CN116179676A

CN116179676A - Kit for diagnosing Alzheimer disease and application thereof

Info

Publication number: CN116179676A
Application number: CN202210984199.0A
Authority: CN
Inventors: 贾龙飞; 任梓烨; 庞亚娜; 蔡慧敏; 傅晓凤
Original assignee: Individual
Current assignee: Individual
Priority date: 2022-08-17
Filing date: 2022-08-17
Publication date: 2023-05-30

Abstract

The present application relates in particular to a kit for diagnosing whether a subject suffers from alzheimer's disease, and the use of a reagent for determining biomarker levels in a biological sample in the preparation of a kit. The method can replace the traditional p-tau/Abeta detection of cerebrospinal fluid, realize diagnosis of AD through 6 kinds of circRNAs, and can distinguish AD patients from normal personnel and AD patients from other dementia patients.

Description

Kit for diagnosing Alzheimer disease and application thereof

Technical Field

The present application relates to the fields of medicine, virology and immunology, in particular immunology diagnostics. The present application relates in particular to a kit for diagnosing whether a subject suffers from alzheimer's disease, and the use of a reagent for determining biomarker levels in a biological sample in the preparation of a kit.

Background

The most common biomarkers of Alzheimer's Disease (AD) are Abeta and tau, which can be examined by cerebrospinal fluid or PET imaging. However, this process is invasive and very expensive and difficult to apply universally to the population. In addition, AD may present overlapping clinical manifestations, pathologies and biomarkers with other types of dementia, such as vascular dementia (vascular dementia, vaD), parkinson's disease dementia (Parkinson disease, PDD), behavior variability frontotemporal dementia (behavioural variant frontotemporal dementia, bvFTD), lewy body dementia (dementia with Lewy bodies, DLB), etc., often resulting in clinical diagnosis difficulties.

The circRNA is a non-coding RNA with a closed loop structure, and plays a biological role by adsorbing miRNA, regulating RNA binding proteins in cells and participating in protein translation. More and more studies confirm the relationship between circRNA and AD. Researches find that the CiRS-7 reduces the clearance effect of the miR-7 on Abeta by downregulating the activity of the miR-7; circ 0000950 causes neuronal damage by exerting a molecular "spongy" effect of miR-103; the circPCCA can inhibit activation of glycogen synthase kinase-3 beta and promote tau protein deposition. However, the previous research sample size is small, and clinical diagnosis is not proved by cerebrospinal fluid core markers, so that clinical transformation is limited.

Based on the above problems, there is a great need for a method for effectively diagnosing AD and distinguishing AD from other types of dementia in clinical treatment of AD.

Disclosure of Invention

The inventors established a diagnostic model of AD by analyzing the differences in the circRNAs of AD patients versus normal persons in two separate datasets and using the differential set of circRNAs. And the diagnosis efficacy of the model is verified in a third independent crowd, and the model consisting of 6 kinds of circRNAs in blood is verified to be capable of accurately diagnosing and differentiating and diagnosing AD. Thus, the circRNAs of the present application contribute to the diagnosis of AD.

Accordingly, in a first aspect, the present application provides a method for diagnosing whether a subject is suffering from alzheimer's disease comprising:

(1) Obtaining a biological sample comprising a biomarker from a subject;

(2) Determining the level of a biomarker in a biological sample; and

(3) Diagnosing whether the subject is suffering from alzheimer's disease based on the level of the biomarker;

wherein the biomarker is hsa_circ_007501, hsa_circ_0022417, hsa_circ_0014356, hsa_circ_0014353, hsa_circ_0074233 and hsa_circ_0089894.

In certain embodiments, the biological sample is selected from whole blood (e.g., peripheral blood), serum, plasma, or cerebrospinal fluid.

In certain embodiments, the biological sample is selected from whole blood (e.g., peripheral blood), serum, and plasma.

In certain embodiments, the biological sample is pre-treated to obtain circRNAs prior to step (2); step (2) is then performed, i.e. the level of the biomarker is determined. Methods for processing biological samples (e.g., whole blood, serum, and plasma) to obtain circRNAs are known to those skilled in the art. For example, circRNAs can be isolated from biological samples (e.g., whole blood, serum, and plasma) using commercial kits (e.g., miRNeasy Serum Kit (Qiagen, USA), ribo-off rRNA Depletion Kit (Vazyme, inc.).

