KR101895853B1 - Association of microRNA polymorphisms 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

The association between the risk of ischemic stroke (cerebral infarction) and the risk of ischemic stroke in a Korean population of microRNA (polymorphism)

The present invention relates to the relationship between the risk of ischemic stroke (cerebral infarction) in Koreans and the microarray apolipoprotein. Specifically, in order to provide information necessary for predicting the risk of ischemic stroke (stroke) in Korean, A method for identifying a single base polymorphism selected from miR-34aC> A (rs6577555), miR-130aC> T (rs731384), miR-150G> A (rs73056059), and miR- (Rs6577555) and miR-150G > A (rs73056059) from a subject's DNA sample in order to provide information necessary for predicting prognosis, and a kit therefor.

Stroke is the second leading cause of death worldwide, with ischemic stroke accounting for about 84% of these deaths. In Korea, stroke is the second most common cause of death following cancer, and it occurs more often than heart disease. Risk factors for stroke include hypertension, diabetes, aging, smoking, hyperlipidemia, hyperhomocysteinemia, and thrombophilia in the blood vessels, and several other diseases can cause ischemic stroke. The most common cause of ischemic stroke is arterial occlusion in the head. Arterial occlusion is mainly caused by atherosclerosis, gradual cholesterol deposition, or thrombosis. Occlusion is mainly caused by thrombosis (53%) or by embolism (31%). Thrombus formation can occur due to excessive platelet production, and much research has been conducted on the relationship between platelets and ischemic stroke. Platelets are involved in both normal hemostasis and thrombosis. Platelets form in the cytoplasm of megakaryocytes (MKs), their precursor cells in the bone marrow.

MicroRNAs (microRNAs, miRNAs, miRs) belong to small endogenous non-coding RNA molecules that regulate gene expression endogenously and physiologically. miRNA binds to the 3'-untranslated region of mRNA and inhibits the expression of the protein, thereby efficiently controlling the expression of the gene. miRNA plays 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 associated with progression of ischemia in the cerebral artery. Recent studies have shown a significant association between miRNA single nucleotide polymorphism (SNP) and ischemic stroke. As previously reviewed by Edelstein et al. On the regulated function of megakaryocytes, various miRNAs play an important role in this process as well as in platelet development.

The present inventors have investigated the relationship between the incidence of ischemic stroke and miRNA and used it for the prediction or diagnosis of ischemic stroke. Four miRNA polymorphisms related to the formation of megakaryocytes such as miR-34a C> A (rs6577555), miR The relationship between the risk of ischemic stroke and that of miR-150 G> A (rs73056059) and miR-155 T> A (rs767649) was investigated in Koreans.

As a result, it was confirmed that the above-mentioned four miRNA polymorphisms are each 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: A 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.

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

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

According to one aspect of the present invention, there is provided a method for diagnosing ischemic stroke in a human, comprising the steps of: detecting miR-34a C> A (rs6577555), miR-130a C> (Rs731384), miR-150 G> A (rs73056059) and miR-155 T> A (rs767649).

In the method of the present invention, if the subject is at least one of the following 1) to 6), the subject is classified as a high risk group of ischemic stroke, and if the subject is 7), it can be classified as a safety group.

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

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

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

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

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

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

7) When 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 among the age, sex, diabetes, whether or not suffering from hyperlipidemia, whether or not suffering from hypertension, smoking, homocysteine level in blood and folate level in blood Based on this information, the subject can be classified as a high-risk group of ischemic stroke if it meets one or more of the following 8) to 22).

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

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

10) If the subject is not suffering from diabetes and is miR-34a CA or AA genotype

11) If the subject has hyperlipidemia and is miR-150 GA or AA genotype

12) If the subject has hypertension and is miR-34a CA or AA genotype

13) If the subject has hyperlipemia and is miR-34a CA or AA genotype

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

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

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

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

18) When the blood level of the subject is less than 3.56 nmol / L and the miR-130a CT or TT genotype

19) If the subject has hypertension and is miR-150 GA or AA genotype

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

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

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

In accordance with another aspect of the present invention, the present invention provides a method of inhibiting miR-34a C> A (rs6577555), miR-130a C> T (rs731384), miR- 150G> A (rs73056059) And a means for detecting a single nucleotide polymorphism selected from the group consisting of the nucleotide polymorphism and the nucleotide polymorphism.

