Methods for determining whether a biological sample originates from liver tissue

Abstract

The present invention relates to a method for determining whether a biological sample of unknown origin is derived from liver tissue and a composition containing a liver tissue-specific DNA methylation marker for implementing the method. The liver tissue-specific methylation marker can very accurately determine whether the source of the biological sample is liver tissue, because normal liver tissue and liver cancer tissue have high methylation levels, while tissues other than liver tissue have low methylation levels.

Description

Technical Field

[0001] The present invention relates to a method for determining whether a biological sample of unknown origin is derived from liver tissue, and a composition containing liver tissue-specific DNA methylation markers for implementing the method. Background Art

[0002] The cells in the human body have the same genetic information, but the function and shape of each cell are very diverse. This is because specific genes are expressed in each cell, so the cell differentiation process is different, resulting in different cell phenotypes. Several factors, such as DNA methylation, histone modification, and tissue-specific transcription factors are involved in the expression of these specific genes. Specifically, DNA methylation, especially methylation of CpG sites, is an essential element of cell-specific gene expression, and it is known that each cell or tissue has a unique DNA methylation signature. Therefore, the origin of a cell or tissue can be easily identified using the DNA methylation signature.

[0003] Identifying the origin of cells or tissues can enable early diagnosis of diseases and improve the accuracy of diagnosis. For example, circulating cell-free DNA (cfDNA) exists in the blood circulating in the living body, in which there may be circulating tumor DNA (ctDNA) flowing out from tumor cells. Even if the presence of ctDNA is detected by liquid biopsy, in order to accurately diagnose cancer, the tissue from which the ctDNA originated needs to be additionally confirmed, and in this process, the diagnosis of cancer will be delayed. However, if the tissue from which the cancer originated can be identified while detecting the corresponding ctDNA, early diagnosis of cancer is possible, and there is an increasing need for a method to identify the origin of biological samples for early diagnosis of other diseases as well as cancer.

[0004] The present inventors studied a method for determining the origin of tissues and cells of unknown origin based on DNA methylation data, and as a result, identified a marker with a high methylation level specific to liver tissue, thereby completing the present invention. Summary of the invention

[0005] Technical issues

[0006] An object of the present invention is to provide a method for determining whether a biological sample of unknown origin is derived from liver tissue and a composition for implementing the method.

[0007] Technical Solution

[0008] In order to achieve the above object, one aspect of the present invention provides a method for determining whether a biological sample is derived from liver tissue, comprising the following steps:

[0009] (a) isolating DNA from an isolated biological sample of a subject; and

[0010] (b) The methylation levels of the CpG sites of the sequences represented by SEQ ID NO: 1 and SEQ ID NO: 2 in the separated DNA were measured.

[0011] In a specific embodiment of the present invention, the subject may be a human, and the biological sample includes tissues, tissue fragments, cells, cell fragments, blood, plasma, body fluids, feces, urine, etc. separated from the subject, but is not limited thereto. Tissues, tissue fragments, cells, cell fragments, etc. may be separated from blood, plasma, body fluids, urine, etc. collected from the subject. In addition, DNA may be DNA separated from tissues, cells, etc., or may be cell-free DNA (cfDNA) floating in blood, plasma, body fluids, etc., or circulating tumor DNA (ctDNA) flowing out of tumor cells.

[0012] The term "methylation" used in this specification refers to the binding of a methyl group (-CH3) to a base constituting DNA, preferably refers to the methylation occurring on a cytosine at a specific CpG site of a specific DNA. The term "methylation level" refers to the quantitative evaluation of the methylation state of a CpG site present in a specific DNA sequence, and the methylation state refers to whether 5-methyl-cytosine is present at one or more CpG sites in a DNA sequence.

[0013] The term "CpG site" used in this specification refers to a sequence where cytosine (C) and guanine (G) are linked by a phosphate group, which can be present in a DNA sequence including a promoter region, a protein coding region (open reading frame, ORF) and a termination region, etc. Methylation of CpG sites is known to be involved in maintaining genome stability and regulating gene expression, etc.

[0014] The present inventors attempted to discover liver tissue-specific methylation markers, and as a result, discovered cg12137206 (SEQ ID NO: 1) and cg03792768 (SEQ ID NO: 2) markers that have high methylation levels in normal liver tissue and liver cancer tissue samples and low methylation levels in other tissues including blood. The target methylation sites of the two markers are CpG sites located at nucleotide position 61 in the sequences shown by SEQ ID NO: 1 and SEQ ID NO: 2, respectively. However, since methylation may occur even in CpG sites other than the target methylation site, in the present invention, the methylation levels of the total CpG sites present in the sequences shown by SEQ ID NO: 1 and 2 including the target CpG site can be measured.

