WO2012070510A1 - Procédé pour le test du risque d'infarctus cardiaque - Google Patents

Procédé pour le test du risque d'infarctus cardiaque Download PDF

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WO2012070510A1
WO2012070510A1 PCT/JP2011/076738 JP2011076738W WO2012070510A1 WO 2012070510 A1 WO2012070510 A1 WO 2012070510A1 JP 2011076738 W JP2011076738 W JP 2011076738W WO 2012070510 A1 WO2012070510 A1 WO 2012070510A1
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risk
polymorphism
alleles
nucleic acid
repeats
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横田　充弘
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Yokota Mitsuhiro
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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 risk test method for myocardial infarction, and a reagent and kit used for the risk test method.
  • Myocardial infarction is the most mortal disease in Western countries, even if not fatal, preventing heart failure, angina pectoris, refractory arrhythmia and significantly reducing the patient's quality of life It goes without saying that it is important to do. Not only environmental factors but also genetic factors have a great influence on the onset of myocardial infarction. If a myocardial infarction susceptibility gene can be identified, it will greatly contribute to the prevention of myocardial infarction. So far, gene loci that are associated with myocardial infarction have been found (see, for example, Non-Patent Document 1).
  • An object of the present invention is to provide a highly accurate and clinically useful myocardial infarction risk test method (determination method for susceptibility), and reagents and kits used therefor.
  • susceptibility genes for lifestyle-related diseases do not contribute significantly to the onset of the disease (odds ratio ⁇ 2), and there are multiple susceptibility genes, so identification is not easy. Furthermore, genetic mutations with a relatively high frequency in the population are not necessarily involved in disease development (common disease common variant hypothesis). disease rare variant hypothesis). Therefore, for the identification of susceptibility genes for lifestyle-related diseases, candidate gene association analysis, genome-wide linkage analysis (GWLS), and large-scale genome-wide association analysis (GWAS) were used for large-scale populations with excellent quality and quantity. A multifaceted approach is necessary.
  • the present inventors performed a genome-wide linkage analysis (GWLS) capable of measuring 660,000 SNPs covering the entire chromosomal region for sibling pairs affected by coronary artery disease. Specific linkage region was found on chromosome 2. Next, high-density SNP-related analysis (case-control-related analysis) was performed on the linked region. As a result, a group of SNPs located in the vicinity of the ALMS1 gene was found to be significantly related, indicating the presence of a myocardial infarction sensitive region. Subsequently, re-sequencing of the ALMS1 region and a large-scale association analysis were performed on some polymorphisms of the ALMS1 region.
  • GWLS genome-wide linkage analysis
  • a risk test method for myocardial infarction including a step of detecting a genetic polymorphism of any of the following (1) to (5) for a nucleic acid sample collected from a subject: (1) a polymorphism of a region encoding a glutamate repeat in the ALMS1 gene; (2) single nucleotide polymorphism identified by the registration number rs2037814 in the SNP database of the National Center for Biotechnology Information (NCBI); (3) Single nucleotide polymorphism identified by registration number rs7598901 in the SNP database of the National Center for Biotechnology Information (NCBI); (4) Single nucleotide polymorphism identified by registration number rs1052161 in the SNP database of the National Center for Biotechnology Information (NCBI); (5) Single nucleotide polymorphism identified by the registration number rs6706562 in the SNP database of the National Center for Biotechnology Information (NCBI).
  • a reagent for examining the risk of myocardial infarction which contains any of the following nucleic acids (i) to (v): (i) a nucleic acid for detecting a polymorphism in a region encoding a glutamate repeat in the ALMS1 gene, comprising a sequence complementary to a certain region including the polymorphic site, and specifically hybridizing to the region Soy nucleic acid; (ii) a nucleic acid for detecting a single nucleotide polymorphism identified by the registration number rs2037814 in the SNP database of the National Center for Biotechnology Information (NCBI), and comprising a sequence complementary to a certain region containing the polymorphic site A nucleic acid that specifically hybridizes to said region; (iii) A nucleic acid for detecting a single nucleotide polymorphism identified by the registration number rs7598901 in the SNP database of the National Center for Biotechnology Information (NCBI), and comprising a sequence
  • MI myocardial infarction patient
  • OR odds ratio
  • CI confidence interval.
