Methods for determining the genetic risk of vascular aging

Abstract

This provides a means to accurately and easily determine an individual's genetic predisposition to vascular aging. [Solution] A method for determining the genetic risk of vascular aging, comprising the steps of: detecting alleles of single nucleotide polymorphisms (SNPs) at the 51st base of one or more sequences identified in (A) to (E) below from a DNA-containing sample taken from a subject; and determining that the subject is prone to developing symptoms related to vascular aging if the detected allele's base is a risk allele. (A)SNP:ID rs17108108 (B)SNP:ID rs2271987 (C)SNP:ID rs6470860 (D)SNP:ID rs2284972 (E)SNP:ID rs12028323

Description

Technical Field

[0001] The present invention relates to a method for determining the genetic risk of vascular aging. More specifically, the present invention relates to a method for determining the genetic risk of vascular aging by detecting a single nucleotide polymorphism (SNP) associated with vascular aging, and a kit used for the method.

Background Art

[0002] Blood vessels change structurally and functionally and age with aging. Structural changes include an increase in blood vessel wall thickness due to intimal thickening, arterial wall calcification, and a concomitant decrease in extensibility. Functional changes include a decrease in the production of nitric oxide (NO) in vascular endothelial cells. NO is not only important as a vasodilator, but also acts as an anti-inflammatory and anti-aging substance, such as suppressing the adhesion of inflammatory cells and the migration and proliferation reactions of smooth muscle cells, and having an antioxidant stress effect.

[0003] Vascular aging is caused by various factors such as aging, lifestyle diseases such as hypertension, dyslipidemia, and diabetes, lack of exercise, stress, excessive salt intake, obesity, and smoking. Due to the aging of blood vessels, the blood vessels gradually become hard and lose elasticity, resulting in a decrease in blood flow. This decrease in blood flow is known to be associated with various disorders ranging from physical symptoms such as headache, low back pain, shoulder stiffness, joint pain, muscle pain, numbness in hands and feet, coldness, swelling, anemia, chronic fatigue, and mood swings to mental symptoms.

[0004] However, vascular aging itself cannot be felt, and the physical and mental symptoms caused by vascular aging are diverse and complex, so it is often not immediately recognized that the cause is vascular aging. It is also known that vascular aging has a genetic factor and there are individual differences. Therefore, grasping the state of vascular aging from an individual's genetic background and predicting the presence or absence of future vascular aging risk are important for appropriate prevention and improvement of various physical and mental health disorders caused by the decline of vascular function.

[0005] Technologies are being developed to predict the presence or absence of current diseases and the risk of future disease development by analyzing genes in biological samples. In particular, single nucleotide polymorphisms (SNPs) indicate slight differences in an individual's genetic information and are associated with specific diseases and physical characteristics, making them effective in determining the genetic risk of specific diseases or physical characteristics. For example, regarding pain, there is a method for evaluating disease susceptibility (Patent Document 1) characterized by associating genetic polymorphisms of the HS3ST4 gene, IGF2R gene, or IGF2 gene with an individual's disease (pain) susceptibility; regarding obesity, there is a method for detecting the genetic risk of obesity (Patent Document 2) that uses at least one or more genetic polymorphisms at specific sites of the ACE, GCK, ESR1, APOC3, IRS1, GCLC, ADRB1, F12, and STX1A genes, along with age, as evaluation factors; there is a method for predicting the risk of obesity by analyzing single nucleotide polymorphisms of the UCP1 (uncoupling protein 1) gene or single nucleotide polymorphisms linked to said single nucleotide polymorphism and / or single nucleotide polymorphisms linked to said single nucleotide polymorphism of the ADRB3 (beta3-adrenergic receptor) gene or single nucleotide polymorphisms linked to said single nucleotide polymorphism (Patent Document 3); and there is a method for predicting the risk of developing obesity based on the level of proneurotensin or its fragments (Patent Document 4). Furthermore, regarding mental illnesses, methods have been reported such as an in vitro method for determining whether there is a risk of developing mental illnesses such as depressive disorders based on the expression levels of at least one of the editing enzymes ADAR1a, ADAR1b, and ADAR2 and PDE8A (Patent Document 5), and a method for evaluating the severity of depression in a subject based on the results of an analysis of the expression levels of at least one gene selected from apoptosis-related genes, ATPase-related genes, cell cycle-related genes, cytokine-related genes, heat shock protein-related genes, polymerase-related genes, GTP-binding protein-related genes, protein kinase C-related genes, and mitochondrial cytochrome C oxidase-related genes.

[0006] However, technology to determine the genetic risk of vascular aging, which varies from person to person, had not yet been established. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Japanese Patent Publication No. 2023-133648 [Patent Document 2] Japanese Patent Publication No. 2007-228917 [Patent Document 3] Japanese Patent Publication No. 2012-81 [Patent Document 4] Special Publication No. 2018-511784 [Patent Document 5] Special Publication No. 2019-524115 [Patent Document 6] Japanese Patent Publication No. 2004-208547 [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] The object of this invention is to provide a method for accurately and easily determining an individual's genetic risk of vascular aging. [Means for solving the problem]

[0009] The inventors analyzed single nucleotide polymorphisms (SNPs) in sample providers who met predetermined criteria and gave their consent, using genome-wide association studies (GWAS) or targeted analysis of specific genes involved in NO production. They discovered SNPs in specific genes that were significantly associated with subjective symptoms related to vascular aging, thus completing the present invention.

