KR20210039241A - Method for providing information of prediction and diagnosis of obesity using methylation level of CMIP gene and composition therefor - Google Patents

Abstract

The onset of obesity can be predicted and diagnosed early by detecting the methylation of an obesity-specific marker gene of the present invention. A CMIP methylation biomarker of the present invention can be used for risk assessment, diagnosis, and selection of treatment targets of obesity. A composition for predicting or diagnosing obesity of the present invention comprises a substance capable of detecting methylation of C-Maf inducing protein (CMIP) gene.

Description

Method for providing information of prediction and diagnosis of obesity using methylation level of CMIP gene and composition therefor}

The present invention relates to a method for providing information for predicting or diagnosing obesity using the methylation level of CMIP gene, and a composition therefor, specifically, predicting or diagnosing obesity using the methylation level of CMIP (C-Maf Inducing Protein) gene It relates to a method for providing information for, a composition and a kit for predicting or diagnosing obesity.

Obesity is perceived to be more serious than its own risk because of various complications that can be caused by obesity. Obesity is known to increase the risk of developing metabolic syndromes such as hypertension, hyperlipidemia, diabetes, fatty liver, joint abnormalities, and cancer. According to the World Health Organization's announcement in 2010, the risk of hypertension, diabetes, and dyslipidemia is more than twice as high in obese people as compared to those of normal weight (2.5 times for hypertension, 2 times for diabetes, 2.3 times for hypercholesterolemia, Hypertriglyceridemia 2.4 times) was higher. In addition to the above diseases, it is known that in the case of women, if the body fat is excessive, the balance of sex hormones is broken, and in severe cases, infertility may occur, and the risk of endometrial cancer and breast cancer is increased. In addition, obesity can cause not only physical diseases but also social isolation, alienation, lack of self-confidence, and mental illnesses such as depression, so the need for prevention and treatment of obesity is recognized as very important.

The onset of obesity is caused by a combination of genetic factors, environmental factors, and dietary factors. Among dietary factors, a high-calorie diet, represented by a high-fat diet (HFD), is considered the main cause of obesity. . If you become obese due to incorrect eating habits, you are more likely to develop metabolic syndrome, which is represented by symptoms such as glucose intolerance, high blood pressure, and hyperlipidemia, and also increases the risk of developing non-alcoholic fatty liver.

Metabolic syndrome and non-alcoholic liver disease are generally known to be associated with insulin resistance, but a clear causal mechanism has not yet been identified. In particular, the liver is an organ that is the center of metabolism such as synthesis, decomposition, and storage of carbohydrates, lipids and proteins. In the case of obesity, insulin resistance appears in the liver tissue, and carbohydrate and lipid metabolism in the liver changes, resulting in a risk of non-alcoholic fatty liver It is known to increase. In addition, it has been reported that the accumulation of fat in the liver increases the inflammatory indicators in the liver tissue.

In recent years, as methods and techniques for epigenetic research have been developed, studies to investigate the causes of metabolic disturbances and inflammatory responses in liver tissue due to obesity are increasing in terms of epigenetics. Epigenetic changes can regulate gene expression without altering the DNA base sequence, and are reversible and inherited from generation to generation. In particular, DNA methylation is a major mechanism of epigenetic change, and many researchers are interested in it to clarify the effect of dietary factors on health. Studies on the effect of a high fat diet on changes in DNA methylation patterns have been conducted in liver tissues, adipose tissues, and muscle tissues. Most studies have analyzed the effect of high fat diets on DNA methylation changes at the individual gene level, and studies at the full-length genome-wide level through high-throughput methods are very insufficient.

There is a study that analyzed changes in the DNA methylation pattern at the full-length genomic level when a high-fat diet was consumed continuously from pregnancy to adulthood, but DNA methylation patterns can be affected not only during pregnancy/nursing periods, but also in adulthood. The effect of high-fat diet consumption in adulthood on the change in DNA methylation patterns at the full-length genome also needs to be studied.

The present inventors have tried to find specific methylation biomarkers for prediction or diagnosis of obesity. As a result, genome methylation analysis was performed on samples of the normal group and the obese group to identify the intron site of the CMIP (c-Maf inducing protein) gene, which has a significant difference in the level of methylation between the two groups, and DNA methylation for the site. The present invention was completed by performing sequencing to find out that there is a decrease in the level of methylation at the site in the obese group.

Accordingly, an object of the present invention is to provide a composition for predicting or diagnosing obesity.

Another object of the present invention is to provide a kit for predicting or diagnosing obesity.

Another object of the present invention is to provide a method of providing information for predicting or diagnosing obesity.

