Method for using as an indicator of the risk of amyloid-β accumulation, method for detecting the presence or absence of single nucleotide polymorphisms (SNPs), composition, and kit

Abstract

To provide methods for determining the risk of being likely to develop Alzheimer's dementia even in an early stage before the onset of Alzheimer's dementia, besides methods focusing solely on the ApoE4 gene.SOLUTION: Provided is a method in which the presence or absence of a single nucleotide polymorphism (SNP) rs4907405 on human chromosome region 8q24.3 in a subject-derived nucleic acid is used as an indicator of the presence or absence of amyloid β accumulation risk in the subject.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for use as an indicator of the presence or absence of amyloid-β accumulation risk, a method for detecting the presence or absence of single nucleotide polymorphisms (SNPs), a composition, and a kit. [Background technology]

[0002] Dementia is a disease whose risk of developing increases as the aging population rate increases. There were an estimated 47 million dementia patients worldwide in 2017, and this is expected to increase to 75 million by 2030 and 131 million by 2050. It has also been pointed out that the increase in dementia could cause medical and nursing care costs to reach 2 trillion dollars (globally) in 2030 and 10.7 trillion yen (Japan) in 2025. Japan has a dementia prevalence rate of 2.33% of its total population, the highest rate among OECD member countries (1.48%).

[0003] In Japan, 50% of dementia cases are Alzheimer's type (Alzheimer's disease; AD), 20% are Lewy body type, 15% are vascular type, and 15% are other types. Treatment and prevention of Alzheimer's type dementia, which accounts for the largest proportion, is particularly important.

[0004] There is a condition called mild cognitive impairment (MCI) in which cognitive function can be restored with appropriate prevention and treatment in the pre-dementia stage, and it has been found that improvement at this stage can delay the onset of dementia. It is said that the rate of people who progress from MCI to dementia is 10% per year, or approximately 40% within five years. Furthermore, the Japanese Society of Neurology's Dementia Disease Treatment Guidelines 2017 indicates that with appropriate treatment, 16-41% of people with MCI will recover. Therefore, there is a strong need for early detection of dementia and preventative measures.

[0005] Tests and diagnoses for dementia include memory and language tests, blood tests, brain imaging tests (CT, MRI), cerebral blood flow SPECT (scintigraphy), dopamine transporter scintigraphy (testing for decreased expression levels), and MIBG myocardial scintigraphy (myocardial sympathetic nerve testing).However, methods for diagnosing MCI and methods for testing and diagnosing at earlier stages, such as before the onset of symptoms or in the early stages, are limited.

[0006] In general, the onset of Alzheimer's disease involves both congenital genetic factors and acquired environmental factors.

[0007] The most widely known susceptibility gene for Alzheimer's disease is the apolipoprotein E type 4 (ApoE4) gene, and in Japan, approximately 10% of the population possesses the ApoE4 gene. It is known that those who possess the ApoE4 gene show a clear difference in the amount of amyloid beta (Aβ) accumulation after the age of 60 compared to those who do not, and that this increases the risk of developing dementia (Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Jansen WJ et al. Prevalence of cerebral amyloid pathology in persons without dementia: a meta-analysis. JAMA 313, 1924-1938, (2015). [PubMed: 25988462] Summary of the Invention [Problem to be solved by the invention]

[0009] If we could identify healthy individuals who are at high risk of developing Alzheimer's disease and take preventive measures, we could significantly reduce the number of dementia patients. Therefore, there is strong hope for the development of methods to identify healthy individuals at high risk of developing the disease and preventative foods.

[0010] In Alzheimer's disease, the accumulation of amyloid beta in the brain is prominent. The "amyloid cascade hypothesis" supports the idea that Alzheimer's disease occurs when amyloid beta accumulates in the brain, causing changes in neurofibrillary fibers and shrinking of the hippocampus, which controls memory.

[0011] It is known that amyloid-β accumulation begins approximately 20 years before the onset of clinical dementia, and prevention at an early stage of accumulation is desirable. Considering that Alzheimer's disease develops after a pre-symptomatic period in which amyloid-β gradually accumulates in the brain, it is important to identify genetic factors involved in the early pre-symptomatic stage.

[0012] However, previous efforts to prevent Alzheimer's disease from genetic factors have often focused solely on the ApoE4 gene, and although genetic factors other than the ApoE4 gene have been reported, they have not been thoroughly verified.

[0013] Therefore, the present invention aims to provide a method for determining the risk of developing Alzheimer's disease even in the early stages before the onset of Alzheimer's disease, in addition to methods that focus solely on the ApoE4 gene. [Means for solving the problem]

[0014] The present inventors focused on amyloid β accumulation, which is widely observed even before the onset of Alzheimer's disease, and discovered genetic factors that cause differences in amyloid β accumulation in the early stages before the onset of Alzheimer's disease, leading to the completion of the present invention. More specifically, the present invention provides the following.

[0015] [1] A method in which the presence or absence of single nucleotide polymorphism (SNP) rs4907405 on human chromosomal region 8q24.3 in nucleic acid derived from a subject is used as an indicator of whether or not the subject is at risk of amyloid beta accumulation. [2] The method according to [1] above, wherein the single nucleotide polymorphism (SNP) is the A allele of rs4907405. [3] The method according to [1] or [2] above, wherein the subject is under 50 years of age.