[0010] In other words, the present invention encompasses the following inventions. [1] A method for determining the genetic risk of vascular aging, comprising the steps of detecting one or more single nucleotide polymorphism (SNP) alleles from (A) to (E) below in a DNA-containing sample taken from a subject, and determining that the subject is likely to develop symptoms related to vascular aging if the base of the detected allele is a risk allele. (A) SNP: Identified by ID rs17108108, this SNP is located at the 51st base of the sequence shown in Sequence ID No. 1 (risk allele is minor allele C). (B) SNP: Identified by ID rs2271987, this SNP is located at the 51st base of the sequence shown in Sequence ID No. 2 (risk allele is minor allele T). (C) SNP: Identified by ID rs6470860, this SNP is located at the 51st base of the sequence shown in Sequence ID No. 3 (risk allele is major allele A). (D) SNP: Identified by ID rs2284972, this SNP is located at the 51st base of the sequence shown in Sequence ID No. 4 (risk allele is minor allele G). (E) SNP: Identified by ID rs12028323, this SNP is located at the 51st base of the sequence shown in Sequence ID No. 5 (risk allele is minor allele C). [2] The method according to [1], wherein the single nucleotide polymorphism (SNP) is rs17108108, and the symptoms associated with vascular aging are a tendency to gain weight and difficulty losing weight. [3] The method according to [1], wherein the single nucleotide polymorphism (SNP) is rs2271987 and the symptom associated with vascular aging is lower back pain. [4] The method according to [1], wherein the single nucleotide polymorphism (SNP) is rs6470860 and the symptom associated with vascular aging is numbness in the hands and feet. [5] The method according to [1], wherein the single nucleotide polymorphism (SNP) is rs2284972 and the symptom associated with vascular aging is body pain. [6] The method according to [1], wherein the single nucleotide polymorphism (SNP) is rs12028323 and the symptom associated with vascular aging is a mood disorder. A method for providing food or pharmaceuticals to a subject, based on the results determined by any of the methods in [1] to [6], wherein the subject is provided with food or pharmaceuticals that have a preventive and / or ameliorative effect on vascular aging in proportion to the degree of the subject's genetic risk of vascular aging. [8] An advice method for presenting measures for preventing and / or improving the necessary vascular aging in a subject by using the result determined by the method according to any one of [1] to [6]. [9] A kit for determining the genetic risk of vascular aging, comprising a probe containing a sequence of 10 bases or more including the 51st base of the base sequence shown in any one of SEQ ID NOs: 1 to 5, or its complementary sequence, and / or a primer capable of amplifying a region containing the 51st base of the base sequence shown in any one of SEQ ID NOs: 1 to 5.

[10] A method for screening a substance for preventing and / or improving vascular aging, using the change in expression of one or more genes selected from the group consisting of the KCNMB4 gene, the NOS1 gene, the ADCY8 gene, and the PRMT6 gene as an index.

Effects of the Invention

[0011] According to the method of the present invention, by detecting the allele of a single nucleotide polymorphism (SNP) contained in the DNA present in the biological sample of the subject, the risk of vascular aging in the subject can be accurately and easily determined. Therefore, based on this determination result, it is possible to give notice of the progress of vascular aging that is difficult to feel, and to implement measures for preventing and improving vascular aging at an early stage.

Brief Description of the Drawings

[0012] [Figure 1] FIG. 1 shows a scatter plot of the first and second principal components based on the genotypes of 1996 subjects.

Modes for Carrying Out the Invention

[0013] 1. Method for Determining Genetic Risk of Vascular Aging The method for determining the genetic risk of vascular aging of the present invention includes a step of detecting the allele of one or more single nucleotide polymorphisms (SNPs) of the following (A) to (E) for a DNA-containing sample collected from a subject, and when the base of the detected allele is a risk allele, a step of determining that the subject is likely to develop symptoms related to vascular aging. (A) SNP: SNP at the 51st base of the nucleotide sequence shown in SEQ ID NO: 1, identified by SNP:ID rs17108108 (the risk allele is the minor allele C) (B) SNP: SNP at the 51st base of the nucleotide sequence shown in SEQ ID NO: 2, identified by SNP:ID rs2271987 (the risk allele is the minor allele T) (C) SNP: SNP at the 51st base of the nucleotide sequence shown in SEQ ID NO: 3, identified by SNP:ID rs6470860 (the risk allele is the major allele A) (D) SNP: SNP at the 51st base of the nucleotide sequence shown in SEQ ID NO: 4, identified by SNP:ID rs2284972 (the risk allele is the minor allele G) (E) SNP: SNP at the 51st base of the nucleotide sequence shown in SEQ ID NO: 5, identified by SNP:ID rs12028323 (the risk allele is the minor allele C)

[0014] The nucleotide sequences containing the SNPs (A) to (E) above (the SNPs are represented within parentheses) are shown in Table 1.

[0015] [Table 1]

[0016] As shown in the results of genome-wide association studies (GWAS) or targeted analyses in the examples described below, the SNP rs12028323 had a p-value less than 5.0 E-08 and an odds ratio of 1.8 or higher, while the SNPs rs17108108, rs2271987, rs6470860, and rs2284972 had a p-value less than 8.38 E-05 and an odds ratio of 1.3 or higher (or 0.7 or lower), demonstrating a statistically significant association with symptoms related to vascular aging. While it is possible to determine the genetic risk of vascular aging with just one of the above SNPs, combining two or more can further improve the accuracy of the determination and increase the number of possible determination patterns. Here, "determination" means presenting information such as whether symptoms related to vascular aging are likely to occur, whether the risk of vascular aging is high, whether symptoms related to vascular aging are unlikely to occur, or whether the risk of vascular aging is low. Furthermore, the term "assessment" as used here does not include a diagnosis by a physician. Rather, it refers to assistance in diagnosis, even when a physician's diagnosis is involved in addressing specific symptoms or diseases related to vascular aging. Specifically, it means obtaining data for diagnosis.

[0017] "Genetic risk" refers to the fact that individual genetic differences, such as single nucleotide polymorphisms, can be factors that cause differences in susceptibility to or constitution of a particular disease, increasing the likelihood of developing that specific disease.

[0018] A single nucleotide polymorphism (SNP, hereafter sometimes referred to as "SNP") generally refers to a condition in which the base sequence of a gene differs at only one location, and the site of that difference. A polymorphism, on the other hand, generally refers to two or more alleles that exist at a frequency of 1% or more in a population. In this invention, "SNP" refers to an SNP registered in the National Center for Biotechnology Information (NCBI) SNP database (http: / / www.ncbi.nlm.nih.gov / SNP / ), a publicly available database freely accessible to those skilled in the art, and can be identified by its reference number, the rs number.

