KR20070082700A - Ucp-2 snp makers for the prediction of susceptibility to coronary heart disease and atheriosclerosis - Google Patents

Abstract

UCP-2(uncoupling protein-2) SNP(single nucleotide polymorphism) makers for the prediction of susceptibility to coronary heart disease and atherosclerosis are provided to analyze correlation between the UCP-2 SNP and obesity, especially indexes of blood cholesterol and atherosclerosis. The UCP-2 SNP makers for the prediction of susceptibility to coronary heart disease and atherosclerosis are -1957G>A SNP marker in which a -1957th base from the transcription-starting point of the UCP-2 gene(87th base of SEQ ID NO:1) is G, -866G>A SNP marker in which a -866th base from the transcription-starting point of the UCP-2 gene(63th base of SEQ ID NO:2) is G, +4787C>T SNP marker in which a 4784th base from the transcription-starting point of the UCP-2 gene(53th base of SEQ ID NO:3) is C, and +791_45d>i SNP marker in which 45 bp bases from 7941th base from the transcription-starting point of the UCP-2 gene(45 bp bases from 253th base of SEQ ID NO:4) are added or deleted. A UCP2 haplotype marker for the prediction of susceptibility to coronary heart disease, in which -866th and 4787th bases from the transcription-starting point of the UCP-2 gene are G and C, respectively, is also provided. A method for the prediction of susceptibility to coronary heart disease and atherosclerosis comprises the steps of: (1) isolating the genomic DNA from a blood sample; (2) detecting the UCP-2 SNP site from the isolated genomic DNA; and (3) confirming the detected UCP-2 SNP site.

Description

UCP-2 SNP makers for the prediction of susceptibility to coronary heart disease and atheriosclerosis}

1 shows the locations and allele frequencies of four known polymorphic sites,

1A is a chromosome map showing four polymorphic sites of UCP-2 present at chromosome 11q13,

FIG. 1B is a chart showing the correlation disequilibrium coefficients between the four polymorphisms of UCP-2,

FIG. 1C is a graph showing the association of four polymorphisms of UCP-2 and three haplotypes comprising them with cholesterol levels in blood.

The present invention relates to a human uncoupling protein-2 (UCP-2) polymorphism marker for predicting the risk of atherosclerosis and vascular disease, and more particularly, to a method for predicting the risk of the disease by using the same. The present invention relates to markers for predicting the risk of atherosclerosis and vascular disease by measuring the association between -2 monobasic polymorphism, HDL cholesterol index, and atherosclerosis index.

Uncoupling proteins (UCPs) are transporter proteins that move protons across the mitochondrial membrane and separate oxidative phosphorylation from ATP synthesis (Adams, SH, J. Nutr) . 130: 711-714, 2002). In addition, these mitochondrial proteins have been reported as important regulators of thermoregulation, body composition, metabolism and obesity (Fleury, C., et al ., Nat . Genet . 15: 269-272, 1997; Bouchard, C. , et al ., Hum . Mol . Genet ., 6: 1887-1889, 1997; Gong.DW, et al ., J. Biol . Chem ., 272: 24129-24132, 1997). In particular, UCP-2 is known to be widely expressed in most tissues and is therefore believed to play an important role in overall proton migration (Fleury, C., et al ., Nat . Genet . 15: 269-). 272, 1997; Gimeno. RE, et al ., Diabetes 46: 900-906, 1997).

In humans, the UCP-2 gene is associated with the UCP-3 gene present on chromosome 11q13, which contains several genes in one chromosome associated with hyperinsulinemia and obesity in mice. and forming a cluster) (Walder, K., et al, Hum Mol Genet, 7:......... 1431-1435, 1998; Gong, DW, et al, Biochem Biophys Res Commun 256: 27 -32, 1999). As such, the location and tissue distribution of the UCP-2 gene are thought to affect obesity within the range of energy metabolism.

Many studies have been conducted in vitro and in vivo experiments focusing on the functional effects of the UCP-2 gene. Expression outside the UCP-2 normal site in cultured mammalian cells or transformed yeast was reduced in Δp (Fleury, C., et al ., Nat . Genet . 15: 269 -272, 1997; Gimeno. RE, et al ., Diabetes 46: 900-906, 1997) and an increase in heat generation (Paulik, MA, et al., Pharm. Res., 15: 944-949, 1998). Induced, and this finding is related to the incoherence of mitochondrial respiration induced by UCP-2. UCP-2 expression in brown adipose tissues has been reported to be relatively slow by simultaneous reaction with UCP-1, which is mainly expressed in these tissues, and the resulting heatogenesis (Boss, O). , et al ., FEBS Lett ., 412: 111-114, 1997). UCP-2 expression has also been reported to increase and decrease in hyperthyroid and hypothyroid conditions, respectively (Masaki. T., et al., FEBS Lett., 418: 323-). 326, 1997). Genetic relevance studies of UCP-2 have been conducted mainly by examining the association between UCP-2 polymorphism and obesity and metabolic disorders. For example, the general G> A polymorphism present at the UCP-2 promoter site has been reported to be closely associated with increased adipose tissue mRNA expression in vitro (Esterbauer, H., et al ., Nat . Genet . , 18 (2): 178-183, 2001). In addition, the G / G and G / A genotypes reported more frequent occurrences in the obese population compared to the normal population. In contrast, variants of UCP-2 or UCP-3 have not been reported to be associated with body mass index (BMI) in young castor Indians, except for those over 45 of 790 subjects. Walder, K., et al ., Hum . Mol . Genet ., 7: 1431-1435, 1998). Therefore, further investigation should be carried out in groups other than the above to clarify the correlation between UCP-2 polymorphism and obesity.

