KR102662899B1 - Biomarkers for diagnosing Alzheimer's disease and uses thereof - Google Patents

Abstract

The present invention relates to a composition and kit for diagnosing Alzheimer's disease using a novel miRNA biomarker, and a method of providing information for diagnosing Alzheimer's disease using the same. The present invention relates to a novel biomarker that can predict Alzheimer's disease. By providing compositions and kits for diagnosing Alzheimer's disease, it is possible to quickly and accurately diagnose Alzheimer's disease patients and provide appropriate treatment to Alzheimer's disease patients.

Description

Biomarkers for diagnosing Alzheimer's disease and uses thereof}

The present invention relates to a composition and kit for diagnosing Alzheimer's disease using a novel miRNA biomarker, and a method of providing information for diagnosing Alzheimer's disease using the same.

Dementia is a representative disease of old age, which involves the gradual deterioration of personality caused by acquired loss of intellectual ability, cognitive impairment, and behavioral and psychological symptoms (abnormal behavioral and psychological symptoms such as insomnia, bulimia, anxiety, nervousness, depression, and delusions). It is a degenerative disease that occurs. Any disease that can cause damage to overall brain function can cause dementia. Alzheimer's disease (AD), commonly known as Alzheimer's disease, accounts for about 60% of all dementias and causes brain tissue loss and brain atrophy due to degeneration or gradual decline of nerve cells in the cerebral cortex or hippocampus. In some cases, vascular dementia occurs when brain cells die due to impaired cerebral blood circulation or cerebrovascular abnormalities.

It is estimated that more than 20% of people over the age of 80 are affected by Alzheimer's disease, and the number is rapidly increasing as we become an aging society. However, because Alzheimer's disease is caused by various etiological factors accompanied by accumulation of beta amyloid (β-amyloid) in brain tissue, there is still no clear diagnosis and treatment method. The most common diagnostic methods for Alzheimer's disease include imaging such as magnetic resonance imaging (MRI) and positron emission tomography (PET), as well as indirect methods such as mini mental state examination (MMSE) and questionnaires. , Imaging diagnosis imposes a large burden on patients due to the high cost and limitations of equipment, and the accuracy of diagnosis through MMSE is problematic because the results vary depending on age, education, etc.

Recently, research on blood biomarkers for the diagnosis of Alzheimer's disease has been actively conducted worldwide. However, the results to date are extremely limited, and there are various contradictory opinions and results. In the case of protein biomarkers, quantitative values differ depending on the binding state or aggregation between the countless proteins present in the body. Therefore, research on other types of biomarkers other than proteins is urgently needed.

Republic of Korea Patent Publication No. 10-2018-0067313 Republic of Korea Patent Publication No. 10-2020-0097636

The purpose of the present invention is to provide a composition for diagnosing Alzheimer's disease using a novel miRNA biomarker.

Additionally, the present invention aims to provide a kit for diagnosing Alzheimer's disease, including the composition for diagnosing Alzheimer's disease.

Additionally, the present invention aims to provide a method of providing information for diagnosing Alzheimer's disease using a novel miRNA biomarker.

One aspect of the present invention is hsa-miR-1-3p, hsa-miR-10395-3p, hsa-miR-122-3p, hsa-miR-1273c, hsa-miR-1287-3p, hsa-miR-1296- 3p, hsa-miR-136-3p, hsa-miR-214-3p, hsa-miR-215-5p, hsa-miR-27a-5p, hsa-miR-29a-3p, hsa-miR-301b-5p, hsa-miR-3175, hsa-miR-3179, hsa-miR-323b-3p, hsa-miR-329-3p, hsa-miR-3651, hsa-miR-3667-3p, hsa-miR-375-3p, hsa-miR-376a-2-5p, hsa-miR-376b-5p, hsa-miR-376c-5p, hsa-miR-3920, hsa-miR-409-5p, hsa-miR-410-3p, hsa- miR-4301, hsa-miR-4436b-3p, hsa-miR-4645-3p, hsa-miR-494-3p, hsa-miR-497-5p, hsa-miR-5582-3p, hsa-miR-598- 5p, hsa-miR-642a-5p, hsa-miR-6809-5p, hsa-miR-6842-5p, hsa-miR-6858-3p, hsa-miR-7112-3p, hsa-miR-758-5p, Provided is a composition for diagnosing Alzheimer's disease, including an agent for measuring the expression level of at least one type of miRNA selected from the group consisting of hsa-miR-933, hsa-miR-99a-5p, and Mature_426.

