Application of KLHDC8B in systemic lupus erythematosus diagnosis

Abstract

The invention relates to the technical field of disease diagnosis, in particular to application of KLHDC8B in systemic lupus erythematosus diagnosis. The invention discloses application of a detection agent or component for acquiring the KLHDC8B level in a sample in preparation of a product for diagnosing systemic lupus erythematosus, or application of the KLHDC8B level as a marker in diagnosis of systemic lupus erythematosus, and proves that KLHDC8B nucleic acid or protein can be used as a biomarker for diagnosis of systemic lupus erythematosus and has remarkable clinical value.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] The present invention claims priority to the Chinese patent application with application number: 202310693287.X, filed with the Chinese Patent Office on June 12, 2023, and invention name: “Application of KLHDC8B in the diagnosis of systemic lupus erythematosus”, the entire contents of which are incorporated by reference in the present disclosure. Technical Field

[0003] The present application relates to the field of disease diagnosis technology, and specifically to the application of KLHDC8B in the diagnosis of systemic lupus erythematosus and the preparation of diagnostic products. Background Art

[0004] Systemic lupus erythematosus (SLE) is a common chronic systemic autoimmune disease, with the main clinical features 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.

[0005] At present, a series of progress has been made in the pathogenesis and target research of systemic lupus erythematosus at home and abroad, and the clinical transformation of research results related to systemic lupus erythematosus is advancing with each passing day. Despite this, the field still faces the problem of few available drugs. The new drug Belimumab is only the fourth systemic lupus erythematosus treatment drug officially approved by the FDA. In addition, in the past few decades, glucocorticoids and traditional immunosuppressants have been the main drugs for the treatment of systemic lupus erythematosus. These drugs have large side effects. A considerable number of systemic lupus erythematosus patients do not respond well to these traditional treatments, and long-term traditional drug treatment may cause serious side effects to patients.

[0006] In terms of diagnosis, since systemic lupus erythematosus often affects multiple tissues and organs throughout the body, the clinical manifestations of patients are highly heterogeneous, which brings difficulties to clinical diagnosis. There are deficiencies in the diagnosis of systemic lupus erythematosus in China. The diagnostic process is complicated and mostly invasive: the current clinical diagnosis method is complex, including clinical symptoms and immunology and other tests, and systemic lupus erythematosus is diagnosed after scoring. In addition, lupus nephritis patients need tissue biopsy for diagnosis, which is an invasive test and brings great pain to patients.

[0007] Therefore, continuing to explore the potential therapeutic or diagnostic targets for systemic lupus erythematosus, exploring the mechanism of target action, and promoting new drug development and clinical transformation will be of great significance to the treatment of systemic lupus erythematosus.

[0008] In view of this, this application is filed. SUMMARY OF THE INVENTION

[0010] In order to solve the above technical problems, the present application screened the genes or proteins differentially expressed in peripheral blood mononuclear cells of patients with systemic lupus erythematosus and normal people through a series of bioinformatics methods, and surprisingly found that KLHDC8B can be used as an indication of systemic lupus erythematosus. Therefore, it is proposed that KLHDC8B nucleic acid or KLHDC8B protein can be used as a biomarker for the diagnosis and judgment of systemic lupus erythematosus, which makes up for the shortcomings of existing systemic lupus erythematosus-related indicators and has significant clinical value.

[0011] Therefore, this application has at least the following purposes:

[0012] The first purpose of this application is to seek a new use for the diagnosis of systemic lupus erythematosus;

[0013] The second purpose of this application is to seek a new product suitable for the diagnosis of systemic lupus erythematosus;

[0014] The third purpose of this application is to seek a new method for diagnosing systemic lupus erythematosus.

[0015] To achieve the above objectives, this application proposes the following specific technical solutions:

[0016] The present application first provides an application of KLHDC8B level as a marker in diagnosing systemic lupus erythematosus, or an application of a detection agent or component for obtaining KLHDC8B level in a sample in the preparation of a product for diagnosing systemic lupus erythematosus.

[0017] In some aspects, the product includes, but is not limited to, a kit form, a system device form, a computer readable medium, or a computer system form.

[0018] In some aspects, the KLHDC8B is used as an independent indicator for the diagnosis of systemic lupus erythematosus.

[0019] In some aspects, the levels include nucleic acid levels or protein levels.

[0020] In some aspects, the nucleic acid levels include DNA levels or RNA levels.

[0021] In some aspects, the nucleic acid level or protein level includes but is not limited to the abundance or concentration of nucleic acid or protein; in some aspects, it may also include such as the abundance or concentration of mutations of nucleic acid or protein.

[0022] Furthermore, the nucleic acid level is obtained by sequencing technology, nucleic acid amplification technology, nucleic acid hybridization technology, electrophoresis technology, mass spectrometry technology or chromatography technology.

[0023] 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.

[0024] Furthermore, the protein level is obtained by sequencing technology, immunoassay technology, electrophoresis technology, mass spectrometry technology or chromatography technology.

[0025] 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.

[0026] In some aspects, the product also includes a detection agent or component for obtaining the level of other markers.

[0027] Furthermore, the other markers include but are not limited to one or more of IFI27, ABCB1, ID3, EIF2AK2, C3, C4, dsDNA, HERC6, and PLSCR1.

[0028] In some aspects, the product further includes a sample processing reagent, and the sample processing reagent includes at least one of a sample lysis reagent, a sample purification reagent, and a sample extraction reagent.

[0029] In some aspects, the product further comprises at least one of a standard, a calibrator, a control, and a buffer.

[0030] In some aspects, the sample comprises tissue, cells, body fluid, serum, plasma, whole blood (peripheral blood), urine, semen, saliva, pleural effusion, ascites, cerebrospinal fluid, stool, or synovial fluid.

[0031] In some aspects, the sample comprises peripheral blood mononuclear cells.

[0032] In some aspects, the diagnosis includes, but is not limited to, screening for systemic lupus erythematosus, assisting in diagnosis, confirming the disease, predicting / judging the severity of the disease, assessing disease activity, and monitoring treatment.

