Testing method and test kit

Abstract

To provide a simple method and test kit for assessing the risk of developing epilepsy. [Solution] A testing method for determining the risk of developing or worsening epilepsy in dogs, comprising a step of checking for the presence or absence of a mutation in the dog's MCPH1 gene. The step of checking for the presence or absence of a mutation in the dog's MCPH1 gene includes analyzing the presence or absence of a mutation in the MCPH1 gene in a sample taken from the subject dog, and it is preferable that the mutation in the MCPH1 gene is (1) a mutation in the 601st base of the base sequence shown in Sequence ID No. 1, or (2) a mutation in the base encoding the 756th amino acid of the MCPH1 protein.

Description

Technical Field

[0001] The present invention relates to a testing method and a testing kit, and more particularly, to a testing method and a testing kit for determining the risk of epilepsy onset or exacerbation in dogs.

Background Art

[0002] Epilepsy is a disease that repeatedly causes seizures such as convulsions and loss of consciousness through abnormal nerve cells caused by disturbances in brain electrical signals. It is said that the incidence of epilepsy in dogs kept at home is nearly 1%.

[0003] The main symptom of epilepsy is an epileptic seizure. Epileptic seizures include general seizures (seizures that occur throughout the body) and focal seizures (seizures that appear in a part of the body). General seizures occur when epilepsy affects the entire brain and the seizure appears as a whole-body symptom at once. Typical symptoms include general tonic seizures where the whole body stiffens and convulses, general clonic seizures where the limbs and mouth shake, and general tonic-clonic seizures where these two occur in sequence. In addition, there are also general seizures such as myoclonic seizures where specific muscles contract strongly and convulse, yawn seizures, and weakness seizures, which rarely result in loss of consciousness.

[0004] Focal seizures occur when the part of the brain where epilepsy occurs is limited, and the epileptic seizure appears in a part of the body. The main symptoms include being unable to move as if the body is stiff while conscious, small convulsions in one or more of the limbs, repeating actions such as chewing food, and biting at the air (fly-biting behavior).

[0005] Canine epilepsy is classified into three types based on its cause: idiopathic epilepsy, symptomatic epilepsy (structural epilepsy, secondary epilepsy), and cryptogenic epilepsy. Idiopathic epilepsy is when the cause cannot be identified, and when people generally refer to epilepsy, they are often referring to this type. It is thought to be caused by dysfunction of brain nerve cells, and genetic predisposition is believed to be involved. It often develops at a relatively young age. Symptomatic epilepsy is caused by an abnormality in the brain, and a diagnosis is made when, in addition to epileptic symptoms, a clear disease that damages brain tissue, such as a brain tumor, encephalitis, hydrocephalus, or trauma, is identified. The onset time varies depending on the underlying disease. Cryptogenic epilepsy is sometimes used when symptomatic epilepsy is suspected, but no clear abnormalities are found in various tests, and it appears to be idiopathic epilepsy.

[0006] There is no cure for epilepsy. Treatment for epilepsy primarily involves taking antiepileptic drugs to minimize brain damage caused by seizures. Because antiepileptic drugs have side effects, their use is often limited to reducing the frequency of seizures.

[0007] To determine whether a dog's seizure is an epileptic seizure, a physical examination, blood tests to assess the dog's overall condition, and a neurological examination are generally performed. The information gathered from the owner is crucial for the veterinarian to determine if it is an epileptic seizure. Although epilepsy is a brain disorder, the cause may or may not be detectable as a clear abnormality in the brain. Because of this characteristic, multiple tests may be necessary to rule out non-brain causes. If neurological abnormalities are present or a more definitive diagnosis is needed, an MRI scan may be performed. However, MRI scans require general anesthesia, which can be stressful for dogs.

[0008] Thus, there is no fundamental cure for canine epilepsy, and it must be managed for life. When an epileptic seizure occurs, there is nothing the owner can do but watch as their dog suffers, which places a great physical and mental burden not only on the dog but also on the owner. Antiepileptic drugs used to prevent epileptic seizures have side effects, so they must be administered only after a definitive diagnosis of epilepsy has been made, but diagnosing epilepsy requires a complex series of tests.

[0009] Therefore, there is a need for a simple testing method and test kit that can assess the risk of developing epilepsy.