"Alzheimer's disease" as used in the present invention is a brain pathology in which neuritic plaques and neurofibrillary tangles associated with deposition of beta-amyloid are observed, and overall brain atrophy due to loss of nerve cells is observed with the naked eye. It refers to a degenerative brain disease that shows symptoms. In the beginning, memory decline mainly occurs, but as it progresses, not only cognitive function declines, but also mental behavioral symptoms such as personality changes, agitated behavior, depression, delusions, hallucinations, increased aggression, and sleep disorders become more severe.

In the present invention, miRNA can be used as a biomarker for diagnosing Alzheimer's disease. Here, a biomarker is a substance that can generally be detected in a biological sample and includes all organic biomolecules such as polypeptides, proteins, nucleic acids, genes, lipids, glycolipids, glycoproteins, and sugars that can detect biological changes.

"miRNA" used in the present invention is also called micro RNA, and is a 21 to 23 non-coding RNA that regulates gene expression post-transcriptionally by promoting the degradation of target RNA or suppressing their translation. It means RNA. Generally, miRNA is transcribed into a precursor about 70 to 80 nt (nucleotide) long with a hairpin structure called pre-miRNA, and then cleaved by Dicer, an RNAse III enzyme, to produce a mature form. More than 30% of human miRNAs exist in clusters, and are transcribed as a single precursor and then undergo a cleavage process to form the final mature miRNA.

These miRNAs consist of specific miRNAs that show specific expression level changes in Alzheimer's disease patients identified through next-generation sequencing (NGS), and can be presented as biomarkers for diagnosing Alzheimer's disease.

The NGS has the multiplexing ability to perform hundreds of thousands of reactions simultaneously, and sequencing is possible even with a small amount of sample. NGS has somewhat different specific application techniques depending on the commercialized technology, but generally uses clonal amplification, massively parallel sequencing, and a new sequencing method that has a different mechanism of action from the Sanger method. Commercialized technologies include the 454 GS improved FLX model sequencer released by Roche in 2007, Genome Analyzer HiSeq released by Illumina in 2006, and SOLiD released by Applied Biosystems in 2007. These three platforms have in common the adoption of clone amplification technology, abandoning complex library construction and cloning processes, massively parallel sequencing technology that can process large quantities at once, and cyclic sequencing. By determining the base sequence through sequencing by synthesis, a complicated electrophoresis process was eliminated. In addition, short reads read using the shotgun method are arranged by a computer, and an algorithm is used to find duplicated parts and complete the whole.

These 41 miRNAs for diagnosing Alzheimer's disease include not only previously identified miRNAs but also novel miRNAs first identified as miRNAs by the present inventors.

The new miRNA has not been reported in existing references and corresponds to Mature_426.

According to one embodiment of the present invention, Mature_426 may be represented by SEQ ID NO: 1.

These 41 miRNAs can be used alone or in combination of two or more types as biomarkers for diagnosing Alzheimer's.

More specifically, for a more accurate diagnosis of Alzheimer's disease among the 41 miRNAs, it is desirable to use either a single miRNA or a combination of miRNAs with a statistically significant expression difference (|fc| > 2 and/or p value < 0.01).

According to one embodiment of the present invention, the miRNA is hsa-miR-10395-3p, hsa-miR-122-3p, hsa-miR-1273c, hsa-miR-1296-3p, hsa-miR-29a-3p, hsa-miR-301b-5p, hsa-miR-3651, hsa-miR-3667-3p, hsa-miR-375-3p, hsa-miR-376b-5p, hsa-miR-376c-5p, hsa-miR- 3920, hsa-miR-642a-5p, hsa-miR-6809-5p, hsa-miR-6858-3p, hsa-miR-7112-3p, hsa-miR-758-5p, hsa-miR-933 and Mature_426. It may be one or more types selected from the group consisting of.

In addition, according to one embodiment of the present invention, the miRNA is hsa-miR-10395-3p, hsa-miR-122-3p, hsa-miR-1273c, hsa-miR-376b-5p, hsa-miR-376c- It may be one or more types selected from the group consisting of 5p and hsa-miR-6809-5p.

The expression level of miRNA according to the present invention can be measured in biological samples.

“Biological samples” used in the present invention include cells and tissues as well as organic biomolecules such as nucleic acids, genes, peptides, amino acids, proteins, lipids, sugars, glycolipids, and glycoproteins that are derived from living organisms and can detect biological changes. It refers to the sample containing. In the present invention, there is no limitation as long as the sample contains miRNA.

