Method, support device, and support program for assisting in the detection of arteriosclerosis.

Abstract

To provide a support method, a support apparatus and a support program for supporting detection of the presence / absence of arteriosclerosis by utilizing a measured value of a biomarker in blood plasma.SOLUTION: A method for supporting detection of the presence / absence of arteriosclerosis includes acquiring a measured value of free DNA derived from a cell of a subject in blood plasma collected from the subject. It is suggested, in the case where the measured value is a reference value corresponding to the measured value or higher, that the subject has arteriosclerosis, and / or it is suggested, in the case where the measured value is a reference value corresponding to the measured value or lower, that the subject does not have arteriosclerosis.SELECTED DRAWING: None

Description

Technical Field

[0001] This specification discloses a method, a support device, and a support program for assisting in detecting the presence or absence of arteriosclerosis. This specification also discloses a method, a support device, and a support program for assisting in detecting a subject belonging to a pre-group of ischemic cardiovascular disease. Furthermore, this specification discloses a method, a stratification device, and a stratification program for stratifying the degree of progression of arteriosclerosis in a subject.

Background Art

[0002] One of the main causes of heart failure is arteriosclerosis, and its simple and early diagnosis is important in preventive medicine.

[0003] It is known that oxidative stress is involved in the onset mechanism of arteriosclerosis. Non-Patent Document 1 shows that oxidative stress induces the most lethal double-strand DNA breaks in vascular endothelial cells and vascular smooth muscle cells, and double-strand breaks and oxidative damage to DNA are observed in actual atherosclerotic lesions.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] However, arteriosclerosis is often asymptomatic, and people rarely notice it until it progresses to its end stages, such as myocardial infarction or stroke. Generally, if abnormalities in blood cholesterol levels, LDL cholesterol levels, etc., which are risk factors for arteriosclerosis, are pointed out during a health checkup, then imaging tests of the blood vessel walls are performed to confirm the presence of plaque in the blood vessels before treatment begins. Imaging tests include carotid artery ultrasound using ultrasound equipment, CT scans, and catheterization, but these require specialized equipment and the skills of medical professionals.

[0006] One objective of the present invention is to provide a support method, support device, and support program for assisting in the detection of the presence or absence of arteriosclerosis using measured values ​​of biomarkers in plasma. Another objective of the present invention is to provide a method, support device, and support program for assisting in the detection of subjects belonging to the pre-ischemic cardiovascular disease group using measured values ​​of biomarkers in plasma. Furthermore, another objective of the present invention is to provide a method, stratification device, and stratification program for stratifying the progression of arteriosclerosis in subjects using measured values ​​of biomarkers in plasma. [Means for solving the problem]

[0007] The present invention includes the following embodiments. Section 1. A method for assisting in the detection of the presence or absence of arteriosclerosis, The aforementioned method, This includes obtaining measurements of free DNA derived from the subject's cells in plasma collected from the subject, If the measured value is equal to or greater than the reference value corresponding to the measured value, it is suggested that the subject has arteriosclerosis, and / or if the measured value is lower than the reference value corresponding to the measured value, it is suggested that the subject does not have arteriosclerosis. method. Section 2. The method according to item 1, wherein the free DNA derived from the cells of the subject is mitochondrial DNA. Section 3. A support device for assisting in the detection of the presence or absence of arteriosclerosis, The support device includes a processing unit, The aforementioned processing unit, We obtained measurements of free DNA derived from the subject's cells in plasma collected from the subject. If the measured value is equal to or greater than the reference value corresponding to the measured value, a label is output suggesting that the subject has arteriosclerosis, and / or if the measured value is lower than the reference value corresponding to the measured value, a label is output suggesting that the subject does not have arteriosclerosis. Support equipment. Section 4. When you run it on a computer, the computer will A step of obtaining a measurement of free DNA derived from the subject's cells in plasma collected from the subject, The steps include: outputting a label suggesting that the subject has arteriosclerosis if the measured value is equal to or greater than a reference value corresponding to the measured value, and / or outputting a label suggesting that the subject does not have arteriosclerosis if the measured value is lower than a reference value corresponding to the measured value; A support program that enables the detection of arteriosclerosis. Section 5. A method for assisting in the detection of subjects belonging to the pre-symptomatic group of ischemic cardiovascular disease, The aforementioned method, This includes obtaining measurements of free DNA derived from the subject's cells in plasma collected from the subject, If the measured value is equal to or greater than the corresponding reference value, it is suggested that the subject belongs to the group at risk of ischemic cardiovascular disease, and / or if the measured value is lower than the corresponding reference value, it is suggested that the subject does not belong to the group at risk of ischemic cardiovascular disease. method. Section 6. A support device for assisting in the detection of subjects belonging to the pre-symptomatic group of ischemic cardiovascular disease, The support device includes a processing unit, The aforementioned processing unit, We obtained measurements of free DNA derived from the subject's cells in plasma collected from the subject. If the measured value is equal to or greater than the reference value corresponding to the measured value, a label is output suggesting that the subject belongs to the group at risk of ischemic cardiovascular disease, and / or if the measured value is lower than the reference value corresponding to the measured value, a label is output suggesting that the subject does not belong to the group at risk of ischemic cardiovascular disease. Support equipment. Section 7. When you run it on a computer, the computer will A step of obtaining a measurement of free DNA derived from the subject's cells in plasma collected from the subject, The steps include: outputting a label suggesting that the subject belongs to the pre-ischemic cardiovascular disease group if the measured value is equal to or greater than the reference value corresponding to the measured value, and / or outputting a label suggesting that the subject does not belong to the pre-ischemic cardiovascular disease group if the measured value is lower than the reference value corresponding to the measured value; A support program designed to assist in the detection of subjects who are at risk of ischemic cardiovascular disease. Section 8. A method for supporting the stratification of the progression of arteriosclerosis in subjects, The aforementioned method, To obtain measurements of free DNA derived from the subject's mitochondria in plasma collected from the subject, This includes searching for the reference range to which the measured value belongs from among several reference ranges set according to the progression of arteriosclerosis. The degree of arteriosclerosis progression corresponding to the reference range to which the aforementioned measurement value belongs is suggested as the degree of arteriosclerosis progression in the subject. method. Section 9. A stratification device for supporting the stratification of the progression of arteriosclerosis in a subject, The stratification apparatus includes a processing unit, The aforementioned processing unit, We obtained measurements of free DNA derived from the subject's mitochondria in plasma collected from the subject. Search for the reference range to which the measurement value belongs from a plurality of reference ranges set according to the degree of progression of arteriosclerosis, Output, as a label indicating the degree of progression of arteriosclerosis in the subject, the label of the degree of progression of arteriosclerosis corresponding to the reference range to which the measurement value belongs. Stratification device. Item 10. When causing a computer to execute, cause the computer to Obtain a measurement value of free DNA derived from mitochondria of a subject in plasma collected from the subject; Search for the reference range to which the measurement value belongs from a plurality of reference ranges set according to the degree of progression of arteriosclerosis; Output, as a label indicating the degree of progression of arteriosclerosis in the subject, the label of the degree of progression of arteriosclerosis corresponding to the reference range to which the measurement value belongs; A stratification program for assisting in stratifying the degree of progression of arteriosclerosis in a subject, which causes the above to be executed. Item 11. A biomarker in plasma, wherein the biomarker is free DNA derived from cells of a subject, and the biomarker is for use in detecting the presence or absence of arteriosclerosis or for use in detecting a subject belonging to a pre-group of ischemic cardiovascular diseases. Item 12. A biomarker in plasma, wherein the biomarker is free DNA derived from mitochondria of cells of a subject, and the biomarker is for use in stratifying the degree of progression of arteriosclerosis in the subject.

