KR20030081967A - Mthfr 677tt genotype for prediction or prevention of an ischemic stroke - Google Patents

Abstract

PURPOSE: An MTHFR(methylenetetrahydrofolate reductase) 677TT genotype for the prediction and prevention of ischemic stroke is provided, thereby diagnosing and predicting ischemic stroke in an early stage. CONSTITUTION: An MTHFR(methylenetetrahydrofolate reductase) 677TT genotype for prediction of small-artery disease, multiple infarcts or multiple small-artery diseases caused by hyperhomocysteinemia is provided. A method for prevention of small-artery disease, multiple infarcts or multiple small-artery diseases comprises injecting folic acid or folic acid containing material which can decrease the concentration of homocysteine in blood to a person. A method for prediction of small-artery disease, multiple infarcts or multiple small-artery diseases comprises the steps of: collecting a DNA sample from a patient; amplifying DNA fragment containing 677th base of the MTHFR gene using sense primer and antisense primer; and inspecting the amplified DNA using restriction enzymes to identify the presence of MTHFR 677TT genotype in the DNA sample.

Description

MTHFR 677TT GENOTYPE FOR PREDICTION OR PREVENTION OF AN ISCHEMIC STROKE for predicting or preventing cerebral infarction

The present invention relates to the MTHFR 677TT genotype, which can be used for the prediction or early diagnosis of cerebral infarction and, at the same time, the use of folic acid or folate-containing homocysteine in combination with the intake of a substance that also enables the prevention of cerebral infarction. .

Homocysteine is a thiol containing amino acid produced by demethylation of methionine. Homocysteine is metabolized by remethylation. In the remethylation pathway, homocysteine is converted to methionine by methionine synthase, using vitamin B ₁₂ as cofactor and 5-methyl tetrahydrofolate as donor of methyl groups. Use This remethylation process plays a major role in maintaining homocysteine levels in the blood. The 5-methyl tetrahydrofolate used here is made from 5,10-methylenetetrahydrofolate (5,10-methylenetetrahydrofolate) produced by the ingestion of folic acid by the action of methylenetetrahydrofolate reductase (MTHFR) enzyme. . Therefore, when the function of the MTHFR enzyme is reduced, the amount of co-factor, 5-methyl tetrahydrofolate, is reduced, thereby reducing remethylation of homocysteine to methionine, which causes hyperhomocysteinemia, which increases the concentration of homocysteine in the blood. The C677T gene mutation of MTHFR, an enzyme that regulates the concentration of homocysteine in the blood, is shown by the substitution of alanine for valine as the 677th cytosine is converted to thymine. Among them, a person with 677TT genotype, a homozygous variant of MTHFR, is known to have a 67.6% reduction in MTHFR enzyme function compared to a normal 677CC genotype without mutation. Therefore, it can be seen that the homozygous mutation of the MTHFR gene is a potent causative factor for hyperhomocysteinemia. However, it is known that increasing the concentration of folic acid by increasing the intake of folic acid reduces the homocysteine concentration in the blood by 1/3 (Homocysteine lowering trialists' collaboration.Lowering blood homocysteine with folic acid based supplements: meta-analysis of randomised trials BMJ 1998; 316: 894-898). Malinow et al. (Malinow MR, Nieto FJ, Kruger WD, et al.The effects of folic acid supplementation on plasma total homocysteine are modulated by multivitamin use and methylenetetrahydrofolate reductase genotypes.Arterioscler Thromb Vasc Bio 1997; 17: 1157-1162) Patients with the 677TT genotype reported significantly lower levels of homocysteine in their blood when they increased folate intake than did 677CC or 677CT. Therefore, the administration of folic acid or folic acid-containing multivitamins, which are very inexpensive, easy to take and has few side effects, can be easily reduced in blood homocysteine concentrations by hyperhomocysteinemia. It can be seen that it can be very effective in preventing prophylactic diseases, especially cerebral infarction.

The pathophysiological mechanism by which hyperhomocysteinemia causes angiopathy is not yet clear, but is explained by the following hypotheses. Exposure of high levels of homocysteine to endothelial cells in the vessel wall decreases nitric oxide, increases levels of adhesion molecules, plasminogen activator inhibitor-1, tissue plasmino Progenogulant species such as gene plasminogen activator, C protein, and thrombomodulin are expressed. These actions promote platelet aggregation, leukocyte adsorption and thrombosis. The effect of homocysteine is mediated by the free-reducing form of homocysteine that promotes the production of superoxide, hydrogen peroxide, oxygen-derived free radicals and the like. In addition, homocysteine has been reported to promote the growth of vascular smooth muscle cells and exhibit neurotoxicity such as stimulating N-methyl-D-aspartate receptors. Recent studies have shown that homocysteine thiolactone via protein homocysteine acts on vascular endothelial cells.

