JP2006067866A - Obesity-related gene and its utilization - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a polynucleotide utilizable for genetic testing for obesity having high accuracy and to provide a method for detecting a genetic polymorphism, capable of carrying out genetic testing of obesity having high accuracy and to provide an oligonucleotide set used for the method. <P>SOLUTION: The present inventors found at first that correlation exists between a specific polymorphism of human FABP2 gene, human angiotensinogen gene, human KIR6.2 gene, human RAGE gene, human PPARγ gene and human SUR1 gene in exon 31 and inclination to obesity. Information having high reliability to decide whether or not a person has inclination to obesity is obtained by detecting the polymorphism. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a human gene that can be used for the examination of obesity constitution, a method for detecting a gene polymorphism, and an oligonucleotide used for this method.

  Excessive obesity is an important factor for maintaining health because it is an exacerbating factor such as hypertension, hyperlipidemia, and type II diabetes. Therefore, elucidation of the onset mechanism of obesity and establishment of preventive and therapeutic methods are important medical issues.

  A gene may contain point mutations or mutations such as partial or complete deletion, insertion or substitution of genes. As the obesity-related genes, there are many genes encoding proteins that affect the metabolic rate at rest. If these genes are mutated, the protein will not be expressed or the original gene product protein will not function normally, resulting in a decrease in resting metabolic rate compared to humans with wild-type genes. It is known to increase or decrease. This means that when the same calorie is consumed, the body weight tends to increase or decrease compared to a human having the wild type.

  So far, methods for estimating changes in resting metabolic rate by examining mutations in several genes are known. For example, it is known that when a mutation occurs in the β3-adrenergic receptor gene, UCP (Uncoupling Protein) 1 gene, and β2-adrenergic receptor gene, the metabolic rate changes.

  The present invention provides a polynucleotide that can be used for genetic testing for obesity with high accuracy, a method for detecting a genetic polymorphism that can perform genetic testing for obesity with high accuracy, and an oligonucleotide set used for the method. Let it be an issue.

  In order to solve the above problems, the present inventors conducted research and investigated the presence or absence of the following gene polymorphism and resting metabolic rate in 74 subjects. As a result, it was found that the resting metabolic rate varies significantly as shown in Table 1 below due to the presence of the following gene polymorphism.

  When the resting metabolic rate is decreased due to the polymorphism, it means that even if the same amount of calories is consumed, it is more likely to become fat compared to wild-type humans. If it is increased, it means that even if the same amount of calories is ingested, it is easier to lose weight than a wild type human.

