JP6963271B2 - Genetic diagnosis of canine hereditary gastrointestinal neoplasia - Google Patents

Description

The present invention relates to a test / diagnostic technique in veterinary medicine. More specifically, the present invention relates to a test method for hereditary gastrointestinal neoplasia in dogs and its use (confirmed diagnosis, risk diagnosis, etc.).

Originally, the development of gastrointestinal tumors is very rare in dogs. However, in recent years, the incidence of gastrointestinal tumors has increased in Jack Russell terriers (J.R. terriers), and it has been shown that their pathophysiology is clearly different from that of normal canine gastrointestinal tumors. Due to the low genetic diversity of purebred dogs, many breed-specific hereditary diseases are found in dogs. The entire base sequence of the dog genome has been deciphered (see Non-Patent Document 1).

Lindblad-Toh K et al., Genome sequence, comparative analysis and haplotype structure of the domestic dog. Nature 438 (7069), 803-19. 2005

This disease seen in JR terrier is also considered to have a high possibility of being a hereditary disease, and the development of a genetic diagnostic method for it is desired. As described above, although the entire base sequence of the canine genome has been deciphered, the genetic abnormality that causes this disease is unknown. Therefore, at present, it is not possible to carry out genetic testing for this disease found in JR terrier. Therefore, it is an object of the present invention to clarify a genetic abnormality specifically possessed by a dog suffering from this disease and to establish a genetic test method capable of detecting the abnormality.

In the course of studies to solve the above problems, the present inventors have found that the pathophysiology of the gastrointestinal tumor of JR terrier is familial adenomatous polyposis, which is a human hereditary disease caused by the APC gene (Adenomatous Polyposis Coli). Focusing on the similarity to the disease (FAP, Familial Polyposis Coli), we investigated whether there is an abnormality in the APC gene in dogs affected by this disease. Specifically, the entire coding region of the APC gene was divided into 29 regions, each region was amplified by PCR (polymerase chain reaction), and the nucleotide sequence was analyzed by the direct sequencing method (direct nucleotide sequence determination method) (PCR- Direct sequence method). As a result of analysis of affected dogs and non-affected dogs of JR Terrier, mutations in the APC gene (germline mutation) were found in the affected dogs, indicating that this disease is a hereditary disease caused by the APC gene. It became clear. Furthermore, since the same point mutations (c.462A> T and c.463A> T) were detected in all the affected dogs (5) analyzed, this mutation was detected to detect this disease. It was found that genetic testing and definitive diagnosis using it are possible. The following inventions are based on the findings or results.
[1] Regarding the nucleic acid sample collected from the dog to be inspected, the following (1) germline mutation or (2) germline mutation, or the following (1) germline mutation and (2) Testing for hereditary gastrointestinal neoplasia in dogs, including the step of detecting both germline mutations:
(1) C.462A> T mutation of APC gene;
(2) C.463A> T mutation of APC gene.
[2] When a germline mutation of (1) or a germline mutation of (2) is detected, or both a germline mutation of (1) and a germline mutation of (2) are detected. The test method according to [1], wherein it is determined that a hereditary gastrointestinal neoplasia has developed.
[3] When a germline mutation of (1) or a germline mutation of (2) is detected, or both a germline mutation of (1) and a germline mutation of (2) are detected. The test method according to [1], which determines that there is a high risk of developing hereditary gastrointestinal neoplasia in some cases.
[4] The test method according to any one of [1] to [3], wherein the dog to be tested is a Jack Russell terrier.
[5] A nucleic acid for detecting a c.462A> T mutation of an APC gene, which is a germline mutation, which contains a sequence complementary to a certain region containing the mutation position and is specific to the region. A reagent for testing canine hereditary gastrointestinal neoplasia, which contains a nucleic acid that hybridizes with.
[6] A nucleic acid for detecting a c.463A> T mutation of an APC gene, which is a germline mutation, which contains a sequence complementary to a certain region containing the mutation position and is specific to the region. A reagent for testing canine hereditary gastrointestinal neoplasia, which contains a nucleic acid that hybridizes with.
[7] A kit for testing canine hereditary gastrointestinal neoplasia, which comprises the reagent according to [5] and / or the reagent according to [6].
[8] A canine hereditary gastrointestinal neoplasia biomarker consisting of a c.462A> T mutation or a c.463A> T mutation in the APC gene.
In addition, the present invention can also be realized in the following forms.
[1] A step of detecting the following mutation (1) or (2), or both the following mutations (1) and (2) in a nucleic acid sample collected from a dog to be tested. Testing methods for hereditary gastrointestinal neoplasia, including:
(1) C.462A> T mutation of APC gene;
(2) C.463A> T mutation of APC gene.
[2] Hereditary gastrointestinal neoplasia develops when the mutation of (1) or the mutation of (2) is detected, or when both the mutation of (1) and the mutation of (2) are detected. The inspection method according to [1], which determines that the tumor is present.
[3] Risk of developing hereditary gastrointestinal neoplasia when a mutation of (1) or (2) is detected, or when both a mutation of (1) and a mutation of (2) are detected. The inspection method according to [1], which determines that the value is high.
[4] The test method according to any one of [1] to [3], wherein the dog to be tested is a Jack Russell terrier.
[5] A nucleic acid for detecting the c.462A> T mutation of the APC gene, which contains a sequence complementary to a certain region containing the mutation position and specifically hybridizes to the region. Nucleic acid for testing hereditary gastrointestinal neoplasia, including.
[6] A nucleic acid for detecting the c.463A> T mutation of the APC gene, which contains a sequence complementary to a certain region containing the mutation position and specifically hybridizes to the region. Nucleic acid for testing hereditary gastrointestinal neoplasia, including.
[7] A kit for testing hereditary gastrointestinal tract neoplasia, which comprises the reagent according to [5] and / or the reagent according to [6].
[8] A hereditary gastrointestinal neoplasia marker consisting of a c.463A> T mutation or a c.463A> T mutation of the APC gene.

