Method for examining clonal hematopoiesis and / or clonal hematopoiesis-related disease

Abstract

To provide a biomarker for clonal hematopoiesis.SOLUTION: The present inventors have found that PYY, LEP, SCN3A, FCGR2A, CYTH3, CD8A, TK1, GBP6, DSC2, GP5, IL17D, CD244, SLC4A1, BGN, APOA4, and MMP7 in a body fluid sample are biomarkers for clonal hematopoiesis.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for testing for clonal hematopoiesis and / or clonal hematopoiesis-associated disorders, and the like. [Background technology]

[0002] Recent studies have revealed that cell populations with identical genomic abnormalities expand in the hematopoietic organs of healthy individuals without disease, a phenomenon known as clonal hematopoiesis. Clonal hematopoiesis is considered a precancerous lesion of hematologic tumors, particularly in elderly individuals, and has been detected based on two types of genomic abnormalities: gene mutations and copy number abnormalities. Clonal hematopoiesis increases the risk of multiple diseases, including not only hematologic tumors but also cardiovascular disease and chronic obstructive pulmonary disease. Therefore, testing for clonal hematopoiesis has attracted attention in recent years as a major prognostic factor, especially in elderly individuals. Therefore, testing for clonal hematopoiesis holds promise as a foundation for novel preventive medicine.

[0003] However, detecting clonal hematopoiesis requires genome analysis (Non-Patent Documents 1 and 2), making it difficult to implement as a screening test from a cost perspective. In addition, although detecting clonal hematopoiesis can estimate the risk of developing the disease, there are currently no intervention methods for preventing the onset of the disease. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] The New England Journal of Medicine. 2014 Dec 25;371(26):2477-87. doi: 10.1056 / NEJMoa1409405. Epub 2014 Nov 26. [Non-patent document 2] Nature. 2018 Jul;559(7714):350-355. doi: 10.1038 / s41586-018-0321-x. Epub 2018 Jul 11. Summary of the Invention [Problem to be solved by the invention]

[0005] An objective of the present invention is to provide a biomarker for clonal hematopoiesis. [Means for solving the problem]

[0006] The present inventors have conducted extensive research in light of the above-mentioned problems and have found that PYY, LEP, SCN3A, FCGR2A, CYTH3, CD8A, TK1, GBP6, DSC2, GP5, IL17D, CD244, SLC4A1, BGN, APOA4, and MMP7 in body fluid samples are biomarkers for clonal hematopoiesis. Based on this finding, the present inventors have conducted further research and have completed the present invention. Specifically, the present invention encompasses the following aspects:

[0007] Item 1. (1) A method for testing for clonal hematopoiesis and / or a clonal hematopoietic-associated disorder, comprising the step of detecting at least one protein selected from the group consisting of PYY, LEP, SCN3A, FCGR2A, CYTH3, CD8A, TK1, GBP6, DSC2, GP5, IL17D, CD244, SLC4A1, BGN, APOA4, and MMP7 in a body fluid sample collected from a subject.

[0008] Item 2. The method according to Item 1, wherein the protein comprises PYY and / or LEP.

[0009] Item 3. The method according to Item 1, wherein the protein comprises PYY.

[0010] Item 4. Furthermore, (2) determining the presence or absence of clonal hematopoiesis and / or the presence or absence of a risk of developing and / or aggravating a clonal hematopoietic-associated disorder in the subject based on the amount or concentration of the protein detected in step (1); Item 4. The method according to any one of Items 1 to 3, comprising:

[0011] Item 5. When the protein includes at least one protein (protein A) selected from the group consisting of PYY, FCGR2A, CYTH3, CD8A, TK1, DSC2, IL17D, CD244, APOA4, and MMP7, The step (2) (2Aa) determining that the subject has clonal hematopoiesis and / or a risk of developing and / or aggravating a clonal hematopoietic-associated disorder when the amount or concentration of the protein A detected in the step (1) is equal to or greater than a cutoff value; and / or (2Ab) determining that the subject does not have clonal hematopoiesis and / or does not have a risk of developing and / or aggravating a clonal hematopoietic-associated disorder when the amount or concentration of the protein A detected in the step (1) is equal to or lower than a cutoff value; and / or When the protein includes at least one protein (protein B) selected from the group consisting of LEP, SCN3A, GBP6, GP5, SLC4A1, and BGN, The step (2) (2Aa) determining that the subject has clonal hematopoiesis and / or a risk of developing and / or aggravating a clonal hematopoietic-associated disorder when the amount or concentration of the protein B detected in the step (1) is equal to or lower than a cutoff value; and / or (2Ab) determining that the subject does not have clonal hematopoiesis and / or does not have a risk of developing and / or aggravating a clonal hematopoietic-associated disorder when the amount or concentration of the protein B detected in the step (1) is equal to or greater than a cutoff value; Item 5. The method according to item 4, comprising:

[0012] Item 6. The method according to any one of Items 1 to 3, wherein the clonal hematopoiesis-related disease is at least one selected from the group consisting of hematopoietic tumors, cardiovascular diseases, cerebrovascular diseases, Alzheimer's disease, respiratory diseases, and infectious diseases.

[0013] Item 7. The method according to any one of Items 1 to 3, wherein the body fluid sample is at least one selected from the group consisting of whole blood, serum, and plasma.

[0014] Item 8. The method according to any one of Items 1 to 4, wherein the subject is a human.

[0015] Item 9. A diagnostic agent for clonal hematopoiesis and / or a clonal hematopoietic-related disorder, comprising a binding molecule for at least one protein selected from the group consisting of PYY, LEP, SCN3A, FCGR2A, CYTH3, CD8A, TK1, GBP6, DSC2, GP5, IL17D, CD244, SLC4A1, BGN, APOA4, and MMP7.

[0016] Item 10. A method for screening for an active ingredient of an agent for preventing or treating a clonal hematopoietic disorder, using as an indicator the amount or concentration of at least one protein selected from the group consisting of PYY, LEP, SCN3A, FCGR2A, CYTH3, CD8A, TK1, GBP6, DSC2, GP5, IL17D, CD244, SLC4A1, BGN, APOA4, and MMP7 in a body fluid sample collected from an animal treated with a test substance.

