JP2007282553A - Gene susceptible to arteriosclerosis and use thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for identifying a susceptibility gene of arteriosclerosis or related diseases thereof and judging the crisis risk of a disease by using the gene and to obtain a new administration protocol of prophylactic/therapeutic agent of a disease to a patient judged to have a high risk by the method. <P>SOLUTION: The method for judging genetic susceptibility comprises assaying a subject for the genotype of SNP determining 186th amino acid of HRG protein, in the case when the subject is a homozygote of allele in which an amino acid is Pro, judging that the subject has a high genetic susceptibility to arteriosclerosis or related diseases thereof. The prophylactic/therapeutic agent of arteriosclerosis or related diseases thereof is administered to a patient judged to be a homozygote of allele in which an amino acid is Pro so that a total cholesterol in blood and/or LDL-cholesterol value becomes a target value in a category in risk higher by at least one stage than a category which a patient in a fixed guide line belongs. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a susceptibility gene for arteriosclerosis and related diseases, a method for determining genetic susceptibility to arteriosclerosis and related diseases by detecting a polymorphism of the gene, or a drug efficacy evaluation marker for a prophylactic / therapeutic agent for the disease And a method for diagnosing arteriosclerosis or a related disease using the expression variation of the protein as an index, or a method for evaluating the effect of preventing or treating the disease of a compound.

  Arteriosclerosis is a general term for a condition in which the arterial wall is thickened and loses its elasticity. Acute coronary syndromes (ACS) such as unstable angina (UA) and acute myocardial infarction (AMI), cerebral infarction and cerebral hemorrhage, etc. It is the underlying pathology of serious life-threatening diseases such as cerebrovascular disorder (CVA) and renal failure. Atherosclerosis progresses asymptomatically from the age of teenagers, and clinical symptoms often appear after blood flow has deteriorated and organ damage has started. Therefore, how to predict the onset of an event due to arteriosclerotic disease at an early stage and efficiently prevent it is an extremely important issue.

Atherosclerosis develops due to the accumulation of risk factors such as diabetes, hypertension, hyperlipidemia, smoking, and infection in the genetic background. Common diseases (such as common diseases; CDs), including lifestyle-related diseases such as diabetes, have multiple penetrances (penetrance; the rate at which individuals with mutations in certain genes develop certain diseases) It is considered to be a multifactorial disease that develops through the interaction of disease-related gene mutations and multiple environmental factors such as exercise and nutrition. Hypothesis that disease-related gene mutations in CD are common variants (CVs) and are present in healthy subjects but have a significantly higher prevalence in patients (Common Disease-Common Variant (CD-CV) hypothesis) has been proposed. As a famous example, it is known that a single nucleotide polymorphism (SNP) in the apolipoprotein E gene ε4 correlates with the onset of Alzheimer's disease.
Since fiscal 2000, as part of the Millennium Genome Project, Japan has identified approximately 200,000 SNPs in the gene region of the human genome (they are available as a JSNP database at http://snp.ims.u-tokyo.ac Research is going on to identify genes that define the sensitivity (susceptibility) to CD by analyzing the correlation of these SNPs with typical CDs. A susceptibility gene has also been discovered for myocardial infarction, which is an arteriosclerotic disease (Non-patent Document 1).

Histidine-Rich Glycoprotein (hereinafter abbreviated as “HRG”), which is a glycoprotein that exists in large amounts in blood, interacts with plasmin, suggesting its involvement in the blood coagulation system (for example, patent literature) 2), it has been reported that blood HRG levels are elevated in thrombotic families (Patent Documents 3 to 5). There are two variants of HRG protein, about 75 kDa and about 77 kDa, due to differences in glycosylation, and this difference is related to the polymorphism of the HRG gene (ie, SNP giving Pro / Ser amino acid substitution at the 186th amino acid). This is known to be caused (Non-Patent Document 6). In addition, there are reports that plasma HRG levels are high in humans carrying the Ser ¹⁸⁶ allele (Non-patent Document 7). However, the results of large-scale clinical trials showed that this polymorphism did not correlate with either myocardial infarction (Non-patent document 8) or deep vein thrombosis (Non-patent document 9).
Ozaki et al., "Nature. Genet.", (USA), 32, pp. 650-654, 2002 Jones AL et al., "Immunol. Cell Biol.", (Australia), 83, pp. 106-118, 2005 Engsser L. et al., "British Journal of Hematology (Br. J. Haematol.)", (UK), 67, pp. 355-358, 1987 Falkon L. et al., “Thromb. Res.” (USA), 66, 265-270, 1992 Castaman G. et al., “Thromb. Res.” (USA), 69, 297-305, 1993 Hennis BC et al., "Thromb. Heamost.", (Germany), 74, pp. 1491-1496, 1995 Hennis BC et al., "Thromb. Heamost.", (Germany), 74, pp. 1497-1500, 1995 Herrmann SM et al., "Thromb. Heamost.", (Germany), Vol. 79, pp. 359-361, 1998 Rattink AP et al., "Fibrinolysis and Proteolysis", Vol. 13, pp. 35-38, 1999

  An object of the present invention is to identify a gene that affects genetic susceptibility to arteriosclerosis and related diseases, and to use it to easily determine the risk of developing the disease, and a prophylactic / therapeutic agent for the disease It is to provide a method for selecting a high-risk group in drug efficacy evaluation and administration plan determination. Another object of the present invention is to provide a prophylactic / therapeutic agent for arteriosclerosis and related diseases, which is administered at a dose determined based on the genotype of the gene.

The present inventors searched for blood markers of arteriosclerosis by two-dimensional gel electrophoresis using WHHL (Watanabe Heritable Hyperlipidemic) rabbit plasma samples known to spontaneously develop hypercholesterolemia and arteriosclerosis. As a result, HRG was identified as a protein whose expression increases as the lesion progresses. Next, as a result of confirming the fluctuation of blood HRG level with the progress of arteriosclerosis by Western blot using human plasma, the present inventors surprisingly did not significantly change the expression level. It was found that the band pattern was different from the normal control. As a result of further investigation, many healthy individuals give two bands at about 75 kDa and about 77 kDa, whereas many patients with arteriosclerotic diseases such as myocardial infarction and unstable angina are about 75 kDa. It became clear to give one band at the position. Glycosidase treatment resulted in two bands, giving a band at the same position as the above-mentioned one band treated in the same manner, suggesting that the two bands were due to differences in sugar chain modification. The molecular weight suggested that these two bands were due to the previously reported HRG gene polymorphism (ie, Pro / Ser difference at the 186th amino acid), so genotyping of the gene polymorphism was performed. The two bands matched the Pro ¹⁸⁶ / Ser genotype, and the one band matched the Pro ¹⁸⁶ / Pro (or Ser ¹⁸⁶ / Ser) genotype. Based on these findings, the present inventors have concluded that the HRG gene is a susceptibility gene for arteriosclerosis and related diseases, and as a result of further research, the present invention has been completed.

That is, the present invention
[1] A method for determining a genetic susceptibility of a subject to arteriosclerosis or a related disease, wherein the subject is tested for a SNP genotype that determines the 186th amino acid of the HRG protein. ,
[2] The above [1], wherein when the subject is a homozygote of the allele whose amino acid at 186 is Pro, the subject is determined to have high genetic susceptibility to arteriosclerosis or a related disease. ] Method described,
[3] A method for selecting subjects or patients in a high-risk group in the evaluation of the efficacy of a prophylactic / therapeutic agent for arteriosclerosis or related diseases or in determining the administration plan, and for each subject or patient, the 186th of HRG protein A method comprising testing a genotype of an SNP that determines an amino acid, and selecting a subject or patient who is a homozygote of an allele whose 186th amino acid is Pro as a high-risk group,
[4] The method according to [1] or [3] above, wherein the genotype assay is performed at the nucleic acid level,
[5] The method according to [1] or [3] above, wherein the genotype test is performed at the protein level,
[6] The method according to [5] above, wherein the presence of an HRG protein having an apparent molecular weight of about 75 kDa and an HRG protein of about 77 kDa by SDS-PAGE is assayed.
[7] When only an HRG protein with an apparent molecular weight of about 75 kDa is detected by SDS-PAGE, the subject is judged to be a homozygote of an allele in which the 186th amino acid of the HRG protein is Pro. The method according to [6] above, characterized in that
[8] A kit for determining genetic susceptibility to arteriosclerosis or a related disease, comprising a nucleic acid probe and / or primer pair capable of detecting SNP that determines the 186th amino acid of HRG protein,
[9] The nucleic acid probe is a nucleic acid containing a continuous base sequence of about 15 to about 500 bases including the base of the SNP site that determines the 186th amino acid of the HRG protein, and the nucleic acid primer pair is HRG A partial base sequence of a gene or HRG mRNA, which is a pair of nucleic acids capable of amplifying a continuous base sequence of about 50 to about 1,000 bases including the base of the SNP site that determines the 186th amino acid of the HRG protein, [8] The kit according to
[10] A first reagent capable of detecting an HRG protein having an apparent molecular weight of about 75 kDa by SDS-PAGE and a second reagent capable of detecting an HRG protein having an apparent molecular weight of about 77 kDa by SDS-PAGE A kit for determining genetic susceptibility to arteriosclerosis or related disease and / or selecting a high-risk group of arteriosclerosis or related disease,
[11] The kit according to [10] above, wherein the first reagent and / or the second reagent is an antibody against HRG protein,
[12] The kit according to the above [11], wherein the first and second reagents are the same antibody,
[13] The first reagent is an antibody capable of recognizing only an HRG protein having an apparent molecular weight of about 75 kD by SDS-PAGE, and / or the second reagent is an HRG having an apparent molecular weight of about 77 kDa by SDS-PAGE. The kit of the above-mentioned [11], which is an antibody capable of recognizing only a protein, and [14] an arteriosclerosis or a related disease comprising a substance having a blood total cholesterol and / or LDL-cholesterol lowering action A patient who is a prophylactic / therapeutic agent and is a homozygote of an allele whose HRG protein has the 186th amino acid as a Pro, the blood total cholesterol and / or LDL-cholesterol level belongs to the patient according to the prescribed guidelines. An agent or the like is provided that is administered so as to achieve a target value in a category that is at least one stage higher risk than the category.

