JP4657857B2 - Diagnosis and prevention of metabolic syndrome - Google Patents

Description

  The present invention relates to diagnosis and prevention of metabolic syndrome, and more particularly to diagnosis and prevention of metabolic syndrome using a polymorphism of a gene associated with the onset of metabolic syndrome or a protein that is a translation product thereof.

  Metabolic syndrome (also referred to as “MS”) is the accumulation of fat in the internal organs, which weakens the effect of insulin, a hormone that lowers blood sugar (ie, increases insulin resistance) It represents a disease state in which abnormalities in lipid metabolism occur. In MS, risk factors for arteriosclerosis, “visceral fat obesity”, “hypertension”, “abnormal glucose tolerance (hyperglycemia)”, and “hyperlipidemia” often occur. . All of these symptoms are considered to cause arteriosclerosis. Atherosclerosis is currently the second and third most common cause of death in Japan, and is thought to cause cardio-cerebral vascular disorders, the cause of heart disease and cerebrovascular disease, which accounts for about one third of the national causes of death. . Therefore, MS, which is a pathological condition in which multiple risk factors such as hypertension, diabetes, dyslipidemia, and obesity overlap, is not a direct cause of death, but a disease that causes a high mortality if MS is prolonged. It can be said.

  However, MS can be expected to improve symptoms by exercise therapy and / or diet therapy if it is mild. Therefore, early detection and early treatment of MS are extremely important for extending the healthy life expectancy and improving the quality of life (QOL) of the elderly.

  Such a diagnosis of MS is performed based on, for example, US hyperlipidemia treatment guidelines. In the above US hyperlipidemia treatment guidelines, (a) waist (abdominal circumference) is 102 cm or more for men, 88 cm or more for women, (b) neutral fat is 150 mg / dl or more, and (c) HDL cholesterol is 40 mg / min for men. at least 3 items out of 5 items, less than dl, less than 50 mg / dl in women, (d) blood pressure is 130 mmHg or more at maximum blood pressure or 85 mmHg or more at minimum blood pressure, and (e) fasting blood glucose level is 110 mg / dl or more If it is true, it is diagnosed as MS.

  In Japan, diagnostic criteria were not established in Japan for many years, but in 2005, the diagnostic criteria for MS were first shown in Japan at the Japanese Society of Internal Medicine. According to the diagnostic criteria of MS, the waist is not less than a prescribed value (85 cm for men, 90 cm for women), and (a) the highest blood pressure is 130 or higher, the lowest blood pressure is 85 or higher, (b) hungry It is diagnosed as MS when two or more of the three items of blood glucose level at time 110 or higher, (c) neutral fat 150 or higher, or HDL-cholesterol is lower than 40 are applied. In addition, MS diagnostic criteria include diagnostic criteria by the World Health Organization (WHO).

  The above diagnostic criteria are only for diagnosing MS after the onset of MS, but MS is a disease that can be prevented from occurring by improving lifestyle habits. In other words, if the possibility of becoming MS in the future can be determined before the onset of MS, the determination result can be received to improve the lifestyle and prevent the onset of MS.

Therefore, development of a method for determining the possibility of becoming MS in the future has been demanded, and so far, for example, a method for detecting a polymorphism in a Klotho (also referred to as Kloth) gene has been proposed (patent) Reference 1). Specifically, Patent Document 1 discloses a method for diagnosing a predisposition for MS by determining the genotype of a Klotho gene and determining whether the genotype includes a risk genotype.
JP 2003-514513 A (published on April 22, 2003) Kuro-o et al. Nature, 390, 45-51 (1997) Christopher D. Kontos et al., Tyrosine 1101 of Tie2 is the major site of association of p85 and is required for activation of phosphatidylinositol 3-kinase and Akt, Molecular and Cellular Biology, 18, 4131-4140 (1998)

  As a method for determining the onset of MS or the possibility of developing in the future, if a molecular biological technique as disclosed in Patent Document 1 is used, a rapid and accurate diagnosis can be performed. However, the Klotho gene disclosed in Patent Document 1 has been identified as a gene involved in the suppression of several aging phenotypes in mice (see Non-Patent Document 1). Furthermore, it has been suggested that the human Klotho gene is involved in the progression of senile osteoporosis in postmenopausal women (see Patent Document 1). That is, since the Klotho gene is not a gene whose direct causal relationship with MS has been scientifically proved, it is not sufficient to diagnose the onset possibility of MS only by using this as an index.

  In general, a disease is rarely caused by a single causative gene, and often develops by complicatedly intertwining multiple causative genes. In order to determine this, it is considered effective to use polymorphisms of a plurality of genes as an index. Therefore, it is also required to identify a new gene polymorphism for determining (diagnosis) the onset or possibility of onset for MS.

  The present invention has been made in view of the above problems, and an object of the present invention is to identify a polymorphism of a gene associated with the onset of MS and to provide diagnosis and prevention of MS.

  As a result of intensive studies in view of the above problems, the present inventors have uniquely found that the frequency of developing MS varies depending on the genotype of an angiopoietin-1 gene (hereinafter also referred to as “Ang-1 gene”). The present invention has been completed.

  That is, the method for determining the onset or likelihood of onset of MS according to the present invention uses an angiopoietin-1 protein (hereinafter referred to as “Ang−”) consisting of the amino acid sequence shown in SEQ ID NO: 1 using a sample isolated from a living body. A gene that encodes glycine corresponding to the 269th amino acid of the above-mentioned angiopoietin-1 is included in the gene encoding “a protein” (also referred to as “1 protein”).

  The method for determining the onset or likelihood of onset of MS according to the present invention uses a sample isolated from a living body to convert the base sequence shown in SEQ ID NO: 2 into an open reading frame (hereinafter referred to as “ORF”). The presence or absence of insertion of 3 bases of “GGT” (G and T represent guanine and thymine, respectively) corresponding to the 805th to 807th bases in the base sequence of the Ang-1 gene possessed as a region is determined. It is characterized by including a process.

  Furthermore, the method for determining the onset or likelihood of onset of MS according to the present invention is characterized by including a step of detecting a polymorphism of Ang-1 protein.

  The kit for determining the onset or likelihood of onset of MS according to the present invention includes (a) a region comprising the base corresponding to the 805th to 807th positions in the above-mentioned base sequence of the gene having the base sequence represented by SEQ ID NO: 2 as the open reading frame region (B) a probe that binds to a region containing bases corresponding to positions 805 to 807 in the above-described base sequence of the gene having the base sequence represented by SEQ ID NO: 2 as an open reading frame region, and (c And at least one of an antibody that recognizes only one of the protein consisting of the amino acid sequence shown in SEQ ID NO: 1 or the protein lacking glycine corresponding to the 269th amino acid of the protein, and How to determine the onset or likelihood of the above metabolic syndrome It is characterized by the use of any of the.

