Biomarker for diagnosing lupus nephritis and application thereof

Abstract

The invention relates to the technical field of biomolecular detection, in particular to a biomarker for diagnosing lupus nephritis and application of the biomarker. The invention discloses application of a reagent for detecting the level of one or more biomarkers in preparation of a lupus nephritis diagnosis product, the biomarkers comprise EGLN1, FAM129C and FCRL2, a new biomarker and product are provided for lupus nephritis detection, lupus nephritis patients and normal people can be effectively distinguished, and the application of the reagent for detecting the level of one or more biomarkers is provided. The invention provides a new scheme and approach for diagnosis and treatment of lupus nephritis, and has significant clinical value.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application number 202311205616.8, filed with the Chinese Patent Office on September 18, 2023, and entitled “Biomarkers for Lupus Nephritis and Their Applications”, the entire contents of which are incorporated into this application by application. Technical Field

[0003] The present application relates to the field of biomolecular detection technology, and in particular to biomarkers for lupus nephritis diagnosis and their applications. Background Art

[0004] Systemic lupus erythematosus (SLE) is a common chronic systemic autoimmune disease, with the main clinical characteristics of the production of multiple autoantibodies, multi-organ and multi-system involvement, and high disability rate. With the vigorous development of modern immunology, systemic lupus erythematosus, as the prototype of systemic autoimmune diseases, has become a hot spot and focus of research in the field of rheumatology and immunology. Studies have shown that 30-80% of SLE patients will develop lupus nephritis (Lupus nephritis, LN), which often leads to chronic kidney disease (Chronic Renal Disease, CKD) and end-stage renal disease (End Stage Renal Disease, ESRD). In clinical practice, hormones and immunosuppressants are usually used for treatment, but some LN patients are insensitive to drugs and transform into chronic nephritis, and can only survive for 5-10 years. In addition, hormone therapy can also produce significant side effects. Some patients progress extremely rapidly and survive for only 2-3 years.

[0005] At present, renal histological biopsy is still the gold standard for diagnosing and distinguishing different stages of LN; however, renal biopsy has many disadvantages and problems, such as less tissue is obtained. If the renal lesions are focal or unevenly distributed, it may lead to misdiagnosis due to deviations; and renal histological biopsy may leave trauma after surgery, which is easy to aggravate hematuria, and even cause massive bleeding and death in severe cases. Renal puncture can clarify the pathological type of kidney disease and is helpful for clinical research, but it has little significance for clinical treatment guidance for patients. There are many contraindications, and many patients are not suitable for renal puncture. In 2008, Lane et al. from the Cleveland Clinic in Ohio, USA, reported the results of a systematic review of English literature on renal tumor puncture biopsy published before 2001; in this systematic review, insufficient tissue obtained by puncture for pathological diagnosis was defined as puncture failure, and tissue obtained by puncture was inconsistent with the pathological diagnosis of the surgical resection specimen as inaccurate puncture. The researchers collected 27 relevant articles, including 2474 cases. The results showed that the average false negative rate of biopsy was 4.4% (0-25%), the average false positive rate was 1.2% (0-8.2%), the average puncture failure rate was 8.9% (0-22%), the average pathological diagnosis rate was 5.5% (0-36%), the average diagnostic accuracy was 88.9% (40%-100%), and the incidence of complications of renal tumor biopsy was <5%, including bleeding, infection, arteriovenous fistula, pneumothorax, etc. CT scans after renal puncture revealed that 85%-90% of patients had perirenal bleeding, but only 1%-2% of patients required blood transfusions.

[0006] Currently, there is no clinically established non-invasive method to diagnose and guide the treatment of LN. Traditional SLE biomarkers such as anti-DNA antibodies and complement deficiency are widely accepted as diagnostic tools for assessing SLE activity. So far, double-stranded anti-DNA antibodies (anti-dsDNA) and serum complement are the only non-invasive conventional biomarkers for monitoring renal activity in LN patients. However, these markers are only a reflection of the immune activity of the disease, not a marker of renal damage or poor prognosis.

[0007] Therefore, it is of great significance to the diagnosis and treatment of lupus nephritis to continue to explore the potential therapeutic or diagnostic targets of lupus nephritis, explore the mechanism of target action, and promote the development of new drugs and clinical transformation. Summary of the invention

[0008] The purpose of this application is to provide a new biomarker for lupus nephritis and its application.

[0009] In order to achieve the above objectives, this application adopts the following technical solutions:

[0010] The first aspect of the present application discloses the use of EGLN1, FAM129C and FCRL2 as biomarkers for lupus nephritis; or the use of reagents for detecting the levels of one or more of the following biomarkers in the preparation of lupus nephritis diagnostic products, wherein the biomarkers include EGLN1, FAM129C and FCRL2.

[0011] It should be noted that the key to this application is that the study found that EGLN1, FAM129C and FCRL2 have expression differences between lupus nephritis patients and healthy people, and verified that this expression difference only exists in lupus nephritis patients, but there is no differential expression in systemic lupus erythematosus patients without lupus nephritis. EGLN1, FAM129C and FCRL2 can all be used as independent indications for the diagnosis of lupus nephritis. Therefore, this application creatively proposes to use EGLN1, FAM129C and FCRL2 as biomarkers for lupus nephritis; on this basis, the reagents for detecting EGLN1, FAM129C and FCRL2 can be used as lupus nephritis diagnostic reagents, that is, the detection reagents or components for obtaining the levels of EGLN1, FAM129C or FCRL2 in the sample are used to prepare lupus nephritis diagnostic products.

[0012] In one implementation of the present application, the biomarkers for diagnosing lupus nephritis include one or more of EGLN1, FAM129C, and FCRL2.

[0013] Furthermore, EGLN1, FAM129C and FCRL2 can all serve as independent indicators for the diagnosis of lupus nephritis.

[0014] It should also be noted that the present application uses EGLN1, FAM129C and FCRL2 as biomarkers for lupus nephritis, which can be nucleic acids or proteins. For example, lupus nephritis can be diagnosed based on the nucleic acid expression level of the biomarker, or the protein expression level of the biomarker. Among them, the nucleic acid expression level can be determined by detecting DNA, RNA or mRNA; the protein expression level can be characterized by detecting the level of binding between the protein and its specific polypeptide. In the present application, the analysis object can be a collected sample, for example, lupus nephritis can be diagnosed by detecting the nucleic acid expression level or protein expression level of the biomarker in the collected sample; or, the nucleic acid expression level or protein expression level of the biomarker can be directly analyzed based on the existing data to diagnose lupus nephritis.

[0015] Furthermore, the nucleic acid level or protein level includes but is not limited to the abundance or concentration of nucleic acid or protein, and in some aspects may also include the abundance or concentration of mutations such as nucleic acid or protein.

[0016] Furthermore, the nucleic acid level of the present application is obtained by sequencing technology, nucleic acid amplification technology, nucleic acid hybridization technology, electrophoresis technology, biological mass spectrometry technology or chromatography technology.

[0017] Furthermore, methods for obtaining nucleic acid levels include, but are not limited to, any of the following methods: gene sequencing, polymerase chain reaction, isothermal amplification reaction, gene chip, probe hybridization, gel electrophoresis, Northern blotting, nucleic acid mass spectrometry or liquid chromatography.

[0018] Furthermore, the protein level of the present application is obtained by sequencing technology, immunoassay technology, electrophoresis technology, biomass spectrometry technology or chromatography technology.

[0019] Furthermore, methods for obtaining protein levels include, but are not limited to, any of the following methods: amino acid sequencing, enzyme-linked immunosorbent assay, chemiluminescence, immunochromatography, radioimmunoassay, immunohistochemistry, immunoblotting, flow cytometry, gel electrophoresis, protein profiling or liquid chromatography.

[0020] The second aspect of the present application discloses a product for diagnosing lupus nephritis, wherein the product contains reagents for detecting biomarker nucleic acids and / or reagents for detecting biomarker proteins, wherein the aforementioned biomarkers are selected from one or more of EGLN1, FAM129C and FCRL2.

[0021] In some aspects, the article of manufacture includes, but is not limited to, a kit form, a system device form, a computer readable medium, or a computer system form.