According to one embodiment of the present invention, the biological sample may be one or more types selected from the group consisting of blood, plasma, serum, lymph, saliva, urine, and tissue.

The agent may be a substance that binds to miRNA.

According to one embodiment of the present invention, the agent may be one or more selected from the group consisting of primers, probes, and antisense oligonucleotides that specifically bind to miRNA.

As used in the present invention, “specifically binding” means that the binding ability to the target substance is superior to that of other substances to the extent that the presence of the target substance can be detected by binding.

As used herein, a “primer” is a nucleic acid sequence with a short free 3-terminal hydroxyl group, a single-stranded oligomer that can base pair with a complementary template and serves as a starting point for copying the template strand. It means nucleotide (single strand oligonucleotide). The primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and a reagent for polymerization (i.e., DNA polymerase or reverse transcriptase) at an appropriate buffer solution and temperature. In addition, the primer consists of sense and antisense nucleic acids with a 7 to 50 nucleotide sequence, and contains 15 to 30 nucleotides at a level that does not change the basic properties of the primer, which serves as the starting point for DNA synthesis. It may have a nucleotide sequence.

As used herein, “probe” refers to a linear oligomer of natural or modified monomers or linkages, including deoxyribonucleotides and ribonucleotides, and specifically hybridizing to the target nucleotide sequence. It can exist naturally or be artificially synthesized.

Another aspect of the present invention provides a kit for diagnosing Alzheimer's disease, including the composition for diagnosing Alzheimer's disease described above.

The "Alzheimer's disease diagnostic kit" used in the present invention refers to a substance that can diagnose Alzheimer's disease through biological samples collected from a test subject, and through this, Alzheimer's disease can be diagnosed quickly, accurately, and easily. In the present invention, the kit for diagnosing Alzheimer's disease includes an agent capable of measuring the expression level of miRNA, and the agent preferably includes a primer or primer set.

According to one embodiment of the present invention, the kit may further include a reagent for nucleic acid amplification.

The nucleic acid amplification reagent may include conventional PCR reagents known in the art, and examples may include nucleoside triphosphate, (deoxy)ribonuclease inhibitor, heat-stable polymerase, and reaction buffer. may, but is not limited to this.

The nucleoside triphosphate refers to a molecule in which a purine-based or pyrimidine-based nucleoside is bound to ribose or deoxyribose and is bound to three phosphate groups, (deoxyribose ) Adenosine triphosphate ((deoxy)adenosine triphosphate, ATP or dATP), (deoxy)guanosine triphosphate ((deoxy)guanosine triphosphate, GTP or dGTP), (deoxy)cytidine triphosphate ((deoxy)cytidine triphosphate , CTP or dCTP) and (deoxy)thymidine triphosphate (TTP or dTTP).

The reaction buffer may be Taq polymerase buffer, PCR buffer, etc., and is a compound added to the amplification reaction that changes the stability, activity, and/or longevity of one or more components of the amplification reaction by adjusting the pH of the amplification reaction. This reaction buffer may further contain additives to optimize efficient PCR reaction.

According to one embodiment of the present invention, the kit may be one or more types selected from the group consisting of PCR, RT-PCR kit, real-time PCR kit, and microarray chip kit.

According to one embodiment of the present invention, the kit may further include a reagent for nucleic acid isolation.

The nucleic acid isolation reagent is a typical RNA or miRNA extraction reagent known in the art, and may include salts capable of cell lysis, surfactants, metal ions, sugars, reducing agents (e.g., DTT), etc. .

The kit according to the present invention may include a diagnostic kit based on conventional quantitative gene analysis without limitation.

For example, when the kit is applied to a PCR amplification process, the kit of the present invention may optionally include reagents necessary for PCR amplification, such as buffer, DNA polymerase, DNA polymerase cofactor, and dNTPs. In addition, the kit according to the present invention can be manufactured in a number of separate packaging or compartments containing the above-mentioned reagent components, and the kit according to the present invention is a diagnostic kit containing the essential elements required to perform DNA or RNA chipping. It can be.

