KR101946645B1 - Early diagnostic biomarker for essential hypertension in Korean women - Google Patents

Abstract

(SNP) located in the promoter region of the angiotensin converting enzyme (ACE) gene. The present invention also relates to a composition for diagnosing essential hypertension in a female female. More specifically, the present invention relates to a composition for the diagnosis of essential hypertension in Korean female, characterized by being rs4291, rs4292, and / or rs4363 among the angiotensin converting enzyme (ACE) gene related to the renin-angiotensin system, A diagnostic kit, and an information providing method for prediction. Ultimately, the present invention can provide diagnostic compositions, diagnostic kits, and information providing methods for predicting hypertension that enable early diagnosis of essential female hypertension in Korean, and can be used for early treatment of Korean female essential hypertension It is expected to be able to do.

Description

The purpose of this study was to investigate the biomarkers of essential hypertension in Korean women.

The present invention relates to a composition for early diagnosis of essential hypertension in Korean women comprising single nucleotide polymorphism (SNP) located in a promoter region of angiotensin converting enzyme (ACE) gene, more specifically, A composition for early diagnosis of essential hypertension in Korean female, characterized by being rs4291, rs4292, and / or rs4363 among the angiotensin converting enzyme genes related to the renin-angiotensin system, a diagnostic kit, and an information providing method for early prediction And the like.

The worldwide prevalence of hypertension is expected to rise to 29.2% (about 1.56 billion people) by 2025, and changes in dietary and lifestyle patterns in Korea are likely to increase hypertensive diseases in relation to cardiovascular disease . In 1998, the awareness rate of illness increased from 24.6% in 1998 to 65.8% in 2008, and the cure rate increased from 21.9% in 1998 to 58.7% in 2008. According to the 2007 Annual Statistical Yearbook of Health Insurance published jointly by the National Health Insurance Corporation and the National Health Insurance Corporation, the cost of hypertension related treatment increased to 63.3% in four years compared to 2003 and 2007. In Korea, the interest in hypertension has been steadily increasing, and as the cure rate and awareness have increased, the market for hypertension treatment has steadily increased, and interest in hypertension treatment and hypertension research has been increasing in Korea .

The main causes of hypertension are increased peripheral vascular resistance, action and mechanism of endothelin (ET), increased free radicals and nitric oxide, contraction of blood vessels due to insulin resistance, and renin-angiotensin system (renin-agiotensin system) abnormalities are known. Recently, the renin-angiotensin system, which has been actively researched recently, is a system in which renin converts angiotensinogen to angiotensin I and decreases angiotensin I Angiotensin converting enzyme (ACE) converts angiotensin II, which is an active hormone, to increase blood pressure in the body, sympathetic activation and electrolyte balance (see FIG. 1). The angiotensin converting enzyme, which is an enzyme that converts angiotensin I into angiotensin II, is a very important enzyme that contributes to elevation of blood pressure in blood vessels. The gene of angiotensin converting enzyme includes ACE gene, . The ACE gene has been studied so far, and I / D gene markers are a typical example.

Meanwhile, lifestyle-related diseases such as hypertension such as dietary habits and lifestyle changes are rapidly increasing. However, there are limitations in disease control through environmental factor control. Therefore, in order to effectively overcome them, understanding of underlying genetic factors In recent years, the development of single nucleotide polymorphism (SNP) analysis technology has revealed the linkage of disease outbreaks due to genotypes, and research on this has been actively conducted worldwide. Therefore, in Korea, it is expected that the incidence of diseases according to the genotypes of Koreans can be significantly lowered if the information is obtained.

Based on this background, the inventors of the present invention have conducted studies on a novel single nucleotide polymorphism capable of early diagnosis of the risk of essential hypertension specific to Korean women, and have completed the present invention.

