KR101895855B1 - Association of miR-34a C>A and miR-150 G>A SNP with the risk of ischemic stroke in a Korean population - Google Patents

Abstract

The present invention relates to the relationship between the risk of ischemic stroke in Koreans and the microarray a polymorphism. Specifically, in order to provide information necessary for predicting the risk of ischemic stroke in Korean, miR-34a C A method for identifying a single base polymorphism selected from among A rs6577555, miR-130a C> T (rs731384), miR-150 G> A (rs73056059) and miR-155 T> A (rs767649) (Rs6577555) and miR-150 G > A (rs73056059) from a subject's DNA sample to provide information necessary for predicting prognosis, and a kit therefor .
According to the present invention, based on the above-mentioned four miRNA polymorphisms, it is possible to predict the risk of ischemic stroke in Korean and to provide useful information for predicting the prognosis. In particular, when the kit of the present invention is used, the above information can be provided more easily.

Description

Association of miR-34a C＞A and miR-150 G＞A SNP with the risk of ischemic stroke in Koreans with monobasic polymorphism a Korean population}

The present invention relates to the association between the risk of ischemic stroke (cerebral infarction) and micro-RNA polymorphism in Koreans, and specifically, in order to provide information necessary to predict the risk of ischemic stroke (cerebral infarction) in Koreans, a DNA sample of a subject From miR-34aC>A (rs6577555), miR-130aC>T (rs731384), miR-150G>A (rs73056059) and miR-155T>A (rs767649) selected from a method for determining a monobasic polymorphism selected from, and a kit therefor, And in order to provide information necessary for prognosis prediction, it relates to a method for discriminating a single nucleotide polymorphism selected from miR-34aC>A (rs6577555) and miR-150G>A (rs73056059) from a DNA sample of a subject, and a kit therefor.

Stroke is the second leading cause of death worldwide, with approximately 84% of these deaths being caused by ischemic stroke. In Korea, stroke is the second most frequent cause of death after cancer, and it is more common than heart disease. Risk factors for stroke include high blood pressure, diabetes, aging, smoking, hyperlipidemia, hyperhomocysteinemia, and thrombophilia of blood vessels, and several other diseases can cause ischemic stroke. The most common cause of ischemic stroke is arterial occlusion in the head, which is mainly due to atherosclerosis, gradual cholesterol deposition or thrombosis. Vascular occlusion occurs mainly due to thrombosis (53%) or due to embolism (31%). Thrombus formation can occur due to excessive platelet production, and many studies have been conducted on the relationship between platelets and ischemic stroke. Platelets are involved in both normal hemostasis and thrombosis. Platelets are formed in the cytoplasm of their progenitor cells, megakaryocytes (MKs), in the bone marrow.

MicroRNAs (miRNAs, miRs) belong to small endogenous non-coding RNA molecules that regulate gene expression endogenously physiologically. miRNA efficiently regulates gene expression by binding to the 3'-untranslated region of the mRNA and inhibiting the expression of the protein. miRNAs play an important role in several physiological and pathological processes such as metabolism, hematopoiesis and immune function. In addition, previous studies have reported that transient miRNA regulation may be related to the progression of ischemia in the cerebral artery. In addition, recent studies have shown that there is a significant association between miRNA single nucleotide polymorphism (SNP) and ischemic stroke. As Edelstein et al. previously investigated the regulated function of megakaryocytes, various miRNAs play an important role in this process as well as in platelet generation.

Accordingly, the present inventors investigated the association between the incidence of ischemic stroke and miRNA and attempted to use it for predicting or diagnosing ischemic stroke, and four miRNA polymorphisms related to the formation of megakaryocytes, namely miR-34a C>A(rs6577555), miR The relationship between -130a C>T(rs731384), miR-150 G>A(rs73056059) and miR-155 T>A(rs767649) and the risk of ischemic stroke was investigated in Koreans.

As a result, it was confirmed that the four miRNA polymorphisms were significantly associated with ischemic stroke under specific conditions, and the present invention was completed.

