KR20060119737A - Multiple snp for diagnosing cardiovascular disease, microarray and kit comprising the same, and method for diagnosing cardiovascular disease using the same - Google Patents

Abstract

A multiple SNP for diagnosing cardiovascular disease is provided to be able to diagnose an attack and the possibility of the cardiovascular disease at the initial stage by using specific SNPs. And a method for diagnosing cardiovascular disease is provided to be able to effectively diagnose the existence or danger of the cardiovascular disease by analyzing the SNP related with the cardiovascular disease. The multiple SNP for diagnosing cardiovascular disease comprises at least one polynucleotide or a complementary polynucleotide thereof selected from the group consisting of polynucleotides consisting of at least 10 consecutive nucleotides including each SNP position nucleotide of polynucleotides consisting of SEQ ID : NOs. 1 to 35(76th for the SEQ ID : NO. 4, 85th for the SEQ ID : NO. 8, 51st for SEQ ID : NO. 9, 35th for the SEQ ID : NO. 10, 85th for the SEQ ID : NO. 19, and 101st for number of the remaining SEQ ID). The microarray comprises the polynucleotide, a polynucleotide encoded by the same or a cDNA thereof. The method for diagnosing cardiovascular disease comprises the steps of: (a) separating a nucleic acid sample from an object to be tested; and (b) determining a genotype of the polymorphic portion of the each SNP position nucleotide consisting of SEQ ID : NOs. 1 to 35(76th for the SEQ ID : NO. 4, 85th for the SEQ ID : NO. 8, 51st for the SEQ ID : NO. 9, 35th for the SEQ ID : NO. 10, 85th for the SEQ ID : NO. 19, and 101st for number of the remaining SEQ ID).

Description

Multiple SNP for diagnosing cardiovascular disease, microarray and kit comprising the same, and method for diagnosing cardiovascular disease using the same

The present invention relates to multiple SNPs for diagnosing cardiovascular disease, microarrays and kits including the same, and a method for diagnosing cardiovascular disease using the same.

The genomes of all organisms undergo spontaneous mutations that result in variants in the sequence of progeny during evolution (Gusella, Ann. Rev. Biochem . 55, 831-854, 1986). Variants may be evolutionarily beneficial or disadvantageous or neutral compared to progeny forms. In some cases, variants may be fatally penalized and not passed on to their offspring. In other cases, it gives evolutionary benefit to the species, and eventually DNA is inserted into most of the species, effectively forming a ancestral form. In many cases, these ancestral forms and variants survive and coexist among species. By coexistence of plural forms of sequences, polymorphism occurs.

Such polymorphisms include restriction fragment length polymorphism (RFLP), short tandem repeats (STR), and single nucleotide polymorphism (SNP). Dual SNPs take the form of single nucleotide variations between individuals of the same species. When SNPs occur in the coding region sequence, a defective or mutated protein may be expressed by any of the polymorphic forms. In other cases, SNPs may occur in the non-coding region sequence. Some of these may result in the expression of defective or mutated proteins, for example as a result of defective splicing. Some SNPs may have no effect on the phenotype.

SNPs are known to occur once in about 1,000bp in humans. Where these SNPs affect phenotypes such as disease, polynucleotides comprising such single base polymorphs can be used as primers or probes to diagnose such disease. Monoclonal antibodies that specifically bind to the SNPs can also be used for the diagnosis of diseases. Currently, several research institutes are conducting research on single base analysis and its functional analysis. The sequencing and other experimental results of the SNPs found in this database are made public so that anyone can access and use them for research.

However, these SNPs only discovered the presence of SNPs in the human genome or cDNA, and did not reveal their effect on the phenotype. Some of these functions are known, but most are unknown.

Cardiovascular disease is a leading cause of death in industrialized countries around the world, and has been a leading cause of death in Korea since the 1970s. According to the National Statistical Office, 22,000 people (9087 deaths per 100,000 people), 9.1% of all Korean deaths (246,000) during 2003, died of heart disease and hypertension, leading to cancer (first place) and cerebrovascular disease ( Second place) followed by third place of death cause.

Cardiovascular diseases include myocardial infarction, angina pectoris, atherosclerosis, high blood pressure, heart failure, aneurysms, arteriosclerosis, embolism, stroke and thrombosis.

Coronary artery disease, which is an important part of cardiovascular disease, is usually caused by blockage or narrowing of coronary arteries that supply blood to the heart by arteriosclerosis. The complete blockage of coronary arteries by atherosclerosis is called myocardial infarction and narrowing is called angina. The causes of coronary artery disease are hyperlipidemia (hypercholesterolemia), hypertension, smoking, diabetes, heredity, obesity, lack of exercise, stress and menopause of women. The combination of risk factors increases the risk of illness. Like many other diseases, cardiovascular disease is strongly influenced by genetic factors.

The biggest problem in diagnosing or early predicting complex diseases, including conventional cardiovascular diseases, is that these diseases can be diagnosed using physical methods only to some extent. Currently, cardiovascular disease is diagnosed by X-ray and ultrasound imaging of the inside of the heart and coronary artery using an imaging device, but this can only be diagnosed after onset. However, as recent advances in the development of various molecular biology techniques and studies of the human genome have led to the discovery of genes or genetic variants directly or indirectly related to the disease, which can be used for early diagnosis of the disease. From diagnostic methods that depended on the characteristics of existing phenotypes or external diseases, it is possible to make early diagnosis that can predict the onset of disease with genetic factors.

The inventors of the present invention continue to research SNPs for early diagnosis of the onset of cardiovascular disease and its probability, and individuals with cardiovascular disease have specific SNPs at the same time and use the specific SNPs for cardiovascular disease. The present invention was completed by confirming that the occurrence probability and genetic susceptibility of can be predicted.

It is an object of the present invention to provide multiple SNPs for diagnosing cardiovascular disease.

Another object of the present invention is to provide a polynucleotide that hybridizes with the polynucleotide of the multiple SNP.

It is still another object of the present invention to provide a microarray for diagnosing cardiovascular disease comprising the polynucleotide, a polypeptide encoded by the polypeptide, or a cDNA thereof.

Still another object of the present invention is to provide a kit for diagnosing a cardiovascular disease including the microarray.

Still another object of the present invention is to provide a method for diagnosing cardiovascular disease using the multiple SNPs.

In order to achieve the object of the present invention, the present invention provides a polynucleotide consisting of at least one polynucleotide selected from the polynucleotide consisting of 10 or more consecutive bases comprising each SNP position base in the polynucleotide consisting of SEQ ID NO: 1 to 35 Provided are multiple SNPs for diagnosing cardiovascular disease comprising a teat or complementary polynucleotides thereof.

In order to achieve another object of the present invention, the present invention provides a polynucleotide that hybridizes with the polynucleotide or its complementary polynucleotide.

In order to achieve another object of the present invention, the present invention provides a microarray for diagnosing a cardiovascular disease comprising the polynucleotide or a complementary polynucleotide thereof or a polynucleotide hybridizing thereto, a polypeptide encoded therein or a cDNA thereof. .

In order to achieve another object of the present invention, the present invention provides a cardiovascular disease diagnostic kit comprising the microarray.

In order to achieve another object of the present invention, the present invention comprises the steps of: a) separating the nucleic acid sample from the subject; And b) determining the genotype of each polymorphic site of at least one polynucleotide selected from SEQ ID NOs: 1 to 35.

Hereinafter, the present invention will be described in more detail.

One aspect of the invention relates to multiple SNPs for diagnosing cardiovascular disease.

Multiple SNPs for diagnosing cardiovascular disease according to the present invention are polynucleotides consisting of SEQ ID NOs: 1 to 35, respectively SNP positions (76th for SEQ ID NO: 4, 85th for SEQ ID NO: 8, 51th for SEQ ID NO: 9, One or more polynucleotides selected from polynucleotides consisting of at least 10 consecutive bases comprising the 35th base for SEQ ID NO. 10, the 85th for SEQ ID NO. 19 and the 101st for remaining SEQ ID NO. It is characterized in that it comprises a polynucleotide.

