KR102313453B1 - Marker for diagnosing prodromal Alzheimer’s disease using alteration of DNA methylation of gene - Google Patents

Abstract

The present invention relates to a composition, kit and method for diagnosing Alzheimer's disease mild cognitive impairment or predicting the risk of progression to Alzheimer's disease dementia by detecting the methylation level of a gene CpG region.

Description

Diagnostic marker for Alzheimer's disease mild cognitive impairment using alteration of DNA methylation of gene {Marker for diagnosing prodromal Alzheimer's disease using alteration of DNA methylation of gene}

The present invention relates to a composition, kit and method for diagnosing Alzheimer's disease mild cognitive impairment or predicting the risk of progression to Alzheimer's disease dementia by detecting the methylation level of a gene CpG region.

Korea's rapid economic growth, westernization of lifestyles, and advances in medicine have resulted in an increase in the average life expectancy of Koreans, while Korea is experiencing the fastest aging of the population in the world. As a result, neurodegenerative diseases that occur in conjunction with aging are also rapidly increasing.

Dementia is a representative disease with a large socioeconomic burden, and among them, Alzheimer's disease dementia is the most common type of dementia. Alzheimer's disease dementia is a representative neurodegenerative brain disease that starts with memory loss and progresses gradually over a long period of time with symptoms such as loss of time and space cognitive ability, delusions and personality changes, paralysis of speech and motor functions, and eventually death. am.

Although research for the treatment of Alzheimer's disease dementia has been continuously conducted for the past several decades, there is no fundamental treatment until now, and only a level of treatment that relieves symptoms or slows the progression of the lesion is being performed. Therefore, delaying the onset of dementia through early diagnosis and preemptive treatment for Alzheimer's disease is suggested as the most effective management method.

However, in the case of neurocognitive tests currently used for diagnosing Alzheimer's disease and dementia, the patient's age, education level, or intentional refusal to answer has a disadvantage in that the accuracy of the test results is greatly affected. In this case, since expensive equipment is used, it is difficult to conduct the test in the initial stage when there are no special symptoms.

Accordingly, as another method for the diagnosis of Alzheimer's disease dementia, a method of detecting beta-amyloid protein in cerebrospinal fluid or serum, a method of measuring the concentration of tau protein, a glial fibrillary acidic protein (GFAP)-antibody Biochemical methods such as a detection method have been proposed, but problems such as the efficiency and accuracy of clinical application of the diagnostic method are raised (International Patent Publication No. 92/17152; U.S. Patent No. 4,666,829; International Patent Publication No. 89/ 06242; US Pat. No. 5,231,000, etc.).

Therefore, there is an urgent need for a new diagnostic marker that can represent clinical symptoms or measure the preclinical state before symptoms appear for early diagnosis of Alzheimer's disease dementia.

International Patent Publication WO 1992-017152 (published on Oct. 15, 1992) US Patent Registration US 4,666,829 (Registered on May 19, 1987) International Patent Publication WO1989-006242 (published on July 13, 1989) US Patent Registration US 5,231,000 (Registered on July 27, 1993)

The present inventors confirmed that the CD96 (CD96 molecule) gene is hypomethylated specifically for Alzheimer's disease, specifically Alzheimer's disease mild cognitive impairment, and using this as a biomarker to measure the degree of methylation of the CD96 gene, Alzheimer's disease The present invention was completed by confirming that it is possible to diagnose mild cognitive impairment or predict the risk of progression to Alzheimer's disease dementia.

Accordingly, an embodiment of the present invention provides a composition for diagnosing Alzheimer's disease mild cognitive impairment or predicting the risk of progression to Alzheimer's disease dementia, comprising an agent for measuring the methylation level of the CpG region of the CD96 gene promoter.

Another example provides a kit for diagnosing Alzheimer's disease mild cognitive impairment or predicting the risk of progression to Alzheimer's disease dementia, comprising the composition.

Another example provides a method for diagnosing Alzheimer's disease mild cognitive impairment or predicting the risk of progression to Alzheimer's disease dementia by measuring the level of methylation in the CpG region of the CD96 gene promoter.

Another example provides an information providing method for diagnosing Alzheimer's disease mild cognitive impairment or predicting the risk of progression to Alzheimer's disease dementia, comprising measuring the methylation level of the CpG region of the CD96 gene promoter from a sample of an individual.

Another example provides a method of measuring the level of methylation in the CpG region of a CD96 gene promoter from a sample of an individual to provide information necessary for diagnosing Alzheimer's disease mild cognitive impairment or predicting the risk of progression to Alzheimer's disease dementia.

The present invention is based on the discovery that the CD96 gene is specifically hypomethylated in Alzheimer's disease, specifically Alzheimer's disease mild cognitive impairment. It provides molecular biological diagnostic technology.

Because DNA methylation changes are present in DNA, they are easy to detect, more stable than protein or RNA markers, and have the advantages of easy analysis because they occur at a specific location in a gene.