[0019] In this specification, "allele" refers to two distinct bases that can be present at a given SNP site. Furthermore, in this specification, "risk allele" refers to an allele that frequently appears in a group of subjects at high risk of vascular aging. In this invention, the risk allele is defined based on the odds ratio. The odds ratio is generally the ratio of the proportion of people with a risk factor to the proportion of people without a risk factor in a symptomatic (case) group, i.e., the odds, divided by the odds similarly obtained in an asymptomatic (control) group. It is commonly used in case-control studies. In this invention, the odds ratio is determined based on allele frequency and can be calculated by dividing the ratio of the frequency of one allele to another in the case group by the ratio of the frequencies similarly obtained in the control group. Also, at a given SNP site, there are three types of opposing allele combinations (genotypes): the same allele combination is called homozygous, and the different allele combinations are called heterozygous. For example, the SNP identified by rs17108108 has three types of opposing allele combinations: C / C homozygous, T / C heterozygous, and T / T homozygous.

[0020] In the determination method of the present invention, "detecting an allele of a single nucleotide polymorphism (SNP)" means identifying the type of base of the allele of the SNP, and embodiments of this include detecting one allele of the SNP, detecting both alleles of the SNP, and determining the genotype of the SNP.

[0021] In the determination method of the present invention, if the detected allele base is a risk allele, it is determined that the subject is more likely to develop symptoms related to vascular aging (has a genetic risk of vascular aging). Specifically, for rs17108108 (SNP at the 51st base of the nucleotide sequence shown in SEQ ID NO: 1), rs2271987 (SNP at the 51st base of the nucleotide sequence shown in SEQ ID NO: 2), rs6470860 (SNP at the 51st base of the nucleotide sequence shown in SEQ ID NO: 3), rs2284972 (SNP at the 51st base of the nucleotide sequence shown in SEQ ID NO: 4), and rs12028323 (SNP at the 51st base of the nucleotide sequence shown in SEQ ID NO: 5), if the risk alleles shown in Table 2 below are detected in at least one allele, it can be determined that the subject is more likely to develop symptoms related to vascular aging (has a genetic risk of vascular aging) compared to cases where the risk allele is not detected.

[0022] [Table 2]

[0023] For example, in the case of the SNP identified as rs17108108, if the detected allele base is risk allele C, it indicates a higher likelihood of developing symptoms associated with vascular aging, and the allele combinations C / C, T / C, and T / T indicate a higher likelihood of developing symptoms associated with vascular aging, in that order.

[0024] In this invention, symptoms related to vascular aging include "tendency to gain weight and difficulty losing weight," "lower back pain," "numbness in the hands and feet," "body aches," and "mood disorders."

[0025] rs1710810 is an SNP located on the KCNMB4 gene that is statistically significant in association with "tendency to gain weight and difficulty to lose weight." If the allele at the 51st base of the sequence shown in Sequence ID No. 1 is minor allele C, it can be determined that the individual has a body type that is prone to gaining weight and difficulty to lose weight, and is at high risk of vascular aging.

[0026] rs2271987 is an SNP located on the NOS1 gene that has a statistically significant association with "lower back pain." If the 51st base of the sequence shown in Sequence ID No. 2 is the minor allele T, it can be determined that the individual has a predisposition to lower back pain and a high risk of vascular aging.

[0027] rs6470860 is a SNP located on the ADCY8 gene that has a statistically significant association with "numbness in the hands and feet." If the 51st base of the sequence shown in Sequence ID No. 3 is major allele A, it can be determined that the person has a predisposition to numbness in the hands and feet and a high risk of vascular aging.

[0028] rs2284972 is an SNP located on the PDE9A gene that is statistically significant in association with "physical pain." If the 51st base of the sequence shown in Sequence ID No. 4 is the minor allele G, it can be determined that the individual has a predisposition to physical pain and a high risk of vascular aging.

[0029] rs12028323 is a SNP located on the PRMT6 gene that is statistically significant in association with "mood disorders." If the 51st base of the sequence shown in Sequence ID No. 5 is minor allele C, it can be determined that the individual has a predisposition to mood disorders and a high risk of vascular aging.

[0030] In this invention, "tendency to gain weight and difficulty to lose weight" refers to a state in which weight increases and body shape deteriorates even with the same amount of food and exercise, and weight and body shape do not return to normal even with dieting or exercise.

[0031] In this invention, "lower back pain" refers to a general term for symptoms that involve pain or inflammation in the lower back.

[0032] In this invention, "numbness" refers to an abnormal sensation that can be described as "tingling" or "stinging," and includes decreased sensation (hypensic numbness / loss of sensation), paresthesia, parasensory perception, and hyperesthesia. Specifically, symptoms include "tingling and numbness in the hands and feet," "decreased sensation in the hands and feet," "dryness and tingling sensation on the surface of the skin," "involuntary movement of the hands and fingers," "difficulty exerting force in the hands and feet," and "stiffness in the hands."

[0033] In this invention, "body pain" refers to the sensation that arises when there is injury, inflammation, or other irritation in a part of the body, and is synonymous with "pain." Human pain sensation occurs in most parts of the body other than the skin and mucous membranes (for example, internal organs, pleura, peritoneum, teeth, eyes, ears, etc.), and is all perceived in the brain as electroencephalograms or their fluctuations. Body pain is not particularly limited, but may be pain caused by aging, poor posture, work, excessive exercise, or mental stress. Specifically, examples include headaches, stomachaches, joint pain, muscle pain, menstrual cramps, neuralgia, and shoulder pain.

