CN113308539A

CN113308539A - Product for diagnosing alzheimer's disease

Info

Publication number: CN113308539A
Application number: CN202110826207.4A
Authority: CN
Inventors: 刘新; 孙鹏涛; 王金丽
Original assignee: Second Hospital of Hebei Medical University
Current assignee: Second Hospital of Hebei Medical University
Priority date: 2021-07-21
Filing date: 2021-07-21
Publication date: 2021-08-27
Also published as: CN116219002A

Abstract

Products for diagnosing Alzheimer's disease are disclosed, the products comprising reagents for detecting the expression level of a combination of biomarkers in a sample. ROC curve analysis shows that the biomarker combinations of the present invention have higher AUC values. According to the research results of the present invention, the biomarker combinations of the present invention can be applied to the diagnosis of alzheimer's disease.

Description

Product for diagnosing alzheimer's disease

Technical Field

The invention belongs to the field of biological medicines, and relates to a product for diagnosing Alzheimer disease.

Background

Alzheimer Disease (AD) is a progressive neurodegenerative Disease with occult onset of Disease. Clinically, the overall dementia such as dysmnesia, aphasia, disuse, agnosia, impairment of visual spatial skills, dysfunction in execution, and personality and behavior changes are characterized, and the etiology is unknown. Patients who are older than 65 years are called presenile dementia; the patient after 65 years old is called senile dementia. Patients can usually survive 8 to 10 years after symptoms appear, but the course of the disease can be 1 to 25 years. Death is usually caused by pneumonia, malnutrition or general physical consumption (activity). In 2015, the number of global dementia patients reaches 4680 million, 50% -75% of them are alzheimer patients, about 990 million new dementia patients will be diagnosed, and 1 new person is increased every 3 seconds on average, and 1.31 million persons will be predicted by 2050. Studies have shown that senile dementia is the fourth leading cause of death in the elderly following tumors, heart disease, cerebrovascular disease, and is spent the third in all diseases. With the aging of the population in China, the number of Alzheimer's disease patients is continuously increasing. At present, about 600 thousands of patients exist in China, the number of the patients is the first of the world, and 30 thousands of new cases exist in each year, so that the traditional Chinese medicine is one of the countries with the fastest global acceleration. Alzheimer's disease may be a heterogeneous group of diseases involving many factors, such as genetics, trauma, viral infection, other diseases, etc., but the pathogenesis of alzheimer's disease is not clear, wherein one of the main mechanisms is that β -secretase l (β -site amyloid accusor protein-cleavangenzyme, BACE1) cleaves amyloid plaques formed by amyloid preproprotein (APP) to aggregate in neurons, breaking the equilibrium state of Α β 42/Α β 40, and generating neurotoxicity.

AD and other types of dementia, such as vascular dementia (VaD), Parkinson's Disease Dementia (PDD), behavioral variability frontotemporal dementia (bvFTD), lewy body Dementia (DLB), etc., may have overlapping clinical manifestations, pathology and biomarkers, often leading to difficulties in clinical diagnosis. Based on the above problems, clinical treatment of AD requires an effective method for diagnosing AD.

Disclosure of Invention

The invention provides a biomarker combination, wherein the biomarker comprises LOC653658, CLC, RPL26 and RPL 31.

The invention also provides the application of the detection reagent of the biomarker combination in the preparation of products for diagnosing Alzheimer disease.

In the present invention, the term "biomarker" means a compound, preferably a gene, which is differentially present (i.e. increased or decreased) in a biological sample from a subject or a group of subjects having a first phenotype (e.g. having a disease) compared to a biological sample from a subject or a group of subjects having a second phenotype (e.g. no disease). The term "biomarker" generally refers to the presence/concentration/amount of one gene or the presence/concentration/amount of two or more genes.