MTHFR 677TT genotype was determined by Frosst et al. (Frosst P, Blom HJ, Milos R et al. A candidate genetic factor for vascular disease; a common mutation in methylenetetrahydrofolate reductase.Nature Genetics 1995; 10: 111-113) It is the first time that it is known. It is well known that hyperhomocysteinemia is an important independent risk factor for cerebral infarction, and many studies have shown that the MTHFR 677TT homozygous variant genotype causes hyperhomocysteinemia. Therefore, the MTHFR 677TT genotype was thought to be one of the major causes of cerebral infarction. However, numerous studies have been conducted in the past to confirm that the MTHFR 677TT genotype is a risk factor for cerebral infarction, but the results were negative. In other words, despite the fact that MTHFR 677TT genotype causes hyperhomocysteinemia and hyperhomocysteinemia causes cerebral infarction, most studies show that MTHFR 677TT genotype is not related to cerebral infarction.

However, as described above, the disease caused by hyperhomocysteinemia can be prevented before the onset of the disease through folic acid, which lowers the level of homocysteine in the blood, and thus, MTHFR 677TT genotype and In the present situation where only a relationship with cerebral infarction can be identified, it is possible to predict and prevent the occurrence of breakthrough cerebral infarction. The negation of the relationship between the MTHFR 677TT genotype and cerebral infarction has been a major obstacle to the preliminary prediction and prevention of cerebral infarction using the MTHFR 677TT genotype.

Homocysteine is known for 30 years, MTHFR 677TT genotype is known, but seven years have passed, but in the interest of the pathological diversity of cerebral infarction, as shown in the present invention by classifying the cerebral infarction showing various clinical syndromes into subtypes, homocysteine, MTHFR 677TT genotype And few attempts to confirm association with cerebral infarction.

Cerebral infarction can be characterized as the most heterogeneous clinical syndrome among several brain diseases, because the disease itself, which is called cerebral infarction, varies greatly among patients in terms of symptoms, severity, and prognosis, and causes and mechanisms of cerebral infarction vary widely. In addition, the prognosis and treatment are greatly affected by the etiology of cerebral infarction. Therefore, the present inventors felt the necessity to classify cerebral infarction according to the cause. In particular, since homocysteine is deposited on the vessel wall to cause angiopathy, small-vessel infarction and large-vessel infarction causing cerebral vessels have been of interest. However, the MTHFR 677TT genotype and the classification of small and large blood vessels are described by Eikelboom et al. (Eikelboom JW, Hankey GJ, Anand SS, Lofthouse E, Staples N, Baker RI.Association between high homocyst (e) ine and ischemicstroke due to large-and small-artery disease but not other etiologic subtypes of ischemic stroke.Stroke 2000; 31: 1069-1075). In these cases, homocysteine is associated with large vessel infarction but not with MTHFR 677TT genotype. . If we assume that the pathophysiology of homocysteine is deposited on the vascular wall depending on the size of the blood vessels and causes damage, the MTHFR 677TT genotype, the most important factor that causes hyperhomocysteinemia, is also associated with hypervascular cystosis if hyperhomocysteinemia is associated with large vessel infarction. Should be relevant. However, the results of Eikelboom et al. (2000) show that the two do not match.

As such, the association between the MTHFR 677TT genotype and cerebral infarction still forms an important research theme, and although many articles have been published, there are few experiments or papers demonstrating the association.

However, according to the results of the present inventors, both hyperhomocysteinemia and MTHFR 677TT genotype were significantly higher in small vessel infarction. This is a match between the two in small vessel infarction. If hyperhomocysteinemia is caused by the MTHFR 677TT genotype and angiopathy is caused by hyperhomocysteinemia, the two should be pathologically physiologically consistent with the results of the present inventors, unlike the results of Eikelboom et al. (2000). Conforms. In addition, the results of this study are consistent with previous findings in most papers that hyperhomocysteinemia is associated with cerebral small vessel infarction.

In addition, much attention has been paid to the fact that multivitamins can lower high levels of homocysteine in blood, and recent studies have shown that homocysteine in the blood when taking daily multivitamins containing folic acid 0.5-5 mg and vitamin B ₁₂ 0.5 mg The concentration can be reduced to about one third. AHA is the case of cholesterol and homocysteine in ischemic cerebral infarction by primary prevention guidelines provided by the (American Heart Association), vegetables, fruits, beans, meat, fish, cereals, such as enhanced recommended that the daily intake of folate, vitamin B _12. In this way, large-scale studies have been conducted to reduce the incidence of vascular diseases such as cerebral infarction by lowering homocystein in the blood, and statistically significant results were obtained. The world is paying attention to the prevention and treatment of dementia and cerebral infarction, which can be caused by hyperhomocysteinemia, by taking multivitamins containing folic acid, such as folic acid, which is difficult to take and has little to stop or side effects. .