According to an aspect of the present invention, it relates to a composition for predicting or diagnosing obesity comprising a substance capable of detecting whether or not methylation of a CMIP (C-Maf Inducing Protein) gene is included.

The present inventors have tried to discover specific methylation biomarkers for predicting or diagnosing obesity, and as a result, by performing genome methylation analysis on samples of the normal group and the obese group, CMIP, which has a significant difference in methylation level between the two groups. The intron site of the gene was identified, and DNA methylation sequencing was performed on the site to find out that there is a decrease in the methylation level of the site in the obese group.

The obesity-specific methylation marker gene of the present invention can be used to evaluate, predict, diagnose, and select a therapeutic target for obesity risk.

In the genomic DNA of mammalian cells, in addition to A, C, G, and T, there is 5-methylcytosine (5-mC) with a methyl group attached to the fifth carbon of the cytosine ring. 5-mC always comes only to the C of the CG dinucleotide (5'-mCG-3'), and this CG is often referred to 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 and transposon, and is the most common site of extragenic changes in mammalian cells.

In the present invention, the methylation may be detected in the promoter region, 5'untranslated region, intron or exon region of the CMIP gene, for example, detection in the region represented by SEQ ID NO: 1. It can be.

In the present invention, the substance capable of detecting the methylation is a primer pair capable of amplifying a fragment including the methylated site, a probe capable of hybridizing with the methylated site, and binding to the methylated site. It may be any one or more selected from the group consisting of a methylation-specific binding protein, a methylation-specific binding antibody or aptamer, a sequencing primer, a sequencing bi-synthesis primer, and a sequencing bi-ligation primer.

The methylation is bisulfite sequencing, sequencing by synthesis, sequencing by ligation, methylation specific PCR, real time methylation specific PCR, methylated DNA. It may be detected by a method such as PCR using a specific binding protein, PCR using a methylated DNA specific binding antibody or an aptamer, or a nucleic acid chip, 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 the CpG-in of the sample using a methylation-independent oligonucleotide primer. And amplifying the containing nucleic acid. The agent for modifying the unmethylated cytosine may be bisulfite. The unmethylated cytosine (C) base on the bisulfite-treated DNA is deaminated to change to the uracil (U) base, while 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. The oligonucleotide primer may be characterized by amplifying a nucleic acid without discriminating between the modified methylated and unmethylated nucleic acids. The amplified product may be sequenced by the Sanger method using a sequencing primer or analyzed by a next generation sequencing (NGS) method to detect the methylated nucleic acid.

The next-generation sequencing method may be characterized by a sequencing by synthesis and a sequencing by ligation method. The feature of this method is that instead of creating a bacterial clone, a single DNA fragment is spatially separated, amplified in situ (clonal amplification), and sequenced. At this time, since hundreds of thousands of fragments are read at the same time, it is also referred to as a massive parallel sequencing method. Basically, it is a sequencing bi-synthesis method, and it uses a method of obtaining a signal by sequentially attaching mono or dinucleotides, such as 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. The 454 and Ion PGM use emulsion PCR as a clonal amplification method, and HiSeq uses bridge amplification. The sequencing bi-synthesis method reads the sequence by detecting phosphate, hydrogen ions, or pre-attached fluorescence generated when DNA is synthesized by attaching one nucleotide in 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 decode the sequence by detecting fluorescence.

Sequencing by ligation is a sequencing technique that uses DNA ligase to identify a nucleotide at a specific position in a DNA sequence. Unlike most sequencing techniques that use polymerases, they do not use polymerases, and DNA ligases do not ligate mismatched sequences. This is the SOLiD system. In this technique, two bases are read at intervals, and since each base is independently repeated five times through a primer reset, the accuracy is increased by reading each base twice.

Methylation specific PCR, real-time methylation specific PCR, PCR using methylated DNA specific binding protein, PCR using methylated DNA specific binding antibody or aptamer are methods of detecting methylation based on PCR.

The methylation specific PCR method will be described in detail as follows. When bisulfite is treated on genomic DNA, when the cytosine at the 5'-CpG'-3 site is methylated, it remains as cytosine, and when it is unmethylated, it turns into uracil. Therefore, a PCR primer corresponding to the region where the 5'-CpG-3' nucleotide sequence exists was prepared for the modified nucleotide sequence after bisulfite treatment, and in this case, the PCR primer corresponding to the methylated case and the non-methylated case Prepare the corresponding two types of primers. When the genomic DNA is transformed into bisulfite and then PCR is performed using the above two types of primers, in the case of methylation, a PCR product is made from the primers corresponding to the methylated nucleotide sequence, and conversely, in the case of non-methylation. PCR products are made from those using primers corresponding to non-methylation. Whether methylation can be determined qualitatively by an agarose gel electrophoresis method.