Another aspect of the invention includes a) collecting a biological sample from an individual in need of diagnosis; b) hsa-miR-1-3p, hsa-miR-10395-3p, hsa-miR-122-3p, hsa-miR-1273c, hsa-miR-1287-3p, hsa-miR-1296- in the biological sample. 3p, hsa-miR-136-3p, hsa-miR-214-3p, hsa-miR-215-5p, hsa-miR-27a-5p, hsa-miR-29a-3p, hsa-miR-301b-5p, hsa-miR-3175, hsa-miR-3179, hsa-miR-323b-3p, hsa-miR-329-3p, hsa-miR-3651, hsa-miR-3667-3p, hsa-miR-375-3p, hsa-miR-376a-2-5p, hsa-miR-376b-5p, hsa-miR-376c-5p, hsa-miR-3920, hsa-miR-409-5p, hsa-miR-410-3p, hsa- miR-4301, hsa-miR-4436b-3p, hsa-miR-4645-3p, hsa-miR-494-3p, hsa-miR-497-5p, hsa-miR-5582-3p, hsa-miR-598- 5p, hsa-miR-642a-5p, hsa-miR-6809-5p, hsa-miR-6842-5p, hsa-miR-6858-3p, hsa-miR-7112-3p, hsa-miR-758-5p, Measuring the expression level of one or more types of miRNA selected from the group consisting of hsa-miR-933, hsa-miR-99a-5p, and Mature_426; and c) comparing the measured expression level of the miRNA with a control group.

The above steps a) to c) will be discussed in detail below, and description of content in common with the above-described content will be omitted to avoid excessive complexity.

Step a) is a process of collecting biological samples from a subject who needs to be tested for Alzheimer's disease.

According to one embodiment of the present invention, the biological sample in step a) may be one or more types selected from the group consisting of blood, plasma, serum, lymph, saliva, urine, and tissue.

Step b) is a process of measuring the presence or absence of biomarker miRNA and the expression level in the biological sample.

According to one embodiment of the present invention, the miRNAs in step b) are hsa-miR-10395-3p, hsa-miR-122-3p, hsa-miR-1273c, hsa-miR-1296-3p, hsa-miR- 29a-3p, hsa-miR-301b-5p, hsa-miR-3651, hsa-miR-3667-3p, hsa-miR-375-3p, hsa-miR-376b-5p, hsa-miR-376c-5p, hsa-miR-3920, hsa-miR-642a-5p, hsa-miR-6809-5p, hsa-miR-6858-3p, hsa-miR-7112-3p, hsa-miR-758-5p, hsa-miR- It may be one or more types selected from the group consisting of 933 and Mature_426.

In addition, according to one embodiment of the present invention, the miRNAs in step b) are hsa-miR-10395-3p, hsa-miR-122-3p, hsa-miR-1273c, hsa-miR-376b-5p, hsa- It may be one or more types selected from the group consisting of miR-376c-5p and hsa-miR-6809-5p.

According to one embodiment of the present invention, the level of miRNA expression in step b) is determined by polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), competitive RT-PCR, real-time RT-PCR, nuclease It may be measured by one or more methods selected from the group consisting of nuclease protection assay, in situ hybridization, microarray, and Northern blot.

It is preferable to use the composition or kit for diagnosing Alzheimer's disease according to the present invention described above to determine this level of miRNA expression.

Step c) is a process of determining whether or not there is Alzheimer's disease by comparing the level of miRNA expression measured in the biological sample with the control group.

According to one embodiment of the present invention, the control group may be a normal person, an individual without symptoms suspected of Alzheimer's disease or not diagnosed with Alzheimer's disease, or a sample obtained therefrom.

According to one embodiment of the present invention, step c) is performed in an individual in hsa-miR-1-3p, hsa-miR-122-3p, hsa-miR-136-3p, hsa-miR-215-5p, hsa- miR-3175, hsa-miR-3179, hsa-miR-323b-3p, hsa-miR-329-3p, hsa-miR-3667-3p, hsa-miR-375-3p, hsa-miR-376a-2- 5p, hsa-miR-376b-5p, hsa-miR-376c-5p, hsa-miR-3920, hsa-miR-409-5p, hsa-miR-410-3p, hsa-miR-494-3p, hsa- miR-5582-3p, hsa-miR-6809-5p, hsa-miR-6842-5p, hsa-miR-6858-3p, hsa-miR-7112-3p, hsa-miR-758-5p, hsa-miR- Alzheimer's disease may be diagnosed when the expression level of one or more types of miRNA selected from the group consisting of 933 and hsa-miR-99a-5p is higher than that of the control group.

