KR102421788B1 - Method for providing information of prediction and diagnosis of small vessel occlusion using methylation level of CUEDC2 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 the promoter region methylation level of CUEDC2 (CUE Domain Containing 2) gene, and a composition and kit for prediction or diagnosis of small vessel occlusion, and the CUEDC2 according to the present invention The methylation biomarker can be used not only for the prediction of small vessel occlusion, but also for small vessel occlusion risk assessment, disease identification, disease stage diagnosis, and treatment target selection.

Description

The method for providing information for the prediction or diagnosis of small vessel occlusion using the methylation level of the CUEDC2 gene, and a composition therefor

The present invention relates to a method for providing information for prediction or diagnosis of small vessel occlusion using the methylation level of the CUEDC2 gene and a composition therefor, specifically, CUEDC2 (CUE Domain Containing 2) It relates to a method for providing information for prediction or diagnosis of small vessel occlusion using the methylation level of a promoter region of a gene, and a composition and kit for prediction or diagnosis of small vessel occlusion.

Small vessel occlusion is a cerebral infarction (stroke) caused by blockage of small blood vessels in the brain and not receiving blood through these blood vessels. It is known to occur more frequently in Asians than in Westerners. According to a report analyzing patients with cerebral infarction of all ages from 2011 to 2013 through the Clinical Research Center for Stroke (CRCS) in Korea, 38.3% had atherosclerosis of the major arteries, 22.8% of cardiac embolism, and small vessel occlusion. 14.6% of these, 2.1% of other causes, and 22.2% of unknown causes. Small blood vessels act on our body more diversely and microscopically than large blood vessels. In general, small blood vessel disease is a disease requiring attention in that it can be a precursor symptom of macrovascular disease, that is, a precursor signal that foreshadows macrovascular disease.

In the genomic DNA of mammalian cells, there is a 5th base in addition to A, C, G, and T, which is 5-methylcytosine (5-mC) with a methyl group attached to the 5th carbon of the cytosine ring. 5-mC always occurs only at C of a CG dinucleotide (5'-mCG-3'), and this CG is often denoted as CpG. Most of C in CpG is methylated with a methyl group attached. 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 most frequently occur in mammalian cells. 5-mC of this CpG is naturally deamination and changed to T, and accordingly, CpG in the mammalian genome only occurs at a frequency of 1%, which is much lower than the frequency that should normally appear (1/4 x 1/4 =6.25%). indicates.

Among the CpGs, there are some that appear in exceptional concentration, and these are called CpG islands. CpG islands are 0.2~3kb in length, and the distribution percentage of C and G bases exceeds 50%, and the distribution percentage of CpG indicates a highly concentrated region of 3.75% or more. About 45,000 CpG islands appear in the entire human genome, and are particularly concentrated in the promoter region that regulates gene expression. On the other hand, in normal human somatic cells, the CpG islands of these important gene expression regulatory regions are not methylated, but imprinted genes and inactivated genes on the X chromosome are methylated so that they are not expressed during development. .

A significant number of diseases are caused by abnormalities in genes, and the most common type of genetic abnormalities is a change in the coding sequence of a gene, and this change in the gene itself is called a mutation. When there is a mutation in a gene, the structure and function of the protein encoded by the gene is changed, resulting in disorders and defects, and these mutated proteins cause disease. However, even if there is no mutation in a specific gene, if there is an abnormality in the expression of that gene, disease can be induced. A typical example is methylation in which a methyl group is attached to the cytosine base of the CpG island, a regulatory region of gene transcription. In this case, the expression of the gene is blocked. Such an epigenetic change is called an epigenetic change, and it is transmitted to progeny cells like a mutation, and causes the same effect, ie, loss of expression of the corresponding protein.

On the other hand, Korean Patent No. 2085663 discloses 'information providing method for prediction or diagnosis of small vessel occlusion using methylation level of WRB gene and composition therefor', and Korean Patent No. 2085667 discloses ' GPR160 gene Although the 'method for providing information for prediction or diagnosis of small vessel occlusion using methylation level and a composition therefor' are disclosed, the 'method of providing information for prediction or diagnosis of small vessel occlusion using the methylation level of CUEDC2 gene of the present invention and There is no report on the 'composition for this purpose.

The present invention was derived from the above request, and the present inventors performed WGBS (whole genome bisulfite sequencing) from samples of the normal group and the small vessel occlusion group, and SEQ ID NO: 1 with a significant difference in methylation level between the two groups The promoter region of the CUEDC2 (CUE Domain Containing 2) gene consisting of the nucleotide sequence of By confirming that the level is increased, the present invention was completed.

