KR102045987B1 - Association of single nucleotide polymorphism of DICER, DROSHA and RAN with the risk or prognosis of ischemic stroke in a Korean population - Google Patents

Abstract

The present invention relates to an association between single nucleotide polymorphism of DICER, DROSHA, and RAN and the risk of pathogenesis or prognosis of ischemic stroke in Korean population and, more specifically, to a method for determining single nucleotide polymorphism of rs3742330, rs10719, or rs14035 from a DNA sample of an examinee in order to provide information necessary for predicting the risk of pathogenesis or prognosis of ischemic stroke in Korean population, and to a kit for the same. According to the present invention, it is possible to provide very useful information for predicting the risk of pathogenesis of ischemic stroke in Korean population by determining single nucleotide polymorphism of rs3742330 or rs10719 and also provide useful information for predicting the prognosis of ischemic stroke in Korean population by determining single nucleotide polymorphism of rs14035. In particular, the above information can be more easily provided by using the kit of the present invention.

Description

Association of single nucleotide polymorphism of DICER, DROSHA and RAN with the risk or prognosis of ischemic stroke in a Korean population}

The present invention relates to the relationship between DICER , DROSHA and RAN monobasic polymorphisms and the risk or prognosis of ischemic stroke in Koreans. Specifically, rs3742330 from DNA samples of subjects to provide information necessary for predicting the risk or prognosis of ischemic stroke in Koreans. , a method for determining a single base polymorphism of rs10719 or rs14035, and a kit therefor.

Ischemic stroke can be caused by cerebral infarction in large vessel disease, cerebral infarction in cardiogenic embolism, small vessel disease, or hiatus, depending on the mechanism in which it occurs. It is classified as a cerebral infarction caused by lacunar infarction and other rare causes, and is classified as a transient ischemic attack when symptoms are fully recovered within 24 hours due to short duration.

The most common cause of ischemic stroke is atherosclerosis (arteriosclerosis) in blood vessels that supply blood to the brain due to hypertension, diabetes, hyperlipidemia, etc., which causes the brain blood flow to be blocked. In addition, due to cardiac arrhythmias, heart failure, and aftereffects of myocardial infarction, blood clots (blood clots in the heart or blood vessels) are produced in the heart, and these blood clots travel along the bloodstream and block the cerebral blood vessels, resulting in strokes. There is also. In rare cases, ischemic strokes are caused by extremely rare diseases such as moyamoya disease and homocysteinemia. As described above, early diagnosis is important because the first action is a very important disease when the disease is caused by various mechanisms and causes.

The course of an ischemic stroke is determined by the size of the brain tissue in which the stroke is affected and the location of the stroke. In general, the symptoms of ischemic stroke are most severe immediately after their occurrence and often do not show a clear improvement for about one week. This initial week is the time to be most careful of acute complications from stroke, such as aspiration pneumonia, stroke recurrence and brain edema. Initially, conservative treatment and careful rehabilitation are mainly performed to prevent acute complications.

Ischemic stroke is differentiated from hemorrhagic stroke by imaging tests such as brain computed tomography (brain CT) or brain magnetic resonance imaging (brain MRI), and the location, size and location of occluded blood vessels are confirmed. If acute ischemic stroke arrives within 3 hours of symptom onset, it is diagnosed with brain CT or brain MRI.

These existing methods can be confirmed only when symptoms or images are shown, but the treatment of stroke is limited. Therefore, the development of a method for predicting the onset before the onset of the disease is required. The basic and clinical studies conducted so far suggest that the treatment can be expected only if the treatment is done within a few hours after the onset of stroke, so the need for early diagnosis and treatment has already been proven.

Therefore, early diagnosis and treatment are regarded as the most important research topics in the field of stroke, and the development of effective methods is expected to contribute to the improvement of national health and social welfare, as well as economic compensation. Because of this field, developed countries' health authorities and leading pharmaceutical and medical device companies have been cultivating this field by policy, but there are no developed diagnostic markers or treatment methods.

J Stroke 2017; 19: 166-187. Stroke 2008; 39: 959-966. J Cereb Blood Flow Metab 2009; 29: 675-687.

Therefore, the main object of the present invention is to provide a new marker useful for predicting the risk of ischemic stroke in Koreans and a method of using the same.