Humans with the HRG genotype Pro ¹⁸⁶ / Pro have a higher risk of developing arteriosclerosis and related diseases than humans with the Pro ¹⁸⁶ / Ser genotype. The disease can be effectively prevented. In addition, a high-risk group can be selected from subjects to be administered in the evaluation of the efficacy of a prophylactic / therapeutic agent for the disease and the determination of the administration plan. Furthermore, a novel guideline for the prevention and treatment of arteriosclerotic diseases, in which the genotype is added as an arteriosclerosis risk factor, is provided.

The present invention provides a method of determining a subject's genetic susceptibility to arteriosclerosis or related diseases. The method is characterized by testing the genotype of the subject's HRG gene, specifically, the SNP genotype that determines the 186th amino acid of the HRG protein.
As long as the “subject” as a target of the determination method of the present invention is a human, there are no particular limitations on age, sex, race, past history, and the like. For example, (i) 30 years of age or older, preferably 40 years of age or older, and (ii) serum lipid levels by fasting blood sampling within a certain period (eg, within 30 days, within 60 days, preferably within 90 days) More than the diagnostic criteria of blood serum (for example, serum total cholesterol level is 220 mg / dl or higher, serum LDL-cholesterol level is 140 mg / dl or higher, serum triglyceride (TG) level is 150 mg / dl or higher, preferably 130 mg (iii) diseases and / or conditions that may cause hyperlipidemia (eg obesity, hypothyroidism, nephrotic syndrome, The present invention can be preferably applied to a human who satisfies one or more conditions selected from (iv) a history of myocardial infarction and / or a family history, etc., but is not limited thereto.
Here, the “related disease” of arteriosclerosis is used in the meaning including various diseases caused accompanying the progress of arteriosclerotic lesions and diseases resulting in the development of arteriosclerotic lesions. For example, myocardial infarction, angina pectoris, cerebral infarction, cerebral hemorrhage, cerebral thrombus, cerebral embolism, aortic aneurysm, aortic dissection, nephrosclerosis, renal failure, obstructive arteriosclerosis, post-PCI restenosis, acute coronary syndrome, coronary artery disease, Examples of the disease include peripheral arterial occlusion, but are not limited thereto. In the present specification, arteriosclerosis and related diseases may be collectively referred to as “arteriosclerotic disease”. In addition, here, “genetic susceptibility” means having a characteristic of being susceptible to a certain disease (in the present invention, arteriosclerotic disease) as a genetic background.

  The SNP that determines the 186th amino acid of the HRG protein to be tested by the determination method of the present invention is specifically an SNP that determines whether the 186th amino acid is Pro or Ser. The SNP reported in is a single base substitution in which the first base of the codon (YCC) encoding amino acid 186 is C (encodes Pro) or T (encodes Ser) (therefore, The allele encoding the HRG protein whose 186th amino acid is Pro is called C allele, and the allele encoding the HRG protein whose 186th amino acid is Ser is called T allele).

  In the determination method of the present invention, the genotype of the SNP that determines the 186th amino acid of the HRG protein of the subject is tested. Possible genotypes are C allele homozygotes (CC), heterozygotes (CT) and T allele homozygotes (TT). Such genotyping can be performed using a method known per se (a) at the gene level or (b) at the protein level. Hereinafter, each case will be described in detail.

(a) HRG genotyping at the gene level Any known SNP detection method can be used. As a classic detection method, for example, a biological sample derived from a subject (not particularly limited as long as it contains genomic DNA, but preferably capable of being collected minimally invasively, for example, blood, semen, mucous membrane, skin, hair, etc. Or a partial base sequence of the base sequence represented by GenBank accession number NC_000003.10 (SEQ ID NO: 1), using genomic DNA extracted from the sample as a sample About 15 to about 500 consecutive bases including the base of the SNP site that determines the 186th amino acid of the protein (that is, base Y indicated by base number 6830 (in NC_000003.10, the base is C)) Two kinds of nucleic acids containing a sequence (ie, a nucleic acid completely complementary to the C allele and a nucleic acid completely complementary to the T allele) are used as probes, for example, Wallace et al. (Proc. Natl. Acad. Sci. USA) , 80, 278-282 (1983)) A method of performing a hybridization reaction under conditions in which only the nucleic acid having a sequence completely complementary to the probe can be hybridized with precise control of the stringency, or one of the above nucleic acids is labeled and the other is not Hybridization is performed using the labeled mixed probe while gradually lowering the reaction temperature from the denaturation temperature, and a sequence completely complementary to one probe is first hybridized to prevent cross-reaction with a probe having a mismatch. The method etc. are mentioned. Here, as the labeling agent, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, or the like is used. As the radioisotope, for example, [ ¹²⁵ I], [ ¹³¹ I], [ ³ H], [ ¹⁴ C] and the like are used. As the enzyme, a stable enzyme having a large specific activity is preferable. For example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate and the like are used. As the luminescent substance, for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used.

  Preferably, the detection of the polymorphism is performed by various methods described in, for example, WO 03/023063, such as RFLP method, PCR-SSCP method, ASO hybridization, direct sequencing method, ARMS method, denaturing agent concentration gradient gel electrophoresis. Electrophoresis, RNaseA cleavage method, chemical cleavage method, DOL method, TaqMan PCR method, invader method, MALDI-TOF / MS method, TDI method, molecular beacon method, dynamic allele specific hybridization method, padlock probe method, It can be carried out by the UCAN method, the nucleic acid hybridization method using a DNA chip or DNA microarray, the ECA method, etc. (see WO 03/023063, page 17, line 5 to page 28, line 20). Hereinafter, the TaqMan PCR method, the invader method, and the RFLP method will be described in more detail as representative methods.

(1) TaqMan PCR method
The TaqMan PCR method uses a fluorescently labeled allele-specific oligonucleotide (TaqMan probe) and PCR using Taq DNA polymerase. The TaqMan probe is a partial base sequence of the base sequence represented by SEQ ID NO: 1, and a continuous base sequence of about 15 to about 30 bases including the SNP site base that determines the 186th amino acid of the HRG protein An oligonucleotide consisting of The probe is labeled with a fluorescent dye such as FAM or VIC at the 5 'end and labeled with a quencher (quenching substance) such as TAMRA at the 3' end, and the quencher absorbs the fluorescence energy as it is. Therefore, no fluorescence is detected. Probes are preferably prepared for both alleles and labeled with fluorescent dyes having different fluorescence wavelengths (for example, one allele is FAM and the other is VIC) for batch detection. In addition, the 3 ′ end is phosphorylated so that a PCR extension reaction from the TaqMan probe does not occur. When PCR is performed with a primer designed to amplify a partial sequence of genomic DNA containing a region that hybridizes with the TaqMan probe and Taq DNA polymerase, the TaqMan probe hybridizes with the template DNA and simultaneously extends from the PCR primer. However, when the extension reaction proceeds, the TaqMan probe hybridized by the 5 ′ nuclease activity of Taq DNA polymerase is cleaved, the fluorescent dye is released, and it is not affected by the quencher, and fluorescence is detected. The fluorescence intensity increases exponentially by amplification of the template.
For example, in the detection of a polymorphism at the base represented by base number 6830 in the base sequence represented by SEQ ID NO: 1, an allele-specific oligonucleotide containing about the base (about 15 to about 30 bases in length; C allele is FAM And T alleles are labeled 5 ′ with VIC and 3 ′ ends are labeled with TAMRA), and if the subject's genotype is CC or TT, FAM or VIC, respectively. Strong fluorescence intensity is recognized, and the other fluorescence is hardly recognized. On the other hand, if the subject's genotype is CT, both FAM and VIC fluorescence are detected.

