JP7684545B2 - How to detect mild cognitive impairment - Google Patents

Description

The present invention relates to a method for detecting mild cognitive impairment.

Mild cognitive impairment (MCI) is somewhere between a healthy state and dementia such as Alzheimer's disease, vascular dementia, Lewy body dementia, or frontotemporal dementia. It is a state in which cognitive function has declined but daily life can be carried out without any problems. MCI is defined as: 1. There is memory impairment that cannot be explained solely by age or education level; 2. The individual or a family member complains of forgetfulness; 3. Overall cognitive function is within the normal range; 4. The individual is independent in activities of daily living; and 5. There is no dementia. The number of people with MCI is expected to continue to increase as the population ages.

It has been reported that if MCI is left untreated, on average 5-15% of patients will progress to dementia each year, while cognitive function will recover to normal in approximately 16-41% of patients (Non-Patent Document 1). Furthermore, it has been reported that the recovery rate is higher in patients with mild MCI than in those with more advanced symptoms and a decline in multiple cognitive functions (Non-Patent Document 2). Therefore, while there is no established fundamental treatment for dementia except in some cases where the underlying disease is clear, it is essential to diagnose MCI at an early stage when cognitive decline is mild and to intervene early.

Currently, MCI is diagnosed based on a comprehensive assessment of the results of a medical interview with the patient and their family, cognitive function tests, general physical examinations such as blood tests, head MRI, CT, and cerebral blood flow SPECT. However, this method requires the expert judgment of a doctor. Therefore, there is a strong demand for technology to easily diagnose MCI.

On the other hand, DNA methylation is essential for normal development, regulates gene expression, and is known to be deeply involved in biological phenomena such as cell development, differentiation, aging, and reprogramming. Abnormal DNA methylation is thought to be involved in the onset of various diseases; for example, in cancer, it has been revealed that abnormal DNA methylation of CpG islands in the promoter regions of some genes is involved in carcinogenesis through the inactivation of tumor suppressor genes.

For Alzheimer's disease (AD), one of the main causes of dementia, biomarkers such as amyloid beta (Aβ) peptide, Tau peptide, α-synuclein, and TDP-43 in plasma have been reported (Non-Patent Document 3). It has also been reported that DNA methylation at the CpG site in the promoter region of the NCAPH2/LMF2 gene was significantly reduced in AD patients and amnesic MCI patients compared to healthy subjects (Non-Patent Document 4). However, in Non-Patent Document 4, the number of samples used for screening was small, and the DNA methylation rate at the CpG site was significantly correlated with the Mini-Mental State Examination (MMSE) and Frontal Assessment Battery (FAB) scores, which reflect cognitive impairment, so it is merely a biomarker for AD, including amnesic MCI.

Dementia Disease Treatment Guidelines 2017, supervised by the Japanese Society of Neurology and edited by the Dementia Disease Treatment Guidelines Editorial Committee, 2017 Brodaty H et al., Alzheimers Dement. 2013, 9(3):310-317 Molinuevo JL et al., Acta Neuropathologica, 2018, 136:821-853 Kobayashi N et al., PLoS One, 2016, 11(1):e0146449

The present invention relates to a method for detecting MCI based on DNA methylation rate.

The inventors conducted extensive research into blood samples collected from healthy individuals, MCI patients, and AD patients, and found that methylation at specific CpG sites was significantly increased only in MCI patients compared to healthy individuals and AD patients, and that MCI can be detected using the DNA methylation rate of the CpG site as an indicator, thus completing the present invention.

２）被験者におけるＭＣＩの検出のためのプライマー又はプローブであって、
前記プライマー又はプローブは、少なくとも１０塩基の鎖長を有し、かつバイサルファイト処理された後の標的ＣｐＧサイトを含むＤＮＡ領域、前記標的ＣｐＧサイトの上流のＤＮＡ領域、又は該標的ＣｐＧサイトの下流のＤＮＡ領域にハイブリダイズし、
前記標的ＣｐＧサイトが、上記表１の（１）～（３４）に記載される染色体上の位置又はその近傍に位置するＣｐＧサイトからなる群より選択される少なくとも１つのＣｐＧサイトである、プライマー又はプローブ。 That is, the present invention relates to the following 1) and 2).
1) A method for detecting mild cognitive impairment (MCI) in a subject, comprising measuring a DNA methylation rate of a target CpG site in genomic DNA derived from a biological sample collected from the subject,
The method, wherein the target CpG site is at least one CpG site selected from the group consisting of CpG sites located at or near the chromosomal positions set forth in (1) to (34) of Table 1 below.

2) A primer or probe for detecting MCI in a subject, comprising:
the primer or probe has a chain length of at least 10 bases, and hybridizes to a DNA region including a target CpG site after bisulfite treatment, a DNA region upstream of the target CpG site, or a DNA region downstream of the target CpG site;
The primer or probe, wherein the target CpG site is at least one CpG site selected from the group consisting of CpG sites located at or near the chromosomal positions described in (1) to (34) of Table 1 above.

The method of the present invention makes it possible to easily detect MCI. If MCI can be detected early using the present invention, appropriate treatment can be provided to MCI patients at an early stage.

FIG. 1 shows MCI scores in a healthy control (HC) group, an MCI group, and an AD group, based on a discriminant equation utilizing DNA methylation rates at 20 CpG sites. FIG. 1 shows MCI scores in healthy control (HC), MCI, and AD groups based on a discriminant equation utilizing DNA methylation rates at 34 CpG sites.

