KR102085663B1 - Method for providing information of prediction and diagnosis of small vessel occlusion using methylation level of WRB gene and composition therefor - Google Patents

Abstract

The present invention relates to a method for providing information for prediction or diagnosis of small vessel occlusion using a methylation level of a terminator site of the tryptophan rich basic protein (WRB) gene, and a composition and a kit for prediction or diagnosis of small vessel occlusion. A WRB methylated biomarker according to the present invention can be not only used for prediction of small vessel occlusion, but also used for the assessment of the risk of the small vessel occlusion, disease identification, disease stage diagnosis, and treatment target selection.

Description

Method for providing information of prediction and diagnosis of small vessel occlusion using methylation level of WRB gene and composition therefor}

The present invention relates to a method for providing information for predicting or diagnosing small vessel occlusion using the methylation level of the WRB gene, and a composition therefor. Specifically, WRB (Tryptophan Rich Basic Protein) The present invention relates to a method of providing information for predicting or diagnosing small vessel occlusion using a terminator site methylation level of a gene, and to a composition and kit for predicting or diagnosing small vessel occlusion.

Small vessel occlusion is a cerebral infarction (stroke) caused by blockage of the small vessels in the brain and no blood supply through these vessels, and is known to occur more frequently in Asians than in Westerners. According to a report of patients with cerebral infarction of all ages from 2011 to 2013 through the Korean Clinical Research Center for Stroke (CRCS), 38.3% for atherosclerosis, 22.8% for cardiac embolism, and small vessel occlusion 14.6% of the cases, 2.1% of other causes, and 22.2% of unknown causes. Small blood vessels work in our bodies more diversely and more finely than large blood vessels. In general, small blood vessel disease is a disease that requires attention in that it can be a precursor of large vessel disease, that is, a preceding signal for predicting large vessel disease.

Recently, methods for diagnosing diseases such as cancer by measuring DNA methylation have been proposed. When the CpG island of a particular gene is hypermethylated, its expression becomes gene silencing. This is the major mechanism by which genes lose their function without mutation in the protein-specific coding sequence of genes in vivo, especially in cancers where many tumor suppressor genes lose their function. It is interpreted as a cause. Therefore, the detection of methylation of CpG island of a specific gene is a great help in disease research, and attempts to use it for diagnosis and screening of diseases by examining methylation-specific PCR or automatic base analysis methods have been actively made recently. .

The genomic DNA of mammalian cells contains a fifth base in addition to A, C, G, and T, which is 5-methylcytosine (5-mC) having a methyl group attached to the fifth carbon of the cytosine ring. 5-mC always comes only to C of CG dinucleotide (5'-mCG-3 '), and this CG is often referred to as CpG. Most of C of CpG is methylated by attaching a methyl group. This methylation of CpG inhibits the expression of repetitive sequences in the genome, such as Alu or transposon, and is the site where extragenic changes occur most frequently in mammalian cells. This 5-mC of CpG naturally deaminates and changes to T, so that CpG in the mammalian genome is only 1% less than the frequency at which it should normally appear (1/4 x 1/4 = 6.25%). Indicates.

Among CpGs, there are exceptionally dense ones, which are called CpG islands. CpG islands are 0.2-3 kb in length, and the distribution percentage of C and G bases is over 50%, and the distribution percentage of CpG is concentrated at 3.75% or more. About 45,000 CpG islands appear in the entire human genome, particularly in promoter regions that regulate gene expression. Meanwhile, in somatic cells of normal individuals, CpG islands of these important gene expression control sites are not methylated, but genes imprinted and inactivated on X chromosome are methylated so that they are not expressed during development. .

Many diseases are caused by gene abnormalities, and the most common type of gene abnormality is a change in the coding sequence of a gene. Such genetic changes are called mutations. When a gene is mutated, the protein it encodes changes its structure and function, leads to disorders and defects, and these mutant proteins cause disease. However, even without mutation of a specific gene, if the expression of the gene abnormality can cause disease. A representative example is methylation in which the methyl group is attached to the cytosine base of the regulatory region of the CpG island of gene transcription, in which case the gene is blocked. This is called epigenetic change, which, like mutations, is transmitted to progeny cells, leading to the same effect: loss of expression of the protein.

There have been some techniques for the diagnosis or prediction of specific cancers by analyzing the methylation levels of genes, but no studies have been reported to predict or diagnose small vessel occlusion through analysis of the methylation levels of specific genes. Therefore, the present inventors have attempted to develop specific methylation markers for predicting or diagnosing effective small vessel occlusion that can be diagnosed early.