[0017] Item 11. A method for evaluating the inducibility or exacerbation of a clonal hematopoietic disorder, using as an indicator the amount or concentration of at least one protein selected from the group consisting of PYY, LEP, SCN3A, FCGR2A, CYTH3, CD8A, TK1, GBP6, DSC2, GP5, IL17D, CD244, SLC4A1, BGN, APOA4, and MMP7 in a body fluid sample collected from an animal treated with a test substance. [Effects of the Invention]

[0018] According to the present invention, a biomarker for clonal hematopoiesis can be provided. DETAILED DESCRIPTION OF THE INVENTION

[0019] In this specification, the expressions "contain" and "comprise" include the concepts of "contain", "include", "consist essentially of" and "consist only of".

[0020] As used herein, the "identity" of an amino acid sequence refers to the degree of correspondence between two or more comparable amino acid sequences. Therefore, the greater the correspondence between two amino acid sequences, the greater the identity or similarity between those sequences. The level of identity between amino acid sequences can be determined, for example, using the sequence analysis tool FASTA with default parameters. Alternatively, it can be determined using the BLAST algorithm by Karlin and Altschul (Karlin S, Altschul S F. "Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes," Proc. Natl. Acad. Sci. USA. 87:2264-2268 (1990); Karlin S, Altschul S F. "Applications and statistics for multiple high-scoring segments in molecular sequences," Proc. Natl. Acad. Sci. USA. 90:5873-7 (1993)). A program called BLASTX, based on the BLAST algorithm, has been developed. Specific techniques for these analysis methods are known and can be found on the National Center of Biotechnology Information (NCBI) website (http: / / www.ncbi.nlm.nih.gov / ). The "identity" of nucleotide sequences is also defined in the same manner as above.

[0021] As used herein, "conservative substitution" refers to the substitution of an amino acid residue with an amino acid residue having a similar side chain. For example, substitution between amino acid residues having basic side chains such as lysine, arginine, and histidine constitutes a conservative substitution. Other examples of conservative substitution include substitution between amino acid residues having acidic side chains such as aspartic acid and glutamic acid; amino acid residues having uncharged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues having nonpolar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues having β-branched side chains such as threonine, valine, and isoleucine; and amino acid residues having aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine.

[0022] 1. Methods for testing for clonal hematopoiesis and / or clonal hematopoietic-associated disorders In one aspect, the present invention relates to (1) a method for testing for clonal hematopoiesis and / or clonal hematopoiesis-associated disorders (sometimes referred to herein as the "testing method of the present invention"), which comprises the step of detecting at least one protein selected from the group consisting of PYY, LEP, SCN3A, FCGR2A, CYTH3, CD8A, TK1, GBP6, DSC2, GP5, IL17D, CD244, SLC4A1, BGN, APOA4, and MMP7 in a body fluid sample collected from a subject. This method is described below.

[0023] 1-1. Process (1) Clonal hematopoiesis, the subject of testing, is a state in which a population of hematopoietic stem cells with the same genomic abnormality proliferates. Genomic abnormalities are of two types: gene mutations and copy number abnormalities. For example, clonal hematopoiesis can be determined when the number of hematopoietic stem cell clones with a certain genomic abnormality is, for example, 0.5% or more, 1% or more, 2% or more, 4% or more, 6% or more, or 8% or more of the total number of peripheral blood cells (100%).

[0024] Clonal hematopoiesis has been reported to increase the risk of developing and / or worsening various diseases. Examples of such diseases (clonal hematopoiesis-associated diseases) include hematopoietic tumors (e.g., leukemia, lymphoma, etc.), cardiovascular diseases (e.g., myocardial infarction, heart failure, angina pectoris, aortic dissection, etc.), cerebrovascular diseases (e.g., cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage, etc.), respiratory diseases (e.g., chronic obstructive pulmonary disease, interstitial lung disease, etc.), and infectious diseases (e.g., pneumonia, sepsis, COVID-19, etc.).

[0025] The subject is a living organism that is the target of the testing method of the present invention, and the species of the subject is not particularly limited. Examples of the subject species include various mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits, and preferably humans.

[0026] The condition of the subject is not particularly limited. Examples of the subject include a specimen in which it is unknown whether or not it has clonal hematopoiesis, a specimen already determined by another method to have clonal hematopoiesis, a specimen already determined by another method to not have clonal hematopoiesis, a specimen suspected of having clonal hematopoiesis, a specimen not suspected of having clonal hematopoiesis, and a specimen with a high possibility of clonal hematopoiesis (e.g., an elderly specimen, such as specimens aged 40 years or older, 50 years or older, 60 years or older, 65 years or older, 70 years or older, 75 years or older, or 80 years or older).

[0027] The body fluid is not particularly limited. Examples of the body fluid include blood, ascites, cerebrospinal fluid, saliva, synovial fluid, urine, tissue fluid, sweat, tears, sputum, nasal discharge, and exhaled air, with blood being preferred. The body fluid may be used alone or in combination of two or more.

[0028] 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.

[0029] The body fluid sample may be the body fluid itself, or may be a sample derived from a body fluid (a sample prepared from a body fluid). Examples of body fluid-derived samples include samples obtained by removing certain components from the body fluid, or by concentrating and purifying proteins contained in the body fluid. Examples of body fluid-derived samples include serum and plasma.

[0030] Serum is a portion of whole blood from which blood cells and specific blood coagulation factors have been removed, and can be obtained, for example, as the supernatant after clotting of whole blood. Plasma is a portion of whole blood from which blood cells have been removed, and can be obtained, for example, as the supernatant when whole blood is centrifuged under conditions that do not cause clotting.

[0031] The body fluid sample may be one type alone or two or more types in combination.

[0032] In step (1), a body fluid sample is used as a test sample, and proteins contained in the test sample are detected. The target protein to be detected in step (1) is at least one protein selected from the group consisting of PYY, LEP, SCN3A, FCGR2A, CYTH3, CD8A, TK1, GBP6, DSC2, GP5, IL17D, CD244, SLC4A1, BGN, APOA4, and MMP7. Hereinafter, these proteins may be collectively referred to as "target proteins."