In this specification, "mild cognitive impairment (MCI)" refers to a state between a healthy state and dementia, where cognitive function is impaired but daily life is able to be carried out without hindrance, or the earliest stage of dementia. Specifically, it refers to a state in which the Mini-Mental State Examination (MMSE) score is between 20 and 23, or a state of cognitive function equivalent thereto.

In this specification, "detection" of MCI in a subject means clarifying the presence or absence of MCI in a subject, and can also be referred to as examination, measurement, judgment, or evaluation. Note that in this specification, the terms "judgment" or "evaluation" do not include judgment or evaluation by a physician.

In this specification, "CpG site" refers to a two-base sequence in DNA in which cytosine (C) and guanine (G) are linked by a phosphodiester bond. Regions in which CpG sites occur frequently are called CpG islands. CpG islands are often located near the coding region of a gene, for example in the promoter region, and therefore CpG sites of a gene are often found in CpG islands located near the coding region of the gene or in the promoter region of the gene.

In this specification, "DNA methylation" means a state in which the carbon at position 5 of the pyrimidine ring of cytosine in DNA is methylated. In addition, in this specification, the "DNA methylation rate" of a certain CpG site means the rate at which DNA is methylated in that CpG site. This rate may be expressed as a percentage, with fully methylated being 100% and fully unmethylated being 0%, or may be expressed as fully methylated being 1 and fully unmethylated being 0. In this specification, high and low DNA methylation rates mean that the rate at which DNA is methylated is high and low, respectively.

The present invention relates to a method for detecting MCI in a subject. The method of the present invention includes measuring the DNA methylation rate of a target CpG site in genomic DNA derived from a biological sample collected from the subject. Here, the subject may be a human, for example, a person who complains of cognitive decline, a person who is suspected of cognitive decline, a person who wishes to have their cognitive function evaluated, etc.

The biological sample collected from the subject to be used in the method of the present invention is not particularly limited as long as it contains genomic DNA, and may be a sample collected from the subject (a sample separated from a living body), specifically, collected cells, tissues, blood, blood-derived plasma or serum, oral mucus, etc. Among these, a blood sample is preferred, and a whole blood sample is more preferred.

The genomic DNA derived from a biological sample collected from a subject and used in the method of the present invention includes genomic DNA prepared from the above biological sample. There are no particular limitations on the method for preparing genomic DNA from a biological sample, and any known method can be appropriately selected and used. Known methods for preparing DNA include the phenol-chloroform method, the CTAB method, the alkaline SDS method, or a DNA extraction method using a commercially available DNA extraction kit, such as DNeasy Blood & Tissue Kit (Qiagen), QIAamp DNA Mini kit (Qiagen), Clean Columns (NexTec), AquaPure (Bio-Rad), ZR Plant/Seed DNA Kit (Zymo Research), prepGEM (ZyGEM), BuccalQuick (TrimGen), etc.

Preferably, the prepared genomic DNA is bisulfite-treated. There are no particular limitations on the method for bisulfite-treatment of DNA, and any known method can be appropriately selected and used. Known methods for bisulfite treatment include, for example, EZ DNA Methylation-Gold Kit, EZ DNA Methylation-Lightning Kit (both manufactured by Zymo Research), EpiTect Bisulfite Kit (48) (manufactured by Qiagen), MethylEasy (manufactured by Human Genetic Signatures Pty), Cells-to-CpG Bisulfite Conversion Kit (manufactured by Applied Biosystems), CpGenome Turbo Bisulfite Modification Kit (MERCK Examples include using commercially available kits such as those manufactured by MILLIPORE.

Furthermore, it is preferable to amplify the bisulfite-treated genomic DNA. There are no particular limitations on the amplification method, but PCR is preferably used. The amplification method and conditions can be appropriately selected from known methods and conditions depending on the sequence, length, amount, etc. of the DNA to be amplified.

As shown in the Examples below, the inventors found that the DNA methylation rates at CpG sites (1) to (34) shown in Table 2 were significantly higher only in the MCI patient group compared to the healthy subject group and the Alzheimer's disease (AD) patient group. Furthermore, the group average difference in DNA methylation rates at these CpG sites was very large, at 5% or more. Therefore, the DNA methylation rates at these CpG sites can be used as markers for detecting MCI. In other words, the presence or absence of MCI in a subject can be detected based on the level of DNA methylation rates at these CpG sites.

Therefore, in the method of the present invention, the target CpG site for measuring the DNA methylation rate includes a CpG site located at or near the chromosomal position shown in Table 2. In the method of the present invention, it is sufficient to measure the DNA methylation rate of at least one CpG site selected from the group consisting of CpG sites located at or near the chromosomal position shown in Table 2.
In this specification, the chromosomal positions of CpG sites are expressed based on the positions on the reference human genome sequence, NCBI database Genome Build 37. In addition, in this specification, the names of genes are based on the names of genes registered in the NCBI database.

In this specification, the term "vicinity" of a chromosomal position refers to a region 500 bases upstream (5' side of DNA) to 500 bases downstream (3' side of DNA) from the chromosomal position, preferably 200 bases upstream to 200 bases downstream, more preferably 100 bases upstream to 100 bases downstream, and even more preferably 50 bases upstream to 50 bases downstream. It is known that DNA methylation of a CpG site located near a certain CpG site in genomic DNA often correlates with DNA methylation of the CpG site (see, for example, Bell JT et al., Genome Biology 2011, 12:R10).