[0033] The present application also provides a product for diagnosing systemic lupus erythematosus, including a detection agent or component for obtaining the level of KLHDC8B in a sample.

[0034] In some aspects, the product includes, but is not limited to, a product in the form of a kit, a system device, a computer readable medium, or a computer system.

[0035] In some aspects, the KLHDC8B can be used independently as an indicator for the diagnosis of systemic lupus erythematosus.

[0036] In some aspects, the levels include nucleic acid levels or protein levels.

[0037] In some aspects, the nucleic acid level or protein level includes but is not limited to the abundance or concentration of nucleic acid or protein; in some aspects, it may also include such as the abundance or concentration of mutations of nucleic acid or protein.

[0038] Furthermore, the nucleic acid level is obtained by sequencing technology, nucleic acid amplification technology, nucleic acid hybridization technology, electrophoresis technology, biological mass spectrometry technology or chromatography technology.

[0039] 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.

[0040] Furthermore, the protein level is obtained by sequencing technology, immunoassay technology, electrophoresis technology, mass spectrometry technology or chromatography technology.

[0041] Preferably, the method for obtaining protein levels includes but is 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 spectrum or liquid chromatography.

[0042] In some aspects, the product also includes a detection agent or component for obtaining the level of other markers.

[0043] Furthermore, the other markers include but are not limited to one or more of IFI27, ABCB1, ID3, EIF2AK2, C3, C4, dsDNA, HERC6, and PLSCR1.

[0044] In some aspects, the product further includes a sample processing reagent, and the sample processing reagent includes at least one of a sample lysis reagent, a sample purification reagent, and a sample extraction reagent.

[0045] In some aspects, the product further comprises at least one of a standard, a calibrator, a control, and a buffer.

[0046] In some aspects, the sample comprises tissue, cells, body fluid, serum, plasma, whole blood (peripheral blood), urine, semen, saliva, pleural effusion, ascites, cerebrospinal fluid, stool, or synovial fluid.

[0047] In some aspects, the sample comprises peripheral blood mononuclear cells.

[0048] In some aspects, the diagnosis includes, but is not limited to, screening for systemic lupus erythematosus, assisting in diagnosis, confirming the disease, predicting / judging the severity of the disease, assessing disease activity, and monitoring treatment.

[0049] The present application also provides a method for diagnosing systemic lupus erythematosus in vivo or in vitro, comprising the step of obtaining the level of KLHDC8B in a sample from a subject.

[0050] In some aspects, the method comprises the steps of:

[0051] (i) obtaining the level of KLHDC8B in the sample from the subject;

[0052] (ii) comparing the level of KLHDC8B with that of a control sample; wherein a significant difference in the level of KLHDC8B between the subject sample and the control sample is an indication that the subject suffers from systemic lupus erythematosus; or,

[0053] (ii) comparing with a set threshold absolute amount; wherein the level of the subject's sample being higher than the threshold absolute amount is an indication that the subject suffers from systemic lupus erythematosus.

[0054] In some aspects, the KLHDC8B can be used independently as an indicator for the diagnosis of systemic lupus erythematosus.

[0055] In some aspects, the levels include nucleic acid levels or protein levels.

[0056] In some aspects, the nucleic acid level or protein level includes but is not limited to the abundance or concentration of nucleic acid or protein; in some aspects, it may also include such as the abundance or concentration of mutations of nucleic acid or protein.

[0057] Furthermore, the nucleic acid level is obtained by sequencing technology, nucleic acid amplification technology, nucleic acid hybridization technology, electrophoresis technology, mass spectrometry technology or chromatography technology.

[0058] 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.

[0059] Furthermore, the protein level is obtained by sequencing technology, immunoassay technology, electrophoresis technology, mass spectrometry technology or chromatography technology.

[0060] 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.

[0061] In some aspects, the method further comprises the step of obtaining the levels of other markers in the subject's sample.

[0062] Furthermore, the other markers include but are not limited to one or more of IFI27, ABCB1, ID3, EIF2AK2, C3, C4, dsDNA, HERC6, and PLSCR1.

[0063] In some aspects, the sample comprises tissue, cells, body fluid, serum, plasma, whole blood (peripheral blood), urine, semen, saliva, pleural effusion, ascites, cerebrospinal fluid, stool, or synovial fluid.

[0064] In some aspects, the diagnosis includes, but is not limited to, screening for systemic lupus erythematosus, assisting in diagnosis, confirming the disease, predicting / judging the severity of the disease, assessing disease activity, and monitoring treatment.

[0065] In some aspects, the subject is preferably a human.

[0066] The present application also provides a method for detecting a marker in a subject having or suspected of having systemic lupus erythematosus in vivo or in vitro, the method comprising determining or detecting the level of KLHDC8B in a sample from the subject.

[0067] Furthermore, the method also includes determining or detecting the level of one or more of IFI27, ABCB1, ID3, EIF2AK2, C3, C4, dsDNA, HERC6, and PLSCR1 in a sample from a subject.

[0068] In some aspects, the method can be used for disease diagnosis or non-disease diagnosis.

[0069] The present application may also provide a method for evaluating or screening diagnostic markers for systemic lupus erythematosus, comprising the steps of performing correlation or consistency analysis between potential biomarkers and KLHDC8B, and verifying the diagnostic value of the potential markers through the conclusions of the correlation or consistency.

[0070] The present application also provides a method for in vivo or in vitro screening of a systemic lupus erythematosus therapeutic agent, the method comprising the step of evaluating the KLHDC8B level in a sample treated with the therapeutic agent.

[0071] The present application also provides a marker for diagnosing systemic lupus erythematosus, including KLHDC8B nucleic acid or protein; preferably also including one or more nucleic acids or proteins of ABCB1, ID3 and EIF2AK2.

[0072] The present application also provides a method for treating systemic lupus erythematosus, the method comprising the following steps:

[0073] (1) Obtaining TRIM25 levels in subject samples and comparing TRIM25 levels with those in control samples;

[0074] (2) Compared with the set threshold absolute amount, if the TRIM25 level in the subject's sample is higher than the threshold absolute amount, the subject is given standard oral SLE treatment, or the subject is given a Bruton's tyrosine kinase (BTK) inhibitor or a pharmaceutically acceptable salt thereof, or the subject is given an IRAK4 pathway inhibitor.