The present invention has been completed based on the above findings, and provides the following polynucleotides, gene polymorphism detection methods, and oligonucleotides and primer sets used in the methods.
Item 1. The following polynucleotide (1) or (2):
(1) A polynucleotide comprising 20 to 300 bases including the 163rd nucleotide of the human FABP (Fatty Acid Binding Protein) 2 gene or a variant thereof, wherein the 163rd gene polymorphic site is A.
(2) A polynucleotide complementary to the polynucleotide of (1).
Item 2. Item 5. The polynucleotide according to Item 1, consisting of 20 to 300 bases including the nucleotide of base number 163 of SEQ ID NO: 1, or complementary thereto.
Item 3. An oligonucleotide set for detecting a gene polymorphism comprising the following (a) to (c).
(A) A 20 to 30 nucleotide oligonucleotide capable of hybridizing to a nucleotide sequence containing the polymorphic site of the wild-type human FABP2 gene or its complementary strand whose 163rd gene polymorphic site is G, the gene polymorphism A wild-type oligonucleotide for detection, wherein the nucleotide corresponding to the site is complementary to the nucleotide of the polymorphic site.
(B) a 20-30 base oligonucleotide capable of hybridizing to a nucleotide sequence comprising the polymorphic site of the mutant human FABP2 gene or its complementary strand, wherein the 163rd gene polymorphic site is A, the gene polymorphism An oligonucleotide for detecting a mutation, wherein the nucleotide corresponding to the site is complementary to the nucleotide of the polymorphic site.
(C) An oligonucleotide consisting of 20 to 30 bases that can hybridize to both wild type and mutant human FABP2 genes and can be used in pairs with (a) and (b) in nucleic acid amplification.
Item 4. A gene polymorphism detection primer set comprising the following (a) to (c).
(a) a 20-30 base oligonucleotide capable of hybridizing to a nucleotide sequence comprising the polymorphic site of the human wild-type FABP2 gene or its complementary strand, wherein the 163rd gene polymorphic site is G, the gene polymorphism The nucleotide corresponding to the site is complementary to the nucleotide of the polymorphic site and is present at the 1st to 2nd positions from the 3 'end, and the wild type and mutant human FABP2 gene is located in the upstream 1 to 2 base region adjacent to the nucleotide. A wild type detection oligonucleotide having nucleotides that are not complementary to each other.
(b) a 20 to 30 nucleotide oligonucleotide capable of hybridizing to a nucleotide sequence containing the polymorphic site of the mutant human FABP2 gene or its complementary strand whose 163rd gene polymorphic site is A, the gene polymorphism The nucleotide corresponding to the site is complementary to the nucleotide of the polymorphic site, is present at the 1st to 2nd positions from the 3 ′ end, and is adjacent to the upstream 1 to 2 base region, the wild type and mutant human FABP2 genes A mutant detection oligonucleotide having an oligonucleotide that is not complementary to any of the above.
(c) An oligonucleotide consisting of 20 to 30 bases that can hybridize to both wild type and mutant human FABP2 genes and can be used in pairs with (a) and (b) in nucleic acid amplification.
Item 5. A method for detecting a human FABP2 gene polymorphism that detects a base substitution at position 163 of the human FABP2 gene.
Item 6. Item 6. The method according to Item 5, wherein G to A base substitution at position 163 of the human FABP2 gene is detected.
Item 7. The following polynucleotide (3) or (4):
(3) A polynucleotide comprising 20 to 300 bases comprising the 803th nucleotide of the human angiotensinogen gene or a variant thereof, wherein the 803th gene polymorphism site is C.
(4) A polynucleotide complementary to the polynucleotide of (3).
Item 8. Item 8. The polynucleotide according to Item 7, comprising 20 to 300 bases including the nucleotide of base number 803 of SEQ ID NO: 2, or complementary thereto.
Item 9. An oligonucleotide set for detecting a gene polymorphism comprising the following (e) to (f).
(D) a 20-30 base oligonucleotide capable of hybridizing to a nucleotide sequence comprising the polymorphic site of the wild type human angiotensinogen gene or its complementary strand, wherein the 803th gene polymorphic site is T, A wild-type detection oligonucleotide, wherein a nucleotide corresponding to a polymorphic site is complementary to a nucleotide of the polymorphic site.
(E) a 20-30 base oligonucleotide capable of hybridizing to a mutant human angiotensinogen gene whose 803 gene polymorphic site is C or a nucleotide sequence containing the polymorphic site of its complementary strand, A variant detection oligonucleotide, wherein a nucleotide corresponding to a polymorphic site is complementary to a nucleotide of the polymorphic site.
(F) An oligonucleotide consisting of 20 to 30 bases that can hybridize to both wild-type and mutant human angiotensinogen genes and can be used in pairs with (d) and (v) in nucleic acid amplification.
Item 10. A gene polymorphism detection primer set comprising the following (d) to (f).
(d) a 20-30 base oligonucleotide capable of hybridizing to a human wild-type angiotensinogen gene whose 803th gene polymorphic site is T or a nucleotide sequence containing the polymorphic site of its complementary strand, The base corresponding to the polymorphic site is complementary to the nucleotide of the polymorphic site and is present at the 1st to 2nd positions from the 3 'end, and the wild type and mutant human angio in the region of 1 to 2 bases adjacent to it. A wild type detection oligonucleotide having a nucleotide that is not complementary to any of the tensinogen genes.
(e) a 20-30 base oligonucleotide capable of hybridizing to a mutant human angiotensinogen gene whose 803 gene polymorphic site is C or a nucleotide sequence containing the polymorphic site of its complementary strand, The base corresponding to the polymorphic site is complementary to the nucleotide of the polymorphic site, is present at the 1st to 2nd positions from the 3 ′ end, and is a wild type and mutant human in the region of 1 to 2 bases upstream adjacent to it. A mutant detection oligonucleotide having an oligonucleotide that is not complementary to any of the angiotensinogen genes.
(f) An oligonucleotide consisting of 20 to 30 bases that can hybridize to both wild-type and mutant human angiotensinogen genes and can be used in pairs with (d) and (f) in nucleic acid amplification.
Item 11. A method for detecting a human angiotensinogen gene polymorphism, wherein a base substitution at position 803 of a human angiotensinogen gene is detected.
Item 12. Item 12. The method according to Item 11, wherein a base substitution from T to C at position 803 of the human angiotensinogen gene is detected.
Item 13. The following polynucleotide (5) or (6):
(5) A polynucleotide comprising 20 to 300 bases including the 67th nucleotide of the human KIR (inwardly rectifying potassium channel) 6.2 gene or a variant thereof, wherein the 67th gene polymorphic site is A.
(6) A polynucleotide complementary to the polynucleotide of (5).
Item 14. Item 14. The polynucleotide according to Item 13, consisting of 20 to 300 bases including the nucleotide of SEQ ID NO: 3 and nucleotide number 67, or complementary thereto.
Item 15. An oligonucleotide set for detecting a gene polymorphism comprising the following (ki) to (ke).
(G) a wild type human KIR6.2 gene in which the 67th gene polymorphism site is G or a 20-30 base oligonucleotide capable of hybridizing to the nucleotide sequence containing the polymorphic site of its complementary strand, A wild-type detection oligonucleotide, wherein a nucleotide corresponding to a polymorphic site is complementary to a nucleotide of the polymorphic site.
(H) a 20-30 base oligonucleotide capable of hybridizing to a mutant human KIR6.2 gene whose 67th gene polymorphic site is A or a nucleotide sequence containing the polymorphic site of its complementary strand, A variant detection oligonucleotide, wherein a nucleotide corresponding to a polymorphic site is complementary to a nucleotide of the polymorphic site.
(G) An oligonucleotide consisting of 20 to 30 bases that can hybridize to both wild-type and mutant human KIR6.2 genes and can be used in pairs with (ki) and (ku) in nucleic acid amplification.
Item 16. A method for detecting a human KIR6.2 gene polymorphism that detects a base substitution at position 67 of the human KIR6.2 gene.
Item 17. Item 17. The method according to Item 16, wherein a base substitution from G to A at the 67th position of the human KIR6.2 gene is detected.
Item 18. The following polynucleotide (7) or (8):
(7) A polynucleotide comprising 20 to 300 bases including the 8368th P2 nucleotide of a human RAGE (Receptor for Advanced Glycation End products) gene or a mutant thereof, wherein the polymorphic site of the 8368th gene is T. nucleotide.
(8) A polynucleotide complementary to the polynucleotide of (7).
Item 19. 19. The polynucleotide of claim 18 comprising or complementary to 20-300 bases comprising the nucleotide of SEQ ID NO: 4 base number 8368.
Item 20. An oligonucleotide set for detecting a gene polymorphism, comprising:
(E) a 20-30 base oligonucleotide capable of hybridizing to a nucleotide sequence comprising the polymorphic site of the wild type human RAGE gene or its complementary strand, wherein the 8368th gene polymorphic site is G, the gene polymorphism A wild-type oligonucleotide for detection, wherein the nucleotide corresponding to the site is complementary to the nucleotide of the polymorphic site.