Examples of CpG sites located at or near the chromosomal positions shown in Table 2 include CpG sites contained in the DNA regions shown in Table 3.

In other words, examples of target CpG sites for measuring DNA methylation rates include CpG sites contained in a DNA region consisting of any of the base sequences of SEQ ID NOs: 1 to 34 or their complementary sequences.

Of the CpG sites located at the chromosomal positions shown in Table 2, the CpG sites located at positions 1,594,715, 1,594,678, and 1,594,733 on chromosome 5 (CpG sites (1), (3), and (21) in Table 2) are included in the CpG island (SEQ ID NO: 35) of the SDHAP3 gene located at positions 1,594,238 to 1,595,027 on chromosome 5. In addition, the CpG sites located at positions 857,454 and 857,863 on chromosome 16 (CpG sites (8) and (22) in Table 2) are included in the CpG island (SEQ ID NO: 36) of the PRR25 gene located at positions 857,341 to 858,025 on chromosome 16. Therefore, other examples of CpG sites located at or near the chromosomal positions shown in Table 2 include the CpG site contained in the CpG island at positions 1,594,238 to 1,595,027 on chromosome 5, or the CpG site contained in the CpG island at positions 857,341 to 858,025 on chromosome 16.

In the method of the present invention, the DNA methylation rate of at least one of the target CpG sites described above may be measured. For example, in the method of the present invention, the DNA methylation rate of at least one CpG site selected from the group consisting of CpG sites located at the chromosomal positions shown in Table 2 above may be measured. Alternatively, in the method of the present invention, the DNA methylation rate of at least one CpG site selected from the group consisting of CpG sites contained in the DNA region shown in Table 3 above may be measured. Alternatively, in the method of the present invention, the DNA methylation rate of at least one CpG site selected from the group consisting of CpG sites contained in the DNA region consisting of the nucleotide sequences of SEQ ID NOs: 1 to 36 or their complementary sequences may be measured.

From the viewpoint of improving detection sensitivity, the above-mentioned CpG sites may be combined to form the target CpG site. Examples of such combinations include a combination of any CpG site shown in Table 2 and a CpG site located in the vicinity thereof, a combination of two or more of the CpG sites shown in Table 2, a combination of CpG sites (1) to (20) shown in Table 2, a combination of CpG sites (1) to (34) shown in Table 2, etc. Among these, a combination of any CpG site shown in Table 2 and a CpG site located in the vicinity thereof, a combination of CpG sites (1) to (20) shown in Table 2, and a combination of CpG sites (1) to (34) shown in Table 2 are preferred.

Therefore, when the bisulfite-treated DNA is amplified by PCR or the like as described above in the method of the present invention, the DNA region containing the target CpG site described above is amplified. The size of the amplified DNA region is not particularly limited as long as it contains the target CpG site.

In the present invention, the method for measuring the DNA methylation rate of a CpG site is not particularly limited, and any known method capable of measuring the DNA methylation rate can be used as appropriate. Such known methods include, but are not limited to, the microarray method, the pyrosequencing method, the bisulfite sequencing method, the methylation-specific PCR (MSP) method, the quantitative MSP method, and the Combined Bisulfite Restriction Analysis (COBRA) method.

The microarray method is a method for quantifying DNA methylation at CpG sites by utilizing a single-base extension reaction using a probe constructed to have a base complementary to methylated cytosine or unmethylated cytosine at the 3' end for each CpG site. The method can be carried out, for example, as follows. First, the genomic DNA is treated with bisulfite. Note that this bisulfite treatment converts unmethylated cytosine residues to uracil, but does not convert methylated cytosine residues. Next, the whole genome is amplified using the bisulfite-treated genomic DNA as a template, and the DNA is enzymatically fragmented (usually to about 300 to 600 bp) and dissociated into single strands.

On the other hand, a probe is prepared that hybridizes to the genomic DNA converted by bisulfite treatment, and the base at the 3' end of the probe is a base complementary to the cytosine at the CpG site. That is, if the CpG site is methylated, the base at the 3' end of the probe is guanine, and if the CpG site is not methylated, the base at the 3' end of the probe is adenine.

Then, two types of probes, which differ only in the base at the 3' end complementary to the CpG site, are hybridized with the single-stranded DNA fragment, and a single-base extension reaction is carried out in the presence of a fluorescently labeled base. As a result, if the CpG site of the single-stranded fragment is methylated, a fluorescently labeled base is incorporated into a probe (methylation detection probe) whose 3' end base is guanine by a single-base extension reaction, but a probe (non-methylation detection probe) whose 3' end base is adenine does not incorporate a fluorescently labeled base because of a mismatch of the 3' end bases. On the other hand, if the CpG site of the single-stranded fragment is not methylated, a fluorescently labeled base is incorporated into the non-methylation detection probe, but a fluorescently labeled base is not incorporated into the methylation detection probe. Therefore, the DNA methylation rate can be calculated from the intensity of the fluorescence emitted by the methylation detection probe and/or the non-methylation detection probe.

