SNP as biomarker for high cognitive reserve and uses thereof

Abstract

The present invention relates to a composition, kit, and method for diagnosing individuals with normal cognitive function in Alzheimer's disease or for identifying individuals with high cognitive reserve using genetic polymorphisms.

Description

SNPs as biomarkers of cognitive reserve and their use

[0001] The present invention relates to a composition, a kit, and a method for diagnosing a normal cognitive function group of Alzheimer's disease or selecting a person with high cognitive reserve using genetic polymorphism.

[0002] Korea's rapid economic growth, Westernized lifestyles, and advancements in medicine have led to an increase in the average life expectancy of its citizens. Meanwhile, Korea also has the fastest aging population in the world. Consequently, the incidence of neurodegenerative diseases, often associated with aging, is also rapidly increasing.

[0003] Dementia is a major social and economic burden, and Alzheimer's disease (AD) is the most common type. It is a representative neurodegenerative brain disease that begins with memory loss, progresses gradually over time to symptoms such as loss of spatial and temporal perception, delusions and personality changes, language and motor dysfunction, and ultimately leads to death.

[0004] Although the exact pathogenesis and cause of Alzheimer's disease (AD) remain unknown, the key mechanism is believed to be the excessive production and deposition of a small protein called beta-amyloid in the brain, which detrimentally impacts synaptic homeostasis and neural network connectivity. However, most clinical trials aimed at suppressing amyloid deposition, a hallmark neuropathological change in AD, have failed to yield significant results, leading to calls for addressing other causative factors before amyloid deposition causes irreversible damage.

[0005] Meanwhile, cognitive reserve refers to the brain's ability to slow age-related changes, utilizing alternate brain networks that are not normally used to prepare for aging. Cognitive reserve can delay the progression from normal cognition to mild cognitive impairment (MCI), and higher cognitive reserve is known to be associated with lower rates of cognitive decline and dementia incidence. High cognitive reserve allows for the maintenance of normal cognitive function even when significant amyloid lesions accumulate in the brain and brain atrophy occurs. Cognitive reserve has been reported to be associated with the progression of cognitive disorders such as Alzheimer's disease. Therefore, given the current lack of fundamental treatments, which only alleviate symptoms or slow the progression of lesions, identifying neuroprotective factors and factors contributing to cognitive reserve in older adults with high cognitive reserve will significantly contribute to the development of fundamental treatments for Alzheimer's disease.

[0006] Therefore, there is a need to develop biomarkers that can diagnose populations with high cognitive reserve or select individuals with high cognitive reserve.

[0007] An example of the present invention provides a composition for diagnosing a normal cognitive function group with Alzheimer's disease or screening a person with high cognitive reserve, comprising an agent capable of detecting a single nucleotide polymorphism (SNP) of rs2233369.

[0008] For example, a preparation capable of detecting a SNP may include a polynucleotide consisting of 10 to 100 consecutive bases including the SNP or a complementary polynucleotide thereof; or a probe or primer that specifically hybridizes with a polynucleotide including the SNP.

[0009] Another example provides a kit for diagnosing Alzheimer's disease with normal cognitive function or screening for a person with high cognitive reserve, comprising the composition.

[0010] For example, the kit may be an RT-PCR kit, a microarray chip, or a microfluidic chip kit.

[0011] Another example provides a method for diagnosing a normal cognitive function group or screening a person with high cognitive reserve in Alzheimer's disease, comprising the step of detecting a single nucleotide polymorphism (SNP) of rs2233369 from a sample of an individual.

[0012] Another example provides a method for providing information for diagnosing a person with normal cognitive function in Alzheimer's disease or screening a person with high cognitive reserve, comprising the step of detecting a single nucleotide polymorphism (SNP) of rs2233369 from a sample of an individual.

[0013] Another example provides a method for detecting a single nucleotide polymorphism (SNP) of rs2233369 from a sample of an individual to provide information necessary for diagnosing a normal cognitive function group of Alzheimer's disease or screening for a person with high cognitive reserve.

