KR102735656B1 - Predicting biomarker for progression of Alzheimer’s dementia using alteration in DNA methylation of KRT32 gene - Google Patents

Abstract

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

Description

{Predicting biomarker for progression of Alzheimer’s dementia using alteration in DNA methylation of KRT32 gene}

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

Due to Korea's rapid economic growth, Westernization of lifestyles, and advancements in medicine, the average life expectancy of Koreans has increased, while Korea is also experiencing the fastest aging population in the world. As a result, neurodegenerative diseases that occur in conjunction with aging are also increasing rapidly.

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, progresses gradually over a long period of time with symptoms such as loss of time and space recognition ability, delusions and personality changes, language function paralysis, and motor function paralysis, and eventually leads to death.

Although research on treating Alzheimer's disease has been continuously conducted for the past several decades, there is currently no fundamental treatment, and only treatments that alleviate symptoms or slow the progression of lesions are being performed. Therefore, the most effective management method for Alzheimer's disease dementia is to delay the onset of dementia through early diagnosis and preemptive treatment.

However, the neurocognitive tests currently used to diagnose Alzheimer's disease have the disadvantage that the accuracy of the test results is greatly affected by the patient's age, education level, or intentional refusal to answer, and brain imaging tests using MRI or PET have the disadvantage that they are difficult to perform in the early stages when no particular symptoms appear because they use expensive equipment.

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

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

International Publication Patent WO 1992-017152 (published on October 15, 1992) US Patent Registration US 4,666,829 (registered on May 19, 1987) International Publication Patent No. 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 KRT32 (Keratin 32) gene is specifically hypermethylated in mild cognitive impairment of Alzheimer's disease, and confirmed that the risk of progression to Alzheimer's disease dementia can be predicted by measuring the degree of methylation of the KRT32 gene using this as a biomarker, thereby completing the present invention.

Accordingly, an example of the present invention provides a composition for predicting the risk of progression to Alzheimer's disease dementia, comprising a preparation for measuring the methylation level of a CpG site of a KRT32 gene promoter.

Another example provides a kit for predicting the risk of progression to Alzheimer's disease dementia comprising the composition.

Another example provides a method for predicting the risk of progression to Alzheimer's disease dementia by measuring the methylation level at the CpG site of the KRT32 gene promoter.

Another example provides a method for providing information for predicting the risk of progression to Alzheimer's disease dementia, comprising the step of measuring the methylation level of a CpG site of a KRT32 gene promoter from a sample of an individual.

Another example provides a method for measuring the level of methylation at the CpG site of the KRT32 gene promoter from a sample of an individual to provide information necessary for predicting the risk of progression to Alzheimer's disease dementia.

The present invention is based on the discovery that the KRT32 gene is specifically hypermethylated in mild cognitive impairment of Alzheimer's disease, and provides a molecular biological diagnostic technology for predicting the risk of progression to Alzheimer's disease dementia by measuring the degree of specific DNA hypermethylation occurring at a specific CpG position of the KRT32 gene.

DNA methylation changes have the advantage of being easy to detect because they exist in DNA, being more stable than protein or RNA markers, and being easy to analyze because they occur at a specific location in a gene.

Specifically, in one embodiment of the present invention, genomic DNA was extracted from samples of a group of patients with mild cognitive impairment without amyloid pathology and a group of patients with mild cognitive impairment of Alzheimer's disease, and DNA methylation profiles were analyzed through DNA methylation mutation analysis. In comparison with the group of patients with mild cognitive impairment without amyloid pathology, genes in which DNA methylation was changed by 20% or more in the CpG region of the gene promoter region of the group of patients with mild cognitive impairment of Alzheimer's disease were selected. Among these selected genes, we confirmed that the DNA methylation of the CpG region of the promoter of the KRT32 gene was increased by approximately 21% in the Alzheimer's disease mild cognitive impairment patient group compared to the mild cognitive impairment patient group without amyloid pathology, and through receiver operating characteristics curve (ROC curve) analysis, we confirmed that the hypermethylation of the CpG region of the KRT32 gene promoter can distinguish the Alzheimer's disease mild cognitive impairment patient group from the mild cognitive impairment patient group without amyloid pathology with high accuracy (AUC = 0.8).

These results suggest that disease-specific hypermethylation of the KRT32 gene may serve as a biomarker for noninvasively predicting the risk of progression to Alzheimer's disease dementia.

Accordingly, one specific example provides a composition for predicting the risk of progression to Alzheimer's disease dementia, comprising a formulation that measures the methylation level of a CpG site of the KRT32 gene promoter.

Another specific example provides a kit for predicting the risk of progression to Alzheimer's disease dementia comprising the composition.