A kit for predicting ischemic stroke risk according to the present invention, comprising: a) means for detecting miR-34a C> A single nucleotide polymorphism comprises means for detecting consecutive 50 to 10,000 bp regions comprising a miR-34a C> A single nucleotide polymorphism site And a restriction enzyme capable of cleaving the DNA of any one of the cases where the base of the miR-34a C > A single nucleotide polymorphism site is cytosine or adenine, and the primer- b) the means for detecting the miR-130a C > T single nucleotide polymorphism comprises a primer for amplifying consecutive 50 to 10,000 bp sites comprising the miR-130a C > T single nucleotide polymorphism site and the miR- A primer-restriction enzyme set comprising a restriction enzyme capable of cleaving DNA in any one of cases where the base of the single nucleotide polymorphism site is cytosine or thymine; c) the primer set of the miR-150 G > A single base polymorphism Means a primer for amplifying a consecutive 50 to 10,000 bp region comprising a miR-150 G > A single nucleotide polymorphism site and a primer for amplifying a consecutive 50 to 10,000 bp region comprising miR-150 G > A single nucleotide polymorphism sites if guanine or adenine Wherein the means for detecting the miR-155 T > A single nucleotide polymorphism is a primer-restriction enzyme set comprising a restriction enzyme capable of cleaving DNA of any one of miR-155 T > A single base A primer for amplifying a consecutive 50 to 10,000 bp region containing a polymorphic site and a restriction enzyme capable of cleaving DNA in either of the case where the base of the miR-155 T > A single nucleotide polymorphism site is thymine or adenine The primer of the above a) comprises an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 1 and a nucleotide sequence of SEQ ID NO: Wherein the restriction enzyme of a) is BanII, the primer of b) is selected from the group consisting of an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 3 and an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 4 Wherein the restriction enzyme of b) is NlaIII, the primer of c) comprises an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 5 and an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 6, The restriction enzyme of c) is BccI, the primer of d) comprises an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 7 and an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 8, The enzyme is preferably Tsp45I.

In another embodiment of the present invention, miR-34a C > A (rs6577555) and miR-150 G > are selected from DNA samples of a subject in order to provide information necessary for prediction of ischemic stroke prognosis in Korean, A (rs73056059) and to determine whether the subject is suffering from a small-vessel disease (SVD) or a large-artery disease (LAD) .

In the method of the present invention, when the miR-34a C > A single nucleotide polymorphism is CC genotype among the subjects suffering from small-vessel disease (SVD) GG genotypes of the miR-150 G> A single nucleotide polymorphism among patients with large-artery disease (LAD) can be classified as having a higher survival rate over a longer period than those with GA genotype.

In accordance with another aspect of the present invention, the present invention provides a method of detecting a single nucleotide polymorphism selected from miR-34a C> A (rs6577555) and miR-150 G> A (rs73056059) in a Korean ischemic stroke ) Prognosis prediction kit is provided.

A kit for predicting an ischemic stroke prognosis of the present invention, comprising: a) means for detecting the miR-34a C> A single nucleotide polymorphism comprises a consecutive 50 to 10,000 bp region comprising a miR-34a C> A single nucleotide polymorphism site And a restriction enzyme capable of cleaving the DNA of any one of the cases where the base of the miR-34a C > A single nucleotide polymorphism site is cytosine or adenine; and b is a primer- The means for detecting the miR-150 G > A single nucleotide polymorphism comprises a primer for amplifying consecutive 50 to 10,000 bp sites comprising miR-150 G > A single nucleotide polymorphic sites and a primer for amplifying the miR- A primer-restriction enzyme set comprising a restriction enzyme capable of cleaving DNA in any one of cases where the base in the base polymorphism site is guanine or adenine, and the primer of a) 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 restriction enzyme of a) is BanII, and the primer of b) An oligonucleotide represented by the sequence of SEQ ID NO: 6, and an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 6, wherein the restriction enzyme of b) is preferably BccI.