To investigate the characteristics of single nucleotide polymorphisms in Korean female hypertensive patients using six known single nucleotide polymorphisms (SNP) markers in angiotensin converting enzyme, we selected six nucleotide polymorphic markers registered in the genotypse transgene gene dbSNP A case-control study of rs4291, rs4292, and / or rs4363 in minor recessive models could be used to identify rs4291, rs4292, and / or rs4363 as a composition for the early diagnosis of essential hypertension in Korean women And has reached the present invention. Therefore, the object of the present invention is to provide a composition for diagnosing essential hypertension in Korean female, characterized by being rs4291, rs4292, and / or rs4363 among angene-angiotensin system-related angiotensin converting enzyme (ACE) And a method for providing information for early prediction of a hypertension and a kit.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

The present invention provides a composition for diagnosing or predicting essential hypertension in Korean women comprising a single nucleotide polymorphism located in a promoter region of an angiotensin converting enzyme gene.

The present invention also provides a kit for the diagnosis or prediction of essential hypertension in Korean female, comprising a single nucleotide polymorphism located in the promoter region of angiotensin converting enzyme gene.

The present invention also provides a method of providing information for predicting Korean female essential hypertension, which confirms single nucleotide polymorphism information located in the promoter region of the angiotensin converting enzyme gene.

In one embodiment of the invention, the single base polymorphism is characterized by being rs4291, rs4292, and / or rs4363.

 Among the angiotensin converting enzyme (ACE) genes related to the renin-angiotensin system of the present invention, rs4291, rs4292, or rs4363 have an advantage of early diagnosis of Korean female essential hypertension. In particular, in the present invention, rs4291, rs4292, or rs4363 were specifically associated with essential hypertension in Korean women. Statistically, there was a significant difference in the concentration of angiotensin II in the blood according to the genotype of the single nucleotide polymorphism, It is expected to be a promising composition as well as an early diagnosis of essential hypertension. Ultimately, the present invention provides a method for early diagnosis of essential hypertension in Korean women characterized by rs4291, rs4292, or rs4363 mononucleotide polymorphism in the conversion enzyme (ACE) gene that enables early diagnosis or prediction of Korean female essential hypertension Or predictive compositions, kits and methods for providing information for predicting hypertension, and it is expected that it will be possible to prevent the early treatment of hypertension in Korean women or induce hypertension.

1 is a schematic diagram of a renin-angiotensin system.
Figure 2 shows the association disequilibrium for the 6 single nucleotide polymorphisms of the angiotensin converting enzyme gene (ACE).
FIG. 3 shows the results of the χ ² (Chi square) test showing the association of the single nucleotide polymorphism of the angiotensin converting enzyme gene (ACE) with hypertension.
FIG. 4 shows logistic regression analysis results in dominant modes of minor allele and recessive modes of minor allele of angiotensin converting enzyme gene (ACE).
FIG. 5 shows ELISA results showing the difference in blood concentration of three markers, angiotensin II, which is associated with hypertension among six single nucleotide polymorphisms of the angiotensin converting enzyme gene (ACE).

The present invention provides a composition for early diagnosis of essential hypertension in Korean women comprising a promoter region of an angiotensin converting enzyme (ACE) gene, a single nucleotide polymorphism (SNP) located in an intron and an exon do.

The present inventors investigated the characteristics of specific single nucleotide polymorphisms in Korean hypertensive female patients using 6 single nucleotide polymorphism (SNP) markers known from angiotensin converting enzyme. In the present invention, the linkage disequilibrium relationship between the six markers of the angiotensin converting enzyme gene was analyzed (see Fig. 2), and three markers rs4291 and rs4292 of six single nucleotide polymorphisms registered in the angiotensin converting enzyme gene dbSNP And / or rs4363 can be used as a composition for the diagnosis or prediction of essential hypertension in minor allele-recessive modes (see Example 3). Where rs is the abbreviation of the reference sequence and the numbers after it are the unique numbers representing each single base polymorphism provided by the NCBI dbSNP database. In case-control studies of rs4291, rs4292 or rs4363 among the 6 nucleotide polymorphisms registered in the angiotensin converting enzyme gene (ACE) dbSNP, rs4291, rs4292 or rs4363 single nucleotide polymorphisms were used And thus it is possible to predict or diagnose Korean female essential hypertension. Accordingly, the present invention provides a composition for early diagnosis or prediction of essential hypertension in Korean female comprising a single nucleotide polymorphism (SNP) located in a promoter region of an angiotensin converting enzyme gene.

