KR20130026620A - A genetic polymorphic marker of neuropeptide y for classification of stroke and the use thereof - Google Patents

Abstract

PURPOSE: A genetic polymorphic marker of neuropeptide Y and a use thereof are provided to effectively determine whether or not a stroke patient has dampness-phlegm. CONSTITUTION: A marker for classification of stroke with dampness-phlegm containing a polypeptide or a complementary polypeptide thereof with 20-100 continuous DNA sequences containing 27th base which is C or T. A kit for classification of stroke with dampness-phlegm contains a probe or a primer pair for detecting the marker. The primer pair has base sequences of sequence numbers 6 and 7.

Description

The present invention relates to a neuropeptide Y gene polymorphism marker for stroke classification and a use thereof,

The present invention relates to a neuropeptide Y gene polymorphism marker and a use thereof, and more particularly, to a marker for predicting stroke occurrence risk having a hygroscopicity based on the neuropeptide Y gene polymorphism, A kit for predicting the occurrence of a stroke, a kit for classifying a stroke patient having a microarray or a wet poison, a microarray; And a method for predicting a stroke occurrence risk having a wet poisoning or a stroke classification method having a wet poisoning.

Stroke (cerebrovascular disease) is the most common cause of death and disability among Koreans over 65 years of age. In 2004, the total death rate of Koreans was 506.97 per 100,000 people. About 13.9% of all deaths were caused by stroke (cerebrovascular disease) and its aftermath. , And it is expected that the cost will increase further due to changes in the aging society and lifestyle of society.

The first predictive model for paralysis was the Framingham model, developed by Wolf et al. In 1991, which was developed from 55 to 84 years of age. The Framingham Heart Study has contributed to identifying risk factors for stroke and has developed a multivariate function to predict absolute stroke risk. The risk factors considered in the development of the stroke model in the Framingham study were age, systolic blood pressure, use of antihypertensive therapy, presence of diabetes, smoking, Crdiovascular disease, Atrial fibrillation ), And left ventricular hypertrophy. Among them, Crdiovascular disease, Atrial fibrillation and Left ventricular hypertrophy are factors that can be measured through ultrasound and it is difficult to measure easily by the general public. There are limitations in the application of efficacy. These limitations make it different from other ethnic groups in terms of dietary habits, lifestyle, social environment, genetic distribution, and people from urban suburbs such as Coca- nian or Framingham. In addition, recent studies have reported significant inconsistencies between the incidence of heart disease predicted by Framingham and the actual incidence observed in southern Europe and Chinese populations, and studies in China have shown that in Asian populations at high risk for stroke, And other studies of Asians have failed to provide a prediction of risk.

Due to many differences such as constitution, physical condition, lifestyle, and genetic difference between oriental and western people, a discrimination model for oriental paranasal augmentation was developed by Shin Yang - gyu in 1996 in Korea. From the clinical data of 65 patients collected by a stroke specialist, Shin Yang Kyu research has constructed a mathematical discriminant model that can judge the middle type from the symptoms using the discriminant analysis which is one of the multivariate data analysis. This provides the most useful information as a methodology for the discrimination model for the diagnosis of paranasal sinuses developed in Oriental medicine to date.

However, there is also a limit to the paranoid stimulation (paresis) obtained from the discriminant model for the diagnosis of this stroke. That is, the above discrimination model is made using the standard of diagnosis of paralysis of the paralysis according to the Chinese standard, and thus there is a limit in which the clinical reality of the country can not be completely included. In addition, since the clinical data are collected only in one area, it is not possible to classify them as paresis of Korean paralytic patients, and there is a problem that the reliability of the model made of only 65 clinical data is low.

In this study, we compared the results of 1196 stroke patients who visited the National Oriental Medicine and Oriental Medicine Clinic between 2005 and 2008 from 684 patients who were diagnosed as experts and residents except 512 patients The Korean version of the Korean version of the Korean Patent Registration No. 10-1025930 was used to develop a discrimination model for oriental orthopedic dialysis based on the symptoms and other risk factors. However, in the case of using the medical symptom as an index, it is still required to find more scientific and clear indicators to classify the paresthesias.