In certain embodiments, in step (3), the subject is diagnosed with alzheimer's disease by comparing the level of the biomarker to a reference value.

In certain embodiments, the level of the biomarker is the level of circRNA. In such embodiments, the reference value is the level or range of the circRNA in a biological sample obtained from a normal population. In certain embodiments, the level of a biomarker in the biological sample is determined by quantitative PCR (e.g., real-time fluorescent quantitative PCR).

In certain embodiments, an increase in the level of hsa_circ_0077001, hsa_circ_0022417, hsa_circ_0014356, hsa_circ_0014353, hsa_circ_0074533 in the peripheral blood relative to the reference value indicates that the subject has AD. In certain embodiments, a decrease in the level of hsa_circ_0089894 in peripheral blood relative to a reference value indicates that the subject has AD.

In certain embodiments, in step (3), the levels of the 6 biomarkers are subjected to ridge regression using elastic network regression, thereby obtaining a predictive model; the predictive model is then used to predict the risk of the subject for a neurodegenerative disease.

In certain embodiments, the method further comprises determining the age, sex, and ApoE epsilon 4 genotype of the subject. In certain preferred embodiments, in step (3), the levels of the 6 biomarkers are subjected to ridge regression using elastic network regression and corrected for age, gender, and/or ApoE epsilon 4 genotype, thereby obtaining a predictive model; the predictive model is then used to diagnose whether the subject is suffering from Alzheimer's disease.

In certain embodiments, the subject is a mammal, e.g., a human.

In certain embodiments, the hsa_circ_0077001 has the sequence set forth in SEQ ID NO. 1. In certain embodiments, the hsa_circ_0022417 has the sequence set forth in SEQ ID NO. 2. In certain embodiments, the hsa_circ_0014356 has the sequence set forth in SEQ ID NO. 3. In certain embodiments, the hsa_circ_0014353 is provided as SEQ ID NO. 4. In certain embodiments, the hsa_circ_0074533 is provided as SEQ ID NO. 5. In certain embodiments, the hsa_circ_0089894 has the sequence set forth in SEQ ID NO. 6.

In certain embodiments, the methods are capable of diagnosing whether a subject is suffering from alzheimer's disease.

In a second aspect, there is provided a kit for diagnosing whether a subject has alzheimer's disease, the kit comprising reagents for determining the level of a biomarker in a biological sample, the biomarker being hsa_circ_007501, hsa_circ_0022417, hsa_circ_0014356, hsa_circ_0014353, hsa_circ_0074033 and hsa_circ_0089894.

In certain embodiments, the 2 kit comprises: a first reagent or combination of reagents for determining the hsa_circ_0077001 level of a subject, a second reagent or combination of reagents for determining the hsa_circ_0022417 level of a subject, a third reagent or combination of reagents for determining the hsa_circ_0014356 level of a subject, a fourth reagent or combination of reagents for determining the hsa_circ_0014353 level of a subject, a fifth reagent or combination of reagents for determining the hsa_circ_0074533 level of a subject, a sixth reagent or combination of reagents for determining the hsa_circ_0089894 level of a subject.

In certain embodiments, the agent (e.g., first, second, third, fourth, fifth, and/or sixth agent or combination of agents) determines the level of a biomarker in the biological sample by hybridization techniques.

In certain embodiments, the hybridization technique is selected from the group consisting of Northern blot analysis, microarray techniques, in situ hybridization, liquid chip techniques, and splint ligation.

In certain embodiments, the first reagent or combination of reagents comprises a primer and/or probe capable of quantifying the level of hsa_circ_ 0077001. In certain embodiments, the second reagent or combination of reagents comprises a primer and/or probe capable of quantifying the level of hsa_circ_ 0022417. In certain embodiments, the third reagent or combination of reagents comprises a primer and/or probe capable of quantifying the level of hsa_circ_ 0014356. In certain embodiments, the fourth reagent or combination of reagents comprises a primer and/or probe capable of quantifying the level of hsa_circ_ 0014353. In certain embodiments, the fifth reagent or combination of reagents comprises a primer and/or probe capable of quantifying the level of hsa_circ_ 0074533. In certain embodiments, the sixth reagent or combination of reagents comprises a primer and/or probe capable of quantifying the level of hsa_circ_ 0089894.

