Kit for diagnosis of gastrointestinal or cutaneous graft-versus-host disease, diagnostic device, and method for determining possibility of affliction with the disease

Abstract

To provide a kit for diagnosis of gastrointestinal or cutaneous graft-versus-host disease, the kit utilizing a novel diagnostic method for gastrointestinal or cutaneous graft-versus-host disease.SOLUTION: There is provided a kit for diagnosis of gastrointestinal or cutaneous graft-versus-host disease, comprising: a nucleic acid composed of at least one miRNA selected from the group consisting of (A) miR-638, miR-1246, miR-762, miR-3648, miR-4497, miR-4508, miR-3960, miR-4732-5p, miR-4739, miR-4745-5p, miR-4787-5p, miR-204-3p, miR-1233-5p, miR-5787, miR-6089, miR-6090, miR-6131, miR-6786-5p, miR-6787-5p, miR-6869-5p, miR-7704, and miR-8072, or a base sequence complementary to the miRNA; a nucleic acid capable of specifically binding thereto, or a nucleic acid composed of at least one miRNA selected from the group consisting of(B) miR-1246, miR-762, miR-3648, miR-4488, miR-4497, miR-4508, miR-3960, miR-4732-5p, miR-4739, miR-4745-5p, miR-4787-5p, miR-197-5p, miR-204-3p, miR-1233-5p, miR-5787, miR-6089, miR-6090, miR-6131, miR-6727-5p, miR-6786-5p, miR-6869-5p, and miR-7704, or a base sequence complementary to the miRNA; and a nucleic acid capable of specifically binding thereto.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to a diagnostic kit for gastrointestinal or skin graft-versus-host disease, a diagnostic device for gastrointestinal or skin graft-versus-host disease, a method for determining the possibility of gastrointestinal or skin graft-versus-host disease, etc. The contents of all documents described in this specification are incorporated herein by reference. [Background technology]

[0002] Allogeneic transplantation is the only curative treatment for refractory acute leukemia. Currently, the 5-year survival rates after allogeneic transplantation for standard-risk acute leukemia and refractory acute leukemia are approximately 40% and 20%, respectively. However, approximately 50% of deaths are due to relapse, 25% to graft-versus-host disease (GVHD), and 25% to transplant-related complications including infection. Therefore, new developments in diagnostic and therapeutic methods for these diseases will lead to improved survival rates.

[0003] Previous studies have reported numerous biomarkers for diagnosing and predicting GVHD, but none have been used in clinical practice. Currently, a definitive diagnosis of GVHD requires gastrointestinal endoscopic biopsy, skin biopsy, or liver biopsy. However, these invasive procedures are particularly burdensome in cases where the patient's general condition is poor. Therefore, a clinical diagnosis is often made based on clinical symptoms alone, without biopsy. Furthermore, histopathological diagnosis requires the expertise of a pathologist.

[0004] In recent years, it has been reported that microRNAs (miRNAs) in exosomes can be biomarkers for various diseases. There have been some reports in the field of hematopoietic cell transplantation, but no established results have been obtained.

[0005] As a diagnostic method using such miRNA, Patent Document 1 reports the discovery of a gene from blood that can be used as a prostate cancer detection marker. Specifically, Patent Document 1 describes a method for detecting prostate cancer, which includes measuring the expression level of at least one polynucleotide selected from the group consisting of prostate cancer markers miR-1185-2-3p, miR-1185-1-3p, miR-197-5p, and miR-6076 in a specimen from a subject, and using the measured expression level to evaluate in vitro whether or not the subject is suffering from prostate cancer. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2020 / 032228 Summary of the Invention [Problem to be solved by the invention]

[0007] The present disclosure aims to provide a diagnostic kit and diagnostic device for gastrointestinal or skin graft-versus-host disease, and a method for determining the possibility of suffering from gastrointestinal or skin graft-versus-host disease, using a novel diagnostic method for gastrointestinal or skin graft-versus-host disease. [Means for solving the problem]

[0008] As a result of extensive research conducted by the inventors to achieve the above-mentioned object, they discovered that gastrointestinal GVHD cases can be distinguished from non-GVHD cases by utilizing expression information of 22 miRNAs: miR-638, miR-1246, miR-762, miR-3648, miR-4497, miR-4508, miR-3960, miR-4732-5p, miR-4739, miR-4745-5p, miR-4787-5p, miR-204-3p, miR-1233-5p, miR-5787, miR-6089, miR-6090, miR-6131, miR-6786-5p, miR-6787-5p, miR-6869-5p, miR-7704, and miR-8072. Furthermore, we found that by utilizing expression information of 22 miRNAs, namely miR-1246, miR-762, miR-3648, miR-4488, miR-4497, miR-4508, miR-3960, miR-4732-5p, miR-4739, miR-4745-5p, miR-4787-5p, miR-197-5p, miR-204-3p, miR-1233-5p, miR-5787, miR-6089, miR-6090, miR-6131, miR-6727-5p, miR-6786-5p, miR-6869-5p, and miR-7704, it is possible to distinguish between cases of cutaneous GVHD and cases without GVHD.