[0022] It should be noted that the key to this application is that the research found that EGLN1, FAM129C or FCRL2 can be used as a biomarker for lupus nephritis; therefore, reagents for detecting one or more biomarker nucleic acids and / or proteins selected from EGLN1, FAM129C and FCRL2 can be used as components of lupus nephritis diagnostic products.

[0023] In one implementation of the present application, the reagents for detecting biomarker nucleic acids include specific primers and / or probes designed for EGLN1, FAM129C or FCRL2 nucleic acids; the reagents for detecting biomarker proteins include polypeptides that specifically bind to or recognize EGLN1, FAM129C or FCRL2 proteins.

[0024] It should be noted that the specific primers of the present application refer to primers designed for the biomarker nucleic acids of the present application, such as primers for gene sequencing methods, primers for polymerase chain reaction methods, primers for isothermal amplification reaction methods, or other primers capable of detecting the biomarker nucleic acids of the present application; specific probes refer to probes designed for the biomarker nucleic acids of the present application, such as real-time fluorescence quantitative PCR probes, gene chip detection probes, specific probes for probe hybridization methods, or other probes capable of detecting the biomarker nucleic acids of the present application.

[0025] It should also be noted that the key to the product of the present application is the specific primers and / or probes designed for the biomarker nucleic acid of the present application, or the polypeptides that specifically bind to or recognize the biomarker protein of the present application; for ease of use, the reagents for detecting the biomarker nucleic acid of the present application may also include conventional reagents or components corresponding to the specific detection method used, such as gene sequencing, polymerase chain reaction, isothermal amplification reaction, gene chip, probe hybridization, gel electrophoresis, RNA blotting, nucleic acid mass spectrometry or liquid chromatography conventional reagents or components; similarly, the reagents for detecting the biomarker protein of the present application may also include amino acid sequencing, enzyme-linked immunosorbent assay, chemiluminescence, immunochromatography, radioimmunoassay, immunohistochemistry, immunoblotting, flow cytometry, gel electrophoresis, protein spectrum or liquid chromatography conventional reagents or components. It can be understood that these conventional reagents or components corresponding to the specific detection methods used can be purchased commercially or integrated into the product of the present application.

[0026] In one implementation of the present application, the biomarker nucleic acid of the present application includes the biomarker gene, a DNA fragment extracted from the biomarker gene, and the RNA, mRNA, miRNA or LcRNA corresponding to the biomarker gene or its DNA fragment; the polypeptide includes at least one of an antibody, an antigen-binding fragment, a single-chain antibody and a ligand that specifically binds to the biomarker protein of the present application.

[0027] It should be noted that the key to this application is to detect the level of the biomarker of this application. The detection object can be the biomarker nucleic acid of this application, including various DNA or RNA, or the biomarker protein of this application. When the detection object is a protein, it can be detected by various polypeptides that specifically bind to it, such as but not limited to antibodies, antigen binding fragments, single-chain antibodies, ligands that specifically bind to the biomarker protein of this application, etc.

[0028] In one implementation of the present application, the product of the present application also includes reagents or components for detecting other markers.

[0029] Furthermore, the reagents for detecting the biomarkers of the present application can be used in combination with reagents or components for detecting other markers. More specifically, other markers include but are not limited to one or more of C3, C4, dsDNA, COBLL1, CD79B, HLA-DOB, SLPI, and GPR18.

[0030] In some aspects, the product of the present application also includes a sample processing reagent. The sample processing reagent depends on the specific sample, for example, more specifically, the sample processing reagent includes at least one of a sample lysis reagent, a sample purification reagent, and a sample extraction reagent.

[0031] Furthermore, the product of the present application also includes at least one of a standard substance, a calibrator, a reference substance, and a buffer solution.

[0032] Furthermore, the product of the present application may be supplemented with tubes for mixing the components, microplates, and instruction materials recording the method of use, etc., as needed.

[0033] In some aspects, the sample of the present application includes tissue, cell, urine, saliva, pleural effusion, ascites, feces, synovial fluid or various body fluids. Wherein, body fluid refers to intracellular fluid and extracellular fluid, including tissue fluid, plasma, blood (such as whole blood, serum, or plasma), lymph, cerebrospinal fluid, semen, aqueous humor, etc. In some embodiments, the sample includes peripheral blood mononuclear cells.

[0034] The third aspect of the present application discloses a method for diagnosing lupus nephritis, comprising the following steps:

[0035] (i) obtaining the level of a biomarker in a sample to be tested, wherein the biomarker is selected from one or more of the following: EGLN1, FAM129C and FCRL2;

[0036] (ii) comparing the biomarker level in the test sample with the corresponding biomarker level in the control sample; if there is a significant difference, the subject from which the test sample is derived has an indication of lupus nephritis; or, comparing the biomarker level in the test sample with a set threshold; if there is a significant difference between the biomarker level in the test sample and the threshold, the subject from which the test sample is derived has an indication of lupus nephritis.

[0037] In the present application, for example, the control sample is a sample from a healthy population. At this time, the diagnostic criteria for lupus nephritis are that there is a significant difference between the biomarker level of the present application in the sample to be tested and the corresponding biomarker level in the control sample. Alternatively, a threshold is set in advance according to the biomarker level of the control sample, and the biomarker level of the present application in the sample to be tested is compared with the set threshold. Therefore, in the present application, the threshold is a threshold set according to the biomarker level of the control sample, that is, a threshold is set according to the biomarker level in the sample from a healthy population.

[0038] Furthermore, the biomarker level of the present application includes nucleic acid level or protein level.

[0039] Furthermore, the biomarkers EGLN1, FAM129C and FCRL2 of the present application can all independently serve as indications for the diagnosis of lupus nephritis.

[0040] Furthermore, the biomarker levels of the present application do not change significantly in systemic lupus erythematosus patients without lupus nephritis (NoLN), and cannot distinguish NoLN patients from normal people. Therefore, the biomarker of the present application can not only be used as a marker to distinguish lupus nephritis patients (LN) from healthy people, but also as a marker to distinguish NoLN patients from LN patients.

[0041] In some aspects, the above diagnosis includes but is not limited to screening, auxiliary diagnosis, diagnosis, prediction / judgment of disease severity, assessment of disease activity, and monitoring of treatment for lupus nephritis.

[0042] The fourth aspect of the present application discloses a method for evaluating or screening lupus nephritis markers, comprising performing a correlation or consistency analysis between the candidate biomarkers and the biomarker levels of the present application, and verifying the diagnostic value of the potential markers through the conclusions of the correlation or consistency.

[0043] It can be understood that the present application study found that EGLN1, FAM129C or FCRL2 can be used as lupus nephritis markers; therefore, EGLN1, FAM129C or FCRL2 can be used as positive controls for screening other lupus nephritis markers; for example, based on the correlation or consistency between the candidate biomarkers and the levels of EGLN1, FAM129C or FCRL2, lupus nephritis markers can be screened.

[0044] It should be noted that the biomarkers EGLN1, FAM129C and FCRL2 of the present application can all be independently used as indications for screening other lupus nephritis markers.

[0045] The fifth aspect of the present application discloses a method for evaluating or screening a therapeutic agent for lupus nephritis, comprising evaluating the level of a biomarker in a sample treated with the therapeutic agent, wherein the biomarker is selected from one or more of the following: EGLN1, FAM129C and FCRL2.

[0046] It should be noted that the present application study found that there were expression differences in EGLN1, FAM129C and FCRL2 between lupus nephritis patients and healthy people, so the aforementioned biomarkers were used as lupus nephritis markers; on this basis, it can be understood that if the lupus nephritis patient is successfully treated, the expression levels of EGLN1, FAM129C and FCRL2 will also be different from those before treatment. Therefore, the present application creatively proposes that the levels of EGLN1, FAM129C and FCRL2 be used to evaluate or screen lupus nephritis therapeutic agents. For example, if the levels of EGLN1, FAM129C and FCRL2 of the sample are significantly lower than before treatment with the therapeutic agent after treatment with the therapeutic agent, it is judged that the therapeutic agent has a good therapeutic effect on lupus nephritis; otherwise, it indicates that the lupus nephritis patient has not improved or the effect is not obvious before and after treatment with the therapeutic agent; thereby, the screening of lupus nephritis therapeutic agents or the evaluation of the efficacy of the therapeutic agent are achieved.

