WO2003087360A1 - Method of judging risk of arterial sclerosis, method of measuring risk of arterial sclerosis, microarray for judging risk of arterial sclerosis, device for judging risk of arterial sclerosis and program for judging risk of arterial sclerosis - Google Patents

Abstract

A method of judging the risk of arterial sclerosis in which the tendency to crisis of arterial sclerosis, tendency to progress thereof, etc. can be accurately judged as the risk of arterial sclerosis to thereby enable use of the method in the prevention and treatment of arterial sclerosis; a kit for analysis of genetic polymorphism which is used in the judging of the risk, etc.; a device for judging the risk of arterial sclerosis; etc. The method of judging the risk of arterial sclerosis is characterized in that it comprises a risk determining step wherein the risk of arterial sclerosis attributed to genetic polymorphism is determined from the genetic type of genetic polymorphism of examinee on the basis of the risk of arterial sclerosis peculiar to a combination of a plurality of genetic polymorphisms, and that at least one of the combination of a plurality of genetic polymorphisms exhibits a significant positive association between the combination of a plurality of genetic polymorphisms and a level of thickening of carotid intima/media composite.

Description

 Atherosclerotic disease risk assessment method, Atherosclerotic disease risk measurement method, Microarray for atherosclerotic disease risk assessment, Atherosclerotic disease risk assessment device, Arteriosclerotic disease risk assessment program Technical field

 The present invention relates to the determination of the risk of atherosclerotic disease, and more particularly, to a method of determining the risk of atherosclerotic disease that can be used for prevention, treatment and diagnosis of arteriosclerosis, a method of manifesting factors related to atherosclerotic disease, Disease risk measurement method, gene polymorphism detection method, gene marker, gene polymorphism analysis kit, microarray for arteriosclerotic disease risk assessment, arteriosclerotic disease risk assessment device, and arteriosclerotic disease risk It relates to the judgment program.

Background art

 Environmental requirements such as hypertension, diabetes, hyperlipidemia, obesity, and smoking are known to be risk factors for the development of atherosclerotic disease (ischemic heart disease). Is also one of the risk factors, and the development of molecular biological techniques in recent years has revealed various genetic polymorphisms on genes related to arteriosclerosis, and the involvement in disease Has been studied.

 Using the genetic polymorphism involved in such a disease as a risk factor, information on the genotype of the genetic polymorphism of the subject can be used to determine the so-called arteriosclerotic disease risk, such as susceptibility to disease and progression. If the judgment can be made, the test subject with a high risk can focus on the prevention of the disease from an early stage on a daily basis, and can also predict the degree of progression after the onset, which can be used for more detailed diagnosis and treatment.

 However, in clinically relevant studies of genetic polymorphisms including SNP in atherosclerotic diseases, a single genetic polymorphism was examined and a group of one genotype for the polymorphism was determined. The odds ratio of the susceptibility to myocardial infarction was calculated by examining the ratio of myocardial infarction patients to healthy subjects in each of the genotype populations. With such a survey, most polymorphisms are not significantly different

Confirmation H The risk of disease susceptibility or progression could not be predicted from the polymorphism.

 (Non-patent literature) Yamada Y, Izawa H, Ichihara S, Takatsu F, Ishihara H, Hi rayama H, Sone T, Tanaka M, Yokota M. Prediction of the risk of my o card ial infarction from polymorphisms in candidate genes.

N. Engl. J. Med. 2002; 347 (24): 1916-23 Disclosure of the Invention.

 An object of the present invention is to solve the conventional problem of determining the risk of arteriosclerotic disease and achieve the following objects. That is, an arteriosclerotic disease risk assessment method that can accurately determine the likelihood of onset or progression of arteriosclerotic disease as the risk of atherosclerotic disease, and can be used for prevention and treatment of arteriosclerosis. Method for eliciting arteriosclerotic disease-related factors used for determination of degree, method for measuring risk of arteriosclerotic disease, method for detecting gene polymorphism, gene marker, kit for analyzing gene polymorphism, risk assessment for arteriosclerotic disease It is intended to provide a microarray for use, an arteriosclerotic disease risk judging device, and an arteriosclerotic disease risk judging program.

 The present inventors quantitatively analyzed the relationship between carotid intima-media complex thickening for a large number of gene polymorphisms, and further, when a plurality of the * gene polymorphisms were combined, It has been found that the carotid artery intima-media complex has a significant additive or synergistic effect on the degree of thickening.

 The present invention is based on the above findings of the present inventors, and means for solving the above problems are as follows.

 <1> Based on the risk of arteriosclerosis inherent to a combination of a plurality of gene polymorphisms, a risk determination step of determining the risk of arteriosclerosis due to the gene polymorphism from the genotype of the subject polymorphism is performed. Including

At least one of the combinations of the plurality of polymorphisms has a significant positive association between the combination of the plurality of polymorphisms and the carotid intima-media complex thickness. This is a method for determining the risk of atherosclerotic disease. <2> The combination-specific atherosclerosis risk of multiple gene polymorphisms is set based on whether the combination has a significant positive association with the carotid intima-media thickening <1> The arteriosclerotic disease risk determination method according to <1>.

 <3> The combination specific arteriosclerosis risk of multiple gene polymorphisms is the odds ratio of the frequency at which there is a significant positive association between the combination and carotid intima-media complex thickness An arteriosclerotic disease risk determination method according to <1>, which is set.

 <4> The arteriosclerosis risk risk determination method according to <1>, wherein the risk of arteriosclerosis specific to the combination of a plurality of gene polymorphisms is set by the amount of increase in the media thickness of the atherosclerotic intima. It is.

<5> At least one set of arteriosclerosis-related gene polymorphisms selected from the set of atherosclerosis-related gene polymorphisms described in Tables 11 to 14 below, in which a combination of a plurality of gene polymorphisms is selected. The arteriosclerotic disease risk determination method according to any one of the above <1> to <4>, comprising:

Fiberi O P8AV! One-

Fiber! -

[Table 1-1-3]

In Tables 11 to 14, “Number” represents the genetic polymorphism of the same number in Table 2-1 to Table 2-2 below, and the “Classification” number is the gene type of the genetic polymorphism. Of these, 1 represents the genotype targeted for the combination, 1 is the homozygous polymorphism shown on the left side of the polymorphism names in Table 2-1 to Table 2-2, and 2 is the heterozygous , 3 represent the homologue of the polymorphism shown on the right side, 12 represents the genotype combining the above 1 and 2, and 23 represents the genotype combining the above 2 and 3.

[2 [Table 2—2]

<6> An arteriosclerosis disease case is defined as a case where the thickness of the carotid intima-media complex is at least 0.2 mm thicker than the average thickness of the carotid intima-media complex in healthy volunteers. Is defined as a non-arteriosclerotic disease case,

In at least 150 patients with atherosclerotic disease, 30% had a combination of multiple polymorphisms with a significant positive association with carotid intima-media complex thickness Above

In at least 150 non-atherosclerotic populations, 15% have a combination of multiple polymorphisms that have a significant positive association with carotid intima-media complex thickness So that

The method according to any one of the above <1> to <5>, wherein a combination of a plurality of gene polymorphisms is selected.

 <7> The arteriosclerosis disease risk group according to <6>, wherein the atherosclerosis disease case group and the non-atherosclerosis case group are both diabetic patients and have no history of myocardial infarction. is there.

 <8> The atherosclerotic disease risk according to any one of <1> to <7>, wherein the combination of a plurality of gene polymorphisms is a combination of at least one of 2 to 5 gene polymorphisms. This is a degree determination method. One

 <9> The arteriosclerotic disease risk determination method according to any one of <1> to <8>, wherein the combination of a plurality of gene polymorphisms comprises at least three gene polymorphisms.

<10> A combination of a plurality of gene polymorphisms described in any one of the above <1> Karaku 9> including at least two gene polymorphisms belonging to any of the following groups a) to 1): This is a method for determining the risk of arteriosclerotic disease.

 a) Genetic polymorphisms related to renin and angiotensin system

 b) Genetic polymorphisms related to platelet function and coagulation system

 c) Polymorphisms related to lipids

 d) Polymorphisms related to nitric oxide synthase

e) Polymorphisms related to TNF- _α gene f) Polymorphisms related to IRS-1 gene

g) Polymorphism group related to FAB P2 gene

h) Polymorphisms related to muscle glycogen synthase gene

 i) Polymorphisms related to NAD P · NADPH oxidase p22pho x j) Polymorphisms related to methylenetetrahydrofolate reductase

k) Polymorphisms related to heat shock protein 70-1

 1) Gene polymorphisms related to TGF-J31 gene

 <1 1> Genetic polymorphisms belonging to the polymorphism group related to platelet function and coagulation system and renin. Gene polymorphism group related to angiotensin system, gene polymorphism related to methylenetetrahydrofolate reductase (MTHFR) <10> The method for determining a risk of atherosclerotic disease according to <10>, wherein the method includes at least a type group and a gene polymorphism belonging to at least one of a lipid-related gene polymorphism group.

 <12> The risk assessment of arteriosclerotic disease according to <11>, wherein the polymorphism belonging to the polymorphism group related to platelet function and coagulation system is a polymorphism related to the PA I-1 gene. Is the way.

 <13> The gene polymorphism belonging to the gene polymorphism group related to the renin-angiotensin system is any one of <1> to <1>, which is a gene polymorphism related to the ACE gene. It is an arteriosclerotic disease risk determination method described in the above.

 <14> An arteriosclerotic disease according to any one of <12> to <13>, wherein the gene polymorphism belonging to the lipid-related gene polymorphism group is a gene polymorphism related to the HUMP OM A gene. This is a risk determination method.

 <15> The method further includes a risk determination step of determining an arteriosclerosis risk due to the environmental factor from information on the environmental factor of the test subject based on the arteriosclerosis risk specific to the environmental factor. 14>.

<16> The carotid intima-media thickness is calculated from the carotid intima-media thickness based on the atherosclerosis risk inherent in the carotid-media-media thickness. by The arteriosclerotic disease risk determination method according to any one of <1> to <15>, further including a risk determination step of determining an arteriosclerosis risk.

 <17> The arteriosclerotic disease risk determination method has a plurality of risk determining steps, and calculates the arteriosclerotic disease risk by integrating the arteriosclerotic risk calculated for each of the risk determining steps. The arteriosclerotic disease risk judging method according to any one of the above <1> to <16>, including a step of calculating the risk of arteriosclerotic disease.

 <18> The arteriosclerotic disease risk according to any one of <1> to <17>, further including a detection step of detecting a genotype of a plurality of genetic polymorphisms in the test subject before the manifestation step. This is a degree determination method.

 <19> a detection step of detecting a genotype of a plurality of gene polymorphisms in the test subject, and a plurality of the gene polymorphisms, based on a risk of arteriosclerosis inherent to the combination, of the test subject detected in the detection step. Determining a risk of atherosclerosis due to the genetic polymorphism from the genotype of the genetic polymorphism,

At least one combination of the plurality of gene polymorphisms has a significant positive association between the combination of the plurality of gene polymorphisms and carotid intima-media complex thickness. It is an arteriosclerosis disease risk measurement method.

 <20> By selectively elucidating the genotypes of a plurality of polymorphisms related to the atherosclerosis-related gene polymorphism set among the gene polymorphisms of the test subject, Including a manifestation process for manifesting a set of polymorphisms related to atherosclerosis,

 The arteriosclerosis-related gene polymorphism set is a combination of a plurality of gene polymorphisms having a significant positive association with the carotid intima-media complex thickness, and This is a method for eliciting an arteriosclerotic disease-related factor, which is characterized by the following.

 <21> The manifestation step, wherein the genotypes of a plurality of selectively identified polymorphisms are represented by whether or not a genotype of an arteriosclerosis-related gene polymorphism set is described in (20). This is a method for eliciting atherosclerotic disease-related factors.

<2 2> When the genotype of multiple gene polymorphisms selectively identified in the manifestation process corresponds to the set of arteriosclerosis-related gene polymorphisms, The atherosclerotic disease-related disease according to item 20>, which is expressed by a odds ratio of a frequency having a significant positive association with the thickness of the intimal media-media complex of the splenic artery, which is specific to the type set. This is the factor manifestation method.

 <2 3> When the genotypes of multiple gene polymorphisms selectively identified in the manifestation process correspond to the arteriosclerosis-related gene polymorphism set, they are unique to the arteriosclerosis-related gene polymorphism set. The arteriosclerotic disease-related factor manifestation method described in <20> above, which is represented by the amount of increase in carotid intima-media thickness of the carotid artery.

 <24> At least one atherosclerosis-related gene polymorphism set selected from among the atherosclerosis-related gene polymorphism sets described in Tables 11 to 1 to 4 above. 20. The method for manifesting an atherosclerotic disease-related factor according to any one of the above <20> to <23>.

 <25> If the carotid intima-media thickness is at least 0.2 mm thicker than the healthy carotid intima-media thickness in healthy volunteers, it is defined as an atherosclerotic disease case. Is defined as a non-arteriosclerotic disease case,

In at least 150 atherosclerotic disease patient populations, at least 30% had at least one atherosclerosis-related polymorphism set,

In at least 150 non-atherosclerotic disease populations, at least 15% have at least one atherosclerosis-related polymorphism set,

The method according to any one of the above items <20> to <24>, wherein an atherosclerosis-related gene polymorphism set is selected.

 The arteriosclerotic disease risk assessment according to <25>, wherein both the arteriosclerosis disease case group and the non-arteriosclerosis case group are diabetic patients and have no history of myocardial infarction. Is the way.

 <27> The arteriosclerosis disease-related factor manifestation according to any one of <20> to <26>, wherein the atherosclerosis-related gene polymorphism set comprises at least one of 2 to 5 polymorphisms. Method.

The <28> arteriosclerosis-related gene polymorphism set, wherein the atherosclerotic disease-related factor manifestation according to any one of (20)> (27), which comprises at least three gene polymorphisms. It is a localization method.

 <29> The arteriosclerosis-related gene polymorphism set includes at least two gene polymorphisms belonging to any one of the following groups a) to 1): This is a method for eliciting atherosclerotic disease-related factors.

a) Polymorphisms associated with the renin-angiotensin system

b) Genetic polymorphisms related to platelet function and coagulation system

c) Lipid-related polymorphisms

d) Polymorphisms related to nitric oxide synthase

e) Polymorphisms related to TNF-α gene

 f) Polymorphisms related to IRS-1 gene

g) Polymorphism group related to FABP2 gene

h) Polymorphisms related to muscle glycogen synthase gene

 i) Polymorphisms related to NAD P and NADPH oxidase p22phox j) Polymorphisms related to methylenetetrahydrofolate reductase

k) Polymorphisms related to heat shock protein 70-1

 1) TGF-; 81 Polymorphisms related to 1 gene

 <30> The method for eliciting an arteriosclerotic disease-related factor according to any one of <20> to <29>, further including a detection step for detecting a genotype of a plurality of polymorphisms in the test subject before the elicitation step. It is.