[0015] In a specific embodiment of the present invention, (b) can be implemented by a method 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, quantitative PCR, DNA chip, molecular beacon, MS-HRM (methylation-sensitive high resolution melting), asymmetric PCR, asymmetric PCR MS-HRMA (asymmetric PCR methylation-sensitive high resolution melting analysis), recombinase polymerase amplification, LAMP (loop-mediated isothermal amplification), Eclipse probe, next-generation sequencing panel (NGS panel), pyrophosphate sequencing and disulfide sequencing.

[0016] For example, the methylation level can be identified by a microarray, and the microarray can use a probe immobilized on a solid surface. The probe can include a sequence complementary to a 10 to 100 consecutive nucleotide sequence including a CpG site.

[0017] In a specific embodiment of the present invention, the method may further include (c) comparing the methylation level with the methylation level of a normal control group after (b). For example, when the methylation level of the biological sample is higher than the methylation level of the normal control group, it can be determined that the biological sample is derived from liver tissue, and when the methylation level is lower or similar, it can be determined that the biological sample is derived from tissues other than the liver.

[0018] Another aspect of the present invention provides a method for detecting DNA from liver tissue in a biological sample, comprising:

[0019] (a) isolating DNA from an isolated biological sample of a subject; and

[0020] (b) The methylation levels of the CpG sites of the sequences represented by SEQ ID NO: 1 and SEQ ID NO: 2 in the separated DNA were measured.

[0021] Since this method and the method for determining whether a biological sample is derived from liver tissue use the same sequence of methylation level detection technology, the repeated content between the two methods is omitted to avoid excessive description in the instructions.

[0022] In the present invention, the method for detecting DNA from liver tissue can be used in combination with conventional liver cancer diagnosis methods.

[0023] Another aspect of the present invention provides a composition for determining whether a biological sample is derived from liver tissue, comprising a reagent capable of measuring the methylation level of the sequences represented by SEQ ID NO: 1 and SEQ ID NO: 2.

[0024] In a specific embodiment of the present invention, the number of CpG sites may be one or more, including the CpG site located at the 61st nucleotide in the sequence shown by SEQ ID NO: 1 and SEQ ID NO: 2.

[0025] In a specific embodiment of the present invention, the reagent capable of measuring the methylation level can be a primer, a probe or an antisense nucleic acid that binds to the CpG site of the sequence shown by SEQ ID NO: 1 and SEQ ID NO: 2, and the primer, probe or antisense nucleic acid can be used as a hybridizable array element and can be fixed on a substrate.

[0026] In addition, the sequence shown in SEQ ID NO: 1 or 2 may be genomic DNA, or may be a sequence in which unmethylated cytosine is converted into uracil by bisulfite treatment.

[0027] Substrate is suitable solid or semisolid support, for example, it can include film, filter, chip, slide, wafer, fiber, magnetic or non-magnetic beads, gel, tubing, plate, polymer, microparticle and capillary. Hybridizable array element can be fixed on substrate by chemical binding method, covalent binding method such as UV or linking agent (for example: ethylene glycol oligomer and diamine).

[0028] In a specific embodiment of the present invention, DNA isolated from a biological sample (sample DNA) can be hybridized with array elements when applied to a hybridizable array, and the hybridization conditions can be modified in various ways according to techniques known in the art, and the detection and analysis of the hybridization level can be performed in various ways. In addition, in order to provide a signal that allows confirmation of hybridization, the sample DNA and / or primers, probes or antisense nucleic acids can be labeled and connected to oligonucleotides.

[0029] Labels may include, but are not limited to, fluorophores (e.g., fluorescein, phycoerythrin, rhodamine, lissamine, Cy3 and Cy5 (Pharmacia)), chromophores, chemiluminescent groups, magnetic particles, radioisotopes (P32 and S35), enzymes (alkaline phosphatase or horseradish peroxidase), cofactors, enzyme substrates, heavy metals (e.g., gold), antibodies, streptavidin, biotin, digoxigenin, and haptens with specific binding partners (e.g., chelating groups).