  • Glutamate repeat polymorphism (E repeat polymorphism), rs10191517 polymorphism and rs6720094 polymorphism showing significantly lower P value in the SNP group in linkage disequilibrium block of ALMS1 region, and rs2037814 polymorphism, rs7598901 polymorphism and rs1052161 Result of linkage disequilibrium analysis of polymorphism.
  • the first aspect of the present invention relates to a risk test method for myocardial infarction.
  • “risk of myocardial infarction” refers to the degree of risk (susceptibility) of causing myocardial infarction. Therefore, according to the risk test method of the present invention, it is possible to determine and evaluate the susceptibility of myocardial infarction.
  • the present invention is used for risk determination / evaluation of juvenile myocardial infarction.
  • a “juvenile myocardial infarction” refers to a myocardial infarction that develops before the age of 50 for men and less than 55 for women. Therefore, in the case of this embodiment, a male person under 50 years old and a female person under 55 years old are subjects of the present invention.
  • a specific gene polymorphism is detected in a nucleic acid sample collected from a subject, and the risk is determined and evaluated based on the detection result. Details of the gene polymorphism to be detected will be described later.
  • a nucleic acid sample collected from the subject In the present invention, a nucleic acid sample derived from a person (subject) who needs to determine the risk of myocardial infarction is used.
  • the nucleic acid specimen can be prepared from a subject's blood, saliva, lymph, urine, sweat, skin cells, mucosal cells, hair and the like using a known extraction method and purification method. As long as it contains a polymorphic site to be detected, genomic DNA of any length can be used as a nucleic acid sample. Alternatively, mRNA containing a region corresponding to the polymorphic site or its complementary DNA (cDNA) may be used as the nucleic acid sample.
  • cDNA complementary DNA
  • mRNA which is a transcription product of genomic DNA containing the polymorphic site to be detected
  • cDNA is prepared by reverse transcription.
  • the sequence of the polymorphic portion of the genomic DNA can be determined.
  • the subject is not particularly limited. That is, the present invention can be widely applied to those who need to determine the risk of myocardial infarction.
  • a patient with myocardial infarction, a person suspected of suffering from myocardial infarction, or a healthy person is the subject.
  • the determination results of the former two are useful for determining a more appropriate treatment policy, and promote improvement of treatment effect and improvement of patient's QOL (Quality of Life).
  • the determination result for a healthy person is useful for prevention or early diagnosis of myocardial infarction. That is, if preventive measures and lifestyle improvements are made based on information that the risk is high, the possibility of occurrence of myocardial infarction (morbidity) can be reduced.
  • the “healthy person” means a person who has not been determined to be suffering from myocardial infarction at the time of applying the risk test method of the present invention.
  • the scope of application of the present invention is not limited to Japanese. That is, the present invention can be applied to non-Japanese mongoloids and other races (such as cocaside). However, in consideration of the fact that polymorphism types and frequencies tend to show the same tendency in genetically close populations (for example, Chinese population and Korean population are close to Japanese population)
  • the subject is preferably a Mongoloid (Japanese, Chinese, Korean, etc.), more preferably a Japanese.
  • any one of the following polymorphisms (1) to (5) is detected.
  • Polymorphism in the region encoding glutamate repeat (repeated glutamate) in the ALMS1 gene (hereinafter also referred to as “glutamate repeat polymorphism”)
  • Single nucleotide polymorphism identified by rs2037814 (hereinafter also referred to as “rs2037814 polymorphism”)
  • Single nucleotide polymorphism identified by rs7598901 hereinafter also referred to as “rs7598901 polymorphism”
  • Single nucleotide polymorphism identified by rs1052161 (hereinafter also referred to as “rs1052161 polymorphism”)
  • Single nucleotide polymorphism identified by rs6706562 (hereinafter also referred to as “rs6706562 polymorphism”)
  • Rs numbers in (2) to (5) are registered numbers in the SNP database of the National Center for Biotechnology Information (NCBI).