  The prophylactic agent for MS according to the present invention is for administration of a person having a protein consisting of the amino acid sequence shown in SEQ ID NO: 1, and encodes a protein lacking glycine corresponding to the 269th amino acid of the protein or the protein It is characterized by containing a DNA, a low-molecular compound as an agonist of a receptor protein to which the protein is a ligand, or a modulator of the Ang-1 gene.

  As described above, according to the present invention, it is possible to determine the onset or possibility of onset of MS using the causal relationship between the polymorphism of the Ang-1 gene or its translation product protein and the onset of MS. it can. Therefore, there is an effect that it is possible to easily and accurately determine the onset or possibility of onset of MS. Further, among the polymorphisms of the Ang-1 gene or a translation product thereof, among the polymorphisms of the Ang-1 gene or a translation product thereof, a method for preventing MS and a preventive drug by using the Ang-1 gene or a translation product thereof with a low risk of developing MS Application to the development of is also possible.

  An embodiment of the present invention will be described as follows, but the present invention is not limited to this.

  In the present specification, unless otherwise specified, A, C, G and T represent adenine, cytosine, guanine and thymine bases. For amino acids and amino acid residues, the one-letter code or three-letter code defined by IUPAC and IUB is used.

<1. Angiopoietin-1 gene and angiopoietin-1 protein>
In this specification, “Ang-1 gene” and “Ang-1 protein” are a gene having a base sequence published as an angiopoietin-1 gene on a base sequence database, and a translation product of the gene. It is a general term for a certain protein. The Ang-1 protein includes a protein whose amino acid sequence is disclosed as angiopoietin-1 protein on various databases.

  Examples include (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1, a protein consisting of the amino acid sequence registered in NCBI accession numbers BAB91325 and NP_001137, and genes encoding these proteins.

  The protein consisting of the amino acid sequence shown in SEQ ID NO: 1 is a protein having glycine as the 269th amino acid in the sequence. On the other hand, a protein consisting of an amino acid sequence registered under NCBI accession number BAB91325 is a protein that does not have the glycine.

  The protein consisting of the amino acid sequence shown in SEQ ID NO: 1 is encoded by a gene having the base sequence shown in SEQ ID NO: 2 as the ORF region. In the present specification, the “ORF region” refers to a region from the start codon to the stop codon or a part thereof. The gene has 3 bases “GGT” as the 805th to 807th bases in the base sequence shown in SEQ ID NO: 2. These three bases correspond to the base encoding glycine, which is the 269th amino acid in the amino acid sequence shown in SEQ ID NO: 1. The gene having the base sequence shown in SEQ ID NO: 2 as the ORF region corresponds to the base sequence (accession number: U83508) registered in GenBank as the Ang-1 gene cDNA. The 805th to 807th bases in SEQ ID NO: 2 correspond to the 1114th to 1116th bases of the base sequence (accession number: U83508).

  A protein having an amino acid sequence registered in NCBI accession number BAB91325 is encoded by a gene having a base sequence registered in GenBank (accession number: AB084454) as an ORF region. In the gene, 3 bases “GGT” corresponding to the 805th to 807th bases are deleted from the base sequence shown in SEQ ID NO: 2. As a result, the translation product protein lacks glycine corresponding to the 269th amino acid of the amino acid sequence shown in SEQ ID NO: 1.

  Further, as the Ang-1 gene and Ang-1 protein according to the present invention, (b) one or several amino acids in the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence registered in NCBI accession numbers BAB91325 and NP_001137 Include a protein having a function of Ang-1, which comprises an amino acid sequence substituted, deleted, inserted and / or added, and a gene encoding the protein.

  The above “one or several amino acids are substituted, deleted, inserted, and / or added” means (1) one or several amino acids are substituted, deleted, inserted, and / or naturally. Or (2) the number that can be substituted, deleted, inserted, and / or added by a known mutant peptide production method such as site-directed mutagenesis (preferably 10 or less, more (Preferably 7 or less, more preferably 5 or less) both the meaning of substitution, deletion, insertion and / or addition. That is, the protein (b) can be considered as a mutant protein of the protein (a), or can be considered as a polymorphism of the protein (a).

  In addition, the Ang-1 gene according to the present invention has one or more bases inserted, substituted, and / or inserted into the base sequence (accession number: AB0844454) and a part of the base sequence shown in SEQ ID NO: 2. Alternatively, it may be a gene having a deleted base sequence as an OFR region. Also in this case, the “base sequence in which one or more bases are inserted, substituted, and / or deleted” may be naturally occurring or may be generated by artificial mutagenesis.

  Furthermore, the Ang-1 gene according to the present invention hybridizes under stringent hybridization conditions with a DNA comprising a base sequence complementary to the DNA comprising the base sequence shown in SEQ ID NO: 2, and A gene encoding a protein having a function can be mentioned.

  The above “hybridization under stringent hybridization conditions” means only when at least 90% identity, preferably at least 95% identity, most preferably at least 97% identity exists between sequences. It means that hybridization occurs. Specific examples of “stringent hybridization conditions” include, for example, a hybridization solution (50% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6). ) In 5 × Denhart solution, 10% dextran sulfate, and 20 μg / ml denatured sheared salmon sperm DNA) after overnight incubation at 42 ° C., then filter in 0.1 × SSC at about 65 ° C. The conditions for washing can be mentioned. The hybridization can be performed by a conventionally known method such as the method described in J. Sambrook et al. Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory (1989). It is not limited. Usually, the higher the temperature and the lower the salt concentration, the higher the stringency (harder to hybridize).

  In the present specification, “gene” is used interchangeably with “polynucleotide”, “nucleic acid” or “nucleic acid molecule”. “Polynucleotide” means a polymer of nucleotides. Therefore, the term “gene” in the present specification includes not only double-stranded DNA but also single-stranded DNA and RNA (such as mRNA) such as sense strand and antisense strand constituting the DNA. The antisense strand can be utilized as a probe or as an antisense agent. “DNA” includes, for example, cDNA and genomic DNA obtained by cloning, chemical synthesis techniques, or a combination thereof. That is, the DNA may be a “genomic” type DNA containing a non-coding sequence such as an intron which is a form contained in the genome, or a cDNA obtained through mRNA using a reverse transcriptase or a polymerase, That is, it may be “transcribed” DNA that does not contain non-coding sequences such as introns. In addition to the nucleotide sequence encoding the amino acid sequence described in (a) or (b) above, the gene according to the present invention includes an untranslated region (UTR) sequence and a vector sequence (including an expression vector sequence). It may contain a sequence. Moreover, arbitrary polynucleotides, such as a regulatory sequence and a polyadenyl sequence, may be contained at the terminal and / or inside of the translation region of these mRNA or cDNA. In the present specification, the term “nucleic acid” includes a polynucleotide comprising any simple nucleotide and / or modified nucleotide, such as cDNA, mRNA, total RNA, and hnRNA. “Modified nucleotides” include inosin, acetylcytidine, methylcytidine, methyladenosine, and methylguanosine, as well as nucleotides that can be generated by the action of ultraviolet light or chemical substances.