[0047] It should also be noted that the biomarkers EGLN1, FAM129C and FCRL2 of the present application can all be independently used as indications for evaluating or screening lupus nephritis therapeutic agents.

[0048] In the present application, the level of a biomarker refers to the nucleic acid level or protein level. For example, the nucleic acid level is the expression level of DNA, RNA or mRNA, and the protein level is the level of binding between the protein of the biomarker and its specific polypeptide.

[0049] Furthermore, it also includes one or more nucleic acids or proteins of C3, C4, dsDNA, COBLL1, CD79B, HLA-DOB, SLPI, and GPR18.

[0050] Due to the adoption of the above technical solution, the beneficial effects of this application are:

[0051] The present application provides a new biomarker for lupus nephritis. By analyzing the levels of one or more of EGLN1, FAM129C, and FCRL2, it can effectively distinguish lupus nephritis patients from normal people, and provides a new solution and approach for the diagnosis and treatment of lupus nephritis, making up for the lack of existing clinical non-invasive detection methods for lupus nephritis, and has significant clinical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 contains Figure 1A to Figure 1C , which is the GEO discovery set data analysis result in the embodiment of this application. Figure 1A : Compared with normal controls, the expression of EGLN1 gene in peripheral blood mononuclear cells (PBMC) of patients with lupus nephritis is increased; Figure 1B : Compared with normal people, the expression of FAM129C gene in PBMC of patients with lupus nephritis is reduced; Figure 1C : Compared with normal people, the expression of FCRL2 gene in PBMC of patients with lupus nephritis is reduced.

[0053] Figure 2 contains Figure 2A to Figure 2C , is the ROC curve analysis result of the discovery set in the examples of this application. Figure 2A : The data from the discovery set showed that the EGLN1 gene had a good ability to distinguish patients from normal people; Figure 2B : The data from the discovery set showed that the FAM129C gene had a good ability to distinguish patients from normal people; Figure 2C : The discovery set data showed that the FCRL2 gene had a good ability to distinguish patients from normal people.

[0054] Figure 3 contains Figure 3A to Figure 3C , which is the analysis result of the GEO validation set in the embodiment of this application. Figure 3A : Compared with normal controls, the expression of EGLN1 gene in peripheral blood mononuclear cells (PBMC) of patients with lupus nephritis is increased; Figure 3B : Compared with normal people, the expression of FAM129C gene in PBMC of patients with lupus nephritis is reduced; Figure 3C : Compared with normal people, the expression of FCRL2 gene in PBMC of patients with lupus nephritis is reduced.

[0055] Figure 4 contains Figure 4A to Figure 4C , is the ROC curve analysis result of the validation set in the embodiment of this application. Figure 4A :The validation set data showed that the EGLN1 gene had good discrimination ability between patients and normal people; Figure 4B :The validation set data showed that the FAM129C gene had good discrimination ability between patients and normal people; Figure 4C : The validation set data showed that the FCRL2 gene had good discrimination ability between patients and normal people.

[0056] Figure 5 contains Figure 5A to Figure 5C , which is the GEO discovery set analysis result in the embodiment of this application. Figure 5A : The data from the discovery set showed that the expression of the EGLN1 gene did not change in PBMCs between patients with systemic lupus erythematosus without lupus kidney (NoLN) and normal subjects, and was not a differentially expressed gene; Figure 5B : The data from the discovery set showed that the expression of the FAM129C gene did not change in PBMCs between NoLN patients and normal subjects, and it was not a differentially expressed gene; Figure 5C: The data from the discovery set showed that the expression of the FCRL2 gene did not change in PBMCs between NoLN patients and normal controls, and was not a differentially expressed gene.

[0057] Figure 6 contains Figure 6A to Figure 6C , which is the analysis result of the GEO validation set in the embodiment of this application. Fig. 6A : The validation set data showed that the expression of EGLN1 gene in PBMCs of NoLN patients and normal subjects did not change, and it was not a differentially expressed gene; Figure 6B : The validation set data showed that the expression of FAM129C gene in PBMCs of NoLN patients and normal subjects did not change, and it was not a differentially expressed gene; Figure 6C : The validation set data showed that the expression of FCRL2 gene in PBMCs of NoLN patients and normal subjects did not change, and it was not a differentially expressed gene.

[0058] Figure 7 contains Figure 7A to Figure 7C , which is the GEO discovery set analysis result in the embodiment of this application. Fig. 7A : The data from the discovery set showed that the EGLN1 gene was differentially expressed between NoLN patients and LN patients; Figure 7B : The data from the discovery set showed that the FAM129C gene was differentially expressed in NoLN patients and LN patients; Figure 7C : The discovery set data showed that the FCRL2 gene was differentially expressed between NoLN patients and LN patients.

[0059] Figure 8 contains Figure 8A to Figure 8C , which is the analysis result of the GEO validation set in the embodiment of this application. Fig. 8A : The validation set data showed that the EGLN1 gene was differentially expressed in NoLN patients and LN patients; Figure 8B : The validation set data showed that the FAM129C gene was differentially expressed in NoLN patients and LN patients; Figure 8C : The validation set data showed that the FCRL2 gene was differentially expressed in NoLN patients and LN patients.

[0060] Figure 9 contains Figures 9A to 9C , is the ROC curve analysis result of the discovery set in the examples of this application. Fig. 9A : The data from the discovery set showed that the combined detection of EGLN1+FAM129C+FCRL2 had good discrimination between LN patients and normal people; Fig. 9B : The data from the discovery set showed that the combined detection of FCRL2+CD79B had good discrimination between LN patients and normal people; Fig. 9C : The discovery set data showed that the combined detection of CD79B+COBLL1+EGLN1+FAM129C+FCRL2 had good discrimination between LN patients and normal people.

[0061] Figure 10 contains Figures 10A to 10C , is the ROC curve analysis result of the validation set in the embodiment of this application. Fig. 10A : The validation set data showed that the combined detection of EGLN1+FAM129C+FCRL2 had good discrimination between LN patients and normal people; Fig. 10B :The validation set data showed that the combined detection of FCRL2+CD79B had good discrimination between LN patients and normal people; Fig. 10C : The validation set data showed that the combined detection of CD79B+COBLL1+EGLN1+FAM129C+FCRL2 had good discrimination between LN patients and normal people.

[0062] Figure 11 contains Figures 11A to 11C , is a comparison of the test results of clinical samples of lupus nephritis patients in the embodiments of this application and normal people. Fig.11A : Compared with normal people, the expression level of EGLN1 in patients with lupus nephritis is significantly increased; Fig. 11B : Compared with normal people, the expression level of FAM129C in patients with lupus nephritis is significantly reduced; Fig. 11C : Compared with normal people, the expression level of FCRL2 in patients with lupus nephritis is significantly reduced.

[0063] Figure 12 contains Figures 12A to 12C , is a comparison of the test results of clinical samples of patients with systemic lupus erythematosus and without lupus nephritis in the embodiments of this application and normal people. Fig. 12A :The expression level of EGLN1 in patients with systemic lupus erythematosus and without lupus nephritis was not significantly different from that in normal subjects; Fig. 12B : The expression level of FAM129C in patients with systemic lupus erythematosus and without lupus nephritis was not significantly different from that in normal subjects; Fig. 12C :The expression level of FCRL2 in patients with systemic lupus erythematosus without lupus nephritis has no significant difference from that in normal people. DETAILED DESCRIPTION

[0064] At present, there is no non-invasive detection method for lupus nephritis in clinical practice; therefore, this application creatively downloads 1038 samples from the GeneExpression Omnibus (GEO) database, hoping to screen genes or their proteins that are differentially expressed in peripheral blood mononuclear cells of lupus nephritis patients and normal people through a series of bioinformatics methods. Finally, it was surprisingly found that EGLN1, FAM129C and FCRL2 can be used as indications of lupus nephritis. Therefore, it is proposed to use EGLN1, FAM129C and FCRL2 genes or proteins as biomarkers for the diagnosis of lupus nephritis, which makes up for the shortcomings of existing lupus nephritis-related indicators and has significant clinical value. This application is completed on this basis.