(2) Invader method Unlike the TaqMan PCR method, the invader method does not label the allele-specific oligonucleotide (allele probe) itself, and a sequence (flap) that is not complementary to the template DNA on the 5 'side of the base of the polymorphic site. And a complementary sequence specific to the template on the 3 ′ side. In the invader method, an oligonucleotide having a specific complementary sequence on the 3 ′ side of the polymorphic site of the template (invader probe; the base corresponding to the polymorphic site at the 5 ′ end of the probe is arbitrary), The 5 'side has a sequence that can take a hairpin structure, and when the hairpin structure is formed, the sequence that is continuous 3' from the base paired with the 5 'end base is a sequence complementary to the allele probe flap. FRET (fluorescence resonance energy transfer) probes characterized by this are used. The 5 ′ end of the FRET probe is fluorescently labeled (for example, FAM, VIC, etc.), and a quencher (for example, TAMRA, etc.) is bound in the vicinity thereof, and fluorescence is not detected as it is (hairpin structure).
When the allele probe and the invader probe are reacted with the genomic DNA as a template, the 3 ′ end of the invader probe enters the polymorphic site when the three members complementarily bind. Using the enzyme that recognizes the structure of this polymorphic site (cleavase), the single-stranded part of the allele probe (ie, the 5'-side flap from the base of the polymorphic site) is excised, and the flap is complementary to the FRET probe. And the polymorphic site of the flap enters the hairpin structure of the FRET probe. When this structure is recognized and cleaved by cleavase, the end-labeled fluorescent dye of the FRET probe is released, and the fluorescence is detected without being affected by the quencher. An allele probe whose base at the polymorphic site does not match the template is not cleaved by cleavase, but an allele probe that is not cleaved can also hybridize with the FRET probe, and thus fluorescence is detected in the same manner. However, since the reaction efficiencies are different, the allele probe that matches the base of the polymorphic site has significantly higher fluorescence intensity than the allele probe that does not match.
Usually, before reacting with the three types of probes and cleavase, the template DNA is preferably amplified by PCR using a primer that can amplify a region containing a portion to which the allele probe and invader probe hybridize.

(3) RFLP method
In the HRG gene, the SNP that determines the 186th amino acid of the HRG protein causes a difference in the length of the restriction enzyme fragment. By using this, the polymorphism can be easily detected. That is, the T allele is cleaved by Hpy188III (recognizes TCNNGA and cleaves between C and N), but the C allele is not cleaved. Thus, for example, in the base sequence represented by SEQ ID NO: 1, a 160 bp fragment represented by base numbers 6781 to 6940 is amplified by PCR and digested with Hpy188III, the C allele becomes a single 160 bp fragment. In contrast, the T allele yields two fragments of 51 bp and 109 bp. Therefore, when the digested product is electrophoresed, one band is detected at the 160 bp position when the genotype is CC, and three bands are detected at the 51 bp, 109 bp and 160 bp positions when the genotype is CT. When the genotype is TT, two bands are detected at 51 bp and 109 bp. In this way, the genotype of the HRG gene in the subject can be determined.

(b) HRG genotyping at the protein level
The Pro / Ser polymorphism at amino acid 186 results in a molecular weight difference of about 2 kDa in the gene product. That is, the HRG protein whose amino acid at 186 is Pro has an apparent molecular weight of about 75 kDa in SDS-PAGE, whereas the HRG protein whose amino acid at 186 is Ser has an apparent molecular weight in SDS-PAGE. It is about 77 kDa. When these two variants are subjected to glycosidase treatment and electrophoresed, a band is formed at the same position. Therefore, this difference in molecular weight is considered to be based on a difference in sugar chain modification (HRG represented by SEQ ID NO: 2). Looking at the 186 th amino acid sequence of about amino acid of the amino acid sequence, variants of Ser ¹⁸⁶ is because it has a N- glycosylation consensus sequence Asn-Xaa-Ser / Thr, e.g., sugar chain added to Asn ¹⁸⁴ It is suggested).
As described above, the SNP that determines the 186th amino acid of the HRG protein produces a difference in molecular weight based on the difference in glycosylation in the gene product, so this can be used to detect the SNP at the protein level. Can do. For example, a biological sample collected from a subject (although not particularly limited, a sample that can be collected in a minimally invasive manner is preferable, for example, blood, serum, plasma, saliva, semen, mucous membranes, tears, urine, etc. Or more preferably blood, serum, plasma, more preferably serum or plasma) or a fraction thereof [the difference in molecular weight between the two HRG variants is about Since it is as small as 2 kDa, when separating and detecting both using the difference in molecular weight, remove a large amount of contaminating proteins (eg, albumin in the case of plasma or serum) in advance in the starting sample. It is desirable to extract the protein by a conventional method and detect the presence or absence of bands at positions of about 75 kDa and about 77 kDa by Western blotting using an antibody against the HRG protein. It is possible to detect the type. That is, when a single band is detected at a position of about 75 kDa, the genotype of the HRG gene in the subject is CC, and when two bands are detected at a position of about 75 kDa and about 77 kDa, The genotype of the HRG gene in the subject is CT, and when a single band is detected at a position of about 77 kDa, the genotype of the HRG gene in the subject is TT.

  As a method for removing the contaminating protein from the above-described biological sample, the HRG protein may be purified by subjecting the biological sample collected from the subject to affinity chromatography using an antibody against HRG, and may be subjected to Western blotting. Alternatively, the HRG protein purified as described above can be subjected to gel electrophoresis such as SDS-PAGE, and the presence or absence of bands at about 75 kDa and about 77 kDa can be detected by Coomassie brilliant blue staining or silver staining.

The antibody against HRG used in the above method is not particularly limited as long as it can recognize both the Pro ¹⁸⁶ variant with a molecular weight of about 75 kDa and the Ser ¹⁸⁶ variant with a molecular weight of about 77 kDa. Such an antibody can be prepared, for example, by isolating and purifying the HRG protein from human cells that express it, and immunizing an animal using the protein or a partial peptide thereof as an antigen. Alternatively, based on the known amino acid sequence of HRG (the amino acid sequence represented by SEQ ID NO: 2), the partial peptide can be synthesized by a known peptide synthesis technique and used as an antigen (hapten). . Further, cDNA encoding HRG according to a conventional method (in the nucleotide sequence represented by SEQ ID NO: 1, nucleotide numbers 24 to 206, 2927 to 3043, 3930 to 4020, 5615 to 5781, 6779 to 6859, 9081 to 9182 and The sequence obtained by linking the nucleotide sequences represented by 11039 to 11875 is equivalent to the coding region (CDS) of HRG), the cDNA is incorporated into an appropriate expression vector, introduced into an appropriate host cell, and the resulting transformation Recombinant protein is collected by culturing the body, and HRG is purified using a known protein separation and purification method, or the protein obtained using the cDNA obtained as described above as a template and using a cell-free transcription / translation system. Can be obtained and used as an antigen.

The antibody against HRG may be either a polyclonal antibody or a monoclonal antibody, and can be prepared by a well-known immunological technique. The antibody includes not only a complete antibody molecule but also a fragment thereof, and examples thereof include Fab, F (ab ′) ₂ , ScFv, and minibody.

[Production of monoclonal antibodies]
(A) Preparation of monoclonal antibody-producing cells
HRG is administered to a mammal at a site where antibody production is possible by administration, together with a carrier and a diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production ability upon administration. The administration is usually performed once every 2 to 6 weeks, 2 to 10 times in total. Examples of mammals to be used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep and goats, and mice and rats are preferably used.
When producing monoclonal antibody-producing cells, select a mammal with an antigen titer from an antigen-immunized mammal, such as a mouse, and collect the spleen or lymph nodes 2 to 5 days after the final immunization. Monoclonal antibody-producing hybridomas can be prepared by fusing the antibody-producing cells to be fused with myeloma cells. The antibody titer in the antiserum can be measured, for example, by reacting a labeled HRG described below with the antiserum and then measuring the activity of the labeling agent bound to the antibody. The fusion operation can be carried out according to a known method, for example, the method of Kohler and Milstein [Nature, 256, 495 (1975)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, and PEG is preferably used.
Examples of myeloma cells include NS-1, P3U1, SP2 / 0, etc., and P3U1 is preferably used. The preferred ratio of the number of antibody-producing cells (spleen cells) to be used and the number of myeloma cells is about 1: 1 to 20: 1, and PEG (preferably PEG1000 to PEG6000) is added at a concentration of about 10 to 80%. The cell fusion can be efficiently carried out by incubating at about 20 to 40 ° C., preferably about 30 to 37 ° C. for about 1 to 10 minutes.