  • NCBI National Center for Biotechnology Information
  • the following records in the database namely rs72319667, rs66480235, rs72293473, rs72445008, rs36223922, rs66908601, rs3074417, rs72325073, rs61156725, rs70965731, rs55889738, rs13009043, rs72525315, rs13009604, and rs13009609 are all glutamic acid repeat polytypes. Is.
  • All the polymorphisms are present on the second chromosome.
  • the position on the chromosome (ii) the corresponding position in ALMS1 mRNA, and (iii) the corresponding position (amino acid number) in ALMS1 protein are shown below.
  • the position on the chromosome was identified from the rs number using dbSNP (BUILD132).
  • the corresponding position in mRNA was identified from database accession number NM_015120
  • (iii) the corresponding position in ALMS1 protein was identified from database accession number NP_055935.
  • the full-length sequence of ALMS1 mRNA is shown in SEQ ID NO: 1, and the coding region sequence is shown in SEQ ID NO: 2, respectively.
  • the amino acid sequence of ALMS1 protein is shown in SEQ ID NO: 3.
  • Glutamate repeat polymorphism (i) 73613033-73613083; (ii) 148-198; (iii) 13-29 (17 repeats) rs2037814 polymorphism: (i) 73675669; (ii) 2129; (iii) 673 rs7598901 polymorphism: (i) 73675844; (ii) 2304; (iii) 731 rs1052161 polymorphism: (i) 73828538; (ii) 12203; (iii) 4031 rs6706562 polymorphism: (i) 73548787; (ii) Not applicable; (iii) Not applicable; (iii) Not applicable
  • detecting a polymorphism can be replaced with the term “analyzing a polymorphism” or the term “detecting an allele”. Detection of the polymorphism reveals the state of the polymorphism position (that is, the type of base).
  • the glutamate repeat polymorphism found to be most useful by large-scale association analysis is used as the detection target. According to this aspect, it is possible to perform a risk inspection with higher reliability.
  • the ALMS1 gene has been identified as a causative gene for Alstrom syndrome.
  • Alstrom syndrome is mainly characterized by retinitis pigmentosa, hearing loss, obesity and diabetes. Other symptoms or pathophysiology of Alstrom syndrome have been reported, such as black epidermoma, scoliosis, dilated cardiomyopathy, liver disease, pulmonary fibrosis, renal failure, hyperlipidemia, and thyroid dysfunction. Although multiple polymorphisms have been reported for the ALMS1 gene, there are no reports that these polymorphisms are associated with specific diseases.
  • the risk is determined based on the detection result of the polymorphism, that is, the type of allele detected or the combination of alleles (genotype).
  • the determination can be automatically / mechanically performed without depending on the determination of a person having specialized knowledge such as a doctor or a laboratory technician, as is apparent from the determination criteria.
  • ⁇ Standards for glutamate repeat polymorphism> (1-a) High risk when alleles with 9 to 14 repeats are detected (1-b) High risk when alleles with 14 repeats are detected ⁇ Criteria for rs2037814 polymorphism> (2-a) High risk if an allele with a base of T is detected ⁇ Criteria for rs7598901 polymorphism> (3-a) High risk if an allele with a base of C is detected ⁇ Criteria for rs1052161 polymorphism> (4-a) High risk if an allele with a base G is detected ⁇ Criteria for rs6706562 polymorphism> (5-a) High risk if an allele with a base of C is detected
  • the genotype is determined from the detection result of the polymorphism. Specifically, in the case of glutamic acid repeat polymorphism, the number of repeats of each allele is determined. In the case of rs2037814 polymorphism, T / T type (homozygous type of allele whose base at polymorphic position is T), T / G type (allele whose base at polymorphic position is T and allele whose base at polymorphic position is G) Heterozygote) and GG type (the base at the polymorphic position is homozygous for the G allele).