[0014] Another example provides a method for detecting a single nucleotide polymorphism (SNP) of rs2233369 from a sample of an individual for diagnosing a normal cognitive function group of Alzheimer's disease or screening for individuals with high cognitive reserve.

[0015] As specific examples, methods for detecting single nucleotide polymorphisms (SNPs) may include sequencing, mini-sequencing, automated sequencing, TaqMan assay, pyrosequencing, allele specific PCR, dynamic allelespecific hybridization (DASH), PCR-RELP (restriction fragment length polymorphism), PCR-SSCP (single strand conformation polymorphism), PCR-SSO (specific sequence oligonucleotide), hybridization by microarray, primer extension, Southern blot hybridization, dot hybridization, allele specific oligonucleotide (ASO) hybridization combining PCR-SSO and dot hybridization, rolling circle amplification (RCA), high resolution melting (HRM), or matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF / MS).

[0016] Figure 1 shows the expression frequency of the SNP (rs2233369 G / A, R44Q) of the ABI3 gene in each sample of a normal cognitive group without amyloid pathology (normal control group, “Normal”), a normal cognitive group with amyloid pathology (preclinical Alzheimer’s disease patients, “Preclinical AD”), patients with mild cognitive impairment of Alzheimer’s disease (“MCI”), and patients with dementia of Alzheimer’s disease (“AD”), and the average expression frequency (11%) of the SNP (rs2233369 G / A, R44Q) of the ABI3 gene in Koreans as shown in the dbSNP 155 database.

[0017] The present inventors analyzed samples from a normal control group (a normal cognitive group without amyloid pathology), an Alzheimer's disease normal cognitive function group (a normal cognitive group with amyloid pathology, i.e., a preclinical Alzheimer's disease patient group), Alzheimer's disease mild cognitive impairment patients, and Alzheimer's disease dementia patients, and identified a base of a specific single nucleotide polymorphism (SNP) that exhibits a particularly high expression frequency in the Alzheimer's disease normal cognitive function group (a preclinical Alzheimer's disease patient group). Accordingly, the present invention provides a molecular biological diagnostic technique that utilizes the SNP, which specifically appears in a preclinical Alzheimer's disease patient group that maintains normal cognition despite the presence of amyloid pathology, as a biomarker for selecting individuals with high cognitive reserve.

[0018] The SNPs of this study can be used as biomarkers to select individuals with high cognitive reserve by extracting DNA from blood and tissue collected through a non-invasive method and analyzing the DNA base sequence. This means that they can exhibit neuroprotective effects that suppress cognitive decline as described above, and thus can be utilized in follow-up research to discover neuroprotective factors and identify factors contributing to cognitive reserve.

[0019] Hereinafter, the present invention will be described in more detail.

[0020] Where the terms "comprise," "comprised," or "comprising" are used in this specification (including the claims), they should be construed as specifying the presence of stated features, integers, steps, or components, but not excluding the presence of one or more other features, integers, steps, components, or groups thereof.

[0021] The descriptions of documents, laws, materials, devices, and articles contained herein are included solely to provide context for the present invention. They are not intended to be, in whole or in part, part of the prior art or to suggest or imply that they were common general knowledge in the field to which the present invention pertains prior to the priority date of each claim of this application.

[0022] In one aspect, the present invention provides a composition for diagnosing a normal cognitive function group of Alzheimer's disease or screening a person with high cognitive reserve, and a kit including the same, comprising an agent capable of detecting a single nucleotide polymorphism (SNP) of rs2233369 (NCBI refSNP ID).

[0023] In another aspect, the present invention provides a method for providing information for diagnosing a normal cognitive function group of Alzheimer's disease or screening a person with high cognitive reserve, the method comprising the step of identifying a single nucleotide polymorphism (SNP) of rs2233369 from a sample of an individual.