Effect of the Invention

[0008] <00001​​​

[0009] [Figure 1] The hardware configuration of support devices 10 and 20 is shown. [Figure 2] Figure 2(A) is a flowchart showing the processing flow of support program 1042a. Figure 2(B) is a flowchart showing the processing flow of support program 2042a. [Figure 3] The hardware configuration of the stratification device 30 is shown. [Figure 4] This is a flowchart showing the processing flow of the stratification program 3042a. [Figure 5] Figure 5(A) shows the results of immunostaining for γ-H2AX. Figure 5(A)(a) is a stained image of γ-H2AX in the nucleus of a control (ctl) cell before the addition of CSE. Figure 5(A)(b) is a stained image of γ-H2AX in cells 72 hours after the start of CSE treatment. Figure 5(A)(c) is a graph showing the mean and standard deviation over time, calculated by counting the number of γ-H2AX foci per cell for multiple cells. Figure 5(B) shows the results of immunostaining for 8-oxo-dG. Figure 5(B)(a) is a stained image of 8-oxo-dG in a control (ctl) cell before the addition of CSE. Figure 5(B)(b) is a stained image of 8-oxo-dG in cells 72 hours after the start of CSE treatment. Figure 5(B)(c) is a graph showing the fluorescence intensity of 8-oxo-dG in the nucleus over time. Figures 5(B) and 5(d) are graphs showing the fluorescence intensity of 8-oxo-dG in the cytoplasm over time. Figure 5(C) shows DNA staining (DAPI), mitochondrial staining (Mitotracker), 8-oxo-dG immunostaining (8-oxo-dG), and a merge image of these three stains in one cell 24 hours after CSE addition. [Figure 6]Figure 6(A) shows the increase in mitochondrial DNA (Cytosolic mtDNA) in the cytoplasm on days 1, 3, and 7 after the start of CSE treatment. Figure 6(B) shows the increase in nuclear DNA (Cytosolic nDNA) in the cytoplasm on days 1, 3, and 7 after the start of CSE treatment. [Figure 7] Figure 7(A) shows the increase in mitochondrial DNA (mtDNA) in the culture supernatant of HUVEC cells after CSE treatment. Figure 7(B) shows the increase in nuclear DNA (nDNA) in the same culture supernatant as in Figure 7(A). [Figure 8] Figure 8(A) shows the breakdown of subjects. Figure 8(B) shows the distribution of copy numbers of cfDNA derived from mitochondrial DNA in the subjects' plasma. Figure 8(C) shows the distribution of copy numbers of cfDNA derived from nuclear DNA in the subjects' plasma. [Figure 9] The clinical information of the subject is shown. [Figure 10] Figure 10(A) shows the distribution of copy numbers of cfDNA (mt-cfDNA) derived from mitochondrial DNA in the subject's plasma. Figure 10(B) shows the distribution of copy numbers of cfDNA (n-cfDNA) derived from nuclear DNA in the subject's plasma. [Figure 11] Figure 11(A) shows the distribution of copy numbers of cfDNA (mt-cfDNA) derived from mitochondrial DNA in the subject's plasma. Figure 11(B) shows the distribution of copy numbers of cfDNA (n-cfDNA) derived from nuclear DNA in the subject's plasma. [Figure 12] Figure 12(A) shows the correlation between the copy number of cfDNA (mt-cfDNA) derived from mitochondrial DNA in the subject's plasma and plaque thickness. Figure 12(B) shows the correlation between the copy number of cfDNA (n-cfDNA) derived from nuclear DNA in the subject's plasma and plaque thickness. [Figure 13] Figure 13(A) shows the correlation between the copy number of cfDNA (mt-cfDNA) derived from mitochondrial DNA in the subject's plasma and age. Figure 13(B) shows the correlation between the copy number of cfDNA (n-cfDNA) derived from nuclear DNA in the subject's plasma and age. [Figure 14] Figure 14(A) shows the correlation between the copy number of cfDNA (mt-cfDNA) derived from mitochondrial DNA in the subject's plasma and the serum creatinine concentration. Figure 14(B) shows the correlation between the copy number of cfDNA (n-cfDNA) derived from nuclear DNA in the subject's plasma and the serum creatinine concentration. [Figure 15] The copy number of cfDNA in plasma indicates the accuracy of its use as a biomarker for detecting arteriosclerosis. [Modes for carrying out the invention]

[0010] 1. Support for detecting the presence or absence of arteriosclerosis. 1-1. Biomarkers for detecting the presence or absence of arteriosclerosis, and methods for supporting the detection of arteriosclerosis. One embodiment of the present invention relates to a method for assisting in the detection of the presence or absence of arteriosclerosis. The method includes obtaining a measurement of free DNA derived from the subject's cells in plasma collected from the subject. Free DNA derived from the subject's cells in plasma collected from the subject can be used as a biomarker for detecting the presence or absence of arteriosclerosis.

[0011] In this specification, arteriosclerosis refers to a condition in which the blood vessels of the arteries harden and lose their elasticity. In arteriosclerosis, lipids such as cholesterol are deposited on the walls of the blood vessels (especially arteries) to form plaque, and at the same time inflammation is induced, which changes the composition of the blood vessel walls and causes a loss of elasticity in the blood vessels.

[0012] In this specification, the presence or absence of arteriosclerosis can be determined, for example, by measuring the thickness of carotid plaques using carotid echo and based on the thickness. For example, according to the method described in Handa N, et al. Stroke 1990;21:1567-1572, the thickness of plaques in the left and right carotid arteries is measured by carotid echo, and the sum of the thicknesses is calculated as the plaque score (PS). The classification of PS can be set as follows: PS ≤ 1 mm is normal, 1 mm < PS ≤ 5 mm is mild, 5 mm < PS ≤ 10 mm is moderate, and 10 mm < PS is severe. Also, when PS is 1 mm or less, it can be determined as plaque-free or negative, and when PS exceeds 1 mm, it can be determined as plaque-positive or positive.

[0013] In this specification, the subject is not limited as long as it is an individual to be examined. Examples of the subject include humans, dogs, cats, mice, rats, rabbits, etc. Preferably, the subject is a human.

[0014] The sample for obtaining the measured value of free DNA derived from the cells of the subject is a blood sample collected from the subject. The blood sample may contain plasma and serum. Preferably, the sample is plasma.

[0015] Plasma can be collected according to a conventional method. Blood collection for collecting plasma is not limited as long as anticoagulants are used for blood collection. As anticoagulants, Ethylenediaminetetraacetic acid (EDTA) salts, sodium citrate, CPD solution, ACD solution, heparin salts, etc. can be used. Preferably, the anticoagulant is an EDTA salt, and more preferably, it is sodium EDTA. "Derived from the cells of the subject" is intended to mean that it does not include those derived from pathogens such as viruses, bacteria, and fungi.

[0016] Free DNA refers to DNA released from the cell into the extracellular space of a subject for any reason. In this specification, free DNA is also referred to as "Cell-free DNA: cfDNA". Free DNA may include DNA derived from mitochondria and DNA derived from the cell nucleus (hereinafter sometimes simply referred to as "nucleus"). Free DNA is preferably derived from mitochondria.

[0017] "Obtaining measurements of free DNA" includes measuring the copy number or concentration of free DNA and obtaining the measurement. Furthermore, "Obtaining measurements of free DNA" also includes a computer obtaining previously measured copy number or concentration data of free DNA via a network by reading from a storage medium or inputting from an input device.

[0018] The copy number or concentration of free DNA can be measured, for example, by real-time quantitative PCR, next-generation sequencer, or third-generation sequencer DNA sequencing.

[0019] Methods for recovering free DNA from samples are well known. For example, free DNA can be recovered by column chromatography using commercially available kits such as NucleoSpin™ cfDNA XS (TAKARA, Japan), QIAamp MinElute ccfDNA Kits (QIAGEN), and EZ1&2 ccfDNA Kit (QIAGEN). Alternatively, free DNA may be recovered by the conventional phenol-chloroform-ethanol precipitation method.

[0020] Let's consider an example of measuring the copy number of free DNA using real-time quantitative PCR. For example, if the free DNA is derived from mitochondria, genes encoded on mitochondrial DNA can be targeted and amplified. Examples of genes encoded on mitochondrial DNA include NADH dehydrogenase subunit 1, COX-III (Cytochrome C Subunit III), the non-coding region of human mitochondrial genes, and mtDNA 16S. For example, if the free DNA is derived from the cell nucleus, genes encoded on genomic DNA can be targeted and amplified. Examples of genes encoded on genomic DNA include β-globin, GAPDH, β-actin, beta-2-microglobulin, eukaryotic translation initiation factor 2C1 (EIF2C1), and rhodopsin.