In this regard, identification of genetic factors associated with stroke is important in reducing the risk of stroke and in developing customized medicine to prevent or treat stroke. For the last 10 years, a 5-lipoxygenase activating protein gene (ALOX5AP) and a phospholipase gene have been identified from a genome-wide association study (GWAS) and a control-case association study (Gretarsdottir et al., 2003; Helgadottir et al., 2004) were identified as a genetic risk factor for stroke, including the phosphodiesterase 4D gene (PDE4D).

In a recent study, it was found that the neuropeptide Y gene is located at the chromosomal locus 7p15.1, and it is known that there are 25 gene polymorphisms in the promoter and seven exon regions of this gene. In particular, its L7P gene polymorphism Type 2 diabetes, alcoholism, and obesity. In addition, when the C-399T polymorphic site is allele T, the transcriptional activity is lower than that of allele C, and this gene polymorphism has been reported to be associated with schizophrenia and non-Hodgkin lymphomas . However, there were no reports of differences in C-399T gene polymorphism between paralytic patients with dyspnea and dyspepsia with dyspnea.

Under these circumstances, the present inventors, while studying genetic polymorphisms associated with stroke and its hereditary apoplexy, conducted a confirmatory association analysis of a single-nucleotide polymorphism of the neuropeptide Y gene, C-399T, in a humid- The results showed that the frequency of allele T of C-399T was significantly decreased in the stroke patients compared to the normal group, And can be used as a risk prediction factor.

An object of the present invention is to provide a rs16147 having the nucleotide sequence of SEQ ID NO: 1, wherein the 27th base (-1450th base from the transcription start point of the Neuropeptide Y gene) is C or T, 20-100 And to provide a marker for classification of a paralytic patient having a hygrometry consisting of a polynucleotide consisting of a continuous DNA sequence or a complementary polynucleotide thereof.

It is still another object of the present invention to provide a kit for classifying a paralytic patient having a moisturizing apex that includes a probe or a pair of primers capable of detecting the classifying marker.

It is still another object of the present invention to provide a microarray for classifying a paralytic patient having a wet-mouth syndrome including the marker for classification.

Yet another object of the present invention is a method of treating a paralytic patient comprising: a) obtaining DNA from a paralytic patient; b) amplifying or hybridizing the polymorphic site of the marker for classifying the paralytic patient having the hygroscopicity from the DNA obtained in the step a); And c) determining bases of the amplified or hybridized polymorphic site of step b) to be a paralytic patient having a hydrosphere apex or non-humid apnea, thereby providing a method of classifying a paralytic patient having a humor syndrome will be.

It is still another object of the present invention to provide a polynucleotide comprising rs16147 having the nucleotide sequence of SEQ ID NO: 1, wherein the 27th base is C or T, the 20th to 100th consecutive DNA sequences comprising the 27th base, And to provide a marker for predicting the risk of stroke occurrence having a hygroscopic syndrome consisting of complementary polynucleotides.

It is still another object of the present invention to provide a kit for predicting the risk of stroke occurrence having a wet-bulb irritation including a pair of probes or primers capable of detecting the marker for prediction.

It is still another object of the present invention to provide a microarray for predicting the risk of stroke occurrence having a wet poisoning including the predictive marker.

Still another object of the present invention is a method for producing DNA comprising: a) obtaining DNA from a normal person; b) amplifying or hybridizing a polymorphic site of a marker for predicting the risk of stroke occurrence having a hygroscopicity from the DNA obtained in step a); And c) identifying a base of the amplified or hybridized polymorphic site in step b) to predict the risk of stroke occurrence having a wet poisoning, and providing a method for providing information on the risk of stroke occurrence having a wet poisoning .

In one aspect of the present invention, there is provided a rs16147 having the nucleotide sequence of SEQ ID NO: 1, wherein the 27th base (-1450th base from the transcription start point of the Neuropeptide Y gene) is C or T, Wherein the marker is a polynucleotide consisting of 20-100 consecutive DNA sequences comprising the base sequence of SEQ ID NO: 1 or its complementary polynucleotide.

According to another aspect of the present invention, there is provided a rs16147 having a nucleotide sequence of SEQ ID NO: 1, wherein the 27th base is C or T, and the 20th to 100th There is provided a marker for predicting the risk of stroke occurrence having a hygroscopic syndrome consisting of a polynucleotide consisting of consecutive DNA sequences or a complementary polynucleotide thereof.