Donnan GA, Fisher M, Macleod M and Davis SM: Stroke. Lancet 371: 1612-1623, 2008. Goldstein LB, Adams R, Becker K, Furberg CD, Gorelick PB, Hademenos G, Hill M, Howard G, Howard VJ, Jacobs B, et al: Primary prevention of ischemic stroke: A statement for healthcare professionals from the Stroke Council of the American Heart Association. Circulation 103: 163-182, 2001. Mustacchi P: Risk factors in stroke. West J Med 143: 186-192, 1985. Arboix A: Cardiovascular risk factors for acute stroke: Risk profiles in the different subtypes of ischemic stroke. World J Clin Cases 3: 418-429, 2015. Mohr JP, Caplan LR, Melski JW, Goldstein RJ, Duncan GW, Kistler JP, Pessin MS and Bleich HL: The Harvard Cooperative Stroke Registry: A prospective registry. Neurology 28: 754-762, 1978. Cosemans JM, Angelillo-Scherrer A, Mattheij NJ and Heemskerk JW: The effects of arterial flow on platelet activation, thrombus growth, and stabilization. Cardiovasc Res 99: 342-352, 2013. Shah AB, Beamer N and Coull BM: Enhanced in vivo platelet activation in subtypes of ischemic stroke. Stroke 16: 643-647, 1985. Sharma SC, Vijayan GP, Suri ML and Seth HN: Platelet adhesiveness in young patients with ischaemic stroke. J Clin Pathol 30: 649-652, 1977. O'Malley T, Langhorne P, Elton RA and Stewart C: Platelet size in stroke patients. Stroke 26: 995-999, 1995. George JN: Platelets. Lancet 355: 1531-1539, 2000. Pease DC: An electron microscopic study of red bone marrow. Blood 11: 501-526, 1956.

Therefore, the main object of the present invention is to provide a method of using miRNA polymorphism and a kit therefor to provide information necessary for predicting the risk of ischemic stroke.

Another object of the present invention is to provide a method of using miRNA polymorphism and a kit therefor to provide information necessary for predicting ischemic stroke prognosis.

According to one aspect of the present invention, the present invention provides information necessary for predicting the risk of ischemic stroke in Koreans, miR-34a C>A(rs6577555), miR-130a C> from a DNA sample of a subject. It provides a method of discriminating a monobasic polymorphism selected from T(rs731384), miR-150 G>A(rs73056059), and miR-155 T>A(rs767649).

In the method of the present invention, in the case of one or more of the following 1) to 6), the subject may be classified as a high risk group for ischemic stroke, and in the case of 7), the subject may be classified as a safety group.

1) When the subject is miR-34a CA / miR-150 GA combination type

2) In case the subject is miR-150 GA / miR-155 TA combination type

3) If the subject is miR-34a A / miR-130a C / miR-150 A haplotype

4) If the subject is miR-34a A / miR-150 A haplotype

5) If the subject is miR-34a A / miR-150 A haplotype

6) If the subject is miR-150 A / miR-155 A haplotype

7) If the subject is miR-34a A / miR-130a T / miR-150 G / miR-155 A haplotype

In the method of the present invention, information selected from the age of the subject, sex, whether or not suffering from diabetes, whether suffering from hyperlipidemia, whether suffering from hypertension, whether smoking, homocysteine level in blood, and folic acid level in blood Further investigation is desirable, and based on this information, if one or more of the following 8) to 22) falls under, the subject can be classified as a high risk group for ischemic stroke.

8) If the subject is 63 years of age or older and is of the miR-34a CA or AA genotype

9) If the subject is a female and is of the miR-34a CA or AA genotype

10) If the subject is not diabetic and is of the miR-34a CA or AA genotype

11) If the subject is suffering from hyperlipidemia and is of the miR-150 GA or AA genotype

12) If the subject has high blood pressure and is of the miR-34a CA or AA genotype

13) If the subject is suffering from hyperlipidemia and is of the miR-34a CA or AA genotype

14) If the subject is a smoker and is of the miR-34a CA or AA genotype

15) When the homocysteine level in the subject's blood is 13.92 μmol/ℓ or more and the miR-34a CA or AA genotype

16) If the subject suffers from hypertension and is of the miR-130a CT or TT genotype

17) If the subject has diabetes and is of miR-130a CT or TT genotype

18) If the subject's blood folate level is 3.56 nmol/ℓ or less and is miR-130a CT or TT genotype

19) If the subject has high blood pressure and is of the miR-150 GA or AA genotype

20) If the subject is a smoker and is of the miR-150 GA or AA genotype

21) If the subject has diabetes and is of the miR-155 TA genotype

22) If the subject is suffering from hyperlipidemia and is of the miR-155 TA genotype

According to another aspect of the present invention, the present invention miR-34a C>A (rs6577555), miR-130a C>T (rs731384), miR-150 G>A (rs73056059) and miR-155 T>A (rs767649) It provides a kit for predicting the risk of occurrence of ischemic stroke in Koreans, including a means for detecting a monobasic polymorphism selected from among.