  In the present specification, “base sequence” is used interchangeably with “nucleic acid sequence” and is shown as a sequence of deoxyribonucleotides. Also, a polynucleotide or polynucleotide “base sequence” is intended to be a sequence of deoxyribonucleotides relative to a DNA molecule or polynucleotide, and a corresponding sequence of ribonucleotides relative to an RNA molecule or polynucleotide (wherein Each thymidine deoxynucleotide (T) in the specified deoxynucleotide sequence is intended to be replaced by the ribonucleotide uridine (U)).

  For example, “an RNA molecule having the sequence of SEQ ID NO: 2 or 4” indicated using the abbreviation of deoxyribonucleotide means that each deoxynucleotide A, G, or C of SEQ ID NO: 2 or 4 corresponds to the corresponding ribonucleotide A, G Or is intended to indicate an RNA molecule having a sequence that is replaced by C and deoxynucleotide T is replaced by ribonucleotide U. The “polynucleotide or a fragment thereof containing the base sequence shown in SEQ ID NO: 2 or 4” means a polynucleotide containing the sequence shown by each deoxynucleotide A, G, C and / or T of SEQ ID NO: 2 or 4. Or a fragment part thereof is intended.

<2. Method for determining the onset or likelihood of onset of MS>
The method for determining the onset or possibility of onset of MS according to the present invention (in other words, the method for diagnosing MS) includes the presence or absence of a specific amino acid in the Ang-1 protein, the gene encoding the Ang-1 protein, or Ang- Any method may be used as long as it determines the presence or absence of a specific base in one gene and determines the onset or possibility of onset of MS based on the presence or absence of the amino acid or base. Other specific configurations are not particularly limited. For example, the method shown below is mentioned.

  (I) A method comprising the step of determining the presence or absence of insertion of 3 bases encoding glycine corresponding to the 269th amino acid of the protein consisting of the amino acid sequence shown in SEQ ID NO: 1 in the gene encoding Ang-1 protein.

  (II) A step of determining the presence or absence of insertion of 3 bases of “GGT” corresponding to the 805th to 807th bases in the base sequence of the gene having the base sequence shown in SEQ ID NO: 2 as the ORF region in the Ang-1 gene Including methods.

  (III) A method comprising a step of determining the presence or absence of insertion of glycine corresponding to the 269th amino acid of the protein consisting of the amino acid sequence represented by SEQ ID NO: 1 in the Ang-1 protein.

  The methods (I) to (III) further include a step of determining a genotype of a gene encoding the Ang-1 protein, a step of determining a genotype of the Ang-1 gene, and a polymorphism of the Ang-1 protein Preferably, the method includes a step of detecting each.

  By providing these steps, it is possible to more accurately determine the onset or possibility of onset of MS.

  In the present specification, “gene polymorphism” means that there are several types of base sequences of the same gene. The “protein polymorphism” means that there are several types of amino acid sequences of the same protein.

  Hereinafter, the methods (I) to (III) will be described in detail. The Ang-1 protein and the Ang-1 gene have been described in detail in the above section <1>, and thus description thereof is omitted here.

[Method (I)]
In the gene encoding the Ang-1 protein, the method for determining the presence or absence of insertion of 3 bases encoding glycine corresponding to the 269th amino acid of the protein consisting of the amino acid sequence shown in SEQ ID NO: 1 is particularly limited. In addition, a conventionally known method can be used. Specifically, (A) a method using genomic DNA and (B) a method using mRNA (cDNA) can be used.

(A) Method using genomic DNA Although the genome of a subject can be obtained from all cells of the human body by a conventional method, it can be obtained from, for example, hair, organs, peripheral lymphocytes, synovial cells, etc. . Further, the obtained cells can be obtained by culturing and proliferating. It can also be obtained from peripheral blood.

  The obtained genome can be obtained by, for example, a commonly performed gene amplification method such as PCR (Polymerase Chain Reaction) method, NASBA (Nucleic acid sequence based amplification) method, TMA (Transcription-mediated amplification) method, and SDA (Strand Displacement Amplification) method. Can be amplified and used.

  The method for determining the genotype using genomic DNA is not particularly limited. For example, allyl-specific oligonucleotide probe method, oligonucleotide ligation assay method, PCR-SSCP method, PCR -CFLP method, PCR-HPFA method, invader method, RCA (Rolling Circle Amplification) method, primer oligo base extension (Primer Oligo Base Extension) method, tiling array method, denaturing gradient gel electrophoresis method and the like.

  Specifically, a specific region in the gene encoding the Ang-1 protein from the genome (for example, a region containing 3 bases encoding glycine corresponding to the 269th amino acid of the protein consisting of the amino acid sequence shown in SEQ ID NO: 1) After amplification, the nucleotide sequence of a specific region of the gene can be determined by direct sequencing of the obtained PCR product or by subcloning and then sequencing the PCR product portion. That is, the presence or absence of three bases encoding glycine corresponding to the 269th amino acid of the protein consisting of the amino acid sequence represented by SEQ ID NO: 1 in this embodiment can be determined.

  Further, by using the specific region as an oligonucleotide probe, a difference in the base sequence of the specific region can be detected. That is, the presence or absence of three bases encoding glycine corresponding to the 269th amino acid of the protein consisting of the amino acid sequence represented by SEQ ID NO: 1 in this embodiment can be determined. In this way, as a probe, for example, an oligonucleotide consisting of a base sequence of a specific region of the Ang-1 gene is fixed on a chip to constitute a DNA chip, and the DNA chip is used for genotype analysis. Cases. In this case, the length of the oligonucleotide probe is preferably 7 to 50 nucleotides or 10 to 30 nucleotides including the polymorphic region of the Ang-1 gene, and more preferably 15 to 25 nucleotides.

(B) Method using mRNA (cDNA) When using mRNA, for example, cDNA is prepared from mRNA extracted from the subject's cells by reverse transcription reaction, and after amplification of the gene region, as in (A) above, The genotype can be determined by directly sequencing the base sequence of the amplified fragment, using a DNA chip, or using the RFLP (Restriction Fragment Length Polymorphism) method.