The neuropeptide Y gene is known as GenBank accession number NC_000007, and in the present invention, the sequence number 24323384 to 24323435 in the NC_000007 sequence is represented by SEQ ID NO: 1. The -1450th base from the transcription start point of the promoter region of the neuropeptide Y gene in the polynucleotide is due to a point mutation substituted with T or C, and the polymorphic site is designated as "C-399T ".

In the present invention, the term "polymorphism" refers to a case where two or more alleles exist in one locus. Of the polymorphic sites, only a single base differs from a polymorphism region to a single base polymorphism (single nucleotide polymorphism, SNP).

The term "allele " in the present invention refers to various types of genes that exist on the same locus of a homologous chromosome. Alleles are also used to indicate polymorphism, for example SNPs have two kinds of bialleles.

The term "neuropeptide Y (NPY)" in the present invention belongs to the family of neuroendocrine peptides and includes 36 < RTI ID = 0.0 >Quot; means a peptide consisting of an amino acid. In a recent study, it was found that the neuropeptide Y gene is located at chromosome locus 7p15.1, and it is known that there are 25 gene polymorphisms in the promoter and 7 axon regions of this gene, and some gene polymorphisms are associated with type 2 diabetes , Alcoholism, obesity, schizophrenia, and non-Hodgkin's lymphomas. However, there have been no reports of differences in C-399T gene polymorphic sites among patients with paralytic dyspepsia with damp or non-damping syndrome.

The present inventors first confirmed that the frequency of the allele T of C-399T, a single base polymorphism of the neuropeptide Y gene, was significantly lowered in the dyspepsia group than in the normal group. Using this gene polymorphism as a marker, To a swift or non-swallowing stroke patient. Further, the present invention provides a means for easily and effectively predicting the risk of the occurrence of paralysis with wet or dry non-wetting among healthy persons.

The term "paralyticism" in the present invention refers to a paralytic dialysis diagnostic standardization committee composed of domestic clinical experts, including a review of previous literature related to paralytic dialysis, Dongwoo Bohak, recent paralytic clinical trials, Chinese dialectical standard, , Which means that they are classified according to the matters which are agreed to classify them into five dialectic languages, such as Hwajeong, Hapham, Euphorbiae, Haengheung, or Hwangwol, which is the basic element of the paranoia.

In the five dialects that classify this paralysis, "damp" is expressed as dementia (痰 证) which occurs due to the internal downturn in dental medicine for a long time, , And also called nonspecific depression (脾 经 濕 痰). In addition, in the 5th volume of the introduction to medicine, it is recorded that "most of the occurrences in the spleen are caused by limbs, stomach ache, lethargy and diarrhea," and " It is called "痰 (sputum)" in the spleen. The color is yellow, smooth and easy to come out. Mostly, it is weak and weak. It likes to lie down. The stomach is full and the stomach is blocked and the vein is necessarily loose. (君 君子 湯) is suitable for the huh (虛), and the bamboo flute (和 和 丸) is suitable for the meal. Soosheejang is suitable, and Myojyohwan is suitable if it combines with surprise. It should be categorized on the basis of each spleen. " Thus, dampness includes all the waste products generated by the inability of circulation of nutritional metabolites in the body, for example, sputum, excessive fat mass in the body, and muscle or ligament aggregation.

In general, one of the superior aspects of Oriental medicine is that it can be divided into various dialectic groups for a single disease, so that the individual can be treated appropriately. However, since the judgment of such a medical dialectic is subjectively judged by the oriental hospital specialist on the basis of the patient's symptoms, the scientific and clear index has been demanded. Therefore, by using the marker according to the present invention, it is possible to classify the paralytic patients more scientifically according to the dialect based on the dampness, and it is advantageous that the treatment can be more tailored to the patient than the conventional amblyopia treatment have. In addition, there is an advantage of predicting the risk of the occurrence of paralysis which has wet or no dampness in normal people, thereby preventing paralysis.

In another aspect, the present invention provides a kit for classifying a patient with a wet catheter having a probe or a pair of primers capable of detecting the classifying marker of the present invention.

According to still another aspect of the present invention, there is provided a kit for predicting the risk of stroke occurrence having a hygroscopic apnea including a pair of probes or primers capable of detecting the predictive marker of the present invention.

The kit according to the present invention comprises a polynucleotide or its complementary polynucleotide capable of detecting the polymorphism of the gene at C-399T of the neuropeptide Y gene, a polynucleotide which hybridizes with them, or a cDNA thereof, And can be prepared by conventional methods known to those skilled in the art.