[0010] Non-patent document 1 describes DIRAS family GTPase 1 as a gene mutation associated with symptomatic and idiopathic epilepsy in dogs, but does not disclose the MCPH1 gene.

[0011] Furthermore, Non-Patent Document 2 describes the MCPH1 gene as being associated with microcephaly in humans, but does not disclose its association with epilepsy. [Prior art documents] [Non-patent literature]

[0012] [Non-Patent Document 1] Franziska Wielaendar et al., PNAS, Feb 21, 2017,114 (10) 2669-2674 [Non-Patent Document 2] Sarah Duerinckx et al., Mol Genet Genomic Med.,Sep 9, 2021(9):e1768. [Overview of the project] [Problems that the invention aims to solve]

[0013] Therefore, the present invention aims to provide a testing method and a testing kit that can test the risk of developing epilepsy in a simple manner. [Means for solving the problem]

[0014] The inventors of this invention completed the present invention by comparing the genes of dogs that have developed epilepsy with the genes of dogs that have not developed epilepsy, and by discovering that a mutation occurs in MCPH1 in dogs that have developed epilepsy.

[0015] In other words, the present invention is as follows [1] to

[11] . [1] A test method for determining the risk of developing or worsening epilepsy in dogs, comprising a step of checking for the presence or absence of mutations in the MCPH1 gene of dogs. [2] The step of examining whether or not there is a mutation in the MCPH1 gene of the dog includes analyzing whether or not there is a mutation in the MCPH1 gene in a sample taken from the dog subject, and if the mutation in the MCPH1 gene is, (1) A mutation in the 601st base of the nucleotide sequence shown in Sequence ID No. 1, or (2) Mutation of the base encoding the 756th amino acid of the MCPH1 protein The testing method for [1] is. [3] A method for testing that the mutation at the 601st base of the base sequence shown in Sequence ID No. 1 is a mutation from C to A.[2] [4] A test method for determining the risk of developing or worsening epilepsy in dogs, comprising the step of examining the amino acid length of the MCPH1 gene product in dogs. [5] The test method described in [4] in which the amino acid length of the MCPH1 gene product is shorter than that of the wild type, and the risk of developing epilepsy is determined to be high. [6] A biomarker comprising the canine MCPH1 gene or MCPH1 gene product for testing the risk of developing or worsening epilepsy in dogs. [7] A primer used to amplify a DNA fragment containing the 601st single nucleotide polymorphism site of the base sequence represented by Sequence ID No. 1. A test kit for determining the risk of developing or worsening epilepsy in dogs, comprising the primers [8][7]. [9]An oligonucleotide containing the 601st single nucleotide polymorphism site of the nucleotide sequence represented by SEQ ID NO: 1, which is an oligonucleotide consisting of a partial sequence of 10 to 30 bases of the nucleotide sequence of SEQ ID NO: 1 or a sequence complementary to the partial sequence, or a sequence capable of hybridizing with those sequences under stringent conditions, or a labeled substance thereof, which is a probe for examination for determining the risk of epilepsy onset or exacerbation in dogs. <00,00075>

[10] A DNA chip comprising an immobilized substrate containing the probe of [9] or a labeled substance thereof. <00,00076>

[11] The following (a) and / or (b): <00,00077>(a) A nucleic acid probe or nucleic acid primer capable of specifically detecting a transcription product of the MCPH1 gene <00,00078>(b) An antibody capable of specifically recognizing the MCPH1 gene product <00,00079>A test kit for determining the risk of epilepsy onset or exacerbation in dogs, which contains the above. <00,00080>[Advantages of the Invention] <00,00081><00,00082><00,00083><00,00084>According to the present invention, it becomes possible to provide a test method and a test kit capable of testing the risk of epilepsy onset by a simple method. In addition, by implementing the present invention during breeding, it becomes possible to perform breeding that reduces the incidence of epilepsy. In fact, regarding the genetic disease Degenerative Myelopathy (DM), which frequently occurred in the dog breed Welsh Corgi Pembroke, as a result of the spread of genetic testing and appropriate breeding, the proportion of individuals at risk of onset was 42% in 2017, but it was reported that it decreased to 16% in 2020. <00,00085>[Brief Description of the Drawings] <00,00086><00,00087><00,00088><00,00089><00,00090>It is a graph showing the results of Example 1. <00,00091><00,00092>It is a graph showing the results of Example 2. <00,00093><00,00094>[Modes for Carrying Out the Invention] <00,00095><00,00096><00,00097><00,00098>[Testing Method] The present invention relates to a testing method for determining the risk of developing or worsening epilepsy in dogs, and comprises a step of examining whether or not there is a mutation in the MCPH1 gene in dogs.