The present invention demonstrates the association between the MTHFR C677T genotype and cerebral infarction, which have been previously negated, so that small-artery disease in which the C677T homozygous mutation of the MTHFR gene (MTHFR 677TT genotype) is a subtype of cerebral infarction (small-artery disease) By predicting the cause of multiple infarcts and multiple small-artery disease, which enables the prediction and early diagnosis of cerebral infarction and the prevention of cerebral infarction by concurrently taking multivitamins such as folic acid. It is an object of the present invention to provide a MTHFR 677TT genotype that can be used for the purpose of making it possible.

Figure 1 is an electrophoresis picture showing the results of testing the MTHFR C677T gene polymorphism (MTHFR 677CC, MTHFR 677CT and MTHFR 677TT) by cleaving the MTHFR gene using the restriction enzyme Hin f1 RFRF method.

2 is a schematic showing the metabolic pathway of homocysteine.

Figure 3 shows the adjusted OR and 95% CI of the MTHFR 677TT genotype as a log graph, where 3a compares the MTHFR 677TT homozygous variation with the 677CC (normal) genotype without mutation, and 3b homologates the MTHFR 677TT homozygous variation. All non-conjugated mutations-MTHFR 677CC (normal) and MTHFR 677CT (heterozygous variation) combined are shown.

The present invention relates to the MTHFR 677TT genotype that can be used for early diagnosis, prediction and prevention of cerebral infarction.

According to the present invention, when MTHFR C677T polymorphism is detected, when MTHFR 677TT genotype is found, small-artery disease, multiple infarcts and multiple small-artery disease may occur. The risk was found to be high. All of these patient groups were confirmed to have hyperhomocysteinemia, which was thought to be due to the damage of the inner wall of blood vessels due to the increase of blood homocysteine concentration. As described above, hyperhomocysteinemia can reduce blood homocysteine concentrations by administering folic acid or folic acid-containing substances, thereby reducing vascular damage. Therefore, if homocysteine levels are high among patients with the MTHFR 677TT genotype, folic acid or folic acid may be used. It may be possible to prevent cerebral infarction by administering a containing substance.

Accordingly, according to the present invention, the MTHFR 677TT genotype can be used to predict and diagnose cerebral infarction of the subtypes, and at the same time, the occurrence of cerebral infarction of the subtypes by administration of folic acid or an agent containing folic acid that lowers homocysteine levels in the blood. It is possible to prevent.

Hyperhomocysteinemia is known as an independent risk factor of cerebral infarction. Increasing homocysteine levels in blood is known to be caused by impaired remethylation due to decreased activity of methylenetetrahydrofolate reductase (MTHFR) enzyme. Figure 2 shows the mechanism of action in homocysteine metabolism of MTHFR enzyme. MTHFR is an enzyme involved in the metabolic process of homocysteine to methionine. When the activity of the enzyme decreases, the level of homocysteine remaining without being metabolized to methionine increases, causing hyperhomocysteinemia. This decrease in MTHFR enzyme activity is known to be due to mutations in the MTHFR C677T gene. Chango et al. (Chango A, Boisson F, Barbe F, Quilliot D, Droesch S, Pfister M, et al.The effect of 677C-> T and 1298A-> C mutations on plasma homocysteine and 5,10-methylenetetrahydrofolate reductase activity in healthy subjects.Br J Nutr 2000; 83 (6): 593-596) In experiments with healthy adults, among MTHFR C677T gene mutations, the MTHFR enzyme with the MTHFR 677CT genotype, the MTHFR heterozygous mutation, The function is reduced by 32.4% compared to MTHFR with normal (MTHFR 677CC) genotype, and MTHFR enzyme with homozygous mutation MTHFR 677TT genotype is reduced by 64.4% compared to normal MTHFR enzyme. Thus, it can be seen that among the MTHFR C677T gene mutations, the homozygous mutant genotype (MTHFR 677TT) has a stronger effect on high homocysteine authentication.

As such, it is well known by several reports that the C677T polymorphism of the MTHFR gene (Genebank, accession no. UO9806) is associated with hyperhomocysteinemia, but its association with cerebral infarction has not been reported. It is not clear yet. Many interpretations have been made as to the cause of this phenomenon. One of the causes is that the MTHFR 677TT genotype, a potent factor that increases the level of homocysteine in the blood, is associated with a specific subtype rather than the entire stroke. In other words, because a particular subtype may or may not be related to another subtype, the entire set of all of them may seem irrelevant.