[0065] The following basic terms or definitions are provided only to help understand the application. These definitions should not be construed as having a scope less than that understood by those skilled in the art. Unless defined otherwise hereinafter, the meaning of all technical terms and scientific terms used in the specific embodiments of the application is intended to be the same as that commonly understood by those skilled in the art. Although it is believed that the following terms are well understood by those skilled in the art, the following definitions are still set forth to better explain the application.

[0066] The terms "comprise", "include", "have", "contain" or "involve" used in this application are inclusive or open-ended and do not exclude other unlisted elements or method steps. The term "consisting of" is considered a preferred embodiment of the term "comprising". If a group is defined below as comprising at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists of only these embodiments.

[0067] When referring to a singular noun an indefinite or definite article e.g. "a" or "an", "the" or "an" is used, this includes a plural of that noun.

[0068] The terms "approximately" and "substantially" used in this application represent the accuracy range that can be understood by those skilled in the art to still ensure the technical effect of the feature in question. The term usually represents ±10%, preferably ±5%, of the indicated value.

[0069] The terms "probe" and "primer" used in this application refer to oligonucleotides complementary to the target position of the target nucleic acid, preferably single-stranded deoxyribonucleotides, including naturally occurring dNMPs (dAMP, dGMP, dCMP and dTMP), deformed nucleotides or non-natural nucleotides, and may also contain ribonucleotides. Of course, the "probe" and "primer" can be modified or sequences unrelated to the target nucleic acid can be added as needed; for example, in real-time fluorescence quantitative PCR amplification, the 5' end of the probe can be modified with a fluorescent group and the 3' end can be modified with a fluorescent quenching group; for example, in the sequencing process, a label sequence can be added to the 5' end of the primer or a corresponding adapter sequence can be added according to the sequencing platform.

[0070] In the present application, the meaning of "complementary" refers to the sufficient complementarity that a primer or a probe selectively hybridizes with a target nucleic acid sequence under hybridization conditions, and has the meaning of including both substantially complementary and perfectly complementary, preferably perfectly complementary. The term "substantially complementary sequences" used in the present specification includes not only completely identical sequences, but also sequences that can function as primers on a specific target sequence and are partially inconsistent with the sequence to be compared.

[0071] The sequences of gene probes and primers do not need to have sequences that are completely complementary to a portion of the sequence of the template, as long as they have sufficient complementarity within the range that they can hybridize with the template to exert their inherent effects. Therefore, the gene probes and primers in the present invention do not need to have sequences that are completely complementary to the above-mentioned nucleotide sequences as templates, as long as they have sufficient complementarity within the range that they can hybridize with the template to exert their inherent effects. The design of primers and gene probes is a skill mastered by those skilled in the art, for example, a primer design program (e.g., PRIMER 3 program) can be utilized.

[0072] The term "peptide" used in this application is a compound formed by α-amino acids linked together by peptide bonds. A peptide composed of three or more amino acid molecules is called a polypeptide; a protein is a substance with a certain spatial structure formed by the winding and folding of a polypeptide chain. "Antibody" refers to a protein that specifically recognizes and binds to an antigen protein. "Antigen binding fragment" refers to a polypeptide chain that can specifically bind to an antigen protein. In this application, the antigen protein refers to EGLN1, FAM129C or FCRL2 protein.

[0073] The terms "EGLN1", "FAM129C" and "FCRL2" used in the present application refer to nucleic acids or proteins thereof.

[0074] This application found through differential gene expression analysis that EGLN1 is expressed in the peripheral blood mononuclear cells of patients with lupus nephritis, and FAM129C and FCRL2 are expressed in the peripheral blood mononuclear cells of patients with lupus nephritis. This revealed that the above gene expressions can distinguish LN patients from normal people, thereby making a judgment on LN patients and assisting doctors in making decisions on kidney biopsy. The known functions of these genes are shown in Table 1.

[0075] Therefore, in various aspects of the present application, there is provided a method for diagnosing lupus nephritis (LN) comprising EGLN1, FAM129C and FCRL2 as markers, and a method for preparing a product for diagnosing lupus nephritis by using a detection reagent for detecting EGLN1, FAM129C or FCRL2 in a sample.

[0076] The terms "lupus nephritis" and "LN" used in this application are interchangeable and widely occur in patients with systemic lupus erythematosus. 30-80% of SLE patients will develop lupus nephritis (Lupus nephritis, LN), often leading to chronic kidney disease (Chronic Renal Disease, CKD) and end-stage renal disease (End Stage Renal Disease, ESRD). Poor prognosis and high mortality. The onset is related to factors such as heredity, sex hormones, immunity, and environment. Specifically, it can refer to the criteria for diagnosing LN set by the American College of Rheumatology (ACR) in 1982 and / or the criteria revised by the Systemic Lupus Collaborative Clinic (SLICC) (reviewed in Petri et al. (Arthritis and Rheumatism, 2012, Vol.64, pp. 2677-2686)) characterized by autoimmune diseases.

[0077] The term "diagnosis" as used in this application refers to the process of identifying a medical condition or disease (such as LN) through its signs, symptoms, and especially the results of various diagnostic processes (including detecting the level of FCRL2 in a biological sample (such as serum) obtained from an individual). Moreover, the term "diagnosis" as used herein includes screening for diseases, auxiliary diagnosis, diagnosis (determining whether a disease exists), prediction or judgment of the severity of a disease, assessment of disease activity, monitoring treatment, such as monitoring disease progression or recurrence during treatment, evaluating the efficacy of disease treatment, and the selection of a given treatment regimen, etc. Without limitation, in some specific embodiments, the present application has fully demonstrated that the presence or absence of LN in a subject based on the level of EGLN1, VPREB or FCRL2 can be used to determine, and thus can be used for screening, auxiliary diagnosis, and diagnosis.

[0078] The term "treatment" as used in this application includes any treatment of a disease in a mammal, and includes: (a) preventing the disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, i.e., preventing its development; or (c) alleviating the disease, i.e., causing partial or complete improvement of the disease. The therapeutic agent can be administered before, during, or after the onset of the disease or injury.

[0079] As used herein, the terms "individual," "subject," "subject," and "patient" are used interchangeably herein and refer to any mammalian subject, particularly a human, from whom a biological sample is collected.

[0080] The terms "sample", "sample", "test sample", "subject sample" and the like used in this application include various sample types obtained from a patient, individual or subject and can be used for diagnosis or monitoring determination. Patient samples can be obtained from healthy subjects, sick patients or patients with LN related symptoms. In addition, the sample obtained from the patient can be divided into parts, and only a part can be used for diagnosis. In addition, the sample or a part thereof can be stored under the condition of keeping the sample for subsequent analysis. This definition specifically includes other liquid samples of blood and biological origin (including but not limited to tissue, cells (such as PMBC cells, T cells, etc.), body fluids, serum, plasma, whole blood, urine, semen, saliva, pleural effusion, ascites, cerebrospinal fluid, feces and synovial fluid, etc.). In a specific embodiment, the sample includes a blood sample. In another embodiment, a serum sample is used. This definition also includes samples that are manipulated in any way after the sample is obtained, such as by centrifugation, filtration, precipitation, dialysis, chromatography, treatment with reagents, washing or enrichment of certain cell populations. These terms also include clinical samples, and also include cells, cell supernatants, tissue samples, organs, etc. in culture. Samples may also include fresh frozen and / or formalin fixed, paraffin embedded tissue blocks, such as blocks prepared from clinical or pathological biopsies, prepared for pathological analysis or research by immunohistochemistry. Samples may be tested immediately after collection, stored at RT, 4 degrees Celsius, -20 degrees Celsius, or -80 degrees Celsius, and tested after storage for 24 hours, 1 week, 1 month, 1 year, 10 years, or up to 30 years.