In certain embodiments, the kit has the following features: (1) The first reagent or combination of reagents comprises primers and/or probes capable of quantifying the level of hsa_circ_ 0077001; (2) The second reagent or combination of reagents comprises primers and/or probes capable of quantifying the level of hsa_circ_ 0022417; (3) The third reagent or combination of reagents comprises primers and/or probes capable of quantifying the level of hsa_circ_ 0014356; (4) The fourth reagent or combination of reagents comprises primers and/or probes capable of quantifying the level of hsa_circ_ 0014353; (5) The fifth reagent or combination of reagents comprises primers and/or probes capable of quantifying the level of hsa_circ_ 0074533; (6) The sixth reagent or combination of reagents comprises primers and/or probes capable of quantifying the level of hsa_circ_ 0089894.

In certain embodiments, the kit further comprises means for capturing a biomarker. In certain embodiments, the means for capturing the biomarker is located on a solid support (e.g., nylon membrane, microsphere).

In certain embodiments, wherein the reagent (e.g., first, second, third, fourth, fifth, and/or sixth reagent or combination of reagents) determines the level of a biomarker in the biological sample by an amplification technique.

In certain embodiments, the amplification technique is selected from qPCR, RT-PCR, stem-loop PCR, poly a PCR, RCA, sequencing.

In certain embodiments, the kit further comprises reagents for performing nucleic acid amplification and/or reagents for performing sequencing.

In certain embodiments, the reagents for performing nucleic acid amplification include, working buffers of enzymes (e.g., nucleic acid polymerase), dNTPs (labeled or unlabeled), water, ion-containing (e.g., mg ²⁺ ) Or a single-stranded DNA binding protein, or any combination thereof.

In certain embodiments, wherein the biological sample is whole blood (e.g., peripheral blood), serum, plasma, or cerebrospinal fluid obtained from a subject. In certain embodiments, the biological sample is selected from whole blood (e.g., peripheral blood), serum, and plasma. In certain embodiments, the biological sample is selected from peripheral blood.

In certain embodiments, the subject is a mammal, e.g., a human.

In certain embodiments, the kit further comprises a pretreatment reagent or combination of reagents for pretreatment of a biological sample. In certain embodiments, the pretreatment reagent or combination of reagents is used to pretreat the biological sample (e.g., whole blood, serum, or plasma) to obtain circRNAs. In certain embodiments, the pretreatment reagent or reagents comprise a circRNAs extraction purification solution, and optionally a buffer.

In certain embodiments, the kit is used to distinguish between subjects suffering from alzheimer's disease and normal subjects.

In certain embodiments, the kits are used to distinguish between subjects suffering from alzheimer's disease and subjects suffering from other dementias, e.g., vascular dementia (VaD), parkinson's Disease Dementia (PDD), behavioral variability frontotemporal dementia (bvFTD), dementia with lewy bodies (DLB).

In a third aspect, the present application provides the use of a reagent for determining the level of a biomarker in a biological sample in the manufacture of a kit for diagnosing whether a subject is suffering from alzheimer's disease; wherein the biomarker is hsa_circ_007501, hsa_circ_0022417, hsa_circ_0014356, hsa_circ_0014353, hsa_circ_0074233 and hsa_circ_0089894.

In certain embodiments, the kit comprises: a first reagent or combination of reagents for determining the hsa_circ_0077001 level of a subject, a second reagent or combination of reagents for determining the hsa_circ_0022417 level of a subject, a third reagent or combination of reagents for determining the hsa_circ_0014356 level of a subject, a fourth reagent or combination of reagents for determining the hsa_circ_0014353 level of a subject, a fifth reagent or combination of reagents for determining the hsa_circ_0074533 level of a subject, a sixth reagent or combination of reagents for determining the hsa_circ_0089894 level of a subject.

In certain embodiments, wherein the agent (e.g., first, second, third, fourth, fifth, and/or sixth agent or combination of agents) determines the level of a biomarker in the biological sample by an amplification technique.

In certain embodiments, the subject is a mammal, e.g., a human.

In certain embodiments, the kit further comprises a pretreatment reagent or combination of reagents for pretreatment of a biological sample.

In certain embodiments, the pretreatment reagent or combination of reagents is used to pretreat the biological sample (e.g., whole blood, serum, or plasma) to obtain circRNAs.

In certain embodiments, the pretreatment reagent or reagents comprise a circRNAs extraction purification solution, and optionally a buffer.

In certain embodiments, the kit diagnoses whether the subject is suffering from alzheimer's disease by the methods described above.

