Methods in which presence or absence of amyloid β accumulation risk is used as indicator as well as methods, compositions, and kits for detecting presence or absence of single nucleotide polymorphisms (SNPS)

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 a single nucleotide polymorphism (SNP), a composition, and a kit.

Background Art

[0002] Dementia is a disease with an increasing incidence rate as the aging rate increases. The number of dementia patients worldwide is estimated to be 47 million in 2017, and is expected to increase to 75 million in 2030 and 131 million in 2050. In addition, it has been pointed out that the increase in dementia may lead to medical and nursing care costs reaching $2 trillion (worldwide) in 2030 and 10.7 trillion yen (Japan) in 2025. Japan has the highest prevalence rate of dementia among the OECD member countries (1.48%) at 2.33% of the total population.

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

[0004] There is a state of mild cognitive impairment (MCI) in which cognitive function can be restored by appropriate prevention / treatment at the pre-dementia stage. It is known that if improvement can be achieved at this stage, the onset timing of dementia can be delayed. The proportion of people progressing from MCI to dementia is said to be about 10% per year on average and about 40% over five years. In addition, the Japanese Society of Neurology, Dementia Disease Clinical Practice Guidelines 2017 shows that 16 - 41% of people can recover if appropriate measures are taken for MCI. Therefore, early detection and preventive measures for dementia are strongly demanded.

[0005] As tests and diagnoses for dementia, tests for memory and language, blood tests, brain imaging tests (CT, MRI), cerebral blood flow SPECT (scintigraphy) tests, dopamine transporter scintigraphy (to test for a decrease in expression levels), MIBG myocardial scintigraphy tests (myocardial sympathetic nerve tests), etc. are carried out. However, the diagnostic methods for MCI and the test and diagnostic methods at earlier stages such as before the onset or in the initial stage are limited.

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

[0007] As a susceptibility gene for Alzheimer's disease, the most widely known is the apolipoprotein E gene type 4 (ApoE4 gene). In Japan, about 10% of the entire population carries the ApoE4 gene. In the group carrying the ApoE4 gene, a clear difference in the amyloid-β (Aβ) accumulation amount occurs after the age of 60 compared to the group not carrying it, and it is known that the risk of developing dementia increases (Non-Patent Document 1).

Prior Art Documents

Non-Patent Documents

[0008]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0009] If healthy individuals who are likely to develop Alzheimer's disease can be discriminated and preventive measures can be taken, the number of dementia patients can be significantly reduced. Therefore, there is a strong expectation for the development of a discrimination method for healthy individuals with a high risk of onset and preventive foods.

[0010] In Alzheimer's disease, amyloid-β accumulation in the brain is significantly observed. The "amyloid cascade hypothesis" is supported, which states that Alzheimer's disease occurs due to the accumulation of amyloid-β in the brain, the change of neurofibrils, and the shrinkage of the hippocampus responsible for memory.

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

[0012] However, the prevention of Alzheimer's disease from conventional genetic factors has often focused only on the ApoE4 gene. Regarding genetic factors other than the ApoE4 gene, although reports have been made, verification has not advanced.

[0013] Therefore, an object of the present invention is to provide a method for determining the risk of developing Alzheimer's disease even in the early pre-onset stage of Alzheimer's disease, in addition to methods that mainly focus only on the ApoE4 gene.

Means for Solving the Problems

[0014] The present inventors focused on amyloid-β accumulation widely observed from the pre-onset stage of Alzheimer's disease, found genetic factors that bring differences in amyloid-β accumulation in the early pre-onset stage of Alzheimer's disease, and completed the present invention. More specifically, the present invention provides the following.

[0015] [1] A method using the presence or absence of a single nucleotide polymorphism (SNP) rs4907405 on human chromosome region 8q24.3 in a nucleic acid derived from a subject as an indicator of the presence or absence of amyloid-β accumulation risk in the subject. [2] The method according to [1] above, wherein the single nucleotide polymorphism (SNP) is the A allele at rs4907405. [3] The method according to [1] or [2] above, wherein the subject is less than 50 years old.

