WO2004048572A1

WO2004048572A1 - Type 2 diabetes-associated gene

Info

Publication number: WO2004048572A1
Application number: PCT/JP2003/014936
Authority: WO
Inventors: Shiro Maeda; Yusuke Nakamura; Shuichi Tsukada
Original assignee: Riken
Priority date: 2002-11-22
Filing date: 2003-11-21
Publication date: 2004-06-10
Also published as: JP2004215647A; JP4444576B2; AU2003284640A1

Abstract

As polymorphisms relating to type 2 diabetes, polymorphisms in TFAP-2β gene (for example, substitution of guanine (G) at the 774-position in the first intron of the TFAP-2β gene into thymine (T), and a tandem repeat polymorphism in the first intron) are found out. Since these mutations in the TFAP-2β gene are observed at a significantly higher frequency in patients with type 2 diabetes than in normal subjects, the polymorphisms in the TFAP-2β gene are usable in detecting a subject with a hereditary trait of being liable to suffer from type 2 diabetes.

Description

Specification

Type 2 diabetes-related genes

The present invention relates to genes associated with type 2 diabetes. The present invention further provides a primer or probe for detecting a mutation associated with type 2 diabetes mellitus, a kit for detecting a mutation in a polymorphism associated with type 2 diabetes, and a kit for diagnosis of the onset of type 2 diabetes. And a preliminary method for detecting type 2 diabetes.

Conventional technology

Type 2 diabetes is a disease that affects more than 100 million people worldwide. Type 2 diabetes is thought to be caused by insulin resistance in peripheral tissues and impaired β-cell function in the islets of Langenolehans in the knee, but the exact cause and mechanism are still unknown.

Genetic factors are known to be deeply involved in the onset and progression of diabetes, and so far, such as maturity-onset diabetes of the young (MO DY) and mitochondrial diabetes. Several causative genes of diabetes have been identified that exhibit specific pathologies. However, diabetes, which exhibits a specific condition explained by these genetic mutations, is only a very small case of power. In other words, it is thought that most of the genes that affect whether a person is predisposed to diabetes are not identified. In particular, since the involvement of known diabetes-related genes in Japanese diabetic patients is low, there may be other unknown related genes.

To date, few genes have been identified that have been linked to type 2 diabetes. This is because it is very difficult to identify disease-causing alleles with classical approaches, because the effects of individual genes are so small due to the complex genetic and environmental contexts. This is believed to be the cause. Single nucleotide polymorphisms (SNPs), which are frequently detected in sequenced genomes and are useful markers for a variety of diseases, including diabetes. Using high-throughput SNP gene analysis by a combination of the Invaders method and multiplex PCR, the inventors set out genome-wide related studies using SNPs to identify loci related to diabetes. proceeding.

Many type 2 diabetics are thought to develop a combination of genetic factors and environmental factors (such as obesity, lack of exercise, and a fatty diet). Therefore, by pre-diagnosing the genetic factors involved in diabetes and paying attention to the diet and exercise of those who are more likely to have diabetes before the onset of the disease, the onset of diabetes can be anticipated. Can be prevented. Therefore, detection of polymorphisms associated with type 2 diabetes in order to detect in advance those who are genetically likely to have diabetes and to elucidate the mechanism of diabetes onset genetically Is required.

Disclosure of the invention

The present invention has been achieved in view of the above circumstances, and has as its object to provide information on mutations in a gene associated with type 2 diabetes. It is another object of the present invention to provide a method and a kit for detecting a person who is likely to develop type 2 diabetes before the onset using a gene associated with type 2 diabetes.

We genotyped more than 55,000 single nucleotide polymorphism (SNP) loci on the genome to identify genes associated with type 2 diabetes. As a result, the significant association between polymorphisms in the intron and type 2 diabetes indicates that the gene encoding transcription factor activating protein 28 (TFAP-26) present on chromosome 6p12 is a promising candidate. Led to the present invention. The TFAP-2β gene refers to a region containing not only the coding region of TFAP-2β but also introns, 5, flanking, 3, and flanking.

That is, the present invention provides a method for detecting a person who is susceptible to type 2 diabetes. A polynucleotide comprising the DNA sequence represented by 1 is provided. This polynucleotide is more often found in people with type 2 diabetes, their reserves, or those who are genetically susceptible to type 2 diabetes than in normal individuals who have the first intron region of the TFAP-26 gene. It is thought to have.

In this specification, the term “polynucleotide” is used in a broad sense including what is generally called “oligonucleotide”.

Further, the present invention provides a method for detecting a person susceptible to type 2 diabetes, wherein the 774th guayun (G) of the first intron of the human TFAP-2β gene represented by SEQ ID NO: 2 is replaced with thymine (Τ). Provided is a polynucleotide comprising at least 7 base sequences or the entire sequence of the substituted polynucleotide, wherein the polynucleotide comprises at least a base at position 774. SEQ ID NO: 2 shows the polynucleotide sequence S of the first fftron of human TFAP-28.

Here, the position of the base is counted from the 5 'side to the 3' side of the first intron in the DNA. For example, the 774th base is a base in DNA located 774 bases downstream from the 5 'side of the first intron.

The polynucleotide in which the 774th guayun (G) of the first intron of the human TFAP-26 gene is substituted with a thymine (Τ) has one or several bases at positions other than the 774th base. May be deleted, inserted or substituted.

In addition, this effort is possible, if the 774th base in the first ^ T-tron of human TFAP-26 is thymine (T), a fragment of any length is possible if necessary. The present invention also relates to a probe for detecting the above-mentioned polynucleotide. Furthermore, the present invention relates to a probe of SEQ ID NO: 3 for detecting that the 774th base of the first intron of the human TFAP-28 gene is thymine (T), and a first intron of the human TFAP-26 gene. The probe provides the probe of SEQ ID NO: 4 for detecting that the 774th base is guanine (G). Using these probes, it is possible to detect mutations due to the substitution of guayun (G) for thymine (T>) at the 774th base in the first ^ ftrontron region of human TFAP-28. is there.

The present invention further provides a guanine (G) thymine (Τ) at the 774th base of the first intron of the human TFΑII-2 gene, which comprises the probes of SEQ ID NO: 3 and SEQ ID NO: 4. And a kit for detecting the substitution to the kit. The present invention also provides a pre-type 2 diabetes diagnosis kit comprising such a probe. These kits can detect people who are likely to have type 2 diabetes. These kits may further include the probe of SEQ ID NO: 5. The kit further containing the probe of SEQ ID NO: 5 can be used for invader single-angle analysis, which is a rapid polymorphism detection method without involving DNA amplification.

The kit containing the probe and the probe of the present invention can be obtained by using the polynucleotide of SEQ ID NO: 1 or the first intron of the human TFAP-28 gene represented by SEQ ID NO: 2 as shown above. A polynucleotide comprising 7 or more nucleotides or the entire nucleotide sequence of a polynucleotide in which guanine (G) is substituted by thymine (T), and which is complementary to a polynucleotide comprising at least the 774th base Including those that can detect various polynucleotides.

Furthermore, the present invention provides a primer used for amplifying the above-mentioned polynucleotide. As the primer, any primer having any base length and sequence can be used as long as the target polynucleotide is amplified. Preferably, the primers of SEQ ID NO: 6 and SEQ ID NO: 7 can be used.