[0016] [4] A method for determining whether or not a subject is at risk of amyloid beta accumulation based on the presence or absence of at least one of the following single nucleotide polymorphisms (SNPs) (a) to (d) in nucleic acid derived from the subject: (a) rs17019641 on human chromosome region 2p22.2 (b) rs12493550 on human chromosome region 3q27.1 (c) rs4907405 on human chromosome region 8q24.3 (d) rs74792644 on human chromosome region 20q13.31

[0017] [5] The risk allele of the above single nucleotide polymorphism (SNP) (a) is G, The risk allele of the single nucleotide polymorphism (SNP) (b) is A, The risk allele of the single nucleotide polymorphism (SNP) (c) is A, The risk allele of the single nucleotide polymorphism (SNP) (d) is C, The method according to [4] above, wherein the risk allele is an allele constituting a homozygous type that exhibits a higher positive correlation with the average Composite Biomarker value than the other homozygous type when the average Composite Biomarker values ​​are calculated and compared in a population having each of two types of homozygous types at an SNP site. The Composite Biomarker value represents a score obtained by scaling two values, Aβ1-42 and APP669-711, which are particularly closely related to the onset of amyloid-β-related diseases or conditions, such as MCI, Alzheimer's disease, and other dementias.

[0018] [6] The method according to [4] or [5] above, wherein the subject is 50 years of age or older. [7] The method according to any one of [1] to [6] above, wherein the subject does not have apolipoprotein E genotype 4 (ApoE4 gene).

[0019] [8] The method according to any one of [1] to [7] above, wherein the presence of the single nucleotide polymorphism (SNP) is an indicator of a higher risk of amyloid beta accumulation in the subject compared to when the single nucleotide polymorphism (SNP) is absent. [9] The method according to any one of [1] to [7] above, wherein the absence of the single nucleotide polymorphism (SNP) is an indicator that the risk of amyloid beta accumulation in the subject is lower than when the single nucleotide polymorphism (SNP) is present.

[0020]

[10] The method according to any one of [1] to [9] above, wherein the risk of amyloid β accumulation is the presence of amyloid β accumulation or the risk of amyloid β accumulation.

[11] The method according to any one of [1] to

[10] above, wherein the risk of amyloid beta accumulation is the risk of amyloid beta accumulation before the onset or in an early stage of a disease or condition associated with amyloid beta.

[0021]

[12] The method according to

[11] above, wherein the disease is a neurological disorder selected from the group consisting of mild cognitive impairment (MCI), Alzheimer's disease (AD), Down's syndrome, hereditary cerebral hemorrhage with amyloidosis (Dutch type), cerebrovascular amyloid angiopathy, and Guam Parkinsonism-Dementia Complex, or progressive supranuclear palsy, multiple sclerosis, inclusion body myositis (IBM), Creutzfeldt-Jakob disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), inclusion body myositis (IBM), adult-onset diabetes mellitus (type 2 diabetes), senile cardiac amyloidosis, endocrine tumor, glaucoma, ocular amyloidosis, primary retinal degeneration, macular degeneration, optic nerve drusen, optic neuropathy, optic neuritis, or lattice dystrophy.

[0022]

[13] A method according to any one of [1] to

[12] above, which is used to measure or detect the subject's risk of developing the disease or condition associated with amyloid beta, to detect the disease or condition associated with amyloid beta in the subject, to select a subject having the disease or condition associated with amyloid beta, to measure, evaluate or monitor the subject's response to an intake for preventing or treating the disease or condition associated with amyloid beta, or to measure, evaluate or monitor the progress of the disease or condition associated with amyloid beta in the subject.

[0023]

[14] The method according to any one of [1] to

[13] above, which comprises a detection step of detecting the presence or absence of the single nucleotide polymorphism (SNP).

[0024]

[15] A method for detecting the presence or absence of a single nucleotide polymorphism (SNP) for use in the method according to any one of [1] to

[14] above.

[16] The presence or absence of the above single nucleotide polymorphisms (SNPs) can be determined using scanning probe and nanopore DNA sequencing, pyrosequencing, denaturing gradient gel electrophoresis (DGGE), time-temperature gradient electrophoresis (TTGE), Zn(II)-cyclen polyacrylamide gel electrophoresis, homogeneous fluorescent PCR-based single nucleotide polymorphism analysis, phosphate-affinity polyacrylamide gel electrophoresis, high-throughput SNP genotyping platforms, molecular beacons, 5' nuclease reactions, Taqman assays, MassArray (single-base primer extension coupled with matrix-assisted laser desorption / ionization time-of-flight mass spectrometry), trityl mass tags, and genotyping platforms (Invader Assay (registered trademark), single base primer extension (SBE) assay, PCR amplification (e.g., PCR amplification on magnetic nanoparticles (MNPs)), restriction enzyme analysis of PCR products (RFLP method), allele-specific PCR, multiple primer extension (MPEX), isothermal smart amplification, PCR-SSCP (single-stranded conformation polymorphism analysis), sequencing, ARMS (Amplification Refracting Mutation System), and RNAse A cleavage method.