The term "subject" means any animal, also human and non-human animals. The term "non-human animal" includes all vertebrates, e.g., mammals, such as non-human primates (particularly higher primates), sheep, dogs, rodents (such as mice or rats), guinea pigs, goats, pigs, cats, rabbits, cattle, and any domestic or pet animal; and non-mammals, such as chickens, amphibians, reptiles, and the like.

Biomarkers can be differentially present at any level, but are typically present at levels that are increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, or more; or generally at a level that is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% (i.e., absent).

Preferably, the biomarkers are differentially present at levels of statistical significance (i.e., p-value less than 0.05 and/or q-value less than 0.10, as determined using the Welch's T-Test or the Wilcoxon rank-sum Test.

Further, the reagent comprises a primer, a probe and an antibody.

The term "primer" refers to 7 to 50 nucleic acid sequences capable of forming a base pair (base pair) complementary to a template strand and serving as a starting point for replication of the template strand. The primers are generally synthesized, but naturally occurring nucleic acids may also be used. The sequence of the primer does not necessarily need to be completely identical to the sequence of the template, and may be sufficiently complementary to hybridize with the template. Additional features that do not alter the basic properties of the primer may be incorporated. Examples of additional features that may be incorporated include, but are not limited to, methylation, capping, substitution of more than one nucleic acid with a homolog, and modification between nucleic acids.

The term "nucleic acid" broadly refers to: a segment of a chromosome; fragments or portions of DNA, cDNA and/or RNA. Nucleic acids can be obtained or obtained from a nucleic acid sample that is initially separated from any source (e.g., isolated from, purified from, amplified from, cloned or reverse transcribed from sample DNA or RNA).

The term "probe" refers to a molecule that binds to a specific sequence or subsequence or other portion of another molecule. Unless otherwise indicated, the term "probe" generally refers to a polynucleotide probe that is capable of binding to another polynucleotide (often referred to as a "target polynucleotide") by complementary base pairing. Depending on the stringency of the hybridization conditions, a probe can bind to a target polynucleotide that lacks complete sequence complementarity to the probe. The probe may be directly or indirectly labeled, and includes within its scope a primer. Hybridization modes include, but are not limited to: solution phase, solid phase, mixed phase or in situ hybridization assays.

The invention also provides a product for diagnosing Alzheimer's disease, which comprises a reagent for detecting the gene expression level of the biomarker combination in a sample.

Further, the product comprises a kit, a chip or a test strip.

Further, the reagent comprises a reagent for detecting the expression level of the biomarker combination by PCR, in situ hybridization, a chip, a high-throughput sequencing platform or an immunoassay, preferably, the PCR comprises reverse transcription PCR or real-time quantitative PCR.

In the present invention, the kit further comprises a container, instructions for use, a positive control, a negative control, a buffer, an auxiliary agent or a solvent, and instructions for use with the kit, wherein the instructions describe how to use the kit for detection, and how to use the detection results to determine the development of a disease and select a treatment regimen.

The kit of the present invention may contain a plurality of different reagents suitable for practical use (e.g., for different detection methods), and is not limited to the reagents listed so far, and is included in the scope of the present invention as long as the reagent diagnoses alzheimer's disease based on the detection of the biomarker combination described above.

Further, the product also comprises reagents for processing the sample.

The invention also provides the use of a biomarker for constructing a computational model for predicting alzheimer's disease or a system incorporating said computational model, said biomarker comprising the biomarker combination according to claim 1;

preferably, the calculation model takes the expression level of the biomarker as an input variable, and outputs the risk probability of the disease by performing operation through a bioinformatics method.

As the skilled person will be aware, the step of associating a marker level with a certain likelihood or risk may be carried out and carried out in different ways. Preferably, the measured concentrations of the marker and one or more other markers are mathematically combined and the combined value is correlated to the underlying diagnostic problem. The determination of marker values may be combined by any suitable prior art mathematical method.