Further, the present invention provides a kit for detecting the substitution of guan (G) for thymine (T) at the 774th base of the first intron of the human TFAP-28 gene containing the above primer. . The present invention also provides a pre-onset diagnostic kit for type 2 diabetes comprising the above primer. These kits make it possible to detect people who are likely to have type 2 diabetes. Further, the present invention relates to (GYG G VMKAG) _n (where A is adenine, G is guanine, C is thymine, Y is thymine or cytosine, M is adenine or cytosine, K is guanine or thymine, and V is adenine. Or guanine or cytosine, wherein n is 8 or 9.) provided is a polynucleotide comprising a tandem repeat polymorphism represented by the formula: In addition, a primer for detecting such a tandem repeat polymorphism is provided. Here, the primer can have any base length and sequence, as long as the target tandem repeat is detected, and is not particularly limited.

Then, the present invention discriminates whether the tandem repeat polymorphism of the first intron of the human TFAP-2B gene containing the above primer is (GYG GVMKAG) _{8 to} 9 or (G YG GVMKAG) ιο Provide a kit for Further, the present invention also provides a pre-type 2 diabetes onset diagnostic kit comprising the above probe or primer. These kits can detect individuals genetically susceptible to type 2 diabetes.

Furthermore, the present invention relates to a polymorphism or tandem repeat polymorphism in which one or several bases are deleted, inserted or substituted, wherein the polymorphism has a susceptibility to type 2 diabetes. And a polynucleotide comprising a polymorphism in the human TFAP-26 gene.

The above polymorphisms include, for example, the substitution of a thymine (Τ) for an adenylate (位) from a position 512 in the 5 ′ flanking region of the human TFAP-26 gene, Substitution of guanine (G) to thymine (Τ) at base, 8 or 9 tandem repeats of GYG GVMKAG in the DNA sequence of the first intron, adenine at base 209 of the first intron ( Substitution of A) for cytosine (C), substitution of cytosine (C) for guanine (G) at bases 58 and 58 of the second intron, 514th thymine (T) of third intron For example, the substitution of the third intron with the cytosine (C) at position 234 of the third intron with thymine (T). Here, 5, the base at position 512 of the flanking region refers to a base located 5 ′ upstream 5 ′ from the transcription start point.

In addition, the present invention relates to the substitution of thymine (T) with adenine (A) at the -512 position of the 5 'flanking region in the human TFAP-26 gene, and the 774th base of the first intron. Of guanine (G) to thymine (T) in E. coli, 8 or 9 tandem repeats of GY G GVMKA G in the DNA sequence of the first intron, and adenine of the 209rd base of the first intron Substitution of (A) for cytosine (C), substitution of cytosine (C) for guan (G) at position 58 of the second intron, 514th thymine (T) of third intron And a probe for detecting the substitution of cytosine (C) with thymine (T) at position 214 of the third intron.

Probes for detecting the above substitution are exemplified below.

As a probe for detecting a polynucleotide having a substitution of thymine (T) with adenine (A) at the base at position 512 of the 5 'flanking region, a group consisting of SEQ ID NOS: 44 to 46 One or more probes selected from:

A probe for detecting a polynucleotide having a guanine (G) to thymine (T) substitution at the 774th base of the first ^ f-tron is selected from the group consisting of SEQ ID NOS: 3 to 5. One or more probes.

The probe for detecting a polynucleotide having a substitution of adenine (A) at the 209rd base of the first intron with cytosine (C) is selected from the group consisting of SEQ ID NOS: 7 to 49. One or more probes may be included.

Probes for detecting a polynucleotide having a cytosine (C) to guanine (G) substitution at position 58 of the second intron are selected from the group consisting of SEQ ID NOS: 50 to 52 One or more probes may be included.

As a probe for detecting a polynucleotide having a substitution of cytosine (C) from thymine (T) at position 514 of the third intron, SEQ ID NO: 53-5 One or more probes selected from the group consisting of 5.

Probes for detecting a polynucleotide having a substitution of cytosine (C) at position 2134 of the third intron with thymine (T) are selected from the group consisting of SEQ ID NOS: 56-58 One or more probes are included.

Further, the present invention provides a method for substituting the nucleotide at position −512 in the 5 ′ flanking region of the human TFAP-2B gene with thymine (T) to adenine (A), Substitution of guayun (G) to thymine (T) in base, GY G GVMKAG in tandem repeat of 8 or 9 times in DNA sequence of first intron, adenine of 209th base in first intron ( Substitution of A) for cytosine (C), substitution of cytosine (C) for guanine (G) at bases 58 and 58 of the second intron, and substitution of guanine (G) for the 514th base of the third intron The present invention relates to a primer for detecting the substitution of cytosine (C) with thymine (T) from cytosine (C) at position 214 of the third intron.

Primers for detecting the above-mentioned substitution are exemplified below.

As a primer for detecting a polynucleotide having a substitution of thymine (T) with adenine (A) at the base at position 512 of the 5 'flanking region, a pair consisting of SEQ ID NOS: 59 and 60 was used. Primers.

As a primer for detecting a polynucleotide having a guanine (G) to thymine (T) substitution at the 774th base of the first intron, a pair of primers consisting of SEQ ID NOs: 6 and 7 is mentioned. Can be

Examples of primers for detecting a polynucleotide having 8 or 9 tandem repeats of GYGGVMKAG in the DNA sequence of the first intron include a pair of primers consisting of SEQ ID NOS: 8 and 9.

As a primer for detecting a polynucleotide having a substitution of adenine (A) at the 209rd base of the first intron with cytosine (C), a pair of primers consisting of SEQ ID NOs: 61 and 62 Primers. Examples of a primer for detecting a polynucleotide having a substitution of cytosine (C) with guanine (G) at the 58th base of the second intron include a pair of primers consisting of SEQ ID NOS: 63 and 64. Can be

Examples of primers for detecting a polynucleotide having a substitution of cytosine (C) from thymine (T) at position 514 of the third intron include a pair of primers consisting of SEQ ID NOS: 65 and 66. Can be

As a primer for detecting a polynucleotide having a substitution of cytosine (C) at position 214 of the third intron with thymine (T), a pair of primers consisting of SEQ ID NOs: 67 and 68 was used. No.

The present invention also includes a polynucleotide comprising an antisense sequence or a phase capture chain of the polynucleotide comprising the above mutation.

Furthermore, the present invention provides a method for detecting a mutation associated with the possibility of afflicted with type 2 diabetes by detecting a polynucleotide containing the above mutation, or a polynucleotide containing a complementary strand thereof or an antisense sequence thereof.

The present invention provides a diagnostic kit for the onset of type 2 diabetes mellitus, which comprises a reagent for measuring the expression level of the TFAP-2i3 gene. Using this kit, the expression level of the TFAP-2] 3 gene is measured, and people with higher expression levels than normal individuals are subjected to more precise tests as those who are more likely to develop type 2 diabetes. Can be achieved.

As described below, polymorphisms in the human TFAP-2B gene, particularly those specifically identified above, are closely associated with type 2 diabetes. Prior to the onset of diabetes, the detection method of the present invention detects the presence or absence of mutations in these polymorphisms, and those who are determined to have the mutations are genetically predisposed to type 2 diabetes In addition, it is possible to instruct students to be careful about diet and exercise before the onset of symptoms, and if necessary, take preventive treatment to prevent the development of type 2 diabetes. It is also possible to judge whether the relatives of a diabetic patient have inherited the predisposition to diabetes. In addition, the presence or absence of mutations in other genes suggested to be associated with diabetes Detection will enable the group at higher risk for type 2 diabetes to be detected with greater accuracy.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the polymorphisms identified in the human TFAP-2B gene.