TABLE 1

SNP NCBI Zen Bank Registration Number Polynucleotide with SNP SNP NCBI Zen Bank Registration Number Polynucleotide with SNP rs4459610 SEQ ID NO: 1 rs731710 SEQ ID NO: 19 rs20568 SEQ ID NO: 2 rs991827 SEQ ID NO: 20 rs5050 SEQ ID NO: 3 rs1566307 SEQ ID NO: 21 rs1404396 SEQ ID NO: 4 rs1467523 SEQ ID NO: 22 rs511678 SEQ ID NO: 5 rs378660 SEQ ID NO: 23 rs1916382 SEQ ID NO: 6 rs1546642 SEQ ID NO: 24 rs995717 SEQ ID NO: 7 rs209176 SEQ ID NO: 25 rs2611 SEQ ID NO: 8 rs1056409 SEQ ID NO: 26 rs1805564 SEQ ID NO: 9 rs1859754 SEQ ID NO: 27 rs1469876 SEQ ID NO: 10 rs1527509 SEQ ID NO: 28 rs251692 SEQ ID NO: 11 rs1028140 SEQ ID NO: 29 rs1409765 SEQ ID NO: 12 rs1983628 SEQ ID NO: 30 rs889179 SEQ ID NO: 13 rs557831 SEQ ID NO: 31 rs882432 SEQ ID NO: 14 rs1194029 SEQ ID NO: 32 rs1801 SEQ ID NO: 15 rs388332 SEQ ID NO: 33 rs973126 SEQ ID NO: 16 rs2055018 SEQ ID NO: 34 rs361594 SEQ ID NO: 17 rs1557771 SEQ ID NO: 35 rs734072 SEQ ID NO: 18

SNP (single nucleotide polymorphism) is the most common form of DNA polymorphism due to the difference in single base-pair variation in individual DNA between individuals (approximately 1 / 1kb). ).

In addition, the NCBI genbank registration number of the SNP indicates the sequence and position of each SNP. Those skilled in the art will be able to easily identify the position and sequence of the SNP using the registration number. The specific sequence corresponding to the rs number of the SNP registered in the NCBI may change slightly over time. It will be apparent to those skilled in the art that the scope of the present invention also applies to such altered sequences.

In addition, SEQ ID NO: 1 to 35 is a polynucleotide containing the nucleotide sequence of the SNP site. The properties of the polynucleotide and each SNP present in the polynucleotide are described in Table 2 below.

SEQ ID NOs: 1 to 35 are polymorphic sequences including polymorphic sites. A polymorphic sequence refers to a sequence comprising a polymorphic site representing SNP in a polynucleotide sequence. The polynucleotide sequence may be DNA or RNA.

TABLE 2

SNP NCBI Zen Bank Registration Number Polynucleotide with SNP Multiple SNP Participation Count band gene SNP Role rs4459610 SEQ ID NO: 1 One 17q23.3 ACE Coding exon rs20568 SEQ ID NO: 2 One 3q13.33 ADPRH Coding exon Rs5050 SEQ ID NO: 3 One 1q42.2 AGT Promoter rs1404396 SEQ ID NO: 4 One 7p21.1 ANKMY2 Intron rs511678 SEQ ID NO: 5 One 1q32.2 CR2 Intron rs1916382 SEQ ID NO: 6 One 10q21.3 CTNNA3 Intron rs995717 SEQ ID NO: 7 One 21q22.13 DSCR4 Intron Rs2611 SEQ ID NO: 8 One 10q22.1 FLJ22761 Exon rs1805564 SEQ ID NO: 9 One 3q26.33 FXR1 Intron rs1469876 SEQ ID NO: 10 One 1p31.3 KIAA1573 Intron rs251692 SEQ ID NO: 11 One 19q13.32 LIG1 Exon rs1409765 SEQ ID NO: 12 One 1p31.1 LPHN2 Intron rs889179 SEQ ID NO: 13 One 19p13.2 MGC15716 3 'UTR rs882432 SEQ ID NO: 14 One 22q12.1 MYO18B Intron Rs1801 SEQ ID NO: 15 One 4q24 NFKB1 Intron rs973126 SEQ ID NO: 16 One 4q25 PAPSS1 Coding exon rs361594 SEQ ID NO: 17 One 22q11.21 PEX26 3 'UTR rs734072 SEQ ID NO: 18 One 3p21.31 SCOTIN Intron

Table 2 (continued)

SNP NCBI Zen Bank Registration Number Polynucleotide with SNP Multiple SNP Participation Count band gene SNP Role rs731710 SEQ ID NO: 19 One 16q24.2 SLC7A5 Intron rs991827 SEQ ID NO: 20 One 12q24.32 rs1566307 SEQ ID NO: 21 One 12q24.21 rs1467523 SEQ ID NO: 22 One 14q22.1 rs378660 SEQ ID NO: 23 2 16q23.1 rs1546642 SEQ ID NO: 24 One 2p21 rs209176 SEQ ID NO: 25 One 6p22.1 rs1056409 SEQ ID NO: 26 One 9q33.1 rs1859754 SEQ ID NO: 27 One 7q21.13 rs1527509 SEQ ID NO: 28 One 21q21.1 rs1028140 SEQ ID NO: 29 One 2q14.1 rs1983628 SEQ ID NO: 30 One 20q13.2 rs557831 SEQ ID NO: 31 One 11q24.2 rs1194029 SEQ ID NO: 32 One 12q24.32 rs388332 SEQ ID NO: 33 One 21q22.2 rs2055018 SEQ ID NO: 34 One 11p14.1 rs1557771 SEQ ID NO: 35 One 7p21.1

In Table 2, the number of participation of multiple SNPs represents the number of times each single SNP is cited in the combination of 12 multiple SNPs according to the present invention (see Table 3).

The 'band' refers to a chromosome number where each single SNP is located, a short arm (p) portion or a long arm (q) portion and a band number centered on a centromere on each chromosome. For example, the position of the SNP included in SEQ ID NO: 1 is 17q23.3, which means that it is on the 23.3 band of the arm portion (q) on chromosome 17.

The 'gene' means a gene including each SNP.

The 'role of SNP' means a role of each single SNP in the gene.

In the diagnostic multiple SNP according to the present invention, the one or more polynucleotides in the polynucleotide consisting of SEQ ID NO: 1 to 35 each SNP position (76th for SEQ ID NO: 4, 85 for SEQ ID NO: 8, SEQ ID NO: 9 from polynucleotides consisting of 10 or more consecutive bases, including the 51st, 35th for SEQ ID NO. 10, 85th for SEQ ID NO. 19, and 101th for the remaining SEQ ID NO. It is preferably selected from the group consisting of 1 to 12 selected in combination.

TABLE 3

number Multi SNP One (SEQ ID NO: 2, SEQ ID NO: 25, SEQ ID NO: 3) 2 (SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 35) 3 (SEQ ID NO: 6, SEQ ID NO: 23, SEQ ID NO: 16) 4 (SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 13) 5 (SEQ ID NO: 17, SEQ ID NO: 1, SEQ ID NO: 31) 6 (SEQ ID NO: 5, SEQ ID NO: 29, SEQ ID NO: 8) 7 (SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 32) 8 (SEQ ID NO: 26, SEQ ID NO: 19, SEQ ID NO: 7) 9 (SEQ ID NO: 23, SEQ ID NO: 9, SEQ ID NO: 28) 10 (SEQ ID NO: 21, SEQ ID NO: 14, SEQ ID NO: 4) 11 (SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 33) 12 (SEQ ID NO: 24, SEQ ID NO: 12, SEQ ID NO: 34)

In the multiple SNP for diagnosing cardiovascular disease according to the present invention, each of the SNP positions of the selected polynucleotides (76th for SEQ ID NO: 4, 85th for SEQ ID NO: 8, 51st for SEQ ID NO: 9, and SEQ ID NO: 10) In the case of the 35th, the 85th in the case of SEQ ID NO: 19 and the 101st in the case of the remaining SEQ ID NO :) It is preferable that the genotypes of the bases are as shown in Table 4, respectively.

TABLE 4

number Multi SNP Allele One (SEQ ID NO: 2, SEQ ID NO: 25, SEQ ID NO: 3) (CC, CC, TG) 2 (SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 35) (CC, GC, TT) 3 (SEQ ID NO: 6, SEQ ID NO: 23, SEQ ID NO: 16) (CC, AG, TT or TC) 4 (SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 13) (AA or AC, CG, TT or TG) 5 (SEQ ID NO: 17, SEQ ID NO: 1, SEQ ID NO: 31) (TC or CC, TT or TA, TT or TG) 6 (SEQ ID NO: 5, SEQ ID NO: 29, SEQ ID NO: 8) (GC, TT or TC, GC) 7 (SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 32) (CC, CC, AA or AG) 8 (SEQ ID NO: 26, SEQ ID NO: 19, SEQ ID NO: 7) (TG, TC or CC, GG) 9 (SEQ ID NO: 23, SEQ ID NO: 9, SEQ ID NO: 28) (AG, GG, TC or CC) 10 (SEQ ID NO: 21, SEQ ID NO: 14, SEQ ID NO: 4) (CT, AG or GG, CT or TT) 11 (SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 33) (TT or TC, CG or GG, AC) 12 (SEQ ID NO: 24, SEQ ID NO: 12, SEQ ID NO: 34) (AC, CC or CG, TA)

Multiple SNPs according to the present invention are characterized by 12 multiple SNPs combining each single SNP. Combinations of each of the single SNPs and their genotypes are as described in Tables 3 and 4. In Tables 3 and 4, the 'multiple SNP' column represents the combination of four single SNPs selected, and the 'genotype' column represents the genotype at the SNP position of each single SNP in the same order. For example, in multiple SNP No. 1 of Table 4, genotype of rs20568 is CC, genotype of rs209176 is CC, and genotype of rs5050 is TG.