In order to solve the above problems, the present invention comprises the steps of detecting methylation of the promoter region of the CUEDC2 (CUE Domain Containing 2) gene from the isolated biological sample; And it provides a method of providing information for prediction or diagnosis of small vessel occlusion, comprising the step of comparing the detected methylation with the methylation of the promoter region of the CUEDC2 gene of a normal control sample.

In addition, the present invention provides a composition for predicting or diagnosing small vessel occlusion, comprising a substance capable of detecting whether the CUEDC2 gene promoter region is methylated.

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

It is expected that the onset of small vessel occlusion can be predicted early through the CUEDC2 methylation biomarker according to the present invention. In addition, the CUEDC2 gene of the present invention can be utilized for risk assessment of small vessel occlusion, identification of disease, stage diagnosis of disease, and selection of a treatment target, and may be helpful in epigenetics research of small vessel occlusion. will be.

1 shows the chromosome position and genomic sequence of CUEDC2 .
2 is a result of comparative analysis of the methylation level of the promoter region of the CUEDC2 gene in the normal group (Normal) and the small vessel occlusion group (SVO). FIG. 2A is a comparison of average values of methylation levels of promoter regions of the CUEDC2 gene, and FIG. 2B shows the methylation levels of each position. * indicates a significant difference (p<0.05) in the small vessel occlusion patient group compared to the control group.

In order to achieve the object of the present invention, the present invention comprises the steps of detecting methylation of the promoter region of the CUEDC2 (CUE Domain Containing 2) gene from the isolated biological sample; And it provides a method of providing information for prediction or diagnosis of small vessel occlusion, comprising the step of comparing the detected methylation with the methylation of the promoter region of the CUEDC2 gene of a normal control sample.

The promoter region of the CUEDC2 gene is a sequence including a CpG island, and may consist of the nucleotide sequence of SEQ ID NO: 1, but is not limited thereto. The nucleotide sequence of SEQ ID NO: 1 is It is a base at positions 104,192,980 ~ 104,193,444 of the negative strand of chromosome 10 in the UCSC Genome Browser Human GRCh37/hg19 DNA sequence.

The information providing method for prediction or diagnosis of small vessel occlusion according to the present invention is specifically,

(a) isolating genomic DNA from the isolated biological samples of normal control and suspected individuals;

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

(c) detecting methylation of the promoter region of the CUEDC2 gene in the genomic DNA or a fragment thereof treated with the reagent; and

(d) determining as small vessel occlusion when the promoter region methylation level of the CUEDC2 gene detected from the isolated biological sample of the suspected individual is increased compared to the promoter region methylation level of the CUEDC2 gene of the normal control; It is not limited thereto.

The biological sample of step (a) of the present invention includes all biological fluids of a wide range obtained from an individual, preferably cells, tissues, biopsies, paraffin tissues, blood, It may be one or more selected from the group consisting of serum, plasma, urine, and combinations thereof, and more preferably blood, but is not limited thereto. A method for obtaining a body fluid, tissue, biopsy, etc. from a mammal may use a conventional method known in the art.

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

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

The unmethylated cytosine (C) base on the bisulfite-treated DNA is deamination and is changed to the uracil (U) base, whereas the methylated cytosine base remains as cytosine. That is, they are labeled with different bases so that they can be distinguished from each other according to the presence or absence of methylation of cytosine bases on DNA by bisulfite treatment.

In step (c) of the present invention, the promoter region sequence of the CUEDC2 gene for methylation detection may consist of the nucleotide sequence of SEQ ID NO: 1, but is not limited thereto, and bisulfite and hydrogen sulfite for methylation detection , disulfite, or a nucleotide sequence modified by treatment with a combination thereof may also be included in the scope of the promoter region sequence of the CUEDC2 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 into uracil or another base detected as being different from cytosine in the hybridization process.

In addition, the methylation detection in step (c) is performed by bisulfite sequencing, sequencing by synthesis, sequencing by ligation, methylation specific PCR (methylation specific PCR), real-time methylation specific PCR (real). time methylation specific PCR), PCR using a methylated DNA specific binding protein, PCR using a methylated DNA specific binding antibody or an aptamer, or a nucleic acid chip, etc., but is not limited thereto.