 <31> Genes that constitute at least one atherosclerosis-related gene polymorphism set selected from among the atherosclerosis-related gene polymorphism sets described in Table 1-1 1 Detecting the genotype of the subject for the type,

 This is a gene polymorphism detection method, wherein the detection result is used for determining the risk of arteriosclerotic disease.

 <32> Genetic polymorphisms constituting at least one arteriosclerosis-related gene polymorphism set selected from the set of arteriosclerosis-related gene polymorphisms described in Table 1-1 1 above. Is a genetic marker containing

33> Atherosclerosis-related gene polymorphism sets listed in Table 1-1 to Tables 1-4 above. A primer pair capable of specifically amplifying a gene constituting at least one atherosclerosis-related gene polymorphism set selected from the group consisting of: or a nucleic acid probe capable of specifically hybridizing to the gene;

A genetic polymorphism analysis kit comprising detecting at least one set of arteriosclerosis-related gene polymorphisms described in Tables 11 to 14.

 <34> The genetic polymorphism analysis kit according to <33>, wherein the atherosclerosis-related gene polymorphism set includes at least three gene polymorphisms.

 3 5> It has at least one of the primers and probes for detecting polymorphisms of at least two polymorphisms selected from the polymorphisms listed in Table 2-1 to Table 2-2 ,

 A case in which the thickness of the carotid intima-media complex is 0.2 mm or more thicker than the average thickness of the carotid intima-media complex in a healthy person is defined as an arteriosclerotic disease case. When defined as a sclerosis disease case,

In at least 150 arteriosclerosis disease patient populations, the carotid artery in the set of arteriosclerosis-related gene polymorphisms described in Tables 2-1 to 2-2 above, which can be constituted by the selected gene polymorphisms More than 30% of cases have at least one genotype combination that has a significant positive association with the thickness of the media-media complex,

In at least 150 non-atherosclerotic disease patient populations,

33. The genetic polymorphism analysis kit according to any one of <33> to <34>.

 <36> having a primer or probe for detecting a polymorphism of at least two polymorphisms selected from the polymorphisms described in Tables 2-1 to 2-2,

 A case in which the carotid intima-media thickness is 0.2 mm or more thicker than the average carotid intima-media thickness in a healthy person is defined as an arteriosclerotic disease case, and the other cases are non-arterial When defined as a sclerosis disease case,

In at least 150 arteriosclerosis disease patient populations, the carotid artery in the set of arteriosclerosis-related gene polymorphisms set forth in Tables 11 to 14 above, which can be constituted by the selected gene polymorphisms Transmission with a Significant Positive Association with Membrane-Media Complex Thickness More than 50% of cases have a combination of child types,

The genetic polymorphism analysis kit according to any one of <33> to <35>, which accounts for 15% or less of at least 150 non-atherosclerotic disease patient populations.

 <37> The gene polymorphism analysis kit according to any one of <33> to <36>, wherein the plurality of gene polymorphism detection primers or probes belong to one of the following different groups a) to 1). It is.

a) Gene polymorphisms related to the renin-angiotensin system

b) Genetic polymorphisms related to platelet function and coagulation system

 c) Lipid-related polymorphisms

d) Polymorphisms related to nitric oxide synthase

e) Polymorphisms related to TNF-α gene

 f) Polymorphisms related to IRS-1 gene

g) Polymorphism group related to FAB P2 gene

h) Polymorphisms related to muscle glycogen synthase gene

 i) Polymorphisms related to NADP / NADPH oxidase p22phox j) Polymorphisms related to methylenetetrahydrofolate reductase

k) Polymorphisms related to heat shock protein 70-1

 1) Polymorphisms related to TGF-1 gene

 38> At least one gene polymorphism set that is selected from among the arteriosclerosis-related gene polymorphism sets listed in Tables 11 to 14 above, An arteriosclerotic disease risk determination microarray comprising a gene polymorphism detection probe.

 <39> A computer-based atherosclerotic disease risk determination device, comprising: an atherosclerotic risk data table in which combinations of a plurality of gene polymorphisms and atherosclerotic risks are listed in correspondence. ,

The entered combination of a plurality of gene polymorphisms of the subject is compared with a combination of a plurality of gene polymorphisms in the atherosclerosis risk data table, and when there is a matching combination of the gene polymorphisms, The arteriosclerosis risk corresponding to the combination of polymorphisms An arteriosclerotic disease risk judging device characterized by having a detecting means for detecting.

 <40> Atherosclerosis risk data table, which lists the combinations of multiple gene polymorphisms and the risk of atherosclerosis, has a significant effect on carotid intima-media thickness. 39. The arteriosclerotic disease risk judging device according to <39>, wherein one unit corresponds to a combination of a plurality of gene polymorphisms having a positive association as arteriosclerosis risk.

 4 1> The atherosclerosis risk data table, which lists the combinations of multiple gene polymorphisms and the risk of atherosclerosis, shows a significant difference between the carotid intima-media thickening The combination of a plurality of polymorphisms having a positive association with the odds ratio of the frequency at which the thickness of the carotid intima-media complex exceeds the normal range as the risk of atherosclerosis. > The arteriosclerotic disease risk determination device described in <>.

 4 2> The atherosclerosis risk data table, which lists the combinations of multiple genetic polymorphisms and the risk of atherosclerosis, has a significant difference between the carotid intima-media thickening The arteriosclerosis according to <39>, wherein a combination of a plurality of polymorphisms having a positive association is associated with an increase in carotid intima-media complex thickness as a risk of arteriosclerosis. It is a disease risk determination device.

 4 3> A combination of gene polymorphisms with a significant positive association with carotid intima-media thickness is normal The odds ratio of the frequency exceeding the range is not less than a certain value, and the mean value of the thickness of the intima-media complex of the carotid artery is defined by at least one of the following: The arteriosclerotic disease risk judging device according to any one of to <42>.

 <4 4> Atherosclerosis risk data table that lists the presence or value of environmental factors and the risk of atherosclerosis in correspondence,

The inputted or nonexistent or numerical value of the environmental factor of the test subject is compared with the presence or numerical value of the environmental factor in the atherosclerosis risk data table, and the atherosclerotic risk corresponding to the presence or numerical value of the environmental factor is determined. The arteriosclerotic disease risk judging device according to any one of 3 9> Karaku 4 3>, further comprising a detecting means for detecting. You.

 <45> The arteriosclerotic disease risk judging device has a plurality of detecting means, and judges the arteriosclerotic disease risk based on the sum of the plurality of carotid artery risk detected by the plurality of detecting means. The arteriosclerotic disease risk judging device according to any one of the above <39> to <64>, further comprising a judging means.

 <4 6> Atherosclerosis risk data table in which carotid artery intima-media thickening and arteriosclerosis risk are listed in correspondence,

 The carotid intima-media complex is compared with the carotid intima-media thickness of the test subject and the carotid intima-media thickness in the atherosclerosis risk data table. The arteriosclerotic disease risk judging device according to any one of <39> to <45>, further comprising a detecting means for detecting the arteriosclerosis risk corresponding to the degree of hypertrophy.

 <47> Any one of the carotid risk detected by the detection means and the added value of the carotid risk extracted from the plurality of extraction means, and the input carotid intima media of the subject The arterial sclerosis disease risk judging device according to any one of (39) to (46), further comprising a judging means for judging an arteriosclerotic disease risk based on an added value with the complex thickening degree. It is.

 4 8> Includes a vascular intima pressure measurement means for measuring the carotid intima-media thickness of the subject and measuring the carotid intima-media thickness to the computer. 9> The apparatus for determining risk of arteriosclerotic disease according to any one of <7> and <7>.

 <4 9> An arteriosclerosis risk data table in which combinations of a plurality of gene polymorphisms and arteriosclerosis risk are listed in correspondence with each other is stored in the computer.

The computer compares the combination of the plurality of genetic polymorphisms of the test subject with the combination of the plurality of genetic polymorphisms in the atherosclerosis risk data table, and finds a matching combination of the genetic polymorphisms. And a function of detecting a risk of arteriosclerosis corresponding to the combination of the genetic polymorphisms. <50> Atherosclerosis risk data table, which lists combinations of multiple gene polymorphisms and arteriosclerosis risk, has a significant difference between carotid intima-media thickening. The arteriosclerotic disease risk determination program according to the item <49>, wherein one unit corresponds to a combination of a plurality of gene polymorphisms having a positive association as an arteriosclerosis risk.

 <5 1> The atherosclerosis risk data table, which lists the combination of multiple gene polymorphisms and the risk of arteriosclerosis, has a significant difference between the carotid intima-media thickness and the carotid intima-media thickness. The combination of a plurality of polymorphisms having a positive association with the odds ratio of the frequency at which the thickness of the carotid intima-media complex exceeds the normal range as the risk of arteriosclerosis corresponds to <49. > The arteriosclerotic disease risk determination program described in>.

 <52> Atherosclerosis risk data table, which lists the combinations of multiple gene polymorphisms and the risk of atherosclerosis, shows a significant difference between The arteriosclerotic disease risk according to the item 4 9>, wherein the combination of gene polymorphisms having a positive association is associated with an increase in carotid artery intima-media complex thickness as the risk of arteriosclerosis. This is a degree determination program.

 <5 3> Carotid intima-media complex thickness is within the normal range if it is a gene type combination that has a significant positive association with carotid intima-media thickness. Defined by at least one of the following: the odds ratio of the frequency is more than a certain value; and the average value of the carotid intima-media complex thickness is significantly different. 2> The risk assessment program for arteriosclerotic diseases according to any of <2>.

 <54> If the carotid intima-media thickness is at least 0.2 mm thicker than the average carotid intima-media thickness in healthy volunteers, this is defined as an arteriosclerosis disease case. Is defined as a non-arteriosclerotic disease case,

In at least 150 cases of atherosclerotic disease, at least 30% of patients had at least one unit of risk of atherosclerosis,

In at least 150 non-atherosclerotic disease populations, less than 15% have at least 1 unit at risk of atherosclerosis,

The arteriosclerotic disease risk determination program according to any one of <49> Lamb. BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 is a diagram showing sequences of gene polymorphisms specified in Tables 2-1 to 2-2.

 FIG. 2 is a diagram showing the sequences of the polymorphisms identified in Tables 2-1 to 2-2.

 FIG. 3 is a diagram showing the ratio of matching cases according to the number of polymorphisms constituting a combination in Example 4. BEST MODE FOR CARRYING OUT THE INVENTION

 (Method of determining risk of atherosclerotic disease)

 The arteriosclerotic disease risk determination method of the present invention is based on the atherosclerosis risk inherent in a combination of a plurality of gene polymorphisms. A risk determining step of determining a risk, wherein at least one of the combinations of the plurality of polymorphisms is between the combination of the plurality of polymorphisms and the carotid intima-media complex thickness It is characterized by having a significant positive relationship with any other step, and may include any other step as long as this step is included.

 The arteriosclerotic disease refers to an ischemic disease, and includes angina, myocardial infarction, cerebral infarction, and peripheral arterial occlusion. In addition, the arteriosclerotic disease risk is an index indicating the ease with which the arteriosclerotic disease develops and progresses.

 The gene polymorphism means a diversity in which a plurality of alleles (alleles) exist at one locus. However, the gene referred to here is not limited to the region transcribed as RNA, but includes any DNA that can be identified on the human genome, including promoters, regulatory regions such as enhancers, etc. .

99.9% of human genomic DNA is common among individuals, with the remaining 0.1% responsible for this diversity, including individuals who are susceptible to a particular disease, responsive to drugs or environmental factors. May be involved as a difference. If there is a difference in phenotype even if there is a genetic polymorphism Not necessarily. SNP (nucleotide polymorphism) is also a kind of the gene polymorphism, but the gene polymorphism of the present invention is not limited to this.

 The gene polymorphism used in the present invention is appropriately selected from among known gene polymorphisms that are presumed to be involved in arteriosclerotic disease so as to satisfy the following requirements for a plurality of gene polymorphisms. Can be.

 As a combination of a plurality of gene polymorphisms used in the present invention, at least one arteriosclerosis-related gene selected from the set of arteriosclerosis-related gene polymorphisms described in Tables 1-1 to 14 above. Preferably, it contains a gene polymorphism set. As detailed in Example 4 in Tables 11 to 14, Table 4 shows that in a population of diabetic patients with no history of myocardial infarction, A case-control study was performed on about 195 cases without the control, and the significance level was set at an odds ratio of 10 or more and a chi-square value of 6.635 (P <0.01) or more. This is the result of extracting a combination of a plurality of polymorphisms having a positive association with the degree of membrane thickening. Each line is a set of polymorphisms related to arteriosclerosis. The plurality of gene polymorphisms used in the present invention may include the arteriosclerosis-related gene polymorphism sets shown in Tables 11 to 4 and may be combined with other gene polymorphisms.

Further, as the plurality of gene polymorphisms used in the present invention, gene polymorphisms listed in Table 3 can also be used. Table 4 shows the literature on the genetic polymorphisms in Table 3. In Tables 3 and 4, the same number is assigned to the same gene polymorphism

[Table 3]

[表 4]

[Table 4]

前記複数の遺伝子多型とは、 異なる遺伝子座を有する 2種以上の遺伝子多型を いい、 2種の遺伝子多型であれば、 例えば、 表 3中に記載されている、 SERP I NE 1と ACEとをいい、 3種の遺伝子多型であれば、 例えば SERP I NE 1と、 APOA1と、 APOA2とをいう。

The plurality of gene polymorphisms refers to two or more gene polymorphisms having different loci.If the two gene polymorphisms are, for example, as described in Table 3, SERP I NE 1 It refers to ACE, and if it is three gene polymorphisms, it refers to, for example, SERP I NE1, APOA1, and APOA2.

