WO2004084797A2 - Gene associe au diabete sucre du type 2 et son utilisation - Google Patents

Gene associe au diabete sucre du type 2 et son utilisation Download PDF

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WO2004084797A2
WO2004084797A2 PCT/IB2004/000579 IB2004000579W WO2004084797A2 WO 2004084797 A2 WO2004084797 A2 WO 2004084797A2 IB 2004000579 W IB2004000579 W IB 2004000579W WO 2004084797 A2 WO2004084797 A2 WO 2004084797A2
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gene
site
seq
nucleotide sequence
dna region
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PCT/IB2004/000579
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Japanese (ja)
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WO2004084797A3 (fr
WO2004084797A1 (fr
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Makoto Daimon
Takeo Kato
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Hubit Genomix Inc
Makoto Daimon
Takeo Kato
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Priority to JP2005503973A priority Critical patent/JPWO2004084797A1/ja
Publication of WO2004084797A1 publication Critical patent/WO2004084797A1/fr
Publication of WO2004084797A2 publication Critical patent/WO2004084797A2/fr
Publication of WO2004084797A3 publication Critical patent/WO2004084797A3/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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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

  • Type 2 diabetes-related genes and their use
  • the present invention relates to a gene associated with type 2 diabetes and a method for testing type 2 diabetes using the gene.
  • Type 2 diabetes is a heterogeneous disorder of glucose metabolism characterized by both insulin resistance and impaired kidney cell function.
  • the disease is a major health problem due to the high incidence (3% to 10%) of DM-related vascular complications and the clinical severity.
  • the hereditary pattern of DM suggests the polygenic features of the disease and the complex interactions between genes and environmental factors (see Non-Patent Document 1).
  • the genes involved in the general formation of 'DM have not been known so far. Genome-wide studies using microsatellite mathematics, and analysis of affected sib-pairs have identified DM-related loci in various genomic regions from different populations (see Non-Patent Documents 1 to 7). .
  • DM is a multifactorial disease, affected by environmental factors, and the etiology of the disease among individuals is heterogeneous, and is caused by multiple causative genes (disease susceptibility genes).
  • causative genes disease susceptibility genes
  • Non-Patent Document 1 Barnett, A. H. et al., "Diabetologia”, Ranji, Vol. 20, p. 87-93
  • Non-Patent Document 2 Knowler, W. C., et al., "Am. J. Hum. Genet. J, 1988,
  • Non-Patent Document 3 Bell, G. I. et al., "Proc. Natl. Acad. Sci. USA", 1991, Vol. 88, p. 1484-1488.
  • Non-Patent Document 4 Van den Ouwenl and J. M. W. et al., ("Nature
  • Non-Patent Document 5 Vionnet, N. et al., "Am. J. Hum. Genet. J, 2000,
  • Non-Patent Document 7 Wiltshire, S. et al., "Am. J. Hum. Genet.”, 2001,
  • Non-Patent Document 8 Ghosh, S. et al., "Am. J. Hum. Genet. J, 2000,
  • the present invention has been made in view of such a situation, and an object thereof is to find a gene associated with type 2 diabetes mellitus, and to further identify a polymorphism associated with type 2 diabetes present on the gene. It is intended to provide a method for testing type 2 diabetes.
  • the present inventors have conducted intensive research to solve the above-mentioned problems. First, the present inventors used a case-control test to identify 2039 single nucleotide polymorphisms (SPs) derived from 704 candidate genes in order to identify genes associated with type 2 diabetes (DM). The relationship with M was examined. The study group consisted of 148 cases and 227 control subjects. The results showed that 29 genes were significantly associated with DM.
  • SPs single nucleotide polymorphisms
  • the identification of genes related to DM by the present inventors is expected to greatly elucidate the onset mechanism of DM and to develop a drug for preventing or treating DM.
  • the present inventors succeeded in identifying 29 genes related to DM, SNPs related to DM, and haplotypes, and tested DM using SNPs or haplotypes on the genes as indicators. Completed the method. That is, the present invention relates to type 2 diabetes More specifically, the present invention relates to a method for testing type 2 diabetes using a gene related to, and a polymorphic site or haplotype on the gene as an index.
  • the present inventors succeeded in identifying 29 genes related to DM, SNPs related to DM, and haplotypes, and performed DM detection using SNPs or haplotypes on the genes as indicators. ⁇ Completed the method. That is, the present invention relates to a gene associated with type 2 diabetes, and a method for testing type 2 diabetes using a polymorphic site or a haplotype on the gene as an index, more specifically,
  • a test method for type 2 diabetes which comprises detecting a mutation in a gene according to any of the following (1) to (29) for a subject;
  • the above-mentioned inspection method is, for example, for a test sample obtained from a subject,
  • the above-mentioned inspection method is, for example, for a test sample obtained from a subject,
  • any polymorphic site at position 78 (6a) a site on the ZNF 263 gene or a DNA region near the gene, wherein positions 876, 1552, 3213, 3269, 6401, 6425, and 6425 in the nucleotide sequence of SEQ ID NO: 6 6602th, 9595th, 10001st, 10252th, 10269th, 10294th, 1 1377th, 12469th, 1
  • ALDOB gene or a site on the DNA region in the vicinity of the gene, which is located at positions 99, 380, 396, and 3 in the nucleotide sequence of SEQ ID NO: 11.
  • a site on the VLDLR gene or a DNA region in the vicinity of the gene which corresponds to positions 6297, 6733, 7994, 9242, 9952, and 1 in the nucleotide sequence of SEQ ID NO: 20; 000 1st, 1 1 838, 1 299 5th, 1 3224, 1 3277, 1 702, 188 96, or 192 1 4 Any polymorphic site in position
  • polymorphic sites are polymorphic sites described in the following (lb) to (29b), respectively:
  • (3c) a site on the DNA region near the SLC1A1 gene or the gene, wherein the base species at position 10001 in the base sequence of SEQ ID NO: 3 is G
  • (4c) a site on the DNA region in the vicinity of the SERP I NB2 gene or the gene, wherein the nucleotide species at position 24578 in the nucleotide sequence of SEQ ID NO: 4 is A
  • the base species at position 10001 in the base sequence of SEQ ID NO: 7 is A (8c) THB S3 gene or a site on a DNA region in the vicinity of the gene, and the position 10001 in the base sequence of SEQ ID NO: 8 Base type of C
  • step (b) replacing the base species determined in step (a) with the above-mentioned gene showing a haplotype according to any one of the following (1 ′) to (14 ′) or a polymorphic site in a DNA region near the gene: Process to compare with base type of (polymorphic site on haptic type block)
  • nucleotide types at the polymorphic site at positions 10001, 14258, and 19768 in the nucleotide sequence of SEQ ID NO: 1 are G, C, and C, respectively.
  • the nucleotide type of the polymorphic site at positions 10001, 16198, and 17848 in the nucleotide sequence of SEQ ID NO: 2 is Haplotypes that are G, G, and G respectively, or
  • the nucleotide types of the polymorphic sites at positions 10001, 16198, and 17848 in the nucleotide sequence of SEQ ID NO: 2 are G, C, and G, respectively.
  • nucleotide type of the polymorphic site at positions 10001, 18958, 19354, 19567, and 20971 in the nucleotide sequence of SEQ ID NO: 13 is: Haplotypes that are A, G, T, ⁇ , and C, respectively, or
  • the nucleotide types of the polymorphic sites at positions 20971 and 21213 in the nucleotide sequence of SEQ ID NO: 13 are C and C, respectively.
