JP2007124974A - Method for detecting hereditary predisposition of human epilepsy - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for detecting genetic predisposition of human epilepsy. <P>SOLUTION: The method comprises the detection of the presence of mutation generating the change of amino acid sequence of human KCND2 protein in the base sequence of human KCND2 gene. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for detecting the presence or absence of a genetic predisposition to human epilepsy.

  Epilepsy is caused by excessive synchronous reflex activity of cerebral neurons, resulting in the same type of clinical seizures in the same individual (systemic tonic-clonic seizures, absence seizures, hallucinogenic seizures, partial tonic limbs) It refers to a condition that repeats seizures). It is also accompanied by a wide variety of clinical and laboratory findings.

  Epilepsy is an idiopathic epilepsy with only epileptic seizures and no ventricular lesions, ventricular lesions, refractory / severe symptomatic epilepsy, and no ventricular lesions. Sexual epilepsy is classified into three types of latent epilepsy, which is difficult to classify.

  Idiopathic epilepsy accounts for about 70% of all epilepsy. Based on such factors as family onset and high agreement rate in identical twins (70%), genetic background is assumed for idiopathic epilepsy, and up to 20 genes have been identified to date, most of which are ions. Code the channel.

  In symptomatic epilepsy, many of the causes are due to injury to the brain due to traffic accidents, infections, perinatal disorders, and the like. Less than 10% are hereditary (less than 3% of all epilepsy), but there are more than 200 types and the number of causative genes reported exceeds that of idiopathic epilepsy. To date, in contrast to idiopathic epilepsy, no causative gene encoding an ion channel has been reported.

  Lafora disease is known as a typical hereditary symptomatic epilepsy. Lafora's disease is an inherited symptomatic epilepsy with an autosomal recessive inheritance, with generalized convulsions manifesting as an initial symptom from 6 to 18 years of age, stimulus-induced myoclonus, absence seizures, major seizures, dementia, cerebellum Indicates ataxia. Symptoms are progressive and a very severe and disastrous disease that dies within 10 years (average 5.5 years) after onset. To date, two causative genes, EPM2A (encoding protein phosphatase) and NHLRC1 (encoding ubiquitin ligase), have been reported, and mutations in either gene are detected in most patients with Lafora disease.

  Temporal lobe epilepsy, which is categorized as symptomatic epilepsy, is the most frequent epilepsy in adult refractory epilepsy. It is a complex partial seizure (often starting with a movement stop, staring, automatic symptoms such as mouth, posture Abnormal, consciousness disorder, forgetfulness, recent memory disorder, language disorder, etc.). Many consider the medial temporal region (around the hippocampus) the beginning of the stroke and is considered one of the best indications for surgery. Hippocampal sclerosis is often seen, but the etiology is not yet clear. Temporal lobe epilepsy has previously been seen as an acquired syndrome, but there is a new consensus that certain temporal lobe epilepsy has a genetic component.

  Biological studies have been conducted on temporal lobe epilepsy through experiments using rats and mice. Specifically, recent studies using a rat model of human temporal lobe epilepsy with methylazoxymethanol have shown that inhibitory cell body dendrites type A conducted by the Kv4.2 channel encoded by the KCND2 gene. The loss of current suggests the possibility of promoting the positive feedback loop underlying epileptic seizures (Non-Patent Document 1). There is also a report that KCND2 knockout mice tend to have inducible epileptic seizures (Non-patent Document 2).

  To date, the KCND2 gene encodes Kv4.2, a member of the Shal subfamily of voltage-gated potassium channels, which mediates type A outward potassium current in the brain and neuronal dendrites It is known to play a critical role in the regulation of the backpropagating action potential (b-AP) in (Non-patent Document 3). In addition, a cell expressing a mutant Kv4.2 protein in which the interaction region to the carboxyl terminal filamin of the KCND2 gene is disrupted has a report of a lower current density (Non-patent Document 4). However, these reports are both in non-human model animals or cultured cells and directly demonstrate that the KCND2 gene encoding the Kv4.2 potassium channel is the causative gene for human temporal lobe epilepsy. It was n’t.

The number of epilepsy patients in Japan is approximately 1.2 million, of which refractory patients is a very common disease, reaching 200,000. It is important from the aspect of Furthermore, in symptomatic epilepsy, especially in temporal lobe epilepsy, identifying whether the cause of epileptic seizures is a genetic predisposition or other cause is similarly needed from the perspective of treatment planning, etc. . Therefore, it is desired to develop a method that can accurately and easily detect a genetic predisposition related to epilepsy.
Bernard, C.I. et al. Science 305,532 (2004) Forbes L. et al. American Episode Society 58th annual meeting (2004) Birnbaum S. G. et al. Physiol Rev. 84,803 (2004) Petrecca K.M. J et al. Neurosci 20, 8736 (2000) Engel J. et al. Jr, Neuroscientist, 7 (4), 340 (2001) Kobayashi E .; et al. Arch Neurol. 59, 1891 (2002) Duno M.M. et al. Eur J Hum Genet 12, 738 (2004)

  An object of the present invention is to provide a method for detecting a genetic predisposition of human epilepsy in order to investigate the possibility of diagnosis and treatment of human epilepsy. The method of the present invention comprises detecting the presence of a mutation that causes a change in the amino acid sequence of the human KCND2 protein in the base sequence of the human KCND2 gene.

  The present invention also provides a kit for detecting a genetic predisposition for human epilepsy according to the present invention, the primer used for detecting a mutation that causes a change in the amino acid sequence of the human KCND2 protein in the base sequence of the human KCND2 gene. An object is to provide a probe, a nucleic acid chip, and a kit using them.

  Another object of the present invention is to provide a nucleic acid having a mutation that causes a change in the amino acid sequence of the human KCND2 protein in the base sequence of the human KCND2 gene.

  The present invention also aims to provide non-human knockout mammals for human epilepsy models. The non-human knockout mammal of the present invention is characterized in that part or all of the biological function of the KCND2 protein is inactivated by modification of part or all of the DNA region of the KCND2 gene.

  The present invention is also a method for detecting a genetic predisposition to human epilepsy, wherein the expression level of the human KCND2 gene or a protein thereof is changed in the nucleotide sequence of the human KCND2 gene or a control sequence for the expression thereof. Detecting the presence of the mutation.

  As a result of diligent research to solve the above problems, the inventors have identified the first potassium channel mutation associated with human temporal lobe epilepsy (TLE), the KCND2 partial excision mutation. KCND2 encodes a voltage-gated potassium channel Kv4.2 that mediates type A outward potassium current in the brain. The present invention reveals for the first time a genetic predisposition to TLE, particularly in humans.

  Specifically, the inventors screened all six exons and intron-exon boundaries by direct sequencing for mutations in this gene in individuals diagnosed with TLE who are not 65 relatives of Japanese families. did. This results in a nonsense codon c. A 5 base pair deletion of 2723 — 2727delAAACT was identified in one proband of TLE (FIG. 1). The identified mutation corresponding to the identified amino acid change (N587fsX) was expected to produce a partially excised Kv4.2 protein lacking the last 44 amino acids at the carboxyl terminus. Importantly, this deleted region has an important ERK phosphorylation site and an interaction motif to the cytoskeletal proteins PSD-95 and filamin (Example 1).

  The inventors analyzed the electrophysiological properties of human wild type Kv4.2 channel (WT) and mutant Kv4.2 (delAAACT_N587fsX) channels expressed in HEK293 cells using whole cell patch clamp recordings. . The main finding was that the current density in cells transfected with the mutant channel was dramatically reduced compared to the WT channel cell record (FIG. 3) (Example 2).

  These findings indicate that the KCND2 gene is the causative gene for TLE. Based on the above discovery, the present invention provides a method for detecting the genetic predisposition of human epilepsy, comprising detecting the presence of a mutation that causes a change in the amino acid sequence of the human KCND2 protein in the base sequence of the human KCND2 gene. The inventor came up with the present invention.

(I) Method for Detecting Genetic Predisposition of Human Epilepsy The method of the present invention provides a method for detecting a genetic predisposition for human epilepsy. The method of the present invention comprises detecting the presence of a mutation in the nucleotide sequence of the human KCND2 gene that causes a change in the amino acid sequence of the human KCND2 protein.