  • the C / C type (homozygous type of the allele whose base at the polymorphic position is C) and C / T type (the allele whose base at the polymorphic position is C and the base at the polymorphic position are T) Heterozygous for alleles) or T / T (the base at the polymorphic position is homozygous for the T allele), or the rs1052161 polymorphism is the G / G type (the base at the polymorphic position) Is homozygous for the allele of G), G / A type (heterozygous for the allele of G polymorphic position and the allele of polymorphic position A), A / A type (polymorphic base) Is a homozygote of the allele of A), in the case of the rs6706562 polymorphism, C / C type (homozygote of the allele whose polymorphic position is C) and C / T type (the allele whose base
  • each polymorphism to be detected has a significant difference between the myocardial infarction group and the control group in the additive model analysis (see Examples below) and the dominant model analysis (data not shown). It is what you admit. In view of this fact, in the risk determination / evaluation using the genotype, for example, the following criteria can be adopted.
  • ⁇ Standards for glutamate repeat polymorphism> (1-c) Risk of homozygote for alleles other than 9 to 14 repeats ⁇ Risk of heterozygote for alleles with 9 to 14 repeats and alleles other than 9 to 14 repeats, 9 repeats Risk of homozygotes of alleles up to 14 (1-d) Risk of homozygotes of alleles other than repeats 9 to 14 ⁇ Alleles with repeats 9 to 14 and alleles other than repeats 9 to 14 The risk of homozygotes for alleles with 9 to 14 repeats (1-e) The risk of homozygotes for alleles other than 14 repeats ⁇ 14 with alleles with 14 repeats and 14 repeats Risk of heterozygotes with other alleles, risk of homozygotes of alleles with 14 repeats (1-f) Risk of homozygotes with alleles with a repeat number other than 14 ⁇ Alleles and repeats with 14 repeats Risk of heterozygotes with alleles other than 14
  • the polymorphism detection method is not particularly limited. For example, using an allele-specific primer (and probe), amplification by the PCR method, and detection of the polymorphism of the amplification product by fluorescence or luminescence, PCR-RFLP (restriction fragment length polymorphism) method using PCR (polymerase chain reaction) method, PCR-SSCP (single chain conformation polymorphism) method (Orita, M . Et al., Proc. Natl. Acad.
  • PCR-RFLP restriction fragment length polymorphism
  • PCR-SSCP single chain conformation polymorphism
  • nucleic acid sample is amplified in advance (including amplification of a partial region of the nucleic acid sample) by a nucleic acid amplification method such as a PCR method or a method applying the PCR method, any of the above detection methods can be applied.
  • a nucleic acid amplification method such as a PCR method or a method applying the PCR method
  • allele-specific PCR method When detecting a large number of nucleic acid samples, allele-specific PCR method, allele-specific hybridization method, TaqMan-PCR method, Invader method, FRET-based method, ASP-PCR method, MALDI- using primer extension method It is particularly preferable to use a detection method that can detect a large number of specimens in a relatively short time, such as a TOF / MS (matrix) method, an RCA (rolling cycle amplification) method, or a method using a DNA chip or a microarray.
  • a detection method that can detect a large number of specimens in a relatively short time, such as a TOF / MS (matrix) method, an RCA (rolling cycle amplification) method, or a method using a DNA chip or a microarray.
  • a nucleic acid such as a probe or primer corresponding to each method (also referred to as “polymorphism detection nucleic acid” in the present invention) is used.
  • polymorphism detection nucleic acids used as probes include nucleic acids that specifically hybridize to a chromosomal region (partial chromosomal region) containing the polymorphic position to be detected.
  • the polymorphism to be detected is a glutamate repeat polymorphism, it begins 37 bases downstream (base 148 of SEQ ID NO: 1) from the initiation codon (base 112 of SEQ ID NO: 1), which is the translation start position of the ALMS1 gene.