Specifically, in one embodiment of the present application, genomic DNA is extracted from the samples of the normal control group (normal cognitive function group without amyloid pathology) and Alzheimer's disease mild cognitive impairment patient group, and the DNA methylation profile is analyzed through DNA methylation mutation analysis. and compared with the normal control group, genes with more than 30% DNA methylation change in the CpG region of the gene promoter region of the Alzheimer's disease mild cognitive impairment patient group were selected. Among these selected genes, it was confirmed that the DNA methylation of the CpG region of the promoter was reduced by about 31% in the CD96 gene in the Alzheimer's disease mild cognitive impairment patient group compared to the normal control group, and the receiver operating characteristics curve (ROC curve: receiver operating characteristics curve) ), it was confirmed that hypomethylation of the CpG region of the CD96 gene promoter can distinguish the Alzheimer's mild cognitive impairment patient group with high accuracy (AUC = 0.9706) compared to the normal control group.

These results indicate that disease-specific hypomethylation of the CD96 gene can be used as a biomarker for diagnosing Alzheimer's disease mild cognitive impairment or predicting the risk of progression to Alzheimer's disease dementia in a non-invasive manner.

Accordingly, one embodiment provides a composition for diagnosing Alzheimer's disease mild cognitive impairment or predicting the risk of progression to Alzheimer's disease dementia, comprising an agent for measuring the methylation level of the CpG region of the CD96 gene promoter.

Another embodiment provides a kit for diagnosing Alzheimer's disease mild cognitive impairment or predicting the risk of progression to Alzheimer's disease dementia, comprising the composition.

Another embodiment provides a method of diagnosing Alzheimer's disease mild cognitive impairment or predicting the risk of progression to Alzheimer's disease dementia by measuring the level of methylation in the CpG region of the CD96 gene promoter from a sample of an individual.

Another embodiment provides an information providing method for diagnosing Alzheimer's disease mild cognitive impairment or predicting the risk of progression to Alzheimer's disease dementia, comprising measuring the level of methylation in the CpG region of the CD96 gene promoter from a sample of an individual .

Another embodiment provides a method of measuring the level of methylation in the CpG region of the CD96 gene promoter from a sample of an individual to provide information necessary for the diagnosis of Alzheimer's disease mild cognitive impairment or the prediction of the risk of progression to Alzheimer's disease dementia.

As used herein, the term "methylation" or "methylation" refers to the attachment of a methyl group to a base constituting DNA. Preferably, in the present invention, methylation means whether methylation occurs in the cytosine of the CpG region of a specific gene promoter. In the case of methylation, the binding of transcription factors is disturbed and the expression of a specific gene is suppressed. Conversely, when unmethylation or hypomethylation occurs, the expression of a specific gene is increased.

In the genomic DNA of mammalian cells, in addition to A, C, G and T, there is a fifth base called 5-methylcytosine (5-mC) with a methyl group attached to the fifth carbon of the cytosine ring. do. 5-methylcytosine methylation occurs only at C of a CG dinucleotide called CpG (5'-mCG-3'), and CpG methylation inhibits expression of alu or transposon and genomic repeats. In addition, since 5-mC of CpG is easily deamidated to thymine (T), CpG is a site where most epigenetic changes frequently occur in mammalian cells.

As used herein, the term "measurement of methylation level" refers to measuring the methylation level of the CpG region of a gene, and methylation-specific PCR, for example, methylation-specific polymerase chain reaction (MSP), real-time methylation-specific It can be measured through real time methylation-specific polymerase chain reaction (PCR), PCR using a methylated DNA-specific binding protein, or quantitative PCR. Alternatively, measurement by methods such as automatic sequencing such as pyrosequencing or bisulfite sequencing, or immunoprecipitation using DNA methylation microarray, methylated CpG binding domain or anti-methylcytosine antibody, etc. can be used. However, the present invention is not limited thereto. Diagnosis of Alzheimer's disease mild cognitive impairment or Alzheimer's disease through the specific expression of hypomethylation in the CD96 gene in the patient's sample compared to a non-Alzheimer's human sample or compared to a predetermined cut-off The risk of progression to disease dementia can be predicted.

The human CD96 gene is located on chromosome 3 and is registered as Gene ID: 10225 in the NCBI Entrez database.

In the present invention, the CpG region of a gene refers to a CpG region present on the DNA of a gene. DNA of a gene is a concept that includes all a series of structural units that are necessary for gene expression and are operably linked to each other, for example, a promoter region, a protein coding region (open reading frame, ORF) and a terminator region. include Accordingly, the CpG region of a gene may exist in a promoter region, a protein coding region (open reading frame, ORF) or a terminator region of the gene. Preferably, the CpG region where disease-specific hypomethylation occurs in the CD96 gene may be present in the promoter of the gene.