[0021] Real-time quantitative PCR is not limited as long as it can amplify DNA containing all or part of the target gene and quantify the target DNA. Examples of real-time quantitative PCR include the TaqMan® probe method and the intercalator method using double-stranded DNA-binding fluorescent dyes such as SYBR® green I. The forward primers and reverse primers required for real-time quantitative PCR, the fluorescent dye used in the TaqMan® probe method, and the probe labeled with a quencher for the fluorescent dye can be appropriately set according to the gene to be amplified. Furthermore, the composition of the PCR reaction solution and reaction conditions can also be appropriately set according to the gene to be amplified.

[0022] For example, the sequences of primers for detecting the NADH1 gene derived from mitochondria and the β-globin gene derived from genomic DNA are as follows. "F" indicates a forward primer and "R" indicates a reverse primer. NADH1_F:5'-ATACCCATGGCCAACCTCCT-3' (Sequence ID 1) NADH1_R:5'-GGGCCTTTGCGTAGTTGTAT-3' (Sequence ID 2) β-globin_F:5'-GTGCACCTGACTCCTGAGGAGA-3' (Sequence ID 3) β-globin_R:5'-CCTTGATACCAACCTGCCCAG-3' (SEQ ID NO: 4)

[0023] In real-time quantitative PCR, the copy number of the template DNA contained in the PCR reaction solution is reflected in the Ct (Threshold Cycle) value. Real-time quantitative PCR can convert the Ct value obtained by amplifying the template DNA into a copy number using a standard curve. The curve is created using multiple standard solutions that contain polynucleotides containing the target nucleotide sequence to be amplified, have known copy numbers, and each standard solution contains polynucleotides with different copy numbers. The standard curve is generated from the Ct values ​​and copy numbers of each standard solution obtained by amplifying the multiple standard solutions with template DNA, PCR reagents, and the same PCR conditions. By applying the Ct value obtained by amplifying the template DNA to this standard curve, the copy number of the target DNA contained in the template DNA is determined. The standard curve can be generated by the following method: The concentration of synthetic DNA (polynucleotide containing the target nucleotide sequence to be amplified) is measured with a spectrophotometer, and the DNA concentration is converted to a copy number by multiplying this absorbance by the molecular weight and Avogadro's number. PCR is performed simultaneously with the sample using synthetic DNA with a determined copy number, and a standard curve is generated with the DNA copy number (log) on ​​the x-axis and the Ct value on the y-axis.

[0024] Furthermore, the copy number obtained from the standard curve can be adjusted considering the initial and final elution volumes of the sample using the following formula reported in Rosa HS et al. FASEB J. 2020 Sep;34(9):12278-12288, and expressed as copy number / plasma μL.

number

[0025] When measuring the copy number of free DNA by DNA sequencing, the DNA copy number can be obtained by mapping the sequences of each DNA fragment contained in the sample, obtained by DNA sequencing, to a reference sequence and calculating its frequency of occurrence. The copy number can be converted to concentration from the molecular weight of the target DNA.

[0026] The measured value of free DNA is compared to a corresponding reference value. The "corresponding reference value" is the reference value determined based on the measured value of free mitochondrial DNA if the free DNA originates from mitochondria, or the reference value determined based on the measured value of free nuclear DNA if the free DNA originates from the cell nucleus.

[0027] Reference values ​​are used to distinguish between plaque-negative and plaque-positive groups. Reference values ​​are determined, for example, based on measurements of free DNA obtained from samples of a negative control group (determined to be plaque-negative based on the PS described above) and a positive control group (determined to be plaque-positive based on the PS described above). Reference values ​​can be determined using methods such as the ROC (Receiver Operating Characteristic curve) curve, discriminant analysis, modal method, Kittler method, 3σ method, and p-tile method. Examples of reference values ​​include sensitivity, specificity, negative predictive value, positive predictive value, and first quartile.

[0028] In the above method, if the measured value is equal to or greater than the reference value corresponding to the measured value, it is suggested that the subject has arteriosclerosis. Conversely, if the measured value is lower than the reference value corresponding to the measured value, it is suggested that the subject does not have arteriosclerosis.

[0029] 1-2. Support devices for detecting the presence or absence of arteriosclerosis One embodiment of the present invention relates to a support device 10 (hereinafter also simply referred to as "support device 10") for assisting in the detection of the presence or absence of arteriosclerosis. The hardware configuration of the support device 10 will be explained using Figure 1.

[0030] The support device 10 may be connected to the input device 111, the output device 112, and the media drive 113. Alternatively, the support device 10 may be connected to the analyzer 90 via a wired or wireless network to form a support system 1000 for assisting in the detection of arteriosclerosis. The analyzer 90 may be a real-time quantitative PCR instrument, a next-generation sequencer, a third-generation sequencer, etc.

[0031] In the support device 10, the processing unit 101, memory 102, ROM (read-only memory) 103, storage device 104, communication interface (I / F) 105, input interface (I / F) 106, output interface (I / F) 107, and media interface (I / F) 108 are connected to each other via a bus 109, enabling data communication. The memory 102 and storage device 104 together are sometimes simply referred to as the storage unit. The storage unit stores the measured values ​​and reference values ​​either volatilely or non-volatilely.

[0032] The processing unit 101 is the CPU of the support device 10, and is also called the arithmetic unit. The processing unit 101 may also cooperate with the GPU. The processing unit 101 cooperates with the operating system (OS) 1041 stored in the memory device 104 or ROM 103 to execute the disease information support program 1042a (hereinafter sometimes simply referred to as "support program 1042a"), which will be described later, and processes the acquired data, thereby enabling the computer to function as the support device 10.

[0033] ROM 103 stores the support program 1042a executed by the processing unit 101 and the data used therefor. The processing unit 101 may also be the MPU 101. ROM 103 stores the boot program executed by the processing unit 101 when the support device 10 is started, as well as programs and settings related to the operation of the support device 10's hardware.

[0034] Memory 102 is used to read the support programs 1042a recorded in ROM 103 and storage device 104. Memory 102 is also used as a workspace when the processing unit 101 executes these support programs 1042a.

[0035] The storage device 104 is composed of a hard disk or the like. The storage device 104 stores various support programs 1042a, such as an operating system and application programs, which are to be executed by the processing unit 101, as well as various setting data used to execute the support programs 1042a. Specifically, it non-volatilely stores a reference value database (DB) DB1 that stores reference values.

[0036] The communication interface 105, under the control of the processing unit 101, receives data from the analyzer 90 or other external devices, and transmits or displays information stored or generated by the support device 10 to the analyzer 90 or externally as needed. The communication interface 105 may also communicate with the analyzer 90 or other external devices via a network.

[0037] The input interface 106 accepts text input, clicks, voice input, etc., from the input device 111. The received input is stored in memory 102 or storage device 104.

[0038] The input device 111 consists of a touch panel, keyboard, mouse, pen tablet, microphone, etc., and is used to input text or voice to the support device 10. The input device 111 may be connected from outside the support device 10 or may be integrated with the support device 10.

[0039] The output interface 107 outputs the information generated by the processing unit 101 to the output device 112. The output interface 107 also outputs the information generated by the processing unit 101 and stored in the storage device 104 to the output device 112.

[0040] The output device 112 consists of, for example, a display, a printer, etc., and displays the measurement results transmitted from the analyzer 90, as well as various operation windows and analysis results in the support device 10.

[0041] The media interface 108 reads, for example, application software stored in the media drive 113. The read application software is stored in memory 102 or storage device 104. The media interface 108 also writes information generated by the processing unit 101 to the media drive 113. The media interface 108 writes information generated by the processing unit 101 and stored in storage device 104 to the media drive 113.

[0042] The media drive 113 consists of a flexible disk, CD-ROM, or DVD-ROM, etc. The media drive 113 is connected to the media interface 108 by a flexible disk drive, CD-ROM drive, or DVD-ROM drive, etc. The media drive 113 may also store application programs, etc., for the computer to perform operations.

[0043] The processing unit 101 may obtain application software and various settings necessary for controlling the support device 10 via the network instead of reading them from the ROM 103 or storage device 104. Alternatively, the application program may be stored in a storage device on a server computer on the network, and the support device 10 can access this server computer to download the support program 1042a and store it in the ROM 103 or storage device 104.