[0024] The term "rs number" or "NCBI refSNP ID" herein refers to a unique number of SNPs registered in the SNP Reference Database (http: / www.ncbi.nlm.nih.gov / snp) of the National Center for Biotechnology Information (NCBI) in the United States. This unique number allows access to the database to identify information about the SNP. Therefore, once the rs number is specified herein, those skilled in the art can easily identify information about the SNP, such as the chromosomal location where the SNP exists, the genetic loci, and the polymorphic sequence including the SNP. As an example, Table 1 below shows information about rs2233369 identified through the database.

[0025] NCBI refSNP IDLocation on chromosomeGene siteAllelesrs2233369chr17:49216544 (GRCh38.p14)ABI3 geneG / A

[0026] In the above table, the location on the human genome chromosome is expressed according to GRCh38.p14 (Genome Reference Consortium Human Build 38 patch release 14), but the specific sequence of the human genome chromosome region may be somewhat changed 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, it will be obvious that the scope of the present invention extends to the changed human genome chromosome region 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. Anyone having ordinary skill in the art to which the present invention belongs can easily understand such change. As used herein, the term "Alzheimer's disease (AD)" is a representative neurodegenerative brain disease that starts with memory loss as an initial symptom and then shows a decline in overall cognitive function. Traditionally, Alzheimer's disease was defined based on clinical symptoms and confirmed through brain autopsy. However, recently, as neuropathological changes associated with Alzheimer's disease have been discovered and can be utilized in clinical tests, it has become possible to define Alzheimer's disease by confirming these changes using in vivo biomarkers. Although Alzheimer's disease is still diagnosed through postmortem brain autopsy findings, the pathological accumulation of amyloid proteins and tau proteins, which are known to be representative features of brain autopsy findings, can now be indirectly confirmed through PET imaging or cerebrospinal fluid examination, leading to a dramatic improvement in diagnostic accuracy compared to the past when diagnosis was made solely based on clinical features.Biomarkers for defining Alzheimer's disease include beta-amyloid-related markers (e.g., binding of amyloid-PET ligand in the cerebral cortex or Aβ in the cerebrospinal fluid). 42 ), tau-related markers in the form of neurofibrillary tangles (e.g., increased phosphorylated tau in cerebrospinal fluid or binding of tau-PET ligands in the cerebral cortex), or markers of neurodegeneration (e.g., increased total tau in cerebrospinal fluid or brain atrophy on MRI and brain hypometabolism on FDG-PET).

[0027] 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 Alzheimer's disease diagnostic criteria announced by the National Institute of Aging (NIA) and the Alzheimer Association (AA) in 2011, Alzheimer's disease is included in the diagnostic criteria 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 (AD), prodromal Alzheimer's disease, and symptomatic Alzheimer's disease through a combination of biomarkers and cognitive stages, and explained as a continuum.

[0028] "Alzheimer's disease with normal cognitive function" is 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 with mild cognitive impairment, shows pathological findings of Alzheimer's disease in biological marker tests, but there is mild objective cognitive decline, and the ability to perform independent daily living is maintained. The terms "Alzheimer's disease 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, symptoms of dementia appear, and independent daily living ability is impaired due to objective decline in cognitive function. The terms "Alzheimer's disease with dementia" or "Symptomatic AD" are also used.

[0029] The "Alzheimer's disease with normal cognitive function" group, which is diagnosed at our hospital, is a group that maintains normal cognition despite the presence of amyloid pathology, and is considered to have high cognitive reserve that can delay the progression from normal cognition to mild cognitive impairment. The composition, kit, and method of the present invention can select a patient group with high cognitive reserve by identifying the Alzheimer's disease with normal cognitive function, thereby delaying the worsening of the disease through active preventive management and early treatment, and further contributing to the development of a fundamental treatment for Alzheimer's disease by identifying factors contributing to cognitive reserve and discovering neuroprotective factors. Therefore, if clinically utilized in the future, it can delay the onset of dementia and reduce its prevalence, ultimately improving the quality of life of patients and their families, and further significantly reducing the socioeconomic costs borne by the country.