Β-catenin immunostaining image of tumor tissue of JR terrier. The accumulation of β-catenin in tumor tissue was examined. Left: Gastric tubular papillary adenoma (pylorus), Right: Gastric tubular papillary adenocarcinoma (pylorus). Β-catenin immunostaining image of tumor tissue of JR terrier. The accumulation of β-catenin in tumor tissue was examined. Left: Rectal adenoma, Right: Rectal acinar adenocarcinoma. Summary of β-catenin immunostaining results. The percentage of tumor cells (nucleus-positive cells) that showed β-catenin accumulation in the nucleus was scored in 25% increments. Results of sequence analysis of APC genes. Two point mutations were found in exon 4 in the affected individuals.

1. 1. Testing Method for Hereditary Gastrointestinal Tumor Disease The first aspect of the present invention relates to a method for testing hereditary gastrointestinal tumor disease in dogs (hereinafter, may be abbreviated as "testing method of the present invention"). The studies by the present inventors have identified two point mutations in the APC gene as the cause of gastrointestinal neoplasia. The present invention targets the gene mutation as a detection target. Specifically, in the test method of the present invention, with respect to the nucleic acid sample collected from the dog to be tested, the following mutation (1) or (2), or the following mutation (1) and (2) Detect both mutations.
(1) c.462A> T mutation of APC gene (2) c.463A> T mutation of APC gene

According to convention, in the present specification, each mutation position is specified by the number of the translation start codon reference, that is, the position where A of the translation start codon is numbered as the first (1st base).

The mRNA sequence of the wild-type (normal type) APC gene (Gene ID: 479139) is assigned to SEQ ID NO: 1 (NCBI GenBank ACCESSION XM_014111995, DEFINITION PREDICTED: Canis lupus familiaris adenomatous polyposis coli (APC), mRNA.) Is shown in SEQ ID NO: 2 (NCBI GenPept ACCESSION XP_013967470, DEFINITION PREDICTED: LOW QUALITY PROTEIN: adenomatous polyposis coli protein [Canis lupus familiaris].).