In the present invention, the cardiovascular disease may be selected from the group consisting of myocardial infarction, angina pectoris, atherosclerosis, hypertension, heart failure, aneurysm, arteriosclerosis, embolism, stroke and thrombosis, preferably myocardial infarction.

In one embodiment of the present invention, a series of screening processes were performed to develop a combination of single SNPs, i.e., multiple SNPs, which are highly associated with the development of cardiovascular disease from each single SNP. The multiple SNP screening process was aimed at men. That is, DNA is isolated and amplified from the blood of male cardiovascular disease patients and normal males, and the sequence of the SNP region of the DNA is analyzed, and is specifically shown only in male cardiovascular disease patients, but rarely in male normal people. Specific combinations of SNPs and their genotypes were identified. The SNP combinations and their genotypes identified in the examples of the present invention are shown in Tables 3 and 4. The properties of each multiple SNP are listed in Table 5 below.

TABLE 5

number Patient frequency Frequency of normal group appearance Ozbee 95% confidence interval One 30 0 61.3 3.7 1010 2 30 0 61.3 3.7 1010 3 28 0 56.7 3.4 935.5 4 25 0 50 3 826.5 5 25 0 50 3 826.5 6 24 0 47.8 2.9 790.9 7 23 0 45.6 2.8 755.7 8 28 One 27.7 3.7 205.7 9 33 One 33.5 4.5 247.6 10 35 3 11.9 3.6 39.22 11 29 2 14.4 3.4 60.98 12 34 3 11.5 3.5 37.94

In Table 5, 'number' indicates the number of multiple SNPs, which is the same as the number described in Table 3.

'Patient emergence frequency' and 'normal group appearance frequency' refer to the number of normal persons and patients who were found to have the corresponding multiple SNPs among the 221 patients and 192 normal persons tested, respectively.

The 'odds ratio' represents a ratio of the probability of having the corresponding multiple SNP among the patient group to the probability of having the corresponding multiple SNP in the normal group. That is, the odds ratio is ad / bc, where a represents the frequency of appearance of the patient group in Table 5, and c represents the frequency of appearance of the normal group in Table 5. And b = [(total number of patients)-a] and d = [(total number of normal groups)-c]. The patients tested and the normal group were 221 and 192 respectively, so b = [221-a] and d = [192-c].

When the odds ratio exceeds 1, it indicates that the corresponding multiple SNP is associated with the patient group. The larger the odds ratio, the higher the association. As shown in Table 5, multiple SNPs 1 to 12 according to the present invention have an odds ratio of 11.5 to 61.3. Since this value is much larger than 1, it can be seen that multiple SNPs 1 to 12 according to the present invention have a great correlation with the development of cardiovascular disease.

The '95% confidence interval 'indicates a section having a 95% probability that the odds ratio exists, and is calculated by the following equation. If the confidence interval contains 1, that is, if the confidence interval is less than 1 and the upper bound is 1 or more, the association with disease cannot be considered significant.

), 오즈비×exp(1.960

)) (상기 식에서, V=1/a + 1/b + 1/c +1/d)95% confidence interval = (summer, upper bound) = (oz ratio × exp (-1.960)

), Ozbi × exp (1.960)

)) (Wherein, V = 1 / a + 1 / b + 1 / c + 1 / d)

The multiple SNP for diagnosing cardiovascular disease according to the present invention may be composed of each of the multiple SNPs, more preferably, two or more multiple SNPs, and most preferably, all of the multiple SNPs 1 to 12.

The polynucleotide of each single SNP constituting the multiple SNP for diagnosing cardiovascular disease according to the present invention is preferably 10 or more consecutive bases, more preferably 10 to 100 consecutive bases.

Another aspect of the invention relates to an allele specific polynucleotide for diagnosing cardiovascular disease which hybridizes to a polynucleotide according to the invention or a complementary polynucleotide thereof.

The allele-specific polynucleotide means to hybridize specifically to each allele. That is, it means hybridizing so that the base of the polymorphic site in each polymorphic sequence of SEQ ID NO: 1-35 can be distinguished specifically. The hybridization is performed under stringent conditions. For example, the stringent conditions are carried out at salt concentrations below 1 M and at temperatures above 25 ° C. Alternatively, conditions of 5 × SSPE (750 mM NaCl, 50 mM Na Phosphate, 5 mM EDTA, pH 7.4) and 25-30 ° C. may be suitable for allele specific probe hybridization. The hybridization conditions may be easily modified and used by those skilled in the art.

In the present invention, the allele specific polynucleotide may be a primer. Here primers are the starting point for template-directed DNA synthesis under appropriate conditions (e.g. four different nucleoside triphosphates and polymerizers such as DNA, RNA polymerase or reverse transcriptase) in appropriate buffers and at appropriate temperatures. Refers to a single stranded oligonucleotide that can act. The appropriate length of the primer may vary depending on the purpose of use, but is usually 15 to 30 nucleotides. Short primer molecules generally require lower temperatures to form stable hybrids with the template. The primer sequence need not be completely complementary to the template, but should be sufficiently complementary to hybridize with the template. It is preferred that the primer 3 'end is aligned with the polymorphic site of SEQ ID NO: 1 to 35. The primer hybridizes to the target DNA comprising the polymorphic site and initiates amplification of an allelic form in which the primer shows complete homology. This primer is used in pairs with a second primer that hybridizes to the other side. By amplification the product is amplified from the two primers, which means that certain allelic forms are present. Primers of the invention include polynucleotide fragments used in ligase chain reaction (LCR).

In the present invention, the allele specific polynucleotide may be a probe. Probe (probe) in the present invention means a hybridization probe, it means an oligonucleotide capable of sequence-specific binding to the complementary strand of the nucleic acid. Such probes include peptide nucleic acids described in Nielsen et al., Science 254, 1497-1500 (1991). The probe of the present invention is an allele-specific probe, in which polymorphic sites exist in nucleic acid fragments derived from two members of the same species, and hybridize to DNA fragments derived from one member, but not to fragments derived from other members. . Hybridization conditions in this case show significant differences in hybridization strength between alleles and should be sufficiently stringent to hybridize to only one of the alleles. In the probe of the present invention, the central region (that is, position 7 when the probe is 15 nucleotides and position 8 or 9 when the probe is 16 nucleotides) is preferably aligned with the polymorphic site of the sequence. This can lead to good hybridization differences between different allelic forms. The probe of the present invention can be used for diagnostic methods for detecting alleles. The diagnostic method includes detection methods based on hybridization of nucleic acids such as Southern blotting, or the like, and may be provided in a form that is pre-coupled to a substrate of the microarray in a method using a microarray.

Another aspect of the present invention relates to a microarray for diagnosing a cardiovascular disease comprising a polynucleotide according to the present invention or a complementary polynucleotide thereof or a polynucleotide hybridizing thereto, a polypeptide encoded by the same, or a cDNA thereof.

Microarrays according to the invention are conventional methods known to those skilled in the art using polynucleotides according to the invention or polynucleotides which hybridize with the probes or their complementary polynucleotides, polypeptides encoded by them or cDNAs thereof. It can be prepared by.

For example, the polynucleotide may be immobilized on a substrate coated with an active group selected from the group consisting of amino-silane, poly-L-lysine, and aldehyde. In addition, the substrate may be selected from the group consisting of silicon wafer, glass, quartz, metal and plastic. As a method of immobilizing the polynucleotide on a substrate, a micropipetting method using a piezoelectric method, a method using a pin-shaped spotter, or the like can be used.

Another aspect of the present invention relates to a cardiovascular disease diagnostic kit comprising a microarray according to the present invention.

The kit according to the present invention may further comprise a primer set used to separate and amplify DNA containing the SNP from the subject in addition to the microarray of the present invention. Such suitable primer sets will be readily apparent to those skilled in the art with reference to the sequences of the present invention. For example, those shown in Table 6 may be used as the primer set.

Another aspect of the present invention relates to a method for diagnosing cardiovascular disease using multiple SNPs according to the present invention.

A method for diagnosing cardiovascular disease according to the present invention comprises the steps of: a) separating a nucleic acid sample from a subject; And b) each SNP position of at least one polynucleotide selected from SEQ ID NOs: 1 to 35 (76th for SEQ ID NO: 4, 85th for SEQ ID NO: 8, 51st for SEQ ID NO: 9, 35 for SEQ ID NO: 10), and Second, determining the genotype of the polymorphic site, which is the 85th base for SEQ ID NO: 19 and the 101st base for the remaining SEQ ID NO: 19).

