Diagnostic marker for early minimally invasive diagnosis of Alzheimer's disease and application and product thereof

Abstract

The invention relates to a diagnostic marker for early minimally invasive diagnosis of Alzheimer's disease. A miRNA marker composition comprises eight paired miRNAs. According to the diagnostic marker, serum can be adopted as a detection sample, the expression quantity of the diagnostic marker between an early Alzheimer's disease patient and a normal person has stable difference, and the differential expression quantity of eight paired miRNAs between the early Alzheimer's disease patient and the normal person can be accurately and sensitively detected by training an algorithm model; the AUC of a combined model of eight paired miRNAs can reach 0.907, and the sensitivity and the specificity of the combined model are 82.6% and 81.0% respectively.

Description

Technical Field

[0001] The present invention relates to the field of biological detection markers, and particularly to a miRNA marker composition for early diagnosis of Alzheimer's disease and its preliminary application. Background Art

[0002] Alzheimer's disease (AD) is a degenerative disease of the central nervous system, mainly occurring in the elderly or pre-elderly. The main features of the disease include progressive cognitive impairment and behavioral impairment. At present, there is no effective treatment for Alzheimer's disease, and only early detection and early intervention can delay or even reverse the progression of the disease. Therefore, early diagnosis of Alzheimer's disease is the key to delaying disease progression and improving the quality of life of patients.

[0003] Currently, in clinical practice, the guidelines formulated by the National Institute on Aging - Alzheimer's Association (NIA - AA) in 2011 are mainly followed, and a comprehensive analysis is carried out based on the patient's clinical symptoms or manifestations, neuropsychological scales, and imaging techniques for diagnosis. The only definitive diagnosis method is pathological examination of brain tissue. However, the neuropsychological scales and clinical dementia rating scales commonly used in clinical practice to evaluate cognitive function are not very sensitive to mild dementia; in the field of brain structure imaging, although CT and MRI techniques can clearly show changes in brain tissue structures such as brain atrophy and ventricular enlargement, they are not sufficient to provide reliable diagnostic evidence for mild patients with insignificant brain atrophy.

[0004] In recent years, the detection of AD genes has also been developing rapidly. Currently, the generally recognized AD susceptibility genes include the amyloid precursor protein (APP) gene, presenilin (PS) gene, and apolipoprotein E (ApoE) gene. These genes can be used to screen AD - susceptible populations and analyze family genetic characteristics, but they still cannot be used for diagnosis. At present, there are few types of biomarkers that can diagnose AD, and their sensitivity and specificity are not ideal. Only a few can be used as auxiliary diagnostic methods in clinical practice, such as β - amyloid protein, tau protein, etc. Their expressions in the cerebrospinal fluid of AD patients are significantly abnormal, but due to the invasiveness of cerebrospinal fluid collection, their clinical applications are limited. Whether biomarkers such as β - amyloid protein and tau protein in blood can be used as biomarkers for AD diagnosis is still controversial. The early Aβ and tau detections had low specificity and sensitivity, which limited their clinical applications. Therefore, new biomarkers and biomarker enrichment detection methods are urgently needed.

[0005] In summary, the early diagnosis of Alzheimer's disease remains a clinical challenge. Existing detection methods are either invasive diagnostic methods that cause serious trauma or are not sensitive enough for early detection. Among biomarker detection methods using blood as the detection object, no diagnostic biomarker with good detection specificity and high sensitivity has been screened yet. Summary of the Invention

[0006] The present invention aims to provide a diagnostic biomarker for minimally invasive early diagnosis of Alzheimer's disease (AD), its application, and a product for early diagnosis of AD to address the deficiencies in the prior art. The technical problems to be solved by the present invention are achieved through the following technical solutions.

[0007] The present invention creatively uses serum as the detection sample, solving the problem that existing biomarker diagnoses require cerebrospinal fluid collection and cause relatively large trauma. Through creative efforts, 8 paired miRNAs have been screened. There are stable differences in the expression levels of the 8 paired miRNAs between early Alzheimer's disease patients and normal individuals. By training an algorithm model, the differential expression levels of the 8 paired miRNAs between early Alzheimer's disease patients and normal individuals can be accurately and sensitively detected. The AUC of the combined model of the 8 paired miRNAs can reach 0.907, and its sensitivity and specificity are 82.6% and 81.0% respectively.

