WO2015129018A1 - Méthode et kit pour le diagnostic du glaucome chez les chiens - Google Patents

Méthode et kit pour le diagnostic du glaucome chez les chiens Download PDF

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WO2015129018A1
WO2015129018A1 PCT/JP2014/055019 JP2014055019W WO2015129018A1 WO 2015129018 A1 WO2015129018 A1 WO 2015129018A1 JP 2014055019 W JP2014055019 W JP 2014055019W WO 2015129018 A1 WO2015129018 A1 WO 2015129018A1
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polymorphism
seq
canine
dna
glaucoma
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English (en)
Japanese (ja)
Inventor
正樹 今安
水木信久
目黒明
信行 印牧
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Menicon Co Ltd
School Corp Azabu Veterinary Medicine Educational Inst
Yokohama City University
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Menicon Co Ltd
School Corp Azabu Veterinary Medicine Educational Inst
Yokohama City University
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Priority to PCT/JP2014/055019 priority Critical patent/WO2015129018A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to a method and kit for diagnosing glaucoma in dogs.
  • Glaucoma in dogs is a disease in which the flow of aqueous humor flowing between the cornea and the lens is impaired, an increase in intraocular pressure occurs, the retina and optic nerve are compressed, and visual impairment such as visual field stenosis is caused.
  • ⁇ There is no prevention method for glaucoma, but if it can be detected early, there is a method to suppress the progression of the disease. For example, administration of eye drops (miotics, ⁇ -adrenergic blockers, prostaglandin-related drugs, etc.) and oral drugs (osmotic diuretics, carbonic anhydrase inhibitors, etc.), to reduce production of aqueous humor Surgery, surgery to increase aqueous humor outflow, etc.
  • eye drops miotics, ⁇ -adrenergic blockers, prostaglandin-related drugs, etc.
  • oral drugs osmotic diuretics, carbonic anhydrase inhibitors, etc.
  • canine glaucoma has been diagnosed by measuring intraocular pressure, gonioscopic examination, and fundus examination.
  • Non-patent Document 1 the search for a disease susceptibility gene related to canine glaucoma has been advanced, and Kuchtey et al. Have reported a Gly661Arg variant in the ADAMTS10 gene as a candidate for a glaucoma disease susceptibility gene in beagle dogs (Non-patent Document 1). It has been reported that the mutation was not found in breeds other than beagle dogs (Non-patent Document 3). Mizuki et al. Have reported a plurality of SNPs in the SRBD1 gene as glaucoma susceptibility genes common to dogs and humans (Non-patent Document 2, Patent Document 1).
  • An object of the present invention is to find a disease susceptibility gene effective for diagnosis of glaucoma in dogs and to provide a method for using the gene.
  • the present inventors conducted SNP analysis on glaucoma dog populations and healthy dog populations, and found SNPs that are particularly effective for the diagnosis of glaucoma.
  • the present invention has been completed based on these findings.
  • the gist of the present invention is as follows. (1) A single nucleotide polymorphism present on the canine ADAMTS10 gene, the polymorphism in the 901st site of the DNA sequence represented by SEQ ID NO: 1 and / or a polymorphism in linkage disequilibrium with the polymorphism A method for examining canine glaucoma, comprising identifying a type.
  • a reagent for examining canine glaucoma comprising at least one component selected from the group consisting of the following components (a) and (b): (a) A single nucleotide polymorphism present on the canine ADAMTS10 gene, which is a polymorphism in the 901st site of the DNA sequence represented by SEQ ID NO: 1 and / or a linkage disequilibrium with the polymorphism Primer that can amplify the region containing the type (b) A single nucleotide polymorphism present on the canine ADAMTS10 gene, the polymorphism at the 901st site of the DNA sequence represented by SEQ ID NO: 1 and / or a polymorphism in linkage disequilibrium with the polymorphism
  • the probe according to (2) wherein a probe capable of hybridizing to a region containing a mold (3) is immobilized on a solid phase.
  • a test kit for canine glaucoma comprising the reagent according
  • the present invention makes it possible to diagnose canine glaucoma earlier and more accurately. Individuals who have already developed a definitive diagnosis, and can be actively treated. In addition, it is possible to predict the onset of an unaffected individual, and it is recommended to conduct examinations frequently, leading to early detection.
  • the present invention is a single nucleotide polymorphism existing on the canine ADAMTS10 gene, and is in a state of linkage disequilibrium with the polymorphism at the 901st site of the DNA sequence represented by SEQ ID NO: 1 and / or the polymorphism.