  • probes targeting chromosomal regions containing 9 glutamate repeat sequences, probes targeting chromosomal regions containing 10 glutamate repeat sequences, and targeting chromosomal regions containing 11 glutamate repeat sequences may be designed.
  • the length of the “partial chromosome region” here is, for example, 16 to 500 bases, preferably 18 to 200 bases.
  • the nucleic acid preferably has a sequence complementary to a partial chromosomal region, but may have some mismatch as long as specific hybridization is not hindered.
  • the degree of mismatch is 1 to several, preferably 1 to 5, and more preferably 1 to 3.
  • specific hybridization refers to hybridization with a target nucleic acid (partial chromosomal region) under the hybridization conditions (preferably stringent conditions) usually employed for detection with a nucleic acid probe. It means that while it soy, it does not cause significant cross-hybridization with other nucleic acids.
  • hybridization conditions preferably stringent conditions
  • polymorphism detection nucleic acid used as a primer is a DNA fragment having a sequence complementary to a certain region (partial region of a chromosome) containing the polymorphic position to be detected and containing the polymorphic portion.
  • the nucleic acid designed so that can be specifically amplified can be mentioned.
  • Another example of a polymorphism detection nucleic acid used as a primer is designed to specifically amplify a DNA fragment containing the polymorphic site only when the polymorphic site to be detected is of a risk allele. Nucleic acid sets.
  • a nucleic acid set comprising a sense primer can be exemplified.
  • the length of the DNA fragment to be amplified 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, more preferably 30 to 200 bases long.
  • the polymorphic nucleic acid used as the primer is a template sequence as long as it can specifically hybridize to the amplification target (template) and amplify the target DNA fragment. There may be some mismatch.
  • the degree of mismatch is 1 to several, preferably 1 to 3, and more preferably 1 to 2.
  • the polymorphism detection nucleic acid As the polymorphism detection nucleic acid (probe, primer), DNA, RNA, peptide nucleic acid (PNA: Peptide-nucleic acid) is used as appropriate depending on the detection method.
  • the base length of the nucleic acid for detecting a polymorphism may be any length as long as the function is exhibited. Examples of the base length when used as a probe are 16 to 500 bases, preferably 18 to 200 bases. On the other hand, examples of the base length when used as a primer are 10 to 50 bases, preferably 15 to 40 bases, and more preferably 15 to 30 bases.
  • the polymorphism detection nucleic acid can be synthesized by a known method such as a phosphodiester method.
  • a known method such as a phosphodiester method.
  • nucleic acids for polymorphism detection 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 helpful.
  • the polymorphism detection nucleic acid in the present invention can be labeled with a labeling substance in advance.
  • a labeled nucleic acid for example, a polymorphism can be detected using the labeling amount of the amplification product as an index.
  • two types of primers designed to specifically amplify partial DNA regions in each genotype gene constituting the polymorphism are labeled with different labeling substances, they can be detected from the amplification product.
  • the genotype of the nucleic acid sample can be discriminated by the labeling substance and the labeling amount.
  • nucleic acid primers As a specific example of a detection method using such a labeled primer, two types of nucleic acid primers (allele-specific sense primers) that specifically hybridize to the sense strand of each genotype constituting the polymorphism are fluorescein.
  • allele-specific sense primers In each of the amplification products obtained by labeling with isothiocyanate and Texas Red, amplifying a partial DNA region containing a polymorphic site using these labeled primers and an antisense primer that specifically hybridizes to the antisense strand.
  • a method for detecting the polymorphism by measuring the labeling amount of each fluorescent substance can be mentioned. If the antisense primer here is labeled with, for example, biotin, amplification products can be separated using specific binding between biotin and avidin.
