KR101455284B1

KR101455284B1 - Composition for diagnosing Moyamoya disease using CpG methylation status in promoter region of APOD gene and uses thereof

Info

Publication number: KR101455284B1
Application number: KR1020140090464A
Authority: KR
Inventors: 안정혁; 성혜윤; 김승기; 이지연
Original assignee: 이화여자대학교 산학협력단; 서울대학교산학협력단
Priority date: 2014-07-17
Filing date: 2014-07-17
Publication date: 2014-11-14

Abstract

The present invention relates to a composition, a kit and a method for diagnosing Moyamoya disease by detecting the methylation level of the CpG region of the APOD (apolipoprotein D) gene promoter.

Description

Composition for diagnosis of moyamoya disease using CpG methylation change of APOD gene promoter and use thereof <BACKGROUND OF THE INVENTION 1. Field of the Invention < RTI ID = 0.0 >

Moyamoya disease refers to a chronic cerebrovascular disease in which stenosis and occlusion occur at the distal end or branch of the internal carotid artery supplying blood to the cerebrum without special causes and abnormal blood vessels are observed at the base of the brain. In children, it usually occurs in the form of repeated transient ischemic attacks, and cerebral hemorrhage is common in adults. Moyamoya disease occurs mostly in childhood, causing permanent and serious damage to the brain of children in development, which has a negative impact on intelligence, impaired cognitive functioning, and emotional development. Thus, long-term academic and social life are obstacles.

Cerebral angiography, which is currently used for the diagnosis of moyamoya disease, is an invasive diagnostic technique and is inappropriate for repeated tests. In addition, although MRI / MRA is a non-invasive diagnostic method, it is disadvantageous in that it is not economical for screening purposes because it uses expensive equipment.

Therefore, it is necessary to develop a technique that can diagnose moyamoya disease more easily, accurately and stably.

U.S. Patent Publication No. 2011-0028331 (Feb. 3, 2011)

It is an object of the present invention to provide a composition, a kit and a method for diagnosing Moyamoya disease by detecting the methylation level of the CpG region of the APOD gene promoter.

More specifically, one object of the present invention is to provide a composition for the diagnosis of Moyamoya disease, which comprises an agent for measuring the methylation level of the CpG region of the APOD gene promoter.

It is another object of the present invention to provide a kit for the diagnosis of moyamoya disease comprising the above composition.

Another object of the present invention is to provide a method for detecting the methylation level of the CpG region of the APOD gene promoter from a biological sample of a patient suspected of having Moyamoya disease so as to provide information necessary for diagnosis of Moyamoya disease.

It is another object of the present invention to provide a method for diagnosing Moyamoya disease by detecting the methylation level of the CpG region of the APOD gene promoter.

The present invention is based on the discovery that the specific CpG site of the APOD gene promoter is specifically under-methylated in endothelial progenitor cells (EPC) of patients with Moyamoya disease, and the degree of methylation of the promoter of the genes is biomarker To provide a technique for diagnosing moyamoya disease.

In a specific example of the present invention, genomic DNA and RNA were extracted from vascular endothelial progenitor cells obtained from blood of three patients with Moyamoya disease and two healthy controls, and DNA methylation microarray and mRNA microarray analysis were carried out. The genes involved in gene expression were selected by the methylation change of CpG present in the promoter region of each gene. The selected APOD gene showed 2.6 times higher expression in Moyamoya disease vascular endothelial progenitor cells than control vascular endothelial progenitor cells. At the same time, DNA methylation was 2.0 times higher in two specific CpG sites in the promoter region 1.9 times.

Thus, specific low methylation of DNA methylation occurring at specific CpG sites of the APOD gene in samples from patients with Moyamoya disease can be used as a biomarker to diagnose moyamoya disease.

Accordingly, in one aspect, the present invention relates to a composition for the diagnosis of moyamoya disease, comprising the agent for measuring the methylation level of the CpG region of the APOD gene promoter, and a kit comprising the same.

In the present invention, the APOD gene can identify the sequence information in a known gene database. For example, the nucleic acid sequence of the human APOD gene can be found in Genbank accession number NM_001647.

In the present invention, the term "methylation" refers to the attachment of a methyl group to a base constituting DNA. Preferably, the methylation status in the present invention means whether methylation occurs in the cytosine of a specific CpG site in the promoter region of a specific gene. When methylation occurs, the binding of transcription factors is hindered and the expression of a specific gene is inhibited. On the other hand, when unmethylated or hypomethylated, the expression of a specific gene is increased.