[0034] In this invention, "mood disorder" refers in particular to a condition in which a person experiences distress or has some difficulty in daily life due to a persistent change in mood (emotion) over a certain period of time. Furthermore, "mood disorder" also includes milder symptoms, such as a state of decreased vitality (a state of decreased physical activity) and a state of decreased curiosity (a state of not showing interest in new tasks). Examples of symptoms of a mood disorder include abnormal expression of emotions such as joy, anger, sadness, and pleasure; depressed moods such as feelings of melancholy, sadness, guilt, and despair; psychomotor retardation; anxiety and agitation; sleep disorders; loss of appetite; decreased libido; constipation; dry mouth; and headaches.

[0035] In the determination method of the present invention, the race of the subject is not particularly limited, but is preferably East Asian, and more preferably Japanese.

[0036] The DNA-containing sample used in the determination method of the present invention is not particularly limited, as long as it is a biological sample containing DNA collected from a subject. Examples of DNA-containing samples include any bodily fluid, secretion, tissue, cell, or tissue or cell culture from which genomic DNA can be collected. Specifically, examples include the subject's saliva, blood, urine, sputum, pharyngeal swab, nasal swab, oral (inner cheek) mucosa swab, lacrimal gland secretion, sweat, hair, nails, skin, and mucous membranes. However, saliva is preferred due to its ease of use and minimal invasiveness. The sample can be collected according to methods commonly used in clinical testing and prepared using known extraction and purification methods. A commercially available genomic DNA extraction kit can be used in this process.

[0037] The methods for detecting SNPs and determining SNP types (SNP typing) are not particularly limited and can be carried out by known methods, such as using allele-specific primers (and probes), amplifying by PCR, and detecting polymorphisms of the amplified product by fluorescence or luminescence. Examples include PCR-RFLP (restriction fragment length polymorphism), PCR-SSCP (single-strand conformation polymorphism), PCR-SSO (sequence-specific oligonucleotide), direct sequencing, ASO (Allele Specific Oligonucleotide) hybridization, ASP-PCR (Allele Specific Primer-PCR), Snapshot, ARMS (Amplification Refracting Mutation System), TaqMan PCR, Invader method, MALDI-TOF / MS method, RNase A cleavage method, DOL (Dye-labeled Oligonucleotide Ligation), and TDI (Template-directed Dye-terminator Incorporation). All of the above methods are well known to those skilled in the art, and reagents and kits for determining SNP types are commercially available. For example, TaqMan SNP Genotyping Assays (manufactured by Thermo Fisher Scientific) can be used.

[0038] The results obtained by the above determination method serve as a useful indicator for subjects to select foods, beverages, or pharmaceuticals (including supplements) that have preventive and / or ameliorative effects on symptoms related to vascular aging. For example, subjects who are genetically determined to have a genetic risk of vascular aging can be recommended measures to prevent vascular aging. Therefore, according to another aspect of the present invention, a method for providing foods, beverages, or pharmaceuticals that have preventive and / or ameliorative effects on vascular aging according to the degree of the subject's genetic risk of vascular aging, based on the results obtained by the above determination method, is also provided, as well as an advice method that presents necessary measures for preventing and / or improving vascular aging to the subject. Examples of measures to prevent and / or improve vascular aging include reviewing dietary habits (limiting salt, sugar, and lipids, increasing protein intake, etc.), recommending supplements containing vascular aging inhibitory components and blood flow increasing components, maintaining a moderate exercise habit (aerobic exercise such as walking or jogging, flexibility exercises such as stretching or yoga, strength training, etc.), and improving lifestyle habits (limiting smoking and drinking, improving sleep, etc.).

[0039] 2. Kit for determining the genetic risk of vascular aging The SNP detection and typing methods described above utilize probes and primers appropriate to each method. Such probes and primers are also included within the scope of the present invention and can be provided as a kit for determining the genetic risk of vascular aging.

[0040] Examples of probes include those that include the above-mentioned SNP sites and can distinguish the type of base at the SNP site based on the presence or absence of hybridization. Specifically, examples include probes having a sequence of at least 10 consecutive bases, preferably 15 or more, or a complementary sequence thereof, including rs17108108 (SNP at the 51st base of the base sequence shown in SEQ ID NO: 1), rs2271987 (SNP at the 51st base of the base sequence shown in SEQ ID NO: 2), rs6470860 (SNP at the 51st base of the base sequence shown in SEQ ID NO: 3), rs2284972 (SNP at the 51st base of the base sequence shown in SEQ ID NO: 4), and rs12028323 (SNP at the 51st base of the base sequence shown in SEQ ID NO: 5). The length of the probe is preferably 15 to 40 bases, more preferably 20 to 35 bases. Furthermore, the probe may be labeled with an appropriate labeling substance, such as enzymes (peroxidase, β-galactosidase, alkaline phosphatase, etc.), fluorescent substances (FITC, RITC, Cy3, Cy5, etc.), luminescent substances (luminol, luminol derivatives, luciferin, lucigenin, etc.), radioisotopes ( 3 H, 14 C, 32 P, 125 I, 131 Examples include (I, etc.), biotin, digoxigenin, polypeptides containing a tag sequence, etc. Alternatively, a quencher (quenching substance) that absorbs the fluorescence energy emitted by the fluorescent substance may be further bound to the vicinity of the fluorescent substance.

[0041] Furthermore, the probes may be immobilized on a solid phase (DNA array). A DNA array allows for the simultaneous detection of hybridization to each probe by hybridizing sample DNA with numerous probes arranged on the same plane and scanning the plane. Therefore, DNA arrays are useful for simultaneously analyzing multiple SNP sites. Oligonucleotides, which serve as probes for the array, are usually synthesized in situ. For example, in situ synthesis methods for oligonucleotides using lithography (Thermo Fisher Scientific), inkjet (Agilent), and bead arrays (Illumina) are known.