(Sa) a 20-30 base oligonucleotide capable of hybridizing to a nucleotide sequence comprising the polymorphic site of the mutant human RAGE gene or its complementary strand, wherein the 8368th gene polymorphic site is T, the gene polymorphism A variant detection oligonucleotide, wherein a nucleotide corresponding to a site is complementary to a nucleotide of the polymorphic site.
(F) It consists of 20-30 bases that can hybridize to both wild-type and mutant human RAGE genes and can be used in pairs with (co) and (sa) in nucleic acid amplification, or is complementary thereto. An oligonucleotide.
Item 21. A gene polymorphism detection primer set comprising the following (g) to (i).
(g) a 20-30 base oligonucleotide capable of hybridizing to a nucleotide sequence containing the polymorphic site of the human wild-type RAGE gene or its complementary strand, wherein the 8368th gene polymorphic site is G, the gene polymorphism The base corresponding to the site is complementary to the nucleotide of the polymorphic site and is present at the 1st to 2nd positions from the 3 'end, and the wild type and mutant human RAGE genes are located in the upstream 1 to 2 base region adjacent to it. A wild type detection oligonucleotide having nucleotides that are not complementary to each other.
(h) a 20-30 base oligonucleotide capable of hybridizing to a nucleotide sequence containing the polymorphic site of the mutant human RAGE gene or its complementary strand, wherein the 8368th gene polymorphic site is T, the gene polymorphism The base corresponding to the site is complementary to the nucleotide of the polymorphic site, is present at the 1st to 2nd position from the 3 ′ end, and the wild type and mutant human RAGE genes in the upstream 1 to 2 base region adjacent to it A mutant detection oligonucleotide having an oligonucleotide that is not complementary to any of the above.
(i) An oligonucleotide consisting of 20 to 30 bases that can hybridize to both wild-type and mutant human RAGE genes and can be used in pairs with (g) and (h) in nucleic acid amplification.
Item 22. A method for detecting a human RAGE gene polymorphism that detects a base substitution at position 8368 of a human RAGE gene.
Item 23. Item 23. The method according to Item 22, wherein a base substitution from G to T at the 8368th position of the human RAGE gene is detected.
Item 24. The following polynucleotide (9) or (10):
(9) A polynucleotide comprising 20 to 300 bases including the 34th nucleotide of a PPAR (Peroxisome Proliferator-Activated Receptor) γ gene or a variant thereof, wherein the 34th gene polymorphic site is G.
(10) A polynucleotide complementary to the polynucleotide of (9).
Item 25. 25. The polynucleotide of claim 24, comprising or complementary to 20-300 bases comprising nucleotide number 34 of SEQ ID NO: 5.
Item 26. An oligonucleotide set for detecting a gene polymorphism comprising the following (su) to (so).
(Su) An oligonucleotide of 20 to 30 bases capable of hybridizing to a nucleotide sequence containing the polymorphic site of the wild-type human PPARγ gene or its complementary strand whose 34th gene polymorphic site is C, the gene polymorphism A wild-type oligonucleotide for detection, wherein the nucleotide corresponding to the site is complementary to the nucleotide of the polymorphic site.
(C) a 20-30 base oligonucleotide capable of hybridizing to a nucleotide sequence comprising the polymorphic site of the mutant human PPARγ gene or its complementary strand whose 34th gene polymorphic site is G, the gene polymorphism A variant detection oligonucleotide, wherein a nucleotide corresponding to a site is complementary to a nucleotide of the polymorphic site.
(So) An oligonucleotide consisting of 20 to 30 bases that can hybridize to both wild-type and mutant human PPARγ genes and can be used in pairs with (su) and (se) in nucleic acid amplification.
Item 27. A gene polymorphism detection primer set comprising the following (j) to (l).
(j) a 20-30 base oligonucleotide capable of hybridizing to a nucleotide sequence containing the polymorphic site of the human wild-type PPARγ gene or its complementary strand whose 34th gene polymorphic site is C, the gene polymorphism The base corresponding to the site is complementary to the nucleotide of the polymorphic site, is present at the 1st to 2nd position from the 3 ′ end, and the wild type and mutant human PPARγ genes in the upstream 1 to 2 base region adjacent thereto A wild type detection oligonucleotide having a nucleotide that is not complementary to any of the above.
(k) a 20-30 base oligonucleotide capable of hybridizing to a nucleotide sequence containing the polymorphic site of the mutant human PPARγ gene or its complementary strand whose 34th gene polymorphic site is G, the gene polymorphism The base corresponding to the site is complementary to the nucleotide of the polymorphic site, is present at the 1st to 2nd position from the 3 ′ end, and the wild type and mutant human PPARγ genes in the upstream 1 to 2 base region adjacent thereto A mutant detection oligonucleotide having an oligonucleotide that is not complementary to any of the above.
(l) An oligonucleotide consisting of 20 to 30 bases that can hybridize to both wild type and mutant human PPARγ genes and can be used in pairs with (j) and (k) in nucleic acid amplification.
Item 28. A method for detecting a human PPARγ gene polymorphism, wherein a base substitution at position 34 of the human PPARγ gene is detected.
Item 29. Item 29. The method according to Item 28, wherein a base substitution from C to G at position 34 of the human PPARγ gene is detected.
Item 30. The following polynucleotide (11) or (12):
(11) A polynucleotide comprising 20 to 300 bases including the 1273th nucleotide of exon 31 of human SUR (Sulfonylurea Receptor) 1 gene or a variant thereof, wherein the 1273th gene polymorphism site is A .
(12) A polynucleotide complementary to the polynucleotide of (11).
Item 31. Item 31. The polynucleotide according to item 30, consisting of 20 to 300 bases including the nucleotide of base number 3819 of SEQ ID NO: 6, or complementary thereto.
Item 32. An oligonucleotide set for detecting a gene polymorphism comprising the following (ta) to (tu).
(Ta) An oligonucleotide of 20 to 30 bases that can hybridize to a nucleotide sequence containing the polymorphic site of the wild-type human SUR1 gene or its complementary strand, wherein the 1273th gene polymorphic site of exon 31 is G, A wild-type oligonucleotide for detection, wherein a nucleotide corresponding to a gene polymorphic site is complementary to a nucleotide of the polymorphic site.
(H) an oligonucleotide of 20 to 30 bases capable of hybridizing to a nucleotide sequence containing the polymorphic site of the mutant human SUR1 gene or its complementary strand, wherein the 1273th gene polymorphic site of exon 31 is A, A mutant detection oligonucleotide, wherein a nucleotide corresponding to a gene polymorphic site is complementary to a nucleotide of the polymorphic site.
(Iv) An oligonucleotide consisting of 20 to 30 bases that can hybridize to both wild type and mutant human SUR1 genes and can be used in pairs with (ta) and (h) in nucleic acid amplification.
Item 33. A gene polymorphism detection primer set comprising the following (m) to (o).
(m) a 20-30 base oligonucleotide capable of hybridizing to a nucleotide sequence containing the polymorphic site of the human wild-type SUR1 gene or its complementary strand, wherein the 1273th gene polymorphic site of exon 31 is G, The base corresponding to the gene polymorphic site is complementary to the nucleotide of the polymorphic site, is present first to second from its 3 ′ end, and is adjacent to the upstream 1-2 base region, wild type and mutant type A wild-type detection oligonucleotide having nucleotides that are not complementary to any of the human SUR1 genes.
(n) an oligonucleotide of 20 to 30 bases capable of hybridizing to a nucleotide sequence containing the polymorphic site of the human mutant SUR1 gene or its complementary strand, wherein the 1273th gene polymorphic site of exon 31 is A, The base corresponding to the gene polymorphic site is complementary to the nucleotide of the polymorphic site, is present first to second from its 3 ′ end, and is adjacent to the upstream 1-2 base region, wild type and mutant type A mutant detection oligonucleotide having a nucleotide that is not complementary to any of the human SUR1 genes.
(o) An oligonucleotide consisting of 20 to 30 bases that can hybridize to both wild type and mutant human SUR1 genes and can be used in pairs with (m) and (n) in nucleic acid amplification.
Item 34. A method for detecting a human SUR1 gene polymorphism that detects a base substitution at position 1273 of exon 31 of human SUR1 gene.
Item 35. Item 35. The method according to Item 34, wherein G to A base substitution at position 1273 of human SUR1 gene exon 31 is detected.
Item 36. A method for examining an obesity constitution comprising a step of detecting four or more of the following gene polymorphisms (i) to (ix) for a human gene and a step of determining whether or not the subject is an obesity constitution.
(i) Base substitution at position 163 of human FABP2 gene
(ii) Base substitution at position 803 of the human angiotensinogen gene
(iii) Base substitution at position 67 of human KIR6.2 gene
(iv) Base substitution at the 8368th position of the human RAGE gene
(v) Base substitution at position 34 of human PPARγ gene
(vi) Base substitution at position 1273 of exon 31 of human SUR1 gene
(vii) Base substitution at position 190 in human β3-adrenergic receptor gene
(viii) Base substitution at position 3826 (−3862) upstream from the start codon of human UCP (Uncoupling Protein) 1 gene
(ix) Base substitution at 46th position in human β2-adrenergic receptor gene

