KR101992792B1 - Method for providing information of prediction and diagnosis of obesity using methylation level of AKR1E2 gene and composition therefor - Google Patents

Abstract

The present invention relates to a method for providing information for predicting or diagnosing obesity using a methylation level of a promoter site of aldo-keto reductase family 1 member E2 (AKR1E2) gene, and a composition and kit for predicting or diagnosing obesity. An AKR1E2 methylation biomarker according to the present invention can be used not only for predicting obesity, but also for assessing risk of obesity, diagnosing a disease, diagnosing a disease stage and selecting a therapeutic target.

Description

[0001] The present invention relates to a method for providing information for predicting or diagnosing obesity using the methylation level of AKR1E2 gene and a composition therefor,

The present invention relates to a method and a composition for providing information for predicting or diagnosing obesity using the methylation level of the AKR1E2 gene, and more particularly, to a method and an apparatus for providing information for obesity by using the methylation level of AKR1E2 (Aldo-Keto Reductase Family 1 member E2) A method for providing information for prediction or diagnosis of obesity, a composition and a kit for predicting or diagnosing obesity.

Obesity occurs when the energy intake exceeds the energy consumption over a long period of time and the surplus energy is stored in the fat. In normal conditions, appetite is regulated by peripheral-derived factors such as leptin and insulin (Schwartz et al., Nature, 404: 661-671, 2000). Increased weight increases leptin and insulin levels in the blood, acts on the hypothalamus, which is the feeding center, suppresses the appetite, and promotes energy expenditure to restore the increased body weight. This normal feedback system, which regulates energy balance, is a kind of protection mechanism to prevent weight gain. However, in obesity, the feedback system does not work properly, so obesity worsens and causes concomitant diseases.

A team of researchers from the Institute of Genetics and Pathology (IGE) and Molecular Pathology Research Institute (AME) in Germany in 2013 studied the relationship between postmenopausal genetics and health, especially metabolic abnormalities. The researchers analyzed 1800 volunteers and 457,000 genotypes, and compared the relationship between DNA alterations and metabolic abnormalities, and found that methylation at 28 DNA sites resulted in metabolic abnormalities.

In the genomic DNA of mammalian cells, there is a fifth base besides 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 comes only to C of the CG dinucleotide (5'-mCG-3 '), and this CG is often referred to as CpG. Most of C of CpG is methylated with a methyl group. Such methylation of CpG inhibits the repetitive sequence in the genome, such as Alu and transposon, from being expressed, and is the most common site of extracellular changes in mammalian cells. The 5-mC of this CpG is deaminated naturally to T, and thus the CpG in the mammalian genome has a frequency of 1% which is much lower than the normal frequency (1/4 x 1/4 = 6.25%) .

There are exceptions in CpG that appear to be dense, and this is called CpG island. CpG islands are 0.2 to 3 kb in length, and the distribution percentage of C and G bases is more than 50% and the distribution percentage of CpG is higher than 3.75%. About 45,000 CpG islands are found in the whole human genome, and they are concentrated on promoter regions that regulate gene expression. In somatic cells of normal individuals, CpG islands are not methylated in these important gene expression regulatory regions, but imprinted genes and inactivated genes on X chromosomes are methylated so that they are not expressed during development .

Many diseases are caused by gene abnormalities, and the most common type of gene abnormalities is a change in the coding sequence of a gene, which is called a genetic change. When there is a mutation in a gene, the protein that the gene encodes changes in structure and function, leading to disability and deficiency, and this mutant protein causes disease. However, if there is an abnormality in the expression of the gene without mutation in the gene, the disease can be induced. A typical example is the methylation of methylation at the cytosine base site of the regulatory CpG island of gene transcription, in which case the gene is blocked from expression. This is called epigenetic change, which, like the mutation, is transmitted to progeny cells and causes the same effect, the loss of expression of the protein.