The c.462A> T mutation represents the substitution of the APC gene at position 462 (base 533 in the sequence of SEQ ID NO: 1) from A to T. This mutation results in the substitution of amino acid 154 (glutamic acid: Glu) with aspartic acid (Asp) (missense mutation). On the other hand, the c.463A> T mutation represents the substitution of the APC gene at position 463 (base 534 in the sequence of SEQ ID NO: 1) from A to T. This mutation results in the substitution of amino acid 155 (lysine: Lys) with a stop codon (nonsense mutation).

In the present invention, "mutation detection" is performed to clarify whether or not a nucleic acid sample has a specific mutation, that is, whether it is a wild type or a mutant type with respect to a specific base position. Therefore, the detection or measurement for examining the presence or absence of a mutation is expressed as "mutation detection" or "mutation detection" or the like.

In the test method of the present invention, first, a nucleic acid sample collected from a dog to be tested is prepared. Nucleic acid samples can be prepared using known extraction methods and purification methods from blood, saliva, lymph, urine, sweat, skin cells, mucosal cells, hair, tissues collected or excised by surgery, etc. of the dog to be inspected. Genomic DNA of any length can be used as a nucleic acid sample as long as it contains the mutation position to be detected.

The dog to be tested is not particularly limited, but the preferred breed of dog to be tested is JR Terrier. Further, as described later, since the test method of the present invention is useful for diagnosing hereditary gastrointestinal neoplasia, dogs suspected of developing hereditary gastrointestinal neoplasia (typically, gastrointestinal tract). Dogs with symptoms of neoplasia) are suitable dogs to be tested. When a dog suspected of developing hereditary gastrointestinal neoplasia is regarded as a dog to be tested, the test method of the present invention is typically used for diagnosing hereditary gastrointestinal neoplasia.

On the other hand, the test method of the present invention is also useful for determining the risk of developing hereditary gastrointestinal neoplasia (risk diagnosis). Therefore, a healthy dog, that is, a dog that has not developed gastrointestinal neoplasia is also a suitable dog to be tested. When a healthy dog is the dog to be tested, the test method of the present invention is typically used for risk diagnosis of hereditary gastrointestinal neoplasia. The "healthy dog" here is "healthy" in the sense that it has not developed hereditary gastrointestinal neoplasia, and it does not matter whether or not it has developed other diseases.

The means for detecting the mutation is not particularly limited, for example, amplification by the PCR method using an allergen-specific primer (and probe), a method for detecting the mutation of the amplification product by fluorescence or luminescence, or PCR (polymerase chain reaction). PCR-RFLP (restriction fragment length polymorphism) method using the method, PCR-SSCP (single strand conformation polymorphism) method (Orita, M. et al., Proc) . Natl. Acad. Sci., USA, 86, 2766-2770 (1989), etc.), PCR-SSO (specific sequence oligonucleotide) method, ASO combining PCR-SSO method and dot hybridization method (allele specific oligonucleotides) hybridization method (Saiki, Nature, 324, 163-166 (1986), etc.), TaqMan (registered trademark, Roche Molecular Systems) -PCR method (Livak, KJ, Genet Anal) , 14,143 (1999), Morris, T. et al., J. Clin. Microbiol., 34,2933 (1996)), Real-time PCR method, Invader (registered trademark, Third Wave Technologies) method (Lyamichev V) et al., Nat Biotechnol, 17,292 (1999)), Method using FRET (Fluorescence Resonance Energy Transfer) (Heller, Academic Press Inc, pp. 245-256 (1985), Cardullo et al., Proc. Natl. Acad Sci. USA, 85, 8790-8794 (1988), International Publication No. 99/28500, Japanese Patent Application Laid-Open No. 2004-121232, etc.), ASP-PCR (Allele Specific Primer-PCR) method (International Publication No. 01 / 042498 Gazette MALDI-TOF / MS (matrix) method using primer extension method (Haff LA, Smirnov IP, Genome Res 7,378 (1997)), RCA (rolling cycle amplification) method (Lizardi PM et al., Nat Genet 19,225) (1998)), method using DNA chip or microarray (Wang DG et al., Science 280, 1077 (1998), etc.), primer extension method, Southern blot hybridization method, dot hybridization method (Southern, E., A known method such as J. Mol. Biol. 98, 503-517 (1975)) can be adopted. Further, the mutation position to be detected may be directly sequenced. The mutation may be detected by arbitrarily combining these methods. Further, any of the above detection methods can be applied after the nucleic acid sample is amplified in advance (including the amplification of a part of the nucleic acid sample region) by a nucleic acid amplification method such as a PCR method or a method applying the PCR method.