[0009] The present disclosure was completed based on these findings and further investigations, and includes, for example, the subject matter described in the following sections. Item 1. (A) A nucleic acid capable of specifically binding to at least one miRNA selected from the group consisting of miR-638, miR-1246, miR-762, miR-3648, miR-4497, miR-4508, miR-3960, miR-4732-5p, miR-4739, miR-4745-5p, miR-4787-5p, miR-204-3p, miR-1233-5p, miR-5787, miR-6089, miR-6090, miR-6131, miR-6786-5p, miR-6787-5p, miR-6869-5p, miR-7704, and miR-8072, or a nucleic acid comprising a nucleotide sequence complementary to the miRNA; or (B) a nucleic acid capable of specifically binding to at least one miRNA selected from the group consisting of miR-1246, miR-762, miR-3648, miR-4488, miR-4497, miR-4508, miR-3960, miR-4732-5p, miR-4739, miR-4745-5p, miR-4787-5p, miR-197-5p, miR-204-3p, miR-1233-5p, miR-5787, miR-6089, miR-6090, miR-6131, miR-6727-5p, miR-6786-5p, miR-6869-5p, and miR-7704, or a nucleic acid having a base sequence complementary to the miRNA. A diagnostic kit for gastrointestinal or skin graft-versus-host disease, comprising: Item 2. (A1) A nucleic acid capable of specifically binding to at least one miRNA selected from the group consisting of miR-1246, miR-3648, miR-3960, miR-4732-5p, miR-4787-5p, miR-1233-5p, and miR-7704, or a nucleic acid having a base sequence complementary to the miRNA; or (B1) A nucleic acid capable of specifically binding to at least one miRNA selected from the group consisting of miR-4488, miR-3960, miR-4739, miR-4787-5p, miR-1233-5p, miR-6089, miR-6090, miR-6727-5p, and miR-7704, or a nucleic acid having a base sequence complementary to the miRNA. Item 1. The kit according to Item 1, comprising: Item 3. The kit according to Item 1 or 2, which is used for diagnosing gastrointestinal or skin graft-versus-host disease after allogeneic hematopoietic stem cell transplantation. Item 4. (A) A nucleic acid capable of specifically binding to at least one miRNA selected from the group consisting of miR-638, miR-1246, miR-762, miR-3648, miR-4497, miR-4508, miR-3960, miR-4732-5p, miR-4739, miR-4745-5p, miR-4787-5p, miR-204-3p, miR-1233-5p, miR-5787, miR-6089, miR-6090, miR-6131, miR-6786-5p, miR-6787-5p, miR-6869-5p, miR-7704, and miR-8072, or a nucleic acid comprising a nucleotide sequence complementary to the miRNA; or (B) a nucleic acid capable of specifically binding to at least one miRNA selected from the group consisting of miR-1246, miR-762, miR-3648, miR-4488, miR-4497, miR-4508, miR-3960, miR-4732-5p, miR-4739, miR-4745-5p, miR-4787-5p, miR-197-5p, miR-204-3p, miR-1233-5p, miR-5787, miR-6089, miR-6090, miR-6131, miR-6727-5p, miR-6786-5p, miR-6869-5p, and miR-7704, or a nucleic acid having a base sequence complementary to the miRNA. A device for diagnosing gastrointestinal or skin graft-versus-host disease, comprising: Item 5. (A1) A nucleic acid capable of specifically binding to at least one miRNA selected from the group consisting of miR-1246, miR-3648, miR-3960, miR-4732-5p, miR-4787-5p, miR-1233-5p, and miR-7704, or a nucleic acid having a base sequence complementary to the miRNA; or (B1) A nucleic acid capable of specifically binding to at least one miRNA selected from the group consisting of miR-4488, miR-3960, miR-4739, miR-4787-5p, miR-1233-5p, miR-6089, miR-6090, miR-6727-5p, and miR-7704, or a nucleic acid having a base sequence complementary to the miRNA. Item 5. The device according to item 4, comprising: Item 6. The device according to Item 4 or 5, which is used to diagnose gastrointestinal or skin graft-versus-host disease after allogeneic hematopoietic stem cell transplantation. Item 7. A method for determining the possibility of gastrointestinal or skin graft-versus-host disease, comprising the following steps: (1) In samples derived from body fluids collected from subjects (A) the expression level of at least one miRNA selected from the group consisting of miR-638, miR-1246, miR-762, miR-3648, miR-4497, miR-4508, miR-3960, miR-4732-5p, miR-4739, miR-4745-5p, miR-4787-5p, miR-204-3p, miR-1233-5p, miR-5787, miR-6089, miR-6090, miR-6131, miR-6786-5p, miR-6787-5p, miR-6869-5p, miR-7704, and miR-8072; or (B) Expression level of at least one miRNA selected from the group consisting of miR-1246, miR-762, miR-3648, miR-4488, miR-4497, miR-4508, miR-3960, miR-4732-5p, miR-4739, miR-4745-5p, miR-4787-5p, miR-197-5p, miR-204-3p, miR-1233-5p, miR-5787, miR-6089, miR-6090, miR-6131, miR-6727-5p, miR-6786-5p, miR-6869-5p, and miR-7704. A process of measuring Item 8. (A1) The expression level of at least one miRNA selected from the group consisting of miR-1246, miR-3648, miR-3960, miR-4732-5p, miR-4787-5p, miR-1233-5p, and miR-7704, or (B1) Expression level of at least one miRNA selected from the group consisting of miR-4488, miR-3960, miR-4739, miR-4787-5p, miR-1233-5p, miR-6089, miR-6090, miR-6727-5p, and miR-7704 The method according to item 7, wherein the Item 9. (2) determining the possibility of gastrointestinal or cutaneous graft-versus-host disease based on the expression level measured in step (1); Item 9. The method according to Item 7 or 8, comprising: Item 10. The method according to any one of Items 7 to 9, wherein in the step (1), the expression level of miRNA is measured using the kit according to Item 1 or 2, or the device according to Item 4 or 5. Item 11. The method according to any one of Items 7 to 10, wherein the gastrointestinal or cutaneous graft-versus-host disease is gastrointestinal or cutaneous graft-versus-host disease after allogeneic hematopoietic stem cell transplantation. Item 12. The method according to any one of Items 7 to 11, wherein the body fluid is serum, plasma, saliva, or urine. Item 13. The method according to any one of Items 7 to 12, wherein the body fluid-derived sample is extracellular vesicles derived from body fluid. Item 14. The method according to any one of Items 7 to 13, wherein the subject is a human. [Effects of the Invention]

[0010] The present disclosure provides a novel and effective means for diagnosing gastrointestinal or cutaneous graft-versus-host disease. [Brief explanation of the drawings]

[0011] [Figure 1] Figure 1 shows violin plots of the analysis results of expression information on 22 miRNAs in severe gastrointestinal GVHD (12 control cases on the left, 12 gastrointestinal GVHD cases at stage 3 or higher on the right). [Figure 2] This figure shows the receiver operating characteristic curve (ROC) curve for the classification of severe gastrointestinal GVHD using expression information of 22 miRNAs. The area under the ROC curve (AUC) was 0.94 (95% CI, 0.81-1.00). [Figure 3-1] FIG. 3 shows ROC curves obtained by analyzing the expression levels of each miRNA in the analysis of FIG. 2. [Figure 3-2]FIG. 3 shows ROC curves obtained by analyzing the expression levels of each miRNA in the analysis of FIG. 2. [Figure 4-1] FIG. 2 shows violin plots of the results of the analysis of FIG. 1 for the expression levels of each miRNA. [Figure 4-2] FIG. 2 shows violin plots of the results of the analysis of FIG. 1 for the expression levels of each miRNA. [Figure 5] FIG. 1 shows a violin plot of the analysis results based on expression information of 22 miRNAs in severe cutaneous GVHD (left: 12 cases of cutaneous GVHD at stage 3 or higher, right: 12 control cases). [Figure 6] This figure shows the ROC curve for the case in which severe cutaneous GVHD was discriminated using expression information of 22 miRNAs. The area under the ROC curve (AUC) was 0.81 (95% CI, 0.62-1.00). [Figure 7-1] FIG. 7 shows ROC curves obtained by analyzing the expression levels of each miRNA in the analysis of FIG. 6. [Figure 7-2] FIG. 7 shows ROC curves obtained by analyzing the expression levels of each miRNA in the analysis of FIG. 6. [Figure 8-1] FIG. 6 shows violin plots of the results of the analysis of FIG. 5 for the expression levels of each miRNA. [Figure 8-2] FIG. 6 shows violin plots of the results of the analysis of FIG. 5 for the expression levels of each miRNA. DETAILED DESCRIPTION OF THE INVENTION

[0012] Each embodiment of the present disclosure will be described in more detail below. The present disclosure preferably includes, but is not limited to, a kit for diagnosing gastrointestinal or cutaneous graft-versus-host disease, and the like. The present disclosure includes all that is disclosed herein and that would be recognized by a person skilled in the art.

[0013] In this disclosure, "nucleic acid," "nucleotide," and "polynucleotide" are synonymous and include both DNA and RNA, which may be double-stranded or single-stranded.

[0014] In addition, in this disclosure, "miRNA" refers to a 15-25 base non-coding RNA that is transcribed as a hairpin-like RNA precursor, cleaved by a dsRNA cleaving enzyme with RNase III cleavage activity, incorporated into a protein complex called RISC, and involved in the translational repression of mRNA. "miRNA" not only refers to "miRNA" represented by a specific base sequence, but also encompasses precursors of the "miRNA" (pre-miRNA, pri-miRNA) and miRNAs with biological functions equivalent to the "miRNA."

[0015] In the present disclosure, "extracellular vesicles" refer to tiny vesicles with a lipid bilayer structure that are secreted, released, or otherwise formed by various types of cells. Extracellular vesicles are secreted from various cells and contain functional molecules such as proteins, microRNA, and mRNA, and are known to function as intermediaries for intercellular communication within the body. Examples of extracellular vesicles include exosomes, microvesicles, and apoptotic bodies.

[0016] In this disclosure, "exosome" refers to a lipid bilayer-enclosed vesicle secreted from cells. Exosomes are derived from multivesicular endosomes and may contain biological substances such as RNA, DNA, and proteins when released into the extracellular environment.

[0017] In this disclosure, the term "probe" refers to a nucleic acid used to specifically detect RNA produced by gene expression or a nucleic acid derived therefrom. Also, in this disclosure, the term "primer" refers to a nucleic acid that specifically recognizes and amplifies RNA produced by gene expression or a nucleic acid derived therefrom.

[0018] In the present disclosure, "graft-versus-host disease" refers to an immune response in which a transplanted organ, tissue, or cell attacks, damages, or destroys the cells, tissue, or organ of the recipient.

[0019] In this disclosure, "leukemia" refers to a disease in which tumorigenic hematopoietic cells proliferate indefinitely and appear in the blood. Leukemia in which tumor cells have lost their ability to differentiate is called acute leukemia, while leukemia in which tumor cells retain their ability to differentiate is called chronic leukemia. Furthermore, when the cells that originated the tumor are myeloid cells, it is classified as myeloid leukemia, and when the cells that originated the tumor are lymphocytic cells, it is classified as lymphocytic leukemia.

[0020] In the present disclosure, the term "capable of specifically binding" means that the nucleic acid used in the present disclosure binds to a specific target nucleic acid and is substantially incapable of binding to other nucleic acids.

