DNA methylation biomarker for diagnosis of metabolic dysfunction–associated steatohepatitis and use thereof

Abstract

The present invention relates to: a DNA methylation biomarker for the early diagnosis of metabolic dysfunction–associated steatohepatitis and the diagnosis of high-risk metabolic dysfunction–associated steatohepatitis; and a use thereof. Using the composition and the method of the present invention not only enables the early diagnosis of metabolic dysfunction–associated steatohepatitis but also enables the diagnosis of high-risk metabolic dysfunction–associated steatohepatitis, and makes it possible to obtain data with very stable numerical values based on DNA methylation from small amounts of blood, and can reduce diagnosis costs.

Description

[Revised on 04.12.2024 under Article 91 of the Rules] DNA methylation biomarkers for the diagnosis of metabolic steatohepatitis and their uses

[0001] The present invention relates to a DNA methylation biomarker for early diagnosis of metabolic steatohepatitis and diagnosis of high-risk metabolic steatohepatitis and its use.

[0002] Metabolic dysfunction-associated steatohepatitis (MASH), previously known as non-alcoholic steatohepatitis (NASH), is a disease closely related to metabolic disorders, particularly obesity, insulin resistance, type 2 diabetes, and dyslipidemia. It is a condition in which fat accumulates in the liver, accompanied by inflammation and fibrosis, and if left untreated, can progress to cirrhosis or liver cancer. Metabolic steatohepatitis is one of the most common chronic liver diseases worldwide, and its incidence is steadily increasing alongside the increasing prevalence of metabolic diseases.

[0003] Meanwhile, in 2024, the Korean Association for the Study of the Liver (KASL) officially changed the name of nonalcoholic fatty liver disease (NAFLD) to metabolic dysfunction-associated steatotic liver disease (MASLD). Furthermore, nonalcoholic steatohepatitis (NASH) was also renamed to metabolic dysfunction-associated steatohepatitis (MASH).

[0004] Early detection is crucial for metabolic dysfunction-associated steatohepatitis (MASH), but existing blood tests measuring liver damage markers like ALT and AST are not very accurate. Furthermore, imaging techniques like abdominal ultrasound can be used to measure the degree of liver fibrosis, but this method can lead to significant variability.

[0005] Recently, research results have been reported that liver elasticity testing using FibroScan, a liver fibrosis scan test, can noninvasively predict liver fibrosis; however, liver elasticity test values ​​tend to differ depending on the location of the test.

[0006] In addition, magnetic resonance elastography (MRE) using magnetic resonance imaging and real-time elastography, which tests liver elasticity in real time while performing ultrasound by adding a transducer for liver elasticity testing to ultrasound, are being attempted, but improvements are needed in terms of cost and effectiveness.

[0007] Accordingly, the present inventors have developed a MASH diagnostic technology that not only diagnoses MASH early and more accurately than Fibroscan, but also diagnoses high-risk MASH, and can obtain data with very stable values ​​based on DNA methylation from a small amount of blood, while reducing the diagnostic cost.

[0008] One object of the present invention is to provide a composition for diagnosing fatty liver disease.

[0009] Specifically, the present invention provides a composition for diagnosing high-risk metabolic abnormality steatohepatitis, comprising a preparation for measuring the methylation level of a CpG site of one or more (e.g., 1, 2 or 3) genes selected from the group consisting of OGDHL, PRDM8 and IMPDH2.

[0010] Specifically, the present invention provides a composition for early diagnosis of metabolic steatohepatitis, comprising an agent for measuring the methylation level of a CpG site of one or more (e.g., 1, 2, 3, 4, 5, 6 or 7) genes selected from the group consisting of RNF165, HSPH1, PPT1, RBM46, PPARG, LIMA1 and CLEC2D.

[0011] Another object of the present invention is to provide a kit for diagnosing fatty liver disease.

[0012] Specifically, the present invention provides a kit for diagnosing high-risk metabolic abnormality steatohepatitis, comprising the composition for diagnosing high-risk metabolic abnormality steatohepatitis.

[0013] Specifically, the present invention provides a kit for early diagnosis of metabolic steatohepatitis, including a composition for early diagnosis of metabolic steatohepatitis.

[0014] Another object of the present invention is to provide a method for providing information for diagnosing metabolic steatohepatitis.

[0015] Specifically, the present invention provides a method for providing information for diagnosing high-risk metabolic abnormality steatohepatitis, comprising the steps of: (a) measuring a methylation level of a CpG site of one or more (e.g., 1, 2, or 3) genes selected from the group consisting of OGDHL, PRDM8, and IMPDH2 in a sample isolated from a subject; and (b) comparing the methylation level measured in step (a) with a methylation level of a CpG site of the same gene in a control sample.