[0016] [4] A method for using the presence or absence of at least one of the following single nucleotide polymorphisms (SNPs) (a) to (d) in a nucleic acid derived from a subject as an indicator of the presence or absence of amyloid-β accumulation risk in 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 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 risk allele is an allele that constitutes a homozygous form that shows a positive correlation with the average value of the Composite Biomarker value compared to the other homozygous form when comparing the average values of the Composite Biomarker values in populations each having two types of homozygous forms at the SNP site. Note that the Composite Biomarker value represents a value obtained by scaling and scoring two values, Aβ1-42 and APP669-711, which are particularly closely related to the onset of diseases or conditions related to amyloid-β such as MCI, Alzheimer's disease, and other dementias among amyloid-β.

[0018] [6] The method according to [4] or [5] above, wherein the subject is 50 years old or older. [7] The subject is a method according to any one of [1] to [6] above that does not carry the apolipoprotein E gene type 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 used as an indicator that the amyloid-β accumulation risk in the subject is high compared to the case where 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 used as an indicator that the amyloid-β accumulation risk in the subject is low compared to the case where the single nucleotide polymorphism (SNP) is present.

[0020]

[10] The amyloid-β accumulation risk in the method according to any one of [1] to [9] above is that amyloid-β is accumulating or there is a risk of amyloid-β accumulation.

[11] The amyloid-β accumulation risk in the method according to any one of [1] to

[10] above is the amyloid-β accumulation risk before or at an early stage of the onset of a disease or condition associated with amyloid-β.

[0021]

[12] The disease is a neuropathy 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 (type 2 diabetes), senile cardiac amyloidosis, endocrine tumor, glaucoma, ocular amyloidosis, primary retinal degeneration, macular degeneration, optic disc drusen, optic neuropathy, optic neuritis, or lattice dystrophy selected from the group consisting of mild cognitive impairment (MCI), Alzheimer's disease (AD), Down syndrome, hereditary cerebral hemorrhage with amyloidosis (Dutch type), cerebral amyloid angiopathy, and Guam Parkinson dementia complex. The method according to

[11] above.

[0022]

[13] For measuring or detecting the risk of the subject for the onset of a disease or condition associated with the amyloid β, for detecting a disease or condition associated with the amyloid β in the subject, for selecting a subject having a disease or condition associated with the amyloid β, for measuring, evaluating or monitoring the response of the subject to an intake for preventing or treating a disease or condition associated with the amyloid β, or for measuring, evaluating or monitoring the course of a disease or condition associated with the amyloid β in the subject, the method according to any one of [1] to

[12] above.

[0023]

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

[13] above, comprising 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 the 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 polymorphism (SNP) is detected by at least one technique selected from the group consisting of scanning probes and nanopore DNA sequencing, pyrosequencing, denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TTGE), Zn(II)-cyclen polyacrylamide gel electrophoresis, homogeneous fluorescence PCR-based single nucleotide polymorphism analysis, phosphate-affinity polyacrylamide gel electrophoresis, high-throughput SNP genotyping platform, molecular beacon, 5'-nuclease reaction, Taqman assay, MassArray (single base primer extension combined with matrix-assisted laser desorption / ionization time-of-flight mass spectrometry), trityl mass tag, genotyping platform (such as Invader Assay (registered trademark)), single base primer extension (SBE) assay, PCR amplification (for example, PCR amplification on magnetic nanoparticles (MNP)), restriction enzyme analysis (RFLP method) of PCR products, allele-specific PCR, multiprimer extension (MPEX), isothermal smart amplification, PCR-SSCP (single-stranded DBA higher-order structure polymorphism analysis) method, sequencing method, ARMS (Amplification Refracting Mutation System) method, and RNAseA cleavage method, which is the method described in the above

[14] or

[15] .

[0025]

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

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

[0026]

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

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

[19] The composition according to the above

[17] or the kit according to the above

[18] , wherein the reagent is selected from oligonucleotides, DNA probes, RNA probes, and ribozymes.

Advantages 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 the initial stage before the onset of Alzheimer's disease.

Brief Description of the Drawings

[0028]

Figure 1

Figure 2

Modes for Carrying Out the Invention

[0029] Hereinafter, embodiments of the present invention will be described, but the present invention is not limited thereto.

[0030] <Method 1 as an Indicator of the Presence or Absence of Amyloid-β Accumulation Risk> The first aspect of the present invention is a method for using the presence or absence of a single nucleotide polymorphism (SNP) rs4907405 on human chromosome region 8q24.3 in a nucleic acid derived from a subject as an indicator of the presence or absence of amyloid-β accumulation risk in the subject.

