CN113481293B - Molecular marker for diagnosing diabetic nephropathy and product and application thereof - Google Patents
Molecular marker for diagnosing diabetic nephropathy and product and application thereof Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
- C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/34—Genitourinary disorders
- G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy
Abstract
The molecular marker combination provided by the invention has excellent diagnosis efficiency on diabetic nephropathy, can be used for auxiliary diagnosis of diabetic nephropathy, can be conveniently applied to clinic, can quickly and effectively diagnose the diabetic nephropathy at early stage, and provides support for a clinician to adopt a prevention and treatment scheme in time.
Description
Technical Field
The invention belongs to the technical field of biological medicines, particularly relates to a molecular marker for diagnosing diabetic nephropathy, and more particularly relates to a molecular marker for diagnosing diabetic nephropathy and a product and application thereof.
Background
Diabetes Mellitus (DM) is one of the most common chronic metabolic diseases, and data published by the international Diabetes union in 2017 show that there are about 1.14 billion adult Diabetic patients in china and show a continuous rising trend, and furthermore, it is predicted that adult Diabetic patients worldwide will have up to 6.29 billion (Cho NH, shaw JE, karuranga S, et al. Idf Diabetes Atlas: global Diabetes mellitus of Diabetes mellitus for 2017and subjects for 2045 j. Diabetes Res in all directions, diabetes mellitus is mainly manifested in various persistent complications such as Diabetic retinopathy, diabetic neuropathy, diabetic nephropathy (Diabetes mellitus, diabetes mellitus) and the like, which affect the quality of Diabetic nephropathy, wherein the survival rate of Diabetic nephropathy is one of the most common renal diseases, i.e. the survival rate of Diabetes mellitus is high, and the survival rate of renal dysfunction is high, and the survival rate of Diabetes mellitus is high, and the survival rate of renal dysfunction is high, i.e. (23-7) and the most common cause renal failure, i.e. Diabetes mellitus.
In recent years, a great deal of research has been conducted on diabetic nephropathy at home and abroad, but due to the complexity of the etiology, the molecular mechanism of onset of diabetic nephropathy is still unclear, and especially, the research on early diagnosis and early onset mechanism of diabetic nephropathy, and the treatment for early diabetic nephropathy are urgently needed. The results of relevant statistical studies indicate that the mortality rate of diabetic nephropathy patients in end stage renal patients for 10 years is still above 30% (Fox CS, matsushita K, woodward M, et al associates of kidney disease measures with mortalities and end-stage renal diseases: a meta-analysis [ J ]. Lancet.2012,9854 (380): 1662-73.). At present, the 'gold standard' of diabetic nephropathy diagnosis is a renal biopsy pathology, which is also called renal biopsy, the renal biopsy is used for routine pathological examination such as light microscope, electron microscope, immunopathology and the like, the renal biopsy is an operation providing an important basis for a clinician to determine diagnosis, guide treatment and evaluate prognosis, and the renal biopsy is an important method for finding out the renal disease, especially the renal glomerular disease.
The clinical applicability and patient acceptance of renal biopsy pathology as an invasive diagnostic method is limited, and therefore, the search for new, reliable, noninvasive, rapid diagnostic methods is of great clinical significance for early diagnosis and early intervention treatment of diabetic nephropathy.
Disclosure of Invention
In order to make up for the above-mentioned deficiencies of the prior art, the present invention aims to provide a molecular marker for diagnosing diabetic nephropathy, which can determine whether a subject suffers from diabetic nephropathy and/or risks of diabetic nephropathy by detecting the expression level of the molecular marker in a sample of the subject, thereby providing an early, rapid, noninvasive, and accurate diagnosis method for the field, and having a good clinical application value.
The above purpose of the invention is realized by the following technical scheme:
in the invention, the molecular markers comprise genes PDZD2, B4GALT5 and LPCAT1, and the information of the genes PDZD2, B4GALT5 and LPCAT1 is respectively as follows:
gene PDZD2: PDZ domain stabilizing 2, expressed as Gene ID:23037 typical homo sapiens mRNA and protein sequences can be found in NCBI;
gene B4GALT5: beta-1,4-galactosyltransferase 5, expressed as Gene ID:9334 typical homo sapiens mRNA and protein sequences can be found in NCBI;
gene LPCAT1: lysophosphatelcholine acyltransferase 1, expressed as Gene ID:79888 typical homo sapiens mRNA and protein sequences can be found in NCBI.