In the kit for predicting the risk of ischemic stroke of the present invention, a) the means for detecting the miR-34a C>A mononucleotide polymorphism is a continuous 50 to 10,000 bp site including the miR-34a C>A mononucleotide polymorphism site A primer for amplifying and a primer-restriction enzyme set comprising a restriction enzyme capable of cleaving DNA in any one of cases where the base of the miR-34a C>A mononucleotide polymorphism site is cytosine or adenine; and, b) The means for detecting the miR-130a C>T mononucleotide polymorphism is a primer for amplifying a contiguous 50 to 10,000 bp site including a miR-130a C>T mononucleotide polymorphism site, and the miR-130a C>T A primer-restriction enzyme set comprising a restriction enzyme capable of cleaving DNA in either case where the base of the single nucleotide polymorphism site is cytosine or thymine; and c) the miR-150 G>A single nucleotide polymorphism The means of detection is a primer for amplifying a contiguous 50 to 10,000 bp site including a miR-150 G>A mononucleotide polymorphism site, and when the base of the miR-150 G>A monobasic polymorphism site is guanine or adenine. A primer-restriction enzyme set comprising a restriction enzyme capable of cleaving DNA in any one case; and d) The means for detecting the miR-155 T>A mononucleotide polymorphism is miR-155 T>A mononucleotide polymorphism A primer for amplifying a contiguous 50 to 10,000 bp site including a site and a restriction enzyme capable of cleaving DNA in any case of the case where the base of the miR-155 T>A mononucleotide polymorphism site is thymine or adenine. Primer-restriction enzyme set comprising; preferably, the primer of a) comprises an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 1 and an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 2, wherein the The restriction enzyme of a) is BanII, and the primer of b) is SEQ ID NO: It consists of an oligonucleotide represented by the nucleotide sequence of 3 and an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 4, wherein the restriction enzyme of b) is NlaIII, and the primer of c) is the nucleotide sequence of SEQ ID NO: 5 Consists of including the oligonucleotide represented and the oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 6, wherein the restriction enzyme of c) is BccI, and the primer of d) is an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 7 and It comprises an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 8, and the restriction enzyme of d) is preferably Tsp45I.

In another aspect of the present invention, the present invention provides information necessary for predicting the prognosis of ischemic stroke in Koreans, miR-34a C>A(rs6577555) and miR-150 G> from a DNA sample of a subject. A (rs73056059) is selected from the single base polymorphism is discriminated, and provides a method to investigate whether the subject suffers from small-vessel disease (SVD) or large-artery disease (LAD). .

In the method of the present invention, among subjects suffering from small-vessel disease (SVD), the case of the miR-34a C>A single nucleotide polymorphism of the CC genotype has a higher probability of long-term survival than the case of the CA or AA genotype. , Among the subjects suffering from large-artery disease (LAD), the miR-150 G>A mononucleotide polymorphism of the GG genotype can be classified as having a higher chance of long-term survival than the GA genotype.

According to another aspect of the present invention, the present invention comprises a means for detecting a monobasic polymorphism selected from miR-34a C>A (rs6577555) and miR-150 G>A (rs73056059) ischemic stroke in Koreans. ) Provide a kit for predicting prognosis.

In the kit for predicting the prognosis of ischemic stroke of the present invention, a) the means for detecting the miR-34a C>A mononucleotide polymorphism comprises a continuous 50 to 10,000 bp site including a miR-34a C>A monopolymorphism site. A primer-restriction enzyme set comprising a primer for amplification and a restriction enzyme capable of cleaving DNA in either case where the base of the miR-34a C>A mononucleotide polymorphism site is cytosine or adenine; and, b ) The means for detecting the miR-150 G>A single nucleotide polymorphism is a primer for amplifying a contiguous 50 to 10,000 bp region including a miR-150 G>A mononucleotide polymorphism site, and the miR-150 G>A single Primer-restriction enzyme set comprising a restriction enzyme capable of cleaving DNA in either case of guanine or adenine as the base of the nucleotide polymorphism site; preferably, the primer of a) is the base of SEQ ID NO: 1 Consists of an oligonucleotide represented by a sequence and an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 2, wherein the restriction enzyme of a) is BanII, and the primer of b) is an oligo represented by the nucleotide sequence of SEQ ID NO: 5 It consists of a nucleotide and an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 6, and the restriction enzyme of b) is preferably BccI.

According to the present invention, it is possible to predict the risk of ischemic stroke in Koreans based on the four miRNA polymorphisms and provide useful information for predicting the prognosis. In particular, when the kit of the present invention is used, the above information can be more easily provided.

1 shows a Cox proportional risk regression graph on the survival of small-vessel disease (SVD) patients according to the miR-34a C>A polymorphism. A is a survival graph of miR-34a C>A polymorphism (CC vs. CA, P=0.016), and B is a survival graph of miR-34a C>A polymorphism (CC vs. CA+AA, P=0.019).
Figure 2 shows a Cox proportional risk regression graph on the survival of patients with large-artery disease (LAD) according to the miR-150 G>A polymorphism. This is the survival graph of miR-150 G>A polymorphism (GG vs. GA, P=0.009).