[0016] Specifically, the present invention provides a method related to information for early diagnosis of metabolic steatohepatitis, comprising: (a) measuring a methylation level of a CpG site of one or more (e.g., 1, 2, 3, 4, 5, 6 or 7) genes selected from the group consisting of RNF165, HSPH1, PPT1, RBM46, PPARG, LIMA1 and CLEC2D in a sample isolated from a subject; and (b) comparing the methylation level measured in step (a) with a methylation level of a CpG site of the same gene in a control sample.

[0017] Another object of the present invention is to provide a preparation for measuring the methylation level of a CpG site of a target gene for use in diagnosing metabolic steatohepatitis.

[0018] Specifically, the present invention provides a formulation for use in diagnosing high-risk metabolic abnormality steatohepatitis, which comprises measuring the methylation level of CpG sites of one or more (e.g., 1, 2 or 3) genes selected from the group consisting of OGDHL, PRDM8 and IMPDH2.

[0019] Specifically, the present invention provides a formulation for the early diagnosis of metabolic steatohepatitis, comprising a preparation for measuring the methylation level of CpG sites of one or more (e.g., 1, 2, 3, 4, 5, 6 or 7) genes selected from the group consisting of RNF165, HSPH1, PPT1, RBM46, PPARG, LIMA1 and CLEC2D.

[0020] Another object of the present invention is to provide a use of a preparation for measuring the methylation level of a CpG site of a target gene for the manufacture of a composition or kit for diagnosing metabolic abnormality steatohepatitis.

[0021] Specifically, the present invention provides a use of a formulation for measuring the methylation level of a CpG site of one or more genes selected from the group consisting of OGDHL, PRDM8 and IMPDH2 for the manufacture of a composition or kit for diagnosing high-risk metabolic abnormality steatohepatitis.

[0022] Specifically, the present invention provides a use of a formulation for measuring the methylation level of a CpG site of one or more (e.g., 1, 2, 3, 4, 5, 6 or 7) genes selected from the group consisting of RNF165, HSPH1, PPT1, RBM46, PPARG, LIMA1 and CLEC2D, for the manufacture of a composition or kit for the early diagnosis of metabolic steatohepatitis.

[0023] One aspect of the present invention is a composition for diagnosing metabolic steatohepatitis, comprising a preparation for measuring the methylation level of a CpG site of a target gene.

[0024] Specifically, the composition is a composition for diagnosing high-risk metabolic abnormality steatohepatitis, comprising an agent for measuring the methylation level of a CpG site of one or more genes selected from the group consisting of OGDHL, PRDM8, and IMPDH2.

[0025] In this specification, non-alcoholic fatty liver disease (NAFLD) and metabolic fatty liver disease (MASLD) may be used interchangeably, and non-alcoholic steatohepatitis (NASH) and metabolic steatohepatitis (MASH) may also be used interchangeably. The term "high-risk metabolic steatohepatitis" in this specification means metabolic steatohepatitis that satisfies NAFLD Activity Score (NAFLD) ≥ 4 and fibrosis stage (F; MASH CRN fibrosis staging system) ≥ 2 among metabolic steatohepatitis. NAS is calculated by adding the scores of steatosis (0-3), lobular inflammation (0-2), and hepatocellular ballooning (0-2) (Kleiner, et. al., Hepatology, 2005;41:1313-1321), and the fibrosis stage is divided into no fibrosis (F0), mild fibrosis (F1), significant fibrosis (F2 or more), advanced fibrosis (F3 or more), and hepatic lesions (F4) according to the MASH CRN fibrosis staging system.

[0026] Specifically, the composition is a composition for early diagnosis of metabolic steatohepatitis, comprising an agent for measuring the methylation level of a CpG site of one or more (e.g., 1, 2, 3, 4, 5, 6 or 7) genes selected from the group consisting of RNF165, HSPH1, PPT1, RBM46, PPARG, LIMA1 and CLEC2D. By comparing the methylation levels of the CpG sites of the genes, normal or metabolic fatty liver (NAFL) can be distinguished from metabolic steatohepatitis, thereby enabling early diagnosis of metabolic steatohepatitis.

[0027] In the present invention, "methylation" refers to the attachment of a methyl group to a base constituting DNA. Specifically, methylation in the present invention refers to methylation that occurs at a cytosine at a specific CpG site of a specific gene. When methylation occurs, the binding of transcription factors is hindered, thereby suppressing the expression of a specific gene. Conversely, when unmethylation or hypomethylation occurs, the expression of a specific gene is increased. DNA methylation is the most well-known epigenetic change that occurs when a methyl group is added to the 5'-position of a cytosine at a CpG site. Because DNA methylation changes exist in DNA, they are easy to detect, are more stable than protein or RNA markers, and occur at a specific location in a gene, making analysis easy.