In a first aspect, the invention provides a set of molecular markers associated with diabetic nephropathy.
Further, the molecular markers include PDZD2, B4GALT5, LPCAT1;
preferably, the molecular marker is PDZD2, B4GALT5;
preferably, the molecular marker is B4GALT5, LPCAT1;
preferably, the molecular marker is PDZD2, B4GALT5, LPCAT1.
In a second aspect, the present invention provides the use of a reagent for detecting the level of a molecular marker according to the first aspect of the present invention in a sample for the manufacture of a product for diagnosing diabetic nephropathy.
Further, the product comprises a reagent for detecting the level of the molecular marker according to the first aspect of the invention.
Further, the reagent comprises: an oligonucleotide probe that specifically recognizes the molecular marker of the first aspect of the present invention, or a primer that specifically amplifies the molecular marker of the first aspect of the present invention, or a binding agent that specifically binds to a polypeptide and/or protein encoded by the molecular marker of the first aspect of the present invention.
Further, the sample is selected from blood, serum, plasma, tissue.
In a third aspect, the invention provides a product for diagnosing diabetic nephropathy.
Further, the product comprises a reagent for detecting the level of the molecular marker according to the first aspect of the invention;
preferably, the product comprises a nucleic acid membrane strip, chip or kit.
Furthermore, the chip comprises a gene chip and a protein chip;
preferably, the gene chip comprises primers or oligonucleotide probes specific for the molecular marker according to the first aspect of the invention;
preferably, the protein chip comprises a binding agent that specifically binds to a polypeptide and/or protein encoded by a molecular marker according to the first aspect of the invention.
Further, the kit comprises a gene detection kit and a protein detection kit;
preferably, the gene detection kit comprises primers, oligonucleotide probes or chips specific for the molecular marker of the first aspect of the invention;
preferably, the protein detection kit comprises a binding agent that specifically binds to a polypeptide and/or protein encoded by a molecular marker according to the first aspect of the invention.
In a fourth aspect, the present invention provides a use of the molecular marker of the first aspect of the present invention in constructing a computational model for predicting diabetic nephropathy or a system in which the computational model is embedded;
preferably, the calculation model takes the level of the molecular marker as an input variable, and carries out operation by a bioinformatics method to output the risk probability of the diabetic nephropathy;
preferably, the level of the molecular marker comprises the mRNA level of the molecular marker, the protein level of the molecular marker.
In a fifth aspect, the present invention provides a system.
Further, the system comprises: a diabetic nephropathy evaluation device, information communication terminal devices connected to each other;
preferably, the diabetic nephropathy assessment apparatus comprises a control unit and a storage unit for assessing whether a subject suffers from diabetic nephropathy and/or is at risk of suffering from diabetic nephropathy;
preferably, the information communication terminal devices connected to each other provide data on the level of the molecular marker according to the first aspect of the present invention in a sample from a subject;
more preferably, the control unit of the diabetic nephropathy evaluation apparatus includes:
(1) A data receiving unit: which receives data on the level of the molecular marker in a sample of a subject transmitted from the information communication terminal device;
(2) A discrimination value calculation unit: calculating a discrimination value based on the level of the molecular marker in the subject sample received by the data receiving unit and the discrimination having the level of the molecular marker as an explanatory variable stored in the storage unit;
(3) Discrimination value criterion evaluation unit: evaluating the condition of diabetic nephropathy of the subject based on the discrimination value calculated by the discrimination value calculation unit;
(4) An evaluation result transmitting unit: which 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 a reagent for promoting the expression of the molecular marker PDZD2, a reagent for inhibiting the expression of the molecular marker B4GALT5 and a reagent for inhibiting the expression of the molecular marker LPCAT1 in preparing a pharmaceutical composition for preventing and/or treating diabetic nephropathy;
preferably, said agent promoting the expression of the molecular marker PDZD2 is selected from the group consisting of a mimic of the molecular marker PDZD2, an agonist of the molecular marker PDZD2, a vector overexpressing the molecular marker PDZD 2;
more preferably, the agent promoting the expression of the molecular marker PDZD2 is a mimic of the molecular marker PDZD 2;
preferably, the agent inhibiting the expression of molecular marker B4GALT5, the agent inhibiting the expression of molecular marker LPCAT1 are selected from gapmer, interfering RNA, CRISPR, TALEN, zinc finger nuclease;
more preferably, the agent for inhibiting the expression of the molecular marker B4GALT5 and the agent for inhibiting the expression of the molecular marker LPCAT1 are interfering RNAs.