  • a method for examining canine glaucoma comprising identifying a polymorphism is provided.
  • a single nucleotide polymorphism present on the canine ADAMTS10 gene, the polymorphism at the 901st site of the DNA sequence represented by SEQ ID NO: 1 is SNP (53096346, Val658Val) (CanFam3.1) on the canine ADAMTS10 gene It is.
  • This single nucleotide polymorphism (SNP) exists at position number 53096346 of canine chromosome 20.
  • the single nucleotide polymorphism present on the canine ADAMTS10 gene which is in linkage disequilibrium with the polymorphism at the 901st site of the DNA sequence represented by SEQ ID NO: 1, is represented by the DNA represented by SEQ ID NO: 1.
  • the polymorphism in the LD block of the polymorphism at the 901st position of the sequence is good.
  • the polymorphism that is in linkage disequilibrium with the polymorphism at the 901st site of the DNA sequence represented by SEQ ID NO: 1 is within the region containing the polymorphism at the 901st site of the DNA sequence represented by SEQ ID NO: 1.
  • polymorphisms may have p-values (an indicator of significant differences between glaucoma and healthy individuals) less than 0.05.
  • a single nucleotide polymorphism present on the canine ADAMTS10 gene which is in linkage disequilibrium with the polymorphism at position 901 of the DNA sequence represented by SEQ ID NO: 1, canine chromosome 20 position number 53097497 SNP (rs22878605) and the like can be exemplified, but not limited thereto.
  • the polymorphism in linkage disequilibrium with the polymorphism in the 901st site of the DNA sequence represented by SEQ ID NO: 1 is, for example, a polymorphism having a larger D ′ between these SNPs.
  • LD block is the method of Gabriel et al. Using Haploview software (Barrett JC, Fry B, Maller J, Daly MJ. Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics. 2005; 21 (2): 263-265.) (Gabriel SB, Schaffner SF, Nguyen H, et al. The structure of haplotype blocks in the human genome. Science. 2002; 296 (5576): 2225-2229.).
  • test for glaucoma is a test for determining whether or not a test individual is highly likely to suffer from glaucoma, and if the test individual already suffers from glaucoma It is a concept that includes a test for making a definitive diagnosis.
  • the polymorphism at position 901 of the DNA sequence represented by SEQ ID NO: 1 is a polymorphism of adenine (A) / guanine (G) at position 53096346 in canine chromosome 20, and the base at this position is AA is judged as non-risk, AG is judged as heterozygous, and GG is judged as risk.
  • the SNP to be identified may be one type or may be combined with other SNPs.
  • the sense strand of the gene may be analyzed, or the antisense strand may be analyzed.
  • the polymorphism at position 901 of the DNA sequence represented by SEQ ID NO: 1 is present in exon 15 of the canine ADAMTS10 gene and is a mutation without amino acid substitution, but is in linkage disequilibrium with this polymorphism.
  • Some polymorphisms exist in exons of genes, in regions that control gene expression (eg, promoter regions, enhancer regions, etc.), introns of genes, or in regions before or after linkage disequilibrium with these genes. sell.
  • Examples of polymorphisms include single nucleotide polymorphisms, polymorphisms in which one to several tens of bases (sometimes several thousand bases) are substituted, deleted, inserted, transferred, or inverted. There is no particular limitation.
  • the identification of the base at the polymorphic site can be performed by a known single nucleotide polymorphism analysis method.
  • single nucleotide polymorphism analysis methods include sequence analysis, PCR, PCR-SSCP, hybridization, RFLP method, Taqman-PCR method, invader method, cycleave-PCR method, HRM method, etc. It is not limited.
  • genomic DNA may be extracted from the biological sample of the subject individual.
  • Biological samples include, for example, blood, skin, oral mucosa, tissues or cells collected or excised by surgery, body fluids collected for the purpose of examination (saliva, lymph, airway mucosa, semen, sweat, urine, etc.) ) Etc.
  • the biological sample is preferably whole blood collected from a vein such as one of the extremities or the neck.
  • Genomic DNA can be extracted from a biological sample using a commercially available DNA extraction kit. Then, if necessary, DNA containing the polymorphic site is isolated.
  • the DNA can be isolated by PCR or the like using genomic DNA or RNA as a template, using a primer capable of hybridizing to DNA containing a polymorphic site.