We did not treat cerebral infarction showing various clinical syndromes as one disease, but divided them into subtypes based on the most common international classification of "TOAST", and compared each of these subtypes with the MTHFR 677TT genotype. The TOAST international classification is a method of classification by etiology, in which cerebral infarction is classified into small-artery disease, large-artery disease, cardioembolism, other determined etiology, And five subtypes of undetermined etiology if the cause is not clearly identified. Among them, cerebrovascular involvement is small-vessel infarction and large-vessel infarction (cardiopulmonary infarction is related to heart). Given that the pathophysiology of homocysteine is responsible for vascular damage, the inventors have focused on small vessel infarction and large vessel infarction associated with cerebrovascular.

In one specific example of the present invention, whether the MTHFR C677T homozygous mutation is a subtype of ischemic cerebral infarction or whether it is a risk factor for large-vessel infarction or small-vessel infarction was examined through comparative risk analysis. As a result, when the MTHFR 677TT genotype was compared to all cerebral infarction patients without subclassification, there was no significant difference from the normal control group as in the previous studies. However, when they were subdivided into subtypes and compared, they found significant differences in small vessel infarction. In other words, many previous studies have shown that MTHFR 677TT genotype was not related to cerebral infarction because cerebral infarction itself is a complex of heterogeneous diseases caused by a wide variety of causes, and these MTHFR 677TT genotypes are subtypes of cerebral infarction. One of the causes of small blood vessels infarction was confirmed.

The frequency of the MTHFR 677TT genotype was significantly higher in the small-vessel infarction group than in the large-vessel infarction group, and homocysteine levels were also significantly increased in the small-vessel infarction group (see Table 3). This is because a decrease in the activity of MTHFR enzyme due to genetic mutation increases the concentration of homocysteine in the blood and acts in the intracranial blood vessels, especially penetrating small blood vessels, and promotes the proliferation of vascular smooth muscle cells, thereby leading to cerebral microangiopathy. It is thought to generate.

As described above, the results of Eikelboom et al. (2000) on the association between MTHFR 677TT genotype, hyperhomocysteinemia and macrovascular infarction showed conflicting results, but the present invention found hyperhomocysteinemia in small vessel infarction. The MTHFR 677TT genotype was also significantly higher, indicating a consistent match. This is considered reasonable even when considering the pathophysiology of homocysteine, which is consistent with previous studies. In addition, the results of the present inventors provided another recognition for classifying the causes of vascular pathologies into small vessels and large vessels, and suggested a more advanced method for predicting and preventing cerebral infarction.

In another specific example of the present invention, as a method of classifying a subtype of a second cerebral infarction, brain magnetic resonance imaging (brain magnetic resonance imaging, brain MRI) is used to investigate the number of brain lesions, so that a single cerebral infarction and multiple cerebral infarction The classification method was used. These classification methods were classified using brain computed tomography (brain CT), and were classified by examining either brain computed tomography or brain magnetic resonance imaging. However, brain magnetic resonance imaging has superior discrimination ability compared to brain computerized imaging, and even when it is judged as a single cerebral infarction by brain CT imaging, it is relatively common to change to multiple cerebral infarction by brain magnetic resonance imaging. Was classified into brain MRI only.

The present inventors measured the MTHFR C677T gene polymorphism and blood homocysteine concentration to determine whether it is a risk factor of single cerebral infarction or multiple cerebral infarction classified as subtypes of cerebral infarction through comparative risk analysis. As a result, the risk of multiple cerebral infarction with the 677TT genotype of homozygous mutation of MTHFR was significantly higher than that of the normal control group, and hyperhomocysteinemia was also shown in the same result (see Table 4). However, single cerebral infarction showed no significant results in both hyperhomocysteinemia and MTHFR 677TT genotypes. This confirms a significant increase in the incidence of multiple cerebral infarction in subjects with hyperhomocysteinemia or MTHFR 677TT genotypes. This means that cerebral infarction is severe in people with the MTHFR 677TT genotype.

In another specific example, the present inventors have combined a combination of small-vessel infarction and multiple infarction, which were significant in the two classifications presented above in the analysis of the third subtype. We found that there was a significantly higher comparative risk in multiple small vessel infarction. However, the comparative risk was much higher than that of small vessel infarction or multiple cerebral infarction. Thus, it was confirmed that the MTHFR 677TT genotype is an important independent risk factor in multiple small vessel infarction, a subtype of cerebral infarction (see Table 5). In addition, when small-vessel infarction occurs in multiple cases, it becomes multiple small-vessel infarction and clinically means that vascular dementia occurs. From this point of view, the association with multiple small-vessel cerebral infarction means that vascular dementia may also be a risk factor for the MTHFR 677TT genotype. Thus, testing the MTHFR 677TT genotype may mean early detection of a subject who is more likely to develop vascular dementia. Therefore, if a person with MTHFR 677TT genotype prevents hyperhomocysteinemia from ingesting folic acid, it may be early detection and prevention of vascular dementia.