Recently, methods for diagnosing diseases such as cancer through DNA methylation measurement have been suggested. When the CpG islands of a particular gene are hypermethylated, expression of the gene is silenced. This is a major mechanism in which the function of the gene is lost without mutation in the coding sequence of the gene coding sequence in the living body. In particular, the function of tumor suppressor genes in the cancer is lost It is interpreted as a cause. Therefore, searching methylation of CpG islands of a specific gene is a great help in disease research, and it has recently been actively tried to use such as methylation specific PCR or automatic base analysis for diagnosis and screening of diseases .

Although there exist some techniques for diagnosing or predicting specific cancers by analyzing the level of methylation of genes, there have been no reports of predicting or diagnosing obesity through the analysis of methylation level of a specific gene. Therefore, the present inventors have developed a specific methylation marker for predicting or diagnosing obesity that can be diagnosed early or predicted.

Korean Patent No. 1284521 discloses a biomarker composition for diagnosing obesity or obesity related diseases, a screening method for treating obesity-related diseases, and a method for providing information for diagnosing obesity-related diseases. 1587635 discloses a method for detecting methylation of a thyroid cancer-specific methylation marker gene for diagnosis of thyroid cancer. However, a method for providing information for predicting or diagnosing obesity using the methylation level of the AKR1E2 gene of the present invention and a composition therefor There is no description.

The present invention has been made in view of the above-described needs. The present inventors conducted whole genome bisulfite sequencing (WGBS) from a normal group and an obesity group, and found that the nucleotide sequence of SEQ ID NO: 1 , The promoter region of AKR1E2 (Aldo-Keto Reductase Family 1 member E2) gene was selected and DNA methylation sequencing was performed on the corresponding region. As a result, it was confirmed that the methylation level of the region was decreased in the obese group, .

In order to solve the above problems, the present invention provides a method for detecting methylation of a promoter region of AKR1E2 gene from a separated biological sample; And comparing the detected methylation to promoter region methylation of the AKR1E2 gene of a normal control sample.

The present invention also provides a composition for predicting or diagnosing obesity, which comprises a substance capable of detecting whether or not the promoter region of the AKR1E2 gene has been methylated.

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

It is expected that the onset of obesity can be predicted early through the AKR1E2 methylation biomarker according to the present invention. In addition, the AKR1E2 gene of the present invention can be used for the evaluation of risk of obesity, diagnosis of pathology, diagnosis of stage of disease, and selection of therapeutic target, and may be useful for studying epigenetics of obesity.

1 is a schematic diagram of the present invention.

In order to accomplish the object of the present invention, there is provided a method for detecting methylation of a promoter region of AKR1E2 (Aldo-Keto Reductase Family 1 member E2) gene from a separated biological sample; And comparing the detected methylation to promoter region methylation of the AKR1E2 gene of a normal control sample.

The promoter of the AKR1E2 gene of the present invention is a sequence including a CpG island and may be a nucleotide sequence of SEQ ID NO: 1, but is not limited thereto. The nucleotide sequence of SEQ ID NO: UCSC Genome Browser Human GRCh37 / hg19 DNA sequence It is a base at position 4,867,961 ~ 4,868,359 of chromosome 10.

The method for providing information for predicting or diagnosing obesity according to the present invention may be,

(a) isolating genomic DNA from separate biological samples of a normal control and suspected individual;

(b) treating the separated genomic DNA with a reagent that differentially transforms methylated DNA and unmethylated DNA;

(c) detecting methylation of the promoter region of the AKR1E2 gene in the genomic DNA treated with the reagent or a fragment thereof; And

(d) determining the obesity as a decrease in the methylation level of the promoter region of the AKR1E2 gene as compared to the methylation of the promoter region of the AKR1E2 gene in the normal control group.