  • Canine glands that are subject to glaucoma testing include canine tumors that are susceptible to glaucoma, such as Syvine, Shih Tzu, American Cocker Dogl, Miniature Ducks, Border Collie, Sheepdog, Chihuahua, Dachshund, Rubrador Retriever, Maltese, Examples include, but are not limited to, miniature pinschers, pugs, and corgis.
  • the present invention also provides a reagent for examining canine glaucoma, comprising at least one component selected from the group consisting of the following components (a) and (b).
  • a single nucleotide polymorphism present on the canine ADAMTS10 gene which is a polymorphism in the 901st site of the DNA sequence represented by SEQ ID NO: 1 and / or a linkage disequilibrium with the polymorphism Primer that can amplify the region containing the type
  • the polymorphism at the 901st site of the DNA sequence represented by SEQ ID NO: 1 and the polymorphism in linkage disequilibrium with the polymorphism are as described above. .
  • the present invention provides a test kit for canine glaucoma comprising the reagent described above.
  • the primer and probe that are components of the reagent of the present invention may be an oligonucleotide having a chain length of at least 15 nucleotides.
  • the oligonucleotide When used as a primer, its length is usually 15 to 100 bp, preferably 17 to 30 bp.
  • the primer is not particularly limited as long as it can amplify at least a part of the DNA containing the polymorphic site.
  • the length of DNA that can be amplified by the primer is usually 15 to 1000 bp, preferably 20 to 500 bp, more preferably 20 to 200 bp.
  • the length is usually 5 bp to 200 bp, preferably 7 bp to 100 bp, more preferably 7 bp to 50 bp.
  • the probe is not particularly limited as long as it can hybridize with the DNA containing the polymorphic site.
  • a primer capable of amplifying a region containing a polymorphic site is preferably one that can initiate complementary strand synthesis toward the polymorphic site using a DNA containing the polymorphic site as a template.
  • primer which is a component of the reagent of the present invention
  • the following primer pair consisting of a forward primer and a reverse primer can be mentioned.
  • the base sequence of SEQ ID NO: 2 is the same sequence as the 765th to 784th base sequences in the DNA sequence represented by SEQ ID NO: 1.
  • the base sequence of SEQ ID NO: 3 is a sequence complementary to the 1083th to 1102nd base sequences in the DNA sequence represented by SEQ ID NO: 1.
  • the base sequence of SEQ ID NO: 4 is the same sequence as the 766th to 784th base sequences in the DNA sequence represented by SEQ ID NO: 1.
  • the base sequence of SEQ ID NO: 5 is a sequence complementary to the 1015th to 1033rd base sequences in the DNA sequence represented by SEQ ID NO: 1.
  • the primer has a base sequence that can hybridize to a region containing a polymorphic site, but is the same or complementary to a region shifted by several bases from a region containing a polymorphic site that is identical or complementary to the base sequence of SEQ ID NOs: 2 to 5 It may be a typical arrangement.
  • an arbitrary base sequence can be added to the primer.
  • a primer for a polymorphism analysis method using a type IIs restriction enzyme a primer to which a recognition sequence for a type IIs restriction enzyme is added is used.
  • the primer may be modified.
  • a primer labeled with a fluorescent substance or a binding affinity substance such as biotin or digoxin may be used.
  • the probe that can hybridize to the region containing the polymorphic site may be any probe that can hybridize to the polynucleotide having the base sequence of the region containing the polymorphic site.
  • Those that specifically hybridize to DNA having the base sequence of the region to be included are preferred.
  • “specifically hybridizes” means normal hybridization conditions, preferably stringent hybridization conditions (for example, Sambrook et al., Molecular® Cloning, Cold® Spring® Harbor® Laboratory® Press, New® York, USA, In the condition described in the second edition 1989), it means that cross-hybridization does not occur significantly with DNA other than DNA having the base sequence of the region containing the polymorphic site.
  • a probe containing a polymorphic site in the base sequence of the probe is preferable.
  • the probe may be designed so that the end of the probe corresponds to a base adjacent to the polymorphic site. Therefore, although the polymorphic site is not included in the base sequence of the probe itself, a probe including a base sequence complementary to the region adjacent to the polymorphic site can also be shown as a desirable probe in the present invention.
  • the probe is allowed to modify the base sequence, add the base sequence, or modify the same as the primer.
  • a probe used for the Invader method is added with a base sequence unrelated to the genome constituting the flap.
  • Such a probe is also included in the probe of the present invention as long as it hybridizes to a region containing a polymorphic site.