Therefore, the present inventors have found a correlation between UCP-2 polymorphism, blood cholesterol index and arteriosclerosis index through the polymorphism investigation of the UCP-2 gene, and confirmed that it can be used to predict the risk of atherosclerosis and vascular disease. The invention was completed.

An object of the present invention is to provide a UCP-2 polymorphic marker for predicting the risk of atherosclerosis and vascular disease and a prediction method using the same.

The present invention provides a UCP-2 polymorphic marker for predicting the risk of atherosclerosis and vascular disease.

In addition, the present invention provides a UCP-2 haplotype for predicting the risk of atherosclerosis and vascular disease.

In addition, the present invention provides a kit for predicting the risk of atherosclerosis and vascular disease comprising a primer capable of amplifying a -866G> A or + 4787C> T polymorphic site of the UCP2 gene.

Hereinafter, the present invention will be described in detail.

The present invention provides a UCP-2 polymorphic marker for predicting the risk of atherosclerosis and vascular disease.

The uncoupling protein-2 (UCP-2) polymorphic markers include -1957G> A (SEQ ID NO: 87, G) and -866G> A (SEQ ID NO: 2) of the promoter region of the 5 'adjacent region of the UCP-2 gene. The 63rd sequence is G), + 4787C> T on exon 4 (53rd base of SEQ ID NO: 3 is C), and the base sequence of + 7941_45d> i (SEQ ID NO: 253 base to 45bp base) at exon 8 position Consisting of constructed polynucleotides. In this case, the sequence of the promoter region is included in GeneBank Accession Number NT_033927 (AP003717), and the sequence of NT_033927 (AP003717) number is a genome including a partial coding region of the human UCP-2 gene, 5 'adjacent region, and 3' UTP region. Sequences relating to DNA fragments.

In the polynucleotide, base G of the 1957 bp and 866 bp upstream of the promoter region of the 5 'region of the human UCP-2 gene was replaced by A to G, and the base C of 4787 bp on exon 4 was replaced by A to C. By point mutation and by addition or removal of 45 bp on top of 7941 bp on exon 8, the inventors have identified single nucleotide polymorphisms (SNPs) as -1957G> A and -866G> A, respectively. , + 4787C> T and + 7941_45d> i, respectively.

The genotypes of -1957G> A and -866G> A SNPs identified as described above and two polymorphisms, + 4787C> T and + 7941_45d> i, were analyzed in 794 Korean women. There was no correlation between body mass index, abdominal obesity, body fat weight and percentage body fat mass (see Table 5), but the two SNPs (-866G> A and + 4787C> T) were closely correlated with HDL cholesterol index (co-dominance). P = 0.0139 and p = 0.0108 respectively in subjects (see Table 3). Thus, the -866G> A and + 4787C> T SNPs of the present invention can be used as DNA markers useful in predicting the risk of vascular disease in Korean women.

In the present invention, it is not clear how the variant of the -866G> A SNP A allele reduces the HDL cholesterol index, but the variant of the A allele of -866G> A SNP is a human umbilical vein endothelial cells, HUVEC) and PAZ-6 human adipocyte cell lines have been reported to reduce transcription of reporter genes (Esterbauer. H., et al ., Nat . Genet . 28 (2): 178-183 , 2001; Oberkofler, H., et al ., Arterioscler . Thromb . Vasc . Biol . 25 (3): 604-610, 2005). In addition, a decrease in UCP-2 mRNA expression has been reported to induce a co-dominant increase in the production of reactive oxygen species (ROS) in endothelial and UCP-2 depleted mice (Arsenijevic, D., et. al ., Nat . Genet . 26 (4): 435-439, 2000). Therefore, the A allelic variant of -866G> A SNP is believed to reduce UCP-2 mRNA in tissues to increase free radical species as well as to co-dominantly reduce HDL cholesterol production.

In addition, the present invention provides a UCP-2 haplotype for predicting the risk of atherosclerosis and vascular disease.

In the present invention, the linkage disequilibrium coefficient at -866G> A and + 4787C> T of four SNPs of UCP-2 is very high, which means that the two SNP genes are closely related to each other (FIG. 1). Eventually, haplotype ht1 containing the -866G and + 4787C alleles was closely associated with the HDL cholesterol index (p = 0.0480 in dominant subjects, p = 0.0148 in codominant subjects, see Table 3), and atherosclerosis index. (artherogenic index, AI) has been shown to have a close correlation. Specifically, ht1 showed a relatively low value in the AI and LH ratio represents the atherogenic index (see Table 4) high atherogenic index onset disease (Lewis, GF, and Rader, DJ, Circ. Res. 96 ( 12): 1221-1232, 2005). Since the -866G> A and + 4787C> T of UCP-2 SNPs are known to be important risk factors, myocardial infarction and stroke It has been shown to be closely associated with the risk of vascular disease.

In addition, the present invention

1) separating genomic DNA from blood samples of the subject;

2) detecting the polymorphic site in the marker of claim 1 in genomic DNA isolated in step 1; And

3) It provides a method for predicting the risk of atherosclerosis and vascular disease comprising the step of identifying the polymorphic site detected in step 2.

In this case, the detection method of step 2) may include sequencing analysis, hybridization by microarray, allele specific PCR, dynamic allele-specific hybridization technique, and PCR extension analysis. Or it may be performed by the TaqMan technique, but is not limited thereto.