[0075] In some aspects, the standard oral SLE treatment includes, but is not limited to, one or more of oral corticosteroids, antimalarials, immunosuppressants, and biologics.

[0076] Furthermore, the corticosteroids include, but are not limited to, one or more of hydrocortisone, cortisone, prednisone, prednisolone, methylprednisolone, triamcinolone acetonide, betamethasone, and dexamethasone.

[0077] Furthermore, the antimalarial drugs include but are not limited to hydroxychloroquine.

[0078] Furthermore, the immunosuppressant includes, but is not limited to, one or more of azathioprine, methotrexate, mycophenolate mofetil, mycophenolate sodium, chloroquine, quinacrine, cyclophosphamide, mycophenolate mofetil, cyclosporine, and tacrolimus.

[0079] Furthermore, the biological agent includes but is not limited to one or more of belimumab, rituximab, and tetanuscept.

[0080] In some aspects, the BTK inhibitor is selected from an antibody, a binding polypeptide, a small molecule and / or a polynucleotide, such as the small molecule fenebrutinib.

[0081] In some aspects, the BTK inhibitor or a pharmaceutically acceptable salt thereof is used in an amount of 150 mg-400 mg / day.

[0082] In some aspects, the IRAK4 pathway inhibitor includes but is not limited to one or more of an IRAK4 inhibitor, an IRAK1 inhibitor, a toll-like receptor (TLR) inhibitor, an interleukin-1 receptor (IL-1R) inhibitor, an interleukin-33 receptor (IL-33R) inhibitor, and a myeloid differentiation major response gene 88 (MyD88) inhibitor.

[0083] Beneficial technical effects of this application:

[0084] This application proposes and confirms for the first time that KLHDC8B nucleic acid or protein can be used as a biomarker for the diagnosis of systemic lupus erythematosus by screening genes or their proteins that are differentially expressed in peripheral blood mononuclear cells of systemic lupus erythematosus patients and normal people, which makes up for the shortcomings of existing systemic lupus erythematosus disease diagnosis and has significant clinical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0086] Figure 1 , GEO data discovery set analysis results showed that the gene KLHDC8B was expressed at an elevated level in peripheral blood mononuclear cells (PBMCs) of patients with systemic lupus erythematosus;

[0087] Figure 2 , ROC curve analysis results show that the gene has good discrimination between patients and normal people;

[0088] Figure 3 , GEO validation set analysis results showed that the expression of gene KLHDC8B was increased in peripheral blood mononuclear cells (PBMC) of patients with systemic lupus erythematosus;

[0089] Figure 4 , ROC curve analysis results, validation set data showed that the gene had good discrimination between patients and normal people;

[0090] Figure 5 The results of Spearman correlation analysis revealed that the expression of KLHDC8B in peripheral blood mononuclear cells of patients with systemic lupus erythematosus was positively correlated with the disease activity score (SLEDAI), disease activity (Activity), and double-stranded DNA (dsDNA) of systemic lupus erythematosus (SLE), and negatively correlated with complement C3 and C4, indicating that the expression of this gene is related to the severity of the disease;

[0091] Figure 6 ,The results of logistic regression analysis revealed that the AUC value of KLHDC8B combined with ABCB1, ID3, EIF2AK2, HERC6 and EIF2AK2 in the discovery set data could reach 0.961;

[0092] Figure 7 , The results of ROC curve analysis of the discovery set showed that when KLHDC8B was combined with ABCB1, IFI27, PLSCR1, EIF2AK2, HERC6, and ID3, the AUC values ​​were increased to 0.896, 0.909, 0.904, 0.926, 0.920, and 0.893, respectively.

[0093] Figure 8 , clinical sample test results showed that there was a statistical difference in the expression level of KLHDC8B between patients with systemic lupus erythematosus and normal people. DETAILED DESCRIPTION OF THE INVENTION

[0095] The present application discloses an application of KLHDC8B in the diagnosis of systemic lupus erythematosus. Those skilled in the art can refer to the content of this article to realize its application. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all deemed to be included in this application. The method and application of this application have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the preparation method and application of this article without departing from the content, spirit and scope of this application to realize and apply the technology of this application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by ordinary technicians in the field to which this application belongs.

[0096] 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.

[0097] As used in this application, the terms "comprises", "comprising", "having", "containing" or "involving" are inclusive or open-ended and do not exclude other unrecited 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.

[0098] When referring to a singular noun an indefinite or definite article e.g. "a" or "an", "the" is used, this includes a plural of that noun.

[0099] The terms "approximately" and "substantially" 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% deviation from the indicated value, preferably ±5%.

[0100] In addition, the terms first, second, third, (a), (b), (c), and the like in the specification and claims are used to distinguish similar elements and are not necessarily required to describe a sequential or chronological order. It should be understood that the terms so used are interchangeable under appropriate circumstances, and the embodiments described in this application can be implemented in other sequences than those described or illustrated in this application.

[0101] References to embodiments of the present application will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present application. It will be apparent to those skilled in the art that various modifications and variations may be made to the present application without departing from the scope or spirit of the present application. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment. Therefore, it is intended that the present application covers such modifications and variations that fall within the scope of the appended claims and their equivalents. Other objects, features, and aspects of the present application are disclosed in or apparent from the following detailed description. It will be appreciated by those of ordinary skill in the art that this discussion is merely a description of exemplary embodiments and is not intended to limit the broader aspects of the present application.

[0102] Diagnostic Uses

[0103] This application screened out the protein KLHDC8B that is differentially expressed in peripheral blood mononuclear cells of patients with systemic lupus erythematosus and normal people through the discovery set, and drew the receiver operating characteristic (ROC) curve of KLHDC8B in the discovery data set. It was found that the area under the ROC curve (AUC) value of KLHDC8B in the discovery set was 0.813, indicating that it has good performance in distinguishing patients with systemic lupus erythematosus and normal people. The validation set analysis also found that the expression of KLHDC8B in peripheral blood mononuclear cells of patients with systemic lupus erythematosus was significantly increased; at the same time, the ROC analysis also found that the AUC value of KLHDC8B could reach 0.893.