[0019] (Target dogs) There are no particular limitations on the dogs that can be tested. While the age of the dogs is not a factor, it is preferable to perform the test before the onset of epilepsy, preferably at a younger age than the age at which epilepsy frequently occurs, preferably under one year old, and more preferably under six months old. Furthermore, from the perspective of proper breeding, it is also preferable to perform the test before breeding. When determining the risk of severe epilepsy, it is preferable to target dogs that have already developed epilepsy or have been diagnosed with epilepsy, but even before the onset of epilepsy, it is possible to determine the risk of developing epilepsy along with the possibility of severe epilepsy.

[0020] (Dog breed) The breed of dog to be tested is not particularly limited. It is preferable to target breeds with a high incidence of epilepsy. Examples of breeds with a high incidence of epilepsy include Italian Greyhounds, (Toy) Poodles, Chihuahuas, Siberian Huskies, Boston Terriers, American Cocker Spaniels, Beagles, Dachshunds, Golden Retrievers, Labrador Retrievers, Shetland Sheepdogs, Border Collies, Cavalier King Charles Spaniels, Dalmatians, and Bernese Mountain Dogs.

[0021] (sample) The samples used for testing are not particularly limited and include, for example, dog saliva, blood, urine, bodily fluids, hair, nails, oral cells obtained by swabbing the inside of the mouth with a cotton swab, and oral mucosa. These samples can be processed by known methods to extract and isolate DNA, RNA, peptides, proteins, etc., which can then be used for testing.

[0022] (MCPH1) The MCPH1 gene encodes microcephalin, and in humans, it has been reported to be expressed during fetal brain development. Certain mutations in MCPH1 are known to cause primary microcephaly (a severe reduction in brain size) in humans when homozygous. Mutations include, for example, single nucleotide mutations, deletions, substitutions, insertions, and translocations.

[0023] (A process to check for mutations in the MCPH1 gene) The method for investigating the presence or absence of mutations in the MCPH1 gene is not particularly limited, and known methods can be employed. Examples of such methods include sequencing the base sequence of DNA or RNA and comparing it with a reference sequence, hybridization methods using probes or microarrays (DNA chips) immobilized with such probes, PCR using primers for mutation detection, and allele-specific PCR. More specifically, methods include PCR, NASBA, LCR, SDA, LAMP, methods utilizing restriction fragment length polymorphism (RFLP), denaturing gradient gel electrophoresis (DGGE), methods utilizing chemical cleavage of mismatch sites (CCM), primer extension methods (TaqMan), PCR-SSCP, single-strand conformational polymorphism analysis (SSCP), Invader method, single-nucleotide primer method, SNaPshot, MassArray, Pyrosequncing, SNP-IT, BeadArray, Scorpion, and MADI-TOF / MS, with microarray methods or the TagMan method being preferred.

[0024] Methods for sequencing the base sequences of nucleic acids such as DNA and RNA include, for example, sequencing the surrounding sequence containing the site where the presence or absence of a mutation is to be checked and comparing it with a reference sequence, or performing a sequencing reaction using a primer set at a position of several tens of bases 5' of the site where the presence or absence of a mutation is to be checked, and determining the presence or absence of a mutation from the analysis results.

[0025] One method using probes involves performing hybridization with nucleic acids in a sample isolated from a test dog, or nucleic acid samples amplified by PCR, using probes consisting of oligonucleotides corresponding to part or all of the MCPH1 gene sequence containing the site (location) where the presence or absence of mutations is to be confirmed, or oligonucleotides consisting of sequences that can hybridize to those sequences under stringent conditions.

[0026] The conditions for hybridization with the probe only need to be sufficient to distinguish between cases where the target nucleic acid contains gene mutations such as single nucleotide mutations and cases where it does not. For example, conditions that allow hybridization to occur when gene mutations are present but not when they are not. Examples of stringing conditions include performing hybridization in approximately 5-6 × SSC at approximately 60°C or higher, preferably approximately 65°C or higher, and more preferably approximately 70°C or higher. The stringing conditions may also include performing a washing treatment in approximately 0.1-1 × SSC at approximately 40-70°C, preferably approximately 50-67°C, and more preferably approximately 60-65°C.