[0033] Among the target proteins, at least one protein (protein A) selected from the group consisting of PYY, FCGR2A, CYTH3, CD8A, TK1, DSC2, IL17D, CD244, APOA4, and MMP7 is a protein that increases during clonal hematopoiesis.

[0034] Among the target proteins is at least one protein (protein B) selected from the group consisting of LEP, SCN3A, GBP6, GP5, SLC4A1, and BGN, a protein that is decreased in clonal hematopoiesis.

[0035] Among the target proteins, PYY and LEP are preferred, with PYY being particularly preferred, from the viewpoint of accuracy in determining clonal hematopoiesis.

[0036] PYY is a known protein, and in the case of humans, it is a protein with UniProt Number: P10082. The amino acid sequence of the protein can be determined based on known genetic information. The protein to be detected in step (1) also includes those containing mutations that occur between individuals.

[0037] LEP is a known protein, and in the case of humans, it is the protein with UniProt Number: P41159. The amino acid sequence of the protein can be determined based on known genetic information. The protein to be detected in step (1) also includes those containing mutations that occur between individuals.

[0038] SCN3A is a known protein, and in the case of humans, it is a protein with UniProt Number: Q9NY46. The amino acid sequence of the protein can be determined based on known genetic information. The protein to be detected in step (1) also includes those containing mutations that occur between individuals.

[0039] FCGR2A is a known protein, and in the case of humans, it is a protein with UniProt Number: P12318. The amino acid sequence of the protein can be determined based on known genetic information. The protein to be detected in step (1) also includes those containing mutations that occur between individuals.

[0040] CYTH3 is a known protein, and in the case of humans, it is a protein with UniProt Number: O43739. The amino acid sequence of this protein can be determined based on known genetic information. The protein to be detected in step (1) also includes those containing mutations that occur between individuals.

[0041] CD8A is a known protein, and in the case of humans, it is the protein with UniProt Number: P01732. The amino acid sequence of this protein can be determined based on known genetic information. The protein to be detected in step (1) also includes those containing mutations that occur between individuals.

[0042] TK1 is a known protein, and in the case of humans, it is a protein with UniProt Number: P04183. The amino acid sequence of the protein can be determined based on known genetic information. The protein to be detected in step (1) also includes those containing mutations that occur between individuals.

[0043] GBP6 is a known protein, and in the case of humans, it is the protein with UniProt Number: Q6ZN66. The amino acid sequence of this protein can be determined based on known genetic information. The protein to be detected in step (1) also includes those containing mutations that occur between individuals.

[0044] DSC2 is a known protein, and in the case of humans, it is a protein with UniProt Number: Q02487. The amino acid sequence of this protein can be determined based on known genetic information. The protein to be detected in step (1) also includes those containing mutations that occur between individuals.

[0045] GP5 is a known protein, and in the case of humans, it is a protein with UniProt Number: P40197. The amino acid sequence of this protein can be determined based on known genetic information. The protein to be detected in step (1) also includes those containing mutations that occur between individuals.

[0046] IL17D is a known protein, and in the case of humans, it is a protein with UniProt Number: Q8TAD2. The amino acid sequence of this protein can be determined based on known genetic information. The protein to be detected in step (1) also includes those containing mutations that occur between individuals.

[0047] CD244 is a known protein, and in the case of humans, it is a protein with UniProt Number: Q9BZW8. The amino acid sequence of this protein can be determined based on known genetic information. The protein to be detected in step (1) also includes those containing mutations that occur between individuals.

[0048] SLC4A1 is a known protein, and in the case of humans, it is a protein with UniProt Number: P02730. The amino acid sequence of the protein can be determined based on known genetic information. The protein to be detected in step (1) also includes those containing mutations that occur between individuals.

[0049] BGN is a known protein, and in the case of humans, it is a protein with UniProt Number: P21810. The amino acid sequence of the protein can be determined based on known genetic information. The protein to be detected in step (1) also includes those containing mutations that occur between individuals.

[0050] APOA4 is a known protein, and in the case of humans, it is a protein with UniProt Number: P06727. The amino acid sequence of the protein can be determined based on known genetic information. The protein to be detected in step (1) also includes those containing mutations that occur between individuals.

[0051] MMP7 is a known protein, and in the case of humans, it is a protein with UniProt Number: P09237. The amino acid sequence of this protein can be determined based on known genetic information. The protein to be detected in step (1) also includes those containing mutations that occur between individuals.

[0052] In the step of detecting a target protein, the method for detecting the target protein (preferably a method for measuring the amount or concentration of the target protein) is not particularly limited as long as it is a method that can detect the target protein. Examples of such methods include immunoassays. Immunoassays can be widely used, regardless of whether they are direct, indirect, homogeneous, heterogeneous, competitive, or non-competitive. More specific examples of immunoassays include ELISA (e.g., direct, indirect, sandwich, or competitive), radioimmunoassay (RIA), immunoradiometric assay (IRMA), enzyme immunoassay (EIA), sandwich EIA, immunochromatography, Western blot, immunoprecipitation, slot or dot blot assay, immunohistochemical staining, fluorescent immunoassay, immunoassay using an avidin-biotin or streptavidin-biotin system, and immunoassay using surface plasmon resonance (SPR). Specifically, the target protein can be detected by immunoassay, for example, by contacting a labeled antibody directly or indirectly with a target protein-binding molecule bound to the target protein and quantifying the signal derived from the label of the bound labeled antibody. The labeled antibody used in this case and the antibody acting as an intermediary between the labeled antibody and the target protein-binding molecule or the target protein are not particularly limited, and examples that can be used include antibodies against antibody constant regions and anti-idiotype antibodies.

[0053] The detection method may be adopted singly or in combination of two or more.

[0054] The type of label used in the labeling substance (e.g., labeled antibody) used to detect a target protein is not particularly limited. Examples of labels include fluorescent substances, luminescent substances, dyes, enzymes, gold colloids, and radioisotopes. Among these, enzyme labels such as peroxidase and alkaline phosphatase are preferred from the viewpoints of safety, economy, detection sensitivity, and the like.

[0055] In one embodiment, step (1) can preferably include the steps of: (1a) contacting a target protein-binding molecule with the body fluid sample; and (1b) measuring the amount or concentration of the target protein bound to the target protein-binding molecule.