[0081] The term "ROC" or "ROC curve" used in this application refers to a receiver operating characteristic curve, also known as a sensitivity curve, which can be a graphical representation of the performance of a binary classifier system. For any given method, a ROC curve can be generated by plotting sensitivity for specificity under a variety of threshold settings. In addition, as long as at least one of the three parameters (e.g., sensitivity, specificity, and threshold settings) is provided, and the ROC curve can determine the value or expected value of any unknown parameter, the unknown parameter can be determined using a curve for ROC curve fitting.

[0082] The term "AUC" or "ROC-AUC" used in this application generally refers to the area under the receiver operating characteristic curve, i.e., Area Under Curve. This metric takes into account the sensitivity and specificity of the method and can provide a measurement of the diagnostic utility of the method. Typically, ROC-AUC is in the range of 0.5 to 1.0, where a value closer to 0.5 indicates that the method has limited diagnostic utility (e.g., lower sensitivity and / or specificity), and a value closer to 1.0 indicates that the method has greater diagnostic utility (e.g., higher sensitivity and / or specificity). See, e.g., Pepe et al., "Limitations of the Odds Ratio in Gauging the Performance of a Diagnostic, Prognostic, or Screening Marker", Am. J. Epidemiol 2004, 159 (9): 882-890, which is incorporated herein by reference in its entirety. Other approaches to characterizing diagnostic utility using likelihood functions, odds ratios, information theory, predictive values, calibration (including goodness of fit), and reclassification measures are summarized in Cook, "Use and Misuse of the Receiver Operating Characteristic Curve in Risk Prediction), Circulation 2007, 115:928-935, which is incorporated herein by reference in its entirety.

[0083] The detection agents or components for obtaining the levels of biomarkers in a sample can be understood to include detection agents that directly obtain the levels of biomarkers in a sample, or components that indirectly obtain the levels of biomarkers in a sample (such as a computer program that directly obtains an indicator of the levels of biomarkers in a sample that has already been detected).

[0084] It can be understood that there are many ways to detect nucleic acids in the art, including but not limited to sequencing technology, nucleic acid amplification technology, nucleic acid hybridization technology, electrophoresis technology, biological mass spectrometry technology or chromatography technology, all of which can be used in this application. In some specific embodiments of the present application, it includes but is not limited to any of the following specific methods: gene sequencing, polymerase chain reaction, isothermal amplification reaction, gene chip method, probe hybridization method, gel electrophoresis, Northern blotting, nucleic acid mass spectrometry or liquid chromatography. It can be understood that nucleic acids include DNA or RNA levels.

[0085] For example, in some embodiments, gene sequencing can be transcriptome sequencing or genome sequencing. Genome sequencing can adopt first-generation Sanger sequencing, second-generation sequencing ("NGS") or third-generation sequencing reagents, etc. Second-generation sequencing ("NGS") is different from "Sanger sequencing" (first-generation sequencing), which is based on the electrophoretic separation of chain termination products in a single sequencing reaction. NGS is a revolutionary change to traditional Sanger sequencing technology, and can sequence hundreds of thousands to millions of nucleic acid molecules at a time. The sequencing platform of NGS that can be used for the present application is commercially available, including but not limited to Roche / 454FLX, Illumina / Solexa GenomeAnalyzer and Applied Biosystems SOLID system, etc. Transcriptome sequencing can also quickly and comprehensively obtain almost all transcripts and gene sequences of a certain species' specific cells or tissues in a certain state through the second-generation sequencing platform, which can be used to study gene expression, gene function, structure, alternative splicing and new transcript prediction, etc. In some other embodiments of the present application, the nucleic acid sequencing method can be single-molecule real-time sequencing. Single-molecule DNA sequencing technology is a new generation of sequencing technology developed in the past 10 years, also known as the third-generation sequencing technology, including single-molecule real-time sequencing, true single-molecule sequencing, single-molecule nanopore sequencing and other technologies.

[0086] For another example, in some embodiments, the polymerase chain reaction method includes primers for biomarker amplification, wherein the primer refers to a primer that can specifically amplify a biomarker gene, such as a polynucleotide of a certain length, such as a primer of about 35 nucleotides or longer, which can hybridize with at least a portion of the template sequence and serve as a starting point for synthesizing primer extension products. In some embodiments, the probe hybridization method includes a biomarker probe, wherein the probe refers to a probe that can specifically identify the FCRL2 gene or the transcript of the gene, which is a molecule that can bind to a specific sequence or subsequence or other part of another molecule, and generally refers to a nucleic acid probe that binds to another nucleic acid (i.e., a biomarker-specific sequence as a target nucleotide) through complementary base pairing. In some embodiments, a gene chip refers to a composite structure formed by an array of the aforementioned probes fixed on a substrate material (specifically including but not limited to polymers such as nylon membranes, nitrocellulose membranes, and glass, etc.). In some embodiments, when RNA needs to be detected, cDNA needs to be obtained by reverse transcription, and then the detection is carried out by the primers for biomarker amplification.

[0087] It is understood that there are many protein detection methods in the art, including but not limited to sequencing technology, immunological technology, electrophoresis technology, biological mass spectrometry technology or chromatography technology, all of which can be used in this application. In some specific embodiments of the present application, it includes but is not limited to any of the following specific methods: amino acid sequencing, enzyme-linked immunosorbent assay, chemiluminescence, immunochromatography, radioimmunoassay, immunohistochemistry, immunoblotting, flow cytometry, gel electrophoresis, protein spectrometry or liquid chromatography.

[0088] For example, in some embodiments, the method for quantitatively detecting biomarkers at the protein level includes specific antibodies. Among them, specific antibodies refer to antibodies that can specifically recognize biomarker-encoded proteins, specifically including but not limited to monoclonal antibodies (such as full-length or complete monoclonal antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (such as bispecific antibodies with expected biological activity), nano antibodies, or at least one of certain antibody fragments. It is understood that the antibody can be a human, humanized and / or affinity matured antibody. According to the experimental data of the present application, EGLN1, FAM129C and FCRL2 can be used as independent indications for the diagnosis of lupus nephritis, that is, effective diagnosis of lupus nephritis can be achieved based on any one of the biomarkers EGLN1, FAM129C or FCRL2 alone.

[0089] It is understandable that in order to further enhance the diagnostic effect of lupus nephritis, the biomarkers detected by the reagents can also be other biological materials that can be used as markers, such as nucleic acid fragments, proteins, metabolites, etc., which are well known to those skilled in the art and have diagnostic effects. In this way, a combination of multiple biomarkers can be used, and they can cooperate with each other to achieve a better diagnostic effect on lupus nephritis patients, thereby achieving a more effective diagnostic evaluation. Therefore, in addition to being combined with each other as markers for the joint detection of lupus nephritis, EGLN1, FAM129C and FCRL2 can also be combined with other existing known diagnostic markers for detection, thereby improving the diagnostic efficiency.

[0090] For example, in some specific embodiments, the diagnosis also includes diagnostic reagents for detecting other genes or proteins, such as those mentioned in this application. The combined detection can be a combination of any one or more of them. In a preferred embodiment of this application, when CD79B, COBLL1, EGLN1, FAM129C and FCRL2 are used for the diagnosis and evaluation of lupus nephritis at the same time, it has a higher AUC value of 0.848, which significantly improves the effect of distinguishing lupus nephritis patients from normal people.

[0091] In the present application, the control samples can be selected according to actual needs. For example, in disease diagnosis, the control samples are samples from the normal population, while when used for prognosis assessment, they can be control samples of different prognostic conditions.

[0092] In some specific embodiments, a set value of a biomarker level can be given. For example, the set value can be determined based on the expression level of FCRL2 in normal samples of normal people and / or non-lupus nephritis patients, for example, the average value of the expression level of FCRL2 in normal samples of an appropriate number of samples is selected, or a reasonable multiple is set based on the average value, such as 0.9 times, 0.8 times, 0.7 times, 0.6 times, 0.5 times, etc. When the expression level of FCRL2 in the subject is lower than the set value, it is judged to be lupus nephritis. It is understandable that the set value determined based on the average value, or the multiple of the average value needs to have good classification significance, and the known samples can be tested by using a commonly used statistical test method through the classification based on the set value. When the result is statistically significant, it indicates that the set value can be used as a judgment standard.