The term "primer" in the present invention means a base sequence having a short free 3 'hydroxyl group, capable of forming base pairs with a complementary template and having a starting point for template strand copy Quot; short sequence " Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization (ie, DNA polymerase or reverse transcriptase) at appropriate buffers and temperatures. Preferably, the marker according to the present invention can be amplified using SEQ ID NO: 6 and SEQ ID NO: 7. The PCR conditions, the lengths of the sense and antisense primers can be modified based on what is known in the art.

The term "probe" in the present invention means a nucleic acid fragment such as RNA or DNA corresponding to a short period of a few nucleotides or a few hundreds of nucleotides capable of specifically binding to a nucleic acid, . The probe can be produced in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, or an RNA probe. In the present invention, by observing the polymorphism of the neuropeptide Y gene, it is possible to predict the risk of the occurrence of the paralysis having the hygroscopic or to classify the paralytic patients having the hygrosis.

Preferably, the use of the nucleic acid sequence shown in SEQ ID NO: 14 as a probe, and the selection and hybridization conditions of a suitable probe may be modified based on those known in the art.

Primers or probes of the invention can be synthesized chemically using phosphoramidite solid support methods, or other well known methods. Such nucleic acid sequences may also be modified using many means known in the art. Non-limiting examples of such modifications include, but are not limited to, methylation, "capping ", replacement of natural nucleotides with one or more homologues, and modifications between nucleotides, such as uncharged linkers, such as methylphosphonate, Phosphoamidates, carbamates, etc.) or charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.).

The term "kit" in the present invention means a tool for easily detecting or quantifying a polymorphism of a gene to be detected or quantified in a sample. The kit according to the present invention may contain an artificial base sequence comprising at least one primer and / or probe site specific for C-399T of the neuropeptide Y gene, as well as one or more other component compositions, solutions, or devices suitable for the assay method May be included. In particular, the probe labeled with a labeling substance may be used. Such a labeling substance may be, for example, a second labeling substance such as a radioisotope, a fluorescent reporter molecule, a chemiluminescent reporter molecule, an antibody, an antibody fragment, a hapten, a biotin, a derivative of biotin, photobiotin, iminobiotin, digoxigenin, An electrophile, an enzyme, an acridinium, a sugar, an enzyme, an apo enzyme, a homopolymeric oligonucleotide, a hormone, a ferromagnetic part, a paramagnetic part, a half-magnetic part, a phosphorescent part, a luminescent part, But are not limited to, spin resonance, capacitance, dielectric constant, or portions having electrical conductivity, or combinations thereof.

As a specific example, the kit according to the present invention may be a kit containing essential elements necessary for performing RT-PCR. The RT-PCR kit contains test tubes or other appropriate containers, reaction buffers (varying in pH and magnesium concentrations), deoxynucleotides (dNTPs), Taq-polymer (s), as well as the respective primer pairs specific for C-399T of the neuropeptide Y gene Enzymes such as lyase and reverse transcriptase, DNase, RNAse inhibitor, DEPC-water, sterile water, and the like. It may also contain a primer pair specific for the gene used as a quantitative control.

In addition, preferably, the kit of the present invention may be a kit for predicting the occurrence of a stroke, or a kit for classifying a stroke patient having a wet-mouth syndrome, which has a wet-mouth syndrome containing essential elements necessary for carrying out a DNA chip. The DNA chip kit may include a substrate to which a cDNA corresponding to a gene or a fragment thereof is attached as a probe, and the substrate may include a cDNA corresponding to a quantitative control gene or a fragment thereof.

In yet another aspect, the present invention provides a microarray for classifying paranasal sinuses having a wet-mouth syndrome comprising the marker for classification of the present invention.

According to another aspect of the present invention, there is provided a microarray for predicting the risk of stroke occurrence having a wet poisoning including the predictive marker of the present invention.

The microarray may include DNA or RNA polynucleotides. Methods for preparing microarrays by immobilizing probe polynucleotides on substrates are well known in the art. The probe polynucleotide refers to a polynucleotide capable of hybridization, and refers to an oligonucleotide capable of sequence-specific binding to the complementary strand of a nucleic acid. The probe of the present invention is an allele specific probe in which polymorphic sites exist in nucleic acid fragments derived from two members of the same species, and hybridize to DNA fragments derived from one member, but not to fragments derived from other members. . In this case, the hybridization conditions must be sufficiently stringent to hybridize to only one of the alleles, showing significant differences in the hybridization intensity between alleles. This can lead to good hybridization differences between different allelic forms.