[0021] The method for determining the possibility of gastrointestinal or cutaneous graft-versus-host disease of the present disclosure (hereinafter sometimes referred to as the "determination method of the present disclosure") is characterized by comprising the following steps. (1) In samples derived from body fluids collected from subjects (A) the expression level of at least one miRNA selected from the group consisting of miR-638, miR-1246, miR-762, miR-3648, miR-4497, miR-4508, miR-3960, miR-4732-5p, miR-4739, miR-4745-5p, miR-4787-5p, miR-204-3p, miR-1233-5p, miR-5787, miR-6089, miR-6090, miR-6131, miR-6786-5p, miR-6787-5p, miR-6869-5p, miR-7704, and miR-8072; or (B) Expression level of at least one miRNA selected from the group consisting of miR-1246, miR-762, miR-3648, miR-4488, miR-4497, miR-4508, miR-3960, miR-4732-5p, miR-4739, miR-4745-5p, miR-4787-5p, miR-197-5p, miR-204-3p, miR-1233-5p, miR-5787, miR-6089, miR-6090, miR-6131, miR-6727-5p, miR-6786-5p, miR-6869-5p, and miR-7704. A process of measuring

[0022] The gastrointestinal or cutaneous graft-versus-host disease to be assessed is not particularly limited, and particularly includes gastrointestinal or cutaneous graft-versus-host disease after allogeneic hematopoietic stem cell transplantation. Allogeneic hematopoietic stem cell transplantation is performed, for example, in the treatment of leukemia (particularly refractory leukemia). The stage of the gastrointestinal or cutaneous graft-versus-host disease to be assessed is not particularly limited, and may be any stage, for example, stage I, stage II, stage III, and stage IV.

[0023] The subject is a living organism that is the target of the determination method of the present invention, and the species of the subject is not particularly limited. Examples of the subject include various mammals such as humans, monkeys, chimpanzees, mice, rats, dogs, cats, rabbits, cows, horses, sheep, and goats, and preferably humans.

[0024] The body fluid is not particularly limited, and examples thereof include urine, whole blood, serum, plasma, cerebrospinal fluid, saliva, synovial fluid, tissue fluid, sweat, tears, sputum, and nasal discharge. Preferred are serum, plasma, saliva, and urine, and more preferred is serum.

[0025] The body fluid-derived sample may be the body fluid itself, or may be a sample obtained by subjecting the body fluid to some manipulation (separation, purification, etc.). The body fluid-derived sample is preferably extracellular vesicles derived from body fluid, more preferably exosomes, and even more preferably serum exosomes. The body fluid-derived sample may be used singly or in combination of two or more types.

[0026] Body fluids can be collected from a subject by methods known to those skilled in the art. For example, whole blood can be collected by drawing blood using a syringe or the like. It is desirable that blood be collected by a medical professional such as a doctor or nurse. Serum is a portion of blood from which blood cells and specific blood coagulation factors have been removed, and can be obtained, for example, as the supernatant after blood clotting.

[0027] Extracellular vesicles can be purified, separated, and concentrated from body fluids according to or in accordance with known methods. Methods for purifying, separating, concentrating, etc. of extracellular vesicles include, for example, ultracentrifugation, polymer precipitation, immunoprecipitation, etc. Purification, separation, concentration, etc. of extracellular vesicles can also be performed using commercially available kits. These methods may be used alone or in combination of two or more.

[0028] The method for extracting miRNA from body fluid samples may be the commonly used acid phenol method (Acid Guanidinium-Phenol-Chloroform (AGPC) method), or commercially available reagents and kits such as Trizol (Life Technologies), Isogen (Nippon Gene Co., Ltd.), 3D-Gene (registered trademark) RNA extraction reagent (Toray Industries, Inc.), and miRNeasy (registered trademark) Mini Kit (Qiagen).

[0029] In the determination method of the present disclosure, in order to determine the possibility of gastrointestinal graft-versus-host disease, miR-638, miR-1246, miR-762, miR-3648, miR-4497, miR-4508, miR-3960, miR-4732-5p, miR-4739, miR-4745-5p, miR-4787-5p, miR-204-3p, miR-12 The expression level of at least one miRNA (hereinafter sometimes referred to as "target miRNA (I)") selected from the group consisting of miR-33-5p, miR-5787, miR-6089, miR-6090, miR-6131, miR-6786-5p, miR-6787-5p, miR-6869-5p, miR-7704, and miR-8072 is measured.

[0030] Target miRNAs (I) are human miRNAs (i.e., hsa-miR-638, hsa-miR-1246, hsa-miR-762, hsa-miR-3648, hsa-miR-4497, hsa-miR-4508, hsa-miR-3960, hsa-miR-4732-5p, hsa-miR-4739, hsa-miR-4745-5p, and hsa-miR-478, respectively). Preferably, the miR-1233-5p, hsa-miR-5787, hsa-miR-6089, hsa-miR-6090, hsa-miR-6131, hsa-miR-6786-5p, hsa-miR-6787-5p, hsa-miR-6869-5p, hsa-miR-7704, and hsa-miR-8072).

[0031] In the determination method of the present disclosure, miR-1246, miR-762, miR-3648, miR-4488, miR-4497, miR-4508, miR-3960, miR-4732-5p, miR-4739, miR-4745-5p, miR-4787-5p, miR-197-5p, miR-204-3p, miR-1233-5p, miR-5787, miR-5787, miR-5787-5p ... The expression level of at least one miRNA selected from the group consisting of miR-6089, miR-6090, miR-6131, miR-6727-5p, miR-6786-5p, miR-6869-5p, and miR-7704 (hereinafter, also referred to as "target miRNA(II)", and target miRNA(I) and target miRNA(II) may be collectively referred to as "target miRNA") is measured.

[0032] Target miRNAs (II) were human miRNAs (i.e., hsa-miR-1246, hsa-miR-762, hsa-miR-3648, hsa-miR-4488, hsa-miR-4497, hsa-miR-4508, hsa-miR-3960, hsa-miR-4732-5p, hsa-miR-4739, hsa-miR-4745-5p, and hsa-miR-478, respectively). Preferably, the miR-1 and miR-2 are selected from the group consisting of miR-7-5p, hsa-miR-197-5p, hsa-miR-204-3p, hsa-miR-1233-5p, hsa-miR-5787, hsa-miR-6089, hsa-miR-6090, hsa-miR-6131, hsa-miR-6727-5p, hsa-miR-6786-5p, hsa-miR-6869-5p, and hsa-miR-7704.

[0033] The base sequence etc. of the target miRNA can be identified using a publicly known database (for example, miRBase: http: / / www.mirbase.org / ).

[0034] "hsa-miR-638" as used in the present disclosure has the nucleotide sequence set forth in SEQ ID NO: 1 (miRBase Accession No. MIMAT0003308), and may be a variant of the miRNA having the nucleotide sequence of SEQ ID NO: 1, so long as it has an equivalent biological function. Furthermore, "hsa-miR-638" is known to have a precursor, "hsa-mir-638" (miRBase Accession No. MI0003653, SEQ ID NO: 26), which has a hairpin-like structure.

[0035] "hsa-miR-1246" as used in the present disclosure has the nucleotide sequence set forth in SEQ ID NO: 2 (miRBase Accession No. MIMAT0005898), and may be a variant of the miRNA having the nucleotide sequence of SEQ ID NO: 2, so long as it has an equivalent biological function. Furthermore, "hsa-miR-1246" is known to have a precursor, "hsa-mir-1246" (miRBase Accession No. MI0006381, SEQ ID NO: 27), which has a hairpin-like structure.

[0036] "hsa-miR-762" as used in the present disclosure has the nucleotide sequence set forth in SEQ ID NO: 3 (miRBase Accession No. MIMAT0010313), and may be a variant of the miRNA having the nucleotide sequence of SEQ ID NO: 3, so long as it has an equivalent biological function. Furthermore, "hsa-miR-762" is known to have a precursor, "hsa-mir-762" (miRBase Accession No. MI0003892, SEQ ID NO: 28), which has a hairpin-like structure.