[0093] The level of a biomarker refers to a value derived from direct measurement or further indirect measurement of the biomarker for a subject, which is usually derived at least in part from the abundance or concentration of the biomarker in the subject's sample. Indirectly derived values ​​include those derived by applying a function to the measured value of the biomarker. Directly measured values ​​include, but are not limited to, values ​​of biomarkers determined by at least one of sequencing, hybridization, mass spectrometry, immunoassay, immunofluorescence, flow cytometry, etc.

[0094] According to the core diagnostic use of the present application, the process for detecting the level of the biomarker of the present application can be configured into a corresponding product form for the diagnosis or prediction of lupus nephritis. The product includes reagents or components for detecting the biomarker of the present application. It is understood that such product forms are diverse, including but not limited to kit forms, system devices, computer readable media or computer system forms.

[0095] In some embodiments of the present application, a kit for detecting or analyzing the biomarker level of the present application to predict LN disease is also disclosed. The kit may include reagents for detecting the level of one or more markers and instructions for predicting LN disease based on the detected levels.

[0096] The kit of the present application may include a set of reagents for generating a data set by at least one determination. The set of reagents can detect any biomarker level of the quantitative present application. The set of reagents can also further detect other one or more biomarker levels. In some aspects, the detection reagent is used for the level detection of nucleic acids or proteins.

[0097] When the detection reagent is used for nucleic acid level detection, it can be understood that there are many ways of nucleic acid detection in the art, including but not limited to sequencing technology, nucleic acid amplification technology, nucleic acid hybridization technology, electrophoresis technology, biological mass spectrometry technology or chromatography technology, all of which can be used in this application. In some specific embodiments of the present application, including but not limited to any of the following specific methods: gene sequencing, polymerase chain reaction, isothermal amplification reaction, gene chip method, probe hybridization method, gel electrophoresis, Northern blotting, nucleic acid mass spectrometry or liquid chromatography.

[0098] For example, in some embodiments, the reagent may be a sequencing reagent, such as a first-generation Sanger sequencing reagent, a second-generation sequencing (NGS) reagent, a third-generation sequencing reagent, etc. In other embodiments, the reagent may be a PCR primer reagent, wherein the primer refers to a primer that can specifically amplify a biomarker gene, such as a polynucleotide of a certain length, such as a primer of about 35 nucleotides or longer, which can hybridize with at least a portion of the template sequence and serve as a starting site for synthesizing primer extension products. In other embodiments, the reagent may be a probe reagent, wherein the probe refers to a probe that can specifically identify a biomarker gene or a transcript of the gene, which is a molecule that can bind to a specific sequence or subsequence or other portion of another molecule, generally referring to a nucleic acid probe that binds to another nucleic acid (i.e., a biomarker-specific sequence as a target nucleotide) through complementary base pairing.

[0099] When the detection reagent is used for protein level detection, it can be understood that there are many protein detection methods in the art, including but not limited to sequencing technology, immunoassay, electrophoresis technology, biomass spectrometry technology or chromatography technology, all of which can be used in this application. In some specific embodiments of the present application, it includes but is not limited to any of the following specific methods: amino acid sequencing, enzyme-linked immunosorbent assay, radioimmunoassay, immunohistochemistry, immunoblotting, flow cytometry, gel electrophoresis, protein spectrometry or liquid chromatography.

[0100] For example, in some embodiments, the reagent can be a specific antibody, wherein the specific antibody refers to an antibody that can specifically recognize the protein encoded by the biomarker, specifically including but not limited to monoclonal antibodies (such as full-length or complete monoclonal antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (such as bispecific antibodies with expected biological activity) or at least one of certain antibody fragments. It is understood that the antibody can be a human, humanized and / or affinity matured antibody.

[0101] In some embodiments, the kit may also include a carrier, package or container that is compartmentalized to receive one or more containers, such as vials, tubes, etc., each of which contains one of the individual elements to be used in the method. The kit of the present application may include the above-mentioned containers and one or more other containers containing materials required from the standpoint of a commercial end user, including buffers, diluents, filters, and package inserts with instructions for use.

[0102] In some embodiments, the kit further includes a sample processing reagent, which may include at least one of a sample lysis reagent, a sample purification reagent, and a sample extraction reagent.

[0103] In some embodiments, the kit further includes at least one of a standard, a calibrator, a reference substance, and a buffer. The reference substance is a control substance for testing the effectiveness of the experiment and serves as a control for judging the results. The buffer can be any solution known in the art that can provide appropriate buffer conditions during the detection process.

[0104] In some embodiments, the kit may include instructions for use of a set of reagents. For example, the kit may include instructions for performing at least one assay, such as an immunoassay, a protein binding assay, an antibody-based assay, an antigen binding protein-based assay, a protein-based array, an enzyme-linked immunosorbent assay (ELISA), flow cytometry, a protein array, blots, Western blots, nephelometry, turbidimetry, chromatography, mass spectrometry, enzyme activity, and an immunoassay selected from RIA, immunofluorescence, immunochemiluminescence, immunoelectrochemiluminescence, immunoelectrophoresis, competitive immunoassay, and immunoprecipitation.

[0105] In addition to the above-mentioned components, the kit will further include instructions for implementing the subject method. These instructions can be present in the subject kit in various forms, one or more of which can be present in the kit. One form in which these instructions can exist is that they are printed information on a suitable medium or substrate, for example, one or more sheets of paper on which the information is printed, the paper is in the packaging of the kit or in the form of a package insert, etc.

[0106] In some embodiments of the present application, a system for detecting or analyzing biomarker levels to predict the severity of LN disease is also disclosed. The system may include a set of reagents for detecting the level of one or more markers; a device configured to receive a mixture of one or more reagents and a test sample obtained from a subject to measure the biomarker level of the present application; and a computer system that is coupled to the device in communication to obtain the measured level and determine a score that predicts the likelihood of LN disease severity in LN patients.

[0107] In some embodiments, the device is configured to detect the biomarker level in the mixture of reagent and test sample.For example, the device can determine the biomarker level of the present application by immunoassay or the assay for nucleic acid detection.The mixture of reagent and test sample can be provided to the device by various containers, and the examples of these containers include the hole, bottle or tube of the orifice plate (such as 96 orifice plate).Because of this, the device can have an opening (such as a slot, a cavity, an opening, a sliding tray), which can receive the container for accommodating the reagent test sample mixture and read to generate the quantitative expression value of a soluble medium.The example of the device includes an ELISA reader (such as a luminescent ELISA reader, an absorbent ELISA reader, a fluorescent ELISA reader), a spectrometer and a spectrophotometer.

[0108] In some embodiments, the computer system communicates with the device to receive the quantitative expression values ​​of the soluble mediators, and the computer system analyzes the quantitative expression values ​​and determines the likelihood of a LN disease activity event in the subject by applying a predictive model.

[0109] In some embodiments of the present application, the method or use of the present application can be performed on a computer, including detecting or analyzing the level of the biomarkers of the present application by a computer to predict LN disease.

[0110] For example, the construction and execution of a predictive model for generating a score (e.g., an LFPI score) can be implemented in hardware or software or a combination of both. In one embodiment, a readable storage medium is provided, such as a medium including a data storage material encoded with machine-readable data, which, when used with a machine programmed with instructions of the data, can display any data set and execution and results of the predictive model of the present application. Such data can be used for a variety of purposes, such as LN patient monitoring, diagnosis, treatment considerations, etc. The implementation scheme of the above method can be implemented in a computer program executed on a programmable computer, the computer including a processor, a data storage system, a graphics adapter, a network adapter, at least one input device and at least one output device, etc. The display is coupled to the graphics adapter. The program code is applied to the input data to perform the above functions and generate output information. The output information is applied to one or more output devices in a known manner. The computer can be a personal computer, a microcomputer, or a workstation of conventional design.