[0056] The target protein-binding molecule is not particularly limited as long as it selectively (specifically) recognizes the target protein. Here, "selectively (specifically) recognizes" means that the target protein can be specifically detected, for example, in Western blotting or ELISA, but is not limited thereto, and may be any molecule that enables a person skilled in the art to determine that the detected product is derived from the target protein.

[0057] The target protein-binding molecules include, for example, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single-chain antibodies, and molecules containing antigen-binding portions of the above antibodies, such as Fab fragments and fragments produced by an Fab expression library. The target protein-binding molecules of the present invention also include those that have antigen-binding ability to polypeptides consisting of at least 8, preferably 15, and more preferably 20 consecutive amino acids in the amino acid sequence of the target protein.

[0058] Methods for producing these target protein-binding molecules are already well known, and the target protein-binding molecules of the present invention can also be produced according to these standard methods (Current Protocols in Molecular Biology, Chapters 11.12-11.13 (2000)). Specifically, when the target protein-binding molecule of the present invention is a polyclonal antibody, the target protein can be expressed in Escherichia coli or the like and purified according to standard methods, or an oligopeptide having a partial amino acid sequence of the target protein can be synthesized according to standard methods and used to immunize a non-human animal such as a rabbit, and the antibody can be obtained from the serum of the immunized animal according to standard methods. On the other hand, in the case of monoclonal antibodies, a target protein expressed and purified in Escherichia coli or the like according to standard methods, or an oligopeptide having a partial amino acid sequence of the target protein, is immunized into a non-human animal such as a mouse, and the resulting spleen cells are fused with myeloma cells to prepare hybridoma cells, which can be obtained from the hybridoma cells (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley and Sons. Sections 11.4 to 11.11).

[0059] The target protein used as an immunizing antigen to prepare a target protein-binding molecule can be obtained based on known gene sequence information by DNA cloning, constructing each plasmid, transfecting the host, culturing the transformant, and recovering the protein from the culture. These procedures can be performed according to methods known to those skilled in the art or methods described in the literature (e.g., Molecular Cloning, T. Maniatis et al., CSH Laboratory (1983), DNA Cloning, D.M. Glover, IRL PRESS (1985)).

[0060] Specifically, a recombinant DNA (expression vector) capable of expressing a gene encoding a target protein in a desired host cell is prepared, which is then introduced into the host cell to transform it. The transformant is then cultured, and the target protein is recovered from the resulting culture, thereby obtaining a protein that can be used as an immunogen for producing a target protein-binding molecule of the present invention. Alternatively, a partial peptide of the target protein can be produced by standard chemical synthesis (peptide synthesis) using known gene sequence information.

[0061] Furthermore, the target protein-binding molecules of the present invention may be prepared using oligopeptides having a partial amino acid sequence of the target protein. The oligo(poly)peptides used to produce such target protein-binding molecules do not need to have functional biological activity, but desirably have immunogenic properties similar to those of the target protein. Preferred examples include oligo(poly)peptides that have these immunogenic properties and consist of at least 8, preferably 15, and more preferably 20 consecutive amino acids in the amino acid sequence of the target protein.

[0062] The production of target protein-binding molecules against such oligo(poly)peptides can be enhanced by using various adjuvants depending on the host, including, but not limited to, Freund's adjuvant, mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol, and human adjuvants such as bacilli Calmette-Guerin (BCG) and Corynebacterium parvum.

[0063] The contacting mode in step (1a) is not particularly limited, and an appropriate mode can be selected depending on the type of detection method for the target protein described above (e.g., various immunoassays, etc.). Examples of contacting modes include a mode in which either the protein (antigen) in the body fluid sample or the target protein-binding molecule is contacted while immobilized on a solid phase, or a mode in which neither of them is immobilized on a solid phase. Among these, from the viewpoint of efficiency, a preferred mode is a mode in which at least one of them is contacted while immobilized on a solid phase. When at least one of the molecules is immobilized on a solid phase in the contacting mode in step (1a), it is preferable to wash the solid phase after immobilization.

[0064] The solid phase is not particularly limited as long as it is capable of immobilizing the target protein or target protein-binding molecule. Examples of the solid phase include plates, slides, and membranes containing polystyrene, glass, nitrocellulose, or the like as a main component. The solid phase may be coated with a component that makes it easier to immobilize the target protein or target protein-binding molecule, such as a reactive compound (e.g., a compound having a reactive group, gold colloid, etc.). Compounds having a readily reactive group are groups that can form covalent bonds with proteins, and examples thereof include compounds having a (1H-imidazol-1-yl)carbonyl group, a succinimidyloxycarbonyl group, an epoxy group, an aldehyde group, an amino group, a thiol group, a carboxyl group, an azide group, a cyano group, an active ester group (e.g., a 1H-benzotriazol-1-yloxycarbonyl group, a pentafluorophenyloxycarbonyl group, a paranitrophenyloxycarbonyl group), or a carbonyl halide group (e.g., a carbonyl chloride group, a carbonyl fluoride group, a carbonyl bromide group, or a carbonyl iodide group).

[0065] Examples of compounds having an easily reactive group include epoxysilane and polylysine.

[0066] The solid phase is preferably blocked using a bovine serum albumin (BSA) buffer solution or the like.

[0067] The manner in which the amount or concentration of the target protein is measured in step (1b) is not particularly limited, and an appropriate manner can be selected depending on the type of the target protein detection method (e.g., various immunoassays, etc.) described above. The measurement can be carried out, for example, by quantifying the signal derived from the label of the label used. In a more specific embodiment, the measurement can be carried out, for example, by contacting a labeled antibody with a complex of the target protein and the target protein-binding molecule, and quantifying the signal derived from the label of the bound labeled antibody.