[0027] The probe may be fixed at one end to a substrate and used as a DNA chip (microarray). In this case, the DNA chip may have only one probe corresponding to one gene mutation site, or probes corresponding to multiple gene mutation sites. As the substrate for immobilizing the oligonucleotide, known materials such as glass slides, nitrocellulose membranes, and microbeads can be used. When multiple oligonucleotides are aligned and immobilized on the immobilization substrate, the immobilization substrate can be used as a DNA microarray or DNA chip. Oligonucleotides may be synthesized on the substrate, or synthesized oligonucleotides may be immobilized on the substrate. Oligonucleotide immobilization can be performed by adsorption or covalent bonding. When immobilization is performed by covalent bonding, functional groups such as amino groups and SH groups for covalent bonding should be introduced to the substrate surface and the oligonucleotide. The probe may also be a labeled product labeled with a fluorescent substance, enzyme, radioisotope, chemiluminescent substance, etc., for detection. The labeling substance used for labeling can be a known substance and a known method. Examples of fluorescent substances include Cy3, Cy5, rhodamine, and fluorescein.

[0028] The method using mutation detection primers involves amplifying nucleic acids isolated from the subject using primers corresponding to a portion of the MCPH1 gene sequence containing the mutation site. Probes and primers for detecting mutations in the MCPH1 gene can be appropriately designed based on the MCPH1 sequence information. Such primers are preferably designed so that the mutation site is located at the 3' end. Allele-specific primers used in allele-specific PCR are designed so that the bases at the 3' end of the primer are complementary to the polymorphic or mutation site, and amplification by PCR occurs only with primers that match the bases of the polymorphic or mutation site. Furthermore, to enhance specificity, it is preferable to artificially introduce a mismatch a few bases from the 3' end, for example, 1 to 3 bases from the 3' end.

[0029] The Taqman method, also known as the Taqman probe method, is characterized by the use of a Taqman probe in addition to primers in real-time PCR. In a typical Taqman method, for example, the Taqman probe has a gene-specific sequence and is designed to bind to the target between two PCR primers. A reporter, a fluorescent dye that reports the amplification of the target, is bound to the 5′ end of the Taqman probe. A quencher that quenches the fluorescence from the reporter is bound to the 3′ end of the Taqman probe. The quencher also blocks the 3′ end of the probe, preventing extension by thermostable DNA polymerase. Before the start of PCR, the Taqman probe is unchanged, and because the reporter and quencher are in close proximity, FRET occurs, so the reporter signal is quenched before the PCR reaction. During the PCR process, the primers and probe anneal to the target. When DNA polymerase extends from the primer to the upstream of the probe, if the probe is bound to the correct target, the 5′ nuclease activity of the polymerase cleaves the probe, releasing a fragment containing the reporter dye. Once cleavage occurs, the reporter and quencher are no longer in close proximity to each other, so the released reporter molecule is not quenched. If there is a mismatch, i.e., a mutation, in either the primer binding site or the site where the Taqman probe binds, the reporter will not be quenched, making it possible to detect the mutation. There are several variations of the Taqman method, including methods using allele-specific blockers, which can be used as appropriate.

[0030] (Epilepsy) In this invention, the type and cause of epilepsy are not limited. The risk of developing epilepsy refers to the risk of developing epilepsy in the future, and the risk of severe epilepsy refers to the risk of the epilepsy becoming severe if it develops. Determining the risk of developing or severe epilepsy in a dog preferably means determining that the dog has a high risk of developing or severe epilepsy when an MCPH1 mutation is detected.