[0037] "hsa-miR-3648" as used in the present disclosure has the nucleotide sequence set forth in SEQ ID NO: 4 (miRBase Accession No. MIMAT0018068), and may be a variant of the miRNA having the nucleotide sequence of SEQ ID NO: 4, so long as it has an equivalent biological function. Also known as "hsa-miR-3648" are precursors that have a hairpin-like structure, "hsa-mir-3648-1" (miRBase Accession No. MI0016048, SEQ ID NO: 29) and "hsa-mir-3648-2" (miRBase Accession No. MI0031512, SEQ ID NO: 30).

[0038] "hsa-miR-4497" as used in the present disclosure has the nucleotide sequence set forth in SEQ ID NO: 5 (miRBase Accession No. MIMAT0019032), and may be a variant of the miRNA having the nucleotide sequence of SEQ ID NO: 5, so long as it has an equivalent biological function. Furthermore, "hsa-miR-4497" is known to have a precursor, "hsa-mir-4497" (miRBase Accession No. MI0016859, SEQ ID NO: 31), which has a hairpin-like structure.

[0039] "hsa-miR-4488" as used in the present disclosure has the nucleotide sequence set forth in SEQ ID NO: 6 (miRBase Accession No. MIMAT0019022), and may be a variant of the miRNA having the nucleotide sequence of SEQ ID NO: 6, so long as it has an equivalent biological function. Furthermore, "hsa-miR-4488" is known to have a precursor, "hsa-mir-4488" (miRBase Accession No. MI0016849, SEQ ID NO: 32), which has a hairpin-like structure.

[0040] "hsa-miR-4508" as used in the present disclosure has the nucleotide sequence set forth in SEQ ID NO: 7 (miRBase Accession No. MIMAT0019045), and may be a variant of the miRNA having the nucleotide sequence of SEQ ID NO: 7, so long as it has an equivalent biological function. Furthermore, "hsa-miR-4508" is known to have a precursor, "hsa-mir-4508" (miRBase Accession No. MI0016872, SEQ ID NO: 33), which has a hairpin-like structure.

[0041] "hsa-miR-3960" as used in the present disclosure has the nucleotide sequence set forth in SEQ ID NO: 8 (miRBase Accession No. MIMAT0019337), and may be a variant of the miRNA having the nucleotide sequence of SEQ ID NO: 8, so long as it has an equivalent biological function. Furthermore, "hsa-miR-3960" is known to have a precursor, "hsa-mir-3960" (miRBase Accession No. MI0016964, SEQ ID NO: 34), which has a hairpin-like structure.

[0042] "hsa-miR-4732-5p" as used in the present disclosure has the nucleotide sequence set forth in SEQ ID NO: 9 (miRBase Accession No. MIMAT0019855), and may be a variant of the miRNA having the nucleotide sequence of SEQ ID NO: 9, so long as it has an equivalent biological function. Furthermore, "hsa-miR-4732-5p" is known to have a precursor, "hsa-mir-4732" (miRBase Accession No. MI0017369, SEQ ID NO: 35), which has a hairpin-like structure.

[0043] "hsa-miR-4739" as used in the present disclosure has the nucleotide sequence set forth in SEQ ID NO: 10 (miRBase Accession No. MIMAT0019868), and may be a variant of the miRNA having the nucleotide sequence of SEQ ID NO: 10, so long as it has an equivalent biological function. Furthermore, "hsa-miR-4739" is known to have a precursor, "hsa-mir-4739" (miRBase Accession No. MI0017377, SEQ ID NO: 36), which has a hairpin-like structure.

[0044] "hsa-miR-4745-5p" as used in the present disclosure has the nucleotide sequence set forth in SEQ ID NO: 11 (miRBase Accession No. MIMAT0019878), and may be a variant of the miRNA having the nucleotide sequence of SEQ ID NO: 11, so long as it has an equivalent biological function. Furthermore, "hsa-miR-4745-5p" is known to have a precursor, "hsa-mir-4745" (miRBase Accession No. MI0017384, SEQ ID NO: 37), which has a hairpin-like structure.

[0045] "hsa-miR-4787-5p" as used in the present disclosure has the nucleotide sequence set forth in SEQ ID NO: 12 (miRBase Accession No. MIMAT0019956), and may be a variant of the miRNA having the nucleotide sequence of SEQ ID NO: 12, so long as it has an equivalent biological function. Furthermore, "hsa-miR-4787-5p" has a known precursor, "hsa-mir-4787" (miRBase Accession No. MI0017434, SEQ ID NO: 38), which has a hairpin-like structure.

[0046] "hsa-miR-204-3p" used in the present disclosure has the nucleotide sequence set forth in SEQ ID NO: 13 (miRBase Accession No. MIMAT0022693), and may be a variant of the miRNA having the nucleotide sequence of SEQ ID NO: 13, so long as it has an equivalent biological function. Furthermore, "hsa-miR-204-3p" is known to have a precursor, "hsa-mir-204" (miRBase Accession No. MI0000284, SEQ ID NO: 39), which has a hairpin-like structure.

[0047] "hsa-miR-1233-5p" as used in the present disclosure has the nucleotide sequence set forth in SEQ ID NO: 14 (miRBase Accession No. MIMAT0022943), and may be a variant of the miRNA having the nucleotide sequence of SEQ ID NO: 14, so long as it has an equivalent biological function. Furthermore, "hsa-miR-1233-5p" has known precursors, "hsa-mir-1233-1" (miRBase Accession No. MI0006323, SEQ ID NO: 40) and "hsa-mir-1233-2" (miRBase Accession No. MI0015973, SEQ ID NO: 41), which have a hairpin-like structure.

[0048] "hsa-miR-5787" as used in the present disclosure has the nucleotide sequence set forth in SEQ ID NO: 15 (miRBase Accession No. MIMAT0023252), and may be a variant of the miRNA having the nucleotide sequence of SEQ ID NO: 15, so long as it has an equivalent biological function. Furthermore, "hsa-miR-5787" is known to have a precursor, "hsa-mir-5787" (miRBase Accession No. MI0019797, SEQ ID NO: 42), which has a hairpin-like structure.

[0049] "hsa-miR-6089" as used in the present disclosure has the nucleotide sequence set forth in SEQ ID NO: 16 (miRBase Accession No. MIMAT0023714), and may be a variant of the miRNA having the nucleotide sequence of SEQ ID NO: 16, so long as it has an equivalent biological function. Also known as "hsa-miR-6089" are "hsa-mir-6089-1" (miRBase Accession No. MI0020366, SEQ ID NO: 43) and "hsa-mir-6089-2" (miRBase Accession No. MI0023563, SEQ ID NO: 44), which have hairpin-like structures as precursors.

[0050] "hsa-miR-6090" as used in the present disclosure has the nucleotide sequence set forth in SEQ ID NO: 17 (miRBase Accession No. MIMAT0023715), and may be a variant of the miRNA having the nucleotide sequence of SEQ ID NO: 17, so long as it has an equivalent biological function. Furthermore, "hsa-miR-6090" is known to have a precursor, "hsa-mir-6090" (miRBase Accession No. MI0020367, SEQ ID NO: 45), which has a hairpin-like structure.

[0051] "hsa-miR-6131" as used in the present disclosure has the nucleotide sequence set forth in SEQ ID NO: 18 (miRBase Accession No. MIMAT0024615), and may be a variant of the miRNA having the nucleotide sequence of SEQ ID NO: 18, so long as it has an equivalent biological function. Furthermore, "hsa-miR-6131" is known to have a precursor, "hsa-mir-6131" (miRBase Accession No. MI0021276, SEQ ID NO: 46), which has a hairpin-like structure.

[0052] "hsa-miR-6786-5p" as used in the present disclosure has the nucleotide sequence set forth in SEQ ID NO: 19 (miRBase Accession No. MIMAT0027472), and may be a variant of the miRNA having the nucleotide sequence of SEQ ID NO: 19, so long as it has an equivalent biological function. Furthermore, "hsa-miR-6786-5p" is known to have a precursor, "hsa-mir-6786" (miRBase Accession No. MI0022631, SEQ ID NO: 47), which has a hairpin-like structure.