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.

FIG. 1 shows a Cox proportional hazard regression graph for the survival of patients with small-vessel disease (SVD) 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).
FIG. 2 shows a Cox proportional hazard regression graph for the survival of patients with large-artery disease (LAD) according to the miR-150 G> A polymorphism. 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 polymorphisms used in the prediction of the risk of ischemic stroke and prediction of prognosis in Koreans, Are registered as rs6577555, rs731384, rs73056059 and rs767649 in the Center's single nucleotide polymorphism database (NCBI dbSNP, http: //www.ncbi.nlm.nih.gov/snp/).

The miR-34a C > A single nucleotide polymorphism can be divided into the 26th nucleotide in the part of SEQ ID NO: 9, which may be cytosine or adenine. This single nucleotide polymorphism is the miR-34a CC genotype when both chromosomes are cytosine. When both chromosomes are adenine, the miR-34a AA genotype, and the miR-34a CA genotype when one chromosome is cytosine and the other chromosome is adenine.

The miR-130a C > T single nucleotide polymorphism can be distinguished from the 26th nucleotide in SEQ ID NO: 10, which may be cytosine or thymine. This single nucleotide polymorphism is miR-130a CC genotype when both chromosomes are cytosine, miR-130a TT genotype when both chromosomes are thymine, and miR-130a CT genotype when one chromosome is cytosine and the other chromosome is thymine.

The miR-150 G > A single nucleotide polymorphism can be divided into 26 nucleotides in the sequence shown in SEQ ID NO: 11, which may be guanine or adenine. This single nucleotide polymorphism is the miR-150 GG genotype when both chromosomes are guanine, miR-150 AA genotype when both chromosomes are adenine, miR-150 GA genotype when one chromosome is guanine and the other chromosome is adenine.

The miR-155 T > A single nucleotide polymorphism can be divided into 26 nucleotides in the region represented by SEQ ID NO: 12, which may be thymine or adenine. This single nucleotide polymorphism is the miR-155 TT genotype when both chromosomes are thymine. The miR-155 TA genotype when both chromosomes are adenine. The miR-155 TA genotype when one chromosome is thymine and the other chromosome is adenine.

Such single nucleotide polymorphism discrimination can be performed by PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) method or nucleotide sequence analysis method.

The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method amplifies a target gene or DNA region by performing a polymerase chain reaction, and when treated with a specific restriction enzyme, Deletion, addition, or substitution of bases by distinguishing whether digestion occurs or not, or whether digestion is not carried out because the restriction enzyme is not recognized. DNA amplification and digestion with restriction enzymes can be confirmed by electrophoresis using agarose gel.

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

When the polymorphism is determined by the PCR-RFLP method, the following primers and restriction enzymes for amplification can 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 DNA fragment of 202 bp is amplified by polymerase chain reaction. When the restriction enzyme is treated, a DNA fragment of 180 bp and 22 bp of miR-34a CC and a DNA fragment of miR-34a AA A DNA fragment of 202 bp, 180 bp and 22 bp is generated in the case of miR-34a CA.

When the primers and restriction enzymes of b) are used, a DNA fragment of 203 bp is amplified by a polymerase chain reaction. When the restriction enzyme is treated, a DNA fragment of 203 bp for miR-130 a CC, 179 bp for miR-130 a TT, And DNA fragments of 203 bp, 179 bp and 24 bp in the case of miR-130a CT are produced.

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

When the primers and restriction enzymes of d) are used, a DNA fragment of 294 bp is amplified by a polymerase chain reaction. When the restriction enzyme is treated, a DNA fragment of 155 bp and 139 bp for miR-155 TT and a DNA fragment of miR-155 AA 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 any separate analysis method when each of the combined polymorphisms is homozygous, but if not, it can be determined by performing sequential nucleotide sequence analysis.

According to the present invention, the SNP of the miRNA is discriminated in the same manner as described above, and information for predicting the risk of ischemic stroke in Korean can be provided. This is due to the significant differences identified between the ischemic stroke patients and the control group.