[0031] (Sequence ID 1) The nucleotide sequence corresponding to Sequence ID No. 1 shown in Table 1 below is a part of the canine MCPH1 gene sequence. Specifically, it corresponds to the sequence 57520975-57522175 on canine chromosome 16. The testing method of the present invention preferably includes a step of analyzing whether or not there is a mutation in the 601st base of the base sequence shown in Sequence ID No. 1. The testing method of the present invention preferably further includes a step of detecting whether or not there is a mutation in which C (cytosine) changes to A (adenine) at the 601st base of the base sequence shown in Sequence ID No. 1. The mutation from C to A at the 601st base of the base sequence shown in Sequence ID No. 1 is a change from encoding cysteine ​​to a stop codon, and it is thought that this mutation results in the production of an MCPH1 protein with a shorter amino acid length compared to a normal MCPH1 protein. The 601st base of Sequence ID No. 1 corresponds to the 2268th base in the canine MCPH1 gene Transcript:ENSCAFT00845039419.1. [Table 1]

[0032] Furthermore, the 601st base of Sequence ID No. 1 corresponds to the base encoding the 756th amino acid of the MCPH1 protein. The testing method of the present invention preferably includes a step of analyzing whether or not there is a mutation in the base encoding the 756th amino acid of the MCPH1 protein.

[0033] (In combination with other gene tests) The testing method of the present invention may detect the presence or absence of mutations in other genes in addition to mutations in the MCPH1 gene. In particular, by detecting the presence or absence of mutations in genes that are already known to cause canine epilepsy or genes that have been pointed out to be associated with epilepsy, together with mutations in MCPH1, it is expected that it will be possible to predict and determine the risk of developing epilepsy or the risk of epilepsy becoming more accurate. Examples of such genes that are already known to be associated with the development of epilepsy include LGI2, ADAM23, DIRAS1, and EPM2B.

[0034] [Other methods of inspection] Another aspect of the present invention relates to a testing method for determining the risk of developing or worsening epilepsy in dogs, comprising the step of examining the amino acid length of the canine MCPH1 gene product. The inventors have found that the canine MCPH1 gene is associated with epilepsy. The MCPH1 gene is a gene that codes for a protein called microcephalin. If the amino acid length of the MCPH1 gene product, microcephalin, differs from the amino acid length of normal microcephalin, it can be determined that there is a high risk of developing or worsening epilepsy. In particular, the inventors have found that in dogs with epilepsy, a mutation from an amino acid-coding codon to a stop codon is observed in the MCPH1 gene. When this mutation is present, the amino acid length of the protein, which is the MCPH1 gene product, is shorter compared to a normal MCPH1 gene product. Therefore, in the other aspect of the present invention, if the amino acid length of the MCPH1 gene product is shorter than that of the wild type, it is preferably determined that there is a high risk of developing epilepsy.

[0035] The method for determining the amino acid length of a gene product is not particularly limited, and known methods can be employed. Examples of such methods include amino acid sequence determination methods such as SDS-PAGE, Western blotting, immunoprecipitation, gel filtration chromatography, and Edman degradation, as well as molecular weight measurement methods such as LC-MS, mass spectrometry, and ultracentrifugation.

[0036] [Biomarkers] The biomarker of the present invention is a biomarker for testing the risk of developing or worsening epilepsy in dogs, consisting of the canine MCPH1 gene or the MCPH1 gene product. Preferably, the biomarker of the present invention is the MCPH1 gene product. By measuring the amino acid length and molecular weight of the MCPH1 gene product of a target dog, it is possible to predict the risk of developing or worsening epilepsy. Furthermore, by measuring the amino acid length and molecular weight of the MCPH1 gene product as a biomarker in dogs suspected of having epilepsy, the accuracy of diagnosing epilepsy can be improved.

[0037] [primer] The primer of the present invention is used for amplifying a DNA fragment containing the 601st single nucleotide polymorphism site of the nucleotide sequence represented by Sequence ID No. 1. Preferably, it is designed so that the 601st single nucleotide polymorphism site of the nucleotide sequence represented by Sequence ID No. 1 is located at the 3' end. The following are specific examples of suitable primers. The following primer set is suitable for PCR. MCPH1_F1: GCAACACCCTATGTCCCACA(Sequence ID 2) MCPH1_R1: CCACGACCAAGGTTCTGTGA (Sequence ID 3) Furthermore, the following are specific examples of other suitable primers. The following primer set is suitable for sequencing sequences containing mutation sites. MCPH1_seqF1: ACTCTGCACGCACGTAGAAA (Sequence ID 4) MCPH1_seqR1: TCATCACCAGAACACTAAGATTGGT (Sequence ID 5)

[0038] [Test kit] The test kit of the present invention is a test kit for determining the risk of developing or worsening epilepsy in dogs, comprising the primers of the present invention described above. In addition to the primers, it may also contain reagents and components necessary for PCR. Examples of such reagents include enzymes such as DNA polymerase, reagents for extracting nucleic acids such as DNA, and buffers. In addition to the primers, it may also contain probes such as Taqman probes.