FIG. 2 shows the results of RT-PCR for examining the expression of TFAP-2β in human tissues.

FIG. 3 shows the results of RT-PCR for examining the expression of TFAP-28 in adipocytes after differentiation induction.

FIG. 4 (a) shows the results of RT-PCR for the adivocaine gene in cultured human adipocytes infected with adeno-TFA-28 or adeno-LacZ.

Fig. 4 (b) shows the measurement of TNF-α and hs-CRP in human plasma. The numerical values represent the average soil standard error. In the figure, X represents 8 or 10 repetitions, and Y represents 9 repetitions of VNTR in the first intron of TFAP-28. <0.05 vs. XX.

Figure 5 shows the effect of polymorphism in the first intron of TFAP-2B on enhancer activity.

FIG. 6 shows the results of gel shift assay using TFAP-26 and nuclear extracts of 3T3-L1 cells after induction of differentiation.

FIG. 7 shows the results of gel shift assay using nuclear extract of 3T3-L1 cells after induction of differentiation and TFAP-2B.

FIG. 8 shows the results of gel shift assay using TFAP-28 and a nuclear extract of 3T3-L1 cells after differentiation induction when a competitor was further added. Figure 9 shows insulin-stimulated uptake of 2-dexoxyglucose (2DOG) in differentiated 3T3-L1 adipocytes transfected with adeno-TFAP-2i3 or adeno-LacZ. It is a graph shown. data Was indicated by the average soil standard deviation obtained from six independent experiments. Closed circles are cells transfected with adeno-TFAP-2, and closed squares are cells transfected with adeno-LacZ. * P = 0.008 for adeno-Lac Z.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention relates to polynucleotides having polymorphisms associated with type 2 diabetes. As used herein, a polymorphism refers to a state in which two or more alleles at a certain locus are present in the same population and the frequency is 1% or more. Single nucleotide polymorphism (SNP), restriction enzyme Includes truncated fragment length polymorphism (RFLP), tandem repeat polymorphism, microsatellite polymorphism, insertion / deletion polymorphism, etc.

Specifically, substitution of the thymine (T) at position 612 of the 5 'flanking region of the human TFAP-26 gene with adenine (A), and the nucleotide at position 774 of the first ^ f Substitution of guanine (G) for thymine (T), 8 or 9 tandem repeats of GYGGVMKAG in the DNA sequence of the first intron, adenine (A) at the 2093th base of the first intron to cytosine (C) Substitution, substitution of cytosine (C) with guayun (G) at position 58 of the second intron, substitution of 514th thymine (T) with cytosine (C) at position 514 of the third intron, A polynucleotide which is any one of the substitution of cytosine (C) to thymine (T) at position 2134. The polymorphism at the 774th base of the first intron, the tandem repeat polymorphism of the first intron, and the 514th polymorphism of the third intron were found by the present inventors for the first time. SEQ ID NOs: 73 and 74 show the sequences of eight or nine tandem repeats of GYGGVMKAG in the DNA sequence of the first intron thought to be associated with type 2 diabetes. SEQ ID NO: 75 shows the sequence of 10 tandem repeats of GYGGVMKAG in the DNA sequence of the first intron possessed by a normal individual. The polynucleotide of the present invention is a double-stranded DNA comprising single-stranded DNA and its complementary strand. It may include DNAs containing an antisense strand and partially having a modified base. In addition, it includes those in which one or several bases are deleted, inserted or substituted at positions other than the above polymorphisms.

Such a polynucleotide is one of the causative genes that confer a predisposition to type 2 diabetes. Therefore, probes, primers and kits for determining whether or not they have such a polynucleotide should be used for genetic diagnosis to determine the inheritance of a group who is likely to have type 2 diabetes and relatives with type 2 diabetes. Is possible.

The probe of the present invention is not particularly limited as long as it is a probe substantially complementary to a part of the sequence of the first intron region of the human TFAP-28 gene. Here, “substantially complementary” means that it is sufficient to be complementary to the sequence of the first intron region of the human TFAP-2B gene. In addition, these probes may contain sequences on the 5 'and / or 3' side that are not complementary to those of the first intron region of the human TFAP-26 gene, As long as it can hybridize with the sequence of the region, it is included in the probe of the present invention. For example, a sequence to which an arbitrary sequence is added for easy detection can be used. Further, a 5′-end labeled one can be used for easy detection. Examples of such a label include biotin, fluorescence, and ³² P.

The primer of the present invention is not particularly limited as long as it is a primer pair substantially complementary to a part of the upstream and downstream sequences of the polymorphism of the human TFAP-2β gene. Here, “substantially complementary” means that it is sufficient to be complementary to the sequence of the first intron region of the human TFAP-2β gene to such an extent that it can be annealed. For example, these primers may contain a sequence that is not complementary to the sequence of the first intron region of the human TFAP-26 gene on the 5 ′ side or 3 ′ side, or both, but may have the same sequence as the first intron region. Included in primers of the present invention as far as possible Shall be In addition, a primer having a mismatch sequence that is not complementary to the human TFAP-2B gene can be used to prevent non-specific amplification or to introduce an appropriate restriction enzyme recognition site.

Examples of suitable primers include the primers of SEQ ID NOs: 6 and 7. Using a set of primers of SEQ ID NOS: 6 and 7, a polynucleotide having a mutation in a polymorphism associated with type 2 diabetes can be amplified by PCR or the like.

The method for detecting a polynucleotide having a polymorphism associated with type 2 diabetes is not particularly limited. As a method for detecting a mutation in a polymorphism, for example, the Sanger method, NO. The nucleotide sequence may be determined directly by iro-sequencing, etc., but Southern plotting, PCR, oligonucleotide-specific oligonucleotide hybridization, Invader Atssay, TaqMan PCR, SinPer method, Indirect detection without sequencing like OF method, mass spectrometry, RCA method, DNA chip, ARMS, BAMPER, RFLP, PCR—RFLP, PCR—SSO, PCR—SSCP, allele-specific PCR You may.

The term “detection” as used in the present invention includes not only the case where the target sequence is directly detected but also the indirect detection by detecting a sequence complementary to the target sequence. In the sense, the term "detection" will be used. For example, detection of a substitution of guanine (G) for thymine (T) at base 774 of the first intron would result in the replacement of the base at that position with cytosine (C) to adenine (A). It may be based on detecting that it has been replaced.

The kit of the present invention can specifically hybridize a polynucleotide having a mutation in a polymorphism associated with type 2 diabetes in the human TFAP-2B gene in distinction from a polynucleotide having no such mutation. It includes a probe or a primer capable of amplifying only a polynucleotide having such a mutation. The reagent included in the kit can be appropriately changed by a method capable of detecting base substitution. For example, if a method for detecting a mutation that synthesizes a complementary strand, d Includes dNTPs, dNTPs, DNA synthases, buffers, etc., and contains appropriate restriction enzymes for detection based on cleavage of restriction enzymes such as RFLP.

The kit for detecting the onset of type 2 diabetes according to the present invention detects a mutation in the human TFAP-2B gene, for example, the substitution of the 774th base in the first intron from guan (G) to thymine (T). It is for. The kit for detecting the onset of type 2 diabetes mellitus of the present invention is effective for finding a person who is constitutionally susceptible to type 2 diabetes and for early treatment of type 2 diabetes.