Real-time methylation-specific PCR is a conversion of the methylation-specific PCR method to a real-time measurement method. After bisulfite is treated on genomic DNA, PCR primers corresponding to methylation are designed, and real-time PCR is performed using these primers. Is to perform. At this time, there are two methods of detection using the amplified nucleotide sequence and the complementary TanMan probe and the detection using Sybergreen. Therefore, real-time methylation-specific PCR can selectively quantitatively analyze only methylated DNA. At this time, a standard curve is prepared using an in vitro methylated DNA sample, and for standardization, a gene without a 5'-CpG-3' sequence in the nucleotide sequence is amplified together as a negative control group and the degree of methylation is quantitatively analyzed.

The PCR or DNA chip method using a methylated DNA-specific binding protein can selectively separate only methylated DNA because when a protein that specifically binds only methylated DNA is mixed with DNA, the protein is specifically bound to only methylated DNA. It is using features. After mixing genomic DNA with a methylated DNA-specific binding protein, only methylated DNA can be selectively isolated. These isolated DNAs are amplified using PCR primers corresponding to the intron site, and then methylation is measured by agarose electrophoresis.

In addition, methylation can be measured by quantitative PCR method. The methylated DNA isolated with a methylated DNA-specific binding protein is labeled with a fluorescent dye and hybridized to a DNA chip in which a complementary probe is integrated to measure the methylation status. I can.

According to another aspect of the present invention, the present invention relates to a kit for predicting or diagnosing obesity comprising the composition according to the present invention.

The kit for predicting or diagnosing obesity of the present invention contains a composition containing a substance capable of detecting methylation of the CMIP gene as an active ingredient, so that the methylation of the CMIP gene is performed in a genomic DNA sample isolated from a biological sample. Obesity can be predicted or diagnosed by detection.

It is preferable that the genomic DNA sample contains CpG. 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 first container containing an agent sensitively cleaving unmethylated cytosine, a second container containing a primer for amplifying a CpG-containing nucleic acid, and a cleaved or uncut nucleic acid. It may comprise one or more containers comprising a third container containing means for detecting presence. In one embodiment, the primer is PCR for detecting whether methylation has occurred on the genome, methylation specific PCR, real-time methylation specific PCR, PCR using methylated DNA specific binding protein, methylated DNA specific binding antibody or aptamer. It may be a primer for use in PCR used, quantitative PCR, nucleic acid chip, sequencing, sequencing bi-synthesis, or sequencing bi-ligation.

According to another aspect of the present invention, the present invention relates to a method of providing information for predicting or diagnosing obesity, comprising measuring whether the CMIP (C-Maf Inducing Protein) gene is methylated from an isolated biological sample. will be.

In the present invention, the measurement of methylation may be to measure the methylation of CpG located at any one of CMIP gene promoter, 5'UTR (Untranslated region), intron and exon, for example, SEQ ID NO: 1 It may be to measure the methylation of CpG in the region indicated by.

The step of measuring the methylation is PCR, methylation specific PCR, real time methylation specific PCR, PCR using methylated DNA specific binding protein, quantitative PCR, DNA chip, and pyrosequencing. And bisulfite sequencing.

The biological sample may be one or more selected from the group consisting of cells, tissues, biopsies, paraffin tissues, blood, serum, plasma, and urine derived from a subject suspected of obesity or diagnosis, but is not limited thereto.

According to the present invention, the method may further include determining obesity when the level of methylation of the CMIP gene decreases compared to the methylation of the CMIP gene of the normal control.

By detecting the methylation of the obesity-specific marker gene of the present invention, the onset of obesity can be predicted and diagnosed early. The CMIP methylation biomarker of the present invention can be used to evaluate the risk of obesity, diagnose, and select a treatment target.

1 is a result of measuring the weight gain of a mouse according to a high-fat, high-sugar diet.
Figure 2a is a result of measuring the liver weight of the mouse after supplying a high-fat, high-sugar diet for 12 weeks.
Figure 2b is a result of measuring the weight of the epididymis fat of the mouse after feeding a high-fat high-sugar diet for 12 weeks.
Figure 2c is a result of measuring the liver weight of the mouse after supplying a high-fat, high-sugar diet for 24 weeks.
Figure 2d is a result of measuring the weight of the epididymis fat of the mouse after supplying a high-fat high-sugar diet for 24 weeks.
3 is a result of measuring the DNA methylation level of the Cmip gene in mice fed a high-fat high-sugar diet for 12 weeks and 24 weeks.
4 is a result of methylation sequencing of the liver of mice fed a high-fat high-sugar diet for 12 weeks.
5A is a result of analyzing the expression of the Cmip gene in the liver of mice after supplying a high-fat high-sugar diet for 12 weeks.
5B is a result of analyzing the expression of the Cmip gene in the liver of mice after supplying a high-fat high-sugar diet for 24 weeks.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Example 1. Obesity animal model construction

