A in preparing a kit for determining, a patient's polymorphisms of brain voltage-gated sodium channel alpha subunit SCN2A, which are associated with either increased or decreased likelihood of resistance to either all classes of anti-epileptic drugs (AEDs) or to the class of AEDs that blocks sodium channels.

30. Use of polymorphic alleles or haplotypes of SCN2A which are associated with either increased or decreased likelihood of resistance to either all classes of anti-epileptic drugs (AEDs) or to the class of AEDs that blocks sodium channels in preparing a kit for effectively treating an individual epilepsy patient.

31. Use of polymorphic alleles or haplotypes of SCN2A which are associated with either increased or decreased likelihood of resistance to either all classes of anti-epileptic drugs (AEDs) or to the class of AEDs that blocks sodium channels as a marker for consideration of non-AED therapy or for not prescribing certain AEDs to an epilepsy patient.