Data collection method and kit for determining likelihood of developing Alzheimer's disease
By measuring DNA methylation at specific CpG islands in the COASY gene promoter region, the method offers a non-invasive and cost-effective approach for assessing the risk of Alzheimer's disease, improving early detection and diagnosis.
Patent Information
- Application Number
- JP2024555866
- Authority / Receiving Office
- JP · JP
- Patent Type
- Patents
- Current Assignee / Owner
- Priority Date
- 2022-10-06
- Filing Date
- 2023-10-06
- Publication Date
- 2025-11-26
- Estimated Expiration
- 2043-10-06
Smart Images
Figure 0007775546000005 
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Abstract
Description
[Technical Field]
[0001] The present invention relates to a data collection method and kit for determining the likelihood of developing Alzheimer's disease. This application claims priority based on Japanese Patent Application No. 2022-161607, filed on October 6, 2022, the contents of which are incorporated herein by reference. [Background technology]
[0002] Alzheimer's disease is one of the most common forms of dementia. It is believed that Alzheimer's disease develops when amyloid beta and phosphorylated tau deposit in brain tissue, but the underlying mechanism remains unknown. Genetic factors, aging, and environmental factors such as lifestyle and infection are thought to be involved in epigenetics, and these factors are thought to be involved in the onset of the disease.
[0003] Early detection of Alzheimer's disease is performed by specialists, but accurate diagnosis is difficult and it is not possible to diagnose before the onset of the disease. Furthermore, while methods such as detecting amyloid beta in cerebrospinal fluid and PET (positron emission tomography) diagnosis are used, detecting amyloid beta in cerebrospinal fluid is highly invasive, and PET diagnosis is expensive, only available at limited facilities, and not covered by insurance, making them of little clinical practicality.
[0004] Currently, there are no blood biomarkers in clinical use for diagnosing Alzheimer's disease. The present inventors comprehensively quantified changes in blood DNA methylation levels in healthy individuals, patients with Alzheimer's disease, and patients with mild cognitive impairment (MCI), a precursor to Alzheimer's disease. They found that patients with Alzheimer's disease and mild cognitive impairment had significantly decreased DNA methylation in the promoter region of the NCAPH2 (LMF2) gene, and significantly increased DNA methylation in the promoter regions of the COASY gene and SPINT1 gene. They reported that these biomarkers may be useful for the early diagnosis and differentiation of dementia (Non-Patent Documents 1 to 3). [Prior art documents]
Non-Patent Literature
[0005]
Non-Patent Literature 1
Non-Patent Literature 2
Non-Patent Literature 3
[0006] However, the promoter region of the COASY gene contains multiple CpG islands, and it is unknown which of these CpG islands has methylation associated with Alzheimer's disease. The present inventors discovered that the DNA methylation rate of a specific CpG island among the CpG islands in the promoter region of the COASY gene is useful for determining the likelihood of developing Alzheimer's disease, and thus completed the present invention. Specifically, the present invention aims to provide a technique useful for determining the likelihood of a subject developing Alzheimer's disease by measuring the DNA methylation rate of a specific CpG island among the CpG islands in the promoter region of the COASY gene. [Means for solving the problem]
[0007] The present invention includes the following aspects. [1] A data collection method for determining the likelihood that a subject will develop Alzheimer's disease, comprising measuring the rate of methylation of DNA in a CpG island containing a cytosine residue selected from the group consisting of cytosine residues at positions 24, 56, 75, 81, 98, 103, 115, 126, 134, 142, 153, and 179 in the base sequence of the promoter region of the COASY gene shown in SEQ ID NO: 1 in a biological sample from the subject; wherein a higher rate compared to a control indicates a higher likelihood that the subject will develop Alzheimer's disease. [2] The data collection method according to [1], wherein the biological sample is blood. [3] A kit for determining the likelihood that a subject will develop Alzheimer's disease, comprising a primer or probe for amplifying DNA of a CpG island containing a cytosine residue selected from the group consisting of cytosine residues at positions 24, 56, 75, 81, 98, 103, 115, 126, 134, 142, 153, and 179 in the base sequence of the promoter region of the COASY gene shown in SEQ ID NO: 1. [4] The kit described in [3], wherein the primers for amplifying DNA of a CpG island containing the 24th cytosine residue in the base sequence of the promoter region of the COASY gene shown in SEQ ID NO: 1 are a primer set including a forward primer consisting of the base sequence shown in SEQ ID NO: 2 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 9. [Effects of the Invention]