C57BL/6N male mice of the age of 8 weeks were purchased from a central laboratory animal (Seoul, Korea) and stabilized by providing general feed 98052602 (Research Diets, Inc., NJ, USA) for 1 week. Liver and adipose tissues were separated at 12 and 24 weeks after weight recording while providing a high-fat and high-sucrose diet (D12079B, Research Diets, Inc.) and general feed 98052602 (Research Diets, Inc.) I did.

Compared to the control group, the body weight and liver and epididymal fat weight were significantly increased in the mice fed the high-fat high-sugar diet, and it was confirmed that obesity was induced through this (FIGS. 1, 2a to 2d).

Example 2. Methylation analysis

2-1. RRBS library construction

In order to construct an RRBS library containing MspI and ApeKI, 500 ng of genomic DNA was reacted with MspI (NEB, Ipswich, MA, USA) at 37°C for 7 hours. Then, ApeKI (NEB) was added and reacted at 75° C. for 16 to 20 hours. The product digested with restriction enzyme was purified by MiniElute PCR Purification Kit (Qiagen, Venlo, Netherlands). After purification dA was added and the methylated adapter was connected. After performing electrophoresis, slices having a size of 160-420 bp were separated on an agarose gel. Bisulfite conversion was performed using the ZYMO EZ DNA Methylation-Gold Kit (ZYMO Research, Irvine, CA, USA) according to the manufacturer's instructions.

PCR was performed using PfuTurbo Cx Hotstart DNA polymerase (Agilent technologies, Santa Clara, CA, USA) to obtain a final library. The RRBS library was analyzed with an Agilent 2100 Bioanalyzer (Agilent Technologies). Prior to sequencing the samples, the amount of sequencable library fragments was determined by qPCR. Then, the sample was diluted to 10 nM using elution buffer (QIAGEN). The RRBS library was sequenced with NextSeq500 (Illumina, San Diego, CA, USA).

2-2. RRBS data analysis

To adjust the quality of raw reads, FastQC v0.11.2 (www.bioinformatics.babraham.ac.uk/projects/fastqc/) was implemented, and trim galore v0.4.1 (www.bioinformatics.babraham.ac.uk/projects/) trim_galore/) was used to cut adapter sequencing. The truncated sequence was aligned to the murine reference genome (mm10) using BS-seeker2 v2.0.10 (Guo et al., 2013). To cover the MspI and ApeKI fragments of the RRBS library, the double enzyme MspI (CCGG) and ApeKI (GCWGC) fragments ranging in length from 30 to 500 bp in silico were constructed. The leads were aligned with Bowtie2 in local alignment mode by allowing 4 mismatches per read. In order to improve the mapping ratio, unmapped reads were remapped in a paired-end mode. When two paired-end mates overlap, one mate was removed and the methylation level of each CpG site was measured.

At 12 weeks and 24 weeks, 3 animals were selected from the high-fat high-sugar diet and control diet groups (based on body weight), extracted DNA from liver and adipose tissue, and analyzed the change in DNA methylation of the full-length genome by RRBS method. It was confirmed that methylation was reduced by a high-fat high sugar diet in CpGs located in the Cmip gene intron in liver tissue (q <0.05, reduced by more than 20%) (FIG. 3 ).

2-3. Through statistical processing Cmip Selection of methylation sites

A custom Perl script was used to identify the DMR (100 bp) between groups. Briefly, the level of DNA methylation on the genome was profiled by sliding a fixed size window (100 bp) through the reference genome (mm10) in 50 bp increments. The DNA methylation ratio (0 to 1) of all CpG sites in a given window was compared between the two groups (HFHS vs. control at 12 and 24 weeks) using the Mann-Whitney U test (q <0.05). To filter out unreliable DMR candidates, areas covered with less than 10 readings or where the mean difference between groups <0.2 were removed. The identified DMR was annotated using HOMER (v5.7) with UCSC reference gene annotation (mm10).

Individual CpG methylation changes at the selection site were analyzed using the bisulfite amplicon sequencing (BSAS) method, and it was confirmed using the BSAS method that DNA methylation reduction was induced by a high-fat high sugar diet in 10 CpGs (Fig. 4).