For example, in the present invention, measuring the methylation level of the CpG region of the CD96 gene promoter is the CpG region of the CD96 gene promoter, more specifically, the CpG region appearing in the nucleotide sequences 111541849 to 111541970 of chromosome 3 (SEQ ID NO: 1). It may include measuring the methylation level of cytosine of More specifically, it may include measuring the methylation of cytosine located at the 111541909th base of chromosome 3 (the 61st base of SEQ ID NO: 1).

In the present invention, the nucleotide sequence of the human genome chromosomal region is expressed according to the latest version of the GRCh38 Genome Reference Consortium Human Reference 38 (GRCh38/hg38), but the specific sequence of the human genome chromosomal region is expressed as the genome sequence study results are updated. This may be slightly altered, and the expression of the human genome chromosomal region of the present invention may be different according to such alteration. Therefore, the human genome chromosomal region expressed according to the GRCh38 Genome Reference Consortium Human Reference 38 (GRCh38/hg38) of the present invention has been updated with the human reference sequence since the filing date of the present invention to express the human genome chromosomal region. Even if this is changed differently from now, it will be apparent that the scope of the present invention extends to the altered human genome chromosomal region. These changes can be easily recognized by anyone with ordinary skill in the art to which the present invention pertains.

As used herein, the term "Alzheimer's disease (AD)" is a representative disease of neurodegenerative brain disease, with memory loss as an initial symptom, and then overall cognitive function decline. In the past, Alzheimer's disease was defined based on clinical symptoms and confirmed through an autopsy of the brain. ), it became possible to define Alzheimer's disease by identifying it through biomarkers. Even now, although the diagnosis of Alzheimer's disease is a post-mortem autopsy of the brain, the pathological accumulation of amyloid protein and tau protein, which are known as representative features of autopsy findings in the brain, can be indirectly confirmed by PET image or cerebrospinal fluid test. Compared to the era when diagnosis was based only on modalities, the accuracy of diagnosis has dramatically improved.

Biomarkers for defining Alzheimer's disease include beta-amyloid-related markers (e.g., cortical amyloid-PET ligand binding or _{decreased Aβ 42} in cerebrospinal fluid), neurofibrillary tangible tau-related markers (e.g., For example, an increase in phosphorylated tau in the cerebrospinal fluid or binding of tau-PET ligand in the cerebral cortex), or an indicator of neurodegeneration (e.g., an increase in cerebrospinal fluid total tau or brain atrophy in MRI and decreased brain metabolism in FDG-PET) ) and the like.

The National Institute of Aging (NIA) and Alzheimer's Association (AA) updated the NIA-AA Alzheimer's disease diagnostic criteria published in 2011. , et al., NIA-AA Research Framework: toward a biological definition of Alzheimer's disease. If the above-described Alzheimer's disease biomarker) is observed, it is included in the diagnostic criteria for Alzheimer's disease. Specifically, Alzheimer's disease is largely subdivided into preclinical Alzheimer's disease (AD), prodromal Alzheimer's disease, and Symptomatic Alzheimer's disease through a combination of biomarkers and cognitive stages. Thus, it is explained as a concept of a continuum.

Alzheimer's disease Normal cognitive function shows pathological findings of Alzheimer's disease in biological marker tests, but corresponds to the preclinical stage without clinical symptoms. The next stage, Alzheimer's disease mild cognitive impairment, shows pathological findings of Alzheimer's disease in the biological marker test, but there is a mild objective cognitive decline and independent daily life performance is maintained. The term "AD with mild cognitive impairment" or "prodromal AD" is also used. Alzheimer's disease dementia shows the pathological findings of Alzheimer's disease in biomarker tests, develops dementia symptoms, and decreases objective cognitive function As a result, the ability to function independently is impaired.

Accordingly, in the present invention, the term "alzheimer's disease mild cognitive impairment" is premised on representing the pathological characteristics (biomarkers) of Alzheimer's disease, but there is objective cognitive decline, but the ability to perform daily life is preserved, so it is not dementia It refers to the stage before the onset of Alzheimer's disease dementia.

In contrast, the general "mild cognitive impairment (MCI)" includes Alzheimer's disease, as well as other neurodegenerative diseases or clinically determined cognitive decline due to various factors. Compared to 1~2% of the general elderly population progressing to dementia every year, it is known that 5-20% of the elderly with mild cognitive impairment progress to dementia per year, so mild cognitive impairment is considered a high-risk group for dementia. However, mild cognitive impairment does not progress to Alzheimer's disease, but to other dementia diseases such as frontal temporal dementia or vascular dementia. known to be maintained.

However, the "alzheimer's disease mild cognitive impairment", which is the subject of diagnosis herein, refers to mild cognitive impairment due to Alzheimer's disease, that is, mild cognitive impairment showing the pathological characteristics (biomarkers) of Alzheimer's disease, compared to general mild cognitive impairment. Alzheimer's disease is more likely to progress to dementia. According to a previous report, in the case of mild cognitive impairment not caused by Alzheimer's disease, the rate of progression to Alzheimer's disease dementia within 3 years was only about 5%, but in the case of Alzheimer's mild cognitive impairment according to the NIA-AA diagnostic criteria, Alzheimer's within 3 years The rate of progression to disease dementia was very high, about 59% (Brain 2015: 138; 1327-1338).