The combination of the genetic polymorphisms means that the genotypes of the plurality of genetic polymorphisms are combined. For example, the SERPINE1 polymorphism, which is a promoter polymorphism associated with PAI-1 shown in Table 3, includes 4G and 5G types that differ in the number of G repeats. Alleles are present, of which type 4G is a risk factor. The genotype to be tested for this S ERP I NE1 polymorphism is either 4G / 4G, 4G / 5G or 5GZ5G. Similarly, the ACE polymorphism, which is the 16th intron polymorphism associated with ACE and listed in Table 3, includes an import (type I) and a deletion (type D) allele. Present, of which type D is a risk factor. The genotype to be tested for this ACE polymorphism will be one of D / D, D / I and IZI. Therefore, when the SERP I NE1 polymorphism and the ACE polymorphism are selected as a plurality of gene polymorphisms, the combination of the gene polymorphisms for both polymorphisms is 4G / 4G and DZD. There are 9 cases in total, such as those with / 4G and D / I. For each of these nine cases, the risk can be set individually.For example, the genotype of the subject to be tested for the SERP I NE1 polymorphism is 4 G / 4 G having a homozygous risk allele, Other than 4G and 5G heterozygously or 5G homozygous 5GZ?., And ACE polymorphisms are similarly classified into DZD and I /? When the combination of molds has 4 GZ4G and DZD, 4 G / 4 G and IZ ?, 5 G /? And D / D, and 5 G /? And IZ? They can be combined in four ways to create a combination of genetic polymorphisms. Furthermore, the risk level can be set by integrating the combination of genotypes having homologous risk factors (combination of 4G / 4G and DZD) and other combinations. Further, as described in detail in the fourth embodiment, the risk can be set by integrating the combinations according to a certain rule, and such integration is not particularly limited. At least one of the combinations of the plurality of gene polymorphisms is a plurality of genes having a significant positive association between the combination of the plurality of gene polymorphisms and the degree of carotid-media / media complex thickening. Need to be polymorphic.

 For example, 4G Z4G and Val Va of SERP I NE1 polymorphism with PA I-1 as a related factor, MTHF R polymorphism with MTHFR as a related factor, and ACE polymorphism with ACE as a related factor Significant positive association between the combination of 1 and D / D and carotid intima-media thickness (relevance in the direction of increasing carotid intima-media thickness) ), Those that include this combination of multiple genetic polymorphisms meet this requirement.

 Here, the value measured by the high-resolution ultrasonic tomography apparatus is used as the measurement value of the carotid artery intima-media thickness in determining whether or not there is a significant correlation. The carotid intima-media thickness is the mean value of the carotid intima-media thickness, which is the average of the measured carotid intima-media thickness, and the measured value. One of the maximal hypertrophy (PIMT) values, which is the maximum value of the intima-media complex thickness of the carotid artery in the subject, shall be used.

 Also, having significant relevance is an empirical value of significance when it is significant according to the result of a statistically general hypothesis test with the significance level set to 0.05. In the regression analysis, the increase in mean thickness of the carotid intima-media complex (Δ ΙΜ T) is greater than or equal to 0.2 mm, and also in the regression analysis, which is an empirical value of significance. This means that the increment of the maximum thickness of the carotid intima-media complex (ΔΡ ΙΜΤ) is at least 0.3 mm or more.

In addition, regarding the selection of the combination of the plurality of gene polymorphisms and the selection of the number of sets, the carotid intima-media thickening is more than the carotid intima-media thickening average of a healthy person. At least 0.2 mm thick is defined as an arteriosclerotic disease case, and the other cases are defined as non-arteriosclerotic disease cases. 30% or more, preferably 50% or more, more preferably 60% or more of the cases having a combination of a plurality of gene polymorphisms having a significant positive association with the membrane complex thickness, at least In a population of 150 non-atherosclerotic cases Combinations of multiple polymorphisms such that 15% or less have multiple polymorphism combinations that have a significant positive association with carotid intima-media complex thickness Is preferably selected. Under the same conditions, more than 30% of the cases have a combination of multiple polymorphisms that have a significant positive association with carotid intima-media complex thickness, and at least 30% In a population of 150 non-atherosclerotic cases, less than 10% of patients have a combination of multiple polymorphisms with a significant positive association with carotid intima-media thickness It is also preferred that they be selected such that

 Further, the number of polymorphisms constituting a combination of a plurality of polymorphisms is preferably about 2 to 5. Although a combination of 6 or more gene polymorphisms can be used, the percentage that the total of the combination can cover is not so large in the case of arteriosclerosis, but the percentage that the combination has in the control example However, there is a possibility that the error in the risk level will be rather large. In addition, when the number of polymorphisms constituting a combination increases, the number of combinations having superiority increases remarkably, but the frequency of individual combinations decreases significantly, which is disadvantageous in that analysis becomes complicated. Combinations can also be selected from those consisting of at least three genetic polymorphisms.

 The plurality of gene polymorphisms preferably include at least two gene polymorphisms belonging to any of the following groups a) to 1), and more preferably include three or more gene polymorphisms .

a) Genetic polymorphisms related to renin and angiotensin system

b) Genetic polymorphisms related to platelet function and coagulation system

c) Polymorphisms related to lipids

d) Polymorphisms related to nitric oxide synthase

e) Polymorphisms related to TNF-α gene

 f) Polymorphisms related to IRS-1 gene

g) Polymorphism group related to FABP 2 gene h) Polymorphisms related to muscle glycogen synthase gene

 i) Polymorphisms related to NADP-NADP H oxidase p22phox j) Polymorphisms related to methylenetetrahydrofolate reductase (MTHFR) k) Polymorphisms related to heat shock protein 70-1

 1) TGF-polymorphism group related to i31 gene

 Here, the polymorphism group related to a certain gene is not limited to a polymorphism present in exons and introns of the gene, but exists in a promoter region, a 3 ′ untranslated region, a 5 ′ untranslated region, etc. Polymorphisms are included. In general, a polymorphism in the coding region may change the amino acid sequence or change the expression level of mRNA, and even a polymorphism in the regulatory region may change the expression level of mRNA. In some cases, splicing may be altered, and in either case, the expression level or properties of the protein may be altered.

Specific examples of the gene polymorphism belonging to any of the groups a) to 1) include, for example, the gene polymorphisms described in Tables 1-1 to 14 but are not limited thereto. D) — a gene polymorphism group related to nitric oxide synthase, f) a gene polymorphism group related to IRS-1 gene, h) muscle glycogen synthase gene among the above groups a) to 1) And i) gene polymorphisms related to NADP and NADPH oxidase p22phoX can be considered together as insulin resistance and vascular endothelial function, and e) TNF — Gene polymorphisms related to α gene, k) polymorphisms related to heat shock protein 70-1 and 1) polymorphisms related to TGF-j31 gene are summarized as related to inflammatory response. A) related to the Reen-angiotensin system Genetic polymorphisms can be considered as sympathetic blood pressure-related, b) gene polymorphisms related to platelet function and coagulation system, and j) gene polymorphisms related to methylenetetrahydrofolate reductase. Can be regarded collectively as coagulation-fibrinolysis-related, and c) polymorphisms related to lipids and g) polymorphisms related to FAB P2 gene can be collectively considered as lipid-related. , It can also be classified as follows. (A) Insulin resistance

 (B) Inflammatory response

 (C) Adhesion factor

 (D) Sympathetic blood pressure

 (E) Solidified fibrinolytic system

 (F) Fat

 Genes of each component of the renin-angiotensin system have been identified in blood vessels and myocardium, and are reported to play important roles in arteriosclerosis, cardiac hypertrophy, and remodeling of blood vessels and myocardium . Therefore, polymorphisms related to the genes of each component of the renin'angiotensin system are suitably used in the present invention.

 Genes related to platelet surface GP (glycoprotein) Ib and IX receptors and vWF (von Willebrand factor) involved in the binding of platelets to vascular lesions under endothelial cells, and platelet surface fibrinogen receptor G多 b, polymorphisms related to genes related to Ilia and the like are suitably used in the present invention as polymorphisms belonging to a group of polymorphisms related to platelet function and coagulation system.

 In addition, coagulation factors such as coagulation factor VII and polymorphisms in the fibrinogen β-chain gene, which have been reported to be significantly associated with serum fipurinogen concentration, are also known to be gene-related in platelet function and coagulation system. The polymorphism belonging to the type group is suitably used in the present invention.

 Gene polymorphism group related to nitric oxide synthase, gene polymorphism group related to TNF-α gene, gene polymorphism group related to IRS-1 gene, gene polymorphism related to FABP 2 gene It is known that a group, a gene polymorphism group related to the muscle daricogen synthase gene is involved in insulin resistance.

 MTHFR (methylenetetrahydrofolate reductase) is a homocysteine metabolizing enzyme, and it has been reported that an increase in blood homocysteine concentration is an independent risk factor for cardiovascular disease.

The plurality of gene polymorphisms may include at least two gene polymorphisms belonging to different groups a) to 1) from each other; This is particularly preferable because a synergistic effect is easily generated and the contribution to the risk is large. Further, it is particularly preferable that the plurality of gene polymorphisms include at least three gene polymorphisms belonging to different groups a) to 1).

 The plurality of polymorphisms include a polymorphism belonging to a group of polymorphisms related to platelet function and coagulation system, a polymorphism group related to a renin-angiotensin system, and methylene tetrahydrofolate reductase (MT HFR). It is particularly preferable to include at least a related gene polymorphism group and a gene polymorphism belonging to at least one of the lipid-related gene polymorphism groups in terms of a large contribution to risk. In addition, it is also preferable to include at least a polymorphism group related to methylenetetrahydrofolate reductase (MTHFR) and a polymorphism group related to lipid.

 As the polymorphism belonging to the polymorphism group related to the platelet function / coagulation system, a polymorphism related to the PAI-1 gene is preferably used. In addition, as the polymorphism belonging to the polymorphism group related to the Renin-angiotensin system, a polymorphism related to the ACE gene is preferably used. As the polymorphism belonging to the lipid-related polymorphism group, a polymorphism related to the HUMPONA gene is preferably used.

 The risk of arteriosclerosis inherent to a combination of a plurality of gene polymorphisms can be determined by analyzing the relationship between the risk of atherosclerosis measured in a population and the combination of the plurality of gene polymorphisms. According to the obtained numerical value, an arteriosclerosis risk specific to the combination can be set in advance.

 The risk of arteriosclerosis can be appropriately selected and used from various known indices of carotid arteriosclerosis obtained by measuring the degree of carotid artery hypertrophy and the like. The carotid artery thickening measurement method is not particularly limited, but is a non-invasive and quantitative measurement method of the carotid artery using an ultrasonic tomography apparatus that measures the carotid artery thickening that can be reached ultrasonically. Measurement of the intima-media complex thickness (I MT) is common. The above-described method of measuring the carotid intima-media thickness (IMT) is an example, but the measuring method for determining the risk of arteriosclerosis is not limited thereto.

The ultrasonic tomography apparatus has a linear pulse echo having a center frequency of 7.5 MHz or more. It is desirable to use one with one probe. Since the extracranial carotid artery is located in the subcutaneous shallow layer, a frequency of 7.5 MHz or higher can be used, and high resolution (distance resolution 0.1 mm) can be obtained.

 The blood vessel wall is analyzed on the echo image as a two-layer structure consisting of one layer of low echo brightness on the lumen side of the blood vessel and another layer of high echo brightness. The authors, from observations of 104 healthy cases, confirmed that IMT increased almost linearly with age from the age of 10 to the age of 70, and that the thickness did not exceed 1.1 mm. The IMT of a healthy person is calculated from the age according to the following formula.

 IMT = 0.08 Age + 0.3 (3 <Age <80 yr) [1]

 Various known indices of arteriosclerosis of the carotid artery that can represent the risk of arteriosclerosis include a maximum IMT (MaX-IMT) representing a maximum value of IMT, an average IMT representing an average value of IMT. There are MT (AV g I MT), plaque score (PS), carotid stiffness, etc., and certain indices have not yet been determined. In addition, there are various measurement methods for each index.

The maximum intima-media thickness in each of the anterior oblique, lateral, and posterior oblique longitudinal images is defined as Ma X IMT, and the central side 1 cm and the distal side 1 cm centering on the site showing the Ma XI MT Avg IMT with the average of 3 points in total, and the skin of three longitudinal sections from the left and right common carotid artery (common car otid: CC) to the carotid bifurcation and the internal carotid artery (internal carotid: IC) Of the total thickness of the proximal wall (far wall) and the distal wall (far wall) with respect to the total thickness of 12, the researchers set the maximum value to AV _g IMT. Some researchers call it AV g I MT. In addition, the mean thickness of a certain section of the far wall may be referred to as me an IMT. The index may be the thickening of the far wall 10 mm centrally from the bifurcation of one carotid artery.

The plaque score (ps) refers to the sum of the carotid artery in each of the left and right carotid arteries with a plaque thickness of 1.1 mm or more at each site divided into four sections of 15 mm each based on the bifurcation. Also, the sum of the number of plaques (IMT 1.1 cm or more) in each of the above three or four sections may be referred to as a plaque number (PN) and used as an index. Carotid stiffness is a number measured from the diameter of the carotid artery during systole and diastole. Value.

 The method using the index of the thickening of the far wall 1 Omm centrally from the bifurcation of one carotid artery is easy to measure, and it is said that the measurement error is small because the common carotid artery has few lesions. IMT is an index that indicates the largest lesion of the carotid artery. The PS can show the entire image of the carotid artery with advanced arterial stiffness, but it is disadvantageous in that it is 0 in non-developed cases (thickness less than 1.1 mm). Suitable indicators differ depending on the disease. In the case of diabetes or hyperlipidemia, the carotid wall often thickens relatively evenly, and Av g IMT me an I MT is an important index. , Plaque is often recognized, and PS, PN and Maxl MT are effective indicators. '

The arteriosclerosis risk specific to the combination of the plurality of gene polymorphisms can be set using various known indices of arteriosclerosis of the carotid artery as described above. For example, the combination and the carotid artery Whether the combination has a significant positive association with the intima-media thickness (eg, 1 or 0), and the combination of the combination with the carotid intima-media thickness It is preferable to set a frequency with a significant positive association between the odds ratio and the like. In addition, it is also preferable to set the amount of increase in the degree of thickening of the intima-media complex, as an indicator of the overall risk of atherosclerotic disease. The increase in carotid intima-media complex thickness can be an increase in average IMT (ΔIMT), an increase in maximum IMT (ΔPIMT), and the like. It is particularly preferable as an indicator of a typical arteriosclerotic disease risk. There have been many reports on the relationship between the increase in the thickness of the carotid intima-media complex and arteriosclerotic diseases. In particular, for ΔΙΜΤ, the myocardium is increased every 0.333 mm of ΔΙΜΤ. It is known that the odds of infarction increase by 4.9 times (Yamasaki. Diabetes Care 2000 (9)), and the mode in which ΔIMT is used as the risk of arteriosclerosis is extremely effective in atherosclerotic disease risk. It is to judge the degree. As described above, the increase in the thickness of the intimal media-media complex of the arterial artery can be used as it is as the risk of atherosclerosis to indicate the risk of atherosclerotic disease. The arteriosclerosis risk may be calculated from the increase in the degree using an appropriate function. The increase in carotid intima-media complex thickness (such as ΔIMT and ΔPIMT) is determined by the partial regression coefficient calculated from the IMT value or PIMT value measured from the population by the method of multiple regression analysis. Can be represented.