Another specific example provides a method for predicting the risk of progression to Alzheimer's disease dementia by measuring the methylation level at a CpG site of the KRT32 gene promoter from a sample of an individual.

Another specific example provides a method for providing information for predicting the risk of progression to Alzheimer's disease dementia, comprising the step of measuring the methylation level at a CpG site of a KRT32 gene promoter from a sample of an individual.

Another specific example provides a method of measuring the methylation level at a CpG site of a KRT32 gene promoter from a sample of an individual to provide information necessary for predicting the risk of progression to Alzheimer's disease dementia.

In the present invention, the term "methylation" or "methylation" refers to the attachment of a methyl group to a base constituting DNA. Preferably, in the present invention, methylation refers to methylation occurring at a cytosine of a CpG site of a specific gene promoter. If methylation occurs, binding of a transcription factor is hindered, thereby inhibiting the expression of a specific gene, and conversely, if unmethylation or hypomethylation occurs, the expression of a specific gene increases.

In addition to A, C, G, and T, the genomic DNA of mammalian cells contains a fifth base called 5-methylcytosine (5-mC), which has a methyl group attached to the fifth carbon of the cytosine ring. Methylation of 5-methylcytosine occurs only at the C of the CG dinucleotide (5'-mCG-3') called CpG, and methylation of CpG suppresses the expression of alu or transposons and repetitive sequences in the genome. In addition, since the 5-mC of CpG is easily deaminated naturally to become thymine (T), CpG is the site where most epigenetic changes frequently occur in mammalian cells.

In the present invention, the term "measurement of methylation level" refers to measuring the methylation level of a CpG region of a gene, which can be measured by methylation-specific PCR, for example, methylation-specific PCR (methylation-specific polymerase chain reaction, MSP), real time methylation-specific PCR, PCR using methylated DNA-specific binding protein, or quantitative PCR. Alternatively, it can be measured by a method such as automated base analysis such as pyrosequencing or bisulfite sequencing, or by using a DNA methylation microarray, or an immunoprecipitation method using a methylated CpG binding domain or an anti-methylcytosine antibody, but is not limited thereto. The risk of progression to Alzheimer's disease dementia can be predicted by specifically showing hypermethylation in the KRT32 gene in a patient's sample compared to a sample from a person without Alzheimer's disease or compared to a predetermined threshold (cut-off).

The human KRT32 gene is located on chromosome 17 and is registered in the NCBI Entrez database as Gene ID: 3882.

In the present invention, the CpG site of a gene refers to a CpG site existing on the DNA of the gene. The DNA of the gene is a concept that includes a series of structural units that are necessary for the expression of the gene and are operably linked to each other, for example, a promoter region, a protein coding region (open reading frame, ORF), and a terminator region. Therefore, the CpG site of the gene may exist in the promoter region, the protein coding region (open reading frame, ORF), or the terminator region of the gene. Preferably, the CpG site where disease-specific hypermethylation occurs in the KRT32 gene may exist in the promoter of the gene.

For example, in the present invention, measuring the methylation level of the CpG site of the KRT32 gene promoter may include measuring the methylation level of cytosine in the CpG site of the promoter of the KRT32 gene, more specifically, in the CpG site appearing in the nucleotide sequence at positions 41467764 to 41467885 of chromosome 17 (SEQ ID NO: 1):

TCCCCATGGGAAGAGGCCTGGCTATTTAACAAGGAAATGTGTGGGTGGTAGAAACTGCTC C GATGCTGGCAGCGGCATAAATCCAGCAGAAAGGAACAGGAACCTCAGAGGCTTCCTAACAG (SEQ ID NO: 1)

More specifically, it may include measuring methylation of cytosine located at base 41467824 of chromosome 17 (base 61 of SEQ ID NO: 1).

In the present invention, the base sequence of the human genome chromosome region is expressed according to the latest version, GRCh38 Genome Reference Consortium Human Reference 38 (GRCh38/hg38). However, the specific sequence of the human genome chromosome region may be somewhat changed in expression as the genome sequence research results are updated, and the expression of the human genome chromosome region of the present invention may be different according to such change. Therefore, even if the human reference sequence is updated after the filing date of the present invention and the expression of the human genome chromosome region is changed differently from the present expression, it will be obvious that the scope of the present invention extends to the changed human genome chromosome region. Anyone having ordinary skill in the art to which the present invention pertains can easily understand such change.