Genomic DNA of mammalian cells contains 5th methyl-cytosine (5-methylcytosine, 5-mC) base with a methyl group attached to the fifth carbon of the cytosine ring in addition to A, C, G and T do. Methylation of 5-methylcytosine occurs only in C of a CG dinucleotide (5'-mCG-3 ') called CpG, and methylation of CpG inhibits expression of repeated sequences of alu or transposon and genome. Further, since 5-mC of the CpG is naturally deaminated and is likely to become a thymine (T), CpG is a site where most of the epigenetic changes occur frequently in mammalian cells.

The term " measurement of the methylation level "in the present invention means a measurement of the methylation level of the CpG region of the promoter of the APOD gene and includes methylation-specific PCR such as methylation-specific polymerase chain reaction (MSP) PCR using real-time methylation-specific polymerase chain reaction (PCR), methylated DNA-specific binding protein, or quantitative PCR. Or by automated base analysis such as pyrosequencing and bisulfite sequencing, but are not limited thereto.

Preferably, in the present invention, the level of methylation of the CpG region of the promoter of the APOD gene is determined by measuring the 195583952 to 195584073 base sequence (SEQ ID NO: 1) of chromosome 3 or the 195583956 to 195584077 base sequence (SEQ ID NO: 2) can be measured by measuring the methylation level of cytosine at the CpG site present in the 61st base.

In the present invention, the nucleotide sequence of the human genome chromosome region is expressed according to the latest version of The December 2011 Human reference sequence (GRCh38 / hg38) The specific sequence of the human genome chromosome region may be changed somewhat as the result of the genome sequence study is updated, and the expression of the human genome chromosome region of the present invention may be different according to the modification. Therefore, the human genome chromosome region expressed in accordance with the December 2011 Human reference sequence (GRCh38 / hg38) of the present invention is updated after the filing date of the present invention so that the expression of the human genome chromosome region is updated It will be obvious that the scope of the present invention is extended to the modified human genome chromosome region even if it is changed. Such modifications are readily apparent to those skilled in the art to which the present invention pertains.

As used herein, the term "diagnosis" means identifying the presence or characteristic of a pathological condition. For the purpose of the present invention, the diagnosis is to confirm whether or not the disease has developed, and furthermore, whether the disease progresses or not.

In the present invention, since the abnormal methylation change of vascular endothelial progenitor cells shows considerable similarity to the methylation change of genomic DNA obtained from a biological sample such as tissue, whole blood, serum, plasma, saliva, sputum or urine, When the marker is used, there is an advantage that it is possible to easily diagnose Moyamoya disease through blood or body fluids.

In the present invention, the agent for measuring the methylation level of the CpG region is a compound that modifies an unmethylated cytosine base or a methylation-sensitive restriction enzyme, a primer specific to the methylated allele sequence of the APOD gene, and a non- Specific primers may be included.

The compound capable of modifying the unmethylated cytosine base may be bisulfite or a salt thereof, but is not limited thereto, preferably sodium bisulfite. Methods for detecting the methylation of a promoter by modifying an unmethylated cytosine residue using such bisulfite are well known in the art (WO01 / 26536; US2003 / 0148326A1).

In addition, the methylation sensitive restriction enzyme may be a restriction enzyme capable of specifically detecting the methylation of the CpG site, and may be a restriction enzyme containing CG as a recognition site of the restriction enzyme. For example, Sma I, Sac II, Eag I, Hpa II, Msp I, Bss HII, Bst UI, Not I, and the like. The methylation or non-methylation of the restriction enzyme recognition site at C may or may not be cleaved by restriction enzymes and may be detected by PCR or Southern blot analysis. Other methylation sensitive restriction enzymes than the above restriction enzymes are well known in the art.

Promoter of the APOD gene in patients with suspected Moyamoya disease A representative method for measuring the level of methylation at a specific CpG site is to obtain genomic DNA from a biological sample of a patient and transform the cytosine base that is not methylated in the obtained DNA A compound or a methylation-sensitive restriction enzyme, amplifying the treated DNA by PCR using a primer, and confirming presence or absence of the amplified product.

Thus, the formulations of the invention may include primers specific for the methylated allele sequence of the APOD gene and primers specific for the unmethylated allele sequence. In the present invention, the term "primer" means a short nucleic acid sequence capable of forming a base pair with a complementary template with a nucleic acid sequence having a short free 3-terminal hydroxyl group and serving as a starting point for template strand copying. The primer can initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i. E., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures. In addition, primers can incorporate additional features that do not alter the primer properties of primers that serve as a starting point for DNA synthesis, as sense and antisense nucleic acids with 7 to 50 nucleotide sequences.