Another object of the present invention is to provide a kit useful for predicting the risk of ischemic stroke in Koreans using the markers.

It is still another object of the present invention to provide a novel marker useful for predicting ischemic stroke prognosis in Koreans and a method of using the same.

Still another object of the present invention is to provide a kit useful for predicting ischemic stroke prognosis in Korean using the marker.

According to one aspect of the present invention, the present invention provides a method for determining the single base polymorphism of rs3742330 or rs10719 from a DNA sample of a subject in order to provide information necessary for predicting the risk of ischemic stroke in Koreans.

According to another aspect of the present invention, the present invention provides a kit for predicting the risk of ischemic stroke in Koreans comprising a means for detecting a single base polymorphism of rs3742330 or rs10719.

In the kit of the present invention, a) the means for detecting a single nucleotide polymorphism of rs3742330 is a primer for amplifying a continuous 50 to 10,000 bp region comprising a single nucleotide polymorphism site of rs3742330 on human genomic DNA and the rs3742330 The base of the monobasic polymorphism site is a set of primer-limiting enzymes comprising a restriction enzyme capable of cleaving the DNA of any one of adenine or guanine, b) means for detecting the single nucleotide polymorphism of rs10719 is human A primer for amplifying a contiguous 50 to 10,000 bp site comprising a single nucleotide polymorphism site of rs10719 on the genomic DNA of and a base of either of the cases where the base of the single nucleotide polymorphism site of rs10719 is thymine or cytosine. It is preferred that it is a primer-limiting enzyme set comprising a restriction enzyme.

In the kit of the present invention, the primer of a) comprises an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 4 and an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 5, wherein the restriction enzyme of a) is Ban II, wherein the primer of b) comprises an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 6 and an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 7, wherein the restriction enzyme of b) is Dra III Do.

According to another aspect of the present invention, the present invention provides a method for determining the single base polymorphism of rs14035 from a DNA sample of a subject in order to provide information necessary for predicting ischemic stroke prognosis in Koreans.

According to another aspect of the present invention, the present invention provides a kit for predicting ischemic stroke prognosis in Korean, comprising a means for detecting a single base polymorphism of rs14035.

In the kit of the present invention, the means for detecting the single nucleotide polymorphism of rs14035 is a primer for amplifying a continuous 50 to 10,000 bp region comprising a single nucleotide polymorphism site of rs14035 on human genomic DNA and the single base of the rs14035 It is preferred that the base of the polymorphic site is a primer-limiting enzyme set comprising a restriction enzyme capable of cleaving DNA in either case of cytosine or thymine.

In the kit of the present invention, the primer for detecting a single nucleotide polymorphism of rs14035 comprises an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 8 and an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 9, the rs14035 The restriction enzyme for detecting monobasic polymorphism is preferably Bsl I.

According to the present invention, it is possible to provide very useful information for predicting the risk of ischemic stroke in Koreans by determining the single base polymorphism of rs3742330 or rs10719, and is also useful for predicting the ischemic stroke prognosis of Koreans by determining the single base polymorphism of rs14035. Information can also be provided. In particular, when using the kit of the present invention can be provided with such information more easily.