[0008] According to the present invention, a technique useful for determining the possibility of developing Alzheimer's disease in a subject can be provided. [Brief explanation of the drawings]
[0009] [Figure 1]The nucleotide sequence of the promoter region of the COASY gene shown in SEQ ID NO: 1 and the positions of CpG island numbers 1 to 14 are shown in Figure 1. The shaded DNA (CG) indicates the positions of the CpG islands. [Figure 2A] This graph shows the percentage of DNA methylation in CpG islands 1 to 9 in the promoter region of the COASY gene in non-demented elderly (NC) and patients with Alzheimer's disease (AD) and amnestic mild cognitive impairment (MCI). A to I indicate the percentage of DNA methylation in CpG islands 1 to 9, respectively. [Figure 2B] This graph shows the percentage of DNA methylation in CpG islands 10 to 14 in the promoter region of the COASY gene in non-demented elderly (NC) subjects and patients with Alzheimer's disease (AD) and amnestic mild cognitive impairment (MCI). J to N indicate the percentage of DNA methylation in CpG islands 10 to 14, respectively. [Figure 3A] FIG. 1 shows an amplification plot of TaqMan real-time PCR performed using a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 2, a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 3, and a probe for detecting methylated DNA consisting of the nucleotide sequence shown in SEQ ID NO: 4, and using bisulfite-modified DNA of CpG island number 14 in the promoter region of the COASY gene with methylation rates of 100%, 75%, 50%, 25%, 5%, and 0% as a template. [Figure 3B] FIG. 1 shows an amplification plot of TaqMan real-time PCR performed using a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 2, a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 3, and a probe for detecting unmethylated DNA consisting of the nucleotide sequence shown in SEQ ID NO: 5, and using bisulfite-modified DNA of CpG island number 14 in the promoter region of the COASY gene with methylation rates of 100%, 75%, 50%, 25%, 5%, and 0% as a template. [Figure 4A]FIG. 1 shows an amplification plot of TaqMan real-time PCR performed using a forward primer in which the third and twentieth thymine residues of the base sequence shown in SEQ ID NO: 2 are LNA, a reverse primer in which the eighth and twentieth adenine residues of the base sequence shown in SEQ ID NO: 9 are LNA, and a probe for detecting methylated DNA consisting of the base sequence shown in SEQ ID NO: 4, and using as a template DNA of CpG island number 14 in the promoter region of the COASY gene, which was bisulfite-modified with methylation rates of 100%, 75%, 50%, 25%, 5%, and 0%. [Figure 4B] 1 shows an amplification plot of TaqMan real-time PCR performed using a forward primer in which the third and twentieth thymine residues of the base sequence shown in SEQ ID NO: 2 are LNA, a reverse primer in which the eighth and twentieth adenine residues of the base sequence shown in SEQ ID NO: 9 are LNA, and a probe for detecting unmethylated DNA consisting of the base sequence shown in SEQ ID NO: 5, and using as a template DNA of CpG island number 14 in the promoter region of the COASY gene, which was bisulfite-modified with methylation rates of 100%, 75%, 50%, 25%, 5%, and 0%. [Figure 5] 1 is a graph showing the percentage of DNA methylation at CpG island number 14 in the promoter region of the COASY gene in the blood of non-demented elderly (NC), patients with Alzheimer's disease (AD), and patients with amnestic mild cognitive impairment (MCI). The graph was obtained using a forward primer in which the third and twentieth thymine residues in the base sequence shown in SEQ ID NO: 2 are LNA, a reverse primer in which the eighth and twentieth adenine residues in the base sequence shown in SEQ ID NO: 9 are LNA, and a probe for detecting methylated DNA consisting of the base sequence shown in SEQ ID NO: 4 or a probe for detecting unmethylated DNA consisting of the base sequence shown in SEQ ID NO: 5. DETAILED DESCRIPTION OF THE INVENTION
[0010] [Data collection method] In one embodiment, the present invention provides a data collection method for determining the likelihood that a subject will develop Alzheimer's disease, the data collection method comprising measuring the rate of methylation of DNA in a CpG island containing a cytosine residue selected from the group consisting of cytosine residues at positions 24, 56, 75, 81, 98, 103, 115, 126, 134, 142, 153, and 179 in the base sequence of the promoter region of the COASY gene (NM_001042529) represented by SEQ ID NO: 1, wherein a higher rate compared to a control indicates a higher likelihood that the subject will develop Alzheimer's disease.