[0030] The composition of the present invention is characterized by containing an agent for detecting or confirming the SNP of rs2233369 in a genetic sample isolated from a patient.

[0031] A preparation capable of detecting a SNP comprises a polynucleotide consisting of 10 to 100 consecutive bases including the SNP or a complementary polynucleotide thereof; or a probe or primer that specifically hybridizes with a polynucleotide including the SNP.

[0032] For example, the agent capable of detecting a SNP may be a polynucleotide consisting of 10 to 100 consecutive bases including a SNP or a complementary polynucleotide thereof, which may be, for example, 10 to 100, 10 to 90, 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 10 to 20, 20 to 100, 20 to 90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 20 to 40, 20 to 30, 30 to 100, 30 to 90, 30 to 80, 30 to 70, It may be composed of 30 to 60, 30 to 50, 30 to 40, 40 to 100, 40 to 90, 40 to 80, 40 to 70, 40 to 60, or 40 to 50 consecutive bases, but is not limited thereto, and can be appropriately determined by a person skilled in the art.

[0033] The polynucleotide or its complementary polynucleotide according to the present invention is associated with a polymorphic sequence. A polymorphic sequence refers to a sequence that includes a polymorphic site representing a single nucleotide polynucleotide (SNP) in a nucleotide sequence. A polymorphic site refers to a site in a polymorphic sequence where a SNP exists. In the present invention, the polynucleotide may be DNA or RNA.

[0034] As another example, the agent capable of detecting a SNP herein may be a probe or primer that specifically hybridizes with a polynucleotide containing the SNP, and preferably, these are allele-specific.

[0035] An allele-specific probe or primer refers to a probe or primer that can specifically hybridize to each allele. In other words, it refers to hybridization that can specifically distinguish the bases of a polymorphic site present in a polymorphic sequence. Here, the hybridization conditions must be sufficiently stringent to ensure a significant difference in hybridization intensity between alleles and to hybridize only to a specific allele. Conditions suitable for hybridization can be determined by referring to information commonly known in the art, and can be determined by controlling factors such as temperature, ionic strength (buffer concentration), and the presence of compounds such as organic solvents. These stringent conditions may be determined differently depending on the sequence to be hybridized.

[0036] In the present invention, the term "probe" refers to a nucleic acid fragment capable of sequence-specific binding to a specific nucleic acid. The probe may be a nucleic acid fragment such as RNA or DNA, ranging from a few bases to several hundred bases, and may be labeled to confirm the presence or absence of a specific nucleic acid. The probe may be produced in the form of an oligonucleotide probe, a single-stranded DNA probe, a double-stranded DNA probe, an RNA probe, etc. The probe of the present invention may be a sequence that is completely or partially complementary to a sequence containing a SNP, but a substantially complementary sequence may also be used as long as it does not interfere with specific hybridization. In the present invention, hybridization is performed using a probe complementary to a region containing a SNP of the present invention, and the SNP can be confirmed by hybridization. The selection of an appropriate probe and hybridization conditions may be modified based on those known in the art.

[0037] As used herein, the term "primer" refers to a short nucleic acid sequence having a short free three-terminal hydroxyl group, capable of forming base pairs with a complementary template, and serving as an initiation point for copying the template strand. The primer can initiate DNA synthesis in the presence of a polymerization reagent (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in an appropriate buffer and temperature. Furthermore, the primer typically consists of 7 to 50 or 15 to 30 bases, although the appropriate length of the primer may vary depending on the intended use. The primer may incorporate additional features that do not alter the basic property of the primer as an initiation point for DNA synthesis. The primer sequence need not be perfectly complementary to the template, but must be sufficiently complementary to hybridize with the template. The primer can be used to amplify and detect a DNA fragment containing the polymorphic site by hybridizing to a sequence containing a SNP.