[0044] Furthermore, the ROM 103 or storage device 104 has an operating system installed that provides a graphical user interface environment, such as Windows®, manufactured and sold by Microsoft Corporation in the United States. The application program according to the second embodiment shall run on the operating system. That is, the support device 10 may be a personal computer or the like.

[0045] The support system 1000 does not need to be installed in one location; the support device 10 and the analysis device 90 may be located in separate places and connected by a network. Furthermore, the support device 10 may be a device that does not require an operator, omitting the input device 111 and output device 112.

[0046] 1-3. Processing of support programs to assist in the detection of arteriosclerosis. Using Figure 2(A), we will explain the process performed by the support program 1042a, which assists in the detection of the presence or absence of arteriosclerosis.

[0047] The processing unit 101 of the support device 10 starts support processing to assist in the detection of the presence or absence of arteriosclerosis when the operator inputs the start of processing from the input device 111. In step S11, the processing unit 101 obtains the measured value of free DNA derived from the subject's cells in the plasma collected from the subject from the analyzer 90. The details of obtaining the measured value of free DNA derived from the subject's cells in the plasma collected from the subject are the same as those described in 1-1 above, so the explanation in 1-1 above will be incorporated here.

[0048] Next, in step S12, the processing unit 101 refers to the reference value database DB1 stored in the storage device 104 and compares the measured value with the reference value corresponding to the measured value. In step S12, if the measured value is greater than or equal to the reference value ("YES"), the processing unit 101 proceeds to step S13 and outputs a label indicating that the subject has arteriosclerosis. Alternatively, in step S12, if the measured value is lower than the reference value ("NO"), the processing unit 101 proceeds to step S14 and outputs a label indicating that the subject does not have arteriosclerosis. Or, in step S12, if the measured value is lower than the reference value ("NO"), the processing unit 101 may terminate the process without proceeding to step S14.

[0049] Labels indicating that a subject has arteriosclerosis may be marks such as ×, *, or exclamation marks. They may also be text such as "Yes" or "Risk Present." Labels indicating that a subject does not have arteriosclerosis may be marks such as ○. They may also be text such as "No" or "No Risk." The detailed information regarding the reference values ​​is the same as that described in 1-1 above, so we will refer to the explanation in 1-1 above here.

[0050] 2. Support for the detection of subjects who are at risk of ischemic cardiovascular disease. 2-1. Biomarkers for detecting subjects at risk of ischemic cardiovascular disease, and methods for supporting the detection of subjects at risk of ischemic cardiovascular disease. One embodiment of the present invention relates to a method for assisting in the detection of subjects belonging to the pre-symptomatic stage of ischemic cardiovascular disease. The method includes obtaining a measurement of free DNA derived from the subject's cells in plasma collected from the subject. Free DNA derived from the subject's cells in plasma collected from the subject can be used as a biomarker for detecting subjects belonging to the pre-symptomatic stage of ischemic cardiovascular disease.

[0051] Here, the detailed procedure for obtaining the measurement of free DNA derived from the subject's cells in the plasma collected from the subject is the same as described in 1-1 above, so we will refer to the explanation in 1-1 here.

[0052] If the measured value is above the corresponding reference value, it suggests that the subject belongs to the group at risk of ischemic cardiovascular disease. Conversely, if the measured value is below the corresponding reference value, it suggests that the subject does not belong to the group at risk of ischemic cardiovascular disease.

[0053] Ischemic cardiovascular disease may include vascular occlusion due to thrombi formed at plaque sites, vascular occlusion due to plaque, detached thrombi, or embolus caused by plaque. Furthermore, the group at risk of ischemic cardiovascular disease may include subjects who have never yet experienced ischemic cardiovascular disease, as well as subjects who have previously experienced ischemic cardiovascular disease and are currently under observation after treatment.

[0054] 2-2. Support devices for detecting subjects who are at risk of ischemic cardiovascular disease. One embodiment of the present invention relates to a support device 20 (hereinafter also simply referred to as "support device 20") for assisting in the detection of subjects belonging to the group at risk of ischemic cardiovascular disease. The hardware configuration of the support device 20 will be explained using Figure 1.

[0055] The support device 20 may be connected to the input device 211, the output device 212, and the media drive 213. Alternatively, the support device 20 may be connected to the analyzer 90 via a wired or wireless network to form a support system 2000 for assisting in the detection of arteriosclerosis. The analyzer 90 may be a real-time quantitative PCR instrument, a next-generation sequencer, a third-generation sequencer, etc.

[0056] The hardware configuration of the support device 20 is basically the same as that of the support device 10 described above. Therefore, in the support device 20, the processing unit 101 in the description of the support device 10 is replaced with the processing unit 201, the memory 102 with the memory 202, the ROM (read-only memory) 103 with the ROM 203, the storage device 104 with the storage device 204, the communication interface (I / F) 105 with the communication I / F 205, the input interface (I / F) 106 with the input I / F 206, the output interface (I / F) 107 with the output I / F 207, the media interface (I / F) 108 with the media I / F 208, and the bus 109 with the bus 209, and these descriptions are used herein. The memory 202 and the storage device 204 together are sometimes simply called the storage unit. The storage unit stores the measured values ​​and reference values ​​either volatilely or non-volatilely.

[0057] Furthermore, the memory device 204 stores OS2041 and support program 2042a (hereinafter also simply referred to as support program 2042a) in place of OS1041 and support program 1042a, in order to assist in the detection of subjects belonging to the group at risk of ischemic cardiovascular disease.

[0058] The support system 2000 does not need to be installed in one location; the support device 20 and the analysis device 90 may be located in separate places and connected by a network. Furthermore, the support device 20 may be a device that does not require an operator, omitting the input device 211 and output device 212.

[0059] 2-3. Processing of support programs to assist in the detection of subjects belonging to the pre-ischemic cardiovascular disease group. Using Figure 2(B), we will explain the process performed by support program 2042a, which assists in the detection of subjects belonging to the pre-ischemic cardiovascular disease group.

[0060] The processing unit 201 of the support device 20 starts support processing to assist in the detection of subjects belonging to the pre-ischemic cardiovascular disease group when the operator inputs the start of processing from the input device 211.

[0061] In step S21, the processing unit 201 obtains the measured value of free DNA derived from the subject's cells in the plasma collected from the subject from the analyzer 90. The details of obtaining the measured value of free DNA derived from the subject's cells in the plasma collected from the subject are the same as those described in 1-1 above, so the explanation in 1-1 above will be incorporated here.

[0062] Next, in step S22, the processing unit 201 refers to the reference value database DB1 stored in the storage device 204 and compares the measured value with the reference value corresponding to the measured value. In step S22, if the measured value is greater than or equal to the reference value ("YES"), the processing unit 201 proceeds to step S23 and outputs a label indicating that the subject belongs to the group at risk of ischemic cardiovascular disease. Alternatively, in step S22, if the measured value is lower than the reference value ("NO"), the processing unit 201 proceeds to step S24 and outputs a label indicating that the subject does not belong to the group at risk of ischemic cardiovascular disease. Or, in step S22, if the measured value is lower than the reference value ("NO"), the processing unit 201 may terminate the process without proceeding to step S24.

[0063] Labels suggesting that a subject is at risk of ischemic cardiovascular disease may be symbols such as ○, *, or exclamation marks. They may also be text such as "belongs to" or "at risk." Labels suggesting that a subject does not have arteriosclerosis may be symbols such as ×. They may also be text such as "does not belong to" or "no risk." The detailed information regarding the reference values ​​is the same as that described in 1-1 above, so we will refer to the explanation in 1-1 above here.

[0064] 3. Stratification of the degree of arteriosclerosis progression in the subjects. 3-1. Biomarkers for stratifying the progression of arteriosclerosis in subjects, and methods for supporting the stratification of the progression of arteriosclerosis in subjects. One embodiment of the present invention relates to a method for assisting in the stratification of the progression of arteriosclerosis in a subject. The method includes obtaining a measurement of free DNA derived from the subject's mitochondria in plasma collected from the subject, and searching for the reference range to which the measurement belongs from a plurality of reference ranges set according to the progression of arteriosclerosis. Free DNA derived from the subject's mitochondria in plasma collected from the subject can be used as a biomarker for stratifying the progression of arteriosclerosis in the subject.