The DNA for which the presence or absence of a mutation is detected by the present invention can be obtained from human hair, blood, body fluid, saliva, cultured cells, excised tissue, and the like, and is not particularly limited. The gene expression level can be measured by a method known to those skilled in the art (for example, “New Genetic Engineering Handbook”, 3rd revision, edited by Masami Matsumura, Masaru Yamamoto, September 10, 1999, Yodosha See). For example, Northern blotting, RNase protection assay, quantitative RT-PCR, and the like can be used, but are not limited thereto. In addition, examples of a reagent for measuring the expression level of the TFAP-2 / 3 gene include a probe or primer that hybridizes with the TFAP-2] 3 gene.

Hereinafter, the present invention will be described in more detail with reference to Examples, but these are not intended to limit the present invention.

Example

Example 1

Obtain written informed consent from Japanese patients with type 2 diabetes who regularly visit outpatient clinics at Shiga Medical University, Tokyo Women's Medical University, Juntendo University, Kawasaki Medical University or Iwate Medical University Then, a DNA sample was prepared.

DNA extraction was performed by the following standard phenol-chloroform precipitation method.

About 10 ml of peripheral blood is centrifuged at 3000 rpm for 5 minutes to remove serum, then red blood The sphere lysis solution was added and left at room temperature for 20 minutes. Then, the mixture was centrifuged at 3000 r for 5 minutes to leave a precipitate (white blood cell component), and the supernatant was removed. The precipitate was treated with proteinase K at 37 ° C for 4 hours or more to remove the protein. After adding an equal volume of phenol, the mixture was centrifuged at 3000 rpm for 10 minutes. After centrifugation, the upper layer (aqueous layer) was transferred to a new tube and treated with phenolic-cloth-form. The mixture was further centrifuged, the aqueous layer was transferred to a new tube, subjected to black hole processing, and the upper layer (aqueous layer) was transferred to a new tube, and DNA was precipitated with isopropanol. The DNA precipitated by centrifugation was treated with 70% ethanol and then dissolved in a TE solution.

First, the genotype of 58267 single nucleotide polymorphisms (SNPs) in type 2 diabetic patients was analyzed using 188 of the obtained DNA samples, and the allele frequency or genotype frequency was determined for diabetic patients. Not compared (control). The SNPs for genotype identification experiments were randomly selected from IMS-JST Japan SNP database (http://sn.pims.u-tokyo.ac.jp). The genotype at each SNP locus was determined by Invader Assy.

Before performing the invader assay, the target site was amplified by multiplex PCR (Multiplex PCR). Multiplex P CR is of _{_{16.6 mM (NH4) 2 S0 4}} , 67 mM of ris (pH 8.8), 6.7 mM of MgCl _2, 10 mM 2-mercaptoethanol, 6.7Myumyu of EDTA, 1.5 mM of dNTPs, LOxTaq mix (2.5 U / pl Taq polymerase, 31.25 U / pl Taq antibody), 0% or 10% DMSO, 0.25 μΜ in each primer mixture, denaturation 95 ° C, annealing 60 ° C, The cycle of elongation at 72 ° C was repeated 40 times.

The obtained PCR product was diluted, dispensed into 384 plates, air-dried, and 3 μl of the Invader reaction solution and probe mix were added. After reacting at 63 ° C for 20 minutes, the fluorescence was measured to determine the genotype.

Of the 58267 SNPs analyzed, allele frequencies and / or gene frequencies at 1496 SNP loci were significantly different between diabetic patients and controls (P <0.01). A second invader assay was performed in another group of 758 diabetic patients (not including the above 188) for SNPs showing significant differences between this diabetic patient and the control. As a result, SNPs showing significant differences between diabetic patients and controls in the TFAP-26 gene were identified and genotyped.

For the DNA sequence of the genomic region containing the TFAP-26 gene, PCR primers were designed based on the information of GenBank (Accession No. NT-007592), and the appropriate fragment of the genomic DNA was amplified. SNPs were identified by the sequence method. The primers used are as shown in SEQ ID NOs: 10 to 43. Repetitive elements were excluded from analysis using the REPEAT MASKER computer program according to the method described by Sekί et al. The SNPs identified above were genotyped by Invader Atsey. After amplifying the target site by the PCR method, the VNTR locus was determined using the Applied Biosystems AB IPRISM3700 automatic fDNA sequencer and GeneScan software (Geno Hyperprogram). The analysis was performed by judging the difference between the two. PCR conditions were: 10 mM ris-HCl (pH 8.3), 50 mM KC1, 1.5 mM MgCl ₂ , 250 pM dNTP, 5 U Taq polymerase, denaturation 94 ° C, annealing 60 ° C, The cycle of elongation at 72 ° C was repeated 30 times, and the primers used were those of SEQ ID NO: 8 and SEQ ID NO: 9.

Relevance, haplotype frequency and Hardy ■ Statistical methods for determining Weinberg equilibrium and calculation of LD coefficient (D ') were performed by known methods. In testing transfusion experiments, comparisons between three or more groups are analyzed by one-way analysis of variance and then used to estimate the statistical differences between the two groups. She went the way.

As a result, the genotype distribution of the landmark S Ν ニ in the ninth intron of the TFAP-2β gene on chromosome 6p12 is closely related to type 2 diabetes. (Chi-square value chi ² = 15.2, risk factor ρ = 0.00007). According to the NCB I database, six other genes were predicted to be located 250 kb upstream or downstream of the TFA A-26 gene. Analysis of 18 other SNP loci present in these genes in 188 patients showed no significant association with type 2 diabetes (1> 0. 0 1, data not shown). Therefore, the TFAP-28 gene itself was considered to be a gene closely related to type 2 diabetes.

Screening TFAP-28 for other polymorphisms identified 28 SNPs, 8 insertion / deletion polymorphisms, and 4 tandem repeat polymorphisms. FIG. 1 shows the polymorphisms identified in the TFAP-2β gene. In Figure 1, those marked with * are polymorphisms due to insertion Ζ deletions, those marked with † are tandem repeat polymorphisms, and those marked with j: are genome-wide. The landmark SNPs used for screening and those without symbols are SNPs found in subsequent screen jungs. No SNPs were found in the coding region of the TFAP-28 gene.

In a total of 964 patients and 922 controls, these genotypes were identified using the probes of SEQ ID NOs: 3-5, 4: -58 and the primers of SEQ ID NOs: 6, 7, 59-68 as described above. By identifying such invaders using Atsusei or PCR, it was found that some of these mutations were significantly correlated with type 2 diabetes associated with obesity (IE¾BMI≥25). This correlation has a strong correlation especially with the polymorphism (variable number of tandem repeat: VNTR) due to the difference in the number of repetitions of the sequence in which the 9-base unit sequence is repeated in series (chi square value chi ² = 1 1 0, risk factor ρ = 000 9, odds ratio = 1.53, 95% confidence interval 1.19-1.98), and a strong correlation between the two SNPs in the first intron. (Chi-square value chi 2 = 1 1.2 p = 0.0008, odds ratio = 1.54, 95% confidence interval 1.20 to 1.99. 9 , P = 0.0005, o Odds ratio = 1.56, 95% confidence interval 1.21-2.02). Table 1 summarizes these results.