The bisulfite sequencing method is a method of detecting a nucleic acid containing methylated CpG. The step of contacting a sample containing the nucleic acid with an agent that modifies unmethylated cytosine and methylation-independent oligonucleotide primers are used to detect CpG- and amplifying the containing nucleic acid. Here, the oligonucleotide primer may be characterized in that it amplifies nucleic acids without discriminating between modified methylated and unmethylated nucleic acids. The methylated nucleic acid can be detected by sequencing the amplified product by the Sanger method using sequencing primers or by analyzing the amplified product by the next generation sequencing (NGS) method. Here, the next-generation sequencing method may be characterized by performing a sequencing by synthesis method and a sequencing by ligation method. A characteristic of this method is that, instead of creating a bacterial clone, a single DNA fragment is spatially separated, amplified in situ (clonal amplification), and sequencing is performed. At this time, since hundreds of thousands of fragments are read simultaneously, 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 sequentially attaching mono or dinucleotides is used, and pyrosequencing, ion torrent, and Solexa methods correspond to these methods.

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

Sequencing-by-ligation is a sequencing technique using DNA ligase, and is a technique to identify nucleotides at a specific position in a DNA base sequence. Unlike most sequencing technologies that use a polymerase, a polymerase is not used and DNA ligase does not ligate mismatched sequences. SOLiD systems are one such example. In this technique, two bases are read at intervals, and since each base is independently repeated five times through primer reset, the accuracy is improved by reading each base twice in the end. In the case of sequencing-by-ligation, among the dinucleotide primer sets made of 16 combinations, the dinucleotide primers corresponding to the corresponding nucleotide sequence are sequentially ligated, and the combination of these ligations is finally analyzed to determine the nucleotide sequence of the DNA. is completed

In the method of providing information for prediction or diagnosis of small blood vessel occlusion according to an embodiment of the present invention, the methylation is a result of amplification of a fragment containing a promoter region of the CUEDC2 gene with a primer capable of amplifying whether or not a result is generated Alternatively, it may be detected through a nucleotide sequence change, but is not limited thereto.

In the method of providing information for prediction or diagnosis of small vessel occlusion according to an embodiment of the present invention, the increase in the methylation level of the promoter region of the CUEDC2 gene in step (d) is 145 of the nucleotide sequence of SEQ ID NO: 1, At cytosines 205, 207, 217 and 220, methylation may be increased compared to the normal control (see Table 4; the position is the p value of Table 4 based on the first position (104, 192, 980) of the nucleotide sequence of SEQ ID NO: 1) positions that are less than or equal to 0.05 are sequentially described).

The present invention also provides a composition for predicting or diagnosing small vessel occlusion, comprising a substance capable of detecting whether the promoter region of the CUEDC2 (CUE Domain Containing 2) gene is methylated. The composition of the present invention contains a substance capable of detecting methylation of the promoter region of the CUEDC2 gene as an active ingredient. When the methylation of the promoter region of the CUEDC2 gene is increased compared to the normal control, small vessel occlusion can be determined. The promoter region of the CUEDC2 gene may consist of the nucleotide sequence of SEQ ID NO: 1, but is not limited thereto.

In the composition for prediction or diagnosis of small vessel occlusion of the present invention, the substance capable of detecting whether the promoter region of the CUEDC2 gene is methylated is a primer set capable of amplifying a fragment including the promoter region of the methylated CUEDC2 gene, methylation A probe capable of hybridizing with the promoter region of the CUEDC2 gene, a methylation-specific binding protein capable of binding to the promoter region of the methylated CUEDC2 gene, a methylation-specific binding antibody or aptamer and a sequencing primer, a 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.

In addition, in the composition of the present invention, the detection of whether the promoter region of the CUEDC2 gene is methylated is genomic DNA treated with a reagent that modifies methylated DNA and unmethylated DNA differently to determine whether the promoter region of the CUEDC2 gene is methylated. It may be confirmed by contact with a detectable substance, and the genomic DNA is composed of cells, tissues, biopsies, paraffin tissues, blood, serum, plasma, urine, and combinations thereof derived from an individual suspected or diagnosed with 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 prediction or diagnosis of small vessel occlusion according to the present invention, the methylation is performed by bisulfite sequencing, sequencing by synthesis, sequencing by ligation, or methylation specific PCR (methylation specific PCR). , real time methylation specific PCR (real time methylation specific PCR), PCR using a methylated DNA specific binding protein, PCR using a methylated DNA specific binding antibody or an aptamer, or a nucleic acid chip, etc. 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 contains, as an active ingredient, a composition comprising a substance capable of detecting methylation of the promoter region of the CUEDC2 gene consisting of the nucleotide sequence of SEQ ID NO: 1, from a biological sample. Small vessel occlusion can be predicted or diagnosed by detecting methylation of the promoter region of the CUEDC2 gene in the isolated genomic DNA sample.