[0028] In the present invention, any CpG site within each gene can be used as the site for measuring methylation levels without limitation. For example, methylation of CpGs located in one or more of the promoter, 5' UTR, 3' UTR, intron, and exon of each gene can be measured, but is not limited thereto.

[0029] In one implementation, the methylation level of each gene can be measured using fragments containing the CpG sites of each gene. The fragment containing the CpG site can refer to a fragment that has selectivity and specificity for the gene, such that it can represent the gene among fragments containing the CpG site of the gene. Therefore, by measuring the methylation level of a fragment containing the CpG site of any gene, the methylation level of the gene can be compared, thereby diagnosing the target disease.

[0030] Furthermore, since the locations of CpG sites in any gene are known in the art, those skilled in the art can appropriately select fragments containing CpG sites within a gene to achieve the desired effects of the present invention, or utilize fragments containing CpG sites known to represent any gene in the art. Furthermore, agents that measure the methylation level of these fragments can be purchased and compared to the methylation level of the desired gene.

[0031] In one embodiment, the OGDHL gene may include the base sequence of SEQ ID NO: 1, the PRDM8 gene may include the base sequence of SEQ ID NO: 2, or the IMPDH2 gene may include the base sequence of SEQ ID NO: 3. That is, high-risk metabolic abnormality steatohepatitis may be diagnosed by measuring the methylation level of a CpG site within any one of the amino acid sequences of SEQ ID NOs: 1 to 3.

[0032] As one embodiment, the RNF165 gene may include a base sequence of SEQ ID NO: 4, the HSPH1 gene may include a base sequence of SEQ ID NO: 5, the PPT1 gene may include a base sequence of SEQ ID NO: 6, the RBM46 gene may include a base sequence of SEQ ID NO: 7, the PPARG gene may include a base sequence of SEQ ID NO: 8, the LIMA1 gene may include a base sequence of SEQ ID NO: 9, or the CLEC2D gene may include a base sequence of SEQ ID NO: 10. That is, by measuring the methylation level of a CpG site in any one of the amino acid sequences of SEQ ID NOs: 4 to 10, metabolic steatohepatitis may be diagnosed early.

[0033] The term “measuring methylation level” in this specification refers to measuring the degree of methylation of a nucleic acid sequence, and specifically, may mean measuring the amount of methylation present in a DNA base sequence of a target DNA methylation gene within the entire genomic region or some non-genomic region.

[0034] In one implementation, the methylation level of CpG sites of a gene can be measured at the genome level.

[0035] In one embodiment, the agent for measuring the methylation level of a CpG site of a gene may include, but is not limited to, bisulfite or a salt thereof, a methylation-sensitive restriction enzyme, a primer that specifically binds to a sequence comprising a CpG site of the gene, a probe that can hybridize to a sequence comprising a methylated CpG site of the gene, a methylated CpG binding domain, or an antibody or aptamer that specifically binds to methylcytosine.

[0036] In this specification, a methylation-sensitive restriction enzyme is a restriction enzyme that can specifically detect methylation of a CpG site, and may contain CG as a recognition site of the restriction enzyme. Examples thereof include, but are not limited to, SmaI, SacII, EagI, HpaII, MspI, BssHII, BstUI, NotI, etc. Depending on methylation or unmethylation at C of the above restriction enzyme recognition site, whether or not the restriction enzyme cuts is different, and this can be detected through PCR or Southern blot analysis. Other methylation-sensitive restriction enzymes other than the above restriction enzymes are well known in the art.

[0037] In this specification, primers can be preferably designed according to the sequence of a specific CpG site to be analyzed for methylation, and can be a primer pair that can specifically amplify cytosine that is methylated and not modified by bisulfite, and a primer pair that can specifically amplify cytosine that is not methylated and modified by bisulfite.