In addition, according to one embodiment of the present invention, step c) is performed in an individual in hsa-miR-10395-3p, hsa-miR-1273c, hsa-miR-1287-3p, hsa-miR-1296-3p, hsa- miR-214-3p, hsa-miR-27a-5p, hsa-miR-29a-3p, hsa-miR-301b-5p, hsa-miR-3651, hsa-miR-4301, hsa-miR-4436b-3p, When the expression level of one or more miRNAs selected from the group consisting of hsa-miR-4645-3p, hsa-miR-497-5p, hsa-miR-598-5p, hsa-miR-642a-5p, and Mature_426 is lower than the control group It may be a diagnosis of Alzheimer's disease.

The present invention provides a composition and kit for diagnosing Alzheimer's disease by presenting a new biomarker that can predict Alzheimer's disease, thereby enabling rapid and accurate diagnosis of Alzheimer's disease patients and providing appropriate treatment to Alzheimer's disease patients.

Figure 1 shows the number of reads for a total of 18 samples according to an embodiment of the present invention.
Figure 2 shows smRNA (small RNA) types and their ratios classified in processed data according to an embodiment of the present invention.
Figure 3 shows the number of mature miRNAs commonly observed in all 18 samples according to an embodiment of the present invention.
Figure 4 shows the expression distribution of each sample based on original data (number of reads), log transformation of reads per million (CPM), and TMM normalization according to an embodiment of the present invention.
Figure 5 shows the expression density of each sample based on original data (number of reads), log transformation to reads per million (CPM), and TMM normalization according to an embodiment of the present invention.
Figure 6 shows the similarity between each sample using the Pearson correlation coefficient for normal values according to an embodiment of the present invention.
Figure 7 shows hierarchical clustering analysis using normal values according to an embodiment of the present invention.
Figure 8 shows multidimensional scale analysis using normal values according to an embodiment of the present invention.
Figure 9 shows the number of up- and down-regulated mature miRNAs based on the fold change (fc) between the NC group and the LOAD group according to an embodiment of the present invention.
Figure 10 shows the number of up- and down-regulated mature miRNAs based on the fold change and p value between the NC group and the LOAD group according to an embodiment of the present invention.
Figure 11 shows the log-transformed fold change and p value comparing the NC group and the LOAD group according to an embodiment of the present invention.
Figure 12 shows transcripts with expression differences compared to the NC group according to an embodiment of the present invention.
Figure 13 is a hierarchical clustering analysis of the similarity between 41 mature miRNAs and samples according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail with reference to the attached drawings. However, this description is merely provided as an example to aid understanding of the present invention, and the scope of the present invention is not limited by this example description.

Example: Discovery of new biomarkers for diagnosing Alzheimer's disease

1. Population selection

From April 2020 to February 2021, 9 normal controls (NC) and 9 patients with late-onset Alzheimer's disease (LOAD) who underwent Alzheimer's disease tests including MMSE were selected to form the population (Table One).

division ID gender age MMSE NC 19 female 72 28 25 female 73 30 77 female 73 29 21 female 74 30 7 female 75 28 12 other 75 30 30 female 76 30 26 female 77 27 39 other 79 18 LOAD 86 female 72 25 46 too much 73 25 56 female 73 21 13 female 75 21 48 other 75 29 63 female 75 22 41 female 76 23 16 female 77 15 17 other 79 23

2. miRNA sequence analysis and data processing

NGS analysis was performed to analyze gene expression patterns between normal people and Alzheimer's disease patients. Blood samples collected from the population were used for miRNA sequencing ( Homo sapiens miRNA sequencing) and were requested to Macrogen Co., Ltd. Briefly, RNA present in blood was synthesized into cDNA using PrimeScript reverse transcriptase (RT) and SMARTer® smRNA-Seq Kit for Illumina® (TaKaRa), and then PCR amplification was performed. For data processing after sequence analysis, the quality of the raw sequence reads was evaluated and reads containing adapter sequences were excluded, and trimmed reads and reads without adapter sequences (non-adapter reads) were evaluated for data processing. read) (≥ 50 bp) was selected (Figure 1). Afterwards, reads that were 100% identical in read length, sequence identity, etc. were collected to form a cluster, and then unnecessary rRNA was removed and used for miRNA profiling. The RNA remaining after removing most of the rRNA was short RNA of 18 to 100 nt, and included a trimmed read of 47 bp and mature miRNA of 20 to 25 nt.