Various methods can be used to screen for monoclonal antibody-producing hybridomas. For example, the hybridoma culture supernatant is added to a solid phase (eg, microplate) on which an antigen such as a protein is adsorbed directly or with a carrier, and then radioactive. A method for detecting a monoclonal antibody bound to a solid phase by adding an anti-immunoglobulin antibody labeled with a substance or an enzyme (if the cell used for cell fusion is a mouse, an anti-mouse immunoglobulin antibody is used) or protein A; Examples include a method in which a hybridoma culture supernatant is added to a solid phase on which an anti-immunoglobulin antibody or protein A is adsorbed, a protein labeled with a radioactive substance or an enzyme is added, and a monoclonal antibody bound to the solid phase is detected. .
Selection of the monoclonal antibody can be carried out according to a method known per se or a method analogous thereto, but can usually be carried out in a medium for animal cells to which HAT (hypoxanthine, aminopterin, thymidine) is added. As a selection and breeding medium, any medium may be used as long as the hybridoma can grow. For example, RPMI 1640 medium containing 1 to 20%, preferably 10 to 20% fetal calf serum, GIT medium (Wako Pure Chemical Industries, Ltd.) containing 1 to 10% fetal calf serum, or serum-free for hybridoma culture A culture medium (SFM-101, Nissui Pharmaceutical Co., Ltd.) etc. can be used. The culture temperature is usually 20-40 ° C, preferably about 37 ° C. The culture time is usually 5 days to 3 weeks, preferably 1 to 2 weeks. Culturing can usually be performed under 5% carbon dioxide gas. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the antibody titer in the above antiserum.

(B) Purification of monoclonal antibody Separation and purification of monoclonal antibodies are carried out in the same manner as in the separation and purification of ordinary polyclonal antibodies (eg, salting-out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis). Method, adsorption / desorption method using ion exchanger (eg DEAE), ultracentrifugation method, gel filtration method, antigen-binding solid phase or active adsorbent such as Protein A or Protein G Specific purification method for obtaining antibody].

[Preparation of polyclonal antibody]
The polyclonal antibody against HRG can be produced according to a method known per se or a method analogous thereto. For example, a complex of an immunizing antigen (an antigen such as a protein) and a carrier protein is prepared, and a mammal is immunized or a chicken is immunized in the same manner as the above-described monoclonal antibody production method. It can be produced by collecting the contents and separating and purifying the antibody.
Regarding the complex of immune antigen and carrier protein used to immunize mammals and chickens, the type of carrier protein and the mixing ratio of carrier and hapten are such that the antibody is effective against hapten immunized by cross-linking to carrier. If possible, any material may be crosslinked at any ratio. For example, bovine serum albumin, bovine thyroglobulin, keyhole limpet hemocyanin, etc. are about 0.1 to 20 by weight ratio to hapten 1. Preferably, a method of coupling at a ratio of about 1 to 5 is used.
Various coupling agents can be used for coupling of the hapten and the carrier, but an active ester reagent containing glutaraldehyde, carbodiimide, maleimide active ester, thiol group or dithiobilidyl group is used.
The condensation product is administered to mammals or chickens at the site where antibody production is possible, together with the carrier or diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production ability upon administration. Administration can be performed about once every 2 to 6 weeks, usually about 3 to 10 times in total.
Polyclonal antibodies can be collected from blood, ascites, breast milk, etc. of mammals immunized by the above method, preferably from blood, and in the case of chickens from blood and egg yolk.
The polyclonal antibody titer in the antiserum can be measured in the same manner as the above-described measurement of the antibody titer in the serum. Separation and purification of the polyclonal antibody can be performed according to the same immunoglobulin separation and purification method as the above-described monoclonal antibody separation and purification.

Another method for separating the Pro ¹⁸⁶ variant and the Ser ¹⁸⁶ variant using the difference in molecular weight and detecting the respective proteins is a method using mass spectrometry. For example, the antibody against the HRG is immobilized on the surface of a probe that can be adapted to a mass spectrometer, the test sample is brought into contact with the antibody on the probe, and a biological sample component captured by the antibody is subjected to mass spectrometry. The method of detecting the peak of HRG of each molecular weight is mentioned.

  Alternatively, chromatographic techniques combined with time-of-flight mass spectrometry (TOF-MS) can be captured on chromatographic carriers (eg, cation exchangers, anion exchangers, hydrophobic chromatographic carriers, metal ions, etc.) under certain conditions. A method for measuring the mass of all biological sample components at once, in particular, fixing the chromatographic carrier to the surface of a probe used for time-of-flight mass spectrometry, bringing the probe surface into contact with the test sample, and Examples include a method of measuring the mass of the component captured on the probe surface with a time-of-flight mass spectrometer after washing. Examples of probes that can be adapted to a time-of-flight mass spectrometer include, but are not limited to, various protein chips manufactured by Cyphergen Biosystems. The washing can be performed using water or a buffer solution. For example, the pH is appropriately selected according to the isoelectric point of the target marker of each sample. For example, pH 4-8 Tris-HCl buffer, phosphate buffer, borate buffer, acetate buffer and the like are used. Mass spectrometry is preferably performed by a MALDI method using an appropriate matrix (MALDI-TOFMS). Examples of matrix molecules used include sinapinic acid (SPA), saturated 2,5-dihydroxybenzoic acid (DHB), and indole acrylic. Examples include acid (IAA) and cinnamic acid.

As a method for detecting the presence of each of these variants without using the difference in molecular weight, that is, without separating the Pro ¹⁸⁶ variant and the Ser ¹⁸⁶ variant, there is a method using a reagent that can detect only one of these variants. Can be mentioned. Examples of such a reagent include, but are not limited to, an antibody that reacts specifically with each variant, preferably a monoclonal antibody.

A monoclonal antibody that specifically reacts with each variant is a portion specific to each variant [for example, a partial peptide consisting of the amino acid sequence at the 186th amino acid and its vicinity, or a specific peptide for one variant and the other variant. No sugar chain or partial peptide containing the sugar chain (as described above, the molecular weight difference between the Pro ¹⁸⁶ variant and the Ser ¹⁸⁶ variant is based on the difference in the sugar chain modification. 186 amino acid obtained by limited degradation and the vicinity thereof, for example, a partial peptide containing Asn at 184 can be used))], and a method similar to the method for producing a monoclonal antibody against HRG described above It can be acquired by carrying out. Each variant can be prepared from a human blood having a CC allele and a TT allele using a protein purification method known per se, but DNA encoding each variant is used as a host mammalian cell (preferably a human cell). It is desirable to purify from those in which each variant is overexpressed. In addition, for antibodies that react specifically with each variant, monoclonal antibodies are prepared using each variant itself as an immunogen, the cross-reactivity of each obtained monoclonal antibody against both variants is verified, and only one variant is reactive. It can also be obtained by verifying that it does not have.

The determination method of the present invention using an antibody specific for each variant is not particularly limited, and the amount of antibody, antigen or antibody-antigen complex corresponding to the amount of antigen in the test sample is chemically or Any measurement method may be used as long as it is a measurement method that is detected by physical means and calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephrometry, competition method, immunometric method and sandwich method are preferably used.
As a labeling agent used in a measurement method using a labeling substance, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance or the like is used. As the radioisotope, for example, [ ¹²⁵ I], [ ¹³¹ I], [ ³ H], [ ¹⁴ C] and the like are used. The enzyme is preferably stable and has a large specific activity. For example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate and the like are used. As the luminescent substance, for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used. Furthermore, a biotin-avidin system can also be used for binding of an antibody or antigen and a labeling agent.

For insolubilization of an antigen or antibody, physical adsorption may be used, or a method using a chemical bond usually used to insolubilize or immobilize a protein or an enzyme may be used. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, or glass.
In the sandwich method, a test sample is reacted with an insolubilized HRG variant-specific antibody (primary reaction), and another labeled antibody against HRG (not necessarily variant-specific) is reacted (secondary). After the reaction), the amount of HRG variant in the test sample can be quantified by measuring the amount (activity) of the labeling agent on the insolubilized carrier. The primary reaction and the secondary reaction may be performed in the reverse order, or may be performed simultaneously or at different times.

Monoclonal antibodies against the HRG variant can also be used in measurement systems other than the sandwich method, such as competitive methods, immunometric methods, nephrometry, and the like.
In the competition method, the antigen in the test sample and the labeled antigen are reacted competitively with the antibody, and then the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated. (B / F separation), the amount of labeled B or F is measured, and the amount of antigen in the test sample is quantified. In this reaction method, a soluble antibody is used as an antibody, a B / F separation is performed using polyethylene glycol, a liquid phase method using a secondary antibody against the antibody, and a solid phase antibody is used as a primary antibody, or As the primary antibody, a soluble one is used, and a solid phase method using a solid phase antibody as a secondary antibody is used.
In the immunometric method, the antigen of the test sample and the immobilized antigen are subjected to a competitive reaction with a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated, or the antigen in the test sample is separated from the antigen. After reacting with an excess amount of labeled antibody and then adding a solid phased antigen to bind unreacted labeled antibody to the solid phase, the solid phase and the liquid phase are separated. Next, the amount of label in any phase is measured to quantify the amount of antigen in the test sample.
In nephrometry, the amount of insoluble precipitate produced as a result of the antigen-antibody reaction in a gel or solution is measured. Laser nephrometry using laser scattering is preferably used even when the amount of antigen in the test sample is small and only a small amount of precipitate is obtained.