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

The kit of the present invention comprises a compartmentalized carrier means for holding a sample, a second container containing a primer capable of amplifying a 5'-CpG-3' sequence region of the sample, and a cleaved or cleaved nucleic acid for amplifying a methylated CpG-containing nucleic acid. It may comprise one or more containers comprising a third container containing means for detecting the presence of undissolved nucleic acids. In one embodiment, the primer is PCR for detecting whether methylation has occurred in the genome, methylation specific PCR (methylation specific PCR), real time methylation specific PCR (real time methylation specific PCR), methylated DNA specific binding protein It may be a primer for use in PCR, PCR using a methylated DNA-specific binding antibody or an aptamer, quantitative PCR, nucleic acid chip, sequencing, sequencing-by-synthesis, or sequencing-by-ligation. The carrier means is suitable for containing one or more containers, such as bottles and tubes, each container containing independent components for use in the method of the present invention. In the context of the present invention, a person of ordinary skill in the art can readily dispense the required formulation in a container.

Hereinafter, the present invention will be described in detail by way of Examples. However, the following examples are only illustrative of the present invention, and the content of the present invention is not limited to the following examples.

1. Sample Preparation

The following test subjects were recruited for the development of a DNA methylation marker for prediction or diagnosis of 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, the test subjects were selected based on the criteria in Table 1 below, and a total of 88 patients in the normal group and 46 patients in the small vessel occlusion group were finally selected. Physical characteristics and blood analysis were performed on the selected test subjects (Tables 2 and 3).

test subject normal group People without high blood pressure, diabetes, hyperlipidemia, heart disease, stroke (cognitive or non-cognitive),
non-coagulants,
People with a BMI of 25 (male) or 23 (female) or less and a BMI of 20 or higher.
Excluding metabolic and paralytic diseases.
* Obesity was selected according to the Korean obesity standards (Korean Society for Obesity Standards), and hypertension, diabetes, and hyperlipidemia were selected based on historical data. Small vessel occlusion patient group People without high blood pressure, hyperlipidemia, heart disease,
Those who have not taken blood coagulants, diabetes medications,
Patients diagnosed with arteriosclerosis by MRI;
BMI 25 or less.

In addition, the present invention was carried out with the approval of the Institutional Bioethics Review Committee of the Korea Institute of Oriental Medicine, and the collection of clinical questionnaire data and human material was carried out after obtaining the written consent of the subject.

2. Methylation Analysis

Genomic DNA samples of each of the four subjects were selected from the normal group and small vessel occlusion patient group, and WGBS (whole genome bisulfite sequencing) was performed. WGBS analysis was commissioned by Macrogen (Korea). Through WGBS analysis, regions with different methylation rates were selected between the normal group and the small vessel occlusion group, and 88 samples in the normal group and 46 samples in the small vessel occlusion group were selected. Bisulfite sequencing was performed on the sample for the corresponding position. Bisulfite sequencing was performed by request of LAS (Korea), and Illumina MiSeq. platform was used. Human GRCh37/hg19 was used as the reference sequence used for the analysis in the UCSC Genome Browser (http://genome.ucsc.edu/cgi-bin/hgGateway).

Example 1. Selection of analysis area through WGBS

Through WGBS analysis of genomic DNA samples from the normal group and small vessel occlusion patient group, regions with significant differences in methylation levels between the two groups in the promoter region of the CUEDC2 gene were identified.

Example 2. CUEDC2 Test between normal group and small vessel occlusion patient group through gene promoter region (hg19:chr10: 104,192,980 ~ 104,193,444) methylation analysis

Whether or not methylation of the CUEDC2 gene promoter region was confirmed was confirmed by using the genomic DNA samples of all the collected test subjects. As a result, as shown in Table 4 below, it was confirmed that the methylation level was increased at five cytosine positions in the sample of the small vessel occlusion patient group compared to the normal group ( p < 0.05).

