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WO2003087360A1 - Procede d'evaluation du risque d'arteriosclerose, procede de mesure du risque d'arteriosclerose, microreseau, dispositif et programme pour evaluer le risque d'arteriosclerose - Google Patents

Procede d'evaluation du risque d'arteriosclerose, procede de mesure du risque d'arteriosclerose, microreseau, dispositif et programme pour evaluer le risque d'arteriosclerose Download PDF

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WO2003087360A1
WO2003087360A1 PCT/IB2003/001368 IB0301368W WO03087360A1 WO 2003087360 A1 WO2003087360 A1 WO 2003087360A1 IB 0301368 W IB0301368 W IB 0301368W WO 03087360 A1 WO03087360 A1 WO 03087360A1
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risk
polymorphisms
gene
polymorphism
arteriosclerosis
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Japanese (ja)
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Yoshimitsu Yamasaki
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Osaka Industrial Promotion Organization
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Osaka Industrial Promotion Organization
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Priority to AU2003220759A priority Critical patent/AU2003220759A1/en
Priority to JP2003584302A priority patent/JPWO2003087360A1/ja
Publication of WO2003087360A1 publication Critical patent/WO2003087360A1/fr
Priority to US10/961,043 priority patent/US20050089914A1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • 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.
  • 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.
  • 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.
  • a risk determination step of determining the risk of arteriosclerosis due to the gene polymorphism from the genotype of the subject polymorphism is performed.
  • 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.
  • 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
  • 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
  • ⁇ 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:
  • “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.
  • 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,
  • non-atherosclerotic populations 15% have a combination of multiple polymorphisms that have a significant positive association with carotid intima-media complex thickness So that
  • ⁇ 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.
  • ⁇ 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.
  • MTHFR methylenetetrahydrofolate reductase
  • ⁇ 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.
  • 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.
  • 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>.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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,
  • At least 150 atherosclerotic disease patient populations at least 30% had at least one atherosclerosis-related polymorphism set,
  • non-atherosclerotic disease populations at least 15% have at least one atherosclerosis-related polymorphism set,
  • 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.
  • 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.
  • 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.
  • 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.
  • ⁇ 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.
  • 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,
  • a genetic polymorphism analysis kit comprising detecting at least one set of arteriosclerosis-related gene polymorphisms described in Tables 11 to 14.
  • 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.
  • an arteriosclerotic disease case When defined as a sclerosis disease case,
  • ⁇ 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,
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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
  • 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>.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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>.
  • 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,
  • At least 150 cases of atherosclerotic disease at least 30% of patients had at least one unit of risk of atherosclerosis,
  • non-atherosclerotic disease populations less than 15% have at least 1 unit at risk of atherosclerosis
  • 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.
  • 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.
  • 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.
  • 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. .
  • 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.
  • At least one arteriosclerosis-related gene selected from the set of arteriosclerosis-related gene polymorphisms described in Tables 1-1 to 14 above.
  • it contains a gene polymorphism set.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • the risk can be set individually.
  • 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 /?
  • 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.
  • 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.
  • 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.
  • 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.
  • the increase in mean thickness of the carotid intima-media complex ( ⁇ ⁇ 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 ( ⁇ ⁇ ) is at least 0.3 mm or more.
  • 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.
  • 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
  • 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 .
  • 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.
  • 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.
  • splicing may be altered, and in either case, the expression level or properties of the protein may be altered.
  • 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)
  • i) gene polymorphisms related to NADP and NADPH oxidase p22phoX can be considered together as insulin resistance and vascular endothelial function
  • TNF Gene polymorphisms related to ⁇ 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
  • 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 ⁇ 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.
  • coagulation factors such as coagulation factor VII and polymorphisms in the fibrinogen ⁇ -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- ⁇ 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
  • 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.
  • MTHFR methylenetetrahydrofolate reductase
  • a polymorphism related to the PAI-1 gene is preferably used as the polymorphism belonging to the polymorphism group related to the platelet function / coagulation system.
  • a polymorphism related to the ACE gene is preferably used as the polymorphism belonging to the Renin-angiotensin system.
  • a polymorphism related to the HUMPONA gene is preferably used as the polymorphism belonging to the lipid-related polymorphism group.
  • 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]
  • 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.
  • MaX-IMT maximum IMT
  • AV g I MT plaque score
  • PS carotid stiffness
  • 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)
  • the researchers set the maximum value to AV g IMT.
  • 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 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 ( ⁇ IMT), an increase in maximum IMT ( ⁇ PIMT), and the like. It is particularly preferable as an indicator of a typical arteriosclerotic disease risk.
  • ⁇ IMT average IMT
  • ⁇ PIMT maximum IMT
  • the myocardium is increased every 0.333 mm of ⁇ . It is known that the odds of infarction increase by 4.9 times (Yamasaki. Diabetes Care 2000 (9)), and the mode in which ⁇ IMT is used as the risk of arteriosclerosis is extremely effective in atherosclerotic disease risk. It is to judge the degree.
  • 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 ⁇ IMT and ⁇ PIMT) 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.
  • 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.
  • 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.
  • 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].
  • 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.].
  • age, gender, duration of diabetes, and hemoglobin A 1c level are particularly important.
  • 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.
  • 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.
  • 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.
  • the combination has a significant association between the combination of the plurality of gene polymorphisms and the carotid intima-media complex thickness.
  • any method can be used as long as the method detects the genotype of a plurality of polymorphisms in the subject.
  • a sample containing DNA such as a subject's blood, sputum, skin, bronchoalveolar lavage fluid, other body fluid, or tissue.
  • 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.
  • PCR method only a certain polymorphism is specifically amplified using a primer specific to the polymorphism.
  • A1 lele Specific Primer (ASP)-Like the PCR method the 3' terminal
  • 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.
  • 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.
  • 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.
  • 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.
  • the polymorphism is identified by identifying the type of the added ddNTP.
  • 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.
  • a molecular beacon method, a ligation method and the like can be exemplified as known methods.
  • the same steps as those described in the arteriosclerotic disease risk determination method can be used.
  • 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
  • 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.
  • selectively clarifying means selecting and clarifying a specific one of a myriad of genetic polymorphism combinations.
  • a plurality of the selectively identified gene polymorphisms can be obtained.
  • 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.
  • 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.
  • 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.
  • the atherosclerosis-related gene polymorphism set includes at least two gene polymorphisms belonging to any of the following groups a) to 1).
  • a detection step of detecting genotypes of a plurality of polymorphisms of a test subject may be further included before the revealing step.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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).
  • TGF— polymorphism group related to 31 genes
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the arteriosclerotic disease risk determination device 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.
  • 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.
  • a carotid artery risk detected by the detecting unit 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the detection means for detecting the arteriosclerosis risk corresponding to the combination of the gene polymorphisms is used. It is characterized by functioning with.
  • 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.
  • arteriosclerosis 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.
  • 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.
  • 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.
  • ⁇ IMT and ⁇ IMT specific to the genotype having a homologous risk factor for the single gene polymorphism were calculated by a linear multiple regression analysis technique.
  • ⁇ represents the increment of the average thickness of the intimal media complex of the arterial artery
  • ⁇ IMT represents the fraction of the maximum thickness of the intima-media complex of the carotid artery.
  • the units of PIMMT and ⁇ IMT are millimeters (mm).
  • the “number” is assigned to represent the polymorphism of the same number in Tables 3 and 4.
  • ⁇ in the case of having a genotype having a homologous risk factor for both PON1 and MTHFR was * 0.30 lmm.
  • ⁇ IMT was 0.16 mm.
  • the gene type is a homologous type containing a risk factor
  • 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.
  • 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.
  • 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.
  • 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.
  • the risk of arterial sclerosis in the test subject is determined to be 0.771.
  • this value can be directly used as the risk of arteriosclerotic disease of the subject.
  • these ⁇ IMT 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.
  • 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 ⁇ .
  • the obtained partial regression coefficients are shown below.
  • the method for determining risk of disease can further include a risk determination step based on these factors.
  • the environmental factor includes a risk determination step based on age and gender
  • 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.
  • 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.
  • VNTR variable number of tandem repeats
  • Example 2 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). ⁇ / ⁇ ⁇ -iP8 OAV-
  • 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.
  • the 49 polymorphisms that could explain early arteriosclerosis did not exist.
  • 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.
  • 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.
  • 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.
  • 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.