The probe of the present invention can be used for a method of predicting the risk of occurrence of a paralytic wind having a wet seam or a method of classifying a paralytic patient having a wet seam by detecting alleles. Such methods include detection methods based on hybridization of nucleic acids such as Southern blots, and may be provided in a form preliminarily bonded to a substrate of a DNA chip in a method using a DNA chip. For example, the polynucleotide can be immobilized on a substrate coated with an activator selected from the group consisting of amino-silane, poly-L-lysine and aldehyde. In addition, the substrate may be selected from the group consisting of silicon wafer, glass, quartz, metal and plastic.

The method of immobilizing the probe polynucleotide associated with the parasitic wind of the present invention on a substrate may be a micropipetting method using a piezo electric method, a spotter method using a pin, And the like. In addition, hybridization of nucleic acids on a microarray and detection of hybridization results are well known in the art. The detection is accomplished by labeling a nucleic acid sample with a labeling substance capable of generating a detectable signal comprising, for example, a fluorescent substance such as Cy3 and Cy5, and then hybridizing onto a microarray and generating from the labeling substance. The hybridization result can be detected by detecting the signal.

In another aspect, the present invention provides a method for classifying a stroke patient having a wet poisoning based on the polymorphism of the neuropeptide Y gene, or a method for providing information on the risk of stroke occurrence having a wet poisoning.

Specifically, the classification method according to the present invention comprises the steps of: a) obtaining DNA from a paralytic patient; b) amplifying or hybridizing a polymorphic site of a marker for classifying a paralytic patient having a hygroscopicity from the DNA obtained in the step a); And c) determining the base of the amplified or hybridized polymorphic site of step b) to be a paralytic patient having a wet or elongated apnea syndrome.

Preferably, when the genotype of the 27th nucleotide of SEQ ID NO: 1 identified in step c) is CC, it can be judged to be a paralytic patient having a wet-bulb aneurysm. In addition, when the 27th base of SEQ ID NO: 1 identified in step c) is T, it can be judged to be a paralytic patient having non-wettability.

Further, a method of providing information according to the present invention comprises the steps of: a) obtaining DNA from a normal person; b) amplifying or hybridizing a polymorphic site of a marker for predicting the risk of stroke occurrence having a hygroscopicity from the DNA obtained in step a); And c) identifying the base of the amplified or hybridized polymorphic site of step b) and predicting the risk of stroke.

Preferably, when the genotype of the 27th nucleotide sequence of SEQ ID NO: 1 identified in step c) is CC, it can be predicted that the risk of the occurrence of paralysis with wetting syndrome is increased.

All biological samples such as whole blood, blood cells, serum, plasma, bone marrow fluid, sweat, urine, tears, saliva, skin, mucous membrane and hair can be used to obtain DNA from the paralytic patient or normal person. In the present invention, by analyzing the polymorphism of the neuropeptide Y gene according to the present invention using blood as a sample, it was confirmed that a wet or non-humid stroke patient can be classified (Table 6).

The method of obtaining the genomic DNA of step a) may be any method known to those skilled in the art.

The marker of step b), that is, rs16147 having the nucleotide sequence of SEQ ID NO: 1, is C or T, and the 20th nucleotide (the -1450th nucleotide from the transcription start point of the NPY gene) The step of amplifying a polynucleotide consisting of 100 consecutive DNA sequences or a complementary polynucleotide thereof can be used in any method known to a person skilled in the art. For example, the target nucleic acid can be amplified by PCR and purified. Other ligase chain reaction (LCR), self-sustained sequence replication, and nucleic acid-based sequence amplification (NASBA) may be used. In addition, the steps of hybridizing with the probe include sequencing analysis, hybridization with a microarray, allele specific PCR, dynamic allele specific hybridization (DASH), PCR extension analysis, SSCP, PCR -RFLP analysis, or TaqMan technique. ≪ / RTI > Preferably, primers represented by SEQ. ID. NO. 6 and 7 can be used, and probes represented by SEQ. ID. NO. 14 can be used as the probes, according to PCR-RFLP analysis.