[0025]

[17] A composition for use in the method according to any one of [1] to

[14] above, the composition comprising a reagent capable of directly or indirectly detecting the presence or absence of the single nucleotide polymorphism (SNP).

[0026]

[18] A kit for use in the method according to any one of [1] to

[14] above, the kit comprising a reagent capable of directly or indirectly detecting the presence or absence of the single nucleotide polymorphism (SNP).

[19] The composition according to

[17] or the kit according to

[18] , wherein the reagent is selected from an oligonucleotide, a DNA probe, an RNA probe, and a ribozyme. [Effects of the Invention]

[0027] According to the present invention, in addition to the method using the ApoE4 gene, it is possible to provide a method for determining the risk of developing Alzheimer's disease at an early stage before the onset of Alzheimer's disease. [Brief explanation of the drawings]

[0028] [Figure 1] FIG. 1 shows the results of Example 1 for a group under 50 years old who do not carry the ApoE4 gene. [Figure 2] FIG. 1 shows the results of a group of people aged 50 or older who do not carry the ApoE4 gene in Example 1. DETAILED DESCRIPTION OF THE INVENTION

[0029] Hereinafter, an embodiment of the present invention will be described, but the present invention is not limited to this.

[0030] <Method 1 for use as an indicator of the risk of amyloid beta accumulation> A first aspect of the present invention is a method for determining whether or not a single nucleotide polymorphism (SNP) rs4907405 on human chromosomal region 8q24.3 in nucleic acid derived from a subject is an indicator of whether or not the subject is at risk of amyloid beta accumulation.

[0031] The method of the first aspect of the present invention focuses on amyloid beta accumulation, which is widely observed even before the onset of diseases or conditions such as Alzheimer's disease, and can be said to be a method for determining genetic predispositions or genetic factors that cause differences in amyloid beta accumulation before the onset of diseases or conditions such as Alzheimer's disease or conditions or in the early stages (including the early stages and MCI).The presence or absence of amyloid beta accumulation risk in a subject can be determined from the presence or absence of genetic predispositions or genetic factors that cause differences in amyloid beta accumulation in the subject.

[0032] Amyloid beta is a partial fragment of the precursor protein APP, which is produced in the brain and secreted by sequential cleavage by beta-secretase and gamma-secretase. It consists of approximately 40 amino acids and there are many peptides, among which Aβ1-40, Aβ1-42 and their precursors APP669-711 are known to be good blood biomarkers for detecting Alzheimer's disease.

[0033] Amyloid-β-related diseases or conditions are thought to be highly correlated with the amount of amyloid-β accumulation in the brain, and the accumulation of amyloid-β in the brain can be estimated, for example, by measuring the amount of amyloid-β in the blood. The amount of amyloid-β in the blood can be estimated, for example, by a composite biomarker value. The method of the first aspect of the present invention may include correlating the presence or absence of the single nucleotide polymorphism (SNP) with the presence or absence of amyloid-β accumulation risk and / or the composite biomarker value.

[0034] In this specification, the risk of amyloid β accumulation means that amyloid β has accumulated or there is a risk of amyloid β accumulating. The method of the first aspect of the present invention can detect the risk of developing amyloid-β-associated diseases or conditions in subjects who are in the pre-onset or early stage (including early stage and MCI) of amyloid-β-associated diseases or conditions, for example, because amyloid-β accumulation has begun but is still minimal. Furthermore, since the method of the first aspect of the present invention can determine the presence or absence of genetic factors, it can also determine the risk of amyloid-β accumulation in subjects who do not have amyloid-β accumulation, or who at least have not yet shown amyloid-β accumulation or whose amyloid-β accumulation is not detectable. Therefore, the method of the first aspect of the present invention is highly significant in terms of early prevention of amyloid-β-associated diseases or conditions. In this regard, the risk of amyloid-β accumulation can also be determined in subjects who are in the pre-onset or early stage of amyloid-β-associated diseases or conditions.

[0035] Specifically, the method of the first aspect of the present invention can be used as an indicator that a subject having the above-mentioned single nucleotide polymorphism (SNP) has a higher risk of amyloid beta accumulation compared to a subject not having the above-mentioned single nucleotide polymorphism (SNP), i.e., it can be determined that the subject has a higher risk of amyloid beta accumulation. Furthermore, for subjects who do not have the above-mentioned single nucleotide polymorphism (SNP), this can be used as an indicator that the risk of amyloid beta accumulation in the subject is lower than for subjects who have the above-mentioned single nucleotide polymorphism (SNP), i.e., it can be determined that the risk of amyloid beta accumulation is low.

[0036] Details of the single nucleotide polymorphism (SNP) rs4907405 on the human chromosomal region 8q24.3 are shown in Table 1 below. In this specification, rs4907405 may be abbreviated as "SNP on the human chromosomal region 8q24.3." As shown in Example 1 below, the allele of the SNP (rs4907405) on the human chromosomal region 8q24.3 is G / A.