[0042] Furthermore, examples of primers include those that can be used in PCR to amplify the above-mentioned SNP sites, or those that can be used for sequence analysis (sequencing) of the above-mentioned SNP sites. Specifically, examples of primers that can amplify or sequence regions containing rs17108108 (SNP at the 51st base of the nucleotide sequence shown in SEQ ID NO: 1), rs2271987 (SNP at the 51st base of the nucleotide sequence shown in SEQ ID NO: 2), rs6470860 (SNP at the 51st base of the nucleotide sequence shown in SEQ ID NO: 3), rs2284972 (SNP at the 51st base of the nucleotide sequence shown in SEQ ID NO: 4), and rs12028323 (SNP at the 51st base of the nucleotide sequence shown in SEQ ID NO: 5). A primer that can be used in PCR to amplify the above-mentioned SNP site is any oligonucleotide that can perform complementary strand synthesis toward the SNP site using the DNA of the region containing the SNP site as a template. The length of such a primer is preferably 10 to 30 bases, and more preferably 15 to 25 bases. The primer can be positioned upstream or downstream of the SNP site in the base sequence shown in any of SEQ ID NOs: 1 to 5.

[0043] Those skilled in the art can design probes and primers according to the analysis method based on the nucleotide sequence information of the surrounding DNA region containing the SNP site. Furthermore, oligonucleotides that serve as probes and primers can be synthesized using commonly used automated DNA synthesizers by methods known in the art for the synthesis of oligonucleotides, such as the phosphoramidite method and the H-phosphonate method.

[0044] The kit of the present invention may contain at least the oligonucleotides used as probes and primers as described above. The kit may also optionally include DNA extraction reagents, PCR reagents such as PCR buffer and DNA polymerase, detection reagents such as stains and electrophoresis gels, immobilization carriers, labeling substances, substrate compounds used for label detection, positive and negative standard samples, and instructions describing how to use the kit. The reagents in the kit may be in solution or lyophilized form.

[0045] 3. Screening method for agents that prevent and / or improve vascular aging The KCNMB4, NOS1, ADCY8, and PRMT6 genes are those in which the SNPs rs17108108, rs2271987, rs6470860, and rs12028323 are present, and the gene expression levels change depending on the symptoms (Table 3).

[0046] [Table 3]

[0047] Therefore, the present invention provides a screening method for agents that prevent and / or improve vascular aging, using changes in the expression of one or more genes selected from the group consisting of KCNMB4, NOS1, ADCY8, and PRMT6 (hereinafter referred to as "vascular aging susceptibility genes") as an indicator.

[0048] The screening method of the present invention is not particularly limited as long as it uses cells suitable for evaluating the expression of vascular aging susceptibility genes, but typically, it involves measuring the expression level of vascular aging susceptibility genes in cells. More specifically, the screening method of the present invention can be carried out by the steps of: culturing cells expressing the above-mentioned vascular aging susceptibility genes in the presence of a test substance and measuring the expression level of the vascular aging susceptibility genes in the cells; measuring the expression level of the vascular aging susceptibility genes in the same cells in the absence of the test substance (control); and selecting the test substance as a candidate substance for preventing and / or improving vascular aging if the expression level measured in the presence of the test substance is significantly increased or decreased compared to the expression level measured in the absence of the test substance.

[0049] In the present invention, the expression levels of vascular aging susceptibility genes in test samples and control samples can be measured by any method known to those skilled in the art, and the measurement should be carried out according to the conventional methods of each method. Gene expression level refers to the amount of mRNA, which is the transcript of the gene. The method for measuring mRNA is not particularly limited as long as it can measure the desired amount of mRNA, and can be appropriately selected from known methods. For example, a gene amplification method using oligonucleotides that hybridize to vascular aging susceptibility genes as primers, or a hybridization method using oligonucleotides that hybridize to vascular aging susceptibility genes as probes can be used. Specifically, examples include RT-PCR, real-time RT-PCR, microarray, Northern plot, and dot blot. The primers and probes used in the above methods can be labeled, and the amount of mRNA can be measured by examining the signal intensity of the label. Among these, real-time RT-PCR is preferred because it allows RNA to be used directly in the sample, and gene quantification can be performed from the number of temperature cycles required for amplification by optically measuring the gene amplification process. The primers and probes used in the above methods can be appropriately designed and prepared by a person skilled in the art based on the nucleotide sequences of vascular aging susceptibility genes obtained from databases such as GenBank. Various protocols have been reported for each method, and a person skilled in the art can carry them out according to known protocols, or by appropriately modifying or changing known protocols.

[0050] Furthermore, in another embodiment of the screening method of the present invention, cells into which a plasmid in which a reporter gene is operably linked to the transcriptional regulatory region of a vascular aging susceptibility gene is introduced may be used as cells expressing a vascular aging susceptibility gene, and the expression level of the reporter gene may be measured. Examples of reporter genes include the GFP gene, GUS gene, LUS gene, etc.

[0051] The test substances used for screening are mainly components that can be used in pharmaceuticals and / or food and beverages, and include, for example, mixtures containing multiple compounds such as extracts of animal and plant tissues or microbial cultures, and purified standards therefrom; naturally occurring molecules (e.g., amino acids, peptides, oligopeptides, polypeptides, proteins, nucleic acids, lipids, steroids, glycoproteins, proteoglycans, etc.); synthetic analogs or derivatives of naturally occurring molecules (e.g., peptide mimetic substances, etc.); and molecules that do not occur naturally (e.g., low-molecular-weight organic compounds produced using combinatorial chemistry technology, etc.); and mixtures thereof. Furthermore, a single test substance may be tested independently, or a mixture of several candidate test substances (including libraries, etc.) may be tested. Examples of libraries containing multiple test substances include synthetic compound libraries and peptide libraries. [Examples]

[0052] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to these examples.

[0053] (Examples) 1. Method (1) Subjects DeNA Life Sciences Inc. publicly recruited participants for a research project (research project title: "Research aimed at realizing a healthy and long-lived society using health big data," hereinafter referred to as the "MYCODE research") conducted using samples and information obtained through its genetic testing service "MYCODE." 2,000 individuals who agreed to participate in this study and met all of the following selection criteria were selected as subjects.

[0054] <Selection Criteria> (a) The participant must be 40 years of age or older at the time of obtaining consent to participate in the study. (b) Having already given written consent to participate in the MYCODE study. (c) The results of the MYCODE genetic test have already been obtained. (d) Having received a full explanation of the purpose and content of this examination, having the capacity to give consent, fully understanding it, and voluntarily volunteering to participate and agreeing to participate in this examination.