[0053] "hsa-miR-6787-5p" as used in the present disclosure has the nucleotide sequence set forth in SEQ ID NO: 20 (miRBase Accession No. MIMAT0027474), and may be a variant of the miRNA having the nucleotide sequence of SEQ ID NO: 20, so long as it has an equivalent biological function. Furthermore, "hsa-miR-6787-5p" has a known precursor, "hsa-mir-6787" (miRBase Accession No. MI0022632, SEQ ID NO: 48), which has a hairpin-like structure.

[0054] "hsa-miR-6727-5p" as used in the present disclosure has the nucleotide sequence set forth in SEQ ID NO: 21 (miRBase Accession No. MIMAT0027355), and may be a variant of the miRNA having the nucleotide sequence of SEQ ID NO: 21, so long as it has an equivalent biological function. Furthermore, "hsa-miR-6727-5p" has a known precursor, "hsa-mir-6727" (miRBase Accession No. MI0022572, SEQ ID NO: 49), which has a hairpin-like structure.

[0055] "hsa-miR-6869-5p" as used in the present disclosure has the nucleotide sequence set forth in SEQ ID NO: 22 (miRBase Accession No. MIMAT0027638), and may be a variant of the miRNA having the nucleotide sequence of SEQ ID NO: 22, so long as it has an equivalent biological function. Furthermore, "hsa-miR-6869-5p" has a known precursor, "hsa-mir-6869" (miRBase Accession No. MI0022716, SEQ ID NO: 50), which has a hairpin-like structure.

[0056] "hsa-miR-7704" as used in the present disclosure has the nucleotide sequence set forth in SEQ ID NO: 23 (miRBase Accession No. MIMAT0030019), and may be a variant of the miRNA having the nucleotide sequence of SEQ ID NO: 23, so long as it has an equivalent biological function. Furthermore, "hsa-miR-7704" is known to have a precursor, "hsa-mir-7704" (miRBase Accession No. MI0025240, SEQ ID NO: 51), which has a hairpin-like structure.

[0057] "hsa-miR-8072" as used in the present disclosure has the nucleotide sequence set forth in SEQ ID NO: 24 (miRBase Accession No. MIMAT0030999), and may be a variant of the miRNA having the nucleotide sequence of SEQ ID NO: 24, so long as it has an equivalent biological function. Furthermore, "hsa-miR-8072" is known to have a precursor, "hsa-mir-8072" (miRBase Accession No. MI0025908, SEQ ID NO: 52), which has a hairpin-like structure.

[0058] "hsa-miR-197-5p" used in the present disclosure has the nucleotide sequence set forth in SEQ ID NO: 25 (miRBase Accession No. MIMAT0022691), and may be a variant of the miRNA having the nucleotide sequence of SEQ ID NO: 25, so long as it has an equivalent biological function. Furthermore, "hsa-miR-197-5p" has a known precursor, "hsa-mir-197" (miRBase Accession No. MI0000239, SEQ ID NO: 53), which has a hairpin-like structure.

[0059] As used herein, the term "mutant" refers to, for example, a nucleic acid consisting of a base sequence that has 70% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the base sequences represented by SEQ ID NOs: 1 to 25.

[0060] The identity (%) of the nucleotide sequence can be calculated using an analytical tool that is commercially available or available via telecommunications lines (Internet). For example, it can be determined using the default settings of a program commonly used in the field, such as BLAST or FASTA.

[0061] In the determination method of the present disclosure, the expression levels of (A) miR-638, miR-1246, miR-762, miR-3648, miR-4497, miR-4508, miR-3960, miR-4732-5p, miR-4739, miR-4745-5p, miR-4787-5p, miR-204-3p, miR-1233-5p, miR-5787, miR-6089, miR-1233-5p ... At least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, or twenty-two types of miRNA selected from the group consisting of miR-6090, miR-6131, miR-6786-5p, miR-6787-5p, miR-6869-5p, miR-7704, and miR-8072.

[0062] Among these, it is particularly preferred to measure the expression levels of at least one, two, three, four, five, six, or seven miRNAs selected from the group consisting of (A1) miR-1246, miR-3648, miR-3960, miR-4732-5p, miR-4787-5p, miR-1233-5p, and miR-7704. It is desirable to measure the expression levels of the miRNAs in (A) above in a form that includes these miRNAs.

[0063] In the determination method of the present disclosure, the expression levels of (B) miR-1246, miR-762, miR-3648, miR-4488, miR-4497, miR-4508, miR-3960, miR-4732-5p, miR-4739, miR-4745-5p, miR-4787-5p, miR-197-5p, miR-204-3p, miR-1233-5p, and miR-5787 are measured. , miR-6089, miR-6090, miR-6131, miR-6727-5p, miR-6786-5p, miR-6869-5p, and miR-7704.

[0064] Among these, it is particularly preferred to measure the expression levels of at least one, two, three, four, five, six, seven, eight, or nine miRNAs selected from the group consisting of (B1) miR-4488, miR-3960, miR-4739, miR-4787-5p, miR-1233-5p, miR-6089, miR-6090, miR-6727-5p, and miR-7704. It is desirable to measure the expression levels of the miRNAs in (B) above in a form that includes these miRNAs.

[0065] In the determination method of the present disclosure, the expression level of miRNA can be measured according to a standard method using a known method for specifically detecting a specific gene, such as hybridization techniques such as Northern blotting, Southern blotting, in situ hybridization, Northern hybridization, and Southern hybridization, quantitative amplification techniques such as quantitative RT-PCR, nucleic acid array technology (nucleic acid arrays are also known as nucleic acid chips (DNA chips or RNA chips), microarrays, etc.), or methods using next-generation sequencers. Furthermore, in the determination method of the present disclosure, the expression level of miRNA can be measured, for example, using the kit or device of the present disclosure described below.

[0066] The determination method of the present disclosure may further include the following steps. (2) determining the possibility of gastrointestinal or cutaneous graft-versus-host disease based on the expression level measured in step (1), where "possibility of gastrointestinal or cutaneous graft-versus-host disease" means "possibility of gastrointestinal or cutaneous graft-versus-host disease at the time of collection of body fluid."

[0067] In the determination method of the present disclosure, the possibility that a subject has gastrointestinal or cutaneous graft-versus-host disease is determined using the expression level of a target miRNA as an index. In this case, for example, if the expression level of the target miRNA is higher than a predetermined or custom-defined cutoff value, or if it is lower than a predetermined or custom-defined cutoff value, the subject is determined to have a possibility of gastrointestinal or cutaneous graft-versus-host disease. The cutoff value can be appropriately set by those skilled in the art from the perspectives of sensitivity, specificity, positive predictive value, negative predictive value, etc., and can be, for example, the average, percentile, or maximum value of the expression level of the target miRNA in a body fluid collected from a subject not suffering from (gastrointestinal or cutaneous) graft-versus-host disease. More specifically, for example, the expression level of the target miRNA in body fluid-derived samples collected from a subject not suffering from (gastrointestinal or skin) GVHD and a subject suffering from GVHD can be measured, and the measured values ​​can be used to perform statistical analysis based on receiver operating characteristic (ROC) curve analysis (more specifically, a method using the Youden index can be exemplified) to set a cutoff value.

[0068] The level of "high" is not particularly limited, and examples thereof include an expression level of the target miRNA that is 2 times or more, 4 times or more, 6 times or more, 8 times or more, or 10 times or more the cutoff value. The level of "low" is not particularly limited, and examples thereof include an expression level of the target miRNA that is 1 / 2 times or less, 1 / 4 times or less, 1 / 6 times or less, 1 / 8 times or less, or 1 / 10 times or less the cutoff value.

[0069] In step (2) of the present disclosure, the expression level of the target miRNA in a body fluid-derived sample collected from the subject is substituted into a discriminant that is prepared using the expression levels of miRNA in a subject known to be affected with gastrointestinal or cutaneous graft-versus-host disease and the expression levels of miRNA in a subject not affected with (gastrointestinal or cutaneous) graft-versus-host disease as teacher samples, and that is capable of differentially determining whether or not the subject is affected with gastrointestinal or cutaneous graft-versus-host disease, thereby determining the possibility of the subject being affected with gastrointestinal or cutaneous graft-versus-host disease.