In the present invention, the term "Alzheimer's disease (AD)" is a representative neurodegenerative brain disease that starts with memory loss as an initial symptom and then shows overall cognitive function decline. In the past, Alzheimer's disease was defined based on clinical symptoms and confirmed through brain autopsy, but recently, as neuropathological changes related to Alzheimer's disease have been discovered and can be utilized as clinical tests, Alzheimer's disease can be defined by confirming these changes through in vivo biomarkers. Even now, Alzheimer's disease is confirmed through postmortem brain autopsy findings, but pathological accumulation of amyloid protein and tau protein, which are known as representative features of brain autopsy findings, can be indirectly confirmed through PET imaging or cerebrospinal fluid examination, and the accuracy of diagnosis has been dramatically improved compared to the era when diagnosis was made based solely on clinical features.

Biomarkers for defining Alzheimer's disease include beta-amyloid-related markers (e.g., decreased amyloid-PET ligand binding in the brain cortex or Aβ ₄₂ in the cerebrospinal fluid), tau-related markers in the form of neurofibrillary tangles (e.g., increased phosphorylated tau in the cerebrospinal fluid or tau-PET ligand binding in the brain cortex), or markers of neurodegeneration (e.g., increased total tau in the cerebrospinal fluid or brain atrophy on MRI and brain hypometabolism on FDG-PET).

According to the 2018 Alzheimer's disease research criteria (Jack CR, et al., NIA-AA Research Framework: toward a biological definition of Alzheimer’s disease. Alzheimers Dement 2018;14:535-562), which are an update of the NIA-AA diagnostic criteria for Alzheimer’s disease announced in 2011 by the National Institute on Aging (NIA) and the Alzheimer Association (AA), Alzheimer’s disease is included in the diagnostic criteria for Alzheimer’s disease if the pathological characteristics of Alzheimer’s disease (Alzheimer’s disease biomarkers as described above) are observed even before the appearance of clinical symptoms of dementia. Specifically, Alzheimer’s disease is broadly categorized into preclinical Alzheimer’s disease, prodromal Alzheimer’s disease, and symptomatic Alzheimer’s disease through a combination of biomarkers and cognitive stages, and explained as a continuum.

Alzheimer's disease normal cognitive function corresponds to the preclinical stage where biological marker tests show pathological findings of Alzheimer's disease but there are no clinical symptoms. The next stage, Alzheimer's disease mild cognitive impairment, is the stage where biological marker tests show pathological findings of Alzheimer's disease but there is mild objective cognitive decline and the ability to perform independent daily living is maintained. The terms "AD with mild cognitive impairment" or "prodromal AD" are also used. Alzheimer's disease dementia shows pathological findings of Alzheimer's disease in biological marker tests, dementia symptoms appear, and independent daily living ability is impaired due to objective decline in cognitive function. The terms "AD with dementia" or "Symptomatic AD" are also used.

Accordingly, in the present invention, the term "Alzheimer's disease mild cognitive impairment" refers to a state before the onset of Alzheimer's disease dementia, in which objective cognitive decline is present but the ability to perform daily living activities is preserved, thus not dementia, while presupposing the pathological characteristics (biomarkers) of Alzheimer's disease.

In contrast, the general "mild cognitive impairment (MCI)" includes cases in which cognitive ability is clinically judged to be declining due to not only Alzheimer's disease but also other neurodegenerative diseases or various factors. While 1-2% of the general elderly population progresses to dementia every year, 5-20% of the elderly with mild cognitive impairment are known to progress to dementia per year, so mild cognitive impairment is considered a high-risk group for dementia. However, mild cognitive impairment may progress to other dementia diseases such as frontotemporal dementia or vascular dementia rather than Alzheimer's disease dementia, and 20-30% are known to recover to normal or not progress to dementia but remain in a similar state of mild cognitive impairment.

However, in this hospital, "Alzheimer's disease mild cognitive impairment" refers to mild cognitive impairment caused by Alzheimer's disease, that is, mild cognitive impairment that exhibits the pathological characteristics (biomarkers) of Alzheimer's disease, and is much more likely to progress to Alzheimer's disease dementia than general mild cognitive impairment. According to previous reports, the rate of progression to Alzheimer's disease dementia within 3 years was only about 5% in the case of mild cognitive impairment not caused by Alzheimer's disease, but in the case of Alzheimer's disease mild cognitive impairment according to the NIA-AA diagnostic criteria, the rate of progression to Alzheimer's disease dementia within 3 years was about 59%, which was much higher (Brain 2015: 138; 1327-1338).

Accordingly, the methylation marker of the present invention is specifically hypermethylated in the Alzheimer's disease mild cognitive impairment patient group compared to the mild cognitive impairment patient group without amyloid pathology findings, and the validity of distinguishing between the mild cognitive impairment patient group without amyloid pathology findings and the Alzheimer's disease mild cognitive impairment patient group was confirmed through ROC curve analysis. Therefore, it can be utilized as a diagnostic marker for predicting Alzheimer's disease dementia that predicts the risk of progression from Alzheimer's disease mild cognitive impairment to Alzheimer's disease dementia in advance.