[0008] Specifically, in the first aspect of the present invention, a diagnostic biomarker for minimally invasive early diagnosis of Alzheimer's disease is provided, wherein the miRNA biomarker composition comprises 8 paired miRNAs, and the 8 paired miRNAs are hsa-miR-451a / hsa-let-7c-5p, hsa-miR-377-3p / hsa-miR-193b-3p, hsa-miR-27b-3p / hsa-miR-146a-5p, hsa-miR-223-3p / hsa-miR-21-5p, hsa-miR-223-3p / hsa-miR-146a-5p, hsa-miR-20a-5p / hsa-miR-15b-3p, hsa-miR-15b-3p / hsa-miR-107, and hsa-miR-146a-5p / hsa-let-7d-3p.

[0009] Preferably, there are differences in the expression levels of the 8 paired miRNAs in the bodies of early Alzheimer's disease patients and healthy individuals.

[0010] Preferably, there are significant differences in the expression ratios of the 8 paired miRNAs in the sera of early Alzheimer's disease patients and healthy individuals.

[0011] In a second aspect of the present invention, there is provided an application of a miRNA diagnostic marker in the preparation of a product for the early diagnosis of Alzheimer's disease, wherein the miRNA diagnostic marker comprises 8 paired miRNAs, hsa-miR-451a / hsa-let-7c-5p, hsa-miR-377-3p / hsa-miR-193b-3p, hsa-miR-27b-3p / hsa-miR-146a-5p, hsa-miR-223-3p / hsa-miR-21-5p, hsa-miR-223-3p / hsa-miR-146a-5p, hsa-miR-20a-5p / hsa-miR-15b-3p, hsa-miR-15b-3p / hsa-miR-107, and hsa-miR-146a-5p / hsa-let-7d-3p.

[0012] In a specific embodiment, the product for the early diagnosis of Alzheimer's disease diagnoses or aids in the diagnosis of early AD patients by detecting the expression levels of 8 paired miRNAs in a serum sample.

[0013] Preferably, the product for the early diagnosis of Alzheimer's disease diagnoses or aids in the diagnosis of early AD patients by detecting the expression level ratios of 8 paired miRNAs in a serum sample.

[0014] Preferably, the product for the early diagnosis of Alzheimer's disease diagnoses or aids in the diagnosis of early AD patients by inputting the expression level ratios of 8 paired miRNAs into a combined diagnostic model.

[0015] In a third aspect of the present invention, there is provided a product for the early diagnosis of Alzheimer's disease, wherein the product comprises a detector for detecting a miRNA diagnostic marker, and the 8 paired miRNAs are respectively hsa-miR-451a / hsa-let-7c-5p, hsa-miR-377-3p / hsa-miR-193b-3p, hsa-miR-27b-3p / hsa-miR-146a-5p, hsa-miR-223-3p / hsa-miR-21-5p, hsa-miR-223-3p / hsa-miR-146a-5p, hsa-miR-20a-5p / hsa-miR-15b-3p, hsa-miR-15b-3p / hsa-miR-107, and hsa-miR-146a-5p / hsa-let-7d-3p.

[0016] Preferably, the product comprises a detector for detecting the expression levels of 8 paired miRNAs in a serum sample.

[0017] Preferably, the product comprises a detection agent for detecting the expression ratio of 8 paired miRNAs in a serum sample.

[0018] Preferably, the detection agent for detecting the expression of 8 paired miRNAs comprises lectin, probe, primer, enzyme and buffer.

[0019] Preferably, the primer comprises a primer having a nucleotide sequence as shown in SEQ ID NO.1-14.

[0020] Preferably, the probe comprises a probe having a nucleotide sequence as shown in SEQ ID NO.15.

[0021] In a specific embodiment, the product for early diagnosis of Alzheimer's disease is a kit.

[0022] In a specific embodiment, the product for early diagnosis of Alzheimer's disease comprises a diagnostic reagent and a diagnostic instrument.