Therefore, the inventors have discovered that the occurrence of these cerebral infarct subtypes can be diagnosed in advance through the examination of the MTHFR 677TT genotype, thereby enabling the prevention thereof.

As such, the present invention revealed by comparative risk analysis that MTHFR 677TT genotype is not related to the overall risk of cerebral infarction but is associated with small vessel infarction, multiple cerebral infarction and multiple small vessel infarction classified as subtypes (Table 3 to Table 5). And MTHFR 677TT genotype, which can be used to diagnose the possibility of cerebral infarction of the above subtypes in advance and to prevent the occurrence of cerebral infarction by administering a substance containing folic acid or folic acid in advance. .

In other words, in contrast to the conventional genetic test, most of the patients who can only be diagnosed after the diagnosis, nothing can be done, in case of predicting cerebral infarction using the MTHFR 677TT genotype provided in the present invention, the cerebral infarction is predicted simultaneously with the cerebral infarction. Since it is directly confirmed that it is due to high homocysteine certification among these various infarction factors, there is an important advantage that the prevention of cerebral infarction can be simultaneously performed by administering a multivitamin such as folic acid. Therefore, the prediction and prevention of cerebral infarction according to the present invention can be said to be completely different from the conventional methods for detecting, preventing and pathogenic general cerebral infarction.

Therefore, according to the present invention, MTHFR 677TT genotype is associated with not only hyperhomocysteinemia but also small vessel infarction, multiple cerebral infarction and multiple small vessel infarction, and after extraction of the patient's DNA, a specific sense primer and antisense primer are used. To amplify a DNA fragment comprising the 677 position of the MTHFR gene, and to treat the presence of the MTHFR677TT genotype by treating a restriction enzyme that specifically cleaves the 677C → T mutation site, thereby detecting small vessel infarction, multiple cerebral infarction and The occurrence of multiple small vessel cerebral infarction can be predicted in advance, and by administering a substance containing folic acid which reduces the concentration of folic acid or homocysteine in the blood, it is possible to predict and prevent it.

Example

1. Research subject

In this study, 195 patients (106 males and 89 females) diagnosed with ischemic cerebral infarction who visited Pocheon Jung-Mun Medical Center Bundang Hospital from November 2000 to December 2001 were selected. A randomly selected 198 controls (111 males and 87 females) were included in the study. Ischemic cerebral infarction was diagnosed by a neurologist in the presence of lesions consistent with clinical symptoms on brain MRI. Patients who were diagnosed by clinical neurologists based on clinical examination findings, echocardiography, cerebrovascular imaging, cerebrovascular ultrasound, and brain imaging were selected. Diagnosis of ischemic cerebral infarction was performed in patients with cerebral infarction who showed clinical symptoms and symptoms due to sudden or partial partial brain injury. . In the present invention, the ischemic cerebral infarction based on clinical symptoms and brain imaging findings is classified into five subtypes of small-vessel infarction, large-vessel cerebral infarction, psychogenic cerebral infarction, other causes, and cases where the cause is not clearly understood. This is based on the "TOAST" classification, which is already widely used internationally and widely used in Korea. According to the above classification, the present invention is 55 patients with psychovascular cerebral infarction, 6 patients with other causes, and 21 patients because the cause was not clearly known, after 55 large vessels infarction 72 Patients with small vessel infarction were performed.

In the study group, hypertension and diabetes were diagnosed according to WHO criteria based on the condition at the time of the test. Smoking habits and smoking volumes were set based on oral responses to all subjects. Among the patients with ischemic cerebral infarction, 132 patients were diagnosed by Brain MRI. They were divided into single cerebral infarction and multiple cerebral infarction, and the lesion size and location were based on the results of this test. Single infarct was defined as one stroke in clinical and brain imaging findings, and multiple infarcts were defined as neurologic deficits due to more than two clinical infarcts or brain imaging findings. It was limited to cases with two or more distinct lesions.

2. Biochemical Analysis of Homocysteine

Blood pretreatment was performed for 8 hours before blood collection and intravenous blood collection was taken from the patient's upper arm before breakfast. Whole blood was placed in a Vacutainer Tube containing trisodium EDTA, cooled in ice, and immediately centrifuged at 2,000 rpm for 5 minutes to store the plasma at 0-4 ^° C. Quantitative determination of homocystein in blood was measured by plasma enzymatic radioactivity (FPIA: fluorescence polarization immunoassay) using IMX kit (Abbott Laboratories).