In addition, the composition, kit, and method of the present invention can predict the risk of Alzheimer's dementia in advance, thereby selecting patients with a high possibility of progressing to Alzheimer's disease dementia and delaying the worsening of the disease through active preventive management and early treatment. In addition, if clinically utilized in the future, it can delay the onset of dementia and reduce the prevalence due to early detection, ultimately improving the quality of life of patients and their families and significantly reducing the socioeconomic costs borne by the country.

In the present invention, "diagnosis" means confirming the existence of a disease, the existence or characteristics of a disease pathological condition. In general, diagnosis may include confirming whether a person currently has a specific disease, measuring a condition before symptoms appear, determining the prognosis of a disease, and further confirming whether a disease is progressing or worsening.

In the present invention, the term "prediction of risk of progression to Alzheimer's disease dementia" means assessing and predicting in advance the risk of developing or progressing to Alzheimer's disease dementia in the future at a stage before symptoms of Alzheimer's disease dementia appear.

In the present invention, the agent for measuring the methylation level of a CpG site may include a compound that modifies an unmethylated cytosine base, a methylation-sensitive restriction enzyme, a primer specific for a methylated allele sequence of a gene, a primer specific for an unmethylated allele sequence, a methylated CpG binding domain, or a methylated DNA antibody that specifically binds to methylated DNA (e.g., an antibody that specifically binds to methylcytosine).

The compound modifying the above unmethylated cytosine base may be, but is not limited to, bisulfite or a salt thereof, and preferably may be sodium bisulfite. Bisulfite-modified DNA can be methylated by various methods such as sequence analysis or methylation-specific PCR, and methods for modifying unmethylated cytosine residues using such bisulfite to detect whether a gene is methylated are widely known in the art (e.g., WO01/26536; US2003/0148326A1).

In addition, the methylation-sensitive restriction enzyme may be a restriction enzyme that can specifically detect methylation of a CpG site and contains CG as a recognition site of the restriction enzyme. Examples thereof include, but are not limited to, Sma I, Sac II, Eag I, Hpa II, Msp I, Bss HII, Bst UI, Not I, etc. Depending on methylation or unmethylation at C of the restriction enzyme recognition site, whether or not the restriction enzyme cuts is different, and this can be detected through PCR or Southern Blot analysis. Other methylation-sensitive restriction enzymes other than the above restriction enzymes are well known in the art.

As a representative example of measuring the methylation level at a specific CpG site of an individual's gene, genomic DNA is obtained from a patient's sample, the obtained DNA is treated with a compound that modifies unmethylated cytosine bases or a methylation-sensitive restriction enzyme, the treated DNA is amplified by PCR using primers, and the presence or absence of the amplified result is confirmed.

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" means a short nucleic acid sequence having a short free 3-terminal hydroxyl group, which can form base pairs with a complementary template and serves as a starting point for copying the template strand. The primer can initiate DNA synthesis in the presence of a reagent for polymerization reaction (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in an appropriate buffer and temperature. In addition, the primer may incorporate additional features that do not change the basic property of the primer to act as a starting point for DNA synthesis as a sense and antisense nucleic acid having a sequence of 7 to 50 nucleotides.

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 can be a primer pair that can specifically amplify a cytosine that is methylated and not modified by bisulfite, and a primer pair that can specifically amplify a cytosine that is not methylated and modified by bisulfite.

Meanwhile, 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 a methylated cytosine in a DNA chain, such as an antibody for methyl cytosine, may be mentioned. In addition, among the methylated DNA antibodies on the market, an antibody that can specifically recognize and specifically bind to DNA in a methylated state as described herein may be mentioned.

Methylated DNA antibodies can be produced using methylated bases, methylated DNA, etc. as antigens by conventional methods. For example, to produce a methylcytosine antibody, an antibody is produced using 5-methylcytidine, 5-methylcytosine, or DNA containing 5-methylcytosine as antigens, and then the antibody can be selected using specific binding to methylcytosine in the DNA as an indicator.

In addition, when measuring the methylation level using a methylated CpG binding domain (MBD) or methylated DNA antibody, the methylated DNA can be immunoprecipitated using these, and then a specific CpG site can be identified through Southern blot, PCR, microarray, or sequencing. In addition, 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 can be used for immunological detection or quantification of the antibody. In the above, the substrate may be a nitrocellulose membrane, a 96-well plate synthesized with polyvinyl resin, a 96-well plate synthesized with polystyrene resin, a glass slide glass, etc., the chromogenic enzyme may be peroxidase, alkaline phosphatase, etc., the fluorescent substance may be FITC, RITC, etc., the chromogenic substrate solution may be ABTS (2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)) or OPD (o-phenylenediamine), TMB (tetramethyl benzidine), and other radioisotope labels, latex bead labels, colloid labels, biotin labels, etc., but are not limited thereto.