Primers used for amplification of a specific region of the gene (for example, a region containing 3 bases encoding glycine corresponding to the 269th amino acid of the protein consisting of the amino acid sequence shown in SEQ ID NO: 1) are not particularly limited However, for example, an amplification reaction using cDNA as a template is possible by the following combination of primers.
Sense primer 1: 5'-CCACCAACAACAGTGTCCTT-3 '(SEQ ID NO: 3)
Sense primer 2: 5'-CAACCTTGTCAATCTTTGC-3 '(SEQ ID NO: 4)
Sense primer 3: 5'-GCTGGCAGTACAATGACAG-3 '(SEQ ID NO: 5)
Antisense primer 1: 5'-TCAAAAATCTAAAGGTCGAAT-3 '(SEQ ID NO: 6)
Antisense primer 2: 5'-CAGCTTGATATACATCTGCACAG-3 '(SEQ ID NO: 7)
The primers and probes used in the present invention can be prepared by a conventional method using a DNA synthesizer or the like.

  The presence or absence of 3 bases encoding glycine corresponding to the 269th amino acid of the protein consisting of the amino acid sequence shown in SEQ ID NO: 1 is cleaved using the above amplified fragment or genomic DNA using an appropriate restriction enzyme. It can also be determined by detecting differences in the size of genome fragments by Southern blotting or the like.

  In the method for determining the onset or the likelihood of onset of MS according to the present invention, depending on the presence or absence of the determined three bases, those who have a gene having the three bases are more likely to develop MS. It can be determined that a person having a deleted gene is less likely to develop MS (see Examples below).

  Further, in the step of determining the genotype of the gene encoding the Ang-1 protein, a gene lacking 3 bases encoding glycine corresponding to the 269th amino acid of the protein consisting of the amino acid sequence represented by SEQ ID NO: 1, Determine whether you have homo, hetero, or no. The genotype of the gene encoding the Ang-1 protein can be determined by using the method for determining the presence or absence of the three bases. In this case, the “specific region in the gene encoding Ang-1 protein” may be read as “polymorphic region in the gene encoding Ang-1 protein”. In the present specification, the “polymorphic region” means a region including a locus or position where the diversity of the gene appears.

  By providing the said process, the determination precision of onset or onset possibility of MS can be improved more. Specifically, when the genotype of the Ang-1 gene is a genotype having the Ang-1 gene lacking the 3 bases (hereinafter also referred to as “3-base deleted Ang-1 gene”) in homology. It can be determined that the possibility of developing MS or the possibility of developing MS in the future is low. On the other hand, when the genotype has the 3-base insertion type Ang-1 gene homozygously, it can be determined that MS is likely to develop or is likely to develop in the future. In the case of a genotype having an Ang-1 gene having 3 bases (hereinafter also referred to as “3-base insertion type Ang-1 gene”) and a 3-base deletion Ang-1 gene in a heterogeneous form, MS is used. It can be determined that the possibility of developing or the possibility of developing in the future is moderate.

  Thus, according to this configuration, the diagnosis (determination of onset or possibility of onset) of the subject's MS can be performed accurately and simply.

[Method (II)]
In the Ang-1 gene, the method for determining the presence or absence of insertion of 3 bases “GGT” corresponding to the 805th to 807th bases in the base sequence of the gene having the base sequence shown in SEQ ID NO: 2 as the ORF region is particularly limited. Is not to be done. For example, a method similar to the method for determining the presence or absence of insertion of 3 bases in the above method (I) can be used. The “specific region of the gene encoding the Ang-1 protein” described in the above method (I) may be read as the specific region of the Ang-1 gene in this embodiment. In the present invention, the specific region of the gene encoding the Ang-1 protein is 3 bases of “GGT” corresponding to the 805th to 807th bases in the base sequence of the gene having the base sequence represented by SEQ ID NO: 2 as the ORF region. It is preferable that it is the area | region containing.

  In the method for determining the onset or the likelihood of onset of MS according to the present invention, depending on the presence or absence of the determined three bases, those who have a gene having the three bases are more likely to develop MS. It can be determined that a person having a deleted gene is less likely to develop MS (see Examples below).

  In the step of determining the genotype of the Ang-1 gene, 3 bases “GGT” corresponding to the 805th to 807th bases in the base sequence of the gene having the base sequence shown in SEQ ID NO: 2 as the ORF region are added. Determine whether the deleted gene is homozygous, heterozygous or not. The genotype of the Ang-1 gene can be determined by using the method for determining the presence or absence of the above three bases. In this case, the “specific region in the gene encoding the Ang-1 protein” may be read as “polymorphic region in the Ang-1 gene”.

  By including the step of determining the genotype of the Ang-1 gene, the determination accuracy of the onset or onset possibility of MS can be further increased. Specifically, when the genotype of the Ang-1 gene is a genotype having the Ang-1 gene lacking the 3 bases (hereinafter also referred to as “3-base deleted Ang-1 gene”) in homology. It can be determined that the possibility of developing MS or the possibility of developing MS in the future is low. On the other hand, if it is a genotype having the Ang-1 gene having 3 bases (hereinafter also referred to as “3-base insertion type Ang-1 gene”) in homology, the possibility of developing MS or future onset It is possible to determine that there is a high possibility of doing. In the case of a genotype having a heterozygous 3 base insertion type Ang-1 gene and a 3 base deletion type Ang-1 gene, it is determined that the possibility of developing MS or the possibility of developing in the future is moderate. can do. In addition, the above “medium” means the possibility in the case of a genotype having a homology with the 3-base deletion Ang-1 gene and the possibility in the case of a genotype having a homology with the 3-base insertion Ang-1 gene. It means between and.

  Thus, according to this configuration, the diagnosis (determination of onset or possibility of onset) of the subject's MS can be performed accurately and simply.

[Method (III)]
In the above Ang-1 protein, the method for determining the presence or absence of insertion of glycine corresponding to the 269th amino acid of the protein consisting of the amino acid sequence shown in SEQ ID NO: 1 is not particularly limited, and a conventionally known method is used. be able to.

  For example, when using a protein prepared from a subject's cells, the amino acid sequence of the Ang-1 protein is determined according to a general protein sequencing method, and the presence or absence of the glycine is detected based on the amino acid sequence. be able to. Further, an antibody that recognizes only the Ang-1 protein having the glycine or the Ang-1 protein not having the glycine is prepared and detected using an immunochemical technique such as ELISA or Western blotting. be able to. In addition, protein is isolated, directly or if necessary, cleaved with an enzyme, etc., detected using a protein sequencer, detected using the isoelectric point of an amino acid as an indicator, and mass difference measured by mass spectrometry The method of detecting from can be used. Among these exemplified methods, since it is simple and highly accurate, the presence / absence of an antibody that recognizes only a specific Ang-1 protein, in particular, glycine corresponding to the 269th amino acid of the protein consisting of the amino acid sequence represented by SEQ ID NO: 1 It is preferable to prepare an antibody that detects the presence of glycine in the Ang-1 protein by ELISA.