3. miRNA profiling

Novel miRNAs were predicted from the processed data using the miRNA analysis software miRDeep2. First, the reference genome, miRBase v22.1 (2,656 human miRNAs) and the non-coding RNA database, RNAcentral 14.0, were sequentially used to identify previously known miRNAs and other RNAs such as tRNA, snRNA, and snoRNA. classified (Figure 2). Afterwards, using the reference Homo sapiens GRCh3, a previously unreported miRNA was determined to be a new miRNA.

4. Differentially expressed miRNA analysis results

Differential expression miRNA analysis was performed between samples in the NC group (n = 9) and the LOAD group (n = 9). In the differentially expressed miRNA analysis, the number of mature miRNA reads obtained from the miRDeep2 Quantifier module was used as raw data.

First, data quality and similarity tests were performed as a data preprocessing process. Of a total of 3,473 mature miRNAs (original data), 359 were commonly observed in all 18 samples, while 1,119 were not observed at all in all samples. 909 mature miRNAs (processed data) commonly observed in more than 9 samples, which is more than half of the total number of samples, were used for analysis (Figure 3). Afterwards, data transformation and normalization were performed to reduce systematic bias in the data. The read size of the original data was estimated using the calcNormFactors method, and the number of reads was log-transformed into counts per million reads (CPM), which was normalized using the Trimmed mean of M-values (TMM) method (Figure 4). Here, 1 was added to the CPM or normalized number of reads to prevent negative values. Then, raw data, log transformation and TMM normalization were visualized to show expression density per sample (Figure 5). To analyze the correlation between each sample, the similarity between each sample was determined using Pearson's coefficient (r) of the normalized value for each sample. The correlation coefficient (r) was found to range from about 0.8 to 1.0 (FIG. 6). Subsequently, in hierarchical clustering analysis, samples with high expression similarity were grouped using the normal value of each sample (FIG. 7). Here, Euclidean distance was used as the distance metric, and Complete Linkage was used as the linkage method. In order to show the variability of the entire data, the similarity between samples was shown in a two-dimensional graph using the normal value of each sample through multidimensional scaling analysis (FIG. 8). In the graph, the presence or absence of samples with outliers and the similarity of expression patterns between groups could be confirmed.

Afterwards, statistical analysis (fold change and exactTest) was performed on 909 mature miRNAs to identify up- and down-regulated mature miRNAs between the NC group and the LOAD group. Among 909, there were 99 mature miRNAs that showed expression differences by fold change (fc) (Figure 9), and 41 mature miRNAs showed significant expression differences by p value along with fold change (Figure 10) . |fc| 41 mature miRNAs that satisfied the conditions of ≥ 2 and exactTest initial p value < 0.05 were selected as significant data (Table 2). The fold change and p value were log-transformed for comparison between the NC group and the LOAD group (FIG. 11). In addition, a scatter plot was used to identify miRNAs with a large expression difference compared to the NC group according to the overall average expression level (Figure 12). For example, a mature miRNA can be said to be more reliable if it is higher than the average logCPM even if there is a more than 2-fold difference in fold change. Taken together, the similarity between mature miRNAs and samples was clustered according to the expression level (normal value) of the core data and displayed in hierarchical clustering analysis (Figure 13).

fold change
> 2 p-value
< 0.05 fold change
> 2 p-value
< 0.05 hsa-miR-1-3p 2.630 0.0169 hsa-miR-376c-5p 3.601 0.0013 hsa-miR-10395-3p -16.137 0.0003 hsa-miR-3920 2.623 0.0097 hsa-miR-122-3p 3.732 0.0084 hsa-miR-409-5p 2.178 0.0471 hsa-miR-1273c -4.437 0.0094 hsa-miR-410-3p 2.432 0.0194 hsa-miR-1287-3p -2.418 0.0347 hsa-miR-4301 -2.602 0.0487 hsa-miR-1296-3p -4.220 0.0419 hsa-miR-4436b-3p -2.059 0.0432 hsa-miR-136-3p 2.287 0.0155 hsa-miR-4645-3p -2.103 0.0365 hsa-miR-214-3p -2.176 0.0280 hsa-miR-494-3p 2.309 0.0188 hsa-miR-215-5p 2.322 0.0380 hsa-miR-497-5p -2.221 0.0182 hsa-miR-27a-5p -2.173 0.0284 hsa-miR-5582-3p 2.854 0.0421 hsa-miR-29a-3p -2.483 0.0028 hsa-miR-598-5p -2.970 0.0313 hsa-miR-301b-5p -2.722 0.0051 hsa-miR-642a-5p -3.564 0.0397 hsa-miR-3175 2.965 0.0451 hsa-miR-6809-5p 3.353 0.0016 hsa-miR-3179 2.148 0.0373 hsa-miR-6842-5p 2.520 0.0101 hsa-miR-323b-3p 2.119 0.0465 hsa-miR-6858-3p 3.550 0.0245 hsa-miR-329-3p 2.223 0.0160 hsa-miR-7112-3p 3.619 0.0215 hsa-miR-3651 -5.177 0.0240 hsa-miR-758-5p 4.976 0.0153 hsa-miR-3667-3p 3.153 0.0258 hsa-miR-933 3.209 0.0299 hsa-miR-375-3p 3.110 0.0212 hsa-miR-99a-5p 2.373 0.0148 hsa-miR-376a-2-5p 2.451 0.0208 Mature_426 -5.677 0.0425 hsa-miR-376b-5p 3.670 0.0022 * hsa-miR is a mature miRNA reported in the reference, and Mature is a sequence predicted to be a new miRNA.