[0104] Therefore, in various aspects of the present disclosure, provided are methods for diagnosing systemic lupus erythematosus (SLE) comprising KLHDC8B levels as a marker, and methods for obtaining KLHDC8B levels in a sample using a detection agent or component in the preparation of a product for diagnosing systemic lupus erythematosus.

[0105] The terms "systemic lupus erythematosus" and "SLE" as used herein are interchangeable, and are an inflammatory disease similar to rheumatoid arthritis in mechanism, and are caused by the imbalance of the human immune system, which causes the human body to form antibodies to fight against its own organs or tissues. Its clinical characteristics are that the clinical manifestations can be varied, and can involve multiple systems and multiple organs, and clinically, the disease remission and aggravation appear alternately. The onset is related to factors such as heredity, sex hormones, immunity, and environment. Specifically, it can refer to the criteria for diagnosing SLE 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.

[0106] As used herein, the terms "KLHDC8B" or "Kelch Domain Containing 8B" are used interchangeably, and mutations in its 5'-UTR can reduce its expression. KLHDC8B has been reported to be associated with classical Hodgkin's lymphoma.

[0107] As used herein, the term "diagnosis" refers to the process of identifying a medical disorder or disease (such as SLE) by its signs, symptoms, and especially the results of various diagnostic processes (including detecting the level of KLHDC8B in a biological sample (such as serum) obtained from an individual). Moreover, as used herein, the term "diagnosis" includes screening, auxiliary diagnosis, diagnosis (determining whether the disease exists), predicting or judging the severity of the disease, evaluating the activity of the disease, monitoring treatment, such as monitoring the deterioration or recurrence of the disease during treatment, evaluating the therapeutic efficacy of the disease, and the selection of a given treatment regimen. Without limitation, in some specific embodiments, the present application has fully confirmed that the level of KLHDC8B can be used to determine whether the subject has SLE, and can then be used for screening, auxiliary diagnosis, and diagnosis; in other specific embodiments, the present application is based on the evaluation of the correlation with SLEDAI, disease activity, dsDNA, C3 or C4, and confirms that the level of KLHDC8B can be used to predict or judge the severity of SLE, evaluate the activity of the disease, and can be used for monitoring patients after treatment. It is expected to be used to determine whether the condition has improved by severity or activity, and it is highly consistent with existing indicators.

[0108] As used herein, "treatment" 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., arresting its development; or (c) alleviating the disease, i.e., causing partial or complete improvement of the disease. The therapeutic agent may be administered before, during, or after the onset of the disease or injury.

[0109] The terms "individual," "subject," "subject," and "patient" are used interchangeably herein and refer to any mammalian subject, particularly a human, in need of diagnosis, treatment, or therapy.

[0110] As used herein, the term "sample", "sample", "test sample", "subject sample" etc. 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 SLE associated 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, cell (such as PMBC cell, T cell, etc.), body fluid, 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 a sample that is manipulated in any way after the sample is obtained, for example, by centrifugation, filtration, precipitation, dialysis, chromatography, treatment with reagents, washing or enrichment of some cell colonies. 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.

[0111] The term "ROC" or "ROC curve" as used herein may refer to a receptor operating characteristic curve. A ROC curve may be a graphical representation of the performance of a binary classifier system. For any given method, a ROC curve may 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 may determine the value or expected value of any unknown parameter. Unknown parameters may be determined using a curve fit for the ROC curve.

[0112] As used herein, the term "AUC" or "ROC-AUC" generally refers to the area under the receiver operating characteristic curve. This metric takes into account the sensitivity and specificity of a method and can provide a measure of the diagnostic utility of a method. Typically, ROC-AUC ranges from 0.5 to 1.0, where values ​​closer to 0.5 indicate that a method has limited diagnostic utility (e.g., lower sensitivity and / or specificity), and values ​​closer to 1.0 indicate that a 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

[0113] Diagnostic, Prognostic, or Screening Marker", Am. J. Epidemiol 2004, 159(9):882-890, which is incorporated herein by reference in its entirety. Other methods of 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.

[0114] The detection agent or component for obtaining the KLHDC8B level in the sample can be understood to include a detection reagent that directly obtains the KLHDC8B level in the sample, or a component that indirectly obtains the KLHDC8B level in the sample (such as a computer program that directly obtains an indicator of the KLHDC8B level in a sample that has been detected).

[0115] According to some embodiments of the present application, the marker level index can be obtained at the nucleic acid level, protein level, etc. The detection method is not limited, and any method that can be used to directly or indirectly evaluate the KLHDC8B nucleic acid or protein level is suitable for the present application.

[0116] It is understood that nucleic acid levels may include DNA levels or RNA levels. There are many ways to detect DNA or RNA levels in this area, 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, gel electrophoresis, Northern blotting, nucleic acid mass spectrometry or liquid chromatography.

[0117] For example, in some embodiments, the gene sequencing method 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.

[0118] For example, in some specific embodiments, the polymerase chain reaction method includes primers for KLHDC8B amplification, wherein the primer refers to a primer that can specifically amplify the KLHDC8B 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 part of the template sequence and serve as the starting point for synthesizing primer extension products. In some specific embodiments, the probe hybridization method includes a probe of KLHDC8B, wherein the probe refers to a probe that can specifically identify the KLHDC8B 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, generally referring to a nucleic acid probe that binds to another nucleic acid (i.e., a specific sequence of KLHDC8B as a target nucleotide) through complementary base pairing. In some specific embodiments, the gene chip refers to a composite structure formed by fixing an array of the aforementioned probes 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 KLHDC8B amplification.

[0119] It can be understood that there are many ways to detect proteins in the art, including but not limited to sequencing technology, immune technology, electrophoresis technology, biomass spectrometry technology or chromatography technology, all of which can be used in the present application; in some specific embodiments of the present application, including but 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.