[0011] As will be described later in the Examples, the method of this embodiment allows for the collection of data for determining whether or not a subject is likely to develop Alzheimer's disease. Note that the data collection method does not include a step in which a doctor makes a judgment. The method of this embodiment can also be described as a method for determining whether or not a subject is likely to develop Alzheimer's disease.
[0012] In the method of this embodiment, the rate of DNA methylation in a CpG island containing a cytosine residue selected from the group consisting of cytosine residues at positions 24, 56, 75, 81, 98, 103, 115, 126, 134, 142, 153, and 179 in the base sequence of the promoter region of the COASY gene shown in SEQ ID NO: 1 is data for determining whether or not a subject is likely to develop Alzheimer's disease.
[0013] In the method of this embodiment, the control is the DNA methylation rate of the CpG island in the promoter region of the COASY gene in a biological sample from a subject who is known in advance to not have Alzheimer's disease. Subjects who are known in advance to not have Alzheimer's disease include healthy individuals, healthy elderly people, etc.
[0014] In the method of this embodiment, the percentage of DNA methylation in one or more CpG islands containing cytosine residues selected from the group consisting of cytosine residues at positions 24, 56, 75, 81, 98, 103, 115, 126, 134, 142, 153, and 179 in the base sequence of the promoter region of the COASY gene shown in SEQ ID NO: 1 in a biological sample from a subject is measured, and if the percentage of DNA methylation in the CpG islands containing the cytosine residues is higher than that in a control, it can be determined that the subject is highly likely to develop Alzheimer's disease.
[0015] In this specification, the numbers of the CpG islands containing the cytosine residues at positions 24, 56, 75, 81, 98, 103, 115, 120, 126, 134, 142, 153, 177, and 179 in the nucleotide sequence of the promoter region of the COASY gene shown in SEQ ID NO: 1 are referred to as CpG island numbers 14 to 1, respectively. Figure 1 shows the nucleotide sequence of the promoter region of the COASY gene shown in SEQ ID NO: 1 and the positions of the CpG islands with CpG island numbers 1 to 14 in the nucleotide sequence. Table 1 shows the relationship between the CpG island numbers and the positions of the cytosine residues in SEQ ID NO: 1.
[0016] [Table 1]
[0017] In the method of this embodiment, the biological sample is not particularly limited as long as it can collect DNA containing data for determining the likelihood of a subject developing Alzheimer's disease, and examples include blood, hair, saliva, skin, etc., but blood is preferred because it is less invasive and contains a large amount of DNA. More specifically, genomic DNA present in the biological sample is extracted, and the DNA methylation rate of the CpG island in the promoter region of the COASY gene is measured.