  • the base sequence constituting the probe of the present invention can be designed according to the analysis method based on the base sequence of the DNA region surrounding the polymorphic site of the present invention in the genome. Examples of the probe that is a component of the reagent of the present invention include Probe-1 and Probe-2 as follows.
  • Probe-1 5'-gccgt (A) gtg-3 '
  • Probe-2 5'-gccgt (G) gt-3 '
  • the base sequence of Probe-1 is the same sequence as the base sequence of the 896th to 904th positions (the 901st base is A) in the DNA sequence represented by SEQ ID NO: 1.
  • the base sequence of Probe-2 is the same sequence as the base sequence of the 896th to 903th (the 901st base is G) in the DNA sequence represented by SEQ ID NO: 1.
  • a person skilled in the art can design primers and probes according to the analysis method based on the base sequence information about the surrounding DNA region including the polymorphic site.
  • the base sequences constituting the primers and probes can be modified as appropriate as well as the base sequences that are completely complementary to the genomic base sequences. Since the polymorphism at the 901st base (polymorphic site) in the polynucleotide consisting of the DNA sequence represented by SEQ ID NO: 1 is a polymorphism that is not found in beagle dogs, beagle glaucoma groups and healthy groups Glaucoma can be detected in many types of dogs, including beagle dogs, by combining with genetic polymorphisms that are statistically significantly different from each other.
  • SNP 53096339, Gly661Arg
  • CanFam3.1 CanFam3.1
  • SNP (53096339, Gly661Arg) (CanFam3.1) on the canine ADAMTS10 gene is a polymorphism of adenine (A) / guanine (G) at the position of position number 53096339 of canine chromosome 20, and the base at this position is AA is judged as non-risk, AG is judged as heterozygous, and GG is judged as risk.
  • primers that can amplify a region containing SNP (53096339, Gly661Arg) (CanFam3.1) on the canine ADAMTS10 gene include the following primer pairs consisting of a forward primer and a reverse primer.
  • the base sequence of SEQ ID NO: 6 is the same sequence as the 765th to 783th base sequences in the DNA sequence represented by SEQ ID NO: 8.
  • the base sequence of SEQ ID NO: 7 is a sequence complementary to the 1019th to 1035th base sequences in the DNA sequence represented by SEQ ID NO: 8. Examples of probes that can hybridize to a region containing SNP (53096339, Gly661Arg) (CanFam3.1) on the canine ADAMTS10 gene include Probe-11 and Probe-12 as follows.
  • Probe-11 5'-gtggac (A) gg-3 '
  • Probe-12 5'-tggac (G) gga-3 '
  • the base sequence of Probe-11 is the same sequence as the base sequence of the 902st to 910th positions (the 908th base is A) in the DNA sequence represented by SEQ ID NO: 8.
  • the base sequence of Probe-12 is the same sequence as the base sequence of the 903th to 911th positions (the 908th base is G) in the DNA sequence represented by SEQ ID NO: 8.
  • Primers and probes can be synthesized by any method based on the base sequences constituting them.
  • a technique for synthesizing an oligonucleotide having the base sequence based on the given base sequence is known.
  • any modification can be introduced into the oligonucleotide using a nucleotide derivative modified with a fluorescent dye (FAM, ROX, etc.) or biotin.
  • FAM fluorescent dye
  • ROX ROX
  • biotin biotin.
  • a method of binding a fluorescent dye or the like to a synthesized oligonucleotide is also known.
  • the probe may be fixed on a solid phase (DNA array).
  • sample DNA or RNA
  • RNA is hybridized to a large number of probes arranged on the same plane, and the hybridization to each probe is detected by scanning the plane. Since responses to many probes can be observed simultaneously, for example, a DNA array is useful for analyzing a large number of polymorphic sites simultaneously.
  • nucleotide immobilization (array) methods include arrays based on oligonucleotides developed by Affymetrix. In an array of oligonucleotides, the oligonucleotides are usually synthesized in situ. For example, in-situ synthesis methods of oligonucleotides by lithography method (Affymetrix), inkjet method (Agilent), bead array method (Illumina), etc. are known.
  • Oligonucleotide is composed of a base sequence complementary to a region containing a polymorphic site to be detected.
  • the length of the nucleotide probe to be bound to the substrate is usually 8 to 100 bp, preferably 8 to 50 bp, more preferably 8 to 25 bp when the oligonucleotide is immobilized.
  • a sample for SNP detection by the DNA array method can be prepared by a method well known to those skilled in the art based on a biological sample collected from a test individual.
  • the biological sample is not particularly limited.