The biological sample of step (a) of the present invention includes a wide range of all biological fluids obtained from individuals, and preferably includes cells, tissues, biopsies, paraffin tissues, blood, serum, And may be at least one selected from the group consisting of plasma, urine, and combinations thereof, and more preferably, it may be a blood sample, but is not limited thereto. Methods for obtaining body fluids, tissues, biopsies and the like from mammals can be performed by conventional methods known in the art.

The subject suspected of being obese according to the present invention may be an individual having no diagnosis of obesity but having a family history of irregular eating habits, obesity or a metabolic disease, but the present invention 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, they are labeled with other bases so that they can be distinguished from each other depending on whether methylation of the cytosine base is present on DNA by bisulfite treatment.

In the step (c) of the present invention, the promoter region sequence of the AKR1E2 gene for methylation detection may be composed of the nucleotide sequence of SEQ ID NO: 1, but is not limited thereto. For detection of methylation, the bisulfite, , Disulfite, or a combination thereof can also be included in the range of the promoter region sequence of the AKR1E2 gene for methylation detection according to the present invention. The nucleotide sequence of SEQ ID NO: 2 may be a nucleotide sequence of SEQ ID NO: 1 that has been treated to convert a cytosine base to uracil or another base that is detected to be different from cytosine in the course of hybridization.

In addition, the detection of methylation in step (c) may be accomplished by a method selected from the group consisting of bisulfite sequencing, sequencing by synthesis, sequencing by ligation, methylation specific PCR, time methylation specific PCR), PCR using methylated DNA-specific binding protein, methylated DNA-specific binding antibody, PCR using aptamer, or nucleic acid chip method.

The bisulfite sequencing method is a method for detecting a nucleic acid containing methylated CpG, comprising the steps of contacting a sample containing nucleic acid with an agent for transforming unmethylated cytosine, and contacting the sample with a CpG- 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 can be sequenced using the Sanger method using a sequencing primer or the next generation sequencing (NGS) method to detect methylated nucleic acid.

Here, the next generation sequencing method may be a sequencing by synthesis method and a sequencing by ligation method. A feature of this method is that instead of making a bacterial clone, a single DNA fragment is spatially separated, amplified in situ (clonal amplification), and sequenced. At this time, it is called a massive parallel sequencing method because it reads hundreds of thousands of fragments at the same time. Basically, this is a sequencing by-synthesis method. In this method, mono or dinucleotides are sequentially attached to obtain a signal, which includes pyrosequencing, 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 finally Pacific BioSciences's PacBio platform. have. 454 and Ion PGM use emulsion PCR as a clonal amplification method and HiSeq use bridge amplification. The sequencing bi-synthase method sequentially attaches one nucleotide and detects the phosphate, hydrogen ion, or pre-attached fluorescence generated when the DNA is synthesized and reads the sequence. In the method of detecting the sequence, 454 uses a pyroseqeuncing method using phosphoric acid, and Ion PGM uses hydrogen ion detection. HiSeq and PacBio detect fluorescence and decode the sequence.

Sequencing by-ligation is a technique for identifying nucleotides at specific positions in a DNA sequence by a sequencing technique using DNA ligase. Unlike polymerase, most sequencing techniques do not use any polymerase, and DNA ligase does not ligate mismatched sequences. This is the SOLiD system. In this technique, two bases are read with spacing, which is repeated five times independently through primer reset, so that the accuracy is improved by reading each base twice in duplicate.

In the method for providing information for predicting or diagnosing obesity according to an embodiment of the present invention, the decrease in the methylation level of the promoter region of the AKR1E2 gene in step (d) may be 58, 120, The methylation of the 125th cytosine may be decreased compared to that of the normal control (see Table 5), the position of which corresponds to the value of p 1 and p 2 in Table 5 based on the first position (4867961) of the nucleotide sequence of SEQ ID NO: 0.05 or less in locus order).

Furthermore, it was confirmed that the three cytosines of the present invention had a correlation with the body mass index (BMI) and the waist circumference (see Table 6), and the methylation analysis of the promoter region of the AKR1E2 gene of the present invention showed that obesity Can provide information for prediction or diagnosis of the disease.