[0018] A CpG island is a region in the genome where the frequency of CpG, a dinucleotide sequence consisting of cytosine followed by guanine, is higher than in other regions. The "p" in CpG represents the phosphodiester bond between cytosine and guanine. Many mammalian genes are known to contain CpG islands within or near their promoters.
[0019] Among the CpG islands in the promoter region of the COASY gene used in the method of this embodiment, the Cg numbers (ID numbers of the probes used in the Illumina Infinium HD Methylation Assay Kit (Illumina)) of CpG islands 7, 6, 5, 4, and 1 are CG15138339, CG19618279, CG05465955, CG25135457, and CG01756799, respectively.
[0020] Detection of methylation of cytosine residues can be suitably carried out by a method utilizing bisulfite (hydrogen sulfite) treatment of DNA fragments. By the bisulfite treatment, cytosine residues are deaminated and converted to uracil residues, while methylated cytosine residues remain. Bisulfite treatment can be carried out using a commercially available kit. Examples of commercially available kits include the EZ DNA Methylation Kit (manufactured by Zymo Research).
[0021] Therefore, methylation of cytosine residues can be detected by sequencing the DNA fragments after bisulfite treatment.The percentage of methylated cytosine residues can be measured by sequencing the base sequence of the entire DNA fragment using a next-generation sequencer.
[0022] Alternatively, methylation of cytosine residues can be detected by PCR using primers that distinguish between cytosine and uracil residues and the bisulfite-treated DNA fragment as a template. Here, quantitative PCR is performed, and the proportion of methylated cytosine residues can be measured by creating a high-resolution melting curve of the PCR product at various reaction temperatures.
[0023] Alternatively, a probe capable of hybridizing to the target region can be designed, and the bisulfite-treated DNA fragments can be subjected to whole genome amplification using an enzyme, followed by enzymatic fragmentation. The fragments are then hybridized to probe-bound beads, and fluorescently labeled nucleotides are incorporated by single-base extension. The fluorescence intensity is then measured using a fluorescent dye-labeled antibody, allowing the proportion of methylated cytosine residues to be determined. This method can be performed using a commercially available kit. For example, the proportion of methylation can be measured by bead analysis using an iScan (Illumina) using the Illumina Infinium HD Methylation Assay Kit (Illumina).
[0024] Alternatively, the bisulfite-treated DNA fragments can be used as templates for PCR amplification of the target region, followed by transcription into RNA, which is then cleaved in a base-specific manner using RNase. Fragments with different molecular weights for methylated and unmethylated DNA can be detected using a time-of-flight mass spectrometer to measure the percentage of methylated cytosine residues. This method can be performed using a commercially available kit. For example, the percentage of methylation can be measured using analysis with MassARRAY EpiTYPER (SEQUENOM).
[0025] Alternatively, methylation of cytosine residues can be detected by PCR using primers that distinguish between cytosine and uracil residues and the bisulfite-treated DNA fragment as a template. Here, the proportion of methylated cytosine residues can be measured by quantitative PCR using probes labeled with two types of fluorescent dyes that can distinguish between cytosine and uracil residues.
[0026] [kit] In one embodiment, the present invention provides a kit for determining the likelihood that a subject will develop Alzheimer's disease, comprising primers or probes for amplifying DNA of a CpG island containing a cytosine residue selected from the group consisting of cytosine residues at positions 24, 56, 75, 81, 98, 103, 115, 126, 134, 142, 153, and 179 in the base sequence of the promoter region of the COASY gene shown in SEQ ID NO: 1.
[0027] The kit of this embodiment can measure the percentage of DNA methylation in a CpG island containing a cytosine residue selected from the group consisting of cytosine residues at positions 24, 56, 75, 81, 98, 103, 115, 126, 134, 142, 153, and 179 in the base sequence of the promoter region of the COASY gene.