 The risk determining step of determining the risk of arteriosclerosis based on the genetic polymorphism may be plural. That is, the arteriosclerosis risk may be determined from each of the plurality of gene polymorphisms in a plurality of sets.

 When the risk determination step is a single step, the risk of arteriosclerosis determined in this step can be directly used as the risk of arteriosclerotic disease. When there are two or more risk determination steps, the risk of arteriosclerosis can be determined by performing a linear operation by integrating the arteriosclerosis risks determined in the steps.

 The risk deciding method of arteriosclerotic disease of the present invention comprises a risk deciding step of deciding a risk of arteriosclerosis due to the environmental factor from information on the environmental factor of the test subject based on a risk of arteriosclerosis inherent to the environmental factor. It can further include.

 As the environmental factors, age, gender, hypertension, diabetes, hyperlipidemia, obesity, smoking, hemoglobin A1c value and the like have been reported.

 Vitelli et al. Reported that in the Atherosclerosis Risk Assessment (AR ICS tudy), 208 patients had carotid thickening (mean IMT, 1.21 mm) and non-diabetic patients had 208 carcinomas without thickening (mean IMT). (0.63 mm) .Comparing non-diabetic subjects, the authors report that a 1% increase in hemoglobin A 1c increases the risk of arteriosclerosis 1.77-fold [Vitelli LL. Diabetes Care 1997; 20: 1454-8].

 Although smoking is considered to be a risk factor for atherosclerosis, the atherosclerosis risk survey (AR ICS tudy) among the residents shows a strong correlation between smoking history and IMT, and smoking is stronger in patients with diabetes or hypertension. It has been shown to be a promoter [Howard G, JAMA 1998; 279: 119-24.].

Sutton—Ty rrell et al. Searched for IMT and plaque lesions in premenopausal and postmenopausal women of the same age, with an average IMT of 0.69 to 0.77 mm due to menopause and 25 to 54 for women with plaque. Report that menopause promotes atherosclerosis in women, with a significant increase to% [Sutton-Tyrrell K, Stroke 1998; 29:11] 16-21].

 The involvement of various infectious diseases is considered as a cause of arteriosclerosis. Nieto and colleagues extracted the advanced and non-developed IMT groups by an arteriosclerosis risk assessment (ARICS tudy) and searched for cytomegalovirus antibody titers. The antibody titers in patients with an antibody titer of 20 or more were determined. The odds ratio for the group of less than 4 was significantly higher at 5.3, suggesting the possibility of cytomegalovirus as a progression factor of atherosclerosis [Meto FJ, Circulation 1996; 94: 922-7].

 The authors previously performed a multiple regression analysis with the IMT of type 1 diabetes, type 2 diabetes, and borderline cases as the dependent variables, and determined the age, duration of diabetes in type 1 diabetic patients (under 30 years old). , That hemoglobin A1c value is an independent risk factor ', age, hemoglobin Ale level, non-HDL cholesterol, systolic blood pressure, and smoking history are independent risk factors for patients with type 2 diabetes (age 30 and older) In addition, in patients with borderline diabetes, they reported that systolic blood pressure and smoking were risk factors in addition to aging [Yamasaki Y, Diabetes 1994; 43: 634-639].

 Among the above environmental factors, age, gender, duration of diabetes, and hemoglobin A 1c level are particularly important. Regarding these environmental factors, the increase in carotid artery intima-media complex thickening caused by these environmental factors can be used as the arteriosclerosis risk.

 The risk determining step for determining the risk of arteriosclerosis based on the environmental factor may be plural.

The method for determining risk of arteriosclerotic disease of the present invention comprises the steps of: determining the risk of carotid intima-media in a test subject based on the risk of arteriosclerosis inherent to carotid intima-media thickening; The method may further include a risk determining step of determining an arteriosclerosis risk based on the carotid artery intima-media complex thickness from the combined thickness. The arteriosclerosis risk inherent in the carotid intima-media complex thickness can also be set, for example, as the subject's arterial-intimal media complex thickness X 1. If the carotid intima-media thickness of the test subject is directly measured, the risk or progress of atherosclerotic disease at the time of the measurement can be determined by itself. More risk due to polymorphic combinations The combination is excellent in that the risk, including the risk of future onset and the ease of progression, can be predicted. In particular, in young subjects who have not progressed to thickening at the time of measurement, predicting the future risk will enable prevention of lifestyle improvement if risk is high, and atherosclerosis It can prevent the onset of the disease.

 The risk deciding method for arteriosclerotic disease includes a risk deciding step for deciding arteriosclerosis risk due to environmental factors, and a risk deciding step for deciding arteriosclerosis risk due to carotid intima-media complex thickening. If there are a plurality of risk determination steps including the arteriosclerosis risk calculated by calculating the risk of arteriosclerosis by linearly integrating the risk of arteriosclerosis calculated for each of the risk determination steps The method may include a disease risk calculating step.

 The risk deciding method of arteriosclerosis according to the present invention is characterized in that even if a single genetic polymorphism does not significantly affect the risk, the risk is determined by combining a plurality of genetic polymorphisms. Based on the findings of the inventors that they have a synergistic effect as well as an additive effect on the atherosclerotic disease risk by combining gene polymorphisms, This enabled highly accurate judgment. The arteriosclerotic disease risk determination method is based on the relationship between the genetic polymorphism and the carotid intima-media complex thickening index, which can be quantitatively measured in healthy subjects as well as in patients with the disease. By scrutinizing, we can decide on a feasible judgment method.

 (Method of measuring arteriosclerotic disease risk)

The method for measuring the risk of arteriosclerotic disease of the present invention comprises the steps of: detecting a genotype of a plurality of genetic polymorphisms in a test subject; A risk determination step of determining an arteriosclerosis risk due to the genetic polymorphism from the genotype of the genetic polymorphism of the test subject detected in the detection step, wherein at least one of the plurality of genetic polymorphisms is included. There is no particular limitation, except that the combination has a significant association between the combination of the plurality of gene polymorphisms and the carotid intima-media complex thickness. In the detection step, any method can be used as long as the method detects the genotype of a plurality of polymorphisms in the subject. As a general method, a sample containing DNA, such as a subject's blood, sputum, skin, bronchoalveolar lavage fluid, other body fluid, or tissue, is used. Many analysis methods are known, and the following are typical ones (Clin. Cem. 43: 1114—112, 1997). The sequence method is a method in which a DNA region containing a polymorphism is directly sequenced. In the PCR method, only a certain polymorphism is specifically amplified using a primer specific to the polymorphism. In the PCR method, it is common to place the nucleic acid of the gene polymorphism at the 3 'end most. However, A1 lele Specific Primer (ASP)-Like the PCR method, the 3' terminal The ability to place the gene polymorphism in the primer, such as the method of placing the primer with the gene polymorphism in the second position, and the design of the primer, such as what kind of nucleic acid sequence to put in addition to the gene to be detected Is not particularly limited as long as a genetic polymorphism can be identified. In the TaqMan method, an allele-specific probe labeled at both ends with a fluorescent dye and a quencher is hybridized to a target site, and a PCR reaction is performed with a primer designed to widen the region including this site. When the PCR reaction from the primer reaches the region where this allele-specific probe has hybridized, the fluorescent dye present at the 5 'end of the hybridized probe is cleaved by the 5-prime nuclease activity of Taq polymerase, and the quencher is cleaved. The separation causes fluorescence. This technique shows how much the allele-specific probe has hybridized. In the Invader Atssay method, an allele probe having a specific sequence on the 5 'side from the type III polymorphism site and a flap sequence on the 3' side, and a three side gene from the type III polymorphism site Using three types of oligonucleotides, an invader probe having a specific sequence and a FRET probe containing a complementary sequence in the flap sequence, which allele probe is used in the same principle as the TadMan method It is possible to identify whether it is a hybrid soybean. In the MALD I-TOF ZMS method, after amplifying this region by creating a primer adjacent to the polymorphic site, only one base of the polymorphic site is increased using ddNTP. Width. Then, using MA LDI-TOFMS, the polymorphism is identified by identifying the type of the added ddNTP. In a method generally called a DNA chip method such as the Hybrigene method, an oligonucleotide probe containing a gene polymorphism is arranged on a microarray, and hybridization with a sample DNA subjected to PCR amplification is detected. In addition, a molecular beacon method, a ligation method and the like can be exemplified as known methods.

 As the risk determination step, the same steps as those described in the arteriosclerotic disease risk determination method can be used.

 (Method of manifesting factors related to atherosclerotic disease)

 The method for revealing atherosclerotic disease-related factors of the present invention comprises selectively elucidating genotypes of a plurality of polymorphisms related to an atherosclerosis-related gene polymorphism set among gene polymorphisms of a test subject, A manifestation step of manifesting a set of atherosclerosis-related gene polymorphisms in the subject's genetic polymorphisms,

 The arteriosclerosis-related gene polymorphism set is a combination of a plurality of gene polymorphisms having a significant positive association with the carotid intima-media complex thickness, and There is no particular limitation other than the above, and the present invention having any other additional steps is included in the present invention as long as it includes a manifestation step.

The human genome has an extremely large number of genetic polymorphisms, and only one of them has a low odds ratio and a limited frequency, so that it is impossible to predict the risk of arteriosclerosis. Therefore, it is not possible to find factors associated with arteriosclerosis that exist as combinations in individual genetic polymorphisms by looking at those genetic polymorphisms separately. Analysis of a large number of populations in the present invention revealed that there are multiple combinations of polymorphisms that have a significant positive association with carotid intima-media complex thickness Based on the above, these polymorphism-related gene polymorphism sets are positioned as arteriosclerotic disease-related factors, and gene polymorphisms related to these specific combinations in the test sample are selectively identified and integrated. For the first time, atherosclerotic disease-related factors can be revealed. The arteriosclerotic disease-related factors that have become apparent are extremely valuable as information for determining the risk of atherosclerotic disease.

 Here, selectively clarifying means selecting and clarifying a specific one of a myriad of genetic polymorphism combinations.

 In addition, as the manifestation step, in addition to simply elucidating a set of the gene types related to the combination of the plurality of gene polymorphisms, a plurality of the selectively identified gene polymorphisms can be obtained. Depending on whether the genotype corresponds to the set of atherosclerosis-related polymorphisms (for example, 0 or 1 force), the genotypes of a plurality of selectively identified polymorphisms can be determined. If it falls under the type set, it is expressed by the odds ratio of the frequency that has a significant positive association with the carotid intima-media complex thickness, which is specific to the atherosclerosis-related gene polymorphism set. In addition, when the genotypes of a plurality of selectively identified polymorphisms correspond to the atherosclerosis-related gene polymorphism set, the types specific to the atherosclerosis-related gene polymorphism set are included. Increase in arterial intima-media thickness The method is not particularly limited as long as it is a method capable of manifesting the arteriosclerosis-related gene polymorphism set in the gene polymorphism of the test subject. It preferably contains at least one atherosclerosis associated gene polymorphism set selected from among the atherosclerosis-related gene polymorphism sets described in Tables 11 to 14 above.

 An arteriosclerosis disease case is defined as a case where the thickness of the carotid artery-media media complex is at least 0.2 mm thicker than the average thickness of the intimal media-media complex of a healthy person. When defined as atherosclerotic disease cases, at least 150% of at least 150 atherosclerosis disease groupings have at least one polymorphic set of atherosclerosis-related genes, and at least 150 It is particularly preferable that the arteriosclerosis-related gene polymorphism set is selected such that the percentage of cases having at least one atherosclerosis-related gene polymorphism set in the non-atherosclerosis disease case population is 15% or less.

The atherosclerosis-related gene polymorphism set preferably comprises at least 2 to 5 gene polymorphisms, and the atherosclerosis-related gene polymorphism set has at least 3 It may be composed of individual gene polymorphisms.

 It is also preferable that the atherosclerosis-related gene polymorphism set includes at least two gene polymorphisms belonging to any of the following groups a) to 1).

a) Polymorphisms related to renin-angiotensin system

b) Genetic polymorphisms related to platelet function and coagulation system

 c) Lipid-related polymorphisms

d) Polymorphisms related to nitric oxide synthase

e) Polymorphisms related to TNF-α gene

 f) Polymorphisms related to the IRS-1 gene ''

g) Polymorphism group related to FAB P 2 gene ''

h) Polymorphisms related to muscle glycogen synthase gene

 i) Polymorphisms related to NAD P · NAD PH oxidase p22phox j) Polymorphisms related to methylenetetrahydrofolate reductase

k) Polymorphisms related to heat shock protein 70-1

 1) TGF— polymorphism group related to ^ 1 gene

 In addition, a detection step of detecting genotypes of a plurality of polymorphisms of a test subject may be further included before the revealing step.

 (Gene polymorphism detection method)

 The method for detecting a gene polymorphism according to the present invention comprises the at least one atherosclerosis-related gene polymorphism set selected from the set of arteriosclerosis-related gene polymorphisms listed in Tables 1-1 to 14 above. There is no particular limitation as long as it includes the step of detecting the genotype of the test subject with respect to the genetic polymorphisms constituting, and the detection results are used for determining the risk of atherosclerotic disease.

 (Gene marker)

The gene marker of the present invention is a gene that constitutes at least one atherosclerosis-associated gene polymorphism set selected from among the atherosclerosis-related gene polymorphism sets described in Tables 11 to 14 above. There is no particular limitation as long as it contains a polymorphism. In this embodiment, the combination of the gene polymorphisms is used as various markers of atherosclerotic disease. (Gene polymorphism analysis kit)

 The gene polymorphism analysis kit of the present invention comprises at least one arteriosclerosis-related gene polymorphism set selected from the arteriosclerosis-related gene polymorphism sets described in Tables 11 to 14 above. A primer pair capable of specifically amplifying the constituent gene or a nucleic acid probe capable of specifically hybridizing to the gene; Any type that detects a set may be used.