  • the nucleotide types of the polymorphic sites at positions 24016, 29469, and 37583 in the nucleotide sequence of SEQ ID NO: 22 are C, G, and C, respectively.
  • the presence or absence of the haplotype (block) described in any of the above (1 ′) to (14,) is used as an index. It is a test method to determine whether you have a predisposition to susceptibility or resistance to type 2 diabetes.
  • a site on the MET gene or a DNA region in the vicinity of the MET gene wherein positions 765, 138, 189, 209, and 289 in the nucleotide sequence of SEQ ID NO: 1 14th, 3293th, 375 2nd, 3774th, 3927th, 4690th, 74190, 8214, 10000, 1st, 103rd, 8th, 137676 1st, 3989th, 1425 8th, 1 5427th, 1 6275, 1 659 7th, 1
  • a site on the CTSD gene or a DNA region in the vicinity of the CTSD gene wherein positions 81, 81, 844, 844, 861, and 954 in the nucleotide sequence of SEQ ID NO: 12; 1st, 1026th, 1159th, 1299th, 5464th, 5600th, 7144th, 7707th, 7726th, 773 9th, 7778th, 7803th,
  • polymorphic site on the SERP I NB2 gene wherein the polymorphic site is at position 10001 or position 24578 in the base sequence of SEQ ID NO: 4;
  • a polymorphic site on the ABCA1 gene wherein the polymorphic site at position 10001 or 11000 in the nucleotide sequence of SEQ ID NO: 71
  • a polymorphic site on the CTSD gene wherein the polymorphic site is at position 10001, position 1 1990, or position 16771 in the nucleotide sequence of SEQ ID NO: 12.
  • polymorphic site on the MYL2 gene wherein the polymorphic site is position 10001, position 18386, position 42589, or position 83500 in the nucleotide sequence of SEQ ID NO: 19;
  • (11) a polymorphic site on the LARGE gene, wherein the polymorphic site is at positions 10001, 13103, 15448, 36690, or 43240 in the nucleotide sequence of SEQ ID NO: 25
  • test method according to any one of [3] to [9], further comprising: preparing a DNA containing a polymorphic site from a biological sample of the subject.
  • present invention provides an oligonucleotide for a type 2 diabetes test for use in the above-mentioned detection method.
  • an oligonucleotide primer or an oligonucleotide probe used in the above-mentioned detection method can be mentioned. More specifically, an oligonucleotide primer or an oligonucleotide probe used in the above-mentioned detection method can be mentioned. More specifically,
  • a type 2 diabetes mellitus comprising an oligonucleotide that hybridizes to DNA containing the polymorphic site according to any one of (la) to (29a) of [4] and has an oligonucleotide length of at least 15 nucleotides.
  • Test reagents test reagents
  • a test for type 2 diabetes comprising a solid phase on which a nucleotide probe that hybridizes with DNA containing the polymorphic site according to any one of (la;) to (29a) in [4] is immobilized.
  • Drugs test reagents
  • a test agent for type 2 diabetes comprising a primer oligonucleotide for amplifying a DNA containing the polymorphic site according to any one of (1a) to (29a) of [4].
  • the present inventors have identified a gene associated with type 2 diabetes (DM) (DM-related gene).
  • DM type 2 diabetes
  • mutations are significantly found in the gene. Therefore, by examining the presence or absence of a mutation in the gene, it is possible to test whether or not the subject is DM or not. is there.
  • Table 1 shows the name of the DM-related gene of the present invention, the position of the gene on the chromosome, and the GenBank accession number of the gene. Information on the nucleotide sequences of these genes and the amino acid sequences of the proteins encoded by the genes can be easily obtained from the GenBank accession numbers shown in Table 1. In addition, those skilled in the art may use the public notation based on the gene notation (gene name) shown in Table 1. It is possible to easily obtain information on the nucleotide sequence of the gene and the amino acid sequence of the protein encoded by the gene from a common gene database or literature database.
  • NPHS1 nephrosis 1 congenital, Finnish type (nephrin) 19q12-q13.1 AG002133.1 solute carrier family 1 (neuronal / epithelial high affinity
  • TNFA tumor necrosis factor (TNF superfamily, member 2) 6p21.3 16441.1
  • ABCA1 ATP-binding cassette sub-family A (ABC1) t member 1 9q31 AF275948.1 serine (or cysteine) proteinase inhibitor, clade B
  • ALDOB aldolase B fructose-bisphosphate 9q21.3-q22.2 M15656.1
  • CTSD cathepsin D (lysosomal aspartyl protease) 11p15.5 AC068580.2
  • ATP-binding cassette sub-family C (CFTR / MRP)
  • ABCC8 member 8 I1p15.1 NT 000564.1 serine (or cysteine; proteinase inhibitor, clade A (.alpha- 1
  • RCV1 recoverin 1 /
  • VLDLR very low density lipoprotein receptor 9p24 AC019222.3
  • P2RX7 purinergic receptor P2X, ligand-gated ion channel, 7 12q24 Z98941.1
  • JEM-1 basic leucine zipper nuclear factor 1
  • LEP leptin (obesity homolog, mouse) 7q31 AG018635.5
  • the present invention is characterized by detecting a mutation in the gene described in any one of the following (1) to (29) for a subject, To provide a method for testing type 2 diabetes.
  • the “gene described in any one of the above” of the present invention means at least one gene of any one of the above (1) to (29). That is, the present invention includes a case where a type 2 diabetes test is performed by detecting mutations in a plurality of genes described in the above (1) to (29).
  • test for type 2 diabetes refers to determining whether a subject is likely or unlikely to have type 2 diabetes, and determining the cause or type of disease if the subject already has DM. Inspection to do is included.
  • the subject When a mutation is detected in each of the genes in (29), the subject is determined to be susceptible or unlikely to have type 2 diabetes. Even in subjects who have already suffered from type 2 diabetes, the cause and the type of type 2 diabetes can be determined, and this can be used for determining a treatment policy.
  • mutation refers to any mutation that alters the expression level of the gene, alters properties such as mRNA stability, or alters the activity of the protein encoded by the gene. Without limitation, examples thereof include addition, deletion, substitution, and insertion mutation of a base.
  • the present inventors have succeeded in finding a polymorphic mutation significantly associated with DM in each of the above-mentioned genes (1) to (29) or a DNA region near the gene in a DM patient. Therefore, DM is detected by using the presence or absence of a mutation as an index (determining the base type) for each of the above-mentioned genes (1) to (29) or a polymorphic site in a DNA region near the gene. It is possible.
  • the above-mentioned “DNA region near the gene” usually refers to a region on the chromosome near the gene. Neighborhood is especially Although not limited, it is usually a DNA region containing the polymorphic site of the present invention, and preferably refers to a region within 10 kb from the end of the gene.
  • Polymorphisms in the 10 kb or 20 kb range are likely to be linked as reported by Gabriel et al. (Gabriel SB, Schaffner SF, Nguyen H et al. The structure of haplotype blocks in the human genome. Science 296 , 2225-9. 2002).
  • SEQ ID NOs: 1 to 29 Each of the above-mentioned genes (1) to (29) and the DNA sequence near the gene are shown in SEQ ID NOs: 1 to 29, respectively. (That is, the nucleotide sequence described in SEQ ID NO: 1 indicates a DNA sequence in the vicinity of the MET gene or the gene.)