Interpretation of the terms

As used herein, the term "neurodegenerative disease" is a progressive disease characterized by a substantial loss of specific neurons. Mainly comprises Parkinson's Disease (PD), alzheimer's Disease (AD), mild cognitive impairment (mild cognitive impairment, MCI) and amyotrophic lateral sclerosis (amyotrophic lateral sclerosis, ALS), etc.

As used herein, the term "Alzheimer's Disease (AD)" is a common neurodegenerative disease of the elderly, characterized by cognitive dysfunction as a major clinical feature. For diagnosis of AD, methods such as magnetic resonance imaging (magnetic resonance imaging, MRI), positron emission tomography (positron emission computed tomography, PET), and biomarker diagnosis can be used.

As used herein, the term "biomarker" refers to a biochemical marker that can mark alterations in system, organ, tissue, cellular and subcellular structures or functions, or alterations that may be suffered from, having a very broad range of uses. Biomarkers can be used for disease diagnosis, for judging disease stage or for evaluating the safety and effectiveness of new drugs or new therapies in a target population.

As used herein, the term "reference value" refers to a predetermined value of a biomarker, which is derived from the level of the biomarker in a control sample (e.g., a biological sample obtained from a normal population). The reference value may be used as a threshold to distinguish between subjects at risk of a disease and subjects not at risk of the disease. The reference values may be relative values, numerical ranges having upper and lower limits, average values, median values, and the like. Suitable control samples can be selected, assayed and reference values obtained by those skilled in the art according to methods disclosed in the prior art. The above-described methods can be found, for example, in Burtis c.a. et al, 2008,Chapter 14,section"statistical treatment of reference values, which is incorporated herein by reference in its entirety.

As used herein, the term "ApoE epsilon 4 genotype" refers to a variant of the ApoE gene, and there are many possible variants of the ApoE gene, e.g., epsilon 2, epsilon 3, epsilon 4, epsilon 2 epsilon 3, epsilon 2 epsilon 4, and epsilon 3 epsilon 4. Several studies have shown that populations carrying variants of the APOE gene epsilon 4 develop more easily into alzheimer's disease in the late years.

As used herein, the term "subject" includes, but is not limited to, various animals, particularly mammals, such as humans.

As used herein, the term "APP (myloid beta (A4) precursor protein) gene" plays an important role in the pathogenesis of early senile dementia. Currently, 6 missense mutations are found in the APP gene.

Advantageous effects of the invention

The method can replace the traditional p-tau/Abeta detection of cerebrospinal fluid, realize diagnosis of AD through 6 kinds of circRNAs, and can distinguish AD patients from normal persons and AD patients from other dementia patients (such as vascular dementia (VaD), parkinson's Disease Dementia (PDD), behavior variability frontotemporal dementia (bvFTD) and dementia with lewy bodies (DLB)). The method can complete detection by taking blood (for example, peripheral blood), and has the following beneficial technical effects compared with the traditional lumbar puncture cerebrospinal fluid detection: (1) has the advantages of almost no wound, low risk and the like; (2) The method has the advantage of low cost, can be completed in communities or simple medical institutions, and detected personnel do not need to be hospitalized; (3) The AD can be screened in a large range of people, so that the screening of a large range of old people is possible; (4) need not be done in a hospital or professional medical facility; (5) low cost.

Drawings

Fig. 1 shows the levels of 22 upregulated and 19 downregulated circrnas detected in AD group compared to control group in data set 1.

FIG. 2 shows that in dataset 2, 12 differentially expressed circRNAs were further validated. Wherein AD is a patient suffering from Alzheimer's disease; FC is a fold change.

Figure 3 shows the establishment of a diagnostic model for alzheimer's disease in dataset 2. FIG. 3A is a graph of the subject's working characteristics (ROC) analysis performed in combination with 6 circRNAs, and FIG. 3B is the result of the ROC analysis for each circRNAs. AUC is the area under the curve.

FIG. 4 shows the measurement results of 6 circRNAs for control group, AD, vaD, PDD, bvFTD and DLB in dataset 3.

Figure 5 shows AUC values predicted for the combination of 6 circRNAs in dataset 3. Fig. 5A shows AD and control, fig. 5B shows AD and other types of dementia, and fig. 5C shows AD and no AD. Absence of AD represents a combination of control and other types of dementia.

Information on the partial sequences to which the present application relates is provided in table 1 below.