The primer of the present invention can be preferably designed according to the sequence of a specific CpG site to be subjected to methylation analysis and includes a pair of primers capable of specifically amplifying cytosine that has been methylated and not modified by bisulfite, And a primer pair that is not methylated and can specifically amplify cytosine modified by bisulfite.

In addition to the above preparation, the composition and kit may further contain a polymerase agarose, a buffer solution necessary for electrophoresis, and the like.

In another aspect, the present invention relates to a method for detecting a mammalian disease by detecting the methylation level of the CpG region of the APOD gene promoter.

The present invention also relates to a method for detecting the methylation level of the CpG region of the APOD gene promoter from a biological sample of a patient suspected of having Moyamoya disease, in order to provide information necessary for the diagnosis of Moyamoya disease.

For example, the present invention provides a method for detecting a methylation level of a CpG region of an APOD gene promoter from a biological sample of a patient suspected of having Moyamoya disease; And comparing the methylation level to the methylation level of the corresponding gene promoter of the control sample.

The term "biological sample " in the present invention includes, but is not limited to, tissues, cells, whole blood, serum, plasma, saliva, sputum, or urine samples that differ in the level of methylation of the APOD gene promoter by Moyamoya disease Do not.

First, in order to obtain genomic DNA from a patient suspected of having Moyamoya disease and to detect the methylation level, the genomic DNA can be obtained by the phenol / chloroform extraction method, SDS extraction method (Tai et al., Plant Mol. Biol. Reporter, 8: 297-303, 1990), Cetyl Trimethyl Ammonium Bromide (Murray et al., Nuc. Res., 4321-4325, 1980) or commercially available DNA extraction kits .

The step of detecting the methylation level of the specific CpG region of the gene promoter comprises, more particularly, (a) treating the genomic DNA in the obtained sample with a compound or methylation-sensitive restriction enzyme that modifies the unmethylated cytosine base; And (b) amplifying the treated DNA by PCR using a primer capable of amplifying a specific CpG site of the gene promoter of interest.

The compound that modifies the unmethylated cytosine base in step (a) may be bisulfite, preferably sodium bisulfite. Methods for detecting the methylation of a promoter by modifying an unmethylated cytosine residue using such a bisulfite are well known in the art.

In addition, as described above, the methylation-sensitive restriction enzyme in step (a) may be a restriction enzyme capable of specifically detecting methylation of a specific CpG site and may be a restriction enzyme containing CG as a recognition site of a restriction enzyme For example, Sma I, Sac II, Eag I, Hpa II, Msp I, Bss HII, Bst UI, Not I, and the like.

In the step (b), the amplification may be performed by a conventional PCR method. As described above, the primers used herein can be desirably designed according to the sequence of a specific CpG site to be subjected to methylation analysis, and can be specifically amplified by specifically amplifying cytosine which has been methylated and not modified by bisulfite And a primer pair capable of specifically amplifying cytosine modified by bisulfite without being methylated.

The step of detecting the methylation level of the specific CpG region of the gene promoter may further include the step of (c) confirming presence or absence of the amplified product in the step (b). The presence or absence of the amplified product in the step (c) may be performed according to a method known in the art, for example, electrophoresis to detect a band at a desired position. For example, in the case of using a compound for modifying an unmethylated cytosine residue, two kinds of primer pairs used in the step (a), that is, a primer capable of specifically amplifying cytosine that has been methylated and not modified by bisulfite The degree of methylation can be determined according to the presence or absence of the PCR product amplified by the pair of primers that can specifically amplify cytosine modified by bisulfite and methylation, respectively. Preferably, the methylation can be determined by treating the sample genomic DNA with bisulfite, amplifying the CpG region of the gene by PCR, and analyzing the base sequence of the amplified region using a bisulfite genomic sequencing method .

Also, in the case of using restriction enzymes, it is judged that the promoter is methylated when there is a PCR result in a DNA treated with a restriction enzyme in a state that the result of PCR is shown in mock DNA, If there is no PCR result in the DNA treated with the enzyme, it can be judged whether or not the promoter is methylated by judging that the promoter is unmethylated, which is obvious to a person skilled in the art. In the above, mock DNA refers to a sample DNA that has been separated from the sample and has not undergone any treatment.

According to such a method, when the CpG region of the APOD gene promoter in the patient sample is detected as a low methylated state, it can be diagnosed or predicted as Moyamoya disease.

Thus, according to the present invention, the hypomethylation of the specific CpG region of the APOD gene promoter is specifically expressed in samples of patients with Moyamoya disease and affects the expression of APOD in patients with moyamoya disease. The use of methylation as a biomarker can be used to diagnose moyamoya disease.