Therefore, the methylation marker of the present invention is specifically hypomethylated in the Alzheimer's disease mild cognitive impairment patient group compared to the normal control group, and the effectiveness of distinguishing the normal group from the patient group through ROC curve analysis was confirmed, so Alzheimer's disease mild cognitive impairment In addition to being able to diagnose, it can be used as a predictive diagnostic marker for Alzheimer's disease, which predicts the risk of progression from mild cognitive impairment to Alzheimer's disease in advance.

In addition, the compositions, kits and methods of the present invention predict the risk of Alzheimer's disease in advance, so that patients with a high probability of progressing to Alzheimer's disease can be selected and the exacerbation of the disease can be delayed through active preventive management and early treatment. In addition, if clinically used in the future, early detection can delay the onset of dementia and reduce the prevalence of dementia, ultimately improving the quality of life of patients and their families, and further reducing the socioeconomic cost of the state. can

In the present invention, "diagnosis" means to confirm the presence of a disease, the presence or characteristics of a disease pathology. In general, diagnosis includes determining whether a person has a specific disease, measuring the condition before symptoms appear, determining the prognosis of the disease, and further determining whether the disease has progressed or worsened. can do.

As used herein, the term "predicting the risk of progression to Alzheimer's disease dementia" means pre-assessing and predicting the risk of developing Alzheimer's disease dementia in the future or progressing to Alzheimer's disease dementia at a stage prior to the appearance of symptoms of Alzheimer's disease dementia.

In the present invention, the agent for measuring the methylation level of the CpG region is a compound that modifies an unmethylated cytosine base or a methylation-sensitive restriction enzyme, a primer specific for the methylated allele sequence of a gene, and a non-methylated allele sequence specific a primer, a methylated CpG binding domain, or a methylated DNA antibody that specifically binds to the methylated DNA (eg, an antibody that specifically binds to methylcytosine), and the like.

The compound that modifies the unmethylated cytosine base may be, but is not limited to, bisulfite or a salt thereof, and preferably sodium bisulfite. Bisulfite-modified DNA can detect methylation through various methods, such as sequencing or methylation-specific PCR. It is well known in the art (eg WO01/26536; US2003/0148326A1).

In addition, the methylation-sensitive restriction enzyme may be a restriction enzyme capable of specifically detecting methylation of a CpG site, and may be a restriction enzyme containing CG as a recognition site of the restriction enzyme. Examples include, but are not limited to, Sma I, Sac II, Eag I, Hpa II, Msp I, Bss HII, Bst UI, Not I, and the like. Depending on the methylation or unmethylation at C of the restriction enzyme recognition site, whether or not cleavage by the restriction enzyme is changed, which can be detected through PCR or Southern blot analysis. Methylation-sensitive restriction enzymes other than the above restriction enzymes are well known in the art.

As a representative example of measuring the level of methylation in a specific CpG region of an individual's gene, genomic DNA is obtained from a patient's sample, and the obtained DNA is treated with a compound that modifies unmethylated cytosine bases or a methylation-sensitive restriction enzyme. Thereafter, the processed DNA can be amplified by PCR using primers and measured by confirming the presence or absence of the amplified product.

Accordingly, the formulation of the present invention may include a primer specific for a methylated allele sequence of a gene and a primer specific for an unmethylated allele sequence. As used herein, the term "primer" refers to a short nucleic acid sequence that is capable of base pairing with a complementary template with a nucleic acid sequence having a short free three-terminal hydroxyl group and serves as a starting point for template strand copying. Primers are capable of initiating DNA synthesis in the presence of reagents for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in appropriate buffers and temperatures. In addition, the primers may incorporate additional features that do not change the basic properties of the primers, which are sense and antisense nucleic acids with a sequence of 7 to 50 nucleotides, which serve as the starting point of DNA synthesis.

The primers of the present invention can be preferably designed according to the sequence of a specific CpG site to be analyzed for methylation, and each methylated primer pair capable of specifically amplifying cytosine that has not been modified by bisulfite; and a primer pair capable of specifically amplifying a cytosine modified by bisulfite because it is not methylated.

On the other hand, a methylated DNA antibody refers to an antibody that specifically binds to a methylated base in DNA. Specifically, an antibody having a property of recognizing and binding to methylated cytosine in a DNA chain, such as an antibody against methylcytosine, is mentioned. Also, among commercially available methylated DNA antibodies, it may be an antibody capable of specifically recognizing and specifically binding to the methylated DNA described herein.