Table 1: description of the sequence

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Example 1: data sets and statistical methods.

1.1 data set of study.

The study included 3 data sets altogether.

During the pre-trial phase (data set 1), subjects were enrolled in the Beijing area (n=40; including 20 control groups, 20 AD patients).

In the predictive model establishment phase (data set 2), subjects (n=124, 61 control groups, 63 AD patients) were recruited from other areas such as the eastern, the henna, and the guangxi provinces.

During the model validation phase (data set 3), subjects were enrolled in beijing area (n=503; including control 58, AD60, vaD60, PDD51, bvFTD52, DLB 50).

The diagnosis standard of AD is based on the American national aging institute-Alzheimer's Association (National Institute on Aging-Alzheimer's Association, NIA-AA) diagnosis standard in 2011. In addition, AD and normal controls were determined from the ratio of cerebrospinal fluid P-tau/Abeta 42 (cut-off taken to 0.14), which was calculated from our previously published data, consistent with other research reports (see, jia L, qia Q, zhang H, chu L, du Y, zhang J, et al Concordance between the assessment of Abeta, T-tau, and P-T181-tau in peripheral blood neuronal-derived exosomes and cerebrospinal fluid. Alzheimer's de 2019; 15:1071-80). According to the ATN framework, low levels of cerebrospinal fluid aβ42 are a critical pathological change in AD. We therefore used the previously reported threshold value 500pg/ml for Abeta42 of cerebrospinal fluid as another standard for determining AD and normal controls (see, jia L, qiaQ, zhang H, chu L, du Y, zhang J, et al Concordance between the assessment of Abeta, T-tau, and P-T181-tau in peripheral blood neuronal-derived exosomes and cerebrospinal fluid. Alzheimer's de. 2019; 15:1071-80). All subjects or their legal guardians had fully informed and signed written consent. The study was approved by the institutional review board of the first medical university Xuan Wu hospital.

1.2 subject characteristics.

Tables 2-4 list the features of the study, as shown below.

Table 2. Characteristics of subjects in dataset 1.

Table 3. Characteristics of subjects in dataset 2.

Table 4. Characteristics of subjects in dataset 3.

Note that: age, age-of-education, estimated year before onset, and MMSE values are expressed as average values (standard deviation). Abbreviations ApoE.epsilon.4, apolipoprotein.epsilon.4; MMSE, simple mental state examination; SD, standard deviation; * P < 0.05 compared to the control group.

As shown in the table above. No significant differences were observed between age and male-female ratio between groups. There was a significant difference in APOE epsilon 4, mmse scale scores between subjects with AD and control in

data sets

1, 2. There was a significant difference in MMSE scale scores between subjects with AD, vaD, PDD, bvFTD and DLB in dataset 3 and the control group.

Example 2: collection and analysis of circRNA.

2.1RNA collection and sequencing.

All subjects were collected with 20ml venous blood in the early morning on an empty stomach (12 hours fasted) and polypropylene tubes containing ethylenediamine tetraacetic acid (EDTA) were used as the collection tubes. To obtain the circRNAs in blood, whole blood samples were processed at a central laboratory at the university of capital medical university Xuan Wu hospital. Blood samples collected in areas other than Beijing were immediately centrifuged at 4200 Xg for 10 minutes at room temperature to obtain plasma, which was then stored at 4℃and transported to a Xuan Wu hospital central laboratory using dry ice for 12 hours. We purified total RNA using serum RNA extraction purification kit. Among them, 1. Mu.g total RNA was prepared into sequencing libraries (micro protein nuclease 8000 and Agilent technology 2100 bioanalyzer [ Agilent, USA ]), and double-stranded and single-stranded DNA in another 3. Mu.g total RNA was degraded using DNase I. We used plant removal rRNA kit (Vazyme, inc.) to remove ribosomal ribonucleic acids and exonuclease (New England Biolabs Inc, USA) to remove linear RNA, agencourt RNAClean XP magnetic beads for further purification. The distribution of fragment sizes was detected using an Agilent technology 2100 bioanalyzer and BMG (OMEGA) quantified libraries. Finally, the library was double-ended using the BGISEQ-500 (BGI-Shenzhen, china) platform.

2.2 analysis of circRNA data.