[0038] In one embodiment, when an antibody or aptamer that specifically binds to a methylated CpG binding domain or methylcytosine is used as a preparation for measuring the methylation level, the methylated DNA can be immunoprecipitated using these, and then the methylation of a specific CpG site can be confirmed through Southern blot, PCR, microarray, or sequencing. In addition, a substrate, an appropriate buffer solution, a chromogenic enzyme or fluorescent substance label, a secondary antibody labeled with a chromogenic enzyme or fluorescent substance, and a chromogenic substrate can be used for immunological detection or quantification of the antibody. In the above, the substrate may be a nitrocellulose membrane, a plate synthesized with polyvinyl resin, a plate synthesized with polystyrene resin, a glass slide glass, etc., the chromogenic enzyme may be peroxidase, alkaline phosphatase, etc., the fluorescent substance may be FITC, RITC, etc., the chromogenic substrate may be ABTS (2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)), OPD (o-phenylenediamine), or TMB (tetramethyl benzidine), and other labeling methods may be used, but are not limited thereto.

[0039] The term “diagnosis” as used herein includes determining a subject’s susceptibility to a particular disease or condition, determining whether a subject has a particular disease or condition, determining a prognosis for a subject having a particular disease or condition, or monitoring the same.

[0040]

[0041] Another aspect of the present invention is a kit for diagnosing fatty liver disease.

[0042] Specifically, the kit is a high-risk metabolic abnormality steatohepatitis diagnostic kit including the composition for diagnosing high-risk metabolic abnormality steatohepatitis.

[0043] Specifically, the kit is a kit for early diagnosis of metabolic steatohepatitis, including a composition for early diagnosis of metabolic steatohepatitis.

[0044] In one embodiment, the kit of the present invention comprises:

[0045] (i) a plurality of primers, probes, or antisense nucleotides capable of hybridizing to a target gene or fragment thereof; a methylated CpG binding domain; and / or an antibody or aptamer that specifically binds to methylcytosine;

[0046] (ii) a vessel suitable for containing a biological sample (e.g., a nucleic acid isolated from the biological sample) and a plurality of primers, probes, or antisense nucleotides capable of hybridizing to a gene of interest or a fragment thereof; a methylated CpG binding domain; and / or an antibody or aptamer that specifically binds to methylcytosine;

[0047] (iii) means for detecting hybridization or binding of (ii); and / or

[0048] Optionally (iv) may include instructions for use and interpretation of kit results.

[0049] The kit of the present invention may also contain other components, including a hybridization solution packaged in a separate container, in which the nucleic acid of the target gene can be hybridized with a plurality of primers, probes, or antisense nucleotides.

[0050] Specifically, the kit of the present invention may be comprised of one or more different component compositions, solutions, or devices suitable for the analytical method. For example, the kit of the present invention may be an RT-PCR (Reverse Transcription Polymerase Chain Reaction) kit or a DNA chip kit. In addition to the above formulations, the kit of the present invention may additionally include a polymerase, agarose, or a buffer solution required for electrophoresis.

[0051] In one embodiment, the kit of the present invention may comprise a nucleic acid chip (CHIP) or gene panel having immobilized probes capable of hybridizing or binding to a gene having a CpG site or a fragment thereof having a CpG site.

[0052] In one embodiment, the kit of the present invention may include a computer having a built-in agent, device, and algorithm for measuring the methylation level of a CpG site of a target gene or a fragment thereof, and the algorithm may be used to correlate the result of measuring the level of the target gene with the diagnosis of the target disease.

[0053]

[0054] Another aspect of the present invention is a method for providing information for diagnosing metabolic fatty liver disease.

[0055] Specifically, the method is a method for providing information for diagnosing high-risk metabolic disorder steatohepatitis, comprising the steps of (a) measuring the methylation level of a CpG site of one or more (e.g., 1, 2, or 3) genes selected from the group consisting of OGDHL, PRDM8, and IMPDH2 in a sample isolated from a subject; and (b) comparing the methylation level measured in step (a) with the methylation level of a CpG site of the same gene in a control sample. The methylation, the site for measuring the methylation level, the measurement of the methylation level of the CpG site, the diagnosis, etc. are as described above.

[0056] Specifically, the method provides a method for providing information for early diagnosis of metabolic steatohepatitis, comprising the steps of: (a) measuring a methylation level of a CpG site of one or more (e.g., 1, 2, 3, 4, 5, 6 or 7) genes selected from the group consisting of RNF165, HSPH1, PPT1, RBM46, PPARG, LIMA1 and CLEC2D in a sample isolated from a subject; and (b) comparing the methylation level measured in step (a) with the methylation level of a CpG site of the same gene in a control sample. The methylation, the site for measuring the methylation level, the measurement of the methylation level of the CpG site, the diagnosis, etc. are as described above.

[0057] In one embodiment, the CpG site for diagnosing high-risk metabolic abnormality steatohepatitis may include, but is not limited to, a CpG site within any one of the amino acid sequences of SEQ ID NOs: 1 to 3.