In the present invention, miR-34a C>A, miR-130a C>T, miR-150 G>A, and miR-155 T>A single nucleotide polymorphism used for predicting the risk of ischemic stroke and prognosis in Koreans The center's monobasic polymorphism database (NCBI dbSNP, http://http://www.ncbi.nlm.nih.gov/snp/) is registered as rs6577555, rs731384, rs73056059 and rs767649, respectively.

The miR-34a C>A single nucleotide polymorphism can be identified by the 26th base in the portion represented by SEQ ID NO: 9, and this base may be cytosine or adenine. If this mononucleotide polymorphism is cytosine on both chromosomes, it is the miR-34a CC genotype, if both chromosomes are adenine, it is the miR-34a AA genotype, and if one chromosome is cytosine and the other is adenine, it is the miR-34a CA genotype.

The miR-130a C>T monobasic polymorphism can be classified by the 26th base in the portion represented by SEQ ID NO: 10, and this base may be cytosine or thymine. If this mononucleotide polymorphism is cytosine on both chromosomes, it is the miR-130a CC genotype, if both chromosomes are thymine, it is the miR-130a TT genotype, and if one chromosome is cytosine and the other is thymine, it is the miR-130a CT genotype.

The miR-150 G>A single nucleotide polymorphism can be identified by the 26th base in the portion represented by SEQ ID NO: 11, which may be guanine or adenine. If this single nucleotide polymorphism is guanine on both chromosomes, it is the miR-150 GG genotype, if both chromosomes are adenine, it is the miR-150 AA genotype, and if one chromosome is guanine and the other is adenine, it is the miR-150 GA genotype.

The miR-155 T>A monobasic polymorphism can be identified by the 26th base in the portion represented by SEQ ID NO: 12, and this base may be thymine or adenine. If both chromosomes are thymine, this polymorphism is miR-155 TT genotype, if both chromosomes are adenine, it is miR-155 AA genotype, and if one chromosome is thymine and the other is adenine, it is miR-155 TA genotype.

The determination of single nucleotide polymorphism as described above can be achieved by performing a polymerase chain reaction-restriction fragment path formation (PCR-RFLP) method or a nucleotide sequence analysis method.

The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method is a restriction enzyme when a target gene or DNA site is amplified by performing a polymerase chain reaction and treated with a specific restriction enzyme. This is a method to distinguish polymorphism, deletion, addition, or substitution of a base by distinguishing whether digestion is performed by recognizing and digestion is not performed because the restriction enzyme does not recognize it. At this time, whether or not DNA is amplified and digested by restriction enzymes can be confirmed through electrophoresis using an agarose gel.

As the nucleotide sequence analysis method, a known method of Maxam-Gilbert or Sanger may be used.

When the polymorphism is determined by the PCR-RFLP method, the following amplification primers and restriction enzymes may be used.

a) miR-34a C>A SNP discrimination: forward primer of SEQ ID NO: 1 and reverse primer of SEQ ID NO: 2, BanII restriction enzyme

b) miR-130a C>T SNP discrimination: forward primer of SEQ ID NO: 3 and reverse primer of SEQ ID NO: 4, NlaIII restriction enzyme

c) miR-150 G>A SNP discrimination: forward primer of SEQ ID NO: 5 and reverse primer of SEQ ID NO: 6, BccI restriction enzyme

d) miR-155 T>A SNP discrimination: forward primer of SEQ ID NO: 7 and reverse primer of SEQ ID NO: 8, Tsp45I restriction enzyme

When the primers and restriction enzymes of a) are used, a 202 bp DNA fragment is amplified through polymerase chain reaction, and when the restriction enzyme is treated, DNA fragments of 180 bp and 22 bp in the case of miR-34a CC, and in the case of miR-34a AA DNA fragments of 202 bp, and DNA fragments of 202 bp, 180 bp and 22 bp in the case of miR-34a CA were generated.

When the primer and restriction enzyme of b) are used, a 203 bp DNA fragment is amplified through polymerase chain reaction, and when the restriction enzyme is treated, a 203 bp DNA fragment for miR-130a CC, 179 bp for miR-130a TT and DNA fragments of 24 bp, and DNA fragments of 203 bp, 179 bp and 24 bp were generated in the case of miR-130a CT.

When the primers and restriction enzymes of c) are used, a 164 bp DNA fragment is amplified through polymerase chain reaction, and when the restriction enzyme is treated, 138 bp and 26 bp DNA fragments in the case of miR-150 GG, and in the case of miR-150 AA DNA fragments of 164 bp, and DNA fragments of 164 bp, 138 bp and 26 bp in the case of miR-150 GA are produced.

When the primer and restriction enzyme of d) are used, a 294 bp DNA fragment is amplified through polymerase chain reaction, and when the restriction enzyme is treated, 155 bp and 139 bp DNA fragments for miR-155 TT, and miR-155 AA DNA fragments of 294 bp, and DNA fragments of 294 bp, 155 bp and 139 bp are generated in the case of miR-155 TA.