The raw sequencing data was filtered and processed as follows: 1) Deleting reads comprising sequencing adaptors; 2) Deleting the base with the proportion (base mass is less than or equal to 5) of more than 20% of low mass base; and 3) removing reads with a ratio of unknown bases ('N' bases) greater than 5%. Thus we obtain clean reads and store them in FASTQ format. The clean reads were then mapped to the reference genome using HISAT2 (v2.0.4). Subsequently, fusion genes and differential splicing genes were detected using Ericscript (v 0.5.5) and RMAS (V3.2.5). We used Bowtie2 (v 2.2.5) to compare the clean reads with the gene set constructed by Beijing genome research located in Shenzhen, which is a database containing known and new coding and non-coding transcripts. Next, we calculated the expression level of the gene using RSEM (v1.2.12). The differential expression analysis adopts DEseq2 with the Q value less than or equal to 0.05 as a default threshold value so as to judge the significance of differential expression.

2.3 primer design.

Primer sequences were designed to confirm altered circRNAs in the validation study. All circRNA primers are listed in Table 5.

Table 5. Sequences of the circRNA primers.

2.4 Cerebrospinal Fluid (CFS) collection, determination of Aβ42, T-tau and P-tau.

Cerebrospinal fluid is collected with reference to international guidelines. In lumbar puncture, the subject was placed in a left lateral position, L3-L5 intervertebral disc space was taken as a puncture site, 15ml of CSF sample was collected, centrifuged at 2,000Xg for 10 minutes at room temperature, and the CSF sample was then split-packed into polypropylene tubes and stored at-80 ℃. All subjects were monitored for at least 12 hours after lumbar puncture.

2.5 Abeta 42, T-tau and P-tau assays.

Abeta 42, total tau (T-tau) and P-tau (phosphorylated tau at the Thr 181 site) levels in cerebrospinal fluid were determined using an enzyme-linked immunosorbent assay (ELISA) kit. Preliminary experiments were first performed to determine the detection range of the ELISA assay. The average value of CD81 levels in each group was set to 1.00 and relative ratios were used for normalization. All detection results are within the detection range of the ELISA kit. All assays were performed using the blind method.

2.6 statistical analysis.

Statistical analysis was performed using SPSS v.22 and Stata 13.0. Data set 1, data set 2, and data set 3 were independently analyzed, respectively. The comparison between the groups of counting data is performed by chi-square test. The differences between the panels of metering data, such as biomarker concentrations, were analyzed using the Welch's t test or analysis of variance (ANOVAs). To identify differentially expressed circrnas, P values were corrected using the pseudo-discovery rate. In datasets 2 and 3, predictions were generated by a binary Logistic regression model with age, gender, education age, and APOE epsilon 4 status as covariates, and then examined with subject work profile analysis. The tolerance, variance expansion factor (variance inflation factor, VIF), eigenvalue and conditional index were used to calculate the multiple collinearity between each protein. All assays were double sided assays, with a level of significant difference set to P <0.05.

2.7 preliminary experimental stage study results.

The preliminary study was a relatively small sample (data set 1). RNA sequencing results 1875 circRNAs were found in AD and control patients. Exclusion of cirrnas reads below 100 did not take part in the next analysis. The AD patient and control group were selected as significant groups of circRNAs with a circRNA change of 1.2 fold or more or 0.80 fold or less; we found 22 up-regulated and 19 down-regulated circRNA samples in AD group (P <0.05, fig. 1).

Example 3: and (5) establishing a prediction model.

A relatively large sample (data set 2) was collected for further confirmation of differential circRNA. 22 up-regulated groups and 19 down-regulated groups in dataset 1 were confirmed in dataset 2, supporting the significance of sequencing data obtained in the earlier study. Subsequently we selected the first 6 cases of circrnas in the up-regulation group (hsa_circ_ 0077001, hsa_circ_0022417, hsa_circ_0014356, hsa_circ_0051000, hsa_circ_0014353, and hsa_circ_ 0074533) and the first 6 cases of circrnas in the down-regulation group (hsa_circ_ 0006940, hsa_circ_0089762, hsa_circ_0089894, hsa_circ_0037139, hsa_circ_0089761, and hsa_circ_ 0079275) (fig. 2) were evaluated by Logistic analysis to assess their ability to distinguish AD patients from the control group.

The diagnosis results (AD and control) were used as dependent variables, and 12 circRNAs were used as covariates. After correction of age, gender, age of education and status of APOEε 4, 6 circRNAs were found (up-regulated-hsa_circ_ 0077001, hsa_circ_0022417, hsa_circ_0014356, hsa_circ_0014353 and hsa_circ_0074533; down-regulated-hsa_circ_ 0089894) to be associated with AD.