[0065] The detailed procedure for obtaining measurements of free DNA derived from the subject's mitochondria in plasma collected from the subject is the same as described in 1-1 above, so the explanation in 1-1 is used here.

[0066] Searching for the reference range to which a measurement belongs is intended to compare the subject's measurement to each of several reference ranges that have been pre-set according to the progression of arteriosclerosis. For example, when classifying the progression of arteriosclerosis based on the PS described above, four categories of reference ranges will be set: normal, mild, moderate, and severe. The reference ranges are set based on the measurement values ​​of free DNA derived from the subject's mitochondria in plasma collected from subjects belonging to each of the following groups: those classified as normal, those classified as mild, those classified as moderate, and those classified as severe. The reference ranges can be set based on the group of measurement values ​​obtained in each category, for example, from hazard ratios, confidence intervals, odds ratios, etc. Alternatively, the reference range may be determined from the median and interquartile range calculated from the group of measurement values ​​in each category. Alternatively, the reference range may be set from the mean, standard deviation (SD), variance (CV), etc., calculated from the group of measurement values ​​in each category.

[0067] If the search results determine the reference range to which the measured value belongs, it is suggested that the degree of arteriosclerosis progression corresponding to the reference range to which the measured value belongs is the degree of arteriosclerosis progression of the subject.

[0068] 3-2. Stratification device to support stratification of the progression of arteriosclerosis in subjects One embodiment of the present invention relates to a stratification device 30 (hereinafter also simply referred to as "stratification device 30") for assisting in the stratification of the progression of arteriosclerosis in a subject. The hardware configuration of the stratification device 30 will be explained using Figure 3.

[0069] The stratification device 30 may be connected to an input device 311, an output device 312, and a media drive 313. Alternatively, the stratification device 30 may be connected to an analyzer 90 via a wired or wireless network to form a stratification system 3000 for stratifying the progression of arteriosclerosis. The analyzer 90 may be a real-time quantitative PCR device, a next-generation sequencer, a third-generation sequencer, etc.

[0070] The hardware configuration of the stratification device 30 is basically the same as that of the support device 10 described above. Therefore, in the stratification device 30, the processing unit 101 in the description of the support device 10 is replaced with the processing unit 301, the memory 102 with the memory 302, the ROM (read-only memory) 103 with the ROM 303, the storage device 104 with the storage device 304, the communication interface (I / F) 105 with the communication I / F 305, the input interface (I / F) 106 with the input I / F 306, the output interface (I / F) 107 with the output I / F 307, the media interface (I / F) 108 with the media I / F 308, and the bus 109 with the bus 309, and these are used in this explanation. The memory 302 and the storage device 304 together are sometimes simply called the storage unit. The storage unit stores the measured values ​​and reference values ​​either volatilely or non-volatilely.

[0071] Furthermore, the memory device 304 stores OS 3041 and stratification program 3042a (hereinafter also simply referred to as stratification program 3042a) for stratifying the progression of arteriosclerosis, instead of OS 1041 and support program 1042a. In addition, the reference range database (DB) DB2 is stored instead of reference value database DB1.

[0072] The stratification system 3000 does not need to be installed in one location; the support device 30 and the analysis device 90 may be located in separate locations and connected by a network. Furthermore, the stratification device 30 may be a device that does not require an operator, omitting the input device 311 and output device 312.

[0073] 3-3. Processing of a stratification program to support stratification of the progression of arteriosclerosis in subjects. Using Figure 3, we will explain the process performed by the stratification program 3042a, which supports the stratification of the progression of arteriosclerosis in the subjects.

[0074] The processing unit 301 of the stratification device 30 starts a stratification process to support the stratification of the progression of arteriosclerosis when the operator inputs the start of processing from the input device 311.

[0075] In step S31, the processing unit 301 obtains a measurement value of free DNA derived from the mitochondria of the subject in the plasma collected from the subject from the analyzer 90. The details of obtaining the measurement value of free DNA derived from the cells of the subject in the plasma collected from the subject are the same as those described in 1-1 above, so the explanation in 1-1 above will be incorporated here.

[0076] Next, in step S32, the processing unit 301 searches for a reference range to which the measured value belongs from a plurality of reference ranges set according to the degree of progression of arteriosclerosis. The plurality of reference ranges are stored in the reference range database DB2, linked to labels indicating the degree of progression of arteriosclerosis.

[0077] Next, in step S33, the processing unit 301 outputs a label associated with the degree of arteriosclerosis progression corresponding to the reference range to which the measured value belongs, as a label indicating the degree of arteriosclerosis progression. The label indicating the degree of arteriosclerosis progression may be text in Japanese, English, etc., such as "normal," "mild," "moderate," or "severe." Furthermore, the label associated with the degree of arteriosclerosis progression and the output label do not necessarily have to be the same, as long as they represent equivalent clinical meaning. In addition, if the measured value falls within the "normal" reference range, step S33 may be omitted and the process may be terminated.

[0078] 4. Storage medium on which the program is stored One embodiment of the present invention relates to a program product such as a media drive that stores support programs 1042a, 2042a and a stratification program 3042a. That is, the support programs 1042a, 2042a and the stratification program 3042a can be stored in a media drive such as a hard disk, a semiconductor memory element such as flash memory, or an optical disc. The media drive may also be a computer such as a server device. The format in which the program is recorded to the media drive is not limited as long as each device can read the program. It is preferable that the recording to the media drive is non-volatile.

[0079] 5. Test kit and test reagents One embodiment of the present invention relates to a test kit used to stratify the degree of arteriosclerosis progression in a subject as described in 3, in order to assist in the detection of the presence or absence of arteriosclerosis as described in 1. above, in order to assist in the detection of subjects belonging to the pre-ischemic cardiovascular disease group as described in 2. above. The test kit is a kit for obtaining a measurement of free DNA derived from the subject's cells in plasma collected from the subject by real-time quantitative PCR.

[0080] When real-time quantitative PCR is performed using the intercalator method, the kit includes the forward primer, reverse primer, and PCR reaction solution necessary for real-time quantitative PCR. The PCR reaction solution contains an intercalator such as SYBR Green I, dNTPs, magnesium ions, and a heat-stable polymerase. The forward primer and reverse primer may be pre-mixed. Alternatively, the forward primer, reverse primer, and PCR reaction solution may be pre-mixed and provided as a test reagent.

[0081] When real-time quantitative PCR is performed using the TaqMan® probe method, the kit includes forward primers, reverse primers, a probe labeled with a fluorescent dye and a quencher for the fluorescent dye, and a PCR reaction solution. The probe hybridizes with the product amplified by the forward primers and the reverse primers. The PCR reaction solution contains dNTPs, magnesium ions, and a thermostable polymerase. The forward primers, reverse primers, and the probe labeled with a fluorescent dye and a quencher for the fluorescent dye may be pre-mixed. Furthermore, the forward primers, reverse primers, the probe labeled with a fluorescent dye and a quencher for the fluorescent dye, and the PCR reaction solution may be pre-mixed and provided as a test reagent.

[0082] The PCR reaction solution may also contain glycerol, β-mercaptoethanol, dithiothreitol, buffer solution, EDTA, etc. The explanation of the gene to be amplified for detecting free DNA is used here. Furthermore, the above kit may include a DNA extraction kit for extracting DNA from plasma.

[0083] 6. Prevention and Treatment Methods One embodiment of the present invention relates to a method for preventing the progression of arteriosclerosis. The method for preventing the progression of arteriosclerosis can be applied to subjects in whom arteriosclerosis was detected in 1. above, or to subjects in 3. above whose arteriosclerosis progression was mild or greater.

[0084] Furthermore, one embodiment of the present invention relates to a method for preventing ischemic cardiovascular disease. The method for preventing ischemic cardiovascular disease can be applied to subjects who have been determined to be at risk of ischemic cardiovascular disease as described in 2. above.