  • labeling substances used for labeling nucleic acids for detecting polymorphisms 7-AAD, Alexa Fluor (registered trademark) 488, Alexa Fluor (registered trademark) 350, Alexa Fluor (registered trademark) 546, Alexa Fluor (registered trademark) 555, Alexa Fluor (registered trademark) 568, Alexa Fluor (registered trademark) 594, Alexa Fluor (registered trademark) 633, Alexa Fluor (registered trademark) 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 (registered trademark)), PE, PE-Cy TM 5, PE-Cy TM 5,
  • the radioisotope and biotin can be exemplified, and the labeling method is alkaline phosphate. 5 'end labeling method using phatase and T4 polynucleotide kinase, 3' end labeling method using T4 DNA polymerase or Klenow fragment, nick translation method, random primer method (Molecular Cloning, Third Edition, Chapter 9, Cold Spring Harbor Laboratory Press, New York).
  • the above polymorphism detection nucleic acid can also be used in a state immobilized on an insoluble support. If the insoluble support used for immobilization is processed into a chip shape, a bead shape or the like, polymorphisms can be detected more easily using these immobilized nucleic acids.
  • Glutamate repeat polymorphism (the number of glutamic acid changes), rs2037814 polymorphism (becomes valine or glycine), and rs1052161 polymorphism (becomes arginine or lysine) are accompanied by amino acid changes.
  • polymorphisms may be detected using peptides or proteins that are gene expression products. In this case, as long as the amino acid corresponding to the polymorphic site is included, the size (length) of the expression product is not particularly limited.
  • Examples of the detection method using a gene expression product include a method of directly detecting an amino acid at a polymorphic site, or a method of immunological analysis using a change in a three-dimensional structure.
  • a well-known amino acid sequence analysis method (method using Edman method) can be used.
  • the latter includes ELISA (enzyme-linked immunosorbent assay), radioimmunoassay, and immunoprecipitation using antibodies that have specific binding activity to the expression products of genes with any of the genotypes that make up the polymorphism.
  • an immunodiffusion method or the like can be used.
  • An antibody against a target (an expression product of a gene having any genotype constituting a polymorphism) can be prepared using an immunological method, a phage display method, a ribosome display method, or the like.
  • the antibody against the target may be polyclonal or monoclonal.
  • Preparation of a polyclonal antibody by an immunological technique can be performed by the following procedure.
  • a target (or part thereof) is prepared and used to immunize animals such as rabbits.
  • the target (or part thereof) one prepared from a biomaterial (natural antigen) or a recombinant antigen can be used.
  • an antigen bound with a carrier protein may be used.
  • KLH KeyholeHLimpet
  • BSA Bovine Serum Albumin
  • OVA Optalbumin
  • a carbodiimide method, a glutaraldehyde method, a diazo condensation method, an MBS (maleimidobenzoyloxysuccinimide) method, or the like can be used for carrier protein binding.
  • an antigen in which CD46 (or a part thereof) is expressed as a fusion protein with GST, ⁇ -galactosidase, maltose-binding protein, histidine (His) tag or the like can also be used.
  • Such a fusion protein can be easily purified by a general method.
  • a monoclonal antibody can be prepared by the following procedure. First, an immunization operation is performed in the same procedure as described above. Immunization is repeated as necessary, and antibody-producing cells are removed from the immunized animal when the antibody titer sufficiently increases. Next, the obtained antibody-producing cells and myeloma cells are fused to obtain a hybridoma. Subsequently, after this hybridoma is monoclonalized, a clone that produces an antibody having high specificity for the target protein is selected.
  • the target antibody can be obtained by purifying the culture medium of the selected clone.
  • the desired antibody can be obtained by growing the hybridoma to a desired number or more, then transplanting it into the abdominal cavity of an animal (for example, a mouse), growing it in ascites, and purifying the ascites.
  • affinity chromatography using protein G, protein A or the like is preferably used.
  • affinity chromatography in which an antigen is immobilized may be used.
  • methods such as ion exchange chromatography, gel filtration chromatography, ammonium sulfate fractionation, and centrifugation can also be used. These methods can be used alone or in any combination.