[0070] In the present disclosure, the discriminant can be created using any discriminant analysis method that can create a discriminant that discriminates between the presence or absence of gastrointestinal or cutaneous graft-versus-host disease, such as principal component analysis, Fisher's discriminant analysis, multiple regression analysis, nonlinear discriminant analysis using Mahalanobis distance, neural network, support vector machine (SVM), logistic regression analysis (particularly, logistic regression analysis using LASSO (Least Absolute Shrinkage and Selection Operator) method), k-nearest neighbor analysis, decision tree, etc., but is not limited to these specific examples.

[0071] In addition, the determination method of the present disclosure does not exclude the use of the expression levels of miRNAs other than the target miRNA in addition to the target miRNA, and the expression levels of miRNAs other than the target miRNA can also be used to determine the possibility of suffering from gastrointestinal or cutaneous graft-versus-host disease.

[0072] According to the determination method of the present disclosure, it is possible to determine the possibility of having gastrointestinal or cutaneous graft-versus-host disease. Furthermore, since the determination method of the present disclosure can determine the possibility of having gastrointestinal or cutaneous graft-versus-host disease with higher sensitivity, the determination method of the present disclosure can more reliably determine that a subject who truly has gastrointestinal or cutaneous graft-versus-host disease "has gastrointestinal or cutaneous graft-versus-host disease" (i.e., it can further reduce the possibility of erroneously determining that a subject "does not have gastrointestinal or cutaneous graft-versus-host disease").

[0073] The judgment method of the present disclosure can also be used before performing a gastrointestinal endoscopy to determine the need for performing a gastrointestinal endoscopy.

[0074] If the disclosed method of determination determines that a subject is highly likely to be suffering from gastrointestinal or cutaneous graft-versus-host disease, the subject can be treated for gastrointestinal or cutaneous graft-versus-host disease, thereby treating the subject for gastrointestinal or cutaneous graft-versus-host disease.

[0075] The diagnostic kit and diagnostic device for gastrointestinal or skin graft-versus-host disease of the present disclosure (hereinafter, sometimes referred to as the "kit of the present disclosure" and the "device of the present disclosure", respectively) comprise: (A) a nucleic acid capable of specifically binding to at least one miRNA selected from the group consisting of miR-638, miR-1246, miR-762, miR-3648, miR-4497, miR-4508, miR-3960, miR-4732-5p, miR-4739, miR-4745-5p, miR-4787-5p, miR-204-3p, miR-1233-5p, miR-5787, miR-6089, miR-6090, miR-6131, miR-6786-5p, miR-6787-5p, miR-6869-5p, miR-7704, and miR-8072, or a nucleic acid comprising a nucleotide sequence complementary to the miRNA; or (B) A nucleic acid capable of specifically binding to at least one miRNA selected from the group consisting of miR-1246, miR-762, miR-3648, miR-4488, miR-4497, miR-4508, miR-3960, miR-4732-5p, miR-4739, miR-4745-5p, miR-4787-5p, miR-197-5p, miR-204-3p, miR-1233-5p, miR-5787, miR-6089, miR-6090, miR-6131, miR-6727-5p, miR-6786-5p, miR-6869-5p, and miR-7704, or a nucleic acid comprising a base sequence complementary to the miRNA (hereinafter, also referred to as the "nucleic acid of the present disclosure"). The present invention is characterized by comprising:

[0076] The target miRNA, gastrointestinal or skin graft-versus-host disease, etc. are defined as above.

[0077] The kit or device of the present disclosure can be suitably used to determine the possibility of gastrointestinal or cutaneous graft-versus-host disease in the above-described determination method of the present disclosure.

[0078] The nucleic acids of the present disclosure can be used as nucleic acid probes and primers for measuring the expression level of a target miRNA, which is a target nucleic acid that is a marker for gastrointestinal or cutaneous graft-versus-host disease.

[0079] Specific examples of nucleic acids of the present disclosure include the following: (a) a nucleic acid containing 15 or more consecutive bases in the base sequence of a target miRNA or a base sequence complementary to the base sequence; (b) A nucleic acid containing 15 or more consecutive bases that hybridizes under stringent conditions with a nucleic acid consisting of the base sequence of a target miRNA or a base sequence complementary to said base sequence.

[0080] A complementary base sequence (complementary strand, reverse strand) refers to a base sequence that is complementary to the full-length sequence of a nucleic acid consisting of the base sequence of a target miRNA, or a partial sequence thereof having a base sequence of at least 15 consecutive bases in length (for convenience, these are also referred to as the "positive strand" herein) based on base pairing such as A:T and G:C. However, such a complementary strand is not limited to a sequence that forms a completely complementary sequence with the base sequence of the target positive strand, but may also have a complementary relationship to the extent that it can hybridize with the target positive strand under stringent conditions. Note that stringent conditions can be determined based on the melting temperature (Tm) of the nucleic acid to which the complex or probe binds, as taught by Berger and Kimmel (1987, Guide to Molecular Cloning Techniques Methods in Enzymology, Vol. 152, Academic Press, San Diego, CA). For example, typical post-hybridization washing conditions include approximately 1x SSC, 0.1% SDS, and 37°C. It is preferable that the complementary strand maintains its hybridization state with the target positive strand even when washed under such conditions. While not particularly limited, more stringent hybridization conditions include approximately 0.5x SSC, 0.1% SDS, and 42°C, and even more stringent hybridization conditions include approximately 0.1x SSC, 0.1% SDS, and 65°C. Specifically, examples of such complementary strands include a strand consisting of a nucleotide sequence that is completely complementary to the nucleotide sequence of the target positive strand, and a strand consisting of a nucleotide sequence that shares at least 90%, 95%, 98%, or 99% identity with the target positive strand.

[0081] The nucleic acids of the present disclosure can be prepared using common techniques such as DNA recombinant technology, PCR, and methods using automated DNA / RNA synthesizers.

[0082] The nucleic acids included in the kits of the present disclosure may be packaged individually or in any combination in different containers.

[0083] The base length of the primer or probe can be set appropriately depending on the application, for example, 15 to 35 bases when used as a primer, and 15 to 35 bases when used as a probe, as described above.

[0084] Primers, probes, etc. can be designed based on the nucleotide sequence of the target miRNA, for example, using various design programs. Specifically, candidate sequences for primers or probes obtained by applying the nucleotide sequence of the target miRNA to a design program, or sequences containing at least a portion of such sequences, can be used as primers or probes.

[0085] The kit of the present disclosure may include a kit for extracting nucleic acids (for example, total RNA) from body fluids, a fluorescent labeling substance, an enzyme for amplifying nucleic acids, a culture medium, instructions for use, and the like.

[0086] An example of a device of the present disclosure is a device in which the nucleic acid of the present disclosure is bound or attached to a solid phase. Examples of materials for the solid phase include plastic, glass, silicon, and paper, with plastic being a preferred solid phase material due to its ease of processing. The shape of the solid phase is not particularly limited, and examples include square, round, rectangular, and film shapes. Examples of devices of the present disclosure include devices for measurement using hybridization techniques, such as nucleic acid arrays (e.g., microarrays, DNA chips, RNA chips, etc.) and blotting devices.

[0087] The present disclosure makes it possible to objectively and non-invasively or minimally invasively diagnose gastrointestinal or cutaneous graft-versus-host disease using body fluids (particularly serum). Currently, there are no non-invasive diagnostic methods for gastrointestinal graft-versus-host disease, so in cases where conditions suggest gastrointestinal graft-versus-host disease clinically, an objective, non-invasive diagnostic method would enable rapid treatment of patients and would be expected to be of high clinical value.

[0088] It should be noted that, in this specification, the term "comprising" includes "consisting essentially of" and "consisting of." Furthermore, the present disclosure encompasses all arbitrary combinations of the constituent elements described in this specification.

[0089] Furthermore, the various characteristics (properties, structures, functions, etc.) described in each embodiment of the present disclosure above may be combined in any way to specify the subject matter encompassed by the present disclosure, i.e., the present disclosure encompasses all subject matter consisting of any combination of the combinable characteristics described herein. [Example]

[0090] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0091] method Among 199 patients who underwent allogeneic hematopoietic stem cell transplantation at Osaka Municipal University Hospital, a prospective cohort study (OCU10-4) was enrolled. Twelve patients developed severe gastrointestinal GVHD (stage 3 or higher), 12 patients developed severe cutaneous GVHD (stage 3 or higher), and 14 patients did not develop GVHD. Serum samples at the time of GVHD were analyzed using ExoQuick (System Biosciences, LLC) treatment with and without treatment. MicroRNA expression in body fluids was analyzed comprehensively using a microarray (3D-Gene® Human miRNA Oligo chip (Toray Industries, Inc.)). PCAUFE (principal components analysis-based unsupervised feature extraction) analysis was used to clarify the characteristics of microRNA expression at the time of GVHD onset (see Taguchi YH. Sci Rep. 2017;7:44016).