  In the present invention, “epilepsy” refers to the occurrence of excessive synchronous reflex activity of cerebral neurons, resulting in the same type of clinical seizures (systemic tonic-clonic seizures, absence seizures, hallucinations). It means a disease state that repeats seizures, tonic seizures of some limbs). The type of epilepsy is not particularly limited as long as it is associated with a genetic cause, and includes idiopathic epilepsy, symptomatic epilepsy, and latent epilepsy. Temporal lobe epilepsy is preferred, but it includes a type in which no lesion in the brain is observed.

  A “genetic predisposition to human epilepsy” is a characteristic of a base sequence in a genomic DNA sequence that is associated with the onset of human epilepsy, compared to a known natural base sequence over a length of one base or more. It has a mutation. Specifically, it refers to a mutation that causes a change in the amino acid sequence of the human KCND2 protein in the base sequence of the human KCND2 gene. Alternatively, it refers to a mutation that causes a change in the expression level of the human KCND2 gene or a protein thereof in the base sequence of the human KCND2 gene or a control sequence for the expression thereof. If a genetic predisposition is detected and determined to have a genetic predisposition, it means that the individual with the genetic predisposition is suffering from or has a high probability of developing human epilepsy .

  The “human KCND2 gene” is a gene encoding a human potassium channel “human KCND2 protein”. “Human KCND2 protein”, also known as Kv4.2, is preferably represented by SEQ ID NO: 2.

  However, in addition to SEQ ID NO: 2, those having mutations that do not change the biological properties of “human KCND2 protein” compared to SEQ ID NO: 2 are included. That is, “mutation that does not change biological properties” means that a defined amino acid may be substituted, for example, with a residue having similar physicochemical properties. Examples of such conservative substitutions are those that replace one aliphatic residue with each other, eg Ile, Val, Leu, or Ala; one such as between Lys and Arg, Glu and Asp, or between Gln and Asn. Substitution from one polar residue to another; or substitution of another aromatic residue, such as substituting one another for Phe, Trp, or Tyr. Other conservative substitutions are well known, for example, substitution of entire regions with similar hydrophobic properties.

  Alternatively, the “mutation that does not change the biological property” includes a mutation having a mutation that does not change the activity as a channel of the KCND2 protein, even if it is other than the conservative substitution described above. The biological property of the KCND2 protein means, for example, a function as a voltage-dependent potassium channel, that is, a function of releasing potassium ions from the cell to the outside in a voltage-dependent manner (by electrical stimulation). The function as a voltage-dependent potassium channel can be determined by, for example, the value of the current density of KCND2 protein, the membrane potential dependence of activation / inactivation, and the like.

  “Human KCND2 gene” is preferably represented by SEQ ID NO: 1, but includes those having a base sequence encoding the amino acid sequence of SEQ ID NO: 2 in addition to SEQ ID NO: 1 due to degeneracy of the gene. Furthermore, even those encoding an amino acid sequence other than SEQ ID NO: 2 that contain mutations in SNPs, haplotypes, or other nucleotide sequences that do not lose the biological function of the KCND2 protein, Of KCND2 gene ”.

(I) Mutation of nucleotide sequence causing change in amino acid sequence of human KCND2 protein By the method of the present invention, a mutant in which the nucleotide sequence of AAACT from 2723 to 2727 in SEQ ID NO: 1 of KCND2 gene is deleted is detected. It was done. Here, in the wild type KCND2 gene, the nucleotide sequence after the 2720th is CTAAACTGTGAA..., And this sequence encodes leucine-asparagine-cysteine-glutamic acid-. On the other hand, in the mutant KCND2 gene of the KCND2 gene, CTTGGAA... Was obtained due to deletion, and TGA was a stop codon. Therefore, it was revealed that the corresponding amino acid was leucine and translation was stopped. That is, it was clarified that a protein lacking 44 amino acids after asparagine was produced due to the nonsense mutation that occurred.

  From the description in Example 2, it was revealed that the cells expressing the mutant Kv4.2 (delAAACT_N587fsX) channel have a significantly reduced current density. Here, mutant Kv4.2 has this phenotype only by lacking 44 amino acids on the C-terminal side. Therefore, even if a mutation other than the nonsense occurs in the human KCND2 gene, it is predicted that the current density will be significantly reduced in the same manner. Accordingly, the nucleotide sequence mutation that causes a change in the amino acid sequence of the human KCND2 protein is not particularly limited, and includes a nucleotide sequence mutation that causes any known amino acid sequence change. For this reason, the nucleotide sequence mutation that causes the change in the amino acid sequence of the human KCND2 protein preferably includes missense, nonsense, frameshift, splicing site formation or deletion, and truncation, and more preferably, the KCDN2 protein organism. Also included are mutations that reduce or substantially eliminate physiologic function.

  “Missense” means that a base sequence in a translation region of a gene is mutated and a codon for one amino acid is replaced with a codon corresponding to another amino acid. “Nonsense” refers to changing a codon corresponding to a certain amino acid to a termination codon among mutations occurring in a base sequence on a gene to interrupt and terminate protein synthesis. “Frame shift” refers to the reading of a gene by causing a mutation in the base sequence in the translation region of the gene and adding or deleting one or several bases (excluding multiples of 3) in DNA. This means that the frame is shifted. “Splicing site formation or deletion” means that, among mutations generated in genomic DNA, a site originally removed by splicing as an intron is not removed, or an exon region is mistakenly removed by splicing. “Truncation” means that a base sequence in a translation region of a gene is mutated and a part of a protein that should be originally formed is lost.

  The “biological function of KCND2 protein” means, for example, a function as a voltage-dependent potassium channel, that is, a function of releasing potassium ions from the cell to the outside in a voltage-dependent manner (by electrical stimulation). The function as a voltage-dependent potassium channel can be determined by, for example, the value of the current density of KCND2 protein, the membrane potential dependence of activation / inactivation, and the like.

  The amount of current density and the membrane potential dependence of activation / deactivation may be measured by any method known to those skilled in the art, for example, the patch clamp method. In the patch clamp method, a channel protein is expressed in cultured cells, and a microelectrode is applied to the channel protein, whereby the current flowing through the cell membrane can be measured. Preferably, this can be achieved by the method of Example 2.

  “Reducing the biological function of the KCND2 protein” means that a change in the amino acid sequence of the KCND2 protein reduces the function of the KCND2 protein as a voltage-dependent potassium channel. For example, in Example 2, the value of the current density of the KCND2 protein is about 66% (75 → 25 pA / pF) when the potential is 0 mV and about 40% when the potential is 40 mV, for example. It decreased by 70% (190 → 60 pA / pF) (FIG. 3). Therefore, in the present invention, the value of the current density of the KCND2 protein is “decreased the biological function of the KCND2 protein” compared to the case where the value of the current density of the KCND2 protein is not mutated. Compared with the case where it does not, it means that it is preferably reduced by 10% or more, more preferably 40%, further preferably 60% or more, and most preferably 70% or more.

  “Substantially lose the biological function of the KCND2 protein” means that a change in the amino acid sequence of the KCND2 protein renders the KCND2 protein substantially non-functional as a voltage-gated potassium channel.

  In both cases of “reducing the biological function of KCDN2 protein” and “substantially losing the biological function of KCND2 protein”, a mutation occurs in the base sequence of the human KCDN2 gene, so that the mutant protein Is expressed. This includes a truncated protein that results from a stop codon within the coding region. Furthermore, although it is known that four molecules are associated with each other and the function of the potassium channel is exhibited, it may be a mutation that prevents this molecular association. Alternatively, a case where the protein is not expressed by a system through a nonsense-mediated mRNA decay (NMD) or the like is also included.

  “Reducing the current density of KCND2 protein” means that the absolute amount of potassium ions that KCND2 protein can pass per unit time decreases. This also includes the case where the mutant protein is expressed and the amount of passing ions per molecule is decreased, and the case where the amount of protein expressed in the cell is decreased due to the change in the base sequence.