In addition, in another embodiment of the method, instead of the methylation detection probe and the unmethylation detection probe, a probe that hybridizes to genomic DNA converted by bisulfite treatment and in which the base at the 3' end of the probe is a base complementary to the guanine at the CpG site may be used. Then, such a probe is hybridized to the single-stranded DNA fragment, and a single-base extension reaction is performed in the presence of guanine labeled with a fluorescent substance and/or adenine labeled with a fluorescent dye different from the fluorescent substance. As a result, if the CpG site is methylated, fluorescently labeled guanine is incorporated into the probe, while if the CpG site is not methylated, fluorescently labeled adenine is incorporated into the probe, so that the DNA methylation rate can be calculated from the intensity of fluorescence emitted by each fluorescent substance incorporated into the probe.

A preferred example of such a method is the bead array method (e.g., the Infinium (registered trademark) Assay).

The pyrosequencing method can be carried out, for example, as follows. First, the genomic DNA is treated with bisulfite. Note that this bisulfite treatment converts unmethylated cytosine residues to uracil, but uracil is shown as thymine in the following extension reaction (sequence reaction). Next, DNA containing at least one of the CpG sites is amplified using the bisulfite-treated genomic DNA as a template. Then, the amplified DNA is dissociated into single strands. Next, only one strand of the dissociated single-stranded DNA is separated. Then, an extension reaction is carried out one base at a time from the vicinity of the base of the CpG site, and the pyrophosphate generated during this is enzymatically made to emit light, and the intensity of the light emission is measured. The intensity of the light emission derived from the methylated cytosine residue obtained in this way (light emission intensity of cytosine) is compared with the intensity of the light emission derived from the unmethylated cytosine residue (light emission intensity of thymine), and the DNA methylation rate at the CpG site is calculated, for example, by the following formula (1).

The bisulfite sequencing method can be carried out, for example, as follows. First, the genomic DNA is subjected to a bisulfite treatment. Next, a direct sequencing reaction is carried out using the bisulfite-converted nucleotides containing the CpG site as a template. Then, the DNA methylation rate at the CpG site is calculated by comparing the fluorescence intensity based on the determined base sequence, i.e., the fluorescence intensity derived from the methylated cytosine residue (fluorescence intensity of cytosine) with the fluorescence intensity derived from the unmethylated cytosine residue (fluorescence intensity of thymine).

The MSP method can be carried out, for example, as follows. First, the genomic DNA is subjected to a bisulfite treatment. Next, a primer set capable of amplifying when the CpG site is methylated and a primer set capable of amplifying when the CpG site is unmethylated are prepared. Then, using the bisulfite-treated genomic DNA as a template, nucleotides containing at least one CpG site are amplified using these primer sets. Then, the amount of the obtained amplification product, i.e., the amount of the amplification product specific to the methylated CpG site and the amount of the amplification product specific to the unmethylated CpG site, is compared to calculate the DNA methylation rate at the CpG site.

The quantitative MSP method can be carried out, for example, as follows. First, the genomic DNA is subjected to a bisulfite treatment. Next, an oligonucleotide probe having a nucleotide capable of hybridizing when the CpG site is methylated and labeled with a reporter fluorescent dye and a quencher fluorescent dye is prepared. In addition, an oligonucleotide probe having a nucleotide capable of hybridizing when the CpG site is not methylated and labeled with a reporter fluorescent color different from the reporter fluorescent dye and a quencher fluorescent dye is prepared. Then, the oligonucleotide probe is hybridized to the bisulfite-treated genomic DNA, and the nucleotides containing the CpG site are amplified using the genomic DNA hybridized with the oligonucleotide probe as a template. Then, the fluorescence emitted by the reporter fluorescent dye is detected due to the decomposition of the oligonucleotide probe accompanying the amplification. The intensity of the fluorescence emitted by the reporter fluorescent dye specific to the methylated cytosine CpG site thus detected is compared with the intensity of the fluorescence emitted by the reporter fluorescent dye specific to the unmethylated cytosine CpG site to calculate the DNA methylation rate at the CpG site.

Examples of such methods include the MethyLight method using TaqMan probes (registered trademark).

The COBRA method can be carried out, for example, as follows. First, the genomic DNA is treated with bisulfite. Next, the region containing the CpG site is amplified by PCR using the bisulfite-converted nucleotides containing the CpG site as a template. Next, the amplified DNA fragment is treated with a restriction enzyme that recognizes a portion of the sequence that differs between when the CpG site is methylated and when it is not. Then, the DNA methylation rate of the CpG site can be calculated by quantitatively analyzing the band densities of the restriction enzyme fragments derived from the methylated CpG site and the restriction enzyme fragments derived from the unmethylated CpG site, which are fractionated by electrophoresis.

In the present invention, the "method for detecting DNA methylation rate" is not limited to the above-mentioned methods, but is preferably a pyrosequencing method, a bisulfite sequencing method, or a quantitative MSP method. In the above-mentioned methods, genomic DNA is subjected to bisulfite treatment. Therefore, the genomic DNA used for measuring the DNA methylation rate of CpG sites in the method of the present invention is preferably bisulfite-treated genomic DNA.

Thus, the DNA methylation rate of the target CpG site of the present invention in genomic DNA derived from a biological sample collected from a subject is measured, and the presence or absence of MCI in the subject is detected based on the DNA methylation rate. A person skilled in the art can appropriately set a specific index (threshold) for detection in accordance with the method for detecting the DNA methylation rate. An embodiment of the detection procedure is described below.