[0092] ·RNA extraction method The RNA extraction method from peripheral blood was performed as follows.

[0093] <Without ExoQuick treatment> 1. Add 1 ml of Qiazol Lysis Reagent to 200 μl of serum and vortex (for 5 minutes at room temperature). 2. Add 200 μl of chloroform and vortex (for 5 minutes at room temperature). 3. Centrifuge at 13,400 rpm (12,000 xg) for 15 minutes at 4°C. 4. Prepare two 1.5 ml tubes with 577.5 μl of 100% ethanol added. 5. Mix 385 μl of the supernatant (colorless and transparent layer) from step 3 into the ethanol in step 4. 6. Put 700 μl of the supernatant from step 5 into the filter unit of a 2 ml tube containing the miRNA Mini Kit filter unit. 7. Centrifuge at 11,000 rpm (8,000 xg) for 15 seconds at room temperature and discard the filtrate. 8. Add 700 μl of RWT buffer, centrifuge at 11,000 rpm (8,000 xg) for 15 seconds at room temperature, and discard the filtrate. 9. Add 500 μl of RPE buffer, centrifuge at 11,000 rpm (8,000 xg) for 15 seconds at room temperature, and discard the filtrate. 10. Add 500 μl of RPE buffer, centrifuge at 11,000 rpm (8,000 xg) for 2 minutes at room temperature, and discard the filtrate. 11. Replace the tube with the filter unit into a new 2 ml tube. 12. Centrifuge at 13,900 rpm (13,000 xg) for 1 minute at room temperature to dry the membrane of the filter unit. 13. Replace with an extraction tube, add 40 (30 - 50) μl of RNase-free water, and centrifuge at 11,000 rpm (8,000 xg) for 1 minute at room temperature. 14. Discard the filter unit and store the tube with the sample in ice. 15. Using 1 μl of the sample, the nucleic acid amount was measured with a Nano drop, and the remaining sample was frozen at -80 °C.

[0094] <With ExoQuick treatment> 1. 900 μl of serum was mixed (vortex) with 225 μl of ExoQuick at a ratio and stored overnight at 4 °C. 2. Centrifugation was performed at 4,700 rpm (1,500 xg) for 30 minutes at 4 °C, and the supernatant was removed. 3. 200 μl of PBS was added each time to dilute, and the exosomes were dispersed by pipetting (tapping). 4. 1 ml of Qiazol Lysis Reagent was added to the exosome solution and shaken (vortex) (5 minutes at room temperature). 5. 200 μl of chloroform was added and shaken (vortex) (5 minutes at room temperature). 6. Centrifugation was performed at 13,400 rpm (12,000 xg) for 15 minutes at 4 °C. 7. Two 1.5 ml tubes were prepared with 577.5 μl of 100% ethanol added. 8. 385 μl of the supernatant from 6 (colorless and transparent layer) was mixed into the ethanol in 7 each. 9. 700 μl of the supernatant from 8 was put into the filter unit of a 2 ml tube containing the filter unit of the miRNA Mini Kit. 10. Centrifugation was performed at 11,000 rpm (8,000 xg) for 15 seconds at room temperature, and the filtrate was discarded. 11. 700 μl of RWT buffer was added, centrifugation was performed at 11,000 rpm (8,000 xg) for 15 seconds at room temperature, and the filtrate was discarded. 12. 500 μl of RPE buffer was added, centrifugation was performed at 11,000 rpm (8,000 xg) for 15 seconds at room temperature, and the filtrate was discarded. 13. 500 μl of RPE buffer was added, centrifugation was performed at 11,000 rpm (8,000 xg) for 2 minutes at room temperature, and the filtrate was discarded. 14. The tube of the filter unit was replaced with a new 2 ml tube. 15. The filter unit membrane was dried by centrifugation at 13,900 rpm (13,000 x g) for 1 minute at room temperature. 16. Replace with an extraction tube, add 40 (30-50) μl of RNase-free water, and centrifuge at 11,000 rpm (8,000 x g) at room temperature for 1 minute. 17. The filter unit was discarded and the sample tube was stored on ice. 18. 1 μl of the sample was used to measure the amount of nucleic acid using a Nano drop, and the remaining sample was frozen at -80°C.

[0095] Comprehensive measurement of miRNA The comprehensive measurement of miRNAs was carried out according to the details described in the following literature. 1) Yamamoto Y, et al.Highly Sensitive Circulating MicroRNA Panel for Accurate Detection of Hepatocellular Carcinoma in Patients With Liver Disease.Hepatol Commun.2019;4(2):284-297. 2) Yokoi A, et al.Integrated extracellular microRNA profiling for ovarian cancer screening.Nat Commun.2018;9(1):4319.

[0096] result (A) Examination of blood microRNA profiles for diagnosing severe gastrointestinal GVHD Using ExoQuick-treated patient serum, a comparison of 12 cases of severe gastrointestinal GVHD and 12 control cases (microRNA data could not be obtained from ExoQuick-treated serum in 2 of the control cases) revealed that 22 microRNAs (hsa-miR-638, hsa-miR-1246, hsa-miR-762, hsa-miR-3648, hsa-miR-4497, hsa-miR-4508, hsa-miR-3960, hsa-miR-4732-5p, hsa-miR-4739, hsa-miR-474 Using expression information for miR-5-5p, hsa-miR-4787-5p, hsa-miR-204-3p, hsa-miR-1233-5p, hsa-miR-5787, hsa-miR-6089, hsa-miR-6090, hsa-miR-6131, hsa-miR-6786-5p, hsa-miR-6787-5p, hsa-miR-6869-5p, hsa-miR-7704, and hsa-miR-8072, we were able to distinguish between gastrointestinal GVHD cases and control cases (those without GVHD), as shown in the violin plot in Figure 1 (Figure 1, P = 1.37*e-05). The discriminative prediction using the 22 microRNA profiles had an average AUC of 0.94 (95% confidence interval: 0.81-1.00) by ROC analysis (Figure 2), a positive predictive value of 92% (12 / 13), a negative predictive value of 100% (11 / 11), and a diagnostic prediction accuracy of 95.8% (23 / 24) (Table 1).

[0097] [Table 1]

[0098] On the other hand, a similar study was performed on serum that had not been treated with ExoQuick, but it was not possible to distinguish between the 12 cases of severe gastrointestinal GVHD and the 14 control cases.

[0099] Next, to develop a simpler diagnostic prediction method using fewer microRNAs, we used receiver operating characteristic (ROC) analysis to examine the individual discriminatory ability of each miRNA constituting the 22 microRNAs in the ExoQuick-treated serum (Figure 3). Violin plots were also used (Figure 4). Based on an average AUC of 0.70 or higher by ROC analysis, seven microRNAs—hsa-miR-1246, hsa-miR-3648, hsa-miR-3960, hsa-miR-4732-5p, hsa-miR-4787-5p, hsa-miR-1233-5p, and hsa-miR-7704—may be used alone or in combination to identify severe gastrointestinal GVHD.