A methylated DNA antibody can be prepared by a conventional method using a methylated base, methylated DNA, or the like as an antigen. For example, in order to produce a methylcytosine antibody, 5-methylcytosine, 5-methylcytosine, or DNA containing 5-methylcytosine is used as an antigen to prepare an antibody, and then the antibody is prepared using methylcytosine in DNA. of specific binding can be selected as an indicator.

In addition, when the methylation level is measured using a methylated CpG binding domain (MBD) or methylated DNA antibody, methylated DNA is immunoprecipitated using these, and then Southern blot, PCR, microarray, or sequence A specific CpG site may be identified through sequencing or the like. In addition, for immunological detection or quantification of the antibody, a substrate, an appropriate buffer solution, a chromogenic enzyme or fluorescent substance label, a secondary antibody labeled with a chromogenic enzyme or fluorescent substance, and a chromogenic substrate may be used. In the above, as the substrate, a nitrocellulose membrane, a 96-well plate synthesized from polyvinyl resin, a 96-well plate synthesized from polystyrene resin, and a glass slide glass may be used, and the chromogenic enzyme is peroxidase, alkaline phosphorus Alkaline phosphatase, etc. may be used, the fluorescent material may be FITC, RITC, etc., and the color developing substrate solution may be ABTS (2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfur). phonic acid)) or OPD (o-phenylenediamine), TMB (tetramethyl benzidine) may be used, and other radioactive isotope labels, latex bead labels, colloidal labels, biotin labels, etc. may be used, but are limited thereto no.

The composition and kit may further include a polymerase agarose, a buffer solution required for electrophoresis, and the like, in addition to the preparation. In addition, the kit may be implemented in the form of a DNA methylation microarray.

In another embodiment, the present invention relates to a method for diagnosing Alzheimer's disease mild cognitive impairment or predicting the risk of progression to Alzheimer's disease dementia by measuring the level of methylation in the CpG region of the CD96 gene promoter.

In addition, the present invention relates to a method for detecting the methylation level of the CpG region of a gene promoter from a sample of an individual in order to provide information necessary for the diagnosis of Alzheimer's disease mild cognitive impairment or the prediction of the risk of progression to Alzheimer's disease dementia.

In addition, the present invention relates to a method of providing information for diagnosing Alzheimer's disease mild cognitive impairment or predicting the risk of progression to Alzheimer's disease dementia, comprising measuring the methylation level in the CpG region of the CD96 gene promoter from a sample of an individual will be.

In one embodiment for this, the present invention comprises the steps of measuring the methylation level of the CpG region of the CD96 gene promoter from a sample of an individual; and comparing the methylation level with the methylation level of the corresponding gene of a non-Alzheimer's disease control sample or with a predetermined threshold (cut-off), the diagnosis of Alzheimer's disease mild cognitive impairment or Alzheimer's disease dementia It relates to a method of providing information for predicting progression risk.

In the present invention, the term "sample" includes samples such as cells, tissues, whole blood, serum, plasma, cerebrospinal fluid, saliva, sputum or urine with different methylation levels of genes due to Alzheimer's disease. Any sample that can be extracted can be used. Preferred embodiments include, but are not limited to, skin tissue or skin cells, for example, skin fibroblasts, and the like.

First, in order to detect methylation level by obtaining genomic DNA from a sample of an individual, phenol/chloroform extraction method, SDS extraction method commonly used in the art (Tai et al., Plant Mol. Biol. Reporter, 8: 297-303, 1990), CTAB separation (Cetyl Trimethyl Ammonium Bromide; Murray et al., Nuc. Res., 4321-4325, 1980), or a commercially available DNA extraction kit.

The step of detecting the methylation level of a specific CpG region of the gene, a method using a restriction enzyme or bisulfite, using a difference between a methylated base and an unmethylated base, a methylated CpG binding domain or an anti-methylcytosine antibody Immunoprecipitation method (eg, MIRA, MeDIP) using a DNA methylation microarray, etc. can be performed. For example, methylation-specific polymerase chain reaction, real time methylation-specific polymerase chain reaction, PCR using methylated DNA-specific binding protein, quantitative PCR, The methylation level may be measured or detected using pyrosequencing, bisulfite sequencing, DNA methylation microarray, or immunoprecipitation using a methylated CpG binding domain or an anti-methylcytosine antibody.

As an example, the method of the present application comprises the steps of (a) treating the genomic DNA in the obtained sample with a compound or a methylation-sensitive restriction enzyme that modifies the unmethylated cytosine base; and (b) amplifying the processed DNA by PCR using a primer capable of amplifying a specific CpG region of the gene.

The compound that modifies the unmethylated cytosine base in step (a) may be bisulfite, preferably sodium bisulfite. A method for detecting whether a gene is methylated by modifying an unmethylated cytosine residue using such a bisulfite is well known in the art.