[0037] Specifically, the method of the first aspect of the present invention can use the presence or absence of the A allele of the SNP (rs4907405) on the human chromosomal region 8q24.3 as an indicator of the risk of amyloid β accumulation in a subject. Specifically, for a subject with the A allele of the SNP (rs4907405) on the human chromosomal region 8q24.3, this can be used as an indicator of a higher risk of amyloid β accumulation in the subject compared to subjects without the A allele of the SNP on the human chromosomal region 8q24.3 or with the G allele of the SNP, i.e., a high risk of amyloid β accumulation can be determined. Furthermore, for a subject without the A allele of the SNP (rs4907405) on the human chromosomal region 8q24.3 or with the G allele of the SNP, this can be used as an indicator of a lower risk of amyloid β accumulation in the subject compared to subjects with the A allele of the SNP on the human chromosomal region 8q24.3, this can be used as an indicator of a lower risk of amyloid β accumulation in the subject, i.e., a low risk of amyloid β accumulation can be determined.

[0038] As used herein, alleles that can indicate a higher risk of amyloid beta accumulation in a subject compared to the presence of other alleles at the SNP site, such as the A allele in the SNP (rs4907405) on the human chromosome region 8q24.3, are also referred to as risk alleles. A "risk allele" is an allele that constitutes a homozygous type that shows a higher positive correlation with the average Composite Biomarker value than the other homozygous type when the average Composite Biomarker values ​​are calculated and compared in populations with two types of homozygous types at the SNP site. For example, in the case of a SNP on the human chromosome region 8q24.3, when comparing a population with the A / A type and a population with the G / G type among the three genotypes, the A allele that constitutes the A / A genotype is referred to as a risk allele.

[0039] Although it is known that the presence or absence of the ApoE4 gene affects the amount of amyloid beta accumulation, even if a subject does not have the ApoE4 gene, the presence or absence of the SNP (rs4907405) on human chromosome region 8q24.3 can be used as an indicator of the presence or absence of amyloid beta accumulation risk in the subject, regardless of the subject's age. The subject may be any age, for example, 40 years or older, 45 years or older, 50 years or older, 55 years or older, 60 years or older, 65 years or older, 70 years or older, etc., or younger than 40 years, 45 years or older, 50 years or older, 55 years or older, 60 years or older, 65 years or older, 70 years or older, etc., but from the perspective of early detection of amyloid beta accumulation risk, subjects under 50 years of age are particularly meaningful. Furthermore, the subject may be either male or female, regardless of gender.

[0040] The subject-derived nucleic acid is not particularly limited and may be, for example, a nucleic acid extract from a biological sample or biological specimen collected from the subject. The biological sample or biological specimen is not particularly limited as long as nucleic acid can be obtained from the biological sample or biological specimen, and examples thereof include body fluid samples such as blood, serum, saliva, sputum, sweat, tears, semen, and cerebrospinal fluid; mucosal exfoliation samples such as buccal cells and cheek swabs; lacrimal gland secretions; and body tissue samples such as tissue specimens.

[0041] The method of the first aspect of the present invention may include a detection step of detecting the presence or absence of the single nucleotide polymorphism (SNP). When the method includes the detection step, specifically, the presence or absence of the SNP (rs4907405) on human chromosomal region 8q24.3 can be determined based on the detection results obtained by the detection step.

[0042] The presence or absence of the above single nucleotide polymorphisms (SNPs) can be determined using scanning probe and nanopore DNA sequencing, pyrosequencing, denaturing gradient gel electrophoresis (DGGE), time-temperature gradient electrophoresis (TTGE), Zn(II)-cyclen polyacrylamide gel electrophoresis, homogeneous fluorescent PCR-based single nucleotide polymorphism analysis, phosphate-affinity polyacrylamide gel electrophoresis, high-throughput SNP genotyping platforms, molecular beacons, 5' nuclease reactions, Taqman assays, MassArray (single-base primer extension coupled with matrix-assisted laser desorption / ionization time-of-flight mass spectrometry), trityl mass tags, and genotyping platforms (Invader Assay (registered trademark), etc.), single base primer extension (SBE) assay, PCR amplification (e.g., PCR amplification on magnetic nanoparticles (MNPs)), restriction enzyme analysis of PCR products (RFLP method), allele-specific PCR, multi-primer extension (MPEX), isothermal smart amplification, PCR-SSCP (single-stranded conformation polymorphism analysis), sequencing, ARMS (Amplification Refracting Mutation System), and RNAse A cleavage method. For example, the detection may be by typing (genotyping) the genotype of one or more single nucleotide polymorphisms (SNPs) including the above-mentioned SNPs.

[0043] Examples of diseases or conditions associated with amyloid beta include dementia, Down's syndrome, hereditary cerebral hemorrhage with amyloidosis (Dutch type), cerebrovascular amyloid angiopathy, and Guam Parkinson-Dementia Complex, including progressive supranuclear palsy or other neurological disorders, multiple sclerosis, inclusion body myositis (IBM), Creutzfeldt-Jakob disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), inclusion body myositis (IBM), adult-onset diabetes mellitus (type 2 diabetes), senile cardiac amyloidosis, endocrine tumors, glaucoma, ocular amyloidosis, primary retinal degeneration, macular degeneration, optic nerve drusen, optic neuropathy, optic neuritis, and lattice dystrophy. Examples of the dementia include mild cognitive impairment (MCI) and Alzheimer's disease (AD). Among these, mild cognitive impairment (MCI) and dementia such as Alzheimer's disease (Alzheimer's disease; AD) are preferred, with mild cognitive impairment (MCI) being preferable.