[0030] In the present invention, the hybridization of primers, probes or antisense nucleic acids with sample DNA depends on various factors, such as reaction temperature, hybridization and washing time, buffer components and their pH and ionic strength, nucleotide length, nucleotide sequence, number of GC sequences, etc. Detailed conditions for hybridization can be found in Joseph Sambrook et al., Molecular Cloning, A laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); and MLM Anderson, Nucleic Acid Hybridization, Springer-Verlag New York Inc. NY (1999).

[0031] After the hybridization reaction, the hybridization signal generated by the hybridization reaction can be detected. For example, when the probe is labeled with an enzyme, hybridization can be confirmed by reacting a substrate of the enzyme with the hybridization reaction product.

[0032] As enzymes and substrates, peroxidases (e.g., horseradish peroxidase) and chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis-N-methylacridinium nitrate), resorufin benzyl ether, luminol, Amplex red reagent (10-acetyl-3,7-dihydroxyphenoxazine), HYR (p-phenylenediamine-HCl and catechol), TMB (tetramethylbenzidine), ABTS (2,2'-azabis[3-ethylbenzothiazoline sulfonate]), o-phenylenediamine (OPD), and naphthol / pyronine can be used; alkaline phosphatase and bromochloroindol phosphate (BCIP), nitro blue tetrazolium chloride (NBT), naphthol-AS-B1-phosphate, and ECF substrate; glucose oxidase and t-NBT (nitro blue tetrazolium chloride) and m-PMS (phenazine methylsulfate).

[0033] In the present invention, liver tissue-specific markers were discovered based on DNA methylation. Specifically, by performing methylation analysis on various cell types in blood and various organ samples, markers with low methylation in organs other than liver tissue and markers with high methylation specific to liver tissue were selected, and the validity of the markers was confirmed by machine learning. The selected markers had high methylation levels in all samples from normal liver tissue to liver cancer in an independent cohort, and low methylation levels in other tissues including blood, and it was confirmed that only two markers had liver specificity with an accuracy of more than 99% or higher. A high trend of liver specificity was also confirmed in the gene expression of the corresponding markers. It has been determined that liver-specific methylation markers can be used as the main markers for confirming tissue origin, and are intended to be used as indicators to improve the accuracy of monitoring and early detection of liver-related diseases in the future.

[0034] Beneficial Effects

[0035] The method for determining whether a biological sample is derived from liver tissue and the composition for implementing the method use a liver tissue-specific DNA methylation marker, and the liver tissue-specific marker has a low methylation level in tissues other than liver tissue and a high methylation level in normal liver tissue and liver cancer tissue, so it can be very accurately determined whether the biological sample is derived from liver tissue.

[0036] BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The process of discovering liver tissue-specific markers is schematically illustrated.

[0038] Figure 2 Shown are the variable importance plots (VarImp plots) of discovered liver tissue specific markers.

[0039] Figure 3 Results confirming the performance according to the number of liver tissue-specific markers found are shown.

[0040] Figure 4 In the figure, A shows the confirmation results of the methylation levels of the finally selected cg12137206 and cg03792768 markers in normal liver tissue (liver N) and liver cancer tissue (liver T), and B shows the confirmation results in normal liver tissue (LIHC_N) and liver cancer tissue (LIHC_T), normal tissue other than the liver (Pan_N) and cancer tissue other than the liver (Pan_T).

[0041] Figure 5 The results of confirmation of the methylation level of the cg12137206 marker in various normal tissues (A) and cancer tissues (B) are shown.

[0042] Figure 6 The results of confirmation of the methylation level of the cg03792768 marker in various normal tissues (A) and cancer tissues (B) are shown.

[0043] Figure 7 The validation results of the methylation levels of cg12137206 and cg03792768 markers in major normal and cancer tissues are shown: bladder (BL), breast (BR), cervix (CE), colon (CO), esophagus (ES), glioblastoma (GB), head and neck (HN), kidney (KI), liver (LI), lung (LU), pancreas (PA), paraganglioma (PC), prostate (PR), rectum (RE), sarcoma (SA), skin (SK), stomach (ST), thymus (TH), uterus (UC), and blood (B). DETAILED DESCRIPTION

[0044] Hereinafter, the present invention will be described in more detail through one or more specific embodiments. However, these embodiments are intended to illustrate one or more specific embodiments, but the scope of the present invention is not limited by these embodiments.