[0120] For example, in some embodiments, the method for quantitatively detecting KLHDC8B at the protein level includes a specific antibody. Among them, the specific antibody refers to an antibody that can specifically recognize the KLHDC8B encoding protein, specifically including but not limited to a monoclonal antibody (e.g., a full-length or complete monoclonal antibody), a polyclonal antibody, a multivalent antibody, a multispecific antibody (e.g., a bispecific antibody with expected biological activity), a nanobody, or at least one of certain antibody fragments. It is understood that the antibody can be a human, humanized and / or affinity matured antibody.

[0121] According to the experimental data of the present application, the KLHDC8B can be used as an independent indication for the diagnosis of systemic lupus erythematosus, and an effective diagnosis of systemic lupus erythematosus can be achieved based only on KLHDC8B.

[0122] It is understandable that in order to further enhance the diagnostic effect of systemic lupus erythematosus, the biomarker detected by the reagent can also be other biological materials that can be used as markers, such as nucleic acid fragments, proteins, metabolites, etc. with diagnostic effects known to those skilled in the art. In this way, a combination of multiple biomarkers can be used, and they cooperate with each other to achieve a better diagnostic effect on systemic lupus erythematosus patients, thereby achieving a more effective diagnostic evaluation. Therefore, in addition to being an independent indication for the diagnosis of systemic lupus erythematosus, the KLHDC8B can also be detected in combination with other existing known diagnostic markers to improve the diagnostic efficiency.

[0123] For example, in some specific embodiments, the diagnosis also includes diagnostic reagents for detecting other genes or proteins, such as IFI27, ABCB1, ID3, EIF2AK2, C3, C4, dsDNA, HERC6, PLSCR1, etc. mentioned in this application. The combined detection can be combined with any one or more of them. In the preferred embodiment of the present application, when KLHDC8B and ABCB1, HERC6, ID3 and EIF2AK2 are used for the diagnosis and evaluation of systemic lupus erythematosus at the same time, they have a higher AUC value (0.961), which significantly improves the effect of distinguishing patients with systemic lupus erythematosus from normal people. In some embodiments of the present application, when KLHDC8B is used in conjunction with IFI27, ABCB1, ID3, EIF2AK2, HERC6, and PLSCR1, the AUC values ​​are increased to 0.909, 0.896, 0.893, 0.926, 0.920, and 0.904, respectively.

[0124] In some embodiments of the present application, the test sample described in the present application can be selected from tissues, cells, body fluids serum, plasma, whole blood (peripheral blood), urine, semen, saliva, pleural effusion, ascites, cerebrospinal fluid, feces and synovial fluid; in a preferred embodiment, the test sample is selected from any one of tissues, cells, serum, plasma, whole blood, urine, semen, and saliva. In some embodiments, the sample is peripheral blood mononuclear cells.

[0125] Diagnostic Methods

[0126] The applicant found that the level of KLHDC8B in peripheral blood monocytes of patients with systemic lupus erythematosus is higher than that in peripheral blood monocytes of normal people. Therefore, a more accurate prediction of the diagnosis of systemic lupus erythematosus can be made based on the level of KLHDC8B in peripheral blood monocytes (such as expression level).

[0127] The core of the diagnostic method includes the steps of detecting or determining KLHDC8B in a sample from a subject;

[0128] In some specific embodiments, the method comprises the following steps:

[0129] (i) obtaining the level of KLHDC8B in the test sample;

[0130] (ii) comparing the level of KLHDC8B with that of a control sample; wherein a significant difference in the level of KLHDC8B between the test sample and the control sample is an indication that the subject suffers from systemic lupus erythematosus;

[0131] or,

[0132] (ii) comparing with a set threshold absolute amount; wherein the level of the subject's sample being higher than the threshold absolute amount is an indication that the subject suffers from systemic lupus erythematosus.

[0133] In some embodiments, the “level of KLHDC8B” or “KLHDC8B level” herein includes but is not limited to the abundance or concentration of KLHDC8B nucleic acid or protein, such as in some specific embodiments, the expression abundance or concentration of the corresponding protein.

[0134] It is understandable that the control sample can be selected according to actual needs. For example, in disease diagnosis, the control sample is a normal population sample, while when used for prognosis assessment, it can be a control sample of different prognostic conditions.

[0135] In some specific embodiments, a set value for the expression level of KLHDC8B can be given, and the set value can be determined according to the expression level of KLHDC8B in normal samples of normal people and / or non-systemic lupus erythematosus patients. For example, the average or median value of the expression level of KLHDC8B of normal samples of an appropriate number of samples is selected, or a reasonable multiple is set based on the average value, median value, etc., such as 0.5 times or more, such as 1.5 times or more, such as 2 times or more, 2.5 times, 2 times, 1.9 times, 1.8 times, 1.7 times, 1.6 times, 1.5 times, 1.4 times, 1.3 times, 1.2 times, 1 times, 0.9 times, 0.8 times, 0.7 times, 0.6 times, 0.5 times, etc. When the expression level of KLHDC8B of the subject is higher than the set value, it is judged as systemic lupus erythematosus. It can be understood that the set value determined based on the mean value, or multiples of the mean value, needs to have good classification significance. The known samples can be tested using commonly used statistical test methods through classification based on the set value. When the result is statistically significant, it indicates that the set value can be used as a judgment criterion.

[0136] Wherein, the level of KLHDC8B refers to a value of this biomarker for a subject that is directly measured or further indirectly derived from direct measurement, which is usually derived at least in part from the abundance or concentration of the biomarker in the sample of the subject. Wherein, the indirectly derived value includes the value derived by applying a function to the measured value of this biomarker. Directly measured values ​​include, but are not limited to, the value of the biomarker determined by at least one of sequencing, hybridization, mass spectrometry, immunoassay, immunofluorescence, flow cytometry, etc.

[0137] product

[0138] According to the core diagnostic use of this application, the process for detecting KLHDC8B levels can be configured into a corresponding product form for diagnosis or prediction of systemic lupus erythematosus. The product includes reagents or components for detecting KLHDC8B biomarkers. 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.