Table 1. Association between TFAP-2 j8 gene polymorphism and type 2 diabetes

5 'Franking' 1st 1st 1st 2nd 3rd 3rd Intron Intron Intron Int. Intron Intron

-512 +774 VNTR +2093 +58 +514 +2134 Wild type / mutant (Τ / Α) (G / T) (10 or (A / C) (C / G) (T / C) (C / T )

8/9)

Type 2 diabetes *

major / major 322 (0.35) 405 (0.46) 406 (0.45) 414 (0.45) 325 (0.43) 381 (0.42) 380 (0.42) major / minor 456 (0.50) 396 (0.44) 412 (0.45) 411 (0.45) 333 (0.44) 424 (0.46) 423 (0.46) minor / minor 139 (0.15) 88 (0.10) 92 (0.10) 93 (0.10) 102 (0.13) 1 12 (0.12) 1 13 (0.12)

. n l 917 889 910 918 760 917 916

Type 2 diabetes (ΒΜΪ ≥25) *

major / major 111 (0.32) 138 (0.41) 141 (0.42) 140 (0.41) 121 (0.41) 131 (0.38) 130 (0.38) major / minor 178 (0.52) 159 (0.48) 160 (0.47) 164 (0.48) 132 (0.45) 166 (0.48) 165 (0.48) minor / minor 53 (0.16) 36 (0.1 1) 37 (0.1 1) 38 (0.1 1) 41 (0.14) 46 (0.13) 47 (0.14)

342 333 338 342 294 343 342 Control *

major / major 374 (0.42) 464 (0.52) 458 (0.52) 472 (0.52) 423 (0.48) 427 (0.48) 426 (0.48) major / minor 402 (0.45) 352 (0.40) 338 (0.39) 361 (0.40) 373 (0.42) 366 (0.41) 369 (0.42) minor / minor 124 (0.1) 73 (0.08) 79 (0.09) 75 (0.08) 93 (0.10) 90 (0.10) 89 (0.10) mouth ηΤ 900 889 875 908 908 889 883 884

5 'Franking' 1st 1st 1st 2nd

Intron Intron Intron Intron Int. Intron

-512 +774 VNTR +2093 +58 +514 +2134 Wild type / mutant (T / A) (G / T) (10 or (A / C) (C / G) (T / C) (C / T ) Is 8/9)

Type 2 diabetes vs. control

2 (2 X 3) 8.0 8.0 10.7 8.9 5.5 8.6 8.6

5.9 7.3 8.2 8.3 5.6 7.9 8.2

X 2 (major / major 8.0 7.8 10.7 8.7 3.8 8.4 8.2 vs.)

χ 2 (minor / minor 0.7 1.5 0.6 1.9 3.4 1.8 2.3 vs.)

p (2 X 3) 0.02 0.02 0.004 0.01 0.07 0.01 0.01

P (aryl) 0.02 0.007 0.004 0.004 0.02 0.005 0.004 p (major / major vs.) 0.005 0.005 0.001 0.003 0.05 0.004 0.004 p (minor / minor vs.) 0.4 0.21 0.43 0.17 0.06 0.17 0.13

Type 2 diabetes (BMI≥25) vs. control

Size 2 (2 X 3) 8.7 11.4 11.0 12.4 4.8 10.7 11.0 2 (aryl) 6.2 10.3 9.1 1 1.4 4.9 10.0 10.7 χ 2 (major / major 8.6 11.2 11.0 12.1 3.7 10.3 10.3 s.)

χ 2 (minor / minor 0.6 2.0 1.0 2.5 2.7 2.6 3.4 vs.)

ρ (2 Χ 3) 0.01 0.003 0.004 0.002 0.09 0.005 0.004 p (aryl) 0.01 0.001 0.002 0.0007 0.03 0.002 0.001 p (major / major vs.) 0.003 0.0008 0.0009 0.0005 0.06 0.001 0.001 p (minor / minor vs.) 0.43 0.15 0.31 0.12 0.10 0.11 0.07

In the table, major / major and major / minor minor / minor represent wild-type homozygous, wild-type and mutant heterozygous, and mutant homozygous polymorphisms, respectively. Numbers in katzko indicate frequency.

From this table, it can be seen that the SNP of the first intron +774 (the 774th in the first intron; ^ group) with the lowest risk factor p, the VNTR of the first intron, and the +2093 SNP of the first ^ f intron ( The first intron (base 2093) was very likely to be closely associated with type 2 diabetes.

Example 2: Expression of TFAP-2B in human tissues

In mice, mTFAP-26 expression is significantly reduced after birth, but transcripts are detected in the kidney even in 2 month old mice. Mice deficient in TFAP-2β die within one or two days of life due to renal failure due to polycysts. In humans, mutations in TFAP-26 cause char syndrome, which is characterized by patent ductus arteriosus and varying degrees of facial morphogenesis and hand abnormalities. These features imply that TFAP-28 plays an important role in embryonic development of various tissues. However, to date, there is no evidence for a role for TFAP-2B in type 2 diabetes.

To investigate the possible involvement of TFAP-2β in type 2 diabetes, the expression pattern of TFA Ρ-2β was determined by reverse transcription (RT) PCR using RNA obtained from multiple human tissues. Investigated by. In addition, human cDNAs derived from multiple tissues were obtained from C1onthechInc. (PaloA1to, California, USA). First- or double-stranded cDNA was amplified by PCR. HAP2RT-F (5'-CCAAATCGTGACTTCTCTAATGA-3,) was used as the forward primer, and hAP2RT-R (5, -GTAACGTGACATTTGCTGCTTTG-3,) was used as the reverse primer (SEQ ID NO: 69, 70).

RT-PCR was performed according to the following procedure. All EA with oligodT primer and reverse transcriptase By reacting for 60 minutes, cDNA was obtained. The resulting cDNA was amplified using primers specific for the target site. The PCR conditions at that time are as follows. lOmM Tris-HCl (H 8.3), 50 mM KC1, 1.5 mM MgCl ₂ , 250 μΜ dNTP,

Denature in a 5 U Taq polymerase reaction 94. The cycle of C, annealing 60 ° C and extension 72 ° C was repeated 30 times. The amplification products were analyzed by agarose gel electrophoresis.

Figure 2 shows the results of RT-PCR. This result closely resembles the pattern previously reported by Moser et al. ("Genes & Dev." 11,. 1983-1948 (1997)), but previous studies, including Moser et al. High levels of TFAP-26 expression were observed in adipose tissues that had not been reported.

Example 3: Expression of TFAP-2B in adipocytes

Murine 3T3-L1 cells were obtained from the Human Science Research Resource Bank (Daiwa, Japan), cultured until confluent, and induced to differentiate into adipocytes. At 0, 3, 5, 7 or 10 days after induction of differentiation, the expression of TFAP-2β was examined by RT-PCR. First-strand cDNA was prepared by reverse transcription of total RNA extract from mouse 3T3-L1 cells using SuperscriptII reverse transcriptase (Invitrogen) by oligo dT priming. MAP 2 RT-F (5, — GCGTCCT CAGAAGAGCCAAATC-3 ') was used as the forward primer, and mA P 2 RT-R (5' -GTGCGTGATGAGACTGAAGTGC-3 ') was used as the reverse primer (SEQ ID NOs: 71, 71, respectively). 72). The results are shown in Figure 3. Expression of mouse TFAP-26 in adipose tissue was found to increase with the degree of differentiation.

Example 4: Enhancer effect of polymorphism

Subsequently, the effects of SNP and VNTR, which are presumed to be associated with diabetes, on 3T3-L1 cells at different stages of differentiation were examined.