In the method of the present invention, the method of separating DNA from the subject can be made through a method known in the art. For example, it can be done by directly purifying DNA from tissue or cells or by specifically amplifying and isolating specific regions using amplification methods such as PCR. In the present invention, DNA is meant to include not only DNA but also cDNA synthesized from mRNA. Obtaining nucleic acids from a subject may include, for example, PCR amplification, ligase chain reaction (LCR) (Wu and Wallace, Genomics 4, 560 (1989), Landegren et al., Science 241, 1077 (1988)), transcription amplification. transcription amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA 86, 1173 (1989)) and self-sustaining sequence replication (Guatelli et al., Proc. Natl. Acad. Sci. USA 87, 1874 (1990)) and Nucleic acid based sequence amplification (NASBA) can be used.

Determination of the nucleotide sequence of the isolated DNA can be made by various methods known in the art. For example, a nucleotide of a polymorphic site is determined by the dideoxy method by hybridizing the probe or complementary probes comprising the sequence of the SNP site or through the determination of the nucleotide sequence of the nucleic acid with the DNA and measuring the degree of hybridization obtained therefrom. Methods for determining sequences may be used. The degree of hybridization may be achieved by, for example, labeling a detectable label on the target DNA to specifically detect only the hybridized target DNA, but other electrical signal detection methods may be used. More preferably, the step b) hybridizes the nucleic acid sample isolated from the subject to a polynucleotide comprising a SNP according to the present invention or a complementary polynucleotide thereof, or a polynucleotide hybridizing thereto. ; And b-2) detecting the hybridization result.

The method for diagnosing cardiovascular disease according to the present invention includes c) subjecting the subject to cardiovascular disease when the genotype of the polymorphic site of at least one polynucleotide selected from SEQ ID NOs: 1 to 35 is at least one of 1 to 12 in Table 4. The method may further include determining that the group belongs to a high risk group.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1

Selection of Multiple SNPs According to the Invention

In this example, DNA was isolated from blood of a Korean patient who was found to be a cardiovascular disease patient and a normal patient without cardiovascular disease symptoms, and the frequency of occurrence of a specific SNP was analyzed. The SNPs selected in this example are published databases (NCBI dbSNP: http://www.ncbi.nlm.nih.gov/SNP/) or realsnp.com (http: //: //www.realsnp.com/) of Sequenom. SNP sequences in the samples were analyzed using primers using sequences around the selected SNP.

1-1. Preparation of DNA Samples

DNA was extracted from the blood of male patients treated with myocardial infarction (221) and normal males (192) without myocardial infarction. Chromosome DNA extraction was performed according to known extraction methods (Molecular cloning: A Laboratory Manual, p 392, Sambrook, Fritsch and Maniatis, 2nd edition, Cold Spring Harbor Press, 1989) and the instructions of the commercial kit (Gentra system, D-50K). Was done. Among the extracted DNA, the purity standard was selected and used only at least 1.7 in the UV measurement ratio (260 / 280nm).

1-2. Amplification of Target DNA

Target DNA, a constant DNA region containing 705 SNPs to be analyzed, was amplified by PCR. PCR proceeded in a conventional manner, the conditions were as follows. First, chromosomal DNA was prepared at 2.5 ng / ml. Next, the following PCR reaction solution was prepared.

2.24 µl of water (HPLC grade)

0.5 μl 10x buffer (with 15 mM MgCl ₂ and 25 mM MgCl ₂ )

0.04 μl dNTP Mix (GIBCO) (25 mM / each)

Taq pol (HotStart) (5U / μl) 0.02μl

0.02 μl of potential / back primer mix (1 μM / each)

1.00 μl DNA

5.00 μl total reaction volume

Here, the forward and reverse primers were selected at appropriate positions from upstream and downstream of the SNPs of known data bases. Some of the 705 primer sets are summarized in Table 6.

Thermal cycling of PCR was maintained at 95 ° C. for 15 minutes, repeated for 30 seconds at 95 ° C., 30 seconds at 56 ° C., 1 minute at 72 ° C., and maintained at 72 ° C. for 3 minutes, followed by 4 ° C. Stored in. As a result, a target DNA fragment having a length of 200 nucleotides or less was obtained.

1-3. Analysis of SNPs in Amplified Target DNA

The analysis of SNPs in the target DNA fragments was carried out using a homogeneous Mass Extend (hME) technique from Sequenom. The principle of the MassEXTEND technique is as follows. First, a primer (also called extension primer) complementary to the base immediately before the SNP in the target DNA fragment is prepared. Next, the primer is hybridized to the target DNA fragment, and a DNA polymerization reaction is caused. At this time, a reagent (Termination mix; for example, ddTTP) to stop the reaction after the base complementary to the first allele base (for example, A allele) among the target SNP alleles is added to the reaction solution. Include it. As a result, when a first allele (eg, A allele) is present in the target DNA fragment, a product is obtained in which only one base (eg, T) complementary to the first allele is added. . On the other hand, if a second allele (eg, G allele) is present in the target DNA fragment, the closest first allele added with a base (eg, C) complementary to the second allele A product extending to the base (eg A) is obtained. By determining the length of the product extended from the primer thus obtained by mass spectrometry, it is possible to determine the type of allele present in the target DNA. Specific experimental conditions were as follows.

First, unbound dNTPs were removed from the PCR product. To this end, 1.53 μl of pure water, 0.17 μl of hME buffer, and 0.30 μl of SAP (shrimp alkaline phosphatase) were added to a 1.5 ml tube to prepare a SAP enzyme solution. The tube was centrifuged at 5,000 rpm for 10 seconds. After that, the PCR (Polymerase Chain Reaction) product was placed in the SAP solution tube, sealed, and kept at 37 ° C. for 20 minutes and 85 ° C. for 5 minutes, and then stored at 4 ° C.

Next, a homogeneous extension reaction was performed using the target DNA product as a template. The reaction solution was as follows.

1.728 μl of water (nano-grade pure water)

0.200 μl hME extension mix (10 × buffer with 2.25 mM d / ddNTPs)

0.054 μL extension primer (100 μM each)

0.018 μl Thermosequenase (32U / μl)

Total volume 2.00 μl

The reaction solution was mixed well, followed by spin down centrifugation. Seal the tube or plate containing the reaction solution well, hold for 2 minutes at 94 ℃, then repeat 5 seconds at 94 ℃, 5 seconds at 52 ℃, 5 seconds at 72 ℃ 40 times, and then 4 ℃ Stored in. The salts were removed from the homogeneous extended reaction product thus obtained using Resin (SpectroCLEAN, Sequenom company # 10053). Some of the 705 extension primers used in the extension reaction are shown in Table 6.

TABLE 6

SEQ ID No. including SNP Target DNA Amplification Primer (SEQ ID NO :) Extension Primer (SEQ ID NO) Potential primer Back primer One 36 37 38 2 39 40 41 3 42 43 44 4 45 46 47 5 48 49 50 6 51 52 53 7 54 55 56 8 57 58 59 9 60 61 62 10 63 64 65 11 66 67 68 12 69 70 71 13 72 73 74 14 75 76 77 15 78 79 80 16 81 82 83 17 84 85 86 18 87 88 89

Table 6 (continued)

SEQ ID No. including SNP Target DNA Amplification Primer (SEQ ID NO :) Extension Primer (SEQ ID NO) Potential primer Back primer 19 90 91 92 20 93 94 95 21 96 97 98 22 99 100 101 23 102 103 104 24 105 106 107 25 108 109 110 26 111 112 113 27 114 115 116 28 117 118 119 29 120 121 122 30 123 124 125 31 126 127 128 32 129 130 131 33 132 133 134 34 135 136 137 35 138 139 140

The obtained extended reaction product was analyzed by using a matrix assisted laser desorption and ionization-time of flight (MALDI-TOF) in the mass spectrometry. The MALDI-TOF is based on the principle of mass analysis by calculating the time taken to fly with an ionized matrix (3-Hydorxypicolinic acid) to fly to the opposite detector under vacuum when the material to be analyzed receives a laser beam. Works. The smaller mass arrives at the detector quickly, and the sequence of SNPs in the target DNA can be determined based on the difference in mass and the known sequence of SNPs.

Some of the results of determining the sequence of the SNP of the target DNA using the MALDI-TOF as described above are shown in Tables 1-2. At this time, each allele may exist in the form of a homozygote or heterozygote in each individual. Mendel's genetic and Hardy-Weinberg law states that the composition of the alleles that make up a group is maintained at a constant frequency and, if statistically significant, can give a biological function meaning. The 705 single SNPs of the present invention appeared in a statistically significant level in patients with cardiovascular disease, it can be seen that it can be used for diagnosis of cardiovascular disease.