In a first embodiment, the detection is performed by comparing the measured DNA methylation rate of the target CpG site with a control DNA methylation rate. Here, the "control DNA methylation rate" may be, for example, the DNA methylation rate of the target CpG site in a healthy individual. The DNA methylation rate of the target CpG site in a healthy individual may be a statistical value (e.g., an average value, etc.) of the DNA methylation rate of the target CpG site measured from a population of healthy individuals. When there are multiple target CpG sites, it is preferable to determine a reference level for each CpG site.
In the first embodiment, when the DNA methylation rate of the target CpG site in the biological sample collected from the subject is higher than the control DNA methylation rate, the DNA methylation rate is judged to exceed the diagnostic threshold, and the subject is judged to have MCI, whereas when the DNA methylation rate is equal to or lower than the control DNA methylation rate, the subject is judged not to have MCI.

In a second embodiment, the detection is performed by detecting an increase in the measured DNA methylation rate of the target CpG site. In this case, the measured DNA methylation rate of the target CpG site is compared with a cutoff value (reference value) of each CpG site. The cutoff value may be appropriately determined based on statistical values such as the average value and standard deviation of the DNA methylation rate based on the DNA methylation rate of the target CpG site in healthy subjects, which is previously obtained as reference data.
In the second embodiment, when the DNA methylation rate of the target CpG site in the biological sample collected from the subject is higher than the cutoff value, the DNA methylation rate is judged to exceed the diagnostic threshold, and the subject is judged to have MCI, whereas when the DNA methylation rate is equal to or lower than the cutoff value, the subject is judged not to have MCI.

In the first and second embodiments, when the DNA methylation rates of multiple CpG sites are measured, the number or percentage of CpG sites whose DNA methylation rates exceed the diagnostic threshold can be used as an index for judgment. For example, if the DNA methylation rates of all examined CpG sites exceed the diagnostic threshold, the subject can be judged to have MCI. Alternatively, if the DNA methylation rates of a certain percentage or more of the examined CpG sites exceed the diagnostic threshold, the subject can be judged to have MCI. Alternatively, if the DNA methylation rates of a certain number or more CpG sites exceed the diagnostic threshold, the subject can be judged to have MCI. On the other hand, subjects who do not meet these criteria can be judged not to have MCI.

In a third embodiment, the detection is carried out by constructing a discriminant (prediction model) for finding MCI patients using the DNA methylation rate of the target CpG site measured in a biological sample collected from an MCI patient and the measured values of the DNA methylation rate of the target CpG site derived from biological samples collected from healthy subjects and/or AD patients, and using the discriminant. The discriminant is constructed, for example, by calculating a logistic regression model based on the actual measured values of the DNA methylation rate of the target CpG site, as shown in the Examples described later.
The DNA methylation rate of the target CpG site is then measured in the same manner for a biological sample collected from the subject, and the obtained DNA methylation rate value is substituted into a discriminant to generate a score. The score is then compared with a cutoff value (reference value), thereby enabling the presence or absence of MCI in the subject to be evaluated.
In the third embodiment, when the value calculated by the discriminant for a biological sample derived from a subject is equal to or greater than the cutoff value, the diagnostic threshold is exceeded, and the subject is determined to have MCI, whereas when the value is less than the cutoff value, the subject is determined not to have MCI.

The method for determining the cutoff value (reference value) is not particularly limited, and can be determined according to a known method. For example, it can be determined from a receiver operating characteristic curve (ROC) curve created using a discriminant. In the ROC curve, the vertical axis plots the probability of a positive result in a positive patient (sensitivity), and the horizontal axis plots the value obtained by subtracting the probability of a negative result in a negative patient (specificity) from 1 (false positive rate). With regard to the "true positive (sensitivity)" and "false positive (1-specificity)" shown in the ROC curve, the value (Youden index) at which "true positive (sensitivity)" - "false positive (1-specificity)" is maximized can be used as the cutoff value (reference value).

In the examples described below, a discriminant (prediction model) for detecting MCI of the following formula (2) was constructed for the target CpG sites shown in Table 2.

In formula (2), α is the intercept, β ₁ to β _k are the coefficients of each target CpG site, x ₁ to x _k are the DNA methylation rate of each target CpG site, and k is the number of target CpG sites. In formula (2), the intercept (α) is multiplied by the coefficients (β ₁ to β _k ) and the DNA methylation rate (x ₁ to x _k ) for each target CpG site, and the products are added to calculate the MCI score (linear predictor). Here, the value of the DNA methylation rate may be a ratio where complete methylation is 1 and complete non-methylation is 0, and may be a so-called β value. In the discriminant when 20 CpG sites of the target CpG sites (1) to (20) shown in Table 2 are used, α is −533.1722, and the coefficients are shown in Table 4. Furthermore, when the 34 target CpG sites (1) to (34) are used, α in the discriminant is −585.9345, and the coefficients are shown in Table 5.
The MCI score calculated by formula (2) is an index for evaluating whether or not a subject has MCI, with the cutoff value being 0. If the calculated MCI score is 0 or greater, the subject is determined to have MCI, whereas if the calculated value is a negative value, the subject is determined not to have MCI.

In this way, according to the present invention, the presence or absence of MCI in a subject can be determined. If MCI patients can be detected early by the method of the present invention, early intervention can be performed, which contributes to improving cognitive function, avoiding or delaying the onset of dementia, improving QOL, etc.
Therefore, the present invention also relates to early intervention in subjects determined to have MCI by the method of the present invention. This aspect of the present invention allows for improvement of cognitive function, prevention, early diagnosis, or early treatment of dementia in subjects with MCI.
The present invention further provides a method for treating MCI, comprising treating a subject diagnosed as having MCI by the method of the present invention. This aspect of the present invention makes it possible to provide an appropriate treatment to a subject with MCI at an earlier stage. Treatment methods include, but are not limited to, exercise therapy, diet therapy, cognitive function training, chemotherapy, etc.

