KR100761005B1 - Methods for the predicting and detecting silent brain infarction ischemic stroke patients using hyperhomocysteinemia by the polymorphism of thymidylate synthase enhancer region gene - Google Patents

Abstract

The present invention relates to a method for predicting or diagnosing asymptomatic cerebral infarction and onset of cerebral infarction by hyperhomocysteinemia using the gene polymorphism factor of the thymidylate synthase enhancer region (TSER).

The present invention relates to a genetic factor for predicting or diagnosing asymptomatic cerebral infarction and cerebral infarction caused by hyperhomocysteinemia including gene polymorphism of the thymidylate synthase enhancer site, and a thymidylate synthase enhancer site from DNA extracted from a subject sample. Thymidylate synthase enhancer resion) provides a method of predicting or diagnosing asymptomatic cerebral infarction and cerebral infarction caused by high homocysteineemia by measuring genotype and analyzing polymorphism from the measured genotype.

The present invention can provide a method that can easily predict or diagnose asymptomatic cerebral infarction and cerebral infarction caused by high homocysteineemia by measuring the presence of an allele comprising a mutation represented by TSER 2R.

Thymidylate synthase enhancer site, gene polymorphism, hyperhomocysteinemia, cerebral infarction, asymptomatic

Description

Methods for the predicting and detecting silent brain infarction & ischemic stroke patients using hyperhomocysteinemia by the polymorphism of thymidylate using polymorphism factor at the thymidylate synthase enhancer site synthase enhancer region gene}

Figure 1 is a schematic diagram showing the mechanism by which methylenetetrahydrofolate reductase and thymidylate synthase regulate the concentration of homocysteine in plasma using folic acid (5, 10-methylene THF),

Figure 2 is a photograph showing the allele according to the genotype of the 28bp repeat nucleotide sequence of the thymidylate synthase enhancer region of the present invention by electrophoresis.

The present invention relates to a method for predicting or diagnosing asymptomatic cerebral infarction and onset of cerebral infarction by hyperhomocysteinemia using the gene polymorphism factor of the thymidylate synthase enhancer region (TSER).

In general, a stroke is a neurological condition in which the blood vessels that supply blood to a part of the brain are blocked or broken, and the brain of the part is damaged, and the circulatory disorder of the brain that causes the stroke includes bleeding and I)

At this time, the hemorrhage includes cerebral hemorrhage, and the pleural blood flow includes cerebral thrombosis (腦血栓 症) and cerebral embolism (腦 塞 栓 症), which is referred to collectively as cerebral infarction (腦梗塞).

Among them, asymptomatic cerebral infarction is brain cell death due to clogging of blood vessels, but 'fortunately, dead brain cells are not so important that they have no symptoms such as paralysis.

As such, asymptomatic cerebral infarction and stroke (infarction) leave irreversible neurological damage once developed, and healing is impossible, so cause analysis and prevention are of paramount importance, and many risk factors are actively studied for this purpose. It's going on.

In addition, hyperhomocysteinemia is known as an independent risk factor for various vascular diseases such as cerebrovascular disease, cardiovascular disease, peripheral vein thrombosis, recurrent natural heritage. In addition, the plasma concentration of homocysteine is influenced by environmental and genetic factors. As a representative gene known to date, methylenetetrahydrofolate reductase (MTHFR) is known. Recently, however, thymidylate synthase, an enzyme that serves to convert deoxyuridylate monophosphate (dUMP) to deoxythymidylate monophosphate (dTMP) in normal people It has been reported that the genetic polymorphism of the enhancer region of TS) is a genetic determinant for controlling homocysteine concentrations in plasma (see FIG. 1).

As shown in FIG. 1, thymidylate synthetase has a competitive relationship using 5-methyltetrahydrofolate as a cofactor, like methylenetetrahydrofolate reductase. It has been proved to be closely related to homocysteine concentration determination.