[0023] In a specific embodiment, the detection agent for detecting miRNA diagnostic markers is a detection agent for detecting miRNA diagnostic markers in a serum glycosylated exosome sample. Specifically, a peripheral blood sample is collected, centrifuged under vacuum to remove cell debris, the supernatant is aspirated, and the GlyExo-Capture method is used for separation and characterization. This method selectively captures glycosylated exosomes through lectin.

[0024] In a specific embodiment, the product for early diagnosis of Alzheimer's disease includes a reagent for extracting glycosylated exosomes from a serum sample.

[0025] In some embodiments, the reagent for extracting glycosylated exosomes from a serum sample includes a macromolecular carrier; and lectin conjugated to the outside of the macromolecular carrier.

[0026] In some embodiments, the lectin includes any one or two or more of: artocarpin, peanut agglutinin, pea agglutinin, concanavalin A, lentil agglutinin, wheat germ agglutinin, soybean agglutinin, kidney bean agglutinin, snail agglutinin.

[0027] In some embodiments, the macromolecular carrier includes any one or two or more of: dextran microspheres, agarose microspheres, resin or epoxy resin microspheres, polystyrene microspheres.

[0028] In some embodiments, the glycosylated exosomes include any one or two or more of: N-glycosylated exosomes, O-glycosylated exosomes, core fucosylated exosomes.

[0029] In the specific embodiments, the analyte for detecting miRNA diagnostic markers is an analyte for detecting the expression ratio of 8 paired miRNAs.

[0030] In some embodiments, the analyte for detecting miRNA diagnostic markers can detect the ratio of the expression levels of 8 paired miRNAs by the following methods: high-throughput sequencing, reverse transcription real-time fluorescence PCR, in situ hybridization, and / or gene chip.

[0031] In some embodiments, the product for early diagnosis of Alzheimer's disease includes reagents and / or instruments for high-throughput sequencing (NGS), reverse transcription real-time fluorescence PCR (qPCR), in situ hybridization, and / or gene chip.

[0032] In some embodiments, the product for early diagnosis of Alzheimer's disease includes buffers, polymerases, ATP, primers with nucleotide sequences as shown in SEQ ID NOs. 1 to 14, and probes with nucleotide sequences as shown in SEQ ID NO. 15 for high-throughput sequencing and / or reverse transcription real-time fluorescence PCR.

[0033] "Diagnosis" as used in the present invention means to find out whether a patient has had, has, or will have a disease or disorder in the past, at the time of diagnosis, or in the future.

[0034] "Early diagnosis" as used in the present invention refers to the stage without any clinical symptoms and the stage with mild symptoms but still in the reversible stage. Specifically, early diagnosis of AD refers to the preclinical stage and the mild cognitive impairment stage (Mild Cognitive Impairment, MCI), that is, the AD stage 0.5 to 1. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 It is a heat map of the NGS data of the expression ratios of 8 paired miRNAs in Example 1 of the present invention;

[0036] Figure 2 It is a box plot of 8 paired miRNAs in the NGS data in Example 1 of the present invention;

[0037] Figure 3 It is an LDA plot of the qPCR data of 8 paired miRNAs in Example 2 of the present invention;

[0038] Figure 4 It is a box plot of the qPCR data of 8 paired miRNAs in Example 2 of the present invention;

[0039] Figure 5 It is an ROC curve graph in Example 3 of the present invention;

[0040] Figure 6Box plot of model scores for Example 3 of the present invention;

[0041] Figure 7 ROC curve of the comparative example. Detailed implementation mode

[0042] It should be noted that, without conflict, the embodiments in this application and the features in the embodiments can be combined with each other. The present invention will be described in detail below with reference to the drawings and in conjunction with the embodiments.

[0043] Example 1 Differential detection of NGS data of 8 paired miRNAs

[0044] 1. Extraction of exosomal miRNAs

[0045] Collect 53 peripheral blood samples, including 25 AD samples and 28 normal samples. Among them, 25 AD samples include 5 stage 0.5 samples (preclinical AD), 14 stage 1 samples (MCI-AD), 5 stage 2 samples (mild AD dementia), and 1 stage 3 sample (moderate to severe AD). Parallel experiments are carried out on all samples.