TaqMan methods (Livak, KJ, Genet . Anal ., 14: 143-149, 1999) include: 1) designing and constructing primers and TaqMan probes to amplify the desired DNA fragments; 2) labeling probes of different alleles with FAM dye and VIC dye (Applied Biosystems); 3) using the DNA as a template and performing PCR using the primers and probes; 4) after the PCR reaction is completed, analyzing and confirming the TaqMan assay plate with a nucleic acid analyzer; And 5) determining the genotype of the polynucleotides of step 1) from the analysis result.

In the above, sequencing analysis can use conventional methods for sequencing and can be performed using an automated genetic analyzer (Snager, F. et al ., Proc . Natl . Acad . Sci . USA ., 74 (12): 5463-5467, 1977; Maxam, AM and Gilbert, W., Proc . Natl . Acad. Sci . USA ., 74 (2): 560-564, 1997). In addition, allele-specific PCR refers to a PCR method of amplifying a DNA fragment in which the SNP is located with a primer set including a primer designed with the base where the SNP is located as the 3 'end. The principle of the method is, for example, when a specific base is substituted from G to A, by designing a primer comprising a G as a 3 'terminal base and a reverse primer capable of amplifying a DNA fragment of a suitable size When the reaction is performed, when the base of the SNP position is G, the amplification reaction is normally performed, and a band of the desired position is observed. When the base is substituted with A, the primer may complementarily bind to template DNA. The amplification reaction was not performed properly because the 3 'end was not complementary (Newton, CR et al ., Nucleic Acids Res ., 17 (1): 2503-2516, 1989). DASH can be performed by a method by Prince et al . (Prince, JA et al ., Genome Res . 11 (1): 152-162, 2001).

PCR extension analysis, on the other hand, first inactivates a DNA fragment containing a base on which a single nucleotide polymorphism is located by amplifying a primer pair, and then deactivating all nucleotides added to the reaction, thereby inactivating the SNP specific extension primer, by adding a dNTP mixture, didioxynucleotide, reaction buffer and DNA polymerase to effect primer extension. In this case, the extension primer is a 3 'end of the base immediately adjacent to the 5' direction of the base where the SNP is located, and the dNTP mixture excludes a nucleic acid having the same base as the didioxynucleotide, and the didioxynucleotide represents the SNP. It is selected from one of the base types. For example, in the case of a G to A substitution, when a mixture of dATP, dCTP and TTP and ddGTP are added to the reaction, the primer at the base where the substitution has occurred is extended by DNA polymerase and after several bases G The primer extension is terminated by ddGTP at the position where the base first appears. If the substitution has not occurred, since the extension reaction is terminated at the position, it is possible to determine the type of base representing the SNP by comparing the length of the extended primer.

In this case, as the detection method, when fluorescently labeling an extension primer or didioxynucleotide, the SNP can be detected by detecting fluorescence using a gene analyzer (for example, Model 3700, manufactured by ABI, etc.) used for determining a general sequence. (Chen, J., Genome Res ., 10 (4): 549-557, 2000), and matrix assisted laser desorption ionization-time of MALDI-TOF when using unlabeled extension primers and didioxynucleotides. The SNP can be detected by measuring molecular weight using the technique of flight (Ross, PL, Anal . Chem , 69 (20): 4197-4202, 1997).

In addition, the present invention provides a kit for predicting the risk of atherosclerosis and vascular disease including a primer capable of amplifying a -1957G> A, -866G> A, + 4787C> T or + 7941_45d> i polymorphic site of the UCP2 gene. do.

The primers in the kit were primers of SEQ ID NOs: 5 and 6, 7 and 8, 9, 10 and 11 and 12, respectively, in order for the -1957G> A, -866G> A, + 4787C> T and + 7941_45d> i polymorphic sites, respectively. By amplifying the polymorphic site, the base of the polymorphic site can be identified to predict the risk of atherosclerosis and vascular disease.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 UCP-2 SNP Identification on Chromosome 11q13

<1-1> Selection of Test Subjects and DNA Sampling from Samples

Genomic DNA was extracted from 794 women who visited Kirin Oriental Hospital (Seoul, South Korea). Specifically, after obtaining a blood sample to be tested under the approval of the Institutional Review Board of Korea Institute of Oriental Medicine (Accuprep ^TM Genomic DNA Extraction Kit, Bioneer Co., Korea) Extracted according to the manufacturer's instructions using. The mean age of the subjects was 27.9 years old and the mean body mass index (BMI) was 25.9 kg / m 2.

<1-2> PCR analysis and SNP identification

UCP-2 genome sequence from the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/) (GeneBank Accession No.:NT_033927, SEQ ID NO: 1 ) Genotype polymorphism sites are shown schematically in FIG. 1A (Esterbauer H et al., Nat Genet. 28 (2): 178-183, 2001; Walder K et al., Hum. Mol. Genet. 7: 1431-1435, 1998)

-1957G> A (SEQ ID NO: 87 is G or A), -866G> A (SEQ ID NO: 63 is G or A), + 4787C> T (SEQ ID NO: 53 is 53) Or T) polymorphism was detected using TaqMan probe PCR for polymorphism and normal PCR for + 7941_45 bp-in / del polymorphism. First, -1957G> A, -866G> A , + 4787C> T polymorphism with respect to, Primer Express (Applied Biosystems) using each of the primers and TaqMan probes (Livak, KJ, Genet Anal used, 14: 143-149, 1999) (Table 1). In addition, the probe of one allele was labeled with FAM dye (Applied Biosystems) and the probe of the other allele was labeled with VIC dye (Applied Biosystems), respectively.