In one embodiment of the present invention, venous blood is collected from a paralytic patient and a normal person, the genomic DNA is purified, and then a single-base extension reaction is performed from the purified genomic DNA to obtain neuropeptide Y The genotype of C-399T of the gene was determined. To investigate the relationship between the gene polymorphism and paresthesias, we used multiple logistic regression analysis of paresthesia with normals and apnea. As a result, the allele T of C-399T of the neuropeptide Y gene was significantly lower than that of the paralytic patients. In particular, in paralytic patients, it was found that paralytic patients with dampness were significantly lower than those with non-damp palsy (Table 6).

In addition, multiple logistic regression analysis was performed as described above after applying various clinical factors to determine the genetic association of C-399T of the neuropeptide Y gene with the paresthesias and their apologies for sex, age, hypertension, It is still valid after applying multiple factors such as hyperlipidemia, ischemic heart disease, smoking, drinking, waist, total cholesterol (TC) and HDL. In other words, it was confirmed that the frequency of allele T of C-399T, a single nucleotide polymorphism of neuropeptide Y gene, was statistically different between general and non-hypersensitive paralytic patients and dyspepsia patients. Therefore, the C-399T single base polymorphism of the present invention can be used as a marker to classify a paralytic patient as a wet or non-humid paralytic patient, and furthermore, to predict the risk of paralysis that has a humorous apology for a normal person.

According to the present invention, it is possible to effectively use for the treatment of paralysis by efficiently determining a paralytic patient having a wet-bulb irritation and a paralytic patient having a non-wet-mouth irritation and providing a low-level therapeutic treatment according to the determined irritation. Furthermore, there is an advantage that the stroke can be effectively prevented by easily and efficiently predicting the risk of the occurrence of the paralysis having a wet-mouth syndrome.

Figure 1 is a chromosome map showing the polymorphic site of neuropeptide Y located on chromosome 7p15.1.

Hereinafter, preferred examples are provided to help understanding of the present invention. The following examples are provided only to more easily understand the present invention, but the contents of the present invention are not limited by the examples.

Example 1. Selection of test subjects

The present inventors have conducted experiments in a variety of fields including Kyung Hee Oriental Medicine Hospital, Kyung Hee Dongseo New Medical School, Dongguk University Ilsan Hospital, Kyungwon University School of Medicine (Seoul and Incheon), Dongseo Oriental Hospital, Daejeon Oriental Medicine Hospital, Dongshin University Oriental Medicine Hospital (Gwangju and Jeonnam) The patients were recruited from 12 hospitals in the Korean Oriental Hospital, Woosuk University Oriental Hospital, and Sangji University Oriental Medicine Hospital. All patients with cerebral infarction (CI) were examined by magnetic resonance imaging (MRI) or magnetic resonance angiography (MRA). Clinical data of patients and data related to chronic diseases were obtained through the Stroke PI case report form, and the patients were judged on the basis of the data of two specialist practitioners. , Transient ischemic attack, diabetes, hyperlipidemia, and heart disease. Patients with a history of paralysis or traumatic paralysis were also excluded.

Based on the above criteria, 498 normal and 599 stroke patients were selected for analysis. Of these, 198 were diagnosed as dyspepsia patients and 401 as non - dyspepsia patients.

Example 2. Analysis of Clinical Characteristics of Subjects

The clinical characteristics of the subjects selected through the method of Example 1 were statistically analyzed. Statistical analysis was performed using SAS software version 9.1.3 (SAS Institute Inc, NC, USA). Categorical variables were compared using the Chi-square test. All continuous variables were subjected to the Kolmogorov-Smirnov normality test. The differences in the continuous variables were determined using parametric tests (Student's t-test and ANOVA) or non-parametric tests (Wilcoxon rank-sum test and Kruskal-Wallis test) Respectively. Hereinafter, statistical analysis was performed in the same manner in the experiment.

As a result, as shown in Table 2 below, the paralytic patient group contained more male and older persons than the normal control group, and hypertension, smoking, and drinking, which are typical paralysis risk factors, were higher in the paralytic patients than in the control group. In addition, the paralytic patient group showed lower total cholesterol and HDL-cholesterol levels than the control group.

Example 3. DNA purification and neuropeptide Y gene polymorphism analysis

The present inventors collected whole blood from the subject and purified the genomic DNA from the whole blood using the GeneAll genomic isolation kit (GeneAll co., Korea) according to the manufacturer's instructions. All exon and promoter regions (2 kb) of the neuropeptide Y gene were analyzed by using 24 ABI PRISM 3700 DNA analyzer (Applied Biosystems, Foster City, CA, USA) to analyze 24 Korean genomic DNA samples to identify the sequence After confirming the polymorphic site of the gene, it is shown in Table 3 and Fig.