Table 2 above shows 41 mature miRNAs corresponding to the core data, including 40 previously reported mature miRNAs and 1 new mature miRNA identified for the first time in this sequence analysis. These miRNAs were selected as candidate biomarkers for diagnosing Alzheimer's disease.

The sequences of the new mature miRNAs are shown in Table 3 below.

Novel mature miRNA Base sequence (5'-3') sequence number Mature_426 GAAGGCAGAGGAAGAATC One

And, as shown in Table 4 below, among the 41 miRNAs, 19 miRNAs with a fold change (fc) in expression level compared to the NC group exceeding 3-fold or a p value of less than 0.01 (fc > 3 or p < 0.01) were used for diagnosing Alzheimer's disease. It was selected as a biomarker, and among these, six miRNAs (fc > 3 and p < 0.01) that satisfy all of the above conditions can be expected to improve the accuracy and reliability of Alzheimer's disease diagnosis.

fold change
> 3 p-value
< 0.01 hsa-miR-10395-3p O O hsa-miR-122-3p O O hsa-miR-1273c O O hsa-miR-1296-3p O - hsa-miR-29a-3p - O hsa-miR-301b-5p - O hsa-miR-3651 O - hsa-miR-3667-3p O - hsa-miR-375-3p O - hsa-miR-376b-5p O O hsa-miR-376c-5p O O hsa-miR-3920 - O hsa-miR-642a-5p O - hsa-miR-6809-5p O O hsa-miR-6858-3p O - hsa-miR-7112-3p O - hsa-miR-758-5p O - hsa-miR-933 O - Mature_426 O -

So far, the present invention has been examined focusing on its preferred embodiments. A person skilled in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the equivalent scope should be construed as being included in the present invention.

<110> BioNano Health Guard Research Center SEOUL NATIONAL UNIVERSITY HOSPITAL <120> Biomarkers for diagnosing Alzheimer's disease and uses it <130> SNUBH <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 18 <212> RNA <213> Artificial Sequence <220> <223> Mature_426 <400> 1 gaaggcagag gaagaatc 18

Claims (12)

