Biomarker composition and application related to diagnosis and prognosis of IgA nephropathy

Abstract

The present invention discloses a biomarker composition and application related to the diagnosis and prognosis of IgA nephropathy, and belongs to the field of biomedicine. The above-mentioned biomarker composition includes at least one of the cytokines FCAR, MBL and MMP-9. After screening and analysis, it was found that FCAR, MBL and MMP-9 were related to the diagnosis of IgA nephropathy, especially to severe IgA nephropathy. The present invention can better distinguish between mild IgA nephropathy patients, severe IgA nephropathy patients and healthy people through protein chips, and has high specificity and sensitivity. The present invention directly detects markers in the blood, making the operation non-invasive and rapid, convenient for diagnosing IgA nephropathy patients and evaluating the condition and treatment effect, and suitable for clinical promotion and application.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a biomarker composition and application related to the diagnosis and prognosis of IgA nephropathy. Background Art

[0002] IgA nephropathy (IgAN) is a chronic glomerulonephritis caused by abnormal deposition of IgA in the glomerular mesangial region. It is the most common primary glomerular disease worldwide and the most important disease leading to end-stage renal disease (ESRD) in my country. The pathogenesis of IgAN has not yet been fully elucidated. Its clinical and renal pathological manifestations are diverse, treatment options are limited, responses to treatment vary, and prognosis varies. Therefore, early diagnosis of IgAN and timely assessment of the degree of disease progression, as well as individualized intervention treatment, are very important for delaying the disease and improving prognosis.

[0003] At present, the diagnosis and evaluation of IgAN still rely on renal puncture biopsy, but due to the invasiveness of this operation, patients have low acceptance of it, and it is inconvenient to repeat the operation to timely evaluate the condition and treatment effect, which greatly limits its clinical application. In recent years, with the development of biomedical technology, domestic and foreign scholars have proposed some biomarkers related to IgAN. The detection of their expression levels in serum and urine alone or in combination may help the diagnosis and evaluation of IgAN. However, the sensitivity and specificity of these biomarkers in clinical practice still need to be further investigated, and more effective biomarkers and biomarker combinations still need to be explored in depth. Summary of the invention

[0004] The purpose of the present invention is to provide a biomarker composition and application related to the diagnosis and prognosis of IgA nephropathy, so as to solve the problems existing in the above-mentioned prior art and achieve high sensitivity and specificity detection of IgA nephropathy, especially severe IgA nephropathy.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a biomarker composition related to the diagnosis and prognosis of IgA nephropathy, wherein the biomarker composition comprises at least one of the cytokines FCAR, MBL and MMP-9.

[0007] Preferably, the biomarker composition consists of cytokines FCAR, MBL and MMP-9.

[0008] The present invention also provides a product for diagnosing IgA nephropathy and / or assessing the prognosis of IgA nephropathy, wherein the product comprises a reagent for detecting the expression level of the biomarker composition.

[0009] Preferably, the score of the biomarker expression level is calculated using the following formula: m = 0.17*FCAR-4.684*MBL-0.162*MMP-9+8.834; wherein m represents the expression level of the biomarker composition, pg / ml; FCAR, MBL and MMP-9 represent their respective concentrations, pg / ml; 8.834 is a constant in the formula calculated using a logistic regression model. According to the results of the model, the probability of severe IgAN P = e m / 1+e m .

[0010] Preferably, the product comprises a protein chip or a kit.

[0011] The present invention also provides the use of a reagent for detecting the biomarker composition in preparing a product for assisting in the diagnosis of IgA nephropathy and / or evaluating the prognosis of IgA nephropathy.

[0012] Preferably, the IgA nephropathy includes severe IgA nephropathy; and the product includes a protein chip or a kit.

[0013] The present invention discloses the following technical effects:

[0014] The present invention has found through screening and analysis that the blood biomarkers urine FCAR, MBL, MMP-9 are related to the diagnosis of IgAN, especially closely related to the diagnosis of severe IgAN; when urine FCAR, MBL and MMP-9 are jointly detected, the AUC for identifying severe IgAN reaches 0.9902, indicating that the combined detection of urine FCAR, MBL and MMP-9 can be used for the diagnosis of IgAN. The present invention also takes protein chips as an example, prepares protein chips for IgAN diagnosis, and verifies the sensitivity and specificity of combined urine FCAR, MBL and MMP-9. The present invention uses the above three biomarkers to detect IgAN, which has the advantages of non-invasive, convenient and fast operation, and has good scalability and practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0016] Figure 1Results of differentially expressed proteins screening and bioinformatics analysis after serum biomarker protein chip detection; A: Concentrations and hierarchical clustering heat map of serum biomarkers in mild IgAN and normal controls; B: Concentrations and hierarchical clustering heat map of serum biomarkers in severe IgAN and normal controls; C: Concentrations and hierarchical clustering heat map of serum biomarkers in mild IgAN and severe IgAN; D: Figure 1 A and Figure 1 The intersection of differentially expressed markers in B (FCAR, MBL, MMP-9);