  Therefore, the present invention relates to human epilepsy in which the result of a mutation in the base sequence that causes a change in the amino acid sequence of human KCND2 protein is one of missense, nonsense, frameshift, splicing site formation or deletion, and truncation. A method of detecting a genetic predisposition of is provided. Furthermore, the present invention detects a genetic predisposition to human epilepsy wherein a mutation that causes a change in the amino acid sequence of the human KCND2 protein is a mutation that reduces or substantially eliminates the biological function of the human KCND2 protein. Provide a method.

(Ii) Sites causing mutations in the human KCND2 gene In the method of the present invention, any mutation that reduces or substantially eliminates the biological function of the human KCND2 protein can be predicted to be associated with epilepsy. . For example, from the description in Example 2, attention can be focused on mutations that decrease the current density of the human KCND2 protein of the KCND2 protein. Therefore, the site causing the mutation of the human KCND2 gene may be any site in the base sequence of the KCND2 gene.

  Without limitation, the following is specified in the order of increasing preference: 3 'from the codon encoding the amino acid immediately after the most transmembrane segment on the C-terminal side; encoding the amino acid corresponding to the 587th asparagine Codon or 3 'from it; within a region encoding one or more moieties selected from an interaction motif to filamin, a MAP / ERK phosphorylation site, or a PSD95 binding domain Or a codon encoding the amino acid corresponding to the 587th asparagine of SEQ ID NO: 2; and AAACT of the base sequence corresponding to the 2723 to 2727 positions of SEQ ID NO: 1.

  “The amino acid immediately after the most transmembrane segment on the C-terminal side” refers to the amino acid next to the most C-terminal amino acid in the sixth transmembrane region of the KCND2 protein, which is a six-transmembrane protein. However, the present invention is not limited to this, and when a protein having a different number of transmembrane regions is generated by splicing variants or other mutations, the most C-terminal of the sixth transmembrane region from the surrounding sequence or the like. An amino acid judged to be equivalent to the next amino acid after the amino acid. The determination of the transmembrane region can be inferred from ordinary knowledge in the field by creating a hydropathy diagram using the hydropathy value of each amino acid. The hydropathic value is an organism representing the hydrophobicity of each amino acid, calculated from fragments of a certain size (usually around 10) before and after each amino acid in the polypeptide chain. Is a scientific variable. Empirically, a region having a positive hydropathic value of approximately 20 to 30 amino acids is estimated to be a transmembrane region. Moreover, you may utilize the software and website which predict a transmembrane region from an amino acid sequence.

  The “amino acid corresponding to the 587th asparagine” preferably refers to asparagine which is the 578th amino acid of the human KCND2 protein of SEQ ID NO: 2. When a protein having a different amino acid sequence from that of SEQ ID NO: 2 is generated by splicing variants or other mutations, it means asparagine that is judged to be equivalent to the asparagine from surrounding sequences.

  In one embodiment of the present invention, the mutation that causes a change in the amino acid sequence of the human KCND2 protein is a mutation that eliminates the entire C-terminal amino acid sequence from the amino acid corresponding to the 587th asparagine of SEQ ID NO: 2.

  The “interaction motif to filamin” refers to the portion corresponding to the amino acid sequence PTPP at positions 601 to 604 of SEQ ID NO: 2, and the “MAP / ERK phosphorylation site” refers to the T, 607 at position 602 of SEQ ID NO: 2. The amino acid corresponding to the T th, the 616 th S, and the “PSD95 binding domain” refers to the portion corresponding to the amino acid sequence VSAL of the 627-630 th sequence of SEQ ID NO: 2, but a splicing variant or other mutation, etc. When a protein having a partly different amino acid as compared with SEQ ID NO: 2 is generated, it means that the site is equivalent to each site from the surrounding sequence.

  In one aspect of the invention, the site that causes mutation of the human KCND2 gene is preferably one or more moieties selected from an interaction motif to filamin, a MAP / ERK phosphorylation site, or a PSD95 binding domain. Existing in or including the same region. More preferably, it includes a region encoding all the three sites.

  “AAACT of the nucleotide sequence corresponding to the 2723th to 2727th” refers to the 2723th to 2727th AAACT of SEQ ID NO: 1, but if the nucleotide sequence differs due to mutation or the like, the 2723th to 2727th from the surrounding sequence It means a sequence determined to be equivalent to AAACT.

  Furthermore, in one aspect of the present invention, the present invention relates to a method for detecting a genetic predisposition for human epilepsy, wherein the expression of the human KCND2 gene or protein thereof is detected in the base sequence of the human KCND2 gene or a control sequence for the expression thereof. Detecting the presence of a mutation that causes a change in amount.

  “KCND2 expression control sequence” is a 5 ′ upstream portion including a start codon, that is, a site containing a nucleic acid sequence for controlling transcription or translation in the genome, for example, a transcription promoter, operator, Alternatively, it refers to a region containing an enhancer, an mRNA ribosome binding site, and other sites that regulate the initiation of transcription and translation.

(Iii) Technique for detecting a mutation that causes a change in the amino acid sequence of a human KCND2 protein In the present invention, a specific technique for detecting a mutation that causes a change in the amino acid sequence of a human KCND2 protein is particularly limited. Rather, it is possible to use a known method for detecting a nucleotide difference using any one of base sequencing, nucleic acid amplification reaction, hybridization, electrophoresis, restriction enzyme treatment, or a combination thereof. Is possible. For example, but not limited to, determination of nucleotide sequence, RFLP (restriction enzyme fragment length polymorphism), system using probe, SSCP method, and the like are included.

  As a method of detecting a mutation, it is possible to sequence the base sequence of KCND2. That is, the KCND2 gene can be sequenced by extracting genomic DNA from a biological sample, for example, a sample derived from blood, hair, or cheek, and amplifying it by an appropriate means such as PCR. Alternatively, mRNA may be purified from a biological sample and sequenced based on cDNA obtained by reverse transcription. The primer group for PCR is designed to amplify all six coding regions and boundary regions in the genomic sequence containing the KCND2 gene. A DNA polymerase used for PCR has a high replication fidelity so that no base mutation occurs during the amplification reaction. Sequencing can be performed by any known method, preferably with a capillary automated sequencing system.

  As another method for detecting a mutation, the presence or absence of a mutation can also be detected using the principle of RFLP (restriction fragment length polymorphism). That is, both ends of a region that is considered to be mutated in genomic DNA are cleaved with an appropriate restriction enzyme. After that, the genomic DNA fragment after the restriction enzyme treatment is denatured into a single strand, a probe for a region that seems to be mutated is added, hybridized, and electrophoresed to detect a difference in nucleotide length.

  As another method of detecting the mutation, it is also possible to detect the presence or absence of the mutation in a system using a probe. That is, detection can also be performed based on the presence or absence of hybridization of a probe that specifically hybridizes to a mutant DNA fragment. In this case, the probe can be easily detected by an appropriate label such as a fluorescent label, a radioactive label, or an enzyme label. Also, a plurality of probes can be immobilized on a carrier on a DNA chip and used as a nucleic acid chip. In another embodiment, the probe may be immobilized on beads or a carrier in a multiwell plate.

As another method for detecting a mutation, the presence or absence of a mutation can also be detected by using an SSCP (single strand formation polymorphism) method. When double-stranded DNA is dissociated into single strands, each strand has a unique higher-order structure depending on its base sequence. When the dissociated DNA is electrophoresed in a non-denaturing polyacrylamide gel, it is migrated to different positions according to the difference of each higher order structure (SSCP analysis). The SSCP method uses this property to perform PCR using a primer labeled with a radioisotope such as ³² P, a fluorescent label, or an enzyme label, or a radioisotope such as a ³² P-labeled nucleotide on one of the reaction substrates. The presence or absence of a mutation can be identified by amplifying a target gene fragment by PCR using a fluorescent label or an enzyme label and conducting SSCP analysis.

(Iv) According to the process example 2 in which it is determined that the genetic predisposition of epilepsy is present, when the mutation occurs in the nucleotide sequence of the KCND2 gene, the amino acid sequence changes, and the current density of the KCND2 protein is significantly reduced, It has also been suggested that epilepsy symptoms can be caused by reducing the membrane potential dependence of activation / deactivation. Therefore, the method for detecting a genetic predisposition for human epilepsy of the present invention preferably causes a change in the amino acid sequence of the human KCND2 protein in the base sequence of the human KCND2 gene in the base sequence of the human KCND2 gene. Determining the presence of a genetic predisposition to epilepsy if the mutation is present.