In applying these individual immunological measurement methods to the quantification method of the present invention, special conditions, operations and the like are not required to be set. What is necessary is just to construct | assemble the measurement system of the protein of this invention, adding the usual technical consideration of those skilled in the art to the usual conditions and operation methods in each method. For details of these general technical means, it is possible to refer to reviews, books and the like.
For example, Hiroshi Irie “Radioimmunoassay” (Kodansha, published in 1974), Hiroshi Irie “Sequel Radioimmunoassay” (published in Kodansha, 1979), “Enzyme Immunoassay” edited by Eiji Ishikawa et al. 53)), edited by Eiji Ishikawa et al. “Enzyme Immunoassay” (2nd edition) (Medical Shoin, published in 1982), edited by Eiji Ishikawa et al. 62), “Methods in ENZYMOLOGY” Vol. 70 (Immunochemical Techniques (Part A)), Id. Vol. 73 (Immunochemical Techniques (Part B)), Id. Vol. 74 (Immunochemical Techniques (Part C)), Id. 84 (Immunochemical Techniques (Part D: Selected Immunoassays)), ibid.Vol. 92 (Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods)), ibid.Vol. 121 (Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies) )) (From Academic Press ) And the like can be referred to.

  In the detection method using the above-described HRG variant-specific antibody, both variants may be detected using specific antibodies, or the specific antibody against one variant is used for detection / quantification of the variant. In addition, HRG is detected and quantified using an antibody reactive to both variants, and the presence or absence of the other variant is determined by comparing (subtracting) the measured values of the former and the latter. You may verify.

If only the Pro ¹⁸⁶ variant is detected as a result of the above measurement, the HRG genotype in the subject is CC, and if both the Pro ¹⁸⁶ variant and the Ser ¹⁸⁶ variant are detected, the subject The HRG genotype is CT, and if only the Ser ¹⁸⁶ variant is detected, the HRG genotype in the subject is TT.

  As a result of genotyping at the above-mentioned (a) gene level or (b) protein level, subjects whose HRG genotype was recognized as CC were compared to subjects whose genotype was recognized as CT, Future arteriosclerotic diseases (eg, myocardial infarction, angina pectoris, cerebral infarction, cerebral hemorrhage, cerebral thrombus, cerebral embolism, aortic aneurysm, aortic dissection, renal sclerosis, renal failure, obstructive arteriosclerosis, post-PCI restenosis, acute Coronary syndrome, coronary artery disease, peripheral arterial occlusion, etc.) can be determined to be at higher risk. In addition, if a subject whose HRG genotype was recognized as CC already had an arteriosclerotic condition or had a related disease, the genotype was recognized as CT It can be diagnosed that stricter disease management (for example, setting the target value of LDL-cholesterol and / or total cholesterol to a lower value) is necessary as compared with patients with atherosclerotic disease. Note that subjects whose genotype is recognized as TT are not subject to determination.

  As described above, humans having the CC genotype for the HRG gene have a significantly higher risk of developing arteriosclerotic diseases than humans having the CT genotype. In addition, it can be said that a patient already suffering from arteriosclerotic disease has a high risk of causing a serious cardio-cerebral vascular event. Therefore, in clinical trials of prophylactic / therapeutic drugs for arteriosclerotic diseases and the evaluation of the prophylactic / therapeutic effects of existing drugs, subjects or patient groups must be assigned to the HRG gene in order to analyze the test / evaluation data more effectively. It is desirable to classify in advance by genotype. Therefore, the present invention also examines for each subject or patient the SNP genotype that determines the 186th amino acid of the HRG protein, and selects the subject or patient whose genotype is CC as the high-risk group. A method for selecting subjects or patients in a high-risk group in evaluating the efficacy of a prophylactic / therapeutic agent for arteriosclerotic diseases or determining a dosage plan. The genotype test can be performed by the genotyping method (a) or (b) described above for the method for determining genetic susceptibility.

The present invention also provides a kit for use in the determination method of the present invention and the method of selecting a high risk group. The kit of the present invention is classified into (a) a kit for genotyping at the gene level and (b) a kit for genotyping at the protein level.
A kit for genotyping at the gene level is characterized by comprising a nucleic acid probe and / or primer pair capable of detecting an SNP that determines the 186th amino acid of the HRG protein.

Specifically, a nucleic acid probe is a nucleic acid that hybridizes with genomic DNA in a region containing the base of the SNP site that determines the 186th amino acid of the HRG protein, is specific for the target site, and is polymorphic. The length (base length of the portion that hybridizes with the genomic DNA) is not particularly limited, for example, about 15 bases or more, preferably about 15 to about 500 bases, more preferably about 15 to about 200 bases, more preferably about 15 to about 50 bases.
The probe may contain additional sequences suitable for detection of polymorphisms (sequences that are not complementary to genomic DNA). For example, the allele probe used in the invader method has an additional sequence called a flap at the 5 ′ end of the base at the polymorphic site.
The probe may be an appropriate labeling agent such as a radioisotope (eg, ¹²⁵ I, ¹³¹ I, ³ H, ¹⁴ C, etc.), an enzyme (eg, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase). , Malate dehydrogenase, etc.), fluorescent substances (eg, fluorescamine, fluorescein isothiocyanate, etc.), luminescent substances (eg, luminol, luminol derivatives, luciferin, lucigenin, etc.) and the like. Alternatively, a quencher (quenching substance) that absorbs fluorescence energy emitted by the fluorescent substance may be further bound in the vicinity of the fluorescent substance (eg, FAM, VIC, etc.). In such an embodiment, the fluorescence is detected by separating the fluorescent substance and the quencher during the detection reaction.

  Preferably, the nucleic acid probe used in the diagnostic kit of the present invention is the base Y of the SNP site represented by the base number 6830 in the base sequence represented by GenBank accession number NC_000003.10 (SEQ ID NO: 1) (ie , C or T), and a nucleic acid containing a continuous base sequence of about 15 to about 500 bases, preferably about 15 to about 200 bases, more preferably about 15 to about 50 bases. Depending on the polymorphism detection method used, a nucleic acid having one of the bases for the SNP site can be used, or two types of nucleic acids corresponding to the C allele and the T allele can be used. In the invader probe used in the above invader method, the base of the polymorphic site (that is, the base at the 3 'end) may be any base.

The nucleic acid primer used in the kit of the present invention is not particularly limited as long as it is designed to specifically amplify the genomic DNA region containing the base of the SNP site that determines the 186th amino acid of the HRG protein. There may be. For example, it is a partial base sequence of the base sequence represented by GenBank accession number NC_000003.10 (SEQ ID NO: 1), which is a complementary strand 5 'to the base of the SNP site that determines the 186th amino acid of the HRG protein A nucleic acid that hybridizes to a part of the sequence and includes a nucleic acid containing a base sequence of about 15 to about 50 bases, preferably about 15 to about 30 bases, and hybridizes to a part of the sequence 3 'to the base of the polymorphic site In combination with a nucleic acid containing a base sequence of about 15 to about 50 bases, preferably about 15 to about 30 bases, and the fragment length of the nucleic acid amplified by them is about 50 to about 1,000 bases, preferably about 50 Examples include a pair of nucleic acids having from about 500 bases, more preferably from about 50 to about 200 bases. In addition, as used in Examples described later, the SNP site to be detected may be included in the genomic DNA sequence to which one of the primer pairs hybridizes.
The primer may contain additional sequences suitable for detection of polymorphisms (sequences that are not complementary to genomic DNA), such as linker sequences.
The primer may be an appropriate labeling agent such as a radioisotope (eg, ¹²⁵ I, ¹³¹ I, ³ H, ¹⁴ C, etc.), an enzyme (eg, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase). , Malate dehydrogenase, etc.), fluorescent substances (eg, fluorescamine, fluorescein isothiocyanate, etc.), luminescent substances (eg, luminol, luminol derivatives, luciferin, lucigenin, etc.) and the like.

  Preferably, the nucleic acid primer used in the kit of the present invention is the base Y (ie, CNP) of the SNP site represented by the base number 6830 in the base sequence represented by GenBank accession number NC_000003.10 (SEQ ID NO: 1). Or a pair of nucleic acids capable of amplifying a continuous base sequence of about 50 to about 1,000 bases, preferably about 50 to about 500 bases, more preferably about 50 to about 200 bases.