Also provided is a kit for diagnosing or predicting essential hypertension in Korean women comprising a single nucleotide polymorphism (SNP) located in the promoter region of the angiotensin converting enzyme gene, and a method for providing information for predicting hypertension.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[ Example ]

Example  One. Angiotensin  Six of the transgene genes On the marker  Composition of a group to analyze the association with hypertension in Korea

Samples for analysis of diagnostic markers were distributed to the present invention in the Genomic Research Center for Cardiovascular Diseases and the National Institutes of Health, Yonsei University Medical Center. For analysis of hypertension diagnosis markers, a total of 1,417 Korean population were composed of 676 control group and 741 hypertensive patient group. Among the 577 men, 244 of the control group and 333 of the hypertensive patients were male, and 432 of the control group and 408 of the hypertensive group were female. Blood pressure was measured by taking a rest for at least 10 minutes and then measuring the blood pressure at two 3-minute intervals. When the systolic blood pressure was greater than 140 mmHg (SBP) or the diastolic blood pressure was greater than 90 mmHg (DBP) Respectively. (BMI), total cholesterol (TCHO), triglyceride (TG), high density lipoprotein cholesterol (HDL), and low density lipoprotein (HDL) were measured by age, sex, smoking and women. Cholesterol (LDL), blood sugar and the like.

Example  2. In the group DNA  Extraction and gene identification

To perform genotyping, each genomic DNA was extracted from the blood of the group constituted in Example 1 using a blood extraction kit (Qiagen, USA), and an angiotensin converting enzyme (ACE) gene Primers were designed to analyze the SNPs of rs4291, rs4292, introne 15 regions of promoter rs12447447, rs4371, rs4343 of exon 16 region and rs4363 of intron 24 region . The primer sequences are shown in Table 1 below. Then, a polymerase chain reaction was performed using the extracted genomic DNA and a primer. The amplified DNA was treated with Xba I, Hinf I and BstUI restriction enzymes, reacted at 37 ° C, The size of the cut DNA fragment was confirmed by electrophoresis.

[Table 1]

In addition, rs12447447 was subjected to genotyping using the product size difference upon electrophoresis after the PCR reaction according to the insert and deletion form, and the primer sequences used were as shown in Table 2 below . rs4371 and rs4363 were genotyped using the SNaPshot assay method (DNAlink, Korea).

[Table 2]

Example  3. Statistical analysis Angiotensin  Six of the transgene genes Marker  Analysis of association with hypertension

Statistical analysis was performed using χ ² (Chi square) test and logistic regression analysis for case-control studies to confirm the association of 6 markers of angiotensin-converting enzyme gene with hypertension. More specifically, in order to analyze the relationship between 6 single nucleotide polymorphisms of angiotensin-converting enzyme genes and hypertension, statistical tools (SAS Ver 9.1, excel, etc.) were used to calculate χ ² Respectively. The results are shown in Fig.

As shown in FIG. 3, among the six markers of the angiotensin converting enzyme gene, three rs4291, rs4292, and rs4363 markers were not found in the male, but in female recessive modes of the minor allele (Rs4291; P = 0.0371, rs4292; P = 0.0371, rs4363; P = 0.0282) were found to be associated with hypertension.

In addition, the risk of each genotype was analyzed by logistic regression analysis. The results are shown in Fig.

As shown in FIG. 4, minor allele-recessive modes of Korean women were analyzed for minor homozygotes of rs4291, rs4292, and rs4363 as risk factors. Both rs4291 and rs4292 were found to be 1.451 times more dangerous (95%; 1.021-2.06, P = 0.0377) than minor homozygotes and 1.495 times more likely to be minor homozygotes (rs4363) (95%; 1.042-2.132, P = 0.0287). There are three types of genotypes of rs4291, rs4292, and rs4363 of the angiotensin converting enzyme genes in the homologous chromosome pair, rs4291 is a homozygous AA (adenine adenine), TT (thymine thymine), and heterozygous AT (adenine thymine) is present. rs4292 has homozygous TT (thymine thymine) and CC (cytosine cytosine), and heterozygote TC (thymine cytosine). rs4363 has homozygous AA (adenine adenine) and GG (guanine guanine), and heterozygote AG (adenine guanine). These genotypes were analyzed in the form of minor dominant modes and minor recessive modes. Minor dominant modes are major isoforms. The minor recessive modes are the major homozygous and heterozygous, and the minor heterozygous. Minor isomeric conjugate.