When detecting mutations in a large number of nucleic acid samples, allergen-specific PCR method, allergen-specific hybridization method, TaqMan (registered trademark) -PCR method, Invader method, FRET-based method, ASP-PCR method, primer extension Use a detection method that can detect a large number of samples in a relatively short time, such as the MALDI-TOF / MS (matrix) method using the method, the RCA (rolling cycle amplification) method, or the method using a DNA chip or microarray. Is particularly preferable.

In the above method, nucleic acids such as probes and primers corresponding to each method (also referred to as “mutation detection nucleic acid” in the present invention) are used. Examples of the mutation detection nucleic acid used as a probe include a nucleic acid that specifically hybridizes to a chromosomal region (partial chromosomal region) containing a mutation position to be detected. If the mutation to be detected is a c.462A> T mutation, the probe may be designed by targeting the chromosomal region containing position 462. The same applies to the c.463A> T mutation. The length of the "partial chromosomal region" here is, for example, 16 to 500 bases, preferably 18 to 200 bases, and more preferably 20 to 50 bases. In addition, the nucleic acid preferably has a sequence complementary to a partial chromosomal region, but there may be some mismatch as long as it does not interfere with specific hybridization. The degree of mismatch is 1 to several, preferably 1 to 3, and more preferably 1 to 2. The term "specific hybridization" as used herein refers to hybridization to a target nucleic acid (partial chromosomal region) under hybridization conditions (preferably stringent conditions) that are usually adopted for detection by a nucleic acid probe. It means that while soybeaning, cross-hybridization with other nucleic acids does not occur significantly. Those skilled in the art can easily set the hybridization conditions by referring to, for example, Molecular Cloning (Third Edition, Cold Spring Harbor Laboratory Press, New York).

As an example of a mutation detection nucleic acid used as a primer, it has a sequence complementary to a chromosomal region (partial chromosomal region) containing the mutation position to be detected, and can specifically amplify a DNA fragment containing the mutation portion. Nucleic acids designed as such can be mentioned. As another example of a mutation detection nucleic acid used as a primer, a primer pair designed to specifically amplify a DNA fragment containing the mutation position only when the mutation position to be detected is any base. Can be mentioned. More specifically, it is a primer pair designed to specifically amplify a DNA fragment containing a mutation position to be detected, and includes the mutation position of the antisense strand whose mutation position is any base. A primer pair consisting of a sense primer that specifically hybridizes to a chromosomal region (partial chromosomal region) and an antisense primer that specifically hybridizes to a partial region of the sense strand (region near the mutation position). Can be exemplified. Here, the length of the amplified DNA fragment is appropriately set within a range suitable for its detection, and is, for example, 15 to 1000 bases long, preferably 20 to 500 bases long, and more preferably 30 to 200 bases long. As in the case of the probe, the mutation detection nucleic acid used as a primer also hybridizes specifically to the amplification target (template) and has a sequence that serves as a template as long as the target DNA fragment can be amplified. On the other hand, there may be some mismatch. The degree of mismatch is 1 to several, preferably 1 to 3, and more preferably 1 to 2.

As the mutation detection nucleic acid (probe, primer), DNA, RNA, peptide nucleic acid (PNA: Peptide nucleic acid) or the like is appropriately used depending on the detection method. The base length of the nucleic acid for mutation detection may be any length as long as the function is exhibited, and an example of the base length when used as a probe is 16 to 500 base lengths, preferably 18 to 200 base lengths, and more preferably. It is 20 to 50 bases long. On the other hand, an example of the base length when used as a primer is 10 to 50 base length, preferably 15 to 40 base length, and more preferably 15 to 30 base length.