In particular, the genetic polymorphism of thymidylate synthase is 2.6-3.6 times more mRNA than 28 repeat twice or three times in the 5'-UTR region, which is the promoter region of the gene, and twice repeated three times. It is known that the amount of expression is high, the protein activity is high. This is because there is a transcription factor binding site called upstream stimulatory factor (USF-1) USF-1 / USF-2) in the repeat sequence, and guanine (G), the 12th base of 28bp, is cytosine. In the case where the mutation is induced by (C), it has also been reported that the individual having three repetitions has the same enzyme activity as the individual having two repetitions.

In addition, the research on TSER is a target enzyme of 5-fluorouracil and 5-fluoropyrimidine-based anticancer agents which are widely used as therapeutic agents for colorectal cancer, gastric cancer and breast cancer. High activity of thymidylate synthase is known to produce resistance to anticancer drugs.

By the way, the frequency of homozygous twice repeated sequence in Western people was about 18-19%, which was about 5-6 times higher than 3.38% in Korean, and the result was pyrimidine synthesis regulation. It is thought that it plays an important role in the determination of homocysteine concentration in the plasma.

In addition, diseases caused by high homocysteinemia are known to be able to be treated and prevented by folic acid preparations.

Therefore, the study on the relation of homocysteine concentration of TSER gene is considered to be important for the cause analysis and prevention of stroke (cerebral infarction), the present invention can easily predict or determine the onset of pneumococcal stroke and asymptomatic cerebral infarction during stroke. To provide a way.

Therefore, the present inventors have attempted to develop a method for easily predicting or diagnosing the onset of ischemic cerebral infarction, that is, asymptomatic cerebral infarction and stroke (cerebral infarction), and the disease caused by high homocysteinemia is a folic acid agent. Based on the report that treatment and prevention are possible, analysis of the relation between the related gene and homocysteine concentration, mutation of the thymidylate synthase enhancer region (TSER) and ischemic stroke The present invention was completed by demonstrating the relevance to the onset and by demonstrating for the first time in the world that TSER is one of the homocysteine metabolism related genes in the control of homocysteine in cerebral infarction patients.

Accordingly, the present invention aims to enable early diagnosis of cerebral infarction and predictive increase in the amount of homocysteine through TSER genotyping, and to be used as a prognostic predictor for predicting the course of treatment of patients according to genotype. .

In order to achieve the above object, the present invention provides a genetic factor for predicting or diagnosing asymptomatic cerebral infarction and cerebral infarction caused by hyperhomocysteinemia including gene polymorphism of thymidylate synthase enhancer site.

In addition, in order to achieve another object, the present invention is to determine the genotype of the thymidylate synthase enhancer (SE) genome from the DNA extracted from the subject sample, and to analyze the polymorphism from the measured genotype to cause high homocysteinemia Provides a method for predicting or diagnosing asymptomatic cerebral infarction and cerebral infarction.

Hereinafter, the present invention will be described in detail.

At this time, if there is no other definition in the technical terms and scientific terms used, it has a meaning commonly understood by those of ordinary skill in the art.

In addition, repeated description of the same technical configuration and operation as in the prior art will be omitted.

According to the present invention, the homocysteine value of asymptomatic cerebral infarction and cerebral infarction patient group with TSER 2R (+) genotype in the correlation between genotype and homocysteine concentration is shown in the thymidylate synthase enhancer region. TSER 2R allele by demonstrating statistically significant comparison with control group and the statistically significant homocysteine value of TSER 2R (+) type compared to TSER 2R (-) type in cerebral infarction control group and patient group. To demonstrate that genotypes, including, acting as a cause of hyperhomocysteinemia, and to easily predict or diagnose hyperhomocysteinemia using TSER's DNA polymorphism as a predictor or diagnostic factor. Perform a precise cause analysis of stroke (infarction) and prevent the onset To make, and will have to appropriate treatment.