The nucleic acid probe or primer used in the kit of the present invention may be DNA or RNA, and may be single-stranded or double-stranded. In the case of a double strand, it may be a double-stranded DNA, a double-stranded RNA, or a DNA / RNA hybrid. Therefore, when describing a nucleic acid having a certain base sequence in the present specification, unless otherwise specified, a single-stranded nucleic acid having the base sequence, a single-stranded nucleic acid having a sequence complementary to the base sequence, and a hybrid thereof It should be understood that the term is used to encompass all double-stranded nucleic acids.
The nucleic acid probe or primer can be synthesized according to a conventional method using a DNA / RNA automatic synthesizer based on the information of the base sequence represented by NC_000003.10, for example.

The above nucleic acid probe and / or primer are each separately (or mixed if possible) in water or in an appropriate buffer (eg, TE buffer) at an appropriate concentration (eg, 2 × to 20 × concentration). 1-50 μM, etc.) and can be stored at about -20 ° C.
Depending on the polymorphism detection method, the kit of the present invention may further include other components necessary for carrying out the method as a component. For example, when the kit is for polymorphism detection by TaqMan PCR method, the kit includes 10 × PCR reaction buffer, 10 × MgCl ₂ aqueous solution, 10 × dNTPs aqueous solution, Taq DNA polymerase (5 U / μL), etc. Can further be included. In addition, when the kit is for polymorphism detection by PCR-RFLP method, the kit may further contain a restriction enzyme Hpy188III, a restriction enzyme reaction buffer, and the like in addition to the above-mentioned reagents.

On the other hand, the genotyping kit at the protein level includes a first reagent capable of detecting an HRG protein (ie, Pro ¹⁸⁶ variant) having an apparent molecular weight of about 75 kDa by SDS-PAGE and an apparent molecular weight by SDS-PAGE. And a second reagent capable of detecting an HRG protein (ie, Ser ¹⁸⁶ variant) that is approximately 77 kDa. In the case where the genotyping method uses a molecular weight difference such as Western blotting to separate two variants and detect each of them, the first reagent and the second reagent may be the same. Examples of such a common reagent include, but are not limited to, an antibody capable of recognizing both variants described above and a protein such as plasmin known to interact with HRG.

  On the other hand, when the genotyping method is a mode that detects two variants without separating them, at least one of the first reagent and the second reagent must be a substance that can detect only one variant. . It is more preferable that both the first reagent and the second reagent do not show cross-reactivity with the other variant in that quantitative measurement is not required. Examples of such first and second reagents include antibodies specific for each variant described above.

  Depending on the polymorphism detection method, the kit of the present invention may further include other components necessary for carrying out the method as a component. For example, when the kit is for polymorphism detection by Western blotting, the kit includes molecular weight markers, transfer membrane, transfer buffer, blocking solution, washing buffer, labeling agent, label detection reagent, etc. Can further be included.

The present invention is also administered in a novel administration protocol (for example, dosage / administration schedule, etc.) for subjects determined to have CC genotype by HRG genotyping or patients with arteriosclerotic disease Provide a preventive / therapeutic agent for arteriosclerotic diseases.
As the active ingredient contained in the prophylactic / therapeutic agent of the present invention, any compound known to be effective in preventing / treating arteriosclerotic diseases can be selected. Preferable examples include compounds showing LDL-cholesterol lowering action that are effective in suppressing foaming. Examples of such compounds include those described in WO 2005/012272 and WO 02/059077. In addition, as already marketed drugs, clofibrate and its derivatives (eg, simfibrate, clinofibrate, bezafibrate, etc.), nicotinic acid and its derivatives (eg, nicomol, niceritrol, tocopherol nicotinate, etc.), dextran sulfate, Anion exchange resins (eg, cholestyramine, etc.), plant sterols (eg, soysterol, γ-oryzanol, etc.), probucol, HMG-CoA reductase inhibitors (eg, pravastatin, simvastatin, etc.) can also be preferably used. .

The compound exhibiting LDL-cholesterol lowering action can be used as a prophylactic / therapeutic agent for arteriosclerotic disease after mixing with a pharmacologically acceptable carrier as necessary to obtain a pharmaceutical composition.
Here, as the pharmacologically acceptable carrier, various organic or inorganic carrier substances commonly used as pharmaceutical materials are used, and excipients, lubricants, binders, disintegrants in solid preparations; solvents in liquid preparations , Solubilizing agents, suspending agents, isotonic agents, buffers, soothing agents and the like. If necessary, preparation additives such as preservatives, antioxidants, colorants, sweeteners and the like can also be used.
Suitable examples of excipients include lactose, sucrose, D-mannitol, D-sorbitol, starch, pregelatinized starch, dextrin, crystalline cellulose, low-substituted hydroxypropylcellulose, sodium carboxymethylcellulose, gum arabic, pullulan, light Anhydrous silicic acid, synthetic aluminum silicate, magnesium aluminate metasilicate and the like can be mentioned.
Preferable examples of the lubricant include magnesium stearate, calcium stearate, talc, colloidal silica and the like.
Preferred examples of the binder include pregelatinized starch, sucrose, gelatin, gum arabic, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, crystalline cellulose, sucrose, D-mannitol, trehalose, dextrin, pullulan, hydroxypropylcellulose, hydroxy Examples thereof include propylmethylcellulose and polyvinylpyrrolidone.
Preferable examples of the disintegrant include lactose, sucrose, starch, carboxymethylcellulose, carboxymethylcellulose calcium, croscarmellose sodium, carboxymethylstarch sodium, light anhydrous silicic acid, low substituted hydroxypropylcellulose and the like.
Preferable examples of the solvent include water for injection, physiological saline, Ringer's solution, alcohol, propylene glycol, polyethylene glycol, sesame oil, corn oil, olive oil, cottonseed oil and the like.
Preferable examples of solubilizers include polyethylene glycol, propylene glycol, D-mannitol, trehalose, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate, sodium salicylate, sodium acetate. Etc.
Suitable examples of the suspending agent include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glyceryl monostearate; for example, polyvinyl alcohol, polyvinyl Examples include hydrophilic polymers such as pyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose; polysorbates, polyoxyethylene hydrogenated castor oil, and the like.
Preferable examples of the isotonic agent include sodium chloride, glycerin, D-mannitol, D-sorbitol, glucose and the like.
Preferable examples of the buffer include buffers such as phosphate, acetate, carbonate and citrate.
Preferable examples of the soothing agent include benzyl alcohol.
Preferable examples of the preservative include p-hydroxybenzoates, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like.
Preferable examples of the antioxidant include sulfite and ascorbate.
Suitable examples of colorants include water-soluble food tar dyes (eg, food red Nos. 2 and 3, food yellow Nos. 4 and 5, food blue Nos. 1 and 2), water-insoluble lake dyes ( Examples thereof include aluminum salts of the aforementioned water-soluble edible tar pigments, natural pigments (eg, β-carotene, chlorophyll, bengara, etc.).
Preferable examples of the sweetening agent include saccharin sodium, dipotassium glycyrrhizinate, aspartame, stevia and the like.

Examples of the dosage form of the pharmaceutical composition include oral preparations such as tablets, capsules (including soft capsules and microcapsules), granules, powders, syrups, emulsions and suspensions; and injections (eg, subcutaneous injection). Preparations, intravenous injections, intramuscular injections, intraperitoneal injections, etc.), external preparations (eg, nasal preparations, transdermal preparations, ointments, etc.), suppositories (eg, rectal suppositories, vaginal suppositories) Etc.), pellets, drops, sustained-release preparations (eg, sustained-release microcapsules, etc.) and the like.
The pharmaceutical composition can be produced by a method commonly used in the field of pharmaceutical technology, such as the method described in the Japanese Pharmacopoeia. Below, the specific manufacturing method of a formulation is explained in full detail. The content of the active ingredient in the pharmaceutical composition varies depending on the dosage form, the dose of the active ingredient, etc., but is, for example, about 0.1 to 100% by weight.
For example, oral preparations contain active ingredients, excipients (eg, lactose, sucrose, starch, D-mannitol, etc.), disintegrants (eg, carboxymethylcellulose calcium, etc.), binders (eg, pregelatinized starch, gum arabic) , Carboxymethylcellulose, hydroxypropylcellulose, polyvinylpyrrolidone, etc.) or lubricant (eg, talc, magnesium stearate, polyethylene glycol 6000, etc.), etc., and then compression molded, and then, if necessary, taste masking, enteric or For the purpose of durability, it is produced by coating with a coating base by a method known per se.