[0031] The method of the first aspect of the present invention focuses on amyloid-β accumulation widely observed from the pre-onset stage of diseases or conditions such as Alzheimer's disease, and determines genetic factors or genetic factors that cause differences in amyloid-β accumulation in the pre-onset or early stage (including the initial stage and MCI) of diseases or conditions such as Alzheimer's disease. It can also be said that the presence or absence of amyloid-β accumulation risk can be determined from the presence or absence of genetic factors or genetic factors that cause differences in amyloid-β accumulation in a subject.

[0032] Amyloid-β is a partial fragment of the precursor protein APP produced in the brain, and is produced and secreted by successive cleavage by β-secretase and γ-secretase. It is composed of around 40 amino acids and there are a number of peptides. Among them, Aβ1-40, Aβ1-42 and their precursor APP669-711 are known to be good blood biomarkers for detecting Alzheimer's lesions.

[0033] Diseases or conditions associated with amyloid-β are likely to be correlated with the amount of amyloid-β accumulated in the brain, and the accumulation of amyloid-β in the brain can be inferred, for example, by measuring the amount of amyloid-β in the blood. The amount of amyloid-β in the blood can be inferred, for example, by the Composite Biomarker value. The method of the first aspect of the present invention may include correlating the presence or absence of the above 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 above amyloid-β accumulation risk means that amyloid-β is accumulated or there is a risk of amyloid-β accumulation. The method of the first aspect of the present invention can, for example, detect the presence or absence of the risk of developing a disease or condition associated with amyloid-β in a subject who is in the pre-onset or early stage (including the initial stage and MCI) of a disease or condition associated with amyloid-β, due to, for example, the accumulation of amyloid-β having just started but still being in trace amounts. Further, since the method of the first aspect of the present invention can determine the presence or absence of genetic factors, in particular, even for a subject in whom there is no accumulation of amyloid-β, or who has not yet shown at least accumulation of amyloid-β or in whom accumulation of amyloid-β is not detected, it is possible to determine the presence or absence of the risk of amyloid-β accumulation. Therefore, the method of the first aspect of the present invention is of great significance in terms of early prevention of diseases or conditions associated with amyloid-β. In that regard, as the above amyloid-β accumulation risk, it is also possible to determine the amyloid-β accumulation risk in the pre-onset or early stage of a disease or condition associated with amyloid-β.

[0035] Specifically, for a subject in whom the above single nucleotide polymorphism (SNP) is present, it can be used as an indicator that the amyloid-β accumulation risk in the subject is high as compared with a subject in whom the above single nucleotide polymorphism (SNP) is not present, that is, it can be determined that the amyloid-β accumulation risk is high. Further, for a subject in whom the above single nucleotide polymorphism (SNP) is not present, it can be used as an indicator that the amyloid-β accumulation risk in the subject is low as compared with a subject in whom the above single nucleotide polymorphism (SNP) is present, that is, it can be determined that the amyloid-β accumulation risk is low.

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

[0037] Specifically, the method of the first aspect of the present invention can use the presence or absence of the A allele at the SNP (rs4907405) on human chromosome region 8q24.3 as an indicator of the presence or absence of amyloid-β accumulation risk in a subject. Specifically, for a subject in whom the A allele at the SNP (rs4907405) on the human chromosome region 8q24.3 is present, compared with a subject in whom the A allele at the SNP on the human chromosome region 8q24.3 is absent or is the G allele at the SNP, it can be an indicator that the amyloid-β accumulation risk in the subject is high, that is, it can be determined that the amyloid-β accumulation risk is high. Also, for a subject in whom the A allele at the SNP (rs4907405) on the human chromosome region 8q24.3 is absent or is the G allele at the SNP, compared with a subject in whom the A allele at the SNP on the human chromosome region 8q24.3 is present, it can be an indicator that the amyloid-β accumulation risk in the subject is low, that is, it can be determined that the amyloid-β accumulation risk is low.