[0068] One example is the ELISA method. Using the ELISA method, a standard curve can be created using a standard antibody, allowing the concentration of a target protein to be quantified. Standard antibodies can be prepared from commercially available antibodies, for example, by affinity purification using biotin-labeled antigen and streptavidin-agarose resin. First, the wells of an appropriate ELISA plate are coated with the antigen or antibody and then blocked with a BSA buffer solution. Next, various concentrations of the standard antibody or the sample are added to the wells and allowed to stand for a certain period of time. The wells are then washed, and a peroxidase-labeled secondary antibody is added to the wells and allowed to stand for a certain period of time. After washing the wells, a peroxidase substrate is added to the wells and allowed to stand for a certain period of time to develop color. After adding a reaction stop solution such as sulfuric acid, the absorbance is measured using a plate reader. After creating a standard curve based on the concentration of the standard antibody and its absorbance, the concentration of the target protein in the sample is quantified based on the standard curve.

[0069] The amount of the target protein can be calculated based on the amount of the signal obtained. For example, in the case of a non-competitive method, the amount of the signal obtained can be directly used as the amount of the target protein. As another example, in the case of a competitive method, the amount of the signal obtained and the amount of the target protein are inversely proportional to each other, so the amount of the target protein can be calculated from the amount of the signal obtained based on this relationship.

[0070] Furthermore, the concentration of the target protein can be calculated by dividing the amount of the target protein by the amount of the body fluid sample or the amount of a component in the body fluid sample (for example, the total amount of protein).

[0071] According to the testing method of the present invention including step (1), it is possible to provide the amount and / or concentration of a target biomarker that is an indicator for testing clonal hematopoiesis and / or clonal hematopoiesis-related diseases, thereby assisting in testing for clonal hematopoiesis and / or clonal hematopoiesis-related diseases.

[0072] 1-2. Process (2) In one embodiment, the testing method of the present invention preferably further comprises the step of (2) determining the presence or absence of clonal hematopoiesis and / or the presence or absence of a risk of developing and / or aggravating a clonal hematopoietic-associated disease in the subject, based on the amount or concentration of the protein detected in the step (1).

[0073] In addition, one aspect of the present invention relates to a determination method comprising steps (1) and (2).

[0074] In a more specific embodiment, preferably When the protein includes at least one protein (protein A) selected from the group consisting of PYY, FCGR2A, CYTH3, CD8A, TK1, DSC2, IL17D, CD244, APOA4, and MMP7, The step (2) (2Aa) determining that the subject has clonal hematopoiesis and / or a risk of developing and / or aggravating a clonal hematopoietic-associated disorder when the amount or concentration of the protein A detected in the step (1) is equal to or greater than a cutoff value; and / or (2Ab) determining that the subject does not have clonal hematopoiesis and / or does not have a risk of developing and / or aggravating a clonal hematopoietic-associated disorder when the amount or concentration of the protein A detected in the step (1) is equal to or lower than a cutoff value; and / or When the protein includes at least one protein (protein B) selected from the group consisting of LEP, SCN3A, GBP6, GP5, SLC4A1, and BGN, The step (2) (2Aa) determining that the subject has clonal hematopoiesis and / or a risk of developing and / or aggravating a clonal hematopoietic-associated disorder when the amount or concentration of the protein B detected in the step (1) is equal to or lower than a cutoff value; and / or (2Ab) determining that the subject does not have clonal hematopoiesis and / or does not have a risk of developing and / or aggravating a clonal hematopoietic-associated disorder when the amount or concentration of the protein B detected in the step (1) is equal to or greater than a cutoff value; Includes:

[0075] The cutoff value is set in advance based on a database that tracks the amount or concentration of a target protein and the presence or absence of clonal hematopoiesis for a population to be evaluated, and based on statistical analysis or ROC analysis of the data on the amount or concentration of the target protein for subjects with and without clonal hematopoiesis. Alternatively, the cutoff value can be set on a case-by-case basis. When the cutoff value is determined by statistical analysis, for example, the median, arithmetic mean, or other average value of the data on the amount or concentration of the target protein for the population to be evaluated can be used. When the cutoff value is determined by ROC analysis, for example, the cutoff value based on ROC analysis can be the amount or concentration of the target protein at the point on the ROC curve where the distance between the point on the vertical axis (sensitivity or true positive) of the ROC curve graph that is 1.0 and the point on the horizontal axis (1 - specificity) that is 0.0 is the shortest. Alternatively, the cutoff value can be derived from the Youden index of the ROC curve (Cancer 1950;3:32-35.). Once established, the database of the population for evaluation may be used to set the cutoff value in the testing method of the present invention without any changes. Alternatively, new subjects may be incorporated into the population for evaluation, and the database of the population for evaluation may be updated as appropriate and used to set the cutoff value. The cutoff value may be, for example, a percentile value of the amount or concentration of the target protein in a body fluid sample from a reference group of subjects, such as any of the 10th to 90th percentile values, any of the 30th to 70th percentile values, or any of the 40th to 60th percentile values.

[0076] 2. Diagnostic agents for clonal hematopoiesis and / or clonal hematopoietic-related disorders In one aspect, the present invention relates to a diagnostic agent for clonal hematopoiesis and / or clonal hematopoiesis-associated disorders (also referred to herein as the "diagnostic agent of the present invention"), which comprises a target protein-binding molecule. This will be described below.

[0077] The diagnostic agent of the present invention is an agent for testing for clonal hematopoiesis and / or clonal hematopoiesis-associated disorders. Preferably, the diagnostic agent of the present invention is used in the testing method / determination method of the present invention.

[0078] The test agent of the present invention may be in the form of a composition containing a target protein-binding molecule. The composition may contain other components as necessary. Examples of other components include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, moisturizers, colorants, fragrances, chelating agents, etc.

[0079] The test agent of the present invention may be in the form of a kit containing a target protein-binding molecule. The kit may also contain instruments, reagents, etc. that can be used to carry out the test method of the present invention.

[0080] The target protein-binding molecule can be immobilized on any solid phase, and thus the test agent of the present invention can be provided in the form of a substrate on which the target protein-binding molecule is immobilized (e.g., a microarray chip on which a probe is immobilized, or another example, an ELISA plate on which an antibody is immobilized).

[0081] The solid phase used for immobilization is not particularly limited as long as it can immobilize antibodies, etc., and examples thereof include glass plates, nylon membranes, microbeads, silicon chips, capillaries, and other substrates. There are no particular limitations on the immobilization of the detection agent to the solid phase.