[0038] Primers or probes can be chemically synthesized using phosphoramidite solid support methods or other well-known methods. These nucleic acid sequences can also be modified using many means known in the art. Non-limiting examples of such modifications include methylation, capping, substitution with one or more homologs of a natural nucleotide, and modification between nucleotides, such as modification with uncharged linkers (e.g., methyl phosphonate, phosphotriester, phosphoroamidate, carbamate, etc.) or charged linkers (e.g., phosphorothioate, phosphorodithioate, etc.).

[0039] In the present invention, the kit may be an RT-PCR kit, a microarray chip, or a microfluidic chip kit. The kit of the present invention may include not only a polynucleotide but also one or more other component compositions, solutions, or devices suitable for the analysis method.

[0040] For example, the kit of the present invention may be a PCR kit. Preferably, it may be a real-time PCR (RT-PCR) kit. Such a kit may include essential elements necessary for performing PCR, such as real-time PCR (RT-PCR). For example, the kit may additionally include each primer pair capable of amplifying a nucleic acid containing a SNP site, a test tube or other suitable container, a reaction buffer, deoxynucleotides (dNTPs), enzymes such as Taq polymerase and reverse transcriptase, DNase, RNAse inhibitors, DEPC water, and sterile water.

[0041] As another example, the kit of the present invention may be a DNA chip kit. Preferably, it may be a microarray chip or microfluidic chip kit. Such a kit may include a substrate on which a polynucleotide comprising the SNP site or its complementary polynucleotide, or a probe or primer that specifically hybridizes with the polynucleotide comprising the SNP, is immobilized.

[0042] The microarray may be a conventional microarray, except that it includes the polynucleotides, primers, or probes of the present invention. Hybridization of nucleic acids on a microarray and detection of hybridization results are well known in the art. For example, the detection can be performed by labeling a nucleic acid sample with a label capable of generating a detectable signal, such as a fluorescent substance such as Cy3 or Cy5, hybridizing the nucleic acid sample onto a microarray, and detecting the signal generated from the label.

[0043] Microarrays can be fabricated using a variety of techniques, including printing using fine-pointed pins on a substrate, photolithography using a prefabricated mask, photolithography using a dynamic micromirror device, inkjet printing, or electrochemistry on a microelectrode array. The substrate of the microarray chip is preferably coated with an active group selected from the group consisting of aminosilane, poly-L-lysine, and aldehyde, but is not limited thereto. In addition, the substrate is preferably selected from the group consisting of slide glass, plastic, metal, silicon, nylon membrane, and nitrocellulose membrane, but is not limited thereto.

[0044] A microfluidic chip is a microfluidic device that uses microfluidic control technology to measure and analyze the interaction of analyte contained in a fluid sample with a biological substance, cell, tissue, or detection device on the chip, and can include processes such as probe / target hybridization, nucleic acid amplification, and capillary electrophoresis reaction in a miniaturized and compartmentalized manner.

[0045] For detection of the genotype of the SNP of the present invention, sequencing, hybridization analysis using microarray, etc., amplification using PCR, etc. can be used. For example, known methods such as sequencing, mini-sequencing, automated sequencing, TaqMan assay, pyrosequencing, allele specific PCR, dynamic allelespecific hybridization (DASH), PCR-RELP (restriction fragment length polymorphism), PCR-SSCP (single strand conformation polymorphism), PCR-SSO (specific sequence oligonucleotide), hybridization by microarray, primer extension, Southern blot hybridization, dot hybridization, ASO (allele specific oligonucleotide) hybridization combining PCR-SSO and dot hybridization, rolling circle amplification (RCA), high resolution melting (HRM), or matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF / MS) can be used, but are not limited thereto.

[0046] In another aspect, the present invention relates to a method for diagnosing a normal cognitive function group or selecting a person with high cognitive reserve for Alzheimer's disease, comprising the step of detecting a single nucleotide polymorphism (SNP) of rs2233369 from a sample of an individual.

[0047] In addition, the present invention relates to a method for providing information for diagnosing a person with normal cognitive function in Alzheimer's disease or selecting a person with high cognitive reserve, comprising a step of detecting a single nucleotide polymorphism (SNP) of rs2233369 from a sample of an individual.