[0139] Kit Format

[0140] In some embodiments of the present application, a kit for detecting or analyzing KLHDC8B levels to predict SLE disease is also disclosed herein. Such a kit may include reagents for detecting the level of one or more markers and instructions for predicting SLE disease based on the detected levels.

[0141] The kit may include a set of reagents for generating a data set by at least one assay. The set of reagents is capable of detecting quantitative KLHDC8B levels. The set of reagents may also further detect one or more other marker levels. In certain aspects, the reagents are detected at the level of nucleic acid or protein.

[0142] When the detection agent is detected at the nucleic acid level, 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, 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.

[0143] Thus, 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 is a PCR primer reagent, wherein the primer refers to a primer capable of specifically amplifying the KLHDC8B 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 is a probe reagent, wherein the probe refers to a probe capable of specifically identifying the KLHDC8B gene or a transcript of the gene, which is a molecule capable of binding 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 specific sequence of KLHDC8B as a target nucleotide) through complementary base pairing.

[0144] When the detection agent is used for detection at the protein level, it can be understood that there are many methods for protein detection 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 the present application; in some specific embodiments of the present application, including but 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.

[0145] Thus, for example, in some embodiments, the reagent is a specific antibody, wherein the specific antibody refers to an antibody that can specifically recognize the KLHDC8B encoded protein, specifically including but not limited to monoclonal antibodies (e.g., full-length or complete monoclonal antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., 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.

[0146] In some embodiments, such a kit may include a carrier, package or container that is compartmentalized to receive one or more containers, such as vials, tubes, etc., each container containing one of the individual elements to be used in the method. The kit of the present application may include a container as described above 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.

[0147] 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.

[0148] In some embodiments, the product further comprises 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.

[0149] 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.

[0150] In addition to the above components, the kit will further include instructions for implementing the subject method. These instructions can be present in the subject kit in a variety of forms, one or more of which can be present in the kit. One form in which these instructions can be present is as printed information on a suitable medium or substrate, for example, one or more sheets of paper on which the information is printed, the paper being in the packaging of the kit in the form of a package insert, etc.

[0151] System Devices

[0152] In some embodiments of the present application, a system for detecting or analyzing KLHDC8B levels to predict SLE disease is also disclosed herein. Such a 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 KLHDC8B level; 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 SLE disease in an SLE patient.

[0153] The device is configured to detect the KLHDC8B level in a mixture of a reagent and a test sample. For example, the device can determine the KLHDC8B level by an immunoassay or an assay for nucleic acid detection. The mixture of reagents and test samples can be provided to the device by various containers, examples of which include holes, vials or tubes of a well plate (e.g., a 96-well plate). Because of this, the device can have an opening (e.g., a slot, a cavity, an opening, a sliding tray) that can receive a container containing a reagent test sample mixture and read to generate a quantitative expression value of a soluble medium. Examples of devices include microplate readers (e.g., luminescent microplate readers, absorbance microplate readers, fluorescent microplate readers), spectrometers and spectrophotometers.

[0154] The computer system communicates with the device to receive the quantitative expression value of the soluble mediator. The computer system analyzes the quantitative expression value by applying a prediction model and determines the likelihood of an SLE disease activity event in the subject.

[0155] Computer system or computer readable medium

[0156] 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 KLHDC8B levels by a computer to predict SLE disease.

[0157] For example, the construction and execution of the prediction model for generating a score (for example, LFPI score) can be realized in hardware or software or a combination of the two. In one embodiment, a readable storage medium is provided, such as a medium including a data storage material encoded with machine-readable data, when using a machine programmed with instructions utilizing the data, the data storage material can display any data set of the prediction model of the application and execution and results. Such data can be used for multiple purposes, such as SLE patient monitoring, diagnosis, treatment considerations, etc. The embodiment of the above method can be realized in a computer program executed on a programmable computer, and the computer includes 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 a graphics adapter. Program code is applied to input data to perform the above functions and generate output information. Output information is applied to one or more output devices in a known manner. The computer can be, for example, a personal computer, a microcomputer or a workstation of traditional design.

[0158] 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 an 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 (e.g., ROM or disk) readable by a general or special programmable computer, for configuring and operating a computer to perform the program described herein when the computer reads the storage medium or device. The system can 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 the computer to operate in a specific and predefined manner to perform the functions described herein.

[0159] Feature patterns and their databases can be provided in various media to facilitate their use. "Medium" refers to a manufactured article 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 can be directly read and accessed by a computer. 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 any currently known computer-readable medium can be used to produce a record containing current database information. "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.

[0160] Other aspects

[0161] It can be understood that on the basis of believing that KLHDC8B can be used as a diagnostic marker for systemic lupus erythematosus, those skilled in the art can evaluate or screen other potential diagnostic markers for systemic lupus erythematosus based on this, and usually evaluate the correlation between potential biomarkers and KLHDC8B, and on the basis of reaching a conclusion of positive correlation, it helps to illustrate the value of the potential diagnostic marker.

[0162] The present application may also relate to a method for detecting a marker in a subject having or suspected of having systemic lupus erythematosus in vivo or in vitro, the method comprising determining or detecting the level of KLHDC8B in a biological sample from the subject, and the method may include uses other than disease diagnosis.

[0163] In addition, the present application may also relate to a method for in vivo or in vitro screening of systemic lupus erythematosus therapeutic agents, the method comprising the step of evaluating the KLHDC8B level of a sample treated with a therapeutic agent, and evaluating the therapeutic effect of the therapeutic agent based on the KLHDC8B level. For example, when the KLHDC8B expression level decreases, the therapeutic effect of the therapeutic agent can be determined.

[0164] The implementation scheme of the present application is described in detail below with reference to examples.

[0165] Dataset

[0166] This application downloads three expression matrix files of peripheral blood mononuclear cell samples of systemic lupus erythematosus patients from the Gene Expression Omnibus (GEO) database, namely GSE24706, GSE61635, and GSE65391. Among them, the GSE65391 data set contains 924 SLE and 72 control samples, the GSE61635 data set contains 99 SLE and 30 control samples, and the GSE24706 data set contains 28 SLE and 20 control samples. After merging and normalizing the GSE24706, GSE61635, and GSE65391 data, all samples are allocated as discovery sets and validation sets in a ratio of 7:3.