The haplotype according to the combination of each single nucleotide polymorphism can be determined without performing a separate analysis method when each of the combined polymorphisms is homozygous, but otherwise, it can be determined by performing a continuous nucleotide sequence analysis method.

According to the present invention, it is possible to determine the SNP of miRNA in the same manner as described above, and provide information for predicting the risk of ischemic stroke in Koreans based on this. This is due to the significant difference confirmed through comparative analysis of ischemic stroke patients and control groups.

In addition, additional information such as age, sex, diabetes, hyperlipidemia, hypertension, smoking, homocysteine level in blood, folic acid level in blood, etc. A method that is considered together can also provide information to predict the risk of developing ischemic stroke. This is due to significant differences identified through stratified analysis and gene-environment binding analysis of ischemic stroke patients and control groups. Additional information as described above can be obtained through methods such as a questionnaire survey, a doctor's diagnosis, and a blood test.

The kit for predicting the risk of ischemic stroke in Koreans of the present invention enables the SNP identification as described above to be easily performed to predict the risk of ischemic stroke in Koreans based on this. It characterized in that it comprises a means for detecting. As a means for detecting miRNA SNPs, there are probes commonly used to detect SNPs of miRNAs, for example, primers for PCR, primers and restriction enzymes for PCR-RFLP, and probes for DNA-DNA hybridization. In the present invention, it is preferable to use PCR-RFLP primers and restriction enzymes as detection means. In particular, miR-34a C>A SNP detection means include the primers and restriction enzymes of a) above, and miR-130a C>T SNPs. As a means of detection, the primer and restriction enzyme of b) above, the primer and restriction enzyme of c) above as a means of detecting miR-150 G>A SNP, and the primer of d) above as means of detecting miR-155 T>A SNP Restriction enzymes are preferred.

The kit for predicting prognosis of the present invention is characterized in that it comprises a means for detecting a SNP selected from miR-34a C>A and miR-150 G>A. At this time, the detection means may also be a primer, a restriction enzyme, or a probe as described above, and as a means for detecting miR-34a C>A SNP, the primer and restriction enzyme of a) above, and as a means for detecting miR-150 G>A SNP, c ) Primers and restriction enzymes are preferred.

The kit for predicting the risk of occurrence and the kit for predicting prognosis of the present invention may further include reagents and instruments used for detecting SNP of miRNA in addition to the detection means as described above.

Hereinafter, the present invention will be described in more detail through examples. Since these examples are for illustrative purposes only, the scope of the present invention is not to be construed as being limited by these examples.

<Example 1>

1-1. Research Group Selection

596 patients with ischemic stroke (mean age ± SD: 63.67 ± 10.42 years, 254 men, 342 women) were organized into the study group. Ischemic stroke was diagnosed on the basis of acute infarction occurring concurrently with neurological symptoms that worsened rapidly through magnetic resonance imaging (MRI) recording. In addition, 404 control groups (mean age ± SD: 63.66 ± 10.47 years old, 173 men, 231 women) were included. The subject of the study was entrusted to the Department of Neurology at CHA Bundang Hospital (Seongnam, Korea) between July 1, 2000 and February 28, 2008. The control group was recruited from people who visited the hospital for general health screening. The exclusion criteria for the control group are as follows: A person with a family history of stroke or experience of non-specific dizziness during the enrollment period, non-substrate headache, or anxiety. All control groups received the same examination as brain imaging (~75% MRI), and no organic brain lesions were found. Interviews were conducted to collect demographic data and medical information on vascular risk factors. Those who had previously experienced cerebral hemorrhage or those with an incomplete medical history were excluded from this study. Patients with ischemic stroke were classified into 3 groups as follows: 202 patients with large-artery disease (LAD), 143 patients with small-vessel disease (SVD), and cardiovascular disease ( Cardioembolism, CE) in 57 patients, 194 patients of unknown cause.

Ischemic stroke is defined as a stroke (specified by clinical symptoms and signs of rapid progressive focal and/or general brain function loss) in a clinically relevant area of the brain based on brain MRI. Based on clinical signs and neuroimaging data, two neurologists classified ischemic stroke into three etiological subgroups using the following Trial of Org 10172 in Acute Stroke Treatment (TOAST) clinical trial criteria: i ) Subtype 1: LAD, with symptoms related to the arterial sphere, with infarct damage of 15 mm or more in diameter by MRI and significant (>50%) of major cerebral arteries or branched cortical arteries through cerebrovascular angiography If stenosis is identified; ii) Subtype 2: SVD, MRI confirms infarct damage less than 15 mm in diameter and more than 5 mm in diameter, and traditional lacunar syndrome without signs of cerebral cortical dysfunction or cardiac cause of embolism; iii) Subtype 3: CE, when arterial obstruction, which appears to be due to heart-derived embolism, is confirmed through evaluation of heart disease. Hypertension, diabetes, hyperlipidemia, homocysteine levels, folic acid levels, vitamin B12 levels, cholesterol, platelet (PLT) levels, prothrombin time (PT), active partial thromboplastin time (aPTT), fibrinogen levels, based on the previously described method. Clinical parameters including antithrombin levels, BUN levels and uric acid levels were measured.