  In the method for determining the onset or possibility of onset of MS according to the present invention, a person having a protein having the glycine is likely to develop MS depending on the presence or absence of the determined glycine, and conversely, the glycine is deleted. It can be determined that those who have proteins are unlikely to develop MS.

  Further, in the step of detecting the polymorphism of the Ang-1 protein, whether the subject has only the Ang-1 protein not having the glycine, only the Ang-1 protein having the glycine, or the glycine It is discriminate | determined whether it has both Ang-1 protein which has this, and Ang-1 protein which does not have the said glycine.

  The method for detecting the polymorphism of the Ang-1 protein is not particularly limited. For example, by using an antibody that recognizes only the Ang-1 protein having the glycine and an antibody that recognizes only the Ang-1 protein not having the glycine, the above-described many immunochemical techniques are used. The type can be detected. Moreover, the polymorphism of the protein can be detected by mass spectrometry.

  By including the step of detecting the polymorphism of the Ang-1 protein, it is possible to further improve the accuracy of determining the onset or possibility of onset of MS. Specifically, when only the glycine-deleted Ang-1 protein (hereinafter also referred to as “glycine-deleted Ang-1 protein”) is detected, the possibility of developing MS or It can be determined that the possibility of developing in the future is low. On the other hand, when only the glycine-inserted type Ang-1 protein (hereinafter, also referred to as “glycine-inserted Ang-1 protein”) is detected, MS may develop or may develop in the future. It can be determined that the property is high. In addition, when Ang-1 protein of both the glycine inserted type and the deleted type is detected, the possibility of developing MS or the possibility of developing in the future is moderate. Can be determined.

  Thus, according to the above configuration, MS diagnosis (determination of onset or possibility of onset thereof) can be performed accurately and simply.

  In addition, the preparation method of the said protein is not specifically limited, It can prepare by a conventionally well-known method from the test subject's all tissues, cells, etc. In particular, it is preferable to prepare from a tissue with a high content of Ang-1 protein.

  In any of the above methods (I) to (III), the subject is not particularly limited, but is a visceral fat-type obese person, impaired glucose tolerance, hypertriglyceridemia, or hypertension Those who suffer from any of the symptoms or those who develop MS are preferred.

  Furthermore, the sample to which the method for determining the onset of MS or the possibility of the onset according to the present invention is applied may be a sample separated from a living body such as a human body, and is not limited to those exemplified above. Absent.

  Further, the method for determining the onset or onset possibility of MS according to the present invention is not only a method for determining the onset or onset possibility of MS, but also by applying it to a patient who has developed MS. It can also be used as a method of determining (predicting). Specifically, when the genotype of the Ang-1 gene is a genotype having a homology with the 3-base deletion Ang-1 gene, it can be determined that the prognosis of MS is good. On the other hand, when the genotype has the 3-base insertion type Ang-1 gene in homology, it can be determined that the prognosis of MS is poor. In the case of a genotype having a 3 base insertion type Ang-1 gene and a 3 base deletion type Ang-1 gene in a heterogeneous form, the prognosis of MS is a genotype having a 3 base insertion type Ang-1 gene in a homology. And a case of a genotype having a 3 base deletion type Ang-1 gene homozygously (medium).

  On the other hand, in the method using protein (the above method (III)), when only glycine-deficient Ang-1 protein is detected, it can be determined that the prognosis of MS is good. On the other hand, when only glycine insertion type Ang-1 protein is detected, it can be determined that the prognosis of MS is poor. In addition, when both types of Ang-1 protein, the glycine inserted type and the deleted type, are detected, the prognosis of MS can be determined to be moderate.

<3. Evaluation kit for the onset of MS or its possibility>
The kit for determining the onset or likelihood of onset of MS according to the present invention (in other words, a diagnostic kit for MS) is the presence or absence of a specific base or amino acid in the above gene or protein, and more preferably the amount of the above gene or protein. It is only necessary to include at least a reagent capable of detecting the type, and other configurations are not particularly limited. Examples of such a reagent include a primer, a probe, and an antibody. These reagents may be contained alone or in a plurality of combinations. Furthermore, the determination kit can be obtained by combining other reagents in addition to the reagents exemplified above.

  Specifically, as a kit for detecting the presence or absence of a specific base in a gene using genomic DNA or mRNA (cDNA), preferably a polymorphism of the gene, a specific region of the gene, preferably a polymorphic region, is used. It includes primers designed to amplify and is used for sequencing, such as probes designed to detect specific bases, preferably polymorphisms, restriction enzymes, Maxam Gilbert and Sanger methods. And a kit in which one or more reagents necessary for detecting a specific base, preferably a polymorphism, are combined. Such a reagent is appropriately selected and employed depending on the detection method employed. For example, reagents used for labeling such as dATP, dUTP, dTTP, dGTP, DNA synthase, RNA synthase, and probes (for example, In the case where the label is biotin, an avidin enzyme conjugate, enzyme substrate and chromophore) and the like can be mentioned. Furthermore, an appropriate buffer solution and washing solution that do not hinder the detection of the polymorphism may be included.

  In the case of a kit including a primer, the primer is not particularly limited as long as it can amplify a specific region, preferably a polymorphic region, of a target gene (for example, Ang-1 gene in the present embodiment), For example, it can be selected from the primers shown in SEQ ID NOs: 3 to 7 above.

  In the case of a kit containing a probe, the probe is particularly limited as long as it can bind to a specific region, preferably a polymorphic region, of a target gene (for example, the Ang-1 gene in the present embodiment). is not.

  Furthermore, the kit for detecting the presence or absence of a specific amino acid in a protein, preferably a polymorphism of the protein includes, for example, an antibody that recognizes only a specific type of the protein according to the present invention, and the antibody A reagent or instrument used for detecting the presence or absence of a specific amino acid in the protein of the present invention, preferably a polymorphism of the protein, for example, a reagent used for labeling an antibody (for example, the label is biotin) In this case, avidin enzyme conjugate and enzyme substrate and chromophore), when the above antibody is used as a primary antibody, a secondary antibody corresponding thereto, a blocking reagent, a titer plate, a kit containing a buffer solution, a washing solution, etc. Can be mentioned. As the antibody, the antibody exemplified in the above item <2> can be used.

By using these determination kits, as described in the section <2> above, MS diagnosis (determination of onset or its onset possibility) can be performed accurately and easily. The subject to which the kit for determining the onset of MS or the possibility of its onset is not particularly limited, is any person with visceral fat type obesity, impaired glucose tolerance, hypertriglyceridemia, or hypertension Those who suffer from MS or those who develop MS are preferred.