[0055] (2) Questionnaire for obtaining information on phenotype (traits) Information on phenotype (traits) was obtained from a questionnaire survey on subjective symptoms answered by the subjects. The questionnaire survey consisted of (2-1) subjective VAS, (2-2) POMS2 mood disorder score (TMD), and (2-3) SP-36 physical pain (BP). For each trait obtained from the questionnaire survey, subjects who possessed a specific trait were designated as the case group, and subjects who did not possess the trait were designated as the control group. The case / control groups were defined accordingly.

[0056] (2-1) VAS questionnaire (emotional index of susceptibility to weight gain and difficulty losing weight, lower back pain, numbness in hands and feet) Participants were asked to complete a questionnaire regarding their subjective symptoms for each of the following items: "I feel like I've gained weight easily and it's harder to lose weight," "I feel lower back pain," and "I feel numbness in my hands and feet." These symptoms were then evaluated using a Visual Analogue Scale (VAS). For each evaluation item, the participant's score was calculated from 0 to 10. For each item, participants were asked to rate their score on a scale of 0 to 10, with 0 representing "I don't feel it at all," 5 representing "neither," and 10 representing "I feel it very strongly."

[0057] For the symptoms "feeling like I've gained weight easily and it's harder to lose weight," "feeling like I have lower back pain," and "feeling like I have numbness in my hands and feet," the top 30-40% of those experiencing these symptoms were designated as the case group, and the rest as the control group (those who answered 4-6, meaning they couldn't determine either, were not included in either the case or control group (NA: Not Applicable)).

[0058] (2-2) POMS2 Mood Disorder Score (TMD) A correlation has been reported between a decrease in FMD (flow-dependent vasodilation), an indicator of vascular endothelial function, and a decrease in the mood disorder score on the POMS (Denise C. Cooper et al., Psychosom Med 73(5):360-9 (2011)).

[0059] POMS2 (Profile of Mood States 2nd Edition) is a test that can assess a subject's mood, anxiety, and depressive state. It evaluates mood within a predetermined time frame using seven scales: "anger-hostility," "confusion-embarrassment," "depression-downcasting," "fatigue-apathy," "tension-anxiety," "vigor-energy," and "friendliness," as well as a "TMD score" that comprehensively represents negative mood states. In this example, mood states were evaluated using the POMS2 TMD score.

[0060] Participants were asked a total of 65 questions about their mood state over the past week, and were asked to respond on one of five options: "0. Not at all," "1. A little," "2. Somewhat," "3. Quite a lot," and "4. Very much." Referring to the Japanese version of the POMS2 manual, a median T-score (standardized score) of 50 points was used as the baseline, with participants scoring 50 points or less designated as the control group and those scoring 51 points or more as the case group.

[0061] (2-3) SF-36 Bodily pain (BP) It has been reported that the SF-36 score increases with improvement in baPWV (brachial-ankle pulse wave velocity), an indicator of vascular elasticity (Hai-ya Wang MD et al., The American Journal of the Medical Sciences (2018)).

[0062] The SF-36 (MOS Short-Form 36-Item Health Survey) is a self-report health questionnaire designed to measure a comprehensive health concept, rather than health conditions specific to particular diseases or symptoms, through 36 questions linked to eight subscales (physical functioning, role functioning (physical), bodily pain, overall health perception, vitality, social functioning, role functioning (mental), and mental health). After some of the choices are weighted, the subscales are scored on a scale of 0 to 100, with higher scores indicating better health. National standard values ​​for each scale have been calculated by gender and age group, and the national standard values ​​for Japanese people are described in detail in the SF-36 Japanese manual (Japanese Journal of Low Back Pain, 8(1):38-43, 2002). In this example, bodily pain was evaluated using "bodily pain (BP)," one of the subscales.

[0063] Participants were asked about physical pain and asked to respond with either "In the past month, I have experienced very severe physical pain that has significantly interfered with my usual work" or "In the past month, I have experienced no physical pain at all, and physical pain has not interfered with my usual work at all." Since the BP score calculated using the SF-36 scoring algorithm showed a significant correlation with the POMS2 TMD T-value, in order to collect participants with more distinctive phenotypes, the control group consisted of individuals with a POMS2 TMD T-value of less than 40 and whose BP score was above the mean within that group, while the case group consisted of individuals with a POMS2 TMD T-value of 60 or higher and whose BP score was below the mean within that group.

[0064] (3) Single nucleotide polymorphism (SNP) data Single nucleotide polymorphism (SNP) data was obtained using information already acquired in the MYCODE study. Specifically, in the MYCODE study, saliva samples were collected in a dedicated saliva collection container, stored mixed with a preservation solution, and then acquired by genotyping using Infinium OmniExpress-24 or Human OmniExpress-24 BeadChip (Illumina) containing approximately 30,000 custom probes.

[0065] (4) Selection of subjects for analysis (Sample-quality control (Sample-QC)) Based on the SNP genotyping data obtained from the MYCODE study, a sample-QC of 2,000 subjects was performed using the open-source genome analysis software PLINK version 1.9 to eliminate potential bias in association analyses and ensure a homogeneous population (Purcell, S. et al. PLINK: a tool set for whole-genome association and population-based linkage analyses. Am. J. Hum. Genet. 81, 559-575 (2007); Chang, CC et al. Second-generation PLINK: rising to the challenge of larger and richer datasets. Gigascience. 4, 7 (2015)). Samples with sex mismatch and samples with low genotyping accuracy (Missing call rate > 1.0%) were checked, but no samples were found that should be excluded. To exclude close relatives, we used PLINK's PI_HAT (IBD estimates) as the IBD (Identity by Descent) evaluation value and checked for pairs that showed a value of 0.1875 or higher, which is a commonly used threshold (a value between second-degree and third-degree kinship). No samples were found that should be excluded.