1-4. Screening of Multiple SNPs

Based on the 705 SNP sequences of 221 cardiovascular disease patients (males) and 192 normal persons (males) analyzed above, a combination of SNPs frequently found in cardiovascular disease patients, ie, multiple SNPs, was selected.

First, it was confirmed that the possible number of multiple SNPs consisting of 1 to 3 combinations of the 705 SNP sequences is approximately 7.3 × 10 ⁹ .

11,582,361 multiple SNPs were selected using a genotype ratio calculated by the following formula based on the primary screening and having a genotype difference of 0.1 or more (the total number of patients, 221).

Genotype ratio = (number of patients with any genotype) / (number of normal persons with any genotype)

-Genotype difference = (number of patients with any genotype)-(number of normal persons with any genotype)

In addition, 5,348 multiple SNPs with p-values less than 10 ⁻⁶ were selected. The p-value represents Fisher's exact test and is a variable used to test more accurate statistical significance (p-value). As described above, in the present invention, when p-value ≦ 10 ⁻⁶ , the genotype acts as a risk factor or a protective factor. In other words, there was a significant correlation with the disease.

The odds ratio, its 95% confidence interval, and 99% confidence interval, used in the second screening criterion, was calculated.

Odds ratio: The number of patients with or without a genotype and normal persons, respectively, is as shown in Table 7 below: Odds ratio = ad / bc (when the odds ratio exceeds 1, the genotype is It is a risk factor for cardiovascular disease.)

TABLE 7

Numbers with specific multiple SNP genotypes Numbers that do not have a specific multiple SNP genotype Patient group frequency a b Normal group frequency c d

), 오즈비×exp(1.960

)) (상기 식에서, V=1/a + 1/b + 1/c +1/d)95% confidence interval of the odds ratio = (odds ratio xexp (-1.960)

), Ozbi × exp (1.960)

)) (Wherein, V = 1 / a + 1 / b + 1 / c + 1 / d)

), 오즈비×exp(2.576

)) (상기 식에서, V=1/a + 1/b + 1/c +1/d)99% confidence interval of the odds ratio = (odds ratio xexp (-2.576)

), Ozbi × exp (2.576

)) (Wherein, V = 1 / a + 1 / b + 1 / c + 1 / d)

For the 5,348 multiple SNPs selected above, the lower bound of the 95% confidence interval of the odds ratio is 2.0 or more, and the odds ratio is selected to be 3.0 or more, and then the odds ratio 2,826 multiple SNPs were selected by selecting the lower summer of the 99% confidence interval (odds ratio) of 2.0 or higher. Although each of these values is statistically significant above 1.0, their baseline values were 2.0, 3.0 and 2.0, respectively, in order to select strong markers.

The number of multiple SNPs is determined using the greedy method (Cormen et al. , "Introduction to Algorithms", MIT Press, 2001), which is one of the optimization methods for the 2,826 multiple SNPs selected above . Twelve multiple SNPs were selected that used less, each had a higher odds ratio, higher coverage for the patient group, and lower coverage for the normal group. The twelve multiple SNPs are shown in Tables 3-4.

Example 2

Fabrication of Multiple SNP-Finished Microarrays According to the Present Invention

The multiple SNPs selected above were fixed on a substrate to prepare a microarray. That is, in the polynucleotide consisting of each sequence number shown in Table 1, each SNP position (76th for SEQ ID NO: 4, 85th for SEQ ID NO: 8, 51st for SEQ ID NO: 9, 35th for SEQ ID NO: 10), A polynucleotide consisting of 20 contiguous bases containing 85 bases for SEQ ID No. 19 and 101 bases for the remaining SEQ ID Nos. (Each SNP is located on 11 of 20) was immobilized on a substrate. . Since there are two alleles at each SNP position, two polynucleotides are immobilized on the substrate for each sequence.

First, the N-terminus of each polynucleotide was substituted with an amine group and then spotted on a silylation slide (Telechem), wherein the spotting buffer was 2 × SSC (pH 7.0). After spotting, the mixture was placed in a dryer to induce binding, followed by washing with 0.2% SDS for 2 minutes and tertiary distilled water for 2 minutes to remove unbound oligos. The slides were treated at 95 ° C. for 2 minutes to induce denaturation, followed by 15 minutes with blocking solution (1.0 g NaBH ₄ , PBS (pH 7.4) 300 mL, EtOH 100 mL), 1 minute with 0.2% SDS solution, 3rd Each was washed with distilled water for 2 minutes and dried at room temperature to prepare a microarray.

Example 3

Cardiovascular disease diagnosis using microarray according to the present invention

Target DNA was isolated and fluorescently labeled from blood of a subject to diagnose the onset or possibility of developing cardiovascular disease using the method described in steps 1-1 and 1-2 of Example 1. Hybridization of the microarray prepared in Example 2 and the fluorescently labeled target DNA under UniHyb hybridization solution (TeleChem) was performed at 42 ° C. for 4 hours. It was washed twice with 2 x SSC for 5 minutes at room temperature and then dried in air. After drying, the slides were scanned with ScanArray 5000 (GSI Lumonics), and the scan results were analyzed with QuantArray (GSI Lumonics) and ImaGene software (BioDiscover), and the subjects were subjected to multiple SNPs according to the present invention. By confirming whether they had all or part, the likelihood of developing cardiovascular disease and susceptibility to cardiovascular disease was measured.

According to the SNP of the present invention, it can be used for applications related to cardiovascular diseases such as diagnosis and treatment of cardiovascular diseases. In addition, the microarray and kit including the SNP of the present invention can effectively diagnose cardiovascular disease. In addition, by the method of analyzing the SNP associated with the cardiovascular disease of the present invention, it is possible to effectively diagnose the presence or risk of the cardiovascular disease.