(II) Primer The present invention provides a primer for detecting the presence of a mutation that causes a change in the amino acid sequence of the human KCND2 protein in the base sequence of the human KCND2 gene.

  The primer of the present invention can be used to amplify nucleic acid from an extracted biological sample, and the amplified nucleic acid is used for a polymorphism detection method by restriction enzyme treatment, or for sequencing by direct sequencing. can do.

  In one embodiment of the detection method of the present invention, when detection is limited to a specific site and / or mutation, for example, a difference in the length of a restriction enzyme fragment of an amplification product by PCR or the like results in a polymorphism. Can be detected using PCR-RFLP (PCR-refraction fragment polymorphism) marker or CAPS (cleaved amplified polymorphic sequences) marker (A. KoniecZn.F. A procedure for mapping Arabidopsis mutations using co-dominant ecotype-specific PCR-based marker, (1993), Plant J. 4 (2) p. 403-410). Therefore, the present invention also provides a primer therefor.

  Although not limited, the primer of the present invention has an amplified region of preferably about 50 bases to about 3000 bases, more preferably about 50 bases to about 2000 bases, more preferably about 100 bases to about 1000 bases. Within the base range.

  In the present invention, nucleic acid amplification reaction is performed using human genomic DNA as a template, using the above primer pair. The nucleic acid amplification reaction is a replication chain reaction (PCR) (Saiki et al., 1985, Science 230, p. 1350-1354).

Primer pairs for direct sequencing can be prepared by a known method based on the base sequence of the KCND2 gene. Specifically, but not limited to, for example, based on the base sequence of the transposable element and / or its adjacent region, the following conditions:
1) Each primer is 15-30 bases in length;
2) The ratio of G + C in the base sequence of each primer is 30-70%;
3) The distribution of A, T, G and C in the base sequence of each primer is not partially largely biased;
4) The length of the nucleic acid amplification product amplified by the primer pair is 50 to 3000 bases, preferably 100 to 2000 bases; and 5) Complementary in the base sequence of each primer itself or between the base sequences of the primers. A single-stranded DNA having the same base sequence as the base sequence of the KCND2 gene or a base sequence complementary thereto, or, if necessary, binding to the KCND2 gene so as to satisfy the absence of a typical sequence portion Primer pairs can be generated by a method comprising producing the single-stranded DNA modified so as not to lose specificity.

  In one embodiment of the detection method of the invention, mutations in KCND2 are amplified by primer pairs in all six coding regions and border regions, and sequenced by direct sequencing. Accordingly, the primers of the present invention include all those that amplify the coding region of the KCND2 gene corresponding to the 6 'coding region of the KCND2 gene and the 5' or 3 'side of at least one region of the border region. That is, the primers used in the present invention can be selected by amplifying and sequencing each exon by selecting an intron region or a 5 'or 3' UTR sandwiching each exon. If these conditions are satisfied, the primer may be selected by amplifying the intron region near the exon-intron boundary and partially sequencing it. When used for sequencing, the base length between primer sets used in the present invention is preferably 800 bases or less, more preferably 600 bases or less.

Accordingly, the present invention includes primers for use in a method for detecting a genetic predisposition for human epilepsy. Preferably, it is represented by SEQ ID NO: 3-18.
A method of amplifying a specific region using a primer is performed using a method known to those skilled in the art. That is, a primer, dNTP, DNA polymerase, buffer solution, etc. are added to the genomic DNA sample taken out from the sample to make a sample amplification mixture. The mixed solution is amplified by a thermal cycler program that repeats a cycle including a step of dissociating double-stranded DNA, an annealing step of template DNA and a primer, and a step of synthesizing DNA from each primer. In the step of dissociating the double-stranded DNA, the double-stranded DNA is easily dissociated into single-stranded DNA by placing it at a temperature of 90 ° C. to 100 ° C. for several tens of seconds. The temperature of the template DNA and primer annealing step is determined by the melting temperature (Tm) for each primer sequence. The melting temperature (Tm) is the temperature at which 50% of the oligonucleotide forms a hybrid with its complementary strand. When the reaction conditions are higher than the melting temperature (Tm), annealing between the template DNA and the primer hardly occurs and amplification efficiency. Decrease. On the other hand, when the reaction conditions are lower than the melting temperature (Tm), the primer anneals to a sequence other than its complementary sequence, and sequences other than the sequence to be amplified are also amplified. In the step of synthesizing DNA from the primer, the reaction is performed at a temperature suitable for DNA polymerase and for a time corresponding to the length of the base sequence to be amplified. For example, amplification is performed by incubating at 94 ° C. for 2 minutes once, at 94 ° C. for 30 seconds, at 59 ° C. for 30 seconds, and at 72 ° C. for 60 seconds 30 times, and finally at 72 ° C. for 5 minutes.

(III) Probe The present invention also provides a probe for detecting the presence of a mutation that causes a change in the amino acid sequence of the human KCND2 protein in the base sequence of the human KCND2 gene. The probe may be DNA or RNA.

  The base sequence of the probe of the present invention has all or part of the sequence corresponding to each exon of the KCND2 gene. As long as it has this sequence, an intron region continuous with each exon or a sequence of a 5'-side or 3'-side UTR may be appropriately linked. An appropriate base length of the probe of the present invention may be any length, but is preferably 10 to 100 bases, more preferably 20 to 100 bases.

  Any known method may be used as the method for producing the probe of the present invention. For example, a synthetic probe or a system based on in vitro transcription may be used. Preferably, it is a synthetic probe.

  In embodiments of the invention, the probes of the invention are labeled with means that facilitate confirmation of hybridization with the human KCND2 gene or fragments thereof, such as fluorescent labels, radioisotope elements (RI), and biotin. May be. In addition, other DNA sequences than those described above may be added to the probe of the present invention, and the added DNA sequence is not related to the human KCND2 gene sequence, that is, has no homology with the gene. May be.

  In the embodiment of the present invention, the probe can hybridize to the mutant DNA fragment of the nucleotide sequence of SEQ ID NO: 1 under stringent conditions, for example, moderately or highly stringent conditions.

“Stringent conditions” means to hybridize under moderately or highly stringent conditions. Specifically, moderately stringent conditions can be easily determined by those skilled in the art having general techniques based on, for example, the length of the DNA. Basic conditions are shown in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, Chapter 6-7, Cold Spring Harbor Laboratory Press, 2001, and for nitrocellulose filters, 5 × SSC, 0.5 Pre-wash solution of% SDS, 1.0 mM EDTA, pH 8.0, about 50% formamide at about 40-50 ° C., 2 × SSC-6 × SSC (or about 50% formamide at about 42 ° C. , Other similar hybridization solutions such as Stark's solution), and the use of washing conditions of about 60 ° C., 0.5 × SSC, 0.1% SDS. Preferably moderately stringent conditions include hybridization conditions of about 50 ° C. and 6 × SSC. High stringency conditions can also be readily determined by one skilled in the art based on, for example, the length of the DNA. In general, these conditions include hybridization at higher temperatures and / or lower salt concentrations than moderately stringent conditions (eg, about 65 ° C., 6 × SSC to 0.2 × SSC, preferably 6 × SSC, More preferably 2 × SSC, most preferably 0.2 × SSC hybridization) and / or washing, eg hybridization conditions as described above, and approximately 68 ° C., 0.2 × SSC, 0.1% Defined with SDS washing. In hybridization and wash buffers, SSC (1 × SSC is 0.15 M NaCl and 15 mM sodium citrate) to SSPE (1 × SSPE is 0.15 M NaCl, 10 mM NaH ₂ PO ₄ , and 1. 25 mM EDTA, pH 7.4) can be substituted, and washing is performed for 15 minutes after hybridization is complete.

(IV) Nucleic Acid Chip The present invention also provides a nucleic acid chip for detecting the presence of a mutation that causes a change in the amino acid sequence of the human KCND2 protein in the base sequence of the human KCND2 gene. In the nucleic acid chip, a plurality of the aforementioned probes may be immobilized on a slide glass.