[0044] That is, as described above, the first aspect of the present invention is a method in which the presence or absence of the single nucleotide polymorphism (SNP) is used as an indicator of the presence or absence of amyloid-β accumulation risk, and specifically, it is a method in which the presence or absence of the single nucleotide polymorphism (SNP) can be used as an indicator of whether a subject has developed or is at risk of developing in the future a disease or condition associated with amyloid-β, such as mild cognitive impairment (MCI), dementia such as Alzheimer's disease (AD), or other such disease or condition.

[0045] The method of the first aspect of the present invention is suitable for, for example, measuring, detecting (including early detection), determining or judging the risk of a subject, such as a healthy individual, for developing a disease or condition associated with amyloid beta; determining, judging or detecting (including early detection) a disease or condition associated with amyloid beta in a subject; selecting (or screening) a subject having a disease or condition associated with amyloid beta; measuring, evaluating or monitoring a subject's response to an ingested substance (e.g., food, supplement, medicine) for preventing or treating a disease or condition associated with amyloid beta (e.g., whether the ingested substance is effective in preventing or treating the disease or condition); or measuring, evaluating or monitoring the progress of a disease or condition associated with amyloid beta in a subject (including after the start of treatment, such as medication).

[0046] The method of the first aspect of the present invention can be used on healthy individuals or individuals who have not yet developed clinical dementia, as described in Example 1 below. Therefore, the subjects can include individuals who will develop Alzheimer's dementia in the future, i.e., individuals who are in the early pre-symptomatic stage of Alzheimer's dementia at the time of application of the method, as well as individuals who will not develop Alzheimer's dementia in the future. Even in populations containing a mixture of these subjects, the present invention was able to detect SNPs that can be statistically concluded to increase the risk of amyloid-β accumulation. Therefore, these SNPs can be used industrially, for example, in group health checkups and individual health examinations, to determine whether or not a subject is at risk for amyloid-β accumulation. Use of the present invention can determine whether a subject is prone to early amyloid-β accumulation and identify individuals who require early measures to prevent amyloid-β accumulation. The method of the present invention is also suitable as a method for diagnosing amyloid-β-related diseases or conditions, a method for pre-diagnosis, or a method for supporting or assisting in these diagnoses.

[0047] Furthermore, the above SNPs are useful in ApoE gene-noncarrying populations, for which it has been difficult to obtain insights into genetic prevention, and therefore have great industrial value. In particular, the SNP (rs4907405) on human chromosome region 8q24.3 can be applied to ApoE gene-noncarrying populations and subjects belonging to such populations, regardless of the age of the subjects when using the method of the first aspect of the present invention.

[0048] <Method 2 for use as an indicator of the risk of amyloid beta accumulation> A second aspect of the present invention is a method for determining whether or not a subject is at risk of amyloid beta accumulation based on the presence or absence of at least one of the following single nucleotide polymorphisms (SNPs) (a) to (d) in nucleic acid derived from the subject: (a) rs17019641 on human chromosome region 2p22.2 (b) rs12493550 on human chromosome region 3q27.1 (c) rs4907405 on human chromosome region 8q24.3 (d) rs74792644 on human chromosome region 20q13.31

[0049] Details of the above SNPs (a) to (d) are shown in Table 1 below. In this specification, the above SNPs (a) to (d) may be abbreviated as "SNP(a)" or "SNP on human chromosomal region 2p22.2," "SNP(b)" or "SNP on human chromosomal region 3q27.1," "SNP(c)" or "SNP on human chromosomal region 8q24.3," and "SNP(d)" or "SNP on human chromosomal region 20q13.31," respectively. The above SNP (c) is identical to the SNP (rs4907405) on human chromosomal region 8q24.3 in the method of the first aspect of the present invention.

[0050] As shown in Example 1 below, the above SNPs are as follows. The alleles of the above SNP (a) are C / G, with G being the risk allele and C being the non-risk allele. The allele of the above SNP (b) is G / A, with A being the risk allele and G being the non-risk allele. The allele of the above SNP (c) is G / A, with A being the risk allele and G being the non-risk allele. The alleles of the above SNP (d) are T / C, with C being the risk allele and T being the non-risk allele.

[0051] Specifically, in the method of the second aspect of the present invention, the presence or absence of a risk allele in at least one of the SNPs (a) to (d) can be used as an indicator of the risk of amyloid β accumulation in a subject. Specifically, for a subject who has a risk allele in at least one of the SNPs (a) to (d), this can be used as an indicator of a higher risk of amyloid β accumulation in the subject compared to a subject who does not have a risk allele in at least one of the SNPs (a) to (d) or has a non-risk allele in that SNP, i.e., a high risk of amyloid β accumulation can be determined. Furthermore, for a subject who does not have a risk allele in at least one of the SNPs (a) to (d) or has a non-risk allele in that SNP, this can be used as an indicator of a lower risk of amyloid β accumulation in the subject compared to a subject who has a risk allele in at least one of the SNPs (a) to (d), i.e., a low risk of amyloid β accumulation can be determined.