PCR reactions were templated with genomic DNA (20 ng) of each study in 384-well format, producing a total of 5 μl of TaqMan Universal Master mix (Applied Biosystems) containing 900 nM of primers and 200 nM of TaqMan MGB-probe. 40 cycles were performed in a thermal cycler (PE 9700, Applied Biosystems) at 50 ° C. for 20 minutes, at 95 ° C. for 10 minutes, at 95 ° C. for 15 seconds, and at 60 ° C. for 1 minute. After the reaction, TaqMan assay intensity was measured with Prism 7900HT (Applied Biosystems) and then analyzed with automated software SDS 2.1 (Applied Biosystems).

The insertion / deletion polymorphism marker of UCP-2 SNP (+7941 45bp-in / del) (bases 253 to 297 of SEQ ID NO: 4) was located at exon 8 using primers set forth in SEQ ID NOs: 11 and 12. Gene fragment (SEQ ID NO: 4) was produced by amplification by PCR reaction. After PCR reaction conditions were denatured at 94 ° C. for 5 minutes, the reaction was repeated 35 times for 30 seconds at 94 ° C., 30 seconds at 65 ° C., and 30 seconds at 72 ° C., and finally amplified at 72 ° C. for 10 minutes.

The resulting product was electrophoresed on 1.5% agarose gel to confirm the size was 691bp or 649bp, respectively, and the sequencing kit (DynamicTM ET Dye Terminator Kit, Amersham Biosciences, UK) and a genetic analyzer (MegaBACE 500 Genetic Analyzer, Sequencing was performed according to the manufacturer's instructions using Amersham Biosciences, UK).

Then, using potential alignment software (ClustalX alignment software, Ver 1, http://bips.u-strasbg.fr/fr/Documentation/ClustalX/), the potential heterozygote positions were marked and present on the 11q13 chromosome. The UCP-2 SNP polymorphic sites are -1957G> A and -866G> A (promoter), one non-conservative polymorphism + 4787C> T (Aal55Val) (exon 4), and one insertion / deletion polymorphic site Four types of + 7941_45bp-in / del (3 ′ untranslated region) were identified (FIG. 1A).

The Hardy-Weinberg equilibrium was calculated to determine equilibrium at each position in the population between individuals, and ┃D'┃ and r ² to measure the association disparity of all pairs that could occur between the two alleles. (Hedric, PW, Genetics , 117: 331-341, 1987). As a result, the linkage disequilibrium coefficient between -866G> A and + 4787C> T was the highest as ┃D'┃ = 0.993, r ² = 0.960, which means that the relationship between the two SNPs is very high (FIG. 1B).

Example 2 Correlation Analysis between UCP-2 SNP and Cholesterol Cholesterol

<2-1> Identifying Characteristics of Research Subjects

The present inventors obtained the blood in Example <1-1> The general characteristics of 794 subjects were reviewed. Specifically, age, height and weight were measured, and body fat mass was measured using a commercially available bio-impedance analysis device (bio-impedance analysis, Inbody 2.0, Biospace Co., Korea). Body mass index (BMI) and waist hip ratio (WHR) were calculated from one height and weight, where body mass index is the mass (kg) divided by the height (m) squared. The pelvic pelvic ratio is the value of the waist circumference divided by the pelvic circumference The general physical characteristics of the study subjects are shown in Table 2. All measured values are expressed as mean ± standard deviation (SD).

Table 2-General physical characteristics of the women studied

Factor (unit) Mean ± S.D. Age (years) 27.88 ± 8.00 Height (cm) 160.66 ± 5.48 Weight (kg) 66.86 ± 11.74 Body mass index (kg / m ² ) 25.89 ± 4.27 % Fat 34.21 ± 6.06 Waist Pelvis 0.88 ± 0.07 Systolic Blood Pressure (mmHg) 115.38 ± 12.84 Diastolic Blood Pressure (mmHg) 72.35 ± 10.28

The age and body mass index-corrected univariate analysis of the variance was performed using a linear model (Ultra-model) to determine the individual correlations between UCP-2 SNP and LDL cholesterol , HDL cholesterol, and total cholesterol indices, depending on the variables to be performed below. general linear model). Statistical significance was established at the level of p <0.05 and all analyzes were performed using statistical analysis software (SAS Institute) for Windows, version 8.2.

<2-2> Analysis of association between SNP and haplotype and cholesterol index

Four SNPs and three haplotypes containing them (FIG. 1C) were corrected by age and body mass index and analyzed for association with blood cholesterol index.

First, in order to measure the blood cholesterol level, blood samples collected from the study subjects fasted for 12 hours or more were centrifuged at 2,000 rpm for 10 minutes, and only serum of the upper layer was obtained to automatically calculate the concentrations of total cholesterol, HDL cholesterol, and LDL cholesterol. It was measured using an auto-biochemical analyzer (SP-4410, Arkray Co., Japan).

As a result, as shown in Table 3, there was a close correlation between -866G> A and + 4787C> T in the UCP-2 SNP and the mean HDL-cholesterol index, which was as follows: GG genotype of -866G> A Mean HDL-cholesterol index is 74.26 mg / dL, type GA is 54.42 mg / dL, type AA is 49.73 mg / dL (co-dominant, p = 0.0139; recessive, p = 0.0035) and + 4787C> T. CC was 54.58 mg / CT, 54.25 mg / dL, and TT was 49.54 mg / dL (co-dominant, p = 0.0180; recessive, p = 0.0028). In addition, ht1 in the haplotype showed reverse association with the mean HDL-cholesterol index, and the mean HDL-cholesterol index was as follows: 50.68 mg / dL for the ht1 noncarrier and 54.04 for the heterozygote. mg / dL, homozygous ht1 carrier was 54.44 mg / dL.