Through the above results, it was thought that small vessel occlusion could be predicted or diagnosed when the methylation level in the promoter region of the CUEDC2 gene was increased, and the CUEDC2 gene of the present invention is a specific methylation biomarker for prediction or diagnosis of small vessel occlusion. was confirmed to be usable as

<110> Korea Institute of Oriental Medicine KOREA FOOD RESEARCH INSTITUTE <120> Method for providing information of prediction and diagnosis of small vessel occlusion using methylation level of CUEDC2 gene and composition there <130> PN20153 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 465 <212> DNA <213> Homo sapiens <400> 1 gctcaggtgg atcctcatcc tggcacctgc taccactcca cctctcccag accacgtcca 60 aaatcccagg ctccaccctc cggagacaca gacctgtagc tcaccccaat ctaggggaga 120 cattgctgtt aagggaatgc aaaaggtaca gacagggtgg ggaagtcagt acctaagaga 180 ccagcctggg ccacgtggca aaaaccctgt ctctacaaaa aaaaaaaaaa aaaaaaaaat 240 tagctcggcg tggtggcacg cgcctgtggt tccagctact ccagaggctg agatgggagg 300 atcacttgaa cccaggaggt cggggctgca gtgagccatg ttcgcactcg tgaacctgca 360 ctccagcctg gggctgtaac agcaagaaaa gaagaaaaag aaagagaagg aaggaaggga 420 gggagggaaa gaaaggaaaa taaagaaaga agaaaagaaa agagg 465

Claims (11)

Detecting the promoter region methylation of the CUEDC2 (CUE Domain Containing 2) gene consisting of the nucleotide sequence of SEQ ID NO: 1 from the separated biological sample; And Comprising the step of comparing the detected methylation with the promoter region methylation of the CUEDC2 gene consisting of the nucleotide sequence of SEQ ID NO: 1 of a normal control sample, a method of providing information for the diagnosis of small vessel occlusion. According to claim 1,
(a) isolating genomic DNA from the isolated biological samples of normal control and suspected individuals;
(b) treating the isolated genomic DNA with a reagent that modifies methylated DNA and unmethylated DNA differently;
(c) detecting methylation of the promoter region of the CUEDC2 gene consisting of the nucleotide sequence of SEQ ID NO: 1 in the genomic DNA or fragment thereof treated with the reagent; and
(d) the promoter region methylation level of the CUEDC2 gene consisting of the nucleotide sequence of SEQ ID NO: 1 detected from the isolated biological sample of the suspected individual compared to the methylation level of the promoter region of the CUEDC2 gene consisting of the nucleotide sequence of SEQ ID NO: 1 of the normal control; Determining small vessel occlusion if increased; Method for providing information for diagnosis of small vessel occlusion, comprising: a. According to claim 2, wherein the biological sample of step (a) is selected from the group consisting of cells, tissues, biopsies, paraffin tissues, blood, serum, plasma, urine, and combinations thereof derived from a subject suspected or diagnosed with small vessel occlusion. A method for providing information for the diagnosis of small vessel occlusion, characterized in that at least one is. The method of claim 2, wherein the reagent in step (b) is bisulfite, disulfite, hydrogen sulfite, or a combination thereof. According to claim 2, wherein the detection of methylation in step (c) is bisulfite sequencing, sequencing by synthesis, sequencing by ligation, methylation specific PCR (methylation specific PCR), real-time Methylation specific PCR (real time methylation specific PCR), PCR using a methylated DNA specific binding protein, PCR using a methylated DNA specific binding antibody or an aptamer, or small vessel occlusion, characterized in that it is performed by the method of a nucleic acid chip How to provide information for diagnosis. The method according to claim 1 or 2, wherein the methylation is detected by the presence or absence of generation of the amplified product with a primer capable of amplifying the fragment including the promoter region of the CUEDC2 gene consisting of the nucleotide sequence of SEQ ID NO: 1 or by changing the nucleotide sequence. A method of providing information for the diagnosis of small vessel occlusion, characterized in that delete A composition for diagnosis of small vessel occlusion, comprising a material capable of detecting whether or not the promoter region of the CUEDC2 (CUE Domain Containing 2) gene consisting of the nucleotide sequence of SEQ ID NO: 1 is methylated. The method of claim 8, wherein the substance capable of detecting whether the promoter region of the CUEDC2 gene is methylated is a primer set capable of amplifying a fragment including the promoter region of the CUEDC2 gene consisting of the methylated base sequence of SEQ ID NO: 1, methylation A probe capable of hybridizing with the promoter region of the CUEDC2 gene consisting of the nucleotide sequence of SEQ ID NO: 1, a methylation-specific binding protein capable of binding to the promoter region of the CUEDC2 gene consisting of the nucleotide sequence of methylation SEQ ID NO: 1, methylation-specific A composition for diagnosis of small vessel occlusion, characterized in that at least one selected from the group consisting of a binding antibody or aptamer, a sequencing primer, a sequencing-by-synthesis primer, and a sequencing-by-ligation primer. delete A kit for diagnosing small vessel occlusion comprising the composition of claim 8 or 9 as an active ingredient.

Images