Position Gene Tissue Genomic region Strand #CpG Chr Loci CpG1 8 117,325,154 Cmip Liver Intron (+) CpG2 8 117,325,176 Cmip Liver Intron (+) CpG3 8 117,325,179 Cmip Liver Intron (+) CpG4 8 117,325,186 Cmip Liver Intron (+) CpG5 8 117,325,224 Cmip Liver Intron (+) CpG6 8 117,325,243 Cmip Liver Intron (+) CpG7 8 117,325,249 Cmip Liver Intron (+) CpG8 8 117,325,271 Cmip Liver Intron (+) CpG9 8 117,325,289 Cmip Liver Intron (+) CpG10 8 117,325,314 Cmip Liver Intron (+)

2-4. Gene expression analysis using real-time RT-PCR

RNA was isolated from the cells using the RNeasy Mini kit (Qiagen) according to the manufacturer's instructions. 500 ng of total RNA and reverse transcriptase (TOYOBO, Osaka, Japan) were reacted at 30° C. for 10 minutes, 42° C. for 20 minutes, and 99° C. for 5 minutes to synthesize cDNA. Using SYBR green super mix (TOYOBO) and thermal cycler (Bio-Rad, Hercules, CA, USA), the reaction was repeated 40 times at 95°C for 10 seconds, 58°C for 10 seconds, and 72°C for 20 seconds, and the final melting curve Steps were taken to amplify the gene.

Cmip gene expression analysis was performed in the liver tissue of the obese animal model, and it was confirmed that Cmip expression was increased by the decrease in DNA methylation (FIGS. 5A and 5B ).

<110> KOREA FOOD RESEARCH INSTITUTE <120> Method for providing information of prediction and diagnosis of obesity using methylation level of CMIP gene and composition therefor <130> PN190266 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 200 <212> DNA <213> Artificial Sequence <220> <223> CMIP Human <400> 1 tacgctggct ttgcggcagg ggtgcagcag cagtgggtgg tgatgcggct gctcttgagt 60 ggcctggtgg ggtcatttca gtttcaaggc cttggagagt gtgcttagca ggtggggcct 120 aagatttgca tttcagctgc ccaggtagtc aagaagcggt atagcaaagc ggctgtgagc 180 ccattctctg gagtcctgct 200 <210> 2 <211> 170 <212> DNA <213> Artificial Sequence <220> <223> CMIP Mouse <400> 2 gatgcggctg ctctgaggag ccctgtcgtc gaactccgcc ctcaggtggc ctcatctggc 60 ctgaggttct ccaacggtca gatgtccaca gcccggagac ggctgtttga actgttgctg 120 gcgtgcacag ccccttggac gtcttatcct tgttgggcct tattcgtatt 170

Claims (8)

A composition for predicting or diagnosing obesity comprising a substance capable of detecting whether or not methylation of CMIP (C-Maf Inducing Protein) gene.
The composition for predicting or diagnosing obesity according to claim 1, wherein the methylation is detected at any one of CMIP gene promoter, 5'UTR (Untranslated region), intron and exon.
The method of claim 1, wherein the material capable of detecting the methylation of whether the probe to site and the hybridization of the primer set, methylated CMIP gene to amplify a fragment containing the portion of the methylated CMIP gene, methylated CMIP Prediction of obesity, which is one or more selected from the group consisting of a methylation-specific binding protein, a methylation-specific binding antibody or aptamer, a sequencing primer, a sequencing bi-synthesis primer, and a sequencing bi-ligation primer capable of binding to a site of a gene. Or a diagnostic composition.
A kit for predicting or diagnosing obesity comprising the composition of any one of claims 1 to 3.
A method of providing information for predicting or diagnosing obesity, comprising measuring methylation of a CMIP (C-Maf Inducing Protein) gene from an isolated biological sample.
The method of claim 5, wherein the method further comprises determining obesity when the level of methylation of the CMIP gene decreases compared to the methylation of the CMIP gene of a normal control, providing information for predicting or diagnosing obesity. Way.
The method of claim 5, wherein the biological sample is one or more selected from the group consisting of cells, tissues, biopsies, paraffin tissue, blood, serum, plasma, and urine derived from an individual suspected of obesity or diagnosis. How to provide information for your needs.
The method of claim 5, wherein the measuring of methylation comprises PCR, methylation specific PCR, real time methylation specific PCR, PCR using methylated DNA-specific binding protein, quantitative PCR, and DNA. A method of providing information for prediction or diagnosis of obesity, which is performed by a method selected from the group consisting of chip, pyro-sequencing, and bisulfite sequencing.

Images