1 is a graph showing the analysis of the effect of the DROSHA rs10719 T> C mutation on the incidence of ischemic stroke controlled by clinical factors. (A) Ischemic stroke with hypertension and DROSHA rs10719 CC genotype (AOR, 4.781), without hypertension and with DROSHA rs10719 CC genotype (AOR, 1.967) or with hypertension and with DROSHA rs10719 TT genotype (AOR, 2.415) Synergistic effects on sensitivity, (B) with diabetes and DROSHA rs10719 CC genotype (AOR, 12.046), without diabetes and DROSHA rs10719 CC genotype (AOR, 1.770) or with diabetes and DROSHA rs10719 TT genotype (AOR , 2.351), and the association of DROSHA rs10719 T> C with increased ischemic stroke prevalence.
Figure 2 is a graph showing the difference between the ratio of aPTT and antithrombin based on DROSHA rs10719 T> C in ischemic stroke patients. Statistical analysis was performed by ANOVA test or student's T-test for each DROSHA rs10719 T> C genotype. (A) aPTT: significant difference in blood coagulation time between DROSHA rs10719 TT (31.07 ± 7.06), TC (31.20 ± 7.30) and CC (36.23 ± 35.28) genotypes ( P = 0.005), (B) Plasma antithrombin ratio: DROSHA rs10719 T> C polymorphism affects antithrombin ratio, DROSHA rs10719 CC genotype (97.32 ± 27.29) shows increased antithrombin ratio compared to DROSHA rs10719 TT genotype (94.67 ± 17.64), DROSHA rs10719 CC genotype shows high antithrombin ratio Related to ( P = 0.017). aPTT: activated partial thromboplastin time, the P * calculated as ANOVA test <0.05, ** The P <0.05 calculated by student's T-test.
FIG. 3 is a graph showing the survival rate of the RAN rs14035 C> T polymorphism in ischemic stroke with a Cox proportional hazard model. (A) RAN rs14035 CC vs. RAN rs14035 TT genotype and (B) RAN rs14035 CC + CT vs. Survival graph of patients grouped into large vessel disease (LAD) subtype based on RAN rs14035 TT genotype, (C) RAN rs14035 CC vs. RAN rs14035 TT genotype and (D) RAN rs14035 CC + CT vs. Survival graph of patients grouped into SVD subtypes based on the RAN rs14035 TT genotype. HR: hazard ratio.

Single base polymorphisms (hereinafter, abbreviated as 'SNP') of rs3742330, rs10719 and rs14035 of the present invention are SNPs that can be expressed in human DICER , DROSHA , and RAN genes, respectively. , http: // http: //www.ncbi.nlm.nih.gov/snp/).

The rs3742330 SNP can be classified as the 26th base in the DNA region of human represented by SEQ ID NO: 1, and this base may be adenine or guanine.

The rs10719 SNP can be identified as the 26th base in the human DNA region represented by SEQ ID NO: 2, which may be adenine, cytosine, or thymine, but is rarely adenine.

The rs14035 SNP can be classified as the 26th base in the human DNA site represented by SEQ ID NO: 3, which may be cytosine or thymine.

The SNP can be determined by performing polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) or sequencing.

Polymerase chain reaction-limiting enzyme fragment length formation method is to amplify a target gene or DNA site by performing polymerase chain reaction, and the restriction enzyme recognizes or digests when treated with a specific restriction enzyme. It is a method of distinguishing polymorphism, deletion, addition or substitution of base by distinguishing whether or not digestion is performed. The DNA amplification and digestion by restriction enzymes can be confirmed by electrophoresis using agarose gel.

As the sequencing method, a known method of Maxam-Gilbert or Sanger can be used.

When determining polymorphism by the PCR-RFLP method, a primer for amplifying a continuous 50 to 10,000 bp region including each SNP region and a restriction enzyme capable of cleaving DNA when each SNP region is a specific base are used. Can be.

More preferably, the following amplification primers and restriction enzymes can be used.

rs3742330 SNP determination: Forward primer of SEQ ID NO: 4 and reverse primer of SEQ ID NO: 5, Ban II restriction enzyme

rs10719 SNP determination: forward primer of SEQ ID NO: 6 and reverse primer of SEQ ID NO: 7, Dra III restriction enzyme

rs14035 SNP determination: Forward primer of SEQ ID NO: 8 and reverse primer of SEQ ID NO: 9, Bsl I restriction enzyme

When using the rs3742330 SNP discrimination primers and restriction enzymes, DNA fragments of 232 bp are amplified by polymerase chain reaction, and the DNA fragments of 232 bp for AA genotypes and 232 bp, 191 bp and 41 bp for AA genotypes when the restriction enzymes are processed. DNA fragments, 191 bp and 41 bp DNA fragments for the GG genotype.

When using the rs10719 SNP discrimination primers and restriction enzymes, 168 bp DNA fragments are amplified by polymerase chain reaction, and when the restriction enzymes are processed, 168 bp DNA fragments in the TT genotype, and 168 bp, 141 bp and 27 bp in the TC genotype DNA fragments, 141 bp and 27 bp DNA fragments are generated for the CC genotype.

When using the rs14035 SNP discrimination primers and restriction enzymes, DNA fragments of 152 bp are amplified by polymerase chain reaction, and DNA fragments of 131 bp and 21 bp for CC genotype, 152 bp, 131 bp for CT genotype when the restriction enzyme is treated A 21 bp DNA fragment, 152 bp DNA fragment, is generated.