[0017] Figure 2 for Figure 1 GO and KEGG analysis of the intersection differentially expressed markers in D;

[0018] Figure 3 ROC curves for separate and combined detection of serum FCAR, MBL, and MMP-9; A: FCAR; B: MBL; C: MMP-9; D: combined detection of FCAR, MBL, and MMP-9; E: sensitivity detection; F: specificity detection. DETAILED DESCRIPTION

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0021] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0022] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0023] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0024] Example 1 Screening of biomarkers related to IgAN diagnosis

[0025] 1. Criteria for screening biomarkers

[0026] (1) In this example, the literature in PubMed (1966-2020.10), Embase (1966-2020.10), Google Scholar, and Cochrane Library databases were searched to preliminarily screen cytokines and proteins that may be related to the diagnosis and prognosis of IgAN.

[0027] Through the above preliminary screening, 20 cytokines and proteins related to the diagnosis and prognosis of IgAN were identified (see Table 1).

[0028] Table 1 Candidate biomarkers

[0029]

[0030]

[0031] (2) Based on the screening results in Table 1, further screening was performed using the following criteria.

[0032] Screening criteria: 1) Stable and measurable in serum; 2) Can be detected using protein chip method to achieve convenience, speed and reduce costs; 3) The quality score of the study's Newcastle-Ottawa Scale (NOS) is greater than or equal to 6 points.

[0033] After further screening, 9 candidate biomarkers with high application value were screened out, namely FCAR, MBL, MMP-7, MMP-9, IL-6, MCP-1, TGF-β1, EGF and FGF-23.

[0034] Example 2 Protein biochip verification of the role of biomarkers

[0035] 1. Sample

[0036] Blood samples from 16 patients with mild IgAN and 16 patients with severe IgAN were collected from the Kidney Center of the First Affiliated Hospital of Zhejiang University. The diagnosis of IgAN was confirmed by renal biopsy. Mild cases were classified as Lee grade I and II in renal biopsy, and severe cases were classified as Lee grade III or greater in renal biopsy. Blood samples from 16 healthy subjects were collected as controls. Sample information is shown in Table 2. The blood samples were centrifuged to obtain the upper serum. The above sample collection was authorized by the patients.

[0037] Table 2 Clinical sample information

[0038]

[0039] 2. Experimental methods

[0040] Using RayBio's QAH-CUST chip ( Custom Arrays, Cat No: QAH-CUST-9, Raybiotech Co, Norcross GA, USA) were used to detect the levels of 9 biomarkers in serum and urine.

[0041] The samples collected in the previous step (serum diluted 2 times for loading, urine stock solution for loading) were added to the protein chip and incubated at 4°C overnight. After washing, the detection antibody mixture was added and incubated at room temperature for 2 hours. After washing, Cy3-conjugated streptavidin was added to the protein chip and incubated in the dark for 2 hours. After washing, the chip was scanned with an InnoScan 300 laser scanner (Innopsys, Parc d'Activités Activestre, 31390 Carbonne-France), and then the data analysis software QAH-CUST from Raybiotech was used for data analysis:

[0042] (1) Processing of raw data and screening of differentially expressed markers

[0043] The raw data obtained by scanning were processed by chip background removal and inter-chip normalization using Raybiotech software chip. The limma data package was used for moderated t-statistics analysis in R / Bioconductor, and the differential proteins were screened using adjusted p value (BH method corrected p value) (or p value) and logFC (expression difference multiple, with 2 as the base), and the selection conditions were: logFC>log2(1.2), adjusted p value (or p value)<0.05, and the results are shown in Figures 3-Table 5.

[0044] Table 3 Concentrations of serum biomarkers in mild IgAN and normal controls

[0045]

[0046] *Differentially expressed markers.

[0047] Table 4 Concentrations of serum biomarkers in severe IgAN and normal controls

[0048]

[0049] *Differentially expressed markers.

[0050] Table 5 Concentrations of serum biomarkers in mild and severe IgAN

[0051]

[0052]

[0053] *Differentially expressed markers.