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

[0039] Although it is known that the presence or absence of the ApoE4 gene affects the amyloid-β accumulation level, even if the subject does not carry the ApoE4 gene, the SNP (rs4907405) on human chromosomal region 8q24.3 can serve as an indicator of the presence or absence of amyloid-β accumulation risk in the subject regardless of the subject's age. The subject can be of any age, for example, 40 years old or older, 45 years old or older, 50 years old or older, 55 years old or older, 60 years old or older, 65 years old or older, 70 years old or older, etc., or less than 40 years old, less than 45 years old, less than 50 years old, less than 55 years old, less than 60 years old, less than 65 years old, less than 70 years old, etc. However, from the perspective of early detection of amyloid-β accumulation risk, it is particularly significant when the subject is less than 50 years old. Also, the subject can be of any gender, male or female.

[0040] The nucleic acid derived from the subject is not particularly limited. For example, it may be a nucleic acid extract from a biological sample or a biological specimen derived from the subject collected from the subject. The biological sample or biological specimen is not particularly limited as long as nucleic acid can be obtained. Examples include body fluid samples such as blood, serum, saliva, sputum, sweat, tears, semen, and cerebrospinal fluid; body tissue samples such as oral cells, mucosal exfoliates such as buccal swabs, lacrimal gland secretions, and 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 above single nucleotide polymorphism (SNP). When including the detection step, specifically, based on the detection result obtained by the detection step, the presence or absence of the SNP (rs4907405) on human chromosomal region 8q24.3 can be determined.

[0042] The presence or absence of the above single nucleotide polymorphism (SNP) can be detected by a technique including at least one selected from the group consisting of scanning probes and nanopore DNA sequencing, pyrosequencing, denaturing gradient gel electrophoresis (DGGE), temporal temperature gradient electrophoresis (TTGE), Zn(II)-cyclen polyacrylamide gel electrophoresis, homogeneous fluorescence PCR-based single nucleotide polymorphism analysis, phosphate-affinity polyacrylamide gel electrophoresis, high-throughput SNP genotyping platform, molecular beacon, 5'-nuclease reaction, Taqman assay, MassArray (single base primer extension combined with matrix-assisted laser desorption / ionization time-of-flight mass spectrometry), trityl mass tag, genotyping platform (such as Invader Assay (registered trademark)), single base primer extension (SBE) assay, PCR amplification (for example, PCR amplification on magnetic nanoparticles (MNP)), restriction enzyme analysis (RFLP method) of PCR products, allele-specific PCR, multiprimer extension (MPEX), isothermal smart amplification, PCR-SSCP (single-stranded DBA higher-order structure polymorphism analysis) method, sequencing method, ARMS (Amplification Refracting Mutation System) method, and RNAseA cleavage method. For example, it may be typing (genotyping) of the genotype of one or more single nucleotide polymorphisms (SNPs) including the above SNP.

[0043] Diseases or conditions associated with amyloid-β include neurological disorders or progressive supranuclear palsy selected from the group consisting of dementia, Down syndrome, hereditary cerebral hemorrhage with amyloidosis (Dutch type), cerebral amyloid angiopathy, and Guam Parkinson dementia complex, multiple sclerosis, inclusion body myositis (IBM), Creutzfeldt-Jakob disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), inclusion body myositis (IBM), adult-onset diabetes (type 2 diabetes), senile cardiac amyloidosis, endocrine tumors, glaucoma, ocular amyloidosis, primary retinal degeneration, macular degeneration, optic disc drusen, optic neuropathy, optic neuritis, or lattice dystrophy, etc. Examples of the above-mentioned dementia typically include mild cognitive impairment (MCI), Alzheimer's type dementia (Alzheimer's disease; AD), etc. Among them, dementias such as mild cognitive impairment (MCI) and Alzheimer's type dementia (Alzheimer's disease; AD) are preferred, and mild cognitive impairment (MCI) is desirable.

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

[0045] The method of the first aspect of the present invention is, for example, for measuring, detecting (including early detection), determining or judging the risk of a subject such as a healthy person regarding the onset of a disease or condition associated with amyloid-β, for determining, judging or detecting (including early detection) a disease or condition associated with amyloid-β in a subject, for selecting (or screening) a subject having a disease or condition associated with amyloid-β, for measuring, evaluating or monitoring the reaction of a subject to an intake (e.g., food, supplement, pharmaceutical) for preventing or treating a disease or condition associated with amyloid-β (e.g., whether the intake has an effect on the prevention or treatment), or for measuring, evaluating or monitoring the course of a disease or condition associated with amyloid-β in a subject (including after the start of treatment such as medication), and is each suitable.