In order to detect at least one atherosclerosis-related gene polymorphism set selected from among the atherosclerosis-related gene polymorphism sets described in Tables 11 to 14, the gene polymorphism set is used. Any of the primers and probes for detecting at least two gene polymorphisms selected from the gene polymorphisms listed in Table 2-1 (Table 2-1). It is necessary to have Even if it contains a gene polymorphism detection primer for one gene polymorphism and a gene polymorphism detection probe for another gene polymorphism, as long as the plurality of gene polymorphisms can be analyzed, It is included in the gene polymorphism analysis kit of the present invention. For detection of each gene polymorphism described in Table 2-1 to Table 2-2, any of the methods described in the above-mentioned gene polymorphism detection step can be used, but the hybrigene method using PCR and the TaqMan method Invader method, ASP-PCR method using a nucleic acid probe that specifically hybridizes to a gene having a genetic polymorphism, and the like can be preferably used. Therefore, the gene polymorphism analysis kit needs to include at least one of a primer and a probe used in the step of detecting these gene polymorphisms. In the PCR method for detecting gene polymorphisms, it is common to place the nucleic acid of the gene polymorphism at the 3'-most side, but Allele Specific Primer (AS P) ~ PCR method The ability to place a gene polymorphism in any region of the primer, such as a method of arranging a primer having a gene polymorphism at the second position from the 3 'end, as well as any nucleic acid other than the gene to be detected There are no particular restrictions on the design of the primers, including the sequence, as long as the polymorphism can be identified. Similarly, in the design of a probe, as long as a genetic polymorphism can be identified, It can be used without limitation.

 The set of atherosclerosis-related gene polymorphisms detected by the gene polymorphism analysis kit may be any of those described in Tables 1-1 to 1-4 above. Groups containing genetic polymorphisms are preferred because they increase the sensitivity of risk detection. Furthermore, it has a primer or probe for detecting at least two gene polymorphisms selected from the gene polymorphisms listed in Tables 2-1 to 2-2, and comprises a cervical artery intima-media. When the thickness of the complex is more than 2 mm thicker than the average thickness of the carotid intima-media complex of a healthy person, it is defined as an arteriosclerosis disease case, and the other cases are defined as non-atherosclerosis disease cases In at least 150 cases of arteriosclerosis disease cases, the carotid artery in the arteriosclerosis-related gene polymorphism set according to Tables 11 to 14 described above, which can be constituted by the selected gene polymorphisms. 3 More than 30% had a combination of genotypes that had a significant positive association with the medial media complex thickness, and in at least 150 non- atherosclerotic disease populations, % To detect atherosclerosis-related gene polymorphism sets DOO, at risk prediction, preferred.

 Further, the carotid intima-media complex has a primer or probe for detecting a polymorphism of at least two polymorphisms selected from the polymorphisms listed in Tables 2-1 to 2-2. A case where the thickness is 0.2 mm or more thicker than the average thickness of the intimal-media intimal-media complex of a healthy person is defined as an arteriosclerosis disease case, and the other cases are defined as non-atherosclerosis disease cases. In at least 150 cases of arteriosclerosis disease case population, the atherosclerosis-related gene polymorphism set described in Table 11 above, which can be constituted by the selected gene polymorphism, More than 50% of patients had at least one combination of genotypes that had a significant positive association with carotid intima-media thickness, and at least 150 non-atherosclerotic patients Assay kits that are less than 15% in the population are dangerous More preferably in the prediction.

It is also preferable that in the analysis kit, a plurality of gene polymorphism detection primers or probes belong to any of the following different groups a) to 1). a) Polymorphisms related to renin and angiotensin system

b) Genetic polymorphisms related to platelet function and coagulation system

c) Polymorphisms related to lipids

d) Polymorphisms related to nitric oxide synthase

e) Polymorphisms related to TNF-α gene

f) Polymorphisms related to IRS-1 gene

g) Polymorphism group related to FABP2 gene

h) Polymorphisms related to muscle glycogen synthase gene

 i) NAD P · NADPH oxidase! ) 22 pho x related polymorphisms j) methylene tetrahydrofolate reductase related polymorphisms

k) Polymorphisms related to heat shock protein 70-1

 1) Polymorphisms related to TGF-131 gene

 (Microarray for risk assessment of atherosclerotic disease)

 The microarray for risk determination of arteriosclerotic disease of the present invention comprises at least one arteriosclerosis-related gene selected from the set of atherosclerosis-associated gene polymorphisms described in Tables 11 to 14 above. Except for having a probe for detecting a gene polymorphism of a gene polymorphism constituting the gene polymorphism set, the material can be appropriately selected from known materials as long as the effects of the present invention are not impaired.

The microarray for risk assessment of atherosclerotic disease is known from a method in which a probe prepared in advance is fixed on a base, and a method by Afmetrix which is synthesized on a substrate. May be used. The substrate on which the probe is fixed is not particularly limited, but a known plate such as a glass plate or a filter can be used. The length of the probe to be immobilized and the type of nucleic acid used are not particularly limited as long as the gene polymorphism can be detected. It is desirable from the viewpoint of sensitivity that the region containing the gene polymorphism is amplified by PCR in advance. In particular, a method for amplifying a region containing a gene polymorphism using a labeled primer can be suitably used in terms of sensitivity, simplicity, and the like. For example, in the case of the Hy brigene method, a gene labeled with biotin is used to contain gene polymorphisms. After amplifying the region, adding it to the microarray and allowing it to hybridize, the nucleic acid that has not hybridized is washed away. The hybridized probe is then detected with an avidin-labeled fluorescent dye. By this method, gene polymorphism can be detected with high sensitivity.

 In the microarray for determining risk of atherosclerotic disease, it is also preferable that the probe for detecting a gene polymorphism is a probe for detecting a gene polymorphism belonging to any of the following different groups a) to 1).

a) Polymorphisms related to the renin-angiotensin system

b) Genetic polymorphisms related to platelet function and coagulation system

c) Lipid-related polymorphisms

d) polymorphisms related to nitric oxide synthase

e) Polymorphisms related to TNF-α gene

 f) Polymorphisms related to IRS-1 gene

g) Polymorphism group related to FAB P2 gene

 ) Gene polymorphisms related to muscle glycogen synthase gene

 i) Polymorphisms related to NADP · NADPH oxidase p22p ox ί) Polymorphisms related to methylenetetrahydrofolate reductase (MTHFR) k) Polymorphisms related to heat shock protein 70-1

 1) TGF—; polymorphism group related to 31 genes

 (Arteriosclerotic disease risk assessment device)

The arteriosclerotic disease risk judging device of the present invention is a computer-based arteriosclerotic disease risk judging device, wherein a combination of a plurality of gene polymorphisms and an arteriosclerotic risk are associated. It has a list of atherosclerosis risk data tables, and is a combination of a plurality of genetic polymorphisms of a subject to be input and a combination of a plurality of polymorphisms in the atherosclerosis risk data table. There is no particular limitation other than having a detection means for detecting the risk level of arteriosclerosis corresponding to the combination of the gene polymorphisms when there is a matching and matching gene polymorphism combination. The detected carotid artery risk can be used as it is as an arteriosclerotic disease risk, or a simple numerical value as appropriate. The arteriosclerotic disease risk can also be used instead.

 The plurality of gene polymorphisms in the atherosclerosis risk data table has a significant association between at least one combination of the plurality of gene polymorphisms and the carotid intima-media complex thickness It is preferred that there are multiple gene polymorphisms.

 In addition, an atherosclerosis risk data table listing the combination of multiple gene polymorphisms and the risk of atherosclerosis is significant, and shows a significant positive correlation between carotid intima-media thickening. Atherosclerosis risk may be assigned to one unit for a combination of multiple related gene polymorphisms.

 In addition, an atherosclerosis risk data table listing the combinations of multiple genetic polymorphisms and the risk of atherosclerosis correspondingly shows a significant difference between the carotid intima-media thickness and carotid intima-media thickness. A combination of a plurality of polymorphisms that have a positive association and a risk ratio of carotid intima-media thickening exceeding the normal range as the risk of atherosclerosis may be associated with the combination. .

 The combination of polymorphisms that have a significant positive association with the carotid intima-media complex thickness indicates that the carotid intima-media complex thickness exceeds the normal range. It can be suitably defined by the fact that the odds ratio is not less than a certain value and that there is a significant difference in the average value of the carotid intima-media complex thickness.

 Further, the arteriosclerotic disease risk judging device of the present invention performs more accurate arteriosclerotic disease risk judgment by inputting information on environmental factors which are risk factors. An arterial sclerosis risk data table in which the arteriosclerosis risk is listed in correspondence,

 The inputted or nonexistent or numerical value of the environmental factor of the test subject is compared with the presence or numerical value of the environmental factor in the atherosclerosis risk data table, and the atherosclerotic risk corresponding to the presence or numerical value of the environmental factor is determined. It may further have a detecting means for detecting.

When the arteriosclerotic disease risk determination device has a plurality of detecting means, the arteriosclerotic disease risk may be determined based on an added value of the plurality of carotid artery risks detected from the plurality of detecting means. it can. Further, the arteriosclerotic disease risk determination device of the present invention includes an arteriosclerosis risk data table in which carotid intima-media thickening and arteriosclerosis risk are listed correspondingly.

 The carotid intima-media complex is compared with the carotid intima-media thickness of the test subject and the carotid intima-media thickness in the atherosclerosis risk data table. A detecting means for detecting the arteriosclerosis risk corresponding to the degree of hypertrophy may be further provided.

 In addition, the arteriosclerotic disease risk determining apparatus of the present invention may further comprise: a carotid artery risk detected by the detecting unit and a carotid artery risk extracted from the plurality of extracting units. When expressed as an increase in membrane complex thickness, the entered carotid intima-media thickness of the subject is used as it is, and the risk of atherosclerotic disease is calculated based on the added value. The degree can also be determined.

 Further, the arteriosclerotic disease risk determining apparatus of the present invention measures a carotid intima-media complex thickness of a test subject and supplies the carotid intima-media complex thickness to the computer. It may further include a film pressure measuring means. As the vascular membrane pressure measuring means, a vascular membrane pressure measuring device different from a computer, a device combining a vascular membrane pressure measuring device with a computer for analysis, and the like are used. The vascular membrane pressure measuring device can be appropriately selected from known vascular membrane pressure measuring devices according to the purpose. When the vascular membrane pressure measuring means includes a computer, the vascular membrane pressure measuring means The computer and the computer for storing the data table and detecting the degree of risk may be integrated.

 (Arteriosclerotic disease risk assessment program)

The arteriosclerosis disease risk determination program of the present invention stores an arteriosclerosis risk data table in which a combination of a plurality of gene polymorphisms and an arteriosclerosis risk are listed in a computer, and The computer compares the combination of the plurality of polymorphisms of the subject to be inputted with the combination of the plurality of polymorphisms in the atherosclerosis risk data table, and finds a matching combination of the polymorphisms. In some cases, the detection means for detecting the arteriosclerosis risk corresponding to the combination of the gene polymorphisms is used. It is characterized by functioning with.

 The arteriosclerosis risk data table in which the combination of a plurality of gene polymorphisms and the arteriosclerosis risk are listed correspondingly has the same function as that described in the arteriosclerosis disease risk determination device. Ones having.

 A case in which the carotid intima-media thickness is at least 0.2 mm thicker than the average carotid / media thickness in healthy volunteers is defined as an arteriosclerosis disease case, and the rest are non-moving. When defined as pulse sclerosis disease cases, at least 30%, preferably 50% or more, more preferably 60 or more, of at least one unit of arteriosclerosis risk in at least 150 cases of arteriosclerosis disease population In at least 15.0 non-atherosclerotic disease group, at least 15% of patients have at least 1 unit of atherosclerotic risk. Very useful from.

 Example

 Hereinafter, examples of the present invention will be described, but the present invention is not limited to these examples.

 (Example 1)

Relationship between genetic polymorphism and IMT

 The carotid intima-media complex thickness was measured in 200 healthy subjects and 200 type I diabetic patients, and the mean IMT and peak IMT (PIMT) were obtained in each subject. On the other hand, DNA was extracted from blood collected from the healthy subjects and diabetic patients by the phenol-chloroform method. The obtained DNA was designated as type 、, and ACE, AGT, SERINE1, APOE, APOB, PON1 shown in Table 3 were obtained using the known ASP-PCR method and primers specific to the polymorphism. , LOC1 13690, MTHFR IRS1, and FABP2 were amplified. Each gene polymorphism was identified by confirming the presence or absence of the PCR product by agarose electrophoresis.

The measurement of carotid intima-media thickening is performed by measuring three or more points in two consecutive directions using a high-resolution ultrasonic tomograph, and averaging the average of the measured values of one subject T, the maximum value of the measured values was taken as the peak IMP. From this data, ΔIMT and ΔΡIMT specific to the genotype having a homologous risk factor for the single gene polymorphism were calculated by a linear multiple regression analysis technique. Here, ΔΙΜΤ represents the increment of the average thickness of the intimal media complex of the arterial artery, and ΔΡIMT represents the fraction of the maximum thickness of the intima-media complex of the carotid artery. The results are shown in Table 5.

[Table 5]

The units of PIMMT and ΔIMT are millimeters (mm).

 In the table, “*” indicates a significant association with IMT (P value less than 0.05). Also

The “number” is assigned to represent the polymorphism of the same number in Tables 3 and 4.

 As a result, it was recognized that a single gene polymorphism was significantly involved in ΔIMT addition for MTHFR and p22phoX.

 Next, with respect to two gene polymorphisms of PON1 and MTHFR, or two gene polymorphisms of PON1 and SERPINE1, a ΔΙΜΤ unique to a combination of genotypes having homologous risk factors was calculated.

 ΔΙΜΤ in the case of having a genotype having a homologous risk factor for both PON1 and MTHFR was * 0.30 lmm. In addition, when both PON1 and SERP INE1 had a genotype having a homologous risk factor, ΔIMT was 0.16 mm.

 As a result, when multiple gene polymorphisms such as PON 1 and MTHFR involved in different functions occur in combination, they may be more involved in ΔIMT than in the case of a single gene polymorphism. It was recognized that it was possible to set a more accurate risk based on this. Also, in the case of the combination of PON 1 and SERPINE 1, the ΔIMT was larger than that of the single gene polymorphism, but did not increase to a significant difference. This combination has a small increase in ΔIMT as compared to the combination of Example 2 described below, etc., because the gene polymorphism belongs to different polymorphism groups in Table 3. However, both are polymorphisms that are closely related to arteriosclerosis, and are considered to be largely in the same category.

 (Example 2)

 Relationship between the combination of three gene polymorphisms and IMT, and a method for determining the risk of atherosclerotic disease

Next, in 200 healthy subjects and 200 type II diabetics, three polymorphisms were similarly detected and IMT was measured, and multiple regression analysis was used to determine the uniqueness of the three genotype combinations. Δ IMT was determined. ! ^ Ding! ! Is a related factor! ^ Ding! ! Gene polymorphisms, ACE polymorphisms with ACE as the relevant factor, and SERP I NE1 polymorphisms with PA I-1 as the relevant factor are selected as a combination of multiple polymorphisms. The genotypes were classified into genotypes containing homologous risk factors (+) and other genotypes (1). Then, the classifications of the three different genotype polymorphisms were combined to calculate ΔIMT unique to the combination of the genotype polymorphisms, and the calculated ΔIMT was defined as the arteriosclerosis risk. The results are shown in Table 6.