[0082] Examples of the apparatus include test tubes, microtiter plates, agarose particles, latex particles, purification columns, epoxy-coated slide glasses, and gold colloid-coated slide glasses.

[0083] Examples of reagents include labeled antibodies and standard samples (positive control, negative control).

[0084] As the labeled antibody, various commercially available antibodies can be used depending on the type (for example, isotype) of the target protein-binding molecule.

[0085] The standard sample used is a target protein, which can be obtained, for example, by culturing cells into which a target protein expression vector has been introduced and purifying the protein from the cells or the culture supernatant.

[0086] 3. Method for screening active ingredients for preventive or therapeutic agents for clonal hematopoietic disorders In one aspect, the present invention relates to a method for screening for an active ingredient of an agent for preventing or treating a clonal hematopoietic disorder, using as an index the amount or concentration of a target protein in a body fluid sample collected from an animal treated with a test substance (also referred to herein as the "active ingredient screening method of the present invention"). This will be described below.

[0087] The species of animal is not particularly limited. Examples of animal species include various mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits. The animal can be, for example, an animal with clonal hematopoiesis. The animal can also be a non-human animal.

[0088] A wide range of test substances can be used, regardless of whether they are naturally occurring or artificially produced. Furthermore, not only purified compounds but also compositions containing a variety of compounds and animal and plant extracts can be used. Compounds are not limited to low-molecular-weight compounds, but also include high-molecular-weight compounds such as proteins, nucleic acids, and polysaccharides.

[0089] When the target protein includes protein A, the method for screening for an active ingredient of the present invention can more specifically include a step of selecting the test substance as an active ingredient for a preventive or therapeutic agent for a clonal hematopoiesis-related disease, for example, when the value of the index for the target protein is lower than the amount or concentration (control value) of the target protein in a body fluid sample collected from an animal that has not been treated with the test substance.

[0090] "Low" means, for example, that the index value is 1 / 2, 1 / 5, 1 / 10, 1 / 20, 1 / 50, or 1 / 100 of the control value.

[0091] When the target protein includes protein B, the method for screening for an active ingredient of the present invention can more specifically include a step of selecting the test substance as an active ingredient for a preventive or therapeutic agent for a clonal hematopoiesis-related disease when the value of the index for the target protein is higher than the amount or concentration (control value) of the target protein in a body fluid sample collected from an animal that has not been treated with the test substance.

[0092] "High" means, for example, that the index value is 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold higher than the control value.

[0093] 4. Method for evaluating the inducibility or exacerbation of clonal hematopoietic disorders In one aspect, the present invention relates to a method for evaluating the inducibility or exacerbation of a clonal hematopoietic disorder, using as an index the amount or concentration of a target protein in a body fluid sample collected from an animal treated with a test substance (also referred to herein as the "toxicity evaluation method of the present invention"). This will be described below.

[0094] The animals and test substances are the same as those in the active ingredient screening method of the present invention.

[0095] More specifically, when the target protein includes protein A, the toxicity evaluation method of the present invention can include a step of determining that the test substance has the ability to induce or exacerbate a clonal hematopoietic-related disorder, for example, when the value of the indicator for the target protein is higher than the amount or concentration (control value) of the target protein in a body fluid sample collected from an animal that has not been treated with the test substance.

[0096] "High" means, for example, that the index value is 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold higher than the control value.

[0097] More specifically, when the target protein includes protein B, the toxicity evaluation method of the present invention can include a step of determining that the test substance has the ability to induce or exacerbate a clonal hematopoietic-related disorder, for example, when the value of the indicator for the target protein is lower than the amount or concentration (control value) of the target protein in a body fluid sample collected from an animal that has not been treated with the test substance.

[0098] "Low" means, for example, that the index value is 1 / 2, 1 / 5, 1 / 10, 1 / 20, 1 / 50, or 1 / 100 of the control value. [Example]

[0099] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0100] Test Example 1. Search for biomarkers of clonal hematopoiesis Genomic DNA extracted from peripheral blood nucleated cells of 1,200 patients with COVID-19 infection was subjected to targeted sequencing using a next-generation sequencer to detect representative gene mutations in clonal hematopoiesis. SNP array analysis was also performed to detect copy number abnormalities associated with clonal hematopoiesis. Additionally, plasma collected from the same 1,200 individuals was analyzed using a proximity extension assay to measure 2,925 plasma proteins. After taking into account subject background factors such as age and gender, the relationship between these genomic abnormalities and the amounts of the 2,925 plasma proteins was evaluated using linear regression. Specifically, the analysis is as follows:

[0101] <1-1. Subject> We enrolled 4,955 hospitalized patients in Japan who were diagnosed with COVID-19 by a physician using clinical symptoms and PCR test results.

[0102] <1-2. Genotyping array and sample filtering> Peripheral blood DNA samples from 4,955 patients were genotyped using the Asian Screening Assay (Illumina). Focusing on variants with a call rate of 99% or higher, the autocorrelation value between the call rate per sample and the B allele frequency (BAF) for each sample was calculated. Samples with a call rate of less than 98% or an autocorrelation of the BAF greater than 3% were excluded as low-quality samples. Additionally, patients with missing clinical information (n = 20), patients who fell outside the Japanese mainland cluster in principal component analysis (n = 209), related or overlapping samples with a PI_HAT > 0.185 (n = 76), and low coverage (n = 1) were excluded. Clonal hematopoiesis (CH) analysis was performed on the remaining 4,541 cases.

[0103] <1-3. Detection of mosaic chromosomal aberrations (mCA)> Copy number analysis was performed on 4,542 Japan COVID-19 Task Force (JCTF) samples based on IDAT intensity data from the genotyping array. IDAT data were converted to GTC genotype files using the Illumina GenCall algorithm and then converted to VCF files containing log2 R ratios (LRRs), B-allele frequencies (BAFs), and unphased genotypes using the bcftools gtc2vcf plugin (https: / / github.com / freeseek / gtc2vcf). Genotyping arrays for JCTF samples were phased using SHAPEIT4 with 54,405 publicly available samples from the Japan Biobank (https: / / humandbs.biosciencedbc.jp / hum0311-v1) to minimize the frequency of phasing errors. Copy number alteration detection was performed using MoChA (https: / / github.com / freeseek / mocha) based on the VCF files containing information on LRRs, BAFs, and phased genotypes.