[0046] The method of the first aspect of the present invention can be directed to healthy persons or those without the appearance of clinical dementia, etc., as in Example 1 described later. Therefore, the subjects can include those who will develop Alzheimer's disease in the future, that is, those in the early stage before the onset of Alzheimer's disease at the time of applying this method, and those who will not develop Alzheimer's disease in the future. Even in a group in which these subjects are mixed, since SNPs that can be statistically concluded to increase the amyloid-β accumulation risk can be detected according to the present invention, it can be said that they are SNPs that can be used for determining the presence or absence of amyloid-β accumulation risk, for example, in mass screening or individual health checkups in the industry. By using the present invention, it is possible to determine whether a subject is of a type that is likely to accumulate amyloid-β early, and it is possible to identify those who need to take early measures for preventing amyloid-β accumulation. The method of the present invention is also suitable as a method for diagnosing a disease or condition associated with amyloid-β, a method for pre-diagnosis, a method for assisting or supplementing these diagnoses, etc.

[0047] In addition, since the above SNP can be used in the ApoE gene non-carrier population, where it has been difficult to obtain insights into prevention from genetic factors, it also has great industrial utility value. In particular, the SNP (rs4907405) on human chromosome region 8q24.3 can be applied to the ApoE gene non-carrier population and subjects belonging to the population without limiting the age of the subjects when using the method of the first aspect of the present invention.

[0048] <Method 2 for using as an indicator of the presence or absence of amyloid-β accumulation> A second aspect of the present invention is a method for using the presence or absence of at least one of the following single nucleotide polymorphisms (SNPs) (a) to (d) in a nucleic acid derived from a subject as an indicator of the presence or absence of amyloid-β accumulation risk in 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 described later. In this specification, the above SNPs (a) to (d) may be abbreviated as "SNP(a)" or "SNP on human chromosome region 2p22.2", "SNP(b)" or "SNP on human chromosome region 3q27.1", "SNP(c)" or "SNP on human chromosome region 8q24.3", "SNP(d)" or "SNP on human chromosome region 20q13.31", respectively. The above SNP(c) is the same as the SNP (rs4907405) on human chromosome region 8q24.3 in the method of the first aspect of the present invention described above.

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

[0051] Specifically, the method of the second aspect of the present invention can use the presence or absence of the risk allele in at least one of the above SNPs (a) to (d) as an indicator of the presence or absence of amyloid-β accumulation risk in a subject. Specifically, for a subject in whom the risk allele exists in at least one of the above SNPs (a) to (d), compared with a subject in whom the risk allele does not exist in at least one of the above SNPs (a) to (d) or who is a non-risk allele in the SNP, it can be used as an indicator that the amyloid-β accumulation risk in the subject is high, that is, it can be determined that the amyloid-β accumulation risk is high. Also, for a subject in whom the risk allele does not exist in at least one of the above SNPs (a) to (d) or who is a non-risk allele in the SNP, compared with a subject in whom the risk allele exists in at least one of the above SNPs (a) to (d), it can be used as an indicator that the amyloid-β accumulation risk in the subject is low, that is, it can be determined that the amyloid-β accumulation risk is low.

[0052] In the method of the second aspect of the present invention, the matters described above for the method of the first aspect of the present invention can be applied.

[0053] The method of the second aspect of the present invention is particularly effective when the subject is 50 years old or older and / or when the subject is a non-carrier of the ApoE4 gene who does not possess the ApoE4 gene. Among them, it is particularly effective when the subject is 50 years old or older and a non-carrier of the ApoE4 gene. Also, the subject may be 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 the 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> A 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> A 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. For example, it may be a DNA microarray, a DNA chip, a gene chip, or the like.

Examples

[0058] Hereinafter, the present invention will be described in more detail by way of 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 following procedure. These healthy subjects (test subjects) may include patients with mild cognitive impairment (MCI), but have not been diagnosed with dementia. 1) Collect a biological sample from the test subject. 2) Extract genomic DNA present in the biological sample. 3) Detect the alleles of single nucleotide polymorphisms (SNPs) contained in the DNA. 4) Determine the early amyloid-β accumulation type based on the combination of the SNP alleles.