A composition for diagnosing Alzheimer's disease that measures the expression level of miRNAs consisting of hsa-miR-10395-3p, hsa-miR-3651, hsa-miR-758-5p, hsa-miR-1273c, hsa-miR-1296-3p, and Mature_426;
The composition includes hsa-miR-1-3p, hsa-miR-122-3p, hsa-miR-1287-3p, hsa-miR-136-3p, hsa-miR-214-3p, hsa-miR-215-5p , hsa-miR-27a-5p, hsa-miR-29a-3p, hsa-miR-301b-5p, hsa-miR-3175, hsa-miR-3179, hsa-miR-323b-3p, hsa-miR-329 -3p, hsa-miR-3667-3p, hsa-miR-375-3p, hsa-miR-376a-2-5p, hsa-miR-376b-5p, hsa-miR-376c-5p, hsa-miR-3920 , hsa-miR-409-5p, hsa-miR-410-3p, hsa-miR-4301, hsa-miR-4436b-3p, hsa-miR-4645-3p, hsa-miR-494-3p, hsa-miR -497-5p, hsa-miR-5582-3p, hsa-miR-598-5p, hsa-miR-642a-5p, hsa-miR-6809-5p, hsa-miR-6842-5p, hsa-miR-6858 -3p, hsa-miR-7112-3p, hsa-miR-933, and hsa-miR-99a-5p, further comprising an agent for measuring the expression level of one or more types of miRNA selected from the group consisting of,
The Mature_426 is a miRNA composed of the base sequence represented by SEQ ID NO: 1,
Composition for diagnosing Alzheimer's disease.
delete delete delete In claim 1,
A composition wherein the agent is at least one selected from the group consisting of primers, probes, and antisense oligonucleotides that specifically bind to miRNA. A kit for diagnosing Alzheimer's disease comprising the composition of any one of claims 1 and 5.
In claim 6,
The kit further includes a reagent for nucleic acid amplification. a) obtaining a biological sample isolated from an individual requiring diagnosis;
b) Measurement of the expression level of miRNAs consisting of hsa-miR-10395-3p, hsa-miR-3651, hsa-miR-758-5p, hsa-miR-1273c, hsa-miR-1296-3p and Mature_426 in the biological samples. steps; and
c) a method of providing information for diagnosing Alzheimer's disease, comprising comparing the measured expression level of the miRNA with a control group;
Step b) is hsa-miR-1-3p, hsa-miR-122-3p, hsa-miR1287-3p, hsa-miR-136-3p, hsa-miR-214-3p, hsa-miR-215-5p. , hsa-miR-27a-5p, hsa-miR-29a-3p, hsa-miR-301b-5p, hsa-miR-3175, hsa-miR-3179, hsa-miR-323b-3p, hsa-miR-329 -3p, hsa-miR-3667-3p, hsa-miR-375-3p, hsa-miR-376a-2-5p, hsa-miR-376b-5p, hsa-miR-376c-5p, hsa-miR-3920 , hsa-miR-409-5p, hsa-miR-410-3p, hsa-miR-4301, hsa-miR-4436b-3p, hsa-miR-4645-3p, hsa-miR-494-3p, hsa-miR -497-5p, hsa-miR-5582-3p, hsa-miR-598-5p, hsa-miR-642a-5p, hsa-miR-6809-5p, hsa-miR-6842-5p, hsa-miR-6858 -3p, hsa-miR-7112-3p, hsa-miR-933, and hsa-miR-99a-5p, which further comprises an agent for measuring the expression level of at least one type of miRNA selected from the group consisting of,
How to provide information for diagnosing Alzheimer's disease.
In claim 8,
A method wherein the biological sample in step a) is one or more selected from the group consisting of blood, plasma, serum, lymph, saliva, urine, and tissue. In claim 8,
The expression level of miRNA in step b) was determined by polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), competitive RT-PCR, real-time RT-PCR, nuclease protection assay, in A method that is measured by one or more methods selected from the group consisting of situ hybridization, microarray, and Northern blot. In claim 8,
Step c) is performed in an individual by performing hsa-miR-1-3p, hsa-miR-122-3p, hsa-miR-136-3p, hsa-miR-215-5p, hsa-miR-3175, and hsa-miR-3179. , hsa-miR-323b-3p, hsa-miR-329-3p, hsa-miR-3667-3p, hsa-miR-375-3p, hsa-miR-376a-2-5p, hsa-miR-376b-5p , hsa-miR-376c-5p, hsa-miR-3920, hsa-miR-409-5p, hsa-miR-410-3p, hsa-miR-494-3p, hsa-miR-5582-3p, hsa-miR -6809-5p, hsa-miR-6842-5p, hsa-miR-6858-3p, hsa-miR-7112-3p, hsa-miR-758-5p, hsa-miR-933 and hsa-miR-99a-5p A method of diagnosing Alzheimer's disease when the expression level of one or more types of miRNA selected from the group consisting of is higher than that of the control group. In claim 8,
Step c) is performed in the individual in hsa-miR-10395-3p, hsa-miR-1273c, hsa-miR-1287-3p, hsa-miR-1296-3p, hsa-miR-214-3p, hsa-miR-27a -5p, hsa-miR-29a-3p, hsa-miR-301b-5p, hsa-miR-3651, hsa-miR-4301, hsa-miR-4436b-3p, hsa-miR-4645-3p, hsa-miR A method of diagnosing Alzheimer's disease when the expression level of one or more types of miRNA selected from the group consisting of -497-5p, hsa-miR-598-5p, hsa-miR-642a-5p, and Mature_426 is lower than that of the control group.