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

In another specific example, the present invention relates to a method for predicting the risk of progression to Alzheimer's disease dementia by measuring the methylation level at a CpG site of the KRT32 gene promoter.

In addition, the present invention relates to a method for detecting the methylation level of a CpG region of a gene promoter from a sample of an individual to provide information necessary for predicting the risk of progression to Alzheimer's disease dementia.

In addition, the present invention relates to a method for providing information for predicting the risk of progression to Alzheimer's disease dementia, comprising a step of measuring the methylation level at a CpG site of the KRT32 gene promoter from a sample of an individual.

As an embodiment for this, the present invention relates to a method for providing information for predicting the risk of progression to Alzheimer's disease dementia, comprising the steps of: measuring a methylation level of a CpG site of a KRT32 gene promoter from a sample of a subject; and comparing the methylation level with the methylation level of the corresponding gene in a control sample that does not have Alzheimer's disease or with a predetermined threshold value (cut-off).

The term "sample" in the present invention includes samples such as cells, tissues, whole blood, serum, plasma, cerebrospinal fluid, saliva, sputum or urine, in which the level of gene methylation is different due to Alzheimer's disease, but is not limited to the above examples and any sample from which DNA can be extracted may be used. Preferred specific examples include skin tissue or skin cells, and include, but are not limited to, skin fibroblasts.

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

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

For example, the method of the present invention may include (a) a step of treating genomic DNA in the obtained sample with a compound that modifies an unmethylated cytosine base or a methylation-sensitive restriction enzyme; and (b) a step of amplifying the treated DNA by PCR using a primer capable of amplifying a specific CpG site of the corresponding gene.

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

In addition, in the step (a), the methylation-sensitive restriction enzyme may be a restriction enzyme that can specifically detect methylation of a specific CpG site as described above and contains CG as a recognition site of the restriction enzyme, and examples thereof include, but are not limited to, Sma I, Sac II, Eag I, Hpa II, Msp I, Bss HII, Bst UI, and Not I.

In the step (b) above, amplification can be performed by a conventional PCR method. The primers used at this time can be preferably designed according to the sequence of a specific CpG site to be analyzed for methylation as described above, and can be a primer pair that can specifically amplify cytosine that is methylated and not modified by bisulfite, and a primer pair that can specifically amplify cytosine that is not methylated and modified by bisulfite.

The step of detecting the methylation level of a specific CpG site of the gene may additionally include a step of (c) confirming the presence or absence of the result amplified in step (b). The presence or absence of the result amplified in step (c) may be determined by a method known in the art, for example, by performing electrophoresis and determining whether a band at a desired position is detected. For example, when a compound that modifies an unmethylated cytosine residue is used, the degree of methylation may be determined based on the presence or absence of a PCR result amplified by each of the two types of primer pairs used in step (a), that is, a primer pair that can specifically amplify a cytosine that is methylated and not modified by bisulfite, and a primer pair that can specifically amplify a cytosine that is not methylated and modified by bisulfite. Preferably, the presence or absence of methylation may be determined by using a bisulfite genome sequencing method that treats sample genomic DNA with bisulfite, amplifies a CpG site of the corresponding gene by PCR, and analyzes the base sequence of the amplified site.

In addition, even in the case of using a restriction enzyme, the methylation can be determined by a method known in the art, for example, if a PCR result is shown in mock DNA, it is judged that CpG is methylated if there is a PCR result in DNA treated with the restriction enzyme, and if there is no PCR result in DNA treated with the restriction enzyme, it is judged that CpG is unmethylated, and this is obvious to those skilled in the art. In the above, mock DNA refers to sample DNA separated from a sample and not treated in any way.

Another example of detecting the methylation level of a specific CpG site of a gene may be an immunoprecipitation method using a methylated DNA antibody, for example, an antibody against a methylated CpG binding domain (MBD) or methylcytosine. 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.

According to this method, if the CpG site of the KRT32 gene promoter in the target sample is detected as being hypermethylated, the risk of progression to Alzheimer's disease dementia can be predicted in advance.

Therefore, according to the present invention, since the methylation change of a specific CpG site of a gene is specifically shown in a sample of an individual, the methylation change of the gene can be usefully used as a biomarker to predict Alzheimer's disease dementia. For example, the hypermethylation of a specific CpG site of the KRT32 gene can be usefully used as a biomarker to predict the risk of progression to Alzheimer's disease dementia.