[0052] The same points as those mentioned above regarding the method of the first aspect of the present invention can be applied to the method of the second aspect of the present invention.

[0053] The method of the second aspect of the present invention is particularly effective when the subject is 50 years of age or older and / or when the subject is an ApoE4 gene non-carrier, and is particularly effective when the subject is 50 years of age or older and an ApoE4 gene non-carrier. Furthermore, the subject may be either male or female, regardless of gender.

[0054] <Method for Detecting the Presence or Absence of SNPs> A method for detecting the presence or absence of the above single nucleotide polymorphism (SNP) for use in the method of the first and / or second aspect of the present invention is also one of the present inventions and is the third aspect of the present invention. As a method for detecting the presence or absence of the above single nucleotide polymorphism (SNP), the detection step for detecting the presence or absence of the above single nucleotide polymorphism (SNP) described above for the method of the first aspect of the present invention or the detection method in the step can be applied.

[0055] <Composition> The fourth aspect of the present invention is a composition for use in the method of the first and / or second aspect of the present invention, the composition comprising a reagent capable of directly or indirectly detecting the presence or absence of the above single nucleotide polymorphism (SNP).

[0056] The above reagent is not particularly limited as long as it can directly or indirectly detect the presence or absence of the above single nucleotide polymorphism (SNP). For example, known reagents can be used, for example, at least one selected from oligonucleotides, DNA probes, RNA probes, and ribozymes can be used.

[0057] <Kit> The fifth aspect of the present invention is a kit for use in the method of the first and / or second aspect of the present invention, the kit comprising a reagent capable of directly or indirectly detecting the presence or absence of the above single nucleotide polymorphism (SNP). As the above reagent, those described above for the composition of the fourth aspect of the present invention can be used. The kit is not particularly limited as long as it contains the above reagent, and may be, for example, a DNA microarray, a DNA chip, a gene chip, or the like.

Examples

[0058] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0059] Example 1 474 healthy subjects (humans) were tested and analyzed according to the procedure outlined below. These healthy subjects (subjects) may include patients with mild cognitive impairment (MCI), but were not diagnosed with dementia. 1) Collect biological samples from subjects. 2) Extracting genome-derived DNA present in the biological sample. 3) Detecting the alleles of single nucleotide polymorphisms (SNPs) contained in the above DNA. 4) The type of early amyloid beta accumulation will be determined based on the combination of the above SNP alleles.

[0060] 1. Measurement of amyloid beta levels Subject plasma was treated with magnetic beads conjugated with anti-amyloid beta monoclonal antibodies, followed by immunoprecipitation (IP). Amyloid beta was eluted from the magnetic beads, and three peptides, Aβ1-40, Aβ1-42, and their precursor APP669-711, were measured by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS). Biomarker values ​​for APP669-711 / Aβ1-42 and Aβ1-40 / Aβ1-422 were calculated. Each value was z-scored (values ​​in the dataset were converted to a mean of 0 and a standard deviation of 1), and values ​​with significantly different scales were scaled. The average value was then used to calculate the composite biomarker value.

[0061] 2. Genotype Measurement Blood samples were collected from the subjects, and DNA was extracted and amplified according to standard methods. The DNA was then run on the "Japonica Array (registered trademark)" (a genetic polymorphism analysis array manufactured by the Tohoku Medical Megabank Organization, Tohoku University) to detect genetic polymorphisms in each subject. The "Japonica Array (registered trademark)" is a genetic polymorphism analysis array containing approximately 660,000 single nucleotide polymorphisms (SNPs) that was developed based on the results of large-scale genome analysis of Japanese people. Polymorphisms in the ApoE4 gene were detected using probe PCR.

[0062] 3. Identification of genes correlated with composite biomarker values Quality control was performed on approximately 660,000 single nucleotide polymorphism (SNP) markers detected using the Japonica Array (registered trademark). As quality control, SNPs with a genotyping success rate of less than 0.99 and a distorted Hardy-Weinberg equilibrium p-value (HWE: p<1.0×10) were selected. 6 ) and 15,308 SNPs with a minor allele frequency (MAF) of less than 0.01 were excluded before the association analysis. A total of 643,607 SNPs that passed quality control were analyzed for association with Composite Biomarker values ​​using PLINK1.9 (http: / / pngu.mgh.harvard.edu / purcell / plink / ). The GWAS analysis revealed that a total of four SNPs located in human chromosome region 2p22.2, human chromosome region 3q27.1, human chromosome region 8q24.3, and human chromosome region 20q13.31 were significant at a genome-wide level (p<1.0×10 -5 ) and showed a significant association with Composite Biomarker values.

[0063] The SNP on human chromosomal region 2p22.2 is rs17019641 C / G, and its association with the onset of Alzheimer's disease or amyloid beta accumulation is unknown. The SNP on human chromosome region 3q27.1 is rs12493550 G / A, and its association with the onset of Alzheimer's disease or amyloid beta accumulation is unknown. The SNP on human chromosome region 8q24.3 is rs4907405 G / A, and its association with the onset of Alzheimer's disease or amyloid beta accumulation is unknown. The SNP on human chromosome region 20q13.31 is rs74792644 T / C, and its association with the onset of Alzheimer's disease or amyloid beta accumulation is unknown. Details of these four SNPs are shown in Table 1 below.