Further, additional information such as age, sex, diabetes, whether or not suffering from hyperlipidemia, whether or not suffering from hypertension, smoking status, homocysteine level in blood, and folate level in blood, Together, they can provide information to predict the risk of ischemic stroke. This is due to the significant differences identified by stratification analysis of the ischemic stroke patients and the control group, and gene-environment binding analysis. Such additional information can be obtained through a survey, a doctor's diagnosis, a blood test, or the like.

The kit for predicting the risk of ischemic stroke of the present invention enables the SNP discrimination to be easily performed and thus predicts the risk of ischemic stroke in Korean. And detecting means for detecting the presence or absence of an abnormality. Examples of miRNA SNP detection means include probes commonly used for detecting 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 that primers and restriction enzymes for PCR-RFLP are used as detection means. In particular, miR-34a C > SN SNP detection means include primers and restriction enzymes of a) Examples of the detection means include primers and restriction enzymes of b), miR-150 G> A SNP detection means include the primers and restriction enzymes of c), miR-155 T> A SNP detection means include the primers of d) Restriction enzymes are preferred.

The prognostic prediction kit of the present invention is characterized by comprising means for detecting SNPs selected from miR-34a C> A and miR-150 G> A. The miR-34a C > A SNP detection means may be primers and restriction enzymes of the above a), miR-150 G > A SNP detection means may be c ) Primers and restriction enzymes are preferable.

The occurrence risk prediction kit and the prognostic prediction kit of the present invention may further include reagents, instruments, etc. used for SNP detection of miRNA in addition to the above detection means.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

≪ Example 1 >

1-1. Study group selection

The study population consisted of 596 ischemic stroke patients (mean age ± SD: 63.67 ± 10.42 years, 254 men, 342 women). Ischemic stroke was diagnosed based on acute infarction that occurred simultaneously with neurological symptoms that rapidly deteriorated through magnetic resonance imaging (MRI) recordings. In addition, a control group of 404 (mean age ± SD: 63.66 ± 10.47 years, 173 men, 231 women) was included. Subjects were enrolled and enrolled in Bundang Cha 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 checks. The control group exclusion criteria were: a family history of stroke or experience of non-specific dizziness during the registration period, non-temperamental headache, or anxiety. All control groups received the same test (~ 75% MRI) as brain imaging, and no underlying brain lesions were found. Interviews were conducted to collect medical information on demographic and vascular risk factors. Patients who had previous hemorrhage or who had an incomplete history were excluded from the study. Patients with ischemic stroke were divided into three groups as follows: 202 patients with large-artery disease (LAD), 143 patients with small-vessel disease (SVD) cardioembolism, CE) and 194 patients with unknown etiology.

Ischemic stroke is defined as stroke with signs of cerebral infarction in the clinically relevant areas of the brain based on brain MRI (specific as clinical signs and symptoms of rapid onset of lesion and / or generalized cerebral dysfunction). Based on clinical signs and neuroimaging data, two neurologists classified the ischemic stroke into three etiologic subgroups using the TOAST (Trial of Org 10172 in Acute Stroke Treatment) clinical trial standard: i ) Subtype 1: LAD, Significant (> 50%) of major cerebral or branchial cortical arteries through a cerebral angiographic examination when an infarction diameter greater than 15 mm was identified via MRI, Stenosis is confirmed; ii) Subtype 2: classic lacunar syndrome with an infarction lesion> 5 mm in diameter identified via SVD, MRI, and without signs or symptoms of brain dysfunction; iii) Subtype 3: CE, cardiopulmonary assessment showing evidence of arterial occlusion seen due to cardiac-induced embolization. Based on the above-described methods, it is possible to provide a method for treating hypertension, diabetes, hyperlipidemia, homocysteine level, folate level, vitamin B12 level, cholesterol, platelet (PLT) level, prothrombin time (PT), active part thromboplastin time (aPTT) Clinical parameters including antithrombin level, BUN level and uric acid level were measured.

1-2. Genetic analysis

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

The 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- (reverse). DNA was amplified for 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 products were digested with BanII restriction enzyme at 37 DEG C for 16 hours and confirmed by 3.5% agarose gel electrophoresis.