  • SEQ ID NO: 30 is represented by SEQ ID NO: 28 The DNA sequence near the FRAP gene and the gene other than the FRAP gene and the DNA region near the gene is shown.
  • a method for detecting type 2 diabetes comprising detecting a single nucleotide polymorphism mutation in the gene according to any one of the above (1) to (29).
  • Polymorphism is generally defined genetically as a change in a certain base in one gene that is present at a frequency of 1% or more in the population. You are not limited to this definition. Examples of the types of polymorphisms in the present invention include single nucleotide polymorphisms and polymorphisms in which several tens (sometimes thousands) of nucleotides are deleted or inserted from — Not limited. Furthermore, the number of polymorphism sites is not limited to one, and may have a plurality of polymorphisms.
  • the method according to any one of (1) to (29) above, or a base species at a polymorphic site in a DNA region near the gene is determined, This is a test method for type 2 diabetes.
  • the “polymorphism site” in the above method of the present invention is not particularly limited as long as it is a polymorphism present on each of the above-mentioned genes (1) to (29) or a DNA region near the gene.
  • the above (1) to (2) The following (la) to (29a) polymorphic sites present on each gene of 9) or a DNA region near the gene can be mentioned. (In the present specification, these polymorphic sites may be simply referred to as “polymorphic sites of the present invention”.
  • 1 4 3 9 3rd place 1 447 5th place, 1 4 5 9 1st place, 1 4 7 2 2nd place, 1 5 2 94th place, 1 5 6 0 1st place, 1 5 6 5 1st place, 1 5 6 5 7th place, 1 6 1 6 0th, 1 6 2 3 0, 1 6 3 6 7, 1 64 1 7, 1 7 6 9 5, 1 8 2 13 3, 1 8 8 1 5, 1 909 3rd, 20082, 2021 1st, 2046 1st, 2 1 547, 2 1995, 22825, or 2390 1 polymorphic site (1 5a) SERP I NA3 gene Or a site on a DNA region near the gene, wherein positions 937, 1145, 1878, 2345, 2559, 3250, and 3250 in the nucleotide sequence of SEQ ID NO: 15 5 1 3rd, 4
  • a polymorphic site at any of positions 6708, 16748, or 19630 (29a) a site on the LEP gene or a DNA region near the gene, wherein position 244 in the nucleotide sequence of SEQ ID NO: 29; 575, 675, 1487, 1644, 2343, 271 5th, 2763, 309 8th, 3589, 3880, 5787, 5865, 6436,
  • the DNA in the genome usually has a double-stranded DNA structure complementary to each other. Therefore, in the present specification, even when a DNA sequence on one strand is shown for convenience, it is understood that a sequence complementary to the sequence (base) is also disclosed. For those skilled in the art, if one DNA sequence (base) is known, the sequence (base) complementary to the sequence (base) is obvious.
  • CFRAP 1 gene or a polymorphic site on a DNA region near the gene
  • the base type at the polymorphic site shown in the above table may indicate a base type that is on the complementary strand side to the sequence shown in the sequence listing, but the base sequence before and after published in the dbSNP and JSNP databases It is easy for those skilled in the art to confirm the difference if it is used. Examining either the plus or minus strand can inevitably determine the other result.
  • those skilled in the art usually use the registration ID number assigned to the polymorphism disclosed in the present specification, for example, the rs number in the dbSNP database, the IMS-JST number in the JSNP database, and the ssj number in the present invention.
  • the actual position on the genome and the sequence before and after the polymorphic site can be easily known.
  • those skilled in the art will appropriately correspond to the polymorphic site based on the information on the nucleotide sequence and the polymorphic site shown in SEQ ID NOs: 1 to 30. It is easy to know the actual position on the genome.
  • the position of the polymorphic site of the present invention on the genome can be known by referring to a published genome database or the like. That is, even if a slight difference in the base sequence between the base sequence listed in the sequence listing and the actual base sequence on the genome is found, the genome is determined based on the base sequence listed in the sequence listing. By performing a homology search with the sequence, it is possible to accurately know the actual genome position of the polymorphic site of the present invention. In addition, even if the position on the genome cannot be identified, it is easy to perform the detection described in the present invention from the sequence listing and information on the polymorphic site described in this specification.
  • the base species at position 10001 in the nucleotide sequence of SEQ ID NO: 5 is A (6c) ZNF 263 gene or in the vicinity of the gene A site on the DNA region, wherein the base species at position 10001 in the nucleotide sequence of SEQ ID NO: 6 is a site on the G (7c) TNF A gene or a DNA region near the gene;
  • the base species at position 10001 in the base sequence described in 7 is the A (8c) THBS 3 gene or a site on the DNA region in the vicinity of the gene, and the base sequence described in SEQ ID NO: 8
  • the nucleotide at position 10001 is the C (9c) AB CA1 gene or a site on the DNA region near the gene, and the nucleotide at position 17283 in the nucleotide sequence of SEQ ID NO: 9 is T (10c)
  • the RCV1 gene or a site on the DNA region near the gene wherein the nucleotide at position 10001 in the nucleotide sequence of SEQ ID NO: 16 is T (17c) TFRC gene or A base on the DNA region near the offspring, wherein the base species at position 10001 in the base sequence set forth in SEQ ID NO: 17 is a site on the G (18c) MMP19 gene or the DNA region near the gene;
  • the base type at position 10001 in the base sequence set forth in SEQ ID NO: 18 is
  • nucleotide at position 18563 in the nucleotide sequence of SEQ ID NO: 23 is G (24c) AQP8 gene or the DNA region in the vicinity of the gene;
  • nucleotide at position 10001 in the nucleotide sequence of SEQ ID NO: 24 is a site on the G (25c) LARGE gene or a DNA region near the gene, and is described in SEQ ID NO: 25.
  • the base type at position 13103 is G
  • the base type at position 15448 is G (26c) BLZF1 gene or a site on a DNA region near the gene, and position 20031 in the base sequence of SEQ ID NO: 26 Base type of
  • the type of diabetes can be tested by determining the base type in the subject for the “near polymorphic site” and comparing the base type with the base type determined in advance. If the base species is the same as the base species determined in advance, the subject is determined to be susceptible to or unlikely to have type 2 diabetes. In the case of a subject already suffering from type 2 diabetes, the cause and type of type 2 diabetes can be determined and used for determining a treatment policy.
  • the case of the MET gene will be described as an example.
  • the base type of the type site for example, the polymorphic site at position 8214 is determined. Assuming that the base type at this site is T in the DM patient group, the subject is examined for the base type of the polymorphic site at position 8214, and if the base type at this site is T, The subject is determined to be susceptible to type 2 diabetes.
  • the DM-related inspection can be performed without imposing an excessive burden on those skilled in the art.
  • a haplotype refers to a combination of the genotypes of individuals with respect to one of them, and each shows how the loci are arranged on one parental chromosome. Since chromosomes are inherited one by one from parents, if recombination does not occur during gametogenesis, genes on one chromosome will always be transmitted to the child together, that is, linked. . However, recombination actually occurs during meiosis, so even genes on a single chromosome are not necessarily linked. However, conversely, even when genetic recombination occurs, loci at close distances on the same chromosome are strongly linked.