As shown in Table 3 and FIG. 1, it was confirmed that the neuropeptide Y gene had three polymorphic sites in the promoter region and no two amino acid changes in the exon region.

In addition, the Hardy-Weinberg equilibrium was calculated to confirm that the neuropeptide Y gene had an equilibrium of each position in the population between the 5 gene polymorphisms, and all pairwise occurrences a linkage disequilibrium was assessed by ┃D'┃ and r ² in order to measure (Hedric, PW, Genetics, 117 : 331-341, 1987). As a result, as shown in Table 4, the association imbalance coefficients between G-1484A, C-399T, and A1201G were the highest with ┃D'┃ = 1 and r ² = 0.862-0.951, indicating that there was a high correlation among three gene polymorphic sites .

In order to identify the identified polymorphic site from normal individuals and patients, a primer set was designed based on sequence information provided from NPY: NC_000007 for sequence analysis of the polymorphic site. The information of the primers used for the amplification and sequencing of the neuropeptide Y gene is shown in Table 5.

Example 4: Genotyping and correlation with clinical factors in patients with stroke

Follow the manufacturer's instructions to identify SNaPshot® genotypes and their association with clinical factors in normal and stroke patients ^. Multiplex system (Applied Biosystems, Foster City, CA, USA). At this time, a primer probe set designed from Example 3 was used, and the results are shown in Table 6 below.

As shown in Table 6, when the values of p = 0.0378 and OR = 0.737 (0.552-0.983) of clinical parameters including sex, age, smoking, drinking and hypertension were applied, the C-399T gene polymorphism The incidence of allele T was 27.27%, which was significantly lower than that of normal controls (33% and 45.96%). Also, when the values of p = 0.0315 and OR = 0.663 (0.456-0.964) of the clinical parameters including sex, age, smoking, drinking and hypertension were applied, the ratio of individuals having allele T in the C-399T polymorphism site In addition, 45.96% of patients with dyspepsia were significantly lower than those of normal controls (53.25% and 56.0% of non-dyspepsia patients) (Table 6).

These results indicate that it is possible to classify the paralytic patients with wet or non-wetting acuity quickly and efficiently using the C-399T single nucleotide polymorphism as a marker, and to predict the risk of stroke.

Example 5 Correlation Analysis of C-399T Genotyping of Neuropeptide Y with Blood Cholesterol

In order to confirm the association of neuropeptide Y with blood cholesterol according to the C-399 genotype, the clinical factor data missing in Example 1 were classified by the type of C-399 genotype of neuropeptide Y obtained in Example 4 And the results are shown in Table 7 below.

As shown in Table 7, it was confirmed that the levels of total cholesterol and LDL-cholesterol of normal individuals having TT type C-399 genotype of neuropeptide Y were significantly lower than those of alleles C (p = 0.0111 and p = 0.0186). These results indicate that the C-399T monoclonal polymorphic site of neuropeptide Y is involved in the alteration of blood cholesterol levels and is associated with dampness, especially dyspnea in paralytic patients.

<110> Korea Institute of Oriental Medicine <120> A genetic polymorphic marker of Neuropeptide Y for classification          of Stroke and the Use thereof <130> PA110489KR <160> 16 <170> Kopatentin 1.71 <210> 1 <211> 52 <212> DNA <213> rs16147 <400> 1 cttgtctcct gccaacagga ctaccayccc actgggtgcc ggagtaggaa gc 52 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> G-1484A forward primer <400> 2 gaatcaagct atgaacctgc tatc 24 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> G-1484A reverse primer <400> 3 gtgtagagtg tgccctgtac aaa 23 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> C-1471T forward primer <400> 4 gaatcaagct atgaacctgc tatc 24 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> C-1471T reverse primer <400> 5 gtgtagagtg tgccctgtac aaa 23 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> C-399T forward primer <400> 6 gaatcaagct atgaacctgc tatc 24 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> C-399T reverse primer <400> 7 gtgtagagtg tgccctgtac aaa 23 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> A1201G forward primer <400> 8 gaatcaagct atgaacctgc tatc 24 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> A1201G reverse primer <400> 9 gtgtagagtg tgccctgtac aaa 23 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> C5325T forward primer <400> 10 cattacccaa ataggagact at 22 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> C5325T reverse primer <400> 11 caccttgagc tctgcaacaa tg 22 <210> 12 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> G-1484A probe <400> 12 aatagaagat gttttgcact tttacc 26 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> C-1471T probe <400> 13 agttgctcgg gggtgcttac 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> C-399T probe <400> 14 tcctgccaac aggactacca 20 <210> 15 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> A1201G probe <400> 15 ggacatggcc agatactact c 21 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> C5325T probe <400> 16 tgaaatcagt gtctctgggc t 21