More specifically, the present inventors found that homocysteine values in asymptomatic cerebral infarction and cerebral infarction patients with TSR 2R (+) genotypes containing TSER 2R allele and 2R3R were statistically significantly higher than those of control group and cerebral infarction. The homocysteine value of TSER 2R (+) was significantly higher than that of TSER 2R (-) in the control and patient groups. This result shows that genotypes containing TSER 2R allele are responsible for the cause of hyperhomocysteinemia. However, unlike the asymptomatic cerebral infarction control group, the homocysteine value of TSER type 3R was significantly higher in the patient group than in TSER type 2R.

In the present invention, as described above, the mutant type of the repeat sequence region of the TSER promoter and its allele type are analyzed, and the indication thereof is TSER 2R3R when the allele of TSER 2R is 3R, and TSER 2R2R when 2R is used. It is written.

In the present invention, the TSER 2R allele (or genotype) is repeated twice a 28bp region of the TSER promoter, and 3R, 4R, 5R and 6R are repeated three, four, five and six times, respectively. Say what it is. And, the base sequence of 28bp is composed of 5'-CCG CGC CAC TTG GCC TGC CTC CGT CCC G-3 '.

Therefore, in the present invention, it is preferable to use an allele including a mutation represented by TSER 2R in TSER gene polymorphism (denoted TSER (+)), and more preferably TSER 2R2R, TSER 2R3R, TSER 2R4R. , Alleles selected from the group consisting of TSER 2R5R and TSER 2R6R are predicted or diagnosed asymptomatic onset of asymptomatic cerebral infarction or cerebral infarction using genetic factors for the prediction or diagnosis of hyperhomocysteinemia.

At this time, the mutant type of TSER 6R in the present invention is found in the world for the first time in the world of Korean (see Fig. 2).

In the present invention, in addition to the TSER 2R and 3R alleles, the 4R and 5R alleles were found to be 0.224% and 0.448% of the alleles in the normal control group, respectively, all of which were found to be less than 1.0% of the mutant type, and in the infarct patients group. The TSER 2R (+) type, which is known to have significantly higher homocysteine concentrations, demonstrated a slight increased risk than the TSER 2R (-) type.

In addition, in the present invention, the average value of homocysteine between the control group and the patient group did not show a significant difference, but in the TSER 2R (+) type, the homocysteine value between the patient group and the control group showed a significant difference (p = 0.0398).

In the present invention, the concentration of folic acid in the plasma concentration of folic acid is confirmed by the binding of TSER 2R (+) type, and the possibility of treatment with folic acid by hyperhomocysteinemia is expected.

Accordingly, the present invention predicts TSER gene polymorphism and the increase in the amount of homocysteine in accordance with the polymorphism and causes the asymptomatic cerebral infarction, thereby enabling the prediction and early diagnosis of asymptomatic cerebral infarction in advance, and to cerebral infarction. It is used to prevent and treat cerebral infarction by concurrently ingesting folic acid before proceeding.

In addition, in the present invention, homocysteine concentration in plasma is usually increased as a result of complex interaction between acquired factors and genetic factors, and as a representative factor, relative deficiency of folic acid, vitamin B6 and vitamin B12 is known. Two-thirds of patients with high homocysteine are caused by the relative deficiency of these vitamins (Selhub et al.), And administration of folate at 500 ug daily to patients at risk for vascular disease significantly reduces homocysteine levels. (Malinow et al.), By measuring TSER gene polymorphism and measuring the concentration of homocysteine including folic acid and folic acid from a subject sample to improve the accuracy of the prediction and diagnosis of asymptomatic cerebral infarction and cerebral infarction caused by high homocysteinemia. It is preferable.