[0066] Subsequently, principal component analysis (PLINK-pca) was performed on the acquired genome-wide SNP data for each subject to examine the existence of population structure. For the principal component analysis, SNPs were shifted to form blocks of 50 SNPs each, taking linkage disequilibrium (LD) into consideration. Only SNPs with a linkage disequilibrium coefficient (R²) of less than 0.5 were extracted (PLINK-indep-pairwise 50 5 0.5). The first and second principal components obtained from the principal component analysis for each sample were plotted. Samples significantly outside the population cluster were visually identified and excluded, and the same principal component analysis was repeated for the remaining samples. Finally, four samples were excluded, and it was confirmed that there were no samples significantly outside the population in the principal component analysis after exclusion.

[0067] Principal component analysis of the 1,996 samples after Sample-QC confirmed that no samples were significantly outside the group cluster (Figure 1). These 1,996 samples were used for the following GWAS and target analysis. The mean age of the samples was 53.5 years (including 3 individuals with no gender). When classified by gender, the male-to-female ratio was 967 individuals (48.4%) / 1,026 individuals (51.4%) (3 individuals with no gender (0.2%)), and the mean age was 54.6 years / 52.4 years (Table 4).

[0068] [Table 4]

[0069] (5) Genome-wide association study (GWAS) After sample-QC, phasing was performed on 694,434 SNPs on autosomes using Eagle v2.4.1, software developed by Autdesk, referencing East Asian samples from the 1KGP (International 1000 Genome Project whole-genome reference panel) to calculate haplotype sequences (allelic combinations). Genotype imputation was performed on the genomic data after phasing using Minimac3, a genotyping algorithm software (Das S. et al. Next-generation genotype imputation service and methods. Nat Genet 48, 1284-1287 (2016)). After genotype imputation, SNPs with low estimation accuracy (estimated correlation coefficient Rsq < 0.7 between the true genotype and the estimated genotype output by Minimac3) were removed from all SNPs. Furthermore, marker-QC was performed on SNPs after genotype imputation using PLINK version 1.9 to adjust for call rates per SNP (exclusion of missing call rates > 1.0%), Hardy-Weinberg equilibrium test thresholds (exclusion of p<1E-06), and Minor Allele Frequency (MAF) (exclusion of MAF<0.05). Finally, a GWAS was performed on 5,246,599 to 5,250,269 SNPs (depending on the case / control population for each trait) after marker-QC. Specifically, logistic regression analysis was performed on the case / control group using PLINK version 1.9, assuming an additive model (the effect of SNPs on phenotype is proportional to the number of alleles). As covariates, age, sex, and the first and second principal components from principal component analysis of SNP data, which are commonly used in GWAS, were used. The p-value for the significance level set in the GWAS was set to a genome-wide significance level of p<5E-08.

[0070] (6) Target analysis Nitric oxide (NO) regulates blood pressure by inducing relaxation of vascular smooth muscle and protects the vascular wall by inhibiting the adhesion and aggregation of platelets and leukocytes to the vascular wall (Mirenayat MS et al., Curr Hypertens Rep 20(11):98 (2018), Barkhidarian B et al., Avicenna J Phytomed 9(1):10-20 (2019)). It also plays a role in maintaining vascular structure by inhibiting vascular smooth muscle proliferation and promoting angiogenesis (Sarkar R et al., Circ Res 78(2):225-230 (1996)). Therefore, NO is known to be an indicator of vascular endothelial function. Consequently, a decrease in NO production capacity is thought to cause a decrease in vascular endothelial function, i.e., vascular aging.

[0071] Therefore, in this study, 20 genes were selected from specific metabolic pathways involved in NO production. Specifically, from the arginine metabolic pathway upstream of NO production, arginase (ARG1, ARG2), argininosuccinate lyase (ASL), argininosuccinate synthase (ASS1), dimethylarginine dimethylaminohydrolase (DDAH1), and nitric oxide synthase (NOS1) were selected. In addition, from the glutamine metabolic pathway upstream of NO production, carbamoyl phosphate synthase (CPS1), glutaminase (GLS), glutamate dehydrogenase (GLUD1), glutamate ammonia ligase (GLUL), and N-acyl-L-amino acid amide hydrolase (ABHD14A-ACY1) were selected. Furthermore, from the NO-cGMP cascade downstream of NO production, we selected the guanylate cyclase soluble subunit (GUCY1A2), phosphodiesterases (PDE1A, PDE2A, PDE3A, PDE5A, PDE9A, PDE10A), potassium-calcium activated channel (KCNMB4), and adenylyl cyclase (ADCY8). It has been reported that alterations in the expression of these genes alter NO production levels (William Durante et al., Clin Exp Pharmacol Physiol 34(9):906-911 (2007), Rodrigo Goncalves Dias et al., Arq Bras Cardiol 96(1):68-75 (2011)).

[0072] In this study, 597 SNPs located on the target genes that are on autosomes, directly typed using the MYCODE microarray chip, and satisfy MAF ≥ 0.05 were selected as the target SNPs for analysis (Table 5). The p-value for the significance level set for the target analysis was set to p < 8.38E-05 (0.05 / 597), taking into account Bonferroni correction.

[0073] [Table 5]

[0074] (7) eQTL analysis (Expression quantitative trait locus analysis) We performed eQTL analysis to examine the association between SNPs and gene expression using the GTEx Analysis Release V8 database [https: / / gtexportal.org / home / ] for related SNPs identified from GWAS and target analysis. We targeted 1M-based cis-eQTLs around transcription start sites (TS S) and referred to the results of the association between significant mutations and gene expression (GTEx_Analysis_v8_eQTL.tar).