In the Logistic model, P values >0.05 due to age, gender and age of education were excluded from further analysis. The multiplex colinear diagnosis results of 6 circRNAs for AD patients and control group showed that all tolerances were >0.1, variance expansion factor <10, eigenvalue >0, and state index <30, indicating no significant multiplex colinear between these 6 circRNAs. To determine the diagnostic capabilities of the six proteins, the combined predictions in the Logistic model were further evaluated by ROC curve analysis. The results showed that the 6 combinations of circRNAs had good diagnostic ability for AD, with the area under the curve (auc=0.968, P < 0.001, fig. 3A) significantly higher than the area under the curve of a single protein (aucs=0.636-0.796, fig. 3B), indicating that it was necessary to combine 6 circRNAs to obtain an effective diagnosis.

Example 4: and (3) model verification stage.

Using a third dataset to assess the diagnostic ability of the model in clinical practice for subjects including control group, AD and other neurodegenerative diseases, such as VaD, PDD, bvFTD and DLB, we obtained similar results as datasets 1, 2: hsa_circ_0077001, hsa_circ_0022417, hsa_circ_0014356, hsa_circ_0014353 and hsa_circ_0074533 are increased in level and hsa_circ_0074533 is decreased (P <0.001; FIGS. 4A-F). The 6 circRNAs were not significantly altered (all P > 0.05) in other types of dementia, such as vascular dementia, parkinson dementia, abnormal frontotemporal dementia, dementia with lewy bodies, indicating that these circRNAs are AD-specific. Further ROC analysis showed high AUC (0.914-0.966, P <0.001, fig. 5A-C), suggesting that 6 circRNAs are very effective in distinguishing AD from healthy controls and other types of neurodegenerative diseases.

Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate that: many modifications and variations of the details are possible in light of the above teachings, and such variations are within the scope of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A kit for diagnosing whether a subject has alzheimer's disease, the kit comprising reagents for determining the level of a biomarker in a biological sample, the biomarker being hsa_circ_007501, hsa_circ_0022417, hsa_circ_0014356, hsa_circ_0014353, hsa_circ_0074033 and hsa_circ_0089894;

preferably, the kit comprises: a first reagent or combination of reagents for determining the hsa_circ_0077001 level of a subject, a second reagent or combination of reagents for determining the hsa_circ_0022417 level of a subject, a third reagent or combination of reagents for determining the hsa_circ_0014356 level of a subject, a fourth reagent or combination of reagents for determining the hsa_circ_0014353 level of a subject, a fifth reagent or combination of reagents for determining the hsa_circ_0074533 level of a subject, a sixth reagent or combination of reagents for determining the hsa_circ_0089894 level of a subject.

2. The kit of claim 1, wherein the reagent (e.g., first, second, third, fourth, fifth, and/or sixth reagent or combination of reagents) determines the level of a biomarker in the biological sample by hybridization techniques; preferably, the hybridization technique is selected from the group consisting of Northern blot analysis, microarray techniques, in situ hybridization, liquid chip techniques, splint ligation;

Preferably, the kit has the following features:

(1) The first reagent or combination of reagents comprises primers and/or probes capable of quantifying the level of hsa_circ_ 0077001;

(2) The second reagent or combination of reagents comprises primers and/or probes capable of quantifying the level of hsa_circ_ 0022417;

(3) The third reagent or combination of reagents comprises primers and/or probes capable of quantifying the level of hsa_circ_ 0014356;

(4) The fourth reagent or combination of reagents comprises primers and/or probes capable of quantifying the level of hsa_circ_ 0014353;

(5) The fifth reagent or combination of reagents comprises primers and/or probes capable of quantifying the level of hsa_circ_ 0074533;

(6) The sixth reagent or combination of reagents comprises primers and/or probes capable of quantifying the level of hsa_circ_ 0089894;

preferably, the kit further comprises means for capturing a biomarker;

more preferably, the means for capturing the biomarker is located on a solid support (e.g., nylon membrane, microsphere).