[0064] [Table 1]

[0065] 4. Identification of genes involved in early amyloid-β accumulation 4.1 Under 50 age group Of the 474 individuals used in the GWAS analysis, 187 individuals under the age of 50 were selected. Because it is known that the presence or absence of the ApoE4 gene affects the amount of amyloid beta accumulation, these 187 individuals were further divided according to the presence or absence of the ApoE4 gene detected using the probe PCR method described above. This left 45 individuals under the age of 50 who were ApoE4 gene carriers, and 142 individuals under the age of 50 who were ApoE gene non-carriers.

[0066] For a group of people under 50 years old who did not carry the ApoE4 gene, odds ratios for the Composite Biomarker values ​​and genotype distributions were calculated for the above SNP sites on human chromosome regions 2p22.2, 3q27.1, 8q24.3, and 20q13.31, and Pearson's chi-square test was performed. Composite Biomarker values ​​were compared between two groups: those with a Composite Biomarker value of 0 or greater (Case) and those with a Composite Biomarker value of less than 0 (Control).

[0067] Figure 1 shows the p-values ​​and sequence information for each SNP extracted as having a statistically significantly higher correlation with the Composite Biomarker value, in order of the odds ratio for the minor type. The numbers in the table represent the number of people. The allele type (risk allele) with the higher risk, either the major or minor type, is on the right side of Table 1, as shown below. (a) Risk allele of rs17019641 on human chromosome region 2p22.2: G (b) Risk allele of rs12493550 on human chromosome region 3q27.1: A (c) Risk allele of rs4907405 on human chromosome region 8q24.3: A (d) Risk allele of rs74792644 on human chromosome region 20q13.31: C

[0068] As shown in Figure 1, in the group under 50 years of age who did not have the ApoE4 gene, for the above SNPs on human chromosome region 8q24.3, subjects with a Composite Biomarker value of 0 or greater and the AA allele had an odds ratio of 9.62, 95% CI (1.25, 74.06), and a p-value calculated by Pearson's chi-squared test of 0.001.

[0069] Therefore, the only SNP site showing p<0.05 was the above SNP site on human chromosome region 8q24.3, and we concluded that the above SNP site (rs4907405) on human chromosome region 8q24.3 is effective for determining the risk of amyloid beta accumulation in people under 50 years of age who do not carry the ApoE4 gene.

[0070] 4.2 Age 50 and over Of the 474 individuals used in the GWAS analysis, 287 individuals aged 50 years or older were selected and further divided by ApoE4 gene carrier status, as in the under-50 group. This resulted in 65 individuals aged 50 years or older who carried the ApoE4 gene, and 222 individuals aged 50 years or older who did not carry the ApoE gene. The results of the analysis of the group aged 50 years or older and not carrying the ApoE4 gene in the same manner as for the group under 50 years old and not carrying the ApoE4 gene are shown in Figure 2.

[0071] As shown in Figure 2, the following was found for the group aged 50 years or older who did not have the ApoE4 gene. For SNPs on human chromosome region 2q22.2, subjects with a Composite Biomarker value of 0 or greater and a G allele (risk allele) had an odds ratio of 2.73, 95% CI (1.25, 6.01), and a p-value calculated by Pearson's chi-squared test of 0.01. For SNPs on human chromosome region 3q27.1, subjects with a Composite Biomarker value of 0 or greater and an A allele (risk allele) had an odds ratio of 3.39, 95% CI (1.32, 8.71), and a p-value calculated by Pearson's chi-squared test of 0.01. For SNPs on human chromosome region 8q24.3, subjects with a Composite Biomarker value of 0 or greater and an A allele (risk allele) had an odds ratio of 2.83, 95% CI (1.12, 7.14), and a p-value calculated by Pearson's chi-squared test of 0.02. For SNPs on human chromosome region 20q13.31, subjects with a Composite Biomarker value of 0 or greater and a C allele (risk allele) had an odds ratio of 6.64, 95% CI (1.57, 28.09), and a p-value calculated by Pearson's chi-squared test of <0.001.

[0072] As described above, all four of the above SNP sites showed p<0.05, and we concluded that all of them are effective in determining the risk of amyloid beta accumulation.

[0073] 5. Discussion In healthy individuals under the age of 50 who do not possess the ApoE4 gene, which has a strong influence on the development of Alzheimer's disease, a SNP on the human chromosome region 8q24.3 was able to determine the likelihood of amyloid beta accumulation. Furthermore, in a group of healthy individuals aged 50 years or older who do not possess the ApoE4 gene, the likelihood of amyloid beta accumulation could be determined using SNPs on human chromosome region 2q22.2, SNPs on human chromosome region 3q27.1, SNPs on human chromosome region 8q24.3, and SNPs on human chromosome region 20q13.31.