[0060] 1. Measurement of the amount of amyloid-β After treating the plasma of the test subject with magnetic beads conjugated with an anti-amyloid-β monoclonal antibody, immunoprecipitation (IP) was performed, and amyloid-β was eluted from the magnetic beads. Then, 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), and two biomarker values, APP669-711 / Aβ1-42 and Aβ1-40 / Aβ1-42, were calculated. After z-scoring (transforming the values of the dataset so that the mean value = 0 and the standard deviation = 1) to scale values with significantly different scales and then averaging, the averaged value was calculated as the Composite Biomarker value.

[0061] 2. Measurement of genotype Blood was collected from the test subject, and DNA was extracted and amplified according to a conventional method. The DNA was then subjected to a "Japonica Array (registered trademark)" (an array for gene polymorphism analysis, manufactured by Tohoku University, Tohoku Medical Megabank Organization) to detect the gene polymorphism of each subject. This "Japonica Array (registered trademark)" is an array for gene polymorphism analysis carrying approximately 660,000 single nucleotide polymorphisms (SNPs) based on the results of large-scale genomic analysis of Japanese people. In addition, the polymorphism of the ApoE4 gene was detected by the probe PCR method.

[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 above-mentioned "Japonica Array (registered trademark)". As quality control, SNPs with a genotyping success rate of less than 0.99, SNPs with a p-value of distorted Hardy-Weinberg equilibrium (HWE: p < 1.0×10 6 ), and 15,308 SNPs with a minor allele frequency (MAF) of less than 0.01 were excluded and then used for association analysis. For the association between the total 643,607 SNPs that passed quality control and the Composite Biomarker values, GWAS analysis was performed using PLINK1.9 (http: / / pngu.mgh.harvard.edu / purcell / plink / ). As a result of the GWAS analysis, a total of 4 SNPs located in human chromosome region 2p22.2, human chromosome region 3q27.1, human chromosome region 8q24.3, and human chromosome region 20q13.31 satisfied the significant genome-wide level (p < 1.0×10 -5 ) and showed a significant association with the Composite Biomarker values.

[0063] The SNP on human chromosome region 2p22.2 is rs17019641 C / G, and its association with the onset of Alzheimer's disease and amyloid-β accumulation is not known. The SNP on human chromosome region 3q27.1 is rs12493550 G / A, and its association with the onset of Alzheimer's disease and amyloid-β accumulation is not known. The SNP on human chromosome region 8q24.3 is rs4907405 G / A, and its association with the onset of Alzheimer's disease and amyloid-β accumulation is not known. The SNP on human chromosome region 20q13.31 is rs74792644 T / C, and its association with the onset of Alzheimer's disease and amyloid-β accumulation is not known. The details of these 4 SNPs are shown in Table 1 below.

[0064]

Table 1

[0065] 4. Identification of Genes Involved in Early Amyloid-β Accumulation 4.1 Population Under 50 Years Old Among the 474 individuals used in the GWAS analysis, 187 individuals who were under 50 years old were selected. Since it is known that the presence or absence of the ApoE4 gene affects the amount of amyloid-β accumulation, the 187 individuals were further divided according to the presence or absence of the ApoE4 gene detected using the above probe PCR method. There were 45 individuals under 50 years old who carried the ApoE4 gene and 142 individuals under 50 years old who did not carry the ApoE gene.

[0066] For the population under 50 years old and without the ApoE4 gene, at the above SNP sites on human chromosome region 2p22.2, human chromosome region 3q27.1, human chromosome region 8q24.3, and human chromosome region 20q13.31, the odds ratio of the Composite Biomarker value and the genotype distribution was calculated, and Pearson's chi-square test was performed. Regarding the Composite Biomarker value, it was compared as two groups: Composite Biomarker value of 0 or more (Case) and Composite Biomarker value less than 0 (Control).

[0067] In Figure 1, for each SNP extracted as having a statistically significantly higher degree of association with the Composite Biomarker value, the p-value and sequence information are shown in the order of the odds ratio for the minor type. The numbers in the table represent the number of people. Among the major type and minor type, the allele type with the higher risk (risk allele) was on the right side in Table 1, that is, as follows. (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 A of rs4907405 on human chromosome region 8q24.3 (d) Risk allele C of rs74792644 on human chromosome region 20q13.31

[0068] As shown in Figure 1, in the population under 50 years old and without the ApoE4 gene, for the above SNP on human chromosome region 8q24.3, in subjects with a Composite Biomarker value of 0 or more and the AA allele, the odds ratio = 9.62, 95% CI (1.25, 74.06), and the p-value calculated by the Pearson Χ2 test = 0.001.