  According to the present invention, a gene polymorphism that causes a significant change in resting metabolic rate in a gene involved in metabolism is provided. Thus, by detecting the presence or absence of the polymorphism, it is possible to accurately test whether or not the subject has an obesity constitution, which can be useful for the prevention and treatment of obesity. Furthermore, according to the present invention, it is expected to contribute to the prevention and treatment of many lifestyle-related diseases such as diabetes caused by obesity, hypertension, cardiovascular disorders, and hyperlipidemia.

  The present invention is described in detail below.

In the present invention, “gene polymorphism” refers to the presence of two or more bases by a human at a specific site in a gene. A frequently occurring type is called a wild type and a less frequently used type is called a mutant type.
Human FABP (Fatty Acid Binding Protein) 2 gene The first polynucleotide of the present invention is the following polynucleotide (1) or (2).
(1) A polynucleotide comprising 20 to 300 bases including the 163rd nucleotide of the human FABP2 gene, wherein the 163rd gene polymorphism site is A.
(2) A polynucleotide complementary to the polynucleotide of (1).

  The human FABP2 gene whose 163rd gene polymorphism site is A typically includes the nucleotide sequence shown in SEQ ID NO: 1, but the bases other than nucleotide number 163 in SEQ ID NO: 1 These genes are also included in the human FABP2 gene in which the 163rd gene polymorphism site is A. There are also gene variations that vary in amino acid sequence among individuals, but gene sequence variations that do not appear as changes in amino acid sequence are often seen between individuals.

The above-mentioned polynucleotide can be used for designing a primer or probe for detecting a gene polymorphism of the human FABP2 gene.
Human angiotensinogen gene The second polynucleotide of the present invention comprises the following polynucleotide (3) or (4).
(3) A polynucleotide comprising 20 to 300 bases comprising the 803th nucleotide of the human angiotensinogen gene or a variant thereof, wherein the 803th gene polymorphism site is C.
(4) A polynucleotide complementary to the polynucleotide of (3).

As a human angiotensinogen gene whose 803th gene polymorphism site is C, a representative example of the gene having the base sequence shown in SEQ ID NO: 2 is mentioned. These genes are also included in the human angiotensinogen gene in which the 803th gene polymorphism site is C.
Human KIR (inwardly rectifying potassium channel) 6.2 gene The third polynucleotide of the present invention is the following polynucleotide (5) or (6).
(5) A polynucleotide comprising 20 to 300 bases including the 67th nucleotide of the human KIR (inwardly rectifying potassium channel) 6.2 gene or a variant thereof, wherein the 67th gene polymorphic site is A.
(6) A polynucleotide complementary to the polynucleotide of (5).

The human KIR gene whose 67th gene polymorphism site is A typically includes the nucleotide sequence shown in SEQ ID NO: 3, but the bases other than nucleotide number 67 in SEQ ID NO: 3 Such genes are also included in the human KIR gene whose 67th gene polymorphism site is A.
Human RAGE (Receptor for Advanced Glycation End products) gene The fourth polynucleotide of the present invention is the following polynucleotide (7) or (8).
(7) A polynucleotide comprising 20 to 300 bases including the 8368th nucleotide of a human RAGE (Receptor for Advanced Glycation End products) gene or a variant thereof, wherein the polymorphic site of the 8368th gene is T .
(8) A polynucleotide complementary to the polynucleotide of (7).

  The human RAGE gene whose 8368th gene polymorphism site is T typically includes the nucleotide sequence shown in SEQ ID NO: 4, but the bases other than nucleotide number 8368 in SEQ ID NO: 4 These genes are also included in the human RAGE gene whose T at the 8368th gene polymorphism site is T.

  SEQ ID NO: 4 is a nucleotide sequence of a mutant human RAGE gene including all introns and exons from 5 ′ UTR. The mutation at base number 8368 is a mutation in an intron.

The base sequences of other genes (SEQ ID NOs: 1-3, 5, 6, 34-36) are base sequences of exons as usual.
PPAR (Peroxisome Proliferator-Activated Receptor) γ gene The fifth polynucleotide of the present invention is the following polynucleotide (9) or (10).
(9) A polynucleotide comprising 20 to 300 bases including the 34th nucleotide of a PPAR (Peroxisome Proliferator-Activated Receptor) γ gene or a variant thereof, wherein the 34th gene polymorphic site is G.
(10) A polynucleotide complementary to the polynucleotide of (9).

The human PPAR gene whose 34th gene polymorphism site is A typically includes the nucleotide sequence shown in SEQ ID NO: 5. For nucleotides other than nucleotide number 34 in SEQ ID NO: 5, These genes are also included in the human PPAR gene whose G polymorphism site is G.
Human SUR (Sulfonylurea Receptor) 1 gene The sixth polynucleotide of the present invention is the following polynucleotide (11) or (12).
(11) A polynucleotide comprising 20 to 200 bases including the 1273th nucleotide of exon 31 of human SUR (Sulfonylurea Receptor) 1 gene or a variant thereof, wherein the polymorphic site of the 1273rd gene is A .
(12) A polynucleotide complementary to the polynucleotide of (11).

As the human SUR gene exon 31 in which the 1273rd gene polymorphic site is A, a representative example of the nucleotide sequence shown in SEQ ID NO: 6 can be mentioned. These genes are also included in the human SUR gene exon 31 in which the 1273rd gene polymorphism site is A.
Method for detecting a gene polymorphism The method for detecting a gene polymorphism of the present invention is a method for detecting the following human gene polymorphism.
(i) A method for detecting a base substitution at position 163 of the human FABP2 gene, particularly a base substitution from G to A. A method for detecting the corresponding base substitution in the complementary strand is also included in the scope of the present invention. This base substitution is particularly obese if this gene polymorphism is detected, as it reduces significantly the resting metabolic rate compared to humans with the wild-type FABP2 gene, especially when it is a homozygous mutation. It is suggested.
(ii) A method for detecting a base substitution at position 803 of the human angiotensinogen gene, in particular, a base substitution from T to C. A method for detecting the corresponding base substitution in the complementary strand is also included in the scope of the present invention. This base substitution significantly reduces resting metabolic rate compared to humans with wild-type angiotensinogen gene, especially when it is a homozygous mutation. It is suggested that
(iii) A method for detecting a base substitution at the 67th position of the human KIR6.2 gene, particularly a base substitution from G to A. A method for detecting the corresponding base substitution in the complementary strand is also included in the scope of the present invention. This base substitution significantly increases resting metabolic rate compared to humans with the wild type KIR6.2 gene, especially when it is a homozygous mutation. It is suggested that the constitution is difficult.
(iv) A method of detecting a base substitution at the 8368th position of the human RAGE gene, particularly a base substitution from G to T. A method for detecting the corresponding base substitution in the complementary strand is also included in the scope of the present invention. This base substitution, especially in the case of both heterozygous and homozygous mutations, significantly reduces resting metabolic rate compared to humans with the wild type RAGE gene, so when this gene polymorphism is detected, It is suggested to be obese.
(v) A method for detecting a base substitution at the 34th position of the human PPRAγ gene, particularly a base substitution from C to G. A method for detecting the corresponding base substitution in the complementary strand is also included in the scope of the present invention. This base substitution significantly reduces resting metabolic rate compared to humans with the wild type PPRAγ gene, especially in both heterozygous and homozygous mutations. It is suggested to be obese.
(vi) A method of detecting a base substitution at the 1273th position of exon 31 of the human SUR1 gene, particularly a base substitution from G to A. A method for detecting the corresponding base substitution in the complementary strand is also included in the scope of the present invention. This base substitution increases the resting metabolic rate significantly compared to humans with the wild-type SUR1 gene, particularly in the case of homozygous mutation. It is suggested that there is.