According to the present invention, the rs3742330 or rs10719 SNP is related to the risk of ischemic stroke in Koreans. Therefore, rs3742330 or rs10719 SNP can be determined by the above method, and based on this, information for predicting the risk of ischemic stroke in Koreans can be provided.

According to the present invention, based on the rs3742330 or rs10719 SNP discrimination results, it is possible to predict the risk of ischemic stroke in Koreans as follows.

-If the subject is rs3742330 AG or GG genotype, the risk of ischemic stroke is higher than that of AA genotype (about 1.4 times).

-If the subject is rs10719 CC genotype, the risk of ischemic stroke is higher than that of TT or TC genotype (approximately 2.04 times).

In addition, according to the present invention, the rs11077 SNP, which is the SNP of the XPO5 gene, is also related to the risk of ischemic stroke in Koreans, and based on the discrimination result, information for predicting the risk of ischemic stroke in Koreans can be provided as follows. .

-If the subject is rs11077 AC genotype, the risk of ischemic stroke is lower than that of AA or CC genotype.

Therefore, using the rs11077 SNP determination result together with the rs3742330 or rs10719 SNP determination result will provide more accurate information.

The rs11077 SNP may be classified as a 26th base in a human DNA site represented by SEQ ID NO: 10, and the base may be adenine or cytosine. In order to determine the SNP, a forward primer of SEQ ID NO: 11, a reverse primer of SEQ ID NO: 12, and a Bsm I restriction enzyme can be used. In this case, 129 bp DNA fragment is amplified by polymerase chain reaction. 129 bp DNA fragment for AA genotype, 129 bp, 102 bp and 27 bp DNA fragment for AC genotype, and 102 bp and 27 bp DNA fragment for CC genotype.

According to the present invention, in the association between rs3742330 or rs10719 and the risk of ischemic stroke in Koreans, the presence or absence of diseases such as small vessel disease (SVD), cardioembolism (CE), hypertension, and diabetes Factors affecting risk Therefore, if the combination of the small vessel disease (SVD), cardioembolism (CE), hypertension or diabetes of the subject with the SNP, it will be possible to provide more accurate information. Considering the presence of these diseases, the risk of ischemic stroke in Koreans can be predicted as follows.

-If the subject is rs3742330 AG genotype and has small vessel disease, the risk of ischemic stroke is higher than that of the AG genotype without small vessel disease.

-If the subject is rs10719 CC genotype and has a psychogenic embolism, the risk of ischemic stroke is higher than that of a CC genotype without a psychogenic embolism (approximately 3.5 times).

-If the subject is rs10719 CC genotype and has hypertension, the risk of ischemic stroke is higher than that of the CC genotype without hypertension (approximately 4.781 times).

-If the subject is rs10719 CC genotype and has diabetes, the risk of ischemic stroke is higher than that of the CC genotype without diabetes (about 12.046 times).

In addition, according to the present invention, the rs14035 SNP is related to the prognosis of ischemic stroke in Koreans. Therefore, rs14035 SNP can be determined by the above method, and based on this, information for predicting the ischemic stroke prognosis of Koreans can be provided.

According to the present invention, the ischemic stroke prognosis of Koreans can be predicted as follows based on the rs14035 SNP determination result.

-If the subject is rs14035 TT genotype, mortality due to ischemic stroke is higher (approximately 6.0-fold) than humans with CC or CT genotype.

According to the present invention, in the association between the rs14035 SNP and the ischemic stroke prognosis of Koreans, the causative disease of the ischemic stroke is also a factor affecting the prognosis. Therefore, if the combination of the disease causing the ischemic stroke of the subject with the SNP, it will be possible to provide more accurate information. Considering the causes of ischemic stroke, the prognosis of ischemic stroke in Koreans can be predicted as follows.

-If the subject is rs14035 TT genotype and ischemic stroke is caused by large vessel disease or small vessel disease, mortality due to ischemic stroke is higher than that of TT genotype who developed ischemic stroke by other causes.