The present invention provides a molecular biological diagnostic method for diagnosing moyamoya disease by measuring the degree of methylation of a specific gene promoter region of a genomic DNA obtained from a biological sample of a patient. This method is simple, noninvasive and economical. In addition, DNA methylation changes are easy to detect and are more stable and easier to analyze than conventional protein or RNA markers.

1 is a schematic diagram showing a process of integrating mRNA and CpG methylation data.
FIG. 2 shows the results of confirming the expression level of APOD gene in vascular endothelial progenitor cells of normal (Normal1, Normal2) and Moyamoya disease patients (MMD1, MMD3, MMD4).
FIG. 3 shows the results of DNA methylation microarray analysis of changes in DNA methylation in the promoter CpG region of the APOD gene in vascular endothelial progenitor cells of normal (Normal1, Normal2) and moyamoya disease patients (MMD1, MMD3, MMD4) .
4 shows the results of DNA methylation microarray analysis of changes in DNA methylation in promoter CpG region of APOD gene in vascular endothelial progenitor cells of normal (Normal1, Normal2) and Moyamoya disease patients (MMD1, MMD3, MMD4) .

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

Example 1. Obtaining and culturing vascular endothelial progenitor cells

Ficoll 1077 g / mL (Histopaque-1077) was diluted 1: 1 with the same volume of RPMI for each donated peripheral blood from three patients who were diagnosed with Moyamoya disease at the Seoul National University Children's Hospital and two patients undergoing surgery. ; Sigma, St. Louis, Mo.) to separate the mononuclear cell layer by density difference method. After separating mononuclear cells, cells were counted and cultured in a 6-well culture dish (BD BioCoat; BD Biosciences, Mountain View, CA) coated with collagen type 1 at 2 × 10 ⁶ cells / CO ₂ Under the incubator conditions, media was changed every 3 days.

Example 2. Total RNA extraction

Total RNA was extracted from the vascular endothelial progenitor cells of two normal subjects and three patients with moyamoya disease using RNeasy mini kit (Qiagen). The extraction method was carried out according to the manufacturer's manual. Extracted total RNA was quantified by spectrophotometer and RNA status was confirmed by electrophoresis on 1% agarose gel.

Example 3. Genomic DNA extraction

Genomic DNA was extracted from the vascular endothelial progenitor cells of two normal subjects and three patients with moyamoya disease using QIAmp DNA mini kit (Qiagen). The extraction method was carried out according to the manufacturer's manual. The extracted genomic DNA was quantitated by spectrophotometer and the DNA status was confirmed by electrophoresis on 1% agarose gel.

Example 4. mRNA Microarray

MRNA microarrays were performed using GeneChip Human Gene 2.0 ST arrays.

Background correction, RMA normalization (Biostatistics. 2003 Apr; 4 (2): 249-64. Exploration, normalization, and summaries of high density oligonucleotide array probe level data), and log2 transformation ) And finally used for statistical analysis. The Bayesian t-test (Limma: Linear Models for Microarray Data, Gordon K. Smyth.) Method was used to identify differentially expressed genes (DEGs) in both groups. Finally, DEG was selected as the gene with p value <0.05 and the absolute value of log2 (fold change) greater than 0.585.

Example 5. DNA Methylation Microarray

A DNA methylation microarray was performed using Infinium (?) Human Methylation 450K BeadChip. The degree of DNA methylation is expressed as a value having a value of 0 to 1, and a value of 0 means that the corresponding CpG site is completely unmethylated and 1 means completely methylated.

The Bayesian t-test method was used to identify differentially methylated genes (DMGs) in both groups. Finally, a CpG site having a p value <0.05 and an absolute value beta difference ≥0.3 was selected as a differentially methylated CpG site (differently methylated CpG site), and the degree of methylation of the CpG site present in the promoter site was changed The gene was selected as DMG.

Example 6. DEG And DMG Integrated analysis of data

Following the procedure of FIG. 1, the final determined DEG and DMG data were combined.