[0058] In one embodiment, the CpG site for early diagnosis of metabolic steatohepatitis may include, but is not limited to, a CpG site within any one of the amino acid sequences of SEQ ID NOs: 4 to 10.

[0059] In one embodiment, the step of measuring the methylation level can be performed by at least one selected from the group consisting of PCR, methylation-specific PCR (methylation-specific polymerase chain reaction), real-time methylation-specific PCR (real-time methylation-specific polymerase chain reaction), MethyLight PCR, MehtyLight digital PCR, EpiTYPER, PCR using methylated DNA-specific binding protein, methyl PNA-PCR, quantitative PCR, DNA chip, pyrosequencing, bisulfite sequencing, Southern blot, RLGS (Restriction landmark genomic scanning), MS-SnuPE (Methylation-sensitive single-nucleotide primer extension), CpG microarray, COBRA (Combined bisulfite-restriction analysis), MIRA (methylated-CpG island recovery assay), mass spectrometry, and immunoprecipitation using a methylated CpG binding domain or an anti-methylcytosine antibody.

[0060] In one embodiment, the method for providing information for diagnosing high-risk metabolic abnormality steatohepatitis of the present invention may further include a step of determining high-risk metabolic abnormality steatohepatitis when the methylation level of the CpG site of the OGDHL gene or the IMPDH2 gene increases compared to the control group, or when the methylation level of the CpG site of the PRDM8 gene decreases compared to the control group.

[0061] In one embodiment, the method may further include a step of determining metabolic steatohepatitis when the methylation level of a CpG site of one or more genes selected from the group consisting of NF165, HSPH1, PPT1, RBM46, and PPARG increases compared to a control group, or when the methylation level of a CpG site of one or more genes selected from the group consisting of LIMA1 and CLEC2D decreases compared to a control group.

[0062] As used herein, the term “increased methylation level of a gene” or “increased methylation level of a CpG site of a gene” means that the methylation level in a sample of a subject is measurably and significantly increased compared to a control group, and may mean, for example, an increase of about 1.1 times or more, for example, 1.1 to 2.5 times, 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, or 2 times or more.

[0063] As used herein, the term “decreased methylation level of a gene” or “decreased methylation level of a CpG site of a gene” means that the methylation level in a sample of a subject is measurably and significantly reduced compared to a control group, for example, by about 0.9 times or less, for example, by 0.1 times to 0.9 times, 0.9 times, 0.8 times, 0.7 times, 0.6 times, 0.5 times, 0.4 times, 0.3 times, 0.2 times, or 0.1 times or less.

[0064] In one embodiment, the diagnostic method of the present invention can be performed using a plurality of panels.

[0065] As an example of implementation, when diagnosed with high-risk metabolic dystrophy steatohepatitis, the control group may be, but is not limited to, normal subjects, patients with low-risk metabolic dystrophy steatohepatitis, or patients with metabolic dystrophy fatty liver.

[0066] As an example of implementation, the control group in the early diagnosis of metabolic steatohepatitis may be, but is not limited to, normal subjects or patients with metabolic steatohepatitis.

[0067] As used herein, the term "subject" includes, without limitation, any subject capable of suffering from metabolic steatohepatitis, but may be, for example, a mammal including a human.

[0068] As used herein, "sample" may refer to a sample obtained from a subject. Depending on the type of analysis being performed, the sample may encompass a wide range of samples obtained from individuals, body fluids, cell lines, tissue cultures, DNA isolation, and the like, including, for example, blood, serum, plasma, saliva, stool, urine, cells, tissues, biopsies, paraffin-embedded tissues, and fine needle aspiration specimens. In one embodiment, the sample may be derived from, but is not limited to, blood, plasma, serum, and / or liver.

[0069]

[0070] Another aspect of the present invention is a use of a preparation for measuring the methylation level of a CpG site of a target gene for diagnosing metabolic fatty liver disease.

[0071] Specifically, the use is for the diagnosis of high-risk metabolic abnormality steatohepatitis, comprising a preparation measuring the methylation level of CpG sites of one or more (e.g., 1, 2 or 3) genes selected from the group consisting of OGDHL, PRDM8 and IMPDH2.

[0072] Specifically, the use is for early diagnosis of metabolic steatohepatitis by a preparation that measures the methylation level of CpG sites of one or more (e.g., 1, 2, 3, 4, 5, 6 or 7) genes selected from the group consisting of RNF165, HSPH1, PPT1, RBM46, PPARG, LIMA1 and CLEC2D.

[0073]

[0074] Another aspect of the present invention is the use of a preparation for measuring the methylation level of a CpG site of a target gene for the manufacture of a composition or kit for diagnosing metabolic abnormality steatohepatitis.