[0045] Example 1: Discovery of liver tissue-specific methylation markers

[0046] DNA methylation data of various cancers and normal tissues were downloaded from The Cancer Genome Atlas (hereinafter referred to as TCGA). From the data downloaded from TCGA, methylated CpG sites were selected from liver cancer tissue samples (n=379), while non-methylated CpG sites were selected from cancer samples of other tissues (n=7260). The standard is to classify as methylated when 50% or more of the samples are methylated, and to classify as unmethylated when 90% or more of the samples are not methylated.

[0047] Then, we further selected the unmethylated CpG sites in 90% of cancer samples (pan-tumor) except liver cancer, and in 90% of normal tissues (pan-normal) except liver.

[0048] exist Figure 1 , the process of discovering the liver tissue-specific marker of the present invention is schematically shown.

[0049] Example 2: Verification of the performance of liver tissue-specific methylation markers

[0050] The variable importance map (VarImp map) of liver tissue-specific markers found in Example 1 above was produced by random forest method, such as Figure 2 As shown. Figure 2In the figure, the mean decrease in accuracy (MeanDecreaseAccuracy) on the X-axis indicates the degree to which each marker helps improve the classification accuracy of liver tissue / other tissues, and the mean decrease in the Gini index (MeanDecreaseGini) on the Y-axis indicates the degree to which each marker helps improve the impurity in the classification of liver tissue / other tissues. In other words, a larger value means that the marker can clearly distinguish liver tissue from other tissues.

[0051] Then, the liver tissue-specific marker was tested by randomly dividing the methylation data of the cancer tissue used in Example 1 into training data and validation data. As a result, as shown in the following Table 1, it can be confirmed that the discovered marker distinguishes liver tissue from other tissues with high efficiency and accuracy. The performance of the liver tissue-specific marker showed an accuracy of 0.9988, a sensitivity of 0.9884, a specificity of 0.9994, and an area under the curve (AUC) of 0.9999.

[0052] [Table 1]

[0053]

[0054] In addition, in order to confirm the performance according to the number of markers, 5 tests were performed with different numbers of markers and the average value was confirmed. The results are shown in Table 2 and Figure 3 As shown, it was found that when two or more markers were used, the performance converged to 0.999.

[0055] [Table 2]

[0056] 1 marker 2 markers 3 markers 4 markers 5 markers 6 markers Accuracy 0.9916 0.9976 0.99736 0.99784 0.99796 0.9982 Kappa 0.9142 0.97526 0.97264 0.97762 0.97886 0.98142 Sensitivity 0.923036 0.969686 0.965034 0.96963 0.971954 0.976718 Specificity 0.995338 0.999118 0.999118 0.99937 0.99937 0.999362 Positive Predicted Value 0.915048 0.983764 0.983764 0.988172 0.988318 0.988428 Negative predictive value 0.995844 0.998364 0.998112 0.998364 0.99849 0.998742 Balanced accuracy 0.959188 0.984402 0.982078 0.9845 0.985664 0.988048

[0057] In addition, the performance of two markers (cg12137206, cg03792768) found to be highly important in the variable importance map was further verified. As a result, as shown in Table 3 below, it can be confirmed that these markers clearly distinguish liver tissue from other tissues. The results showed that the accuracy was 0.9982, the sensitivity was 0.9647, the specificity was 1, and the area under the curve (AUC) was 0.9939.

[0058] [Table 3]

[0059]

[0060] Based on the above results, cg12137206 and cg03792768 markers were finally selected as liver tissue specific markers. In Tables 4 and 5 below, the information and sequences of cg12137206 and cg03792768 markers are described.

[0061] [Table 4]

[0062]

[0063] [Table 5]

[0064]

[0065] - In the table, [CG] refers to the target methylation site for each probe.

[0066] Example 3: Validation of the final selected liver tissue-specific markers

[0067] The methylation levels of the finally selected cg12137206 and cg03792768 markers in living tissues and other tissues were confirmed. Figure 4 As shown in A, it can be seen that in normal liver tissue (liver N) and liver cancer tissue (liver T), the two markers are methylated at high levels, such as Figure 4 As shown in B, it can be seen that in all other cancer tissues and other normal tissues, the two markers have low methylation levels.

[0068] exist Figures 5 to 7 , the methylation levels of the cg12137206 and cg03792768 markers confirmed in liver tissue and other tissues are shown, and the cross-validation results of the markers are described in Tables 6 to 8.