The present invention also provides a composition for predicting or diagnosing obesity, which comprises a substance capable of detecting methylation of AKR1E2 (Aldo-Keto Reductase Family 1 member E2) gene promoter region. The composition of the present invention contains a substance capable of detecting the methylation of the promoter region of the AKR1E2 gene as an active ingredient and can be determined as obesity when the methylation of the promoter region of the AKR1E2 gene is decreased as compared with the normal control. The AKR1E2 gene promoter may be a nucleotide sequence of SEQ ID NO: 1, but is not limited thereto.

In obesity predictive or diagnostic composition of the present invention, the AKR1E2 gene promoter substance which is capable of detecting the methylation of whether the site is methylated AKR1E2 gene promoter region primer set designed to amplify a fragment containing the, methylated AKR1E2 gene promoter region A methylation-specific binding protein capable of binding to a methylated AKR1E2 gene promoter region, a methylation-specific binding antibody or aptamer and a sequencing primer, a sequencing by-synthase primer, and a sequencing by-ligation primer But is not limited to, one or more selected.

Further, in the composition of the present invention, the AKR1E2 detection of methylation whether the gene promoter region is a genomic DNA treated with of different modifications to the methylated DNA and unmethylated DNA reagents to detect methylation if the AKR1E2 gene promoter region And the genomic DNA is selected from the group consisting of obesity suspects or cells, tissues, biopsies, paraffin tissues, blood, serum, plasma, urine, and combinations thereof from the subject to be diagnosed May be isolated from the biological sample, and may be, but is not limited to, genomic DNA, preferably separated from the blood sample.

In the composition for predicting or for diagnosing obesity according to the present invention, the methylation may be selected from the group consisting of bisulfite sequencing, sequencing by synthesis, sequencing by ligation, methylation specific PCR, But are not limited to, those detected by specific PCR (real time methylation specific PCR), PCR using methylated DNA specific binding protein, methylated DNA specific binding antibody or PCR using aptamer or nucleic acid chip method.

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

The kit for predicting or diagnosing obesity of the present invention comprises as an active ingredient a composition comprising a substance capable of detecting whether or not the AKR1E2 gene promoter region comprising the nucleotide sequence of SEQ ID NO: 1 is methylated, DNA samples can be used to predict or diagnose obesity by detecting methylation of the AKR1E2 gene promoter region.

The genomic DNA sample is preferably CpG-containing. Typically, the CpG-containing nucleic acid may include DNA, but not limited thereto, DNA or a sample containing RNA and mRNA. Wherein 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 partitioned carrier means for containing a sample, a second container containing a primer capable of amplifying the 5'-CpG-3 'base sequence region of the sample, and a second container containing a cleaved or truncated nucleic acid to amplify the methylated CpG- And a third container containing means for detecting the presence of nucleic acid that has not been detected. In one embodiment, the primers are selected from the group consisting of PCR for detecting methylation on the genome, methylation specific PCR, real time methylation specific PCR, methylated DNA specific binding protein , Primers for use in PCR, quantitative PCR, nucleic acid chip, sequencing, sequencing biosynthesis, or sequencing by-ligation using PCR, methylated DNA-specific binding antibody or extramammer. The carrier means is suitable for containing one or more containers such as bottles, tubes, and each container contains the independent components used in the method of the present invention. In the context of the present invention, those skilled in the art will readily be able to dispense the necessary formulation in a container.

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

1. Sample Preparation

For the development of DNA methylation markers for the prediction or diagnosis of obesity of the present invention, the following subjects were collected and genomic DNA was extracted from the blood samples and used for methylation analysis. Among the subjects who were recruited, the experimental group was selected based on the criteria shown in Table 1, and a total of 104 normal subjects and 80 obese people were selected. Physical characteristics and blood analysis were performed on selected subjects (Table 2 and Table 3).