[0054] (2) Cluster heat map

[0055] The heatmap.2 function was used in R / Bioconductor using the gplots package. The distance between two samples was calculated as the Euclidean distance; the distance between two clusters was calculated as the complete method, and the inter-cluster distance was defined as the maximum distance between the data of the two clusters.

[0056] (3) GO enrichment analysis of differentially expressed genes and KEGG pathway enrichment analysis

[0057] The clusterProfiler data package was used in R / Bioconductor, and Fisher's exact test was used for analysis. The selection criteria were that the number of proteins with differences in a certain term / GO was >= 2, and p_value < 0.05. The terms / GO obtained in the graph were arranged in descending order according to the Count value, and the first 10 results were taken.

[0058] Serum biomarker analysis results Figure 1-Figure 2 The intersection of the differentially expressed biomarkers between the healthy vs severe and mild vs severe groups was taken to further screen out three biomarkers that significantly distinguished healthy controls from different degrees of IgAN, namely serum FCAR, MBL, and MMP-9.

[0059] 3. ROC curve determines the diagnostic value of biomarkers and their combinations for IgAN

[0060] The ROC curves of the above three biomarkers were drawn when they were detected separately and in combination. According to the serum marker concentrations measured by the protein chip, a logistic regression model was constructed to draw the ROC curves of the above three markers when they were detected separately and in combination. The area under the ROC curve was calculated to evaluate its diagnostic value. The specific results are as follows: Figure 3 shown.

[0061] The results showed that the combined detection of blood biomarkers FCAR, MBL, and MMP-9 had good sensitivity and specificity for the detection of IgAN, especially severe IgAN. When detecting severe IgAN, the combined score of these three blood biomarkers = 0.17*FCAR concentration (pg / ml)-4.684*MBL concentration (ng / ml)-0.162*MMP-9 concentration (ng / ml)+8.834. Its AUC reached 0.9902. When the cutoff value of the combined score was 0.025, the sensitivity of the detection was 93.75% and the specificity was 96.88%, indicating that this biomarker combination has a very high value for the detection of severe IgAN. In comparison, the AUCs of blood FCAR, MBL, and MMP-9 alone for the identification of severe IgAN were 0.6865, 0.9473, and 0.9199, which were all lower than the combined detection of the three markers.

[0062] At present, the clinical diagnosis and prognosis of IgAN are still based on renal biopsy, but its invasiveness and possible complications limit its timely and effective clinical application. Some traditional indicators are also helpful in assessing the severity of IgAN, such as proteinuria, blood pressure and estimated glomerular filtration rate (eGFR) at the time of biopsy. Although the invasiveness is small, they are all non-specific and cannot provide potential pathophysiological information, thereby improving the choice of personalized treatment. Some new biomarkers discovered in recent years cannot replace invasive renal biopsy because of their unsatisfactory specificity and sensitivity. The serum biomarker combination discovered in the present invention provides the possibility for clinical non-invasive diagnosis of IgAN, especially severe IgAN. Based on the above results, the present invention has determined a new biomarker combined detection method for non-invasive diagnosis of IgAN through literature review and preliminary statistical analysis, and further verified it in clinical samples, and found that it has good sensitivity and specificity through ROC curve analysis, which can be used to prepare protein chips for IgAN diagnosis. At the same time, these biomarker combinations can also be applied to products such as commercial kits.

[0063] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A biomarker composition related to the diagnosis and prognosis of IgA nephropathy, It is characterized in that The biomarker composition consists of cytokines FCAR, MBL and MMP-9.

2. A product for diagnosing IgA nephropathy and / or assessing the prognosis of IgA nephropathy, It is characterized in that The product comprises a reagent for detecting the expression level of the biomarker composition of claim 1.

3. The product according to claim 2, It is characterized in that The score of the biomarker expression level was calculated using the following formula: m = 0.17*FCAR-4.684*MBL-0.162*MMP-9+8.834; wherein m represents the expression level of the biomarker composition, pg / ml; FCAR, MBL and MMP-9 represent their respective concentrations, pg / ml; 8.834 is a constant in the formula calculated using the logistic regression model.

4. The product according to claim 2, It is characterized in that The product includes a protein chip or a kit.

5. Use of a reagent for detecting the biomarker composition as claimed in claim 1 in preparing a product for assisting in the diagnosis of IgA nephropathy and / or assessing the prognosis of IgA nephropathy.

6. The use according to claim 5, It is characterized in that The IgA nephropathy includes severe IgA nephropathy; the product includes a protein chip or a kit.

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