The mutation detection nucleic acid (probe, primer) can be synthesized by a known method such as a phosphodiester method. Regarding the design and synthesis of nucleic acids for mutation detection, books (for example, Molecular Cloning, Third Edition, Cold Spring Harbor Laboratory Press, New York and Current protocols in molecular biology (edited by Frederick M. Ausubel et al., 1987)). ) Can be referred to.

The nucleic acid for mutation detection in the present invention can be labeled with a labeling substance in advance. By using such a labeled nucleic acid, for example, a mutation can be detected using the labeled amount of the amplification product as an index. Further, if the probe specific for the partial DNA region in the mutant gene and the probe specific for the partial DNA region in the wild-type gene are labeled with different labeling substances, the labeling substance detected from the amplification product and the labeling substance can be detected. The presence or absence of mutation can be determined by the labeling amount.

Labeling substances used for labeling nucleic acids for mutation detection include FAM ^TM (6-carboxyfluorescein), VIC ^TM , TET ^TM (tetrachlorofluorescein), NED ^TM , HEX ^TM , CAL Fluor Gold 540, CAL Fluor Orange 560 (CFO), and CAL. Fluor Red 590, CAL Fluor Red 610, CAL Fluor Red 635, Quasar 570, Quasar 670, Quasar 705, T (JOE ^TM ), 7-AAD, Alexa Fluor® 488, Alexa Fluor® 350, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 633, Alexa Fluor® 647, Cy ^TM 2 , DsRED, EGFP, EYFP, FITC, ^{PerCP TM} , R-Phycoerythrin, Propidium Iodide, AMCA, DAPI, ECFP, MethylCoumarin, Allophycocyanin (APC), Cy ^TM 3, Cy ^TM 5, Rhodamine-123, Tetramethylrhodamine, Texas Red (Texas) Red®), PE, PE-Cy ^TM 5, PE-Cy ^TM 5.5, PE-Cy ^TM 7, APC-Cy ^TM 7, Oregon Green, Carboxyfluorescein, Carboxyfluorescein Diacetate, Quantum Dot Fluorescent dyes such as ³² P, ¹³¹ I, ¹²⁵ I and other radioactive isotopes and biotin can be exemplified, and labeling methods include 5'terminal labeling method using alkaline phosphatase and T4 polynucleotide kinase, T4 DNA polymerase and Klenow fragment. 3'end labeling method, nick translation method, random primer method (Molecular Cloning, Third Edition, Chapter 9, Cold Spring Harbor Laboratory Press, New York), etc. using

The mutation detection nucleic acid can also be used in a state of being immobilized on an insoluble support. If the insoluble support used for immobilization is processed into a chip shape, a bead shape, or the like, mutations can be detected more easily using these immobilized nucleic acids.

The test method of the present invention can be used for a definitive diagnosis (first aspect) and a risk diagnosis (second aspect) of hereditary gastrointestinal neoplasia in dogs.

<Confirmed diagnosis of hereditary gastrointestinal neoplasia (first aspect)>
In this embodiment, if the mutation in (1) (ie, the c.462A> T mutation in the APC gene) or the mutation in (2) (ie, the c.463A> T mutation in the APC gene) is detected, or ( When both the mutation of 1) and the mutation of (2) are detected, it is determined that hereditary gastrointestinal neoplasia has developed. In this way, an objective index, that is, a determination based on the presence or absence of a specific gene mutation is performed. Therefore, in the field of veterinary medicine, an accurate and highly reliable definitive diagnosis can be made for an affected individual, and appropriate treatment can be performed. It also enables evidence-based medical care and contributes to the advancement of veterinary medicine.

<Risk diagnosis (second aspect)>
In this embodiment, hereditary gastrointestinal neoplasia develops when a mutation in (1) or (2) is detected, or when both a mutation in (1) and a mutation in (2) are detected. It is judged that the risk of developing the disease (probability of onset or susceptibility to onset) is high. Similar to the first aspect, in this aspect as well, since the determination is made based on the objective index, accurate and highly reliable risk diagnosis becomes possible. The information provided by this aspect of testing, i.e. risk information, is expected to be used, for example, by animal breeders. As is well known, there are many hereditary diseases in purebreds, so efforts are currently being made to reduce hereditary diseases at the reproductive stage. The dog pedigree certificate issued by JKC (Japan Kennel Club) describes the evaluation results of ancestral dog diseases for typical dog hereditary diseases such as hip dysplasia and elbow dysplasia. It came to be done. In addition, among breeders, it is expected that this approach will be applied to other hereditary diseases in the future, and there is a good possibility that it will be applied to this disease of JR Terrier. Therefore, it is assumed that the inspection method of the present invention is used for the purpose of inspecting or confirming whether or not the individual used for breeding carries an abnormal gene.