[0167] Example 1. Expression analysis of KLHDC8B in peripheral blood mononuclear cells (PBMC) of patients with systemic lupus erythematosus

[0168] In this example, 945 SLE samples and 85 control samples were used as metadata cohorts for analysis, and differentially expressed genes were screened (screening conditions: difference fold> 1.5, p<0.05). It was found that KLHDC8B was expressed higher in the disease group of the metadata dataset than in the normal group samples. Subsequently, the least absolute shrinkage and selection operator (LASSO) was used to screen potential diagnostic markers, and LASSO analysis was performed. KLHDC8B was also screened out. The results are shown in Figure 2. Figure 1 As shown, the gene KLHDC8B is expressed at an elevated level in peripheral blood mononuclear cells (PBMCs) of patients with systemic lupus erythematosus, and it is inferred that KLHDC8B is a potential diagnostic marker for systemic lupus erythematosus.

[0169] Example 2: ROC-based evaluation analysis

[0170] In order to test the diagnostic efficacy of the candidate marker, this example plotted the receiver operating characteristic (ROC) curve of KLHDC8B in the discovery data set. The results showed that the area under the ROC curve (AUC) value of KLHDC8B in the discovery set was 0.813 (see Figure 2 ), indicating that KLHDC8B has good performance in distinguishing patients with systemic lupus erythematosus from normal people.

[0171] Example 3: Verification of the verification set

[0172] In this example, a statistical analysis was performed on a data set of 371 SLE samples and 38 control samples, and it was found that KLHDC8B expression was also significantly increased in peripheral blood mononuclear cells of patients with systemic lupus erythematosus (see Figure 3 ); At the same time, ROC analysis in this cohort found that the AUC of KLHDC8B could reach 0.893 (see Figure 4 ), fully demonstrating the specificity and sensitivity of KLHDC8B in the diagnosis of systemic lupus erythematosus.

[0173] Example 4: Evaluation of correlation with SLEDAI, disease activity, dsDNA, C3, and C4

[0174] The systemic lupus erythematosus disease activity index (SLEDAI) measurement can indicate whether the SLE patient has not experienced a clinical attack, is experiencing a mild clinical attack, is experiencing a moderate clinical attack, or is experiencing a severe clinical attack. This example intends to detect the correlation between KLHDC8B expression and SLEDAI, and then determine the relationship between KLHDC8B expression and the severity of systemic lupus erythematosus.

[0175] The target antigen of anti-dsDNA antibodies is the double-base-paired DNA double helix structure, which is mainly found in systemic lupus erythematosus. It is currently recognized as a highly specific antibody and is also listed as one of the diagnostic criteria for lupus. The positive rate of anti-dsDNA antibodies in lupus is 60-90%, and the positive rate of lupus in the active stage is generally higher, while that in the inactive stage is correspondingly lower. Therefore, anti-dsDNA antibodies are closely related to the activity of lupus disease, and the antibody titer rises and falls with the activity and remission of the disease.

[0176] Complement is a group of heat-inlaborative proteins that exist in human or vertebrate serum and tissue fluid and have enzymatic activity after activation. It includes more than 30 soluble proteins and membrane-bound proteins, so it is called the complement system. It is widely involved in the body's anti-infection defense response and immune regulation, and can also mediate the damaging response of immunopathology. It is an effect amplification system with important biological effects in the body. Overactivation of the complement system and low complement levels are common pathological changes in SLE. Complement C3 and C4 are important biologically active proteins of the complement system, which are mainly synthesized by hepatocytes and macrophages. They participate in the classical complement activation pathway and the bypass activation pathway through activation and lysis, thereby mediating the complement cascade reaction, which is an important immune activity in the body.

[0177] From a dataset containing 85 normal subjects and 945 SLE peripheral blood mononuclear cell samples; Spearman correlation analysis was performed between the expression of KLHDC8B and SLE activity SLEDAI, disease activity, dsDNA, C3, and C4. The results showed that the expression of KLHDC8B was positively correlated with SLEDAI (correlation coefficient r = 0.24, p = 5.6e-14), positively correlated with anti-dsDNA antibodies (correlation coefficient r = 0.29, p = 2.2e-13), and negatively correlated with complement C3 and C4 (correlation coefficient r = -0.29, p = 4.6e-14; r = -0.22, p = 3.3e-08) (see Figure 5 ).

[0178] This result not only confirms the diagnostic value of KLHDC8B, but also shows that KLHDC8B expression is related to the severity of the disease and can be used to judge the severity of the disease. It also indicates that it can be used for post-treatment patient monitoring and prognosis assessment, and even for medication guidance.

[0179] Example 5: Evaluation of correlation with existing systemic lupus erythematosus diagnostic markers

[0180] This example tests whether the addition of KLHDC8B has a reinforcing effect on the detection of known systemic lupus erythematosus markers. KLHDC8B was combined with four reported diagnostic markers for systemic lupus erythematosus (applicable to Chinese patients), ABCB1, ID3, EIF2AK2 and HERC6, and the effect of the combination in distinguishing systemic lupus erythematosus from normal people was tested (AUC value evaluation). Similarly, subsequent analysis was performed in the metadata cohort of Example 1. The experiment found that when KLHDC8B was used in combination with ABCB1, ID3, EIF2AK2 and HERC6, the AUC value was 0.961 (see Figure 6 ), the combined use improved the differentiation effect between patients with systemic lupus erythematosus and normal people, further confirming the diagnostic value of KLHDC8B.

[0181] In addition, this example also combined with other systemic lupus erythematosus diagnostic markers IFI27, ABCB1, ID3, EIF2AK2, HERC6, and PLSCR1 to detect the effect of the combination in distinguishing systemic lupus erythematosus from normal people (AUC value evaluation). Similarly, subsequent analysis was performed in the metadata queue of Example 1. The experiment found that when KLHDC8B was used in combination with IFI27, ABCB1, ID3, EIF2AK2, HERC6, and PLSCR1, the AUC values ​​were increased to 0.909, 0.896, 0.893, 0.926, 0.920, and 0.904, respectively (see Figure 7 ). The combined use improved the differentiation effect between patients with systemic lupus erythematosus and normal subjects, further confirming the diagnostic value of KLHDC8B.