Examples of the coating base include sugar coating base, water-soluble film coating base, enteric film coating base, sustained-release film coating base and the like.
As the sugar coating base, sucrose is used, and one or more selected from talc, precipitated calcium carbonate, gelatin, gum arabic, pullulan, carnauba wax and the like may be used in combination.
Examples of the water-soluble film coating base include cellulose polymers such as hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, and methylhydroxyethylcellulose; polyvinyl acetal diethylaminoacetate, aminoalkyl methacrylate copolymer E [Eudragit E (trade name), Rohm Pharma Co., Ltd.], synthetic polymers such as polyvinylpyrrolidone; polysaccharides such as pullulan.
Examples of enteric film coating bases include cellulose-based polymers such as hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate, carboxymethylethylcellulose, and cellulose acetate phthalate; methacrylic acid copolymer L [Eudragit L (trade name), Acrylic polymers such as Rohm Pharma Co.], methacrylic acid copolymer LD [Eudragit L-30D55 (trade name), Rohm Pharma Co., Ltd.], and methacrylic acid copolymer S [Eudragit S (trade name), Rohm Pharma Co., Ltd.]; Examples include natural products such as shellac.
Examples of sustained-release film coating bases include cellulose polymers such as ethyl cellulose; aminoalkyl methacrylate copolymer RS [Eudragit RS (trade name), Rohm Pharma Co., Ltd.], ethyl acrylate / methyl methacrylate copolymer suspension Examples thereof include acrylic acid polymers such as suspensions (Eudragit NE (trade name), Rohm Pharma).
Two or more of the coating bases described above may be mixed and used at an appropriate ratio. Moreover, you may use light-shielding agents, such as a titanium oxide, ferric oxide, etc. in the case of coating.

  Injections contain active ingredients as dispersants (eg, polysorbate 80, polyoxyethylene hydrogenated castor oil 60, polyethylene glycol, carboxymethyl cellulose, sodium alginate, etc.), preservatives (eg, methyl paraben, propyl paraben, benzyl alcohol, chlorobutanol, Phenol, etc.), isotonic agents (eg, sodium chloride, glycerin, D-mannitol, D-sorbitol, glucose, etc.) and other aqueous solvents (eg, distilled water, physiological saline, Ringer's solution, etc.) or oily solvents (eg, , Olive oil, sesame oil, cottonseed oil, vegetable oils such as corn oil, propylene glycol, etc.) and the like. At this time, additives such as a solubilizing agent (eg, sodium salicylate, sodium acetate, etc.), a stabilizer (eg, human serum albumin, etc.), a soothing agent (eg, benzyl alcohol, etc.) may be used as desired. The injection solution is usually filled in a suitable ampoule.

Usually, the dose of the drug is set so that the patient's blood total cholesterol and / or LDL-cholesterol level is controlled within the range of the target value in the category to which the patient belongs according to predetermined guidelines. For example, according to the Japanese Society for Arteriosclerosis “Clinical Guidelines for Atherosclerotic Disease 2002”, major coronary risk factors other than LDL-C [aging, hypertension without coronary artery disease (myocardial infarction, angina pectoris) , Diabetes, smoking, family history of coronary artery disease, low HDL-C blood pressure], 0, 1, 2, 3, ≧ 4 patients are classified into patient categories A and B1 to B4, respectively, and patients with coronary artery disease are classified into patient category C For patient categories A, B1-B2, B3-B4, and C, TC is <240, <220, <200, <180 (mg / dl), and LDL-C is <160, <140, respectively. Target values of <120 and <100 (mg / dl) (hereinafter referred to as management categories I, II, III, and IV for convenience) are set.
However, none of the conventional guidelines consider the genotype of the HRG gene as a risk factor. However, according to the present invention, the risk of developing arteriosclerotic disease in humans whose HRG genotype is CC is comparable to that of humans with a diagnosis of hypertension or hyperlipidemia as long as the results of Examples below are followed. To do. Therefore, the prophylactic / therapeutic agent of the present invention is a blood total within the range of the target value in at least one stage (for example, the first stage, the second stage, the third stage) higher risk category than the category to which the patient belongs according to the predetermined guidelines. Administered to control cholesterol and / or LDL-cholesterol levels. For example, in the guidelines of the Japanese Arteriosclerosis Society, if a patient belonging to management category I has a CC genotype, it is treated as management category II (or III or IV) and the target value of TC is <220 mg / dl (or <200 mg / dl or <180 mg / dl), the target value of LDL-C is set to <140 mg / dl (or <120 mg / dl or <100 mg / dl).

In the present specification and drawings, bases, amino acids, and the like are represented by abbreviations based on abbreviations by IUPAC-IUB Commission on Biochemical Nomenclature or conventional abbreviations in the field, examples of which are described below. In addition, when there is an optical isomer with respect to an amino acid, the L form is shown unless otherwise specified.
DNA: deoxyribonucleic acid cDNA: complementary deoxyribonucleic acid A: adenine T: thymine G: guanine C: cytosine RNA: ribonucleic acid mRNA: messenger ribonucleic acid dATP: deoxyadenosine triphosphate dTTP: deoxythymidine triphosphate dGTP: deoxyguanosine tri Phosphate dCTP: Deoxycytidine triphosphate ATP: Adenosine triphosphate EDTA: Ethylenediaminetetraacetic acid SDS: Sodium dodecyl sulfate

Gly: Glycine Ala: Alanine Val: Valine Leu: Leucine Ile: Isoleucine Ser: Serine Thr: Threonine Cys: Cysteine Met: Methionine Glu: Glutamic acid Asp: Aspartic acid Lys Argin P : Tryptophan Pro: Proline Asn: Asparagine Gln: Glutamine pGlu: Pyroglutamic acid Me: Methyl group Et: Ethyl group Bu: Butyl group Ph: Phenyl group TC: Thiazolidine-4 (R) -carboxamide group

  EXAMPLES The present invention will be described in more detail with reference to the following examples, but these do not limit the scope of the present invention.

(sample)
Rabbits: We used plasma from WHHL rabbits, which are used worldwide for research such as high cholesterol and arteriosclerosis. In rabbits, the area of aorta lesion (arteriosclerosis) was calculated by intravascular ultrasound (IVUS). A score of 0-5 was assigned from light to heavy lesions, and a group with IVUS score 0 was used as a control for expression comparison.
Human: Human serum was used to confirm the expression pattern of HRG. All human sera were obtained from BioClinical Partners, USA, and blood collection was performed on men and women over 40 years of age. At the time of blood collection, those that meet any of the following criteria were registered as disease groups:
1. Those who have been diagnosed with acute myocardial infarction by EKG test within 21 days from the day of blood collection.
2. Patients diagnosed with coronary artery occlusion by coronary angiography on the day of blood collection.
Donors who do not meet the above criteria and who meet the following criteria were registered as normal:
1. Fasting serum triglyceride measured within 90 days is 130 mg / dl or less and HDL cholesterol is 40 mg / dl or more.
2. There is no diagnosis of obesity, hypothyroidism, nephrotic syndrome, and diabetes that can cause hyperlipidemia.
3. No history of myocardial infarction or family history.
Blood collection was performed at a hospital affiliated with BioClinical Partners. Serum was secured by centrifugation for 30 to 60 minutes after blood collection and quickly frozen and stored.
(Pretreatment of plasma and serum)
In order to reduce the influence of highly expressed proteins in blood during protein expression analysis, plasma and serum were pretreated by any of the following methods.
1. Albumin / IgG removal: Rabbit plasma was treated with Blue sepharose and Protein A columns to remove albumin and IgG in the plasma.
2. Removal of highly expressed protein in blood: Rabbit plasma is treated with an antibody affinity column to remove transferrin, hemopexin, alpha 1 antitrypsin and fibrinogen in plasma, and albumin, IgG, antitrypsin, IgA, transferrin and haptoglobin in human serum Removal.
3. Lectin fractionation: Rabbit plasma is treated with a lectin column. Select the fraction with the least effect of highly expressed protein.
(Protein expression analysis by two-dimensional electrophoresis)
Rabbit plasma proteins obtained by each pretreatment and fractionation method were developed on a two-dimensional electrophoresis gel (one-dimensional: isoelectric point, two-dimensional: molecular weight). The gel for expression analysis was stained with Cy dye (Amersham Bioscience), and the gel for protein identification was stained with SyproRuby (Molecular Probe). Protein expression analysis was performed with Decerser software from Amersham.
(Protein identification)
Identification of protein spots with significant expression differences by two-dimensional gel analysis was performed by mass spectrometry. The expression variation spot was cut out from the SyproRuby stained gel, and the protein was digested with trypsin in the gel. Then, it measured by MS and MS / MS with the mass spectrometer (Q-TOF type). The mass spectrometry data obtained by this method was analyzed by the following product ion retrieval method. That is, the protein database was searched based on the mass of the parent peptide, and the MS / MS spectrum was collated with the hit candidate amino acid sequence to determine the score. This method is effective when proteins are registered in the database, but identification of unregistered proteins is difficult.
(Western blot)
The expression pattern of HRG in human serum was confirmed by Western blotting. The plasma protein subjected to the treatment for removing the highly expressed protein was developed on SDS-PAGE, and then transferred to a PVDF membrane. HRG on the membrane was recognized by an anti-HRG antibody (R & D Systems), incubated with an HRP-labeled secondary antibody, and then reacted and detected.
(Glycosidase treatment)
Glycosidase treatment was used for confirmation of glycosylation modification. The plasma protein that had been subjected to the removal of the highly expressed protein was subjected to deglycosylation treatment with glycopeptidase F (Takara Bio Inc.) and accompanying reagents, and then developed on SDS-PAGE to perform Western blotting.
(Polymorphism analysis by PCR)
The genotype of the HRG gene of the blood donor was confirmed by PCR. One of two 20-base sequences containing either cytosine or thymine at the SNP site that determines the 186th amino acid of the HRG protein and about 400 residues downstream from the genomic gene of the blood donor PCR was performed using a combination of base sequences as a probe. Then, the presence or absence of the band amplified by the agarose gel was confirmed. At this time, if a band was detected, the donor possessed a specific SNP.