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Abstract

Procédé pour évaluer le risque d'artériosclérose, qui permet d'évaluer précisément la tendance aux crises d'artériosclérose, la tendance de cette maladie à évoluer, etc., et est ainsi utile dans la prévention et le traitement de l'artériosclérose ; une trousse pour analyser le polymorphisme génétique servant à évaluer le risque, etc. ; un dispositif pour évaluer le risque d'artériosclérose, etc.. Le procédé est caractérisé en ce qu'il comporte une étape de détermination du risque, qui consiste à déterminer le risque d'artériosclérose attribué au polymorphisme génétique à partir du type génétique de polymorphisme du sujet, sur la base du risque d'artériosclérose propre à une combinaison formée par une pluralité de polymorphismes génétiques ; et qu'au moins une de ces combinaisons de polymorphismes génétiques présente une association positive significative avec un niveau d'épaississement du composite média/intima carotidienne.
PCT/IB2003/001368 2002-04-12 2003-04-14 Procede d'evaluation du risque d'arteriosclerose, procede de mesure du risque d'arteriosclerose, microreseau, dispositif et programme pour evaluer le risque d'arteriosclerose Ceased WO2003087360A1 (fr)

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EP1531180A4 (fr) * 2002-06-21 2006-03-15 Inst Nagoya Ind Science Res Methode de diagnostic du risque d'infarctus du myocarde
US7521181B2 (en) 2002-06-21 2009-04-21 Nagoya Industrial Science Research Institute Methods of diagnosing risk of myocardial infarction by detection of polymorphisms in connexin and NADH/NADPH oxidase genes
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JP2009524404A (ja) * 2002-12-20 2009-07-02 アプレラ コーポレイション 心筋梗塞に関連する遺伝的多型、その検出方法および使用
WO2006126618A1 (fr) * 2005-05-26 2006-11-30 Signpost Corporation Procédé de détermination de polymorphisme de gène pour évaluer le niveau de risque de maladie, procédé d’évaluation du niveau de risque de maladie et matrice d'évaluation correspondante
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WO2008018542A1 (fr) * 2006-08-11 2008-02-14 The New Industry Research Organization Polymorphisme génique utile pour l'aide et le traitement visant à l'arrêt du tabac
JP5216982B2 (ja) * 2006-08-11 2013-06-19 公益財団法人新産業創造研究機構 禁煙支援治療に有用な遺伝子多型
JP2020178560A (ja) * 2019-04-23 2020-11-05 ジェネシスヘルスケア株式会社 動脈硬化のリスクを判定する方法
JP7165098B2 (ja) 2019-04-23 2022-11-02 ジェネシスヘルスケア株式会社 動脈硬化のリスクを判定する方法

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