[Table 6]

That is, for each of the polymorphisms of MTHFR, ACE, and SERPINE1, the risk of arteriosclerosis inherent to a combination of genotypes containing homologous risk factors was set to 0.771. For both MTHFR and ACE polymorphisms, genotypes that include a risk factor in a homozygous form, and for SERPINE 1, the risk of arteriosclerosis inherent to a combination of genotypes that do not include a risk factor in a homozygous form. Set to 0.451. Similarly, for both the MTHFR and SERP I NE1 polymorphisms, the gene type is a homologous type containing a risk factor, and the ACE is unique to a combination of genotypes not including the risk factor in a homozygous type. The arteriosclerosis risk was set at 0.318. Similarly, ACE and SERP For both polymorphisms with INE1, a genotype that contains a risk factor homozygously, and for MTH FR, the arteriosclerosis risk specific to a combination of genotypes that do not contain a risk factor homozygous is 0.127 Was set. In addition, the genotype-specific arteriosclerosis risk that is homozygous for only one of MTHFR, ACE and S ERP I NE1 is 0.089, 0.018, respectively. It was set to 0.145. Finally, the risk of atherosclerosis, which is unique to the combination of genotypes in which none of the three polymorphisms contains a homologous risk factor, was set to zero. The subject's risk of arteriosclerosis was determined by applying the genotype of the subject's ACE, SERPINE1, and MTH FR polymorphisms to the preset risk of arteriosclerosis. That is, if all of the three polymorphisms in the test subject are genotypes containing homologous risk factors, the risk of arterial sclerosis in the test subject is determined to be 0.771. When there is one risk determination step, this value can be directly used as the risk of arteriosclerotic disease of the subject.

 From these results, in the arteriosclerotic disease risk determination method and the like of the present invention, these ΔIMT values can be preset as an arteriosclerosis risk unique to a combination of a plurality of gene polymorphisms, The risk of arterial sclerosis can be determined from the genotypes of the plurality of genetic polymorphisms of the test subject, and the risk of arteriosclerotic disease can be determined with high accuracy.

 (Example 3)

Relationship between risk factors other than gene polymorphisms and IMT

 Age, gender, duration of diabetes, and hemoglobin A Ic value were analyzed by multiple regression analysis for 200 healthy subjects and 200 type I diabetes patients, respectively, with respect to ΔΙΜΤ. The obtained partial regression coefficients are shown below.

Environmental factor Partial regression coefficient

Age + 0.01 5 mm / y r

Male + 0.178 mm

Diabetes duration + 0.006 mm / y r

Hemoglobin A 1 c value + 0.024 mm /%

Each of these environmental factors independently becomes a risk factor, and the arteriosclerotic disease of the present invention The method for determining risk of disease can further include a risk determination step based on these factors.

 For example, if the environmental factor includes a risk determination step based on age and gender, if the subject to be judged is 30 years old, the arteriosclerosis risk specific to age is age X 0.015, Judgment The carotid effect risk according to the age of the test subject is 0.45. If the test subject is a male, the gender-specific risk of arteriosclerosis among environmental factors is 0.178 (0 for females).

 The subject to be determined is a genotype that includes a risk factor homozygous for both the polymorphisms of MTHFR and S ERP IE1 for the three gene polymorphisms of Example 2, and a homologous risk factor for ACE. If the genotype is not included in the combination, the risk of arteriosclerosis inherent to this combination is 0.318 (Table 6). Desired.

0.3 1 8 + 0.45 + 0.17 8 = 0.94 6

 (Example 4)

 Next, based on the above results, in a larger population, we examined various combinations of a wide range of known gene polymorphisms that are presumed to be related to arteriosclerosis, etc. The decision was made and the possibility of predicting disease onset was examined.

 The polymorphisms searched were 57 gene polymorphisms, excluding those with a polymorphism frequency of 1% or less, and finally 49 gene polymorphisms, and 4 7 polymorphisms listed in Tables 2-1 to 2-2. Genes were searched.

These gene polymorphisms were grouped according to the causes of atherosclerosis as shown in (A) to (G) below, and they were classified as shown in Tables 7-1 to 7-2. Una breakdown.

Insulin resistance · Five types related to vascular endothelial function (A)

Inflammatory response 9 species (B)

4 adhesion factors (C)

8 types of sympathetic blood pressure (D)

1 1 type (E) Fat 8 types (F) Other 4 types (G)

89

89CT0 / C0ai / X3d 09 80 / £ 0 OAV [Table 7-2]

However, the reference names corresponding to the reference numbers in Table 2-1 to Table 2-2 and Table 7-1 are shown below.

 1. Yoshimura M, Nakayama M, Shimasaki Y, Oga a H, Kugiyama K, Nakamura S, Ito T, Mizuno Y, Harada E, Yasue H, Miyamoto Y, Saito Y, Nakao K. AT— 786-> C mutation in the 5 '-flanking region of the endothelial nitric oxide synthase gene and coronary arterial vasomotility.Am J Cardiol. 2000 85 (6): 710-4

 2. Shimomura H, Sanke T, Ueda K, Hanabusa T, Sakagashira S, Nan jo K. A missense mutation of the muscle glycogen synthase gene (M416V) is associated itn insulin resistance in the Japanese population. Diabetologia. 40 (8): 947-52 ·

 3. Renner W, Schallmoser K, Gall ippi P, rauss C, Toplak H, Wascher TC, Pilger E. C242T polymorphism of the p22 phox gene is not associated with peripheral arterial occlusive disease. Atherosclerosis. 152 (1): 175-9 .

 4. Rosmond R, Chagnon M, Bouchard C, Bjorntorp P. Increased Abdominal Obesity, Insulin and Glucose Levels in Nondiabetic Subjects with a T29C Polymorphism of the Transforming Growth Fact or beta (1) Gene. Horm Res. 59 (4): 191 -Four.

 5. Blankenberg S, Rupprecht HJ, Poirier 0, Bickel C, Smieja M, Hafner G, Meyer JT, Cambien F, riret L. Plasma concentrations and genetic variation of matrix metalloproteinase 9 and prognosis of patients with cardiovascular disease. Circulation. 107 ( 12): 1579-85.

6. Hofmann MA, Drury S, Hudson BI, Gleason MR, Qu W, Lu Y, Lalla E _? Chitnis S, Monteiro J, Stickland MH, Bucciarelli LG, Moser B, Moxley G, Itescu S, Grant PJ _S Gregersen PK, Stern DM, Schmidt AM.RAGE and arthritis: the G82S polymorphism amplifies the inflammatory response.Genes Immun. 3 (3): 123--35.

7. Omori K, Kazama JJ, Song J, Goto S, Takada T, Saito N, Sakatsume M, Narita I, Gejyo F. Association of the MCP-1 gene polymorphism A— 2518G with carpal-tunnel syndrome in hemodialysis patients.Amyloid. 9 (3): 175-82.

Cascorbi I, Henning S, Brockmoller J, Gephart J, Meisel C, Muller JM, Loddenkemper R, Roots I. Substantially reduced risk of cancer of the aerodigestive tract in subjects with variants—463A of the myeloperoxidase gene. Cancer Res. (3 ): 644— 9.

Rauchhaus M, Gross M, Schulz S, Francis DP, Greiser P, Norig A ₃ Weidhase L, Coats AJ, Dietz R, Anker SD, Glaser C. The E— selectin SER128ARG gene polymorphism and restenosis after successful coronary angioplasty. Int J Cardiol. 83 (3): 249- 57.

Reinhardt D, Sigusch HH, Vogt SF, Zeiss C, Farker, Hoffmann A, Muller S. A common variant of the angiotensinogen gene and the risk of coronary artery disease in a German population.Pharmazie. 55 (1): 69 71.

Kroll H, Gardemann A, Fechter A, Haberbosch W, Santoso S. The impact of the glycoprotein la collagen receptor subunit A1648G gene polymorphism on coronary artery disease and acute myocardial infarction. Thromb Haemost. 83 (3): 392-6.

von Beckerath N, Koch W, Mehilli J, Bottiger C, Schomig A, Kastrati A. Glycoprotein la gene C807T polymorphism and risk for major adverse cardiac events within the first 30 days after coronary-artery stenting. Blood. 95 (11): 3297 -301.

Park HY, Nabika T, Jang Y, Kwon HM, Cho SY, Masuda J. Association of G—33A polymorphism in the thrombomodulin gene with myocardial infarction in Koreans.Hypertens Res. 25 (3): 389-94.

Blake GJ, Schmitz C, Lindpaintner K, Ridker PM. Mutation in the promoter region of the beta-fibrinogen gene and the risk of future myocardial infarction, stroke and venous thrombosis. Eur Heart J. 22 (24): 2262-6.

Jeng JR. Association of PAI-1 gene promoter 4g / 5g polymorphism with plasma PAI-1 activity in Chinese patents with and without hypertension. Am J Hypertens. 16 (4): 290-6.

Cenarro A, Artieda M, Castillo S, Mozas P, Reyes G _s Tejedor D, Alonso R, Mat a P, Pocovi M, Civeira F. A common variant in the ABCAl gene is associated with a lower risk for premature coronary heart disease in J Med Genet. 40 (3): 163-8. Jans en H, Chu G, Ehnholm C, Dallongeville JT, Nicaud V, Talmud PJ, The T allele of the hepatic lipase promoter variant C— 480T is associated wi th increased fasting lipids and HDL and increased preprandial and postprandial LpCIII: B: European Atherosclerosis Research Study (EARS)] Arterioscler Thromb Vase Biol. 19 (2): 303- 8.

Yamada Y, Ando F, Niino N, Ohta S, Shimokata H, Association of polymorphisms of the estrogen receptor alpha gene with bone mineral density of the femoral neck in elderly Japanese women.J Mol Med. 80 (7): 452-60 .

Bjork JM, Moeller FG, Dougherty DM, Sw recitation AC, Machado MA, Hanis CL. Serotonin 2a receptor T102C polymorphism and impaired impulse control.Am J Med Genet. 114 (3): 336-9.

Jormsjo S, What ling C _? Walter DH, Zeiher AM, Hams ten A, Eriksson P. Allele-specific regulation of matrix meta 丄 丄 oproteinase— 7 promoter activity is associated with coronary artery luminal dimensions among hypercholesterolemic patients.Arterioscler Thromb Vase Biol . 21 (11): 1834-9.

Yeh HI, Chou Y, Liu HF, Chang SC, Tsai CH. Connexin37 gene polymorphism and coronary artery disease in Taiwan.Xnt J Cardiol. 81 (2-3): 251-5. Zheng F, Chevalier JA, Zhang LQ, Virgil D, Ye SQ, Kwiterovich PO.An Hphl polymorphism in the E-select in gene is associated with premature coronary artery disease.Clin Genet. 59 (1): 58-64

fang AY, Chan JC, Wang M, Poon E, Lui SF, Li PK, Sanderson J. Cardiac hypertrophy and remodeling in relation to ACE and angiotensinogen genes genotypes in Chinese dialysis patients. Kidney Int. 63 (5): 1899-907.

Pihusch R, Buchholz T, Lohse P, Rubs amen H, Rogenhofer N, Hasbargen U, Hiller E, Thaler and J. Thrombophilic gene mutations and recurrent spontaneous abortion: prothrombin mutation increases the risk in the first trimester. Am J Reprod Immunol. 46 (2): 124 31.

Krikovszky D, Vasarhelyi B, Toth-He n P, Korner A, Tulassay T, Madacsy L. Association between G-308A polymorphism of the turaor necrosis factor-alpha gene and 24 ~ hor ambulatory blood pressure values in type 1 diabetic adolescents. Clin Genet. 62 (6): 474— 7.

Cao H, Hegele RA. Human C-reactive protein (CRP) 1059G / C polymorphism. J Hum Genet. 45 (2): 100-1.

Okumura K, Matsui H, Ogawa Y, Takahashi R, Matsubara K, Imai H, Imamura A, Mizuno T, Tsuzuki M, Kitamura Y. The polymorphism of the be "ta3-adrenergic receptor gene is associated with reduced low-density lipoprotein particle size. Metabolism. 52 (3): 356-61.

Simsek S, Bleeker PM, van der Schoot CE, von dem Borne AE. Association of a variable number of tandem repeats (VNTR) in glycoprotein lb alpha and HP A-2 alloantigens. Thromb Haemost. 72 (5): 757-61.

Carter AM, Catto AJ, ohler HP, Ariens RA, Stickland MH, Grant PJ. Alpha-fibrinogen Thr312Ala polymorphism and venous thromboembolism. Blood. 96 (3): 1177— 9.

Huber G, 'Marz W, Martin JR, Malherbe P, Richards JG, Sueoka N, Ohm T, Hoffmann MM. Characterization of transgenic mice expressing apolipoprotein E4 (C112R) and apolipoprotein E4 (L28P; C112R). Neuroscience. 101 (1): 211-8.

 31. Couture P, Otvos JD, Cupples LA, Wilson PW, Schaefer EJ, Ordovas J.

 Absence of association between genetic variation in the promoter of the microsomal triglyceride transfer protein gene and plasma lipoproteins in the Framingham Offspring Study.Atherosclerosis.148 (2): 337-43.

 32. Nakamura S, Kugiyama K, Sugiyama S, Miyamoto S, Koide S, Fukushima H, Honda 0, Yoshimura M, Ogawa H. Polymorphism in the 5 '-flanking region of human glutamate-cysteine ligase modifier subunit gene is associated with myocardial. infarction. Circulation. 25; 105 (25): 2968-73. The gene sequences corresponding to the sequence numbers shown in Table 2-1 to Table 2-2 and Table 7-1 to Table 7-2 are shown in FIG. And as shown in 2.

 In a population of diabetics with no history of myocardial infarction, a case-control study was performed on about 433 patients with early arteriosclerosis and about 195 patients without early arteriosclerosis as controls. The search for the population without a history of myocardial infarction was based on the fact that, in the population with a history of myocardial infarction, the detection of genes associated with myocardial infarction, which is related to myocardial infarction, led to a gene polymorphism specific to atherosclerosis. This is to prevent the sensitivity of the mold set from deteriorating. This provided a polymorphic set of atherosclerosis-related genes that is specific to atherosclerosis and can predict the risk of atherosclerosis at a high level.