Preferably, the mathematical algorithm applied in the marker combination is a logarithmic function. Preferably, the result of applying such a mathematical algorithm or such a logarithmic function is a single value. Such values can be easily correlated, for example, to an individual's risk for alzheimer's disease or to other diagnostic purposes of interest that are helpful in assessing alzheimer's patients, in light of underlying diagnostic problems. In a preferred manner, such a logarithmic function is obtained as follows: a) classifying individuals into groups, e.g., normal persons, individuals at risk of alzheimer's disease, patients with alzheimer's disease, etc., b) identifying markers that differ significantly between these groups by univariate analysis, c) logarithmic regression analysis to assess independent difference values of the markers that can be used to assess these different groups, and d) constructing a logarithmic function to combine the independent difference values. In this type of analysis, the markers are no longer independent, but represent a combination of markers.

The logarithmic function used to correlate marker combinations with disease preferably employs algorithms developed and obtained by applying statistical methods. For example, suitable statistical methods are Discriminant Analysis (DA) (i.e., linear, quadratic, regular DA), Kernel methods (i.e., SVM), nonparametric methods (i.e., k-nearest neighbor classifiers), PLS (partial least squares), tree-based methods (i.e., logistic regression, CART, random forest methods, boosting/bagging methods), generalized linear models (i.e., logistic regression), principal component-based methods (i.e., SIMCA), generalized additive models, fuzzy logic-based methods, neural network-and genetic algorithm-based methods. The skilled person will not have problems in selecting a suitable statistical method to evaluate the marker combinations of the invention and thereby obtain a suitable mathematical algorithm.

The present invention also provides a system, comprising:

(1) an Alzheimer's disease assessment apparatus including a control unit and a storage unit for assessing whether a subject has Alzheimer's disease; and

(2) information communication terminal devices communicatively connected to each other, which provide data on gene expression levels of the aforementioned biomarker combinations in a sample from a subject;

wherein the control unit of the Alzheimer's disease assessment apparatus comprises:

1) a data receiving unit that receives data on expression levels of the aforementioned biomarker combinations of the sample transmitted from the information communication terminal device;

2) a discrimination value calculation unit that calculates a discrimination value based on the discrimination of the expression level of the aforementioned biomarker combination in the sample received by the data reception unit and the expression level of the aforementioned biomarker combination having the explanatory variable stored in the storage unit;

3) a discrimination value criterion evaluation unit that evaluates the condition of alzheimer's disease in the subject based on the discrimination value calculated by the discrimination value calculation unit; and

4) an evaluation result transmitting unit that transmits the evaluation result of the subject obtained by the discrimination value reference evaluation unit to the information communication terminal device.

The invention also provides the application of the biomarker combination in screening candidate drugs for treating Alzheimer disease.

The invention has the following advantages and beneficial effects:

the invention discovers for the first time that whether a subject suffers from Alzheimer disease can be predicted or diagnosed by detecting the expression levels of LOC653658, CLC, RPL26 and RPL 31.

LOC653658 Gene ID in NCBI 653658.

Gene ID 1178 of CLC in NCBI.

Gene ID 6154 of RPL26 in NCBI.

Gene ID 6160 of RPL31 in NCBI.

LOC650276 Gene ID:650276 in NCBI, also known as RPL7P6, Gene ID: 90193.

Gene ID 6232 of RPS27 in NCBI.

Drawings

FIG. 1 shows the ROC plot of LOC653658 for the diagnosis of Alzheimer's disease;

FIG. 2 shows ROC plots for CLC diagnosis of Alzheimer's disease;

FIG. 3 shows a ROC plot of RPL26 for diagnosis of Alzheimer's disease;

FIG. 4 shows a ROC plot of RPL31 diagnosis of Alzheimer's disease;

FIG. 5 shows the ROC plot of LOC650276 for diagnosing Alzheimer's disease;

FIG. 6 shows a ROC plot of RPS27 diagnosis of Alzheimer's disease;

FIG. 7 shows ROC plots for the joint diagnosis of Alzheimer's disease by LOC653658, CLC, RPL26 and RPL 31;

FIG. 8 shows ROC plots for the combined diagnosis of Alzheimer's disease by CLC, LOC650276, RPS27, RPL 26.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention.