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

Figure 1 shows the difference in the degree of DNA methylation of the KRT32 gene between the mild cognitive impairment patient group without amyloid pathology findings (“MCI (non-AD)”) and the mild cognitive impairment patient group with Alzheimer’s disease (“Prodromal AD”) in the DNA methylation mutation analysis.
Figure 2 shows the difference in the degree of DNA methylation of the KRT32 gene between the group of patients with mild cognitive impairment without amyloid pathology ("MCI (non-AD)") and the group of patients with mild cognitive impairment with Alzheimer's disease ("Prodromal AD") in pyrosequencing analysis.
Figure 3 shows the results of a receiver operating characteristics curve (ROC curve) analysis to evaluate the effectiveness of using DNA methylation changes in the KRT32 gene to distinguish between a group of patients with mild cognitive impairment without amyloid pathology (“MCI (non-AD)”) and a group of patients with mild cognitive impairment due to Alzheimer’s disease (“Prodromal AD”).

Hereinafter, the present invention will be described in detail by examples. However, the following examples are only intended to illustrate the present invention, and the present invention is not limited to the following examples.

Example 1. Research subjects

The Alzheimer’s disease patient group includes three groups: preclinical Alzheimer’s disease (AD), prodromal Alzheimer’s disease, and symptomatic Alzheimer’s disease. The control group includes three groups: normal control (normal cognitive function, no amyloid pathology), mild cognitive impairment (MCI) due to non-AD, and dementia due to non-AD.

Diagnosis and classification of the above six groups of research subjects were performed according to the following four steps:

In the first step, a medical history was collected through a structured interview by a neurologist with the subjects and their guardians.

In the second step, the degree of decline in current cognitive function and decline in daily life ability were determined through neuropsychological tests. Through these two steps, the three stages of cognitive function decline, namely normal cognitive function, mild cognitive impairment, and dementia, were determined. In the case of the normal cognitive function group, regardless of subjective complaints of cognitive decline, all items of the objective neuropsychological test showed test results above the standard score (z score ≥-1.5), so there were no objective findings of cognitive decline and no impairment in daily life ability. In the case of mild cognitive impairment, there was a complaint of memory decline by the patient or informant, and the standard score of 1.5 or lower (z score <-1.5) was shown in the neuropsychological test, so there was objective memory decline, but overall cognitive function and daily life ability were maintained. The dementia group showed objective findings of decline (standard score 1.5 or lower) in two or more cognitive areas, and the clinical symptoms progressed slowly, and the group required care from others due to impairment in daily life ability.

In the third step, blood tests and structural brain imaging (computed tomography, CT or magnetic resonance imaging, MRI) and functional brain imaging (functional MRI, amyloid positron emission tomography, amyloid PET) were performed to exclude other causes of cognitive decline and to check for accumulation of amyloid pathology in the brain. Excluding other causes of cognitive decline is an important step in diagnosing Alzheimer's disease. It is important to exclude these possibilities by checking for thyroid hormone dysfunction, vitamin B12 or folate deficiency, and neurosyphilis in blood tests. In addition, alcoholism, drug addiction, major depressive disorder, bipolar disorder, schizophrenia, and convulsive disorders were also excluded because they may be associated with cognitive decline. In addition, structural brain abnormalities, such as normal pressure hydrocephalus, stroke, and brain tumors, were excluded in brain imaging tests, and other neurodegenerative diseases such as Parkinson's disease, dementia with Lewy bodies, and vascular dementia were also excluded. According to the 2018 Diagnostic Criteria for Research on Alzheimer's Disease, cases with positive amyloid PET results (brain amyloid plaque load, BAPL 2 or 3) were considered to have Alzheimer's disease and were classified into one of the following groups: preclinical, prodromal, and symptomatic AD, based on clinical symptoms. Cases with negative amyloid PET results (BAPL 1) were considered the control group and, based on clinical symptoms, were classified into one of the following groups: normal control group (normal cognitive function and no amyloid pathology), mild cognitive impairment (MCI) due to non-AD, and dementia due to non-AD. Fourth, the clinical information of these three stages was synthesized to make a final decision on six groups.

Example 2. Skin biopsy collection and skin fibroblast culture

A skin biopsy was collected from the patient's inner thigh using a cylindrical blade with a 2 mm diameter. The collected skin biopsy was divided into six equal parts and placed in a 24-well cell culture plate coated with 0.1% gelatin with enough medium (DMEM/20% FBS) to submerge the skin biopsy pieces. Cells were observed to extend from the edges of the skin biopsy pieces for 7 days. The medium was replenished every 2-3 days to prevent the skin biopsy pieces from drying. After 7 days, the amount of medium was increased to 500 μL and the medium was replaced every 2-3 days. After 14 days, when the cells had grown around the skin biopsy pieces and filled the outer edges of the culture wells, the cells were transferred to a 35-mm culture dish and the concentration of fetal bovine serum (FBS) in the medium was reduced from 20% to 10%, and cell culture was continued. Subculture was performed when the cells filled 80-90% of the 35-mm culture dishes. After repeating the subculture 2-3 times and selecting only skin fibroblasts, the expression of SERPHINH1, a fibroblast marker, was confirmed using the SERPHINH1 antibody.