Meanwhile, Korean Patent No. 1069593 discloses a marker for diagnosing acute myocardial infarction, and Korean Patent No. 1587635 discloses a method for detecting methylation of a thyroid cancer specific methylation marker gene for diagnosing thyroid cancer. There is no description of a method for providing information for predicting or diagnosing small vessel occlusion using the methylation level of the WRB gene and a composition thereof.

The present invention was derived by the above-described requirements, and the present inventors performed whole genome bisulfite sequencing (WGBS) from samples of the normal group and the small vessel occlusion group, and SEQ ID NO: 1 having a significant difference in the methylation level between the two groups. By selecting the terminator region of the WRB (Tryptophan Rich Basic Protein) gene consisting of the nucleotide sequence of and performing DNA methylation sequencing on the region, the present invention is confirmed by reducing the methylation level of the region in the group of patients with small vessels. Completed.

In order to solve the above problems, the present invention comprises the steps of detecting the terminator site methylation of the Tryptophan Rich Basic Protein ( WRB ) gene from the separated biological sample; And comparing the detected methylation with the terminator site methylation of the WRB gene in a normal control sample, providing a method for providing information for predicting or diagnosing small vessel occlusion.

The present invention also provides a composition for predicting or diagnosing small vessel occlusion, comprising a substance capable of detecting the methylation of the WRB gene terminator.

The present invention also provides a kit for predicting or diagnosing small vessel occlusion comprising the composition as an active ingredient.

The WRB methylated biomarker according to the present invention is expected to predict the development of small vessel occlusion early. In addition, the WRB gene of the present invention may be used for risk assessment, identification of disease, diagnosis of disease stage, and selection of treatment target for small vessel occlusion, and may be useful for epigenetics research of small vessel occlusion. will be.

1 is a schematic diagram of the present invention.

In order to achieve the object of the present invention, the present invention comprises the steps of detecting the terminator site methylation of the Tryptophan Rich Basic Protein ( WRB ) gene from an isolated biological sample; And comparing the detected methylation with the terminator site methylation of the WRB gene in a normal control sample, providing a method for providing information for predicting or diagnosing small vessel occlusion.

The terminator portion of the WRB gene is a sequence including a CpG island (island), but may be composed of the nucleotide sequence of SEQ ID NO: 1, but is not limited thereto. The base sequence of SEQ ID NO: 1 is a base at position 40,770,064 to 40,770,318 of chromosome 21 in the UCSC Genome Browser Human GRCh37 / hg19 DNA sequence.

Specifically, the method of providing information for predicting or diagnosing small vessel occlusion according to the present invention includes

(a) separating genomic DNA from separated biological samples of normal control and suspect individuals;

(b) treating the isolated genomic DNA with a reagent that differently modifies the methylated and unmethylated DNA;

(c) detecting methylation of the terminator region of the WRB gene in genomic DNA or fragment thereof treated with the reagent; And

(d) determining the terminator site methylation level of the WRB gene detected from an isolated biological sample of the suspect subject as compared with the terminator site methylation level of the WRB gene of the normal control group to determine as small vessel occlusion; This is not restrictive.

The biological sample of step (a) of the present invention includes a wide range of all biological fluids obtained from an individual, preferably cells, tissues, biopsies, paraffin tissue, blood, from suspected or diagnosed small vessel occlusion It may be one or more selected from the group consisting of serum, plasma, urine and combinations thereof, more preferably blood, but is not limited thereto. Methods for obtaining body fluids, tissues, biopsies, and the like from mammals may use conventional methods known in the art.

The subject suspected of small vessel occlusion according to the present invention may be a subject having a hereditary factor of small vessel occlusion due to a family history of cerebral infarction disease including small vessel occlusion but not diagnosed with small vessel occlusion, but is not limited thereto.

The reagent of step (b) of the present invention may be bisulfite, disulfite, hydrogen sulfite or a combination thereof, but is not limited thereto.

Unmethylated cytosine (C) bases on bisulfite treated DNA are deaminated and converted to uracil (U) bases, while methylated cytosine bases remain cytosine intact. That is, the bisulfite treatment is labeled with different bases so that they can be distinguished from each other depending on the presence or absence of methylation of the cytosine base on the DNA.

In the step (c) of the present invention, the terminator region sequence of the WRB gene for methylation detection may be composed of the nucleotide sequence of SEQ ID NO: 1, but is not limited thereto, and bisulfite and hydrogen sulfite for methylation detection The base sequence modified with disulfite or a combination thereof may also be included in the range of the terminator region sequence of the WRB gene for methylation detection according to the present invention. The modified nucleotide sequence may be processed to convert the cytosine base in the nucleotide sequence of SEQ ID NO: 1 to other bases detected as being different from cytosine during uracil or hybridization.