3. Genetic Analysis of MTHFR

DNA isolation was isolated from leukocytes extracted from the patient's blood using a DNA extraction kit (QIAmp blood kit, Qiagen) according to the manufacturer's protocol. The isolated DNA samples were amplified with a GeneAmp PCR machine (Perkin Elmer 9600) using a primer set (sense primer (5'-TGAAGGAGAAGGTGTCTGCGGGA-3 ') and antisense primer (5'-AGGACGGTGCGGTGAGAGTG-3'). In order to amplify the 198 bp product generated by the PCR amplification as described above, after denaturing at 95 ^o C for 60 seconds, annealing the primer for 90 seconds at 62 ^o C, and then 60 seconds at 72 ^o C The procedure of performing the primer extension reaction was repeated 35 cycles. The amplified fragments were digested at 37 ^° C. for 3-4 hours with a restriction enzyme Hin f1 (10 unit / reaction mixture, purchased from MBI Fermentas) that can recognize 677C → T mutations in the amplified fragments. The 198 bp fragment obtained from the A (Ala) allele was not digested with Hin f1, while the fragments of the same length obtained from the V (Val) allele were digested into 175 bp and 23 bp fragments. The fragments treated with Hin f1 were then electrophoresed with 2.5% agarose gel and stained with EtBr (ethidium bromide) to observe the mutation. The electrophoresis results are shown in FIG. 1.

4. Statistical Analysis

In order to measure the comparative risk in subtypes of cerebral infarction, the authors used odd odds ratio (OR) and 95 percent confidence intervals (95 percent CI). To analyze the general characteristics of the patient and control groups, a chi-square test was used for categorical data (gender, hypertension, diabetes, hypercholesterolemia and smoking) and continuous data (continuous) Data: Two-sample t-test was used for age and fasting plasma homocysteine concentrations. Logistic regression analysis was used for multivariate analysis, and adjusted OR was calculated by adjusting the possible confounders (age, sex, hypertension, diabetes and smoking). The statistical package used SPSS for Windows, version 9.0 (SPSS Inc., Chicago, Illinois, USA).

5. Results

Table 1 compares the basic characteristics and the usual risk factors in the ischemic cerebral infarction patient group and the control group. The cerebral infarction group showed a significantly higher frequency of vascular risk factors except hypercholesterolemia than the control group, which is consistent with the known results.

Cerebral infarction patient group (n = 195) Control group (n = 198) Crude OR 95% CI p * Age (mean ± SD) 61.4 ± 10.9 60.8 ± 11.2 … … 0.570 Male count (ratio) 106 (54.4%) 111 (56.1%) … … 0.735 High blood pressure 127 (65.1%) 78 (39.4%) 2.87 1.91, 4.33 <0.001 diabetes 62 (31.8%) 44 (22.2%) 1.63 1.04, 2.56 0.033 Hypercholesterolemia 45 (23.1%) 39 (19.7%) 1.22 0.75, 1.98 0.414 smoking 57 (29.2%) 36 (18.2%) 1.72 1.07, 2.78 0.025

* Chi-square test for categorical data and 2-sample t-test for continuous data.

In addition, the plasma homocysteine concentrations in the control group, the whole cerebral infarction patient group, and each group classified into the cerebral infarction subtype are shown in Table 2. As can be seen in Table 2, the homocysteine concentrations in the whole cerebral infarction group, small vessel infarction, multiple cerebral infarction and multiple small vessel infarction showed a significant increase compared to the control group ( p <0.05). However, homocysteine levels in the blood vessels in large-vessel infarction, single-stroke and single-vessel infarction were not significantly increased compared to the control group ( p > 0.05).

Homocysteine Concentration (μmol / L) p * Control group (n = 198) 8.99 ± 2.57 … Total Cerebral Infarction Patient Group (n = 195) 10.38 ± 6.18 0.004 Small vessel infarction (n = 72) 11.27 ± 6.00 0.003 Large vessel infarction (n = 55) 9.15 ± 6.86 0.870 Single cerebral infarction (n = 54) 8.62 ± 4.22 0.541 Multiple Cerebral Infarction (n = 78) 11.66 ± 7.27 0.002 Single small vessel infarction (n = 32) 9.59 ± 4.87 0.504 Multiple Small Blood Vessel Infarction (n = 40) 12.61 ± 6.52 0.001

Table 3 shows the association between the MTHFR 677TT genotype, total cerebral infarction patients, and small-vessel infarction patients and large-vessel infarction according to the MTHFR gene mutation. When measuring the risk of comparison, the first was to compare the 677TT homozygous variant genotype with the 677CC genotype without mutations, and the second was to compare the 677TT homozygous variant genotype with all non-homozygous mutations-677CC genotype (normal) and 677CT Genotypes (heterozygous mutations) were compared to-. In the total cerebral infarction group, the 677TT genotype showed no significant value in both comparisons. There was no significant difference in both comparisons in the group of patients with combined small vessel infarction and large vessel infarction. However, in the small-vessel infarction group, all of them showed a significantly higher comparative risk. However, there was no significant difference in all of the patients with large vessel infarction (Table 3).