On the other hand, as a form of use of information (risk information) brought about by this type of inspection method, use related to pet insurance (animal medical insurance) is also assumed. In recent years, the number of animal pet insurance (animal medical insurance) subscribers has increased, and the market has expanded rapidly, with more than 10 companies currently entering the market. It is possible that having a genetic test for this disease (especially in the case of JR Terrier) is a condition for enrollment.

Risk information is also useful for prevention and early diagnosis of hereditary gastrointestinal neoplasia.

2. Reagents and Kits for Testing Hereditary Gastrointestinal Tumor Disease The second aspect of the present invention provides reagents and kits used for testing canine hereditary gastrointestinal tumor disease. The reagent of the present invention comprises a nucleic acid for detecting the c.462A> T mutation of the APC gene or a nucleic acid for detecting the c.463A> T mutation of the APC gene.

The nucleic acid for mutation detection, which is the reagent of the present invention, is a detection method to which it is applied (method using PCR method using allyl-specific nucleic acid or the like described above, PCR-RFLP method, PCR-SSCP method, TaqMan (registration). Trademark)-The PCR method, Invader (registered trademark) method, DigiTag2 method, etc.) are appropriately designed. The details of the mutation detection nucleic acid are as described above, but a specific example of a mutation detection nucleic acid or a set of mutation detection nucleic acids that can be used as a component of the kit is shown below.
(1) An unlabeled or labeled nucleic acid having a sequence complementary to the chromosomal region (partial chromosomal region) of the APC gene, where the base at the mutation position is T.
(2) An unlabeled or labeled nucleic acid having a sequence complementary to the chromosomal region (partial chromosomal region) of the APC gene, where the base at the mutation position is A.
(3) Combination of nucleic acid of (1) and nucleic acid of (2)
(4) A nucleic acid set designed to specifically amplify a DNA fragment containing the mutation site only when the base at the mutation position is T.
(5) A nucleic acid set designed to specifically amplify a DNA fragment containing the mutation site only when the base at the mutation position is A.
(6) Combination of the nucleic acid set of (4) and the nucleic acid set of (5)
(7) A nucleic acid set designed to specifically amplify a DNA fragment containing a mutation position, wherein the base of the mutation position is T, and the chromosomal region (partial chromosomal region) of the APC gene containing the mutation position. ), And / or the base of the mutation position is A, and specifically hybridizes to the chromosomal region (partial chromosomal region) of the APC gene containing the mutation position. A nucleic acid set consisting of a sense primer and an antisense primer that specifically hybridizes to a region in the vicinity of the partial chromosomal region.

The kit of the present invention contains the reagent of the present invention (nucleic acid for detecting mutation). Both the reagent for the c.462A> T mutation and the reagent for the c.463A> T mutation may be included in the kit. Reagents (DNA polymerase, restriction enzymes, buffers, color-developing reagents, etc.), containers, instruments, etc. required for using the nucleic acid for mutation detection (that is, for detecting mutations) may be included in the kit of the present invention. An instruction manual is usually attached to the kit of the present invention.