1-2. Genetic analysis

Genomic DNA was extracted from blood leukocytes using a G-DEX blood extraction kit (Intron Inc., Seongnam, Korea). Four of the miRNAs (miR-34a rs6577555C>A, miR-130a rs731384C>T, miR-150 rs73056059G>A and miR-155 rs767649T>A) were determined through record investigation. All SNP sequences were obtained through the HapMap database (www.hapmap.org) and dbSNP- (www.ncbi.nlm.nih.gov/projects/SNP/). Nucleotide substitution was determined through PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) analysis using the extracted genomic DNA as a template.

PCR for miR-34a rs6577555C>A polymorphism was performed using the following primers: 5'-CCT GGT TAA CAT AGC CAG AGC-3' (forward) and 5'-GCA GAC ATG CTG ACT TTT CAA-3' (reverse). DNA was amplified over 35 cycles (denaturation at 95°C for 30 sec, annealing at 56°C for 30 sec, and extension at 72°C for 35 sec). The PCR product was digested by treatment with BanII restriction enzyme at 37° C. for 16 hours, and confirmed by 3.5% agarose gel electrophoresis.

To confirm the miR-130a rs731384C>T polymorphism, 5'-GAT GCT CAG TCC TCA AAG AAC A-3' (forward) and 5'-TGA GGC CTA GAG CTC TGC TTT AT-3' (reverse) primers were used. . DNA was amplified over 35 cycles (denaturation at 95°C for 30 sec, annealing at 58°C for 30 sec, and extension at 72°C for 35 sec). The PCR product was digested by treatment with NlaIII (New England Biolabs) at 37° C. for 16 hours and confirmed by 3% agarose gel electrophoresis.

For miR-150 rs73056059G>A polymorphism, 5'-GTT CCT GCC AGA GGA AGT G-3' (forward) and 5'-CCT CTG GAG TCC ACA CTC CAT-3' (reverse) primers were used. DNA was amplified over 35 cycles (denaturation at 95°C for 30 sec, annealing at 60°C for 35 sec, and extension at 72°C for 40 sec). The amplified product was treated with BccI (New England Biolabs) at 37° C. for 16 hours and confirmed by 4% gel electrophoresis.

For miR-155 rs767649T>A polymorphism, 5'-CCT GTA TGA CAA GGT TGT GTT TG-3' (forward) and 5'-GCT GGC ATA CTA TTC TAC CCA TAA-3' (reverse) primers were used. DNA was amplified over 35 cycles (denaturation at 95°C for 35 sec, annealing at 56°C for 30 sec, and extension at 72°C for 35 sec). The PCR product was treated with Tsp45I (New England Biolabs) at 37° C. for 16 hours and confirmed by 3% agarose gel electrophoresis.

1-3. Statistical analysis

Clinical features were compared using Student's unpaired t-test. The association between ischemic stroke and the four miRNA genotypes was analyzed based on the calculation of odds ratio using Fisher's exact test and 95% confidence intervals (CIs). Adjusted ORs (AORs) of miRNA polymorphisms were determined using multiple logistic regression analysis based on gender, age, diabetes, hypertension, hyperlipidemia and smoking. The genotype distribution of each polymorphism was evaluated by Hardy-Weinberg equilibrium deviation, and the difference in genotype and allele frequency between groups was evaluated by the χ2 test. A value of P<0.05 was considered to represent a statistically significant difference. Stratified analysis was used to differentiate stroke subgroups based on the size of the obstructed blood vessels. One-way analysis of variance (ANOVA) was performed to compare average homocysteine concentration levels between different genotypes. Stats Direct Statistical Software (version 2.4.4; StatsDirect Ltd., Altrincham, UK) was used to calculate the adjusted OR and 95% CI. Survival curves were created using Cox proportional hazards regression, and the significance of differences between groups was evaluated using log-rank test. Cox's regression model was used to analyze the independent prognostic significance of various markers, and the results were adjusted for age, sex, diabetes, hypertension, hyperlipidemia and smoking. Hazard ratios (HRs) are presented with 95% CI. In addition, in silico analysis was performed using Alibaba 2.1 (Grabe 2002), an online bioinformatics tool, to identify potential transcription factor binding sites in the miRNA promoter region.