  Furthermore, if the determination kit of the onset or onset possibility of MS according to the present invention is applied to a patient who has developed MS like the above-described determination method of onset or onset of MS, the prognosis of MS It can be used as a determination kit (in other words, a prognosis kit for MS).

<4. Method for Preventing Onset of MS and Preventive Drug>
As shown in the examples described later, the Ang-1 protein includes an Ang-1 protein that is likely to develop MS and an Ang-1 protein that is less likely to cause MS. Among them, the protein that is the translation product of the 3-base insertion type Ang-1 gene shown in the Examples described later, that is, the glycine insertion type Ang-1 protein is a protein that contributes to the onset of MS. On the other hand, a protein that is a translation product of a 3-base deletion Ang-1 gene, that is, a glycine deletion Ang-1 protein has an effect of suppressing the onset of MS. Therefore, the onset of MS can be prevented by utilizing the polymorphism of the protein according to the present invention. That is, the present invention provides (A) a method for preventing MS and (B) a preventive agent for MS.

(A) MS prophylaxis method The MS prophylaxis method according to the present invention is intended to prevent MS from occurring in a person who has a type of Ang-1 protein that easily develops MS, for example, a glycine insertion type Ang-1 protein. It is applied for the purpose of alleviating symptoms when delayed or onset. Specifically, for example, a glycine-deficient Ang-1 protein or DNA encoding the protein, or a low-molecular compound as an agonist of a receptor protein having the glycine-deficient Ang-1 protein as a ligand is complemented. A method is mentioned. Moreover, the method of administering the modulator of the gene concerning this invention can also be used.

  Examples of the gene modulators according to the present invention include, but are not limited to, compounds and substances that affect gene expression and the activity and / or amount of the expressed gene product. Common modulators suitable for use in the present invention include, for example, agonists and / or antagonists of any of the genes of the present invention or gene products thereof; macromolecules or small molecules for the expression or function of the genes of the present invention. An inhibitor; an antisense sequence comprising an antisense oligonucleotide to the gene or transcript according to the invention; an antibody; binding of the gene product according to the invention comprising a dominant negative form of the gene product involved in oligomerization. Proteins; and kinases of gene products according to the invention, or fragments, variants and derivatives thereof.

  Furthermore, as the modulator according to the present invention, an antisense oligonucleotide that binds to the mRNA of the gene according to the present invention and prevents or reduces its transcription can be used. The antisense oligonucleotide is mixed with, for example, mRNA of the gene according to the present invention produced by a cell to form a double chain. These duplexes can then block both further transcription of the mRNA and translation thereof. Therefore, the activity and / or amount of the protein according to the present invention can be reduced.

  The protein, DNA, low molecular weight compound, and modulator described above may be administered prophylactically, that is, before diagnosis of MS, or may be administered after diagnosis of MS. The administration route is not particularly limited, but general administration routes include oral, rectal, intravenous, parenteral, intramuscular and subcutaneous routes. In addition, when administered as either DNA or RNA, that is, when administered in the form of gene therapy, directly in the cell using conventionally known methods such as liposomes, viral vectors or coated particles (gene guns). Can be transported to.

(B) Prophylactic agent for MS The prophylactic agent for MS according to the present invention is a prophylactic agent for MS, such as a glycine-inserted Ang-1 protein that is susceptible to developing MS. It is a prophylactic agent used for the purpose of symptom relief when delayed or onset. For example, a glycine-deficient Ang-1 protein or a DNA encoding the glycine-deficient Ang-1 protein, or a low molecular compound as an agonist of a receptor protein having the glycine-deficient Ang-1 protein as a ligand And a drug containing the modulator of the gene according to the present invention exemplified in the above-mentioned “(A) MS prevention method”. The administration method of these drugs is not particularly limited, and the method exemplified in the above “(A) MS prevention method” can be used.

  In addition, examples of the receptor protein in which the protein according to the present invention is a ligand include a Tie-2 receptor. The fact that Ang-1 protein is a ligand for the Tie-2 receptor is disclosed in Non-Patent Document 2, for example. Therefore, it is considered that administering a low molecular weight compound acting as an agonist of the Tie-2 receptor into the body by oral or intravenous injection is effective as a method for preventing MS. The low molecular compound referred to here includes proteins such as peptides.

  Moreover, the glycine-deficient Ang-1 protein or the DNA encoding the glycine-deficient Ang-1 protein and the low-molecular compound as an agonist of the receptor protein are only used alternatively as preventive drugs. It is also possible to use them in combination.

  In addition, the subject to whom the prevention method or preventive agent for the onset of MS according to the present invention is applied is not particularly limited, but a person diagnosed with MS, a person diagnosed with the possibility of MS Or a person who has been determined to be a genotype having a high possibility of developing MS by the determination method according to the present invention.

  The present invention is not limited to the configurations exemplified above, and various modifications are possible within the scope shown in the claims, and the technical means disclosed in different embodiments are appropriately combined. The obtained embodiment is also included in the technical scope of the present invention.

  Although this invention is demonstrated more concretely based on an Example and FIGS. 1-8, this invention is not limited to this. Those skilled in the art can make various changes, modifications, and alterations without departing from the scope of the present invention. In addition, the genotype of the Ang-1 gene in the following examples was evaluated as follows.

[Method for evaluating genotype of Ang-1 gene]
(1) Sequence analysis EDTA blood was collected from the peripheral blood of the subject by a known method. Total RNA was isolated from the collected peripheral blood using RNA isolation kit (GENTRA SYSTEMS, MN). A reverse transcription reaction with an Oligo dT primer was performed from the isolated total RNA by a known method to synthesize cDNA. And the amplification fragment | piece was obtained by RT-PCR method using the primer set from the exon 4 to 5 of Ang-1 gene. In addition, the primer used for said reaction is as showing below.
Sense primer F2-2: 5′-CCACCAACAACAGTGTCCTT-3 ′ (SEQ ID NO: 3)
Sense primer F2-4: 5′-CAACCTTGTCAATCTTTGC-3 ′ (SEQ ID NO: 4)
Antisense primer R1186: 5′-CAGCTTGATATACATCTGCACAG-3 ′ (SEQ ID NO: 7)
Moreover, the setting positions of the sense primers F2-2 and F2-4 and the antisense primer R1186 are as shown in FIG.

  Furthermore, the amplification conditions in the RT-PCR are as follows. First, fragment amplification by RT-PCR was performed using the synthetic cDNA as a template and the sense primer F2-2 and the antisense primer R1186 as primers.

The reaction solution in the RT-PCR is cDNA, 1 × PCR-Buffer II (Applied Biosystems), 1.5 mM MgCl ₂ , 0.2 mM dATP, dTTP, dGTP and dCTP, 200 nM each primer, 2.5 U Prepared to contain AmpliTaq Gold DNA-Polymerase (Applied Biosystems).