Ischemic stroke risk prediction kit and ischemic stroke prognosis prediction kit of the present invention to facilitate the SNP discrimination as described above to be able to predict the risk of ischemic stroke onset or ischemic stroke prognosis on the basis of Korean As a means for detecting SNPs, materials commonly used for detecting SNPs, for example, PCR primers, primers for PCR-RFLP and restriction enzymes, DNA-DNA hybridization probes, and the like may be used. In the present invention, it is preferable to use the PCR-RFLP primer and restriction enzyme as a detection means, and the above-mentioned SNP determination primer and restriction enzyme are particularly preferable.

In addition to the above detection means, the kit of the present invention may further include reagents, instruments, and the like used for detecting SNPs.

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

EXAMPLE

1. How to

1-1. Select research group

All research plans were reviewed and approved by the Institutional Review Board of Bundang Hospital and followed the recommendations of the Declaration of Helsinki. Research subjects were recruited between 2000 and 2008 in Seoul and Gyeonggi-do. The Institutional Review Board of Bundang Cha Hospital approved the study in June 2000 (IRB No. 2013-09-073) and obtained informed consent from participants.

585 patients with ischemic stroke were recruited. Ischemic stroke was defined as a stroke that shows evidence of cerebral infarction in clinically relevant areas of the brain, based on magnetic resonance imaging (MRI) scans (a clinical syndrome characterized by fast-running clinical symptoms and signs of local or global loss of brain function). . Based on clinical symptoms and neuroimaging data, two neurologists used the Org 10172 assay (Trial of Org 10172) under the Acute Stroke Treatment (TOAST) criteria to cause all four ischemic strokes: Subtypes were: (1) characterized by infarct lesions greater than 15 mm in diameter recorded by MRI, and narrowing (> 50%) of the major cerebral or cortical arteries as reported by cerebral angiography with symptoms related to arterial dysfunction. Large artery disease (LAD); (2) Infarct lesions less than 15 mm in diameter and more than 5 mm in diameter as recorded by MRI, and small vessel disease characterized by classic lacunar symptoms with no evidence of cerebral cortical disorder or a potentially detectable heart cause for embolism. (small vessel disease, SVD); (3) cardioembolism (CE) or arterial obstruction that appears to be due to cardiac embolism found by heart disease assessment; (4) The cause of the stroke cannot be determined with some certainty or the cause of the illness is not clear because more than two causes are involved. Single and multiple (two or more lesions) SVDs were distinguished by brain MRI scan. The size and location of cerebral infarction were recorded using MRI only. A total of 403 controls with sex ratios and age (within 5 years) were selected (see Table 1). The control group was selected from subjects who visited the hospital during the same period for medical examination, including biochemical examination, electrocardiogram and brain MRI. The control group had no history of cerebrovascular disease or myocardial infarction.

1-2. Genotyping

DNA was extracted from white blood cells using G-DEX II Genomic DNA Extraction kit (Intron Biotechnology, Seongnam) according to the manufacturer's instructions. 3'-UTR SNPs ( DICER rs13078 A> T, rs3742330 A> G, DROSHA rs10719 T> C, rs6877842 G> C, RAN , the six best studied single nucleotide polymorphisms (SNPs) in miRNA biosynthesis genes rs14035 C> T and XPO5 rs11077 A> C) were selected. miRNA biosynthesis gene polymorphism was analyzed by PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) method. PCR conditions for miRNA biosynthesis gene polymorphism analysis are shown in Table 2. To verify RFLP discovery, 30% of the PCR assays for each polymorph were randomly selected and repeated, followed by DNA sequencing. Sequencing was performed using an ABI 3730xl DNA analyzer (Applied Biosystems, Foster City, CA, USA). The conformity of RFLP results of these QC samples was 100%.

1-3. Mortality Analysis After Stroke

Survival was tracked from stroke onset to death to assess the association between miRNA biosynthesis gene polymorphism and long-term prognosis after ischemic stroke. The death certificate of the National Statistical Office was used to confirm the death date of each stroke patient (n = 585). Patients who lived until December 31, 2013, were excluded from the study.