  In addition, by detecting a plurality of the 6-gene polymorphisms, the accuracy of testing whether or not the subject is obese is improved accordingly.

In addition, there are the following three genes that have been known to correlate with polymorphisms and changes in resting metabolic rate.
-A heterozygous or homozygous mutation from the 190th T to C of the β3-adrenergic receptor gene reduces the resting metabolic rate. This is considered to be an obese constitution.
-Restoration of resting metabolism is reduced by homo-mutation from A to G at the 3826th position (-3826th position) upstream from the start codon of UCP (Uncoupling Protein) gene. This is considered to be an obese constitution.
・ The 46th A-to-G homo mutation of the β2-adrenergic receptor gene reduces resting metabolic rate. This is considered to be an obese constitution.

  The base sequence of the β3-adrenergic receptor gene (mutant form in which the 190th is C) is the sequence described in SEQ ID NO: 34. The base sequence of the UCP gene is the sequence described in SEQ ID NO: 35. The base sequence of the β2-adrenergic receptor gene (a mutant in which the 46th is G) is the sequence set forth in SEQ ID NO: 36.

  Thus, since there are a plurality of gene polymorphisms correlated with obesity, drug metabolizing enzyme activity fluctuates when gene polymorphism occurs in several places. Accordingly, at least any 9 gene polymorphism of the above 6 gene polymorphism whose relationship with obesity constitution has been clarified in the present invention and the above 3 gene polymorphism whose relationship with obesity constitution has been conventionally known is at least arbitrary. By detecting the 4-gene polymorphism, a reliable determination as to whether or not the subject is obese is obtained.

From Table 2 of the Examples described later, it can be seen that the resting metabolic rate generally changes as follows by the above gene mutations.
.Beta.3-adrenergic receptor gene: -200 kcal
・ UCP gene: -100kcal
・ Β2-adrenergic receptor gene: + 100kcal
・ FABP gene: -100kcal
・ Angiotensinogen gene: -200kcal
・ KIR6.2 gene: + 150kcal
・ RAGE gene: -350kcal
・ PRRAγ gene: -150kcal
・ SUR1 exon 31: + 350kcal
For the examined genes, the above-mentioned changes in resting metabolic rate predicted by the mutation are totaled. If it becomes negative, it can be determined to be obese, and if it becomes 0 or positive, it is determined not to be obese. can do.
Method for detecting gene polymorphism The method for detecting the presence or absence of a gene polymorphism is not particularly limited, and any known method can be used without limitation. The following methods can be exemplified as a method for detecting the presence or absence of a known gene polymorphism.
<Nucleic acid sequencing method (sequencing method)>
The nucleic acid sequencing method is a method for detecting and identifying a base sequence contained in a test nucleic acid sequence. The polymorphism can be detected by reading the base sequence of the test nucleic acid using an automatic sequencer or the like and examining the base sequence near the polymorphic site to be examined.
<Southern blot method>
After amplifying the test nucleic acid, it is immobilized on a nylon membrane, and a labeled probe that specifically hybridizes to the wild type or a specific mutant type is allowed to act on the test nucleic acid on the nylon membrane to detect the presence or absence of hybridization. Southern blotting can also be used.
<Sandwich hybridization method>
A complementary probe immobilized on a solid support, the test nucleic acid acting on a probe that specifically hybridizes to a wild-type nucleic acid or polymorphic nucleic acid, and then the detection nucleic acid is allowed to act so that the test nucleic acid is supplemented The polymorphism can be detected by detecting whether or not it has been hybridized.
<Method using DNA polymerase activity>
Using the 5 'exonuclease activity of DNA polymerase, PCR is performed using a pair of oligonucleotides and oligonucleotides that are fluorescently labeled at both ends that bind complementarily to the mutation site, and the released fluorescent substance is detected. Thus, the presence or absence of the polymorphism can be detected.
<Method using melting of nucleic acid>
Nucleic acid released by binding the test nucleic acid, which is previously single-stranded, to a carrier on which an oligonucleotide that binds complementarily to the region containing the predicted mutation site is immobilized, and gradually raising the temperature of the binding carrier It is possible to examine whether or not the test nucleic acid has a predicted mutation.

  In the gene polymorphism detection method exemplified above, it is generally desirable to use a test nucleic acid that has been amplified in advance. As the amplification method, known methods such as PCR, NASBA, LCR, SDA, RCA, and TMA can be used without limitation.

  These nucleic acid amplification methods are well known, but in brief, PCR is performed by allowing oligonucleotides, four types of deoxynucleoside triphosphates (dNTPs), and DNA polymerase to act on target nucleic acids that have been denatured into single strands. Then, an oligonucleotide extension reaction using the target nucleic acid as a template occurs, and a complementary strand of the nucleic acid sequence is synthesized. By repeating this reaction, the target nucleic acid is amplified.

  In the NASBA method and the TMA method, a single-stranded DNA is synthesized from RNA by reverse transcription reaction, and then RNA synthesis reaction is performed using oligonucleotide and RNA polymerase using the DNA as a template. This reverse transcription reaction and RNA synthesis reaction A method of amplifying a target nucleic acid by repeating a cycle consisting of:

  In the LCR method, in the presence of two kinds of oligonucleotides, deoxynucleoside triphosphate (dNTP), a ligase is allowed to act on the target nucleic acid, and the reaction for binding the two kinds of oligonucleotides on the target nucleic acid is repeated. Is a method of amplifying the signal.

  The SDA method is a method that substitutes and amplifies nucleic acids by combining a restriction enzyme and a polymerase. A DNA polymerase that uses a restriction enzyme to cut a nucleic acid to be detected and sequentially replaces the DNA fragments having the cut. This is a method of amplifying a nucleic acid using the action of.

The RCA method is a method of amplifying a target nucleic acid continuously while separating a newly generated oligonucleotide from a template by allowing a complementary oligonucleotide and DNA polymerase to act on the circular target nucleic acid. is there.
<Gene amplification method>
As a method for detecting an expected point mutation, a gene point using a gene amplification method such as a PCR (polymerase chain reaction) method (Japanese Patent Publication No. 4-67960 and Japanese Patent Publication No. 4-67957) has been conventionally used. Mutation detection methods are known. By detecting the presence or absence of amplification by PCR and the difference in amplification rate, the presence or absence of a polymorphism can be detected.
<< PCR-SSPC method >>
After amplification of the site containing the mutation site by PCR using a pair of oligonucleotides, electrophoresis is performed as a single-stranded nucleic acid, and the presence or absence of a polymorphism is determined using the fact that the migration distance of electrophoresis varies depending on the presence or absence of mutation. Can be detected.
<< PCR-RFLP method >>
The PCR-RFLP method uses a pair of oligonucleotides to amplify a site containing a mutation site, treat with a restriction enzyme, separate by electrophoresis, and detect different fragment sizes depending on the presence or absence of mutation. The presence or absence can be detected.
<< ASP (Allyl Specific) -PCR >>
In addition, a method of determining the presence or absence of mutation from the amplification rate or the presence or absence of amplification using a gene amplification method such as PCR can also be used.

  That is, a nucleic acid amplification reaction using an oligonucleotide that easily amplifies the wild-type gene and is difficult to amplify the mutant type, and an oligonucleotide common to the wild-type gene and the mutant type gene; There is a method for examining which type of gene a test nucleic acid has by performing a nucleic acid amplification reaction using an oligonucleotide that is difficult to amplify a type gene and an oligonucleotide common to a wild type gene and a mutant type gene. is there.

  Specifically, in the wild type detection oligonucleotide, the nucleotide corresponding to the gene polymorphism site is complementary to the nucleotide of the wild type gene, and the mutation detection oligonucleotide has the nucleotide corresponding to the gene polymorphism. It is complementary to the nucleotide of the mutant gene.

  Further, in both oligonucleotides for wild type detection and mutation type detection, a nucleotide complementary to the gene polymorphism site is present near the 3 ′ end. In particular, it is preferably about 1 to 2 from the 3 ′ end, more preferably 2nd.