  • linkage disequilibrium Observing such phenomena in a group and finding the independence of Aryl is called linkage disequilibrium. For example, when observing a three ⁇ child seat, haplotypes present that there is no linkage complaints ⁇ between them are predicted are two 3, each frequency value predicted from the frequency of each locus made but absent fewer haplotypes than are two 3 when there is linkage disequilibrium, the frequency also becomes results showing the predicted value different.
  • haplotypes have been shown to be useful for linkage disequilibrium analysis.
  • the type 2 diabetes can be tested by detecting the haplotype.
  • the present inventors have made intensive studies and succeeded in finding a haplotype associated with type 2 diabetes.
  • the present invention provides a gene associated with the gene according to any one of the following (1) to (14) and / or a type 2 diabetes present in a DNA region near the gene:
  • a “haplotype associated with type 2 diabetes” when a “haplotype associated with type 2 diabetes” is detected for a subject, it is determined that the subject is susceptible / susceptible to type 2 diabetes. In the case of a subject already suffering from type 2 diabetes, the cause and type of the onset can be determined and used to determine a treatment policy.
  • haplotype associated with type 2 diabetes can specifically indicate the following haplotypes.
  • susceptible means that the subject is susceptible to type 2 diabetes
  • protective means that the subject is less susceptible to type 2 diabetes.
  • nucleotide types of the polymorphic sites at positions 10001, 14258, and 19768 in the nucleotide sequence of SEQ ID NO: 1 are G, C, and C, respectively.
  • the nucleotide types of the polymorphic sites on the haplotype or the MET gene, the polymorphic sites at positions 19768 and 24209 in the nucleotide sequence of SEQ ID NO: 1, are T and T, respectively.
  • the base types of the polymorphic sites at positions 10001, 14258, and 19768 in the base sequence of SEQ ID NO: 1 are G, T, and
  • the haplotype ⁇ is susceptible to type 2 diabetes, and the haplotypes whose base species are G, C, and C, respectively, are resistant to type 2 diabetes.
  • a polymorphic site on the MET gene Haplotypes in which the base species at the polymorphic site at positions 19768 and 24209 in the nucleotide sequence are T and T, respectively, are susceptible to type 2 diabetes.
  • the polymorphic site on the NPHS1 gene wherein the base species at the polymorphic positions 10001, 1698, and 17848 in the base sequence set forth in SEQ ID NO: 2 are G, G, and G, respectively.
  • the nucleotide types at positions 10001, 16198, and 17848 in the nucleotide sequence of SEQ ID NO: 2 are G, G, and G, respectively.
  • haplotypes G and C are susceptible to type 2 diabetes
  • C and G are susceptible to type 2 diabetes
  • haplotypes whose base types are A, C and A Is resistant to type 2 diabetes.
  • polymorphic site on the SERP I NB2 gene wherein the nucleotides at the polymorphic site at positions 10001 and 24578 in the nucleotide sequence of SEQ ID NO: 4 Haplotypes whose species are C and A, respectively, are susceptible to type 2 diabetes, and haplotypes whose base species are T and T are resistant to type 2 diabetes.
  • the base type of the polymorphic site at positions 10001, 15824, 17283, 2051 5 and 28564 in the nucleotide sequence of SEQ ID NO: 9 is:
  • the haplotypes C, A, C, T, and G, respectively, are resistant to type 2 diabetes, are polymorphic sites on the ABCA1 gene, and are located at position 10001 in the nucleotide sequence of SEQ ID NO: 71.
  • Haplotypes in which the bases at the polymorphic site at position 11000 are G and C, respectively, are susceptible to type 2 diabetes mellitus.
  • the nucleotide types at the 10001, 11990, and 16771 positions in the nucleotide sequence of SEQ ID NO: 12 are C and G, respectively.
  • Haplotypes that are,, and T are resistant to type 2 diabetes.
  • nucleotide species at the polymorphic site at position 20971 and the polymorphic site at position 211 in the nucleotide sequence of SEQ ID NO: 13 are C and C, respectively.
  • the STE gene is a polymorphic site on the STE gene, and is represented by the nucleotide sequence of SEQ ID NO: 13 at positions 10001, 18958, 19354, 19567 , And 2097
  • the haplotype in which the base type at the polymorphic site at position 1 is A, G, T, ⁇ , and C, respectively, is susceptible to type 2 diabetes.
  • the nucleotide types of the polymorphic sites at positions 20971 and 221 in the nucleotide sequence of SEQ ID NO: 13 are C and C, respectively. Haplotypes are susceptible to type 2 diabetes.
  • a haplotype that is a polymorphic site and has a base type A of the polymorphic site at position 14475 in the nucleotide sequence described in SEQ ID NO: 14 is susceptible to type 2 diabetes.
  • polymorphism site is a polymorphism site on the MYL2 gene, Haplotypes in which the base species at the polymorphic sites at positions 000 1, 1838 6, 4258 9 and 8350 are C, ⁇ , ⁇ , and C, respectively, are susceptible to type 2 diabetes.
  • a polymorphic site on the DLG5 gene, wherein the polymorphic site at positions 240, 169, and 36983 in the nucleotide sequence set forth in SEQ ID NO: 22 Are the haplotypes of C, G, and C, respectively, or a polymorphic site on the DLG5 gene, wherein the base sequence of SEQ ID NO.
  • Haplotypes in which the bases at the polymorphic positions at positions 469 and 37583 are C, G, and T, respectively. More specifically, they are polymorphic sites on the DLG5 gene, : A haplotype in which the base species at the polymorphic site at positions 241016, 294969, and 37583 in the nucleotide sequence described in 22 are C, G, and C, respectively. Is susceptible to type 2 diabetes, and haplotypes whose base species are C, G, and T, respectively, are resistant to type 2 diabetes.
  • polymorphic site on the LDLC gene wherein the nucleotide types of the polymorphic site at position 1 001 and position 185 in the nucleotide sequence of SEQ ID NO: 23 are respectively Haplotypes G and G are susceptible to type 2 diabetes, and haplotypes whose base species are G and ⁇ ⁇ ⁇ ⁇ are resistant to type 2 diabetes.
  • (11,) base type of the polymorphic site on the LARGE gene which is the polymorphic site at positions 10001, 13103, 15448, 36690, and 43240 in the base sequence of SEQ ID NO: 25 Haplotypes are T, G, G, T, and ⁇ ⁇ ⁇ ⁇ , respectively
  • polymorphic site on the LARGE gene More specifically, it is a polymorphic site on the LARGE gene, and the nucleotide type of the polymorphic site at positions 10001, 13103, 15448, 36690, and 43240 in the nucleotide sequence described in SEQ ID NO: 25
  • haplotypes with T, G, G, T, and ⁇ ⁇ ⁇ ⁇ , respectively, are susceptible to type 2 diabetes.
  • the haplotype is a polymorphic site on the BLZF1 gene, wherein the nucleotide types of the polymorphic site at position 10001 and 20031 in the nucleotide sequence shown in SEQ ID NO: 26 are G and T, respectively. Is resistant to type 2 diabetes.
  • a haplotype which is a polymorphic site on the FRAP1 gene and whose base type at the polymorphic site at position 17020 in the nucleotide sequence shown in SEQ ID NO: 28 is G is susceptible to type 2 diabetes.
  • the haplotype which is a polymorphic site on the LEP gene and whose nucleotide types at positions 10001 and 10503 in the nucleotide sequence of SEQ ID NO: 29 are G and A, respectively, is 2 I am susceptible to type 2 diabetes.