  • Labeling substances include 7-AAD, Alexa Fluor (registered trademark) 488, Alexa Fluor (registered trademark) 350, Alexa Fluor (registered trademark) 546, Alexa Fluor (registered trademark) 555, Alexa Fluor (registered trademark) 568, Alexa Fluor.
  • the reagent of the present invention comprises a nucleic acid (polymorphic detection nucleic acid) for detecting a polymorphism to be detected (glutamate repeat polymorphism, rs2037814 polymorphism, rs7598901 polymorphism, rs1052161 polymorphism or rs6706562 polymorphism).
  • the polymorphism detection nucleic acid which is the reagent of the present invention, is applied to the detection method to which it is applied (method using the PCR method using the above-described allele-specific nucleic acid, PCR-RFLP method, PCR-SSCP, TaqMan (registered) (Trademark) -PCR method, Invader (registered trademark) method, etc.).
  • the polymorphism detection nucleic acid are as described above, but specific examples of glutamate repeat polymorphism detection nucleic acid (or a glutamate repeat polymorphism detection nucleic acid set) that are particularly effective as components of the kit are as follows. Show.
  • Unlabeled or labeled nucleic acid having a sequence complementary to the partial DNA region containing the glutamic acid repeat polymorphic site of the ALMS1 gene having 9 glutamic acid repeats (2) The glutamic acid repeat of the ALMS1 gene having 10 glutamic acid repeats Unlabeled or labeled nucleic acid having a sequence complementary to the partial DNA region containing the polymorphic site (3) Having a sequence complementary to the partial DNA region containing the glutamic acid repeat polymorphic site of the ALMS1 gene having 11 glutamate repeats Unlabeled or labeled nucleic acid (4) Unlabeled or labeled nucleic acid having a sequence complementary to the partial DNA region containing the glutamic acid repeat polymorphic site of the ALMS1 gene having 12 glutamic acid repeats (5) The number of glutamic acid repeats is 13 Unlabeled or labeled nucleic acid having a sequence complementary to the partial DNA region containing the glutamate repeat polymorphic site of the ALMS1 gene (6) Glutamate
  • the DNA fragment containing the polymorphic site is Nucleic acid set designed to specifically amplify (21) Only when the number of repeats in the glutamate repeat polymorphic region is 14, the DNA fragment containing the polymorphic site is specific Nucleic acid set designed to amplify (22) A nucleic acid set designed to specifically amplify a DNA fragment containing the polymorphic site only when the number of repeats of the glutamate repeat polymorphic region is 15 ( 23) A nucleic acid set designed to specifically amplify a DNA fragment containing the polymorphic site only when the number of repeats of the glutamate repeat polymorphic region is 16.
  • the number of repeats of the glutamate repeat polymorphic region Is a nucleic acid set designed to specifically amplify a DNA fragment containing the polymorphic site only when the number of repeats in the glutamic acid repeat polymorphic region is 18.
  • nucleic acid set designed to specifically amplify the contained DNA fragment Only when the number of repeats in the glutamate repeat polymorphic region is 20, so that the DNA fragment containing the polymorphic site is specifically amplified
  • the kit of the present invention contains the reagent of the present invention (nucleic acid for polymorphism detection).
  • Reagents DNA polymerase, restriction enzyme, buffer solution, coloring reagent, etc.
  • containers, instruments, etc. necessary for using polymorphism detection nucleic acid ie, polymorphism detection
  • an instruction manual is attached to the kit of the present invention.
  • polymorphism groups existing in the region near the ALMS1 gene (rs6546820, rs10191517, rs7560272, rs7604588, rs6718864, rs6706179, rs7573719, rs6720094, rs6740173, rs6748040, rs6546829, rs1881246, rs12996463, rs2178154, rs1528169, rs1406105, rs6749680, rs1246105, rs780395, rs1246096, rs3820700, rs7598660, rs11685372, rs6744697, rs13008860, rs17349804, rs7566315, rs2948441, rs4852937, rs7570014, rs42
  • ⁇ Selection criteria for 3072 SNPs> (i) Select SNPs with MAF (Minor Allele Frequency)> 0.1. (ii) If the RNA is registered and the SNP is sparse, lower the threshold to MAF> 0.05 and add SNP. (iii) If the interval between adjacent SNPs is 2 Kb or less, delete one of them. (iv) The SNP density is lowered in the region where RNA is not registered.