3'(forward) and 5'-TGA GGC CTA GAG CTC TGC TTT AT-3 '(reverse) primers were used to confirm miR-130a rs731384C> T polymorphism . DNA was amplified at 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 with NlaIII (New England Biolabs) at 37 ° C for 16 hours and confirmed by 3% agarose gel electrophoresis.

5'-GTT CCT GCC AGA GGA AGT G-3 '(forward) and 5'-CCT CTG GAG TCC ACA CTC CAT-3' (reverse) primers were used for miR-150 rs73056059G> A polymorphism. DNA was amplified for 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 primer were used. DNA was amplified by 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 products were 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 four miRNA genotypes was analyzed based on 95% confidence intervals (CIs) and odd ratio calculations using Fisher's exact test. Modified ORs (AORs) of miRNA polymorphisms were determined using multiple logistic regression analysis based on sex, age, diabetes, hypertension, hyperlipidemia and smoking. The genotypic distribution of each polymorphism was assessed by Hardy-Weinberg equilibrium deviation, and the difference between the genotypes and allele frequencies of the groups was evaluated by the χ2 test. P <0.05 was considered to be statistically significant. The stratified analysis was used to distinguish stroke subgroups based on blocked blood vessel size. 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 adjusted OR and 95% CI. Survival curves were generated using Cox proportional hazards regression and the significance of differences between groups was assessed using a log-rank test. The Cox regression model was used to analyze the independent prognostic significance of the various markers and the results were adjusted for age, sex, diabetes, hypertension, hyperlipidemia, and smoking. Hazard ratios (HRs) were presented with 95% CI. In silico analysis was also 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

The demographic characteristics and clinical variables of the ischemic stroke group and the control group are shown in Table 1. Comparing the ischemic stroke group to the control group, the stroke patients had a significantly higher incidence of hypertension and diabetes, significantly higher homocysteine and fibrinogen levels, and significantly lower folate levels and aPTT (all P <0.05) (See Table 1).

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

^a P -value was calculated by two-sided t-test for continuous variables and 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. At first, there was no polymorphism frequency difference between ischemic stroke and control group (see Table 2). However, stratifying and analyzing the ischemic stroke group into subgroups, we were able 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-values were adjusted for the false discovery rate (FDR) (see Table 2). There was no significant difference in the miR-34a C> A, miR-130a C> T, and miR-155 T> A polymorphisms between patients with stroke and the control group.

Age, sex, hypertension, diabetes, hyperlipidemia, smoking, folate levels and homocysteine levels were analyzed. (AOR, 1.309, 95% CI, 1.010-2.062), female (AOR, 1.459; 95% CI, 1.026-2.076) and non-diabetic patients (AOR, % CI, 1.013-1.827), respectively. The miR-150 GA + AA genotype was also found to increase in hyperlipidemia patients (AOR, 6.060; 95% CI, 1.358-27.04) (see Table 3).

[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) for testing multiple hypotheses using the Benjamini-Hochberg method - adjusted P-value

[Table 3]

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

^a Adjusted odds ratio based on risk factors such as age, sex, hypertension, diabetes, hyperlipemia, smoking

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

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

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

In the gene-environment binding assay, several genotypes were found to be associated with risk factors for ischemic stroke. The miR-34a CA + AA genotype was associated with 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% 2.665) and high homocysteine levels (AOR, 2.230; 95% CI, 1.236-4.024). (AOR, 2.390; 95% CI, 1.483-3.850), diabetes (AOR, 2.205; 95% CI, 1.043-4.661) and low folate levels (AOR, 4.702; 95% CI, 1.341-16.49) had a higher incidence of stroke. The genotype of miR-150 GA + AA was significantly higher in hypertension (AOR, 2.871; 95% CI, 1.416-5.824), hyperlipidemia (AOR, 7.215; 95% CI, 1.655-31.45) 10.20) had a higher incidence of stroke. The miR-155 TA genotype showed a higher incidence of stroke in patients with diabetes (AOR, 2.945; 95% CI, 1.659-5.228) and hyperlipidemia (AOR, 1.734; 95% CI, 1.049-2.865).