[0085] Methods for preventing the progression of arteriosclerosis and for preventing ischemic cardiovascular disease are well known. Both methods may involve administering drugs to improve hypertension, drugs to improve diabetes, drugs to improve hyperlipidemia, etc., as well as lifestyle guidance such as smoking cessation and dietary therapy. For example, drugs to improve hypertension include beta-blockers (carvedilol, bisoprolol, metoprolol, atenolol, etc.), calcium channel blockers (nifedipine, amlodipine besylate, etc.), angiotensin II receptor antagonists (losartan potassium, valsartan, irbesartan, candesartan cilexetil, telmisartan, olmesartan medoxomil, azilsartan, etc.), and antihypertensive diuretics (trichlormethiazide, hydrochlorothiazide, indapamide, etc.). Drugs used to improve diabetes include biguanides (metformin, buformin, etc.), DPP-4 inhibitors (sitagliptin, vildagliptin, alogliptin, linagliptin, teneligliptin, anagliptin, saxagliptin, trelagliptin, omaligliptin, etc.), sulfonylureas (acetohexamide, glyclopyramide, chlorpropamide, glibenclamide, gliclazide, glimepiride, etc.), SGLT2 inhibitors (ipragliflozin, dapagliflozin, luseogliflozin, tofogliflozin, canagliflozin, empagliflozin, etc.), and insulin. Drugs used to improve hyperlipidemia include statins, fibrates, and small intestinal cholesterol transporter inhibitors (ezetimibe, etc.). The dosage of each drug should follow the package insert for that drug.

[0086] Furthermore, one embodiment of the present invention relates to a treatment method for improving arteriosclerosis. The treatment method is applied to a subject in which arteriosclerosis is detected as described in 1. above, a subject in which it is determined to belong to the group at risk of ischemic cardiovascular disease as described in 2. above, or a subject in which the progression of arteriosclerosis is mild or greater as described in 3. above. In the treatment method, a drug that causes arteriosclerotic plaque regression, such as a statin, is administered to the subject.

[0087] Furthermore, for subjects in whom arteriosclerosis is detected in 1. above, subjects determined to be at risk of ischemic cardiovascular disease in 2. above, or subjects in 3. above whose arteriosclerosis progression is mild or greater, angiography may be performed to determine the severity of arteriosclerosis. For subjects in whom angiography confirms that arteries, especially coronary arteries, carotid arteries, and lower extremity arteries, are narrowed or occluded due to plaque, a balloon or stent may be inserted into the affected area via catheter to dilate the narrowed area or reperfusion of blood flow. Furthermore, for subjects in whom no improvement is seen with balloon or stent treatment, surgical procedures such as vascular replacement or vascular reconstruction may be applied to the affected blood vessels. [Examples]

[0088] The present invention will be described in more detail with reference to examples. However, the present invention is not to be interpreted as being limited to the examples. Furthermore, the experiments using patient specimens in these examples were conducted with the approval of the Ethics Committee of Hiroshima University, a national university corporation.

[0089] 1. Verification of DNA damage by Cigarette Smoke Extracts Human umbilical vein endothelial cells (HUVECs) were treated with Cigarette Smoke Extracts (CSE), and double-strand DNA breaks (DSBs) were detected by immunostaining for phosphohistone H2AX (γ-H2AX) according to the method described in Non-Patent Literature 1. Oxidative DNA damage was detected by immunostaining for 8-oxo-2'-deoxyguanosine (8-oxo-dG). CSE was prepared by dissolving the smoke from 8 cigarettes in 15 ml of PBS and adding it to the culture medium to a volume of 1 / 200 of the culture medium. Data are expressed as mean ± SEM. Statistical significance was evaluated by Student's t-test or Mann-Whitney U-test.

[0090] The results are shown in Figure 5. Figure 5(A) shows the results of immunohistochemistry for γ-H2AX. Figure 5(A)(a) is a stained image of γ-H2AX in the nucleus of the control (ctl) before the addition of CSE. No dot-like foci of γ-H2AX were observed. Figure 5(A)(b) is a stained image of γ-H2AX in cells 72 hours after the start of CSE treatment. Foci of γ-H2AX were confirmed in the nucleus. Figure 5(A)(c) is a graph showing the mean and standard deviation over time, calculated by counting the number of γ-H2AX foci per cell for multiple cells. The number of γ-H2AX foci per cell increased over time, and a significant difference compared to the control was observed 72 hours after the start of CSE treatment (p<0.05).

[0091] Figure 5(B) shows the results of immunostaining for 8-oxo-dG. Figure 5(B)(a) is an image of 8-oxo-dG staining in control (ctl) cells before the addition of CSE. No dot-like foci of 8-oxo-dG were observed. Figure 5(B)(b) is an image of 8-oxo-dG staining in cells 72 hours after the start of CSE treatment. Foci of 8-oxo-dG were confirmed in the nucleus and cytoplasm. Figure 5(B)(c) is a graph showing the fluorescence intensity of 8-oxo-dG in the nucleus over time. Figure 5(B)(d) is a graph showing the fluorescence intensity of 8-oxo-dG in the cytoplasm over time. The fluorescence intensity of Ctl is set to 1, and the change in fluorescence intensity is shown as a relative value. As a result, it was shown that the fluorescence intensity of 8-oxo-dG in the nucleus increased with time. Furthermore, the fluorescence intensity of 8-oxo-dG in the cytoplasm increased significantly over time, even more so than in the nucleus.

[0092] Figure 5(C) shows DNA staining (DAPI), mitochondrial staining (Mitotracker), 8-oxo-dG immunostaining (8-oxo-dG), and a merge image of these three stains in a single cell 24 hours after CSE addition. The fluorescence signal of 8-oxo-dG was detected not only in the nucleus but also in the mitochondria.

[0093] These results suggest that, in CSE-induced DNA damage, DSBs occur significantly in the nucleus, but oxidative DNA damage occurs not only in the nucleus but also in mitochondrial DNA.

[0094] 2. Identification of DNA accumulated in the cytoplasm To identify whether the DNA accumulated in cells originates from the nucleus or from mitochondria, the following experiment was performed.

[0095] (1) Isolation and quantification of cytoplasmic DNA Cytoplasmic DNA isolation and quantification were performed as follows: HUVEC cells were collected using a 0.05 w / v% Trypsin-0.53 mmol / l EDTA·4Na solution, divided into two equal parts, and centrifuged at 1000 rpm for 3 minutes to pellet the cells. One part was resuspended in 200 μl of 50 μmol / L NaOH and boiled for 30 minutes to solubilize the DNA. This supernatant contained the DNA of all cells. The pH was neutralized by adding 20 μl of 1 mol / L Tris-HCl pH 8, and these extracts were used as a standardization control for cytoplasmic DNA. The other part was resuspended in 200 μl buffer containing 150 mmol / L NaCl, 50 mmol / L HEPES pH 7.4, and 25 μg / mL digitonin (TCI, JAPAN). After incubating the homogenate for 10 minutes, it was centrifuged at 17,000 g for 10 minutes, and the supernatant was transferred to a new tube. The supernatant is the cytoplasmic fraction, which does not contain the nucleus, mitochondria, or endoplasmic reticulum. Next, DNA was isolated from both extracts using QIAQuick Nucleotide Removal Columns (QIAGEN). Quantitative PCR was performed on both the whole cell extract and the cytoplasmic fraction using nuclear DNA (nDNA) primers (β-globin) or mitochondrial DNA (mtDNA) primers (NADH Dehydrogenase Subunit 1, hereafter also referred to as NADH1).

[0096] Real-time quantitative PCR was performed using the SYBR Green method, a type of intercalator PCR. THUNDERBIRD® SYBR® qPCR Mix® (TOYOBO Cat.No.: QPS-201) was used as the amplification reagent. The reagent composition followed the manufacturer's protocol. The primers are as follows: NADH1_F:5'-ATACCCATGGCCAACCTCCT-3' (Sequence ID 1) NADH1_R:5'-GGGCCTTTGCGTAGTTGTAT-3' (Sequence ID 2) β-globin_F:5'-GTGCACCTGACTCCTGAGGAGA-3' (Sequence ID 3) β-globin_R:5'-CCTTGATACCAACCTGCCCAG-3' (SEQ ID NO: 4) The amplification conditions are as follows: i. First denature: 95℃ 60sec ii. Denature: 95℃ 5sec iii. Anneling 60℃ 10sec iv. Extension 72℃ 30sec ii. to iv. for 40 cycles The Ct values ​​obtained from whole cell extracts were used as a normalized control for the Ct values ​​obtained from the cytoplasmic fraction. Data are expressed as mean ± SEM. Statistical significance was assessed using Student's t-test or Mann-Whitney U-test.