<110> Samsung Electronics Co., Ltd <120> Multiple SNP for diagnosing cardiovascular disease, microarray          and kit comprising the same, and method for diagnosing          cardiovascular disease using the same <130> PN067732 <160> 140 <170> KopatentIn 1.71 <210> 1 <211> 201 <212> DNA <213> Homo sapiens <400> 1 caccacatct cctgccccca tcccaagcaa gaggaacaag ggaagcccca gtgtacatgt 60 caaagagggc tgcaagctct gggcctcctg gaagccctaa wcttcctcca ggcaagaatc 120 tcttgcttct tgtcctttgt aaatctcacc tccttgcttt tagagatcca ggtttctgcc 180 ctctccctct cagcaaatct t 201 <210> 2 <211> 201 <212> DNA <213> Homo sapiens <400> 2 tggtgctgag tgcagctgga gatgccctgg ggtactacaa tgggaagtgg gagttcctcc 60 aggatgggga gaagatacac cggcagttgg cccagctggg yggcttggat gccctagacg 120 tgggaaggtg gagagttagt gacgacacag tgatgcactt ggccacagca gaagctcttg 180 tggaagctgg gaaagcccct a 201 <210> 3 <211> 201 <212> DNA <213> Homo sapiens <400> 3 tgagagagac aagaccgaga aggagctgag ggggcccccg gcttaccttc tgctgtagta 60 cccagaacaa cggcagcttc ttcccccggc cgggtcacga kgccctattt atagctgagg 120 ggtggggatg gagctgttcc caggctgcct gtgcacaggc tggagaggag ggttacatca 180 cttggccaga ccacaggctg g 201 <210> 4 <211> 176 <212> DNA <213> Homo sapiens <400> 4 gttctatatc tttatatttt caagtgacat gaaaacttct ttcttctctt gatcaaacag 60 ttgccatgac agagcygtct gtttgcctca ctggcactta tgtactaaat atgatgatcc 120 ctttagaaac ttactcggat ttgtattgtt tattatttgc tgaataagga caattt 176 <210> 5 <211> 201 <212> DNA <213> Homo sapiens <400> 5 gaccttgtga tctgccgccc tcagcctcct gaagtgctgg gattataggc gtgagccact 60 gtgcctggtc ctttttactt tttatttaca ttctctggac stcctagtct gtagtcttat 120 gccaacttta cagaagaaag gctagagttt cagaggagtt gtacaagtgg tccaaggtct 180 cacactagta agcagtagag a 201 <210> 6 <211> 170 <212> DNA <213> Homo sapiens <400> 6 tttccctttc tgggccagtg agaatcttcc aggccacacc catagtacaa ggacttcacc 60 agtcacggcc ttcctcatct gtttctctag cctcctcttg mtagcgtcca tgctctcatt 120 tcatcatcca tgggacttct tgtatgcctt tccctcttcc tgaaccttcc 170 <210> 7 <211> 201 <212> DNA <213> Homo sapiens <400> 7 ttcaatggta atgcaaagct aaatatcaat taattaaaga gttctggggg tggaaaaggg 60 gacaagggaa aggaaccact cttggttctt acaagatgga rtttgcaaga gaacttaaat 120 tacaaaggaa aaatggcatt aaattaatat ctaatttcta agctagttga ccaaattatt 180 tgaggcttat tggcgttctc t 201 <210> 8 <211> 185 <212> DNA <213> Homo sapiens <400> 8 tgcaaagttt attggaaaat caaaattttt ttattaattt actggatttc ctatacctaa 60 caatccttaa aacaactatc aacasctgca acacaaacca caggcaaaat gaaaaacaga 120 tgccccagac agcaccccac cacatggcac acacttaata aggaacaaaa tcctacaggg 180 tgctg 185 <210> 9 <211> 101 <212> DNA <213> Homo sapiens <400> 9 cttttttccc ctcctcttac cccgtatagg cagaagacac acatttggcc rtaagtgtga 60 attgccattg atgagtttca aagtcagtta tcatgccttg a 101 <210> 10 <211> 135 <212> DNA <213> Homo sapiens <400> 10 caaattcaaa gtctgtgtgc tcatgcactc gctgyggtgc ctcagacaaa ccaaacctaa 60 aagaagctgc aatggtggct tgaaatatta aaatatgaca aatcacacag gctgagaatt 120 tctgttaaca taatc 135 <210> 11 <211> 201 <212> DNA <213> Homo sapiens <400> 11 gctgtggatt ttgagagtca ggggaggggt gtgtgtgtga gggggtggct tactccggag 60 tctgggattc atcccgtcat ttctttcaat aaataattat yggatagcta ctttgtgaca 120 gggtctgtgc tgggctctgg gggcaaagct gtcatgggtg tcacctgcat agcatgtagc 180 catgtagcca tggcctagga a 201 <210> 12 <211> 201 <212> DNA <213> Homo sapiens <400> 12 ggaggattaa atgcccctta gttgcttagt aaataaacaa tagatattgt tagttttaga 60 ccagaaaaaa cacacattct ttaaaaggcc accagataat sctacatagg ttgtttaggg 120 taaatccttg tatgtcttta ataaggtaag tttggggaaa ctaacaaaac atatttgtct 180 tgctgttttg tattaataga g 201 <210> 13 <211> 201 <212> DNA <213> Homo sapiens <400> 13 gggacagccc ttcctgtgtg tacagcgggc ccacggccac cacactaccc tctggagtga 60 actgactgcc gcgaggcctg gggagctggc tcaccacctg kcctccgccg ttcaccctgt 120 ccactgcaga aacgacctcc taccctcaag ccggccccca acccccatgc acaggagccc 180 agccctcttg ggccccggct c 201 <210> 14 <211> 201 <212> DNA <213> Homo sapiens <400> 14 tcaaggagca aagctagtct gtgagcaaca agctacagca tcaaaagata caagcaagtg 60 tgggtttggg agccaggttt ctgcctggtg tgtgagtctg rgaaagtcag ttcctttcct 120 tgagccttgg tttcttcatt tttcttgtga ggattagagg agattagtga ctggcacata 180 ataagcagcc agcaaatggc c 201 <210> 15 <211> 201 <212> DNA <213> Homo sapiens <400> 15 gcatagtact cagactcatt gaaagtttat agaatttggc ctgtgtggaa aactctgtgc 60 tccaagtaca acaactaact gaattctcta atttaaacaa stttgaaggg aagtgaaagg 120 ttataaaatg atacagactc ataccgcaga atcaccttaa aatgcagctg ttggagaaaa 180 aaaaaaagga agctttatgc t 201 <210> 16 <211> 201 <212> DNA <213> Homo sapiens <400> 16 agaggcggcg gctccggaac gagctgctcc cacagcccct ccgctcctcg ccgtctcgcc 60 ttcgtcccca gcttacccag ttctgcgcgt tattgctcag yttgactttc ttgcacaggc 120 tcccggggat ctccatgacc gcggagcgcg ctgagcagcc ggggttctct gcgccgggan 180 ggtagcaaga ggagggcagg c 201 <210> 17 <211> 151 <212> DNA <213> Homo sapiens <400> 17 gattacaggc gtgagccacg gcacctggag agaaaaaagc ttctttaatg acgacctcaa 60 cataatttac atggagaagt ttcttaggtg gaaaaccgat ytgtatggtc ctcagaatgt 120 aacctacagg ttcttgccct cagaaaaatg c 151 <210> 18 <211> 201 <212> DNA <213> Homo sapiens <400> 18 cctccaaagg ggaaaccctg ccctcctggg cagacctttt ctttaagcgg cttctagatc 60 caccagggaa gaggggagac agcaagtgga acagacccag scaggaaagc ccgccctagt 120 ggctgcacac ccattccacc tccaggaaga tgtcacaact cctgttacgg tggcaataac 180 acaggccgga gcccagccaa g 201 <210> 19 <211> 185 <212> DNA <213> Homo sapiens <400> 19 ctgctttttc tcttggggct ggtagaagga ataaagagag tgagtgtgaa acagcccccg 60 cccctttgca cctgtgttct ctgtygacgt ggactgacac agttggtatt ttgctgggtg 120 tctctggacc ctttaacaca cactccatag atctgtgttc tgtgtctgcc tgcagagcct 180 ccacc 185 <210> 20 <211> 201 <212> DNA <213> Homo sapiens <400> 20 tgatttgatt ttatccagtg ttgcaaactg ggtttcgtaa gtgactcttt tgtaaggagt 60 ttcagtgcac cccgtctgca acccatgcat atcttattta mccccaagct tctaaggtag 120 aatcctactt ttttcaagtg ctattgatta gttgcctgtt tagacttttg ttatatgatt 180 taaaatttta aaaatgataa t 201 <210> 21 <211> 201 <212> DNA <213> Homo sapiens <400> 21 gggtatgttg gtcaagtatc ttgagcagat aaattttact aatagaaaag gaacaataat 60 gatactgttg tatgtgtgtg cacgcacaca cactaacacg ygcacaggaa agaaagaaaa 120 atgtactctg gcccatccat tctgtgccaa tttcccctgt tgggacttag tttaatccag 180 ccttttggct tgttcatcat a 201 <210> 22 <211> 201 <212> DNA <213> Homo sapiens <400> 22 tttttctgtt ctccatttcc tttaagtttg tttttagata cactgcattg caagtatgtg 60 tggtacagta aaatgttgct aattgtatct gcgctgataa satgccattt gctctaggtc 120 agtttcctac cccctccccc aactattcct agccagtggt agcatgcttc ttacttccaa 180 atatatgtgt gtgtgtgtgt g 201 <210> 23 <211> 201 <212> DNA <213> Homo sapiens <400> 23 gaagtattgg gaaaagatgt ccttaaccga aattataagc acatttagaa ggaaacaaaa 60 taaaaagagt tttcactcat tccagaattt tacaggcaat rgctattttc taatataatt 120 attttagcaa ataaatgttt gatttggatg tgctatgaca gtatcttcca tatttcattt 180 tattcattac atatttctga t 201 <210> 24 <211> 201 <212> DNA <213> Homo sapiens <400> 24 gctccatgga cttagtacct gggtgaccac cgtcagcaga gaagcatgtt tggaagacag 60 agaaccaatg agggatgttt cacactcttg aggagtttgc mgtcaaaaag caaagcagga 120 taagcaaaaa atgcatgtcc tcaaacccca gataacaaac acaaaacatc atagatagaa 180 ccagagctga ggtagataca c 201 <210> 25 <211> 201 <212> DNA <213> Homo sapiens <400> 25 ttaggaaatt ttcaatggag aaaataagtt ccaaaagtca ttctttctca gattaagatg 60 tcttgggttt cagtttactt ctttaaagaa agattcagag ytagtactga ggacccagac 120 tttcttgatg tgggaaatag ataattttct gttctgttga cattttttct ctctcttctc 180 tcattttcaa agtacagttc c 201 <210> 26 <211> 201 <212> DNA <213> Homo sapiens <400> 26 tagattaatt ggtgatttcg tacaataatt tgggagaaat taacattgca tttttatgtc 60 tttcgacgtg ataaatctcc tgatactcaa atgttctgtt ktgtacttca acaagtttca 120 taattttgtt cttatttaaa attcatgtaa atctttttct ggacttattt cttcatattg 180 tatagctgat gttactatca t 201 <210> 27 <211> 201 <212> DNA <213> Homo sapiens <400> 27 ataccattcc aaaaaatcta aaatatgcat tactgaagat tcttttggat aagaaaaaaa 60 aggaaatgta ctctgggcat gaacaataat ttttctgaag mcttaattct ttctaaaatg 120 gaaaactatt ggaaaactaa cctgcatggt tctaagaaca ttttccacaa aaaaaaaaaa 180 taataattca attcaaagat c 201 <210> 28 <211> 201 <212> DNA <213> Homo sapiens <400> 28 aaagctgagt aagcacctat agtaacaggt aacaaagtat tctagggatc agctgctgtg 60 agttagttca caagtgcttt aagtacgtgt taatcaaaga ygatgtattt ttcatttctg 120 acaataaaca tctggggcat cctacatttt tcctgggcaa tttgccttta tcattatatt 180 ttaatctctg atgcaatctt t 201 <210> 29 <211> 201 <212> DNA <213> Homo sapiens <400> 29 atgggagaga acaatagctc cctaggcaag aaaaagttac atatttgcaa aagccaagtt 60 ccagtgaagg agaataggta cagggattgc tctgagaagt ygtttgggag actggagaat 120 ttgctgtaca tgaagactac aatgagttgt gtttaagagt ctggtgcttt ggaatcagaa 180 agtttgagtt taaatcttta t 201 <210> 30 <211> 201 <212> DNA <213> Homo sapiens <400> 30 tgatatgact tcttgctagt gatttgtttc caggatctcc tcctagtata tccaaggcaa 60 tgtactattt catgggaggg gaatggcttt ataaagcttt ygtatctact ctctcacagt 120 aatttctggg gagtctgggg atttggatga caactttctt catttccaaa tctgaatcag 180 cattctggtt tcttggctat c 201 <210> 31 <211> 201 <212> DNA <213> Homo sapiens <400> 31 acagggatgg ggtcagattt ttccatcctc tggtggcagg gttggaggcc tgacaagaga 60 tcctctcttt ccctccttcc ccccttccca aggagcttca kccaagctga ggtcacctgg 120 agctgtgctc tgcactgtgc aagctcagca gccgggccct ggggcaggta aagctgtgga 180 ttcccaggtt ctggtgcctg c 201 <210> 32 <211> 201 <212> DNA <213> Homo sapiens <400> 32 cagagacctg gtcctaaatc tgtgtgactt ttgcgaaatc cttttgcctc acagagctct 60 aggttcttat ctgtcaaaca ggaaccagac catcagatcc rtagagatcc aaccaatgaa 120 ggagacttcg aaaacttcac attgccgtgt caatgtgagg ttgctggatg gttatgacaa 180 gaaatcacat taaattcact g 201 <210> 33 <211> 201 <212> DNA <213> Homo sapiens <400> 33 aaacccacag aaccaggcaa gagaccacct aaaattttgt tcacctaaaa ttctagagtt 60 ctttgagcaa accaaatgag tggttgtatc attttaaatt macaggaata gataggaagt 120 ctaggtcatt aacataattt gtgctctcag ttcatttttc aagtgggctt cctattgcat 180 ttttcttctg attcagttga g 201 <210> 34 <211> 201 <212> DNA <213> Homo sapiens <400> 34 gtatagaatg tctttccttc ccctacccaa cctgacaagc tcttctgctg cctccaagac 60 ccatttcaga tgacttctct tccagaaaaa ccttccttag wtcctgagac cgagctagtc 120 tgtcctttct gtgtgctccc acaaagcttt gttaaacact agtcacctca actatatgat 180 attctctctc tgactacctc a 201 <210> 35 <211> 201 <212> DNA <213> Homo sapiens <400> 35 gcaccattct caccctccta catcactctt gccagaaatt tgatgtacat ttgctattta 60 taagtaccct ttaggcactg tggattaaaa aaatacaaaa wgtgatggac agtgaactaa 120 agaacatttg gccaatgatt ccaaggttca atgaagctta gtacagttat tattattcaa 180 aatttagtac tttgtctttc a 201 <210> 36 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 36 acgttggatg ccccagtgta catgtcaaag 30 <210> 37 