  The nucleic acid chip of the present invention is characterized in that nucleic acids having all or part of the sequence corresponding to each exon of the KCND2 gene are aligned on a substrate such as glass. As long as it has the sequence of each exon, an intron region continuous with each exon or a sequence of 5 ′ side or 3 ′ side UTR may be appropriately linked. The nucleic acid chip of the present invention may be a type that synthesizes nucleic acids on a substrate or a type that is prepared by attaching nucleic acids on a substrate. An appropriate base length of the nucleic acid used in the nucleic acid chip of the present invention may be any length, but is preferably 10 to 100 bases, more preferably 20 to 100 bases.

  Any known method may be used as a method for producing a nucleic acid used in the nucleic acid chip of the present invention. For example, a synthetic nucleic acid, a system by in vitro transcription, or cDNA by PCR or the like may be used. . Preferably, it is a synthetic nucleic acid.

  Nucleic acid chips can be prepared by methods common to those skilled in the art. For example, see “New Genetic Engineering Handbook 4th Edition” Yodosha, 289-294 (2003). Specifically, it can be prepared by spotting the above-mentioned probe or the like on a glass substrate coated with aminosilane using a commercially available arrayer, for example, Affymetrix 417 Arrayer. Specifically, the “arrayer” refers to a device that operates a pin tip or a slide holder in the XYZ axial directions under computer control, carries a nucleic acid sample on the surface of a glass substrate, and repeats the steps of washing and drying.

  The nucleic acid chip can be used by a method common to those skilled in the art. As a specific example, a method for detecting a mutation in the base sequence of an mRNA sample with a nucleic acid chip is shown below. First, the mRNA sample and the random primer are annealed by mixing the mRNA sample with a random primer, setting in a thermal cycler, and incubating at 99 ° C. for 3 minutes, 70 ° C. for 10 minutes, and 42 ° C. for 10 minutes. Next, together with DTT, buffer solution, and dNTP, a detectable dye such as cyanine 5 (or 3) -dUTP and a conjugate of dNTP and a reverse transcriptase such as SuperScript III are mixed to prepare a premix. After the sample of the thermal cycler is kept at 15 ° C. for 5 minutes, the premix is mixed and reacted at 42 ° C. for 2 hours to label the cDNA produced thereby together with the reverse transcription reaction. After degrading mRNA, a cDNA sample labeled with cyanine 5 (or 3) is purified by a centrifuge tube with a nucleic acid purification filter such as Micropure EZ or Microcon YM-30. Next, the sample is dropped onto a nucleic acid chip, sealed in a hybridization chamber, and left at 42 ° C. for about 16 to 18 hours. Then, it is washed with 2 × SSC / 0.1% SDS solution, 1 × SSC, 0.2 × SSC, milliQ water at room temperature. The moisture of the nucleic acid chip is removed by, for example, light centrifugation, and an image of the signal intensity of each spot is taken into a computer with a scanner, and the presence or absence of mutation is analyzed appropriately.

(V) Kit for detecting genetic predisposition of human epilepsy The present invention provides a kit for detecting genetic predisposition of human epilepsy.
The kit of the present invention includes a primer, a probe, and / or a nucleic acid chip for detecting a mutation that causes a change in the amino acid sequence of the human KCND2 protein in the base sequence of the human KCND2 gene. The kit of the present invention may further contain human genomic DNA, mRNA extraction means, DNA amplification means, reaction vessels, positive and negative control samples, instructions, packages and the like as appropriate.

(VI) Nucleic acid The present invention contains a nucleic acid having any of the mutations described above in the KCND2 gene. That is, this mutation is in the order of increasing preference: a mutation that causes a change in the amino acid sequence of the human KCND2 protein; a result of a mutation in the base sequence that causes a change in the amino acid sequence of the human KCND2 protein is a missense or nonsense. Mutations that are either frameshift, splicing site formation or deletion, or truncation; mutations that reduce or substantially eliminate the biological function of the human KCND2 protein.

  In addition, any of the above-described sites also includes a site that causes mutation. That is, the mutation may be caused in any site listed in the item “(ii) Site causing mutation of human KCND2 gene”.

  As used herein, “nucleic acid” includes synthetic DNA and synthetic RNA in addition to DNA, RNA and cDNA. Furthermore, vectors containing these are also within the scope of the present invention. Unless specifically stated, either double-stranded or single-stranded embodiments are included. In the case of a double strand, when a base sequence is referred to in this specification, it is intended to include its complementary base sequence.

(VII) Non-human knockout mammal The present invention relates to a human epilepsy model in which part or all of the biological function of KCND2 protein is inactivated by modification of part or all of the DNA region of KCND2 gene. A non-human knockout mammal is provided. In the present invention, since the relationship between human epilepsy and KCND2 protein has been clarified, it has become possible to provide a non-human knockout mammal in which part or all of the DNA region of the KCND2 gene has been modified.

  Non-human knockout mammals in the present invention include not only non-human mammals in which KCND2 in genomic DNA has been partially or wholly deleted due to homologous recombination or the like, but also mechanisms such as RNAi Also included are transgenic non-human mammals that express double-stranded RNA against the KCND2 gene that are suppressed by.

  The non-human knockout mammal of the present invention is a non-human mammal, preferably a mouse, rat, goat, cow, pig, rabbit, sheep or the like. Most preferred are mice that are easy to handle and have established experimental systems. The knock-out mammal of the present invention includes not only adults but also ES cells, fertilized eggs, embryos from the 8-cell stage to blastocyst formation and just before birth, eggs, sperm, tissue / organ cultures, chimeric animals, etc. Includes all aspects. These may be in a frozen state as necessary for storage.

The following shows how to create a knockout mouse.
1) Preparation of targeting vector A targeting vector is used for homologous recombination of part or all of the genomic DNA region of the KCND2 gene. Therefore, the targeting vector for producing the knockout mouse of the present invention has a 5 'and 3' sequence of the KCND2 gene in mouse genomic DNA, and a form in which all or part of the exon region of the KCND2 gene is deleted. Built in. At the same time, genes for selection such as positive selection and negative selection are appropriately introduced so that screening of homologous recombinants that must be performed in the subsequent steps can be performed more easily. By this targeting vector, homologous recombination is induced by the 5 ′ and 3 ′ sides of the KCND2 gene and can be incorporated into genomic DNA together with the modified KCND2 gene gene.

Positive selection means that only embryonic stem cells into which the targeting vector has been introduced into the chromosome are selected. Generally, the probability that the introduced gene DNA is integrated into the chromosome by the electroporation method used for transformation is about one in 10 ³ -10 ⁴ cells, so only the cells into which the gene DNA has been introduced You must first consider the sequence (positive selection) to select

  Examples of positive selection marker genes include neomycin resistance gene (selected by G418), hygromycin B phosphotransferase (BPH) gene (selected by hygromycin-B), blasticidin S deaminase gene (selected by blasticidin S), puromycin Resistance genes (selected with puromycin) can be used. The neomycin resistance gene is most preferred.

  Negative selection means selective removal of embryonic stem cells that have undergone non-homologous recombination. This can be used particularly when the target gene is not expressed in ES cells or the expression level is low. While homologous recombination occurs at the homologous region, non-homologous recombination occurs at both ends of the introduced DNA, which is highly linear. Negative selection is performed by adding a gene that is toxic to cells when incorporated into one end of the introduced DNA. As a negative selection, a method using a herpes simplex virus (HSV) thymidine kinase (tk) gene, a diphtheria toxin A fragment (DT-A) gene, or the like is known. Compared to intracellular thymidine kinase (TK), HSV-derived TK phosphorylates nucleic acid analogs such as FIAU and cancer cyclovia at a higher rate. The tk selection utilizes the fact that when an HSVtk gene is introduced by non-homologous recombination, FIAU or the like is phosphorylated and incorporated into chromosomal DNA to inhibit DNA synthesis. DT-A inhibits protein synthesis by polyADP ribosylation of elongation factor II in the cell. If a DT-A gene linked to a promoter having high expression activity in ES cells is used, it is expected that cells that have been integrated into the chromosome by non-homologous recombination will die. There is an advantage that a selection agent is not required in the selection of the translation level.