Subject Normal group People without hypertension, diabetes, hyperlipidemia, heart disease, paralysis (cognitive, non-cognitive)
Blood coagulant users,
A person with a BMI of 25 (male) or 23 (female) or less, and a BMI of 20 or more.
Excluding metabolic and paralytic diseases.
* The criteria for obesity were selected according to Korean obesity criteria (Korean Standards for Obesity), and hypertension, diabetes, and hyperlipidemia were selected based on past history data Obesity group People without diabetes, hyperlipidemia, heart disease, paralysis (cognitive, non-cognitive)
Blood coagulant users,
BMI 25 (male) or 23 (female) exceeded

In addition, the present invention was conducted under the approval of the Korea Institute of Oriental Medicine, Korea Bioethics Review Committee, and clinical questionnaire data collection and human body collection were conducted after obtaining the subject's written consent.

2. Methylation analysis

Whole genome bisulfite sequencing (WGBS) was performed by selecting 4 genomic DNA samples from the normal and obese groups. Analysis of WGBS was performed with Macrogen (Korea). Analysis of WGBS showed that regions with different methylation ratios in normal and obese groups were selected, Bisulfite sequencing was performed. Bysulfite sequencing was commissioned by LEE Co., Ltd. (Korea), and Illumina MiSeq. Platform. The reference sequence used in the analysis was Human GRCh37 / hg19 from the UCSC Genome Browser (http://genome.ucsc.edu/cgi-bin/hgGateway).

Example 1. Selection of analysis area through WGBS

WGBS analysis of genomic DNA samples from normal and obese groups showed that there was a significant difference in the methylation level between the two groups at the promoter region of AKR1E2 gene (Table 4).

Example 2. AKR1E2  Promoter region (hg19: chr10: 4867961-4868359) Methylation analysis was performed between normal and obese groups

The genomic DNA samples collected from the promoter region of the AKR1E2 gene were used to confirm methylation. As a result, methylation increased or decreased at a significant level relative to the normal group at 8 cytosine sites was confirmed in the obese group as shown in Table 5 below.

In order to confirm the correlation between the methylation level of the promoter region and the obesity phenotype of the AKR1E2 gene, weight, BMI, waist circumference, and WHR (Waist-Hip ratio) were analyzed .

As a result, cytosine at 4868018, 4868080, and 4868085 sites, which were found to have decreased methylation in the obese group, among the eight cytosine positions in which the methylation level was found to be significant between the normal group and the obese group as shown in Table 6, And waist circumference, and that the cytosines at 4868080 and 4868085 were also correlated with abdominal obesity rates.

From the above results, it is considered that obesity can be predicted or diagnosed when the methylation level is reduced in the promoter region of the AKR1E2 gene. It can be confirmed that AKR1E2 of the present invention can be used as a specific methylation biomarker for predicting or diagnosing obesity there was.

<110> Korea Institute of Oriental Medicine          KOREA FOOD RESEARCH INSTITUTE <120> Method for providing information of prediction and diagnosis of          obesity using methylation level of AKR1E2 gene and composition          therefor <130> PN17480 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 399 <212> DNA <213> Homo sapiens <400> 1 gcttcctgac tggctgctgg tgtcgcgcgc cgggcactga ctggctggtt gtattaagga 60 cgggcacccc gattggctgg aggcgcagag ccagctggag tcccaaatgg ctggaagcac 120 cgcgctctgg acatcctgat tggctggcgg catagacgac ggaattttcg attggcttgc 180 agagccacgc aggagcaggg aaggttttac aatcggctgg cattgccgct cacgccccca 240 ccttaggtgt gacctcctgg ttgccttctc cctcgcaccg ggtgcatatg cgactgaggg 300 tagggcagag gtgctaggtt gatgggcaga gagtcttcac cgttccaggg cctcgccatg 360 gccgggccat cctagggcaa gctctagggt cagggtgca 399 <210> 2 <211> 399 <212> DNA <213> Artificial Sequence <220> <223> Bisulfite-converted AKR1E2 <400> 2 gttttttgat tggttgttgg tgtygygygt ygggtattga ttggttggtt gtattaagga 60 ygggtattty gattggttgg aggygtagag ttagttggag ttttaaatgg ttggaagtat 120 ygygttttgg atattttgat tggttggygg tatagaygay ggaattttyg attggtttgt 180 agagttaygt aggagtaggg aaggttttat aatyggttgg tattgtygtt taygttttta 240 ttttaggtgt gattttttgg ttgttttttt tttygtatyg ggtgtatatg ygattgaggg 300 tagggtagag gtgttaggtt gatgggtaga gagtttttat ygttttaggg tttygttatg 360 gtygggttat tttagggtaa gttttagggt tagggtgta 399