[Table 4]

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

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

An allele combination analysis was performed using a multifactor dimensionality reduction (MDR) method that compared controls with ischemic stroke patients (see Table 5). The following allele combinations were found to be significantly associated with the incidence of stroke: miR-34a C> A / miR-130a C> T / miR-150 G> A / miR- (OR, 4.285; 95% CI, 1.255-14.62) of miR-34a C> A / miR-130a C> T / miR- ), the AA allele combination (OR, 3.814; 95% CI, 1.106-13.15) of miR-34a C> A / miR-150 G> Allelic combinations (OR, 1.970; 95% Cl, 1.013-3.831). Genotype combination analysis was also performed. (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) And an increase in the incidence of stroke (see Table 6).

* 95% CI, 95% confidence interval

^a Fisher's exact test

[Table 6 Description]

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

^b ORs and 95% CIs for each specific genotype calculated with reference to the frequency 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 < 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). The miR-155 thermolabile model (TT + TA vs. AA) was significantly higher than the vitamin B12 (TT + TA vs. AA: TT + TA, 748.94 + 631.87; AA, 716.23 + 580.51; P = 0.030) and fibrinogen levels (TT vs. TA, AA: TT, 97.42 ± 39.69; TA; TT) were associated with AA, TT + TA, 423.93 ± 177.56; AA, 400.34 ± 117.63; P = , 91.92 ± 17.99; AA, 92.29 ± 16.25; P = 0.049) (Table 7).

[Table 7]

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

Calculated using ^a ANOVA

^b Kruskal-Wallis test.

^c Mann-Whitney test

The association between miRNA polymorphisms and the survival of ischemic stroke patients is shown in Figures 1 and 2. A proportional analysis of Cox showed that the miR-34a CA genotype was significantly associated with survival in the SVD subgroup (CC vs CA, P = 0.016) (CC vs CA + AA, P = 0.019) (see Fig. 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 microRNA polymorphisms with the risk of ischemic          stroke in a Korean population <130> PA-D16284 <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 (12)