Example 1 No. 774 of the first intron, which was suggested to be closely related to type 2 diabetes A fragment containing a portion corresponding to the SNP of the base of the eye and VNTR (GYGGCVMKAG) of the first intron was prepared. The prepared fragment contains a sequence containing one or three copies of the sequence corresponding to nucleotides 766 to 785 of the first intron of a normal human, and a thymine (T) at nucleotide 774 which is thought to be related to diabetes. 3 OO bp sequence containing 1 copy of the sequence corresponding to bases 766 to 785 of the first ^ f intron and 10 repeats of VNT R of the first intron, and 774th This sequence contains one copy of the thymine (T) SNP and nine repeats of the VNTR of the first intron.

Various DNA fragments of the prepared TFAP-28 gene were subcloned into a PGL3_ promoter vector (Promega) upstream of the SV40 promoter. 3 T 3—L 3 or 5 days after the induction of differentiation of the cells, ribosome transfection (FuGene 6, Roche) was used to control p RL— TK (P rome Each construct was introduced into cells together with ga). Forty-eight hours after transfection, luciferase activity was measured by Dua1 LuciferaseReporterSySystem (Promega).

Fig. 5 shows the results. In Fig. 5, the activity value of luciferase is represented by the mean ± | * Is ρ <0.01 vs promoter alone, # is ρ <0.001 vs other constructs. In differentiated adipocytes, a fragment containing one copy of the 774th thymine (Τ) SNP and nine repeats of the VNTR of the first intron was shown to have higher enhancer activity than the other fragments. In the undivided cells, there was no significant difference in the enhancer activity between the normal human fragment and the diabetes-related allele.

Example 5: Gelshift Atsee

Since the fragment corresponding to the SNP identified in Example 1 was similar to the E2F binding motif, a gel shift assay was performed to examine the binding of the protein to this region. Was.

Nuclear extracts obtained from 3T3-L1 cells 0, 3, 5 or 7 days after differentiation induction were prepared according to the method of Sadowski et al. Nuclear extracts were preincubated with lpg of poly (dAdT) for 10 minutes at room temperature.

The probe contains a 20 bp nucleotide sequence from 766 to 785 of the first intron of the human TFAP-2B gene, a T probe whose 774th base is thymine (T), and a 774th base. Two types of G probes, which are guanine (G), were prepared. The T probe and the G probe are double-stranded probes, of which the base sequences of one strand are shown in SEQ ID NO: 76 and SEQ ID NO: 77, respectively. After incubating the T probe or G probe with the nuclear extract for an additional 15 minutes, the samples were electrophoresed on a 4% polyatarylamide gel in 0.5 x TBE. After autoradiography, four bands were observed that may reflect specific binding of the protein to DNA. Fig. 6 shows the results. The four bands are designated α, β, γ and S. Interestingly, the α band was only observed when the 774 probe containing thymine at position 774 was incubated with a nucleic acid extract obtained from differentiated 3T3-L1 cells. Figure 7 shows the results of a longer electrophoresis (4 hours) using the Τ-prop to separate the _α and β bands. From the results in Figures 6 and 7, the 774th base of the first intron is involved in the regulation of TFAP-28 expression, and thereby other genes induced by TFAP-2β in differentiated adipocytes May have been affected. Single or multiple copies of this 20 bp fragment did not show enhancer activity in 3T3-L1 cells (FIG. 5). This indicates that the 20 bp fragment containing the base at position 774 does not function alone, but may cooperate with the VNTR to regulate the expression of other genes.

In another experiment, various competitor DNAs were further added before adding the ³² P-labeled fragment of TFAP-28. From the induction of 3 T 3— L 1 cell differentiation After 7 days, nuclear extracts are preincubated with a 100-fold excess of unlabeled T-probe or G-probe corresponding to the E2F-binding consensus motif, followed by ³² P-labeled T-probe or G-probe. After electrophoresis, autoradiography was performed. Fig. 8 shows the results.

Since a 100-fold excess of unlabeled oligonucleotide corresponding to the E2F binding consensus motif could not compete with the formation of the protein-DNA complex, one or more proteins other than E2F Is thought to regulate the expression of TFAP-28.

Example 6: 2- Dokishi-D-C ³ H] glucose uptake

To investigate the role of the TFAP-20 gene in type 2 diabetes, the effect of overexpression of the TFAP-2 gene on the insulin responsiveness of differentiated adipocytes was investigated. Differentiated 3 T 3 cells more transfected Hue transfected into Adeno TFAP- 2 or Adeno L ac Z, after 50 hours, the addition of insulin at various concentrations to the culture medium, the 2-Dokishi one D- ^[3 H] glucose uptake It was measured.

The uptake of 2-deoxy D— [ ³ H] glucose was determined according to the method described in J. Biol. Chem. 277, 38863-38869. Briefly, after culturing for 16 hours in a medium containing 0.4% 8SA under starvation conditions, the cells were cultured for 1 hour with 0.4% BSA without insulin or with various concentrations of insulin. Insulin was added and the cells were cultured at 37 ° C for 1 hour. 10 1 of the substrate (2 Dokishi one D- ^[3 H] Darco one scan: 0. 2 C i, the final concentration (0. lmmo 1/1)) was added 2-Dokishi one D one ^[3 H] glucose Was measured for 5 minutes at room temperature. Protein concentration was determined by the Bradford method. Incorporation was measured four times for each experiment. The results are shown in FIG. Increased uptake of 2-dexoxyglucose in response to insulin at 2 ° ηΜ was significantly reduced in cells overexpressing TFAP-2 | 3 compared to control cells (basal TFAP-2 / 3 And L ac Z, respectively, 16 1 ± 12% vs. 19 3 ± 18%, p = 0.008). Example 7: Effect of overexpression of TFAP-2B gene on adivocaine gene expression

To understand the role of the TFAP-2B gene in the pathogenesis of type 2 diabetes, the effect of the overexpression of the TFA P-26 gene on the expression of the adivocaine gene in human adipocytes was examined. Leptin and TNF-spike genes were used as adipokine genes. The GAPDH (glyceraldehyde 3-phosphate dehydrogenase) gene was used as a control.

From the white adipocytes of humans infected with Adeno-LacZ or Adeno-TFAP-2β, total RΝ was prepared, and real-time quantitative RT-PCR was performed. The primers used are as follows. Human TNF—Q !: Sense, 5'—AGGTGCAATGCACAGCCTTC-3 '; Antisense, 5'-AGC TTCCCACACTGGGTCC—3', Human leptin: Sense, 5'—TC CAGAAAGTCCAGGATGACACCAA-3 '; Antisense, 5'-Sense 5'-AGGTGAAGGTCGGAGTCAACG-3 '; antisense 5, one GCTCCTGGAAGATGGTGATGG-3. Amplification was performed using 1 XE XT aq buffer, 200 riM dNTP blend, 800 nM each primer, 0.05 U EX T a q DNA polymerase, 2.75 ng T aq Start antibody (C 1 ontech) and Performed with 25 1 reaction volumes, including 5 ng of template. The reaction temperatures and times are as follows. 40 cycles of 2 minutes at 50 ° C, 10 minutes at 95 ° C, followed by 30 seconds at 95, 30 seconds at 63 ° C, and 30 seconds at 72 ° C.