The present invention further provides a primer or probe for detecting MCI. The primer or probe has a chain length of at least 10 bases and hybridizes to a DNA region containing a target CpG site after bisulfite treatment, a DNA region upstream of the target CpG site, or a DNA region downstream of the target CpG site.

An example of the primer or probe of the present invention is a primer or probe that hybridizes to a DNA region containing a target CpG site contained in any of the DNA regions shown in Table 3 (e.g., a DNA region consisting of any of the nucleotide sequences of SEQ ID NOs: 1 to 34 or their complementary sequences) or a DNA region consisting of the nucleotide sequence of SEQ ID NO: 35 or 36 or their complementary sequences, a DNA region upstream of the target CpG site, or a DNA region downstream of the target CpG site.

A preferred example of the primer or probe of the present invention is a primer or probe consisting of any one of the nucleotide sequences of SEQ ID NOs: 1 to 36 or a complementary sequence thereof. Another preferred example of the primer or probe of the present invention is a primer or probe that has 95% or more identity with any one of the nucleotide sequences of SEQ ID NOs: 1 to 36 or a complementary sequence thereof and hybridizes to the same CpG site as the CpG site to which the sequence hybridizes. Furthermore, fragments of the primer or probe that hybridize to the same CpG site as the CpG site to which the primer or probe hybridizes can also be used as the primer or probe of the present invention.

Another preferred example of the primer or probe of the present invention is a probe that hybridizes to a DNA fragment containing the bisulfite-treated target CpG site (e.g., a fragment containing a nucleotide sequence of any one of SEQ ID NOs: 1 to 36 or a complementary strand thereof) and has a base complementary to methylated cytosine or unmethylated cytosine at its 3' end (e.g., a probe that can be used in the microarray method described above). Another preferred example of the primer or probe of the present invention is a probe that hybridizes to a DNA fragment containing the bisulfite-treated target CpG site (e.g., a fragment containing a nucleotide sequence of any one of SEQ ID NOs: 1 to 36 or a complementary strand thereof) and has a base complementary to guanine at the target CpG site at its 3' end (e.g., a probe that can be used in the microarray method described above).

Another preferred example of the primer or probe of the present invention is a primer (sequencing primer) that can carry out an extension reaction of one base at a time from the vicinity of the base of the bisulfite-treated target CpG site (for example, a primer that can be used in the pyrosequencing method described above).

Another preferred example of the primer or probe of the present invention is a primer set capable of specifically amplifying a region containing the methylated or unmethylated target CpG site in a DNA fragment (e.g., a fragment containing any of the nucleotide sequences of SEQ ID NOs: 1 to 36 or a complementary strand thereof) that has been bisulfite-treated and contains the target CpG site (e.g., a primer set that can be used for PCR amplification in the aforementioned MSP method, quantitative MSP method, or COBRA method).

Another preferred example of the primer or probe of the present invention is a probe that specifically hybridizes to a DNA fragment (e.g., a fragment containing any of the nucleotide sequences of SEQ ID NOs: 1 to 36 or its complementary strand) containing the target CpG site that has been bisulfite-treated when the CpG site is methylated or unmethylated, and is labeled with a reporter fluorescent dye and a quencher (e.g., a probe that can be used in the quantitative MSP method described above).

The length of the primer or probe of the present invention may be at least 10 bases, but is preferably at least 15 bases, more preferably at least 20 bases, and even more preferably 15 to 200 bases. In the case of a probe, the length is preferably 100 to 200 bases, more preferably 100 to 150 bases. In the case of a PCR primer, the length is preferably 10 to 60 bases, more preferably 15 to 40 bases. The primer or probe of the present invention may be labeled (e.g., fluorescently labeled). The primer or probe of the present invention is preferably a primer or probe that can be used in any of the microarray method, the pyrosequencing method, the bisulfite sequencing method, the MSP method, the quantitative MSP method, and the COBRA method, and more preferably a primer or probe that can be used in the pyrosequencing method, the bisulfite sequencing method, or the quantitative MSP method.

The present invention further provides a kit for detecting MCI, comprising the primer or probe of the present invention. Preferably, the kit of the present invention is used for detecting MCI by any of the microarray method, pyrosequencing method, bisulfite sequencing method, MSP method, quantitative MSP method, and COBRA method, more preferably by pyrosequencing method, bisulfite sequencing method, or quantitative MSP method.

The kit of the present invention may contain components other than the primer or probe of the present invention. Examples of such components include, but are not limited to, reagents required for bisulfite treatment (e.g., sodium bisulfite solution, etc.), reagents required for PCR reaction (e.g., deoxyribonucleotides and heat-resistant DNA polymerase, etc.), reagents required for Infinium Assay (e.g., nucleotides labeled with fluorescent substances), reagents required for pyrosequencing (e.g., ATP sulfurylase, adenosine-5'-phosphosulfate, luciferase, luciferin for detecting pyrophosphate, streptavidin for separating single-stranded DNA, etc.), reagents required for detecting labels (e.g., substrates and enzymes, positive and negative control samples), reagents required for COBRA method (e.g., restriction enzymes, etc.), buffer solutions used for diluting and washing samples (e.g., genomic DNA derived from tissues, etc.), etc. The kit may also include instructions for use.