  • a preferred embodiment of the above method is a method for testing type 2 diabetes, comprising the following steps (a) and (b).
  • step (b) converting the base species determined in the step (a) to the gene showing the haplotype according to any one of the above (1 ′) to (14 ′) or a polymorphic site in a DNA region near the gene; Process to compare with base type of
  • the polymorphic site in the above step (a) preferably, the following polymorphic sites can be shown.
  • a MET gene or a site on a DNA region near the gene wherein positions 765, 1383, 1890, 2099, 2814, 3293, 3752, and 3752 in the nucleotide sequence described in SEQ ID NO: 1; 3774, 392 7th, 4690, 7419, 8214, 10001, 10318, 13766, 13989, 14258, 15427, 162 75, 16597, 16932, 19768, 19835, Two
  • nucleotide sequence according to SEQ ID NO: 12 is at positions 813, 815, 844,
  • ABCC 8 A site on a gene or a DNA region near the gene, wherein positions 6978, 7358, 7359, 7649, 7649, 7755, and 778 in the nucleotide sequence of SEQ ID NO: 14 806 8th, 8 2 14th, 8 2 1 5th,
  • the LARGE gene or a site on a DNA region near the gene wherein positions 1816, 3116, 3117, 3118, 3154, 3157, and 3191 in the nucleotide sequence of SEQ ID NO: 25 , 3197,
  • polymorphic site a site on the LEP gene or a DNA region near the gene, wherein positions 244, 575, 675, 1487, and 1644 in the nucleotide sequence of SEQ ID NO: 29; , 2343, 2 7 1 5, 276 3, 3 980, 3 589, 3880, 5 787, 58 6 5, 6436, 73 92, 79 9 5 1st, 8 1 6 1st, 8 544th, 9 230th, 979 5th, 1 000 1st, 10 19 9th, 10503, 1 1975, 14 98 1st, 1 5 6 8 1 or 1 9 300 polymorphic site More preferably, the following polymorphic site can be shown as the polymorphic site in the step (a).
  • polymorphic site on the NPHS 1 gene wherein the polymorphic site is at position 10001, 16198, or 17848 in the nucleotide sequence of SEQ ID NO: 2
  • polymorphic site on the SERP I NB2 gene wherein the polymorphic site is at position 10001 or 24578 in the nucleotide sequence of SEQ ID NO: 4;
  • a polymorphic site on the ABCA1 gene wherein the polymorphic site at position 10001 or 11000 in the nucleotide sequence set forth in SEQ ID NO: 71
  • polymorphic site on the CTS D gene wherein the polymorphic site is at position 10001, position 1 1990, or position 16771 in the nucleotide sequence of SEQ ID NO: 12
  • polymorphic site on the STE gene wherein the polymorphic site is at positions 10001, 18958, 19354, 19567, 20971, or 21213 in the nucleotide sequence of SEQ ID NO: 13.
  • a polymorphic site on the MYL2 gene wherein the polymorphic site is at position 10001, position 18386, position 42589, or position 83500 in the nucleotide sequence of SEQ ID NO: 19;
  • a polymorphic site on the DLG5 gene wherein the polymorphic site is at position 10001, 24016, 29469, or 37583 in the nucleotide sequence of SEQ ID NO: 22
  • (11) a polymorphic site on the LARGE gene, wherein the polymorphic site at positions 10001, 13103, 15448, 36690, or 43240 in the nucleotide sequence of SEQ ID NO: 25
  • polymorphic site on the FRAP1 gene, wherein the polymorphic site is at position 10001 or 17020 in the nucleotide sequence of SEQ ID NO: 28
  • the determination of the base type at the polymorphic site of the present invention can be performed by those skilled in the art by various methods. As an example, it can be carried out by directly determining the nucleotide sequence of the DNA containing the polymorphic site of the present invention.
  • a DNA sample is prepared from a subject.
  • the DNA sample is, for example, chromosomal DNA extracted from blood, skin, oral mucosa, tissues or cells collected or excised by surgery, body fluids collected for the purpose of examination, or the like, or It can be prepared based on RNA.
  • the DNA containing the polymorphic site of the present invention is then isolated.
  • the DNA can be isolated by PCR using chromosomal DNA or RNA as a ⁇ type, using a primer that hybridizes to the DNA containing the polymorphic site of the present invention.
  • the base sequence of the isolated DNA is determined. Determination of the nucleotide sequence of the isolated DNA can be easily performed by those skilled in the art using a DNA sequencer or the like.
  • the permutation of the base species at that site has already been clarified.
  • “determination of a base type” does not necessarily mean that the polymorphic site is determined to be any of A, G, T, and C base types. For example, if it is known that the variation of the base type is A or G for a certain polymorphic site, it is sufficient to determine that the base type at that site is “not A” or “not G”. It is.
  • the method for determining the base species of the present invention is not particularly limited.
  • TaqMan PCR method, Acyclo Prime method, MALDI-TOF / MS method, and the like have been put into practical use as analysis methods applying the PCR method.
  • the Invader method and the RCA method are known as methods for determining the base type independent of PCR.
  • the base type can be determined using a DNA array. The following briefly describes these methods. The method described here can be applied to the determination of the base species at the polymorphic site in the present invention, both in terms of deviation and deviation.
  • the principle of the TaqMan PCR method is as follows.
  • the TaqMan PCR method is an analysis method using a primer set capable of amplifying a region containing an allele and a TaqMan probe. TaqMan probes are designed to hybridize to the region containing the allele amplified by this primer set.
  • the hybridization efficiency of the TaqMan probe is significantly reduced due to the difference of one base.
  • extension reaction from primers The mis-hybridized TaqMan probe is reached.
  • the TaqMan probe is degraded from its 5 'end by the 5'-3' exonuclease activity of DNA polymerase. If the TaqMan probe is labeled with a reporter dye and quencher, the degradation of the TaqMan probe can be tracked as a change in the fluorescent signal.
  • the TaqMan probe is degraded, the reporter dye is released and the distance to the quencher is increased, generating a fluorescent signal. If the hybridization of the TaqMan probe is reduced due to a single base difference, the degradation of the TaqMan probe does not proceed, and no fluorescent signal is generated.
  • TaqMan probes corresponding to the polymorphisms and further generating different signals by decomposition of each probe, it is possible to simultaneously determine the base type.
  • a reporter dye 6-carboxy-fluorescein (FAM) is used for TaqMan probe of allele A of a certain allele
  • VIC is used for probe of allele B.
  • FAM 6-carboxy-fluorescein
  • the quencher suppresses the generation of the fluorescent signal of the reporter dye. If each probe hybridizes to the corresponding allele, a fluorescent signal corresponding to the hybridization is observed. That is, if the signal of either FAM or VIC is stronger than that of the other, it is determined that it is homozygous for allele A or allele B.
  • the TaqMan PCR method is useful as a method for determining the base type of many subjects.
  • the Acyclo Prime method has also been put into practical use as a method for determining base types using the PCR method.
  • the Acyclo Prime method one set of primers for genome amplification and one primer for polymorphism detection are used.
  • the region containing the polymorphic site in the genome is amplified by PCR. I do. This step is the same as for normal genomic PCR.
  • the obtained PCR product is annealed with a primer for detecting a polymorphism, and an extension reaction is performed.
  • Primers for polymorphism detection are designed to anneal to the region adjacent to the polymorphic site to be detected.