  Moreover, both the wild-type detection oligonucleotide and the mutant-type detection oligonucleotide have nucleotides that are not complementary to both the wild-type and mutant-type genes. Due to the presence of this mismatch site, in the wild type detection oligonucleotide, inhibition of gene amplification is enhanced when acting on the mutant gene, and in the mutant detection oligonucleotide, the gene is detected when acting on the wild type gene. Inhibition of amplification is enhanced. The mismatch nucleotide may be upstream (5 ′ end side) of the nucleotide corresponding to the polymorphic site. Sometimes, gene amplification can be reliably inhibited by being present at a position close to the nucleotide corresponding to the polymorphic site. In particular, the mismatch nucleotide is preferably present in a region of about 1-2 bases upstream of the nucleotide complementary to the polymorphic site, and more preferably present in one base upstream. Most preferably, it is adjacent to the nucleotide complementary to the polymorphic site and at its 5 ′ end. As a result, when a nucleic acid sequence that does not expect an extension reaction is used as a template, a mismatch of two bases continuously exists together with the nucleotide polymorphism site, so that the extension reaction downstream thereof is strongly inhibited.

  The other region of the oligonucleotide only needs to have complementarity to the extent that it can hybridize to the gene to be detected. The length of the oligonucleotide is usually about 13 to 35 bases, preferably about 16 to 30 bases.

  That is, in a typical wild-type detection oligonucleotide, the nucleotide complementary to the polymorphic site of the wild type gene is located second from the 3 ′ end, and the third nucleotide from the 3 ′ end is the wild type and the mutant type. It is not complementary to both genes.

  The other oligonucleotide used as a pair is an oligonucleotide common to the wild type and the mutant type, and nucleic acid amplification can be performed in both genes.

In the present invention, the wild-type oligonucleotide and the mutant-type detection oligonucleotide are allowed to act on the sample separately or simultaneously.

The oligonucleotide may be extended by a known method. Usually, a oligonucleotide containing a specific nucleotide polymorphism site denatured into a single strand or a fragment thereof, together with four types of deoxynucleoside triphosphates (dNTP) and a DNA polymerase, a wild type detection oligonucleotide, and a mutant type detection The oligonucleotides are extended using the target nucleic acid as a template by allowing the oligonucleotides for use to act simultaneously or separately.

This extension reaction may be performed by a known method, for example, according to the method described in Molecular Cloning, A Laboratory Manual (Sambrook et al., 1989). Usually, a reverse primer common to four types of deoxynucleoside triphosphates (dNTP), DNA polymerase, wild-type and mutant-type genes on a chromosome containing a specific nucleotide polymorphism site denatured into a single strand or a fragment thereof In addition, the target nucleic acid is allowed to act between the forward primer and the reverse primer by using the wild type detection oligonucleotide and the single type detection oligonucleotide (corresponding to the forward primer) simultaneously or separately. Amplified.
Examples of nucleic acid amplification methods include PCR, NASBA (Nucleic acid sequence-based amplification method; Nature, Vol. 350, page 91 (1991)), LCR (International Publication No. 89/12696, JP-A-2-2934), SDA (Strand Displacement Amplification: Nucleic acid research Vol. 20, 1691 (1992)), RCR (International Publication No. 90/1069), TMA (Transcription mediated amplification method; J. Clin. Microbiol. Vol. 31, Vol. 31) 3270 (1993)) can be used.

  In the method using the nucleic acid amplification method as described above, when a wild-type nucleic acid can be amplified and a sample nucleic acid is amplified using a wild-type detection oligonucleotide, the reaction occurs if the sample nucleic acid is wild-type. In the mutant type, no reaction occurs. On the other hand, when the amplification reaction of the sample nucleic acid is performed using the mutant detection oligonucleotide, the reaction occurs if the sample nucleic acid is a mutant type, but does not occur if the sample nucleic acid is a wild type.

  Therefore, one sample is divided into two parts, one is reacted with the wild-type detection oligonucleotide and the other is reacted with the mutant detection oligonucleotide to check whether the reaction has occurred. This makes it possible to clearly know whether the sample nucleic acid is a wild type or a mutant type.

  In particular, higher organisms such as humans have one gene each derived from a father and one gene derived from a mother for one type of gene. According to this method, a sample gene is a wild-type homolog. It can also be distinguished whether it is a homozygous variant or a heterozygous variant. That is, in the case of heterozygous mutation, both a wild type gene and a mutant gene exist, and therefore an amplification reaction occurs both when a wild type detection oligonucleotide is used and when a mutation type detection oligonucleotide is used.

  When an amplification reaction is performed using a wild-type detection oligonucleotide and a mutation-type detection oligonucleotide simultaneously, the wild-type gene, homo-mutation gene, and hetero-mutation gene can be distinguished by using two sample nucleic acids after the amplification reaction. After reacting with the strand nucleic acid-specific binding substance, the amount of oligonucleotide can be determined or compared.

A labeled oligonucleotide can also be used. For example, each oligonucleotide may be labeled in advance with an enzyme, biotin, fluorescent substance, hapten, antigen, antibody, radioactive substance or luminophore, and the label of the reacted oligonucleotide may be detected after the extension or amplification reaction. .
Examples of the enzyme include alkaline phosphatase and peroxidase. Examples of the fluorescent substance include FITC, 6-FAM, HEX, TET, Texas Red (TxR), Cy3, and Cy5. Examples of the double-stranded nucleic acid specific binding substance include ethylene bromide and SYBR GreenI. Examples of haptens include biotin and digoxigenin. Examples of radioactive substances include ³² P and ³⁵ S. Examples of the luminophore include ruthenium. These labeling substances may be bound to any position of the oligonucleotide as long as they do not affect the extension reaction of the oligonucleotide. Preferably, it may be bound to the 5 ′ site.

When different labels are used for the wild-type detection oligonucleotide and the mutant-type detection oligonucleotide, the detection can be performed even if the amplification reaction is performed in one reaction vessel.
EXAMPLES Hereinafter, the present invention will be described more specifically based on examples, but the present invention is not limited to the following examples.

  Whether or not gene amplification was performed on the genome prepared from the human blood sample was detected as follows.

Detection of polymorphism of β3-adrenergic receptor gene Trp64Arg 2 mM dNTPs 2.5 µl, PCR Buffer 2.5 µl, Primer 5 pmol, Taq DNA polymerase 1.25 u, Mg to a final concentration of 2.5 mM per 18 µl milliQ water per sample A final volume of 25 μl was prepared, 1 μl of 20 ng / μl genome was added, and the reaction at 95 ° C. for 30 seconds, 65 ° C. for 30 seconds, 72 ° C. for 30 seconds was repeated 35 times to obtain an amplification product. PCR was performed using an allele-specific PCR method using Primer (w) for detecting the wild type and Primer (m) for detecting the mutant type. At this time, the Forward (w) Primer used was FITC-labeled at the 5 ′ end, the Forward (m) Primer used was labeled with TxR at the 5 ′ end, and the Reverse Primer used was labeled with biotin at the 5 ′ end.
Primer array
Forward (w): FITC-GCTATCGTGGCCATCGCGTG (SEQ ID NO: 7)
Forward (m): TxR-GCTATCGTGGCCATCGCACG (SEQ ID NO: 8)
Reverse: Biotin-ACGAACACGTTGGTCATGGTCT (SEQ ID NO: 9)

Detection of polymorphism of UCP1 gene Ala3826Gly 25μl of 2mM dNTPs 2.5μl, PCR Buffer 2.5μl, Primer 5pmol, Taq DNA polymerase 1.25u, Mg final concentration 2.5mM per sample 18μl of milli-Q water 1 μl of 20 ng / μl genome was added, and the reaction at 95 ° C. for 30 seconds, 60 ° C. for 30 seconds, and 72 ° C. for 30 seconds was repeated 35 times to obtain an amplification product. PCR was performed using an allele-specific PCR method using Primer (w) for detecting the wild type and Primer (m) for detecting the mutant type. At this time, the Forward Primer was labeled with Biotin at the 5 ′ end, the Reverse (w) Primer was labeled with FITC at the 5 ′ end, and the Reverse (m) Primer was labeled with TxR at the 5 ′ end.
Primer array
Forward: Biotin-AGCGATTTCTGATTGACCACA (SEQ ID NO: 10)
Reverse (w): FITC-AACACATTAACAAATGCACATG (SEQ ID NO: 11)
Reverse (m): TxR-AACACATTAACAAATGCACGCG (SEQ ID NO: 12)