[0069] [Table 6]

[0070]

[0071] [Table 7]

[0072] organize TCGA Sample size Accuracy_N Accuracy_T Bladder (BL) BLCA 434 1 1 Breast (BR) BRCA 869 1 1 Cervix (CE) CESC 312 1 1 Colon (CO) COAD 335 1 1 Rectal (RE) READ 106 1 1 Esophagus (ES) ESCA 202 1 1 Glioblastoma (GB) GBM 154 1 1 Head and Neck (HN) HNSC 580 1 1 Kidney (KI) KIRC 480 1 1 Kidney (KI) KIRP 321 1 1 Lung (LU) LUAD 492 1 1 Lung (LU) LUSC 412 1 1 Pancreas (PA) PAAD 195 1 1 Paraganglioma (PC) PCPG 187 1 1 Prostate (PR) PRAD 549 1 1 Stomach (ST) STAD 397 1 1 Sarcoma (SA) SARC 269 1 1 Skin (SK) SKCM 475 1 1 Thymus (TH) THCA 574 1 1 Thymus (TH) THYM 126 1 1 Uterus (UC) UCEC 466 1 1

[0073] [Table 8]

[0074]

[0075] From the current results, it can be seen that the cg12137206 and cg03792768 markers have high methylation levels in liver tissue and low methylation levels in other tissues, so these two markers can be used as liver tissue-specific markers.

Claims

1. Use of a reagent in the preparation of a composition for performing a method for determining whether a biological sample is derived from liver tissue, wherein: The reagents include a reagent capable of measuring the methylation level of a CpG site located at nucleotides 61-62 in the sequence shown in SEQ ID NO: 1 and a reagent capable of measuring the methylation level of a CpG site located at nucleotides 61-62 in the sequence shown in SEQ ID NO: 2, wherein the method includes: (a) isolating DNA from a biological sample isolated from a subject; and (b) Measuring the methylation level of the CpG site located at nucleotides 61-62 in the sequence shown in SEQ ID NO: 1 and the methylation level of the CpG site located at nucleotides 61-62 in the sequence shown in SEQ ID NO: 2 in the separated DNA.

2. The use according to claim 1, wherein The biological sample is selected from the group consisting of tissue, tissue fragments, cells, cell fragments, blood, plasma, feces and urine isolated from a subject.

3. The use according to claim 1, wherein The biological sample is a bodily fluid isolated from a subject.

4. The use according to claim 1, wherein (b) is performed by a method selected from the group consisting of PCR, EpiTYPER, DNA chip, molecular beacon, MS-HRM (methylation-sensitive high resolution melting), recombinase polymerase amplification, Eclipse probe, next generation sequencing panel (NGS panel), pyrosequencing and disulfide sequencing.

5. The use according to claim 4, wherein The PCR is methylation-specific PCR or quantitative PCR.

6. The use according to claim 4, wherein The PCR is MehtyLight PCR, asymmetric PCR, LAMP (loop-mediated isothermal amplification) or real-time methylation-specific PCR.

7. The use according to claim 4, wherein The PCR was MehtyLight digital PCR.

8. The use according to claim 4, wherein The MS-HRM is asymmetric PCR MS-HRMA (asymmetric PCR methylation-sensitive high resolution melting analysis).

9. Use of a reagent in the preparation of a composition for performing a method for detecting DNA derived from liver tissue in a biological sample, wherein: The reagents include a reagent capable of measuring the methylation level of a CpG site located at nucleotides 61-62 in the sequence shown in SEQ ID NO: 1 and a reagent capable of measuring the methylation level of a CpG site located at nucleotides 61-62 in the sequence shown in SEQ ID NO: 2, wherein the method includes: (a) isolating DNA from a biological sample isolated from a subject; and (b) Measuring the methylation level of the CpG site located at nucleotides 61-62 in the sequence shown in SEQ ID NO: 1 and the methylation level of the nucleotides 61-62 in the sequence shown in SEQ ID NO: 2 in the separated DNA.

10. A composition for determining whether a biological sample is derived from liver tissue, comprising a reagent capable of measuring the methylation level of nucleotides at positions 61-62 in the sequence shown in SEQ ID NO: 1 and a reagent capable of measuring the methylation level of a CpG site at positions 61-62 in the sequence shown in SEQ ID NO:

2.

11. The composition for determining whether a biological sample is derived from liver tissue according to claim 10, wherein: The reagent capable of measuring the methylation level is a primer, a probe or an antisense nucleic acid.

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