Claims (18)

In rs16147 having the nucleotide sequence of SEQ ID NO: 1, the 27th base (-1450th base from the transcription start point of the Neuropeptide Y gene) is C or T and is composed of 20-100 consecutive DNA sequences including the 27th base. Marker for classifying paralytic patients with ecstatic dialectic consisting of a polynucleotide consisting of or a complementary polynucleotide thereof.
A kit for classifying palsy patients with eclampsia comprising a probe or primer pair capable of detecting a marker for classifying palsy patients with schizophrenia of claim 1.
3. The kit according to claim 2, wherein the primer pair has the nucleotide sequence of SEQ ID NOs: 6 and 7.
3. The kit according to claim 2, wherein the probe has the nucleotide sequence of SEQ ID NO: 14.
3. The kit according to claim 2, wherein the kit is an RT-PCR kit or a DNA chip kit.
In rs16147 having the nucleotide sequence of SEQ ID NO: 1, the 27th base (-1450th base from the transcription start point of the Neuropeptide Y gene) is C or T, and is represented by 20-100 consecutive DNA sequences including the 27th base. A microarray for classifying palsy patients with ecstatic symptoms comprising a polynucleotide or a complementary polynucleotide thereof.
a) obtaining DNA from a paralytic patient;
b) amplifying or hybridizing with a probe a polymorphic site of a marker for classifying a paralytic patient having the ecstasy of claim 1 from the DNA obtained in step a); And
c) identifying the base of the amplified or hybridized polymorphic site of step b) and judging as a palsy patient with eclampsia or non-diagnosis.
The method of claim 7, wherein the probe has a nucleotide sequence of SEQ ID NO: 14.
The method according to claim 7, wherein the genotype of the 27th base of SEQ ID NO: 1 identified in step c) is CC, and is judged as a stroke patient with eclampsia.
The method of claim 7, wherein when the 27th base of SEQ ID NO: 1 identified in step c) is T, the classification method is determined to be a paralytic patient with non-wetness dialectic.
In rs16147 having the nucleotide sequence of SEQ ID NO: 1, the 27th base is C or T and a polynucleotide or a complementary polynucleotide consisting of 20-100 consecutive DNA sequences comprising the 27th base. Marker for predicting the risk of stroke caused by dialectic.
A kit for predicting the risk of stroke, having a phlegm, comprising a pair of probes or primers capable of detecting a marker for predicting the risk of stroke having a sputum episode of claim 11.
According to claim 12, wherein the primer pair has a nucleotide sequence of SEQ ID NO: 6 and 7 kit for predicting the risk of stroke caused by wet phlegm.
According to claim 12, wherein the probe has a nucleotide sequence of SEQ ID NO: 14 is a kit for predicting the risk of palsy having ecstasy.
According to claim 12, wherein the kit is RT-PCR kit or DNA chip kit is a kit for predicting the risk of stroke caused by schizophrenia.
In rs16147 having the nucleotide sequence of SEQ ID NO: 1, the 27th base (-1450th base from the transcription start point of NPY gene) is C or T and consists of 20-100 consecutive DNA sequences containing the 27th base Microarray for predicting the risk of stroke caused by ecstatic symptoms comprising a polynucleotide or a complementary polynucleotide thereof.
a) obtaining DNA from a normal person;
b) amplifying or hybridizing the polymorphic site of the marker for predicting the risk of stroke, having the weeding symptom of claim 11, from the DNA obtained in step a); And
c) identifying the base of the amplified or hybridized polymorphic site of step b) to predict the risk of stroke occurrence with schizophrenia.
18. The method of claim 17, wherein the risk of developing a stroke with eczematism is increased when the genotype of the 27th base of SEQ ID NO: 1 identified in step c) is CC.

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