Claims (11)

Detecting methylation of the promoter region of the AKR1E2 (Aldo-Keto Reductase Family 1 member E2) gene consisting of the nucleotide sequence of SEQ ID NO: 1 from the separated biological sample; And comparing said detected methylation to promoter region methylation of an AKR1E2 gene consisting of a nucleotide sequence of SEQ ID NO: 1 of a normal control sample. The method according to claim 1,
(a) isolating genomic DNA from separate biological samples of a normal control and suspected individual;
(b) treating the separated genomic DNA with a bisulfite, disulfite, hydrogen sulfite or a combination thereof that differentially transforms methylated DNA and unmethylated DNA;
(c) detecting methylation of the promoter region of the AKR1E2 gene consisting of the nucleotide sequence of SEQ ID NO: 1 in the genomic DNA treated with the bisulfite, disulphite, hydrogen sulfite or a combination thereof or a fragment thereof; And
(d) determining the obesity as a decrease in the methylation level of the AKR1E2 gene promoter region comprising the nucleotide sequence of SEQ ID NO: 1 as compared to the methylation of the AKR1E2 gene promoter region comprising the nucleotide sequence of SEQ ID NO: 1 of the normal control group Characterized in that it provides information for the prediction or diagnosis of obesity. The method 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, Or more of the obesity. delete The method according to claim 2, wherein the detection of methylation in step (c) is performed by a method selected from the group consisting of bisulfite sequencing, sequencing by synthesis, sequencing by ligation, methylation specific PCR, A method using 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 method, Or providing information for diagnosis. 3. The method according to claim 1 or 2, wherein the methylation is detected by the presence or absence of nucleotide sequence change or the result of amplification with a primer capable of amplifying a fragment containing the AKR1E2 gene promoter region comprising the nucleotide sequence of SEQ ID NO: &Lt; / RTI &gt; wherein the information is for prediction or diagnosis of obesity. delete 1. A composition for predicting or diagnosing obesity comprising a substance capable of detecting the methylation of an AKR1E2 gene promoter region comprising the nucleotide sequence of SEQ ID NO: 1. The method of claim 8, wherein the substance capable of detecting methylation whether AKR1E2 gene promoter region consisting of the nucleotide sequence of the Sequence ID No. 1 is designed to amplify a fragment containing the AKR1E2 gene promoter region consisting of the nucleotide sequences of the methylation SEQ ID NO: 1 methylation-specific binding protein can be combined with the primer set consisting of base sequences of that AKR1E2 gene consisting of the nucleotide sequences of the methylation SEQ ID NO: 1, the promoter region and to the hybridization probe, a methylation SEQ ID NO: 1 AKR1E2 gene promoter region, which , A methylation-specific binding antibody or aptamer, a sequencing primer, a sequencing bi-synthase primer, and a sequencing bi-ligation primer. delete 8. A kit for predicting or diagnosing obesity comprising the composition of claim 8 or 9 as an active ingredient.

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