MTHFR Control group (n = 198) Total Cerebral Infarction Patient Group (n = 195) Large vessel infarction + small vessel infarction ^* (n = 127) Large vessel cerebral infarction (n = 55) Small vessel infarction (n = 72) 677CC (Ratio): Normal 73 (36.9) 62 (31.8) 40 (31.5) 20 (36.4) 20 (27.8) 677CT (ratio): heterozygous mutation 100 (50.5) 96 (49.2) 62 (48.8) 30 (54.5) 32 (44.4) 677TT (ratio): homozygous variation 25 (12.6) 37 (19.0) 25 (19.7) 5 (9.1) 20 (27.8) OR (95% CI) TT vs CC 1.00 0.94 (0.42, 2.09) 2.15 (0.82, 5.62) 1.29 (0.31, 5.34) 2.92 (1.01, 8.48) OR (95% CI) TT vs CC / CT 1.00 0.96 (0.51, 1.80) 1.95 (0.93, 4.12) 0.91 (0.28, 2.97) 2.85 (1.24, 6.54)

OR (95% CI): Age, sex, hypertension, diabetes and smoking

Large vessel infarction + small vessel infarction ^* : The sum of the number of patients with large vessel infarction and those with small vessel infarction.

Table 4 shows the association between MTHFR 677TT and single cerebral infarction and multiple cerebral infarction according to MTHFR gene mutation. According to the number of lesions on the MR images, they were classified into single cerebral infarction (one lesion) and multiple cerebral infarction (two or more lesions). There was no significant difference in both the patients with combined infarction and single infarction compared with the non-mutated MTHFR 677CC genotype and with all nonhomozygous mutations. The same result was observed with a single cerebral infarction. However, in multiple cerebral infarctions, both comparisons showed significantly higher risks (Table 4).

MTHFR Control group (n = 198) Single Infarction + Multiple Infarction ^* (n = 132) Single cerebral infarction (n = 54) Multiple Cerebral Infarction (n = 78) 677CC (Ratio): Normal 73 (36.9) 41 (31.1) 22 (40.7) 19 (24.4) 677CT (ratio): heterozygous mutation 100 (50.5) 65 (49.2) 28 (51.9) 37 (47.4) 677TT (ratio): homozygous variation 25 (12.6) 26 (19.7) 4 (7.4) 22 (28.2) OR (95% CI) TT vs CC 1.00 2.10 (0.82, 5.34) 0.52 (0.13, 2.13) 4.82 (1.59, 14.57) OR (95% CI) TT vs CC / CT 1.00 1.85 (0.89, 3.82) 0.59 (0.17, 2.07) 3.16 (1.39, 7.16)

OR (95% CI): Age, sex, hypertension, diabetes and smoking

Single Cerebral Infarction + Multiple Cerebral Infarction ^* : The sum of the number of patients with a single cerebral infarction and those with multiple cerebral infarction.

Table 5 shows the association between the MTHFR 677TT genotype according to MTHFR gene mutation, single small vessel infarction, and multiple small vessel infarction. This analysis was based on the combination of the previously mentioned significantly higher risks of subtypes: small vessel infarction and multiple infarction. Among the multiple small-vessel infarctions, there was a significant association, and the comparison risk was not significantly higher in the single-vessel infarction. However, the comparative risk in multiple small vessel infarction was higher than that in either small or multiple infarction.

MTHFR Control group (n = 198) Single small vessel infarction (n = 32) Multiple small vessel infarction (n = 40) 677CC (Ratio): Normal 73 (36.9) 11 (34.4) 9 (22.5) 677CT (ratio): heterozygous mutation 100 (50.5) 17 (53.1) 15 (37.5) 677TT (ratio): homozygous variation 25 (12.6) 4 (12.5) 16 (40.0) OR (95% CI) TT vs CC 1.00 1.19 (0.27, 5.35) 6.90 (1.70, 27.99) OR (95% CI) TT vs CC / CT 1.00 1.14 (0.31, 4.17) 5.57 (1.97, 15.72)

OR (95% CI): Age, sex, hypertension, diabetes and smoking

Comparing the results shown in Tables 3 to 5, the comparative risk for cerebral infarction of the MTHFR 677TT genotype with homozygous variation was shown in a log graph in FIG. 3 compared to the case where no mutation occurred and all non-homozygous mutations. . 3A compares the MTHFR 677TT homozygous variant with the 677CC (normal) genotype without mutation, and 3b shows the MTHFR 677TT homozygous variant in all non-homozygous variants-MTHFR 677CC (normal) and MTHFR 677CT (heterozygous variant) ) Is a comparison with-. In indicating the comparative risk, the adjusted OR (odds ratio) and 95% CI calculated based on factors such as the patient's age, sex, hypertension, diabetes and smoking were shown. Here, "*" indicates a statistically significant value, and "■" indicates adjusted OR.