Example 1 Analysis of expression fluctuation by two-dimensional gel electrophoresis
Plasma (n = 4) collected from J-0 and J-4,5 rabbits was treated with a Blue sepharose / Protein A column and then with an antibody affinity column, followed by lectin fractionation, and then with a different Cy dye. As a result of labeling and two-dimensional electrophoresis on the same gel, protein spots whose expression increased with the progress of arteriosclerosis were found. The expression variation for one of these spots is shown in FIG.

Example 2 Identification of Protein The partial amino acid sequence of the protein obtained in Example 1 was determined by a product ion search method. As a result, it was revealed that the protein obtained from the spot obtained in Example 1 was HRG (the amino acid sequence of rabbit HRG is shown in FIG. 2). The amino acid sequence of the part identified by the amino acid sequence analysis of the separated protein is underlined in FIG.

Example 3 Analysis of Human HRG by Western Blot The phenomenon that HRG increased in association with arteriosclerosis obtained in Examples 1 and 2 was confirmed by Western blot using normal and patient-derived human sera. As a result, it was revealed that two types of HRG having molecular weights of about 77 kDa and 75 kDa were detected in humans (FIG. 3 (a)). Furthermore, it was revealed that both 77 kDa and 75 kDa HRG were detected in many normal subjects, whereas only 75 kDa HRG was present in sera from most disease groups (FIG. 3 (b)).

Example 4 Human HRG after glycosidase treatment
In order to verify whether the difference in the molecular weight of human HRG obtained in Example 3 was due to the difference in sugar chain, human serum from which the highly expressed protein had been removed was subjected to glycosidase and then Western blotted. Regardless of whether one or both of 77 kDa and 75 kDa HRG were present, a single band with almost the same molecular weight was detected (FIG. 4). From this, it became clear that the difference between two HRGs with different molecular weights was due to the difference in sugar chains.

Example 5 Analysis of human HRG gene polymorphism It was confirmed by PCR of the genomic gene that the sugar chain modification to HRG shown in Example 4 was caused by the gene polymorphism resulting in the substitution of the 186th amino acid of HRG. As a result, as shown in FIG. 5 (a), when the 186th amino acid of the HRG protein has an allele that becomes Pro, a band is detected by a primer whose SNP site is c, and has an allele that becomes Ser. In some cases, a band was detected with the primer t. FIG. 5 (b) shows a comparison between the normal group and the HRG gene of the disease group, and it was revealed that there are many heterozygotes in the normal group and only in the disease group. This result is consistent with the 77 kDa and 75 kDa bands detected by Western blot, indicating that the phenotype of this protein reflects the gene polymorphism.

  According to the present invention, genetic susceptibility to arteriosclerotic diseases can be easily determined noninvasively using a blood sample of a patient, etc. This is useful in determining the dosage regimen for disease patients. In addition, the present invention can provide new and useful management guidelines for arteriosclerotic diseases based on the HRG gene genotype.

It is a figure which shows the fluctuation | variation of the magnitude | size of the two-dimensional gel electrophoresis spot which shows the expression of HRG in the plasma extract | collected from the WHHL rabbit of each score (aorta lesion area ratio: J-0, J-4,5). The vertical axis shows Standardized Log Abundance. In the figure, a circle indicates each measured value (n = 4), and a + indicates an average value. It is a figure which shows the amino acid sequence and rabbit HRG amino acid sequence of the protein isolate | separated from the spot corresponding to HRG detected by the mass spectrometry. The underlined portion indicates the same sequence as the amino acid sequence of the detected peptide. It is a figure which shows the result of the Western blot analysis with respect to HRG in the human serum after a high expression protein removal process. (a) is a stained image of the membrane. In the figure, N represents a normal person, MI represents a myocardial infarction patient, UA represents an unstable angina patient, and the numbers represent subject numbers in each group. (b) is a panel showing the determination result of one or two detected HRG bands. In the figure, MI represents a myocardial infarction patient, UA represents an unstable angina patient, and No. represents the subject number of each group. The shaded area in column A represents a single band of approximately 75 kDa, and the shaded area in column B represents a double band. A blank in both A and B indicates that it cannot be determined. It is a figure which shows the result of the Western blot analysis with respect to HRG of the human serum protein after a glycosidase process. Regardless of the pattern before treatment, one band with the same molecular weight is detected, indicating that the difference in molecular weight seen in the sample before treatment is due to the difference in sugar chain. In the figure, N represents a normal person, MI represents a myocardial infarction patient, UA represents an unstable angina patient, and the numbers represent subject numbers in each group. (-) Represents no glycosidase treatment, and (+) represents a protein sample treated with glycosidase. It is the figure which showed the polymorphism analysis by PCR of a human HRG gene. (A) shows a part of the HRG gene amplified by PCR detected on an agarose gel. In the figure, N represents a normal person, MI represents a myocardial infarction patient, UA represents an unstable angina patient, and the numbers represent subject numbers in each group. c and t indicate the base of the SNP site incorporated in the primer. (B) shows the genotype of the SNP site that determines the 186th amino acid in the panel. In the figure, MI represents a myocardial infarction patient, UA represents an unstable angina patient, and No. represents the subject number of each group.

Claims (14)

  A method for determining a genetic susceptibility of a subject to arteriosclerosis or a related disease, wherein the subject is tested for a genotype of SNP that determines the 186th amino acid of the HRG protein.   The method according to claim 1, wherein when the subject is a homozygote of an allele whose amino acid at 186 is Pro, the subject is determined to have high genetic susceptibility to arteriosclerosis or a related disease. .   A method for selecting subjects or patients in a high-risk group in the evaluation of the efficacy of a prophylactic / therapeutic agent for arteriosclerosis or related diseases or in determining the administration plan, and determining the 186th amino acid of the HRG protein for each subject or patient A test or a patient who is a homozygote of an allele whose 186th amino acid is Pro is selected as a high-risk group.   The method according to claim 1 or 3, characterized in that the genotype assay is performed at the nucleic acid level.   4. A method according to claim 1 or 3, characterized in that the genotype assay is performed at the protein level.   6. The method according to claim 5, wherein the presence of an HRG protein having an apparent molecular weight of about 75 kDa and an HRG protein of about 77 kDa by SDS-PAGE is assayed.   When only an HRG protein having an apparent molecular weight of about 75 kDa is detected by SDS-PAGE, the subject is determined to be a homozygote of an allele in which the 186th amino acid of the HRG protein is Pro. The method according to claim 6.   A kit for determining genetic susceptibility to arteriosclerosis or a related disease, comprising a nucleic acid probe and / or a primer pair capable of detecting SNP that determines the 186th amino acid of HRG protein.   The nucleic acid probe is a nucleic acid containing a continuous base sequence of about 15 to about 500 bases including the base of the SNP site that determines the 186th amino acid of the HRG protein, and the nucleic acid primer pair is an HRG gene or HRG The partial base sequence of mRNA, which is a pair of nucleic acids capable of amplifying a continuous base sequence of about 50 to about 1,000 bases including the base of the SNP site that determines the 186th amino acid of the HRG protein. Kit.   A first reagent capable of detecting an HRG protein having an apparent molecular weight of about 75 kDa by SDS-PAGE, and a second reagent capable of detecting an HRG protein having an apparent molecular weight of about 77 kDa by SDS-PAGE. A kit for determining genetic susceptibility to arteriosclerosis or a related disease and / or selecting a high risk group of arteriosclerosis or a related disease.   The kit according to claim 10, wherein the first reagent and / or the second reagent is an antibody against the HRG protein.   The kit according to claim 11, wherein the first and second reagents are the same antibody.   The first reagent is an antibody capable of recognizing only the HRG protein having an apparent molecular weight of about 75 kDa by SDS-PAGE, and / or the second reagent is only an HRG protein having an apparent molecular weight of about 77 kDa by SDS-PAGE. The kit according to claim 11, wherein the kit is a recognizable antibody.   A prophylactic / therapeutic agent for arteriosclerosis or related diseases, comprising a substance having a blood total cholesterol and / or LDL-cholesterol lowering action, wherein the homologue of an allele of which the 186th amino acid of HRG protein is Pro It is characterized by administering to a patient who is a zygote so that the blood total cholesterol and / or LDL-cholesterol level is a target value in a category at least one stage higher risk than the category to which the patient belongs according to a predetermined guideline. Agent.