[0075] Specifically, the use is for the preparation of a composition or kit for diagnosing high-risk metabolic abnormality steatohepatitis, which measures the methylation level of a CpG site of one or more (e.g., 1, 2 or 3) genes selected from the group consisting of OGDHL, PRDM8 and IMPDH2.

[0076] Specifically, the purpose is the use of a preparation for measuring the methylation level of a CpG site of one or more (e.g., 1, 2, 3, 4, 5, 6 or 7) genes selected from the group consisting of RNF165, HSPH1, PPT1, RBM46, PPARG, LIMA1 and CLEC2D, for the manufacture of a composition or kit for the early diagnosis of metabolic steatohepatitis.

[0077] The composition and method of the present invention not only enable early diagnosis of metabolic steatohepatitis but also enable diagnosis of high-risk metabolic steatohepatitis, and enable acquisition of data with very stable values ​​based on DNA methylation from a small amount of blood, and have the effect of reducing diagnostic costs.

[0078] Figure 1 is a diagram showing the process of searching for blood DNA methylation markers for detection of high-risk MASH.

[0079] Figure 2 is a diagram showing the correlation between OGDHL and high-risk MASH.

[0080] A shows the analysis of the methylation level of OGDHL in blood samples from high-risk MASH patients and low-risk MASH patients (left figure) and in blood samples from MASL patients, MASH_F0-F2 patients, and MASH_F3-F4 patients (right figure).

[0081] B shows the analysis of the methylation level of OGDHL in liver samples from high-risk MASH patients and low-risk MASH patients (left figure) and in liver samples from MASL patients, MASH_F0-F2 patients, and MASH_F3-F4 patients (right figure).

[0082] C is a diagram analyzing the methylation level of OGDHL in blood samples from high-risk MASH patients (H) and low-risk MASH or metabolic fatty liver patients (L) using pyrosequencing.

[0083] Figure 3 is a diagram showing the ROC (Receiver Operating Characteristics) curve for OGDHL.

[0084] Figure 4 is a diagram showing the correlation between OGDHL methylation and OGDHL expression.

[0085] Figure 5 is a diagram showing the process of searching for blood DNA methylation markers for early diagnosis of NASH.

[0086] Figure 6 is a diagram showing the correlation between RNF165 and NASH.

[0087] Figure 7 is a diagram showing the correlation between HSPH1 and NASH.

[0088] Figure 8 is a diagram showing the correlation between PPT1 and NASH.

[0089] Figure 9 is a diagram showing the correlation between RBM46 and NASH.

[0090] Figure 10 is a diagram showing the correlation between PPARG and NASH.

[0091] Figure 11 is a diagram showing the correlation between LIMA1 and NASH.

[0092] Figure 12 is a diagram showing the correlation between CLEC2D and NASH.

[0093] Figure 13 is a diagram showing the ROC curve (NAFL vs NASH) for RNF165, HSPH1, PPT1, RBM46, PPARG, LIMA1, and CLEC2D.

[0094] Hereinafter, the present invention will be described in more detail through examples. These examples are intended merely to illustrate the present invention and are not to be construed as limiting the scope of the present invention.

[0095]

[0096] Experimental Example 1. DNA Methylation Microarray Experiment and Data Analysis Method

[0097]

[0098] The EPIC BeadChip (Illumina) was used for methylation analysis experiments according to the manufacturer's instructions. Specifically, 1 μg of genomic DNA collected from whole blood was treated with 20 μL of sodium bisulfite solution included in the EZ DNA Methylation-Gold Kit (Zymo Research, Orange, CA). The bisulfite-modified DNA (4 μL) was amplified using the InfiniumMethylation Assay kit (Illumina). The amplified DNA was hybridized to the EPIC BeadChip and scanned with the Illumina iSCAN system. CpG methylation values ​​were calculated as mean-β values ​​using the minfipackage (version 1.26.2) in R software, and functional normalization was used to eliminate technical variations. Measurements with a detection p-value less than 0.05 were considered to have a significantly higher signal than background.

[0099]

[0100] Experimental Example 2. DNA methylation analysis using pyrosequencing

[0101]

[0102] Pyrosequencing technology was used to verify the selected DNA methylation markers. Specifically, 1 μg of total DNA collected from whole blood was subjected to bisulfite conversion using the EZ DNA Methylation-Gold Kit (Zymo Research, California, USA). Each sample was eluted with 20 μL of the kit's elution buffer. Next, 2 μL of the bisulfite-modified DNA was added to 15 μL of a PCR mixture containing the primer set and a 2x master mix (Thermo Scientific, Massachusetts, USA) and amplified using a SimpliAmp Thermal Cycler (AppliedBiosystems, MA, USA).