In addition, as described above, the methylation-sensitive restriction enzyme in step (a) is a restriction enzyme capable of specifically detecting methylation of a specific CpG site, and may be a restriction enzyme containing CG as a recognition site of the restriction enzyme. , For example, Sma I, Sac II, Eag I, Hpa II, Msp I, Bss HII, Bst UI, Not I, and the like, but are not limited thereto.

The amplification in step (b) may be performed by a conventional PCR method. As described above, the primer used at this time can be preferably designed according to the sequence of a specific CpG site to be analyzed for methylation, and specifically amplifies cytosine that has been methylated and has not been modified by bisulfite. and a primer pair capable of specifically amplifying a cytosine modified by bisulfite because it is not methylated.

The step of detecting the methylation level of the specific CpG region of the gene may further include (c) confirming the presence or absence of the amplified product in step (b). The presence or absence of the amplified product in step (c) may be performed according to a method known in the art, for example, by performing electrophoresis to detect a band at a desired position. For example, when a compound that modifies an unmethylated cytosine residue is used, two types of primer pairs used in step (a), that is, a primer capable of specifically amplifying methylated cytosine that has not been modified by bisulfite The degree of methylation can be determined according to the presence or absence of a PCR product amplified by a pair and a primer pair capable of specifically amplifying a cytosine modified by bisulfite because it is not methylated. Preferably, methylation can be determined using a bisulfite genome sequencing method in which the sample genomic DNA is treated with bisulfite, the CpG region of the corresponding gene is amplified by PCR, and the base sequence of the amplified region is analyzed. .

In addition, even when a restriction enzyme is used, it is determined that CpG is methylated by a method known in the art, for example, if there is a PCR product in the DNA treated with a restriction enzyme in the state where the PCR product is shown in mock DNA, If there is no PCR product in the DNA treated with the enzyme, whether CpG is methylated can be determined according to the determination that it is unmethylated, which is apparent to those skilled in the art. In the above, mock DNA refers to sample DNA separated from the sample and untreated.

As another example of detecting the methylation level of a specific CpG region of a gene, an immunoprecipitation method using a methylated DNA antibody, for example, an antibody against a methylated CpG binding domain (MBD) or methylcytosine can be exemplified. . For example, after immunoprecipitation using an antibody that specifically recognizes a methylated CpG binding domain or 5-methylcytosine, a specific CpG site can be identified through Southern blot, PCR, microarray, or sequencing. can

According to this method, when the CpG region of the CD96 gene promoter in the target sample is detected in a hypomethylated state, the risk of diagnosing Alzheimer's disease mild cognitive impairment or progressing to Alzheimer's disease dementia can be predicted in advance.

Therefore, according to the present invention, since the change in methylation of a specific CpG region of a gene is specifically shown in a sample of an individual, the change in methylation of a gene as a biomarker can be usefully used for diagnosis or prediction of Alzheimer's disease. . For example, by using the hypomethylation of a specific CpG region of the CD96 gene as a biomarker, it can be usefully used for diagnosing Alzheimer's disease mild cognitive impairment or predicting the risk of progression to Alzheimer's disease dementia.

The present invention provides a molecular biological diagnostic method for diagnosing or predicting Alzheimer's disease mild cognitive impairment or Alzheimer's disease dementia by measuring the degree of methylation of a specific gene CpG region of genomic DNA collected from a patient's sample, which is simple, non-invasive and economical There are advantages to being In addition, DNA methylation changes are easy to detect, stable and easy to analyze compared to conventional protein or RNA markers.

1 shows the difference in the degree of DNA methylation of the CD96 gene in the normal control group and the Alzheimer's disease mild cognitive impairment group.
2 is a receiver operating characteristics curve (ROC curve: receiver operating characteristics curve) analysis results for evaluating the effectiveness of distinguishing the Alzheimer's disease mild cognitive impairment patient group and the normal control group using the change in DNA methylation of the CD96 gene. .

Hereinafter, the present invention will be described in detail by way of Examples. However, the following examples are merely illustrative of the present invention, and the present invention is not limited by the following examples.

Example 1. Study subject

The Alzheimer's disease patient group includes three groups: Alzheimer's disease preclinical Alzheimer's disease (AD), Alzheimer's disease mild cognitive impairment (Alzheimer's disease), and Symptomatic Alzheimer's disease. Controls include a normal control group (a group with normal cognitive function and no amyloid pathology), mild cognitive impairment due to non-AD, and dementia not caused by Alzheimer's disease (dementia). It is composed of three groups: due to non-AD).

Diagnosis and classification of study subjects in group 6 were performed according to the following four steps:

As a first step, patients who visited Ewha Womans University Mokdong Hospital from 2018 to 2019, along with the study subjects and their guardians, listened to their medical history through a structured interview with a neurologist.