  • a nucleotide derivative (terminator) labeled with a fluorescent polarizing dye and blocking 3'-0H is used as a nucleotide substrate for the extension reaction.
  • FP fluorescence polarization
  • the incorporation of the nucleotide derivative into the primer can be detected by an increase in fluorescence polarization (FP) due to an increase in molecular weight. If two types of labels with different wavelengths are used as fluorescent polarization dyes, it is possible to identify whether a particular SNP is the difference or shift between the two types of bases. Since the level of fluorescence polarization can be quantified, a single analysis can determine whether an allele is homozygous or heterozygous.
  • the base type can also be determined by analyzing the PCR product with MALDI-TOF / MS. Since MALDI-TOF / MS can determine the molecular weight with extremely high accuracy, it can be used in various fields as an analysis method that can clearly discriminate small differences in protein amino acid sequences and DNA base sequences. It's being used.
  • MALDI-TOF / MS In order to determine the nucleotide type by MALDI-TOF / MS, first, a region containing the allele to be analyzed is amplified by PCR. The amplification product is then isolated and its molecular weight is determined by MALDI-TOF / MS. Since the base sequence of the allele is known in advance, the base sequence of the amplification product is uniquely determined based on the molecular weight.
  • Determining the base type using MALDI-T0F / MS requires a step of separating the PCR products. However, accurate determination of base type can be expected without using labeled primers or labeled probes.
  • the present invention can also be applied to simultaneous detection of polymorphisms at a plurality of locations. 2 9
  • the base type of a polymorphic site is determined using a lis type restriction enzyme.
  • a primer having a recognition sequence for a lis type restriction enzyme is used for PCR.
  • General restriction enzymes (type II) used for genetic recombination recognize specific nucleotide sequences and cleave specific sites in the nucleotide sequences.
  • lis-type restriction enzymes recognize specific nucleotide sequences and cut sites away from the recognized nucleotide sequence. The number of bases between the recognition sequence and the cleavage site is determined by the enzyme.
  • the amplification product can be cleaved at the polymorphic site by the lis-type restriction enzyme. it can.
  • a cohesive end containing the bases of SNPs is formed at the end of the amplification product cleaved with the lis type restriction enzyme.
  • an adapter consisting of a base sequence corresponding to the cohesive end of the amplification product is ligated.
  • the adapter consists of different base sequences containing bases corresponding to the polymorphic mutation, and can be labeled with different fluorescent dyes.
  • the amplification product is labeled with a fluorescent dye corresponding to the base at the polymorphic site.
  • the amplification product can be fluorescently labeled and immobilized using the capture primer.
  • the amplification product can be captured on avidin-conjugated beads. By tracking the fluorescent dye of the amplification product thus captured, the base type can be determined.
  • a probe having a base complementary to the polymorphic site of each allele at the end is immobilized on the magnetic fluorescent beads. Both are combined so that each fluorescent allele corresponds to a magnetic fluorescent bead having a unique fluorescent signal.
  • the probe immobilized on the magnetic fluorescent beads hybridizes to the complementary sequence, a fluorescently labeled oligo DNA having a complementary base sequence in an adjacent region on the allele is prepared.
  • the region containing the allele is amplified by asymmetric PCR, the above-mentioned magnetic fluorescent bead-immobilized probe and the fluorescent-labeled oligo DNA are hybridized, and both are ligated.
  • the end of the probe for immobilizing magnetic fluorescent beads has a base sequence complementary to the base at the polymorphic site, ligation is performed efficiently.
  • the terminal bases are different due to polymorphism, the ligation efficiency of both will decrease.
  • fluorescently labeled oligo DNA is bound to each magnetic fluorescent bead only when the sample is a base species complementary to the magnetic fluorescent bead.
  • the base species is determined by collecting the magnetic fluorescent beads by magnetism and detecting the presence of fluorescently labeled oligo-DNA on each magnetic fluorescent bead.
  • Magnetic fluorescent beads can analyze the fluorescent signal of each bead using a flow cytometer. Even if gas fluorescent beads are mixed, the signal can be easily separated. In other words, “multiplexing” is achieved, in which multiple types of polymorphic sites are analyzed in parallel in a single reaction vessel.
  • the determination of the base type is realized using only three types of oligonucleotides, an allele probe, an invader probe, and a FRET probe, and a special nuclease called cleavase. Of these probes, only the FRET probe needs to be labeled.
  • Allele probes are designed to hybridize to the region adjacent to the allele to be detected.
  • a flap consisting of a nucleotide sequence unrelated to hybridization is linked to the 5 'side of the allele probe.
  • the allele probe hybridizes to the 3 'side of the polymorphic site and has a structure linked to a flap above the polymorphic site.
  • the invader probe is composed of a base sequence that hybridizes to the 5 ′ side of the polymorphic site.
  • the nucleotide sequence of the invader probe is designed by hybridization so that the 3 'end corresponds to the polymorphic site.
  • the base at the position corresponding to the polymorphic site in the Invader probe may be arbitrary. In other words, the nucleotide sequences of both the invader probe and the allele probe are designed such that they hybridize adjacent to each other with the polymorphic site therebetween.
  • the cleavase cleaves the penetrated side strand of the thus formed oligonucleotide that has formed the penetrated structure. Since the cut occurs on the intrusion structure, the flap of the allele probe is cut off as a result. Will be done. On the other hand, if the base at the polymorphic site is not complementary to the base of the allele probe, there is no competition between the invader probe and the allele probe at the polymorphic site, and no invasion structure is formed. Therefore, the cleavage of the flap by cleavase does not occur.
  • the FRET probe is a probe for detecting the flap thus separated.
  • the FRET probe has a self-complementary sequence at the 5 'end and constitutes a hairpin loop in which a single-stranded portion is located at the 3' end.
  • the single-stranded portion located on the 3 'end side of the FRET probe has a nucleotide sequence complementary to the flap, and the flap can hybridize here. Both base sequences are designed so that when the flap hybridizes to the FRET probe, a structure is formed in which the 3 'end of the flap invades the 5' end of the self-complementary sequence of the FRET probe. Cleavase recognizes and cuts invasive structures.
  • cleavage of the FRET probe can be detected as a change in the fluorescent signal.
  • the flap should hybridize to the FRET probe even if it is not cleaved.
  • the flap should hybridize to the FRET probe even if it is not cleaved.
  • two types of allele probes containing base sequences complementary to each of allele A and allele B may be prepared. At this time, the base sequences of both flaps are different base sequences. If two types of FRET probes for detecting flaps are prepared and each reporter dye can be identified, the base type can be determined by the same concept as the TacMan PCR method.
  • the advantage of the Invader method is that the only oligonucleotide that needs to be labeled is the FRET probe. For the FRET probe, the same oligonucleotide can be used regardless of the nucleotide sequence to be detected. Therefore, mass production is possible. On the other hand, since the allele probe and the invader probe do not need to be labeled, after all, reagents for dienotyping can be produced at low cost.
  • An RCA method can be cited as a method for determining a base type independent of the PCR method.
  • a method of amplifying DNA based on a reaction in which a DNA polymerase having a strand displacement action converts a circular single-stranded DNA into a type I and synthesizes a long complementary strand is called Rolling Circle.
  • RCA Amplification
  • the RCA method utilizes a DNA polymerase having a strand displacement action. Therefore, the portion that has become double-stranded by complementary strand synthesis is replaced by a complementary strand synthesis reaction started from another primer that is annealed to the 5 'side.