In addition, the methylation detection of the step (c) is bisulfite sequencing, sequencing by synthesis, sequencing by ligation, methylation specific PCR, real time methylation specific PCR time methylation specific PCR), PCR using methylated DNA specific binding proteins, PCR using methylated DNA specific binding antibodies or aptamers, or nucleic acid chips, but are not limited thereto.

The bisulfite sequencing method detects nucleic acids containing methylated CpG, which comprises contacting a sample containing nucleic acid with an agent that modifies non-methylated cytosine and using a methylation-independent oligonucleotide primer to sample CpG- Amplifying the containing nucleic acid. Here, the oligonucleotide primer may be characterized by amplifying the nucleic acid without distinguishing the modified methylated and unmethylated nucleic acid. The amplified product may be sequenced by a Sanger method using sequencing primers or analyzed by next generation sequencing (NGS) to detect methylated nucleic acids.

Here, the next generation sequencing method may be characterized by sequencing by synthesis and sequencing by ligation. The unique feature of this method is that instead of producing bacterial clones, a single DNA fragment is spatially separated, in situ amplified, and sequenced. In this case, since several hundreds of thousands of fragments are read at the same time, it is also called a massive parallel sequencing method. Basically, it is a sequencing by synthesis method, and a method of obtaining a signal by attaching mono or dinucleotides sequentially includes pyro sequencing, ion torrent, and Solexa methods.

NGS equipment based on sequencing by synthesis includes Roche's 454 platform, Illumina's HiSeq platform, Life Technology's Ion PGM platform, and Pacific BioSciences' PacBio platform. have. 454 and Ion PGM use emulsion PCR as a clonal amplification method, and HiSeq uses bridge amplification. The sequencing by synthesis method reads a sequence by detecting phosphate, hydrogen ions, or pre-attached fluorescence generated when DNA is synthesized by sequentially attaching one nucleotide. In the method of detecting a sequence, 454 uses a pyroseqeuncing method using phosphoric acid, and Ion PGM uses hydrogen ion detection. HiSeq and PacBio decode sequences by detecting fluorescence.

Sequencing by ligation is a sequencing technique that uses DNA ligase to identify nucleotides at specific positions in the DNA sequence. Unlike most sequencing techniques that use polymerases, they do not use polymerases and take advantage of the feature that DNA ligase does not ligation mismatch sequences. This is the case with SOLiD systems. In this technique, two bases are read at intervals, which are repeated five times independently through a primer reset, so that each base is read twice twice to increase accuracy.

In the case of sequencing by ligation, among 16 sets of dinucleotide primer sets, dinucleotide primers corresponding to the corresponding nucleotide sequences are sequentially ligated, and the combination of these ligations is finally analyzed to determine the base sequence of the corresponding DNA. Is completed.

In the method for providing information for predicting or diagnosing small vessel occlusion according to an embodiment of the present invention, the methylation presence or absence of the production of the result of amplification with a primer capable of amplifying a fragment containing a terminator portion of the WRB gene Or it may be detected through a nucleotide sequence change, but is not limited thereto.

In the method for providing information for predicting or diagnosing small vessel occlusion according to an embodiment of the present invention, the reduction in the methylation level of the terminator portion of the WRB gene of step (d) is 29, 39, 101, 129, 152 and 165 cytosine may be reduced methylation compared to the normal control (see Table 4; the position is shown in Table 4 based on the first position (40,770,064) of the nucleotide sequence of SEQ ID NO: 1) where the p value is 0.00).

The present invention also provides a composition for predicting or diagnosing small vessel occlusion, comprising a substance capable of detecting whether or not methylation of the terminator region of the WRB gene. The composition of the present invention includes a substance capable of detecting methylation of the terminator region of the WRB gene as an active ingredient, and may be determined as small vessel occlusion when the methylation of the terminator region of the WRB gene is reduced compared to a normal control group. Terminator portion of the WRB gene may be composed of the nucleotide sequence of SEQ ID NO: 1, but is not limited thereto.

In the predictive or diagnostic composition of the small vessel occlusion according to the present invention, a substance capable of detecting a methylation if the terminator portion of the WRB gene is a primer set designed to amplify a fragment containing a terminator region of the methylated WRB gene, methylation of which can hybridize with the terminator portion of the WRB gene probe, that can be used with the terminator region of the methylated WRB gene methylation-specific binding protein, the methylation-specific binding antibody or aptamer, and sequencing primer, sequencing-by-synthesis primer and a sequencing-by- It may be one or more selected from the group consisting of ligation primers, but is not limited thereto.