2) Homologous recombination using a targeting vector Homologous recombination is performed using the targeting vector prepared by the above method. In the currently established gene targeting method, a system using ES cells is desirable. At present, ES cells have been established in a plurality of mammals such as human, mink, hamster, pig, cow, marmoset, rhesus monkey and the like. If similar ES cells are established in a wider variety of animals in the future, genetically modified animals can be produced using the same method.

  An ES cell line for producing a genetically modified mouse may be prepared by itself using a known manual, but an existing established ES cell line is available and is preferred. Mouse-derived ES cells that can be used at present include TT2 cells, AB-1 cells, and the like. Each desired ES cell line can be obtained from a specific research institution as needed.

  A targeting vector obtained by modifying part or all of the genomic DNA region of the prepared KCND2 gene is introduced into ES cells. Homologous recombination can be generated by utilizing the homology between the genomic DNA sequence of the KCND2 gene and the sequence of the unmodified portion in the targeting vector. As a method for introducing the targeting vector into ES cells, known methods such as an electroporation method, a calcium phosphate method, a DEAE-dextran method can be used.

3) Screening for homologous recombinants The obtained recombinant ES cells are plated on neomycin-resistant feeder cells. Then, selective culture, which is a screening, is performed using a positive selection marker and / or a negative selection marker introduced into ES cells by homologous recombination in an ES culture solution. That is, the culture solution contains, for example, neomycin depending on the type of the selectable marker gene. Colonies that survive on days 6-8 of the selective culture are picked as resistant clones. In addition, in order to reliably select transformants that have undergone homologous recombination, the presence or absence of homologous recombination is appropriately confirmed by PCR, Southern blot hybridization, or the like.

4) Preparation of KCND2 genetically modified animal Next, the recombinant ES cells obtained as a result of homologous recombination are transplanted into the 8-cell stage or blastocyst embryo. A chimeric animal can be prepared by transplanting the embryo transferred with ES cells into the uterus of a pseudopregnant foster mother and giving birth.

  As a method for transplanting ES cells into an embryo, for example, a microinjection method, an aggregation (aggregation) method, and the like are known, and any of them may be used. In the case of a mouse, first, a female mouse subjected to superovulation treatment with a hormonal agent (for example, using PMSG having an FSH-like action and hCG having an LH action) is mated with a male mouse. Thereafter, early embryos are collected from the uterus on day 2.5 after fertilization when using an 8-cell stage embryo, and on day 3.5 after fertilization when using a blastocyst. The embryos thus collected are injected in vitro with ES cells that have undergone homologous recombination using a targeting vector to produce chimeric embryos.

  On the other hand, a pseudopregnant female mouse for use as a foster parent can be obtained by mating a female mouse having a normal cycle with a male mouse castrated by vagina ligation or the like. A chimeric animal can be prepared by transplanting the chimeric embryo produced by the above-mentioned method into the uterus to the produced pseudopregnant mouse, and allowing it to become pregnant and give birth. It is desirable to create female mice that collect fertilized eggs and pseudopregnant mice that become foster mothers from a group of female mice in the same sexual cycle in order to ensure that implantation and pregnancy of the chimeric embryo occur. .

  From such chimeric mice, male mice derived from embryos transplanted with ES cells are selected. After the male chimeric mouse derived from the selected embryo of ES cell transplantation has matured, this mouse is mated with a female mouse of a pure mouse strain, and the ES cell-derived coat color appears in the next-generation offspring. It can be confirmed that it has been introduced into the germ line of chimeric mice. In order to confirm that the ES cell has been introduced into the germ line, various traits can be used as an index, but it is preferable to use a coat color in consideration of ease of confirmation. As described above, an individual in which a target gene is deficient can be obtained by selecting an animal in which a recombinant ES cell transplanted into an embryo is introduced into the germ line and breeding the chimeric animal. By crossing the KCND2 gene hetero-deficient mice thus obtained, KCND2-deficient homozygous mice can be obtained. From the viewpoint of unity of genetic background, those that have undergone as many backcrossings as possible are preferred as knockout mice.

(VIII) Methods for Gene Therapy of Epilepsy With the discovery of the present invention, human epilepsy can be overcome by gene therapy. Gene therapy is a method in which when a genetic predisposition causes the onset of a disease, a specific gene clone is introduced into a somatic cell of a patient, and the introduced gene exerts its function to treat it. There are ex vivo methods to remove the patient's stem cells, lymphocytes, bone marrow cells, etc. from the body and introduce the gene clone, and then return to the patient's body, and in vivo method to introduce the gene clone directly into the patient's diseased tissue . Since the in vivo method has a problem in safety, the ex vivo method is general, but any method may be used.

The gene therapy method by ex vivo method is described below.
The KCND2 gene encodes a voltage-gated potassium channel Kv4.2 that mediates type A outward potassium current in the brain. Therefore, the cells used for the gene therapy of the present invention are stem cells that can differentiate into nerves, for example, neural stem cells. Neural stem cells have the proliferative ability, self-renewal ability, pluripotency, and tissue repair ability, which are the characteristics of stem cells, and have been found to exist throughout life from the developmental stage in the living body. So far, neural stem cells are thought to be present in the lower part of the granule cell layer of the dentate gyrus of the hippocampus and the subventricular zone of the forebrain, and can be separated from these regions.

  Several methods have been established so far for isolating neural stem cells. For example, it is a method of collecting neural stem cells using an antibody against a surface antigen. Neural stem cells can be separated by a combination of antibodies such as cholera toxin B subunit (ChTx), tetanus toxin fragment C (TnTx), and A2B5.

  The isolated neural stem cells can induce cell proliferation by adding EGF or FGF-2, and can be induced to differentiate by administering various factors. Therefore, a large amount of neural stem cells can be secured from one neural stem cell, and these cells are isolated from the patient, and thus can be autotransplanted without causing an immune response.

  As a method for introducing a gene into a neural stem cell, any of a method using a viral vector (such as a retrovirus or a lentivirus), a chemical method such as lipofection, or a physical method such as electroporation is used. Is possible. In the present invention, these gene introduction methods are used to introduce a KCND2 gene having no mutation. A normal KCND2 gene can be differentiated into a nerve cell by inducing a neural stem cell into which the KCND2 gene has been stably introduced with an appropriate nerve differentiation-inducing factor, or transplanted into the brain without differentiation. It is expected that epilepsy can be treated by the presence of nerves with

  Therefore, the present invention provides a method for gene therapy so as to express KCND2 having no mutation in a patient in which the presence of a mutation causing a change in the amino acid sequence of the human KCND2 protein in the nucleotide sequence of the human KCND2 gene is detected. Including.

  For the first time, the present invention has revealed the relationship between epilepsy and KCND2 gene mutation. From the present invention, 4 patterns of mutations were found in 65 patients (Example 1). From this, it is expected that the relationship between new epilepsy and KCND2 gene mutation will become clear in the future. In addition, the cause of the onset of epilepsy patients can be accurately identified, providing meaningful information for clinical treatment.

  Hereinafter, the present invention will be described by way of examples. However, the examples are for illustrative purposes and do not limit the present invention. The scope of the present invention is determined based on the description of the scope of claims. Furthermore, those skilled in the art can easily make corrections and changes based on the description of the present specification.

Example 1 Mutational analysis of the KCND2 gene in patients with temporal lobe epilepsy
Probands and Mutation Analysis The inventors have directly mutated mutations in KCND2 in individuals diagnosed with temporal lobe epilepsy (TLE), who are not relatives of 65 Japanese families, directly in all six coding regions and border regions. Screened by manual sequencing. Genomic DNA was extracted from heparinized blood samples using QIAamp DNA Blood Mini Kit (Qiagen) and hair and cheek samples using ISOHAIR Kit (Nippon Gene). All genomic DNA samples were initially amplified by PCR, and PCR products were analyzed by direct sequencing using the ABI 3700 capillary automated sequencing system (PE Applied Biosystems).