  Further, the RT-PCR reaction conditions were 95 ° C for 12 minutes for 1 cycle, 94 ° C for 30 seconds, 50 ° C for 30 seconds, and 72 ° C for 1 minute for 30 cycles.

  After performing RT-PCR under the above conditions, 1 μl of the obtained PCR product was used as a template, and PCR was performed using the sense primer F2-4 and antisense primer R1186 as primers and the above reaction mixture composition and reaction conditions. The amplified fragment was obtained.

  As a result, the obtained amplified fragment was purified by ethanol precipitation. Next, a sample for sequence analysis was prepared from the purified amplified fragment by the dye terminator method using the sense primer F2-4 and BigDye Terminator (Applied Biosystems, CA). Thereafter, the samples were subjected to sequence analysis using a fluorescent DNA sequencer (ABI377, Applied Biosystems). It was decided to evaluate the genotype of the Ang-1 gene by comparing the sequence of the amplified fragment determined by sequence analysis with the base sequence shown in SEQ ID NO: 1 (see the right panel in FIG. 2). When duplication of the sequence pherogram (waveform signal) is recognized from the base number 805, it was decided that the 3 base insertion type Ang-1 gene and the 3 base deletion type Ang-1 gene exist in heterogeneity (FIG. 2). (See bottom right panel).

  In FIG. 2, “− / −”, “+ / +”, and “+/−” respectively represent a three-base insertion type Ang when the three-base deletion type Ang-1 gene is detected homozygously. Means that the -1 gene is detected in a homozygous manner and that the 3-base deleted Ang-1 gene and the 3-base inserted Ang-1 gene are detected in a heterogeneous manner.

(2) Chain length analysis An amplified fragment was obtained in the same manner as in the case of the sequence analysis described above. However, in the chain length analysis, the sense primer F2-4 used was labeled with TET fluorescence. Chain length analysis was performed using a solution containing the amplified fragment obtained by diluting the final PCR reaction product 10-fold. For the chain length analysis, 1 μl of the above solution containing the amplified fragment is used as a sample, and 3 μl of a formamide solution containing 0.3 μl of 5 mg / ml Blue Dextran, 2.5 mM EDTA and molecular weight marker GS500 TAMRA (Applied Biosystems) is used as a sample. This was performed using a sequencer.

  In the chain length analysis, the genotype of the Ang-1 gene was determined based on whether a 101-base or 98-base peak appeared (see the left panel in FIG. 2). In addition, when peaks of both 101 bases and 98 bases appeared, it was determined to be heterogeneous (see the lower panel on the left side of FIG. 2).

[Example 1]
The above sequence analysis and chain length analysis were performed using peripheral blood collected from MS patients by the above method. FIG. 3 is a diagram showing the results of polymorphism analysis of the Ang-1 gene by sequence analysis. As shown in FIG. 3, it was found that there are two polymorphisms in the Ang-1 gene: a 3-base insertion type and a 3-base deletion type. The figure (left) shows the analysis result of the 3-base insertion type, and the figure (right) shows the analysis result of the 3-base deletion type. In other words, it can be confirmed that the 3-base insertion of “GGT” occurs in the 3-base insertion type (see FIG. 3 (left)), and in the 3-base deletion type, these 3 bases are deleted. Was confirmed (see FIG. 3 (right)).

  Of these, SEQ ID NO: 2 shows a 3-base insertion type gene sequence. In this three-base insertion type, as shown in SEQ ID NO: 2, three bases “GGT” are inserted as the 805th to 807th bases in the sequence.

  In addition, as shown in SEQ ID NO: 1, the protein that is the translation product of the three-base insertion gene has glycine inserted as the 269th amino acid in the amino acid sequence. On the other hand, the 269th glycine is missing in the protein that is the translation product of the three-base deletion gene.

[Example 2]
Peripheral blood collected from patients with rheumatoid arthritis (hereinafter also referred to as “RA”), diabetic retinopathy (hereinafter also referred to as “DR”), mixed connective tissue disease (hereinafter also referred to as “MCTD”), and healthy subjects FIG. 4 shows the results of examining the genotype of the Ang-1 gene by using the above method.

  In FIG. 4, n represents the total number of various subjects, and a group of patients having homozygous 3 base deletion type Ang-1 gene (shown in the figure and hereinafter referred to as “− / −”), 3 base deletions Group of patients having heterozygous type-1 Ang-1 gene and 3-base insertion type Ang-1 gene (in the figure and hereinafter referred to as "+/-"), and group of patients having 3-base-insertion type Ang-1 gene homozygously The numbers (shown as “+ / +” in the figure and hereinafter) are shown in the respective columns. Furthermore, in parentheses, the ratio of each of the above-mentioned subjects among various subjects is shown.

As shown in FIG. 4, in healthy individuals, “− / −” was 11.9%, “+/−” was 78.0%, and “+ / +” was 10.1%. In RA patients, “− / −” was 5.1%, “+/−” was 79.0%, and “+ / +” was 24.5%. In DR patients, “− / −” was 25.8%, “+/−” was 36.5%, and “+ / +” was 37.9%. Furthermore, in MCTD patients, “− / −” was 10.5%, “+/−” was 34.2%, and “+ / +” was 55.3%. Furthermore, about the person who has 3 base insertion type Ang-1 gene by homo, the significant difference between a healthy subject group, RA patient group, and DR patient group was analyzed by the chi-square test. As a result, the P value between the healthy group and the RA patient group is 10 ⁻² , the P value between the healthy group and the DR patient group is 10 ⁻⁴ , and between the healthy group and the MCTD patient group. The P value was 10 ⁻⁶ .

Example 3
DR patients were examined for age, hemoglobin A _1c (HbA1c), fasting blood glucose (FBS), and duration of disease. HbA1c and FBS were measured according to a conventional method using a fasting blood sample. As a result, for each measurement item, there was no difference due to the genotype of the Ang-1 gene.

  In FIG. 5, a group of patients having homozygous 3 base deletion type Ang-1 gene (in the figure and hereinafter referred to as “− / −”), 3 base deletion type Ang-1 gene and 3 base insertion type A group of patients having a heterozygous Ang-1 gene (in the figure and hereinafter referred to as “+/−”) and a group of patients having a homologous 3-base insertion type Ang-1 gene (in the figure and hereinafter, “+ / +”) The measurement results of the above various measurement items are indicated in the respective columns.

Example 4
The DR patients were further classified into simple type DR, preproliferative type DR, and proliferative type DR according to the degree of progression, and the genotype of the Ang-1 gene was examined in patients at each advanced stage. Moreover, the genotype of the Ang-1 gene was also examined for patients who were diagnosed as having diabetes and who did not develop DR as a complication. The result is shown in FIG. In FIG. 6, SDR represents a simple DR patient, PPDR represents a preproliferative DR patient, PDR represents a proliferative DR patient, and DR (−) represents a diagnosis of diabetes. Represents a patient who has received DR and has not developed DR as a complication.