[0104] From the obtained mCA candidates, germline copy number variations (CNPs) that satisfy at least one of the following conditions: (1) Overpalling common CNPs listed in the integrated SV map of 1000 Genome project phase 3 (https: / / www.internationalgenome.org / category / phase-3 / ). (2) Bdev ≧ 0.1, n50_hets ≧ 2.0×10 5 , and lod_baf_conc ≦ 10.0 (3) lod_baf_phase ≦ 10.0 and congenital duplications that meet at least one of the following criteria: (1) >5Mb mCAs with Bdev > 0.15 (2) 0.5-5 Mb mCAs with relative coverage >2.5 and Bdev > 0.1 (3) <0.5 Mb mCAs with relative coverage >2.1 and Bdev > 0.05 was removed.

[0105] <1-4. Target Capture Sequence> Targeted capture sequencing was performed on peripheral blood DNA samples from 4,542 JCTF patients. Thirty-nine genes / regions were captured using the Lotus DNA Library Prep Kit and xGen lockdown probes (Integrated DNA Technologies) according to the manufacturer's instructions, followed by massively parallel sequencing in standard 150-bp paired-end mode using the DNBSEQ-G400 (MGI) as previously described.

[0106] <1-5. Detection of gene mutations> Sequence reads were aligned to the human genome reference (hg19) using Burrows-Wheeler Aligner v.0.7.8 (https: / / sourceforge.net / projects / bio-bwa / ) with default parameters. Variant calling was performed using Genomon2 pipeline v.2.6.2 (https: / / genomon.readthedocs.io / ja / latest), picard-tools v.1.39 (http: / / picard.sourceforge.net / ), and GenomonMutationFilter v.0.2.1 (https: / / github.com / Genomon-Project / GenomonMutationFilter) as previously described. Extracted SNVs / indels were annotated using ANNOVAR (https: / / annovar.openbioinformatics.org / en / latest / ). Variants meeting the following criteria were then included: 1. Number of variant reads ≧10 2. Depth of coverage ≧50 3. VAF ≧ 2% 4. Nonsynonymous variants within coding sequence or splice-site variants Only base calls with a mapping quality score ≥ 40 and a base quality score ≥ 20 were counted for calculation of read count, depth, and VAF.

[0107] To further exclude false-positive calls due to sequencing artifacts, the site-specific error rate was modeled as a beta-binomial distribution using the R package VGAM 1.1.3 (https: / / cran.r-project.org / web / packages / VGAM / index.html). The parameters of the beta-binomial distribution of the error rate were determined by maximum likelihood based on the read counts of all samples. Mutation calls whose VAF deviated significantly from the background error distribution (Pbeta -binomial ≦ 10 -6 ) was considered as a genuine candidate.

[0108] Candidates were further curated to minimize contamination by germline polymorphisms. For mutations in NPM1, MPL, MYD88, GNB1, GNAS, JAK2, KRAS, NRAS, IDH1, IDH2, SF3B1, SRSF2, U2AF1, KIT, WT1, PTPN11, CALR, FLT3, STAT3, CEBPA, and CBL, only established hotspot mutations were called. For mutations in other genes, candidates that fulfilled at least one of criteria A and B were considered driver mutations: Standard A Variants with a POPMAX frequency of less than 1% in The Exome Aggregation Consortium (ExAC) database (https: / / gnomad.broadinstitute.org / ). Standard B 1. At least 15 counts of candidate amino acid substitutions in the "Hematopoietic and lymphoid" category registered in the Catalogue of Somatic Mutations in Cancer v.91 database (https: / / cancer.sanger.ac.uk / cosmic). 2. Candidates with VAF<40, ≥60 or <90% 3. Nonsense, frameshift, and splice site candidates of DNMT3A, TET2, ASXL1, PPM1D, and TP53.

[0109] Finally, the resulting set of candidates was manually reviewed with Integrated Genome Viewer 2.4.6 ( http: / / software.broadinstitute.org / software / igv / ).

[0110] <1-6. Calculation of cell fraction for gene mutation> Basically, the cellular fraction of gene mutations (CF 遺伝子変異)は2 x VAF 遺伝子変異と However, if the mutation is on the X chromosome in a male patient, CF 遺伝子変異は VAF 遺伝子変異と Furthermore, combinations of gene mutations and mCA, which likely coexist within the same cells, were repeatedly observed in ATM, DNMT3A, JAK2, TET2, and TP53 (n=23). For these gene mutations, the effect of mCA was also taken into account in calculating the cell fraction as follows: CNN-LOH:CF 遺伝子変異 =VAF 遺伝子変異 ×2-CF mCA Deletion: CF 遺伝子変異 = VAF 遺伝子変異 × ( 2 - CF mCA ) Duplicate: CF 遺伝子変異 = VAF 遺伝子変異 × ( 1 + CF mCA ) × 2 - CF mCA <1-7. Plasma proteome analysis> Proteomic analysis was performed using plasma samples from 1,200 of the 4,542 JCTF subjects. The library (Olink Explore 3072) consisted of 2,925 unique proteins and was divided into eight 384-plex panels focused on inflammation, tumor, cardiometabolic, and neuronal proteins. Each of the four 384-plex panels included overlapping assays for IDO1, LMOD1, SCRIB, IL-6, IL-8 (CXCL8), and TNF for quality control (QC) purposes. PEA uses oligonucleotide-conjugated monoclonal or polyclonal antibodies (PEA probes) to bind to target proteins in a pairwise manner, preventing any cross-reactivity. Upon binding, the oligonucleotides come into close proximity, hybridize, and then elongate, generating a unique sequence used for digital identification of specific protein assays. For the immunoassay, 2.8 mL of sample was mixed with the PEA probe and incubated overnight at 4°C. The combined extension and preamplification mix is ​​then added to the samples, which have been incubated at room temperature, and PCR is performed. The PCR products are pooled before a second PCR step, which follows the addition of index sequences from each sample. All samples are then pooled, bead-purified, and the libraries are QC'd on a Bioanalyzer. Finally, sequencing is performed on a NovaSeq 6000 system. Counts of known sequences are converted to normalized protein expression (NPX) using a QC and normalization process developed and provided by Olink.