[0103] For pyrosequencing, forward, reverse, and sequencing primers were designed using PyroMark Assay Design 2.0 (Qiagen, Hilden, Germany). Standard pyrosequencing was then performed. Specifically, 10 μL of PCR product was immobilized on 3 μL of PyroMark Q48 Magnetic Beads (Qiagen, Hilden, Germany) and annealed with sequencing primers at 80°C for 10 min. Sequence analysis was performed on a PyroMark Q48 Autoprep instrument using the PyroMark Q48 Advanced CpG Reagent Kit according to the manufacturer's instructions (Qiagen, Hilden, Germany). Finally, the generated pyrograms were analyzed using the PyroMark Q48 Autoprep.

[0104]

[0105] Example 1: Exploration of DNA methylation markers in blood for detection of high-risk MASH.

[0106]

[0107] DNA methylation was analyzed in blood derived from normal controls (n=50), patients with metabolic fatty liver disease (MASL) (n=55), MASH_F0-F2 patients (n=79), and MASH_F3-F4 patients (n=16) of the metabolic steatohepatitis (MASH) cohort (Table 1) according to Experimental Example 1. Among the 150 patient samples, 43 patients were in the high-risk group, satisfying NAFLD Activity Score (NAFLD) ≥4 and fibrosis stage (F; MASH CRN fibrosis staging system) ≥2, and 107 patients were in the low-risk group.

[0108]

[0109] Based on the integrated analysis of the generated DNA methylation data, transcriptome data, and clinical data (NAS, Fibrosis, etc.), DNA methylation markers with significantly increased or decreased methylation of CpG sites in DNA specific to high-risk MASH were selected (Tables 2 and 3).

[0110]

[0111]

[0112] Specifically, OGDHL and IMPDH2 showed increased methylation in high-risk MASH compared to low-risk MASH, and PRDM8 showed decreased methylation in high-risk MASH compared to low-risk MASH (Fig. 1). The AUROC for the combination of these three markers was 0.9 or higher, demonstrating excellent diagnostic performance (Fig. 1).

[0113]

[0114] Example 2: Correlation Analysis between OGDHL and High-Risk MASH

[0115]

[0116] For the OGDHL selected in Example 1, the degree of methylation was analyzed using the Infinium 850K methylation array in blood and liver samples. As a result, it was confirmed that the degree of methylation of OGDHL increased in blood and liver samples in high-risk MASH compared to low-risk MASH (Figs. 2A and B). In addition, the results of pyrosequencing using blood similarly confirmed that the degree of methylation increased in high-risk MASH (Fig. 2C). The area under the curve (AUROC), which indicates the diagnostic performance of the marker, was confirmed to have excellent diagnostic ability with a value of 0.8 or higher (Fig. 3).

[0117] Additionally, it was confirmed that as the degree of OGDHL methylation increased, OGDHL gene expression significantly decreased (Fig. 4).

[0118]

[0119] Example 3: Exploration of DNA methylation markers in blood for early diagnosis of NASH.

[0120]

[0121] To identify markers for early diagnosis of NASH that can be distinguished from simple fatty liver disease (NAFL) in blood, DNA methylation was analyzed in blood derived from normal controls (n=50), non-alcoholic fatty liver disease (NAFL) patients (n=55), NASH_F0-F2 patients (n=79), and NASH_F3-F4 patients (n=16) according to Experimental Example 1.

[0122]

[0123] Based on the integrated analysis of the generated DNA methylation data, transcriptome data, and clinical data (NAS, Fibrosis, etc.), DNA methylation markers with significantly increased or decreased methylation of CpG sites in DNA specific to NASH were selected (Tables 4 and 5).

[0124]

[0125]

[0126] Specifically, RNF165, HSPH1, PPT1, RBM46, and PPARG showed increased methylation in NASH compared to NAFL (Figs. 6 to 10), and LIMA1 and CLEC2D showed decreased methylation in NASH compared to NAFL (Figs. 11 and 12). The AUROCs of RNF165, HSPH1, PPT1, RBM46, PPARG, LIMA1, and CLEC2D were 0.67, 0.66, 0.63, 0.61, 0.62, 0.68, and 0.65, respectively (Figs. 6 to 12), and the AUROC for the combination of the seven markers was 0.76 or higher, confirming excellent diagnostic ability (Fig. 13).

[0127]

[0128] From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical concept or essential characteristics. In this regard, it should be understood that the experimental examples and embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as encompassing all changes or modifications derived from the meaning and scope of the following claims and their equivalent concepts, rather than the detailed description above.