[0028] Among the cytosine residues, examples of primers for amplifying DNA in a CpG island (CpG island number 14) containing the 24th cytosine residue in the nucleotide sequence of the promoter region of the COASY gene shown in SEQ ID NO: 1 include a primer set shown in Table 2 below, which includes a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 2 and a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 9. The primers may contain locked nucleic acid (LNA) in all or part of the primer. By incorporating LNA into the primer, the melting temperature (Tm) of the DNA double strand can be increased, thereby enhancing the binding affinity to the target. For example, the forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 2 preferably contains LNA at the third and twentieth thymine residues. Furthermore, the reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 9 preferably contains LNA at the eighth and twentieth adenine residues.
[0029] [Table 2]
[0030] Among the above cytosine residues, examples of probes that amplify DNA of a CpG island (CpG island number 14) containing the 24th cytosine residue in the base sequence of the promoter region of the COASY gene shown in SEQ ID NO: 1 include, for example, a probe that detects methylated DNA consisting of the base sequence shown in SEQ ID NO: 4 and a probe that detects unmethylated DNA consisting of the base sequence shown in SEQ ID NO: 5, as shown in Table 3 below.
[0031] [Table 3]
[0032] The kit of this embodiment may contain, in addition to the above-described primers or probes, a bisulfite, and may further contain various reagents for DNA extraction, hybridization, ligation, PCR, etc., restriction enzymes, etc.
[0033] [Other embodiments] In one embodiment, the present invention provides a method for treating Alzheimer's disease, comprising measuring a methylation rate of DNA in a CpG island containing a cytosine residue selected from the group consisting of cytosine residues at positions 24, 56, 75, 81, 98, 103, 115, 126, 134, 142, 153, and 179 in the base sequence of the promoter region of the COASY gene as shown in SEQ ID NO: 1 in a biological sample from the subject; and administering treatment for Alzheimer's disease to the subject if the methylation rate is higher than that of a control.
[0034] As described above, the present inventors have demonstrated that the percentage of DNA methylation in CpG islands containing cytosine residues selected from the group consisting of cytosine residues at positions 24, 56, 75, 81, 98, 103, 115, 126, 134, 142, 153, and 179 in the nucleotide sequence of the promoter region of the COASY gene shown in SEQ ID NO: 1 is significantly higher in biological samples from subjects at a high risk of developing Alzheimer's disease than in controls. Therefore, if the percentage of methylation of these cytosine residues is significantly higher than that in controls, it can be determined that the subject is at a high risk of developing Alzheimer's disease.
[0035] In the treatment method of this embodiment, the control, biological sample, method for measuring DNA methylation in CpG islands, etc. are the same as those described above.
[0036] Methods for treating Alzheimer's disease include drug therapies such as Aricept (registered trademark) and Reminyl (registered trademark), as well as known methods such as non-drug therapies for the patient's behavioral and psychological symptoms. [Example]
[0037] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.
[0038] [Experimental Example 1] Peripheral blood DNA from the subject was bisulfite-treated, and the promoter region of the COASY gene (SEQ ID NO: 1) was amplified by PCR using the bisulfite-treated DNA as a template by pyrosequencing. The primers used were a biotin-modified forward primer (5'-[Biotin]-GATTATGGGATAGGAGAAGTGTT-3') in which the 5' end of the nucleotide sequence (SEQ ID NO: 6) was biotin-modified, a reverse primer (5'-CCTAATCCAAAATCCCTCTTAC-3') consisting of the nucleotide sequence (SEQ ID NO: 7), and a sequencing primer (5'-ACCACAAACTACTCCTAA-3') consisting of the nucleotide sequence (SEQ ID NO: 8). The resulting PCR amplification product was adsorbed onto beads and sequenced from the 3' end of the bisulfite-treated COASY promoter sequence using a PyroMark Q24 Advanced (Qiagen). There are 14 CpG islands in this sequence, with the first CpG island assigned a CpG island number of 1 and the last CpG island assigned a CpG island number of 14. Figure 1 shows the nucleotide sequence of the promoter region of the COASY gene and the locations of the CpG islands with CpG island numbers 1 to 14.