[0069] Therefore, the SNPs site showing p < 0.05 is only the above SNP site on human chromosome region 8q24.3, and it was concluded that the SNPs site (rs4907405) on human chromosome region 8q24.3 is effective for amyloid-β accumulation risk determination in the population without the ApoE4 gene and under 50 years old.

[0070] 4.2 Population over 50 years old Among the above 474 subjects used in the GWAS analysis, 287 subjects corresponding to 50 years old or more were selected and further divided according to the presence or absence of the ApoE4 gene in the same way as the population under 50 years old. There were 65 subjects over 50 years old and with the ApoE4 gene, and 222 subjects over 50 years old and without the ApoE gene. For the population over 50 years old and without the ApoE4 gene, the results of the same analysis as the population under 50 years old and without the ApoE4 gene are shown in Figure 2.

[0071] As shown in Figure 2, for the population over 50 years old and without the ApoE4 gene, the following was found. For the SNP on human chromosome region 2q22.2, in subjects with a Composite Biomarker value of 0 or more and the G allele (risk allele), the odds ratio = 2.73, 95% CI (1.25, 6.01), and the p-value calculated by the Pearson Χ2 test = 0.01. For SNPs on human chromosomal region 3q27.1, in subjects with a Composite Biomarker value of 0 or higher and the A allele (risk allele), the odds ratio was 3.39, 95% CI (1.32, 8.71), and the p-value calculated by Pearson Χ² test was 0.01. For SNPs on human chromosomal region 8q24.3, in subjects with a Composite Biomarker value of 0 or higher and the A allele (risk allele), the odds ratio was 2.83, 95% CI (1.12, 7.14), and the p-value calculated by Pearson Χ² test was 0.02. For SNPs on human chromosomal region 20q13.31, in subjects with a Composite Biomarker value of 0 or higher and the C allele (risk allele), the odds ratio was 6.64, 95% CI (1.57, 28.09), and the p-value calculated by Pearson Χ² test was <0.001.

[0072] As described above, since all four of the above SNP sites showed p < 0.05, it was concluded that all were effective for determining the risk of amyloid-β accumulation.

[0073] 5. Discussion In a group of healthy individuals without the ApoE4 gene, which has a strong influence on the onset of Alzheimer's disease, and who are under 50 years old, the SNP on human chromosomal region 8q24.3 was able to determine the likelihood of amyloid-β accumulation. Also, in a group of healthy individuals without the ApoE4 gene and who are 50 years old or older, the SNPs on human chromosomal regions 2q22.2, 3q27.1, 8q24.3, and 20q13.31 were able to determine the likelihood of amyloid-β accumulation.

Claims

Claim 1 A method for in vitro determination of the amyloid-β accumulation risk, comprising using the presence of at least one risk allele from the group consisting of the following single nucleotide polymorphisms (SNPs) (b), (a), and (d) in a nucleic acid derived from a subject as an indicator that the amyloid-β accumulation risk in the subject is high, wherein the risk allele is an allele that constitutes a homozygous form showing a higher positive correlation with the average value of the Composite Biomarker value, which is obtained by scaling and scoring two values of Aβ1-42 and APP669-711, which are particularly closely related to the onset of a disease or condition associated with amyloid-β in a population having each of two homozygous forms at the SNP site, than the other homozygous form when comparing the average values, the method. (b) rs12493550 on human chromosome region 3q27.1 (a) rs17019641 on human chromosome region 2p22.2 (d) rs74792644 on human chromosome region 20q13.31 Claim 2 The risk allele of the 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) (d) is C, wherein the risk allele is an allele that constitutes a homozygous form showing a higher positive correlation with the average value of the Composite Biomarker value, which is obtained by scaling and scoring two values of Aβ1-42 and APP669-711, which are particularly closely related to the onset of a disease or condition associated with amyloid-β in a population having each of two homozygous forms at the SNP site, than the other homozygous form when comparing the average values; the method according to Claim 1. Claim 3 The method according to Claim 1 or 2, wherein the subject is 50 years of age or older. Claim 4 The method according to any one of Claims 1 to 3, wherein the subject does not carry the apolipoprotein E gene type 4 (ApoE4 gene). Claim 5 The method according to any one of Claims 1 to 4, wherein the presence of the single nucleotide polymorphism (SNP) is used as an indicator that the amyloid-β accumulation risk in the subject is high as compared with the case where the single nucleotide polymorphism (SNP) is absent. Claim 6 The method according to any one of claims 1 to 4, wherein the absence of the single nucleotide polymorphism (SNP) is used as an indicator that the risk of amyloid-β accumulation in the subject is lower compared to the case where the single nucleotide polymorphism (SNP) is present.