Example 5
DR patients were examined for the presence of total cholesterol (T-Cho), HDL cholesterol (HDL-C), triglycerides (TG), and hypertension. In addition, total cholesterol (T-Cho), HDL cholesterol (HDL-Cho), and triglyceride (TG) were measured according to a conventional method using a fasting blood sample. The result is shown in FIG. In FIG. 7, a group of patients having a homology with the 3-base deletion type Ang-1 gene (in the figure and hereinafter referred to as “− / −”), a 3-base deletion type Ang-1 gene and a 3-base insertion type A group of patients having a heterozygous Ang-1 gene (in the figure and hereinafter referred to as “+/−”) and a group of patients having a homologous 3-base insertion type Ang-1 gene (in the figure and hereinafter, “+ / +”) The measurement results of the above various measurement items are indicated in the respective columns. As shown in FIG. 7, the HDL-Cho value is 67.9 ± 3.9 mg / dl in the “− / −” patient group and 55.1 ± 6.5 mg in the “+/−” patient group. / Dl, for patients with “+ / +”, it was 56.3 ± 12.7 mg / dl. Further, regarding the HDL-Cho value, a significant difference between the “− / −” patient group, the “+/−” patient group, and the “+ / +” patient group was analyzed by chi-square test. As a result, the P value between the “− / −” patient group and the “+/−” patient group was 0.039, and the “− / −” patient group and the “+ / +” patient group were The P value between them was 0.024. From this, it was found that the “− / −” patient group had a significantly higher HDL-Cho value than the “+/−” patient group and the “+ / +” patient group.

  The TG value is 148.6 ± 43.1 mg / dl in the patient group of “− / −”, 182.6 ± 87.4 mg / dl in the patient group of “+/−”, and “+ / +”. ”Was 193.6 ± 68.2 mg / dl. Moreover, regarding the TG value, a significant difference between the patient group of “− / −” and the patient group of “+ / +” was analyzed by chi-square test. As a result, the P value between the “− / −” patient group and the “+ / +” patient group was 0.047. From this, it was found that the “− / −” patient group had a significantly lower TG value than the “+ / +” patient group.

  From the above results, it has been clarified that having a 3 base deletion type Ang-1 gene ameliorates abnormal human lipid metabolism observed in MS and the like.

Example 6
Among the diagnostic criteria for MS based on US hyperlipidemia guideline, Ang-1 gene genotype for those who correspond to 2 or more of 3 items of hyperTGemia, HDL cholesterolemia and hypertension (See FIG. 8). The results are shown in the table in FIG. As shown in the table of FIG. 8, among the above three items, those who correspond to two or more items are 15.4% for “− / −”, 26.1% for “+/−”, In "+ / +", it was 40.0%. From this, it was clarified that those who have the 3 base deletion type Ang-1 gene are less likely to have MS than those who do not have the 3 base deletion type Ang-1 gene.

  The present invention is not limited to the configurations described above, and various modifications are possible within the scope of the claims, and technical means disclosed in different embodiments and examples, respectively. Embodiments and examples obtained by appropriately combining the above are also included in the technical scope of the present invention. Moreover, all the academic literatures and patent literatures described in this specification are incorporated herein by reference.

  As described above, in the present invention, the possibility of the onset of MS can be predicted by utilizing the Ang-1 protein or the polymorphism of the Ang-1 gene. Therefore, it can be widely used not only in the field of diagnostic medicine typified by a method for judging the onset or possibility of onset of MS, but also in the field of health medicine. Furthermore, by using an Ang-1 protein or an Ang-1 gene that is unlikely to develop MS, it can be used as a method for preventing the onset of MS or a preventive drug. Furthermore, the gene and protein according to the present invention can be used for alleviation of symptoms of MS after onset of MS, that is, treatment.

It is the schematic which showed typically a part of cDNA of Ang-1 gene concerning this invention. In a present Example, it is a figure which shows the polymorphism analysis (sequence analysis and chain length analysis) of Ang-1 gene. In a present Example, it is a figure which shows the result of the polymorphism analysis of the Ang-1 gene by sequence analysis. In a present Example, it is a table | surface which shows the result of having investigated the genotype of the Ang-1 gene in RA patient, DR patient, MCTD patient, and a healthy subject. In a present Example, it is a table | surface which shows the result of having investigated age, HbA1c, FBS, and the illness period about DR patient. In a present Example, it is a table | surface which shows the result of having investigated the relationship between the progress degree of DR, and the genotype of Ang-1 gene. In a present Example, it is a table | surface which shows the result of having investigated the presence or absence of T-Cho, HDL-Cho, TG, and hypertension about DR patient. In a present Example, it is a figure which shows the diagnostic criteria of MS based on the US hyperlipidemia guideline, and the result of having investigated the genotype of Ang-1 gene about the patient diagnosed with MS in the said diagnostic criteria.

Claims (4)

  Using a sample isolated from a living body, a gene encoding angiopoietin-1 protein having the amino acid sequence shown in SEQ ID NO: 1 encodes glycine corresponding to the 269th amino acid of the angiopoietin-1 protein. A data acquisition method for determining the possibility of developing metabolic syndrome, comprising a step of determining whether or not three bases are inserted.   Using a sample isolated from a living body, “GGT” (G and G) corresponding to the 805th to 807th bases in the above base sequence of the angiopoietin-1 gene having the base sequence shown in SEQ ID NO: 2 as an open reading frame region A method for obtaining data for determining the likelihood of developing metabolic syndrome, comprising the step of determining whether or not three bases are inserted (T represents guanine and thymine, respectively). An angiopoietin-1 protein comprising the step of detecting a polymorphism depending on the presence or absence of insertion of glycine corresponding to the 269th amino acid of the protein consisting of the amino acid sequence shown in SEQ ID NO: 1 is capable of developing metabolic syndrome Data acquisition method for sex determination. (A) a primer for amplifying a region containing a base corresponding to positions 805 to 807 in the above-mentioned base sequence of the gene having the base sequence represented by SEQ ID NO: 2 as an open reading frame region;
(B) a probe that binds to a region containing bases corresponding to positions 805 to 807 in the above-described base sequence of the gene having the base sequence represented by SEQ ID NO: 2 as an open reading frame region; and
(C) at least one of an antibody that recognizes only one of the protein consisting of the amino acid sequence shown in SEQ ID NO: 1 or the protein lacking glycine corresponding to the 269th amino acid of the protein, And,
Data collected by the data acquisition kit for obtained for the determination of the likelihood of developing metabolic syndrome according to any one of claims 1 to 3.

Images