[0182] Example 6: Clinical sample testing

[0183] In this example, 29 samples of human whole blood were collected, including 14 samples from patients with systemic lupus erythematosus and 15 samples from normal subjects. Inclusion criteria for systemic lupus erythematosus patients: patients with a SLE disease activity index (SLEDAI) greater than 5. The levels of KLHDC8B in normal subjects and patients with systemic lupus erythematosus were detected.

[0184] After all the whole blood samples were collected, 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 cDNA synthesis superMix for qPCR (One-step gDNA removal) kit (TransGen Biotech, AT341-01). qPCR detection was performed using the PerfectStart Green qPCR SuperMix kit (TransGen Biotech, AQ601-02). The forward primer for KLHDC8B was 5′-TAGCTAGCCGAGGCGGAACGGCG-3′, and the reverse primer was 5′-GTAAGCTTGACCCCACGCAGACACAG-3′. The forward primer for the internal reference gene GAPDH was 5-TGACTTCAACAGCGACACCCA-3, and the reverse primer was 5-CACCCTGTTGCTGTAGCCAAA-3. IFI27 was used as a positive control. The relative expression of genes was calculated using the 2-ΔCT method. Unpaired T test statistical method was used. p value

[0185] <0.05 was considered statistically significant.

[0186] The results of clinical sample testing showed that the expression level of KLHDC8B in the whole blood of patients with systemic lupus erythematosus was increased, and there was a statistically significant difference in the expression level of KLHDC8B between patients with systemic lupus erythematosus and normal subjects (p = 0.011) (see Figure 8 ), and the expression of KLHDC8B was positively correlated with SLEDAI and disease activity. The results showed that KLHDC8B can well distinguish patients with systemic lupus erythematosus from normal people, and KLHDC8B is an ideal diagnostic marker for systemic lupus erythematosus.

[0187] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, a person of ordinary skill in the art should understand that the technical solutions described in the aforementioned embodiments can still be modified, or some or all of the technical features therein can be replaced by equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. Use of a detection agent or component for obtaining the level of KLHDC8B in a sample in the preparation of a product for diagnosing systemic lupus erythematosus, or use of KLHDC8B level as a marker in the diagnosis of systemic lupus erythematosus.

2. A product for diagnosing systemic lupus erythematosus, It is characterized in that Contains a detection agent or component for obtaining the level of KLHDC8B in a sample.

3. The use according to claim 1 or the product according to claim 2, It is characterized in that The level includes nucleic acid level or protein level.

4. The use or product according to claim 3, It is characterized in that The nucleic acid level is obtained by sequencing technology, nucleic acid amplification technology, nucleic acid hybridization technology, electrophoresis technology, biological mass spectrometry technology or chromatography technology; preferably, the nucleic acid level acquisition method includes but is not limited to any one of the following methods: gene sequencing method, polymerase chain reaction method, isothermal amplification reaction method, gene chip method, probe hybridization method, gel electrophoresis method, Northern blotting method, nucleic acid mass spectrometry or liquid chromatography.

5. The use or product according to claim 3, It is characterized in that The protein level is obtained by sequencing technology, immunoassay technology, electrophoresis technology, biomass spectrometry technology or chromatography technology; preferably, the protein level acquisition method includes but is 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 spectrometry or liquid chromatography.

6. The use according to claim 1 or the product according to claim 2, It is characterized in that The product also includes detection agents or components for obtaining the levels of other markers; preferably, the other markers include but are not limited to one or more of IFI27, ABCB1, ID3, EIF2AK2, C3, C4, dsDNA, HERC6, and PLSCR1.

7. The use according to claim 1 or the product according to claim 2, It is characterized in that The sample includes one or more of tissue, cells, body fluid, serum, plasma, whole blood, urine, semen, saliva, pleural effusion, ascites, cerebrospinal fluid, stool or synovial fluid.

8. A method for diagnosing systemic lupus erythematosus in vivo or in vitro, It is characterized in that The method comprises the steps of obtaining the level of KLHDC8B in the subject sample; Preferably, the method comprises the following steps: (i) obtaining the level of KLHDC8B in the sample from the subject; (ii) comparing the KLHDC8B level with that of a control sample; wherein a significant difference in the KLHDC8B level between the subject sample and the control sample is an indication that the subject suffers from systemic lupus erythematosus; or, (ii) comparing with a set threshold absolute amount; wherein the level of the subject's sample being higher than the threshold absolute amount is an indication that the subject suffers from systemic lupus erythematosus; Preferably, the method further comprises the step of obtaining the levels of other markers in the subject's sample. Preferably, the other markers include but are not limited to one or more of IFI27, ABCB1, ID3 and EIF2AK2, C3, C4, dsDNA, HERC6, and PLSCR1.

9. A method for detecting a marker in a subject having or suspected of having systemic lupus erythematosus in vivo or in vitro, It is characterized in that The method comprises determining or detecting the level of KLHDC8B in a sample from a subject; Preferably, the method further comprises determining or detecting the level of one or more of IFI27, ABCB1, ID3, EIF2AK2, C3, C4, dsDNA, HERC6, PLSCR1 in a sample from the subject.

10. A method for evaluating or screening diagnostic markers for systemic lupus erythematosus, It is characterized in that The method comprises the steps of performing correlation or consistency analysis between potential biomarkers and KLHDC8B.

11. A method for in vivo or in vitro screening of a therapeutic agent for systemic lupus erythematosus, It is characterized in that The method includes the step of evaluating the level of KLHDC8B in a sample treated with a therapeutic agent.

12. A marker for diagnosing systemic lupus erythematosus, It is characterized in that The markers include KLHDC8B nucleic acid or protein; Preferably, it also includes one or more nucleic acids or proteins of IFI27, ABCB1, ID3, EIF2AK2, C3, C4, dsDNA, HERC6, and PLSCR1.

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