On the other hand, unlike the high correlation between UCP-2 SNP and HDL-cholesterol index, it was confirmed that there is no correlation between UCP-2 SNP and total cholesterol and LDL-cholesterol index (Table 3).

Table 3-Correlation between UCP-2 Polymorphism and Blood Cholesterol in Korean Women

Phenotype location C / C C / R R / R P Dominant dominant zeal Total-cholesterol -1959G> A 390 (196.39 ± 54.44) 328 (196.33 ± 62.27) 66 (204.70 ± 57.59) 0.6049 0.6073 0.3284 -866G> A 224 (197.46 ± 55.13) 379 (196.49 ± 60.32) 191 (198.08 ± 58.07) 0.9971 0.9736 0.9578 + 4787C> T 232 (198.16 ± 55.43) 370 (196.05 ± 60.11) 189 (197.80 ± 58.65) 0.9723 0.8163 0.9670 + 7941_45d / i 523 (198.29 ± 58.78) 213 (195.72 ± 56.62) 32 (189.63 ± 42.40) 0.4649 0.4282 0.2513 ht1 220 (198.37 ± 59.28) 358 (196.20 ± 59.75) 216 (197.46 ± 54.94) 0.9855 0.8716 0.9923 ht2 418 (196.50 ± 55.88) 311 (196.29 ± 61.54) 65 (205.43 ± 57.72) 0.5767 0.6361 0.2989 ht3 555 (198.61 ± 60.03) 209 (193.92 ± 55.65) 30 (192.53 ± 41.20) 0.4405 0.2727 0.3359 LDL-cholesterol -1959G> A 390 (106.93 ± 29.31) 328 (104.94 ± 31.10) 66 (112.58 ± 30.88) 0.4578 0.9093 0.2506 -866G> A 224 (106.49 ± 30.66) 379 (105.28 ± 29.70) 191 (108.73 ± 30.60) 0.8021 0.9657 0.5460 + 4787C> T 232 (106.50 ± 31.00) 370 (105.25 ± 29.05) 189 (108.27 ± 31.10) 0.8965 0.9915 0.6664 + 7941_45d / i 523 (107.45 ± 31.18) 213 (104.73 ± 25.58) 32 (114.89 ± 34.00) 0.4628 0.5962 0.3745 ht1 220 (106.80 ± 31.23) 358 (105.84 ± 29.17) 216 (107.11 ± 30.86) 0.9649 0.8568 0.8015 ht2 418 (105.85 ± 29.86) 311 (105.82 ± 30.47) 65 (113.44 ± 30.52) 0.3882 0.5064 0.1756 ht3 555 (106.59 ± 31.47) 209 (104.34 ± 25.75) 30 (117.07 ± 32.45) 0.3798 0.8134 0.2212 HDL-cholesterol -1959G> A 390 (54.08 ± 17.58) 328 (52.98 ± 16.27) 66 (49.75 ± 15.48) 0.1829 0.1777 0.1287 -866G> A 224 (54.26 ± 16.37) 379 (54.42 ± 17.53) 191 (49.73 ± 15.97) 0.0139  0.3237 0.0035 + 4787C> T 232 (54.58 ± 17.04) 370 (54.25 ± 17.22) 189 (49.54 ± 15.90) 0.0108 0.1485 0.0028 + 7941_45d / i 523 (53.39 ± 15.80) 213 (53.68 ± 18.40) 32 (46.07 ± 12.13) 0.1090 0.4999 0.0352 ht1 220 (50.68 ± 17.43) 358 (54.04 ± 16.88) 216 (54.44 ± 16.32) 0.0480 0.0148 0.2016 ht2 418 (54.26 ± 18.36) 311 (52.67 ± 15.01) 65 (49.28 ± 15.23) 0.1238 0.0908 0.1025 ht5 555 (53.57 ± 16.49) 209 (53.48 ± 18.47) 30 (46.35 ± 12.44) 0.1804 0.4091 0.0668

(Mean ± standard deviation) and P values are obtained by linear model analysis corrected with age.

The ratio of C / C, C / R, and R / R means homozygotes divided into common (C) alleles, heterozygotes and homozygotes divided into rare (R) alleles.

ND means a value that is too small for statistical analysis.

Gothic figures indicate a statistically significant association with a P value of 0.05 or less.

Example 3 Correlation Analysis between UCP-2 SNP and Arteriosclerosis Index

The atherosclerotic index (AI), LH ratio, and TH ratio were calculated according to the following equations 1, 2, and 3, respectively, based on the blood cholesterol measurement values measured in Example 2-2. Takasaki, Y., J. Physiol . Anthropol.Human Sci ., 24 (4): 511-515, 2005).

Atherosclerosis index (AI) = (total cholesterol-HDL cholesterol) / HDL cholesterol

LH Ratio = LDL Cholesterol / HDL Cholesterol

AI (TH Ratio) = Total Cholesterol / HDL Cholesterol

<3-1> Analysis of association between polymorphism and arteriosclerosis index

First, Hardy-Weinberg's equilibrium was calculated to see if it had equilibrium at each position in the population between individuals, and D '측정 and to determine all pair association disparities that could occur between the two alleles. evaluated as r ² (Hedric, PW, Genetics , 117: 331-341, 1987). As a result, it was found that the atherosclerotic index (AI) was closely related to arterogenic index (AI). Specifically, ht1 carriers had very low atherosclerotic index (AI) and LH ratio (Equation 2). (Table 4).