[0046] The peripheral blood samples are centrifuged at 1800x g for 10 minutes at room temperature within 1 hour after collection; the obtained serum is further centrifuged at 3000x g for 10 minutes at room temperature to remove cell debris; after sucking the supernatant, the samples are aliquoted and stored at -80°C until use.

[0047] Exosomes are selectively captured by lectin to capture glycosylated exosomes using the GlyExo-Capture method, and glycosylated exosomes in serum samples are isolated and extracted using the GlyExo-Capture extraction reagent (Beijing Kaijing Gene Technology Co., Ltd., product number: GlyE01-24).

[0048] 2. High-throughput sequencing (NGS) and bioinformatics analysis:

[0049] Use an RNA extraction kit (Qiagen, Germany, product number: 217004) to extract the total RNA of glycosylated exosomes, and perform 75-bp single-end sequencing on the Illumina NextSeq550 sequencing system. Subsequently, fastQC was used to evaluate the quality of the sequencing data, and Cutadapt software was used to remove primers and adapters. Bowtie2 was used to align the sequences with the human reference genome (HG38). Then HT-seq was used to annotate the matched data, where the gff3 file used for annotation came from miRBase version 23. After annotation, NGS sequencing data was obtained, the miRNA expression level was quantified, and a miRNA expression matrix was constructed.

[0050] Feature screening was performed using single factor and genetic algorithms, and 8 paired miRNAs (hsa-miR-451a / hsa-let-7c-5p, hsa-miR-377-3p / hsa-miR-193b-3p, hsa-miR-27b-3p / hsa-miR-146a-5p, hsa-miR-223-3p / hsa-miR-21-5p, hsa-miR-223-3p / hsa-miR-146a-5p, hsa-miR-20a-5p / hsa-miR-15b-3p, hsa-miR-15b-3p / hsa-miR-107, hsa-miR-146a-5p / hsa-let-7d-3p) were screened out. A heat map of the ratios of the expression levels of these 8 paired miRNAs was plotted on the NGS sequences, as shown in Figure 1 , Figure 1 The heat map showed that AD samples were mainly concentrated on the left side and healthy samples were mainly concentrated on the right side, and the 8 paired miRNAs showed significant differences in the NGS data.

[0051] Furthermore, a box plot of the ratios of the expression levels of the 8 paired miRNAs was plotted on the NGS data sequences, as shown in Figure 2 shown, Figure 2 The box plot of the NGS data indicated that there were significant differences in the 8 paired miRNAs between healthy samples and AD samples.

[0052] Example 2 Verification of the differences in qPCR data of 8 paired miRNAs

[0053] 1. Extraction of exosomal miRNAs

[0054] Forty-eight peripheral blood samples were collected, including 48 samples in the 2nd - 3rd stages of AD, 29 samples in the 1st stage of AD, and 69 control samples. Among them, the 69 control samples included 27 patients with other dementias and 42 patients with other neurological diseases. Parallel experiments were carried out on the collected samples according to the following method.

[0055] The method for collecting glycosylated exosomes from peripheral blood samples was the same as that in Example 1.

[0056] 2. Verification by fluorescence quantitative PCR

[0057] For the 8 paired miRNAs, primers were designed for different miRNAs, and detection and analysis were carried out using the probe-based real-time fluorescence quantitative PCR method.

[0058] The reaction process of RT-qPCR was as follows:

[0059] First, the synthesis of the first-strand cDNA was carried out, and the reaction system was as follows:

[0060] Reaction system Volume (μL) Reverse transcription primer (20 μM) 0.5 5× Reverse transcription buffer 2 Poly A polymerase (5 U / μL) 0.5 Reverse transcriptase (200 U / μL) 0.5 ATP (10 mM) 0.5 dNTP (10 mM) 1 RNA template 5 Total volume 10

[0061] The reaction conditions are as follows:

[0062] Reaction conditions Time (minutes) 42℃ 15 85℃ 1 4℃ ∞

[0063] Then, miRNA qPCR reactions were performed on an ABI 7500 real-time fluorescence quantitative PCR system, and the reaction system is as follows:

[0064] Reaction system Volume (μL) 2× qPCR reaction solution 12.5 Forward primer (10 μM) 2 Reverse primer (10 μM) 2 Probe (10 μM) 1 Rox 0.5 Nuclease-free water 2 cDNA 5 Total volume 25

[0065] The reaction conditions are as follows:

[0066]

[0067] The primer and probe sequences are as follows in the table:

[0068]

[0069] Fluorescence quantitative PCR verification used the Ct value (the number of cycles when the fluorescence signal first exceeded the background noise (threshold line) during PCR amplification) as a characterization index for the miRNA expression level, and LDA (linear discriminant analysis) was performed on the Ct values. The LDA graph is as Figure 3 shown, and it can be seen that the samples in AD2 - 3 stages and AD stage 1 samples can all be distinguished from the control.

[0070] Furthermore, standardized data analysis was performed on the qPCR data of 8 paired miRNAs, and box plots of the qPCR data of 8 paired miRNAs were prepared. The box plots of the qPCR data are shown in Figure 4 as Figure 4 shown, indicating that there are differences in 8 paired miRNAs between healthy samples and AD samples. By comparing with the box plots of NGS data in Example 1, it can be seen that the trends of AD samples in NGS and qPCR data relative to healthy samples are consistent.

[0071] Verification of the diagnostic efficacy of the combined diagnostic model in Example 3

[0072] The qPCR cohort obtained from the detection in Example 2 was randomly divided into a training set and a test set at a ratio of 7:3. The training set was used to train the model, and the test set was used to evaluate the model performance. A random forest model (the model prototype was provided by Beijing Hotgen Biotechnology Co., Ltd.) was established, and an ROC curve was plotted based on the Ct value data of qPCR to detect the performance of the training set and the test set. The ROC curve graph is as Figure 5As shown, the AUC (Area Under the ROC Curve) of the combined model of 8 paired miRNAs (hsa-miR-451a / hsa-let-7c-5p, hsa-miR-377-3p / hsa-miR-193b-3p, hsa-miR-27b-3p / hsa-miR-146a-5p, hsa-miR-223-3p / hsa-miR-21-5p, hsa-miR-223-3p / hsa-miR-146a-5p, hsa-miR-20a-5p / hsa-miR-15b-3p, hsa-miR-15b-3p / hsa-miR-107, hsa-miR-146a-5p / hsa-let-7d-3p) on the test set can reach 0.907, and its sensitivity and specificity are 82.6% and 81.0% respectively.

[0073] Draw a score box plot for the qPCR data of AD2-3 stage samples, AD1 stage samples and control samples, as Figure 6 shown, showing that there are significant differences in the model scores among AD2-3 stage samples, AD1 stage samples and control samples, and the discrimination effect is good, all with significant differences.

[0074] Comparison of the diagnostic efficacy of the combined diagnostic model in Comparative Example 1

[0075] 1. Detection and extraction of tau181, tau217 and total tau

[0076] Collect 55 plasma samples, including 15 AD2-3 stage samples, 20 AD1 stage samples, and 20 control samples (the control samples include samples of other types of dementia, other types of neurological diseases, etc.).

[0077] Use a phosphorylated tau181 protein assay kit (enzyme-linked immunosorbent assay) (Human tau181 ELISA Kit, product number XZK-807) to detect the expression level of p-Tau217 protein in serum;

[0078] Use a phosphorylated Tau217 protein assay kit (enzyme-linked immunosorbent assay) (Human tau217 ELISA Kit, product number HB-P99253) to detect the expression level of p-Tau217 protein in serum;

[0079] Use an enzyme-linked immunosorbent assay kit (Human Tau (Total) ELISA Kit, product number KHB0041) to detect the expression level of total tau protein.

[0080] 2. Verification by fluorescence quantitative PCR

[0081] The expression levels of the biomarkers detected in Step 1 were input into the combined diagnostic model described in Example 3 according to the method described in Example 3 for training, and an ROC curve was plotted to detect the performance of the training set and the test set. The ROC curve is as shown in Figure 7 Figure [not provided]. The AUC values for the discrimination of samples in AD Stages 2 - 3, AD Stage 1, and control samples by the detection results of tau181 protein, tau217 protein, and total tau protein were 0.677, 0.687, and 0.679 respectively. Compared with the AUC of 0.907 for the biomarker combination selected in the present invention in Example 3, the detection sensitivity and specificity of the present invention are significantly higher than those of the tau protein biomarkers.