Claims

1. A method for determining a risk of amyloid beta accumulation in vitro, comprising: the method comprises using the presence of a risk allele of single nucleotide polymorphism (SNP) rs4907405 on human chromosomal region 8q24.3 in nucleic acid derived from a subject as an indicator of a high risk of amyloid beta accumulation in the subject; the risk allele is an allele constituting a homozygous type that, when average values ​​of Composite Biomarker values ​​obtained by scaling and scoring two values ​​of Aβ1-42 and APP669-711 that are particularly closely related to the onset of amyloid-β-associated diseases or conditions in a population having each of two types of homozygous types at the SNP site are calculated and compared, shows a higher positive correlation with the average Composite Biomarker value than the other homozygous type; method.

2. 2. The method of claim 1, wherein the single nucleotide polymorphism (SNP) is the A allele in rs4907405.

3. 3. The method of claim 1 or 2, wherein the subject is under 50 years of age.

4. The method of any one of claims 1 to 3, wherein the subject is 50 years of age or older.

5. The method according to any one of claims 1 to 4, wherein the subject does not have apolipoprotein E genotype 4 (ApoE4 gene).

6. The method according to any one of claims 1 to 5, wherein the presence of the single nucleotide polymorphism (SNP) is used as an indicator that the risk of amyloid beta accumulation in the subject is higher than when the single nucleotide polymorphism (SNP) is absent.

7. The method according to any one of claims 1 to 5, wherein the absence of the single nucleotide polymorphism (SNP) is used as an indicator that the risk of amyloid beta accumulation in the subject is lower than when the single nucleotide polymorphism (SNP) is present.

8. The method according to any one of claims 1 to 7, wherein the risk of amyloid beta accumulation is the presence of amyloid beta accumulation or the risk of amyloid beta accumulation.

9. The method according to any one of claims 1 to 8, wherein the risk of amyloid beta accumulation is a risk of amyloid beta accumulation before the onset or in an early stage of a disease or condition associated with amyloid beta.

10. 10. The method of claim 9, wherein the disease is a neurological disorder selected from the group consisting of mild cognitive impairment (MCI), Alzheimer's dementia (AD), Down's syndrome, hereditary cerebral hemorrhage with amyloidosis (Dutch type), cerebrovascular amyloid angiopathy, and Guam Parkinsonism-Dementia Complex or progressive supranuclear palsy, multiple sclerosis, inclusion body myositis (IBM), Creutzfeldt-Jakob disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), inclusion body myositis (IBM), adult-onset diabetes mellitus (type 2 diabetes), senile cardiac amyloidosis, endocrine tumors, glaucoma, ocular amyloidosis, primary retinal degeneration, macular degeneration, optic nerve drusen, optic neuropathy, optic neuritis, or lattice dystrophy.

11. The method according to any one of claims 1 to 10, which is used to measure or detect the subject's risk of developing the disease or condition associated with amyloid beta, to detect the disease or condition associated with amyloid beta in the subject, to select a subject having the disease or condition associated with amyloid beta, to measure, evaluate or monitor the subject's response to an intake for preventing or treating the disease or condition associated with amyloid beta, or to measure, evaluate or monitor the progress of the disease or condition associated with amyloid beta in the subject.

12. The method according to any one of claims 1 to 11, comprising a detection step of detecting the presence or absence of the single nucleotide polymorphism (SNP).

13. A method for detecting the presence or absence of a single nucleotide polymorphism (SNP) for use in the method according to any one of claims 1 to 12.

14. The presence or absence of the single nucleotide polymorphisms (SNPs) can be determined by scanning probe and nanopore DNA sequencing, pyrosequencing, denaturing gradient gel electrophoresis (DGGE), time-temperature gradient electrophoresis (TTGE), Zn(II)-cyclen polyacrylamide gel electrophoresis, homogeneous fluorescent PCR-based single nucleotide polymorphism analysis, phosphate-affinity polyacrylamide gel electrophoresis, high-throughput SNP genotyping platforms, molecular beacons, 5' nuclease reactions, Taqman assays, MassArray (single-base primer extension coupled with matrix-assisted laser desorption / ionization time-of-flight mass spectrometry), trityl mass tags, genotyping platforms (Invader, etc.), and the like.

14. The method of claim 12 or 13, wherein the nucleotide sequence is detected by at least one technique selected from the group consisting of PCR amplification (e.g., PCR Amplification on Magnetic Nanoparticles (MNPs)), single base primer extension (SBE) assay, PCR amplification (e.g., PCR amplification on magnetic nanoparticles (MNPs)), restriction enzyme analysis of PCR products (RFLP method), allele-specific PCR, multiple primer extension (MPEX), isothermal smart amplification, PCR-SSCP (single-stranded conformation polymorphism analysis), sequencing, ARMS (Amplification Refracting Mutation System), and RNAse A cleavage method.

15. A composition for use in the method of any one of claims 1 to 12, comprising a reagent capable of directly or indirectly detecting the presence or absence of the single nucleotide polymorphism (SNP); The reagent is one or more selected from an oligonucleotide, a DNA probe, an RNA probe, and a ribozyme. composition.

16. A kit for use in the method according to any one of claims 1 to 12, comprising a reagent capable of directly or indirectly detecting the presence or absence of the single nucleotide polymorphism (SNP); The reagent is one or more selected from an oligonucleotide, a DNA probe, an RNA probe, and a ribozyme. kit.

Images