The results of RT-PCR are shown in Fig. 4 (a). There was a clear difference between cells overexpressing TFAP-2β and control cells. In other words, cells overexpressing TFAP-2B compared to control cells showed no change in leptin expression but significantly increased TNF-Q! Expression. Was. This Therefore, increased expression of adipokine genes such as TNF-α may be caused by increased expression of TFAP-2β in differentiated adipocytes, indicating that type 2 diabetes in patients with disease-sensitive alleles Was suggested to be one of the etiologies of the disease.

Example 8

Measurement of TNF-α and highly sensitive C-responsive peptide (hs-CR—) in plasma

In order to examine in more detail the association between the overexpression of the TFAP-28 gene and the type 2 diabetes confirmed in Example 7, the plasma levels of TNF-α and hs-CRP in 225 patients were determined, and TFAP was determined. — The association with 2β genotype was investigated. hs-CRP has been reported to be involved in potential inflammation under conditions of chronically elevated adivocaine.

Measurement of plasma TNF-α was performed by enzyme-linked immunosorbent analysis using an ELISA kit (R & D systems). The measurement of hs-CRP was performed by latex immunoturbidimetry using a BN-II analyzer (Dade Behring).

The results are shown in FIG. 4b. Plasma TNF—human hs_CRP was significantly elevated in patients with disease-susceptible alleles. Although body mass has been reported to affect these parameters, BMI values did not differ between groups (data not shown).

Interestingly, in particular, plasma TNF-α and hs-CRP were significantly elevated in patients with disease susceptibility alleles. This result strongly supports the hypothesis that increased expression of adipokine genes such as TNF- is one of the causes of type 2 diabetes in patients with disease susceptibility alleles. The above experiments by the present inventors have shown that examining the polymorphism of the TFAP-26 gene is effective for diagnosing a person susceptible to type 2 diabetes. Therefore, according to the present invention, early detection of type 2 diabetes patients and the possibility of developing type 2 diabetes Can be detected at a higher frequency than previously reported. By detecting the mutation in the polymorphism of the present invention, it is possible to detect a group having a high possibility of developing type 2 diabetes before diagnosis of diabetes, and to carry out prevention or early treatment before diagnosis of diabetes. In addition, in combination with the detection of mutations in genes that have been implicated in other diabetes, it is possible to detect with greater accuracy those who are more likely to have type 2 diabetes. Using the type 2 diabetes diagnosis kit or method of the present invention, even before the onset of diabetes is confirmed, it is examined whether or not the subject has the mutation according to the present invention, even if the onset of diabetes is confirmed. This is useful because it can be estimated whether the trait is inherited.

Furthermore, according to the present invention, it was shown for the first time that TFAP-2B is expressed in differentiated adipocytes, that is, cells well known as a target of insulin and a central tissue of insulin resistance. TFAP-28 is specifically expressed in an adipocyte, a tissue important for insulin resistance, in a differentiation-dependent manner. In in vitro functional analysis, the DNA fragment containing the allele associated with type 2 diabetes found in the present invention showed higher enhancer activity in differentiated adipocytes as compared to undifferentiated preadipocytes. These results indicate that the TFAP-28 gene itself, or a gene upstream or downstream thereof, or a transcription pathway involving the TFAP-2β protein, regulates the expression of other genes in differentiated adipocytes by regulating the expression of other genes. It is thought to be involved in the pathogenesis of type 2 diabetes. Furthermore, overexpression of the TFAP-2β gene in adipocytes reduced saccharide absorption in response to insulin stimulation. From this, there is a correlation between the increase in the expression level of TFAP-2] 3 and the onset of type 2 diabetes, and a preliminary diagnosis of type 2 diabetes can be made by measuring the expression level of TFAP-2 (3). It is also suggested that suppressing the expression of TFAP-2] 3 can improve type 2 diabetes.

Thus, TFAP-28 may be a promising target for the treatment or prevention of type 2 diabetes. So you have already been diagnosed with type 2 diabetes Analysis of the mutation in the polymorphism of the present invention can be useful for the treatment of patients as well, even in patients.

Prior art document information related to this application includes the following.

1 · Zimmet, P "Alberti, K G.M.M., Shaw, J" Global and societal implications of the diabetes epidemic. "Nature" 414, p. 782-786 (2001)

2 Kahn _? BB, T e 2 diabetes- When insulin secretion fails to compensate for insulin resistance. "Cell" 92, p. 593-596 (1988)

3. Saltiel, A.R., New perspectives into the molecular pathogenesis and treatment of type 2 diabetes. "Cell" 104, 517-529 (2001)

4. Fajaiis, S.S., Bell, G.I. 'Polonsky K.S-, Molecular mechanisms and clinical pathophysiology of maturity-onset diabetes of the yong "" N. Engl. J. Med. "345, p. 971-980 (2001)

5. Kadowaki, T. et al "A subtype of diabetes mellitus associated with a mutation of mitochondorial DNA." N. Engl. J. Med. "P. 330, 962-968 (1994)

6. International Human Genome Sequencing Consortium. Initial sequencing and the analysis of human genome. "Nature" 409, p. 860-921 (2001)

7. Olinisni, Y et al., A high-throughput SNP typing system for genome-wide association studies. "J. Hum. Genet." 46, p. 471-477 (2001).

8 'Moser, M. et al "Cloning and characterization of a second AP-2 transcription factor: Ap-2b." Development "121, p. 2779-2788 (1995)

9, Moser, M. et aL, Enhanced apoptotic cell death of renal epithelial cells in mice lacking transcription factor AP-2b. "Genes & Dev." 11, p, 1983-1948 (1997)

Satoda, M. et al "Mutation in TFAP2B cause Char syndrome, a familial form of patent ductus arteriosus." Nature Genet. "25, p. 42-46 (2000) 1 1Horikawa, Y. et al" Genetic variation in the gene encoding- calpain-10 is associated with type 2 diabetes mellitus, "Nature Genet." 26, p. 163-175 (2000)

1 2. Ong _? KK 丄. Et al., The iasulin gene VNTR, type 2 diabetes and birth weight. "Nature Genet." 21, ρ262-263 (1999)

1 3. Spigiman Β · Μ ·, Flier J.S., Adipogenesis and Obesity: Rounding out the big picture. "Cell" 87, p. 377 389 (1996)

1 4. Mats zawa, Y "Funahashi, T" Nakam ra, T "Molecular mechanism of metabolic syndrome X: contribution of adipocytokies, adipocyte-deriverd bioactive substances." Ann. NY Acad. Sci. "892, p. 146 154 (1999)

15 Kroeger, KM, Abraham LJ Indentification of an AP-2 element in the -323 to -285 region of the TNF-alpha gene. "Biochem. Mol. Biol." Int. 40, p. 43-51 (1996 )

16 丄 sse, N. et al. Structural organization and cliromosomal assignment of the human obese gene. "J. Biol. Chem. ' ¹ 270, p. 27728-27733 (1995)

1 7. Takahashi, Μ · et al. Genomic structure and mutations in adipose-spedfic gene, adiponectin. "Int. J. Obes." 24, p. 861-868 (2000)

1 8. Hirakawa, M. et al. JSNP: a database of common gene variations in the Japanese population. "Nucleic Acids Res." 30, p. 158-162 (2002)

1 9. SeM, T "Tanaka _; T" Nakamura, Y. Genomic structure and multiple "suigle-nucleotide polymorphisms (SNPs) of the thiopurine S-methyltransferase (TPMT) gene." J. Hum. Genet. "45, p. 299-302 (2000)

2 O, Saito, S. et al. Identification of 197 genetic variations in six humanmethyltransferase genes in the Japanese population "J. Hum. Genet." 46, p.529-537 (2001)

2 1. Kishida, K. et al. Genomic structure and insulin-mediated repression of the Aquaporin adipose (AQPap) _? Adipose-specific glycerol channel. "J. Biol. Chem." 275, p. 20896-20902 (2000) 2 2. Sadowski, HB, Gilman M. Ζ ·, Cell-free activation of a DNA-binding protein by epidermal growth factor. "Nature" 362, p. 79-83 (1993)

23, Yamada, R. et al. Association between a single-nucleotide polymorphismia the human interlexikin-3 gene and rheumatoid arthritis in Japanese patients, and maximum-likelifood estimation of combinational effect that two genetic loci have on susceptibility to the disease. "J. Hum. Genet." 68, p. 674-685 (2001).