[0082] It should be noted that the above detailed description is exemplary and is intended to provide further illustration of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs.

[0083] Note that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments described according to the present application. As used herein, unless the context clearly indicates otherwise, the singular forms are also intended to include the plural forms. In addition, it should also be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0084] It should be noted that the terms "first", "second", etc. in the specification, claims, and above-mentioned drawings of this application are used to distinguish similar objects and do not necessarily describe a specific order or sequence. It should be understood that such terms can be interchanged under appropriate circumstances so that the embodiments of the present application described herein can be implemented in an order other than those illustrated or described herein.

[0085] In addition, the terms "include" and "have" and any variations thereof are intended to cover non-exclusive inclusion. For example, a process, method, system, product, or device that includes a series of steps or units is not necessarily limited to those steps or units clearly listed, but may include other steps or units not clearly listed or inherent to these processes, methods, products, or devices.

[0086] In the above detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, like symbols typically identify like components, unless the context indicates otherwise. The illustrated embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be used and other changes may be made without departing from the spirit or scope of the subject matter presented herein.

[0087] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various changes and modifications. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A diagnostic marker for minimally invasive early diagnosis of Alzheimer's disease, characterized in that: The miRNA marker composition comprises 8 paired miRNAs, and the 8 paired miRNAs are hsa-miR-451a / hsa-let-7c-5p, hsa-miR-377-3p / hsa-miR-193b-3p, hsa-miR-27b-3p / hsa-miR-146a-5p, hsa-miR-223-3p / hsa-miR-21-5p, hsa-miR-223-3p / hsa-miR-146a-5p, hsa-miR-20a-5p / hsa-miR-15b-3p, hsa-miR-15b-3p / hsa-miR-107 and hsa-miR-146a-5p / hsa-let-7d-3p.

2. The diagnostic marker for early minimally invasive diagnosis of Alzheimer's disease according to claim 1, characterized in that: There are differences in the expression levels of the eight paired miRNAs in the serum of early-stage Alzheimer's disease patients and healthy subjects.

3. The diagnostic marker for early minimally invasive diagnosis of Alzheimer's disease according to claim 2, characterized in that: There is a significant difference in the expression ratio of the 8 paired miRNAs in the serum of early-stage Alzheimer's disease patients and healthy subjects.

4. Use of a miRNA diagnostic marker in the preparation of a product for diagnosis or early diagnosis of Alzheimer's disease, characterized in that: The miRNA diagnostic marker comprises the eight paired miRNAs of claims 1 to 3. Preferably, the product for diagnosing or early-stage diagnosis of Alzheimer's disease performs diagnosis by detecting the expression ratio of the eight paired miRNAs in the serum of early-stage Alzheimer's disease patients and healthy subjects. More preferably, the product for diagnosing or early-stage diagnosis of Alzheimer's disease performs diagnosis by inputting the detected expression ratio of the eight paired miRNAs into a joint diagnostic model.

5. A product for early diagnosis or auxiliary diagnosis of Alzheimer's disease, characterized in that: The product comprises a detection substance for detecting the eight paired miRNAs according to claims 1 to 3.

6. The product according to claim 6, characterized in that The product comprises a detection substance for detecting the expression levels of the eight paired miRNAs according to claims 1 to 3.

7. The product according to claim 6, characterized in that The product comprises a detection substance for detecting the expression ratio of the eight paired miRNAs described in claims 1 to 3 in a serum sample.

8. The product according to claim 6, characterized in that The detection objects for detecting the expression levels of 8 paired miRNAs include lectin, probes, primers, enzymes and buffer.

9. The product according to claim 8, characterized in that The primers include primers with nucleotide sequences as shown in SEQ ID NOs. 1-14.

10. The product according to claim 8, characterized in that The probe comprises a probe whose nucleotide sequence is shown as SEQ ID NO.15.

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