[0097] (2) Results The results are shown in Figure 6. Figure 6(A) shows the increase in mitochondrial DNA (Cytosolic mtDNA) in the cytoplasm on days 1, 3, and 7 after the start of CSE treatment. The increase is shown as a relative value to the amount of mitochondrial DNA in the cytoplasm of the control (Ctl) before the start of CSE treatment. It was shown that mitochondrial DNA accumulated in the cytoplasm on days 1, 3, and 7 after the start of CSE treatment.

[0098] Figure 6(B) shows the increase in cytoplasmic nuclear DNA (Cytosolic nDNA) on days 1, 3, and 7 after the start of CSE treatment. The increase is shown as a relative value to the amount of nuclear DNA in the cytoplasm of the control (Ctl) before the start of CSE treatment. It was shown that nuclear DNA accumulated in the cytoplasm on days 1 and 3 after the start of CSE treatment. On the other hand, the amount of nuclear DNA accumulated in the cytoplasm had decreased by day 7 after treatment. These results indicate that CSE treatment leads to the accumulation of both mitochondrial-derived DNA and nuclear-derived DNA in the cytoplasm during the first half of the treatment period.

[0099] 3. Measurement of free DNA using cultured cells It has been reported that a portion of the DNA accumulated in the cytoplasm is released outside the cell. This DNA released outside the cell is called cell-free DNA (cfDNA). As shown in section 2 above, since nuclear-derived DNA and mitochondrial-derived DNA accumulate in the cytoplasm, it was considered possible that a portion of this DNA is also released outside the cell. To verify this, the following experiment was conducted.

[0100] (1) Measurement of cfDNA in the culture supernatant HUVEC cells were cultured with CSE for 2 days. The culture medium was then transferred to a microtube, and cells were removed by centrifugation (1000 × g, 10 min). NucleoSpin cfDNA XS (TAKARA, JAPAN) was used to isolate DNA from the supernatant culture medium, following the manufacturer's guidelines with a starting sample volume of 720 μL and an elution volume of 20 μL. Total cellular DNA was also extracted using the same method. Quantitative PCR was performed on both cfDNA in the culture medium and total cellular DNA using nDNA primers (β-globin) and mtDNA primers (NADH1). The Ct values ​​obtained from total cellular DNA were used as a normalized control of the Ct values ​​obtained from cfDNA in the culture medium.

[0101] (2) Results The results are shown in Figure 7. Figure 7(A) shows the increase in mitochondrial DNA (mtDNA) in the culture supernatant of HUVEC cells. Figure 7(B) shows the increase in nuclear DNA (nDNA) in the same culture supernatant as in Figure 7(A). The results are shown as relative amounts, with the copy number of mitochondrial DNA in the culture supernatant of the control (Ctl) before CSE treatment set to 1. An increase in the amount of mitochondrial DNA and nuclear DNA was observed in the culture supernatant of HUVEC cells treated with CSE. This indicates that CSE treatment releases mitochondrial DNA and nuclear DNA into the culture supernatant as cfDNA.

[0102] 4. Measurement of free DNA using plasma Next, to verify that smoking actually releases cfDNA in vivo, we measured cfDNA using plasma from smokers and non-smokers.

[0103] (1) Subject information The breakdown of subjects is shown in Figure 8(A). Continuous data are expressed as median and interquartile range. Categorical data are expressed as number and percentage. The Mann-Whitney U test was applied to the significance test for continuous data, and the Chi-square test was applied to the significance test for categorical data.

[0104] (2) Determination of cfDNA in plasma Peripheral blood was collected in EDTA-2Na tubes, centrifuged at 3000 rpm at 20°C for 10 minutes to separate the plasma, and immediately stored at -20°C. For DNA separation from the plasma fraction, NucleoSpin cfDNA XS (TAKARA, Japan) was used, following the manufacturer's protocol with a starting sample volume of 240 μL and an elution volume of 30 μL. The copy numbers of nuclear-derived cell-free DNA (n-cfDNA) and mitochondrial-derived cell-free DNA (mt-cfDNA) were determined by absolute quantification using real-time qPCR with nDNA primers (β-globin) and mtDNA primers (NADH1). For absolute quantification, a five-point standard curve consisting of known mitochondrial and nuclear DNA amplicons (Integrated DNA Technologies, USA) was applied redundantly to each plate. The copy number obtained from the standard curve was adjusted using the following formula, taking into account the initial and final elution volumes of the sample, and expressed as copy number / plasma μL.

number

[0105] (3) Results The results are shown in Figs. 8(B) and (C). Fig. 8(B) shows the distribution of the copy number of cfDNA derived from mitochondrial DNA in the plasma of the subject. Fig. 8(C) shows the distribution of the copy number of cfDNA derived from nuclear DNA in the plasma of the subject. Both mitochondrial DNA and nuclear DNA could be detected from the plasma. Also, compared with the non-smoker group, in the smoker group, the copy numbers in the plasma were significantly higher for both mitochondrial DNA and nuclear DNA.

[0106] From these results, it was shown that in vivo as well, in the smoker group, mitochondrial DNA and nuclear DNA were released as cfDNA from cells.

[0107] 5. Measurement of cfDNA in Plasma Collected from Patients with Arteriosclerosis (1) Information on Subjects The subjects were 83 healthy check examinees and outpatients with lifestyle-related diseases. Information on the subjects is shown in Fig. 9.

[0108] The diagnosis of arteriosclerosis was performed by carotid echo. The degree of progression of arteriosclerosis was classified with the total plaque thickness in the left and right carotid arteries as the plaque score according to Handa N, et al. Stroke 1990;21:1567-1572. Specifically, the classification of the plaque score (PS) was Normal for PS ≤ 1 mm, Mild for 1 mm < PS ≤ 5 mm, Moderate for 5 mm < PS ≤ 10 mm, and Severe for 10 mm < PS. Also, when PS was 1 mm or less, it was defined as plaque negative (-), and when PS exceeded 1 mm, it was defined as plaque positive (+). Continuous data were represented by the median and the interquartile range. Categorical data were represented by the number and the percentage. The Steel-Dwass test was applied for the significance test of continuous data, and the Chi-square test was applied for the significance test of categorical data. In the figure, * indicates p < 0.05 vs normal, and † indicates p < 0.05 vs mild. The groups with moderate and severe arteriosclerosis were significantly older, and Cr was significantly higher in the severe group. No significant differences were observed for the other items.

[0109] (2) Determination of cfDNA in plasma The quantification was performed according to the method described in 4.(2) above.

[0110] (3) Results Figure 10 shows the distribution of cfDNA copy numbers per unit volume in plasma for subjects with no plaque (Normal) and subjects with plaque (Plaque). Figure 10(A) shows the distribution of cfDNA copy numbers derived from mitochondrial DNA (mt-cfDNA) in the subjects' plasma. Figure 10(B) shows the distribution of cfDNA copy numbers derived from nuclear DNA (n-cfDNA) in the subjects' plasma. In the figures, ● indicates smokers and ○ indicates non-smokers. Comparing the healthy subjects with no plaque with the subjects with plaque, both mt-cfDNA and n-cfDNA were higher in the plaque-positive subjects (mt-cfDNA: **p<0.01 Normal vs Plaque; n-cfDNA: *p<0.05 Normal vs Plaque). Note that this study included smokers in the subjects. However, in this study, both smokers and non-smokers in the plaque-positive group showed similar distributions. Therefore, plasma cfDNA was considered a biomarker that can be used to detect plaque in vascular endothelial tissue, regardless of whether the individual smokes or not. For reference, the cutoff values ​​for distinguishing between plaque-negative and plaque-positive groups were 69.28 copies / μl for mt-cfDNA and 15.3369.28 copies / μl for n-cfDNA.

[0111] It should be noted that in the study described in 4.(3) above, the effects of smoking were relatively more apparent because plasma from younger generations was used. However, since the subjects in this study were older, it is thought that the presence or absence of plaque had a greater impact on the copy number of cfDNA in the plasma than the effects of smoking. Therefore, the following study will not consider the presence or absence of smoking.