Example 1 screening of differentially expressed genes associated with Alzheimer's disease

GSE63060 is downloaded from a GEO database, and is subjected to difference analysis by using an R language edgeR package to obtain 69 differentially expressed genes, wherein the screening standard is as follows: pvaluel <0.05, | logFC | > 0.5.

GSE63060 data set control AD 104:145 (population ratio)

Among them, LOC653658, CLC, RPL26, RPL31, LOC650276, RPS27 genes were significantly down-regulated in the blood level of alzheimer patients compared to normal controls, with the difference having statistical significance (P value <0.05), and the results are shown in fig. 1A, fig. 2A, fig. 3A, fig. 4A, fig. 5A, fig. 6A.

Example 2 diagnostic Performance validation

Receiver Operating Curves (ROCs) were plotted using the R package "pROC" (version 1.15.0), AUC values, sensitivity and specificity were analyzed, and the diagnostic efficacy of the markers alone or in combination was judged. When the diagnosis efficiency of the index combination is judged, logistic regression is carried out on the expression level of each gene, the probability of whether each individual suffers from cancer is calculated through a fitted regression curve, different probability division threshold values are determined, and the sensitivity, specificity, accuracy and the like of the combined detection scheme are calculated according to the determined probability division threshold values. The single gene and multiple gene combined diagnostic potency data are shown in table 1 and fig. 1B, 2B, 3B, 4B, 5B, 6B, 7, 8.

TABLE 1 diagnostic efficacy

Gene	AUC value
		LOC653658	0.772
CLC	0.670
		RPL26	0.755
RPL31	0.700
		LOC650276	0.724
RPS27	0.701
		LOC653658+CLC+RPL26+RPL31	0.822
CLC+LOC650276+RPS27+RPL26	0.753

According to experimental results, the diagnosis effect of the combination of LOC653658, CLC, RPL26 and RPL31 on Alzheimer disease is better than that of a single marker, and the diagnosis effect is better.

The description of the embodiments is only intended to serve for understanding the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

Claims

1. A biomarker combination, wherein the biomarkers comprise LOC653658, CLC, RPL26 and RPL 31.

2. Use of a detection reagent of the biomarker combination according to claim 1 in the preparation of a product for diagnosing alzheimer's disease.

3. The use of claim 2, wherein said reagents comprise primers, probes, antibodies.

4. A product for diagnosing alzheimer's disease comprising reagents for detecting the gene expression level of the biomarker panel of claim 1 in a sample.

5. The product of claim 4, wherein the product comprises a kit, chip or strip.

6. The product of claim 4, wherein the reagents comprise reagents for detecting the expression level of the biomarker combinations by PCR, in situ hybridization, chip, high throughput sequencing platform, or immunoassay, preferably wherein the PCR comprises reverse transcription PCR or real time quantitative PCR.

7. A product according to any of claims 4 to 6, further comprising reagents for processing the sample.

8. Use of a biomarker for constructing a computational model for predicting alzheimer's disease or a system incorporating said computational model, wherein said biomarker comprises the biomarker panel of claim 1;

9. A system, comprising:

(2) information communication terminal devices communicatively connected to each other, which provide data on the gene expression level of the biomarker combination according to claim 1 in a sample from a subject;

1) a data receiving unit that receives data on the expression level of the biomarker combination according to claim 1 for the sample transmitted from the information communication terminal device;

2) a discrimination value calculation unit that calculates a discrimination value based on discrimination of an expression level of the biomarker combination of claim 1 in the sample received by the data reception unit and an expression level of the biomarker combination of claim 1 with an explanatory variable stored in the storage unit;

10. Use of the biomarker combination according to claim 1 for screening a candidate drug for the treatment of alzheimer's disease.