Example 3. Genomic DNA extraction

In Example 2, genomic DNA was extracted from cultured skin fibroblasts using 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 status was checked for degradation by electrophoresis on a 1% agarose gel.

Example 4. DNA methylation mutation analysis

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

To identify differentially methylated genes (DMGs) between the patient group and the control group, the independent t-test method was used. Finally, promoter CpG sites with p value<0.05 and absolute value β difference≥0.3 were selected as differentially methylated CpG sites, and among these, genes with changed methylation levels of CpG sites were selected as DMGs.

Example 5. Pyrosequencing analysis

Pyrosequencing analysis was performed to measure the methylation level of a specific CpG site (cg25598400, Chr17: 41467824) of the KRT32 (Keratin 32) gene. PCR was performed using bisulfite-treated gDNA as a template. At this time, PCR primers were designed using the PSQ assay design program (Qiagen, USA). The base sequences of the designed PCR primers are as follows:

Forward primer, 5'- AGTAGTGGTTGTTTTTTTTAATGAAAGTAT -3' (SEQ ID NO: 2)

Biotinylated reverse primer, 5'- CCTCTAAAATTCCTATTCCTTTCTACT -3' (SEQ ID NO: 3)

The PCR reaction mixture was prepared in a total volume of 20 μl containing less than 20 ng of bisulfite-converted gDNA, 10 μl 2' Hot/Start PCR premix (Enzynomics), 1 μl forward primer (10 pmole/μl), and 1 μl biotinylated reverse primer (10 pmole/μl). The PCR reaction was performed with an initial denaturation step at 95°C for 10 min, followed by denaturation step at 95°C for 30 sec, annealing step at 56°C for 30 sec, and extension step at 72°C for 30 sec, which were repeated 50 times. Finally, a final extension step was performed at 72°C for 10 min. After the PCR reaction was completed, 2 μl of the reaction solution was taken and electrophoresed on a 2.5% agarose gel, and the base sequence size of the final PCR product was confirmed through ethidium bromide staining.

The PCR final product was prepared as an ssDNA template using streptavidin Sepharose HP beads (Amersham Biosciences). The reaction solution was prepared for a total volume of 80 μl, including 1-2 μl beads, 40 μl binding buffer, 16-18 μl PCR product, and high-purity water, for each sample, and dispensed into each well of a PCR plate. After sealing the plate, it was mixed well using an orbital shaker at 1400 rpm for 15-20 minutes. The sequencing primers were diluted to a concentration of 0.3 μM using PyroMark annealing buffer, and 25 μl was dispensed into each well, and the PCR plate was mounted on the workstation of the PyroMark Q48 (Qiagen) instrument to perform sequencing analysis. The base sequence of the sequencing primer used at this time is as follows: Sequencing primer 5'- GGGTGGTAGAAATTGT -3' (SEQ ID NO. 4)

The DNA methylation percentage of a specific CpG site (cg25598400, Chr17:41467824) of the KRT32 gene was calculated as follows. The base sequence of the generated PCR product was 5'- AGTAGTGGTTGTTTTTTTTAATGAAAGTATTTTTTATGGGAAGAGGTTTGGTTATTTAATAAGGAAATGTGTGGGTGGTAGAAATTGTTT Y GATGTTGGTAGCGGTATAAATTTAGTAGAAAGGAATAGGAATTTTAGAGG -3' (SEQ ID NO: 5), and the ratio of cytosine base at the position indicated by Y (C or T) was calculated as a percentage: C/C+T *100 (%)

Experimental Results

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

From 2018 to 2019, genomic DNA was extracted from skin fibroblasts cultured from the skin tissues of 20 patients with mild cognitive impairment without amyloid pathology and 28 patients with mild cognitive impairment of Alzheimer's disease who visited Ewha Womans University Mokdong Hospital. DNA methylation profiles were analyzed using the Illumina Human MethylationEPIC bead chip, and genes with DNA methylation changes of more than 20% at specific CpG sites in the gene promoter of the skin fibroblasts of the Alzheimer's disease mild cognitive impairment patient group were selected compared to the mild cognitive impairment patient group without amyloid pathology. Among these selected genes, the KRT32 (Keratin 32) gene showed a 21% increase in DNA methylation at a specific promoter CpG site in the skin fibroblasts of the Alzheimer's disease mild cognitive impairment patient group compared to the mild cognitive impairment patient group without amyloid pathology.

gene Target ID β value
(difference) ¹⁾ Methylation status p value KRT32 cg25598400 0.214804 High methylation 0.000393 1) The value obtained by subtracting the β value of the normal control cells from the β value of the patient group cells.