Table 4-Correlation between UCP-2 Polymorphism and Atherosclerosis Index in Korean Women.

Phenotype location* C / C C / R R / R P Co-dominance dominant zeal AI -1957G> A 390 (2.63 ± 1.27) 328 (2.62 ± 1.33) 66 (2.94 ± 1.29) 0.3856 0.4614 0.1806 -866G> A 224 (2.60 ± 1.23) 379 (2.55 ± 1.24) 191 (2.90 ± 1.47) 0.0584 0.5024 0.0175 + 4787C> T 232 (2.59 ± 1.23) 370 (2.55 ± 1.24) 189 (2.90 ± 1.48) 0.0639 0.3511 0.0190 + 7941_45d / i 523 (2.64 ± 1.21) 213 (2.61 ± 1.47) 32 (3.23 ± 1.34) 0.1912 0.5303 0.0689 ht1 220 (2.83 ± 1.47) 358 (2.57 ± 1.24) 216 (2.60 ± 1.22) 0.1439 0.0494 0.3977 ht2 418 (2.61 ± 1.28) 311 (2.63 ± 1.33) 65 (2.98 ± 1.27) 0.3006 0.3332 0.1448 ht3 555 (2.62 ± 1.22) 209 (2.62 ± 1.48) 30 (3.27 ± 1.33) 0.1953 0.4248 0.0732 LH-B -1957G> A 390 (2.22 ± 1.03) 328 (2.23 ± 1.17) 66 (2.48 ± 1.10) 0.3923 0.3860 0.2023 -866G> A 224 (2.17 ± 0.99) 379 (2.18 ± 1.03) 191 (2.47 ± 1.30) 0.0433 0.2542 0.0127 + 4787C> T 232 (2.17 ± 0.99) 370 (2.19 ± 1.03) 189 (2.47 ± 1.31) 0.0465 0.1721 0.0154 + 7941_45d / i 523 (2.23 ± 1.01) 213 (2.25 ± 1.26) 32 (2.70 ± 1.12) 0.2166 0.3132 0.0929 ht1 220 (2.41 ± 1.28) 358 (2.20 ± 1.03) 216 (2.17 ± 0.98) 0.1107 0.0440 0.1884 ht2 418 (2.21 ± 1.04) 311 (2.25 ± 1.17) 65 (2.51 ± 1.09) 0.2868 0.2626 0.1570 ht3 555 (2.22 ± 1.02) 209 (2.26 ± 1.27) 30 (2.74 ± 1.10) 0.1865 0.2331 0.0890 TH-B -1957G> A 390 (3.60 ± 1.30) 328 (3.62 ± 1.33) 66 (3.87 ± 1.38) 0.5201 0.4048 0.3150 -866G> A 224 (3.58 ± 1.26) 379 (3.54 ± 1.25) 191 (3.88 ± 1.50) 0.0801 0.4854 0.0248 + 4787C> T 232 (3.57 ± 1.25) 370 (3.54 ± 1.25) 189 (3.88 ± 1.51) 0.0855 0.3414 0.0269 + 7941_45d / i 523 (3.62 ± 1.24) 213 (3.59 ± 1.49) 32 (4.23 ± 1.34) 0.1857 0.5417 0.0664 ht1 220 (3.81 ± 1.49) 358 (3.56 ± 1.25) 216 (3.58 ± 1.24) 0.1702 0.0613 0.3627 ht2 418 (3.59 ± 1.31) 311 (3.63 ± 1.33) 65 (3.90 ± 1.37) 0.4147 0.2977 0.2648 ht3 555 (3.61 ± 1.24) 209 (3.60 ± 1.50) 30 (4.27 ± 1.33) 0.1933 0.4421 0.0716

(Mean ± standard deviation) and P values are obtained by age-corrected linear model analysis.

The ratio of C / C, C / R, and R / R means the homozygotes divided into general alleles, the heterozygotes and homozygotes divided into lean alleles, respectively.

ND means a value that is too small for statistical analysis.

Body mass index is (cholesterol-HDL cholesterol) divided by HDL cholesterol, LH ratio is LDL cholesterol divided by HDL cholesterol, TH ratio means total cholesterol divided by HDL cholesterol.

Gothic figures indicate a statistically significant association with a P value of 0.05 or less.

Example 4 Correlation Analysis between UCP-2 SNP and Obesity Phenotype

Since the cholesterol index in the blood is closely associated with the obesity phenotype, two SNPs (-1957C> A and -866G> A) and HDL-cholesterol index among UCP-2 SNPs, although body mass index was corrected by statistical analysis It can be considered that the obesity phenotype is directly related. To confirm this, body mass index, waist hip ratio (WHR) and body fat mass index of all subjects were used as an index of UCP-2 polymorphism (Table 5).

As a result, none of the UCP-2 SNPs examined had a significant effect on the subject's body mass index, lumbar pelvic pelvis, or body fat mass index. It means that.