1-4. Statistical analysis

The frequency of genotype and allele combinations in ischemic stroke patients and controls was compared using a multivariate logistic regression model and Fisher exact test, respectively. Allele frequencies were calculated from the Hardy-Weinberg equilibrium with P = 0.05 as the threshold to identify deviations. Odds ratios (OR), adjusted odds ratios (AOR) and 95% Cls (confidence intervals) were used to determine the association between various genotypes and ischemic stroke. The association between miRNA biosynthetic gene SNP and mortality after stroke was assessed using Cox proportional risk regression analysis. The proportional risk hypothesis was tested using a log-surveal plot and interactions for additional time in a time-dependent Cox regression model. Logistic regression analysis was used to adjust for possible disturbances such as age, sex, hypertension, diabetes, hyperlipidemia and smoking for multivariate analysis. Statistical significance was acknowledged at P <0.05.

2. Results

2-1. Basic characteristics

The demographics of 585 stroke patients and 403 controls are shown in Table 1. 41.5% and 41.7% of the strokes and controls, respectively, were male, and the mean age was 62.7 ± 10.9 years and 62.8 ± 10.6 years, respectively.

2-2. Genotype Frequency of miRNA Biosynthetic Gene Polymorphism

Tables 3 and 4 show genotype distributions of six miRNA biosynthetic gene polymorphisms in ischemic stroke patients and controls. DICER rs3742330 A> G polymorphisms have been shown to be associated with a high risk of ischemic stroke (AA vs. GG: AOR, 1.459; 95% CI, 1.000 to 2.126; P = 0.050; and AA vs. AG + GG: AOR, 1.360; 95% CI, 1.024 to 1.807; P = 0.034). The DROSHA rs10719 T> C polymorphism was also associated with a high risk of ischemic stroke (TT vs. CC: AOR, 2.038; 95% CI, 1.113 to 3.730; P = 0.021; and TT + TC vs. CC: AOR, 2.001; 95% CI, 1.106 to 3.621; P = 0.022). In contrast, the XPO5 rs11077 A> C polymorphism was associated with a low risk of stroke (AA vs. CC: AOR, 0.101; 95% CI, 0.011 to 0.951; P = 0.045; and AA vs. AC + CC: AOR , 0.669; 95% CI, 0.473 to 0.945; P = 0.023). The incidence of DICER1 rs13078 A> T, DROSHA rs6877842 C> G and RAN rs14035 C> T polymorphisms was not significantly different between stroke cases and controls. Stroke patients were divided into three subgroups (LAD, SVD, and CE) according to the TOAST classification to investigate whether the effects of each polymorphism were localized to specific subtypes (see Tables 5 and 6). Comparison of control with single vs. multiple SVD patients was also performed (see Tables 7 and 8). There was no significant association between the polymorphism investigated and LAD. However, DICER1 rs3742330 A> G polymorphism was found to be significantly associated with SVD (AA vs. AG: AOR, 1.705; 95% CI, 1.073 to 2.710; P = 0.024; and AA vs. AG + GG: AOR, 1.616 95% CI, 1.041 to 2.509; P = 0.032). In addition, the DROSHA rs10719 T> C polymorphism was found to be significantly associated with CE (TT vs. CC: AOR, 3.451; 95% CI, 1.264 to 9.422; P = 0.016; and TT + TC vs. CC: AOR, 3.499 95% CI, 1.348 to 9.082; P = 0.010).

2-3. miRNA biosynthesis gene polymorphism and clinical factors

The stratification analysis of each clinical factor was performed to determine the effect of clinical factors on ischemic stroke incidence. However, no significant clinical factors were found to influence the risk of ischemic stroke (see Table 9). In order to confirm the effects of stroke and genotype on the prevalence of ischemic stroke, a composite effect analysis was conducted. As a result, a synergistic effect on the ischemic stroke prevalence between clinical factors (hypertension and diabetes) and miRNA biosynthetic gene polymorphism was found (FIG. 1). Reference). The DROSHA rs10719 CC genotype has been shown to be associated with stroke in patients with hypertension (AOR, 4.781; 95% CI, 1.981 to 11.54). The combination of DROSHA rs10719 CC genotype and diabetes was found to be the most significant association with stroke (AOR, 12.05; 95% CI, 1.541 to 94.19). The combination of other genes and clinical factors did not appear to be significantly associated with ischemic stroke (see Tables 10-12). Platelet ratio, prothrombin time, activated partial thromboplastin time (aPTT), fibrinogen and antithrombin were measured to examine the effect of miRNA biosynthetic genotype on blood coagulation (see Table 13). The DROSHA rs10719 CC genotypes were aPTT (TT vs. CC: P = 0.007; TC vs. CC: P = 0.019) (see A in FIG. 2) and antithrombin (TC vs. CC: P = 0.039) (B in FIG. 2). There was a significant correlation with the rise. Other coagulation factors did not show a statistically significant association with any of the genotypes investigated.