The present invention, as described above, by confirming that the presence of MTHFR 677TT genotype homozygous mutations among the MTHFR C677T gene polymorphism is significantly high in the incidence of small-vessel infarction, multiple cerebral infarction and multiple small-vessel infarction, which has been previously negated The association between MTHFR 677TT genotype and cerebral infarction was demonstrated. Therefore, according to the present invention, by examining the presence of the MTHFR 677TT genotype, it is possible to predict or early diagnose the onset of small vessel infarction, multiple cerebral infarction and multiple small vessel infarction. In addition, subtypes of cerebral infarction as described above—small vessel infarction, multiple cerebral infarction and multiple small vessel infarction—significantly increased blood homocysteine concentrations as compared to normal controls. Therefore, when predicting or diagnosing the cerebral infarction of the above subtypes by the presence of the MTHFR 677TT genotype according to the present invention, it is directly confirmed that such cerebral infarction is caused by hyperhomocysteinemia, thereby lowering the homocysteine concentration in blood such as folic acid. By administering an acting substance, cerebral infarction can be prevented in parallel with the prediction or diagnosis of cerebral infarction.

By examining mutations of the MTHFR C677T gene in accordance with the present invention and by examining the presence of MTHFR 677TT, it is possible to predict in advance the onset of small vessel infarction, multiple cerebral infarction and multiple small vessel infarction, which are subtypes of cerebral infarction, and at the same time include folic acid and the like. This can be prevented by lowering the concentration of homocysteine in the blood by administration of a multivitamin complex. Therefore, using the MTHFR 677TT genotype provided for the prediction of small vessel infarction, multiple cerebral infarction and multiple small vessel infarction, the disease can be predicted, diagnosed early and prevented at the same time. In addition, the MTHFR 677TT genotype can be used in kits for the prediction and diagnosis of small vessel infarction, multiple cerebral infarction and multiple small vessel infarction.

Claims (4)

Increasing the concentration of homocysteine in the blood, leading to small-artery disease, multiple infarcts or multiple small-artery diseases caused by hyperhomocysteinemia among subtypes of cerebral infarction The MTHFR 677TT genotype, which is the 677TT homozygous variant genotype of the MTHFR gene (Genebank, accession no. UO9806), used in advance to predict or diagnose the development of small vessel infarction, multiple infarction, or multiple small vessel infarction. The incidence of small vessel infarction, multiple cerebral infarction or multiple small vessel infarction by increasing the concentration of homocysteine in the blood, thereby inducing induction of small vessel infarction, multiple cerebral infarction or multiple small vessel infarction caused by hyperhomocysteinemia among subtypes of cerebral infarction By predicting in advance and directly indicating that the cause of the occurrence is high homocysteine, administration of a substance that lowers the concentration of homocysteine in the blood containing folic acid or folic acid enables the prevention of small vessel infarction, multiple cerebral infarction or multiple small vessel infarction MTHFR 677TT genotype, which is the 677TT homozygous variant genotype of the MTHFR gene, used to A DNA sample of the patient was taken to amplify a DNA fragment containing the 677th base of the MTHFR gene using a sense primer and an antisense primer, and then the amplified DNA was examined using a restriction enzyme, and the MTHFR gene was 677TT homozygous. A method for predicting or diagnosing the onset of small vessel infarction, multiple cerebral infarction or multiple small vessel infarction caused by hyperhomocysteinemia by examining the presence of the MTHFR 677TT genotype according to claim 1. A DNA sample of the patient was taken to amplify a DNA fragment containing the 677th base of the MTHFR gene using a sense primer and an antisense primer, and then the amplified DNA was examined using a restriction enzyme, and the MTHFR gene was 677TT homozygous. Investigating the presence of the MTHFR 677TT genotype according to claim 2, administering folic acid or folic acid-containing substances to patients suspected of developing small vessel infarction, multiple cerebral infarction or multiple small vessel infarction caused by hyperhomocysteinemia Thereby reducing the concentration of homocysteine in the blood, thereby preventing the development of small vessel infarction, multiple cerebral infarction or multiple small vessel infarction.