  The primers used for amplification of the genomic DNA are SEQ ID NOs: 3 to 18, and each primer set amplifies each region of the cDNA of the KCND2 gene (SEQ ID NO: 1). That is, the primer sets of SEQ ID NO: 3 and SEQ ID NO: 4 are 843-1388 (part of the first exon) of SEQ ID NO: 1, and the primer sets of SEQ ID NO: 5 and SEQ ID NO: 6 are 1138-1699 of SEQ ID NO: 1 (first SEQ ID NO: 7 and SEQ ID NO: 8 are the primer sets of SEQ ID NO: 1 648-2080 (part of the first exon), SEQ ID NO: 9 and SEQ ID NO: 10 are the primer sets of SEQ ID NO: 1 2021 to 2243 (second exon) of SEQ ID NO: 11 and SEQ ID NO: 12 are set to 2244 to 2339 (third exon) of SEQ ID NO: 1, and primer sets of SEQ ID NO: 13 and SEQ ID NO: 14 are set to SEQ ID NO: 1. 2340-2432 (exon 4) of SEQ ID NO: 15 and SEQ ID NO: 16 are the primer sets of SEQ ID NO: 1 2433-2680 (5th Existing), primer set of SEQ ID NO: 17 and SEQ ID NO: 18 a 2681-2976 (exon 6) of SEQ ID NO: 1, respectively amplify. However, since SEQ ID NOs: 6 to 15 are sequences complementary to the intron region of the KCND2 gene, a part of the intron region is also amplified.

  For PCR reactions, Pwo (Roche), Pyrobest (Takara), and Blend Taq Plus (Toyobo) DNA polymerase systems were used according to product instructions. All 65 TLE patients met established clinical criteria for disease and were diagnosed and treated at Shizuoka Epilepsy and Neuromedical Center, Shizuoka, Japan. Informed consent was obtained from all individuals tested with the present invention.

Results A total of 4 different nucleotide changes were identified in the inventors' pool of 65 Japanese TLE patients.

  Heterozygote creating a frameshift that results in a stop codon with a premature 5 base pair deletion c. One of these, which is a 2723_2727delAAACT mutation, is expected to contain an amino acid change (N587fsX). The 44 carboxyl terminal amino acids deleted in this mutein are the interaction motif to filamin, amino acids 601-604 (PTPP); 3MAP / ERK phosphorylation sites, amino acids T602, T607, S616; and the PSD95 binding domain, amino acids 627-630 (VSAL). DNA from hair and cheek specimen samples along with lymphocytes from the proband's asymptomatic father were examined by direct sequencing and heterozygous c. The 2723_2727delAAACT mutation was represented (FIG. 2).

  A heterozygous non-coding region SNP, IVS3 + 40T> C, was found in 2 TLE patients but was not present in a pool of 224 chromosomes obtained from unrelated healthy individuals. Heterozygous coding region silent mutation, c. 756 C> T (C252C) was found in only one TLE patient and was also found in one unrelated healthy individual. c. Both 756 C> T (C252C) and IVS3 + 40T> C were not listed in either the NCBI or Japanese SNP database.

Another heterozygous non-coding region SNP, IVS3 + 68T> C, is listed in the NCBI database as NCBI refSNP ID: rs7795370.
These data are important when viewed in the context of the NCBI SNP database, which lists approximately 1000 SNPs of the KCND2 gene, which has only one silent mutation in the coding region .

  That is, c. The 2723_2727delAAACT mutation appeared as an amino acid change in the KCND2 protein, suggesting that it is directly associated with epilepsy. On the other hand, all three other mutations (c. 756 C> T (C252C), IVS3 + 40T> C, IVS3 + 68T> C) are not mutations that appear in the amino acid change of KCND2, and are also found in healthy individuals. However, it is thought that the relationship with the disease is weak.

Example 2 Patch clamp
2-A Construction of Wild Type and Mutant KCND2 Expression Vector Wild type human KCND2 cDNA was synthesized from normal human total brain poly (A) RNA (Clontech) using SuperScript III reverse transcriptase (Invitrogen). The resulting cDNA was then amplified by PCR using a 5 ′ oligonucleotide primer containing a BglII site and a 3 ′ primer containing an EcoRI site. The resulting amplicon was first inserted into pBluntII-TOPO (Invitrogen) and then subcloned between the BglII and EcoRI sites of pIRES2-EGFP (Clontech). Mutant vectors were generated from wild type vectors using the QuikChange Site-Directed Mutagenesis kit (Stratagene). All constructs were confirmed by sequencing.

2-B cell culture and transfection HEK293 cells supplemented with 10% (v / v) heat-immobilized fetal calf serum, penicillin (100 U / mL), and streptomycin (100 mg / mL) Dulbecco's modified Eagle medium Grown in and maintained at 37 ° C. in a humidified 5% CO ₂ incubator. For whole cell patch clamp, HEK293 cells are first seeded into polylysine-coated 6-well dishes and then according to product instructions using Fugene 6 (Roche) and 1 μg pKCND2-IRES2-EGFP and / Or pKCND2 (N587fsX) -IRES2-EGFP was transfected the next day. For patch clamp analysis, HEK293 cells were plated on polylysine-coated coverslips (Sumitomo Bakerite Co., Tokyo, Japan) 24 hours after transfection.

Using a 2-C patch clamp analysis inverted microscope (IX70, Olympus, Tokyo, Japan), GFP positive transfected HEK293 cells were selected for electrical recording. To minimize the space clamp problem, GFP positive cells in the syncytia were not taken into account and only strong fluorescent cells were selected. External solution was adjusted to pH7.4 with NaOH, and containing the following in _{(mM): NaCl, 135;} KCl, 4.0; CaCl 2, 1.0; MgCl 2, 2.0; glucose, 10 And Hepes, 10. The pipette electrode is from a borosilicate glass capillary tube (inner diameter 0.8-1.0 mm, Hilgenberg GmbH, Marsford, Germany) using a multi-step horizontal puller (P-97, Sutter Instrument Co. Canada, Novoto). Created. The pipette solution was adjusted to pH7.2 with KOH, and consisting of _{(mM): KCl, 135;} MgCl 2, 1.0; EGTA, 10; glucose, 5.0; and HEPES, 10. When filled with the internal pipette solution, the pipette resistance was 1.0-3.5 MΩ. Except for the very tip, the outer wall of the pipette was coated with dental wax to reduce stray volume (GC Corporation, Tokyo, Japan). The reference electrode was an Ag-AgCl pellet in the wall connected to a tank using an agar bridge filled with 150 mM NaCl. Current was recorded using an Axopatch 200B amplifier (Axon Instruments, Burlingame, Canada). Current and voltage signals were filtered using a low-pass Bessel filter that cuts at a frequency of 10 kHz and stored on a V450JS2 (Iiyama, Nagoya, Japan) hard disk; "pclamp 8.0" software was used throughout (Axon Instruments, Canada, Burlingame). All pulse protocol values were programmed on a computer. The capacitance of the cells was calculated using “pclamp 8.0” software. Capacitive current and series resistance were supplemented electrically. All experiments were performed at room temperature (22 ° C.). For comparison of current density, the peak amplitude of K ⁺ transient current was measured and normalized to total cell capacitance.

After introducing the whole cell setting, recording started when the holding current was <200 pA; this routinely required 5 minutes of intracellular perfusion. FIG. 4 showed that K ⁺ transient current was drawn from the -80 mV holding potential by a depolarization voltage step (-70 to 70 mV) in wild type Kv4.2 channel cells. K ⁺ transients peaked within 25 ms and were gradually deactivated. Exogenous K ⁺ transients was confirmed by the absence of K ⁺ transients in HEK293 cells not transfected. Similar K ⁺ transients were observed in cells expressing the delAAACT_N587fsX mutant channel (data not shown), and the surface expression level of the mutant channel was also similar to the wild-type channel level (data shown) Absent). In addition, at the holding potential of −80 mV, no K ⁺ transient current was observed in any of the transfected cells. However, the current density represented by cells in the delAAACT_N587fsX channel was significantly lower than that of cells with wild type channels. Specifically, the value of the current density of the KCND2 protein is about 66% (75 → 25 pA / pF) when the potential is 0 mV, and about 70% when the potential is 40 mV, compared to the case where there is no mutation. (190 → 60 pA / pF) decreased (FIG. 3). In cells expressing both wild type and delAAACT_N587fsX channels, the current density fell between the values recorded in either the wild type channel or the mutant channel (FIG. 3).