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

Example 1 Identification of DNA methylation markers capable of detecting MCI (1) Samples Whole blood samples from 24 healthy subjects (HC), 8 mild cognitive impairment (MCI), and 14 Alzheimer's disease (AD) patients stored in the Juntendo University DNA Bank were used. Details of each sample are shown in Table 6. The Mini-Mental State Examination (MMSE), a type of cognitive function test, had scores of 20≦MMSE≦23 in the MCI group and MMSE≦19 in the AD group. The HC group, MCI group, and AD group were arranged so that the male-female ratio and age distribution were almost equal. Patients with early-onset AD and strong familial AD were excluded.
Genomic DNA was prepared from each whole blood sample using a QIAamp DNA Blood Maxi kit (QIAGEN) according to the protocol attached to the kit.

(2) Measurement of DNA methylation rate at CpG site The obtained genomic DNA was treated with sodium sulfite for 16 hours and then sulfonated. PCR amplification was performed using the bisulfite-treated genomic DNA as a template. As a result, unmethylated cytosine (C) was converted to uracil (U) and recognized as T through PCR amplification. This step was performed using EZ Methylation Gold kit (Zymo, Cat# D5001/D5002), and treatment was performed under the conditions for Illumina Infinium (registered trademark) Methylation Assay according to the Appendix described in the protocol.
A bead array for measuring DNA methylation (Infinium (registered trademark) Methylation EPIC BeadChip, manufactured by Illumina) was used to measure the DNA methylation rate at approximately 850,000 CpG sites. Specifically, PCR-amplified DNA fragments were hybridized with the probes on the array. Fluorescently labeled bases were incorporated into the hybridized DNA by a single-base extension reaction, and fluorescent labeling was performed according to the methylation or non-methylation of each CpG site. Then, the fluorescent signal was measured using an iScan Reader (manufactured by Illumina), and the obtained data was analyzed using R bioconductor package ChAMP (https://bioconductor.org/packages/release/bioc/html/ChAMP.html). For each CpG site, the relative ratio of the fluorescent signal from the methylation detection probe to the sum of the signals from the methylation detection probe and the unmethylation detection probe was calculated. The DNA methylation rate of each CpG site was calculated as a so-called β value. The β value is expressed as 0 (completely unmethylated) to 1 (completely methylated). The β value normalized using ChAMP was used in the following test.

(3) Extraction of CpG sites with different DNA methylation rates between groups Logistic regression analysis was performed on the DNA methylation rate data for each CpG site obtained in (2), and CpG sites with significantly different DNA methylation rates between (i) the HC group and the MCI group, and (ii) the MCI group and the AD group were extracted, with p<0.05 as significance. As a result, 2219 CpG sites with significantly higher DNA methylation rates in the MCI group compared to the HC group and the AD group were found in each of the comparisons (i) and (ii).

(4) Confirmation of the effect of blood cell ratio on DNA methylation The effect of blood cell ratio on DNA methylation rate was confirmed for the 2219 CpG sites obtained in (3). Specifically, methylation between blood cell types was compared using DNA methylation data from the DNA methylation array HumanMethylation 450K (a platform similar to MethylationEPIC with approximately half of the common CpG probes) of 25 blood cell types stored in the database of EWAS Atlas (https://bigd.big.ac.cn/ewas). When compared with the average DNA methylation rate of each CpG site of the 25 cell types, the 1410 CpG sites that could be compared showed a pattern similar to that of whole blood in many cell types, but only naive CD4+ T cells were highly methylated at 613 CpG sites (50% or more in naive CD4+ T cells, 20% or less in WBC). 797 CpG sites showed no difference between naive CD4+ T cells and whole blood.

(5) Selection of CpG sites Among the 2219 CpG sites obtained in (3), 34 CpG sites were found in which the difference in the group average of DNA methylation rate was 5% or more (β value of 0.05 or more) (Table 7). The regions in which these CpG sites were located were regions highly related to a group of transcription factors such as GREB1, RUNX1, SFF4, and KDM5B. In addition, of the 34 CpG sites, only 4 CpG sites were CpG sites that showed naive CD4+ T cell-specific high methylation (CD4Naive in Table 7). On the other hand, 14 CpG sites showed no difference in methylation between naive CD4+ T cells and whole blood (Others in Table 7). This suggests that the selection of the 34 CpG sites is highly likely not based on naive CD4+ T cell-specific methylation.

Example 2: Construction of a discriminant for detecting MCI Twenty or thirty-four CpG sites were selected from the 34 CpG sites obtained in (5) of Example 1, and a logistic regression model was calculated based on the actual measurement data of the DNA methylation rate of each CpG site (proportion in which complete methylation is 1 and complete unmethylation is 0), to construct a discriminant (prediction model) for detecting MCI, as shown in the following formula (2).

In formula (2), α is the intercept, β ₁ to β _k are the coefficients of each target CpG site, x ₁ to x _k are the DNA methylation rate of each target CpG site, and k is the number of target CpG sites. In formula (2), the intercept (α) is multiplied by the coefficients (β ₁ to β _k ) and DNA methylation rates (x ₁ to x _k ) for each CpG site, and the products are added to calculate the MCI score (linear predictor). In the discriminant when 20 CpG sites (1) to (20) shown in Table 7 are used, α is −533.1722, and the coefficients are shown in Table 8. In addition, in the discriminant when 34 CpG sites (1) to (34) are used, α is −585.9345, and the coefficients are shown in Table 9. The cutoff value for the MCI score is 0, and if the calculated MCI score is equal to or greater than 0, the subject is determined to have MCI, whereas if the calculated value is a negative number, the subject is determined not to have MCI.