[0111] Each program can be implemented in a high-level program or object-oriented programming language to communicate with a computer system. However, if desired, the program can be implemented in assembly or machine language. In any case, the language can be a compiled language or an interpreted language. Each such computer program is preferably stored in a storage medium or device (such as a ROM or disk) readable by a general or special programmable computer, for configuring and operating the computer to execute the program of the present application when the computer reads the storage medium or device.

[0112] The system may also be considered to be implemented in the form of a computer-readable storage medium configured with a computer program, wherein the storage medium so configured causes a computer to operate in a specific and predefined manner to perform the functions of the present application.

[0113] In some embodiments, feature patterns and their databases can be provided in various media to facilitate their use. The term "medium" refers to a manufactured product containing feature pattern information of the present application. The database of the present application can be recorded on a computer-readable medium, such as any medium that a computer can directly read and access. Such media include, but are not limited to: magnetic storage media, such as floppy disks, hard disk storage media, and magnetic tapes; optical storage media, such as CD ROMs; electronic storage media such as RAM and ROM; and hybrids of these categories, such as magnetic / optical storage media. Those skilled in the art can easily understand how to use any currently known computer-readable medium to produce a record containing current database information. The term "record" refers to the process of storing information on a computer-readable medium using any such method known in the art. Based on the method used to access the stored information, any convenient data storage structure can be selected. A variety of data processor programs and formats can be used for storage, such as word processing text files, database formats, etc.

[0114] It can be understood that, based on the belief that the biomarkers of the present application can be used as diagnostic markers for lupus nephritis, technical personnel in this field can evaluate or screen other potential diagnostic markers for lupus nephritis based on this, and usually evaluate the potential biomarkers with the biomarkers of the present application through correlation, and on the basis of reaching the conclusion of positive correlation, it helps to illustrate the value of the potential markers.

[0115] The present application also relates to a method for detecting a marker in a subject suffering from or suspected of suffering from lupus nephritis in vivo or in vitro, the method comprising determining or detecting the level of the biomarker of the present application in a biological sample from the subject, and may include uses other than disease diagnosis.

[0116] In addition, the present application further relates to a method for in vivo or in vitro screening of lupus nephritis therapeutic agents, the method comprising the step of evaluating the biomarker level of the present application on a sample treated with the therapeutic agent, and evaluating the therapeutic effect of the therapeutic agent based on the biomarker level. For example, when the expression level of FCRL2 decreases, the therapeutic effect of the therapeutic agent can be determined.

[0117] The present application is further described in detail below through specific examples. The following examples are only used to further illustrate the present application and should not be construed as limiting the present application.

[0118] This application downloads 2 expression matrix files of peripheral blood mononuclear cell samples of lupus nephritis patients from the Gene Expression Omnibus (GEO) database, namely GSE99967 and GSE65391. Among them, the GSE99967 data set contains 29 SLE samples with LN and 13 SLE samples without LN, and the GSE65391 data set contains 924 SLE patients with or without LN and 72 control samples. After merging and normalizing the GSE99967 and GSE65391 data, all samples are allocated as discovery set and validation set according to 7:3.

[0119] Example 1. Expression analysis of EGLN1, FAM129C and FCRL2 in peripheral blood mononuclear cells (PBMC) of patients with lupus nephritis

[0120] The screening conditions for differentially expressed genes in this example are: multiple difference > 1.5, p < 0.05. It was found that FCRL2 was expressed lower in the disease group of the discovery set than in the normal group samples, that is, 541 LN samples and 49 control samples. The results are shown in Figure 1. The gene EGLN1 ( Figure 1A ) is upregulated in peripheral blood mononuclear cells (PBMCs) of patients with lupus nephritis, and FAM129C ( Figure 1B ) and FCRL2( Figure 1C ) were expressed at a low level in peripheral blood mononuclear cells (PBMCs) of patients with lupus nephritis, indicating that the above three genes can all be used as diagnostic markers for potential lupus nephritis.

[0121] Example 2: ROC-based evaluation analysis

[0122] In order to test the diagnostic efficacy of the candidate markers, the present example plotted the receiver operating characteristic (ROC) curves of EGLN1, FAM129C and FCRL2 in the discovery data set, and the results are shown in FIG2 .

[0123] The results in Figure 2 show that in the discovery set, the area under the ROC curve (AUC) value of EGLN1 is 0.641 ( Figure 2A ), the AUC value of FAM129C was 0.683 ( Figure 2B ), the AUC value of FCRL2 was 0.680( Figure 2C ), indicating that EGLN1, FAM129C and FCRL2 all have good performance in distinguishing lupus nephritis patients from normal people.

[0124] Example 3: Verification of the verification set

[0125] In this example, 154 LN samples and 22 control sample data cohorts were used for statistical analysis, and the screening conditions for differentially expressed genes were: difference fold>1.5, p<0.05, and the results are shown in Figure 3. ROC curves of EGLN1, FAM129C, and FCRL2 in the validation set were drawn, and the results are shown in Figure 4.

[0126] The results in Figure 3 show that the gene EGLN1 ( Figure 3A ) was significantly upregulated in PBMCs of patients with lupus nephritis, and FAM129C ( Figure 3B ) and FCRL2( Figure 3C ) was significantly downregulated in PBMCs of patients with lupus nephritis; at the same time, as shown in Figure 4, ROC analysis in this cohort revealed that the AUC of EGLN1 could reach 0.665 ( Figure 4A ), the AUC value of FAM129C was 0.776 ( Figure 4B ), the AUC value of FCRL2 was 0.695( Figure 4C ); fully demonstrated the specificity and sensitivity of EGLN1, FAM129C and FCRL2 in the diagnosis of lupus nephritis.

[0127] Example 4: Analysis of expression in patients with systemic lupus erythematosus without lupus nephritis (NoLN)

[0128] 4.1 Differences in gene expression between NoLN patients and normal subjects

[0129] In this example, 189 NoLN samples and 49 control sample data cohorts were used for expression difference statistical analysis, and the screening conditions were: difference fold>1.5, p<0.05. The results are shown in Figure 5. In the discovery set, EGLN1 ( Figure 5A )、FAM129C( Figure 5B ) and FCRL2( Figure 5C ) in PBMCs of NoLN patients and normal controls did not express more than 1.5 times, and p>0.05, indicating that EGLN1, FAM129C and FCRL2 are not diagnostic markers of NoLN.

[0130] In order to further verify the diagnostic effect of the marker, a statistical analysis was performed on a data cohort of 67 NoLN samples and 22 control samples. The results are shown in Figure 6. In the validation set, EGLN1 ( Fig. 6A )、FAM129C( Figure 6B ) and FCRL2( Figure 6C ) in the PBMCs of NoLN patients and normal controls did not express more than 1.5 times, and p>0.05, which fully proved that EGLN1, FAM129C and FCRL2 are not diagnostic markers of NoLN.

[0131] 4.2 Differences in gene expression between NoLN patients and LN patients

[0132] The expression difference statistical analysis was performed using 189 NoLN samples and 541 LN sample data cohorts. The results are shown in Figure 7. In the disease group of the discovery set, EGLN1 ( Fig. 7A )、FAM129C( Figure 7B ) and FCRL2( Figure 7C ) were differentially expressed between NoLN patients and LN patients (p<0.05), demonstrating that EGLN1, FAM129C, and FCRL2 can distinguish LN from NoLN patients.

[0133] In order to further verify the diagnostic effect of the marker, the expression difference statistical analysis was performed on the data cohorts of 67 NoLN samples and 154 LN samples. The results are shown in Figure 8. In the disease group of the validation set, EGLN1 ( Fig. 8A )、FAM129C( Figure 8B ) and FCRL2( Figure 8C ) were differentially expressed between NoLN patients and LN patients (p<0.05), which fully demonstrated that EGLN1, FAM129C and FCRL2 can distinguish LN and NoLN patients.