When the IMT measured by the same measurement method as in Example 1 was 0 • 2 mm or more (SD-0.1) thicker than the average IMT of healthy subjects, it was classified as a group having early arteriosclerosis. Atherosclerosis-related gene polymorphism sets were determined by setting the significance level to an odds ratio of 10 or more and a chi-square value of 6.635 (P-0.01) or more (Table 8-1 to Table 8-4). π / ο ー -iP8 OAV-

[Table 8-3]

 Mouth mouth mouth

 Descendants "^ 5" Classification Classification Classification Frequency Odd ratio Kai square value

I T 5 1 20 3 23 3 31 3 0.038 99 7.25

IMT 5 1 20 3 37 2 1 3 39 1 1 2 0.063 12.5 9.92

IMT 5 1 12 1—2 25 1 44 1 0.037 99 6.68

IMT 5 1 25 1 39 1_2 45 1 0.035 99 6.65

IMT 6 1—2 1 1 3 37 2 1 3 39 1_2 0.038 99 6.99

IMT 6 1_2 1 1 3 39 1—2 43 3 0.043 99 7.97

IMT 6 1 15 1 17 3 25 1 0.037 99 6.73

IMT 6 1 17 3 33 1_2 40 1—2 0.06 1 1.5 8.97

IMT 6 1 1 1 3 20 1 42 1_2 0.043 99 7.95

IMT 6 1 20 1 23 3 40 1_2 0.036 99 6.77

IMT 6 2 1 3 9 1 22 2 1 3 25 1 0.039 99 7.08

IMT 6 2 1 3 22 2—3 25 1 42 1_2 0.042 99 7.65

IMT 6 2—3 20 3 25 1 46 1_2 0.052 99 9.83

IMT 7 3 9 1—2 20 3 38 3 0.054 99 10.03

IMT 7 3 13 3 20 3 38 3 0.05 99 9.47

IMT 7 3 14 3 20 3 38 3 0.053 99 10.09

IMT 7 3 20 3 22 1 1 2 38 3 0.053 99 9.45

IMT 7 3 20 3 23 3 38 3 0.046 99 8.55

IMT 7 3 20 3 27 3 38 3 0.055 99 10.35

IMT 7 3 20 3 29 1 38 3 0.042 99 7.57

IMT 7 3 20 3 30 1 38 3 0.053 99 10.07

IMT 7 3 20 3 33 1_2 40 1_2 0.062 99 12.1 1

IMT 7 3 20 3 35 1 38 3 0.046 99 8.58

IMT 7 3 20 3 38 3 49 1 1 2 0.053 99 10.04

IMT file 2_3 17 2 1 3 33 1 40 1_2 0.036 99 6.68

IMT 9 1 12 1—2 34 3 36 1 1 2 0.042 99 7.44

IMT 9 1 20 3 31 3 36 1—2 0.048 99 9.13

IMT 9 1 31 3 33 1 1 2 40 1_2 0.045 99 8.61

IMT 9 1 12 1—2 24 1 34 3 0.039 99 6.93

IMT 9 1 2 2 25 1 .39 1 1 2 45 1 0.04 99 7.56

IMT θ 1 1 2 25 1 39 1 1 2 46 1 1 2 0.038 99 7.21

IMT 3 16 1 31 2_3 39 1_2 0.038 99 7.29

IMT 3 17 1−2 31 2 1 3 39 1_2 0.042 99 7.78

IMT 3 18 1 31 2—3 39 1_2 0.036 99 6.73

IMT 3 22 1_2 31 2 1 3 39 1 1 2 0.045 99 8.1

IMT 11 3 23 2 1 3 31 2 1 3 39 1 1 2 0.039 99 7.26

IMT 3 31 2 1 3 36 1_2 39 1_2 0.041 99 7.66

IMT 3 39 1_2 42 1_2 43 3 0.045 99 8.42

IMT 3 39 1_2 43 3 47 1 1 2 0.042 99 7.87

IMT 12 1 1 2 20 3 23 3 36 1_2 0.048 99 8.76

IMT 12 1−2 23 3 32 1_2 41 1 0.039 99 7.05

IMT 12 1_2 40 1_2 42 1 47 1_2 0.041 99 7.63

IMT 12 1_2 25 1 34 1_2 44 1 0.046 99 8.16

IMT 13 3 25 1 1 2 38 3 39 1 2 0.041 99 7.64

Here, the odds ratio (Odd) is an index of how likely the corresponding event is to occur in comparison to the control group, and an odds ratio of 2 indicates that, for example, arteriosclerosis is twice as likely. An odds ratio of 99 indicates that no event occurred in the control group. The chi-square value (Ka 1) is an index that indicates the significant difference in occurrence of the event. A value of 6.635 or more is equivalent to <0.01.

 In the analysis of only one polymorphism (49 x 4 = 196 genotypes), the 49 polymorphisms that could explain early arteriosclerosis (Odd> 10 and Kai> 6.635) Did not exist. Here, as a method for classifying genotypes of the polymorphisms, for example, when MPO (G463A) is described, the homozygous (GG) of the polymorphism on the left side is defined as genotype 1, and the heterozygous (GA) ) As genotype 2 and the homozygous (AA) of the polymorphism on the right as genotype 3, and analyzed four classifications: genotype 1, genotypes 1 and 2, genotypes 2 and 3, and genotype 3. However, when combined with the genotypes of other polymorphisms, the classification that produces a minimum positive significance was adopted. For example, if both genotype 1 and genotypes 1 and 2 show significance, the more significant one was adopted.

 When a combination of two polymorphisms was searched (49 x 48Z2 x 4 x 4 = 17186 polymorphism set), O dd> 10 and Ka i> 6. 635 could explain the association with early atherosclerosis One set of atherosclerosis-related gene polymorphisms was extracted, and 14 out of 437 early arteriosclerosis cases were explained by this set. On the other hand, none of the 1.95 non-arteriosclerotic cases showed this polymorphism set. The combination of the groups was ag.

When three gene polymorphism combinations were searched (49 x 48 x 47/6 x 4 x 4 x 4 <500,000 gene polymorphism sets), O dd> 10 could explain the association with early atherosclerosis In addition, 72 atherosclerosis-related gene polymorphism sets with Kai> 6.635 were extracted, and 233 out of 437 early arteriosclerosis cases were explained by this polymorphism set. On the other hand, only 14 out of 95 non-early arteriosclerosis cases showed this polymorphism set. Note that those containing the gene polymorphism set already extracted by the combination of two gene polymorphisms were not extracted by the combination of three gene polymorphisms. Ma In addition, among the combinations of each group, 21 out of the 34 combinations incorporated only the gene polymorphisms of each of the above groups (A) to (G).

 When four gene polymorphism combinations were searched (49 x 48 x 47 x 46Z24 x 4 x 4 x 4 x 4 and 15,000,000 polymorphism sets), it could explain early arteriosclerosis (Od A set of 103 atherosclerosis-related gene polymorphisms with d> 10 and Kai> 6.6 3 5) was extracted, and 283 out of 43 early arteriosclerosis cases were described. On the other hand, only 19 out of 95 non-early arteriosclerosis cases showed this polymorphism set. In addition, those containing the gene polymorphism set already extracted by the combination of 2 and 3 gene polymorphisms were not extracted by the combination of 4 gene polymorphisms. In addition, among the combinations of each group, 200 out of 706 combinations incorporated only the 1 gene polymorphism of each group from (A) to (G).

 FIG. 3 shows the percentage of early arteriosclerosis cases that can be explained by the atherosclerosis-related gene polymorphism set and the percentage that non-early atherosclerosis cases have the atherosclerosis-related gene polymorphism set and cause no symptoms. Looking at Figure 3, the number of complex gene polymorphisms can reach equilibrium with up to 5 sets. Therefore, the required number of polymorphism combinations of 3-5 is sufficient. ADVANTAGE OF THE INVENTION According to the present invention, the onset of arteriosclerosis, the likelihood of progression, etc., can be accurately determined as the risk of arteriosclerosis, and the risk of arteriosclerosis can be used for prevention and treatment of atherosclerosis Method, arteriosclerotic disease-related factor manifestation method used for determining the risk, etc., arteriosclerotic disease risk measuring method, gene polymorphism detection method, gene marker, gene polymorphism analysis kit, arteriosclerosis The present invention can provide a microarray for determining the risk of dermatological disease, an apparatus for determining the risk of atherosclerotic disease, and a program for determining the risk of atherosclerotic disease.

Claims

The scope of the claims 1. Includes a risk determination step of determining the risk of arteriosclerosis due to the genetic polymorphism from the genotype of the subject genetic polymorphism based on the combination-specific arteriosclerosis risk of a plurality of genetic polymorphisms. ,  At least one of the combinations of the plurality of polymorphisms has a significant positive association between the combination of the plurality of polymorphisms and the carotid intima-media complex thickness. To determine the risk of atherosclerotic disease.  2. The combination-specific arteriosclerosis risk of multiple gene polymorphisms is determined by whether there is a significant positive association between the combination and cervical artery intima-media thickness. The method for determining the risk of atherosclerotic disease according to claim 1.  3. The combination-specific arteriosclerosis risk of multiple genetic polymorphisms is set as the odds ratio of the frequency with which there is a significant positive association between the combination and cervical artery intima-media thickening. 2. The method for determining risk of arteriosclerotic disease according to claim 1, wherein the risk is determined.  4. The arteriosclerosis risk assessment method according to claim 1, wherein the risk of arteriosclerosis inherent to the combination of a plurality of gene polymorphisms is set by the increase in carotid intima-media thickness. . 5. At least one set of arteriosclerosis-related gene polymorphisms selected from the set of arteriosclerosis-related gene polymorphisms described in Tables 11 to 1 to 4 below, in which a combination of a plurality of gene polymorphisms is selected. The method for determining a risk of arterial sclerosis disease according to any one of claims 1 to 4, comprising: [Table 1-1]

O / AVεζ-80 One Fiber ー-- CO

no // al13d--- O

In Table 1-1 to Table 1-4, “Number” indicates the polymorphism of the same number in Table 2-1 to Table 2-2 below, and “Classification” indicates the genotype of the polymorphism. , Represents the genotype targeted for the combination, 1 is the homozygous polymorphism appearing on the left side of the polymorphism name in Table 2_1 to Table 2-2, 2 is heterologous, 3 Represents a polymorphic homology shown on the right side, 12 represents a genotype combining the above 1 and 2, and 23 represents a genotype combining the above 2 and 3.

[Table 2—2]