  • ALMS1 region resequencing and linkage disequilibrium analysis In addition to the polymorphism group in the ALMS1 region rebalancing block shown in the previous patent application (WO 2010/010697 pamphlet) by ALMS1 region resequencing , Rs2037814, rs7598901, rs1052161 were newly discovered as polymorphisms within this linkage disequilibrium block and in linkage disequilibrium with the glutamate repeat polymorphism (FIG. 10). These polymorphisms are also useful as risk markers for myocardial infarction.
  • the risk test method of the present invention provides highly accurate clinically useful risk information (information on the possibility of occurrence) regarding myocardial infarction. Risk information can be used to reduce the likelihood of myocardial infarction, prevent or early diagnosis of myocardial infarction, determine a more appropriate treatment policy, improve the therapeutic effect, improve the patient's quality of life (QOL), etc. Useful. It is also expected to contribute to the medical economy by preventing unnecessary medical practices.
  • the risk test method of the present invention is particularly useful in evaluating the risk for juvenile myocardial infarction.
  • the five polymorphisms to be detected in the present invention are in linkage disequilibrium. Polymorphisms in linkage disequilibrium with these polymorphisms are also useful as risk markers for myocardial infarction.

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  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'objectif de la présente invention est de fournir : un procédé de test du risque de (un procédé de détermination de la susceptibilité à) un infarctus cardiaque, qui est hautement précis et est cliniquement utile ; et un réactif et une trousse, chacun desquels pouvant être utilisé dans le procédé. L'invention concerne un procédé de test du risque d'infarctus cardiaque qui met en jeu une étape de détection de la présence de l'un quelconque des polymorphismes géniques choisis parmi les éléments (1) à (5) mentionnés ci-dessous dans un échantillon d'acide nucléique prélevé à partir d'un sujet : (1) un polymorphisme pour une région codant pour une répétition de l'acide glutamique dans le gène ALMS1 ; (2) un polymorphisme de nucléotide unique qui est identifié sous le numéro d'enregistrement rs2037814 dans la base de données de SNP du Centre National Américain pour l'information biotechnologique (NCBI) ; (3) un polymorphisme de nucléotide unique qui est identifié sous le numéro d'enregistrement rs7598901 dans la base de données de SNP du Centre National Américain pour l'information biotechnologique (NCBI) ; (4) un polymorphisme de nucléotide unique qui est identifié sous le numéro d'enregistrement rs1052161 dans la base de données de SNP du Centre National Américain pour l'information biotechnologique (NCBI) ; et (5) un polymorphisme de nucléotide unique qui est identifié sous le numéro d'enregistrement rs6706562 dans la base de données de SNP du Centre National Américain pour l'information biotechnologique (NCBI).
PCT/JP2011/076738 2010-11-22 2011-11-19 Procédé pour le test du risque d'infarctus cardiaque WO2012070510A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010010697A1 (fr) * 2008-07-24 2010-01-28 Yokota Mitsuhiro Méthode d’analyse du risque d’infarctus du myocarde

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010010697A1 (fr) * 2008-07-24 2010-01-28 Yokota Mitsuhiro Méthode d’analyse du risque d’infarctus du myocarde

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SEIKO MIYATA ET AL.: "Genome Ekigaku Kenkyu to Rinsho eno Tenkai Junkanki Bun'ya ni Okeru Kobetsuka Iryo eno Tenkai -Shinkin Kosoku Kanjusei Idenshi no Tansaku", SAISHIN IGAKU, vol. 62, no. 9, September 2007 (2007-09-01), pages 2265 - 2275 *

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