[0048] In addition, the present invention relates to a method for detecting a single nucleotide polymorphism (SNP) of rs2233369 from a sample of an individual to provide information necessary for diagnosing a normal cognitive function group of Alzheimer's disease or screening a person with high cognitive reserve.

[0049] In addition, the present invention relates to a method for detecting a single nucleotide polymorphism (SNP) of rs2233369 from a sample of an individual for diagnosing a normal cognitive function group of Alzheimer's disease or screening a person with high cognitive reserve.

[0050] The term "sample" in the present invention refers to a biological material isolated from a subject. The sample may include any biological material from which a desired SNP can be detected. For example, the sample may include genetic material, such as DNA, genomic DNA, complementary DNA (cDNA), RNA, heterogeneous nuclear RNA (hnRNA), mRNA, etc. Such samples include, but are not limited to, samples such as cells, tissues, whole blood, serum, plasma, cerebrospinal fluid, saliva, sputum, or urine, and any sample from which DNA can be extracted may be used. Preferred specific examples include skin tissue or skin cells, such as, but not limited to, skin fibroblasts.

[0051] First, the acquisition of genomic DNA from a sample of an individual can be performed 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.

[0052] Detection of SNP genotypes can be accomplished using sequencing, hybridization analysis using microarrays, amplification using PCR, etc. For example, it can be performed by a known method such as sequencing, mini-sequencing, automated sequencing, TaqMan analysis, pyrosequencing, allele specific PCR, dynamic allelespecific hybridization (DASH), PCR-RELP (restriction fragment length polymorphism), PCR-SSCP (single strand conformation polymorphism), PCR-SSO (specific sequence oligonucleotide), hybridization by microarray, primer extension, Southern blot hybridization, dot hybridization, ASO (allele specific oligonucleotide) hybridization combining PCR-SSO and dot hybridization, rolling circle amplification (RCA), high resolution melting (HRM), or matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF / MS).

[0053] The method may further include a step of comparing the SNP detected in the subject's sample with the SNP of a normal control group (a normal cognitive group without amyloid pathology findings), a patient with mild cognitive impairment of Alzheimer's disease, and / or a patient with dementia of Alzheimer's disease. For example, if the SNP detected in the subject's sample shows a different genotype from the SNP of a normal control group (a normal cognitive group without amyloid pathology findings), a patient with mild cognitive impairment of Alzheimer's disease, and / or a patient with dementia of Alzheimer's disease, the patient may be determined to have high cognitive reserve. Alternatively, the method may further include a step of determining that the patient has high cognitive reserve if the SNP genotype of rs2233369 is confirmed or detected to be A.

[0054] Therefore, according to the present invention, it can be usefully used to diagnose a normal cognitive function group of Alzheimer's disease or to select a person with high cognitive reserve by detecting the presence of the above-mentioned specific SNP.

[0055] The present invention will be described in more detail below with reference to the following examples. However, these examples are intended solely to illustrate the invention, and the scope of the invention is not limited by these examples. It will be apparent to those skilled in the art that modifications to the examples described below may be made without departing from the essential spirit of the invention.

[0056]

[0057] Example 1. Classification of patient and control groups in a study cohort for the development of an Alzheimer's disease diagnostic marker using skin fibroblasts.

[0058] 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 the normal control group (normal cognitive function and no amyloid pathology), mild cognitive impairment due to non-AD, and dementia due to non-AD.

[0059] Diagnosis and classification of the above six study subjects were performed according to the following four steps:

[0060] In the first step, the medical histories of patients who visited Ewha Womans University Mokdong Hospital and Ewha Womans University Seoul Hospital from 2018 to 2020 were collected through structured interviews with neurologists, together with the subjects and their guardians.