Figure 1 shows the MCI scores of each group when a discriminant using 20 CpG sites, target CpG sites (1) to (20), was used. Wilcoxon rank sum test showed p-value = 1.901e-07 for the HC group vs. MCI group, and p-value = 6.254e-06 for the AD group vs. MCI group. Figure 2 shows the MCI scores of each group when a discriminant using 34 CpG sites, target CpG sites (1) to (34), was used. Wilcoxon rank sum test showed p-value = 1.901e-07 for the HC group vs. MCI group, and p-value = 6.254e-06 for the AD group vs. MCI group. Such a discriminant using the DNA methylation rates of multiple CpG sites can accurately detect whether a subject has MCI.

Claims (7)

A method for detecting mild cognitive impairment (MCI) in a subject, comprising: measuring a DNA methylation rate at a target CpG site in genomic DNA derived from a biological sample collected from the subject ; and substituting the DNA methylation rate into the following formula (1); and determining that the subject has mild cognitive impairment (MCI) if the obtained value is 0 or greater, and that the subject does not have MCI if the obtained value is a negative value ,
The method, wherein the target CpG site is a CpG site located at a chromosomal position described in (1) to (20) of Table 1 below.

(In formula (1), α is −533.1722, β ₁ is 446.3352, β ₂ is −450.7728, β ₃ is −244.6187, β ₄ is 385.7751, β ₅ is 204.7351, β ₆ is 54.8807, β ₇ is 504.2445, β ₈ is −139.5432, β ₉ is 332.2435, β ₁₀ is −350.7700, β ₁₁ is 114.3273, β ₁₂ is 232.6809, β ₁₃ is −189.6994, β ₁₄ is 66.6434, β ₁₅ is −12.0331, β ₁₆ is 9.2219, β _{β 17} is −47.9527, β ₁₈ is 152.8140, β ₁₉ is −67.5121, and β ₂₀ is −19.4151, and x ₁ to x ₂₀ respectively indicate the DNA methylation rates of the CpG sites located at the chromosomal positions described in (1) to (20) of Table 3 above.) 1. A method for detecting MCI in a subject, comprising: measuring a DNA methylation rate at a target CpG site in genomic DNA derived from a biological sample collected from the subject; and substituting the DNA methylation rate into the following formula (2); and determining that the subject has MCI when the obtained value is 0 or more, and that the subject does not have MCI when the obtained value is a negative value,
The method, wherein the target CpG site is a CpG site located at a chromosomal position described in (1) to (34) of Table 2 below.

(In formula (1), α is −585.9345, and β ₁ is 725.2845, β ₂ is -629.1517, β ₃ is -396.5470, β ₄ is 350.7335, β ₅ is 316.9368, β ₆ is 291.4193, β ₇ is 235.9880, β ₈ is -227.1130, β ₉ is 203.5180, β ₁₀ is -193.7436, β ₁₁ is 183.5877, β ₁₂ is 182.7943, β ₁₃ is -177.1248, β ₁₄ is 159.0189, β ₁₅ is 136.6889, β ₁₆ is -134.3153, β ₁₇ is -117.9282, β ₁₈ is 115.0902, β ₁₉ is -113.9686, β ₂₀ is -111.6214, β _{twenty one} is -108.6566, β _{twenty two} is 104.5547, β _{twenty three} is -100.0736, β _{twenty four} is 96.2618, β _{twenty five} is 96.1807, β ₂₆ is -89.5214, β ₂₇ is 87.8011, β ₂₈ is -57.1841, β ₂₉ is 56.5640, β ₃₀ is -50.0396, β ₃₁ is -42.3310, β ₃₂ is 29.2738, β ₃₃ is -28.2257, β ₃₄ indicates -25.8969, and x ₁ ～x ₃₄ indicates the DNA methylation rate of the CpG sites located on the chromosomes at the positions listed in (1) to (34) of Table 4 above, respectively.) The method according to claim 1 or 2 , wherein the genomic DNA is bisulfite-treated genomic DNA. The method according to claim 3 , wherein the DNA methylation rate is measured using a microarray method, a pyrosequencing method, a bisulfite sequencing method, or a quantitative methylation-specific PCR (MSP) method. The method according to any one of claims 1 to 4 , wherein the biological sample is a whole blood sample. A kit for use in the method according to any one of claims 1 and 3 to 5, comprising:
The primers or probes for measuring DNA methylation rates for each of the target CpG sites located at the chromosomal positions listed in (1) to (20) of Table 1 above are included,
the primer or probe has a chain length of at least 10 bases and hybridizes to a DNA region including the target CpG site after bisulfite treatment, a DNA region upstream of the target CpG site, or a DNA region downstream of the target CpG site ;
kit . A kit for use in the method according to any one of claims 2 to 5, comprising:
The primers or probes for measuring DNA methylation rates for each of the target CpG sites located at the chromosomal positions listed in (1) to (34) of Table 2 above are included,
the primer or probe has a chain length of at least 10 bases and hybridizes to a DNA region including the target CpG site after bisulfite treatment, a DNA region upstream of the target CpG site, or a DNA region downstream of the target CpG site;
kit.

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