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 37 acgttggatg agcaagagat tcttgcctgg 30 <210> 38 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 38 cctcctggaa gccctaa 17 <210> 39 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 39 acgttggatg tgtgtcgtca ctaactctcc 30 <210> 40 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 40 acgttggatg agaagataca ccggcagttg 30 <210> 41 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 41 acgtctaggg catccaagcc 20 <210> 42 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 42 acgttggatg tgtagtaccc agaacaacgg 30 <210> 43 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 43 acgttggatg agcctgggaa cagctccatc 30 <210> 44 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 44 ttcccccggc cgggtcacga 20 <210> 45 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 45 acgttggatg ctcttgatca aacagttgcc 30 <210> 46 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 46 acgttggatg gtacataagt gccagtgagg 30 <210> 47 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 47 agttgccatg acagagc 17 <210> 48 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 48 acgttggatg cctttcttct gtaaagttgg c 31 <210> 49 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 49 acgttggatg ctgtgcctgg tcctttttac 30 <210> 50 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 50 tggcataaga ctacagacta gga 23 <210> 51 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 51 acgttggatg aggaagaggg aaaggcatac 30 <210> 52 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 52 acgttggatg cctcatctgt ttctctagcc 30 <210> 53 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 53 tgagagcatg gacgcta 17 <210> 54 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 54 acgttggatg gaaccactct tggttcttac 30 <210> 55 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 55 acgttggatg ggtcaactag cttagaaatt 30 <210> 56 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 56 tggttcttac aagatgga 18 <210> 57 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 57 acgttggatg cattttgcct gtggtttgtg 30 <210> 58 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 58 acgttggatg ctggatttcc tatacctaac 30 <210> 59 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 59 gcctgtggtt tgtgttgcag 20 <210> 60 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 60 acgttggatg tttttcccct cctcttaccc 30 <210> 61 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 61 acgttggatg ctttgaaact catcaatggc 30 <210> 62 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 62 aagacacaca tttggcc 17 <210> 63 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 63 acgttggatg tttcaagcca ccattgcagc 30 <210> 64 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 64 acgttggatg tcaaagtctg tgtgctcatg 30 <210> 65 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 65 ggtttgtctg aggcacc 17 <210> 66 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 66 acgttggatg cagaccctgt cacaaagtag 30 <210> 67 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 67 acgttggatg gggattcatc ccgtcatttc 30 <210> 68 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 68 gtcacaaagt agctatcc 18 <210> 69 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 69 acgttggatg cacattcttt aaaaggccac c 31 <210> 70 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 70 acgttggatg gttagtttcc ccaaacttac c 31 <210> 71 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 71 aaaggccacc agataat 17 <210> 72 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 72 acgttggatg cctctggagt gaactgactg 30 <210> 73 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 73 acgttggatg tcgtttctgc agtggacagg 30 <210> 74 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 74 gggagctggc tcaccacctg 20 <210> 75 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 75 acgttggatg acaagcaagt gtgggtttgg 30 <210> 76 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 76 acgttggatg caaggctcaa ggaaaggaac 30 <210> 77 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 77 ctgcctggtg tgtgagtctg 20 <210> 78 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 78 acgttggatg ctgcggtatg agtctgtatc 30 <210> 79 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 79 acgttggatg ctctgtgctc caagtacaac 30 <210> 80 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 80 aacctttcac ttcccttcaa a 21 <210> 81 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 81 acgttggatg ctccgcggtc atggagatc 29 <210> 82 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 82 acgttggatg ttacccagtt ctgcgcgtta 30 <210> 83 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 83 ctgtgcaaga aagtcaa 17 <210> 84 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 84 acgttggatg tttctgaggg caagaacctg 30 <210> 85 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 85 acgttggatg tggagaagtt tcttaggtgg 30 <210> 86 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 86 ttacattctg aggaccatac a 21 <210> 87 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 87 acgttggatg ggagacagca agtggaacag 30 <210> 88 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 88 acgttggatg atcttcctgg aggtggaatg 30 <210> 89 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 89 agcaagtgga acagacccag 20 <210> 90 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 90 acgttggatg gagacaccca gcaaaatacc 30 <210> 91 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 91 acgttggatg agagtgagtg tgaaacagcc 30 <210> 92 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 92 caactgtgtc agtccacgtc 20 <210> 93 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 93 acgttggatg gtaggattct accttagaag c 31 <210> 94 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 94 acgttggatg taaggagttt cagtgcaccc 30 <210> 95 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 95 accttagaag cttgggg 17 <210> 96 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 96 acgttggatg ctgttgtatg tgtgtgcacg 30 <210> 97 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 97 acgttggatg attggcacag aatggatggg 30 <210> 98 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 98 gcacacacac taacacg 17 <210> 99 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 99 acgttggatg tgcaagtatg tgtggtacag 30 <210> 100 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 100 acgttggatg gtaggaaact gacctagagc 30 <210> 101 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 101 aattgtatct gcgctgataa 20 <210> 102 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 102 acgttggatg agagttttca ctcattccag 30 <210> 103 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 103 acgttggatg gatactgtca tagcacatcc 30 <210> 104 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 104 tccagaattt tacaggcaat 20 <210> 105 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 105 acgttggatg gggtttgagg acatgcattt 30 <210> 106 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 106 acgttggatg cagagaacca atgagggatg 30 <210> 107 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 107 ctgctttgct ttttgac 17 <210> 108 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 108 acgttggatg tcaagaaagt ctgggtcctc 30 <210> 109 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 109 acgttggatg ctcagattaa gatgtcttgg g 31 <210> 110 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 110 ctgggtcctc agtacta 17 <210> 111 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 111 acgttggatg cgtgataaat ctcctgatac 30 <210> 112 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 112 acgttggatg gtccagaaaa agatttacat g 31 <210> 113 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 113 tcctgatact caaatgttct gtt 23 <210> 114 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 114 acgttggatg ggaaatgtac tctgggcatg 30 <210> 115 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 115 acgttggatg tgttcttaga accatgcagg 30 <210> 116 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 116 catgaacaat aatttttctg aag 23 <210> 117 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 117 acgttggatg aatgtaggat gccccagatg 30 <210> 118 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 118 acgttggatg cagctgctgt gagttagttc 30 <210> 119 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 119 ttgtcagaaa tgaaaaatac atc 23 <210> 120 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 120 acgttggatg gtacagcaaa ttctccagtc 30 <210> 121 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 121 acgttggatg aagccaagtt ccagtgaagg 30 <210> 122 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 122 tctccagtct cccaaac 17 <210> 123 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 123 acgttggatg ccagactccc cagaaattac 30 <210> 124 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 124 acgttggatg tgtactattt catgggaggg 30 <210> 125 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 125 ttactgtgag agagtagata c 21 <210> 126 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 126 acgttggatg atcctctctt tccctccttc 30 <210> 127 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 127 acgttggatg ttgcacagtg cagagcacag 30 <210> 128 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 128 ccccttccca aggagcttca 20 <210> 129 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 129 acgttggatg caaacaggaa ccagaccatc 30 <210> 130 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 130 acgttggatg agcaacctca cattgacacg 30 <210> 131 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 131 accagaccat cagatcc 17 <210> 132 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 132 acgttggatg tgttaatgac ctagacttcc 30 <210> 133 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <133> 133 acgttggatg tgagcaaacc aaatgagtgg 30 <210> 134 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 134 tagacttcct atctattcct gt 22 <210> 135 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 135 acgttggatg caagacccat ttcagatgac 30 <210> 136 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 136 acgttggatg cacagaaagg acagactagc 30 <210> 137 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 137 ttccagaaaa accttcctta g 21 <210> 138 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 138 acgttggatg gaaccttgga atcattggcc 30 <139> <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 139 acgttggatg taagtaccct ttaggcactg 30 <210> 140 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 140 agttcactgt ccatcac 17