[0061] In the second step, neuropsychological testing was used to assess the degree of cognitive decline and the presence of impairment in daily living skills. Through these two steps, three stages of cognitive decline were determined: normal cognitive function, mild cognitive impairment, and dementia. The normal cognitive function group, regardless of subjective complaints of cognitive decline, demonstrated objective neuropsychological test scores of ≥-1.5 (z score ≥-1.5), indicating no objective evidence of cognitive decline and no impairment in daily living skills. Mild cognitive impairment refers to patients or informants complaining of memory decline and neuropsychological test scores of 1.5 or lower (z score <-1.5), indicating objective memory decline, but maintaining overall cognitive function and daily living skills. The dementia group demonstrated objective decline (z score ≤1.5) in two or more cognitive domains, with a slowly progressive clinical picture and impairment in daily living skills, requiring the care of others.

[0062] 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 the 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 seizure disorders were also excluded as they may be associated with cognitive decline. In addition, structural brain abnormalities such as normal pressure hydrocephalus, stroke, and brain tumors were excluded from 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 research diagnostic criteria for Alzheimer's disease, cases with a positive amyloid PET result (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, depending on clinical symptoms. Cases with a negative amyloid PET result (BAPL 1) were considered the control group and, depending on clinical symptoms, were classified into one of the following groups: normal control group (normal cognitive function and no amyloid pathology), mild cognitive impairment due to non-AD, and dementia due to non-AD.Fourth, by synthesizing the clinical information of these three stages, we made a final decision on six groups.

[0063]

[0064] Example 2. Skin biopsy collection and dermal fibroblast culture

[0065] A skin biopsy was obtained from the patient's inner thigh using a 2-mm diameter cylindrical blade. The 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 medium volume was increased to 500 μL and replaced every 2-3 days. After 14 days, when cells had grown around the skin biopsy pieces and filled the culture wells, the cells were transferred to 35-mm culture dishes 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 reached 80-90% confluence in the 35-mm culture dish. 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.

[0066]

[0067] Example 3. Genomic DNA extraction

[0068] Genomic DNA was extracted from cultured skin fibroblasts in Example 2 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 status was confirmed for degradation by electrophoresis on a 1% agarose gel.

[0069]

[0070] Example 4. Single nucleotide polymorphism (SNP) genotyping analysis

[0071] All equipment and resources required for the automated Axiom 2.0 Assay were performed according to the Axiom 2.0 Assay Automated Workflow User Guide (P / N 702963, http: / / www.thermofisher.com / kr / ko / home.html). Approximately 200 ng of genomic DNA was amplified using the Axiom 2.0 Reagent Kit (96 reactions, P / N 901758) and randomly fragmented into 25–125 base pair (bp) fragments. An additional fragmentation step further fragmented the amplified products into approximately 25–50 bp segments, which were then final labeled using biotinylated nucleotides. After labeling, the samples were denatured and transferred to hyb trays, and hybridization was performed on a GeneTitan MC Instrument (Affymetrix). Hybridization steps were performed using the Axiom BiobankPlus Genotyping Array KNIHv1.0 according to the GeneTitan Multichannel Instrument User's Manual (P / N 08-0306). After ligation, the arrays were stained and imaged on a GeneTitan MC Instrument (Affymetrix). The acquired images were analyzed according to the Affymetrix GeneChip Command Console Software User Manual (P / N 702569, http: / www.thermofisher.com / kr / ko / home.html). Genotype data were hybridized to the Affymetrix Axiom KORV1_1.r1 array available through the K-CHIP Consortium, and 827,783 SNPs were genotyped.

[0072] The acquired SNPs were annotated with LJB*(NSFP) to extract non-synonymous variants. 130 SNPs in 36 genes related to the WAVE1 complex involved in amyloid processing were analyzed using SIFT scores, PolyPhen2 HDIV scores, PolyPhen2 HVAR scores, LRT scores, MutationTasterscores, MutationAssessorscore, FATHMM scores, GERP++ scores, PhyloPscores, and SiPhyscores.