Detection of polymorphism of β2-adrenergic receptor gene Arg16Gly 2 mM dNTPs 2.5 µl, PCR Buffer 2.5 µl, Primer 5 pmol, Taq DNA polymerase 1.25 u, Mg to a final concentration of 2.5 mM per 18 µl milliQ water per sample A final volume of 25 μl was prepared, 1 μl of 20 ng / μl genome was added, and the reaction at 95 ° C. for 30 seconds, 60 ° C. for 30 seconds, and 72 ° C. for 30 seconds was repeated 35 times to obtain an amplification product. PCR was performed using an allele-specific PCR method using Primer (w) for detecting the wild type and Primer (m) for detecting the mutant type. At this time, the Forward (w) Primer used was FITC-labeled at the 5 ′ end, the Forward (m) Primer used was labeled with TxR at the 5 ′ end, and the Reverse Primer used was labeled with biotin at the 5 ′ end.
Primer array
Forward (w): FITC-TCTTGCTGGCACCCAAAAG (SEQ ID NO: 13)
Forward (m): TxR-CTTGCTGGCACCCAAGGG (SEQ ID NO: 14)
Reverse: Biotin-CTGCGTGACGTCGTGGTC (SEQ ID NO: 15)

Detection of polymorphism of FABP2 gene Ala54Thr per sample 18 μl of milliQ water 2 μm dNTPs 2.5 μl, PCR Buffer 2.5 μl, each Primer 5 pmol, Taq DNA polymerase 1.25 u, Mg final concentration 25 μl to a final concentration of 2.5 mM 1 μl of 20 ng / μl genome was added, and the reaction at 95 ° C. for 30 seconds, 62.5 ° C. for 30 seconds, and 72 ° C. for 30 seconds was repeated 35 times to obtain an amplification product. PCR was performed using an allele-specific PCR method using Primer (w) for detecting the wild type and Primer (m) for detecting the mutant type. At this time, the Forward (w) Primer used was FITC-labeled at the 5 ′ end, the Forward (m) Primer used was labeled with TxR at the 5 ′ end, and the Reverse Primer used was labeled with biotin at the 5 ′ end.
Primer array
Forward (w): FITC-TCACAGTCAAAGAATCAAGTGC (SEQ ID NO: 16)
Forward (m): TxR-ATTCACAGTCAAAGAATCAAGAAC (SEQ ID NO: 17)
Reverse: Biotin-CAAAAACAACTTCAATGTTTCGA (SEQ ID NO: 18)

Detection of polymorphism of Angiotensinogen gene Met235Thr 25ml of 2mM dNTPs 2.5µl, PCR Buffer 2.5µl, Primer 5pmol, Taq DNA Polymerase 1.25u, Mg final concentration 2.5mM 1 μl of 20 ng / μl genome was added, and the reaction at 95 ° C. for 30 seconds, 60 ° C. for 30 seconds, and 72 ° C. for 30 seconds was repeated 35 times to obtain an amplification product. PCR was performed using an allele-specific PCR method using Primer (w) for detecting the wild type and Primer (m) for detecting the mutant type. At this time, the Forward Primer was labeled with Biotin at the 5 ′ end, the Reverse (w) Primer was labeled with FITC at the 5 ′ end, and the Reverse (m) Primer was labeled with TxR at the 5 ′ end.
Primer array
Forward: Biotin-GGCTGTGACAGGATGGAAGACT (SEQ ID NO: 19)
Reverse (w): FITC-CTGTCCACACTGGCTCCGAT (SEQ ID NO: 20)
Reverse (m): TxR-GTCCACACTGGCTCCAGT (SEQ ID NO: 21)

Detection of polymorphism of KIR6.2 gene Glu23Lys 2 mM dNTPs 2.5 μl, PCR Buffer 2.5 μl, Primer 5 pmol, Taq DNA polymerase 1.25 u, Mg final solution to 2.5 mM per sample in 18 μl milliQ water An amount of 25 μl was prepared, 1 μl of 20 ng / μl genome was added, and the reaction at 95 ° C. for 30 seconds, 62.5 ° C. for 30 seconds, and 72 ° C. for 30 seconds was repeated 35 times to obtain an amplification product. PCR was performed using an allele-specific PCR method using Primer (w) for detecting the wild type and Primer (m) for detecting the mutant type. At this time, the Forward (w) Primer used was FITC-labeled at the 5 ′ end, the Forward (m) Primer used was labeled with TxR at the 5 ′ end, and the Reverse Primer used was labeled with biotin at the 5 ′ end.
Primer array
Forward (w): FITC-GGCAGAGGACCCTGCGGA (SEQ ID NO: 22)
Forward (m): TxR-GGCAGAGGACCCTGCAAA (SEQ ID NO: 23)
Reverse: Biotin-CCTTTCTTGGACACAAAGCG (SEQ ID NO: 24)

Detection of polymorphism of RAGE gene Gly1704Thr 25ml of 2mM dNTPs 2.5µl, PCR Buffer 2.5µl, Primer 5pmol, Taq DNA polymerase 1.25u, Mg final concentration 2.5mM per sample 1 μl of 20 ng / μl genome was added, and the reaction at 95 ° C. for 30 seconds, 60 ° C. for 30 seconds, and 72 ° C. for 30 seconds was repeated 35 times to obtain an amplification product. PCR was performed using an allele-specific PCR method using Primer (w) for detecting the wild type and Primer (m) for detecting the mutant type. At this time, the Forward (w) Primer used was FITC-labeled at the 5 ′ end, the Forward (m) Primer used was labeled with TxR at the 5 ′ end, and the Reverse Primer used was labeled with biotin at the 5 ′ end.
Primer array
Forward (w): FITC-GGTAGGGTGAACCATAACTGGC (SEQ ID NO: 25)
Forward (m): TxR-GGTAGGGTGAACCATAACTTTC (SEQ ID NO: 26)
Reverse: Biotin-TTTCCCTCGTTAGCCCTCTG (SEQ ID NO: 27)

Detection of polymorphism of PPARγ gene Pro12Ala 2μm dNTPs 2.5μl, PCR Buffer 2.5μl, Primer 5pmol, Taq DNA polymerase 1.25u, Mg final concentration 25μl to 18μl of milliQ water per sample 1 μl of 20 ng / μl genome was added, and the reaction at 95 ° C. for 30 seconds, 62.5 ° C. for 30 seconds, and 72 ° C. for 30 seconds was repeated 35 times to obtain an amplification product. PCR was performed using an allele-specific PCR method using Primer (w) for detecting the wild type and Primer (m) for detecting the mutant type. At this time, the Forward (w) Primer used was FITC-labeled at the 5 ′ end, the Forward (m) Primer used was labeled with TxR at the 5 ′ end, and the Reverse Primer used was labeled with biotin at the 5 ′ end.
Primer array
Forward (w): FITC-GGGAGATTCTCCTATTGATCC (SEQ ID NO: 28)
Forward (m): TxR-TGGGAGATTCTCCTATTGAGGC (SEQ ID NO: 29)
Reverse: Biotin-ACAGTGTATCAGTGAAGGAATCG (SEQ ID NO: 30)

Detection of polymorphism of SUR1 Exon31 gene AGG1273AGA 2ml of dNTPs 2.5µl, PCR Buffer 2.5µl, Primer 5pmol, Taq DNA polymerase 1.25u, Mg final concentration 2.5mM per sample 18ml of milliQ water 25 μl was prepared, 1 μl of 20 ng / μl genome was added, and the reaction at 95 ° C. for 30 seconds, 65 ° C. for 30 seconds, 72 ° C. for 30 seconds was repeated 35 times to obtain an amplification product. PCR was performed using an allele-specific PCR method using Primer (w) Primer (w) for detecting the wild type and Primer (m) for detecting the mutant type. At this time, the Forward Primer was labeled with Biotin at the 5 ′ end, the Reverse (w) Primer was labeled with FITC at the 5 ′ end, and the Reverse (m) Primer was labeled with TxR at the 5 ′ end.
Primer array
Forward: Biotin-TGACCTCCATCTCCAACTCC (SEQ ID NO: 31)
Reverse (w): FITC-AGGCCAGCAGAGAGCTGCC (SEQ ID NO: 32)
Reverse (m): TxR-AGGCCAGCAGAGAGCTATC (SEQ ID NO: 33)

Detection with avidin-binding magnetic particles Whether or not the gene was amplified in Examples 1 to 9 was examined as follows.

10 μl of the amplification reaction solution obtained in Examples 1 to 9 was added to 40 μl of a solution of 500 mM Tris buffer (pH 7.5), 2 M NaCl, 1 mM EDTA, 1.25 μg avidin-coupled magnetic particles (Genovision), and brought to room temperature. For 15 minutes. Thereby, the amplified gene fragment and the biotin-labeled oligonucleotide are captured by the avidin-bound magnetic particles. Next, the magnetic particles are separated with a magnet, and the supernatant containing the unreacted fluorescently labeled oligonucleotide is mixed with 100 μl of 7.5 mM NaOH. After standing at room temperature for 10 minutes, the fluorescence intensity was measured with a fluorescent plate reader (Dainippon Pharmaceutical Co., Ltd.) in a dark room. The time required was about 0.5 hours. From the obtained fluorescence intensity amount, the fluorescence intensity amount of the sample was calculated using the following calculation formula.
FL (fluorescence intensity of sample) = FLb-FLs
FLb: Negative Control (no sample added) fluorescence intensity
FLs: Fluorescence intensity of each sample

Genetic polymorphisms were detected as in Examples 4 to 9 above for genes collected from 72 humans 40 years of age or older who had a BMI (body mass index) of 25 or more.