[0039] Another embodiment of the present invention is a test kit, (a) and / or (b) below: (a) A nucleic acid probe or nucleic acid primer capable of specifically detecting the transcript of the MCPH1 gene. (b) Antibodies that specifically recognize the MCPH1 gene product This is a test kit for determining the risk of developing or worsening epilepsy in dogs, containing the following: It may also contain reagents or components other than nucleic acid probes, primers, or antibodies, as described above.

[0040] [probe] The probe of the present invention is an oligonucleotide containing the 601st single nucleotide polymorphism site of the nucleotide sequence represented by SEQ ID NO: 1, and comprises an oligonucleotide or label thereof consisting of a subsequence of 10 to 30 nucleotides of the nucleotide sequence of SEQ ID NO: 1, a sequence complementary to that subsequence, or a sequence that can hybridize with those sequences under stringent conditions, and is a test probe for determining the risk of developing or worsening epilepsy in dogs. The hybridization conditions and stringent conditions are the same as described above. The subsequence of 10 to 30 nucleotides of the nucleotide sequence of SEQ ID NO: 1, or a sequence complementary to that subsequence, is preferably a contiguous subsequence of 10 to 30 nucleotides from the sequence represented by SEQ ID NO: 1, or a sequence complementary to that subsequence.

[0041] [DNA chip] The DNA chip of the present invention is a DNA chip comprising an immobilized substrate containing the above-described probe or label thereof. The DNA chip is preferably a microarray. The method of immobilizing the probe or label thereof onto the substrate is not particularly limited, and known methods can be employed. [Examples]

[0042] [Example 1] (1) Specimen Samples containing oral cells were collected using oral swabs from 16 Italian Greyhounds diagnosed with epilepsy (epilepsy group) and 7 Italian Greyhounds not diagnosed with epilepsy and showing no symptoms of epilepsy (non-epilepsy group, control).

[0043] (2) DNA extraction Genomic DNA was extracted from each sample using DNAdvance from Beckman Coulter.

[0044] (3) Preparation of the WES library For each genomic DNA sample, a WES library was prepared using the Twist Library Preparation Kit from Twist Bioscience.

[0045] (4) Whole exome sequencing (WES) The WES library prepared as described above was subjected to whole exome sequencing (WES) using an Illumina Novaseq 6000 sequencer. The exome panel used was the Twist Alliance Canine Exome from Twist Bioscience.

[0046] (5) Detection of mutations The obtained sequencing data was mapped to a reference sequence using Illumina's DRAGEN to detect mutations. ROS_Cfam_1.0 was used as the reference sequence for mutation detection.

[0047] (6) Results We discovered a mutation in the MCPH1 gene that showed a significant difference in frequency between epilepsy and non-epileptic groups in Italian Greyhounds. Fisher's Exact Test was used to test for differences in allele frequency between the groups, and a statistically significant difference was found (p=0.00716). The mutation in question was a change from C to A at position 601 (position 2268 in the full-length MCPH1 gene) in the partial sequence of the MCPH1 gene represented by Sequence ID No. 1. The results are shown in Figure 1. As shown in the graph in Figure 1, in the epilepsy group, 15 out of 16 patients were affected by the MCPH1 gene mutation, and 1 patient was a carrier of the MCPH1 mutation. On the other hand, in the non-epileptic group (control), 2 out of 7 individuals were affected and 5 were carriers. Affected means that both of the two sets of genes are mutated, while carrier means that one of the two sets of genes is mutated. Compared to the epileptic group, the proportion of affected individuals was significantly lower in the non-epileptic group. The presence of affected individuals in the non-epileptic group suggests that, although the non-epileptic group consisted of individuals who had not yet developed epilepsy at the time of sample collection, it is possible that some of them may have the potential to develop epilepsy in the future.