The difference in the degree of DNA methylation of the KRT32 gene between the mild cognitive impairment patient group without amyloid pathology and the Alzheimer's disease mild cognitive impairment patient group is shown in a scatter dot plot in Figure 1, and the mean ± standard error (mean ± SEM) value is presented.

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

Using pyrosequencing analysis, the degree of DNA methylation at the specific CpG site (cg25598400) of the KRT32 gene was measured. Similar to the Human MethylationEPIC bead results, it was confirmed that DNA methylation at the specific promoter CpG site (cg25598400) of the KRT32 gene was increased by 21% in the Alzheimer's disease mild cognitive impairment patient group compared to the mild cognitive impairment patient group without amyloid pathology (Fig. 2).

In addition, we verified whether the receiver operating characteristics curve (ROC curve) could be used to distinguish between the MCI patient group without amyloid pathology and the Alzheimer's disease MCI patient group. In general, the ROC curve is classified as non-informative (AUC=0.5), less accurate (0.5<AUC≤0.7), moderately accurate (0.7<AUC≤0.9), very accurate (0.9<AUC<1.0), and perfect test (AUC=1.0) based on the area under the curve (AUC) value of the ROC curve.

In the case of KRT32, it was found to be highly methylated in the Alzheimer's disease mild cognitive impairment patient group, and this hypermethylation was confirmed to be able to distinguish between the mild cognitive impairment patient group without amyloid pathology and the Alzheimer's disease mild cognitive impairment patient group with high accuracy (AUC = 0.8) (Fig. 3).

These results indicate that the disease-specific hypermethylation of the KRT32 gene in skin fibroblasts from patients with mild cognitive impairment (MCI) of Alzheimer's disease is a valid diagnostic marker for predicting Alzheimer's disease dementia in advance by noninvasively predicting whether patients with mild cognitive impairment (MCI) of Alzheimer's disease will progress to Alzheimer's disease dementia.

From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without changing the technical idea or essential characteristics thereof. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive. The scope of the present invention should be interpreted as including all changes or modifications derived from the meaning and scope of the patent claims described below rather than the above detailed description and their equivalent concepts within the scope of the present invention.

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Claims (10)

A composition for predicting the risk of progression to Alzheimer's disease dementia, comprising a preparation for measuring the methylation level of a CpG site of the KRT32 (Keratin 32) gene promoter. In the first paragraph,
A composition for measuring the methylation level of a CpG site of the gene promoter, the composition comprising a compound or a methylation-sensitive restriction enzyme that modifies an unmethylated cytosine base, a primer specific for a methylated sequence of the CpG site of the gene promoter, a primer specific for an unmethylated sequence, a methylated CpG binding domain, or an antibody specifically binding to methylcytosine. In the second paragraph,
A composition wherein the compound that modifies the above unmethylated cytosine base is bisulfite or a salt thereof. In the second paragraph,
A composition wherein the above 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 predicting the risk of progression to Alzheimer's disease dementia, comprising a composition according to any one of claims 1 to 4. A method for providing information for predicting the risk of progression to Alzheimer's disease dementia, comprising the step of measuring the methylation level of a CpG site of a KRT32 (Keratin 32) gene promoter from a sample of an individual. In Article 6,
A method further comprising the step of comparing said methylation level with the methylation level in the corresponding gene in a control sample that does not have Alzheimer's disease. In Article 6,
The step of measuring the methylation level of the CpG site of the above gene promoter is
(a) a step of treating the genomic DNA in the obtained sample with a compound that modifies unmethylated cytosine bases or a methylation-sensitive restriction enzyme; and
(b) A method comprising a step of amplifying the treated DNA by PCR using a primer capable of amplifying a CpG region of the KRT32 gene promoter. In Article 8,
A method wherein the compound that modifies the above unmethylated cytosine base is bisulfite or a salt thereof. In Article 6,
A method for detecting the above methylation level is a method selected from the group consisting of methylation-specific polymerase chain reaction, real time methylation-specific polymerase chain reaction, PCR using a methylated DNA-specific binding protein, quantitative PCR, pyrosequencing, bisulfite sequencing, DNA methylation microarray, and immunoprecipitation using a methylated CpG binding domain or an anti-methylcytosine antibody.