The invention also provides the application of the molecular marker of the first aspect in screening candidate drugs for preventing and/or treating diabetic nephropathy;
the method for screening the candidate drug comprises the following steps:
(1) Treating a system expressing or comprising a molecular marker according to the first aspect of the invention with a substance to be screened;
(2) Detecting the level of expression of the molecular marker of the first aspect of the invention in the system of step (1);
if the substance to be screened can improve the expression level of the molecular marker PDZD2 and reduce the expression level of the molecular markers B4GALT5 and LPCAT1, the substance to be screened is a candidate drug for preventing and/or treating diabetic nephropathy.
Further, the system includes (but is not limited to): a cell system, a subcellular system, a solution system, a tissue system, an organ system, or an animal system.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and some of the terms are to be interpreted as follows:
as used herein, the terms "diagnose," "diagnosing," "diagnosed," and variations of these terms, refer to the discovery, judgment, or cognition of an individual's health state or condition based on one or more symptoms, data, or other information associated with the individual. The health status of an individual can be diagnosed as healthy/normal (i.e., no disease or condition is present), or as unhealthy/abnormal (i.e., a disease or condition is present), and the terms "diagnose," "diagnosing," and variations thereof include early detection of a disease associated with a particular disease or condition; the nature or classification of the disease; the discovery of the progression, cure, or recurrence of a disease; the finding of a response to a disease after treatment or therapy of an individual, in the present invention, the diagnosis of diabetic nephropathy includes the distinction between an individual not suffering from diabetic nephropathy and an individual suffering from diabetic nephropathy.
The terms "molecular marker", "marker", as used herein, refer to a molecular indicator having a specific biological property, biochemical characteristic, or other characteristic that can be used to determine the presence or absence of a particular disease or condition and/or the severity of a particular disease or condition. In the present invention, the molecular marker includes PDZD2, B4GALT5, LPCAT1, PDZD2+ B4GALT5, PDZD2+ LPCAT1, B4GALT5+ LPCAT1, PDZD2+ B4GALT5+ LPCAT1, preferably PDZD2+ B4GALT5, B4GALT5+ LPCAT1, PDZD2+ B4GALT5+ LPCAT1.
The term "probe" as used herein refers to a nucleic acid fragment such as RNA or DNA as short as a few to as long as several hundred bases that can establish specific binding to mRNA and can determine the presence of a particular mRNA by the Labeling action. The probe can be prepared in the form of an oligonucleotide probe, a single-stranded DNA probe, a double-stranded DNA probe, an RNA probe, or the like.
The term "primer" as used herein, refers to 7-50 nucleic acid sequences capable of forming a base pair complementary to a template strand and serving as a starting point for replication of the template strand. The primer is generally synthesized by a method known to those skilled in the art, but a naturally occurring nucleic acid may be used, and the sequence of the primer does not necessarily have 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 terms "differentially expressed", "differential expression", as used herein, refer to the presence of a difference between the expression levels of the same one or more molecular markers in a second sample as compared to the expression levels of the same one or more molecular markers in the second sample. Differential expression can be determined as described herein and by methods well known to those skilled in the art. The term "differential expression" or "change in expression level" means an increase or decrease in the measurable expression level of a given molecular marker in a sample as measured by the amount of RNA as compared to the measurable expression level of the given molecular marker in a second sample. The term "differential expression" or "change in expression level" may also mean that an increase or decrease in the measurable expression level of a given molecular marker in a sample population is compared to the measurable expression level of the molecular marker in a second sample population.