Table 5-Correlation between UCP-2 Polymorphism and Obesity Phenotype in Korean Women

Phenotype location* C / C C / R R / R P Co-dominance dominant zeal BMI -1957G> A 390 (25.82 ± 4.32) 328 (25.91 ± 4.24) 66 (26.17 ± 4.20) 0.7672 0.5215 0.5952 -866G> A 224 (25.89 ± 4.39) 379 (25.79 ± 4.35) 191 (26.07 ± 3.95) 0.8742 0.9509 0.6147 + 4787C> T 232 (25.89 ± 4.38) 370 (25.73 ± 4.34) 189 (26.14 ± 3.98) 0.6617 0.9933 0.3948 + 7941_45d / i 523 (25.96 ± 4.36) 213 (25.73 ± 4.13) 32 (26.65 ± 3.93) 0.5960 0.6483 0.4641 ht1 220 (26.00 ± 3.99) 358 (25.78 ± 4.35) 216 (25.94 ± 4.43) 0.9450 0.8299 0.8754 ht2 418 (25.79 ± 4.30) 311 (25.93 ± 4.25) 65 (26.25 ± 4.17) 0.6116 0.3745 0.4762 ht3 555 (25.93 ± 4.34) 209 (25.65 ± 4.12) 30 (26.82 ± 3.97) 0.4377 0.6229 0.3232 WHR -1957G> A 390 (0.88 ± 0.07) 328 (0.88 ± 0.07) 66 (0.88 ± 0.07) 0.9722 0.8125 0.9508 -866G> A 224 (0.88 ± 0.07) 379 (0.88 ± 0.07) 191 (0.88 ± 0.06) 0.9662 0.8202 0.9667 + 4787C> T 232 (0.88 ± 0.07) 370 (0.88 ± 0.07) 189 (0.88 ± 0.06) 0.9772 0.9810 0.8353 + 7941_45d / i 523 (0.88 ± 0.07) 213 (0.88 ± 0.07) 32 (0.89 ± 0.06) 0.7628 0.7854 0.5678 ht1 220 (0.88 ± 0.06) 358 (0.88 ± 0.07) 216 (0.88 ± 0.07) 0.9757 0.8342 0.9903 ht2 418 (0.88 ± 0.07) 311 (0.88 ± 0.07) 65 (0.88 ± 0.07) 0.8563 0.5775 0.8427 ht3 555 (0.88 ± 0.07) 209 (0.88 ± 0.07) 30 (0.90 ± 0.06) 0.6425 0.7510 0.4525 Fat mass -1957G> A 390 (23.41 ± 8.21) 328 (23.51 ± 7.85) 66 (23.13 ± 7.58) 0.9175 0.9332 0.7203 (Kg) -866G> A 224 (23.48 ± 8.17) 379 (23.45 ± 8.30) 191 (23.25 ± 7.15) 0.9252 0.8795 0.6934 + 4787C> T 232 (23.45 ± 8.12) 370 (23.38 ± 8.33) 189 (23.36 ± 7.18) 0.9914 0.9150 0.9122 + 7941_45d / i 523 (23.56 ± 8.11) 213 (23.05 ± 7.94) 32 (24.87 ± 7.41) 0.4194 0.6247 0.3065 ht1 220 (23.24 ± 7.17) 358 (23.44 ± 8.34) 216 (23.55 ± 8.24) 0.8839 0.6343 0.7526 ht2 418 (23.37 ± 8.16) 311 (23.51 ± 7.88) 65 (23.26 ± 7.54) 0.9388 0.8331 0.8418 ht3 555 (23.51 ± 8.03) 209 (22.94 ± 7.94) 30 (25.11 ± 7.55) 0.3028 0.5864 0.2226 Fat(%) -1957G> A 390 (34.16 ± 6.22) 328 (34.36 ± 6.00) 66 (33.94 ± 5.69) 0.8005 0.7478 0.6512 100 -866G> A 224 (34.13 ± 5.99) 379 (34.32 ± 6.29) 191 (34.11 ± 5.71) 0.8618 0.7848 0.7299 + 4787C> T 232 (34.16 ± 6.01) 370 (34.25 ± 6.29) 189 (34.15 ± 5.70) 0.9662 0.8899 0.8747 + 7941_45d / i 523 (34.29 ± 6.03) 213 (34.08 ± 6.30) 32 (34.84 ± 5.95) 0.7738 0.7821 0.5836 ht1 220 (34.12 ± 5.60) 358 (34.30 ± 6.36) 216 (34.16 ± 6.04) 0.8954 0.7274 0.8745 ht2 418 (34.11 ± 6.16) 311 (34.40 ± 6.02) 65 (34.02 ± 5.69) 0.7310 0.5654 0.7474 ht3 555 (34.25 ± 5.97) 209 (34.02 ± 6.30) 30 (34.88 ± 6.07) 0.7331 0.7717 0.5396

(Mean ± standard deviation) and P values are determined by age-corrected linear model analysis.

Wanted.

The ratio of C / C, C / R, and R / R means the homozygotes divided into general alleles, the heterozygotes and homozygotes divided into lean alleles, respectively.

ND means a value that is too small for statistical analysis.

As described above, -866G> A SNP of the UCP-2 gene according to the present invention is closely related to cholesterol and arteriosclerosis index in blood, and haplotype ht1 including the same has a significant correlation with HDL cholesterol index. The association of can be useful for effectively predicting the risk of polymorphism and atherosclerosis and vascular disease of the UCP-2 gene.