1-4. result

Table 1 shows the demographic characteristics and clinical parameters of the ischemic stroke group and the control group. Comparing the ischemic stroke group to the control group, stroke patients had a significantly higher incidence of hypertension and diabetes, significantly higher homocysteine and fibrinogen levels, and significantly lower folic acid and aPTT levels (all P<0.05). (See Table 1).

SD, standard deviation; PLT, platelet; PT, prothrombin time; aPTT, activated partial thromboplastin time; BUN, blood urea nitrogen

^a P -value was calculated by a two-sided t-test for continuous variables and a chi-square test for categorical variables.

Four miRNA polymorphisms (miR-34a rs6577555C>A, miR-130a rs731384C>T, miR-150 rs73056059G>A and miR-155 rs767649T>A) and their association with ischemic stroke were investigated. All observed polymorphic frequencies were consistent with the Hardy-Weinberg equilibrium. Initially, no difference in frequency of polymorphism was found between the ischemic stroke and control groups (see Table 2). However, as a result of stratifying and analyzing the ischemic stroke group into subgroups, it was possible to observe the association between miRNA polymorphism and each stroke subtype. LAD was significantly associated with the miR-150 GA genotype (GG vs. AA: AOR, 1.922; 95% CI, 1.003-3.681). CE was also significantly associated with miR-150 (GG vs. AA: AOR, 2.996; 95% CI, 1.293-6.939). On the other hand, these differences disappeared when the P-value was adjusted for FDR (false discovery rate) (see Table 2). The miR-34a C>A, miR-130a C>T, and miR-155 T>A polymorphisms were not significantly different between stroke patients and controls.

A stratified analysis according to age, sex, hypertension, diabetes, hyperlipidemia, smoking, folic acid level and homocysteine level was performed. The miR-34a CA+AA genotype is 63 years old or older (AOR, 1.443; 95% CI, 1.010-2.062), female (AOR, 1.459; 95% CI, 1.026-2.076) and patients without diabetes (AOR, 1.360; 95). % CI, 1.013-1.827) showed an increase in the appearance rate. The miR-150 GA+AA genotype was also found to increase in prevalence in hyperlipidemia patients (AOR, 6.060; 95% CI, 1.358-27.04) (see Table 3).

[Description of Table 2]

AOR, adjusted odd ratio; 95% CI, 95% confidence interval; LAD, large-artery disease; SVD, small-vessel disease; CE, cardioembolism; N/A, not applicable

^a Adjusted for age, sex, hypertension, diabetes, hyperlipidemia and smoking

^b False discovery rate (FDR)-adjusted P-value for testing multiple hypotheses using the Benjamini-Hochberg method.

[Description of Table 3]

AOR, adjusted odd ratio; 95% CI, 95% confidence interval

^a Odds ratio adjusted based on risk factors such as age, sex, high blood pressure, diabetes, hyperlipidemia, and smoking

^b False discovery rate (FDR)-adjusted P-value for testing multiple hypotheses using the Benjamini-Hochberg method.

^c Folic acid 3.56 nmol/L is the lower 15% cut-off folic acid level in ischemic stroke patients and controls.

^d Homocysteine 13.92 μmol/L is the top 15% cut-off homocysteine level in stroke patients and controls.

Gene-environmental coupling analysis has shown that several genotypes are associated with clinical factors related to the risk of ischemic stroke. miR-34a CA+AA genotype is hypertension (AOR, 3.088; 95% CI, 2.089-4.566), hyperlipidemia (AOR, 1.570; 95% CI, 1.010-2.443), smokers (AOR, 1.651; 95% CI, 1.023- 2.665) and high homocysteine levels (AOR, 2.230; 95% CI, 1.236-4.024). The miR-130a CT+TT genotype also includes hypertension (AOR, 2.390; 95% CI, 1.483-3.850), diabetes (AOR, 2.205; 95% CI, 1.043-4.661) and low folic acid levels (AOR, 4.702; 95% CI, 1.341-16.49) was found to have a high incidence of stroke. The miR-150 GA+AA genotype was hypertension (AOR, 2.871; 95% CI, 1.416-5.824), hyperlipidemia (AOR, 7.215; 95% CI, 1.655-31.45) and smoking (AOR, 3.594; 95% CI, 1.267- 10.20) showed a high incidence of stroke in patients. The miR-155 TA genotype was found to have a high stroke prevalence in diabetic (AOR, 2.945; 95% CI, 1.659-5.228) and hyperlipidemia (AOR, 1.734; 95% CI, 1.049-2.865) patients (see Table 4).

[Description of Table 4]

^a Folic acid 3.56 nmol/L is the lower 15% cut-off folate level in ischemic stroke patients and controls.

^b Homocysteine 13.92 μmol/L is the top 15% cut-off homocysteine level in stroke patients and controls.