Claims (16)

In the polynucleotides consisting of SEQ ID NOs: 1 to 35, each SNP position (76 th for SEQ ID NO: 4, 85 th for SEQ ID NO: 8, 51 th for SEQ ID NO: 9, 35 th for SEQ ID NO: 10, SEQ ID NO: Multiple SNPs for diagnosing cardiovascular disease comprising one or more polynucleotides selected from polynucleotides consisting of at least 10 consecutive bases comprising the 85th for 19 and the 101th for the remaining SEQ ID NOs) or complementary polynucleotides thereof . The method of claim 1, Wherein the at least one polynucleotide polynucleotide consisting of SEQ ID NOS: 1 to 35 each SNP position (76th for SEQ ID NO: 4, 85th for SEQ ID NO: 8, 51th for SEQ ID NO: 9, of SEQ ID NO: 10 The 35th, the 85th for SEQ ID NO: 19 and the 101st for the remaining SEQ ID NO: 19) in the group consisting of 1 to 12 selected from the combination of Table 3 from the polynucleotides consisting of 10 or more consecutive bases Multiple SNP for diagnosing cardiovascular disease, characterized in that the selected. TABLE 3 number Multi SNP One (SEQ ID NO: 2, SEQ ID NO: 25, SEQ ID NO: 3) 2 (SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 35) 3 (SEQ ID NO: 6, SEQ ID NO: 23, SEQ ID NO: 16) 4 (SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 13) 5 (SEQ ID NO: 17, SEQ ID NO: 1, SEQ ID NO: 31) 6 (SEQ ID NO: 5, SEQ ID NO: 29, SEQ ID NO: 8) 7 (SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 32) 8 (SEQ ID NO: 26, SEQ ID NO: 19, SEQ ID NO: 7) 9 (SEQ ID NO: 23, SEQ ID NO: 9, SEQ ID NO: 28) 10 (SEQ ID NO: 21, SEQ ID NO: 14, SEQ ID NO: 4) 11 (SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 33) 12 (SEQ ID NO: 24, SEQ ID NO: 12, SEQ ID NO: 34) The method of claim 2, The respective SNP positions of the selected polynucleotides (76th for SEQ ID NO: 4, 85th for SEQ ID NO: 8, 51st for SEQ ID NO: 9, 35th for SEQ ID NO: 10, 85th for SEQ ID NO: 19, and In the case of the remaining SEQ ID NO: 101) multiple SNP for diagnosing cardiovascular disease, wherein the alleles of the bases are shown in Table 4, respectively. TABLE 4 number Multi SNP Allele One (SEQ ID NO: 2, SEQ ID NO: 25, SEQ ID NO: 3) (CC, CC, TG) 2 (SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 35) (CC, GC, TT) 3 (SEQ ID NO: 6, SEQ ID NO: 23, SEQ ID NO: 16) (CC, AG, TT or TC) 4 (SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 13) (AA or AC, CG, TT or TG) 5 (SEQ ID NO: 17, SEQ ID NO: 1, SEQ ID NO: 31) (TC or CC, TT or TA, TT or TG) 6 (SEQ ID NO: 5, SEQ ID NO: 29, SEQ ID NO: 8) (GC, TT or TC, GC) 7 (SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 32) (CC, CC, AA or AG) 8 (SEQ ID NO: 26, SEQ ID NO: 19, SEQ ID NO: 7) (TG, TC or CC, GG) 9 (SEQ ID NO: 23, SEQ ID NO: 9, SEQ ID NO: 28) (AG, GG, TC or CC) 10 (SEQ ID NO: 21, SEQ ID NO: 14, SEQ ID NO: 4) (CT, AG or GG, CT or TT) 11 (SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 33) (TT or TC, CG or GG, AC) 12 (SEQ ID NO: 24, SEQ ID NO: 12, SEQ ID NO: 34) (AC, CC or CG, TA) The method of claim 1, The cardiovascular disease is multiple SNP for diagnosing cardiovascular disease, characterized in that selected from the group consisting of myocardial infarction, angina pectoris, atherosclerosis, hypertension, heart failure, aneurysm, arteriosclerosis, embolism, stroke and thrombosis. The method of claim 1, The cardiovascular disease is myocardial infarction, characterized in that multiple SNP for cardiovascular disease diagnosis. The method of claim 2, Multiple SNP for diagnosing cardiovascular disease, characterized in that consisting of the multiple SNP 1 to 12. The method of claim 1, The 10 or more consecutive base is a multi-SNP for diagnosing cardiovascular disease, characterized in that 10 to 100 consecutive base. A polynucleotide hybridizing with the polynucleotide of claim 1 or a complementary polynucleotide thereof. A microarray for diagnosing cardiovascular disease, comprising the polynucleotide of claim 1 or 8, a polypeptide encoded by the same, or a cDNA thereof. The method of claim 9, The polynucleotide microarray for diagnosing cardiovascular disease, characterized in that the polynucleotide is immobilized on a substrate coated with an active group selected from the group consisting of amino-silane, poly-L- lysine and aldehyde. The method of claim 10, The substrate is a microarray for diagnosing cardiovascular disease, characterized in that selected from the group consisting of silicon wafer, glass, quartz, metal and plastic. Cardiovascular disease diagnostic kit comprising the microarray of claim 9. a) separating the nucleic acid sample from the subject; And b) each SNP position of at least one polynucleotide selected from SEQ ID NOs: 1 to 35 (76 th for SEQ ID NO: 4, 85 th for SEQ ID NO: 8, 51 th for SEQ ID NO: 9, 35 th for SEQ ID NO: 10) , 85th in the case of SEQ ID NO: 19 and 101th in the case of the remaining SEQ ID NO: 19) determining the genotype of the polymorphic site that is the base; cardiovascular disease diagnostic method comprising a. The method of claim 13, B) hybridizing the nucleic acid sample isolated from the subject with the polynucleotide of claim 1 or claim 8; And b-2) detecting the hybridization result. The method of claim 13, c) determining that the subject belongs to a high risk group for cardiovascular disease when the genotype of the polymorphic site of at least one polynucleotide selected from SEQ ID NOs: 1 to 35 is at least one of 1 to 12 of Table 4. Additionally comprising. The method of claim 13, Wherein said subject is a male.