Since the gastrointestinal tumor that develops in the JR terrier shows a characteristic pathological condition that is not seen in other dog breeds, it is assumed that it is a dog breed-specific hereditary disease, and humans whose causative gene is the APC gene. We focused on its resemblance to the hereditary disease familial adenomatous polyposis (FAP). FAP is an autosomal dominant inheritance syndrome in which multiple adenomas occur in the large intestine, and if the adenomas are left untreated, almost 100% adenocarcinoma develops. In FAP, mutations in the APC gene are thought to activate the classical Wnt signaling pathway and cause β-catenin accumulation. In normal cells, β-catenin is degraded by these proteins, including APC, and remains low. When Wnt binds to the receptor as a ligant, this degradation is suppressed and β-catenin is not degraded and begins to act as a transcription factor involved in gene expression in the nucleus, resulting in cell proliferation and the like. Thus, in normal cells, transcription is regulated and balanced by Wnt. Here, when a mutation is introduced into APC involved in regulation, the degradation of β-catenin is inhibited, and the transmission pathway is constantly activated even in the absence of a ligand. In addition, this change can be histologically regarded as the accumulation of β-catenin. Therefore, it was examined by immunostaining whether β-catenin accumulation was observed in the tumor of JR terrier. As a result, in adenomas, accumulation in the cytoplasm was remarkable at the same time as localization in the cell membrane observed in normal cells, and accumulation in the nucleus was also observed (Fig. 1, left). In adenocarcinoma, the number of tumor cells (nucleus-positive cells) that accumulated in the nucleus increased further (Fig. 1, right). On the other hand, similar changes were observed in the large intestine (Fig. 2).

As summarized in the table of FIG. 3, only positive cell membranes were observed in hyperplasia, and β-catenin accumulation in the cytoplasm and nucleus was observed in adenomas and adenocarcinomas. In addition, especially in colorectal adenocarcinoma, the positive rate of nuclei was high, and many tumors accounted for 50% or more. These results suggest that mutations in the APC gene are present.

Based on the above results, we decided to analyze the APC gene in affected and non-affected dogs.
A. Method 1. DNA extraction
(1) 2 ml of blood was collected from 5 JR terriers with hereditary gastrointestinal tumors and 6 JR terriers with other diseases.
(2) The collected blood was placed in a blood collection tube (Terumo Corporation) containing EDTA / 2NA (ethylenediaminetetraacetic acid disodium) and subjected to anticoagulant treatment.
(3) DNA was extracted with Wizard® Genomic DNA Purification Kit (Promega).

2. Analysis of DNA base sequence The coding region of the APC gene was analyzed using the DNA sample prepared by the above method. Specifically, the entire coding region of the APC gene was divided into 29 regions, each region was amplified by PCR, and the nucleotide sequence was determined by the direct sequence method. Below, the analysis method of the region containing exon 4 is shown. The same analysis was performed for other regions.

(1) PCR (Polymerase Chain Reaction) was performed using TaKaRa Ex Taq Hot Start Version (Takara Bio Inc.). Using the DNA extracted from blood as a template, the 385 bp region containing exon 4 of the canine APC gene was amplified by PCR. In order to amplify the region containing exon 4 of the canine APC gene by PCR, the forward primer (primer sequence: 5'-agtcccaccttcaaaaatcc-3': SEQ ID NO: 3) is attached to the intron 3, and the reverse primer (primer sequence: 5'-aactaaaaatgcaattatcttgaatg- 3': SEQ ID NO: 4) was designed for intron 4. PCR was performed using Thermal Cycler Dice (Model TP600) (Takara Bio Inc.) under the following conditions.

<Reaction solution>
Taq enzyme 0.15 μl
10X Ex Taq Buffer (Mg ²⁺ plus) 2.0 μl
dNTPs 2.0 μl
Forward primer (15 μM) 0.2 μl
Reverse primer (15 μM) 0.2 μl
Distilled water 14.45 μl
Template 1.0 μl
(Total capacity 20.0 μl)

<Amplification condition>
94 ℃ ・ 5 minutes: 1 cycle
95 ℃ ・ 30 seconds → 58 ℃ ・ 30 seconds → 72 ℃ ・ 30 seconds: 35 cycles
72 ℃ ・ 5 minutes: 1 cycle

(2) The PCR product was electrophoresed on a 3% agarose gel containing ethidium bromide (100 V, 30 minutes).
(3) Using a UV transilluminator (UVP), the agarose gel after electrophoresis was irradiated with ultraviolet rays having a wavelength of 365 nm, and a gel containing 385 bp of DNA was selectively cut out.
(4) DNA was extracted from the cut gel with the QIAquick Gel Extraction Kit (QIAGEN).
(5) Using the Big Dye (registered trademark) Terminator v3.1 Cycle sequencing Kit (Thermo Fisher Scientific), a sequencing reaction was performed using the DNA extracted from the gel as a template. The sequence reaction was carried out using a T100 Thermal Cycler (Bio-Rad) under the following conditions.