Experiment result

1. Vascular endothelial progenitor cells Epigenetic Analysis of change and gene expression integration

In contrast to normal vascular endothelial progenitor cells, genes expressing differences in DNA methylation and gene expression in vascular endothelial progenitor cells of patients with Moyamoya disease were selected and integration analysis was performed. APOD was selected as a gene presumed to affect gene expression by the methylation change of CpG present in the promoter region of each gene (Table 1).

gene Genbank Registration Number Expression logFC Expression P. Value Beta difference Beta P. Value APOD NM_001647 1.394194355 0.040378983 0.38107182 0.049852699 APOD NM_001647 1.394194355 0.040378983 0.42796088 0.001644122

2. Moyamoya disease vascular endothelial progenitor cells APOD Gene promoter CpG Methylation Changes and changes in gene expression

The expression of the APOD gene in vascular endothelial progenitor cells of patients with moyamoya disease was increased compared to normal vascular endothelial progenitor cells (Fig. 2).

As a result of the DNA methylation microarray analysis, the DNA methylation of specific CpG sites (Chr3: 195583952-195584073; and Chr3: 195583956-195584077) in the promoter of the APOD gene was remarkably lowered 4).

These results indicate that the increased expression of APOD gene in Moyamoya disease vascular endothelial progenitor cells is regulated by hypomethylation of specific CpG sites in the APOD promoter region and is a phenomenon that is specific to moyamoya disease.

<110> Ewha University - Industry Collaboration Foundation SNU R & DB FOUNDATION <120> Composition for diagnosing Moyamoya disease using CpG methylation status in promoter region of APDO gene and uses thereof <130> DPP20142115KR <160> 2 <170> Kopatentin 1.71 <210> 1 <211> 122 <212> DNA <213> Homo sapiens <220> <221> promoter &Lt; 222 > (1) .. (122) <223> CpG in promoter region of APOD gene (Chr3: 195583952-195584073) <400> 1 tctcccaagc tattttataa gcttcttcag gtctctgatg tttacttttc atgcatgcca 60 cgcccgccgc ccccatccag tcaggagggt tggcctaaaa gcttagtctg tatcgaaaca 120 ca 122 <210> 2 <211> 122 <212> DNA <213> Homo sapiens <220> <221> promoter &Lt; 222 > (1) .. (122) <223> CpG in promoter region of APOD gene (Chr3: 195583956-195584077) <400> 2 ccaagctatt ttataagctt cttcaggtct ctgatgttta cttttcatgc atgccacgcc 60 cgccgccccc atccagtcag gagggttggc ctaaaagctt agtctgtatc gaaacacaaa 120 ga 122

Claims

A composition for diagnosing moyamoya disease comprising an agent for measuring the methylation level of the CpG region of the APOD (apolipoprotein D) gene promoter.

2. The method according to claim 1, wherein the agent for measuring the methylation level of the CpG region of the gene promoter comprises
Compounds or methylation sensitive restriction enzymes that modify unmethylated cytosine bases;
A primer specific for the methylated sequence of the CpG region of the APOD gene promoter; And
Wherein the primer comprises a primer specific for the unmethylated sequence.

3. The composition of claim 2, wherein the compound that modifies the unmethylated cytosine base is bisulfite or a salt thereof.

3. The composition of claim 2, wherein the methylation sensitive restriction enzyme is Sma I, Sac II, Eag I, Hpa II, Msp I, Bss HII, Bst UI or Not I.

2. The method according to claim 1, wherein the CpG site of the gene promoter comprises CpG which appears in the nucleotide sequence of SEQ ID NO: 1 (195583952 to 195584073th of chromosome 3) or SEQ ID NO: 2 (195583956 to 195584077th of chromosome 3) / RTI >

A kit for diagnosing a moyamoya disease, comprising the composition of any one of claims 1 to 5.

A method of detecting the methylation level of the CpG region of the APOD gene promoter from a biological sample of suspected moyamoya disease in order to provide information necessary for diagnosis of Moyamoya disease.

8. The method according to claim 7, wherein the method for detecting the methylation level of the CpG region of the gene promoter comprises:
(a) treating the genomic DNA in the obtained sample with a compound or methylation-sensitive restriction enzyme that transforms the unmethylated cytosine base; And
(b) amplifying the treated DNA by PCR using a primer capable of amplifying the CpG region of the APOD gene promoter.

The method according to claim 7, wherein the CpG site of the gene promoter comprises CpG that appears in the nucleotide sequence of SEQ ID NO: 1 (195583952 to 195584073th of chromosome 3) or SEQ ID NO: 2 (195583956 to 195584077th of chromosome 3) / RTI >

9. The method of claim 8, wherein the compound that modifies the unmethylated cytosine base is bisulfite or a salt thereof.

8. The method according to claim 7, wherein the methylation level is detected by a methylation-specific polymerase chain reaction, a real-time methylation-specific polymerase chain reaction, Wherein the method is selected from the group consisting of PCR using affinity binding proteins, quantitative PCR, pyrosequencing and bisulfite sequencing.