[0073]

[0074] Experimental results

[0075] 1. Expression frequency of ABI3 gene SNP (rs2233369 G / A, R44Q) specific to preclinical Alzheimer's disease patients

[0076] As described above, genomic DNA was extracted from skin tissues of 19 normal controls (normal cognitive function group without amyloid pathology), 5 Alzheimer's disease normal cognitive function group (normal cognitive function group with amyloid pathology, i.e., preclinical Alzheimer's disease patients), 29 Alzheimer's disease mild cognitive impairment patients, and 16 Alzheimer's disease dementia patients by culturing skin fibroblasts, and genotyping 827,783 single nucleotide polymorphisms (SNPs) using Axiom_KORV1_1.r1, and the obtained SNPs were annotated to LJB*(NSFP) to extract non-synonymous variants. The expression frequency of non-synonymous variants of 36 genes related to the WAVE1 complex was analyzed in the normal control group, preclinical Alzheimer's disease patient group, Alzheimer's disease mild cognitive impairment patient group, and Alzheimer's disease dementia patient group. As a result, the expression frequency of SNP (rs2233369 G / A, R44Q) of the ABI3 gene was 40% in the preclinical Alzheimer's disease patient group, which was more than twice as high as that in the normal control group (17%), Alzheimer's disease mild cognitive impairment patient group (21%), and Alzheimer's disease dementia patient group (19%) (Fig. 1). The SNP rs2233369 of the ABI3 gene is a nonsynonymous variant that changes the 44th amino acid of the ABI3 protein from arginine (R) to glutamine (Q) when the G genotype changes to the A genotype. In addition, the frequency of the ABI3 gene SNP (rs2233369 G / A, R44Q) appearing in the preclinical Alzheimer's disease patient group was 3.6 times higher than the average frequency (11%) in Koreans shown in the dbSNP 155 database (Fig. 1).

[0077] These results indicate that the SNP (rs2233369 G / A, R44Q) in the ABI3 gene, a non-synonymous variant of ABI3 that is specifically found in a group of preclinical Alzheimer's disease patients who maintain normal cognition despite the presence of amyloid pathology, can be used as a biomarker to select individuals with high cognitive reserve through DNA sequencing.

[0078] From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering the technical concept or essential characteristics thereof. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as encompassing all changes or modifications derived from the meaning and scope of the following claims and their equivalent concepts, rather than the detailed description above.

Claims

1. A composition for diagnosing a normal cognitive function group of Alzheimer's disease or screening a person with high cognitive reserve, comprising a preparation capable of detecting a single nucleotide polymorphism (SNP) of rs2233369.

2. In the first paragraph, the agent capable of detecting the SNP is A polynucleotide consisting of 10 to 100 consecutive bases including the above SNP or a complementary polynucleotide thereof; or A composition comprising a probe or primer that specifically hybridizes with a polynucleotide containing the above SNP.

3. A kit for diagnosing a person with normal cognitive function in Alzheimer's disease or screening a person with high cognitive reserve, comprising the composition of clause 1 or clause 2.

4. In the third paragraph, the kit is an RT-PCR kit, a microarray chip, or a microfluidic chip.

5. A method for diagnosing a normal cognitive function group or selecting a person with high cognitive reserve in Alzheimer's disease, comprising the step of detecting a single nucleotide polymorphism (SNP) of rs2233369 from a sample of an individual.

6. In the 5th paragraph, the detection of the single nucleotide polymorphism (SNP) is performed by sequencing, mini-sequencing, automated sequencing, TaqMan analysis, pyrosequencing, allele specific PCR, dynamic allelespecific hybridization (DASH), PCR-RELP (restriction fragment length polymorphism), PCR-SSCP (single strand conformation polymorphism), PCR-SSO (specific sequence oligonucleotide), hybridization by microarray, primer extension, Southern blot hybridization, dot hybridization, ASO (allele specific oligonucleotide) hybridization combining PCR-SSO and dot hybridization, RCA (rolling circle amplification), HRM (high resolution melting), or MALDI-TOF / MS (matrix-assisted laser desorption ionization-time of flight mass spectrometry).

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