[0111] <1-8.Statistical analysis> All statistical analyses were performed using the R statistical platform (https: / / www.r-project.org / ) v.4.2.1. All statistical tests were two-sided. Benjamini-Hochberg multiple testing correction was applied where appropriate.

[0112] <1-9. Analysis of proteome data> The effects of COVID-19 and CH severity on protein expression were investigated. The association with severe COVID-19 was investigated using linear regression with the following model: Protein: NPX ~ severe_COVID19 + age + age 2 + sex + experimental_batch, Analysis of CH was performed using linear regression with the following model: Protein: NPX ~ CH+ age + age 2 + sex + experimental_batch + Severity_of_COVID19, Here, patients with genetic mutations and / or mCA with a clone size ≥ 10% (percentage of mutant cells in peripheral blood) were compared with patients without genetic mutations or mCA. COVID-19 severity was incorporated into the model to assess the independent effect of CH on protein expression.

[0113] For pathway analysis, proteins were sorted by z-score calculated by linear regression for their association with severe COVID-19 or CH. Based on the resulting protein ordering, gene set enrichment analysis (GSEA) was performed on 50 hallmark gene sets from MSigDB (http: / / www.gsea-msigdb.org / gsea / msigdb / collections.jsp) using the R package ClusterProfiler 4.6.0 (https: / / bioconductor.org / packages / release / bioc / html / clusterProfiler.html).

[0114] <1-10.Results> Plasma protein expression correlates with the following four: (a) Association with clonal hematopoiesis (clone size ≥ 10%); (b) Clonal hematopoiesis (clone size ≥ 10%) and associated with genetic mutations (c) Clonal hematopoiesis (clone size ≥ 10%) and associated with copy number variation; (d) The association between clonal hematopoiesis (clone size ≥ 10%), gene mutations, and copy number mutations was plotted in a volcano plot (vertical axis: -log10(P), horizontal axis: effect size). Results showing a significant association (P<1e-05) are shown in Table 1.

[0115] [Table 1] After judging including non-significant results for each association, PYY and LEP (especially PYY) were biomarkers with good accuracy for determining clonal hematopoiesis.

Claims

1. (1) A method for testing for clonal hematopoiesis and / or clonal hematopoiesis-related disorders, comprising the step of detecting at least one protein selected from the group consisting of PYY, LEP, SCN3A, FCGR2A, CYTH3, CD8A, TK1, GBP6, DSC2, GP5, IL17D, CD244, SLC4A1, BGN, APOA4, and MMP7 in a body fluid sample collected from a subject.

2. The method of claim 1 , wherein the protein comprises PYY and / or LEP.

3. The method of claim 1 , wherein the protein comprises PYY.

4. Furthermore, (2) determining the presence or absence of clonal hematopoiesis and / or the presence or absence of a risk of developing and / or aggravating a clonal hematopoietic-associated disease in the subject based on the amount or concentration of the protein detected in the step (1); The method according to any one of claims 1 to 3, comprising:

5. When the protein includes at least one protein (protein A) selected from the group consisting of PYY, FCGR2A, CYTH3, CD8A, TK1, DSC2, IL17D, CD244, APOA4, and MMP7, The step (2) (2Aa) determining that the subject has clonal hematopoiesis and / or a risk of developing and / or aggravating a clonal hematopoietic-associated disorder when the amount or concentration of the protein A detected in the step (1) is equal to or greater than a cutoff value; and / or (2Ab) determining that the subject does not have clonal hematopoiesis and / or does not have a risk of developing and / or aggravating a clonal hematopoietic-associated disorder when the amount or concentration of the protein A detected in the step (1) is equal to or lower than a cutoff value; and / or When the protein includes at least one protein (protein B) selected from the group consisting of LEP, SCN3A, GBP6, GP5, SLC4A1, and BGN, The step (2) (2Aa) determining that the subject has clonal hematopoiesis and / or a risk of developing and / or aggravating a clonal hematopoietic-associated disorder when the amount or concentration of the protein B detected in the step (1) is equal to or lower than a cutoff value; and / or (2Ab) determining that the subject does not have clonal hematopoiesis and / or does not have a risk of developing and / or aggravating a clonal hematopoietic-associated disorder when the amount or concentration of the protein B detected in the step (1) is equal to or greater than a cutoff value; The method of claim 4, comprising:

6. The method according to any one of claims 1 to 3, wherein the clonal hematopoiesis-related disease is at least one selected from the group consisting of hematopoietic tumors, cardiovascular diseases, cerebrovascular diseases, Alzheimer's disease, respiratory diseases, and infectious diseases.

7. The method according to any one of claims 1 to 3, wherein the body fluid sample is at least one selected from the group consisting of whole blood, serum, and plasma.

8. The method according to any one of claims 1 to 4, wherein the subject is a human.

9. A diagnostic agent for clonal hematopoiesis and / or clonal hematopoiesis-related diseases, comprising a binding molecule for at least one protein selected from the group consisting of PYY, LEP, SCN3A, FCGR2A, CYTH3, CD8A, TK1, GBP6, DSC2, GP5, IL17D, CD244, SLC4A1, BGN, APOA4, and MMP7.

10. A method for screening for an active ingredient of a preventive or therapeutic agent for a clonal hematopoietic-related disease, using as an indicator the amount or concentration of at least one protein selected from the group consisting of PYY, LEP, SCN3A, FCGR2A, CYTH3, CD8A, TK1, GBP6, DSC2, GP5, IL17D, CD244, SLC4A1, BGN, APOA4, and MMP7 in a body fluid sample collected from an animal treated with a test substance.

11. A method for evaluating the inducibility or exacerbation of a clonal hematopoietic-related disorder, using as an indicator the amount or concentration of at least one protein selected from the group consisting of PYY, LEP, SCN3A, FCGR2A, CYTH3, CD8A, TK1, GBP6, DSC2, GP5, IL17D, CD244, SLC4A1, BGN, APOA4, and MMP7 in a body fluid sample collected from an animal treated with a test substance.

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