  • a complementary strand synthesis reaction using a circular DNA as a ⁇ type does not end in one round.
  • the complementary strand synthesis continues while replacing the previously synthesized complementary strand, producing a long single-stranded DNA.
  • the second primer is annealed to the long single-stranded DNA generated as a circular DNA as a type II, and the complementary strand synthesis is started.
  • the single-stranded DNA produced by the RCA method is a circular DNA of type II, its base sequence is a repeat of the same base sequence.
  • the continuous generation of a long single strand results in a continuous annealing of the second primer.
  • a single-stranded portion where the primer can be annealed is continuously produced without going through a denaturation step.
  • DNA amplification is achieved.
  • the base type can be determined using the RCA method. For this purpose, a linear, single-stranded padlock probe is used.
  • the padlock probe has complementary nucleotide sequences on both sides of the polymorphic site to be detected at the 5, 5 and 3 'ends. These base sequences are linked by a part consisting of a special base sequence called a packbone. If the polymorphic site is a nucleotide sequence complementary to the terminal of the padlock probe, the terminal of the padlock probe hybridized to the allele can be ligated by DNA ligase. As a result, the linear padlock probe is circularized and the RCA reaction is triggered. The reaction of DNA ligase greatly reduces the efficiency of the reaction if the terminal portion to be ligated is not completely complementary. Therefore, by confirming the presence or absence of ligation by the RCA method, it is possible to determine the nucleotide type at the polymorphic site.
  • the RCA method can amplify DNA, but does not generate a signal as it is. In addition, if only the presence or absence of amplification is used as an index, it is usually impossible to determine the base type unless a reaction is performed for each allele. Methods that improve these points for determination of base species are known.
  • a molecular beacon can be used to determine the base type in one tube based on the RCA method.
  • the molecular beacon is a probe for signal generation using a fluorescent dye and quencher as in the TaqMan method.
  • the 5'-end and 3'-end of the molecular beacon are composed of complementary nucleotide sequences and form a hairpin structure by themselves.
  • both ends are labeled with a fluorescent dye and a quencher, no fluorescent signal can be detected while the hairpin structure is formed.
  • a part of the molecular beacon is used as a base sequence complementary to the RCA amplification product, the molecular beacon will hybridize to the RCA amplification product. The fluorescence signal is generated because the hairpin structure is eliminated by the hybridization.
  • An advantage of the molecular beacon is that by using the base sequence of the backbone portion of the padlock probe, the base sequence of the molecular beacon can be shared regardless of the detection target. By changing the base sequence of the backbone for each allele and combining two types of molecular beacons with different fluorescence wavelengths, the base type can be determined in one tube.
  • RFLP utilizes the fact that mutations in the recognition site of a restriction enzyme or insertion or deletion of a base in a DNA fragment caused by treatment with a restriction enzyme can be detected as a change in the size of the fragment generated after the treatment with the restriction enzyme. If there is a restriction enzyme that recognizes the base sequence containing the polymorphism to be detected, the base at the polymorphic site can be known by the RFLP principle.
  • PCR-SSCP utilizes the fact that the secondary structure of single-stranded DNA reflects the difference in its nucleotide sequence (Cloning and
  • polymerase chain reaction single-strand conformation polymorphism analysis of anonymous Alu repeats on chromosome 11.Genomics. 1992 Jan 1; 12 (1): 139-146., Detection of p53 gene mutations in human brain tumors by single— strand conformation poation ymorpnism analysis of polymerase chain reaction products. Oncogene. 1991 Aug 1; 6 (8): 1313-1318.,
  • PCR-SSCP analysis with postlabeling., PCR Methods Appl. 1995 Apr 1; 4 (5): 275-282.
  • the PCR-SSCP method is performed by dissociating PCR products into single-stranded DNA and separating them on a non-denaturing gel. Since the mobility on the gel varies depending on the secondary structure of the single-stranded DNA, if there is a difference in base at the polymorphic site, it can be detected as a difference in mobility.
  • DGGE method denaturant gradient gel electrophoresis
  • the DGGE method is a method in which a mixture of DNA fragments is electrophoresed in a polyacrylamide gel having a concentration gradient of a denaturing agent, and the DNA fragments are separated based on differences in instability.
  • an unstable DNA fragment with a mismatch migrates to a certain denaturant concentration in the gel, the DNA sequence around the mismatch is partially dissociated into single strands due to the instability.
  • the mobility of the partially dissociated DNA fragment becomes very slow, and differs from that of the complete double-stranded DNA without the dissociated part, so that the two can be separated.
  • a region containing a polymorphic site is amplified by a PCR method or the like.
  • the amplification product is hybridized with a probe DNA whose base sequence is known to form a double strand.
  • This is electrophoresed in a polyacrylamide gel, which gradually increases as the concentration of a denaturant such as urea moves, and compared with a control.
  • a denaturant such as urea
  • the base type can be determined using a DNA array (Cell Engineering Separate Volume “DNA Microarrays and the Latest PCR Method”, Shujunsha, Apr. 20, 2000, pp97-103 “SNP Analysis Using Oligo DNA Chip”) , Shinichi Kajie).
  • a DNA array hybridizes sample DNA (or RNA) to a number of probes arranged on the same plane, and scans the plane to detect the hybridization of each probe. Will be issued. Since the reaction to many probes can be observed at the same time, a DNA array is useful for, for example, simultaneously analyzing a large number of polymorphic sites.
  • DNA arrays are composed of thousands of nucleotides printed on a substrate at high density. Usually, these DNAs are printed on the surface of a non-porous substrate. The surface of the substrate is typically glass, but a permeable membrane, such as a nitrocellulose membrane, may be used.
  • an array based on oligonucleotides developed by Affymetrix can be exemplified.
  • Oligonucleotides are usually synthesized in vitro in yeast arrays.
  • photolithographic technology Affymetrix ⁇
  • in situ synthesis of oligonucleotides by ink jet Rosetta Inpliarmatics
  • Oligonucleotides are composed of base sequences complementary to the region containing the SNPs to be detected.
  • the length of the nucleotide probe to be bound to the substrate is generally 10 to 100 bases, preferably 10 to 50 bases, and more preferably 15 to 25 bases when immobilizing oligonucleotides.
  • mismatched probes are generally used to avoid errors due to cross-hybridization (non-specific hybridization).
  • the mismatch probe forms a pair with an oligonucleotide having a nucleotide sequence completely complementary to the target nucleotide sequence.
  • Oligonucleotides consisting of a base sequence that is completely complementary to the mismatch probe are called perfect match (PM) probes.
  • a sample for dienotyping by the DNA array method can be prepared based on a biological sample collected from a subject by a method well known to those skilled in the art.
  • Biological test The fee is not particularly limited.
  • a DNA sample can be prepared from chromosomal DNA extracted from tissues or cells such as peripheral blood leukocytes, skin, or oral mucosa, tears, saliva, urine, feces, or hair of a subject.
  • Specific regions of chromosomal DNA are amplified using primers to amplify the region containing the polymorphic site to be determined.
  • a plurality of regions can be simultaneously amplified by the multiplex PCR method.
  • Multiplex PCR is a PCR method that uses multiple primer sets in the same reaction mixture. When analyzing multiple polymorphic sites, multiplex PCR is useful.
  • a DNA sample is amplified by a PCR method and the amplified product is labeled.