7. The method according to any one of claims 1 to 6, wherein the risk of amyloid-β accumulation is that amyloid-β is accumulating or there is a risk of amyloid-β accumulation.

8. The method according to any one of claims 1 to 7, wherein the risk of amyloid-β accumulation is the risk of amyloid-β accumulation before or at an early stage of the onset of a disease or condition associated with amyloid-β.

9. The disease is selected from the group consisting of mild cognitive impairment (MCI), Alzheimer's disease (AD), Down syndrome, hereditary cerebral hemorrhage with amyloidosis (Dutch type), cerebral amyloid angiopathy, and Guam Parkinson 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 (type 2 diabetes), senile cardiac amyloidosis, endocrine tumors, glaucoma, ocular amyloidosis, primary retinal degeneration, macular degeneration, optic disc drusen, optic neuropathy, optic neuritis, or lattice dystrophy. The method according to claim 8.

10. For measuring or detecting the risk of the subject for the onset of a disease or condition associated with amyloid-β, for detecting a disease or condition associated with amyloid-β in the subject, for selecting a subject having a disease or condition associated with amyloid-β, for measuring, evaluating or monitoring the response of the subject to an intake for preventing or treating a disease or condition associated with amyloid-β, or for measuring, evaluating or monitoring the course of a disease or condition associated with amyloid-β in the subject. The method according to any one of claims 1 to 9.

11. The method according to any one of claims 1 to 10, comprising a detection step of detecting the presence or absence of the single nucleotide polymorphism (SNP).

12. A method for detecting the presence or absence of the single nucleotide polymorphism (SNP) for use in the method according to any one of claims 1 to 11.

13. The presence or absence of the single nucleotide polymorphism (SNP) is detected by at least one technique selected from the group consisting of scanning probe and nanopore DNA sequencing, pyrosequencing, denaturing gradient gel electrophoresis (DGGE), temporal temperature gradient electrophoresis (TTGE), Zn(II)-cyclen polyacrylamide gel electrophoresis, homogeneous fluorescence PCR-based single nucleotide polymorphism analysis, phosphate-affinity polyacrylamide gel electrophoresis, high-throughput SNP genotyping platform, molecular beacon, 5'-nuclease reaction, Taqman assay, MassArray (single base primer extension combined with matrix-assisted laser desorption / ionization time-of-flight mass spectrometry), trityl mass tag, genotyping platform (such as Invader Assay (registered trademark)), single base primer extension (SBE) assay, PCR amplification (for example, PCR amplification on magnetic nanoparticles (MNP)), restriction enzyme analysis (RFLP method) of PCR products, allele-specific PCR, multiprimer extension (MPEX), isothermal smart amplification, PCR-SSCP (single-stranded DNA higher-order structure polymorphism analysis) method, sequencing method, ARMS (Amplification Refracting Mutation System) method, and RNaseA cleavage method, the method according to claim 11 or 12.

14. A composition for use in the method according to any one of claims 1 to 11, the composition comprising a reagent capable of directly or indirectly detecting the presence or absence of the single nucleotide polymorphism (SNP), wherein the reagent is one or more selected from oligonucleotides, DNA probes, RNA probes, and ribozymes, Composition.

15. A kit for use in the method according to any one of claims 1 to 11, the kit comprising a reagent capable of directly or indirectly detecting the presence or absence of the single nucleotide polymorphism (SNP), wherein the reagent is one or more selected from oligonucleotides, DNA probes, RNA probes, and ribozymes, Kit.

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