  • a DNA sample can be prepared from genomic DNA extracted from tissues or cells of blood, skin, oral mucosa, etc., tears, saliva, urine, feces, or hair of a subject.
  • a specific region of genomic DNA is amplified using a primer for amplifying a region containing a polymorphic site to be determined.
  • a plurality of regions can be simultaneously amplified by the multiplex PCR method.
  • the multiplex PCR method is a PCR method using a plurality of primer sets in the same reaction solution. When analyzing multiple polymorphic sites, the multiplex PCR method is useful.
  • a DNA sample is amplified by the PCR method and the amplified product is labeled.
  • a labeled primer is used for labeling the amplification product.
  • genomic DNA is first amplified by PCR using a primer set specific to the region containing the polymorphic site.
  • biotin-labeled DNA is synthesized by a labeling PCR method using a biotin-labeled primer.
  • the biotin-labeled DNA synthesized in this way is hybridized to the oligonucleotide probe on the chip.
  • the hybridization reaction solution and reaction conditions can be appropriately adjusted according to conditions such as the length of the nucleotide probe immobilized on the solid phase and the reaction temperature.
  • One skilled in the art can design appropriate hybridization conditions.
  • avidin labeled with a fluorescent dye is added.
  • the array is analyzed with a scanner, and the presence or absence of hybridization is confirmed using fluorescence as an index.
  • An example of the procedure for carrying out the test method of the present invention using the DNA array method is as follows. After preparing a solid phase on which a DNA and nucleotide probe containing a polymorphic site prepared from a test individual are immobilized, The solid phase is contacted. Subsequently, the base species of the polymorphic site is determined by detecting DNA hybridized to the nucleotide probe immobilized on the solid phase.
  • solid phase means a material capable of immobilizing nucleotides.
  • the solid phase is not particularly limited as long as nucleotides can be immobilized, and specific examples include a solid phase containing microplate wells, plastic beads, magnetic particles, a substrate, and the like.
  • a substrate generally used in DNA array technology can be preferably used.
  • the “substrate” means a plate-like material capable of fixing nucleotides.
  • the nucleotide includes oligonucleotides and polynucleotides.
  • an allele-specific oligonucleotide (Aligonucleotide / ASO) hybridization method can be used to detect a base at a specific site.
  • An allele-specific oligonucleotide (ASO) is composed of a base sequence that hybridizes to a region where a polymorphic site to be detected exists.
  • ASO is hybridized to sample DNA, the hybridization efficiency decreases if a mismatch occurs at the polymorphic site due to the polymorphism.
  • Mismatches can be detected by Southern blotting or a method that uses the property of quenching by intercalating a special fluorescent reagent into the hybrid gap. Mismatches can also be detected by the ribonuclease A mismatch cleavage method.
  • the reagents and kits of the present invention can contain various enzymes, enzyme substrates, buffers, and the like depending on the base identification method.
  • the enzyme include enzymes necessary for the various analysis methods exemplified as the base identification method, such as DNA polymerase, DNA ligase, or IIs restriction enzyme.
  • the buffer solution a buffer solution suitable for maintaining the activity of the enzyme used for these analyzes is appropriately selected.
  • the enzyme substrate for example, a substrate for complementary strand synthesis is used.
  • a control in which the base at the polymorphic site is clear can be attached to the reagent and kit of the present invention.
  • genomic DNA or a fragment of genomic DNA in which the base type of the polymorphic site is known in advance can be used.
  • Genomic DNA extracted from cells may be attached as a control, or a cell or a fraction of cells may be attached as a control, and a user may extract genomic DNA therefrom. If a cell is used as a control, the result of the control can prove that the genomic DNA extraction operation was performed correctly.
  • DNA comprising a base sequence containing a polymorphic site can be used as a control.
  • a YAC vector or a BAC vector containing a genome-derived DNA whose base type at the polymorphic site has been clarified may be used as a control.
  • a vector in which only tens to hundreds of bp corresponding to the polymorphic site are excised and inserted can be used as a control.
  • Example 1 Diagnosis of canine glaucoma and DNA purification 1) A total of 406 animals including 90 Shivaine, 115 Shih Tzu, 41 Beagle, 46 American Cocker Dogl, 47 Miniature Dachshunds, 39 Border Collies, 28 Sheep Dogs who visited Azabu University Hospital . 2) Benoxeal was instilled into both eyes and local anesthesia was performed. 3) The intraocular pressure in both eyes was measured with tonopen, and individuals with an intraocular pressure of 25 mmHg or higher were determined to be glaucoma, and individuals with an intraocular pressure of less than 25 mmHg were determined to be healthy.