On the other hand, the present invention is a sample according to the measurement items from cells, tissues, serum, blood, plasma, saliva, feces, vaginal secretion, lymph, cerebrospinal fluid, mucosal secretion, peritoneal fluid, ascites, umbilical cord blood, chorionic villus and wool of the subject. It can be prepared and used in different ways, double folic acid, homocysteine and gene can be analyzed at the same time, it is preferable to use a plasma that is easy to extract from the subject.

Hereinafter, the present invention will be described in more detail with reference to specific examples. However, the present invention is not limited to the following examples, and it will be apparent to those skilled in the art that various changes or modifications can be made within the spirit and scope of the present invention.

EXAMPLE

1. Purpose of Experiment

In general, high homocysteinemia in plasma is determined by the complex interaction of acquired and genetic factors. For example, relative deficiencies of folic acid, vitamin B6, and vitamin B12 are known. Enzymatic abnormalities such as cystathionine β-synthase (CBS), methionine synthase (MTR), methionine synthase reductase (MTRR), and methylenetetrahydrofolate reductase (MTHFR) are known.

Therefore, in this embodiment, the correlation between the TSER gene polymorphism of the present invention and folate concentration and MTHFR gene mutation among the above-mentioned factors causing high homocysteinemia was analyzed in patients with asymptomatic cerebral infarction and cerebral infarction, To determine the predictive or diagnostic effect of asymptomatic cerebral infarction and onset of cerebral infarction.

2. Subject

The test subjects used in this example were 275 patients diagnosed with Ischemic Stroke and cerebral infarction or other thrombosis among 498 patients who visited the Bundang Medical Center Neurology Department from March 1999 to February 2001. The mean age of the patients was 61.04 years (age: 27-91) and the control group was 60.13 years (age: 31-91).

The subjects were 99 patients (46 males, 53 females) diagnosed with asymptomatic cerebral infarction, and 92 controls (45 males and 47 females) without asymptomatic cerebral infarction or other thrombotic disease.

3. Experimental method

1) Homocysteine Measurement

Samples were collected from blood in a tube containing EDTA anticoagulant and centrifuged at 1,000 g for 15 minutes to use plasma. If the test was not possible immediately, it was stored at -70 ℃. Samples were to be taken after 12 hours of fasting. Homocysteine was measured using an IMx (Abbott, USA) instrument by FPIA (fluorescent polarizing immunoassay) method.

2) TSER and MTHFR Gene Analysis

DNA isolation was isolated from leukocytes extracted from the patient's blood by a DNA extraction kit (QIAmp blood kit, Qiagen) according to the manufacturer's protocol. TSER gene analysis shows that genomic DNA is stored in 10 mM Tris / 1 mM EDTA pH 8.0. ATG GCG CGG-3 ') was used to analyze by GeneAmp PCR machine (Perkin Elmer 2400). PCR-RFLP method for TSER was used (Horie et al.). For the MTHFR C677T polymorphism analysis, a known PCR-RFLP method was used.

4. Statistical Analysis

 The significance test of MTHFR and TSER gene mutations at each homocysteine level was performed using the Cochran-Mantel-Haenszel assay. The significance of the combination of MTHFR C677T, A1298C and TSER was analyzed using the statistical package SAS 8.2 for Windows using ANOVA.

5. Results

The incidence of TSER 28bp repetition sequence and MTHFR C677T mutant type in 275 patients with cerebral infarction and 223 normal controls were examined. The results were as follows.

(1) Relationship between MTHFR genotype and homocysteine concentration

As shown in Table 1, homocysteine (tHcy) concentrations were 9.89 ± 4.09 μM / mL and 10.72 ± 5.63 μM / mL in the control and patient groups, respectively, and the difference was not statistically significant (p = 0.0610). Folate concentrations also did not differ between the two groups (p = 0.8506).

(2) Frequency analysis of alleles according to genotype of TSER 28bp repeat sequence of Korean

As shown in Table 2, the frequency of TSER 2R allele was 20.5% and 17.9%, and the frequency of TSER 3R allele was 78.8% and 81.4%, respectively. TSER 4R and 5R alleles were observed with very low frequencies of 0.2%, 0.6%, 0.2% and 0.5% in patient and control groups, respectively. The frequency of these TSER genotypes was in the Hardy-Weinberg equilibrium in the Korean population.