<Reaction solution>
Big Dye 1.0 μl
5X Buffer 4.0 μl
Template 5.0 μl
Primer (forward or reverse primer) (1 μM) 5.0 μl
Sterilized distilled water 5.0 μl
(Total capacity 20.0 μl)

<Reaction conditions>
96 ℃ ・ 1 minute: 1 cycle
96 ℃ ・ 10 seconds → 50 ℃ ・ 5 seconds → 60 ℃ ・ 4 minutes: 25 cycles
4 ℃

(6) After the sequence reaction, DNA was purified from the reaction solution.
(7) 10.0 μl of Hi-Di formamide (Thermo Fisher Scientific) was added to the purified DNA, treated with T100 Thermal Cycler (Bio-Rad) for 2 minutes at 95 ° C, and then cooled to 4 ° C.
(8) The base sequence of DNA was analyzed using ABI Prism 3130 Genetic Analyzer (Applied Biosystems).

B. Results As a result of the above analysis, all five JR terriers with gastrointestinal tumors (adenocarcinoma of the stomach or large intestine) had gene mutations (c.462A> T and c.463A> T in exon 4 of the APC gene. ) Was observed (Fig. 4). On the other hand, the above mutation was not observed in 6 JR terriers with diseases other than gastrointestinal tumors.

C. Summary We succeeded in identifying a gene mutation (germline mutation) peculiar to the JR terrier that affected the gastrointestinal tumor, and revealed that this disease is a hereditary disease caused by the APC gene.

According to the present invention, it is possible to make a definitive diagnosis and a risk diagnosis of hereditary gastrointestinal neoplasia in dogs based on an objective index of the presence or absence of a specific gene mutation. The former enables appropriate treatment of affected dogs. On the other hand, the information provided by the latter is useful, for example, in animal breeding and pet insurance. As described above, the present invention can be used in the fields of veterinary medicine and the pet industry.

The present invention is not limited to the description of the embodiments and examples of the above invention. Various modifications are also included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims. The contents of the papers, published patent gazettes, patent gazettes, etc. specified in this specification shall be cited by reference in their entirety.

SEQ ID NO: 3: Description of artificial sequence: Primer SEQ ID NO: 4: Description of artificial sequence: Primer

Claims (7)

Regarding the nucleic acid sample collected from the dog to be tested, the following (1) germline mutation or (2) germline mutation, or the following (1) germline mutation and (2) germline mutation Testing for hereditary gastrointestinal neoplasia in dogs, including the step of detecting both mutations:
(1) C.462A> T mutation of APC gene;
(2) C.463A> T mutation of APC gene. Inherited when (1) germline mutation or (2) germline mutation is detected, or when both (1) germline mutation and (2) germline mutation are detected The test method according to claim 1, wherein it is determined that a germline gastrointestinal tumor has developed. Inherited when (1) germline mutation or (2) germline mutation is detected, or when both (1) germline mutation and (2) germline mutation are detected The test method according to claim 1, wherein the risk of developing germline gastrointestinal neoplasia is determined to be high. The inspection method according to any one of claims 1 to 3, wherein the dog to be inspected is a Jack Russell terrier. A germline mutation, a nucleic acid for detecting a c.462A> T mutation in the APC gene, which contains a sequence complementary to a certain region containing the mutation position and specifically hybridizes to the region. A test reagent for hereditary gastrointestinal neoplasia in dogs, which contains nucleic acid to soy. A germline mutation, a nucleic acid for detecting a c.463A> T mutation in the APC gene, which contains a sequence complementary to a certain region containing the mutation position and specifically hybridizes to the region. A test reagent for hereditary gastrointestinal neoplasia in dogs, which contains nucleic acid to soy. A kit for testing canine hereditary gastrointestinal neoplasia, comprising the reagent according to claim 5 and / or the reagent according to claim 6.

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