3. The kit of claim 1, wherein the reagents (e.g., first, second, third, fourth, fifth, and/or sixth reagents or reagent combinations) determine the level of a biomarker in the biological sample by an amplification technique;

Preferably, the amplification technique is selected from qPCR, RT-PCR, stem-loop PCR, poly a PCR, RCA, sequencing;

preferably, the kit has the following features:

preferably, the kit further comprises reagents for performing nucleic acid amplification and/or reagents for performing sequencing;

preferably, the reagents for performing nucleic acid amplification include, working buffer for enzymes (e.g., nucleic acid polymerase), dNTPs (labeled or unlabeled), water, a reagent containing ions (e.g., mg ²⁺ ) Is a solution of (a) and single-stranded DNA binding eggWhite, or any combination thereof.

4. The kit of any one of claims 1-3, wherein the biological sample is whole blood (e.g., peripheral blood), serum, plasma, or cerebrospinal fluid obtained from a subject;

preferably, the biological sample is selected from whole blood (e.g., peripheral blood), serum, and plasma;

more preferably, the biological sample is selected from peripheral blood;

preferably, the subject is a mammal, such as a human.

5. The kit of any one of claims 1-4, further comprising a pretreatment reagent or combination of reagents for pretreatment of a biological sample;

preferably, the pretreatment reagent or combination of reagents is used to pretreat the biological sample (e.g., whole blood, serum, or plasma) to obtain circRNAs;

preferably, the pretreatment reagent or reagents comprise a circRNA extraction purification solution, and optionally a buffer;

preferably, the kit is for distinguishing between a subject suffering from alzheimer's disease and a normal subject;

preferably, the kit is used to distinguish between subjects suffering from alzheimer's disease and subjects suffering from other dementias, e.g. vascular dementia (vascular dementia, vaD), parkinson's disease dementia (Parkinson disease, PDD), behavior variability frontotemporal dementia (behavioural variant frontotemporal dementia, bvFTD), dementia with lewy bodies (dementia with Lewy bodies, DLB).

6. Use of a reagent for determining the level of a biomarker in a biological sample in the manufacture of a kit for diagnosing whether a subject is suffering from alzheimer's disease; wherein the biomarker is hsa_circ_007501, hsa_circ_0022417, hsa_circ_0014356, hsa_circ_0014353, hsa_circ_0074233 and hsa_circ_0089894;

preferably, the kit comprises a first reagent or combination of reagents for determining the hsa_circ_0077001 level of the subject, a second reagent or combination of reagents for determining the hsa_circ_0022417 level of the subject, a third reagent or combination of reagents for determining the hsa_circ_0014356 level of the subject, a fourth reagent or combination of reagents for determining the hsa_circ_0014353 level of the subject, a fifth reagent or combination of reagents for determining the hsa_circ_0074533 level of the subject, a sixth reagent or combination of reagents for determining the hsa_circ_0089894 level of the subject.

7. The use of claim 6, wherein the reagent (e.g., first, second, third, fourth, fifth, and/or sixth reagent or combination of reagents) determines the level of a biomarker in the biological sample by hybridization techniques;

Preferably, the hybridization technique is selected from the group consisting of Northern blot analysis, microarray techniques, in situ hybridization, liquid chip techniques, splint ligation;

preferably, the kit has the following features:

preferably, the kit further comprises means for capturing a biomarker;

8. The kit of claim 6, wherein the reagents (e.g., first, second, third, fourth, fifth, and/or sixth reagents or reagent combinations) determine the level of a biomarker in the biological sample by an amplification technique;

preferably, the kit has the following features:

Preferably, the reagents for performing nucleic acid amplification include, working buffer for enzymes (e.g., nucleic acid polymerase), dNTPs (labeled or unlabeled), water, a reagent containing ions (e.g., mg ²⁺ ) Or a single-stranded DNA binding protein, or any combination thereof.

9. The use of any one of claims 6-8, wherein the biological sample is whole blood (e.g., peripheral blood), serum, plasma, or cerebrospinal fluid obtained from a subject;

more preferably, the biological sample is selected from peripheral blood;

preferably, the biological sample comprises circRNAs;

preferably, the subject is a mammal, such as a human.

10. The use of any one of claims 6-9, wherein the kit further comprises a pretreatment reagent or combination of reagents for pretreatment of a biological sample;

preferably, the kit is used to distinguish between subjects suffering from alzheimer's disease and subjects suffering from other dementias, e.g., vascular dementia (VaD), parkinson's Disease Dementia (PDD), behavior variability frontotemporal dementia (bvFTD), dementia with lewy bodies (DLB).