<110> Korea Institute of Oriental Medicine <120> UCP-2 SNP makers for the diagnosis of susceptibility to coronary          heart disease and atheriosclerosis <160> 12 <170> KopatentIn 1.71 <210> 1 <211> 162 <212> DNA <213> -1957G> A polymorphism <400> 1 gggtgcagcg tttgtatgtc tataggattg ctcagatctg cccccaccct gaaagaattt 60 aagagaattt cttgaggcca ggcacagtgg ctcacacctg taattccagt actgtgagag 120 tccgaggtca gaggactgct tgaggccagg agttcaagag ca 162 <210> 2 <211> 100 <212> DNA <213> -866G> A polymorphism <400> 2 ttctactccc caaacgcacg tgtttgtccc ggccagaggg cccaattgtt ggctgttcac 60 gcgtcagtta cccccacagg acgggtcagc caattaaagg 100 <210> 3 <211> 105 <212> DNA <213> + 4787C> T polymorphism <400> 3 gtcttggcct tgcagatcca aggagaaagt caggggccag tgcgcgctac agccagcgcc 60 cagtaccgcg gtgtgatggg caccattctg accatggtgc gtact 105 <210> 4 <211> 692 <212> DNA <213> + 7941_45del> ins polymorphism <400> 4 gaggtgcccc catgacctgt gatttttctc ctctaggttc atgccctcct ttctccgctt 60 gggttcctgg aacgtggtga tgttcgtcac ctatgagcag ctgaaacgag ccctcatggc 120 tgcctgcact tcccgagagg ctcccttctg agcctctcct gctgctgacc tgatcacctc 180 tggctttgtc tctagccggg ccatgctttc cttttcttcc ttctttctct tccctccttc 240 ccttctctcc ttccctcttt ccccacctct tccttccgct cctttaccta ccaccttdcc 300 ctctttctac attctcatct actcattgtc tcagtgctgg tggagttgac atttgacagt 360 gtgggaggcc tcgtaccagc caggatccca agcgtcccgt cccttggaaa gttcagccag 420 aatcttcgtc ctgcccccga cagcccagcc tagcccactt gtcatccata aagcaagctc 480 aaccttggcg tctcctccct ctcttgtagc tcttaccaga ggtcttggtc caatggcctt 540 tttggtacct ggtgggcagg ggaggaacca cctgactttg aaaatgggtg tgatccacct 600 tccacctcca gcatccaatc tgaagcccgt gtaggtcatc tggtccattt ctctctagac 660 ccaggccctg tactaacatg gggagtgcag ga 692 <210> 5 <211> 21 <212> DNA <213> -1957G> A forward primer <400> 5 gggtgcagcg tttgtatgtc t 21 <210> 6 <211> 20 <212> DNA <213> -1957G> A reverse primer <400> 6 tgctcttgaa ctcctggcct 20 <210> 7 <211> 20 <212> DNA <213> -866G> A forward primer <400> 7 ttctactccc caaacgcacg 20 <210> 8 <211> 21 <212> DNA <213> -866G> A reverse primer <400> 8 cctttaattg gctgacccgt c 21 <210> 9 <211> 20 <212> DNA <213> + 4787C> T (A55V) forward primer <400> 9 gtcttggcct tgcagatcca 20 <210> 10 <211> 22 <212> DNA <213> + 4787C> T (A55V) reverse primer <400> 10 agtacgcacc atggtcagaa tg 22 <210> 11 <211> 17 <212> DNA <213> + 7941_45del> ins forward primer <400> 11 gaggtgcccc catgacc 17 <210> 12 <211> 21 <212> DNA <213> + 7941_45del> ins reverse <400> 12 tcctgcactc cccatgttag t 21

Claims (11)

UCP-2 polymorphic marker for predicting the risk of atherosclerosis and vascular disease. The DNA sequence of claim 1, wherein the marker comprises -1957 base G from the transcription start point of the UCP-2 gene and G-8757 sequence of SEQ ID NO: 1 comprises the -1957 base. -1957G> A polymorphic marker, characterized in that the polynucleotide consisting of. According to claim 1, wherein the marker is 20-100 contiguous DNA from the start point of transcription of the UCP-2 gene is -866 base G (SEQ ID 63 is the 63rd sequence G) and the -866 base -866G> A polymorphic marker, characterized in that the polynucleotide consists of a sequence. According to claim 1, wherein the marker is from the start of the transcription of the UCP-2 gene 4784 base C (53 53 base of SEQ ID NO: 3) and 20-100 consecutive DNA sequence comprising the 4784 base + 4787C> T polymorphic marker, characterized in that the polynucleotide consists of. According to claim 1, wherein the marker is added to or removed 45 bp base (from base 253 of SEQ ID NO: 4 to bp 45 base) from the 7941 base from the transcription start point of the UCP-2 gene 20 before and after the addition or removal site + 7941_45d> i polymorphic marker characterized in that the polynucleotide consists of -100 consecutive DNA sequences. UCP-2 haplotype marker for predicting the risk of coronary artery disease. 7. The haplotype marker according to claim 6, wherein the marker is -866 base G and 4787 base C from the transcription start point of the UCP-2 gene. 1) separating genomic DNA from blood samples of the subject; 2) detecting the polymorphic site in the marker of claim 1 in genomic DNA isolated in step 1; And 3) A method for predicting the risk of atherosclerosis and vascular disease comprising identifying a polymorphic site detected in step 2. The method of claim 8, wherein step 2) comprises sequencing analysis, hybridization by microarray, allele specific PCR, dynamic allele-specific hybridization (DASH), PCR extension Method for predicting the risk of atherosclerosis and vascular disease, characterized in that performed by the analysis or TaqMan technique. Kit for predicting the risk of atherosclerosis and vascular disease comprising a primer pair capable of amplifying each of the -1957G> A, -866G> A, + 4787C> T or + 7941_45d> i polymorphic regions of the UCP2 gene. 10. The method of claim 9, wherein the primers are SEQ ID NOs: 5 and 6, 7 and 8, 9 and 10 and 11, respectively, in order for the -1957G> A, -866G> A, + 4787C> T and + 7941_45d> i polymorphic sites, respectively. Atherosclerosis and vascular disease risk prediction kit, characterized in that the primers and 12.

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