Claims

The scope of the claims

1. (i) a polynucleotide comprising the DNA sequence represented by SEQ ID NO: 1,

(ii) 7 or more nucleotide sequences of the polynucleotide in which guanine (G) at position 774 of the first intron of the human TFAP-2B gene represented by SEQ ID NO: 2 is substituted with thymine (T) or A polynucleotide comprising the entire sequence, wherein the polynucleotide comprises at least the 774th base; and

(iii) a probe having a sequence complementary to the above (i) or (ii), a probe used for detecting a polynucleotide associated with the possibility of suffering from type 2 diabetes selected from the group consisting of:

2. Human TFAP—A probe of SEQ ID NO: 3 that detects thymine (T) at position 774 of the first intron of the 26 gene.

3. Human TFAP—A probe of SEQ ID NO: 4 that detects guanine (G) at position 774 of the first intron of the 2β gene.

4. The 774th intron of the first intron of the human TFAP-26 gene, which is associated with the possibility of developing type 2 diabetes, comprising the probe according to claim 1 or the probe of SEQ ID NO: 3 and SEQ ID NO: 4. Kit for detecting the substitution of guanine (G) for thymine (Τ) at the bases

5. A diagnostic kit for the onset of type 2 diabetes, comprising the probe according to claim 1 or the probes of SEQ ID NO: 3 and SEQ ID NO: 4.

6. The kit according to claim 4 or 5, further comprising a probe of SEQ ID NO: 5.

7. (i) a polynucleotide containing the DNA sequence represented by SEQ ID NO: 1; (ii) the 774th guan of the first intron of the human TFAP-2β gene represented by SEQ ID NO: 2 (G) Is a polynucleotide comprising seven or more nucleotide sequences or all of the polynucleotides substituted by thymine (Τ), A polynucleotide comprising at least the 774th base; and

(iii) A primer used for detecting a polynucleotide associated with a possibility of suffering from type 2 diabetes selected from the group consisting of polynucleotides having a sequence complementary to (i) or (ii) above.

8. Human TFAP—Detects a polynucleotide associated with a possible type 2 diabetes in which guanine (G) at position 774 of the first intron of the 26 gene is replaced with thymine (Τ) Primers set forth in SEQ ID NO: 6 and SEQ ID NO: 7 to perform

9. A guanine (G) to thymine at the 774th base of the first intron of the human TFAP-2β gene, which comprises the primer of claim Ί or claim 8, which is associated with the possibility of developing type 2 diabetes. Kit for detecting substitution to (Τ).

10. A pre-onset diagnosis kit for type 2 diabetes, comprising the primer according to claim 7 or 8.

1 1. GYGGVMKAG) _η (where Α represents adenine, G represents guanine, C represents thymine, Y represents thymine or cytosine, M represents adenine or cytosine, K represents guanine or thymine, and V represents adenine or guayun or cytosine. N is 8 or 9.) A polynucleotide comprising the tandem repeat polymorphism or a polynucleotide complementary to the polynucleotide.

1 2. A primer for detecting the polynucleotide according to claim 11.

13. The primer according to SEQ ID NO: 8 and SEQ ID NO: 9 for detecting the polynucleotide according to claim 11.

14. Whether the tandem repeat polymorphism of the first intron of the human TFAP-126 gene containing the primer according to claim 12 or claim 13 is (GYGGVMKAG) _{8 to} 9 (GYGGVMKAG) Kit to determine if there is.

1 5. A diagnostic kit for onset of type 2 diabetes, comprising the primer according to claim 12 or 13.

16. In the DNA sequence of the first intron of the human TFAP-26 gene

(i) substitution of guanine (G) for thymine (T) at base 774, (ii) GYGGVMKAG (where A is adenine, G is guanine, C is thymine, Y is thymine or cytosine, and M is 8 or 9 tandem repeats of adenine or cytosine, K represents guanine or thymine, V represents adenine or guanine or cytosine), or

(iiii) Both (i) and (ii) above

A method for detecting a mutation associated with the possibility of suffering from type 2 diabetes, characterized by detecting a mutation.

17.A mutation due to a polymorphism or tandem repeat polymorphism in which one or several bases are deleted, inserted, or substituted, and the presence of the mutation indicates that the mutation has a susceptibility to type 2 diabetes. A polynucleotide comprising a mutation in the human TFAP-2B gene, characterized in that:

18. The above mutation resulted in the substitution of thymine (T) at position 512 in the 5 'flanking region of the human TFAP-28 gene with adenine (A), and the guanine (G) at position 774 of the first intron. ) To thymine (T), 8 or 9 tandem repeats of GYGGVMKAG in the DNA sequence of the first intron, adenine (A) of the 2093th base of the 1st intron to cytosine (C) Substitution of cytosine (C) at position 58 of the second intron with guanine (G), substitution of thymine (T) at position 514 of the third intron with cytosine (C), 18. The polynucleotide comprising the mutation according to claim 17, wherein the intron is any one of substitution of cytosine (C) at position 2134 with thymine (T).

19. One or more probes selected from the group consisting of SEQ ID NOS: 3-5 and SEQ ID NOs: 44-58 for detecting the polynucleotide of claim 18.

20. SEQ ID NOS: 6 and 7 for detecting the polynucleotide of claim 18 A pair of primers selected from the group consisting of SEQ ID NOs: 8 and 9, SEQ ID NOs: 59 and 60, SEQ ID NOs: 61 and 62, SEQ ID NOs: 63 and 64, SEQ ID NOs: 65 and 66, SEQ ID NOs: 67 and 68.

21. A polynucleotide comprising a DNA sequence represented by SEQ ID NO: 1 for detecting a person susceptible to type 2 diabetes.

22. guanine (G) at position 774 of the first intron of the TFAP-2B gene of human is replaced with thymine (T) to detect people who are susceptible to type 2 diabetes A polynucleotide comprising at least 7 base sequences or the entire sequence of the polynucleotide, wherein the polynucleotide comprises at least the 774th base.

23. The polynucleotide according to claim 22, wherein one or several bases are deleted, inserted, or substituted at a position other than the 23.774 base.

24. A polynucleotide comprising a complementary strand or an antisense sequence of the polynucleotide according to any of claims 17, 18, 21 to 23.

25. A method for detecting a mutation associated with a possibility of developing type 2 diabetes by detecting the polynucleotide according to any one of claims 17, 18, 21 to 24.

26. A type 2 diabetes pre-onset diagnostic kit comprising a reagent for measuring the expression level of the TFAP-2 j3 gene.