2-4. miRNA biosynthesis gene polymorphism and survival after stroke

Cox regression analysis was performed on 585 patients with total ischemic stroke according to TOAST subtype to assess the association between miRNA biosynthetic gene polymorphism and stroke mortality (see FIG. 3, Tables 14 and 15). During the average follow-up of 4.80 ± 2.11 years, 99 of the stroke patients died. In the multivariate Cox proportional hazards regression model, there was a significant association between RAN rs14035 and survival of LAD patients with ischemic stroke (CC vs. TT: adjusted risk ratio [HR], 5.978; P = 0.015; and CC +). CT vs. TT: adjusted HR, 3.946; P = 0.034) (see Figure 3). A significant association between RAN rs14035 and SVD was also found in ischemic stroke subtype analysis (CC vs. TT: adjusted HR, 9.403; P = 0.015; and CC + CT vs. TT: adjusted HR, 5.223; P =). 0.039) (see FIG. 3). On the other hand, survival analysis by Cox proportional risk regression based on the stepwise method to confirm the covariate effect showed that mortality in the SVD subtype of ischemic stroke is associated with age and RAN rs14039 polymorphism (see Table 16). ).

2-5. Genetic analysis

Gene-gene interaction analyzes of miRNA biosynthetic gene polymorphisms were performed to identify gene combinations with a synergistic effect on stroke risk, showing associations in some variant and allele combinations (see Tables 17-19).

<110> SUNGKWANG MEDICAL FOUNDATION <120> Association of single nucleotide polymorphism of DICER, DROSHA          and RAN with the risk or prognosis of ischemic stroke in a Korean          population <130> PA-D18137 <160> 12 <170> KoPatentIn 3.0 <210> 1 <211> 51 <212> DNA <213> Homo sapiens <400> 1 cttcaatctt gtgtaaaggg attagacacc ctaacagagc aagatccaat a 51 <210> 2 <211> 51 <212> DNA <213> Homo sapiens <400> 2 gcctagtttt cctgcagaca atgaatgaag tgtgctcatt gaaataaaat a 51 <210> 3 <211> 51 <212> DNA <213> Homo sapiens <400> 3 aagtaatcat gttttaatgt agaacctcaa acaggatgga acatcagtgg a 51 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for rs3742330 <400> 4 ggtctcagtt tggtggcttc 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for rs3742330 <400> 5 cctgccttga caacatgaaa 20 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for rs10719 <400> 6 ctagttttcc tgcagacaat gca 23 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for rs10719 <400> 7 gtaatgcaca ttcaccaaag tca 23 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for rs14035 <400> 8 gaagcacttg ctcaaaatct gtgac 25 <210> 9 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for rs14035 <400> 9 tgccatccac tgatgttcca tc 22 <210> 10 <211> 51 <212> DNA <213> Homo sapiens <400> 10 agtacctcca aggaccaggg ctgggaagtc tttagtgcta acatcccctt t 51 <210> 11 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for rs11077 <400> 11 tgctttgggc aagaatctgg tcac 24 <210> 12 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for rs11077 <400> 12 taaaggggat gttagcacta aagaat 26

Claims (8)

delete delete delete delete A method for determining the single nucleotide polymorphism of rs14035 from DNA samples of subjects in order to provide information necessary to predict the prognosis of ischemic stroke in Koreans. A kit for predicting ischemic stroke prognosis in Koreans comprising a means for detecting a single base polymorphism of rs14035. The method of claim 6,
The means for detecting the single nucleotide polymorphism of rs14035 is a primer for amplifying a continuous 50 to 10,000 bp region comprising a single nucleotide polymorphism site of rs14035 on human genomic DNA and the base of the single nucleotide polymorphism site of rs14035 is cytosine or A kit comprising a primer-limiting enzyme set comprising a restriction enzyme capable of cleaving DNA of any one of thymine. The method of claim 7, wherein
The primer comprises an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 8 and an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 9, wherein the restriction enzyme is a kit, characterized in that Bsl I.

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