[0075] 2. Test Results (1) Subjective VAS Questionnaire <Tendency to gain weight, difficulty to lose weight> Target analysis revealed that the subjective VAS questionnaire (whether participants felt they gained weight more easily and lost weight more difficult) (Case: participants felt they gained weight more easily and lost weight more difficult / Control: participants did not feel they gained weight more easily and lost weight more difficult) showed an association with the SNP rs17108108 (MAF=0.1769) on the KCNMB4 gene. A tendency was observed for individuals with minor allele C to feel they gained weight more easily and lost weight more difficult (p=3.89E-05, odds ratio (OR)=1.61). Furthermore, eQTL analysis using the GTEx Analysis Release V8 database also showed that the ALT allele of rs17108108 increased the expression levels of the KCNMB4 gene in fibroblasts, whole blood, lower limb skin (sun exposure), and suprapubic skin (non-sun exposure).

[0076] <Back pain> Target analysis revealed that the subjective VAS questionnaire (whether or not one experiences lower back pain) (Case: experiences lower back pain / Control: does not experience lower back pain) showed an association with the SNP rs2271987 (MAF=0.1568) on the NOS1 gene, and a tendency to experience lower back pain was observed in individuals with the minor allele T (p=8.34E-05, odds ratio (OR)=1.43).

[0077] <Numbness in the hands and feet> Target analysis revealed that the subjective VAS questionnaire (whether or not numbness is felt in the hands and feet) (Case: numbness is felt in hands and feet / Control: no numbness is felt in hands and feet) showed an association with the SNP rs6470860 (ALT_frq=0.7104) on the ADCY8 gene, and a tendency to feel numbness in the hands and feet was observed in those with major allele A (p=8.10E-05, odds ratio (OR)=0.623). Furthermore, eQTL analysis showed that the major allele rs6470860 of the ADCY8 gene reduced its expression level in testicular tissue.

[0078] (2) Physical pain (SF-36 BP) Target analysis revealed that in SF-36 BP (cases; high physical pain score / control; low physical pain score), the SNP rs2284972 (MAF=0.4201) on the PDE9A gene showed a significant association, and a tendency for higher physical pain scores was observed in individuals with minor allele G (p=6.39E-05, odds ratio (OR)=1.338).

[0079] (3) Mood Disorder Score (POMS-TMD) GWAS results showed that in POMS2-TMD (cases: high mood disorder score / controls: low mood disorder score), the SNP rs12028323 (MAF = 0.09619) on the PRMT6 gene was significantly associated with a higher mood disorder score in individuals with minor allele C (p = 2.43E-08, odds ratio (OR) = 1.89).

[0080] A summary of the above results is shown in Table 6 below.

[0081] [Table 6]

[0082] From the above analysis, five SNPs were identified as being associated with symptoms related to vascular aging and capable of determining an individual's risk of vascular aging (Table 6). The SNP ID (NCBI dsSNP), the name of the gene in which the SNP exists, the symptoms, p-value, alternative (mutant) allele (ALT), reference allele (REF), alternative allele frequency (ALT_frq), minor allele frequency (MAF), and odds ratio (OR) for each identified SNP are shown. In the p-value column, each p-value is expressed in base-10 exponential form, and the numbers before and after the sign E indicate the mantissa and exponent, respectively, when the p-value is expressed in exponential form. Furthermore, ALT corresponds to the mutant allele, REF corresponds to the base form allele, and ALT represents the risk allele. [Industrial applicability]

[0083] This invention can be used in the field of genetic testing services related to vascular aging.

Claims

1. A method for determining the genetic risk of vascular aging, comprising the steps of detecting one or more single nucleotide polymorphism (SNP) alleles from (A) to (E) below in a DNA-containing sample taken from a subject, and determining that the subject is prone to developing symptoms related to vascular aging if the base of the detected allele is a risk allele. (A) SNP: Identified by ID rs17108108, this SNP is located at the 51st base of the sequence shown in Sequence ID No. 1 (risk allele is minor allele C). (B) SNP: Identified by ID rs2271987, this SNP is located at the 51st base of the sequence shown in Sequence ID No. 2 (risk allele is minor allele T). (C) SNP: Identified by ID rs6470860, this SNP is located at the 51st base of the sequence shown in Sequence ID No. 3 (risk allele is major allele A). (D) SNP: Identified by ID rs2284972, this SNP is located at the 51st base of the sequence shown in Sequence ID No. 4 (risk allele is minor allele G). (E) SNP: Identified by ID rs12028323, this SNP is located at the 51st base of the sequence shown in Sequence ID No. 5 (risk allele is minor allele C).

2. The method according to claim 1, wherein the single nucleotide polymorphism (SNP) is rs17108108, and the symptoms associated with vascular aging are a tendency to gain weight and difficulty losing weight.

3. The method according to claim 1, wherein the single nucleotide polymorphism (SNP) is rs2271987, and the symptom associated with vascular aging is lower back pain.

4. The method according to claim 1, wherein the single nucleotide polymorphism (SNP) is rs6470860, and the symptom associated with vascular aging is numbness in the hands and feet.

5. The method according to claim 1, wherein the single nucleotide polymorphism (SNP) is rs2284972, and the symptom associated with vascular aging is body pain.

6. The method according to claim 1, wherein the single nucleotide polymorphism (SNP) is rs12028323, and the symptom associated with vascular aging is a mood disorder.

7. A method for providing food or pharmaceuticals, comprising providing a subject with food or pharmaceuticals that have a preventive and / or ameliorative effect on vascular aging in proportion to the degree of the subject's genetic risk of vascular aging, based on the results determined by the method according to any one of claims 1 to 6.

8. An advice method for presenting measures necessary for preventing and / or improving vascular aging in a subject, using the results determined by the method according to any one of claims 1 to 6.

9. A kit for determining the genetic risk of vascular aging, comprising a probe containing a sequence of 10 or more bases including the 51st base of the base sequence shown in any of SEQ ID NOs: 1 to 5, or a complementary sequence thereof, and / or a primer capable of amplifying the region containing the 51st base of the base sequence shown in any of SEQ ID NOs: 1 to 5.

10. A screening method for substances that prevent and / or improve vascular aging, using changes in the expression of one or more genes selected from the group consisting of the KCNMB4 gene, NOS1 gene, ADCY8 gene, and PRMT6 gene as an indicator.

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