(Rs6577555), miR-130a C> T (rs731384) and miR-150 G> A (rs6577555) from the subject's DNA samples in order to provide information necessary for predicting the risk of ischemic stroke in Koreans (rs73056059) and miR-155 T > A (rs767649). The method according to claim 1,
The method according to any of claims 1 to 6, wherein the subject is classified as a high-risk group of ischemic stroke, and the subject is classified as a safe group if it corresponds to 7).
1) If the subject is a miR-34a CA / miR-150 GA combination
2) When the subject is miR-150 GA / miR-155 TA combination type
3) When the subject is miR-34a A / miR-130a C / miR-150A haplotype
4) If the subject is miR-34a A / miR-150 A haplotype
5) When the subject is miR-34a A / miR-150 A haplotype
6) If the subject is miR-150 A / miR-155 A haplotype
7) When the subject is miR-34a A / miR-130a T / miR-150 G / miR-155 A haplotype The method according to claim 1,
Wherein the information is further selected from information on the age, sex, diabetes, whether or not suffering from hyperlipidemia, whether or not suffering from hypertension, smoking, homocysteine level in blood and folate level in blood Way. The method of claim 3,
The method according to any of claims 1 to 6, 8) to 22), wherein the subject is classified as a high risk group of ischemic stroke, and if the subject is classified as a safe group, the group is classified as a safe group.
1) If the subject is a miR-34a CA / miR-150 GA combination
2) When the subject is miR-150 GA / miR-155 TA combination type
3) When the subject is miR-34a A / miR-130a C / miR-150A haplotype
4) If the subject is miR-34a A / miR-150 A haplotype
5) When the subject is miR-34a A / miR-150 A haplotype
6) If the subject is miR-150 A / miR-155 A haplotype
7) When the subject is miR-34a A / miR-130a T / miR-150 G / miR-155 A haplotype
8) If the subject is 63 years of age or older and has miR-34a CA or AA genotype
9) If the subject is female and miR-34a is CA or AA genotype
10) If the subject is not suffering from diabetes and is miR-34a CA or AA genotype
11) If the subject has hyperlipidemia and is miR-150 GA or AA genotype
12) If the subject has hypertension and is miR-34a CA or AA genotype
13) If the subject has hyperlipemia and is miR-34a CA or AA genotype
14) If the subject is a smoker and is miR-34a CA or AA genotype
15) When the homocysteine level in the blood of the subject is 13.92 μmol / ℓ or more and miR-34a CA or AA genotype
16) If the subject has hypertension and is miR-130a CT or TT genotype
17) If the subject has diabetes and is miR-130a CT or TT genotype
18) When the blood level of the subject is less than 3.56 nmol / L and the miR-130a CT or TT genotype
19) If the subject has hypertension and is miR-150 GA or AA genotype
20) If the subject is a smoker and is miR-150 GA or AA genotype
21) If the subject has diabetes and is miR-155 TA genotype
22) If the subject is suffering from hyperlipidemia and the miR-155 TA genotype Means for detecting a single base polymorphism selected from miR-34a C> A (rs6577555), miR-130a C> T (rs731384), miR-150 G> A (rs73056059) and miR-155 T> A (rs767649) Kits for the prediction of the risk of ischemic stroke in Koreans. 6. The method of claim 5,
wherein the means for detecting the miR-34a C > A single nucleotide polymorphism comprises an oligonucleotide primer represented by the nucleotide sequence of SEQ ID NO: 1, an oligonucleotide primer represented by the nucleotide sequence of SEQ ID NO: 2, and a BanII restriction enzyme Primer-restriction enzyme set;
b) the means for detecting the miR-130a C > T single nucleotide polymorphism comprises an oligonucleotide primer represented by the nucleotide sequence of SEQ ID NO: 3, an oligonucleotide primer represented by the nucleotide sequence of SEQ ID NO: 4 and an NlaIII restriction enzyme Primer-restriction enzyme set;
c) the means for detecting the miR-150 G > A single nucleotide polymorphism comprises an oligonucleotide primer represented by the nucleotide sequence of SEQ ID NO: 5, an oligonucleotide primer represented by the nucleotide sequence of SEQ ID NO: 6, and a BccI restriction enzyme Primer-restriction enzyme set;
d) means for detecting the miR-155 T > A single nucleotide polymorphism comprises an oligonucleotide primer represented by the nucleotide sequence of SEQ ID NO: 7, an oligonucleotide primer represented by the nucleotide sequence of SEQ ID NO: 8, and a Tsp45I restriction enzyme Primer-restriction enzyme &lt; RTI ID = 0.0 &gt; set. &Lt; / RTI &gt; delete In order to provide information necessary for prediction of ischemic stroke prognosis in Koreans, a single base polymorphism selected from miR-34a C> A (rs6577555) and miR-150 G> A (rs73056059) , Whether the subject is suffering from a small-vessel disease (SVD) or a large-artery disease (LAD). 9. The method of claim 8,
Among the subjects with small-vessel disease (SVD), the miR-34a C> A single nucleotide polymorphism was more likely to survive than the CA or AA genotype for the CC genotype,
Wherein the GG genotype of the miR-150 G > A single nucleotide polymorphism is classified as having a higher probability of prolonged survival than that of the GA genotype among the subjects suffering from a large-artery disease (LAD). A kit for predicting ischemic stroke prognosis in Korean comprising means for detecting a single base polymorphism selected from miR-34a C> A (rs6577555) and miR-150 G> A (rs73056059). 11. The method of claim 10,
wherein the means for detecting the miR-34a C > A single nucleotide polymorphism comprises an oligonucleotide primer represented by the nucleotide sequence of SEQ ID NO: 1, an oligonucleotide primer represented by the nucleotide sequence of SEQ ID NO: 2, and a BanII restriction enzyme Primer-restriction enzyme set;
b) the means for detecting the miR-150 G > A single nucleotide polymorphism comprises an oligonucleotide primer represented by the nucleotide sequence of SEQ ID NO: 5, an oligonucleotide primer represented by the nucleotide sequence of SEQ ID NO: 6, and a BccI restriction enzyme Primer-restriction enzyme &lt; RTI ID = 0.0 &gt; set. &Lt; / RTI &gt;
delete

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