Example  4. Angiotensin II Wow Angiotensin  Correlation analysis with converting enzyme genotype

In order to confirm the correlation between the amount of angiotensin II produced by angiotensin converting enzyme activity, which is known as a substance inducing an increase in blood pressure, and the genotype of angiotensin converting enzyme, the relationship with hypertension confirmed in Example 3 The enzyme-linked immunosorbent assay (ELISA) was performed using an Angiotensin II EIA kit (phoenix) to identify the concentration of angiotensin II in the blood. The rs4291 genotypes consisted of 12 AA genotypes, 13 AT genotypes, and 15 TT genotypes according to the rs4291 genotype. Twelve TT genotypes, 13 genotypes and 15 CC genotypes were identified according to the rs4292 genotype. A total of 40 persons were composed. According to the rs4363 genotype, there were 14 AA genotypes, 11 AG genotypes and 14 GG genotypes. The results are shown in Fig.

As shown in FIG. 5, when blood concentration of angiotensin II was measured by the enzyme binding assay in the general population, it was confirmed that the concentration of angiotensin II in blood was different according to the genotype in the control group. In the minor recessive modes, the minor heterozygotes were found to have a high blood concentration of angiotensin II. In rs4291, the genotype AA + AT was 0.1445 ng / mL, the TT type was 0.2325 ng / mL (P = 0.0220), the rT4292 TT + TC was 0.1445 ng / mL and the CC type was 0.2325 ng / mL P = 0.0220). In addition, in rs4363, the AA + AG type was 0.1465 ng / mL and the GG type was 0.2434 ng / mL (P = 0.0150), and the difference in blood concentration of angiotensin II between AT genotype and TT genotype was statistically significant .

These results indicate that single nucleotide polymorphisms of rs4291, rs4292, and / or rs4363 can be used as markers for early diagnosis of essential hypertension in Korean women.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

<110> CATHOLIC UNIVERSITY INDUSTRY ACADEMIC COOPERATION FOUNDATION <120> Early diagnostic biomarker for essential hypertension in Korean          women <130> PB12-10488 <150> KR 10-2011-0067056 <151> 2011-07-06 <160> 8 <170> Kopatentin 2.0 <210> 1 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> rs4291 forward primer <400> 1 tgtcactccg gaggcgggag gct 23 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> rs4291 reverse primer <400> 2 gagaaagggc ctcctctctg t 21 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> rs4292 forward primer <400> 3 accatggcct ggtgaagaag ccc 23 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> rs4292 reverse primer <400> 4 cggctctgcc ccttctcctg cgc 23 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> rs4343 forward primer <400> 5 ctgacgaatg tgatggccac 20 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> rs4343 reverse primer <400> 6 cctcctcaag gcccgacatg t 21 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> rs12447447 forward primer <400> 7 ctggagacca ctcccatcct ttct 24 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> rs12447447 reverse primer <400> 8 gatgtggcca tcacattcgt cagat 25

Claims (6)

Wherein the polynucleotide comprises at least one single nucleotide polymorphism selected from the group consisting of rs4363 located in the intron 24 region of the angiotensin converting enzyme gene, rs4291 located in the promoter region, and rs4292 located in the promoter region. For the diagnosis or prediction. delete A kit for diagnosing or predicting the onset of essential hypertension in Koreans comprising a polynucleotide comprising the polynucleotide of single nucleotide polymorphism of claim 1 and a polynucleotide hybridizing therewith. delete For gene samples from patients,
Identifying at least one single nucleotide polymorphism selected from the group consisting of rs4363 located in the intron 24 region of the angiotensin converting enzyme gene, rs4291 located in the promoter region, and rs4292 located in the promoter region, A method of providing information for predicting essential hypertension. 6. The method of claim 5,
When the rs4363 genotype was GG, the risk of Korean female essential hypertension was higher than that of AA or AG,
When the rs4291 genotype is TT, the risk of developing essential hypertension is higher than that of AA or AT,
The method of providing information for predicting that the genotype of rs4292 is higher than that of TT or TC in the case of CC is higher in the risk of Korean essential hypertension.

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