Allele combination analysis was performed using a multifactor dimensionality reduction (MDR) method comparing ischemic stroke patients and controls (see Table 5). The following allele combinations were found to have a significant correlation with the incidence of stroke: miR-34a C>A / miR-130a C>T / miR-150 G>A / miR-155 T>A ATGA allele combination ( OR, 0.052; 95% CI, 0.003-0.921), miR-34a C>A / miR-130a C>T / miR-150 G>A combination of ACA alleles (OR, 4.285; 95% CI, 1.255-14.62 ), miR-34a C>A / miR-150 G>A AA allele combination (OR, 3.814; 95% CI, 1.106-13.15), and miR-150 G>A / miR-155 T>A AA Allele combination (OR, 1.970; 95% CI, 1.013-3.831). In addition, genotyping combination analysis was performed. The miR-34a CA / miR-150 GA genotype (AOR, 5.470; 95% CI, 1.580-18.932) and the miR-150 GA / miR-155 TA genotype (AOR, 3.265; 95% CI, 1.426-7.474) were both ischemic. It was associated with an increase in the incidence of stroke (see Table 6).

* 95% CI, 95% confidence interval

^a Fisher's exact test

[Description of Table 6]

^a Adjusted for age, sex, hypertension, diabetes, hyperlipidemia and smoking

^b ORs and 95% CIs of each specific genotype calculated with reference to the frequencies of all others

The miR-130a genotype was associated with fibrinogen levels (CC vs. CT vs. TT: CC, 411.65±124.83; CT, 462.34±147.50; TT, 429.80±94.44; P<0.001), and miR-150 dominant model (GG) vs. GA+AA) was significantly associated with an increase in platelet count (GG vs. GA+AA: GG, 244.44±66.30; GA+AA, 266.53±58.58; P=0.002). miR-155 recessive model (TT+TA vs. AA) included vitamin B12 (TT+TA vs. AA: TT+TA, 748.94±631.87; AA, 716.23±580.51; P=0.030) and fibrinogen levels (TT+TA vs. AA) and fibrinogen levels (TT+TA vs. AA). AA: TT+TA, 423.93±177.56; AA, 400.34±117.63; P=0.036), and miR-155 genotype was associated with antithrombin level (TT vs. TA vs. AA: TT, 97.42±39.69; TA , 91.92±17.99; AA, 92.29±16.25; P=0.049) (see Table 7).

[Description of Table 7]

SD, standard deviation; PLT, platelet; PT, prothrombin time; aPTT, activated partial thromboplastin time

^{a was} calculated using ANOVA

^b Calculated using the Kruskal-Wallis test

^c Calculated using Mann-Whitney test

The association between miRNA polymorphism and survival of patients with ischemic stroke is shown in FIGS. 1 and 2. Cox's proportional analysis revealed that the miR-34a CA genotype had a significant association with survival in the SVD subgroup (CC vs. CA, P=0.016) (see Fig. 1A), (CC vs. CA+AA, P). =0.019) (see Figure 1B). The miR-150 GA genotype was associated with survival in the LAD subgroup (GG vs. GA, P=0.009) (see Fig. 2).

<110> SUNGKWANG MEDICAL FOUNDATION <120> Association of miR-34a C>A and miR-150 G>A SNP with the risk of ischemic stroke in a Korean population <130> PA-D18196 <160> 12 <170> KoPatentIn 3.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for miR-34a C>A <400> 1 cctggttaac atagccagag c 21 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for miR-34a C>A <400> 2 gcagacatgc tgacttttca a 21 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for miR-130a C>T <400> 3 gatgctcagt cctcaaagaa ca 22 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for miR-130a C>T <400> 4 tgaggcctag agctctgctt tat 23 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for miR-150 G>A <400> 5 gttcctgcca gaggaagtg 19 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for miR-150 G>A <400> 6 cctctggagt ccacactcca t 21 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for miR-155 T>A <400> 7 cctgtatgac aaggttgtgt ttg 23 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for miR-155 T>A <400> 8 gctggcatac tattctaccc ataa 24 <210> 9 <211> 51 <212> DNA <213> Homo sapiens <400> 9 ccagcctggt taacatagcc agacccccac cttcagcaac tgtctctaca a 51 <210> 10 <211> 51 <212> DNA <213> Homo sapiens <400> 10 cttgatgctc agtcctcaaa gaaaacggct attagcaatc cccaaagtct g 51 <210> 11 <211> 51 <212> DNA <213> Homo sapiens <400> 11 cagtagagaa ccagcaaggg gcaaagaggg agtgtggact ccagagggag g 51 <210> 12 <211> 52 <212> DNA <213> Homo sapiens <400> 12 atataacaca ttatcaaaaa cactgatcac ttttctgagt gctctaatca gg 52

Claims (1)

(Rs6577555) and miR-150 G> A (rs73056059) from DNA samples of a subject to determine the risk of ischemic stroke in Korean patients Way.

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