  • a labeled primer is used to label the amplification product.
  • genomic DNA is amplified by PCR using a primer set specific to the region containing the polymorphic site.
  • a biotin-labeled DNA is synthesized by labeling PCR using a biotin-labeled primer.
  • the biotin-labeled DNA thus synthesized is hybridized to an oligonucleotide probe on the chip.
  • the reaction conditions of the reaction solution of the hybridization can be appropriately adjusted depending on conditions such as the length of the nucleotide probe immobilized on the substrate and the reaction temperature.
  • Avidin labeled with a fluorescent dye is added to detect the hybridized DNA.
  • the array is analyzed with a scanner, and the presence or absence of hybridization is checked using fluorescence as an index.
  • the method includes obtaining a DNA containing the polymorphic site of the present invention prepared from a subject, and a solid phase on which nucleotide probes are immobilized. Make contact. Further, the base species of the polymorphic site of the present invention 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 of the present invention is not particularly limited as long as nucleotides can be immobilized. Specifically, a solid phase including microplate wells, plastic beads, magnetic particles, substrates, and the like can be used. Can be exemplified.
  • a substrate generally used in DNA technology can be suitably used.
  • substrate means a plate-like material to which nucleotides can be immobilized.
  • the nucleotide includes an oligonucleotide and a polynucleotide.
  • an allele-specific oligonucleotide (Allele Specific 01 igonuc 1 eotide / ASO) hybridization method can be used to detect a base at a specific site.
  • the allele-specific oligonucleotide (AS0) is composed of a nucleotide sequence that hybridizes to the region where the polymorphic site to be detected exists. When AS0 is hybridized to sample DNA, the efficiency of hybridization decreases if a mismatch occurs at the polymorphic site due to the polymorphism.
  • the mismatch can be detected by a Southern blot method, a method utilizing a property that a special fluorescent reagent is quenched by interlacing with a hybrid gap, and the like.
  • the mismatch can also be detected by the ribonuclease A mismatch cleavage method.
  • the present invention also provides a test agent for type 2 diabetes, comprising an oligonucleotide that hybridizes to DNA containing the polymorphic site of the present invention and has a chain length of at least 15 nucleotides. These are used for tests using gene expression as an index or tests using gene polymorphism as an index.
  • the oligonucleotide specifically hybridizes to the DNA containing the polymorphic site of the present invention.
  • the term "specifically hybridizes" as used herein means under ordinary hybridization conditions, preferably under stringent hybridization conditions (for example, Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, NY). USA, in conditions) described in the second edition 1 9 89, other data It means that cross-hybridization with DNA encoding the protein is not significantly caused. As long as specific hybridization is possible, the oligonucleotide does not need to be completely complementary to each of the nucleotide sequences described in (1) to (29) above.
  • the oligonucleotide can be used as a probe-primer in the above-described detection method of the present invention.
  • the oligonucleotide is used as a primer, its length is usually 15 bp to! OObp, preferably 17 bp 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 of the present invention.
  • the present invention provides a primer for amplifying a region containing the polymorphic site of the present invention, and a probe that hybridizes to a DNA region containing the polymorphic site.
  • primers for amplifying a region containing a polymorphic site include a primer capable of initiating complementary strand synthesis toward a polymorphic site by using DNA containing the polymorphic site as a ⁇ type. Is also included.
  • the primer can also be expressed as a primer for providing a replication origin on the third side of the polymorphic site in DNA containing the polymorphic site. The space between the region where the primer hybridizes and the polymorphic site is arbitrary.

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Abstract

Pour identifier un gène de sensibilité au diabète sucré du type 2, on examine la relation au diabète sucré du type 2 à l'aide d'un essai de cas témoin au moyen de polymorphismes simples de nucléotide (SNP) dérivés de 704 gènes candidats. Ainsi, on a identifié avec succès 29 gènes associés au diabète sucré du type 2. Selon la présence ou l'absence de polymorphismes simples du nucléotide sur ces gènes, on peut examiner le diabète sucré de type 2.
PCT/IB2004/000579 2003-02-28 2004-02-20 Gene associe au diabete sucre du type 2 et son utilisation WO2004084797A2 (fr)

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006063703A2 (fr) * 2004-12-13 2006-06-22 F.Hoffmann-La Roche Ag Polymorphisme a simple nucleotide (snp)
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WO2007032496A1 (fr) * 2005-09-16 2007-03-22 The University Of Tokushima Procédé de détermination de risque de diabète de type 2
JP2008536474A (ja) * 2004-12-09 2008-09-11 パーレジェン・サイエンシズ・インク． メタボリックシンドローム、肥満及びインスリン抵抗性のマーカー
EP2069768A2 (fr) * 2006-09-01 2009-06-17 American Type Culture Collection Compositions et procédés pour le diagnostic et le traitement du diabète de type 2
US7951382B2 (en) 2006-09-01 2011-05-31 American Type Culture Collection Methods for treatment of type 2 diabetes
US7951776B2 (en) 2006-09-01 2011-05-31 American Type Culture Collection Methods for treatment of type 1 diabetes
CN104846069A (zh) * 2015-03-25 2015-08-19 刘坤 Q-pcr法检测aqp7基因的snp的多样本多位点的扩增引物和探针及其应用
CN112094905A (zh) * 2020-10-15 2020-12-18 连步生物科技(南京)有限公司 一种用于检测糖尿病肾病相关易感基因aldob的snp的引物组和试剂盒

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008536474A (ja) * 2004-12-09 2008-09-11 パーレジェン・サイエンシズ・インク． メタボリックシンドローム、肥満及びインスリン抵抗性のマーカー
WO2006063703A2 (fr) * 2004-12-13 2006-06-22 F.Hoffmann-La Roche Ag Polymorphisme a simple nucleotide (snp)
WO2006063703A3 (fr) * 2004-12-13 2006-10-26 Hoffmann La Roche Polymorphisme a simple nucleotide (snp)
JP2008522597A (ja) * 2004-12-13 2008-07-03 エフ．ホフマン−ラ ロシュ アーゲー Ｉｉ型糖尿病に関連する単一ヌクレオチド多型（ｓｎｐ）
WO2006072654A1 (fr) * 2005-01-05 2006-07-13 Oy Jurilab Ltd Nouveaux genes et marqueurs associes au diabete sucre de type 2
WO2007032496A1 (fr) * 2005-09-16 2007-03-22 The University Of Tokushima Procédé de détermination de risque de diabète de type 2
EP2069768A2 (fr) * 2006-09-01 2009-06-17 American Type Culture Collection Compositions et procédés pour le diagnostic et le traitement du diabète de type 2
EP2069768A4 (fr) * 2006-09-01 2010-01-20 American Type Culture Collecti Compositions et procédés pour le diagnostic et le traitement du diabète de type 2
US7951382B2 (en) 2006-09-01 2011-05-31 American Type Culture Collection Methods for treatment of type 2 diabetes
US7951776B2 (en) 2006-09-01 2011-05-31 American Type Culture Collection Methods for treatment of type 1 diabetes
CN104846069A (zh) * 2015-03-25 2015-08-19 刘坤 Q-pcr法检测aqp7基因的snp的多样本多位点的扩增引物和探针及其应用
CN112094905A (zh) * 2020-10-15 2020-12-18 连步生物科技(南京)有限公司 一种用于检测糖尿病肾病相关易感基因aldob的snp的引物组和试剂盒

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