Further, in the composition of the present invention, detection of the methylated if the terminator portion of the WRB gene is a methylation whether the genomic DNA treated with of different modifications to the methylated DNA and unmethylated DNA reagent terminator region of the WRB gene The genomic DNA may consist of cells, tissues, biopsies, paraffin tissue, blood, serum, plasma, urine, and combinations thereof from suspected or diagnosed small vessel occlusion. It may be isolated from a biological sample selected from the group, preferably genomic DNA isolated from a blood sample, but is not limited thereto.

In the composition for predicting or diagnosing small vessel occlusion according to the present invention, the methylation is bisulfite sequencing, sequencing by synthesis, sequencing by ligation, methylation specific PCR (methylation specific PCR) , May be detected by a method such as real time methylation specific PCR, PCR using methylated DNA specific binding protein, PCR using methylated DNA specific binding antibody or aptamer, or nucleic acid chip. It is not limited.

The present invention also provides a kit for predicting or diagnosing small vessel occlusion comprising the composition according to the present invention as an active ingredient.

The kit for predicting or diagnosing small vessel occlusion of the present invention includes a composition containing a substance capable of detecting the methylation of the terminator portion of the WRB gene consisting of the nucleotide sequence of SEQ ID NO: 1 as an active ingredient, In isolated genomic DNA samples, methylation of the terminator region of the WRB gene can be detected to predict or diagnose small vessel occlusion.

The genomic DNA sample is preferably CpG-containing. Typically, a CpG-containing nucleic acid is DNA, but is not limited to this, and may include a sample containing DNA or RNA, including DNA and mRNA. Wherein the DNA or RNA may be single stranded or double stranded, or may contain a DNA-RNA hybrid.

Kits of the invention include a compartmentalized carrier means for holding a sample, a second vessel containing a primer capable of amplifying the 5'-CpG-3 'sequencing site of the sample and a truncated or cleaved for amplifying methylated CpG containing nucleic acids. And one or more containers including a third container containing means for detecting the presence of a nucleic acid that is not present. In one embodiment, the primer is a PCR for detecting whether methylation has occurred in the genome, methylation specific PCR, real time methylation specific PCR, methylated DNA specific binding protein Primers for use in PCR, methylated DNA specific binding antibodies or PCR with aptamers, quantitative PCR, nucleic acid chips, sequencing, sequencing by synthesis or sequencing by ligation. The carrier means is suitable for containing one or more containers, such as bottles, tubes, each container containing independent components used in the method of the invention. In the context of the present invention, one of ordinary skill in the art can readily dispense the required formulation in the container.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

1. Sample Preparation

The following experimental subjects were recruited for the development of DNA methylation markers for predicting or diagnosing small vessel occlusion of the present invention, and genomic DNA was extracted from their blood samples and used for methylation analysis. Among the recruited subjects, experimental subjects were selected based on the criteria in Table 1 below, and a total of 104 patients were selected from the normal group and 58 patients from the small vessel occlusion group. Physical and blood analyzes were performed on the selected subjects (Tables 2 and 3).

Test subject Normal High blood pressure, diabetes, hyperlipidemia, heart disease, people without stroke (cognitive or non-cognitive),
Non-coagulants,
A person with BMI 25 (male) or 23 (female) or less, and BMI 20 or greater.
Excludes metabolic and paralytic diseases.
* The criteria for obesity were selected according to the Korean Obesity Standard (Korean Association for Obesity). Artery occlusion group People without high blood pressure, diabetes, hyperlipidemia, heart disease,
Non-coagulants,
Patients diagnosed with arterial occlusion by MRI,
BMI 25 or less.

In addition, the present invention proceeded with the approval of the Institutional Review Board of the Korean Institute of Oriental Medicine, and clinical questionnaire data collection and human-derived material collection proceeded after obtaining the subject's written consent.

2. Methylation Analysis

Whole genome bisulfite sequencing (WGBS) was performed by selecting genomic DNA samples of each of the four subjects of the normal group and the small vessel occlusion group. The analysis of WGBS was performed by Macrogen (Korea). By analyzing WGBS, the regions with different methylation rates were selected from normal and small vessel occlusion patients, and 97 normal samples and 56 patients with small vessel occlusion. Bisulfite sequencing was performed on the sample for the site. Bisulfite sequencing was performed by LS Co., Ltd., Korea. The platform was used. The reference sequence used for analysis was Human GRCh37 / hg19 in UCSC Genome Browser (http://genome.ucsc.edu/cgi-bin/hgGateway).