6. If the carotid intima-media thickening is at least 0.2 mm thicker than the healthy carotid intima-media thickening in healthy volunteers, it is defined as an arteriosclerotic disease case. When defined as non-atherosclerotic cases, More than 30% of at least 150 atherosclerotic disease populations have a combination of multiple polymorphisms that have a significant positive association with carotid intima-media complex thickness Becomes In at least 150 non-atherosclerotic populations, 15% have a combination of multiple polymorphisms that have a significant positive association with carotid intima-media complex thickness So that 6. The arteriosclerotic disease risk determination method according to claim 1, wherein a combination of a plurality of gene polymorphisms is selected.  7. The arteriosclerotic disease risk assessment method according to claim 6, wherein the arteriosclerotic disease case group and the non-arteriosclerotic case group are both diabetic patients and have no history of myocardial infarction.  8. The arteriosclerotic disease risk determination according to any one of claims 1 to 7, wherein the combination of a plurality of gene polymorphisms is a combination of at least one of 2 to 5 gene polymorphisms. Method.  9. The method for determining risk of arteriosclerotic disease according to any one of claims 1 to 8, wherein a combination of a plurality of gene polymorphisms comprises at least three gene polymorphisms. 10. The combination according to any one of claims 1 to 9, wherein the combination of a plurality of gene polymorphisms includes at least two gene polymorphisms belonging to any of the following groups a) to 1). For determining the risk of arteriosclerotic disease. a) Polymorphisms related to the renin-angiotensin system b) Genetic polymorphisms related to platelet function and coagulation system c) Polymorphisms related to lipids d) Polymorphisms related to nitric oxide synthase e) Polymorphisms related to TNF-α gene f) Polymorphisms related to IRS-1 gene g) Polymorphism group related to FAB P2 gene h) Polymorphisms related to muscle glycogen synthase gene  i) Polymorphisms related to NAD P and NAD PH oxidase p22phox j) Polymorphisms related to methylenetetrahydrofolate reductase k) Polymorphisms related to heat shock protein 70_1  1) Polymorphisms related to TGF-1 gene  1 1. Polymorphisms belonging to the polymorphism group related to platelet function and coagulation system, gene polymorphism group related to renin / angiotensin system, and gene polymorphism related to methylenetetrahydrofolate reductase (MTHFR) 11. The arteriosclerotic disease risk determination method according to claim 10, wherein the method includes at least a type group and a gene polymorphism belonging to at least one of the gene polymorphism groups related to lipids.  12. The risk of arterial sclerosis disease according to claim 11, wherein the gene polymorphism belonging to the gene polymorphism group related to platelet function and coagulation system is a gene polymorphism related to the PAI-11 gene. Judgment method.  13. The gene according to any one of claims 11 and 12, wherein the polymorphism belonging to the polymorphism group related to the renin-angiotensin system is a polymorphism related to the ACE gene. An arteriosclerotic disease risk determination method.  14. The atherosclerotic disease risk according to any one of claims 12 and 13, wherein the polymorphism belonging to the lipid-related polymorphism group is a HUMPOMA gene-related polymorphism. Degree judgment method.  15. The method according to claim 1, further comprising a risk determination step of determining an arteriosclerosis risk due to the environmental factor from information on the environmental factor of the subject based on the arteriosclerosis risk specific to the environmental factor. 15. The method for judging risk of atherosclerotic disease according to any one of claim 14. 16. Based on the risk of arteriosclerosis inherent to the carotid intima-media thickness, the carotid intima-media thickness can be calculated from the carotid intima-media thickness according to the carotid intima-media thickness. The arteriosclerotic disease risk determining method according to any one of claims 1 to 15, further comprising a risk determining step of determining a risk of sclerosis. 17. The arteriosclerotic disease risk determination method includes a plurality of risk determining steps, and calculates the arteriosclerotic disease risk by integrating the arteriosclerotic risk calculated for each of the risk determining steps. The arteriosclerotic disease risk determination method according to any one of claims 1 to 16, further comprising an arteriosclerotic disease risk calculating step.  18. The risk of arteriosclerotic disease according to any one of claims 1 to 17, further comprising a detection step of detecting genotypes of a plurality of genetic polymorphisms in the test subject before the manifestation step. Degree judgment method.  1 9. A detection step of detecting a genotype of a plurality of gene polymorphisms in the test subject, and a gene of the test subject detected in the detection step based on a risk of arteriosclerosis inherent to the combination of the plurality of gene polymorphisms Determining a risk of atherosclerosis due to the genetic polymorphism from the genotype of the polymorphism, At least one combination of the plurality of gene polymorphisms has a significant positive association between the combination of the plurality of gene polymorphisms and carotid intima-media complex thickness. Atherosclerotic disease risk measurement method.  20. Among the genetic polymorphisms of the test subjects, the genotypes of a plurality of polymorphisms related to the atherosclerosis-related gene polymorphism set are selectively identified, whereby the arteriosclerosis-related genetic polymorphisms of the test subjects are identified. Including a manifestation step of manifesting the gene polymorphism set,  The arteriosclerosis-related gene polymorphism set is a combination of a plurality of gene polymorphisms having a significant positive association with the carotid intima-media complex thickness, and A method for revealing an atherosclerotic disease-related factor, which is characterized in that: 21. The claim 20 wherein the manifestation step is represented by whether or not the genotypes of a plurality of selectively identified polymorphisms correspond to an atherosclerosis-related gene polymorphism set. The method for eliciting atherosclerotic disease-related factors according to the above. 22. If the genotype of the multiple polymorphisms selectively identified in the manifestation step corresponds to the atherosclerosis-related gene polymorphism set, the genotype specific to the atherosclerosis-related gene polymorphism set The arteriosclerotic disease-related according to claim 20, wherein the carotid intima-media complex has a significant positive association with the thickness of the intima-media complex, and is represented by a frequency odds ratio. Factor manifestation method.  2 3. When the genotype of multiple gene polymorphisms selectively revealed in the manifestation process corresponds to the atherosclerosis-related gene polymorphism set, it is unique to the arteriosclerosis-related gene polymorphism set. 21. The method for manifesting an arteriosclerotic disease-related factor according to claim 20, wherein the method is expressed by an increase in carotid intima-media thickness of the carotid artery.  24. The atherosclerosis-related gene polymorphism set is at least one atherosclerosis-related gene polymorphism set selected from the atherosclerosis-related gene polymorphism sets described in Tables 11 to 4 above. 24. The method for revealing an atherosclerotic disease-related factor according to any one of claims 20 to 23, wherein the method comprises:  25. If the carotid intima-media thickness is at least 0.2 mm thicker than the average carotid intima-media thickness in healthy volunteers, we define it as an arteriosclerotic disease case. When defined as non-atherosclerotic cases, In at least 150 atherosclerotic disease patient populations, at least 30% had at least one atherosclerosis-related polymorphism set, In at least 150 non-atherosclerotic disease populations, at least 15% have at least one atherosclerosis-related polymorphism set, 25. The method for revealing an arteriosclerotic disease-related factor according to any one of claims 20 to 24, wherein an arteriosclerosis-related gene polymorphism set is selected.  26. The arterial sclerosis disease group according to claim 25, wherein the arteriosclerosis disease case population and the non-arteriosclerosis case population are all diabetic patients and have no history of myocardial infarction. Disease risk determination method.  27. The arteriosclerotic disease-related gene according to any one of claims 20 to 26, wherein the atherosclerosis-related gene polymorphism set comprises at least 2 to 5 gene polymorphisms. Factor manifestation method.  28. The arteriosclerosis disease-related factor manifestation method according to any one of claims 20 to 27, wherein the atherosclerosis-related gene polymorphism set comprises at least three gene polymorphisms. 2 9. Atherosclerosis-related gene polymorphism set is classified into one of the following groups a) to 1) 29. The method for eliciting an arteriosclerotic disease-related factor according to any one of claims 20 to 28, wherein the method comprises at least two gene polymorphisms. a) Polymorphisms related to the renin-angiotensin system b) Genetic polymorphisms related to platelet function and coagulation system c) Polymorphisms related to lipids · d) Polymorphisms related to nitric oxide synthase e) Polymorphisms related to TNF_α gene  f) Polymorphisms related to IRS-1 gene g) Polymorphism group related to FAB P2 gene h) Polymorphisms related to muscle glycogen synthase gene  i) Polymorphisms related to NAD P · NADPH oxidase p22pho x j) Polymorphisms related to methylenetetrahydrofolate reductase k) Polymorphisms related to heat shock protein 70-1  1) Polymorphisms related to TGF_j3 1 gene  30. The manifestation of an atherosclerotic disease-related factor according to any one of claims 20 to 29, further comprising a detection step of detecting a genotype of a plurality of polymorphisms in the test subject before the manifestation step. Method.  3 1. Regarding the gene polymorphisms constituting at least one atherosclerosis-related gene polymorphism set selected from among the atherosclerosis-related gene polymorphism sets described in Tables 11 to 14 above, Detecting the genotype of the test subject,  A method for detecting a genetic polymorphism, wherein the detection result is used for determining the risk of atherosclerotic disease.  32. A gene containing a gene polymorphism that constitutes at least one atherosclerosis-related gene polymorphism set selected from among the atherosclerosis-related gene polymorphism sets described in Tables 1-1 to 1-4 above. marker. 33. Specifically, at least one gene selected from the set of atherosclerosis-related gene polymorphisms listed in Tables 11 to 14 is selected. Specific hybridization for primer pairs or genes that can be amplified A kit for analyzing a gene polymorphism, comprising a nucleic acid probe which can be used as a primer and detecting at least one of the arteriosclerosis-related gene polymorphism sets described in Tables 11 to 14.  34. The kit for gene polymorphism analysis according to claim 33, wherein the polymorphism set for atherosclerosis-related gene contains at least three gene polymorphisms. 35. It has at least one of a primer and a probe for detecting a polymorphism of at least two polymorphisms selected from the polymorphisms listed in Tables 2-1 to 2-2,  A case in which the carotid intima-media thickness is 0.2 mm or more thicker than the average carotid intima-media thickness in a healthy person is defined as an arteriosclerotic disease case, and the other cases are non-arterial When defined as a sclerosis disease case, In at least 150 arteriosclerosis disease patient populations, the carotid artery in the set of arteriosclerosis-related gene polymorphisms described in Tables 2-1 to 2-2 above, which can be constituted by the selected gene polymorphisms More than 30% of cases have at least one genotype combination that has a significant positive association with the thickness of the media-media complex, The genetic polymorphism analysis kit according to any one of Claims 33 to 34, wherein the kit is 15% or less in at least 150 non-arteriosclerotic disease cases. 36. Carotid intima-media complex having primers or probes for detecting polymorphisms of at least two polymorphisms selected from the polymorphisms listed in Table 2-1 to Table 2-2 Cases in which the body hypertrophy is 0.2 mm or more thicker than the average thickness of the carotid intima-media complex of a healthy person is defined as an arteriosclerosis disease case, and other cases are defined as non-arteriosclerosis disease cases To In at least 150 arteriosclerosis disease patient populations, the carotid artery in the set of arteriosclerosis-related gene polymorphisms set forth in Tables 11 to 14 above, which can be constituted by the selected gene polymorphisms More than 50% of cases have a combination of genotypes that have a significant positive association with the thickness of the In at least 150 non-atherosclerotic cases, 36. The gene polymorphism analysis kit according to any one of items 33 to 35. 37. The gene polymorphism analysis method according to any one of claims 33 to 36, wherein the plurality of gene polymorphism detection primers or probes belong to any one of the following different groups a) to 1). kit. a) Polymorphisms related to the renin-angiotensin system b) Genetic polymorphisms related to platelet function and coagulation system c) Lipid-related polymorphisms d) Polymorphisms related to nitric oxide synthase e) Polymorphisms related to TNF-α gene  f) Polymorphisms related to IRS-1 gene g) Polymorphism group related to FABP2 gene h) Polymorphisms related to muscle glycogen synthase gene  i) Polymorphisms related to NAD P. NAD PH oxidase p22phox j) Polymorphisms related to methylenetetrahydrofolate reductase k) Polymorphisms related to heat shock protein 70-1  1) Polymorphisms related to TGF-1 gene  38. At least one gene polymorphism set that is selected from the set of arteriosclerosis-related gene polymorphisms listed in Table 1-1 1 A micro T-ray for determining the risk of atherosclerotic disease, comprising a probe for detecting a gene polymorphism.  39. A computer-based atherosclerotic disease risk assessment device, comprising: an arterial sclerosis risk data table in which a combination of a plurality of gene polymorphisms and an atherosclerotic risk are listed. ,  The entered combination of a plurality of gene polymorphisms of the subject is compared with a combination of a plurality of gene polymorphisms in the atherosclerosis risk data table, and when there is a matching combination of the gene polymorphisms, An arteriosclerotic disease risk judging device, comprising: detecting means for detecting the arteriosclerosis risk corresponding to a combination of polymorphisms. 40. Combinations of multiple genetic polymorphisms and the risk of atherosclerosis Atherosclerosis risk data table corresponds to one unit of atherosclerosis risk for combinations of multiple polymorphisms that have a significant positive association with carotid intima-media thickness An arteriosclerotic disease risk assessment according to claim 39 4 1. The atherosclerosis risk data table, which lists the combinations of multiple genetic polymorphisms and the risk of atherosclerosis, shows a significant positive correlation between the carotid intima-media thickness and the carotid intima-media thickness. The combination of a plurality of genetic polymorphisms having a relationship of at least the odds ratio of the frequency at which the thickness of the intima-media complex of the cervical artery exceeds the normal range as the risk of arteriosclerosis. Item 9. The arteriosclerotic disease risk determination device according to item 9. 4 2. The atherosclerosis risk data table, which lists the combinations of multiple genetic polymorphisms and the risk of atherosclerosis, shows a significant positive correlation between the carotid intima-media thickening. The combination of a plurality of gene polymorphisms that are associated with each other with an increase in the thickness of the cervical artery intima-media complex as the risk of arteriosclerosis. Sclerosis disease risk assessment device.  4 3. A combination of gene polymorphisms with a significant positive association with carotid intima-media complex thickness is beyond the normal range of carotid intima-media complex thickness Claims 39 to 39 defined by at least one of a frequency odds ratio being equal to or higher than a certain value and / or a significant difference in the average value of the thickness of the carotid intima-media complex. 43. The arteriosclerotic disease risk judging device according to any of paragraph 42. 4 4. An arteriosclerosis risk data table that lists the presence or value of environmental factors and the arteriosclerosis risk in correspondence with each other,  The inputted or nonexistent or numerical value of the environmental factor of the test subject is compared with the presence or numerical value of the environmental factor in the atherosclerosis risk data table, and the atherosclerotic risk corresponding to the presence or numerical value of the environmental factor is determined. 43. The arteriosclerotic disease risk assessment device according to any one of claims 39 to 43, further comprising a detection means for detecting. 4 5. The arteriosclerotic disease risk judging device has a plurality of detecting means, and the arteriosclerotic disease risk is determined based on the sum of the plurality of carotid artery risk detected by the plurality of detecting means. 51. The arteriosclerotic disease risk judging device according to claim 39, further comprising a judging means for judging a degree.  4 6. An arteriosclerosis risk data table in which the carotid intima-media complex thickness and the arteriosclerosis risk are listed correspondingly.  The carotid intima-media complex is compared with the carotid intima-media thickness of the test subject and the carotid intima-media thickness in the atherosclerosis risk data table. The arteriosclerotic disease risk assessment according to any one of claims 39 to 45, further comprising a detection means for detecting the arteriosclerosis risk corresponding to the degree of hypertrophy. 47. Any of the carotid risk detected by the detection means and the sum of the carotid risk extracted from the plurality of extraction means, and the input carotid artery intima-media composite of the subject The arteriosclerotic disease risk judging device according to any one of claims 39 to 46, further comprising a judging means for judging an arteriosclerotic disease risk based on an added value to the body hypertrophy. .  48. A vascular intimal pressure measuring means for measuring the carotid artery intima-media thickness of the subject and supplying the carotid intima-media thickness to the computer. Item 9. The device for judging risk of arteriosclerotic disease according to any one of Items 9 to 47. 4 9. The computer stores an arteriosclerosis risk data table in which combinations of a plurality of gene polymorphisms and arteriosclerosis risk are listed in correspondence with each other.  The computer compares the combination of the plurality of polymorphisms of the test subject with the combination of the plurality of polymorphisms in the atherosclerosis risk data table, and if there is a matching combination of the polymorphisms, An arteriosclerotic disease risk determination program, which functions as detection means for detecting the arteriosclerosis risk corresponding to the combination of the gene polymorphisms. 50. The atherosclerosis risk data table, which lists the combinations of multiple gene polymorphisms and the risk of atherosclerosis, shows a significant positive correlation between the carotid intima-media thickening. The arteriosclerotic disease risk assessment according to claim 49, wherein one unit is assigned as the arteriosclerosis risk to a combination of a plurality of genetic polymorphisms having Fixed program.  5 1. The atherosclerosis risk data table, which lists combinations of multiple genetic polymorphisms and the risk of atherosclerosis, shows that there is a significant positive correlation between the carotid intima-media thickness and the carotid intima-media thickness. 4. The combination of a plurality of gene polymorphisms having a relationship of (1) to (2), wherein the odds ratio of the frequency at which the thickness of the intima-media complex of the cervical artery exceeds the normal range as the risk of atherosclerosis. Item 9. The arteriosclerotic disease risk assessment program according to item 9. 5 2. The atherosclerosis risk data table, which lists the combinations of multiple polymorphisms and the risk of atherosclerosis, shows a significant positive correlation between the carotid intima-media thickness and the carotid intima-media thickness. The arteriosclerosis according to claim 49, wherein the combination of gene polymorphisms having an association with the increase corresponds to an increase in carotid intima-media thickening as the risk of arteriosclerosis. Disease risk assessment program.  5 3. The frequency of carotid intima-media complex thickness exceeding the normal range may be a combination of genotypes that have a significant positive association with carotid intima-media thickness. Claims 49 to 52 defined by at least one of the following: an odds ratio of not less than a certain value, and a significant difference in average value of carotid intima-media complex thickness. The program for determining risk of arteriosclerotic disease according to any one of the above items. 5 4. If the carotid intima-media thickening is at least 0.2 mm thicker than the average carotid intima-media thickening in healthy volunteers, it is defined as an arteriosclerotic disease case. When defined as non-atherosclerotic cases, In at least 150 cases of atherosclerotic disease, at least 30% of patients had at least one unit of risk of atherosclerosis, In at least 150 non-atherosclerotic disease populations, less than 15% have at least 1 unit at risk of atherosclerosis, An arteriosclerotic disease risk determination program according to any one of claims 49 to 53.