Example 3 Proband of KCND2 partial excision mutation
Proband c. A 31-year-old female proband with the 2723_2727delAAACT (N587fsX) mutation has a complex history of medically refractory seizures that began at age 13. There were no signs, her seizure types ranged from complex seizures with convulsion and oral autophagy to static gaze and autophagia following general spasm. Epilepsy is significantly drug resistant, as only very limited seizure modulation is observed with valproate and carbamazepine therapy. A complete cessation of seizures was only achieved at the age of 28 after two consecutive surgical treatments.

  Standard EEG analysis showed that epileptic seizure discharge spreads from the left frontal temporal region to the right. Although these studies and other functional studies using the magnetoencephalography method suggested that the left temporal region was involved in epileptogenesis, neuroimaging data was not remarkable. Therefore, the EEG assessment within the invasive skull was used to map the left meso-basal temporal region as a source of anomalous discharge activity. In fact, all 11 recorded seizures began in the left central basal temporal region.

  These findings indicate that, at the age of 27, the first abdominal-hippocamptomy to remove epilepsy-producing tissue by excising the amygdala and hippocampus but not touching the temporal neocortex Made the attempt easier. Unfortunately, a difficult-to-resolve seizure recurs 6 months after surgery, which is a second larger surgical practice, ie the left anterior temporal lobe, to ultimately eradicate epileptic seizure activity. Left aerial temporal lobe was required.

Discussion The mesial TLE (mTLE) is often cited as the most common type of human epilepsy and is almost unchanged with hippocampal sclerosis, the single most frequently encountered epilepsy-causing factor in patients with epilepsy. It occurs at the same time (Non-Patent Document 5). Along with electrodiagnostic findings where discharges originate from the central basement structure, the clinical signs represented by the proband are consistent with the diagnosis of mTLE, but no obvious lesions were evident in the tissue excised from the proband. While this seems paradoxical, hippocampal atrophy, as found by the discovery of such abnormal characteristics in asymptomatic first-degree relatives of familial mTLE (Non-Patent Document 6) Is not directly related to overt epilepsy symptoms. Nevertheless, the surgically treatable outcome of staying without seizures for 3 years after the patient's left anterior temporal lobe resection is important for epileptogenesis and is inherent in the resected tissue Shows pathology.

  The proband's asymptomatic father also had a heterogeneous N587fsX mutation in the lymphocyte genomic DNA. Most epilepsy has a complex form of heredity, which is now believed to have a multifactorial etiology of developmental, environmental, and genetic factors (5). ), TLE will also apply. Many mutagenic interactions often lead to incomplete penetrance, and this may explain the discovery of the N587fsX mutation in the asymptomatic father of the proband. Similarly, the phenotypic discrepancy between the father and proband is that the difference in expression is that the father was less serious and could have a phenotypic shape that could escape clinical diagnosis. May be an indicator. Such variability often arises from allelic imbalances due to differentially regulated expression levels of mutant versus normal mRNA in individuals affected or not. Importantly, allelic imbalance has been described for at least one channelopathy (7) and may play a role here. Other possibilities include mosaicism, that is, the paternal N587 fsX mutation may be limited to certain cell lines.

A 5 base pair deletion of KCND2 causing a frameshift resulting in a heterologous, premature stop codon (TGA), c. 2723_2727delAAACT, was identified in a TLE proband. Asymptomatic fathers were also heterozygous for this mutation. A 5-base deletion corresponding to the N587fsX amino acid change, predicted to produce a partially excised Kv4.2 protein lacking amino acids 587-630. Voltage-dependent potassium channel currents were recorded from HEK293 expressing only wild-type human Kv4.2 channel (WT), WT and mutant Kv4.2 (WT + delAAACT_N587fsX) channel, and mutant Kv4.2 channel only (delAAACT_N587fsX). . Current-voltage relationship of K ⁺ current in HEK293 cells. Cells expressing only the mutant Kv4.2 protein show significantly lower current density (p = 1.49 × 10 ⁻⁵ ) than cells in the WT channel. The data point represents the average current density of the pooled data. Voltage-dependent potassium transient in WT channel. The current was caused by a depolarization step (-70 mV to 70 mV, pulse interval 15 seconds) from a holding current of -80 mV.

Claims (23)

A method for detecting a genetic predisposition to human epilepsy, comprising:
The above method comprising detecting the presence of a mutation that causes a change in the amino acid sequence of the human KCND2 protein in the base sequence of the human KCND2 gene.   The method according to claim 1, wherein the base sequence of the human KCND2 gene is represented by SEQ ID NO: 1.   The method according to claim 1 or 2, wherein the mutation of the base sequence causing a change in the amino acid sequence of human KCND2 protein results in any of missense, nonsense, frameshift, splicing site formation or deletion, and truncation.   The method according to any one of claims 1 to 3, wherein the mutation that causes a change in the amino acid sequence of the human KCND2 protein is a mutation that reduces or substantially loses the biological function of the human KCND2 protein.   The method according to any one of claims 1 to 4, wherein the site causing the mutation of the human KCND2 gene is 3 'to the codon encoding the amino acid immediately after the most transmembrane segment at the C-terminal side.   The site for causing a mutation in the human KCND2 gene is a codon encoding an amino acid corresponding to the 587th asparagine of SEQ ID NO: 2, or 3 ′ of the codon. One way.   The mutation that causes a change in the amino acid sequence of human KCND2 protein is a mutation that lacks all of the C-terminal amino acid sequence from the amino acid corresponding to the 587th asparagine of SEQ ID NO: 2. One way.   The site causing mutation of the human KCND2 gene is present in a region encoding one or more parts selected from an interaction motif for filamin, a MAP / ERK phosphorylation site, or a PSD95 binding domain. 8. A method according to any one of claims 1 to 7, comprising a region.   The method according to any one of claims 1 to 8, wherein the site causing the mutation of the human KCND2 gene is a codon encoding an amino acid corresponding to the 587th asparagine of SEQ ID NO: 2.   The method according to any one of claims 1 to 9, wherein the site causing the mutation of the human KCND2 gene is AAACT having a base sequence corresponding to the 2723th to 2727th positions of SEQ ID NO: 1.   The method according to claim 10, wherein the mutation that causes a change in the amino acid sequence of the human KCND2 protein is a mutation in which AAACT of the nucleotide sequence corresponding to positions 2723 to 2727 of SEQ ID NO: 1 is deleted.   12. The method according to any one of claims 1 to 11, wherein the human epilepsy is an epilepsy included in the symptomatic epilepsy group.   13. The method of claim 12, wherein the human epilepsy is temporal lobe epilepsy.   The step of detecting a mutation that causes a change in the amino acid sequence of the human KCND2 protein is performed using any one of base sequencing, nucleic acid amplification reaction, hybridization, electrophoresis, restriction enzyme treatment, or a combination thereof. 14. The method according to any one of claims 1 to 13, wherein:   15. The step of detecting a mutation that causes a change in the amino acid sequence of human KCND2 protein comprises examining the base sequence of genomic DNA in a sample derived from blood, hair, or cheek. One way. 16. The method according to any one of claims 1 to 15, further comprising determining that there is a genetic predisposition to epilepsy when there is a mutation in the nucleotide sequence of the human KCND2 gene that causes a change in the amino acid sequence of the human KCND2 protein. Or one way.   A primer for use in the method of any one of claims 1-16.   A probe for use in the method of any one of claims 1-16.   A nucleic acid chip for use in the method of any one of claims 1-16. A kit for detecting a genetic predisposition of human epilepsy,
The kit comprising a primer, a probe, and / or a nucleic acid chip for detecting a mutation that causes a mutation that causes a change in the amino acid sequence of a human KCND2 protein in the base sequence of the human KCND2 gene.   A nucleic acid having a mutation that causes a change in the amino acid sequence of a human KCND2 protein in the base sequence of the human KCND2 gene.   A non-human knockout mammal for a human epilepsy model in which a part or all of the biological function of the KCND2 protein is inactivated by modification of part or all of the DNA region of the KCND2 gene. A method for detecting a genetic predisposition to human epilepsy, comprising:
Detecting the presence of a mutation that causes a change in the expression level of the human KCND2 gene or a protein thereof in the base sequence of the human KCND2 gene or a control sequence for the expression thereof.

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