In addition, alleles according to the genotype of TSER 28bp repeat nucleotide sequence are shown in FIG. 2 by electrophoresis.

(3) Comparison of Homocysteine Concentration According to TSER Genotype

As shown in Table 3 below, the odd ratio values were calculated by dividing the TSER gene into a TSER 2R (+) group including the TSER 2R allele and a TSER 2R (-) group not including the TSER 2R. The odds ratio (OR) of type 2R (-) to type TSER 2R (+) was 0.834 and 95% confidence interval (CI) was 0.575-1.210, slightly 1.2 than TSER 2R (-). 2 times higher risk. However, there was no statistical significance (p> 0.05).

※ AOR is adjusted odd ratio

As shown in Table 4 below, homocysteine values were significantly different between TSER 2R (+) type and TSER 2R (+) type when compared to control group and patient group. = 0.0398).

In addition, as shown in Table 5 below, 99 patients diagnosed with asymptomatic cerebral infarction and 92 normal controls without asymptomatic cerebral infarction or other thrombotic diseases were divided into TSER 2R3R (2R + type) and TSER 3R3R (2R-type). When comparing the control group and the patient group, homocysteine values were higher in the patient group in both genotypes. In the control group, TSER 2R3R type was higher than 3R3R type, but in the patient group, TSER 3R3R type was higher in TSER 3R type than in 2R3R type (p = 0.006). This is a different result from the infarction group. As a result, 50-80% of asymptomatic infarction patients progress to cerebral infarction, and further studies on the association between genotype and homocysteine are required.

And, as shown in Table 6, 491 participants except for 8 who do not have a homocysteine measurement of the 498 participants participated in the experiment was compared to make a combination between the TSER and MTHFR genotype difference of the homocysteine value. Comparing the MTHFR 677CC / TSER 2R (-) combination with the lowest homocysteine value, the MTHFR 677CT / TSER 2R (-) combination did not show any difference, but all the other combinations showed a significant difference in homocysteine.

The amount of homocysteine in the MTHFR genotypes differed by at least 1.0 ng / ml, depending on the TSER genotype. In addition, when compared with the MTHFR 677CC / TSER 2R (-) combination type with the lowest homocysteine value in folic acid concentrations of 263 patients, only the MTHFR 677CC / TSER 2R (+) combination type showed a significant difference. There was no significant difference.

As described above, the present invention provides a method for simply predicting or diagnosing asymptomatic cerebral infarction and cerebral infarction caused by high homocysteinemia by measuring the presence or absence of an allele including a mutation represented by TSER 2R in TSER gene polymorphism. I can do it.

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Claims (7)

delete delete Genetic polymorphism predicting or diagnosing asymptomatic cerebral infarction and cerebral infarction caused by high homocysteinemia consisting of a mutant gene in which the thymidylate synthase enhancer region (TSER) represented by the nucleotide sequence of SEQ ID NO: 1 is repeated twice and a mutant allele thereof factor. The method of claim 3, wherein Said allele is an allele selected from the group consisting of TSER 2R2R, TSER 2R3R, TSER 2R4R, TSER 2R5R, and TSER 2R6R. A gene polymorphism factor for predicting or diagnosing asymptomatic cerebral infarction and cerebral infarction by hyperhomocysteinemia. Here, TSER 2R represents a mutated genotype in which the nucleotide sequence of the thymidylate synthase enhancer region (TSER) is repeated twice, and 2R, 3R, 4R, 5R, and 6R shown after TSER 2R represent the thymidylate synthesis. The base sequence of the enzyme enhancer region shows an allelic type repeated two, three, four, five and six times, respectively. delete delete delete

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