The invention has the advantages and beneficial effects that:
the molecular marker combination PDZD2+ B4GALT5, B4GALT5+ LPCAT1, PDZD2+ B4GALT5+ LPCAT1 provided by the invention has extremely high association degree with diabetic nephropathy, shows extremely good diagnosis efficiency on the diabetic nephropathy in training set and verification set, has high accuracy, sensitivity and specificity, can be used for early discovery of the diabetic nephropathy, and further performs timely and effective intervention treatment in the early stage of the diabetic nephropathy, and improves the survival quality of patients.
Drawings
Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
fig. 1 shows a boxplot of PDZD2 gene differential expression, wherein, panel a: training set, B panel: a verification set;
fig. 2 shows a boxplot of the differential expression of the B4GALT5 gene, in which panel a: training set, B panel: verifying the set;
FIG. 3 is a boxplot showing differential expression of the LPCAT1 gene, in which panel A: training set, B picture: a verification set;
fig. 4 shows ROC plot of the gene combination PDZD2+ B4GALT5, where a plot: training set, B panel: a verification set;
FIG. 5 shows ROC plot of gene combination PDZD2+ LPCAT1, in which, A plot: training set, B panel: a verification set;
FIG. 6 shows ROC plots for gene combination B4GALT5+ LPCAT1, where panel A: training set, B picture: a verification set;
FIG. 7 shows ROC plots of the gene combination PDZD2+ B4GALT5+ LPCAT1, where panel A: training set, B picture: and (5) verifying the set.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention. As will be understood by those of ordinary skill in the art: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents. The following examples are examples of experimental methods in which specific conditions are not specified, and the detection is usually carried out according to conventional conditions or according to conditions recommended by the manufacturers.
Example 1 screening of genes differentially expressed in diabetic nephropathy
1. Data sources
The data come from GEO database, download diabetic nephropathy data set GSE142153 from GEO database as training set, sample size is Control: case =10:23; and downloading a diabetic nephropathy data set GSE30122 from a GEO database to serve as a verification set, wherein the sample size is Control: case =50:19.
2. data pre-processing
And standardizing the original data of the training set and the verification set downloaded from the GEO database, annotating the standardized gene expression matrix through a Platform file, and taking an average value of a plurality of probes corresponding to the same gene as the expression quantity of the gene.
3. Differential expression analysis
And respectively carrying out differential expression analysis on the data in the data sets GSE142153 and GSE30122 by using a 'limma' packet in R language software, wherein the screening standard of the differential expression genes is as follows: value <0.05, | logFC | >0.5.
4. Results of the experiment
The result shows that 1391 differential expression genes are obtained in the training set by screening, 740 differential expression genes are obtained in the verification set, and 84 common differential expression genes are obtained by intersecting the differential expression genes in the two groups of data sets, wherein the differential expression conditions of the screened differential expression genes PDZD2, B4GALT5 and LPCAT1 in the training set and the verification set are respectively shown in figures 1-3, the expression conditions of PDZD2, B4GALT5 and LPCAT1 in diabetic nephropathy are respectively down-regulated, up-regulated and up-regulated, and the differences have statistical significance (P < 0.05).
Example 2 diagnostic efficacy validation
1. Experimental methods
For the differentially expressed genes PDZD2, B4GALT5, LPCAT1 screened in example 1, a receiver operating curve (ROC curve) was plotted using R package "pROC" (version 1.15.0), and the sensitivity, specificity, AUC values were analyzed to determine their diagnostic potency in the training set and the validation set, alone and in combination;
when the diagnosis effectiveness of individual indexes in a training set and a verification set is judged, the expression quantity of genes is directly used for analysis, the level corresponding to the point with the maximum Youden index is selected as the cutoff value, and genes with AUC of 0.5-AUC-0.8 are used for combined analysis;
and when the judgment indexes are combined with the diagnosis efficiency in the training set and the verification set, performing Logitics regression on the expression level of each gene, calculating the probability of whether each individual is ill or not through a fitted regression curve, determining different probability division thresholds, and calculating the sensitivity, specificity, accuracy and the like of the combined diagnosis scheme according to the determined probability division thresholds.