In the second step, the degree of current cognitive decline and daily living ability were judged through a neuropsychological test. Through these two stages, three stages of cognitive decline, ie, normal cognitive function, mild cognitive impairment, and dementia, were determined. In the case of the normal cognitive function group, apart from complaints of subjective cognitive decline, all items of the objective neuropsychological test showed test results higher than the standard score (z score ≥-1.5), and there were no objective findings of cognitive decline and daily living ability. is undamaged. In the case of mild cognitive impairment, the patient or information provider complains of memory loss, and the neuropsychological test shows a result of 1.5 or less (z score <-1.5) in the standard score, and there is a decrease in objective memory, but overall cognitive function and It is a group in which daily living ability is maintained. The dementia group shows objective deterioration (standard score of 1.5 or less) in two or more cognitive domains, and is a group that requires the care of others due to impairment of daily living ability, along with a slowly progressing clinical picture.

As a third step, blood tests, structural imaging of the brain (computed tomography, CT or magnetic resonance imaging, MRI), and functional imaging of the brain (functional MRI, amyloid positron emission tomography, amyloid PET) are performed to reduce cognitive decline. Other possible causes were excluded, and the accumulation of amyloid pathological findings in the brain was checked. Excluding other causes that may cause cognitive decline is an important step in the diagnosis of Alzheimer's disease. In addition, alcoholism, drug addiction, major depressive disorder, bipolar disorder, schizophrenia, and convulsive disease were excluded because they may be associated with cognitive decline. In addition, on brain imaging, structural brain abnormalities such as normostatic hydrocephalus, stroke, brain tumors, etc. were excluded, and other neurodegenerative diseases such as Parkinson's disease, Lewy body dementia, and vascular dementia were also excluded. According to the 2018 Alzheimer's disease research diagnostic criteria, positive amyloid PET (Brain amyloid Plaque load, BAPL 2 or 3) was judged to have Alzheimer's disease, and one of preclinical, prodromal, and symptomatic AD was determined according to clinical symptoms. group was judged. Negative amyloid PET (BAPL 1) is judged as a control group, and according to clinical symptoms, normal control group (group with normal cognitive function and no amyloid pathology), mild cognitive impairment due to not Alzheimer's disease non-AD), it was decided as one of the dementia due to non-AD groups. Fourth, by synthesizing the clinical information from these three stages, a final judgment was made into six groups.

Example 2. Skin biopsy collection and skin fibroblast culture

A skin biopsy was collected from the inside of the patient's thigh using a cylindrical blade with a diameter of 2 mm. Divide the collected skin biopsy into 6 equal parts and place medium (DMEM/20% FBS) sufficient to submerge the skin biopsy pieces in a 24-well cell culture plate coated with 0.1% gelatin, and wait for 7 days to prevent the cells from protruding from the edges of the skin biopsy pieces. observed. Medium was replenished every 2-3 days to prevent the skin biopsy pieces from drying out. After 7 days, the amount of medium was increased to 500 μL, and the medium was changed every 2-3 days. After 14 days, when the cells grew around the skin biopsy piece and filled to the outside of the culture well, it was transferred to a 35 mm culture dish and the right side of the medium was removed. After lowering the concentration of fetal serum (FBS) from 20% to 10%, cell culture was continued. When the cells were filled to about 80-90% of the 35 mm culture dish, subculture was performed. When subculture was repeated 2-3 times and only skin fibroblasts were selectively cultured, the expression of SERPHINH1, a fibroblast marker, was confirmed using the SERPHINH1 antibody.

Example 3. Genomic DNA Extraction

Genomic DNA from the skin fibroblasts cultured in Example 2 was extracted using the QIAmp DNA mini kit (Qiagen). The extraction method was performed according to the manufacturer's manual. The extracted genomic DNA was quantified using a spectrophotometer, and the DNA state was checked for degradation by electrophoresis on a 1% agarose gel.

Example 4. DNA methylation mutation analysis

DNA methylation mutation analysis is performed by extracting DNA from the skin fibroblasts of the patient group and control group, converting unmethylated cytosine to uracil by performing bisulfite conversion, and using Infinium MethylationEPIC bead chip (illumina) The degree of methylation was measured for 850,000 CpG sites. The degree of DNA methylation is expressed as a β value with a value of 0 to 1. A β value of 0 means that the corresponding CpG site is completely unmethylated, and 1 means that the corresponding CpG site is completely methylated.

An independent t-test method was used to identify differentially methylated genes (DMGs) in the patient group and the control group. Finally, a promoter CpG site with a p value <0.05 and an absolute value β difference ≥ 0.3 was selected as a differentially methylated CpG site, and among them, a gene with a changed degree of methylation of the CpG site was selected as DMG. .

Experiment result

1. Confirmation of disease-specific DNA methylation changes in Alzheimer's disease mild cognitive impairment patient group

As a result of analyzing DNA methylation changes in 4 normal controls (normal cognitive function group without amyloid pathology) and 17 patients with mild cognitive impairment with Alzheimer's disease, the DNA methylation level was differentially changed in 12 patients with Alzheimer's disease mild cognitive impairment. DMGs of the species were selected, and the results of CD96 among them are described in Table 1 below.

gene Target ID β value
(difference) ^One) methylation status p value CD96 cg05655806 -0.308234 hypomethylation 2.75E-05 1) The value obtained by subtracting the β value of the normal control group from the β value of the cells of the patient group

The difference between the DNA methylation degree of the selected gene in the normal control group and the Alzheimer's disease mild cognitive impairment patient group is shown as a scatter dot plot in FIG. 1, and the mean ± standard error (mean ± SEM) values are indicated.