[0039] Next, 0.5 to 1.0 μg of peripheral blood DNA from 173 patients with amnesic mild cognitive impairment (MCI) and Alzheimer's disease (AD) and 200 healthy elderly people (NC) was treated with bisulfite using the EZ DNA Methylation Kit (Zymo Research). The bisulfite-treated DNA was sequenced by pyrosequencing as described above, and the DNA methylation rate of CpG islands 1 to 14 in the promoter region of the COASY gene was measured.
[0040] The results are shown in Figures 2A and 2B. As shown in Figures 2A and 2B, in the nucleotide sequence of the promoter region of the COASY gene, the DNA methylation rates of CpG islands with CpG island numbers 1, 3 to 6, and 8 to 14 (excluding CpG island numbers 2 and 7) were significantly higher in Alzheimer's disease (AD) patients and amnesic mild cognitive impairment (MCI) patients compared with non-demented elderly (NC). The CpG islands with CpG island numbers 1, 3 to 6, and 8 to 14 contain the cytosine residues at positions 24, 56, 75, 81, 98, 103, 115, 126, 134, 142, 153, and 179, respectively, in the nucleotide sequence of the promoter region of the COASY gene shown in SEQ ID NO: 1. Next, the Area Under the Curve (AUC) of the ROC curve separating NCs from AD and MCI patients was calculated for CpG islands with CpG island numbers 1 to 14. The results are shown in Table 4. As shown in Table 4, the AUC value for the DNA methylation level of the CpG island with CpG island number 14 was the highest.
[0041] [Table 4]
[0042] [Experimental Example 2] Next, we developed a method for measuring the DNA methylation level of CpG island number 14 in the promoter region of the COASY gene. TaqMan real-time PCR was performed using a primer set consisting of a forward primer with the nucleotide sequence shown in SEQ ID NO: 2 (5'-GATTATGGGATAGGAGAAGTG-3') and a reverse primer with the nucleotide sequence shown in SEQ ID NO: 3 (5'-AACTACTCCTAAACCAAAATACAC-3'). The primer set also included a probe for detecting methylated DNA with the nucleotide sequence shown in SEQ ID NO: 4 (hereafter referred to as the HsCOASY_m_probe) or a probe for detecting unmethylated DNA with the nucleotide sequence shown in SEQ ID NO: 5 (hereafter referred to as the HsCOASY_um_probe). The DNA of the CpG island (CpG island number 14) in the promoter region of the COASY gene, with bisulfite methylation rates of 100%, 75%, 50%, 25%, 5%, and 0%, was used as a template. Figures 3A and 3B show amplification plots using the HsCOASY_m_probe and HsCOASY_um_probe, respectively. As shown in Figure 3A, the HsCOASY_m_probe, which detects methylated DNA, was able to amplify samples with a 100% methylation rate, but no amplification was observed in other samples. Furthermore, as shown in Figure 3B, the HsCOASY_um_probe, which detects unmethylated DNA, was able to amplify samples with a 0% methylation rate, but no amplification was observed in other samples. This suggests that the amplification efficiency of the primer set used was poor. Therefore, we incorporated LNA into the primers, using a forward primer (hereafter referred to as COASY_F_LNA) in which the 3rd and 20th thymine residues of the nucleotide sequence shown in SEQ ID NO: 2 were replaced with LNA, and a reverse primer (hereafter referred to as COASY_R_LNA) in which the 8th and 20th adenine residues of the nucleotide sequence shown in SEQ ID NO: 9 were replaced with LNA, thereby increasing the melting temperature (Tm) of the duplex. Figures 4A and 4B show the amplification plots when the HsCOASY_m_probe and HsCOASY_um_probe were used, respectively. As shown in Figure 4A, the HsCOASY_m_probe, which detects methylated DNA, was able to amplify samples with methylation rates of 100%, 75%, and 50%. Furthermore, as shown in Figure 4B, the HsCOASY_um_probe, which detects unmethylated DNA, was able to amplify samples with methylation rates of 0%, 5%, 25%, and 50%. Although the PCR in this example was not performed using digital PCR, digital PCR could improve measurement sensitivity. Based on these results, it is believed that all samples can be amplified by digital PCR using the HsCOASY_m_probe and HsCOASY_um_probe.