2. Results of the experiment
The results are shown in tables 1-3 and fig. 4-7, and the results show that the gene combinations PDZD2+ B4GALT5, B4GALT5+ LPCAT1 and PDZD2+ B4GALT5+ LPCAT1 have better diagnostic effect on diabetic nephropathy than a single marker, have higher sensitivity and specificity, and indicate that the gene combinations can be applied to the diagnosis of diabetic nephropathy.
TABLE 1 statistics of diagnostic potency of individual genes
| Gene | Training set AUC | Verification set AUC |
| PDZD2 | 0.765 | 0.652 |
| B4GALT5 | 0.778 | 0.728 |
| LPCAT1 | 0.796 | 0.762 |
TABLE 2 diagnostic Performance statistics of Gene combinations in training sets
| Gene | AUC | Sensitivity of the composition | Specificity of |
| PDZD2+B4GALT5 | 0.839 | 0.783 | 0.900 |
| PDZD2+LPCAT1 | 0.843 | 0.739 | 0.900 |
| B4GALT5+LPCAT1 | 0.861 | 0.696 | 1.000 |
| PDZD2+B4GALT5+LPCAT1 | 0.891 | 0.739 | 1.000 |
TABLE 3 diagnostic Performance statistics of Gene combinations in validation set
| Gene | AUC | Sensitivity of the composition | Specificity of |
| PDZD2+B4GALT5 | 0.838 | 0.684 | 0.920 |
| PDZD2+LPCAT1 | 0.774 | 0.684 | 0.840 |
| B4GALT5+LPCAT1 | 0.823 | 0.632 | 0.940 |
| PDZD2+B4GALT5+LPCAT1 | 0.865 | 0.789 | 0.900 |
The above description of the embodiments is only intended to illustrate 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 (3)
1. The application of a reagent for detecting the level of a molecular marker combination in a sample in preparing a product for diagnosing diabetic nephropathy is characterized in that the molecular marker combination is PDZD2, B4GALT5 and LPCAT1.
2. The use according to claim 1, wherein the agent comprises: an oligonucleotide probe that specifically recognizes the combination of molecular markers, or a primer that specifically amplifies the combination of molecular markers, or a binding agent that specifically binds to a polypeptide and/or protein encoded by the combination of molecular markers.
3. Use according to claim 1, wherein the sample is selected from blood, serum, plasma, tissue.
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Citations (3)
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|---|---|---|---|---|
| CN106676167A (en) * | 2017-03-13 | 2017-05-17 | 天津医科大学代谢病医院 | MiR-192 serving as specificity biomarker for early diagnosis of diabetic kidney disease and application of MiR-192 |
| CN109765380A (en) * | 2018-12-27 | 2019-05-17 | 郑州大学第一附属医院 | A kind of marker and application method detecting early diabetic nephropathy |
| CN110499364A (en) * | 2019-07-30 | 2019-11-26 | 北京凯昂医学诊断技术有限公司 | A kind of probe groups and its kit and application for detecting the full exon of extended pattern hereditary disease |
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| CN106676167A (en) * | 2017-03-13 | 2017-05-17 | 天津医科大学代谢病医院 | MiR-192 serving as specificity biomarker for early diagnosis of diabetic kidney disease and application of MiR-192 |
| CN109765380A (en) * | 2018-12-27 | 2019-05-17 | 郑州大学第一附属医院 | A kind of marker and application method detecting early diabetic nephropathy |
| CN110499364A (en) * | 2019-07-30 | 2019-11-26 | 北京凯昂医学诊断技术有限公司 | A kind of probe groups and its kit and application for detecting the full exon of extended pattern hereditary disease |
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| Title |
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| B4GALT5 - Diabetic nephropathy;GEO Profiles;《NCBI》;20040401;第1-3页 * |
| LPCAT1 - Diabetic nephropathy;GEO Profiles;《NCBI》;20040401;第1-3页 * |
| PDZD2 - Diabetic nephropathy;GEO Profiles;《NCBI》;20040401;第1-3页 * |
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