2. DNA Selection of predictive diagnostic markers for Alzheimer's disease dementia using methylation changes

For 12 types of DMG with differentially changed DNA methylation levels in the Alzheimer's disease mild cognitive impairment patient group, whether the patient group and the normal control group can be distinguished using the receiver operating characteristics curve (ROC curve) Confirmed. In general, non-informative (AUC=0.5), less accurate (0.5<AUC≤0.7), moderately accurate (0.7<AUC≤0.9), and very Classified as accurate (0.9<AUC<1.0) and complete test (AUC=1.0).

In the case of CD96, it was confirmed that it was hypomethylated in the Alzheimer's disease mild cognitive impairment patient group, and the degree of hypomethylation could distinguish the Alzheimer's mild cognitive impairment patient group with high accuracy (AUC = 0.9706) compared to the normal control group (FIG. 2). These results show that the disease-specific hypomethylation of the CD96 gene in the skin fibroblasts of patients with mild cognitive impairment with Alzheimer's disease can be diagnosed with Alzheimer's disease mild cognitive impairment by a non-invasive method, or Alzheimer's disease dementia among patients with Alzheimer's disease mild cognitive impairment. It indicates that it is effective as a predictive diagnostic marker for Alzheimer's disease and dementia, which predicts the progression of the disease in advance.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be implemented in other specific forms without changing the technical spirit or essential characteristics thereof. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the claims described below and their equivalents.

<110> Ewha University - Industry Collaboration Foundation <120> Marker for diagnosing prodromal Alzheimer's disease using alteration of DNA methylation of gene <130> DPP20200591KR <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 122 <212> DNA <213> Homo sapiens <220> <221> promoter <222> (1)..(122) <223> ch3: 111541849-111541970 <400> 1 tgttaaatga ctttgtgctc ttgaaaaaaa tgtataaaaa gcacattgcc aactgctctg 60 cgtgatatcc tttgtttgga ttttagacgc agcttgactg acacagagat cctgaataag 120 ag 122

Claims (10)

A composition for diagnosing Alzheimer's disease mild cognitive impairment or predicting the risk of progression to Alzheimer's disease dementia, comprising an agent for measuring the methylation level of the CpG region of the CD96 (CD96 molecule) gene promoter,
CpG of the CD96 gene promoter is SEQ ID NO: 1 (111541849 to 111541970 of chromosome 3) will include CpG appearing in the nucleotide sequence, the composition. According to claim 1, wherein the agent for measuring the methylation level of the CpG region of the gene promoter, bisulfite (bisulfite) or a salt thereof, or a methylation sensitive restriction enzyme, specific for the methylated sequence of the CpG region of the gene promoter A composition comprising a primer, a primer specific for an unmethylated sequence, a methylated CpG binding domain, or an antibody that specifically binds to methylcytosine. The composition of claim 2, wherein the methylation sensitive restriction enzyme is Sma I, Sac II, Eag I, Hpa II, Msp I, Bss HII, Bst UI or Not I. A kit for diagnosing Alzheimer's disease mild cognitive impairment or predicting the risk of progression to Alzheimer's disease dementia, comprising the composition of any one of claims 1 to 3. A method of providing information for diagnosing Alzheimer's disease mild cognitive impairment or predicting the risk of progression to Alzheimer's disease dementia, comprising measuring the methylation level of the CpG region of the CD96 gene promoter from a sample of an individual,
CpG of the CD96 gene promoter is to include CpG appearing in the nucleotide sequence of SEQ ID NO: 1 (111541849 to 111541970 of chromosome 3). 6. The method of claim 5, further comprising comparing the level of methylation with the level of methylation in the gene of interest in a control sample that is not Alzheimer's disease. According to claim 5, wherein the step of measuring the methylation level of the CpG region of the gene promoter
(a) treating the genomic DNA in the obtained sample with bisulfite or a salt thereof, or a methylation-sensitive restriction enzyme; and
(b) amplifying the processed DNA by PCR using a primer capable of amplifying the CpG region of the CD96 gene promoter. The method of claim 5, wherein the method for detecting the methylation level is a methylation-specific polymerase chain reaction, a real time methylation-specific polymerase chain reaction, and a methylation DNA-specific reaction. selected from the group consisting of PCR, quantitative PCR, pyrosequencing, bisulfite sequencing, DNA methylation microarray, and immunoprecipitation using a methylated CpG binding domain or anti-methylcytosine antibody using an antigen binding protein how to be. delete delete

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