  In addition, resting metabolic rate, body weight, body mass index (BMI: value obtained by gradually grading body weight (kg) by the square of height (cm)), and Δ (kg) were measured. The results are summarized in Table 2 below.

In Table 2, RMR is the resting metabolic rate expressed by the following formula: Resting metabolic rate = ([total exercise metabolic rate]-[basal metabolic rate] x 1.2) / basal metabolic rate Also in Table 2, BW (kg) indicates body weight, and BMI is a value obtained by gradually grading body weight (kg) by the square of height (m), and indicates obesity. Δ (kg) indicates the amount of change in body weight.

  As can be seen from Table 2, significant changes in resting metabolic rate were observed due to mutations in specific sites of each gene, and these gene polymorphisms enable a reliable test as to whether or not they are obese. I understand.

Claims (36)

The following polynucleotide (1) or (2):
(1) A polynucleotide comprising 20 to 300 bases including the 163rd nucleotide of the human FABP (Fatty Acid Binding Protein) 2 gene or a variant thereof, wherein the 163rd gene polymorphic site is A.
(2) A polynucleotide complementary to the polynucleotide of (1). The polynucleotide according to claim 1, which consists of or is complementary to 20 to 300 bases including the nucleotide of base number 163 of SEQ ID NO: 1. An oligonucleotide set for detecting a gene polymorphism comprising the following (a) to (c).
(A) A 20 to 30 nucleotide oligonucleotide capable of hybridizing to a nucleotide sequence containing the polymorphic site of the wild-type human FABP2 gene or its complementary strand whose 163rd gene polymorphic site is G, the gene polymorphism A wild-type oligonucleotide for detection, wherein the nucleotide corresponding to the site is complementary to the nucleotide of the polymorphic site.
(B) a 20-30 base oligonucleotide capable of hybridizing to a nucleotide sequence comprising the polymorphic site of the mutant human FABP2 gene or its complementary strand, wherein the 163rd gene polymorphic site is A, the gene polymorphism An oligonucleotide for detecting a mutation, wherein the nucleotide corresponding to the site is complementary to the nucleotide of the polymorphic site.
(C) An oligonucleotide consisting of 20 to 30 bases that can hybridize to both wild type and mutant human FABP2 genes and can be used in pairs with (a) and (b) in nucleic acid amplification. A gene polymorphism detection primer set comprising the following (a) to (c).
(a) a 20-30 base oligonucleotide capable of hybridizing to a nucleotide sequence comprising the polymorphic site of the human wild-type FABP2 gene or its complementary strand, wherein the 163rd gene polymorphic site is G, the gene polymorphism The nucleotide corresponding to the site is complementary to the nucleotide of the polymorphic site and is present at the 1st to 2nd positions from the 3 'end, and the wild type and mutant human FABP2 gene is located in the upstream 1 to 2 base region adjacent to the nucleotide. A wild type detection oligonucleotide having nucleotides that are not complementary to each other.
(b) a 20 to 30 nucleotide oligonucleotide capable of hybridizing to a nucleotide sequence containing the polymorphic site of the mutant human FABP2 gene or its complementary strand whose 163rd gene polymorphic site is A, the gene polymorphism The nucleotide corresponding to the site is complementary to the nucleotide of the polymorphic site, is present at the 1st to 2nd positions from the 3 ′ end, and is adjacent to the upstream 1 to 2 base region, the wild type and mutant human FABP2 genes A mutant detection oligonucleotide having an oligonucleotide that is not complementary to any of the above.
(c) An oligonucleotide consisting of 20 to 30 bases that can hybridize to both wild type and mutant human FABP2 genes and can be used in pairs with (a) and (b) in nucleic acid amplification. A method for detecting a human FABP2 gene polymorphism that detects a base substitution at position 163 of the human FABP2 gene. The method according to claim 5, wherein a base substitution from G to A at the 163rd position of the human FABP2 gene is detected. The following polynucleotide (3) or (4):
(3) A polynucleotide comprising 20 to 300 bases comprising the 803th nucleotide of the human angiotensinogen gene or a variant thereof, wherein the 803th gene polymorphism site is C.
(4) A polynucleotide complementary to the polynucleotide of (3). The polynucleotide according to claim 7, comprising 20 to 300 bases including the nucleotide of base number 803 of SEQ ID NO: 2, or being complementary thereto. An oligonucleotide set for detecting a gene polymorphism comprising the following (e) to (f).
(D) a 20-30 base oligonucleotide capable of hybridizing to a nucleotide sequence comprising the polymorphic site of the wild type human angiotensinogen gene or its complementary strand, wherein the 803th gene polymorphic site is T, A wild-type detection oligonucleotide, wherein a nucleotide corresponding to a polymorphic site is complementary to a nucleotide of the polymorphic site.
(E) a 20-30 base oligonucleotide capable of hybridizing to a mutant human angiotensinogen gene whose 803 gene polymorphic site is C or a nucleotide sequence containing the polymorphic site of its complementary strand, A variant detection oligonucleotide, wherein a nucleotide corresponding to a polymorphic site is complementary to a nucleotide of the polymorphic site.
(F) An oligonucleotide consisting of 20 to 30 bases that can hybridize to both wild-type and mutant human angiotensinogen genes and can be used in pairs with (d) and (v) in nucleic acid amplification. A gene polymorphism detection primer set comprising the following (d) to (f).
(d) a 20-30 base oligonucleotide capable of hybridizing to a human wild-type angiotensinogen gene whose 803th gene polymorphic site is T or a nucleotide sequence containing the polymorphic site of its complementary strand, The base corresponding to the polymorphic site is complementary to the nucleotide of the polymorphic site and is present at the 1st to 2nd positions from the 3 'end, and the wild type and mutant human angio in the region of 1 to 2 bases adjacent to it. A wild type detection oligonucleotide having a nucleotide that is not complementary to any of the tensinogen genes.
(e) a 20-30 base oligonucleotide capable of hybridizing to a mutant human angiotensinogen gene whose 803 gene polymorphic site is C or a nucleotide sequence containing the polymorphic site of its complementary strand, The base corresponding to the polymorphic site is complementary to the nucleotide of the polymorphic site, is present at the 1st to 2nd positions from the 3 ′ end, and is a wild type and mutant human in the region of 1 to 2 bases upstream adjacent to it. A mutant detection oligonucleotide having an oligonucleotide that is not complementary to any of the angiotensinogen genes.
(f) An oligonucleotide consisting of 20 to 30 bases that can hybridize to both wild-type and mutant human angiotensinogen genes and can be used in pairs with (d) and (f) in nucleic acid amplification. A method for detecting a human angiotensinogen gene polymorphism, wherein a base substitution at position 803 of a human angiotensinogen gene is detected. The method of Claim 11 which detects the base substitution from T to C in the 803rd of a human angiotensinogen gene. The following polynucleotide (5) or (6):
(5) A polynucleotide comprising 20 to 300 bases including the 67th nucleotide of the human KIR (inwardly rectifying potassium channel) 6.2 gene or a variant thereof, wherein the 67th gene polymorphic site is A.
(6) A polynucleotide complementary to the polynucleotide of (5). The polynucleotide according to claim 13, comprising 20 to 300 bases including the nucleotide of base number 67 of SEQ ID NO: 3, or complementary thereto. An oligonucleotide set for detecting a gene polymorphism comprising the following (ki) to (ke).
(G) a wild type human KIR6.2 gene in which the 67th gene polymorphism site is G or a 20-30 base oligonucleotide capable of hybridizing to the nucleotide sequence containing the polymorphic site of its complementary strand, A wild-type detection oligonucleotide, wherein a nucleotide corresponding to a polymorphic site is complementary to a nucleotide of the polymorphic site.
(H) a 20-30 base oligonucleotide capable of hybridizing to a mutant human KIR6.2 gene whose 67th gene polymorphic site is A or a nucleotide sequence containing the polymorphic site of its complementary strand, A variant detection oligonucleotide, wherein a nucleotide corresponding to a polymorphic site is complementary to a nucleotide of the polymorphic site.
(G) An oligonucleotide consisting of 20 to 30 bases that can hybridize to both wild-type and mutant human KIR6.2 genes and can be used in pairs with (ki) and (ku) in nucleic acid amplification. A method for detecting a human KIR6.2 gene polymorphism that detects a base substitution at position 67 of the human KIR6.2 gene. The method according to claim 16, wherein a G-to-A base substitution at position 67 of the human KIR6.2 gene is detected. The following polynucleotide (7) or (8):
(7) A polynucleotide comprising 20 to 300 bases including the 8368th P2 nucleotide of a human RAGE (Receptor for Advanced Glycation End products) gene or a mutant thereof, wherein the polymorphic site of the 8368th gene is T. nucleotide.
(8) A polynucleotide complementary to the polynucleotide of (7). 19. The polynucleotide of claim 18 comprising or complementary to 20-300 bases comprising the nucleotide of SEQ ID NO: 4 base number 8368. An oligonucleotide set for detecting a gene polymorphism, comprising:
(E) a 20-30 base oligonucleotide capable of hybridizing to a nucleotide sequence comprising the polymorphic site of the wild type human RAGE gene or its complementary strand, wherein the 8368th gene polymorphic site is G, the gene polymorphism A wild-type oligonucleotide for detection, wherein the nucleotide corresponding to the site is complementary to the nucleotide of the polymorphic site.
(Sa) a 20-30 base oligonucleotide capable of hybridizing to a nucleotide sequence comprising the polymorphic site of the mutant human RAGE gene or its complementary strand, wherein the 8368th gene polymorphic site is T, the gene polymorphism A variant detection oligonucleotide, wherein a nucleotide corresponding to a site is complementary to a nucleotide of the polymorphic site.
(F) It consists of 20-30 bases that can hybridize to both wild-type and mutant human RAGE genes and can be used in pairs with (co) and (sa) in nucleic acid amplification, or is complementary thereto. An oligonucleotide. A gene polymorphism detection primer set comprising the following (g) to (i).
(g) a 20-30 base oligonucleotide capable of hybridizing to a nucleotide sequence containing the polymorphic site of the human wild-type RAGE gene or its complementary strand, wherein the 8368th gene polymorphic site is G, the gene polymorphism The base corresponding to the site is complementary to the nucleotide of the polymorphic site and is present at the 1st to 2nd positions from the 3 'end, and the wild type and mutant human RAGE genes are located in the upstream 1 to 2 base region adjacent to it. A wild type detection oligonucleotide having nucleotides that are not complementary to each other.
(h) a 20-30 base oligonucleotide capable of hybridizing to a nucleotide sequence containing the polymorphic site of the mutant human RAGE gene or its complementary strand, wherein the 8368th gene polymorphic site is T, the gene polymorphism The base corresponding to the site is complementary to the nucleotide of the polymorphic site, is present at the 1st to 2nd position from the 3 ′ end, and the wild type and mutant human RAGE genes in the upstream 1 to 2 base region adjacent to it A mutant detection oligonucleotide having an oligonucleotide that is not complementary to any of the above.
(i) An oligonucleotide consisting of 20 to 30 bases that can hybridize to both wild-type and mutant human RAGE genes and can be used in pairs with (g) and (h) in nucleic acid amplification. A method for detecting a human RAGE gene polymorphism that detects a base substitution at position 8368 of a human RAGE gene. The method according to claim 22, wherein a base substitution from G to T at the 8368th position of the human RAGE gene is detected. The following polynucleotide (9) or (10):
(9) A polynucleotide comprising 20 to 300 bases including the 34th nucleotide of a PPAR (Peroxisome Proliferator-Activated Receptor) γ gene or a variant thereof, wherein the 34th gene polymorphic site is G.
(10) A polynucleotide complementary to the polynucleotide of (9). 25. The polynucleotide of claim 24, comprising or complementary to 20-300 bases comprising nucleotide number 34 of SEQ ID NO: 5. An oligonucleotide set for detecting a gene polymorphism comprising the following (su) to (so).
(Su) An oligonucleotide of 20 to 30 bases capable of hybridizing to a nucleotide sequence containing the polymorphic site of the wild-type human PPARγ gene or its complementary strand whose 34th gene polymorphic site is C, the gene polymorphism A wild-type oligonucleotide for detection, wherein the nucleotide corresponding to the site is complementary to the nucleotide of the polymorphic site.
(C) a 20-30 base oligonucleotide capable of hybridizing to a nucleotide sequence comprising the polymorphic site of the mutant human PPARγ gene or its complementary strand whose 34th gene polymorphic site is G, the gene polymorphism A variant detection oligonucleotide, wherein a nucleotide corresponding to a site is complementary to a nucleotide of the polymorphic site.
(So) An oligonucleotide consisting of 20 to 30 bases that can hybridize to both wild-type and mutant human PPARγ genes and can be used in pairs with (su) and (se) in nucleic acid amplification. A gene polymorphism detection primer set comprising the following (j) to (l).
(j) a 20-30 base oligonucleotide capable of hybridizing to a nucleotide sequence containing the polymorphic site of the human wild-type PPARγ gene or its complementary strand whose 34th gene polymorphic site is C, the gene polymorphism The base corresponding to the site is complementary to the nucleotide of the polymorphic site, is present at the 1st to 2nd position from the 3 ′ end, and the wild type and mutant human PPARγ genes in the upstream 1 to 2 base region adjacent thereto A wild type detection oligonucleotide having a nucleotide that is not complementary to any of the above.
(k) a 20-30 base oligonucleotide capable of hybridizing to a nucleotide sequence containing the polymorphic site of the mutant human PPARγ gene or its complementary strand whose 34th gene polymorphic site is G, the gene polymorphism The base corresponding to the site is complementary to the nucleotide of the polymorphic site, is present at the 1st to 2nd position from the 3 ′ end, and the wild type and mutant human PPARγ genes in the upstream 1 to 2 base region adjacent thereto A mutant detection oligonucleotide having an oligonucleotide that is not complementary to any of the above.
(l) An oligonucleotide consisting of 20 to 30 bases that can hybridize to both wild type and mutant human PPARγ genes and can be used in pairs with (j) and (k) in nucleic acid amplification. A method for detecting a human PPARγ gene polymorphism, wherein a base substitution at position 34 of the human PPARγ gene is detected. The method according to claim 28, wherein a base substitution from C to G at position 34 of the human PPARγ gene is detected. The following polynucleotide (11) or (12):
(11) A polynucleotide comprising 20 to 300 bases including the 1273th nucleotide of exon 31 of human SUR (Sulfonylurea Receptor) 1 gene or a variant thereof, wherein the 1273th gene polymorphism site is A .
(12) A polynucleotide complementary to the polynucleotide of (11). 31. The polynucleotide of claim 30, comprising or complementary to 20-300 bases comprising the nucleotide of SEQ ID NO: 6 base number 3819. An oligonucleotide set for detecting a gene polymorphism comprising the following (ta) to (tu).
(Ta) An oligonucleotide of 20 to 30 bases that can hybridize to a nucleotide sequence containing the polymorphic site of the wild-type human SUR1 gene or its complementary strand, wherein the 1273th gene polymorphic site of exon 31 is G, A wild-type oligonucleotide for detection, wherein a nucleotide corresponding to a gene polymorphic site is complementary to a nucleotide of the polymorphic site.
(H) an oligonucleotide of 20 to 30 bases capable of hybridizing to a nucleotide sequence containing the polymorphic site of the mutant human SUR1 gene or its complementary strand, wherein the 1273th gene polymorphic site of exon 31 is A, A mutant detection oligonucleotide, wherein a nucleotide corresponding to a gene polymorphic site is complementary to a nucleotide of the polymorphic site.
(Iv) An oligonucleotide consisting of 20 to 30 bases that can hybridize to both wild type and mutant human SUR1 genes and can be used in pairs with (ta) and (h) in nucleic acid amplification. A gene polymorphism detection primer set comprising the following (m) to (o).
(m) a 20-30 base oligonucleotide capable of hybridizing to a nucleotide sequence containing the polymorphic site of the human wild-type SUR1 gene or its complementary strand, wherein the 1273th gene polymorphic site of exon 31 is G, The base corresponding to the gene polymorphic site is complementary to the nucleotide of the polymorphic site, is present first to second from its 3 ′ end, and is adjacent to the upstream 1-2 base region, wild type and mutant type A wild-type detection oligonucleotide having nucleotides that are not complementary to any of the human SUR1 genes.
(n) an oligonucleotide of 20 to 30 bases capable of hybridizing to a nucleotide sequence containing the polymorphic site of the human mutant SUR1 gene or its complementary strand, wherein the 1273th gene polymorphic site of exon 31 is A, The base corresponding to the gene polymorphic site is complementary to the nucleotide of the polymorphic site, is present first to second from its 3 ′ end, and is adjacent to the upstream 1-2 base region, wild type and mutant type A mutant detection oligonucleotide having a nucleotide that is not complementary to any of the human SUR1 genes.
(o) An oligonucleotide consisting of 20 to 30 bases that can hybridize to both wild type and mutant human SUR1 genes and can be used in pairs with (m) and (n) in nucleic acid amplification. A method for detecting a human SUR1 gene polymorphism that detects a base substitution at position 1273 of exon 31 of human SUR1 gene. 35. The method according to claim 34, wherein a base substitution from G to A at the 1273th position of human SUR1 gene exon 31 is detected. A method for examining an obesity constitution comprising a step of detecting four or more of the following gene polymorphisms (i) to (ix) for a human gene and a step of determining whether or not the subject is an obesity constitution.
(i) Base substitution at position 163 of human FABP2 gene
(ii) Base substitution at position 803 of the human angiotensinogen gene
(iii) Base substitution at position 67 of human KIR6.2 gene
(iv) Base substitution at the 8368th position of the human RAGE gene
(v) Base substitution at position 34 of human PPARγ gene
(vi) Base substitution at position 1273 of exon 31 of human SUR1 gene
(vii) Base substitution at position 190 in human β3-adrenergic receptor gene
(viii) Base substitution at position 3826 (−3862) upstream from the start codon of human UCP (Uncoupling Protein) 1 gene
(ix) Base substitution at 46th position in human β2-adrenergic receptor gene