[0100] (B) Examination of blood microRNA profiles for diagnosing severe cutaneous GVHD Similarly, when ExoQuick-treated patient serum was used, a comparison of 12 cases of severe skin GVHD and 12 control cases (microRNA data could not be obtained from ExoQuick-treated serum in 2 of the control cases) revealed that 22 microRNAs (hsa-miR-1246, hsa-miR-762, hsa-miR-3648, hsa-miR-4488, hsa-miR-4497, hsa-miR-4508, hsa-miR-3960, hsa-miR-4732-5p, hsa-miR-4739, hsa-miR-4740, hsa-miR-4741, hsa-miR-4742, hsa-miR-4743, hsa-miR-4744, hsa-miR-4745, hsa-miR-4746, hsa-miR-4747, hsa-miR-4748, hsa-miR-4749, hsa-miR-4749, hsa-miR-4749, hsa-miR-4746, hsa-miR-4749 ... Using the expression information of miR-45-5p, miR-4787-5p, miR-197-5p, miR-204-3p, miR-1233-5p, miR-5787, miR-6089, miR-6090, miR-6131, miR-6727-5p, miR-6786-5p, miR-6869-5p, and miR-7704, we were able to distinguish between cutaneous GVHD cases and control cases (non-GVHD cases) as shown in the violin plot in Figure 5 (Figure 5, P = 1.38*e-03). The discriminative prediction using the 22 microRNA profiles had an average AUC of 0.81 (95% confidence interval: 0.62-1.00) by ROC analysis (Figure 6), a positive predictive value of 90% (9 / 10), a negative predictive value of 79% (11 / 14), and a diagnostic prediction accuracy of 83.3% (20 / 24) (Table 2).

[0101] [Table 2]

[0102] On the other hand, a similar study was performed on serum that had not been treated with ExoQuick, but it was not possible to distinguish between 11 cases of severe skin GVHD (microRNA data could not be obtained from serum that had not been treated with ExoQuick in one of the severe skin GVHD cases) and 14 control cases.

[0103] Next, to develop a simpler diagnostic prediction method using fewer microRNAs, we used receiver operating characteristic (ROC) analysis to examine the individual discriminatory ability of each of the 22 microRNAs in the ExoQuick-treated serum (Figure 7). Violin plots were also used (Figure 8). Based on an average AUC of 0.70 or higher by ROC analysis, nine microRNAs—hsa-miR-4488, hsa-miR-3960, hsa-miR-4739, hsa-miR-4787-5p, hsa-miR-1233-5p, hsa-miR-6089, hsa-miR-6090, hsa-miR-6727-5p, and hsa-miR-7704—may be used alone or in combination to identify severe gastrointestinal GVHD.

Claims

1. (A) miR-638, miR-1246, miR-762, miR-3648, miR-4497, miR-4508, miR-3960, miR-4732 -5p, miR-4739, miR-4745-5p, miR-4787-5p, miR-204-3p, miR-1233-5p, miR-5787, miR a nucleic acid capable of specifically binding to at least one miRNA selected from the group consisting of miR-6089, miR-6090, miR-6131, miR-6786-5p, miR-6787-5p, miR-6869-5p, miR-7704, and miR-8072, or a nucleic acid having a base sequence complementary to the miRNA; (B) miR-1246, miR-762, miR-3648, miR-4488, miR-4497, miR-4508, miR-3960, miR-473 2-5p, miR-4739, miR-4745-5p, miR-4787-5p, miR-197-5p, miR-204-3p, miR-1233-5p, A nucleic acid capable of specifically binding to at least one miRNA selected from the group consisting of miR-5787, miR-6089, miR-6090, miR-6131, miR-6727-5p, miR-6786-5p, miR-6869-5p, and miR-7704, or a nucleic acid having a base sequence complementary to the miRNA. A diagnostic kit for gastrointestinal or skin graft-versus-host disease, comprising:

2. (A1) a nucleic acid capable of specifically binding to at least one miRNA selected from the group consisting of miR-1246, miR-3648, miR-3960, miR-4732-5p, miR-4787-5p, miR-1233-5p, and miR-7704, or a nucleic acid having a base sequence complementary to the miRNA; or (B1) A nucleic acid capable of specifically binding to at least one miRNA selected from the group consisting of miR-4488, miR-3960, miR-4739, miR-4787-5p, miR-1233-5p, miR-6089, miR-6090, miR-6727-5p, and miR-7704, or a nucleic acid having a base sequence complementary to the miRNA. The kit of claim 1 , comprising:

3. The kit according to claim 1 or 2, which is for diagnosing gastrointestinal or cutaneous graft-versus-host disease after allogeneic hematopoietic stem cell transplantation.

4. (A) miR-638, miR-1246, miR-762, miR-3648, miR-4497, miR-4508, miR-3960, miR-4732 -5p, miR-4739, miR-4745-5p, miR-4787-5p, miR-204-3p, miR-1233-5p, miR-5787, miR a nucleic acid capable of specifically binding to at least one miRNA selected from the group consisting of miR-6089, miR-6090, miR-6131, miR-6786-5p, miR-6787-5p, miR-6869-5p, miR-7704, and miR-8072, or a nucleic acid having a base sequence complementary to the miRNA; (B) miR-1246, miR-762, miR-3648, miR-4488, miR-4497, miR-4508, miR-3960, miR-473 2-5p, miR-4739, miR-4745-5p, miR-4787-5p, miR-197-5p, miR-204-3p, miR-1233-5p, A nucleic acid capable of specifically binding to at least one miRNA selected from the group consisting of miR-5787, miR-6089, miR-6090, miR-6131, miR-6727-5p, miR-6786-5p, miR-6869-5p, and miR-7704, or a nucleic acid having a base sequence complementary to the miRNA. A device for diagnosing gastrointestinal or skin graft-versus-host disease, comprising:

5. (A1) a nucleic acid capable of specifically binding to at least one miRNA selected from the group consisting of miR-1246, miR-3648, miR-3960, miR-4732-5p, miR-4787-5p, miR-1233-5p, and miR-7704, or a nucleic acid having a base sequence complementary to the miRNA; or (B1) A nucleic acid capable of specifically binding to at least one miRNA selected from the group consisting of miR-4488, miR-3960, miR-4739, miR-4787-5p, miR-1233-5p, miR-6089, miR-6090, miR-6727-5p, and miR-7704, or a nucleic acid having a base sequence complementary to the miRNA. The device of claim 4 , comprising:

6. The device according to claim 4 or 5, which is for diagnosing gastrointestinal or cutaneous graft-versus-host disease after allogeneic hematopoietic stem cell transplantation.

7. A method for determining the possibility of suffering from gastrointestinal or cutaneous graft-versus-host disease, comprising the steps of: (1) In a body fluid-derived sample collected from a subject (A) the expression level of at least one miRNA selected from the group consisting of miR-638, miR-1246, miR-762, miR-3648, miR-4497, miR-4508, miR-3960, miR-4732-5p, miR-4739, miR-4745-5p, miR-4787-5p, miR-204-3p, miR-1233-5p, miR-5787, miR-6089, miR-6090, miR-6131, miR-6786-5p, miR-6787-5p, miR-6869-5p, miR-7704, and miR-8072; or (B) Expression level of at least one miRNA selected from the group consisting of miR-1246, miR-762, miR-3648, miR-4488, miR-4497, miR-4508, miR-3960, miR-4732-5p, miR-4739, miR-4745-5p, miR-4787-5p, miR-197-5p, miR-204-3p, miR-1233-5p, miR-5787, miR-6089, miR-6090, miR-6131, miR-6727-5p, miR-6786-5p, miR-6869-5p, and miR-7704. A process of measuring

8. (A1) the expression level of at least one miRNA selected from the group consisting of miR-1246, miR-3648, miR-3960, miR-4732-5p, miR-4787-5p, miR-1233-5p, and miR-7704; or (B1) Expression level of at least one miRNA selected from the group consisting of miR-4488, miR-3960, miR-4739, miR-4787-5p, miR-1233-5p, miR-6089, miR-6090, miR-6727-5p, and miR-7704 The method according to claim 7, wherein

9. (2) determining the possibility of gastrointestinal or cutaneous graft-versus-host disease based on the expression level measured in the step (1); 9. The method of claim 7 or 8, comprising:

10. The method according to claim 7 or 8, wherein in the step (1), the expression level of miRNA is measured using the kit according to claim 1 or 2, or the device according to claim 4 or 5.

11. The method according to claim 7 or 8, wherein the gastrointestinal or cutaneous graft-versus-host disease is gastrointestinal or cutaneous graft-versus-host disease after allogeneic hematopoietic stem cell transplantation.

12. 9. The method of claim 7 or 8, wherein the body fluid is serum, plasma, saliva or urine.

13. The method of claim 7 or 8, wherein the body fluid-derived sample is extracellular vesicles derived from body fluid.

14. The method of claim 7 or 8, wherein the subject is a human.

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