Example 1.Selection of analysis region through WGBS

WGBS analysis of genomic DNA samples from normal and small vessel occlusion patients revealed regions with significant differences in methylation levels between the two groups at the terminator region of the WRB gene.

Example 2. WRB  Gene Terminator site (hg19: chr21: 40,770,064-40,770,318) Test between normal and small vessel occlusion patients by methylation analysis

The genomic DNA samples of all the test subjects recruited to the terminator region of the WRB gene were checked for methylation. As a result, it was confirmed that the methylation level was reduced in the six cytosine positions compared to the normal group in the sample of the group of patients with small vessels as shown in Table 4 below ( p <0.05).

Based on the above results, it is thought that when the level of methylation at the terminator of the WRB gene is reduced, it is possible to predict or diagnose small vessel occlusion, and the specific methylation biomarker for predicting or diagnosing small vessel occlusion of the WRB gene of the present invention. It could be confirmed that it can be used as.

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Claims (11)

Detecting terminator site methylation of the Tryptophan Rich Basic Protein ( WRB ) gene consisting of the nucleotide sequence of SEQ ID NO: 1 from the isolated biological sample; And comparing the detected methylation with the terminator site methylation of the WRB gene consisting of the nucleotide sequence of SEQ ID NO: 1 of a normal control sample, providing information for predicting or diagnosing small vessel occlusion. Way. The method of claim 1,
(a) separating genomic DNA from separated biological samples of normal control and suspect individuals;
(b) treating the isolated genomic DNA with bisulfite, disulfite, hydrogen sulfite, or a combination thereof that differently modifies methylated and unmethylated DNA;
(c) detecting methylation of the terminator region of the WRB gene consisting of the nucleotide sequence of SEQ ID NO: 1 in genomic DNA or fragment thereof treated with bisulfite, disulfite, hydrogen sulfite or a combination thereof; And
(d) the detected from the separated biological sample SEQ terminator region methylation level of the WRB gene consisting of the nucleotide sequence of one of the suspect object as compared to SEQ ID NO: 1 WRB terminator region methylation level of a gene consisting of the nucleotide sequence of the normal control group Determining if it is reduced to small vessel occlusion; a method for providing information for the prediction or diagnosis of small vessel occlusion. The method of claim 2, wherein the biological sample of step (a) is selected from the group consisting of cells, tissues, biopsies, paraffin tissue, blood, serum, plasma, urine and combinations thereof from suspected or diagnosed small vessel occlusion Method for providing information for the prediction or diagnosis of small vessel occlusion, characterized in that at least one. delete The method of claim 2, wherein the detection of methylation in step (c) is performed by bisulfite sequencing, sequencing by synthesis, sequencing by ligation, methylation specific PCR, and real time. Real time methylation specific PCR (PCR), PCR using methylated DNA specific binding proteins, PCR using methylated DNA specific binding antibodies or aptamers, or by methods of nucleic acid chips, How to provide information for prediction or diagnosis. The method according to claim 1 or 2, wherein the methylation is detected by the presence or absence of a nucleotide sequence change of the result of the amplification of a fragment containing a terminator portion of the WRB gene consisting of the nucleotide sequence of SEQ ID NO: 1 with a primer that can be amplified Method for providing information for the prediction or diagnosis of small vessel occlusion, characterized in that. delete A composition for predicting or diagnosing small vessel occlusion, comprising a substance capable of detecting the methylation of a terminator portion of a Tryptophan Rich Basic Protein ( WRB ) gene consisting of the nucleotide sequence of SEQ ID NO: 1. According to claim 8, wherein the substance capable of detecting the methylation of the terminator portion of the WRB gene consisting of the base sequence of SEQ ID NO: 1 fragment containing a terminator portion of the WRB gene consisting of the nucleotide sequence of SEQ ID NO: 1 A primer set that can be amplified, a probe that can hybridize with the terminator site of the WRB gene consisting of the nucleotide sequence of methylated SEQ ID NO: 1, and a methylation that can bind to the terminator site of the WRB gene, which consists of the nucleotide sequence of methylated SEQ ID NO: 1 A specific binding protein, methylated specific binding antibody or aptamer, sequencing primers, sequencing bi-synthesis primers and sequencing bi-ligation primers, characterized in that at least one selected from the group consisting of predicting or diagnosing small vessels. delete A kit for predicting or diagnosing small vessel occlusion comprising the composition of claim 8 or 9 as an active ingredient.

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