  • CycleavePCR was performed to reconfirm the two SNP genotypes of the canine ADAMTS gene. The conditions of CycleavePCR are shown below.
  • ⁇ Reagent> CycleavePCR Reaction Mix (Takara Bio) 12.5 ⁇ L Forward Primer (10 ⁇ M) (Takara Bio) 0.5 ⁇ L Reverse Primer (10 ⁇ M) (Takara Bio) 0.5 ⁇ L Probe-1 (5 ⁇ M) (Takara Bio) 1 ⁇ L Probe-2 (5 ⁇ M) (Takara Bio) 1 ⁇ L Template 1 ⁇ L dH 2 O 8.5 ⁇ L Total volume 25 ⁇ L ⁇ CycleavePCR conditions> (95 °C 10 seconds) 1 cycle (95 °C 5 seconds, 55 °C 10 seconds, 72 °C 20 seconds) 45 cycles 13) SNP was analyzed with the analysis software (Thermal Cycler Dice Real Time System Ver 3.00) attached to the real-time PCR device (Takara Bio TP800).
  • Canine ADAMTS10 gene SNP (53096346, Val658Val) (CanFam3.1), when FAM fluorescence intensity is strong, non-risk type (non-risk), when ROX fluorescence intensity is strong, risk type (risk), The case where both fluorescence intensities were strong was judged as a hetero type (hetero).
  • Canine ADAMTS10 gene (53096339, Gly661Arg) (CanFam3.1), when ROX fluorescence intensity is strong, non-risk type (non-risk), when FAM fluorescence intensity is strong, risk type (risk), both The case where the fluorescence intensity was strong was determined to be hetero.
  • ADAMTS10 Gly661Arg
  • the risk allele frequency in the glaucoma group and the healthy group were both 0%, and this SNP did not exist, but in Beagle, the risk allele frequency in the glaucoma group was 50.0%, The risk allele frequency in the healthy group was 21.8%, and when the two groups were compared, the risk of glaucoma when holding the risk allele was 4.57 times higher.
  • the present invention can be used for veterinary medicine, diagnosis, and the like.
  • SEQ ID NO: 2 shows the base sequence of Forward Primer used for polymorphic site detection (sequence) of SNP (53096346, Val658Val) (CanFam3.1) on canine ADAMTS10 gene.
  • SEQ ID NO: 3 shows the base sequence of Reverse Primer used for polymorphic site detection (sequence) of SNP (53096346, Val658Val) (CanFam3.1) on canine ADAMTS10 gene.
  • SEQ ID NO: 4 shows the base sequence of Forward Primer used for polymorphic site detection (real-time PCR) of SNP (53096346, Val658Val) (CanFam3.1) on canine ADAMTS10 gene.
  • SEQ ID NO: 5 shows the nucleotide sequence of Reverse Primer used for polymorphic site detection (real-time PCR) of SNP (53096346, Val658Val) (CanFam3.1) on canine ADAMTS10 gene.
  • SEQ ID NO: 6 shows the base sequence of Forward Primer used for polymorphic site detection (real-time PCR) of SNP (53096339, Gly661Arg) (CanFam3.1) on canine ADAMTS10 gene.
  • SEQ ID NO: 7 shows the base sequence of Reverse Primer used for polymorphic site detection (real-time PCR) of SNP (53096339, Gly661Arg) (CanFam3.1) on canine ADAMTS10 gene.
  • SEQ ID NO: 8> SEQ ID NO: 8 shows a 1801-base long base sequence containing the polymorphic site of SNP (53096339, Gly661Arg) (CanFam3.1) on the canine ADAMTS10 gene at the 908th position (r A / G).

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Abstract

　L'invention concerne un gène de susceptibilité au glaucome canin et un procédé d'utilisation de celui-ci. Par analyse et comparaison du polymorphisme mononucléotidique chez les chiens atteints de glaucome et un chien normal, on a découvert qu'un SNP (53096346, Val658Val) (CanFam3.1) sur le gène canin ADAMTS10 nouvellement découvert est efficace dans le diagnostic.
PCT/JP2014/055019 2014-02-28 2014-02-28 Méthode et kit pour le diagnostic du glaucome chez les chiens Ceased WO2015129018A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020158503A1 (fr) * 2019-01-31 2020-08-06 株式会社シード Procédé permettant de déterminer le risque d'apparition de la myopie

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