[0043] [Experimental Example 3] Using COASY_F_LNA as the forward primer and COASY_R_LNA as the reverse primer, and HsCOASY_m_ probe for detecting methylated DNA and HsCOASY_um_ probe for detecting unmethylated DNA, we measured the DNA methylation level of CpG island number 14 in the promoter region of the COASY gene in the blood of five patients (combined AD and MCI patients) and nine NCs. Specifically, a standard curve was created using bisulfite-modified DNA with methylation rates of 100%, 75%, 50%, 25%, 5%, and 0%. For each sample, the amount of methylated DNA was quantified using the HsCOASY_m_ probe, and the amount of unmethylated DNA was quantified using the HsCOASY_um_ probe. The sum of the methylated and unmethylated DNA amounts was normalized to 100%. The results are shown in Figure 5. As shown in Figure 5, the DNA methylation rate of CpG island number 14 was significantly higher in the combined AD and MCI patients compared to the NCs (P<0.0001, Welch's t test). [Industrial Applicability]
[0044] According to the present invention, a technique for determining the possibility of developing Alzheimer's disease can be provided.
Claims
1. 1. A data collection method for determining a subject's likelihood of developing Alzheimer's disease, comprising: measuring the percentage of methylation of "CG" dinucleotides containing cytosine residues at positions 24, 56, 75, 81, 98, 103, 115, 126, 134, 142, 153, and 179 in the base sequence of the promoter region of the COASY gene shown in SEQ ID NO: 1 in a biological sample from the subject; A data collection method wherein a higher rate compared to a control indicates that the subject is more likely to develop Alzheimer's disease.
2. A data collection method for determining the likelihood that a subject will develop Alzheimer's disease, comprising: measuring the percentage of methylation of a "CG" dinucleotide containing a 24th cytosine residue in the base sequence of the promoter region of the COASY gene shown in SEQ ID NO: 1 using a primer for amplifying the "CG" dinucleotide containing the 24th cytosine residue, a probe for detecting methylated DNA, and a probe for detecting unmethylated DNA; A data collection method, wherein a higher rate compared to a control indicates that the subject is likely to develop Alzheimer's disease, comprising: the primers are a primer set comprising a forward primer having the nucleotide sequence shown in SEQ ID NO: 2, in which the 3rd and 20th thymine residues are LNA, and a reverse primer having the nucleotide sequence shown in SEQ ID NO: 9, in which the 8th and 20th adenine residues are LNA, The data collection method as described above, wherein the probe for detecting methylated DNA consists of the base sequence shown in SEQ ID NO: 4, and the probe for detecting unmethylated DNA consists of the base sequence shown in SEQ ID NO:
5.
3. The data collection method of claim 1 , wherein the biological sample is blood.
4. A kit for determining the possibility that a subject will develop Alzheimer's disease, comprising a primer for amplifying a "CG" dinucleotide containing the 24th cytosine residue in the base sequence of the promoter region of the COASY gene shown in SEQ ID NO: 1, a probe for detecting methylated DNA, and a probe for detecting unmethylated DNA, the primers are a primer set comprising a forward primer having the nucleotide sequence shown in SEQ ID NO: 2, in which the 3rd and 20th thymine residues are LNA, and a reverse primer having the nucleotide sequence shown in SEQ ID NO: 9, in which the 8th and 20th adenine residues are LNA, The above kit, wherein the probe for detecting methylated DNA consists of the base sequence shown in SEQ ID NO: 4, and the probe for detecting unmethylated DNA consists of the base sequence shown in SEQ ID NO: 5.