[0134] Example 5: Multi-marker combined detection

[0135] In this example, a statistical analysis was performed on a data cohort of 541 LN samples and 49 control samples, and ROC curves of three combined detection combinations of (1) EGLN1+FAM129C+FCRL2, (2) FCRL2+CD79B, and (3) CD79B+COBLL1+EGLN1+FAM129C+FCRL2 were plotted in the discovery set. The results are shown in FIG9 . In this cohort, the AUC value of the combined detection of EGLN1+FAM129C+FCRL2 was found to be 0.719 ( Fig. 9A ), the AUC value of FCRL2+CD79B combined detection can reach 0.833 ( Fig. 9B), the AUC value of CD79B+COBLL1+EGLN1+FAM129C+FCRL2 combined detection can reach 0.848 ( Fig. 9C ); It was demonstrated that EGLN1, FAM129C and FCRL2 can be combined with each other as markers for the joint detection of lupus nephritis, and can also be combined with other existing known diagnostic markers.

[0136] In order to further verify the effect of combined detection, statistical analysis was performed on the data cohort of 154 LN samples and 22 control samples, and the ROC curves of the three combined detection combinations of (1) EGLN1+FAM129C+FCRL2, (2) FCRL2+CD79B, and (3) CD79B+COBLL1+EGLN1+FAM129C+FCRL2 in the validation set were plotted. The results are shown in Figure 10. In this cohort, the AUC value of EGLN1+FAM129C+FCRL2 combined detection was found to be 0.799 ( Fig. 10A ), the AUC value of FCRL2+CD79B combined detection can reach 0.815( Fig. 10B ), the AUC value of CD79B+COBLL1+EGLN1+FAM129C+FCRL2 combined detection can reach 0.842 ( Fig. 10C ); It is fully proved that EGLN1, FAM129C and FCRL2 can be combined with each other as markers for the combined detection of lupus nephritis, and can also be combined with other existing known diagnostic markers for combined detection. When combined detection is performed, a higher AUC value can be obtained, which significantly improves the diagnostic efficiency.

[0137] Example 6: Clinical sample testing

[0138] In this case, 60 whole blood samples were collected from people, including 20 samples from patients with systemic lupus erythematosus and lupus nephritis, and 20 samples from patients with systemic lupus erythematosus and without lupus nephritis; at the same time, 20 whole blood samples from healthy people were collected as controls. The inclusion criteria for lupus nephritis patients in this case were: patients with a SLE disease activity index (SLEDAI) greater than 5.

[0139] 6.1 Detection of gene expression levels

[0140] After collecting all whole blood specimens, peripheral blood mononuclear cells were isolated, dissolved with Trizol (Beyotime, R-0016), and stored at -80°C. Total RNA was isolated from peripheral blood mononuclear cells according to the manufacturer's instructions. mRNA was reverse transcribed using the transScript all-in-one-first-strand cDNAsynthesis superMix for qPCR (One-step gDNAremoval) kit (TransGen Biotech, AT341-01). qPCR detection was performed using the PerfectStart Green qPCRSuperMix kit (TransGen Biotech, AQ601-02). The relative expression of genes was calculated using the 2-ΔCT method. p value < 0.05 was considered statistically significant.

[0141] The test results showed that the EGLN1 expression levels of 20 samples from patients with systemic lupus erythematosus and lupus nephritis were significantly higher than those of 20 control samples ( Fig.11A ), the expression levels of FAM129C and FCRL2 were significantly lower than those of 20 samples used as control samples ( Fig. 11B , Fig. 11C ); however, the expression levels of EGLN1, FAM129C and FCRL2 in the 20 samples of systemic lupus erythematosus without lupus nephritis were not significantly different from those in the 20 control samples ( Fig. 12A , Fig. 12B , Fig. 12C ). The results showed that there were statistical differences in the expression levels of EGLN1, FAM129C and FCRL2 genes between lupus nephritis patients and healthy subjects, and they can be used as biomarkers for lupus nephritis.

[0142] 6.2 Protein level expression detection

[0143] To detect FCRL2 protein in plasma samples, the Bradford method was first used to prepare the standard curve protein sample, and FCRL2 protein was used as the standard. A standard curve was usually drawn between 20 and 150 μg / 100 μL, and the concentration of the sample to be tested was calculated based on the standard curve. Next, SDS-PAGE electrophoresis was performed and transfected onto a PVDF membrane. Finally, the transfected membrane was blocked and incubated overnight with a monoclonal FCRL2-specific antibody, washed and incubated with an HRP-labeled secondary antibody for 1 hour, and DAB was used to develop the brown protein band. The results showed that there was a statistical difference in the FCRL2 protein level between lupus nephritis patients and healthy people, and it can be used as a biomarker for lupus nephritis.

[0144] It can be understood that although this example is a study on the expression level of the FCRL2 gene; however, it is foreseeable that there is a statistical difference in the expression level of the FCRL2 gene between lupus nephritis patients and healthy people, and there must be a statistical difference in the FCRL2 protein level between lupus nephritis patients and healthy people; therefore, FCRL2, as a biomarker for lupus nephritis, can be the FCRL2 gene or the FCRL2 protein, and lupus nephritis can be diagnosed based on the expression level of the FCRL2 gene or the expression level of the FCRL2 protein. On this basis, detection primers and / or probes can be designed based on the FCRL2 gene, or specific detection polypeptides can be designed based on the FCRL2 protein, and these primers, probes or polypeptides can be used as reagents for diagnosing lupus nephritis.

[0145] The protein level expression detection of other markers EGLN1 and FAM129C in the present application refers to the above method.

[0146] The above contents are further detailed descriptions of the present application in combination with specific implementation methods, and it cannot be determined that the specific implementation of the present application is limited to these descriptions. For ordinary technicians in the technical field to which the present application belongs, several simple deductions or substitutions can be made without departing from the concept of the present application.

[0147] Table 1. Gene-encoded proteins and functions

[0148]

Claims

1. Use of a reagent for detecting the level of one or more of the following biomarkers in the preparation of a lupus nephritis diagnostic product, wherein the biomarkers include EGLN1, FAM129C and FCRL2.

2. A product for the diagnosis of lupus nephritis, wherein: The product contains reagents for detecting the levels of one or more of the following biomarkers, including EGLN1, FAM129C and FCRL2.

3. The use according to claim 1 or the product according to claim 2, wherein: The biomarker level includes nucleic acid level or protein level; Optionally, the reagents for detecting the nucleic acid level include specific primers and / or probes designed for the nucleic acid of the biomarker; or The reagents for detecting the protein level include polypeptides that specifically bind to or recognize the biomarker protein.

4. The product according to claim 3, wherein: The nucleic acid includes the biomarker gene, a DNA fragment extracted from the biomarker gene, or RNA, mRNA, miRNA or LcRNA corresponding to the biomarker gene or its DNA fragment; or The polypeptide includes at least one of an antibody, an antigen binding fragment, a single-chain antibody, and a ligand that specifically binds to the biomarker protein.

5. The use according to claim 1 or the product according to any one of claims 2 to 4, wherein: The product may also include reagents or components for detecting other markers.

6. The use or product according to claim 5, wherein: The other markers include C3, C4, dsDNA, COBLL1, CD79B, HLA-DOB, SLPI, and GPR18.

7. A method for diagnosing lupus nephritis, wherein: The following steps are involved: (i) obtaining the level of a biomarker in a test sample, wherein the biomarker is selected from one or more of the following: EGLN1, FAM129C and FCRL2; (ii) comparing the biomarker level in the test sample with the corresponding biomarker level in the control sample; if there is a significant difference, the subject from which the test sample is derived has an indication of lupus nephritis; or, comparing the biomarker level in the test sample with a set threshold; if there is a significant difference between the biomarker level in the test sample and the threshold, the subject from which the test sample is derived has an indication of lupus nephritis.

8. A method for evaluating or screening lupus nephritis markers, wherein: This includes correlation or concordance analysis of candidate biomarkers with EGLN1, FAM129C, or FCRL2 levels.

9. A method for evaluating or screening a therapeutic agent for lupus nephritis, wherein: The method comprises evaluating the level of a biomarker in a sample after treatment with a therapeutic agent, wherein the biomarker is selected from one or more of the following: EGLN1, FAM129C and FCRL2.

10. The method according to any one of claims 7 to 9, wherein: The biomarker level includes nucleic acid level or protein level; Optionally, the nucleic acid level is the expression level of DNA, RNA or mRNA; or The protein level refers to the level of binding between the protein serving as a marker and its specific polypeptide.

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