[0048] [Example 2] Samples containing oral cells were collected from 110 Toy Poodles diagnosed with epilepsy (epilepsy group) and 50 Toy Poodles not diagnosed with epilepsy and showing no symptoms of epilepsy (non-epilepsy group, control group) using oral swabs. DNA extraction was performed in the same manner as in Example 1 above. Next, 71 of the epilepsy group were sequenced (Sanger sequencing) using the following primers. For 39 of the animals, WGS libraries were prepared for each genome sample using Illumina DNA PCR-Free Prep and Tagmentation, and whole-genome sequencing was performed using Illumina Novaseq 6000 sequencers to detect mutations. In addition, six of the control group underwent sequencing (Sanger sequencing) using the following primers. For the 44 animals, WGS libraries were prepared for each genome sample using Illumina DNA PCR-Free Prep and Tagmentation, and whole-genome sequencing was performed using Illumina Novaseq 6000 sequencers to detect mutations. The obtained sequencing data was mapped to a reference sequence using Illumina DRAGEN to detect mutations. ROS_Cfam_1.0 was used as the reference sequence for mutation detection. MCPH1_seqF1: ACTCTGCACGCACGTAGAAA (Sequence ID 4) MCPH1_seqR1: TCATCACCAGAACACTAAGATTGGT (Sequence ID 5) As a result, similar to the Italian Greyhound, there was a significant difference between the epilepsy group and the non-epileptic group in the frequency of the mutation in which the 601st C (2268th position in the full-length MCPH1 gene) of the partial sequence represented by Sequence ID No. 1 becomes A. Fisher's Exact Test was used to test for differences in allele frequencies between the groups, and a significant difference was found (p=0.04638). The results are shown in Figure 2. As shown in the graph in Figure 2, in the epilepsy group, out of 110 animals, 5 were affected by the MCPH1 gene mutation, 23 were carriers of the MCPH1 mutation, and 82 were clear. Clear means that neither of the two sets of genes has a mutation. On the other hand, in the non-epileptic group (control), out of 50 dogs, 0 were affected, 7 were carriers, and 43 were clear. Compared to the epileptic group, the proportion of carriers and affected dogs was significantly lower in the non-epileptic group.

Claims

1. A testing method for determining the risk of developing or worsening epilepsy in dogs, comprising a step of examining whether or not there is a mutation in the MCPH1 gene in dogs.

2. The step of investigating whether or not there is a mutation in the MCPH1 gene of the dog includes analyzing whether or not there is a mutation in the MCPH1 gene in a sample taken from the subject dog, and if the mutation in the MCPH1 gene is, (1) A mutation in the 601st base of the base sequence shown in Sequence ID No. 1, or (2) Mutation of the base encoding the 756th amino acid of the MCPH1 protein The inspection method according to claim 1.

3. The testing method according to claim 2, wherein the mutation at the 601st base of the base sequence shown in Sequence ID No. 1 is a mutation from C to A.

4. A testing method for determining the risk of developing or worsening epilepsy in dogs, comprising the step of examining the amino acid length of the MCPH1 gene product in dogs.

5. The testing method according to claim 4, wherein if the amino acid length of the MCPH1 gene product is shorter than that of the wild type, it is determined that there is a high risk of developing epilepsy.

6. A biomarker comprising the canine MCPH1 gene or MCPH1 gene product for testing the risk of developing or worsening epilepsy in dogs.

7. A primer used to amplify DNA fragments containing the single nucleotide polymorphism site at position 601 of the base sequence represented by Sequence ID No.

1.

8. A test kit for determining the risk of developing or worsening epilepsy in dogs, comprising the primer described in claim 7.

9. A probe for testing the risk of developing or worsening epilepsy in dogs, comprising an oligonucleotide containing the 601st single nucleotide polymorphism site of the nucleotide sequence represented by SEQ ID NO: 1, and comprising an oligonucleotide or label thereof consisting of a subsequence of 10 to 30 nucleotides of the nucleotide sequence of SEQ ID NO: 1, a sequence complementary to that subsequence, or a sequence that can hybridize with those sequences under stringent conditions.

10. A DNA chip comprising an immobilized substrate containing the probe or label thereof according to claim 9.

11. (a) and / or (b) below: (a) A nucleic acid probe or nucleic acid primer capable of specifically detecting the transcript of the MCPH1 gene. (b) Antibodies that specifically recognize the MCPH1 gene product A test kit containing the following for determining the risk of developing or worsening epilepsy in dogs.

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