[0112] Next, the subjects were divided into four groups according to the severity of arteriosclerosis: plaque-free (Normal), mild, moderate, and severe. The distribution of cfDNA copy numbers in plasma was then compared. Figure 11 shows the results. Figure 11(A) shows the distribution of cfDNA copy numbers derived from mitochondrial DNA (mt-cfDNA) in the subjects' plasma. Figure 11(B) shows the distribution of cfDNA copy numbers derived from nuclear DNA (n-cfDNA) in the subjects' plasma. The mt-cfDNA copy number tended to increase with increasing severity of arteriosclerosis (in the figure, * indicates p<0.05 vs normal, and ** indicates p<0.01 vs normal). On the other hand, no significant difference was observed in the n-cfDNA copy number.

[0113] To further verify these results, the correlation between each subject's plaque thickness and the copy number of mt-cfDNA or n-cfDNA in the plasma was evaluated using Pearson correlation analysis. The results are shown in Figure 12. Figure 12(A) shows the correlation between the copy number of cfDNA derived from mitochondrial DNA (mt-cfDNA) in the subject's plasma and plaque thickness. Figure 12(B) shows the correlation between the copy number of cfDNA derived from nuclear DNA (n-cfDNA) in the subject's plasma and plaque thickness. A correlation was observed between the copy number of mt-cfDNA in plasma and plaque thickness, with r=0.350 and p=0.001. On the other hand, no correlation was observed between the copy number of n-cfDNA in plasma and plaque thickness, with r=0.177 and p=0.109.

[0114] Furthermore, the correlation between the clinical data (age and serum creatinine concentration) that showed a significant difference in Figure 9 and the copy number of mt-cfDNA or n-cfDNA in plasma was evaluated using Pearson correlation analysis. The correlation between age and cfDNA copy number is shown in Figure 13. Figure 13(A) shows the correlation between the copy number of cfDNA derived from mitochondrial DNA (mt-cfDNA) in the subject's plasma and age. Figure 13(B) shows the correlation between the copy number of cfDNA derived from nuclear DNA (n-cfDNA) in the subject's plasma and age. No correlation was observed between the copy number of mt-cfDNA or n-cfDNA in plasma and age, with r=0.185 and p=0.093, and r=0.092 and p=0.408, respectively. The correlation between serum creatinine concentration and cfDNA copy number is shown in Figure 14. Figure 14(A) shows the correlation between the copy number of cfDNA derived from mitochondrial DNA (mt-cfDNA) in the subject's plasma and serum creatinine concentration. Figure 14(B) shows the correlation between the copy number of cfDNA derived from nuclear DNA (n-cfDNA) in the subject's plasma and serum creatinine concentration. No correlation was observed between the copy number of mt-cfDNA or n-cfDNA in plasma and age, with r=0.197 and p=0.075, and r=0.083 and p=0.455, respectively.

[0115] These results indicate that mt-cfDNA in plasma correlates only with the plaque thickness of each subject. Therefore, the copy number of mt-cfDNA in plasma can be used as a biomarker for stratifying the severity of arteriosclerosis.

[0116] 5.ROC analysis To verify the accuracy of cfDNA copy number in plasma as a biomarker for detecting arteriosclerosis, an evaluation was performed using the receiver operating characteristic curve (ROC curve). The ROC curves are shown in Figure 15. In the figure, the symbol a represents the ROC curve for mt-cfDNA copy number, and the symbol b represents the ROC curve for n-cfDNA copy number. The AUC value in the ROC curve for mt-cfDNA copy number was 0.78, and the AUC value in the ROC curve for n-cfDNA copy number was 0.68. This indicates that mt-cfDNA copy number is particularly favored as a biomarker for arteriosclerosis. [Explanation of symbols]

[0117] 101,201,301 Processing Unit 10,20 Support equipment 30 Stratification device

Claims

1. A method for assisting in the detection of the presence or absence of arteriosclerosis, The aforementioned method, This includes obtaining measurements of free DNA derived from the subject's mitochondria in plasma collected from the subject, If the measured value is equal to or greater than the reference value corresponding to the measured value, it is suggested that the subject has arteriosclerosis, and / or if the measured value is lower than the reference value corresponding to the measured value, it is suggested that the subject does not have arteriosclerosis. method.

2. A support device for assisting in the detection of the presence or absence of arteriosclerosis, The support device includes a processing unit, The aforementioned processing unit, We obtained measurements of free DNA derived from the subject's mitochondria in plasma collected from the subject. If the measured value is equal to or greater than the reference value corresponding to the measured value, a label is output suggesting that the subject has arteriosclerosis, and / or if the measured value is lower than the reference value corresponding to the measured value, a label is output suggesting that the subject does not have arteriosclerosis. Support equipment.

3. When you run it on a computer, the computer will A step of obtaining a measurement of free DNA derived from the mitochondria of the subject in plasma collected from the subject, The steps include: outputting a label suggesting that the subject has arteriosclerosis if the measured value is equal to or greater than a reference value corresponding to the measured value, and / or outputting a label suggesting that the subject does not have arteriosclerosis if the measured value is lower than a reference value corresponding to the measured value; A support program that enables the detection of arteriosclerosis.

4. A method for assisting in the detection of subjects belonging to the pre-symptomatic group of ischemic cardiovascular disease, The aforementioned method, This includes obtaining measurements of free DNA derived from the subject's mitochondria in plasma collected from the subject, If the measured value is equal to or greater than the reference value corresponding to the measured value, it is suggested that the subject belongs to the group at risk of ischemic cardiovascular disease, and / or if the measured value is lower than the reference value corresponding to the measured value, it is suggested that the subject does not belong to the group at risk of ischemic cardiovascular disease. method.

5. A support device for assisting in the detection of subjects belonging to the pre-symptomatic group of ischemic cardiovascular disease, The support device includes a processing unit, The aforementioned processing unit, We obtained measurements of free DNA derived from the subject's mitochondria in plasma collected from the subject. If the measured value is equal to or greater than the reference value corresponding to the measured value, a label is output suggesting that the subject belongs to the group at risk of ischemic cardiovascular disease, and / or if the measured value is lower than the reference value corresponding to the measured value, a label is output suggesting that the subject does not belong to the group at risk of ischemic cardiovascular disease. Support equipment.

6. When you run it on a computer, the computer will A step of obtaining a measurement of free DNA derived from the mitochondria of the subject in plasma collected from the subject, The steps include: outputting a label suggesting that the subject belongs to the pre-ischemic cardiovascular disease group if the measured value is equal to or greater than the reference value corresponding to the measured value, and / or outputting a label suggesting that the subject does not belong to the pre-ischemic cardiovascular disease group if the measured value is lower than the reference value corresponding to the measured value; A support program designed to assist in the detection of subjects who are at risk of ischemic cardiovascular disease.

7. A method for supporting the stratification of the progression of arteriosclerosis in subjects, The aforementioned method, To obtain measurements of free DNA derived from the subject's mitochondria in plasma collected from the subject, This includes searching for the reference range to which the measured value belongs from among several reference ranges set according to the progression of arteriosclerosis. The degree of arteriosclerosis progression corresponding to the reference range to which the aforementioned measurement value belongs is suggested as the degree of arteriosclerosis progression in the subject. method.

8. A stratification device for supporting the stratification of the progression of arteriosclerosis in a subject, The stratification apparatus includes a processing unit, The aforementioned processing unit, We obtained measurements of free DNA derived from the subject's mitochondria in plasma collected from the subject. From among multiple reference ranges set according to the progression of arteriosclerosis, the reference range to which the measured value belongs is searched. The label indicating the degree of arteriosclerosis progression corresponding to the reference range to which the measured value belongs is output as a label indicating the degree of arteriosclerosis progression of the subject. Stratification device.

9. When you run it on a computer, the computer will A step of obtaining a measurement of free DNA derived from the mitochondria of the subject in plasma collected from the subject, The steps include searching for the reference range to which the measured value belongs from among several reference ranges set according to the degree of progression of arteriosclerosis, The steps include outputting a label indicating the degree of arteriosclerosis progression corresponding to the reference range to which the measured value belongs, as a label indicating the degree of arteriosclerosis progression of the subject, A stratification program designed to assist in stratifying the degree of arteriosclerosis progression in subjects.

Images