Claims

1. A composition for diagnosing high-risk metabolic abnormality steatohepatitis, comprising a preparation for measuring the methylation level of a CpG site of one or more genes selected from the group consisting of OGDHL, PRDM8, and IMPDH2.

2. In paragraph 1, The OGDHL gene contains the base sequence of sequence number 1, or The PRDM8 gene contains the base sequence of sequence number 2, or A composition comprising the IMPDH2 gene having a base sequence of sequence number 3.

3. In the first paragraph, a composition comprising a preparation for measuring the methylation level of the CpG site of the gene, bisulfite or a salt thereof, a methylation-sensitive restriction enzyme, a primer that specifically binds to a sequence including the CpG site of the gene, a probe that can hybridize to a sequence including the methylated CpG site of the gene, a methylated CpG binding domain, or an antibody or aptamer that specifically binds to methylcytosine.

4. A composition for early diagnosis of metabolic steatohepatitis, comprising a preparation for measuring the methylation level of a CpG site of one or more genes selected from the group consisting of RNF165, HSPH1, PPT1, RBM46, PPARG, LIMA1, and CLEC2D.

5. In paragraph 4, The RNF165 gene contains the base sequence of sequence number 4, or The HSPH1 gene contains the base sequence of sequence number 5, or The PPT1 gene contains the base sequence of sequence number 6, or The RBM46 gene contains the base sequence of sequence number 7, or The PPARG gene contains the base sequence of sequence number 8, or The LIMA1 gene contains the base sequence of sequence number 9, or A composition comprising a CLEC2D gene having a base sequence of sequence number 10.

6. In the fourth paragraph, a composition comprising a preparation for measuring the methylation level of the CpG site of the gene, bisulfite or a salt thereof, a methylation-sensitive restriction enzyme, a primer that specifically binds to a sequence including the CpG site of the gene, a probe that can hybridize to a sequence including the methylated CpG site of the gene, a methylated CpG binding domain, or an antibody or aptamer that specifically binds to methylcytosine.

7. A kit for diagnosing high-risk metabolic abnormality fatty liver disease, comprising a composition according to any one of claims 1 to 3.

8. A kit for early diagnosis of metabolic fatty liver disease, comprising a composition according to any one of claims 4 to 6. 9.(a) a step of measuring the methylation level of a CpG site of one or more genes selected from the group consisting of OGDHL, PRDM8 and IMPDH2 in a sample isolated from the subject; and (b) a method for providing information for diagnosing high-risk metabolic abnormality steatohepatitis, comprising a step of comparing the methylation level measured in step (a) with the methylation level of a CpG site of the same gene in a control sample.

10. A method for determining high-risk metabolic abnormality steatohepatitis in the 9th paragraph, when the methylation level of the CpG site of the OGDHL gene or the IMPDH2 gene increases compared to the control group, or when the methylation level of the CpG site of the PRDM8 gene decreases compared to the control group. 11.(a) a step of measuring the methylation level of a CpG site of one or more genes selected from the group consisting of RNF165, HSPH1, PPT1, RBM46, PPARG, LIMA1 and CLEC2D in a sample isolated from a subject; and (b) A method for providing information for early diagnosis of metabolic steatohepatitis, comprising a step of comparing the methylation level measured in step (a) with the methylation level of a CpG site of the same gene in a control sample.

12. A method for determining metabolic steatohepatitis when the methylation level of the CpG site of one or more genes selected from the group consisting of RNF165, HSPH1, PPT1, RBM46, and PPARG increases compared to the control group, or when the methylation level of the CpG site of one or more genes selected from the group consisting of LIMA1 and CLEC2D decreases compared to the control group in paragraph 11.

13. A method according to any one of claims 9 to 12, wherein the step of measuring the methylation level is performed by at least one selected from the group consisting of PCR, methylation-specific PCR, real time methylation-specific PCR, MethyLight PCR, MehtyLight digital PCR, EpiTYPER, PCR using methylated DNA-specific binding protein, methyl PNA-PCR, quantitative PCR, DNA chip, pyrosequencing, bisulfite sequencing, Southern blot, RLGS (Restriction landmark genomic scanning), MS-SnuPE (Methylation-sensitive single-nucleotide primer extension), CpG microarray, COBRA (Combined bisulfite-restriction analysis), MIRA (methylated-CpG island recovery assay), mass spectrometry, and immunoprecipitation using a methylated CpG binding domain or an anti-methylcytosine antibody.

14. A method according to any one of claims 9 to 12, wherein the sample is derived from blood, plasma, serum or liver of the subject.

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